Anther Development Research Articles

Identification of heat-induced proteomes in meiotic pollen mother cells of tomato 'Maxifort' using single-cell-type tandem mass tag (TMT) proteomics

Heat stress (HS) poses a significant challenge to tomato production due to disruption of the reproductive organs, especially the male gametophytes. This study reports HS-induced proteome changes in meiotic pollen mother cells during early stages of anther development. Tomato (<em>Solanum lycopersicum</em> L. × <em>S. habrochaites</em>) &#39;Maxifort&#39; were grown in a heated polytunnel in Nashville, Tennessee, USA. Plants at flowering stage were subjected to heat treatment at 40 ± 2 °C for 4 hr (11:00−15:00 HR); and the non-heat-treated control was at 30 ± 2 °C (day/night) at the same period of time for 10 d. The size of the flower buds containing meiotic pollen mother cells was determined based on the histology of DAPI stained cross section of anthers. Flower buds were embedded in optimal cutting temperature solutions (OCT) and then cut into sections of 20 µm thickness. Sections containing meiotic pollen mother cells were collected using laser capture microdissection (LCM). A protein extraction procedure was optimized for the LCM collected pollen samples which yielded 25−30 μg protein from 150,000−200,000 pollen cells. The heat-induced proteomes of meiotic pollen mother cells were quantified using tandem mass tag (TMT) quantitative proteomics analysis. Among the 6,343 quantified proteins, 254 differentially expressed proteins (DEPs) showed significant differences in abundance level from heat treated to non-heat-treated control conditions. The heat-up-regulated-DEPs (96 proteins) include heat shock proteins, calreticulin and exocytosis (synaptobrevin) which are involved in protein folding/refolding/targeting/removal and secretion of aggregated and damaged proteins/peptides. The heat-down-regulated-DEPs (158 proteins) were involved in pathways of ubiquitin-mediated protein degradation, antioxidant mechanism, and metabolic processes of carbohydrates and lipids. Proteins affecting apoptotic programmed cell death and pollen mother cell meiotic activity were significantly changed under HS. The identified proteins and the affected biological processes could represent the major heat tolerance mechanisms during early developmental stages of male gametophyte when exposed to daily periods of above 40 °C HS condition.

Integrated analysis of small RNA, transcriptome and degradome sequencing reveals that micro-RNAs regulate anther development in CMS cotton

Cytoplasmic male sterility (CMS) is an important phenotypic trait for heterosis utilization in cotton (Gosspium hirsutum L.). Micro-RNAs (miRNAs) play crucial roles in the pollen development of plants. To elucidate the roles of miRNA-target gene pairs in the CMS cotton line ganmian15A and its maintainer line ganmian15B, an integrated small RNA (sRNA), transcriptome, and degradome sequencing analysis of these lines at the pollen abortion stage was performed in the present research. A total of 339 known miRNAs and 961 conserved miRNAs were identified through high-throughput sequencing, among which 124 were differentially expressed between ganmian15A and ganmian15B. By comprehensive analysis of miRNA, transcriptome, and degradome sequencing, we identified 11 pairs of miRNAs/target genes at the pollen abortion stage. Among them, 9 miRNAs (4 novel and 5 known) were found for the first time to be associated with the occurrence of male sterility in cotton. The target genes of miRna-38 and miRna-23 were linked to cell wall development at the pollen stage. They may play an imperative role in CMS cotton by delaying the degradation of tapetal cells. Our findings revealed new insights into the genetic control of CMS occurrence, which will support the breeding of new sterile lines of cotton in the future.

Combined Transcriptome and Proteome Analysis of Anthers of AL-type Cytoplasmic Male Sterile Line and Its Maintainer Line Reveals New Insights into Mechanism of Male Sterility in Common Wheat.

