Microarray analysis of laser-microdissected tissues indicates the biosynthesis of suberin in the outer part of roots during formation of a barrier to radial oxygen loss in rice (Oryza sativa).

Internal aeration is crucial for root growth in waterlogged soil. A barrier to radial oxygen loss (ROL) can enhance long- distance oxygen transport via the aerenchyma to the root tip; a higher oxygen concentration at the apex enables root growth into anoxic soil. The ROL barrier is formed within the outer part of roots (OPR). Suberin and/or lignin depos- ited in cell walls are thought to contribute to the barrier, but it is unclear which compound is the main constituent. This study describes gene expression profiles during ROL barrier formation in rice roots to determine the relative responses of suberin and/or lignin biosyntheses for the barrier. OPR tissues were isolated by laser microdissection and their transcripts were analysed by microarray. A total of 128 genes were significantly up- or downregulated in the OPR during the barrier formation. Genes associated with suberin biosynthesis were strongly upregulated, whereas genes associated with lignin biosynthesis were not. By an ab initio analysis of the promoters of the upregulated genes, the putative cis-elements that could be associated with transcription factors, WRKY, AP2/ERF, NAC, bZIP, MYB, CBT/DREB, and MADS, were elucidated. They were particularly associated with the expression of transcrip- tion factor genes containing WRKY, AP2, and MYB domains. A semiquantitative reverse-transcription PCR analysis of genes associated with suberin biosynthesis (WRKY, CYP, and GPAT) confirmed that they were highly expressed during ROL barrier formation. Overall, these results suggest that suberin is a major constituent of the ROL barrier in roots of rice. 23-d-old plants were either continued in aerated solution or transplanted into N2-flushed or stagnant deoxygenated solution for 9 h. After treating the roots of plants in aerated, stagnant, or N2-flushed conditions for 9h, the basal parts (12.5 -22.5mm below the root - shoot junction) of the adventitious roots were collected. Cells in OPR (including exodermis and sclerenchyma) were isolated using laser microdissection. RNA extracted from the isolated OPR was analysed with a 44k rice oligo-DNA microarray. Total RNAs were labeled with a Quick Amp Labeling Kit (Agilent Technologies) according to the manufacturer’s instructions. Aliquots of Cy5-labeled and Cy3-labeled cRNA (10 ng each) were used for hybridization in a rice 44K oligo-DNA microarray.

根系内部通气对渍水土壤中的根系生长至关重要。径向氧损失（radial oxygen loss, ROL）屏障可通过通气组织增强氧气向根尖的长距离运输；根尖处更高的氧浓度可使根系得以在缺氧土壤中生长。该径向氧损失屏障形成于根系外层（outer part of roots, OPR）中。人们认为沉积于细胞壁中的木栓质和/或木质素参与了该屏障的形成，但尚不明确二者中哪种是该屏障的主要组成成分。本研究旨在明确木栓质与木质素生物合成在径向氧损失屏障形成过程中的相对贡献，对水稻根系径向氧损失屏障形成过程中的基因表达谱进行了分析。研究人员通过激光显微切割分离得到根系外层组织，并利用基因微阵列（microarray）对其转录本进行了分析。在屏障形成过程中，根系外层组织中共计128个基因出现了显著的上调或下调表达。与木栓质生物合成相关的基因出现了显著上调，而与木质素生物合成相关的基因则无明显表达变化。通过对上调基因的启动子进行从头预测分析，本研究明确了可能与WRKY、AP2/ERF、NAC、bZIP、MYB、CBT/DREB及MADS家族转录因子结合的潜在顺式作用元件（cis-elements）。这些顺式作用元件尤其与携带WRKY、AP2及MYB结构域的转录因子基因的表达密切相关。针对木栓质生物合成相关基因（WRKY、CYP及GPAT）的半定量逆转录PCR（reverse-transcription PCR, RT-PCR）分析证实，这些基因在径向氧损失屏障形成过程中呈高表达。综合以上结果，本研究表明木栓质是水稻根系径向氧损失屏障的主要组成成分。将生长23天的水稻植株分别置于通气溶液中继续培养，或移栽至充氮（N₂）溶液及静置缺氧溶液中处理9小时。在通气、静置及充氮条件下对植株根系处理9小时后，采集不定根的基部区域（根冠接合点下方12.5~22.5毫米处）。通过激光显微切割分离得到根系外层组织（包括外皮层及厚壁组织）中的细胞。利用44K水稻寡聚DNA基因微阵列对分离得到的根系外层组织中的RNA进行检测分析。总RNA按照试剂盒说明书，使用Quick Amp标记试剂盒（安捷伦科技，Agilent Technologies）进行标记。取等量（各10 ng）经Cy5及Cy3标记的cRNA用于水稻44K寡聚DNA基因微阵列的杂交反应。