Mature Pollen Grains Research Articles

Heat stress during reproductive development inhibits fertilization in olives

In this study, we investigated the effect of heat exposure during olive inflorescence on reproductive development. We hypothesized that male development processes during this period are particularly sensitive to high temperatures. We characterized the development of flowers and inflorescences under natural conditions in the cultivars 'Arbequina' and 'Koroneiki', and defined eight phenological stages, beginning with the emergence of reproductive buds until anthesis (international standard BBCH scale of the phenological inflorescence development phases 53–60). In addition, we examined the response of olive inflorescence and flowering, to two heat scenarios: prolonged exposure to moderately high temperatures and a two-hour concentrated heat shock treatment. Analysis of our data revealed that both treatments had a detrimental effect on the development and functioning of the tapetum, the innermost layer of the anther, which is essential for the development and functioning of pollen. Additionally, we found that the 'Arbequina' cultivar displayed relative tolerance to heat compared to 'Koroneiki' in terms of pollen germinability after heat stress. In contrast to the response of the above cultivars, the 'Souri' exhibited an exceptionally high sensitivity to heat. An assessment of pollen viability after in vitro heat shock treatment of 54 cultivars revealed that pollen from seven varieties, 'Niedda de Oliana', 'Lechín de Sevilla', 'Tonda Oliana', 'Tamir', 'Sigoise', 'Taggiasca', and 'Carrasquenha', showed exceptional tolerance to heat. These cultivars show a greater degree of suitability for cultivation in warm climates, (or in areas susceptible to extreme hot-spells). This study demonstrated that heat has a negative impact on the male reproductive system in olives, during early flower development and in mature pollen grains, which are relatively resistant to heat. Heat stress impaired pollen tube elongation capacity, and germination rate. Additionally, heat stress affected the ultrastructure of the external wall of the pollen grain, the exine, potentially impairing its ability to adhere to the stigma and fertilize the ovule. The sensitivity of olive reproductive capacity to heat is a genotype-dependent trait, allowing for the identification of heat-resistant cultivars for use as pollen donors and for development of new heat-resistant cultivars.

Micromorphological and Phytochemical Evaluation of Heliotropium rariflorum Stocks From Karak, Pakistan.

Micromorphological and phytochemical studies play a major role in quality control and standardization of traditional or herbal medications. In the present research, micromorphological assessment of Heliotropium rarifloum stocks was performed through light and scanning electron microscopies (LM & SEM). The anatomy of leaves, stem and root showed salient histological features. Both surfaces of the leaves had setose glandular trichomes measuring 20-38 × 6-15 µm. The lower epidermis had comparatively a maximum anomocytic stomata (16-35) and stomatal index (12-33). The mature pollen grains were small (74 µm) and spheroidal shaped, with psilate exine (2 μm) sculpturing. Vein termination and vein islet number of the upper epidermis were 5-20 and 5-15, respectively. The palisade ratio of the leaf lamina for the upper and lower epidermis was 2-10 and 2-8. LM and SEM of the powdered samples displayed crystals, phloem fibers, xylem, vessels, sieve tube elements, companion cells, and tracheids. Extractive values determination, fluorescence, and phytochemical analysis were employed for quality control according to the World Health Organization (WHO) guidelines. Phytochemical screening revealed various secondary metabolites. It is suggested that H. rariflorum might be a reliable source of nutrients and secondary metabolites and might be more medically effective. The current findings confirm its standardization and validation.

Morphogenesis, megagametogenesis, and microgametogenesis in Actinidia arguta flower buds

