Male Sterile Rice Research Articles

Genome-wide identification and biochemical characterization of glycoside hydrolase gene family members in Tilletia Horrida.

Rice kernel smut, caused by Tilletia horrida, is becoming an increasingly serious disease in hybrid rice planting, leading to production losses and quality decline of male-sterile rice varieties. Successful infection requires an efficient energy source that the pathogen obtains from rice plants, such as carbohydrates. Glycoside hydrolases (GHs), one of the largest sub-families in the cell wall-degrading enzyme family, play a key role in the infection progress of pathogens. To investigate their roles in facilitating infection, in this study, we identified and characterized genes encoding GH family proteins of T. horrida and further explored the functions and structures of these genes. Through genome-wide sequencing and bioinformatics analyses, 52 GH genes were identified from T. horrida, named ThGhd_1 to ThGhd_52. The subcellular location, conserved motifs, and structures of ThGhds were identified by bioinformatics analyses. Phylogenetic analysis revealed that ThGhds with similar domains clustered together, although some proteins clustered in different branches, which might reflect functional diversity. Protein-protein interaction network analysis revealed that ThGhds interact with partner proteins involved in reactive oxygen species signaling, protein kinase activity, and plant hormone signal transduction pathways. RNA-sequencing analysis showed that the expression of ThGhd genes responded differently at different infection time points, with dynamic changes detected during the T. horrida infection process, indicating that these genes are involved in interactions with rice and have potential roles in pathogenic mechanisms. The results of this study provide valuable resources for the structure elucidation of GH family proteins of T. horrida and can help to further elucidate their roles in pathogenesis.

A new gene for restoring wild abortive-type cytoplasmic male sterility in rice.

The WA-type CMS in rice is widely used in Asia for its stability and adaptability. Li Li's team found that the Rf20 gene enhances the fertility restoration of Rf4. It is recommended to introduce the H3 haplotype of the Rf20 gene into indica or Basmati rice, and high-temperature fertility issues in CMS-WA can be addressed through Rf20 gene editing.

The E3 ligase OsHel2 impedes readthrough of stalled mRNAs to regulate male fertility in thermo-sensitive genic male sterile rice.

Heterosis is extensively utilized in the two-line hybrid breeding system. Photo-/thermo-sensitive genic male sterile (P/TGMS) lines are key components of two-line hybrid rice. TGMS lines containing tms5 have significantly advanced two-line hybrid rice breeding. We cloned the TMS5 gene and found that TMS5 is a tRNA cyclic phosphatase that can remove 2',3'-cyclic phosphate (cP) from cP-ΔCCA-tRNAs for efficient repair to ensure maintenance of mature tRNA levels. tms5 mutation causes increased cP-ΔCCA-tRNAs and reduced mature tRNAs, leading to male sterility at the restrictive temperatures. However, the regulatory network of tms5-mediated TGMS remains to be elucidated. Here, we identified that an E3 ligase OsHel2 cooperates with TMS5 to regulate TGMS at the restrictive temperatures. Consistently, both the accumulation of 2',3'-cP-ΔCCA-tRNAs and insufficiency of mature tRNAs in tms5 mutant were largely recovered in the tms5 oshel2-1 mutant. A lesion in OsHel2 results in partial readthrough of the stalled sequences, thereby evading ribosome-associated protein quality control (RQC) surveillance. Our findings reveal a mechanism by which the OsHel2 impede readthrough of stalled mRNA sequences to regulate male fertility in TGMS rice, thus providing a paradigm for investigating how disorders in the components of the RQC pathway impair cellular functions and lead to diseases or defects in other organisms.

The Residual Activity of Fatty Acyl-CoA Reductase Underlies Thermo-Sensitive Genic Male Sterility in Rice.

