A 2-oxoglutarate-dependent dioxygenase, GLUCORAPHASATIN SYNTHASE 1 (GRS1) is a major determinant for different aliphatic glucosinolates between radish and Chinese cabbage

A 2-oxoglutarate-dependent dioxygenase, GLUCORAPHASATIN SYNTHASE 1 (GRS1) is a major determinant for different aliphatic glucosinolates between radish and Chinese cabbage | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article A 2-oxoglutarate-dependent dioxygenase, GLUCORAPHASATIN SYNTHASE 1 (GRS1) is a major determinant for different aliphatic glucosinolates between radish and Chinese cabbage Peter Choi, Adji Baskoro Dwi Nugroho, Heewon Moon, Dong-Hwan Kim This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4895273/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 10 Dec, 2024 Read the published version in Plant Molecular Biology → Version 1 posted 6 You are reading this latest preprint version Abstract Glucosinolates (GSLs) are secondary metabolites in Brassicaceae plants and play a defensive role against a variety of abiotic and biotic stresses. Also, it exhibits anti-cancer activity against cancer cell in human. Different profiles of aliphatic GSL compounds between radish and Chinese cabbage were previously reported. However, molecular details underlying the divergent profile between two species were not clearly understood. In this study, we found that major difference of aliphatic GSLs profiles between two species is determined by the dominantly expressed genes in first step of the secondary modification phase, which are responsible for enzymatic catalysis of methylthioalkyl-glucosinolate. For instance, active expression of GLUCORAPHASATIN SYNTHASE 1 ( GRS1 ) gene in radish play an important role in the production of glucoraphasatin (GRH) and glucoraphenin (GRE), a major aliphatic GSLs in radish. Meanwhile, Chinese cabbage was found to merely produce glucoraphasatin (GRH), instead producing glucoraphanin (GRA) and gluconapin (GNP) due to the mere expression of GRS1 homologs and abundant expressions of FLAVIN-CONTAINING MONOOXYGENASES ( FMO GS-OX) homologs in Chinese cabbage. In addition, we noticed that wounding treatment on leaf tissues substantially enhanced the production of aliphatic and indolic GSLs in both Chinese cabbage and radish, indicating that GSLs are wound-induced defensive compounds in both Chinese cabbage and radish plants. Glucosinolates radish Chinese cabbage GLUCORAPHASATIN SYNTHASE 1 FLAVIN-CONTAINING MONOOXYGENASES Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Introduction Glucosinolates (GSLs) are a group of nitrogen and sulfur-containing compounds found in the Brassicale plants like Chinese cabbage, cabbage, radish, broccoli, kale and so on. GSLs and their hydrolyzed products play a defensive role against a variety of abiotic and biotic stresses. Intensive studies using Arabidopsis plant have identified most of transcription factors and catalytic enzyme genes related to the production of GSLs (Sonderby et al. 2010). For instance, a subgroup of R2R3-type MYB genes like MYB28, MYB29, and MYB76 regulate biosynthesis of aliphatic GSLs (Gigolashvili et al. 2008; Hirai et al. 2007; Sonderby et al. 2007). Meanwhile, other three R2Re-type MYB genes such as MYB34, MYB51, and MYB122 are involved in the production of indolic GSL compounds (Celenza et al. 2005; Gigolashvili et al. 2007; Malitsky et al. 2008). Based on amino acid precursors, GSLs can be grouped into three categories: aliphatic, indolic, and aromatic GSLs. Aliphatic GSLs are produced from alanine, leucine, isoleucine, valine, and methionine, whereas indolic and aromatic GSLs are generated from tryptophan and phenylalanine or tyrosine, respectively (Fahey et al. 2001). These MYB TFs regulate the downstream catalytic pathways for GSLs, which is mainly composed of three major stages: 1) chain elongation, 2) core structure formation, 3) secondary modification (Sonderby et al. 2010). Production of aliphatic GSLs requires these three stages, whereas indolic and aromatic GSLs does not require the chain elongation stage (Petersen et al. 2019). For the production of the aliphatic GSLs, amino acid precursor (i.e. methionine) are catalyzed to a corresponding elongated 2-oxo acid through several catalytic steps including deamination step by BRANCHED CHAIN AMINO ACID TRANSFERASE 4 (BCAT4), condensation step by METHYLTHIOALKYL MALATE SYNTHASES (MAMs), isomerization step by ISOPROPYLMALATE ISOMERASES (IPMIs), and oxidative decarboxylation step by ISOPROPYLMALATE DEHYDROGENASES (IPMDHs) in the “chain elongation” stage (Petersen et al. 2019). Elongated 2-oxo acid were subsequently transaminated by BRANCHED CHAIN AMINO ACID TRANSFERASE 3 (BCAT3) and enters into the second stage, “core structure formation” (Halkier and Gershenzon 2006). In the second stage, “core structure formation”, several consecutive reactions are catalyzed to generate the desulfo-aliphatic GSLs from the corresponding elongated 2-oxo acid. It is initiated from the oxidation step by CYTOCHROME P450 MONOOXYGENASE family proteins such as CYP79F1, CYP79F2, and CYP83A1, then, conjugation step by GLUTATHIONE-BY-GLUTATHIONE S-TRANSFERASE F11 (GSTF11) and GLUTATHIONE S-TRANSFERASE TAU20 (GSTU20). It is further hydrolyzed by GAMMA-GLUTAMYL PEPTIDASE 1 (GGP1), cleaved by C-S LYASE (SUR1), the undergoes the glycosylation step by UDP-GLUCOSYLTRANSFERASE 74B1 (UGT74B1), and sulfation step by SULFOTRANSFERASE (SOT17/18) (Halkier and Gershenzon 2006). Third stage, “secondary modification”, is responsible for the production of various types of GSLs via oxidation, alkylation, methoxylation, and desaturation step in Brassicale plants (Rask et al. 2000; Mikkelsen et al. 2002; Grubb and Abel 2006). For example, in B. rapa , FLAVIN MONOOXYGENASES (FMO GS-OXs) catalyzes the conversion of methylthioalkyl GSLs to methylsulfinylalkyl GSLs, then methylsulfinylalkyl GSLs is further converted to alkenyl GSLs by ALKENYLHYDROXALKYL-PRODUCING 2 (AOP2) (Hansen et al. 2008). In case of the indolic GSL pathway, CYP81F enzymes catalyze the hydroxylation of indolic GSLs. For example, CYP81F2 catalyzes the production of 4-hydroxyindolic GSL (Sonderby et al. 2010; Grubb and Abel 2006). These hydroxyindolic GSLs can be further methylated by the enzymatic activity of INDOLE GLUCOSINOLATE METHYLTRANSFERASES 1 and 2 (IGMT1 and IGMT1) (Pfalz et al. 2011). Radish ( Raphanus sativus L.) is an edible tuberous root vegetable, which is consumed globally. Radish is reported as a rich source of aliphatic GSLs due to the presence of glucoraphasatin (GRH), which occupies approximately 60–90% proportion of aliphatic GSLs in radish (Kang et al. 2020; Nugroho et al. 2021). A study reported that GLUCORAPHASATIN SYNTHASE 1 ( GRS1 ) encoding 2-oxoglutarate-dependent dioxygenase is responsible for the conversion of glucoerucin (GER) to GRH through its dehydrogenase activity (Fig. 1 ) (Kakizaki et al., 2017). Further conversion of GRH to glucoraphenin (GRE) is catalyzed by a subgroup of FLAVIN-CONTAINING MONOOXYGENASE (FMO GS-OXs) family proteins (Fig. 1 ). Chinese cabbage ( Brassica rapa L.) is also one of the most important dietary vegetable crops in South Asia, which contains beneficial nutrients including GSLs. Intensive studies have also been conducted to elucidate the biosynthetic processes of aliphatic GSLs in Chinese cabbage (Zang et al. 2009). Even radish and Chinese cabbage share similar biosynthetic pathways for the production of GSLs, radish contains a large amount of aliphatic GSLs, including glucoraphasatin (GRH) and glucoraphenin (GRE), while Chinese cabbage contains alkenyl GSLs, such gluconapin (GNP) and glucobrassicanapin (GBN) (Baek et al. 2016; Nugroho et al. 2020; Nugroho et al. 2021). Molecular details underlying different profiles of GSLs between radish and Chinese cabbage were not fully understood. In this study, we investigated the molecular relationship between aliphatic GSLs profiles and expression patterns of the genes involved in the aliphatic GSLs production between radish and Chinese cabbage. We believe the results presented herein provide better understanding on the molecular details of aliphatic GSL biosynthesis between radish and Chinese cabbage. RESULTS GSLs are more abundant in radish than Chinese cabbage It was reported that radish and Chinese cabbage contains a different profile of GSLs (Nugroho et al. 2020). To understand the molecular details underlying different profiles of GSLs between two species, eight week-old seedlings of radish and Chinese cabbage were subjected to the quantification of GSLs from leaves and roots. In case of total GSLs (amounts of total aliphatic GSLs + total indolic GSLs). We noticed that radish ‘Jinjudaepyung (JD)’ has more abundant amounts of total GSLs than those of Chinese cabbage ‘Bulam-3 (BL3)’ in both leaves and roots (Fig. 2 A). Two groups of GSLs, aliphatic and indolic GSLs were all more abundant in radish than Chinese cabbage (Fig. 2 B and 2 C). In addition, we noticed that root tissue of radish contained more amounts of total GSLs than those of leaf tissue, whereas Chinese cabbage contained lower amounts of total GSLs in root tissue in comparison to that of leaf tissue. In detail, based on HPLC chromatogram results, we identified seven GSLs (GER, GRH, GRE, GNP, GAS, GBN, GNA) from radish and eleven GSLs (PGT, GRA, GAS, GAFN, GNP, 4-HGB, GBN, GBS, 4-MTGB, GNT, and NGB) from Chinese cabbage (Fig. 3 A ~ 3D). In radish, both leaf and root tissues contained four aliphatic GSLs and three indolic GSLs. Among them, GRH and to a lesser extent, GRE were major GSL compounds (Fig. 3 A and 3 B). Meanwhile, Chinese cabbage had seven aliphatic and five indolic GSLs in both leaf and root tissues (Fig. 3 C ~ 3D). Among aliphatic GSL compounds, GBN and to lesser extent, GRA were most abundant GSL compounds. For indolic GSL compounds, NGB was most abundant in our detection condition. Radish and Chinese cabbage have different profiles of aliphatic GSLs In leaf tissues, amount of GER, which is a precursor GSL of GRA and GRH, was not detected from Chinese cabbage in our detection condition, whereas greatly detected in radish (148.39 nmol/g DW) (Fig. 3 A, 3 E and Supplementary Table S1). Because GRH is synthesized from GER, biosynthesis of high amount of GER in leaf of radish might contribute to the high content of GRH (698.33 nmol/g DW) in radish. Similar to GRH, GRE which is converted from GRH was also abundant in the leaf tissues of radish (Fig. 3 A). On the contrary, GER was not detected in Chinese cabbage, possibly being fully converted to GRA in leaf tissue of Chinese cabbage (Fig. 3 C, 3 E, and Supplementary Table S1). As expected, GRA in leaf tissue of Chinese cabbage was substantially detected (175.26 nmol/g DW). GNP, which is converted from GRA was most abundantly detected among GSLs compounds (1,389.12 nmol/g DW). In root tissues of radish, amounts of aliphatic GSLs were higher than those of leaf tissues (Fig. 2 B, and Supplementary Table S1). Similar to leaf tissue, GRH (8,701.35 nmol/g DW) and GBN (742.3 nmol/g DW) were most abundant GSLs in the root tissues of radish and Chinese cabbage, respectively (Fig. 3 E and Supplementary Table S1). To a lesser extent, GRE (3,527.95 nmol/g DW) and GRA (89.19 nmol/g DW) were 2nd most highly produced in radish and Chinese cabbage, respectively (Fig. 3 E, and Supplementary Table S1). In particular, we noticed that in radish, GSLs were more highly accumulated in root tissue than leaf tissue. It might implicate that even though glucose, an output molecule of photosynthesis and also a substrate for production of GSLs is actively synthesized in the leaf