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.

MassARRAY and SABER Analyses of SNPs in Embryo DNA Reveal the Abscission of Self-Fertilised Progeny during Fruit Development of Macadamia (Macadamia integrifolia Maiden & Betche).

Yield in many crops is affected by abscission during the early stages of fruitlet development. The reasons for fruitlet abscission are often unclear but they may include genetic factors because, in some crops, self-pollinated fruitlets are more likely to abscise than cross-pollinated fruitlets. Pollen parentage can also affect final fruit size and fruit quality. Here, we aimed to understand the effects of pollen parentage on fruitlet retention and nut quality in orchards of macadamia (Macadamia integrifolia Maiden & Betche). We identified the pollen parent of macadamia 'cultivar '816' embryos by analysing single nucleotide polymorphisms (SNPs) in their DNA using customised MassARRAY and Single Allele Base Extension Reaction (SABER) methods. This allowed us to determine the proportions of self-fertilised and cross-fertilised progeny during premature fruit drop at 6 weeks and 10 weeks after peak anthesis, as well as at nut maturity. We determined how pollen parentage affected nut-in-shell (NIS) mass, kernel mass, kernel recovery, and oil concentration. Macadamia trees retained cross-fertilised fruitlets rather than self-fertilised fruitlets. The percentage of progeny that were cross-fertilised increased from 6% at 6 weeks after peak anthesis to 97% at nut maturity, with each tree producing on average 22 self-fertilised nuts and 881 cross-fertilised nuts. Three of the four cross-pollen parents provided fruit with significantly higher NIS mass, kernel mass, or kernel recovery than the few remaining self-fertilised fruit. Fruit that were cross-fertilised by '842', 'A4', or 'A203' had 16-29% higher NIS mass and 24-44% higher kernel mass than self-fertilised fruit. Nuts that were cross-fertilised by 'A4' or 'A203' also had 5% or 6% higher kernel recovery, worth approximately $US460-540 more per ton for growers than self-fertilised nuts. The highly selective abscission of self-fertilised fruitlets and the lower nut quality of self-fertilised fruit highlight the critical importance of cross-pollination for macadamia productivity.

Overriding Mendelian inheritance in Arabidopsis with a CRISPR toxin-antidote gene drive that impairs pollen germination.

Synthetic gene drives, inspired by natural selfish genetic elements and transmitted to progeny at super-Mendelian (>50%) frequencies, present transformative potential for disseminating traits that benefit humans throughout wild populations, even facing potential fitness costs. Here we constructed a gene drive system in plants called CRISPR-Assisted Inheritance utilizing NPG1 (CAIN), which uses a toxin-antidote mechanism in the male germline to override Mendelian inheritance. Specifically, a guide RNA-Cas9 cassette targets the essential No Pollen Germination 1 (NPG1) gene, serving as the toxin to block pollen germination. A recoded, CRISPR-resistant copy of NPG1 serves as the antidote, providing rescue only in pollen cells that carry the drive. To limit potential consequences of inadvertent release, we used self-pollinating Arabidopsis thaliana as a model. The drive demonstrated a robust 88-99% transmission rate over two successive generations, producing minimal resistance alleles that are unlikely to inhibit drive spread. Our study provides a strong basis for rapid genetic modification or suppression of outcrossing plant populations.

Characterization of organelle DNA degradation mediated by DPD1 exonuclease in the rice genome-edited line.

