S5H/DMR6 Encodes a Salicylic Acid 5-HydroxylaseThat 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 and Kewei Zhanga,3

aInstitute of Plant Genetics and Developmental Biology, College of Chemistry and Life Sciences, ZhejiangNormal University, Jinhua, Zhejiang 321004, ChinabPlant Biology Section, School of Integrative Plant Science, Cornell University, Ithaca, New York 14853cDepartment of Biosciences, Brookhaven National Laboratory, Upton, New York 11973

ORCID IDs: 0000-0002-7475-103X (Y.J.Z.); 0000-0001-6189-8756 (C.-J.L.); 0000-0002-0844-1121 (K.W.Z.).

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 isassumed 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 notwell understood. Here, we report the discovery and functional characterization of DMR6 as a salicylic acid 5-hydroxylase (S5H) thatcatalyzes the formation of 2,5-DHBA by hydroxylating SA at the C5 position of its phenyl ring in Arabidopsis. S5H/DMR6 specificallyconverts SA to 2,5-DHBA in vitro and displays higher catalytic efficiency (Kcat/Km = 4.96 3 104 M

21 s21) than the previously reportedS3H (Kcat/Km = 6.09 3 103 M

21 s21) for SA. Interestingly, S5H/DMR6 displays a substrate inhibition property that may enableautomatic control of its enzyme activities. The s5h mutant and s5hs3h double mutant overaccumulate SA and display phenotypessuch as a smaller growth size, early senescence, and a loss of susceptibility to Pseudomonas syringae pv tomato DC3000. S5H/DMR6 issensitively induced by SA/pathogen treatment and is expressed widely from young seedlings to senescing plants, whereas S3H ismore specifically expressed at the mature and senescing stages. Collectively, our results disclose the identity of the enzyme requiredfor 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 aplant hormone that not only mediates plant defenseresponses against biotic and abiotic stresses but alsoplays a crucial role in regulating many physiologicaland biochemical processes during the entire plant life-span (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 indifferent members of the same family (Raskin, 1992). Forexample, in the model plant Arabidopsis (Arabidopsisthaliana), the basal levels of total SA range from 0.24 to1 mg g21 fresh weight (Nawrath and Métraux, 1999;Wildermuth et al., 2001; Brodersen et al., 2005), while inOryza sativa, its basal levels range from 0.01 to 37.19mg g21

freshweight (Silverman et al., 1995; Yang et al., 2004). SA isgeneratedvia twodifferent pathways in plants, namely thephenylalanine ammonia lyase (PAL) pathway and theisochorismate synthase (IC) pathway; both pathways re-quire the primary metabolite chorismate that is synthe-sized 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 seriesof enzymatic reactions that are initially catalyzed by PAL.However, in the IC pathway, chorismate is converted intoSA through an intermediate, isochorismate, produced byICs (Dempsey et al., 2011; Widhalm and Dudareva, 2015).Two ICs, ICS1 and ICS2, have been detected in Arabi-dopsis (Wildermuth et al., 2001; Garcion et al., 2008). TheIC pathway appears to be responsible for 90% of SA pro-duction when Arabidopsis is stimulated with a pathogenor UV light, and the PAL pathway may play a comple-mentary role in SA biosynthesis (Garcion et al., 2008).

Controlled SA levels are required for optimal reactiveoxygen species content and redox homeostasis (Lamband Dixon, 1997; Gapper and Dolan, 2006; Mateo et al.,

1 This work was supported by grants from the Zhejiang ProvincialOutstanding Young Scientist Award Fund (LR15C020001), the Na-tional Science Foundation of China (31670277 and 31470370), the1000-Talents Plan for Young Researchers of China to K.W.Z., andthe Zhejiang Provincial Public Welfare Project 2015C32043 to Y.J.Z.C.J.L. was supported by the Division of Chemical Sciences, Geoscien-ces, and Biosciences, Office of Basic Energy Sciences of the U.S. De-partment of Energy (DOE) grant DEAC0298CH10886 (BO-169).

2 These authors contributed equally to the article.3 Address correspondence to kwzhang@zjnu.edu.cn.The author responsible for distribution of materials integral to the

findings presented in this article in accordance with the policy de-scribed in the Instructions for Authors (www.plantphysiol.org) is:Kewei Zhang (kwzhang@zjnu.edu.cn).

K.W.Z. conceived and designed the experiments; Y.J.Z., L.Z., J.Z.Z., Y.J.L., J.B.W., and R.G. performed the experiments; K.W.Z., Y.J.Z.,L.Z., J.Z.Z., S.S.G., and C.-J.L. analyzed the data; K.W.Z., Y.J.Z., C.-J.L., and S.S.G. wrote and revised the article; all authors discussed theresults and collectively edited the article.

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2006). SA homeostasis directly affects its biologicalfunctions, such as development, photosynthesis, andpathogen responses (Mateo et al., 2006; Liu et al., 2010).Plants generally maintain hormone homeostasis byfine-tuning the balance between the biosynthesis andcatabolism of the hormone. ICS1 and ICS2 are two keygenes in the SA biosynthesis pathway that are regulatedby multiple factors, such as pathogen infections, eth-ylene signaling, and circadian rhythms (Chen et al.,2009; Wang et al., 2015; Zheng et al., 2015). Although asmall amount of SA produced in planta remains in afree state, most SA is subjected to biologically relevantchemical modifications by different enzymes aftersynthesis (Dempsey et al., 2011). SA glucosyltransfer-ases convert SA into salicylic acid O-b-glucoside (SAG)or salicyloyl Glc ester, which is transported from thecytosol into the vacuole for storage in Arabidopsis(Dean et al., 2005; Dean and Delaney, 2008). The ben-zoic acid/salicylic acid carboxyl methyltransferase1(BSMT1) catalyzes the formation of the SA methyl estermethyl salicylate, which renders SA inactive but in-creases its membrane permeability and facilitates thelong-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 evenhigher levels than SA and SA glycosides in Arabi-dopsis; therefore, SA hydroxylation is suggested to bethe major pathway for SA catabolism (Bartsch et al.,2010; Zhang et al., 2013).2,5-DHBA is one of the most widely produced aro-