tissue, there might be a transportation system of synthesized GSLs into root tissue, possibly for the storage of GSLs. Two homologs of GRS1 , are merely expressed in Chinese cabbage Most dramatic difference of aliphatic GSLs profile was derived from the production of GRH and GBN from radish and Chinese cabbage, respectively (Fig. 3 E). In radish, while GRH was most highly produced, merely detected in Chinese cabbage. Vice versa, GBN was not detected in radish, most abundantly detected in Chinese cabbage. Because it was previously reported that GRH is synthesized from GER by GRS1, a 2-oxoglutarate dependent dioxygenase enzyme in radish (Kakizaki et al. 2017), we decided to check expression of GRS1 from radish and Chinese cabbage. When we performed BLAST search using the amino acid sequence of radish GRS1 ( RsGRS1 , Rs002010), two homologs of GRS1 (named as BrGRS1.1 and BrGRS1.2 ) were detected in the Brassica rapa genome database ( www.brassicadb.cn/# ) (Fig. 4 A and Supplementary Fig. S1). Multiple sequence alignment analysis between RsGRS1 and two copies of BrGRS1 (BrGRS1.1 and BrGRS1.2) indicated that function domains (2-oxoglutarate binding, Fe2 + binding, and dioxygenase activity) of them were well conserved. It indicated that catalytic activity as a dioxygenase enzyme might operate normally. We also conducted genomic synteny analysis using genomic sequence of RsGRS1 against Brassica rapa genome. As expected, two copies of RsGRS1 homologs (Bra033396 and Bra033397) were detected in the Brassica rapa genome (Fig. 4 B). Interestingly, Bra033396 (named as BrGRS1.1 ) and Bra033397 ( BrGRS1.2 ) were very closely located each other, suggesting that radish GRS1 homolog might be duplicated during evolution process in Chinese cabbage. Expression of GRS1 is a major cause of different profile of GSLs between radish and Chinese cabbage Variation of aliphatic GSL compounds is affected by the catalytic enzymes belonging to the ‘secondary modification’ phase such as FMO GS-OXs (FLAVIN-CONTAINING MONOOXYGENASES) or GRS1 in Brassicaceae family plants (Fig. 1 ). For example, GER can be either converted to GRH or GRA in radish and Chinese cabbage, respectively by the catalytic activity of GRS1 and FMO GS-OXs. These catalytic enzymes might play a critical role in the divergent production of aliphatic GSLs between radish and Chinese cabbage. Thus, we examined how many FMO GS-OX genes exist in the Brassica rapa and radish genome. Using amino acid sequences from four Arabidopsis FMO GS-OX, we performed the BLAST search at Brassica rapa genome database ( www.brassicadb.cn/# ) and radish genome database ( http://www.radish-genome.org/ ), respectively. Total seven radish FMO GS-OXs (RsFMO GS-OX1 ~ RsFMO GS-OX7) and five Chinese cabbage FMO GS-OX (BrFMO GS-OX2 ~ BrFMO GS-OX4, BrFMO GS-OX6 ~ BrFOM GS-OX7) were found (Supplementary Fig. S2). Expression of these FMO GS-OX homologs from radish and Chinese cabbage were examined by the quantitative RT-PCR (qRT-PCR) analysis. Among five FMO GS-OX homologs in Chinese cabbage, three FMO GS-OXs ( BrFMO GS-OX5 ~ BrFMO GS-OX7 ) were dominantly expressed in the five-week-old seedling plants (Fig. 5 A). It indicated that these three FMO GS-OX genes ( BrFMO GS-OX5 , BrFMO GS-OX6 , and BrFMO GS-OX7 ) might play an important role in the conversion of GER to GRA. Meanwhile, transcripts of two RsGRS1 homologs ( RsGRS1.1 and RsGRS1.2 ) were merely detected in the Chinese cabbage in our qRT-PCR analysis (Fig. 5 A). In an agreement of this observation, these two RsGRS1 homologs were not detected in our RNA-seq dataset, possibly filtered out due to the low expression of these BrGRS1 homologs. The fact that BrGRS1.1 and BrGRS1.2 were merely expressed in Chinese cabbage indicated that they cannot impact on the first catalytic step of ‘secondary modification’ stage of GSLs biosynthesis in Chinese cabbage. Hence, a majority of aliphatic GSL compounds seems to be destined to enter GRA-GNP production path, but not GRH-GRE path. In case of radish, total seven FMO GS-OX homologs were found in the radish genome. Interestingly, all seven radish FMO GS-OX homologs ( RsFMO GS-OX1 ~ RsFMO GS-OX7 ), were merely expressed in radish young plants (Fig. 5 B). It suggested that mere expression of all these RsFMO GS-OX genes resulted in the low abundance of GER-GRA-GNP path in radish. Meanwhile, a 2-oxoglutarate-dioxygenase, RsGRS1 was substantially expressed in radish (Fig. 4 B). It indicated that active expression of RsGRS1 directed the first catalytic step of ‘secondary modification’ stage of GSLs biosynthesis toward path for the substantial production of GRH in radish. BrGRS1.1 is constantly silenced, but , BrGRS1.2 is epigenetically regulated in Chinese cabbage Though there are two copies of GRS1 homologs in Chinese cabbage genome, they were merely expressed. Histone modification contexts of gene is highly correlated with the status of expression of the gene (Dong and Weng 2013). To understand the molecular reason on the mere expressions of BrGRS1 homologs, we analyzed a recently published B. rapa epigenome dataset containing enrichment profiles of four histone marks like H3K4me2, H3K36me3, H3K27me3, and H3K9me2. While H3K36me3 mark represent a histone mark closely correlated with active transcription, other three marks like H3K4me2, H3K27me3 and H3K9me2 represent histone marks related to the gene repression. When we looked into four histone enrichment profiles, genomic region of BrGRS1.1 was not enriched with any histone marks, implying that BrGRS1.1 is constantly silenced (Fig. 6 left). Meanwhile, BrGRS1.2 was substantially enriched with two repressive histone marks, H3K4me2 and H3K27me3, suggesting that BrGRS1.2 is in a state of epigenetic suppression context (Fig. 6 right). It is worthy to note that a small amount of GRH was detected in the root tissue of Chinese cabbage. It is possible that BrGRS1.2 is a bit expressed in the root tissue of Chinese cabbage and contribute to the small production of GRH in Chinese cabbage. Alternatively, it is also possible that because a bit expression of BrGRS1.2 was detected in the leaf tissue of Chinese cabbage (Fig. 5 A), BrGRS1.2 might contribute to the production of GRH in the leaf tissue, then synthesized GRH in the leaf might be subsequently translocated to root tissue by GTR transport system in Chinese cabbage, thus not showing detection of GRH in the leaf tissue of Chinese cabbage (Fig. 3 C). This hypothesis needs further investigation. Taken together, constantly silenced BrGRS1.1 and epigenetically suppressed BrGRS1.2 might explain why these two BrGRS1 homologs were merely expressed in Chinese cabbage and GRH was not detected in the leaf tissue of Chinese cabbage. Aliphatic and indolic GSLs were induced by wounding in both radish and Chinese cabbage It was reported that GSLs are induced upon abiotic (i.e. salt and drought) and biotic stresses (i.e. insects and herbivores) in some Brassicaceae family plants (Muthusamy and Lee 2023; Nephali et al. 2020). To examine whether radish aliphatic GSLs are induced by stress like wounding, we treated wounding on leaves of radish and Chinese cabbage and measured the amounts of aliphatic and indolic GSLs along wounding time points (0h, 24h, 72h, and 120h after wounding). Amounts of total GSLs were substantially increased after wounding in both radish, ‘JD’ and Chinese cabbage, ‘BL3’ line (Fig. 7 A and Supplementary Table S5). In case of aliphatic GSLs, total amounts were dramatically increased from the 0h sample to 120h sample after wounding in both radish and Chinese cabbage (Fig. 7 B and Supplementary Table S5). For instance, in radish, total aliphatic GSLs at 0h (254.7 nmol/g∙DW) was increased to 1,515.0 nmol/g∙DW at 120h after wounding (5.9 times increase). In case of Chinese cabbage, total aliphatic GSLs at 0h (39.72 nmol/g∙DW) was increased to 202.03 nmol/g∙DW at 120h sample after wounding (5.1 times increase). Similar to the case of aliphatic GSLs, wounding treatment significantly increased amounts of indolic GSLs after wounding in both radish and Chinese cabbage (Fig. 7 C and Supplementary Table S5). In case of radish, amount of total indolic GSLs was moderately (1.6 times) increased from 0h (115.5 nmol/g∙DW) to 120h (180.0 nmol/g∙DW). Meanwhile, total indolic GSLs in Chinese cabbage was more drastically (4.9 times) induced from 0h (21.56 nmol/g∙DW) to 120h (102.87 nmol/g∙DW) after wounding. Collectively, these data indicate that GSL compounds are wound-responsively synthesized in both radish and Chinese cabbage. Next, we examined whether wound-induced increase of GSL compounds was resulted from the triggered expression of GSLs pathway genes. Expression profiles of total 10 and 11 GSL pathway genes from radish and Chinese cabbage, respectively were examined along wounding time course. Resultantly, most of tested aliphatic and indolic GSL pathway genes commonly exhibited increased levels of transcription after wounding, even dynamic expression profiles were detected in both radish and Chinese cabbage (Fig. 8 A ~ 8D). Based on these observations, we came up with schematic model on GRS1 action in the divergent profile of GSL compounds between radish and Chinese cabbage (Fig. 9 ). In brief, major difference of aliphatic GSLs profiles between two species seems to be determined by the dominantly expressed genes in the first catalytic step of the ‘secondary modification’ stage, which are responsible for enzymatic catalysis of methylthioalkyl-GSLs (Fig. 9 ). For example, in Chinese cabbage, FMO GS-OXs catalyze the oxidation of 4-carbon methylthioalkyl-GSLs (GER) or 5-carbon methylthioalkyl-GSLs (GBT) to 4-methylsulfinylalkyl-GSLs (GRA) or 5- methylsulfinylalkyl-GSLs (GAS), respectively. Methylsulfinylalkyl-GSLs can be further converted to alkenyl-GSLs, which is catalyzed by AOPs (2-oxoglutarate-dependent dioxygenase) like AOP2 and AOP3 (indicated with orange color boxes, Fig. 9 ). Interestingly, we found that in radish, RsGRS1 is dominantly expressed in radish, instead of FMO GS-OXs . Dominantly expressed RsGRS1 shift a direction towards the conversion of a precursor, glucoerucin (GER) into glucoraphasatin (GRH), a type of methylthioalkyl-glucosinolate (indicated with blue color box, Fig. 9 ). This different expression profile of FMO GS-OXs and GRS1 resulted in the divergent entry of aliphatic GSLs biosynthesis in the first step of ‘secondary modification’ stage between Chinese cabbage and radish. In summary, presence of active RsGRS1 gene in radish play an important role in the production of glucoraphasatin (GRH) and glucoraphenin (GRE), a major aliphatic GSLs in radish. Meanwhile, Chinese cabbage was found to merely produce glucoraphasatin (GRH), instead producing glucoraphanin (GRA) and gluconapin (GNP) due to the absence of expression of RsGRS1 homologs in Chinese cabbage. DISCUSSION Over 130 different glucosinolates can by synthesized in Brassicaceae family plants (Nguyen et al. 2020). Composition of GSL compounds varies along different tissues and developmental stages. Besides, different species has unique GSLs composition. Production of diverse GSLs compounds can be derived from the different profile of GSL biosynthetic genes as well as GSL transport from one tissue to others. Long-distance transport of GSLs was reported from a study using Arabidopsis plants, in which it is mediated by two nitrate/peptide transporter proteins named as GTR1 and GTR2 (Andersen et al. 2013; Nour-Eldin et al. 2012). In our study, we noticed that root tissue of radish contained more substantial amounts of GRH than those of leaf tissue, whereas Chinese