A Hydrogen-Sulfide-Repressed Methionine Synthase SlMS1 Acts as a Positive Regulator for Fruit Ripening in Tomato

Ethylene is a key phytohormone that regulates the ripening of climacteric fruits, and methionine is an indirect precursor of ethylene. However, whether methionine synthase plays a role in fruit ripening in Solanum lycopersicum (tomato) is still unknown. In this study, we find that a tomato methionine synthase (named SlMS1), which could be repressed at the transcriptional level by hydrogen sulfide (H2S), acts as a positive regulator for tomato fruit ripening. By a bioinformatics analysis, it is found that SlMS1 and SlMS2 in tomato are highly homologous to methionine synthases in Arabidopsis thaliana. The expression pattern of SlMS1 and SlMS2 is analyzed in tomato, and SlMS1 expression is up-regulated during fruit ripening, suggesting its potential role in regulating fruit ripening. A potential bipartite nuclear localization signal is found in the amino acid sequence of SlMS1; thus, SlMS1 is tagged with GFP and observed in the leaves of Nicotiana benthamiana. Consistently, SlMS1-GFP shows strong nuclear localization and also cytoplasmic localization. The role of SlMS1 in regulating fruit ripening is investigated in tomato fruit by transient silencing (virus-induced gene silencing, VIGS) and transient overexpression. The results show that SlMS1 silencing causes delayed fruit ripening, evidenced by more chlorophyll and less carotenoid accumulation, while SlMS1 overexpression accelerates fruit ripening significantly compared with control. Further investigation shows that SlMS1 overexpression could up-regulate the expression of carotenoid-synthesis-related genes (PSY1, PDS, ZDS), chlorophyll-degradation-related genes (NYC1, PAO, PPH, SGR1), cell-wall-metabolism-related genes (CEL2, EXP, PG, TBG4, XTH5) and ethylene-synthesis-pathway-related genes (ACO1, ACO3, ACS2), while SlMS1 silencing causes the opposite results. The correlation analysis indicates that SlMS1 expression is negatively correlated with chlorophyll content and positively correlated with carotenoid and ripening-related gene expressions. Taken together, our data suggest that SlMS1 is a positive regulator of tomato fruit ripening and a possible target gene for the ripening-delaying effect of H2S.


Introduction
Tomato fruit is rich in nutrients and thus a popular healthy vegetable. With a clear genetic background and short growth cycle, tomato is a model organism for the research on plant growth and fruit ripening and senescence. Tomato is a typical climacteric fruit, and ethylene plays a key role in tomato fruit ripening [1]. Tomato fruit ripening is a complex and orderly physiological and biochemical process, accompanied by fruit softening, chlorophyll decomposition and carotenoid accumulation. At the level of the subcellular structure, the transition from chloroplast to chromoplast occurs, and the thylakoid structure of chloroplast disintegrates and loses the ability of photosynthesis [2]. Fruit degreening is a visible sign during tomato fruit ripening. The degradation of chlorophylls begins with the conversion of chlorophyll b to chlorophyll a [3]. Subsequently, the chlorophyll degradation pathway could be further catalyzed by the Pheophorbide a monooxygenase (PAO) pathway and the Pheophytin pheophorbide hydrolase (PPH) pathway. In addition, SGR (STAY-GREEN) proteins could promote chlorophyll degradation during tomato fruit ripening [4,5]. During the transition from chloroplast to chromoplast, a large amount of carotenoid accumulates in the chromoplast mediated by phytoene synthase (PSY), phytoene desaturase (PDS), ζ-carotene isomerase (ZISO), ζ-carotene desaturase (ZDS), etc. [6]. Moreover, fruit softening during the ripening process involves a coordinated expression of a large number of genes. Many cell-wall-modifying enzymes cooperate to induce changes in texture, leading to cell wall loosening and hydration of cell wall polymers. The cell-wall-modifying enzymes include pectinesterase (PE), pectate lyase (PL), polygalacturonase (PG) and β-galactosidase (β-GAL), and the hemicellulose/cellulosemodifying enzymes: 1,4-glucanase, xyloglucan transglycosylase/hydrolase (XTH) and expansin (EXP), etc. [6]. Expansins promote cellulose microfibrils to slide over one another, and PG reduces the chain length of pectins [7]. Due to the strong respiration, texture softening and the consumption of fruit nutrients, the shelf life of tomato fruit is shortened during the postharvest storage, resulting in a substantial waste of resources. Therefore, further exploration of the regulation mechanism of tomato fruit ripening and senescence will be helpful for the research on the mechanism of tomato fruit development.
Tomato is a typical climacteric fruit, as it undergoes a rapid rise in respiration and a burst of ethylene production at the onset of the ripening process. The pathway of ethylene biosynthesis involves the conversion of S-adenosylmethionine (SAM) to 1-aminocyclopropane-1-carboxylic acid (ACC) by ACC synthase (ACS) and then by ACC oxidase (ACO) to ethylene [8,9]. In tomato, ACO1 and ACO3 expression increases strongly and correlates well with the autocatalytic rise in ethylene production, suggesting their important role in regulating fruit ripening [10].
Methionine is an indirect precursor for ethylene biosynthesis, and we propose that its influx into the ethylene biosynthesis pathway is critical for ethylene burst during fruit ripening. SAM is generated with methionine as the substrate under the catalyzation of S-adenosylmethionine synthase [11]. Given that ethylene is a key phytohormone regulating tomato fruit ripening and senescence, we believe that methionine is a key metabolite controlling the ethylene pathway. During de novo methionine synthesis, methionine synthase in plants converts homocysteine to methionine using N5-methyltetrahydrofolate as a methyl donor [12,13]. In tomato, the functions of methionine synthase are not well studied. Methionine synthase ER69, i.e., SlMS1, corresponding to Solanum lycopersicum methionine synthase in this work, was found to be an ethylene-regulated gene, suggesting that methionine synthase may regulate tomato fruit ripening [14].
Hydrogen sulfide (H 2 S) can alleviate ripening and senescence of multiple postharvest fruits, such as strawberry, kiwifruit and tomato, by activating the antioxidant system and antagonizing the ethylene pathway [15][16][17]. However, the key point gene for the target of H 2 S in mediating its ripening delay remains unclear. As methionine synthase is the determinant for the influx to the ethylene pathway, we propose that methionine synthase genes may be the targets for H 2 S signal. In this study, we studied the role of SlMS1 in regulating fruit ripening by transient silencing (virus-induced gene silencing, VIGS) and transient overexpression of SlMS1 in tomato fruits. We found that SlMS1 showed both nucleus and cytoplasm localization, and it is a positive regulator for tomato fruit ripening.

