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. 2017 Sep 12;175(3):1082–1093. doi: 10.1104/pp.17.00695

S5H/DMR6 Encodes a Salicylic Acid 5-Hydroxylase That Fine-Tunes Salicylic Acid Homeostasis1,[OPEN]

Yanjun Zhang a,2, Li Zhao a,2, Jiangzhe Zhao a, Yujia Li a, Jinbin Wang a, Rong Guo a, Susheng Gan b, Chang-Jun Liu c, Kewei Zhang a,3
PMCID: PMC5664462  PMID: 28899963

SALICYLIC ACID 5-HYDROXYLASE catalyzes the formation of 2,5-DHBA both in vitro and in vivo and fine-tunes SA homeostasis in Arabidopsis.

Abstract

The phytohormone salicylic acid (SA) plays essential roles in biotic and abiotic responses, plant development, and leaf senescence. 2,5-Dihydroxybenzoic acid (2,5-DHBA or gentisic acid) is one of the most commonly occurring aromatic acids in green plants and is assumed to be generated from SA, but the enzymes involved in its production remain obscure. DMR6 (Downy Mildew Resistant6; At5g24530) has been proven essential in plant immunity of Arabidopsis (Arabidopsis thaliana), but its biochemical properties are not well understood. Here, we report the discovery and functional characterization of DMR6 as a salicylic acid 5-hydroxylase (S5H) that catalyzes the formation of 2,5-DHBA by hydroxylating SA at the C5 position of its phenyl ring in Arabidopsis. S5H/DMR6 specifically converts SA to 2,5-DHBA in vitro and displays higher catalytic efficiency (Kcat/Km = 4.96 × 104 m−1 s−1) than the previously reported S3H (Kcat/Km = 6.09 × 103 m−1 s−1) for SA. Interestingly, S5H/DMR6 displays a substrate inhibition property that may enable automatic control of its enzyme activities. The s5h mutant and s5hs3h double mutant overaccumulate SA and display phenotypes such as a smaller growth size, early senescence, and a loss of susceptibility to Pseudomonas syringae pv tomato DC3000. S5H/DMR6 is sensitively induced by SA/pathogen treatment and is expressed widely from young seedlings to senescing plants, whereas S3H is more specifically expressed at the mature and senescing stages. Collectively, our results disclose the identity of the enzyme required for 2,5-DHBA formation and reveal a mechanism by which plants fine-tune SA homeostasis by mediating SA 5-hydroxylation.

Salicylic acid (SA or 2-hydroxy benzoic acid) is a plant hormone that not only mediates plant defense responses against biotic and abiotic stresses but also plays a crucial role in regulating many physiological and biochemical processes during the entire plant lifespan (Vlot et al., 2009; Rivas-San Vicente and Plasencia, 2011). SA has a broad distribution, and its basal levels can vary up to 100-fold in different plant species, even in different members of the same family (Raskin, 1992). For example, in the model plant Arabidopsis (Arabidopsis thaliana), the basal levels of total SA range from 0.24 to 1 μg g−1 fresh weight (Nawrath and Métraux, 1999; Wildermuth et al., 2001; Brodersen et al., 2005), while in Oryza sativa, its basal levels range from 0.01 to 37.19 μg g−1 fresh weight (Silverman et al., 1995; Yang et al., 2004). SA is generated via two different pathways in plants, namely the phenylalanine ammonia lyase (PAL) pathway and the isochorismate synthase (IC) pathway; both pathways require the primary metabolite chorismate that is synthesized from the shikimate pathway (Dempsey et al., 2011; Widhalm and Dudareva, 2015). In the PAL pathway, chorismate-derived l-Phe is converted into SA via a series of enzymatic reactions that are initially catalyzed by PAL. However, in the IC pathway, chorismate is converted into SA through an intermediate, isochorismate, produced by ICs (Dempsey et al., 2011; Widhalm and Dudareva, 2015). Two ICs, ICS1 and ICS2, have been detected in Arabidopsis (Wildermuth et al., 2001; Garcion et al., 2008). The IC pathway appears to be responsible for 90% of SA production when Arabidopsis is stimulated with a pathogen or UV light, and the PAL pathway may play a complementary role in SA biosynthesis (Garcion et al., 2008).

Controlled SA levels are required for optimal reactive oxygen species content and redox homeostasis (Lamb and Dixon, 1997; Gapper and Dolan, 2006; Mateo et al., 2006). SA homeostasis directly affects its biological functions, such as development, photosynthesis, and pathogen responses (Mateo et al., 2006; Liu et al., 2010). Plants generally maintain hormone homeostasis by fine-tuning the balance between the biosynthesis and catabolism of the hormone. ICS1 and ICS2 are two key genes in the SA biosynthesis pathway that are regulated by multiple factors, such as pathogen infections, ethylene signaling, and circadian rhythms (Chen et al., 2009; Wang et al., 2015; Zheng et al., 2015). Although a small amount of SA produced in planta remains in a free state, most SA is subjected to biologically relevant chemical modifications by different enzymes after synthesis (Dempsey et al., 2011). SA glucosyltransferases convert SA into salicylic acid O-β-glucoside (SAG) or salicyloyl Glc ester, which is transported from the cytosol into the vacuole for storage in Arabidopsis (Dean et al., 2005; Dean and Delaney, 2008). The benzoic acid/salicylic acid carboxyl methyltransferase1 (BSMT1) catalyzes the formation of the SA methyl ester methyl salicylate, which renders SA inactive but increases its membrane permeability and facilitates the long-distance transport of SA signals (Chen et al., 2003). Two dihydroxybenzoates, 2,5-DHBA and 2,3-DHBA, exist as glycoside conjugates and are produced at even higher levels than SA and SA glycosides in Arabidopsis; therefore, SA hydroxylation is suggested to be the major pathway for SA catabolism (Bartsch et al., 2010; Zhang et al., 2013).

