GhWRKY33 Interacts with GhTIFY10A to Synergistically Modulate Both Ageing and JA-Mediated Leaf Senescence in Arabidopsis

WRKY transcription factors play critical roles in the modulation of transcriptional changes during leaf senescence, but the underlying mechanisms controlled by them in this progress still remain enigmatic. In this study, Gossypium hirsutum WRKY DNA-binding protein 33 (GhWRKY33) was characterized as a negative regulator of both ageing and JA-mediated leaf senescence. The overexpression of GhWRKY33 in Arabidopsis greatly delayed leaf senescence, as determined by elevated chlorophyll content, lower H2O2 content, and reduced expression of several senescence-associated genes (SAGs). An electrophoretic mobility shift assay (EMSA) and transient dual–luciferase reporter assay revealed that GhWRKY33 could bind to the promoters of both AtSAG12 and Ghcysp and suppress their expression. Yeast two-hybrid (Y2H) and firefly luciferase complementation imaging (LUC) assays showed that GhWRKY33 could interact with GhTIFY10A. Similarly, the overexpression of GhTIFY10A in Arabidopsis also dramatically delayed leaf senescence. Furthermore, both GhWRKY33 and GhTIFY10A negatively regulate JA-mediated leaf senescence. In addition, a transientdual-luciferase reporter assay indicated that GhWRKY33 and GhTIFY10A could function synergistically to inhibit the expression of both AtSAG12 and Ghcysp. Thus, our work suggested that GhWRKY33 may function as a negative regulator to modulate both ageing and JA-mediated leaf senescence and also contributes to a basis for further functional studies on cotton leaf senescence.


Introduction
Plant senescence causes a series of active degenerative alterations at the cellular, tissue, organ, and organism levels and often accompanies color changes and the shedding of leaves in autumn. At the growth and maturation stages, leaves are the primary photosynthetic organ for energy harvesting and nutrient production. When a leaf initiates senescing, it then serves as a source for mobilizable nutrients to increase reproductive success [1]. Leaf senescence is a programmed cell death process controlled by a highly regulated genetic network. This process represents one of the external manifestations of plant growth and development in response to the adverse environment. Normally, plant senescence contributes to survival under various adverse environmental conditions. Senescence is normally initiated in an ageing-dependent manner, but environmental signals and multiple phytohormones can also trigger it. Several phytohormones, such as jasmonic acid (JA),

Generation of Transgenic Overexpression Lines
To generate the 35S:GhWRKY33 and 35S:GhTIFY10A, the cDNA fragments containing the full coding sequence were cloned into the same restriction sites of the Agrobacterium transformation vector pOCA30 in the sense orientation driven by the CaMV 35S promoter. The floral dip procedure performed the Arabidopsis transformation. The seeds were collected from transformed plants and selected on 1/2 Murashige & Skoog medium containing 50 µg/mL kanamycin. Kanamycin-resistant plants were transferred to soil 8 d after germination and were grown in a growth chamber. The primers used for identifying transgenic overexpression lines are listed in Table S1.

Gene Expression Analysis
For RT-qPCR analysis, total RNA was extracted using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) and was treated with RNase-free DNase, according to the manufacturer's instructions. Total RNA (1 µg) was reverse-transcribed in a 20 µL reaction mixture using Superscript II (Invitrogen, Carlsbad, CA, USA). After the reaction, 1µL aliquots were used as templates for RT-qPCR. Half-reactions (10 µL each) were performed with the Light Cycler FastStart DNA Master SYBR Green I Kit on a Light Cycler 480 real-time PCR machine (Roche, Mannheim, Germany), according to the manufacturer's instructions. AtActin2 and GhActin were used as controls in RT-qPCR [15,19]. Analysis was conducted following the minimum information for publication of quantitative Real-Time PCR experiments guidelines [20]. Primers used for RT-qPCR analysis are listed in Table S1.

