Characteristics Of The Post-transcriptional Processing Of Glycine Betaine Biosynthesis Genes In Cereal Crops

Among the few classes of organic compounds that are commonly employed as osmoprotectants, the quaternary ammonium elements (e.g. glycine betaine) are widely distributed in bacteria, algae, higher plants, and animals. In plant, glycine betaine is known to provide a tolerance under stresses, such as salinity, high temperature and freezing. During photosynthesis, it stabilizes both photosystem II complexes and Rubisco. In the known biological systems, glycine betaine is synthesized from choline. The two-enzyme pathway is commonly found across a wide range of plants, in which glycine betaine is formed as the result of a two-step oxidation of choline, catalyzed by choline monooxygenase (CMO) and betaine aldehyde dehydrogenase (BADH), respectively.The presence of two BADH homologues has been widely reported in many plants species under investigations. In plants, genes encoding for glycine betaine biosynthesis have been identified. However, the level of accumulated glycine betaine is completely different. Rice, one of the most important crops, has been claimed as a salt-sensitive species and does not have the ability to accumulate glycine betaine. In this study, we extensively examined differential transcriptional products of the BADH genes in rice derived from different treatments that had deletion(s) of 5' exon material or insertion of exogenous gene sequences. The observed events took place at the 5' exonic region, leading to the frame-shifts with premature termination by introducing stop codon, removal of translation initiation codon and deletion of functional domain. Similar processing events were observed in BADH homologues of other cereal crops including wheat, maize and barley. By contrast, the BADH homologues from dicotyledonous species spinach, Arabidopsis, tomato and a distantly related BADH-like gene in rice were spliced correctly.Rice also contains a CMO in addition to two BADH genes. From comparison of the coding proteins between rice and spinach CMOs, rice CMO potentially shares two conservative motifs including a Rieske-type [2Fe-2S] cluster and a mononuclear non-heme Fe binding sequence. These motifs are considered to be essential for the function of CMO. Previous study revealed that the CMO activity was not detected in rice. Here in this study, we examined differential transcriptional products derived from CMO gene in rice under different treatments. Sequence analysis unraveled that a large number of rice CMO transcripts contained deletions of 5' coding sequences, together with multiple alternative splicing events mainly including retention of intron 2. Sequence analysis revealed that abnormally processed transcripts resulted in frame-shifts with premature termination by introducing stop codon, removal of translation initiation codon and deletion of functional domain. Similar result was obtained in maize CMO. However, this phenomenon did not occur in spinach CMO. The differentiation of post-transcriptional processing in CMO and BADH genes potentially contributes to the variation of glycine betaine synthesizing capacities among various plant species.Comprehensive sequence analyses demonstrated that extensive sequence similarities (named as short-direct repeats, SDR) are of paired presence surrounding the junctions of both the deletion/insertion sites in the unusual BADH and CMO transcripts. The sites selection for the deletion/insertion was altered in response to the stress conditions. This indicates that the sequence elements of SDRs are probably required for the recognition of the deletion/insertion sites in this processing.Some candidate genes containing SDRs sequences were selected by using sequences around SDRs derived from rice BADHs and CMO to BLAST against rice database. RT-PCR and sequencing approaches were conducted to determine the processing patterns of these genes from various rice tissues. The results showed that, similar post-transcriptional processing occur in majority of the candidate genes. And similar SDRs are present in the deletion/insertion sites. In addition, trans-acting events taking place in different molecules derived from the same locus were observed. For Arabidopsis, a glycine-rich protein gene, one of the two candidates, underwent splicing, alternative splicing, deletion mediated by SDRs and partial exonic material repetition. However, the deletion and repetition does not exist at genomic level.The characteristics of the unusual post-transcriptional processing in the tested genes were analyzed. The unusual processing showed preference to some genes based on their primary structure, and might be regulated. The analysis of dinucleotide boundaries within the deletion region demonstrated that a different recognition mechanism from alternative splicing might be evolved. The similarity among nearly identical SDR sequences derived from different genes or plant species embodied the principle of evolution.