Quantitative Proteomics and Transcriptomics Reveals Differences in Proteins During Anthers Development in Oryza longistaminata

Oryza longistaminata is an African wild rice species that possesses special traits for breeding applications. Self-incompatibility is the main cause of sterility in O. longistaminata, but here we demonstrated that its pollen vitality are normal. Lipid and carbohydrate metabolism were active throughout pollen development. In this study, we used I2-KI staining and TTC staining to investigate pollen viability. Aniline-blue-stained semithin sections were used to investigate important stages of pollen development. Tandem mass tags (TMT)-based quantitative analysis was used to investigate the profiles of proteins related to lipid and carbohydrate metabolism in 4-, 6-, and 8.5-mm O. longistaminata spikelets before flowering. Pollen was found to germinate normally in vitro and in vivo. We documented cytological changes throughout important stages of anther development, including changes in reproductive cells as they formed mature pollen grains through meiosis and mitosis. A total of 31,987 RNA transcripts and 8,753 proteins were identified, and 6,842 of the proteins could be quantified. RNA-seq and proteome association analysis indicated that fatty acids were converted to sucrose after the 6-mm spikelet stage, based on the abundance of most key enzymes of the glyoxylate cycle and gluconeogenesis. The abundance of proteins involved in pollen energy metabolism was further confirmed by combining quantitative real-time PCR with parallel reaction monitoring (PRM) analyses. In conclusion, our study provides novel insights into the pollen viability of O. longistaminata at the proteome level, which can be used to improve the efficiency of male parent pollination in hybrid rice breeding applications.

QTL Mapping of a Novel Genomic Region Associated with High Out-Crossing Rate Derived from Oryza longistaminata and Development of New CMS Lines in Rice, O. sativa L.

High seed cost due to poor seed yield severely limits the adoption of hybrid rice by farmers. Increasing the out-crossing rate is one of the key strategies to increase hybrid seed production. Out-crossing rate is highly influenced by the size of female floral traits, which capture pollen grains from male donor plants. In the current study, we identified 14 QTLs derived from the perennial wild rice Oryza longistaminata by composite interval mapping for five key floral traits: stigma length (five), style length (three), stigma breadth (two), stigma area (one), and pistil length (three). QTL analysis and correlation studies revealed that these stigma traits were positively correlated and pleiotropic to the stigma length trait. We selected the major-effect QTL qSTGL8.0 conferring long stigma phenotype for further fine mapping and marker-assisted selection. The qSTGL8.0 (~ 3.9 Mb) was fine mapped using newly developed internal markers and was narrowed down to ~ 2.9 Mb size (RM7356–RM256 markers). Further, the flanking markers were validated in a segregating population and in progenies from different genetic backgrounds. The markers PA08-03 and PA08-18 showed the highest co-segregation with the stigma traits. The qSTGL8.0 was introgressed into two cytoplasmic male sterile (CMS) lines, IR58025A and IR68897A, by foreground, background, and trait selection approaches. The qSTGL8.0 introgression lines in CMS backgrounds showed a significantly higher seed setting rate (2.5–3.0-fold) than the original CMS lines in test crosses with their corresponding maintainer lines. The newly identified QTLs especially qSTGL8.0, will be quite useful for increasing out-crossing rate and this will contribute to increase seed production and decrease seed cost.

Strategies and Structure Feature of the Aboveground and Belowground Microbial Community Respond to Drought in Wild Rice (Oryza longistaminata)

Background Drought is global environmental stress that limits crop yields. Plant-associated microbiomes play a crucial role in determining plant fitness in response to drought, yet the fundamental mechanisms for maintaining microbial community stability under drought disturbances in wild rice are poorly understood. We make explicit comparisons of leaf, stem, root and rhizosphere microbiomes from the drought-tolerant wild rice (Oryza longistaminata) in response to drought stress. Results We find that the response of the wild rice microbiome to drought was divided into aboveground–underground patterns. Drought reduced the leaf and stem microbial community diversity and networks stability, but not that of the roots and rhizospheres. Contrary to the aboveground microbial networks, the drought-negative response taxa exhibited much closer interconnections than the drought-positive response taxa and were the dominant network hubs of belowground co-occurrence networks, which may contribute to the stability of the belowground network. Notably, drought induces enrichment of Actinobacteria in belowground compartments, but not the aboveground compartment. Additionally, the rhizosphere microbiome exhibited a higher proportion of generalists and broader habitat niche breadth than the microbiome at other compartments, and drought enhanced the proportion of specialists in all compartments. Null model analysis revealed that both the aboveground and belowground-community were governed primarily by the stochastic assembly process, moreover, drought decreased ‘dispersal limitation’, and enhanced ‘drift’. Conclusions Our results provide new insight into the different strategies and assembly mechanisms of the above and belowground microbial community in response to drought, including enrichment of taxonomic groups, and highlight the important role of the stochastic assembly process in shaping microbial community under drought stress.

