Os10g0561400

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Annotated Information

OsMYBS3 is a single DNA-binding repeat MYB transcription factor to mediate sugar signaling and plays a critical role in cold adaptation in rice.

Function

OsMYBS3 acts as a repressor, in sugar starvation–induced α-amylase gene expression. As a repressor, whether OsMYBS3 plays a negative role in the sugar regulation ofα-amylase gene expression is not clear. OsMYBS3 is most abundant in senescent leaves, in which sugars are depleted as a result of their transport to other tissues. OsMYBS3 may preventα-amylase genes from being induced by the low sugar level in senescent leaves, which would be a wasteful process in dying cells . Yeast one-hybrid experiments demonstrated that OsMYBS1 and OsMYBS2 bind to the TATCCA element and transactivate a promoter containing the TATCCA element in vivo. Transient expression assays with barley half-seeds showed that OsMYBS1 and OsMYBS2 transactivate a promoter containing the TATCCA element when sugar is provided, whereas OsMYBS3 represses transcription of the same promoter under sugar starvation. Transient expression assays also showed that these three OsMYBSs cooperate with a GA-regulated transcription factor, HvMYBGa, in the transactivation of a low-pI barley α-amylase gene promoter in the absence of GA.

Expression

The open reading frames of OsMYBS1, OsMYBS2, and OsMYBS3 encode polypeptides of 306, 276, and 318 amino acid residues, respectively. The three OsMYBSs contain a highly conserved single DNA binding domain that is very similar to the DNA binding domains of other mammalian, Drosophila, and plant. OsMYBS3 is expressed in all tissues, with the highest level in senescent leaves. The levels of OsMYBS3 mRNAs are low in the presence of Suc but increase in the absence of Suc which were parallel to the mRNA levels of α-Amy3 and another rice α-amylase gene, α-Amy8. OsMYBS3 is suppressed in the presence of Glc or GA but remains unchanged in the presence of abscisic acid. OsMYBS3 doesn't significantly enhance luciferase activity in the presence of Glc and repressed luciferase activity in the absence of Glc. OsMYBS3 may serve as a repressor, for transcription of the promoter containing SRS . OsMYBS3 in rice are enhanced by ABA and abiotic stresses and cold stress conditions , respectively.OsMYBS3 transgenic rice plants acquire cold tolerance with no penalty in terms of yield under normal field conditions. In addition, OsMYBS3 in rice regulates the expression of cold-related genes such as WRKY77 and Glu decarboxylase, both of which contain a TATCCA element in their promoters .The barley half-seeds were cobombarded with the effector and reporter plasmids. The bombarded half-seeds were divided into two halves, each half was incubated with 0.3 M Glc or 0.3 M mannitol for 24 h, and luciferase activity was determined. Figure 9B shows that OsMYBS1 significantly enhanced luciferase activity in the presence of Glc to the same level as in the absence of Glc. Although OsMYBS2 and OsMYBS3 seemed to enhance luciferase expression slightly in the presence of Glc, they significantly suppressed luciferase activity in the absence of Glc. These results indicate that OsMYBS1 activates, whereas OsMYBS2 and OsMYBS3 repress, the transcription of a promoter containing only the TATCCA element and adjacent flanking sequences.

Evolution

OsMYBS3 is rich in both Pro (19 of 166 amino acids, or 11%) and acidic amino acids (22 of 166 amino acids, or 13%). Some 1R-Myb proteins, including CCA1, OsMYBS3, and PSR1, are 30 to 40% identical in the Myb domain with each other and possess a SHAQK(Y/F)F motif. It is not clear why OsMYBS2 and OsMYBS3 do not form homodimers by themselves and why the three Os-MYBSs do not form heterodimers with each other . Barley HvMYBS3, rice OsMYBS3 and maize ZmMYBSt1 are probably orthologous, because, in addition to the 100% identity of their DBD, they share 84.6% and 80.9% identical residues, respectively, along their whole protein sequences (WPS) . Sequence comparison of single MYB-like domain TFs, MYB-like domain proteins and StMYB1R-1. The 1R sequence of OsMYBS3 is most homologous with that of StMYB1 (92% identity) and is somewhat homologous with that of OsMYBS1 (87% identity) and OsMYBS2 (85% identity), and the 1R sequences of OsMYBS1 and OsMYBS2 are least homologous (77% identity) with each other. There is very low homology among the N- and C-terminal regions outside of the 1R regions of all of the MYBs with 1R DNA binding domains, except that OsMYBS3 and StMYB1 have 71% identity at the N-terminal 90–amino acid region and 62% identity at the C-terminal 70–amino acid region.

