The phosphorylated pathway of serine biosynthesis affects sperm, embryo, and sporophyte development, and metabolism in Marchantia polymorpha.

Serine metabolism is involved in various biological processes. Here we investigate primary functions of the phosphorylated pathway of serine biosynthesis in a non-vascular plant Marchantia polymorpha by analyzing knockout mutants of MpPGDH encoding 3-phosphoglycerate dehydrogenase in this pathway. Growth phenotypes indicate that serine from the phosphorylated pathway in the dark is crucial for thallus growth. Sperm development requires serine from the phosphorylated pathway, while egg formation does not. Functional MpPGDH in the maternal genome is necessary for embryo and sporophyte development. Under high CO2 where the glycolate pathway of serine biosynthesis is inhibited, suppressed thallus growth of the mutants is not fully recovered by exogenously-supplemented serine, suggesting the importance of serine homeostasis involving the phosphorylated and glycolate pathways. Metabolomic phenotypes indicate that the phosphorylated pathway mainly influences the tricarboxylic acid cycle, the amino acid and nucleotide metabolism, and lipid metabolism. These results indicate the importance of the phosphorylated pathway of serine biosynthesis in the dark, in the development of sperm, embryo, and sporophyte, and metabolism in M. polymorpha.

Retrograde sulfur flow from glucosinolates to cysteine in Arabidopsis thaliana.

Specialized (secondary) metabolic pathways in plants have long been considered one-way routes of leading primary metabolite precursors to bioactive end products. Conversely, endogenous degradation of such "end" products in plant tissues has been observed following environmental stimuli, including nutrition stress. Therefore, it is of general interest whether specialized metabolites can be reintegrated into primary metabolism to recover the invested resources, especially in the case of nitrogen- or sulfur-rich compounds. Here, we demonstrate that endogenous glucosinolates (GLs), a class of sulfur-rich plant metabolites, are exploited as a sulfur source by the reallocation of sulfur atoms to primary metabolites such as cysteine in Arabidopsis thaliana Tracer experiments using 34S- or deuterium-labeled GLs depicted the catabolic processing of GL breakdown products in which sulfur is mobilized from the thioglucoside group in GL molecules, potentially accompanied by the release of the sulfate group. Moreover, we reveal that beta-glucosidases BGLU28 and BGLU30 are the major myrosinases that initiate sulfur reallocation by hydrolyzing particular GL species, conferring sulfur deficiency tolerance in A. thaliana, especially during early development. The results delineate the physiological function of GL as a sulfur reservoir, in addition to their well-known functions as defense chemicals. Overall, our findings demonstrate the bidirectional interaction between primary and specialized metabolism, which enhances our understanding of the underlying metabolic mechanisms via which plants adapt to their environments.

Diversified amino acid-mediated allosteric regulation of phosphoglycerate dehydrogenase for serine biosynthesis in land plants.

The phosphorylated pathway of serine biosynthesis is initiated with 3-phosphoglycerate dehydrogenase (PGDH). The liverwort Marchantia polymorpha possesses an amino acid-sensitive MpPGDH which is inhibited by l-serine and activated by five proteinogenic amino acids, while the eudicot Arabidopsis thaliana has amino acid-sensitive AtPGDH1 and AtPGDH3 as well as amino acid-insensitive AtPGDH2. In this study, we analyzed PGDH isozymes of the representative land plants: the monocot Oryza sativa (OsPGDH1-3), basal angiosperm Amborella trichopoda (AmtriPGDH1-2), and moss Physcomitrium (Physcomitrella) patens (PpPGDH1-4). We demonstrated that OsPGDH1, AmtriPGDH1, PpPGDH1, and PpPGDH3 were amino acid-sensitive, whereas OsPGDH2, OsPGDH3, AmtriPGDH2, PpPGDH2, and PpPGDH4 were either sensitive to only some of the six effector amino acids or insensitive to all effectors. This indicates that PGDH sensitivity to effectors has been diversified among isozymes and that the land plant species examined, except for M. polymorpha, possess different isozyme types in terms of regulation. Phylogenetic analysis suggested that the different sensitivities convergently evolved in the bryophyte and angiosperm lineages. Site-directed mutagenesis of AtPGDH1 revealed that Asp538 and Asn556 residues in the ACT domain are involved in allosteric regulation by the effectors. These findings provide insight into the evolution of PGDH isozymes, highlighting the functional diversification of allosteric regulation in land plants.

