Whole-genome landscape of histone H3K4me3 modification during sperm cell lineage development in tomato.

BACKGROUND: During male gametogenesis of flowering plants, sperm cell lineage (microspores, generative cells, and sperm cells) differentiated from somatic cells and acquired different cell fates. Trimethylation of histone H3 on lysine 4 (H3K4me3) epigenetically contributes to this process, however, it remained unclear how H3K4me3 influences the gene expression in each cell type. Here, we conducted chromatin immunoprecipitation sequencing (ChIP-seq) to obtain a genome-wide landscape of H3K4me3 during sperm cell lineage development in tomato (Solanum lycopersicum). RESULTS: We show that H3K4me3 peaks were mainly enriched in the promoter regions, and intergenic H3K4me3 peaks expanded as sperm cell lineage differentiated from somatic cells. H3K4me3 was generally positively associated with transcript abundance and served as a better indicator of gene expression in somatic and vegetative cells, compared to sperm cell lineage. H3K4me3 was mutually exclusive with DNA methylation at 3' proximal of the transcription start sites. The microspore maintained the H3K4me3 features of somatic cells, while generative cells and sperm cells shared an almost identical H3K4me3 pattern which differed from that of the vegetative cell. After microspore division, significant loss of H3K4me3 in genes related to brassinosteroid and cytokinin signaling was observed in generative cells and vegetative cells, respectively. CONCLUSIONS: Our results suggest the asymmetric division of the microspore significantly reshapes the genome-wide distribution of H3K4me3. Selective loss of H3K4me3 in genes related to hormone signaling may contribute to functional differentiation of sperm cell lineage. This work provides new resource data for the epigenetic studies of gametogenesis in plants.

Morpho-Anatomical, Physiological and Biochemical Adjustments in Response to Heat and Drought Co-Stress in Winter Barley.

This study aimed to investigate the combined effect of high temperatures 10 °C above the optimum and water withholding during microgametogenesis on vegetative processes and determine the response of winter barley genotypes with contrasting tolerance. For this purpose, two barley varieties were analyzed to compare the effect of heat and drought co-stress on their phenology, morpho-anatomy, physiological and biochemical responses and yield constituents. Genotypic variation was observed in response to heat and drought co-stress, which was attributed to differences in anatomy, ultrastructure and physiological and metabolic processes. The co-stress-induced reduction in relative water content, total soluble protein and carbohydrate contents, photosynthetic pigment contents and photosynthetic efficiency of the sensitive Spinner variety was significantly greater than the tolerant Lambada genotype. Based on these observations, it has been concluded that the heat-and-drought stress-tolerance of the Lambada variety is related to the lower initial chlorophyll content of the leaves, the relative resistance of photosynthetic pigments towards stress-triggered degradation, retained photosynthetic parameters and better-preserved leaf ultrastructure. Understanding the key factors underlying heat and drought co-stress tolerance in barley may enable breeders to create barley varieties with improved yield stability under a changing climate.

Tomato POLLEN DEFICIENT 2 encodes a G-type lectin receptor kinase required for viable pollen grain formation.

Pollen development is a crucial biological process indispensable for seed set in flowering plants and for successful crop breeding. However, little is known about the molecular mechanisms regulating pollen development in crop species. This study reports a novel male-sterile tomato mutant, pollen deficient 2 (pod2), characterized by the production of non-viable pollen grains and resulting in the development of small parthenocarpic fruits. A combined strategy of mapping-by-sequencing and RNA interference-mediated gene silencing was used to prove that the pod2 phenotype is caused by the loss of Solanum lycopersicum G-type lectin receptor kinase II.9 (SlG-LecRK-II.9) activity. In situ hybridization of floral buds showed that POD2/SlG-LecRK-II.9 is specifically expressed in tapetal cells and microspores at the late tetrad stage. Accordingly, abnormalities in meiosis and tapetum programmed cell death in pod2 occurred during microsporogenesis, resulting in the formation of four dysfunctional microspores leading to an aberrant microgametogenesis process. RNA-seq analyses supported the existence of alterations at the final stage of microsporogenesis, since we found tomato deregulated genes whose counterparts in Arabidopsis are essential for the normal progression of male meiosis and cytokinesis. Collectively, our results revealed the essential role of POD2/SlG-LecRK-II.9 in regulating tomato pollen development.

