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Björn H. Karlsson and Jiwan P. Palta

Recent studies suggest cold-regulated heat-stable proteins mitigate the potential damaging effects of low water activity associated with freezing. A proposed function of these proteins is stabilization of enzymes during exposure of plants to subzero temperatures. To test this hypothesis for tuber-bearing Solanum L. species we determined the quantitative expression of heat-stable proteins, the qualitative changes in dehydrin proteins, and the capacity of heat-stable proteins to cryoprotect a freeze-thaw labile enzyme lactate dehydrogenase (LDH). We used five tuber-bearing Solanum species (S. tuberosum L. `Red Pontiac', S. acaule Bitter, S. sanctae rosea Hawkes, S. commersonii Dunal, and S. cardiophyllum Bitter), which vary in nonacclimated relative freezing tolerance (NA RFT), acclimated relative freezing tolerance (AC RFT), and acclimation capacity (ACC). The protein fraction containing a mixture of heat-stable proteins demonstrated cryoprotective capacities greater or equal to other cryoprotective compounds (bovine serum albumin, polyethylene glycol, glycerol, and sucrose). Heat-stable proteins extracted from acclimated S. commersonii had superior cryoprotective capacity than those extracted from nonacclimated S. commersonii plants. Interestingly, in the presence of these proteins extracted from acclimated plants (in S. commersonii and S. sanctae rosea), LDH activity was elevated above that of unfrozen controls. No quantitative relationships were found between heat-stable protein concentration and NA RFT, AC RFT, or ACC among the five species. This was also true for dehydrin protein expression. Cold acclimation treatment resulted in increased dehydrin expression for acclimating and nonacclimating species. In three of the cold acclimating species (S. acaule, S. sanctae rosea, and S. commersonii), an increase in dehydrin expression may play a role in increased freezing tolerance during cold acclimation. In the cold sensitive, nonacclimating species (S. tuberosum and S. cardiophyllum), however, an increase in dehydrin level maybe related to the response of these species to changed (perhaps stressful) environment during cold treatment. By exploiting the genetic variation in NA RFT and ACC for five tuber-bearing species, we were able to gain new insight into the complexity of the relationship between heat-stable protein and cold response.

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Reeser C. Manley and Rita L. Hummel

The index of injury (It) and tissue ionic conductance (gTi) formulas for analyzing electrolyte leakage data from freeze-stressed tissues of cabbage (Brassica oleracea L. Capitata group) were compared. The two formulas produced similar results in calculating the relative freezing responses of stem pith, lamina, and petiole tissues. Disagreement occurred only with lamina tissues when the magnitude of ion leakage was low. Vital staining of pith and petiole tissues with triphenyl tetrazolium chloride indicated that the tissue TK50 (the temperature resulting in 50% injury), derived from It data, was a reliable indicator of the freeze-killing point. These results support the use of the simpler It method for analyzing electrolyte leakage data in studies of cabbage freezing tolerance.

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Robert K. Stevenson and Karen K. Tanino

Dogwood (Cornus sericea L.) clonal ecotypes from northern latitudes (Northwest Territories “NWT”) and more southern latitudes (Massachusetts, Utah, and Chalk River, Ont.) were allowed to acclimate naturally in a shade house (52°07') beginning in early July and continuing through the middle of October. The NWT ecotype began to attain vegetative maturity by the second week of September, whereas the southern ecotypes did not attain any significant degree of VM before the first lethal frost.

Defoliation tests in controlled environment chambers paralleled shade house results. Under VM-inducing conditions (20/15°C, 8h), NWT ecotype attained VM after 40-50 days. Conversely, after 80 days Utah ecotype had not attained full VM.

Chilling requirement will be compared among ecotypes and ABA levels will be quantified using HPLC and ELISA systems. The results will be compared with date of VM attainment, subsequent freezing tolerance and satisfaction of the chilling requirement.

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Patrick P. Moore and Rita L. Hummel

Days to bud break and freezing tolerance of `Chilcotin', `Chilliwack', `Meeker' and `Willamette' red raspberry were measured during the 1990-1991 winter and at monthly intervals from mid-September 1991 through mid-March 1992. Canes were harvested from the field and cut into two-bud samples which were either frozen in laboratory tests or held with cut stem ends in water in a controlled environment chamber and monitored daily until bud growth was observed. Viability was estimated by visual browning after exposure to controlled laboratory freezing treatments. In general, freeze test results indicated `Meeker' and `Willamette' were not as hardy as `Chilliwack' and `Chilcotin' in late fall and midwinter but retained their hardiness longer in spring. Results for 1990-1991 indicated the greatest delay in days to bud break occurred in midwinter.

