Prolyl 4‐hydroxylases (P4H; EC 1.14.11.2) have been studied extensively in various organisms and recently in relation to oxygen deficiency while little is known about their role in response to similar stresses in plants. To this direction, we investigated the expression patterns of Arabidopsis P4Hs under hypoxia, anoxia and mechanical wounding. The 13 Arabidopsis P4Hs are low‐abundance transcripts with differential patterns of expression in response to two hypoxic, 1.5% and 5% O2, anoxic conditions and mechanical wounding. The majority of AtP4Hs showed higher levels of expression in roots compared with shoots under hypoxia and anoxia. Hypoxia of 1.5% O2 induced the expression of six AtP4Hs, while hypoxia of 5% O2 and anoxia induced the expression of three and two AtP4Hs, respectively. Moreover, we identified in silico, 308 Arabidopsis genes among the differentially expressed genes under hypoxia that contain proline hydroxylation motifs. It is likely that the AtP4Hs hydroxylate proline residue(s) of the 308 polypeptides suggest involvement in their post‐translational modification process. This modification might serve as an alternative level of regulation for the function of these polypeptides in the context of hypoxic response. Furthermore, six AtP4Hs were upregulated in response to mechanical wounding, suggesting involvement in this stress response.

Summary• By comparing cDNA populations derived from chromium-stressed primary leaves of barley ( Hordeum vulgare L.) with controls, differentially expressed cDNA fragments could be identified. The deduced amino acid sequence of one of these cDNAs [named 'C2 domain 1' ( HvC2d1 )] exhibits a motif that is similar to the known C2 domain and a nuclear localization signal (NLS).• Expression of this member of a novel class of plant C2 domain-like proteins was studied using real-time PCR, and subcellular localization was investigated using green fluorescent protein (GFP) fusion constructs. Calcium binding was analysed using a 45 Ca 2+ overlay assay.• HvC2d1 was transiently induced after exposure to different heavy metals and its mRNA accumulated during the phase of leaf senescence. HvC2d1 expression responded to changes in calcium levels caused by the calcium ionophore A23187 and to treatment with methylviologen resulting in the production of reactive oxygen species (ROS). Using overexpressed and purified HvC2d1, the binding of calcium could be confirmed. Chimeric HvC2d1-GFP protein was localized in onion epidermal cells at the plasma membrane, cytoplasm and the nucleus. After addition of calcium ionophore A23187 green fluorescence was only visible in the nucleus.• The data suggest a calcium-dependent translocation of HvC2d1 to the nucleus. A possible role of HvC2d1 in stress-and development-dependent signalling in the nucleus is discussed.

Arabidopsis prolyl 4 hydroxylases (P4Hs) catalyze an important post-translational modification in plants, though the only information on their patterns of expression is solely based on Arabidopsis microarray analysis data. In addition, the expression patterns of plants P4Hs in response to hypoxia, anoxia and other abiotic stresses such as mechanical wounding have never been studied extensively, despite their central role in hypoxic response of several other organisms through the regulation of stability of the HIF-1alpha transcription factor, the global regulator of hypoxic response. The 13 putative Arabidopsis P4Hs are low abundance transcripts with differential patterns of expression in response to two hypoxic, 1.5% and 5% O(2), anoxic conditions and mechanical wounding. Hypoxia of 1.5% O(2) induced the expression of six At-P4Hs while hypoxia of 5% O(2) and anoxia induced the expression of three and two At-P4Hs, respectively. Moreover, 308 Arabidopsis genes including 25 transcription factors were identified in silico among the differentially expressed genes under hypoxia that contain proline hydroxylation motifs. These results suggest involvement of this post-translational modification in the processing of hypoxia induced proteins providing an alternative level of regulation for responses to oxygen deficiency conditions.

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