Two Chalcone Synthase Isozymes Participate Redundantly in UV-Induced Sakuranetin Synthesis in Rice

International Journal of Molecular Sciences

Article Two Chalcone Synthase Isozymes Participate Redundantly in UV-Induced Sakuranetin Synthesis in Rice

1, 1 1 2 1, Hye Lin Park †, Youngchul Yoo , Seong Hee Bhoo , Tae-Hoon Lee , Sang-Won Lee * and Man-Ho Cho 1,*

1 Graduate School of Biotechnology and Department of Genetic Engineering, Kyung Hee University, Yongin 17104, Korea; [email protected] (H.-L.P.); [email protected] (Y.Y.); [email protected] (S.H.B.) 2 Department of Applied Chemistry, Kyung Hee University, Yongin 17104, Korea; [email protected] * Correspondence: [email protected] (S.-W.L.); [email protected] (M.-H.C.) Present address; Department of Botany and Plant Pathology, and Center for Plant Biology, † Purdue University, West Lafayette, IN 47907, USA

Received: 28 April 2020; Accepted: 25 May 2020; Published: 27 May 2020

Abstract: Chalcone synthase (CHS) is a key enzyme in the flavonoid pathway, participating in the production of phenolic phytoalexins. The rice genome contains 31 CHS family genes (OsCHSs). The molecular characterization of OsCHSs suggests that OsCHS8 and OsCHS24 belong in the bona fide CHSs, while the other members are categorized in the non-CHS group of type III polyketide synthases (PKSs). Biochemical analyses of recombinant OsCHSs also showed that OsCHS24 and OsCHS8 catalyze the formation of naringenin chalcone from p-coumaroyl-CoA and malonyl-CoA, while the other OsCHSs had no detectable CHS activity. OsCHS24 is kinetically more efficient than OsCHS8. Of the OsCHSs, OsCHS24 also showed the highest expression levels in different tissues and developmental stages, suggesting that it is the major CHS isoform in rice. In oschs24 mutant leaves, sakuranetin content decreased to 64.6% and 80.2% of those in wild-type leaves at 2 and 4 days after UV irradiation, respectively, even though OsCHS24 expression was mostly suppressed. Instead, the OsCHS8 expression was markedly increased in the oschs24 mutant under UV stress conditions compared to that in the wild-type, which likely supports the UV-induced production of sakuranetin in oschs24. These results suggest that OsCHS24 acts as the main CHS isozyme and OsCHS8 redundantly contributes to the UV-induced production of sakuranetin in rice leaves.

Keywords: chalcone synthase; sakuranetin; rice; sakuranetin biosynthesis; phytoalexin; UV

1. Introduction Phytoalexins are antimicrobial secondary metabolites, and their production is induced by pathogen infections and environmental stress [1]. Rice produces a variety of diterpenoid and phenolic phytoalexins in response to pathogen attacks as well as UV stress [2–7]. The flavonoid sakuranetin is a well-known phenolic phytoalexin in rice, which is the 7-O-methylated form of naringenin [3,6,7]. Sakuranetin was first isolated from UV-irradiated rice leaves, and was also detected in blast-infected rice leaves [3]. Naringenin O-methyltransferase (OsNOMT) for sakuranetin synthesis was purified from the UV-treated rice oscomt1 mutant and the corresponding gene was identified [8]. The expression of OsNOMT was found to be induced in response to UV irradiation prior to sakuranetin accumulation [5]. Microarray and phytochemical analyses of UV-treated rice leaves revealed that the expressions of phenylpropanoid and flavonoid pathway genes including CHS and chalcone isomerase (CHI) genes are induced by UV and participate in sakuranetin production [5–7].

Int. J. Mol. Sci. 2020, 21, 3777; doi:10.3390/ijms21113777 www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2020, 21, 3777 2 of 13

CHS is the first committed enzyme in the flavonoid pathway, which catalyzes the formation of naringenin chalcone from one p-coumaroyl-CoA and three malonyl-CoAs [9–11]. Naringenin chalcone is converted to naringenin by CHI to provide C6-C3-C6 backbones for a wide range of flavonoids [12]. The CHS superfamily is known as the plant type III PKS superfamily [9,10,13]. In addition to CHS, plant type III PKSs include diverse biosynthetic enzymes—such as stilbene synthase (STS), curcumin synthase, acridone synthase, bibenzyl synthase, and benzophenone synthase—providing backbones for a variety of plant secondary metabolites [9,10]. Type III PKSs are homodimeric enzymes of two identical subunits and have the Cys-His-Asn catalytic triad at the active site [9,10]. The crystal structures of CHSs, including Medicago sativa CHS2 (MsCHS2), reveal that CHS retains catalytic Cys-His-Asn residues [14,15]. The number of CHS family genes is highly variable among plant species. Eight copies of CHS genes were identified in bread wheat (Triticum aestivum)[16]. In maize and soybean, the CHS family consists of 14 genes [17,18]. A moss, Physcomitrella patens, contains 17 CHS members [19]. Citrus species were found to have 77 CHS family genes [20]. Homology searches of databases using the MsCHS2 sequence as a query have shown that the rice genome has more than 27 CHS family genes [21,22]. Several studies have reported the biochemical functions of rice CHS family genes rather than CHS, such as curcuminoid synthase (CUS) and alkylresorcylic acid synthase (ARAS) [21,23,24]. In the present study, we performed the molecular and biochemical characterization of OsCHSs and found that OsCHS8 and OsCHS24 encode the functional CHSs in rice. Analyses of sakuranetin accumulation and the expressions of OsCHS24 and OsCHS8 in a UV-treated oschs24 mutant revealed their roles in sakuranetin biosynthesis under UV stress conditions.

2. Results

2.1. The Rice CHS Family A search of the MSU RGAP Database revealed 31 genes that were annotated as putative CHSs and/or STSs, which together comprise the CHS family in rice (Table1). This includes 27 previously identified OsPKSs [22]. CHS is a homodimer of two 40–45 kDa polypeptides [10,11]. Theoretical molecular masses of most OsCHSs were comparable with those of the functional CHSs. OsCHS26–28 and 31 have very short open reading fames (ORFs) of 417–636 base pairs (bp) (Table1). The ORFs of OsCHS5, 19, 20, and 21 were also significantly shorter than the typical lengths of CHSs, leading to a large deletion in the N-terminal region of CHS (Figure S1). Therefore, these OsCHSs are unlikely to encode functional CHS family enzymes. In contrast, OsCHS11 has a long ORF of 1365 bp encoding a protein of 49.8 kDa, leading to an insertion of 50 amino acids in the middle of the N-terminal region of CHS (Figure S1). Multiple alignments of the amino acid sequences of OsCHSs and other plant CHSs showed that OsCHS8 and OsCHS24 are highly homologous to other CHSs, showing similarities of 71–94% (Table S1). Phylogenetic analysis also indicated that OsCHS8 and OsCHS24 were closely related to the bona fide CHSs (Figure1). The other OsCHSs showed similarities of 17–66% to CHSs (Table S1) and were categorized into the non-CHS group of type III PKSs (Figure1), suggesting that they likely play different metabolic roles than CHS. OsCHS9, OsCHS16, and OsCHS18 showed 55–62% similarities to CHSs (Table S1). OsCHS9 was previously identified as CUS [23]. OsCHS16 and OsCHS18 were demonstrated to encode ARASs [21]. Phylogenetic analysis revealed that OsCHS10 and OsCHS22 are closely related to AtPKSA and AtPKSB, which are involved in the formation of the outer pollen wall (Figure1)[25,26]. Int. J. Mol. Sci. 2020, 21, 3777 3 of 13

Table 1. Rice CHS family. The MSU RGAP Database search revealed 31 genes that were annotated as putative CHSs and/or STSs, which comprise the CHS family in rice.

