Note: Descriptions are shown in the official language in which they were submitted.
CA 02754916 2011-09-08
WO 2010/108836 PCT/EP2010/053470
TRANSGENIC PLANTS WITH ALTERED REDOX MECHANISMS AND INCREASED
YIELD
FIELD OF THE INVENTION
[00011 This application claims priority benefit of U.S. provisional patent
application
serial number 61/162,427, filed March 23, 2009, the entire contents of which
are incorporated
herein by reference.
BACKGROUND OF THE INVENTION
[0002] Population increases and climate change have brought the possibility of
global
food, feed, and fuel shortages into sharp focus in recent years. Agriculture
consumes 70% of
water used by people, at a time when rainfall in many parts of the world is
declining. In
addition, as land use shifts from farms to cities and suburbs, fewer hectares
of arable land are
available to grow agricultural crops. Agricultural biotechnology has attempted
to meet
humanity' s growing needs through genetic modifications of plants that could
increase crop
yield, for example, by conferring better tolerance to abiotic stress responses
or by increasing
biomass.
[0003] Crop yield is defined herein as the number of bushels of relevant
agricultural
product (such as grain, forage, or seed) harvested per acre. Crop yield is
impacted by abiotic
stresses, such as drought, heat, salinity, and cold stress, and by the size
(biomass) of the
plant. Traditional plant breeding strategies are relatively slow and have in
general not been
successful in conferring increased tolerance to abiotic stresses. Grain yield
improvements by
conventional breeding have nearly reached a plateau in maize. The harvest
index, i.e., the
ratio of yield biomass to the total cumulative biomass at harvest, in maize
has remained
essentially unchanged during selective breeding for grain yield over the last
hundred years.
Accordingly, recent yield improvements that have occurred in maize are the
result of the
increased total biomass production per unit land area. This increased total
biomass has been
achieved by increasing planting density, which has led to adaptive phenotypic
alterations, such
as a reduction in leaf angle, which may reduce shading of lower leaves, and
tassel size, which
may increase harvest index.
[0004] When soil water is depleted or if water is not available during periods
of drought,
crop yields are restricted. Plant water deficit develops if transpiration from
leaves exceeds the
supply of water from the roots. The available water supply is related to the
amount of water
held in the soil and the ability of the plant to reach that water with its
root system.
Transpiration of water from leaves is linked to the fixation of carbon dioxide
by photosynthesis
through the stomata. The two processes are positively correlated so that high
carbon dioxide
influx through photosynthesis is closely linked to water loss by
transpiration. As water
transpires from the leaf, leaf water potential is reduced and the stomata tend
to close in a
hydraulic process limiting the amount of photosynthesis. Since crop yield is
dependent on the
fixation of carbon dioxide in photosynthesis, water uptake and transpiration
are contributing
factors to crop yield. Plants which are able to use less water to fix the same
amount of carbon
dioxide or which are able to function normally at a lower water potential have
the potential to
conduct more photosynthesis and thereby to produce more biomass and economic
yield in
many agricultural systems.
[0005] Agricultural biotechnologists have used assays in model plant systems,
greenhouse studies of crop plants, and field trials in their efforts to
develop transgenic plants
CA 02754916 2011-09-08
WO 2010/108836 PCT/EP2010/053470
2
that exhibit increased yield, either through increases in abiotic stress
tolerance or through
increased biomass. For example, water use efficiency (WUE), is a parameter
often correlated
with drought tolerance. Studies of a plant' s response to desiccation, osmotic
shock, and
temperature extremes are also employed to determine the plant' s tolerance or
resistance to
abiotic stresses.
[0006] An increase in biomass at low water availability may be due to
relatively
improved efficiency of growth or reduced water consumption. In selecting
traits for improving
crops, a decrease in water use, without a change in growth would have
particular merit in an
irrigated agricultural system where the water input costs were high. An
increase in growth
without a corresponding jump in water use would have applicability to all
agricultural systems.
In many agricultural systems where water supply is not limiting, an increase
in growth, even if it
came at the expense of an increase in water use also increases yield.
[0007] Agricultural biotechnologists also use measurements of other parameters
that
indicate the potential impact of a transgene on crop yield. For forage crops
like alfalfa, silage
corn, and hay, the plant biomass correlates with the total yield. For grain
crops, however,
other parameters have been used to estimate yield, such as plant size, as
measured by total
plant dry weight, above-ground dry weight, above-ground fresh weight, leaf
area, stem volume,
plant height, rosette diameter, leaf length, root length, root mass, tiller
number, and leaf
number. Plant size at an early developmental stage will typically correlate
with plant size later
in development. A larger plant with a greater leaf area can typically absorb
more light and
carbon dioxide than a smaller plant and therefore will likely gain a greater
weight during the
same period. There is a strong genetic component to plant size and growth
rate, and so for a
range of diverse genotypes plant size under one environmental condition is
likely to correlate
with size under another. In this way, a standard environment is used to
approximate the
diverse and dynamic environments encountered at different locations and times
by crops in the
field.
[0008] Harvest index is relatively stable under many environmental conditions,
and so
a robust correlation between plant size and grain yield is possible. Plant
size and grain yield
are intrinsically linked, because the majority of grain biomass is dependent
on current or stored
photosynthetic productivity by the leaves and stem of the plant. As with
abiotic stress
tolerance, measurements of plant size in early development, under standardized
conditions in
a growth chamber or greenhouse, are standard practices to measure potential
yield
advantages conferred by the presence of a transgene.
[0009] Plants cannot move to find sources of energy or to avoid predation or
stress. As
a result, plants have evolved various biochemical pathways and networks to
respond to their
environment that maintain the supply of energy to the developing plant under
diverse
environmental conditions. One of the challenges to plants under these adverse
conditions,
such as drought, temperature extremes and exposure to heavy metals, is that
some metabolic
products are highly toxic. In the case of oxidative stress, these toxins
include the highly
reactive oxygen species (ROS) of superoxide, peroxide, hydroxyl radicals, and
organic
derivatives thereof. ROS, are highly reactive towards organic molecules such
as unsaturated
lipids, nucleic acids and proteins. ROS abstract hydrogen from these organic
molecules,
leading to the formation of reduced oxygen (water or a reduced organic
product) and a second
organic ROS, which perpetuates a chain reaction leading to the continuous
destruction of
CA 02754916 2011-09-08
WO 2010/108836 PCT/EP2010/053470
3
cellular components until the ROS is scavenged. Scavenging of ROS involves the
formation of
a non-reactive end product that is not a ROS species. A number of hydrogen
donors that act
as ROS scavengers are known to function in plant cells, including tocopherol,
ascorbate,
gluthione, and thioredoxin. These diverse ROS scavengers share two common
characteristics;
their oxidized form is not reactive to other organic compounds, and the
oxidized form can be
reduced by metabolic reactions in the cell to regenerate the reduced form of
the scavenger in
a cyclic reaction drawing reducing equivalents directly or indirectly from
NAD(P)H.
[0010] Oxidative stress occurs in plants under adverse environmental
conditions when
the production of ROS formed as by-products of metabolism exceeds the capacity
of the
plant' s scavenging systems to dissipate ROS into stable end-products. To cope
with
oxidative stress, the plant cell must contain adequate quantities of
scavengers or enzymes
capable of inactivating ROS. In addition, the cell also requires an adequate
supply of reducing
equivalents in the form of NAD(P)H to regenerate the active form of the
scavenger. If either is
inadequate, the titer of ROS increases and the cell suffers oxidative damage
to lipids, nucleic
acids or proteins. In severe cases, this damage may lead to cell death,
necrosis and loss of
productivity.
[0011] Glutathione has been detected in nearly all plant cell compartments,
such as the
cytosol, chloroplasts, endoplasmic reticulum, vacuoles, and mitochondria.
Glutathione is the
major source of non-protein thiols in plant cells; it is the chemical
reactivity of the thiol group
that makes glutathione involved in many biochemical functions. Glutathione is
water-soluble,
stable and in addition to detoxifying ROS, it also protects against other
stresses such as heavy
metals, organic chemicals, and pathogens. The soluble enzyme, " classic"
glutathione
peroxidase, converts reduced monomeric glutathione (GSH) with H202 to its
oxidized form,
disulfide glutathione (GSSG) and H2O. The cellular redox balance of a cell is
indicative of the
GSH/GSSG ratio, and has been suggested to be involved in ROS perception and
signaling. A
second form of glutathione peroxidase, phospholipid hydroperoxide glutathione
peroxidase
(PHGPx), can be membrane-associated. PHGPx is associated with diverse
functions, such as
signaling and cellular differentiation, and may be linked to the thioredoxin
pathway. PHGPx
also reduces lipid hydroperoxides esterified to membranes. Thus, PHGPx has
been
associated with repair of membrane lipid peroxidation.
[0012] Glutathione is also involved in glutathionylation, which modifies
proteins by
protecting specific cysteine residues from irreversible oxidation, thereby
regulating activity of
certain proteins. The enzyme isocitrate lyase is deactivated through
glutathionylation.
Isocitrate lyase catalyzes the formation of succinate and glyoxylate from
isocitrate, part of the
glyoxylate cycle, which converts two molecules of acetyl-CoA to one succinate
molecule.
[0013] Glutathione can also be degraded by the action of gamma-
glutamyltranspeptidase, which catalyzes the transfer of the gamma-glutamyl
moiety of
glutathione to an acceptor that may be an amino acid, a peptide or water.
Based on homology
to animal GGTs, four genes have been found in Arabidopsis: GGT1, GGT2, GGT3,
and
GGT4. GGT1 accounts for 80-99% of the activity, except in seeds, where GGT2
accounts for
50% activity. Knockouts of GGT2 and GGT4 show no apparent phenotype, but GGT1
knockouts had premature senescence of rosettes shortly after flowering.
Knockouts of GGT3
show reduced number of siliques and reduced seed yield.
[0014] Reduction-oxidation (redox) reactions occur when atoms undergo a change
in
CA 02754916 2011-09-08
WO 2010/108836 PCT/EP2010/053470
4
their oxidative state, by an electron-transfer reaction. Oxidation describes a
gain of oxidation
state by losing hydrogen or gaining oxygen. Reduction describes a loss of
oxidation state by
gaining hydrogen or losing oxygen. In biology, many important energy storing
or releasing
pathways involve redox reactions. Cellular respiration oxidizes glucose to
C02, and reduces
02 to water. In photosynthesis, C02 is reduced to sugars and H2O is oxidized
to 02 in
Photosystem II. In Photosystem I, the electron gradient reduces cofactor NAD+
to NADH. A
proton gradient is produced, driving the synthesis of ATP, as what occurs in
the respiratory
chain, which pumps H+ out; the H+ transporting ATP synthase couples H+ uptake
to ATP
synthesis. In non-photosynthetic organisms such as E. coli, redox reactions
can exchange
electrons and utilize hydrogen as an energy source to allow anaerobic growth,
which require
the action of hydrogenases.
[0015] The redox state of a cell is mainly reflective of the ratio of
NAD+/NADH or
NADP+/NADPH. This balance is reflected in the amount of metabolites such as
pyruvate and
lactate. Plant growth requires a supply of carbon, ATP, NADH and NADPH. These
requirements are met by glycolysis and the pentose phosphate pathway, which
provides an
oxidative route for regenerating NADPH as well as a non-oxidative route for
producing ribose
and other pentoses from the hexoses enocuountered in metabolism. Transaldolase
is an
enzyme in the non-oxidative pentose phosphate pathway that catalyzes the
reversible transfer
of a three-carbon ketol unit from sedoheptulose-7-phosphate to glyceraldehyde-
3-phosphate
to form erythrose-4-phosphate and fructose-6-phosphate. Transaldolase,
together with
transketolase, provides a link between the glycolytic and pentose phosphate
pathways.