Regulatory dynamics of gene expression in the developing male gametophyte of Arabidopsis.

Sexual reproduction in angiosperms requires the production and delivery of two male gametes by a three-celled haploid male gametophyte. This demands synchronized gene expression in a short developmental window to ensure double fertilization and seed set. While transcriptomic changes in developing pollen are known for Arabidopsis, no studies have integrated RNA and proteomic data in this model. Further, the role of alternative splicing has not been fully addressed, yet post-transcriptional and post-translational regulation may have a key role in gene expression dynamics during microgametogenesis. We have refined and substantially updated global transcriptomic and proteomic changes in developing pollen for two Arabidopsis accessions. Despite the superiority of RNA-seq over microarray-based platforms, we demonstrate high reproducibility and comparability. We identify thousands of long non-coding RNAs as potential regulators of pollen development, hundreds of changes in alternative splicing and provide insight into mRNA translation rate and storage in developing pollen. Our analysis delivers an integrated perspective of gene expression dynamics in developing Arabidopsis pollen and a foundation for studying the role of alternative splicing in this model.

The DC1 Domain Protein BINUCLEATE POLLEN is Required for POLLEN Development in Arabidopsis thaliana.

The development of the male gametophyte is a tightly regulated process that requires the precise control of cell division and gene expression. A relevant aspect to understand the events underlying pollen development regulation constitutes the identification and characterization of the genes required for this process. In this work, we showed that the DC1 domain protein BINUCLEATE POLLEN (BNP) is essential for pollen development and germination. Pollen grains carrying a defective BNP alleles failed to complete mitosis II and exhibited impaired pollen germination. By yeast two-hybrid analysis and bimolecular fluorescence complementation assays, we identified a set of BNP-interacting proteins. Among confirmed interactors, we found the NAC family transcriptional regulators Vascular Plant One-Zinc Finger 1 (VOZ1) and VOZ2. VOZ1 localization changes during pollen development, moving to the vegetative nucleus at the tricellular stage. We observed that this relocalization requires BNP; in the absence of BNP in pollen from bnp/BNP plants, VOZ1 nuclear localization is impaired. As the voz1voz2 double mutants showed the same developmental defect observed in bnp pollen grains, we propose that BNP requirement to complete microgametogenesis could be linked to its interaction with VOZ1/2 proteins. BNP could have the role of a scaffold protein, recruiting VOZ1/2 to the endosomal system into assemblies that are required for their further translocation to the nucleus, where they act as transcriptional regulators.

Anther development in Brachiaria brizantha (syn. Urochloa brizantha) and perspective for microspore in vitro culture.

Brachiaria, a genus from the Poaceae family, is largely cultivated as forage in Brazil. Among the most cultivated varieties of Brachiaria spp., B. brizantha cv. Marandu (syn. Urochloa brizantha) is of great agronomical importance due to the large areas cultivated with this species. This cultivar is apomictic and tetraploid. Sexual diploid genotype is available for this species. The difference in levels of ploidy among sexual and apomictic plants contributes to hindering Brachiaria breeding programs. The induction of haploids and double haploids is of great interest for the generation of new genotypes with potential use in intraspecific crosses. A key factor for the success of this technique is identifying adequate microspore developmental stages for efficient embryogenesis induction. Knowledge of the morphological changes during microsporogenesis and microgametogenesis and sporophytic tissues composing the anther is critical for identifying the stages in which microspores present a higher potential for embryogenic callus and somatic embryo through in vitro culture. In this work, morphological markers were associated with anther and pollen grain developmental stages, through histological analysis. Anther development was divided into 11 stages using morphological and cytological characteristics, from anther with archesporial cells to anther dehiscence. The morphological characteristics of each stage are presented. In addition, the response of stage 8 anthers to in vitro culture indicates microspores initiating somatic embryogenic pathway.

