Melatonin delayed leaf senescence induced by WRKY75 in Arabidopsis thaliana

Melatonin delayed leaf senescence induced by WRKY75 in Arabidopsis thaliana | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Melatonin delayed leaf senescence induced by WRKY75 in Arabidopsis thaliana Ke-yu Wang, Jing-jing Mao, Wen-hui Chen, De-qiang Li, Zi-zhong Tang, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5326045/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Leaf senescence, highly regulated by plant hormones and environmental factors, represents the final stage of leaf development. Therefore, the strategies to delay leaf senescence might extent the limitation of growth and yield for crop. Although previous studies had demonstrated the potential of melatonin to delay leaf senescence, its mechanism remained many mysteries. Here, we reported the role of melatonin in delaying WRKY75-induced leaf senescence. Exogenous melatonin increased chlorophyll content and reduced the accumulation of ROS in plants. In addition, it up-regulated the expression of the SNAT gene and increased its melatonin level. Exogenous melatonin also decreased SA level and down-regulated SID2 gene expression. Furthermore, the expression of SAG13 and WRKY75 , both positive senescence-related genes, was found to decrease after melatonin treatment. These findings suggest that melatonin counteracts the effects of a network structure comprising SA, ROS, and WRKY75 on senescence, thereby regulating various events related to plant senescence and delaying leaf senescence. leaf senescence melatonin WRKY75 salicylic acid ROS Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 1. Introduction Leaf senescence has always been a prominent topic in the field of plant physiology. It is a complex and highly coordinated process, controlled by both external and internal factors(Gan and Amasino 1997 ; Guo et al. 2017 ; Lim et al. 2007 ). The external environmental factors include abiotic stress and biological stress, internal factors include plant age, plant hormones levels and ROS (Beers and McDowell 2001 ; Sun et al. 2016 ). Leaf senescence is associated with the transfer of nutrients from leaves to seeds. This underlined the significance of leaf senescence as the final stage of plant senescence(Woo et al. 2013 ). Effectively controlling leaf senescence could regulate the degradation process of lipids, proteins, chlorophyll, and other substances in plants, and greatly enhance crop yield and (Buchanan-Wollaston et al. 2003 ). Therefore, understanding and managing leaf senescence is vital for improving agricultural productivity. Numerous studies had shown that salicylic acid (SA) promoted the natural senescence of leaves (He et al. 2022 ; Lim et al. 2007 ; Rivas-San Vicente and Plasencia 2011 ). Morris et al. found that SA induced senescence by enhancing leaf senescence-related gene expression (Morris et al. 2000 ). Mutants with defects in SA biosynthesis or SA signal, such as npr1 ( nonexpresser of pathogenes related genes 1 ), pad4 ( phytoalexin deficiency 4 ), and NahG ( Naphthalene hydroxylase G ), exhibited a senescence-delayed phenotype, accompanied by a significant reduction in the expression of senescence-associated genes (SAGs) (Lim et al. 2007 ; Morris et al. 2000 ). SAGs played crucial roles in the regulation of plant senescence. Among them, SAG13 was considered a marker of early developmental senescence and programmed cell death, and was also involved in hypersensitivity responses following pathogen infection and plant cell death (Dhar et al. 2020 ). During leaf senescence, SA induced the accumulation of hydrogen peroxide (H 2 O 2 ) by reducing the activity of H 2 O 2 metabolism enzymes, consequently lead to oxidative damage (Durner and Klessig 1995 ; Durner and Klessig 1996 ; Ruiz-Sáenz et al. 2022 ). Because the levels of SA and H 2 O 2 gradually increased as the leaf senescence process proceeds, the leaves' capacity to scavenge ROS was diminished. Disruption of ROS balance ultimately leads to the increase of the protein and lipid peroxidation (Guo et al. 2017 ; Khanna-Chopra 2012 ). Moreover, Leon et al. identified that H 2 O 2 could stimulate the biosynthesis of SA in tobacco, further highlighting the interconnectedness of H 2 O 2 and SA signaling pathways (Leon et al. 1995 ). WRKY transcription factors (WRKY-TFs) are a prominent family of transcription factors implicating in leaf senescence(Guo et al. 2004 ). In Arabidopsis thaliana , several WRKY-TFs, including WRKY6, WRKY42, WRKY46, WRKY55, and WRKY75 were involved in the leaf senescence through SA signaling pathways (Niu et al. 2020 ; Wang et al. 2020 ; Zhang et al. 2021a ). Notably, the WRKY75 silent mutant has showed to delay age-dependent leaf senescence, whereas the overexpression mutant accelerated this process (Lim et al. 2007 ). The interaction of WRKY75 with the SID2 promoter enhanced SA biosynthesis, ultimately triggering the activation of the SA signaling pathway and consequent promoting plant cell death, autophagy, and leaf senescence (Zhang et al. 2017 ). Guo et al. unveiled that WRKY75 was induced by SA and ROS (Guo et al. 2017 ). Furthermore, WRKY75 promoted SA biosynthesis by activating SID2 transcription and promoted H 2 O 2 accumulation by inhibiting CATALASE2 ( CAT2 ) transcription (Guo et al. 2017 ). Consequently, during leaf senescence, WRKY75, SA, and ROS underwent a gradual yet irreversible rise, and propelled each other by a positive feedback loop mechanism (Guo et al. 2017 ). Melatonin (MT), chemical name is N -acetyl-5-methoxytryptamine, was first identified and named in 1958 by Lerner et al. (Lerner et al. 1958 ). Melatonin played various regulatory functions in plant growth and development, with significant crosstalk with other plant hormones such as SA (Yu et al. 2022 ; Zhang et al. 2016 ; Zhao et al. 2021 ). Moreover, interactions between melatonin and WRKY-TFs had been identified. For instance, it has been reported that WRKY17 activated the transcription of ASMT7 , which was a key gene of melatonin biosynthesis in apples (Song et al. 2023 ). Weeda et al. showed that exogenous melatonin could increase the expression of WRKY75 in A. thaliana (Weeda et al. 2014 ). Upon treating A. thaliana with melatonin for 16 hours, Gao et al. observed the gene expression of WRKY6 , WRKY22 , WRKY31 , WRKY50 , WRKY51 , WRKY54 , WRKY70 , and WRKY75 were upgregulated more than twice, and the expression of many genes related to SA and other hormones in response to biotic and abiotic stresses also changed (Gao et al. 2018 ). In this study, we investigated whether melatonin was involved in leaf senescence through a cyclic model composed of WRKY75, SA, and ROS. The experimental results demonstrated that melatonin and SA, ROS, and WRKY75 antagonize each other on senescence — melatonin downregulates the accumulation of SA and ROS, leading to a decrease in the expression of WRKY75 , thereby regulating plant senescence related processes and delaying leaf senescence in A. thaliana . This study may provide ideas for applying melatonin to regulate crop leaf senescence and improve photosynthesis for yield and quality improvement. 2 Materials and methods 2.1 Plant materials and growth conditions Arabidopsis thaliana Col-0 and T-DNA inserted mutants ( snat-2 , sid2 , and wrky75 ) were obtained from ABRC, and WRKY75 overexpression mutants ( WOE-3 and WOE-6 ) were presented by Diqiu Yu and Ligang Chen, Xishuangbanna Tropical Botanical Garden, Chinese Academy of Sciences. All the seeds were soaked in distilled water at 4°C for 2 d and then sown in sterilized mixed nutrient soil (soil: perlite: vermiculite = 4: 1: 1). The plants were subsequently grown under controlled conditions at 120 µM photon·m − 2 ·s − 1 , 22 ± 2°C, 60% humidity, 16h light /8h dark cycle. Upon reaching 20 days of growth, plants in the same growth state were irrigated with 100 µM melatonin or distilled water (as control) once every three days. Leaf samples from the 3rd-5th true leaves were collected at 20 d, 27 d, 34 d, 41 d, and 48 d (W1-W5) for subsequent experiments. Except for the first collection time (W1) before treatment, each subsequent collection time (W2-W5) corresponded to the second day after melatonin treatment. After collection, the leaves were promptly treated with liquid nitrogen and then preserved in a -80°C refrigerator. 2.2 Mutant identification and screening The 36 d leaves of A. thaliana were selected to identify and screen the mutants by the three-primers method. Plant Genomic DNA Kit (TIANGEN Biotech, China) was used to extract the DNA template, and TIANGEN masterMix II (TIANGEN Biotech, China) was used in PCR. Detailed primer sequences can be found in Table S1 , and the PCR amplification program is outlined in Table S2. 2.3 Determination of chlorophyll content Acetone (80%, v/v) was used to extract chlorophyll from 0.1 g of fresh leaves. The chlorophyll concentration was determined by a UV spectrophotometer (Shimadzu, Japan, UV-1750) as outlined by Porra et al. (Porra et al. 1989 ). 2.4 Quantification of melatonin Melatonin was extracted according to Sturtz et al. (Stürtz et al. 2011 ). 1.0 g leaves were collected and ground with liquid nitrogen. Subsequently, 2 mL of acetone was used for ultrasonic extraction for 30 min at 25°C, centrifugating at 4,000 × g for 10 min. The supernatant was then freeze-dried and then redissolved in 2 mL of distilled water for collection by using the C18 separation column (Agela Technologies, China). The separation column was washed with 2 mL of 10% methanol, then eluted with 1 mL of methanol, and determined the melatonin content by HPLC-MS/MS (Agilent Technologies, USA, HPLC-1290 Series and 6470 Triple Quadrupole MS/MS). HPLC-MS/MS parameters were set as Han et al ., detect ion m/z 159.0 (Han et al. 2017 ). 2.5 Quantification of SA 0.5 g leaves were ground with liquid nitrogen, then 3 mL of 90% methanol was added. This mixture underwent ultrasonic extraction for 20 min, followed by centrifugation at 12,000 × g for 15 min. The pellet was subsequently treated with 2 mL of methanol, centrifuged at 12,000 × g for 15 min. The two supernatants were combined, and dried in vacuum. It was redissolved with 5 mL of 5 mM sodium acetate buffer, and followed by shock extraction with equal volumes extracting solution (ethyl acetate, cyclopentane, and isopropanol 100: 99:1, v/v) for 10 min. The aqueous phase was collected, dried in vacuum, and dissolved with 3 mL of methanol. SA content determined by HPLC (Agilent Technologies, USA, HPLC-1260 Series),as described by He et al. , UV detection wavelength was 306 nm (He et al. 2007 ). 2.6 Evaluation of the redox equilibrium In situ staining of ROS was performed with reference to Yang et al. (Yang et al. 2004 ). For H 2 O 2 staining, rosette leaves were immersed in 2 mg·mL − 1 DAB, with vacuum infiltration at room temperature for 12 h. For O 2 ·− staining, the leaves were washed with decolorization solution (80% ethanol) and immersed in 0.5 mg·mL − 1 NBT solution, and decolorized after vacuum infiltration for 30 min. After leaves was decolorized, then saved images using a scanner. Detection H 2 O 2 content was referred to Velikova et al. (Velikova et al. 2000 ). 