In Prunus, self-incompatibility (SI) is controlled by S-RNases and pollen-expressed F-box proteins, whereas the molecular processes governing S-RNase regulation in pollen remain incompletely understood. Here, we characterized PavSDJ, a novel pollen protein from sweet cherry (Prunus avium), as a candidate factor involved in pollen-side S-RNase-associated processes. Sequence and structural analyses identified PavSDJ as a type III J-protein. Phylogenetic analyses placed PavSDJ within a distinct SDJ-like sublineage of the type III J-protein group, separate from a closely related sister lineage. Consistent with this divergence, PavSDJ was strongly expressed in anthers and pollen, whereas its sister gene was broadly expressed across organs. Transient expression assays showed that PavSDJ–GFP exhibited a predominantly cell-peripheral fluorescence pattern consistent with intracellular localization. Biochemical analyses showed that PavSDJ associated with recombinant PavS-RNases in pollen extracts and in reconstituted pull-down assays, without obvious allele preference. Proteomic analysis of PavSDJ co-immunoprecipitants from pollen extracts identified a complex including PavSLFL6 and PavSSK1. Reconstitution assays further showed that PavSDJ promoted the co-precipitation of PavSLFL6 with S-RNase. These findings identify PavSDJ as a candidate pollen-side factor in the Prunus SI pathway and provide evidence that a specialized J-protein may contribute to SI-related protein complex assembly. PavSDJ is a pollen-expressed type III J-protein that binds S-RNase and promotes recruitment of SLFL6 to an S-RNase-associated complex in Prunus. It may function as a general modifier involved in the GSI system of Prunus.
The formation of unisexual florets in Poaceae occurs through diverse developmental pathways. However, the morphogenetic mechanisms controlling sex determination in dioecious grasses remain poorly understood. Here, we investigated the developmental basis of floral unisexuality in the dioecious forage species Poa lanigera (section Dioicopoa) to determine whether its developmental trajectory conforms to previously described patterns in grasses or represents a distinct pathway. We also compared stamen arrest in pistillate florets of P. lanigera with suppression of the third stamen in the model grass Brachypodium distachyon to assess potential similarities in organ arrest mechanisms. By using scanning electron microscopy and histological analyses, we reconstructed the sequence of floret organ initiation and differentiation in both species. Florets were initially established as perfect and followed a common sequence of organ initiation, with divergence arising shortly after ovary closure, stylar branch initiation, and the transition of anthers to the tetralobed stage. In pistillate florets of P. lanigera, androecium abortion involved early disorganization of hypodermal anther tissues, which prevented sporogenous differentiation and microsporogenesis, whereas, in staminate florets, gynoecium abortion occurred after sporogenous cell formation through localized tissue degeneration, which disrupted megasporogenesis. These developmental routes differ from previously described patterns in Poaceae, indicating a distinct variant of floral unisexuality. In contrast, suppression of the third stamen in B. distachyon occurred at the primordium stage without tissue degeneration, suggesting a mechanistically different process. Together, these findings provide a developmental framework to investigate the genetic regulation of sex determination in P. lanigera, with potential applications for pasture improvement.
In most angiosperm plants, seeds are produced through a sexual process. Some angiosperms have developed an alternative reproductive strategy termed apomixis which involves producing seeds without the key steps of sexual development (i.e. meiosis, fertilization) and results in progeny genetically identical to the mother plant. Despite significant research advances, the evolutionary origin and molecular nature of apomixis remain unclear. Recent findings suggest that apomixis may have emerged from a sexual pathway deregulated by genetic and epigenetic modifications and stress signals (both endogenous and exogenous) that led to the alteration or omission of selected stages of sexual development. Apomixis, as a natural phenomenon that enables clonal reproduction by seeds, is a desirable trait with great potential in plant breeding, especially in terms of preserving hybrid vigor and gene combinations of elite phenotypes over subsequent generations. Notwithstanding, apomixis does not occur in major crops and therefore research programs focus on how to introgress, induce, or mimic apomixis in agronomically significant sexual species. The present review outlines the history of research on apomixis in flowering plants, its mechanisms, and a summary of the latest and most impactful research advances that may pave the way for the introduction of apomixis to sexually reproducing crops. The reader’s attention is also drawn to the partially explained issue of intercellular communication in ovules during early apomictic processes in the context of the cell wall’s changed chemical composition in cells that enter the apomictic developmental pathway. The review summarizes what we already know about apomixis, starting from early research and ending with the latest reports that present the genetic basis of apomixis and outline future perspectives.
