Note: Descriptions are shown in the official language in which they were submitted.
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Control of Plastid Associated Protein Degradation I
This invention relates to plants with improved yield-related traits, including
delayed leaf
senescence, improved seedling survival, fruit ripening, grain size/starch
content and/or
stress tolerance. Such plants have plastids, e.g. chloroplasts, which are
altered in their
membrane protein composition so that transport of proteins in and out of the
plastids is
modified and controlled. Such modified plants may be made by mutagenesis and
selection, genetic engineering or gene editing. The invention therefore also
concerns
isolated nucleic acids, expression vectors or gene editing constructs and
associated host
cells and methods of using any of these to produce modified plants and
germplasm. Such
modified plant material has potential application in the generation and
breeding of new
plants.
BACKGROUND
Plastids are a diverse family of plant organelles. The family includes
chloroplasts ¨ the
organelles responsible for photosynthesis ¨ as well as a range of non-
photosynthetic
variants such as starch-containing amyloplasts in seeds, tubers and roots,
carotenoid-rich
chromoplasts in flowers and fruits, and chloroplast-precursor organelles in
dark-grown
plants called etioplasts (see Jarvis, P. and L6pez-Juez, E. (2013) Biogenesis
and
homeostasis of chloroplasts and other plastids Nat. Rev. Mol. Cell Biol. 14:
787-802.
Most plastid proteins are encoded by the nuclear genome and synthesized in the
cytosol
as precursors with N-terminal targeting signals called transit peptides.
Import of
precursors into chloroplasts is mediated by the TOC and TIC (Translocon at
the Outer/Inner envelope membrane of Chloroplasts) complexes. (See Jarvis, P.
(2008)
Targeting of nucleus-encoded proteins to chloroplasts in plants (Tansley
Review) New
Phytol. 179: 257-285 for more detail).
From Ling, Q., Huang, W., Baldwin, A. and Jarvis, P. (2012) Chloroplast
biogenesis is
regulated by direct action of the ubiquitin-proteasome system Science 338: 655-
659 and
Ling, Q. and Jarvis, P. (2013) Dynamic regulation of endosymbiotic organelles
by
ubiquitination Trends Cell Biol. 23: 399-408, it is known that plastid
biogenesis is directly
regulated by the ubiquitin-proteasome system (UPS). A screen of extragenic
suppressors
of the Arabidopsis plastid protein import mutation ppi1 - which is a knockout
mutant of a
translocon at the outer membrane of chloroplasts (Toc33) - identified
SUPPRESSOR
OF PPI1 LOCUS1 (SP1). SP1 encodes a RING-type ubiquitin E3 ligase in the
plastid
.. outer membrane that selectively targets the TOC machinery for
ubiquitination and
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degradation. By controlling the levels of different TOO receptor isoforms, SP1
regulates
which proteins are imported, and this in turn controls the plastid's proteome,
functions and
developmental fate (i.e., which type of plastid is formed and when and at what
point in time
of plant development).
W02014/037735 Al UNIVERSITY OF LEICESTER discloses transgenic plants with
altered expression of the SP1 gene or cDNA encoding SP1 (AT1G59560.1). Also,
transgenic plants in which the activity of SP1 protein is altered by
expression of a nucleic
acid encoding by a mutant SP1 gene or cDNA encoding a mutated SP1 protein.
Plastid
development can be accelerated by these changes in expression. For example, in
a
seedling the transition from etioplasts to chloroplasts is accelerated, or in
a seed the
transition to amyloplasts may be accelerated. Alternatively, inactivation of
SP1 such as by
downregulation of SP1 expression results in delaying of plastid development.
So, for
example, the transition from chloroplast to gerontoplast is delayed whereby a
"stay-green"
phenotype may be achieved. The RING domain of SP1 is identified as a
particular site for
.. mutation. Also disclosed are nucleic acid expression vectors and host cells
containing
such vectors. Further disclosed are methods of increasing plant yield by
increasing,
inactivating, repressing or down-regulating regulating the expression of a
nucleic acid
comprising the SP1 gene or cDNA encoding SPI, or a functional homologue or
variant
thereof; or introducing and expressing in a plant a nucleic acid comprising a
gene or cDNA
encoding a mutant SP1. Other identified phenotypes include improved seedling
emergence, growth and survival; alteration of fruit ripening; increased
tolerance to stress
such as salinity, osmotic stress and/or oxidative stress.
Also revealed was the role of SP1 in plant responses to abiotic stress. SP1 is
activated
under stress to deplete the TOO apparatus, thereby reducing the import of new
photosynthetic machinery components, attenuating photosynthetic activity, and
reducing
the potential for overaccumulation of harmful reactive oxygen species (see
Ling, Q., etal.,
(2012) supra. The vitally important nature of the functions of SP1, during
development and
stress, suggest agricultural applications linked to crop improvement.
Knowing that SP1 is an E3 ligase of the chloroplast outer membrane and
operates to
degrade TOO complexes, thus enabling reconfiguration of the import machinery
essential
for organellar protein changes occurring during development, this does not
explain fully
how the SP1 pathway in chloroplasts might work. The inventors undertook basic
research
in order to try and find suspected, but as yet unknown, further components of
the SP1
pathway.
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BRIEF SUMMARY OF THE DISCLOSURE
Accordingly, the present invention provides a modified plant cell, modified
plant or part
thereof, wherein the expression and/or activity of plastid SP2 protein
comprising an amino
acid sequence of SEQ ID NO: 3, or a variant or homologue thereof, is altered
compared to
the expression and/or activity of plastid 5P2 in an unmodified control or wild-
type (VVT)
plant cell, plant or part thereof.
The expression and/or activity of plastid 5P2 protein or variant or homologue
thereof may
be increased compared to expression and/or activity of plastid 5P2 in the
unmodified
control or VVT.
Alternatively, the expression and/or activity of plastid 5P2 protein or
variant or homologue
thereof may be decreased or eliminated compared to expression and/or activity
of plastid
5P2 in the unmodified control or VVT.
A "variant or homologue" of 5P2 may be defined as being a protein which
comprises an
amino acid sequence of at least 50% identity to reference sequence SEQ ID NO:
3;
optionally at least 55% identity therewith. Additionally, such definition of
"variant or
homologue" may include the biological function of 5P2 as further described
herein, so that
functional variants are disclosed herein, including such functional variants
as defined by
percentage sequence identity to the reference SEQ ID NO: 3.
Modified plant cells, modified plants or parts thereof as defined herein are
preferably
genetically modified, which may mean genetically engineered, e.g. transgenic,
or it may
mean gene edited, for example whereby a naturally occurring plant is modified
with, for
example, Cas9 gene editing, to alter from as few as a single nucleotide base
in a genomic
sequence. Plants arising from mutagenesis and screening are also included
amongst
what is meant by "modified plants". Further included in the invention are
modified plant
cells, plants or plant parts where the expression and/or activity levels of
5P2 (and the
optional SP1 described later) may be achieved solely by epigenetic changes,
preferably
heritable and stable epigenetic changes.
Modified plant cells, modified plants or parts thereof as defined herein may
be stably
transformed with additional genetic material. Such additional genetic material
is preferably
under the control of at least one regulatory sequence, but a multiplicity of
control points
may be built in, whether using native of modified regulatory sequences.
The regulatory sequence may be a promoter; optionally an inducible promoter,
preferably
then one which may be induced by an external stress condition. In the
alternative, a
constitutive promoter may be employed, e.g. cauliflower mosaic 35S.
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The regulatory sequence may optionally be tissue specific and/or
developmentally
regulated.
In modified plant cells, modified plants or parts thereof as defined herein,
when SP2 is
overexpressed there is preferably at least one copy of the additional genetic
material
compared to the unmodified or VVT.
Such additional genetic material as is described herein may comprise a
polynucleotide
comprising the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 2, or a
sequence of
at least 50% identity therewith, or a fragment thereof of at least 17
contiguous nucleotides
thereof.
A polynucleotide sequence or fragment thereof may be operably linked to a
promoter in a
sense or an antisense orientation if it is desired to employ an RNA-based
suppression or
knockdown of expression, of which there are many types known in the art.
In some modified plant cells, modified plants or parts thereof in accordance
with any
aspect of the invention, at least some of the expressed 5P2 has itself altered
activity
compared to unmodified control or VVT. This alteration of activity by way of
mutation or by
gene editing may be combined with increasing or decreasing the level of
expression of
5P2 compared to unmodified or VVT.
The activity of 5P2 which may be altered is preferably that of the 5P2 protein
association
with plastid protein SP1 or with Toc proteins; wherein SP1 may comprise an
amino acid
sequence of SEQ ID NO: 6 or a sequence of at least 55% identity therewith.
The 5P2 protein of altered activity is preferably lacking at least one, and up
to 27 of the N-
terminal amino acids of the protein sequence, e.g. SEQ ID NO: 3 or variants or
homologs
thereof.
As well as modified plant cells, modified plants or parts thereof based on
altered 5P2
expression and/or activity as hereinbefore defined, the invention includes the
combination
of all these possible aforementioned aspects and variations, together with an
altered
expression and/or activity of plastid SP1 protein. The SP1 protein comprises
an amino
acid sequence of SEQ ID NO: 6, or a sequence of at least 55% identity
therewith, and is
altered in expression and/or activity compared to plastid SP1 protein in an
unmodified
.. control or VVT plant.
In these combined SP1 and 5P2 aspects of the invention, expression and/or
activity of
SP1 may be increased compared to the expression and/or activity of SP1 in an
unmodified
control or VVT plant.
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Alternatively, the expression and/or activity of SP1 is decreased or
eliminated compared to
the expression and/or activity of SP1 in an unmodified control or VVT plant.
Where there is a polynucleotide encoding SP1 involved, this may comprise the
nucleotide
sequence of SEQ ID NO: 4 or SEQ ID NO: 5, or a sequence of at least 50%
identity
therewith, or a fragment of at least 17 contiguous nucleotides thereof.
In some aspects, the SP1 polynucleotide sequence or fragment thereof may be
operably
linked to a promoter in a sense or an antisense orientation if it is desired
to employ an
RNA-based suppression or knockdown of expression, of which there are many
types
known in the art. Possible within the scope of the invention for example is
for differing
methods of expression control for each of SP1 and 5P2. So SP1 may be altered
in
expression by one method, e.g. mutation in a regulatory element for SP1, and
5P2 may be
altered in expression by an RNA-based suppression approach.
At least some of the expressed SP1 may have an altered SP1 activity compared
to
unmodified control or VVT. This may be achieved by genetic modification, e.g.
known
transgenic approaches or by gene editing usually performed using Cas9.
In particularly preferred aspects, the activity of the SP1 protein which is
altered may be (a)
reduced or abolished interaction with plastid 5P2 protein; and/or (b) reduced
or abolished
E3 ligase activity; and/or (c) reduced or no association with plastid Toc
proteins.
In any of the aforementioned possibilities of the invention for modified plant
cells, modified
plants or parts thereof, plastids may be altered in at least some cells, when
compared to
an unmodified control or VVT. In this context, embodiments of the invention
are concerned
with spatial and/or temporal alterations in plastids and so considerations are
for tissue
specific and development specific controls being involved in making the
alterations
compared to VVT.
The alteration may for example be in the numbers and/or type(s) and/or
functions of plastid
in the cell(s). This can also serve to better adapt an existing plant by
modifying it to
tolerate better particular environmental conditions such as light quality and
level,
temperature fluctuations, minima and/or maxima, and/or water, saline or
osmotic stresses.
In accordance with the invention, the 5P2 including any variant or homologue
as herein
defined by way of the percentage identities of sequence, may be from a
different species,
genus, family or order of plant. Where SP1 is altered in expression and/or
activity in
combination with 5P2 alterations, then similarly this SP1 can independently be
selected
from a different species, genus, family or order of plant. The 5P2 and SP1
genes being
altered can be taken from the same or different plant species
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The invention therefore provides a modified plant as herein defined, which has
increased
expression and/or activity of SP2 protein; optionally also increased
expression and/or
activity of SP1 protein, in at least a particular cell type, and preferably
wherein there is (a)
increased tolerance to a stress condition compared to an unmodified control or
VVT plant;
more preferably wherein the stress condition is one or more of saline stress,
osmotic
stress or oxidative stress; and/or (b) accelerated fruit ripening; and/or (c)
the cell type is at
a seed setting stage and there is an increase in seed/grain size or starch
content; and/or
(d) the cell type is at seedling stage and there is increased seedling
survival, increased
seedling growth and/or emergence
A plant as mentioned above may have a decreased or no expression and/or
decreased or
no activity of SP2 protein; optionally decreased or no expression and/or no
decreased or
no activity of SP1 protein, in at least a particular cell type; preferably
wherein (a) the cell
type is green photosynthetic and there is a delay of senescence; or (b) the
plant is at seed
setting stage and there is an increase in seed/grain size or starch content;
or (c) the cell
type is at fruiting stage and there is a delaying of fruit ripening.
In particularly preferred aspects the modified plants of the invention
described herein are
crop plants, biofuel plants or horticultural plants.
In another aspect, the present invention provides an isolated polynucleotide
construct
comprising a promoter and:
a. a polynucleotide comprising a nucleotide sequence: (i) of SEQ ID NO: 1 or
a sequence of at least 50% identity thereto; or (ii) of SEQ ID NO: 2 or a
sequence of at least 50% identity thereto; or (iii) encoding a protein of
amino acid sequence SEQ ID NO: 3 or a sequence of at least 50% identity
thereto;
b. a polynucleotide comprising a fragment of at least 17 contiguous
nucleotides of: (i) SEQ ID NO: 1 or a sequence of at least 50% identity
thereto; or (ii) SEQ ID NO: 2 or a sequence of at least 50% identity thereto.
Such polynucleotide constructs will be of assistance to persons of average
skill in the
making of altered plant cells, plants and parts thereof, wherein the 5P2 gene
is altered in
expression and/or activity. The constructs of the invention lend themselves to
the full
range of known gene modification and gene expression modulation methods,
including
gene editing using Cas9 or Cpf1, for example
In modification of the above, there may also be provided an isolated
polynucleotide
construct which additionally comprises:
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a. a polynucleotide comprising a nucleotide sequence: (i) of SEQ ID NO: 4 or a
sequence of at least 55% identity thereto; or (ii) of SEQ ID NO: 5 or a
sequence of at least
55% identity thereto; or (iii) encoding a protein of amino acid sequence SEQ
ID NO: 6 or a
sequence of at least 55% identity thereto;
b. a polynucleotide comprising a fragment of at least 17 contiguous
nucleotides of: (i) SEQ ID NO: 4 or a sequence of at least 55% identity
thereto; or (ii) SEQ
ID NO: 5 or a sequence of at least 55% identity thereto.
The 5P2 encoding polynucleotide in the above may be downstream of the
promoter, and
the SP1 encoding polynucleotide may be downstream of the 5P2 encoding
polynucleotide.
Alternatively, these positions of 5P2 and SP1 may be reversed.
Described herein is also an isolated polynucleotide construct which comprises
a promoter
and:
a. a polynucleotide comprising a nucleotide sequence: (i) of SEQ ID NO: 4 or
a sequence of at least 55% identity thereto; or (ii) of SEQ ID NO: 5 or a
sequence of at least 55% identity thereto; or (iii) encoding a protein of
amino acid sequence SEQ ID NO: 6 or a sequence of at least 55% identity
thereto;
b. a polynucleotide comprising a fragment of at least 17 contiguous
nucleotides of: (i) SEQ ID NO: 4 or a sequence of at least 55% identity
thereto; or (ii) SEQ ID NO: 5 or a sequence of at least 55% identity
thereto.
The invention also includes the aforementioned isolated polynucleotides as
separate
polynucleotides, each with their own same or different promoters and optional
regulatory
and other features, forming a portion of a kit of parts for use in a binary
rather than single
construct approach to the alteration of 5P2 and SP1 protein activity and/or
expression in
plant cells, plants or parts thereof.
Such polynucleotides as aforementioned include Ti plasmids of Agrobacterium
tumefaciens which are well known in the art.
Included in the invention are vectors comprising a polynucleotide as
aforementioned. This
includes Agrobacterium tumefaciens, tobacco mosaic virus (TMV), potato virus X
and
cowpea mosaic virus.
Included therefore in the invention as another aspect are host cells
comprising a
polynucleotide or a vector as hereinbefore described. Such cells may not
necessarily be
plant cells when cloning, in vitro expression or genetic manipulation
procedures are being
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carried out as part of a series of experimental or developmental steps to
yield altered plant
material. So, such host cells may include bacteria, e.g. Agrobacterium or
Escherichia coli,
or yeast, e.g. Saccharomyces cerevisiae.
In further aspect, the invention provides a method of altering plastids in a
plant cell,
comprising:
a. increasing, decreasing or eliminating the expression in the cell of plastid
protein 5P2 comprising an amino acid sequence of SEQ ID NO: 3 or a
sequence of at least 50% identity therewith when compared to a control or
WT plant cell; and/or
b. increasing, decreasing or eliminating the activity in the cell of plastid
protein
5P2 comprising an amino acid sequence of SEQ ID NO: 3 or a sequence of
at least 50% identity therewith when compared to a control or VVT plant cell,
wherein the activity of 5P2 is that of (i) 5P2 association with plastid SP1
protein of SEQ ID NO: 6 or a sequence of at least 55% identity therewith,
and/or (ii) 5P2 association with Toc proteins.
In such a method of altering plastids, this may further (additionally)
comprise:
a. increasing, decreasing or eliminating the expression in the cell of plastid
protein SP1 comprising an amino acid sequence of SEQ ID NO: 6 or a
sequence of at least 55% identity therewith when compared to a control or
WT plant cell; and/or
b. increasing, decreasing or eliminating the activity in the cell of plastid
protein
SP1 comprising an amino acid sequence of SEQ ID NO: 6 or a sequence of
at least 55% therewith when compared to a control or VVT plant cell,
wherein the activity of SP1 is that of SP1 association with plastid 5P2
protein of SEQ ID NO: 3 or a sequence of at least 50% identity therewith,
and/or (ii) SP1 association with Toc proteins, and/or (iii) SP1 alteration of
E3 ligase activity.
In these aforementioned methods of altering plastids in a plant cell, the
expression of 5P2;
optionally also the expression of SP1, may be increased by the editing of a
nucleotide
sequence of at least one native 5P2 regulatory element in the cell; optionally
also at least
one SP1 native regulatory element in the cell, and/or by the insertion of a
polynucleotide
construct or a vector as hereinbefore defined, into the cell.
In some aforementioned methods of altering plastids in a plant cell, plastid
development
may be accelerated; optionally wherein the cell is (a) a seedling cell and the
transition of
etioplasts into chloroplasts is accelerated; or (b) a seed cell and the
transition to
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amyloplasts is accelerated; or (c) a fruit cell and the transition (a) from
chloroplasts, and/or
(b) to chromoplasts, is accelerated.
Alternatively, in other methods of altering plastids in plant cells, the
expression of 5P2;
optionally also the expression of SP1, may be decreased or eliminated by the
editing of a
.. nucleotide sequence of at least one native SP2 regulatory element in the
cell; optionally
also at least one SP1 native regulatory element in the cell, and/or by the
insertion of a
polynucleotide construct or a vector as hereinbefore defined, into the cell.
In methods of altering plastids where plastid development is delayed; the cell
is optionally:
(a) a green photosynthetic cell and the transition of chloroplasts into
another type of
plastid, e.g. gerontoplast, is delayed; or (b) a fruit cell and the transition
from chloroplasts
is delayed.
In any aspect of the invention herein, not excluded is the possibility that
SP2 expression
and/or activity may be increased whilst SP1 expression and/or activity may be
decreased,
with respect to a control or VVT. And vice versa, not excluded, SP2 expression
and/or
activity may be decreased whilst SP1 expression and/or activity may be
increased, with
respect to a control or VVT.
What is also possible in the present invention is that the ratio of SP2:SP1
expression or
activity may be altered compared to a control or VVT. This ratio may increase
or decrease
compared to control of VVT. Ratios may be a ratio in the range of
possibilities between 10:1
to 1:10.
Also included as part of the invention are uses of the SP2 gene of SEQ ID NO:
1 or cDNA
of SEQ ID NO: 2, or a polynucleotide sequence of at least 50% identity
therewith, or an at
least 17 nucleotide fragment thereof, for altering plastid protein composition
in plant cells.
Such uses therefore provide for alteration of plastid type, number, size,
protein content,
ultrastructural features, e.g. grana or inter-granal spaces. The alterations
are referenced
to unmodified control or VVT. The alterations in plastids may be quantitative
and/or
qualitative, temporal and/or spatial in a plant, so as to realise advantageous
physiological
and phenotypic changes, e.g. stress tolerance and/or yield increase. Also, to
achieve
particular desired changes in plant growth and development, e.g. seedling
emergence
rates, de-etiolation rates, fruit ripening and starch accumulation. Where a
method of the
invention is disclosed this may be interpreted as being equivalent to "use" of
the 5P2
material for the state purpose(s).