cabbage contained lower amounts of total GSLs in root tissue than those of leaf tissue (Fig. 2 ). Although RsGRS1 was highly expressed in the leaves of radish (Supplementary Fig S5), GRH was found in a greater amount in the roots, not in the leaves. This discrepancy might imply that GRH synthesized in leaves might be actively translocated to roots in radish. This observation is in a line with a recent report proposing that long distance transport might resulted in the accumulation of GRH in radish roots (Kakizaki et al., 2017). In detail, a wild type radish scion grafted to a mutant grs1 root stock evidently displayed a high accumulation of GRH in the root tissue of the grs1 mutant. Meanwhile, a non-grafted radish grs1 mutant did not show accumulation of GRH in root tissues. Thus, even though high amounts of GSLs were produced in leaf tissue, it is highly plausible to be translocated and subsequently accumulated in root tissues in Brassicaceae family plants including radish (Sotelo et al. 2016; Touw et al. 2019). It might be an interesting topic to investigate the translocation of GRH from shoot to root tissues possibly by glucosinolate transporters in radish. It was previously suggested that the GRS1 and its product, GRH, originally exist only in radish and not in other Brassica plants (Ishida et al. 2014; Kakizaki et al. 2017). However, a more recent studies of GSLs reported that a small amount of GRH were detected in several brassica plants including Chinese cabbage, broccoli, and choysum (Liang et al. 2018; Nugroho et al. 2020). In this study, albeit a low amount was detected, we also observed that GRH is produced in Chinese cabbage, particularly in root tissue by the virtue of BrGRS1.2 (Fig. 3 D). However, expression of BrGRS1.1 was constantly suppressed and merely expressed even in wounding stress, implying that BrGRS1.1 might be stably silenced. Furthermore, we found that chromatin of BrGRS1.2 is epigenetically regulated, that is, in a state of repressive histone mark context. Genomic region of BrGRS1.2 was shown to be enriched with an two repressive histone marks, H3K4me2 and H3K27me3. Repressive histone mark context of BrGRS1.2 might imply that BrGRS1.2 is delicately regulated in an epigenetic manner in a certain condition. In a similar line, we previously reported that radish RsGRS1 is also epigenetically regulated during vernalization, highly enriched with H3K27me3 histone mark by long-term cold in radish (Nugroho et al. 2021). These results suggest that some of GSL biosynthetic genes including GRS1 in Brassicaceae family crop plants might be under the delicate control of transcription in an epigenetic manner. MATERIALS AND METHODS Plant materials and growth condition Seeds of radish (Jinjudaepyung’) and Chinese cabbage (‘Bulam3’) were purchased from the Coupang online retailer and used in this study. Seeds were sterilized and plated on a half-strength solid Murashige and Skoog (MS) media and incubated at 4 o C for at least 3 days under the dark. Radish and Chinese cabbage seedlings with a 2–4 mm radicle were transplanted into the soil in the growth room at 22 o C under long day light (16h light:8h dark) condition. After growth of seedling for certain period of weeks (5 ~ 8 weeks), shoots or roots of all seedling plants were sampled for the measurement of glucosinolates (GSLs) and further molecular analyses like qRT-PCR analysis. Extraction and analysis of GSLs For harvested shoots and roots tissues, eight-week-old seedlings were sampled and directly lyophilized using a vacuum freeze dryer (Ilshin Biobase, Korea) and ground into a fine powder for the HPLC analysis. DS-GSLs (desulfo glucosinolates) were isolated and analyzed as described previously (Nugroho et al. 2021). For wounding samples, shoots tissue of five-week-old seedlings were sampled and used for quantification of GSLs. Individual DS-GSLs were analyzed with high performance liquid chromatography (Vanquish HPLC System, Thermo scientific). The DS-GSLs were separated on a C18 reverse phase column (Zorbax XDB-C18, 4.6 x 250mm, 5µm particle size, Agilent, USA) with a water and acetonitrile gradient system. Total 20 µL of samples were injected in a flow rate of 1.0 mL min − 1 . Individual peak was identified using standard compounds (Phytoplan, Germany) (Supplementary Table S2), and sinigrin compound was used for relative quantification (Brown et al., 2003). The contents were analyzed independently with three replicates and presented in nmol g − 1 on a dry weight (DW) basis. Quantitative RT-PCR analysis After growth of seedlings for 5 weeks in soil pots, shoots of plants were sampled for the extraction of total RNAs. Total RNAs were isolated from each tissue sample using the RNeasy Plant mini kit (QIAGEN, USA) according to the manufacturer’s instructions. To get rid of residual DNAs, total RNAs were treated with DNase I (New England Biolabs, USA), and then used for the cDNA synthesis using EasyScript reverse transcriptase (Transgen Biotech, China). RT-qPCR analysis was performed using BioFACT™ 2X Real-Time PCR Mix (BioFACT, South Korea) in a LineGene 9600 Plus Real-Time PCR system (BioER, China). RsTEF2a (Rs419480) and BrPP2Aa (Bra012474 ) were respectively used as a reference gene for gene expression normalization from radish and Chinese cabbage plants (Duan et al., 2017). Three biological replicates were used for each quantitative RT-PCR analysis. One-way ANOVA with Tukey’s post-hoc test was used for statistical analysis. Information on the primer sequences used in the RT-qPCR analysis was shown in the Supplementary Table S3. ChIP-seq dataset analysis Raw FASTQ files of ChIP-seq epigenome data analyzed in this study were downloaded from the DNA Data Bank of Japan (DDBJ) database. FASTQ reads were first trimmed and quality-filtered using FASTQC before alignment to the Brassica rapa reference genome using STAR aligner (Dobin et al. 2013). Integrative Genomics Viewer (IGV) was used for visualization of aligned reads (Robinson et al. 2011). Bigwig files for IGV visualization were generated using bamCoverage command in the deepTools package (Ramirez et al. 2014). List of public ChIP-seq dataset was shown in Supplementary Table S4. Phylogenetic analysis The amino acid sequences of FMO GS-OXs and GRS1 homologs from radish, Chinese cabbage, and Arabidopsis thaliana were aligned together with other amino acid from corresponding organism ( Arabidopsis thaliana ) using MUSCLE method in MEGA version 7 program. Phylogenetic tree was constructed using maximum likelihood method, and bootstrap values were set at 1000 replications. Statistical Analysis One-way analysis of variance (ANOVA) and post-hoc Tukey’s test (p < 0.05) were used to analyze statistical differences. Data were analyzed using a statistical software package (SAS; version 9.4; SAS Institute Inc., Cary, NC, USA) and presented as the mean ± standard deviation (SD) of three biological replicates. Declarations ACKNOWLEDGEMENTS This work was supported by a grant from the National Research Foundation of Korea (NRF) (2021R1F1A1047822) toD-HK. AUTHOR CONTRIBUTIONS PC prepared all plant materials and performed molecular experiments; HM analyzed ChIP-seq dataset. PC and ABDN performed HPLC analysis. D-HK conceived and designed the study; PC and D-HK analyzed the data and wrote the manuscript. CONFLICT OF INTEREST The authors declare they have no conflicts of interest DATA AVAILABILITY STATEMENT All data supporting the findings of this study are available within the paper and its Supplementary Information. The data that support the findings of this study are also available from the corresponding author upon reasonable request. 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Metabolites 10 (12). doi:10.3390/metabo10120505 Nguyen VPT, Stewart J, Lopez M, Ioannou I, Allais F (2020) Glucosinolates: Natural Occurrence, Biosynthesis, Accessibility, Isolation, Structures, and Biological Activities. Molecules 25 (19). doi:10.3390/molecules25194537 Nour-Eldin HH, Andersen TG, Burow M, Madsen SR, Jorgensen ME, Olsen CE, Dreyer I, Hedrich R, Geiger D, Halkier BA (2012) NRT/PTR transporters are essential for translocation of glucosinolate defence compounds to seeds. Nature 488 (7412):531-534. doi:10.1038/nature11285 Nugroho ABD, Han N, Pervitasari AN, Kim DH, Kim J (2020) Differential expression of major genes involved in the biosynthesis of aliphatic glucosinolates in intergeneric Baemoochae (Brassicaceae) and its parents during development. Plant Mol Biol 102 (1-2):171-184. doi:10.1007/s11103-019-00939-2 Nugroho ABD, Lee SW, Pervitasari AN, Moon H, Choi D, Kim J, Kim DH (2021) Transcriptomic and metabolic analyses revealed the modulatory effect of vernalization on glucosinolate metabolism in radish (Raphanus sativus L.). Sci Rep 11 (1):24023. doi:10.1038/s41598-021-03557-5 Petersen A, Hansen LG, Mirza N, Crocoll C, Mirza O, Halkier BA (2019) Changing substrate specificity and iteration of amino acid chain elongation in glucosinolate biosynthesis through targeted mutagenesis of Arabidopsis methylthioalkylmalate synthase 1. Biosci Rep 39 (7). doi:10.1042/BSR20190446 Pfalz M, Mikkelsen MD, Bednarek P, Olsen CE, Halkier BA, Kroymann J (2011) Metabolic engineering in Nicotiana benthamiana reveals key enzyme functions in Arabidopsis indole glucosinolate modification. Plant Cell 23 (2):716-729. doi:10.1105/tpc.110.081711 Ramirez F, Dundar F, Diehl S, Gruning BA, Manke T (2014) deepTools: a flexible platform for exploring deep-sequencing data. Nucleic Acids Res 42 (Web Server issue):W187-191. doi:10.1093/nar/gku365 Rask L, Andreasson E, Ekbom B, Eriksson S, Pontoppidan B, Meijer J (2000) Myrosinase: gene family evolution and herbivore defense in Brassicaceae. Plant Mol Biol 42 (1):93-113 Robinson JT, Thorvaldsdottir H, Winckler W, Guttman M, Lander ES, Getz G, Mesirov JP (2011) Integrative genomics viewer. Nat Biotechnol 29 (1):24-26. doi:10.1038/nbt.1754 Sonderby IE, Burow M, Rowe HC, Kliebenstein DJ, Halkier BA (2010) A complex interplay of three R2R3 MYB transcription factors determines the profile of aliphatic glucosinolates in Arabidopsis. Plant Physiol 153 (1):348-363. doi:10.1104/pp.109.149286 Sonderby IE, Hansen BG, Bjarnholt N, Ticconi C, Halkier BA, Kliebenstein DJ (2007) A systems biology approach identifies a R2R3 MYB gene subfamily with distinct and overlapping functions in regulation of aliphatic glucosinolates. Plos One 2 (12):e1322. doi:10.1371/journal.pone.0001322 Sotelo T, Velasco P, Soengas P, Rodriguez VM, Cartea ME (2016) Modification of Leaf Glucosinolate Contents in Brassica oleracea by Divergent Selection and Effect on Expression of Genes Controlling Glucosinolate Pathway. Front Plant Sci 7:1012. doi:10.3389/fpls.2016.01012 Touw AJ, Verdecia Mogena A, Maedicke A, Sontowski R, van Dam NM, Tsunoda T (2019) Both Biosynthesis and Transport Are Involved in Glucosinolate Accumulation During Root-Herbivory in Brassica rapa. Front Plant Sci 10:1653. doi:10.3389/fpls.2019.01653 Zang YX, Kim HU, Kim JA, Lim MH, Jin M, Lee SC, Kwon SJ, Lee SI, Hong JK, Park TH, Mun JH, Seol YJ, Hong SB, Park BS (2009) Genome-wide identification of glucosinolate synthesis genes in Brassica rapa. FEBS J 276 (13):3559-3574. doi:10.1111/j.1742-4658.2009.07076.x Supplementary Files SupplementaryInformation.docx Cite Share Download PDF Status: Published Journal Publication published 10 Dec, 2024 Read the published version in Plant Molecular Biology → Version 1 posted Editorial decision: Minor revisions 26 Sep, 2024 Reviewers agreed at journal 20 Aug, 2024 Reviewers invited by journal 18 Aug, 2024 Editor invited by journal 12 Aug, 