Sporophytic Tissues Research Articles

Analysis of the cytohistological phenotype of anthers in sunflower with ASI-AG3 in the context of the PET1 mytotype and the allelic genotype for the Rf1 gene

Microsporogenesis and male gametogenesis are processes that, although under tight morphogenetic control, can often be affected by deviations of internal or external stimuli causing androsterility. The article examines the cytohistological phenotypes of sunflower anthers with gibberellin induced androsterility in plants with different mitotype and allelic genotype for the Rf1 gene, in comparison with the cytohistological phenotypes of fertile and sterile anthers (ASC-PET1). The treatment with gibberellic acid (AG3, 0.01%) was carried out by spraying the inflorescence at the budding stage. Cytological samples and those with semi-thin anther sections were analysed by light microscopy. It was determined that the gametocidal effects induced by AG3 associated with degenerative and early apoptotic events in tapetum cells were more severe in plants with sterile cytoplasm and nuclear Rf1 genes compared to those with fertile cytoplasm and no fertility-restoring genes. In plants with ASC, the exogenous gibberellin stimulus does not cause any visible compensatory events of the sterile phenotype, on the contrary, the effects of microsporogenesis inhibition are more temporally advanced, with sporogenous and sporophytic tissue cells showing various lesions in the premeiosis. The general common and differentiation characteristics found at the androsteril phenotypes suggest interactions between orfH522 and Rf1 gene expression products of negative feedback type in untreated plants and positive feedback type in AG3-treated ones. The research is relevant in the context of elucidating the physiological regulatory processes of the sensitivity of male gametophyte development to various stress conditions, which prospectively makes possible regulatory intervention in plants with different androsterility/androfertility control systems.

Maternal nitric oxide homeostasis impacts female gametophyte development under optimal and stress conditions.

In adverse environments, the number of fertilizable female gametophytes (FGs) in plants is reduced, leading to increased survival of the remaining offspring. How the maternal plant perceives internal growth cues and external stress conditions to alter FG development remains largely unknown. We report that homeostasis of the stress signaling molecule nitric oxide (NO) plays a key role in controlling FG development under both optimal and stress conditions. NO homeostasis is precisely regulated by S-nitrosoglutathione reductase (GSNOR). Prior to fertilization, GSNOR protein is exclusively accumulated in sporophytic tissues and indirectly controls FG development in Arabidopsis (Arabidopsis thaliana). In GSNOR null mutants, NO species accumulated in the degenerating sporophytic nucellus, and auxin efflux into the developing FG was restricted, which inhibited FG development, resulting in reduced fertility. Importantly, restoring GSNOR expression in maternal, but not gametophytic tissues, or increasing auxin efflux substrate significantly increased the proportion of normal FGs and fertility. Furthermore, GSNOR overexpression or added auxin efflux substrate increased fertility under drought and salt stress. These data indicate that NO homeostasis is critical to normal auxin transport and maternal control of FG development, which in turn determine seed yield. Understanding this aspect of fertility control could contribute to mediating yield loss under adverse conditions.

Understanding megasporogenesis through model plants: contemporary evidence and future insights.

The megasporangium serves as a model system for understanding the concept of individual cell identity, and cell-to-cell communication in angiosperms. As development of the ovule progresses, three distinct layers, the epidermal (L1), the subepidermal or the hypodermal (L2) and the innermost layers (L3) are formed along the MMC (megaspore mother cell). The MMC, which is the primary female germline cell, is initiated as a single subepidermal cell amongst several somatic cells. MMC development is governed by various regulatory pathways involving intercellular signaling, small RNAs and DNA methylation. The programming and reprograming of a single nucellar cell to enter meiosis is governed by 'permissive' interacting processes and factors. Concomitantly, several nucellar sister cells are prevented from germline fate also by a set of 'repressive' factors. However, in certain angiosperms, anomalies in development of the female gametophyte have been observed. The sporophytic tissue surrounding the female gametophyte affects the gametophyte in multiple ways. The role of genes and transcription factors in the development of the MMC and in the regulation of various processes studied in selected model plants such as Arabidopsis is explained in detail in this paper. However, as angiosperms display enormous diversity, it is important to investigate early stages of megasporogenesis in other plant systems as well. Such studies provide valuable insights in understanding the regulation of megasporogenesis and the evolution of the female gametophyte from gymnosperms to flowering plants.