The invention in various aspects opens up a practical approach to increasing
plant yields
by extending the duration of active photosynthesis. "Stay-green" is a term
that is used to
describe mutant and transgenic plants or cultivars with the trait of
maintaining their leaves
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for a longer period of time than the wild-type or crosses from which they are
derived. The
sp2 mutants of the present invention and SP2 overexpressing plants may provide
the
necessary regulation of chloroplast longevity and thereby regulation of leaf
senescence
which allows the extension of duration of active photosynthesis in plants;
ideally crop
plants.
BRIEF DESCRIPTION OF THE DRAWINGS
Embodiments of the invention are further described hereinafter with reference
to the
examples and accompanying drawings, in which:Figure 1A is a photograph showing
phenotypes of 30-day-old sp2 suppressor and control plants grown on soil.
Figure 1B is a chart showing Chlorophyll contents of 10-day-old sp2 suppressor
and
control seedlings grown in vitro.
Figure 1C shows micrographs of cotyledon chloroplasts in 10-day-old sp2
suppressor and
control plants grown in vitro. Scale bar, 2 pm.
Figure 1D is estimated chloroplast cross-sectional area from Figure 1C.
Figure lE is thylakoid development estimated from Figure 1C.
Figure 1F is 2 shows protein import analysis using chloroplasts isolated from
sp2
suppressor and control plants, and corresponding quantification of the
maturation (mat) of
35S-labelled Rubisco small subunit precursor protein (pre).
Figure 1G is a domain map of the 5P2 protein. Grey box, 13-barrel domain;
black boxes,
predicted transmembrane spans. The sites of amino acid substitutions in two
sp2 mutant
alleles are indicated with grey triangles.
Figure 1H is an immunoblot analyses of total leaf protein extracts from the
indicated
genotypes, including sp2 ppil suppressors.
Figure 11 shows protein abundances of immunoblot of Figure 1H.
Figure 1J is an immunoblot analyses of total leaf protein extracts from the
indicated
genotypes of sp2 and toc75-III-3 single and double mutants.
Figure 1K shows protein abundances of immunoblot of Figure 1J.
Figure 1L is an immunoblot analyses of total leaf protein extracts from 5P2
overexpressors
(OX).
Figure 1M shows protein abundances of immunoblot of Figure 1L.
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Figure 1N is a photograph showing leaf senescence analysis of the indicated
genotypes
employing mature rosette leaves induced to senesce by covering with aluminium
foil.
Typical control (uncovered) and senescent (covered) leaves are shown (left).
Photochemical efficiency of photosystem II (Fv/Fm) was measured to estimate
the extent of
senescence (right).
Figure 10 shows the abiotic stress tolerance analysis of the indicated
genotypes
employing 14-day-old plants grown in vitro on NaCI medium. Typical plants
(left) and
chlorophyll contents (right) are shown. All values are means SEM (n 3
experiments or
samples).
Figure 2A is a photograph of VVT and indicated genotype plants grown on agar.
Figure 2B is a chart showing chlorophyll content of the plants of Figure 2A.
Figure 3A is a shows genetic mapping of the sp2 locus.
Figure 3B shows a 40bp alignment of of sp2-1 ppi1 reads to the ppi1 reference
genome.
Figure 30 shows a schematic representation of the SP2 (At3g44160) gene,
annotated with
the positions of the sp2 mutations.
Figure 3D shows analysis of SP2 mRNA expression in each of the sp2 mutant
alleles by
RT-PCR.
Figure 4A shows a Bayesian inference phylogenetic analysis of SP2 and OEP80.
Figure 4B shows structural models for the Arabidopsis SP2 and OEP80 proteins.
Figure 5 is an immunoblot showing P2 chloroplast localization and topology.
Figure 6 is an immunoblot showing enrichment of SP2 in isolated chloroplasts,
and
analysis of its interaction with TOO proteins by co-immunoprecipitation.
Figure 7A is a photograph of soil grown plants of the identified genotypes
used to
demonstrate the effect of the sp2 mutation on other TOO mutants.
Figure 7B is a chart showing the chlorophyll content of the plants in Figure
7A.
Figure 70 is a photograph of agar grown plants of the identified genotypes
used to
demonstrate the effect of the sp2 mutation on other TOO mutants.
Figure 7D is a chart showing the chlorophyll content of the plants in Figure
70.
Figure 8A is a photograph of agar grown plants of the identified genotypes
used to
.. demonstrate specificity of suppression mediated by the sp2 mutation.
Figure 8B is a chart showing the chlorophyll content of the plants in Figure
8A.
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Figure 9 is a chart of transcript levels for identified genotypes to show how
SP2 does not
affect TOO component transcript levels.
Figure 9B is a chart of transcript levels for SP2 overexpressor plants to show
how SP2
does not affect TOO component transcript levels.
Figure 10A is a photograph of agar grown plants of identified genotype used to
demonstrate phenotypes of sp2 single-mutant and SP2 overexpressor plants.
Figure 10B is a chart showing the chlorophyll content of the plants of Figure
10A.
Figure 100 is a gel showing semi-quantitative RT-PCR analysis of SP2
expression in wild-
type and SP2-0X plants.
Figure 10D is a chart quantitating the result of Figure 100.
Figure 11A is a photograph of agar grown plants of identified genotype,
including triple
mutants.
Figure 11B is a chart showing the chlorophyll content of the plants in Figure
11A.
Figure 110 is an immunoblot showing abundance of Toc75 protein in the plants
of Figure
11A.
Figure 11D is chart quantitating the protein abundance found in the immunoblot
of Figure
110.
Figure 11E is a photograph of agar grown SP2 overexpressing plants.
Figure 11F is an immunoblot of extracts of plants of Figures 11E.
Figure 11G is a chart quantitating the protein abundance found in the
immunoblot of Figure
11F.
Figure 11G is an immunoblot analysing TOO protein depletion in WT, sp2 mutant
and SP2
overexpressing plants.
Figure 11G is a chart quantitating the protein abundance found in the
immunoblot of Figure
11G.
Figure 13A is two-dimensional (2D)-blue native (BN)/SDS-PAGE analysis of SP1
and SP2
which shows how SP1 and SP2 proteins associate to form a complex.
Figure 13B is an in vitro pull-down analysis of the association between SP1
and SP2.
Figure 14A is a photograph of agar grown plants where the sp2 mutation is
introduced into
the SP1-0X ppil background.
Figure 14B is a chart showing the chlorophyll content of the plants of Figure
14A.
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Figure 15A is a table of sequence alignments using SP1 (At1g63900.2) as the
reference
sequence and whereby sequences were retrieved from EnsemblPlants by BLAST
(TBLASTN), and they were aligned (without any editing) using ClustalV in
DNAstar
Lasergene.
Figure 15B is a table of sequence alignments using 5P2 (At3g44160) as the
reference
sequence and whereby sequences were retrieved from EnsemblPlants by BLAST
(TBLASTN), and they were aligned (without any editing) using ClustalW in
DNAstar
Lasergene.
Figure 16 is a model diagram for SP1-5P2 action in the chloroplast membrane.
DETAILED DESCRIPTION
In the following passages, different aspects of the invention are explained in
more detail.
Each aspect explained or defined may be combined with any other aspect or
aspects,
unless explicitly indicated to the contrary. In particular, any feature
indicated as being
preferred or advantageous may be combined with any other feature or features
indicated
as being preferred or advantageous.
Conventional techniques of botany, microbiology, tissue culture, molecular
biology,
chemistry, biochemistry and recombinant DNA technology, gene editing and
bioinformatics
for use in employing the present invention are all readily known and available
to a person
of average skill in the art. Specific techniques are explained more fully in
the referenced
literature.
Described herein is a modified plant cell, modified plant or part thereof,
wherein the
expression and/or activity of plastid 5P2 protein comprising an amino acid
sequence of
SEQ ID NO: 3, or a variant or homologue thereof, is altered compared to the
expression
and/or activity of plastid 5P2 in an unmodified control or wild-type (VVT)
plant cell, plant or
part thereof.
The terms "peptide", "polypeptide" and "protein" are used interchangeably
herein and refer
to amino acids in a polymeric form of any length, linked together by peptide
bonds.
The term "plastid" refers to any plant plastid, including etioplasts,
chloroplasts,
amyloplasts, elaioplasts, chromoplasts or gerontoplasts. Preferably, the
plastid is a
chloroplast. Preferably, the development is the transition from one type of
plastid to
another. Preferably, plastid development is chloroplast development and more
preferably
refers to the transition of an etioplast into a chloroplast or the transition
of a chloroplast into
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a gerontoplast. In another embodiment, plastid development is the transition
from a
chloroplast into a chromoplast.
The expression and/or activity of plastid SP2 protein or variant or homologue
thereof may
be increased compared to expression and/or activity of plastid SP2 in the
unmodified
control or VVT.
Alternatively, the expression and/or activity of plastid SP2 protein or
variant or homologue
thereof may be decreased or eliminated compared to expression and/or activity
of plastid
SP2 in the unmodified control or VVT.
The terms "altered", "changed" and "modified" may be used interchangeably
herein. A
control plant as used herein is a plant which has not been modified.
Accordingly, the
control plant has not been genetically modified to alter either expression of
a nucleic acid
encoding SP2 or activity of a SP2 peptide as described herein. The control
plant may be a
wild type (VVT) plant. Even if a plant were transgenic, but not in respect of
SP2 (or
additionally SP1) then it could function as a control plant. The VVT or
control need not be
too specific, so long as it may provide a reliable reference against which SP2
expression
and/or activity can be measured in a modified plant material. The control
plant may be a
transgenic plant that does not have altered expression of SP2 or altered
activity of a SP2
peptide, but expresses a transgene that does not comprise a SP2 nucleic acid.
The terms "increase", "improve" or "enhance" are used interchangeably herein.
Yield of
SP2 expression levels for example are increased by at least a 3%, 4%, 5%, 6%,
7%, 8%,
9% or 10%, preferably at least 15% or 20%, more preferably 25%, 30%, 35%, 40%
or 50%
or more in comparison to a control plant. SP2 expression levels can be
measured by
routine methods in the art and compared to control plants.
The terms "reduce" or "decrease" are also used interchangeably herein. A
decrease, for
example in SP2 expression may be 3%, 4%, 5%, 6%, 7%, 8%, 9% or 10%, preferably
at
least 15% or 20%, more preferably 25%, 30%, 35%, 40% or 50% or more in
comparison to
a control plant.
Alternatively, a control plant may carry an expression vector only or carries
a mutant SP2
gene expressing a non-functional SP2 peptide. The control plant is typically
of the same
plant species, preferably having the same genetic background as the modified
plant.
The term "functional variant" as used herein refers to a variant gene or
peptide sequence
or part of the gene or peptide sequence which retains the biological function
of the full non-
variant SP2 sequence, for example confers altered plastid development when
expressed
in a plant. A functional variant also comprises a variant of the gene of
interest encoding a
peptide which has sequence alterations that do not affect function of the
resulting protein,
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for example in non-conserved residues. Also encompassed is a variant that is
substantially identical, i.e. has only some sequence variations, for example
in non-
conserved residues, to the wild type sequences as shown herein and is
biologically active,
for example complements the A. thaliana sp2 mutant.
A "variant or homologue" of SP2 may be defined as being a protein which
comprises an
amino acid sequence of at least 50% identity to reference sequence SEQ ID NO:
3;
optionally at least 55% identity therewith. Additionally, such definition of
"variant or
homologue" may include the biological function of 5P2 as further described
herein, so that
functional variants are disclosed herein, including such functional variants
as defined by
percentage sequence identity to the reference SEQ ID NO: 3.
The term "homologue" as used herein also designates an 5P2 orthologue from
other plant
species. A homologue (or variant) of AtSP2 polypeptide has, in increasing
order of
preference, at least 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%,
36%,
37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%,
52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%,
67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%,
82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,
97%, 98%, or at least 99% overall sequence identity to the amino acid
represented by
SEQ ID NO: 3. (The "at least" prefixes each and every percentage identity
listed above.)
Preferably, overall sequence identity is more than 49%, and in increasing
order of
preference more than 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%,
61%, 62%, 63%, 64%, 65%, 66%, 67%, 68% or more than 69%. (The "more than"
prefixes each and every percentage identity listed here.)
Preferably, overall sequence identity for 5P2 homologues or variants is at
least 70%, 71%,
72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%,
87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, most
preferably 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99%.
The overall sequence identity may be determined using a global alignment
algorithm
known in the art, such as the Needleman Wunsch algorithm in the program GAP
(GCG
Wisconsin Package, Accelrys).
Suitable homologues can be identified by sequence comparisons and
identifications of
conserved domains. There are predictors in the art that can be used to
identify such
sequences. The function of the homologue can be identified as described herein
and a
skilled person would thus be able to confirm the function when expressed in a
plant. Thus,
one of skill in the art will recognize that analogous amino acid substitutions
listed above
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with reference to SEQ ID NO: 3 can be made in 5P2 from other plants by
aligning the 5P2
polypeptide sequence to be mutated with the AtSP2 polypeptide sequence as set
forth in
SEQ ID NO: 3.
Modified plant cells, modified plants or parts thereof as defined herein may
be genetically
modified, which may mean genetically engineered, e.g. transformed so as to be
transgenic, or it may mean gene edited, for example whereby a naturally
occurring plant is
modified with, for example, Cas9 gene editing, to alter from a few as a single
nucleotide
base in a genomic sequence. Plant arising from mutagenesis and screening are
also
included amongst what is meant by "modified plants". Further included in the
invention are
modified plant cells, plants or plant parts where the expression and/or
activity levels of 5P2
(and the optional SP1 described later) may be achieved solely by epigenetic
changes,
preferably heritable and stable epigenetic changes.
The invention includes a method of producing a mutant plant expressing a 5P2
variant and
which is characterised by one of the phenotypes described herein, wherein said
method
uses mutagenesis and Targeting Induced Local Lesions in Genomes (TILLING) to
target
the gene expressing a 5P2 polypeptide. The method comprises mutagenising a
plant
population and selecting a plant with altered plastid development and
identifying the 5P2
variant. For example, mutagenesis is carried out using TILLING where
traditional chemical
mutagenesis is flowed by high-throughput screening for point mutations. The
plants are
screened for one of the phenotypes described herein, for example a plant that
shows
delayed/accelerated plastid development or improved yield. A 5P2 locus is then
analysed
to identify a specific 5P2 mutation responsible for the phenotype observed.
Plants can be
bred to obtain stable lines with the desired phenotype and carrying a mutation
in a 5P2
locus. In one embodiment, germplasm is screened.
Thus, plants with different genotypes, induced through artificial means, or
alternatively
having originated through natural sequence divergence, may be screened for the
expression of the endogenous 5P2 gene to identify germ plasm or plants with
particular
plastid development or yield characteristics.
In one embodiment, the method used to create and analyse mutations is
targeting induced
local lesions in genomes. In this method, seeds are mutagenised with a
chemical
mutagen. The mutagen may be fast neutron irradiation or a chemical mutagen,
for
example selected from the following non-limiting list: ethyl methanesulfonate
(EMS),
methylmethane sulfonate (MMS), N-ethyl-N-nitrosurea (EN U), triethylmelamine
(1 EM), N-
methyl-N-nitrosourea (MNU), procarbazine, chlorambucil, cyclophosphamide,
diethyl
sulfate, acrylamide monomer, melphalan, nitrogen mustard, vincristine,
dimethylnitosamine, N-methyl-N'-nitro-nitrosoguanidine (MN NG),
nitrosoguanidine, 2-
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aminopurine, 7,12 dimethyl-benz(a)anthracene (DM BA), ethylene oxide,
hexamethylphosphoramide, bisulfan, diepoxyalkanes (diepoxyoctane (DEO),
diepoxybutane (BEB), and the like), 2-methoxy-6-chloro-9 [3-(ethyl-2-
chloroethyl)aminopropylamino]acridine dihydrochloride (ICR-170) or
formaldehyde.
The resulting M1 plants are self-fertilised and the M2 generation of
individuals is used to
prepare DNA samples for mutational screening. DNA samples are pooled and
arrayed on
microtiter plates and subjected to gene specific PCR. The PCR amplification
products
may be screened for mutations in the SP2 target gene using any method that
identifies
heteroduplexes between wild-type and mutant genes. For example, but not
limited to,
denaturing high pressure liquid chromatography (dHPLC), constant denaturant
capillary
electrophoresis (C DOE), temperature gradient capillary electrophoresis
(TGCE), or by
fragmentation using chemical cleavage. Preferably the PCR amplification
products are
incubated with an endonuclease that preferentially cleaves mismatches in
heteroduplexes
between wild-type and mutant sequences. Cleavage products are electrophoresed
using
an automated sequencing gel apparatus, and gel images are analyzed with the
aid of a
standard commercial image-processing program. Any primer specific to the SP2
gene
may be utilized to amplify the SP2 genes within the pooled DNA sample.
Preferably, the
primer is designed to amplify the regions of the SP2 gene where useful
mutations are most
likely to arise, specifically in the areas of the SP2 gene that are highly
conserved and/or
confer activity. To facilitate detection of PCR products on a gel, the PCR
primer may be
labelled using any conventional labelling method.
Rapid high-throughput screening procedures thus allow the analysis of
amplification
products for identifying a mutation conferring the reduction or inactivation
of the expression
of the SP2 gene as compared to a corresponding non-mutagenised wild-type
plant. Once
a mutation is identified in a gene of interest, the seeds of the M2 plant
carrying that
mutation are grown into adult M3 plants and screened for the phenotypic
characteristics
associated with the SP2 gene. Loss of and reduced function mutants with
increased yield
or increased/delayed plastid development compared to a control plant can thus
be
identified.
For example, a "plant part" may be green tissue, for example a leaf. In using
green tissue
the transition from chloroplast to gerontoplast might be desired to be
delayed. A plant part
may be a fruit and the transition from chloroplast to chromoplast might be
desired to be
delayed.
When employed in the invention herein, "transgenic", "transgene" or
"recombinant" means
with regard to, for example, a nucleic acid sequence, an expression cassette,
gene
construct or a vector comprising the nucleic acid sequence or an organism
transformed
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with the nucleic acid sequences, expression cassettes or vectors according to
the
invention, all those constructions brought about by recombinant methods in
which either
(a) the nucleic acid sequences encoding proteins useful in the methods of the
invention, or
(b) genetic control sequence(s) which is operably linked with the nucleic acid
sequence
according to the invention, for example a promoter, or (c) a) and b) are not
located in their
natural genetic environment or have been modified by recombinant methods, it
being
possible for the modification to take the form of, for example, a
substitution, addition,
deletion, inversion or insertion of one or more nucleotide residues. The
natural genetic
environment is understood as meaning the natural genomic or chromosomal locus
in the
original plant or the presence in a genomic library. In the case of a genomic
library, the
natural genetic environment of the nucleic acid sequence is preferably
retained, at least in
part. The environment flanks the nucleic acid sequence at least on one side
and has a
sequence length of at least 50 bp, preferably at least 500 bp, especially
preferably at least
1000 bp, most preferably at least 5000 bp. A naturally occurring expression
cassette - for
example the naturally occurring combination of the natural promoter of the
nucleic acid
sequences with the corresponding nucleic acid sequence encoding a polypeptide
useful in
the methods of the present invention, as defined above - becomes a transgenic
expression
cassette when this expression cassette is modified by non-natural, synthetic
("artificial")
methods such as, for example, mutagenic treatment. Suitable methods are
described, for
example, in US 5,565,350 or WO 00/15815 both incorporated by reference.
Where the invention may provide a transgenic plant, the nucleic acids used in
the method
of the invention are not at their natural locus in the genome of said plant,
it being possible
for the nucleic acids to be expressed homologously or heterologously. Thus,
the plant
expresses a transgene. However, as mentioned, in certain embodiments,
transgenic may
means that, while the nucleic acids according to the different embodiments of
the invention
are at their natural position in the genome of a plant, the sequence has been
modified with
regard to the natural sequence, and/or that the regulatory sequences of the
natural
sequences have been modified, for example by mutagenesis.
Transgenic is preferably understood as meaning the expression of the nucleic
acids
according to the invention at an unnatural locus in the genome, i.e.
homologous or,
preferably, heterologous expression of the nucleic acids takes place.
According to the
invention, the transgene is stably integrated into the plant and the plant is
preferably
homozygous for the transgene.
Modified plant cells, modified plants or parts thereof as defined herein may
be stably
transformed with additional genetic material. Such additional genetic material
is preferably
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under the control of at least one regulatory sequence, but a multiplicity of
control points
may be built in, whether using native of modified regulatory sequences.
Transformation of plants is now a routine technique in many species.
Advantageously,
any of several transformation methods may be used to introduce the gene of
interest into a
suitable ancestor cell. The methods described for the transformation and
regeneration of
plants from plant tissues or plant cells may be utilized for transient or for
stable
transformation. Transformation methods include the use of liposomes,
electroporation,
chemicals that increase free DNA uptake, injection of the DNA directly into
the plant,
particle gun bombardment, transformation using viruses or pollen and
microprojection.
Methods may be selected from the calcium/polyethylene glycol method for
protoplasts,
electroporation of protoplasts, microinjection into plant material, DNA or RNA-
coated
particle bombardment, infection with (non-integrative) viruses and the like.