2024 Editor assigned by journal 12 Aug, 2024 First submitted to journal 11 Aug, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4895273","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":341805012,"identity":"d0039add-f663-4639-8433-3c0b953f54da","order_by":0,"name":"Peter Choi","email":"","orcid":"","institution":"Chung-Ang University","correspondingAuthor":false,"prefix":"","firstName":"Peter","middleName":"","lastName":"Choi","suffix":""},{"id":341805013,"identity":"1265fe9d-b548-4d9c-9f16-0ed485cd9b15","order_by":1,"name":"Adji Baskoro Dwi Nugroho","email":"","orcid":"","institution":"Chung-Ang University","correspondingAuthor":false,"prefix":"","firstName":"Adji","middleName":"Baskoro Dwi","lastName":"Nugroho","suffix":""},{"id":341805014,"identity":"7256f1d3-059b-44c7-849e-c56480c43ab7","order_by":2,"name":"Heewon Moon","email":"","orcid":"","institution":"Chung-Ang University","correspondingAuthor":false,"prefix":"","firstName":"Heewon","middleName":"","lastName":"Moon","suffix":""},{"id":341805015,"identity":"6d36004a-8f20-4546-89b6-7168b74daaaf","order_by":3,"name":"Dong-Hwan Kim","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAyUlEQVRIiWNgGAWjYDCCG2wMBxgqbORgfAMitZxJMyZNCwNj2+HEBqK18N1uSzx0s405vb/9jAHDjxoGY/MGAlok7xw7cDjnHFvujDM5Bow9xxjMZA4Q0GJwI73hcE4ZT+4GhhwDBt4GBhsJQg6DaGGTSDfgf2PA+Jc4LWlAh7UZJBhI5BgwA20xI6hF8kZawuGcMwmGM248Kzgsc0zCmKAWvhtpxp9zKv7L8/cnb3z4psbGcAYhLSjgAAMDQTtGwSgYBaNgFBADAK5OQcTazIsxAAAAAElFTkSuQmCC","orcid":"https://orcid.org/0000-0003-3348-4948","institution":"Chung Ang University - Anseong Campus","correspondingAuthor":true,"prefix":"","firstName":"Dong-Hwan","middleName":"","lastName":"Kim","suffix":""}],"badges":[],"createdAt":"2024-08-11 13:01:51","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4895273/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4895273/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s11103-024-01537-7","type":"published","date":"2024-12-10T15:57:38+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":64460381,"identity":"da29736d-2f10-4e4f-b266-71b4db77fa6f","added_by":"auto","created_at":"2024-09-13 12:38:22","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":339155,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSchematic pathway showing biosynthetic pathway of major aliphatic GSLs.\u003c/strong\u003e Three major stages like ‘side-chain elongation’, ‘core structure formation’, and ‘secondary modification’ were indicated on the left panel with vertical words of the picture. Aliphatic GSLs biosynthesis starts from precursor amino acids like methionine. Methionine udergoes side-chain elongation process in the Chloroplast by enzymes indicated with orange color ovals in the picture. Next, chain-elongated methionine enters the ‘core structure formation’ which occurs mainly in the endoplasmic reticulum and cytosol within plant cell. Enzymes in the ‘core structure formation’ were indicated with gray color ovals. After the ‘core structure formation’ stage, methylthioalkyl-glucosinolate like glucoerucin (GER, indicated by blue letters) enters last stage, ‘secondary modification’ in the cytosol within plant cell. Enzymes involved in the ‘secondary modification’ like ‘FMO GS-OXs’, ‘GRS1’, and ‘AOPs’ were indiclated with blue, pale orange, and pale green color ovals, respectively. Different catalytic activity of these enzymes in the first step of ‘secondary modification’ stage of GSL biosynthesis determines diverse types of aliphatic GSL compounds in Brassicaceae family crop plant.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-4895273/v1/5763eab233df8457682dc67c.png"},{"id":64461061,"identity":"d131e6d4-ba08-4557-9cbd-98337ef7194d","added_by":"auto","created_at":"2024-09-13 12:46:20","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":154255,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eComparison of the total GSLs contents of leaf and root tissues between Chinese cabbage and radish. A)\u003c/strong\u003eAmounts of total GSLs (combining aliphatic and indolic GSLs) of the leaves and roots between Chinese cabbage and radish \u003cstrong\u003eB)\u003c/strong\u003e Amounts of total aliphatic GSLs of the leaves and roots between Chinese cabbage and radish \u003cstrong\u003eC)\u003c/strong\u003eAmounts of total indolic GSLs of the leaves and roots between Chinese cabbage and radish. Data were presented as mean ± standard deviation (SD) (n=3). Statistically significant differences were determined by one-way ANOVA and Tukey’s post hoc test (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05).\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-4895273/v1/03963a4d4c2796d18bece862.png"},{"id":64460371,"identity":"4d43956e-de12-4fa9-9692-7cf241469c3f","added_by":"auto","created_at":"2024-09-13 12:38:19","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":465552,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eHPLC chromatogram of GSLs in leaves and roots of radish and Chinese cabbage.\u003c/strong\u003e \u003cstrong\u003eA)\u003c/strong\u003e a HPLC chromatogram of GSLs in leaf of radish. \u003cstrong\u003eB)\u003c/strong\u003e a HPLC chromatogram of GSLs in root of radish. \u003cstrong\u003eC)\u003c/strong\u003e a HPLC chromatogram of GSLs in leaf of Chinese cabbage. \u003cstrong\u003eD)\u003c/strong\u003e a HPLC chromatogram of GSLs in leaf of Chinese cabbage. \u003cstrong\u003eA)\u003c/strong\u003e~\u003cstrong\u003eD)\u003c/strong\u003e a peak of glucoraphasatin (GRH, 9) was indicated by dashed red lines. 1, progoitrin (PGT); 2, glucoraphanin (GRA); 3, glucoalyssin (GAS); 4, gluconapoleiferin (GNPF); 5, gluconapin (GNP); 6, 4-hydroxyglucobrassicin (4-HGB); 7, glucobrassicannapin (GBN); 8, glucoerucin (GER); 9, glucoraphasatin (GRH); 10, glucobrassicin (GBS); 11, 4-methoxyglucobrassicin (4-MTGB); 12; gluconasturtiin (GNT); 13, Neoglucobrassicin (NGB); 14, glucoraphenin (GRE). \u003cstrong\u003eE) \u003c/strong\u003eComparison of major aliphatic GSL compounds detected in leaf and root tissues between Chinese cabbage and radish. Three aliphatic GSL compounds like glucoraphasatin (GRH), glucoerucin (GER), and glucoraphenin (GRE) were dominantly detected in radish, whereas other three aliphatic GSL compounds such as glucoraphanin (GRA), gluconapin (GNP), and glucobrassicanapin (GBN) was solely detected in Chinese cabbage, not in radish.\u003cstrong\u003e \u003c/strong\u003eData were presented as mean ± standard deviation (SD) (n=3). Statistically significant differences were determined by one-way ANOVA and Tukey’s post hoc test (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05).\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-4895273/v1/150aa5cebcd9e0b16ed9a647.png"},{"id":64460372,"identity":"25dc9db5-8708-496b-a0f4-728adfbab19c","added_by":"auto","created_at":"2024-09-13 12:38:19","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":684349,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTwo copies of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eGRS1\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e homologs were found in Chinese cabbage genome. A) \u003c/strong\u003eMultiple sequence alignment between radish RsGRS1 and two copies of \u003cem\u003eBrGRS1\u003c/em\u003e (\u003cem\u003eBrGRS1.1\u003c/em\u003e and \u003cem\u003eBrGRS1.2\u003c/em\u003e) in Chinese cabbage. Conserved residues between radish RsGRS1 and two copies of \u003cem\u003eBrGRS1\u003c/em\u003e (\u003cem\u003eBrGRS1.1\u003c/em\u003e and \u003cem\u003eBrGRS1.2\u003c/em\u003e) in Chinese cabbage were indicated with red letters. A 2-oxoglutarate dioxygenase domain was indicated by pale green box. A 2-oxoglutarate dioxygenase domain was highly conserved between radish RsGRS1 and Chinese cabbage BrGRS1.1 and BrGRS1.2. Amino acid residues responsible for binding to Fe\u003csup\u003e2+\u003c/sup\u003e were indicated by green asterisks.\u003cstrong\u003e\u0026nbsp; B) \u003c/strong\u003eMicro-synteny analysis between radish and Chinese cabbage genome sequence contigs containing \u003cem\u003eRsGRS1\u003c/em\u003e and its two homologs in Chinese cabbage.\u003cstrong\u003e \u003c/strong\u003eHorizontal red and yellow bars indicate the radish and Chinese cabbage genome sequence contigs, respectively. White and grey boxes in each bar indicate the exons and introns, respectively. Similar regions in the compared sequences are connected with green lines. The degree of similarity is depicted using a color gradient as indicated in the right upper panel.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-4895273/v1/87fed43b70894a8b084c30a3.png"},{"id":64461062,"identity":"8b3b0d77-ae75-43e0-9a59-8df7fa23997e","added_by":"auto","created_at":"2024-09-13 12:46:22","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":112942,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eResult of qRT-PCR analysis of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eFMO GS-OXs\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e and \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eGRS1\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e homologs of Chinese cabbage and radish. A) \u003c/strong\u003eQuantification of transcript levels of five \u003cem\u003eB. rapa FMO GS-OXs\u003c/em\u003e homologs (\u003cem\u003eBrFMO GS-OX2, BrFMO GS-OX3, BrFMO GS-OX5, BrFMO GS-OX6\u003c/em\u003e, and \u003cem\u003eBrFMO GS-OX7\u003c/em\u003e)\u003cstrong\u003e \u003c/strong\u003eand two \u003cem\u003eGRS1\u003c/em\u003e homologs (\u003cem\u003eBrGRS1.1\u003c/em\u003eand \u003cem\u003eBrGRS1.2\u003c/em\u003e) in Chinese cabbage\u003cstrong\u003e B) \u003c/strong\u003eQuantification of transcript levels of five \u003cem\u003eradish FMO GS-OXs\u003c/em\u003e homologs (\u003cem\u003eRsFMO GS-OX1~ RsFMO GS-OX7\u003c/em\u003e)\u003cstrong\u003e \u003c/strong\u003eand a \u003cem\u003eGRS1\u003c/em\u003e gene (\u003cem\u003eRsGRS1\u003c/em\u003e) in radish\u003cstrong\u003e. A) ~ B) \u003c/strong\u003eWhole seedling plants of Chinese cabbage and radish grown for 5 weeks were harvested and used for this analysis.\u003cstrong\u003e \u003c/strong\u003eOne-way analysis of variance (ANOVA) and post-hoc Tukey’s test (p \u0026lt; 0.05) were used to analyze statistical differences. Mean ± standard deviation (SD) of three biological replicates were presented.\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-4895273/v1/fadcf863c55a974faaa17617.png"},{"id":64460382,"identity":"0ae1e01e-f90b-487b-812b-1fe2303cf1f5","added_by":"auto","created_at":"2024-09-13 12:38:22","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":128428,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDifferent epigenetic feature of silenced two \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eBrGRS1\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003ehomologs in Chinese cabbage. \u003c/strong\u003eIntegrative Genomic Viewer (IGV) illustration showing epigenomic profiles of four different histone marks, H3K4me2, H3K36me3, H3K27me3, and H3K9me2 on \u003cem\u003eBrGRS1.1\u003c/em\u003e (left panel) and \u003cem\u003eBrGRS1.2\u003c/em\u003e homologs (right panel). Each row represents the normalized ChIP-seq read density of histone mark. Ranged numbers in parenthesis in y-axis on individual track indicate the amplitude of the ChIP-seq read signal. Input, control Input DNA; H3, H3 histone; H3K4me2, di-methylated H3 histone at Lys (K) 4 residue; H3K36me3, tri-methylated H3 histone at Lys (K) 36 residue; H3K27me3, tri-methylated H3 histone at Lys (K) 27 residue; H3K9me2, di-methylated H3 histone at Lys (K) 9 residue; Detailed information regarding the genome-wide data was provided in Supplementary Table S4. Transcriptional start site of each \u003cem\u003eBrGRS1 \u003c/em\u003ehomolog was indicated by arrows on the bottom.