2,5-DHBA is one of the most widely produced aromatic acids in green plants (Griffiths, 1958; Ibrahim and Towers, 1959). Similar to other hydroxybenzoates, 2,5-DHBA accumulates as glycoconjugates in plants, primarily as 2,5-DHBA 5-O-β-d-glucosides, 2,5-DHBA 5-O-β-d-xylosides, or 2,5-DHBA 2-O-β-d-xylosides (Dean and Delaney, 2008; Tárraga et al., 2010; Li et al., 2014). 2,5-DHBA accumulates in response to different types of plant-pathogen interactions in much higher levels than SA (Bellés et al., 1999, 2006; Campos et al., 2014). The exogenous application of 2,5-DHBA to tomato (Solanum lycopersicum), cucumber (Cucumis sativus), and Gynura auriantiaca induces the expression of a distinct subset of PR genes compared with the genes induced by SA (Bellés et al., 1999, 2006); 2,5-DHBA also induces RNA silencing-related genes and resistance to RNA pathogens in tomato and G. auriantiaca (Campos et al., 2014). Moreover, 2,5-DHBA itself displays antibacterial activity in the harvested fruits (Lattanzio et al., 1996). Although the enzymes UGT89A2 and GAGT, which are responsible for the conversion of 2,5-DHBA to its glycosides, have been well characterized (Lim et al., 2002; Tárraga et al., 2010; Li et al., 2014) and the in vivo feeding of a 14C-labeled SA tracer in Gaultheria procumbens suggested that 2,5-DHBA was formed from the substrate SA half a century ago (Ibrahim and Towers, 1959), the enzymes responsible for the formation of 2,5-DHBA are still a mystery.

In a previous study screening for mutants with a loss of susceptibility to the downy mildew Hyaloperonospora parasitica, dmr6-1 and dmr6-3, two alleles were identified to have impaired susceptibility to the downy mildew H. parasitica and later were shown to have wide resistance to the bacterial pathogen Pseudomonas syringae and the oomycete pathogen Phytophthora capsici (van Damme et al., 2008; Zeilmaker et al., 2015). Map-based gene cloning disclosed that DMR6 is a putative 2-oxoglutarate/Fe(II)-dependent dioxygenase [2-oxoglutarate-Fe(II) oxygenase] belonging to the 2-oxoglutarate-dependent dioxygenase superfamily (van Damme et al., 2008; Kawai et al., 2014). Another 2-oxoglutarate-Fe(II) oxygenase, salicylic acid 3-hydroxylase (S3H; also named DLO1), which is encoded by a homolog gene of DMR6, was characterized to be able to catalyze the formation of 2,3-DHBA and mediate leaf senescence and pathogen responses in Arabidopsis (Zhang et al., 2013). The double mutant dmr6-3dlo1 of DMR6 and S3H (DLO1) displayed a dwarf phenotype, a substantial increase in SA levels, and a complete loss of susceptibility to the downy mildew Hyaloperonospora arabidopsidis (Zeilmaker et al., 2015). However, the underlying biochemical mechanism of DMR6 was still not revealed.

To characterize the enzyme that catalyzes the formation of 2,5-DHBA, we assayed a subset of the 2-oxoglutarate-Fe(II) oxygenase family in Arabidopsis using a previously developed approach (Zhang et al., 2013). Here, we report the identification of DMR6 (At5g24530) as an S5H capable of converting SA to 2,5-DHBA both in vitro and in vivo and characterize its essential roles in mediating SA homeostasis during plant development, leaf senescence, and pathogen responses in Arabidopsis.

RESULTS

2,5-DHBA Exists in a Wide Range of Plant Species

We used HPLC to quantify the benzoate derivatives in the adult and senescing leaves of 10 plant species, Capsicum frutescens, Bambusa multiplex, Solanum lycopersicum, Glycine max, Arachis hypogaea, Brassica campestris, Sorghum bicolor, Vinca major, Oryza sativa, and Arabidopsis, to identify the SA and SA hydroxylation profiles in different plant species (Supplemental Table S1). The total SA (the sum of free SA and SA glucosides) levels varied in different species, ranging from 0.73 μg g−1 fresh weight in S. bicolor to 115.38 μg g−1 fresh weight in B. multiplex, and the SA concentration increased in the senescing leaves of all species analyzed. Free 2,5-DHBA and 2,3-DHBA were not detected by HPLC in our experiments, indicating that the levels of those free SA derivatives are much lower than the free SA levels. Although total 2,3-DHBA was detected only in Arabidopsis and V. major, total 2,5-DHBA was detected in all species analyzed. These data suggest that 2,5-DHBA is a widely distributed hydroxylated product of SA in planta, consistent with the observations of Ibrahim and Towers (1959). Interestingly, we found much higher 2,5-DHBA accumulated in senescing leaves than that in young leaves, suggesting that 2,5-DHBA may be involved in leaf senescence. We also quantified the benzoate derivatives in Arabidopsis inoculated by the pathogen Pseudomonas syringae pv tomato DC3000 (Pst DC3000). Similar to SA and 2,3-DHBA, 2,5-DHBA production in Arabidopsis was induced by the pathogen Pst DC3000 at 1, 2, 4, and 6 d post inoculation (dpi; Supplemental Fig. S1), indicating that 2,5-DHBA may be involved in plant immunity.

Identification of an S5H in Vitro and in Vivo

We postulated that S5H is a 2-oxoglutarate-Fe(II) oxygenase and that the gene encoding this enzyme is induced by SA. Using an established enzymatic assay procedure (Zhang et al., 2013), we biochemically screened five members of the Arabidopsis 2-oxoglutarate-Fe(II) oxygenase family (Supplemental Fig. S2), which were all induced significantly by SA (Supplemental Fig. S3). We finally identified one recombinant protein encoded by At5g24530, a gene characterized previously as DMR6 (van Damme et al., 2008), responsible for 2,5-DHBA formation (Fig. 1), but the other four recombinant proteins did not possess the activity. The recombinant protein encoded by At5g24530 specifically catalyzed the formation of a compound exhibiting the same retention time and the fluorescence emission spectra as the 2,5-DHBA authentic standard from SA (Fig. 1, B and C), indicating that the recombinant enzyme possesses 5-hydroxylation activity on SA in vitro. Therefore, the enzyme was designated as S5H. According to a comparison of the sequence similarity, S5H/DMR6 and S3H are closely evolutionarily related (Supplemental Fig. S2), sharing 50.57% similarity at their amino acid levels (Supplemental Fig. S4). A homozygous mutant line with a T-DNA insertion in the third exon of At5g24530 (SK19807) was further characterized as a knockout line and was named s5h (Supplemental Fig. S5). The 2,5-DHBA level in the s5h mutant was reduced to 12% of the wild-type level, and the levels were restored by introducing the expression of the At5g24530 gene in the s5h mutant background (i.e. S5H-OX lines; Supplemental Fig. S6), indicating that the protein encoded by At5g24530 functions as an S5H in vivo.

Figure 1.

Figure 1.

