SupplementalText
An example: glucosinolate metabolism
Spatiotemporal regulation of accumulation is essential for biologically active metabolites to fulfill their functions. In the case of phenylpropanoids, regulationof accumulation is governed mainly by the gene expression profiles of biosynthetic enzymes (high M-G similarity). In contrast,regulation of accumulation of glucosinolates (GSLs), secondary metabolites specific to the plants of the order Capparales,seemsto be governed by other factors (no M-G similarity).
Among GSLs found in Arabidopsis1-3, aliphatic GSLs are produced from methionine through three biosynthetic stages: side chain elongation, core structure synthesis, and side chain modification (Supplementary Fig S2-1). MethylthioalkylGSLs (MT-GSLs) with various side chain lengths (C3 to C8) are formed viathe reactions of the first two stages. Then side chain modification of MT-GSLs takes place to form methylsulfinylalkyl- (MS-), hydroxyalkyl- (OH-), and benzoyloxyalkyl- (Bz-) GSLs. The BL-SOM result indicates high G-G similarity amongst the genes involved in chain elongation and core structure synthesis (Supplementary Fig S2-2a, shown in red), supporting previous reportsclaiming that these genes are coordinately expressed under the control of a small number of Myb gene family members4-9.While these genes show preferable expression in the leaves and the internodes (Supplementary Fig. S2-2b), GSLs accumulate predominantly in the dry seeds (Supplementary Fig. S2-2A and B, shown in orange) and the flowers (Supplementary Fig S2-2A and B, shown in green). To our knowledge, this is the first report of highGSL accumulation in the flowers of Arabidopsis. Aliphatic GSLs are clearly divided into two classes, a seed-accumulation class and a flower/leaf-accumulation class.The former is composed of MT-, OH-, and Bz-GSLs, while the latter is composed of MS-GSLs. From the seed-accumulation class, the seed/root-accumulation subclass was derived (Supplementary Fig S2-2A and B, shown in pale blue) based on the accumulation in the roots. MT-GSL with C8 chain was the only member of this subclass. The fact that GSL profiles are different in the seeds (embryos) and in the maternal organs (flowers/leaves) raises a question of how the structure-dependent GSL accumulation pattern is controlled, because MS-GSLs are formed from MT-GSLs10,and then converted into OH- and Bz-GSLs11,12(Supplementary Fig S2-1).Because the known genes involved in chain elongation and core structure synthesis are poorly express in the seeds (Supplementary Fig S2-2b), de novo GSL synthesis does not seem to occur in the seeds. Based on this finding, two hypotheses were formulated13; in the first hypothesis, MS-GSLs formed in the maternal organs are transported into the embryos and reduced to MT-GSLs by as yet unidentified enzymes. In the second hypothesis, MT-GSLs synthesized in the maternal organs are selectively transported to the embryos by as yet unidentified transporters. To date, five FMOGS-OX genes exhibiting different expression patterns (Supplementary Fig S2-3)have been characterized, and their recombinant proteins have been shown to convert MT-GSLs to MS-GSLs in vitro10,14.However, it remains unclear which FMOGS-OX is responsible for the formation of MS-GSLs accumulated in the seeds.Accumulation of OH- and Bz-GSLs in the seeds seemed to be dependent on the expression pattern of the genes, AOP3 and BZO1, encoding side chain modification enzymes.AOP3encodes a2-oxoglutarate-dependent dioxygenase responsible for the formation of OH-GSLs from MS-GSLs12, while BZO1encodes a benzoyl-CoA ligase which provides benzoyl-CoA as a substrate of Bz-GSLs formation11.
Among the genes responsible for chain elongation and core structure synthesis, MAM3 exhibited different expression pattern (strong expression in the roots) (Supplementary Fig S2-2A and Supplementary Fig S2-4). As the root-preferential pattern of MAM3 expression was similar to that of MTc8 accumulation (M-G similarity), we considered that MAM3 may play an important role in MTc8 biosynthesis in the roots. As is the case with MAM1 and MAM3, CYP79F1 and CYP79F2 catalyze the same chemical reaction with different substrate specificity (Supplementary Fig S2-1). Because CYP79F1 and CYP79F2 are cross-hybridized to the same probe sets on Affymetrix the ATH1 array used in AtGenExpress, we analyzed the expression patterns of CYP79F1 and CYP79F2 separately in silico using Tile-Viz( in which developmental transcriptome data were obtained using Arabidopsis tiling arrays. As shown in Supplementary Fig S2-5, CYP79F1 and CYP79F2 exhibited different expression patterns. Interestingly, the expression pattern of CYP79F2 was quite similar to that of MAM3, suggesting that CYP79F2 also may play a role in MTc8 biosynthesis in theroots.
Another finding of this study relates to the specific accumulation of isothiocyanates (ITCs). It is known that GSLs and their degradation enzyme myrosinase are stored separately in different types of cells. When tissues are damaged by an attack of herbivore insects, GSLs get contact with myrosinase and are degraded into toxic volatiles such as ITCs. We found that ITCs derived from MS-GSLs (MS-ITCs) were specifically accumulated in the stage 5 seeds/siliques (Supplementary Figs. S2-2a and b). ITC detectionin the plant tissues was not considered to be an artifact due to homogenization of the tissues, because no ITCs were detected in GSL-rich organs other than the stage 5 seeds/siliques. Maturing seeds/siliques-specific accumulation of MS-ITCs leads to another scenario relating to the control of structure-dependent GSL accumulation pattern. That is, MS-GSLs transported to the embryos from the maternal organs are partly degraded into MS-ITCs, whereas the rest are subjected to chain modification to form OH- and Bz-GSLs. Further quantitative analyses of GSLs and ITCs would assist our understanding of the spatiotemporal control of GSL accumulation.
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Supplementary Figure S2-1 Biosynthetic pathway of methionine-derived glucosinolates (GS). MT-GSL: methylthioglucosinolate; MS-GSL: methylsulfinylglucosinolate; OH-GSL: hydroxyalkylglucosinolate; Bz-GSL; benzoyloxyalkylglucosinolate.
Supplementary Figure S2-2Integrated analysis of transcriptome (AtGenExpress) and metabolome (AtMetExpress) data. (a) Mapping of glucosinolate biosynthesis related genes (red and purple) and metabolites (blue, green, and orange) on the Batch learning self-organizing map (BL-SOM) shown in Fig 2. (b) Accumulation and expression patterns of OHc4, MAM1, AOP3, MSc8, MTc8 and MSc8-ITC. Gene expression and metabolite accumulation patterns were obtained from Bar eFP Browser16 and AtMetExpress database, respectively.
Supplementary Figure S2-3Mapping of five flavin-monooxygenase (FMO) family genes encoding glucosinolate S-oxygenase (GS-OX) (FMOGS-OX1-5, At1g65860, At1g62540, At1g62560, At1g62570, and At1g12140, respectively) on the Batch learning self-organizing map (BL-SOM) shown in Fig 2.
Supplementary Figure S2-4 Comparison of expression/accumulation patterns of MAM3 (left panel) and MTc8 (right panel) across 36 tissues. Gene expression and metabolite accumulation patterns were obtained from Bar eFP Browser16 and AtMetExpress database, respectively.
Supplementary Figure S2-5 Expression of At1g16400 (CYP79F2), At1g16410 (CYP79F1), At5g23010 (MAM1), and At5g23020 (MAM3) genes during Arabidopsis development. The figure were obitained from TileViz( 15