Note: Descriptions are shown in the official language in which they were submitted.
CA 02589145 2007-05-18
METHOD OF CONFERRING MULTIPLE STRESS TOLERANCE AND EARLY
FLOWERING IN PLANTS
FIELD
The present invention relates to a method of enhancing plant tolerance to
abiotic stresses and/or
promoting early flowering in plants, by introducing genes that confer stress
tolerance or early
flowering into plant cells, seeds or plants.
BACKGROUND
Plants encounter a wide range of environmental stresses including drought,
salinity, temperature
extremes, flooding and ultraviolet radiation, which severely limit crop
productivity worldwide
(Boyer 1982). Abiotic stresses can occur simultaneously and could affect
multiple stages of
plant growth and development (Chinnusamy et al., 2004)_leading to serious
morphological,
physiological, biochemical and molecular changes and can cause concomitant
reduction in the
average yield of most major crops by more than 50% (Boyer 1982; Bray et al.,
2000; Wang et
al., 2003). Soil salinity alone can lead to a significant drop in the yield,
affecting as much as 7%
of the world's arable land (Hasegawa et al., 2000; Zhu 2003). Furthermore,
increased
salinization is expected to reduce agricultural land by an estimated 30% by
the year 2025 and up
to 50% by the middle of the 21st century (Wang et al., 2003). In the extreme
cold conditions of
the Canadian Prairies as well as in other parts of the world, successful
growth of many crops is
based on their ability to germinate and survive better in the cold temperature
(Coursolle et al.,
1998).
The phytohormone abscisic acid (ABA) plays a crucial role in response to
environmental stresses
such as desiccation, salinity and cold and it is also involved in regulating
events such as
dormancy and maturation during late seed development (McCarty 1995; Leung and
Giraudat
1998). Many ABA-inducible genes share the cis-regulatory elements (i.e., ABA-
responsive
elements; ABREs; Guiltinan et al., 1990) and have been demonstrated to be
involved in the
regulation of plant stress responses. Constitutive expression of ABF3 in
Arabidopsis (Kang et
al., 2002) and rice (Oh et al., 2005) enhanced tolerance to drought stress as
well as tolerance to
multiple stresses in Arabidopsis (Kim et al., 2004).
CA 02589145 2007-05-18
Several gene products homologous to ABA-responsive proteins (also known as
dehydrins and
late embryogenesis abundant or LEA proteins) have been identified in different
plant systems
(Skriver and Mundy 1990; Close et al., 1993). It has been suggested that
dehydrins (members of
LEA proteins) and small heat shock proteins (sHSPs) may protect cells from the
deleterious
effects of dehydration (Pneuli et al., 2002). Expression of the HVA1 gene of
barley, which
encodes a LEA protein, conferred tolerance to salinity and water-deficit
stresses (Xu et al.,
1996), a tomato Le25 gene in yeast increased freezing and salinity tolerance
(Imai et al., 1996),
and a wheat chloroplast LEA-like protein (WCS 19) in Arabidopsis (Ndong et
al., 2002) resulted
in increased freezing tolerance.
Enhancing plant tolerance to abiotic stresses by introducing genes is a
desirable method for many
crop plants. For example, the over-expression of vacuolar Na+/H+ antiporter
AtNHXl promoted
growth and development in saline conditions (up to 200 mM NaCI) in Arabidopsis
(Apse et al.,
1999) and in B. napus (Zhang et al., 2001). With respect to tolerance to cold
temperatures,
transgenic plants over-expressing various transcription factors have been
demonstrated to have a
higher tolerance. For example, in Arabidopsis, significant improvement of
freezing stress
tolerance was demonstrated by the over-expression of the transcription factor
CBFl (Jaglo-
Ottosen et al., 1998); enhancement of drought and freezing tolerance by CBF4
(Haake et al.,
2002) and increased tolerance to freezing, water and salinity stress by over-
expression of
DREBIA gene (Kasuga et al., 1999), whereas transgenic rice over-expressing
CBF3
demonstrated elevated tolerance to drought and salinity but very low freezing
tolerance (Oh et
al., 2005). It appears, therefore, that these transcription factors are able
to enhance tolerance to a
variety of stresses however such enhancement may be species-specific (Oh et
al., 2005).
The availability of high throughput genomics and proteomics technologies
enables scientists to
conduct a comprehensive study of the genome, the transcriptome and the
proteome under
different abiotic and biotic stress conditions (Ramonell and Somerville 2002;
Agrawal et al.,
2005). An analysis of salinity-induced changes in pea root proteome
demonstrated an increase in
the levels of several members of group 10 family of pathogenesis-related
proteins (PR 10; Kav et
al., 2004), including PR 10.1 and the ABA-responsive protein ABR17 (PR 10.4),
in response to
stress. The PR proteins are part of a multicomponent defense response in many
plants that
respond to various abiotic and biotic stresses (Walter et al., 1990). They are
grouped into 14
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different families based on their serological relations, homology at the
nucleotide/amino acid
sequence level and similarities in biological functions (van Loon et al.,
1994; 1999).
Proteins belonging to the PR 10 family have been detected in a variety of
angiosperms, monocots
as well as dicot plants (Biesiadka et al., 2002). PR 10 proteins are small (15-
18 kDa), acidic and
intracellular, unlike other PR proteins that are extracellular (Walter et al.,
1990; van Loon et al.,
1994). These intracellular PR (IPR) proteins were first described in cultured
parsley cells upon
elicitor treatment (Somssich et al., 1988) and as described earlier, have
subsequently been
detected in many species. PR 10 genes are known to be induced by pathogens
(Fristensky et al.
1985; McGee et al. 2001; Borsics and Lados 2002) as well as other stresses
including salinity,
drought, wounding and darkness (Osmark et al. 1998; Hashimoto et al., 2004;
Kav et al., 2004).
In addition to the induction by stimulus, PR 10 proteins occur in high
concentrations in roots,
flowers and pollen (Biesiadka et al., 2002 and references therein). These
observations suggest
that in addition to their role(s) in plant stress response, they could play an
important role during
the normal growth and development of plants (Wu et al., 2003).
The pea ABA-responsive protein ABR17 is similar to pea disease resistance
response proteins; it
is produced late in seed development, and is induced by exogenous application
of ABA
(Iturriaga et al., 1994; Colditz et al., 2004). Many proteins with significant
homology to the pea
ABA-responsive protein include an IPR protein from bean (Walter et al., 1990),
garden pea
(Fristensky et al., 1988), parsley (Somssich et al., 1988), major birch pollen
allergen Bet vl
(Breiteneder et al., 1989), potato (Constabel and Brisson, 1992), and SAM22
from soybean
(Crowell et al., 1992). Several gene products homologous to ABR17, proteins
also known as
dehydrins and late embryogenesis abundant (LEA) related proteins, have been
identified in
different plant systems (Skriver and Mundy, 1990; Close et al., 1993; Goday et
al., 1994).
Srivastava et al., (2004) reported that the constitutive expression of a PR 10
(PR 10.1) gene from
pea in Brassica napus ameliorates the effects of salinity stress during
germination and early
seedling growth.
Despite the identification of proteins with significant similarities to ABR17
(and other PR 10) in
many species, a direct role for these proteins in mediating plant responses to
abiotic stresses has
not been demonstrated.
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SUMMARY OF THE INVENTION
In one aspect, the invention described herein is directed to transgenic plants
that are more
tolerant to environmental stresses than untransformed plants. Demonstrated
herein is the use of
the pea ABR17 (Abscisic acid responsive 17) to enhance germination of plants
such as
Arabidopsis sp. and Brassica sp. while under one or more stresses, and to
enhance the tolerance
of these plants to these stresses. Three independently derived Arabidopsis
transgenic lines,
containing ABR17, germinated better in the presence of salt, cold temperature
or both.
Furthermore, the transgenic plants also exhibited enhanced tolerance to
freezing temperature or
extreme heat, suggesting the potential utility of the ABR17 gene to engineer
multiple stress
tolerance. Two independently derived transgenic Brassica lines germinated
better than the wild-
type strain, in the presence of salt, salt and cold temperature, or darkness
and salt.
In one aspect, this invention comprises an expression vector for
transformation of a plant cell
comprising:
(a) a nucleic acid sequence encoding abscisic responsive protein 17 (ABR 17)
or a
biologically active portion or variant thereof;
(b) regulatory elements operatively linked to the nucleic acid sequence
encoding
ABR17 or a biologically active portion or variant thereof, such that the
nucleic acid
sequence is expressed in the plant cell,
wherein said expression results in expression of the protein encoded by the
polynucleotide, in
said plant cell. In one embodiment, the nucleic acid sequence in the vector
encodes the amino
acid sequence of SEQ ID NO:2. In one embodiment, the nucleic acid sequence
comprises SEQ
ID NO: 1 or a variant or portion thereof, for example, nucleotides 20 to 493
of SEQ ID NO: 1.
In another aspect, the invention comprises a transgenic plant cell transformed
with the expression
vector. In one embodiment, the transgenic plant cell is transformed with an
expression vector
comprising a nucleic acid sequence that encodes the amino acid sequence of SEQ
ID NO: 2.
In another aspect, the invention comprises a transgenic plant seed comprising
a transgenic plant
cell transformed by the expression vector, and transgenic plants grown from a
transgenic plant
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cell that is transformed with the expression vector. In one embodiment, the
transgenic plant is
grown from a plant cell or seed that is transformed with an expression vector
comprising a
nucleic acid sequence that encodes the amino acid sequence of SEQ ID NO: 2.
The transgenic
plant may be monocot or a dicot plant, for example, a transgenic Arabidopsis
sp. or a transgenic
Brassica sp.
In another aspect, the invention comprises a method of increasing the
tolerance of a plant to at
least one environmental stress, comprising the steps of:
(a) transfecting cells of said plant with a nucleic acid sequence encoding
ABR17,
(b) selecting and maintaining from said cells a transgenic cell line that
expresses a
protein encoded by said polynucleotide, and
(c) producing a plant from the transgenic cell line;
(d) wherein the increased tolerance to the at least one environmental stress
is
demonstrated by one or more of enhanced germination, greater rate of
flowering, earlier
flowering, greater plant height, increased root length, increased shoot
length, overall
plant health as compared to a control plant.
In one embodiment, the nucleic acid sequence encoding ABR17 encodes an amino
acid sequence
which comprises SEQ ID NO: 2. In one embodiment, the nucleic acid sequence
encoding
ABR17 comprises SEQ ID NO:1. In one embodiment, the cells used in step (a) are
from
Arabidopsis sp or the cells used in step (a) are from Brassica sp.
In another aspect, the invention may comprise a method of promoting early
flowering in a plant
under normal conditions, or under one or more abiotic stresses, comprising the
steps of:
(a) transfecting cells of said plant with a nucleic acid sequence encoding
ABR17,
(b) selecting and maintaining from said cells a transgenic cell line that
expresses a
protein encoded by said polynucleotide, and
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(c) producing a plant from the transgenic cell line.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1: Expression of ABR17 cDNA in transgenic A. thaliana. (a) RT-PCR
analysis of
ABR17 and 18s RNA expression demonstrating the presence of ABR17 transcript in
the 3
transgenic lines (6.9, 14.9, 25.20) and its absence in the wild type (WT) as
well as the presence
of 18s transcript throughout. (b) Western blot analysis of protein extracts
(from 2 week-old
seedlings) demonstrating the presence of a unique band corresponding to the
molecular weight of
ABR17 in the three transgenic lines.
Figure 2: (a-e) demonstrate the effects of NaCl and low temperature on the
germination of wild
type (WT) seeds and seeds from three transgenic Arabidopsis lines (6.9, 14.9
and 25.20). Room
temperature and 75 mM NaCI (a) or 150 mM NaCI (b), or 10 C and no NaCI (c), 75
mM NaCI
(d) or 150 mM NaCI (e). Experiments were repeated three times (n = 42) and the
bars indicate
the standard errors.
Figure 3: Effects of salinity on seedlings germinated and grown at room
temperature.
Morphology of seedlings germinated after (a) 1 and (b) 2 weeks of growth at 0,
75 and 150 mM
NaCl; (c) total chlorophyll and (d) carotenoid levels following two weeks of
growth at 0 and 75
mM NaCl.
Figure 4: Effects of salinity on the seedlings germinated and grown at 10 C.
(a) and (b) are
panels showing the growth of wild-type (WT) seedlings and seedlings of three
transgenic
Arabidopsis lines (6.9, 14.9 and 25.20), respectively, after two weeks (a) and
three weeks (b) of
growth at 0, 75 and 150 mM NaCl.
Figure 5: Freezing-tolerance of ABR17 transgenic plants. (a-d) are panels
showing the growth
of seedlings from wild-type (WT) and three transgenic Arabidopsis lines (6.9,
14.9 and 25.20)
lines carrying the ABR17 gene, one day after exposure to cold stress (-5 C)
for 4 h.
Experiments were repeated three times (n = 28).
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Figure 6: Heat-tolerance of ABR17 transgenic plants. (a-d) are panels showing
the growth of
wild-type (WT) seedlings and seedling from three transgenic Arabidopsis lines
(6.9, 14.9 and
25.20) carrying the ABR17 gene, one day after exposure to heat stress (48 C)
for 2 h.
Experiments were repeated three times (n = 28).
Figure 7: Images of two-dimensional gels of protein extracted from wild type
and transgenic
plants. (a) representative images of whole gels and (b) a closer view of
changes in the intensities
of the spots selected for identification.