Hydroxyproline-O-Galactosyltransferases Synthesizing Type II Arabinogalactans Are Essential for Male Gametophytic Development in Arabidopsis.

In flowering plants, male reproductive function is determined by successful development and performance of stamens, pollen grains, and pollen tubes. Despite the crucial role of highly glycosylated arabinogalactan-proteins (AGPs) in male gamete formation, pollen grain, and pollen tube cell walls, the underlying mechanisms defining these functions of AGPs have remained elusive. Eight partially redundant Hyp-galactosyltransferases (named GALT2-GALT9) genes/enzymes are known to initiate Hyp-O-galactosylation for Hyp-arabinogalactan (AG) production in Arabidopsis thaliana. To assess the contributions of these Hyp-AGs to male reproductive function, we used a galt2galt5galt7galt8galt9 quintuple Hyp-GALT mutant for this study. Both anther size and pollen viability were compromised in the quintuple mutants. Defects in male gametogenesis were observed in later stages of maturing microspores after meiosis, accompanied by membrane blebbing and numerous lytic vacuoles. Cytological and ultramicroscopic observations revealed that pollen exine reticulate architecture and intine layer development were affected such that non-viable collapsed mature pollen grains were produced, which were devoid of cell content and nuclei, with virtually no intine. AGP immunolabeling demonstrated alterations in cell wall architecture of the anther, pollen grains, and pollen tube. Specifically, the LM2 monoclonal antibody (which recognized ß-GlcA epitopes on AGPs) showed a weak signal for the endothecium, microspores, and pollen tube apex. Pollen tube tips also displayed excessive callose deposition. Interestingly, expression patterns of pollen-specific AGPs, namely AGP6, AGP11, AGP23, and AGP40, were determined to be higher in the quintuple mutants. Taken together, our data illustrate the importance of type-II AGs in male reproductive function for successful fertilization.

Pollen Development and Viability in Diploid and Doubled Diploid Citrus Species.

Seedlessness is one of the most important agronomic traits in mandarins on the fresh fruit market. Creation of triploid plants is an important breeding strategy for development of new commercial varieties of seedless citrus. To this end, one strategy is to perform sexual hybridizations, with tetraploid genotypes as male parents. However, while seed development has been widely studied in citrus, knowledge of key steps such as microsporogenesis and microgametogenesis, is scarce, especially in polyploids. Therefore, we performed a study on the effect of ploidy level on pollen development by including diploid and tetraploid (double diploid) genotypes with different degrees of pollen performance. A comprehensive study on the pollen ontogeny of diploid and doubled diploid "Sanguinelli" blood orange and "Clemenules" clementine was performed, with focus on pollen grain germination in vitro and in planta, morphology of mature pollen grains by scanning electron microscopy (SEM), cytochemical characterization of carbohydrates by periodic acid-Shiff staining, and specific cell wall components revealed by immunolocalization. During microsporogenesis, the main difference between diploid and doubled diploid genotypes was cell area, which was larger in doubled diploid genotypes. However, after increase in size and vacuolization of microspores, but before mitosis I, doubled diploid "Clemenules" clementine showed drastic differences in shape, cell area, and starch hydrolysis, which resulted in shrinkage of pollen grains. The loss of fertility in doubled diploid "Clemenules" clementine is mainly due to lack of carbohydrate accumulation in pollen during microgametogenesis, especially starch content, which led to pollen grain abortion. All these changes make the pollen of this genotype unviable and very difficult to use as a male parent in sexual hybridization with the objective of recovering large progenies of triploid hybrids.

From birth to function: Male gametophyte development in flowering plants.

