Difference between revisions of "Os10g0542100"
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− | + | The rice '''''Os10g0542100''''' was first identified as '''''OsMT-II-1a''''' in 2005 by the researchers from Tsinghua University <ref name="ref1" />. | |
==Annotated Information== | ==Annotated Information== | ||
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Department of Biological Sciences and Biotechnology, Tsinghua University | Department of Biological Sciences and Biotechnology, Tsinghua University | ||
− | ==References== | + | == References == |
− | Gong-Ke | + | <references> |
− | + | <ref name="ref1"> | |
− | + | Zhou, Gong-Ke, Yu-Feng Xu, and Jin-Yuan Liu. "Characterization of a rice class II metallothionein gene: tissue expression patterns and induction in response to abiotic factors." Journal of plant physiology 162.6 (2005): 686-696.</references> | |
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==Structured Information== | ==Structured Information== |
Revision as of 15:59, 23 June 2016
The rice Os10g0542100 was first identified as OsMT-II-1a in 2005 by the researchers from Tsinghua University [1].
Contents
Annotated Information
This genomic fragment encoding a characteristic metallothionein (MT) protein, and its full-length cDNA was isolated from rice developing seeds by RT-PCR. This cDNA, designated OsMT-II-1a, contains an open reading frame of 264 bp encoding a protein of 87 amino acid residues. The predicted amino acid sequence was shown to have structural features characteristic of plant class II MT proteins. Accumulation of OsMT-II-1a mRNA is specifically abundant in developing seeds and 2-day glumes after pollination, and OsMT-II-1a transcription can markedly be induced by H2O2, paraquat, SNP, ethephon, ABA and SA, but barely by metal ions or other exogenous abiotic factors such as low temperature and PEG. The processes of pollination and seed development might be mediated, at least in part, by expression of the OsMT-II-1a gene that is regulated by several abiotic factors.
Function
Plant MTs have been suggested to play important roles in maintaining the homoeostasis of essential transition metals, detoxification of toxic metals, and protecting against intercellular oxidative stress. Moreover, the diverse expression patterns of MT genes suggest that MT-like protein isoforms may differ in sequence, as well as in the functions that these perform in specific tissues. A number of investigations demonstrated these to have distinct but overlapping functions in the multigene family in single species. Recently, several studies indicated that plant MT-like proteins, especially the characterized plant class II MT-like proteins, are also involved in important developmental processes showed that the 50-flanking genomic DNA for the wheat EcMT gene contains a core sequence in the 50-flanking region known to be an ABA-responsive element (ABARE), but does not contain metal-responsive elements seen in animal MT genes. Consistent with this, accumulation of the EcMT mRNA is strongly induced during development of immature embryos but not during germination of mature wheat embryos unless ABA is added to the germination medium. Also, supplementation of the medium with zinc does not induce accumulation of EcMT mRNA in germination embryos. Accumulation of OsMT-II-1a mRNA is specifically abundant in developing seeds and 2-day glumes after pollination. However, embryogenesis is a complex process regulated by the developmental program and aimed at the formation of viable seeds able to germinate under appropriate environmental conditions. ABA and active oxygen species are postulated to play an important role in many processes of embryogenesis. Also, a hallmark of MT gene regulation is their inducibility by different endogenous and exogenous factors acting directly or indirectly on multiple cis-acting motifs in the regulatory regions of MT genes. Therefore, to investigate whether embryogenesis-related signal molecules (such as ABA and active oxygen species) or other exogenous factors (such as PEG, SA, ethylene, heat shock, low temperature, wounding and etiolation) are involved in the regulation of the OsMT-II-1a gene, rice seedlings treated with the above stress factors were harvested, and the transcriptional levels of OsMT-II-1a gene were determined by northern analysis. The effects of ABA, paraquat, SA and SNP were most dramatic in roots, whereas the transcript levels of OsMT-II-1a were low after treatments of H2O2, ethephon, heat shock and etiolation. In shoots, OsMT-II-1a mRNA levels were elevated markedly by treatments with H2O2, ethephon, ABA and SA, but were only slightly induced by treatments with SNP, paraquat, heat shock, low temperature and etiolation. Interestingly, the expression of OsMT-II-1a can be induced, markedly by embryogenesis-related signal molecules (e.g. H2O2 and ABA) or related reagents (e.g. SNP and paraquat), slightly by heat shock, low temperature and etiolation, but hardly by other exogenous factors (e.g. PEG and wounding). These results suggest that the OsMT-II-1a protein may play a role during embryogenesis, but not in response to environmental stresses. On the basis of the above results and previous studies, we further propose that the OsMT-II-1a protein not