Abscisic acid-mediated induction of FLAVIN-CONTAINING MONOOXYGENASE 2 leads to reduced accumulation of methylthioalkyl glucosinolates in Arabidopsis thaliana.

Side-chain modification contributes to the structural diversity of aliphatic glucosinolates (GSLs), a class of sulfur-containing secondary metabolites found in Brassicales. The first step in side-chain modification of aliphatic GSLs is the S-oxygenation of the methylthioalkyl (MT) moiety to the methylsulfinylalkyl (MS) moiety. This reaction is catalyzed by flavin-containing monooxygenase (FMOGS-OX), which is encoded by seven genes in Arabidopsis thaliana. Therefore, the regulation of FMOGS-OX gene expression is key to controlling side-chain structural diversity. In this study, we demonstrated that the expression of FMOGS-OX2 and FMOGS-OX4 was induced by glucose treatment, independent of MYB28/29 and MYC2/3/4, the transcription factors that positively regulate aliphatic GSL biosynthesis. Glucose treatment of the abscisic acid (ABA)-related mutants indicated that glucose-triggered upregulation of FMOGS-OX2 and FMOGS-OX4 was partially regulated by ABA through the key negative regulators ABI1 and ABI2, and the positive regulator SnRK2, but not via the transcription factor ABI5. In wild-type plants, glucose treatment drastically reduced the accumulation of 4-methylthiobutyl (4MT) GSL, whereas a decrease in 4MT GSL was not observed in the fmogs-ox2, abi1-1, abi2-1, aba2-1, or aba3-1 mutants. This result indicated that the decreased accumulation of 4MT GSL by glucose treatment was attributed to upregulation of FMOGS-OX2 via the ABA signaling pathway.

The cytochrome P450 CYP77A4 is involved in auxin-mediated patterning of the Arabidopsis thaliana embryo.

Metabolism often plays an important role in developmental control, in addition to supporting basal physiological requirements. However, our understanding of this interaction remains limited. Here, we performed quantitative phenome analysis of Arabidopsis thaliana cytochrome P450 mutants to identify a novel interaction between development and metabolism. We found that cyp77a4 mutants exhibit specific defects in cotyledon development, including asymmetric positioning and cup-shaped morphology, which could be rescued by introducing the CYP77A4 gene. Microscopy revealed that the abnormal patterning was detected at least from the 8-cell stage of the cyp77a4 embryos. We next analysed auxin distribution in mutant embryos, as the phenotypes resembled those of auxin-related mutants. We found that the auxin response pattern was severely perturbed in the cyp77a4 embryos owing to an aberrant distribution of the auxin efflux carrier PIN1. CYP77A4 intracellularly localised to the endoplasmic reticulum, which is consistent with the notion that this enzyme acts as an epoxidase of unsaturated fatty acids in the microsomal fraction. We propose that the CYP77A4-dependent metabolic pathway is an essential element for the establishment of polarity in plant embryos.

Autopolyploidization, geographic origin, and metabolome evolution in Arabidopsis thaliana.

PREMISE OF THE STUDY: Autopolyploidy, or whole-genome duplication, is a recurrent phenomenon in plant evolution. Its existence can be inferred from the presence of massive levels of genetic redundancy revealed by comparative plant phylogenomics. Whole-genome duplication is theoretically associated with evolutionary novelties such as the development of new metabolic reactions and therefore contributes to the evolution of new plant metabolic profiles. However, very little is yet known about the impact of autopolyploidy on the metabolism of recently formed autopolyploids. This study provides a better understanding of the relevance of this evolutionary process. METHODS: In this study, we compared the metabolic profiles of wild diploids, wild autotetraploids, and artificial autotetraploids of Arabidopsis thaliana using targeted ultra-high performance liquid chromatography-triple quadrupole- mass spectrometry (UPLC-QqQ-MS) metabolomics. KEY RESULTS: We found that wild and artificial A. thaliana autotetraploids display different metabolic profiles. Furthermore, wild autotetraploids display unique metabolic profiles associated with their geographic origin. CONCLUSIONS: Autopolyploidy might help plants adapt to challenging environmental conditions by allowing the evolution of novel metabolic profiles not present in the parental diploids. We elaborate on the causes and consequences leading to these distinct profiles.

ACR11 is an Activator of Plastid-Type Glutamine Synthetase GS2 in Arabidopsis thaliana.

