Glycine betaine accumulation and induction of cold tolerance in
strawberry (
Fragaria
X
ananassa
Duch.) plants
C.B. Rajashekar
a,*, H. Zhou
a, K.B. Marcum
b, O. Prakash
c aDi6ision of Horticulture,Kansas State Uni6ersity,Manhattan,KS66506,USAbDepartment of Plant Science,Uni6ersity of Arizona,Tucson,AZ85721,USA cDepartment of Biochemistry,Kansas State Uni6ersity,Manhattan,KS66506,USA Received 1 March 1999; received in revised form 9 July 1999; accepted 12 July 1999
Abstract
Endogenous glycine betaine levels in the leaves of strawberry (FragariaXananassaDuch.) plants were determined during cold acclimation and in response to exogenous abscisic acid (ABA) application. Glycine betaine levels in the leaves increased nearly two-fold after 4 weeks of cold acclimation treatment during which the cold tolerance of leaves increased from −5.8 to −17°C. Exogenous application of ABA (100mM) to plants triggered glycine betaine accumulation in unhardened plants. It also increased
cold tolerance of leaves in both unhardened and cold-hardened plants. Similar to ABA, exogenous glycine betaine was effective in inducing cold tolerance in unhardened and cold-hardening plants. Exogenous application of glycine betaine (2 mM) to unhardened plants increased the cold tolerance of leaves almost two-fold within 72 h of application. In addition, it improved freezing survival and regrowth in whole plants. The results suggest that glycine betaine is involved in inducing cold tolerance both in response to exogenous ABA and during natural cold acclimation of strawberry plants. © 1999 Published by Elsevier Science Ireland Ltd. All rights reserved.
Keywords:FragariaXananassa; Strawberry; Cold tolerance; Cold hardiness; Cold acclimation; Glycine betaine; Abscisic acid
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1. Introduction
Glycine betaine is known to accumulate in a wide range of plants under environmental stress conditions [1]. By far, most of the work on glycine betaine in plants is focused on its possible role in relation to osmotic and water stress [1,2]. Both osmotic and drought stress can trigger significant accumulation of glycine betaine in many plants. This in part has lead the researchers to propose that the accumulation of such compatible solute in plants is an adaptive mechanism against these stresses [1,3,4]. Glycine betaine is known to accu-mulate in the cytoplasm as an osmolyte and may have a stabilizing and protective action on proteins, enzymes and membranes under
unfavor-able and stressful conditions [5 – 10].
In addition to its role in osmotic and drought stress, glycine betaine appears to be involved in low temperature stress in plants as well. Glycine betaine has been shown to enhance the growth of bacteria at chilling temperatures [11]. Further-more, it has been shown to protect thylakoid membranes against low temperatures in spinach [12] and membrane integrity during freezing in alfalfa [13]. Glycine betaine has been known to accumulate during cold acclimation in rye, barley, and wheat [14 – 16]. Although there have been some studies dealing with protective effects of glycine betaine against low temperatures, its role in plant cold tolerance and cold acclimation pro-cesses is not clear.
Considerable attention has been devoted to elu-cidate the role of ABA in cold tolerance of plants. ABA has been associated with the cold
acclima-* Corresponding author. Tel.:+1-785-532-1427; fax:+ 1-785-532-6949.
E-mail address:[email protected] (C.B. Rajashekar)
tion process in a number of plant species [17,18] and, when exogenously applied, it has been shown to induce cold tolerance of plant cell cultures [19 – 21] as well as whole plants [17,18,22]. In fact, exogenously applied ABA is known to induce greater cold tolerance more readily than the accli-mating low temperatures do [18,19,23].
In this study we attempt to characterize the role of glycine betaine in cold acclimation and in in-duction of cold tolerance in strawberry plants.
2. Materials and methods
2.1. Plant materials
Strawberry (FragariaXananassaDuch. cv. Ear-liglow) plants were grown in the greenhouse at 23/18°C day/night temperatures under natural day light in 15 or 22.5 cm pots containing a sterile growing medium of peat, perlite, and soil (2:2:1 v/v). Plants were irrigated once every two days to field capacity and fertilized weekly with Peat-Lite Special (Scotts-Sierra Horticultural Products Co., Marysville, OH) with N-P-K of 20-10-20 (ammo-nium nitrate, potassium phosphate and potassium nitrate) at 250 ppm of nitrogen in irrigation water.