Photoperiod/thermo-sensitive genic male sterility (P/TGMS) is critical for rice two-line hybrid system. Previous studies showed that slow development of pollen is a general mechanism for sterility-to-fertility conversion of TGMS in Arabidopsis. However, whether this mechanism still exists in rice is unknown. Here, we identified a novel rice TGMS line, ostms16, which exhibits abnormal pollen exine under high temperature and fertility restoration under low temperature. In mutant, a single base mutation of OsTMS16, a fatty acyl-CoA reductase (FAR), reduced its enzyme activity, leading to defective pollen wall. Under high temperature, the mOsTMS16M549I couldn't provide sufficient protection for the microspores. Under low temperature, the enzyme activity of mOsTMS16M549I is closer to that of OsTMS16, so that the imperfect exine could still protect microspore development. These results indicated whether the residual enzyme activity in mutant could meet the requirement in different temperature is a determinant factor for fertility conversion of P/TGMS lines. Additionally, we previously found that res2, the mutant of a polygalacturonase for tetrad pectin wall degradation, restored multiple TGMS lines in Arabidopsis. In this study, we proved that the osres2 in rice restored the fertility of ostms16, indicating the slow development is also suitable for the fertility restoration in rice.

Disruption of Energy Metabolism and Reactive Oxygen Species Homeostasis in Honglian Type-Cytoplasmic Male Sterility (HL-CMS) Rice Pollen

Honglian type-cytoplasmic male sterility (HL-CMS) is caused by the inter-communication between the nucleus and mitochondria. However, the mechanisms by which sterility genes regulate metabolic alterations and changes in mitochondrial morphology in the pollen of HL-CMS remains unclear. In this study, we compared the morphological differences between the pollen of the male sterile line YA and the near-isogenic line NIL-Rf6 using hematoxylin and eosin staining as well as 4ʹ,6-diamidino- 2-phenylindole (DAPI) staining. HL-CMS is characterized by gametophytic sterility, where the aborted pollen grains are empty and the tapetal layer remains intact. Transmission electron microscopy was employed to observe mitochondrial morphological changes at the microspore stage, revealing that significant mitochondrial alternations, characterized by the formation of 'large spherical mitochondria', occurred at the binucleate stage in the YA line. Additionally, metabolomics analysis revealed decreased levels of metabolites associated with the carbohydrate and flavonoid pathways. Notably, the decrease in flavonoids was found to contribute to an elevation in reactive oxygen species (ROS) levels. Therefore, we propose a model in which rice fertility is modulated by the levels of pollen carbohydrates and flavonoid metabolites, with impaired mitochondrial energy production and reduced flavonoid biosynthesis being the main causes of ROS accumulation and pollen abortion in rice.

Impaired 2',3'-cyclic phosphate tRNA repair causes thermo-sensitive genic male sterility in rice.

Hybrid rice, widely planted in Asia, is pathogen resistant and has superior yields, making it a major contributor to global food security. The two-line hybrid rice system, which utilizes mutants exhibiting photo-/thermo-sensitive genic male sterility (P/TGMS), is the leading hybrid rice breeding technology. Mutations in THERMO-SENSITIVE GENIC MALE STERILE 5 (TMS5) accounts for over 95% of current TGMS lines. We previously found that tms5 carries a mutation in ribonuclease ZS1. Despite its importance for breeding robust rice lines, the mechanism underlying tms5-mediated TGMS remains elusive. Here, we demonstrate that TMS5 is a tRNA 2',3'-cyclic phosphatase. The tms5 mutation leads to accumulation of 2',3'-cyclic phosphate (cP)-ΔCCA-tRNAs (tRNAs without 3'CCA ended with cP), which is exacerbated by high temperatures, and reduction in the abundance of mature tRNAs, particularly alanine tRNAs (tRNA-Alas). Overexpression of tRNA-Alas in the tms5 mutant restores male fertility to 70%. Remarkably, male fertility of tms5 mutant is completely restored at high temperatures by knocking out OsVms1 which encodes the enzyme for cP-ΔCCA-tRNA generation. Our study reveals the mechanism underlying tms5-mediated TGMS in rice and provides mechanistic insight into the further improvement of TGMS in hybrid crop development.

Temperature change regulates pollen fertility of a PTGMS rice line PA64S by modulating the ROS homeostasis and PCD within the tapetum.