Disruptions of sugar utilization and carbohydrate metabolism in rice developing anthers aggravated heat stress-induced pollen abortion

High temperature (HT) stress at reproductive stage is one of most important environment negatively affecting spikelet fertility and rice yield. In this study, the effect of HT exposure on the sugar composition and carbohydrate metabolism in developing anthers and its relation to floret fertility and pollen viability were investigated by different temperature regimes under well-controlled climatic condition. Result showed that HT exposure during microspore development significantly reduced the starch deposition in developing anther and evidently disrupted the spatial distribution of sugar and starch concentrations in different compartments of rice anther, with the higher ratio of sucrose to hexose concentrations in HT-stressed anthers relative to the control ones. Under HT exposure, the amount of starch deposition in the fraction of sporophytic tissues dropped evidently, while the concentrations of sucrose and starch in anther wall tissue enhanced significantly, suggesting that HT exposure impaired the translocation of sucrose from the anther wall tissue to the sporophytic tissues inside rice anther. Furthermore, we presented possible contribution of various genes and key enzymes involving in sugar conversion and carbohydrate metabolism in developing anther to the formation of HT-induced pollen abortion by disrupting the sugar utilization in HT-stressed anther. HT exposure suppressed the activities of cell wall and vacuolar invertase, hexokinase, and ADP-glucose pyrophosphorylase in developing anther, while it was opposite for the effect of HT exposure on sucrose synthase and fructokinase. HT-induced suppression of OsCWIN3 in the anther walls might be strongly responsible for the HT-induced impairments of sugar utilization in HT-stressed anthers.

PXL1 and SERKs act as receptor–coreceptor complexes for the CLE19 peptide to regulate pollen development

Gametophyte development in angiosperms occurs within diploid sporophytic structures and requires coordinated development; e.g., development of the male gametophyte pollen depends on the surrounding sporophytic tissue, the tapetum. The mechanisms underlying this interaction remain poorly characterized. The peptide CLAVATA3/EMBRYO SURROUNDING REGION-RELATED 19 (CLE19) plays a “braking” role in preventing the harmful overexpression of tapetum transcriptional regulators to ensure normal pollen development in Arabidopsis. However, the CLE19 receptor is unknown. Here, we show that CLE19 interacts directly with the PXY-LIKE1 (PXL1) ectodomain and induces PXL1 phosphorylation. PXL1 is also required for the function of CLE19 in maintaining the tapetal transcriptional regulation of pollen exine genes. Additionally, CLE19 induces the interactions of PXL1 with SOMATIC EMBRYOGENESIS RECEPTOR-LIKE KINASE (SERK) coreceptors required for pollen development. We propose that PXL1 and SERKs act as receptor and coreceptor, respectively, of the extracellular CLE19 signal, thereby regulating tapetum gene expression and pollen development.

Stomatal regulators are co-opted for seta development in the astomatous liverwort Marchantia polymorpha.

The evolution of special types of cells requires the acquisition of new gene regulatory networks controlled by transcription factors (TFs). In stomatous plants, a TF module formed by subfamilies Ia and IIIb basic helix-loop-helix TFs (Ia-IIIb bHLH) regulates stomatal formation; however, how this module evolved during land plant diversification remains unclear. Here we show that, in the astomatous liverwort Marchantia polymorpha, a Ia-IIIb bHLH module regulates the development of a unique sporophyte tissue, the seta, which is found in mosses and liverworts. The sole Ia bHLH gene, MpSETA, and a IIIb bHLH gene, MpICE2, regulate the cell division and/or differentiation of seta lineage cells. MpSETA can partially replace the stomatal function of Ia bHLH TFs in Arabidopsis thaliana, suggesting that a common regulatory mechanism underlies setal and stomatal formation. Our findings reveal the co-option of a Ia-IIIb bHLH TF module for regulating cell fate determination and/or cell division of distinct types of cells during land plant evolution.