\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-4895273/v1/33c8309337aca559759bf4ee.png"},{"id":64460410,"identity":"17e1ffff-a6fb-4dd6-b88e-42ee07a0cf42","added_by":"auto","created_at":"2024-09-13 12:38:22","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":195787,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMeasurement of GSL compounds along wounding time points between radish ‘JD’ and Chinese cabbage ‘BL3’ A) \u003c/strong\u003eAmounts of total GSLs (combining amounts of total aliphatic GSLs and indolic GSLs) along four different wounding time points (0h, 24h, 72h, and 120h after wounding). \u003cstrong\u003eB)\u003c/strong\u003eAmounts of aliphatic GSLs along four different wounding time points (0h, 24h, 72h, and 120h after wounding). \u003cstrong\u003eC)\u003c/strong\u003e Amounts of indolic GSLs along four different wounding time points (0h, 24h, 72h, and 120h after wounding). \u0026nbsp;\u003cstrong\u003eA) \u003c/strong\u003e~ \u003cstrong\u003eC)\u003c/strong\u003e Leaf tissues of 5-week-old seedlings were wounded with scissors, and then harvested along different wounding time points. One-way analysis of variance (ANOVA) and post-hoc Tukey’s test (p \u0026lt; 0.05) were used to analyze statistical differences. Mean ± standard deviation (SD) of three biological replicates were presented. JD: ‘Jinjudaepyung’ radish, BL3: ‘Bulam-3’ Chinese cabbage.\u003c/p\u003e","description":"","filename":"floatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-4895273/v1/bb1173352becc258f690ccef.png"},{"id":64460376,"identity":"3af4aee7-e495-429e-b2a0-8f61d65a10b2","added_by":"auto","created_at":"2024-09-13 12:38:20","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":576898,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eResult of qRT-PCR analysis of aliphatic and indolic GSL pathway genes detected from leaf tissue of radish and Chinese cabbage along wounding time course. A) \u003c/strong\u003eExpression patterns of seven genes related to aliphatic GSL biosynthesis\u003cstrong\u003e \u003c/strong\u003ein radish along wounding time points (0h, 24h, 72h, and 120h after wounding treatment) \u003cstrong\u003eB) \u003c/strong\u003eExpression patterns of three genes related to indolic GSL biosynthesis\u003cstrong\u003e \u003c/strong\u003ein radish along wounding time points (0h, 24h, 72h, and 120h after wounding treatment). \u003cstrong\u003eC)\u003c/strong\u003e Expression patterns of seven genes related to aliphatic GSL biosynthesis\u003cstrong\u003e \u003c/strong\u003ein Chinese cabbage along wounding time points (0h, 24h, 72h, and 120h after wounding treatment). \u003cstrong\u003eD)\u003c/strong\u003e Expression patterns of four genes related to indolic GSL biosynthesis\u003cstrong\u003e \u003c/strong\u003ein Chinese cabbage along wounding time points (0h, 24h, 72h, and 120h after wounding treatment). \u003cstrong\u003eA) ~ D) \u003c/strong\u003eOne-way analysis of variance (ANOVA) and post-hoc Tukey’s test (p \u0026lt; 0.05) were used to analyze statistical differences. Mean ± standard deviation (SD) of three biological replicates were presented.\u003c/p\u003e","description":"","filename":"floatimage8.png","url":"https://assets-eu.researchsquare.com/files/rs-4895273/v1/7390efc77b2b9bba3fc0bc17.png"},{"id":64460363,"identity":"8a0c294c-7804-40b4-b046-9c733154c1dc","added_by":"auto","created_at":"2024-09-13 12:38:18","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":170442,"visible":true,"origin":"","legend":"\u003cp\u003eSchematic illustration of biosynthetic pathway genes and corresponding GSLs compounds in the ‘secondary modification’ phase for aliphatic GSL compounds between Chinese cabbage and radish plants. Methylthioalkyl-GSLs derived from the ‘core structure formation’ phase can be either glucoerucin (GER, 4-carbon GSL) or glucoberteroin (GBT, 5-carbon GSL). In case of Chinese cabbage, a 4-carbon GSL, GER can be processed to generate glucoraphanin (GRA) and gluconapin (GNP) by enzymatic activity of FMO GS-OXs and AOP2, respectively. In case of a 5-carbon GSL, GBT can be processed to GAS and GBN by FMO GS-OXs and AOP2, respectively. Two divergent pathway occurring in Chinese cabbage were indicated by orange color boxes. In case of radish, a 4-carbon GSL, GER can be converted to glucoraphasatin (GRH) by enzymatic activity of GRS1, then further processed to produce glucoraphenin (GRE) by FMO GS-OXs. A pathway occurring in radish was indicated by blue color box.\u003c/p\u003e","description":"","filename":"floatimage9.png","url":"https://assets-eu.researchsquare.com/files/rs-4895273/v1/a62e4b08128faa9b8c31df24.png"},{"id":71552448,"identity":"81756182-f743-4ebd-a7a9-b1be1555cc0c","added_by":"auto","created_at":"2024-12-16 16:06:14","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3611553,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4895273/v1/42648341-2cf7-43e8-9e28-27293faaf545.pdf"},{"id":64460408,"identity":"2ba9ea13-b137-4668-9494-fe0957d40813","added_by":"auto","created_at":"2024-09-13 12:38:22","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":1281412,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryInformation.docx","url":"https://assets-eu.researchsquare.com/files/rs-4895273/v1/f4e2fe006e478cdc4695837c.docx"}],"financialInterests":"","formattedTitle":"A 2-oxoglutarate-dependent dioxygenase, GLUCORAPHASATIN SYNTHASE 1 (GRS1) is a major determinant for different aliphatic glucosinolates between radish and Chinese cabbage","fulltext":[{"header":"Introduction","content":"\u003cp\u003eGlucosinolates (GSLs) are a group of nitrogen and sulfur-containing compounds found in the Brassicale plants like Chinese cabbage, cabbage, radish, broccoli, kale and so on. GSLs and their hydrolyzed products play a defensive role against a variety of abiotic and biotic stresses. Intensive studies using Arabidopsis plant have identified most of transcription factors and catalytic enzyme genes related to the production of GSLs (Sonderby et al. 2010). For instance, a subgroup of R2R3-type MYB genes like MYB28, MYB29, and MYB76 regulate biosynthesis of aliphatic GSLs (Gigolashvili et al. 2008; Hirai et al. 2007; Sonderby et al. 2007). Meanwhile, other three R2Re-type MYB genes such as MYB34, MYB51, and MYB122 are involved in the production of indolic GSL compounds (Celenza et al. 2005; Gigolashvili et al. 2007; Malitsky et al. 2008). Based on amino acid precursors, GSLs can be grouped into three categories: aliphatic, indolic, and aromatic GSLs. Aliphatic GSLs are produced from alanine, leucine, isoleucine, valine, and methionine, whereas indolic and aromatic GSLs are generated from tryptophan and phenylalanine or tyrosine, respectively (Fahey et al. 2001).\u003c/p\u003e \u003cp\u003eThese MYB TFs regulate the downstream catalytic pathways for GSLs, which is mainly composed of three major stages: 1) chain elongation, 2) core structure formation, 3) secondary modification (Sonderby et al. 2010). Production of aliphatic GSLs requires these three stages, whereas indolic and aromatic GSLs does not require the chain elongation stage (Petersen et al. 2019). For the production of the aliphatic GSLs, amino acid precursor (i.e. methionine) are catalyzed to a corresponding elongated 2-oxo acid through several catalytic steps including deamination step by BRANCHED CHAIN AMINO ACID TRANSFERASE 4 (BCAT4), condensation step by METHYLTHIOALKYL MALATE SYNTHASES (MAMs), isomerization step by ISOPROPYLMALATE ISOMERASES (IPMIs), and oxidative decarboxylation step by ISOPROPYLMALATE DEHYDROGENASES (IPMDHs) in the \u0026ldquo;chain elongation\u0026rdquo; stage (Petersen et al. 2019). Elongated 2-oxo acid were subsequently transaminated by BRANCHED CHAIN AMINO ACID TRANSFERASE 3 (BCAT3) and enters into the second stage, \u0026ldquo;core structure formation\u0026rdquo; (Halkier and Gershenzon 2006).\u003c/p\u003e \u003cp\u003eIn the second stage, \u0026ldquo;core structure formation\u0026rdquo;, several consecutive reactions are catalyzed to generate the desulfo-aliphatic GSLs from the corresponding elongated 2-oxo acid. It is initiated from the oxidation step by CYTOCHROME P450 MONOOXYGENASE family proteins such as CYP79F1, CYP79F2, and CYP83A1, then, conjugation step by GLUTATHIONE-BY-GLUTATHIONE S-TRANSFERASE F11 (GSTF11) and GLUTATHIONE S-TRANSFERASE TAU20 (GSTU20). It is further hydrolyzed by GAMMA-GLUTAMYL PEPTIDASE 1 (GGP1), cleaved by C-S LYASE (SUR1), the undergoes the glycosylation step by UDP-GLUCOSYLTRANSFERASE 74B1 (UGT74B1), and sulfation step by SULFOTRANSFERASE (SOT17/18) (Halkier and Gershenzon 2006).\u003c/p\u003e \u003cp\u003eThird stage, \u0026ldquo;secondary modification\u0026rdquo;, is responsible for the production of various types of GSLs via oxidation, alkylation, methoxylation, and desaturation step in Brassicale plants (Rask et al. 2000; Mikkelsen et al. 2002; Grubb and Abel 2006). For example, in \u003cem\u003eB. rapa\u003c/em\u003e, FLAVIN MONOOXYGENASES (FMO GS-OXs) catalyzes the conversion of methylthioalkyl GSLs to methylsulfinylalkyl GSLs, then methylsulfinylalkyl GSLs is further converted to alkenyl GSLs by ALKENYLHYDROXALKYL-PRODUCING 2 (AOP2) (Hansen et al. 2008). In case of the indolic GSL pathway, CYP81F enzymes catalyze the hydroxylation of indolic GSLs. For example, CYP81F2 catalyzes the production of 4-hydroxyindolic GSL (Sonderby et al. 2010; Grubb and Abel 2006). These hydroxyindolic GSLs can be further methylated by the enzymatic activity of INDOLE GLUCOSINOLATE METHYLTRANSFERASES 1 and 2 (IGMT1 and IGMT1) (Pfalz et al. 2011).\u003c/p\u003e \u003cp\u003eRadish (\u003cem\u003eRaphanus sativus\u003c/em\u003e L.) is an edible tuberous root vegetable, which is consumed globally. Radish is reported as a rich source of aliphatic GSLs due to the presence of glucoraphasatin (GRH), which occupies approximately 60\u0026ndash;90% proportion of aliphatic GSLs in radish (Kang et al. 2020; Nugroho et al. 2021). A study reported that \u003cem\u003eGLUCORAPHASATIN SYNTHASE 1\u003c/em\u003e (\u003cem\u003eGRS1\u003c/em\u003e) encoding 2-oxoglutarate-dependent dioxygenase is responsible for the conversion of glucoerucin (GER) to GRH through its dehydrogenase activity (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) (Kakizaki et al., 2017). Further conversion of GRH to glucoraphenin (GRE) is catalyzed by a subgroup of FLAVIN-CONTAINING MONOOXYGENASE (FMO GS-OXs) family proteins (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eChinese cabbage (\u003cem\u003eBrassica rapa\u003c/em\u003e L.) is also one of the most important dietary vegetable crops in South Asia, which contains beneficial nutrients including GSLs. Intensive studies have also been conducted to elucidate the biosynthetic processes of aliphatic GSLs in Chinese cabbage (Zang et al. 2009). Even radish and Chinese cabbage share similar biosynthetic pathways for the production of GSLs, radish contains a large amount of aliphatic GSLs, including glucoraphasatin (GRH) and glucoraphenin (GRE), while Chinese cabbage contains alkenyl GSLs, such gluconapin (GNP) and glucobrassicanapin (GBN) (Baek et al. 2016; Nugroho et al. 2020; Nugroho et al. 2021). Molecular details underlying different profiles of GSLs between radish and Chinese cabbage were not fully understood. In this study, we investigated the molecular relationship between aliphatic GSLs profiles and expression patterns of the genes involved in the aliphatic GSLs production between radish and Chinese cabbage. We believe the results presented herein provide better understanding on the molecular details of aliphatic GSL biosynthesis between radish and Chinese cabbage.