matic acids in green plants (Griffiths, 1958; Ibrahimand Towers, 1959). Similar to other hydroxybenzoates,2,5-DHBA accumulates as glycoconjugates in plants,primarily as 2,5-DHBA 5-O-b-D-glucosides, 2,5-DHBA5-O-b-D-xylosides, or 2,5-DHBA 2-O-b-D-xylosides(Dean and Delaney, 2008; Tárraga et al., 2010; Li et al.,2014). 2,5-DHBA accumulates in response to differenttypes of plant-pathogen interactions in much higherlevels than SA (Bellés et al., 1999, 2006; Campos et al.,2014). The exogenous application of 2,5-DHBA to to-mato (Solanum lycopersicum), cucumber (Cucumis sat-ivus), andGynura auriantiaca induces the expression of adistinct subset of PR genes compared with the genesinduced by SA (Bellés et al., 1999, 2006); 2,5-DHBA alsoinduces RNA silencing-related genes and resistance toRNA pathogens in tomato and G. auriantiaca (Camposet al., 2014). Moreover, 2,5-DHBA itself displays anti-bacterial activity in the harvested fruits (Lattanzio et al.,1996). Although the enzymes UGT89A2 and GAGT,which are responsible for the conversion of 2,5-DHBAto its glycosides, have been well characterized (Limet al., 2002; Tárraga et al., 2010; Li et al., 2014) and thein vivo feeding of a 14C-labeled SA tracer in Gaultheriaprocumbens suggested that 2,5-DHBA was formed fromthe substrate SA half a century ago (Ibrahim andTowers, 1959), the enzymes responsible for the forma-tion of 2,5-DHBA are still a mystery.In a previous study screening formutantswith a loss of

susceptibility to the downy mildew Hyaloperonosporaparasitica, 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 tothe bacterial pathogen Pseudomonas syringae and theoomycete pathogenPhytophthora capsici (vanDammeet al.,2008; Zeilmaker et al., 2015). Map-based gene cloningdisclosed that DMR6 is a putative 2-oxoglutarate/Fe(II)-dependent dioxygenase [2-oxoglutarate-Fe(II) oxygenase]belonging to the 2-oxoglutarate-dependent dioxygenasesuperfamily (van Damme et al., 2008; Kawai et al., 2014).Another 2-oxoglutarate-Fe(II) oxygenase, salicylic acid3-hydroxylase (S3H; also namedDLO1), which is encodedby a homolog gene ofDMR6, was characterized to be ableto catalyze the formation of 2,3-DHBA and mediate leafsenescence andpathogen responses inArabidopsis (Zhanget al., 2013). The double mutant dmr6-3dlo1 of DMR6 andS3H (DLO1) displayed a dwarf phenotype, a substantialincrease in SA levels, and a complete loss of susceptibilityto the downy mildew Hyaloperonospora arabidopsidis(Zeilmaker et al., 2015). However, the underlying bio-chemical mechanism of DMR6 was still not revealed.

To characterize the enzyme that catalyzes the for-mation of 2,5-DHBA, we assayed a subset of the2-oxoglutarate-Fe(II) oxygenase family in Arabidopsisusing 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 itsessential roles in mediating SA homeostasis duringplant development, leaf senescence, and pathogen re-sponses in Arabidopsis.

RESULTS

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

We used HPLC to quantify the benzoate derivativesin the adult and senescing leaves of 10 plant species,Capsicum frutescens, Bambusa multiplex, Solanum lyco-persicum, Glycine max, Arachis hypogaea, Brassica cam-pestris, Sorghum bicolor, Vinca major, Oryza sativa, andArabidopsis, to identify the SA and SA hydroxylationprofiles in different plant species (Supplemental TableS1). The total SA (the sum of free SA and SA glucosides)levels varied in different species, ranging from 0.73 mgg21 fresh weight in S. bicolor to 115.38 mg g21 freshweight in B. multiplex, and the SA concentration in-creased in the senescing leaves of all species analyzed.Free 2,5-DHBA and 2,3-DHBA were not detected byHPLC in our experiments, indicating that the levels ofthose free SA derivatives are much lower than the freeSA levels. Although total 2,3-DHBA was detected onlyin Arabidopsis and V. major, total 2,5-DHBA wasdetected in all species analyzed. These data suggest that2,5-DHBA is a widely distributed hydroxylated pro-duct of SA in planta, consistent with the observations ofIbrahim and Towers (1959). Interestingly, we foundmuch higher 2,5-DHBA accumulated in senescingleaves than that in young leaves, suggesting that 2,5-DHBA may be involved in leaf senescence. We also

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quantified the benzoate derivatives in Arabidopsis inoc-ulated by the pathogen Pseudomonas syringae pv tomatoDC3000 (Pst DC3000). Similar to SA and 2,3-DHBA, 2,5-DHBA production in Arabidopsis was induced by thepathogen Pst DC3000 at 1, 2, 4, and 6 d post inoculation(dpi; Supplemental Fig. S1), indicating that 2,5-DHBAmay be involved in plant immunity.

Identification of an S5H in Vitro and in Vivo

We postulated that S5H is a 2-oxoglutarate-Fe(II) oxy-genase and that the gene encoding this enzyme is inducedby SA. Using an established enzymatic assay procedure(Zhang et al., 2013), we biochemically screened fivemembers of the Arabidopsis 2-oxoglutarate-Fe(II) oxy-genase family (Supplemental Fig. S2), which were all in-duced significantly by SA (Supplemental Fig. S3). Wefinally identified one recombinant protein encoded byAt5g24530, a gene characterized previously as DMR6(van Damme et al., 2008), responsible for 2,5-DHBA for-mation (Fig. 1), but the other four recombinant proteinsdid not possess the activity. The recombinant proteinencoded by At5g24530 specifically catalyzed the forma-tion of a compound exhibiting the same retention timeand the fluorescence emission spectra as the 2,5-DHBAauthentic standard from SA (Fig. 1, B and C), indicatingthat the recombinant enzyme possesses 5-hydroxylationactivity on SA in vitro. Therefore, the enzyme was des-ignated as S5H.According to a comparisonof the sequence

similarity, S5H/DMR6 and S3H are closely evolutionarilyrelated (Supplemental Fig. S2), sharing 50.57% similarity attheir amino acid levels (Supplemental Fig. S4). A homo-zygous mutant line with a T-DNA insertion in the thirdexon of At5g24530 (SK19807) was further characterized asa knockout line and was named s5h (Supplemental Fig.S5). The 2,5-DHBA level in the s5hmutant was reduced to12% of the wild-type level, and the levels were restored byintroducing the expression of the At5g24530 gene in thes5h mutant background (i.e. S5H-OX lines; SupplementalFig. S6), indicating that the protein encoded by At5g24530functions as an S5H in vivo.

