Summary: Legume iso/flavonoids have been implicated in the nodulation process, but questions remain as to their specific role(s), and no unequivocal evidence exists showing that these compounds are essential for nodulation. Two hypotheses suggest that the primary role of iso/flavonoids is their ability to induce rhizobial nod gene expression and/or their ability to modulate internal root auxin concentrations. The present work provides direct, genetic evidence that isoflavones are essential for nodulation of soybean roots because of their ability to induce the nodulation genes of Bradyrhizobium japonicum. Expression of isoflavone synthase (IFS), a key enzyme in the biosynthesis of isoflavones, is specifically induced by B. japonicum. When IFS was silenced using RNA interference in soybean hairy root composite plants, these plants had severely reduced nodulation. Surprisingly, pre‐treatment of B. japonicum or exogenous application to the root system of either of the major soybean isoflavones, daidzein or genistein, failed to restore normal nodulation. Silencing of chalcone reductase led to very low levels of daidzein and increased levels of genistein, but did not affect nodulation, suggesting that the endogenous production of genistein was sufficient to support nodulation. Consistent with a role for isoflavones as endogenous regulators of auxin transport in soybean roots, silencing of IFS resulted in altered auxin‐inducible gene expression and auxin transport. However, use of a genistein‐hypersensitive B. japonicum strain or purified B. japonicum Nod signals rescued normal nodulation in IFS‐silenced roots, indicating that the ability of isoflavones to modulate auxin transport is not essential to nodulation.

Small RNAs regulate a number of developmental processes in plants and animals. However, the role of small RNAs in legume-rhizobial symbiosis is largely unexplored. Symbiosis between legumes (e.g. soybean) and rhizobia bacteria (e.g. Bradyrhizobium japonicum) results in root nodules where the majority of biological nitrogen fixation occurs. We sought to identify microRNAs (miRNAs) regulated during soybean-B. japonicum symbiosis.

Summary: Flavonoids play critical roles in legume–rhizobium symbiosis. However, the role of individual flavonoid compounds in this process has not yet been clearly established. We silenced different flavonoid‐biosynthesis enzymes to generate transgenic Medicago truncatula roots with different flavonoid profiles. Silencing of chalcone synthase, the key entry‐point enzyme for flavonoid biosynthesis led to flavonoid‐deficient roots. Silencing of isoflavone synthase and flavone synthase led to roots deficient for a subset of flavonoids, isoflavonoids (formononetin and biochanin A) and flavones (7,4′‐dihydroxyflavone), respectively. When tested for nodulation by Sinorhizobium meliloti, flavonoid‐deficient roots had a near complete loss of nodulation, whereas flavone‐deficient roots had reduced nodulation. Isoflavone‐deficient roots nodulated normally, suggesting that isoflavones might not play a critical role in M. truncatula nodulation, even though they are the most abundant root flavonoids. Supplementation of flavone‐deficient roots with 7, 4′‐dihydroxyflavone, a major inducer of S. meliloti nod genes, completely restored nodulation. However, the same treatment did not restore nodulation in flavonoid‐deficient roots, suggesting that other non‐nod gene‐inducing flavonoid compounds are also critical to nodulation. Supplementation of roots with the flavonol kaempferol (an inhibitor of auxin transport), in combination with the use of flavone pre‐treated S. meliloti cells, completely restored nodulation in flavonoid‐deficient roots. In addition, S. meliloti cells constitutively producing Nod factors were able to nodulate flavone‐deficient roots, but not flavonoid‐deficient roots. These observations indicated that flavones might act as internal inducers of rhizobial nod genes, and that flavonols might act as auxin transport regulators during nodulation. Both these roles of flavonoids appear critical for symbiosis in M. truncatula.

RNA interference-mediated silencing of the key flavonoid and isoflavone biosynthesis enzyme, respectively, by two different research groups has provided direct genetic evidence for the essential roles that these compounds play in nodulation. Anton Wasson et al. have shown that flavonoids are essential for localized auxin transport inhibition during nodulation in the indeterminate legume Medicago truncatula. By contrast, Senthil Subramanian et al. have shown that isoflavones are essential for endogenous nod gene induction in the determinate legume soybean.

Flavonoids and isoflavonoids are major plant secondary metabolites that mediate diverse biological functions and exert significant ecological impacts. These compounds play important roles in many essential physiological processes. In addition, flavonoids and isoflavonoids have direct but complex effects on human health, ranging from reducing cholesterol levels and preventing certain cancers to improving women's health. In this study, we cloned and functionally characterized five soybean (Glycine max) chalcone isomerases (CHIs), key enzymes in the phenylpropanoid pathway that produces flavonoids and isoflavonoids. Gene expression and kinetics analysis suggest that the soybean type I CHI, which uses naringenin chalcone as substrate, is coordinately regulated with other flavonoid-specific genes, while the type II CHIs, which use a variety of chalcone substrates, are coordinately regulated with an isoflavonoid-specific gene and specifically activated by nodulation signals. Furthermore, we found that some of the newly identified soybean CHIs do not require the 4′-hydroxy moiety on the substrate for high enzyme activity. We then engineered yeast (Saccharomyces cerevisiae) to produce flavonoid and isoflavonoid compounds. When one of the type II CHIs was coexpressed with an isoflavone synthase, the enzyme catalyzing the first committed step of isoflavonoid biosynthesis, various chalcone substrates added to the culture media were converted to an assortment of isoflavanones and isoflavones. We also reconstructed the flavonoid pathway by coexpressing CHI with either flavanone 3β-hydroxylase or flavone synthase II. The in vivo reconstruction of the flavonoid and isoflavonoid pathways in yeast provides a unique platform to study enzyme interactions and metabolic flux.