MORE FLORET1 controls anther development by negatively regulating key tapetal genes in both diploid and tetraploid rice.

Polyploid hybrid rice (Oryza sativa) has great potential for increasing yields. However, hybrid rice depends on male fertility and its regulation, which is less well studied in polyploid rice than in diploid rice. We previously identified an MYB transcription factor, MORE FLORET1 (MOF1), whose mutation causes male sterility in neo-tetraploid rice. MOF1 expression in anthers peaks at anther Stage 7 (S7) and progressively decreases to low levels at S10. However, it remains unclear how the dynamics of MOF1 expression contribute to male fertility. Here, we carefully examined anther development in both diploid and tetraploid mof1 rice mutants, as well as lines ectopically expressing MOF1 in a temporal manner. MOF1 mutations caused delayed degeneration of the tapetum and middle layer of anthers and aberrant pollen wall organization. Ectopic MOF1 expression at later stages of anther development led to retarded cytoplasmic reorganization of tapetal cells. In both cases, pollen grains were aborted and seed production was abolished, indicating that precise control of MOF1 expression is essential for male reproduction. We demonstrated that 5 key tapetal genes, CYP703A3 (CYTOCHROME P450 HYDROXYLASE 703A3), OsABCG26 (O. sativa ATP BINDING CASSETTE G26), PTC1 (PERSISTENT TAPETAL CELL1), PKS2 (POLYKETIDE SYNTHASE 2), and OsABCG15 (O. sativa ATP BINDING CASSETTE G15), exhibit expression patterns opposite to those of MOF1 and are negatively regulated by MOF1. Moreover, DNA affinity purification sequencing (DAP-seq), luciferase activity assays, and electrophoretic mobility shift assays indicated that MOF1 binds directly to the PKS2 promoter for transcriptional repression. Our results provide a mechanistic basis for the regulation of male reproduction by MOF1 in both diploid and tetraploid rice. This study will facilitate the development of polyploid male sterile lines, which are useful for breeding of polyploid hybrid rice.

OsmiR5519 regulates grain size and weight and down-regulates sucrose synthase gene RSUS2 in rice (Oryza sativa L.).

MAIN CONCLUSION: The up-regulation of OsmiR5519 results in the decrease of grain size, weight and seed setting rate. OsmiR5519 plays important roles in the process of grain filling and down-regulates sucrose synthase gene RSUS2. MicroRNAs (miRNAs) are one class of small non-coding RNAs that act as crucial regulators of plant growth and development. In rice, the conserved miRNAs were revealed to regulate the yield components, but the function of rice-specific miRNAs has been rarely studied. The rice-specific OsmiR5519 was found to be abundantly expressed during reproductive development, but its biological roles remain unknown. In this study, the function of rice-specific OsmiR5519 was characterized with the miR5519-overexpressing line (miR5519-OE) and miR5519-silenced line (STTM5519). At seedling stage, the content of sucrose, glucose and fructose was obviously lower in the leaves of miR5519-OE lines than those of wild-type (WT) line. The grain size and weight were decreased significantly in miR5519-OE lines, compared to those of WT rice. The cell width of hull in miR5519-OE was smaller than that in WT. The seed setting rate was notably reduced in miR5519-OE lines, but not in STTM5519 lines. Cytological observation demonstrated that the inadequate grain filling was the main reason for the decline of seed setting rate in miR5519-OE lines. The percentage of the defects of grain amounted to 40% in miR5519-OE lines, which almost equaled to the decreased value of seed setting rate. Furthermore, the sucrose synthase gene RSUS2 was identified as a target of OsmiR5519 via RNA ligase-mediated 3'-amplification of cDNA ends (3'-RLM-RACE), dual luciferase assays and transient expression assays. In summary, our results suggest that OsmiR5519 regulates grain size and weight and down-regulates RSUS2 in rice.

Natural allelic variation in GRAIN SIZE AND WEIGHT 3 of wild rice regulates the grain size and weight.