Kinetic Parameters of Recombinant S5H/DMR6

The biochemical properties of recombinant S5H/DMR6were investigated further. The recombinant S5H/DMR6protein was purified, and the preferred temperature andpH value were optimized (Supplemental Fig. S7). Enzy-matic activity increased as the temperature increased from4°C to 40°C and decreased as the temperature increasedfrom 40°C to 50°C, suggesting that the optimal tempera-ture for S5H/DMR6 activity is approximately 40°C(Supplemental Fig. S7B). The effect of pH on S5H/DMR6activity also was evaluated, and the optimal pH of theenzyme was 6.8 under our conditions (Supplemental Fig.S7C). At optimal pH and temperature conditions, the cal-culated apparentKmvalue of the recombinant S5H/DMR6enzymeonSAwas approximately 5.156 1.44mM,which is

Figure 1. Conversion of SA to 2,5-DHBA bythe recombinant S5H/DMR6protein in vitro.A,Biochemical reaction catalyzed by S5H/DMR6in vitro. B,HPLCprofiles of the 30-min reactionof the recombinant S5H/DMR6 protein (S5H)and empty vector extracts (EV) incubated withSA. Authentic 2,5-DHBA was used as a stan-dard. C, The fluorescence emission spectra ofthe enzymatic product 2,5-DHBA is identical tothat of the 2,5-DHBA standard. D, Kinetics ofthe recombinant S5H/DMR6 protein (SA as thesubstrate). The data are presented asmeans6 SE

(n = 3).

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much lower than the Km of S3H (58.29 mM; Zhang et al.,2013). The Kcat value of S5H/DMR6 (0.255 s21) is approx-imately 71.9% of that of S3H (0.355 s21), indicating thatS5H/DMR6 possesses a slightly lower turnover rate to SAthan S3H. Overall, the calculated Kcat/Km for S5H/DMR6was 4.963 104 M

21 s21, which is 8-fold higher than that forS3H (6.09 3 103 M

21 s21), indicating that S5H/DMR6 dis-plays a much higher catalytic efficiency than S3H to SAin vitro. Additionally, in contrast to S3H, S5H/DMR6wasinhibited substantially by its substrate SA, with a calcu-lated Ksi value of 6.056 1.46 mM, which leads to a velocitycurve that surged to its maximum and then descended asthe substrate concentration increased (Fig. 1D).

Spatial and Temporal Expression Patterns of S5H/DMR6and Its Responses to SA and Pathogens

The expression patterns of S5H/DMR6, togetherS3H, were evaluated under different conditions byquantitative reverse transcription (qRT)-PCR and GUSreporter gene expression to obtain a better under-standing of the potential physiological and molecularroles of the genes (Figs. 2 and 3). S5H/DMR6 was con-stitutively expressed in young and mature leaves, andits expression levels increased significantly in leaves inearly and late senescence (Figs. 2A and 3, A, C, E, G, andI). In contrast, the S3H gene was nearly undetectablein young leaves (Figs. 2A and 3, B and D), detectedat moderate levels in mature leaves (Fig. 3F), andexpressed abundantly in leaves in early and late se-nescence (Figs. 2A and 3,H and J). The expression levelsof both S5H/DMR6 and S3Hwere reduced dramatically

in the leaves ofNahG transgenic plants (NahG encodes anSA hydroxylase that degrades SA to catechol; van WeesandGlazebrook, 2003) comparedwithwild-type leaves atthe same stages, indicating that the expression levels ofthe two geneswere highly dependent on SA (Fig. 2A).Wethen quantified S5H/DMR6 and S3H expression in wild-type plants treated with 0.1 to 6 mM SA (Fig. 2B). S5H/DMR6 and S3H expressionwere both induced by SA, andthe relative S5H/DMR6 expression levels were muchhigher (up to 10-fold) than the S3H levels, indicating thatS5H/DMR6wasmore sensitive to theSA treatment (Fig. 2B).

S5H/DMR6 and S3H expression also were investi-gated in response to the pathogen Pst DC3000 at 1, 2, 4,and 6 dpi (Fig. 2, C and D). Both S5H/DMR6 and S3Hexpression were induced by the pathogen, and theirexpression levels peaked on day 6, immediately afterthe total SA reached its peak value (Supplemental Fig.S1), implying that SA hydroxylation plays a role in thedetoxification of excessive SA after the initiation of thepathogen response. A comparison of the expression ofthe S5H/DMR6 and S3H genes with the internal stan-dard ACTIN2 revealed that the highest levels of S5H/DMR6 expression were apparently 3-fold higher thanthe highest levels of S3H expression, suggesting thatS5H is more sensitive to pathogen induction at thetranscriptional level (Fig. 2, C and D). The GUS stainingwas more intensive in the leaves of both S5Hpro::GUSand S3Hpro::GUS transgenic plants that were inocu-lated 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 ex-pression patterns of S5H/DMR6 and S3H revealed byqRT-PCR analyses (Fig. 2, C and D).

Figure 2. SA-induced, pathogen-induced,and senescence-associated patterns of S5H/DMR6 and S3H expression. A, Expression ofthe S5H/DMR6 and S3H genes during leafsenescence in wild-type (WT) and NahGtransgenic plants. YL, Young leaves; ML, ma-ture leaves; ES, early senescence leaves; LS,late senescence leaves. B, S5H/DMR6 andS3H expression were both induced by differ-ent concentrations of SA after a 6-h treatment,and S5H/DMR6 was expressed at 10-foldhigher levels than S3H. C and D, S5H/DMR6(C) and S3H (D) expression inwild-type plantsinoculated with Pst DC3000 at 1, 2, 4, and6 dpi; 0 represents untreated wild-type plants,and M represents the mock treatment. Thedata are presented as means 6 SE (n = 3).

