Identification And Functional Characterization Of Rice OsMPEC Gene And Stress-regulated MicroRNAs In Arabidopsis Thaliana

OsMPEC is a new gene that is isolated from rice. Its homologous genes exist widely throughout evolution of photosynthetic organisms, being present in photosynthetic bacteria, algae and higher plants. The conserved regions included a leucine zipper domain and two copies of EXn DEXRH motif. It was reported to encode the Mg-protoporphyrin IX monomethyl ester cyclase (MPEC) that converts Mg-protoporphyrin IX monomethyl ester (MgPME) to protochlorophyllide (Pchlide) in plants. However, the study of this enzyme remains an area of relative ignorance and there have been considerable problems with attempts to connect genetic and biochemical data.We have previously isolated several cDNA fragments of MPEC from various plants, such as PNZIP and NTZIP, which exhibit high similarity to CHL27 and Xanthal-l. Here, we isolated and characterized a MPEC coding gene OsMPEC in rice. We also investigated the functional mechanism of OsMPEC and the main results were as follows:1. It was a single-copy gene in the whole rice genome. The full-length OsMPEC gene consists of 2559 nucleotides containing a 1227-nucleotide open reading frame (ORF) and four introns. The 5'-upstream and the 3'-downstream untranslated regions are 38 bp and 679 bp, respectively. The ORF is predicted to encode a 409-amino acid polypeptide with a calculated molecular mass of 47.3 kD.The deduced amino acid sequence of OsMPEC shares 79.17%, 78.19%, 78.83% and 87.08% sequence homology with PNZIP, NTZIP, CHL27 and Xantha-l, respectively. Comparative alignment analysis of the plant MPEC sequences suggests that two copies of the EXn DEXRH motif and a conserved leucine zipper domain are present in these proteins2. To determine the pattern of OsMPEC expression in different rice organs, a northern blot hybridization analysis was performed. The results showed that OsMPEC transcripts were relatively more abundant in leaves; they were less abundant in stems and could not be detected at all in the roots.3. The OsMPEC expression was induced by both low temperature and high temperature. The OsMPEC promoter sequence was isolated and analyzed.We found that several known light responsive elements appeared in the promoter region as expected. In addition, some stress-responsive cis-acting elements were also involved in the OsMPEC promoter, such as the ABREs (ABA-response elements), AREs (anaerobic induction elements) and TC-rich repeats (defense and stress responsive elements), indicating that it could be regulated by temperature.4. OsMPEC transcripts were less detected in the copper-excessive condition, and were hardly detected in the iron- or oxygen-deficient condition when compared to the control plants, indicating the proper Fe, Cu and oxygen tension were required to maintain the expression of the gene product.5. The antisense transformants showed a broad variety of phenotypes with reduced green pigmentation, from pale green to yellow. The seeds of the antisense lines germinated slowly and some seedlings displayed a decreased growth rate when compared with the WT control. The difference became apparent about four weeks after germination, and some lines were completely bleached and died within a few days after being transferred to soil. The levels of Chl in the leaves of the antisense transgenic plants were 35% less than those of the WT plants. However, most of the sense transgenic lines did not show much significant difference in growth, seed yield, germination and Chl content compared to the WT lines when grown under normal greenhouse conditions.6. To determine the effect of the OsMPEC gene on rice photosynthetic efficiency, the maximal photochemical efficiency of PSII and the quantum yield of PSII electron transport were measured in the WT, sense and antisense transgenic plants. Our data showed that the Fv/Fm andΦPSII of the sense, antisense and WT plants showed no obvious difference.In addition, a significant reduction in the oxidizableΔI/I0 was observed in all antisense lines. Taken together, these results demonstrated that the PSI but not the PSII was abnormal in the antisense transgenic plants and the OsMPEC was required to maintain the stable PSI in rice. High salinity, drought and low temperature are three common environmental stress factors that seriously influence plant growth and development worldwide. Recently, microRNAs (miRNAs) have emerged as a class of gene expression regulators that have also been linked to stress responses. With the discovery of large numbers of miRNAs in both plants and animals, the important roles of these special small RNAs have been widely recognized. Many processes, such as leaf development, auxin signaling, phase transition, flowering and genome maintenance, are regulated in similar ways by different miRNAs. Recently, there has been strong evidence leading to the proposal that miRNAs are hypersensitive to abiotic or biotic stress as well as to diverse physiological processes. Hence, efforts to identify novel stress-regulated miRNAs and determine their expression patterns could improve our understanding of their functions in stress adaptation. The main results were as follows:1. We identified 14 stress-inducible miRNAs using microarray data in which the effects of three abiotic stresses were surveyed in Arabidopsis thaliana. Among them, ten high-salinity-, four drought- and ten cold-regulated miRNAs were detected. miR165, miR319 and miR393 were upregulated by both high salinity and cold, miR167 was induced by both high salinity and drought, and miR168, miR171 and miR396 were observed to respond remarkably to all three stresses.2. To determine which locus is responsive to stress conditions, we performed semiquantitative RT-PCR analysis using specific primers designed to amplify fold-back precursor transcripts in Arabidopsis, and our RT-PCR results were ultimately consistent with the microarray data for all miRNAs tested. Expression profiling by RT-PCR analysis showed great crosstalk among the high-salinity, drought and cold stress signaling pathways.3. To further elucidate the inducibility of these products, we analyzed the 1000 bp upstream promoter sequence of 20 miRNAs by using the PlantCARE database. We identified several known stress responsive elements, such as the ABREs (ABA-response elements), AREs (anaerobic induction elements), MBS (MYB binding site involved in drought-inducibility), HSEs (heat stress responsive elements), LTRs (low-temperature responsive elements) and TC-rich repeats (defense and stress responsive elements)The existence of stress-related elements in miRNA promoter regions provided further evidence supporting our results. 4. Analysis of transgenic plants with the gus reporter gene which under the control of different sequences upstream miRNA precursors shows that all these sequences can function as miRNA promoters. In addition, the activity of miR159a promoter is very high which is almost 6 times higher than that of the CaMV35S promoter.5. The promoter of miR159a is a constitutive promoter, and miR398a is mainly expressed in root, stem and leaves of Arabidopsis. However, miR393a and miR443 are specific expressed miRNA which might be under the control of their promoters due to their different functions.