Grain size is important for yield in rice (Oryza sativa L.). Although many genes involved in grain size have been isolated, few can be used in breeding due to their interactions and phenotypic effects. Here, we describe natural variation in the granule-type quantitative trait locus GRAIN SIZE AND WEIGHT 3 (GSW3) located on chromosome 3 in wild rice (Oryza rufipogon Griff.) that encodes a GTPase-regulated protein and negatively regulates grain length, grain width, and 1,000-grain weight. The insertion of a 232-bp fragment of the genomic sequence in the wild rice, a natural allelic variant gene (GSW3), increased the expression levels and reduced the grain length and width and 1,000-grain weight. Knockout of GSW3 in the wild rice inbred line Huaye 3 increased the grain length and width and 1,000-grain weight. Introducing GSW3Huaye3 into cultivated rice line KJ01 and overexpressing GSW3Huaye3 in Huaye 3 resulted in reduced grain length and width and 1,000-grain weight, and grain size and 1,000-grain weight changes were closely related to GSW3 expression levels. GSW3 regulated the grain length and width simultaneously by promoting grain glume cell division and longitudinal and transverse cell growth. GSW3 was also involved in regulating the gibberellic acid signaling pathway and negatively regulated plant growth. Furthermore, a critical SNP in the GSW3 coding region was obviously correlated with grain size variation in a core collection of cultivated rice. This SNP resulted in an amino acid substitution from Gln to Arg at position 161 in GSW3, which reduced the grain size. Our study shows that GSW3 negatively regulates the grain shape, which could explain different grain shapes in modern cultivars and wild rice. GSW3 may also be used for breeding rice varieties with improved grain shapes and higher yield.

OsRH52A, a DEAD-Box protein, is required for embryo sac development by regulating functional megaspore specification in rice.

The development of the embryo sac is an important factor affecting seed setting in rice. Numerous genes associated with embryo sac (ES) development have been identified in plants. However, the function of the DEAD-box RNA helicase family genes on ES is poorly known in rice. Here, we characterized a rice DEAD-box protein, OsRH52A, which was localized in the nucleus and cytoplasm and highly expressed in the floral organs in rice. The knockout mutant, rh52a, displayed partial ES sterility, including degenerated ES (21.0%) and the presence of double-female-gametophyte (DFG) structure (11.8%). The DFG developed from two functional megaspores (FM) near the chalazal end in one ovule, and 3.4% of DFG could fertilize via the sac near the micropylar pole in rh52a. OsRH52A was found to interact with OsMFS1 and ZIP4, both of which play a role in homologous recombination in rice meiosis. RNA-seq identified 234 down-regulated differentially expressed genes (DEGs) associated with reproductive development, including the two genes, OsMSP1 and HSA1b, required for female germline cell specification. Taken together, our study demonstrated that OsRH52A is essential for the development of the embryo sac and provided cytological evidence regarding the formation of DFG.

Inhibiting HIF-1 signaling alleviates HTRA1-induced RPE senescence in retinal degeneration.

BACKGROUND: Age-related macular degeneration (AMD), characterized by the degeneration of retinal pigment epithelium (RPE) and photoreceptors, is the leading cause of irreversible vision impairment among the elderly. RPE senescence is an important contributor to AMD and has become a potential target for AMD therapy. HTRA1 is one of the most significant susceptibility genes in AMD, however, the correlation between HTRA1 and RPE senescence hasn't been investigated in the pathogenesis of AMD. METHODS: Western blotting and immunohistochemistry were used to detect HTRA1 expression in WT and transgenic mice overexpressing human HTRA1 (hHTRA1-Tg mice). RT-qPCR was used to detect the SASP in hHTRA1-Tg mice and ARPE-19 cells infected with HTRA1. TEM, SA-ß-gal was used to detect the mitochondria and senescence in RPE. Retinal degeneration of mice was investigated by fundus photography, FFA, SD-OCT and ERG. The RNA-Seq dataset of ARPE-19 cells treated with adv-HTRA1 versus adv-NC were analyzed. Mitochondrial respiration and glycolytic capacity in ARPE-19 cells were measured using OCR and ECAR. Hypoxia of ARPE-19 cells was detected using EF5 Hypoxia Detection Kit. KC7F2 was used to reduce the HIF1α expression both in vitro and in vivo. RESULTS: In our study, we found that RPE senescence was facilitated in hHTRA1-Tg mice. And hHTRA1-Tg mice became more susceptible to NaIO3 in the development of oxidative stress-induced retinal degeneration. Similarly, overexpression of HTRA1 in ARPE-19 cells accelerated cellular senescence. Our RNA-seq revealed an overlap between HTRA1-induced differentially expressed genes associated with aging and those involved in mitochondrial function and hypoxia response in ARPE-19 cells. HTRA1 overexpression in ARPE-19 cells impaired mitochondrial function and augmented glycolytic capacity. Importantly, upregulation of HTRA1 remarkably activated HIF-1 signaling, shown as promoting HIF1α expression which mainly located in the nucleus. HIF1α translation inhibitor KC7F2 significantly prevented HTRA1-induced cellular senescence in ARPE-19 cells, as well as improved the visual function in hHTRA1-Tg mice treated with NaIO3. CONCLUSIONS: Our study showed elevated HTRA1 contributes to the pathogenesis of AMD by promoting cellular senescence in RPE through damaging mitochondrial function and activating HIF-1 signaling. It also pointed out that inhibition of HIF-1 signaling might serve as a potential therapeutic strategy for AMD. Video Abstract.