0.1 g fresh leaves ground with liquid nitrogen and extracted with 1 mL 10% trichloroacetic acid (TCA) (w/v), centrifuged at 13,800 × g at 4°C for 10 min, then the supernatant was added with 10 mM potassium phosphate buffer (pH = 7.0) and 1M KI (1: 1: 2, v/v/v), and incubated for 20 min in dark. The absorbance at 390 nm was recorded. Detection O 2 ·− content was referred to Nahar's method (Nahar et al. 2015 ). 2.7 Determination of CAT activity 0.2 g fresh leaves were ground in 4 mL of ice-cold buffer (150 mM sodium phosphate, pH 7.8), and then centrifuged at 12,000 × g at 4°C for 20 min. CAT activity was determined using Wang’s method (Wang 1995 ). 2.8 Detection of cell death To characterize the degree of cell death, trypan blue staining was employed as described by Huang et al. (Huang et al. 2019 ). The leaves were soaked in a trypan blue staining solution (10 mL of lactic acid, 10 mL of glycerol, 10 g of phenol, 20 mg of trypan blue, and 10 mL of distilled water). The leaves were incubated at 65°C for 1 min. And then, the leaves were washed with distilled water and decolorized with 80% ethanol for 1 h. Finally, the results imaged with a scanner. 2.9 Gene expression analysis The total RNA was extracted using the CTAB method, and reverse transcription was performed using the PrimeScript™ RT reagent Kit with gDNA Eraser (Takara Bio, Japan). Gene expression was detected on CFX96 Touch™ Real-Time PCR Detection Systems (Bio-Rad, USA) using SYBR Premium Ex Taq™ II dye (Takara Bio, Japan). The qPCR procedure was shown in Table S3, ACT2 as a reference. The primer sequences were in Table S4. Adopt 2 −ΔΔCT method was used to analyze the relative expression amount (Livak and Schmittgen 2001 ). 2.10 Data statistics and analysis All data from the above experiments were shown as the mean with SD. Each experiment was repeated at least three independent times. SPSS Statistics 26.0 software (IBM Inc., USA) was used for the analysis of variance (student’s t -test), GraphPad Prism 9.0.0 software (GraphPad Software, USA) was used to draw histograms, and Adobe Photoshop 2022 software (Adobe, USA) was used to create plates. 3. Results 3.1 Identification of mutants The mutant was identified using a three-primer method (Fig. S1 ). Plant 1 in the figure is a heterozygote, while plant 2 is a homozygous deletion mutant, and plants 3 and 4 are of the wild type. The seeds of the homozygous deletion wrky75 mutant were subsequently collected and propagated for experimental purposes. qPCR was used to detect the gene expression levels of each mutant (Fig. S2). The results indicated a significant decrease in the corresponding gene expression levels of the T-DNA insertion mutants when compared to Col-0. Conversely, the WRKY75 gene expression level in WOE-3 was observed to be 14.5 times higher than that in Col-0 (Fig. S2b). 3.2 Melatonin delayed the senescence process of plant leaves In the W1 phase, snat-2 exhibited a significantly smaller size than Col-0 (Fig. S3). Moreover, in the W3 phase, it was observed that the color of snat-2 treated with melatonin leaves was comparable to that of Col-0 (Fig. S3). The application of melatonin resulted in greater growth of snat-2 . During the W5 phase, the senescence process of snat-2 was slower than that of Col-0. It was established that the exogenous melatonin significantly mitigated leaf senescence in both Col-0 and snat-2 . Exogendous melatonin delayed the senescence phenotype caused by endogenous melatonin deficiency. Additionally, in the W4 phase, it was evident that wrky75 had a lower degree of yellowing, while the leaves of WRKY75 overexpression mutants showed a more degree of yellowing compared to Col-0 (Fig. 1 ). This observation indicates that the WRKY75 has a positive regulatory effect on plant senescence. The application of exogenous melatonin alleviated leaf yellowing caused by senescence, especially in WRKY75 overexpressed mutants, indicating its potential to mitigate senescence induced by WRKY75 overexpression. The alleviation of senescence due to melatonin might be dependent on the presence of WRKY75 . It was also noted that melatonin had a more pronounced effect on WOE-3 compared to WOE-6 , leading to the selection of WOE-3 as a representative WRKY75 overexpression strain for subsequent experiments. 3.3 Melatonin increased chlorophyll content in leaves In the W1, W3, and W5 phases, the 3 rd -5th true leaves were collected to assess the impact of exogenous melatonin on chlorophyll (Fig. 2 ). The total chlorophyll content continuously decreased in the senescence process. Notably, in the W3 phase, the exogenous melatonin mitigated the reduction of chlorophyll. However, in the W5 phase, melatonin had less effect on chlorophyll content. Comparative analysis demonstrated that the chlorophyll content of snat-2 was significantly higher than that of Col-0, especially in the W5 phase, where snat-2 exhibited approximately 7 times higher chlorophyll content, possibly attributed to the slower development of snat-2 . Exogenous melatonin increased chlorophyll content of snat-2 , especially in the W3 phase. This indicates the mitigating effect of melatonin on snat-2 senescence. In correlation with the phenotypic results, wrky75 exhibited the slowest decline rate of chlorophyll content, followed by Col-0, while WOE-3 showed the fastest decline rate (Fig. 2 ), indicating that WRKY75 can accelerate leaf senescence. Exogenous melatonin decelerated the decline rate of chlorophyll in Col-0, wrky75 , and WOE-3 , although it could not prevent senescence. 3.4 Exogenous melatonin increased the content of endogenous melatonin and promoted SNAT gene expression The endogenous melatonin content in the 3rd − 5th true leaves of in the Col-0 exhibited a significant decrease during the plant senescence process, while the changes in endogenous melatonin content of snat-2 were not substantial (Fig. 3 a). Furthermore, in the W3 phase, the SNAT gene expression level of snat-2 was found to be lower compared to Col-0. Following treatment with exogenous melatonin, the expression of SNAT in Col-0 was significantly upregulated, leading to a twofold increase in endogenous melatonin content. Even though SNAT expression was not detected in snat-2 , there was a significantly increase in the endogenous melatonin content. These findings suggested that exogenous melatonin is capable of elevating endogenous melatonin levels by augmenting the expression of SNAT , which in turn delays plant leaf senescence. Additionally, due to the lack of activated melatonin synthesase SNAT , snat-2 cannot continue to produce melatonin. As exogenous melatonin entered the plant was degraded, resulting in lower levels of melatonin detected. The endogenous melatonin content of wrky75 and WOE-3 was equivalent, and much lower than that of Col-0 (Fig. 3 a). Following the application of exogenous melatonin, both wrky75 and WOE-3 exhibited a increase in endogenous melatonin content, especially in wrky75 . Comparison with Col-0 revealed that the SNAT gene expression level of WOE-3 in the W3 phase was lower than wrky75 (Fig. 3 b). Exogenous melatonin led to a lower SNAT expression increase level in wrky75 compared to Col-0 and WOE-3. This demonstrated that exogenous melatonin can enhance the accumulation of endogenous melatonin by upregulating SNAT expression, although the regulatory amplitude is influenced by WRKY75 and the plant's senescence. 3.5 Melatonin alleviates oxidative damage in plant leaves In the process of senescence, there was an increase in the ROS staining of A. thaliana rosette leaves, as indicated by the deepening of tissue staining and the significant rise in H 2 O 2 and O 2 ·− content (Fig. 4 a, Fig. S4). Col-0 exhibits more pronounced staining compared to snat-2 , however, the application of exogenous melatonin resulted in lighter staining in Col-0. snat-2 has less accumulation of ROS due to growth and development delay. Exogenous melatonin could also reduce the ROS accumulation of snat-2 , especially in the W3 phase (Fig. 4 c, 4 d). In the W4 phase, the WOE-3 strain exhibited the darkest color, followed by Col-0, and wrky75 has the lightest color (Fig. 4 a, 4 b). The content of H 2 O 2 and O 2 ·− increased with senescence in plants, and WOE-3 increased the most, followed by Col-0 (Fig. 4 c, 4 d), indicating that WRKY75 positively regulates the accumulation of ROS during senescence. Exogenous melatonin slowed down the rate of increase in ROS levels, especially in WOE-3 . These results suggested that exogenous melatonin can delay senescence progresses by reducing H 2 O 2 and O 2 ·− , however, this effect was negatively regulated by WRKY75. Plants contain various antioxidant enzymes and CAT is mainly responsible for clearing H 2 O 2 (Guo et al. 2017 ). The CAT activity of Col-0 3rd -5th true leaves initially increased and then decreased, indicating that senescence at the early stage stimulates the increase of CAT activity (Fig. 5 ). However, as the plants enter the late stage of senescence, their metabolism slows down, resulting in a decrease in CAT activity. Conversely, the CAT activity in snat-2 consistently decreased during the senescence process. The CAT activity increased by exogenous melatonin, particularly in the W3 phase, indicating that melatonin could delay leaf senescence by increasing CAT activity to eliminate H 2 O 2 . Furthermore, the CAT activity of wrky75 was notably higher than that of Col-0, especially in the W3 phase. The overall trend of WOE-3 was slightly higher than that of Col-0, especially in the W1 phase. Following the application of exogenous melatonin, there was a slight increase in CAT activity in Col-0 and all mutants, albeit not significant. The absence of WRKY75 was found to enhance CAT activity, suggesting that WRKY75 has a suppressive effect on CAT activity. 3.6 Exogenous melatonin reduced SA accumulation by decreasing SID2 expression In the W3 phase, the SA content in Col-0 was higher than that in snat-2 (Fig. 6 a). Similar to the result, the expression level of SID2 in Col-0 was higher than that in snat-2 (Fig. 6 b), suggesting that the reason for this situation was the slow development of snat-2 . The SID2 expression and SA content were significantly reduced by exogenous melatonin. Compared with Col-0, wrky75 showed a decrease in SA content and SID2 expression, while WOE-3 showed a increase. The content of SA and the expression of SID2 in Col-0 and all mutants was significantly downregulated by exogenous melatonin, but wrky75 showed the smallest decrease. It indicated that WRKY75 has a promoting effect on the regulation of SID2 , and exogenous melatonin can negatively affect the regulatory function of WRKY75 on SID2 and SA. Exogenous melatonin can effectively alleviate the increase in SID2 expression caused by WRKY75, reduce SA content, and alleviate plant senescence. 3.7 Melatonin downregulates the expression of plant senescence-associated genes WRKY75 is a positive regulator of senescence, and the expression level of SAG13 increases during the senescence of the plant. It was observed that the expression levels of these genes were higher in Col-0 than in snat-2 (Fig. 7 ). Upon the application of exogenous melatonin, the expression levels of both genes decreased in Col-0. This trend was also evident in other mutants following melatonin application. Interestingly, WOE-3 expressed SAG13 to a greater extent than other plants, and the expression of both genes decreased substantially after exogenous melatonin application. Exogenous melatonin did not markedly down-regulate the expression of the SAG13 gene in wrky75 . The results of the expression analysis of these two genes support the conclusion that melatonin may mitigate plant senescence by down-regulating WRKY75 and SAG13 . 