Figure 8. Expression of ABR17 cDNA in transgenic B. napus. (A) Analysis of
ABR17 and 18s
RNA expression by RT-PCR. The presence of ABR17 transcript in the transgenic
lines (3.15 and
9.5) and its absence in the wild type (WT) as well as 18s RNA transcript in
both lines is
visualized by staining with ethidium bromide. (B) Analysis of protein extracts
from two-week
old seedlings by Western blot. The presence of a unique, immunoreactive band
in both of the
transgenic lines (3.15 and 9.5) at the expected molecular weight indicates the
presence of
ABR17.
Figure 9. Appearance of WT and ABR17 transgenic B. napus seedlings and plants.
Appearance
of (A) 7-day, (B) 14 day- and 37 day-old seedlings (C and E). The appearance
of 42-day old
plants demonstrating early flowering in the transgenic lines is shown in
panels D and F.
Figure 10. Effect of NaC1 on germination, root length and shoot length of WT
and ABR17
transgenic B. napus seedlings. (A) Effect of 275 mM NaCI on germination, (B)
root length (cm),
and (C) shoot length (cm) of 7 day-old seedlings.
Figure 11. Effect of low temperature and NaCl on the germination of WT and
ABR17
transgenic B. napus seedlings. (A) The effect of 5 C, (B) 10 C and (C) 10 C
plus 75 mM NaCl
on the germination of the WT and transgenic lines 3.15 and 9.5.
Figure 12. Flowering and height of ABR17 transgenic and WT B. napus adult
plants 42 days
after planting (DAP). (A) First day of flowering, (B) rate of flowering, (C)
plant height of ABR17
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transgenic and WT B. napus adult plants and (D) the relative expression of
ABR17 in the
transgenic lines (3.15 and 9.5).
DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS
Throughout this disclosure, various publications, patents and published patent
specifications are
referenced. Where permissible, the disclosures of these publications, patents
and published
patent specifications are hereby incorporated by reference in their entirety
into the present
disclosure to more fully describe the state of the art. Unless otherwise
indicated, the disclosure
encompasses conventional techniques of molecular biology, microbiology, cell
biology and
recombinant DNA, which are within the skill of the art- see e.g., Maniatis
(1989); Sambrook and
Russell (2001); Ausubel et al. (1987) and (2005).
Unless otherwise noted, technical terms are used according to conventional
usage. Definitions of
common terms in molecular biology may be found in Lewin (2000); Kendrew et al.
(1994);
Meyers (1995); Ausubel et al. (1987) and (2005); Sambrook and Russell (2001).
The results described here demonstrate, surprisingly and unexpectedly, that:
(1) constitutive
expression of pea (Pisum sativum) ABR17 gene in Arabidopsis enhances
germination and early
seedling growth in the presence of salt or cold or when both stresses are
combined, (2) two
week-old transgenic Arabidopsis plants exhibit increased tolerance to multiple
environmental
stresses (3) transgenic Brassica comprising pea ABR17 gene have enhanced
germination and
early seedling growth in the presence of cold, salt and cold, or salt and
darkness, and (4)
transgenic Brassica comprising pea ABR17 exhibited early flowering under
normal conditions.
Therefore, the applicants demonstrate that pea ABR17 is capable of protecting
plants from
multiple abiotic stresses, particularly cold and salinity. This protection is
evident during the
germination of the transgenic seeds in the presence of salt or a combination
of salt and cold and
also when older plants, such as seedlings, are subjected to freezing or heat
stresses. Furthermore,
pea ABR17 is capable of promoting early flowering, a trait which is very
crucial for early
maturity of Brassica sp. in short growing seasons.
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The proteome of a transgenic A. thaliana line 6.9 expressing pea ABR17 gene,
when compared to
its wild type counterpart, demonstrates that the levels of several proteins
involved in
photosynthesis or primary metabolic pathways (Table 2) were significantly
(P<0.01) affected in
the transgenic Arabidopsis line. The proteins showing increased levels in the
transgenic line are:
(a) PSI-E (spot 2; Table 2), a component of photosystem I (PSI); (b) oxygen-
evolving protein,
PSBO-2/PSBO2 (spot 12; Table 2), which showed - 3 fold up-regulation; (c)
rubisco activase,
which showed -2-fold increase and (d) glycine-rich RNA-binding proteins (GR-
RBP; spots 16
and 17; Table 2). Therefore, it is possible that the increase of one or more
of these proteins in
ABR17 transgenic lines may contribute to the observed increase in stress
tolerance.
The level of another enzyme involved in primary metabolism, phosphopyruvate
hydratase (spot
14; Table 2) also known as enolase (2-phospho-D-glycerate hydrolase) was
decreased in the
transgenic line. The observed reduction in enolase level in the ABR17
transgenic A. thaliana line
may be important for its tolerance to cold temperature stress.
To facilitate understanding of the invention, a number of terms are defined
below.
"Stress" refers to a factor that externally causes a change in the growth of
plants.
"Environmental stress" and "environmental stresses" refer to a stress, or
stresses as the case may
be, provided by a change in an external environment, including salt
concentration, high osmotic
pressure, drying, high temperature, low temperature, intense light, air
pollution, and the like.
"Expression" refers to transcription or translation, or both, as context
requires.
The terms "modified", "mutant" or "variant" are used interchangeably herein,
and refer to: (a) a
nucleotide sequence in which one or more nucleotides have been added or
deleted, or substituted
with different nucleotides or modified bases (e.g., inosine, methylcytosine)
or to (b) a protein,
peptide or polypeptide in which one or more amino acids have been added or
deleted, or
substituted with a different amino acid. A variant may be naturally occurring,
or may be created
experimentally by one of skill in the art. A variant of SEQ ID NO: 1 or SEQ ID
NO: 2 may be a
protein, peptide, polypeptide or polynucleotide that differs (i.e., an
addition, deletion or
substitution) in one or more amino acids or nucleotides from the sequence
presented as SEQ ID
NO:1 or SEQ ID NO: 2.
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A variant protein may include analogues or homologues having similar or
identical biologic
activity native in other plant species. The present invention is intended to
encompass all
homologues, paralogues and analogues of acyl CoA binding protein, and
biologically active
portions and variants thereof.
In this regard, it is well understood in the art that certain alterations
inclusive of mutations,
additions, deletions and substitutions can be made to a reference nucleic acid
or protein, to result
in a modified, mutant or variant nucleic acid or protein that retains a
particular biological
function or activity, or perhaps displays an altered but nevertheless useful
activity. Some
deletions, insertions and substitutions will not produce radical changes in
the characteristics of
the protein having the sequence SEQ ID NO:2 or in the nucleic acid represented
by SEQ ID
NO:1. However, while it may be difficult to predict the exact effect of the
substitution, deletion
or insertion in advance of doing so, one skilled in the art will appreciate
that the effect can be
evaluated by routine screening assays. For example whether a variant of the
protein having the
sequence SEQ ID NO:2 enhances germination, confers stress tolerance or
promotes early
flowering, can be determined by following the methods disclosed in the
Examples disclosed
herein. Modifications of protein properties such as redox or thermal
stability, hydrophobicity,
susceptibility to proteolytic degradation, or the tendency to aggregate with
carriers or into
multimers may be assayed by methods well known to one of skill in the art.
Variants may be created experimentally using random mutagenesis,
oligonucleotide-mediated (or
site-directed) mutagenesis, PCR mutagenesis and cassette mutagenesis.
Oligonucleotide-
mediated mutagenesis is well known in the art as, for example, described by
Adelman (1983)
using vectors that are either derived from bacteriophage M13, or that contain
a single-stranded
phage origin of replication as described by Viera et al. (1987). Production of
single-stranded
template is described, for example, in Sambrook (2001). Alternatively, the
single-stranded
template may be generated by denaturing double-stranded plasmid (or other DNA)
using
standard techniques.
Alternatively, linker-scanning mutagenesis of DNA may be used to introduce
clusters of point
mutations throughout a sequence of interest that has been cloned into a
plasmid vector. For
example, reference may be made to Ausubel et al. (1987) and (2005). Region-
specific
CA 02589145 2007-05-18
mutagenesis and directed mutagenesis using PCR may also be employed to
construct variants
according to the invention. In this regard, reference may be made, for
example, to Ausubel et al.
(1987) and (2005). With regard to random mutagenesis, methods include
incorporation of dNTP
analogs (Zaccolo et al., (1996)) and PCR-based random mutagenesis such as
described in
Stemmer (1994) and Shafikhani et al. (1997).
A variant protein, polypeptide or peptide may, for example, have an amino acid
sequence
substantially identical to the specific sequences disclosed herein. By the
term "substantially
identical" it is meant that two polypeptide sequences preferably are at least
70% identical, and
more preferably are at least 85% identical and most preferably at least 95%
identical, for
example 96%, 97%, 98% or 99% identical. In order to determine the percentage
of identity
between two polypeptide sequences the amino acid sequences of such two
sequences are aligned,
using for example the alignment method of Needleman and Wunsch (J. Mol. Biol.,
1970, 48:
443), as revised by Smith and Waterman (Adv. Appl. Math., 1981, 2: 482) so
that the highest
order match is obtained between the two sequences and the number of identical
amino acids is
determined between the two sequences. Methods to calculate the percentage
identity between
two amino acid sequences are generally art recognized and include, for
example, those described
by Carillo and Lipton (SIAM J. Applied Math., 1988, 48:1073) and those
described in
Computational Molecular Biology, Lesk, e.d. Oxford University Press, New York,
1988,
Biocomputing: Informatics and Genomics Projects. Generally, computer programs
will be
employed for such calculations. Computer programs that may be used in this
regard include, but
are not limited to, GCG (Devereux et al., Nucleic Acids Res., 1984, 12: 387)
BLASTP,
BLASTN and FASTA (Altschul et al., J. Molec. Biol., 1990: 215: 403). A
particularly preferred
method for determining the percentage identity between two polypeptides
involves the Clustal W
algorithm (Thompson, JD, Higgines, DG and Gibson TJ, 1994, Nucleic Acid Res
22(22): 4673-
4680 together with the BLOSUM 62 scoring matrix (Henikoff S & Henikoff, JG,
1992, Proc.
Natl. Acad. Sci. USA 89: 10915-10919 using a gap opening penalty of 10 and a
gap extension
penalty of 0.1, so that the highest order match obtained between two sequences
wherein at least
50% of the total length of one of the two sequences is involved in the
alignment.
The term "biologically active" when made in reference to a variant or portion
of an ABR17
protein, refers to a protein, polypeptide or peptide possessing the ability to
enhance germination
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CA 02589145 2007-05-18
or confer stress tolerance to a plant species that is transfected with a
nucleic acid encoding that
protein, polypeptide or peptide. Whether germination is enhanced in, whether
the stress
tolerance is conferred to transgenic plants, or whether early flowering is
promoted may be
determined, for example, by using the methods disclosed herein.
The term "portion" when used in reference to a protein refers to fragments of
that protein. The
fragments may range in size from four amino acid residues to the entire amino
acid sequence of
the protein, minus one amino acid.
An "expression vector" refers to a recombinant DNA molecule containing the
appropriate control
nucleotide sequences (e.g., promoters, enhancers, repressors, operator
sequences and ribosome
binding sites) necessary for the expression of an operably linked nucleotide
sequence in a
particular host cell. By "operably linked/linking" or "in operable
combination" is meant that the
nucleotide sequence is positioned relative to the control nucleotide sequences
to initiate, regulate
or otherwise direct transcription and/or the synthesis of the desired protein
molecule.
As used herein, an "ABR17 protein" is a pea abscisic acid-responsive protein
and includes a
protein having the amino acid sequence of SEQ ID NO:2, and includes
biologically active
portions or variants thereof.
The ABR17 mRNA has the following nucleotide sequence (Accession # Z15128) [SEQ
ID
NO:1 ] where the coding region begins at nucleotide 20 and ends at nucleotide
493:
tttttttttt ttttttatca tgggtgtctt tgtttttgat gatgaatacg tttcaactgt
tgcaccacct aaactctaca aagctctcgc aaaagatgct gacgaaatcg tcccaaaggt
gatcaaggaa gcacaaggag tcgaaattat cgaaggaaat ggaggtccag gaaccatcaa
gaagctatcc attcttgaag atggaaaaac caactatgtg ctacacaaac tagacgcagt
tgatgaagca aactttggtt acaactacag cttagtagga ggaccagggc tacatgaaag
tttagagaaa gttgcattcg agacaattat tttggctggt tctgacggtg gatccatcgt
taagatatct gtgaaatatc acaccaaagg tgatgcagct ctatctgatg cagttcgtga
tgaaacaaag gccaaaggaa ctggacttat caaggccata gaaggttacg ttttggcaaa
tcctggttac taattagttg tataatcttc tacttggttt ggttttgtta tgcgaataat
gaatgaataa agtgttgtga tatggttttt taatttacat gtgtgaggct atgttgtcaa
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gtgtgaacta gtgtcggttt gagtgtggtt ttgggagaat ttggttgggt tgatgatgag
gttttgtact atatgtattt ctctaataaa taatgcaaaa gaaaagttcc tagtaaaaaa
aaaaaaaaaa a
The ABR17 protein has the following amino acid sequence [SEQ ID NO:2]:
MGVFVFDDEYVSTVAPPKLYKALAKDADEIVPKVIKEAQGVEIIEGNGGPGTIKK
LSILEDGKTNYVLHKLDAVDEANFGYNYSLVGGPGLHESLEKVAFETIILAGSDG
GS IV KIS V KYHTKGDAALSDA VRDETKAKGTGLIKAIEGY V LANPGY
The term "nucleic acid sequence" as used herein refers to a sequence of
nucleoside or nucleotide
monomers consisting of naturally occurring bases, sugars and intersugar
(backbone) linkages.