Mitochondria and plastids, originated as ancestral endosymbiotic bacteria, contain their own DNA sequences. These organelle DNAs (orgDNAs) are, despite the limited genetic information they contain, an indispensable part of the genetic systems but exist as multiple copies, making up a substantial amount of total cellular DNA. Given this abundance, orgDNA is known to undergo tissue-specific degradation in plants. Previous studies have shown that the exonuclease DPD1, conserved among seed plants, degrades orgDNAs during pollen maturation and leaf senescence in Arabidopsis. However, tissue-specific orgDNA degradation was shown to differ among species. To extend our knowledge, we characterized DPD1 in rice in this study. We created a genome-edited (GE) mutant in which OsDPD1 and OsDPD1-like were inactivated. Characterization of this GE plant demonstrated that DPD1 was involved in pollen orgDNA degradation, whereas it had no significant effect on orgDNA degradation during leaf senescence. Comparison of transcriptomes from wild-type and GE plants with different phosphate supply levels indicated that orgDNA had little impact on the phosphate starvation response, but instead had a global impact in plant growth. In fact, the GE plant showed lower fitness with reduced grain filling rate and grain weight in natural light conditions. Taken together, the presented data reinforce the important physiological roles of orgDNA degradation mediated by DPD1.

Uncovering genes involved in pollinator-driven mating system shifts and selfing syndrome evolution in Brassica rapa.

Shifts in pollinator occurrence and their pollen transport effectiveness drive the evolution of mating systems in flowering plants. Understanding the genomic basis of these changes is essential for predicting the persistence of a species under environmental changes. We investigated the genomic changes in Brassica rapa over nine generations of pollination by hoverflies associated with rapid morphological evolution toward the selfing syndrome. We combined a genotyping-by-sequencing (GBS) approach with a genome-wide association study (GWAS) to identify candidate genes, and assessed their functional role in the observed morphological changes by studying mutations of orthologous genes in the model plant Arabidopsis thaliana. We found 31 candidate genes involved in a wide range of functions from DNA/RNA binding to transport. Our functional assessment of orthologous genes in A. thaliana revealed that two of the identified genes in B. rapa are involved in regulating the size of floral organs. We found a protein kinase superfamily protein involved in petal width, an important trait in plant attractiveness to pollinators. Moreover, we found a histone lysine methyltransferase (HKMT) associated with stamen length. Altogether, our study shows that hoverfly pollination leads to rapid evolution toward the selfing syndrome mediated by polygenic changes.

Selfing Promotes Spread and Introgression of Segregation Distorters in Hermaphroditic Plants.

Segregation distorters (SDs) are genetic elements that distort the Mendelian segregation ratio to favor their own transmission and are able to spread even when they incur fitness costs on organisms carrying them. Depending on the biology of the host organisms and the genetic architecture of the SDs, the population dynamics of SDs can be highly variable. Inbreeding is considered an effective mechanism for inhibiting the spread of SDs in populations, and can evolve as a defense mechanism against SDs in some systems. However, we show that inbreeding in the form of selfing in fact promotes the spread of SDs acting as pollen killers in a toxin-antidote system in hermaphroditic plants by two mechanisms: (i) By reducing the effective recombination rate between killer and antidote loci in the two-locus system and (ii) by increasing the proportion of SD alleles in individual flowers, rather than in the general gene-pool. We also show that in rice (Oryza sativa L.), a typical hermaphroditic plant, all molecularly characterized SDs associated with pollen killing were involved in population hybridization and have introgressed across different species. Paradoxically, these loci, which are associated with hybrid incompatibility and can be thought of as Bateson-Dobzhansky-Muller incompatibility loci are expected to reduce gene-flow between species, in fact cross species boundaries more frequently than random loci, and may act as important drivers of introgression.

Microinjection of the CRISPR/Cas9 editing system through the germ pore of a wheat microspore induces mutations in the target Ms2 gene.