Photoperiod and temperature-sensitive male sterility rice is an important line for two-line hybrid rice, and the changes in the cultivation temperature strictly control its pollen fertility. However, the mechanism by which temperature variation regulates pollen fertility is still unclear. This study obtained stable fertile PA64S(F) and sterile PA64S(S) rice from PA64S by controlling temperature changes. PA64S(F) shows a normal anther development and fertile pollen under low temperature (21°C), and PA64S(S) shows delayed degradation of the tapetum cells, leading to abnormal pollen wall formation and ubisch development under normal temperature (28°C). The accumulation of reactive oxygen species (ROS) positively correlates with the programmed cell death (PCD) process of tapetum cells. The delayed accumulation of ROS in the PA64S(S) tapetum at early stages leads to a delayed initiation of the PCD process. Importantly, we localized ascorbic acid (ASA) accumulation in the tapetum cells and determined that ASA is a major antioxidant for ROS homeostasis. ROS-inhibited accumulation plants (PA64S-ASA) demonstrated pollen sterility, higher ASA and lower ROS accumulation in the tapetum, and the absence of PCD processes in the tapetum cell. Abnormal changes in the tapetum of PA64S(S) rice disrupted metabolic pathways such as lipid metabolism, cutin and wax synthesis, sugar accumulation, and phenylpropane, affecting pollen wall formation and substance accumulation, suggesting that the timely accumulation of ROS is critical for male fertility. This study highlights the central role of ROS homeostasis in fertility alteration and also provides an avenue to address the effect of environmental temperature changes on pollen fertility in rice.

The agronomic characteristics and combining ability of glutinous Thermo-sensitive Genic Male Sterile rice (Oryza sativa) lines

Glutinous rice (Oryza sativa), despite its unique culinary qualities, typically yields less than other rice varieties, posing agricultural and economic challenges. The construction of a glutinous thermo-sensitive genic male sterile (TGMS) rice variety could indeed be a way to improve the yield and cultivation efficiency of glutinous rice. Among them, the two-line system operates by crossing a TGMS line, which is sterile at high temperatures but fertile at lower temperatures, with a normal fertile line. In this study, we conducted to access morphological characteristics and combining ability of some glutinous TGMS lines. The parental lines were characterized on 13 agronomic traits including growth duration, plant height, flag leaf width, flag leaf length, panicle length, rachis length, number of primary branches, number of secondary branches, number of panicles per plant, percentage of filled grains, P1000 seeds, grain yield, and amylose content. The obtained results revealed significant variations in growth traits, yield, and amylose content of studied rice lines. Hierarchical clustering based on principal component analysis of rice lines on all measured traits showed three major clusters with high genetic diversity. Furthermore, fifteen glutinous TGMS rice lines and three testers were used in a Line x Tester mating system to produce 45 hybrids. The 45 hybrids were evaluated on 6 traits related to grain yield and morphology in a randomized complete block design experiment with three replications. A line x tester analysis was conducted to estimate the combining ability, genetic variance, and the contribution of parental lines to genetic variation in hybrids. These findings were valuable for rice breeders to orient the strategy for breeding of hybrid glutinous rice varieties with high efficiency.

GLUTAMYL-tRNA SYNTHETASE 1 deficiency confers thermosensitive male sterility in rice by affecting reactive oxygen species homeostasis.

Temperature is one of the key environmental factors influencing crop fertility and yield. Understanding how plants sense and respond to temperature changes is, therefore, crucial for improving agricultural production. In this study, we characterized a temperature-sensitive male sterile mutant in rice (Oryza sativa), glutamyl-tRNA synthetase 1-2 (ers1-2), that shows reduced fertility at high temperatures and restored fertility at low temperatures. Mutation of ERS1 resulted in severely delayed pollen development and meiotic progression at high temperatures, eventually leading to male sterility. Moreover, meiosis-specific events, including synapsis and crossover formation, were also delayed in ers1-2 compared with the wild type. However, these defects were all mitigated by growing ers1-2 at low temperatures. Transcriptome analysis and measurement of ascorbate, glutathione, and hydrogen peroxide (H2O2) contents revealed that the delayed meiotic progression and male sterility in ers1-2 were strongly associated with changes in reactive oxygen species (ROS) homeostasis. At high temperatures, ers1-2 exhibited decreased accumulation of ROS scavengers and overaccumulation of ROS. In contrast, at low temperatures, the antioxidant system of ROS was more active, and ROS contents were lower. These data suggest that ROS homeostasis in ers1-2 is disrupted at high temperatures but restored at low temperatures. We speculate that ERS1 dysfunction leads to changes in ROS homeostasis under different conditions, resulting in delayed or rescued meiotic progression and thermosensitive male fertility. ers1-2 may hold great potential as a thermosensitive material for crop heterosis breeding.