only shares similar roles with wheat EcMT protein in storing metal ions that are required during germination, but might also be involved in providing metal ions to maturing pollen grains since some metals, such as Cu, Zn and Fe, are essential micronutrient elements required for a variety of processes in cellular metabolism and serve as structural and catalytic components of proteins and enzymes. The latter proposal is further supported by results showing that the transcript abundance of OsMT-II-1a can be detected by treatment with ethephon, but it is barely detectable in the rachises, stems, sheaths, leaves and roots. It should be noted that ethylene can also modulate the expression of OsMT-II-1a. What is the relationship between metal ions, OsMT-II-1a expression and ethylene during embryogenesis? Results from recent studies indicated that metal ions are involved in ethylene perception and signal transduction by binding to the receptor. In our opinion, it is possible that application of exogenous ethephon may result in an increased usage of metal ions, thereby lowering the cellular metal ion pool. This in turn increases OsMT-II-1a expression and enhances the transport of metal ions that is required during germination and maturation of pollen grains. On the other hand, ethylene can enhance the production of active oxygen species, which in turn can induce the OsMT-II-1a gene expression. Whether ethylene modulates the expression of OsMT-II-1a alone or in conjunction with other signaling molecules such as active oxygen species remains to be further investigated. Furthermore, a variety of oxidants such as H2O2, SNP and paraquat are involved in the regulation of MT gene expression by inducing disulfide formation. Comparison with the presence of putative ARE in the 50-flanking region raises the question of whether oxidants induce the expression of OsMT-II-1a by oxidizing MT cysteines, by ARE, or by both of them together. Further analysis is required to evaluate the relationship between oxidants and OsMT-II-1a expression. Additionally, SA treatments can markedly enhance H2O2 production, which suggests that SA-mediated H2O2 production may result in OsMT-II-1a expression. Previous studies indicated that accumulation of EcMT mRNA can be induced strongly by ABA. Our results presented here are in line with their findings, showing that in both roots and shoots, the transcription of OsMT-II-1a gene is markedly induced by ABA. Taken together and comparing with the presence of two putative ABARE (ACGTGCCC), one putative ARE (GCCAAGTCACC) and one putative GCC-box (GCCGCC) found in many pathogen-responsive genes as ethylene-responsive elements in the 50-flanking region of OsMT-II-1a gene, it might be probable that the predicted cis-elements may be involved in the regulation of OsMT-II-1a expression. On the other hand, metal ions have been shown to up-regulate class I MT-like gene expression in plants, and it has also been observed that wheat EcMT protein provides a mechanism for storing zinc that is required during germination. We are interested in whether the expression of class II MT gene is affected by metal ions. To test the effects of metal ions on the gene, OsMT-II-1a transcript accumulation was investigated in shoots of 10-day-old rice seedling exposed to different metal ions at different concentrations. six metal ions, including Fe, Cu, Al, Pb, Cd and Zn, failed to induce distinctly the accumulation of OsMT-II-1a mRNA in shoots, as well as in roots (data not shown). These results coincide with the prediction of existing regulatory cis-elements in its 50-flanking region, indicating that the expression of OsMT-II-1a might not be appreciably induced by metal ions, which is different from class I MT-like genes. On the basis of the above results, it would appear that the involvement of OsMT-II-1a protein in a general process of metal detoxification or tolerance in developing seeds and 2-day glumes after pollination cannot be attributed, unlike Arabidopsis metallothioneins with the function of metal detoxification, since it is more probable that this kind of mechanism would be accomplished in other parts of rice (e.g. in the leaves or roots) treated with metal ions. In this study, we described and characterized a rice class II MT-like gene OsMT-II-1a; its putative regulatory elements may be present to support its roles in transcriptional regulation processing. Northern blot analysis clearly showed that accumulation of OsMT-II-1a mRNA may occur at specific stages of development (e.g. pollination), in specific tissues (e.g. developing seeds) and under treatments with embryogenesis-related signal molecules (e.g. ABA and H2O2, etc.). These results suggest that the processes of pollination and seed development might be mediated, at least in part, by the expression of the OsMT-II-1a gene that is regulated by ABA and H2O2, etc. Therefore, our results here will provide a framework for continued studies on the transduction pathway linking the environmental signals known to affect pollen embryogenesis, the gene regulated by these signals, and the developmental response of pollination and seed development. It is expected that this work will shed some light on the comprehension of the physiological role of class II MT in plants.