In dioecious plants, understanding sexual differentiation is important for improving cultivation techniques. Flower bud morphogenesis and gametophyte formation underlie the mechanism of sexual differentiation. Actinidia arguta (hardy kiwi) is a unisexual plant, and there have been few studies on its flower bud differentiation or gametophyte formation. We studied flower bud morphogenesis and gametophyte formation in A. arguta using paraffin sections and found that the morphogenesis of male and female flower buds was divided into six stages: the undifferentiated stage, inflorescence initials differentiation stage, calyx primordium differentiation stage, petal primordium differentiation stage, stamen primordium differentiation stage, and pistil primordium differentiation stage. Morphogenesis in the male inflorescence can reach quaternary flower primordia, and morphogenesis in the female inflorescence can reach the tertiary flower primordia, however, no Stage 4 lateral flowers in the male inflorescence or Stage 3 lateral flowers in the female inflorescence were found in natural situation. The embryo sac in female plants was a monocellular Polygonum type, and the embryo sac development in male plants was incomplete; abortion occurred before the archesporial cells developed. The male gametophyte differentiation process in the male plant was normal, with pollen grains similar in shape to a rugby ball and arranged neatly in the anther. The pollen grain differentiation in female plants was abnormal; abortion occurred at the uninucleate microspore stage, and mature pollen grains were irregularly spherical.

The receptor kinase OsANX limits precocious flowering and inflorescence over-branching and maintains pollen tube integrity in rice

CrRLK1L subfamily members are involved in diverse growth- and development-related processes in Arabidopsis. However, the functions of their counterparts in rice are unknown. Here, OsANX expression was detected in developing inflorescences, mature pollen grains, and growing pollen tubes, and it was localized to the plasma membrane in pollen grains and tobacco epidermal cells. Homozygous osanx progeny could not be segregated from the CRISPR/Cas9-edited mutants osanx-c1+/- and osanx-c2+/-, and such progeny were segregated only occasionally from osanx-c3+/-. Further, all three alleles showed osanx male but not female gamete transmission defects, in line with premature pollen tube rupture in osanx-c3. Additionally, osanx-c3 exhibited precocious flowering, excessively branched inflorescences, and an extremely low seed setting rate of 1.4 %, while osanx-c2+/- and osanx-c3+/- had no obvious defects in inflorescence development or the seed setting rate compared to wild-type Nipponbare (Nip). Consistent with this, the complemented line pPS1:OsANX-GFP/osanx-c2 (PSC), in which the lack of OsANX expression was inflorescence-specific, showed slightly earlier flowering and overly-branched panicles. Multiple inflorescence meristem transition-related and inflorescence architecture-related genes were expressed at higher levels in osanx-c3 than in Nip; thus, they may partially account for the aforementioned mutant phenotypes. Our findings broaden our understanding of the biological functions of OsANX in rice.

Morphology of flower bud differentiation and floral organ specialization in Caucasian clover

AbstractFlower bud differentiation is the commencement of plant reproductive development. Flower organ specialization follows flower bud differentiation. This biological mechanism controls whether plants reproduce successfully via seed. Caucasian clover (Trifolium ambiguum) is used in mixed temperate pastures. However, its low natural seed setting rate contributes to a low seed yield. This study aimed to elucidate how the reproductive biology of Caucasian clover could affect seed production. Caucasian clover flower bud differentiation and flower organ development were systematically examined. Flower bud differentiation has six consecutive stages: pre‐differentiation, differentiation of the inflorescence, the floret, the perianth, the stamen, and finally the pistil primordium. The anther has two chambers. Pollen grains have six development phases: the tetraspore, early‐uninucleate, late‐uninucleate, vegetative cell and germ‐cell form, binucleate, and maturation phase. The microspore tetrad is tetrahedral, and the mature pollen grains are bicellular. Pollen grain viability was found 35%. The Caucasian clover monocarpelate ovary contains 1–2 ovules that are covered with a 2‐layer integument. The ovule is anatropous. The embryo sac development type is a polygonum. The floral formula is “⚥↑K(5) C1+1+2 A(9)+1 G(1:1:1−2)”. These genetic traits of two pollen sacs and low viability pollen grains may be responsible for the low natural seed setting. Poor seed setting may also be due to embryo sac dysplasia that leads to ovule sterility. Therefore, the breeding aim should be to improve pollen viability, to decrease embryo sac dysplasia and to reduce embryo abortion that could be targeted for genetic improvement to enhance seed yields.

Tetrad stage transient cold stress skews auxin-mediated energy metabolism balance in Chinese cabbage pollen.

