|
PULSe Home > Faculty Members A-C > Clint Chapple
Clint Chapple
Current Research Interests:
The general 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 mutants that are defective in the sythesis 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 monoooxygenase (P450). P450s belong to a superfamily of heme-containing enzymes most of which catalyze NADPH- and O2 -dependent hydroxylation relations. 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 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 hyperfluouescent under UV. Althought the predominant brt1 phenotype is hyperfluorescent phenotype. All of the fah1-2 mutant. The morphology of the ref1-1, ref2-1, and brt1-1 rosettes is similar to wild-type rosettes whereas ref3-2 and ref4-3 rosettes are reduced in size and display aberrant leaf shapes. These observations suggest that either the 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 and ref4 mutations lead to a substantial decrease in rachis lignin content, while the other mutations had little effect. These results suggest that REF and BRT genes will be a major fucus 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 and sng2 for sinapoylglucose accumulators. Plants homozygous for the sng1 and sng2 mutations fail to accumulate sinapoylmalate in leaves and sinapoylcholine in seeds, respectively, but instead accumulate their biosynthetic precursor, sinapoylglucose. We have cloned the SNG1 and SNG2 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 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 presumed evolutionary progenitors.
Selected Publications:
Weng JK, Tanurdzic M, Chapple C. (2005) Functional analysis and comparative genomics of expressed sequence tags from the lycophyte Selaginella moellendorffii. BMC Genomics 6:85
Fraser CM, Rider LW, Chapple C (2005) An expression and bioinformatics analysis of the Arabidopsis serine carboxypeptidase-like gene family. Plant Physiol. 138: 1136-1148.
Humphreys JM, Chapple C (2004) Immunodetection and quantification of cytochromes P450 using epitope tagging: immunological, spectroscopic and kinetic analysis of cinnamate 4-hydroxylase. J Immun Methods 292: 97-107
Hemm MR, Rider SD, Ogas J, Murry DJ, Chapple C (2004) Light induces phenylpropanoid metabolism in Arabidopsis roots. Plant J 38: 765-778
Nair RB, Bastress KL, Ruegger MO, Denault JW, Chapple C (2004) The Arabidopsis REF1 gene encodes an aldehyde dehydrogenase involved in ferulic acid and sinapic acid biosynthesis. Plant Cell 16: 544-554
Huntley SK, Ellis D, Gilbert M, Chapple C, Mansfield SD (2003) Significant increases in pulping efficiency in C4H-F5H-transformed poplars: improved chemical savings and reduced environmental toxins. J Agric Food Chem 51: 6178-6183
Shirley AM, Chapple C (2003) Biochemical Characterization of sinapoylglucose:choline sinapoyltransferase, a serine carboxypeptidase-like protein that functions as an acyltransferase in plant secondary metabolism. J Biol Chem 278: 19870-19877
Franke R, Hemm MR, Denault JW, Ruegger MO, Humphreys JM, Chapple C. (2002) Changes in secondary metabolism and deposition of an unusual lignin in the ref8 mutant of Arabidopsis. Plant J. 30: 47-59.
Franke R, Humphreys JM, Hemm MR, Denault JW, Ruegger MO, Cusumano JC, Chapple C. (2002) The Arabidopsis REF8 gene encodes the 3-hydroxylase of phenylpropanoid metabolism. Plant J. 30: 33-45.
Hause B, Meyer K, Viitanen PV, Chapple C, Strack D. (2002)Immunolocalization of sinapoylglucose : malate sinapoyltransferase in Arabidiopsis thaliana. Planta 215: 26-32.
Ruegger M, Chapple C. (2001) Mutations that reduce sinapoylmalate accumulation in Arabidopsis define loci with diverse roles in phenylpropanoid metabolism. Genetics 159: 1741-1749.
Shirley AM, McMichael CM, Chapple C. (2001) The sng2 mutant of Arabidopsis is defective in the gene encoding the serine carboxypeptidase-like protein sinapoylglucose: choline sinapoyltransferase. Plant J 28: 83-94.
Ralph J, Lapierre C, Marita JM, Kim H, Lu F, Hatfield RD, Ralph S, Chapple C, Franke R, Hemm MR, Doorsselaere JV, Sederoff RR, O'Malley DM, Scott JT, MacKay JJ, Yahiaoui N, Boudet A, Pean M, Pilate G, Jouanin L, Boerjan W. (2001) Elucidation of new structures in lignins of CAD- and COMT-deficient plants by NMR. Phytochemistry 57: 993-1003.
Lehfeldt C, Shirley AM, Meyer K, Ruegger MO, Cusumano JC, Chapple C. (2000) Cloning of the SNG1 gene of Arabidopsis reveals a role for a serine carboxypeptidase-like protein as an acyltransferase in secondary metabolism. Plant Cell 12: 1295-1306.
Franke R, McMichael Cm, Shirley AM, Meyer K, Cusumano JC, Chapple C. (2000) Modified lignin in tobacco and popular plants overexpressing the Arabidopsis gene encoding ferulate 5-hydroxylase. Plant J 22: 223-234.
Marita JM, Ralph J, Hatfield RD, Chapple C. (1999) NMR characterization of lignin in Arabidopsis altered in the activity of ferulate 5-hydroxylase. Proc Natl Acad Sci USA 96: 12328-12332.
Humphreys JM, Hemm MR, Chapple C. (1999) New routes for lignin biosynthesis defined by biochemical characterization of recombinant ferulate 5-hydroxylase, a multifunctional cytochrome P450-dependent monooxygenase. Proc Natl Acad Sci USA 96: 10045-10050
|