Sorus developmental biology of hybrid cultivar in Saccharina japonica: Environmental and endogenous regulation

The induction and regulation of sporogenesis in the kelp Saccharina japonica is particularly important due to the necessity of obtaining spores to produce “summer seedlings” in the kelp hatchery. However, some hybrid cultivars of S. japonica with excellent agronomic traits are late-maturing, which means the hybrid cultivars cannot produce spore at the time of “summer seedling” production and limits their application in practice. The effects of temperature, irradiance, nutrition level, and exogenous hormones on the sorus formation were investigated in this study, aiming to clarify the optimum inducible conditions for sorus formation of S. japonica. Based on the number of individuals forming sorus and the percentages of the sorus area on the sporophyte tissue discs, it was proposed that the optimal conditions for sorus formation were at 17 °C, 5 mg L−1 nitrate nitrogen, 0.5 mg L−1 phosphorus, 10−6 mol L−1 abscisic acid (ABA), and irradiance of 60-100 μmol photons m−2 s−1. Then, the vegetative tissue discs of S. japonica sporophyte were cultured under the above optimal conditions to induce the sorus formation, and the duration from vegetative tissue to sours formation and zoospores release was divided into five stages. The anatomical structure changes, antioxidant activity, reactive oxygen species (ROS) level, MDA content, and endogenous hormone levels were determined at the five stages during sorus formation. Significant accumulation of ROS was observed but the content of malondialdehyde stayed at a stable level indicating that ROS may have an important role in the regulation of sorus formation rather than their potential cytotoxic effects. For the endogenous hormone analysis, indole-3-acetic acid (IAA) was lowered and ABA was elevated in the early stages during sorus formation. The GA3 trend was consistent with IAA. The contents of dihydrozeatin, isopentenyladenine, and isopentenyladenosine were significantly lower in vegetative tissue than that in sorus tissue. These results indicated ROS and hormones may play an important role in the endogenous regulation of sorus formation in S. japonica. The relevant results herein can lay a foundation for studying the external conditions and endogenous regulation of sorus formation for the kelp hybrids of late-maturing.

Anther development in Arabidopsis thaliana involves symplastic isolation and apoplastic gating of the tapetum-middle layer interface.

During flowering plant reproduction, anthers produce pollen grains, the development of which is supported by the tapetum, a nourishing maternal tissue that also contributes non-cell-autonomously to the pollen wall, the resistant external layer on the pollen surface. How the anther restricts movement of the tapetum-derived pollen wall components, while allowing metabolites such as sugars and amino acids to reach the developing pollen, remains unknown. Here, we show experimentally that in arabidopsis thaliana the tapetum and developing pollen are symplastically isolated from each other, and from other sporophytic tissues, from meiosis onwards. We show that the peritapetal strip, an apoplastic structure, separates the tapetum and the pollen grains from other anther cell layers and can prevent the apoplastic diffusion of fluorescent proteins, again from meiosis onwards. The formation and selective barrier functions of the peritapetal strip require two NADPH oxidases, RBOHE and RBOHC, which play a key role in pollen formation. Our results suggest that, together with symplastic isolation, gating of the apoplast around the tapetum may help generate metabolically distinct anther compartments.

Endosperm and Maternal-specific expression of EIN2 in the endosperm affects endosperm cellularization and seed size in Arabidopsis.

