Note: Descriptions are shown in the official language in which they were submitted.
CA 02209~91 1997-07-03
Title of the invention
Methods for improving cold or freezing tolerance, reducing the growth rate, or inhibiting
the growth of plants, and for improving the germination rate of plant seeds.
Field of the invention
The invention relates to a method to increase the cold or freezing tolerance of
plants by cold acclimating the plants and/or by treating the same with betaines.This invention also relates to the inhibition of the growth or the reduction of the
growth rate of plants by treating them with betaines.
This invention further relates to the improvement of the germination rate of plant
10 seeds at cold temperatures by treating the same with betaines.
Background of the Invention
Betaine is a non-toxic osmolyte that is thought to play a role in the protectionagainst environmental stresses in particular salinity and drought stress (1, 2). This
compound is mostly synthesized in the chloroplast by the enzymes choline
15 monooxygenase and betaine aldehyde dehydrogenase (1). It may accumulate in
different cellular compartments to adjust the osmotic balance (3) and increase the
stability of protein tertiary structure thus protecting proteins from denaturation (4). In
vitro studies have shown that betaine can protect membranes of Befa vulgaris roots
against heat denaturation (5). Several higher plant enzymes were also shown to be
20 protected by betaine from denaturation caused by heat (6) NaCI or KCI (7). Betaine
can also stabilize the photosynthetic activity of isolated chloroplasts over time (8) and
protect photosystem ll against the inhibitory effect of NaCI (9). Interestingly, it was
shown that an exogenous application of 25 mM betaine on barley leaves improves
recovery after an osmotic stress imposed by polyethylene glycol (-10 bar) (10).
Because betaines have been shown to provide some protection to plants
from stressful environmental conditions they have been used to treat soils, plants
and seeds.
WO 95/35022 discloses a method for treating seeds with betaine to
enhance seedling growth and protect seeds against adverse environmental
30 conditions. The seeds may be soaked and dried or coated with betaine. The
adverse conditions enumerated are water stress, excess NaCI, extreme
temperature or pH and heavy metal toxicity. What is not taught are the temperature
extremes and the benefits with respect to the rate of germination at low
temperatures.
WO 96/07320 discloses the application of betaine to improve the yield of
grapevines the temperature extremes are between 3~C to 30~C.
CA 02209~91 1997-07-03
In WO 96/41530, different compositions of betaine are disclosed for use in
protecting wheat, potato and grapevines against adverse conditions including
temperatures between 3~C and 30~C.
These references are silent with regards to freezing temperatures and cold
5 acclimation.
In France Allard's thesis, it is taught that in the wheat cultivar Fredrick, cold
acclimation for three days 6 ~C/2 ~C (day/night) combined with the addition of
1000 mM betaine resulted in the improvement in the freezing tolerance of the
plants as tested by measuring the survival rate at -10 ~C. The survival rate wasshown to be 83%, whereas, when treated with only 1 000mM betaine, the survival
rate was 51%, when compared to controls. This reference does not teach on the
optimal conditions for increasing freezing tolerance. The high amount of betaineused (1 000mM) was shown to have a toxic effect to the plant since it was found
that it produces chlorosis of the leaves. In addition the cultivar Fredrick having a
LT50 (lethal temperature where 50% of the plants die) at -17 ~C, testing the plants
at -10 ~C does not teach how more freezing tolerant is the plant when treated with
the combined treatment. It further does not teach the effect on cultivars that
genotypically exhibit less freezing tolerance. The decrease in plant growth rate is
also described and it is shown to be directly proportional to the amount of betaine
administered. When the maximal amount of 1000mM was applied to the cultivar
Fredrick, a 29% decreased growth rate occurred when compared to control plants.
Finally, in this document there were also teachings relating to the protein WCOR410. It was taught that the protein WCOR 410 accumulates when plants are
subjected to cold-acclimation or by increasing amounts of exogenous betaine.
This reference however does not appear to teach a period of cold-acclimation
which is sufficient for optimally inducing the expression of the Wcor 410 gene and
improving freezing tolerance.