Male germline development in flowering plants involves two distinct and successive phases, microsporogenesis and microgametogenesis, which involve one meiosis followed by two rounds of mitosis. Many aspects of distinctions after mitosis between the vegetative cell and the male germ cells are seen, from morphology to structure, and the differential functions of the two cell types in the male gametophyte are differentially needed and required for double fertilization. The two sperm cells, carriers of the hereditary substances, depend on the vegetative cell/pollen tube to be delivered to the female gametophyte for double fertilization. Thus, the intercellular communication and coordinated activity within the male gametophyte probably represent the most subtle regulation in flowering plants to guarantee the success of reproduction. This review will focus on what we have known about the differentiation process and the functional diversification of the vegetative cell and the male germ cell, the most crucial cell types for plant fertility and crop production.

Microgametophyte Development in Cannabis sativa L. and First Androgenesis Induction Through Microspore Embryogenesis.

Development of double haploids is an elusive current breeding objective in Cannabis sativa L. We have studied the whole process of anther and pollen grain formation during meiosis, microsporogenesis, and microgametogenesis and correlated the different microgametophyte developmental stages with bud length in plants from varieties USO31 and Finola. We also studied microspore and pollen amyloplast content and studied the effect of a cold pretreatment to excised buds prior to microspore in vitro culture. Up to 476,903 microspores and pollen grains per male flower, with in vivo microspore viability rates from 53.71 to 70.88% were found. A high uniformity in the developmental stage of microspores and pollen grains contained in anthers was observed, and this allowed the identification of bud length intervals containing mostly vacuolate microspores and young bi-cellular pollen grains. The starch presence in C. sativa microspores and pollen grains follows a similar pattern to that observed in species recalcitrant to androgenesis. Although at a low frequency, cold-shock pretreatment applied on buds can deviate the naturally occurring gametophytic pathway toward an embryogenic development. This represents the first report concerning androgenesis induction in C. sativa, which lays the foundations for double haploid research in this species.

Classification and Computational Analysis of Arabidopsis thaliana Sperm Cell-Specific F-Box Protein Gene 3p.AtFBP113.

In plants, F-box proteins (FBPs) constitute one of the largest superfamilies of regulatory proteins. Most F-box proteins are shown to be an integral part of SCF complexes, which carry out the degradation of proteins and regulate diverse important biological processes. Anthers and pollen development have a huge importance in crop breeding. Despite the vast diversity of FBPs in Arabidopsis male reproductive organs, their role in anther and pollen development is not much explored. Moreover, a standard nomenclature for naming FBPs is also lacking. Here, we propose a standard nomenclature for naming the FBPs of Arabidopsis thaliana uniformly and carry out a systematic analysis of sperm cell-specific FBP gene, i.e., 3p.AtFBP113 due to its reported high and preferential expression, for detailed functional annotation. The results revealed that 3p.AtFBP113 is located on the small arm of chromosome and encodes 397 amino acid long soluble, stable, and hydrophilic protein with the possibility of localization in various cellular compartments. The presence of the C-terminal F-box associated domain (FBA) with immunoglobulin-like fold anticipated its role in protein binding. Gene ontology based functional annotation and tissue-specific gene co-expression analysis further strengthened its role in protein binding and ubiquitination. Moreover, various potential post/co-translational modifications were anticipated and the predicted tertiary structure also showed the presence of characteristic domains and fold. Thus, the outcomes of the study will be useful in developing a better understating of the function of 3p.AtFBP113 during the process of pollen development, which will be helpful for targeting the gene for manipulation of male fertility that has immense importance in hybrid breeding.

Differential Expression Profiling Reveals Stress-Induced Cell Fate Divergence in Soybean Microspores.