The term also includes modified or substituted sequences comprising non-
naturally occurring
monomers or portions thereof. The nucleic acid sequences of the present
invention may be
deoxyribonucleic acid sequences (DNA) or ribonucleic acid sequences (RNA) and
may include
naturally occurring bases including adenine, guanine, cytosine, thymidine and
uracil. The
sequences may also contain modified bases. Examples of such modified bases
include aza and
deaza adenine, guanine, cytosine, thymidine and uracil; and xanthine and
hypoxanthine. The
term "nucleotide" refers to a ribonucleotide or a deoxyribonucleotide.
"Nucleic acid" refers to a
polymer of nucleotides and may be single- or double-stranded. "Polynucleotide"
refers to a
nucleic acid that is twelve (12) or more nucleotides in length. A nucleic
sequence "encodes" or
"codes for" a protein, polypeptide or peptide if the nucleotide sequence can
be translated to the
amino acid sequence of the protein, polypeptide or peptide.
The term "nucleic acid sequence encoding ABR17" refers to any and all nucleic
acid sequences
encoding an ABR17 protein. Nucleic acid sequences encoding an ABR17 protein
further include
any and all nucleic acid sequences which (i) encode polypeptides that are
substantially identical
to a ABR17 protein; or (ii) hybridize to any nucleic acid sequences encoding
an ABR17 protein
under at least moderately stringent hybridization conditions or which would
hybridize thereto
under at least moderately stringent conditions but for the use of synonymous
codons.
By the phrase "at least moderately stringent hybridization conditions", it is
meant that conditions
are selected which promote selective hybridization between two complementary
nucleic acid
13
CA 02589145 2007-05-18
molecules in solution. Hybridization may occur to all or a portion of a
nucleic acid sequence
molecule. The hybridizing portion is typically at least 15 (e.g. 20, 25, 30,
40 or 50) nucleotides in
length. Those skilled in the art will recognize that the stability of a
nucleic acid duplex, or
hybrids, is determined by the Tm, which in sodium containing buffers is a
function of the sodium
ion concentration and temperature (Tn, = 81.5 C - 16.6 (Logio [Na+]) +
0.41(%(G+C) - 600/1),
or similar equation). Accordingly, the parameters in the wash conditions that
determine hybrid
stability are sodium ion concentration and temperature. In order to identify
molecules that are
similar, but not identical, to a known nucleic acid molecule a 1% mismatch may
be assumed to
result in about a 1 C decrease in Tm, for example if nucleic acid molecules
are sought that have a
>95% identity, the final wash temperature will be reduced by about 5 C. Based
on these
considerations those skilled in the art will be able to readily select
appropriate hybridization
conditions. In preferred embodiments, stringent hybridization conditions are
selected. By way of
example the following conditions may be employed to achieve stringent
hybridization:
hybridization at 5 x sodium chloride/sodium citrate (SSC)/5 x Denhardt's
solution/1.0% SDS at
Tm (based on the above equation) - 5 C, followed by a wash of 0.2 x SSC/0.1%
SDS at 60 C.
Moderately stringent hybridization conditions include a washing step in 3 x
SSC at 42 C. It is
understood however that equivalent stringencies may be achieved using
alternative buffers, salts
and temperatures. Additional guidance regarding hybridization conditions may
be found in:
Current Protocols in Molecular Biology, John Wiley & Sons, N.Y., 1989, 6.3.1. -
6.3.6 and in:
Sambrook et al., Molecular Cloning, a Laboratory Manual, Cold Spring Harbor
Laboratory
Press, 1989, Vol.3.
Preparation of recombinant expression vectors comprising chimeric nucleic acid
sequences
encoding ABR17 and a nucleic acid sequence capable of controlling expression
in a plant
cell
The term "chimeric" as used herein in the context of nucleic acid sequences
refers to at least two
linked nucleic acid sequences which are not naturally linked. Chimeric nucleic
acid sequences
include linked nucleic acid sequences of different natural origins. For
example a nucleic acid
sequence constituting a plant promoter linked to a nucleic acid sequence
encoding an ABR17 is
considered chimeric. Chimeric nucleic acid sequences also may comprise nucleic
acid sequences
of the same natural origin, provided they are not naturally linked. For
example a nucleic acid
14
CA 02589145 2007-05-18
sequence constituting a promoter obtained from a particular cell-type may be
linked to a nucleic
acid sequence encoding a polypeptide obtained from that same cell-type, but
not normally linked
to the nucleic acid sequence constituting the promoter. Chimeric nucleic acid
sequences also
include nucleic acid sequences comprising any naturally occurring nucleic acid
sequence linked
to any non-naturally occurring nucleic acid sequence.
The nucleic acid sequences encoding an ABR17 protein that may be used in
accordance with the
methods and compositions provided herein may be any nucleic acid sequence
encoding an
ABR17 protein.
Alterations to the nucleic acid sequence encoding ABR17 to prepare ABR17
analogs may be
made using a variety of nucleic acid modification techniques known to those
skilled in the art,
including for example site directed mutagenesis, targeted mutagenesis, random
mutagenesis, the
addition of organic solvents, gene shuffling or a combination of these and
other techniques
known to those of skill in the art (Shraishi et al., 1988, Arch. Biochem.
Biophys, 358: 104-115;
Galkin et al., 1997, Protein Eng. 10: 687-690; Carugo et al., 1997, Proteins
28: 10-28; Hurley et
al., 1996, Biochem, 35: 5670-5678; Holmberg et al., 1999, Protein Eng. 12: 851-
856).
In accordance herewith the nucleic acid sequence encoding ABR17 is linked to a
nucleic acid
sequence capable of controlling expression of the ABR17 protein in a plant
cell. Accordingly,
the present invention also comprises a nucleic acid sequence encoding
ABR171inked to a
promoter capable of controlling expression in a plant cell. Nucleic acid
sequences capable of
controlling expression in plant cells that may be used herein include any
plant derived promoter
capable of controlling expression of polypeptides in plants. Generally,
promoters obtained from
dicotyledonous plant species will be used when a dicotyledonous plant is
selected in accordance
herewith, while a monocotyledonous plant promoter will be used when a
monocotyledonous
plant species is selected. Constitutive promoters that may be used include,
for example, the 35S
cauliflower mosaic virus (CaMV) promoter (Rothstein et al., 1987, Gene 53: 153-
161), the rice
actin promoter (McElroy et al., 1990, Plant Ce112:163-171; US Patent
6,429,357), a ubiquitin
promoter, such as the corn ubiquitin promoter (US Patents 5,879,903;
5,273,894), and the
parsley ubiquitin promoter (Kawalleck, P. et al., 1993, Plant Mol. Biol.
21:673-684).
Certain genetic elements capable of enhancing expression of the ABR17 protein
may be used
herein. These elements include the untranslated leader sequences from certain
viruses, such as
CA 02589145 2007-05-18
the AMV leader sequence (Jobling and Gehrke, 1987, Nature, 325: 622-625) and
the intron
associated with the maize ubiquitin promoter (US Patent 5,504,200). Generally
the chimeric
nucleic acid sequence will be prepared so that genetic elements capable of
enhancing expression
will be located 5' to the nucleic acid sequence encoding the ABR17 protein.
In accordance with the present invention the chimeric nucleic acid sequences
comprising a
promoter capable of controlling expression in plant linked to a nucleic acid
sequence encoding
an ABR17 protein can be integrated into a recombinant expression vector which
promotes good
expression in the cell. Accordingly, the present invention includes
recombinant expression
vectors comprising the chimeric nucleic acid sequences of the present
invention, wherein the
expression vector is suitable for expression in a plant cell. The term
"suitable for expression in a
plant cell" means that the recombinant expression vector comprises a chimeric
nucleic acid
sequence of the present invention linked to genetic elements required to
achieve expression in a
plant cell. Genetic elements that may be included in the expression vector in
this regard include a
transcriptional termination region, one or more nucleic acid sequences
encoding marker genes,
one or more origins of replication and the like. In preferred embodiments, the
expression vector
further comprises genetic elements required for the integration of the vector
or a portion thereof
in the plant cell's nuclear genome, for example the T-DNA left and right
border sequences which
facilitate the integration into the plant's nuclear genome in embodiments of
the invention in
which plant cells are transformed using Agrobacterium.
In one embodiment, the recombinant expression vector generally comprises a
transcriptional
terminator which besides serving as a signal for transcription termination
further may serve as a
protective element capable of extending the mRNA half life (Guameros et al.,
1982, Proc. Natl.
Acad. Sci. USA, 79: 238-242). The transcriptional terminator is generally from
about 200
nucleotides to about 1000 nucleotides and the expression vector is prepared so
that the
transcriptional terminator is located 3' of the nucleic acid sequence encoding
ABR17.
Termination sequences that may be used herein include, for example, the
nopaline termination
region (Bevan et al., 1983, Nucl. Acids. Res., 11: 369-385), the phaseolin
terminator (van der
Geest et al., 1994, Plant J. 6: 413-423), the arcelin terminator (Jaeger GD,
et al., 2002, Nat.
Biotechnol . 20:1265-8), the terminator for the octopine synthase genes of
Agrobacterium
16
CA 02589145 2007-05-18
tumefaciens or other similarly functioning elements. Transcriptional
terminators may be obtained
as described by An (An, 1987, Methods in Enzym. 153: 292).
In one embodiment, the expression vector may further comprise a marker gene.
Marker genes
that may be used include all genes that allow the distinction of transformed
cells from non-
transformed cells, including all selectable and screenable marker genes. A
marker gene may be a
resistance marker such as an antibiotic resistance marker against, for
example, kanamycin (US
Patent 6,174,724), ampicillin, G418, bleomycin, hygromycin or spectinomycin
which allows
selection of a trait by chemical means or a tolerance marker against a
chemical agent, such as the
normally phytotoxic sugar mannose (Negrotto et al., 2000, Plant Cell Rep. 19:
798-803). Other
convenient markers that may be used herein include markers capable of
conveying resistance
against herbicides such as glyphosate (US Patents 4,940,935; 5,188,642),
phosphinothricin (US
Patent 5,879,903) or sulphonyl ureas (US Patent 5,633,437). Resistance
markers, when linked in
close proximity to nucleic acid sequence encoding the apolipoprotein
polypeptide, may be used
to maintain selection pressure on a population of plant cells or plants that
have not lost the
nucleic acid sequence encoding the ABR17 protein. Screenable markers that may
be employed to
identify transformants through visual inspection include 0-glucuronidase (GUS)
(US Patents
5,268,463 and 5,599,670) and green fluorescent protein (GFP) (Niedz et al.,
1995, Plant Cell
Rep., 14: 403).
Recombinant vectors suitable for the introduction of nucleic acid sequences
into plants include
Agrobacterium and Rhizobium based vectors, such as the Ti and Ri plasmids,
including for
example pBIN19 (Bevan, Nucl. Acid. Res., 1984, 22: 8711-8721), pGKB5 (Bouchez
et al.,
1993, C R Acad. Sci. Paris, Life Sciences, 316:1188-1193), the pCGN series of
binary vectors
(McBride and Summerfelt, 1990, Plant Mol. Biol., 14:269-276) and other binary
vectors (e.g. US
Patent 4,940,838).
The recombinant expression vectors of the present invention may be prepared in
accordance with
methodologies well known to those skilled in the art of molecular biology.
Such preparation will
typically involve the bacterial species Escherichia coli as an intermediary
cloning host. The
preparation of the E. coli vectors as well as the plant transformation vectors
may be
accomplished using commonly known techniques such as restriction digestion,
ligation,
gelectrophoresis, DNA sequencing, the Polymerase Chain Reaction (PCR) and
other
17
CA 02589145 2007-05-18
methodologies. A wide variety of cloning vectors is available to perform the
necessary steps
required to prepare a recombinant expression vector. Among the vectors with a
replication
system functional in E. coli, are vectors such as pBR322, the pUC series of
vectors, the M13mp
series of vectors, pBluescript etc. Typically, these cloning vectors contain a
marker allowing
selection of transformed cells. Nucleic acid sequences may be introduced in
these vectors, and
the vectors may be introduced in E. coli grown in an appropriate medium.
Recombinant
expression vectors may readily be recovered from cells upon harvesting and
lysing of the cells.
Further, general guidance with respect to the preparation of recombinant
vectors may be found
in, for example: Sambrook et al., Molecular Cloning, a Laboratory Manual, Cold
Spring Harbor
Laboratory Press, 1989, Vol.3.
The expression vector comprising a nucleic acid sequence encoding ABR 17 is
then transfected
into the appropriate plant cell. Examples of how this can be accomplished are
provided in the
Examples herein. As non-limiting examples, the expression vector may be
transfected into
Arabidopsis, Brassica or cells from other plants, including monocots and
dicots, including
wheat, barley, rice, maize, potato and cotton.
Methodologies to introduce plant recombinant expression vectors into a plant
cell, also referred
to herein as "transformation", are well known to the art and typically vary
depending on the plant
cell that is selected. General techniques to introduce recombinant expression
vectors in cells
include, electroporation; chemically mediated techniques, for example CaCl2
mediated nucleic
acid uptake; particle bombardment (biolistics); the use of naturally infective
nucleic acid
sequences, for example virally derived nucleic acid sequences, or
Agrobacterium or Rhizobium
derived sequences, polyethylene glycol (PEG) mediated nucleic acid uptake,
microinjection and
the use of silicone carbide whiskers.