[0016] Galactose metabolism plays a part in cellular metabolism by providing
glucose
for fructose and mannose metabolism, nucleotide sugar metabolism, and
glycolysis. The
transformation of galactose into glucose-1 -phosphate requires the action of
three enzymes by
the Leloir pathway: galactokinase, galactose-1-phosphate uridylyltransferase,
and UDP-
galactose 4-epimerase. Galactokinase specifically phosphorylates galactose
using ATP to
form galactose-1 -phosphate in the first step of the pathway.
[0017] Although some genes that are involved in stress responses, water use,
and/or
biomass in plants have been characterized, but to date, success at developing
transgenic crop
plants with improved yield has been limited, and no such plants have been
commercialized.
There is a need, therefore, to identify additional genes that have the
capacity to increase yield
of crop plants.
SUMMARY OF THE INVENTION
[0018] The present inventors have discovered that alterations to the
expression of
genes related to the ROS scavenging system in plants can improve plant yield.
When targeted
as described herein, the polynucleotides and polypeptides set forth in Table 1
are capable of
improving yield of transgenic plants.
Table 1
Gene Name Organism Polynucleotide Amino acid
SEQ ID NO SEQ ID NO
b0757 Escherichia coli 1 2
GM59594085 Glycine max 3 4
GM59708137 G. max 5 6
CA 02754916 2011-09-08
WO 2010/108836 PCT/EP2010/053470
Gene Name Organism Polynucleotide Amino acid
SEQ ID NO SEQ ID NO
ZMBFb0152K10 Zea mays 7 8
b2464 E. coli 9 10
BN43182918 Brassica napus 11 12
GM48926546 G. max 13 14
b2990 E. coli 15 16
Saccaromyces
YER065C cerevisiae 17 18
YI R037W S. cerevisiae 19 20
BN42261838 B. napus 21 22
BN43722096 B. napus 23 24
BN51407729 B. napus 25 26
GM50585691 G. max 27 28
GMsa56cO7 G. max 29 30
GMsp82f11 G. max 31 32
GMss66fO3 G. max 33 34
HA03MC1446 Helianthus anuus 35 36
HV03MC9784 Hordeum vulgare 37 38
OS34914218 Oryza sativa 39 40
ZM61990487 Z. mays 41 42
ZM68466470.rOl Z. mays 43 44
sIr1269 Synechocystis sp. 45 46
SLL1323 Synechocystis sp. 47 48
Gmsb38bO4 G. max 49 50
YMR015C S. cerevisiae 51 52
GMso65hO7 G. max 53 54
[0019] In one embodiment, the invention provides a transgenic plant
transformed with
an expression cassette comprising, in operative association, an isolated
polynucleotide
encoding a promoter; an isolated polynucleotide encoding a chloroplast transit
peptide; and an
5 isolated polynucleotide encoding a full-length galactokinase polypeptide;
wherein the
transgenic plant demonstrates increased yield as compared to a wild type plant
of the same
variety which does not comprise the expression cassette.
[0020] In another embodiment, the invention provides a transgenic plant
transformed
with an expression cassette comprising, in operative association, an isolated
polynucleotide
encoding a promoter and an isolated polynucleotide encoding a full-length
transaldolase A
polypeptide; wherein the transgenic plant demonstrates increased yield as
compared to a wild
type plant of the same variety which does not comprise the expression
cassette.
[0021] In another embodiment, the invention provides a transgenic plant
transformed
with an expression cassette comprising, in operative association, an isolated
polynucleotide
encoding a promoter and an isolated polynucleotide encoding a full-length
hydrogenase-2
accessory polypeptide; wherein the transgenic plant demonstrates increased
yield as
CA 02754916 2011-09-08
WO 2010/108836 PCT/EP2010/053470
6
compared to a wild type plant of the same variety which does not comprise the
expression
cassette.
[0022] In another embodiment, the invention provides a transgenic plant
transformed
with an expression cassette comprising, in operative association, an isolated
polynucleotide
encoding a promoter capable of enhancing gene expression in leaves; an
isolated
polynucleotide encoding a mitochondrial transit peptide; and an isolated
polynucleotide
encoding a full-length isocitrate lyase polypeptide; wherein the transgenic
plant demonstrates
increased yield as compared to a wild type plant of the same variety which
does not comprise
the expression cassette.
[0023] In another embodiment, the invention provides a transgenic plant
transformed
with an expression cassette comprising, in operative association, an isolated
polynucleotide
encoding a promoter; an isolated polynucleotide encoding a chloroplast transit
peptide; and an
isolated polynucleotide encoding a full-length phospholipid hydroperoxide
glutathione
peroxidase polypeptide; wherein the transgenic plant demonstrates increased
yield as
compared to a wild type plant of the same variety which does not comprise the
expression
cassette.
[0024] In another embodiment, the invention provides a transgenic plant
transformed
with an expression cassette comprising, in operative association, an isolated
polynucleotide
encoding a promoter and an isolated polynucleotide encoding a full-length
gamma-
glutamyltranspeptidase polypeptide; wherein the transgenic plant demonstrates
increased
yield as compared to a wild type plant of the same variety which does not
comprise the
expression cassette.
[0025] In another embodiment, the invention provides a transgenic plant
transformed
with an expression cassette comprising, in operative association, an isolated
polynucleotide
encoding a promoter; an isolated polynucleotide encoding a mitochondrial
transit peptide; and
an isolated polynucleotide encoding a full-length ATP synthase subunit B'
polypeptide; wherein
the transgenic plant demonstrates increased yield as compared to a wild type
plant of the
same variety which does not comprise the expression cassette.
[0026] In another embodiment, the invention provides a transgenic plant
transformed
with an expression cassette comprising, in operative association, an isolated
polynucleotide
encoding a promoter; an isolated polynucleotide encoding a chloroplast transit
peptide; and an
isolated polynucleotide encoding a full-length C-22 sterol desaturase
polypeptide; wherein the
transgenic plant demonstrates increased yield as compared to a wild type plant
of the same
variety which does not comprise the expression cassette.
[0027] In a further embodiment, the invention provides a seed produced by the
transgenic plant of the invention, wherein the seed is true breeding for a
transgene comprising
the expression vectors described above. Plants derived from the seed of the
invention
demonstrate increased tolerance to an environmental stress, and/or increased
plant growth,
and/or increased yield, under normal and/or stress conditions as compared to a
wild type
variety of the plant.
[0028] In a still another aspect, the invention concerns products produced by
or from
the transgenic plants of the invention, their plant parts, or their seeds,
such as a foodstuff,
feedstuff, food supplement, feed supplement, fiber, cosmetic or
pharmaceutical.
[0029] The invention further provides certain isolated polynucleotides
identified in Table
CA 02754916 2011-09-08
WO 2010/108836 PCT/EP2010/053470
7
1, and certain isolated polypeptides identified in Table 1. The invention is
also embodied in a
recombinant vector comprising an isolated polynucleotide of the invention.
[0030] In yet another embodiment, the invention concerns a method of producing
the
aforesaid transgenic plant, wherein the method comprises transforming a plant
cell with an
expression vector comprising an isolated polynucleotide of the invention, and
generating from
the plant cell a transgenic plant that expresses the polypeptide encoded by
the polynucleotide.
Expression of the polypeptide in the plant results in increased tolerance to
an environmental
stress, and/or growth, and/or yield under normal and/or stress conditions as
compared to a
wild type variety of the plant.
[0031] In still another embodiment, the invention provides a method of
increasing a
plant' s tolerance to an environmental stress, and/or growth, and/or yield.
The method
comprises the steps of transforming a plant cell with an expression cassette
comprising an
isolated polynucleotide of the invention, and generating a transgenic plant
from the plant cell,
wherein the transgenic plant comprises the polynucleotide.
BRIEF DESCRIPTION OF THE DRAWINGS
[0032] Figure 1 shows an alignment of the amino acid sequences of the
galactokinases
designated b0757 (SEQ ID NO: 2), GM59594085 (SEQ ID NO: 4), GM59708137 (SEQ ID
NO:
6), and ZMBFb0152K10 (SEQ ID NO: 8). The alignment was generated using Align X
of
Vector NTI.
[0033] Figure 2 shows an alignment of the amino acid sequences of the
transaldolase
A proteins designated b2464 (SEQ ID NO: 10), BN43182918 (SEQ ID NO: 12), and
GM48926546 (SEQ ID NO: 14). The alignment was generated using Align X of
Vector NTI.
[0034] Figure 3 shows an alignment of the amino acid sequences of the
phospholipid
hydroperoxide glutathione peroxidases designated YIR037W (SEQ ID NO: 20),
BN42261838
(SEQ ID NO: 22), BN43722096 (SEQ ID NO: 24), BN51407729 (SEQ ID NO: 26),
GM50585691 (SEQ ID NO: 28), GMsa56cO7 (SEQ ID NO: 30), GMsp82f11 (SEQ ID NO:
32),
GMss66fO3 (SEQ ID NO: 34), HA03MC1446 (SEQ ID NO: 36), HV03MC9784 (SEQ ID NO:
38), OS34914218 (SEQ ID NO: 40), ZM61990487 (SEQ ID NO: 42), and
ZM68466470.rOl
(SEQ ID NO: 44). The alignment was generated using Align X of Vector NTI.
[0035] Figure 4 shows an alignment of the amino acid sequences of the ATP
synthase
subunit B' proteins designated SLL1323 (SEQ ID NO: 48) and Gmsb38bO4 (SEQ ID
NO: 50).
The alignment was generated using Align X of Vector NTI.
[0036] Figure 5 shows an alignment of the amino acid sequences of the C-22
sterol
desaturases designated YMR015C (SEQ ID NO: 52) and GMso65hO7 (SEQ ID NO: 54).
The
alignment was generated using Align X of Vector NTI.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0037] Throughout this application, various publications are referenced. The
disclosures of all of these publications and those references cited within
those publications in
their entireties are hereby incorporated by reference into this application in
order to more fully
describe the state of the art to which this invention pertains. The
terminology used herein is for
the purpose of describing specific embodiments only and is not intended to be
limiting. As
used herein, " a" or " an" can mean one or more, depending upon the context in
which it is
CA 02754916 2011-09-08
WO 2010/108836 PCT/EP2010/053470
8
used. Thus, for example, reference to " a cell" can mean that at least one
cell can be used.
[0038] In one embodiment, the invention provides a transgenic plant that
overexpresses an isolated polynucleotide identified in Table 1 in the
subcellular compartment
and tissue indicated herein. The transgenic plant of the invention
demonstrates an improved
yield as compared to a wild type variety of the plant. As used herein, the
term "improved yield"
means any improvement in the yield of any measured plant product, such as
grain, fruit or
fiber. In accordance with the invention, changes in different phenotypic
traits may improve
yield. For example, and without limitation, parameters such as floral organ
development, root
initiation, root biomass, seed number, seed weight, harvest index, tolerance
to abiotic
environmental stress, leaf formation, phototropism, apical dominance, and
fruit development,
are suitable measurements of improved yield. Any increase in yield is an
improved yield in
accordance with the invention. For example, the improvement in yield can
comprise a 0.1%,
0.5%, 1%, 3%, 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or greater
increase in any measured parameter. For example, an increase in the bu/acre
yield of
soybeans or corn derived from a crop comprising plants which are transgenic
for the
nucleotides and polypeptides of Table 1, as compared with the bu/acre yield
from untreated
soybeans or corn cultivated under the same conditions, is an improved yield in
accordance
with the invention.
[0039] As defined herein, a " transgenic plant" is a plant that has been
altered using
recombinant DNA technology to contain an isolated nucleic acid which would
otherwise not be
present in the plant. As used herein, the term " plant" includes a whole
plant, plant cells,
and plant parts. Plant parts include, but are not limited to, stems, roots,
ovules, stamens,
leaves, embryos, meristematic regions, callus tissue, gametophytes,
sporophytes, pollen,
microspores, and the like. The transgenic plant of the invention may be male
sterile or male
fertile, and may further include transgenes other than those that comprise the
isolated
polynucleotides described herein.
