mM glycine betaine solution and the leaf washing was incubated for up to 72 h at 23°C. Incubated
leaf washings were used to determine the glycine betaine content to ascertain whether glycine be-
taine applied to the foliage was broken down by the microflora.
2
.
5
. Determination of glycine betaine content Samples consisting of leaves or aliquots of solu-
tion were frozen in liquid nitrogen immediately after collection. Glycine betaine was quantified
using NMR methods as outlined by Jones et al. [25] with some modifications. Thawed leaf samples
were used to express sap using a Carver Labora- tory Press Fred S. Carver, Inc., IN. Ten ml of the
extract was dried under a stream of nitrogen in a desiccator, and the final volume was made up to 1
ml with D
2
O. Glycine betaine was quantified using a Varian UNITY Plus 500 NMR spectrometer
operating at 11.75 T 499.869 MHz for
1
H NMR and spectra were measured at 30°C using a 5 mm
triple-resonance inverse detection probe. One di- mensional proton spectra were acquired with 16
transients, using 19.2 K data points, a pulse repeti- tion rate of 2.0 s, a flip angle of 60°, and a spectral
width of 8,000 Hz centered on the water peak. The free induction decays were zero filled to 64 K prior
to Fourier transformation using Varian NMR software VNMR 4.3b. The peak intensities were
measured digitally using the spectrometer’s inte- gration software.
3. Results
3
.
1
. Cold tolerance and endogenous glycine betaine
Glycine betaine levels increased in the leaves of strawberry plants during cold acclimation. Signifi-
cant increase in glycine betaine levels in the leaves began during the first week of cold acclimation
treatment Fig. 1. A nearly two-fold increase in the leaf endogenous glycine betaine level after 4
weeks of cold acclimation was observed. A roughly linear increase in endogenous glycine be-
taine levels was observed up to 3 weeks of cold acclimation.
Cold tolerance of leaves increased nearly three- fold after 3 weeks of cold acclimation. Most of the
increase in cold tolerance of leaves was observed during the first week of cold acclimation, about
2.4 times over that of the unhardened plants and a small increase during the third week of cold accli-
mation. No further increase in cold tolerance was noted beyond 4 weeks of cold acclimation. Plants
maintained their cold tolerance at this level as long as they were under acclimating conditions data
not shown.
3
.
2
. Glycine betaine accumulation in response to exogenous ABA
Exogenous application of ABA to unhardened and cold-hardened plants resulted in a marked rise
in glycine betaine levels in the leaves and the increase in the endogenous glycine betaine level
was about 35 over that in the untreated controls Fig. 2a. Application of ABA to strawberry
plants produced a rather rapid response of in- creased glycine betaine levels in the leaves. In
unhardened plants, a significant increase in the leaf glycine betaine levels was observed only 48 h
after ABA application. In addition, exogenous ABA also increased the endogenous glycine be-
taine levels in the cold-hardened plants, although to a much less extent than in the unhardened
plants data not shown.
Fig. 1. Changes in leaf cold tolerance and endogenous glycine betaine levels of strawberry leaves during cold acclimation.
Strawberry plants were cold acclimated at 42°C daynight with 10 h photoperiod. The data for glycine betaine levels are
means n = 4 with SE. LSD
0.05
for cold tolerance was 0.377.
Fig. 2. Endogenous glycine betaine levels and leaf cold toler- ance of strawberry plants treated with exogenous ABA. ABA
100mM was applied as a foliar spray to unhardened plants and cold-hardened plants. Time course increase in endoge-
nous glycine betaine levels in leaves of unhardened plants after ABA application is shown in a. Cold tolerance of
leaves of unhardened U and cold-hardened H plants was determined before and 72 h after ABA application b. Plants
were cold-hardened for 4 weeks as outlined in materials and methods. The data for glycine betaine levels are mean values
n = 4 with SE. LSD
0.05
for cold tolerance was 0.374.
ing the significant increase in the leaf endogenous glycine betaine levels.
3
.