In preferred embodiments, a transformation methodology is selected which will
allow the
integration of the chimeric nucleic acid sequence in the plant cell's genome,
and preferably the
plant cell's nuclear genome. The use of such a methodology is preferred as it
will result in the
transfer of the chimeric nucleic acid sequence to progeny plants upon sexual
reproduction.
Transformation methods that may be used in this regard include biolistics and
Agrobacterium
mediated methods.
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CA 02589145 2007-05-18
Transformation methodologies for dicotyledenous plant species are well known.
Generally,
Agrobacterium mediated transformation is used because of its high efficiency,
as well as the
general susceptibility by many, if not all, dicotyledenous plant species.
Agrobacterium
transformation generally involves the transfer of a binary vector, such as one
of the hereinbefore
mentioned binary vectors, comprising the chimeric nucleic acid sequence of the
present
invention from E. coli to a suitable Agrobacterium strain (e.g. EHA101 and
LBA4404) by, for
example, tri-parental mating with an E. coli strain carrying the recombinant
binary vector and an
E. coli strain carrying a helper plasmid capable of mobilizing the binary
vector to the target
Agrobacterium strain, or by DNA transformation of the Agrobacterium strain
(Hofgen et al.,
Nucl. Acids. Res., 1988, 16:9877). Other techniques that may be used to
transform
dicotyledenous plant cells include biolistics (Sanford, 1988, Trends in
Biotechn. 6:299-302);
electroporation (Fromm et al., 1985, Proc. Natl. Acad. Sci. USA., 82:5824-
5828); PEG mediated
DNA uptake (Potrykus et al., 1985, Mol. Gen. Genetics, 199:169-177);
microinjection (Reich et
al., Bio/Techn., 1986, 4:1001-1004); and silicone carbide whiskers (Kaeppler
et al., 1990, Plant
Cell Rep., 9:415-418) or in planta transformation using, for example, a flower
dipping
methodology (Clough and Bent, 1998, Plant J., 16:735-743).
Monocotyledonous plant species may be transformed using a variety of
methodologies including
particle bombardment (Christou et al., 1991, Biotechn. 9:957-962; Weeks et
al., Plant Physiol.,
1993, 102:1077-1084; Gordon-Kamm et al., Plant Cell, 1990, 2:5603-618); PEG
mediated DNA
uptake (European Patents 0292 435; 0392 225) or Agrobacterium mediated
transformation
(Goto-Fumiyuki et al., 1999, Nature-Biotech. 17:282-286).
The exact plant transformation methodology may vary somewhat depending on the
plant species
and the plant cell type (e.g. seedling derived cell types such as hypocotyls
and cotyledons or
embryonic tissue) that is selected as the cell target for transformation. As
mentioned above, in a
particularly preferred embodiment, Brassica napus is used. A methodology to
obtain safflower
transformants is described in Baker and Dyer (Plant Cell Rep., 1996, 16:106-
110). Additional
plant species specific transformation protocols may be found in: Biotechnology
in Agriculture
and Forestry 46: Transgenic Crops I (Y.P.S. Bajaj ed.), Springer-Verlag, New
York (1999), and
Biotechnology in Agriculture and Forestry 47: Transgenic Crops II (Y.P.S.
Bajaj ed.), Springer-
Verlag, New York (2001).
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CA 02589145 2007-05-18
Following transformation, the plant cells are grown and upon the emergence of
differentiating
tissue, such as shoots and roots, mature plants are regenerated. Typically a
plurality of plants is
regenerated. Methodologies to regenerate plants are generally plant species
and cell type
dependent and will be known to those skilled in the art. Further guidance with
respect to plant
tissue culture may be found in, for example: Plant Cell and Tissue Culture,
1994, Vasil and
Thorpe Eds., Kluwer Academic Publishers; and in: Plant Cell Culture Protocols
(Methods in
Molecular Biology 111), 1999, Hall Eds, Humana Press.
EXAMPLES
The following examples are intended only to illustrate and describe the
invention rather than
limit the claims that follow.
EXAMPLE 1: Arabidopsis thaliana
Plant expression vectors
A pea ABR17 cDNA clone was amplified using as a forward primer having SEQ ID
NO: 3: 5'-
GTGGTCGAAGCTTATGGGTGTCTTTGTTTTTGATGATGAATAC-3'. This primer,
beginning with the ATG (underlined) corresponds to nucleotides 20-49
inclusive, of SEQ ID
NO: 1. The sequence of the reverse primer follows, and is SEQ ID NO: 4: 5'-
TATATAGCTCGAGTTAGTAACCAGGATTTGCCAAAACGTAACC-3'. This primer,
beginning with the TTA (underlined) corresponds to the reverse complement of
nucleotides 493-
464 inclusive, of SEQ ID NO: 1.
The restriction enzyme sites Hind III and Xho I are highlighted on the forward
and reverse
primers, respectively. The amplified cDNA fragment was ligated between the
CaMV35S
promoter and the rbcS3' terminator in the binary vector pKYLX71 (Schald et
al., 1987) and the
resulting gene construct was sequenced to ensure ligation of the cDNA in the
correct orientation
and the absence of any mutations/rearrangement.
The gene construct was subsequently introduced into the disarmed Agrobacterium
tumefaciens
strain GV3101 through a triparental mating technique using E. coli strain
HB101 carrying the
CA 02589145 2007-05-18
helper plasmid pRK2013. Transconjugant A. tumefaciens was selected on solid
medium
containing rifampicin, gentamicin and tetracycline. Plasmid DNA was extracted
from A.
tumefaciens and analyzed by restriction digestion to ensure that no
rearrangements had taken
place during the introduction into this bacterium.
Transformation of Arabidopsis thaliana
A. thaliana ecotype WS was transformed using the floral dip method (Clough and
Bent, 1998).
Briefly, plants were grown in growth chambers at 25 C until flowering with a
16h light/8h dark
photoperiod. Primary racemes were clipped to encourage proliferation of
secondary bolts. A.
tumefaciens cells at mid-log stage of growth were centrifuged and resuspended
to a density of
~0.8 (Abs600) in 5% sucrose solution containing 0.05% silwet L-77. Above-
ground parts of A.
thaliana plants were dipped in this Agrobacterium solution with gentle
agitation. Dipped plants
were covered with plastic wrap (for 16-24 h) to maintain high humidity and
were returned to
growth chambers. Harvested, dry seeds (TO) were surface sterilized and placed
on 1/2 strength
MS plates containing 50 mg/L kanamycin. The seeds were cold treated at 4 C for
at least 2 d,
and grown at 25 C with 16h light/8h dark, 100 E light for 7-10 d. Plants that
survived on the
kanamycin plates were transferred to soil and maintained in growth cabinets. A
number of
independent transgenic lines were tested for their abilities to germinate on
MS plates containing
75 or 150 mM NaCl and, those with enhanced germination were selected for the
production of
homozygous plants.
The number of copies of the ABR17 cDNA in these lines was estimated based on
their
segregation on kanamycin plates. Essentially, seeds from the transgenic plants
(T1 seeds) were
screened on kanamycin plates to determine segregation ratios and those with a
monogenic (3:1)
segregation ratio and exhibiting enhanced germination in the presence of NaCl
were used for
homozygous T2 seed production. Bulked homozygous seeds from T2 plants were
used in all
subsequent experiments.
Three transgenic lines (6.9, 14.9 and 25.20) with single insertions of the
ABR17 cDNA that
showed best germination in preliminary experiments under saline conditions
were selected for
the production of homozygous lines and further characterization.
21
CA 02589145 2007-05-18
RT-PCR analysis
Total RNA, isolated from wild type and transgenic seedlings, was reverse
transcribed and
subsequently amplified to detect ABR17 transcripts. RNA was isolated using the
QIAGEN
RNeasy Plant Mini Kit (Qiagen, Mississauga, ON, Canada) from pooled 2 week-old
seedling
tissue. The isolated RNA was treated with RNase-free DNase (Qiagen) to ensure
the complete
removal of DNA prior to RT-PCR. Reverse transcription and first strand cDNA
synthesis of
total RNA (50 ng) was performed using the iScript cDNA synthesis kit (Bio-Rad,
Hercules, CA,
USA). PCR reactions were carried out using the newly synthesized cDNA (2 L)
as the template
and as the forward primer 5'-GGTGATCAAGGAAGCACAAGG-3' [SEQ ID NO: 5] and as
the
reverse primer 5'-TTTGGCCTTTGTTTCATCACG-3' [SEQ ID NO: 6] specific to the pea
ABR17 coding sequence using a PCR Master Mix (Promega, MD, USA). The ABR1 7
transcript
was amplified using the following thermocycling parameters: 94 C, 2 min; 35
cycles at 94 C, 1
min; 62 C, 1 min; 72 C, 1 min; and a final extension of 72 C, 10 min. Plant
18s rRNA primers,
with the forward being 5'-CCAGGTCCAGACATAGTAAG-3' [SEQ ID NO: 7] and reverse
being 5'-GTACAAAGGGCAGGGACGTA -3' [SEQ ID NO: 8] (Duval et al., 2002), were
used
as an internal controls. PCR products were separated by electrophoresis on a
1.2% agarose gel
and visualized under UV light after staining with ethidium bromide.
The presence of an ABR17-specific transcript was confirmed by this RT-PCR
analysis, which
produced the expected 319 bp amplification product that was absent in the wild
type as well as
the negative control (Figure 1(a)). The quality of mRNA, cDNA and the
amplification reactions
was confirmed by the successful amplification of the expected 18s rRNA product
in all samples
(Figure 1 (a)).
Western blot analysis
Two week-old pooled Arabidopsis seedlings were crushed in liquid N2 and 500 L
extraction
buffer (0.5 M Tris-HCI, pH 6.8; containing 10% glycerol; 10% SDS and 60 mM
DTT) was
added to 200 mg ground tissue. The tubes were vortexed and a small amount of
protamine
sulfate on the tip of a spatula was added and incubated at RT for 15 min prior
to centrifugation at
12 500 g for 15 min. Ice-chilled acetone containing 0.07% DTT (5 equal
volumes) was added to
the supernatants and they were centrifuged once more as described above. The
pellets were
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CA 02589145 2007-05-18
dried for 15 min under vacuum and resuspended in 100 L of 50 mM Tris pH 6.8
containing
0.5% SDS. The concentration of protein in the resuspended samples was
determined using
modified Bradford assay and the samples stored at -20 C until subjected to
Western blot
analysis.
Protein (30 g) was applied to polyacrylamide gels (15%) and subjected to
electrophoresis
according to Laemmli (1970) using a Mini PROTEAN 3 vertical slab system (Bio-
Rad) at
constant 160 V until the dye front reached the bottom of the gel. After
electrophoresis the gel
was equilibrated in transfer buffer (48 mM Tris, pH 9.2 containing 39 mM
glycine, 20%
methanol and 1.3 mM SDS) for 15 min and transferred to polyvinylidene fluoride
(PVDF)
membrane for 25 min at 15 V using Trans Blot SD, semi-dry transfer apparatus
(Bio-Rad).
Following transfer, the membrane was blocked with TBS (10 mM Tris-HCI, pH 7.5;
150 mM
NaCI) containing 5% non-fat skim milk powder for 2 h and rinsed with TTBS (TBS
containing
0.05% Tween 20) for 10 min. The primary antibody solution (rabbit anti-PR 10.4
in TTBS;
1:20,000) was added to the membrane and, after 1 h incubation, the membrane
was washed 3
times for 5 min each with TTBS. The membrane was then incubated with secondary
antibody
solution [goat anti-rabbit immunoglobulin G conjugated with horseradish
peroxidase (Abcam,
Massachusetts, USA) in TTBS; 1:10,000] for 1 h and membrane after which the
membrane was
washed 3 times for 5 min each with TTBS followed by a 5 min wash with TBS.
Bands on the
blot were visualized by staining the membrane with TMB peroxidase substrate
kit (Vector
laboratories Inc., California, USA) according to manufacturer's instructions.
The presence of the ABR17 protein in all the three transgenic lines was
confirmed by Western
blot analysis with PR 10.4-specific polyclonal antibodies. Protein extracts
prepared from the
transgenic plants revealed the presence of a strongly reacting band with the
molecular weight
corresponding to that of ABR17, whereas extracts from the wild type plants did
not show the
presence of this band (Figure 1(b)).
Plant growth and germination experiments
Wild type and transgenic lines (6.9, 14.9, 25.20) of A. thaliana, ecotype WS
seeds were grown
on 1/2 strength MS medium containing 1.5% sucrose, 0.8% agar, pH 5.7 and
various salt
concentrations (0, 75 and 150 mM NaCI) in 25 x 100 mm Petri dishes. Seeds were
surface
23
CA 02589145 2007-05-18
sterilized by immersing in 70% ethanol for 1 min followed by rinsing twice
with sterile
deionized water. The seeds were then placed in a solution of 20% bleach for 15
min with
occasional mixing, after which they were rinsed four times (5 min each) with
sterile deionized
water. At least 5 plates per treatment and 14 seeds per plate were used in
these experiments and
plates were incubated under continuous light either at 22 C or 10 C.
Germination of seeds was
monitored every day and the numbers of germinated seeds were recorded daily
for two weeks.
Those seeds where the radicles had emerged were considered to have germinated.
Each
experiment was repeated at least three times and the results are shown in
Figure 2.
At room temperature, in the absence of NaCl, 100% germination in the wild type
as well as the
three transgenic lines, was observed (data not shown). At the lower
temperature (10 C), in the
absence of NaC1, while the germination in all the lines was not 100%, there
were no significant
differences in the numbers of germinated seeds on any day (Figure 2 (c)).