2.2. Cold acclimation
Four to five-week old plants grown in the green-house were transferred to walk-in cold chambers set at 4/2°C (day/night) with a 10 h photoperiod (200mE m−2s−1) and were held for up to 4 weeks
for cold acclimation. Soil mix was always kept moist during the cold acclimation treatment to avoid any water stress to the plants.
2.3. E6aluation of cold tolerance
Cold tolerance in leaves was determined follow-ing the procedure described previously [24]. Whole leaves were first wrapped in a moist paper towel and subsequently, partially wrapped with alu-minum foil and placed in 2.5×20 cm test tubes. The samples were cooled at 2°C/h in a pro-grammable freezer (Tenney Engineering, NJ) with ice nucleation of samples between −1 and −2°C. Samples were removed from the freezer after reaching various test temperatures and allowed to thaw at 4°C for 12 h. The thawed samples were
evaluated for injury using electrolyte leakage. Conductivity was measured in the leachate using a YSI conductance meter (Model 32, YSI Co., OH) after incubating samples in a 10 – 15 ml aliquot of distilled water for 10 h. The total electrolytes were released from the leaves by autoclaving at 121°C for 15 min. The final conductivity measurement was made after incubating the samples for 10 h. The midpoint in the transition of electrolyte leak-age was considered the killing temperature as out-lined elsewhere [24]. The experiment was conducted with four replications in a completely randomized design.
Survival and regrowth experiments were con-ducted using unhardened, whole plants. Plants were subjected to cooling at 2°C/h in a pro-grammable freezer. Plants were seeded with ice between −1 and −2°C. After reaching the test temperatures, plants were removed from the freezer and allowed to thaw at 4°C for 12 h. Plants were subsequently transferred to a growth cham-ber at 20 – 18°C with a 12 h photoperiod for recovery and regrowth. Freezing injury was evalu-ated on the shoots two days after the freezing tests. Evaluation of plant survival was based on lethal injury to the shoots which typically did not reflect the survival of regenerative crowns. Evalua-tion of regrowth was conducted periodically over a 4 week period.
2.4. Exogenous ABA and glycine betaine treatments
Plants were sprayed with 100 mM (9) ABA
mixed isomers (Sigma Chemical Co., MO) in 0.1 mM CaCl2 with 0.02% Tween-20 until the shoots
and foliage were covered completely with the solu-tion. Control plants were treated with a solution containing 0.1 mM CaCl2 and 0.02% Tween-20
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). Tenml of the
extract was dried under a stream of nitrogen in a desiccator, and the final volume was made up to 1 ml with D2O. Glycine betaine was quantified using
a Varian UNITY Plus 500 NMR spectrometer operating at 11.75 T (499.869 MHz for 1H 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. 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. LSD0.05for 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 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).
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 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
Glycine betaine levels increased readily in re-sponse to cold acclimation conditions in straw-berry plants. Plants acquired most of their cold (Fig. 3). In addition, it is interesting to note that a
rapid increase in the leaf glycine betaine levels preceded the large increase in cold tolerance of leaves, which occurred 48 h after glycine betaine application. A marked increase in the glycine be-taine levels occurred 24 h before significant in-crease in cold tolerance was observed suggesting
Table 1
Freezing survival of strawberry plants treated with glycine betainea
aPlants were treated with 2 mM glycine betaine as a foliar
spray. Potted plants were cooled at 2°C/h to various test temperatures and were grown in a growth chamber after thawing at 4°C. Data on survival of shoots were collected after 2 days of the freezing test while those on regrowth were collected over a 2–4 week period.
bPlants produced flowers after 2 weeks of freezing. cRegrowth of new shoots from the crown was observed in
tolerance, over 80%, during the first week of cold acclimation. The endogenous leaf glycine betaine levels increased during the first 3 weeks of cold acclimation treatment. Both endogenous glycine betaine levels and cold tolerance of leaves in-creased during the first 3 weeks of cold acclima-tion. The results suggest that cold acclimation in strawberry plants is associated with the increase in the endogenous glycine betaine levels. This is con-sistent with previous reports which show higher glycine betaine levels in the leaves of rye, barley, and wheat in response to cold acclimation
[13,15,16]. In barley, cold acclimation was associ-ated with increased levels of betaine aldehyde de-hydrogenase [26], one of the enzymes involved in the synthesis of glycine betaine.