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Conversion of SA to 2,5-DHBA by the recombinant S5H/DMR6 protein in vitro. A, Biochemical reaction catalyzed by S5H/DMR6 in vitro. B, HPLC profiles of the 30-min reaction of the recombinant S5H/DMR6 protein (S5H) and empty vector extracts (EV) incubated with SA. Authentic 2,5-DHBA was used as a standard. C, The fluorescence emission spectra of the enzymatic product 2,5-DHBA is identical to that of the 2,5-DHBA standard. D, Kinetics of the recombinant S5H/DMR6 protein (SA as the substrate). The data are presented as means ± se (n = 3).

Kinetic Parameters of Recombinant S5H/DMR6

The biochemical properties of recombinant S5H/DMR6 were investigated further. The recombinant S5H/DMR6 protein was purified, and the preferred temperature and pH value were optimized (Supplemental Fig. S7). Enzymatic activity increased as the temperature increased from 4°C to 40°C and decreased as the temperature increased from 40°C to 50°C, suggesting that the optimal temperature for S5H/DMR6 activity is approximately 40°C (Supplemental Fig. S7B). The effect of pH on S5H/DMR6 activity also was evaluated, and the optimal pH of the enzyme was 6.8 under our conditions (Supplemental Fig. S7C). At optimal pH and temperature conditions, the calculated apparent Km value of the recombinant S5H/DMR6 enzyme on SA was approximately 5.15 ± 1.44 μm, which is much lower than the Km of S3H (58.29 μm; Zhang et al., 2013). The Kcat value of S5H/DMR6 (0.255 s−1) is approximately 71.9% of that of S3H (0.355 s−1), indicating that S5H/DMR6 possesses a slightly lower turnover rate to SA than S3H. Overall, the calculated Kcat/Km for S5H/DMR6 was 4.96 × 104 m−1 s−1, which is 8-fold higher than that for S3H (6.09 × 103 m−1 s−1), indicating that S5H/DMR6 displays a much higher catalytic efficiency than S3H to SA in vitro. Additionally, in contrast to S3H, S5H/DMR6 was inhibited substantially by its substrate SA, with a calculated Ksi value of 6.05 ± 1.46 μm, which leads to a velocity curve that surged to its maximum and then descended as the substrate concentration increased (Fig. 1D).

Spatial and Temporal Expression Patterns of S5H/DMR6 and Its Responses to SA and Pathogens

The expression patterns of S5H/DMR6, together S3H, were evaluated under different conditions by quantitative reverse transcription (qRT)-PCR and GUS reporter gene expression to obtain a better understanding of the potential physiological and molecular roles of the genes (Figs. 2 and 3). S5H/DMR6 was constitutively expressed in young and mature leaves, and its expression levels increased significantly in leaves in early and late senescence (Figs. 2A and 3, A, C, E, G, and I). In contrast, the S3H gene was nearly undetectable in young leaves (Figs. 2A and 3, B and D), detected at moderate levels in mature leaves (Fig. 3F), and expressed abundantly in leaves in early and late senescence (Figs. 2A and 3, H and J). The expression levels of both S5H/DMR6 and S3H were reduced dramatically in the leaves of NahG transgenic plants (NahG encodes an SA hydroxylase that degrades SA to catechol; van Wees and Glazebrook, 2003) compared with wild-type leaves at the same stages, indicating that the expression levels of the two genes were highly dependent on SA (Fig. 2A). We then quantified S5H/DMR6 and S3H expression in wild-type plants treated with 0.1 to 6 mm SA (Fig. 2B). S5H/DMR6 and S3H expression were both induced by SA, and the relative S5H/DMR6 expression levels were much higher (up to 10-fold) than the S3H levels, indicating that S5H/DMR6 was more sensitive to the SA treatment (Fig. 2B).

Figure 2.

Figure 2.

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SA-induced, pathogen-induced, and senescence-associated patterns of S5H/DMR6 and S3H expression. A, Expression of the S5H/DMR6 and S3H genes during leaf senescence in wild-type (WT) and NahG transgenic plants. YL, Young leaves; ML, mature leaves; ES, early senescence leaves; LS, late senescence leaves. B, S5H/DMR6 and S3H expression were both induced by different concentrations of SA after a 6-h treatment, and S5H/DMR6 was expressed at 10-fold higher levels than S3H. C and D, S5H/DMR6 (C) and S3H (D) expression in wild-type plants inoculated with Pst DC3000 at 1, 2, 4, and 6 dpi; 0 represents untreated wild-type plants, and M represents the mock treatment. The data are presented as means ± se (n = 3).

Figure 3.

Figure 3.

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Spatial and temporal patterns of S5H/DMR6 and S3H expression were determined by GUS activities in S5Hpro::GUS and S3Hpro::GUS transgenic plants. A and B, C and D, E and F, G and H, and I and J show transgenic S5Hpro::GUS and S3Hpro::GUS plants at 10, 21, 28, 35, and 49 DAG, respectively. K and L present mock and Pst DC3000-inoculated rosette leaves from S5Hpro::GUS transgenic plants. M and N show mock and Pst DC3000-inoculated rosette leaves from S3Hpro::GUS transgenic plants. The arrows point to the area where the GUS staining changed significantly between K and L and between M and N. Bars = 1 mm (A and B), 1 cm (C–J), and 2 mm (K–N).

S5H/DMR6 and S3H expression also were investigated in response to the pathogen Pst DC3000 at 1, 2, 4, and 6 dpi (Fig. 2, C and D). Both S5H/DMR6 and S3H expression were induced by the pathogen, and their expression levels peaked on day 6, immediately after the total SA reached its peak value (Supplemental Fig. S1), implying that SA hydroxylation plays a role in the detoxification of excessive SA after the initiation of the pathogen response. A comparison of the expression of the S5H/DMR6 and S3H genes with the internal standard ACTIN2 revealed that the highest levels of S5H/DMR6 expression were apparently 3-fold higher than the highest levels of S3H expression, suggesting that S5H is more sensitive to pathogen induction at the transcriptional level (Fig. 2, C and D). The GUS staining was more intensive in the leaves of both S5Hpro::GUS and S3Hpro::GUS transgenic plants that were inoculated with the pathogen Pst DC3000 (Fig. 3, L and N) compared with the mock treatments (Fig. 3, K and M), which is consistent with the pathogen-induced expression patterns of S5H/DMR6 and S3H revealed by qRT-PCR analyses (Fig. 2, C and D).

