EIN3 and ORE1 Accelerate Degreening during Ethylene-Mediated Leaf Senescence by Directly Activating Chlorophyll Catabolic Genes in Arabidopsis

Abstract

Degreening, caused by chlorophyll degradation, is the most obvious symptom of senescing leaves. Chlorophyll degradation can be triggered by endogenous and environmental cues, and ethylene is one of the major inducers. ETHYLENE INSENSITIVE3 (EIN3) is a key transcription factor in the ethylene signaling pathway. It was previously reported that EIN3, miR164, and a NAC (NAM, ATAF, and CUC) transcription factor ORE1/NAC2 constitute a regulatory network mediating leaf senescence. However, how this network regulates chlorophyll degradation at molecular level is not yet elucidated. Here we report a feed-forward regulation of chlorophyll degradation that involves EIN3, ORE1, and chlorophyll catabolic genes (CCGs). Gene expression analysis showed that the induction of three major CCGs, NYE1, NYC1 and PAO, by ethylene was largely repressed in ein3 eil1 double mutant. Dual-luciferase assay revealed that EIN3 significantly enhanced the promoter activity of NYE1, NYC1 and PAO in Arabidopsis protoplasts. Furthermore, Electrophoretic mobility shift assay (EMSA) indicated that EIN3 could directly bind to NYE1, NYC1 and PAO promoters. These results reveal that EIN3 functions as a positive regulator of CCG expression during ethylene-mediated chlorophyll degradation. Interestingly, ORE1, a senescence regulator which is a downstream target of EIN3, could also activate the expression of NYE1, NYC1 and PAO by directly binding to their promoters in EMSA and chromatin immunoprecipitation (ChIP) assays. In addition, EIN3 and ORE1 promoted NYE1 and NYC1 transcriptions in an additive manner. These results suggest that ORE1 is also involved in the direct regulation of CCG transcription. Moreover, ORE1 activated the expression of ACS2, a major ethylene biosynthesis gene, and subsequently promoted ethylene production. Collectively, our work reveals that EIN3, ORE1 and CCGs constitute a coherent feed-forward loop involving in the robust regulation of ethylene-mediated chlorophyll degradation during leaf senescence in Arabidopsis.

Spatiotemporal Transcriptome Analysis Provides Insights into Bicolor Tepal Development in Lilium “Tiny Padhye”

“…Many previous studies have identified the TFs responsible for controlling Chl metabolism in leaves (Lim, 2003; Lin et al, 2015; Wen et al, 2015). Three TFs, ANAC046, ETHYLENE INSENSITIVE3, and ORE1, positively regulate Chl degradation by binding to the promoter regions of NYC, SGR1 , and PaO (Qiu et al, 2015; Oda-Yamamizo et al, 2016). FLU, a negative regulator of Chl biosynthesis in A. thaliana , may mediate its regulatory effect through interaction with enzymes (GSA and CHlH) involved in Chl biosynthesis (Meskauskiene et al, 2001).…”

Dark‐induced leaf senescence: new insights into a complex light‐dependent regulatory pathway

“…b,d), and, further, PIFs, ORE1, EIN3 and/or ABI5 together regulating chlorophyll catabolism genes (Fig. e) (Sakuraba et al ., ; Song et al ., ; Qiu et al ., ).…”

Programming of Plant Leaf Senescence with Temporal and Inter-Organellar Coordination of Transcriptome in Arabidopsis1

“…Combining molecular genetic analysis with systems biology approaches has also revealed complex gene regulatory networks governing leaf senescence, such as a trifurcate feedforward loop including ETHYLENE-INSENSITIVE 2, miR164, and ORESARA1/ANAC092 ). EIN3 and additional NAC transcription factors (TFs) were further found to be involved in the leaf senescence network (Balazadeh et al, 2011;Hickman et al, 2013;Li et al, 2013;Kim et al, 2014a;Qiu et al, 2015). On the other hand, comprehensive profiling of molecular programs has been revealed by time-course transcriptome analysis during early and late leaf development (Beemster et al, 2005;Efroni et al, 2008;Breeze et al, 2011;Andriankaja et al, 2012;Thatcher et al, 2015).…”