Female Gametophyte Development Research Articles

Reproductive Traits in Two Alpine Endemic Species of Campanulaceae

Alpine plants are well adapted to environments with extreme conditions – low temperatures, a short growing season, increased air humidity and precipitation, and are therefore of particular interest for research. The aim of the present study is to reveal the main reproductive characteristics of two endemic species of the family Campanulaceae, namely Campanula orbelica (Balkan endemic) and Jasione bulgarica (Bulgarian endemic). The main features of the reproductive biology (embryological features, type of reproduction) were established based on the study of micro- and macrosporogenesis and the development of male and female gametophytes. The established features of the reproductive structures and processes characterized C. orbelica and J. bulgarica as sexually reproducing species. Despite the extreme conditions of existence, the studied species presented normal generative progresses leading to the formation of pollen and embryo, which ensures their successful reproduction, populations stability and their distribution in the subalpine environment. In addition, the genome size of C. orbelica was studied by flow cytometry. The estimated genome size varied in the range 1C = 1.34–1.38 pg. No deviation in the ploidy level was found in 22 specimens fresh leaves of which were collected from different parts of the Rila and Pirin Mts.

Polycomb proteins RING1A/B promote H2A monoubiquitination to regulate female gametophyte development in Arabidopsis.

A functional female gametophyte is the basis of successful sexual reproduction in flowering plants. During female gametophyte development, the megaspore mother cell (MMC), differentiated from a single subepidermal somatic cell in the nucellus, undergoes meiosis to produce four megaspores; only the one at the chalazal end, referred to as functional megaspore (FM), undergoes three rounds of mitosis and develops into a mature embryo sac. Here, we reported that RING1A and RING1B (RING1A/B), two functionally redundant Polycomb proteins in Arabidopsis, are critical for female gametophyte development. The mutations of RING1A/B resulted in defects in MMC and FM's specification and subsequent mitosis of FM, thereby leading to aborted ovules. Gene expression analysis revealed several genes essential for female gametophyte development, including Argonaute (AGO) family genes and critical transcription factors, were ectopically expressed in ring1a ring1b. Furthermore, RING1A/B bound some of these genes to promote H2A monoubiquitination (H2Aub) deposition. Together, RING1A/B promote H2Aub modification at genes essential for female gametophyte development, suppressing their expression to ensure the progression of female gametophyte development.

Sexual reproduction: Is the genetic pathway for female germ cell specification conserved in land plants?

Land plants share several core factors responsible for female gametophyte development, despite their differing structures and developmental programs. New work providing molecular dissection of reproductive phases in non-angiosperm plants is a powerful tool for elucidating the underlying genetic network.

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 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.

An ATP-binding cassette transporter, OsABCB24, is involved in female gametophyte development and early seed growth in rice.

Female gametogenesis has been rarely studied due to gametophyte lethality and the unavailability of related genetic resources. In this study, we identified a rice ATP-binding cassette transporter, OsABCB24, whose null function displayed a significantly reduced seed setting rate by as much as 94%-100% compared with that of the wild type (WT). The reciprocal cross of WT and mutant plants demonstrated that the female reproductive organs in mutants were functionally impaired. Confocal microscopy observations revealed that, although megasporogenesis remained unaffected in CRISPR/Cas9 osabcb24 mutants, the formation of female gametophytes was interrupted. Additionally, the structure of the syncytial nucleus was impaired during the initial stages of endosperm formation. Histochemical analysis showed that OsABCB24 was preferentially expressed at the conjunction of receptacle and ovary, spanning from the functional megaspore stage to the two-nucleate embryo sac stage. Further, OsABCB24 was identified as an endoplasmic reticulum membrane-localized protein. Notably, the overexpression of OsABCB24 triggered a 1.5- to 2-fold increase in grain production compared to the WT. Our findings showed that OsABCB24 plays a key role in both female gametophyte development and the early development of seeds.