Land plants alternate between multicellular haploid and diploid phases, requiring a tight coordination between vegetative growth and sexual reproduction. We investigated SWI3A/B, an ancient core subunit of the SWI/SNF complex, of the non-vascular liverwort Marchantia polymorpha. A mutation in the promoter of MpSWI3A/B affected gametangiophore development and spermiogenesis in males, revealing its male-specific role in reproductive development. The MpSWIA/B mutant line amplifies vegetative propagation in males under conditions that would normally induce reproductive growth. The phenotype was underpinned by transcriptomic changes, showing that MpSWI3A/B modulates key regulators of gametangiophore initiation (e.g. BONOBO, GLID), sperm development and motility (e.g. DUO1, PKAR), and asexual reproduction (e.g. KAI2). We propose that, as a chromatin-level regulator, MpSWI3A/B may contribute to balancing vegetative and reproductive phases and highlight the protein's potential when exploring ancient epigenetic functions that coordinate developmental phase transitions in land plants.
In subtropical rangelands of America, the grasses dominate the landscape, but native leguminous plants could be source of nutritional quality as well as benefit suppliers of environment adaptations. Species of Macroptilium have been included in forage improvement programs and characterization of genes involved in plant architecture could contribute to its improvement. TFL1 is responsible for repressing the onset of flowering, sustaining the indeterminate growth and conditioning the complete plant structure. In this study, we report the cloning of candidate genes for TFL1 functional homology from Macroptilium lathyroides (MlTFL1) and M. erythroloma (MeTFL1) and their sequences and phylogenetic analyses. The genes structure was conserved, the deduced amino acid sequences showed also conservation of key residues and structures for function, and the phylogenetic reconstructions resolved them as a sister clade of Phaseolus spp. and near to Vigna spp. sequences. We also described the functional characterization of MlTFL1 through assays of Arabidopsis tfl1 mutant complementation with severe tfl1-1/tfl1-1 or mild tfl1-11/tfl1-11 genotypes. In both mutant backgrounds, the expression of MlTFL1 rescued the determinate growth, the early flowering time, the reduction in number of rosette leaves and the increase of rosette branches of mutants. Besides, MlTFL1 accomplished intermediate compensation of the height reduction. By PCA analyses of traits, longer vegetative phase and more rosette leaves associated to less rosette branches. Moreover, more height and more cauline branches had less weight in their associations with the other three variables. We consider that MlTFL1 of Macroptilium lathyroides behave as a TFL1 functional homolog and its characterization could contribute to increase the knowledge of this species looking to the improvement of this native forage leguminous plant.
Low temperature is a major environmental stress factor that limits male reproductive success in plants; however, the mechanistic basis of pollen responses to different degrees of low temperature stress remains poorly understood. In this study, we investigated the cytological, biochemical, and molecular responses of tea (Camellia sinensis) pollen grains to low-temperature stress by germinating pollen in vitro at 15, 10, and 5 °C. Although pollen germination rate and pollen tube length were reduced under all low-temperature treatments compared with the control, the underlying regulatory responses differed significantly with the severity of low-temperature stress. At 15 °C, decreases in non-enzymatic antioxidants and stress-related proteins indicated an overall metabolic weakening, while the concomitant accumulation of callose, cellulose, and methyl-esterified pectins reflected an early structural adjustment of the pollen tube cell wall. At 10 °C, the increasing stress load was partially counterbalanced by the induction of enzymatic antioxidant activities, and continued cell wall reinforcement identified this temperature as a transitional state between metabolic limitation and coordinated defense activation. In contrast, exposure to 5 °C resulted in pronounced metabolic suppression, together with a shift of stress-related proteins toward membrane fractions and enhanced deposition of callose, cellulose, and particularly de-esterified acidic pectins, leading to increased cell wall rigidity and mechanical restriction of pollen tube elongation. Overall, this study demonstrates that low-temperature stress constrains pollen tube growth not through a single limiting factor but via a temperature-dependent reorganization of interconnected cytological, biochemical, and molecular mechanisms. Low temperature reshapes tea pollen tube growth by coordinating cell wall remodeling, antioxidant defenses, and stress protein dynamics, revealing temperature specific mechanisms underlying reproductive stress tolerance.