Glutamine synthetase (GS) is an important enzyme for nitrogen assimilation, and GS2, encoded by GLN2, is the only plastid-type GS in Arabidopsis thaliana. A co-expression analysis suggested that the expression level of the gene encoding a uridylyltransferase-like protein, ACR11, is strongly correlated with GLN2 expression levels. Here we showed that the recombinant ACR11 protein increased GS2 activity in vitro by reducing the Km values of its substrate glutamine. A T-DNA insertion mutant of ACR11 exhibited a reduced GS activity under low nitrate conditions and reduced glutamine levels. Biochemical analyses revealed that ACR11 and GS2 interacted both in vitro and in vivo. These data demonstrate that ACR11 is an activator of GS2, giving it a mechanistic role in the nitrogen assimilation of A. thaliana.

Modification of photosynthetic electron transport and amino acid levels by overexpression of a circadian-related histidine kinase hik8 in Synechocystis sp. PCC 6803.

Cyanobacteria perform oxygenic photosynthesis, and the maintenance of photosynthetic electron transport chains is indispensable to their survival in various environmental conditions. Photosynthetic electron transport in cyanobacteria can be studied through genetic analysis because of the natural competence of cyanobacteria. We here show that a strain overexpressing hik8, a histidine kinase gene related to the circadian clock, exhibits an altered photosynthetic electron transport chain in the unicellular cyanobacterium Synechocystis sp. PCC 6803. Respiratory activity was down-regulated under nitrogen-replete conditions. Photosynthetic activity was slightly lower in the hik8-overexpressing strain than in the wild-type after nitrogen depletion, and the values of photosynthetic parameters were altered by hik8 overexpression under nitrogen-replete and nitrogen-depleted conditions. Transcripts of genes encoding Photosystem I and II were increased by hik8 overexpression under nitrogen-replete conditions. Nitrogen starvation triggers increase in amino acids but the magnitude of the increase in several amino acids was diminished by hik8 overexpression. These genetic data indicate that Hik8 regulates the photosynthetic electron transport, which in turn alters primary metabolism during nitrogen starvation in this cyanobacterium.

Seawater cultivation of freshwater cyanobacterium Synechocystis sp. PCC 6803 drastically alters amino acid composition and glycogen metabolism.

Water use assessment is important for bioproduction using cyanobacteria. For eco-friendly reasons, seawater should preferably be used for cyanobacteria cultivation instead of freshwater. In this study, we demonstrated that the freshwater unicellular cyanobacterium Synechocystis sp. PCC 6803 could be grown in a medium based on seawater. The Synechocystis wild-type strain grew well in an artificial seawater (ASW) medium supplemented with nitrogen and phosphorus sources. The addition of HEPES buffer improved cell growth overall, although the growth in ASW medium was inferior to that in the synthetic BG-11 medium. The levels of proteins involved in sugar metabolism changed depending on the culture conditions. The biosynthesis of several amino acids including aspartate, glutamine, glycine, proline, ornithine, and lysine, was highly up-regulated by cultivation in ASW. Two types of natural seawater (NSW) were also made available for the cultivation of Synechocystis cells, with supplementation of both nitrogen and phosphorus sources. These results revealed the potential use of seawater for the cultivation of freshwater cyanobacteria, which would help to reduce freshwater consumption during biorefinery using cyanobacteria.

Alteration of cyanobacterial sugar and amino acid metabolism by overexpression hik8, encoding a KaiC-associated histidine kinase.

Cyanobacteria possess circadian clocks consisting of KaiABC proteins, and circadian rhythm must closely relate to the primary metabolism. A histidine kinase, SasA, interacts with KaiC to transduce circadian signals and widely regulates transcription in Synechococcus sp. PCC 7942, although the involvement of SasA in primary metabolism has not been demonstrated at metabolite levels. Here, we generated a strain overexpressing hik8 (HOX80), an orthologue of SasA in Synechocystis sp. PCC 6803. HOX80 grew slowly under light conditions and lost viability under continuous dark conditions. Transcript levels of genes related to sugar catabolism remained higher in HOX80 under dark conditions. Metabolomic analysis revealed that under light conditions, glycogen was undetectable in HOX80, and there were decreased levels of metabolites of sugar catabolism and increased levels of amino acids, compared with those in the wild-type strain. HOX80 exhibited aberrant degradation of SigE proteins after a light-to-dark transition and immunoprecipitation analysis revealed that Hik8 directly interacts with KaiC1. The results of this study demonstrate that overexpression of hik8 widely alters sugar and amino acid metabolism, revealing the involvement of Hik8 in primary metabolism under both light and dark conditions in this cyanobacterium.

