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The seasonal pattern of dehydrin accumulation was characterized during cold acclimation and deacclimation in the xylem tissues of genetically related (sibling) deciduous and evergreen peach (Prunus persica L.). Immunological studies indicate that a 60-kD polypeptide in peach xylem tissues is a dehydrin protein. Comparison of its accumulation pattern with seasonal fluctuations in cold hardiness indicate that dehydrin accumulated to high levels during the peak of cold acclimation. However, its accumulation was only weakly associated with cold hardiness during early stages of cold acclimation and during deacclimation. Our results indicate that factors related to supercooling rather than dehydrin accumulation may be primarily responsible for determining levels of cold hardiness during transition periods.

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.

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.

Freezing is a major environmental stress during an annual cycle of overwintering, temperate-zone perennials. The timing and extent of seasonal cold acclimation (development of freezing tolerance in the fall) and deacclimation (loss of acquired freezing tolerance in response to warm temperatures) are of critical importance for winter survival, particularly in view of the climate change, i.e., unpredictable extreme weather occurrences. For example, plants may acclimate inadequately if exposed to a milder fall climate and may be damaged by sudden frosts. Alternatively, they may deacclimate prematurely as a result of unseasonable, midwinter warm spells and be injured by the cold that follows. Efficient cold acclimation ability, high deacclimation resistance, and efficient reacclimation capacity are, therefore, important components of winter survival in overwintering perennials. These components should be evaluated separately for a successful breeding program focused on improving winter-hardiness. Another layer of complexity that should be carefully considered is that endodormant status (shallow versus deep) of the reproductive/vegetative apices can significantly impact these components of winter-hardiness. Winter survival, especially by woody evergreens, requires tolerance of light stress, which can result in photo-oxidative damage at cold temperatures when biochemistry of photosynthesis is somewhat compromised but light harvesting is unaffected. Accumulation of Elips (early light-induced proteins) in overwintering evergreens during winter represents a relatively novel strategy to cope with such light stress, and investigations on the precise cellular mechanism and genetic control of this strategy deserve research in the future. Investigations into the mechanisms for cold acclimation use laboratory-based, artificial acclimation protocols that often do not closely approximate conditions that plants are typically exposed to in nature. To draw meaningful conclusions about the biology of cold acclimation and ultimately improve freeze resistance under field conditions, one should also include in cold acclimation regimens parameters such as exposure to subfreezing temperatures and realistic diurnal temperature fluctuations and light levels to simulate natural conditions. One of the main objectives of this article is to highlight two areas of research that we believe are important in the context of plant cold-hardiness but, so far, have not received much attention. These are: 1) to understand the biology of deacclimation resistance and reacclimation capacity, two important components of freeze-stress resistance (winter-hardiness) in woody perennials; and 2) to investigate the cellular basis for various strategies used by broad-leaved evergreens for photoprotection during winter. Our emphasis, in this context, is on a family of proteins, called Elips. The second objective of this article is to draw attention of the cold-hardiness research community to the importance of using realistic cold acclimation protocols in controlled environments that will approximate natural/field conditions to be better able to draw meaningful conclusions about the biology of cold acclimation and ultimately improve freeze resistance. Results from our work with Rhododendron (deciduous azaleas and broad-leaved evergreens), blueberry, and that of other researchers are discussed to support these objectives.

Seasonal pattern of cold tolerance and proteins were studied in the leaves of sibling deciduous and evergreen peach (Prunus persica). In contrast to deciduous peach that undergoes endodormancy in fall, evergreen peach does not (leaves are retained and shoot tips elongate under favorable conditions) (Arora et al., Plant Physiol. 99:1562-1568). Cold tolerance (LT₅₀) was assessed using electrolyte leakage method. Proteins were separated by SDS-PAGE. Electroblots were probed with anti-dehydrin (Dr. T. Close) and anti-19 kD, peach bark storage protein (BSP) antibodies. LT₅₀ of leaves successively increased from about -7C (18 Aug.) to -15C and -11.5C (23 Oct.) in deciduous and evergreen genotypes, respectively. The most apparent change in the protein profiles was the accumulation of a 60-kD protein during cold acclimation in the leaves of deciduous trees; however, it did not change significantly in evergreen peach. Immunoblots indicate that 60-kD protein is a dehydrin protein. PAGE and immunoblots indicated that 19-kD BSP disappeared progressively during summer through fall in the leaves of deciduous peach, but accumulated to large amounts in bark tissues. Similar inverse relationship for its accumulation in leaf vs. bark tissue was not evident in evergreen peach. Results indicate that BSP expression may be regulated by altered source/sink relationship.

Seasonal changes in cold tolerance and proteins were studied in the leaves of sibling deciduous and evergreen peach [Prunus persica (L.) Batsch]. Freezing tolerance [defined as the subzero temperature at which 50% injury occurred (LT₅₀)] was assessed using electrolyte leakage. Proteins were separated by sodium dodecyl sulfate polyacrylamide-gel electrophoresis. Electroblots were probed with anti-dehydrin and anti-19-kD peach bark storage protein (BSP) antibodies. Leaf LT₅₀ decreased successively from -5.8 °C on 18 Aug. to -10.3 °C in the evergreen genotype and from -7.0 °C to -15.0 °C in the deciduous genotype by 14 Oct. Protein profiles and immunoblots indicated the accumulation of a 60- and 30-kD protein during cold acclimation in the leaves of deciduous trees; however, levels of these proteins did not change significantly in the evergreen trees. Immunoblots indicate that the 60-kD protein is a dehydrin-like protein. Gel-electrophoresis and immunoblots also indicated that the 19-kD BSP progressively disappeared from summer through fall in leaves of deciduous peach but accumulated to a high level in bark tissues. A similar inverse relationship was not evident in evergreen peach.