OsmiRNA5488 Regulates the Development of Embryo Sacs and Targets OsARF25 in Rice (Oryza sativa L.).

Small RNAs are a class of non-coding RNAs that typically range from 20 to 24 nucleotides in length. Among them, microRNAs (miRNAs) are particularly important regulators for plant development. The biological function of the conserved miRNAs has been studied extensively in plants, while that of the species-specific miRNAs has been studied in-depth. In this study, the regulatory role of a rice-specific OsmiRNA5488 (OsmiR5488) was characterized with the miR5488-overexpressed line (miR5488-OE) and miR5488-silenced line (STTM-5488). The seed-setting rate was notably reduced in miR5488-OE lines, but not in STTM-5488 lines. Cytological observation demonstrated the different types of abnormal mature embryo sacs, including the degeneration of embryo sacs and other variant types, in miR5488-OE lines. The percentage of the abnormal mature embryo sacs accounted for the reduced value of the seed-setting rate. Furthermore, OsARF25 was identified as a target of OsmiR5488 via RNA ligase-mediated 3'-amplifification of cDNA ends, dual luciferase assays, and transient expression assays. The primary root length was decreased with the increases in auxin concentrations in miR5488-OE lines compared to wild-type rice. Summarily, our results suggested that OsmiR5488 regulates the seed-setting rate and down-regulates the targeted gene OsARF25.

Effect of icon size, icon position and sex on clicking motion when operating smartphones with single hand.

Nowadays, increasingly more situations exist where smartphones are operated with one hand, requiring an in-depth understanding of human-computer interaction in single-hand scenarios. 104 volunteers (57 men, 47 women) participated in this study. We aimed to explore thumb movements with the right and left hand on smartphone touchscreens at different icon sizes (50, 80, 110 and 140 rpx) in different operation areas (a 4*7 icon matrix). The results partially conformed to Fitts' Law. The movement time (MT) significantly increased as the icon size decreased, but this effect was not found over 110 rpx. The MT increased with distance in the vertical direction, but icons with the same horizontal distance had different MTs, indicating that one-handed operation restricted the click on the same side. Additionally, subjects rated 140 rpx better than other sizes, and men clicked faster than women. Suggestions regarding one-handed interface design for different hands of different sexes are provided.Practitioner summary: This study investigated how icon size, position and sex influenced one-thumb click usability on touch-screen mobile phones with different hands of different sexes. The results indicate single-hand operation partially conformed to Fitts' Law. We suggested the most economical and comfortable size and the fast operation area in one-handed interface design.Abbreviations: MT: movement time; ID: index of difficulty; RPX: responsive pixel; NASA-TLX: task load index of National Aeronautics and Space Administration; ANOVA: analysis of variance.

Autotetraploid rice: challenges and opportunities.

Autotetraploid rice is a type of germplasm developed from the whole genome duplication of diploid rice, leading to large grains, high nutrient content, and resistance. However, its low fertility has reduced yield and hampered commercialization. To address this issue, a new type of high fertility tetraploid rice was developed, which may serve as a useful germplasm for polyploid rice breeding. In this review, we summarize the progress made in understanding the cellular and molecular genetic mechanisms underlying the low fertility of autotetraploid rice and its F1 hybrid, as well as the main types of new tetraploid rice with high fertility. Lastly, the idea of utilizing the multi-generation heterosis of neo-tetraploid rice in the future is proposed as a reference for polyploid rice breeding.