Changing ambient temperature often impairs plant development and sexual reproduction, particularly pollen ontogenesis. However, mechanisms underlying cold stress-induced male sterility are not well understood. Here, we exposed Chinese cabbage (Brassica campestris) to different cold conditions during flowering and demonstrated that the tetrad stage was the most sensitive. After completion of pollen development at optimal conditions, transient cold stress at the tetrad stage still impacted auxin levels, starch and lipid accumulation, and pollen germination, ultimately resulting in partial male sterility. Transcriptome and metabolome analyses and histochemical staining indicated that the reduced pollen germination rate was due to the imbalance of energy metabolism during pollen maturation. The investigation of β-glucuronidase (GUS)-overexpressing transgenic plants driven by the promoter of DR5 (DR5::GUS report system) combined with cell tissue staining and metabolome analysis further validated that cold stress during the tetrad stage reduced auxin levels in mature pollen grains. Low-concentration auxin treatment on floral buds at the tetrad stage before cold exposure improved the cold tolerance of mature pollen grains. Artificially changing the content of endogenous auxin during pollen maturation by spraying chemical reagents and loss-of-function investigation of the auxin biosynthesis gene YUCCA6 by artificial microRNA technology showed that starch overaccumulation severely reduced the pollen germination rate. In summary, we revealed that transient cold stress at the tetrad stage of pollen development in Chinese cabbage causes auxin-mediated starch-related energy metabolism imbalance that contributes to the decline in pollen germination rate and ultimately seed set.

Morphological study of sexual reproductive disorders in Ligusticum chuanxiong

Chuanxiong Rhizoma is a well-known Sichuan-specific herbal medicine. Its original plant, Ligusticum chuanxiong, has been cultivated asexually for a long time. L. chuanxiong has sexual reproductive disorders, which restricts its germplasm innovation. However, there is little research on the reproductive system of L. chuanxiong. This study is based on a comparative anatomical research approach, using morphological dissection, paraffin sectioning, staining and compression, and combined with scanning electron microscopy technology, to observe and compare the flowers, fruits, and seeds at various stages of reproductive growth of L. chuanxiong and its wild relative L. sinense. The results showed that the meiosis of pollen mother cells is abnormal in L. chuanxiong anthers, and the size and number of microspores are uneven and inconsistent in the tetrad stage. tapetum cells are not completely degenerated during anther development. During the pollen ripening stage, there are fine cracks in the anther wall, while most anthers could not release pollen normally. The surface of mature pollen grains is concave and partially deformed, and the pollens are all inactive and cannot germinate in vitro. The starch, polysaccharides, and lipids in the pollen were insufficient. The filaments of L. chuanxiong are short at the flowering stage and recurved downward. Double-hanging fruits were observed in the fruiting stage, being wrinkled; with shriveled seeds. Compared with L. sinense at the same stage, the anthers of L. sinense developed normally, and the pollen grains are vigorous and can germinate in vitro. The double-hanging fruits of L. sinense are full and normal; at the flowering period, the filaments are long and erect, significantly higher than the stigma. Mature blastocysts are visible in the ovary of both L. chuanxiong and L. sinense, and there is no significant difference in stigmas. The conclusion is that during the development of L. chuanxiong stamens, the meiosis of pollen mother cells is abnormal, and tetrad, tapetum, filament and other pollen structures develop abnormally. L. chuanxiong has the characteristic of male infertility, which is an important reason for its sexual reproductive disorders.

The transcription factors and pathways underpinning male reproductive development in Arabidopsis.

As Arabidopsis flowers mature, specialized cells within the anthers undergo meiosis, leading to the production of haploid microspores that differentiate into mature pollen grains, each containing two sperm cells for double fertilization. During pollination, the pollen grains are dispersed from the anthers to the stigma for subsequent fertilization. Transcriptomic studies have identified a large number of genes expressed over the course of male reproductive development and subsequent functional characterization of some have revealed their involvement in floral meristem establishment, floral organ growth, sporogenesis, meiosis, microsporogenesis, and pollen maturation. These genes encode a plethora of proteins, ranging from transcriptional regulators to enzymes. This review will focus on the regulatory networks that control male reproductive development, starting from flower development and ending with anther dehiscence, with a focus on transcription factors and some of their notable target genes.