Transgenic
plants, including transgenic crop plants, are preferably produced via
Agrobacterium
tumefaciens mediated transformation.
Transformation methods are well known in the art. Thus, according to the
various aspects
of the invention, a nucleic acid comprising a SP2 nucleic acid, for example
SEQ ID NO: 1
or 2, or a functional variant or homolog thereof, is introduced into a plant
and expressed as
a transgene. The nucleic acid sequence is introduced into said plant through a
process
called transformation. The term "introduction" or "transformation" as referred
to herein
encompasses the transfer of an exogenous polynucleotide into a host cell,
irrespective of
the method used for transfer. Plant tissue capable of subsequent clonal
propagation,
whether by organogenesis or embryogenesis, may be transformed with a genetic
construct
of the present invention and a whole plant regenerated there from. The
particular tissue
chosen will vary depending on the clonal propagation systems available for,
and best
suited to, the particular species being transformed. Exemplary tissue targets
include leaf
disks, pollen, embryos, cotyledons, hypocotyls, megagametophytes, callus
tissue, existing
meristematic tissue (e.g., apical meristem, axillary buds, and root
meristems), and induced
meristem tissue (e.g., cotyledon meristem and hypocotyl meristem). The
polynucleotide
may be transiently or stably introduced into a host cell and may be maintained
non-
integrated, for example, as a plasmid. Alternatively, it may be integrated
into the host
genome. The resulting transformed plant cell may then be used to regenerate a
transformed plant in a manner well known in the art.
To select transformed plants, plant material obtained in the transformation
is, as a rule,
subjected to selective conditions so that transformed plants can be
distinguished from
untransformed plants. For example, seeds obtained in the above-described
manner can
be planted and, after an initial growing period, subjected to a suitable
selection by
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spraying. A further possibility is growing the seeds, if appropriate after
sterilization, on
agar plates using a suitable selection agent so that only the transformed
seeds can grow
into plants. Alternatively, the transformed plants are screened for the
presence of a
selectable marker such as the ones described above. Following DNA transfer and
regeneration, putatively transformed plants may also be evaluated, for
instance using
Southern analysis, for the presence of the gene of interest, copy number
and/or genomic
organisation. Alternatively or additionally, expression levels of the newly
introduced DNA
may be monitored using Northern and/or Western analysis, both techniques being
well
known in the art.
The generated transformed plants may be propagated by a variety of means, such
as by
clonal propagation or classical breeding techniques. For example, a first
generation (or Ti)
transformed plant may be selfed and homozygous second-generation (or T2)
transformants selected, and the T2 plants may then further be propagated
through
classical breeding techniques. The generated transformed organisms may take a
variety
of forms. For example, they may be chimeras of transformed cells and non-
transformed
cells; clonal transformants (e.g., all cells transformed to contain the
expression cassette);
grafts of transformed and untransformed tissues (e.g., in plants, a
transformed rootstock
grafted to an untransformed scion).
The regulatory sequence may be a promoter; optionally an inducible promoter,
preferably
.. then one which may be induced by an external stress condition. In the
alternative, a
constitutive promoter may be employed, e.g. cauliflower mosaic 35S.
The regulatory sequence may optionally be tissue specific.
The term "regulatory element" as used herein may be considered interchangeably
with
"control sequence" and "promoter" and all terms are to be taken in a broad
context to refer
to regulatory nucleic acid sequences capable of effecting expression of the
sequences to
which they are ligated. The term "promoter" typically refers to a nucleic acid
control
sequence located upstream from the transcriptional start of a gene and which
is involved
binding of RNA polymerase and other proteins, thereby directing transcription
of an
operably linked nucleic acid. Encompassed by the aforementioned terms are
.. transcriptional regulatory sequences derived from a classical eukaryotic
genomic gene
(including the TATA box which is required for accurate transcription
initiation, with or
without a CCAAT box sequence) and additional regulatory elements (i.e.
upstream
activating sequences, enhancers and silencers) which alter gene expression in
response
to developmental and/or external stimuli, or in a tissue- specific manner.
Also included
within the term is a transcriptional regulatory sequence of a classical
prokaryotic gene, in
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which case it may include a -35 box sequence and/or -10 box transcriptional
regulatory
sequences.
The term "regulatory element" also encompasses a synthetic fusion molecule or
derivative
that confers, activates or enhances expression of a nucleic acid molecule in a
cell, tissue
or organ.
A "plant promoter" comprises regulatory elements, which mediate the expression
of a
coding sequence segment in plant cells. Accordingly, a plant promoter need not
be of
plant origin, but may originate from viruses or micro-organisms, for example
from viruses
which attack plant cells. The "plant promoter" can also originate from a plant
cell, e.g. from
the plant which is transformed with the nucleic acid sequence to be expressed.
This also
applies to other "plant" regulatory signals, such as "plant" terminators. The
promoters
upstream of the nucleotide sequences useful in the methods of the present
invention can
be modified by one or more nucleotide substitution(s), insertion(s) and/or
deletion(s)
without interfering with the functionality or activity of either the
promoters, the open reading
frame (ORF) or the 3'-regulatory region such as terminators or other 3'
regulatory regions
which are located away from the ORF. Also possible is that the activity of the
promoters is
increased by modification of their sequence, or that they are replaced
completely by more
active promoters, even promoters from heterologous organisms. For expression
in plants,
the nucleic acid molecule is, as described above, preferably linked operably
to or
comprises a suitable promoter which expresses the gene at the right point in
time and with
the required spatial expression pattern. For the identification of
functionally equivalent
promoters, the promoter strength and/or expression pattern of a candidate
promoter may
be analysed for example by operably linking the promoter to a reporter gene
and assaying
the expression level and pattern of the reporter gene in various tissues of
the plant.
Suitable well-known reporter genes are known to the skilled person and include
for
example beta-glucuronidase or beta-galactosidase.
The term "operably linked" as used herein refers to a functional linkage
between the
promoter sequence and the gene of interest, such that the promoter sequence is
able to
initiate transcription of the gene of interest.
For example, the nucleic acid sequence may be expressed using a promoter that
drives
overexpression. Overexpression according to the invention means that the
transgene is
expressed at a level that is higher than expression of endogenous counterparts
driven by
their endogenous promoters. For example, overexpression may be carried out
using a
strong promoter, such as a constitutive promoter. A "constitutive promoter"
refers to a
promoter that is transcriptionally active during most, but not necessarily
all, phases of
growth and development and under most environmental conditions, in at least
one cell,
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tissue or organ. Examples of constitutive promoters include the cauliflower
mosaic virus
promoter (CaMV35S or 19S), rice actin promoter, maize ubiquitin promoter,
rubisco small
subunit, maize or alfalfa H3 histone, OCS, SAD1 or 2, GOS2 or any promoter
that gives
enhanced expression. Alternatively, enhanced or increased expression can be
achieved
by using transcription or translation enhancers or activators and may
incorporate
enhancers into the gene to further increase expression. Furthermore, an
inducible
expression system may be used, where expression is driven by a promoter
induced by
environmental stress conditions (for example the pepper pathogen-induced
membrane
protein gene CaPIMPI or promoters that comprise the dehydration-responsive
element
(DRE), the promoter of the sunflower HD-Zip protein genes Hahbl or Hahb4,
which is
inducible by water stress, high salt concentrations and ABA or a chemically
inducible
promoter (such as steroid- or ethanol-inducible promoter system). The promoter
may also
be tissue-specific. The types of promoters listed above are described in the
art. Other
suitable promoters and inducible systems are also known to a person of average
skill.
A promoter specific for seed development (e.g. HaFAD2-1 from sunflower or a
seed
storage protein promoter, such as zein, glutenin or hordein) or seed
maturation (e.g.
soybean pm36) may be used, or one specific for seed germination (e.g. barley
or wheat
alpha-amylase or carboxypeptidase) or a seedling-specific promoter (such as
the Pyk10
promoter) may be used. The patatin promoter may be used for tubers.
A green tissue-specific promoter may be used. For example, a green tissue-
specific
promoter may be selected from the maize orthophosphate kinase promoter, maize
phosphoenolpyruvate carboxylase promoter, rice phosphoenolpyruvate carboxylase
promoter, rice small subunit rubisco promoter, rice beta expansin EXBO9
promoter,
pigeonpea small subunit rubisco promoter or pea RBS3A promoter.
The promoter may be a constitutive or strong promoter. In a preferred
embodiment, the
regulatory sequence is an inducible promoter or a stress inducible promoter.
The stress
inducible promoter is selected from the following non limiting list: the HaHB1
promoter,
RD29A (which drives drought inducible expression of DREB1A), the maize rab17
drought-
inducible promoter, P5CS1 (which drives drought inducible expression of the
proline
biosynthetic enzyme P5CS1), ABA- and drought-inducible promoters of
Arabidopsis clade
A PP2Cs (ABM , ABI2, HAB1 , PP2CA, HA11 , HAI2 and HAI3) or their
corresponding
crop orthologues.
In modified plant cells, modified plants or parts thereof as defined herein,
when SP2 is
overexpressed there is preferably at least one copy of the additional genetic
material
compared to the unmodified or WT.
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Such additional genetic material as is described herein may comprise a
polynucleotide
comprising the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 2, or a
sequence of
at least 50% identity therewith, or a fragment thereof of at least 17
contiguous nucleotides
thereof.
In the various aspects of the invention, including the methods and uses,
encompass not
only a 5P2 nucleic acid or protein, but also a fragment or part thereof. By
"fragment" or
"part" is intended a portion of the nucleotide sequence or a portion of the
amino acid
sequence and hence of the protein encoded thereby. Fragments of a nucleotide
sequence
may encode protein fragments that retain the biological activity of the native
protein.
A polynucleotide sequence or fragment thereof may be operably linked to a
promoter in a
sense or an antisense orientation if it is desired to employ an RNA-based
suppression or
knockdown of expression, of which there are many types known in the art as
will be
described in more detail below.
Gene silencing may be used to achieve inactivation, repression or down-
regulation of 5P2
(and additionally SP1). For example, RNA-mediated gene suppression or RNA
silencing.
"Gene silencing" is generally used to refer to suppression of expression of a
gene via
sequence-specific interactions that are mediated by RNA molecules. The degree
of
reduction may be so as to totally abolish production of the encoded gene
product, but
more usually the abolition of expression is partial, with some degree of
expression
remaining. The term should not therefore be taken to require complete
"silencing" of
expression.
Transgenes may be used to suppress endogenous plant genes. This was discovered
originally when chalcone synthase transgenes in petunia caused suppression of
the
endogenous chalcone synthase genes and indicated by easily visible
pigmentation
changes. Subsequently it has been described how many, if not all plant genes
can be
"silenced" by transgenes. Gene silencing requires sequence similarity between
the
transgene and the gene that becomes silenced. This sequence homology may
involve
promoter regions or coding regions of the silenced target gene. When coding
regions are
involved, the transgene able to cause gene silencing may have been constructed
with a
promoter that would transcribe either the sense or the antisense orientation
of the coding
sequence RNA. It is likely that the various examples of gene silencing involve
different
mechanisms that are not well understood. In different examples there may be
transcriptional or post-transcriptional gene silencing and both may be used
according to
the methods of the invention.
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The mechanisms of gene silencing and their application in genetic engineering,
which
were first discovered in plants in the early 1990s and then shown in
Caenorhabditis
elegans are extensively described in the literature.
RNA-mediated gene suppression or RNA silencing according to the methods of the
invention includes co-suppression wherein over-expression of the SP2 (and
optionally
SP1) sense RNA or mRNA leads to a reduction in the level of expression of the
genes
concerned. RNAs of the transgene and homologous endogenous gene are co-
ordinately
suppressed. Other techniques used in the methods of the invention include
antisense
RNA to reduce transcript levels of the endogenous SP2 gene (optionally
additionally SP1
gene) in a plant. In this method, RNA silencing does not affect the
transcription of a gene
locus, but only causes sequence-specific degradation of target mRNAs. An
"antisense"
nucleic acid sequence comprises a nucleotide sequence that is complementary to
a
"sense" nucleic acid sequence encoding a SP2 protein, or a part of a SP2
protein, i.e.
complementary to the coding strand of a double-stranded cDNA molecule or
complementary to an mRNA transcript sequence. The antisense nucleic acid
sequence is
preferably complementary to the endogenous SP2 gene to be silenced. The
complementarity may be located in the "coding region" and/or in the "non-
coding region" of
a gene. The term "coding region" refers to a region of the nucleotide sequence
comprising
codons that are translated into amino acid residues. The term "non-coding
region" refers
to 5' and 3' sequences that flank the coding region that are transcribed but
not translated
into amino acids (also referred to as 5' and 3' untranslated regions).
Antisense nucleic acid sequences can be designed according to the rules of
Watson and
Crick base pairing. The antisense nucleic acid sequence may be complementary
to the
entire SP2 nucleic acid sequence, but may also be an oligonucleotide that is
antisense to
only a part of the nucleic acid sequence (including the mRNA 5' and 3' UTR).
For
example, the antisense oligonucleotide sequence may be complementary to the
region
surrounding the translation start site of an mRNA transcript encoding a
polypeptide. The
length of a suitable antisense oligonucleotide sequence is known in the art
and may start
from about 50, 45, 40, 35, 30, 25, 20, 15 or 10 nucleotides in length or less.
An antisense
nucleic acid sequence according to the invention may be constructed using
chemical
synthesis and enzymatic ligation reactions using methods known in the art. For
example,
an antisense nucleic acid sequence (e.g., an antisense oligonucleotide
sequence) may be
chemically synthesized using naturally occurring nucleotides or variously
modified
nucleotides designed to increase the biological stability of the molecules or
to increase the
physical stability of the duplex formed between the antisense and sense
nucleic acid
sequences, e.g., phosphorothioate derivatives and acridine-substituted
nucleotides may be
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used. Examples of modified nucleotides that may be used to generate the
antisense
nucleic acid sequences are well known in the art. The antisense nucleic acid
sequence
can be produced biologically using an expression vector into which a nucleic
acid
sequence has been subcloned in an antisense orientation (i.e., RNA transcribed
from the
inserted nucleic acid will be of an antisense orientation to a target nucleic
acid of interest).
Preferably, production of antisense nucleic acid sequences in plants occurs by
means of a
stably integrated nucleic acid construct comprising a promoter, an operably
linked
antisense oligonucleotide, and a terminator.
Nucleic acid molecules used in a silencing method of the invention hybridize
with or bind to
mRNA transcripts and/or insert into genomic DNA encoding a polypeptide to
thereby inhibit
expression of the protein, e.g., by inhibiting transcription and/or
translation. The
hybridization can be by conventional nucleotide complementarity to form a
stable duplex,
or, for example, in the case of an antisense nucleic acid sequence which binds
to DNA
duplexes, through specific interactions in the major groove of the double
helix. Antisense
nucleic acid sequences may be introduced into a plant by transformation or
direct injection
at a specific tissue site. Alternatively, antisense nucleic acid sequences can
be modified to
target selected cells and then administered systemically. For example, for
systemic
administration, antisense nucleic acid sequences can be modified such that
they
specifically bind to receptors or antigens expressed on a selected cell
surface, e.g., by
linking the antisense nucleic acid sequence to peptides or antibodies which
bind to cell
surface receptors or antigens. The antisense nucleic acid sequences can also
be
delivered to cells using vectors.
RNA interference (RNAi) is another post-transcriptional gene-silencing
phenomenon which
may be used according to the methods of the invention. This is induced by
double-
stranded RNA in which mRNA that is homologous to the dsRNA is specifically
degraded.
It refers to the process of sequence-specific post-transcriptional gene
silencing mediated
by short interfering RNAs (siRNA). The process of RNAi begins when the enzyme,
DICER, encounters dsRNA and chops it into pieces called small-interfering RNAs
(siRNA).
This enzyme belongs to the RNase III nuclease family. A complex of proteins
gathers up
these RNA remains and uses their code as a guide to search out and destroy any
RNAs in
the cell with a matching sequence, such as target mRNA.
Artificial and/or natural microRNAs (miRNAs) may be used to knock out gene
expression
and/or mRNA translation. MicroRNAs (miRNAs) miRNAs are typically single
stranded
small RNAs typically 19-24 nucleotides long. Most plant miRNAs have perfect or
near-
perfect complementarity with their target sequences. However, there are
natural targets
with up to five mismatches. They are processed from longer non-coding RNAs
with
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characteristic fold-back structures by double-strand specific RNases of the
Dicer family.
Upon processing, they are incorporated in the RNA-induced silencing complex
(RISC) by
binding to its main component, an Argonaute protein. miRNAs serve as the
specificity
components of RISC, since they base-pair to target nucleic acids, mostly
mRNAs, in the
cytoplasm. Subsequent regulatory events include target mRNA cleavage and
destruction
and/or translational inhibition. Effects of miRNA overexpression are thus
often reflected in
decreased mRNA levels of target genes. Artificial microRNA (amiRNA) technology
has
been applied in Arabidopsis thaliana and other plants to efficiently silence
target genes of
interest. The design principles for amiRNAs have been generalized and
integrated into a
Web-based tool (http://wmd.weigelworld.org).
A plant may be transformed to introduce a RNAi, snRNA, dsRNA, siRNA, miRNA, ta-
siRNA, amiRNA or cosuppression molecule that has been designed to target the
expression of an SP1 gene and selectively decreases or inhibits the expression
of the
gene or stability of its transcript. Preferably, the RNAi, snRNA, dsRNA,
siRNA, miRNA,
amiRNA, ta-siRNA or cosuppression molecule used according to the various
aspects of
the invention comprises a fragment of at least 17 nt, preferably 22 to 26 nt
and can be
designed on the basis of the information shown in SEQ I D No: 1. Guidelines
for designing
effective siRNAs are known to the skilled person. Briefly, a short fragment of
the target
gene sequence (e.g., 19-40 nucleotides in length) is chosen as the target
sequence of the
siRNA of the invention. The short fragment of target gene sequence is a
fragment of the
target gene mRNA. In preferred embodiments, the criteria for choosing a
sequence
fragment from the target gene mRNA to be a candidate siRNA molecule include 1)
a
sequence from the target gene mRNA that is at least 50-100 nucleotides from
the 5' or 3'
end of the native mRNA molecule, 2) a sequence from the target gene mRNA that
has a
G/C content of between 30% and 70%, most preferably around 50%, 3) a sequence
from
the target gene mRNA that does not contain repetitive sequences (e.g., AAA,
CCC, GGG,
TTT, AAAA, CCCC, GGGG, TTTT), 4) a sequence from the target gene mRNA that is
accessible in the mRNA, 5) a sequence from the target gene mRNA that is unique
to the
target gene, 6) avoids regions within 75 bases of a start codon. The sequence
fragment
from the target gene mRNA may meet one or more of the criteria identified
above. The
selected gene is introduced as a nucleotide sequence in a prediction program
that takes
into account all the variables described above for the design of optimal
oligonucleotides.
This program scans any mRNA nucleotide sequence for regions susceptible to be
targeted
by siRNAs. The output of this analysis is a score of possible siRNA
oligonucleotides. The
highest scores are used to design double stranded RNA oligonucleotides that
are typically
made by chemical synthesis. In addition to siRNA which is complementary to the
mRNA
target region, degenerate siRNA sequences may be used to target homologous
regions.
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siRNAs according to the invention can be synthesized by any method known in
the art.
RNAs are preferably chemically synthesized using appropriately protected
ribonucleoside
phosphoramidites and a conventional DNA RNA synthesizer. Additionally, siRNAs
can be
obtained from commercial RNA oligonucleotide synthesis suppliers.
siRNA molecules may be double stranded. A double stranded siRNA molecule may
comprise blunt ends. Or, a double stranded siRNA molecule may comprise
overhanging
nucleotides (e.g., 1 -5 nucleotide overhangs, preferably 2 nucleotide
overhangs). In some
examples, the siRNA may be a short hairpin RNA (shRNA); and the two strands of
the
siRNA molecule may be connected by a linker region (e.g., a nucleotide linker
or a non-
nucleotide linker). siRNAs described herein may contain one or more modified
nucleotides
and/or non-phosphodiester linkages. Chemical modifications well known in the
art are
capable of increasing stability, availability, and/or cell uptake of the
siRNA. A person of
average skill will be well aware of other types of chemical modification which
may be
incorporated into RNA molecules.
In some modified plant cells, modified plants or parts thereof in accordance
with any
aspect of the invention, at least some of the expressed SP2 has itself altered
activity
compared to unmodified control or WT. This alteration of activity by way of
mutation or by
gene editing may be combined with increasing or decreasing the level of
expression of
SP2 compared to unmodified or WT.
The activity of SP2 which may be altered is preferably that of the SP2 protein
association
with plastid protein SP1; wherein SP1 may comprise an amino acid sequence of
SEQ ID
NO: 6 or a sequence of at least 55% identity therewith.
The 5P2 protein of altered activity is preferably lacking at least one, and up
to 27 of the N-
terminal amino acids of the protein sequence, e.g. SEQ ID NO: 3 or variants or
homologs
thereof.