Identification and Bioinformatics Analyses of Methionine Synthase Genes in Tomato Genome
The amino acid sequence of AtMS1 (AT3G03780) obtained from NCBI was used as a query to obtain homologous proteins in A. thaliana and S. lycopersicum. Five methionine synthase proteins were identified and, as shown in the phylogenetic tree in Figure ??A, the identified methionine synthases could be classified into two groups, in which AtMS3 forms a single branch, and the other proteins, AtMS1, AtMS2, SlMS1 and SlMS2, group into a single branch. Moreover, we analyzed the functional domain and found that all methionine synthases in Arabidopsis and tomato show two conserved domains, i.e., Meth_synt_1 and Meth_synt_2. Meth_synt_1 could be found in Cobalamin-independent methionine synthase, MetE, which catalyzes the synthesis of the amino acid methionine by the transfer of a methyl group from methyltetrahydrofolate to homocysteine. The Meth_synt_2 domain is referred to as the N-terminal half and C-terminal half of MetE in E. coli, which show some sequence similarity, indicating that the MetE gene has evolved from an ancestral MetE gene by duplication. Therefore, sequence alignment was performed, as shown in Figure S1, and it can be found that methionine synthases are highly conserved in A. thaliana and S. lycopersicum. Additionally, the tertiary structures of methionine synthases in A. thaliana and S. lycopersicum are predicted and shown in Figure S2. The dimensional structures are very similar among the proteins, and a cleft can be found between the two domains. The cleft may be the site for the reaction of 5-methyltetrahydrofolate + L-homocysteine to generate tetrahydrofolate + L-methionine.
The analysis of the physicochemical properties of AtMS1 showed that AtMS1, AtMS2 and SlMS1 showed the same 765 amino acids and similar pI values, which were 6.09, 6.09 and 6.01, respectively. Consistently, AtMS1, AtMS2 and SlMS1 with similar physicochemical characteristics show more intimate phylogenetic relations.

Expression Patterns of Tomato MS Gene in Different Tissues of Tomato Plants and in Response to Hydrogen Sulfide Treatment
To explore the expression patterns of MS genes in tomato plant, the transcription data were explored in tomato roots, stems, leaves, flower buds, flowers and fruits at different ripening stages in a public database. As shown in Figure ??C, there were significant differences in the expression of MS genes in different tissues of tomato. SlMS1 showed an increasing trend of expression until the mature green stage, followed by decline, suggesting that SlMS1 might play an important role in regulating tomato fruit ripening. H 2 S could act as a repressive signal for fruit ripening by antagonizing the effect of ethylene in tomatoes. Thus, we attempt to establish whether H 2 S could repress the expression of SlMS1 and SlMS2 in tomato fruit. We explore previous published transcriptome data, where mature green tomato fruits were treated with H 2 S donor NaHS harvested at 0, 1 and 3 days after storage [18]. As shown in Figure ??D, H 2 S application repressed the expression of SlMS1 and SlMS2 at 1 and 3 days of treatment and the original transcriptome data were presented in Table S1. As shown in the q-PCR result of Figure ??E, the expression of SlMS1 in H 2 Streated fruit was less than half of the control at 1 and 3 days of treatment, suggesting that the expression of SlMS1 was repressed by H 2 S.