EMSA Assays
The full-length GhWRKY33 CDS was cloned into pGEX-4T-1. All plasmids were introduced into Escherichia coli BL21 cells, and Glutathione S-transferase (GST), GST-GhWRKY33 protein expression was induced by 0.5 mM Isopropyl β-D-1-thiogalactopyranoside for 24 h at 16 • C. Soluble GST and GST-GhWRKY33 were extracted and immobilized to glutathione beads (Thermo Fisher Scientific, Waltham, MA, USA). The purified GST-GhWRKY33 protein was confirmed by SDS-PAGE and used for EMSA. The EMSA assay was conducted using a Chemiluminescent EMSA Kit (Beyotime) following the manufacturer's protocol. The DNA fragments of the AtSAG12 or Ghcysp promoter were synthesized, and biotin was labeled to the 5 terminal of DNA at Beijing Genomics Institute (Beijing, China). Biotinunlabeled fragments of the same sequences or mutated sequences were used as competitors, and the GST protein alone was used as the negative control.

Yeast Two-Hybrid Screening and Confirmation
The full-length CDS of GhWRKY33 was cloned into the bait vector pGBKT7 and then transformed into the yeast strain Y 2 HGold (Clontech, Mountain View, CA, USA). The two-hybrid screening was performed via the mating protocol described in Clontech's Matchmaker Gold Yeast Two-Hybrid user manual. To confirm protein-protein interactions, the full-length CDS of GhTIFY10A were cloned into the prey vector pGADT7.

Luciferase Complementation Imaging Assay (LCI)
The full-length CDS of GhWRKY33 and GhTIFY10A were fused with pCAMBIA1300-cLUC and pCAMBIA1300-nLUC, respectively. Then, the assays were performed as described previously [21].

Leaf Senescence Assays
Leaves from four-week-old plants were used for the JA-induced leaf senescence assay. The detached leaves were placed into dishes filled with distilled water or water with 100 µM MeJA and then kept in weak light (20 µmol m −2 s −1 photosynthetic photon flux density) at 22 • C. Chlorophyll was then extracted with 80% acetone from detached leaves, and its content was determined at 663 and 645 nm, according to Lichtenthaler (1987) [22]. Membrane ion leakage was determined as described in Jiang et al., 2007 [23]. The H 2 O 2 content was measured with the H 2 O 2 content detection Kit (BC3595, Solarbio, Beijing, China) using the titanium sulfate colorimetric method.

Transient Expression Assay
The promoter regions of both AtSAG12 and Ghcysp were amplified and cloned into the pGreenII 0800-LUC vector as reporters. Full-length CDS of GhWRKY33, GhTIFY10A, and GFP were amplified and cloned into the pGreenII 62-SK vector as effectors. Then the transient expression assay was performed according to Wang et al., 2016 [24]. Agrobacterium tumefaciens GV3101 harboring the above constructs was infiltrated into five-week-old Nicotiana benthamiana leaves using a needleless syringe for transactivation analyses. After growing for 48 h under the condition of 16 h of light and 8 h of dark, leaves were injected with 0.94 mM luciferin, and the resulting luciferase signals were captured using the Tanon-5200 image system. These experiments were repeated at least three times with similar results. Quantitative analysis was performed using ImageJ software.

Overexpression of GhWRKY33 Delayed Leaf Senescence in Transgenic Arabidopsis Plants
During our studies about the role of GhWRKY members, we found that GhWRKY33 may act as a potential candidate that plays a role in leaf senescence. Then, to confirm the role of GhWRKY33 in leaf senescence, four homozygous transgenic Arabidopsis lines (T 3 ) heterologously expressing GhWRKY33 under the control of the CaMV 35S promoter (GhWRKY33-OE-3#, GhWRKY33-OE-5#, GhWRKY33-OE-13# and GhWRKY33-OE-16#) were used for further study ( Figure 1A and Figure S1A). We observed that the four lines all showed a similar phenotype to wild-type in terms of overall development and flowering time. Interestingly, ageing-triggered leaf senescence was delayed in GhWRKY33-overexpressing plants compared with wild-type in a dose-independent manner of GhWRKY33 (Figures 1B-D and S1B). Transgenic plants can survive about 15 more days than wild-type plants, on average. The transgenic plants also displayed a significantly elevated chlorophyll content, lower ion leakage, significantly reduced H 2 O 2 content, and reduced expression of several SAGs but the enhanced expression of the photosynthetic genes (AtCAB1 and AtRBCS1A) than wild-type plants ( Figure 1E-N). Thus, the constitutive overexpression of GhWRKY33 in Arabidopsis led to delayed leaf senescence.