3
. Cold tolerance in response to exogenous glycine betaine and ABA
When plants were treated with glycine betaine as a foliar spray, it was taken up readily by the
leaves. A rapid linear increase in the leaf glycine betaine levels occurred after one day of glycine
betaine application, reaching its highest level of 0.178 mg g
− 1
d.w. 72 h after application Fig. 3. To determine if foliar applied glycine betaine is
broken down before it is absorbed by the leaves, leaf washings of exogenously-applied glycine be-
taine were collected and analyzed for glycine be- taine content after incubating for up to 72 h at
23°C. The results showed no breakdown of foliar- applied glycine betaine over a 3 day period Fig. 3
inset. Also, microscopic examination of leaf washings incubated up to 72 h did not reveal any
microbial growth data not presented.
Cold tolerance of leaves of unhardened plants was significantly increased by exogenous applica-
tion of glycine betaine. The increase in cold toler- ance, 80 over that in the untreated controls,
occurred within 72 h of glycine betaine application
Fig. 3. Leaf glycine betaine levels and cold tolerance in strawberry plants treated with exogenous glycine betaine.
Unhardened plants were treated with glycine betaine 2 mM as a foliar spray at 4°C. Leaf glycine betaine levels
and
cold tolerance were measured following the application of glycine betaine. Glycine betaine content in leaf washings
incubated up to 72 h was also measured inset.
We also examined the ability of exogenous ABA to induce cold tolerance in both unhardened and
cold-hardened plants, which were cold acclimated for 4 weeks. When ABA was applied to the leaves,
a significant increase in cold tolerance was ob- served both in unhardened and cold-hardened
plants. Although the ability of exogenous ABA to induce cold tolerance in unhardened and cold-
hardened plants is about the same, about 5°C, it actually represents a two-fold increase in cold
tolerance in the unhardened plants Fig. 2b. Inter- estingly, the increase in the cold tolerance was
observed about 72 h after ABA application in both unhardened and cold-hardened plants follow-
Fig. 4. Leaf cold tolerance increase in strawberry plants after exogenous glycine betaine application during cold acclima-
tion. Glycine betaine was applied as a foliar spray to unhard- ened plants and plants during the cold acclimation treatment.
Cold tolerance in the leaves was evaluated 72 h after glycine betaine application to plants. Increase in cold tolerance over
that in unhardened and cold acclimating plants that did not receive exogenous glycine betaine is presented.
that the elevated levels of glycine betaine in the leaves may be associated with the induction of
cold tolerance. However, when glycine betaine was applied to
cold-hardening plants during the 4-week cold ac- climation period, plants were much less respon-
sive. Exogenous application of glycine betaine did not produce any significant increase in cold toler-
ance during the first 2 weeks of cold acclimation Fig. 4. However, after 2 weeks of cold acclima-
tion it did increase the cold tolerance of plants significantly over that induced by cold acclimating
conditions alone, nonetheless the increase clearly was much smaller than that in unhardened plants.
3
.
4
. Glycine betaine and whole plant sur6i6al and regrowth
Exogenous glycine betaine, applied as a foliar treatment to unhardened plants, increased the cold
tolerance of whole plants Table 1. Observations on survival were made on the shoots shoot mor-
tality after 2 days of the freezing tests. Glycine betaine treatment to plants increased the percent-
age of freezing survival at various temperatures. For
example, glycine
betaine treated
plants showed 80 survival as against 50 in the un-
treated plants at − 8°C. Similarly, 30 of glycine betaine treated plants survived whereas 5 of the
untreated plants survived when frozen to − 10°C. Although some survival 20 was noted in
glycine betaine-treated plants at − 12°C, a greater percentage over 50 of the plants showed re-
growth 2 weeks after the freezing tests. No re- growth was observed in untreated plants frozen
below − 10°C and typically, fewer untreated plants regrew after freezing than did the glycine
betaine treated plants at temperatures where shoot mortality occurred. An example of survival and
regrowth in glycine betaine-treated plants after freezing is shown in Fig. 5. Also, we observed that
subjecting plants to freezing tends to induce flow- ering in both glycine betaine-treated and the un-
treated plants Table 1.
4. Discussion