Expression
Reynolds and Crawford further showed that ABA biosynthesis is accompanied by increased expression of the EcMT gene transcript concommitant with the differentiation of pollen embryoids in wheat anther cultures, and suggested that the EcMT gene plays an important role in pollen embryogenesis. To date, however, it is not known if different class II MT genes have specific functions in different organs, or at different developmental stages besides embryogenesis. Also their response to the different exogenous factors except zinc remain unclear. Therefore, we were very interested in the relationship between expression of plant class II MT genes and various developmental or environmental signals. The expression of class I MT-like genes has been characterized in many kinds of tissues. These reports suggest that class I MT-like genes are different in structure and are likely to play diverse roles and functions in plants in order to cope with complex developmental and environmental cues. To date, however, the expression pattern of class II MT genes has only been reported during wheat embryogenesis and in the developing seeds of Arabidopsis. To investigate the expression pattern of the OsMTII- 1a gene in different organs and at different developmental stages, a more detailed analysis of OsMT-II-1a mRNA accumulation in mature plants was carried out. Total RNA from tissues of roots, stems, rachises, glumes before pollination, 2-day glumes after pollination, developing seeds, young leaves, mature leaves, old leaves, young sheaths, mature sheaths and old sheaths were subjected to Northern blot analysis. Our results here show that the transcripts of OsMT-II-1a were specifically abundant not only in developing seeds but also in glumes, whereas the highest expression was detected only in 2-day glumes after pollination, in which levels were 2 times higher than in developing seeds. In addition, the hybridization signal was barely detected in roots, old leaves, mature sheaths and old sheaths, but a very weak hybridization signal was detected in glumes before pollination, young leaves, mature leaves, old leaves and young sheaths. These results further support the fact that expression of OsMT-II-1a is only restricted to a particular developmental stage (e.g. pollination) and specific tissue (e.g. developing seeds). Taken together, the special expression pattern of OsMT-II-1a is consistent with the presence of the predicted embryogenesis-related cis-elements in the 50-flanking region of OsMT-II-1a, which suggests that these cis-elements might be involved in the regulation of OsMT-II-1a during pollination and seed development.
Evolution
The coding region of OsMT-II-1a, when translated, is demonstrated to contain 17 cysteine residues arranged into three groups of 6, 6 and 5 cysteines, which are separated by two interdomain regions of 13 and 15 cysteine-free amino acid residues, respectively. The abundance (about 15%) and the distribution of cysteines in OsMT-II-1a were shown to have structural features characteristic of the class II MT-like proteins. An alignment of the deduced OsMT-II-1a protein with all known class II MT-like proteins is shown in Fig. It has high homology with the plant class II MT-like proteins, with the overall sequence similarity varying substantially from 82% to 49%. We discovered that, unlike class I MT-like genes with several members, the members of class II MT-like genes are no more than 2 in a single species, and the arrangement patterns of cysteines are the same in each member. These results imply that this class of proteins might be conserved in structure and may play a special role in plants. Compared to those proteins from dicots, class II proteins from monocots lack 8–13 amino acids in the N-terminal domain before the first cysteine residue, but have a few additional amino acids in the C-terminal domain, thus maintaining the similar size of class II proteins between monocots and dicots. A phylogenetic tree of all known plant class II MT-like proteins was constructed, which showed two distinct groups corresponding to monocots and dicots. Thus, the differences in the structures of class II MT-like proteins may suggest specific functions for monocots and dicots. Several DNA motifs were identified in the promoter of OsMT-II-1a that are homologous to various previously reported regulated elements, which might be important in the transcriptional regulation of this gene. Two putative ABA responsive elements (ABARE) were found (one in a forward orientation and the other in reverse), which were also found in the wheat Em gene and rice rab21 gene. One putative ethylene-responsive element identified was the GCC box, GCCGCC, which was found in many ethylene inducible pathogenesis-related genes. An antioxidant response element (ARE), four low-temperature responsive elements (LTRE), four ABARE-like sequences and four CCAAT boxes were also found in the promoter of OsMT-II-1a. In a word, the presence of these homologous sequences may be related with the effects of various stress treatments on the expression of OsMT-II-1a. Also, unlike animal and most plant class I MT genes containing the metal-responsive element TGCRCNC (in which N is not A, and R is A or G) and/or copper-responsive element CTGCCA, the promoter of OsMT-II-1a did not contain any known metal-responsive elements or metal regulatory motifs. These findings suggested that the expression of OsMT-II-1a gene might not be modulated by metal ions. Additionally, we also found many cis-elements related with embryo-, pollen- and endosperm-specific gene transcription such as legumin box, ACGT motif, AGAAA motif and (CA)n element. These data hint that OsMT-II-1a protein might play some important role during embryogenesis. In addition, one 86-bp length intron divided the coding sequence of OsMT-II- 1a into two fragments with sizes of 59 and 205 bp. The sequences bordering the inton/exon conform to the GT/AG but not AT/AC rule for splice junctions, which is consistent with other MT-like gene sequences in rice (personal communication).
Labs working on this gene
Laboratory of Molecular Biology and MOE Laboratory of Protein Science; Department of Biological Sciences and Biotechnology, Tsinghua University
References
- ↑ Cite error: Invalid
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