拟南芥微丝相关蛋白SCRP1调节气孔运动的功能研究

发布时间：2018-11-07 07:02

【摘要】：微丝蛋白是一类普遍存在于真核生物细胞中的丝状网络结构,始终处于解聚聚合的动态变化中。植物中的微丝动态变化参与多种细胞过程的调控,如细胞分裂,细胞形态建成,细胞运动,细胞极性生长等。近年来的研究表明微丝骨架的动态变化也参与调控植物对生物及非生物胁迫的响应。细胞内的微丝动态变化受到多种微丝结合蛋白及微丝相关因子的调控。保卫细胞的开关运动是植物对外界刺激响应的重要调节机制。保卫细胞内微丝的重排参与调控保卫细胞的运动过程。越来越多的研究表明保卫细胞中微丝的排列方向及丝束的粗细、密度等性状特征与保卫细胞的开度密切相关。但是关于参与保卫细胞内微丝动态变化调节的具体蛋白因子我们了解的并不多。本文运用正向遗传学方法,通过对ABRC订购的T-DNA突变体株系施行离体叶片失水实验分析,筛选得到一个失水快的突变体scrp1。该突变体在拟南芥SCRP1基因上有T-DNA插入,造成SCRP1基因的功能缺失。SCRP1基因编码一个丝/苏氨酸蛋白激酶。我们的研究结果表明SCRP1基因的表达受ABA的诱导表达上调,参与ABA诱导的气孔关闭的调节。SCRP1与微丝蛋白具有相关性,在细胞内与微丝蛋白共定位,在保卫细胞内具有稳定微丝的作用,且参与保卫细胞内微丝动态变化的调节。但SCRP1并不直接结合微丝蛋白,且对微丝的聚合解聚过程没有直接作用。我们研究发现SCRP1能够与微丝解聚因子ADF4有互作,而且能够磷酸化ADF4,抑制ADF4的微丝解聚活性。SCRP1的缺失造成植物体具有失水快,对ABA诱导的气孔关闭反应迟缓,气孔中的微丝表现为再聚合的程度下降的表型。同时我们还发现ADF4功能缺失突变体对失水胁迫和ABA处理都表现为不敏感,气孔中的微丝聚合成束的程度严重。ADF4的过表达转基因植株气孔关闭表型与scrp1突变体表型相似,都表现为气孔关闭对ABA处理不敏感。在scrp1突变体背景下敲除ADF4可以部分恢复scrp1突变体失水快和ABA处理后气孔关闭迟缓的表型。我们的研究表明SCRP1参与气孔关闭过程中的微丝动态变化的调节,这个过程是通过与ADF4互作,磷酸化ADF4,抑制ADF4微丝解聚活性来实现的。本文的主要创新点及意义:1.前人的研究表明微丝解聚因子ADF是一类主要的微丝解聚蛋白,它的活性受磷酸化的调控,但在拟南芥中已知的能够磷酸化ADF的磷酸激酶并不多,本文首次报道了SCRP1也能够磷酸化ADF,这对于ADF的功能探索意义重大。2.目前的研究对能够调节保卫细胞内微丝动态的激酶知道的并不多,本文首次报道了SCRP1作为一类新的微丝相关磷酸激酶,通过磷酸化ADF,抑制ADF微丝解聚活性,调控微丝动态变化,从而调节气孔运动。这对于研究植物细胞骨架如何通过调节保卫细胞的运动来响应外界信号刺激具有重要的贡献。
[Abstract]:Microfilaments are a kind of filamentous network which is ubiquitous in eukaryotic cells and is always in the dynamic process of depolymerization. The dynamic changes of microfilaments in plants are involved in the regulation of many cell processes, such as cell division, cell morphogenesis, cell movement, cell polarity growth and so on. Recent studies have shown that the dynamic changes of microfilament skeleton are also involved in the regulation of plant responses to biotic and abiotic stresses. The dynamic changes of microfilaments in cells are regulated by various microfilament binding proteins and microfilament related factors. The switch movement of guard cells is an important regulation mechanism of plant response to external stimuli. The rearrangement of microfilaments in guard cells is involved in the regulation of the movement of guard cells. More and more studies have shown that the orientation of microfilaments, the thickness and density of filaments in guard cells are closely related to the opening of guard cells. However, we do not know much about the specific protein factors involved in the regulation of microfilaments in guard cells. In this paper, by using the method of forward genetics, a mutant scrp1. with fast loss of water was obtained by the experimental analysis of leaf water loss of T-DNA mutants ordered by ABRC. The mutant has T-DNA insertion into the Arabidopsis SCRP1 gene, resulting in a functional deletion of the SCRP1 gene. The SCRP1 gene encodes a silk / threonine protein kinase. Our results showed that the expression of SCRP1 gene was up-regulated by ABA, which was involved in the regulation of stomatal closure induced by ABA. SCRP1 was associated with microfilament protein and was colocated with microfilament protein in cells. It has the function of stabilizing the microfilaments in the guard cells and participates in the regulation of the dynamic changes of the microfilaments in the guard cells. However, SCRP1 does not directly bind to microfilament proteins and has no direct effect on the polymerization and depolymerization of microfilaments. We found that SCRP1 could interact with microfilament depolymerization factor ADF4, and phosphorylated ADF4, could inhibit the depolymerization activity of ADF4. The absence of SCRP1 resulted in fast water loss and slow response to ABA induced stomatal closure. The microfilaments in the stomata showed the phenotype of decreasing degree of repolymerization. At the same time, we also found that ADF4 functional deletion mutants were insensitive to water loss stress and ABA treatment, and the microfilament aggregation in stomata was serious. The stomatal closure phenotype of ADF4 overexpression transgenic plants was similar to that of scrp1 mutant. Both showed that stomatal closure was insensitive to ABA treatment. Knockout of ADF4 in the background of scrp1 mutants could partially restore the phenotype of fast water loss of scrp1 mutants and delayed stomatal closure after ABA treatment. Our results show that SCRP1 is involved in the regulation of the dynamic changes of microfilaments during stomatal closure, which is achieved by interacting with ADF4 and phosphorylating ADF4, to inhibit the depolymerization activity of ADF4 microfilaments. The main innovation points and significance of this paper are as follows: 1. Previous studies have shown that microfilament depolymerization factor (ADF) is a major type of microfilament depolymerizing protein, and its activity is regulated by phosphorylation, but there are few phosphokinase known to phosphorylate ADF in Arabidopsis thaliana. This paper reports for the first time that SCRP1 can also phosphorylate ADF, which is of great significance for exploring the function of ADF. 2. Little is known about kinases that regulate the dynamics of microfilaments in guard cells. SCRP1, as a new type of microfilament-associated phosphokinase, has been reported for the first time to inhibit the depolymerization of ADF microfilaments by phosphorylated ADF,. Adjust the dynamic change of microfilament, thus adjust the stomatal movement. This is an important contribution to the study of how plant cytoskeleton responds to external signal stimuli by regulating the movement of guard cells.
【学位授予单位】：山东师范大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：Q945

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