| Rice is a staple food crop in the world. Food shortage remains a severe threat due to decreasing farmland area and increasing population. Making use of greater heterosis between indica and japonica subspecies is difficult because of hybrid sterility. It has been a critical subject to study the wid compatibility genes and utilize the heterosis between indica and japonica. S5is a major locus regulating embryo-sac sterility in indica-japonica hybrids. Our lab cloned ORF5in S5locus in2008. And Yang Jiangyi proved that ORF5+acts as a killer, ORF4+acts as a partner, and ORF3+acts as a protector. However, functions and pathway of ORF3, ORF4and ORF5remain elusive.ORF3+and ORF3-both encode heat shock protein70proteins. Because of a13-bp deletion in ORF3-coding sequence, ORF3-has a shorter carboxyl-terminal sequence compared with ORF3+. ORF4+and ORF4-both encode unknown proteins. There is a transmembrane domain in ORF4+, but lacking in ORF4-due to its11-bp deletion in coding sequense. ORF5+and ORF5-encode aspartic proteases which are composed of signal peptides, propeptides and the Asp domains, whereas ORF5n in wide-compatibility varieties lacks the signal peptide and the propeptide.We transiently expressed green fluoresecnt protein (GFP) fusion proteins of ORF3+, ORF3-, ORF4+and ORF4-in tobacco and rice protoplasts, and proved that ORF3+, ORF3-and ORF4-are located to endoplasmic reticulum (ER), while ORF4+is located to plasma membrane and Golgi. We identified the subcellular localizations of ORF5+, ORF5-and ORF5n by immuno-labelling transmission electron microscope (TEM). The results showed that ORF5+and ORF5-are located to cell wall, while ORF5n is in cytoplasm. We confirmed the results in transgenic rice, and got the accordant conclusions.We studied the ORF5processing in ORF5-GFP and ORF5-c-myc transgenic BY-2cells. ORF5is in the conventional secretory pathway which is processed by ER, Golgi and trans-Golgi network (TGN) successively, and is secreted outside ultimately. We found that the signal peptide, the propeptide and another unknown peptide are processed, producing the mature form of ORF5with the molecular weight of about37kDa.In order to characterize ORF4+, we created ORF4+-GFP transgenic BY-2suspension cells and ORF4+-GFP transgenic rice. In BY-2cells,ORF4+-GFP is located to plasma membrane and Golgi. ORF4+-GFP-labeled organelles show aggregation in protein transport inhibitor brefeldin A (BFA) treatment, but insensitive to PI3K inhibitor wortmannin. ORF4+-GFP-labeled organelles are partially co-localized with the endocytic marker FM4-64, indicating that ORF4+is in the endocytic/recycling pathway. We got the accordant results in ORF4+-GFP transgenic rice. We tried to express ORF4+in Pichia pastors expression system, but failed to purify ORF4+due to low expression level.We observed the female gametogenesis of BL, BLORF5+and BL(BL/NJ). We found different patterns of embryo-sac sterility in BLORF5+and BL(BL/NJ). After meiosis, the nucellar cells show morphologic abnormity in BLORF5+, and degradation of nucellar cells and the functional megaspore are observed subsequently. Nucellar cells are all nobrmal in BL(BL/NJ). Differences between BLORF5+and BL(BL/NJ) demonstrate the effects of ORF3, ORF4and ORF5genotype in sporophyte and gametophyte.To study the expression profile of ORF3and ORF4, we analysed the microarray data and performed RNA in situ as well. The results showed similar expression profile between ORF4and ORF5. Both of them are specifically expressed in pistils, but not in anthers. ORF3is expressed in various tissues, but has highest expression signals in pistils and anthers.In order to investigate the molecular mechanism of abortion induced by ORF4+and ORF5+, and protect of ORF3+, we analysed the differentially expressed genes in BLORF5+, BL(NJ/DL)ORF4+and BL(NJ/NJ)ORF4+microarrays at the functional megaspore stage. The results showed that ORF4+and ORF5+induce differentially expression of ER stress and may cause programmed cell death (PCD). While under the protect of ORF3+, few genes in relation to ER stress or PCD are differentially expressed. We performed a TUNEL assay, and found anomalous PCD in nucellar cells and the founctional megaspore in BLORF5+.We observed callose deposition in ovulles of BLORF5+and BL, and found callose deposition in the cell walls of founctional megaspore and nucellar cells in BLORF5+, but not in BLIn conclusion, we propose and prove a killer-protector system at S5locus that regulates indica-japonica hybrid embryo-sac fertility. And we prove the pathway that ORF5+and ORF4+induce ER stress and PCD, and ORF3+prevent ER stress. However, more details are needed to understand the pathway. The results of our study are important to cultivate WCVs, and can greatly increase rice yields. |
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