Sulfoxidation Regulation of Musa acuminata Calmodulin (MaCaM) Influences the Functions of MaCaM-Binding Proteins

Sulfoxidation Regulation of Musa acuminata Calmodulin (MaCaM) Influences the Functions of... Abstract Sulfoxidation of methionine in proteins by reactive oxygen species can cause conformational alteration or functional impairment, and can be reversed by methionine sulfoxide reductase (Msr). Currently, only a few potential Msr substrates have been confirmed in higher plants. Here, we investigated Msr-mediated sulfoxidation regulation of calmodulin (CaM) and its underlying biological significance in relation to banana fruit ripening and senescence. Expression of MaCaM1 and MaMsrA7 was up-regulated with increased ripening and senescence. We verified that MaCaM1 interacts with MaMsrA7 in vitro and in vivo, and sulfoxidated MaCaM1 could be partly repaired by MaMsrA7 (MaMsrA7 reduces oxidized residues Met77 and Met110 in MaCaM1). Furthermore, we investigated two known CaM-binding proteins, catalase (MaCAT1) and MaHY5-1. MaHY5-1 acts as a transcriptional repressor of carotenoid biosynthesis-related genes (MaPSY1, MaPSY2 and MaPSY3) in banana fruit. MaCaM1 could enhance the catalytic activity of MaCAT1 and the transcriptional repression activity of MaHY5-1 toward MaPSY2. Mimicked sulfoxidation in MaCaM1 did not affect the physical interactions of the protein with MaHY5-1 and MaCAT1, but reduced the catalytic activity of MaCAT1 and the transcriptional repression activity of MaHY5-1. Our data suggest that sulfoxidation modification in MaCaM1 by MaMsrA7 regulates antioxidant response and gene transcription, thereby being involved in regulation of ripening and senescence of banana fruit. Introduction Fruits, the seed-bearing structures of flowering plants, are important economic crops and provide humans with nutrition. Ripening and senescence are crucial for the development and maintenance of fruit quality. A better understanding of fruit ripening and senescence may help in developing strategies to improve nutritional and sensorial qualities of fruit and reduce post-harvest losses. Fruits are classified as climacteric or non-climacteric in terms of their ripening or senescence attributes. Climacteric fruits, such as banana and mango, undergo ripening after harvest. Ethylene plays a key role in the ripening of climacteric fruits (Lin et al. 2009). Non-climacteric fruits, such as citrus and strawberry, undergo no ripening process after harvest. No dramatic increases in respiration are observed, and ethylene biosynthesis remains at very low levels (Alexander and Grierson 2002). Many studies have linked ABA to ripening and senescence of non-climacteric fruits (Ji et al. 2012, Wu et al. 2014). Therefore, recent advances in understanding fruit ripening and senescence have mainly been associated with hormone signal transduction and action. Other mechanisms underlying fruit ripening and senescence are still largely unknown. Reactive oxygen species (ROS) are unavoidably produced as by-products of cellular metabolism, and they function as important signaling molecules that are involved in a broad range of processes (Pitzschke et al. 2006, Mittler 2017). However, under senescence or stress, excessive accumulation of ROS can cause oxidative damage to macromolecules such as DNA, proteins, polysaccharides and lipids, which, in turn, results in loss of structure and function or even cell death (Pitzschke et al. 2006, Moller et al. 2007, Choudhury et al. 2017, Rey et al. 2017). To combat oxidative stress, organisms have evolved several protective mechanisms, such as antioxidant enzyme systems and macromolecule repair systems (Levine et al. 2000). Proteins are the major targets of ROS, which lead to the oxidation of the peptide backbone and amino acid side chains. Elimination of oxidized proteins is crucial for maintaining the integrity of the cell (Moller et al. 2007, Qin et al. 2009). Among the amino acids, cysteine and methionine (Met) are most vulnerable to ROS. Interestingly, oxidation products of cysteine and Met in proteins can be repaired by protein repair systems, thioredoxin (Trx)/thioredoxin reductase and methionine sulfoxide reductase (Msr), respectively. Met oxidation, i.e. sulfoxidation, results in the production of two diastereomers of methionine sulfoxide, Met-S-O and Met-R-O, which can be reversed back to Met by the methionine sulfoxide reductases, MsrA and MsrB, respectively. Previous studies on Msr have mainly focused on its role in aging and oxidative stress (Laugier et al. 2010, Chatelain et al. 2013, Lee et al. 2014). However, much evidence suggests that Msr may play a critical role in regulating protein function by modifying the sulfoxidation of proteins, thereby controlling various biological processes (Bigelow and Squier 2011, Gennaris et al. 2015). To date, research on the repair of oxidized proteins by Msr and Msr-mediated physiological functions has mainly focused on animals and microorganisms. Only a few potential Msr substrates and Msr-mediated physiological processes have been investigated in higher plants. Banana is one of the most economically important fruits. The fruit undergoes rapid ripening and senescence once harvested, which results in a short shelf life. Studies on banana fruit ripening and senescence have generally focused on biochemical changes and transcriptional regulation (Elitzur et al. 2016, Gao et al. 2016, Han et al. 2016, Kuang et al. 2017, Yuan et al. 2017). Other mechanisms underlying regulation of ripening and senescence of banana fruit are still largely unknown. Calmodulin (CaM), a calcium-binding protein in all eukaryotic cells, plays a key role in growth, development, stress response and defense (Reddy et al. 2011, Abbas et al. 2014, Zhu et al. 2015, Zhu et al. 2017). CaM acts as an inhibitor or activator to regulate its target proteins. It has been shown that CaM genes from tomato are differentially expressed during fruit development and ripening, and transiently overexpressing SlCaM2 in mature green fruit delays ripening (Yang et al. 2014). CaM is highly susceptible to oxidation by ROS because of its high Met content (Snijder et al. 2011, Lubker et al. 2015). Oxidation of Met in CaM might influence its interactions with target proteins (Slaughter et al. 2007, Boschek et al. 2008). However, little information is available on the physiological involvement of redox modification of Met in CaM by Msr. In the present study, we validated Musa acuminata CaM (MaCaM1) as a specific substrate of the methionine sulfoxide reductase MaMsrA7; the sulfoxidation status of MaCaM1 was regulated by MsMsrA7. Expression of MaCaM1 and MaMsrA7 was up-regulated with increased ripening and senescence. Mimicked sulfoxidation in MaCaM1 did not affect its binding to two CaM-binding proteins, MaHY5-1 and MaCAT1, but reduced the transcriptional repression activity of MaHY5-1 and the catalytic activity of MaCAT1. These results expand our knowledge of the involvement of post-translational modification in the regulation of harvested banana fruit ripening and senescence. Results Ripening and senescence, redox status and expression profiles of MaMsrA7 and MaCaM1 genes Peel turning yellow is one of the most important characteristics of banana fruit after harvest. As Fig. 1A shows, the hue angle of control fruit decreased rapidly after 17 d of storage, indicating the degradation of Chl and the initiation of ripening and senescence. Relative electrolyte leakage is an index of cellular membrane integrity and fruit senescence. Consistent with the change in hue angle, the relative electrolyte leakage of control fruit greatly increased after 17 d of storage. Protein carbonyl is usually used to evaluate oxidative stress in organisms. A rapid increase of protein carbonyl content in control fruit preceded the loss of cell membrane integrity and the accumulation of hydrogen peroxide. Ethylene plays an important role in initiating the ripening and senescence of climacteric fruit and leaves. As shown in Fig. 1, ethylene accelerated the accumulation of hydrogen peroxide and protein carbonyl, ripening and senescence of harvested banana fruit. These results imply that protein oxidation caused by ROS might be involved in the regulation of ripening and senescence of harvested banana fruit. Fig. 1 View largeDownload slide Ripening and senescence parameters, and redox status of harvested banana fruit treated without (control) or with ethylene at 25°C. (A) Hue angle; (B) membrane permeability; (C) protein carbonyl content; (D) H2O2 content. Each value represents the mean ± SE of three biological replicates. Fig. 1 View largeDownload slide Ripening and senescence parameters, and redox status of harvested banana fruit treated without (control) or with ethylene at 25°C. (A) Hue angle; (B) membrane permeability; (C) protein carbonyl content; (D) H2O2 content. Each value represents the mean ± SE of three biological replicates. Based on RNA sequencing (RNA-Seq) data from four ripening stages of banana fruit in a transcriptome database, one MsrA and one CaM gene, designated MaMsrA7 and MaCaM1, were identified in Musa acuminata (data not shown). The banana genomes possess five predicted MsrA genes, which was similar to those of Arabidopsis and rice (Rouhier et al. 2006, Tarrago et al. 2009). Multiple alignment of MaMsrA7 protein with other plant Msr proteins revealed that high similarity was found among MaMsrA7 and OsMsrA4.1 (Supplementary Figs. S1, S2). The transcript levels of MaMsrA7 and MaCaM1 during fruit senescence were investigated by quantitative real-time PCR (qRT-PCR). It was observed that the transcript levels of both genes significantly increased in peel and pulp tissues of harvested banana fruit upon natural (control) and ethylene-induced ripening and senescence. Moreover, the expression of MaCaM1 was up-regulated before the up-regulation of MaMsrA7 during ripening and senescence of harvested fruit (Fig. 2). We speculated that redox modification of Met in CaM by Msr might be implicated in the regulation of banana fruit ripening and senescence. Fig. 2 View largeDownload slide Expression of MaMsrA7 and MaCaM1 genes in harvested banana fruit treated without (control) or with ethylene at 25°C. (A) Peel with ethylene-induced senescence; (B) peel with natural senescence; (C) pulp with ethylene-induced senescence; (D) pulp with natural senescence. The expression levels are expressed as a ratio relative to that at the harvest time (day 0 for control fruit), which was set as 1. Each value represents the mean ± SE of three biological replicates. Fig. 2 View largeDownload slide Expression of MaMsrA7 and MaCaM1 genes in harvested banana fruit treated without (control) or with ethylene at 25°C. (A) Peel with ethylene-induced senescence; (B) peel with natural senescence; (C) pulp with ethylene-induced senescence; (D) pulp with natural senescence. The expression levels are expressed as a ratio relative to that at the harvest time (day 0 for control fruit), which was set as 1. Each value represents the mean ± SE of three biological replicates. MaMsrA7 physically interacts with MaCaM1 in vitro and in vivo To test this hypothesis, we first investigated whether MaCaM1 is a direct substrate of MaMsrA7. Two methods, yeast two-hybrid (Y2H) assay (in vitro) and biomolecular fluorescence complementation (BiFC) assay (in vivo), were applied. Yeast cells co-transformed with DNA-binding domain (DBD)–MaCaM1 and activation domain (AD)–MaMsrA7 grew well on minimal synthetic defined quadruple drop-out (QDO) medium (Fig. 3A), suggesting a specific interaction between MaMsrA7 and MaCaM1. BiFC analysis confirmed the interaction between MaCaM1 and MaMsrA7 in Arabidopsis mesophyll protoplasts (Fig. 3B). Fig. 3 View largeDownload slide Interaction between MaMsrA7 and MaCaM1 in vitro and in vivo. (A) Interaction between MaMsrA7 and MaCaM1 in yeast two-hybrid (Y2H) assay. AD, activation domain; DBD, DNA-binding domain; DDO, double drop-out medium; QDO, quadruple drop-out medium. (B) The interaction between MaMsrA7 and MaCaM1 in living cells indicated by bimolecular fluorescence complementation in Arabidopsis mesophyll protoplasts. A yellow signal indicates