Cytoplasmic male sterility (CMS) plays an essential role in hybrid seeds production. In wheat, orf279 was reported as a CMS gene of AL-type male sterile line (AL18A), but its sterility mechanism is still unclear. Therefore, transcriptomic and proteomic analyses of the anthers of AL18A and its maintainer line (AL18B) were performed to interpret the sterility mechanism. Results showed that the electron transport chain and ROS scavenging enzyme expression levels changed in the early stages of the anther development. Biological processes, i.e., fatty acid synthesis, lipid transport, and polysaccharide metabolism, were abnormal, resulting in pollen abortion in AL18A. In addition, we identified several critical regulatory genes related to anther development through combined analysis of transcriptome and proteome. Most of the genes were enzymes or transcription factors, and 63 were partially homologous to the reported genic male sterile (GMS) genes. This study provides a new perspective of the sterility mechanism of AL18A and lays a foundation to study the functional genes of anther development.

Integrated Isoform Sequencing and Dynamic Transcriptome Analysis Reveals Diverse Transcripts Responsible for Low Temperature Stress at Anther Meiosis Stage in Rice.

Low temperatures stress is one of the important factors limiting rice yield, especially during rice anther development, and can cause pollen sterility and decrease grain yield. In our study, low-temperature stress decreased pollen viability and spikelet fertility by affecting the sugar, nitrogen and amino acid contents of anthers. We performed RNA-seq and ISO-seq experiments to study the genome-wide transcript expression profiles in low-temperature anthers. A total of 4,859 differentially expressed transcripts were detected between the low-temperature and control groups. Gene ontology enrichment analysis revealed significant terms related to cold tolerance. Hexokinase and glutamate decarboxylase participating in starch and sucrose metabolism may play important roles in the response to cold stress. Using weighted gene co-expression network analysis, nine hub transcripts were found that could improve cold tolerance throughout the meiosis period of rice: Os02t0219000-01 (interferon-related developmental regulator protein), Os01t0218350-00 (tetratricopeptide repeat-containing thioredoxin), Os08t0197700-00 (luminal-binding protein 5), Os11t0200000-01 (histone deacetylase 19), Os03t0758700-01 (WD40 repeat domain-containing protein), Os06t0220500-01 (7-deoxyloganetin glucosyltransferase), Pacbio.T01382 (sucrose synthase 1), Os01t0172400-01 (phospholipase D alpha 1), and Os01t0261200-01 (NAC domain-containing protein 74). In the PPI network, the protein minichromosome maintenance 4 (MCM4) may play an important role in DNA replication induced by cold stress.

Comparative Transcriptome Analysis of the Anthers from the Cytoplasmic Male-Sterile Pepper Line HZ1A and Its Maintainer Line HZ1B

Cytoplasmic male-sterility (CMS) is important for the utilization of crop heterosis and study of the molecular mechanisms involved in CMS could improve breeding programs. In the present study, anthers of the pepper CMS line HZ1A and its maintainer line HZ1B were collected from stages S1, S2, and S3 for transcriptome sequencing. A total of 47.95 million clean reads were obtained, and the reads were assembled into 31,603 unigenes. We obtained 42 (27 up-regulated and 15 down-regulated), 691 (346 up-regulated and 345 down-regulated), and 709 (281 up-regulated and 428 down-regulated) differentially expressed genes (DEGs) in stages S1, S2, and S3, respectively. Through Gene Ontology (GO) analysis, the DEGs were found to be composed of 46 functional groups. Two GO terms involved in photosynthesis, photosynthesis (GO:0015986) and photosystem I (GO:0009522), may be related to CMS. Through Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis, oxidative phosphorylation (ko00190) and phenylpropanoid biosynthesis (ko00940) were significantly enriched in the S1 and S2 stages, respectively. Through the analysis of 104 lipid metabolism-related DEGs, four significantly enriched KEGG pathways may help to regulate male sterility during anther development. The mitochondrial genes orf470 and atp6 were identified as candidate genes of male sterility for the CMS line HZ1A. Overall, the results will provide insights into the molecular mechanisms of pepper CMS.