BACKGROUND: Microinjection is a direct procedure for delivering various compounds via micropipette into individual cells. Combined with the CRISPR/Cas9 editing technology, it has been used to produce genetically engineered animal cells. However, genetic micromanipulation of intact plant cells has been a relatively unexplored area of research, partly due to the cytological characteristics of these cells. This study aimed to gain insight into the genetic micromanipulation of wheat microspores using microinjection procedures combined with the CRISPR/Cas9 editing system targeting the Ms2 gene. METHODS AND RESULTS: Microspores were first reprogrammed by starvation and heat shock treatment to make them structurally suitable for microinjection. The large central vacuole was fragmented and the nucleus with cytoplasm was positioned in the center of the cell. This step and an additional maltose gradient provided an adequate source of intact single cells in the three wheat genotypes. The microcapillary was inserted into the cell through the germ pore to deliver a working solution with a fluorescent marker. This procedure was much more efficient and less harmful to the microspore than inserting the microcapillary through the cell wall. The CRISPR/Cas9 binary vectors injected into reprogrammed microspores induced mutations in the target Ms2 gene with deletions ranging from 1 to 16 bp. CONCLUSIONS: This is the first report of successful genome editing in an intact microspore/wheat cell using the microinjection technique and the CRISPR/Cas9 editing system. The study presented offers a range of molecular and cellular biology tools that can aid in genetic micromanipulation and single-cell analysis.

Abnormal Degraded Tapetum 1 (ADT1) is required for tapetal cell death and pollen development in rice.

The timely degradation of tapetum, the innermost somatic anther cell layer in flowering plants, is critical for pollen development. Although several genes involved in tapetum development have been characterized, the molecular mechanisms underlying tapetum degeneration remain elusive. Here, we showed that mutation in Abnormal Degraded Tapetum 1 (ADT1) resulted in overaccumulation of Reactive Oxygen Species (ROS) and abnormal anther development, causing earlier tapetum Programmed Cell Death (PCD) and pollen abortion. ADT1 encodes a nuclear membrane localized protein, which is strongly expressed in the developing microspores and tapetal cells during early anther development. Moreover, ADT1 could interact with metallothionein MT2b, which was related to ROS scavenging and cell death regulation. These findings indicate that ADT1 is required for proper timing of tapetum PCD by regulating ROS homeostasis, expanding our understanding of the regulatory network of male reproductive development in rice.

Paternal Inheritance of Mitochondrial DNA May Lead to Dioecy in Conifers.

In angiosperms cytoplasmic DNA is typically passed on maternally through ovules. Genes in the mtDNA may cause male sterility. When male-sterile (female) cytotypes produce more seeds than cosexuals, they pass on more copies of their mtDNA and will co-occur with cosexuals with a neutral cytotype. Cytoplasmic gynodioecy is a well-known phenomenon in angiosperms, both in wild and crop plants. In some conifer families (e.g. Pinaceae) mitochondria are also maternally inherited. However in some other families (e.g. Taxaceae and Cupressaceae) mtDNA is paternally inherited through the pollen. With paternal mtDNA inheritance, male cytotypes that produce more pollen than cosexuals are expected to co-occur with cosexuals. This is uncharted territory. An ESS model shows that the presence of male cytotypes selects for more female allocation in the cosexual, i.e. for sexual specialisation. An allele that switches sex from male to female can then invade. This leads to rapid loss of the neutral cytotype of the cosexual, fixation of the male cytotype and dioecy with 50% males and 50% females. The models suggest that paternal inheritance of mtDNA facilitates the evolution dioecy. Consistent with this hypothesis the Pinaceae are 100% monoecious, while dioecy is common in the Taxaceae family and in the genus Juniperus (Cupressaceae). However, no reliable data are yet available on both mode of inheritance of mtDNA and gender variation of the same species. When cosexuals benefit from reproductive assurance (high selfing rate, low inbreeding depression, low fertilisation) they maintain themselves next to males and females. This predicted pattern with three sex types present in the same population is observed in conifers in nature.

Integrated transcriptomic and metabolomic analysis provides insight into the pollen development of CMS-D1 rice.