Subcellular Localization of SlMS1
Subcellular localizations of methionine synthases in A. thaliana and S. lycopersicum were predicted by an online tool, and the results are shown in Table 1. It can be found that AtMS1 shows cyto: 7, chlo: 4, mito: 2, nucl: 1, AtMS2 cyto: 5, mito: 4, chlo: 3, nucl: 1, pero: 1 AtMS3 mito: 9, chlo: 5, SlMS1 cyto: 7, chlo: 3, mito: 2, nucl: 1, pero: 1, SlMS2 mito: 9, chlo: 5. As SlMS1 might play a role in regulating fruit ripening, the potential nuclear localization signal in SlMS1 was further predicted by NLS Mapper with a cut-off score of 5. As shown in Figure 2A, two potential bipartite NLS sites were found, where one score was 6, and the other was 5.3. Basic amino acids, such as K or R, are important amino acids that define NLS. The first NLS sites contained KR and KK separated by a linker composed of 19 amino acids, and the NLS localized in the N-terminal region of SlMS1, all suggesting that the first NLS is a real bipartite NLS. Then, the recombinant SlMS1-GFP plasmids were expressed in tobacco leaves, and fluorescence was observed by a co-focal microscope. As shown in Figure 2B, SlMS1-GFP co-localized with the nucleus stain Hoechst 33342, indicating that the localization of SlMS1 was mainly in the nucleus. Moreover, SlMS1-GFP also showed cytoplasm localization.

Effects of Transient Silencing and Gene Expression of SlMS1 on Tomato Fruit Ripening
To investigate the role of SlMS1 in regulating tomato fruit ripening, the expression of SlMS1 was transiently silenced by VIGS, and gene expression was achieved by the transient expression of pSAK277-SlMS1 in tomato fruit. The recombinant plasmids TRV-SlMS1 and pSAK277-SlMS1 were transformed into Agrobacterium to infect tomato fruits at 15 days after anthesis and mature green fruits, respectively. As shown in Figure 3A, the phenotype of SlMS1-silenced fruit in tomato was observed at 14, 21 and 27 days post infection (DPI). At 21 DPI, the color of the fruits of the control group began to change to yellow, but no color change occurred with TRV-SlMS1. At 27 DPI, the fruits of the control group almost turned red, while TRV-SlMS1 were still in the breaker color stage. To confirm the successful silencing of SlMS1 by VIGS, the expression of SlMS1 was examined by RT-qPCR ( Figure 3B), and it could be found that SlMS1 was greatly silenced by VIGS compared with the control, suggesting the efficiency of gene silencing. Thus, the above results indicated that the silencing of SlMS1 delayed tomato fruit ripening, implying that SlMS1 is a positive regulator of fruit ripening. showed that the fruit of SlMS1 pSAK277-SlMS1 showed earlier fruit ripening than the control group in the injection chamber at 5 and 10 DPI, indicating that SlMS1 may play a role in promoting fruit ripening. The efficiency of gene overexpression of pSAK277-SlMS1 was analyzed by RT-qPCR. The data in Figure 3D indicate that the expression of SlMS1 was increased significantly in pSAK277-SlMS1 infected fruit. Therefore, SlMS1 is a positive regulator of tomato fruit ripening. The degradation of chlorophyll and the accumulation of carotenoids are important characteristics of the ripening process of tomato fruit. In order to further study the mechanism of SlMS1 gene silencing and transient expression in affecting tomato fruit ripening, Additionally, the tomato fruit chamber was injected with Agrobacterium harboring pSAK277-SlMS1, which increases the expression of SlMS1 by the 35S promoter. Figure 3C showed that the fruit of SlMS1 pSAK277-SlMS1 showed earlier fruit ripening than the control group in the injection chamber at 5 and 10 DPI, indicating that SlMS1 may play a role in promoting fruit ripening. The efficiency of gene overexpression of pSAK277-SlMS1 was analyzed by RT-qPCR. The data in Figure 3D indicate that the expression of SlMS1 was increased significantly in pSAK277-SlMS1 infected fruit. Therefore, SlMS1 is a positive regulator of tomato fruit ripening.

Effect of Gene Silencing and Overexpression of SlMS1 on the Metabolism of Chlorophyll and Carotenoid in Tomato Fruit
The degradation of chlorophyll and the accumulation of carotenoids are important characteristics of the ripening process of tomato fruit. In order to further study the mechanism of SlMS1 gene silencing and transient expression in affecting tomato fruit ripening, the carotenoid and chlorophyll contents were determined in control fruit, TRV-SlMS1 and pSAK277-SlMS1 infected fruit. As shown in Figure 4A, the content of total chlorophyll decreased slightly in both control and TRV-SlMS1 at 21 and 27 DPI during fruit ripening, whereas the content in SlMS1-silenced fruit was about two-fold of that in the control. The data of chlorophyll a and chlorophyll b showed a similar trend to total chlorophyll content ( Figure 4B,C). The carotenoid content in SlMS1-silenced fruit at 21 DPI was higher than that of the control group, but at 27 DPI, the content in the control was slightly higher than that of the SlMS1-silenced fruit ( Figure 4D). The carotenoid content in pSAK277-SlMS1 infected fruit at 27 DPI was significantly higher than that of the control group, while the chlorophyll in the fruit of the pSAK277-SlMS1 plant was much lower than that of the control group ( Figure 4E-H). The above results indicated that SlMS1 silencing decreased the accumulation of carotenoid, while SlMS1 overexpression improved the biosynthesis of carotenoid.