Considering the effect of exogenous glycine be-taine on the cold tolerance of strawberry leaves, it is reasonable to conclude that a rise in the endoge-nous glycine betaine levels during cold acclimation may trigger cold tolerance in these plants. Exoge-nous application of ABA to plants also induced an increase in glycine betaine levels in unhardened strawberry plants. These results support the
vations by Ishitani et al. [26], who found increased betaine aldehyde dehydrogenase mRNA in barley, suggesting elevated levels of glycine betaine, in response to exogenous ABA. A time-course study on the accumulation of glycine betaine indicated that most of its accumulation in the leaves oc-curred from 48 to 72 h of ABA application sug-gesting a rapid response to ABA.
We found that exogenous application of ABA to unhardened and cold-hardened strawberry plants can also induce cold tolerance. ABA has been shown to be associated with cold acclimation and can induce cold tolerance in a wide range of plant species and cell cultures [18,19,22,27]. In addition, in our studies ABA application to straw-berry plants resulted in a more rapid induction of cold tolerance in the leaves than did the cold acclimation treatment. Thus, ABA was effective in inducing cold tolerance in both unhardened and cold-hardened plants. This is consistent with sev-eral studies in which ABA was found to be more effective in many cell cultures and whole plants in inducing cold tolerance than the cold acclimation treatment alone [18,20,23].
Results from the exogenous application of glycine betaine to plants showed that glycine be-taine can induce cold tolerance in leaves which appear to take up exogenous glycine betaine 24 h after its application. Byerrum et al. [28] observed that most of the exogenously applied glycine be-taine (over 98%) was taken up by tobacco plants in a solution culture. Recently, Gibon et al. [29] observed that rape leaf discs took up large amounts of exogenous glycine betaine and they suggested that the uptake of glycine betaine by the leaves may involve specific transporters.
In our studies when glycine betaine was applied to the leaves, it was not affected by the microflora on the leaves as suggested by the unchanged con-centration of glycine betaine and a lack of micro-bial presence in the leaf washings that were incubated for up to 3 days. Also, Xing [30] ob-served that exogenous glycine betaine application toArabidopsis thaliana, grown under sterile condi-tions, increased the cold tolerance of plants. Thus, these results suggest that glycine betaine, when exogenously applied, is rapidly taken up by the plants and can induce cold tolerance.
The fact that leaf cold tolerance of unhardened plants was nearly doubled by foliar application of glycine betaine shows the possible involvement of
glycine betaine on the cold tolerance in strawberry plants. This is further supported by the data on time-course accumulation of glycine betaine in the plants which suggest that glycine betaine is in-volved in the induction of cold tolerance in straw-berry leaves.
Glycine betaine has been suggested to provide protection to membranes against freezing in alfalfa [13]. Sakai and Yoshida [31] found that glycine betaine can increase the freezing resistance of cab-bage cells. However, in both studies higher levels of glycine betaine were used than in these studies. In preliminary studies we did not observe levels higher than 2 mM to have any additional effect in inducing cold tolerance in strawberries.
Many studies have documented the ability of ABA to induce cold tolerance in plants. Results from our study show that glycine betaine accumu-lates not only in response to exogenous ABA but also to the natural cold acclimation process in strawberries. Thus, ABA may induce cold toler-ance by triggering the synthesis of glycine betaine in strawberry plants. Such a mode of action is supported further by the findings of Ishitani et al. [26], who observed higher levels of expression of betaine aldehyde dehydrogenase, an enzyme cata-lyzing the final step in the biosynthesis of glycine betaine, in response to exogenous ABA.
