3.8 Melatonin enhances autophagic activity through promoting MdWRKY33-mediated transcriptional enhancement of MdATG18a
Among these 48 DEGs, MdATG18a (MD11G1303400) was significantly upregulated in OE-3 and OE-4 lines (Figure 5D). Huo et al. (2020) found that overexpressing MdATG18a in apple plants leads to stronger autophagy activity, thereby enhancing tolerance to heat stress (Huo et al. , 2020). Consequently, the MdATG18a expression pattern and autophagic activity in WT and MdASMT9 -OE lines under heat stress was determined. qRT-PCR showed that OE-3 and OE-4 lines had remarkably higher MdATG18a expression than the WT after 2 h of heat treatment (Figure 9A). TEM was used to observe the formation of autophagosome in response to high temperature (Figure 9B and 9C). The results showed that there were fewer autophagosome in all plants under control conditions, but more autophagosome were accumulated in OE lines than in WT plants under heat stress.
Previous studies found that silencing of WRKY33 reducedATGs gene expression and autophagosome accumulation, and compromised tomato heat tolerance (Zhou et al. , 2014). In this study, the correlation analysis of MdWRKY33 and MdATG18aexpression levels found that the expression of MdWRKY33 was significantly positively correlated with the expression ofMdATG18a (R=0.78, p <0.001) (Figure 9D). After exposure to heat stress, EV plants pretreated with 100 μM MT had higher expression level of MdATG18a than those without MT treatment, but there was no significant difference in MdATG18a expression level between TRV-MdWRKY33 plants pretreated with MT and TRV-MdWRKY33 plants not treated with MT (Figure 9E). As shown in Figure 9F, OE-3/EV and OE-4/MdWRKY33 had higher MdATG18aexpression levels than WT/EV under heat treatment. However,MdATG18a expression levels of OE-3/MdWRKY33 and OE-4/MdWRKY33 is not significantly different from that of WT/MdWRKY33 . The above results indicate that exogenous MT and overexpression of MdASMT9 enhanced autophagic activity through promoting MdWRKY33-mediated transcriptional enhancement ofMdATG18a .
DISCUSSION
MT, an essential antioxidant, plays multifarious roles in the stress tolerance of plants (Arnao and Hernández-Ruiz, 2015). Earlier research has demonstrated that MT helps plants alleviate the stress caused by high temperatures. Xu et al. (2016) suggested that heat stress could increase the endogenous MT content in tomato plants. MT pretreatment has alleviated heat-induced damage by promoting antioxidant defense mechanisms and regulate the biosynthesis of polyamine and nitric oxide (Jahan et al. , 2019). Exogenous MT application andASMT overexpression enhance thermotolerance and protect cellular protein in tomato by inducing HSPs and autophagy (Xu et al. , 2016). The relationship between endogenous MT and heat tolerance has only been described in model plants, while the physiological mechanisms of how endogenous MT responds to heat stress, particularly in perennial fruit trees, remain elusive. Accordingly, exploring the functions of MT biosynthesis genes in non-model plants such as apple would help us better understand the multiple functions of MT, thus furthering our knowledge in this area.
In the present study, apple plants overexpressing MdASMT9 were utilized to explore the role of MdASMT9 in high-temperature response. The findings indicated that MdASMT9 overexpression leads to greater heat tolerance in apple. MdASMT9 -OE lines experienced less damage than the WT under heat stress, as manifested by lower ion leakage and reduced MDA and ROS levels. High-temperature stress causes an imbalance of the osmotic potential and excessive ROS production in plant tissues (Choudhury et al. , 2017; de Pintoet al. , 2015). High concentrations of ROS can be detrimental to plants, resulting in oxidative damage due to the oxidation of crucial cellular components such as DNA, proteins, and lipids (Farmer and Mueller, 2013). Previous studies have shown that an antioxidant system is necessary for plants to prevent oxidative damage under high temperatures (Dong et al. , 2021; Huo et al. , 2020). In this study, MdASMT9 -OE apple lines maintained lower ROS accumulation under high temperatures than WT. The ROS regulatory system was also assessed, and it was discovered that the MdASMT9 -OE lines had significantly high SOD, POD, and CAT levels compared to WT under high-temperature stress.
