14-3-3 proteins act as scaffolds for GmMYB62 and GmMYB176 and regulate their intracellular localization in soybean

Isoflavonoids are plant natural compounds predominantly found in leguminous plant. They play important functions in both nitrogen fixation and stress resistance. Many clinical studies have linked dietary intake of isoflavonoids to human health benefits. Binding of 14-3-3 proteins to GmMYB176, an isoflavonoid regulator, modulates expression of key isoflavonoids gene expression and its biosynthesis. We have recently demonstrated that the interaction of 14-3-3 proteins with GmMYB176 regulates nuclear-cytoplasmic localization of GmMYB176 thereby affecting target gene expression. Here, we report GmMYB62 as a new R1 MYB client protein of soybean 14-3-3s that may function together with GmMYB176 for gene regulation in soybean.     

Modification of phenolic metabolism in soybean hairy roots through down regulation of chalcone synthase or isoflavone synthase

Soybean hairy roots, transformed with the soybean chalcone synthase (CHS6) or isoflavone synthase (IFS2) genes, with dramatically decreased capacity to synthesize isoflavones were produced to determine what effects these changes would have on susceptibility to a fungal pathogen. The isoflavone and coumestrol concentrations were decreased by about 90% in most lines apparently due to gene silencing. The IFS2 transformed lines had very low IFS enzyme activity in microsomal fractions as measured by the conversion of naringenin to genistein. The CHS6 lines with decreased isoflavone concentrations had 5 to 20-fold lower CHS enzyme activities than the appropriate controls. Both IFS2 and CHS transformed lines accumulated higher concentrations of both soluble and cell wall bound phenolic acids compared to controls with higher levels found in the CHS6 lines indicating alterations in the lignin biosynthetic branch of the pathway. Induction of the soybean phytoalexin glyceollin, of which the precursor is the isoflavone daidzein, by the fungal pathogen Fusarium solani f. sp. glycines (FSG) that causes soybean sudden death syndrome (SDS) showed that the low isoflavone transformed lines did not accumulate glyceollin while the control lines did. The (iso)liquritigenin content increased upon FSG induction in the IFS2 transformed roots indicating that the pathway reactions before this point can control isoflavonoid synthesis. The lowest fungal growth rate on hairy roots was found on the FSG partially resistant control roots followed by the SDS sensitive control roots and the low isoflavone transformants. The results indicate the importance of phytoalexin synthesis in root resistance to the pathogen. Names are necessary to report factually on available data; however, the USDA neither guarantees nor warrants the standard of the product, and the use of the name by USDA implies no approval of the product to the exclusion of others that may also be suitable.     

Molecular characterization of the C‐glucosylation for puerarin biosynthesis in Pueraria lobata

C‐glycosyltransferases (CGTs) are important enzymes that are responsible for the synthesis of the C‐glycosides of flavonoids and isoflavonoids. Flavonoid CGTs have been molecularly characterized from several plant species; however, to date, no gene encoding an isoflavonoid CGT has been reported from any plant species. A significant example of an isoflavonoid C‐glycoside is puerarin, a compound that contributes to the major medicinal effects of Pueraria lobata. Little is known about the C‐glucosylation that occurs during puerarin biosynthesis. One possible route for puerarin synthesis is via the C‐glucosylation of daidzein. This study describes the molecular cloning and functional characterization of a novel glucosyltransferase (PlUGT43) from P. lobata. Biochemical analyses revealed that PlUGT43 possesses an activity for the C‐glucosylation of daidzein to puerarin; it shows activity with the isoflavones daidzein and genistein, but displays no activity towards other potential acceptors, including flavonoids. To validate the in vivo function of PlUGT43, the PlUGT43 gene was over‐expressed in soybean hairy roots that naturally synthesize daidzein but that do not produce puerarin. The expression of PlUGT43 led to the production of puerarin in the transgenic soybean hairy roots, confirming a role for PlUGT43 in puerarin biosynthesis.     

Characterization of the soybean GmALMT family genes and the function of GmALMT5 in response to phosphate starvation

A potential mechanism to enhance utilization of sparingly soluble forms of phosphorus (P) is the root secretion of malate, which is mainly mediated by the ALMT gene family in plants. In this study, a total of 34 GmALMT genes were identified in the soybean genome. Expression patterns diverged considerably among GmALMTs in response to phosphate (Pi) starvation in leaves, roots and flowers, with expression altered by P availability in 26 of the 34 GmALMTs. One root‐specific GmALMT whose expression was significantly enhanced by Pi‐starvation, GmALMT5, was studied in more detail to determine its possible role in soybean P nutrition. Analysis of GmALMT5 tissue expression patterns, subcellular localization, and malate exudation from transgenic soybean hairy roots overexpressing GmALMT5, demonstrated that GmALMT5 is a plasma membrane protein that mediates malate efflux from roots. Furthermore, both growth and P content of transgenic Arabidopsis overexpressing GmALMT5 were significantly increased when sparingly soluble Ca‐P was used as the external P source. Taken together, these results indicate that members of the soybean GmALMT gene family exhibit diverse responses to Pi starvation. One member of this family, GmALMT5, might contribute to soybean P efficiency by enhancing utilization of sparingly soluble P sources under P limited conditions.     