Disruption of S5H/DMR6 Results in Growth Retardation, Early Senescence, and a Loss of Susceptibility to Pst DC3000

The s5hs3h double mutant was generated for a detailed phenotypic analysis to further investigate the biological functions of S5H/DMR6 in plant development, leaf senescence, and plant immunity (Supplemental Fig. S5). The s5h, s3h, and s5hs3h mutants and the representative S5H/DMR6 overexpression lines S5H-OX3 in the wild-type background and S5H-OX18 in the s5h background were grown in parallel in the same tray for the phenotypic analysis. At 21 d after germination (DAG), the diameters of the rosette leaves of s5h and s5hs3h were 86% and 60% of the wild type, respectively. The S5H-OX18 line in the s5h background restored the phenotype of the s5h mutant to the wild-type phenotype, and the S5H-OX3 line in the wild-type background displayed even larger leaves (up to 114% of the wild type; Fig. 4B), indicating that S5H/DMR6 plays an important role in the early development of Arabidopsis.

Figure 4.

Figure 4.

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Phenotypes of the s3h, s5h, s5hs3h, and S5H-OX lines. A, Representative images of the s3h, s5h, and s5hs3h mutants and overexpression lines S5H-OX3 and S5H-OX18 at 21, 35, and 49 DAG. Bars = 2 cm. B, Quantification of the diameters of rosette leaves from the indicated plants at 21 DAG. C, Quantification of total chlorophyll contents in the fifth and sixth leaves from the plants shown in A at 49 DAG. The data are presented as means ± se (n ≥ 10). FW, Fresh weight. D, Disease symptoms of 30-DAG plants from the wild-type (WT), s3h, s5h, s5hs3h, S5H-OX3, and S5H-OX18 lines 3 d after Pst DC3000 suspension (OD600 = 0.001) infiltration. Bar = 1 cm. E, The s5h and s5hs3h mutants at 30 DAG showed significantly higher resistance to Pst DC3000 suspension (OD600 = 0.001) infiltration than the wild type. F, The overexpression line S5H-OX3 in the wild-type background was more susceptible to Pst DC3000 suspension (OD600 = 0.0001) infiltration than the wild type, while the overexpression line S5H-OX18 in the s5h background rescued the loss-of-susceptibility phenotype of s5h to the wild type. CFU, Colony-forming units. The data are presented as means ± sd (n = 8). *, P < 0.05 and **, P < 0.01 (Student’s t test).

At 35 and 49 DAG, the s5h mutant displayed an early senescence phenotype, similar to the s3h mutant, and the s5hs3h mutant showed a more dramatic early senescence phenotype. In contrast, the S5H/DMR6 overexpression lines S5H-OX3 and S5H-OX18 exhibited a delayed leaf senescence phenotype (Fig. 4A). Consistent with the visible phenotypes, the chlorophyll contents in the fifth to sixth leaves of the s5h and s3h mutants were reduced to 57% and 55% of the wild-type contents at 49 DAG, respectively; in contrast, the chlorophyll contents of the S5H/DMR6 overexpression lines S5H-OX3 and S5H-OX18 were 40% and 25% higher than in the wild-type line at 49 DAG, respectively (Fig. 4C). Thus, S5H/DMR6, in cooperation with S3H, plays an essential role in the leaf senescence process in Arabidopsis.

Since S5H/DMR6 and 2,5-DHBA were sensitively induced by SA and Pst DC3000 pathogens, we assessed the pathogen resistance of the wild type, the s3h, s5h, and s5hs3h mutants, and the S5H/DMR6 overexpression lines S5H-OX3 and S5H-OX18 by inoculating them with Pst DC3000. Compared with the wild type and s3h mutant lines, the s5h and s5hs3h mutants showed a loss of susceptibility, the S5H-OX3 line displayed enhanced susceptibility to Pst DC3000 over that of the wild type, while the S5H-OX18 line rescued the loss-of-susceptibility phenotype of the s5h mutant (Fig. 4, D–F), consistent with the phenotypes of the 35S:DMR6 lines (Zeilmaker et al., 2015). These results further support the hypothesis that the disruption of S5H/DMR6 leads to a loss of susceptibility to Pst DC3000 in Arabidopsis.

A Transcriptional Analysis Reveals the Complementary Roles of S5H/DMR6 and S3H

We further analyzed S5H/DMR6 and S3H expression in the rosette leaves of the s5h, s3h, and s5hs3h mutants at 21, 35, and 49 DAG by qRT-PCR to understand the relation between S5H/DMR6 and S3H at the transcriptional level (Fig. 5). Compared with the wild type, the S3H expression level increased significantly to 125-, 1.86-, and 1.8-fold in the s5h mutant at 21, 35, and 49 DAG, respectively (Fig. 5A). Similarly, S5H/DMR6 expression was increased by 3.95-, 2.47-, and 1.43-fold in the s3h mutant at 21, 35, and 49 DAG, respectively (Fig. 5B). Based on these results, S5H/DMR6 and S3H display complementary functions at the transcript level in Arabidopsis.

Figure 5.

Figure 5.

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Transcriptional analysis of wild-type (WT), s3h, s5h, and s5hs3h plants by qRT-PCR. A, S3H expression was increased significantly 125-, 1.86-, and 1.8-fold in the s5h mutant compared with the wild-type plants at 21, 35, and 49 DAG, respectively. B, Similarly, S5H/DMR6 expression was increased significantly 3.95-, 2.47-, and 1.43-fold in the s3h mutant at 21, 35, and 49 DAG, respectively. The data are presented as means ± se (n = 3). *, Not detected or detected at low levels.

A Metabolite Analysis Reveals the Cooperation of S5H/DMR6 and S3H in Regulating SA Homeostasis

Subsequently, the metabolites of SA, SAG, and their hydroxyl products were quantified in the wild-type, s3h, s5h, and s5hs3h lines at 21, 35, and 49 DAG, which represent young, mature, and senescence stages, respectively (Fig. 6). At the young stage (21 DAG), the free SA and total SA contents were increased significantly by 43% and up to 239% in the s5h mutant and by 178% and up to 2,333% in the s5hs3h double mutant, respectively (Fig. 6, A and B), but they did not show obvious changes in the s3h mutant compared with the wild-type line (Fig. 6, A and B), suggesting that S5H/DMR6 regulates SA homeostasis at the early growth stage. At 35 DAG, although the free SA and total SA contents were increased by 66% and 193% in s3h, the free SA and total SA contents in the wild type and s5h mutant lines were not obviously different. However, in the s3h background, the free SA and total SA contents in the s5hs3h mutant were increased 164% and 308% compared with the s3h single mutant (Fig. 6, A and B), indicating the S3H and S5H/DMR6 have redundant functions at the mature stage. At 49 DAG, the free SA and total SA contents were similar in the s3h and s5hs3h mutants (Fig. 6, A and B), indicating that S3H plays the essential role at the late senescence stage.