Heteranthery is widespread among angiosperms and is classically linked to nectarless flowers that offer pollen as a reward, where the division of labour in the androecium helps to decrease the consumption of viable pollen grains. However, heteranthery also occurs in lineages with non-pollen-rewarding flowers. This is the case of some neotropical species of Malpighiaceae, which depend on oil-collecting bees for pollination, and offer floral oils. This study analyzes the androecium of Mamedea pulchella to assess the presence of division of labour and whether such specialization functions primarily in pollinator attraction rather than pollen economy. Anther traits from flowers of different populations were measured and analyzed statistically, complemented by anatomical and histochemical studies. Fertile stamens show significantly bigger anther area while sterile stamens bear antherodes with a significantly larger glandular connective. Occasional abortive anthers in usually fertile stamens and fertile anthers in usually sterile stamens were observed. Histochemical reactions indicate that connective glands secrete mainly terpenes. Differences between anthers and antherodes are consistent across populations, suggesting that heteranthery is a stable condition in M. pulchella, with a clear division of labour: anthers of large stamens supply pollen for fertilization whereas antherodes of smaller stamens act as osmophores, emitting scents that attract and guide oil-collecting bees to the elaiophores. This case highlights a potential alternative role for heteranthery in oil-rewarding flowers, where stamen specialization may contribute to pollinator attraction rather than solely mitigating the "pollen dilemma", suggesting that heteranthery can evolve through multiple functional pathways in pollination systems.
Female gametophyte (FG) development is essential for plant reproduction since it is necessary for pollen tube guidance, fertilization, and maternal control of seed development. FG development entails dynamic cellular and developmental changes including multiple cell divisions and cell differentiations with biologically distinct roles in fertilization, a critical process for grain yield in cereal crops. Rice (Oryza sativa) is a model monocotyledonous plant with invaluable relevance for food security as it is one of the most important staple food crops. Therefore, to study rice FG development, a robust and precise staging method is required to explore the molecular dynamics of this important organ. Here, we provide a pistil length-based staging framework for defining developmental stages in rice ovules. We systematically analyzed FG developmental progress using several reproductive morphological parameters. Six unique developmental stages that are morphologically distinguishable and encompass the entirety of FG development were identified. Using transcriptional expression of developmental marker genes that are temporally regulated during FG development we validated this staging method. Our method provides a systematic and accurate staging framework that could be used to explore temporally and developmentally dynamic molecular events during FG development, including transcriptional, epigenetic, and metabolic regulation.
The link between apomictic reproduction and polyploidy in plants has generated numerous hypotheses. Psidium cattleyanum is a valuable model for investigating this relationship, as it is a polyploid woody species that exhibits pseudogamous gametophytic apomixis. In this study, we aim to determine the predominant reproductive mode in natural populations of Psidium cattleyanum f. lucidum. We assessed its association with ploidy level, genetic diversity, and population structure. We analyzed populations representing the four ploidy levels (2C = 5x, 6x, 7x, 8x) present in Uruguay. Reproductive pathways were inferred by combining flow cytometric seed screening (FCSS) with microsatellite (SSR) genotyping of progeny arrays, and population-level differences were evaluated across both datasets. Diplosporous pseudogamous apomixis was identified as the predominant reproductive mode across populations. Importantly, the relative contributions of apomictic versus sexual reproduction as well as the frequencies of different seed progeny types varied markedly among populations and ploidy levels. Genotyping revealed predominantly clonal seed progeny, yet high within-population genetic variability and strong genetic structure were maintained. Moreover, gamete ploidy levels and their combinations were characteristic of each population, resulting in distinct patterns of seed formation among populations. Overall, facultative pseudogamous apomixis predominates in natural populations of P.c. f. lucidum. Our findings demonstrate previously unrecognized variation in reproductive pathways among populations, linked to differences in ploidy level and population genetic structure. Psidium cattleyanum f. lucidum wild populations predominantly reproduce through facultative pseudogamous apomixis. Gamete ploidy and their combinations vary among populations, shaping population-specific reproductive strategies that are associated with ploidy level and genetic structure.
