Photoperiod-sensitive Genic Male-sterile Rice Research Articles

Proteome alterations of reverse photoperiod-sensitive genic male sterile rice (Oryza sativa L.) at fertility transformation stage

The two-line system has been widely used in hybrid rice seed production, and the reverse photoperiod-sensitive genic male sterile line is a new germplasm with an opposite phenotype compared with normal photoperiod-sensitive genic male sterile rice. To better understand the molecular mechanisms of fertility regulation in reverse photoperiod-sensitive genic male sterile rice, a comparative proteomic approach was used to analyze the protein profiles of three different tissues (young panicles, flag leaves and leaf sheaths) of D52S during the sensitive period of pollen fertility transformation under sterile and fertile conditions. By quantitative analysis, 66 protein spots were identified to be significantly changed in the three tissues. Bioinformatics analyses revealed that in sterile rice, a number of proteins involved in lignin-flavonoid biosynthesis pathway were down-accumulated in panicles. The majority of proteins associated with energy metabolism were down-accumulated in leaf sheaths while the proteins up-accumulated in leaves and leaf sheaths were exclusively photosynthesis and defense related. Based on the proteomics data, a short-day induced male sterility protein network was proposed. In addition, the genes of selected protein spots were further analyzed by qPCR. These findings provide data for better understanding the regulation of pollen fertility in reverse photoperiod-sensitive genic male sterile rice, which could assist in the development of practical reverse photoperiod-sensitive genic male sterile rice for large-scale crop breeding programs.

Optimization of the Cry2Aa gene and development of insect-resistant and herbicide-tolerant photoperiod-sensitive genic male sterile rice

In this study, an optimized Cry2Aa gene was obtained after codon optimization based on the preferred codons in rice. The novel fusion gene Cry2Aa<sup>#</sup> was designed by adding the sequence coding the signal peptide of PR1a at the 5' end and the endoplasmic reticulum retention signal peptide KDEL at the 3' end of the optimized Cry2Aa gene, respectively. The Cry2Aa<sup>#</sup> and Bar genes were transformed into 4008S, a photoperiod-sensitive genic male sterile (PGMS) line in rice, using the Agrobacterium transfer method. A total of 65 regenerated plantlets confirmed by PCR were produced, in which eight transgenic lines had single-copy insertions as confirmed by Southern blot analysis. The high variability of Cry2Aa<sup># </sup>gene expression was observed among independent transgenic lines with single-copy insertion, and the spatiotemporal difference of Cry2Aa protein expression was discovered in each transgenic line. The results showed that the transgenic lines were highly resistant to glufosinate, rice leaf roller and striped stem borer, which not only confirmed the effective optimization of the Cry2Aa gene but also produced a useful germplasm for breeding insect-resistant and herbicide-tolerant hybrid rice varieties.

English

In photoperiod-sensitive genic male sterile rice (Oryza sativa L.) pollen abort in long day and high temperature, and revert to fertility in short day length and low temperature growth conditions. This type of control of rice plant fertility can facilitate production of hybrid rice using two lines (photoperiod-sensitive genic male sterile (PGMS) and restorer) system. Our objective in this study was to determine anatomical changes in PGMS rice pollen cells induced by long day length and high temperature growth conditions that are responsible for the cell abortion. All materials in this study were sown at Zhejiang University research field at Hangzhou, China 30°15’ N. Seeds of ZAU11S106, a PGMS rice line, were sown so as to obtain two bulks, 1) Extreme sterile pollen bulk and 2) fertile pollen bulk. Rice plants for extreme sterile pollen bulk were sown on March 18th and those for fertile pollen bulk on July 15th, so that they flowered under 13 h or more and under less than 13 h respectively of day light. Transmission electronic microscopes were used to study histological sections of anthers while light microscopes were used to examine whole pollen cells. The tapetal layers and the cytoplasm of the sectioned ZAU11S106 rice anthers from extreme sterile pollen bulk were deformed but those from fertile pollen bulk were normal when compared to the control rice lines ZAU11F121 and Xusui163. Pollen cells from anthers of extreme sterile pollen bulk had eroded exine and intine, missing or damaged nucleus, and disintegrating cytoplasm that lead to cell abortion. Our conclusion is that, cell abortion observed in extreme sterile pollen bulk of photoperiod genic male sterile rice, display “programmed cell death–like behavior”.   Key words: Pollen cell abortion, photoperiod genic male sterile rice, programmed cell death-like, tapetum.