\u003c/p\u003e"},{"header":"RESULTS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eGSLs are more abundant in radish than Chinese cabbage\u003c/h2\u003e \u003cp\u003eIt was reported that radish and Chinese cabbage contains a different profile of GSLs (Nugroho et al. 2020). To understand the molecular details underlying different profiles of GSLs between two species, eight week-old seedlings of radish and Chinese cabbage were subjected to the quantification of GSLs from leaves and roots. In case of total GSLs (amounts of total aliphatic GSLs\u0026thinsp;+\u0026thinsp;total indolic GSLs). We noticed that radish \u0026lsquo;Jinjudaepyung (JD)\u0026rsquo; has more abundant amounts of total GSLs than those of Chinese cabbage \u0026lsquo;Bulam-3 (BL3)\u0026rsquo; in both leaves and roots (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). Two groups of GSLs, aliphatic and indolic GSLs were all more abundant in radish than Chinese cabbage (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB and \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). In addition, we noticed that root tissue of radish contained more amounts of total GSLs than those of leaf tissue, whereas Chinese cabbage contained lower amounts of total GSLs in root tissue in comparison to that of leaf tissue.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn detail, based on HPLC chromatogram results, we identified seven GSLs (GER, GRH, GRE, GNP, GAS, GBN, GNA) from radish and eleven GSLs (PGT, GRA, GAS, GAFN, GNP, 4-HGB, GBN, GBS, 4-MTGB, GNT, and NGB) from Chinese cabbage (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA\u0026thinsp;~\u0026thinsp;3D). In radish, both leaf and root tissues contained four aliphatic GSLs and three indolic GSLs. Among them, GRH and to a lesser extent, GRE were major GSL compounds (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). Meanwhile, Chinese cabbage had seven aliphatic and five indolic GSLs in both leaf and root tissues (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC\u0026thinsp;~\u0026thinsp;3D). Among aliphatic GSL compounds, GBN and to lesser extent, GRA were most abundant GSL compounds. For indolic GSL compounds, NGB was most abundant in our detection condition.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eRadish and Chinese cabbage have different profiles of aliphatic GSLs\u003c/h2\u003e \u003cp\u003eIn leaf tissues, amount of GER, which is a precursor GSL of GRA and GRH, was not detected from Chinese cabbage in our detection condition, whereas greatly detected in radish (148.39 nmol/g DW) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA, \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE and Supplementary Table S1). Because GRH is synthesized from GER, biosynthesis of high amount of GER in leaf of radish might contribute to the high content of GRH (698.33 nmol/g DW) in radish. Similar to GRH, GRE which is converted from GRH was also abundant in the leaf tissues of radish (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). On the contrary, GER was not detected in Chinese cabbage, possibly being fully converted to GRA in leaf tissue of Chinese cabbage (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC, \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE, and Supplementary Table S1). As expected, GRA in leaf tissue of Chinese cabbage was substantially detected (175.26 nmol/g DW). GNP, which is converted from GRA was most abundantly detected among GSLs compounds (1,389.12 nmol/g DW).\u003c/p\u003e \u003cp\u003eIn root tissues of radish, amounts of aliphatic GSLs were higher than those of leaf tissues (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB, and Supplementary Table S1). Similar to leaf tissue, GRH (8,701.35 nmol/g DW) and GBN (742.3 nmol/g DW) were most abundant GSLs in the root tissues of radish and Chinese cabbage, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE and Supplementary Table S1). To a lesser extent, GRE (3,527.95 nmol/g DW) and GRA (89.19 nmol/g DW) were 2nd most highly produced in radish and Chinese cabbage, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE, and Supplementary Table S1). In particular, we noticed that in radish, GSLs were more highly accumulated in root tissue than leaf tissue. It might implicate that even though glucose, an output molecule of photosynthesis and also a substrate for production of GSLs is actively synthesized in the leaf tissue, there might be a transportation system of synthesized GSLs into root tissue, possibly for the storage of GSLs.\u003c/p\u003e \u003cp\u003e \u003cb\u003eTwo homologs of\u003c/b\u003e \u003cb\u003eGRS1\u003c/b\u003e, \u003cb\u003eare merely expressed in Chinese cabbage\u003c/b\u003e\u003c/p\u003e \u003cp\u003eMost dramatic difference of aliphatic GSLs profile was derived from the production of GRH and GBN from radish and Chinese cabbage, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE). In radish, while GRH was most highly produced, merely detected in Chinese cabbage. Vice versa, GBN was not detected in radish, most abundantly detected in Chinese cabbage. Because it was previously reported that GRH is synthesized from GER by GRS1, a 2-oxoglutarate dependent dioxygenase enzyme in radish (Kakizaki et al. 2017), we decided to check expression of \u003cem\u003eGRS1\u003c/em\u003e from radish and Chinese cabbage. When we performed BLAST search using the amino acid sequence of radish GRS1 (\u003cem\u003eRsGRS1\u003c/em\u003e, Rs002010), two homologs of \u003cem\u003eGRS1\u003c/em\u003e (named as \u003cem\u003eBrGRS1.1\u003c/em\u003e and \u003cem\u003eBrGRS1.2\u003c/em\u003e) were detected in the \u003cem\u003eBrassica rapa\u003c/em\u003e genome database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ewww.brassicadb.cn/#\u003c/span\u003e\u003cspan address=\"http://www.brassicadb.cn/#\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA and Supplementary Fig. S1). Multiple sequence alignment analysis between RsGRS1 and two copies of BrGRS1 (BrGRS1.1 and BrGRS1.2) indicated that function domains (2-oxoglutarate binding, Fe2\u0026thinsp;+\u0026thinsp;binding, and dioxygenase activity) of them were well conserved. It indicated that catalytic activity as a dioxygenase enzyme might operate normally. We also conducted genomic synteny analysis using genomic sequence of \u003cem\u003eRsGRS1\u003c/em\u003e against \u003cem\u003eBrassica rapa\u003c/em\u003e genome. As expected, two copies of \u003cem\u003eRsGRS1\u003c/em\u003e homologs (Bra033396 and Bra033397) were detected in the \u003cem\u003eBrassica rapa\u003c/em\u003e genome (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). Interestingly, Bra033396 (named as \u003cem\u003eBrGRS1.1\u003c/em\u003e) and Bra033397 (\u003cem\u003eBrGRS1.2\u003c/em\u003e) were very closely located each other, suggesting that radish GRS1 homolog might be duplicated during evolution process in Chinese cabbage.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eExpression of\u003c/b\u003e \u003cb\u003eGRS1\u003c/b\u003e \u003cb\u003eis a major cause of different profile of GSLs between radish and Chinese cabbage\u003c/b\u003e\u003c/p\u003e \u003cp\u003eVariation of aliphatic GSL compounds is affected by the catalytic enzymes belonging to the \u0026lsquo;secondary modification\u0026rsquo; phase such as FMO GS-OXs (FLAVIN-CONTAINING MONOOXYGENASES) or GRS1 in Brassicaceae family plants (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). For example, GER can be either converted to GRH or GRA in radish and Chinese cabbage, respectively by the catalytic activity of GRS1 and FMO GS-OXs. These catalytic enzymes might play a critical role in the divergent production of aliphatic GSLs between radish and Chinese cabbage. Thus, we examined how many \u003cem\u003eFMO GS-OX\u003c/em\u003e genes exist in the \u003cem\u003eBrassica rapa\u003c/em\u003e and radish genome. Using amino acid sequences from four \u003cem\u003eArabidopsis\u003c/em\u003e FMO GS-OX, we performed the BLAST search at \u003cem\u003eBrassica rapa\u003c/em\u003e genome database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e\u003ca href=\"http://www.brassicadb.cn/#\" target=\"_blank\"\u003ewww.brassicadb.cn/#\u003c/a\u003e\u003c/span\u003e\u003cspan address=\"http://www.brassicadb.cn/#\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) and radish genome database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.radish-genome.org/\u003c/span\u003e\u003cspan address=\"http://www.radish-genome.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), respectively. Total seven radish FMO GS-OXs (RsFMO GS-OX1\u0026thinsp;~\u0026thinsp;RsFMO GS-OX7) and five Chinese cabbage FMO GS-OX (BrFMO GS-OX2\u0026thinsp;~\u0026thinsp;BrFMO GS-OX4, BrFMO GS-OX6\u0026thinsp;~\u0026thinsp;BrFOM GS-OX7) were found (Supplementary Fig. S2).\u003c/p\u003e \u003cp\u003eExpression of these \u003cem\u003eFMO GS-OX\u003c/em\u003e homologs from radish and Chinese cabbage were examined by the quantitative RT-PCR (qRT-PCR) analysis. Among five \u003cem\u003eFMO GS-OX\u003c/em\u003e homologs in Chinese cabbage, three \u003cem\u003eFMO GS-OXs\u003c/em\u003e (\u003cem\u003eBrFMO GS-OX5\u0026thinsp;~\u0026thinsp;BrFMO GS-OX7\u003c/em\u003e) were dominantly expressed in the five-week-old seedling plants (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). It indicated that these three \u003cem\u003eFMO GS-OX\u003c/em\u003e genes (\u003cem\u003eBrFMO GS-OX5\u003c/em\u003e, \u003cem\u003eBrFMO GS-OX6\u003c/em\u003e, and \u003cem\u003eBrFMO GS-OX7\u003c/em\u003e) might play an important role in the conversion of GER to GRA. Meanwhile, transcripts of two \u003cem\u003eRsGRS1\u003c/em\u003e homologs (\u003cem\u003eRsGRS1.1\u003c/em\u003e and \u003cem\u003eRsGRS1.2\u003c/em\u003e) were merely detected in the Chinese cabbage in our qRT-PCR analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). In an agreement of this observation, these two \u003cem\u003eRsGRS1\u003c/em\u003e homologs were not detected in our RNA-seq dataset, possibly filtered out due to the low expression of these \u003cem\u003eBrGRS1\u003c/em\u003e homologs. The fact that \u003cem\u003eBrGRS1.1\u003c/em\u003e and \u003cem\u003eBrGRS1.2\u003c/em\u003e were merely expressed in Chinese cabbage indicated that they cannot impact on the first catalytic step of \u0026lsquo;secondary modification\u0026rsquo; stage of GSLs biosynthesis in Chinese cabbage. Hence, a majority of aliphatic GSL compounds seems to be destined to enter GRA-GNP production path, but not GRH-GRE path.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn case of radish, total seven \u003cem\u003eFMO GS-OX\u003c/em\u003e homologs were found in the radish genome. Interestingly, all seven radish \u003cem\u003eFMO GS-OX\u003c/em\u003e homologs (\u003cem\u003eRsFMO GS-OX1\u0026thinsp;~\u0026thinsp;RsFMO GS-OX7\u003c/em\u003e), were merely expressed in radish young plants (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). It suggested that mere expression of all these \u003cem\u003eRsFMO GS-OX\u003c/em\u003e genes resulted in the low abundance of GER-GRA-GNP path in radish. Meanwhile, a 2-oxoglutarate-dioxygenase, \u003cem\u003eRsGRS1\u003c/em\u003e was substantially expressed in radish (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). It indicated that active expression of \u003cem\u003eRsGRS1\u003c/em\u003e directed the first catalytic step of \u0026lsquo;secondary modification\u0026rsquo; stage of GSLs biosynthesis toward path for the substantial production of GRH in radish.