OsCBL5-CIPK1-PP23 module enhances rice grain size and weight through the gibberellin pathway.

Grain size is a key factor in determining rice (Oryza sativa) yield, and exploring new pathways to regulate grain size has immense potential to improve yield. In this study, we report that OsCBL5 encodes a calcineurin B subunit protein that significantly promotes grain size and weight. oscbl5 plants produced obviously smaller and lighter seeds. We further revealed that OsCBL5 promotes grain size by affecting cell expansion in the spikelet hull. Biochemical analyses demonstrated that CBL5 interacts with CIPK1 and PP23. Furthermore, double and triple mutations were induced using CRISPR/Cas9 (cr) to analyze the genetic relationship. It was found that the cr-cbl5/cipk1 phenotype was similar to that of cr-cipk1 and that the cr-cbl5/pp23, cr-cipk1/pp23, and cr-cbl5/cipk1/pp23 phenotype was similar to that of cr-pp23, indicating that OsCBL5, CIPK1, and PP23 act as a molecular module influencing seed size. In addition, the results show that both CBL5 and CIPK1 are involved in the gibberellic acid (GA) pathway and significantly affect the accumulation of endogenous active GA4 . PP23 participates in GA signal transduction. In brief, this study identified a new module that affects rice grain size, OsCBL5-CIPK1-PP23, which could potentially be targeted to improve rice yield.

Genome Resequencing for Autotetraploid Rice and Its Closest Relatives Reveals Abundant Variation and High Potential in Rice Breeding.

Polyploid rice and its reverted diploid show rich phenotypic variation and strong heterosis, showing great breeding value. However, the genomic differences among tetraploids, counterpart common diploids, tetraploid-revertant diploids, and hybrid descendants are unclear. In this work, we bred a new excellent two-line hybrid rice variety, Y Liang You Duo Hui 14 (HTRM12), using Haitian tetraploid self-reverted diploid (HTRM2). Furthermore, we comparatively analyzed the important agronomic traits and genome-wide variations of those closest relatives, Haitian diploid (HT2), Haitian tetraploid (HT4), HTRM2, and HTRM12 in detail, based on multiple phenotypic investigations, genome resequencing, and bioinformatics analysis. The results of agronomic traits analysis and genome-wide variation analysis of single nucleotide polymorphism (SNP), insertion-deletion (InDel), and copy number variation (CNV) show that HT4 and HTRM2 had abundant phenotypic and genomic variations compared to HT2. HTRM2 can inherit important traits and variations from HT4. This implies that tetraploid self-reverted diploid has high potential in creating excellent breeding materials and in breeding breakthrough hybrid rice varieties. Our study verifies the feasibility that polyploid rice could be used as a mutation carrier for creating variations and provides genomic information, new breeding materials, and a new way of application for tetraploid rice breeding.

Full-length transcriptome reconstruction reveals genetic differences in hybrids of Oryza sativa and Oryza punctata with different ploidy and genome compositions.

BACKGROUND: Allopolyploid breeding is an efficient technique for improving the low seed setting rate of autotetraploids in plant breeding and one of the most promising breeding methods. However, there have been few comprehensive studies of the posttranscriptional mechanism in allopolyploids. RESULTS: By crossing cultivated rice (Oryza sativa, genome AA) with wild rice (Oryza punctata, genome BB), we created hybrid rice lines with different ploidy and genome compositions [diploid hybrid F01 (AB), allotetraploid hybrid F02 (AABB) and F03 (AAAB)]. The genetic differences of the hybrids and the mechanism of allopolyploid breeding dominance were revealed through morphological and cytological observations and single molecule real-time sequencing techniques. The tissues and organs of allotetraploid hybrid F02 exhibited "gigantism" and the highest levels of fertility. The numbers of non-redundant transcripts, gene loci and new isoforms in the polyploid rice lines were higher and the isoform lengths greater than those of the diploid line. Moreover, alternative splicing (AS) events occurred twice as often in the polyploid rice lines than the diploid line. During these events, intron retention dominated. Furthermore, a large number of new genes and isoforms specific to the lines of different ploidy were discovered. CONCLUSIONS: The results indicated that alternative polyadenylation (APA) and AS events contributed to the complexity and superiority of polyploids in the activity of translation regulators, nucleic acid binding transcription factor activities and the regulation of molecular function. Therefore, these APA and AS events in allopolyploid rice were found to play a role in regulation. Our study provides new germplasm for polyploid rice breeding and reveals complex regulatory mechanisms that may be related to heterosis and fertility.

Polyploidization Increases the Lipid Content and Improves the Nutritional Quality of Rice.