In the patent publication CA 2,104,142, the present inventors disclose the
isolation and sequence of three genes responsive to cold temperature. One of these
genes is Wcor410. However what is not taught is that the protein WCOR410 is
induced by betaine in a manner proportional to the amount of betaine applied and that
this protein is involved in promoting freezing tolerance in some plants. It does not
teach the benefits of combining cold acclimation and betaine administration.
CA 02209~91 1997-07-03
Statement of the invention
There is now provided a method of increasing cold or freezing tolerance in a
plant, which comprises the steps of:
- acclimating said plant to a temperature higher than about 0~C but not
lower than the coldest temperature that said plant is capable to
withstand, for a time suffficient to induce an optimal cold or freezing
tolerance, in said plant, and
- administering betaine or a derivative thereof such as glycine betaine to
said plant, in a dosage regimen suffficient to induce the same or different
optimal cold or freezing tolerance in said plant;
whereby combined steps of cold-acclimating and administering betaine or
derivative thereof increase cold or freezing tolerance of said plant over and above the
optimal cold or freezing tolerance induced by each step alone.
Preferably, the dosage regimen does not provide an unacceptable toxicity, more
preferably, it is non-toxic to said plant.
Any plant could benefit from such a method, preferably, gramineae and
grasses, more preferably, barley or wheat.
In the two latter plants, the time for cold-acclimating is about four weeks, andthe dosage regimen is growing the plants in the presence of a solution of glycine
betaine having a concentration lower than about 500 mM, preferably about 250 mM.In the spring wheat variety Glenlea, in which the optimal freezing tolerance,
expressed as the temperature where fifty percent of a plant population die (LTso) is
about -8 ~C for each step alone, the combined treatment resulted in an increase of
freezing tolerance by about 6~C to reach a LTso of about -14~C and further resulted in
improving photosynthetic capacity and overall physiology of the plants at cold or
freezing temperatures.
The optimal freezing tolerance induced by said each step alone and/or in
combination is due at least in part to an increased expression of the gene Wcor410.
This invention also relates to the reduction of the growth rate of a plant by atleast 30%, which comprises the step of treating the plant with an effective dosage
regimen of betaine or derivative thereof which is not lethal, preferably non-toxic to the
plant.
When growing the spring wheat variety Glenlea, in the presence of 500 mM of
glycine betaine for four days, the growth rate thereof was reduced by about 75%.Another aspect of the present invention is a method of inhibiting the growth of
a plant, which comprises the step of treating said plant with a high dose regimen of
betaine or derivative thereof, which may even result in a herbicidal effect.
Another aspect of the present invention is a method of improving the
germination rate of plant seeds at a temperature which is higher than about 0~C but
CA 02209~91 1997-07-03
not lower than the coldest temperature that said plant seeds can withstand, which
comprises the steps of administering to said seeds an effective dosage regimen of
betaine or derivative thereof, and allowing said seeds to germinate at said
temperature.
5 Description of the invention
This invention is described hereinbelow by way of specific embodiments and
appended figures, which purpose is to illustrate the invention rather than to limit its
scope.
Brief description of figures
10 Figure 1. Effect of betaine on FT in the cultivar Glenlea.
Plant survival was determined by the regrowth test as described by Perras and Sarhan
(25).
A: The survival was evaluated after freezing at -8~C.
B: The LTso was evaluated after freezing different samples to various temperatures
NA,12 day-old control non-acclimated plants; 100, 250, and 500, plants treated for 4
days with 100, 250, and 500 mM betaine at 25~C respectively; CA, plants
cold-acclimated at 6/2~C for 30 days; CA100 and CA250; plants cold acclimated at6/2~C for 30 days in the presence of 100 and 250 mM betaine respectively. Standard
deviation did not exceed i 10%.
Figure 2. Effect of betaine on LTso in the cultivar Glenlea
A: The freezing test was performed at -8~C.
