GABA代谢负调控叶片细胞内复制发生的机制研究

Mechanism study of negative regulation of GABA metabolism on endoreplication in Arabidopsis thaliana leaf development

γ-氨基丁酸(GABA)是植物发育的代谢性信号分子。GABA合成突变体的第一对真叶面积不同程度大于野生型,且突变体的叶表皮细胞的面积大于野生型。流式细胞分析表明GABA合成突变体叶片中多倍体细胞出现早于且比例高于野生型。为研究GABA合成突变体叶片细胞增大和内复制的相关性,对内复制调控因子D类细胞周期蛋白基因(CYCD3;1、CYCD3;2、CYCD3;3和CYCD4;1)和细胞周期蛋白依赖的激酶CDKA;1进行了荧光定量PCR分析。结果表明,GABA合成突变体中CDKA;1的表达量呈现下调趋势。CCS52A(cell cycle switch 52A)在细胞周期退出和内复制进入过程中发挥了重要作用。gad2和gad1/gad2突变体中,CCS52A2的相对表达量明显高于野生型。参与激酶活性抑制的SIM(siamese)、SMR(siamese-related)的表达水平显著高于野生型。为进一步研究GABA合成代谢异常与内复制调控的潜在关系,用活性氧(ROS)的荧光探针分析了叶片保卫细胞中的ROS水平。结果表明,GABA合成突变体中ROS的水平明显高于对照。本研究证明了GABA合成突变体叶片面积增大过程中,周期蛋白、周期蛋白依赖的激酶以及激酶的抑制蛋白在转录水平协调参与了叶片内复制相关的叶片面积调控。本研究首次揭示了GABA正常代谢是阻止叶片细胞早熟分化、保证叶片正常发育相关的内复制发生的负反馈调节因子。

γ-Aminobutyric acid(GABA)is a metabolic signaling molecule for plant development.The area of the first pair of true leaf in GABA synthetic mutants differed was bigger than that in wild type to a greater extent,and the area of the leaf epidermal cells of the mutant was larger than that of wild type.DNA polyploid analysis showed the percentage of polyploidy cells in GABA biosynthetic mutants were earlier and higher than that in wild leaf.To check the correlation between cell size and endoreplication,the expression of the factors involving the endocycles,including D-type cyclin gene(CYCD3;1,CYCD3;2,CYCD3;3 and CYCD4;1)and the kinase CKDA;1 were analyzed by qRT-PCR.The results showed that CKDA;1 in GABA biosynthetic mutants was downward regulated,and four types of CYCDs showed different expression patterns in different GABA biosynthetic mutants.In consistent with this,CCS52 A(CELL CYCLE SWITCH 52 A)(controlling the endocycle entry)in gad2 and gad1/gad2 mutants,the expression of CCS52 A2 was significantly higher than that in wild type(P<0.05).The expression levels of SIM(SIAMESE)and SMR(SIAMESE-RELATED)involved in kinase activity inhibition were also higher expressed than those of control.To further study the possible potential relationship between GABA metabolism and endoreplication,we analyzed the ROS levels in guard cells using Reactive Oxygen Species(ROS)fluorescent probes.The results showed that the ROS level in GABA biosynthetic mutants was significantly higher than that of the control(P<0.05).This study demonstrates that in the regulation of the increase in leaf area of GABA-synthesized mutants in Arabidopsis thaliana,cyclin,cyclin-dependent kinase,and kinase inhibitory proteins are coordinately involved in the regulation of leaf area associated with endoreplication control at the transcriptional level,and this study revealed for the first time that GABA normal metabolism is a negative feedback regulator on endoreplication to prevent the premature differentiation of leaf cells and to ensure normal leaf development.