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

Abstract

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.

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

“…The criteria for evaluating these characteristics were based on Guo et al (2017). Data analysis was performed according to Kamara et al (2022).…”

“…Total RNA of each sample was extracted according to Chen et al (2018). The variations in gene expression between the samples were recorded according to Kamara et al (2022).…”

“…The Roche Transcriptor First Strand cDNA Synthesis Kit was used to perform the reverse transcription. The qRT-PCR experiment was executed according to the procedure defined by Kamara et al (2022). The gene expression patterns were estimated by 2 -DDCt method (Livak and Schmittgen, 2001).…”

“…A cross-section of the ny2 and WT anthers at diverse pollen development phases was analyzed according to Kamara et al (2022) to unveil the cytological abnormalities in anther pollen development. Stage 7 transverse sections of ny2 and WT anthers showed no discernible differences, and both showed a typical structure of all layers (Figures 3A, E).…”

Cytological observation and RNA-seq analysis reveal novel miRNAs high expression associated with the pollen fertility of neo-tetraploid rice

“…Neo-tetraploid lines display over 80% seed set and high heterosis, harbor wide-compatibility and neutral pollen fertility genes, and novel specific alleles [ 19 , 21 , 24 – 27 ]. So far, more than 10 genes have been found to participate in the fertility of neo-tetraploid rice, including kin7l , bzr3 , nrfg4 , MOF1a , HSP101–1 , NY1 , and NY2 [ 23 , 25 , 26 , 28 , 29 ]. However, little information about fertility regulation by microRNAs (miRNAs) is available in neo-tetraploid rice.…”