GhWRKY33 Was Repressed in Senescing Leaves
Since GhWRKY33 appears to act as a negative regulator in leaf senescence, we speculate that GhWRKY33 may show altered expression in cotton senescing leaves. Then cotton leaves at different senescence stages were collected, and the expression pattern of GhWRKY33 in these leaves was analyzed in Zhongzhimian 2 ( Figure 2A). As shown in Figure 2B, compared with non-senescent leaves (NS), the expression level of GhWRKY33 was dramatically lower in early senescent leaves (ES). It was further repressed during the late senescent leaves (LS). The results showed that the accumulation of GhWRKY33 transcript was reduced during cotton leaf senescence. Based on Plant Public RNA-seq Database (PPRD, http://ipf.sustech. edu.cn/pub/plantrna/, accessed on 22 June 2022), we also observed repressed expression of GhWRKY33 in senescing leaves in both TX2094 and Lumianyan 28 ( Figure S2) [25]. Furthermore, as an orthologous gene of GhWRKY33, AtWRKY70 has been demonstrated to function as a negative regulator of leaf senescence in Arabidopsis thaliana [26,27]. Thus, the results indicated that GhWRKY33 may play a role in leaf senescence.  script was reduced during cotton leaf senescence. Based on Plant Public RNA-seq Database (PPRD, http://ipf.sustech.edu.cn/pub/plantrna/, accessed on 22 June 2022 ), we also observed repressed expression of GhWRKY33 in senescing leaves in both TX2094 and Lumianyan 28 ( Figure S2) [25]. Furthermore, as an orthologous gene of GhWRKY33, At-WRKY70 has been demonstrated to function as a negative regulator of leaf senescence in Arabidopsis thaliana [26,27].Thus, the results indicated that GhWRKY33 may play a role in leaf senescence. RT-qPCR analysis of GhWRKY33 transcript levels in wild-type leaves at different developmental stages. Transcript levels of GhWRKY33 in NS leaves were arbitrarily set to 1. Data from three biological replicates were analyzed by ANOVA, and asterisks indicate significant differences compared with NS leaves (*** p < 0.001).

GhWRKY33 Binds Directly to the Promoters of SAGs and Suppresses Their Expression
The WRKY TFs participated in various physiological processes by specifically binding to W-boxes (T/CTGACC/T) in the promoters of their target genes [28,29]. The above results showed that constitutive overexpression of GhWRKY33 in Arabidopsis can delay leaf senescence. Therefore, we speculated that GhWRKY33 might participate in leaf senescence by regulating the expression of SAGs. To test this hypothesis, we then analyzed the promoter sequence of Ghcysp and its homologue gene, AtSAG12, in Arabidopsis. Interestingly, W-box elements were found in the promoter sequence of both Ghcysp and AtSAG12, suggesting that GhWRKY33 may directly regulate their expression during leaf senescence. Then, we performed EMSAs with the GST-GhWRKY33 recombinant protein to determine the in vitro binding of GhWRKY33 to both Ghcysp and AtSAG12 promoters ( Figure 3A). As shown in Figure 3B, GhWRKY33 could bind the probes containing the W-box sequence. The binding signals decreased after the addition of unlabeled WT competitors. In contrast, the GhWRKY33 protein did not bind to the mutant probe carrying a mutated W-box ( Figure 3B). The GST protein alone also did not bind to both Ghcysp and AtSAG12 promoters ( Figure 3B).
suggesting that GhWRKY33 may directly regulate their expression during leaf senescence. Then, we performed EMSAs with the GST-GhWRKY33 recombinant protein to determine the in vitro binding of GhWRKY33 to both Ghcysp and AtSAG12 promoters ( Figure 3A). As shown in Figure 3B, GhWRKY33 could bind the probes containing the W-box sequence. The binding signals decreased after the addition of unlabeled WT competitors. In contrast, the GhWRKY33 protein did not bind to the mutant probe carrying a mutated Wbox ( Figure 3B). The GST protein alone also did not bind to both Ghcysp andAtSAG12 promoters ( Figure 3B).  These data suggest that GhWRKY33 may directly bind to the promoters of both Ghcysp and AtSAG12 to modulate leaf senescence.
To further elucidate the direct regulation GhWRKY33 on the expression of both Ghcysp and AtSAG12, a transient dual-luciferase assay was conducted in Nicotiana benthamiana leaves. Then both the promoters of Ghcysp and AtSAG12 were fused with LUC gene as reporters (Ghcysp:LUC and AtSAG12:LUC) ( Figure 3C). At the same time, the full-length CDS of GhWRKY33 was driven by the CaMV35S promoter as an effector ( Figure 3C). Coexpression of a reporter with effector plasmid in N. benthamiana leaves led to the repression of LUC compared with the control ( Figure 3D-G and Figure S3). This result indicates that GhWRKY33 can inhibit the expression of both Ghcysp and AtSAG12.

