Wei Hui, Dhanaraj Anik L, Arora Rajeev, Rowland Lisa J, Fu Yan, Sun Li
Department of Horticulture, Iowa State University, Ames, IA 50011, USA.
Plant Cell Environ. 2006 Apr;29(4):558-70. doi: 10.1111/j.1365-3040.2005.01432.x.
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 approximately 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, down-regulation 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.
我们之前分析了非驯化（NA）和冷驯化（CA）杜鹃叶片的表达序列标签（EST），并鉴定出可能参与冷驯化的高丰度互补DNA（cDNA）。这些EST数据集一个潜在重要但相对未被探索的应用是对中度丰度cDNA的研究，比如那些在每个包含约430个EST的杜鹃EST文库中仅被挑选1 - 3次的cDNA。通过统计测试和Northern印迹，我们确定基于EST数据的中度丰度cDNA差异表达的概率确实是其“真正”上调或下调的合理准确预测指标，因为所研究的13个cDNA中有11个（85%）符合这一标准。分析还揭示了杜鹃叶片组织冷驯化的四个方面。首先，CA叶片组织中长链酰基辅酶A（酰基 - CoA）合成酶、CTP：胆碱磷酸胞苷转移酶和δ - 12脂肪酸去饱和酶的伴随上调表明磷脂生物合成和去饱和是杜鹃抗寒锻炼的重要组成部分。其次，CA组织中质体烟酰胺腺嘌呤二核苷酸磷酸苹果酸酶（NADP - ME）的上调表明苹果酸是冷驯化期间用于脂肪酸生物合成的乙酰 - CoA的重要来源。第三，CA组织中质膜内在蛋白（PIP）2 - 1水通道蛋白的下调和外向整流钾离子通道（GORK）的上调可能与保护越冬叶片免受冻害诱导的细胞脱水有关。第四，香豆酸3 - 羟化酶的上调可能与CA组织中的细胞壁增厚有关。我们讨论了这些结果的生理学意义，这些结果揭示了越冬木本常绿植物冷驯化潜在的新调控机制。这项工作强调了在基因组研究中除了研究高丰度EST外，还研究低/中度丰度EST的重要性，因为它提供了一种识别胁迫相关基因的有效策略，特别是在无法进行大规模cDNA测序/微阵列研究的情况下。