3.8 Melatonin reduces the degree of cell death The ultimate result of plant cell senescence is death. The trypan blue staining results of snat-2 in the W3 phase were lighter than those of Col-0 staining, possibly due to the lack of melatonin in regulating growth and development in snat-2 , resulting in delayed development and mild cell death (Fig. 8 ). After the application of exogenous melatonin, both snat-2 and Col-0 staining were slightly lighter, and cell death was alleviated. 4. Discussion The mechanism of melatonin alleviates the natural senescence of plant leaves might depend on its potent antioxidant properties. Currently, there is no clear signaling pathway to elucidate the anti-senescence effect of melatonin. The research of leaf senescence provides a new direction for further understanding the regulatory role of melatonin. 4.1 Melatonin can delay leaf senescence in A. thaliana Leaf senescence is characterized by the degradation of chlorophyll and the subsequent yellowing of plant leaves, a process in which melatonin plays a ragulatory role. Our study demonstrated a gradual decrease in chlorophyll content during senescence. The effect of melatonin application as evident from the observed decrease in the significant deceleration of leaf yellowing (Fig. 1 , S3) and the chlorophyll content downward trend (Fig. 2 ). This observation was consistent with previous studies, indicating that melatonin application can alleviate the decline in chlorophyll content induced by stress or senescence in plants, as demonstrated by the work of Yang et al. (Yang et al. 2022 ). Ye et al. found that melatonin-treated wheat leaves had higher chlorophyll content compared to the control group during the senescence process (Ye et al. 2020 ). As plants undergo senescence, a substantial amount of ROS is produced, which is effectively scavenged by the antioxidant system, preserving ROS balance and enhancing cell membrane stability. Our results indicated that ROS levels were gradually elevated CAT activity increased and then decreased with the senescence process (Fig. 4 , S4, 5 ). It was postulated that the modest fluctuation in CAT activity may be attributed to the stimulation of increased ROS in the early stages of senescence. Melatonin exhibited a modest upregulation of CAT activity and served as a potent antioxidant, markedly reducing ROS levels during leaf senescence (Fig. 4 c, 4 d), in line with previous research (Yang et al. 2022 ; Zhao et al. 2021 ). Notably, our results found that melatonin had varying degrees effects on increased CAT activity and decreased levels of ROS (Fig. 5 ). We deduce that the disparity in enzyme activity and ROS regulation instigated by melatonin can be attributed to its own profound ability to neutralize ROS. Endogenous melatonin levels vary across different plant species, organs, growth stages, and environmental conditions (Murch et al. 2009 ). Our findings revealed that the melatonin content in Col-0 was higher than in snat-2 , and exogenous melatonin supplementation can stimulate endogenous melatonin accumulation (Fig. 3 ). Moreover, exogenous melatonin inhibited the decline in accumulation of ROS (Fig. 4 ), upregulation of SA accumulation and SID2 expression(Fig, 6), the upregulation of WRKY75 and SAG13 expression (Fig. 7 ) and cell death (Fig. 8 ), consequently delaying the senescence of snat-2 leaves. Prior studies had identified snat-2 as a crucial enzyme in melatonin synthesis (Zhao et al. 2021 ). Our study corroborated this finding, as snat-2 mutants exhibited markedly lower melatonin content compared to Col-0 (Fig. S2) and failed to increase endogenous melatonin levels following exogenous melatonin application (Fig. 3 a). Moreover, the breakdown of exogenous melatonin in snat-2 by metabolic enzymes lead to a sustained decline in endogenous melatonin levels. The absence of endogenous melatonin in snat-2 mutants delayed their growth and development and impeded the onset of senescence-related factors, including inhibiting chlorophyll decomposition and reducing the expression of WRKY75 and SAG13 . 4.2 Melatonin can delay leaf senescence by reducing SA accumulation Several studies had demonstrated that SA plays a significant role in promoting natural leaf senescence(Li et al. 2016 ; Lim et al. 2007 ; Morris et al. 2000 ; Rivas-San Vicente and Plasencia 2011 ). SA and ROS are two clearly defined factors inducing leaf senescence (Guo et al. 2017 ). During the process of plant leaf senescence, the expression level of SID2 (a key gene for SA synthase) increases, leading to a gradual rise in the endogenous SA content, which in turn accelerates the senescence process of A. thaliana leaves (Lim et al. 2007 ; Morris et al. 2000 ). Our study showed that the deficiency of endogenous melatonin contributed to reduced SA accumulation, while the application of exogenous melatonin further downregulated SA accumulation by affecting the expression of SID2 gene (Fig. 6 ). This phenomenon had also been observed by Rafique et al. (Rafique et al. 2023 ). These findings lead us to speculate that the slow senescence phenotype and low SA content of snat-2 may be attributed to slower growth and development. 4.3 Melatonin delay leaf senescence by down-regulating the expression of the WRKY75 transcription factor WRKY75 plays a crucial role in various signaling pathways of plant hormones such as gibberellin, jasmonic acid, SA, and it has been implicated in the regulation of seed germination, abiotic stress tolerance, flowering, and senescence in plants (Chen et al. 2020 ; Zhang et al. 2021b ; Zhang et al. 2018 ). Previous studies had demonstrated that WRKY75 promotes leaf senescence (Buchanan-Wollaston et al. 2003 ; Guo et al. 2017 ). Additionally, WRKY75 was known to bind to the W-box in the promoters of target genes, thereby modulating their transcriptional activity, and this function in regulating senescence had been linked to its interaction with melatonin (Wei et al. 2018 ). Specifically, Weeda et al. observed that exogenous melatonin application would increase the expression of WRKY75 in A. thaliana (Weeda et al. 2014 ). Wei et al reported that overexpression of MeWRKY75 in cassava ( Manihot esculenta ) leads to an increase in the accumulation of melatonin in leaves (Wei et al. 2018 ). This intricated interaction between WRKY75 and melatonin warranted further investigation and formed the basis of this study. Our experimental findings supported the crucial role of WRKY75 in plant senescence. The overexpression of WRKY75 accelerated plant senescence, as evidenced by reduced chlorophyll content (Fig. 2 ), increased ROS and SA accumulation (Fig. 4 , 6 ), and up-regulated the expression of the senescence-related gene SAG13 (Fig. 7 ). Remarkably, the detrimental effects of WRKY75 overexpression on senescence were mitigated by exogenous melatonin. Moreover, our observations indicated a partial dependence of exogenous melatonin action on WRKY75, as evidenced by a larger degree of senescence in the WOE-3 mutant and a smaller effect on wrky75 compared to Col-0 after treatment with exogenous melatonin. Melatonin counteract the senescence-promoting effect of WRKY75. Furthermore, our investigation into the endogenous melatonin content and the SNAT expression levels of wrky75 and WOE-3 revealed interesting insights. Notably, the expression of SNAT in the W3 phase showed a higher level in the wrky75 , while its expression in WOE-3 was notably lower (Fig. 2 a). Conversely, the endogenous melatonin content of wrky75 and WOE-3 mutants was found to be similar, but substantially lower than that of Col-0 (Fig. 2 b). The melatonin content of Col-0 increased significantly after the application of exogenous melatonin, while wrky75 and WOE-3 showed only slight increases. These results suggested that exogenous melatonin can up-regulate SNAT expression and enhance the endogenous melatonin content, with the up-regulation influenced by WRKY75. Notably, the regulation of melatonin on the down-regulation of WRKY75 expression was dependent on the content of endogenous SA (Fig. 6 a). Weeda et al. 's genome analysis results indicated that exogenous melatonin increases the expression of WRKY75 in A. thaliana (Weeda et al. 2014 ). Conversely, Guo et al. found that SA and ROS promote WRKY75 expression (Guo et al. 2017 ). Consequently, it could be hypothesized that while melatonin can promote WRKY75 expression, it may also reduce WRKY75 expression by down-regulating SA and ROS accumulation. These two pathways seem to compete and antagonize each other, leading to the ultimate reduction in WRKY75 expression by melatonin (Fig. 9 ). 5. Conclusion Exogenous melatonin can delay leaf senescence, dependent on the salicylic acid pathway and the transcription factor WRKY75. Melatonin can downregulate the accumulation of ROS and SA, which subsequently reduces the expression of WRKY75 . It was observed that melatonin counteracts the effects of a network structure comprising SA, ROS, and WRKY75 on senescence, thereby modulating plant senescence-related processes and delaying leaf senescence in A. thaliana . Declarations All authors declare no conflict of interest. Acknowledgments This work was funded by the State Key Laboratory Foundation of Crop Gene Exploration and Utilization in Southwest China (SKL-KF202318), and the Science and Technology Planning Project in 2022 of Dazhu County (2022CGG008). Author Contributions Statement M.Y. proposed and drafted the research project. KY.W., JJ.M. and WH.C. planned and designed the experiments. KY.W. and JJ.M. conducted the experiments necessary for the research. KY.W., JJ.M. analyzed the statistical data for the experimental results. WH.C. prepared all the figures. 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Plant Physiol 173:2294–2307 Zhao Y, Zhang Z, Chen Y, Ding C, Yuan S, Reiter RJ, Yuan M (2021) Melatonin: A potential agent in delaying leaf senescence. Crit Rev Plant Sci 40:1–22 Supplementary Files Supplementalmaterial.