Plant polyploidization is frequently associated with changes in nutrient contents. However, the possible contribution of metabolites to this change has not been investigated by characterizing the metabolite contents of diploid and tetraploid forms of rice (Oryza sativa L.). We compared the metabolites of a group of diploid-tetraploid japonica brown rice and a group of diploid-tetraploid indica brown rice based on liquid chromatography-tandem mass spectrometry. In total, 401 metabolites were identified; of these, between the two diploid-tetraploid groups, 180 showed opposite expression trends, but 221 showed the same trends (147 higher abundance vs. 74 lower abundance). Hierarchical cluster analysis of differential metabolites between diploid and tetraploid species showed a clear grouping pattern, in which the expression abundance of lipids, amino acids and derivatives, and phenolic acids increased in tetraploids. Further analysis revealed that the lipids in tetraploid rice increased significantly, especially unsaturated fatty acids and phospholipids. This study provides further basis for understanding the changes in rice nutritional quality following polyploidization and may serve as a new theoretical reference for breeding eutrophic or functional rice varieties via polyploidization.

DNA methylation in transposable elements buffers the connection between three-dimensional chromatin organization and gene transcription upon rice genome duplication.

INTRODUCTION: Polyploidy is a major force in plant evolution and the domestication of cultivated crops. OBJECTIVES: The study aimed to explore the relationship and underlying mechanism between three-dimensional (3D) chromatin organization and gene transcription upon rice genome duplication. METHODS: The 3D chromatin structures between diploid (2C) and autotetraploid (4C) rice were compared using high-throughput chromosome conformation capture (Hi-C) analysis. The study combined genetics, transcriptomics, whole-genome bisulfite sequencing (WGBS-seq) and 3D genomics approaches to uncover the mechanism for DNA methylation in modulating gene transcription through 3D chromatin architectures upon rice genome duplication. RESULTS: We found that 4C rice presents weakened intra-chromosomal interactions compared to its 2C progenitor in some chromosomes. In addition, we found that changes of 3D chromatin organizations including chromatin compartments, topologically associating domains (TADs), and loops, are uncorrelated with gene transcription. Moreover, DNA methylations in the regulatory sequences of genes in compartment A/B switched regions and TAD boundaries are unrelated to their expression. Importantly, although there was no significant difference in the methylation levels in transposable elements (TEs) in differentially expressed gene (DEG) and non-DEG promoters between 2C and 4C rice, we found that the hypermethylated TEs across genes in compartment A/B switched regions and TAD boundaries may suppress the expression of these genes. CONCLUSION: The study proposed that the rice genome doubling might modulate TE methylation to buffer the effects of chromatin architecture on gene transcription in compartment A/B switched regions and TAD boundaries, resulting in the disconnection between 3D chromatin structure alteration and gene transcription upon rice genome duplication.

A New Way of Rice Breeding: Polyploid Rice Breeding.

Polyploid rice, first discovered by Japanese scientist Eiiti Nakamori in 1933, has a history of nearly 90 years. In the following years, polyploid rice studies have mainly focused on innovations in breeding theory, induction technology and the creation of new germplasm, the analysis of agronomic traits and nutritional components, the study of gametophyte development and reproduction characteristics, DNA methylation modification and gene expression regulation, distant hybridization and utilization among subspecies, species and genomes. In recent years, PMeS lines and neo-tetraploid rice lines with stable high seed setting rate characteristics have been successively selected, breaking through the bottleneck of low seed setting rate of polyploid rice. Following, a series of theoretical and applied studies on high seed setting rate tetraploid rice were carried out. This has pushed research on polyploid rice to a new stage, opening new prospects for polyploid rice breeding.

Unique Glutelin Expression Patterns and Seed Endosperm Structure Facilitate Glutelin Accumulation in Polyploid Rice Seed.

BACKGROUND: Rice is not only an essential food but also a source of high quality protein. Polyploidy is an evolutionary trajectory in plants, and enhancing glutelin by polyploidization is an attractive strategy for improving the nutritional value of rice seeds and presents a great potential for enhancing the commercial value of rice. Elucidating the mechanisms underlying glutelin synthesis and accumulation in tetraploid rice is of great significance. RESULTS: To enhance the nutritional value of rice, we developed tetraploid rice and evaluated the contents of various nutrient elements in mature seeds. The results revealed a significant increase in protein contents, including the total seed storage proteins, glutelins, and amino acids in tetraploid rice when compared with those in diploid rice. Tandem mass tag-based quantitative proteomic analyses of seeds revealed that glutelins regulated by several glutelin genes in 9311-4x were significantly up-regulated (≥1.5-fold), which was further verified by immunoblot analyses. In addition, temporal expression patterns of various glutelin subunits in different rice lines were investigated. The results revealed significant differences in the expression patterns between diploid and tetraploid rice seeds. Cytohistological analyses results revealed that the thickness of aleurone cell layers increased significantly by 32% in tetraploid rice, the structures of protein storage vacuoles (PSVs) in sub-aleurone cells were more diverse and abundant than those of diploid rice. Temporal expression and proteomic analyses results revealed that protein disulfide isomerase-like 1-1 expression levels were higher in tetraploid rice than in diploid rice, and that the gene responded to oxidative folding with increased levels of proglutelin and appropriate distribution of seed glutelins in tetraploid rice. CONCLUSION: The results of the present study revealed that polyploidization increased glutelin content by influencing glutelin biosynthesis, transport, and deposition, while variations in glutelin accumulation between tetraploid and diploid rice were largely manifested in the initial time, duration, and relative levels of various glutelin gene expressions during seed filling stages. These findings provide novel insights into improving the protein quality and nutritional value of rice seeds by polyploid breeding.