ORF Length Protein Theoretical Locus ID. Name Gene Description in the RGAP DB (bp) Size (aa) Mass (kDa) Os01g41834 OsCHS1 Chalcone synthase, putative 1200 399 41.8 Os04g01354 OsCHS2 Chalcone synthase, putative 1182 393 42.7 Os04g23940Int. J. Mol. Sci.OsCHS3 2020, 21, x FOR PEERChalcone REVIEW synthase, putative 1122 3733 39.9 of 14 Os05g12180 OsCHS4 Chalcone synthase, putative 1179 392 42.6 Os05g12190Os10g07040OsCHS5 OsCHS16 ChalconeChalcone synthase, synthase, putative putative 9391197 398 312 43.2333 Os05g12210Os10g07616OsCHS6 OsCHS17 ChalconeChalcone synthase, synthase, putative putative 11791197 398 392 43.2 42.6 Os05g12240Os10g08620OsCHS7 OsCHS18 ChalconeChalcone and synthase, stilbene putativesynthase, putative 11791200 399 392 43.2 42.7 Os07g11440Os10g08670OsCHS8 OsCHS19 ChalconeChalcone synthase, synthase, putative putative 12121032 343 403 37.5 43.9 Os07g17010 OsCHS9 Chalcone synthase, putative 1209 402 43.2 Os10g08710 OsCHS20 Chalcone synthase, putative 885 294 31.6 Os07g22850 OsCHS10 Chalcone and stilbene synthase, putative 1290 429 46.5 Os10g09860 OsCHS21 Chalcone synthase, putative 1092 363 39.2 Os07g31750 OsCHS11 Chalcone synthase, putative 1365 454 49.8 Os10g34360 OsCHS22 Stilbene synthase, putative 1170 389 42.2 Os07g31770 OsCHS12 Chalcone synthase, putative 1218 405 42.8 Os11g32620 OsCHS23 Chalcone synthase, putative 1224 407 42.6 Os07g34140 OsCHS13 Chalcone synthase, putative 1197 398 43 Os07g34190Os11g32650OsCHS14 OsCHS24Chalcone andChalcone stilbene synthase, synthase, putative putative 11971197 398 43.4 42.6 Os07g34260Os11g35930OsCHS15 OsCHS25Chalcone andChalcone stilbene synthase, synthase, putative putative 12001200 399 42.9 42.4 Os10g07040Os03g47000OsCHS16 OsCHS26 ChalconeChalcone synthase, synthase putative 1, putative 1197417 138 398 14.6 43.2 Os10g07616Os05g41645OsCHS17 OsCHS27 ChalconeChalcone synthase, synthase, putative putative 1197438 145 398 15.6 43.2 Os10g08620Os11g32540OsCHS18 OsCHS28Chalcone andChalcone stilbene synthase, synthase, putative putative 1200636 212 399 22.2 43.2 Os10g08670Os11g32580OsCHS19 OsCHS29 ChalconeChalcone synthase, synthase putative 10321242 413 343 43.9 37.5 Os10g08710Os11g32610OsCHS20 OsCHS30 ChalconeChalcone and synthase, stilbene putativesynthases, putative 8851206 401 294 42.4 31.6 Os10g09860Os12g07690OsCHS21 OsCHS31 ChalconeChalcone synthase, synthase, putative putative 1092426 142 363 14.9 39.2 Os10g34360 OsCHS22 Stilbene synthase, putative 1170 389 42.2 Os11g32620MultipleOsCHS23 alignments of theChalcone amino synthase, acid sequences putative of OsCHSs and 1224 other plant CHSs 407 showed 42.6that Os11g32650 OsCHS24 Chalcone synthase, putative 1197 398 43.4 OsCHS8 and OsCHS24 are highly homologous to other CHSs, showing similarities of 71–94% (Table Os11g35930 OsCHS25 Chalcone synthase, putative 1200 399 42.9 Os03g47000S1). PhylogeneticOsCHS26 analysis alsoChalcone indicated synthase that OsCHS8 1, putative and OsCHS24 417were closely related 138 to the bona 14.6 Os05g41645fide CHSs OsCHS27(Figure 1). The otherChalcone OsCHSs synthase, showed putative similarities of 17–66% 438 to CHSs (Table 145 S1) and were 15.6 Os11g32540categorizedOsCHS28 into the non-CHSChalcone group synthase, of type IIIputative PKSs (Figure 1), suggesting 636 that 212 they likely 22.2 play Os11g32580different metabolicOsCHS29 roles than CHS.Chalcone OsCHS9, synthase OsCHS16, and OsCHS18 1242 showed 55 413–62% similarities 43.9 Os11g32610to CHSs (TableOsCHS30 S1). OsCHS9Chalcone was and previously stilbene synthases, identified putative as CUS [23].1206 OsCHS16 and 401 OsCHS18 were 42.4 Os12g07690 OsCHS31 Chalcone synthase, putative 426 142 14.9 demonstrated to encode ARASs [21]. Phylogenetic analysis revealed that OsCHS10 and OsCHS22 are closely related to AtPKSA and AtPKSB, which are involved in the formation of the outer pollen wall (Figure 1) [25,26]. OsCHS24 HvCHS ZmCHS OsCHS8 AtCHS CHS MsCHS2 PnCHS EaCHS AmQNS CsOLS RpALS PsSTS WtPKS1 MdBIS1 RdORS OsCHS1 OsCHS2 OsCHS3 OsCHS25 OsCHS26 OsCHS28 OsCHS22 AtPKSA AtPKSB OsCHS10 OsCHS27 OsCHS31 Non-CHS OsCHS29 OsCHS30 type III PKS OsCHS23 OsCHS9 (CUS) OsCHS13 OsCHS14 OsCHS15 OsCHS11 OsCHS12 OsCHS21 OsCHS4 OsCHS7 OsCHS6 OsCHS5 OsCHS20 OsCHS16 (ARAS1) OsCHS17 OsCHS18 (ARAS2) OsCHS19