Plants’ heat stress tolerance is related to the accumulation of soluble carbohydrates and amino acids (Akter and Rafiqul Islam, 2017; El Habtiet al. , 2020). Under environmental stress, plant soluble sugar accumulation can promote photosynthesis and delay senescence, and involve intracellular signal transduction and transcriptional regulation (Xalxo et al., 2020). Amino acids are the basis of protein synthesis and play a critical role in cell stress response and signal transduction (Häusler et al. , 2014; Zeier, 2013). Under high temperatures, arginine increased the activities of antioxidant enzymes and reduced lipid peroxidation (Khalil et al., 2009). Spraying glutamic acid on leaves inhibited leaf senescence induced by high temperature by inhibiting chlorophyll degradation, promoting amino acid metabolism, and maintaining nitrogen balance (Rossi et al. , 2021). MT can regulate soluble carbohydrates and amino acids levels under high-temperature stress (Iqbal et al. , 2021; Li et al. , 2020). 100 μM MT increases the amount of soluble sugar in wheat (Triticum aestivum L.) and improves its resistance to heat stress (Iqbal et al. , 2021). Exogenous MT application also increased the levels of amino acid, polyphenol, and caffeine, regulated photosynthesis, and promoted the growth and development of tea (Camellia sinensis L.) plants (Li et al. , 2020). In the present study, MdASMT9 overexpression in apple plants exposed to heat stress significantly increase the accumulation of sorbitol, galactose, sucrose, and all measured amino acids (histidine, arginine, aspartic acid, proline, glutamine, and glycine). However, many complex factors can influence carbohydrate and amino acid metabolism. The mechanism by which MT affects plant carbohydrate and amino acid accumulation under high-temperature stress requires further investigation.
Stomata can facilitate leaf cooling through transpiration (Gommers, 2020). Kostaki et al. (2020) showed that stomata rapidly opened once exposed to heat stress, resulting in a significant decrease in leaf surface temperature. Transgenic apple plants overexpressingMdATG18a showed greater stomatal pore aperture than WT plants and improved basal thermotolerance (Huo et al. , 2020). ABA is a key hormone for stomatal closure and plant tolerance to biotic and abiotic stress (Nambara and Marion-Poll, 2005). Li G et al. (2020) demonstrated that ABA negatively modulates heat tolerance by increasing leaf temperature and reducing transpiration. ABA-treated plants exhibited lower stomatal conductance than controls at 40°C (Feller, 2007). Exogenous MT application confers heat tolerance through increased cytokinin (CK) levels, whereas it decreases ABA levels in Lolium perenne (Zhang et al. , 2017). Jahan et al. (2021) also showed that MT inhibited leaf senescence induced by heat stress by inhibiting ABA biosynthesis and activating GA biosynthesis pathways in tomato. Furthermore, we found that MdASMT9 overexpression was associated with decreased ABA content and increased stomatal aperture under heat stress, which may enable leaves to keep a steady rate of transpiration and maintain a suitable temperature.
ABA biosynthesis occurs mainly through several enzymatic steps requiring NCED, zeaxanthin epoxidase (ZEP), and aldehyde oxidase (AO) (Nambara and Marion-Poll, 2005). Among them, NCED is a critical rate-limiting enzyme in ABA biosynthesis. In soybean (Glycine max L.), the positive effects of MT treatment during heat stress were associated with the decrease in ABA levels and down-regulated gmNCED3 expression (Imran et al. , 2021). In the present study, MdASMT9 -OE lines showed significantly decreased expressions of MdNCED1 andMdNCED3 . In a previous study, WRKY33 acted on the NCED3and NCED5 promoters, repressing their expression, thereby negatively regulating ABA biosynthesis (Liu et al. , 2015). Zhouet al. (2014) showed that WRKY33 expression increased after heat treatment, and silencing WRKY33 reduced the heat tolerance in tomato plants. MdWRKY33 expression was significantly upregulated in transcriptome data of transgenic apple plants overexpressing MdASMT9 . EMSA and LUC reporter assays provided evidence that MdWRKY33 binds to MdNCED1 and MdNCED3promoters suppresses their expression. These results suggested thatMdASMT9 overexpression promoted a WRKY33-mediated decrease inMdNCED1 and MdNCED3 expression.
