An expressed sequence tag (EST) analysis approach was undertaken to identify major genes involved in cold acclimation of Rhododendron, a broad-leaf, woody evergreen species. Two cDNA libraries were constructed, one from winter-collected (cold-acclimated, CA; leaf freezing tolerance -53 degrees C) leaves, and the other from summer-collected (non-acclimated, NA; leaf freezing tolerance -7 degrees C) leaves of field-grown Rhododendron catawbiense plants. A total of 862 5'-end high-quality ESTs were generated by sequencing cDNA clones from the two libraries (423 from CA and 439 from NA library). Only about 6.3% of assembled unique transcripts were shared between the libraries, suggesting remarkable differences in gene expression between CA and NA leaves. Analysis of the relative frequency at which specific cDNAs were picked from each library indicated that four genes or gene families were highly abundant in the CA library including early light-induced proteins (ELIP), dehydrins/late embryogenesis abundant proteins (LEA), cytochrome P450, and beta-amylase. Similarly, seven genes or gene families were highly abundant in the NA library and included chlorophyll a/b-binding protein, NADH dehydrogenase subunit I, plastidic aldolase, and serine:glyoxylate aminotransferase, among others. Northern blot analyses for seven selected abundant genes confirmed their preferential expression in either CA or NA leaf tissues. Our results suggest that osmotic regulation, desiccation tolerance, photoinhibition tolerance, and photosynthesis adjustment are some of the key components of cold adaptation in Rhododendron.

No abstract

Our laboratory has been working toward increasing our understanding of the genetic control of cold hardiness in blueberry (Vaccinium section Cyanococcus) to ultimately use this information to develop more cold hardy cultivars for the industry. Here, we report using cDNA microarrays to monitor changes in gene expression at multiple times during cold acclimation under field and cold room conditions. Microarrays contained over 2,500 cDNA inserts, approximately half of which had been picked and single-pass sequenced from each of two cDNA libraries that were constructed from cold acclimated floral buds and non-acclimated floral buds of the fairly cold hardy cv. Bluecrop (Vaccinium corymbosum L.). Two biological samples were examined at each time point. Microarray data were analyzed statistically using t tests, ANOVA, clustering algorithms, and online analytical processing (OLAP). Interestingly, more transcripts were found to be upregulated under cold room conditions than under field conditions. Many of the genes induced only under cold room conditions could be divided into three major types: (1) genes associated with stress tolerance; (2) those that encode glycolytic and TCA cycle enzymes, and (3) those associated with protein synthesis machinery. A few of the genes induced only under field conditions appear to be related to light stress. Possible explanations for these differences are discussed in physiological context. Although many similarities exist in how plants respond during cold acclimation in the cold room and in the field environment, there are major differences suggesting caution should be taken in interpreting results based only on artificial, cold room conditions.

Loss of freeze tolerance, or deacclimation, is an integral part of winter survival in woody perennials because untimely mid-winter or spring thaws followed by a hard freeze can cause severe injury to dehardened tissues. This study was undertaken to investigate deacclimation kinetics, particularly the timing and speed, of five blueberry (Vaccinium L.) cultivars (`Bluecrop', `Weymouth', `Ozarkblue', `Tifblue', and `Legacy'), with different germplasm compositions and mid-winter bud hardiness levels, in response to an environmentally controlled temperature regime. Based upon bud cold hardiness evaluations in 2000 and 2001, `Tifblue', a Vaccinium ashei Reade cultivar, was one of the least hardy and the fastest to deacclimate; `Bluecrop', a predominantly V. corymbosum L. cultivar, was the most hardy and the slowest to deacclimate; and `Ozarkblue', a predominantly V. corymbosum cultivar but including southern species V. darrowi Camp. and V. ashei, was intermediate in speed of deacclimation. `Weymouth' (predominantly V. corymbosum) and `Legacy' (73.4% V. corymbosum and 25% V. darrowi) were slow to intermediate deacclimators. Deacclimation rates did not correlate strictly with mid-winter bud hardiness. Data suggest that the southern germplasm component V. ashei may be responsible for the observed faster deacclimation whereas both southern species, V. darrowi and V. ashei, may contribute genes for cold sensitivity. Strong positive correlations between stage of bud opening and bud cold hardiness existed in both years (r = 0.90 and 0.82 in 2000 and 2001 study, respectively). Previously identified major blueberry dehydrins, 65-, 60-, and 14-kDa, progressively decreased in their abundance during incremental dehardening in `Bluecrop', `Weymouth', and `Tifblue'. However, down-regulation of the 14-kDa dehydrin most closely mirrored the loss in cold hardiness during deacclimation, and, therefore, may be involved in regulation of bud dehardening. Because differences in deacclimation rate were clearly evident among the genotypes studied, rate of deacclimation of the flower buds of blueberry should be an important consideration in breeding to improve winter survival.

We have previously analysed expressed sequence tags (ESTs) from non-acclimated (NA) and cold-acclimated (CA) Rhododendron leaves, 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 each Rhododendron EST 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 in Rhododendron leaf 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 in Rhododendron . Secondly, upregulation of plastidic nicotinamide adenine dinucleotide phosphatemalic 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.