Figure 1. PhylogeneticFigure 1. Phylogenetic tree of OsCHSs tree of and OsCHSs other and plant other CHS plant family CHS members. family members. The amino The aminoacid sequences acid were aligned withsequences Clustal-W were and aligned the with neighbor-joining Clustal-W and treethe neighbor was built-joining with tree MEGA was built ver. with 6. Amino MEGA ver. acid 6. sequences Amino acid sequences used were AtCHS (AAB35812), ZmCHS (NP_001142246.1) EaCHS (Q9MBB1), used wereHvCHS AtCHS (CAA41250), (AAB35812), MsCHS2 ZmCHS (P30074), (NP_001142246.1) PnCHS (BAA87922), EaCHS AmQNS (Q9MBB1), (AGE44110 HvCHS), CsOLS (CAA41250), MsCHS2 (P30074),(B1Q2B6), PnCHS RpALS (BAA87922),(AAS87170), PsSTS AmQNS (CAA43165), (AGE44110), WtPKS1 CsOLS (AAW50921), (B1Q2B6), MdBIS1 RpALS (NP_001315967), (AAS87170), PsSTS (CAA43165),RdORS WtPKS1 (BAV83003), (AAW50921), AtPKSA (O23674) MdBIS1, and (NP_001315967), AtPKSB (Q8LDM2). RdORS QNS, OLS, (BAV83003), ALS, BIS, and AtPKSAORS stand (O23674),

and AtPKSB (Q8LDM2). QNS, OLS, ALS, BIS, and ORS stand for quinolone synthase, olivetol synthase, aloesone synthase, 3,5-dihydroxybiphenol synthase, and orcinol synthase, respectively. Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 4 of 14 Int. J. Mol. Sci. 2020, 21, 3777 4 of 13 for quinolone synthase, olivetol synthase, aloesone synthase, 3,5-dihydroxybiphenol synthase, and orcinol synthase, respectively. 2.2. Analyses of the Conserved Residues and Motifs in the CHS Family 2.2.CHS Analyses contains of the three Conserved conserved Residues residues, and Motifs Cys in164 the, CHS His303 Fa,mily and Asn336 (all numbering of residues followsCHS that contains of MsCHS2), three which conserved form residues, a catalytic Cys triad164, His in303 the, and type Asn III336 PKSs (all [ numbering9,10,14]. These of residues catalytic residuesfollows are that well of MsCHS2), conserved which in most form OsCHSs a catalytic (Figure triad 2in and the Figuretype III S1).PKSs OsCHS19, [9,10,14]. These 26, 27, catalytic and 31, however,residues lack are more well thanconserved one residue in most of OsCHSs the catalytic (Figure triad 2 and (Figure Figure S1). S1). Two OsCHS19, Phe residues 26, 27, (Phe and215 31,and Phehowever,265) have lack been more shown than to one be residue conserved of the in catalytic CHSs [9 triad,10,14 (].Figure Phe215 S1).is Two mostly Phe conserved residues (Phe in OsCHSs215 and exceptPhe265 OsCHS27) have been that shown lacks to the be residue conserved (Figure in CHSs2 and [9,10,14 Figure]. S1). Phe Phe215 is265 mostlyis conserved conserved in OsCHS8in OsCHSs and OsCHS24,except OsCHS27 whereas that it is lacks not strictly the residue conserved (Figure in 2 the and non-CHS Figure S1). group Phe265 of is OsCHSs conserved (Figure in OsCHS82 and Figure and S1).OsCHS24, In addition whereas to the it catalyticis not strictly triad, conserved 13 residues in the shaping non-CHS the group geometry of OsCHSs of the ( activeFigure site2 and have Figure been shownS1). In to addition be highly to conserved the catalytic in triad, CHS enzymes13 residues [14 shaping,27]. These the geometry residues wereof the absolutely active site conserved have been in shown to be highly conserved in CHS enzymes [14,27]. These residues were absolutely conserved in about half of OsCHSs, including OsCHS8 and OsCHS24 (Figure2 and Figure S1). Signiﬁcant numbers about half of OsCHSs, including OsCHS8 and OsCHS24 (Figure 2 and Figure S1). Significant numbers of these residues are missing in OsCHS19, 26, 27 and 31. CHSs have highly conserved motifs of of these residues are missing in OsCHS19, 26, 27 and 31. CHSs have highly conserved motifs of “RLMMYQQGCFAGGTVLR” and “GVLFGFGPGL” [28–30]. These motifs are absolutely conserved in “RLMMYQQGCFAGGTVLR” and “GVLFGFGPGL” [28–30]. These motifs are absolutely conserved OsCHS8 and OsCHS24, but vary in other OsCHSs (Figure2 and Figure S1). In particular, OsCHS19, in OsCHS8 and OsCHS24, but vary in other OsCHSs (Figure 2 and Figure S1). In particular, OsCHS19, 26,26, and and 31 31 were were found found to to be be missing missing one one or orboth both ofof thesethese motifs. Based Based on on the the unusual unusual ORF ORF leng lengthsths andand variations variations in in the the conserved conserved residues residues andand motifs,motifs, OsCHS5, 11, 11, 19, 19, 20, 20, 21, 21, 26, 26, 27, 27, and and 31 31 were were not not expectedexpected to to encode encode functional functional CHS CHS family family proteins.proteins.

FigureFigure 2. Multiple2. Multiple alignments alignments of of amino amino acidacid sequencessequences of OsCHSs and and other other plant plant CHSs. CHSs. The The amino amino acidacid sequences sequences were were aligned aligned with with Clustal-W. Clustal- AminoW. Amino acid acid residues residues that arethat identical are identical or similar or similar are shaded are in blackshaded and in black gray, respectively.and gray, respectively. Red circles Red indicate circles catalyticindicate catalytic triad residues triad residues in CHSs. in ResiduesCHSs. Residues marked withmarked red triangles with red are triangles gatekeeper are gatekeeper Phe residues. Phe Blueresidues. triangles Blue triangles are conserved are conserved residues residues shaping shaping the active sitethe geometry active site of geometry CHS. The of blue CHS. circles The blue indicate circles residues indicate important residues important in the binding in the of binding the coumaroyl of the moietycoumaroyl and the moiety speciﬁcity and th ofe cyclization specificity reactionsof cyclization in CHSs. reactions The in conserved CHSs. The motifs conserved in CHSs motifs are underlined in CHSs and their consensus sequences indicated. Amino acid sequences of other plant CHSs used were MsCHS2 (P30074), AtCHS (AAB35812), ZmCHS (NP_001142246), and MpCHS (CAD42328). Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 5 of 14