B: The freezing test was performed at -13~C.
C,12 day-old control non-acclimated plants; 250 plants treated for 4 days with 250 mM
betaine. CA, plants cold-acclimated at 6/2~C for 30 days; CA250; plants cold
acclimated at 6/2~C for 30 days in the presence of 250 mM betaine.
Figure 3. Accumulation of the WCOR410 protein in response to different betaine
concentrations in the spring wheat cultivar Glenlea.
Total proteins (5 ,ug) were separated by SDS-PAGE, transferred to a nitrocellulose
membrane and probed with the anti-WCOR410 antibody. NA, 12 day old control
non-accii~ ~ lated plants; 100, 250 and 500, plants treated for 4 days with 100, 250, and
500 mM betaine respectively; CA, Cold acclimated plants.
Betaine refers to amino acids where the nitrogen is fully or partly methylated.
Betaines are natural products present in plants and animals with a probable function
as an osmolyte regulator that protect the cell from osmotic stress. Betaine have the
general formula:
(CH3)X- N - (CH2)y - COO-
CA 02209~91 1997-07-03
where x may be 1 and preferably 2 for cyclic betaine or 3 for straight chain betaines,
and y is at least 1. The most common betaine is a glycine derivative where the three
methyl groups are attached to the nitrogen of a glycine molecule.
Other betaines that are known of which some that are available commercially
5are presented in Table 1.
TABLE I
NAMES OTHER NAMES
Glycinebetaine Oxyneurin, betaine
~-alaninebetaine Homobetaine
1 02-trimethylamino-6-ketoheptanoate
Prolinebetaine Stachydrine
Proline
N-methyl-L-proline
Trans-4-hydroxy-N-methyl-L-proline
1 5Cis-3-hydroxy-N-methyl-L-proline
(-)4-hydroxyproline betaine Betonicine
(+)4-hydroxyprolinebetaine Turicine
3-hydroxyprolinebetaine 3-oxystachydrine
Histidinebetaine Herzynine, Ercinine
20Tryptophanbetaine Hypaphorine
2-mercaptohistidine-betaine Ergothioneine
Pipecolabetaine Homostachydrine
Nicotinic acid betaine Trigonelline
Using two wheat cultivars that differ in their levels of freezing tolerance (FT),
25 the role of endogenous betaine was investigated during cold acclil,ldlion. In addition,
studies on the effect of an exogenous application of betaine on FT alone and in
combination with cold acclimation, on the expression of low temperature-responsive
genes and on photosynthetic activity have been conducted.
To determine if betaine accumulation is associated with increased FT, the
30 betaine contents were determined in two wheat varieties differing in their FT (cv
Glenlea, LTso (lethal temperature for 50% of the plants) of -8~C and cv Fredrick, LTso
of -1 7~C). In both cultivars, betaine content decreases during growth at the
non-acclimated temperature of 24/20~C while it increases during growth at the
cold-acclimating conditions of 6/2~C. The basal betaine level is 30% higher in the more
35 tolerant cultivar Fredrick before cold acclimation (8.5 ,umol/g FW in Fredrick compared
CA 02209~91 1997-07-03
to 6.5,umol/g FW in Glenlea). At the end of the acclimation period (where maximal LTso
has been reached) cv Fredrick has accumulated 21.3,umol/g FW of betaine comparedto 15.3,umol/g FW for cv Glenlea. On a dry weight basis, cv Fredrick has accumulated
106.5 ,umol/g DW compared to Glenlea which has accumulated 82.7 ,umol/g DW. This5 result suggests that the increase in betaine content is associated with the dcvelopr"ent
of FT of the two cultivars. A similar increase in betaine was correlated with the FT of
different barley cultivars (11). If we calculate the contribution of betaine to the total
osmolality of the cell, we find that betaine accounts for only 3.6% and 4.5% of the
osmolality after 30 days of cold acclimation for Glenlea and Fredrick respectively. This
10 result demonstrates that betaine contribution to the total osmolality is very low.
