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.

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.

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.

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.

Superoxide accumulates during early stigma papillae growth stages in Arabidopsis. Highly specialized stigma papillae cells play a critical role in plant reproduction. Their main purpose is to catch and interact with pollen, to mediate compatibility responses, to regulate pollen germination, and to guide pollen tubes to the transmitting tract so that the sperm cells carried in the pollen can be delivered to the female gametophyte to achieve double fertilization. In Arabidopsis thaliana, the stigma consists of single-celled stigma papillae that emerge from the apex of the fused carpels. Despite their critical function in plant reproduction, the molecular mechanisms that govern growth and maturation of stigma papillae remain poorly understood. Reactive Oxygen Species (ROS) have been implicated in stigma receptivity, but their roles in papillae development are less explored. Here we show that reactive oxygen species (ROS) also play different roles in stigma papillae development, with superoxide accumulating during the initiation and growth phase and hydrogen peroxide accumulating in mature papillae that are receptive to pollen. Reducing superoxide levels in the stigma by pharmacological treatments or over-expressing superoxide dismutase enzymes under an early stigma promoter inhibited stigma papillae growth, suggesting that ROS homeostasis is critical to papillae growth and differentiation for optimal pollination.

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.

TubeTracker provides a method to partially automate analysis of pollen tube growth using live imaging. Pollen function is critical for successful plant reproduction and crop productivity and it is important to develop accessible methods to quantitatively analyze pollen performance to enhance reproductive resilience. Here we introduce TubeTracker as a method to quantify key parameters of pollen performance such as, time to pollen grain germination, pollen tube tip velocity and maintenance of pollen tube integrity. TubeTracker integrates manual and automatic image processing routines and the graphical interface allows the user to interact with the software to make manual corrections of automated steps. TubeTracker does not depend on training data sets required to implement machine learning approaches and thus can be immediately implemented using readily available imaging systems. Furthermore, TubeTracker is an excellent tool to produce the pollen performance data sets necessary to take advantage of emerging AI-based methods to fully automate analysis. We tested TubeTracker and found it to be accurate in measuring pollen tube germination and pollen tube tip elongation across multiple cultivars of tomato.

Arabidopsis stigma papillae grow by a diffuse growth mechanism rather than by tip growth. 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.

Ultrastructural and cytochemical analyses of the megaspore, embryo sac, and synergid haustoria reveal their roles in nutrition, contributing to the successful development of the megagametophyte in R. pallida. In this paper, we present the first cytochemical and ultrastructural analysis of the megaspores, embryo sac, and synergid haustoria in Rosularia pallida (Schott & Kotschy) Stapf (Crassulaceae) are presented. The haustoria in the ovule of R. pallida primarily function to provide nutrition during megasporogenesis and megagametogenesis. Cytochemical staining reveals a significant increase in the accumulation of insoluble polysaccharides, lipids, and proteins within the megaspores and embryo sac. This increase occurs alongside the progressive degradation of nucellar cells and the growth of haustoria towards the integuments. The direction of haustorial growth within sporophyte tissues and the distribution of nutrients within the ovule complement each other, collectively contributing to efficient nutrition for the developing female gametophyte. Callose is present in the walls of both the megaspores and their haustoria. The functional megaspore (FM) haustorium is the only one that extends beyond the nucellus into the integuments during megasporogenesis. The disappearance of callose in the micropylar portion of the FM haustorium enables apoplasmic transport, particularly in this region. These findings suggest that the FM haustorium supports the development of a specific megaspore in the tetrad, indirectly influencing FM selection through nutrient provision. Furthermore, the removal of callose on the chalazal side of the tetrad likely facilitates the development of the embryo sac from the chalazal megaspore. Ultrastructural analyses of the megaspore, embryo sac, and synergid haustoria reveal the presence of transfer-wall ingrowths. No plasmodesmata were detected in the haustorial walls. Additionally, ultrastructural observations of the synergids indicate that their haustorium significantly elongates toward the micropyle and becomes metabolically active.

