Pollen Number and Ribosome Gene Expression Altered in a Genome-Editing Mutant of REDUCED POLLEN NUMBER1 Gene

The number of pollen grains varies within and between species. However, little is known about the molecular basis of this quantitative trait, in contrast with the many studies available on cell differentiation in the stamen. Recently, the first gene responsible for pollen number variation, REDUCED POLLEN NUMBER1 (RDP1), was isolated by genome-wide association studies of Arabidopsis thaliana and exhibited the signature of natural selection. This gene encodes a homolog of yeast Mrt4 (mRNA turnover4), which is an assembly factor of the large ribosomal subunit. However, no further data were available to link ribosome function to pollen development. Here, we characterized the RDP1 gene using the standard A. thaliana accession Col-0. The frameshift mutant, rdp1-3 generated by CRISPR/Cas9 revealed the pleiotropic effect of RDP1 in flowering, thus demonstrating that this gene is required for a broad range of processes other than pollen development. We found that the natural Col-0 allele conferred a reduced pollen number against the Bor-4 allele, as assessed using the quantitative complementation test, which is more sensitive than transgenic experiments. Together with a historical recombination event in Col-0, which was identified by sequence alignment, these results suggest that the coding sequence of RDP1 is the candidate region responsible for the natural phenotypic variation. To elucidate the biological processes in which RDP1 is involved, we conducted a transcriptome analysis. We found that genes responsible for ribosomal large subunit assembly/biogenesis were enriched among the differentially regulated genes, which supported the hypothesis that ribosome biogenesis is disturbed in the rdp1-3 mutant. Among the pollen-development genes, three key genes encoding basic helix-loop-helix (bHLH) transcription factors (ABORTED MICROSPORES (AMS), bHLH010, and bHLH089), as well as direct downstream genes of AMS, were downregulated in the rdp1-3 mutant. In summary, our results suggest a specialized function of ribosomes in pollen development through RDP1, which harbors natural variants under selection.

Calreticulin expression and localization in relation to exchangeable Ca2+ during pollen development in Petunia

Background Pollen development in the anther in angiosperms depends on complicated cellular interactions associated with the expression of gametophytic and sporophytic genes which control fundamental processes during microsporo/gametogenesis, such as exo/endocytosis, intracellular transport, cell signaling, chromatin remodeling, and cell division. Most if not all of these cellular processes depend of local concentration of calcium ions (Ca2+). Work from our laboratory and others provide evidence that calreticulin (CRT), a prominent Ca2+-binding/buffering protein in the endoplasmic reticulum (ER) of eukaryotic cells, may be involved in pollen formation and function. Here, we show for the first time the expression pattern of the PhCRT1 gene and CRT accumulation in relation to exchangeable Ca2+ in Petunia hybrida developing anther, and discuss probable roles for this protein in the male gametophyte development. Results Using northern hybridization, western blot analysis, fluorescent in situ hybridization (FISH), immunocytochemistry, and potassium antimonate precipitation, we report that PhCRT1 is highly expressed in the anther and localization pattern of the CRT protein correlates with loosely bound (exchangeable) Ca2+ during the successive stages of microsporo/gametogenesis. We confirmed a permanent presence of both CRT and exchangeable Ca2+ in the germ line and tapetal cells, where these factors preferentially localized to the ER which is known to be the most effective intracellular Ca2+ store in eukaryotic cells. In addition, our immunoblots revealed a gradual increase in CRT level from the microsporocyte stage through the meiosis and the highest CRT level at the microspore stage, when both microspores and tapetal cells show extremely high secretory activity correlated with the biogenesis of the sporoderm. Conclusion Our present data provide support for a key role of CRT in developing anther of angiosperms – regulation of Ca2+ homeostasis during pollen grains formation. This Ca2+-buffering chaperone seems to be essential for pollen development and maturation since a high rate of protein synthesis and protein folding within the ER as well as intracellular Ca2+ homeostasis are strictly required during the multi-step process of pollen development.

TaEXPB5 is responsible for male fertility in thermo-sensitive male-sterility wheat with Aegilops kotschyi cytoplasm

Thermo-sensitive male sterility is of vital importance to heterosis, or hybrid vigor in crop production and hybrid breeding. Therefore, it is meaningful to study the function of the genes related to pollen development and male sterility, which is still not fully understand currently. Here, we conducted comparative analyses to screen fertility related genes using RNA-seq, iTRAQ, and PRM-based assay. A gene encoding expansin protein in wheat, TaEXPB5, was isolated in KTM3315A, which was in the cell wall and preferentially upregulated expression in the fertility anthers. The silencing of TaEXPB5 displayed pollen abortion, the declination or sterility of fertility. Further, cytological investigation indicated that the silencing of TaEXPB5 induced the early degradation of tapetum and abnormal development of pollen wall. These results revealed that the silencing of TaEXPB5 could eliminate the effects of temperature on male fertility, and resulting in functional loss of fertility conversion, which implied that TaEXPB5 may be essential for anther or pollen development and male fertility of KTM3315A. These findings provide a novel insight into molecular mechanism of fertility conversion for thermo-sensitive cytoplasmic male-sterility wheat, and contribute to the molecular breeding of hybrid wheat in the future.

bHLH Transcription Factor NtMYC2a Regulates Carbohydrate Metabolism during the Pollen Development of Tobacco (Nicotiana tabacum L. cv. TN90)

