Gene regulatory cascade of senescence-associated NAC transcription factors activated by ETHYLENE-INSENSITIVE2-mediated leaf senescence signalling in Arabidopsis

Abstract

Leaf senescence is a finely tuned and genetically programmed degeneration process, which is critical to maximize plant fitness by remobilizing nutrients from senescing leaves to newly developing organs. Leaf senescence is a complex process that is driven by extensive reprogramming of global gene expression in a highly coordinated manner. Understanding how gene regulatory networks involved in controlling leaf senescence are organized and operated is essential to decipher the mechanisms of leaf senescence. It was previously reported that the trifurcate feed-forward pathway involving EIN2, ORE1, and miR164 in Arabidopsis regulates age-dependent leaf senescence and cell death. Here, new components of this pathway have been identified, which enhances knowledge of the gene regulatory networks governing leaf senescence. Comparative gene expression analysis revealed six senescence-associated NAC transcription factors (TFs) (ANAC019, AtNAP, ANAC047, ANAC055, ORS1, and ORE1) as candidate downstream components of ETHYLENE-INSENSITIVE2 (EIN2). EIN3, a downstream signalling molecule of EIN2, directly bound the ORE1 and AtNAP promoters and induced their transcription. This suggests that EIN3 positively regulates leaf senescence by activating ORE1 and AtNAP, previously reported as key regulators of leaf senescence. Genetic and gene expression analyses in the ore1 atnap double mutant revealed that ORE1 and AtNAP act in distinct and overlapping signalling pathways. Transient transactivation assays further demonstrated that ORE1 and AtNAP could activate common as well as differential NAC TF targets. Collectively, the data provide insight into an EIN2-mediated senescence signalling pathway that coordinates global gene expression during leaf senescence via a gene regulatory network involving EIN3 and senescence-associated NAC TFs.

Keywords: Arabidopsis; EIN2-mediated senescence signalling; EIN3; NAC transcription factor.; gene.

Fig. 1.

Identification of the six senescence-associated NAC TFs as potential downstream components of EIN2. (A) Expression of 29 senescence-associated NAC TF genes in Col, ein2, ore9, and ore12 mutant leaves at the mature stage (12-day-old third and fourth rosette leaves). Transcript levels of each TF gene were examined by qRT–PCR. For qRT–PCR, ACT2 was used as an internal control. Transcript abundance of the NAC TF genes in each mutant was determined relative to that in wild-type leaves. The error bars represent the standard deviation (SD; n=4). The six NAC TF genes whose expression was decreased by >50% in the mature leaves of ein2 mutants compared with the wild-type are highlighted by grey text. (B–G) Age-dependent changes in the expression of candidate NAC TF genes downstream of EIN2. Transcript levels of ANAC019 (B), AtNAP (C), ANAC047 (D), ANAC055 (E), ORS1 (F), and ORE1 (G) were analysed by qRT–PCR in the third and fourth rosette leaves from wild-type and ein2 plants at the indicated ages. Transcript levels of each gene during leaf ageing were determined relative to levels in wild-type 12-day-old leaves. Error bars represent the SD (n=4).

Fig. 2.

EIN3 is necessary and sufficient to induce the expression of the ORE1 and AtNAP genes. (A) Changes in chlorophyll content in the third and fourth rosette leaves of Col and ein3 eil1 mutant plants during leaf ageing. The photographs show representative leaves at the indicated age. Chlorophyll content is compared with the values from each genotype at day 12. (B and C) Expression of the six NAC TF genes in the third and fourth leaves of wild-type and ein3 eil1 mutants at 12 d (B) and 28 d (C) of leaf age. For qRT–PCR, ACT2 was used as an internal control. Transcript levels of each gene were determined relative to levels in wild-type 12-day-old leaves. Error bars represent the SD (n=4). (D) Expression of ORE1, AtNAP, and ANAC055 in transgenic ein3 eil1 ebf1 ebf2 plants overexpressing estradiol-inducible EIN3 (iE/qm). Three-week-old iE/qm transgenic plants were treated with 20 μM estradiol for 6h, and protein and RNA were isolated from the third and fourth leaves. The tagged EIN3 protein was visualized by immunoblot analysis using an anti-FLAG antibody. (E) For qRT–PCR, ACT2 was used as an internal control. Transcript levels of each gene after estradiol treatment were determined relative to the mock treatment. The error bars represent the SD (n=4).

Fig. 3.

The ORE1 or AtNAP loss-of-function mutant suppresses the early senescence phenotypes of an EIN3 overexpressor during dark-induced leaf senescence. (A) Representative leaves of Col, ore1, atnap, EIN3OX, EIN3OX ore1, and EIN3OX atnap plants after incubation in darkness for 6 d. (B and C) Analysis of chlorophyll content (B) and photochemical efficiency of PSII (C) of detached leaves of the indicated genotypes at 6 d after dark incubation (Student’s t-test, **P<0.01). Error bars indicate the SD (n >20).

Fig. 4.