GhWRKY33 Physically Interacts with GhTIFY10A
Increasing evidence suggests that WRKY proteins function by forming protein complexes with other interactors [29]. We employed the yeast two-hybrid system to search for potential partners of the GhWRKY33 protein. GhWRKY33 was fused with the BD domain of the pGBKT7 vector as bait. Yeast cells harboring the bait were transformed with a specific library containing GhTIFYs inserts for prey proteins fused to GAL4-AD. We found that GhWRKY33 can interact with GhTIFY6B and GhTIFY10A (Figures 4A and S4). In this study, we pay attention to the possible role of GhTIFY10A in leaf senescence. GhTIFY10A contained both TIFY-and Jas-conserved domains and belongs to the JAZ subfamily of the TIFY protein family ( Figure S5).

Overexpression of GhTIFY10A Delayed Leaf Senescence in Transgenic Arabidopsis Plants
Given that GhWRKY33 and GhTIFY10A physically interact, and GhWRKY33 transgenic plants have a delayed senescence phenotype, we speculate that GhTIFY10A may also play a role in leaf senescence. We first determined the expression of GhTIFY10A in cotton senescing leaf. As shown in Figure 5A, similar to GhWRKY33, the expression level of GhTIFY10A was also dramatically lower in early senescent leaves (ES) and was further repressed in late senescent leaves (LS) than that of non-senescent leaves (NS). These results showed that GhTIFY10A was repressed during cotton leaf senescence, indicating that GhTIFY10A may also play a role in leaf senescence. To determine whether GhWRKY33 interacts with GhTIFY10A in planta, we conducted firefly luciferase (LUC) complementation imaging (LCI) assays in N. benthamiana leaves. In these experiments, GhWRKY33 was fused to the C-terminal half of LUC (cLUC) to produce GhWRKY33-cLUC, whereas GhTIFY10A was fused to the N-terminal half of LUC (nLUC) to produce nLUC-GhTIFY10A. N. benthamiana cells co-expressing GhWRKY33-cLUC and nLUC-GhTIFY10A displayed strong luminescence signals, whereas those coexpressing nLUC and GhWRKY33-cLUC or nLUC-GhTIFY10A and cLUC displayed no signal, confirming that the GhWRKY33-GhTIFY10A interaction occurs in vivo ( Figure 4B).

Overexpression of GhTIFY10A Delayed Leaf Senescence in Transgenic Arabidopsis Plants
Given that GhWRKY33 and GhTIFY10A physically interact, and GhWRKY33 transgenic plants have a delayed senescence phenotype, we speculate that GhTIFY10A may also play a role in leaf senescence. We first determined the expression of GhTIFY10A in cotton senescing leaf. As shown in Figure 5A, similar to GhWRKY33, the expression level of GhTIFY10A was also dramatically lower in early senescent leaves (ES) and was further repressed in late senescent leaves (LS) than that of non-senescent leaves (NS). These results showed that GhTIFY10A was repressed during cotton leaf senescence, indicating that GhTIFY10A may also play a role in leaf senescence.  To further confirm the role of GhTIFY10A in leaf senescence, four homozygous transgenic Arabidopsis lines (T 3 ) heterologously expressing GhTIFY10A under the control of the CaMV 35S promoter (GhTIFY10A-OE-6#, GhTIFY10A-OE-8#, GhTIFY10A-OE-10# and GhTIFY10A-OE-11#) were used for further study Figures 5B and S6A). Interestingly, ageingtriggered leaf senescence was clearly delayed in GhTIFY10A-overexpressing plants compared with wild-type ( Figures 5C,D and S6B). The transgenic plants also displayed a significantly elevated chlorophyll content, lower ion leakage, significantly reduced H 2 O 2 content, and enhanced the expression of the photosynthetic genes (AtCAB1 and AtRBCS1A) but reduced expression of several SAGs than wild-type plants ( Figure 5E-N). Thus, constitutive overexpression of GhTIFY10A in Arabidopsis led to delayed leaf senescence.