[0040] As used herein, the term " variety" refers to a group of plants within
a species
that share constant characteristics that separate them from the typical form
and from other
possible varieties within that species. While possessing at least one
distinctive trait, a variety
is also characterized by some variation between individuals within the
variety, based primarily
on the Mendelian segregation of traits among the progeny of succeeding
generations. A
variety is considered " true breeding" for a particular trait if it is
genetically homozygous for
that trait to the extent that, when the true-breeding variety is self-
pollinated, a significant
amount of independent segregation of the trait among the progeny is not
observed. In the
present invention, the trait arises from the transgenic expression of one or
more isolated
polynucleotides introduced into a plant variety. As also used herein, the term
" wild type
variety" refers to a group of plants that are analyzed for comparative
purposes as a control
plant, wherein the wild type variety plant is identical to the transgenic
plant (plant transformed
with an isolated polynucleotide in accordance with the invention) with the
exception that the
wild type variety plant has not been transformed with an isolated
polynucleotide of the
invention. The term " wild type" as used herein refers to a plant cell, seed,
plant component,
plant tissue, plant organ, or whole plant that has not been genetically
modified with an isolated
polynucleotide in accordance with the invention.
[0041] The term " control plant" as used herein refers to a plant cell, an
explant,
CA 02754916 2011-09-08
WO 2010/108836 PCT/EP2010/053470
9
seed, plant component, plant tissue, plant organ, or whole plant used to
compare against
transgenic or genetically modified plant for the purpose of identifying an
enhanced phenotype
or a desirable trait in the transgenic or genetically modified plant. A "
control plant" may in
some cases be a transgenic plant line that comprises an empty vector or marker
gene, but
does not contain the recombinant polynucleotide of interest that is present in
the transgenic or
genetically modified plant being evaluated. A control plant may be a plant of
the same line or
variety as the transgenic or genetically modified plant being tested, or it
may be another line or
variety, such as a plant known to have a specific phenotype, characteristic,
or known
genotype. A suitable control plant would include a genetically unaltered or
non-transgenic plant
of the parental line used to generate a transgenic plant herein.
[0042] As defined herein, the term " nucleic acid" and " polynucleotide" are
interchangeable and refer to RNA or DNA that is linear or branched, single or
double stranded,
or a hybrid thereof. The term also encompasses RNA/DNA hybrids. An " isolated"
nucleic
acid molecule is one that is substantially separated from other nucleic acid
molecules which
are present in the natural source of the nucleic acid (i.e., sequences
encoding other
polypeptides). For example, a cloned nucleic acid is considered isolated. A
nucleic acid is
also considered isolated if it has been altered by human intervention, or
placed in a locus or
location that is not its natural site, or if it is introduced into a cell by
transformation. Moreover,
an isolated nucleic acid molecule, such as a cDNA molecule, can be free from
some of the
other cellular material with which it is naturally associated, or culture
medium when produced
by recombinant techniques, or chemical precursors or other chemicals when
chemically
synthesized. While it may optionally encompass untranslated sequence located
at both the
3' and 5' ends of the coding region of a gene, it may be preferable to remove
the
sequences which naturally flank the coding region in its naturally occurring
replicon.
[0043] As used herein, the term " environmental stress" refers to a sub-
optimal
condition associated with salinity, drought, nitrogen, temperature, metal,
chemical, pathogenic,
or oxidative stresses, or any combination thereof. As used herein, the term "
drought" refers
to an environmental condition where the amount of water available to support
plant growth or
development is less than optimal. As used herein, the term " fresh weight"
refers to
everything in the plant including water. As used herein, the term " dry
weight" refers to
everything in the plant other than water, and includes, for example,
carbohydrates, proteins,
oils, and mineral nutrients.
[0044] Any plant species may be transformed to create a transgenic plant in
accordance with the invention. The transgenic plant of the invention may be a
dicotyledonous
plant or a monocotyledonous plant. For example and without limitation,
transgenic plants of
the invention may be derived from any of the following diclotyledonous plant
families:
Leguminosae, including plants such as pea, alfalfa and soybean; Umbelliferae,
including
plants such as carrot and celery; Solanaceae, including the plants such as
tomato, potato,
aubergine, tobacco, and pepper; Cruciferae, particularly the genus Brassica,
which includes
plant such as oilseed rape, beet, cabbage, cauliflower and broccoli); and A.
thaliana;
Compositae, which includes plants such as lettuce; Malvaceae, which includes
cotton;
Fabaceae, which includes plants such as peanut, and the like. Transgenic
plants of the
invention may be derived from monocotyledonous plants, such as, for example,
wheat, barley,
sorghum, millet, rye, triticale, maize, rice, oats and sugarcane. Transgenic
plants of the
CA 02754916 2011-09-08
WO 2010/108836 PCT/EP2010/053470
invention are also embodied as trees such as apple, pear, quince, plum,
cherry, peach,
nectarine, apricot, papaya, mango, and other woody species including
coniferous and
deciduous trees such as poplar, pine, sequoia, cedar, oak, and the like.
Especially preferred
are Arabidopsis thaliana, Nicotiana tabacum, rice, oilseed rape, canola,
soybean, corn (maize),
5 cotton, and wheat.
[0045] In one embodiment, the invention provides a transgenic plant
transformed with
an expression cassette comprising, in operative association, an isolated
polynucleotide
encoding a promoter; an isolated polynucleotide encoding a chloroplast transit
peptide; and an
isolated polynucleotide encoding a full-length galactokinase polypeptide;
wherein the
10 transgenic plant demonstrates increased yield as compared to a wild type
plant of the same
variety which does not comprise the expression cassette. As demonstrated in
Example 2
below, transgenic Arabidopsis plants containing the E. coli gene b0757 (SEQ ID
NO: 1)
targeted to the chloroplast demonstrate increased yield as compared to control
Arabidopsis
plants. The b0757 gene encodes galactokinase and is characterized, in part, by
the presence
of the signature sequences GHMP_kinases_C (Pfam: PF08544) and GHMP_kinases_N
(PF00288). Such signature sequences are exemplified in the galactokinase
proteins set forth
in Figure 1.
[0046] The transgenic plant of this embodiment may comprise any polynucleotide
encoding a galactokinase polypeptide. Preferably, the transgenic plant of this
embodiment
comprises a polynucleotide encoding a full-length polypeptide having
galactokinase activity,
wherein the polypeptide comprises at least one signature sequence selected
from both a
GHMP_kinases_C and a GHMP_kinases_N signature sequence, wherein the
GHMP_kinases_C signature sequence is selected from the group consisting of
amino acids
278 to 362 of SEQ ID NO: 2; amino acids 378 to 426 of SEQ ID NO: 4; amino
acids 326 to 404
of SEQ ID NO: 6; and amino acids 391 to 473 of SEQ ID NO: 8; and wherein the
GHMP_kinases_N signature sequence is selected from the group consisting of
amino acids
114 to 182 of SEQ ID NO: 2; amino acids 152 to 219 of SEQ ID NO: 4; amino
acids 138 to 205
of SEQ ID NO: 6; and amino acids 159 to 226 of SEQ ID NO: 8. Preferably the
polypeptide
comprises both a GHMP_kinases_C signature sequence and a GHMP_kinases_N
signature
sequence. Most preferably, the transgenic plant of this embodiment comprises a
polynucleotide encoding a galactokinase polypeptide having a sequence selected
from the
group consisting of amino acids 1 to 382 of SEQ ID NO: 2; amino acids 1 to 460
of SEQ ID
NO: 4; amino acids 1 to 431 of SEQ ID NO: 6; and amino acids 1 to 504 of SEQ
ID NO: 8.
[0047] In another embodiment, the invention provides a transgenic plant
transformed
with an expression cassette comprising, in operative association, an isolated
polynucleotide
encoding a promoter; and an isolated polynucleotide encoding a full-length
transaldolase A
polypeptide; wherein the transgenic plant demonstrates increased yield as
compared to a wild
type plant of the same variety which does not comprise the expression
cassette. As
demonstrated in Example 2 below, transgenic Arabidopsis plants containing the
E. coli gene
b2464 (SEQ ID NO: 9), which encodes a transadolase A polypeptide, and the
transgenic
plants of this embodiment demonstrate increased yield as compared to control
Arabidopsis
plants. Transaldolase A polypeptides are characterized, in part, by the
presence of a
Transaldolase (PF00923) signature sequence. Such signature sequences are
exemplified in
the transaldolase A proteins set forth in Figure 2.
CA 02754916 2011-09-08
WO 2010/108836 PCT/EP2010/053470
11
[0048] The transgenic plant of this embodiment may comprise any polynucleotide
encoding a transaldolase A protein. Preferably, the transgenic plant of this
embodiment
comprises a polynucleotide encoding a full-length polypeptide having
transaldolase A activity,
wherein the polypeptide comprises a Transaldolase signature sequence selected
from the
group consisting of amino acids 12 to 312 of SEQ ID NO: 10; amino acids 1 to
275 of SEQ ID
NO: 12; and amino acids 1 to 277 of SEQ ID NO: 14. Most preferably, the
transgenic plant of
this embodiment comprises a polynucleotide encoding a transaldolase A
polypeptide having a
sequence selected from the group consisting of amino acids 1 to 316 of SEQ ID
NO: 10;
amino acids 1 to 284 of SEQ ID NO: 12; and amino acids 1 to 283 of SEQ ID NO:
14.
[0049] In another embodiment, the invention provides a transgenic plant
transformed
with an expression cassette comprising, in operative association, an isolated
polynucleotide
encoding a promoter; and an isolated polynucleotide encoding a full-length
hydrogenase-2
accessory polypeptide; wherein the transgenic plant demonstrates increased
yield as
compared to a wild type plant of the same variety which does not comprise the
expression
cassette. As demonstrated in Example 2 below, transgenic Arabidopsis plants
containing the
E. coli gene b2990 (SEQ ID NO: 15) demonstrate increased yield as compared to
control
Arabidopsis plants. The b2990 gene encodes a hydrogenase-2 accessory protein.
In E. coli
under anaerobic conditions, this protein is a chaperone-like protein which is
required for the
generation of active hydrogenase 2, which is an uptake [NiFe] hydrogenase
that, along with
hydrogenase 1, couples H2 oxidation to fumarate reduction. Hydrogenase-2
accessory proteins
are characterized, in part, by the presence of a HupF_HypC (PF01455) signature
sequence.
[0050] The transgenic plant of this embodiment may comprise any polynucleotide
encoding a hydrogenase-2 accessory protein. Preferably, the transgenic plant
of this
embodiment comprises a polynucleotide encoding a full-length polypeptide
having
hydrogenase assembly chaperone activity, wherein the polypeptide comprises a
HupF_HypC
signature sequence comprising amino acids 1 to 79 of SEQ ID NO: 16. Most
preferably, the
transgenic plant of this embodiment comprises a polynucleotide encoding a
hydrogenase-2
accessory protein having a sequence comprising amino acids 1 to 82 of SEQ ID
NO: 16.
[0051] In another embodiment, the invention provides a transgenic plant
transformed
with an expression cassette comprising, in operative association, an isolated
polynucleotide
encoding a promoter capable of enhancing gene expression in leaves; an
isolated
polynucleotide encoding a mitochondrial transit peptide; and an isolated
polynucleotide
encoding a full-length isocitrate lyase polypeptide; wherein the transgenic
plant demonstrates
increased yield as compared to a wild type plant of the same variety which
does not comprise
the expression cassette. As demonstrated in Example 2 below, transgenic
Arabidopsis plants
containing the S. cerevisiae gene YER065C (SEQ ID NO: 17), which encodes
isocitrate lyase,
targeted to the mitochondria demonstrate increased yield as compared to
control Arabidopsis
plants. Isocitrate lyases are characterized, in part, by the presence of an
ICL (PF00463)
signature sequence.