An uncharacterized protein NY1 targets EAT1 to regulate anther tapetum development in polyploid rice.

BACKGROUND: Autotetraploid rice is a useful germplasm for the breeding of polyploid rice; however, low fertility is a major hindrance for its utilization. Neo-tetraploid rice with high fertility was developed from the crossing of different autotetraploid rice lines. Our previous research showed that the mutant (ny1) of LOC_Os07g32406 (NY1), which was generated by CRISPR/Cas9 knock-out in neo-tetraploid rice, showed low pollen fertility, low seed set, and defective chromosome behavior during meiosis. However, the molecular genetic mechanism underlying the fertility remains largely unknown. RESULTS: Here, cytological observations of the NY1 mutant (ny1) indicated that ny1 exhibited abnormal tapetum and middle layer development. RNA-seq analysis displayed a total of 5606 differentially expressed genes (DEGs) in ny1 compared to wild type (H1) during meiosis, of which 2977 were up-regulated and 2629 were down-regulated. Among the down-regulated genes, 16 important genes associated with tapetal development were detected, including EAT1, CYP703A3, CYP704B2, DPW, PTC1, OsABCG26, OsAGO2, SAW1, OsPKS1, OsPKS2, and OsTKPR1. The mutant of EAT1 was generated by CRISPR/Cas9 that showed abnormal tapetum and pollen wall formation, which was similar to ny1. Moreover, 478 meiosis-related genes displayed down-regulation at same stage, including 9 important meiosis-related genes, such as OsREC8, OsSHOC1, SMC1, SMC6a and DCM1, and their expression levels were validated by qRT-PCR. CONCLUSIONS: Taken together, these results will aid in identifying the key genes associated with pollen fertility, which offered insights into the molecular mechanism underlying pollen development in tetraploid rice.

An overview of sucrose transporter (SUT) genes family in rice.

INTRODUCTION: Photosynthesis provides the energy basis for the life activities of plants by producing organic compounds, mainly sugar. As the main energy form of photosynthesis, sugar affects the growth and development of plants. During long-distance transportation, sucrose is the main form of transportation. The rate of sugar transport and the allocation of carbohydrates affect the biomass of crops and are closely related to the reproductive growth of crops. MAIN TEXT: The transportation of sugar is divided into active transportation and passive transportation. So how does the sucrose transporters (SUT) genes, which are the main carriers of sucrose in active transportation, affect the performance of rice agronomic traits is still to be explored. In this article, we describe the structure of inflorescence and review the transport forms and metabolic processes of sucrose in rice, such as how CO2 is fixed, carbohydrate assimilation, and transport of organic matter. Sucrose transporters exhibited remarkable effects on the development of reproductive organs in rice. CONCLUSIONS: Here, the effects of different factors, such as the effects of anthers morphology on starch enrichment of pollen, effects of biotic and abiotic factors on sucrose transporters, effects of changes in trace elements on sucrose transporters, were discussed. Moreover, the regulation of transcription or translation level provides ideas for future research on sucrose transporters.

Association between polymorphisms of cytokine genes and diabetic nephropathy: A comprehensive systematic review and meta-analysis.

AIM: Diabetic nephropathy (DN) is one of the microvascular complications of diabetes, leading to renal failure. In this study, we sought to systematically investigate the cytokine gene polymorphisms association with DN. METHODS: A structured bibliographic search on PubMed, Scopus, and EMBASE databases has been performed to identify related papers. The odds ratio and corresponding 95% confidence intervals (CIs) were calculated to estimate the association. RESULTS: Overall, the pooled results showed that the dominant models of TNF-α rs1800629, IL-1ß rs16944, IL-8 rs4073, and IL-10 rs1800896 were associated with increased susceptibility to DN. Also, the pooled analyses of the mutant allele vs wild allele of TNF-α rs1800629, rs1799964, IL-1ß rs16944, and IL-8 rs4073 were associated with increased susceptibility to DN. Rs1800629, rs16944, rs4073, and rs1800896 polymorphisms were significantly associated with DN susceptibility, suggesting its potential use as a genetic risk marker in the population.

