Chalcone synthase (CHS) catalyzes the first and key regulatory step in the branch pathway of phenylpropanoid biosynthesis specific for synthesis of ubiquitous flavonoid pigments and UV protectants. In bean (Phaseolus vulgaris L.) and other members of the Leguminoseae, chalcone synthase is also involved in the synthesis of the isoflavonoid-derived phytoalexin antibiotics characteristic of this family. We have demonstrated that the haploid genome of bean contains a family of about six to eight CHS genes, some of which are tightly clustered. Treatment of bean cells with fungal elicitor activates several of these genes leading to the accumulation of at least five and probably as many as nine distinct CHS transcripts encoding a set of CHS isopolypeptides of Mr 42-43 kDa but with differing pI in the range pH 6-7. In elicited cells specific transcripts and encoded polypeptides are differentially induced with respect to both the extent and kinetics of accumulation. Wounding or infection of hypocotyl tissue also activates several CHS genes with marked differences in the pattern of accumulation of specific transcripts and encoded polypeptides in wounded compared to infected tissue or elicited cells, indicating operation of more than one cue for defense gene activation. Illumination induces accumulation of a different set of CHS transcripts including only one of the set hitherto demonstrated to be induced by biological stress. The organization and differential regulation of the CHS gene family in bean are discussed in relation to the functions of this enzyme in adaptative and protective responses to diverse environmental stresses.

Hydroxyproline-rich glycoproteins (HRGPs) are important structural components ofplant cell walls and also accumulate in response to infection as an apparent defense mechanism. Accumulation of HRGP mRNA in biologically stressed bean (Phaseolus vulgaris L.) cells was monitored by blot hybridization with 32P-labeled tomato genomic HRGP sequences. Elicitor treatment of suspension-cultured cells caused a marked increase in hybridizable HRGP mRNA. The response was less rapid but more prolonged than that observed for mRNAs encoding enzymes of phytoalexin biosynthesis. HRGP mRNA also accumulated during race:cultivar-specific interactions between bean hypocotyls and the partially biotrophic fungus Colletotrichum lindemuthianum, the causal agent of anthracnose. In an incompatible interaction (host resistant) there was an early increase in HRGP mRNA correlated with expression of hypersensitive resistance; whereas, in a compatible interaction (host susceptible), marked accumulation of HRGP mRNA occurred as a delayed response at the onset of lesion formation. In both interactions, mRNA accumulation was observed in uninfected cells distant from the site of fungal inoculation, indicating intercellular transmission of an elicitation signal.

ABSTRACIChitinase, which catalyzes the hydrolysis of 0-1,4 N-acetylglucosamine linkaes of the funpl cell wall polymer chitin, is a component of the inducible defenses of plants. We show that chitinase synthesis is stimulated in bean (Phaseolus vulgars L.) cell suspension cultures treated with fungal cell wall elicitors and in hypocotyls in response to infection with the fungus Colktotrichum lindemuthianum. Chitinase cDNA clones were isolated by antibody screening of a Agtll cDNA library containing sequences complementary to poly A RNA from elicited cells. The identity of these clones was confirmed by nucleotide sequence analysis and comparison of the deduced amino acid sequence with that determined for the amino-terminal sequence of bean chitinase. Elicitor causes a very rapid activation of chitinase transcription with a 10-fold stimulation after 5 minutes and 30-fold increase within 20 minutes. This leads to a marked, traient accumulation of chitinase transcripts with maximum levels 2 hours after elicitor treatment, concomitant with the phase ofrapid enzyme synthesis. Chitinase transcripts also markedly accumulate in wounded and infected hypocotyls. Chitinase cDNA sequences hybridize to several genomic fragments suggesting there are several chitinase genes in the bean genome.

DNAs complementary to poly(A)+ RNA present in elicitor-treated cells of Phaseolus vulgaris L. were inserted into pBR325 and used to transform Escherichia coli strain JA221. A clone was identified that contained sequences complementary to mRNA encoding chalcone synthase, a regulatory enzyme of phenylpropanoid biosynthesis, which catalyzes the first reaction of a branch pathway specific to flavonoid and isoflavonoid biosynthesis. Rapid, marked but transient increases in chalcone synthase mRNA in response to elicitor treatment were observed by RNA blot hybridization with 32P-labeled chalcone synthase cDNA sequences. Induction of chalcone synthase mRNA governs the rate of enzyme synthesis throughout the phase of rapid increase in enzyme activity at the onset of accumulation of isoflavonoid-derived phytoalexins. The data are consistent with the hypothesis that elicitor causes a rapid transient stimulation of transcription of chalcone synthase gene(s) as an early event in the expression of the phytoalexin defense response.

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