The vase life of roses grown in coal bottom ash (CBA)-amended media was evaluated. CBA is enriched in calcium, a nutrient implicated in delaying senescence. Two rose cultivars, Cara Mia and Dakota, were grown (from started eye plants) in four media: a 50% CBA medium and a peat:vermiculite medium amended with calcitic and dolomitic lime (1:1) were used as “high calcium” media, whereas a 25% CBA medium and a peat:vermiculite medium amended with dolomitic lime only were used as “low calcium” media. Vase life of the freshly harvested roses was evaluated. Elemental analysis of the leaves showed that roses grown in the “high calcium” media had greater calcium in the leaf tissue as well as longer vase lives (12.6 and 13.5 days) when compared to those grown in the “low calcium” media (12.1 and 10.9 days). However, petal tissue Ca was not affected by media and was not correlated with vase life. Petal tissue calcium was ≈15 times lower than leaf tissue calcium. Calcium and magnesium increased in the petal tissue over the vase life of the senescing petals. A comparison of `Cara Mia' roses (vase life of 14 days) and `Dakota' roses (vase life of 8.5 days) showed that the longer-lived `Cara Mia' had lower leaf and petal calcium levels. Both varieties followed a similar kinetics of electrolyte leakage (total E.C. and K) during their respective vase lives.

Intron length polymorphisms were used to investigate relationships among eight Rhododendron L. species (R. catawbiense Michaux., R. minus Michaux., R. ponticum L., R. keiskei Miquel., R. arboreum Sm., R. dichroanthum Diels., ssp. scyphocalyx Cowan., R. maximum L. and R. dauricum L.) and two hybrid cultivars [i.e., R. `PJM' (R. minus var. minus × R. dauricum) and R. `Chionoides' (R. ponticum × unknown)]. A total of 27 of these markers were used to estimate phylogenetic relationships among the species and draw inferences about the parentage of the cultivars, which is partially unknown. In general the expressed sequence tag-polymerase chain reaction (EST-PCR) marker-based phylogenetic map of the eight species is congruent with the currently accepted morphology-based classification of these species at the subgenus as well as the section level. However, the constructed phylogenetic tree revealed that, at the subsection level, two species, R. arboreum (subsection Arborea Sleum.) and R. dichroanthum (subsection Neriiflora Sleum.), are grouped under the same “clade” (80% bootstrap score), suggesting that these species are more closely related than indicated in the current classification system that places them in separate subsections/clades. Moreover, our phylogenetic analysis of the three species belonging to section Ponticum G. Don. demonstrated a closer phylogenetic relationship between R. ponticum and R. maximum (bootstrap score of 74%) than between these species and R. catawbiense; such observation is consistent with a recent phylogenetic analysis of section Ponticum by Milne (2004) using the sequences of a chloroplast gene. Parentage analysis for the two cultivars confirmed the interspecific lineage of R. `PJM' and provided genetic support for the speculated R. ponticum and R. maximum parentage of R. `Chionoides'. Our results indicate that, in addition to their use in mapping studies, intron-flanking EST-based PCR markers are valuable tools for conducting phylogenetic and parentage analyses and/or gene flow studies.

Studies with herbaceous crops have indicated a similarity in the types of proteins that accumulate in response to environmental stresses and ABA. Many of these proteins belong to a group called dehydrins. We have identified a 60 kDa dehydrin-related protein (PCA 60) in peach associated with cold hardiness. Our objective was to determine if seasonal induction of dehydrins are a common feature in a wide array of woody plants Bark tissues from eight species of woody plants were collected monthly for 1.5 years. The species included: Prunus persica `Loring'; Malus domestica `Golden Delicious'; Rubus sp. `Chester'; Populus sp.; Salix babylonica; Cornus florida; Sassafras albidum, and Robinia Pseudo-acacia. Protein extraction, SDSPAGE, and immunoblotting were performed as previously reported. Immunoblots were probed with a polyclonal antibody recognizing a conserved region of dehydrin proteins (provided by Timothy Close). Although some proteins, immunologically related to dehydrins, appeared lo be constitutive, distinct seasonal patterns associated with winter acclimation were observed in all species. The molecular weights of these proteins varied, although there were similarities in related species (willow and poplar). Although this study represents a precursory examination of dehydrins, the results indicate that these proteins are common to woody plants and that further research to characterize their function is warranted.

Deciduous fruit trees undergo endo-dormancy during fall at which time they also attain maximum cold hardiness (CH). Because these two processes occur simultaneously it is difficult to study them independently. We have been able to overcome this limitation with the use of genetically related (sibling) deciduous and evergreen peach trees. Using this system we conducted a time course study to characterize the seasonal fluctuations in CH and proteins in bark and xylem tissues. Cold hardiness (LT₅₀) was assessed using electrolyte leakage method. Polypeptides were separated using SDS-PAGE. The data indicated that 1) CH of bark increased from -5°C (in August) to -49°C (in January) and from -3°C to -22°C for deciduous and evergreen trees, respectively. In January, under favorable conditions, evergreen trees were actively growing. 2) CH of xylem successively increased from -11°C to -36°C in deciduous trees and from -7°C to -16°C (in November) in evergreen trees and then plateaued. 3) LT₅₀ of xylem in both genotypes closely approximated the mid-point of low temperature exotherms determined by differential thermal analysis. 4) As CH increased several qualitative and quantitative differences in polypeptides were noted between two genotypes. These changes during cold acclimation will be compared with those during de-acclimation.