However, as suggested by Wyn Jones et al (12), such a low concentration would
require compartmentation in order to play a significant role as osmoprotectant. Studies
performed by Matho et al (13) have shown that betaine is excluded from vacuoles of
spinach leaf cells and is mostly found in the cytoplasm and chloroplasts. It wasesli" ,ated that betaine concentration can reach 300 mM in spinach (14) and Sueda (8)
chloroplasts when plants are submitted to salt stress. This concentration is
approximately 20 fold greater than the average betaine leaf concentration. Betaine
compartmentation was not determined in wheat but if we consider a similar
concentration factor in the chloroplasts during cold acclimation, the actual
20 concentration of betaine could be very significant. Since we have estimated that
betaine accounts for 4.5% of the osmolality in cold-acclimated Fredrick, a twenty fold
higher concentration of betaine in the chloroplast would mean that betaine contributes
for approximately 90% of the chloroplasts' osmolality (or 612 mOsm). Such a
concentration could have a great impact on chloroplast function since in vitro studies
25 have shown that betaine can increase the thermal stability of photosystem ll (PSII; (5))
and can protect against the inhibitory effect of NaCI (9). Krall et al (16) have shown
that betaine can stabilize the active tetrameric form of phosphoenolpyruvate
carboxylase which normally forms inactive dimers when exposed to low temperature.
Betaine accumulation in the chloroplasts may be an important factor that could play a
30 significant role in maintaining chloroplast function at low temperature. It is worth noting
that hardy cereals such as rye, wheat, and barley have higher basal levels of betaine
compared to sensitive species such as rice, millet, and sorghum (11).
To determine whether exogenous betaine could play a role in improving FT and
photosynthesis at low temperature, we first evaluated the plant's capacity to
35 accumulate betaine. In the first experiment, we incubated plants in 500 mM betaine
and determined the osmolality of the leaves at different periods. The osmolality was
found to increase rapidly during the first two days and Icve"Ed off thereafter (result not
shown). We repeated the experiment using different concentrations of betaine andquantified the amount of betaine accumulated in the leaves after a four day period. The
CA 02209~91 1997-07-03
method described in (17) was used to extract betaine from 1 g of leaf tissue.
Quantitation was performed according to Lerma et al (18). For accurate evaluation, an
internal standard was added before the extraction procedure. The betaine content was
expressed in mOsm/kg H2O considering the tissue water content for each sample (an
5 average of 82% water content was obtained). Osmolality was measured from leaf
tissue after grinding with a mortar and pestle. The liquid obtained was centrifuged at
12,000 g for 10 min at 4~C. The osmolality was evaluated in the supernatant using a
Wide Range Osmometer. We found that betaine accumulated efficiently at all
concentrations used. The accumulated betaine (expressed in mOsm/Kg H2O) was
10 equivalent to 62% of the external betaine when exposed to betaine concentrations
ranging from 118 to 590 mM (100 to 500 mM). Betaine could accumulate even more
at higher concentrations, however, signs of chlorosis at the leaf tips became evident
at 500 mM. Chlorosis became even more extensive when higher betaine
concentrations were used.
Betaine accumulation reduced the growth rate in a manner proportional to the
amount of exogenous betaine applied. At the highest concentration used, the growth
was reduced by 75% over the 4 day incubation period compared to control plants. The
reduction in growth and more importantly, the increase in cellular betaine content was
found to be associated with a substantial increase in survival rate after freezing
20 compared to control non-acclimated plants (Fig.1A). Interestingly, both cultivars are
protected by betaine with only a slight advantage in the more tolerant cultivar at all
concentrations used (not shown). Plant survival is increased even when a relatively low
concentration of betaine is used. At 100mM, survival improved by 5-6 fold compared
to the untreated plants (Fig.1A). Treating with 250 mM betaine alone was sufficient to
25 increase the FT of the spring cultivar Glenlea from -3~C to -8~C. This value
corresponds to the maximal FT achieved by this cultivar after 4 weeks of cold
acclimation (Fig.1 B). Increasing the concentration of betaine to a higher concentration
resulted in a slightly higher survival rate (corresponding to 55% survival at 500 mM
betaine; Fig. 1A) but due to the toxicity, of higher betaine concentrations, the latter
30 were eliminated in other experiments. We have examined whether treatment withbetaine during cold acclimation could improve FT in the less tolerant cultivar Glenlea
submitted to cold-acclimating conditions. Figs. 1A and 1B show that the survival of
plants treated with betaine during cold acclimation were dramatically improved over
plants that are cold acclimated in the absence of betaine. Fig. 2 shows the results of
35 a typical experiment for plants treated with betaine at 25~C or during cold acclimation.