Effect of Gene Silencing and Overexpression of SlMS1 on the Expression of Carotenoid Synthesis Genes and Chlorophyll Degradation Genes
To explore the mechanism of differences in carotenoid metabolism in SlMS1 silenced and overexpressed fruit, the expression levels of carotenoid-synthesis-related genes PSY1, PDS and ZDS were analyzed by RT-qPCR. As shown in Figure 5A-C, the expression of PSY1, PDS and ZDS in control increased gradually from 21 to 27 DPI, whereas their

Effect of Gene Silencing and Overexpression of SlMS1 on the Expression of Carotenoid Synthesis Genes and Chlorophyll Degradation Genes
To explore the mechanism of differences in carotenoid metabolism in SlMS1 silenced and overexpressed fruit, the expression levels of carotenoid-synthesis-related genes PSY1, PDS and ZDS were analyzed by RT-qPCR. As shown in Figure 6A-C, the expression of PSY1, PDS and ZDS in control increased gradually from 21 to 27 DPI, whereas their expression was greatly attenuated in SlMS1 silenced fruit. Additionally, the expression of PSY1, PDS and ZDS was examined in SlMS1 overexpressed fruit mediated by pSAK277-SlMS1. As shown in Figure 6D-F, SlMS1 overexpression for 10 days induced an increased transcription of PSY1, PDS and ZDS significantly compared with the control fruit. For instance, the expression of PSY1 in SlMS1 overexpressed fruit was about 150 times that of the control. Therefore, SlMS1 increased the accumulation of carotenoid possibly by increasing the transcription of carotenoid-synthesis-related genes PSY1, PDS and ZDS.
Chlorophyll degradation is an important indicator of tomato fruit ripening. In order to explore the relations between SlMS1 and the changes in chlorophyll content during the ripening process, we analyzed the key genes NYC1, PPH, PAO and SGR1 in the chlorophyll degradation pathway. Whether it was at 21 DPI or 27 DPI, the contents of PAO, PPH, NYC1 and SGR1 in the gene-silencing fruit TRV-SlMS1 were much lower than those in the control group, indicating that chlorophyll degradation may be slowed by decreased expression of chlorophyll degradation related genes compared to that of the control (Figure 7A-D). In contrast, the expressions of PAO, PPH, NYC1 and SGR1 in pSAK277-SlMS1 were significantly higher than those in control fruit, of which SGR1 was 6.2 times higher than that of control, indicating that the transient expression of SlMS1 tomato fruit accelerated chlorophyll degradation by increasing the expression of chlorophyll degradation related genes ( Figure 7E-H). PSY1, PDS and ZDS was examined in SlMS1 overexpressed fruit mediated by pSAK277-SlMS1. As shown in Figure 5D-F, SlMS1 overexpression for 10 days induced an increased transcription of PSY1, PDS and ZDS significantly compared with the control fruit. For instance, the expression of PSY1 in SlMS1 overexpressed fruit was about 150 times that of the control. Therefore, SlMS1 increased the accumulation of carotenoid possibly by increasing the transcription of carotenoid-synthesis-related genes PSY1, PDS and ZDS. Chlorophyll degradation is an important indicator of tomato fruit ripening. In order to explore the relations between SlMS1 and the changes in chlorophyll content during the ripening process, we analyzed the key genes NYC1, PPH, PAO and SGR1 in the chlorophyll degradation pathway. Whether it was at 21 DPI or 27 DPI, the contents of PAO, PPH, NYC1 and SGR1 in the gene-silencing fruit TRV-SlMS1 were much lower than those in the control group, indicating that chlorophyll degradation may be slowed by decreased expression of chlorophyll degradation related genes compared to that of the control (Figure 6A-D). In contrast, the expressions of PAO, PPH, NYC1 and SGR1 in pSAK277-SlMS were significantly higher than those in control fruit, of which SGR1 was 6.2 times highe than that of control, indicating that the transient expression of SlMS1 tomato fruit acce erated chlorophyll degradation by increasing the expression of chlorophyll degradatio related genes ( Figure 6E-H).

