Flavonoid-induced calcium signalling in Rhizobium leguminosarum bv. viciae

Rhizobium leguminosarum bv. viciae strain 300 (Johnston &Beringer, 1975) and R. leguminosarum strain 8401 (which

lacks the Sym plasmid and carries either the pIJ1477plasmid (nodC–lacZ fusion) or the pIJ1478 plasmid

(nodD–lacZ fusion) (kindly provided by A. Downie,Norwich, UK)) were grown at 28 C in Tryptone-Yeast

(TY) medium containing antibiotics (30 lg ml )1 ofkanamy-cin and 2 lg ml )1of tetracycline), whereappropriate. Escherichia coli JM109 (Yanish-Perron et al.,1985) was grown at 37 C in Luria–Bertani (LB) mediumcontaining 30 lg ml)1 of kanamycin, where appropriate.

 

of the transient increase in [Ca2+]cyt, we constructed an apoaequorin-expressing strain of R. leguminosarum bv. viciae.
Fig. 1(a) shows that root exudates of V. sativa subsp. nigrado indeed elicit a transient elevation of [Ca2+]cyt. The effects
of the major flavonoids that induce nod genes (Zaat et al.,1987; Begum et al., 2001) were then tested. Naringenin
(10 lM) triggered a rapid elevation in the level of intracellular Ca2+ that was absent in buffer-only controls(Fig. 1b). The naringenin-specific [Ca2+]cyt transientpeaked at 1.40 ± 0.22 lM after about 100 s and slowlydecreased to basal levels within 15 min. The RT-PCRanalyses of gene expression confirmed the well-known activation by naringenin of the common nodABC genes in these
apoaequorin-expressing cells (Fig. 1b). Luteolin andhesperetin (10 lM), which are also active inducers of nodgene expression in R. leguminosarum, generated transientelevations in [Ca2+]cyt with kinetics similar to those inducedby naringenin (Fig. 1c). By contrast, in apoaequorinexpressing E. coli, none of the flavonoids tested elicited anychange in Ca2+, although the same cells were found to beresponsive to other stimuli, such as oxidative stress (10 mMH2O2), hypo-osmotic shock (three volumes of distilled
water) and 10 mM external Ca2+ (Fig. S1).The isoflavones daidzein and genistein (10 lM) wereunable to promote induction of the nod gene in this speciesof Rhizobium and were found to be equally unable toinduce any detectable transient increase in Ca2+ (Fig. 2a).No effect on either Ca2+ homeostasis or nod gene activationwas observed with increasing concentrations (up to 50 lM)
of the above isoflavones (data not shown).As noninducing flavonoids have been demonstrated, inSinorhizobium meliloti, to act as competitive inhibitors ofinducers, preventing the transcriptional activator NodDfrom promoting nod gene induction (Peck et al., 2006), wedetermined whether inducers and noninducers compete alsoin the generation of the Ca2+ signal. Competition experiments were carried out by examining the effect of increasing.