Figure 6.

Figure 6.

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Accumulation of free or total SA, 2,3-DHBA, and 2,5-DHBA at different developmental stages. A, The free SA levels in the s5h mutant and the s5hs3h double mutant were significantly higher than the wild-type (WT) level, whereas the S5H/DMR6 overexpression line S5H-OX18 in the s5h background restored the SA level in the s5h mutant at 21 DAG; the free SA levels in the s3h and s5hs3h mutants were significantly higher than the wild-type levels at 35 and 49 DAG; the free SA levels were reduced significantly in the S5H/DMR6 overexpression lines S5H-OX3 and S5H-OX18 at 49 DAG. B, The total SA levels were increased significantly in the s5h and s5hs3h mutants at 21 DAG and in the s3h and s5hs3h mutants at 35 and 49 DAG; the total SA levels were decreased in the S5H-OX3 and S5H-OX18 lines at 35 and 49 DAG. C, The total 2,3-DHBA content was detected in the s5h mutant at 21, 35, and 49 DAG but was only detected at 49 DAG in the wild-type and S5H-OX18 lines. D, The total 2,5-DHBA levels were increased in the s3h mutant at 21, 35, and 49 DAG; the total 2.5-DHBA levels were decreased significantly in the s5h and s5hs3h mutants at 21, 35, and 49 DAG and in the S5H-OX lines at 49 DAG. The data are presented as means ± se (n = 4). Significant differences (Student’s t test) compared with the wild-type plants are indicated with asterisks: *, P < 0.05 and **, P < 0.01. FW, Fresh weight.

In contrast to the continuous light condition (Zhang et al., 2013), 2,3-DHBA was undetectable at 21 and 35 DAG in the wild-type line under a 16-h-light/8-h-dark photoperiod but was detected in the s5h mutant under the same conditions, suggesting that S3H plays a complementary role in maintaining SA homeostasis in the absence of S5H/DMR6 (Fig. 6C), consistent with the overexpression of the S3H gene in the s5h mutant (Fig. 5A). 2,3-DHBA was detected in the wild-type line until 35 DAG, a maturation stage at which the plant is starting to undergo senescence, and high levels were detected at 49 DAG, a senescence stage, suggesting that the main physiological function of S3H occurs during leaf senescence (Fig. 6C).

In contrast to 2,3-DHBA, 2,5-DHBA was detected at 21 DAG (Fig. 6D), and its content was increased as the SA concentration increased in the wild-type plants. In the s3h mutant, the 2,5-DHBA content increased 97% compared with the wild-type line at 49 DAG, indicating that S5H/DMR6 partially complemented the mutant phenotype in the s3h line in the absence of S3H, consistent with the overexpression of the S5H/DMR6 gene in the s3h mutant (Fig. 5B). Interestingly, 2,5-DHBA was not completely absent in the s5h and s5hs3h mutants but was decreased to 52% and 7% of the wild-type levels, respectively, indicating that S5H/DMR6 is the major, but probably not the sole, enzyme responsible 2,5-DHBA formation (Fig. 6D).

DISCUSSION

The elucidation of the molecular mechanism governing SA homeostasis is essential for understanding the developmental and defense-related processes mediated by this hormone. 2,5-DHBA is the major catabolic product of SA and is detected in a wide variety of plant species (Supplemental Table S1). In this study, we identified S5H/DMR6 as an additional 2-oxoglutarate-Fe(II) oxygenase that is responsible for converting SA to 2,5-DHBA. Interestingly, plants have evolved an elegant feedback regulatory mechanism involving S5H/DMR6 and S3H, which are complementarily and cooperatively responsible for SA catabolism and maintain SA homeostasis during development, leaf senescence, and immunity of Arabidopsis.

The characterization of S5H/DMR6 as the major enzyme catalyzing the formation of 2,5-DHBA adequately addresses the biochemical mechanism of dmr6 and further solves a puzzle in the pathway of SA catabolism (Fig. 7). DMR6 was reported recently as a homolog of the maize (Zea mays) flavone synthase ZmFNSI-1, which catalyzes the formation of apigenin from naringenin (Falcone Ferreyra et al., 2015). Under our assay conditions, only low S5H/DMR6 activity was detected when naringenin was used as the substrate; and the specific activity of S5H/DMR6 toward the SA substrate was approximately 179- and 1,429-fold higher than its activity toward naringenin at 30°C and 40°C, respectively (Supplemental Fig. S8). Based on these data, S5H/DMR6 prefers SA over naringenin as its natural substrate. Thus, we conclude that the strong pathogen resistance of dmr6 was most likely caused by enhanced SA levels but was unlikely due to the absence of apigenin.

Figure 7.

Figure 7.

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Simplified schematic of the pathways for SA modification and catabolism in Arabidopsis. The expression of the newly discovered S5H/DMR6, indicated in boldface, is induced by accumulated SA (the encoded S5H/DMR6 is indicated by the red arrow), and the enzyme catalyzes the formation of 2,5-DHBA from the substrate SA. SA also is converted to 2,3-DHBA by an enzyme encoded by another SA-induced gene, S3H (the encoded S3H is indicated by the black arrow), as reported previously. 2,5-DHBA and 2,3-DHBA are conjugated subsequently by UGT89A2 to produce 2,5-DHBA- or 2,3-DHBA-sugar conjugates for storage in vacuoles. SA is converted to its storage form, salicylate-sugar conjugates, by the UGT74F1 and UGT74F2 enzymes. The methyltransferase BSMT1 is responsible for the production of the functional form methylsalicylic acid from SA, and methylesterases 1, 2, 7, and 9 are responsible for the reverse reaction from methylsalicylic acid to SA. The question marks near GH3.5/WES1 and SOT12 indicate that the enzymes possess the activities to catalyze the formation of salicyloyl-l-Asp and SA-2-sufonate, respectively, from SA in vitro, but their functions in plants remain to be determined. The dotted arrow and question mark represent uncertainty regarding whether the 2,5-DHBA accumulated in the s5h mutant is produced from the SA substrate by an unknown enzyme.