YFP fluorescence; a red signal indicates Chl autofluorescence; the merged images represent a digital combination of the Chl autofluorescence and YFP fluorescent images. YFP fluorescence was excited at 488 nm, and Chl autofluorescence was excited at 543 nm. Scale bar = 10 µm. (C) Oxidation of Met in calmodulin (CaM) by H2O2 leads to a mobility shift of the oxidized protein (CaMox) on SDS–PAGE: compare lane 3 with lane 2. Incubation of CaMox with different concentrations of MsrA7 and a reducing system involving dithionite (lanes 4, 2 µM MaMsrA7 and lanes 5, 4 µM MaMsrA7). (D) Methionine sulfoxide reductase A (MaMsrA7) activity determined using MaCaM1 as substrate. (E) MsrA7 can reduce MetO in CaMox. The oxidation state of peptides containing Met77, Met110 or Met125 was determined by LC-MS/MS. Each bar represents the mean ± SE of three biological replicates. Fig. 3 View largeDownload slide Interaction between MaMsrA7 and MaCaM1 in vitro and in vivo. (A) Interaction between MaMsrA7 and MaCaM1 in yeast two-hybrid (Y2H) assay. AD, activation domain; DBD, DNA-binding domain; DDO, double drop-out medium; QDO, quadruple drop-out medium. (B) The interaction between MaMsrA7 and MaCaM1 in living cells indicated by bimolecular fluorescence complementation in Arabidopsis mesophyll protoplasts. A yellow signal indicates YFP fluorescence; a red signal indicates Chl autofluorescence; the merged images represent a digital combination of the Chl autofluorescence and YFP fluorescent images. YFP fluorescence was excited at 488 nm, and Chl autofluorescence was excited at 543 nm. Scale bar = 10 µm. (C) Oxidation of Met in calmodulin (CaM) by H2O2 leads to a mobility shift of the oxidized protein (CaMox) on SDS–PAGE: compare lane 3 with lane 2. Incubation of CaMox with different concentrations of MsrA7 and a reducing system involving dithionite (lanes 4, 2 µM MaMsrA7 and lanes 5, 4 µM MaMsrA7). (D) Methionine sulfoxide reductase A (MaMsrA7) activity determined using MaCaM1 as substrate. (E) MsrA7 can reduce MetO in CaMox. The oxidation state of peptides containing Met77, Met110 or Met125 was determined by LC-MS/MS. Each bar represents the mean ± SE of three biological replicates. MaMsrA7 regulates the redox status of MaCaM1 and reduces oxidized MaCaM1 We prepared recombinant MaCaM1 and MaMsrA7 proteins from Escherichia coli as described in the Materials and Methods. Purified MaCaM1 was subjected to oxidation by H2O2. After oxidation, the band representing MaCaM1 in SDS–PAGE was clearly shifted to a higher molecular weight. The oxidized MaCaM1 was partially reduced by MsrA7 (Fig. 3C). Moreover, oxidized recombinant MaCaM1 was assayed as Msr substrate; the activity was measured as the initial rate of NADPH oxidation in the presence of MaMsrA7 and the Trx reducing system. Almost no activity was detected when using non-oxidized MaCaM1 (Fig. 3D); however, incubation of oxidized MaCaM1 resulted in a specific activity of 2838 nM oxidized NADPH min−1 mg−1 MSRA7. In addition, oxidized MaCaM1 and MaMsrA7-repaired oxidized MaCaM1 were trypsin digested and the resulting peptides were analyzed by liquid chromatography–tandem mass spectrometry (LC-MS/MS). Two peptides, containing Met77 and Met110, respectively, showed significant decreases in the amount of oxidized Met when subjected to repair by MaMsrA7. However, MaMsrA7 had no significant effect on oxidized Met125 (Fig. 3E). Together, these results confirmed that oxidized MaCaM1 was a direct substrate of MaMsrA7. MaCaM1 physically interacts with MaCAT1 or MaHY5-1 in vitro and in vivo To better understand the role of MaCaM1 in regulating ripening and senescence of banana fruit, we performed Y2H screening using MaCaM1 as bait to identify interacting proteins from a banana cDNA expression library. Two cDNAs from positive colonies, corresponding to MaCAT1 and MaHY5-1, were selected for further study. Y2H analysis verified the interactions between MaCaM1 and MaCAT1, and MaCaM1 and MaHY5-1 (Fig. 4A). BiFC analysis confirmed the interactions in Arabidopsis mesophyll protoplasts (Fig. 4B). Subcellular localization analysis showed that MaCaM1, MaCAT1 and MaMsrA7 were all mainly located in the nucleus and cytosol, while MaHY5-1 localized exclusively to the nucleus (Supplementary Fig. S3). In addition, the transcript levels of MaHY5-1 tended to decrease as senescence proceeded, whereas the level of MaCAT1 significantly increased prior to the ripening of harvested banana fruit (Supplementary Fig. S4). Together, these results indicated that MaCAT1 and MaHY5-1 might be implicated in the regulation of banana fruit ripening and senescence. Fig. 4 View largeDownload slide The interaction between MaCaM1 or MaCaM1(M) and MaCAT1 or MaHY5-1 in vitro and in vivo. MaCaM1(M) is a mutated form of MaCaM1 in which all the Met residues except the initiator were mutated to glutamine. (A) The interaction between MaCaM1 or MaCaM1(M) and MaCAT1 or MaHY5-1 indicated by Y2H assay. AD, activation domain; DBD, DNA-binding domain; DDO, double drop-out medium; QDO, quadruple drop-out medium. (B) The interaction between MaCaM1 or MaCaM1(M) and MaCAT1 or MaHY5-1 in bimolecular fluorescence complementation assay. Fig. 4 View largeDownload slide The interaction between MaCaM1 or MaCaM1(M) and MaCAT1 or MaHY5-1 in vitro and in vivo. MaCaM1(M) is a mutated form of MaCaM1 in which all the Met residues except the initiator were mutated to glutamine. (A) The interaction between MaCaM1 or MaCaM1(M) and MaCAT1 or MaHY5-1 indicated by Y2H assay. AD, activation domain; DBD, DNA-binding domain; DDO, double drop-out medium; QDO, quadruple drop-out medium. (B) The interaction between MaCaM1 or MaCaM1(M) and MaCAT1 or MaHY5-1 in bimolecular fluorescence complementation assay. Mimicked sulfoxidation in MaCaM1 does not affect its interactions with MaCAT1 or MaHY5-1 We speculated that redox modification of MaCaM1 might influence its interactions with MaCAT1 and MaHY5-1. To test this hypothesis, we first mimicked Met oxidation by mutating all the Met residues to glutamine in MaCaM1 (Supplementary Fig. S5) except for the initiator Met, and then performed Y2H analysis using the MaCaM1 mutant [MaCaM1(M)] and MaCAT1 or MaHY5-1. MaCaM1(M) interacted with MaCAT1 and MaHY5-1 (Fig. 4A). We also mimicked the effect of Met oxidation in MaCaM1 and tested the interaction between MaCaM1 and MaCAT1 or MaHY5-1 in BiFC assay. The visible yellow fluorescence in the cytoplasm and nucleus, respectively, indicated interactions of MaCaM1(M) with MaCAT1 and MaHY5-1 (Fig. 4B;Supplementary Fig. S6). Collectively, these results demonstrated that Met oxidation in MaCaM1 did not affect its physical interaction with MaCAT1 or MaHY5-1. Mimicked sulfoxidation in MaCaM1 affects the catalase activity of MaCAT1 CaM plays a role in numerous intracellular responses by regulating the function of CaM-binding proteins (CBPs). As mentioned above, MaCAT1 is a CBP of MaCaM1. Sulfoxidation modification of MaCaM1 might influence the catalase activity of MaCAT1. To test this hypothesis, MaCaM1 and the mutant MaCaM1(M) were each co-expressed with MaCAT1 in Arabidopsis mesophyll protoplasts and the catalase activity was analyzed. Compared with MaCAT1 alone, co-expression of MaCAT1 with MaCaM1 significantly increased the catalase activity, while obvious decreased CAT activity was observed when MaCaM1(M) was co-expressed with MaCAT1. Furthermore, a significantly increased catalase activity was also observed by expressing MaCaM1 alone (i.e. without MaCAT1), which might be due to the activation of endogenous CAT enzyme activity by MaCaM1 (Fig. 5). These results indicate that mimicked sulfoxidation of MaCaM1 decreased the catalase activity of MaCAT1. Fig. 5 View largeDownload slide Methionine oxidation in MaCaM1 affects the catalytic activity of MaCAT1. The combination of Myc-CAT1 and GFP–MaCaM1/MaCaM1(M) constructs was co-transformed into Arabidopsis mesophyll protoplasts as described before (Yoo et al., 2007). After incubation overnight at 22°C, the protoplasts were collected to analyze catalase activity. Repression or activation effects of MaCaM1 or MaCaM1(M) on the catalytic activity of MaCAT1 were determined relative to activity of the control (empty vector; 80 U mg–1 protein set as 100%). Each value represents the mean ± SE of three biological replicates. Fig. 5 View largeDownload slide Methionine oxidation in MaCaM1 affects the catalytic activity of MaCAT1. The combination of Myc-CAT1 and GFP–MaCaM1/MaCaM1(M) constructs was co-transformed into Arabidopsis mesophyll protoplasts as described before (Yoo et al., 2007). After incubation overnight at 22°C, the protoplasts were collected to analyze catalase activity. Repression or activation effects of MaCaM1 or MaCaM1(M) on the catalytic activity of MaCAT1 were determined relative to activity of the control (empty vector; 80 U mg–1 protein set as 100%). Each value represents the mean ± SE of three biological replicates. Mimicked sulfoxidation in MaCaM1 affects the transcriptional regulation of PHYTOENE SYNTHASE genes ELONGATED HYPOCOTYL5 (HY5), a member of the bZIP transcription factor family, plays multiple roles in plant growth and development. HY5 regulates the expression of the carotenoid biosynthetic PHYTOENE SYNTHASE (PSY) genes. We cloned the promoters of MaPsy1, MaPsy2 and MaPsy3 from banana fruit (Supplementary Fig. S7). Electrophoretic mobility shift assays (EMSAs) showed that MaHY5-1 bound to the promoters of carotenoid biosynthesis-related genes MaPsy1, MaPsy2 and MaPsy3 (Fig. 6A–C), indicating that MaHY5-1 might be involved in transcriptional regulation of these genes. We also performed a transient dual-luciferase assay in tobacco leaves to examine whether Met oxidation in MaCaM1 affects the expression of MaPsy1, MaPsy2 and MaPsy3. Compared with the control, co-expression of MaHY5-1 with the MaPSY1, MaPSY2 or MaPSY3 promoter significantly decreased the LUC/REN ratio (Fig. 6D–F;Supplementary Fig. S8), suggesting that MaHY5-1 transrepressed these carotenoid biosynthesis-related genes. In addition, MaCaM1 transrepressed the MaPsy2 promoter, but had no effect on the MaPSY1 and MaPSY3 promoters. Mimicked sulfoxidation in MaCaM1 significantly increased the LUC/REN ratio, suggesting that MaCaM1(M) transactivated MaPsy1, MaPsy2 and MaPsy3. Furthermore, the transrepression activity of MaHY5-1 toward MaPsy1, MaPsy2 and MaPsy3 was decreased when MaCaM1(M) was co-expressed, which might be an aggregate effect of MaHY5-1 and MaCaM1(M) (Fig. 6D–F;Supplementary Fig. S8). These data indicated that sulfoxidation in MaCaM1 decreases the transrepression activity of MaHY5-1 toward carotenoid biosynthesis-related genes. Fig. 6 View largeDownload slide Methionine oxidation in MaCaM1 affects the transcriptional regulatory activity of MaHY5-1. Electrophoretic mobility shift assay showed that MaHY5-1 bound to the biotin-labeled probe present in the MaPsy1 (A), MaPsy2 (B) and MaPsy3 (C) promoters. Purified recombinant His-MaHY5-1 protein was mixed with biotin-labeled probes, and the DNA–protein complexes were separated in 6% native polyacrylamide gels. ++ and +++ indicate increasing amounts of unlabeled probes for competition. Dual-luciferase reporter assay shows that methionine oxidation in MaCaM1 affects the transcriptional regulatory activity of MaHY5-1. Repression or activation activity of MaHY5-1, MaCaM1 or MaCaM1(M) toward the MaPsy1 (D), MaPsy2 (E) and MaPsy3 (F) promoter was shown by the ratio of LUC to REN. The ratio of LUC to REN of the empty vector plus promoter vector was used as a reference (set as 1). Each bar and error bar represents the mean ± SE of six biological replicates. Fig. 6 View largeDownload slide Methionine oxidation in MaCaM1 affects the transcriptional regulatory activity of MaHY5-1. Electrophoretic mobility shift assay showed that MaHY5-1 bound to the biotin-labeled probe present in the MaPsy1 (A), MaPsy2 (B) and MaPsy3 (C) promoters. Purified recombinant His-MaHY5-1 protein was mixed with biotin-labeled probes, and the DNA–protein complexes were separated in 6% native polyacrylamide gels. ++ and +++ indicate increasing amounts of unlabeled probes for competition. Dual-luciferase reporter assay shows that methionine oxidation in MaCaM1 