[0052] The transgenic plant of this embodiment may comprise any polynucleotide
encoding an isocitrate lyase. Preferably, the transgenic plant of this
embodiment comprises a
polynucleotide encoding a full-length polypeptide having isocitrate lyase
activity, wherein the
polypeptide comprises an ICL signature sequence comprising amino acids 22 to
550 of SEQ
ID NO: 18. Most preferably, the transgenic plant of this embodiment comprises
a
CA 02754916 2011-09-08
WO 2010/108836 PCT/EP2010/053470
12
polynucleotide encoding an isocitrate lyase having a sequence comprising amino
acids 1 to
557 of SEQ ID NO: 18.
[0053] In another embodiment, the invention provides a transgenic plant
transformed
with an expression cassette comprising, in operative association, an isolated
polynucleotide
encoding a promoter; an isolated polynucleotide encoding a chloroplast transit
peptide; and an
isolated polynucleotide encoding a full-length phospholipid hydroperoxide
glutathione
peroxidase polypeptide; wherein the transgenic plant demonstrates increased
yield as
compared to a wild type plant of the same variety which does not comprise the
expression
cassette. As demonstrated in Example 2 below, transgenic Arabidopsis plants
containing the
S. cerevisiae gene YIR037W (SEQ ID NO: 19) targeted to the chloroplast
demonstrate
increased yield as compared to control Arabidopsis plants. The YIR037W gene
encodes
encodes a phospholipid hydroperoxide glutathione peroxidase protein, which
functions as a
sensor for intracellular hyperoxide levels, and a transducer of the redox
signal to the
transcription factor Yap1, which regulates hyperoxide levels in S. cerevisiae.
Phospholipid
hydroperoxide glutathione peroxidases are characterized, in part, by the
presence of a GSHPx
(PF00255) signature sequence representative of the glutathione peroxidase
family of genes.
Such signature sequences are exemplified in the phospholipid hydroperoxide
glutathione
peroxidases set forth in Figure 3.
[0054] The transgenic plant of this embodiment may comprise any polynucleotide
encoding a phospholipid hydroperoxide glutathione peroxidase. Preferably, the
transgenic
plant of this embodiment comprises a polynucleotide encoding a full-length
polypeptide having
phospholipid hydroperoxide glutathione peroxidase activity, wherein the
polypeptide comprises
a GSHPx signature sequence selected from the group consisting of amino acids 4
to 111 of
SEQ ID NO: 20; amino acids 10 to 118 of SEQ ID NO: 22; amino acids 37 to 145
of SEQ ID
NO: 24; amino acids 9 to 117 of SEQ ID NO: 26; amino acids 9 to 117 of SEQ ID
NO: 28;
amino acids 9 to 117 of SEQ ID NO: 30; amino acids 12 to 120 of SEQ ID NO: 32;
amino acids
12 to 120 of SEQ ID NO: 34; amino acids 11 to 119 of SEQ ID NO: 36; amino
acids 12 to 120
of SEQ ID NO: 38; amino acids 9 to 117 of SEQ ID NO: 40; amino acids 12 to 120
of SEQ ID
NO: 42; and amino acids 24 to 132 of SEQ ID NO: 44. Most preferably, the
transgenic plant of
this embodiment comprises a polynucleotide encoding a phospholipid
hydroperoxide
glutathione peroxidase having a sequence selected from the group consisting of
amino acids 1
to 163 of SEQ ID NO: 20; amino acids 1 to 169 of SEQ ID NO: 22; amino acids 1
to 201 of
SEQ ID NO: 24; amino acids 1 to 169 of SEQ ID NO: 26; amino acids 1 to 166 of
SEQ ID NO:
28; amino acids 1 to 166 of SEQ ID NO: 30; amino acids 1 to 170 of SEQ ID NO:
32; amino
acids 1 to 170 of SEQ ID NO: 34; amino acids 1 to 185 of SEQ ID NO: 36; amino
acids 1 to
176 of SEQ ID NO: 38; amino acids 1 to 166 of SEQ ID NO: 40; amino acids 1 to
170 of SEQ
ID NO: 42; and amino acids 1 to 182 of SEQ ID NO: 44.
[0055] In another embodiment, the invention provides a transgenic plant
transformed
with an expression cassette comprising, in operative association, an isolated
polynucleotide
encoding a promoter; and an isolated polynucleotide encoding a full-length
gamma-
glutamyltranspeptidase polypeptide; wherein the transgenic plant demonstrates
increased
yield as compared to a wild type plant of the same variety which does not
comprise the
expression cassette. Optionally, the expression cassette further comprises an
isolated
polynucleotide encoding a chloroplast transit peptides in operative
association with the isolated
CA 02754916 2011-09-08
WO 2010/108836 PCT/EP2010/053470
13
polynucleotide encoding a promoter and the isolated polynucleotide encoding a
full-length
gamma-glutamyltranspeptidase polypeptide. As demonstrated in Example 2 below,
transgenic
Arabidopsis plants containing the Synechocystis sp. gene s1r1269 (SEQ ID NO:
45), which
encodes a gamma-glutamyltranspeptidase polypeptide, demonstrate increased
yield as
compared to control Arabidopsis plants. Gamma-glutamyltranspeptidases are
characterized, in
part, by the presence of a G_glu_transpept (PF01019) signature sequence.
[0056] The transgenic plant of this embodiment may comprise any polynucleotide
encoding a gamma-glutamyltranspeptidase. Preferably, the transgenic plant of
this
embodiment comprises a polynucleotide encoding a full-length polypeptide
having gamma-
glutamyltranspeptidase activity, wherein the polypeptide comprises a
G_glu_transpept
signature sequence comprising amino acids 21 to 511 of SEQ ID NO: 46. Most
preferably, the
transgenic plant of this embodiment comprises a polynucleotide encoding a
gamma-
glutamyltranspeptidase having a sequence comprising amino acids 1 to 518 of
SEQ ID NO:
46.
[0057] In another embodiment, the invention provides a transgenic plant
transformed
with an expression cassette comprising, in operative association, an isolated
polynucleotide
encoding a promoter; an isolated polynucleotide encoding a mitochondrial
transit peptide; and
an isolated polynucleotide encoding a full-length ATP synthase subunit B'
polypeptide; wherein
the transgenic plant demonstrates increased yield as compared to a wild type
plant of the
same variety which does not comprise the expression cassette. As demonstrated
in Example 2
below, transgenic Arabidopsis plants containing the Synechocystis sp. gene
SLL1323 (SEQ ID
NO: 47) targeted to the mitochondria demonstrate increased yield as compared
to control
Arabidopsis plants. The SLL1323 gene encodes an ATP synthase subunit B'
protein. Subunits
B and B' are from the FO complex in F-ATPases found in chloroplasts and in
bacterial plasma
membranes and form part of the peripheral stalk that links the F1 and FO
complexes together.
ATP synthase subunit B' proteins are characterized, in part, by the presence
of an ATP-synt_B
(PF00430) signature sequence representative of the ATP synthase B/ B' CF(0)
family of
genes. Such signature sequences are exemplified in the ATP synthase subunit B'
proteins set
forth in Figure 4.
[0058] The transgenic plant of this embodiment may comprise any polynucleotide
encoding an ATP synthase subunit B' protein. Preferably, the transgenic plant
of this
embodiment comprises a polynucleotide encoding a full-length polypeptide
having ATP
synthase subunit B' activity, wherein the polypeptide comprises a ATP-synt_B
signature
sequence selected from the group consisting of amino acids 7 to 138 of SEQ ID
NO: 48 and
amino acids 82 to 213 of SEQ ID NO: 50. Most preferably, the transgenic plant
of this
embodiment comprises a polynucleotide encoding a ATP synthase subunit B'
protein having a
sequence comprising amino acids 1 to 143 of SEQ ID NO: 48 and amino acids 1 to
215 of
SEQ ID NO: 50.
[0059] In another embodiment, the invention provides a transgenic plant
transformed
with an expression cassette comprising, in operative association, an isolated
polynucleotide
encoding a promoter; an isolated polynucleotide encoding a chloroplast transit
peptide; and an
isolated polynucleotide encoding a full-length C-22 sterol desaturase
polypeptide; wherein the
transgenic plant demonstrates increased yield as compared to a wild type plant
of the same
variety which does not comprise the expression cassette. Gene YMR015C (SEQ ID
NO: 51)
CA 02754916 2011-09-08
WO 2010/108836 PCT/EP2010/053470
14
encodes C-22 sterol desaturase, which is a cytochrome P450 enzyme (ERG5) that,
in yeast,
catalyzes the formation of the C-22(23) double bond in the sterol side chain
in ergosterol
biosynthesis. C-22 sterol desaturase enzymes are characterized, in part, by
the presence of a
K-helix motif (xExxR), a PERF consensus sequence (PxRx) and an FGRCG motif
surrounding
the protoporphyrin IX heme cysteine ligand near the C-terminus. Such conserved
motifs are
exemplified in the C-22 sterol desaturase polypeptides set forth in Figure 5.
[0060] The transgenic plant of this embodiment may comprise any polynucleotide
encoding a C-22 sterol desaturase. Preferably, the transgenic plant of this
embodiment
comprises a polynucleotide encoding a full-length polypeptide having C-22
sterol desaturase
activity, wherein the polypeptide comprises a domain comprising a K-helix
motif, a PERF motif
and a FGRCG motif, wherein the K-helix motif has a sequence selected from the
group
consisting of amino acids 395 to 398 of SEQ ID NO: 52 and amino acids 365 to
368 of SEQ ID
NO: 54; the PERF motif has a sequence selected from the group consisting of
amino acids
450 to 453 of SEQ ID NO: 52 and amino acids 418 to 421 of SEQ ID NO: 54; and
the FGRCG
motif has a sequence selected from the group consisting of amino acids 469 to
478 of SEQ ID
NO: 52 and amino acids 438 to 447 of SEQ ID NO: 54. More preferably, the
polynucleotide
encodes a full-length polypeptide having C-22 sterol desaturase activity,
wherein the
polypeptide comprises a domain selected from the group consisting of amino
acids 61 to 529
of SEQ ID NO: 52 and amino acids 27 to 498 of SEQ ID NO: 54. Most preferably,
the
transgenic plant of this embodiment comprises a polynucleotide encoding a C-22
sterol
desaturase comprising amino acids 1 to 538 of SEQ ID NO: 52 and amino acids 1
to 513 of
SEQ ID NO: 54.
[0061] The invention further provides a seed which is true breeding for the
expression
cassettes (also referred to herein as " transgenes" ) described herein,
wherein transgenic
plants grown from said seed demonstrate increased yield as compared to a wild
type variety of
the plant. The invention also provides a product produced by or from the
transgenic plants
expressing the polynucleotide, their plant parts, or their seeds. The product
can be obtained
using various methods well known in the art. As used herein, the word "
product" includes,
but not limited to, a foodstuff, feedstuff, a food supplement, feed
supplement, fiber, cosmetic or
pharmaceutical. Foodstuffs are regarded as compositions used for nutrition or
for
supplementing nutrition. Animal feedstuffs and animal feed supplements, in
particular, are
regarded as foodstuffs. The invention further provides an agricultural product
produced by any
of the transgenic plants, plant parts, and plant seeds. Agricultural products
include, but are not
limited to, plant extracts, proteins, amino acids, carbohydrates, fats, oils,
polymers, vitamins,
and the like.