This study investigated the physiological and molecular mechanisms underlying the formation of single-bud versus multi-bud corms in Amorphophallus muelleri foliar bulbils. Hormonal quantification revealed that multi-bud corms possessed significantly higher Cytokinin (CTK) levels and a lower Auxin/CTK ratio compared to single-bud corms. Conversely, branching inhibitors such as Brassinosteroids (BR) and Strigolactones (SLs) were significantly higher in single-bud corms. Metabolomic analysis showed a specific enrichment of fructose in multi-bud corms, suggesting high energy demand. Transcriptome sequencing identified 742 differentially expressed transcription factors and significant enrichment in hormone signaling pathways. Data reliability was validated by qRT-PCR, which confirmed the upregulation of CTK receptors (AmCRE1) and sucrose metabolism genes (AmSPS, AmINV), and the downregulation of SL signaling genes (AmD14, AmD53) in multi-bud corms. Crucially, exogenous application of 1.0 mg/L 6-BA verified the inductive role of CTK, achieving a 100
In plant cells, lipids serve various roles facilitating membrane bilayer formation, energy storage and signaling molecules. Acyl lipids are the most common in distinct plant cell compartments. Lipids regulated by key genes encoding fatty acid desaturases, diacylglycerol acyltransferase, 3-ketoacyl-CoA synthase and acyl-CoA-binding proteins (ACBPs) are deemed crucial during floral development. ACBPs, along with long-chain acyl-CoA synthase, acetyl-CoA carboxylase, fatty acid synthase, acyl-acyl carrier protein desaturases, acyl-ACP thioesterases and the ATP-binding cassette transporter subfamily A, contribute to fatty acid (FA) production, lipid transport and seed oil accumulation, making them bioengineering targets. To investigate lipid function, it is important to use appropriate analytical strategies because different lipid classes contain distinct FA patterns. These well-developed techniques include advanced lipidomic studies using multi-dimensional liquid chromatography-mass spectrometry, matrix-assisted laser desorption/ionization mass spectrometry imaging, lipid-binding assays and x-ray crystallography. As these techniques continue to evolve, further updates on lipid function are expected to rapidly materialize.
The male gametophyte in flowering plants, pollen, both performs the critical role of fertilization and represents a unique and accessible system for interrogating plant cell mechanics. A key component in this robust mechanical system is MscS-Like 8 (MSL8), a mechanosensitive ion channel. We previously proposed that MSL8 serves as an "osmotic safety valve", regulating pressure in the germinating pollen tube by releasing anions in response to plasma membrane tension. However, we subsequently identified defects in the cell walls of msl8 mutant pollen that suggested a role independent of osmoregulation. Here, we show that pollen tubes lacking MSL8 channel function by genetic knockout or channel-blocking point mutation lose major growth pauses, have altered pectin esterification patterns, and are sensitive to pectin crosslinking. Together, these data suggest a mechanism whereby anion efflux through mechanosensitive channels regulates cell wall composition and growth dynamics.
Loss of SPL10 and SPL11 increases penetrance of abnormal phenotypes in dcl1 embryos. The first division of the Arabidopsis zygote is asymmetric, resulting in an apical cell lineage that generates most of the embryo proper, and a basal cell lineage that produces the root meristem and the extraembryonic suspensor. Loss of function mutations in the microRNA processing enzyme genes DICER-LIKE 1 (DCL1) and SERRATE (SE) show cell division defects in the embryo proper, hypophyseal cell, and suspensor. Previous transcriptome analyses showed that the microRNA156-targeted transcription factor genes SQUAMOSA PROMOTER BINDING PROTEIN-LIKE2 (SPL2), SPL3 and SPL11 were upregulated in both globular stage dcl1 and se embryos, while SPL10 was upregulated in dcl1. It was previously proposed that upregulation of SPL10 and SPL11 could explain some abnormal phenotypes in dcl1 embryos. In this work, we used T-DNA and CRISPR-Cas9-induced loss of function alleles to further explore the function of SPL2, SPL3, SPL10 and SPL11 in early embryogenesis and their contribution to the dcl1 phenotype. On their own, spl2, spl3, spl10, and spl11 single mutants and an spl10 spl11 double mutant showed no abnormal cell divisions in early embryogenesis. In the dcl1/+ background, loss of function of SPL2 or SPL3 did not change the proportion of cell division defects in hypophyseal cells or suspensors observed in dcl1/+. Loss of spl10 or spl11 in dcl1/+ resulted in a slight decrease or increase (respectively) in the penetrance of abnormal suspensor divisions in heart stage embryos, while the spl10 spl11 double mutant caused a small increase in the penetrance of abnormal hypophyseal divisions in dcl1 embryos. The differences between our results and previous studies are likely due to genetic redundancy of miR156-targeted SPL genes, variable environmental conditions or the effect of genetic background on the penetrance of the dcl1 phenotype. In the future, analysis of higher order mutations in SPL and MIR156 genes will help to better understand the role of these important developmental regulators in early embryo development.