Knowledge Extension

To determine whether OsMYBS gene expression was regulated by sugars, total RNA was isolated from rice suspension cells cultured in the presence or absence of Suc for 48h and subjected to gel blot analysis using OsMYBS-specific DNAs as probes. Figure 2 shows that the levels of OsMYBS1 and OsMYBS3 mRNAs were low in the presence of Suc (lanes 1 to 8) but increased in the absence of Suc (lanes 9 to 16), which were parallel to the mRNA levels of α-Amy3 and another rice α-amylase gene, α-Amy8. By contrast, the levels of OsMYBS2 were higher in the presence than in the absence of Suc. Transient expression assays with barley half-seeds were used as a system to study the function of OsMYBSs in sugar and hormone regulation in later experiments. To determine whether OsMYBSs are expressed in barley aleurone cells, total RNA was isolated from barley half-seeds and subjected to gel blot analysis using OsMYBS-specific DNAs as probes. Figure 3C shows that all of these OsMYBSs were expressed in barley aleurone cells in the absence of Glc. The expression levels of OsMYBS1 and OsMYBS3 were suppressed in the presence of Glc or GA but remained unchanged in the presence of abscisic acid. The expression of OsMYBS2 was enhanced in the presence of Glc and slightly suppressed by abscisic acid treatment. OsMYBSs Act Independently of or Cooperatively with Other Protein Factors for Promoter Transactivation MYB, with one repeat, may bind to DNA as a dimer, as has been proposed (Jin and Martin, 1999), although this has not been proved experimentally. In the present study, we showed that OsMYBS1 is capable of forming a homodimer in vivo. It is not clear why OsMYBS2 and OsMYBS3 do not form homodimers by themselves and why the three Os-MYBSs do not form heterodimers with each other. The differential ability of OsMYBS in dimerization may lead to differential transactivation ability. Both OsMYBS1 and OsMYBS2 transactivated the promoter containing an SRS in barley aleurone cells under repressed conditions; however, only OsMYBS1 transactivated the promoter containing the TATCCA element with adjacent flanking sequences (TATCCA+F) under repressed conditions. Furthermore, OsMYBS2 repressed the transcription of the promoter containing TATCCA+F even under derepressed conditions in a manner similar to OsMYBS3. These results suggest that additional sequences within SRS might be required for OsMYBS2 to function as a weak activator. GARC contains the GARE sequence and three to four additional cis-acting promoter elements, including the TATCCA element, that are necessary for the GA activation of gene expression. In the present study, a consensus GARE sequence was not found in the 1.1-kb rice αAmy3 promoter or in SRS, and the αAmy3 promoter–Luc or SRS–CaMV35S minimal promoter–Luc chimeric gene did not respond to GA in barley aleurone cells. The 331-bp barley Amy32b promoter contains a GARE sequence but does not contain a complete SRS-like sequence except for a putative G-box and the TATCCA element. The Amy32b promoter–Luc chimeric gene did not respond to sugar in barley aleurone cells. It appears that a complete sugar response complex or GARC sequence may determine whether an α-amylase gene promoter has the potential to respond to sugar or GA. OsMYBS1 and OsMYBS2 act as activators, whereas OsMYBS3 acts as a repressor, in sugar starvation–induced α-amylase gene expression; however, they all cooperate with HvMYBGa in high-level, GA-induced α-amylase gene expression. These studies suggest that OsMYBSs are multifunctional depending on the target promoter sequences and their interactions with other protein factors in response to different signals. The three OsMYBS genes are expressed in various tissues of the rice plant, indicating that they may play certain, not yet identified, physiological roles in these tissues.

References

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Labs working on this gene

• Engineering Research Center for Plant Biotechnology and Germplasm Utilization, Ministry of Education, State Key Laboratory of Hybrid Rice, College of Life Sciences,Wuhan University, Wuhan,China
• Institute of Crop Sciences/National Key Facility for Crop Gene Resources and Genetic Improvement, Chinese Academy of Agricultural Sciences, Beijing, China
• Key Laboratory of Plant Resources, Institute of Botany, The Chinese Academy of Sciences, Beijing 100093, PR China
• Bio-crop Development Division, National Academy of Agricultural Science, Rural Development Administration, Suwon 441–857, Republic of Korea
• Institute of Biotechnology, National Cheng Kung University, Tainan 701, Taiwan, Republic of China
• Department of Biology, Washington University, St. Louis, Missouri 63130
• Division of Integrated Life Science, Graduate School of Biostudies, Kyoto University, Oiwake-cho, Sakyo-ku, Kyoto, 606-8502, Japan