Both GhWRKY33 and GhTIFY10A Negatively Regulate JA-Induced Leaf Senescence
Given that GhWRKY33 and GhTIFY10A physically interact and both negatively regulate leaf senescence, we speculated that they might also participate in the regulation of JA-induced leaf senescence. Interestingly, both GhWRKY33 and GhTIFY10A are induced by MeJA treatment, implying their possible involvement in JA-induced leaf senescence ( Figure S7). Then, the detached leaves of GhWRKY33-OE lines, GhTIFY10A-OE lines, and WT were used for JA-induced leaf senescence assays. After MeJA treatment, compared with WT, GhWRKY33-OE and GhTIFY10A-OE leaves showed less severe yellowing ( Figure 6A,J). Furthermore, the measurement of chlorophyll content also showed that chlorophyll was lost more quickly in the leaves of WT than in the GhWRKY33-OE and GhTIFY10A-OE lines upon MeJA treatment ( Figure 6B,K). Consistently, after treatment with MeJA, the transgenic plants also showed reduced expression of several SAGs but the enhanced expression of the photosynthetic genes (AtCAB1 and AtRBCS1A) than wild-type plants ( Figure 6C-I,L-R).These results suggested that both GhWRKY33 and GhTIFY10A play negative roles in JA-induced leaf senescence. GhTIFY10A-OE-11#) were used for further study (Figures 5B and S6A). Interestingly, ageing-triggered leaf senescence was clearly delayed in GhTIFY10A-overexpressing plants compared with wild-type ( Figures 5C,D and S6B). The transgenic plants also displayed a significantly elevated chlorophyll content, lower ion leakage, significantly reduced H2O2 content, and enhanced the expression of the photosynthetic genes (AtCAB1 and AtRBCS1A) but reduced expression of several SAGs than wild-type plants ( Figure 5E-N). Thus, constitutive overexpression of GhTIFY10A in Arabidopsis led to delayed leaf senescence.

Both GhWRKY33 and GhTIFY10A Negatively Regulate JA-Induced Leaf Senescence
Given that GhWRKY33 and GhTIFY10A physically interact and both negatively regulate leaf senescence, we speculated that they might also participate in the regulation of JA-induced leaf senescence. Interestingly, both GhWRKY33 and GhTIFY10A are induced by MeJA treatment, implying their possible involvement in JA-induced leaf senescence ( Figure S7).Then, the detached leaves of GhWRKY33-OE lines,GhTIFY10A-OE lines, and WT were used for JA-induced leaf senescence assays. After MeJA treatment, compared with WT, GhWRKY33-OE and GhTIFY10A-OE leaves showed less severe yellowing (Figure 6A,J). Furthermore, the measurement of chlorophyll content also showed that chlorophyll was lost more quickly in the leaves of WT than in the GhWRKY33-OE and GhTIFY10A-OE lines upon MeJA treatment ( Figure 6B,K). Consistently, after treatment with MeJA, the transgenic plants also showed reduced expression of several SAGs but the enhanced expression of the photosynthetic genes (AtCAB1 and AtRBCS1A) than wildtype plants( Figure 6C-I,L-R).These results suggested that both GhWRKY33 and GhTIFY10A play negative roles in JA-induced leaf senescence.