\u003c/p\u003e \u003cp\u003e \u003cb\u003eBrGRS1.1\u003c/b\u003e \u003cb\u003eis constantly silenced, but\u003c/b\u003e, \u003cb\u003eBrGRS1.2\u003c/b\u003e \u003cb\u003eis epigenetically regulated in Chinese cabbage\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThough there are two copies of GRS1 homologs in Chinese cabbage genome, they were merely expressed. Histone modification contexts of gene is highly correlated with the status of expression of the gene (Dong and Weng 2013). To understand the molecular reason on the mere expressions of \u003cem\u003eBrGRS1\u003c/em\u003e homologs, we analyzed a recently published \u003cem\u003eB. rapa\u003c/em\u003e epigenome dataset containing enrichment profiles of four histone marks like H3K4me2, H3K36me3, H3K27me3, and H3K9me2. While H3K36me3 mark represent a histone mark closely correlated with active transcription, other three marks like H3K4me2, H3K27me3 and H3K9me2 represent histone marks related to the gene repression. When we looked into four histone enrichment profiles, genomic region of \u003cem\u003eBrGRS1.1\u003c/em\u003e was not enriched with any histone marks, implying that \u003cem\u003eBrGRS1.1\u003c/em\u003e is constantly silenced (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e left). Meanwhile, \u003cem\u003eBrGRS1.2\u003c/em\u003e was substantially enriched with two repressive histone marks, H3K4me2 and H3K27me3, suggesting that \u003cem\u003eBrGRS1.2\u003c/em\u003e is in a state of epigenetic suppression context (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e right). It is worthy to note that a small amount of GRH was detected in the root tissue of Chinese cabbage. It is possible that \u003cem\u003eBrGRS1.2\u003c/em\u003e is a bit expressed in the root tissue of Chinese cabbage and contribute to the small production of GRH in Chinese cabbage. Alternatively, it is also possible that because a bit expression of \u003cem\u003eBrGRS1.2\u003c/em\u003e was detected in the leaf tissue of Chinese cabbage (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA), BrGRS1.2 might contribute to the production of GRH in the leaf tissue, then synthesized GRH in the leaf might be subsequently translocated to root tissue by GTR transport system in Chinese cabbage, thus not showing detection of GRH in the leaf tissue of Chinese cabbage (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). This hypothesis needs further investigation. Taken together, constantly silenced \u003cem\u003eBrGRS1.1\u003c/em\u003e and epigenetically suppressed \u003cem\u003eBrGRS1.2\u003c/em\u003e might explain why these two \u003cem\u003eBrGRS1\u003c/em\u003e homologs were merely expressed in Chinese cabbage and GRH was not detected in the leaf tissue of Chinese cabbage.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eAliphatic and indolic GSLs were induced by wounding in both radish and Chinese cabbage\u003c/h2\u003e \u003cp\u003eIt was reported that GSLs are induced upon abiotic (i.e. salt and drought) and biotic stresses (i.e. insects and herbivores) in some \u003cem\u003eBrassicaceae\u003c/em\u003e family plants (Muthusamy and Lee 2023; Nephali et al. 2020). To examine whether radish aliphatic GSLs are induced by stress like wounding, we treated wounding on leaves of radish and Chinese cabbage and measured the amounts of aliphatic and indolic GSLs along wounding time points (0h, 24h, 72h, and 120h after wounding). Amounts of total GSLs were substantially increased after wounding in both radish, \u0026lsquo;JD\u0026rsquo; and Chinese cabbage, \u0026lsquo;BL3\u0026rsquo; line (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA and Supplementary Table S5).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn case of aliphatic GSLs, total amounts were dramatically increased from the 0h sample to 120h sample after wounding in both radish and Chinese cabbage (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB and Supplementary Table S5). For instance, in radish, total aliphatic GSLs at 0h (254.7 nmol/g∙DW) was increased to 1,515.0 nmol/g∙DW at 120h after wounding (5.9 times increase). In case of Chinese cabbage, total aliphatic GSLs at 0h (39.72 nmol/g∙DW) was increased to 202.03 nmol/g∙DW at 120h sample after wounding (5.1 times increase). Similar to the case of aliphatic GSLs, wounding treatment significantly increased amounts of indolic GSLs after wounding in both radish and Chinese cabbage (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eC and Supplementary Table S5). In case of radish, amount of total indolic GSLs was moderately (1.6 times) increased from 0h (115.5 nmol/g∙DW) to 120h (180.0 nmol/g∙DW). Meanwhile, total indolic GSLs in Chinese cabbage was more drastically (4.9 times) induced from 0h (21.56 nmol/g∙DW) to 120h (102.87 nmol/g∙DW) after wounding. Collectively, these data indicate that GSL compounds are wound-responsively synthesized in both radish and Chinese cabbage.\u003c/p\u003e \u003cp\u003eNext, we examined whether wound-induced increase of GSL compounds was resulted from the triggered expression of GSLs pathway genes. Expression profiles of total 10 and 11 GSL pathway genes from radish and Chinese cabbage, respectively were examined along wounding time course. Resultantly, most of tested aliphatic and indolic GSL pathway genes commonly exhibited increased levels of transcription after wounding, even dynamic expression profiles were detected in both radish and Chinese cabbage (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eA\u0026thinsp;~\u0026thinsp;8D).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eBased on these observations, we came up with schematic model on \u003cem\u003eGRS1\u003c/em\u003e action in the divergent profile of GSL compounds between radish and Chinese cabbage (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003e). In brief, major difference of aliphatic GSLs profiles between two species seems to be determined by the dominantly expressed genes in the first catalytic step of the \u0026lsquo;secondary modification\u0026rsquo; stage, which are responsible for enzymatic catalysis of methylthioalkyl-GSLs (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003e). For example, in Chinese cabbage, FMO GS-OXs catalyze the oxidation of 4-carbon methylthioalkyl-GSLs (GER) or 5-carbon methylthioalkyl-GSLs (GBT) to 4-methylsulfinylalkyl-GSLs (GRA) or 5- methylsulfinylalkyl-GSLs (GAS), respectively. Methylsulfinylalkyl-GSLs can be further converted to alkenyl-GSLs, which is catalyzed by AOPs (2-oxoglutarate-dependent dioxygenase) like AOP2 and AOP3 (indicated with orange color boxes, Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003e). Interestingly, we found that in radish, \u003cem\u003eRsGRS1\u003c/em\u003e is dominantly expressed in radish, instead of \u003cem\u003eFMO GS-OXs\u003c/em\u003e. Dominantly expressed \u003cem\u003eRsGRS1\u003c/em\u003e shift a direction towards the conversion of a precursor, glucoerucin (GER) into glucoraphasatin (GRH), a type of methylthioalkyl-glucosinolate (indicated with blue color box, Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003e). This different expression profile of \u003cem\u003eFMO GS-OXs\u003c/em\u003e and \u003cem\u003eGRS1\u003c/em\u003e resulted in the divergent entry of aliphatic GSLs biosynthesis in the first step of \u0026lsquo;secondary modification\u0026rsquo; stage between Chinese cabbage and radish.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn summary, presence of active \u003cem\u003eRsGRS1\u003c/em\u003e gene in radish play an important role in the production of glucoraphasatin (GRH) and glucoraphenin (GRE), a major aliphatic GSLs in radish. Meanwhile, Chinese cabbage was found to merely produce glucoraphasatin (GRH), instead producing glucoraphanin (GRA) and gluconapin (GNP) due to the absence of expression of \u003cem\u003eRsGRS1\u003c/em\u003e homologs in Chinese cabbage.\u003c/p\u003e \u003c/div\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eOver 130 different glucosinolates can by synthesized in Brassicaceae family plants (Nguyen et al. 2020). Composition of GSL compounds varies along different tissues and developmental stages. Besides, different species has unique GSLs composition. Production of diverse GSLs compounds can be derived from the different profile of GSL biosynthetic genes as well as GSL transport from one tissue to others. Long-distance transport of GSLs was reported from a study using \u003cem\u003eArabidopsis\u003c/em\u003e plants, in which it is mediated by two nitrate/peptide transporter proteins named as GTR1 and GTR2 (Andersen et al. 2013; Nour-Eldin et al. 2012). In our study, we noticed that root tissue of radish contained more substantial amounts of GRH than those of leaf tissue, whereas Chinese cabbage contained lower amounts of total GSLs in root tissue than those of leaf tissue (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Although \u003cem\u003eRsGRS1\u003c/em\u003e was highly expressed in the leaves of radish (Supplementary Fig S5), GRH was found in a greater amount in the roots, not in the leaves. This discrepancy might imply that GRH synthesized in leaves might be actively translocated to roots in radish. This observation is in a line with a recent report proposing that long distance transport might resulted in the accumulation of GRH in radish roots (Kakizaki et al., 2017). In detail, a wild type radish scion grafted to a mutant \u003cem\u003egrs1\u003c/em\u003e root stock evidently displayed a high accumulation of GRH in the root tissue of the \u003cem\u003egrs1\u003c/em\u003e mutant. Meanwhile, a non-grafted radish \u003cem\u003egrs1\u003c/em\u003e mutant did not show accumulation of GRH in root tissues. Thus, even though high amounts of GSLs were produced in leaf tissue, it is highly plausible to be translocated and subsequently accumulated in root tissues in \u003cem\u003eBrassicaceae\u003c/em\u003e family plants including radish (Sotelo et al. 2016; Touw et al. 2019). It might be an interesting topic to investigate the translocation of GRH from shoot to root tissues possibly by glucosinolate transporters in radish.\u003c/p\u003e \u003cp\u003eIt was previously suggested that the \u003cem\u003eGRS1\u003c/em\u003e and its product, GRH, originally exist only in radish and not in other \u003cem\u003eBrassica\u003c/em\u003e plants (Ishida et al. 2014; Kakizaki et al. 2017). However, a more recent studies of GSLs reported that a small amount of GRH were detected in several brassica plants including Chinese cabbage, broccoli, and choysum (Liang et al. 2018; Nugroho et al. 2020). In this study, albeit a low amount was detected, we also observed that GRH is produced in Chinese cabbage, particularly in root tissue by the virtue of \u003cem\u003eBrGRS1.2\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD). However, expression of \u003cem\u003eBrGRS1.1\u003c/em\u003e was constantly suppressed and merely expressed even in wounding stress, implying that \u003cem\u003eBrGRS1.1\u003c/em\u003e might be stably silenced. Furthermore, we found that chromatin of \u003cem\u003eBrGRS1.2\u003c/em\u003e is epigenetically regulated, that is, in a state of repressive histone mark context. Genomic region of \u003cem\u003eBrGRS1.2\u003c/em\u003e was shown to be enriched with an two repressive histone marks, H3K4me2 and H3K27me3. Repressive histone mark context of \u003cem\u003eBrGRS1.2\u003c/em\u003e might imply that \u003cem\u003eBrGRS1.2\u003c/em\u003e is delicately regulated in an epigenetic manner in a certain condition. In a similar line, we previously reported that radish \u003cem\u003eRsGRS1\u003c/em\u003e is also epigenetically regulated during vernalization, highly enriched with H3K27me3 histone mark by long-term cold in radish (Nugroho et al. 2021). These results suggest that some of GSL biosynthetic genes including \u003cem\u003eGRS1\u003c/em\u003e in Brassicaceae family crop plants might be under the delicate control of transcription in an epigenetic manner.