Investigation of the interaction of a papain-like cysteine protease (RD19c) with selenium-binding protein 1 (SBP1) in Arabidopsis thaliana

AtRD19c is a member of the papain-like cysteine proteases known for its participation in anther development after its maturation by βVPE (vacuolar processing enzyme). This papain-like cysteine protease was identified as an interacting protein of AtSBP1 (selenium binding protein 1) in a yeast two-hybrid screening. To confirm this interaction, we studied AtRD19c with respect to its expression and ability to interact with AtSBP1. The highest gene expression levels of AtRD19c were observed in the roots of 10-day-old seedlings, whereas minimum levels appeared in the hypocotyls of 10-day-old seedlings and flowers. AtRD19c expression was upregulated by selenium, and analysis of its promoter activity showed colocalization of a reporter gene (GUS) with AtSBP1. Additionally, the AtRD19c expression pattern was upregulated in the presence of selenite, indicating its participation in the Se response network. Confocal fluorescence microscopy revealed that AtRD19c localizes in the root tip, lateral roots, and leaf trichomes. Finally, we confirmed the physical interaction between AtRD19c and AtSBP1 and showed the importance of the first 175 aa of the AtSBP1 polypeptide in this interaction. Importantly, the AtRD19c-AtSBP1 interaction was also demonstrated in planta by employing bimolecular fluorescent complementation (BiFC) in a protoplast system.

Disruption of the bHLH transcription factor Abnormal Tapetum 1 causes male sterility in watermelon

Although male sterility has been identified as a useful trait for hybrid vigor utilization and hybrid seed production, its underlying molecular mechanisms in Cucurbitaceae species are still largely unclear. Here, a spontaneous male-sterile watermelon mutant, Se18, was reported to have abnormal tapetum development, which resulted in completely aborted pollen grains. Map-based cloning demonstrated that the causal gene Citrullus lanatus Abnormal Tapetum 1 (ClATM1) encodes a basic helix-loop-helix (bHLH) transcription factor with a 10-bp deletion and produces a truncated protein without the bHLH interaction and functional (BIF) domain in Se18 plants. qRT–PCR and RNA in situ hybridization showed that ClATM1 is specifically expressed in the tapetum layer and in microsporocytes during stages 6–8a of anther development. The genetic function of ClATM1 in regulating anther development was verified by CRISPR/Cas9-mediated mutagenesis. Moreover, ClATM1 was significantly downregulated in the Se18 mutant, displaying a clear dose effect at the transcriptional level. Subsequent dual-luciferase reporter, β-glucuronidase (GUS) activity, and yeast one-hybrid assays indicated that ClATM1 could activate its own transcriptional expression through promoter binding. Collectively, ClATM1 is the first male sterility gene cloned from watermelon, and its self-regulatory activity provides new insights into the molecular mechanism underlying anther development in plants.

DCET1 Controls Male Sterility Through Callose Regulation, Exine Formation, and Tapetal Programmed Cell Death in Rice.

In angiosperms, anther development comprises of various complex and interrelated biological processes, critically needed for pollen viability. The transitory callose layer serves to separate the meiocytes. It helps in primexine formation, while the timely degradation of tapetal cells is essential for the timely callose wall dissolution and pollen wall formation by providing nutrients for pollen growth. In rice, many genes have been reported and functionally characterized that are involved in callose regulation and pollen wall patterning, including timely programmed cell death (PCD) of the tapetum, but the mechanism of pollen development largely remains ambiguous. We identified and functionally characterized a rice mutant dcet1, having a complete male-sterile phenotype caused by defects in anther callose wall, exine patterning, and tapetal PCD. DCET1 belongs to the RNA recognition motif (RRM)-containing family also called as the ribonucleoprotein (RNP) domain or RNA-binding domain (RBD) protein, having single-nucleotide polymorphism (SNP) substitution from G (threonine-192) to A (isoleucine-192) located at the fifth exon of LOC_Os08g02330, was responsible for the male sterile phenotype in mutant dcet1. Our cytological analysis suggested that DCET1 regulates callose biosynthesis and degradation, pollen exine formation by affecting exine wall patterning, including abnormal nexine, collapsed bacula, and irregular tectum, and timely PCD by delaying the tapetal cell degeneration. As a result, the microspore of dcet1 was swollen and abnormally bursted and even collapsed within the anther locule characterizing complete male sterility. GUS and qRT-PCR analysis indicated that DCET1 is specifically expressed in the anther till the developmental stage 9, consistent with the observed phenotype. The characterization of DCET1 in callose regulation, pollen wall patterning, and tapetal cell PCD strengthens our knowledge for knowing the regulatory pathways involved in rice male reproductive development and has future prospects in hybrid rice breeding.