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Sandra E. Vega-Semorile, John B. Bamberg and Jiwan P. Palta

Frost damage to the foliage is a common problem where potatoes are grown, and results in significant reductions in tuber yield. Frost injury also limits the cultivation of high-yielding S. tuberosum cultivars in the mountain regions of Central and South America, where potato is a staple crop. Recent studies have shown that some wild potato species possess a high degree of non-acclimated frost tolerance (growing in normal conditions) as well as high cold acclimation capacity (able to increase frost tolerance upon exposure to cold). Natural frosts affecting potatoes are of two types: a) late spring or early fall frost, where the minimum temperature during the frost episode can be very low; b) frost during the growing season, where the minimum temperature during the frost episode is not as low. It is expected that potato species able to acclimate rapidly would survive better from the latter type of frosts, whereas species having higher acclimation capacity might have a great chance to survive better from the former type of frosts. The objective of this study was to find out if there is genetic variability for the speed of acclimation among different tuber-bearing wild potato species. The species used were: S. acaule, S. commersonii, S. megistacrolobum, S. multidissectum, S. polytrichon, S. sanctae-rosae, and S. toralapanum. Relative freezing tolerance of these species was measured during cold acclimation. Preliminary results suggest that there are differences in the speed of acclimation among these species. We found that these species can be divided into four groups: i) non-acclimators; ii) rapid acclimators, with low to medium acclimation capacity; iii) slow acclimators, with low to medium acclimation capacity; iv) slow acclimators, with high acclimation capacity. We plan to use this information in our breeding program aimed at improving the freezing tolerance of potatoes.

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Rajeev Arora and Chon-Chong Lim

Many reports have shown the accumulation of specific proteins associated with cold acclimation in plants. However, there is a scarcity of data on the physiological and/or biochemical changes associated with deacclimation process. This study was initiated to determine protein changes specifically associated with deacclimation in Rhododendron. Current-year leaves were collected from three Rhododendron cultivars (`Chionoides', `Grumpy Yellow', and `Vulcanís Flame'; ≈4-year-old rooted cuttings) during natural non-acclimated (June), cold-acclimated (January), and deacclimated (May) state. Leaf freezing tolerance was evaluated using controlled freezing protocol (Lim et al. 1998, J. Amer. Soc. Hort. Sci. 123:246–252). Seasonal SDS-PAGE profiles exhibited a distinct accumulation of 27 kDa protein in deacclimated and nonacclimated tissues, but this protein was essentially undetectable in cold acclimated tissues of all three cultivars. Further characterization of this polypeptide, labeled as RhDAP27 (for rhododendron deacclimation protein), revealed that it has an iso-electric point of 6.5, has a compositional bias for Glu/Gln (13.9%), His (11.4%), Gly (11%), Ala (10%), Lys (8.3%), and Asp/Asn (8.1%)—hydrophilic amino acids constitutedabout 54% of the total amino acids while 40% were nonpolar, aliphatic amino acids (Gly, Ala, Val, Leu, Ile, Pro) and only 6% were aromatic amino acids (Phe and Tyr). Micro-sequencing of the four peptides produced by partial cleavage of RhDAP27 revealed a striking homology of RhDAP27 with two proteins (from Mesembryanthemum crystallinum and Pinus taeda) that belong to the family of ABA stress ripening/water deficit stress inducible proteins.

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Sandra E. Vega and Jiwan P. Palta

Previous studies in our laboratory both in pine needles and potato leaves have shown evidence of an increase in 18: 2 (linoleate) in the purified plasma membrane fraction during cold acclimation. This increase was reversible on deacclimation, thereby suggesting a link between the accumulation of 18: 2 and acquisition of freezing tolerance. These studies suggest that the activity of specific desaturases may be modulated during cold acclimation. This study was aimed at studying the possible involvement of stearoyl-ACP desaturase (delta9) in potato cold acclimation response. Our approach was to study the induction of delta9 desaturase at the transcript level by using potato delta9 desaturase gene specific primers and reverse transcriptase. For this purpose, mRNA from S. tuberosum (cold sensitive, unable to acclimate) and S. commersonii (cold tolerant, able to cold acclimate) was extracted before and after acclimation. Sequence analysis confirmed that the amplified band was delta9 desaturase. Our results show that there is an increase in delta9 desaturase gene transcripts during cold acclimation and that this increase is associated with the cold acclimation response in potato. These results together with previous reports on the increase in 18: 2 in the plasma membrane during cold acclimation give more evidence toward the involvement of stearoyl-ACP desaturase (delta9) in the potato cold response.