Metabolomic analysis reveals rewiring of Synechocystis†sp. PCC 6803 primary metabolism by ntcA overexpression.

NtcA is a cAMP receptor protein-type transcription factor conserved among cyanobacteria and is essential for gene expression in response to nitrogen status. NtcA has been widely studied; however, no metabolomic analysis has been conducted using the ntcA mutant. Here, we generated a strain that overexpresses ntcA in Synechocystis sp. PCC 6803, named NOX10, and performed physiological, transcriptomic and metabolomic analyses. NOX10 grew faster than the wild-type strain under photoautotrophic conditions, but slower under light-activated heterotrophic conditions. Transcriptome analysis revealed that the expression of genes related to primary metabolism was altered by ntcA overexpression particularly under nitrogen-depleted conditions. Metabolomic analysis revealed that metabolite levels in sugar, purine/pyrimidine nucleotide, organic acid and amino acid metabolism were widely altered by ntcA overexpression. The protein levels of nitrogen-regulated transcriptional regulators were altered by ntcA overexpression during nitrogen starvation. These results demonstrate the alteration of primary metabolism by genetic engineering of NtcA, and they contribute to the current understanding of metabolic regulation of unicellular cyanobacteria.

Pathway-level acceleration of glycogen catabolism by a response regulator in the cyanobacterium Synechocystis species PCC 6803.

Response regulators of two-component systems play pivotal roles in the transcriptional regulation of responses to environmental signals in bacteria. Rre37, an OmpR-type response regulator, is induced by nitrogen depletion in the unicellular cyanobacterium Synechocystis species PCC 6803. Microarray and quantitative real-time polymerase chain reaction analyses revealed that genes related to sugar catabolism and nitrogen metabolism were up-regulated by rre37 overexpression. Protein levels of GlgP(slr1367), one of the two glycogen phosphorylases, in the rre37-overexpressing strain were higher than those of the parental wild-type strain under both nitrogen-replete and nitrogen-depleted conditions. Glycogen amounts decreased to less than one-tenth by rre37 overexpression under nitrogen-replete conditions. Metabolome analysis revealed that metabolites of the sugar catabolic pathway and amino acids were altered in the rre37-overexpressing strain after nitrogen depletion. These results demonstrate that Rre37 is a pathway-level regulator that activates the metabolic flow from glycogen to polyhydroxybutyrate and the hybrid tricarboxylic acid and ornithine cycle, unraveling the mechanism of the transcriptional regulation of primary metabolism in this unicellular cyanobacterium.

Capillary electrophoresis-mass spectrometry reveals the distribution of carbon metabolites during nitrogen starvation in Synechocystis sp. PCC 6803.

Nitrogen availability is one of the most important factors for the survival of cyanobacteria. Previous studies on Synechocystis revealed a contradictory situation with regard to metabolism during nitrogen starvation; that is, glycogen accumulated even though the expressions of sugar catabolic genes were widely upregulated. Here, we conducted transcript and metabolomic analyses using capillary electrophoresis-mass spectrometry on Synechocystis sp. PCC 6803 under nitrogen starvation. The levels of some tricarboxylic acid cycle intermediates (succinate, malate and fumarate) were greatly increased by nitrogen deprivation. Purine and pyrimidine nucleotides were markedly downregulated under nitrogen depletion. The levels of 19 amino acids changed under nitrogen deprivation, especially those of amino acids synthesized from pyruvate and phosphoenolpyruvate, which showed marked increases. Liquid chromatography-mass spectrometry analysis demonstrated that the amount of NADPH and the NADPH/NADH ratio decreased under nitrogen depletion. These data demonstrate that there are increases in not only glycogen but also in metabolites downstream of sugar catabolism in Synechocystis sp. PCC 6803 under nitrogen starvation, resolving the contradiction between glycogen accumulation and induction of sugar catabolic gene expression in this unicellular cyanobacterium.

Pleiotropic effect of sigE over-expression on cell morphology, photosynthesis and hydrogen production in Synechocystis sp. PCC 6803.