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5326045","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":371076915,"identity":"4217ef82-4bb6-4098-8555-efb4d234529e","order_by":0,"name":"Ke-yu Wang","email":"","orcid":"","institution":"Sichuan Agricultural University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Ke-yu","middleName":"","lastName":"Wang","suffix":""},{"id":371076916,"identity":"c288de71-f1d5-4bcd-95d7-34cd10ef1957","order_by":1,"name":"Jing-jing Mao","email":"","orcid":"","institution":"Sichuan Agricultural University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Jing-jing","middleName":"","lastName":"Mao","suffix":""},{"id":371076917,"identity":"67e8dba0-73bd-4111-b0a8-6bb388d573b7","order_by":2,"name":"Wen-hui Chen","email":"","orcid":"","institution":"Sichuan Agricultural University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Wen-hui","middleName":"","lastName":"Chen","suffix":""},{"id":371076918,"identity":"264dfad2-840c-45d9-aed7-439062b0d18d","order_by":3,"name":"De-qiang Li","email":"","orcid":"","institution":"State Key Laboratory of Crop Gene Exploration and Utilization in Southwest China","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"De-qiang","middleName":"","lastName":"Li","suffix":""},{"id":371076919,"identity":"7416d065-478a-4661-bad2-7fd9fc5dcf98","order_by":4,"name":"Zi-zhong Tang","email":"","orcid":"","institution":"Sichuan Agricultural University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Zi-zhong","middleName":"","lastName":"Tang","suffix":""},{"id":371076920,"identity":"64d3c57f-874e-4fa4-8aa8-f8fd7d6ec3ca","order_by":5,"name":"Yang-er Chen","email":"","orcid":"","institution":"Sichuan Agricultural University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Yang-er","middleName":"","lastName":"Chen","suffix":""},{"id":371076921,"identity":"c91abc07-1d58-4444-a366-009699f90e3b","order_by":6,"name":"Shu Yuan","email":"","orcid":"","institution":"Sichuan Agricultural University - 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Each value represents the mean ± SD. Diferent letters denote signifcant differences (student’s \u003cem\u003et\u003c/em\u003e-test. “ns”, \u003cem\u003ep\u003c/em\u003e \u0026gt; 0.05; *, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05; **, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.005; ***, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.0005).\u003c/p\u003e","description":"","filename":"OnlineFigure2.png","url":"https://assets-eu.researchsquare.com/files/rs-5326045/v1/f8ac0d0ba26ecebc52a6f7fa.png"},{"id":68481603,"identity":"c5709ace-656a-494a-bbf2-8efaa37963a9","added_by":"auto","created_at":"2024-11-07 17:28:58","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":95444,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of exogenous melatonin on endogenous melatonin content and \u003cem\u003eSNAT\u003c/em\u003eexpression. (a) The endogenous melatonin content in W3 and W5 phase. 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(a) Detection of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e by DAB staining in W2 and W4 phase. (b) Detection of O\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e·-\u003c/sup\u003e by NBT staining in W3 phase (c) Quantitative analysis of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e (d) Quantitative analysis of O\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e·-\u003c/sup\u003e. “+MT” indicates exogenous melatonin application, “-MT” as the control. Each value represents the mean ± SD. Diferent letters denote signifcant differences (student’s \u003cem\u003et\u003c/em\u003e-test. “ns”, \u003cem\u003ep\u003c/em\u003e \u0026gt; 0.05; *, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05; **, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.005; ***, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.0005).\u003c/p\u003e","description":"","filename":"OnlineFigure4.png","url":"https://assets-eu.researchsquare.com/files/rs-5326045/v1/d38857e4efaa9d8bee4f8309.png"},{"id":68481600,"identity":"1ed18457-0d72-4830-8625-c6ab3f9457ec","added_by":"auto","created_at":"2024-11-07 17:28:58","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":49183,"visible":true,"origin":"","legend":"\u003cp\u003eDetermination of catalase activity. “+” indicates the application of exogenous melatonin, “-” as the control. Each value represents the mean ± SD.\u003c/p\u003e","description":"","filename":"OnlineFigure5.png","url":"https://assets-eu.researchsquare.com/files/rs-5326045/v1/63d55865f8538ac619e532ae.png"},{"id":68481609,"identity":"194a0d23-e525-4250-b5ff-f793cc5caf6a","added_by":"auto","created_at":"2024-11-07 17:28:58","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":83720,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of exogenous melatonin on SA content and \u003cem\u003eSID2\u003c/em\u003e expression. (a) The content of endogenous SA (b). The \u003cem\u003eSID2\u003c/em\u003e relative expression. “+” indicates the application of exogenous melatonin, “-” as the control. Each value represents the mean ± SD. Diferent letters denote signifcant differences (student’s \u003cem\u003et\u003c/em\u003e-test. “ns”, \u003cem\u003ep\u003c/em\u003e \u0026gt; 0.05; *, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05; **, \u003cem\u003ep\u003c/em\u003e\u0026lt; 0.005; ***, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.0005).\u003c/p\u003e","description":"","filename":"OnlineFigure6.png","url":"https://assets-eu.researchsquare.com/files/rs-5326045/v1/638ca49cf5dca816e7b90caa.png"},{"id":68481806,"identity":"8868ee15-a5cf-465b-9b8f-c1442d56f4f1","added_by":"auto","created_at":"2024-11-07 17:36:58","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":83224,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of exogenous melatonin on \u003cem\u003eWRKY75\u003c/em\u003eand \u003cem\u003eSAG13 \u003c/em\u003egene expression in W3 phase. (a) The \u003cem\u003eWRKY75\u003c/em\u003e relative expression. (b) The \u003cem\u003eSAG13\u003c/em\u003e relative expression. “+” indicates the application of exogenous melatonin, “-” as the control. Each value represents the mean ± SD. Diferent letters denote signifcant differences (student’s \u003cem\u003et\u003c/em\u003e-test. “ns”, \u003cem\u003ep\u003c/em\u003e \u0026gt; 0.05; *, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05; **, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.005; ***, \u003cem\u003ep\u003c/em\u003e\u0026lt; 0.0005).\u003c/p\u003e","description":"","filename":"OnlineFigure7.png","url":"https://assets-eu.researchsquare.com/files/rs-5326045/v1/19002003f2399a7aaffe65c3.png"},{"id":68481605,"identity":"57a0fa0e-e481-457a-a4d0-c743e51db45b","added_by":"auto","created_at":"2024-11-07 17:28:58","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":171618,"visible":true,"origin":"","legend":"\u003cp\u003eTrypan blue staining for Col-0 and \u003cem\u003esnat-2\u003c/em\u003ein W4 phase. “+MT” indicates the application of exogenous melatonin, “-MT” as the control.\u003c/p\u003e","description":"","filename":"OnlineFigure8.png","url":"https://assets-eu.researchsquare.com/files/rs-5326045/v1/11e11497ada15159c3132d8c.png"},{"id":68482405,"identity":"4f4b931d-4e4e-49c1-b47e-832dbc18d012","added_by":"auto","created_at":"2024-11-07 17:44:58","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":72604,"visible":true,"origin":"","legend":"\u003cp\u003eSchematic model of melatonin delaying leaf senescence induced by WRKY75 in \u003cem\u003eArabidopsis thaliana\u003c/em\u003e.\u003c/p\u003e","description":"","filename":"OnlineFigure9.png","url":"https://assets-eu.researchsquare.com/files/rs-5326045/v1/1e1bd88434ccf089bee26f34.png"},{"id":68890621,"identity":"8d30e376-040d-47cc-af71-fe0a820e6bcc","added_by":"auto","created_at":"2024-11-13 07:38:32","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4014928,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5326045/v1/57b4d076-6041-4caf-a8d7-cd9d84b5854c.pdf"},{"id":68481807,"identity":"e7ef798f-155e-4ada-9398-0fc084796bf6","added_by":"auto","created_at":"2024-11-07 17:36:58","extension":"docx","order_by":13,"title":"","display":"","copyAsset":false,"role":"supplement","size":8449119,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementalmaterial.docx","url":"https://assets-eu.researchsquare.com/files/rs-5326045/v1/866431de5c2367e10dbe94f4.docx"}],"financialInterests":"","formattedTitle":"Melatonin delayed leaf senescence induced by WRKY75 in Arabidopsis thaliana","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eLeaf senescence has always been a prominent topic in the field of plant physiology. It is a complex and highly coordinated process, controlled by both external and internal factors(Gan and Amasino \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e1997\u003c/span\u003e; Guo et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Lim et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). The external environmental factors include abiotic stress and biological stress, internal factors include plant age, plant hormones levels and ROS (Beers and McDowell \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2001\u003c/span\u003e; Sun et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Leaf senescence is associated with the transfer of nutrients from leaves to seeds. This underlined the significance of leaf senescence as the final stage of plant senescence(Woo et al. \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Effectively controlling leaf senescence could regulate the degradation process of lipids, proteins, chlorophyll, and other substances in plants, and greatly enhance crop yield and (Buchanan-Wollaston et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2003\u003c/span\u003e). Therefore, understanding and managing leaf senescence is vital for improving agricultural productivity.\u003c/p\u003e \u003cp\u003eNumerous studies had shown that salicylic acid (SA) promoted the natural senescence of leaves (He et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Lim et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Rivas-San Vicente and Plasencia \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). Morris \u003cem\u003eet al.\u003c/em\u003e found that SA induced senescence by enhancing leaf senescence-related gene expression (Morris et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2000\u003c/span\u003e). Mutants with defects in SA biosynthesis or SA signal, such as \u003cem\u003enpr1\u003c/em\u003e (\u003cem\u003enonexpresser of pathogenes related genes 1\u003c/em\u003e), \u003cem\u003epad4\u003c/em\u003e (\u003cem\u003ephytoalexin deficiency 4\u003c/em\u003e), and \u003cem\u003eNahG\u003c/em\u003e (\u003cem\u003eNaphthalene hydroxylase G\u003c/em\u003e), exhibited a senescence-delayed phenotype, accompanied by a significant reduction in the expression of senescence-associated genes (SAGs) (Lim et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Morris et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2000\u003c/span\u003e). SAGs played crucial roles in the regulation of plant senescence. Among them, \u003cem\u003eSAG13\u003c/em\u003e was considered a marker of early developmental senescence and programmed cell death, and was also involved in hypersensitivity responses following pathogen infection and plant cell death (Dhar et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eDuring leaf senescence, SA induced the accumulation of hydrogen peroxide (H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e) by reducing the activity of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e metabolism enzymes, consequently lead to oxidative damage (Durner and Klessig \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e1995\u003c/span\u003e; Durner and Klessig \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e1996\u003c/span\u003e; Ruiz-S\u0026aacute;enz et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Because the levels of SA and H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e gradually increased as the leaf senescence process proceeds, the leaves' capacity to scavenge ROS was diminished. Disruption of ROS balance ultimately leads to the increase of the protein and lipid peroxidation (Guo et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Khanna-Chopra \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). Moreover, Leon \u003cem\u003eet al.