As well as modified plant cells, modified plants or parts thereof based on
altered 5P2
expression and/or activity as hereinbefore defined, the invention includes the
combination
of all these possible aforementioned aspects and variations, together with an
altered
expression and/or activity of plastid SP1 protein. The SP1 protein comprises
an amino
acid sequence of SEQ ID NO: 6, or a sequence of at least 55% identity
therewith, and is
altered in expression and/or activity compared to plastid SP1 protein in an
unmodified
control or WT plant.
In these combined SP1 and 5P2 aspects of the invention, expression and/or
activity of
SP1 may be increased compared to the expression and/or activity of SP1 in an
unmodified
control or VVT plant.
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Alternatively, the expression and/or activity of SP1 is decreased or
eliminated compared to
the expression and/or activity of SP1 in an unmodified control or VVT plant.
Where there is a polynucleotide encoding SP1 involved, this may comprise the
nucleotide
sequence of SEQ ID NO: 4 or SEQ ID NO: 5, or a sequence of at least 50%
identity
therewith, or a fragment of at least 17 contiguous nucleotides thereof.
In some aspects, the SP1 polynucleotide sequence or fragment thereof may be
operably
linked to a promoter in a sense or an antisense orientation if it is desired
to employ a gene
silencing approach, of which there are many types known in the art. Possible
within the
scope of the invention for example is for differing methods of expression
control for each of
SP1 and 5P2. So SP1 may be altered in expression by one method, e.g. mutation
in a
regulatory element for SP1, and 5P2 may be altered in expression by an RNA-
based
suppression approach.
In all of the above where there may be gene silencing of 5P2, the same applies
where
optionally expression and/or activity of SP1 being altered as well.
At least some of the expressed SP1 may have an altered SP1 activity compared
to
unmodified control or VVT. This may be achieved by genetic modification, e.g.
known
transgenic approaches or by gene editing usually performed using Cas9.
In particularly preferred aspects, the activity of the SP1 protein which is
altered may be (a)
reduced or no interaction with plastid 5P2 protein; and/or (b) reduced or no
E3 ligase
activity; and/or (c) reduced or no association with plastid Toc proteins.
In any of the aforementioned possibilities of the invention for modified plant
cells, modified
plants or parts thereof, plastids may be altered in at least some cells, when
compared to
an unmodified control or VVT. In this context, embodiments of the invention
are concerned
with spatial and/or temporal alterations in plastids and so considerations are
for tissue
specific and development specific controls being involved in making the
alterations
compared to VVT.
Altered plants in accordance with the invention advantageously may provide
better yield
characteristics. These may be designed into the alterations being made. Yield
characteristics, also known as yield traits may comprise one or more of the
following non-
!imitative list of features: early flowering time, yield, biomass, seed yield,
seed viability and
germination efficiency, seed/grain size, starch content of grain, early
vigour, greenness
index, increased growth rate, delayed senescence of green tissue. The term
"yield" in
general means a measurable produce of economic value, typically related to a
specified
crop, to an area, and to a period of time. Individual plant parts directly
contribute to yield
based on their number, size and/or weight, or the actual yield is the yield
per square meter
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for a crop and year, which is determined by dividing total production
(includes both
harvested and appraised production) by planted square metres. The term "yield"
of a plant
may relate to vegetative biomass (root and/or shoot biomass), to reproductive
organs,
and/or to propagules (such as seeds) of that plant. Thus, according to the
invention, yield
comprises one or more of and can be measured by assessing one or more of:
increased
seed yield per plant, increased seed filling rate, increased number of filled
seeds,
increased harvest index, increased viability/germination efficiency, increased
number or
size of seeds/capsules/pods, increased growth or increased branching, for
example
inflorescences with more branches, increased biomass or grain fill.
Preferably, increased
yield comprises an increased number of grains/seeds/capsules/pods, increased
biomass,
increased growth, increased number of floral organs and/or increased floral
branching.
Yield is usually measured relative to a control plant.
The alteration in plants of the invention may for example be in the numbers
and/or type(s)
of plastid in the cell(s). This can also serve to better adapt an existing
plant by modifying it
to tolerate better particular environmental conditions such as light quality
and level,
temperature fluctuations, minima and/or maxima, and/or water, saline or
osmotic stresses.
(This may also tie in with better yield traits, as described above).
In accordance with the invention, the SP2 including any variant or homologue
as herein
defined by way of the percentage identities of sequence, may be from different
species,
genus, family or order of plant. Where SP1 is altered in expression and/or
activity in
combination with SP2 alterations, then similarly this SP1 can independently be
selected
from a different species, genus, family or order of plant. The SP2 and SP1
genes being
altered can be taken from the same or different plant species
The invention therefore provides a modified plant as herein defined, which has
increased
expression and/or activity of SP2 protein; optionally also increased
expression and/or
activity of SP1 protein, and which has (a) increased tolerance to a stress
condition
compared to an unmodified control or VVT plant; preferably wherein the stress
condition is
one or more of saline stress, osmotic stress or oxidative stress; and/or (b)
accelerated fruit
ripening.
Where the plant stress condition is concerned, this may be one or more of
salinity, osmotic
stress and/or oxidative stress. The tolerance of plants to different types of
abiotic stresses
is not necessarily conferred through related mechanisms and indeed occurs via
different
signal transduction pathways. Thus, it cannot be expected that a gene that
confers, when
expressed, one type of stress, could also confer a different type of stress.
Stress can thus refer to moderate or severe salt stress and is present when
the soil is
saline. Soils are generally classified as saline when the ECe is 4 dS/m or
more, which is
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equivalent to approximately 40 mM NaCI and generates an osmotic pressure of
approximately 0.2 MPa. Most plants can however tolerate and survive about 4 to
8 dS/m
although this will impact on plant fitness and thus yield. For example, in
rice, soil salinity
beyond ECe - 4 dS/m is considered moderate salinity while more than 8 dS/m
becomes
high. Similarly, pH 8.8 - 9.2 is considered as non-stress while 9.3 - 9.7 as
moderate stress
and equal or greater than 9.8 as severe stress. Thus, salt stress as used
herein refers to
an ECe of 4 dS/m or more, for example about 4 to about 8 dS/m or about 40 mM
NaCI or
more, for example about 40 mM NaCI to about 100 mM NaCI or about 40 mM NaCI to
200
mM NaCI. Exposure to high levels of NaCI not only affects plant water
relations but also
creates ionic stress in the form of cellular accumulation of CI- and, in
particular, Na + ions.
Salt stress also changes the homeostasis of other ions such as Ca2+, K+, and
NO3- levels
(water deficit, ion toxicity, nutrient imbalance, and oxidative stress), and
at least two main
responses can be expected: a rapid protective response together with a long-
term
adaptation response. During initial exposure to salinity, plants experience
water stress,
which in turn reduces leaf expansion. During long-term exposure to salinity,
plants
experience ionic stress, which can lead to premature senescence of adult
leaves, and thus
a reduction in the photosynthetic area available to support continued growth.
Thus, by
increasing tolerance to salt stress, plant yield is increased.
When plant cells are under environmental stress, several chemically distinct
reactive
oxygen species (ROS) are generated by partial reduction of molecular oxygen
and these
can cause oxidative stress damage or act as signals. Oxidative stress can be
induced by
various environmental and biological factors such as hyperoxia, light,
drought, high salinity,
cold, metal ions, pollutants, xenobiotics, toxins, reoxygenation after anoxia,
experimental
manipulations, pathogen infection and aging of plant organs. Auto-oxidation of
components of the photosynthetic electron transport chain leads to the
formation of
superoxide radicals and their derivatives, hydrogen peroxide and hydroxyl
radicals. These
compounds react with a wide variety of biomolecules including DNA, causing
cell stasis
and death. Thus, by increasing tolerance to oxidative stress, plant yield is
increased.
The invention therefore permits increasing or enhancing plant response to
oxidative stress,
caused for example by extreme temperatures, drought UV light, irradiation,
high salinity,
cold, metal ions, pollutants, toxins, or pathogen infection by bacteria,
viruses or fungi or a
combination thereof.
Osmotic adjustment plays a fundamental role in water stress responses and
growth in
plants. Drought, salinity and freeze-induced dehydration constitute direct
osmotic
stresses; chilling and hypoxia can indirectly cause osmotic stress via effects
on water
uptake and loss. By increasing tolerance to osmotic stress, plant yield is
increased.
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Osmotic stress in accordance with the invention refers to osmotic stress
caused by salinity,
freezing or chilling and drought.
According to the invention, plant stress responses may be increased, enhanced
or
improved. This is understood to mean an increase compared to the level as
found in a
control, for example a wild-type plant. A skilled person will appreciate that
such stress
responses can be measured and the increase can be 2- to 10-fold.
The stress may be severe or preferably moderate stress. In Arabidopsis
research, stress
is often assessed under severe conditions that are lethal to wild-type plants.
For example,
drought tolerance is assessed predominantly under quite severe conditions in
which plant
.. survival is scored after a prolonged period of soil drying. However, in
temperate climates,
limited water availability rarely causes plant death, but restricts biomass
and seed yield.
Moderate water stress, that is suboptimal availability of water for growth can
occur during
intermittent intervals of days or weeks between irrigation events and may
limit leaf growth,
light interception, photosynthesis and hence yield potential. Leaf growth
inhibition by water
stress is particularly undesirable during early establishment. There is a need
for methods
for making plants with increased yield under moderate stress conditions. In
other words,
whilst plant research in making stress tolerant plants is often directed at
identifying plants
that show increased stress tolerance under severe conditions that will lead to
death of a
wild-type plant, these plants do not perform well under moderate stress
conditions and
often show growth reduction which leads to unnecessary yield loss.
So, in pursuing certain aspect of the invention, plant yield may be improved
under
moderate stress conditions. The terms moderate or mild stress/stress
conditions are used
interchangeably and refer to non-severe stress. In other words, moderate
stress, unlike
severe stress, does not lead to plant death. Under moderate, that is non-
lethal, stress
conditions, wild-type plants are able to survive, but show a decrease in
growth and seed
production and prolonged moderate stress can also result in developmental
arrest. The
decrease can be at least 5%-50% or more. Tolerance to severe stress is
measured as a
percentage of survival, whereas moderate stress does not affect survival, but
growth rates.
The precise conditions that define moderate stress vary from plant to plant
and also
between climate zones, but ultimately, these moderate conditions do not cause
the plant to
die. With regard to high salinity for example, most plants can tolerate and
survive about 4
to 8 dS/m. Specifically, in rice, soil salinity beyond ECe - 4 dS/m is
considered moderate
salinity while more than 8 dS/m becomes high. Similarly, pH 8.8 - 9.2 is
considered as
non-stress while 9.3 - 9.7 as moderate stress and equal or greater than 9.8 as
higher
stress.
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So, in accordance with the invention are methods relating to increasing
resistance to
moderate (non-lethal) stress or severe stress. Modified plants according to
the invention
show increased resistance to stress and therefore, the plant yield is not or
less affected by
the stress compared to wild type yields which are reduced upon exposure to
stress. In
other words, an improve in yield under moderate stress conditions can be
observed.
Preferred homologues of AtSP2 peptides are 5P2 peptides from crop plants, for
example
cereal crops. In one embodiment, preferred homologues include 5P2 in maize,
rice,
wheat, sorghum, sugar cane, oilseed rape (canola), soybean, cotton, potato,
tomato,
tobacco, grape, barley, pea, bean, field bean or other legumes, lettuce,
sunflower, alfalfa,
sugar beet, broccoli or other vegetable brassicas or poplar.
Preferably, the plant is a crop plant. By crop plant is meant any plant which
is grown on a
commercial scale for human or animal consumption or use. In a preferred
embodiment,
the plant is a cereal or legume.
A plant according to the various aspects of the invention, including the
transgenic plants,
methods and uses described herein may be a monocot or a dicot plant.
The term "plant" as used herein encompasses whole plants, ancestors and
progeny of the
plants and plant parts, including seeds, fruit, shoots, stems, leaves, roots
(including
tubers), flowers, and tissues and organs, wherein each of the aforementioned
comprise
the gene/nucleic acid of interest. The term "plant" also encompasses plant
cells,
suspension cultures, callus tissue, embryos, meristematic regions,
gametophytes,
sporophytes, pollen and microspores, again wherein each of the aforementioned
comprises the gene/nucleic acid of interest.
A monocot plant may, for example, be selected from the families Arecaceae,
Amatyllidaceae or Poaceae. For example, the plant may be a cereal crop, such
as wheat,
.. rice, barley, maize, oat, sorghum, rye, millet, buckwheat, turf grass,
Italian rye grass,
sugarcane or Festuca species, or a crop such as onion, leek, yam or banana.
A dicot plant may be selected from the families including, but not limited to
Asteraceae,
Brassicaceae (e.g. Brassica napus), Chenopodiaceae, Cucurbitaceae, Leguminosae
(Caesalpiniaceae, Aesalpiniaceae Mimosaceae, Papilionaceae or Fabaceae),
Malvaceae,
Rosaceae or Solanaceae. For example, the plant may be selected from lettuce,
sunflower,
Arabidopsis, broccoli, spinach, water melon, squash, cabbage, tomato, potato,
yam,
capsicum, tobacco, cotton, okra, apple, rose, strawberry, alfalfa, bean,
soybean, field
(fava) bean, pea, lentil, peanut, chickpea, apricots, pears, peach, grape
vine, bell pepper,
chilli or citrus species. In one embodiment, the plant is oilseed rape.
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Also included are biofuel and bioenergy crops such as rape/canola, sugar cane,
sweet
sorghum, Panicum virgatum (switchgrass), linseed, lupin and willow, poplar,
poplar
hybrids, Miscanthus or gymnosperms, such as loblolly pine. Also included are
crops for
silage (maize), grazing or fodder (grasses, clover, sanfoin, alfalfa), fibres
(e.g. cotton, flax),
building materials (e.g. pine, oak), pulping (e.g. poplar), feeder stocks for
the chemical
industry (e.g. high erucic acid oil seed rape, linseed) and for amenity
purposes (e.g. turf
grasses for golf courses), ornamentals for public and private gardens (e.g.
snapdragon,
petunia, roses, geranium, Nicotiana sp.) and plants and cut flowers for the
home (African
violets, Begonias, chrysanthemums, geraniums, Coleus spider plants, Dracaena,
rubber
plant).
Most preferred plants are maize, rice, wheat, oilseed rape/canola, sorghum,
soybean,
sunflower, alfalfa, potato, tomato, tobacco, grape, barley, pea, bean, field
bean, lettuce,
cotton, sugar cane, sugar beet, broccoli or other vegetable brassicas or
poplar.
A plant as mentioned above may have a decreased or no expression and/or
decreased or
no activity of SP2 protein; optionally decreased or no expression and/or no
decreased or
no activity of SP1 protein, in at least a particular cell type; optionally
wherein (a) the cell
type is at seedling stage and there is increased seedling survival, increased
seedling
growth and/or emergence; or (b) the cell type is green photosynthetic and
there is a delay
of senescence; or (c) the cell type is at seed setting stage and there is an
increase in
seed/grain size or starch content; or (d) the cell type is at fruiting stage
and there is a
delaying of fruit ripening.
In particularly preferred aspects the modified plants of the invention
described herein are
crop plants, biofuel plants or horticultural plants.
In another aspect, the present invention provides an isolated polynucleotide
construct
comprising a promoter and:
a. a polynucleotide comprising a nucleotide sequence of: (i) SEQ ID NO: 1 or
a sequence of at least 50% identity thereto; or (ii) SEQ ID NO: 2 or a
sequence of at least 50% identity thereto; or
b. a polynucleotide comprising a fragment of at least 17 contiguous
nucleotides of: (i) SEQ ID NO: 1 or a sequence of at least 50% identity
thereto; or (ii) SEQ ID NO: 2 or a sequence of at least 50% identity thereto.
As used herein, the terms "nucleic acid", "nucleic acid sequence",
"nucleotide", "nucleic
acid molecule" or "polynucleotide" are intended to include DNA molecules
(e.g., cDNA or
genomic DNA), RNA molecules (e.g., mRNA), naturally occurring, mutated,
synthetic DNA
or RNA molecules, and analogues of the DNA or RNA generated using nucleotide
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analogues. They can be single-stranded or double-stranded. Such nucleic acids
or
polynucleotides include, but are not limited to, coding sequences of
structural genes, anti-
sense sequences, and non-coding regulatory sequences that do not encode mRNAs
or
protein products. These terms also encompass a gene.
As to a "gene" or "gene sequence", these broadly to refer to a DNA nucleic
acid
associated with a biological function. Thus, genes may include introns and
exons as in the
genomic sequence, or may comprise only a coding sequence as in cDNAs, and/or
may
include cDNAs in combination with regulatory sequences.
If orthologues of nucleotide sequences encoding 5P2 are included in the
aforementioned,
then they may have, in increasing order of preference, an identity at least
25%, 26%, 27%,
28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%,
43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%,
58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%,
73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%,
88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% overall
sequence identity to the nucleic acid represented by SEQ ID NO: 1 or 2. (The
"at least"
prefixes each and every percentage identity listed above.)
Preferably though, in the context of polynucleotides of the invention, overall
sequence
identity may be in order of increasing preference, more than 49%, 50%, 51%,
52%, 53%,
54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68% or
more than 69%. (The "more than" prefixes each and every percentage identity
listed
here.)
Preferably though, overall sequence identity with SEQ ID NO: 1 or SEQ ID NO:
2, in
increasing order of preference is at least 50%, 51%, 52%, 53%, 54%, 55%, 56%,
57%,
58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%,
73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%,
88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% overall
sequence identity to the nucleic acid represented by SEQ ID NO: 1 or 2. (The
"at least"
prefixes each and every percentage identity listed above.)
.. As may be estimated from Figure 15A, a preferred limit of percentage
identity which
defines SP1 may be at least 65% compared to SEQ ID NO: 6. A similar preferred
limit of
variation for full length genomic and cDNA may also apply, so at least 65%
identity to SEQ
ID NO: 4 or SEQ ID NO: 5. Each and every individual percentage limit as
recited above
from at least 56% to at least 99% identity with the reference sequences is
contemplated.
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As may be estimated from Figure 15B, the preferred limit of percentage
identity which
defines SP2 is at least 78% compared to SEQ ID NO: 3. A similar preferred
limit of
variation for full length genomic and cDNA may also apply, so at least 78%
identity to SEQ
ID NO: 1 or SEQ ID NO: 2. Optionally the percentage identity for these
reference
sequences may be at least 80% or at least 85%; preferably at least 90%.
However, each
and every individual percentage limit as recited above from at least 78% to at
least 99%
identity with the reference sequences is contemplated.
The degree of sequence identity of polynucleotides of the invention may,
instead of being
expressed as a percentage identity to reference sequence, may instead be
defined in
terms of hybridization to a polynucleotide of reference sequence SEQ ID NO: 1
or SEQ ID
NO: 2. Hybridization of such sequences may be carried out under stringent
conditions. By
"stringent conditions" or "stringent hybridization conditions" is intended
conditions under
which a probe will hybridize to its target sequence to a detectably greater
degree than to
other sequences (e.g., at least 2-fold over background). Stringent conditions
are
sequence dependent and will be different in different circumstances. By
controlling the
stringency of the hybridization and/or washing conditions, target sequences
that are 100%
complementary to the probe can be identified (homologous probing).
Alternatively,
stringency conditions can be adjusted to allow some mismatching in sequences
so that
lower degrees of similarity are detected (heterologous probing). Generally, a
probe is less
than about 1000 nucleotides in length, preferably less than 500 nucleotides in
length.
Typically, stringent conditions will be those in which the salt concentration
is less than
about 1 .5 M Na ion, typically about 0.01 to 1 .0 M Na ion concentration (or
other salts) at
pH 7.0 to 8.3 and the temperature is at least about 30 C for short probes
(e.g., 10 to 50
nucleotides) and at least about 60 C for long probes (e.g., greater than 50
nucleotides).
Duration of hybridization is generally less than about 24 hours, usually about
4 to 12.
Stringent conditions may also be achieved with the addition of destabilizing
agents such as
formamide.
Thus, a nucleotide sequence as described herein can be used to isolate
corresponding
sequences from other organisms, particularly other plants, for example crop
plants. In this
manner, methods such as PCR, hybridization, and the like can be used to
identify such
sequences based on their sequence homology to the sequences described herein.
Topology of the sequences, e.g. for 5P2 the characteristic 16 x TMD pattern
and N-
terminal 27 amino acids of 5P2 can also be considered when identifying and
isolating 5P2
homologues for example. Sequences may be isolated based on their sequence
identity to
the entire sequence or to fragments thereof. In hybridization techniques, all
or part of a
known nucleotide sequence is used as a probe that selectively hybridizes to
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corresponding nucleotide sequences present in a population of cloned genomic
DNA
fragments or cDNA fragments (i.e., genomic or cDNA libraries) from a chosen
plant. The
hybridization probes may be genomic DNA fragments, cDNA fragments, RNA
fragments,
or other oligonucleotides, and may be labelled with a detectable group, or any
other
detectable marker. Thus, for example, probes for hybridization can be made by
labelling
synthetic oligonucleotides based on the ABA-associated sequences of the
invention.
