No abstract

Cell suspension cultures of alfalfa (Medicago sativa L.) accumulated phenolic secondary metabolites in a pattern similar to that seen in alfalfa roots. Upon treatment with a crude elicitor preparation from the bean pathogen Colletotrichum lindemuthianum, the pterocarpan phytoalexin medicarpin accumulated in cells and culture medium. The extractable activities of six enzymes involved in medicarpin biosynthesis (including three cytochrome P450 activities) were induced by treatment with elicitor, and their induction kinetics correlated with the rate of medicarpin accumulation. However, protoplasts prepared from these cultures accumulated neither medicarpin nor other secondary products after treatment with elicitor. The cytochrome P450 activities were induced during the preparation of the protoplasts, but could be further induced by treatment with fungal elicitor. The results are discussed in relation to the use of alfalfa protoplasts as a system for functional analysis of cloned defense genes.

A chimeric gene consisting of a bean (Phaseolus vulgaris L.) chalcone synthase (CHS) promoter fused to a bacterial chloramphenicol acetyltransferase (CAT) reporter gene was strongly expressed, and further induced by fungal elicitor, when electroporated into alfalfa (Medicago sativa L.) suspension cell protoplasts. Functional analysis of 5' deletions of the CHS promoter-CAT construct in these protoplasts indicated that the region between -326 and -130 contained both activator and silencer elements. Co-electroporation experiments confirmed that these cis-acting elements were binding sites for functionally active trans factors. In vitro DNase I footprinting revealed four potential binding sites for alfalfa suspension cell nuclear proteins between positions -326 and -130 of the CHS promoter. These sites mapped to regions shown to contain functional cis-acting elements on the basis of the deletion analysis. Three of these sites mapped to previously identified binding sites for bean nuclear proteins. Competition gel retardation analysis using oligonucleotide probes containing binding site sequences revealed sequence-specific binding of alfalfa nuclear proteins to an AT-rich element and a putative GT-1 factor consensus binding sequence. Our results define cis elements and their cognate trans factors functionally active in determining the quantitative expression of a defense response gene in a heterologous transient expression system.

A chimeric gene construct containing a bean chalcone synthase (CHS) promoter fused to the chloramphenicol acetyltransferase (CAT) reporter gene was strongly expressed when electroporated into alfalfa protoplasts that were then exposed to a fungal elicitor. Low concentrations (5 x 10(-6) to 10(-4) M) of exogenously applied trans-cinnamic acid (CA), the first intermediate of the phenylpropanoid pathway, slightly stimulated elicitor-induced CAT expression, whereas high concentrations (greater than 10(-4) M) severely reduced expression to below the levels observed in the absence of elicitor. In contrast, trans-p-coumaric acid (4-CA, the second intermediate in the pathway) stimulated expression from the CHS promoter up to 4.5-fold at 5 x 10(-4) M. Expression of CAT driven by the promoters of other elicitor-inducible defense response genes was not markedly affected by CA or 4-CA. Stimulation of CHS promoter expression by low concentrations of CA and 4-CA was completely abolished by 5' deletion to position -130, but not -174. When the -180 to -130 region of the CHS15 promoter was coelectroporated into elicited protoplasts on a separate plasmid along with the intact -326 CHS-CAT construct, the decreased CAT expression as a function of CA or 4-CA concentration was consistent with the coelectroporated sequence competing in trans with the intact promoter for the binding of a factor(s) involved in the up regulation of CHS transcription by 4-CA and low concentrations of CA. Our data support the hypothesis that phenylpropanoid compounds may act as natural and specific regulators of plant gene expression and define the location of a cis-acting element in the CHS15 promoter involved in the induction by phenylpropanoid pathway intermediates.