過量表現OsADF3可提升水稻滲透壓逆境耐受性

Abstract

肌動蛋白去聚合因子 (Actin depolymerizing factor, ADF) 為調節植物肌動蛋白細胞骨架動態變化的重要蛋白。它會與絲狀的肌動蛋白結合，將其分解成球狀肌動蛋白。前人研究顯示，肌動蛋白去聚合因子具有調控花粉管生長、賀爾蒙分布、細胞極性等功能，卻很少有在非生物逆境下的相關研究。過去研究顯示水稻肌動蛋白去聚合因子家族中的OsADF3會被離層酸、乾旱與鹽逆境誘導。在阿拉伯芥中，過量表現水稻OsADF3，可使阿拉伯芥具有乾旱逆境的抗性，並提高水通道蛋白 (Aquaporin) 和脫水蛋白 (Dehydrin) 等耐旱相關基因的表現。為了進一步探討OsADF3在水稻中是否也有調控滲透壓或乾旱逆境抗性的功能，我們建立OsADF3的過量表現系，並且進行外表型分析，包括株高和根長，同時利用聚乙二醇 (PEG-6000) 滲透壓逆境模擬乾旱，調查活性氧族 (Reactive oxygen species, ROS)、丙二醛 (MDA)、離子滲漏程度與過氧化氫酶活性在轉殖株內的表現。此外也分析乾旱相關基因表現情形，並且利用RNA定序資料進行轉錄體分析。從外表型分析結果中，我們發現轉殖株之株高與對照組沒有顯著差異，但其根長顯著短於對照組。在PEG處理下，過量表現OsADF3水稻具有較強的耐受性。生理分析結果顯示，過量表現OsADF3可增加水稻滲透壓逆境下過氧化氫酶活性，並且減少活性氧族與丙二醛累積。在乾旱相關基因表現量與轉錄體分析中，我們發現過量表現OsADF3會提高OsDREB2A、OsbZIP23、OsDHN4、OsDHN8和脯胺酸合成基因OsP5CS1的表現，另外也會增加碳水化合物生合成、光合作用、碳固定等相關基因途徑之表現。這些結果顯示OsADF3會參與水稻對滲透壓逆境的調控，但其中詳細之分子機理仍有待進一步研究。

Actin‐depolymerizing factor (ADF) is an essential protein that regulates the actin cytoskeleton dynamics in the plant cells. ADF can bind to the filamentous actin (F-actin) and depolymerize it into globular actins (G-actin). Previous studies showed that ADF can participate in regulating plant pollen tube growth, hormone distribution, cell polarity, etc., but is hardly shown to associate with plant abiotic stress tolerance. Proteomic analysis ever showed that an individual member of the rice ADF family, OsADF3, can be induced by ABA, drought, and salt stress. Heterologous overexpression of OsADF3 in Arabidopsis thaliana can enhance drought resistance and increase the expression level of drought tolerant-related genes such as aquaporin and dehydrin. To further understand the role of OsADF3 in rice, we produced OsADF3 overexpression (OE) transgenic rice. Phenotypic analysis including plant height and root length was performed. Polyethylene glycol (PEG-6000) causing the osmotic potential change to mimic drought stress was used to investigate the accumulation of reactive oxygen species (ROS), malondialdehyde (MDA) content, ion leakage and catalase activity in OE rice. Besides, we also analyzed the expression of drought response-related genes and conducted RNA-seq analysis. Phenotypic analysis showed that the root length of the OE lines was significantly shorter than the wild type (WT). Under PEG treatment, the OE lines were more tolerant to osmotic-induced drought stress. The physiological analysis showed that the OE lines increased the activity of catalase under stress, and reduced the accumulation of ROS and MDA. Analysis of RNA-seq data and the expression of drought response-related genes revealed that the expression of OsDREB2A, OsbZIP23, OsDHN4, OsDHN8, and OsP5CS1 were upregulated in the OE lines. Also, the carbohydrate biosynthesis, photosynthesis, and carbon fixation pathway were all upregulated. Our result indicated that OsADF3 may be involved in the response of osmotic-induced drought stress generated by PEG. The detailed molecular mechanism remains to be clarified.