Glycine betaine application resulted in increased cold tolerance of whole plants as well. In addition to better survival, plants treated with glycine be-taine showed better regrowth 2 weeks after the freezing tests than did the untreated plants. In fact, at lower temperatures only glycine betaine-treated plants showed regrowth, despite the low survival of shoots, suggesting that glycine betaine may protect dormant crown tissue, which is criti-cal for regrowth in the spring. Similar observa-tions were made on alfalfa crowns in which glycine betaine treatment resulted in better freezing toler-ance [13]. Thus, these results show that glycine betaine can have a favorable effect on the cold tolerance of strawberry crowns. Recently, Nolte et al. [32] have suggested the possibility of improving cold tolerance in citrus by bioengineering plants with elevated levels of glycine betaine and other osmoprotectants.
levels and induce cold tolerance in the leaves. In addition, exogenous application of glycine betaine can induce cold tolerance in both unhardened and cold acclimating plants. Both exogenous ABA and glycine betaine could induce an additional increase in cold tolerance over that resulting from cold acclimation treatment alone.
Acknowledgements
We thank Dr Andrew Hanson for providing valuable suggestions during the study. The study was supported by the Kansas Agricultural Experi-ment Station (Contribution No. 96-313-J).
References
[1] R.G. Wyn Jones, R. Storey, Betaines, in: L.G. Paleg, D. Aspinall (Eds.), The Physiology and Biochemistry of Drought Resistance in Plants, Academic Press, Sydney, 1981, pp. 171 – 204.
[2] K.F. McCue, A.D. Hanson, Drought and salt tolerance: towards understanding and application, Trends Biotech. 8 (1990) 358 – 362.
[3] A.D. Hanson, W.D. Hitz, Metabolic responses of meso-phytes to plant water deficits, Annu. Rev. Plant Physiol. 33 (1982) 163 – 203.
[4] D. Rhodes, A.D. Hanson, Quarternary ammonium and tertiary sulfonium compounds in higher plants, Annu. Rev. Plant Physiol. Plant Mol. Biol. 44 (1993) 357 – 384. [5] A. Incharoensakadi, T. Takabe, T. Akazawa, Effect of betaine on enzyme activity and subunit interaction of ribulose-1, 5-bisphosphate carboxylase/oxygenase from
Aphanothece halophytica, Plant Physiol. 81 (1986) 1044 – 1049.
[6] Y. Jolivet, F. Larher, J. Hamelin, Osmoregulation in halophytic higher plants: The protective effect of glycine betaine against the heat destabilization of membranes, Plant Sci. Lett. 25 (1982) 193 – 201.
[7] S. Laurie, G.R. Stewart, The effects of compatible so-lutes on the heat stability of glutamine synthetase from chickpeas grown under different nitrogen and tempera-ture regimes, J. Exp. Bot. 41 (1990) 1415 – 1422. [8] L.G. Paleg, G.R. Stewart, J.W. Bradbeer, Proline and
glycine betaine influence protein solvation, Plant Physiol. 75 (1984) 974 – 978.
[9] A.S. Rudolph, J.H. Crowe, L.M. Crowe, Effects of three stabilizing agent- proline, betaine, and trehalose-on membrane phospholipids, Arch. Biochem. Biophys. 245 (1986) 134 – 143.
[10] C.L. Winzor, D.J. Winzor, L.G. Paleg, G.P. Jones, B.P. Naidu, Rationalization of the effects of compatible so-lutes on protein stability in terms of thermodynamic nonideality, Arch. Biochem. Biophys. 296 (1992) 102 – 107.
[11] R. Ko, L.T. Smith, G.M. Smith, Glycine betaine confers enhanced osmotolerance and cryotolerance on Listeria monocytogenes, J. Bacteriol. 176 (1994) 426 – 431. [12] S.J. Coughlan, U. Heber, The role of glycine betaine in
the protection of spinach thylakoids against freezing stress, Planta 156 (1982) 62 – 69.
[13] Y. Zhao, D. Aspinall, L.G. Paleg, Protection of mem-brane integrity inMedicago sati6aL. by glycine betaine
against the effects of freezing, J. Plant Physiol. 140 (1992) 541 – 543.
