Genistein-Specific G6DT Gene for the Inducible Production of Wighteone in Lotus japonicus

Abstract

Prenylated isoflavonoids have been found in several legume plants, and they possess various biological activities that play important roles in both plant defense and human health. However, it is still unknown whether prenylated isoflavonoids are present in the model legume plant Lotus japonicus. In the present study, we found that the prenylated isoflavonoid wighteone was produced in L. japonicus when leaf was supplemented with genistein. Furthermore, a novel prenyltransferase gene, LjG6DT, was identified, which shared high similarity with and was closely related to several known prenyltransferase genes involved in isoflavonoid biosynthesis. The recombinant LjG6DT protein expressed in yeast exhibited prenylation activity toward genistein as an exclusive substrate, which produced wighteone, a prenylated genistein at the C-6 position that occurs normally in legume plants. The LjG6DT-green fluorescent protein (GFP) fusion protein is targeted to plastids. The transcript level of LjG6DT is induced by glutathione, methyl jasmonate and salicylic acid, implying that LjG6DT is involved in stress response. Overexpression of LjG6DT in L. japonicus hairy roots led to increased accumulation of wighteone when genistein was supplied, indicating that LjG6DT is functional in vivo. Feeding assays with the upstream intermediate naringenin revealed that accumulation of wighteone in L. japonicus was dependent on genistein supplementation, and accumulation of wighteone is competed by genistein methylation. This study demonstrated that phytoalexin wighteone is inducibly produced in L. japonicus, and it provides new insight into the biosynthesis and accumulation of prenylated isoflavonoids in legume plants.

Flavonoids and Isoflavonoids Biosynthesis in the Model Legume Lotus japonicus; Connections to Nitrogen Metabolism and Photorespiration

“…A recent work has established that there is a differential regulation of flavonoid and isoflavonoid biosynthetic pathways in L. japonicus in relation to nitrogen metabolism and in response to different stress conditions [ 62 ]. An increase in the level of expression of several genes of the isoflavonoid pathway was observed in response to drought or active photorespiration, which was much more noticeable in a Ljgln2-2 photorespiratory mutant lacking the plastidic isoform of glutamine synthetase (GS2) [ 1 , 62 ] ( Figure 2 ). Therefore, important changes in phenolic metabolism as a result of GS2 deficiency were observed in L. japonicus plants in response to stress.…”

“…Both photorespiration and phenylpropanoid metabolism are mainly related to carbon metabolism, but also to nitrogen metabolism because of the use of amino acids as precursors. Therefore, the use of photorespiratory mutants allowed us to understand the cross-interaction between carbon and nitrogen metabolisms as well as (iso)flavonoid metabolism [ 1 , 94 ]. Previous works have clearly established how a GS2 defect in nitrogen assimilation affects carbon metabolism in this plant [ 1 , 94 , 95 , 96 ].…”

“…Therefore, the use of photorespiratory mutants allowed us to understand the cross-interaction between carbon and nitrogen metabolisms as well as (iso)flavonoid metabolism [ 1 , 94 ]. Previous works have clearly established how a GS2 defect in nitrogen assimilation affects carbon metabolism in this plant [ 1 , 94 , 95 , 96 ]. In addition, different transcription factors were detected that may be important in the carbon/nitrogen (C/N) balance in L. japonicus plants [ 94 ].…”

“…Different processes exist in plants, which give rise to the production of endogenous sources of ammonium which have to be efficiently re-assimilated by secondary ammonium assimilation. These processes include photorespiration, the biosynthesis of phenylpropanoids, as well as ureide, nucleotide and amino acid catabolism [ 1 ]. Phenylpropanoid metabolism represents an important metabolic pathway from which originates a wide number of secondary metabolites derived from phenylalanine or tyrosine, including monolignols, flavonoids and isoflavonoids, various phenolic acids, and stilbenes [ 2 ].…”

Cytotoxic, Anti-bacterial, and Wound-healing Activity of Prenylated Phenols from the Kurdish Traditional Medicinal Plant Onobrychis Carduchorum (Fabaceae)

“…In addition, various non-prenylated flavonoids were present in the plant extract, but they were generally much less active than their prenylated counterparts. It has already been reported that prenylation increases the lipophilicity of flavonoids, and hence membrane permeability, enhancing the binding affinity of flavonoids to target biomolecules [35].…”