Tapetal 3-Ketoacyl-Coenzyme A Synthases Are Involved in Pollen Coat Lipid Accumulation for Pollen-Stigma Interaction in Arabidopsis.

Pollen coat lipids form an outer barrier to protect pollen itself and play essential roles in pollen-stigma interaction. However, the precise molecular mechanisms underlying the production, deposition, regulation, and function of pollen coat lipids during anther development remain largely elusive. In lipid metabolism, 3-ketoacyl-coenzyme A synthases (KCS) are involved in fatty acid elongation or very-long-chain fatty acid (VLCFA) synthesis. In this study, we identified six members of the Arabidopsis KCS family expressed in anther. Among them, KCS7, KCS15, and KCS21 were expressed in tapetal cells at anther stages 8–10. Further analysis demonstrated that they act downstream of male sterility 1 (MS1), a regulator of late tapetum development. The kcs7/15/21 triple mutant is fertile. Both cellular observation and lipid staining showed pollen coat lipid was decreased in kcs7/15/21 triple mutant. After landing on stigma, the wild-type pollen grains were hydrated for about 5 min while the kcs7/15/21 triple mutant pollen took about 10 min to hydrate. Pollen tube growth of the triple mutant was also delayed. These results demonstrate that the tapetum-localized KCS proteins are involved in the accumulation of pollen coat lipid and reveal the roles of tapetal-derived pollen coat lipid for pollen-stigma interaction.

Gene expression profiling by cDNA‐AFLP reveals potential candidate genes for imazapyr‐induced male sterility in sunflower

AbstractChemically induced male sterility (CIMS) is a valuable tool for hybrid breeding in crops. In this context, the acetohydroxyacid synthase (AHAS)‐inhibiting imazapyr has been proposed to induce male sterility in Imisun sunflower (Helianthus annuus L.) when applied during early reproductive development in a two‐fold higher than recommended field dose. This study aimed to perform a comparative transcriptomic analysis to find out the molecular bases of the gametocidal effect of imazapyr on two Imisun genotypes differing in their herbicide resistance level: completely resistant and intermediate resistant. The CIMS of treated plants was confirmed at physiological and anatomical levels. A total of 948 differentially expressed transcript‐derived fragments (TDFs) were identified by complementary DNA amplified fragment‐length polymorphism (cDNA‐AFLP) analysis at the three most informative developmental stages of anther development. Several genes related to xenobiotic metabolism and stress, including cytochrome P450 monooxygenases and ATP‐binding cassette transporters, were identified in the completely resistant genotype. Interestingly, a group of genes, including protein kinases, sulfonylurea receptor 1, aspartic signal peptide‐peptidase, and specific membrane transporters, were recognized from the differential TDFs simultaneously detected in both genotypes. Some of these transcripts were validated by quantitative real‐time polymerase chain reaction (qPCR) in both genotypes. Therefore, these transcripts could be exclusively related to the CIMS in sunflower. Based on the results, we suggest a probable model of action for the gametocidal effect of imazapyr. Our research provides new insights into the molecular mechanisms of male sterility induction in sunflower.

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.

Molecular Analysis Uncovers the Mechanism of Fertility Restoration in Temperature-Sensitive Polima Cytoplasmic Male-Sterile Brassica napus.