EIN3 binds to the promoters of ORE1 and AtNAP, and induces their transcription. (A) Binding of EIN3 to the promoters of ORE1 and AtNAP in Y1H assay. An effector plasmid containing EIN3 and a reporter plasmid (pORE1-lacZ or pAtNAP-lacZ) were co-transformed into the EGY48 yeast strain. The growth of a blue yeast colony on selective medium containing X-gal indicates a positive interaction. The effector plasmid without EIN3 (vector alone) plus the reporter plasmid served as a negative control. (B) Protein levels of EIN3 in 5-week-old iE/qm transgenic plants treated or not with 100 μM estradiol for 6h. The tagged EIN3 protein was visualized by immunoblot analysis using an anti-FLAG antibody. An asterisk indicates non-specific bands detected by the anti-FLAG antibody. (C) Schematic diagram of the ORE1 and AtNAP promoters. P1–P4 represent the positions of amplicons used for ChIP-qPCR analysis. P1–P4 were chosen because these regions contain putative EIN3 binding sites (EBS, TACAT or TTCAAA). (D) Enrichment of EIN3-associated fragments after ChIP-qPCR. Chromatin from the leaves of 5-week-old iE/qm transgenic plants treated with 100 μM estradiol for 6h was immunoprecipitated with an anti-FLAG antibody. Enrichment was quantified by qPCR using specific primers. Fold changes in enrichment were normalized to ACT2. The promoter of miR164A was used as a positive control. The error bars represent the SD (n=4). (E) Transactivation of the ORE1 and AtNAP promoters by EIN3 in Arabidopsis protoplasts. Protoplasts were co-transfected with the pORE1-LUC or pAtNAP-LUC reporter and an effector plasmid expressing EIN3–GFP (green fluorescent protein). Luciferase activity was determined relative to that in protoplasts that were transfected with the reporter plasmid and an effector plasmid expressing GFP only. Relative expression of ORE1-LUC or AtNAP-LUC was normalized to that of 35Sp:RLuc (internal control). Error bars represent the SD (n=8).

Fig. 5.

The ore1 atnap double mutant exhibited a stronger delay in age-dependent leaf senescence than either mutant alone. (A) Whole-plant phenotypes of Col, ore1, atnap, and ore1 atnap mutant plants at 45 d after germination. The scale bar represents 1cm. (B) Age-dependent senescence phenotype of the third and fourth rosette leaves of Col, ore1, atnap, and ore1 atnap mutant plants at different ages. (C) The photochemical efficiency (F _v/F _m) of PSII was measured from the third and fourth leaves starting at 16 d of leaf age (Student’s t-test, **P<0.01). Error bars indicate the SD (n=12). (D) Age-dependent changes in SAG12 gene expression by qRT–PCR analysis. ACT2 was used as an internal control for qRT–PCR. The transcript level of SAG12 in wild-type at 12-day-old leaves was set at 1 (Student’s t-test, **P<0.01). The error bars represent the SD (n=4).

Fig. 6.

ORE1 and AtNAP play partially additive roles in regulating artificially induced leaf senescence. (A) Phenotypes of Col, ore1, atnap, and ore1 atnap leaves after dark incubation for the indicated times. DAT, days after treatment. (B and C) Changes in photochemical efficiency (F _v/F _m) of PSII (B) and chlorophyll content (C) during dark-induced leaf senescence. Levels of photochemical efficiency and chlorophyll content on the days indicated were determined relative to those before dark incubation (Student’s t-test, **P<0.01). Error bars indicate the SD (n=6). (D and E) Changes in the chlorophyll content (D) and photochemical efficiency of PSII (E) during ACC-induced leaf senescence. (F and G) Changes in the chlorophyll content (F) and photochemical efficiency of PSII (G) during MeJA-induced leaf senescence. Levels of two senescence markers on the days indicated were determined relative to those before ACC or MeJA treatment (Student’s t test, *P<0.05 and **P<0.01). Error bars indicate the SD (n=6).

Fig. 7.

AtNAP and ORE1 control common as well as differential NAC TF genes. (A) Expression of 27 senescence-associated NAC TF genes in Col and ore1 atnap double mutant leaves at 16 d of leaf age. Transcript levels of each NAC TF gene were examined by qRT–PCR. ACT2 was used as an internal control for qRT–PCR. Transcript levels of the NAC TF genes in the ore1 atnap mutant were determined relative to levels in wild-type leaves. The error bars represent the SD (n=4). The nine NAC TF genes whose expression was significantly decreased in the mature leaves of the ore1 atnap mutant, compared wirh levels in wild-type leaves, are highlighted by grey text. (B and C) Transactivation of the promoters of the selected NAC TF genes by ORE1 (B) and AtNAP (C) in Arabidopsis protoplasts. Protoplasts were co-transfected with each NAC TF promoter-LUC reporter and an effector plasmid expressing ORE1-HA or AtNAP-HA. Luciferase activity was determined relative to that in protoplasts that were transfected with the reporter plasmid and an effector plasmid expressing HA only. The relative expression of each NAC TF promoter-LUC was normalized to 35Sp:RLuc (internal control) (Student’s t-test, *P<0.05 and **P<0.01). Error bars represent the SD (n=6).

Fig. 8.

A plausible model for the EIN2–EIN3–NAC TFs regulatory cascade in the control of leaf senescence. EIN2-mediated senescence signalling, triggered by various senescence-inducing factors including age, hormones, and environmental stresses, activates EIN3. EIN3 directly induces the expression of two key positive regulators of leaf senescence, ORE1 and AtNAP. Simultaneously, EIN3 directly suppresses the expression of miR164 (Li et al., 2013), which negatively regulates ORE1 at the post-transcriptional level. ORE1 and AtNAP activate the expression of common as well as distinct downstream NAC TF genes. In addition, EIN2-mediated senescence signal is transduced to four NAC TFs (ANAC019, ANAC047, ANAC055, and ORS1) via an EIN3-independent pathway.