Premature Tapetum Degeneration: a Major Cause of Abortive Pollen Development in Photoperiod Sensitive Genic Male Sterility in Rice

Photoperiod-sensitive genic male-sterile (PSGMS) rice (Oryza sativa L.), a natural mutant found in the rice cultivar Nongken 58, is very useful for the development of hybrid rice cultivars. Despite its widespread use in breeding programs, the initial stage of the abortive development of PSGMS rice and the possible cytological mechanisms of pollen abortion have not been determined. In the present study, a systematic cytological comparison of the anther development of PSGMS rice with its normal fertile counterpart is conducted. The results show that pollen abortion in PSGMS rice first occurs before the pollen mother cell (PMC) stage, and continues during the entire process of pollen development until pollen degradation. The abortive process was closely associated with the abnormal behavior of the tapetum. Although tapetum degeneration in PSGMS rice initiates already at the PMC stage, it proceeds slowly and does not complete until the breakdown of the pollen. Such cytological observations were supported by the results of the TUNEL (TdT-mediated dUTP Nick End Labeling) assay, which detects DNA fragmentation resulting from programmed cell death (PCD), indicating that the premature tapetum degeneration is in the process of PCD.

Cytological study and PCD assay on pollen development of photoperiod sensitive genic male sterile rice

A systematic cytological comparison of the anther development of photoperiod sensitive genic male sterile (PSGMS) rice with its normal fertility counterpart was conducted. The results showed that pollen abortion in PSGMS rice occurred first no later than the pollen mother cell (PMC) stage and continued during the entire process of pollen development till pollen degradation. This abortive process was closely associated with the abnormal behavior of tapetum. Although tapetum degeneration in the PSGMS rice initiated as early as at the PMC stage, it proceeded slowly and did not complete until the breakdown of the pollen, in sharp contrast to the rapid disintegration of the tapetal layer during the late microspore to the bicellular pollen stage in the fertile rice. Such cytological observation was supported by the results of the TUNEL (TdT2 mediated dU TP Nick End Labeling) assay that detectsDNA fragmentation resulting from programmed cell death (PCD), indicating that the tapetum degeneration occurs in the process of PCD.

Daylength responses of various morphogenetic events after panicle initiation in late japonica rice (Oryza sativa L. ssp. japonica)

Plant morphogenesis is an intimate cooperation between environmental signals and a genetically-encoded developmental programme. Although all stages subsequent to floral induction may be influenced by daylength, the information available has come from diverse species during different morphogenetic events. This makes it difficult to investigate how photoperiodic signalling is integrated into various steps of the developmental programme. Here, we report that, in late japonica rice lines, morphogenetic events, including 12th leaf, axis of the main panicle, awn of the glum, anthers, elongation rate of internodes, and pollen fertility of the photoperiod-sensitive genic male-sterile (PGMS) rice, are significantly affected by daylength after panicle initiation. These data indicate that daylength affects many morphologic events, and that changes in morphology are mainly determined by the characteristics of the events themselves. These findings lay a foundation for future investigations into how potentially common photoperiodic signalling system(s) are integrated with diversified developmental events. In addition, we discussed the essential nature of PGMS rice.

Structural analysis and identification of cis-elements of rice osRACD gene.

The osRACD gene correlated with fertility transformation in the photoperiod sensitive genic male sterile rice (PGMR), Nongken 58S, encoded a rice (Oryza sativa L. ssp. japonica) small GTPase belonging to the Rac/Rho family. Inverse PCR was performed to amplify a fragment about 1.4 kb in 5' upstream region of the osRACD promoter. Deletion mutation and gel mobility shift assay characterized two fragments (-799 to -686 nt, and -686 to -431 nt) in the osRACD promoter that could be involved in its transcriptional regulation. When these two deletion fragments were used as probe respectively, a retarded band appeared in the nuclear extracts of fertile 58S rice under short day (58S-SD). Whereas no retarded band was shown in the nuclear extracts of sterile 58S rice under long day (58S-LD). Competition assay indicated that the factors in the retarded bands binding to these two fragments were the same trans-acting factor (termed rice factor, RF). The binding affinity of RF was affected by phosphorylation and was higher in SD-growth rice than that of LD-growth rice.