[0062] The invention also provides an isolated polynucleotide which has a
sequence
selected from the group consisting of SEQ ID NO: 3; SEQ ID NO: 5; SEQ ID NO:
7; SEQ ID
NO: 11; SEQ ID NO: 13; SEQ ID NO: 21; SEQ ID NO: 23; SEQ ID NO: 25; SEQ ID NO:
27;
SEQ ID NO: 29; SEQ ID NO: 31; SEQ ID NO: 33; SEQ ID NO: 35; SEQ ID NO: 37; SEQ
ID
NO: 39; SEQ ID NO: 41; SEQ ID NO: 43; SEQ ID NO: 49; and SEQ ID NO: 53. Also
encompassed by the isolated polynucleotide of the invention is an isolated
polynucleotide
encoding a polypeptide having an amino acid sequence selected from the group
consisting of
SEQ ID NO: 4; SEQ ID NO: 6; SEQ ID NO: 8; SEQ ID NO: 12; SEQ ID NO: 14; SEQ ID
NO:
22; SEQ ID NO: 24; SEQ ID NO: 26; SEQ ID NO: 28; SEQ ID NO: 30; SEQ ID NO: 32;
SEQ
CA 02754916 2011-09-08
WO 2010/108836 PCT/EP2010/053470
ID NO: 34; SEQ ID NO: 36; SEQ ID NO: 38; SEQ ID NO: 40; SEQ ID NO: 42; SEQ ID
NO: 44;
SEQ ID NO: 50; and SEQ ID NO: 54. A polynucleotide of the invention can be
isolated using
standard molecular biology techniques and the sequence information provided
herein, for
example, using an automated DNA synthesizer.
5 [0063] The isolated polynucleotides of the invention include homologs of the
polynucleotides of Table 1. " Homologs" are defined herein as two nucleic
acids or
polypeptides that have similar, or substantially identical, nucleotide or
amino acid sequences,
respectively. Homologs include allelic variants, analogs, and orthologs, as
defined below. As
used herein, the term " analogs" refers to two nucleic acids that have the
same or similar
10 function, but that have evolved separately in unrelated organisms. As used
herein, the term
" orthologs" refers to two nucleic acids from different species, but that have
evolved from a
common ancestral gene by speciation. The term homolog further encompasses
nucleic acid
molecules that differ from one of the nucleotide sequences shown in Table 1
due to
degeneracy of the genetic code and thus encode the same polypeptide.
15 [0064] To determine the percent sequence identity of two amino acid
sequences (e.g.,
one of the polypeptide sequences of Table 1 and a homolog thereof), the
sequences are
aligned for optimal comparison purposes (e.g., gaps can be introduced in the
sequence of one
polypeptide for optimal alignment with the other polypeptide or nucleic acid).
The amino acid
residues at corresponding amino acid positions are then compared. When a
position in one
sequence is occupied by the same amino acid residue as the corresponding
position in the
other sequence then the molecules are identical at that position. The same
type of
comparison can be made between two nucleic acid sequences.
[0065] Preferably, the isolated amino acid homologs, analogs, and orthologs of
the
polypeptides of the present invention are at least about 50-60%, preferably at
least about 60-
70%, and more preferably at least about 70-75%, 75-80%, 80-85%, 85-90%, or 90-
95%, and
most preferably at least about 96%, 97%, 98%, 99%, or more identical to an
entire amino acid
sequence identified in Table 1. In another preferred embodiment, an isolated
nucleic acid
homolog of the invention comprises a nucleotide sequence which is at least
about 40-60%,
preferably at least about 60-70%, more preferably at least about 70-75%, 75-
80%, 80-85%,
85-90%, or 90-95%, and even more preferably at least about 95%, 96%, 97%, 98%,
99%, or
more identical to a nucleotide sequence shown in Table 1.
[0066] For the purposes of the invention, the percent sequence identity
between two
nucleic acid or polypeptide sequences is determined using Align 2.0 (Myers and
Miller,
CABIOS (1989) 4:11-17) with all parameters set to the default settings or the
Vector NTI 9.0
(PC) software package (Invitrogen, 1600 Faraday Ave., Carlsbad, CA92008). For
percent
identity calculated with Vector NTI, a gap opening penalty of 15 and a gap
extension penalty of
6.66 are used for determining the percent identity of two nucleic acids. A gap
opening penalty
of 10 and a gap extension penalty of 0.1 are used for determining the percent
identity of two
polypeptides. All other parameters are set at the default settings. For
purposes of a multiple
alignment (Clustal W algorithm), the gap opening penalty is 10, and the gap
extension penalty
is 0.05 with blosum62 matrix. It is to be understood that for the purposes of
determining
sequence identity when comparing a DNA sequence to an RNA sequence, a
thymidine
nucleotide is equivalent to a uracil nucleotide.
[0067] Nucleic acid molecules corresponding to homologs, analogs, and
orthologs of
CA 02754916 2011-09-08
WO 2010/108836 PCT/EP2010/053470
16
the polypeptides listed in Table 1 can be isolated based on their identity to
said polypeptides,
using the polynucleotides encoding the respective polypeptides or primers
based thereon, as
hybridization probes according to standard hybridization techniques under
stringent
hybridization conditions. As used herein with regard to hybridization for DNA
to a DNA blot,
the term " stringent conditions" refers to hybridization overnight at 60 C in
10X Denhart' s
solution, 6X SSC, 0.5% SDS, and 100 g/ml denatured salmon sperm DNA. Blots
are washed
sequentially at 62 C for 30 minutes each time in 3X SSC/0.1 % SDS, followed by
1X SSC/0.1 %
SDS, and finally 0.1X SSC/0.1% SDS. As also used herein, in a preferred
embodiment, the
phrase " stringent conditions" refers to hybridization in a 6X SSC solution at
65 C. In
another embodiment, " highly stringent conditions" refers to hybridization
overnight at 65 C
in 1OX Denhart' s solution, 6X SSC, 0.5% SDS and 100 g/ml denatured salmon
sperm DNA.
Blots are washed sequentially at 65 C for 30 minutes each time in 3X SSC/0.1 %
SDS,
followed by 1X SSC/0.1 % SDS, and finally 0.1X SSC/0.1 % SDS. Methods for
performing
nucleic acid hybridizations are well known in the art.
[0068] The isolated polynucleotides employed in the invention may be
optimized, that
is, genetically engineered to increase its expression in a given plant or
animal. To provide
plant optimized nucleic acids, the DNA sequence of the gene can be modified
to: 1) comprise
codons preferred by highly expressed plant genes; 2) comprise an A+T content
in nucleotide
base composition to that substantially found in plants; 3) form a plant
initiation sequence; 4) to
eliminate sequences that cause destabilization, inappropriate polyadenylation,
degradation
and termination of RNA, or that form secondary structure hairpins or RNA
splice sites; or 5)
elimination of antisense open reading frames. Increased expression of nucleic
acids in plants
can be achieved by utilizing the distribution frequency of codon usage in
plants in general or in
a particular plant. Methods for optimizing nucleic acid expression in plants
can be found in
EPA 0359472; EPA 0385962; PCT Application No. WO 91/16432; U.S. Patent No.
5,380,831;
U.S. Patent No. 5,436,391; Perlack et al., 1991, Proc. NatI. Acad. Sci. USA
88:3324-3328; and
Murray et al., 1989, Nucleic Acids Res. 17:477-498.
[0069] The invention further provides a recombinant expression vector which
comprises an expression cassette selected from the group consisting of a) an
expression
cassette comprising, in operative association, an isolated polynucleotide
encoding a promoter;
an isolated polynucleotide encoding a chloroplast transit peptide; and an
isolated
polynucleotide encoding a full-length galactokinase polypeptide; b) an
expression cassette
comprising, in operative association, an isolated polynucleotide encoding a
promoter; and an
isolated polynucleotide encoding a full-length transaldolase A polypeptide; c)
an expression
cassette comprising, in operative association, an isolated polynucleotide
encoding a promoter;
and an isolated polynucleotide encoding a full-length hydrogenase-2 accessory
polypeptide; d)
an expression cassette comprising, in operative association, an isolated
polynucleotide
encoding a promoter; an isolated polynucleotide encoding a mitochondrial
transit peptide; and
an isolated polynucleotide encoding a full-length isocitrate lyase
polypeptide; e) an expression
cassette comprising, in operative association, an isolated polynucleotide
encoding a promoter;
an isolated polynucleotide encoding a chloroplast transit peptide; and an
isolated
polynucleotide encoding a full-length phospholipid hydroperoxide glutathione
peroxidase
polypeptide; f) an expression cassette comprising, in operative association,
an isolated
polynucleotide encoding a promoter; and an isolated polynucleotide encoding a
full-length
CA 02754916 2011-09-08
WO 2010/108836 PCT/EP2010/053470
17
gamma-glutamyltranspeptidase polypeptide; g) an expression cassette
comprising, in
operative association, an isolated polynucleotide encoding a promoter; an
isolated
polynucleotide encoding a mitochondrial transit peptide; and an isolated
polynucleotide
encoding a full-length ATP synthase subunit B' polypeptide; and h) an
expression cassette
comprising, in operative association, an isolated polynucleotide encoding a
promoter; an
isolated polynucleotide encoding a chloroplast transit peptide; and an
isolated polynucleotide
encoding a full-length C-22 sterol desaturase polypeptide.
[0070] In another embodiment, the recombinant expression vector of the
invention
comprises an isolated polynucleotide having a sequence selected from the group
consisting of
SEQ ID NO: 3; SEQ ID NO: 5; SEQ ID NO: 7; SEQ ID NO: 11; SEQ ID NO: 13; SEQ ID
NO:
21; SEQ ID NO: 23; SEQ ID NO: 25; SEQ ID NO: 27; SEQ ID NO: 29; SEQ ID NO: 31;
SEQ
ID NO: 33; SEQ ID NO: 35; SEQ ID NO: 37; SEQ ID NO: 39; SEQ ID NO: 41; SEQ ID
NO: 43;
SEQ ID NO: 49; and SEQ ID NO: 53. In addition, the recombinant expression
vector of the
invention comprises an isolated polynucleotide encoding a polypeptide having
an amino acid
sequence selected from the group consisting of SEQ ID NO: 4; SEQ ID NO: 6; SEQ
ID NO: 8;
SEQ ID NO: 12; SEQ ID NO: 14; SEQ ID NO: 22; SEQ ID NO: 24; SEQ ID NO: 26; SEQ
ID
NO: 28; SEQ ID NO: 30; SEQ ID NO: 32; SEQ ID NO: 34; SEQ ID NO: 36; SEQ ID NO:
38;
SEQ ID NO: 40; SEQ ID NO: 42; SEQ ID NO: 44; SEQ ID NO: 50; and SEQ ID NO: 54.
[0071] The recombinant expression vector of the invention also include one or
more
regulatory sequences, selected on the basis of the host cells to be used for
expression, which
is in operative association with the isolated polynucleotide to be expressed.
As used herein
with respect to a recombinant expression vector, " in operative association"
or " operatively
linked" means that the polynucleotide of interest is linked to the regulatory
sequence(s) in a
manner which allows for expression of the polynucleotide when the vector is
introduced into
the host cell (e.g., in a bacterial or plant host cell). The term " regulatory
sequence" is
intended to include promoters, enhancers, and other expression control
elements (e.g.,
polyadenylation signals).
[0072] As set forth above, certain embodiments of the invention employ
promoters that
are capable of enhancing gene expression in leaves. In some embodiments, the
promoter is a
leaf-specific promoter. Any leaf-specific promoter may be employed in these
embodiments of
the invention. Many such promoters are known, for example, the USP promoter
from Vicia
faba (Baeumlein et al. (1991) Mol. Gen. Genet. 225, 459-67), promoters of
light-inducible
genes such as ribulose-1.5-bisphosphate carboxylase (rbcS promoters),
promoters of genes
encoding chlorophyll a/b-binding proteins (Cab), Rubisco activase, B-subunit
of chloroplast
glyceraldehyde 3-phosphate dehydrogenase from A. thaliana, (Kwon et al. (1994)
Plant
Physiol. 105,357-67) and other leaf-specific promoters such as those
identified in Aleman, I.