Involvement of chalcone reductase in the soybean isoflavone metabolon: identification of GmCHR5, which interacts with 2‐hydroxyisoflavanone synthase

Soybean (Glycine max) 5‐deoxyisoflavonoids (daidzein and its conjugates) are precursors of glyceollin phytoalexins. They are also converted to equol by microbes in the human intestine, resulting in health benefits. 5‐Deoxyisoflavonoids accumulate in the roots (93% mol/mol of the total root isoflavonoids) and seeds of unstressed soybean plants. Chalcone reductase (CHR) is a key enzyme mediating 5‐deoxyisoflavonoid biosynthesis because it catalyzes the production of 6′‐deoxychalcone through its effects on the chalcone synthase (CHS)‐catalyzed reaction. The soybean genome encodes at least 11 CHR‐related homologs, but it is unclear which ones are functionally important for daidzein accumulation in unstressed plants. Among the CHR homologs, the temporal and spatial expression patterns of GmCHR5 were the most correlated with the distribution patterns of 5‐deoxyisoflavonoids. The CHR activity of GmCHR5 was confirmed in vitro and in planta. In the in vitro assays, the ratio of CHR products (6′‐deoxychalcone) to total CHS products (R value) was dependent on GmCHR5 and CHS concentrations, with higher concentrations resulting in higher R values (i.e. approaching 90%). Subcellular localization analyses revealed that GmCHR5 was present in the cytoplasm and nucleus. Protein–protein interaction assays indicated that GmCHR5, but not GmCHR1 and GmCHR6, interacted with 2‐hydroxyisoflavanone synthase (IFS) isozymes. The CHS isozymes also interacted with IFS isozymes but not with GmCHR5. The proposed micro‐compartmentalization of isoflavone biosynthesis through the formation of an IFS‐mediated metabolon is probably involved in positioning GmCHR5 close to CHS, resulting in an R value that is high enough for the accumulation of abundant 5‐deoxyisoflavonoids in soybean roots.     

Rapid induction of phenylalanine ammonia-lyase and chalcone synthase mRNAs during fungus infection of soybean (Glycine max L.) roots or elicitor treatment of soybean cell cultures at the onset of phytoalexin synthesis

The differential regulation of the activities and amounts of mRNAs for two enzymes involved in isoflavonoid phytoalexin biosynthesis in soybean was studied during the early stages after inoculation of primary roots with zoospores from either race 1 (incompatible, host resistant) or race 3 (compatible, host susceptible) of Phytophthora megasperma f.sp. glycinea, the causal fungus of root rot disease. In the incompatible interaction, cloned cDNAs were used to demonstrate that the amounts of phenylalanine ammonia-lyase and chalcone synthase mRNAs increased rapidly at the time of penetration of fungal germ tubes into epidermal cell layers (1–2 h after inoculation) concomitant with the onset of phytoalxxin accumulation; highest levels were reached after about 7 h. In the compatible interaction, only a slight early enhancement of mRNA levels was found and no further increase occurred until about 9 h after inoculation. The time course for changes in the activity of chalcone synthase mRNA also showed major differences between the incompatible and compatible interaction. The observed kinetics for the stimulation of mRNA expression related to phytoalexin synthesis in soybean roots lends further support to the hypothesis that phytoalexin production is an early defense response in the incompatible plant-fungus interaction. The kinetics for the enhancement of mRNA expression after treatment of soybean cell suspension cultures with a glucan elicitor derived from P. megasperma cell walls was similar to that measured during the early stages of the resistant response of soybean roots.Abbreviations cDNA copy DNA - CHS chalcone synthase - PAL phenylalanine ammonia-lyase     

Effects of Agrobacterium rhizogenes strains and other parameters on production of isoflavonoids in hairy roots of Pueraria candollei Grah. ex Benth. var. candollei

Using several explants of Pueraria candollei Grah. ex Benth. var. candollei and two strains of Agrobacterium rhizogenes (ATCC 15834 and 43057), hairy root cultures were established. Including 100???M acetosyringone in the culture medium enhanced frequency of hairy root induction by up to 58?%. Subsequently, effects of inoculum size (IS) and temperature on growth and production of isoflavonoids in hairy roots were determined. Conditions of 1?%?IS and 32?°C promoted the highest accumulation of total isoflavonoid content, up to 31.0?±?22.6?mg/g dry weight (DW), in hairy roots. Moreover, culture of hairy roots at 32?°C decreased browning of hairy roots. Furthermore, this temperature promoted accumulation of the secondary metabolite daidzein; whereas, hairy root cultures at the stationary phase accumulated higher amounts of the isoflavonoid puerarin rather than daidzein.