Rapid generation of fragrant thermo-sensitive genic male sterile rice with enhanced disease resistance via CRISPR/Cas9.

The three, by mutagenesis produced genes OsPi21, OsXa5, and OsBADH2, generated novel lines exhibiting desired fragrance and improved resistance. Elite sterile lines are the basis for hybrid rice breeding, and rice quality and disease resistance become the focus of new sterile lines breeding. Since there are few sterile lines with fragrance and high resistance to blast and bacterial blight at the same time in hybrid rice production, we here integrated the simultaneous mutagenesis of three genes, OsPi21, OsXa5, and OsBADH2, into Zhi 5012S, an elite thermo-sensitive genic male sterile (TGMS) variety, using the CRISPR/Cas9 system, thus eventually generated novel sterile lines would exhibit desired popcorn-like fragrance and improved resistance to blast and bacterial blight but without a loss in major agricultural traits such as yield. Collectively, this study develops valuable germplasm resources for the development of two-line hybrid rice with disease resistance, which provides a way to rapid generation of novel TGMS lines with elite traits.

The vesicle trafficking gene, OsRab7, is critical for pollen development and male fertility in cytoplasmic male-sterility rice

Rice cytoplasmic male sterility (CMS) provides an exceptional model for studying genetic interaction within plant nuclei given its inheritable trait of non-functional male gametophyte. Gaining a comprehensive understanding of the genes and pathways associated with the CMS mechanism is imperative for improving the vigor of hybrid rice agronomically, such as its productivity. Here, we observed a significant decrease in the expression of a gene named OsRab7 in the anther of the CMS line (SJA) compared to the maintainer line (SJB). OsRab7 is responsible for vesicle trafficking and loss function of OsRab7 significantly reduced pollen fertility and setting rate relative to the wild type. Meanwhile, over-expression of OsRab7 enhanced pollen fertility in the SJA line while a decrease in its expression in the SJB line led to the reduced pollen fertility. Premature tapetum and abnormal development of microspores were observed in the rab7 mutant. The expression of critical genes involved in tapetum development (OsMYB103, OsPTC1, OsEAT1 and OsAP25) and pollen development (OsMSP1, OsDTM1 and OsC4) decreased significantly in the anther of rab7 mutant. Reduced activities of the pDR5::GUS marker in the young panicle and anther of the rab7 mutant were also observed. Furthermore, the mRNA levels of genes involved in auxin biosynthesis (YUCCAs), auxin transport (PINs), auxin response factors (ARFs), and members of the IAA family (IAAs) were all downregulated in the rab7 mutant, indicating its impact on auxin signaling and distribution. In summary, these findings underscore the importance of OsRab7 in rice pollen development and its potential link to cytoplasmic male sterility.

Heterosis for Resistance to Insect Herbivores in a 3-Line Hybrid Rice System.

Three-line hybrid rice is produced by crossing male sterile (A line) rice with a fertility-restorer (R line). Fertile lines (B lines) are also required to maintain A line seed for breeding programs. We used a range of hybrids and their parental lines to assess the frequency and nature of heterosis for resistance to the whitebacked planthopper (Sogatella furcifera), brown planthopper (Nilaparvata lugens) and yellow stemborer (Scirpophaga incertulas). Heterosis is defined as trait improvement above the average of the parental lines as a result of outbreeding. Based on the results from a greenhouse study that challenged hybrids and their parental lines with each herbivore species, we found that susceptibility to planthoppers was associated with one of the eight A lines tested, but resistance was improved by crossing with a relatively resistant restorer. Higher frequencies of heterosis for susceptibility in comparisons between hybrids and their B lines suggest that susceptibility was not related to the cytoplasmic genomes of the associated sterile A lines. Furthermore, because none of the parental lines possessed currently effective resistance genes, improved resistance against planthoppers was probably due to quantitative resistance. In a related field trial, hybrids had generally higher yields than their fertile parents and often produced larger grain; however, they were often more susceptible to stemborers, leaffolders (Cnaphalocrocis medinalis) and other caterpillars (Rivula atimeta). This was largely a consequence of hybrid heterosis for plant biomass and was strongly affected by crop duration. We make a series of recommendations to improve hybrid breeding to reduce the risks of herbivore damage.

