Background: Salinity is a stressful environmental factor that limits the productivity of crop plants, and roots form the major interface between plants and various abiotic stresses. Rice is a salt-sensitive crop and its polyploid shows advantages in terms of stress resistance. The objective of this study was to investigate the effects of genome duplication on rice root resistance to salt stress. Results: Both diploid rice (HN2026-2x and Nipponbare-2x) and their corresponding tetraploid rice (HN2026-4x and Nipponbare-4x) were cultured in half-strength Murashige and Skoog medium with 150 mM NaCl for 3 and 5 days. Accumulations of proline, soluble sugar, malondialdehyde (MDA), Na⁺ content, H⁺ (proton) flux at root tips, and the microstructure and ultrastructure in rice roots were examined. We found that tetraploid rice showed less root growth inhibition, accumulated higher proline content and lower MDA content, and exhibited a higher frequency of normal epidermal cells than diploid rice. In addition, a protective gap appeared between the cortex and pericycle cells in tetraploid rice. Next, ultrastructural analysis showed that genome duplication improved membrane, organelle, and nuclei stability. Furthermore, Na⁺ in tetraploid rice roots significantly decreased while root tip H⁺ efflux in tetraploid rice significantly increased. Conclusions: Our results suggest that genome duplication improves root resistance to salt stress, and that enhanced proton transport to the root surface may play a role in reducing Na⁺ entrance into the roots.

Low temperature germination (LTG) is an important agronomic trait for direct-seeding cultivation of rice (Oryza sativa). Both OsMYB30 and OsTPP1 regulate the cold stress response in rice, but the function of OsMYB30 and OsTPP1 in regulating LTG and the underlying molecular mechanism remain unknown. Employing transcriptomics and functional studies revealed a sugar signaling pathway that regulates seed germination in response to low temperature. Expression of OsMYB30 and OsTPP1 was induced by low temperature during seed germination, and overexpressing either OsMYB30 or OsTPP1 delayed seed germination and increased sensitivity to low temperature during seed germination. Transcriptomics and qPCR revealed that expression of OsTPP1 was upregulated in OsMYB30-overexpressing lines but downregulated in OsMYB30-knockout lines. In vitro and in vivo experiments revealed that OsMYB30 bound to the promoter of OsTPP1 and regulated the abundance of OsTPP1 transcripts. Over accumulation of trehalose was found in both OsMYB30- and OsTPP1-overexpressing lines, resulting in inhibition of α-amylase 1a (OsAMY1a) gene during seed germination. Both low temperature and exogenous trehalose treatments suppressed the expression of OsAMY1a, and the osamy1a mutant was not sensitive to exogenous trehalose during seed germination. Overall, we concluded that OsMYB30 expression was induced by low temperature to activate the expression of OsTPP1 and increase trehalose content, which thus inhibited α-Amylase activity and seed germination. This study identified a phytohormone-independent pathway that integrates environmental cues with internal factors to control seed germination.

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.

The meiotic processes of most polyploid rice (Oryza sativa) are genetically abnormal, leading to low pollen fertility, which results in low seed set rates. Some polyploid meiosis stability (PMeS) lines with high seed set rates have been bred but their meiotic mechanisms remain unknown. In this study, we investigated the function of OsMND1 regulated polyploid rice meiosis. OsMND1 localized in the nucleus, and its expression level in panicles of PMeS line HN2026-4x was higher than in HN2026-2x and the other lines without the PMeS background. OsMND1’s overexpression improved pollen fertility and viability, early normal embryo development and the seed set rate in Balilla-4x. However, OsMND1 RNAi in PMeS line HN2026-4x impeded pollen and embryo development significantly. The results of chromosome behavior analyses indicated that OsMND1 participates in stabilizing meiosis by maintaining the balance of pairing, synapsis and recombination during early meiosis. Many univalent, trivalent, and even multivalent systems appeared in the OsMND1 RNAi line, resulting in the presence of many lagging chromosomes. The outcome indicated that OsMND1 plays a critical role in stabilizing meiosis, improving pollen fertility and reducing early embryo abortions, ultimately increasing the seed set rate. Additionally, OsMND1 affected some key meiosis-related gene expression levels. These results raise interesting issues in polyploid breeding theory and application, which require integrated solutions in the future.

SUMMARY: 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.