(2001) Isolation and characterization of leaf-specific promoters from alfalfa
(Medicago sativa),
Masters thesis, New Mexico State University, Los Cruces, NM.
[0073] In other embodiments of the invention, a root- or shoot-specific
promoter is
employed. For example, the Super promoter provides high level expression in
both root and
shoots (Ni et al. (1995) Plant J. 7: 661-676). Other root-specific promoters
include, without
limitation, the TobRB7 promoter (Yamamoto et al. (1991) Plant Cell 3, 371-
382), the rolD
promoter (Leach et al. (1991) Plant Science 79, 69-76); CaMV 35S Domain A
(Benfey et al.
(1989) Science 244, 174-181), and the like.
CA 02754916 2011-09-08
WO 2010/108836 PCT/EP2010/053470
18
[0074] In other embodiments, a constitutive promoter is employed. Constitutive
promoters are active under most conditions. Examples of constitutive promoters
suitable for
use in these embodiments include the parsley ubiquitin promoter described in
W02003/102198; the CaMV 19S and 35S promoters, the sX CaMV 35S promoter, the
Sept
promoter, the rice actin promoter, the Arabidopsis actin promoter, the maize
ubiquitin
promoter, pEmu, the figwort mosaic virus 35S promoter, the Smas promoter, the
super
promoter (U.S. Patent No. 5, 955,646), the GRP1-8 promoter, the cinnamyl
alcohol
dehydrogenase promoter (U.S. Patent No. 5,683,439), promoters from the T-DNA
of
Agrobacterium, such as mannopine synthase, nopaline synthase, and octopine
synthase, the
small subunit of ribulose biphosphate carboxylase (ssuRUBISCO) promoter, and
the like.
[0075] In accordance with the invention, a chloroplast transit sequence refers
to a
nucleotide sequence that encodes a chloroplast transit peptide. Examples of a
chloroplast
transit peptide include the group consisting of chlorophyll a/b binding
protein transit peptide,
small subunit of ribulose bisphosphate carboxylase transit peptide, EPSPS
transit peptide, and
dihydrodipocolinic acid synthase transit peptide. As defined herein, a
mitochondrial transit
sequence refers to a nucleotide sequence that encodes a mitochondrial
presequence and
directs the protein to mitochondria. Examples of mitochondrial presequences
include groups
consisting of ATPase subunits, ATP synthase subunits, Rieske-FeS protein,
Hsp60, malate
dehydrogenase, citrate synthase, aconitase, isocitrate dehydrogenase, pyruvate
dehydrogenase, malic enzyme, glycine decarboxylase, serine hydroxymethyl
transferase and
superoxide dismutase.
[0076] Such transit peptides are known in the art. See, for example, Von
Heijne et al.
(1991) Plant Mol. Biol. Rep. 9:104-126; Clark et al. (1989) J. Biol. Chem.
264:17544-17550;
Della-Cioppa et al. (1987) Plant Physiol. 84:965-968; Romer et al. (1993)
Biochem. Biophys.
Res. Commun. 196:1414-1421; and Shah et al. (1986) Science 233:478-481.
Chloroplast
targeting sequences are known in the art and include the chloroplast small
subunit of ribulose-
1,5-bisphosphate carboxylase (Rubisco) (de Castro Silva Filho et al. (1996)
Plant Mol. Biol.
30:769-780; Schnell et al. (1991) J. Biol. Chem. 266(5):3335-3342); 5-
(enolpyruvyl)shikimate-
3-phosphate synthase (EPSPS) (Archer et al. (1990) J. Bioenerg. Biomemb.
22(6):789-810);
tryptophan synthase (Zhao et al. (1995) J. Biol. Chem. 270(11):6081-6087);
plastocyanin
(Lawrence et al. (1997) J. Biol. Chem. 272(33):20357-20363); chorismate
synthase (Schmidt
et al. (1993) J. Biol. Chem. 268(36):27447-27457); and the light harvesting
chlorophyll a/b
binding protein (LHBP) (Lamppa et al. (1988) J. Biol. Chem. 263:14996-14999).
See also Von
Heijne et al. (1991) Plant Mol. Biol. Rep. 9:104-126; Clark et al. (1989) J.
Biol. Chem.
264:17544-17550; Della-Cioppa et al. (1987) Plant Physiol. 84:965-968; Romer
et al. (1993)
Biochem. Biophys. Res. Commun. 196:1414-1421; and Shah et al. (1986) Science
233:478-
481.
[0077] In a preferred embodiment of the present invention, the polynucleotides
listed in
Table 1 are expressed in plant cells from higher plants (e.g., the
spermatophytes, such as crop
plants). A polynucleotide may be " introduced" into a plant cell by any means,
including
transfection, transformation or transduction, electroporation, particle
bombardment,
agroinfection, and the like. Suitable methods for transforming or transfecting
plant cells are
disclosed, for example, using particle bombardment as set forth in U.S. Pat.
Nos. 4,945,050;
5,036,006; 5,100,792; 5,302,523; 5,464,765; 5,120,657; 6,084,154; and the
like. More
CA 02754916 2011-09-08
WO 2010/108836 PCT/EP2010/053470
19
preferably, the transgenic corn seed of the invention may be made using
Agrobacterium
transformation, as described in U.S. Pat. Nos. 5,591,616; 5,731,179;
5,981,840; 5,990,387;
6,162,965; 6,420,630, U.S. patent application publication number 2002/0104132,
and the like.
Transformation of soybean can be performed using for example any of the
techniques
described in European Patent No. EP 0424047, U.S. Patent No. 5,322,783,
European Patent
No.EP 0397 687, U.S. Patent No. 5,376,543, or U.S. Patent No. 5,169,770. A
specific
example of wheat transformation can be found in PCT Application No. WO
93/07256. Cotton
may be transformed using methods disclosed in U.S. Pat. Nos. 5,004,863;
5,159,135;
5,846,797, and the like. Rice may be transformed using methods disclosed in
U.S. Pat. Nos.
4,666,844; 5,350,688; 6,153,813; 6,333,449; 6,288,312; 6,365,807; 6,329,571,
and the like.
Canola may be transformed, for example, using methods such as those disclosed
in U.S. Pat.
Nos.5,188,958; 5,463,174; 5,750,871; EP1566443; WO02/00900; and the like.
Other plant
transformation methods are disclosed, for example, in U.S. Pat. Nos.
5,932,782; 6,153,811;
6,140,553; 5,969,213; 6,020,539, and the like. Any plant transformation method
suitable for
inserting a transgene into a particular plant may be used in accordance with
the invention.
[0078] According to the present invention, the introduced polynucleotide may
be
maintained in the plant cell stably if it is incorporated into a non-
chromosomal autonomous
replicon or integrated into the plant chromosomes. Alternatively, the
introduced polynucleotide
may be present on an extra-chromosomal non-replicating vector and may be
transiently
expressed or transiently active.
[0079] The invention is also embodied in a method of producing a transgenic
plant
comprising at least one polynucleotide listed in Table 1, wherein expression
of the
polynucleotide in the plant results in the plant' s increased growth and/or
yield under normal
or water-limited conditions and/or increased tolerance to an environmental
stress as compared
to a wild type variety of the plant comprising the steps of: (a) introducing
into a plant cell an
expression cassette described above, (b) regenerating a transgenic plant from
the transformed
plant cell; and selecting higher-yielding plants from the regenerated plant
sells. The plant cell
may be, but is not limited to, a protoplast, gamete producing cell, and a cell
that regenerates
into a whole plant. As used herein, the term " transgenic" refers to any
plant, plant cell,
callus, plant tissue, or plant part, that contains the expression cassette
described above. In
accordance with the invention, the expression cassette is stably integrated
into a chromosome
or stable extra-chromosomal element, so that it is passed on to successive
generations.
[0080] The effect of the genetic modification on plant growth and/or yield
and/or stress
tolerance can be assessed by growing the modified plant under normal and/or
less than
suitable conditions and then analyzing the growth characteristics and/or
metabolism of the
plant. Such analytical techniques are well known to one skilled in the art,
and include
measurements of dry weight, wet weight, seed weight, seed number, polypeptide
synthesis,
carbohydrate synthesis, lipid synthesis, evapotranspiration rates, general
plant and/or crop
yield, flowering, reproduction, seed setting, root growth, respiration rates,
photosynthesis rates,
metabolite composition, and the like.
[0081] The invention is further illustrated by the following examples, which
are not to be
construed in any way as imposing limitations upon the scope thereof.
CA 02754916 2011-09-08
WO 2010/108836 PCT/EP2010/053470
EXAMPLE 1
Characterization of Genes
[0082] Lead genes b0757 (SEQ ID NO: 1), b2464 (SEQ ID NO: 9), b2990 (SEQ ID
NO:
5 15), SLL1323 (SEQ ID NO: 47), slr1269 (SEQ ID NO: 45), YER065C (SEQ ID NO:
17),
YIR037W (SEQ ID NO: 19), and YMR015C (SEQ ID NO: 51) were cloned using
standard
recombinant techniques. The functionality of each lead gene was predicted by
comparing the
amino acid sequence encoded by the gene with other genes of known
functionality. Homolog
cDNAs were isolated from proprietary libraries of the respective species using
known methods.
10 Sequences were processed and annotated using bioinformatics analyses.
[0083] The b0757 gene (SEQ ID NO: 1) from E. coli encodes a galactokinase. The
full-
length amino acid sequence of b0757 (SEQ ID NO: 2) was blasted against a
proprietary
database of cDNAs at an e value of e-10 (Altschul et al., supra). Two homologs
from soybean
and one homolog from maize were identified. The amino acid relatedness of
these
15 sequences is indicated in the alignments shown in Figure 1.
[0084] The b2464 gene (SEQ ID NO: 9) from E. coli encodes transaldolase A. The
full-
length amino acid sequence of b2464 (SEQ ID NO: 10) was blasted against a
proprietary
database of cDNAs at an e value of e-10 (Altschul et al., supra). One homolog
from canola and
one homolog from soybean were identified. The amino acid relatedness of these
sequences
20 is indicated in the alignments shown in Figure 2.
[0085] The YIR037W gene (SEQ ID NO: 19) from S. cerevisiae encodes
phospholipid
hydroperoxide glutathione peroxidase. The full-length amino acid sequence of
YIR037W (SEQ
ID NO: 20) was blasted against a proprietary database of cDNAs at an e value
of e-10 (Altschul
et al., supra). Three homologs from canola, four homologs from soybean, one
homolog from
sunflower, one homolog from barley, one homolog from rice, and two homologs
from maize
were identified. The amino acid relatedness of these sequences is indicated in
the alignments
shown in Figure 3.
[0086] The SLL1323 gene (SEQ ID NO: 47) from Synechocystis sp. encodes ATP
synthase subunit B' . The full-length amino acid sequence of SLL1323 (SEQ ID
NO: 48) was
blasted against a proprietary database of cDNAs at an e value of e-10
(Altschul et al., supra).
One homolog from soybean was identified. The amino acid relatedness of these
sequences
is indicated in the alignments shown in Figure 4.
[0087] The YMR015C gene (SEQ ID NO: 51) from S. cerevisiae encodes C-22 sterol
desaturase. The full-length amino acid sequence of YMR015C SEQ ID NO: 52) was
blasted
against a proprietary database of cDNAs at an e value of e-10 (Altschul et
al., supra). One
homolog from soybean was identified. The amino acid relatedness of these
sequences is
indicated in the alignments shown in Figure 5.