In the presence of 75 and 150 mM NaCl at both temperatures tested, the
transgenic seedlings
germinated earlier. For instance, the transgenic lines 6.9 and 14.9 initiated
germination by day 1
in presence of 75 mM NaCl whereas no wild type or 25.20 transgenic seeds had
germinated at
this point (Figure 2 (a)). Similarly, in the presence of 150 mM NaCI, all
three transgenic lines
had initiated germination by day 3 whereas no significant germination in the
wild type had
occurred at this time (Figure 2 (b)). In the presence of 75 mM NaCI the
maximum germination
observed at room temperature at the conclusion of the experiment (on day 14)
in the wild type
was -74%, compared to that of 99%, 99%, 90% for the transgenic lines 6.9, 14.9
and 25.20,
respectively (Figure 2 (a)). In the case of 150 mM NaCl, the maximum
germination in the wild
type was 47% compared to 85%, 89% and 60% in the transgenic lines 6.9, 14.9
and 25.20,
respectively.
When the germination of the seeds was assessed at 10 C, in the presence of
either 75 or 150 mM
NaCI, the differences in germination rates of the transgenic seeds were
obvious with the
transgenic lines clearly performing better than the wild type (Figures 2 (d) &
(e)). At 10 C on
day 5, in the presence of 75 mM NaCl, all three transgenic lines had
significantly higher
germination rate compared to wild type, with the transgenic line 6.9 appearing
to be the best
among the transgenics (Figure 2 (d)). The trend remained the same throughout
the entire period
24
CA 02589145 2007-05-18
of the experiment (14 days) for 75 mM NaC1 treatments. In the presence of 75
mM NaC1 and
C, at the end of the 14-day experiment, 70% of the wild type seeds had
germinated, whereas
the germination rates were 94%, 94% and 86% in the three transgenic lines 6.9,
14.9 and 25.20,
respectively (Figure 2 (d)). In the presence of 150 mM NaCI, the difference
between the wild
5 type and the three transgenics was apparent on day 6, and was very clear on
day 8 (Figure 2 (e)).
At this salt concentration, only 17% of the wild type seeds had germinated by
day 14, whereas
the germination rate was 83%, 67% and 50% in the three transgenic lines 6.9,
14.9 and 25.20,
respectively. Once again, the transgenic line 6.9 appeared to be better than
the other two lines
(Figure 2 (e)). All of the observed differences between the germination rates
of the transgenic
10 and wild type seeds were statistically significant (P < 0.05).
Appearance of seedlings germinated and grown in the presence of NaCI
The appearance of the wild type and transgenic seedlings grown on MS plates
containing 0, 75
or 150 mM NaCI at room temperature, after one and two weeks, is shown in
Figures 3 (a) & (b),
respectively. There are no obvious differences between the wild type and 3
transgenic lines in
the absence of salt after one week of growth (Figure 3 (a)). However, after 2
weeks, in the
absence of salt, it appears as though both roots and shoots of the transgenic
seedlings are better
developed compared to the wild type (Figure 3 (b)). As previously described
for the germination
experiments, the differences between the wild type and transgenic seedlings
were more apparent
in the presence of NaC1 (Figure 3), with the deleterious effects on root and
shoot development
being more pronounced at the higher (150 mM) NaCI concentration. It is clear
from the
appearance of the seedlings (Figure 3) that the deleterious effects of 150 mM
NaC1 at both 1 and
2 weeks in the transgenic lines are considerably less compared to those on the
wild type
seedlings. These observations were confirmed by evaluating the effect of the
salinity treatments
on the chlorophyll and carotenoids levels, which were measured in 2 week-old
control as well as
75 mM NaCI treated tissues.
Measurement of chlorophyll, carotenoid and ion-leakage
The chlorophyll and carotenoid content as well as ion-leakage were used to
assess plant damage
due to the stresses. Total chlorophyll and carotenoid was extracted from
pooled tissue of 2
week-old plants grown on MS plates, using a procedure modified from Kirk and
Allen (1965).
CA 02589145 2007-05-18
Pooled leaf tissue (0.05 g) was homogenized in 5 mL of ice-cold 80% acetone
and incubated at
room temperature for 10 min in the dark. Samples were then centrifuged at 2
500 g for 15 min.
The supernatant was decanted and absorbance measured at 663, 645 and 480 nm.
Total
chlorophyll (a+b) was estimated using a nomogram (Kirk 1968) and the
carotenoid levels were
calculated using the formula (Kirk and Allen, 1965):
DACAR480 =AAas0 + 0.114 DA663 - 0.638 DA645 where, A is absorbance and CAR is
carotenoid
content.
Membrane damage was assessed by measuring ion-leakage from cold-stressed
leaves
(Vettakkorumakankav et al., 1999). Leaves from control and stressed plants
were incubated in
20 mL of distilled water and agitated at room temperature for an hour
following which the
conductivity of the solution (initial value) was determined using a
conductivity meter (HI 8733,
Hanna Instrument, Rhode Island, USA). The solutions were incubated at 4 C
overnight and
subsequently autoclaved to release total ions and the final conductivity
values were determined.
Ion leakage is expressed as a percentage of the initial to final values.
The chlorophyll levels were significantly (P < 0.05) reduced in the wild type
seedlings whereas
the levels in the transgenics grown in 75 mM NaC1 were not significantly
different from the
untreated samples (Figure 3 (c)). The effect of 75 mM NaCl was more pronounced
on the
carotenoid levels of the wild type seedlings, with the salt treatment reducing
the carotenoid
levels significantly (P < 0.05) compared to the transgenics (Figure 3 (d)). It
is also apparent that
the carotenoid levels in the untreated transgenic seedlings were higher
compared to that of the
untreated wild type seedlings (Figure 3 (d)). These elevated levels of
carotenoids may be
contributing to the better tolerance to NaCI exhibited by the transgenic
lines.
Appearance of seedlings germinated and grown at 10 C
The appearance of seedlings after two and three weeks at 10 C in the presence
or absence of
NaCI is shown in Figure 4. It is evident that at this lower temperature, in
the presence or absence
of NaCl, the transgenic seedlings appear to be healthier than the wild type
after 2 weeks (Figure
4 (a)) and 3 weeks (Figure 4 (b)) of growth, suggesting that they are able to
better tolerate the
lower temperature stress. Therefore, the appearance of the seedlings also
support the data
26
CA 02589145 2007-05-18
obtained from the experiments that assessed the abilities of the transgenic
lines to germinate
under these stress conditions.
Imposition of stresses
Wild-type and transgenic Arabidopsis lines (6.9, 14.9 and 25.20) were
evaluated for tolerance to
cold and high temperature stresses as described by Kim et al. (2004). For the
imposition of the
cold stress open MS plates containing the plants were placed at -5 C (freezing
temperature) for 4
h after which the plates were returned to room temperature and photographed
after 24 h of
recovery. In the case of heat stress, 2 week-old plants in closed MS plates
were placed in an
incubator set at 48 C for 2 h after which they were returned to room
temperature and
photographed after 1 and 3 days of recovery.
The appearance of plants grown on MS plates 24 h after a 4 h treatment at -5 C
is shown in
Figure 5. It is apparent that almost all the wild type plants collapsed,
whereas the transgenic
lines appeared healthier indicating that these lines were more tolerant to the
imposed stress
(Figure 5). In order to confirm that this was indeed the case, the percent ion
leakage in the wild
type and transgenic seedlings after 4 h of stress and 4 h of incubation at 4
C, as well as after the
24 h recovery at room temperature (Table 1), was determined.
Table 1 Effects of cold stress on ion leakage, chlorophyll and carotenoid
content.
27
CA 02589145 2007-05-18
Percentage Ion leakage
Cold stress -5 C for 4 h and 4 h incubation at 4 C
WT 61.69 3.32
6.9 37.17 3.66
14.9 47.22 1.49
25.20 44.84 1.17
Percentage Ion leakage
1 day recovery after cold stress -5 C for 4 h
VVT 51.36 4.17
6.9 38.26 4.41
14.9 44.57 6.94
25.20 38.31 7.40
Chlorophyll (pg/g FW)
1 day recovery after cold stress -5 C for 4 h
WT 72.45 1.02
6.9 98.47 18.93
14.9 81.83 4.29
25.20 74.41 15.64
Carotenoid (pg/g FW)
1 day recovery after cold stress -5 C for 4 h
WT 2.50 0.05
6.9 2.95 0.56
14.9 2.68 0.16
25.20 2.37 0.56
Table 1 indicates that the percent ion leakage, which is indicative of
membrane damage, after the
stress and 4 C incubation, was significantly less in all three transgenic
lines compared to the
wild type, even though it appears as if line 6.9 suffered the least amount of
membrane damage
when compared to the other two transgenic lines. These observations are also
supported by the
ion leakage observed after the 24 h recovery period where, once again, it was
observed that line
6.9 had the least amount of membrane damage. Even though the appearance, as
well as the ion
leakage data, indicated that the transgenic plants were significantly more
tolerant to the cold
stress, there were no significant differences in the chlorophyll or
carotenoids content when
measured after the stress as well as after the recovery period (Table 1).
The enhanced germination of the transgenic lines in the presence of NaCI and
the increased
tolerance of these lines to cold temperature stress prompted testing of
whether the ABR1 7
transgenic plants would exhibit enhanced tolerance to heat stress. The
appearance of these plants
after the one-day recovery period is shown in Figure 6. It is evident that all
three transgenic lines
28
CA 02589145 2007-05-18
appeared healthier compared to the wild type after one day of recovery
suggesting that ABR1 7
expression could confer multiple stress tolerance.
Two-dimensional electrophoresis
In order to probe the physiological basis for the observed enhancement of
stress tolerance in
transgenic A. thaliana seedlings expressing the pea ABR17 gene we performed
two-dimensional
electrophoresis to compare proteome-level changes brought about by the
transgene expression.
Protein extracts for two-dimensional electrophoresis were prepared according
to the method
described by Subramanian et al. (2005) with some modifications. Wild-type and
transgenic
seedlings (two week-old) from MS plates were homogenized to a fine powder in
liquid nitrogen.
Homogenized tissue (0.3 g) was further homogenized in acetone containing 10%
(w/v) TCA and
0.07% DTT, transferred to eppendorf tubes and the volume was adjusted to 1.5
mL with acetone
containing 10% (w/v) TCA and 0.07% DTT. Samples were incubated at -17 C for 1
h,
centrifuged at 13 000 g for 15 min and the supernatants discarded. The pellets
were washed by
resuspending them in ice-cold acetone containing 0.07% DTT and centrifuged as
described
above. This was step was repeated four additional times, the pellets were
dried at room
temperature in a speed vac for 30 min and resuspended in 400 L
Rehydration/Sample buffer
(Bio-Rad, Ontario, Canada) containing 8 M urea, 2% w/v CHAPS, 40 mM DTT, 0.2%
Bio-lyte
3-10 and 3 L of 200 mM tributylphosphine (TBP). The samples were mixed
vigorously,
incubated overnight at 4 C and centrifuged at 4 C for 15 min at 13 000 g. The
supernatants were
placed in fresh tubes and protein concentrations determined using a modified
Bradford assay
(Bio-Rad) using BSA as standard.
Two-dimensional electrophoresis of protein extracts was performed as
previously described
(Subramanian et al., 2005). Briefly, immobilized pH gradient (IPG) strips (17
cm, Bio-Rad)
were passively rehydrated overnight with 300 g of protein in 300 L of
Rehydration buffer (8
M urea, 2% CHAPS, 40 mM DTT, 0.2% Bio-Lyte and 2 mM TBP). Isoelectric focusing
(IEF)
was performed using a Bio-Rad PROTEAN IEF unit to provide an optimum, maximum
field
strength of 600 V/cm and a 50 A limit/IPG strip at 10000 V for 60000 VH.
Prior to second
dimension separation, proteins in the rehydrated strips were reduced by
incubating them twice in
a solution (5mL/ strip) containing 6 M urea, 2% sodium dodecyl sulfate (SDS),
0.375 M Tris-
29
CA 02589145 2007-05-18
HCI, pH 8.8, 20% glycerol, and 130 mM DTT for 10 min each. The strips were
then incubated
in the above solution containing 135 mM iodoacetamide instead of DTT twice for
10 min each in
order to alkylate the reduced proteins. SDS-PAGE separation of proteins was
performed on 13%
polyacrylamide gels (20 X 20 cm, 1 mm thickness) using a PROTEAN II XI system
(Bio-Rad) at
constant voltage (90 V) until the dye front reached the bottom of the gel.
Protein spots were
visualised using a Colloidal Blue Staining Kit (Invitrogen, California, USA)
according to the
instructions provided. Two-dimensional electrophoresis was performed at least
three times with
the extracted protein samples.
Image analysis
Images of the two-dimensional gels were acquired using a GS-800 calibrated
densitometer (Bio-
rad) and analyzed using the PDQuest software (Bio-rad). Three gels each of
wild type and
transgenic (6.9) samples were used to generate the match-sets and individual
spots were matched
using automated detection and matching feature of the software followed by
manual refinements
in order to eliminate artifacts and include spots that were missed by the
automated detection
process. In order to identify the protein spots whose levels were
significantly different between
the transgenic and wild type seedlings, the match sets from 3 replicate gels
of wild type and 6.9
samples were analyzed using Student's t-test feature of PDQuest software as
described by the
manufacturer. Those spots which were reproducibly altered in all three
replicates and exhibited
significant (P<0.01) difference were excised from the gels using sterile
scalpels and subjected to
ESI-Q-TOF-MS/MS analysis.
ESI-Q-TOF MS/MS analysis
Tandem MS was performed at the Institute for Biomolecular Design, University
of Alberta, on
protein extracted from isolated gel spots as previously described (Subramanian
et al., 2005).