Cultured Arabidopsis flowers inmedium produce diploid microspores due to occurrence of defective meiotic cytokinesis, which can be partially rescued by anexogenous application of sucrose. Live-imaging microscopy technology has been increasingly applied for meiosis study in plants, which largely relies on the set up of a healthy ex vivo culture system for inflorescences ensuring that the captured chromosomes dynamics approaches the natural features of meiosis. Here, we report that Arabidopsis thaliana flowers cultivated in a culture medium (CCM) composed of the half-strength Murashige and Skoog basal salt, MES, Myo-inositol, sucrose and agar produce diploid microspores due to occurrence of meiotic restitution. Cytological studies revealed adjacent nuclei distribution and incomplete cytokinesis at late meiosis II in meiocytes within the CCM flowers. Immunolocalization of α-tubulin and the microtubule-associated protein MAP65-3 showed that the orientation of spindles at metaphase II and the organization of radial microtubule arrays at the tetrad stage are interfered, which explains the production of meiotically-restituted microspores. Moreover, the CCM flowers showed a gradually impaired expression of Aborted Microspores (AMS), a key transcription factor regulating tapetum development and meiotic cytokinesis. Interestingly, an increased supply of sucrose or its metabolite fructose in culture medium promoted the expression of AMS and partially rescued haploid microspore formation in the CCM flowers. Taken together, this study suggests a role of sucrose in facilitating meiotic cytokinesis and gametophytic ploidy stability in plants.
Recruitment of RALFs and LLGs for assembly at the apical plasma membrane of pollen tubes is orchestrated by ANXs/BUPSs through endomembrane trafficking. Pollen tube growth requires precise regulation of cell wall integrity, which is maintained by ANX/BUPS-RALF-LLG signaling complexes. While structural and biochemical studies have revealed physical interactions between these components, their spatial organization and assembly dynamics in growing pollen tubes remain unclear. Here, we systematically investigated the subcellular localization and endomembrane trafficking of ANX/BUPS-RALF-LLG complex components through transient expression studies in tobacco pollen tubes. We demonstrate that each component exhibits distinct subcellular distribution patterns: RALF4/19 peptide ligands predominantly localize after secretion to the cell wall, ANXs/BUPSs receptors are enriched at the apical plasma membrane (PM), while GPI-anchored LLG2/3 co-receptors exclusively co-localize to cytosolic vesicles. Through co-expression studies, we show that ANXs/BUPSs recruit both RALFs and LLGs that localize to the same vesicles to the PM, indicating their role as master organizers and scaffolds of signaling complex assembly during pollen tube growth. RALFs stabilize ANX/BUPS-LLG interactions at the PM. Disruption of endocytosis by Brefeldin A treatment severely disrupts PM localization of ANXs/BUPSs and causes cytoplasmic aggregation of LLGs, while Wortmannin leads to partial trapping of proteins in pre-vacuolar compartments. Altogether, these findings indicate that ANX/BUPS-RALF-LLG complex assembly is highly dynamic and depends on proper endomembrane trafficking to maintain pollen tube integrity during growth.