Methods for preparation of probes for hybridization and for construction of
cDNA and
genomic libraries are generally known in the art and are disclosed in
Sambrook, etal.,
(1989) Molecular Cloning: A Library Manual (2nd ed., Cold Spring Harbor
Laboratory
Press, Plainview, New York).
Such polynucleotide constructs will be of assistance to persons of average
skill in the
making of altered plant cells, plants and parts thereof, wherein the 5P2 gene
is altered in
expression and/or activity. The constructs of the invention lend themselves to
the full
range of known gene modification and gene expression modulation methods,
including
gene editing using Cas9 or Cpf1, for example.
Recombinant DNA constructs may be made and used as described in US 6635805,
incorporated herein by reference.
A silencing RNA molecule may be introduced into a plant using conventional
methods, for
example a vector and Agrobacterium-mediated transformation. Stably transformed
plants
are generated and expression of the 5P2 gene compared to a wild type control
plant is
analysed.
Silencing of the 5P2 gene may also be achieved using virus-induced gene
silencing.
A modified plant cell, plant or part thereof of the invention may express a
nucleic acid
construct comprising a RNAi, snRNA, dsRNA, siRNA, miRNA, ta-siRNA, amiRNA or
co-
suppression molecule that targets the 5P2 gene as described herein and reduces
expression of the endogenous 5P2 gene. A gene is targeted when, for example,
the
RNAi, snRNA, dsRNA, siRNA, miRNA, ta-siRNA, amiRNA or cosuppression molecule
selectively decreases or inhibits the expression of the gene compared to a
control plant.
Alternatively, a RNAi, snRNA, dsRNA, siRNA, miRNA, ta-siRNA, amiRNA or
cosuppression molecule targets 5P2 when the RNAi, snRNA, dsRNA, siRNA, miRNA,
ta-
siRNA, amiRNA or cosuppression molecule hybridises under stringent conditions
to the
gene transcript. VVithin the context of the invention, preferably, to
specifically target 5P2,
the RNA must comprise at least the same seed sequence. Thus, any RNA that
targets
5P2 is preferably identical in positions 2-8 of the antisense strand.
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Gene silencing may also occur if there is a mutation on an endogenous gene
and/or a
mutation on an isolated gene/nucleic acid subsequently introduced into a
plant. The
reduction or substantial elimination may be caused by a non-functional
polypeptide. For
example, the polypeptide may bind to various interacting proteins; one or more
mutation(s)
and/or truncation(s) may therefore provide for a polypeptide that is still
able to bind
interacting proteins (such as receptor proteins) but that cannot exhibit its
normal function
(such as signalling ligand).
A further approach to gene silencing is by targeting nucleic acid sequences
complementary to the regulatory region of the gene (e.g., the promoter and/or
enhancers)
to form triple helical structures that prevent transcription of the gene in
target cells. Other
methods, such as the use of antibodies directed to an endogenous polypeptide
for
inhibiting its function in planta, or interference in the signalling pathway
in which a
polypeptide is involved are well known. In particular, manmade molecules may
be useful
for inhibiting the biological function of a target polypeptide, or for
interfering with the
signalling pathway in which the target polypeptide is involved.
In modification of the above, there may also be provided an isolated
polynucleotide
construct which additionally comprises:
a. a polynucleotide comprising a nucleotide sequence of: (i) SEQ ID NO: 4 or a
sequence of at least 55% identity thereto; or (ii) SEQ ID NO: 5 or a sequence
of at least
.. 55% identity thereto; or
b. a polynucleotide comprising a fragment of at least 17 contiguous
nucleotides of: (i) SEQ ID NO: 4 or a sequence of at least 55% identity
thereto; or (ii) SEQ
ID NO: 5 or a sequence of at least 55% identity thereto.
This is for achieving a dual alteration of expression and/or activity of 5P2
and SP1 in a
plant, plant cell or plant part. There are various possibilities whereby
single or binary
constructs can be used. In a single construct, the 5P2 encoding polynucleotide
in the
above may be downstream of the promoter, and the SP1 encoding polynucleotide
may be
downstream of the 5P2 encoding polynucleotide. Alternatively, these positions
of 5P2 and
SP1 may be reversed.
Described herein is also an isolated polynucleotide construct which comprises
a promoter
and:
a. a polynucleotide comprising a nucleotide sequence of: (i) SEQ ID NO: 4 or
a sequence of at least 55% identity thereto; or (ii) SEQ ID NO: 5 or a
sequence of at least 55% identity thereto; or
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b. a polynucleotide comprising a fragment of at least 17 contiguous
nucleotides of: (i) SEQ ID NO: 4 or a sequence of at least 55% identity
thereto; or (ii) SEQ ID NO: 5 or a sequence of at least 55% identity
thereto.
The invention also includes the aforementioned isolated polynucleotides as
separate
polynucleotides, each with their own same or different promoters and optional
regulatory
and other features, forming a portion of a kit of parts for use in a binary
rather than single
construct approach to the alteration of 5P2 and SP1 protein activity and/or
expression in
plant cells, plants or parts thereof.
Such polynucleotides as aforementioned include Ti plasmids of Agrobacterium
tumefasciens which are well known in the art.
In further aspect is a single polynucleotide construct comprising a regulatory
sequence,
e.g. promoter, and comprising Included in the invention are vectors comprising
a
polynucleotide as aforementioned. This includes Agrobacterium tumefasciens,
tobacco
mosaic virus (TMV), potato virus X and cowpea mosaic virus. Preferably, the
vector
further comprises a regulatory sequence which directs the desired expression
of the
nucleic acid, spatially or temporally, or possibly in reaction to an inducer
or stress
condition.
Included therefore in the invention as another aspect are host cells
comprising a
polynucleotide or a vector as hereinbefore described. Such cells may not
necessarily be
plant cells when cloning, in vitro expression or genetic manipulation
procedures are being
carried out as part of a series of experimental or developmental steps to
yield altered plant
material. So, such host cells may include isolated plant cells or protoplasts,
bacteria, e.g.
Agrobacterium or Escherichia coli, or yeast, e.g. Saccharomyces cerevisiae.
Also included in the invention is a culture medium or kit comprising a culture
medium and
an isolated host cell as described herein.
In further aspect, the invention provides a method of altering plastids in a
plant cell,
comprising:
a. increasing, decreasing or eliminating the expression in the cell of plastid
protein 5P2 comprising an amino acid sequence of SEQ ID NO: 3 or a
sequence of at least 50% identity therewith when compared to a control or
WT plant cell; and/or
b. increasing, decreasing or eliminating the activity in the cell of plastid
protein
5P2 comprising an amino acid sequence of SEQ ID NO: 3 or a sequence of
at least 50% identity therewith when compared to a control or VVT plant cell,
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wherein the activity of SP2 is that of SP2 association with plastid SP1
protein of SEQ ID NO: 6 or a sequence of at least 55% identity therewith.
In such a method of altering plastids, this may further (additionally)
comprise:
a. increasing, decreasing or eliminating the expression in the cell of plastid
protein SP1 comprising an amino acid sequence of SEQ ID NO: 6 or a
sequence of at least 55% identity therewith when compared to a control or
WT plant cell; and/or
b. increasing, decreasing or eliminating the activity in the cell of plastid
protein
SP1 comprising an amino acid sequence of SEQ ID NO: 6 or a sequence of
at least 55% therewith when compared to a control or VVT plant cell,
wherein the activity of SP1 is that of SP1 association with plastid 5P2
protein of SEQ ID NO: 3 or a sequence of at least 50% identity therewith.
In these aforementioned methods of altering plastids in a plant cell, the
expression of 5P2;
optionally also the expression of SP1, may be increased by the editing of a
nucleotide
sequence of at least one native 5P2 regulatory element in the cell; optionally
also at least
one SP1 native regulatory element in the cell, and/or by the insertion of a
polynucleotide
construct or a vector as hereinbefore defined, into the cell.
In some aforementioned methods of altering plastids in a plant cell, plastid
development
may be accelerated; optionally wherein the cell is (a) a seedling cell and the
transition of
etioplasts into chloroplasts is accelerated; or (b) a seed cell and the
transition to
amyloplasts is accelerated; or (c) a fruit cell and the transition from
chloroplasts is
accelerated.
Alternatively, in other methods of altering plastids in plant cells, the
expression of 5P2;
optionally also the expression of SP1, may be decreased or eliminated by the
editing of a
nucleotide sequence of at least one native 5P2 regulatory element in the cell;
optionally
also at least one SP1 native regulatory element in the cell, and/or by the
insertion of a
polynucleotide construct or a vector as hereinbefore defined, into the cell.
In methods of altering plastids where plastid development is delayed; the cell
is optionally:
(a) a green photosynthetic cell and the transition of chloroplasts into
another type of
plastid, e.g. gerontoplast, is delayed; or (b) a fruit cell and the transition
from chloroplasts
is delayed.
In any aspect of the invention herein, not excluded is the possibility that
5P2 expression
and/or activity may be increased whilst SP1 expression and/or activity may be
decreased,
with respect to a control or VVT. And vice versa, not excluded, 5P2 expression
and/or
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activity may be decreased whilst SP1 expression and/or activity may be
increased, with
respect to a control or VVT.
What is also possible in the present invention is that the ratio of SP2:SP1
expression or
activity may be altered compared to a control or VVT. This ratio may increase
or decrease
compared to control of VVT. Ratios may a ratio in the range of possibilities
between 10:1 to
1:10.
Some aspects of the invention involve recombinant DNA technology and in
preferred
embodiments exclude embodiments that are solely based on generating plants by
traditional breeding methods.
The various aspects of the invention described herein clearly extend to any
plant cell or
any plant produced, obtained or obtainable by any of the methods described
herein, and to
all plant parts and propagules thereof unless otherwise specified. The present
invention
extends further to encompass the progeny of a primary transformed or
transfected cell,
tissue, organ or whole plant that has been produced by any of the
aforementioned
methods, the only requirement being that progeny exhibit the same genotypic
and/or
phenotypic characteristic(s) as those produced by the parent in the methods
according to
the invention.
The invention also extends to harvestable parts of a plant of the invention as
described
above such as, but not limited to seeds, leaves, fruits, flowers, stems,
roots, rhizomes,
tubers and bulbs. The invention furthermore relates to products derived,
preferably directly
derived, from a harvestable part of such a plant, such as dry pellets or
powders, oil, fat and
fatty acids, starch or proteins. The invention also relates to food products
and food
supplements comprising the plant of the invention or parts thereof.
In another aspect, the invention relates to a plant obtainable or obtained by
a method as
described herein.
In all embodiments of methods where 5P2 expression and/or activity is
modified, then
these apply equally to situations where SP1 expression and/or activity is in
addition
modified
EXAMPLES
Hereinafter, reference numbers in parenthesis correspond to the references
listed at the
end of the description.
Chloroplasts are plant organelles responsible for the bulk of photosynthetic
primary
production, and they evolved via endosymbiosis from a cyanobacterial organism
more
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than 1 Bya (1). The modern chloroplast proteome comprises -3000 proteins, most
of
which are nucleus-encoded and imported post-translationally by translocases in
the
chloroplast envelope membranes (2-5). Turnover of internal chloroplast
proteins is
governed by several prokaryotic-type proteases inherited from the endosymbiont
(6). In
contrast, chloroplast outer envelope membrane (OEM) proteins are degraded by
the
cytosolic ubiquitin-proteasome system (UPS) via poorly understood mechanisms
(1).
The RING-type ubiquitin E3 ligase SP1 is located in the chloroplast OEM where
it
mediates the ubiquitination of OEM components of the chloroplast protein
import
machinery (so-called TOO proteins; these act in conjunction with TIC
translocases in the
inner membrane (2-5)), thereby promoting their degradation by the cytosolic
26S
proteasome (7). The TOO components affected by SP1 include the receptors
Toc159 and
Toc33, and the channel protein Toc75. Such SP1- mediated regulation of the TOO
apparatus changes the organellar proteome, which in turn influences the
developmental
fate and functions of the organelle (e.g., enabling plant adaptation to
abiotic stress) (7, 8).
While the role of SP1 in marking proteins for degradation is clear, other
aspects of this
chloroplast protein degradation system have remained obscure. Because TOO
proteins
are integral membrane components, additional factors are most likely required
to
overcome the energetic barrier to their extraction from the membrane, prior to
degradation
in the cytosol, as is the case in other membrane-associated proteolytic
systems (9-11).
The inventors have discovered mutants with lesions at a locus unlinked to sp1.
These are
termed suppressor of ppi1 locus2 (5p2). Double-mutant sp2 ppi1 plants were
found by the
inventors to be larger and greener than the ppi1 progenitor and exhibited
substantial
improvements in chloroplast development and protein import capacity.
Example: Identification of SP2 and its role in plastid development
Genetic Resources
Seeds of Arabidopsis thaliana ecotype Columbia-0 (Col-0), SALK_137135 were
obtained
on 9th April 2014 from Nottingham Arabidopsis Stock Centre (NASC), School of
Biosciences, University of Nottingham, Sutton Bonington Campus, Loughborough,
LE12
5RD, United Kingdom.
Seeds of Arabidopsis thaliana ecotype Landsberg erecta (Ler) have been in
possession of
the inventors since earlier than 12th October 2014 from the laboratory of
Joanne Chory at
the Salk Institute 10010 N Torrey Pines Rd., La Jolla, CA 92037, California,
USA.
The sp2-4 (SALK_137135) mutant was obtained from the Salk Institute Genomic
Analysis
Laboratory.
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Materials and methods:
Plant material and growth conditions
All Arabidopsis thaliana plants were of the Columbia-0 (001-0) ecotype, except
the ppil
.. line used for the genetic mapping of sp2 which was introgressed into
Landsberg erecta
(Ler) through seven outcrosses. The sp1-1, sp1-3, ppil, t1c40-4, hsp93-V-1,
pp12-3 (ftsl),
t0c75-III-3 (marl) mutants, and the 35S promoter-driven SP1 overexpressor (SP1-
0X)
transgenic line, have all been described previously (7, 12, 16, 19, 30, 31).
The sp2-4
(SALK_137135) mutant was obtained from the Salk Institute Genomic Analysis
Laboratory,
.. and confirmed by PCR and RT-PCR analysis, as described previously (32);
this mutant
was phenotypically similar to the three chemically-induced sp2 alleles
identified in this
study, and unlike a previously-described T-DNA mutant (33). For in vitro
growth, seeds
were surface sterilized, sown on Murashige-Skoog (MS) agar medium in petri
plates, cold-
treated at 4 C, and thereafter kept in a growth chamber, as described
previously (34). All
plants were grown under a long-day cycle (16 h light, 8 h dark).
Physiological studies
Chlorophyll measurements were performed by using a Konica-Minolta SPAD-502
meter
(35), or by photometric quantification following extraction in N,N'-
dimethylformamide (DMF)
as described previously (36).
Dark treatments for the induction of senescence were conducted as previously
described
(7, 37). Developmentally-equivalent leaves of 28-day-old plants were wrapped
in
aluminium foil whilst still attached to the plant, and then left under
standard growth
conditions for 5 days. Photochemical efficiency of photosystem II (Fv/Fm) was
determined
by measuring chlorophyll fluorescence using a CF Imager (Technologica, UK) as
described previously (38). Three experiments were performed, and approximately
five
leaves (each one from a different plant) were analysed per genotype in each
experiment.
Salt stress experiments were conducted as described previously with minor
modifications
(8). All seeds of the different genotypes used in this work were harvested at
the same
time. Seeds were germinated directly on MS agar medium (supplemented with 1%
sucrose) containing 150-170 mM NaCI. Stress tolerance was assessed by
measuring
chlorophyll accumulation after 14 days. Three experiments were performed, and -
25
seedlings per genotype were analysed in each experiment.
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Detection of hydrogen peroxide was performed by staining with 3,3'-
diaminobenzidine
(DAB) (Sigma) as previously described (39). The plants were left to grow for
further 2 days
before initiating DAB staining. Each experiment used approximately 5 seedlings
per
genotype. Three experiments were performed with the same result, and typical
images
are presented. The area of staining was quantified using ImageJ as described
previously
(8).
Identification of the sp2 mutants and genetic mapping
The original sp2 mutants (sp2-1 [sp2-310], sp2-2 [sp2-416] and sp2-3 [sp2-
555]) were
identified by screening the M2 progeny of 7,000 M1 ppi1 seeds that had been
treated with
100 mM ethyl methanesulfonate for 3 h using a published procedure (7, 40).
Initial
mapping of sp2 was conducted by analysing the greenest plants in F2
populations from
crosses between sp2-1 ppi1 (001-0) and ppi1 introgressed into the Ler ecotype,
using PCR
markers that detect Col-O/Ler polymorphisms. In a mapping population of 190
such F2
plants, six were heterozygous for the marker F21A17 at position 12285000 on
the upper
arm of chromosome 3, but homozygous for Col-0 downstream of that, suggesting
that the
suppressor mutation was in the downstream Col-0 region; F3 seedlings from
these six
plants were grown and verified visually to be non-segregating, as expected for
a
homozygous sp2-1 ppi1 double mutant. In a second mapping population of 192
plants, the
sp2 mutation was further mapped to the south of a more southerly marker, MJI6-
2 at
position 12597802 on the upper arm of chromosome 3. However, it was not
possible to
determine the position of the sp2 locus precisely owing to the persistence of
an "island" of
Col-0 DNA in the Ler-introgressed ppi1 line, near the sp2 locus (around the
chromosome 3
centromere). Thus, final identification of the gene was achieved by whole-
genome
sequencing.
Whole-genome sequencing and assembly
Approximately 100 mg of plant inflorescence tissue from each of the original
sp2 alleles
(sp2-1 ppi1, sp2-2 ppi1, and sp2-3 ppi1), and from ppi1-1, was harvested and
flash-frozen
in liquid nitrogen. Total genomic DNA was then extracted using an E.Z.N.A.
Plant DNA Kit
(Omega Bio-tek) following the manufacturer's guidelines. The DNA samples were
quantified by comparison with standards.
Library preparation and sequencing were conducted at the Earlham Institute
(Norwich,
UK). Approximately 1-5 pg genomic DNA per sample at a minimum concentration of
20
ng/pl was used in sequencing library preparation. Individual barcoded Illumina
TruSeq
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DNA libraries were generated for each genotype. The four samples were then
sequenced
on one lane of IIlumina HiSeq 2000, which generated between 32.2-42.3 million
100 bp
paired-end reads for each sample. The first five bases of the 5' ends of the
reads were
removed using fast( trimmer vØ0.14 (httplihannonlab.cshi.eduifastx_todkM,
and any
bases with a Phred quality score below 15 were removed from the 3' end using
cutadapt
(v.1.3) (41). I !lumina TruSeq adaptors where also removed using cutadapt,
where a
minimum overlap of 10 bases with the adaptors, a maximum error rate of 0.1,
and a
minimum final read length of 50 bases were set.
Finally, fastq_quality_filter v0Ø14 (http://hannoNab.cshi.eduifastxioolkitt)
was used to
remove sequences with a Phred score below 20 in more than 5% of the bases.
Read
pairs were identified using pairSeq.py (https:iigithub comitopel-research-
groupipairSeq).
The reads from the ppi1 single mutant were mapped to the TAIR10 Arabidopsis
thaliana
reference genome (ftp://ftp.jgi-
psf.org/pub/compgen/phytozome/v9.0/Athaliana/assembly/Athaliana_167.fa.gz) of
the
Phythozome v.9.0 release (httplAvww.phytozome.neti) using cic_mapper v.
4Ø13.86165
(https://www.giagenbioinformatics.com/). A consensus sequence in FASTA format
was
then generated using cic_find_variations v. 4Ø13.86165. Also, the transcript
sequences
from the TAIR10 release were aligned to the reference genome in order to
facilitate
manual examination of identified mutations and visualization of whether a
particular
mutation occurs in an exon, intron, etc.
The three datasets from the individual sp2 double mutants were independently
aligned to
the ppi1 reference genome using cic_mapper v. 4Ø13.86165. The ppi1 reads
were also
aligned to the same reference in order to identify any variable sites
resulting from allelic
variation in the ppi1 line.
In silico identification of mutations
The mutagen ethyl methanesulfonate (EMS) used to generate the three sp2
mutants
reacts with guanine in the DNA molecule and is likely to (1) cause point
mutations that
change guanine to adenosine (or cytosine to thymidine on the reverse strand).
The
-- respective mutations affecting the three sp2 mutants were furthermore
expected to (2)
occur in the same gene (or corresponding promotor region) in (3) all three
mutants but (4)
not necessarily in the exact same position. These four search criteria were
implemented in
the program "find_5p2.py" (https://github.com/topel-research-group/5p2) which
takes as
input a gff3 file with gene coordinates and the SNP variant output from
cic_mapper, and
outputs a list of names of mutated genes from each dataset. Genes found to be
mutated
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in all the sp2 datasets were then manually examined by visualizing the
alignment data
using the genome viewer IGV (v.2.3) (42).
