ABF2, ABF3, and ABF4 Promote ABA-Mediated Chlorophyll Degradation and Leaf Senescence by Transcriptional Activation of Chlorophyll Catabolic Genes and Senescence-Associated Genes in Arabidopsis

ABF2, ABF3和 ABF4通过叶绿素代谢以及衰老相关基因的转录激活而促进ABA介导的拟南芥叶绿素降解和叶片衰老

Shan Gao^1,3, Jiong Gao^1,3, Xiaoyu Zhu¹, Yi Song¹, Zhongpeng Li¹, Guodong Ren¹, Xin Zhou^1,*

and Benke Kuai^1,2,*

1State Key Laboratory of Genetic Engineering, School of Life Sciences

2Ministry of Education Key Laboratory for Biodiversity Science and Ecological Engineering, Institute of Biodiversity Science

Fudan University, Shanghai 200438, China

3These authors contributed equally to this article.

*Correspondence: Xin Zhou (zhouxin@fudan.edu.cn), Benke Kuai (bkkuai@fudan.edu.cn)

http://dx.doi.org/10.1016/j.molp.2016.06.006

Chlorophyll (Chl) degradation is an integral process of leaf senescence, and NYE1/SGR1 has been demonstrated as a key regulator of Chl catabolism in diverse plant species. In this study, using yeast one-hybrid screening, we identified three abscisic acid (ABA)-responsive element (ABRE)-binding transcription factors, ABF2 (AREB1), ABF3, and ABF4 (AREB2), as the putative binding proteins of the NYE1 promoter. Through the transactivation analysis, electrophoretic mobility shift assay, and chromatin immunoprecipitation, we demonstrated that ABF2, ABF3, and ABF4 directly bound to and activated the NYE1 promoter in vitro and in vivo. ABA is a positive regulator of leaf senescence, and exogenously applied ABA can accelerate Chl degradation. The triple mutant of the ABFs, abf2abf3abf4, as well as two ABA-insensitive mutants, abi1-1 and snrk2.2/2.3/2.6, exhibited stay-green phenotypes after ABA treatment, along with decreased induction of NYE1 and NYE2 expression. In contrast, overexpression of ABF4 accelerated Chl degradation upon ABA treatment. Interestingly, ABF2/3/4 could also activate the expression of two Chl catabolic enzyme genes, PAO and NYC1, by directly binding to their promoters. In addition, abf2abf3abf4 exhibited a functional stay-green phenotype, and senescence-associated genes (SAGs), such as SAG29 (SWEET15), might be directly regulated by the ABFs. Taken together, our results suggest that ABF2, ABF3, and ABF4 likely act as key regulators in mediating ABA-triggered Chl degradation and leaf senescence in general in Arabidopsis.

叶绿素的降解是植物叶片衰老的过程，在许多植物中，NYE1/SGR1被证实是叶绿素降解的关键调节因子。我们利用酵母单杂交的方法筛选到了3个ABA反应元件转录因子ABF2 (AREB1), ABF3, and ABF4 (AREB2)，并且假设其可以结合在NYE1的启动子上。进一步利用转录活性分析，电泳迁移率变动分析，和染色质免疫沉淀等方法证明，无论在体内还是体外ABF2、ABF3 和 ABF4直接结合并激活了NYE1启动子。ABA正向调节叶片衰老，且外源喷施ABA加速了叶绿素的降解， ABA处理后，abf2abf3abf4和ABA不敏感型突变体abi1-1 、 snrk2.2/2.3/2.6都表现出持绿特征，且NYE1和 NYE2 的表达量降低。与之相反，过表达ABF4加速了叶绿素的降解。ABF2/3/4能够结合到叶绿素代谢相关基因PAO 和 NYC1的启动子上并提高了其表达量。此外，ABFs可能直接调节衰老相关基因例如SAG29的表达。总之，我们的结果表明，ABF2、ABF3和 ABF4在调节ABA引起的叶绿素以及叶片衰老过程中扮演者重要作用。