Cytological Observations and Bulked-Segregant Analysis Coupled Global Genome Sequencing Reveal Two Genes Associated with Pollen Fertility in Tetraploid Rice.

Neo-tetraploid rice with high fertility is a useful germplasm for polyploid rice breeding, which was developed from the crossing of different autotetraploid rice lines. However, little information is available on the molecular mechanism underlying the fertility of neo-tetraploid rice. Here, two contrasting populations of tetraploid rice, including one with high fertility (hereafter referred to as JG) and another with low fertility (hereafter referred to as JD), were generated by crossing Huaduo 3 (H3), a high fertility neo-tetraploid rice that was developed by crossing Jackson-4x with 96025-4x, and Huajingxian74-4x (T452), a low fertility autotetraploid rice parent. Cytological, global genome sequencing-based bulked-segregant (BSA-seq) and CRISPR/Cas9 technology were employed to study the genes associated with pollen fertility in neo-tetraploid rice. The embryo sacs of JG and JD lines were normal; however, pollen fertility was low in JD, which led to scarce fertilization and low seed setting. Cytological observations displayed low pollen fertility (25.1%) and approximately 31.3 and 27.2% chromosome lagging at metaphase I and II, and 28.8 and 24.8% chromosome straggling at anaphase I and II in JD, respectively. BSA-seq of F2-3 generations and RNA-seq of F4 generation detected a common fragment, i.e., 18,915,234-19,500,000, at chromosome 7, which was comprised of 78 genes associated with fertility. Among 78 genes, 9 genes had been known to be involved in meiosis and pollen development. Two mutants ny1 (LOC_Os07g32406) and ny2 (LOC_Os07g32040) were generated by CRISPR/Cas9 knockout in neo-tetraploid rice, and which exhibited low pollen fertility and abnormal chromosome behavior. Our study revealed that two unknown genes, LOC_Os07g32406 (NY1) and LOC_Os07g32040 (NY2) play an important role in pollen development of neo-tetraploid rice and provides a new perspective about the genetic mechanisms of fertility in polyploid rice.

MircroRNA Profiles of Early Rice Inflorescence Revealed a Specific miRNA5506 Regulating Development of Floral Organs and Female Megagametophyte in Rice.

The developmental process of inflorescence and gametophytes is vital for sexual reproduction in rice. Multiple genes and conserved miRNAs have been characterized to regulate the process. The changes of miRNAs expression during the early development of rice inflorescence remain unknown. In this study, the analysis of miRNAs profiles in the early stage of rice inflorescence development identified 671 miRNAs, including 67 known and 44 novel differentially expressed miRNAs (DEMs). Six distinct clusters of miRNAs expression patterns were detected, and Cluster 5 comprised 110 DEMs, including unconserved, rice-specific osa-miR5506. Overexpression of osa-miR5506 caused pleiotropic abnormalities, including over- or under-developed palea, various numbers of floral organs and spikelet indeterminacy. In addition, the defects of ovaries development were frequently characterized by multiple megasporocytes, ovule-free ovary, megasporocyte degenerated and embryo sac degenerated in the transgenic lines. osa-miR5506 targeted REM transcription factor LOC_Os03g11370. Summarily, these results demonstrated that rice-specific osa-miR5506 plays an essential role in the regulation of floral organ number, spikelet determinacy and female gametophyte development in rice.

Heterosis analysis and underlying molecular regulatory mechanism in a wide-compatible neo-tetraploid rice line with long panicles.