Int. J. Mol.are Sci. underlined2020, 21, 3777and their consensus sequences indicated. Amino acid sequences of other plant CHSs 5 of 13 used were MsCHS2 (P30074), AtCHS (AAB35812), ZmCHS (NP_001142246), and MpCHS (CAD42328). 2.3. Cloning and Heterologous Expression of OsCHSs 2.3. Cloning and Heterologous Expression of OsCHSs To identify the genes encoding functional CHS proteins, we attempted to clone OsCHSs other than theTo expectedidentify the non-functional genes encodingOsCHS functionals. The CHS cDNAs proteins, of weOsCHS1–4 attempted, 7–9 to, clone12, 15 OsCHS, 16, 18s other, 22, 24, andthan25 thewere expected successfully non-functional cloned from OsCHS rices. leaves. The cDNAs Each ofOsCHS OsCHScDNA1–4, 7– was9, 12 inserted, 15, 16, 18 into, 22 the, 24 expression, and 25 vectorwere pET28a,successfully and cloned then thefrom resulting rice leaves. constructs Each OsCHS were cDNA transformed was inserted in to intoEscherichia the expression coli BL21 vector (DE3). pET28a, and then the resulting constructs were transformed in to Escherichia coli BL21 (DE3). The The recombinant OsCHS protein containing the N-terminal His-tag was produced in E. coli under recombinant OsCHS protein containing the N-terminal His-tag was produced in E. coli under various various growth and induction conditions (Figure S2). OsCHS2, 7, 9, 12, 15, 16, 18, 22, 24, and 25 were growth and induction conditions (Figure S2). OsCHS2, 7, 9, 12, 15, 16, 18, 22, 24, and 25 were successfully expressed as soluble forms in E.coli by 0.1 mM isopropyl β-D-thiogalactopyranoside successfully expressed as soluble forms in E.coli by 0.1 mM isopropyl β-D-thiogalactopyranoside (IPTG) under an induction temperature of 25 C. OsCHS4 and 8 were produced in E.coli at 18 C and (IPTG) under an induction temperature of 25 °C◦ . OsCHS4 and 8 were produced in E.coli at 18 °C ◦and atat 0.1 0.1 mM mM IPTG IPTG concentration. concentration. OsCHS1 and and 3 3 were were produced produced only only as as insoluble insoluble forms forms under under various various growthgrowth temperatures temperatures and and IPTGIPTG concentrations.concentrations. The The recombinant recombinant OsCHS OsCHS proteins proteins were were purified puriﬁed with with 2+ NiNi2+a affinityﬃnity chromatographychromatography ( (FigureFigure S2). S2).

2.4.2.4. CHS CHS Activity Activity and and KineticKinetic ParametersParameters of the the Reco Recombinantmbinant OsCHSs OsCHSs TheThe CHS CHS activity activity ofof thethe recombinant OsCHS OsCHS proteins proteins was was assayed assayed with with p-coumaroylp-coumaroyl-CoA-CoA and and malonyl-CoAmalonyl-CoA as as substrates. substrates. Naringenin chalcone chalcone synthesized synthesized by by CHS CHS is isspontaneously spontaneously converted converted to to naringeninnaringenin in in aqueous aqueous solutions solutions [[31,32].31,32]. Therefore, Therefore, the the CHS CHS activity activity of of the the recombinant recombinant OsCHSs OsCHSs was was determineddetermined by by analyzing analyzing thethe accumulationaccumulation o off naringenin naringenin using using HPLC. HPLC. In In accordance accordance with with the the strong strong conservationconservation of ofthe the important important residues residues of CHS of CHS activity, activity, among among 12 recombinant 12 recombinant OsCHS OsCHS proteins proteins examined, OsCHS8examined, and OsCHS24OsCHS8 and exhibited OsCHS24 CHS exhibited activity (Figure CHS 3activity and Figure (Figure S3). 3 All and examined Figure recombinantS3). All examined OsCHSs belongedrecombinant in the OsCHSs non-CHS belonged group showed in the non no detectable-CHS group CHS showed activity no (Figure detecta S3).ble ThisCHS resultactivity suggests (Figure that amongS3). ThisOsCHS resultfamily suggests members, thatOsCHS24 among OsCHSand OsCHS8 familyencode members, biochemically OsCHS24functional and OsCHS8 CHS encode proteins. biochemically functional CHS proteins. mAU 10 (a) 8 6 4 2 0

0 5 10 15 20 mAU 10 (b) 8 6 4 2 0 0 5 10 15 20 mAU 500 (c) 400 300 200 100 0 0 5 10 15 20 Time (min)

Figure 3. CHS activity assay of recombinant OsCHS8 (a) and OsCHS24 (b). The tetrahydroxychalcone was formed from p-coumaroyl-CoA and malonyl-CoA by OsCHS8 and OsCHS24. The resulting chalcone was spontaneously converted to naringenin, which was analyzed by reversed-phase HPLC. (c) HPLC chromatogram of the authentic naringenin. Int. J. Mol. Sci. 2020, 21, 3777 6 of 13 The kinetic parameters of recombinant OsCHS8 and OsCHS24 were determined towards the p- coumaroylThe kinetic- and malonyl parameters-CoA of substrates recombinant (Table OsCHS8 2). The and KM OsCHS24 values of wereOsCHS24 determined and OsCHS8 towards for the p- coumaroyl-CoA were 45.44 and 27.64 µM, respectively (Table 2). Both OsCHS24 and OsCHS8 p-coumaroyl- and malonyl-CoA substrates (Table2). The KM values of OsCHS24 and OsCHS8 for showedp-coumaroyl-CoA similar affinity were 45.44 for and malonyl 27.64-µCoA,M, respectively with KM values (Table 2 of). Both 47.42 OsCHS24 and 59.38 and µ OsCHS8M, respectively. showed OsCHS24 showed a higher catalytic efficiency than OsCHS8, with kcat/KM values of 1137.05 and 539.81 similar affinity for malonyl-CoA, with KM values of 47.42 and 59.38 µM, respectively. OsCHS24 showed a M−1 min−1, respectively. 1 1 higher catalytic efficiency than OsCHS8, with kcat/KM values of 1137.05 and 539.81 M− min− , respectively.