BACKGROUND: Cytoplasmic male sterility (CMS) has greatly improved the utilization of heterosis in crops due to the absence of functional male gametophyte. The newly developed sporophytic D1 type CMS (CMS-D1) rice exhibits unique characteristics compared to the well-known sporophytic CMS-WA line, making it a valuable resource for rice breeding. RESULTS: In this research, a novel CMS-D1 line named Xingye A (XYA) was established, characterized by small, transparent, and shriveled anthers. Histological and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assays conducted on anthers from XYA and its maintainer line XYB revealed that male sterility in XYA is a result of delayed degradation of tapetal cells and abnormal programmed cell death (PCD) of microspores. Transcriptome analysis of young panicles revealed that differentially expressed genes (DEGs) in XYA, compared to XYB, were significantly enriched in processes related to chromatin structure and nucleosomes during the microspore mother cell (MMC) stage. Conversely, processes associated with sporopollenin biosynthesis, pollen exine formation, chitinase activity, and pollen wall assembly were enriched during the meiosis stage. Metabolome analysis identified 176 specific differentially accumulated metabolites (DAMs) during the meiosis stage, enriched in pathways such as α-linoleic acid metabolism, flavone and flavonol biosynthesis, and linolenic acid metabolism. Integration of transcriptomic and metabolomic data underscored the jasmonic acid (JA) biosynthesis pathway was significant enriched in XYA during the meiosis stage compared to XYB. Furthermore, levels of JA, MeJA, OPC4, OPDA, and JA-Ile were all higher in XYA than in XYB at the meiosis stage. CONCLUSIONS: These findings emphasize the involvement of the JA biosynthetic pathway in pollen development in the CMS-D1 line, providing a foundation for further exploration of the molecular mechanisms involved in CMS-D1 sterility.

Lipid and sugar metabolism play an essential role in pollen development and male sterility: a case analysis in Brassica napus.

AIMS: The genic male sterility (GMS) system is an important strategy for generating heterosis in plants. To better understand the essential role of lipid and sugar metabolism and to identify additional candidates for pollen development and male sterility, transcriptome and metabolome analysis of a GMS line of 1205AB in B. napus was used as a case study. DATA RESOURCES GENERATED: To characterize the GMS system, the transcriptome and metabolome profiles were generated for 24 samples and 48 samples of 1205AB in B. napus, respectively. Transcriptome analysis yielded a total of 156.52 Gb of clean data and revealed the expression levels of 109,541 genes and 8,501 novel genes. In addition, a total of 1,353 metabolites were detected in the metabolomic analysis, including 784 in positive ion mode and 569 in negative ion mode. KEY RESULTS: A total of 15,635 differentially expressed genes (DEGs) and 83 differential metabolites (DMs) were identified from different comparison groups, most of which were involved in lipid and sugar metabolism. The combination of transcriptome and metabolome analysis revealed 49 orthologous GMS genes related to lipid metabolism and 46 orthologous GMS genes related to sugar metabolism, as well as 45 novel genes. UTILITY OF THE RESOURCE: The transcriptome and metabolome profiles and their analysis provide useful reference data for the future discovery of additional GMS genes and the development of more robust male sterility breeding systems for use in the production of plant hybrids.

The different response of Brassica napus genotypes to microspore embryogenesis induced by heat shock and trichostatin A is not determined by changes in cell wall structure and composition but by different stress tolerance.

During microspore embryogenesis, microspores are induced to develop into haploid embryos. In Brassica napus, microspore embryogenesis is induced by a heat shock (HS), which initially produces embryogenic structures with different cell wall architectures and compositions, and with different potentials to develop into embryos. The B. napus DH4079 and DH12075 genotypes have high and very low embryo yields, respectively. In DH12075, embryo yield is greatly increased by combining HS and the histone deacetylase (HDAC) inhibitor trichostatin A (TSA). However, we show that HS + TSA inhibits embryogenesis in the highly embryogenic DH4079 line. To ascertain why TSA has such different effects in these lines, we treated DH4079 and DH12075 microspore cultures with TSA and compared the cell wall structure and composition of the different embryogenic structures in both lines, specifically the in situ levels and distribution of callose, cellulose, arabinogalactan proteins and high and low methyl-esterified pectin. For both lines, HS + TSA led to the formation of cell walls unfavorable for embryogenesis progression, with reduced levels of arabinogalactan proteins, reduced cell adhesion of inner walls and altered pectin composition. Thus, TSA effects on cell walls cannot explain their different embryogenic response to TSA. We also applied TSA to DH4079 cultures at different times and concentrations before HS application, with no negative effects on embryogenic induction. These results indicate that DH4079 microspores are hypersensitive to combined TSA and HS treatments, and open up new hypotheses about the causes of such hypersensitivity.