\u003c/p\u003e"},{"header":"MATERIALS AND METHODS","content":"\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003ePlant materials and growth condition\u003c/h2\u003e \u003cp\u003eSeeds of radish (Jinjudaepyung\u0026rsquo;) and Chinese cabbage (\u0026lsquo;Bulam3\u0026rsquo;) were purchased from the Coupang online retailer and used in this study. Seeds were sterilized and plated on a half-strength solid Murashige and Skoog (MS) media and incubated at 4\u003csup\u003eo\u003c/sup\u003eC for at least 3 days under the dark. Radish and Chinese cabbage seedlings with a 2\u0026ndash;4 mm radicle were transplanted into the soil in the growth room at 22\u003csup\u003eo\u003c/sup\u003eC under long day light (16h light:8h dark) condition. After growth of seedling for certain period of weeks (5\u0026thinsp;~\u0026thinsp;8 weeks), shoots or roots of all seedling plants were sampled for the measurement of glucosinolates (GSLs) and further molecular analyses like qRT-PCR analysis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eExtraction and analysis of GSLs\u003c/h2\u003e \u003cp\u003eFor harvested shoots and roots tissues, eight-week-old seedlings were sampled and directly lyophilized using a vacuum freeze dryer (Ilshin Biobase, Korea) and ground into a fine powder for the HPLC analysis. DS-GSLs (desulfo glucosinolates) were isolated and analyzed as described previously (Nugroho et al. 2021). For wounding samples, shoots tissue of five-week-old seedlings were sampled and used for quantification of GSLs. Individual DS-GSLs were analyzed with high performance liquid chromatography (Vanquish HPLC System, Thermo scientific). The DS-GSLs were separated on a C18 reverse phase column (Zorbax XDB-C18, 4.6 x 250mm, 5\u0026micro;m particle size, Agilent, USA) with a water and acetonitrile gradient system. Total 20 \u0026micro;L of samples were injected in a flow rate of 1.0 mL min\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. Individual peak was identified using standard compounds (Phytoplan, Germany) (Supplementary Table S2), and sinigrin compound was used for relative quantification (Brown et al., 2003). The contents were analyzed independently with three replicates and presented in nmol g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e on a dry weight (DW) basis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eQuantitative RT-PCR analysis\u003c/h2\u003e \u003cp\u003eAfter growth of seedlings for 5 weeks in soil pots, shoots of plants were sampled for the extraction of total RNAs. Total RNAs were isolated from each tissue sample using the RNeasy Plant mini kit (QIAGEN, USA) according to the manufacturer\u0026rsquo;s instructions. To get rid of residual DNAs, total RNAs were treated with DNase I (New England Biolabs, USA), and then used for the cDNA synthesis using EasyScript reverse transcriptase (Transgen Biotech, China). RT-qPCR analysis was performed using BioFACT\u0026trade; 2X Real-Time PCR Mix (BioFACT, South Korea) in a LineGene 9600 Plus Real-Time PCR system (BioER, China). \u003cem\u003eRsTEF2a\u003c/em\u003e (Rs419480) and BrPP2Aa (Bra012474\u003cb\u003e)\u003c/b\u003e were respectively used as a reference gene for gene expression normalization from radish and Chinese cabbage plants (Duan et al., 2017). Three biological replicates were used for each quantitative RT-PCR analysis. One-way ANOVA with Tukey\u0026rsquo;s post-hoc test was used for statistical analysis. Information on the primer sequences used in the RT-qPCR analysis was shown in the Supplementary Table S3.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eChIP-seq dataset analysis\u003c/h2\u003e \u003cp\u003eRaw FASTQ files of ChIP-seq epigenome data analyzed in this study were downloaded from the DNA Data Bank of Japan (DDBJ) database. FASTQ reads were first trimmed and quality-filtered using FASTQC before alignment to the \u003cem\u003eBrassica rapa\u003c/em\u003e reference genome using STAR aligner (Dobin et al. 2013). Integrative Genomics Viewer (IGV) was used for visualization of aligned reads (Robinson et al. 2011). Bigwig files for IGV visualization were generated using bamCoverage command in the deepTools package (Ramirez et al. 2014). List of public ChIP-seq dataset was shown in Supplementary Table S4.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003ePhylogenetic analysis\u003c/h2\u003e \u003cp\u003eThe amino acid sequences of FMO GS-OXs and GRS1 homologs from radish, Chinese cabbage, and \u003cem\u003eArabidopsis thaliana\u003c/em\u003e were aligned together with other amino acid from corresponding organism (\u003cem\u003eArabidopsis thaliana\u003c/em\u003e) using MUSCLE method in MEGA version 7 program. Phylogenetic tree was constructed using maximum likelihood method, and bootstrap values were set at 1000 replications.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eStatistical Analysis\u003c/h2\u003e \u003cp\u003eOne-way analysis of variance (ANOVA) and post-hoc Tukey\u0026rsquo;s test (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) were used to analyze statistical differences. Data were analyzed using a statistical software package (SAS; version 9.4; SAS Institute Inc., Cary, NC, USA) and presented as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD) of three biological replicates.\u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eACKNOWLEDGEMENTS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by a grant from the National Research Foundation of Korea (NRF) (2021R1F1A1047822) toD-HK.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAUTHOR CONTRIBUTIONS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePC prepared all plant materials and performed molecular experiments; HM analyzed ChIP-seq dataset. PC and ABDN performed HPLC analysis. D-HK conceived and designed the study; PC and D-HK analyzed the data and wrote the manuscript.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCONFLICT OF INTEREST\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare they have no conflicts of interest\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDATA AVAILABILITY STATEMENT\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data supporting the findings of this study are available within the paper and its Supplementary Information. The data that support the findings of this study are also available from the corresponding author upon reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAndersen TG, Nour-Eldin HH, Fuller VL, Olsen CE, Burow M, Halkier BA (2013) Integration of biosynthesis and long-distance transport establish organ-specific glucosinolate profiles in vegetative Arabidopsis. 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FEBS J 276 (13):3559-3574. doi:10.1111/j.1742-4658.2009.07076.x\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"plant-molecular-biology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"plan","sideBox":"Learn more about [Plant Molecular Biology](https://www.springer.com/journal/11103)","snPcode":"11103","submissionUrl":"https://submission.nature.com/new-submission/11103/3","title":"Plant Molecular Biology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Glucosinolates, radish, Chinese cabbage, GLUCORAPHASATIN SYNTHASE 1, FLAVIN-CONTAINING MONOOXYGENASES","lastPublishedDoi":"10.21203/rs.3.rs-4895273/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4895273/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eGlucosinolates (GSLs) are secondary metabolites in \u003cem\u003eBrassicaceae\u003c/em\u003e plants and play a defensive role against a variety of abiotic and biotic stresses. Also, it exhibits anti-cancer activity against cancer cell in human. Different profiles of aliphatic GSL compounds between radish and Chinese cabbage were previously reported. However, molecular details underlying the divergent profile between two species were not clearly understood. In this study, we found that major difference of aliphatic GSLs profiles between two species is determined by the dominantly expressed genes in first step of the secondary modification phase, which are responsible for enzymatic catalysis of methylthioalkyl-glucosinolate. For instance, active expression of \u003cem\u003eGLUCORAPHASATIN SYNTHASE 1\u003c/em\u003e (\u003cem\u003eGRS1\u003c/em\u003e) gene in radish play an important role in the production of glucoraphasatin (GRH) and glucoraphenin (GRE), a major aliphatic GSLs in radish. Meanwhile, Chinese cabbage was found to merely produce glucoraphasatin (GRH), instead producing glucoraphanin (GRA) and gluconapin (GNP) due to the mere expression of \u003cem\u003eGRS1\u003c/em\u003e homologs and abundant expressions of \u003cem\u003eFLAVIN-CONTAINING MONOOXYGENASES\u003c/em\u003e (\u003cem\u003eFMO GS-OX)\u003c/em\u003e homologs in Chinese cabbage. In addition, we noticed that wounding treatment on leaf tissues substantially enhanced the production of aliphatic and indolic GSLs in both Chinese cabbage and radish, indicating that GSLs are wound-induced defensive compounds in both Chinese cabbage and radish plants.\u003c/p\u003e","manuscriptTitle":"A 2-oxoglutarate-dependent dioxygenase, GLUCORAPHASATIN SYNTHASE 1 (GRS1) is a major determinant for different aliphatic glucosinolates between radish and Chinese cabbage","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-09-13 12:38:12","doi":"10.21203/rs.3.rs-4895273/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Minor revisions","date":"2024-09-26T11:02:51+00:00","index":"","fulltext":""},{"type":"reviewerAgreed","content":"","date":"2024-08-20T13:58:59+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-08-18T22:29:29+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"Plant Molecular Biology","date":"2024-08-12T07:05:22+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-08-12T04:41:53+00:00","index":"","fulltext":""},{"type":"submitted","content":"Plant Molecular Biology","date":"2024-08-11T09:01:35+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"plant-molecular-biology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"plan","sideBox":"Learn more about [Plant Molecular Biology](https://www.springer.com/journal/11103)","snPcode":"11103","submissionUrl":"https://submission.nature.com/new-submission/11103/3","title":"Plant Molecular Biology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"3a1fcbc6-9223-4dee-9ca0-872cc22ce8a1","owner":[],"postedDate":"September 13th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2024-12-16T16:01:20+00:00","versionOfRecord":{"articleIdentity":"rs-4895273","link":"https://doi.org/10.1007/s11103-024-01537-7","journal":{"identity":"plant-molecular-biology","isVorOnly":false,"title":"Plant Molecular Biology"},"publishedOn":"2024-12-10 15:57:38","publishedOnDateReadable":"December 10th, 2024"},"versionCreatedAt":"2024-09-13 12:38:12","video":"","vorDoi":"10.1007/s11103-024-01537-7","vorDoiUrl":"https://doi.org/10.1007/s11103-024-01537-7","workflowStages":[]},"version":"v1","identity":"rs-4895273","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4895273","identity":"rs-4895273","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}