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Selected flavonoids that are known as inducers and a suppressor of nodulation (nod) genes of the symbiotic bacterium Rhizobium leguminosarum bv. viciae were tested for their effect on symbiosis formation with garden pea as the host. A solid substrate was omitted from the hydroponic growing system in order to prevent losses of flavonoids due to adsorption and degradation. The presumed interaction of the tested flavonoids with nod genes has been verified for the genetic background of strain 128C30. A stimulatory effect of a nod gene inducer naringenin on symbiotic nodule number formed per plant 14 d after inoculation was detected at concentrations of 0.1 and 1 micro g ml(-1) nutrient solution. At 10 micro g ml(-1), the highest concentration tested, naringenin was already inhibitory. By contrast, nodulation was negatively affected by a nod gene suppressor, quercetin, at concentrations above 1 micro g ml(-1), as well as by another tested nod gene inducer, hesperetin. The deleterious effect of hesperetin might be due to its toxicity or to the toxicity of its degradation product(s) as indicated by the inhibition of root growth. Both the stimulatory effect of naringenin and the inhibitory effect of quercetin on nodule number were more pronounced at earlier stages of nodule development as revealed with specific staining of initial nodules. The lessening of the flavonoid impact during nodule development was ascribed to the plant autoregulatory mechanisms. Feedback regulation of nodule metabolism might also be responsible for the fact that the naringenin-conditioned increase in nodule number was not accompanied by any increase in nitrogenase activity. By contrast, the inhibitory action of quercetin and hesperetin on nodule number was associated with decreases in total nitrogenase activity. Naringenin also stimulated root hair curling (RHC) as one of the earliest nodulation responses at concentrations of 1 and 10 microg ml(-1), however, the same effect was exerted by the nod gene suppressor, quercetin, suggesting that feedback regulatory mechanisms control RHC in the range of nodulation-inhibiting high flavonoid concentrations. The comparison of the effect of the tested flavonoids in planta with nod gene activity response showed a two orders of magnitude shift to higher concentrations. This shift is explained by the absorption and degradation of flavonoids by both the symbionts during 3 d intervals between hydroponic solution changes. The losses were 99, 96.4, and 90% of the initial concentration of 10 micro g ml(-1) for naringenin, hesperetin, and quercetin, respectively.

The root exudates of 24 pea (Pisum sativum L.) non-nodulating mutants, originating from cv. ′Finale′, were tested for Rhizobium nodulation ( nod) gene-inducing activity to reveal potential differences from the wild type. The interaction with flavonoid receptor NodD of R. leguminosarum bv. viciae strain 248 was monitored as the activity of the regulated operon nodABC fused to the reporter gene lacZ. In the uninoculated plants, the only decreased exudate activity was found in the line Risnod17 and ascribed to the impaired root growth in this line. Consequently, constitutive deficiency in the flavonoid nod gene inducers is not a cause of the asymbiotic phenotypes in the studied set. On the other hand, line Risnod5 had constitutively higher nod gene-inducing activity which might be responsible for its conditional nodulation and Fix– phenotype when the nodules are present. Monitoring of the response of wild-type plants to inoculation revealed an increase in nod gene-inducing activity (Ini response) starting first day after inoculation. The chosen mutant lines Risnod22 (putative locus sym9) and Risnod25 (putatively sym8) showed Ini as well indicating that the flavonoid burst can be mutationally dissociated from nodule initiation. On the other hand, absence of Ini in Risnod20 (assumed sym19) suggests branching of the signal transduction pathway downstream of this mutation and places it upstream of the two former mutations in the transduction of the bacterial Nod factor signal. More intense Ini response observed in the supernodulating mutant RisfixC, included in the study for its contrasting symbiotic phenotype, suggests common regulation of nodulation and Ini response via systemic shoot factor. The time shift in the exudation of isoflavonoid phytoalexin pisatin compared to Ini, its increased level in Risnod25 and lowered level in RisfixC are consistent with the regulatory role of plant defence in nodule initiation, which might be mediated directly by isoflavonoids as negative regulators. [ABSTRACT FROM AUTHOR]