ClBBM and ClPLT2 function redundantly during both male and female gametophytes development in watermelon

As a prerequisite for sexual reproduction, gametophyte development is an interesting process involving cell proliferation, differentiation and specialization. Gametogenesis has been extensively explored in model plants, but the regulatory mechanism of gametophyte development largely remains unknown in Cucurbitaceae species. In present study, we have shown that watermelon ClBBM and ClPLT2, two AP2/ERF transcription factors, participated in both male and female gametophyte development. Clbbm and Clplt2 single mutants resembled wild-type phenotypes in both vegetative and reproductive development. But Clbbm/Clplt2 double mutant showed partial pollen abortion and bore less seeds comparing to WT. Our results indicated that the abnormal pollen grains were caused by premature tapetum degeneration, and reduced seed-set was due to faulty embryo sac development. ClBBM and ClPLT2 were expressed in FG4 embryo sacs, and their transcripts were also detectable in the tapetum and microspore of stage 9 anthers, which was consistent with developmental stages of defective phenotypes observed in double mutant. The expression of genes essential for tapetum development, ClATM1, ClAMS, ClMS1 and ClMS188, was decreased in Clbbm/Clplt2 double mutants. Moreover, the transcriptome analysis indicated that ClBBM and ClPLT2 participated in tapetum and pollen wall development by regulate cell cycle, transmembrane transport, glucan and cellulose metabolic process. Collectively, ClBBM and ClPLT2 were functionally redundant in regulating gametophyte development in watermelon, and their functions differ from their homologous genes in model plant Arabidopsis.

Abnormalities in Stamen and Ovary Development Responsible for Low Seed Set of Dendrocalamus hamiltonii Nees et Arn. ex Munro

Bamboo propagation and seed collection are severely limited by a protracted and unpredictable flowering cycle. Dendrocalamus hamiltonii Nees et Arn. ex Munro was renowned for its delectable, bitter-free bamboo shoots, making it an exceptional choice for culinary purposes, which has significant economic value in China. To date, no fruit has been collected, and there are currently no comprehensive studies available on the floral morphology and embryology of D. hamiltonii. The morphological and anatomical characteristics of floral organs were described, and the developmental process of male and female gametophytes was elucidated, through anatomical observation. It was found that the floret of D. hamiltonii was composed of one lemma, palea, pistil, and six stamens, but lacked lodicules. The single ovule, possessing a parietal placenta, was anatropous and featured a double integument. The floral organs showed normal in external morphology, whereas male and female gametophyte development was abnormal, such as pollen grain shrinkage, hollow anther chambers, and underdeveloped ovules, which might be the important factors of its low seed setting rate. This study provided important information on the developmental stages of D. hamiltonii flowers and offered a theoretical basis for understanding the low seed setting rate of bamboo.

Arabidopsis Sar1 isoforms play redundant roles in female gametophytic development.

Functional loss of Arabidopsis Sar1b with that of either Sar1a or Sar1c inhibits mitosis of functional megaspores, leading to defective embryo sac formation and reduced fertility. Vesicular trafficking among diverse endomembrane compartments is critical for eukaryotic cells. Anterograde trafficking from endoplasmic reticulum (ER) to the Golgi apparatus is mediated by coat protein complex II (COPII) vesicles. Among five cytosolic components of COPII, secretion-associated Ras-related GTPase 1 (Sar1) mediates the assembly and disassembly of the COPII coat. Five genes in Arabidopsis encode Sar1 isoforms, whose different cargo specificities and redundancy were both reported. We show here that Arabidopsis Sar1a, Sar1b, and Sar1c mediate the development of female gametophytes (FGs), in which Sar1b plays a major role, whereas Sar1a and Sar1c play a minor role. We determined that female transmission of sar1a;sar1b or sar1c;sar1b was significantly reduced due to defective mitosis of functional megaspores. Half of ovules in sar1a;sar1b/+ or sar1c;sar1b/+ plants failed to attract pollen tubes, leading to fertilization failure. The homozygous sar1a;sar1b or sar1c;sar1b double mutant was obtained by introducing either UBQ10:GFP-Sar1b or UBQ10:GFP-Sar1c, supporting their redundant function in FG development.

Seasonal gene expression signatures of delayed fertilization in Fagaceae.

In the family Fagaceae, fertilization is delayed by several weeks to 1 year after pollination, leading to 1- or 2-year fruiting species depending on whether fruiting occurs in the same or the next year after flowering. To investigate physiological responses underlying the regulation of delayed fertilization, we monitored seasonal changes in genome-wide gene expression in tissues including leaves and buds over 2 years under natural conditions in one- (Quercus glauca) and 2-year fruiting species (Lithocarpus edulis). Genes associated with metabolic changes in response to winter cold, photosynthesis and cell proliferation, which are essential for survival and growth, showed highly conserved seasonal expression profiles between species. However, seasonal expression profiles diverged between species in genes associated with pollination, an important process contributing to the origin and maintenance of the reproductive barrier between plant species. By comparing seasonal progression of ovule development and gene expression in pistillate flowers, we revealed that ovules started developing after winter in the 2-year fruiting species, which could be linked to the activation of genes involved in fertilization and female gametophyte development after winter. These findings suggest that the 2-year fruiting species may have evolved a requirement of winter cold to prevent fertilization before winter and facilitate fertilization and embryo development in the following spring when temperature rises. This study offers new possibilities to explore the evolution of reproductive strategies in Fagaceae.