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

The s5hs3h double mutant was generated for a detailedphenotypic analysis to further investigate the biologicalfunctions of S5H/DMR6 in plant development, leaf se-nescence, and plant immunity (Supplemental Fig. S5).The s5h, s3h, and s5hs3h mutants and the representativeS5H/DMR6 overexpression lines S5H-OX3 in the wild-type background and S5H-OX18 in the s5h backgroundwere grown in parallel in the same tray for the phenotypicanalysis. At 21 d after germination (DAG), the diametersof the rosette leaves of s5h and s5hs3hwere 86% and 60%of the wild type, respectively. The S5H-OX18 line in thes5h background restored the phenotype of the s5hmutantto the wild-type phenotype, and the S5H-OX3 line in thewild-type backgrounddisplayed even larger leaves (up to114% of the wild type; Fig. 4B), indicating that S5H/DMR6 plays an important role in the early developmentof Arabidopsis.

At 35 and 49 DAG, the s5hmutant displayed an earlysenescence phenotype, similar to the s3h mutant, andthe s5hs3h mutant showed a more dramatic early se-nescence phenotype. In contrast, the S5H/DMR6 over-expression lines S5H-OX3 and S5H-OX18 exhibited adelayed leaf senescence phenotype (Fig. 4A). Consis-tent with the visible phenotypes, the chlorophyll con-tents in the fifth to sixth leaves of the s5h and s3hmutants were reduced to 57% and 55% of the wild-type contents at 49 DAG, respectively; in contrast, the

chlorophyll contents of the S5H/DMR6 overexpressionlines S5H-OX3 and S5H-OX18 were 40% and 25%higher than in the wild-type line at 49 DAG, respec-tively (Fig. 4C). Thus, S5H/DMR6, in cooperation withS3H, plays an essential role in the leaf senescence pro-cess in Arabidopsis.

Since S5H/DMR6 and 2,5-DHBA were sensitivelyinduced by SA and PstDC3000 pathogens, we assessedthe pathogen resistance of the wild type, the s3h,s5h, and s5hs3h mutants, and the S5H/DMR6 over-expression lines S5H-OX3 and S5H-OX18 by inoculat-ing them with Pst DC3000. Compared with the wildtype and s3h mutant lines, the s5h and s5hs3h mutantsshowed a loss of susceptibility, the S5H-OX3 line dis-played enhanced susceptibility to Pst DC3000 over thatof the wild type, while the S5H-OX18 line rescued theloss-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 fur-ther support the hypothesis that the disruption of S5H/DMR6 leads to a loss of susceptibility to Pst DC3000 inArabidopsis.

A Transcriptional Analysis Reveals the ComplementaryRoles of S5H/DMR6 and S3H

We further analyzed S5H/DMR6 and S3H expressionin the rosette leaves of the s5h, s3h, and s5hs3h mu-tants at 21, 35, and 49 DAG by qRT-PCR to under-stand the relation between S5H/DMR6 and S3H at the

Figure 3. Spatial and temporal patterns ofS5H/DMR6 and S3H expression were de-termined by GUS activities in S5Hpro::GUS and S3Hpro::GUS transgenic plants.A and B, C and D, E and F, G and H, and Iand J show transgenic S5Hpro::GUS andS3Hpro::GUS plants at 10, 21, 28, 35, and49 DAG, respectively. K and L presentmock and Pst DC3000-inoculated ro-sette leaves from S5Hpro::GUS trans-genic plants. M and N show mock andPst DC3000-inoculated rosette leavesfrom S3Hpro::GUS transgenic plants. Thearrows point to the area where the GUSstaining changed significantly between Kand L and betweenMandN.Bars = 1mm(A and B), 1 cm (C–J), and 2 mm (K–N).

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transcriptional level (Fig. 5). Compared with the wildtype, the S3H expression level increased significantly to125-, 1.86-, and 1.8-fold in the s5h mutant at 21, 35, and49 DAG, respectively (Fig. 5A). Similarly, S5H/DMR6expression was increased by 3.95-, 2.47-, and 1.43-foldin the s3h mutant at 21, 35, and 49 DAG, respectively(Fig. 5B). Based on these results, S5H/DMR6 and S3Hdisplay complementary functions at the transcript levelin Arabidopsis.

A Metabolite Analysis Reveals the Cooperation ofS5H/DMR6 and S3H in Regulating SA Homeostasis

Subsequently, the metabolites of SA, SAG, and theirhydroxyl products were quantified in the wild-type,s3h, s5h, and s5hs3h lines at 21, 35, and 49 DAG, whichrepresent young, mature, and senescence stages, re-spectively (Fig. 6). At the young stage (21DAG), the freeSA and total SA contents were increased significantlyby 43% and up to 239% in the s5h mutant and by 178%and up to 2,333% in the s5hs3h double mutant, respec-tively (Fig. 6, A and B), but they did not show obviouschanges in the s3hmutant compared with the wild-typeline (Fig. 6, A and B), suggesting that S5H/DMR6 reg-ulates SA homeostasis at the early growth stage. At35 DAG, although the free SA and total SA contentswere increased by 66% and 193% in s3h, the free SA andtotal SA contents in the wild type and s5h mutant lineswere not obviously different. However, in the s3hbackground, the free SA and total SA contents in thes5hs3h mutant were increased 164% and 308% com-pared with the s3h single mutant (Fig. 6, A and B), in-dicating the S3H and S5H/DMR6 have redundantfunctions at the mature stage. At 49 DAG, the free SAand total SA contents were similar in the s3h and s5hs3hmutants (Fig. 6, A and B), indicating that S3H plays theessential role at the late senescence stage.

In contrast to the continuous light condition (Zhanget al., 2013), 2,3-DHBA was undetectable at 21 and35 DAG in the wild-type line under a 16-h-light/8-h-dark photoperiod but was detected in the s5h mutantunder the same conditions, suggesting that S3H plays acomplementary role in maintaining SA homeostasis inthe absence of S5H/DMR6 (Fig. 6C), consistent with theoverexpression of the S3H gene in the s5h mutant (Fig.5A). 2,3-DHBA was detected in the wild-type line until35 DAG, a maturation stage at which the plant isstarting to undergo senescence, and high levels weredetected at 49 DAG, a senescence stage, suggesting thatthe main physiological function of S3H occurs duringleaf senescence (Fig. 6C).

