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 C-4 NADP-malic enzyme species in which the C-4 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 C-4 system. Using differential display, mRNA populations from C-3 and C-4 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 C-4 leaves. Northern and semi-quantitative RT-PCR analyses were used for confirmation. From these screenings, 13 C-4 upregulated genes were identified. Among these one encoded a previously recognized C-4 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 C-4 system that lacks Kranz anatomy, a better understanding of how H. verticillata operates may facilitate the design of a transgenic C-4 system in a C-3 crop species.