Seed size is related to plant evolution and crop yield and is affected by genetic mutations, imprinting, and genome dosage. Imprinting is a widespread epigenetic phenomenon in mammals and flowering plants. ETHYLENE INSENSITIVE2 (EIN2) encodes a membrane protein that links the ethylene perception to transcriptional regulation. Interestingly, during seed development EIN2 is maternally expressed in Arabidopsis and maize, but the role of EIN2 in seed development is unknown. Here, we show that EIN2 is expressed specifically in the endosperm, and the maternal-specific EIN2 expression affects temporal regulation of endosperm cellularization. As a result, seed size increases in the genetic cross using the ein2 mutant as the maternal parent or in the ein2 mutant. The maternal-specific expression of EIN2 in the endosperm is controlled by DNA methylation but not by H3K27me3 or by ethylene and several ethylene pathway genes tested. RNA-seq analysis in the endosperm isolated by laser-capture microdissection show upregulation of many endosperm-expressed genes such as AGAMOUS-LIKEs (AGLs) in the ein2 mutant or when the maternal EIN2 allele is not expressed. EIN2 does not interact with DNA and may act through ETHYLENE INSENSITIVE3 (EIN3), a DNA-binding protein present in sporophytic tissues, to activate target genes like AGLs, which in turn mediate temporal regulation of endosperm cellularization and seed size. These results provide mechanistic insights into endosperm and maternal-specific expression of EIN2 on endosperm cellularization and seed development, which could help improve seed production in plants and crops.

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.

Highlighted articles for June 2022

Highlighted articles for June 2022

Sporophytic control of pollen meiotic progression is mediated by tapetum expression of ABORTED MICROSPORES.

Pollen development is dependent on the tapetum, a sporophytic anther cell layer surrounding the microspores that functions in pollen wall formation but is also essential for meiosis-associated development. There is clear evidence of crosstalk and co-regulation between the tapetum and microspores, but how this is achieved is currently not characterized. ABORTED MICROSPORES (AMS), a tapetum transcription factor, is important for pollen wall formation, but also has an undefined role in early pollen development. We conducted a detailed investigation of chromosome behaviour, cytokinesis, radial microtubule array (RMA) organization, and callose formation in the ams mutant. Early meiosis initiates normally in ams, shows delayed progression after the pachytene stage, and then fails during late meiosis, with disorganized RMA, defective cytokinesis, abnormal callose formation, and microspore degeneration, alongside abnormal tapetum development. Here, we show that selected meiosis-associated genes are directly repressed by AMS, and that AMS is essential for late meiosis progression. Our findings indicate that AMS has a dual function in tapetum-meiocyte crosstalk by playing an important regulatory role during late meiosis, in addition to its previously characterized role in pollen wall formation. AMS is critical for RMA organization, callose deposition, and therefore cytokinesis, and is involved in the crosstalk between the gametophyte and sporophytic tissues, which enables synchronous development of tapetum and microspores.

Identification of key regulatory genes involved in the sporophyte and gametophyte development in Ginkgo biloba ovules revealed by in situ expression analyses

PremiseIn Arabidopsis thaliana, the role of the most important key genes that regulate ovule development is widely known. In nonmodel species, and especially in gymnosperms, the ovule developmental processes are still quite obscure. In this study, we describe the putative roles of Ginkgo biloba orthologs of regulatory genes during ovule development. Specifically, we studied AGAMOUS (AG), AGAMOUS‐like 6 (AGL6), AINTEGUMENTA (ANT), BELL1 (BEL1), Class III HD‐Zip, and YABBY Ginkgo genes.MethodsWe analyzed their expression domains through in situ hybridizations on two stages of ovule development: the very early stage that corresponds to the ovule primordium, still within wintering buds, and the late stage at pollination time.Results GBM5 (Ginkgo ortholog of AG), GbMADS8 (ortholog of AGL6) and GbC3HDZ1‐2‐3 were expressed in both the stages of ovule development, while GbMADS1, GbAGL6‐like genes (orthologs of AGL6), GbBEL1‐2 and YABBY Ginkgo orthologs (GbiYAB1B and GbiYABC) seem mostly involved at pollination time. GbANTL1 was not expressed in the studied stages and was different from GbANTL2 and GbBEL1, which seem to be involved at both stages of ovule development. In Ginkgo, the investigated genes display patterns of expression only partially comparable to those of other studied seed plants.ConclusionsThe expression of most of these regulatory genes in the female gametophyte region at pollination time leads to suggest a communication between the sporophytic maternal tissue and the developing female gametophyte, as demonstrated for well‐studied model angiosperms.

