A novel PLS-DYW type PPR protein OsASL is essential for chloroplast development in rice.

Oryza longistaminata (OL), an AA-genome African wild rice which can propagate clonally via rhizome, is an important germplasm for improvement of Asian cultivated rice, however recessive lethal alleles can hitchhike clonal propagation in heterozygous state. Selfing of OL is difficult due to its self-incompatibility, but simple selfing of hybrid progeny between OL and O. sativa is effective to disclose and eliminate recessive lethal alleles. Here, we identified an exhibited albino-lethal phenotype mutant, from an F2 population between OL and O. sativa, named it albino seedling-lethal (asl). The leaves of asl mutant showed abnormal chloroplast development. The albino characteristics of asl were determined to be governed by a set of recessive nuclear genes through genetic analysis. Map-based cloning experiments found that a single nucleotide variation (G to A) was detected in the exon of OsASL in OL, which causes a premature stop codon. OsASL encodes a PLS-type PPR protein with 12 pentratricopeptide repeat domains, and is translocalized to chloroplasts. Complementation and knockout transgenic experiments further confirmed that OsASL is responsible for the albino-lethal phenotype. Loss-of-function OsASL (i.e. osasl) resulted in devoid of intron splicing of chloroplast RNA atpF, ndhA, rpl2 and rps12, and also RNA editing of ndhB, but facilitates the RNA editing of rpl2 in the plastid. Transcriptome sequencing showed that OsASL was mainly involved in chlorophyll synthesis pathway. The expression of Chlorophyll-associated genes were significantly decreased in asl plants, especially PEP (plastid-encoded RNA polymerase)-mediated genes. Our results suggest that OsASL is crucial for RNA editing, RNA splicing of chloroplast RNA group II genes, and plays an essential role in chloroplast development during early leaf development in rice.

Temperature Effect on Rhizome Development in Perennial rice.

Traditional agriculture is becoming increasingly not adapted to global climate change. Compared with annual rice, perennial rice has strong environmental adaptation and needs fewer natural resources and labor inputs. Rhizome, a kind of underground stem for rice to achieve perenniallity, can grow underground horizontally and then bend upward, developing into aerial stems. The temperature has a great influence on plant development. To date, the effect of temperature on rhizome development is still unknown. Fine temperature treatment of Oryza longistaminata (OL) proved that compared with higher temperatures (28-30 ℃), lower temperature (17-19 ℃) could promote the sprouting of axillary buds and enhance negative gravitropism of branches, resulting in shorter rhizomes. The upward growth of branches was earlier at low temperature than that at high temperature, leading to a high frequency of shorter rhizomes and smaller branch angles. Comparative transcriptome showed that plant hormones played an essential role in the response of OL to temperature. The expressions of ARF17, ARF25 and FucT were up-regulated at low temperature, resulting in prospectively asymmetric auxin distribution, which subsequently induced asymmetric expression of IAA20 and WOX11 between the upper and lower side of the rhizome, further leading to upward growth of the rhizome. Cytokinin and auxin are phytohormones that can promote and inhibit bud outgrowth, respectively. The auxin biosynthesis gene YUCCA1 and cytokinin oxidase/dehydrogenase gene CKX4 and CKX9 were up-regulated, while cytokinin biosynthesis gene IPT4 was down-regulated at high temperature. Moreover, the D3 and D14 in strigolactones pathways, negatively regulating bud outgrowth, were up-regulated at high temperature. These results indicated that cytokinin, auxins, and strigolactones jointly control bud outgrowth at different temperatures. Our research revealed that the outgrowth of axillary bud and the upward growth of OL rhizome were earlier at lower temperature, providing clues for understanding the rhizome growth habit under different temperatures, which would be helpful for cultivating perennial rice.

Elective nodal radiotherapy with a gapless radiation field junction for oligorecurrent prostate cancer after previous radiotherapy.

