拟南芥DNA糖基化酶TAG1调控叶片衰老及株高的分子机制

Leaf senescence is a developmentally programmed degeneration process that constitutes the final step of plant life and is controlled by multiple developmental and environmental signals. By screening mutant libraries, we identified an Arabidopsis mutant tag1, which exhibited the earlier senescence and dwarf phenotypes. TAG1 encodes a 3-methyladenine DNA glycosylase. tag1 is hypersensitive to methyl methanesulfonate (MMS), an alkylating agent and a carcinogen, that produces DNA damage, suggesting that TAG1 is involved in DNA repair in Arabidopsis. RNA-Seq analysis revealed that mutation in TAG1 globally affected the gene expression. Interestingly, genes involved in GA biosynthesis were markedly down-regulated in tag1 mutant compared with that in wild-type. Moreover, GA contents, such as GA4 and GA53, were significantly reduced in tag1 compared with wild-type. Application of GA3 or overexpression of TAG1 can rescue the earlier senescence and dwarf phenotypes of tag1. By performing whole-genome bisulfite sequencing (WGBS) analysis, we found that loss-of-function of TAG1 leads to locus specific hypermethylation. By contrast, overexpression of TAG1 results in a global hypomethylaiton at the CHH and CHG loci, suggesting that TAG1 possibly is involved in DNA repair and regulation of gene expression, and then controls leaf senescence process and height in Arabidopsis. However, the molecular mechanism is largely unclear. In this project, we aim to investigate the underlying regulatory mechanism of TAG1 in regulating leaf senescence process by using genetic, biochemical, structural biology and bioinformatic approaches.

衰老是植物叶片发育的最后阶段，延缓衰老与农作物增产及品质改良密切相关。通过筛选拟南芥突变体库，获得一个叶片早衰且植株矮小的突变体tag1。TAG1编码一种DNA-3-甲基腺嘌呤糖基化酶。tag1突变体对能够引起DNA损伤的试剂（如甲基磺酸甲酯）超敏感，表明TAG1在修复DNA损伤中具有重要作用。RNA-Seq分析发现TAG1基因缺失引起赤霉素（GA）合成途径的关键基因表达水平下调，并且发现tag1突变体内GA含量显著降低。外源施加GA或过表达TAG1恢复了tag1的表型。亚硫酸氢钠测序发现tag1植株DNA的特定位点甲基化水平升高，而过表达TAG1显著减少DNA甲基化水平，暗示TAG1可能通过调节DNA甲基化从而调控基因表达，进而影响叶片衰老及株高等过程，但机制不清楚。本项目通过遗传学和生物化学等方法研究TAG1的功能，从而揭示其调控衰老及株高的分子机制，为分子育种提供有价值的理论指导。

叶片衰老是植物发育的最后阶段，是受遗传调控的高度协调的生物学过程。虽然几百个基因已被证明参与调节叶片衰老过程，但是叶衰老的分子机制仍不清楚。为了鉴定新的叶片衰老调节因子，我们进行了反向遗传学筛选，获得一个拟南芥T-DNA插入突变体tag1。TAG1基因编码一个DNA糖基化酶，在茎尖、根尖和花中高表达。TAG1功能缺失导致年龄依赖的叶片早衰老，并且株高也显著降低，而过表达TAG1则延迟叶片衰老。研究发现，TAG1功能缺失导致赤霉素（GA）含量降低，外源施加GA3完全恢复了tag1突变体的半矮表型。转录组测序分析表明，与野生型相比，tag1突变体中GA生物合成途径受到抑制。全基因组亚硫酸氢盐测序（WGBS）检测DNA甲基化水平，发现TAG1的功能缺失导致特定基因位点超甲基化。相比之下，过表达TAG1显著降低了基因体和转座子元件中的DNA甲基化水平。亚硫酸氢盐测序表明，TAG1功能缺失会导致GA生物合成所需的基因GERANYL DIPHOSPHATE SYNTHASE 1（GPS1）的启动子区域甲基化水平升高，并抑制GPS1基因表达。遗传分析表明，ddm1-2或met1-3可以恢复tag1的半矮表型并恢复GPS1的基因表达水平。尽管TAG1不能在体外直接去除或结合5-甲基胞嘧啶（5mC），但发现TAG1直接与RdDM途径的关键基因AtMORC6 / DMS11直接相互作用。总之，我们的工作发现了一个新的调控叶片衰老和植物高度的重要基因-DNA糖基化酶TAG1，并且阐明了TAG1通过调控表观遗传过程从而调节植物生长发育的分子机制，为通过分子育种培育高产作物提供了分子基础。