Dynamic organelle changes and autophagic processes in lily pollen germination

Pollen germination is a crucial process in the life cycle of flowering plants, signifying the transition of quiescent pollen grains into active growth. This study delves into the dynamic changes within organelles and the pivotal role of autophagy during lily pollen germination. Initially, mature pollen grains harbor undifferentiated organelles, including amyloplasts, mitochondria, and the Golgi apparatus. However, germination unveils remarkable transformations, such as the redifferentiation of amyloplasts accompanied by starch granule accumulation. We investigate the self-sustained nature of amylogenesis during germination, shedding light on its association with osmotic pressure. Employing BODIPY 493/503 staining, we tracked lipid body distribution throughout pollen germination, both with or without autophagy inhibitors (3-MA, NEM). Typically, lipid bodies undergo polarized movement from pollen grains into elongating pollen tubes, a process crucial for directional growth. Inhibiting autophagy disrupted this essential lipid body redistribution, underscoring the interaction between autophagy and lipid body dynamics. Notably, the presence of tubular endoplasmic reticulum (ER)-like structures associated with developing amyloplasts and lipid bodies implies their participation in autophagy. Starch granules, lipid bodies, and membrane remnants observed within vacuoles further reinforce the involvement of autophagic processes. Among the autophagy inhibitors, particularly BFA, significantly impede germination and growth, thereby affecting Golgi morphology. Immunogold labeling substantiates the pivotal role of the ER in forming autophagosome-like compartments and protein localization. Our proposed speculative model of pollen germination encompasses proplastid differentiation and autophagosome formation. This study advances our understanding of organelle dynamics and autophagy during pollen germination, providing valuable insights into the realm of plant reproductive physiology.

Antifungal activity of copper oxide nanoparticles derived from Zizyphus spina leaf extract against Fusarium root rot disease in tomato plants

Incorporating green chemistry concepts into nanotechnology is an important focus area in nanoscience. The demand for green metal oxide nanoparticle production has grown in recent years. The beneficial effects of using nanoparticles in agriculture have already been established. Here, we highlight some potential antifungal properties of Zizyphus spina leaf extract-derived copper oxide nanoparticles (CuO-Zs-NPs), produced with a spherical shape and defined a 13–30 nm particle size. Three different dosages of CuO-Zs-NPs were utilized and showed promising antifungal efficacy in vitro and in vivo against the selected fungal strain of F. solani causes tomato root rot disease, which was molecularly identified with accession number (OP824846). In vivo results indicated that, for all CuO-Zs-NPs concentrations, a significant reduction in Fusarium root rot disease occurred between 72.0 to 88.6% compared to 80.5% disease severity in the infected control. Although treatments with either the chemical fungicide (Kocide 2000) showed a better disease reduction and incidence with (18.33% and 6.67%) values, respectively, than CuO-Zs-NPs at conc. 50 mg/l, however CuO-Zs-NPs at 250 mg/l conc. showed the highest disease reduction (9.17 ± 2.89%) and lowest disease incidence (4.17 ± 3.80%). On the other hand, CuO-Zs-NPs at varied values elevated the beneficial effects of tomato seedling vigor at the initial stages and plant growth development compared to either treatment with the commercial fungicide or Trichoderma Biocide. Additionally, CuO-Zs-NPs treatments introduced beneficial results for tomato seedling development, with a significant increase in chlorophyll pigments and enzymatic activity for CuO-Zs-NPs treatments. Additionally, treatment with low concentrations of CuO-Zs-NPs led to a rise in the number of mature pollen grains compared to the immature ones. however the data showed that CuO-Zs-NPs have a unique antifungal mechanism against F. solani, they subsequently imply that CuO-Zs-NPs might be a useful environmentally friendly controlling agent for the Fusarium root rot disease that affects tomato plants.Graphical

Transcriptomics reveals a core transcriptional network of K-type cytoplasmic male sterility microspore abortion in wheat (Triticum aestivum L.)