TableTable 2. SteadySteady-state-state kinetic parameters of recombinant OsCHS24 and OsCHS8 p-Coumaroyl-CoA Malonyl-CoA p-Coumaroyl-CoA Malonyl-CoA OsCHS KM Vmax kcat kcat/KM KM OsCHS V (nmol k /K (M 1 max −1 −1 1 −1 cat M −1 − −1 K(μMM)(µ M) (nmol1 min1 mg k) cat (min(min− ) ) (M 1 min ) KM (µM)(μM) min− mg− ) min− ) OsCHS8 27.64 ± 4.21 0.352 ± 0.02 0.0149 539.81 59.38 ± 13.83 OsCHS8 27.64 4.21 0.352 0.02 0.0149 539.81 59.38 13.83 ± ± ± OsCHS24OsCHS24 45.44 45.44 ± 2.942.94 1.2181.2180.05 ± 0.05 0.05170.0517 1137.051137.05 47.42 47.428.21 ± 8.21 ± ± ± 2.5. In Silico and qRT-PCR Analyses of OsCHS Expression 2.5. In Silico and qRT-PCR Analyses of OsCHS Expression The in silico expression analysis revealed that among OsCHSs, OsCHS24 showed the highest The in silico expression analysis revealed that among OsCHSs, OsCHS24 showed the highest expression levels at different developmental stages (Figure S4). The expression levels of the other expression levels at different developmental stages (Figure S4). The expression levels of the other OsCHSs were very low at all developmental stages (Figure S4). The quantitative real-time PCR (qRT- OsCHSs were very low at all developmental stages (Figure S4). The quantitative real-time PCR PCR) analysis also showed that OsCHS24 is highly expressed in shoot tissues at an early growth stage (qRT-PCR) analysis also showed that OsCHS24 is highly expressed in shoot tissues at an early growth and in the leaf sheath, stem, and panicle tissues in adult rice plants (Figure 4). Hu et al. [22] examined stage and in the leaf sheath, stem, and panicle tissues in adult rice plants (Figure4). Hu et al. [ 22] the OsCHS expressions in root, stem, leaf, young flower, and adult flower tissues and found that examined the OsCHS expressions in root, stem, leaf, young flower, and adult flower tissues and found OsCHS24 was expressed in the rice stem tissue. It was also reported that OsCHS24 was expressed in that OsCHS24 was expressed in the rice stem tissue. It was also reported that OsCHS24 was expressed rice seedlings [33]. The transgenic expression of OsCHS24 was also shown to restore flavonoid in rice seedlings [33]. The transgenic expression of OsCHS24 was also shown to restore flavonoid accumulation in the Arabidopsis transparent testa 4 mutant [33]. These findings suggest that OsCHS24 accumulation in the Arabidopsis transparent testa 4 mutant [33]. These findings suggest that OsCHS24 acts as the major CHS isoform in rice. The expression of OsCHS8, another gene encoding a functional acts as the major CHS isoform in rice. The expression of OsCHS8, another gene encoding a functional CHS, was very low at all examined developmental stages and tissues under normal growth CHS, was very low at all examined developmental stages and tissues under normal growth conditions. conditions. 0.5 OsCHS8 OsCHS24

0.4 OsUBQ5 0.3

0.2

0.1 Relativeexpression/ 0 Shoot Root Leaf Leaf Stem Panicle Tissue sheath

Developmental Seedling Vegetative Reproductive stage growth growth

Figure 4. Quantitative real-time PCR analysis of OsCHS24 and OsCHS8 gene expression in rice seedlings Figure 4. Quantitative real-time PCR analysis of OsCHS24 and OsCHS8 gene expression in rice and different adult tissues. Shoot and root samples were collected from seven-day-old rice seedlings. seedlings and different adult tissues. Shoot and root samples were collected from seven-day-old rice Leaf, leaf sheath, stem, and panicle samples were obtained from rice plants in vegetative and reproductive seedlings. Leaf, leaf sheath, stem, and panicle samples were obtained from rice plants in vegetative stages. An ubiquitin 5 gene (OsUBQ5) was used as an internal control. Expression levels of each OsCHS and reproductive stages. An ubiquitin 5 gene (OsUBQ5) was used as an internal control. Expression gene are presented as relative expression compared to OsUBQ5 mRNA level. qRT-PCR analysis was performed on the triplicated biological samples. The results represent mean standard deviation. ± Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 7 of 14

Int. J. Mol.levels Sci. of20 20each, 21 ,OsCHS x FOR PEER gene REVIEW are presented as relative expression compared to OsUBQ5 mRNA level.7 of 14 qRT-PCR analysis was performed on the triplicated biological samples. The results represent mean ± levelsstandard of each deviation. OsCHS gene are presented as relative expression compared to OsUBQ5 mRNA level. qRT-PCR analysis was performed on the triplicated biological samples. The results represent mean ± Int.2.6. J. Sakuranetinstandard Mol. Sci. 2020 deviation. ,Accumulation21, 3777 and OsCHS24 and OsCHS8 Expression in the UV-Irradiated oschs24 7 of 13

2.6. SakuranetinTo ascertain Accumulation the role of and OsCHS24 OsCHS24 in and sakuranetin OsCHS8 accumulation,Expression in the we UV characterized-Irradiated oschs24 the oschs24 2.6.mutant Sakuranetin isolated Accumulationfrom the rice and T-DNAOsCHS24 insertionand OsCHS8mutant populationExpression in[34]. the The UV-Irradiated oschs24 mutantoschs24 has the To ascertain the role of OsCHS24 in sakuranetin accumulation, we characterized the oschs24 T-DNATo ascertaininsertion the in rolethe 3 of’ UTROsCHS24 regionin sakuranetinof OsCHS24 accumulation,(Figure 5a). In we homozygous characterized oschs24 the oschs24 mutants,mutant the mutant isolated from the rice T-DNA insertion mutant population [34]. The oschs24 mutant has the isolatedexpression from of theOsCHS24 rice T-DNA was mostl insertiony suppressed mutant population by the insertion [34]. The of oschs24 T-DNAmutant (Figure has 5b). the OsCHS24 T-DNA T-DNA insertion in the 3’ UTR region of OsCHS24 (Figure 5a). In homozygous oschs24 mutants, the insertionexpression in was the 3’increased UTR region in wild of OsCHS24-type plants(Figure by 5UVa). Inirradiation, homozygous whereasoschs24 it wasmutants, not induced the expression in UV- expression of OsCHS24 was mostly suppressed by the insertion of T-DNA (Figure 5b). OsCHS24 oftreatedOsCHS24 oschs24was leaves mostly (Figure suppressed 6a). Theby the sakuranetin insertion of content T-DNA in (Figure UV-treate5b). dOsCHS24 leaves ofexpression oschs24 were was expression was increased in wild-type plants by UV irradiation, whereas it was not induced in UV- increasedanalyzed to in elucidate wild-type the plants effect by of UV the irradiation, suppressed whereas expression it was of OsCHS24 not induced. The in sakuranetin UV-treated contentoschs24 treated oschs24 leaves (Figure 6a). The sakuranetin content in UV-treated leaves of oschs24 were leaveswas not (Figure drastically6a). The decreased sakuranetin in UV content-treated in UV-treated leaves of leavesoschs24 of (Figureoschs24 5c)were, although analyzed the to OsCHS24 elucidate analyzed to elucidate the effect of the suppressed expression of OsCHS24. The sakuranetin content theexpression eﬀect of in the oschs24 suppressed was mostly expression suppressed of OsCHS24 even . under The sakuranetin UV stress conditions content was (Figure not drastically 6a). The was not drastically decreased in UV-treated leaves of oschs24 (Figure 5c), although the OsCHS24 decreasedsakuranetin in content UV-treated in oschs24 leaves ofwasoschs24 64.6%(Figure and 80.2%5c), althoughof that in the the OsCHS24wild-typeexpression at 2 and 4 indaysoschs24 afterwas UV expression in oschs24 was mostly suppressed even under UV stress conditions (Figure 6a). The mostlyirradiation, suppressed respectively evenunder (Figure UV 5c stress). conditions (Figure6a). The sakuranetin content in oschs24 was sakuranetin content in oschs24 was 64.6% and 80.2% of that in the wild-type at 2 and 4 days after UV 64.6% and 80.2% of that in the wild-type at 2 and 4 days after UV irradiation, respectively (Figure5c). irradiation,(a) respectively (Figure 5c). (c) oschs24 20 WT (a) (c) oschs24 oschs24+2857 20