Identification of an 85-kb DNA fragment containing pms1, a locus for photoperiod-sensitive genic male sterility in rice.

Photoperiod-sensitive genic male-sterile rice has a number of desirable characteristics for hybrid rice production. Previous studies identified pms1, located on chromosome 7, as a major locus for photoperiod-sensitive genic male sterility. The objective of this study was to localize the pms1 locus to a specific DNA fragment by genetic and physical mapping. Using 240 highly sterile individuals and a random sample of 599 individuals from an F2 population of over 5000 individuals from a cross between Minghui 63 and 32001S, we localized the pms1 locus by molecular marker analysis to a genetic interval of about 4 cM, 0.25 cM from RG477 on one side and 3.8 cM from R1807 on the other side. A contig map composed of seven BAC clones spanning approximate 500 kb in length was constructed for the pms1 region by screening a BAC library of Minghui 63 DNA using RFLP markers and chromosomal walking. Analysis of recombination events in the pms1 region among the highly sterile individuals reduced the length of the contig map to three BAC clones. Sequencing of one BAC clone, 2109, identified two SSR markers located 85 kb apart in the clone that flanked the pms1 locus on both sides, as indicated by the distribution of recombination events. We thus concluded that the pms1 locus was located on the fragment bounded by the two SSR markers.

Genetic bases of instability of male sterility and fertility reversibility in photoperiod-sensitive genic male-sterile rice

Photoperiod-sensitive genetic male-sterile (PSGMS) rice, with its male fertility regulated by photoperiod length, is very useful for hybrid rice development. However, breeding for new PSGMS lines has faced two major difficulties - the stability of male sterility and the reversibility of male fertility. In this study we assessed the genetic bases of stability of sterility and fertility reversibility using a molecular marker-based approach. A cross was made between two newly bred PSGMS lines: Peiai 64S, which has a stable sterility but is difficult to reverse to fertility, and 8902S, which has a unstable sterility but is easy to reverse to fertility. The fertility of the parents and of the F(1) and F(2) populations was repeatedly examined under 11 different long-day and short-day conditions. The genetic effects were assayed by interval mapping and two-way analyses of variance using the F(2) data of 128 polymorphic loci representing all the 12 rice chromosomes. The analyses resolved a number of single-locus QTLs and two-locus interactions under both long-day and short day conditions. The interactions involved a large number of loci, most of which were not detectable on a single-locus basis. The results showed that the genetic bases of both stability of sterility and reversibility of fertility are the joint effects of the additive effects of the QTLs and additive-by-additive components of two-locus interactions. The implications of these findings in hybrid rice development are also discussed.

Isolation of cDNA fragment of fertility-related gene in photoperiod-sensitive genic male sterile rice

The photoperiod-sensitive genic male sterile rice (PGMR) is particularly useful to take advantage of heterosis in rice. mRNA differential display was used to isolate the fertility-relative genes in rice. After establishing an optimized mRNA differential display system, one of the differential cDNA fragments that maybe related to the development and maturation of rice panicle was cloned from a PGMR Nongken 58s.

Effect of End-of-day far-red light exposures on fertility alteration and flowering in photoperiod-sensitive genic male-sterile rice

The rice photoperiod-sensitive genic male-sterile mutant (PGMR) is sterile under long days, but fertile in short days. Phytochrome is involved in the photoperiod-induced male-sterile process. To investigate the mechanisms, of phytochrome action in PGMR, end-of-day (EOD) experiments were carried out. Flowering in PGMR was delayed considerably by EOD far-red light exposures following a short day of 10 hr, whereas its fertility decreased to the same extent as the original line. This result suggests that photoperiod response mediating fertility alteration in PGMR somewhat differed from that in flowering,i.e., fertility alteration and flowering might be under the separate phytochrome signaling control.