Basic helix-loop-helix (bHLH) transcription factor MYC2 regulates plant growth and development in many aspects through the jasmonic acid (JA) signaling pathway, while the role of MYC2 in plant carbohydrate metabolism has not been reported. Here, we generated NtMYC2a-overexpressing (NtMYC2a-OE) and RNA-interference-mediated knockdown (NtMYC2a-RI) transgenic plants of tobacco (Nicotiana tabacum L. cv. TN90) to investigate the role of NtMYC2a in carbohydrate metabolism and pollen development. Results showed that NtMYC2a regulates the starch accumulation and the starch-sugar conversion of floral organs, especially in pollen. The RT-qPCR analysis showed that the expression of starch-metabolic-related genes, AGPs, SS2 and BAM1, were regulated by NtMYC2a in the pollen grain, anther wall and ovary of tobacco plants. The process of pollen maturation was accelerated in NtMYC2a-OE plants and was delayed in NtMYC2a-RI plants, but the manipulation of NtMYC2a expression did not abolish the pollen fertility of the transgenic plants. Intriguingly, overexpression of NtMYC2a also enhanced the soluble carbohydrate accumulation in tobacco ovaries. Overall, our results demonstrated that the bHLH transcription factor NtMYC2a plays an important role in regulating the carbohydrate metabolism during pollen maturation in tobacco.

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.

Identification of miRNAs and their target genes in genic male sterility lines in Brassica napus by small RNA sequencing

Background Brassica napus is the third leading source of edible oil in the world. Genic male sterility (GMS) lines provide crucial material for harnessing heterosis for rapeseed. GMS lines have been used successfully for rapeseed hybrid production in China. MicroRNAs (miRNAs) play crucial regulatory roles in various plant growth, development, and stress response processes. However, reports on miRNAs that regulate the pollen development of GMS lines in B. napus are few. Results In this study, 12 small RNA and transcriptome libraries were constructed and sequenced for the flower buds from the fertile and sterile lines of two recessive GMS (RGMS) lines, namely, “6251AB” and “6284AB”. At the same time, 12 small RNA and transcriptome libraries were also constructed and sequenced for the flower buds from the fertile and sterile lines of two dominant GMS (DGMS) lines, namely, “4001AB” and “4006AB”. Based on the results, 46 known miRNAs, 27 novel miRNAs on the other arm of known pre-miRNAs, and 44 new conserved miRNAs were identified. Thirty-five pairs of novel miRNA-3p/miRNA-5p were found. Among all the identified miRNAs, fifteen differentially expressed miRNAs with over 1.5-fold change between flower buds of sterile and fertile lines were identified, including six differentially expressed miRNAs between “4001A” and “4001B”, two differentially expressed miRNAs between “4006A” and “4006B”, four differentially expressed miRNAs between “6251A” and “6251B”, and ten differentially expressed miRNAs between “6284A” and “6284B”. The correlation analysis of small RNA and transcriptome sequencing was conducted. And 257 candidate target genes were predicted for the 15 differentially expressed miRNAs. The results of 5′ modified RACE indicated that BnaA09g48720D, BnaA09g11120D, and BnaCnng51960D were cleaved by bna-miR398a-3p, bna-miR158-3p and bna-miR159a, respectively. Among the differentially expressed miRNAs, miR159 was chosen to analyze its function. Overexpression of bna-miR159 in Arabidopsis resulted in decreased seed setting rate, and shortened siliques, illustrating that miR159 may regulate the fertility and silique development in rapeseed. Conclusions Our findings provide an overview of miRNAs that are potentially involved in GMS and pollen development. New information on miRNAs and their related target genes are provided to exploit the GMS mechanism and reveal the miRNA networks in B. napus.

Grain Dispersal Mechanism In Cereals Arose From a Genome Duplication Followed By Changes in Spatial Expression of Genes Involved In Pollen Development

One of the most critical events in the process of cereal domestication was the loss of the natural mode of grain dispersal. Grain dispersal in barley is controlled by two major genes, Btr1 and Btr2, which affect the thickness of cell walls around the disarticulation zone. The barley genome also encodes Btr1-like and Btr2-like genes, which have been shown to be the ancestral copies. While Btr and Btr-like genes are non-redundant, the biological function of Btr-like genes is unknown. We explored the potential biological role of the Btr-like genes by surveying their expression profile across 212 publicly available transcriptome datasets representing diverse organs, developmental stages and stress conditions. We found that Btr1-like and Btr2-like are expressed exclusively in immature anther samples throughout Prophase I of meiosis within the meiocyte. The similar and restricted expression profile of these two genes suggests they are involved in a common biological function. Further analysis revealed 141 genes co-expressed with Btr1-like and 122 genes co-expressed with Btr2-like, with 105 genes in common, supporting Btr-like genes involvement in a shared molecular pathway. We hypothesize that the Btr-like genes play a crucial role in pollen development by facilitating the formation of the callose wall around the meiocyte or in the secretion of callase by the tapetum. Our data suggest that Btr genes retained an ancestral function in cell wall modification and gained a new role in grain dispersal due to changes in their spatial expression becoming spike specific after gene-duplication.

A 31-bp indel localised in the 5' untranslated region of OsSUT3 affects the gene expression and rice (Oryza sativa L.) pollen development

OsSUT genes have been demonstrated to be relevant for diverse biological processes in rice. In this study, we identified the close relationship between a 31-bp insertion in a 5' untranslated region (5' UTR) of the OsSUT3 gene and higher OsSUT3 expression in rice panicles by qRT-PCR and transgenic research. Statistically significant results (P < 0.01) were found for this 31-bp insertions/deletions (indels) in the rice pollen development and other panicle traits, such as the pollen number, pollen fertility, seeding rate, and grain length. An evolution analysis showed that the proportion of the 31-bp insertion significantly increases in rice domestication. Therefore, the 31-bp Indel could be considered as a convenient molecular marker to screen more pollen and better panicle traits in rice breeding.