Regulatory Functions of Cellular Energy Sensor SNF1-Related Kinase1 for Leaf Senescence Delay through ETHYLENE- INSENSITIVE3 Repression

Abstract

Aging of living organisms is governed by intrinsic developmental programs, of which progression is often under the regulation of their cellular energy status. For example, calorie restriction is known to slow down aging of heterotrophic organisms from yeasts to mammals. In autotrophic plants cellular energy deprivation by perturbation of photosynthesis or sugar metabolism is also shown to induce senescence delay. However, the underlying molecular and biochemical mechanisms remain elusive. Our plant cell-based functional and biochemical assays have demonstrated that SNF1-RELATED KINASE1 (SnRK1) directly interacts, phosphorylates, and destabilizes the key transcription factor ETHYLENE INSENSITIVE3 (EIN3) in senescence-promoting hormone ethylene signaling. Combining chemical manipulation and genetic validation using extended loss-of-function mutants and gain-of-function transgenic lines, we further revealed that a SnRK1 elicitor, 3-(3,4-dichlorophenyl)-1,1-dimethylurea enables to slow down senescence-associated leaf degreening through the regulation of EIN3 in Arabidopsis. Our findings enlighten that an evolutionary conserved cellular energy sensor SnRK1 plays a role in fine-tuning of organ senescence progression to avoid sudden death during the last step of leaf growth and development.

Figure 1

AKIN10 interacts directly with EIN3. (a) Structure modeling of protein kinase domain of Snf1 in S. pombe and AKIN10 in A. thaliana. Individual protein structures were generated using SWISS-MODEL (http://swissmodel.expasy.org) and visualized in a superimposed image using PyMOL (http://www.pymol.org). (b) Binary protein-protein interaction of AKIN10 and EIN3 was analyzed using a yeast two-hybrid system. (c) Protein-protein interaction of AKIN10 and EIN3 was confirmed by co-immunoprecipitation using Arabidopsis protoplasts transfected with a combination of AKIN10 and EIN3 constructs. (d) Binary protein-protein interactions between AKIN10 with MYB2, MYC3, or MYC4 were analyzed using a yeast two-hybrid system. (e) AKIN10-dependent EIN3 phosphorylation in vitro was shown with GST-EIN3 fragments as substrate. Coomassie blue staining was used for protein substrate visualization. All experiments were repeated with consistent results.

Figure 2

DCMU-inducible AKIN10 represses EIN3-dependent gene expression. (a,b) DIN6 promoter (DIN6p) and EBS element (EBSp) activities were measured with expression of AKIN10 (a) or EIN3 (b) as well as in the presence of DCMU. (c,d) DIN6p (c) and EBSp (d) activities were measured in combination of active or inactive forms of AKIN10 with and without EIN3. (e) EIN3 protein level was measured as EIN3-fLUC activity with gradient expression of AKIN10 in ein3-1 protoplasts. The fLUC activity was used as a cellular response control. (f) EIN3 protein level was measured as EIN3-fLUC activity in the presence of DCMU in ein3-1 protoplasts. The rLUC activity served as control. All protoplast experiments were repeated three times with consistent results. The means of at least three replicates are shown with standard-error bars. ***P < 0.001, **P < 0.01, and *P < 0.05. (g,h) Protein levels of EIN3-GFP (g) and SAP3-GFP (h) were determined in Arabidopsis protoplasts in the absence and presence of DCMU by protein blot analysis using anti-GFP antibody. All experiments were repeated with consistent results.

Figure 3

RNA sequencing-based analysis of differentially expressed genes (DEG) by EIN3 and AKIN10. (a,b) Comparative analysis of DEG up-regulated by EIN3 and down-regulated by AKIN10 (a) and those down-regulated by EIN3 and up-regulated by AKIN10 (b). (c,d) Expression of genes up-regulated by EIN3 and down-regulated by AKIN10 (c) and those down-regulated by EIN3 and up-regulated by AKIN10 (d) were measured using Arabidopsis protoplasts in combination of EIN3 and AKIN10 using RT-qPCR. (e,f) Comparative analysis of DEG up-regulated by both EIN3 and AKIN10 (e) and those down-regulated by both EIN3 and AKIN10 (f). (g) Meta-analysis of DEG up-regulated by both EIN3 and AKIN10 with those by hypoxia. (h) Expression of genes up-regulated by both EIN3 and AKIN10 was measured in combination of EIN3 and AKIN10 using RT-qPCR. (c,d,h) Values are means of triplicates with standard error bars (*p < 0.001; **p < 0.01; ***p < 0.05). All experiments were repeated with consistent results.

Figure 4

An AKIN10 elicitor DCMU delays EIN3-dependent ethylene inducible leaf senescence. (a,b) Anthocyanin accumulation was observed (a) and measured (b) in detached mature leaves in combination of sucrose and DCMU under light (n = 25). (c,d) Leaf degreening (c) was monitored and chlorophyll contents (d) were measured with detached mature leaves in combination of ACC and DCMU under darkness (n = 25). Values are means of triplicates with standard error bars (*p < 0.001; **p < 0.01; ***p < 0.05). (e) Expression of marker genes for senescence and ethylene signaling was monitored using quantitative RT-qPCR. EIF4a served as a control. (f) Leaf degreening was observed for Col-0, ethylene insensitive ein3-1 and ethylene oversensitive EIN3-expressing transgenic lines (EIN3) in the absence and presence of DCMU. (g) Fluorescence signals were observed in root tips of EIN3-GFP expressing transgenic Col-0 in the presence of ACC and/or DCMU under confocal microscopy. (h) Fluorescence signals were observed in root tips of transgenic lines expressing EIN3-GFP and AKIN10 ^WT or AKIN10 ^IN in the presence of ACC under confocal microscopy. All experiments were repeated with consistent results. Representative results were shown.

Figure 5

AKIN10 activity negatively modulates dark inducible leaf senescence. (a,b) Dark-inducible leaf degreening (a) was observed and chlorophyll contents (b) were measured for Col-0, ein3-1, transgenic Col-0 expressing EIN3, AKIN10 ^WT, or AKIN10 ^IN and transgenic ein3-1 expressing AKIN10 ^WT or AKIN10 ^IN. Values were means of triplicates with standard errors (n = 5). Experiments were repeated with consistent results. (c,d) Expression of marker genes for early (NAC2) (c) and late (SAG12) (d) senescence responses was monitored using quantitative RT-qPCR. Values were means of triplicates with standard-error bars.