Briefly, gel pieces were de-stained, reduced with 10 mM DTT, alkylated with 55
mM
iodoacetamide and digested with 6 ng/ l trypsin (Promega Sequencing Grade
Modified) in 50
mM ammonium bicarbonate (25 l), for 5 h at 37 C in a fully automated fashion
on a Mass Prep
Station (Micromass, Manchester, UK). The tryptic peptides were subjected to
LC/MS/MS
analysis on a Micromass Q-ToF-2 mass spectrometer (Micromass) coupled with a
Waters
CapLC capillary HPLC (Waters Corp., USA). Peptides were separated on a
PicoFrit capillary
CA 02589145 2007-05-18
reversed-phase column (5 BioBasic C18, 300 Angstrom pore size, 75 ID x 10
cm, 15 tip
(New Objectives, MA, USA), using a linear water/acetonitrile gradient (0.2%
Formic acid), after
desalting on a 300 x 5 mm PepMap C18 column (LC Packings, California, USA).
Eluent was
introduced directly to the mass spectrometer by electrospray ionization at the
tip of the capillary
column and data dependent MS/MS acquisition was performed for peptides with a
charge state
of 2 or 3. Proteins were identified from the MS/MS data by searching the NCBI
non-redundant
database with Mascot Daemon (Matrix Science, UK) including
carbamidomethylation of
cysteine, possible oxidation of methionine and one missed cleavage per peptide
as search
parameters.
Representative images of two-dimensional gels obtained from wild type and
transgenic protein
extracts are shown in Figure 7(a). The intensities of 24 protein spots (Figure
7(b)) were
observed to be significantly (P<0.01) and reproducibly altered in transgenic
line (6.9) compared
to the wild type. Out of these 24 protein spots, the intensities of 7 were
greater, 15 were lower
and two spots were unique in the transgenic line. The identities of these 24
spots were
established using tandem MS and are presented in Table 2.
Table 2 Details of proteins identified by ESI-Q-TOF MS/MS
Spot Protein identity MS/MS ESI-Q-ToF ' Access. No. Mr/pI Fold
No. a score Sequence change
PM
%a
1 DRT112 14% 88 NNAGYPHNVVFDEDEIPSGVDVAK gi1166696 17089/ 3.49
[Arabidopsis (>48) 5.06 0.57
thaliana] T
2 Putative 23% 153 AAEDPAPASSSSK gil24030202 14756/ 18.17
photosystem I (>49) RESYWFK 9.92 10.23
subunit PSI-E NVGSVVAVDQDPK T
protein
[Arabidopsis
thaliana]
3 GDCH 15% 147 FCEEEDAAH gi115226973 18050/ 0.14
[Arabidopsis (>49) VKPSSPAELESLMGPK 5.24 0.13
thaliana]
4 Unknown protein 7% 85 VEVTEAEVELGFK gi118422918 17603/ 0.68
[Arabidopsis (>42) 4.80 0.07
thaliana]
5 Basl protein 27% 140 YVILFFYPLDFTFVCPTEITAFSDR giJ861010 23398/ 0.47
[Hordeum vulgare] (>48) SGGLGDLKYPLVSDVTK 5.48 0.02
EGVIQHSTINNLGIGR
[
31
CA 02589145 2007-05-18
6 LHB1B2; 59% 483 GPSGSPWYGSDR gi118403546 28093/ 0.42
chlorophyll (>49) YLGPFSGEPPSYLTGEFPGDYGWDT 5.28 0.11
binding AGLSADPETFAR J
[Arabidopsis WAMLGALGCVFPELLAR
thaliana] FGEAVWFK
LAMFSMFGFFVQAIVTGK
GPLENLADHLADPVNNNAWAFAT
NFVPGK
YLGPFSGEPPSYLTGEFPGDYGWDT
AGLSADPETFAR
LHB1B1; 60% 445 ASKPTGPSGSPWYGSDR giI18403549 28209/
chlorophyll (>49) YLGPFSGEPPSYLTGEFPGDYGWDT 5.15
binding AGLSADPETFAR
[Arabidopsis WAMLGALGCVFPELLAR
thaliana] FGEAVWFK
VAGDGPLGEAEDLLYPGGSFDPLGL
ATDPEAFAELK
LAMFSMFGFFVQAIVTGK
GPLENLADHLADPVNNNAWAFAT
NFVPGK
7 Cp29 27% 499 VNAGPPPPK giJ681904 34602/ 0.86
[Arabidopsis (>49) SSYGSGSGSGSGSGSGNR 5.23 0.05
thaliana] LYVGNLSWGVDDMALENLFNEQG J
K
GFGFVTLSSSQEVQK
AINSLNGADLDGR
VSEAEARPPR
Inorganic 22% 326 VQEEGPAESLDYR gi115242465 33644/
diphosphatase/ (>49) VFFLDGSGK 5.71
magnesium ion VSPWHDIPLTLGDGVFNFIVEIPK
binding / IVAISLDDPK
pyrophosphatase HFPGTLTAIR
[Arabidopsis IPDGKPANR
thaliana]
8 Putative aspartyl 9% 177 GDLASESILLGDTK gi112324588 47745/ 0.47
protease (>49) SSVSLVSQTLK 6.06 0.10
[Arabidopsis V IYDTTQER
thaliana] LGIVGENCR
ribosomal protein 8% 151 VSDIATVLQPGDTLK giI18060 45044/
S1 [Spinacia (>49) AEEMAQTFR 5.41
oleracea] IAQAEAMAR
9 Ribose-5- 53% 727 AVEAIKPGMVLGLGTGSTAAFAVD gi115229349 29401/ 1.46
phosphate (>49) QIGK 5.72 0.09
isomerase LLSSGELYDIVGIPTSK
[Arabidopsis SLGIPLVGLDTHPR
thaliana] IDLAIDGADEVDPNLDLVK
EKMVEAVADK
MVEAVADK
FIV V ADDTK
V DGDG KPYV TDNS NYI IDLYFK
FQGV V EHGLFLGMATS V IIAGK
NGVEVMTK
32
CA 02589145 2007-05-18
Chlorophyll a/b 67% 516 GPSGSPWYGSDR gi116374 25036/ 0.46
binding protein (>49) YLGPFSGESPSYLTGEFPGDYGWDT 5.12 0.02
(LHCP AB 180) AGLSADPETFAR 1
[Arabidopsis WAMLGALGCVFPELLAR
thaliana] FGEAVWFK
VAGNGPLGEAEDLLYPGGSFDPLGL
ATDPEAFAELK
LAMFSMFGFFVQAIVTGK
GPIENLADHLADPV NNNAW AFATN
FVPGK
LHBIB2; 31% 376 GPSGSPWYGSDR gi118403546 28093/
chlorophyll (>49) WAMLGALGCVFPELLAR 5.28
binding FGEAVWFK
[Arabidopsis LAMFSMFGFFVQAIVTGK
thaliana] GPLENLADHLADPVNNNAWAFAT
NFVPGK
11 2-cys 31% 334 AQADDLPLVGNK giJ9758409 29714/ 0.8
peroxiredoxin-like (>41) APDFEAEAVFDQEFIK 5.55 0.04
protein LNTEVLGVSVDSVFSHLAWVQTDR [
[Arabidopsis SGGLGDLNYPLVSDITK
thaliana] EGVIQHSTINNLGIGR
12 PSBO-2/PSBO2; 31% 451 RLTYDEIQSK gi115230324 35226/ 2.92
oxygen evolving (>42) GTGTANQCPTIDGGSETFSFK 5.92 0.30
[Arabidopsis FCFEPTSFTV K T
thaliana] VPFLFTV K
GGSTGYDNAVALPAGGR
NTAASVGEITLK
SKPETGEVIGVFESLQPSDTDLGAK
33 kDa oxygen- 31% 430 RLTYDEIQSK giJ22571 35285/
evolving protein (>42) FCFEPTSFTVK 5.68
[Arabidopsis NAPPEFQNTK
thaliana] VPFLFTV K
GGSTGYDNAVALPAGGR
GDEEELVKENVK
NTAASVGEITLK
SKPETGEVIGVFESLQPSDTDLGAK
13 Chlorophyll a/b 59% 393 GPSGSPWYGSDR gi116374 25036/ 0.29
binding protein (>41) YLGPFSGESPSYLTGEFPGDYGWDT 5.12 0.03
(LHCP AB 180) AGLSADPETFAR
[Arabidopsis WAMLGALGCVFPELLAR
thalianal FGEAVWFK
VAGNGPLGEAEDLLYPGGSFDPLGL
ATDPEAFAELK
GPIENLADHLADP V NNNA W AFATN
FVPGK
LHB1B2; 24% 259 GPSGSPWYGSDR gi118403546 28093/
chlorophyll (>41) WAMLGALGCVFPELLAR 5.28
binding FGEAVWFK
[Arabidopsis GPLENLADHLADPVNNNAWAFAT
thaliana] NFVPGK
33
CA 02589145 2007-05-18
14 Phosphopyruvate 20% 416 SAVPSGASTGIYEALELR giI15221107 51841/ 0.41
hydratase (>42) NQADVDALMLELDGTPNK 5.79 0.13
[Arabidopsis IGMDVAASEFFMK I
thaliana] AAGWGVMVSHR
SGETEDNFIADLSVGLASGQIK
IEEELGNVR
YAGEAFR
15 TUA3 16% 363 QLFHPEQLISGK gi115241168 50250/ 0.66
[Arabidopsis (>41) EDAANNFAR 4.95 0.06
thaliana] SLDIERPTYTNLNR [
LISQIISSLTTSLR
IHFMLSSYAPVISAAK
DVNAAVGTIK
16 ATGRP8 44% 515 CFVGGLAWATNDEDLQR gi115235002 16626/ 2.23 t
(GLYCINE-RICH (>41) TFSQFGDVIDSK 5.58 0.02
PROTEIN 8); GFGFVTFK
RNA binding / GFGFVTFKDEK
nucleic acid VITVNEAQSR
binding SGGGGGYSGGGGGGYSGGGGGGY
[Arabidopsis ER
thaliana]
17 Glycine-rich RNA 53% 581 CFVGGLAWATDDR giJ21553354 16934/ 1.73
binding protein 7 (>42) ALETAFAQYGDVIDSK 5.85 0.15
[Arabidopsis GFGFVTFK
thaliana] SGGGGGYSGGGGSYGGGGGR
EGGGGYGGGEGGGYGGSGGGGGW
18 AT4g38970/F19H 36% 650 LDSIGLENTEANR gi116226653 43029/ 0.65
22_70 (>41) TLLVSAPGLGQYVSGAILFEETLYQ 6.79 0.02
[Arabidopsis STTEGKK I
thaliana] MVDVLVEQNIVPGIK
TAAYYQQGAR
TVVSIPNGPSALAVK
YAAISQDSGLVPIVEPEILLDGEHDI
DR
ATPEQVAAYTLK
ALQNTCLK
YTGEGESEEAK
19 RCA (RUBISCO 22% 620 LVVHITK gi118405145 52347/ 2.22
ACTIVASE) (>42) VPLILGIWGGK 5.87 0.37
[Arabidopsis SFQCELVMAK T
thaliana] SFQCELVMAK
MCCLFINDLDAGAGR
IKDEDIVTLV DQFPGQSIDFFGALR
LMEYGNMLVMEQENVK
VQLAETYLSQAALGDANADAIGR
20 Glutathione S- 24% 288 QEAHLALNPFGQIPALEDGDLTLFE giJ20197312 24119/ 0.55
transferase (GST6) (>41) SR 6.09 0.02
[Arabidopsis GMFGMTTDPAAVQELEGK [
thaliana] VLFDSRPK
21 Unknown protein 10% 169 LSVIVAPVLR giJ2829916 29986/ 0.44
[Arabidopsis (>41) FADNLGDDVK 6.40 0.06
thaliana] IENIGQPAK J
22 Unknown protein 13% 257 SAVADNDNGESQVSDVR gil6437556 31235/ 0.67
[Arabidopsis (>41) GKDPIVSGIEDK 5.91 0.03
thaliana] LSTWTFLPK
34
CA 02589145 2007-05-18
23 ABA-responsive 48% 487 GVFVFDDEYVSTVAPPK giJ20631 16619/ Unique
protein (>41) LDAVDEANFGYNYSLVGGPGLHES 5.07
[Pisum sativum] LEK
VAFETIILAGSDGGSIVK
GDAALSDAVR
GDAALSDAVRDETK
24 ABA-responsive 39% 250 DADEIVPK giJ20631 16619/ Unique
protein (>41) EAQGVEIIEGNGGPGTIK 5.07
[Pisum sativum] LSILEDGK
VAFETIILAGSDGGSIVK
GDAALSDAVR
a Percentage identity between the amino-acids present in MS/MS tag and the
sequences in
databases.
b Ion score is -10 Log (P), where P is the probability that the observed match
is a random event.
Individual ion scores > value indicate identity or extensive homology
(p<0.05).
Accession number is Mascot search result using NCBI and other databases.
In all cases the Mascot searches of the NCBI non-redundant database generated
significant hits
with scores above the threshold value and in most cases these were a result of
multiple peptide
matches (Table 2). These scores are based on individual ion scores where the
ion score is -
10*log (P) and P is the probability that the observed match is a random event.
A score above the
threshold value indicates sequence identity or extensive homology (P<0.05).
