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

doi:10.21203/rs.3.rs-4895273/v1

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

https://doi.org/10.21203/rs.3.rs-4895273/v1

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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

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.

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. 2A). Two groups of GSLs, aliphatic and indolic GSLs were all more abundant in radish than Chinese cabbage (Fig. 2B and 2C). 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. 3A ~ 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. 3A and 3B). Meanwhile, Chinese cabbage had seven aliphatic and five indolic GSLs in both leaf and root tissues (Fig. 3C ~ 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. 3A, 3E 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. 3A). On the contrary, GER was not detected in Chinese cabbage, possibly being fully converted to GRA in leaf tissue of Chinese cabbage (Fig. 3C, 3E, 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. 2B, 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. 3E 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. 3E, 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. 3E). 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. 4A 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. 4B). 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. 5A). 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. 5A). 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. 5B). 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. 4B). 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. 5A), 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. 3C). 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. 7A 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. 7B 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. 7C 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. 8A ~ 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.

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. 3D). 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.

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^oC 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^oC 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.

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

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Abstract

Figures

Introduction

RESULTS

GSLs are more abundant in radish than Chinese cabbage

Radish and Chinese cabbage have different profiles of aliphatic GSLs

Aliphatic and indolic GSLs were induced by wounding in both radish and Chinese cabbage

DISCUSSION

MATERIALS AND METHODS