Characterization and validation of TaAGL66, a gene related to fertility conversion of wheat in the presence of Aegilops kotschyi cytoplasm.

MAIN CONCLUSION: TaAGL66, a MADS-box transcription factor highly expressed in fertile anthers of KTM3315A, regulates anther and/or pollen development, as well as male fertility in wheat with Aegilops kotschyi cytoplasm. Male sterility, as a string of sophisticated biological processes in higher plants, is commonly regulated by transcription factors (TFs). Among them, MADS-box TFs are mainly participated in the processes of floral organ formation and pollen development, which are tightly related to male sterility, but they have been little studied in the reproductive development in wheat. In our study, TaAGL66, a gene that was specifically expressed in spikes and highly expressed in fertile anthers, was identified by RNA sequencing and the expression profiles data of these genes, and qRT-PCR analyses, which was localized to the nucleus. Silencing of TaAGL66 under fertility condition in KTM3315A, a thermo-sensitive male sterile line with Ae. kotschyi cytoplasm, displayed severe fertility reduction, abnormal anther dehiscence, defective pollen development, decreased viability, and low seed-setting. It can be concluded that TaAGL66 plays an important role in wheat pollen development in the presence of Ae. kotschyi cytoplasm, providing new insights into the utilization of male sterility.

Arabidopsis Sar1b is critical for pollen tube growth.

Pollen tube growth is an essential step leading to reproductive success in flowering plants, in which vesicular trafficking plays a key role. Vesicular trafficking from endoplasmic reticulum to the Golgi apparatus is mediated by the coat protein complex II (COPII). A key component of COPII is small GTPase Sar1. Five Sar1 isoforms are encoded in the Arabidopsis genome and they show distinct while redundant roles in various cellular and developmental processes, especially in reproduction. Arabidopsis Sar1b is essential for sporophytic control of pollen development while Sar1b and Sar1c are critical for gametophytic control of pollen development. Because functional loss of Sar1b and Sar1c resulted in pollen abortion, whether they influence pollen tube growth was unclear. Here we demonstrate that Sar1b mediates pollen tube growth, in addition to its role in pollen development. Although functional loss of Sar1b does not affect pollen germination, it causes a significant reduction in male transmission and of pollen tube penetration of style. We further show that membrane dynamics at the apex of pollen tubes are compromised by Sar1b loss-of-function. Results presented provide further support of functional complexity of the Sar1 isoforms.

OsSRF8 interacts with OsINP1 and OsDAF1 to regulate pollen aperture formation in rice.

In higher plants, mature male gametophytes have distinct apertures. After pollination, pollen grains germinate, and a pollen tube grows from the aperture to deliver sperm cells to the embryo sac, completing fertilization. In rice, the pollen aperture has a single-pore structure with a collar-like annulus and a plug-like operculum. A crucial step in aperture development is the formation of aperture plasma membrane protrusion (APMP) at the distal polar region of the microspore during the late tetrad stage. Previous studies identified OsINP1 and OsDAF1 as essential regulators of APMP and pollen aperture formation in rice, but their precise molecular mechanisms remain unclear. We demonstrate that the Poaceae-specific OsSRF8 gene, encoding a STRUBBELIG-receptor family 8 protein, is essential for pollen aperture formation in Oryza sativa. Mutants lacking functional OsSRF8 exhibit defects in APMP and pollen aperture formation, like loss-of-function OsINP1 mutants. OsSRF8 is specifically expressed during early anther development and initially diffusely distributed in the microsporocytes. At the tetrad stage, OsSRF8 is recruited by OsINP1 to the pre-aperture region through direct protein-protein interaction, promoting APMP formation. The OsSRF8-OsINP1 complex then recruits OsDAF1 to the APMP site to co-regulate annulus formation. Our findings provide insights into the mechanisms controlling pollen aperture formation in cereal species.