Effects of light quality, nitrogen source and Fe3+ concentration on female gametophyte development of Costaria costata

The female gametophytes of Costaria costata can be porpagated vegetatively by withholding inductive conditions, and also can be induced to produce gametes by providing the appropriate conditions. The change from vegetative to reproductive development has been attributed to various environmental factors, including light quality and nutrients. The experiment was conducted to test the combined effects of light quality (white light and blue light), nitrogen source (NaNO3 and Urea) and Fe3+ concentration (0, 0.1 and 0.5 mg L−1) in order to evaluate development performance of female gametophytes. When the female gametophytes and male gametophytes (mass ratio 2:1) mixed culture after 10 days, the number of female gametophytic cells remained constant and the apical part of cells elongated, while the male gametophytic cells remained in vegetative growth. The surface area of female and male gametophytes had increased in all treatments, ranging from 0.60 to 1.05 mm2 cm−2 and reaching a maximum under white light, CO(NH2)2 and Fe3+ 0.1 mg L−1. After 16 days, the female gametophytes developed oogonia and released eggs on the tip of the oogonia, while the male gametophytes developed spermatangia. The fraction of female gametophytes releasing eggs or forming sporophytes was determined as a measure of gametophyte phenology. A considerable proportion of female gametophytes (2.85%∼62.42%) had already formed oogonia and eggs at Fe3+ 0.1 mg L−1 and Fe3+ 0.5 mg L−1，particularly under blue light. The highest proportion with oogonia and eggs were observed at Fe3+ 0.5 mg L−1, blue light and CO(NH2)2 (22.12% with oogonia, 3.45% with eggs and 16.8% with sporophytes). On the 26th day, almost all female gametophytes cultivated at Fe3+ 0.1 mg L−1 and Fe3+ 0.5 mg L−1 in blue light developed a high portion of sporophytes (30.98%∼94.03%), while a significant lower portion of sporophyte at these Fe3+ concentrations in white light ranged between 10.97% and 42.59%. The density of sporophytes were maximal at Fe3+ 0.5 mg L−1, blue light and CO(NH2)2, but the length of sporophytes were maximal at Fe3+ 0.1 mg L−1, blue light and CO(NH2)2. In conclusion, the combination of blue light, Fe3+ 0.5 mg L−1 and CO(NH2)2 were favorable for female gametophyte development, which accelerated the process of gametophyte development and increased the final sporophyte growth.

Embryo sac development relies on symplastic signals from ovular integuments in Arabidopsis.

Ovules are female reproductive organs of angiosperms, consisting of sporophytic integuments surrounding female gametophytes, that is, embryo sacs. Synchronization between integument growth and embryo sac development requires intracellular communication. However, signaling routes through which cells of the two generations communicate are unclear. We report that symplastic signals through plasmodesmata (PDs) of integuments are critical for the development of female gametophytes. Genetic interferences of PD biogenesis either by functional loss of CHOLINE TRANSPORTER-LIKE1 (CTL1) or by integument-specific expression of a mutated CALLOSE SYNTHASE 3 (cals3m) compromised PD formation in integuments and reduced fertility. Close examination of pINO:cals3m or ctl1 ovules indicated that female gametophytic development was either arrested at various stages after the formation of functional megaspores. In both cases, defective ovules could not attract pollen tubes, leading to the failure of fertilization. Results presented here demonstrate a key role of the symplastic route in sporophytic control of female gametophytic development.

RLI2 regulates Arabidopsis female gametophyte and embryo development by facilitating the assembly of the translational machinery

Eukaryotic protein translation is a complex process that requires the participation of different proteins. Defects in the translational machinery often result in embryonic lethality or severe growth defects. Here, we report that RNase L inhibitor 2/ATP-BINDING CASSETTE E2 (RLI2/ABCE2) regulates translation in Arabidopsis thaliana. Null mutation of rli2 is gametophytic and embryonic lethal, whereas knockdown of RLI2 causes pleiotropic developmental defects. RLI2 interacts with several translation-related factors. Knockdown of RLI2 affects the translational efficiency of a subset of proteins involved in translation regulation and embryo development, indicating that RLI2 has critical roles in these processes. In particular, RLI2 knockdown mutant exhibits decreased expression of genes involved in auxin signaling and female gametophyte and embryo development. Therefore, our results reveal that RLI2 facilitates assembly of the translational machinery and indirectly modulates auxin signaling to regulate plant growth and development.