Temperature-sensitive male sterility is a heritable agronomic trait affected by genotype-environment interactions. In rapeseed (Brassica napus), Polima (pol) temperature-sensitive cytoplasmic male sterility (TCMS) is commonly used for two-line breeding, as the fertility of pol TCMS lines can be partially restored at certain temperatures. However, little is known about the underlying molecular mechanism that controls fertility restoration. Therefore, we aimed to investigate the fertility conversion mechanism of the pol TCMS line at two different ambient temperatures (16 °C and 25 °C). Our results showed that the anthers developed and produced vigorous pollen at 16 °C but not at 25 °C. In addition, we identified a novel co-transcript of orf224-atp6 in the mitochondria that might lead to fertility conversion of the pol TCMS line. RNA-seq analysis showed that 1637 genes were significantly differentially expressed in the fertile flowers of 596-L when compared to the sterile flower of 1318 and 596-H. Detailed analysis revealed that differentially expressed genes were involved in temperature response, ROS accumulation, anther development, and mitochondrial function. Single-molecule long-read isoform sequencing combined with RNA sequencing revealed numerous genes produce alternative splicing transcripts at high temperatures. Here, we also found that alternative oxidase, type II NAD(P)H dehydrogenases, and transcription factor Hsfs might play a crucial role in male fertility under the low-temperature condition. RNA sequencing and bulked segregant analysis coupled with whole-genome sequencing identified the candidate genes involved in the post-transcriptional modification of orf224. Overall, our study described a putative mechanism of fertility restoration in a pol TCMS line controlled by ambient temperature that might help utilise TCMS in the two-line breeding of Brassica crops.

The proteome of developing barley anthers during meiotic prophase I

Flowering plants reproduce sexually by combining a haploid male and female gametophyte during fertilization. Male gametophytes are localized in the anthers, each containing reproductive (meiocyte) and non-reproductive tissue necessary for anther development and maturation. Meiosis, where chromosomes pair and exchange their genetic material during a process called recombination, is one of the most important and sensitive stages in breeding, ensuring genetic diversity. Most anther development studies have focused on transcript variation, but very few have been correlated with protein abundance. Taking advantage of a recently published barley anther transcriptomic (BAnTr) dataset and a newly developed sensitive mass spectrometry-based approach to analyse the barley anther proteome, we conducted high-resolution mass spectrometry analysis of barley anthers, collected at six time points and representing their development from pre-meiosis to metaphase. Each time point was carefully staged using immunocytology, providing a robust and accurate staging mirroring our previous BAnTr dataset. We identified >6100 non-redundant proteins including 82 known and putative meiotic proteins. Although the protein abundance was relatively stable throughout prophase I, we were able to quantify the dynamic variation of 336 proteins. We present the first quantitative comparative proteomics study of barley anther development during meiotic prophase I when the important process of homologous recombination is taking place.

Anatomical Characteristics of Petalized Anther Abortion in Male Sterile Camellia oleifera Plants

Petalized anther abortion is an important characteristic of male sterility in plants. The male sterile plants (HB-21) evincing petalized anther abortion previously discovered in a clone population of the Camellia oleifera cultivar Huashuo by our research group were selected as the experimental material in this study. Using plant microscopy and anatomic methods and given the correspondence between external morphology and internal structure, we studied the anatomic characteristics of petalized anther abortion (with a fertile plant as the control group) in various stages, from flower bud differentiation to anther maturity, in hopes of providing a theoretical basis for research on and applications of male sterile C. oleifera plants, a new method for the selection of male sterile C. oleifera cultivars, and improvements in the yield and quality of C. oleifera. In this study, the development of anthers in C. oleifera was divided into 14 stages. Petalized anther abortion in male sterile plants was mainly initiated in the second stage (the stage of sporogenous cells). Either the petalized upper anther parts did not form pollen sacs, or the entire anthers did not form pollen sacs. The lower parts of some anthers could form deformed pollen sacs and develop, and these anthers could be roughly divided into two types: fully and partially petalized anthers. Abnormal callose and the premature degradation of the tapetum occurred in the pollen sacs formed by partially petalized anthers during the development process, resulting in the absence of inclusions in the pollen grains formed. Small quantities of mature pollen grains withered inward from the germinal furrows, exhibiting obvious abortion characteristics. The relative in vitro germination rate of the pollen produced by the partially petalized anthers of sterile plants was 11.20%, and the relative activity of triphenyltetrazolium chloride was 3.24%, while the fully petalized anthers did not generate pollen grains. Either the petalized anthers in male sterile plants did not produce pollen, or the vitality of the small amounts of pollen produced by sterile plants was very low compared with that of fertile plants. Such male sterile plants could be used to select correct clones and have good prospects for application in production.