Spatiotemporal Distribution of Homogalacturonans and Hemicelluloses in the Placentas, Ovules and Female Gametophytes of Utricularia nelumbifolia during Pollination.

Utricularia nelumbifolia is a large carnivorous plant that is endemic to Brazil. It forms an extra-ovular female gametophyte, which surpasses the entire micropylar canal and extends beyond the limit of the integument and invades the placenta tissues. Due to the atypical behavior of the female gametophyte, it is interesting to determine the interaction between the gametophyte and sporophytic tissue. Therefore, the aim of this study was to evaluate the role of the placenta, the ovular tissues, the hypertrophied central cell and the integument in guiding the pollen tube in Utricularia nelumbifolia Gardner by studying the distribution of homogalacturonans and hemicelluloses. It was also determined whether the distribution of the homogalacturonans (HG) and hemicelluloses in Utricularia are dependent on pollination. The antibodies directed against the wall components (anti-pectin: JIM5, JIM7, LM19, LM20 and the anti-hemicelluloses: LM25, LM11, LM15, LM20, LM21) were used. Because both low- and high-esterified HG and xyloglucan were observed in the placenta, ovule (integument, chalaza) and female gametophyte of both pollinated and unpollinated flowers, the occurrence of these cell-wall components was not dependent on pollination. After fertilization, low methyl-esterified HGs were still observed in the cell walls of somatic cells and female gametophyte. However, in the case of high-esterified HG, the signal was weak and occurred only in the cell walls of the somatic cells. Because xyloglucans were observed in the cell walls of the synergids and egg cells, this suggests that they play a role in sexual reproduction. Utricularia nelumbifolia with an extra ovule-female gametophyte is presented as an attractive model for studying the male-female dialogue in plants.

Dual and opposing roles of EIN3 reveal a generation conflict during seed growth

Seed size critically affects grain yield of crops and hence represents a key breeding target. The development of embryo-nourishing endosperm is a key driver of seed expansion. We here report unexpected dual roles of the transcription factor EIN3 in regulating seed size. These EIN3 functions have remained largely undiscovered because they oppose each other. Capitalizing on the analysis of multiple ethylene biosynthesis mutants, we demonstrate that EIN3 represses endosperm and seed development in a pathway regulated by ethylene. We, in addition, provide evidence that EIN3-mediated synergid nucleus disintegration promotes endosperm expansion. Interestingly, synergid nucleus disintegration is not affected in various ethylene biosynthesis mutants, suggesting that this promoting function of EIN3 is independent of ethylene. Whereas the growth-inhibitory ethylene-dependent EIN3 action appears to be encoded by sporophytic tissue, the growth-promoting role of EIN3 is induced by fertilization, revealing a generation conflict that converges toward the key signaling component EIN3.

A dormant resource for genome size estimation in ferns: C-value inference of the Ophioglossaceae using herbarium specimen spores.

PremiseThe great variation of genome size (C‐value) across land plants is linked to various adaptative features. Flow cytometry (FCM), the standard approach to estimating C‐values, relies mostly on fresh materials, performing poorly when used with herbarium materials. No fern C‐value reports have been derived from herbarium specimens; however, the herbarium spores of some ferns remain highly viable for decades and are thus promising for further investigation. To explore this possibility, we evaluated herbarium spore collections of Ophioglossaceae ferns using FCM.MethodsFlow cytometry was conducted on 24 spore samples, representing eight of the 12 genera of the Ophioglossaceae, using specimens ranging in age from 2.6 to 111 years obtained from five herbaria.ResultsRegardless of the genus or the source herbarium, high‐quality C‐value data were generated from 17 samples, with the oldest being 26 years old. Estimates of the C‐values from sporophytic tissues of known ploidy did not reveal any evidence of apomixis for the species surveyed here. We also detected a pronounced genome downsizing in Sceptridium polyploids.DiscussionThe recent success of FCM for C‐value estimation using spores provides a much more convenient method of utilizing “dry” refrigerated materials. We demonstrate here that herbarium spores of some ferns are also promising for this use, even for older specimens.