This analysis showed that each sp2 allele contains a G-to-A point mutation
within the
At3g44160 gene, just to the south of the chromosome 3 centromere. In sp2-1, a
mutation
was detected at the splice junction preceding the final exon; this was later
shown to cause
mis-splicing, frame-shifts, and premature termination, implying that sp2-1 is
a knockout
allele. In sp2-2 and sp2-3, the detected mutations were both predicted to
cause an amino-
acid substitution. The transmembrane beta-strands and the three-dimensional
structure of
the SP2 protein were predicted using Phyre2, with a model based on structures
for
bacterial TamA and BamA proteins (c4c00a, c5ekqA, c4k3bA, c4n75A, c4k3cA)
(43).
Phylogenetic analysis
Sequences were obtained by BLAST searches of the Phytozome 12 database (44)
(table
51). Sequences were aligned by multiple alignment using fast Fourier transform
(MAFFT)
(45), and manual alignment adjustments were made using Mesquite 1.12
(Tangient).
Phylogeny was inferred using MrBayes 3.2 software (46). Two runs were
performed in
parallel, with each using 8 MCMC chains for 8 million generations and the
temperature set
to 0.2. The standard deviation of split frequencies (StdDev) was 0.001228 at
the end of
the analysis and therefore assumed to have converged. Trees were sampled every
1000
generations, reaching a total of 8000 trees. Burn-in was set to 25%, and so
the first 2000
trees were discarded. The resulting phylogeny was a minimum 50% consensus of
the
remaining 6000 sampled trees. Parameters not mentioned were retained at the
default
setting.
Gene identifiers
The following gene sequences from Arabidopsis thaliana were employed
experimentally in
this study: 5P2 (At3g44160); Toc33 (At1g02280); CDKA1 (At3g48750); Toc159
(At4g02510); OEP7 (At3g52420); OEP80 (At5g19620); SSU (At1g67090).
Plasmid constructs
All primers used are listed in the tables below:
(A) Primers used to generate various constructs and mutations.
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Primer Primer sequence (5' to 3') Nucleotides underlined Used to SEQ ID
name do not correspond to the target gene, but instead generate ... NO:
correspond to linker sequences or mutation sites.
SP1- GGATCCATGATTCCTTGGGGTGGAG SP1 CDS 7
BamHI for
-F cloning
SP1- GCGGCCGCGTGACGATATGTCTTAACC into pE3c 8
ns- vector
Notl-R
SP2- GGGTTAACTCTGATCAATGGGAGCTCAGAAGAGT SP2 CDS 9
BcII-F ATCCA for
SP2- AAACCGGTCTGCGGCCGCTGTTGATGAAGCAAGA cloning 10
ns- TTGGTG into pE3c
Notl-R vector
SP2- AAAAAGCAGGCTCCATGGGAGCTCAGAAGAGTAT SP2 CDS 11
CDS-F CCA for plant
SP2- AGAAAGCTGGGTTTGTTGATGAAGCAAGATTGGT transform 12
CDS-R G ation
Toc33- AAAAAGCAGGCTCCATGGGGTCTCTCGTTCGTG T0C33 13
CDS-F CDS for
Toc33- AGAAAGCTGGGTTTTAAAGTGGCTTTCCACTTG cloning 14
CDS-R into
NTAPi
vector
CDC48 AAGGTACCATGTCTACCCCAGCTGAATC CDC48 15
-Kpnl-F CDS for
CDC48 AACCCGGGATTGTAGAGATCATCATCGTCC cloning 16
-ns- into
Xmal- pSAT4A-
cEYFP-
N1 vector
Toc159 AAGAATTCAATGGACTCAAAGTCGGTT T0C159 17
CDS for
EcoRI- cloning
into
Toc159 AAGTCGACTTAGTACATGCTGTACTT pSAT4- 18
-Sall-R nEYFP-
C1 vector
OEP7- AAGAATTCATGGGAAAAACTTCGGGA OEP7 19
EcoRI- CDS for
cloning
OEP7- TTGTCGACACAAACCCTCTTTGGATGT into 20
ns- pSAT4A-
Sall-F nEYFP-
N1 vector
CDKA1 CTCGAGATGGATCAGTACGAGAAAG CDKA1 21
-Xhol-F CDS for
CDKA1 GAATTCAGGCATGCCTCCAAGATC cloning 22
-ns- into
EcoRI- pSAT4A-
cEYFP-
N1
SP2- GGTACCATGTCTTTAATGTTTCCTGCTTTCAGGG ...SP2 CDS 23
Kpnl-F for
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SP2-R CTATGTTGATGAAGCAAGATTGG cloning 24
into
pBluescri
pt SK
vector
OEP80 GGTACCATGCATTGTCACAACGATG OEP80 25
-Kpnl-F CDS for
OEP80 TTAGTTCCGCAGACCAAC cloning 26
-R into
pBluescri
pt SK
vector
attB1 GGGGACAAGTTTGTACAAAAAGCAGGCT ...
complete 27
attB2 GGGGACCACTTTGTACAAGAAAGCTGGGT Gateway 28
recombin
ation sites
for
cloning
into
pDONR2
01
(B) Primers used in RT-PCR experiments.
Primer Primer sequence (5' to 3') SEQ ID
name NO:
SP2- GGTTGTGTCCAAGTGGCTTA 29
RT1-F
SP2- TACAGCTTCTCCTTGGACTGT 30
RT1-R
SP2- GGGTTCTCTTCATGGTTGATTCTTCTCT 31
RT2-F
SP2- CCATTCAGGTCTAGGTCTCCTAAAA 32
RT2-R
SP2-F2 GGTTGTGTCCAAGTGGCTTA 33
SP2-R2 CTATGTTGATGAAGCAAGATTGGTG 34
Tool 59- AACTCTTGAAGTGGCTAATAAGT 35
QRT-F
Tool 59- ACAACCTCTGGCTCTACA 36
QRT-R
Toc33- AATGGTGAAGCGTGGATC 37
QRT-F
Toc33- TGCTCCTTGAATCATCTTAACG 38
QRT-R
Toc75- TCGCATCTCCACTCAATC 39
III-QRT-
Toc75- GTCTCTGTATCTCGGTTAGG 40
III-QRT-
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GAPDH- GTGGTGGATTTGGCTCACCT 41
GAPDH- CTCATCAGCCGGGTTTGTCT 42
SP1-F GGTACAAGATAGTGCGTTGATG 43
SP1-R CTGCAGTCAGTGACGATATGTCTTAAC 44
ACTIN2- TCAGATGCCCAGAAGTCTTGTTCC 45
ACTIN2- CCGTACAGATCCTTCCTGATATCC 46
elF4E1- AAACAATGGCGGTAGAAGACACTC 47
elF4E1- AAGATTTGAGAGGTTTCAAGCGGTGTAAG 48
(C) Primers used to genotype mutant plants by dCAPS (derived Cleaved Amplified
Polymorphic Sequence) analysis. The PCR products amplified using dCAPS primers
were
digested with restriction enzyme, and thereafter resolved on 3% agarose gels.
Genotypes
were determined by comparing the sizes of the bands with controls
corresponding to wild
type and the homozygous mutant.
Primer Mutant Primer sequence (5' to 3') Restriction SEQ ID
name Nucleotides underlined do not enzyme NO:
correspond to the target gene, but
instead correspond to linker
sequences or mutation sites.
5P2-dC1- sp2-1 TGTTGCGGAATTGGTTTCAT 49
Enzyme: Ddel
5P2-dC1- GGTTGTGTCCAAGTGGCTTA Digests: VVT 50
5P2-dC2- sp2-2 51
CATTGGTGGGCTAGGCAGTGATC Enzyme: Bc11
Digests: sp2-
5P2-dC2- CATGCAACAGCTCCACGTACCAA 2 52
5P2-dC3- sp2-3 CCACAATCCAAGAAGATGGTAC 53
Enzyme: Kpnl
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SP2-dC3- GCCTTGTTGCCAATACAGAA
Digests: VVT 54
Toc75-11I- toc75-M- GCAAATCACAGGTGGATCTG 55
3-dCAPS- 3
Enzyme: Ddel
Digests:
Toc75-11I- TGCCCATGGAGGACTAGAAC 56
toc75-M-3 ¨
3-dCAPS-
(D) Primers used to genotype T-DNA insertion mutant plants.
Primer Primer sequence (5' to 3') Comments
name
Lbal TGGTTCACGTAGTGGGCCATCG Lbal and SP2- 57
R3 to identify the
SP2-R3 tacagcttctccttggactgt 58
T-DNA;
SP2-F3 CGTGGAGCTGTTGCATGAT 59
SP2-F3 and SP2-
R3 to identify the
SP2 gene region
flanking T-DNA
The SP1-HA, YFP-HA, SP1-YFP, GST-SP1flex and YFP-Toc33 constructs have all
been
described previously (7, 47). All other Arabidopsis CDSs were PCR-amplified
from Col-0
cDNA. The Gateway cloning system (Invitrogen) was used to make most of the
constructs, and all entry clones were verified by DNA sequencing. To generate
C-terminal
6xMyc tag fusion proteins, the SP1 and SP2 CDSs were cloned into the pE3c
vector (49),
and then subcloned into the p2GVV7 35S-driven expression vector (50) for
protoplast
transfection (generating the SP1-Myc and SP2-Myca constructs). The SP2 CDS,
with and
without the Myc tag, was cloned into the pB2GVV7 binary 35S-driven
overexpression
vector (50) for stable plant transformation (generating the SP2-0X and SP2-Myc
constructs). To generate N-terminally TAP-tagged Toc33, the corresponding CDS
was
cloned into the NTAPi binary vector (52) (generating the TAP-Toc33 construct).
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Transient assays and stable plant transformation
Protoplast isolation and transient assays were carried out as described
previously (7, 54).
When required, MG132 (Sigma), epoxomicin (Merck), bortezomib (Selleckchem)
(all three
chemicals prepared as a 10 mM stock solution in DMSO), or E-64 (Me!ford)
(prepared as a
10 mM stock solution in water) was added to the protoplast culture medium at
15 h
following transfection, to a final concentration of 1-30 pM, 1-10 pM, 5 pM, or
1-10 pM,
respectively; subsequently, the culture was incubated for a further 2-3 h
before analysis.
For XFP fluorescence and immunoprecipitation assays, 0.1 ml (105) or 1 ml
(106) aliquots
of protoplasts were transfected with 5 pg or 100 pg of DNA, respectively, and
the
fluorescence signals were analysed after 15-18 h.
Transgenic lines carrying the SP2-0X and SP2-Myc constructs were generated by
Agrobacterium-mediated transformation (16, 32). Transformants were selected
using MS
medium containing phosphinothricin for these 5P2 constructs. At least 12 T2
lines for
each transformation were analysed, and at least two lines with a single T-DNA
insertion
(which showed a 3:1 segregation on selective MS medium in the T2 generation)
were
chosen for further analysis.
Microscopy
Transmission electron microscopy was performed as described previously (16).
Measurements were recorded using at least 30 different plastids per genotype,
and were
representative of three individuals per genotype. Chloroplast cross-sectional
area was
estimated as described previously (16, 30), using the equation: 11 x 0.25 x
length x width.
Numbers of thylakoid lamellae per granal stack, and of interconnections
between granal
stacks, were counted as previously described (7, 16) in at least 96 resolvable
grana across
three individuals per genotype.
All fluorescence microscopy and BiFC experiments were conducted at least twice
with the
same results, and typical images are presented. For the imaging of CFP, YFP
and
chlorophyll fluorescence signals, in most cases protoplasts were examined
using a Zeiss
LSM 510 META laser- scanning confocal microscope (Carl Zeiss Ltd.), as
described
previously (8). To visualize signals associated with chloroplasts without
interference from
cytosolic signals, protoplasts were ruptured by gently tapping the cover
glass; this enabled
the release of the cytosol and of intact chloroplasts. Fluorescence images
were captured
using a Nikon Eclipse TE-2000E inverted microscope as described previously
(32).
In vitro translation and in vitro pull-down analysis
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The SP2 and OEP80 CDSs were cloned into pBlueScript II SK- using a single Smal
restriction site and verified by DNA sequencing. The preSSU construct was
described
previously, as was the in vitro transcription/translation procedure (16, 55).
The GST-SP1flex and GST proteins were purified from bacteria as described
previously,
as was the procedure employed for in vitro pull-down analysis (7).
Chloroplast isolation, protein import, and topology analysis
Chloroplasts were isolated from 14-day-old in vitro grown plants (or, when
stated, from
protoplasts). Isolations, protein import, and protease treatments were
performed as
described previously (16, 34, 56-59). The presented chloroplast protein import
data are
representative of three independent experiments.
lmmunoblotting, immunoprecipitation and blue native (BN) PAGE
lmmunoblotting was performed as previously described (30, 60) with minor
modifications.
Primary antibodies were as follows. To identify TOO proteins or components of
the
translocon at the inner envelope membrane of chloroplasts (TIC), we employed:
anti-
atToc75-III antibody (32); anti-atToc159 antibody (61); anti-atToc33 (G-
domain) antibody
(32); anti-atTic110 antibody (62, 63); and anti-atTic40 antibody (32). To
identify non-TOO
outer envelope membrane proteins, we employed: anti-OEP80 antibody (64); and
anti-
SFR2 antibody (65). To identify chloroplast stromal proteins, we employed:
anti-cpHsc70
(AgriSera, AS08 348) (66); anti-Hsp93 (heat shock protein, 93 kD) antibody
(16, 67); and
anti- PRPL35 antibody (7). To identify proteins of other cellular
compartments, we
employed: anti-S1p1 (mitochondria) (68); anti-calreticulin (ER) (69, 70); and
anti-H3 histone
(Abcam; nucleus) (32). Other primary antibodies we employed were: anti-HA tag
(Sigma);
anti-c-Myc tag (Sigma); anti-GFP (detects both GFP and YFP; Sigma); and anti-
FLAG tag
(Sigma).
Secondary antibodies were anti-rabbit IgG conjugated with horseradish
peroxidase (Santa
Cruz Biotechnology), or, in the case of anti-c-Myc and anti-FLAG, anti-mouse
IgG
conjugated with horseradish peroxidase (GE Healthcare). Chemiluminescence was
detected using ECL Plus Western Blotting Detection Reagents (GE Healthcare)
and an
LAS-4000 imager (Fujifilm). Band intensities were quantified using Aida
software
(Raytest). Quantification data were based on results from at least three
experiments all
showing a similar trend. Typical images are shown in all figures.
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For the immunoprecipitation of HA-tagged proteins, total protein (-500 mg) was
extracted
from protoplasts in IP buffer (25 mM Tris-HCI, pH 7.5, 150 mM NaCI, 1 mM EDTA,
1%
Triton X-100) containing 0.5% plant protease inhibitor cocktail (PPIC, Sigma),
and
centrifuged at 20,000g for 10 min at 4 C. The clear lysate was then incubated
with 50 pl
EZview Red Anti-HA Affinity Gel (Sigma) for 2 h to overnight at 4 C with slow
rotation. After
six washes with 500 pl IP-washing buffer (25 mM Tris-HCI, pH7.5, 150 mM NaCI,
1 mM
EDTA, 0.5% Triton X-100), bound proteins were eluted by boiling in 2x SDS-
PAGE
loading buffer (50 mM Tris-HCI, pH 6.8, 20% glycerol, 1% sodium dodecyl
sulphate [SDS],
and 0.1 M DTT) for 5 min, and analysed by SDS-PAGE and immunoblotting. A
similar
procedure was adopted for the immunoprecipitation of Myc-tagged proteins,
except that 50
pl EZview Red Anti-c-Myc Affinity Gel (Sigma) was used instead of the anti-HA
gel. When
detecting ubiquitinated proteins, the IP buffer also contained 10 mM N-
ethylmaleimide
(NEM; Sigma).
Two-dimensional BN-PAGE was performed using a procedure described previously
(71).
Tandem affinity purification (TAP) and mass spectrometry
Chloroplasts were isolated from a complemented ppi1 mutant line carrying the
TAP:Toc33
construct, and then used as starting material for TAP. The TAP procedure was
performed
as described previously (72), omitting the secondary affinity purification
step which was not
essential for our analysis. The Tobacco Etch Virus (TEV) nuclear-inclusion-a
endopeptidase eluates were concentrated 1:10 by using Vivaspin 500
ultrafiltration spin
columns (Sartorius Stedim Biotech), boiled with 1 volume 2x SDS- PAGE loading
buffer,
and loaded on SDS-PAGE gels for analysis. Silver staining was used to
visualize proteins
and estimate their sizes and migration positions. For identification of CDC48,
the 75-100
kD region of a Coomassie Brilliant Blue-stained SDS-PAGE gel slice was
subjected to in-
gel trypsin digestion and liquid chromatography-tandem mass spectrometry (LC-
MS/MS)
analysis. Scaffold (Proteome Software) and Mascot database searches were used
to
interpret the results.
In vivo retrotranslocation assays
The method used was adapted and modified from similar approaches commonly
applied in
ERAD studies (21). First, SP1-HA and ubiquitin were transiently overexpressed
in 106
protoplasts for each genotype to increase detection sensitivity for higher
molecular weight
(ubiquitinated) forms of SP1 substrate (73). The transformed protoplasts were
incubated
for 15 h, and then bortezomib was applied to a final concentration of 5 pM
before an
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additional 3 h incubation. Subsequent fractionation steps to produce separate
chloroplast
and cytosol samples were all carried out on ice or at 4 C, and used previously
described
procedures with modifications (56, 74). Protoplasts were pelleted by
centrifugation at 100g
for 2 min, and gently resuspended with protoplast-washing buffer (500 mM
mannitol, 4 mM
4-morpholineethanesulfonic acid [MES]-KOH, pH 5.6). Then, the protoplasts were
pelleted
again and resuspended by gentle agitation in 500 pl HS buffer (50 mM 4-(2-
hydroxyethyl)piperazine-1-ethanesulfonic acid [HEPES]-NaOH, pH 8.0, 0.3 M
sorbitol)
containing 0.5% PPIC and 5 pM bortezomib, and gently forced twice through 10
pm nylon
mesh to release chloroplasts. The collected flow-through was centrifuged at
1,000g for 5
min to produce a chloroplast-containing pellet and a cytosol-containing
supernatant (51).
The pellet was gently resuspended in 500 pl HS buffer, and the chloroplasts
were purified
by a two-step Percoll (Fisher Scientific) gradient (34). Intact chloroplasts
were washed with
500 pl HS buffer, and then pelleted by centrifugation at 1,000g for 5 min. The
51 sample
was centrifuged at 10,000g for 15 min. The resulting supernatant (S10) was
recovered
and ultracentrifuged at 100,000g for 1 h, producing a further supernatant
(S100) that was
concentrated to 50 pl by using Vivaspin 500 ultrafiltration spin columns; this
was the
cytosolic fraction. The pelleted chloroplasts were lysed in 100 pl denaturing
buffer (25 mM
Tris-HCI, pH 7.5, 150 mM NaCI, 5 mM EDTA, 10 mM NEM, 1% SDS, 2% Sarcosyl, 5 mM
dithiothreitol [DTT]) containing PPIC, while the cytosolic fraction was mixed
with 50 pl
2xdenaturing buffer containing PPIC. Finally, the SP1-HA protein was purified
by
immunoprecipitation using a previously described procedure to improve
sensitivity of
detection of the ubiquitinated protein (7). Experiments were repeated three
times, and
similar results were obtained.
Statistical analysis
Statistical calculations (mean, standard error of the mean, t-test) were
performed using
Microsoft Excel software. Statistical significance of differences between two
experimental
groups was assessed by using a two-tailed Student's t-test. Differences
between two
datasets were considered significant at p <0.05.
Results
As shown in Figures 1A ¨ F, and Figures 2A and B, double-mutant sp2 ppi1
plants were
larger and greener than the ppi1 progenitor and exhibited substantial
improvements in
chloroplast development and protein import capacity. Three independent mutant
alleles of
sp2 suppress the ppi1 mutation, in similar fashion to sp2-1. The sp2-2 ppi1
and sp2-3 ppi1
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mutants were identified using the same EMS mutagenesis screen as sp2-1 ppi1.
Allelism
tests confirmed that all of these mutations are allelic. All three sp2 ppi1
mutants were
backcrossed to ppi1 three times before analysis. Subsequently, the sp2-4
mutant
(SALK_137135) was obtained from SIGnAL via the Nottingham Arabidopsis Stock
Centre.
It was crossed with ppi1, and the sp2-4 ppi1 double mutant was selected and
verified by
phenotype analysis and using PCR-based genotyping. All sp2 ppi1 and control
plants
were grown under standard conditions on MS agar medium for 10 days before
photography (Figure 2A). Chlorophyll was measured photometrically following
DMF
extraction using plants similar to those shown in Figure 2A. About 10
seedlings per
.. genotype were measured in each experiment, and the values shown are means
SEM
derived from four experiments per genotype. In Figure 2B the data indicate
nmol total
chlorophyll per mg tissue fresh weight. The chlorophyll value for sp2-2 was
highly
statistically significantly different from that for sp2-1 (Student's t-test, p
<0.003), suggesting
that sp2-2 is a weak allele.