BACKGROUND: Neo-tetraploid rice, which is a new germplasm developed from autotetraploid rice, has a powerful biological and yield potential and could be used for commercial utilization. The length of panicle, as a part of rice panicle architecture, contributes greatly to high yield. However, little information about long panicle associated with heterosis or hybrid vigor is available in neo-tetraploid rice. RESULTS: In the present study, we developed a neo-tetraploid rice line, Huaduo 8 (H8), with long panicles and harboring wide-compatibility genes for pollen and embryo sac fertility. All the hybrids generated by H8 produced significant high-parent yield heterosis and displayed long panicles similar to H8. RNA-seq analysis detected a total of 4013, 7050, 6787 and 6195 differentially expressed genes uniquely belonging to F1 and specifically (DEGFu-sp) associated with leaf, sheath, main panicle axis and spikelet in the two hybrids, respectively. Of these DEGFu-sp, 279 and 89 genes were involved in kinase and synthase, and 714 cloned genes, such as GW8, OsGA20ox1, Ghd8, GW6a, and LP1, were identified and validated by qRT-PCR. A total of 2925 known QTLs intervals, with an average of 1~100 genes per interval, were detected in both hybrids. Of these, 109 yield-related QTLs were associated with seven important traits in rice. Moreover, 1393 non-additive DEGs, including 766 up-regulated and 627 down-regulated, were detected in both hybrids. Importantly, eight up-regulated genes associated with panicle were detected in young panicles of the two hybrids compared to their parents by qRT-PCR. Re-sequencing analysis depicted that LP (a gene controlling long panicle) sequence of H8 was different from many other neo-tetraploid rice and most of the diploid and autotetraploid lines. The qRT-PCR results showed that LP was up-regulated in the hybrid compared to its parents at very young stage of panicle development. CONCLUSIONS: These results suggested that H8 could overcome the intersubspecific autotetraploid hybrid rice sterility caused by embryo sac and pollen sterility loci. Notably, long panicles of H8 showed dominance phenomenon and played an important role in yield heterosis, which is a complex molecular mechanism. The neo-tetraploid rice is a useful germplasm to attain high yield of polyploid rice.

Comparative Cytological and Transcriptome Analysis Revealed the Normal Pollen Development Process and Up-Regulation of Fertility-Related Genes in Newly Developed Tetraploid Rice.

Autotetraploid rice is a useful germplasm for polyploid rice breeding; however, low seed setting is a major hindrance for its utilization. Here, we reported the development of a new tetraploid rice, Huoduo1 (H1), which has the characteristic of high fertility, from crossing generations of autotetraploid rice. Cytological observations displayed the high fertility of the pollen (95.62%) in H1, a lower percentage of pollen mother cell (PMC) abnormalities, and stable chromosome configurations during the pollen development process compared with its parents. Using RNA-seq analysis, we detected 440 differentially expressed genes (DEGs) in H1 compared with its parents. Of these DEGs, 193 were annotated as pollen fertility-related genes, and 129 (~66.8%) exhibited significant up-regulation in H1 compared with the parents, including three environmentally sensitive genic male sterility genes (TMS9-1, TMS5, and CSA), one meiosis gene (RAD51D), and three tapetal-related genes (MIL2, OsAP25, and OsAP37), which were validated by qRT-PCR in this study. Two genes, TMS9-1 and TMS5, were knocked out using CRISPR/Cas9 technology, and their mutants displayed low fertility and the abnormal development of pollen. Our findings provide evidence for the regulatory mechanisms of fertility in tetraploid rice and indicated that the up-regulation of pollen fertility-related genes may contribute to the high fertility in new tetraploid rice.

Transcriptome and Gene Editing Analyses Reveal MOF1a Defect Alters the Expression of Genes Associated with Tapetum Development and Chromosome Behavior at Meiosis Stage Resulting in Low Pollen Fertility of Tetraploid Rice.

Autotetraploid rice is a useful rice germplasm for polyploid rice breeding. However, low fertility limits its commercial production. A neo-tetraploid rice with high fertility was developed from the progenies of crossing between autotetraploid lines by our research group. Our previous study showed that a myeloblastosis (MYB) transcription factor, MOF1, might be associated with the pollen development in tetraploid rice. However, little information is available about its role in pollen development in tetraploid rice. Here, we identified a new haplotype of MOF1 from neo-tetraploid rice and marked it as MOF1a. Transcriptome and qRT-PCR analysis demonstrated that MOF1a highly expressed in anthers, and displayed differential expression in neo-tetraploid rice compared to tetraploid rice line with low pollen fertility. The mutant (mof1a) of MOF1a, which was generated by CRISPR/Cas9 knockout in neo-tetraploid rice, showed low pollen fertility, and also exhibited abnormal tapetum and middle layer development, and defective chromosome behaviors during meiosis. A total of 13 tapetal related genes were found to be up-regulated in meiotic anthers of MOF1a compared with wild type plants by RNA-seq analysis, including CYP703A3, PTC1, and OsABCG26, which had been demonstrated to affect tapetal development. Moreover, 335 meiosis-related genes displayed differential expression patterns at same stage, including nine important meiosis-related genes, such as metallothionein OsMT1a. These results demonstrated that MOF1a plays an important role in pollen development and provides a foundation for understanding the molecular mechanism underlying MOF1a in reproduction of tetraploid rice.

