Chill-responsive dehydrins in blueberry: Are they associated with cold hardiness or dormancy transitions?

To survive winters, woody perennials of temperate zones must enter into endodormancy. Resumption of spring growth requires sufficient exposure to low temperature (chill units, CUs) in winter (chilling requirement), which also plays a role in the development of cold hardiness (cold acclimation). Physiological studies on dormancy breaking have focused on identifying markers, such as appearance or disappearance of proteins in response to varying degrees of chill unit accumulation. However, whether these changes are associated with dormancy transitions or cold acclimation is not clear. In the present study, greenhouse-grown blueberry (Vaccinium section Cyanococcus) plants were used to address this question. Three blueberry cultivars, Bluecrop, Tifblue, and Gulfcoast having chilling requirement of approximately 1 200, 900 and 600 CUs, respectively, were first exposed to 4°C for long enough to provide chill units equivalent to one-half of their respective chilling requirement. This treatment was expected to result in cold acclimation. A fraction of plants was then subjected to a 15/12°C (light/dark) regime for 2 weeks, a treatment expected to be “dormancy-neutral” but cause deacclimation. Before and after each treatment, cold hardiness and dormancy status of floral buds were determined; proteins were extracted from the buds collected on the same sampling date, and separated by one-dimensional SDS-PAGE. Dehydrin-like proteins were identified by immunoblotting, using anti-dehydrin antiserum. Results indicate that the chilling treatment resulted in cold acclimation as indicated by increased bud hardiness in all three cultivars. Data also indicate a distinct accumulation of three dehydrin-like proteins of 65, 60, and 14 kDa during cold acclimation. The cold hardiness and levels of dehydrin proteins decreased during the exposure to 15/12°C for 2 weeks. Results also confirmed that this treatment had no negative effect on chill unit accumulation. Densitometric scans of protein gels indicated a close association between the abundance of dehydrins and degree of cold hardiness in these cultivars. In addition, levels of the dehydrin proteins and cold hardiness remained about the same between 100% and >100% satisfaction of chilling requirement. These results suggest that changes in dehydrin expression are more closely associated with cold hardiness than with dormancy transitions.     

Transcript profiling in Vitis riparia during chilling requirement fulfillment reveals coordination of gene expression patterns with optimized bud break

Endodormant grapevine buds require a period of chilling before they break and begin to grow. Custom Vitis bud cDNA microarrays (9,216 features) were used to examine gene expression patterns in overwintering Vitis riparia buds during 2,000 h of 4°C chilling. Three-node cuttings collected concurrently with buds were monitored to determine dormancy status. Chilling requirement was fulfilled after 1,500 h of chilling; however, 2,000 h of chilling significantly increased the rate of bud break. Microarray analysis identified 1,469 significantly differentially expressed (p value < 0.05) array features when 1,000, 1,500, and 2,000 h of chilling were compared to 500 h of chilling. Functional classification revealed that the majority of genes were involved in metabolism, cell defense/stress response, and genetic information processing. The number of significantly differentially expressed genes increased with chilling hour accumulation. The expression of a group of 130 genes constantly decreased during the chilling period. Up-regulated genes were not detected until the later stages of chilling accumulation. Hierarchical clustering of non-redundant expressed sequence tags revealed inhibition of genes involved in carbohydrate and energy metabolism and activation of genes involved in signaling and cell growth. Clusters with expression patterns associated with increased chilling and bud break were identified, indicating several candidate genes that may serve as indicators of bud chilling requirement fulfillment. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Abscisic acid catabolism enhances dormancy release of grapevine buds

The molecular mechanism regulating dormancy release in grapevine buds is as yet unclear. It was formerly proposed that dormancy is maintained by abscisic acid (ABA)‐mediated repression of bud–meristem activity and that removal of this repression triggers dormancy release. It was also proposed that such removal of repression may be achieved via natural or artificial up‐regulation of VvA8H‐CYP707A4, which encodes ABA 8′‐hydroxylase, and is the most highly expressed paralog in grapevine buds. The current study further examines these assumptions, and its experiments reveal that (a) hypoxia and ethylene, stimuli of bud dormancy release, enhance expression of VvA8H‐CYP707A4 within grape buds, (b) the VvA8H‐CYP707A4 protein accumulates during the natural transition to the dormancy release stage, and (c) transgenic vines overexpressing VvA8H‐CYP707A4 exhibit increased ABA catabolism and significant enhancement of bud break in controlled and natural environments and longer basal summer laterals. The results suggest that VvA8H‐CYP707A4 functions as an ABA degrading enzyme, and are consistent with a model in which the VvA8H‐CYP707A4 level in the bud is up‐regulated by natural and artificial bud break stimuli, which leads to increased ABA degradation capacity, removal of endogenous ABA‐mediated repression, and enhanced regrowth. Interestingly, it also hints at sharing of regulatory steps between latent and lateral bud outgrowth.     

