Identification of C4 responsive genes in the facultative C4 plant Hydrilla verticillata
The aquatic monocot Hydrilla verticillata (L.f.) Royle is a well-documented facultative C4 NADP-malic enzyme species in which the C4 and Calvin cycles operate in the same cell with the specific carboxylases confined to the cytosol and chloroplast, respectively. Several key components had already been characterized at the molecular level, thus the purpose of this study was to begin to identify other, less obvious, elements that may be necessary for a functional single-cell C4 system. Using differential display, mRNA populations from C3 and C4 H. verticillata leaves were screened and expression profiles compared. From this study, 65 clones were isolated and subjected to a customized macroarray analysis; 25 clones were found to be upregulated in C4 leaves. Northern and semi-quantitative RT-PCR analyses were used for confirmation. From these screenings, 13 C4 upregulated genes were identified. Among these one encoded a previously recognized C4 phosphoenolpyruvate carboxylase, and two encoded distinct pyruvate orthophosphate dikinase isoforms, new findings for H. verticillata. Genes that encode a transporter, an aminotransferase and two chaperonins were also upregulated. Twelve false positives, mostly housekeeping genes, were determined from the Northern/semi-quantitative RT-PCR analyses. Sequence data obtained in this study are listed in the dbEST database (DV216698 to DV216767). As a single-cell C4 system that lacks Kranz anatomy, a better understanding of how H. verticillata operates may facilitate the design of a transgenic C4 system in a C3 crop species.
KeywordsC4 cycle genes/proteins differential display facultative C4 photosynthesis functional genomics Hydrilla verticillata macroarray
- DD-RT PCR
pyruvate orthophosphate dikinase
serine glyoxylate aminotransferase
triose phosphate isomerase
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The authors thank Professors C.P. Vance (Department of Agronomy and Plant Genetics, University of Minnesota, St. Paul, MN, USA) and M. Matsuoka (BioScience Center, Nagoya University, Chikusa, Nagoya 461–8601, Japan) for their generous gifts of antisera to the alfalfa PEPC and maize PPDK, respectively. The project was supported in part by the US Department of Agriculture National Research Initiatives Competitive Grants Program, grant nos. 98-35306-6449 and 2002-35318-12540.
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