In contrast to 2,3-DHBA, 2,5-DHBA was detected at21 DAG (Fig. 6D), and its content was increased as theSA concentration increased in the wild-type plants. Inthe s3h mutant, the 2,5-DHBA content increased 97%comparedwith the wild-type line at 49 DAG, indicatingthat S5H/DMR6 partially complemented the mutantphenotype in the s3h line in the absence of S3H, con-sistent with the overexpression of the S5H/DMR6 gene

Figure 4. Phenotypes of the s3h, s5h, s5hs3h, and S5H-OX lines. A,Representative images of the s3h, s5h, and s5hs3h mutants and over-expression lines S5H-OX3 and S5H-OX18 at 21, 35, and 49DAG. Bars =2 cm. B, Quantification of the diameters of rosette leaves from the indi-cated plants at 21DAG. C, Quantification of total chlorophyll contents inthe fifth and sixth leaves from the plants shown in A at 49 DAG. The dataare presented as means 6 SE (n $ 10). FW, Fresh weight. D, Diseasesymptoms 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 s5hs3hmutants at30DAG showed significantly higher resistance to PstDC3000 suspension(OD600 = 0.001) infiltration than the wild type. F, The overexpression lineS5H-OX3 in the wild-type background was more susceptible to PstDC3000 suspension (OD600 = 0.0001) infiltration than the wild type,while the overexpression line S5H-OX18 in the s5h background rescuedthe loss-of-susceptibility phenotype of s5h to the wild type. CFU, Colony-forming units. The data are presented as means6 SD (n = 8). *, P , 0.05and **, P , 0.01 (Student’s t test).

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in the s3hmutant (Fig. 5B). Interestingly, 2,5-DHBAwasnot completely absent in the s5h and s5hs3hmutants butwas 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 gov-erning SA homeostasis is essential for understandingthe developmental and defense-related processes me-diated by this hormone. 2,5-DHBA is the major cata-bolic product of SA and is detected in a wide variety ofplant species (Supplemental Table S1). In this study, weidentified S5H/DMR6 as an additional 2-oxoglutarate-Fe(II) oxygenase that is responsible for converting SA to2,5-DHBA. Interestingly, plants have evolved an ele-gant feedback regulatory mechanism involving S5H/DMR6 and S3H, which are complementarily and co-operatively responsible for SA catabolism andmaintainSA homeostasis during development, leaf senescence,and immunity of Arabidopsis.

The characterization of S5H/DMR6 as the majorenzyme catalyzing the formation of 2,5-DHBA ade-quately addresses the biochemical mechanism of dmr6and further solves a puzzle in the pathway of SA ca-tabolism (Fig. 7). DMR6 was reported recently as ahomolog of the maize (Zea mays) flavone synthaseZmFNSI-1, which catalyzes the formation of apigeninfrom naringenin (Falcone Ferreyra et al., 2015). Underour assay conditions, only low S5H/DMR6 activitywasdetected when naringenin was used as the substrate;and the specific activity of S5H/DMR6 toward the SAsubstrate was approximately 179- and 1,429-fold higherthan its activity toward naringenin at 30°C and 40°C,respectively (Supplemental Fig. S8). Based on thesedata, S5H/DMR6 prefers SA over naringenin as itsnatural substrate. Thus, we conclude that the strongpathogen resistance of dmr6 was most likely caused byenhanced SA levels but was unlikely due to the absenceof apigenin.

2,5-DHBA is an important aromatic compound pro-duced in planta, and the identification of the S5H/DMR6

enzyme enabled us to investigate the biochemical mech-anism of 2,5-DHBA formation. 2,5-DHBA and 2,3-DHBAwere suggested previously to be synthesized by a non-enzymatic reaction in which SA scavenged hydroxylradicals (Maskos et al., 1990; Chang et al., 2008). Theglycosides of 2,5-DHBA and 2,3-DHBA were distributedabundantly in Arabidopsis and were suggested to beformed predominantly from the substrate SA via the ICpathway (Bartsch et al., 2010). We previously character-ized S3H, which converts SA to 2,3-DHBA (Zhang et al.,2013). As shown in this study, S5H/DMR6 is responsiblefor 2,5-DHBA formation both in vivo and in vitro. Com-pared with S3H, which converts SA to both 2,3-DHBAand 2,5-DHBA in vitro, S5H/DMR6 more specificallyconverts SA to 2,5-DHBA in vitro (Fig. 1). The apparentKm for S5H/DMR6 toward SA is 5.15 mM, which is muchlower than the Km for S3H (58.29 mM; Zhang et al., 2013),suggesting that S5H/DMR6 may have a higher bindingaffinity for the SA substrate than S3H. Although S5H/DMR6displayed a slightly lowerKcat for SA than S3H, thecatalytic efficiency of S5H/DMR6 was much higher thanthat of S3H, based on the Kcat/Km values of the two en-zymes. Comparedwith the other known enzymes (Fig. 7)involved in SA modification, including UGT74F1/F2(Km = 230/190mM), BSMT1 (Km = 16mM),MES1 (Km = 57–147.1mM), and SOT12 (Km=440mM;Dempsey et al., 2011),theKm of S5H/DMR6 is the lowest; the lowKmmay allowS5H/DMR6 to compete with the other SA-utilizing en-zymes in Arabidopsis, indicating its essential roles in SAcatabolism. Meanwhile, the biochemical properties ofS5H/DMR6 allow it to catalyze SA at low concentrationsand, thus, to fine-tune the SA level during the early de-velopment of Arabidopsis.