The SP2 locus was identified using a combination of genetic mapping and whole-
genome
sequencing. Figure 3A shows genetic mapping of the sp2 locus. Initial analysis
of an F2
population of 190 individuals from crosses between sp2-1 ppi1 (Col-0 ecotype)
and ppi1
(introgressed into the Ler ecotype) placed the sp2 locus to the south of
marker F21A17 at
position 12285000 on the upper arm of chromosome 3. A second mapping
population of
192 plants placed the sp2 mutation to the south of a more southerly marker,
MJI6-2 at
position 12597802 on the upper arm of chromosome 3. However, the genetic
mapping
failed to define a southern boundary concerning the location of sp2, owing to
the existence
of an "island" of Col-0 DNA in the Ler-introgressed ppi1 line, around the
chromosome 3
centromere. Numbers of recombinants at key markers are shown.
Figure 3B shows Identification of the sp2 mutations by whole-genome
sequencing. The
figure shows 40 bp of the alignment of sp2-1 ppi1 reads to the ppi1 reference
genome.
The vertical grey lines represent the read coverage at each base (this is 34
at the mutated
site), and the horizontal grey lines indicate individual sequence reads.
Positions in
different shading in the centres of the reads show the mutated site (C-to-T on
the strand
shown; the brightness of the red colour indicates the mapping quality) that is
responsible
for the sp2 phenotype. The SP2 (At3g44160) gene is coded on the reverse strand
relative
to the reference genome, and so the complement of the SP2 gene, in the ppi1
reference
sequence, is shown at the bottom. Genomic position in the ppi1 reference
genome is
indicated at the top of the figure. Similar results were obtained for the sp2-
2 and sp2-3
alleles, although the mutation sites within the gene differed.
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Figure 30 is a schematic representation of the SP2 (At3g44160) gene, annotated
with the
positions of the sp2 mutations. Information on the nature and consequence of
each
mutation is shown. The positions of PCR primers used in D are indicated with
arrows.
Black boxes and interconnecting black lines, exons and introns, respectively;
white boxes,
untranslated regions; LB, left border sequence of the SALK T-DNA insertion.
Figure 3D is an analysis of SP2 mRNA expression in each of the sp2 mutant
alleles by RT-
PCR. The primer pairs used in each case are indicated at left, and their
positions are
shown in Figure 30. Amplicon sizes are indicated at right. The sp2-1 allele
carries a
splice site mutation and exhibited slicing defects. Two different sp2-1
amplicons were
detected, and sequenced: the larger, minor amplicon retains a portion of
intron 10, and the
corresponding transcript is predicted to encode a truncated protein; the
smaller, more
abundant amplicon carries a single guanine nucleotide deletion (at position
924 of the
CDS, in exon 10), causing a frameshift such that the corresponding transcript
is also
predicted to encode a truncated protein. This indicated that sp2-1, like sp2-
4, is most likely
a null allele. In contrast, sp2-2 and sp2-3 may be weak alleles, which is in
line with the
chlorophyll data shown in Figures 2A - F. The encoded protein (At3g44160;
Figure 1G) is
a member of the 0mp85 superfamily of beta-barrels involved in protein
transport, which
are widely distributed in the outer membranes of bacteria, mitochondria and
chloroplasts
(13, 14).
The 5P2 protein is of unknown function, but Figure 4A shows that it is broadly
conserved
in the angiosperms and closely related to the chloroplast outer membrane
protein OEP80
(Toc75-V) (15); the function of OEP80 is also uncertain (15), although it has
been
proposed to mediate outer membrane protein biogenesis (16, 17) by analogy with
well-
characterized homologues in bacteria (BamA) and mitochondria (5am50/Tob55)
(13, 14).
For Figure 4A, Bayesian inference phylogenetic analysis of 5P2 and OEP80 was
undertaken. Predicted amino acid sequences homologous to Arabidopsis 5P2 and
OEP80 from a variety of plants and algae were retrieved from the Phytozome 12
database
(44). Bacterial 0mp85 sequences were also obtained and were included in the
analysis to
act as an outgroup. Sequences were aligned using the MAFFT algorithm (45), and
the
phylogeny was inferred using MrBayes 3.2 software (46). The tree represents a
50%
consensus of 6000 trees generated from two runs, each using 8 Markov chain
Monte-
Carlo (MCMC) chains for 8 million generations (the first 2000 trees were
discarded as
burn-in) where standard deviation of split frequencies was 0.001 at the end of
the analysis.
Posterior probability values are shown. Scale bar, 0.3 changes per site.
As shown in figures 5 and 6, 5P2 is located in the chloroplast OEM, and it was
previously
shown to form a membrane channel (18). 5P2 CDS was fused with sequence
encoding a
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C-terminal 6xMyc tag and cloned downstream of the strong, constitutive CaMV
35S
promoter in the pB2GVV7 binary vector (50), and then the resultant SP2- Myc
construct
was used to stably transform sp2-4 ppil and sp2-4 plants. Approximately 12 T2
transform ants for each genotype were analysed, and from these, representative
single-
locus lines were selected for further investigation based on segregation of
the resistance
phenotype on petri plates containing phosphinothricin, SP2 mRNA expression,
and
phenotype analysis. The SP2-Myc construct could fully complement the phenotype
of sp2-
4 ppil, indicating that the Myc tag does not affect the function of SP2.
Therefore, SP2-
Myc sp2-4 plants selected in this way were analysed as shown in in Figure 5).
Chloroplast
localization and topology of SP2 were analysed by chloroplast isolation and
subsequent
protease treatment. Chloroplasts were prepared from 14-day-old wild-type (VVT)
and SP2-
Myc sp2-4 plants. The isolated SP2-Myc sp2-4 chloroplasts were treated: with
thermolysin
or trypsin at different concentrations (100 or 500 pg/ml); with either
protease plus 1%
Triton X-100 detergent (TX100); or, with buffer lacking protease (Mock). Then,
the
samples (together with a wild-type chloroplast control; Mock) were analysed by
SDS-
PAGE and immunoblotting using antibodies against the Myc tag and a number of
chloroplast proteins. lmmunoblotting analysis showed that SP2-Myc is localized
in
chloroplasts. Specificity of the Myc signal was verified by comparing the VVT
and 5P2-Myc
Mock chloroplasts. Partial resistance to thermolysin indicated that the C-
terminal tag of
5P2-Myc is oriented towards the intermembrane space, as this protease does not
penetrate the outer membrane (so that only protein domains exposed at the
cytosolic
surface are fully sensitive to thermolysin) (58); assuming that 5P2 has an
even number of
transmembrane beta-strands, one may infer that its N-terminus is also located
in the
intermembrane space. In this assay, 5P2 behaved similarly to Toc75, which is
as expected
as our Toc75 antibody reacts with the N-terminal POTRA domain that is
localized in the
intermembrane space (77, 78). Toc159 has a large cytosolic domain and was
almost
completely degraded by thermolysin, confirming efficacy of the treatments.
Unlike
thermolysin, trypsin can penetrate the outer membrane to the intermembrane
space (58).
Increasing trypsin concentrations progressively depleted 5P2-Myc, like Toc75,
which
indicated that 5P2 is an outer membrane protein with similar topology as
Toc75. In
contrast, TIC-associated proteins located largely or wholly in the stroma
(Tic40 and Hsp93,
respectively) were not sensitive to trypsin, indicating that the protease was
behaving as
expected. When co-applied with Triton X-100 to solubilize the envelope
membranes, both
proteases completely degraded 5P2-Myc, confirming that the signals observed
were due
to protection by the membranes and not a result of intrinsic protease-
resistance.
In more detail, for Figure 6, chloroplasts were isolated from 14-day-old wild-
type and 5P2-
Myc sp2-4 plants, and subsequently lysed with hypotonic buffer (25 mM HEPES,
pH 8.0, 4
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mM MgCl2); the chloroplast pellet was broken up with a sterile plastic pestle
and then the
sample was rotated at 4 C for 1 h to ensure efficient lysis. The membranes
were
recovered by ultracentrifugation at 110,000g for 1 h at 4 C, and were then
subjected to
immunoprecipitation using anti-c-Myc antibody. Input (i.e., before
immunoprecipitation
was initiated; Chloroplast lysate), flow-through, and immunoprecipitated
(Elution) samples
were then analysed by immunoblotting using antibodies against: the Myc tag, to
verify the
enrichment of SP2-Myc; the TOO components Toc159, Toc75 and Toc33, to detect
putative SP2-partner interactions; and, Tic110 and Tic40, to assess whether
the above
interactions are specific. In addition, whole-plant protein extracts (Total
protein) were
prepared from wild-type and SP2-Myc sp2-4 plants (equivalent to those from
which the
chloroplasts were isolated), and analysed in parallel. Enrichment of SP2-Myc
in the
chloroplast lysates (relative to the total protein samples), in a similar way
to other
chloroplast proteins, further confirmed that SP2 is localized in chloroplasts.
The SP2-Myc
sp2-4 plants analysed here are as described in relation to Figure 5.
.. Unlike OEP80, SP2 lacks an N-terminal POTRA domain (such domains typically
mediate
protein interactions), as shown in Figure 1G, suggesting that the two proteins
have
functionally diverged. Figure 4B shows structural models for the Arabidopsis
SP2 and
OEP80 proteins. Three-dimensional models for SP2 and OEP80 were derived by
homology modelling using the crystal structures of bacterial TamA and BamA
proteins,
using the Phyre2 server (43). Both protein models are oriented with the N-
terminal domain
facing downwards. The beta-barrel and polypeptide transport associated (POTRA;
OEP80
only) domains are indicated. OEP80 and SP2 have opposing effects on TOO
protein
abundance, as discussed previously (0EP80 knockdown depletes TOO proteins
(16)) and
below.
.. Referring to Figures 7 and 8, in addition to ppil, two other TOO mutations
(hypomorphic
alleles of the genes encoding Toc159 and Toc75) (16, 19) were suppressed by
sp2,
whereas mutations that cause chlorosis for other reasons were not suppressed.
This
implies a close functional relationship between SP2 and the TOO apparatus, a
notion that
is supported by the restored accumulation of Toc75 in sp2 toc double mutants
(see Figure
1, H - K, and Figure 9). In all of these respects, the sp2 mutants were
phenotypically very
similar to spl mutants (7).
In more detail for Figure 7, panels A and C shows assessment of the effect of
sp2 on the
chlorotic phenotypes of two additional TOO mutants. The sp2-4 mutation was
introduced
into the t0c75-III-3 (marl; affecting Toc75) (16) and pp12-3 (ftsl; affecting
Toc159) (19)
mutant backgrounds by crossing. Double mutants were identified as described in
relation
to Figure 2, and subsequently grown alongside wild-type and single-mutant
controls under
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standard conditions on soil for 22 days (A), or on MS agar medium for 10 days
(C), prior to
photography; typical plants are shown. Both TOO mutations were suppressed by
sp2,
indicating that the effect of the latter is not specific to ppi1. In Figures
7B and D,
chlorophyll contents in the genotypes shown in Figures 7A and C were measured
photometrically following DMF extraction. About ten 10-day-old seedlings per
genotype
were measured in each experiment, and the values shown are means SEM derived
from
four experiments per genotype. The data indicate nmol total chlorophyll per mg
tissue
fresh weight.
In more detail for Figure 8, panel A shows assessment of the effect of sp2 on
the chlorotic
phenotypes of two mutants with protein import defects linked to the TIC
apparatus of the
inner envelope membrane. The sp2-4 mutation was introduced into the hsp93-V-1
background (30) by crossing. The sp2-1 mutation was isolated from the ppi1
background
by crossing to wild type followed by PCR genotyping, and then introduced into
the t1c40-4
background (30) by further crossing. Double mutants were obtained as described
in
relation to Figure 2, and subsequently grown alongside appropriate control
genotypes
under standard conditions on MS agar medium for 10 days prior to photography;
typical
plants are shown. Neither TIC-associated mutant was suppressed by sp2,
indicating that
the suppression effect of the latter is specific. Figure 8B shows chlorophyll
contents in the
genotypes shown in Figure 8A were measured photometrically following DM F
extraction.
About ten 10-day-old seedlings per genotype were measured in each experiment,
and the
values shown are means SEM derived from four experiments per genotype. For
comparison purposes, the individual measured values (nmol per mg tissue fresh
weight)
were expressed as percentages of the corresponding wild-type value.
In more detail for Figure 9, this shows total RNA samples isolated from 10-day-
old plants
of the indicated genotypes analysed by quantitative real-time PCR using gene-
specific
primers for atTOC75-III (A), or atTOC75-III, atTOC159 and atTOC33 (B).
Expression data
for the TOC genes were normalized using equivalent data for two reference
genes:
GAPDH and ACTIN2 (ACT2). For each genotype tested, four biological replicates
were
analysed with three technical replicates of each, and the values shown are
means SEM
(n = 4 biological replicates).
Overexpression of 5P2 triggered the specific depletion of TOO proteins (see
Figures 1L
and M), resembling closely the effect of SP1 overexpression (7). Like SP1, 5P2
interacted
physically with TOO components (see Figure 6). Further similarities with SP1
were
observed when sp2 mutant and 5P2 overexpressor plants were analysed
physiologically,
in relation to leaf senescence (see Figure 1N) and abiotic stress tolerance
(see Figure 10
and Figure 10). Activity of SP1 promotes both of these processes (which it
does by
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reconfiguring the chloroplast protein import machinery to produce the
necessary organellar
proteome changes) (7, 8), and a similar pattern was observed here for SP2 (see
Figures
1N and 0).
In more detail for Figure 10A - B, these show phenotypic analysis of sp2
single-mutant and
.. SP2 overexpressor (OX) plants. The three original sp2 mutants were isolated
from the ppil
background by crossing to wild type, followed by PCR genotyping. The SP2 CDS
was
cloned downstream of the 35S promoter in the pB2GVV7 binary vector (50), and
the
resultant construct (SP2-0X) was used to stably transform wild-type plants.
Using
procedures similar to those described in relation to Figure 5, representative
single-locus
.. lines were chosen for further analysis, based on the segregation of
phosphinothricin
resistance and the level of SP2 mRNA overexpression (see Figure 100 and D);
note that
the selected transformants are also shown in Figure 1L (#12 and #15), and in
Figures 11A
¨ G and Figure 12G and H (#12 only). Images of typical plants (Figure 11A) and
chlorophyll content measurements determined photometrically following
extraction in DM F
.. (Figure B) are shown. In each case 10- day-old seedlings grown under
standard
conditions on MS agar medium were analysed. For the chlorophyll assays, about
10
seedlings per genotype were measured in each experiment, and the values shown
are
means SEM derived from four experiments per genotype. The data indicate nmol
total
chlorophyll per mg tissue fresh weight.
In more detail for Figures 100 ¨ D, these show semi-quantitative RT-PCR
analysis of SP2
expression in wild-type and 5P2-0X plants. Total RNA samples isolated from 10-
day-old
plants were analysed by RT-PCR using gene-specific primers for SP2 and the
reference
gene ACT2. Amplifications employed a limited number of cycles to avoid
saturation, and
products were analysed by agarose gel electrophoresis and staining. A
representative gel
image is shown (Figure 100), along with quantification of four biological
replicates (Figure
10D). The amplicon bands were quantified using Aida software, and the data
obtained for
SP2 were normalized relative to equivalent data for ACT2. Values shown are
means
SEM (n = 4).
In more detail for Figures 11A ¨ D, these show analysis of an spl sp2 ppil
triple mutant.
Triple mutant plants were compared with both sp ppil single mutants in
relation to the
extent of suppression of ppil. No phenotypic additivity in the triple mutants
was apparent
upon analysing visible phenotypes (Figure 11A), chlorophyll contents (Figure
11B), or the
abundance of Toc75 protein by immunoblotting (Figures 110 and D). In Figures
11E ¨ G,
there is analysis of plants simultaneously overexpressing (OX) both SP1 and
5P2. Double
OX plants were compared with both single SP-OX genotypes, revealing a
synergistic
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interaction in relation to visible phenotype (Figure 11E, right), chlorophyll
content (Figure
11E, left), and TOO protein depletion as analysed by immunoblotting (Figures
11F and G).
In more detail for Figures 12G and H, these show analysis of the role of SP2
in
retrotranslocation of SP1-HA substrate. In vivo retrotranslocation assays were
performed
using protoplasts from wild-type, sp2 mutant, and SP2- OX plants. Typical
immunoblotting
results are shown (Figure 12G), along with quantification (Figure 12H). All
values are
means SEM (n = 3-4 experiments).
Figure 13 shows how SP1 and SP2 proteins associate to form a complex. Figure
13A
shows a two-dimensional (2D)-blue native (BN)/SDS-PAGE analysis of SP1 and
SP2. The
SP2 CDS was fused with sequence encoding a C-terminal 6xMyc tag and cloned
downstream of the 35S promoter in the p2GVV7 vector. The resulting SP2-Myca
construct,
together with the SP1-HA construct (7), were used to co-transform Arabidopsis
protoplasts. Chloroplasts were isolated from the transformed cells as
described previously
(56), solubilized in BN-PAGE sample buffer containing 1% DDM, and subjected to
2D-
BN/SDS-PAGE followed by immunoblotting using the indicated antibodies.
Molecular
weights (MW) of standards analysed in the first dimension are shown at the
bottom in kD.
The SP1-HA and SP2-Myc proteins co-migrated at two different positions: the
first (>669
kD) overlapped with the TOO complex (80, 81) (and may indicate an intermediate
step
preceding TOO component degradation); the second (232-440 kD) may correspond
to a
resting or inactive core complex for CHLORAD (see discussion below).
Figure 13B shows an In vitro pull-down analysis of the association between SP1
and SP2.
In vitro translated (IVT), 35S-radiolabelled SP2 or OEP80 (as a control OEM
protein) were
used as "prey" in pull-down assays with bacterially-expressed, purified GST-
SP1flex or an
excess of GST (negative control) as the "baits". Eluted GST proteins, along
with any
associated partner proteins, were resolved by SDS-PAGE and then analysed by
phosphorimaging (to detect the radiolabelled "prey" proteins) or Coomassie
Brilliant Blue
staining (to detect the GST "baits"). The result showed that SP1 specifically
associates
with SP2 in vitro.
An absence of phenotypic additivity in sp1 sp2 double mutants (in the ppi1
background), in
relation to plant greening and Toc75 protein accumulation (Figure 12A - D),
supports SP1
and SP2 functioning together. Indeed, SP2 is essential for SP1 action, as the
sp2
mutation abrogated the effect of SP1 overexpression, as shown in Figure 14.
In more detail for Figure 14, the sp2 mutation was introduced into the SP1-0X
ppi1
background (7) by crossing the latter with the sp2-4 ppi1 double mutant. A
resultant F2
population was grown on phosphinothricin plates to select for the SP1-0X
construct, and
the phosphinothricin-resistant plants were PCR genotyped to identify those
that were sp2-
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4 homozygous. Individual F3 families were grown on phosphinothricin plates to
select
those that were homozygous for the SP1-0X construct. The SP1-0X sp2-4 ppi1
plants
thus identified were then, together with corresponding control genotypes,
grown on MS
agar medium for 10 days before photography (Figure 14A) and photometric
chlorophyll
content analysis following DMF extraction (Figure 14B). About 10 seedlings per
genotype
were measured in each experiment, and the values shown are means SEM derived
from
four experiments per genotype and indicate nmol total chlorophyll per mg
tissue fresh
weight. As reported before (7), SP1-0X enhanced the chlorotic phenotype of
ppi1 by
making the plants even paler. This effect of SP1-0X on ppi1 was abolished by
the sp2-4
mutation, indicating that SP1 action is dependent on 5P2.
Moreover, whereas the overexpression of neither SP1 nor SP2 individually
affected plant
greening in the wild-type background under standard conditions (see Figure
10), the
simultaneous overexpression of both genes caused strong chlorosis linked to
severe
depletion of TOO proteins (see Figure. 3E - G), indicating functional
interdependency
between SP1 and 5P2. Such interactions are often observed where the components
interact physically, and may arise through mutual stabilization (26, 27).
Indeed, SP1 and
5P2 co-migrated (with each other and with the TOO apparatus) on native gels
and
interacted specifically in vitro (see Figure 13).
Chloroplast-Associated Protein Degradation (CHLORAD).
Without wishing to be bound by any particular hypothesis, the inventors
consider that SP1
and 5P2 exist at the core of a system for chloroplast protein removal,
designated
Chloroplast-Associated Protein Degradation (CHLORAD). This system targets
chloroplast
substrates for degradation, either as a homeostatic, quality-control process
for the removal
damaged proteins under stress, or as a regulatory mechanism to control plastid
development and functions. Figure 16 is a detailed diagram showing the
proposed model
of action of the SP1 and 5P2 proteins that the inventors have.