Coexistence field trials between MON810 and conventional maize in Mallorca as a basis for a regional regulatory proposal based on scientific evidence in the times of genome editing.

This paper reports the first coexistence field trials between transgenic and conventional maize carried out under Mediterranean island conditions. Their purpose was to assess the local validity of pollen barriers and sowing delays as coexistence strategies as a basis for a regional regulation on the subject. Two field trials were performed in two agricultural states of Alcudia and Palma, in Mallorca (Spain). In the first one, two adjacent plots were synchronously sown with conventional and transgenic maize, respectively. In the second trial, the previous design was replicated, and two additional plots sown with GM maize were added, paired with their respective conventional recipient plots sown 2 and 4 weeks later. All conventional plots were located downwind from their respective GM plots. Of the two conventional plots in sowing synchrony, only one of them required a 2.25 m pollen barrier to meet the 0.9% labeling threshold. A 4-week sowing delay between GM and non-GM plots proved to be enough to keep the GM content of the recipient plots below the legal threshold. However, with a 2-week sowing delay additional coexistence measures such as pollen barriers might be needed, as suggested in the literature. Results are consistent with previous research conducted in the northeast of Spain, thus validating in the island's agroclimatic conditions a model successfully tested in that peninsular region which allows to accurately estimate the need and width of pollen barriers. The results presented here could perhaps be extrapolated to other islands, coastal areas, and regions with stable prevailing winds during the maize flowering season.

The Dark Side of the pollen: BSA-seq identified genomic regions linked to male sterility in globe artichoke.

Globe artichoke (Cynara cardunculus var. scolymus; 2n = 2x = 34) is a food crop consumed for its immature flower heads. Traditionally, globe artichoke varietal types are vegetatively propagated. However, seed propagation makes it possible to treat the crop as annual, increasing field uniformity and reducing farmers costs, as well as pathogens diffusion. Despite globe artichoke's significant agricultural value and the critical role of heterosis in the development of superior varieties, the production of hybrids remains challenging without a reliable system for large-scale industrial seed production. Male sterility (MS) presents a promising avenue for overcoming these challenges by simplifying the hybridization process and enabling cost-effective seed production. However, within the Cynara genus, genic male sterility has been linked to three recessive loci in globe artichoke, with no definitive genetic mechanism elucidated to date. A 250 offsprings F2 population, derived from a cross between a MS globe artichoke and a male fertile (MF) cultivated cardoon (C. cardunculus var. altilis) and fitting a monogenic segregation model (3:1), was analyzed through BSA-seq, aiming at the identification of genomic regions/genes affecting male sterility. Four QTL regions were identified on chromosomes 4, 12, and 14. By analyzing the sequence around the highest pick on chromosome 14, a cytochrome P450 (CYP703A2) was identified, carrying a deleterious substitution (R/Q) fixed in the male sterile parent. A single dCAPS marker was developed around this SNP, allowing the discrimination between MS and MF genotypes within the population, suitable for applications in plant breeding programs. A 3D model of the protein was generated by homology modeling, revealing that the mutated amino acid is part of a highly conserved motif crucial for protein folding.

GhTKPR1_8 functions to inhibit anther dehiscence and reduce pollen viability in cotton.