The biological function of an enzyme is determinednot only by the catalytic parameters of the enzyme butalso is attributed to its spatial and temporal expressionpattern. The expression pattern of S5H/DMR6 partiallyoverlapped with but was largely distinct from that ofS3H. Similar to S3H, S5H/DMR6 expression was in-duced by pathogens, leaf senescence, and SA. SA andpathogens increased S5H/DMR6 expression 10- and3-fold compared with S3H, respectively (Fig. 2, B–D),supporting the conclusion that S5H/DMR6 maintainsSA homeostasis at low concentrations, whereas S3Hmaintains homeostasis at high concentrations. After

Figure 5. Transcriptional analysis of wild-type (WT), s3h, s5h, and s5hs3h plantsby qRT-PCR. A, S3H expression was in-creased significantly 125-, 1.86-, and 1.8-fold in the s5hmutant compared with thewild-type plants at 21, 35, and 49 DAG,respectively. B, Similarly, S5H/DMR6 ex-pressionwas increased significantly 3.95-,2.47-, and 1.43-fold in the s3hmutant at21, 35, and 49 DAG, respectively. Thedata are presented as means6 SE (n = 3).*, Not detected or detected at low levels.

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comparing the temporal expression patterns, S5H/DMR6is expressed from young to senescence stages, but S3H isexpressed specifically at the mature and senescing stagesin Arabidopsis (Zhang et al., 2013), suggesting that S5H/DMR6 is evolutionarily a housekeeping gene and S3H is aspecific senescence-associated gene that are coopera-tively involved in maintaining SA homeostasis duringArabidopsis development. Regarding spatial expres-sion, S5H/DMR6 was expressed in mesophyll cells,whereas S3H was expressed mainly around the vas-cular tissue in the early stage and in mesophyll cells insenescence (Fig. 3). The inducible spatial and temporalexpression patterns of S5H/DMR6 and S3H, along withthe distribution of 2,5-DHBA and 2,3-DHBA glycosidesin pathogen-stimulated young and senescing leaves,support the hypothesis that S5H/DMR6 and S3H co-operatively maintain SA homeostasis through a feed-back mechanism.

As an endogenous signal, SA is a key signalingmolecule in various plant functions, such as diseaseresistance, development, and leaf senescence (Vlotet al., 2009). At the early development stage, SA ho-meostasis was impaired in the s5h mutant and thes5hs3h double mutant and the SA levels were increased43% and 178%, respectively, resulting in a significantreduction of the rosette leaf size, indicating that SA af-fects plant size. This conclusion was supported by thesuppression of the dwarf phenotype by crossing thedmr6-3dlo1 mutant to the sid2 background (Zeilmakeret al., 2015). Similar phenomena have been observed inother SA overaccumulation mutants, such as cpr1-1,cpr5-1, cpr6-1, and dnd1-1, which showed dwarf phe-notypes (Jirage et al., 2001). On the contrary, theSA-deficient plants of NahG transgenic Arabidopsis,the sid2 mutant and the S3H overexpression lineS3HOE1, similar to the overexpression line of S5H/DMR6,

Figure 6. Accumulation of free or total SA, 2,3-DHBA, and 2,5-DHBA at different developmental stages. A, The free SA levels in thes5h mutant and the s5hs3h double mutant were significantly higher than the wild-type (WT) level, whereas the S5H/DMR6 over-expression line S5H-OX18 in the s5h background restored the SA level in the s5hmutant at 21DAG; the free SA levels in the s3h ands5hs3hmutantswere significantly higher than thewild-type levels at 35 and 49DAG; the free SA levelswere reduced significantly inthe S5H/DMR6 overexpression lines S5H-OX3 and S5H-OX18 at 49 DAG. B, The total SA levels were increased significantly in thes5h and s5hs3hmutants at 21 DAG and in the s3h and s5hs3hmutants at 35 and 49 DAG; the total SA levels were decreased in theS5H-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, and49DAG 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 thes3hmutant at 21, 35, and 49 DAG; the total 2.5-DHBA levels were decreased significantly in the s5h and s5hs3hmutants at 21, 35,and 49DAGand in the S5H-OX lines at 49DAG. The data are presented asmeans6 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.

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exhibited larger rosette leaves (Fig. 4, A and B; Abreu andMunné-Bosch, 2009; Zhang et al., 2013). In contrast to thes5h mutant, the s3h mutant showed no obvious mor-phological difference comparedwith the wild-type line atthe early development stage but exhibited accelerated leafsenescence at later growth stages, suggesting that S3Hhasmore specific functions in leaf senescence under normalconditions. Although the expression of the S5H/DMR6and S3H genes was induced significantly by pathogenstimulation (Fig. 2, C and D), the s5h mutant exhibitedmuch stronger pathogen resistance than the s3h mutant,as supported by the detailed analyses of the dmr6mutant(van Damme et al., 2008; Zeilmaker et al., 2015), which ispossibly attributed to its more predominant gene ex-pression response and higher binding affinity for SA.

In summary, both S5H/DMR6 and S3H expressionwere strongly induced by SA and convert SA to either

2,5-DHBA or 2,3-DHBA, respectively, thus forming afeedback mechanism for SA catabolism in which S5H/DMR6 and S3H maintain SA homeostasis in Arabi-dopsis (Fig. 7). S5H/DMR6 and S3H corporately bal-ance SA homeostasis at different development stagesand under various stress conditions. In addition toS5H/DMR6, plants may express another enzyme orutilize another mechanism to produce 2,5-DHBA; fur-ther studies are needed to examine these possibilities.Furthermore, the mechanisms regulating S5H/DMR6and S3H at the transcriptional and posttranscriptionallevels are unknown. An understanding of the regula-tory mechanisms will help researchers decipher thephysiological roles of SA and facilitate the developmentof methods for mediating plant development andpathogen resistance by manipulating SA metabolismin crops.

Figure 7. Simplified schematic of the pathways for SA modification and catabolism in Arabidopsis. The expression of the newly dis-covered S5H/DMR6, indicated in boldface, is induced by accumulated SA (the encoded S5H/DMR6 is indicated by the red arrow), andthe enzyme catalyzes the formation of 2,5-DHBA from the substrate SA. SA also is converted to 2,3-DHBA by an enzyme encoded byanother SA-induced gene, S3H (the encoded S3H is indicated by the black arrow), as reported previously. 2,5-DHBAand 2,3-DHBAareconjugated subsequently byUGT89A2 to produce 2,5-DHBA- or 2,3-DHBA-sugar conjugates for storage in vacuoles. SA is converted toits storage form, salicylate-sugar conjugates, by the UGT74F1 and UGT74F2 enzymes. The methyltransferase BSMT1 is responsible forthe production of the functional form methylsalicylic acid from SA, and methylesterases 1, 2, 7, and 9 are responsible for the reversereaction from methylsalicylic acid to SA. The question marks near GH3.5/WES1 and SOT12 indicate that the enzymes possess theactivities to catalyze the formation of salicyloyl-L-Asp and SA-2-sufonate, respectively, from SA in vitro, but their functions in plantsremain to be determined. The dotted arrow and question mark represent uncertainty regarding whether the 2,5-DHBA accumulated inthe s5h mutant is produced from the SA substrate by an unknown enzyme.

