Functional Studies Of Osj10gBTF3, OsCROC-1,and OsUbc13Differentially Expressed During Development Of Rice Somatic Shoot And Root

Osj10gBTF3, OsCROC-1and OsUbcl3are3out of nearly100somatic root and shoot development-associated genes that were determined by proteomic analysis previously carried out in our laboratory using in vitro root-and shoot-specific regeneration systems (Dr. Zhang xiaobo’s thesis,2010). The present study used these genes as investigation objects to go for expression analyses and functional confirmation based on the principle of reverse genetics. The protein clustering, subcellular localization and protein-protein interaction analysis were also carried out for the genes of Osj10gBTF3and OsUbcl3. The main results and conclusions obtained were as follows:(1) Osj10gBTF3is a β-subunit of nascent-polypeptide-associated complex (PNAC), and it contains a NAC domain, a putative mitochondrial matrix targeting sequence, a putative nuclear localization signal, and a putative ER-retention/retrieval signal. Phylogenetic analysis indicated that BTF3homologs from different species form three clades:animals, plants, and yeasts. Endogenous Osj10gBTF3was constitutively expressed in all the tissues examined such as root, stem, leaf, stamen, pistil, lemma, palea, with the highest level of abundance in the root. Osj10gBTF3can be generally modulated by salt, high temperature, and exogenous phytohormone stress. Osj10gBTF3:GFP was localized to both nucleus and cytoplasm, indicating that Osj10gBTF3is possibly associated with both cytosolic and nuclear functions. Transformants over-expressing Osj10gBTF3showed no obvious phenotypic differences from the wild type while those harboring RNAi construct of Osj10gBTF3leads to significant plant miniaturization and typical pollen abortion, indicating that Osj10gBTF3plays important role in rice plant growth and development;(2) The plant miniaturization caused by RNA interference of Osj10gBTF3were as follows:shortened length, narrowed width, tapered thickness, and/or reduced number of all observed tissues or organs, such as the crown and small lateral roots, culms or tiller stems, leaf sheath and blade, and panicles. The reduction rates on all these examined tissues or organs were about25-52%. Further microscope observation with the tranverse sections of the root, stem, and leaf revealed that all tissues from Osj10gBTF3Ri plants were much smaller or with fewer cells compared to the corresponding tissues from control plants. These differences were observed in the cortex, vascular bundle and xylem of root, vascular bundle, parenchyma, aerenchyma and vessel of stem, vascular bundle, aerenchyma, parenchyma between2aerenchymas, vessel, mesophyll in midrib, and lateral vein of the leaf. With respect to the longitudinal sections of root and stem, changes in cell size were more obvious. The reduction in cell size was similar to that observed for plant phenotypes, ranging from24%to68%. These results hence demonstrated that Osj10gBTF3-RNAi-induced plant miniaturization was accomplished via the reduction in cell size and number of different tissues or organs.(3) Further protein protein interaction analysis revealed that the typical pollen abortion caused by RNA interference of Osj10gBTF3was due to the altered expression of2Rf-like genes that encode pentatricopeptide repeat-containing proteins OsPPR676and OsPPR920, translational initiation factors OseIF3e and OseIF3h, and the heat shock protein OsHSP82, which have been verified by semi-quantitative and quantitative RT-PCR detection of transcripts of above-memtioned genes. The expression of OsPPR676, OsPPR920, OsHSP82and OseIF3h detected in Osj10gBTF3Ri plants were downregulated at different levels, in contrast, that of OseIF3e was upregulated, compared to the wild plant controls. What is more, OsHsp82, OsPPR920, OsPPR676,OseIF3e, OseIF3h and Osj10gBTF3were expressed in stamen of rice。(4). OsCROC-1and OsUbcl3:OsCROC-1encodes an ubiquitin-congugating enzyme (Ubc)-like protein (Ubc-like protein), which is also called ubiquitin-conjugating E2enzyme variant. Neither over-expression nor repression of OsCROC-1can caused obvious affection in the growth and development of rice. The seed germination and survival rate of either OsCROC-1OE or OsCROC-1Ri plants were still kept in a normal level even in the presence of MMS. However, silencing of ubiquitin-congugating enzyme OsUbcl3that is interacted with OsCROC-1caused leaf yellowing, sterile and death. The interaction between OsUbcl3and OsVDAC. OsB22EL8. OsMADS1which are associated with programmed cell death, elimination of free radical, development of floral organs was already verified by the yeast two-hybrid assay. In addition, this study also confirmed that OsUbcl3like Osj10gBTF3was constitutively expressed in all the tissues of rice, and can take the initiative in response to external stress.In conclusion, Osj10gBTF3and OsUbcl3are essential in the growth and development of rice. Once their expressions were repressed, rice plants displayed pleiotropic defects throughout the whole development process of rice. On the contrary, OsCROC-1could not exert obvious impacts on rice.