EXAMPLE 2
Overexpression of Lead Genes in Plants
[0088] The polynucleotides of Table 1 were ligated into an expression cassette
using
known methods. Three different promoters were used to control expression of
the transgenes
in Arabidopsis: the USP promoter (" USP" ) from Vicia faba (SEQ ID NO: 61 or
SEQ ID NO:
62); the super promoter (" Super" ; SEQ ID NO: 63); and the parsley ubiquitin
promoter
CA 02754916 2011-09-08
WO 2010/108836 PCT/EP2010/053470
21
(" PCUbi" ; SEQ ID NO: 64). For targeted expression, a mitochondrial transit
peptide (SEQ
ID NO: 56 or SEQ ID NO: 58; designated " Mito" in Tables 2-9) or a chloroplast
transit
peptide (SEQ ID NO: 60; designated" Plastid" in Tables 2-10) was used.
[0089] The Arabidopsis ecotype C24 was transformed with constructs containing
the
lead genes described in Example 1 using known methods. Seeds from T2
transformed plants
were pooled on the basis of the promoter driving the expression, gene source
species and
type of targeting (chloroplast, mitochondrial, or no targeting). The seed
pools were used in the
primary screens for biomass under well watered and water limited growth
conditions. Hits from
pools in the primary screen were selected, molecular analysis performed and
seed collected.
The collected seeds were then used for analysis in secondary screens where a
larger number
of individuals for each transgenic event were analyzed. If plants from a
construct were
identified in the secondary screen as having increased biomass compared to the
controls, it
passed to the tertiary screen. In this screen, over 100 plants from all
transgenic events for that
construct were measured under well watered and drought growth conditions. The
data from
the transgenic plants were compared to wild type Arabidopsis plants or to
plants grown from a
pool of randomly selected transgenic Arabidopsis seeds using standard
statistical procedures.
[0090] Plants that were grown under well watered conditions were watered to
soil
saturation twice a week. Images of the transgenic plants were taken at 17 and
21 days using a
commercial imaging system. Alternatively, plants were grown under water
limited growth
conditions by watering to soil saturation infrequently which allowed the soil
to dry between
watering treatments. In these experiments, water was given on days 0, 8, and
19 after sowing.
Images of the transgenic plants were taken at 20 and 27 days using a
commercial imaging
system.
[0091] Image analysis software was used to compare the images of the
transgenic and
control plants grown in the same experiment. The images were used to determine
the relative
size or biomass of the plants as pixels and the color of the plants as the
ratio of dark green to
total area. The latter ratio, termed the health index, was a measure of the
relative amount of
chlorophyll in the leaves and therefore the relative amount of leaf senescence
or yellowing and
was recorded at day 27 only. Variation exists among transgenic plants that
contain the various
lead genes, due to different sites of DNA insertion and other factors that
impact the level or
pattern of gene expression. To show this effect the data tables indicate the
number of plants
that were positive and negative for the trait.
[0092] Tables 2 to 9 show the comparison of measurements of the Arabidopsis
plants.
" CD" indicates that the plants were grown under cycling drought conditions; "
WW"
indicates well-watered conditions. A number after an abbreviation indicates
multiple
independent experiments under the same conditions. Percent change indicates
the
measurement of the transgenic relative to the control plants as a percentage
of the control
non-transgenic plants; p value is the statistical significance of the
difference between
transgenic and control plants based on a T-test comparison of all independent
events where
NS indicates not significant at the 5% level of probabilty; No. of events
indicates the total
number of independent transgenic events tested in the experiment; No. of
positive events
indicates the total number of independent transgenic events that were larger
than the control in
the experiment; No. of negative events indicates the total number of
independent transgenic
events that were smaller than the control in the experiment.
CA 02754916 2011-09-08
WO 2010/108836 PCT/EP2010/053470
22
A. Galactokinase
[0093] The galactokinase gene b0757 (SEQ ID NO: 1) was expressed in
Arabidopsis
under control of the Super promoter with targeting to the chloroplast. Table 2
sets forth
biomass and health index data obtained from the Arabidopsis plants transformed
with these
constructs and tested under well-watered and cycling drought conditions.
CA 02754916 2011-09-08
WO 2010/108836 PCT/EP2010/053470
23
Table 2
...............................................................................
...............................................................................
...............................................................................
........................ .
Assay Gene Promoter Target Trait Percent pValue Valid Positive Negative
Type Change Events Events Events
WW b0757 Super Plastid Biomass at -1.5 NS 7 3 4
Day 17
WW b0757 Super Plastid Biomass at 0.1 NS 7 3 4
Day 21
................; .................................................;
........................:.
WW b0757 Super Plastid Health -2.0 NS 7 3 4
Index
...............................................................................
....................
CD b0757 Super Plastid Biomass at 8.4 0.014 4 4 0
Day 20
CD b0757 Super Plastid Biomass at 8.0 0.026 4 4 0
Day 27
CD b0757 Super Plastid Health -0.9 NS 4 2 2
Index
[0094] Table 2 shows that Arabidopsis plants expressing the b0757 gene
targeted to
the chloroplast resulted in plants that were larger under water limiting
conditions, but not under
well-watered conditions. In these experiments, all independent transgenic
events expressing
the b0757 gene were larger than the controls indicating better adaptation to
the stress
environment.
B. Transaldolase A
[0095] The transaldolase A gene b2464 (SEQ ID NO: 9) was expressed in
Arabidopsis
under control of the USP or the Super promoter with no subcellular targeting.
Table 3 sets forth
biomass and health index data obtained from the Arabidopsis plants transformed
with these
constructs and tested under well-watered and cycling drought conditions.
Table 3
Assay Gene Promoter Target Trait Percent pValue Valid Positive Negative
Type Change Events Events Events
WW b2464 USP None Biomass at 27.4 0.000 6 6 0
Day 17
.............................................
...............................................................................
.......
WW
b2464 USP None Biomass at 14.2 0.000 6 6 0
Day 21
.............................. .....................
...........................................................
.............................................. ....................
........................
WW b2464 USP None Health 6.0 NS 6 4 2
Index
CD b2464 Super None Biomass at 18.8 0.000 5 4 1
Day 20
CD b2464 Super None Biomass at 11.2 0.000 5 4 1
Day 27
......................
...............................................................................
....................... ................... ................. ................
CD b2464 Super None Health 2.5 NS 5 2 3
Index
......................:..................................................:.....
................:.........................................................:....
.........................................:.
......................................................
[0096] Table 3 shows that Arabidopsis plants expressing the b2464 gene under
control
CA 02754916 2011-09-08
WO 2010/108836 PCT/EP2010/053470
24
of the Super promoter were larger under water limiting conditions. Variation
does exist among
transgenic plants that contain the b2464 gene, due to different sites of DNA
insertion and other
factors that impact the level or pattern of gene expression. In these
experiments, the majority
of independent transgenic events expressing the b2464 gene were larger than
the controls
indicating better adaptation to the stress environment. Additionally,
expression of the b2464
gene under control of the USP promoter resulted in plants that were larger
under well-water
conditions. In these experiments, all transgenic events expressing the b2464
gene were larger
than the controls.
C. Hydrogenase-2 accessory protein
[0097] The hydrogenase-2 accessory protein gene b2990 (SEQ ID NO: 15) was
expressed in Arabidopsis under control of the Super promoter with no
subcellular targeting.
Table 4 sets forth biomass and health index data obtained from the Arabidopsis
plants
transformed with these constructs and tested under well-watered and cycling
drought
conditions.
Table 4
...............................................................................
...............................................................................
...............................................................................
........
Assa Gene Promoter Target Trait Percen pValue Valid Positive Negative
y t Events Events Events
Type Chang
e
WW b2990 Super None Biomass 9.3 0.0084 6 5 1
at Day 17
..................;.....................;......................................
........................................ ..................
................... ................. ...................
........................
WW b2990 Super None Biomass 11.1 0.0001 6 5 1
at Day 21
.................... ........................
WW b2990 Super None Health -7.4 0.0120 6 0 6
Index
CD b2990 Super None Biomass 19.4 0.0000 6 6 0
at Day 20
CD b2990 Super None Biomass 21.9 0.0000 6 6 0
at Day 27
...................... ...............................
................................................ .................
.................... ........................
CD b2990 Super None Health 1.9 NS 6 5 1
Index
[0098] Table 4 shows that Arabidopsis plants expressing the b2990 gene were
larger
under both well-watered and water limiting conditions. Variation does exist
among transgenic
plants that contain the b2990 gene, due to different sites of DNA insertion
and other factors
that impact the level or pattern of gene expression. In these experiments, the
majority of
independent transgenic events expressing the b2990 gene were larger than the
controls
indicating better adaptation to the stress environment. Under well-watered
conditions,
expression of the b2990 gene resulted in plants with reduced health index;
this effect was not
seen under water limiting conditions.
D. Isocitrate lyase
CA 02754916 2011-09-08
WO 2010/108836 PCT/EP2010/053470
[0099] The isocitrate lyase gene YER065C (SEQ ID NO: 17) was expressed in
Arabidopsis under control of the USP promoter with targeting to the
mitochondria. Table 5
sets forth biomass and health index data obtained from the Arabidopsis plants
transformed
with these constructs and tested under well-watered conditions.
5 Table 5
...............................................................................
...............................................................................
...............................................................................
.................................. .
Assay Gene Promoter Target Trait Percent pValue Valid Positive Negative
Type Change Events Events Events
WW1 YER065C USP Mito Biomass at 7.5 0.0136 7 5 2
Day 17
WW1 YER065C USP Mito Biomass at 0.9 NS 7 4 3
Day 21
.................
...............................................................
.................................
.................................................... , ........
................ .............................
WW1 YER065C USP Mito Health 1.3 NS 7 3 4
Index
................. ................................................
............. ........................ ......... ........................
......... ............... ......... ....
WW2 YER065C USP Mito Biomass at 30.6 0.0000 8 8 0
Day 17
WW2 YER065C USP Mito Biomass at 22.1 0.0000 8 8 0
Day 21
WW2 YER065C USP Mito Health 14.6 0.0000 8 7 1
Index
[00100] Table 5 shows that Arabidopsis plants expressing the YER065C gene were
larger under well-watered conditions. Variation does exist among transgenic
plants that contain
the YER065C gene, due to different sites of DNA insertion and other factors
that impact the
10 level or pattern of gene expression. In these experiments, the majority of
the independent
transgenic events expressing the YER065C gene were larger than the controls.
E. Phospholipid hydroperoxide glutathione peroxidase
[00101] The phospholipid hydroperoxide glutathione peroxidase gene YIR037W
(SEQ
15 ID NO: 19) was expressed in Arabidopsis under control of the USP or the
PCUbi promoter with
targeting to the chloroplast or to the mitochondria. Table 6 sets forth
biomass and health index
data obtained from the Arabidopsis plants transformed with these constructs
and tested under
well-watered or water limiting conditions.
20 Table 6
...............................................................................
...............................................................................
...............................................................................
................................... .
Assay Gene Promoter Target Trait Percent pValue Valid Positive Negative
Type Change Events Events Events
......... ......................... ...................................
................. ................. ............... .....................
WW YIR037 PCUbi Plastid Biomass 4.9 NS 6 4 2
W at Day 17
WW____ YIR037 PCUbi Plastid Biomass -1.8 NS 6 33
W at Day 21
WWYIR037 PCUbi Plastid Health 11.3 0.006 6 6 0
W Index
................. ......... .. ...................................
................. ................. ............... .....................
WW YIR037 USP Plastid Biomass -12.1 0.003 6 1 5
W at Day 17
CA 02754916 2011-09-08
WO 2010/108836 PCT/EP2010/053470
26
WW YIR037 USP Plastid Biomass -8.1 0.017 6 1 5
W at Day 21
WW YIR037 USP Plastid Health -7.5 0.000 6 0 6
W Index
......... .................. .. ................ ...............
.............. ................. .
CD YIR037 PCUbi Plastid Biomass 12.2 0.004 6 5 1
W at Day 20
................. ......... ....................