Among the proteins that were identified in this study, two were unique and the
identities of both
were established as pea ABR17 (Figure 7; Table 2). Although only one protein
corresponding to
ABR17 was expected, the fact that there are two suggests the possibility that
the additional spot
may be the result of post-translational modification. Most of the proteins
identified in this study
were associated with photosynthesis and primary metabolic pathways including
putative
photosystem I subunit PSI-E protein, chlorophyll binding proteins, Cp29, PSBO-
2/PSBO2;
oxygen evolving protein, Ribose-5-phosphate isomerase, phosphopyruvate
hydratase and
RUBISCO activase. Other interesting proteins identified in this study were
DRT112, which is
thought to be involved in DNA damage repair and glycine-rich proteins which
are involved in
post-transcriptional gene regulation.
Statistical analysis
CA 02589145 2007-05-18
All analyses were performed using the mixed model procedure of SAS version 8e
(Statistical
Analysis System, 1985). Mixed model methodology was employed to perform
analysis of
variance and to estimate least square means and standard errors of genotype.
10
EXAMPLE 2: Brassica napus (Canola)
Constitutive expression of ABR17 cDNA enhances germination under abiotic
stress
conditions and promotes early flowering in canola (Brassica napus)
The effect of constitutive expression of Pisum sativum ABA-responsive 17 (ABR1
7/ PR 10.4)
cDNA on DH (doubled haploid) canola (Brassica napus) line was tested to
determine its effects
on germination as well as other characteristics. We observed increased
germination in the
transgenic line in salt stress (275 mM NaCI), cold stress (5 C), and when both
the stress
conditions were combined (10 C + 75 mM NaCI) as compared to the wild type
("WT"). In
addition, we observed a greater rate of flowering, earlier flowering and
greater height in the
transgenic line when compared to the wild type at 42 days after planting.
These results
demonstrate that (1) ABR17 enhances germination under saline and cold
conditions in canola
and (2) ABR17 promotes germination and early flowering in canola when compared
to the wild
type. Results from this study demonstrate the utility of engineering enhanced
germination under
36
CA 02589145 2007-05-18
abiotic stress conditions and early flowering in a crop species. Plants with
an ability to be seeded
earlier, to germinate quicker, germinate in marginal soils and to flower
faster will ultimately
benefit agricultural production.
1. Transformation of Brassica napus
Pea ABR17 cDNA (provided by Dr. Trevor Wang, Department of Metabolic Biology,
John Innes Centre, UK) was amplified and inserted into the pKLYX-71 plant
expression vector
and used to transform Agrobacterium tumefaciens as described by Srivastava et
al. 2006. B.
napus was transformed using the procedure described by Moloney et al. (1998).
Embryos from
Tl seeds of successful transformants were screened on kanamycin plates (50
.g/ml) and those
with a 3:1 segregation ratio were used to raise homozygous T2 lines and
subsequent seed
production. The segregation ratio was determined based on the number of green
to bleached
embryos. Twelve independent transgenic lines contained the ABR17 construct;
from these
parental lines (Tl) 18 daughter lines (T2) were selected for further
screening. These 18
transgenic lines were tested for their abilities to germinate on half strength
Murashige-Skoog
plates ("MS plates") (Murashige and Skoog 1962) solidified with 0.8% agarose,
supplemented
with 1.5% sucrose, pH 5.7, and containing 250 or 275 mM NaCl. Those with
enhanced
germination relative to their WT were selected for further screening.
2. Plant growth and germination
Seeds (WT and transgenic) were surface sterilized by soaking in 70% ethanol
for 1 minute and in
20% bleach for 20 minutes after which they were rinsed four times (5 minutes
each) with sterile
deionized water. Surface sterilized seeds from WT and transgenic lines (3.15
and 9.5) of DH B.
napus were placed on Whatman filter paper moistened with 5 ml of sterilized
deionized water in
Petri plates and germinated in complete darkness at room temperature, that
being 21 2 C
("RT"), in order to compare the appearance of these seedlings. For evaluating
the ability of these
genotypes to germinate in the presence of NaCl, lower temperatures or both,
seeds were
germinated and grown on half strength MS plates solidified with 0.8% agarose,
supplemented
with 1.5% sucrose, pH 5.7. Seeds were germinated on these MS plates in the
presence or
absence of 275 mM NaC1 in order to assess the effects of NaCl on germination
of these lines; to
investigate the effects of a combination of NaCl and lower temperature
stresses, plates
37
CA 02589145 2007-05-18
containing either 0 or 75 mM NaC1 were placed at 5 C or 10 C in the presence
of light. For all
germination experiments, seed germination was recorded daily for 7 days (at
RT) or for 14 days
(at 5 C and 10 C). Seeds were considered to have germinated if radicle
emergence had
occurred. In all experiments at least 5 plates per treatment and 5 seeds per
plate were used and
each experiment was repeated at least three times.
Seeds from the WT and transgenic lines (3.15 and 9.5) were germinated and
grown under
greenhouse conditions (22 C day/18 C night; 16 h photoperiod) for root (14
days) and whole
plant observations (37 days or full maturity). For root observations, seeds
were planted in
Turface AthleticsTM (100% calcined clay; Profile Products LLC, 111, USA) and
for whole plant
observations, seeds were planted in Metro Mix O 290 (vermiculite and peat
moss; Grace
Horticultural Products, ON, Canada) and fertilized once every 2 weeks with 200
ppm Peters
20-20-20. All observations were made in at least two different batches of
plants planted
independently and each batch consisted of at least 5 plants.
3. Expression analysis
(a) Reverse Transcription (RT)-PCR analysis
Total plant RNA was isolated (QIAGEN RNeasy Plant Mini Kit, Qiagen,
Mississauga, ON,
Canada) from pooled 5 week-old leaf tissue from WT and transgenic plants which
were flash-
frozen in liquid nitrogen. To ensure the complete removal of DNA prior to RT-
PCR an RNase-
free DNase treatment of the RNA samples was performed as per manufacturer's
instructions.
cDNA synthesis was performed using the iScript cDNA synthesis kit (Bio-Rad,
Hercules, CA,
USA) and total RNA (50 ng) as recommended by the manufacturer. PCR reactions
were carried
out with 2 l cDNA as the template and pea ABR17 using specific forward primer
sequence
[SEQ ID NO 3] 5'-GTGGTCGAAGCTTATGGGTGTCTTTGTTT"TTGATGATGAATAC-3'
and reverse primer sequence [SEQ ID NO 4]: 5'-
TATATAGCTCGAGTTAGTAACCAGGATTTGCCAAAACGTAACC-3'using a PCR Master
Mix (Promega, MD, USA) under the following thermocycling parameters: 94 C, 2
min; 35
cycles for 1 min, 94 C; 1 min, 62 C; 1 min, 72 C; and an extension for 10
min, 72 C. Plant
18s rRNA was used in these experiments as internal control using forward
primer sequence: [SEQ
38
CA 02589145 2007-05-18
ID NO 7]: 5' CCAGGTCCAGACATAGTAAG-3'and reverse primer sequence [SEQ ID NO
8]:5'-GTACAAAGGGCAGGGACGTA -3', these primers being specific for this gene
(Duval et
al. 2002). Amplification products were separated on a 1% agarose gel and
visualized under UV
light after staining with ethidium bromide.
(b) Quantitative Real-Time-PCR (qRT-PCR) analysis
Quantitative real-time polymerase chain reaction (qRT-PCR) was performed to
investigate the
relative levels of expression of ABR17 using the expression of actin gene as
the internal control.
The sequences of the ABR17-specific primers and probes were as follows. The
forward primer
sequence was 5'-AAATGGAGGTCCAGGAACCAT-3' [SEQ ID NO 9] and the reverse primer
sequence was 5'- AGCACATAGTTGGTTTTTCCATCTT-3' [SEQ ID NO 10]. The probe
sequence was 5'-AGAAGCTATCCATTCTT-3' [SEQ ID NO 11]. For the amplification of
the
actin gene, the following primers and probes were used. The forward primer
sequence was 5'-
GCCATTCAGGCCGTTCTTT-3' [SEQ ID NO 12] and the reverse primer sequence was 5'-
ATCGAGCACAATACCGGTTGT-3' SEQ ID NO 13]. The probe sequence was 5'-
TCTATGCCAGTGGTCG-3' [SEQ ID NO 14].
qRT-PCR primer sets were designed using Primer Premier software (Applied
Biosystems Inc.,
CA, USA) to generate amplification products that were approximately 70-80 bp
in size. RNA
isolation and cDNA preparation was performed as described above. PCR reactions
contained 2
l of cDNA (5x dilution), 5 pmol of probe, 22.5 pmol of each primer and lx
TaqMan
UniversPCR Master Mix (Roche, NJ, USA). The SNP RT template program was used
for real-
time quantification and was performed in an ABI prism 7700 Sequence detector
(Applied
Biosystems). The delta-delta method was used to determine relative expression
using the
following formula: Relative Expression = 2-[ect sample - ACt conaol], where Ct
is the threshold cycle
(Livak and Schmittgen 2001). The relative expression of ABR17 in the
transgenic lines was
normalized against expression in the WT which was considered to be 1. The
experiment was
repeated at least three times.
(c)Western blot analysis
39
CA 02589145 2007-05-18
Pooled two week-old B. napus seedlings (0.2 g) were crushed in liquid N2 and
500 l extraction
buffer (0.5 M Tris-HCI, pH 6.8; containing 10% glycerol; 10% SDS and 60 mM
DTT) was
added. The tubes were vortexed and then boiled for 5 minutes in a water bath.
After cooling, a
few crystals of protamine sulfate were added and the tubes were incubated at
RT for 15 min and
then centrifuged at 12,500g for 15 min. Ice-cold acetone containing 0.07% DTT
was added to
the supernatant and the tubes were centrifuged as described above. The pellets
were vacuum
dried for 15 min and resuspended in 150 1 of 50 mM Tris pH 6.8 containing
0.5% SDS and
centrifuged a final time as above. The concentration of protein in the samples
was determined
using a modified Bradford assay and the samples stored at -20 C.
Proteins (25 g) were separated by SDS-polyacrylamide gel (15%)
electrophoresis (Laemmli
1970) using a vertical slab system (Mini PROTEAN 3, Bio-Rad) at constant 150 V
until the dye
front reached the bottom of the gel. After electrophoresis, the gel was placed
in transfer buffer
(48 mM Tris, pH 9.2 containing 39 mM glycine, 20% methanol and 1.3 mM SDS) for
15 min
and subsequently transferred to polyvinylidene difluoride (PVDF) membrane for
25 min at 15 V
using Trans Blot SD, semi-dry transfer apparatus (Bio-Rad). The PVDF membrane
was
incubated with TBS (10 mM Tris-HC1, pH 7.5; 150 mM NaCI) containing 5% non-fat
skim milk
powder. The membrane was subsequently blocked for 2 hours and then rinsed with
TTBS (TBS
containing 0.05% Tween 20) for 10 min. The membrane was then incubated for one
hour in the
primary antibody solution (rabbit anti-ABR17; Srivastava et al. 2006) diluted
1:20 000 in TTBS.
The membrane was washed 3 times for 5 minutes each with TTBS after which the
membrane
was incubated for 1 hour in with the secondary antibody (goat anti-rabbit
immunoglobulin G-
horseradish peroxidase conjugate (Abcam, MASS, USA) which was diluted 1:10 000
in TTBS.
After this incubation, the membrane was washed as described earlier, followed
by a 5 minutes
final wash with TBS. Immunoreactive bands on the membrane were visualized by
staining with
TMB peroxidase substrate kit (Vector Laboratories Inc., CA, USA) according to
manufacturer's
instructions.
4. Statistical analysis
For statistical analysis, analysis of variance was conducted by using proc
mixed SAS (version 8e,
Statistical Analysis System 1985) where genotype was considered as fixed
effect and replicate
CA 02589145 2007-05-18
was considered as random effect for all plant characteristics (germination,
root and shoot length,
height and flowering). The values presented on the graphs are based on the LS
means estimates.
Since the transgene had a significant effect on plant height and flowering we
hypothesized that
the early flowering observed in the transgenic plants might be due to the
effect of height. In
order to test this hypothesis we conducted an analysis of covariance on
flowering as dependent
variable with proc mixed (SAS) where genotype was considered as fixed effect,
replicate was
considered as random effect and height was considered a covariant.
Results
1. Generation of transgenic Brassica napus and confirmation of gene expression
Transgenic DH B. napus plants containing one copy of ABR17 were chosen based
on their
segregation ratios on plates containing kanamycin. Seeds from 18 transgenic
lines containing a
single copy of the cDNA were screened on half strength MS media containing 250
mM NaC1.
Two lines, showing the best relative germination (lines 3.15 and 9.5), were
selected for further
studies and homozygous T2 seeds were used for all experiments. Transgene
expression was
confirmed using reverse transcriptase-polymerase chain reaction (RT-PCR) with
pea ABR17-
specific primers with 18s rRNA gene as the internal control. Figure 8A shows
analysis of ABR1 7
and 18S RNA expression by RT-PCR. As shown in Figure 8A, the 417 bp
amplification product
of ABR17 verified expression in the transgenic lines (3.15 and 9.5) and was
absent in the WT
whereas the amplification of the 18s rRNA gene was present in both WT and
transgenic
plants(visualized by staining with ethidium bromide). Figure 8B shows analysis
of protein
extracts from two-week old seedlings by Western Blot. As shown in Figure 8B,
the presence of
pea ABR17 protein was also confirmed in these transgenic plants by Western
blot analysis which
revealed an immunoreactive band at the expected molecular weight (-16-17 kDa)
in both
transgenic lines (3.15 and 9.5) but not in the WT. Results from these
experiments confirmed
that the pea ABR17 cDNA is integrated, transcribed and translated in B. napus.