FW) 15 WT 1 189 1844 2852 +2857 oschs24

ATG TGA g/g

μ (

FW) 15 1 189 1844 2852 10

ATG TGA g/g

μ ( (b) oschs24 10 WT 5 1 2 3 4 (b) oschs24 Sakuranetin OsCHS24 WT 5 1 2 3 4 0 Sakuranetin Sakuranetin Control 1 2 4 OsCHS24OsUBQ5 0 Time after UV treatment (Day) Control 1 2 4 OsUBQ5 Figure 5. Characterization of the oschs24 mutant. (a) The gene structureTime of OsCHS24after UV treatment showing (Day) the T- DNA insertion position of the oschs24 mutant. Black boxes, gray boxes, and lines between boxes Figure 5. Characterization of the oschs24 mutant. (a) The gene structure of OsCHS24 showing the Figureindicate 5. Characterization exons, UTRs and of introns, the oschs24 respectively. mutant. (a A) The triangle gene representsstructure of the OsCHS24 position showing of the T the-DNA T- T-DNA insertion position of the oschs24 mutant. Black boxes, gray boxes, and lines between boxes DNAinsertion insertion in oschs24 position. (b) RT of -PCR the oschs24 analysis mutant. of OsCHS24 Black expression boxes, gray in the boxes, leaves and of lines homozygous between oschs24 boxes indicate exons, UTRs and introns, respectively. A triangle represents the position of the T-DNA insertion indicatemutants exons,. The homozygous UTRs and introns,oschs24 respectively.plants showed A the triangle suppressed represents expression the position of OsCHS24 of the. OsUBQ5 T-DNA in oschs24.(b) RT-PCR analysis of OsCHS24 expression in the leaves of homozygous oschs24 mutants. insertionwas used in as oschs24 an internal. (b) RT control.-PCR analysis (c) Accumulation of OsCHS24 of expressionsakuranetin in in the wild leaves-type of and homozygous oschs24 mutant oschs24 in The homozygous oschs24 plants showed the suppressed expression of OsCHS24. OsUBQ5 was used muresponsetants. The to UV homozygous irradiation. oschs24 The leaf plants samples showed were the collected suppressed from expressionUV-treated of rice OsCHS24 plants .1, OsUBQ5 2, and 4 as an internal control. (c) Accumulation of sakuranetin in wild-type and oschs24 mutant in response wasdays used after as UVan internal irradiation. control. Analyses (c) Accumulation of the sakuranetin of sakuranetin contents in wild were-type performed and oschs24 on mutant triplicate in to UV irradiation. The leaf samples were collected from UV-treated rice plants 1, 2, and 4 days after responsebiological to samples. UV irradiation. The results The represent leaf samples the meanwere collected± standard from deviation. UV-treated FW; freshrice plantsweight. 1, 2, and 4 daysUV irradiation. after UV irradiation.Analyses of the Analyses sakuranetin of the contents sakuranetin were performed contents were on triplicate performed biological on triplicate samples. The results represent the mean standard deviation. FW; fresh weight. biological samples. The results represent± the mean ± standard deviation. FW; fresh weight. (a) (b) 0.05 WT 0.0003 WT (a) oschs24 (b) oschs24

0.050.04 WT 0.0003 WT OsUBQ5 oschs24 OsUBQ5 0.0002 oschs24

0.040.03 OsUBQ5 OsUBQ5 0.0002

0.030.02 expression/ expression/ 0.0001

0.020.01 expression/

expression/ 0.0001 Relative Relative 0.010 Relative 0

Control 1 24 48 Control 1 24 48 Relative Relative 0 Time after UV treatment (h) Relative 0 Time after UV treatment (h) Control 1 24 48 Control 1 24 48 Figure 6. AnalysisTime of after the UVOsCHS24 treatment(a (h)) and OsCHS8 (b) expressionTime in oschs24 after UVin treatment response (h) to UV irradiation. Transcript levels of OsCHS24 and OsCHS8 in the wild-type an oschs24 mutants were analyzed by qRT-PCR. An ubiquitin 5 gene (OsUBQ5) was used as an internal control. Expression levels of each OsCHS gene are presented as relative expression compared to the OsUBQ5 mRNA level.

qRT-PCR analysis was performed on triplicate biological samples. The results represent mean ± standard deviation. Int. J. Mol. Sci. 2020, 21, 3777 8 of 13

Unlike OsCHS24, the expression of OsCHS8, which encode another functional CHS isozyme, was immediately increased by UV irradiation. The induced expression levels were maintained for 48 h after UV irradiation (Figure6b). The OsCHS8 expression, however, reached its peak at 1 h after UV-treatment and then decreased to non-UV-treated levels in the wild-type (Figure6b). The small decrease of sakuranetin contents and the maintained induction of OsCHS8 in UV-treated oschs24 leaves suggest that OsCHS8 contributes to sakuranetin biosynthesis in oschs24 under UV stress conditions.