BackgroundCytoplasmic male sterility (CMS) plays a crucial role in hybrid production. K-type CMS, a cytoplasmic male sterile line of wheat with the cytoplasms of Aegilops kotschyi, is widely used due to its excellent characteristics of agronomic performance, easy maintenance and easy restoration. However, the mechanism of its pollen abortion is not yet clear.ResultsIn this study, wheat K-type CMS MS(KOTS)-90-110 (MS line) and it’s fertile near-isogenic line MR (KOTS)-90-110 (MR line) were investigated. Cytological analysis indicated that the anthers of MS line microspore nucleus failed to divide normally into two sperm nucleus and lacked starch in mature pollen grains, and the key abortive period was the uninucleate stage to dinuclear stage. Then, we compared the transcriptome of MS line and MR line anthers at these two stages. 11,360 and 5182 differentially expressed genes (DEGs) were identified between the MS and MR lines in the early uninucleate and binucleate stages, respectively. Based on GO enrichment and KEGG pathways analysis, it was evident that significant transcriptomic differences were “plant hormone signal transduction”, “MAPK signaling pathway” and “spliceosome”. We identified 17 and 10 DEGs associated with the IAA and ABA signal transduction pathways, respectively. DEGs related to IAA signal transduction pathway were downregulated in the early uninucleate stage of MS line. The expression level of DEGs related to ABA pathway was significantly upregulated in MS line at the binucleate stage compared to MR line. The determination of plant hormone content and qRT-PCR further confirmed that hormone imbalance in MS lines. Meanwhile, 1 and 2 DEGs involved in ABA and Ethylene metabolism were also identified in the MAPK cascade pathway, respectively; the significant up regulation of spliceosome related genes in MS line may be another important factor leading to pollen abortion.ConclusionsWe proposed a transcriptome-mediated pollen abortion network for K-type CMS in wheat. The main idea is hormone imbalance may be the primary factor, MAPK cascade pathway and alternative splicing (AS) may also play important regulatory roles in this process. These findings provided intriguing insights for the molecular mechanism of microspore abortion in K-type CMS, and also give useful clues to identify the crucial genes of CMS in wheat.

Pollen Development and Stainability in Vicia faba L. and Lupinus angustifolius L.

Commercially, leguminous crops (Fabaceae) are the second most important group of cultivated plants, just after grasses (Poaceae). This study focuses on the analysis of pollen development and stainability in two species belonging to the Fabaceae family: Vicia faba L. and Lupinus angustifolius L. Morphological analysis of the anthers at various stages of flower development allowed us to trace the processes of microsporogenesis and microgametogenesis. Nine different cell staining protocols with diverse mechanisms of action, including acetocarmine, Alexander’s dye, aniline blue in lactophenol, Calcein AM, FDA, MTT, TTC, Lugol’s iodine, and aceto-orcein, were tested for their suitability in assessing the viability of microspores as well as pollen grains in both species. Among the applied dyes, four allowed for the discrimination between viable and nonviable microspores in V. faba, and six dyes allowed for this in L. angustifolius. For mature pollen grains, all dyes enabled differentiation between viable and nonviable cells in both species. The highest viability indications for V. faba microspores were obtained with acetocarmine (94.6%), while for mature pollen, aniline blue in lactophenol, MTT, and aceto-orcein yielded the highest viability indications (90.8–96.3%). In L. angustifolius, the highest percentages of viable microspores (64.9–66.5%) were obtained with the acetocarmine, aniline blue in lactophenol, and TTC dyes. For mature pollen, the highest viability indications (83.4%–92.9%) were obtained with acetocarmine, aniline blue in lactophenol, Lugol’s iodine, and aceto-orcein. The viability of V. faba pollen grains in an in vitro germination test showed that the highest pollen germination (61.3%) was observed on the BK medium (rich in minerals with 10% sucrose). In L. angustifolius, the highest pollen germination was observed on the media containing boric acid and 5% sucrose (70.5%) and on the medium containing 10% sucrose only (74.2%).