Effect of Gene Silencing and Overexpression of SlMS1 on the Expression of Ethylene Biosynthesis Genes
Ethylene is an important phytohormone in the ripening process of tomato fruit. In order to study the relationship between SlMS1 and ethylene synthesis, the expression levels of ethylene-synthesis-related genes ACS2, ACO1 and ACO3 were determined in SlMS1 silenced fruit and SlMS1 overexpressed fruit. As shown in Figure 8A-C, the expression of ACS2, ACO1 and ACO3 increased gradually in both TRV-SlMS1 and control fruit from 21 to 27 DPI, whereas TRV-SlMS1 attenuated their expression greatly, whether at 21 DPI or 27 DPI, compared with control fruit; therefore, it could be concluded that SlMS1 gene silencing resulted in a significant down-regulation of ethylene synthesis related genes. The expression level of ACS2, ACO1 and ACO3 in pSAK277-SlMS1 at 10 DPI was much higher than that in the control group, among which ACS2 was about 35 times higher than the control. These results indicate that SlMS1 overexpression enhanced the transcription of genes involved in the ethylene synthesis pathway. ACS2, ACO1 and ACO3 increased gradually in both TRV-SlMS1 and control fruit from 21 to 27 DPI, whereas TRV-SlMS1 attenuated their expression greatly, whether at 21 DPI or 27 DPI, compared with control fruit; therefore, it could be concluded that SlMS1 gene silencing resulted in a significant down-regulation of ethylene synthesis related genes. The expression level of ACS2, ACO1 and ACO3 in pSAK277-SlMS1 at 10 DPI was much higher than that in the control group, among which ACS2 was about 35 times higher than the control. These results indicate that SlMS1 overexpression enhanced the transcription of genes involved in the ethylene synthesis pathway.

Effect of Gene Silencing and Overexpression of SlMS1 on the Expression of Cell-Wall-Metabolism-Related Genes in Tomato Fruit
Tomato fruit is usually accompanied by a decrease in fruit firmness during the ripening process, and the softening of the pericarp is regulated by genes or enzymes related (E) and ACO3 (F) in control (pSAK277) and SlMS1-overexpressed (pSAK277-SlMS1) fruit at 10 days post inoculation. The symbols * and ** stand for p < 0.05 and p < 0.01, as determined by Student's t-test, respectively.

Effect of Gene Silencing and Overexpression of SlMS1 on the Expression of Cell-Wall-Metabolism-Related Genes in Tomato Fruit
Tomato fruit is usually accompanied by a decrease in fruit firmness during the ripening process, and the softening of the pericarp is regulated by genes or enzymes related to cell wall metabolism. Therefore, the expression levels of genes CEL2, EXP, PG, TBG4 and XTH5 encoding cell wall metabolism related enzymes were analyzed in SlMS1 silenced fruit and SlMS1 overexpressed fruit. As shown in Figure 9A-E, compared with the control group, the expressions of CEL2, EXP, PG, TBG4 and XTH5 in TRV-SlMS1 fruits were significantly down-regulated at both 21 and 27 DPI. Additionally, the expression levels of CEL2, EXP, PG, TBG4 and XTH5 in pSAK277-SlMS1 fruits were significantly higher than those in the control group, and the expression levels of CEL2 and PG were about 80 and 150 times higher than those in the control group ( Figure 9F-J). In conclusion, the expression of genes related to fruit cell wall metabolism was significantly increased by the overexpression of SlMS1.
. Mol. Sci. 2022, 23, x FOR PEER REVIEW 12 of silenced fruit and SlMS1 overexpressed fruit. As shown in Figure 8A-E, compared wi the control group, the expressions of CEL2, EXP, PG, TBG4 and XTH5 in TRV-SlMS1 fru were significantly down-regulated at both 21 and 27 DPI. Additionally, the expressi levels of CEL2, EXP, PG, TBG4 and XTH5 in pSAK277-SlMS1 fruits were significant higher than those in the control group, and the expression levels of CEL2 and PG we about 80 and 150 times higher than those in the control group ( Figure 8F-J). In conclusio the expression of genes related to fruit cell wall metabolism was significantly increas by the overexpression of SlMS1.

Correlation Analysis among Different Physiological Indices and Ripening-Related Gene Expression
To reveal the potential relation between SlMS1 and fruit ripening, correlation w analyzed among SlMS1, the content of chlorophyll and carotenoids, and genes related fruit ripening, including NYC1, PAO, PPH, SGR1, PSY1, PDS, ZDS, ACS2, ACO1, ACO

Correlation Analysis among Different Physiological Indices and Ripening-Related Gene Expression
To reveal the potential relation between SlMS1 and fruit ripening, correlation was analyzed among SlMS1, the content of chlorophyll and carotenoids, and genes related to fruit ripening, including NYC1, PAO, PPH, SGR1, PSY1, PDS, ZDS, ACS2, ACO1, ACO3, SlMS1, PG, XTH5, TBG4, EXP and CEL2, based on the data determined. As shown in Figure 10, SlMS1 is positively correlated with fruit-ripening-related genes and negatively correlated with chlorophyll, suggesting that SlMS1 is indeed a positive regulator of fruit ripening. Moreover, total chlorophyll showed high negative correlation with the expression of chlorophyll degradation related genes NYC1, PAO, PPH and SGR1.