\u003c/em\u003e identified that H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e could stimulate the biosynthesis of SA in tobacco, further highlighting the interconnectedness of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e and SA signaling pathways (Leon et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e1995\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eWRKY transcription factors (WRKY-TFs) are a prominent family of transcription factors implicating in leaf senescence(Guo et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2004\u003c/span\u003e). In \u003cem\u003eArabidopsis thaliana\u003c/em\u003e, several WRKY-TFs, including WRKY6, WRKY42, WRKY46, WRKY55, and WRKY75 were involved in the leaf senescence through SA signaling pathways (Niu et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Wang et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Zhang et al. \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2021a\u003c/span\u003e). Notably, the \u003cem\u003eWRKY75\u003c/em\u003e silent mutant has showed to delay age-dependent leaf senescence, whereas the overexpression mutant accelerated this process (Lim et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). The interaction of WRKY75 with the \u003cem\u003eSID2\u003c/em\u003e promoter enhanced SA biosynthesis, ultimately triggering the activation of the SA signaling pathway and consequent promoting plant cell death, autophagy, and leaf senescence (Zhang et al. \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Guo \u003cem\u003eet al.\u003c/em\u003e unveiled that WRKY75 was induced by SA and ROS (Guo et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Furthermore, WRKY75 promoted SA biosynthesis by activating \u003cem\u003eSID2\u003c/em\u003e transcription and promoted H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e accumulation by inhibiting \u003cem\u003eCATALASE2\u003c/em\u003e (\u003cem\u003eCAT2\u003c/em\u003e) transcription (Guo et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Consequently, during leaf senescence, WRKY75, SA, and ROS underwent a gradual yet irreversible rise, and propelled each other by a positive feedback loop mechanism (Guo et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2017\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eMelatonin (MT), chemical name is \u003cem\u003eN\u003c/em\u003e-acetyl-5-methoxytryptamine, was first identified and named in 1958 by Lerner \u003cem\u003eet al.\u003c/em\u003e(Lerner et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e1958\u003c/span\u003e). Melatonin played various regulatory functions in plant growth and development, with significant crosstalk with other plant hormones such as SA (Yu et al. \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Zhang et al. \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Zhao et al. \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Moreover, interactions between melatonin and WRKY-TFs had been identified. For instance, it has been reported that WRKY17 activated the transcription of \u003cem\u003eASMT7\u003c/em\u003e, which was a key gene of melatonin biosynthesis in apples (Song et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Weeda \u003cem\u003eet al.\u003c/em\u003e showed that exogenous melatonin could increase the expression of \u003cem\u003eWRKY75\u003c/em\u003e in \u003cem\u003eA. thaliana\u003c/em\u003e(Weeda et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Upon treating \u003cem\u003eA. thaliana\u003c/em\u003e with melatonin for 16 hours, Gao \u003cem\u003eet al.\u003c/em\u003e observed the gene expression of \u003cem\u003eWRKY6\u003c/em\u003e, \u003cem\u003eWRKY22\u003c/em\u003e, \u003cem\u003eWRKY31\u003c/em\u003e, \u003cem\u003eWRKY50\u003c/em\u003e, \u003cem\u003eWRKY51\u003c/em\u003e, \u003cem\u003eWRKY54\u003c/em\u003e, \u003cem\u003eWRKY70\u003c/em\u003e, and \u003cem\u003eWRKY75\u003c/em\u003e were upgregulated more than twice, and the expression of many genes related to SA and other hormones in response to biotic and abiotic stresses also changed (Gao et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2018\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn this study, we investigated whether melatonin was involved in leaf senescence through a cyclic model composed of WRKY75, SA, and ROS. The experimental results demonstrated that melatonin and SA, ROS, and WRKY75 antagonize each other on senescence \u0026mdash; melatonin downregulates the accumulation of SA and ROS, leading to a decrease in the expression of \u003cem\u003eWRKY75\u003c/em\u003e, thereby regulating plant senescence related processes and delaying leaf senescence in \u003cem\u003eA. thaliana\u003c/em\u003e. This study may provide ideas for applying melatonin to regulate crop leaf senescence and improve photosynthesis for yield and quality improvement.\u003c/p\u003e"},{"header":"2 Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Plant materials and growth conditions\u003c/h2\u003e \u003cp\u003e \u003cem\u003eArabidopsis thaliana\u003c/em\u003e Col-0 and T-DNA inserted mutants (\u003cem\u003esnat-2\u003c/em\u003e, \u003cem\u003esid2\u003c/em\u003e, and \u003cem\u003ewrky75\u003c/em\u003e) were obtained from ABRC, and \u003cem\u003eWRKY75\u003c/em\u003e overexpression mutants (\u003cem\u003eWOE-3\u003c/em\u003e and \u003cem\u003eWOE-6\u003c/em\u003e) were presented by Diqiu Yu and Ligang Chen, Xishuangbanna Tropical Botanical Garden, Chinese Academy of Sciences.\u003c/p\u003e \u003cp\u003eAll the seeds were soaked in distilled water at 4\u0026deg;C for 2 d and then sown in sterilized mixed nutrient soil (soil: perlite: vermiculite\u0026thinsp;=\u0026thinsp;4: 1: 1). The plants were subsequently grown under controlled conditions at 120 \u0026micro;M photon\u0026middot;m\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e\u0026middot;s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, 22\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;C, 60% humidity, 16h light /8h dark cycle. Upon reaching 20 days of growth, plants in the same growth state were irrigated with 100 \u0026micro;M melatonin or distilled water (as control) once every three days. Leaf samples from the 3rd-5th true leaves were collected at 20 d, 27 d, 34 d, 41 d, and 48 d (W1-W5) for subsequent experiments. Except for the first collection time (W1) before treatment, each subsequent collection time (W2-W5) corresponded to the second day after melatonin treatment. After collection, the leaves were promptly treated with liquid nitrogen and then preserved in a -80\u0026deg;C refrigerator.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Mutant identification and screening\u003c/h2\u003e \u003cp\u003eThe 36 d leaves of \u003cem\u003eA. thaliana\u003c/em\u003e were selected to identify and screen the mutants by the three-primers method. Plant Genomic DNA Kit (TIANGEN Biotech, China) was used to extract the DNA template, and TIANGEN masterMix II (TIANGEN Biotech, China) was used in PCR. Detailed primer sequences can be found in Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e, and the PCR amplification program is outlined in Table S2.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Determination of chlorophyll content\u003c/h2\u003e \u003cp\u003eAcetone (80%, v/v) was used to extract chlorophyll from 0.1 g of fresh leaves. The chlorophyll concentration was determined by a UV spectrophotometer (Shimadzu, Japan, UV-1750) as outlined by Porra \u003cem\u003eet al.\u003c/em\u003e (Porra et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e1989\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Quantification of melatonin\u003c/h2\u003e \u003cp\u003eMelatonin was extracted according to Sturtz \u003cem\u003eet al.\u003c/em\u003e (St\u0026uuml;rtz et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). 1.0 g leaves were collected and ground with liquid nitrogen. Subsequently, 2 mL of acetone was used for ultrasonic extraction for 30 min at 25\u0026deg;C, centrifugating at 4,000 \u0026times;\u003cem\u003eg\u003c/em\u003e for 10 min. The supernatant was then freeze-dried and then redissolved in 2 mL of distilled water for collection by using the C18 separation column (Agela Technologies, China). The separation column was washed with 2 mL of 10% methanol, then eluted with 1 mL of methanol, and determined the melatonin content by HPLC-MS/MS (Agilent Technologies, USA, HPLC-1290 Series and 6470 Triple Quadrupole MS/MS). HPLC-MS/MS parameters were set as Han \u003cem\u003eet al\u003c/em\u003e., detect ion m/z 159.0 (Han et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2017\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5 Quantification of SA\u003c/h2\u003e \u003cp\u003e0.5 g leaves were ground with liquid nitrogen, then 3 mL of 90% methanol was added. This mixture underwent ultrasonic extraction for 20 min, followed by centrifugation at 12,000 \u0026times;\u003cem\u003eg\u003c/em\u003e for 15 min. The pellet was subsequently treated with 2 mL of methanol, centrifuged at 12,000 \u0026times;\u003cem\u003eg\u003c/em\u003e for 15 min. The two supernatants were combined, and dried in vacuum. It was redissolved with 5 mL of 5 mM sodium acetate buffer, and followed by shock extraction with equal volumes extracting solution (ethyl acetate, cyclopentane, and isopropanol 100: 99:1, v/v) for 10 min. The aqueous phase was collected, dried in vacuum, and dissolved with 3 mL of methanol. SA content determined by HPLC (Agilent Technologies, USA, HPLC-1260 Series),as described by He \u003cem\u003eet al.\u003c/em\u003e, UV detection wavelength was 306 nm (He et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2007\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6 Evaluation of the redox equilibrium\u003c/h2\u003e \u003cp\u003e \u003cem\u003eIn situ\u003c/em\u003e staining of ROS was performed with reference to Yang \u003cem\u003eet al.\u003c/em\u003e (Yang et al. \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2004\u003c/span\u003e). For H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e staining, rosette leaves were immersed in 2 mg\u0026middot;mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e DAB, with vacuum infiltration at room temperature for 12 h. For O\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026middot;\u0026minus;\u003c/sup\u003e staining, the leaves were washed with decolorization solution (80% ethanol) and immersed in 0.5 mg\u0026middot;mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e NBT solution, and decolorized after vacuum infiltration for 30 min. After leaves was decolorized, then saved images using a scanner. Detection H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e content was referred to Velikova \u003cem\u003eet al.\u003c/em\u003e (Velikova et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2000\u003c/span\u003e). 