Microsporogenesis, microgametogenesis, and pollen grain morphology of Fouquieria fasciculata (Fouquieriaceae, Ericales)

Background: Embryology of the male reproductive structures in Fouquieriaceae has been poorly studied. The ontogeny of the anther wall, microsporogenesis, microgametogenesis, and pollen morphology were described in detail for Fouquieria fasciculata.
 Questions: How microsporogenesis and microgametogenesis are carried out in Fouquieria fasciculata? How is the morphology of mature pollen grain?
 Study species: Fouquieria fasciculata (Fouquieriaceae).
 Study site and date: Río Estórax Canyon, near the El Plátano, Querétaro, Mexico.
 Methods: Floral buds and flowers at different stages of development were collected and processed for further analysis with light and scanning electron microscopy.
 Results: The development of the anther wall is of Dicotyledonous type. The anther wall consists of a single-layered epidermis covered by a thick cuticle, an endothecium with U-shaped fibrous thickenings, one middle layer and a secretory tapetum with uni or binucleate cell. Two orbicules types were characterized: doughnut-shape orbicules and aggregated orbicules with ruminate appearance. Microsporogenesis is successive, the microspore tetrads are tetrahedral. Pollen grains are prolate, tricolporate with reticulate-heterobrochate exine, and are shed in the two-celled stage.
 Conclusions: This research is the first comprehensive study of male structures ontogeny of Fouquieriaceae. Remarkable features were described in Fouquieria fasciculata, such as the differentiation of a secretory tapetum and the presence of orbicules, which are described for the first time and contribute to characterize the embryology of Fouquieriaceae.

Assembly and Comparative Analysis of the Complete Mitochondrial Genome of Two Species of Calla Lilies (Zantedeschia, Araceae).

In this study, the mitochondrial genomes of two calla species, Zantedeschia aethiopica Spreng. and Zantedeschia odorata Perry., were assembled and compared for the first time. The Z. aethiopica mt genome was assembled into a single circular chromosome, measuring 675,575 bp in length with a 45.85% GC content. In contrast, the Z. odorata mt genome consisted of bicyclic chromosomes (chromosomes 1 and 2), measuring 719,764 bp and exhibiting a 45.79% GC content. Both mitogenomes harbored similar gene compositions, with 56 and 58 genes identified in Z. aethiopica and Z. odorata, respectively. Analyses of codon usage, sequence repeats, gene migration from chloroplast to mitochondrial, and RNA editing were conducted for both Z. aethiopica and Z. odorata mt genomes. Phylogenetic examination based on the mt genomes of these two species and 30 other taxa provided insights into their evolutionary relationships. Additionally, the core genes in the gynoecium, stamens, and mature pollen grains of the Z. aethiopica mt genome were investigated, which revealed maternal mitochondrial inheritance in this species. In summary, this study offers valuable genomic resources for future research on mitogenome evolution and the molecular breeding of calla lily.

Arabidopsis pollen-specific glycerophosphodiester phosphodiesterase-like genes are essential for pollen tube tip growth.

In angiosperms, pollen tube growth is critical for double fertilization and seed formation. Many of the factors involved in pollen tube tip growth are unknown. Here, we report the roles of pollen-specific GLYCEROPHOSPHODIESTER PHOSPHODIESTERASE-LIKE (GDPD-LIKE) genes in pollen tube tip growth. Arabidopsis thaliana GDPD-LIKE6 (AtGDPDL6) and AtGDPDL7 were specifically expressed in mature pollen grains and pollen tubes and GFP-AtGDPDL6 and GFP-AtGDPDL7 fusion proteins were enriched at the plasma membrane at the apex of forming pollen tubes. Atgdpdl6 Atgdpdl7 double mutants displayed severe sterility that was rescued by genetic complementation with AtGDPDL6 or AtGDPDL7. This sterility was associated with defective male gametophytic transmission. Atgdpdl6 Atgdpdl7 pollen tubes burst immediately after initiation of pollen germination in vitro and in vivo, consistent with the thin and fragile walls in their tips. Cellulose deposition was greatly reduced along the mutant pollen tube tip walls, and the localization of pollen-specific CELLULOSE SYNTHASE-LIKE D1 (CSLD1) and CSLD4 was impaired to the apex of mutant pollen tubes. A rice pollen-specific GDPD-LIKE protein also contributed to pollen tube tip growth, suggesting that members of this family have conserved functions in angiosperms. Thus, pollen-specific GDPD-LIKEs mediate pollen tube tip growth, possibly by modulating cellulose deposition in pollen tube walls. This article is protected by copyright. All rights reserved.