3. Discussion

3.1. OsCHS24 and OsCHS8 Encode Functional CHSs in Rice Although the CHS families are comprised of multiple genes, a few of them act as bona fide CHSs and the others participate in different metabolic processes [20,26,35]. Arabidopsis contains four CHS family genes, of which one gene (AtCHS) has been identified to participate in flavonoid biosynthesis [26,36]. Two Arabidopsis CHS family members (AtPKSA and AtPKSB) have been shown to encode hydroxyalkyl α-pyrone synthases, which are involved in the synthesis of sporopollenin, the major constituent of exine in the outer pollen wall [25,26]. In soybean, two CHS genes were found to be closely related to bona fide CHSs [18]. Of the OsCHSs, OsCHS24 and OsCHS8 were shown to be closely related to other bona fide CHSs (Figures1 and2). Each subunit of the CHS homodimer contains an independent active site catalyzing the elongation of the ketide chain on the p-coumaroyl starter molecule and cyclization of the tetraketide intermediate to form the chalcone [9,14]. In CHSs, two gatekeeper Phe residues (Phe215 and Phe265) positioned in the lower portion of the CoA-binding tunnel and the active site cavity were shown to facilitate substrate loading and the proper folding of the tetraketide intermediate in the cyclization reaction [9,10,14]. Along with Cys164, His303, and Asn336, Phe215 were known to be conserved in all CHSs and other type III PKSs [9,14]. Phe265 is important in substrate specificity and is conserved in CHSs, whereas it varies in other type III PKSs [9,10,37]. OsCHS9 includes the substitution of Phe265 with Gly and has no detectable CHS activity (Figures S1 and S3). Instead of CHS, OsCHS9 was shown to encode CUS [23,24]. OsCHS24 and OsCHS8 contain both Phe residues and indeed, exhibited CHS activity. These findings suggest that OsCHS24 and OsCHS8 encode functional CHSs in rice (Figures2 and3). Divergent Type III PKSs differ in their preferences for starter molecules, degree of polyketide elongation, and intramolecular cyclization patterns. Subtle changes in the structure of their active sites lead to alterations in their kinetic properties and substrate/product specificities of type III PKSs [9,37,38]. Thr132, Ser133, Glu192, Thr194, Thr197, Gly256, Phe265, and Ser338 in CHS are important in the binding of the coumaroyl moiety and the cyclization reaction [9,10,14]. All of these residues are conserved in OsCHS24, and some of them are substituted in OsCHS8 (Figure2). In OsCHS8, Ser 133, Thr194, Thr197, and Ser338 are substituted for Asn, Met, Ala, and Ala, respectively (Figure2), which likely leads to the lower catalytic efficiency of OsCHS8 compared to OsCHS24. In Grewia asiatica, two isoforms of CHSs, GaCHS1 and GaCHS2, were characterized to have CHS activity and showed different catalytic efficiencies towards p-coumaroyl-CoA [30]. Although important residues for the CHS function are highly conserved in both GaCHSs, Thr132 and Ser133 are substituted for His and Ala in GaCHS1, respectively, which may lead to differences in the kinetic properties of the isozymes. These residues have shown to be specifically substituted in other type III PKSs and are thought to define the substrate and product specificity of the enzyme [10,37]. The recombinant OsCHS9 protein was shown to have CUS activity catalyzing the formation of bisdemethoxycurcumin from two p-coumaroyl-CoAs and one malonyl-CoA [23,24]. In OsCHS9, Thr132, Thr197, Gly256, and Phe265 are replaced with Asn, Tyr, Met, and Gly, respectively (Figure S1), which enlarges the entrance and downward pocket of the CUS active site to accommodate the p-coumaroyldiketide intermediate and the second coumaroyl unit [24,37]. OsCHS16 and OsCHS18 were identified as ARAS1 and ARAS2, respectively, which catalyze the formation of alkylresorcylic acids from fatty acyl-CoAs and three malonyl-CoAs [21]. In OsCHS16 and OsCHS18, Thr132, Thr197, and Gly256 are substituted with Tyr, Cys, and Met, respectively (Figure S1). Int. J. Mol. Sci. 2020, 21, 3777 9 of 13

Thr132, Gly256, and Phe265 are conserved in both OsCHS24 and OsCHS8, whereas they are mostly substituted with other amino acids in OsCHSs of the non-CHS group—such as OsCHS9, OsCHS16, and OsCHS18 (Figure2 and Figure S1)—suggesting that these residues are critical for the substrate and product speciﬁcity of CHSs.

3.2. OsCHS24 and OsCHS8 Redundantly Contribute to the UV-Induced Accumulation of Sakuranetin in Rice Leaves As the first committed enzyme for flavonoid biosynthesis, CHS plays an important role in plant defense [11]. It has been demonstrated that the expressions of CHS genes are induced by diverse stresses, which leads to the production of defensive compounds including phytoalexins [11]. The constitutive expression in different developmental stages (Figure S4) and strong CHS activity (Table2) of OsCHS24 suggest that it is the major CHS isoform in rice. Along with UV-induced sakuranetin accumulation (Figure5c), OsCHS24 expression was stimulated in rice leaves in response to UV irradiation (Figure6a). In a previous study, we suggested that the expression of OsCHS24 was induced by UV and is relevant to sakuranetin accumulation in the UV-irradiated rice leaves [5,6]. The oschs24 mutant was analyzed to confirm the role of OsCHS24 in the UV-induced production of sakuranetin. Unexpectedly, the sakuranetin content was not significantly decreased in the UV-treated leaves of oschs24, despite the suppression of OsCHS24 expression (Figure5c). Although OsCHS8 encodes functional CHS, its expression levels were very low in different developmental stages and tissues (Figure4 and Figure S4). Similarly, Shih et al. [ 33] reported that OsCHS8 expression was not detected in rice seedlings. In wild-type rice leaves, OsCHS8 expression was found to be induced immediately after UV irradiation and then decreased to the non-UV-treated level (Figure6b). Interestingly, OsCHS8 was more strongly induced in oschs24 leaves than in wild-type leaves, and the induced transcript levels were maintained for 48 h after UV irradiation (Figure6b), which likely complements the sakuranetin production in oschs24 under UV stress conditions. This finding, along with a small decrease of the sakuranetin content in UV-treated oschs24 leaves, suggests that OsCHS24 acts as the main CHS isoform and OsCHS8 contributes redundantly to the UV-induced production of sakuranetin in rice leaves.

4. Materials and Methods

4.1. Plant Growth, UV Treatment, and Materials The seeds of oschs24 mutant (line no. 2C-70073) and its Dongjin background rice (Oryza sativa L. spp. Japonica cv. Dongjin) were obtained from the rice T-DNA insertion mutant population in Crop Biotech Institute, Kyung Hee University, Korea [34]. The wild-type and the oschs24 mutant seeds were sterilized and germinated on Murashige and Skoog medium (Duchefa, Harlem, The Netherlands) in a growth chamber at 28 ◦C. Seven-day-old seedlings were transferred to soil, and grown in a greenhouse for 6–7 weeks to analyze sakuranetin content. UV-treatment of rice plants was performed according to the methods described by Park et al. [5]. For the expression analysis of OsCHSs, root and shoot samples were collected from seven-day-old seedlings. Rice stem and leaf samples were collected from 10-week-old wild type rice plants, and panicles were collected from 14-week-old rice plants. Malonyl-CoA was purchased from Sigma-Aldrich (St. Louis, MO, USA). p-Coumaroyl-CoA was synthesized by the method described by Beuerle and Pichersky [39]. Other reagents used in this study were purchased from Sigma-Aldrich, Thermo-Fisher Scientiﬁc (Waltham, MA, USA), Duchefa, and Samchun Chemicals (Seoul, Korea).

4.2. Multiple Sequence Alignment and Phylogenetic Analysis Protein sequences of CHSs and other type III PKSs were obtained from the MSU RGAP database (http://rice.plantbiology.msu.edu/)[40] and the National Center for Biotechnological Information Int. J. Mol. Sci. 2020, 21, 3777 10 of 13

(https://www.ncbi.nlm.nih.gov/) database. The retrieved sequences were aligned using Clustal-W [41], and the phylogenetic tree was built by the neighbor-joining method using MEGA ver. 6 [42].

