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.

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.

Comparative cytological and transcriptome analyses of ny2 mutant delayed degeneration of tapetal cells and promotes abnormal microspore development in neo-tetraploid rice.

We aimed to investigate the genetic defects related to pollen development and infertility in NY2, a novel tetraploid rice germplasm known as Neo-tetraploid rice. This rice variety was created through the crossbreeding and selective breeding of various autotetraploid rice lines and has previously shown high fertility. Our previous research has revealed that the NY2 gene, encoding a eukaryotic translation initiation factor 3 subunit E, regulates pollen fertility. However, the underlying mechanism behind this fertility is yet to be understood. To shed light on this matter, we performed a combined cytological and transcriptome analysis of the NY2 gene. Cytological analysis indicated that ny2 underwent abnormal tapetal cells, microspore, and middle layer development, which led to pollen abortion and ultimately to male sterility. Genetic analysis revealed that the F1 plants showed normal fertility and an obvious advantage for seed setting compared to ny2. Global gene expression analysis in ny2 revealed a total of 7545 genes were detected at the meiosis stage, and 3925 and 3620 displayed upregulation and downregulation, respectively. The genes were significantly enriched for the gene ontology (GO) term "carbohydrate metabolic process. Moreover, 9 genes related to tapetum or pollen fertility showed down-regulation, such as OsABCG26 (ATP Binding Cassette G26), TMS9-1 (Thermosensitive Male Sterility), EAT1 (Programmed cell death regulatory), KIN14M (Kinesin Motor), OsMT1a (Metallothionein), and OsSTRL2 (Atypical strictosidine synthase), which were validated by qRT-PCR. Further analyses of DEGs identified nine down-regulated transcription factor genes related to pollen development. NY2 is an important regulator of the development of tapetum and microspore. The regulatory gene network described in this study may offer important understandings into the molecular processes that underlie fertility control in tetraploid rice.

Nitrogen sources affected the biosynthesis of 2-acetyl-1-pyrroline, cooked rice elongation and amylose content in rice.

Many studies have been carried out on N sources effect on fragrant rice; however, their impact on rice grain quality is largely unclear. In this study, we evaluated the effects of different types of N sources on rice growth, yield, 2-acetyl-1-pyrroline (2AP), amylose and cooked rice elongation. Two indica rice cultivars, Basmati 385 (B385), Xiangyaxiangzhan (XYXZ) and two japonica cultivars, Yunjingyou (YJY), Daohuaxiang (DHX) were grown in experimental pots with six replications under four N sources: Potassium nitrate (KNO3), ammonium bicarbonate (NH4HCO3), urea (H2NCONH2) and sodium nitrate (NaNO3) in 2019 and 2020 early seasons. Our results showed that N dynamics regulated the number of panicles, 1000-grain weight, grain yield, 2-acetyl-1-pyrroline, amylose and cooked rice elongation across all the four treatments. The NH4HCO3 treatment significantly increased the number of panicles and grain yield across the four rice varieties compared with KNO3, H2NCONH2 and NaNO3 N sources in both 2019 and 2020 early season, The KNO3 treatment significantly showed higher 1000-grain weight in B-385, YJY, XYXZ and DHX compared to other N sources. Compared with other N sources treatment, the NH4HCO3 treatments significantly increased the 2AP contents in heading stage leaves, matured leaves and grains of B-385, YJY, XYXZ and DHX respectively. Cooked rice elongation percentage also showed significant difference in all treatments studied with KNO3 recorded the highest across the four varieties. Analysis of major enzymes and compounds such as P5C, P5CS, PDH, Pyrroline, proline and Methylglyoxal showed remarkable differences in each cultivar at heading and maturity stages with higher activity in NH4HCO3 and H2NCONH2 treatments. Similarly, in all treatments, we also observed significant increase in amylose content percentage, with NH4HCO3 having greater percentage of amylose.