Discussion
The sulfur-containing amino acid methionine is essential for all organisms as a building block of proteins and as a precursor for the universal activated methyl donor SAM [19]. Ethylene biosynthesis starts with the conversion of SAM to ACC by ACS and then by ACO to ethylene [20]. Therefore, we propose that methionine content determines the influx to the ethylene pathway, and methionine synthase may participate in regulating fruit ripening. In the present work, the bioinformatics of methionine synthases in Arabidopsis and tomato were explored. All methionine synthases of Arabidopsis and tomato contained the Meth_synt_1 and Meth_synt_2 domains, and the amino acid sequence alignment and tertiary structure analysis suggested that they were highly conserved. Then, the expression patterns of MS genes in tomato were explored, and SlMS1 showed an increasing pattern of expression during the early stage fruit ripening, suggesting that SlMS1 might play an important role in regulating tomato fruit ripening. Previous research

Discussion
The sulfur-containing amino acid methionine is essential for all organisms as a building block of proteins and as a precursor for the universal activated methyl donor SAM [19]. Ethylene biosynthesis starts with the conversion of SAM to ACC by ACS and then by ACO to ethylene [20]. Therefore, we propose that methionine content determines the influx to the ethylene pathway, and methionine synthase may participate in regulating fruit ripening. In the present work, the bioinformatics of methionine synthases in Arabidopsis and tomato were explored. All methionine synthases of Arabidopsis and tomato contained the Meth_synt_1 and Meth_synt_2 domains, and the amino acid sequence alignment and tertiary structure analysis suggested that they were highly conserved. Then, the expression patterns of MS genes in tomato were explored, and SlMS1 showed an increasing pattern of expression during the early stage fruit ripening, suggesting that SlMS1 might play an important role in regulating tomato fruit ripening. Previous research indicates that methionine synthase ER69, i.e., SlMS1 in this work, is an ethylene-regulated gene, implying that methionine synthase may regulate tomato fruit ripening [14]. H 2 S is a negative regulator of fruit ripening and senescence in multiple postharvest fruits by antagonizing the effect of ethylene [15][16][17]. Thus, we proposed that SlMS1 may be the target for the signaling role of H 2 S. By analyzing previously published transcriptome data [18], it was found that the expression of SlMS1 in tomato fruit is greatly attenuated by H 2 S treatment, suggesting that H 2 S may delay fruit ripening by repressing the expression of SlMS1.
The first two reactions for de novo Met synthesis consist of the conversion of cysteine into homocysteine (Hcy) by the enzymes cystathionine γ-synthase and cystathionine β-lyase, and both enzymes are only present in the chloroplasts [12]. Plant methionine synthase seems to be present only in the cytosol [12,21,22], thus implying that Hcy has to exit the chloroplast to be converted into Met. Ravanel et al. [19] reported that AtMS1 and AtMS2 are cytosolic proteins, and the AtMS3-GFP fusion protein in protoplasts resulted in a pattern of green fluorescence, which colocalized with the red autofluorescence of chlorophyll. The potential localization of MS proteins is analyzed by an online tool, and the data in Table 1 suggest that SlMS1 shows cyto: 7, chlo: 3, mito: 2, nucl: 1, pero: 1, suggesting that SlMS1 primarily localizes in the cytoplast and, to a lesser extent, in the nucleus. Then, the detailed NLS of SlMS1 is predicted by NLS Mapper, and a bipartite NLS characterized by the basic amino acids KR and KK is found in the N-terminus of SlMS1, suggesting it may be a genius NLS. Consistent with the localization prediction, the fluorescence of recombinant SlMS1-GFP is observed mainly in the nucleus and partially in the cytoplasm in tobacco leaves. Therefore, we provide solid evidence that SlMS1 shows both nucleus and cytoplasm localization, possibly due to the bipartite NLS. Accumulating evidence suggests that the active sulfur-related metabolism occurs in the cell nucleus. For instance, S-adenosylmethionine synthase MAT4, which is required for the modification of histone H3K9me2 and DNA methylation levels, also showed nuclear localization [23]. L-cysteine desulfhydrase SlLCD1, which is required for H 2 S, shows specific nuclear localization, and the nuclear localization is dependent on the NLS signal in its N-terminus [18]. The O-acetylserine(thiol)lyases SlOAS2, 4 and 6, were found to localize in the membrane, cytosol and nucleus, suggesting that cysteine could be synthesized in the nucleus [24]. A recent report indicates that methionine synthase METS1 in Arabidopsis acting as a nexus between the folate pathway and plant immunity and overexpression of METS1 represses plant immunity and is accompanied by a genome-wide global increase in DNA methylation [25], suggesting that methionine synthase is also involved in regulating plant immunity.
Methionine is a precursor for ethylene biosynthesis, and therefore, we propose that methionine synthase may participate in regulating fruit ripening. In the present work, we silence the expression of SlMS1 in tomato fruit by VIGS and overexpress its expression by pSAK277 plasmid. The results indicate that the silencing of SlMS1 delays tomato fruit ripening, while SlMS1 overexpression promotes fruit ripening, implying that SlMS1 is a positive regulator of fruit ripening. Moreover, the silencing of SlMS1 reduces the accumulation of carotenoid and delays the degradation of chlorophyll. Then, the genes related to carotenoid biosynthesis and chlorophyll degradation are analyzed at the transcriptional level. The data indicate that SlMS1 silencing caused a decreased expression of PSY1, PDS and ZDS compared with control during fruit ripening, whereas SlMS1 overexpression for 10 days induces an increased transcription of PSY1, PDS and ZDS significantly. Further, SlMS1 overexpression enhances the expression of PAO, PPH, NYC1 and SGR1, while SlMS1 silencing reduces their expressions. Thus, the work indicates that SlMS1 overexpression in tomato fruit accelerates chlorophyll degradation and carotenoid biosynthesis by increasing the expression of chlorophyll degradation related genes and carotenoid biosynthesis re-lated genes. Additionally, we find that SlMS1 overexpression enhances the transcription of ethylene-synthesis-related genes, ACS2, ACO1 and ACO3, and genes CEL2, EXP, PG, TBG4 and XTH5, which encode cell wall metabolism related enzymes. To reveal the potential relation between SlMS1 and fruit ripening, the correlation was analyzed among SlMS1, the content of chlorophyll and carotenoids and genes related to fruit ripening. The result suggests that SlMS1 is positively correlated with fruit-ripening-related genes and negatively correlated with chlorophyll, suggesting that SlMS1 acts as a positive regulator of fruit ripening.
Methionine synthase is responsible for methionine synthesis, and SAM accounts for about 80% of methionine metabolism, whereas the synthesis of proteins drives about 20% of methionine [11,19]. Thus, the expression of SlMS1 is critical for initiating fruit ripening, as SlMS1 may determine the content of methionine and also ethylene precursor, SAM. Finally, we propose a model of methionine and methionine synthase regulating fruit ripening, as shown in Figure 11. For methionine biosynthesis, CGS and CBL catalyze the pathway from cysteine to homocysteine via cystathionine. Then, methionine synthase (MS, encoded by ripening related gene SlMS1) methylates homocysteine to yield Met. Methionine generates methyl donor SAM under the action of SAM synthase, and then, ACC synthase (ACS) and ACC oxidase (ACO) catalyze the biosynthesis of ethylene. Therefore, SlMS1 acts as a positive regulator of tomato fruit ripening, possibly through the synthesis of methionine, which leads to the influx into the ethylene biosynthesis pathway. Methionine synthase is responsible for methionine synthesis, and SAM accounts for about 80% of methionine metabolism, whereas the synthesis of proteins drives about 20% of methionine [11,19]. Thus, the expression of SlMS1 is critical for initiating fruit ripening, as SlMS1 may determine the content of methionine and also ethylene precursor, SAM. Finally, we propose a model of methionine and methionine synthase regulating fruit ripening, as shown in Figure 10. For methionine biosynthesis, CGS and CBL catalyze the pathway from cysteine to homocysteine via cystathionine. Then, methionine synthase (MS, encoded by ripening related gene SlMS1) methylates homocysteine to yield Met. Methionine generates methyl donor SAM under the action of SAM synthase, and then, ACC synthase (ACS) and ACC oxidase (ACO) catalyze the biosynthesis of ethylene. Therefore, SlMS1 acts as a positive regulator of tomato fruit ripening, possibly through the synthesis of methionine, which leads to the influx into the ethylene biosynthesis pathway. Figure 10. Metabolic diagram of methionine regulating fruit ripening. For methionine biosynthesis, CGS catalyzes the generation of cystathionine from cysteine and O-phosphohomoserine, and then, homocysteine is produced by CBL. The last step of Met synthesis is catalyzed by methionine synthase (MS encoded by SlMS1), which methylates homocysteine to form Met using N5-methyltetrahydrofolate as a methyl group donor. Methionine generates methyl donor SAM under the action of S-adenosylmethionine synthase, and then, ACC synthase (ACS) and ACC oxidase (ACO) catalyze the biosynthesis of ethylene. MS, methionine synthase; CBL, cystathionine β-lyase; CGS, cystathionine γ-synthase; SAM, S-adenosylmethionine; ACC, 1-aminocyclopropane-1-carboxylate.