The molecular chaperone mtHSC70-1 interacts with DjA30 to regulate female gametophyte development and fertility in Arabidopsis.

Arabidopsis mitochondria-targeted heat shock protein 70 (mtHSC70-1) plays important roles in the establishment of cytochrome c oxidase-dependent respiration and redox homeostasis during the vegetative growth of plants. Here, we report that knocking out the mtHSC70-1 gene led to a decrease in plant fertility; the fertility defect of the mutant was completely rescued by introducing the mtHSC70-1 gene. mtHSC70-1 mutants also showed defects in female gametophyte (FG) development, including delayed mitosis, abnormal nuclear position, and ectopic gene expression in the embryo sacs. In addition, we found that an Arabidopsis mitochondrial J-protein gene (DjA30) mutant, j30+/- , had defects in FG development and fertility similar to those of mtHSC70-1 mutant. mtHSC70-1 and DjA30 had similar expression patterns in FGs and interacted in vivo, suggesting that these two proteins might cooperate during female gametogenesis. Further, respiratory chain complex IV activity in mtHSC70-1 and DjA30 mutant embryo sacs was markedly downregulated; this led to the accumulation of mitochondrial reactive oxygen species (ROS). Scavenging excess ROS by introducing Mn-superoxide dismutase 1 or catalase 1 gene into the mtHSC70-1 mutant rescued FG development and fertility. Altogether, our results suggest that mtHSC70-1 and DjA30 are essential for the maintenance of ROS homeostasis in the embryo sacs and provide direct evidence for the roles of ROS homeostasis in embryo sac maturation and nuclear patterning, which might determine the fate of gametic and accessory cells.

The mutation of CaCKI1 causes seedless fruits in chili pepper (Capsicum annuum)

The seedless mutant tn-1 in chili pepper is caused by a mutation in CaCKI1 (CA12g21620), which encodes histidine kinase involving female gametophyte development. An amino acid insertion in the receiver domain of CaCKI1 may be the mutation responsible for tn-1. Seedlessness is a desirable trait in fruit crops because the removal of seeds is a troublesome step for consumers and processing industries. However, little knowledge is available to develop seedless chili peppers. In a previous study, a chili pepper mutant tn-1, which stably produces seedless fruits, was isolated. In this study, we report characterization of tn-1 and identification of the causative gene. Although pollen germination was normal, confocal laser microscopy observations revealed deficiency in embryo sac development in tn-1. By marker analysis, the tn-1 locus was narrowed down to a 313kb region on chromosome 12. Further analysis combined with mapping-by-sequencing identified CA12g21620, which encodes histidine kinase as a candidate gene. Phylogenetic analysis revealed CA12g21620 was the homolog of Arabidopsis CKI1 (Cytokinin Independent 1), which plays an important role in female gametophyte development, and CA12g21620 was designated as CaCKI1. Sequence analysis revealed that tn-1 has a 3-bp insertion in the 6th exon resulting in one lysine (K) residue insertion in receiver domain of CaCKI1, and the sequence nearby the insertion is widely conserved among CKI1 orthologs in various plants. This suggested that one K residue insertion may reduce the phosphorylation relay downstream of CaCKI1 and impair normal development of female gametophyte, resulting in seedless fruits production in tn-1. Furthermore, we demonstrated that virus-induced gene silencing of CaCKI1 reducednormally developed female gametophyte in chili pepper. This study describes the significant role of CaCKI1 in seed development in chili pepper and the possibility of developing seedless cultivars using its mutation.

