Characterization of prolyl 4-hydroxylase genes utilizing in silico and in vitro approaches

Hydroxyproline-rich glycoproteins (HRGPs) are abundant structural components of the cell wall implicated in plant growth and development. Prolyl 4-hydroxylases (P4H) are responsible for catalyzing the conversion of specific proline residues present in various proteins to hydroxyprolines, a posttranslational modification for the cell wall structural proteins. Although 13 putative P4H genes have been identified in Arabidopsis, their functional characterization has not been studied in detail. The purpose of this study was two folded: a) The characterization of the Arabidopsis p4h9 expression using a GUS reporter gene approach, and b) The clustering of Arabidopsis P4Hs expression patterns with putative substrate proteins comprising the experimentally identified proline hydroxylation motif. In this context, agro-transformed Arabidopsis plants with the p4h9 promoter-gus fusion construct were tested for construct presence and integrity by PCR and then GUS staining assay was utilized for the assessment of p4h9 expression after being subjected to abiotic stress. Arabidopsis genes were surveyed using search engines to find out the genes comprising the proline hydroxylation motif; [AVSTD]-Pro-[AVST]-[GAVPSTC]-[APS or acidic], and the O-glycosylation motif; [AVSG]-Pro-[AVST]-[AVPSThC]-[APS]. Subsequently, the expression profiles of the motif containing genes together with the P4Hs were studied using oligonucleotide microarray data of plants subjected to diverse types of abiotic stress treatments. The data were analyzed using computational biology tools in order to determine the co-expressed genes, aid in the functional annotation of novel genes, also de novo identification of transcription factor binding sites. This study will help demonstrate the importance of proline hydroxylation in plant stress and could be used as a mechanism for regulating stress responses in plants through regulation of proline hydroxylation events.