2. Appearance of ABR17 transgenic B. napus seedlings and plants
41
CA 02589145 2007-05-18
Figure 9 shows the appearance of WT and ABR17 transgenic B. napus seedlings
and plants.
Appearance of (A) 7-day, (B) 14 day- and 37 day-old seedlings (C and E). The
appearance of 42-
day old plants demonstrating early flowering in the transgenic lines is shown
in panels D and F.
As shown in Figures 9A and 9B, the appearance of WT and transgenic seedlings
(3.15 and 9.5)
grown on filter paper in sterile water for 7 days and in clay growth medium
for 14 days did not
demonstrate any visible difference at these stages. Within the WT and
transgenic lines there was
a variation in root length and shoot length at both stages; however, there was
no distinct
difference between the WT and transgenic lines. As shown in Figures 9C, 9D and
9F, at 37
days after planting (DAP) plants grown in soil under greenhouse conditions
started exhibiting
differences between the WT and transgenic line. Transgenic line 3.15 had a
longer internode
length and more advanced floral bud formation when compared to the WT.
Transgenic line 9.5
did not demonstrate any difference from its WT at this stage. As shown in
Figures 9D and 9F, at
42 DAP there was a visual difference between the WT and transgenic lines 3.15
and 9.5. Both
transgenic lines were developmentally ahead of the WT and had a greater number
of flowers per
plant.
3. Effects of salinity and low temperature stresses on germination of ABR17-
transgenic
Brassica napus
The ability of the transgenic lines to germinate in the presence of 275 mM
NaCI was evaluated at
room temperature (RT; 21 2 C) and the results are presented in Fig. 10.
Specifically Figure 10
shows thee effect of NaCI on germination, root length and shoot length of WT
and ABR17
transgenic B. napus seedlings. Figure 10A shows the effect of 275 mM NaCI on
germination.
Figure lOB shows root length (cm). Figure 10C shows shoot length (cm) of 7 day-
old seedlings.
The transgenic line 3.15 demonstrated significantly (P <0.05) higher percent
germination on all
days except days 1 and 7 with the germination on day 3 the being more that 25%
higher in the
transgenic line 3.15 compared to the WT (Fig. l0A). Although not significant,
on day 1 line
3.15 had started germinating while lines 9.5 and the WT had not. Furthermore,
line 3.15 showed
significantly longer shoot and root length when compared to the WT and line
9.5, indicating that
germination as well as initial emergence and growth was greater in line 3.15
during the first
seven days compared to the other lines (Fig. lOB and lOC).
42
CA 02589145 2007-05-18
The ability of ABR17-transgenic B. napus and WT seeds was evaluated for
germination at 10 C
and 5 C alone as well as in combination with these low temperature stresses
and salinity. Figure
11 shows the effect of low temperature and NaC1 on the germination of WT and
ABR1 7
transgenic B. napus seedlings. Figure 11A shows the effect of 5 C. Figure 11B
shows the effect
of 10 C. Figure 11C shows the effect of 10 C plus 75 mM NaCI on the
germination of the WT
and transgenic lines 3.15 and 9.5. Once again, at 5 C, line 3.15 showed
significantly greater
germination than WT on day 1(Fig. 11A). Although line 9.5 was not
significantly ahead of the
WT at this time, it had started germinating while the WT had not. By day two
the trend in
greater germination demonstrated by line 9.5 on day 1 had become significant.
On day 2, 3, and
4 both lines 3.15 and 9.5 were significantly ahead of the WT. In fact, by the
third day, transgenic
lines 3.15 and 9.5 demonstrated between 5 and 10% greater germination,
respectively, than the
other lines tested. At 10 C, although there were no significant differences
in percent
germination between the two transgenic lines that exhibited higher germination
at 5 C (3.15 and
9.5), the transgenic line 3.15 displayed a similar trend of increased
germination on days 1-3 (Fig.
11B). In addition to investigating the ability of ABR17-transgenic seeds to
germinate at the
lower temperatures described above, we also investigated the effects of a
combination of lower
temperature and NaCl on the ability of these lines to germinate by testing the
effect of 75 mM
NaC1 on the ability of B. napus to germinate at 10 C. Under these conditions,
significantly more
seeds of the transgenic line 9.5 germinated than the WT on days 8 and 12 (Fig.
11C).
Salt tolerance in the Brassicas is often associated with ion exclusion. Salt
exposed B. napus
plants have greater K+/Na+ ratios in their tissues indicating that they are
able to preferentially
accumulate K+ over Na+, avoiding much of the nutritional stress associated
with increased Na+
uptake under saline conditions. The documented tolerance exhibited by B. napus
(Francois
1994; Steppuhn and Raney, 2005) was evident in the DH line we were testing, as
salinity
tolerance during germination (radicle emergence) was demonstrated up to 200 mM
NaCI in the
WT and both transgenic lines 3.15, and 9.5 constitutively expressing ABR17
(data not shown).
However, line 3.15 demonstrated increased germination at 275 mM NaCI when
compared to the
WT. Generally, low levels of salinity will slow germination down, but not
affect the final
number of seedlings that germinate, while at higher levels of salinity, the
speed of germination
43
CA 02589145 2007-05-18
will be further reduced with overall germination percentage also decreasing
(Bernstein and
Hayward 1958). The WT and the transgenic line 9.5 demonstrated reduced
germination, while
the high performing transgenic line 3.15 was ahead in germination after 48 h
and continued to be
ahead for the first five days after seeding. The increased germination at 275
mM NaCI indicates
that the transgenic line does show enhanced germination under saline
conditions. Although root
growth does not show the effects of salt stress as much as shoot growth (Munns
2002), line 3.15
had significantly greater root growth than line 9.5 and the WT. Shoot growth
in transgenic line
3.15 was also significantly longer than the WT. The greater root length in
3.15 may give this
line the added benefit that rapidly dividing root cells have in providing more
area for salt
sequestration (Bartels and Sunkar 2005) limiting the concentration of salt in
the plant (Munns
2002). Furthermore, as the seedlings mature a higher root to shoot ratio
ultimately results in
better exploitation of soil resources (Pasternak 1987).
Although seedling stages are often regarded as the most salt sensitive growth
stage, Bernstein
and Hayward (1958) state that the poor emergence in saline soils may be a
result of the soil in
the upper portion of the field being more saline, due to evapotranspiration,
field morphology and
low moisture, rather than low tolerance. Theoretically, in the same soil, the
adult plant could
avoid the saline regions of the soil profile with their more extensive root
system and appear more
tolerant. As a result, using one stage of growth to determine the salt
tolerance of a specific crop
may not be the best approach in determining if a plant is indeed salt
tolerant. The early
germination seen in our transgenic lines is important, because as Steppuhn and
Raney (2005)
demonstrated germination and early seedling growth are important parameters in
determining the
future success of canola under saline soil conditions. They demonstrated that
the canola varieties
used in their study were comparable to salt tolerant Harrington barley if they
displayed good
emergence (Steppuhn and Raney 2005). It is important to determine salt
tolerance during early
growth stages, so that we can establish initial salt tolerance; however, it
will be equally important
in future studies to evaluate the performance of the transgenic line as it
matures under saline
conditions.
Low temperature stress, like salinity stress, impacts crop productivity. Cold
temperatures during
germination can delay the onset of germination and reduce crop yields;
however, the mechanism
44
CA 02589145 2007-05-18
of how low temperature impacts germination is poorly understood (Salaita et
al. 2005). There
have been numerous germination studies on impact of low temperature stress in
tomato
(reviewed in Salatia et al. 2005), Arabidopsis (Srivastava et al. 2006,
Salaita et al. 2005) and
other plants. Cold temperature studies have been performed on Arabidopsis
seedlings and a
number of COR genes have been found to be involved in cold tolerance that
encode Late Embryo
Abundant (LEA) -like proteins (reviewed in Mahajan and Tuteja 2005) which are
synthesized in
the seedlings in response to dehydration stress. The promoter elements of COR
genes contain
DRE (dehydration responsive elements) or CRT (C-repeats) and some of them
contain ABRE
(ABA- responsive element) (Stockinger 1997; Yamaguchi-Shinozaki 1994). The
ability to
germinate under cold conditions is important in the northern hemisphere,
because cold
temperatures in the spring may negatively affect crop germination, early
seedling growth, and
ultimately, stand establishment. Furthermore, the short growing season and
moisture limitations
later in the growing season, particularly in the prairie regions, make it
important to seed canola
crops early so they are able to reach maturity without experiencing a
reduction in yield (Agandi
et al. 2004). Enhanced germination under salt and cold due to overexpression
of ABR17 cDNA
may indicate that molecular responses common to both stresses are responsible
for the observed
effects mediated by ABR17. Our observation that the constitutive expression of
pea ABR17
cDNA in B. napus enhances its germination under cold as well as saline
conditions may have
utility in the genetic engineering of crops.
4. ABR17 expression leads to early flowering in B. napus
The transgenic lines tested in this study exhibited dramatic differences in
development when
visually compared to the WT at 42 DAP, as can be seen in Figures 9D and 9F,
specifically with
regard to number of plants flowering and the amount of flowers per plant.
Figure 12 shows flowering and height of ABR17 transgenic and WT B. napus adult
plants 42
days after planting (DAP). Figure 12A shows first day of flowering. Figure 12B
shows rate of
flowering. Figure 12C shows plant height of ABR17 transgenic and WT B. napus
adult plants.
Figure 12D shows the relative expression of ABR17 in the transgenic lines
(3.15 and 9.5). The
transgenic line 3.15 flowered significantly (P < 0.05) earlier when compared
to the WT and the
transgenic line 9.5 (Fig. 12A). Line 3.15 also displayed a significantly
faster rate of plants
CA 02589145 2007-05-18
flowering per day when compared to line 9.5 and the WT (Fig. 12B).
Furthermore, line 3.15 at
42 DAP was significantly taller than the WT (Fig. 12C). When the effect of
height was removed
from the analysis it was apparent that the height did not contribute to the
difference in flowering
time observed between the WT and line 3.15. A possible reason for the lack of
significant early
flowering by the transgenic line 9.5 could be that the transgene was
integrated in different
locations on the B. napus genome leading to differential expression which
affected its ability to
flower earlier.
Similar to germination, flowering is an important developmental process that
contributes to
determining crop productivity. The timing of flowering becomes extremely
important in
northern latitudes because of the shorter growing season, which reduces the
time for reproductive
growth (Chandler et al. 2005). This can be the case in Alberta, especially the
Peace Region
where the growing season is short, and in the southern part of the province
where adequate
precipitation is a problem. For example, if the plant switches from vegetative
to reproductive
growth too early seed production can be limited, because the plants do not
have enough leaves
and roots to provide energy to the developing flowers and seeds. On the other
hand, if the switch
to reproduction is delayed, even though the plant is large enough to gather
photosynthates and
nutrients, it may not have enough time to produce mature seeds (Franke et al.
2006). Numerous
studies on the transition from reproductive growth to flowering have been
undertaken (reviewed
in Bernier and Perilleux 2005). Aside from environmental cues, like
photoperiod and
vernalization, cytokinins (CK) and gibberellins (GA) also affect the
transition to flowering.
Application of both phytohormones on the shoot apical meristem (SAM) of
Sinapsis alba
activate the SaMADS A gene that may be involved in the transition to flowering
(Bonhomme et
al. 2000). For example, Chaudhury et al. (1993) found that Arabidopsis ampl
mutant with high
CK levels also demonstrated early flowering. Furthermore, Dewitte et al.
(1999) reported that
organ formation, including flowering is correlated with increased endogenous
CK levels in
tobacco. Indeed, we have demonstrated previously (Srivastava et al. 2006) that
transgenic A.
thaliana plants expressing pea ABR17 cDNA flower earlier and that this may be
the result of
enhanced, endogenous CK levels in that species (Srivastava et al. 2007). The
earlier flowering
observed in ABR1 7-transgenic lines investigated in this study may also be the
result of enhanced
46
CA 02589145 2007-05-18
CK levels although this suggestion must be verified through the determination
of endogenous
CKs in these lines.
5. Relative expression of ABR17 cDNA in transgenic lines
Even though RT-PCR experiments revealed the expression of the pea ABR17 cDNA,
the relative
levels of expression in these lines could not be ascertained using that
technique. In order to
determine whether differences in the relative levels of expression between the
two transgenic
lines were contributing to the observed differences in germination as well as
flowering, we
performed qRT-PCR experiments. These experiments revealed that line 3.15 had
the highest
level of ABR17 expression when compared to line 9.5 as seen in Fig. 12D. These
results
correlate well with the observed responses of these lines with respect to
germination as well as
earlier flowering. For example, the transgenic line 3.15 was observed to be
the best when tested
for germination in the presence of 275 mM NaCI, had the best developed roots
and shoots (Figs.
lOB and 10C) and flowered earlier (Figs. 9D and 12A) compared to line 9.5. The
excellent
correlation observed between the levels of ABR17 expression, which was
approximately 2-fold
higher in line 3.15 (Fig. 12D), and the phenotypic characteristics of this
line clearly indicate that
the observed phenomena are the result of the transgene expression. The
differences between the
two transgenic lines with respect to the relative levels of expression may be
the result of
differences in the location of transgene integration i.e. positional effects.
While the invention has been described in conjunction with the disclosed
embodiments, it will be
understood that the invention is not intended to be limited to these
embodiments. On the
contrary, the invention is intended to cover alternatives, modifications and
equivalents, which
may be included within the spirit and scope of the invention as defined by the
appended claims.
Various modifications will remain readily apparent to those skilled in the
art.
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