This study provides insights into the molecular and hormonal control of cleistogamy in pigeonpea, focusing on bHLH transcription factors and jasmonic acid pathway. Pigeonpea, an annual diploid (2n = 22) grain legume, holds significant nutritional value in cereal-dominated diets. The chasmogamous flowers of pigeonpea have a typical 9 + 1 diadelphous stamen where flowers open pre-fertilization resulting in cross-pollination. In contrast, a cleistogamous genotype characterized by polyadelphous stamens and flowers that open post-fertilization ensuring seed purity was analyzed for identifying causal pathways. Subsequent analysis focused on a set of transcription factors and their interaction with the hormonal networks associated with cleistogamy. Genes of the Jasmonic acid (JA) signaling pathway have been established to play a significant role in inducing cleistogamy and one of the key regulators of the JA pathway is bHLH (basic helix loop helix). A genome-wide survey identified 176 bHLH genes in the pigeonpea genome. Phylogenetic analysis classified 176 bHLH genes into 21 subfamilies distributed randomly across the genome. Gene ontology, cis-motifs analysis in the upstream region, and protein-protein interaction network implied the involvement of these genes in various biological processes. Expression analysis of key genes of the jasmonic acid pathway which includes MYC2 (Cc_bHLH135) along with its interacting partners TIFY/JAZ in chasmogamous and cleistogamous floral tissues revealed their potential role in flower opening. The results of UHPLC-MS/MS quantitation of Jasmonic acid and its bioactive form JA-Ile align with the expression analysis. The congruence of gene expression and hormone profiling highlights the involvement of the JA pathway in regulating flower opening, implying their potential role in cleistogamy in pigeonpea.

Overview of the current understanding of PG development, PT growth and the role of AGPs in these processes. The pollen grain (PG) is a complex structure composed of three cells: the vegetative cell which develops into a pollen tube (PT) and two sperm cells that will fuse with the egg cell and central cell, giving rise to the embryo and endosperm, respectively. This resilient gametophyte is constantly subjected to selective pressures, leading to a diverse range of characteristics, with one of its defining features being the pollen cell wall. In this review, we closely examine the developmental stages of PG formation and PT growth, with a specific focus on the dynamic roles of arabinogalactan-proteins (AGPs) throughout these processes. AGPs are initially present in pollen mother cells and persist throughout PT growth. In the early stages, AGPs play a crucial role in primexine anchoring, followed by nexine and intine formation as well as cellulose deposition, thereby providing essential structural support to the PG. As PGs mature, AGPs continue to be essential, as their absence often leads to the collapse of PGs before they reach full maturity. Moreover, the absence of AGPs during PT growth leads to abnormal growth patterns, likely due to disruptions of cellulose, callose, and F-actin deposition, as well as perturbations in calcium ion (Ca) signalling. Understanding the intricate interplay between AGPs and PG development sheds light on the underlying mechanisms that drive reproductive success and highlights the indispensable role of AGPs in ensuring the integrity and functionality of PGs.

SHATTERPROOF 2 regulates TAA1 expression for the establishment of the gynoecium valve margins. Gynoecium development and patterning play a crucial role in determining the ultimate structure of the fruit and, thus, seed production. The MADS-box transcription factor SHATTERPROOF 2 (SHP2) contributes to valve margin differentiation and plays a major role in fruit dehiscence and seed dispersal. Despite the acknowledged contribution of auxin to gynoecium development, its precise role in valve margin establishment remains somewhat enigmatic. Our study addresses this gap by uncovering the role of SHP2 as a positive regulator of key auxin biosynthetic genes, TAA1 and YUCCA 4. Genetic and molecular analyses revealed that SHP2 directly regulates the expression of TAA1 in the valve margins of a stage 12 gynoecium with known regulators of flower and ovule development, such as AGAMOUS, SEEDSTICK, and SEPATALA 3. Collectively, our findings define a previously unrecognized function of SHP2 in the regulation of auxin biosynthetic genes during gynoecium development and raise the possibility that the auxin produced under SHP2 regulation may contribute significantly to the valve margin establishment.

