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Waterlogging, caused by flooding, long periods of rain, and poor drainage, is a serious abiotic stress determining crop productivity worldwide alongside with drought, salt salinity and extreme temperatures. Depletion of oxygen is a major feature of waterlogging in the roots of plant species. To elucidate how plants respond to hypoxia, it is important to identify the sensor of oxygen change and the signal cascades involved in waterlogging stress. Despite many studies, the basis for sensing and signaling of hypoxia stress remains enigmatic in plants. In animals, prolyl 4-hydroxylases (P4Hs) are regarded as oxygen sensors under hypoxia stress, and available data suggests that alternative splicing of P4Hs is involved in the response to a hypoxic environment in animals. However, the role of P4H in the response to hypoxia is little known in plants. Based on the present research, the goal of this study is to determine whether AS was involved in the regulation of P4H in the response to waterlogging in plants by studying P4H genes in Arabidopsis and maize. The main results achieved are as following:1. In Arabidopsis,13 members of P4H family were reported that AtP4Hs might be involved in the early response to hypoxia. Here, we described alternative splicing (AS) forms of 4 AtP4H genes, including AtP4Hl, AtP4H7 and AtP4H13, designated AtP4H1-2, AtP4H1-3, AtP4H7-1 and AtP4H13-1, suggesting that AS may be a common phenomenon in plant P4Hs. The retained introns, sequence deletions and using of alternative splicing sites detected in these isoforms changed the reading frame and produced premature terminal codons. The abundance of these variants generated from AS was very low indicated by RT-PCR using samples from roots of Arabidopsis both under control and waterlogging, and this result might suggest that the AS forms appeared to be substrates of non sense mediated decay of mRNA (NMD)2. There was little information available concerning maize HIF P4Hs. In the present study, we have characterized 17 splice isoforms of three zmP4H genes, including 5 variants for zmP4Hl,6 variants for zmP4H2 and 6 variants for zmP4H3. We showed that these isoforms occur in roots of HZ32 under both control and waterlogging. RT-PCR experiments indicated that zmP4Hl, zmP4H2 and zmP4H3 had different expression profiles under waterlogging, which, for the first time, suggested regulation by AS under waterlogging in maize. Eleven transcript variants contain premature termination codons (PTCs), which made them potential candidates for nonsense-mediated RNA degradation (NMD). Interestingly, one transcript, zmP4H2-1 is relatively abundant under both control and waterlogging, suggesting that NMD is not a "default" mechanism for PTC-containing RNA transcripts generated by alternative splicing. However, we analyzed the abundances of the variant of zmP4H2-1 after NMD that was inhibited by CHX for control and waterlogging of roots of HZ32 to determine if the PTC-containing transcripts zmP4H2-1 was degraded by NMD. Our results showed that the inhibition of NMD system led to accumulation of PTC-containing zmP4H2-1 variant, indicating that zmP4H2-1 variant with a PTC was the targets of degradation by NMD. These data indicate that alternative splicing of zmP4H as well as the mRNA quality control mechanism by NMD are complex and regulated under waterlogging in roots of maize.In conclusion, our results provide a framework for future dissection of the function of the emerging P4H family and suggest that AS might have an important role in the regulation of the expression profile of this gene family under waterlogging stress.
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