affects the transcriptional regulatory activity of MaHY5-1. Repression or activation activity of MaHY5-1, MaCaM1 or MaCaM1(M) toward the MaPsy1 (D), MaPsy2 (E) and MaPsy3 (F) promoter was shown by the ratio of LUC to REN. The ratio of LUC to REN of the empty vector plus promoter vector was used as a reference (set as 1). Each bar and error bar represents the mean ± SE of six biological replicates. Discussion Redox regulation is a process wherein free radicals, via reversible modification of functional protein cysteine and Met residues, act as molecular switches to regulate protein functions (Drazic et al. 2013, Gennaris et al. 2015, Song et al. 2016, Jiang et al. 2017, Kneeshaw et al. 2017, Yin et al. 2017). In recent years, redox regulation has emerged as an important mechanism of signaling and regulation in plant development (Drazic et al. 2013, Li et al. 2016, Viola et al. 2016, Kneeshaw et al. 2017). However, the participation of redox mechanisms in regulation of fruit ripening and senescence is still largely unknown. Msr catalyzes the reduction of MetO in proteins back to Met, which represents an important antioxidant defense mechanism. There is growing evidence that Msr plays an important role in protecting cells against oxidative damage. In animals, Msr expression and Msr activity tend to decrease during aging in different organs (Petropoulos et al. 2001, Picot et al. 2004, Novoselov et al. 2010). Enhanced or inhibited expression of Msr by genetic modification increases or decreases life span or resistance to oxidative stress (Moskovitz et al. 2001, Ruan et al., 2002, Koc et al. 2004, Minniti et al. 2009). Limited information is available on Msrs in relation to plant senescence. Chatelain et al. (2013) found a positive correlation between Msr capacity and longevity in Arabidopsis thaliana seeds. Our previous research showed that expression of Msr genes is down-regulated in harvested litchi fruit with increased senescence (Jiang et al. 2017). In the present study, ROS accumulation and protein oxidation increased during banana fruit ripening and senescence (Fig. 1), and the expression of the gene MsrA7 was up-regulated (Fig. 2). Similarly, up-regulated expression of Trx genes, the genes encoding another class of oxidized protein repair-related proteins, was also found during fruit ripening and senescence (Hartman et al. 2014, Wang et al. 2014, Wang et al. 2017) or when plants were subjected to stress conditions (Kneeshaw et al. 2014, Li et al. 2016, Wu et al. 2016, H. Zhang et al. 2018). These results suggest that the oxidation of protein Met residues, i.e. sulfoxidation, occurs on a large scale. The increased expression of MsrA7 was possibly beneficial for reducing the sulfoxides. Msrs mainly reverse sulfoxide back to Met in proteins. Previous studies on Msr functions mainly focused on repair of oxidized Met residues in proteins and the involvement of Msr in the resistance of cells, tissues and organisms to oxidative stress both in vitro and in vivo (Gustavsson et al. 2002, Khor et al. 2004, Laugier et al. 2010, Tarrago et al. 2012, Chatelain et al. 2013, Jacques et al. 2015). However, growing evidence suggests that Msrs might play a critical role in regulating protein functions by post-translational modification of proteins susceptible to sulfoxidation, i.e. Msrs reversibly regulate the redox status of Met residues in proteins, thereby controlling biological processes. Previous work reported that hypochlorous acid (HOCl) treatment led to Met oxidation and inactivation of a chaperone protein in E. coli, and the activity could be recovered by MsrA/B (Khor et al. 2004). A reversible redox modification of Met155 in Mge1, a co-chaperone of mitochondrial Hsp70, mediated by Msrs in Saccharomyces cerevisiae is associated with the activity of Mge1 in vitro and in vivo (Allu et al. 2015). Recently, inactivation of a primary periplasmic chaperone SurA by Met oxidation was shown in E. coli, while the activity was restored by MsrPQ-based reduction (Gennaris et al. 2015). Erickson et al. (2008) reported that CaMKII (Ca2+/calmodulin-dependent protein kinase II), a regulator of calcium flux, could be activated by the oxidation of two consecutive Met residues in mice, while this autoactivation could be reversed by MsrA; this effect was related to ameliorating a cardiotoxic effect of aldosterone on myocardial infarction and subsequent cardiac rupture. Similarly, it was reported that Met sulfoxidation of HypT, a hypochlorite-responsive transcription factor, leads to its activation, but the transcriptional activity is inactivated through reduction by MsrA/B in E. coli (Drazic et al. 2013). Therefore, Msr enzymes play an important role in controlling biological processes as a regulator or switch of protein function. In the present study, we verified MaCaM1 as a substrate of MaMsrA7 in vitro and in vivo (Fig. 3). Moreover, oxidized MaCaM1 could be repaired by MaMsrA7 (Fig. 3C). Similarly, CaM1 has been reported to be a specific substrate of MsrA1/B1 in litchi fruit (Jiang et al. 2017). It appears that sulfoxidation regulation of CaM1 by Msr is conserved in plants. Furthermore, we also found that MaMsrA7 had specificity toward particular oxidized Met residues in MaCaM1 (Fig. 3E). CaM is a multifunctional intermediate calcium-binding messenger protein in all eukaryotic cells, the primary Ca2+ sensor and part of calcium signal transduction pathways. CaM plays a crucial role in plant growth, development, stress response and defense by inhibiting or activating the functions of CBPs. However, there is a paucity of information on the involvement of CaM in ripening and senescence of harvested fruit. A previous study suggested that tomato CaMs could participate in ethylene-co-ordinated rapid ripening after the ethylene burst (Yang et al. 2014). In the present study, the expression levels of MaCaM1 were rapidly up-regulated during the ripening and senescence of harvested banana fruit (Fig. 2), implying that MaCaM1 is involved in the regulation of senescence of the fruit. CBPs are abundant in plant cells, and include membrane proteins, metabolic enzymes, kinases/phosphatases, RNA-binding proteins, transcription factors and vacuolar cation proton antiporters, which are involved in ion homeostasis, metabolism, hormone biosynthesis, phosphorylation and gene expression. Studies have shown that the redox status of Met residues in CaM could influence the function of its targeted CBPs. Slaughter et al. (2007) reported that the oxidation of Met144 and Met145 residues in CaM disrupts its interaction with CaMKII in E. coli. The function of a CaM-interacting partner, adenylate cyclase, was found to be dependent on the redox status of specific Met residues within CaM (Vougier et al. 2004). Our previous study also showed that mimicked sulfoxidation in Litchi chinensis CaM1 (LcCaM1) did not affect its physical interactions with two LcCaM1-binding senescence-related transcription factors, LcNAC13 and LcWRKY1, but it enhanced their DNA binding activities (Jiang et al. 2017). In the present study, one antioxidant enzyme, MaCAT1, and one plant-specific transcription factor, MaHY5-1, were identified as MaCaM1-binding proteins. Catalases are key regulators of ROS homeostasis in plant cells, and play an important role in plant growth, development and response to stress (Mhamdi et al. 2012). The regulation of catalase activity is not well understood. Recent studies have revealed that the H2O2 detoxification capacity of catalase is boosted by Nucleoredoxin 1 (NRX1), NO CATALASE ACTIVITY1 (NCA1), LESION SIMULATING DISEASE1 (LSD1), glycolate oxidase (GLO) and CaM, thereby protecting the plant cell from oxidative stress (Yang and Poovaiah 2002, Li et al. 2013, Li et al. 2015, Zhang et al. 2016, Kneeshaw et al. 2017). In the present work, we found that CaM binds to MaCAT1 and enhances its activity, which was similar to the findings of a previous report (Yang and Poovaiah 2002). In addition, we found that mimicked sulfoxidation in MaCaM1 did not affect MaCaM1 binding to MaCAT1 (Fig. 5), but it did reduce the catalytic activity of MaCAT1. Based on these results, it is proposed that Ca2+/CaM plays a critical role in controlling H2O2 homeostasis during banana fruit ripening and senescence. HY5, a member of the bZIP transcription factor family, is implicated in different processes such as pigment accumulation, abiotic stress, ROS and light signaling pathways (Catala et al. 2011, Gangappa and Botto 2016, Wang et al. 2016, Nawkar et al. 2017), acting as both a transcriptional activator and repressor (Zhang et al. 2011). The transcriptional regulatory activity of HY5 is achieved by physical interaction with signaling intermediates, including both regulatory proteins and other transcription factors, to form enhanceosome or repressosome complexes (Abbas et al. 2014, Nguyen et al. 2015, Gangappa and Botto 2016, Gangappa and Kumar 2017, X. Zhang et al. 2017). Apart from interactions with CBPs to regulate its functions, CaM can also interact with DNA and serve as a transcription factor. AtCaM7 has been reported to interact directly with the AtHY5 promoter and act as a transcription factor (Kushwaha et al. 2008, Abbas et al. 2014). In addition, the crystal structure of AtCaM7, and molecular docking simulations of AtCaM7 with DNA containing a Z-box, suggest that Arg127 determines its DNA binding ability (Kumar et al. 2016). In addition to Arg127, Met125 and Ile126 are expected to contribute to the binding to the Z-box. Moreover, Ca2+-defective mutants of AtCaM7 lost DNA binding activity, which might be due to the removal of Ca2+ from CaM, disrupting its structural integrity (Kushwaha et al. 2008, Abbas et al. 2014). In this work, we found that MaHY5-1 acts as a transcriptional repressor of carotenoid biosynthesis-related genes (MaPSY1, MaPSY2 and MaPSY3). Y2H and BiFC assays demonstrated that MaCaM1 and MaHY5-1 physically interact with each other. One plausible mechanism of the interaction between MaCaM1 and MaHY5-1 is that MaCaM1 acts as a modulator of MaHY5-1-mediated regulation of carotenoid biosynthesis-related gene expression and thereby regulates banana fruit ripening and senescence. Transient dual-luciferase assays showed that MaCaM1 could enhance the transcriptional repression activity of MaHY5-1 on the MaPSY2 gene. Furthermore, mimicked sulfoxidation in MaCaM1 did not affect its physical interaction with MaHY5-1, but reduced the transcriptional repression activity of MaHY5-1 toward carotenoid biosynthesis-related genes, which might be a combined effect of MaHY5-1 and MaCaM1(M) (Fig. 6D–F). Collectively, this study demonstrates that sulfoxidation modifications of CaM by Msrs indirectly regulate the catalytic activity of MaCAT1 and the transcriptional activity of MaHY5-1, thereby influencing the antioxidant response and ripening and senescence processes of harvested banana fruit. We describe a possible mechanism by which Msrs are indirectly implicated in the regulation of climacteric fruit ripening and senescence (Fig. 7). Oxidized MaCaM1 is a substrate of MaMsrA7. ROS accumulation in harvested banana fruit results in increased protein oxidation. Consequently, the accelerated oxidation of MaCaM1 reduces the transcriptional repression activity of MaHY5-1 and the catalytic activity of MaCAT1, thereby regulating antioxidant and carotenoid biosynthesis-related responses of harvested banana fruit. Fig. 7 View largeDownload slide Proposed model of the involvement of MaMsrA7-mediated redox modifications of methionine in MaCaM1 in regulating the ripening and senescence of harvested banana fruit. Oxidized MaCaM1 is a substrate of MaMsrA7. Reactive oxygen species (ROS) accumulation in harvested banana fruit results in increased protein oxidation. Consequently, the accelerated oxidation of MaCaM1 reduces the transcriptional repression activity of MaHY5-1 and the catalytic activity of MaCAT1, thereby regulating antioxidant and carotenoid biosynthesis-related responses of harvested banana fruit. Fig. 7 View largeDownload slide Proposed model of the involvement of MaMsrA7-mediated redox modifications of methionine in MaCaM1 in regulating the ripening and senescence of harvested banana