Over-expression of sigE, a gene encoding an RNA polymerase sigma factor in the unicellular cyanobacterium Synechocystis sp. PCC 6803, is known to activate sugar catabolism and bioplastic production. In this study, we investigated the effects of sigE over-expression on cell morphology, photosynthesis and hydrogen production in this cyanobacterium. Transmission electron and scanning probe microscopic analyses revealed that sigE over-expression increased the cell size, possibly as a result of aberrant cell division. Over-expression of sigE reduced respiration and photosynthesis activities via changes in gene expression and chlorophyll fluorescence. Hydrogen production under micro-oxic conditions is enhanced in sigE over-expressing cells. Despite these pleiotropic phenotypes, the sigE over-expressing strain showed normal cell viability under both nitrogen-replete and nitrogen-depleted conditions. These results provide insights into the inter-relationship among metabolism, cell morphology, photosynthesis and hydrogen production in this unicellular cyanobacterium.

Novel bioresources for studies of Brassica oleracea: identification of a kale MYB transcription factor responsible for glucosinolate production.

Plants belonging to the Brassicaceae family exhibit species-specific profiles of glucosinolates (GSLs), a class of defence compounds against pathogens and insects. GSLs also exhibit various human health-promoting properties. Among them, glucoraphanin (aliphatic 4-methylsulphinylbutyl GSL) has attracted the most attention because it hydrolyses to form a potent anticancer compound. Increased interest in developing commercial varieties of Brassicaceae crops with desirable GSL profiles has led to attempts to identify genes that are potentially valuable for controlling GSL biosynthesis. However, little attention has been focused on genes of kale (Brassica oleracea var. acephala). In this study, we established full-length kale cDNA libraries containing 59 904 clones, which were used to generate an expressed sequence tag (EST) data set with 119 204 entries. The EST data set clarified genes related to the GSL biosynthesis pathway in kale. We specifically focused on BoMYB29, a homolog of Arabidopsis MYB29/PMG2/HAG3, not only to characterize its function but also to demonstrate its usability as a biological resource. BoMYB29 overexpression in wild-type Arabidopsis enhanced the expression of aliphatic GSL biosynthetic genes and the accumulation of aliphatic GSLs. When expressed in the myb28myb29 mutant, which exhibited no detectable aliphatic GSLs, BoMYB29 restored the expression of biosynthetic genes and aliphatic GSL accumulation. Interestingly, the ratio of methylsulphinyl GSL content, including glucoraphanin, to that of methylthio GSLs was greatly increased, indicating the suitability of BoMYB29 as a regulator for increasing methylsulphinyl GSL content. Our results indicate that these biological resources can facilitate further identification of genes useful for modifications of GSL profiles and accumulation in kale.

Increased bioplastic production with an RNA polymerase sigma factor SigE during nitrogen starvation in Synechocystis sp. PCC 6803.

Because cyanobacteria directly harvest CO2 and light energy, their carbon metabolism is important for both basic and applied sciences. Here, we show that overexpression of the sigma factor sigE in Synechocystis sp. PCC 6803 widely changes sugar catabolism and increases production of the biodegradable polyester polyhydroxybutyrate (PHB) during nitrogen starvation. sigE overexpression elevates the levels of proteins implicated in glycogen catabolism, the oxidative pentose phosphate pathway, and polyhydroxyalkanoate biosynthesis. PHB accumulation is enhanced by sigE overexpression under nitrogen-limited conditions, yet the molecular weights of PHBs synthesized by the parental glucose-tolerant and sigE overexpression strain are similar. Although gene expression induced by nitrogen starvation is changed and other metabolites (such as GDP-mannose and citrate) accumulate under sigE overexpression, genetic engineering of this sigma factor altered the metabolic pathway from glycogen to PHB during nitrogen starvation.

Novel insights into the function of Arabidopsis R2R3-MYB transcription factors regulating aliphatic glucosinolate biosynthesis.

Arabidopsis transcription factors, MYB28, MYB29 and MYB76, positively regulate aliphatic glucosinolate (AGSL) biosynthesis. Mutual transcriptional regulation among these MYB genes makes it difficult to elucidate their individual function simply by analyzing knock-out mutants or ectopically overexpressing lines of these genes. In this study, we constructed transgenic lines expressing each MYB gene driven by its own promoter in the myb28myb29 background, where the expression of the endogenous MYB28, MYB29 and MYB76 was repressed with no AGSL accumulation. In leaves, transgenic MYB28 expression activated AGSL biosynthetic genes and restored accumulation of AGSLs with short side chains. Transgenic MYB29 expression activated the same biosynthetic pathway, but induction of the genes involved in side chain elongation was weaker than that by MYB28, resulting in a weaker recovery of AGSLs. Neither MYB28 nor MYB29 recovered long-chain AGSL accumulation. MYB76 was considered to require both MYB28 and MYB29 for its normal level of expression in leaves, and could not activate AGSL biosynthesis on its own. Interestingly, the accumulation in seeds of long- and short-chain AGSLs was restored by transgenic expression of MYB28 and MYB76, respectively. A sulfur stress experiment indicated that MYB28 expression was induced by sulfur deficiency, while the expression levels of MYB29 and MYB76 were positively correlated with sulfur concentration. This study illustrated how the individual MYBs work in regulating AGSL biosynthesis when expressed alone under normal transcriptional regulation.