Identification of Causal Gene-Specific SNP Markers for the Development of Gynoecious Hybrids in Cucumber (Cucumis sativa L.) Using the PathoLogic Algorithm

Although the genome sequence of cucumber is publicly available, only a limited number of functional markers are in store for developing gynoecious hybrids using Indian genotypes. This study reported novel SNPs and InDels in the exonic regions of genes involved in gynoecy using two parents and their hybrid with genotyping-by-sequencing (GBS) by generating 3.547 Gb of raw data. Using NSDC reference genome GCA_000004075.2, a total of 40,143, 181,008 and 43,612 SNPs were identified, among which 514 were polymorphic between male and female parents but monomorphic between the male parent and the hybrid (confirming hybridity). We further identified that, out of those 514 SNPs, 74 were within the exonic regions of the sex-specific genes. The most interesting functional marker in this study was SNP 2,13,85,488, identified in the gene CsaV3_6G037780 G3I-38214 on chromosome 6, encoding 1-aminocyclopropane-1-carboxylate oxidase 1 (ACS1), which plays a key role in female flower production, as indicated in CuCyc with the PathoLogic algorithm. The InDel analysis also identified a variation inside the gene CsaV3_6G304050 G3I-37940, encoding histone lysine N-methyl transferase, involved in flowering and female gametophyte development. Thus, this study has identified gynoecy-specific functional markers; upon further validation, these markers will accelerate the evolution of gynoecious hybrids in India and global cucumber breeding programs.

Molecular mapping of genomic regions and identification of possible candidate genes associated with gynoecious sex expression in bitter gourd

Bitter gourd is an important vegetable crop grown throughout the tropics mainly because of its high nutritional value. Sex expression and identification of gynoecious trait in cucurbitaceous vegetable crops has facilitated the hybrid breeding programme in a great way to improve productivity. In bitter gourd, gynoecious sex expression is poorly reported and detailed molecular pathways involve yet to be studied. The present experiment was conducted to study the inheritance, identify the genomic regions associated with gynoecious sex expression and to reveal possible candidate genes through QTL-seq. Segregation for the gynoecious and monoecious sex forms in the F2 progenies indicated single recessive gene controlling gynoecious sex expression in the genotype, PVGy-201. Gynoecious parent, PVGy-201, Monoecious parent, Pusa Do Mausami (PDM), and two contrasting bulks were constituted for deep-sequencing. A total of 10.56, 23.11, 15.07, and 19.38 Gb of clean reads from PVGy-201, PDM, gynoecious bulk and monoecious bulks were generated. Based on the ΔSNP index, 1.31 Mb regions on the chromosome 1 was identified to be associated with gynoecious sex expression in bitter gourd. In the QTL region 293,467 PVGy-201 unique variants, including SNPs and indels, were identified. In the identified QTL region, a total of 1019 homozygous variants were identified between PVGy1 and PDM genomes and 71 among them were non-synonymous variants (SNPS and INDELs), out of which 11 variants (7 INDELs, 4 SNPs) were classified as high impact variants with frame shift/stop gain effect. In total twelve genes associated with male and female gametophyte development were identified in the QTL-region. Ethylene-responsive transcription factor 12, Auxin response factor 6, Copper-transporting ATPase RAN1, CBL-interacting serine/threonine-protein kinase 23, ABC transporter C family member 2, DEAD-box ATP-dependent RNA helicase 1 isoform X2, Polygalacturonase QRT3-like isoform X2, Protein CHROMATIN REMODELING 4 were identified with possible role in gynoecious sex expression. Promoter region variation in 8 among the 12 genes indicated their role in determining gynoecious sex expression in bitter gourd genotype, DBGy-1. The findings in the study provides insight about sex expression in bitter gourd and will facilitate fine mapping and more precise identification of candidate genes through their functional validation.

RUVBL proteins are involved in plant gametophyte development.

The proper development of male and female gametophytes is critical for successful sexual reproduction and requires a carefully regulated series of events orchestrated by a suite of various proteins. RUVBL1 and RUVBL2, plant orthologues of human Pontin and Reptin, respectively, belong to the evolutionarily highly conserved AAA+ family linked to a wide range of cellular processes. Previously, we found that RUVBL1 and RUVBL2A mutations are homozygous lethal in Arabidopsis. Here, we report that RUVBL1 and RUVBL2A play roles in reproductive development. We show that mutant plants produce embryo sacs with an abnormal structure or with various numbers of nuclei. Although pollen grains of heterozygous mutant plants exhibit reduced viability and reduced pollen tube growth in vitro, some of the ruvbl pollen tubes are capable of targeting ovules in vivo. Similarly, some ruvbl ovules retain the ability to attract wild-type pollen tubes but fail to develop further. The activity of the RUVBL1 and RUVBL2A promoters was observed in the embryo sac, pollen grains, and tapetum cells and, for RUVBL2A, also in developing ovules. In summary, we show that the RUVBL proteins are essential for the proper development of both male and particularly female gametophytes in Arabidopsis.