fruit. Oxidized MaCaM1 is a substrate of MaMsrA7. Reactive oxygen species (ROS) accumulation in harvested banana fruit results in increased protein oxidation. Consequently, the accelerated oxidation of MaCaM1 reduces the transcriptional repression activity of MaHY5-1 and the catalytic activity of MaCAT1, thereby regulating antioxidant and carotenoid biosynthesis-related responses of harvested banana fruit. Materials and Methods Plant materials and treatments Banana (Musa acuminata L. AAA group, cv. Brazilian) fruit at approximately 80% maturity was harvested from a local commercial orchard in Guangzhou, China. Fruit with uniform weight, shape and maturity, and free from visual defects were selected, dipped in 0.05% Sportak® (Prochloraz, Bayer) solution for 3 min, and then air-dried. The dried fruit were randomly divided into two groups. One group was fumigated with 500 p.p.m. C2H4 in a sealed box for 12 h. The other group in the same volume was sealed in the box for 12 h as a control. After treatments, the fruit were packed into 0.015 mm thick polyethylene bags (three fruits per bag), and stored at 22°C and 85–90% relative humidity. During storage, the senescence parameters were evaluated and peel tissues were collected, frozen in liquid nitrogen and stored at −80°C for further analysis. Fruit senescence parameters Fruit color was measured using a Chroma meter (Konica Minolta, CR-400) according to the method as described by McGuire (1992). Membrane permeability was expressed as relative electrolyte leakage. Thirty discs (10 mm in diameter) from the equatorial region of 30 fruit peels were washed three times in deionized water, dried with filter paper and then incubated in 20 ml of 0.3 mol l−1 mannitol solution for 30 min at 25°C. Initial electrolyte leakage rate was determined using a conductivity meter (model DDS-11A; Shanghai Scientific Instruments). Total electrolyte leakage was then determined after boiling for 20 min and cooling rapidly to 25°C. The relative leakage was expressed as the percentage of the initial electrolytes of the total electrolytes. Total protein was extracted from banana peel using a phenol extraction protocol. The protein concentration was determined using a Bio-Rad Protein Assay Kit. The protein carbonyl content was spectrophotometrically quantified using a carbonyl-specific reagent, 2,4-dinitrophenylhydrazine. The H2O2 content was visualized using a Hydrogen Peroxide Assay Kit (Nanjing Jiancheng Biochemical Reagent Co.) in accordance with the manufacturer’s instructions. RNA extraction, gene isolation and sequence analyses Total RNA was extracted from banana fruit using the hot borate method (Wan and Wilkins 1994) and cleaned with DNase I (TAKARA BIO INC.). DNA-free RNA was used as the template for reverse transcription–PCR. The first-strand cDNA was used for PCR amplification. MaCaM1, MaCAT1, MaMsrA7 and MaHY5-1 were isolated from a transcriptome database obtained using a SolexaHiSeq™ 2000 sequencing system. The gene-specific primers used for gene cloning are listed in Supplementary Table S1. The PCR products were subcloned into a pMD20-T vector (TAKARA), and then transformed into E. coli. DH5a (TAKARA) in accordance with the manufacturer’s protocol. The sequences were verified by further cloning and resequencing. Sequence alignments were carried out using ClustalX (version 1.83). Quantitative real-time PCR analysis DNA-free RNA was reverse-transcribed for first-strand cDNA synthesis. The gene-specific oligonucleotide primers were used for qRT-PCR analysis (Supplementary Table S1). The qRT-PCRs were carried out in the ABI 7500 Real-Time PCR System (Applied Biosystems) with SYBR Green Real-Time PCR Master Mix (TOYOBO Co., Ltd.) in accordance with the manufacturer’s instructions under the following conditions: 30 s at 95°C, 40 cycles of 5 s at 95°C and 34 s at 58°C. MaRPS2 was selected as the reference gene (Chen et al. 2011). qRT-PCRs were normalized using the Ct value corresponding to that of the reference gene. The relative expression levels of target genes were calculated using the formula 2−ΔΔCT. Three independent biological replicates were used in the analysis. Site-directed mutagenesis of Met residues to glutamine To mimic Met sulfoxidation, all the Met residues in MaCaM1, expect for the initiator Met residue, were mutated to glutamine by site-directed mutagenesis using a QuikChange site-directed mutagenesis kit (Stratagene). The full-length mutated MaCaM1 was subcloned into the pET-28a vector (Novagen). The mutations were verified by DNA sequencing. Purification of recombinant proteins The cDNA fragments encoding the mature proteins of MaCaM1, MaMsrA7, MaHY5-1 and MaCaM1(M) were inserted into the pET-28a vector (Novagen) to construct vectors for expressing the recombinant protein. The His fusion proteins were induced and expressed in the E. coli BL21 (DE3) strain. The recombinant proteins were purified with nickel–nitrilotriacetic acid agarose (Qiagen), following the manufacturer’s instructions. Electrophoretic mobility shift assay (EMSA) An EMSA was performed using the EMSA kit (Thermo) in accordance with the manufacturer’s instructions. Oligonucleotide probes from the potential targets were labeled using the Pierce™ DNA 3' End Biotinylation Kit (Thermo Fisher Scientific). The unlabeled DNA fragment was used as a competitor. Protein–DNA complexes were separated in 6% native polyacrylamide gels. The DNA fragments were transferred from the gel to a nitrocellulose membrane. After cross-linking, the biotin-labeled probes were detected by the chemiluminescence method according to the manufacturer’s protocol on a ChemiDoc MP Imaging System (Bio-Rad). Y2H assay Y2H assays were performed using the Matchmaker Gold Yeast Two-Hybrid System (Clontech). The coding sequences of MaCaM1, MaCAT1, MaMsrA7, MaHY5-1 and MaCaM1(M) were subcloned into the pGBKT7 or pGADT7 vector to fuse with the DBD or AD, respectively, to create bait and prey constructs. These constructs then were co-transformed into yeast strain gold Y2H by the lithium acetate method, and grown on minimal synthetic defined double dropout (DDO; −Leu/−Trp) medium according to the manufacturer’s protocol (Clontech) for 3 d. Transformed colonies were plated onto QDO medium to test the possible interactions in terms of their growth status. The ability of yeast cells to grow on QDO medium was scored as a positive interaction. BiFC assay Coding sequences of MaCaM1, MaCAT1, MaMsrA7, MaHY5-1 and MaCaM1(M) without stop codons were subcloned into pUC-pSPYNE or pUC-pSPYCE vectors. The resulting constructs were used for transient assays through a polyethylene glycol transfection of Arabidopsis mesophyll protoplasts, as described earlier (Yoo et al. 2007). The transformed protoplasts were then incubated at 22°C for 24–48 h. Yellow fluorescent protein (YFP) fluorescence was observed using a fluorescence microscope (Zeiss 510 Meta). Oxidation and reduction of MaCaM1 CaM was oxidized using H2O2 (50 mM) for 3 h at 37°C in 20 mM Tris–HCl (pH 7.5, 1 mM diethylenetriaminepentaacetic acid). H2O2 was removed by gel filtration through a NAP-5 Sephadex G-25 column (GE Healthcare). In vitro repair of oxidized MaCaM1 (MaCaM1ox) was performed by incubating oxidized proteins (2 µM MaCaM1ox) with purified MaMsrA7 (2 or 4 µM), 10 mM benzyl viologen and 50 mM sodium dithionite at 37°C for 1 h. The reaction was stopped by adding trifluoroacetic acid. The CaM samples were collected and subjected to SDS–PAGE. Protein bands corresponding to different redox status were in-gel digested with trypsin (Promega). The resulting peptides were analyzed by LC-MS/MS on a C18 reverse-phase column. Relative abundances of every Met-containing peptide with different redox status were obtained by integration of peak area intensities, taking into account the extracted ion chromatogram of both double- and triple-charged ions. Measurement of MaMsr activity Msr activity was measured according to Tarrago et al (2012). The assay was performed by incubating 20 µM protein (MaCaM1 or MaCaM1ox) with purified MaMsrA7 (5 µM), following NADPH oxidation at 340 nm in the presence of a Trx reducing system. For the Trx system, recombinant Dimocarpus longan Trx1 (DlTrx1, a Trxh protein) and NTR1 (DlNTR1) were used for reducing MsrA7. The reactions were carried out at 25°C in 250 µl of 30 mM Tris–HCl (pH 8.0) and the kinetics were recorded using a Smartspec™ plus spectrophotometer (Bio-Rad). Measurement of catalase activity The coding sequences of MaCAT1 with a Myc tag and MaCaM1/MaCaM1(M) with a green fluorescent protein (GFP) tag were cloned into the pBA002 vector and pCAMBIA1302 vector under control of the 35S promoter, respectively. The combinations of Myc-CAT1 and GFP–MaCaM1/MaCaM1(M) constructs were co-transformed into Arabidopsis mesophyll protoplasts as described before (Yoo et al. 2007). After incubation overnight at 22°C, the protoplasts were collected to analyze catalase activity. The CAT activity was determined with a CAT assay kit according to the manufacturer’s instructions (Beyotime). Dual-luciferase reporter assay The dual-luciferase reporter system was used to analyze the transient reporter expression as described by Hellens et al. (2005). The pGreenII 0800-LUC reporter vector and pGreenII 62-SK effector vector were used. The coding sequences of MaCaM1, MaHY5-1 and MaCaM1(M) were amplified and fused to pGreenII 62-SK as effector plasmids. Genomic DNA was extracted from banana pericarp tissues using the DNeasy Plant Mini Kit (Qiagen). The MaPSY1/2/3 promoters were amplified using a GenomeWalker Kit (Clontech) according to the manufacturer’s instructions and inserted into pGreenII 0800-LUC as reporter plasmids. The constructed effector and reporter plasmids were co-transformed into tobacco leaves by Agrobacterium tumefaciens strain GV3101. The activities of LUC and REN luciferase were measured using the Dual-Luciferase® Reporter Assay kit (Promega) 3 d after co-transformation. The analysis was carried out on a Luminoskan Ascent Microplate Luminometer (Thermo Fisher Scientific) in accordance with the manufacturer’s instructions. The ratio of LUC to REN was calculated to reflect the final transcriptional activity. At least six biological replicates were assayed for each combination. Data handling Data were expressed as the mean ± SE. Differences among different treatments were compared using SPSS version 7.5 (SPSS, Inc.). Supplementary Data Supplementary data are available at PCP online. Funding This work was supported by the National Natural Science Foundation of China [31772041, 31501545 and 31322044]; the National Basic Research Program of China [2013CB127104 and 2013CB127102]; Guangdong Natural Science Foundation [2014A030310294]; China Postdoctoral Science Foundation [2014M560681]; Science and Technology Planning Project of Guangdong Province [2015B090901058]; Science and Technology Planning Project of Guangzhou [201604020048]; and Talent Program of Guangdong Province [2014TX01N049]. Disclosures The authors have no conflicts of interest to declare. References Abbas N. , Maurya J.P. , Senapati D. , Gangappa S.N. , Chattopadhyay S. 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Google Scholar CrossRef Search ADS PubMed Abbreviations Abbreviations AD activation domain BiFC bimolecular fluorescence complementation CaM calmodulin CaMKII Ca2+/calmodulin-dependent protein kinase II DBD DNA-binding domain DDO double drop-out EMSA electrophoretic mobility shift assay GFP green fluorescent protein LC-MS/MS liquid chromatography–tandem mass spectrometry Met methionine MetO methionine sulfoxide Msr methionine sulfoxide reductase QDO quadruple drop-out qRT-PCR quantitative real-time PCR ROS reactive oxygen species Trx thioredoxin YFP yellow fluorescent protein Y2H yeast two-hybrid © The Author(s) 2018. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices) http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Plant and Cell Physiology Oxford University Press

Sulfoxidation Regulation of Musa acuminata Calmodulin (MaCaM) Influences the Functions of MaCaM-Binding Proteins

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Oxford University Press