Autophagy is an evolutionarily conserved protein degradation pathway, which helps plants alleviate heat stress (Huo et al. , 2020). Huoet al. (2020) found that overexpressing MdATG18a in apple plants leads to stronger autophagy activity, thereby enhancing tolerance to heat stress (Huo et al. , 2020). In previous studies, silencing of WRKY33 reduced ATGs gene expression and autophagosome accumulation, and compromised tomato heat tolerance (Zhou et al. , 2014). Lai et al. (2011) found that WRKY33 plays a positive role in autophagy activity, and in the wrky33 mutant, the induction of autophagy and ATG18a by Botrytis cinerea was impaired. In this study, the correlation analysis found that the expression ofMdWRKY33 was significantly positively correlated with the expression of MdATG18a . Exogenous MT application and overexpression of MdASMT9 significantly increased expression ofMdATG18a and enhanced relative autophagic activity. However, but there was no significant difference in MdATG18a expression between TRV-MdWRKY33 plants with versus without exogenous MT application. MdATG18a expression levels of OE-3/MdWRKY33and OE-4/MdWRKY33 was not also significantly different from that of WT/MdWRKY33 under heat stress. Therefore, melatonin enhances autophagic activity and MdATG18a expression through an MdWRKY33-mediated pathway.
HSFs act as key components of signal transduction in heat tolerance of plants, which are divided into three subfamilies (HSFA, HSFB, and HSFC) (Zeng et al. , 2021). A lot of HSFAs play active regulatory roles in response to heat stress in plants (Friedrich et al. , 2021; Mishra et al. , 2002; Zhang et al. , 2022). HsfA1denhanced high-temperature resistance by regulating the expression of heat-stress-responsive genes in Thellungiella salsuginea (Higashiet al. , 2013). In kiwifruit plants, AcHsfA2–1overexpression upregulated transcripts of multiple genes and onferred enhanced heat tolerance. Overexpression of MdHSFA9b in Arabidopsis improves heat tolerance of plants (Zhang et al. , 2022). In this study, transcriptome data showed that HSFA1d ,HSFA2-like and HSFA9b expression levels was higher inMdASMT9 -OE lines. In contrast with HSFA , the HSFBhas been reported as a a negative regulator in responses to heat stress (Tan et al. , 2021; Xie et al. , 2023). Under higher temperature, overexpression of MdASMT9 inhibited the expression of HSFB1 and HSFB2b . These suggested that MdASMT9overexpression enhanced heat tolerance by promoting the expression ofHSFAs (HSFA1d , HSFA2-like , HSFA9b ) and inhibiting the transcription of HSFBs (HSFB1 andHSFB2b ), negative regulators in heat response.
In conclusion, we have determined the mechanism ofMdASMT9 -mediated MT biosynthesis in promoting thermotolerance (Figure 10). Heat stress induces the expression of MdASMT9 and the accumulation of MT. Endogenous MT can eliminate excessive ROS by prompting activities of antioxidant enzymes (SOD, CAT, and POD) and improved soluble sugars and amino acids contents under heat stress. Transcriptome sequencing and qRT-PCR indicates that MdASMT9overexpression promoting the expression of HSFA1d ,HSFA2-like , and HSFA9b , and inhibiting the transcription of HSFB1 and HSFB2b . Furthermore, application of MT andMdASMT9 overexpression promotes the MdWRKY33transcription. MdWRKY33 directly targeted and inhibit the expression of MdNCED1 and MdNCED3, reducing ABA accumulation and promoting stomatal openings under heat stress to facilitate plant transpiration and dissipation. In addition, melatonin enhances autophagic activity by promoting the transcription of