We have previously analysed expressed sequence tags (ESTs) from non‐acclimated (NA) and cold‐acclimated (CA)Rhododendronleaves, and identified highly abundant complementary DNAs (cDNAs) possibly involved in cold acclimation. A potentially significant, but relatively unexplored, application of these EST data sets is the study of moderately abundant cDNAs, such as those picked only 1–3 times from eachRhododendronEST library containing ∼ 430 ESTs. Using statistical tests and Northern blots, we established that the probability of differential expression of moderately abundant cDNAs based on the EST data is, indeed, a reasonably accurate predictor of their ‘true’ upregulation or downregulation as 11 out of 13 cDNAs (85%) studied fit this criterion. The analyses also revealed four aspects of cold acclimation inRhododendronleaf tissues. Firstly, the concomitant upregulation of long‐chain acyl‐coenzyme A (acyl‐CoA) synthetase, CTP:cholinephosphate cytidylyltransferase and delta‐12 fatty acid desaturase in CA leaf tissues suggests that phospholipid biosynthesis and desaturation are important components of cold hardening inRhododendron. Secondly, upregulation of plastidic nicotinamide adenine dinucleotide phosphate‐malic enzyme (NADP‐ME) in CA tissues suggests that malate is an important source of acetyl‐CoA used for fatty acid biosynthesis during cold acclimation. Thirdly, downregulation of plasma membrane intrinsic protein (PIP)2‐1 aquaporin and upregulation of gated outward rectifying K⁺channel (GORK) in CA tissues may be associated with the protection of overwintering leaves from freeze‐induced cellular dehydration. Fourthly, upregulation of coumarate 3‐hydroxylase may be associated with cell wall thickening in CA tissues. Physiological implications of these results, which reveal potentially novel regulations of cold acclimation in overwintering woody evergreens, are discussed. This work highlights the importance of also investigating low/moderately abundant ESTs (in addition to highly abundant ones) in genomic studies, in that it offers an effective strategy for identifying stress‐related genes, especially when large‐scale cDNA sequencing/microarray studies are not possible.

Dehydrins (DHNs) are typically induced in response to abiotic stresses that impose cellular dehydration. As extracellular freezing results in cellular dehydration, accumulation of DHNs and development of desiccation tolerance are believed to be key components of the cold acclimation (CA) process. The present study shows that RcDhn5, one of the DHNs from Rhododendron catawbiense leaf tissues, encodes an acidic, SK₂ type DHN and is upregulated during seasonal CA and downregulated during spring deacclimation (DA). Data from in vitro partial water loss assays indicate that purified RcDhn5 protects enzyme activity against a dehydration treatment and that this protection is comparable with acidic SK_n DHNs from other species. To investigate the contribution of RcDhn5 to freezing tolerance (FT), Arabidopsis plants overexpressing RcDhn5 under the control of 35S promoter were generated. Transgenic plants exhibited improved ‘constitutive’ FT compared with the control plants. Furthermore, a small but significant improvement in FT of RcDhn5‐overexpressing plants was observed after 12 h of CA; however, this gained acclimation capacity was not sustained after a 6‐day CA. Transcript profiles of cold‐regulated native Arabidopsis DHNs (COR47, ERD10 and ERD14) during a CA time–course suggests that the apparent lack of improvement in cold‐acclimated FT of RcDhn5‐overexpressing plants over that of wild‐type controls after a 6‐day CA might have been because of the dilution of the effect of RcDhn5 overproduction by a strong CA‐induced expression of native Arabidopsis DHNs. This study provides evidence that RcDhn5 contributes to freezing stress tolerance and that this could be, in part, because of its dehydration stress‐protective ability.

Significant advances have been made in our understanding of the regulation of cold hardiness. The existence of numerous biophysical and biochemical adaptive mechanisms in perennial woody plants and the complexity their regulation has made the development of methods for managing and improving cold hardiness in perennial woody plants has been very difficult. This may be partially attributed to viewing cold hardiness as a single dimensional response, rather than as a complex phenomenon, involving different mechanisms (avoidance and tolerance), different stages (mid-winter vs. late winter), and having an intimate overlap with the genetic regulation of dormancy. In particular separating the molecular regulation of cold hardiness from growth processes has been challenging. ICE and C-repeat binding factor (CBF), transcription factors (Inducer of CBF expression and CRT-binding factor) have been shown to be an important aspect in the regulation of cold-induced gene expression. Evidence has emerged, however, that they are also intimately involved in the regulation of growth, flowering, dormancy, and stomatal development. This evidence includes the presence of CBF binding motifs in genes regulating these processes, or through cross-talk between the pathways that regulate them. Recent changes in climate that have resulted in erratic episodes of unseasonal warming followed by more seasonal patterns of low temperatures has also highlighted the need to better understand the genetic and molecular regulation of deacclimation, a topic of research that is only more recently being addressed. Environmentally-induced epigenetic regulation of stress responses and seasonal processes such as cold acclimation, deacclimation, and dormancy have been documented but are still poorly understood. Advances in the ability to efficiently generate large DNA and RNA datasets and genetic transformation technologies have greatly increased our ability to explore the regulation of gene expression and explore genetic diversity. Greater knowledge of the interplay between epigenetic and genetic regulation of cold hardiness, along with the application of advanced genetic analyses, such as genome-wide-association-studies (GWAS), are needed to develop strategies for addressing the complex processes associated with cold hardiness in woody plants. A cautionary note is also indicated regarding the time-scale needed to examine and interpret plant response to freezing temperatures if progress is to be made in developing effective approaches for manipulating and improving cold hardiness.