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© The Author(s) 2018. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com
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0032-0781
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1471-9053
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10.1093/pcp/pcy057
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Abstract

Abstract Sulfoxidation of methionine in proteins by reactive oxygen species can cause conformational alteration or functional impairment, and can be reversed by methionine sulfoxide reductase (Msr). Currently, only a few potential Msr substrates have been confirmed in higher plants. Here, we investigated Msr-mediated sulfoxidation regulation of calmodulin (CaM) and its underlying biological significance in relation to banana fruit ripening and senescence. Expression of MaCaM1 and MaMsrA7 was up-regulated with increased ripening and senescence. We verified that MaCaM1 interacts with MaMsrA7 in vitro and in vivo, and sulfoxidated MaCaM1 could be partly repaired by MaMsrA7 (MaMsrA7 reduces oxidized residues Met77 and Met110 in MaCaM1). Furthermore, we investigated two known CaM-binding proteins, catalase (MaCAT1) and MaHY5-1. MaHY5-1 acts as a transcriptional repressor of carotenoid biosynthesis-related genes (MaPSY1, MaPSY2 and MaPSY3) in banana fruit. MaCaM1 could enhance the catalytic activity of MaCAT1 and the transcriptional repression activity of MaHY5-1 toward MaPSY2. Mimicked sulfoxidation in MaCaM1 did not affect the physical interactions of the protein with MaHY5-1 and MaCAT1, but reduced the catalytic activity of MaCAT1 and the transcriptional repression activity of MaHY5-1. Our data suggest that sulfoxidation modification in MaCaM1 by MaMsrA7 regulates antioxidant response and gene transcription, thereby being involved in regulation of ripening and senescence of banana fruit. Introduction Fruits, the seed-bearing structures of flowering plants, are important economic crops and provide humans with nutrition. Ripening and senescence are crucial for the development and maintenance of fruit quality. A better understanding of fruit ripening and senescence may help in developing strategies to improve nutritional and sensorial qualities of fruit and reduce post-harvest losses. Fruits are classified as climacteric or non-climacteric in terms of their ripening or senescence attributes. Climacteric fruits, such as banana and mango, undergo ripening after harvest. Ethylene plays a key role in the ripening of climacteric fruits (Lin et al. 2009). Non-climacteric fruits, such as citrus and strawberry, undergo no ripening process after harvest. No dramatic increases in respiration are observed, and ethylene biosynthesis remains at very low levels (Alexander and Grierson 2002). Many studies have linked ABA to ripening and senescence of non-climacteric fruits (Ji et al. 2012, Wu et al. 2014). Therefore, recent advances in understanding fruit ripening and senescence have mainly been associated with hormone signal transduction and action. Other mechanisms underlying fruit ripening and senescence are still largely unknown. Reactive oxygen species (ROS) are unavoidably produced as by-products of cellular metabolism, and they function as important signaling molecules that are involved in a broad range of processes (Pitzschke et al. 2006, Mittler 2017). However, under senescence or stress, excessive accumulation of ROS can cause oxidative damage to macromolecules such as DNA, proteins, polysaccharides and lipids, which, in turn, results in loss of structure and function or even cell death (Pitzschke et al. 2006, Moller et al. 2007, Choudhury et al. 2017, Rey et al. 2017). To combat oxidative stress, organisms have evolved several protective mechanisms, such as antioxidant enzyme systems and macromolecule repair systems (Levine et al. 2000). Proteins are the major targets of ROS, which lead to the oxidation of the peptide backbone and amino acid side chains. Elimination of oxidized proteins is crucial for maintaining the integrity of the cell (Moller et al. 2007, Qin et al. 2009). Among the amino acids, cysteine and methionine (Met) are most vulnerable to ROS. Interestingly, oxidation products of cysteine and Met in proteins can be repaired by protein repair systems, thioredoxin (Trx)/thioredoxin reductase and methionine sulfoxide reductase (Msr), respectively. Met oxidation, i.e. sulfoxidation, results in the production of two diastereomers of methionine sulfoxide, Met-S-O and Met-R-O, which can be reversed back to Met by the methionine sulfoxide reductases, MsrA and MsrB, respectively. Previous studies on Msr have mainly focused on its role in aging and oxidative stress (Laugier et al. 2010, Chatelain et al. 2013, Lee et al. 2014). However, much evidence suggests that Msr may play a critical role in regulating protein function by modifying the sulfoxidation of proteins, thereby controlling various biological processes (Bigelow and Squier 2011, Gennaris et al. 2015). To date, research on the repair of oxidized proteins by Msr and Msr-mediated physiological functions has mainly focused on animals and microorganisms. Only a few potential Msr substrates and Msr-mediated physiological processes have been investigated in higher plants. Banana is one of the most economically important fruits. The fruit undergoes rapid ripening and senescence once harvested, which results in a short shelf life. Studies on banana fruit ripening and senescence have generally focused on biochemical changes and transcriptional regulation (Elitzur et al. 2016, Gao et al. 2016, Han et al. 2016, Kuang et al. 2017, Yuan et al. 2017). Other mechanisms underlying regulation of ripening and senescence of banana fruit are still largely unknown. Calmodulin (CaM), a calcium-binding protein in all eukaryotic cells, plays a key role in growth, development, stress response and defense (Reddy et al. 2011, Abbas et al. 2014, Zhu et al. 2015, Zhu et al. 2017). CaM acts as an inhibitor or activator to regulate its target proteins. It has been shown that CaM genes from tomato are differentially expressed during fruit development and ripening, and transiently overexpressing SlCaM2 in mature green fruit delays ripening (Yang et al. 2014). CaM is highly susceptible to oxidation by ROS because of its high Met content (Snijder et al. 2011, Lubker et al. 2015). Oxidation of Met in CaM might influence its interactions with target proteins (Slaughter et al. 2007, Boschek et al. 2008). However, little information is available on the physiological involvement of redox modification of Met in CaM by Msr. In the present study, we validated Musa acuminata CaM (MaCaM1) as a specific substrate of the methionine sulfoxide reductase MaMsrA7; the sulfoxidation status of MaCaM1 was regulated by MsMsrA7. Expression of MaCaM1 and MaMsrA7 was up-regulated with increased ripening and senescence. Mimicked sulfoxidation in MaCaM1 did not affect its binding to two CaM-binding proteins, MaHY5-1 and MaCAT1, but reduced the transcriptional repression activity of MaHY5-1 and the catalytic activity of MaCAT1. These results expand our knowledge of the involvement of post-translational modification in the regulation of harvested banana fruit ripening and senescence. Results Ripening and senescence, redox status and expression profiles of MaMsrA7 and MaCaM1 genes Peel turning yellow is one of the most important characteristics of banana fruit after harvest. As Fig. 1A shows, the hue angle of control fruit decreased rapidly after 17 d of storage, indicating the degradation of Chl and the initiation of ripening and senescence. Relative electrolyte leakage is an index of cellular membrane integrity and fruit senescence. Consistent with the change in hue angle, the relative electrolyte leakage of control fruit greatly increased after 17 d of storage. Protein carbonyl is usually used to evaluate oxidative stress in organisms. A rapid increase of protein carbonyl content in control fruit preceded the loss of cell membrane integrity and the accumulation of hydrogen peroxide. Ethylene plays an important role in initiating the ripening and senescence of climacteric fruit and leaves. As shown in Fig. 1, ethylene accelerated the accumulation of hydrogen peroxide and protein carbonyl, ripening and senescence of harvested banana fruit. These results imply that protein oxidation caused by ROS might be involved in the regulation of ripening and senescence of harvested banana fruit. Fig. 1 View largeDownload slide Ripening and senescence parameters, and redox status of harvested banana fruit treated without (control) or with ethylene at 25°C. (A) Hue angle; (B) membrane permeability; (C) protein carbonyl content; (D) H2O2 content. Each value represents the mean ± SE of three biological replicates. Fig. 1 View largeDownload slide Ripening and senescence parameters, and redox status of harvested banana fruit treated without (control) or with ethylene at 25°C. (A) Hue angle; (B) membrane permeability; (C) protein carbonyl content; (D) H2O2 content. Each value represents the mean ± SE of three biological replicates. Based on RNA sequencing (RNA-Seq) data from four ripening stages of banana fruit in a transcriptome database, one MsrA and one CaM gene, designated MaMsrA7 and MaCaM1, were identified in Musa acuminata (data not shown). The banana genomes possess five predicted MsrA genes, which was similar to those of Arabidopsis and rice (Rouhier et al. 2006, Tarrago et al. 2009). Multiple alignment of MaMsrA7 protein with other plant Msr proteins revealed that high similarity was found among MaMsrA7 and OsMsrA4.1 (Supplementary Figs. S1, S2). The transcript levels of MaMsrA7 and MaCaM1 during fruit senescence were investigated by quantitative real-time PCR (qRT-PCR). It was observed that the transcript levels of both genes significantly increased in peel and pulp tissues of harvested banana fruit upon natural (control) and ethylene-induced ripening and senescence. Moreover, the expression of MaCaM1 was up-regulated before the up-regulation of MaMsrA7 during ripening and senescence of harvested fruit (Fig. 2). We speculated that redox modification of Met in CaM by Msr might be implicated in the regulation of banana fruit ripening and senescence. Fig. 2 View largeDownload slide Expression of MaMsrA7 and MaCaM1 genes in harvested banana fruit treated without (control) or with ethylene at 25°C. (A) Peel with ethylene-induced senescence; (B) peel with natural senescence; (C) pulp with ethylene-induced senescence; (D) pulp with natural senescence. The expression levels are expressed as a ratio relative to that at the harvest time (day 0 for control fruit), which was set as 1. Each value represents the mean ± SE of three biological replicates. Fig. 2 View largeDownload slide Expression of MaMsrA7 and MaCaM1 genes in harvested banana fruit treated without (control) or with ethylene at 25°C. (A) Peel with ethylene-induced senescence; (B) peel with natural senescence; (C) pulp with ethylene-induced senescence; (D) pulp with natural senescence. The expression levels are expressed as a ratio relative to that at the harvest time (day 0 for control fruit), which was set as 1. Each value represents the mean ± SE of three biological replicates. MaMsrA7 physically interacts with MaCaM1 in vitro and in vivo To test this hypothesis, we first investigated whether MaCaM1 is a direct substrate of MaMsrA7. Two methods, yeast two-hybrid (Y2H) assay (in vitro) and biomolecular fluorescence complementation (BiFC) assay (in vivo), were applied. Yeast cells co-transformed with DNA-binding domain (DBD)–MaCaM1 and activation domain (AD)–MaMsrA7 grew well on minimal synthetic defined quadruple drop-out (QDO) medium (Fig. 3A), suggesting a specific interaction between MaMsrA7 and MaCaM1. BiFC analysis confirmed the interaction between MaCaM1 and MaMsrA7 in Arabidopsis mesophyll protoplasts (Fig. 3B). Fig. 3 View largeDownload slide Interaction between MaMsrA7 and MaCaM1 in vitro and in vivo. (A) Interaction between MaMsrA7 and MaCaM1 in yeast two-hybrid (Y2H) assay. AD, activation domain; DBD, DNA-binding domain; DDO, double drop-out medium; QDO, quadruple drop-out medium. (B) The interaction between