Co-ordinated gene expression during phases of dormancy release in raspberry (Rubus idaeus L.) buds

Bud break in raspberry (Rubus idaeus L.) is often poor and uneven, with many of the subapical buds remaining in a dormant state. In order to determine the dormancy status of raspberry buds, an empirical measure of bud burst in a growth-permissive environment following exposure to chilling (4 degrees C cold storage) was developed. For cv. Glen Ample, percentage bud burst in intact canes and isolated nodes was recorded after 14 d. Isolated nodes (a measure of endodormancy) achieved 100% bud burst after approximately 1500 h chilling whereas buds on intact plants (combined endo- and paradormancy) required an additional 1000 h chilling. A microarray approach was used to follow changes in gene expression that occurred during dormancy transition. The probes for the microarrays were obtained from endodormant and paradormant raspberry bud cDNA libraries. The expression profiles of 5300 clones from these libraries were subjected to principal component analysis to determine the most significant expression patterns. Sequence analysis of these clones, in many cases, enabled their functional categorization and the development of hypotheses concerning the mechanisms of bud dormancy release. Thus a set of novel candidates for key dormancy-related genes from raspberry buds have been identified. Bud dormancy is fundamental to the study of plant developmental processes and, in addition, its regulation is of significant economic importance to fruit and horticultural industries.     

MORPHOLOGICAL AND ULTRASTRUCTURAL DEVELOPMENT AND STARCH ACCUMULATION DURING CHILLING OF SOUR CHERRY FLOWER BUDS

Floral tissue of dormant sour cherry (Prunus cerasus L.) flower buds was examined by light and electron microscopy during field development and laboratory chilling. Differentiation of flower parts ceased by early November and there was no further morphological development until visible signs of budbreak occurred after the end of rest. Pollen meiosis occurred at the half green budbreak stage. No ultrastructural changes accompanied chilling accumulation except development of numerous starch grains in plastids. Buds on cut shoots held at the non-chilling temperature of 15 C developed more mitochondria but acquired no starch when compared with buds on cut shoots held at a 5 C chilling temperature. Nuclei and nucleoli enlarged with chilling in ovules and sporogenous cells but not in ovary parenchyma. Accumulation of starch with chilling in most bud tissues was demonstrated histochemically.     

Freezing exposure releases bud dormancy in Betula pubescens and B. pendula

Bud dormancy in woody plants is released by long-term exposure to non-freezing chilling temperatures, whereas freezing temperatures have been considered to have little or no effect. However, the present results demonstrate that short-term exposure to freezing can release bud dormancy in Betula pubescens (Ehrh.) and B. pendula (Roth). Short-term freezing during the dormancy induction phase improved the release of bud dormancy only if an adequate level of dormancy had been reached. In fully dormant or chilled plants both the percentage and the speed of bud-burst increased, the more so the lower the temperature. Our results rule out the possibility that endogenous abscisic acid could be directly involved in the physiological control of bud dormancy release. The fast, easily applicable method presented here for bud dormancy release could further investigations into the biochemical and biophysical background to the process. The mechanisms of bud dormancy release and its relationship to cold acclimation are discussed in the light of these results, as also are the implications of the findings for modelling of bud dormancy.     

Epigenetic repressor-like genes are differentially regulated during grapevine (Vitis vinifera L.) development

Grapevine sexual reproduction involves a seasonal separation between inflorescence primordia (flowering induction) and flower development. We hypothesized that a repression mechanism implicating epigenetic changes could play a role in the seasonal separation of these two developmental processes in grapevine. Therefore, the expression of five grapevine genes with homology to the Arabidopsis epigenetic repressor genes FERTILIZATION INDEPENDENT ENDOSPERM (FIE), EMBRYONIC FLOWER 2 (EMF2), CURLY LEAF (CLF), MULTICOPY SUPPRESSOR OF IRA 1 (MSI1) and SWINGER (SWN) was analyzed during the development of buds and vegetative and reproductive organs. During bud development, the putative grapevine epigenetic repressor genes VvCLF, VvEMF2, VvMSI1, VvSWN and VvFIE are mainly expressed in latent buds at the flowering induction period, but also detected during bud burst and inflorescence/flower development. The overlapping expression patterns of grapevine PcG-like genes in buds suggest that chromatin remodeling mechanisms could be operating during grapevine bud development for controlling processes such as seasonal flowering, dormancy and bud burst. Furthermore, the expression of grapevine PcG-like genes was also detected in fruits and vegetative organs, suggesting that epigenetic changes could be at the basis of the regulation of various proliferation–differentiation cell transitions that occur during grapevine development.     