Comparative analysis morphology, anatomical structure and transcriptional regulatory network of chlorophyll biosynthesis in Oryza longistaminata, O. sativa and their F1 generation

Oryza longistaminata, a perennial wild species, is widely distributed in the African continent. It has strong tolerance to biotic and abiotic stresses, and high biomass production on poor soils. Chlorophyll biosynthesis is important for photosynthesis in rice. However, the chlorophyll biosynthesis and related gene profiles of O. longistaminata and its descendants remained unclear. Here, the F1 generation of O. sativa and O. longistaminata were obtained. Then, the comparative analysis morphology, anatomical structure, and transcriptional regulatory networks of chlorophyll biosynthesis were detected and analyzed. Results showed that the F1 generation has obvious long awn, similar with that of the male parent. The purple color of the long awn is different from that of the male parent. Microstructural results showed that the flag leaves of F1 have large mesophyll cell gaps in the upper- and lower-positions, small mesophyll cell gaps in the middle position, and more chloroplasts. Increased chlorophyll content was also observed in the F1 generation. In the lower-position flag leaves, the total chlorophyll contents of F1 were 1.55 and 1.5 times those of O. sativa and O. longistaminata, respectively. POR, MgCH and HEMA1 showed higher expression levels than the other related genes selected in the chlorophyll biosynthesis pathway. The HEMA1 expression level in the middle-position flag leaves of O. longistaminata was the highest, and it was 2.83 and 2.51 times that of O. sativa and F1, respectively. The expression level of DVR gene in lower-position flag leaves of F1 were 93.16% and 95.06% lower than those of O. sativa and O. longistaminata, respectively. This study provided a potential reference for studying the photosynthesis and heterosis utilization of O. longistaminata.

Systemic regulation of nitrogen acquisition and use in Oryza longistaminata ramets under nitrogen heterogeneity

Oryza longistaminata, a wild rice, vegetatively reproduces and forms a networked clonal colony consisting of ramets connected by rhizomes. Although water, nutrients, and other molecules can be transferred between ramets via the rhizomes, inter-ramet communication in response to spatially heterogeneous nitrogen availability is not well understood. We studied the response of ramet pairs to heterogeneous nitrogen availability by using a split hydroponic system that allowed each ramet root to be exposed to different conditions. Ammonium uptake was compensatively enhanced in the sufficient-side root when roots of the ramet pairs were exposed to ammonium-sufficient and deficient conditions. Comparative transcriptome analysis revealed that a gene regulatory network for effective ammonium assimilation and amino acid biosynthesis was activated in the sufficient-side roots. Allocation of absorbed nitrogen from the nitrogen-sufficient to the deficient ramets was rather limited. Nitrogen was preferentially used for newly growing axillary buds on the sufficient-side ramets. Biosynthesis of trans-zeatin, a cytokinin, was up-regulated in response to the nitrogen supply, but trans-zeatin appears not to target the compensatory regulation. Our results also implied that the O. longistaminata ortholog of OsCEP1 plays a role as a nitrogen-deficient signal in inter-ramet communication, providing compensatory up-regulation of nitrogen assimilatory genes. These results provide insights into the molecular basis for efficient growth strategies of asexually proliferating plants growing in areas where nitrogen distribution is spatially heterogeneous.

Nitrogen Nutrition Promotes Rhizome Bud Outgrowth via Regulation of Cytokinin Biosynthesis Genes and an Oryza longistaminata Ortholog of FINE CULM 1

Oryza longistaminata, a wild rice, can propagate vegetatively via rhizome formation and, thereby, expand its territory through horizontal growth of branched rhizomes. The structural features of rhizomes are similar to those of aerial stems; however, the physiological roles of the two organs are different. Nitrogen nutrition is presumed to be linked to the vegetative propagation activity of rhizomes, but the regulation of rhizome growth in response to nitrogen nutrition and the underlying biological processes have not been well characterized. In this study, we analyzed rhizome axillary bud growth in response to nitrogen nutrition and examined the involvement of cytokinin-mediated regulation in the promotion of bud outgrowth in O. longistaminata. Our results showed that nitrogen nutrition sufficiency promoted rhizome bud outgrowth to form secondary rhizomes. In early stages of the response to nitrogen application, glutamine accumulated rapidly, two cytokinin biosynthesis genes, isopentenyltransferase, and CYP735A, were up-regulated with accompanying cytokinin accumulation, and expression of an ortholog of FINE CULM1, a negative regulator of axillary bud outgrowth, was severely repressed in rhizomes. These results suggest that, despite differences in physiological roles of these organs, the nitrogen-dependent outgrowth of rhizome axillary buds in O. longistaminata is regulated by a mechanism similar to that of shoot axillary buds in O. sativa. Our findings provide a clue for understanding how branched rhizome growth is regulated to enhance nutrient acquisition strategies.