0.1 g fresh leaves ground with liquid nitrogen and extracted with 1 mL 10% trichloroacetic acid (TCA) (w/v), centrifuged at 13,800 \u0026times;\u003cem\u003eg\u003c/em\u003e at 4\u0026deg;C for 10 min, then the supernatant was added with 10 mM potassium phosphate buffer (pH\u0026thinsp;=\u0026thinsp;7.0) and 1M KI (1: 1: 2, v/v/v), and incubated for 20 min in dark. The absorbance at 390 nm was recorded. Detection O\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026middot;\u0026minus;\u003c/sup\u003e content was referred to Nahar's method (Nahar et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2015\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.7 Determination of CAT activity\u003c/h2\u003e \u003cp\u003e0.2 g fresh leaves were ground in 4 mL of ice-cold buffer (150 mM sodium phosphate, pH 7.8), and then centrifuged at 12,000 \u0026times;\u003cem\u003eg\u003c/em\u003e at 4\u0026deg;C for 20 min. CAT activity was determined using Wang\u0026rsquo;s method (Wang \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e1995\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.8 Detection of cell death\u003c/h2\u003e \u003cp\u003eTo characterize the degree of cell death, trypan blue staining was employed as described by Huang \u003cem\u003eet al.\u003c/em\u003e (Huang et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). The leaves were soaked in a trypan blue staining solution (10 mL of lactic acid, 10 mL of glycerol, 10 g of phenol, 20 mg of trypan blue, and 10 mL of distilled water). The leaves were incubated at 65\u0026deg;C for 1 min. And then, the leaves were washed with distilled water and decolorized with 80% ethanol for 1 h. Finally, the results imaged with a scanner.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e2.9 Gene expression analysis\u003c/h2\u003e \u003cp\u003eThe total RNA was extracted using the CTAB method, and reverse transcription was performed using the PrimeScript\u0026trade; RT reagent Kit with gDNA Eraser (Takara Bio, Japan). Gene expression was detected on CFX96 Touch\u0026trade; Real-Time PCR Detection Systems (Bio-Rad, USA) using SYBR Premium Ex Taq\u0026trade; II dye (Takara Bio, Japan). The qPCR procedure was shown in Table S3, \u003cem\u003eACT2\u003c/em\u003e as a reference. The primer sequences were in Table S4. Adopt 2\u003csup\u003e\u0026minus;ΔΔCT\u003c/sup\u003e method was used to analyze the relative expression amount (Livak and Schmittgen \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2001\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e2.10 Data statistics and analysis\u003c/h2\u003e \u003cp\u003eAll data from the above experiments were shown as the mean with SD. Each experiment was repeated at least three independent times. SPSS Statistics 26.0 software (IBM Inc., USA) was used for the analysis of variance (student\u0026rsquo;s \u003cem\u003et\u003c/em\u003e-test), GraphPad Prism 9.0.0 software (GraphPad Software, USA) was used to draw histograms, and Adobe Photoshop 2022 software (Adobe, USA) was used to create plates.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e3.1 Identification of mutants\u003c/h2\u003e \u003cp\u003eThe mutant was identified using a three-primer method (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). Plant 1 in the figure is a heterozygote, while plant 2 is a homozygous deletion mutant, and plants 3 and 4 are of the wild type. The seeds of the homozygous deletion \u003cem\u003ewrky75\u003c/em\u003e mutant were subsequently collected and propagated for experimental purposes. qPCR was used to detect the gene expression levels of each mutant (Fig. S2). The results indicated a significant decrease in the corresponding gene expression levels of the T-DNA insertion mutants when compared to Col-0. Conversely, the \u003cem\u003eWRKY75\u003c/em\u003e gene expression level in \u003cem\u003eWOE-3\u003c/em\u003e was observed to be 14.5 times higher than that in Col-0 (Fig. S2b).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e3.2 Melatonin delayed the senescence process of plant leaves\u003c/h2\u003e \u003cp\u003eIn the W1 phase, \u003cem\u003esnat-2\u003c/em\u003e exhibited a significantly smaller size than Col-0 (Fig. S3). Moreover, in the W3 phase, it was observed that the color of \u003cem\u003esnat-2\u003c/em\u003e treated with melatonin leaves was comparable to that of Col-0 (Fig. S3). The application of melatonin resulted in greater growth of \u003cem\u003esnat-2\u003c/em\u003e. During the W5 phase, the senescence process of \u003cem\u003esnat-2\u003c/em\u003e was slower than that of Col-0. It was established that the exogenous melatonin significantly mitigated leaf senescence in both Col-0 and \u003cem\u003esnat-2\u003c/em\u003e. Exogendous melatonin delayed the senescence phenotype caused by endogenous melatonin deficiency.\u003c/p\u003e \u003cp\u003eAdditionally, in the W4 phase, it was evident that \u003cem\u003ewrky75\u003c/em\u003e had a lower degree of yellowing, while the leaves of WRKY75 overexpression mutants showed a more degree of yellowing compared to Col-0 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). This observation indicates that the WRKY75 has a positive regulatory effect on plant senescence. The application of exogenous melatonin alleviated leaf yellowing caused by senescence, especially in \u003cem\u003eWRKY75\u003c/em\u003e overexpressed mutants, indicating its potential to mitigate senescence induced by \u003cem\u003eWRKY75\u003c/em\u003e overexpression. The alleviation of senescence due to melatonin might be dependent on the presence of \u003cem\u003eWRKY75\u003c/em\u003e. It was also noted that melatonin had a more pronounced effect on \u003cem\u003eWOE-3\u003c/em\u003e compared to \u003cem\u003eWOE-6\u003c/em\u003e, leading to the selection of \u003cem\u003eWOE-3\u003c/em\u003e as a representative WRKY75 overexpression strain for subsequent experiments.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e3.3 Melatonin increased chlorophyll content in leaves\u003c/h2\u003e \u003cp\u003eIn the W1, W3, and W5 phases, the 3\u003csup\u003erd\u003c/sup\u003e-5th true leaves were collected to assess the impact of exogenous melatonin on chlorophyll (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). The total chlorophyll content continuously decreased in the senescence process. Notably, in the W3 phase, the exogenous melatonin mitigated the reduction of chlorophyll. However, in the W5 phase, melatonin had less effect on chlorophyll content. Comparative analysis demonstrated that the chlorophyll content of \u003cem\u003esnat-2\u003c/em\u003e was significantly higher than that of Col-0, especially in the W5 phase, where \u003cem\u003esnat-2\u003c/em\u003e exhibited approximately 7 times higher chlorophyll content, possibly attributed to the slower development of \u003cem\u003esnat-2\u003c/em\u003e. Exogenous melatonin increased chlorophyll content of \u003cem\u003esnat-2\u003c/em\u003e, especially in the W3 phase. This indicates the mitigating effect of melatonin on \u003cem\u003esnat-2\u003c/em\u003e senescence.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn correlation with the phenotypic results, \u003cem\u003ewrky75\u003c/em\u003e exhibited the slowest decline rate of chlorophyll content, followed by Col-0, while \u003cem\u003eWOE-3\u003c/em\u003e showed the fastest decline rate (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e), indicating that WRKY75 can accelerate leaf senescence. Exogenous melatonin decelerated the decline rate of chlorophyll in Col-0, \u003cem\u003ewrky75\u003c/em\u003e, and \u003cem\u003eWOE-3\u003c/em\u003e, although it could not prevent senescence.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e3.4 Exogenous melatonin increased the content of endogenous melatonin and promoted \u003cem\u003eSNAT\u003c/em\u003e gene expression\u003c/h2\u003e \u003cp\u003eThe endogenous melatonin content in the 3rd \u0026minus;\u0026thinsp;5th true leaves of in the Col-0 exhibited a significant decrease during the plant senescence process, while the changes in endogenous melatonin content of \u003cem\u003esnat-2\u003c/em\u003e were not substantial (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea). Furthermore, in the W3 phase, the \u003cem\u003eSNAT\u003c/em\u003e gene expression level of \u003cem\u003esnat-2\u003c/em\u003e was found to be lower compared to Col-0. Following treatment with exogenous melatonin, the expression of \u003cem\u003eSNAT\u003c/em\u003e in Col-0 was significantly upregulated, leading to a twofold increase in endogenous melatonin content. Even though \u003cem\u003eSNAT\u003c/em\u003e expression was not detected in \u003cem\u003esnat-2\u003c/em\u003e, there was a significantly increase in the endogenous melatonin content. These findings suggested that exogenous melatonin is capable of elevating endogenous melatonin levels by augmenting the expression of \u003cem\u003eSNAT\u003c/em\u003e, which in turn delays plant leaf senescence. Additionally, due to the lack of activated melatonin synthesase \u003cem\u003eSNAT\u003c/em\u003e, \u003cem\u003esnat-2\u003c/em\u003e cannot continue to produce melatonin. As exogenous melatonin entered the plant was degraded, resulting in lower levels of melatonin detected.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe endogenous melatonin content of \u003cem\u003ewrky75\u003c/em\u003e and \u003cem\u003eWOE-3\u003c/em\u003e was equivalent, and much lower than that of Col-0 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea). Following the application of exogenous melatonin, both \u003cem\u003ewrky75\u003c/em\u003e and \u003cem\u003eWOE-3\u003c/em\u003e exhibited a increase in endogenous melatonin content, especially in \u003cem\u003ewrky75\u003c/em\u003e. Comparison with Col-0 revealed that the \u003cem\u003eSNAT\u003c/em\u003e gene expression level of \u003cem\u003eWOE-3\u003c/em\u003e in the W3 phase was lower than \u003cem\u003ewrky75\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb). Exogenous melatonin led to a lower \u003cem\u003eSNAT\u003c/em\u003e expression increase level in \u003cem\u003ewrky75\u003c/em\u003e compared to Col-0 and \u003cem\u003eWOE-3.\u003c/em\u003e This demonstrated that exogenous melatonin can enhance the accumulation of endogenous melatonin by upregulating \u003cem\u003eSNAT\u003c/em\u003e expression, although the regulatory amplitude is influenced by WRKY75 and the plant's senescence.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003e3.5 Melatonin alleviates oxidative damage in plant leaves\u003c/h2\u003e \u003cp\u003eIn the process of senescence, there was an increase in the ROS staining of \u003cem\u003eA. thaliana\u003c/em\u003e rosette leaves, as indicated by the deepening of tissue staining and the significant rise in H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e and O\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026middot;\u0026minus;\u003c/sup\u003e content (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea, Fig. S4). Col-0 exhibits more pronounced staining compared to \u003cem\u003esnat-2\u003c/em\u003e, however, the application of exogenous melatonin resulted in lighter staining in Col-0. \u003cem\u003esnat-2\u003c/em\u003e has less accumulation of ROS due to growth and development delay. Exogenous melatonin could also reduce the ROS accumulation of \u003cem\u003esnat-2\u003c/em\u003e, especially in the W3 phase (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec, \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ed). In the W4 phase, the \u003cem\u003eWOE-3\u003c/em\u003e strain exhibited the darkest color, followed by Col-0, and \u003cem\u003ewrky75\u003c/em\u003e has the lightest color (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea, \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb). The content of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e and O\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026middot;\u0026minus;\u003c/sup\u003e increased with senescence in plants, and \u003cem\u003eWOE-3\u003c/em\u003e increased the most, followed by Col-0 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec, \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ed), indicating that WRKY75 positively regulates the accumulation of ROS during senescence. Exogenous melatonin slowed down the rate of increase in ROS levels, especially in \u003cem\u003eWOE-3\u003c/em\u003e. These results suggested that exogenous melatonin can delay senescence progresses by reducing H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e and O\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026middot;\u0026minus;\u003c/sup\u003e, however, this effect was negatively regulated by WRKY75.