GhTIFY10A and GhWRKY33 Acts Synergistically to Suppress Both AtSAG12 and Ghcysp Expression
Having demonstrated that GhTIFY10A physically interacts with GhWRKY33, we speculated that it might affect the transcriptional function of GhWRKY33. To test this possibility, a transient dual-luciferase assay was performed in tobacco leaves to elucidate the functional role of GhWRKY33 and GhTIFY10A in regulating the expression of SAGs in vivo. The experiment was conducted using a double reporter plasmid, pGreenII0800-LUC, containing the LUC luciferase driven by the AtSAG12 and Ghcysp promoters. In addition, the assay includes effecters plasmid, pGreenII62-SK, expressing the GhWRKY33 and GhTIFY10A ( Figure 7A). The results showed that the expression of GhWRKY33 or GhTIFY10A resulted in reduced LUC signals compared with the reporters alone. More importantly, coexpression of GhWRKY33 with GhTIFY10A further reduced the LUC signals compared with the expression of GhWRKY33 or GhTIFY10A alone ( Figure 7B-E).These results support the hypothesis that GhWRKY33 and GhTIFY10A act synergistically to suppress both AtSAG12 and Ghcysp expression. The wild-type leaves showed a more severe JA-induced senescence phenotype than both GhWRKY33 and GhTIFY10A overexpression lines upon 100 μM MeJA treatment. (B,K) Chlorophyll content in detached full-grown rosette leaves of the indicated genotypes. (C-I,L-R) Transcript levels of AtSAGs, AtCAB1, and AtRBCS1A in the indicated genotypes. Date from three biological replicates were analyzed by ANOVA, and asterisks indicate significant differences compared with WT (** p < 0.01).

GhTIFY10A and GhWRKY33 Acts Synergistically to Suppress Both AtSAG12 and Ghcysp Expression
Having demonstrated that GhTIFY10A physically interacts with GhWRKY33, we speculated that it might affect the transcriptional function of GhWRKY33. To test this possibility, a transient dual-luciferase assay was performed in tobacco leaves to elucidate the functional role of GhWRKY33 and GhTIFY10A in regulating the expression of SAGs in vivo. The experiment was conducted using a double reporter plasmid, pGreenII0800-LUC, containing the LUC luciferase driven by the AtSAG12 and Ghcysp promoters. In addition, the assay includes effecters plasmid, pGreenII62-SK, expressing the GhWRKY33 and GhTIFY10A ( Figure 7A). The results showed that the expression of GhWRKY33 or GhTIFY10A resulted in reduced LUC signals compared with the reporters alone. More importantly, coexpression of GhWRKY33 with GhTIFY10A further reduced the LUC signals compared with the expression of GhWRKY33 or GhTIFY10A alone ( Figure 7B-E).These results support the hypothesis that GhWRKY33 and GhTIFY10A act synergistically to suppress both AtSAG12 and Ghcysp expression.