MRNA cleavage by 21-nucleotide phasiRNAs determines temperature-sensitive male sterility in rice.

Temperature-sensitive male sterility is one of the core components for hybrid rice (Oryza sativa) breeding based on the 2-line system. We previously found that knockout of ARGONAUTE 1d (AGO1d) causes temperature-sensitive male sterility in rice by influencing phased small interfering RNA (phasiRNA) biogenesis and function. However, the specific phasiRNAs and their targets underlying the temperature-sensitive male sterility in the ago1d mutant remain unknown. Here, we demonstrate that the ago1d mutant displays normal female fertility but complete male sterility at low temperature. Through a multiomics analysis of small RNA (sRNA), degradome, and transcriptome, we found that 21-nt phasiRNAs account for the greatest proportion of the 21-nt sRNA species in rice anthers and are sensitive to low temperature and markedly downregulated in the ago1d mutant. Moreover, we found that 21-nt phasiRNAs are essential for the mRNA cleavage of a set of fertility- and cold tolerance-associated genes, such as Earlier Degraded Tapetum 1 (EDT1), Tapetum Degeneration Retardation (TDR), OsPCF5, and OsTCP21, directly or indirectly determined by AGO1d-mediated gene silencing. The loss of function of 21-nt phasiRNAs can result in upregulation of their targets and causes varying degrees of defects in male fertility and grain setting. Our results highlight the essential functions of 21-nt phasiRNAs in temperature-sensitive male sterility in rice and suggest their promising application in 2-line hybrid rice breeding in the future.

An epi-allele of SMS causes Sanming dominant genic male sterility in rice.

Male sterility is an important trait in rice for hybrid rice (Oryza sativa) breeding. However, the factors involved in dominant male sterility are largely unknown. Here, we identified a gene from Sanming dominant genic male sterile rice, named Sanming dominant male sterility (SMS), and reported that an epi-allele of this locus contributes to male sterility. Segregation analysis attributed dominant male sterility to a single locus, SMS, which we characterized using a male-sterile near isogenic line (NIL) of rice cultivar 93-11. The SMS locus was heterozygous in the male-sterile 93-11 NIL, containing an epi-allele identical to that in 93-11, and an epi-allele identical to that in rice cultivar Nipponbare, which we refer to as SMS9 and SMSN, respectively. SMS9 is silent and hyper-methylated, whereas SMSN is expressed and hypo-methylated in the 93-11 NIL. Overexpressing SMSN led to male sterility. Mutations in SMS rescued the male sterility of the 93-11 NIL. Interestingly, we observed the duplication of SMSN in Nipponbare, but did not observe the duplication of SMS9 in 93-11. Together, these findings suggest that the reduced methylation and enhanced expression of the SMSN epi-allele in the 93-11 NIL is responsible for its role in conferring dominant male sterility.

Estimation of the Thermosensitive Period of the Thermosensitive Genic Male Sterile Rice Line ‘Norin-PL12’

Estimation of the Thermosensitive Period of the Thermosensitive Genic Male Sterile Rice Line ‘Norin-PL12’

Anther-specific expression of OsRIP1 causes dominant male sterility in rice.

Anther-specific expression of OsRIP1 causes dominant male sterility in rice.

Spontaneous movement of a retrotransposon generated genic dominant male sterility providing a useful tool for rice breeding.