Characterization of germline development and identification of genes associated with germline specification in pineapple

Understanding germline specification in plants could be advantageous for agricultural applications. In recent decades, substantial efforts have been made to understand germline specification in several plant species, including Arabidopsis, rice, and maize. However, our knowledge of germline specification in many agronomically important plant species remains obscure. Here, we characterized the female germline specification and subsequent female gametophyte development in pineapple using callose staining, cytological, and whole-mount immunolocalization analyses. We also determined the male germline specification and gametophyte developmental timeline and observed male meiotic behavior using chromosome spreading assays. Furthermore, we identified 229 genes that are preferentially expressed at the megaspore mother cell (MMC) stage during ovule development and 478 genes that are preferentially expressed at the pollen mother cell (PMC) stage of anther development using comparative transcriptomic analysis. The biological functions, associated regulatory pathways and expression patterns of these genes were also analyzed. Our study provides a convenient cytological reference for exploring pineapple germline development and a molecular basis for the future functional analysis of germline specification in related plant species.

PsEND1 Is a Key Player in Pea Pollen Development Through the Modulation of Redox Homeostasis.

Redox homeostasis has been linked to proper anther and pollen development. Accordingly, plant cells have developed several Reactive Oxygen Species (ROS)-scavenging mechanisms to maintain the redox balance. Hemopexins constitute one of these mechanisms preventing heme-associated oxidative stress in animals, fungi, and plants. Pisum sativum ENDOTHECIUM 1 (PsEND1) is a pea anther-specific gene that encodes a protein containing four hemopexin domains. We report the functional characterization of PsEND1 and the identification in its promoter region of cis-regulatory elements that are essential for the specific expression in anthers. PsEND1 promoter deletion analysis revealed that a putative CArG-like regulatory motif is necessary to confer promoter activity in developing anthers. Our data suggest that PsEND1 might be a hemopexin regulated by a MADS-box protein. PsEND1 gene silencing in pea, and its overexpression in heterologous systems, result in similar defects in the anthers consisting of precocious tapetum degradation and the impairment of pollen development. Such alterations were associated to the production of superoxide anion and altered activity of ROS-scavenging enzymes. Our findings demonstrate that PsEND1 is essential for pollen development by modulating ROS levels during the differentiation of the anther tissues surrounding the microsporocytes.

Transcriptomic analysis reveals candidate genes for male sterility in Prunus sibirica.

BackgroundThe phenomenon of male sterility widely occurs in Prunus sibirica and has a serious negative impact on yield. We identified the key stage and cause of male sterility and found differentially expressed genes related to male sterility in Prunus sibirica, and we analyzed the expression pattern of these genes. This work aimed to provide valuable reference and theoretical basis for the study of reproductive development and the mechanisms of male sterility in Prunus sibirica.MethodThe microstructures of male sterile flower buds and male fertile flower buds were observed by paraffin section. Transcriptome sequencing was used to screen genes related to male sterility in Prunus sibirica. Quantitative real-time PCR analysis was performed to verify the transcriptome data.ResultsAnther development was divided into the sporogenous cell stage, tetrad stage, microspore stage, and pollen maturity stage. Compared with male fertile flower buds, in the microspore stage, the pollen sac wall tissue in the male sterile flower buds showed no signs of degeneration. In the pollen maturity stage, the tapetum and middle layer were not fully degraded, and anther development stopped. Therefore, the microspore stage was the key stage for anther abortion , and the pollen maturity stage was the post stage for anther abortion. A total of 4,108 differentially expressed genes were identified by transcriptome analysis. Among them, 1,899 were up-regulated, and 2,209 were down-regulated in the transcriptome of male sterile flower buds. We found that “protein kinase activity”, “apoptosis process”, “calcium binding”, “cell death”, “cytochrome c oxidase activity”, “aspartate peptidase activity”, “cysteine peptidase activity” and other biological pathways such as “starch and sucrose metabolism” and “proteasome” were closely related to male sterility in Prunus sibirica. A total of 331 key genes were preliminarily screened.ConclusionThe occurrence of male sterility in Prunus sibirica involved many biological processes and metabolic pathways. According to the results of microstructure observations, related physiological indexes determination and transcriptome analysis, we reveal that the occurrence of male sterility in Prunus sibirica may be caused by abnormal metabolic processes such as the release of cytochrome c in the male sterile flower buds, the imbalance of the antioxidant system being destroyed, and the inability of macromolecular substances such as starch to be converted into soluble small molecules at the correct stage of reproductive development, resulting in energy loss. As a result, the tapetum cannot be fully degraded, thereby blocking anther development, which eventually led to the formation of male sterility.

