Sporophytic Cells Research Articles

Reflections on the absence of stomata on the gametophyte generation of extant land plants: a focus on poikilohydry

Abstract The recent realization that bryophyte sporophytes are homoiohydric enabled the present analysis of morphological features specifically associated with poikilohydry. The only morphological feature absolutely diagnostic of poikilohydry is schizolytic intercellular spaces. In vascular plants these are gas-filled from the outset, but in bryophytes are liquid-filled initially. They remain in this condition in liverwort gametophytes but become gas-filled following stomatal opening in hornworts and mosses. These have fixed apertures and are completely unresponsive to environmental cues. Stomata in Devonian fossils may have had similarly fixed apertures. Unistratose lamellae, characteristic of moss and liverwort leaves and pteridophyte gametophytes, are optimal structures for reversible cell shrinkage and recovery accompanying de- and rehydration. In 1 M sucrose, gametophytic tissues and filmy fern leaves undergo shrinkage, whereas sporophyte cells plasmolyse. Under extreme desiccation, hydroids are the only bryophyte cells to undergo cavitation. Like bryophytes, desiccation-tolerant streptophyte algae undergo reversible cell shrinkages. Mucilage secretion is unimportant in bryophyte desiccation biology and developmental differences rule out homology between the mucilage clefts and stomata in hornworts and Blasiales. Elaborate placental walls in basal liverwort lineages and a stomatal toolkit in the capsule walls of Haplomitrium suggest that liverworts’ ancestors may have had more complex sporophytes than those in extant taxa.

S-acylation of YKT61 modulates its unconventional participation in the formation of SNARE complexes in Arabidopsis

Hetero-tetrameric soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) complexes are critical for vesicle-target membrane fusion within the endomembrane system of eukaryotic cells. SNARE assembly involves four different SNARE motifs, Qa, Qb, Qc, and R, provided by three or four SNARE proteins. YKT6 is an atypical R-SNARE that lacks a transmembrane domain and is involved in multiple vesicle-target membrane fusions. Although YKT6 is evolutionarily conserved and essential, its function and regulation in different phyla seem distinct. Arabidopsis YKT61, the yeast and metazoan YKT6 homologue, is essential for gametophytic development, plays a critical role in sporophytic cells, and mediates multiple vesicle-target membrane fusion. However, its molecular regulation is unclear. We report here that YKT61 is S-acylated. Abolishing its S-acylation by a C195S mutation dissociates YKT61 from endomembrane structures and causes its functional loss. Although interacting with various SNARE proteins, YKT61 functions not as a canonical R-SNARE but coordinates with other R-SNAREs to participate in the formation of SNARE complexes. Phylum-specific molecular regulation of YKT6 may be evolved to allow more efficient SNARE assembly in different eukaryotic cells.

Pollen Mitosis is a Good Choice for Gametophytic Chromosome Study in Higher Plants—Investigation with <i>Allium cepa</i> L. and <i>Aloe vera</i> L.

In higher groups of plants, the somatic cells are conventionally used for karyomorphological studies, which contain a diploid (2n) set of chromosomes. Similar to diploid sporophytic cells, a chromosomal study in haploid (n) gametophytic cells is also necessary as it unfolds the karyomorphological status of germ cells. The mature male gametophytic plant body (i.e., pollen grain) is only two- or three-celled structures. This study focused on the first mitotic division in a microspore, popularly known as ‘pollen mitosis-I.’ Allium cepa L. and Aloe vera L. are two model species for karyomorphological study. Microspores of these species have been stained with 2.0% acetocarmine. The present finding reveals that the cell division in microspores of these two species is completely normal without any significant irregularities. As expected, the chromosome number of the microspores of Allium cepa is n=8 and Aloe vera is n=7, exactly half the number of their sporophytic plants. The haploid karyotype of Allium cepa is the symmetric type with karyotype formula ‘6m+2sm,’ whereas that of Aloe vera is between symmetric and asymmetric types with karyotype formula ‘3sm+4st.’ This study not only enriches the chromosomal database of gametophytes of Allium cepa and Aloe vera, but also will encourage further research on this extremely neglected topic—chromosome study in the gametophytic generation of flowering plants. As Allium cepa and Aloe vera are easily available and model species for karyomorphological study, and with two contrast karyotypes (i.e., symmetric and asymmetric), this article also helps numerous school/college teachers to teach their students about the structure and behavior of gametophytic chromosomes.