Stress-induced microspore embryogenesis is a widely employed method to achieve homozygosity in plant breeding programs. However, the molecular mechanisms that govern gametophyte de- and redifferentiation are understood poorly. In this study, RNA-Seq was used to evaluate global changes across the microspore transcriptome of soybean (Glycine max [L.] Merrill) as a consequence of pretreatment low-temperature stress. Expression analysis revealed more than 20,000 differentially expressed genes between treated and control microspore populations. Functional enrichment illustrated that many of these genes (e.g., those encoding heat shock proteins and cytochrome P450s) were upregulated to maintain cellular homeostasis through the mitigation of oxidative damage. Moreover, transcripts corresponding to saccharide metabolism, vacuolar transport, and other pollen-related developmental processes were drastically downregulated among treated microspores. Temperature stress also triggered cell wall modification and cell proliferation-characteristics that implied putative commitment to an embryonic pathway. These findings collectively demonstrate that pretreatment cold stress induces soybean microspore reprogramming through suppression of the gametophytic program while concomitantly driving sporophytic development.

Distribution of plastids and mitochondria during male gametophyte formation in Tinantia erecta (Jacq.) Fenzl.

During meiosis in microsporogenesis, autonomous cellular organelles, i.e., plastids and mitochondria, move and separate into daughter cells according to a specific pattern. This process called chondriokinesis is characteristic for a given plant species. The key criterion for classification of the chondriokinesis types was the arrangement of cell organelles during two meiosis phases: metaphase I and telophase I. The autonomous organelles participate in cytoplasmic inheritance; therefore, their precise distribution to daughter cells determines formation of identical viable microspores. In this study, the course of chondriokinesis during the development of the male gametophyte in Tinantia erecta was analyzed. The study was conducted using optical and transmission electron microscopes. During microsporogenesis in T. erecta, autonomous cell organelles moved in a manner defined as a neutral-equatorial type of chondriokinesis. Therefore, metaphase I plastids and mitochondria were evenly dispersed around the metaphase plate and formed an equatorial plate between the daughter nuclei in early telophase I. Changes in the ultrastructure of plastids and mitochondria during pollen microsporogenesis were also observed.

Low temperature-induced aberrations in male and female reproductive organ development cause flower abortion in chickpea.

Chickpea (Cicer arietinum L.) is susceptible to low temperature (LT) at reproductive stage. LT causes flower abortion and delays pod set in chickpea until terminal drought becomes an issue, thereby decreasing yield potential. In chickpea, flower and anther/pollen development as well as LT-induced abnormalities on anther and pollen development are described inadequately. In the present manuscript, we report flower development stages, anther development stages, and aberrations in male gamete formation in chickpea under LT. Flower length was linearly correlated to flower and anther stages and can be used to predict these stages in chickpea. LT affected male gamete development in a flower/anther age-dependent manner where outcome ranged from no pollen formation to pollen sterility or no anther dehiscence to delayed dehiscence. In anthers, LT inhibited microsporogenesis, microgametogenesis, tapetum degeneration, breakage of septum and stomium, and induced pollen sterility. Whereas disruption of male function was the prime cause of abortion in flowers below vacuolated pollen stage, flower abortion was due to a combination of male and female reproductive functions in flowers with mature pollen. The study will help in elucidating mechanisms governing flower development, anther and pollen development, and tolerance/susceptibility to LT.

Evidence for a specific and critical role of mitogen-activated protein kinase 20 in uni-to-binucleate transition of microgametogenesis in tomato.

Mitogen-activated protein kinases (MAPKs) regulate diverse aspects of plant growth. However, their potential role in reproductive development remains elusive. Here, we discovered an unique role of SlMPK20, a plant-specific group D MAPK, in pollen development in tomato. RNAi-mediated suppression of SlMPK20 or its knockout using CRISPR/Cas9 significantly reduced or completely abolished pollen viability, respectively, with no effects on maternal fertility. Cell biology and gene expression analyses established that SlMPK20 exerts its role specifically at the uni-to-binucleate transition during microgametogenesis. This assertion is based on the findings that the transgenic pollen was largely arrested at the binucleate stage with the appearance of subcellular abnormality at the middle uninucleate microspore stage; and SlMPK20 mRNA and SlMPK20-GUS signals were localized in the tetrads, uninuclear microspores and binuclear pollen grains but not in microspore mother cells or mature pollen grains. Transcriptomic and proteomic analyses revealed that knockout of SlMPK20 significantly reduced the expression of a large number of genes controlling sugar and auxin metabolism and signaling in anthers. Finally, protein-protein interaction assays identified SlMYB32 as a putative target protein of SlMPK20. We conclude that SlMPK20 specifically regulates post-meiotic pollen development through modulating sugar and auxin metabolism and signaling.