MaMsrA7 and MaCaM1 in living cells indicated by bimolecular fluorescence complementation in Arabidopsis mesophyll protoplasts. A yellow signal indicates YFP fluorescence; a red signal indicates Chl autofluorescence; the merged images represent a digital combination of the Chl autofluorescence and YFP fluorescent images. YFP fluorescence was excited at 488 nm, and Chl autofluorescence was excited at 543 nm. Scale bar = 10 µm. (C) Oxidation of Met in calmodulin (CaM) by H2O2 leads to a mobility shift of the oxidized protein (CaMox) on SDS–PAGE: compare lane 3 with lane 2. Incubation of CaMox with different concentrations of MsrA7 and a reducing system involving dithionite (lanes 4, 2 µM MaMsrA7 and lanes 5, 4 µM MaMsrA7). (D) Methionine sulfoxide reductase A (MaMsrA7) activity determined using MaCaM1 as substrate. (E) MsrA7 can reduce MetO in CaMox. The oxidation state of peptides containing Met77, Met110 or Met125 was determined by LC-MS/MS. Each bar represents the mean ± SE of three biological replicates. Fig. 3 View largeDownload slide Interaction between MaMsrA7 and MaCaM1 in vitro and in vivo. (A) Interaction between MaMsrA7 and MaCaM1 in yeast two-hybrid (Y2H) assay. AD, activation domain; DBD, DNA-binding domain; DDO, double drop-out medium; QDO, quadruple drop-out medium. (B) The interaction between MaMsrA7 and MaCaM1 in living cells indicated by bimolecular fluorescence complementation in Arabidopsis mesophyll protoplasts. A yellow signal indicates YFP fluorescence; a red signal indicates Chl autofluorescence; the merged images represent a digital combination of the Chl autofluorescence and YFP fluorescent images. YFP fluorescence was excited at 488 nm, and Chl autofluorescence was excited at 543 nm. Scale bar = 10 µm. (C) Oxidation of Met in calmodulin (CaM) by H2O2 leads to a mobility shift of the oxidized protein (CaMox) on SDS–PAGE: compare lane 3 with lane 2. Incubation of CaMox with different concentrations of MsrA7 and a reducing system involving dithionite (lanes 4, 2 µM MaMsrA7 and lanes 5, 4 µM MaMsrA7). (D) Methionine sulfoxide reductase A (MaMsrA7) activity determined using MaCaM1 as substrate. (E) MsrA7 can reduce MetO in CaMox. The oxidation state of peptides containing Met77, Met110 or Met125 was determined by LC-MS/MS. Each bar represents the mean ± SE of three biological replicates. MaMsrA7 regulates the redox status of MaCaM1 and reduces oxidized MaCaM1 We prepared recombinant MaCaM1 and MaMsrA7 proteins from Escherichia coli as described in the Materials and Methods. Purified MaCaM1 was subjected to oxidation by H2O2. After oxidation, the band representing MaCaM1 in SDS–PAGE was clearly shifted to a higher molecular weight. The oxidized MaCaM1 was partially reduced by MsrA7 (Fig. 3C). Moreover, oxidized recombinant MaCaM1 was assayed as Msr substrate; the activity was measured as the initial rate of NADPH oxidation in the presence of MaMsrA7 and the Trx reducing system. Almost no activity was detected when using non-oxidized MaCaM1 (Fig. 3D); however, incubation of oxidized MaCaM1 resulted in a specific activity of 2838 nM oxidized NADPH min−1 mg−1 MSRA7. In addition, oxidized MaCaM1 and MaMsrA7-repaired oxidized MaCaM1 were trypsin digested and the resulting peptides were analyzed by liquid chromatography–tandem mass spectrometry (LC-MS/MS). Two peptides, containing Met77 and Met110, respectively, showed significant decreases in the amount of oxidized Met when subjected to repair by MaMsrA7. However, MaMsrA7 had no significant effect on oxidized Met125 (Fig. 3E). Together, these results confirmed that oxidized MaCaM1 was a direct substrate of MaMsrA7. MaCaM1 physically interacts with MaCAT1 or MaHY5-1 in vitro and in vivo To better understand the role of MaCaM1 in regulating ripening and senescence of banana fruit, we performed Y2H screening using MaCaM1 as bait to identify interacting proteins from a banana cDNA expression library. Two cDNAs from positive colonies, corresponding to MaCAT1 and MaHY5-1, were selected for further study. Y2H analysis verified the interactions between MaCaM1 and MaCAT1, and MaCaM1 and MaHY5-1 (Fig. 4A). BiFC analysis confirmed the interactions in Arabidopsis mesophyll protoplasts (Fig. 4B). Subcellular localization analysis showed that MaCaM1, MaCAT1 and MaMsrA7 were all mainly located in the nucleus and cytosol, while MaHY5-1 localized exclusively to the nucleus (Supplementary Fig. S3). In addition, the transcript levels of MaHY5-1 tended to decrease as senescence proceeded, whereas the level of MaCAT1 significantly increased prior to the ripening of harvested banana fruit (Supplementary Fig. S4). Together, these results indicated that MaCAT1 and MaHY5-1 might be implicated in the regulation of banana fruit ripening and senescence. Fig. 4 View largeDownload slide The interaction between MaCaM1 or MaCaM1(M) and MaCAT1 or MaHY5-1 in vitro and in vivo. MaCaM1(M) is a mutated form of MaCaM1 in which all the Met residues except the initiator were mutated to glutamine. (A) The interaction between MaCaM1 or MaCaM1(M) and MaCAT1 or MaHY5-1 indicated by Y2H assay. AD, activation domain; DBD, DNA-binding domain; DDO, double drop-out medium; QDO, quadruple drop-out medium. (B) The interaction between MaCaM1 or MaCaM1(M) and MaCAT1 or MaHY5-1 in bimolecular fluorescence complementation assay. Fig. 4 View largeDownload slide The interaction between MaCaM1 or MaCaM1(M) and MaCAT1 or MaHY5-1 in vitro and in vivo. MaCaM1(M) is a mutated form of MaCaM1 in which all the Met residues except the initiator were mutated to glutamine. (A) The interaction between MaCaM1 or MaCaM1(M) and MaCAT1 or MaHY5-1 indicated by Y2H assay. AD, activation domain; DBD, DNA-binding domain; DDO, double drop-out medium; QDO, quadruple drop-out medium. (B) The interaction between MaCaM1 or MaCaM1(M) and MaCAT1 or MaHY5-1 in bimolecular fluorescence complementation assay. Mimicked sulfoxidation in MaCaM1 does not affect its interactions with MaCAT1 or MaHY5-1 We speculated that redox modification of MaCaM1 might influence its interactions with MaCAT1 and MaHY5-1. To test this hypothesis, we first mimicked Met oxidation by mutating all the Met residues to glutamine in MaCaM1 (Supplementary Fig. S5) except for the initiator Met, and then performed Y2H analysis using the MaCaM1 mutant [MaCaM1(M)] and MaCAT1 or MaHY5-1. MaCaM1(M) interacted with MaCAT1 and MaHY5-1 (Fig. 4A). We also mimicked the effect of Met oxidation in MaCaM1 and tested the interaction between MaCaM1 and MaCAT1 or MaHY5-1 in BiFC assay. The visible yellow fluorescence in the cytoplasm and nucleus, respectively, indicated interactions of MaCaM1(M) with MaCAT1 and MaHY5-1 (Fig. 4B;Supplementary Fig. S6). Collectively, these results demonstrated that Met oxidation in MaCaM1 did not affect its physical interaction with MaCAT1 or MaHY5-1. Mimicked sulfoxidation in MaCaM1 affects the catalase activity of MaCAT1 CaM plays a role in numerous intracellular responses by regulating the function of CaM-binding proteins (CBPs). As mentioned above, MaCAT1 is a CBP of MaCaM1. Sulfoxidation modification of MaCaM1 might influence the catalase activity of MaCAT1. To test this hypothesis, MaCaM1 and the mutant MaCaM1(M) were each co-expressed with MaCAT1 in Arabidopsis mesophyll protoplasts and the catalase activity was analyzed. Compared with MaCAT1 alone, co-expression of MaCAT1 with MaCaM1 significantly increased the catalase activity, while obvious decreased CAT activity was observed when MaCaM1(M) was co-expressed with MaCAT1. Furthermore, a significantly increased catalase activity was also observed by expressing MaCaM1 alone (i.e. without MaCAT1), which might be due to the activation of endogenous CAT enzyme activity by MaCaM1 (Fig. 5). These results indicate that mimicked sulfoxidation of MaCaM1 decreased the catalase activity of MaCAT1. Fig. 5 View largeDownload slide Methionine oxidation in MaCaM1 affects the catalytic activity of MaCAT1. The combination of Myc-CAT1 and GFP–MaCaM1/MaCaM1(M) constructs was co-transformed into Arabidopsis mesophyll protoplasts as described before (Yoo et al., 2007). After incubation overnight at 22°C, the protoplasts were collected to analyze catalase activity. Repression or activation effects of MaCaM1 or MaCaM1(M) on the catalytic activity of MaCAT1 were determined relative to activity of the control (empty vector; 80 U mg–1 protein set as 100%). Each value represents the mean ± SE of three biological replicates. Fig. 5 View largeDownload slide Methionine oxidation in MaCaM1 affects the catalytic activity of MaCAT1. The combination of Myc-CAT1 and GFP–MaCaM1/MaCaM1(M) constructs was co-transformed into Arabidopsis mesophyll protoplasts as described before (Yoo et al., 2007). After incubation overnight at 22°C, the protoplasts were collected to analyze catalase activity. Repression or activation effects of MaCaM1 or MaCaM1(M) on the catalytic activity of MaCAT1 were determined relative to activity of the control (empty vector; 80 U mg–1 protein set as 100%). Each value represents the mean ± SE of three biological replicates. Mimicked sulfoxidation in MaCaM1 affects the transcriptional regulation of PHYTOENE SYNTHASE genes ELONGATED HYPOCOTYL5 (HY5), a member of the bZIP transcription factor family, plays multiple roles in plant growth and development. HY5 regulates the expression of the carotenoid biosynthetic PHYTOENE SYNTHASE (PSY) genes. We cloned the promoters of MaPsy1, MaPsy2 and MaPsy3 from banana fruit (Supplementary Fig. S7). Electrophoretic mobility shift assays (EMSAs) showed that MaHY5-1 bound to the promoters of carotenoid biosynthesis-related genes MaPsy1, MaPsy2 and MaPsy3 (Fig. 6A–C), indicating that MaHY5-1 might be involved in transcriptional regulation of these genes. We also performed a transient dual-luciferase assay in tobacco leaves to examine whether Met oxidation in MaCaM1 affects the expression of MaPsy1, MaPsy2 and MaPsy3. Compared with the control, co-expression of MaHY5-1 with the MaPSY1, MaPSY2 or MaPSY3 promoter significantly decreased the LUC/REN ratio (Fig. 6D–F;Supplementary Fig. S8), suggesting that MaHY5-1 transrepressed these carotenoid biosynthesis-related genes. In addition, MaCaM1 transrepressed the MaPsy2 promoter, but had no effect on the MaPSY1 and MaPSY3 promoters. Mimicked sulfoxidation in MaCaM1 significantly increased the LUC/REN ratio, suggesting that MaCaM1(M) transactivated MaPsy1, MaPsy2 and MaPsy3. Furthermore, the transrepression activity of MaHY5-1 toward MaPsy1, MaPsy2 and MaPsy3 was decreased when MaCaM1(M) was co-expressed, which might be an aggregate effect of MaHY5-1 and MaCaM1(M) (Fig. 6D–F;Supplementary Fig. S8). These data indicated that sulfoxidation in MaCaM1 decreases the transrepression activity of MaHY5-1 toward carotenoid biosynthesis-related genes. Fig. 6 View largeDownload slide Methionine oxidation in MaCaM1 affects the transcriptional regulatory activity of MaHY5-1. Electrophoretic mobility shift assay showed that MaHY5-1 bound to the biotin-labeled probe present in the MaPsy1 (A), MaPsy2 (B) and MaPsy3 (C) promoters. Purified recombinant His-MaHY5-1 protein was mixed with biotin-labeled probes, and the DNA–protein complexes were separated in 6% native polyacrylamide gels. ++ and +++ indicate increasing amounts of unlabeled probes for competition. Dual-luciferase reporter assay shows that methionine oxidation in MaCaM1 affects the transcriptional regulatory activity of MaHY5-1. Repression or activation activity of MaHY5-1, MaCaM1 or MaCaM1(M) toward the MaPsy1 (D), MaPsy2 (E) and MaPsy3 (F) promoter was shown by the ratio of LUC to REN. The ratio of LUC to REN of the empty vector plus promoter vector was used as a reference (set as 1). Each bar and error bar represents the mean ± SE of six biological replicates. Fig. 6 View largeDownload slide Methionine oxidation in MaCaM1 affects the transcriptional regulatory activity of MaHY5-1. Electrophoretic mobility shift assay showed that MaHY5-1 bound to the biotin-labeled probe present in the MaPsy1 (A), MaPsy2 (B) and MaPsy3 (C) promoters. Purified recombinant His-MaHY5-1 protein was mixed with biotin-labeled probes, and the DNA–protein complexes were separated in 6% native polyacrylamide gels. ++ and +++ indicate increasing amounts of unlabeled probes for competition. Dual-luciferase reporter assay shows that methionine oxidation in MaCaM1 affects the transcriptional regulatory activity of MaHY5-1. Repression or activation activity of MaHY5-1, MaCaM1 or MaCaM1(M) toward the MaPsy1 (D), MaPsy2 (E) and MaPsy3 (F) promoter was shown by the ratio of LUC to REN. The ratio of LUC to REN of the empty vector plus promoter vector was used as a reference (set as 1). Each bar and error bar represents the mean ± SE of six biological replicates. Discussion Redox regulation is a process wherein free radicals, via reversible modification of functional protein cysteine and Met residues, act as molecular switches to regulate protein functions (Drazic et al. 2013, Gennaris et al. 2015, Song et al. 2016, Jiang et al. 2017, Kneeshaw et al. 2017, Yin et al. 2017). In recent years, redox regulation has emerged as an important mechanism of signaling and regulation in plant development (Drazic et al. 2013, Li et al. 2016, Viola et al. 2016, Kneeshaw et al. 2017). However, the participation of redox mechanisms in regulation of fruit ripening and senescence is still largely unknown. Msr catalyzes the reduction of MetO in proteins back to Met, which represents an important antioxidant defense mechanism. There is growing evidence that Msr plays an important role in protecting cells against oxidative damage. In animals, Msr expression and Msr activity tend to decrease during aging in different organs (Petropoulos et al. 2001, Picot et al. 2004, Novoselov et al. 2010). Enhanced or inhibited expression of Msr by genetic modification increases or decreases life span or resistance to oxidative stress (Moskovitz et al. 2001, Ruan et al., 2002, Koc et al. 2004, Minniti et al. 2009). Limited information is available on Msrs in relation to plant senescence. Chatelain et al. (2013) found a positive correlation between Msr capacity and longevity in Arabidopsis thaliana seeds. Our previous research showed that expression of Msr genes is down-regulated in harvested litchi fruit with increased senescence (Jiang et al. 2017). In the present study, ROS accumulation and protein oxidation increased during banana fruit ripening and senescence (Fig. 1), and the expression of the gene MsrA7 was up-regulated (Fig. 2). Similarly, up-regulated expression of Trx genes, the genes encoding another class of oxidized protein repair-related proteins, was also found during fruit ripening and senescence (Hartman et al. 2014, Wang et al. 2014, Wang et al. 2017) or when plants were subjected to stress conditions (Kneeshaw et al. 2014, Li et al. 2016, Wu et al. 2016, H. Zhang et al. 2018). These results suggest that the oxidation of protein Met residues, i.e. sulfoxidation, occurs on a large scale. The increased expression of MsrA7 was possibly beneficial for reducing the sulfoxides. Msrs mainly reverse sulfoxide back to Met in proteins. Previous studies on Msr functions mainly focused on repair of oxidized Met residues in proteins and the involvement of Msr in the resistance of cells, tissues and organisms to oxidative stress both in vitro and in vivo (Gustavsson et al. 2002, Khor et al. 2004, Laugier et al. 2010, Tarrago et al. 2012, Chatelain et al. 2013, Jacques et al. 2015). However, growing evidence suggests that Msrs might play a critical role in regulating protein functions by post-translational modification of proteins susceptible to sulfoxidation, i.e. Msrs reversibly regulate the redox status of Met residues in proteins, thereby controlling biological processes. Previous work reported that hypochlorous acid (HOCl) treatment led to Met oxidation and inactivation of a chaperone protein in E. coli, and the activity could be recovered by MsrA/B (Khor et al. 2004). A reversible redox modification of Met155 in Mge1, a co-chaperone of mitochondrial Hsp70, mediated by Msrs in Saccharomyces cerevisiae is associated with the activity of Mge1 in vitro and in vivo (Allu et al. 2015). Recently, inactivation of a primary periplasmic chaperone SurA by Met oxidation was shown in E. coli, while the activity was restored by MsrPQ-based reduction (Gennaris et al. 2015). Erickson et al. (2008) reported that CaMKII (Ca2+/calmodulin-dependent protein kinase II), a regulator of calcium flux, could be activated by the oxidation of two consecutive Met residues in mice, while this autoactivation could be reversed by MsrA; this effect was related to ameliorating a cardiotoxic effect of aldosterone on myocardial infarction and subsequent cardiac rupture. Similarly, it was reported that Met sulfoxidation of HypT, a hypochlorite-responsive transcription factor, leads to its activation, but the transcriptional activity is inactivated through reduction by MsrA/B in E. coli (Drazic et al. 2013). Therefore, Msr enzymes play an important role in controlling biological processes as a regulator or switch of protein function. In the present study, we verified MaCaM1 as a substrate of MaMsrA7 in vitro and in vivo (Fig. 3). Moreover, oxidized MaCaM1 could be repaired by MaMsrA7 (Fig. 3C). Similarly, CaM1 has been reported to be a specific substrate of MsrA1/B1 in litchi fruit (Jiang et al. 2017). It appears that sulfoxidation regulation of CaM1 by Msr is conserved in plants. Furthermore, we also found that MaMsrA7 had specificity toward particular oxidized Met residues in MaCaM1 (Fig. 3E). CaM is a multifunctional intermediate calcium-binding messenger protein in all eukaryotic cells, the primary Ca2+ sensor and part of calcium signal transduction pathways. CaM plays a crucial role in plant growth, development, stress response and defense by inhibiting or activating the functions of CBPs. However, there is a paucity of information on the involvement of CaM in ripening and senescence of harvested fruit. A previous study suggested that tomato CaMs could participate in ethylene-co-ordinated rapid ripening after the ethylene burst (Yang et al. 2014). In the present study, the expression levels of MaCaM1 were rapidly up-regulated during the ripening and senescence of harvested banana fruit (Fig. 2), implying that MaCaM1 is involved in the regulation of senescence of the fruit. CBPs are abundant in plant cells, and include membrane proteins, metabolic enzymes, kinases/phosphatases, RNA-binding proteins, transcription factors and vacuolar cation proton antiporters, which are involved in ion homeostasis, metabolism, hormone biosynthesis, phosphorylation and gene expression. Studies have shown that the redox status of Met residues in CaM could influence the function of its targeted CBPs. Slaughter et al. (2007) reported that the oxidation of Met144 and Met145 residues in CaM disrupts its interaction with CaMKII in E. coli. The function of a CaM-interacting partner, adenylate cyclase, was found to be dependent on the redox status of specific Met residues within CaM (Vougier et al. 2004). Our previous study also showed that mimicked sulfoxidation in Litchi chinensis CaM1 (LcCaM1) did not affect its physical interactions with two LcCaM1-binding senescence-related transcription factors, LcNAC13 and LcWRKY1, but it enhanced their DNA binding activities (Jiang et al. 2017). In the present study, one antioxidant enzyme, MaCAT1, and one plant-specific transcription factor, MaHY5-1, were identified as MaCaM1-binding proteins. Catalases are key regulators of ROS homeostasis in plant cells, and play an important role in plant growth, development and response to stress (Mhamdi et al. 2012). The regulation of catalase activity is not well understood. Recent studies have revealed that the H2O2 detoxification capacity of catalase is boosted by Nucleoredoxin 1 (NRX1), NO CATALASE ACTIVITY1 (NCA1), LESION SIMULATING DISEASE1 (LSD1), glycolate oxidase (GLO) and CaM, thereby protecting the plant cell from oxidative stress (Yang and Poovaiah 2002, Li et al. 2013, Li et al. 2015, Zhang et al. 2016, Kneeshaw et al. 2017). In the present work, we found that CaM binds to MaCAT1 and enhances its activity, which was similar to the findings of a previous report (Yang and Poovaiah 2002). In addition, we found that mimicked sulfoxidation in MaCaM1 did not affect MaCaM1 binding to MaCAT1 (Fig. 5), but it did reduce the catalytic activity of MaCAT1. Based on these results, it is proposed that Ca2+/CaM plays a critical role in controlling H2O2 homeostasis during banana fruit ripening and senescence. HY5, a member of the bZIP transcription factor family, is implicated in different processes such as pigment accumulation, abiotic stress, ROS and light signaling pathways (Catala et al. 2011, Gangappa and Botto 2016, Wang et al. 2016, Nawkar et al. 2017), acting as both a transcriptional activator and repressor (Zhang et al. 2011). The transcriptional regulatory activity of HY5 is achieved by physical interaction with signaling intermediates, including both regulatory proteins and other transcription factors, to form enhanceosome or repressosome complexes (Abbas et al. 2014, Nguyen et al. 2015, Gangappa and Botto 2016, Gangappa and Kumar 2017, X. Zhang et al. 2017). Apart from interactions with CBPs to regulate its functions, CaM can also interact with DNA and serve as a transcription factor. AtCaM7 has been reported to interact directly with the AtHY5 promoter and act as a transcription factor (Kushwaha et al. 2008, Abbas et al. 2014). In addition, the crystal structure of AtCaM7, and molecular docking simulations of AtCaM7 with DNA containing a Z-box, suggest that Arg127 determines its DNA binding ability (Kumar et al. 2016). In addition to Arg127, Met125 and Ile126 are expected to contribute to the binding to the Z-box. Moreover, Ca2+-defective mutants of AtCaM7 lost DNA binding activity, which might be due to the removal of Ca2+ from CaM, disrupting its structural integrity (Kushwaha et al. 2008, Abbas et al. 2014). In this work, we found that MaHY5-1 acts as a transcriptional repressor of carotenoid biosynthesis-related genes (MaPSY1, MaPSY2 and MaPSY3). Y2H and BiFC assays demonstrated that MaCaM1 and MaHY5-1 physically interact with each other. One plausible mechanism of the interaction between MaCaM1 and MaHY5-1 is that MaCaM1 acts as a modulator of MaHY5-1-mediated regulation of carotenoid biosynthesis-related gene expression and thereby regulates banana fruit ripening and senescence. Transient dual-luciferase assays showed that MaCaM1 could enhance the transcriptional repression activity of MaHY5-1 on the MaPSY2 gene. Furthermore, mimicked sulfoxidation in MaCaM1 did not affect its physical interaction with MaHY5-1, but reduced the transcriptional repression activity of MaHY5-1 toward carotenoid biosynthesis-related genes, which might be a combined effect of MaHY5-1 and MaCaM1(M) (Fig. 6D–F). Collectively, this study demonstrates that sulfoxidation modifications of CaM by Msrs indirectly regulate the catalytic activity of MaCAT1 and the transcriptional activity of MaHY5-1, thereby influencing the antioxidant response and ripening and senescence processes of harvested banana fruit. We describe a possible mechanism by which Msrs are indirectly implicated in the regulation of climacteric fruit ripening and senescence (Fig. 7). Oxidized MaCaM1 is a substrate of MaMsrA7. ROS accumulation in harvested banana fruit results in increased protein oxidation. Consequently, the accelerated oxidation of MaCaM1 reduces the transcriptional repression activity of MaHY5-1 and the catalytic activity of MaCAT1, thereby regulating antioxidant and carotenoid biosynthesis-related responses of harvested banana fruit. Fig. 7 View largeDownload slide Proposed model of the involvement of MaMsrA7-mediated redox modifications of methionine in MaCaM1 in regulating the ripening and senescence of harvested banana fruit. Oxidized MaCaM1 is a substrate of MaMsrA7. Reactive oxygen species (ROS) accumulation in harvested banana fruit results in increased protein oxidation. Consequently, the accelerated oxidation of MaCaM1 reduces the transcriptional repression activity of MaHY5-1 and the catalytic activity of MaCAT1, thereby regulating antioxidant and carotenoid biosynthesis-related responses of harvested banana fruit. Fig. 7 View largeDownload slide Proposed