Plant development: Suspensors as a battlefield for parental tug-of-war?

Parental contributions to zygotes can influence early embryogenesis and may regulate the distribution of maternal resources to progeny. A new study in Arabidopsis thaliana has demonstrated that signaling components from maternal sporophytic tissues and paternal gametes converge in zygotes to promote elongation of the extraembryonic suspensor, which supports the developing embryo proper.

The Rab Geranylgeranyl Transferase Beta Subunit Is Essential for Embryo and Seed Development in Arabidopsis thaliana.

Auxin is a key regulator of plant development affecting the formation and maturation of reproductive structures. The apoplastic route of auxin transport engages influx and efflux facilitators from the PIN, AUX and ABCB families. The polar localization of these proteins and constant recycling from the plasma membrane to endosomes is dependent on Rab-mediated vesicular traffic. Rab proteins are anchored to membranes via posttranslational addition of two geranylgeranyl moieties by the Rab Geranylgeranyl Transferase enzyme (RGT), which consists of RGTA, RGTB and REP subunits. Here, we present data showing that seed development in the rgtb1 mutant, with decreased vesicular transport capacity, is disturbed. Both pre- and post-fertilization events are affected, leading to a decrease in seed yield. Pollen tube recognition at the stigma and its guidance to the micropyle is compromised and the seed coat forms incorrectly. Excess auxin in the sporophytic tissues of the ovule in the rgtb1 plants leads to an increased tendency of autonomous endosperm formation in unfertilized ovules and influences embryo development in a maternal sporophytic manner. The results show the importance of vesicular traffic for sexual reproduction in flowering plants, and highlight RGTB1 as a key component of sporophytic-filial signaling.

Sporophytic control of anther development and male fertility by glucose-6-phosphate/phosphate translocator 1 (OsGPT1) in rice

Coordination between the sporophytic tissue and the gametic pollen within anthers is tightly controlled to achieve the optimal pollen fitness. Glucose-6-phosphate/phosphate translocator (GPT) transports glucose-6-phosphate, a key precursor of starch and/or fatty acid biosynthesis, into plastids. Here, we report the functional characterization of OsGPT1 in the rice anther development and pollen fertility. Pollen grains from homozygous osgpt1 mutant plants fail to accumulate starch granules, resulting in pollen sterility. Genetic analyses reveal a sporophytic effect for this mutation. OsGPT1 is highly expressed in the tapetal layer of rice anther. Degeneration of the tapetum, an important process to provide cellular contents to support pollen development, is impeded in osgpt1 plants. In addition, defective intine and exine are observed in the pollen from osgpt1 plants. Expression levels of multiple genes that are important to tapetum degeneration or pollen wall formation are significantly decreased in osgpt1 anthers. Previously, we reported that AtGPT1 plays a gametic function in the accumulation of lipid bodies in Arabidopsis pollen. This report highlights a sporophytic role of OsGPT1 in the tapetum degeneration and pollen development. The divergent functions of OsGPT1 and AtGPT1 in pollen development might be a result of their independent evolution after monocots and dicots diverged.

Function of Small Peptides During Male-Female Crosstalk in Plants.

Plant peptides secreted as signal molecular to trigger cell-to-cell signaling are indispensable for plant growth and development. Successful sexual reproduction in plants requires extensive communication between male and female gametophytes, their gametes, and with the surrounding sporophytic tissues. In the past decade, it has been well-documented that small peptides participate in many important reproductive processes such as self-incompatibility, pollen tube growth, pollen tube guidance, and gamete interaction. Here, we provide a comprehensive overview of the peptides regulating the processes of male-female crosstalk in plant, aiming at systematizing the knowledge on the sexual reproduction, and signaling of plant peptides in future.