2,5-DHBA is an important aromatic compound produced in planta, and the identification of the S5H/DMR6 enzyme enabled us to investigate the biochemical mechanism of 2,5-DHBA formation. 2,5-DHBA and 2,3-DHBA were suggested previously to be synthesized by a nonenzymatic reaction in which SA scavenged hydroxyl radicals (Maskos et al., 1990; Chang et al., 2008). The glycosides of 2,5-DHBA and 2,3-DHBA were distributed abundantly in Arabidopsis and were suggested to be formed predominantly from the substrate SA via the IC pathway (Bartsch et al., 2010). We previously characterized S3H, which converts SA to 2,3-DHBA (Zhang et al., 2013). As shown in this study, S5H/DMR6 is responsible for 2,5-DHBA formation both in vivo and in vitro. Compared with S3H, which converts SA to both 2,3-DHBA and 2,5-DHBA in vitro, S5H/DMR6 more specifically converts SA to 2,5-DHBA in vitro (Fig. 1). The apparent Km for S5H/DMR6 toward SA is 5.15 μm, which is much lower than the Km for S3H (58.29 μm; Zhang et al., 2013), suggesting that S5H/DMR6 may have a higher binding affinity for the SA substrate than S3H. Although S5H/DMR6 displayed a slightly lower Kcat for SA than S3H, the catalytic efficiency of S5H/DMR6 was much higher than that of S3H, based on the Kcat/Km values of the two enzymes. Compared with the other known enzymes (Fig. 7) involved in SA modification, including UGT74F1/F2 (Km = 230/190 μm), BSMT1 (Km = 16 μm), MES1 (Km = 57–147.1 μm), and SOT12 (Km = 440 μm; Dempsey et al., 2011), the Km of S5H/DMR6 is the lowest; the low Km may allow S5H/DMR6 to compete with the other SA-utilizing enzymes in Arabidopsis, indicating its essential roles in SA catabolism. Meanwhile, the biochemical properties of S5H/DMR6 allow it to catalyze SA at low concentrations and, thus, to fine-tune the SA level during the early development of Arabidopsis.

The biological function of an enzyme is determined not only by the catalytic parameters of the enzyme but also is attributed to its spatial and temporal expression pattern. The expression pattern of S5H/DMR6 partially overlapped with but was largely distinct from that of S3H. Similar to S3H, S5H/DMR6 expression was induced by pathogens, leaf senescence, and SA. SA and pathogens increased S5H/DMR6 expression 10- and 3-fold compared with S3H, respectively (Fig. 2, B–D), supporting the conclusion that S5H/DMR6 maintains SA homeostasis at low concentrations, whereas S3H maintains homeostasis at high concentrations. After comparing the temporal expression patterns, S5H/DMR6 is expressed from young to senescence stages, but S3H is expressed specifically at the mature and senescing stages in Arabidopsis (Zhang et al., 2013), suggesting that S5H/DMR6 is evolutionarily a housekeeping gene and S3H is a specific senescence-associated gene that are cooperatively involved in maintaining SA homeostasis during Arabidopsis development. Regarding spatial expression, S5H/DMR6 was expressed in mesophyll cells, whereas S3H was expressed mainly around the vascular tissue in the early stage and in mesophyll cells in senescence (Fig. 3). The inducible spatial and temporal expression patterns of S5H/DMR6 and S3H, along with the distribution of 2,5-DHBA and 2,3-DHBA glycosides in pathogen-stimulated young and senescing leaves, support the hypothesis that S5H/DMR6 and S3H cooperatively maintain SA homeostasis through a feedback mechanism.

As an endogenous signal, SA is a key signaling molecule in various plant functions, such as disease resistance, development, and leaf senescence (Vlot et al., 2009). At the early development stage, SA homeostasis was impaired in the s5h mutant and the s5hs3h double mutant and the SA levels were increased 43% and 178%, respectively, resulting in a significant reduction of the rosette leaf size, indicating that SA affects plant size. This conclusion was supported by the suppression of the dwarf phenotype by crossing the dmr6-3dlo1 mutant to the sid2 background (Zeilmaker et al., 2015). Similar phenomena have been observed in other SA overaccumulation mutants, such as cpr1-1, cpr5-1, cpr6-1, and dnd1-1, which showed dwarf phenotypes (Jirage et al., 2001). On the contrary, the SA-deficient plants of NahG transgenic Arabidopsis, the sid2 mutant and the S3H overexpression line S3HOE1, similar to the overexpression line of S5H/DMR6, exhibited larger rosette leaves (Fig. 4, A and B; Abreu and Munné-Bosch, 2009; Zhang et al., 2013). In contrast to the s5h mutant, the s3h mutant showed no obvious morphological difference compared with the wild-type line at the early development stage but exhibited accelerated leaf senescence at later growth stages, suggesting that S3H has more specific functions in leaf senescence under normal conditions. Although the expression of the S5H/DMR6 and S3H genes was induced significantly by pathogen stimulation (Fig. 2, C and D), the s5h mutant exhibited much stronger pathogen resistance than the s3h mutant, as supported by the detailed analyses of the dmr6 mutant (van Damme et al., 2008; Zeilmaker et al., 2015), which is possibly attributed to its more predominant gene expression response and higher binding affinity for SA.

In summary, both S5H/DMR6 and S3H expression were strongly induced by SA and convert SA to either 2,5-DHBA or 2,3-DHBA, respectively, thus forming a feedback mechanism for SA catabolism in which S5H/DMR6 and S3H maintain SA homeostasis in Arabidopsis (Fig. 7). S5H/DMR6 and S3H corporately balance SA homeostasis at different development stages and under various stress conditions. In addition to S5H/DMR6, plants may express another enzyme or utilize another mechanism to produce 2,5-DHBA; further studies are needed to examine these possibilities. Furthermore, the mechanisms regulating S5H/DMR6 and S3H at the transcriptional and posttranscriptional levels are unknown. An understanding of the regulatory mechanisms will help researchers decipher the physiological roles of SA and facilitate the development of methods for mediating plant development and pathogen resistance by manipulating SA metabolism in crops.