Identification, Phylogenetic Analysis and Data Exploration of Methionine Synthases in Plants
With the protein sequence of AtMS1 in Arabidopsis as a query, the homologous genes of methionine synthase in tomato and Arabidopsis were identified in https://phytozome-next.jgi.doe.gov/blast-search, accessed on 11 August 2022 by the program BLASTP-protein query to protein database. The protein molecular weights and theoretical pI values were computed using ProtParam (http://web.expasy.org/protparam/, accessed on 15 August 2022). The predicted NLSs were obtained from NLS mapper (http://nls-mapper.iab.keio.ac.jp/cgi-bin/NLS_Mapper_form.cgi, accessed on 12 August 2022) [26]. The subcellular localization of proteins was predicted on the website https://wolfpsort.hgc.jp/, ac- Figure 11. Metabolic diagram of methionine regulating fruit ripening. For methionine biosynthesis, CGS catalyzes the generation of cystathionine from cysteine and O-phosphohomoserine, and then, homocysteine is produced by CBL. The last step of Met synthesis is catalyzed by methionine synthase (MS encoded by SlMS1), which methylates homocysteine to form Met using N5-methyltetrahydrofolate as a methyl group donor. Methionine generates methyl donor SAM under the action of S-adenosylmethionine synthase, and then, ACC synthase (ACS) and ACC oxidase (ACO) catalyze the biosynthesis of ethylene. MS, methionine synthase; CBL, cystathionine β-lyase; CGS, cystathionine γ-synthase; SAM, S-adenosylmethionine; ACC, 1-aminocyclopropane-1-carboxylate.