.................................. ................. ................
................ ....................
CD YIR037 PCUbi Plastid Biomass 11.2 0.000 6 6 0
W at Day 27
CD YIR037 PCUbi Plastid Health 10.2 0.011 6 5 1
W Index
CD ....... YIR037 USP Mito....._ Biomass.....-6.1 NS...... 6 2....... 4
W at Day 20
................. ......... ................. ....................
............................_ ... ................. ................
................ ....................
CD YIR037 USP Mito Biomass -6.0 NS 6 1 5
W at Day 27
CD YIR037 USP Mito Health 1.2 NS 6 4 2
W Index
CD YIR037 USP Plastid Biomass -7.9 0.015 6 1 5
W at Day 20
CD YIR037 USP Plastid Biomass -8.9 0.007 6 06
W at Day 27
.................... ....................................
..................... ............... .............. ................. .
CD YIR037 USP Plastid Health -1.0 NS 6 1 5
W Index
[00102] Table 6 shows that Arabidopsis plants expressing the YIR037W gene
controlled
by the PCUbi promoter when targeted to the chloroplast were larger than
controls under water
limiting conditions, indicating better adaptation to the stress environment.
In addition, the
transgenic plants expressing YIR037W were darker green in color than the
controls under both
well-watered and water limiting conditions as shown by the increased health
index. This
suggests that the YIR037W transgenic plants produced more chlorophyll or had
less
chlorophyll degradation compared to the control plants.
[00103] When expression of gene YIR037W was controlled by the USP promoter and
targeted to the chloroplast, YIR037W transgenic plants were smaller than
control plants under
both well-watered and water limiting conditions. Additionally, YIR037W
transgenic plants were
less green than control plants under well-watered conditions as shown by the
decreased
health index. This suggests that the YIR037W transgenic plants with this
specific construct
produced less chlorophyll or had more chlorophyll degradation compared to the
control plants.
If the targeting of YIR037W gene was the mitochondria under the control of the
USP promoter,
no significant difference in biomass or health index was seen when comparing
YIR037W
transgenic and control plants.
H. Gamma-glutamyltranspeptidase
[00104] The gamma-glutamyltranspeptidase gene s1r1269 (SEQ ID NO: 45) was
expressed in Arabidopsis under control the PCUbi promoter with targeting to
the chloroplast, to
the mitochondria, or no subcellular targeting. Table 7 sets forth biomass and
health index data
CA 02754916 2011-09-08
WO 2010/108836 PCT/EP2010/053470
27
obtained from the Arabidopsis plants transformed with these constructs and
tested under well-
watered or cycling drought conditions.
Table 7
....... ............ ............ . ........... ............
......................... ............ ............. ............ ............
............. ............ ............ ............. .......................
Assay Gene Promoter Target Trait Percent pValue Valid Positive Negative
Type Change Events Events Events
..................................................... ....................
....... .................... .......................
WW s1r1269 PCUbi Mito Biomass 0.6 NS 6 3 3
at Day 17
........................................................
WW s1r1269 PCUbi Mito Biomass 0.2 NS 6 2 4
at Day 21
WW s1r1269 PCUbi Mito Health -10.6 0.002 6 2 4
Index
WW s1r1269 PCUbi None Biomass 6.1 0.060 7 4 3
at Day 17
................: .....................................................:
.......................................................
WW s1r1269 PCUbi None Biomass 0.1 NS 7 3 4
at Day 21
...............................................................................
............................... .................... ...................
....................
...
WW s1r1269 PCUbi None Health -3.1 NS 7 3 4
Index
WW s1r1269 PCUbi Plastid Biomass 4.4 0.056 6 4 2
at Day 17
WW s1r1269 PCUbi Plastid Biomass 2.8 NS 6 5 1
at Day 21
...................... ......................................................
......................... ................................
.......................... .................... ...................
.................... .......................
WW s1r1269 PCUbi Plastid Health -7.5 0.034 6 2 4
Index
.........
...................:...........................................................
.......:..............
CD s1r1269 PCUbi Mito Biomass -14.5 0.000 6 1 5
at Day 20
CD s1r1269 PCUbi Mito Biomass -10.8 0.000 6 2 4
at Day 27
CD s1r1269 PCUbi Mito Health -10.2 0.002 6 0 6
Index
.................: .....................................................:
.......................................................
CD s1r1269 PCUbi None Biomass 23.4 0.000 7 6 1
at Day 20
...................... ......................... .............................
......................... ................................ ..............
....... ................ ................ ....................
.......................
CD s1r1269 PCUbi None Biomass 12.2 0.006 7 4 3
at Day 27
CD s1r1269 PCUbi None Health 19.4 0.000 7 7 0
Index
CD s1r1269 PCUbi Plastid Biomass -4.3 NS 5 2 3
at Day 20
CD s1r1269 PCUbi Plastid Biomass -6.8 0.018 5 2 3
at Day 27
.................: .....................................................:
..................................................
CD s1r1269 PCUbi Plastid Health -1.9 NS 5 2 3
Index
[00105] Table 7 shows that Arabidopsis plants expressing the slr1269 gene
targeted to
the mitochondria were smaller than controls under water limiting conditions.
Additionally,
CA 02754916 2011-09-08
WO 2010/108836 PCT/EP2010/053470
28
sIr1269 transgenic plants were less green than control plants in both well-
watered and water
limiting conditions, as shown by the decreased health index. This suggests
that the sIr1269
transgenic plants with targeting to the mitochondria produced less chlorophyll
or had more
chlorophyll degradation compared to the control plants. Similar results were
seen when
expression of gene sIr1269 was targeted to the chloroplast. Under water-
limiting conditions,
sIr1269 transgenic plants were smaller than controls. Under well-watered
conditions, sIr1269
transgenic plants were less green than controls, as indicated by the decreased
health index.
[00106] When expression of the s1r1269 gene had no subcellular targeting,
s1r1269
transgenic plants were larger than control plants under water limiting
conditions, indicating
better adaptation to the stress environment. In addition, the transgenic
plants expressing
sIr1269 were darker green in color than the controls under water limiting
conditions as shown
by the increased health index. This suggests that the sIr1269 transgenic
plants produced more
chlorophyll or had less chlorophyll degradation compared to the control
plants.
G. ATP synthase subunit B'
[00107] The ATP synthase subunit B' gene SLL1323 (SEQ ID NO: 47) was expressed
in Arabidopsis under control the PCUbi promoter with targeting to the
mitochondria. Table 8
sets forth biomass and health index data obtained from the Arabidopsis plants
transformed
with these constructs and tested under well-watered or cycling drought
conditions.
Table 8
...............................................................................
...............................................................................
...............................................................................
................................... .
Assay Gene Promoter Target Trait Percent pValue Valid Positive Negative
Type Change Events Events Events
WWSLL1323 PCUbi Mito Biomass at 14.1 0.0001 6 5
Day 17
WWSLL1323 PCUbi Mito Biomass at 11.2 0.0000 6 5
Day 21
WW SLL1323 PCUbi Mito Health 2.4 NS 6 3 3
Index
................. ......... .............................
..................................:..................... .........
.................. ................ .
CD SLL1323 PCUbi Mito Biomass at 27.2 0.0000 6 6 0
Day 20
CD SLL1323 PCUbi Mito Biomass at 23.6 0.0000 6 60
Day 27
CD SLL1323 PCUbi Mito Health 6.9 0.0061 6 5____
Index
[00108] Table 8 shows that Arabidopsis plants expressing the SLL1323 gene
resulted in
plants that were larger under both well-watered and water limiting conditions.
Variation does
exist among transgenic plants that contain the SLL1323 gene, due to different
sites of DNA
insertion and other factors that impact the level or pattern of gene
expression. In these
experiments, the majority of independent transgenic events expressing the
SLL1323 gene
were larger than the controls indicating better adaptation to the stress
environment. In addition,
the transgenic plants expressing SLL1323 were darker green in color than the
controls under
water limiting conditions as shown by the increased health index. This
suggests that the plants
CA 02754916 2011-09-08
WO 2010/108836 PCT/EP2010/053470
29
produced more chlorophyll or had less chlorophyll degradation during stress
than the control
plants.
H. C-22 sterol desaturase
[00109] The YMR015C gene (SEQ ID NO: 51), which encodes C-22 sterol
desaturase,
was expressed and targeted to the chloroplast in Arabidopsis using three
constructs. In one,
transcription is controlled by the PCUbi promoter. In another, trancription is
controlled by the
Super promoter. Transcription of YMR015C in the third construct is controlled
by the USP
promoter. Table 9 sets forth biomass and health index data obtained from
Arabidopsis plants
transformed with these constructs and tested under well-watered and water-
limiting conditions.
Table 9
...............................................................................
.........
Assay Gene 1 Promoter Target Measure Percent p- Valid Positive Negative
Type ment Change Value Events Events Events
-------------------------- -------------------------------- -------- ----------
------------------ ------------------- ------------------------ ---------------
------------ --------------------------
----------------------------- -------------- ----------------------------
CD YMR015C PCUbi Plasti Biomass 9.5 0.015 6 4 2
d at day 20 0
------------------- -------------------- ------------------- ------------------
---------
-------------- -------------------
CD YMR015C PCUbi Plasti Biomass 17.1 0.001 6 5 1
d at day 27 9
----------------------------- --------------
CD YMR015C PCUbi Plasti Health 7.8 0.041 6 4 2
d index 6
CD YMR015C Super Plasti Biomass 10.2 0.001 6 4 2
d at day 20 3
CD YMR015C Super Plasti Biomass -1.7 NS 6 2 4
d at day 27
.....................
....... ......
CD YMR015C Super Plasti Health 9.4
0.000 6 4 2
d index 3
--------------- :.................. ......................................
....................
..........................................................
WW YMR015C PCUbi Plasti Biomass -16.0 0.000 8 0 8
d at day 20 0
WW YMR015C 1 PCUbi Plasti Biomass -10.7 0.000 8 1 7
d at day 27 3
------------------------ --------------------------- -------------------
WW YMR015C PCUbi Plasti Health -8.7 0.014 8 3 5
d index 4
-------------- -------------------
WW YMR015C Super Plasti Biomass -30.8 0.000 6 0 6
d at day 20 0
----------------------------- --------------
WW YMR015C Super Plasti Biomass -20.1 0.000 6 0 6
d at day 27 0
WW YMR015C Super Plasti Health -13.5
0.004 6 1 5
d index 5
..............................................................:................
.. .............. ............... ....................
..........................................................
WW YMR015C USP Plasti Biomass -39.5 0.000 4 0 4
d at day 20 0
........... .....................
WW YMR015C USP Plasti Biomass -28.7 0.000 4 0 4
d at day 27 0
--------------- :.................. ......................................
....................
..........................................................
WW YMR015C USP Plasti Health -16.8 0.000 4 1 3
d index 6
..._..._................................
[00110] Table 9 shows that Arabidopsis plants with the PCUbi promoter
controlling
CA 02754916 2011-09-08
WO 2010/108836 PCT/EP2010/053470
expression of YMR015C were significantly larger than the control plants when
the protein was
also targeted to the chloroplast. In addition, these transgenic plants and
those with the Super
promoter controlling expression of YMR015C were darker green in color than the
controls.
These data indicate that the plants produced more chlorophyll or had less
chlorophyll
5 degradation during stress than the control plants. Table 9 also shows that
the majority of
independent transgenic events were larger than the controls.
[00111] Table 9 shows that Arabidopsis plants grown under well-watered
conditions with
the either the PCUbi promoter or the Super promoter controlling expression of
YMR015C were
significantly smaller than the control plants when the protein was also
targeted to the
10 chloroplast. Table 9 also shows that the majority of independent transgenic
events were
smaller than the controls. In addition, both of these constructs significantly
reduced the
amount of green color of the plants when grown under well-watered conditions.