MATERIALS AND METHODS

Plant Materials and Growth Conditions

Arabidopsis (Arabidopsis thaliana) ecotype Columbia-0 was used as the wild-type line in all experiments. The s3h mutant was described previously (Zhang et al., 2013). The s5h mutant (SK19807) was obtained from the Arabidopsis Biological Resource Center (Robinson et al., 2009). Seeds were sown on petri dishes containing Murashige and Skoog medium with 0.25% (w/v) phytogel and appropriate antibiotics and incubated at 4°C for 3 d before being moved to a growth chamber. Seedlings with two true leaves were transplanted to soil under a 16-h-light/8-h-dark photoperiod at approximately 60% humidity, unless noted otherwise. The light intensity was approximately 110 μmol m−1 s−1. Wild-type, mutant, and/or transgenic plants were grown in parallel in the same tray to minimize possible variations in growth conditions. Capsicum frutescens, Bambusa multiplex, Solanum lycopersicum, Glycine max, Arachis hypogaea, Brassica campestris, Sorghum bicolor, Vinca major, and Oryza sativa were harvested from a field in Jinhua, Zhejiang, China, in summer 2016.

Identification of Mutants

Gene-specific primers S5H-M1 (5′-GGAAATAGTAAGTAAATACAGTAG-3′), S5H-M2 (5′-TTAGTTGTTTAGAAAATTCTCGA-3′), and S5H-M3 (5′-TATGTTGATGACAAAAGCAT-3′) and the T-DNA border primer TB1 (5′-TGGACGTGAATGTAGACACGTCG-3′) were used to identify homozygous s5h mutant plants. Gene-specific primers S3H-M1 (5′-ATGGCAACTTCTGCAATATC-3′) and S3H-M2 (5′-TTAGGTTGTTGGAGCTTTGA-3′) and the T-DNA border primer TB2 (5′-TGGTTCACGTAGTGGGCCATCG-3′) were used to identify homozygous s3h mutant plants.

SA and Pathogen Treatment

Arabidopsis plants (25 DAG) were sprayed with different concentrations of SA in 0.005% (v/v) Silwet L-77 or with 0.005% (v/v) Silwet L-77 alone (mock). Pathogen inoculation was performed by spraying or injection as described previously (Katagiri et al., 2002). All rosette leaves of individual plants were collected at different time points after treatment for RNA extraction or bacterial counting. The leaf discs (0.28 cm2) were excised from leaves with a smaller cork borer and used for bacterial counting.

Protein Expression and Purification

The S5H/DMR6 coding sequence was PCR amplified using a pair of primers, S5H_BamHI (5′-TTTAAGGATCCATGGCGGCAAAGCTGATATC-3′) and S5H_SalI (5′-CATGGTCGACTTAGTTGTTTAGAAAATTCTCGA-3′), and cloned into pET28a (Novagen) to form pET28a-S5H and produce the His-tagged recombinant S5H/DMR6 protein. The pET28a-S5H construct was introduced into Escherichia coli BL21 (DE3, pLys3; Invitrogen). Bacterial cells containing pET28a-S5H were grown in Luria-Bertani medium containing 50 mg L−1 kanamycin at 37°C to an optical density of approximately 0.6 at 600 nm, induced with 0.5 mm isopropyl β-d-1-thiogalactoside, and then incubated at 18°C for 24 h. The recombinant S5H protein was purified by Ni-NTA affinity chromatography using a previously described method (Zhang et al., 2013). DTT (final concentration, 2 mm) was added to the enzyme solution, and the protein was immediately stored at −80°C.

Enzyme Assays

The enzyme assay was performed according to a previously described method (Zhang et al., 2013). The reaction mixture (100 μL) contained 5 mm DTT, 4 mm sodium ascorbate, 1 mm 2-oxoglutaric acid, 0.4 mm FeSO4, 0.1 mg mL−1 catalase, 50 mm Tris-HCl (pH 8) or other buffer, 1 to 15 μg of recombinant protein, and various concentrations of SA. The protein was incubated with 200 μm SA substrate in phosphate buffer (pH 6.8) at different temperatures for 30 min, or 200 μm SA substrate in citrate buffer (pH 5.3), phosphate buffer (pH 6, 6.5, 7, or 7.5), or Tris-HCl buffer (pH 8) at 28°C for 30 min, to determine the optimal temperature and pH for S5H/DMR6 enzyme activity. Various SA substrate concentrations ranging from 2 to 60 μm were used for the enzyme kinetics analysis, and the incubation proceeded at 40°C for 1 min (2–8 μm) or 3 min (10–60 μm) at pH 6.8. All reactions were started by adding the enzyme and stopped by adding 100 μL of 50% (v/v) acetonitrile and were then heated in boiling water for 1 min to denature the protein. After centrifugation at the top speed for 10 min, the supernatant was analyzed by HPLC. Substrate inhibition kinetics were analyzed using a previously reported equation (Kutsuno et al., 2013), where Ksi is the constant describing the substrate inhibition interaction.

Gene Overexpression

The coding region of the S5H/DMR6 gene was amplified using the primers S5H_BamHI (5′-TTTAAGGATCCATGGCGGCAAAGCTGATATC-3′) and S5H_PstI (5′-CTGCAGTTAGTTGTTTAGAAAATTCTCGA-3′) to construct a binary S5H-OX vector for S5H/DMR6 overexpression. The PCR product was cloned into pGEM-T (Promega) to generate pGEM-S5H. The coding sequence was then released from the vector by digestion with BamHI and PstI and was subcloned into the BamHI and PstI sites of pGL800 (a modified 35S overexpression vector with the pZP211 backbone; Zhang and Gan, 2012) to generate pGL800-S5H, which was transformed into the s5h and wild-type lines to generate S5H overexpression lines in the s5h and wild-type backgrounds, respectively. More than 20 independent transgenic plants were generated, and the representative lines S5H-OX3 in the wild-type background and S5H-OX18 in the s5h background were presented. All constructs described above were confirmed by sequencing.

Metabolite Extraction

All rosette leaves from wild-type, s3h, s5h, s5hs3h, and S5H-OX transgenic plants were collected at different developmental stages for the metabolite analysis. SA was extracted using a previously described method for the extraction of phenolic compounds (Zhang et al., 2012), with some modifications. The rosette leaves were ground in liquid nitrogen. Approximately 100 mg of the powders was added to 1 mL of 80% methanol containing 50 μm methyl salicylate (used as an internal standard) in a 2-mL Eppendorf tube. The Eppendorf tube was agitated for 2 h at 4°C and then centrifuged at 13,000g for 10 min at 4°C. The supernatant was transferred into a new Eppendorf tube, and the pellet was reextracted with 500 μL of 100% methanol. Both extracts were combined and air dried with nitrogen gas before being dissolved in 500 μL of sodium acetate (0.1 m, pH 5.5). Half of the suspension was used for the HPLC analysis of the free SA, 2,3-DHBA, and 2,5-DHBA contents. The other half was treated with 10 μL of β-glucosidase (1 unit μL−1) and hydrolyzed in a 37°C water bath for 2 h. After the hydrolysate was denatured in boiling water for 5 min and centrifuged at 13,000g for 10 min at 4°C to pellet the protein, the supernatant was used for the HPLC analysis of the total SA, 2,3-DHBA, and 2,5-DHBA contents.