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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 (Zhanget al., 2013). The s5h mutant (SK19807) was obtained from the ArabidopsisBiological Resource Center (Robinson et al., 2009). Seeds were sown on petridishes containing Murashige and Skoog medium with 0.25% (w/v) phytogeland appropriate antibiotics and incubated at 4°C for 3 d before being moved toa growth chamber. Seedlings with two true leaves were transplanted to soilunder a 16-h-light/8-h-dark photoperiod at approximately 60% humidity,unless noted otherwise. The light intensity was approximately 110mmolm21 s21.Wild-type, mutant, and/or transgenic plants were grown in parallel in the sametray to minimize possible variations in growth conditions. Capsicum frutescens,Bambusa multiplex, Solanum lycopersicum, Glycine max, Arachis hypogaea, Brassicacampestris, Sorghum bicolor, Vinca major, and Oryza sativa were harvested from afield in Jinhua, Zhejiang, China, in summer 2016.

Identification of Mutants

Gene-specific primers S5H-M1 (59-GGAAATAGTAAGTAAATACAGTAG-39),S5H-M2 (59-TTAGTTGTTTAGAAAATTCTCGA-39), and S5H-M3 (59-TATGTT-GATGACAAAAGCAT-39) and the T-DNA border primer TB1 (59-TGGACGT-GAATGTAGACACGTCG-39) were used to identify homozygous s5h mutantplants. Gene-specific primers S3H-M1 (59-ATGGCAACTTCTGCAATATC-39) andS3H-M2 (59-TTAGGTTGTTGGAGCTTTGA-39) and the T-DNA border primer TB2(59-TGGTTCACGTAGTGGGCCATCG-39) were used to identify homozygous s3hmutant plants.

SA and Pathogen Treatment

Arabidopsis plants (25 DAG) were sprayed with different concentrations ofSA 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 describedpreviously (Katagiri et al., 2002). All rosette leaves of individual plants werecollected at different time points after treatment for RNA extraction or bacterialcounting. The leaf discs (0.28 cm2) were excised from leaves with a smaller corkborer and used for bacterial counting.

Protein Expression and Purification

The S5H/DMR6 coding sequencewas PCRamplifiedusing apair of primers,S5H_BamHI (59-TTTAAGGATCCATGGCGGCAAAGCTGATATC-39) andS5H_SalI (59-CATGGTCGACTTAGTTGTTTAGAAAATTCTCGA-39), andcloned into pET28a (Novagen) to form pET28a-S5H and produce the His-tagged recombinant S5H/DMR6 protein. The pET28a-S5H construct was in-troduced into Escherichia coli BL21 (DE3, pLys3; Invitrogen). Bacterial cellscontaining pET28a-S5Hwere grown in Luria-Bertani medium containing 50mgL21 kanamycin at 37°C to an optical density of approximately 0.6 at 600 nm,induced with 0.5 mM isopropyl b-D-1-thiogalactoside, and then incubated at18°C for 24 h. The recombinant S5H protein was purified by Ni-NTA affinitychromatography using a previously describedmethod (Zhang et al., 2013). DTT(final concentration, 2 mM) was added to the enzyme solution, and the proteinwas immediately stored at 280°C.

Enzyme Assays

The enzyme assay was performed according to a previously describedmethod (Zhang et al., 2013). The reactionmixture (100mL) contained 5mMDTT,4mMsodiumascorbate, 1mM2-oxoglutaric acid, 0.4mMFeSO4, 0.1mgmL21 catalase,50 mM Tris-HCl (pH 8) or other buffer, 1 to 15 mg of recombinant protein, andvarious concentrations of SA. The protein was incubated with 200 mM SAsubstrate in phosphate buffer (pH 6.8) at different temperatures for 30 min, or200 mM SA substrate in citrate buffer (pH 5.3), phosphate buffer (pH 6, 6.5, 7, or7.5), or Tris-HCl buffer (pH 8) at 28°C for 30 min, to determine the optimaltemperature and pH for S5H/DMR6 enzyme activity. Various SA substrateconcentrations ranging from 2 to 60 mM were used for the enzyme kineticsanalysis, and the incubation proceeded at 40°C for 1 min (2–8 mM) or 3 min (10–60 mM) at pH 6.8. All reactions were started by adding the enzyme and stoppedby adding 100 mL 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 for10 min, the supernatant was analyzed by HPLC. Substrate inhibition kineticswere 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 primersS5H_BamHI (59-TTTAAGGATCCATGGCGGCAAAGCTGATATC-39) andS5H_PstI (59-CTGCAGTTAGTTGTTTAGAAAATTCTCGA-39) to construct abinary S5H-OX vector for S5H/DMR6 overexpression. The PCR product wascloned into pGEM-T (Promega) to generate pGEM-S5H. The coding sequencewas then released from the vector by digestion with BamHI and PstI and wassubcloned into the BamHI and PstI sites of pGL800 (a modified 35S over-expression vector with the pZP211 backbone; Zhang and Gan, 2012) to generatepGL800-S5H, which was transformed into the s5h and wild-type lines to gen-erate S5H overexpression lines in the s5h and wild-type backgrounds, respec-tively. More than 20 independent transgenic plants were generated, and therepresentative lines S5H-OX3 in the wild-type background and S5H-OX18 inthe s5h background were presented. All constructs described above were con-firmed by sequencing.