Calcium distribution in fertile and sterile anthers of a photoperiod-sensitive genic male-sterile rice

Potassium antimonate was used to locate Ca2+ in fertile and sterile anthers of a photoperiod-sensitive genic male-sterile rice (Oryza sativa L. japonica). During the development of fertile anthers, abundant calcium precipitates accumulated in the anther walls and on the surface of pollen grains and Ubish bodies at the late developmental stage of the microspore, but not in the cytoplasm of pollen grains. Following the accumulation of starch grains in pollen, calcium precipitates on pollen walls diminished and increased in parenchymatous cells of the connective tissue. In sterile anthers, calcium precipitates were abundant in the middle layer and endothecium, but not in the tapetum, as was found in fertile anthers. A special cell wall was observed between the tapetum and middle layer of sterile anthers that appeared to relate to distinctive calcium accumulation patterns and poor pollen wall formation in the loculi. The formation of different patterns of antimonate-induced calcium precipitates in the anthers of photoperiod-sensitive genic male-sterile rice indicates that anomalies in the distribution of calcium accumulation correlate with the failure of pollen development and pollen abortion.

Characters of plants regenerated from protoplasts of photoperiod-sensitive genic male sterile rice

Characters of plants regenerated from protoplasts of photoperiod-sensitive genic male sterile rice

Conditions inducing fertility alteration and ecological adaptation of photoperiod-sensitive genic male-sterile rice

The pollen fertility of photo-sensitive genic male-sterile (PGMS) lines is regulated interactively by photoperiod and temperature. There is a temperature range within which the pollen fertility alteration of PGMS is regulated by photoperiod, being sterile at long days and fertile at short days. The critical values of day length and temperature vary as the PGMS gene(s) is (are) transferred into different genic backgrounds. According to the temperature reactions in response to fertility alterations, the current sterile lines fall into four photothermal types. The climatic adaptation of these is different. Lines of the high-low (a high critical fertile temperature with a low critical sterile temperature point) group have both stable sterile and fertile periods in the low-altitude subtropical rice-growing areas, which are the major rice-production areas of the world. We could exploit them in hybrid rice production there by a two-line method, because there is no need for both male-sterile and maintainer lines. At high altitudes of the subtropical and tropical rice-growing areas, lines of this group are unstable in their sterility. The low-low group is stable in sterility in long-day conditions at all temperatures, and at high temperatures despite day length. They could be used for hybrid seed production from the tropical through to temperate rice-growing conditions. However, their pollen fertility, and hence, seed multiplication, is easily lost in short days if high temperatures occur. The high-high group of sterile lines can be conveniently multiplied in short days, but their sterility is not stable in long days if temperature decreases in the critical fertility alteration period. The low-high group is stable at higher temperature irrespective of photoperiod, and this could be used in the tropics, but their sterilities are not stable in long days if lower temperatures are encountered. Their multiplication in short-day conditions may also be affected by high temperature.

Two Photoperiodic‐Reactions in Photoperiod‐Sensitive Genic Male‐Sterile Rice

Plant photoperiodism is an important principle explaining the growth and developmental processes of many plants, including rice (Oryza sativa L.). Most researchers currently believe that the effect of photoperiodism in rice plants is restricted to accelerating or delaying heading and flowering. This paper discusses a second effect of photoperiodism on certain lines of rice, the effect of inducing pollen sterility. It summarizes nearly 10 yr of study on photoperiod‐sensitive genic male‐sterile (PGMS) rice. There are two photoperiodic reactions existing simultaneously in the growth and development of PGMS rice. The first photoperiodic reaction (FPR) accelerates or delays both panicle differentiation and heading and is common to all rice lines. The second photoperiodic reaction (SPR) is novel and determines whether pollen will be sterile or fertile. Results show that FPR can be affected by broad changes in photoperiod, light intensity, and temperature and is controlled by one to two pairs of genes. On the other hand, the SPR requires a more critically timed photoperiod, higher light intensity, and higher temperature than does FPR. The SPR is also controlled by one or two pairs of genes and is inherited independently of the genes that modify the FPR. Several lines of PGMS rice have been produced with Nongken58S as a donor parent. Five growth and development phases comprise the life cycle of PGMS rice. The effects of environment on PGMS rice described in this review may guide the practice of breeding and use of PGMS rice in a newly‐developed two‐line production system for hybrid rice.