4.3. Cloning of OsCHSs The first strand cDNA was synthesized using SuPrimeScript RT premix (GeNet Bio, Daejeon, Korea) with an oligo dT primer from total RNA extracted from eight-week-old rice leaves with RNAiso (Takara, Shiga, Japan). Cloning primers for OsCHSs and polymerase chain reaction (PCR) conditions are summarized in Table S2. OsCHSs were amplified from the first strand cDNA with SolgTM Pfu DNA Polymerase (SolGent, Daejeon, Korea). The resulting PCR products were subcloned into the pGEMTM-T Easy vector (Promega, Madison, WI, USA) or the pJET 1.2 blunt cloning vector (Thermo-Fisher Scientific), and then their sequences were confirmed. Each OsCHS was cut with the appropriate restriction enzymes and inserted into the pET28a (+) vector (Novagen, Madison, WI, USA). The resulting OsCHS/pET28a(+) constructs were individually transformed into E. coli BL21(DE3) cells for heterologous expression of OsCHSs.

4.4. Expression and Puriﬁcation of Recombinant OsCHSs

E. coli cells bearing the OsCHS/pET28a(+) construct were grown at 37 ◦C until an OD600 of ~0.6 in LB medium containing kanamycin (25 µg/mL). To induce the production of OsCHSs, 0.1 mM IPTG was added into the culture, followed by an additional incubation for 16 h at 18 or 25 ◦C. After induction, the cells were harvested by centrifugation (5000 g for 15 min). The harvested cells were resuspended × in phosphate-buffered saline (PBS, 137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 2 mM KH2PO4) supplemented with 1 mg/mL lysozyme and 1 mM phenylmethylsulfonyl fluoride. The cells were suspended in PBS and sonicated on ice, and then centrifuged at 15,900 g for 20 min at 4 C. The crude × ◦ extract was mixed with Ni-NTA agarose beads (Qiagen, Hilden, Germany) and incubated at 4 ◦C for 2 h with agitation. The mixtures were packed into a chromatography column and washed three times with a five-column volume of 20 mM imidazole in Tris buffer (50 mM Tris, pH 8.0, 300 mM NaCl). The recombinant OsCHSs were eluted with 50 to 100 mM imidazole in Tris buffer. The eluted proteins were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

4.5. CHS Activity Assay and Steady-State Kinetics OsCHS activity was measured according to the method of Zuubier et al. [43]. The enzyme activities of the purified recombinant OsCHSs were examined with p-coumaroyl- and malonyl-CoAs as substrates. The standard enzyme assay consisted of 80 µM p-coumaroyl-CoA and 160 µM malonyl-CoA in 0.1 M phosphate buffer (pH 7.2) with 30 µg of recombinant OsCHSs in total volume of 500 µL. The mixtures were incubated at 30 ◦C for 1h, and extracted twice with ethyl acetate. The extracts were evaporated, and the resulting residues were dissolved in methanol. The reaction products were analyzed using a reversed-phase high-performance liquid chromatography (HPLC) equipped with a Sunfire C18 column (Waters, Milford, MA, USA) following the elution and detection conditions described by Park et al., (2013). To determine the steady-state kinetic parameters of the CHS reactions, the p-coumaroyl-CoA and malonyl-CoA concentrations used were 5–100 µM and 10–100 µM, respectively. The assays were performed in triplicate and results represented as mean standard deviation. ± 4.6. In Silico and Quantitative Real-Time PCR Analysis of the OsCHS Gene Expression Microarray data for OsCHSs at different development stages of rice were downloaded from the Genevestigator plant biology database (https://genevestigator.com/gv/doc/intro_plant.jsp)[44]. The normalized data were used to generate heatmap expression patterns using the Multi Experiment Viewer program (http://www.tm4.org/mev.html). cDNAs synthesized from different tissues and developmental stages of rice were used as a template for qRT-PCR using a Prime Q-Mastermix (GeNet Bio, Daejeon, Korea) on an AriaMx real-time PCR system (Agilent, Santa Clara, CA, USA). Transcript levels were normalized by rice ubiquitin 5 Int. J. Mol. Sci. 2020, 21, 3777 11 of 13

(OsUBQ5) transcripts as a control. The ∆Ct method was applied to calculate expression levels. We used primers that showed a single peak in melting curve data. The sequences and annealing temperatures of primers for qRT-PCR are listed in Table S3.

4.7. Analysis of Sakuranetin Content Rice leaf samples collected from UV-treated and untreated rice plants were ground in liquid nitrogen and extracted with 70% methanol-water for 1 h with agitation. The aqueous methanol extracts were fractionated with ethyl acetate to enrich phenolic compounds. The ethyl acetate phases were dried in vacuo and the residues were dissolved in a small volume of methanol. The phenolic enriched fractions were used to measure sakuranetin contents using HPLC with the method described above.

Supplementary Materials: Supplementary materials can be found at http://www.mdpi.com/1422-0067/21/11/ 3777/s1. Figure S1. Multiple alignments of amino acid sequences of OsCHSs and other plant CHSs. Figure S2. Purification of the recombinant OsCHSs heterologously expressed in E. coli. Figure S3. CHS activity assay of recombinant OsCHSs. Figure S4. In silico expression analysis of OsCHSs in different developmental stages of rice plants. Table S1. Amino acid sequence similarity percentage between the OsCHS family members and other plant CHSs. Table S2. Primer sequences and PCR conditions for OsCHS cloning. Table S3. Primer sequences for quantitative real-time PCR analysis. Author Contributions: Conceptualization, M.H.C., S.H.B., and S.-W.L.; Investigation, H.-L.P., Y.Y., T.-H.L., and M.H.C.; Writing—original draft preparation, M.-H.C. and H.-L.P.; Writing—review and editing, M.-H.C., H.-L.P., S.H.B., T.-H.L., and S.-W.L. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by the Mid-career Researcher Program (NRF-2016R1A2B4014276 and NRF-2019R1A2B5B01070202) through NRF grant funded by the Ministry of Education, Science and Technology, Republic of Korea. Conflicts of Interest: The authors declare no conflict of interest.

Abbreviations

CHS chalcone synthase PKS polyketide synthase OsCHS rice chalcone synthase OsNOMT rice naringenin O-methyltransferase CHI chalcone isomerase STS stilbene synthase MsCHS2 Medicago sativa chalcone synthase 2 CUS curcuminoid synthase ARAS alkylresorcylic acid synthase ORF open reading frame bp base pair IPTG isopropyl β-D-thiogalactopyranoside PBS phosphate-buﬀered saline qRT-PCR quantitative real-time polymerase chain reaction HPLC high-performance liquid chromatography OsUBQ5 rice ubiquitin 5

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