Genetics, Institute of Plant Biotechnology, Technical University of Cartagena, Campus Muralla del Mar, 30202 Cartagena, Murcia, Spain.
Plant Physiol. 2011 Aug;156(4):1978-89. doi: 10.1104/pp.111.179275. Epub 2011 Jun 15.
The cactus Opuntia ficus-indica is a constitutive Crassulacean acid metabolism (CAM) species. Current knowledge of CAM metabolism suggests that the enzyme phosphoenolpyruvate carboxylase kinase (PPCK) is circadian regulated at the transcriptional level, whereas phosphoenolpyruvate carboxylase (PEPC), malate dehydrogenase (MDH), NADP-malic enzyme (NADP-ME), and pyruvate phosphate dikinase (PPDK) are posttranslationally controlled. As little transcriptomic data are available from obligate CAM plants, we created an expressed sequence tag database derived from different organs and developmental stages. Sequences were assembled, compared with sequences in the National Center for Biotechnology Information nonredundant database for identification of putative orthologs, and mapped using Kyoto Encyclopedia of Genes and Genomes Orthology and Gene Ontology. We identified genes involved in circadian regulation and CAM metabolism for transcriptomic analysis in plants grown in long days. We identified stable reference genes for quantitative polymerase chain reaction and found that OfiSAND, like its counterpart in Arabidopsis (Arabidopsis thaliana), and OfiTUB are generally appropriate standards for use in the quantification of gene expression in O. ficus-indica. Three kinds of expression profiles were found: transcripts of OfiPPCK oscillated with a 24-h periodicity; transcripts of the light-active OfiNADP-ME and OfiPPDK genes adapted to 12-h cycles, while transcript accumulation patterns of OfiPEPC and OfiMDH were arrhythmic. Expression of the circadian clock gene OfiTOC1, similar to Arabidopsis, oscillated with a 24-h periodicity, peaking at night. Expression of OfiCCA1 and OfiPRR9, unlike in Arabidopsis, adapted best to a 12-h rhythm, suggesting that circadian clock gene interactions differ from those of Arabidopsis. Our results indicate that the evolution of CAM metabolism could be the result of modified circadian regulation at both the transcriptional and posttranscriptional levels.
仙人掌 Opuntia ficus-indica 是一种组成性的景天酸代谢（CAM）物种。目前对 CAM 代谢的认识表明，磷酸烯醇丙酮酸羧激酶（PPCK）酶在转录水平上受到昼夜节律的调节，而磷酸烯醇丙酮酸羧化酶（PEPC）、苹果酸脱氢酶（MDH）、NADP-苹果酸酶（NADP-ME）和丙酮酸磷酸二激酶（PPDK）则受到翻译后调控。由于强制性 CAM 植物的转录组数据很少，我们创建了一个源自不同器官和发育阶段的表达序列标签数据库。对序列进行组装，与国家生物技术信息中心非冗余数据库中的序列进行比较，以识别推定的同源物，并使用京都基因与基因组百科全书（KEGG）同源和基因本体论（GO）进行映射。我们鉴定了参与昼夜节律调节和 CAM 代谢的基因，用于在长日下生长的植物中转录组分析。我们确定了用于定量聚合酶链反应的稳定参考基因，并发现 OfiSAND 与其在拟南芥（Arabidopsis thaliana）中的对应物一样，以及 OfiTUB 通常适合用于定量测定 O. ficus-indica 中基因表达。发现了三种表达谱：OfiPPCK 的转录物以 24 小时的周期性波动；OfiNADP-ME 和 OfiPPDK 基因的光活性转录物适应 12 小时周期，而 OfiPEPC 和 OfiMDH 的转录物积累模式则无节律。类似于拟南芥的昼夜节律时钟基因 OfiTOC1 的表达呈 24 小时周期性波动，峰值出现在夜间。OfiCCA1 和 OfiPRR9 的表达与拟南芥不同，最适应 12 小时的节律，这表明昼夜节律时钟基因的相互作用与拟南芥不同。我们的研究结果表明，CAM 代谢的进化可能是转录和转录后水平上昼夜节律调节改变的结果。