Cytological and transcriptome analyses reveal OsPUB73 defect affects the gene expression associated with tapetum or pollen exine abnormality in rice.

BACKGROUND: As one of the main crops in the world, sterility of rice (Oryza sativa L.) significantly affects the production and leads to yield decrease. Our previous research showed that OsPUB73, which encodes U-box domain-containing protein 73, may be associated with male sterility. However, little information is available on this gene that is required for anther development. In the present study, we knocked out OsPUB73 by using the CRISPR/Cas9 system and studied the cytological and transcriptome of the gene-defect associated with pollen development and sterility in the rice variety (Taichung 65). RESULTS: The sequence analysis indicated that OsPUB73 was comprised of 3 exons and 2 introns, of which CDS encoded 586 amino acids including a U-box domain. The expression pattern of OsPUB73 showed that it was highly expressed in the anther during meiosis stage. The ospub73 displayed low pollen fertility (19.45%), which was significantly lower than wild type (WT) (85.37%). Cytological observation showed tapetum vacuolated at the meiosis stage and pollen exine was abnormal at the bi-cellular pollen stage of ospub73. RNA-seq analysis detected 2240 down and 571 up-regulated genes in anther of ospub73 compared with WT during meiosis stage. Among of 2240 down-regulated genes, seven known genes were associated with tapetal cell death or pollen exine development, including CYP703A3 (Cytochrome P450 Hydroxylase703A3), CYP704B2 (Cytochrome P450 Hydroxylase704B2), DPW (Defective Pollen Wall), PTC1 (Persistant Tapetal Cell1), UDT1 (Undeveloped Tapetum1), OsAP37 (Aspartic protease37) and OsABCG15 (ATP binding cassette G15), which were validated by quantitative real-time polymerase chain reaction (qRT-PCR). These results suggested OsPUB73 may play an important role in tapetal or pollen exine development and resulted in pollen partial sterility. CONCLUSION: Our results revealed that OsPUB73 plays an important role in rice male reproductive development, which provides valuable information about the molecular mechanisms of the U-box in rice male reproductive development.

Cytological and transcriptome analyses reveal abrupt gene expression for meiosis and saccharide metabolisms that associated with pollen abortion in autotetraploid rice.

Autotetraploid rice is a useful germplasm that has four chromosome sets and strong biological advantages; however, low fertility limits its commercial utilization. Little information is available about the DNA variation and differential gene expressions associated with low fertility in autotetraploid rice. In the present study, 81 SNPs and 182 InDels were identified in T449 (an autotetraploid rice line with low fertility) compared to E249 (diploid counterpart) by whole-genome re-sequencing. We detected only three non-synonymous SNPs and six large-effect InDels, which were associated with three and six genes, respectively. A total of 75 meiosis-related differentially expressed genes were detected during the meiosis stage by transcriptome analysis, including OsMTOPVIB, which is essential for meiotic DSB formation, and OsMOF, which takes part in homologous chromosome pairing and synapsis. Approximately 20.69% lagging chromosome at metaphase I and 4.65% abnormal tetrad were observed in T449. Moreover, transcriptome analysis revealed down-regulation of a sucrose transporter (OsSUT5) and two monosaccharide transporters (OsMST1 and OsMST8) in T449 at the single microspore stage, and their expression levels were verified by qRT-PCR. Cytological observation of saccharide distribution showed abnormal accumulation of saccharides in T449 and the contents of fructose and glucose were markedly higher in T449 than E249 at the single microspore stage. Our results suggested that polyploidy not only induces abrupt expression changes in the meiosis-related genes that lead to abnormal chromosome behavior, but also causes changes in the saccharide distribution and expression patterns of saccharide-related genes, which jointly causes sterility in the autotetraploid rice.