Discussion
To increase survival and fitness in their given ecological niches, plants always trigger leaf senescence to relocate mobile nutrients and energy from aging leaves to reproducing seeds [30]. Senescence is the final stage of leaf development and is tightly controlled by a sophisticated transcriptional regulatory network in which transcription factors are widely participating. Notably, numerous WRKY transcription factors show strong expression in senescing leaves, implying their potential involvement in senescence-associated transcriptional reprogramming [12]. The functional studies identified several WRKY TFs as critical regulators in leaf senescence, but their specific biological functions in this process remain elucidated. In particular, the functional elucidation of most WRKY genes in cotton represents a major challenge.
Interestingly, recent studies have demonstrated that WRKY proteins often function as key components of various phytohormone-mediated leaf senescence. For example, AtWRKY45 interacts with DELLA protein RGL1 to positively regulates GA-mediated leaf senescence, while AtWRKY75 directly promotes SA INDUCTIONDEFICIENT2 (SID2) expression to promote SA production and finally accelerates leaf senescence [4,31]. Recently, OsWRKY53 was shown to accelerate leaf senescence through the promotion of ABA accumulation, and AtWRKY75 can also play a role in ABA-mediated leaf senescence [21,32]. Although several cotton WRKY members, including GbWRKY27, GhWRKY42, and GhWRKY91, have been shown to play important roles in leaf senescence [15][16][17], it is still unclear whether they can regulate leaf senescence through interaction with certain phytohormones. Here, we provide further evidence to reveal that GhWRKY33 may function as a new component that regulates both ageing and JA-mediated leaf senescence.
We found that GhWRKY33 is repressed in senescing leaves and transgenic Arabidopsis plants overexpressing GhWRKY33 delayed both ageing and JA-triggered leaf senescence (Figures 1, 2 and 6). WRKY TFs specifically bind to the W-boxes of the promoters of their target genes and activate/repress their expression to regulate a variety of physiological processes, such as plant growth and development, defense response, and leaf senescence [4,14,29,33]. In our study, GhWRKY33 was also able to bind to the promoters of both AtSGA12 and Ghcysp, and inhibit their expression. Taken together, these results imply that GhWRKY33 may function as a negative regulator to modulate both age and JA-mediated leaf senescence. A recent study also revealed that GhWRKY33 can function as a negative regulator to mediate plant response to drought stress [28]. Thus, GhWRKY33 may mainly function as a repressor to modulate both plant growth and stress responses. However, it is still interesting to determine whether GhWRKY33 can function as an activator in certain physiological processes.
As a lipid-derived plant hormone, jasmonates (JAs) were revealed to play crucial roles in both plant defense responses and various developmental processes [34]. Studies have demonstrated that the JA signal is perceived by the F-box protein CORONATINE INSENSITIVE1 (COI1), which subsequently recruits the JASMONATEZIM-DOMAIN (JAZ) proteins for ubiquitination and degradation [35][36][37] [38][39][40][41][42]. Recently, AtWRKY57 and AtWRKY75 were also identified as targets of JAZs to modulate JA-mediated leaf senescence and plant defense against necrotrophs, respectively [3,43]. Until now, it remains unclear whether certain GhJAZs can interact with GhWRKYs to regulate leaf senescence. Here, we provide evidence that GhWRKY33 may negatively regulate leaf senescence through the JA pathway.
About 50 GhTIFYs members were recently identified in upland cotton (G. hirsutum) [44], and their functional elucidation remains a big challenge. Furthermore, there is also no report about their involvement in leaf senescence. In this study, we reveal that GhTIFY10A may function as a negative regulator to modulate both ageing and JA-triggered leaf senescence. Similar to GhWRKY33, GhTIFY10A was repressed in cotton senescing leaves, and transgenic Arabidopsis plants overexpressing GhTIFY10A delayed both ageing and JA-triggered leaf senescence (Figures 5 and 6). The yeast two-hybrid (Y2H) and firefly luciferase complementation imaging (LUC) assays showed that GhWRKY33 could interact with GhTIFY10A ( Figure 4). Further analysis using the LUC assay demonstrated that GhTIFY10A enhances the transrepression activity of GhWRKY33 and subsequently synergistically repressed the expression of AtSGA12 and Ghcysp, and finally, negatively modulated both ageing and JA-mediated leaf senescence (Figure 7). Thus, our results indicated that JAZ-targeted Gh-WRKY33 may negatively modulate plant leaf senescence by directly targeting senescenceassociated genes.
The early and abnormal senescence shortens plant lifespan and decreases crop yield and quality. Therefore, it is essential to study the underlying mechanisms and signaling pathways involved in leaf senescence, which will be highly useful for crop genetic breeding. Here, we demonstrate the molecular mechanisms underlying the regulation of both ageing and JA-triggered leaf senescence by GhWRKY33. They indicate that GhWRKY33 may function as a novel component of the cotton senescence regulatory network in both ageing and JA-mediated leaf senescence via interaction with GhTIFYs. Thus, our results represent a new insight into the roles of GhWRKY proteins in senescence-associated signaling and transcriptional reprogramming, and also lay a foundation for further functional studies on cotton leaf senescence.
Supplementary Materials: The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/cells11152328/s1, Figure S1: GhWRKY33 delayed leaf senescence in transgenic Arabidopsis plants; Figure S2: The expression level of GhWRKY33 in leaves of different senescence stages of the cotton variety TX2094 and Lumianyan 28; Figure S3: GhWRKY33 suppressed the expression of both AtSAG12 and Ghgysp; Figure S4: GhWRKY33 physically interacts with GhTIFY6B; Figure S5: Analysis of conservative motif TIFY and Jas in GhTIFY10A; Figure S6: GhTIFY10A delayed leaf senescence in transgenic Arabidopsis plants; Figure S7: Both GhWRKY33 and GhTIFY10A were induced by MeJA treatment; Table S1: Primers used in this study.  Data Availability Statement: The datasets generated during and/or analysed during the currentstudy are available from the corresponding author on a reasonable request.

Conflicts of Interest:
The authors declare no conflict of interest.