Comparative Fertility Proteome Analysis Of Photoperiod-sensitive Genie Male Sterile Rice And Short Photoperiod-sensitive Genie Male Sterile Rice

Hybrid rice has greatly contributed to the increase in rice productivity in China. The discovery and successful utilisation of Photoperiod/Thermo -sensitive genic male sterile lines has resulted in a two-line hybrid breeding system which has been used in large-scale crop breeding programs. Great achievements have been made in China in food security using two-line hybrid rice. Intensive studies of the Photoperiod/Thermo -sensitive genic male sterile lines established that its pollen fertility is regulated by day length/ temperature. For hybrid rice breeding, Photoperiod/Thermo -sensitive genic male sterile rice can be used to propagate itself under fertile conditions, and to produce hybrid seeds by interplanting it with normal fertile lines under sterile conditions. The mechanistic understanding of fertility transformation in Photoperiod/Thermo -sensitive genic male sterile lines could greatly facilitate the development of Photoperiod/Thermo -sensitive genic male sterile rice in two-line hybrid breeding programs. Photoperiod/Thermo-sensitive genic male sterile (sterile under long-day length and/or high temperature but are fertile under short-day length and/or low temperature) and reverse Photoperiod/Thermo-sensitive genic male sterile (fertile under long-day length and/or high temperature but are fertile under short-day length and/or low temperature) rice are two major types of environmentally sensitive genetic male sterile germplasm resources, which could serve as special material for the research of fertility transformation in Photoperiod/Thermo-sensitive genic male sterile rice.In the past forty years, considerable researches have been made in the studies of Photoperiod/Thermo -sensitive genic male sterile lines, including genetics, molecular biology, biochemistry and cell biology. However, the molecular mechanisms underlying fertility remain unclear. Short photoperiod-sensitive genic male sterile rice is a new germplasm, and few reports have been published concerning the molecular control of the photoperiod regulation of its fertility. In the current study, we investigated the proteomic alterations in photoperiod-sensitive genic male sterile rice NK58S and short photoperiod-sensitive genic male sterile rice D52S by systemically analysing the variations in protein patterns in panicles, flag leaves and leaf sheaths during the sensitive period of pollen fertility transformation under sterile and fertile conditions using a 2-DE coupled to MS, qPCR and Western Blot strategy. We hope that these results will contribute to a more comprehensive understanding of the mechanisms of fertility regulations in photoperiod-sensitive genic male sterile rice and short photoperiod-sensitive genic male sterile rice. The main obtained results are as follows:1. Pollen fertility and self-seed setting rate was examined at the critical stages for photoperiod induction after diverse photoperiodic cycles(14.5 h long-day; 10 h short-day) in D52S and NK58S at the same natural temperature conditions in July, Wuhan. Pollen abortion was induced in D52S under short-day conditions with nearly 100% sterility. In contrast, under long-day conditions, most of the pollen grains were fertile (89.06%), resulting in 54.84% self-seed setting rate. Pollen abortion was induced in NK58S under long-day conditions with nearly 100% sterility. In contrast, under short-day conditions, most of the pollen grains were fertile (84.68%), resulting in 25.43% self-seed setting rate. These results showed that the short photoperiod-sensitive genic male sterile rice D52S have an opposite phenotype in fertility transformation compared with normal photoperiod-sensitive genic male sterile rice NK58S.2. To identify the proteomic variation in the tissues of young panicles, flag leaves and leaf sheaths of D52S and NK58S under different day-lengths during the most sensitive phase of fertility transformation (stage 6), 2-DE coupled to MS strategy were obtained. The quantitative image analysis and MALDI-TOF/TOF-MS analysis revealed that fifty-three and forty protein spots from three tissues in D52S and NK58S were successfully identified, respectively. Thirteen protein spots were selected both in D52S and NK58S. The expression tendency of these thirteen protein spots in sterile and fertile plants was consistent both in D52S and NK58S. An investigation of the bioinformatics and functional annotations of the identified protein spots showed the ninety-three protein spots could be classified into 8 functional categories, including metabolism, secondary metabolism, energy associated, disease/defence, protein biosynthesis and processing, signal transduction, cell structure/growth and function unknown. Based on the proteomics data form three tissues, a possible fertility-alteration protein network induced by photoperiod-cycles in (reverse) photoperiod-sensitive genic male sterile rice was proposed with most of the protein spots that were identified.3. The expression patterns of flavonoid biosynthesis-related genes CHS,4CLL9 and carbohydrate metabolism-related genes UGPase, SUS1 were analysed using qPCR. Observations from mRNA levels of 4CLL9 and CHS in our study showed that their expression patterns were different between sterile and fertile plants both in D52S and NK58S, suggesting that these two proteins are intimately associated with fertility transformation.4. Western Blot was conducted to identify the expression patterns of CHS and 4CLL9 in young panicles under different day-lengths during the most sensitive phase of fertility transformation (stage 6). The results revealed that protein levels of CHS and 4CLL9 in fertile panicles under long-day conditions were significantly higher than those levels in sterile panicles under short-day conditions in D52S. The protein levels of 4CLL9 in fertile panicles under short-day conditions were also higher than those levels in sterile panicles under long-day conditions in NK58S. Comparative analysis of flavonoid contents at stage 6 and stage 7 in D52S and NK58S between sterile and fertile panicles revealed that flavonoid contents in sterile panicles were higher than those contents in fertile panicles both in D52S and NK58S at stage 6. When specific to stage 7, the flavonoid contents in fertile panicles were significantly higher than those contents in sterile panicles both in D52S and NK58S. Combined with the 2D, qPCR and Western Blot results, in fertile panicles, protein levels of CHS,4CLL9 may have the positive proportion with flavonoid contents, suggesting that the CHS and 4CLL9 were essential for fertility transformation.Our study provide a large number of differentially accumulated protein spots between sterile and fertile rice plants at the critical stages for photoperiod induction after diverse photoperiodic cycles in Photoperiod -sensitive genic male sterile rice and short Photoperiod -sensitive genic male sterile rice, which may facilitate further investigations concerning fertility transformation regulated by day-length.