Cloning and characterization of a functional flavanone-3ß-hydroxylase gene from Medicago truncatula

Abstract

As a key enzyme in the biosynthesis of flavonols, anthocyanidins and proanthocyanidins, flavanone-3ß-hydroxylase (F3H) plays very important roles in plant stress response. A putative flavanone-3ß-hydroxylase gene from Medicago truncatula (MtF3H), a model legume species, was identified from a bio-data analysis platform. It was speculated to be induced by salt stress based on the outcomes of the analysis platform. The complementary DNA (cDNA) consists of 1499 bp with an open reading frame (ORF) of 1098 bp, which encodes a putative protein of 365 amino acids with a molecular weight of about 41.36 kDa and an isoelectric point of 5.60. To measure the catalytic activity of the protein, the MtF3H gene was ligated to pYES2 vector and heterologously expressed in yeast. The recombinant protein converted naringen into dihydrokaempferol and displayed different enzymatic efficiencies with other flavanones, confirming that MtF3H coding a functional flavanone-3ß-hydroxylase. The expression pattern of the MtF3H gene was analyzed by comparative quantitative RT-PCR and a higher level of expression was observed in the roots than was observed in stems and leaves. Furthermore, the expression was induced by salt stress in the roots, and to a greater extent in the stems, but the response of the gene activity to salt stress in the stems was slower in the first 12 h following treatment when compared to the roots.

Variable salinity responses of 12 alfalfa genotypes and comparative expression analyses of salt-response genes

“…Similar observations were made in Arabidopsis where several regulatory genes indicated upregulation in roots exposed to salt treatment whereas shoots showed upregulation under osmotic stress 51 . Also, M. truncatula exposed to salt stress for 12 and 24 hours presented higher expression of the flavanone 3 hydroxylase gene in roots compared to leaves and stems 52 . Seven genes SOS1, SOS2, SOS3, HKT1, OTS1, SGF29 , and SAL1 were consistently upregulated in the roots of the genotypes that performed well for biomass yield and physiological parameters ( Fig.…”

Small RNA deep sequencing reveals the important role of microRNAs in the halophyte Halostachys caspica

“…OSCP is often considered to be a part of the F 0 sector of the ATPase and associated with the energy generation (Horak et al ., ). Favanone 3β‐hydroxylase (F3H), as a key enzyme in the biosynthesis of flavonols, anthocyanidins and proanthocyanidins, plays very important roles in plant stress response (Shen et al ., ). Both of the genes could be induced by salt stress in our experiment, and the two target genes and miRNAs had certain correlation at the level of post‐transcription, indicating that miRNAs can regulate energy synthesis and reduction oxidation.…”

De novo biosynthesis of garbanzol and fustin in Streptomyces albus based on a potential flavanone 3‐hydroxylase with 2‐hydroxylase side activity

“…Moreover, the bryophyte enzyme also generated kaempferol from dihydrokaempferol, demonstrating that FNSIs can also act as FLS enzymes by recognizing not only flavanones but also flavanonols (Han et al, 2014). In relation to the catalytic activity of the F3H enzyme used in the present work, it has been shown that it lacks FNS or FLS activity and cannot generate apigenin or kaempferol from naringenin or dihydrokaempferol, respectively (Wellmann et al, 2004;Shen et al, 2010); however, as far as we know, there are no references to its suspected F2H activity. Therefore, the close evolutionary relationship between F3H and FNSI, along with the F2H activity described for an FNSI, leads us to hypothesize that our F3H (encoded in pGR and pDF recombinant vectors) can have an F2H side activity allowing the formation of 2hydroxyliquiritigenin and 2-hydroxynaringenin (spectrum signals coming from putative 2-hydroxydihydrokaempferol were also detected, but no commercial standard was available).…”