Metabolite Extraction

All rosette leaves from wild-type, s3h, s5h, s5hs3h, and S5H-OX transgenicplants were collected at different developmental stages for the metaboliteanalysis. SA was extracted using a previously described method for the ex-traction of phenolic compounds (Zhang et al., 2012), with some modifications.The rosette leaves were ground in liquid nitrogen. Approximately 100mg of thepowders was added to 1 mL of 80% methanol containing 50 mM methyl salic-ylate (used as an internal standard) in a 2-mL Eppendorf tube. The Eppendorftube was agitated for 2 h at 4°C and then centrifuged at 13,000g for 10 min at4°C. The supernatant was transferred into a new Eppendorf tube, and the pelletwas reextracted with 500 mL of 100% methanol. Both extracts were combinedand air dried with nitrogen gas before being dissolved in 500 mL of sodiumacetate (0.1 M, pH 5.5). Half of the suspensionwas used for the HPLC analysis ofthe free SA, 2,3-DHBA, and 2,5-DHBA contents. The other half was treated with10 mL of b-glucosidase (1 unit mL21) and hydrolyzed in a 37°C water bath for2 h. After the hydrolysate was denatured in boiling water for 5 min andcentrifuged at 13,000g for 10 min at 4°C to pellet the protein, the supernatantwas used for the HPLC analysis of the total SA, 2,3-DHBA, and 2,5-DHBAcontents.

HPLC

An Agilent 1260 HPLC system (Agilent Technologies) coupled with a diodearray detector and a fluorescence detector and a Zorbax SB-C18 column (4.6 3250mm, 5mm;Agilent Technologies) were used for themetabolite analysis. TheHPLC method was based on a previously described method (Marek et al.,2010), with some modifications. The mobile phases were composed of sodiumacetate (0.2 M, pH 5.5) and methanol. The initial methanol gradient was main-tained at 3% (v/v) for 12 min, linearly increased to 7% (v/v) at 12.5 min, andmaintained until 38 min. After 1 min, the initial conditions were restored andthe system was allowed to equilibrate for 7 min before the next injection. Theflow rate was maintained at 0.8 mL min21 throughout the process. SA or 2,5-DHBAwas detectedwith a 296-nm excitationwavelength and 410-nm emissionwavelength or with a 320-nm excitation wavelength and 449-nm emissionwavelength using a fluorescence detector. 2,3-DHBAwas detectedwith a diodearray detector at 223 nm. The concentration was calculated by determining theHPLC peak area according to a standard curve.

Real-Time qRT-PCR Analyses

Total RNAwas 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)onanABIPRISM7700system(AppliedBiosystems).ACTIN2wasusedasaninternal control in the analysis of the qRT-PCR data fromwhole leaves. The qRT-PCRprimers included S3HQ1 (59-TTCATCGTCAATATCGGCGAC-39) and S3HQ2 (59-ATCGATAACCGCTCGTTCTCG-39) for S3H, S5HQ1 (59-TACCTGCTCA-TACCGACCCAAA-39) and S5HQ1 (59-ATTAACGGCGAACCACTGACC-39)

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for S5H/DMR6, and ActinQ1 (59-GGTAACATTGTGCTCAGTGGTGG-39) andActinQ2 (59-CTCGGCCTTGGAGATCCACATC-39) 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 (59-GCAGGCTCCGAATTCTCCCAAACCATGATGGCACC-39) and S5H-P2 (59-AAGCTGGGTCGAATTCCAGAAAATTGAAGAAGAATC-39) primers andcloned into the Gateway entry vector pCR8 (Thermo Fisher Scientific) via thesequence-and-ligation-independent cloning method (Jeong et al., 2012) to formpCR8-S5Hpro. The S5H/DMR6promoter in pCR8-S5Hprowas introduced intopMDC163 via LR reactions to construct S5Hpro::GUS binary vectors. The 2.6-kbS3H promoter was amplified with S3H-P1 (59-GGGGACAAGTTTGTACAA-AAAAGCAGGCTTCAACTCTTATACTCACTTTCAGCA-39) and S3H-P2 (59-GGGGACCACTTTGTACAAGAAAGCTGGGTTAATGTAATTTTGTAATGTCA-39)primers and cloned into the Gateway entry vector pDONR207 via a BP reaction toform pDONR207-S3Hpro. The S3H promoter in pDONR207-S3HPro was intro-duced into pMDC163 via LR reactions to generate the S3Hpro::GUS binary vector.

The histochemical GUS assays were performed using a previously describedmethod (Jefferson et al., 1987). Samples were infiltrated with 90% (v/v) acetonefor 20 min on ice and then washed with ultrapure water three times. Sampleswere then infiltrated under vacuum for 10 min and incubated with stainingbuffer (0.5 mgmL21 5-bromo-4-chloro-3-indolyl glucuronide in 0.1 MNa2HPO4,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 wasremoved, the samples were cleared with 70% ethanol. All observations with alight microscope were recorded with a Canon EOS60D camera. Chlorophyllwas extracted and quantified as described previously (He and Gan, 2002).

Accession Numbers

Sequence data from this article are found in the GenBank/EMBL data librariesunder 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.

Supplemental Figure S1. Accumulation of free or total SA, 2,3-DHBA, and2,5-DHBA in rosette leaves after Pst DC3000 inoculation.

Supplemental Figure S2. Phylogenic tree of S5H/DMR6 and its homologsin the Arabidopsis genome.

Supplemental Figure S3. SA-inducible expression patterns of five SA5-hydroxylase candidate genes.

Supplemental Figure S4. Amino acid alignment of deduced S3H andS5H/DMR6.

Supplemental Figure S5. Characterization of s5h and s5hs3h mutants.

Supplemental Figure S6. Quantification of SA, 2,5-DHBA, and their sugarconjugates in the wild type, s5h, and S5H/DMR6 overexpression lines inthe s5h background by HPLC.

Supplemental Figure S7. Purification and reaction optimization of the re-combinant S5H/DMR6 protein in E. coli.

Supplemental Figure S8. Specific activities of recombinant S5H/DMR6 onsubstrate naringenin and SA at 30°C and 40°C.

Supplemental Table S1. Quantification of benzoate derivatives related toSA from 10 plant species by HPLC.

ACKNOWLEDGMENTS

We thank Dr. Jianmin Zhou (Institute of Genetics and DevelopmentalBiology, 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 andZhiyingWang (ZhejiangNormal University) fortheir facility assistance.

Received May 24, 2017; accepted September 8, 2017; published September 12,2017.

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SA 5-Hydroxylase Fine-Tunes SA Homeostasis

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