Betaine treatment at 25~C for 4 days allowed the plants to reach an LTso of -8~C (the
maximal LTso normally achieved by this cultivar) while those treated with betaine during
cold acclimation were barely affected by a temperature of -13~C (the average LTso was
e~li",aled as -14~C in Fig.1 B). These results demonstrate that the improvement in FT
CA 02209~91 1997-07-03
observed in control plants exposed to betaine is additive in cold-acclimated plants. This
finding is of crucial importance since it is the first time that the normal genotypic
potential to tolerate freezing has been improved so dramatically. We have also
evaluated the capacity of betaine to improve FT in barley which was also shown to
5 accumulate betaine upon cold acclimation and found that the combined treatment of
low temperature and betaine was as effficient in this species as in wheat to improve FT.
It is therefore expected that the improvement in cold or freezing tolerance willbe observed in almost all plants, particularly gramineae or grasses, more particularly
cereals such as rice, corn, rye, wheat, barley and oat. The conditions at which such
10 improvement will occur are set as follows. The plants are acclimated at the coldest
temperature that they can withstand. Betaine is administered before, during and at the
end of the cold acclimation. The dosage regimen of betaine is determined on testplants in order to evaluate the doses at which betaine is toxic, in such a way that
unacceptable toxic doses will be further avoided. We expect that even tropical plants
15 which are very cold-sensitive will benefit form a combined treatment.
Furthermore, since the growth rate of the plants were reduced down to 25% the
control plants, it is readily apparent that, if betaine is applied at the end of cold
acclimation (in field, that would mean during fall wherein the maximal growth isattained), this effect would not be deleterious to the plants. Moreover, this property
20 may be advantageously used at the beginning or during the growing period, to slow
down the growth of many plants. Thus, it could be used as a growth-retarding
substance for several agronomical applications. A specific useful application would be
found for golf courses to reduce the maintenance costs (it would reduce the number
of times one has to mow the grass during a season). At higher toxic or lethal doses,
25 betaine could event be used as a herbicide to inhibit the growth of undesirable plants
or kill them.
Treatments with other osmolytes such as NaCI or mannitol allowed the
osmolality to increase as much as with betaine treatment. However, the FT was not
significantly improved when these osmolytes were used indicating that betaine
30 specifically improves FT. These results suggest that the improvement in LT50 of 6~C
cannot be explained solely by the osmolyte role of betaine (at a concentration of 250
mM, betaine would depress the freezing point of water by 0.32~C). We thus
investigated whether betaine can induce a number of genes known to be associatedwith the development of FT. We examined the expression of three different proteins
35 induced by low temperature using specific antibodies and immunoblot analysis as well
as the expression of three other low temperature-induced genes using northern
analysis. Our results showed that the protein WCOR410 (Genbank accession no.