ANTHER STRUCTURE IN <i>CRASSULA ERICOIDES</i>, <i>C. INTERMEDIA</i> AND <i>C. MULTICAVA</i> (CRASSULACEAE)

The development and structure of the anther wall in Crassula intermedia, C. multicava and C. ericoides was studied for the first time. In the studied species, the presence of basic features of the Crassulaceae family associated with reproduction processes was confirmed. These include: tetrasporangiate anther, parietal tapetum, simultaneous microsporogenesis, predominantly tetrahedral tetrads of microspores, 2-celled and 3-colporate mature pollen grains. The microsporangium wall is 4-layered (epidermis, endothecium, middle layer and tapetum) and its development occurs within a typical variation of the centrifugal type. Tannins accumulate in epidermal cells: in C. intermedia, along the periphery of the cell protoplast, and in C. multicava and C. ericoides, the cell protoplasts are completely filled with tannins. The tapetum develops without reorganization (typical parietal tapetum). The study has shown that Crassula species are characterized by a number of unique features. They form an eccentric connective displaced from the central position towards the abaxial microsporangia, while the connective tissue does not develop between the adaxial microsporangia. Fibrous thickenings were found not only in the endothecium cells, but also in the cells around the vascular bundle of the connective tissue. The number of cell layers with fibrous thickenings and their location differ in the studied species. In C. ericoides, they are arranged in one layer in the form of a ring in the anther part completely fused with the filament. In C. intermedia and C. multicava, 2–3 layers are formed there. As regards the anther part free from the filament, in C. ericoides cells with fibrous thickenings are formed only in the endothecium, while in other two species not only in the endothecium, but also in 2–3 underlying layers of the connective tissue. In the structure of the stamen and anther of Crassula species, there are features common with Sedum and Aeonium species (the presence of cells with fibrous thickenings in the anther part free from the filament) and Kalanchoe (the stamen is wide at the base and narrow at the junction with the anther). Our data indicate that additional studies are needed, and the position of the Crassula clade continues to be controversial on the phylogenetic tree of the Crassulaceae family.

RHO OF PLANT proteins are essential for pollen germination in Arabidopsis.

Pollen germination is a process of polarity establishment, through which a single and unique growth axis is established. Although most of the intracellular activities associated with pollen germination are controlled by RHO OF PLANTs (ROPs) and increased ROP activation accompanies pollen germination, a critical role of ROPs in this process has not yet been demonstrated. Here, by genomic editing of all four Arabidopsis (Arabidopsis thaliana) ROPs that are preferentially expressed in pollen, we showed that ROPs are essential for polarity establishment during pollen germination. We further identified and characterized two ROP effectors in pollen germination (REGs) through genome-wide interactor screening, Boundary of ROP domain (BDR) members BDR8 and BDR9, whose functional loss also resulted in no pollen germination. BDR8 and BDR9 were distributed in the cytosol and the vegetative nucleus of mature pollen grains but re-distributed to the plasma membrane (PM) of the germination site and to the apical PM of growing pollen tubes. We demonstrated that the PM re-distribution of BDR8 and BDR9 during pollen germination relies on ROPs but not vice versa. Furthermore, enhanced expression of BDR8 partially restored germination of rop1 pollen but had no effects on that of the quadruple rop pollen, supporting their genetic epistasis. Results presented here demonstrate an ROP signaling route essential for pollen germination, which supports evolutionarily conserved roles of Rho GTPases in polarity establishment.

Changes in the content of pollen total lipid and TAG in Arabidopsis thaliana DGAT1 mutant as11.