Self-incompatibility decays with age in plants of Physalis acutifolia, and plants that have transitioned to selfing produce fewer seeds but with comparable viability. Self-compatibility in this system is closely related to flower size, which is in turn dependent on the direction of the cross, suggesting parental effects on both morphology and compatibility. The sharpleaf groundcherry, Physalis acutifolia, is polymorphic for self-compatibility, with naturally occurring self-incompatible (SI) and self-compatible (SC) populations. Moreover, SI individuals have been documented to transition to SC with age, at least in greenhouse conditions. Here we tested whether this within-lifespan transition occurs predictably (developmental decay of SI) or could result from a lack of pollination (a plastic response). Using greenhouse crosses, we demonstrated that SI P. acutifolia plants transition to SC after 70 days, regardless of pollination treatment, consistent with predictable developmental decay. This loss of SI corresponds to a loss of pollen inhibition, with self-pollen often reaching the ovary after 24 h. The originally SI plants that transition to SC can produce viable seeds from self crosses, albeit significantly fewer than from outcrosses of SI plants or from lines fixed for SC. Throughout the experiment, we observed that flower size, which differs between SI and SC populations, was highly correlated with the compatibility phenotype. These findings suggest that the mechanisms leading to the loss of SI during a lifespan are similar to those involved in fixed losses of SI, but that older plants that transition to SC do not present the same reproductive capacity as fixed selfers.

This review covers the latest developments on the regulation of early seed development by phytohormones. The development of seeds in flowering plants starts with the fertilization of the maternal gametes by two paternal sperm cells. This leads to the formation of two products, embryo and endosperm, which are surrounded by a tissue of maternal sporophytic origin, called the seed coat. The development of each of these structures is under tight genetic control. Moreover, several phytohormones have been shown to modulate the development of all three seed compartments and have been implicated in the communication between them. This is particularly relevant, as embryo, endosperm, and seed coat have to coordinate their development for successful seed formation. Here, we review the latest advances on the hormonal regulation of early seed development in the model plant species Arabidopsis thaliana, with a focus on the endosperm and the seed coat. Moreover, we highlight how phytohormones serve as mechanisms of non-cell autonomous communication between these two compartments and how they are determinant in shaping seed formation.

We describe transcriptional dynamics at the rice shoot apex by integrating time resolved single nuclei RNA-seq with bulk RNA-seq data. In rice, short days trigger floral transition and the transcriptional reprogramming of the shoot apex to become reproductive. We integrated time-resolved bulk RNA-seq with single nuclei RNA-seq analysis to gain a refined understanding of the transcriptional programs induced at the shoot apex during floral transition. Our analyses highlighted technological and conceptual differences between single nuclei RNA-seq and bulk RNA-seq data and described previously uncharacterized transcriptional programs associated with the early steps of floral induction in rice.

Induction of parthenogenesis (embryo formation from unfertilized egg cells) by embryogenic transcription factors is associated with twin formation at high frequencies, and involves two distinct mechanisms. Synthetic apomixis has been achieved through the induction of parthenogenesis by ectopic expression of the Baby Boom family of transcription factors. An associated phenomenon from this process is the formation of polyembryony including twin progeny at high frequencies, but the underlying mechanisms have not been explored. Here, we provide a brief description of the phenomenon, discuss potential mechanisms for twin formation in flowering plants, propose two possible models for their occurrence, and evaluate the available evidence from both dizygotic and monozygotic twins in relation to these models. The two proposed models are independent, but they can operate in combination. We conclude that both models are required to explain the types of twins and triplets that we and others have observed. These models provide future directions for basic research, as well as suggest possible approaches towards reducing polyembryony when incorporating synthetic apomixis into crop plants such as maize where twinning is not desirable.