Different Chilling Responses of Brassinosteroid-biosynthetic Genes in Arabidopsis and Mung Bean Seedlings

低溫逆境下阿拉伯芥及綠豆幼苗中油菜固醇生合成基因的不同反應

黃斌(Bin Huang) ； 陳淑玲(Shu-Ling Chen) ； 陳益明(Yih-Ming Chen)

《作物、環境與生物資訊》 3卷2期 (2006/06) Pp. 123-134

https://doi.org/10.30061/CEB.200606.0002

摘要

低溫是影響植物生長的重要因子之一。除離層酸(abscisic acid, ABA)外，亦有文獻報導油菜固醇(brassinosteroid, BR)與植物的低溫耐性相關。油菜固醇具有極高促進細胞生長的功能，其生合成途徑及訊息傳導的基因皆已被發現。CYP90A2是第一個在綠豆中被選殖出來的細胞色素P450基因，與阿拉伯芥中合成油菜固醇的細胞色素P450基因-CPD/CYP90A1具有77%的高相似度。經由南方點墨法判斷CYP90A2在綠豆基因組中只有一個拷貝(copy)。在生理反應方面，CYP90A2基因的表現隨著幼苗的生長而增加，以葉部為主，且呈現日夜變化。利用回饋抑制分析發現油菜固醇終產物24-epibrassinolide(EBS)會抑制CYP90A2的表現，而油菜固醇生合成抑制劑Brz220則會增加其表現。這些生理現象與阿拉伯芥的CPD基因皆相同，因此推測CYP90A2在綠豆中可能擔任合成油菜固醇的角色。以低溫10℃處理耐寒植物-阿拉伯芥與低溫敏感性植物－ 綠豆， 發現阿拉伯芥持續生長，且CYP85A1, CPD/CYP90A1, DWF4/CYP90B1, ROT3/CYP90C1及CYP90D1等負責合成油菜固醇的細胞色素基因亦持續表現;反觀綠豆，除生長受抑制外，其CYP90A2的表現亦明顯降低。由此推測油菜固醇應與低溫生長有關。因此，以EBS前處理綠豆幼苗，發現可以提高其在低溫下的生長及存活率。雖然目前尚無直接證據得以證明CYP90A2是否參與油菜固醇的合成，及此荷爾蒙在低溫下是否會降低生成量，本研究係第一篇以分子層次探討油菜固醇與低溫耐性的報告，期待可以進一步瞭解植物耐寒性的形成。

關鍵字

油菜固醇 ； 寒害 ； 綠豆 ； 細胞色素

並列摘要

Chilling stress is an inevitable environmental factor that deeply affects plants growth and development. In addition to abscisic acid involved in chilling tolerance, increasing attentions have been focused on brassinosteroid (BR) as a chilling protectant. BR, a newly identified phytohormone that exhibits the highest promotion effect on cell growth has been reported, and the components involved in its biosynthesis and signaling are also identified in Arabidopsis. In mung bean, CYP90A2 is the first clone that shares high homologous (77%) to Arabidopsis CPD/CYP90A1, which is the most important enzyme in BR biosynthesis. There is one copy of CYP90A2 per haploid genome in mung bean. The transcriptions of CYP90A2 are abundantly detected in leaves that increase following seedlings development and exhibit diurnal variation. In the feedback regulation assay, CYP90A2 is down regulated in the existence of 24-epibrassinolide (EBS) and up regulated by Brz220, a BR biosynthetic inhibitor. All of these physiological responses are similar to Arabidopsis CPD gene. Therefore, CYP90A2 is postulated to be involved in BR biosynthesis in mung bean. Comparing the growth of two plant species when exposured to 10℃ chilling stress, Arabidopsis, a chilling-tolerant plant, shows higher growth and survival rate than mung bean, which is regarded as a chilling-sensitive plant. In monitoring the regulations of BR-biosynthetic genes under chilling temperature, we found that the reported genes CYP85A1, CPD/CYP90A1, DWF4/CYP90B1, ROT3/CYP90C1 and CYP90D1, involved in BR biosynthesis in Arabidopsis maintained in a constant level, while mung bean CYP90A2 was strongly down regulated. Thus, brassinosteroid (BR) is considered related to chilling growth. With the prediction that chilling decreases endogenous BR level and leads to a chilling-sensitivity of mung bean, the exogenous spraying of EBS is performed and seedlings are survived from chilling injury. In spite of the deficiency of direct evidences in determining the functional role of CYP90A2 and the uncertainty of endogenous level of BR decreasing under chilling stress, to our knowledge, this was the first study applying molecular approaches to elucidate the correlation between BR and chilling response, with which the mechanisms of chilling tolerance could be further explained.

並列關鍵字

Brassinosteroid ； Chilling ； Mung bean ； Cytochrome P450 ； CPD