In mature pollen grains, lipids are primarily stored in the form of lipid droplets that provide energy and act as a carbon source for normal pollen development and germination. Triacylglycerol (TAG) is the major form of stored plant lipids. Diacylglycerol transferase, which is encoded by DGAT1 in Arabidopsis thaliana, is an important enzyme regulating triacylglycerol synthesis. Within the seeds of the DGAT1 mutant as11, the content of TAG is significantly decreased and the fatty acid composition also differs from the wild type. Transcriptome data of mature anthers showed that the genes involved in the TAG synthesis pathway were downregulated in as11. Analysis of gene expression patterns via transcriptome data also revealed that the expression of PDAT1, which functions in a manner complementary to the DGAT1 gene, was significantly decreased in as11, whereas the amylopectin synthase genes SS1 and SS2 were upregulated in mutant as11. We also detected lower total lipid, TAG and fatty acid contents in mature as11 pollen, with palmitic acid (C16:0) and linolenic acid (C18:3) being the major fatty acids in mature pollen. The cytological results showed that the lipid droplet content was reduced in mature as11 pollen. In the binuclear pollen grain II stage, WT pollen contained lipid droplets that were primarily accumulated around the generative nucleus, whereas the pollen in the mutant as11 was rich in starch grains that were primarily distributed around the vegetative nucleus. Ultrastructural analysis indicated that during pollen development in as11, the amount of endoplasmic reticulum in tapetal cells and pollen grains decreased, whereas the Golgi body content increased, which directly or indirectly led to a decrease in the levels of lipidosomes and an increase in the starch content in as11. Changes in the lipid content and fatty acid composition of the mutant as11 differ from those in the wild type during pollen development.

Duplicate mutations of GhCYP450 lead to the production of ms5m6 male sterile line in cotton.

The duplicated male sterile genes ms5m6 in cotton were map-based cloned and validated by the virus-induced gene silencing assays. Duplicate mutations of the GhCYP450 gene encoding a cytochrome P450 protein are responsible for the male sterility in cotton. The utilization of male sterility in cotton plays a vital role in improving yield and fiber quality. A complete male sterile line (ms5ms6) has been extensively used to develop hybrid cotton worldwide. Using Zhongkang-A (ZK-A) developed by transferring Bt and ms5ms6 genes into the commercial cultivar Zhongmiansuo 12, the duplicate genes were map-based cloned and confirmed via the virus-induced gene silencing (VIGS) assays. The duplicate mutations of GhCYP450 genes encoding a cytochrome P450 protein were responsible for producing male sterility in ms5ms6 in cotton. Sequence alignment showed that GhCYP450-Dt in ZK-A differed in two critical aspects from the fertile wild-type TM-1: GhCYP450-Dt has three amino acid (D98E, E168K, G198R) changes in the coding region and a 7-bp (GGAAAAA) insertion in the promoter domain; GhCYP450-At appears to be premature termination of GhCYP450 translation. Further morphological observation and cytological examination of GhCYP450-silenced plants induced by VIGS exhibited shorter filaments and no mature pollen grains. These results indicate that GhCYP450 is essential for pollen exine formation and pollen development for male fertility. Investigating the mechanisms of ms5ms6 male sterility will deepen our understanding of the development and utilization of heterosis.

Cell-type-specific alternative splicing in the Arabidopsis germline.

During sexual reproduction in flowering plants, the two haploid sperm cells (SCs) embedded within the cytoplasm of a growing pollen tube are carried to the embryo sac for double fertilization. Pollen development in flowering plants is a dynamic process that encompasses changes at transcriptome and epigenome levels. While the transcriptome of pollen and SCs in Arabidopsis (Arabidopsis thaliana) is well documented, previous analyses have mostly been based on gene-level expression. In-depth transcriptome analysis, particularly the extent of alternative splicing (AS) at the resolution of SC and vegetative nucleus (VN), is still lacking. Therefore, we performed RNA-seq analysis to generate a spliceome map of Arabidopsis SCs and VN isolated from mature pollen grains. Based on our de novo transcriptome assembly, we identified 58,039 transcripts, including 9,681 novel transcripts, of which 2,091 were expressed in SCs and 3,600 in VN. Four hundred and sixty-eight genes were regulated both at gene and splicing levels, with many having functions in mRNA splicing, chromatin modification, and protein localization. Moreover, a comparison with egg cell RNA-seq data uncovered sex-specific regulation of transcription and splicing factors. Our study provides insights into a gamete-specific AS landscape at unprecedented resolution.