The full length genomic sequence of SP2 At3g44160 [SEQ ID NO:1] is:
1 AAAAATATCC AAAGCATCAA ATCCTTAACC TCTCTGCTAA TTCATTCACT
51 CCTGAAGAAG AAGAAAGAAG AAATAAGTAA ATAAAAATTC CTCCTTTTTC
101 TGGTCATTGC TTGTCTAATG CCAATTCCTA AATTGGGTTC TCTTCATGGT
151 TGATTCTTCT CTCATTCCAT CGCCATGGGA GCTCAGAAGA GTATCCACGC
201 TGGTAGAGGT CATCACTCTT ATCTTAGATT CTCGATTTTT CGAATTTTGT
251 GTTGTTGCTG GATCAGTAGG TAATGTGATA CTGTGCTGGA AATGGTACGA
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301 TTCTGGAATT TGATTGTGGA TTTAACCTTA ACCAATGGAG GGCTTTGCTT
351 TGTTTCCATT TTTCTTCTAT TATCTTACAT TCATTTTCAA TGTATAAGAT
401 ATGATTCATT TCATTTGTAT AGTTATGATG ATGTTTATGT TTGTGGTAAT
451 TGATGAATTA GAATGTTTTA GGCATGAGTA CACATTGTTT TTTTTGGTAA
.. 501 AAGAAACATT TTTCTAAGAC TTTTGTTACC AACCATTATG TGTGATACGT
551 CTTGCTTTTG GGTTTTGTGA AAGATTGGTT TTGATATGTG GATTTCCTAA
601 ATGTCTTTTT GGGGAAAAGT ATCGATTTTG ATCTGAATGT TGTAACTTTT
651 TCTGTATCTT TTTGTTACTA ACTCATCATG TCGAATATGT CTTTCTGTTG
701 AATATAGTGA AAGATTAAGA TCGGACTGGT TTGCAATTGC AATGTAGTTT
.. 751 TCTAGCAGCG GCATTGATTT TGATATTCTT GTGTTGTTGA TTGCAGCCAA
801 GATTGATGTT AATGTGGACT TCACTCACAA GCTTTGTACT TCTTTAATGT
851 TTCCTGCTTT CAGGTTTGCT CTCATCATCC ATACTTTGAC GTCACTTACA
901 TTTTAGCTAA TACTCATGAA ACTGTACCGC CTTTAGCTTT CTCGACGTAT
951 TAAAGCTCAG TTTGTTACTT TTTGAACAGG GACACTAGCA GTCCTCTTTC
1001 TCTAGTGATT GGCAGGCAAG TTTCGAGACT TAATGCTACA GCTTACAGAA
1051 ATATTGTTGG TGTTCATATA ATCTGCAGAA CTAAATTTTC ATATCTTTCT
1101 TTTCGATTCA AGTGTTCATT TGGATCTGTT TGTTGCAGCC TCTGTATCAA
1151 ACATCCAAAT TTGTTTGGAG GAAGCGAGAA GCTTGATGTA TCATGGGATA
1201 AAGGATTGTA TGATTCCAAT GTACTTGTGG CTTTTAGGAG ACCTAGACCT
1251 GAATGGCGTC CACAACAGTG TTTCTTCATA CAGGTACTCA ATGCTTATTT
1301 GTTATTGTAC TGATCTGTGA AGCTATCTTA GAAAGTGAAT TTTAAAGCTA
1351 TGTTACATTT AAATTTGTTA ATCTGGCGCA GCATTCTCTC TCACCCGAGA
1401 TAGGGGTCCA CGGCACCCCA GTCGACAACT TTTCTCGGTC AGGAAGTGGA
1451 GGTGTAAATC TGTCTAAATT GGCTCTTGGT TTAGACTTGA GTGAGCCAGC
1501 GAGTTCAAAA TGGAGCAGCA CAACCAGCAT AAAGTTTGAG GTGCCCGCAC
1551 ATTACCTTCT TCAGACATTG TAGAACAATA TTTTTCTCTT TGCTGTTTTG
1601 CTTTGGTATA AAAGAGTAAT ATTTTCCATT GGTGCAGCAT GTGCGTCCGA
1651 TTAACGATGA TGGACGCGCG ATAACCAGAG ATCTGGACGG ATTTCCTATA
1701 ACATGCAGGT GATATTAGAT CTCGATTCCC TTAATTTGTT TCTTTAAGTA
1751 CTAGCATAAA ACTGATATTT ACATGTGTAG AATTCTCTGC TATGCAGTGG
1801 AAATACCCAT GACAGTATGG TAGTTTTAAA GCAAGAATCC CGGTTTGCAA
1851 AGGCTACCGA CCAAGGTCTT TCTCATGTAA GAGACTCTTC ATTTCTGTTT
1901 TATAGCCTAG GCAAACCACA CAGCCATTTT TGCAGTAACT TTGTCAACGT
1951 TTCTTTTCTT TCATACTGGT TTCTTGCTTT TTCAGTTTAG CATGCAAATA
2001 GAACAAGGTA TTCCGGTTGT GTCCAAGTGG CTTATCTTCA ACCGTTTCAA
2051 ATTTGTTGCA TCAAAAGGTG TCAGGTTTGG ACCGGCTTTT CTCTTAGCAA
2101 GGTACTGACA GAATCGTACA CACTTGATCT AGAAAACTAC ACGTAGAAGA
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2151 TGCTTTGTAA AGATGTCCTA GTTTTGCCGT GGTTCTTACC TCTTCTGTGA
2201 CAGCTTGACA GGTGGTTCAA TTGTAGGAGA CATGGCACCT TATCAAGCAT
2251 TTGCCATTGG TGGGCTAGGC AGTGTTCGCG GATATGGTGA GGGTGCTGTT
2301 GGATCCGGTC GGTCATGCCT TGTTGCCAAT ACAGAATTGG CGTTACCTTT
2351 GGTACGTGGA GCTGTTGCAT GATTTGGTCA TCAGAACCAT TAAATGGTTT
2401 CCCCTGTTAA GCTTTCATTG AGCTTTGGTG TCTTTTTGTG TACAGAACAA
2451 GATGACAGAA GGGACCATCT TCTTGGATTG TGGAACAGAC CTAGGCTCAA
2501 GCCGCCTTGT CCCCGGTAAG TTCACTTTAC CTTCTCCGAA CAAGAATCAT
2551 AAAGAAGTTG AGACACAAAA ATGATAAAGC TGTTGCGGAA TTGGTTTCAG
2601 GAAACCCTTC AATGAGACAA GGGAAGCCAG GGTTTGGGTA TGGATTCGGA
2651 TATGGGCTAC GGTTCAAGTC TCCATTGGGT CACCTTCAGG TTGACTATGC
2701 CATAAATGCT TTCAACCAGA AGACTCTTTA CTTCGGTGTC ACCAATCTTG
2751 CTTCATCAAC ATAGTCAAAT AGAATAAAGC AATCAAGAAA AGCACATATT
2801 CAATGTCTTG ATTCAAAGAT ATATTTGTGT TTGCGTTGGA ACCAGAAACG
2851 CCACAAGATG AGGCAAACAC AGTCCAAGGA GAAGCTGTAT ATGACAGAGA
2901 TCTTGAGAAG ATAAATGTAG TGTTGTCATT AGAAATCATG TAATATTACA
2951 CGGGTATAAG TTTTCATTGT TTTGGTATAT ACAGCTACCA GAGTTTTGTC
3001 TGAAAAGCTG CAGGTTTCAT AGAGAAGAGA ACACACATTT GATTTGATGG
3051 TGTTGCACTG TTGCGAATTA GTATCTATTG CTTTTACATT TGTACGTATA
3101 TCTATGGCTC GACTCTCGAC TAACTCTGAA TACAAGTAGT GGTTGAACC
The full-length cDNA sequence of SP2 [SEQ ID NO: 2] is:
1 AAAAATATCC AAAGCATCAA ATCCTTAACC TCTCTGCTAA TTCATTCACT
51 CCTGAAGAAG AAGAAAGAAG AAATAAGTAA ATAAAAATTC CTCCTTTTTC
101 TGGTCATTGC TTGTCTAATG CCAATTCCTA AATTGGGTTC TCTTCATGGT
151 TGATTCTTCT CTCATTCCAT CGCCATGGGA GCTCAGAAGA GTATCCACGC
201 TGGTAGAGCC AAGATTGATG TTAATGTGGA CTTCACTCAC AAGCTTTGTA
251 CTTCTTTAAT GTTTCCTGCT TTCAGGGACA CTAGCAGTCC TCTTTCTCTA
301 GTGATTGGCA GCCTCTGTAT CAAACATCCA AATTTGTTTG GAGGAAGCGA
351 GAAGCTTGAT GTATCATGGG ATAAAGGATT GTATGATTCC AATGTACTTG
401 TGGCTTTTAG GAGACCTAGA CCTGAATGGC GTCCACAACA GTGTTTCTTC
451 ATACAGCATT CTCTCTCACC CGAGATAGGG GTCCACGGCA CCCCAGTCGA
501 CAACTTTTCT CGGTCAGGAA GTGGAGGTGT AAATCTGTCT AAATTGGCTC
551 TTGGTTTAGA CTTGAGTGAG CCAGCGAGTT CAAAATGGAG CAGCACAACC
601 AGCATAAAGT TTGAGCATGT GCGTCCGATT AACGATGATG GACGCGCGAT
651 AACCAGAGAT CTGGACGGAT TTCCTATAAC ATGCAGTGGA AATACCCATG
701 ACAGTATGGT AGTTTTAAAG CAAGAATCCC GGTTTGCAAA GGCTACCGAC
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751 CAAGGTCTTT CTCATTTTAG CATGCAAATA GAACAAGGTA TTCCGGTTGT
801 GTCCAAGTGG CTTATCTTCA ACCGTTTCAA ATTTGTTGCA TCAAAAGGTG
851 TCAGGTTTGG ACCGGCTTTT CTCTTAGCAA GCTTGACAGG TGGTTCAATT
901 GTAGGAGACA TGGCACCTTA TCAAGCATTT GCCATTGGTG GGCTAGGCAG
.. 951 TGTTCGCGGA TATGGTGAGG GTGCTGTTGG ATCCGGTCGG TCATGCCTTG
1001 TTGCCAATAC AGAATTGGCG TTACCTTTGA ACAAGATGAC AGAAGGGACC
1051 ATCTTCTTGG ATTGTGGAAC AGACCTAGGC TCAAGCCGCC TTGTCCCCGG
1101 AAACCCTTCA ATGAGACAAG GGAAGCCAGG GTTTGGGTAT GGATTCGGAT
1151 ATGGGCTACG GTTCAAGTCT CCATTGGGTC ACCTTCAGGT TGACTATGCC
1201 ATAAATGCTT TCAACCAGAA GACTCTTTAC TTCGGTGTCA CCAATCTTGC
1251 TTCATCAACA TAGTCAAATA GAATAAAGCA ATCAAGAAAA GCACATATTC
1301 AATGTCTTGA TTCAAAGATA TATTTGTGTT TGCGTTGGAA CCAGAAACGC
1351 CACAAGATGA GGCAAACACA GTCCAAGGAG AAGCTGTATA TGACAGAGAT
1401 CTTGAGAAGA TAAATGTAGT GTTGTCATTA GAAATCATGT AATATTACAC
1451 GGGTATAAGT TTTCATTGTT TTGGTATATA CAGCTACCAG AGTTTTGTCT
1501 GAAAAGCTGC AGGTTTCATA GAGAAGAGAA CACACATTTG ATTTGATGGT
1551 GTTGCACTGT TGCGAATTAG TATCTATTGC TTTTACATTT GTACGTATAT
1601 CTATGGCTCG ACTCTCGACT AACTCTGAAT ACAAGTAGTG GTTGAACC
The amino acid sequence of the SP2 protein [SEQ ID NO: 3] is:
1 MGAQKSIHAG RAKIDVNVDF THKLCTSLMF PAFRDTSSPL SLVIGSLCIK
51 HPNLFGGSEK LDVSWDKGLY DSNVLVAFRR PRPEWRPQQC FFIQHSLSPE
101 IGVHGTPVDN FSRSGSGGVN LSKLALGLDL SEPASSKWSS TTSIKFEHVR
151 PINDDGRAIT RDLDGFPITC SGNTHDSMVV LKQESRFAKA TDQGLSHFSM
201 QIEQGIPVVS KVVLIFNRFKF VASKGVRFGP AFLLASLTGG SIVGDMAPYQ
251 AFAIGGLGSV RGYGEGAVGS GRSCLVANTE LALPLNKMTE GTIFLDCGTD
301 LGSSRLVPGN PSMRQGKPGF GYGFGYGLRF KSPLGHLQVD YAINAFNQKT
351 LYFGVTNLAS ST
The full length genomic sequence of SP1 At1g63900 [SEQ ID NO: 4] exons are
marked in
upper case is:
ATGATTCCTTGGGGTGGAGTTACTTGCTGCCTCAGCGCCGCTGCTCTTTATCTTCT
CGGCCGGAGTAGTGGCAGgtttgtctgatctcttttatatttcatcttcccaaagagattatcaatcaatcaaatcc
tttcttatccttttgagtgcagGGATGCTGAAGTACTCGAAACAGTCACTAGGGTTAATCAGCT
CAAGGAGTTAGgtaatcttcttctcccctgattgcttcatctactctcaggatgaagttttgatcatgttttctgattg
ttctgt
atgtgtagCTCAATTGCTAGAATTAGATAGCAAGATTCTGCCTTTCATTGTTGCGGTAT
CAGGAAGAGTCGGCTCTGAGACACCTATCAAATGCGAGCATAGTGGCATACGCG
GTGTTATTGTTGAAGAAACGgtatgttgtagactgatgattagcgcatggaacttagtttgttttctggtttaatcg
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attaggttttatgtgaaactctagtaattgcatgatcttttcagGCGGAACAACATTTCCTGAAACATAATG
AGACGGGTTCTTGGGTACAAGATAGTGCGTTGATGCTATCAATGAGCAAAGAGGT
TCCTTGGTTTCTG gtaagtctagtctagtag catagtgtttg aaacaactgtg attgg
atgttttcttaataccatttcc
aaaactgtttgggatagGACGATGGGACAAGCCGTGTCCATGTAATGGGAGCTCGTGGTG
CG ACG G GTTTTG CCTTG ACTGTTG GTAGTG AAGTTTTTG AAG AGTCAG
GACGCTCGCTTGTACGAGGAACACTTGATTATCTTCAAGGACTTAAGgtttttacttttctttccgg
ttctttgtttgttggcttctctatttgcttaagcggccattgttttgtttcagATGCTTGGAGTTAAGCGCATTGAGCG
T
GT TCTTCCAACTGGAATACCGCTAACAATTGTTGGTGAGgtatgtcgtattctcagtgttttcgggtcct
ctcttttgcttaagttgtaactgttgatagagatacatagcacactaactccttcatcagtctggtatttgcctcttga
aattttctc
aaagttcctttaatagcaatatttgtaggaagtgggattgatctatgtatagaggcttaccgatgagtttaaatctaat
ttgtgtt
gctgccatgtataacagGCTGTCAAGGACGATATTGGAGAATTCAGGATTCAAAAACCTGA
CAGGGGCCCTTTCTACGTCTCTTCTAAATCACTCGATCAGCTCATTTCTAATTTGG
GAAAATGGTCAAG gtcgtgtctctctcctctctcg gttcttctcctatactcttgtag aaaaacg gcaatg ag
ccaaa
ctgattgagaagagtataatttacagGTTGTACAAATATGCCTCCATGGGTTTTACTGTTCTTGG
TGTGTTCCTAATTACGAAGCATGTCATTGACTCTGTTCTAGAGAGAAGACGGCGG
AGACAGTTACAAAAAAG gtatgtcacag atttgtctgtctaaaagtg aataaccgttctcaagcatgagtactag
atcggcttgtttctctcgaaactatgtacacacaaaattaagtagtcagctgtttttgcagAGTGCTTGACGCAGC
AGCAAAGAGAGCTGAGCTAGAGAGTGAAGgtatccattggtgaatctctttattctacatataggttgca
ctggctctgactacaatctcttctgaccagGTTCAAACGGGACACGTGAGAGCATTTCAGATTCTA
CCAAGAAAGAAGACGCTGTTCCTGATCTCTGTGTGATATGCCTAGAGCAGGAGTA
CAACGCTGTGTTTGTCCCgtaagcattcttccgccatttttggttgattctgcatttgcaacttgctaaaatgcttgt
ggttggtactcgcagGTGTGGTCATATGTGCTGCTGCACCGCATGCTCCTCCCACTTGACCA
GCTGTCCACTTTGTCGGAGACGAATAGATCTGGCGGTTAAGACATATCGTCAC TGA
The full-length cDNA sequence of SP1 [SEQ ID NO: 5] is:
atgagaatattgagagagatcgaagcaaaggatcattcaattccaaccctctgaatcttttaatttcccctttcgaaat
tctcc
tcttctttcactgcttctagtttctaattcttcaaactcttcctcgattcatactcataactctcattagctaatttcg
catgatcttcttc
catctctctgtgttctaaatccagattcgtttcactcccatctctatttcattcaattcgctgcatccagattcaaaac
ctacctcta
tctctctgctcatcaataacttcaaaggtattgttgttcttctgcaaacaagtaagagtgacttcagagtctgatgatt
ccttggg
gtggagttacttgctgcctcagcgccgctgctctttatcttctcggccggagtagtggcagggatgctgaagtactcga
aac
agtcactagggttaatcagctcaaggagttagctcaattgctagaattagatagcaagattctgcctttcattgttgcg
gtatc
aggaagagtcggctctgagacacctatcaaatgcgagcatagtggcatacgcggtgttattgttgaagaaacggcgga
acaacatttcctgaaacataatgagacgggttcttgggtacaagatagtgcgttgatgctatcaatgagcaaagaggtt
cc
ttggtttctggacgatgggacaagccgtgtccatgtaatgggagctcgtggtgcgacgggttttgccttgactgttggt
agtg
aagtttttgaagagtcaggacgctcgcttgtacgaggaacacttgattatcttcaaggacttaagatgcttggagttaa
gcg
cattgagcgtgttcttccaactggaataccgctaacaattgttggtgaggctgtcaaggacgatattggagaattcagg
attc
aaaaacctgacaggggccctttctacgtctcttctaaatcactcgatcagctcatttctaatttgggaaaatggtcaag
gttgt
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acaaatatgcctccatgggttttactgttcttggtgtgttcctaattacgaagcatgtcattgactctgttctagagag
aagacg
gcggagacagttacaaaaaagagtgcttgacgcagcagcaaagagagctgagctagagagtgaaggttcaaacgg
gacacgtgagagcatttcagattctaccaagaaagaagacgctgttcctgatctctgtgtgatatgcctagagcaggag
ta
caacgctgtgtttgtcccgtgtggtcatatgtgctgctgcaccgcatgctcctcccacttgaccagctgtccactttgt
cggag
acgaatagatctggcggttaagacatatcgtcactgaacaacaactcaggcctcagaaacattctctacttgagtcttg
tct
gtaaataccgcaaaatcaaaacattacacagtttagcgttcgatattccctttggtttgatttcgacaacaaaacattt
tgaatt
atatagaaacataaggtgtttactcgatttgcaaaacagtacattcgtgtttacttattcgtgttgttgccaatgccat
gaggtg
The amino acid sequence of the SP1 protein [SEQ ID NO: 6] is
MIPWGGVTCCLSAAALYLLGRSSGRDAEVLETVTRVNQLKELAQLLELDSKILPFIVAV
SGRVGSETPIKCEHSGIRGVIVEETAEQHFLKH NETGSVVVQDSALMLSMSKEVPWFL
DDGTSRVHVMGARGATGFALTVGSEVFEESGRSLVRGTLDYLQGLKMLGVKRIERVL
PTGIPLTIVGEAVKDDIGEFRIQKPDRGPFYVSSKSLDQLISNLGKWSRLYKYASMGFT
VLGVFLITKHVIDSVLERRRRRQLQKRVLDAAAKRAELESEGSNGTRESISDSTKKEDA
VPDLCVICLEQEYNAVFVPCGHMCCCTACSSHLTSCPLCRRRIDLAVKTYRH
Throughout the description and claims of this specification, the words
"comprise" and
"contain" and variations of them mean "including but not limited to", and they
are not
intended to (and do not) exclude other moieties, additives, components,
integers or steps.
Throughout the description and claims of this specification, the singular
encompasses the
plural unless the context otherwise requires. In particular, where the
indefinite article is
used, the specification is to be understood as contemplating plurality as well
as singularity,
unless the context requires otherwise.
Features, integers, characteristics, compounds, chemical moieties or groups
described in
conjunction with a particular aspect, embodiment or example of the invention
are to be
understood to be applicable to any other aspect, embodiment or example
described herein
unless incompatible therewith. All of the features disclosed in this
specification (including
any accompanying claims, abstract and drawings), and/or all of the steps of
any method or
process so disclosed, may be combined in any combination, except combinations
where at
least some of such features and/or steps are mutually exclusive. The invention
is not
restricted to the details of any foregoing embodiments. The invention extends
to any novel
one, or any novel combination, of the features disclosed in this specification
(including any
accompanying claims, abstract and drawings), or to any novel one, or any novel
combination, of the steps of any method or process so disclosed.
The reader's attention is directed to all papers and documents which are filed
concurrently
with or previous to this specification in connection with this application and
which are open
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to public inspection with this specification, and the contents of all such
papers and
documents are incorporated herein by reference.
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