Analysis of small RNAs revealed differential expressions during pollen and embryo sac development in autotetraploid rice.

BACKGROUND: Partial pollen and embryo sac sterilities are the two main reasons for low fertility in autotetraploid rice. Our previous study revealed that small RNAs changes may associate with pollen fertility in autotetraploid rice. However, knowledge on comparative analysis between the development of pollen and embryo sac by small RNAs in autotetraploid rice is still unknown. In the present study, WE-CLSM (whole-mount eosin B-staining confocal laser scanning microscopy) and high-throughput sequencing technology was employed to examine the cytological variations and to analyze small RNAs changes during pollen and embryo sac development in autotetraploid rice compared with its diploid counterpart. RESULTS: A total of 321 and 368 differentially expressed miRNAs (DEM) were detected during pollen and embryo sac development in autotetraploid rice, respectively. Gene Ontology enrichment analysis on the targets of DEM associated with embryo sac and pollen development revealed 30 prominent functional gene classes, such as cell differentiation and signal transduction during embryo sac development, while only 7 prominent functional gene classes, such as flower development and transcription factor activity, were detected during pollen development in autotetraploid rice. The expression levels of 39 DEM, which revealed interaction with meiosis-related genes, showed opposite expression patterns during pollen and embryo sac development. Of these DEM, osa-miR1436_L + 3_1ss5CT and osa-miR167h-3p were associated with the female meiosis, while osa-miR159a.1 and osa-MIR159a-p5 were related with the male meiosis. 21 nt-phasiRNAs were detected during both pollen and embryo sac development, while 24 nt-phasiRNAs were found only in pollen development, which displayed down-regulation in autotetraploid compared to diploid rice and their spatial-temporal expression patterns were similar to osa-miR2275d. 24 nt TEs-siRNAs were found to be up-regulated in embryo sac but down-regulated in pollen development. CONCLUSION: The above results not only provide the small RNAs changes during four landmark stages of pollen and embryo sac development in autotetraploid rice but also have identified specifically expressed miRNAs, especially meiosis-related miRNAs, pollen-specific-24 nt-phasiRNAs and TEs-siRNAs in autotetraploid rice. Together, these findings provide a foundation for understanding the effect of polyploidy on small RNAs expression patterns during pollen and embryo sac development that may lead to different abnormalities in autotetraploid rice.

Polyploidy Enhances F1 Pollen Sterility Loci Interactions That Increase Meiosis Abnormalities and Pollen Sterility in Autotetraploid Rice.

Intersubspecific autotetraploid rice (Oryza sativa ssp. indica × japonica) hybrids have greater biological and yield potentials than diploid rice. However, the low fertility of intersubspecific autotetraploid hybrids, which is largely caused by high pollen abortion rates, limits their commercial utility. To decipher the cytological and molecular mechanisms underlying allelic interactions in autotetraploid rice, we developed an autotetraploid rice hybrid that was heterozygous (S(i)S(j)) at F1 pollen sterility loci (Sa, Sb, and Sc) using near-isogenic lines. Cytological studies showed that the autotetraploid had higher percentages (>30%) of abnormal chromosome behavior and aberrant meiocytes (>50%) during meiosis than did the diploid rice hybrid control. Analysis of gene expression profiles revealed 1,888 genes that were differentially expressed between the autotetraploid and diploid hybrid lines at the meiotic stage, among which 889 and 999 were up- and down-regulated, respectively. Of the 999 down-regulated genes, 940 were associated with the combined effect of polyploidy and pollen sterility loci interactions (IPE). Gene Ontology enrichment analysis identified a prominent functional gene class consisting of seven genes related to photosystem I (Gene Ontology 0009522). Moreover, 55 meiosis-related or meiosis stage-specific genes were associated with IPE in autotetraploid rice, including Os02g0497500, which encodes a DNA repair-recombination protein, and Os02g0490000, which encodes a component of the ubiquitin-proteasome pathway. These results suggest that polyploidy enhances epistatic interactions between alleles of pollen sterility loci, thereby altering the expression profiles of important meiosis-related or meiosis stage-specific genes and resulting in high pollen sterility.