BAG6-A from Fragaria viridis pollen modulates gametophyte development in diploid strawberry

Male and female gametophyte development processes are essential steps in the life cycles of all land plants. Here, we characterized a gene, FviBAG6-A, screened from the Fragaria viridis (2 n = 2x=14) pollen cDNA library and physically interacted with S-RNase. Ubiquitinated of Sa-RNase might be determined by the interaction of FviBAG6-A in the ubiquitin-proteasome system during fertilization. We found that overexpression of FviBAG6-A in Arabidopsis caused shorter silique length, and decreased silique number. Moreover, overexpression of FviBAG6-A in Fragaria vesca (2 n = 2x=14) led to a greatly reduced seed number, with nearly 80% of the seeds aborted. Analyses of paraffin sections and reactive oxygen species (ROS) content revealed that the majority of severe pollen defects were likely due to the early degradation of the tapetum and middle layer as a result of ROS accumulation and abnormal development of the uninucleate megaspore mother. Moreover, the FviBAG6-A interact with the E3 ligase SIZ1 and contribute to the SUMOylation of FviBAG6-A , which may be induced by the high level of ROS content, further promoting gametophyte abortion in strawberry transgenic lines. This study characterized the FviBAG6-A and reveals its novel function in gametophyte development.

Sporogenesis, gametophyte development and embryogenesis in Glehnia littoralis

BackgroundGlehnia littoralis is an economic herb with both medicinal and edible uses. It also has important ecological value and special phylogenetic status as it is a monotypic genus species distributing around beach. Little information on its reproductive biology has been reported so far, which has hindered conservation and application of this species. In this study, we observed morphological changes from buds emergence to seeds formation and internal changes during sporogenesis, gametophyte development and embryo and endosperm development of G. littoralis using paraffin-embedded-sectioning and stereo microscope.ResultsThe results showed that the stages of internal development events of G. littoralis corresponded to obvious external morphological changes, most of developmental features were consistent with other Apiaceae species. The development of male and female gametophytes was not synchronized in the same flower, however, exhibited temporal overlap. From mid-late April to mid-May, the anther primordial and ovule primordial developed into the trinucleate pollen grain and eight-nuclear embryo sac, respectively. From late-May to mid-July, the zygote developed into mature embryo. In addition, some defects in gynoecium or ovule development and abnormal embryo and endosperm development were found. We induced that the possible causes of abortion in G. littoralis were as follows: nutrient limitation, poor pollination and fertilization, and bad weather.ConclusionsThis study revealed the whole process and morphological characteristics of the development of reproductive organ in G. littoralis, which not only provided important data for the study of systematic and conservation biology, but also provided a theoretical basis for cross breeding.

Spatiotemporal formation of the large vacuole regulated by the BIN2-VLG module is required for female gametophyte development in Arabidopsis.

In Arabidopsis thaliana, female gametophyte (FG) development is accompanied by the formation and expansion of the large vacuole in the FG; this is essential for FG expansion, nuclear polar localization, and cell fate determination. Arabidopsis VACUOLELESS GAMETOPHYTES (VLG) facilitates vesicular fusion to form large vacuole in the FG, but the regulation of VLG remains largely unknown. Here, we found that gain-of-function mutation of BRASSINOSTEROID INSENSITIVE2 (BIN2) (bin2-1) increases VLG abundance to induce the vacuole formation at stage FG1, and leads to abortion of FG. Loss-of-function mutation of BIN2 and its homologs (bin2-3 bil1 bil2) reduced VLG abundance and mimicked vlg/VLG phenotypes. Knocking down VLG in bin2-1 decreased the ratio of aberrant vacuole formation at stage FG1, whereas FG1-specific overexpression of VLG mimicked the bin2-1 phenotype. VLG partially rescued the bin2-3 bil1 bil2 phenotype, demonstrating that VLG acts downstream of BIN2. Mutation of VLG residues that are phosphorylated by BIN2 altered VLG stability and a phosphorylation mimic of VLG causes similar defects as did bin2-1. Therefore, BIN2 may function by interacting with and phosphorylating VLG in the FG to enhance its stability and abundance, thus facilitating vacuole formation. Our findings provide mechanistic insight into how the BIN2-VLG module regulates the spatiotemporal formation of the large vacuole in FG development.