Dynamics of Calcium during In vitro Microspore Embryogenesis and In vivo Microspore Development in Brassica napus and Solanum melongena.

Calcium is widely known to have a role as a signaling molecule in many different processes, including stress response and activation of the embryogenic program. However, there are no direct clues about calcium levels during microspore embryogenesis, an experimental process that combines a developmental switch toward embryogenesis and the simultaneous application of different stressing factors. In this work, we used FluoForte, a calcium-specific fluorescent vital dye, to track by confocal microscopy the changes in levels and subcellular distribution of calcium in living rapeseed (B. napus) and eggplant (S. melongena) microspores and pollen grains during in vivo development, as well as during the first stages of in vitro-induced microspore embryogenesis in rapeseed. During in vivo development, a clear peak of cytosolic Ca2+ was observed in rapeseed vacuolate microspores and young pollen grains, the stages more suitable for embryogenesis induction. However, the Ca2+ levels observed in eggplant were dramatically lower than in rapeseed. Just after in vitro induction, Ca2+ levels increased specifically in rapeseed embryogenic microspores at levels dramatically higher than during in vivo development. The increase was observed in the cytosol, but predominantly in vacuoles. Non-embryogenic forms such as callus-like and pollen-like structures presented remarkably different calcium patterns. After the heat shock-based inductive treatment, Ca2+ levels progressively decreased in all cases. Together, our results reveal unique calcium dynamics in in vivo rapeseed microspores, as well as in those reprogrammed to in vitro embryogenesis, establishing a link between changes in Ca2+ level and subcellular distribution, and microspore embryogenesis.

The DC1-domain protein VACUOLELESS GAMETOPHYTES is essential for development of female and male gametophytes in Arabidopsis.

In this work we identified VACUOLELESS GAMETOPHYTES (VLG) as a DC1 domain-containing protein present in the endomembrane system and essential for development of both female and male gametophytes. VLG was originally annotated as a gene coding for a protein of unknown function containing DC1 domains. DC1 domains are cysteine- and histidine-rich zinc finger domains found exclusively in the plant kingdom that have been named on the basis of similarity with the C1 domain present in protein kinase C (PKC). In Arabidopsis, both male and female gametophytes are characterized by the formation of a large vacuole early in development; this is absent in vlg mutant plants. As a consequence, development is arrested in embryo sacs and pollen grains at the first mitotic division. VLG is specifically located in multivesicular bodies or pre-vacuolar compartments, and our results suggest that vesicular fusion is affected in the mutants, disrupting vacuole formation. Supporting this idea, AtPVA12 - a member of the SNARE vesicle-associated protein family and previously related to a sterol-binding protein, was identified as a VLG interactor. A role for VLG is proposed mediating vesicular fusion in plants as part of the sterol trafficking machinery required for vacuole biogenesis in plants.

Ultrastructural aspects of pollen ontogeny in an endangered plant species, Pancratium maritimum L. (Amaryllidaceae).

Pollen ontogeny in Pancratium maritimum L. was studied from the sporogenous cell to mature pollen grain stages using transmission electron, scanning electron, and light microscopy to determine whether the pollen development in P. maritimum follows the basic scheme in angiosperms or not. In the course of microsporogenesis and microgametogenesis, special attention was given to the considerable ultrastructural changes that are observed in the cytoplasm of microsporocytes, microspores, and mature pollen grains throughout the successive stages of pollen development. Microsporocyte differentiation concerning number and ultrastructure of organelles facilitates the transition of microsporocytes from the sporophytic phase to the gametophytic phase. However, cytoplasmic differentiation of generative and vegetative cells supports their functional distinctness and pollen maturation. Although microsporogenesis and microgametogenesis in P. maritimum generally follow the usual angiosperm pattern, abnormalities such as formation of unreduced gametes were observed. During normal microsporogenesis, meiocytes undergo meiosis and successive cytokinesis, resulting in the formation of isobilateral, decussate, and linear tetrads. Subsequent to the development of free and vacuolated microspores, the first mitotic division occurs and bicellular monosulcate pollen grains are produced. Pollen grains are shed from the anther at binucleate stage. During pollen ontogeny, three periods of vacuolization were observed: in meiocytes, in mononucleate free microspores, and in the generative cell.