Generative cell internalisation establishes distinctive ‘cell-within-a-cell’ pollen organization in angiosperms. Comparative and transgenic expression analyses provide valuable insights into the cytological and genetic regulation underlying generative cell internalisation in Arabidopsis. For double fertilisation in flowering plants, a three-celled male gametophyte (pollen grain) delivers two non-motile sperm cells to the female gametes through a rapidly elongating pollen tube. This process is facilitated by a distinctive'cell-within-a-cell' pollen organisation, where the smaller generative cell becomes internalised into the larger vegetative cytoplasm through precisely regulated morphogenetic process and callose dynamics. However, the genetic mechanisms underlying generative cell internalisation remain unclear. Therefore, this study aims to comparatively analyse Arabidopsis wild-type with two different mutants that exhibit immobilised generative cells from the loss-of-function of DUF707 STICKY GENERATIVE CELL (SGC) and bHLH transcription factors BONOBO (BNB1) and BNB2, respectively. The results show that wild-type generative cells internalise through four successive steps. In contrast, bnb1;bnb2 mutants fail to progress to step 3 due to incomplete dissolution of the hemispherical callose and sgc-1 mutant proceeds to step 3 but arrests before step 4 with the intine-side callose dissolution disturbed. Moreover, transgenic analyses employing SGC promoter reporters and various BNB expression constructs revealed that SGC expression is positively regulated by BNB transcription factors. These findings enhance the understanding of the cytological and genetic regulation involving BNB transcription factors and downstream components, including SGC, during generative cell internalisation.
The stigma papilla cells of Arabidopsis thaliana control water transport to pollen by regulating the morphology of vacuoles in papilla cells after pollination. Pollen hydration is the first crucial response after pollination for successful fertilization. In the Brassicaceae family, papilla cells on the stigma supply water to pollen. In pollinated papilla cells, cellular responses essential for pollen hydration are induced. However, it remains unclear how papilla cells release water from inside the cells to the pollen. Here, we set up a live-cell imaging system for observing vacuole dynamics in Arabidopsis thaliana papilla cells and investigated the role of vacuole morphology in these cells in the regulation of water transfer to pollen. Before pollination, vacuoles in the papilla cells changed their morphology through fusion and constriction; however, after pollination, they formed larger vacuoles and exhibited reduced movement. Additionally, when the morphological variation of vacuoles in the papilla cells was inhibited by wortmannin treatment, the pollen hydration rate decreased in a concentration-dependent manner. In contrast, the vacuoles tended to be less constricted even before pollination and showed less variation than wild-type after pollination in Rho-like GTPase from plants 2 (ROP2) mutant papilla cells, where the pollen hydration rate is faster. We propose that the regulation of vacuole morphology in papilla cells is involved in water transfer to pollen during pollination.
Key message Arabidopsis stigma papillae grow by a diffuse growth mechanism rather than by tip growth. Abstract In angiosperms, the stigma is the first point of contact between the pollen (male) and pistil (female) during pollination. The stigma facilitates pollen capture and adhesion, compatibility responses, pollen germination, and pollen tube guidance to the transmitting tract. In Arabidopsis thaliana , the stigma is composed of single-celled stigma papillae that initiate from the apex of the carpels. Despite their critical function in plant reproduction, little is known about the cell and molecular mechanisms that govern stigma papillae growth and development. Using morphometric analysis of stigma papillae growth during different stages of floral development, we show that A. thaliana stigma papillae grow via a diffuse growth mechanism. Consistent with this conclusion, several mutants with reduced growth anisotropy in vegetative tissues due to defective cellulose or microtubule function likewise reduce anisotropy in stigma papillae.
We link key aspects of land plant reproductive evolution and detail how successive molecular changes leading to novel tissues and organs require co-evolution of communication systems between tissues. The transition of water-dependent reproduction of algae to mechanisms with very limited water dependence in many land plant lineages allowed plants to colonize diverse terrestrial environments, leading to the vast variety of extant plant species. The emergence of modified cell types, novel tissues, and organs enabled this transition; their origin is associated with the co-evolution of novel or adapted molecular communication systems and gene regulatory networks. In the light of an increasing number of genome sequences in combination with the establishment of novel genetic model organisms from diverse green plant lineages, our knowledge and understanding about the origin and evolution of individual traits that arose in a concerted way increases steadily. For example, novel members of gene families in signaling pathways emerged for communication between gametes and gametophytes with additional tissues surrounding the gametes. Here, we provide a comprehensive overview on the origin and evolution of reproductive novelties such as pollen grains, immobile sperms, ovules and seeds, carpels, gamete/gametophytic communication systems, double fertilization, and the molecular mechanisms that have arisen anew or have been co-opted during evolution, including but not limited to the incorporation of phytohormones, reactive oxygen species and redox signaling as well as small RNAs in regulatory modules that contributed to the evolution of land plant sexual reproduction.