Sporopollenin, as the main component of the pollen exine, is a highly resistant polymer that provides structural integrity under unfavourable environmental conditions. Tetraketone α-pyrone reductase 1 (TKPR1) is essential for sporopollenin formation, catalyzing the reduction of tetraketone carbonyl to hydroxylated α-pyrone. The functional role of TKPR1 in male sterility has been reported in flowering plants such as maize, rice, and Arabidopsis. However, the molecular cloning and functional characterization of TKPR1 in cotton remain unaddressed. In this study, we identified 68 TKPR1s from four cotton species, categorized into three clades. Transcriptomics and RT-qPCR demonstrated that GhTKPR1_8 exhibited typical expression patterns in the tetrad stage of the anther. GhTKPR1_8 was localized to the endoplasmic reticulum. Moreover, ABORTED MICROSPORES (GhAMS) transcriptionally activated GhTKPR1_8 as indicated by luciferase complementation tests. GhTKPR1_8-knockdown inhibited anther dehiscence and reduced pollen viability in cotton. Additionally, overexpression of GhTKPR1_8 in the attkpr1 mutant restored its male sterile phenotype. This study offers novel insights into the investigation of TKPR1 in cotton while providing genetic resources for studying male sterility.

Targeted suppression of siRNA biogenesis in Arabidopsis pollen promotes triploid seed viability.

In plants, small-interfering RNAs (siRNAs) mediate epigenetic silencing via the RNA-directed DNA methylation (RdDM) pathway, which is particularly prominent during reproduction and seed development. However, there is limited understanding of the origins and dynamics of reproductive siRNAs acting in different cellular and developmental contexts. Here, we used the RNaseIII-like protein RTL1 to suppress siRNA biogenesis in Arabidopsis pollen, and found distinct siRNA subsets produced during pollen development. We demonstrate that RTL1 expression in the late microspore and vegetative cell strongly impairs epigenetic silencing, and resembles RdDM mutants in their ability to bypass interploidy hybridization barriers in the seed. However, germline-specific RTL1 expression did not impact transgenerational inheritance of triploid seed lethality. These results reveal the existence of multiple siRNA subsets accumulated in mature pollen, and suggest that mobile siRNAs involved in the triploid block are produced in germline precursor cells after meiosis, or in the vegetative cell during pollen mitosis.

Osmosensor-mediated control of Ca2+ spiking in pollen germination.

Higher plants survive terrestrial water deficiency and fluctuation by arresting cellular activities (dehydration) and resuscitating processes (rehydration). However, how plants monitor water availability during rehydration is unknown. Although increases in hypo-osmolarity-induced cytosolic Ca2+ concentration (HOSCA) have long been postulated to be the mechanism for sensing hypo-osmolarity in rehydration1,2, the molecular basis remains unknown. Because osmolarity triggers membrane tension and the osmosensing specificity of osmosensing channels can only be determined in vivo3-5, these channels have been classified as a subtype of mechanosensors. Here we identify bona fide cell surface hypo-osmosensors in Arabidopsis and find that pollen Ca2+ spiking is controlled directly by water through these hypo-osmosensors-that is, Ca2+ spiking is the second messenger for water status. We developed a functional expression screen in Escherichia coli for hypo-osmosensitive channels and identified OSCA2.1, a member of the hyperosmolarity-gated calcium-permeable channel (OSCA) family of proteins6. We screened single and high-order OSCA mutants, and observed that the osca2.1/osca2.2 double-knockout mutant was impaired in pollen germination and HOSCA. OSCA2.1 and OSCA2.2 function as hypo-osmosensitive Ca2+-permeable channels in planta and in HEK293 cells. Decreasing osmolarity of the medium enhanced pollen Ca2+ oscillations, which were mediated by OSCA2.1 and OSCA2.2 and required for germination. OSCA2.1 and OSCA2.2 convert extracellular water status into Ca2+ spiking in pollen and may serve as essential hypo-osmosensors for tracking rehydration in plants.