Confirmation of a Gametophytic Self-Incompatibility in Oryza longistaminata

Oryza longistaminata, a wild species of African origin, has been reported to exhibit self-incompatibility (SI). However, the genetic pattern of its SI remained unknown. In this study, we conducted self-pollination and reciprocal cross-pollination experiments to verify that O. longistaminata is a strictly self-incompatible species. The staining of pollen with aniline blue following self-pollination revealed that although pollen could germinate on the stigma, the pollen tube was unable to enter the style to complete pollination, thereby resulting in gametophytic self-incompatibility (GSI). LpSDUF247, a S-locus male determinant in the gametophytic SI system of perennial ryegrass, is predicted to encode a DUF247 protein. On the basic of chromosome alignment with LpSDUF247, we identified OlSS1 and OlSS2 as Self-Incompatibility Stamen candidate genes in O. longistaminata. Chromosome segment analysis revealed that the Self-Incompatibility Pistil candidate gene of O. longistaminata (OlSP) is a polymorphic gene located in a region flanking OlSS1. OlSS1 was expressed mainly in the stamens, whereas OlSS2 was expressed in both the stamens and pistils. OlSP was specifically highly expressed in the pistils, as revealed by RT-PCR and qRT-PCR analyses. Collectively, our observations indicate the occurrence of GSI in O. longistaminata and that this process is potentially controlled by OlSS1, OlSS2, and OlSP. These findings provide further insights into the genetic mechanisms underlying self-compatibility in plants.

Comparison of wild rice (Oryza longistaminata) tissues identifies rhizome-specific bacterial and archaeal endophytic microbiomes communities and network structures

Compared with root-associated habitats, little is known about the role of microbiota inside other rice organs, especially the rhizome of perennial wild rice, and this information may be of importance for agriculture. Oryza longistaminata is perennial wild rice with various agronomically valuable traits, including large biomass on poor soils, high nitrogen use efficiency, and resistance to insect pests and disease. Here, we compared the endophytic bacterial and archaeal communities and network structures of the rhizome to other compartments of O. longistaminata using 16S rRNA gene sequencing. Diverse microbiota and significant variation in community structure were identified among different compartments of O. longistaminata. The rhizome microbial community showed low taxonomic and phylogenetic diversity as well as the lowest network complexity among four compartments. Rhizomes exhibited less phylogenetic clustering than roots and leaves, but similar phylogenetic clustering with stems. Streptococcus, Bacillus, and Methylobacteriaceae were the major genera in the rhizome. ASVs belonging to the Enhydrobacter, YS2, and Roseburia are specifically present in the rhizome. The relative abundance of Methylobacteriaceae in the rhizome and stem was significantly higher than that in leaf and root. Noteworthy type II methanotrophs were observed across all compartments, including the dominant Methylobacteriaceae, which potentially benefits the host by facilitating CH4-dependent N2 fixation under nitrogen nutrient-poor conditions. Our data offers a robust knowledge of host and microbiome interactions across various compartments and lends guidelines to the investigation of adaptation mechanisms of O. longistaminata in nutrient-poor environments for biofertilizer development in agriculture.

QTL Fine Mapping of a Novel Genomic Region Associated With High Out-crossing Rate Derived From Oryza Longistaminata and Development of New CMS Lines in Rice, O. Sativa L.

Background: High seed cost due to poor seed yield severely limits the adoption of hybrid rice by farmers. Increasing the out-crossing rate is one of the key strategies to increase hybrid seed production. Out-crossing rate is highly influenced by the size of female floral traits, which capture pollen grains from male donor plants. Results: In the current study, we identified 14 QTLs derived from the perennial wild rice Oryza longistaminata by composite interval mapping for five key floral traits: stigma length (five), style length (three), stigma breadth (two), stigma area (one), and pistil length (three). QTL analysis and correlation studies revealed that these stigma traits were positively correlated and pleiotropic to the stigma length trait. We selected the major-effect QTL qSTGL8.0 conferring long stigma phenotype for further fine mapping and marker-assisted selection (MAS). The qSTGL8.0 (~3.9 Mb) was fine mapped using newly developed internal markers and was narrowed down to ~2.9 Mb size (RM7356‒RM256 markers). Further, the flanking markers were validated in a segregating population and in progenies from different genetic backgrounds. The markers PA08-03 and PA08-18 showed the highest co-segregation with the stigma traits. The qSTGL8.0 was introgressed into two cytoplasmic male sterile (CMS) lines, IR58025A and IR68897A, by foreground, background, and trait selection approaches. Conclusion: The qSTGL8.0 introgression lines in CMS backgrounds showed a significantly higher seed setting rate (2.5‒3.0-fold) than the original CMS lines in testcrosses with their corresponding maintainer lines. The newly identified QTLs especially qSTGL8.0, will be quite useful for increasing out-crossing rate and this will contribute to increase seed production and decrease seed cost.