Is GABA-shunt functional in endodormant grapevine buds under respiratory stress?

It has been suggested that a respiratory stress is part of the mechanism through which the dormancy-breaking compounds, hydrogen cyanamide (HC) and sodium azide, induce the release of buds from the endodormancy (ED) in grapevines. The accumulation of metabolites like succinate, alanine (Ala) and γ-amino butyric acid (GABA), together with the activation of the GABA-shunt pathway, is a general feature of plants in response to oxygen deprivation and to respiratory stress. Unexpectedly, in a previous study, we found that GABA applied exogenously to grapevine buds, down-regulated the expression of most genes encoding for antioxidant enzymes, suggesting that its accumulation under respiratory stress conditions could be deleterious for the bud. In order to analyze whether GABA accumulates under respiratory stress conditions in grapevine buds, we analysed in this study, the effect of hypoxia, the respiration inhibitor KCN and the dormancy breaker compound HC, on the level of GABA, and on the expression levels of the GABA-shunt genes (VvGAD, VvGABA-T, VvSSADH). Additionally, genes from the Ala fermentative pathway (VvAlaAT, VvAspAT) were also analysed. The results revealed that although the three treatments mentioned above, up-regulated the expression of VvGAD1, the content of GABA remained constant, while Ala content increased. The lack of GABA accumulation under respiratory stress is an important physiological fact in grapevine buds, since it avoids the down-regulation of antioxidant genes, and promotes the incorporation of succinate into the TCA cycle, a fact that would be important in the release of buds from the ED.     

The Cold Awakening of <Emphasis Type="Italic">Doritaenopsis</Emphasis> ‘Tinny Tender’ Orchid Flowers: The Role of Leaves in Cold-induced Bud Dormancy Release

Massive flowering of tropical Phalaenopsis orchids is coordinated by the cold-induced release of reproductive bud dormancy. Light and temperature are the two key factors integrated by the dormancy mechanism to both stop and reactivate the meristem development of many other angiosperm species, including fruit trees and ornamental plants. It is well established that leaves and roots play a major role in inducing flower development; however, currently, knowledge of molecular events associated with reproductive bud dormancy release in organs other than the bud is limited. Using differential gene expression, we have shown that the leaves of a hybrid of Phalaenopsis species, Doritaenopsis ‘Tinny Tender’, undergo major metabolic modifications. These changes result in the production of sucrose and amino acids, both of which can sustain bud outgrowth, and auxin and ethylene, which may play important roles in awaking the dormant buds. Intake of abscisic acid and synthesis of the hormone jasmonate may also explain the inhibition of vegetative growth that coincides with bud growth. Interestingly, many genes that were upregulated by cold treatment are homologous for genes involved in flower induction and vernalization in Arabidopsis, indicating that processes regulating flowering induction and those regulating reproductive bud dormancy release may use similar pathways and effector molecules.     

Expressional regulation of PpDAM5 and PpDAM6, peach (Prunus persica) dormancy-associated MADS-box genes, by low temperature and dormancy-breaking reagent treatment

The present study investigated the expressional regulation of PpDAM5 and PpDAM6, two of the six peach (Prunus persica) dormancy-associated MADS-box genes, in relation to lateral bud endodormancy. PpDAM5 and PpDAM6 were originally identified as homologues of Arabidopsis SHORT VEGETATIVE PHASE/AGAMOUS-LIKE 24 identified in the EVERGROWING locus of peach. Furthermore, PpDAM5 and PpDAM6 have recently been suggested to be involved in terminal bud dormancy. In this study, seasonal expression analyses using leaves, stems, and lateral buds of high-chill and low-chill peaches in field conditions indicated that both genes were up-regulated during the endodormancy period and down-regulated with endodormancy release. Controlled environment experiments showed that the expression of both PpDAM5 and PpDAM6 were up-regulated by ambient cool temperatures in autumn, while they were down-regulated by the prolonged period of cold temperatures in winter. A negative correlation between expression levels of PpDAM5 and PpDAM6 and bud burst percentage was found in the prolonged cold temperature treatment. Application of the dormancy-breaking reagent cyanamide to endo/ecodormant lateral buds induced early bud break and down-regulation of PpDAM5 and PpDAM6 expression at the same time. These results collectively suggest that PpDAM5 and PpDAM6 may function in the chilling requirement of peach lateral buds through growth-inhibiting functions for bud break.