A small Rho GTPase OsRacB is required for pollen germination in rice

Plant Rho small GTPases (Rop/Rac) are versatile molecular switches regulating many plant developmental processes. Particularly, their important functions in regulating pollen development have been demonstrated in Arabidopsis. A group of conserved Rop/Rac activators RopGEFs were recently reported to regulate rice (Oryza sativa) pollen tube germination, indicating that rice and Arabidopsis may have a conserved Rop/Rac mediated signaling pathway in regulating pollen tube growth. However, the Rop/Rac activated by the rice pollen specific RopGEFs remains to be identified. Here we demonstrated a Rop/Rac gene, OsRacB, co‐expressed with the mature pollen expressed OsRopGEF2/3/6/8. The knockout mutants were normal in anther and pollen development but defective in the pollen grain germination, suggesting a specific and non‐redundant role of OsRacB in the mature pollen. We further demonstrated that OsRacB is directly activated by the pollen specific expressing OsRopGEFs in vitro. Together with the previous study, we establish a RopGEF‐Rop/Rac regulon which plays essential roles in rice pollen grain germination. Our data encourage further identification of the upstream and downstream players of RopGEF‐Rop/Rac signaling in pollen germination and have agricultural implications for breeding robust seed yielding cultivars.

Genome-Wide Identification and Characterization of Polygalacturonase Gene Family in Maize (Zea mays L.).

Polygalacturonase (PG, EC 3.2.1.15) is a crucial enzyme for pectin degradation and is involved in various developmental processes such as fruit ripening, pollen development, cell expansion, and organ abscission. However, information on the PG gene family in the maize (Zea mays L.) genome and the specific members involved in maize anther development are still lacking. In this study, we identified 55 PG family genes from the maize genome and further characterized their evolutionary relationship and expression patterns. Phylogenetic analysis revealed that ZmPGs are grouped into six Clades, and gene structures of the same Clade are highly conserved, suggesting their functional conservation. The ZmPGs are randomly distributed across maize chromosomes, and collinearity analysis showed that many ZmPGs might be derived from tandem duplications and segmental duplications, and these genes are under purifying selection. Furthermore, gene expression analysis provided insights into possible functional divergence among ZmPGs. Based on the RNA-seq data analysis, we found that many ZmPGs are expressed in various tissues while 18 ZmPGs are highly expressed in maize anther, and their detailed expression profiles in different anther developmental stages were further investigated by using RT-qPCR analysis. These results provide valuable information for further functional characterization and application of the ZmPGs in maize.

Molecular basis of cytoplasmic male sterility and fertility restoration in rice.

Cytoplasmic male sterility (CMS) is a maternally inherited trait that causes dysfunctions in pollen and anther development. CMS is caused by the interaction between nuclear and mitochondrial genomes. A product of a CMS-causing gene encoded by the mitochondrial genome affects mitochondrial function and the regulation of nuclear genes, leading to male sterility. In contrast, the RESTORER OF FERTILITY gene (Rf gene) in the nuclear genome suppresses the expression of the CMS-causing gene and restores male fertility. An alloplasmic CMS line is often bred as a result of nuclear substitution, which causes the removal of functional Rf genes and allows the expression of a CMS-causing gene in mitochondria. The CMS/Rf system is an excellent model for understanding the genetic interactions and cooperative functions of mitochondrial and nuclear genomes in plants, and is also an agronomically important trait for hybrid seed production. In this review article, pollen and anther phenotypes of CMS, CMS-associated mitochondrial genes, Rf genes, and the mechanism that causes pollen abortion and its agronomical application for rice are described.