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003ePlants contain various antioxidant enzymes and CAT is mainly responsible for clearing H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e (Guo et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). The CAT activity of Col-0 3rd -5th true leaves initially increased and then decreased, indicating that senescence at the early stage stimulates the increase of CAT activity (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). However, as the plants enter the late stage of senescence, their metabolism slows down, resulting in a decrease in CAT activity. Conversely, the CAT activity in \u003cem\u003esnat-2\u003c/em\u003e consistently decreased during the senescence process. The CAT activity increased by exogenous melatonin, particularly in the W3 phase, indicating that melatonin could delay leaf senescence by increasing CAT activity to eliminate H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e. Furthermore, the CAT activity of \u003cem\u003ewrky75\u003c/em\u003e was notably higher than that of Col-0, especially in the W3 phase. The overall trend of \u003cem\u003eWOE-3\u003c/em\u003e was slightly higher than that of Col-0, especially in the W1 phase. Following the application of exogenous melatonin, there was a slight increase in CAT activity in Col-0 and all mutants, albeit not significant. The absence of \u003cem\u003eWRKY75\u003c/em\u003e was found to enhance CAT activity, suggesting that WRKY75 has a suppressive effect on CAT activity.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003e3.6 Exogenous melatonin reduced SA accumulation by decreasing \u003cem\u003eSID2\u003c/em\u003e expression\u003c/h2\u003e \u003cp\u003eIn the W3 phase, the SA content in Col-0 was higher than that in \u003cem\u003esnat-2\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea). Similar to the result, the expression level of \u003cem\u003eSID2\u003c/em\u003e in Col-0 was higher than that in \u003cem\u003esnat-2\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eb), suggesting that the reason for this situation was the slow development of \u003cem\u003esnat-2\u003c/em\u003e. The \u003cem\u003eSID2\u003c/em\u003e expression and SA content were significantly reduced by exogenous melatonin. Compared with Col-0, \u003cem\u003ewrky75\u003c/em\u003e showed a decrease in SA content and \u003cem\u003eSID2\u003c/em\u003e expression, while \u003cem\u003eWOE-3\u003c/em\u003e showed a increase. The content of SA and the expression of \u003cem\u003eSID2\u003c/em\u003e in Col-0 and all mutants was significantly downregulated by exogenous melatonin, but wrky75 showed the smallest decrease. It indicated that WRKY75 has a promoting effect on the regulation of \u003cem\u003eSID2\u003c/em\u003e, and exogenous melatonin can negatively affect the regulatory function of WRKY75 on \u003cem\u003eSID2\u003c/em\u003e and SA. Exogenous melatonin can effectively alleviate the increase in \u003cem\u003eSID2\u003c/em\u003e expression caused by WRKY75, reduce SA content, and alleviate plant senescence.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003e3.7 Melatonin downregulates the expression of plant senescence-associated genes\u003c/h2\u003e \u003cp\u003eWRKY75 is a positive regulator of senescence, and the expression level of \u003cem\u003eSAG13\u003c/em\u003e increases during the senescence of the plant. It was observed that the expression levels of these genes were higher in Col-0 than in \u003cem\u003esnat-2\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). Upon the application of exogenous melatonin, the expression levels of both genes decreased in Col-0. This trend was also evident in other mutants following melatonin application. Interestingly, \u003cem\u003eWOE-3\u003c/em\u003e expressed \u003cem\u003eSAG13\u003c/em\u003e to a greater extent than other plants, and the expression of both genes decreased substantially after exogenous melatonin application. Exogenous melatonin did not markedly down-regulate the expression of the \u003cem\u003eSAG13\u003c/em\u003e gene in \u003cem\u003ewrky75\u003c/em\u003e. The results of the expression analysis of these two genes support the conclusion that melatonin may mitigate plant senescence by down-regulating \u003cem\u003eWRKY75\u003c/em\u003e and \u003cem\u003eSAG13\u003c/em\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003e3.8 Melatonin reduces the degree of cell death\u003c/h2\u003e \u003cp\u003eThe ultimate result of plant cell senescence is death. The trypan blue staining results of \u003cem\u003esnat-2\u003c/em\u003e in the W3 phase were lighter than those of Col-0 staining, possibly due to the lack of melatonin in regulating growth and development in \u003cem\u003esnat-2\u003c/em\u003e, resulting in delayed development and mild cell death (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e). After the application of exogenous melatonin, both \u003cem\u003esnat-2\u003c/em\u003e and Col-0 staining were slightly lighter, and cell death was alleviated.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eThe mechanism of melatonin alleviates the natural senescence of plant leaves might depend on its potent antioxidant properties. Currently, there is no clear signaling pathway to elucidate the anti-senescence effect of melatonin. The research of leaf senescence provides a new direction for further understanding the regulatory role of melatonin.\u003c/p\u003e \u003cdiv id=\"Sec23\" class=\"Section2\"\u003e \u003ch2\u003e4.1 Melatonin can delay leaf senescence in \u003cem\u003eA. thaliana\u003c/em\u003e\u003c/h2\u003e \u003cp\u003eLeaf senescence is characterized by the degradation of chlorophyll and the subsequent yellowing of plant leaves, a process in which melatonin plays a ragulatory role. Our study demonstrated a gradual decrease in chlorophyll content during senescence. The effect of melatonin application as evident from the observed decrease in the significant deceleration of leaf yellowing (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, S3) and the chlorophyll content downward trend (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). This observation was consistent with previous studies, indicating that melatonin application can alleviate the decline in chlorophyll content induced by stress or senescence in plants, as demonstrated by the work of Yang \u003cem\u003eet al.\u003c/em\u003e (Yang et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Ye \u003cem\u003eet al.\u003c/em\u003e found that melatonin-treated wheat leaves had higher chlorophyll content compared to the control group during the senescence process (Ye et al. \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eAs plants undergo senescence, a substantial amount of ROS is produced, which is effectively scavenged by the antioxidant system, preserving ROS balance and enhancing cell membrane stability. Our results indicated that ROS levels were gradually elevated CAT activity increased and then decreased with the senescence process (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e, S4, \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). It was postulated that the modest fluctuation in CAT activity may be attributed to the stimulation of increased ROS in the early stages of senescence. Melatonin exhibited a modest upregulation of CAT activity and served as a potent antioxidant, markedly reducing ROS levels during leaf senescence (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec, \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ed), in line with previous research (Yang et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Zhao et al. \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Notably, our results found that melatonin had varying degrees effects on increased CAT activity and decreased levels of ROS (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). We deduce that the disparity in enzyme activity and ROS regulation instigated by melatonin can be attributed to its own profound ability to neutralize ROS.\u003c/p\u003e \u003cp\u003eEndogenous melatonin levels vary across different plant species, organs, growth stages, and environmental conditions (Murch et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). Our findings revealed that the melatonin content in Col-0 was higher than in \u003cem\u003esnat-2\u003c/em\u003e, and exogenous melatonin supplementation can stimulate endogenous melatonin accumulation (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Moreover, exogenous melatonin inhibited the decline in accumulation of ROS (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e), upregulation of SA accumulation and \u003cem\u003eSID2\u003c/em\u003e expression(Fig, 6), the upregulation of \u003cem\u003eWRKY75\u003c/em\u003e and \u003cem\u003eSAG13\u003c/em\u003e expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e) and cell death (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e), consequently delaying the senescence of \u003cem\u003esnat-2\u003c/em\u003e leaves. Prior studies had identified \u003cem\u003esnat-2\u003c/em\u003e as a crucial enzyme in melatonin synthesis (Zhao et al. \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Our study corroborated this finding, as \u003cem\u003esnat-2\u003c/em\u003e mutants exhibited markedly lower melatonin content compared to Col-0 (Fig. S2) and failed to increase endogenous melatonin levels following exogenous melatonin application (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea). Moreover, the breakdown of exogenous melatonin in \u003cem\u003esnat-2\u003c/em\u003e by metabolic enzymes lead to a sustained decline in endogenous melatonin levels. The absence of endogenous melatonin in \u003cem\u003esnat-2\u003c/em\u003e mutants delayed their growth and development and impeded the onset of senescence-related factors, including inhibiting chlorophyll decomposition and reducing the expression of \u003cem\u003eWRKY75\u003c/em\u003e and \u003cem\u003eSAG13\u003c/em\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec24\" class=\"Section2\"\u003e \u003ch2\u003e4.2 Melatonin can delay leaf senescence by reducing SA accumulation\u003c/h2\u003e \u003cp\u003eSeveral studies had demonstrated that SA plays a significant role in promoting natural leaf senescence(Li et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Lim et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Morris et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2000\u003c/span\u003e; Rivas-San Vicente and Plasencia \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). SA and ROS are two clearly defined factors inducing leaf senescence (Guo et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). During the process of plant leaf senescence, the expression level of \u003cem\u003eSID2\u003c/em\u003e (a key gene for SA synthase) increases, leading to a gradual rise in the endogenous SA content, which in turn accelerates the senescence process of \u003cem\u003eA. thaliana\u003c/em\u003e leaves (Lim et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Morris et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2000\u003c/span\u003e). Our study showed that the deficiency of endogenous melatonin contributed to reduced SA accumulation, while the application of exogenous melatonin further downregulated SA accumulation by affecting the expression of \u003cem\u003eSID2\u003c/em\u003e gene (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). This phenomenon had also been observed by Rafique \u003cem\u003eet al.