Identification, Phylogenetic Analysis and Data Exploration of Methionine Synthases in Plants
With the protein sequence of AtMS1 in Arabidopsis as a query, the homologous genes of methionine synthase in tomato and Arabidopsis were identified in https://phytozomenext.jgi.doe.gov/blast-search, accessed on 11 August 2022 by the program BLASTP-protein query to protein database. The protein molecular weights and theoretical pI values were computed using ProtParam (http://web.expasy.org/protparam/, accessed on 15 August 2022). The predicted NLSs were obtained from NLS mapper (http://nls-mapper.iab.keio. ac.jp/cgi-bin/NLS_Mapper_form.cgi, accessed on 12 August 2022) [26]. The subcellular localization of proteins was predicted on the website https://wolfpsort.hgc.jp/, accessed on 15 August 2022. The tertiary structure of proteins was predicted on the website of SWISS-MODEL (expasy.org).
The phylogenetic relationships among methionine synthases from Arabidopsis thaliana and tomato were investigated using MEGA 7.0 [27] with the neighbor-joining method and 1000 bootstrap replicates. Multiple sequence alignment was performed on the website https: //espript.ibcp.fr/ESPript/ESPript/, accessed on 14 August 2022. The domain composition of proteins was predicted on SMART: Main page (embl-heidelberg.de). For the exploration of transcription of methionine synthase genes in different tissues of tomato, the original expression data were obtained from the online tool http://tomexpress.toulouse.inra.fr/ query, accessed on 11 August 2022, and the tomato cultivar was Micro Tom.

Subcellular Localization Analysis
The CDS of SlMS1 without a stop codon was inserted into the p1300-35S-GFP vector for SlMS1-GFP expression. Agrobacterium tumefaciens GV3101 containing the recombinant vectors was infiltrated into 5-week-old N. benthamiana leaves. Then, the tobacco was cultured at 22 • C under a 16 h light/8 h dark photoperiod for 2 days. The location of the nucleus was indicated by treatment with Hoechst 33342 (5 µg/mL) for 5 min. The excitation/emission wavelengths were 488 nm/507 nm for GFP and 346 nm/460 nm for Hoechst 33342. All fluorescence signals were detected using a Zeiss LSM710NLO confocal laser scanning microscope. The primers for the construction of SlMS1-GFP were shown in Table S2.

VIGS of SlMS1 and Transient Overexpression of SlMS1 in Tomato Fruit
A fragment of 400 bp corresponding to nt 1563-1964 of the SlMS1 sequence was amplified from the cDNA of tomato by PCR and inserted into pTRV2 to yield pTRV2-SlMS1 at restriction sites of EcoRI and BamHI. Agrobacterium tumefaciens GV3101 containing the vectors was cultured at 28 • C for 16 h in Luria-Bertani medium containing 20 µM AS, 10 mM MES and 50 µg/mL each of the antibiotics kanamycin, gentamycin and rifampicin, according to the method of Fu et al. [28]. Cells resuspended with the pTRV1 and pTRV2 or pTRV2-SlMS1 vector were then mixed together at a ratio of 1:1 and infected into the pedicels of Micro Tom tomato plants. Tomato petioles infiltrated with pTRV2 without the insert were used as controls. Tomato plants were then stored at 16 • C for 24 h and transferred to normal culture conditions at 22 • C under a 16 h light/8 h dark photoperiod thereafter. The images of fruits were taken at 14, 21 and 27 days post injection. The samples without seeds were sampled at 21 and 27 days post injection and stored in a -80 • C refrigerator for subsequent measurement.
For transient overexpression of SlMS1, the coding sequence of SlMS1 was cloned into the plasmid pSAK277 under the 35S promoter at the restriction sites of XbaI and EcoRI. Agrobacterium tumefaciens GV3101 containing pSAK277-SlMS1 or the empty vector cultured overnight was diluted into 20 µM AS (Acetosyringone), 10 mM MES (2-Morpholinoethanesulphonic acid) to OD 600 0.8-1.0. After 4 h of culture, the Agrobacterium was injected into one chamber of the tomato fruit at the green mature stage. The infected plant was stored at 16 • C for 24 h in the dark and then placed in normal growth conditions. The images of fruits were taken at 5 and 10 days post injection. The samples without seeds were sampled at 10 days post injection and stored in a -80 • C refrigerator for subsequent measurement. The primers for the construction of pTRV2-SlMS1 and pSAK277-SlMS1 were shown in Table S2.