Widely targeted metabolomics and coexpression analysis as tools to identify genes involved in the side-chain elongation steps of aliphatic glucosinolate biosynthesis.

Amino acid and glucosinolate biosynthesis are two interdependent pathways; amino acid synthesis as a part of primary metabolism provides the precursors for glucosinolate biosynthesis in secondary metabolism. In our previous studies, the combination of coexpression analysis and metabolite profiling led to the identification of genes and key regulators involved in glucosinolate biosynthesis. Moreover, the integration of transcriptome and metabolome data of sulphur-deprived Arabidopsis plants revealed coordinate changes in the expression profiles of genes involved in glucosinolate and amino acid metabolism.This review provides an overview of our recent studies involving Arabidopsis mutant plants that exhibit impairment in the side-chain elongation process occurring during aliphatic glucosinolate biosynthesis by means of coexpression analysis and a novel metabolite profiling approach based on ultra-performance liquid chromatography coupled with tandem quadrupole mass spectrometry (UPLC-TQMS) (Sawada et al. 2009a). Thus, this review highlights the advantages of the omics-based approach in identifying genes involved in glucosinolate biosynthesis.

Arabidopsis bile acid:sodium symporter family protein 5 is involved in methionine-derived glucosinolate biosynthesis.

Glucosinolates (GSLs) are a group of plant secondary metabolites that have repellent activity against herbivore insects and pathogens, and anti-carcinogenic activity in humans. They are produced in plants of the Brassicaceae and other related families. Biosynthesis of GSLs from precursor amino acids takes place in two subcellular compartments; amino acid biosynthesis and side chain elongation occur mainly in the chloroplast, whereas the following core structure synthesis takes place in the cytosol. Although the genes encoding biosynthetic enzymes of GSLs are well known in Arabidopsis thaliana, the transporter genes responsible for translocation of biosynthetic intermediates between the chloroplast and cytosol are as yet unidentified. In this study, we identified the bile acid:sodium symporter family protein 5 (BASS5) gene in Arabidopsis as a candidate transporter gene involved in methionine-derived GSL (Met-GSL) biosynthesis by means of transcriptome co-expression analysis. Knocking out BASS5 resulted in a decrease of Met-GSLs and concomitant increase of methionine. A transient assay using fluorescence fusion proteins indicated a chloroplastic localization of BASS5. These results supported the idea that BASS5 plays a role in translocation across the chloroplast membranes of the biosynthetic intermediates of Met-GSLs.

Omics-based approaches to methionine side chain elongation in Arabidopsis: characterization of the genes encoding methylthioalkylmalate isomerase and methylthioalkylmalate dehydrogenase.

Glucosinolates (GSLs) are secondary metabolites in Brassicaceae plants synthesized from amino acids. Methionine-derived GSLs (Met-GSLs) with diverse side chains of various lengths are the major GSLs in Arabidopsis. Methionine chain elongation enzymes are responsible for variations in chain length in Met-GSL biosynthesis. The genes encoding methionine chain elongation enzymes are considered to have been recruited from the leucine biosynthetic pathway in the course of evolution. Among them, the genes encoding methylthioalkylmalate synthases and aminotransferases have been identified; however, the remaining genes that encode methylthioalkylmalate isomerase (MAM-I) and methylthioalkylmalate dehydro-genase (MAM-D) remain to be identified. In a previous study based on transcriptome co-expression analysis, we identified candidate genes for the large subunit of MAM-I and MAM-D. In this study, we confirmed their predicted functions by targeted GSL analysis of the knockout mutants, and named the respective genes MAM-IL1/AtleuC1 and MAM-D1/AtIMD1. Metabolic profiling of the knockout mutants of methionine chain elongation enzymes, conducted by means of widely targeted metabolomics, implied that these enzymes have roles in controlling metabolism from methionine to primary and methionine-related secondary metabolites. As shown here, an omics-based approach is an efficient strategy for the functional elucidation of genes involved in metabolism.