HPLC

An Agilent 1260 HPLC system (Agilent Technologies) coupled with a diode array detector and a fluorescence detector and a Zorbax SB-C18 column (4.6 × 250 mm, 5 μm; Agilent Technologies) were used for the metabolite analysis. The HPLC method was based on a previously described method (Marek et al., 2010), with some modifications. The mobile phases were composed of sodium acetate (0.2 m, pH 5.5) and methanol. The initial methanol gradient was maintained at 3% (v/v) for 12 min, linearly increased to 7% (v/v) at 12.5 min, and maintained until 38 min. After 1 min, the initial conditions were restored and the system was allowed to equilibrate for 7 min before the next injection. The flow rate was maintained at 0.8 mL min−1 throughout the process. SA or 2,5-DHBA was detected with a 296-nm excitation wavelength and 410-nm emission wavelength or with a 320-nm excitation wavelength and 449-nm emission wavelength using a fluorescence detector. 2,3-DHBA was detected with a diode array detector at 223 nm. The concentration was calculated by determining the HPLC peak area according to a standard curve.

Real-Time qRT-PCR Analyses

Total RNA was extracted using a TaKaRa minibest universal RNA extraction kit, and cDNAs were synthesized using HiScript QRT supermix for quantitative PCR (+gDNA wiper; R123-01; Vazyme). qRT-PCR was performed using SYBR Green (TaKaRa) on an ABI PRISM 7700 system (Applied Biosystems). ACTIN2 was used as an internal control in the analysis of the qRT-PCR data from whole leaves. The qRT-PCR primers included S3HQ1 (5′-TTCATCGTCAATATCGGCGAC-3′) and S3HQ2 (5′-ATCGATAACCGCTCGTTCTCG-3′) for S3H, S5HQ1 (5′-TACCTGCTCATACCGACCCAAA-3′) and S5HQ1 (5′-ATTAACGGCGAACCACTGACC-3′) for S5H/DMR6, and ActinQ1 (5′-GGTAACATTGTGCTCAGTGGTGG-3′) and ActinQ2 (5′-CTCGGCCTTGGAGATCCACATC-3′) for ACTIN2. The S3H or S5H/DMR6 transcript levels were normalized to the levels of the ACTIN2 transcript.

Histochemical GUS Staining and Chlorophyll Assay

The 2.4-kb S5H/DMR6 promoter was amplified with S5H-P1 (5′-GCAGGCTCCGAATTCTCCCAAACCATGATGGCACC-3′) and S5H-P2 (5′-AAGCTGGGTCGAATTCCAGAAAATTGAAGAAGAATC-3′) primers and cloned into the Gateway entry vector pCR8 (Thermo Fisher Scientific) via the sequence-and-ligation-independent cloning method (Jeong et al., 2012) to form pCR8-S5Hpro. The S5H/DMR6 promoter in pCR8-S5Hpro was introduced into pMDC163 via LR reactions to construct S5Hpro::GUS binary vectors. The 2.6-kb S3H promoter was amplified with S3H-P1 (5′-GGGGACAAGTTTGTACAAAAAAGCAGGCTTCAACTCTTATACTCACTTTCAGCA-3′) and S3H-P2 (5′-GGGGACCACTTTGTACAAGAAAGCTGGGTTAATGTAATTTTGTAATGTCA-3′) primers and cloned into the Gateway entry vector pDONR207 via a BP reaction to form pDONR207-S3Hpro. The S3H promoter in pDONR207-S3HPro was introduced into pMDC163 via LR reactions to generate the S3Hpro::GUS binary vector.

The histochemical GUS assays were performed using a previously described method (Jefferson et al., 1987). Samples were infiltrated with 90% (v/v) acetone for 20 min on ice and then washed with ultrapure water three times. Samples were then infiltrated under vacuum for 10 min and incubated with staining buffer (0.5 mg mL−1 5-bromo-4-chloro-3-indolyl glucuronide in 0.1 m Na2HPO4, pH 7, 10 mm Na2EDTA, 0.5 mm potassium ferricyanide/ferrocyanide, and 0.1% (v/v) Triton X-100) at 37°C for different times. After the staining buffer was removed, the samples were cleared with 70% ethanol. All observations with a light microscope were recorded with a Canon EOS60D camera. Chlorophyll was extracted and quantified as described previously (He and Gan, 2002).

Accession Numbers

Sequence data from this article are found in the GenBank/EMBL data libraries under the following accession numbers: AT5G24530 (S5H/DMR6), AT4G10500 (S3H), AT3G18780 (ACTIN2), AT1G74710 (ICS1), AT1G18870 (ICS2), AT5G03490 (UGT89A2), AT3G11480 (BSMT1), AT2G43840 (UGT74F1), AT2G43820 (UGT74F2), AT2G23620 (AtMES1), AT2G03760 (SOT12), GRMZM2G099467 (ZmFNSI-1), AT4G12560 (CPR1), AT5G64930 (CPR5), AT5G15410 (DND1), AT1G74710 (CPR6), and AJ889012 (GAGT).

Supplemental Data

The following supplemental materials are available.

Acknowledgments

We thank Dr. Jianmin Zhou (Institute of Genetics and Developmental Biology, Chinese Academy of Sciences) for providing the Pst DC3000 strains and Dr. Shunping Yan (Huazhong Agricultural University) for useful discussions. We also thank Pinghua Zhang and Zhiying Wang (Zhejiang Normal University) for their facility assistance.

Footnotes

1

This work was supported by grants from the Zhejiang Provincial Outstanding Young Scientist Award Fund (LR15C020001), the National Science Foundation of China (31670277 and 31470370), the 1000-Talents Plan for Young Researchers of China to K.W.Z., and the Zhejiang Provincial Public Welfare Project 2015C32043 to Y.J.Z. C.J.L. was supported by the Division of Chemical Sciences, Geosciences, and Biosciences, Office of Basic Energy Sciences of the U.S. Department of Energy (DOE) grant DEAC0298CH10886 (BO-169).

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