Purpose: To evaluate the feasibility of subsequent elective nodal radiotherapy (ENRT) for nodal recurrences after previous radiotherapy with a defined planning approach for a gapless radiation field junction. Methods: Patients with 1) previous radiotherapy of prostate or prostatic fossa and subsequent pelvic ENRT or 2) previous pelvic radiotherapy and subsequent ENRT to paraaortic lymph nodes (LN) and gapless junction of both radiation fields were analyzed. The cumulative maximum dose (Dmax-cum) and the maximum cumulative dose in 1 cc (D1cc-cum) were estimated. Absolute toxicity and the toxicity exceeding baseline were evaluated. Results: Twenty-two patients with PSMA-PET/CT-staged nodal oligorecurrence after prior radiotherapy were treated with pelvic (14 patients) or paraaortic ENRT (9 patients). One patient was treated sequentially at both locations. Median time between first and second RT was 20.2 months. Median doses to the lymphatic pathways and to PET-positive LN were 47.5 Gy and 64.8 Gy, respectively. The planning constraint of an estimated Dmax-cum ≤ 95 Gy and of D1cc-cum < 90 Gy were achieved in 23/23 cases and 22/23 cases, respectively. Median follow-up was 33.5 months. There was no additional acute or late toxicity ≥ grade 3. Worst acute toxicity exceeding baseline was grade 1 in 68.2% and grade 2 in 22.7% of patients. Worst late toxicity exceeding baseline was grade 1 in 31.8% and grade 2 in 18.2% of patients. Conclusion: ENRT for nodal recurrences after a previous radiotherapy with gapless junction of radiation fields seems to be feasible, applying the dose constraints Dmax-cum ≤ 95 Gy and D1cc-cum < 90 Gy without grade 3 acute or late toxicities exceeding baseline.

Sucrose Facilitates Rhizome Development of Perennial Rice (Oryza longistaminata).

Compared with annual crops, perennial crops with longer growing seasons and deeper root systems can fix more sunlight energy, and have advantages in reducing soil erosion and saving water, fertilizer and pesticide inputs. Rice is one of the most important food crops in the world. Perennial rice can be of great significance for protecting the ecological environment and coping with the shortage of young farmers due to urbanization. Oryza longistaminata (OL) is a rhizomatous wild rice with an AA genome and has strong biotic and abiotic resistances. The AA genome makes OL easy to cross with cultivated rice, thus making it an ideal donor material for perennial rice breeding. Sucrose plays an important role in the development and growth of plants. In this study, OL seedlings were cultured in medium with different concentrations of sucrose, and it was found that sucrose of appropriate concentrations can promote the sprout of basal axillary buds and the subsequent development of rhizomes. In order to explore the molecular mechanism, comparative transcriptome analysis was carried out with OL cultured under two concentrations of sucrose, 20 g/L and 100 g/L, respectively. The results showed that the boost of sucrose to rhizome elongation may be due to the glucose and fructose, hydrolyzed from the absorbed sucrose by vacuolar acid invertase. In addition, the consequent increased osmotic pressure of the cells would promote water absorption, which is benefit for the cell elongation, eventually causing the rhizome elongation. These results may provide a reference for elucidating the regulatory mechanism of sucrose on the rhizome development of OL.

Genetic Analysis of S5-Interacting Genes Regulating Hybrid Sterility in Rice.

BACKGROUND: Asian cultivated rice (Oryza sativa L.) comprises two subspecies, O. sativa subsp. indica and subsp. japonica, and the hybrids between them display strong heterosis. However, hybrid sterility (HS) limits practical use of the heterosis between these two subspecies. S5 is a major-effect locus controlling the HS of female gametes in rice, consisting of three closely-linked genes ORF3, ORF4 and ORF5 that act as a killer-protector system. The HS effects of S5 are inconsistent for different genetic backgrounds, indicating the existence of interacting genes within the genome. RESULTS: In the present study, the S5-interacting genes (SIG) and their effects on HS were analyzed by studying the hybrid progeny between an indica rice, Dular (DL) and a japonica rice, BalillaORF5+ (BLORF5+), with a transgenic ORF5+ allele. Four interacting quantitative trait loci (QTL): qSIG3.1, qSIG3.2, qSIG6.1, and qSIG12.1, were genetically mapped. To analyze the effect of each interacting locus, four near-isogenic lines (NILs) were developed. The effect of each specific locus was investigated while the other three loci were kept DL homozygous (DL/DL). Of the four loci, qSIG3.1 was the SIG with the greatest effects in which the DL allele was completely dominant. Furthermore, the DL allele displayed incomplete dominance at qSIG3.2, qSIG6.1, and qSIG12.1. qSIG3.1 will be the first choice for further fine-mapping. CONCLUSIONS: Four S5-interacting QTL were identified by genetic mapping and the effect of each locus was analyzed using advanced backcrossed NILs. The present study will facilitate elucidation of the molecular mechanism of rice HS caused by S5. Additionally, it would provide the basis to explore the origin and differentiation of cultivated rice, having practical significance for inter-subspecific hybrid rice breeding programs.

Interactions Among Multiple Quantitative Trait Loci Underlie Rhizome Development of Perennial Rice.