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Milton E. Tignor, Frederick S. Davies, Wayne B. Sherman and John M. Davis

Poncirus trifoliata (L.) Raf. seeds were germinated in perlite under intermittent mist at about 25 °C and natural daylight in a greenhouse. Two-week-old seedlings were then transferred into a growth chamber at 25 °C and 16-hour daylength for 1 week. Tissue samples were collected at 0, 6, 24, 168, and 504 hours after temperature equilibration at 10 °C. Freezing tolerance at –6.7 °C, as determined by electrolyte leakage, and stem (leaves attached) water potential (ψx), measured using a pressure chamber, was recorded for a subset of seedlings for each time interval. Red coloration (apparently anthocyanin) developed at the petiole leaflet junction and buds after 48 hours at 10 °C and gradually occurred throughout the leaves during further exposure. Complementary DNA clones for phenylalanine ammonia lyase (PAL), 4-coumarate: coA ligase (4CL), and chalcone synthase (CHS) were used to probe RNA isolated from the leaves. No increase in steady-state messenger RNA level was detected. Increases in freeze hardiness occurred within 6 hours in the leaves, and continued for up to 1 week. Water potential initially decreased from –0.6 to –2.0 MPa after 6 hours, then returned to –0.6 MPa after 1 week. Thus, Poncirus trifoliata seedlings freeze-acclimate significantly after only 6 hours at 10 °C.

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Steven P. Castagnoli, Leslie H. Fuchigami, Tony H. H. Chen and Liping Zhen

Studies were performed on the development of dormancy, cold hardiness, and desiccation tolerance, and the effect of manual defoliation timing on performance of `Fuji' and `Braeburn' apple nursery stock. Dormancy development, response to defoliation, and desiccation tolerance of apple differed from those reported for other temperate woody plant species. Dormancy development in `Fuji' was approximately two weeks ahead of `Braeburn', and was strongly regulated by temperature. Photoperiod had no influence on dormancy development of `Fuji'. Desiccation tolerance of both varieties was greatest just prior to the onset of dormancy and early dormancy. This pattern in the seasonal development of tolerance to desiccation is not typical of temperate woody plant species. Early defoliation was detrimental to performance of `Braeburn', but had little effect on `Fuji'. Early defoliation promoted earlier spring budbreak in `Fuji'. Development of freezing tolerance in both apple varieties was typical of other woody plants, and coincided with the onset of dormancy. Maximum hardiness was achieved after the requirements for dormancy were completely satisfied.

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Rajeev Arora and Lisa J. Rowland

To survive winters, woody perennials of temperate zone must enter into endodormancy. Resuming spring growth requires sufficient exposure to low temperature or chill units (CUs) in winter, referred to as chilling requirement (CR), which also plays a role in the development of freezing tolerance (cold acclimation; CA). Physiological studies on the breaking of dormancy have focused on identifying markers, such as appearance or disappearance of proteins in response to varying degrees of CU accumulation. However, whether these changes are associated with breaking dormancy or CA is not clear. We conducted a study, using greenhouse blueberry (Vaccinium section Cyanococcus) plants, to address this question Three blueberry cultivars (`Bluecrop', `Tifblue', and `Gulfcoast'), having CRs of ≈1200, 600, and 400 CUs, respectively, first were exposed to 4° for long enough to provide CUs equivalent to one-half of their respective CRs. This treatment resulted in CA. Plants were then transferred to 15C for 2 weeks (a treatment which should not negate CU accumulation but did result in deacclimation). Before and after each treatment cold hardiness (using a controlled freezing bath) and dormancy status (observe budbreak after placing shoots in water at 20C for 2 to 3 weeks) of floral buds were determined. Proteins were extracted from buds collected, simultaneously and separated by SDS-PAGE. To determine the association of dehydrin-like proteins with dormancy or CA, electroblots were probed with anti-dehydrin antibody. The relationship of protein and western blots data to cold acclimation and dormancy are presented.