Characterization Of Flavonoid Specific Malonyltransferases From Soybean (Glycine Max Merr.)

As unique to legume plants, isoflavonoids co-exist with flavonoids in many tissues and organs in legumes with various biological functions. Isoflavonoids have vital role to response against different biotic and abiotic stresses in plant defense. The major isoflavonoids in soybean plants are daidzein, genistein, and glycitein aglycones and their glucoside and malonate derivatives. The biosynthesis of isoflavonoids through phenylpropanoid pathway have been extensively studied, and genes involved in early steps have been characterized clearly, yet genes involved in late steps, particularly, in extensive modifications, transport, and storage of isoflavones, as well as the complex mechanisms underlying the regulation of isoflavone biosynthesis, are not fully understood.The extensive malonylation of isoflavone glucosides may have physiological functions yet to be identified, although the malonylation may stabilize (iso)flavonoids, enhance their solubility, or facilitate their transport or storage in plants. The malonyltransferase enzymes were responsible for the conjugation of isoflavone 7-O-?-D-glucoside(GmIF7GT) to isoflavone 7-0-(6"-malonyl-?-D-glucoside (GmIF7MaT) in soybean. The physiological function of genes responsible for their biosyntheses is not well understood.Here we report a new BAHD family isoflavone glucoside malonyltransferase GmIMaTl,and GmIMaT3, an allele to the previously characterized GmMT7 and GmIF7MaT. The main research result of this study is as follows:Four homologous (GmIMaTl, GmIMaT2, GmIMaT3 and GmIMaT4) of MtMaTl from soybean have been cloned on homology base. These genes were selected on the basis of their expression level in different tissues. Biochemical studies showed that among four homologous, only two recombinant GmIMaT1 and GmIMaT3 enzymes used malonyl-CoA and several isoflavone 7-O-glucosides as substrates.Soybean transgenic hairy roots were generated with overexpression and RNAi of GmIMaT1 and GmIMaT3. qRT-PCR analyses showed increased transcript level of both genes in overexpressed and decreased level in RNAi transgenic hairy roots than GUS.Transgenic hairy roots overexpressing both GmIMaTs had increased levels of malonyldaidzin and malonylgenistin, and contents of daidzin and glycitin increased only in GmIMaT1-overexpression lines. The increased daidzein and genestein contents were detected only in GmIMaT3-overexpression lines. Knockdown of GmIMaT1 and GmIMaT3 reduced malonyldaidzin and malonylgenistin contents, and affected other isoflavonoids differently. Gene localization analyses showed that GmIMaTl is primarily localized to the ER while GmIMaT3 is primarily in the cytosol.The isoflavone quantification and expression of these two GmIMaTs in different tissues have been done in response to different stresses like heat, cold, drought, aluminium,acidic conditions, abscisic acid (ABA) and methyl jasmonate (MeJA). Isoflavone accumulation response differently against different stresses and expression level of these two GmIMaTs was consistent with malonylisoflavone accumulation; we probed the possible functions of GmIMaTs. Two GmIMaTs displayed distinct tissue expression patterns and respond differently to various factors in modifying isoflavone 7-O glucosides under various stresses.The role of GmIMaTs was also explored in soybean nodulation. Soybean chimerical plants were generated with GmIMaTs-OE and -RNAi transgenic hairy roots. GmIMaTs-OE increased nodule ratio significantly while GmIMaTs-RNAi decreased significantly than control GUS. The expression level of both GmIMaTs along with some nodule biosynthesis genes was also increased in overexpressed and decreased in RNAi hairy roots. Significantly increased malonylisoflavone was obtained in high nodule containing hairy roots while decreased malonylisoflavone was observed in less nodule containing hairy roots than GUS control. GmIMaTl transcript increased markedly after 6 and 12 h post B. japonica strain USDA110 inaculation, whereas the GmIMaT3 transcript was increased slowly after 6 and 12 h, then start to decrease and reached to its initial position after 48 h. Both genes functions genetically in malonyldaidzin and malonylgenistin accumulation and improve the nodule development because malonylgenistin is the main activator of all nod box-associated genes.These insights not only help to understand how these malonylisoflavones are synthesized and play role in nodulation process, but also their putative physiological roles in soybean plants in response to various stresses.