Phosphatidylinositol 4-phosphate 5-kinases 1 and 2 are involved in the regulation of vacuole morphology during Arabidopsis thaliana pollen development.

The pollen grains arise after meiosis of pollen mother cells within the anthers. A series of complex structural changes follows, generating mature pollen grains capable of performing the double fertilization of the female megasporophyte. Several signaling molecules, including hormones and lipids, have been involved in the regulation and appropriate control of pollen development. Phosphatidylinositol 4-phophate 5-kinases (PIP5K), which catalyze the biosynthesis of the phosphoinositide PtdIns(4,5)P2, are important for tip polar growth of root hairs and pollen tubes, embryo development, vegetative plant growth, and responses to the environment. Here, we report a role of PIP5Ks during microgametogenesis. PIP5K1 and PIP5K2 are expressed during early stages of pollen development and their transcriptional activity respond to auxin in pollen grains. Early male gametophytic lethality to certain grade was observed in both pip5k1(-/-) and pip5k2(-/-) single mutants. The number of pip5k mutant alleles is directly related to the frequency of aborted pollen grains suggesting the two genes are involved in the same function. Indeed PIP5K1 and PIP5K2 are functionally redundant since homozygous double mutants did not render viable pollen grains. The loss of function of PIP5K1 and PIP5K2results in defects in vacuole morphology in pollen at the later stages and epidermal root cells. Our results show that PIP5K1, PIP5K2 and phosphoinositide signaling are important cues for early developmental stages and vacuole formation during microgametogenesis.

The Rice Eukaryotic Translation Initiation Factor 3 Subunit f (OseIF3f) Is Involved in Microgametogenesis.

Microgametogenesis is the post-meiotic pollen developmental phase when unicellular microspores develop into mature tricellular pollen. In rice, microgametogenesis can influence grain yields to a great degree because pollen abortion occurs more easily during microgametogenesis than during other stages of pollen development. However, our knowledge of the genes involved in microgametogenesis in rice remains limited. Due to the dependence of pollen development on the regulatory mechanisms of protein expression, we identified the encoding gene of the eukaryotic translation initiation factor 3, subunit f in Oryza sativa (OseIF3f). Immunoprecipitation combined with mass spectrometry confirmed that OseIF3f was a subunit of rice eIF3, which consisted of at least 12 subunits including eIF3a, eIF3b, eIF3c, eIF3d, eIF3e, eIF3f, eIF3g, eIF3h, eIF3i, eIF3k, eIF3l, and eIF3m. OseIF3f showed high mRNA levels in immature florets and is highly abundant in developing anthers. Subcellular localization analysis showed that OseIF3f was localized to the cytosol and the endoplasmic reticulum in rice root cells. We further analyzed the biological function of OseIF3f using the double-stranded RNA-mediated interference (RNAi) approach. The OseIF3f-RNAi lines grew normally at the vegetative stage but displayed a large reduction in seed production and pollen viability, which is associated with the down-regulation of OseIF3f. Further cytological observations of pollen development revealed that the OseIF3f-RNAi lines showed no obvious abnormalities at the male meiotic stage and the unicellular microspore stage. However, compared to the wild-type, OseIF3f-RNAi lines contained a higher percentage of arrested unicellular pollen at the bicellular stage and a higher percentage of arrested unicellular and bicellular pollen, and aborted pollen at the tricellular stage. These results indicate that OseIF3f plays a role in microgametogenesis.