Perennial crops have some advantages over annuals in soil erosion prevention, lower labor and water requirements, carbon sequestration, and maintenance of thriving soil ecosystems. Rhizome, a kind of root-like underground stem, is a critical component of perenniality, which allows many grass species to survive through harsh environment. Identification of rhizome-regulating genes will contribute to the development of perennial crops. There have been no reports on the cloning of such genes until now, which bring urgency for identification of genes controlling rhizomatousness. Using rhizomatous Oryza longistaminata and rhizome-free cultivated rice as male and female parents, respectively, genetic populations were developed to identify genes regulating rhizome. Both entire population genotyping and selective genotyping mapping methods were adopted to detect rhizome-regulating quantitative trait loci (QTL) in 4 years. Results showed that multiple genes regulated development of rhizomes, with over 10 loci related to rhizome growth. At last, five major-effect loci were identified including qRED1.2, qRED3.1, qRED3.3, qRED4.1, and qRED4.2. It has been found that the individual plant with well-developed rhizomes carried at least three major-effect loci and a certain number of minor-effect loci. Both major-effect and minor-effect loci worked together to control rhizome growth, while no one could work alone. These results will provide new understanding of genetic regulation on rhizome growth and reference to the subsequent gene isolation in rice. And the related research methods and results in this study will contribute to the research on rhizome of other species.

The underlying pathway involved in inter-subspecific hybrid male sterility in rice.

f5 locus in rice (Oryza sativa L.) confers significant effects on hybrid male sterility and segregation distortion. BC14F2 plants with f5-i/i, f5-j/j and f5-i/j genotypes were used to dissect the underlying pathway of f5-caused hybrid male sterility via comparative transcriptome analysis. A total of 350, 421, and 480 differentially expressed genes (DEGs) were identified from f5-i/j vs f5-j/j, f5-j/j vs f5-i/i, and f5-i/j vs f5-i/i, respectively. 145 DEGs were identified simultaneously in f5-i/j vs f5-j/j and f5-i/j vs f5-i/i. Enrichment analysis indicated that stress and cell control related processes were enriched. The expression of ascorbate peroxidase (APX) and most of the heat shock proteins (HSPs) were decreased, which might result in higher sensitivity to various stresses in pollen cells. A model was proposed to summarize the underlying process for f5-caused hybrid male sterility. These results would provide significant clues to further dissecting the molecular mechanism of f5-caused inter-subspecific reproductive isolation.

Genetic and molecular characterization of photoperiod and thermo-sensitive male sterility in rice.

KEY MESSAGE: A review on photoperiod and temperature-sensitive genic male sterility in rice. Male sterility in plants, facilitating the development of hybrid crops, has made great contribution to crop productivity worldwide. Environment-sensitive genic male sterility (EGMS), including photoperiod-sensitive genic male sterility (PGMS) and temperature-sensitive genic male sterility (TGMS), has provided a special class of germplasms for the breeding of "two-line" hybrids in several crops. In rice, the finding of the PGMS NK58S mutant in 1973 started the journey of research and breeding of two-line hybrids. Genetic and molecular characterization of these germplasms demonstrated diverse genes and molecular mechanisms of male sterility regulation. Two loci identified from NK58S, PMS1 and PMS3, both encode long noncoding RNAs. A major TGMS locus, TMS5, found in the TGMS line Annong S-1, encodes an RNase Z. A reverse PGMS mutant carbon starved anther encodes an R2R3 MYB transcription factor. Breeding efforts in the last three decades have resulted in hundreds of EGMS lines and two-line hybrids released to rice production, which have greatly elevated the yield potential and grain quality of rice varieties. The enhanced molecular understanding will offer new strategies for the development of EGMS lines thus further improving two-line hybrid breeding of rice as well as other crops.

PMS1T, producing phased small-interfering RNAs, regulates photoperiod-sensitive male sterility in rice.

Phased small-interfering RNAs (phasiRNAs) are a special class of small RNAs, which are generated in 21- or 24-nt intervals from transcripts of precursor RNAs. Although phasiRNAs have been found in a range of organisms, their biological functions in plants have yet to be uncovered. Here we show that phasiRNAs generated by the photopheriod-sensetive genic male sterility 1 (Pms1) locus were associated with photoperiod-sensitive male sterility (PSMS) in rice, a germplasm that started the two-line hybrid rice breeding. The Pms1 locus encodes a long-noncoding RNA PMS1T that was preferentially expressed in young panicles. PMS1T was targeted by miR2118 to produce 21-nt phasiRNAs that preferentially accumulated in the PSMS line under long-day conditions. A single nucleotide polymorphism in PMS1T nearby the miR2118 recognition site was critical for fertility change, likely leading to differential accumulation of the phasiRNAs. This result suggested possible roles of phasiRNAs in reproductive development of rice, demonstrating the potential importance of this RNA class as regulators in biological processes.