[14] B.P. Naidu, L.G. Paleg, D. Aspinall, A.C. Jennings, G.P. Jones, Amino acid and glycine betaine accumulation in cold-stressed wheat seedlings, Phytochem. 30 (1991) 407 – 409.
[15] S. Kishitani, K. Watanabe, S. Yasuda, K. Arakawa, T. Takabe, Accumulation of glycine betaine during cold acclimation and freezing tolerance in leaves of winter and spring barley plants, Plant Cell Environ. 17 (1994) 89 – 95.
[16] K.L. Koster, D.V. Lynch, Solute accumulation and com-partmentation during cold acclimation of Puma rye, Plant Physiol. 98 (1992) 108 – 113.
[17] H.H. Chen, P.H. Li, M.L. Brenner, Involvement of abscisic acid in potato cold acclimation, Plant Physiol. 71 (1983) 362 – 365.
[18] V. Lang, P. Heino, E.T. Palva, Low temperature accli-mation and treatment with exogenous abscisic acid in-duce common polypepetides inArabidopsis thaliana(L.) Heynh, Theor. Appl. Gen. 77 (1989) 729 – 734.
[19] T.H.H. Chen, L.V. Gusta, Abscisic acid-induced freezing resistance in cultured plant cells, Plant Physiol. 73 (1983) 71 – 75.
[20] C.N. Keith, B.D. McKersie, The effect of abscisic acid on the freezing tolerance of callus cultures of Lotus corniculatusL, Plant Physiol. 80 (1986) 766 – 770. [21] M.J.T. Reaney, L.V. Gusta, Factors influencing the
in-duction of freezing tolerance by abscisic acid in cell suspension cultures of Bromus inermis Leyss and
Medicago sati6aL, Plant Physiol. 83 (1987) 423 – 427.
[22] R.W. Wilen, L.V. Gusta, B. Lei, S.R. Abrams, B.E. Ewan, Effects of abscisic acid (ABA) and ABA analogs on freezing tolerance, low-temperature growth, and flow-ering in rapeseed, J. Plant Growth Regul. 13 (1994) 235 – 241.
[23] A.J. Robertson, L.V. Gusta, M.J. Reaney, M. Ishikawa, Protein synthesis in bromegrass (Bromus inermis Leyss) cultured cells during the induction of frost tolerance by abscisic acid or low temperature, Plant Physiol. 84 (1987) 1331 – 1336.
[24] C.B. Rajashekar, P.H. Li, J.V. Carter, Frost injury and heterogeneous ice nucleation in leaves of tuber-bearing
Solanum species; Ice nucleation activity of external source of nucleants, Plant Physiol. 71 (1983) 749 – 755. [25] G.P. Jones, B.P. Naidu, R.K. Starr, L.G. Paleg,
Esti-mates of solutes accumulating in plants by 1H nuclear
magnetic resonance spectroscopy, Aus. J. Plant Physiol. 13 (1986) 649 – 658.
[27] A.M. Johnson – Flanagan, Z. Huiwen, M.R. Thi-agarajah, H.S. Saini, Role of abscisic acid in the induction of freezing tolerance in Brassica napus sus-pension-cultured cells, Plant Physiol. 95 (1991) 1044 – 1048.
[28] R.U. Byerrum, C.S. Sato, C.D. Ball, Utilization of be-taine as a methyl group in tobacco, Plant Physiol. 31 (1956) 374 – 377.
[29] Y. Gibon, M.A. Bessieres, F. Larher, Is glycine betaine a non-compatible solute in higher plants that do not accumulate it?, Plant Cell Environ. 20 (1997) 329 – 340.
[30] W. Xing, Studies on the role of glycine betaine and pathogenesis-related proteins in cold and drought toler-ance of plants, Ph.D. Thesis, Kansas State University, 1997.
[31] A. Sakai, S. Yoshida, The role of sugars and related compounds in variations of freezing resistance, Cry-obiol. 5 (1968) 160 – 174.
[32] K.D. Nolte, A.D. Hanson, D.A. Gage, Proline accu-mulation and methylation to proline betaine in Citrus: Implications for genetic engineering of stress resistance, J. Amer. Soc. Hort. Sci. 122 (1997) 8 – 13.