model of the involvement of MaMsrA7-mediated redox modifications of methionine in MaCaM1 in regulating the ripening and senescence of harvested banana fruit. Oxidized MaCaM1 is a substrate of MaMsrA7. Reactive oxygen species (ROS) accumulation in harvested banana fruit results in increased protein oxidation. Consequently, the accelerated oxidation of MaCaM1 reduces the transcriptional repression activity of MaHY5-1 and the catalytic activity of MaCAT1, thereby regulating antioxidant and carotenoid biosynthesis-related responses of harvested banana fruit. Materials and Methods Plant materials and treatments Banana (Musa acuminata L. AAA group, cv. Brazilian) fruit at approximately 80% maturity was harvested from a local commercial orchard in Guangzhou, China. Fruit with uniform weight, shape and maturity, and free from visual defects were selected, dipped in 0.05% Sportak® (Prochloraz, Bayer) solution for 3 min, and then air-dried. The dried fruit were randomly divided into two groups. One group was fumigated with 500 p.p.m. C2H4 in a sealed box for 12 h. The other group in the same volume was sealed in the box for 12 h as a control. After treatments, the fruit were packed into 0.015 mm thick polyethylene bags (three fruits per bag), and stored at 22°C and 85–90% relative humidity. During storage, the senescence parameters were evaluated and peel tissues were collected, frozen in liquid nitrogen and stored at −80°C for further analysis. Fruit senescence parameters Fruit color was measured using a Chroma meter (Konica Minolta, CR-400) according to the method as described by McGuire (1992). Membrane permeability was expressed as relative electrolyte leakage. Thirty discs (10 mm in diameter) from the equatorial region of 30 fruit peels were washed three times in deionized water, dried with filter paper and then incubated in 20 ml of 0.3 mol l−1 mannitol solution for 30 min at 25°C. Initial electrolyte leakage rate was determined using a conductivity meter (model DDS-11A; Shanghai Scientific Instruments). Total electrolyte leakage was then determined after boiling for 20 min and cooling rapidly to 25°C. The relative leakage was expressed as the percentage of the initial electrolytes of the total electrolytes. Total protein was extracted from banana peel using a phenol extraction protocol. The protein concentration was determined using a Bio-Rad Protein Assay Kit. The protein carbonyl content was spectrophotometrically quantified using a carbonyl-specific reagent, 2,4-dinitrophenylhydrazine. The H2O2 content was visualized using a Hydrogen Peroxide Assay Kit (Nanjing Jiancheng Biochemical Reagent Co.) in accordance with the manufacturer’s instructions. RNA extraction, gene isolation and sequence analyses Total RNA was extracted from banana fruit using the hot borate method (Wan and Wilkins 1994) and cleaned with DNase I (TAKARA BIO INC.). DNA-free RNA was used as the template for reverse transcription–PCR. The first-strand cDNA was used for PCR amplification. MaCaM1, MaCAT1, MaMsrA7 and MaHY5-1 were isolated from a transcriptome database obtained using a SolexaHiSeq™ 2000 sequencing system. The gene-specific primers used for gene cloning are listed in Supplementary Table S1. The PCR products were subcloned into a pMD20-T vector (TAKARA), and then transformed into E. coli. DH5a (TAKARA) in accordance with the manufacturer’s protocol. The sequences were verified by further cloning and resequencing. Sequence alignments were carried out using ClustalX (version 1.83). Quantitative real-time PCR analysis DNA-free RNA was reverse-transcribed for first-strand cDNA synthesis. The gene-specific oligonucleotide primers were used for qRT-PCR analysis (Supplementary Table S1). The qRT-PCRs were carried out in the ABI 7500 Real-Time PCR System (Applied Biosystems) with SYBR Green Real-Time PCR Master Mix (TOYOBO Co., Ltd.) in accordance with the manufacturer’s instructions under the following conditions: 30 s at 95°C, 40 cycles of 5 s at 95°C and 34 s at 58°C. MaRPS2 was selected as the reference gene (Chen et al. 2011). qRT-PCRs were normalized using the Ct value corresponding to that of the reference gene. The relative expression levels of target genes were calculated using the formula 2−ΔΔCT. Three independent biological replicates were used in the analysis. Site-directed mutagenesis of Met residues to glutamine To mimic Met sulfoxidation, all the Met residues in MaCaM1, expect for the initiator Met residue, were mutated to glutamine by site-directed mutagenesis using a QuikChange site-directed mutagenesis kit (Stratagene). The full-length mutated MaCaM1 was subcloned into the pET-28a vector (Novagen). The mutations were verified by DNA sequencing. Purification of recombinant proteins The cDNA fragments encoding the mature proteins of MaCaM1, MaMsrA7, MaHY5-1 and MaCaM1(M) were inserted into the pET-28a vector (Novagen) to construct vectors for expressing the recombinant protein. The His fusion proteins were induced and expressed in the E. coli BL21 (DE3) strain. The recombinant proteins were purified with nickel–nitrilotriacetic acid agarose (Qiagen), following the manufacturer’s instructions. Electrophoretic mobility shift assay (EMSA) An EMSA was performed using the EMSA kit (Thermo) in accordance with the manufacturer’s instructions. Oligonucleotide probes from the potential targets were labeled using the Pierce™ DNA 3' End Biotinylation Kit (Thermo Fisher Scientific). The unlabeled DNA fragment was used as a competitor. Protein–DNA complexes were separated in 6% native polyacrylamide gels. The DNA fragments were transferred from the gel to a nitrocellulose membrane. After cross-linking, the biotin-labeled probes were detected by the chemiluminescence method according to the manufacturer’s protocol on a ChemiDoc MP Imaging System (Bio-Rad). Y2H assay Y2H assays were performed using the Matchmaker Gold Yeast Two-Hybrid System (Clontech). The coding sequences of MaCaM1, MaCAT1, MaMsrA7, MaHY5-1 and MaCaM1(M) were subcloned into the pGBKT7 or pGADT7 vector to fuse with the DBD or AD, respectively, to create bait and prey constructs. These constructs then were co-transformed into yeast strain gold Y2H by the lithium acetate method, and grown on minimal synthetic defined double dropout (DDO; −Leu/−Trp) medium according to the manufacturer’s protocol (Clontech) for 3 d. Transformed colonies were plated onto QDO medium to test the possible interactions in terms of their growth status. The ability of yeast cells to grow on QDO medium was scored as a positive interaction. BiFC assay Coding sequences of MaCaM1, MaCAT1, MaMsrA7, MaHY5-1 and MaCaM1(M) without stop codons were subcloned into pUC-pSPYNE or pUC-pSPYCE vectors. The resulting constructs were used for transient assays through a polyethylene glycol transfection of Arabidopsis mesophyll protoplasts, as described earlier (Yoo et al. 2007). The transformed protoplasts were then incubated at 22°C for 24–48 h. Yellow fluorescent protein (YFP) fluorescence was observed using a fluorescence microscope (Zeiss 510 Meta). Oxidation and reduction of MaCaM1 CaM was oxidized using H2O2 (50 mM) for 3 h at 37°C in 20 mM Tris–HCl (pH 7.5, 1 mM diethylenetriaminepentaacetic acid). H2O2 was removed by gel filtration through a NAP-5 Sephadex G-25 column (GE Healthcare). In vitro repair of oxidized MaCaM1 (MaCaM1ox) was performed by incubating oxidized proteins (2 µM MaCaM1ox) with purified MaMsrA7 (2 or 4 µM), 10 mM benzyl viologen and 50 mM sodium dithionite at 37°C for 1 h. The reaction was stopped by adding trifluoroacetic acid. The CaM samples were collected and subjected to SDS–PAGE. Protein bands corresponding to different redox status were in-gel digested with trypsin (Promega). The resulting peptides were analyzed by LC-MS/MS on a C18 reverse-phase column. Relative abundances of every Met-containing peptide with different redox status were obtained by integration of peak area intensities, taking into account the extracted ion chromatogram of both double- and triple-charged ions. Measurement of MaMsr activity Msr activity was measured according to Tarrago et al (2012). The assay was performed by incubating 20 µM protein (MaCaM1 or MaCaM1ox) with purified MaMsrA7 (5 µM), following NADPH oxidation at 340 nm in the presence of a Trx reducing system. For the Trx system, recombinant Dimocarpus longan Trx1 (DlTrx1, a Trxh protein) and NTR1 (DlNTR1) were used for reducing MsrA7. The reactions were carried out at 25°C in 250 µl of 30 mM Tris–HCl (pH 8.0) and the kinetics were recorded using a Smartspec™ plus spectrophotometer (Bio-Rad). Measurement of catalase activity The coding sequences of MaCAT1 with a Myc tag and MaCaM1/MaCaM1(M) with a green fluorescent protein (GFP) tag were cloned into the pBA002 vector and pCAMBIA1302 vector under control of the 35S promoter, respectively. The combinations of Myc-CAT1 and GFP–MaCaM1/MaCaM1(M) constructs were co-transformed into Arabidopsis mesophyll protoplasts as described before (Yoo et al. 2007). After incubation overnight at 22°C, the protoplasts were collected to analyze catalase activity. The CAT activity was determined with a CAT assay kit according to the manufacturer’s instructions (Beyotime). Dual-luciferase reporter assay The dual-luciferase reporter system was used to analyze the transient reporter expression as described by Hellens et al. (2005). The pGreenII 0800-LUC reporter vector and pGreenII 62-SK effector vector were used. The coding sequences of MaCaM1, MaHY5-1 and MaCaM1(M) were amplified and fused to pGreenII 62-SK as effector plasmids. Genomic DNA was extracted from banana pericarp tissues using the DNeasy Plant Mini Kit (Qiagen). The MaPSY1/2/3 promoters were amplified using a GenomeWalker Kit (Clontech) according to the manufacturer’s instructions and inserted into pGreenII 0800-LUC as reporter plasmids. The constructed effector and reporter plasmids were co-transformed into tobacco leaves by Agrobacterium tumefaciens strain GV3101. The activities of LUC and REN luciferase were measured using the Dual-Luciferase® Reporter Assay kit (Promega) 3 d after co-transformation. The analysis was carried out on a Luminoskan Ascent Microplate Luminometer (Thermo Fisher Scientific) in accordance with the manufacturer’s instructions. The ratio of LUC to REN was calculated to reflect the final transcriptional activity. At least six biological replicates were assayed for each combination. Data handling Data were expressed as the mean ± SE. Differences among different treatments were compared using SPSS version 7.5 (SPSS, Inc.). Supplementary Data Supplementary data are available at PCP online. Funding This work was supported by the National Natural Science Foundation of China [31772041, 31501545 and 31322044]; the National Basic Research Program of China [2013CB127104 and 2013CB127102]; Guangdong Natural Science Foundation [2014A030310294]; China Postdoctoral Science Foundation [2014M560681]; Science and Technology Planning Project of Guangdong Province [2015B090901058]; Science and Technology Planning Project of Guangzhou [201604020048]; and Talent Program of Guangdong Province [2014TX01N049]. Disclosures The authors have no conflicts of interest to declare. References Abbas N. , Maurya J.P. , Senapati D. , Gangappa S.N. , Chattopadhyay S. 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Google Scholar CrossRef Search ADS PubMed Abbreviations Abbreviations AD activation domain BiFC bimolecular fluorescence complementation CaM calmodulin CaMKII Ca2+/calmodulin-dependent protein kinase II DBD DNA-binding domain DDO double drop-out EMSA electrophoretic mobility shift assay GFP green fluorescent protein LC-MS/MS liquid chromatography–tandem mass spectrometry Met methionine MetO methionine sulfoxide Msr methionine sulfoxide reductase QDO quadruple drop-out qRT-PCR quantitative real-time PCR ROS reactive oxygen species Trx thioredoxin YFP yellow fluorescent protein Y2H yeast two-hybrid © The Author(s) 2018. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices)

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Plant and Cell PhysiologyOxford University Press

Published: Mar 15, 2018

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