Clint Chapple

Head of Department, Distinguished Professor of Biochemistry

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	Area	Biochemistry and molecular biology of plant secondary metabolism
	Office	WSLR B030
	Phone	(765) 494-0494
	Email	chapple@purdue.edu
	Degree	Ph.D. University of Guelph, Canada, 1989

The phenylpropanoid pathway gives rise to a wide array of soluble metabolites in plants.  These compounds participate in many plant defense responses and absorb potentially-damaging UV-B radiation.  The pathway also generates the monomers required for lignin biosynthesis:  ferulic acid and sinapic acid.  Lignin is integrated into the plant secondary cell wall where it provides structural rigidity to plant tissues and enables tracheary elements to withstand the tension generated during transpiration.  The analysis of this pathway in Arabidopsis using the tools of biochemistry, molecular biology and genetics is the focus of our laboratory.

Sinapoylmalate biosynthesis as a marker for phenylpropanoid biosynthesis in Arabidopsis.  Our laboratory has isolated mutants that are defective in the synthesis of sinapoylmalate, one of the major soluble phenylpropanoid secondary metabolites in Arabidopsis.   In wild type, sinapoylmalate is accumulated in the adaxial leaf epidermis and the distribution of this blue-fluorescent secondary metabolite can be exploited as a rapid method for isolating mutants defective in genes encoding enzymes or regulatory factors of the phenylpropanoid pathway.  Mutants that lack sinapoylmalate can be readily identified by their red chlorophyll fluorescence under UV light among a population of blue fluorescent wild type plants.  Using this novel mutant screen, we have isolated a variety of mutants that are permitting us to clone genes of the phenylpropanoid pathway that have not previously been characterized.

The fah1 mutant.  The fah1 mutant is blocked at the step of the general phenylpropanoid pathway catalyzed by ferulate-5-hydroxylase (F5H), a cytochrome P450-dependent monooxygenase (P450).  P450s belong to a superfamily of heme-containing enzymes, most of which catalyze NADPH- and O₂-dependent  hydroxylation reactions.  In animals, P450s have been carefully studied because of their importance in the breakdown of foreign compounds such as pharmaceuticals and carcinogens.  Plant P450s are involved in a wide array of biosynthetic pathways including those giving rise to lignin, alkaloids, cyanogenic glycosides, sterols, and plant growth regulators such as gibberellins, jasmonic acid and brassinosteroids. Using the fah1 mutant, we cloned the gene encoding F5H by T-DNA tagging, an approach that circumvented the requirement of protein purification. Because P450s have relatively low turnover numbers and F5H is required for the biosynthesis of syringyl lignin, we tested the hypothesis that this enzyme catalyzes the rate-limiting step in the biosynthesis of syringyl lignin.  We found that over-expression of the F5H gene has demonstrated that F5H expression limits syringyl lignin accumulation both quantitatively and developmentally.  The identification of this control point in lignin monomer composition is likely to have a significant agricultural and industrial impact.  We have now begun a series of studies aimed at the characterization of the wild type F5H protein, and the proteins encoded by various fah1 alleles.  It will be very interesting to determine in what way the plant P450s differ from their animal counterparts.  The use of F5H as a paradigm for plant P450s will contribute significantly to our understanding of these "linchpin" enzymes of plant metabolism.

The ref and brt mutants of Arabidopsis.  We recently identified a number of new sinapoylmalate-deficient mutants named red-fluorescent leaves (ref1-ref8).  A second class of mutants, bright trichomes (brt), have trichomes that are hyperfluorescent under UV.  Although the predominant brt1 phenotype is hyperfluorescent trichomes, the non-trichome epidermal cells of brt1 have a red-fluorescent phenotype. All of the ref mutants display a UV phenotype that is intermediate between wild type and the null fah1-2 mutant.  The morphology of the ref1, ref2, and brt1 rosettes is similar to wild-type rosettes whereas ref3 and ref4 rosettes are reduced in size and display aberrant leaf shapes.  These observations suggest that either the REF3 and REF4 genes have roles in plant development, while the functions of REF1, REF2, and BRT1 are limited to phenylpropanoid biosynthesis, or it may indicate that phenylpropanoid products downstream of REF3 and REF4 action are required for normal plant growth.  The ref3 andref4 mutations lead to a substantial decrease in rachis lignin content, while the other mutations had little effect.  These results suggest that REF3 and REF4 act at a point in the phenylpropanoid pathway common to both sinapate ester and lignin biosynthesis. In depth analysis of these mutants and the isolation of the REF and BRT genes will be a major focus of our laboratory in the coming years.

The sng mutants of Arabidopsis.  We have also isolated two mutants defective in the later stages of sinapoylmalate biosynthesis which we call sng1 andsng2 forsinapoylglucose accumulators.  Plants homozygous for the sng1 andsng2 mutations fail to accumulate sinapoylmalate in leaves and sinapoylcholine in seeds, respectively, but instead accumulate their biosynthetic precursor, sinapoylglucose.  We have cloned the SNG1 andSNG2 genes and have found that they encode serine carboxypeptidase-like proteins.  This finding suggests that the enzymes of sinapate ester biosynthesis have been recruited from recruited from those involved in protein turnover and have acquired new functions over evolutionary time.  We are currently conducting experiments to understand how these enzymes have acquired the ability to catalyze transacylation reactions as opposed to the hydrolytic reactions catalyzed by their evolutionary progenitors.