Joanne C. Cusumano

Cloning of the SNG1 Gene of Arabidopsis Reveals a Role for a Serine Carboxypeptidase-like Protein as an Acyltransferase in Secondary Metabolism

Serine carboxypeptidases contain a conserved catalytic triad of serine, histidine, and aspartic acid active-site residues. These enzymes cleave the peptide bond between the penultimate and C-terminal amino acid residues of their protein or peptide substrates. The Arabidopsis Genome Initiative has revealed that the Arabidopsis genome encodes numerous proteins with homology to serine carboxypeptidases. Although many of these proteins may be involved in protein turnover or processing, the role of virtually all of these serine carboxypeptidase-like (SCPL) proteins in plant metabolism is unknown. We previously identified an Arabidopsis mutant, sng1 (sinapoylglucose accumulator 1), that is defective in synthesis of sinapoylmalate, one of the major phenylpropanoid secondary metabolites accumulated by Arabidopsis and some other members of the Brassicaceae. We have cloned the gene that is defective in sng1 and have found that it encodes a SCPL protein. Expression of SNG1 in Escherichia coli demonstrates that it encodes sinapoylglucose:malate sinapoyltransferase, an enzyme that catalyzes a transesterification instead of functioning like a hydrolase, as do the other carboxypeptidases. This finding suggests that SCPL proteins have acquired novel functions in plant metabolism and provides an insight into the evolution of secondary metabolic pathways in plants.

Cinnamate-4-hydroxylase expression in arabidopsis. Regulation in response to development and the environment

Cinnamate-4-hydroxylase (C4H) is the first Cyt P450-dependent monooxygenase of the phenylpropanoid pathway. To study the expression of this gene in Arabidopsis thaliana, a C4H cDNA clone from the Arabidopsis expressed sequence tag database was identified and used to isolate its corresponding genomic clone. The entire C4H coding sequence plus 2.9 kb of its promoter were isolated on a 5.4-kb HindIII fragment of this cosmid. Inspection of the promoter sequence revealed the presence of a number of putative regulatory motifs previously identified in the promoters of other phenylpropanoid pathway genes. The expression of C4H was analyzed by RNA blot hybridization analysis and in transgenic Arabidopsis carrying a C4H-[beta]-glucuronidase transcriptional fusion. C4H message accumulation was light-dependent, but was detectable even in dark-grown seedlings. Consistent with these data, C4H mRNA was accumulated to light-grown levels in etiolated det1–1 mutant seedlings. C4H is widely expressed in various Arabidopsis tissues, particularly in roots and cells undergoing lignification. The C4H-driven [beta]-glucuronidase expression accurately reflected the tissue-specificity and wound-inducibility of the C4H promoter indicated by RNA blot hybridization analysis. A modest increase in C4H expression was observed in the tt8 mutant of Arabidopsis.

QUANTITATIVE EFFECTS OF IRON CHELATORS ON HYDROXYL RADICAL PRODUCTION BY THE SUPEROXIDE-DRIVEN FENTON REACTION

Iron bound to certain chelators is known to promote the conversion of superoxide radicals (O2-) to hydroxyl radicals (HO.) by the superoxide-driven Fenton reaction. The production of HO. by various iron chelates was studied using the reaction of dimethyl sulfoxide and HO. to produce methane sulphinic acid. Methane sulphinic acid was quantified by use of a simple colorimetric assay and used to determine the amounts of HO. produced. Superoxide was generated from 200 microM hypoxanthine and 0.05 U/ml xanthine oxidase in the presence of 0-100 microM iron and 100 microM of each chelator. The results of this preliminary investigation illustrate that, at physiological pH, the superoxide-driven Fenton reaction is significantly promoted by iron chelated to EDTA, nitrilotriacetate, and citrate, but is not promoted by the other anions studied.

Tissue specific specialization of the nanoscale architecture of Arabidopsis.

The Arabidopsis stem is composed of five tissues - the pith, xylem, phloem, cortex and epidermis - each of which fulfills specific roles in support of the growth and survival of the organism. The lignocellulosic scaffolding of cell walls is specialized to provide optimal support for the diverse functional roles of these layers, but little is known about this specialization. X-ray scattering can be used to study this tissue-specific diversity because the cellulosic components of the cell walls give rise to recognizable scattering features interpretable in terms of the underlying molecular architecture and distinct from the largely unoriented scatter from other constituents. Here we use scanning X-ray microdiffraction from thin sections to characterize the diversity of molecular architecture in the Arabidopsis stem and correlate that diversity to the functional roles the distinct tissues of the stem play in the growth and survival of the organism.