Male sterility in plants provides valuable breeding tools in germplasm innovation and hybrid crop production. However, genetic resources for dominant genic male sterility, which hold great promise to facilitate breeding processes, are extremely rare in natural germplasm. Here we characterized the Sanming Dominant Genic Male Sterility in rice and identified the gene SDGMS using a map-based cloning approach. We found that spontaneous movement of a 1978-bp long terminal repeat (LTR) retrotransposon into the promoter region of the SDGMS gene activates its expression in anther tapetum, which causes abnormal programmed cell death of tapetal cells resulting in dominant male sterility. SDGMS encodes a ribosome inactivating protein showing N-glycosidase activity. The activation of SDGMS triggers transcription reprogramming of genes responsive to biotic stress leading to a hypersensitive response which causes sterility. The results demonstrate that an ectopic gene activation by transposon movement can give birth to a novel trait which enriches phenotypic diversity with practical utility.

A review of rice male sterility types and their sterility mechanisms

Male sterility plays an important role in the utilization of heterosis in rice. The establishment of male sterile lines in rice is one of the key technologies in hybrid rice production systems. The currently widely used male sterile line breeding systems mainly include: three-line hybrid rice based on cytoplasmic male sterility, two-line hybrid rice based on environmental sensitive gene male sterility, and third-generation hybrid rice based on nuclear gene male sterility Seed production system. This study reviewed the types and mechanisms of male sterility in rice, and looked forward to the development direction of hybrid rice.

Genetic Variability of Floral and Agronomic Characteristics that Influence Outcrossing Rate Percentage of Cytoplasmic Male Sterile Rice

Developing high-yielding hybrid rice varieties is much needed to ensure food security to meet the requirement of the increasing population across the world. Cytoplasmic male genetic sterility system is a valuable technology for the exploitation of heterosis and production of high-yielding hybrid rice with better grain quality. Large-scale commercial exploitation of hybrid vigor in rice largely depends on the availability of good CMS (Cytoplasmic Male Sterile) lines. A total of eighteen traits of five CMS lines of rice were evaluated to study the variability and correlation between floral and other yield-contributing traits with outcrossing rate using Randomized Complete Block Design with three replications. High significant variation was observed among the genotypes for all the studied traits. High GCV (Genotypic Coefficient of Variance) and PCV (phenotypic Coefficient of Variance) coupled with high heritability and high genetic advance in percent of mean were observed for number of panicles per plant, filled grain, flag leaf breadth, stigma length and breadth, filament length and outcrossing rate which indicated the broad genetic base, less environmental influence, and these traits are under the control of additive gene action. character association and path analysis indicated that number of grains per panicle, filled grains per panicle, stigma length, and breadth possessed both positive significant correlation and positive direct effects on outcrossing rate therefore, selection for the traits mentioned above will certainly bring in the improvement of the outcrossing percentage of the lines, which in turn enhances the seed quantity of the hybrid.

Base Editing of EUI1 Improves the Elongation of the Uppermost Internode in Two-Line Male Sterile Rice Lines

The use of male sterile lines (MSLs) of rice is essential for heterosis utilization. However, MSLs have a common defect in the elongation of the uppermost internode, eventually leading to incomplete panicle exsertion, blocking pollination, and reducing the hybrid rice seed yield. Previously, the elongated uppermost internode 1 (EUI1) was identified as an active gibberellin-deactivating enzyme that plays a key role in panicle exsertion from the flag leaf sheath in rice (Oryza sativa L.). We used an adenine base editor to edit EUI1 and obtained two types of homozygous transgenic plants (eui1-1 and eui1-2). The transcription and translation levels of EUI1 in the two mutants were significantly lower than in the wild type, as was the oxidation activity of EUI1 to active gibberellins (GAs), which also decreased. The contents of the plant hormones GA1, GA3, and GA4 in eui1-1 (1.64, 1.55, and 0.92 ng/g) and eui1-2 (0.85, 0.64, and 0.65 ng/g) panicles were significantly higher than the wild type (0.70, 0.57, and 0.42 ng/g). The uppermost internode lengths of the mutant were 26.5 and 23.6 cm, which were significantly longer than that of the wild type (18.0 cm), and the cell lengths of the mutant were 161.10 and 157.19 μm, which were longer than that of the wild type (89.28 μm). Our results indicate that the adenine base editing system could increase the content of endogenous bioactive GAs in young panicles by fine-tuning EUI1 activity, reduce the defect of panicle enclosure in MSLs and increase the yield of hybrid rice seed production.