\u003c/em\u003e (Rafique et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). These findings lead us to speculate that the slow senescence phenotype and low SA content of \u003cem\u003esnat-2\u003c/em\u003e may be attributed to slower growth and development.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec25\" class=\"Section2\"\u003e \u003ch2\u003e4.3 Melatonin delay leaf senescence by down-regulating the expression of the WRKY75 transcription factor\u003c/h2\u003e \u003cp\u003eWRKY75 plays a crucial role in various signaling pathways of plant hormones such as gibberellin, jasmonic acid, SA, and it has been implicated in the regulation of seed germination, abiotic stress tolerance, flowering, and senescence in plants (Chen et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Zhang et al. \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2021b\u003c/span\u003e; Zhang et al. \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Previous studies had demonstrated that WRKY75 promotes leaf senescence (Buchanan-Wollaston et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2003\u003c/span\u003e; Guo et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Additionally, WRKY75 was known to bind to the W-box in the promoters of target genes, thereby modulating their transcriptional activity, and this function in regulating senescence had been linked to its interaction with melatonin (Wei et al. \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Specifically, Weeda \u003cem\u003eet al.\u003c/em\u003e observed that exogenous melatonin application would increase the expression of \u003cem\u003eWRKY75\u003c/em\u003e in \u003cem\u003eA. thaliana\u003c/em\u003e (Weeda et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Wei \u003cem\u003eet al\u003c/em\u003e reported that overexpression of \u003cem\u003eMeWRKY75\u003c/em\u003e in cassava (\u003cem\u003eManihot esculenta\u003c/em\u003e) leads to an increase in the accumulation of melatonin in leaves (Wei et al. \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). This intricated interaction between WRKY75 and melatonin warranted further investigation and formed the basis of this study.\u003c/p\u003e \u003cp\u003eOur experimental findings supported the crucial role of WRKY75 in plant senescence. The overexpression of \u003cem\u003eWRKY75\u003c/em\u003e accelerated plant senescence, as evidenced by reduced chlorophyll content (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e), increased ROS and SA accumulation (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e, \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e), and up-regulated the expression of the senescence-related gene \u003cem\u003eSAG13\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). Remarkably, the detrimental effects of \u003cem\u003eWRKY75\u003c/em\u003e overexpression on senescence were mitigated by exogenous melatonin. Moreover, our observations indicated a partial dependence of exogenous melatonin action on WRKY75, as evidenced by a larger degree of senescence in the \u003cem\u003eWOE-3\u003c/em\u003e mutant and a smaller effect on \u003cem\u003ewrky75\u003c/em\u003e compared to Col-0 after treatment with exogenous melatonin. Melatonin counteract the senescence-promoting effect of WRKY75.\u003c/p\u003e \u003cp\u003eFurthermore, our investigation into the endogenous melatonin content and the \u003cem\u003eSNAT\u003c/em\u003e expression levels of \u003cem\u003ewrky75\u003c/em\u003e and \u003cem\u003eWOE-3\u003c/em\u003e revealed interesting insights. Notably, the expression of \u003cem\u003eSNAT\u003c/em\u003e in the W3 phase showed a higher level in the \u003cem\u003ewrky75\u003c/em\u003e, while its expression in \u003cem\u003eWOE-3\u003c/em\u003e was notably lower (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea). Conversely, the endogenous melatonin content of \u003cem\u003ewrky75\u003c/em\u003e and \u003cem\u003eWOE-3\u003c/em\u003e mutants was found to be similar, but substantially lower than that of Col-0 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb). The melatonin content of Col-0 increased significantly after the application of exogenous melatonin, while \u003cem\u003ewrky75\u003c/em\u003e and \u003cem\u003eWOE-3\u003c/em\u003e showed only slight increases. These results suggested that exogenous melatonin can up-regulate \u003cem\u003eSNAT\u003c/em\u003e expression and enhance the endogenous melatonin content, with the up-regulation influenced by WRKY75. Notably, the regulation of melatonin on the down-regulation of \u003cem\u003eWRKY75\u003c/em\u003e expression was dependent on the content of endogenous SA (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea). Weeda \u003cem\u003eet al.\u003c/em\u003e's genome analysis results indicated that exogenous melatonin increases the expression of \u003cem\u003eWRKY75\u003c/em\u003e in \u003cem\u003eA. thaliana\u003c/em\u003e (Weeda et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Conversely, Guo \u003cem\u003eet al.\u003c/em\u003e found that SA and ROS promote \u003cem\u003eWRKY75\u003c/em\u003e expression (Guo et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Consequently, it could be hypothesized that while melatonin can promote \u003cem\u003eWRKY75\u003c/em\u003e expression, it may also reduce \u003cem\u003eWRKY75\u003c/em\u003e expression by down-regulating SA and ROS accumulation. These two pathways seem to compete and antagonize each other, leading to the ultimate reduction in \u003cem\u003eWRKY75\u003c/em\u003e expression by melatonin (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"5. Conclusion","content":"\u003cp\u003eExogenous melatonin can delay leaf senescence, dependent on the salicylic acid pathway and the transcription factor WRKY75. Melatonin can downregulate the accumulation of ROS and SA, which subsequently reduces the expression of \u003cem\u003eWRKY75\u003c/em\u003e. It was observed that melatonin counteracts the effects of a network structure comprising SA, ROS, and WRKY75 on senescence, thereby modulating plant senescence-related processes and delaying leaf senescence in \u003cem\u003eA. thaliana\u003c/em\u003e.\u003c/p\u003e"},{"header":"Declarations","content":" \u003cp\u003eAll authors declare no conflict of interest.\u003c/p\u003e\u003ch2\u003eAcknowledgments\u003c/h2\u003e \u003cp\u003eThis work was funded by the State Key Laboratory Foundation of Crop Gene Exploration and Utilization in Southwest China (SKL-KF202318), and the Science and Technology Planning Project in 2022 of Dazhu County (2022CGG008).\u003c/p\u003e \u003cp\u003e \u003cb\u003eAuthor Contributions Statement\u003c/b\u003e \u003c/p\u003e \u003cp\u003eM.Y. proposed and drafted the research project. KY.W., JJ.M. and WH.C. planned and designed the experiments. KY.W. and JJ.M. conducted the experiments necessary for the research. KY.W., JJ.M. analyzed the statistical data for the experimental results. WH.C. prepared all the figures. KY.W. and JJ.Mao. wrote the manuscript together, and DQ.L., ZZ.T., YE.C., S.Y., GH.L., and M.Y. revised and edited it to a suitable format. All authors reviewed the manuscript.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eBeers EP, McDowell JM (2001) Regulation and execution of programmed cell death in response to pathogens, stress and developmental cues. Curr Opin Plant Biol 4:561\u0026ndash;567\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBuchanan-Wollaston V, Earl S, Harrison E, Mathas E, Navabpour S, Page T, Pink D (2003) The molecular analysis of leaf senescence \u0026ndash; A genomics approach. 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Plant Physiol 173:2294\u0026ndash;2307\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhao Y, Zhang Z, Chen Y, Ding C, Yuan S, Reiter RJ, Yuan M (2021) Melatonin: A potential agent in delaying leaf senescence. Crit Rev Plant Sci 40:1\u0026ndash;22\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"leaf senescence, melatonin, WRKY75, salicylic acid, ROS","lastPublishedDoi":"10.21203/rs.3.rs-5326045/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5326045/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eLeaf senescence, highly regulated by plant hormones and environmental factors, represents the final stage of leaf development. Therefore, the strategies to delay leaf senescence might extent the limitation of growth and yield for crop. Although previous studies had demonstrated the potential of melatonin to delay leaf senescence, its mechanism remained many mysteries. Here, we reported the role of melatonin in delaying WRKY75-induced leaf senescence. Exogenous melatonin increased chlorophyll content and reduced the accumulation of ROS in plants. In addition, it up-regulated the expression of the \u003cem\u003eSNAT\u003c/em\u003e gene and increased its melatonin level. Exogenous melatonin also decreased SA level and down-regulated \u003cem\u003eSID2\u003c/em\u003e gene expression. Furthermore, the expression of \u003cem\u003eSAG13\u003c/em\u003e and \u003cem\u003eWRKY75\u003c/em\u003e, both positive senescence-related genes, was found to decrease after melatonin treatment. These findings suggest that melatonin counteracts the effects of a network structure comprising SA, ROS, and WRKY75 on senescence, thereby regulating various events related to plant senescence and delaying leaf senescence.\u003c/p\u003e","manuscriptTitle":"Melatonin delayed leaf senescence induced by WRKY75 in Arabidopsis thaliana","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-11-07 17:28:53","doi":"10.21203/rs.3.rs-5326045/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"bd416800-fa44-4c47-8e13-76bccf125a2b","owner":[],"postedDate":"November 7th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-11-13T07:30:21+00:00","versionOfRecord":[],"versionCreatedAt":"2024-11-07 17:28:53","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-5326045","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5326045","identity":"rs-5326045","version":["v1"]},"buildId":"7rjqhiLT3MXkJMwkYKINL","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}