離層酸處理水稻根尖轉錄體及水稻肌動蛋白去聚合因子基因家族之功能性分析

研究水稻根系結構與環境間的影響，對於水稻的生長與發育很重要。環境逆境會導致水稻根部發生形態改變現象，先前文獻也證實外加離層酸會促使水稻根尖部位發生膨大、且根毛增生以及發生側根原基的形成。為了瞭解離層酸對水稻根尖形態改變之影響以及與非生物性逆境間的關聯性，本論文以外加10 μM離層酸處理0、6、12與24小時之TCN1水稻根尖RNA進行NSF45K之微陣列分析。總共篩選出917個離層酸調控表現具顯著差異性的基因探針數。GO enrichment分析顯示與氧化逆境、蛋白質水解及脂質運送有關。KO enrichment分析顯示極長鏈脂肪酸延長、黃酮類生合成、與醣解作用相關路徑的酵素基因具顯著誘導表現。此外，涉及脂質運送、細胞壁修飾與氧化逆境反應相關之酵素基因群亦具有顯著性表現。超氧陰離子自由基與過氧化氫的原位螢光染色結果，說明活化氧族可能參與水稻根尖於離層酸作用下的生長分化過程。而外加離層酸也造成根尖荷爾蒙代謝與訊息傳導相關基因群表現的顯著性改變。水稻根尖轉錄體之微陣列分析指出離層酸可誘導水稻根尖形態的變化以適應環境變動。除此之外，於水稻根尖受離層酸調控差異性表現之基因群中，發現肌動蛋白去聚合因子基因，OsADF3，表現量增加且亦受乾旱及高鹽逆境所影響。肌動蛋白去聚合因子(Actin Depolymerizing Factor, ADFs)為參與細胞骨架動態變化相關之肌動結合蛋白。ADFs已知與植物細胞之形狀、分裂，生長發育、訊息傳遞及生物性逆境相關。但ADFs是否參與植物之非生物性逆境耐受性及其功能則仍有待探討。本論文首先以RT-PCR分析水稻OsADF家族的基因表現，結果指出OsADF家族各成員於不同的組織、發育期及非生物性逆境處理下之基因表現皆具差異性。OsADF1與OsADF3之GFP融合蛋白表現於細胞核中。啟動子OsADF1與OsADF3::GUS活性皆表現於維管束組織。在非生物性逆境與ABA處理下，啟動子OsADF3 :: GUS活性誘導表現於側根和根尖。異位過度表現OsADF3可增加阿拉伯芥轉殖株對於滲透壓及乾旱逆境的耐受性。在添加不同濃度甘露醇培養基中，阿拉伯芥OsADF3-OE轉殖株的萌芽率高於野生型，且轉殖株幼苗根長比野生型不受甘露醇影響抑制生長。乾旱逆境下OsADF3-OE轉殖株比野生型具存活率，且轉殖株比野生型顯著被誘導與耐旱反應相關之基因表現(RD22, ABF4, DREB2A, RD29A, PIP1; 4 and PIP2; 6)。這些結果指出OsADF3基因可能參與植物逆境反應或耐受性。總而言之，本研究以微陣列分析探討了離層酸於水稻根尖形態變化上扮演之可能角色；亦對OsADF3與非生物性逆境之關連提出了佐證。

關鍵字

水稻, 根尖組織, 離層酸, 基因表現, 生長與分化, 肌動蛋白去聚合因子, 基因槍, GUS染色, 異源基因表現, 非生物性逆境

並列摘要

The establishment of root architecture in response to environmental stresses is important for rice growth and development. Environmental stresses caused rice root tips morphological alteration. Exogenously applied abscisic acid has been shown to cause rice root tips swollen, root hair and lateral root formation. To understand the effect of ABA on the alteration of rice root architecture, we conducted a NSF45K microarray analysis for a large-scale gene expression profiling with 10 μM ABA-treated rice root tip at different time points (0, 6, 12 and 24hr). More than 917 differentially expressed probes which affected by ABA were identified. Transcriptome analyses of ABA-treated TCN1 rice root tips through Gene Ontology (GO) enrichment analysis showed that those genes involved in very long-chain fatty acid elongation, flavonoid biosynthesis, and glycolysis are significantly induced. Moreover, the transcripts of genes that predominantly participated in lipid transport, cell wall modification, antioxidants and protective enzymes were also increased. The in situ staining of superoxide and hydrogen peroxide in root tip after ABA treatment suggesting that ROS may play a role in adjusting the growth and development of rice root. The gene expression patterns of phytohormones biosynthesis and signaling were also changed. Taken together, these results provide an initial step to understand how ABA leads to the morphological and physiological changes of rice root. Besides, microarray analysis pointed out OsADF3 gene was induced by ABA and also regulated by drought and salt stresses. Actin depolymerizing factors (ADFs) are small actin-binding proteins that known to involve in plant growth, development and pathogen defense. However, little is known about the role of OsADFs under abiotic stresses. Therefore, to understand the physiological function of OsADF gene family, first we determined the gene expression profile of the OsADF gene family by RT-PCR. The OsADF genes showed distinct and overlapping gene expression patterns at different growth stages, tissues and abiotic stresses. We found that both OsADF1 and OsADF3 proteins were localized in the nucleus. OsADF1 and OsADF3::GUS activity were preferentially expressed in vascular tissues. Under ABA or abiotic stress treatments, OsADF3::GUS activity was induced in lateral roots and root tips. Ectopically overexpressed OsADF3 enhanced the mannitol- and drought-stress tolerance of transgenic Arabidopsis seedlings by increasing germination rate, root length and survival rate. Several drought-tolerance responsive genes (RD22, ABF4, DREB2A, RD29A, PIP1; 4 and PIP2; 6) were regulated in transgenic Arabidopsis under drought stress. These results suggested that OsADF3 may participate in plant abiotic stresses response or tolerance. In conclusion, within this study we highlighted the role of ABA in alteration of rice root tip change using microarray analysis and demonstrated the relationship of OsADF3 with drought stress tolerance.

並列關鍵字

Rice (Oryza sativa L.), root tips, Abscisic Acid, Gene expression profiling, development and growth, Actin depolymerizing factor, Particle bombardment, GUS staining, Heterologus gene expression, Abiotic stresses

參考文獻

Aroca R, Porcel R, Ruiz-Lozano JM. (2012) Regulation of root water uptake under abiotic stress conditions. J Exp. Bot. 63:43-57

Google Scholar

Bach L, Faure JD. (2010) Role of very-long-chain fatty acids in plant development, when chain length does matter. C. R. Biol. 333:361-370

Google Scholar

Beaudoin F, Wu X, Li F, Haslam RP, Markham JE, Zheng H, Napier JA, Kunst L. (2009) Functional characterization of the Arabidopsis beta-ketoacyl-coenzyme A reductase candidates of the fatty acid elongase. Plant Physiol. 150:1174-1191

Google Scholar

Brady SM, Orlando DA, Lee JY, Wang JY, Koch J, Dinneny JR, Mace D, Ohler U, Benfey PN. (2007) A high-resolution root spatiotemporal map reveals dominant expression patterns. Science 318:801-806

Google Scholar

Brown DE, Rashotte AM, Murphy AS, Tague BW, Peer WA, Taiz L and Mudat GK. (2001) Flavonoids act as negative regulators of auxin transport in vivo in Arabidopsis thaliana. Plant Physiol. 126: 524-535

Google Scholar

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離層酸處理水稻根尖轉錄體及水稻肌動蛋白去聚合因子基因家族之功能性分析

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