Genome duplication effects on pollen development and the interrelated physiological substances in tetraploid rice with polyploid meiosis stability.

The breeding of polyploid rice made no breakthrough for a long time because of low seed set. The discovery and application of polyploid meiosis stability (PMeS) material played a pivotal role in solving this problem. Our results indicated that genome duplication led to different outcomes in different rice cultivars in terms of pollen fertility, viability, and the accumulation of important physiological substances such as free proline and endogenous hormones. Pollen from the PMeS HN2026-4X lines showed a high fertility and viability similar to those of HN2026-2X (4X indicates tetraploid while 2X indicates the diploid), whereas both rates decreased dramatically in Balilla-4X. The results of pollen microstructure and ultrastructure investigations suggested that the pollen development pattern in HN2026-4X appeared normal at all stages, but a lot of changes were discovered in Balilla-4X. Stable meiosis, timely tapetum degradation, and normal mitochondria development were critical factors insuring the high frequency pollen fertility of PMeS rice. The free proline content increased markedly in HN2026-4X as compared to HN2026-2X, but it was decreased for Balilla-4X. Genome duplication effects on regulating endogenous hormones accumulation in pollen were evident, resulting in the clear difference between PMeS HN2026-4X and Balilla-4X. The accumulation of IAA, ZR, and GA in mature pollen distinguished HN2026-4X from Balilla-4X, which was linked to normal pollen development. In particular, the excessive accumulation of ABA at the meiosis stage may be correlated to disorganized meiosis in Balilla-4X. All the results provided unequivocal evidence that genome duplication played specific roles in the normal pollen development of PMeS HN2026-4X.

Integration of small RNA, degradome and proteome sequencing in Oryza sativa reveals a delayed senescence network in tetraploid rice seed.

Seed of rice is an important strategic resource for ensuring the security of China's staple food. Seed deterioration as a result of senescence is a major problem during seed storage, which can cause major economic losses. Screening among accessions in rice germplasm resources for traits such as slow senescence and increased seed longevity during storage is, therefore, of great significance. However, studies on delayed senescence in rice have been based mostly on diploid rice seed to date. Despite better tolerance have been verified by the artificial aging treatment for polyploid rice seed, the delayed senescence properties and delayed senescence related regulatory mechanisms of polyploid rice seed are rarely reported, due to the lack of polyploid rice materials with high seed set. High-throughput sequencing was applied to systematically investigate variations in small RNAs, the degradome, and the proteome between tetraploid and diploid rice seeds. Degradome sequencing analysis of microRNAs showed that expression of miR-164d, which regulates genes encoding antioxidant enzymes, was changed significantly, resulting in decreased miRNA-mediated cleavage of target genes in tetraploid rice. Comparisons of the expression levels of small RNAs (sRNAs) in the tetraploid and diploid libraries revealed that 12 sRNAs changed significantly, consistent with the findings from degradome sequencing. Furthermore, proteomics also showed that antioxidant enzymes were up-regulated in tetraploid rice seeds, relative to diploids.

The breeding of two polyploid rice lines with the characteristic of polyploid meiosis stability.

Polyploidization is a basic feature of plant evolution. Nearly all of the main food, cotton and oil crops are polyploid. When ploidy levels increase, yields double; this phenomenon suggested a new strategy of rice breeding that utilizes wide crosses and polyploidization dual advantages to breed super rice. Because low seed set rates in polyploid rice usually makes it difficult to breed, the selection of Ph-liked gene lines was emphasized. After progenies of indica-japonica were identified and selected, two polyploid lines, PMeS-1 and PMeS-2 with Polyploid Meiosis Stability (PMeS) genes were bred. The procedure included seven steps: selecting parents, crossing or multiple crossing, back-crossing, doubling chromosomes, identifying the polyploid, and choosing plants with high seed set rates that can breed themselves into stable lines. The characteristics of PMeS were determined by observing meiotic behaviors and by cross-identification of seed sets. PMeS-1 and PMeS-2, (japonica rice), have several characteristics different from other polyploid rice lines, including a higher rate of seed set (more than 65%, increasing to more than 70% in their F1 offspring); and stable meiotic behaviors (pairing with bivalents and quarivalents nearly without over-quarivalent in prophase, nearly without lagging chromosomes in metaphase and without micronuclei in anaphase and telophase). The latter was obviously different from control polyploid line Dure-4X, which displayed abnormal meiotic behaviors including a higher rate of multivalents, univalents and trivalents in prophase, lagging chromosomes in metaphase and micronuclei in anaphase and telophase. There were also three differences of the breeding method between PMeS lines and normal diploid lines: chromosomes doubling, polyploidism identifying and higher seed set testing. The selection of PMeS lines is the first step in polyploid rice breeding; their use will advance the progress of polyploid rice breeding, which will in turn offer a new way to breed super rice.