L29152) accumulates in a concentration-dependent manner when plants are exposed
to varying amounts of betaine (Figure 3). When we examined the expression of the
CA 02209~91 1997-07-03
WCS120 (Genbank accession no. M93342) protein family or of the WCS19 (Genbank
~ccession no. L13437) protein, we found that these proteins did not accumulate upon
exposure to betaine (not shown). Since these two genes are known to be specifically
induced by low temperature and that the gene WCOR410 was inducible by low
temperature, salinity, and water stress (19), we examined the possibility that other
genes induced by drought or salinity are also induced by the presence of betaine. The
genes WCOR80 (Genbank accession no. U73212), WCOR413 (Genbank accession
no. U73216), and WCOR825 (Genbank accession no. U73215) were only slightly
inducible by betaine (not shown). These results suggest that the WCOR410 gene is10 specifically induced to high levels by betaine exposure and may be a major contributor
to the improvement of FT above the level that could be explained solely by the role of
betaine as an osmolyte. The WCOR410 protein was found to be associated with the
plasma membrane. This is the first abundant protein found in the membrane fraction
of the vascular tissues (20), a region of the seedling known to be the most freezing
15 sensitive tissue in wheat (21).
Betaine was shown to protect thylakoid membranes from freezing stress in
vitro (22). Furthermore, in glycine-betaine deficient maize lines, high temperature
decreases membrane stability and the resistance to photoinhibition as well as the
steady-state yield of electron transport over PSII (23). These results suggest that
20 betaine may confer greater membrane stability under both low and high temperature.
It can also protect the photosystem ll against salt stress in vitro. Since betaine is
normally synthesized in the chloroplast, it may accumulate to a higher concentration
in this organelle as suggested (8,14). Thus, we evaluated the effect of the exogenous
supply of betaine on the resistance to photoinhibition and oxygen evolution.
During steady-state photosynthesis at the prevailing growth conditions,
exposure of spring and winter wheat to 250 mM betaine resulted in small but
consistently higher levels of qP and higher yields of PSII electron transport (~>e) than
non-treated controls (Table 1). Thus, betaine treated spring and winter wheat seedlings
appeared to exhibit a greater capacity to prevent the reduction of PSII reaction centres
30 than non-treated controls. The increased capacity to keep PSII reaction centres
oxidized was correlated with a decreased susceptibility to low temperature (5~C)photoinhibition (measured according to 24) in betaine-treated seedlings. The Fv/Fm
ratio of 250 mM betaine-treated plants submitted to high light (2h at 5~C and 1600
,umol m~2.s~1) was of 84 i 4% of non-photoinhibited control plants compared to 72 i 2%
35 for plants not treated with betaine. This effect of betaine on the photosynthetic
machinery may improve the physiology and performance of the plants at low
temperature and thus provide the plants with a greater capacity to use the available
energy to develop FT.
CA 02209~91 1997-07-03
- 10-
Based on the above results and based on the partial teachings of WO
95/35022, it is further expected that the germination rate of plant seeds can beimproved at cold temperature. This method will comprise the steps of administering
to the seeds an effective dosage regimen of betaine or derivative thereof, and allowing
5 the same to germinate at cold temperatures. Such cold temperatures would be above
about 0~C but not lower than the coldest temperature that the one plant seeds can
withstand.
An important conclusion one can draw from these observations is that it may
be possible to increase FT by using only one or two of the major genes (such as
10 Wcor410) associated with this multigenic trait. Thus, it may be possible to increase the
FT of cold sensitive species by transforming plants with this gene. Furthermore, our
results suggest that FT could be improved in an additive manner in plants that already
possess some degree of FT by overexpressing the most important genes involved inthe process of FT. For example, overexpressing betaine dehydrogenase and choline15 monooxygenase under a low temperature promoter may allow the accumulation of
betaine at the time where it could help against freezing stress. Such manipulations of
betaine production could have a great agronomic and economic impact not only forprotection against drought stress as previously suggested but also for FT as shown in
this report. In addition, the results presented in this report indicate that exogenous
20 application of betaine before a predicted frost or sudden decrease in temperature may
be exploited on a short-term basis to increase the resistance of cold sensitive plants
to low temperature stress.
This invention has been described in details hereinabove, and it is readily
apparent that modifications thereto can be made, without departing from the spirit of
25 the invention and from the above teachings. These modifications fall under the scope
of the invention as defined in the appended claims.
CA 02209~91 1997-07-03
- 11 -
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