Ferric Chloride Controls Citrus Anthracnose by Inducing the Autophagy Activity of Colletotrichum gloeosporioides

Colletotrichum gloeosporioides causes citrus anthracnose, which seriously endangers the pre-harvest production and post-harvest storage of citrus due to its devastating effects on fruit quality, shelf life, and profits. However, although some chemical agents have been proven to effectively control this plant disease, little to no efforts have been made to identify effective and safe anti-anthracnose alternatives. Therefore, this study assessed and verified the inhibitory effect of ferric chloride (FeCl3) against C. gloeosporioides. Our findings demonstrated that FeCl3 could effectively inhibit C. gloeosporioides spore germination. After FeCl3 treatment, the germination rate of the spores in the minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC) groups decreased by 84.04% and 89.0%, respectively. Additionally, FeCl3 could effectively inhibit the pathogenicity of C. gloeosporioides in vivo. Optical microscopy (OM) and scanning electron microscopy (SEM) analyses demonstrated the occurrence of wrinkled and atrophic mycelia. Moreover, FeCl3 induced autophagosome formation in the test pathogen, as confirmed by transmission electron microscopy (TEM) and monodansylcadaverine (MDC) staining. Additionally, a positive correlation was identified between the FeCl3 concentration and the damage rate of the fungal sporophyte cell membrane, as the staining rates of the control (untreated), 1/2 MIC, and MIC FeCl3 treatment groups were 1.87%, 6.52%, and 18.15%, respectively. Furthermore, the ROS content in sporophyte cells increased by 3.6%, 29.27%, and 52.33% in the control, 1/2 MIC, and MIC FeCl3 groups, respectively. Therefore, FeCl3 could reduce the virulence and pathogenicity of C. gloeosporioides. Finally, FeCl3-handled citrus fruit exhibited similar physiological qualities to water-handled fruit. The results show that FeCl3 may prove to be a good substitute for the treatment of citrus anthracnose in the future.

Epigenetic mechanisms of sexual reproduction in Angiosperms

Epigenetic mechanisms, such as DNA methylation, RNA interference, posttranslational histone modifications and rearrangements of chromatin structure play an important role during genome reprogramming in both animals and plants. The correct epigenetic pattern of eu- and heterochromatin marks allows for maintaining chromatin in an active or transcriptionally silenced state. In the life cycle of angiosperms, epigenetic mechanisms participate in genome reprogramming during: 1) differentiation of sporophyte cells into spore mother cells (SMC) that undergo meiosis, 2) development of female and male gametophytes, within which the gametes differentiate and 3) after double fertilization during the embryo and endosperm development. SMC speciation and control of meiosis, followed by reprogramming of the sperm cells and egg cell genome, are non-cell-autonomous and require RdDM pathway. These processes involve companion cells, which produce „mobile” siRNAs signal molecules. Epigenetic control of gene expression through siRNAs also participates in maintenance of gametes and embryo genome integrity and in the parental imprinting.

Brassinosteroid signaling regulates female germline specification in Arabidopsis

Unlike in humans and animals, plant germlines are specified de novo from somatic cells in the reproductive organs of the flower. In most flowering plant ovules, the female germline starts with the differentiation of one megaspore mother cell (MMC), which initiates a developmental program distinct from adjoining cells. Phytohormones act as a key player in physiological processes during plant development, in particular by providing positional information that supports localized differentiation events. However, little is known about the role of phytohormones for female germline initiation and establishment. Using Arabidopsis as a flowering plant model, we show that brassinosteroid (BR) biosynthesis and signaling components are accumulated in sporophytic cells of ovule primordia but not in the megaspore mother cell representing the precursor of the female germline. We further demonstrate that BR signaling restricts multiple sub-epidermal cells in the distal nucellus region of ovule primordia from acquiring MMC-like cell identity by transiently activating the WRKY23 transcription factor, expressed exclusively in L2 layer cells adjacent to the MMC. This activation is regulated through the BRI1 receptor and directly by the BZR1 transcriptional repressor family. Mutations in BR biosynthesis or signaling components and ectopic activation of BR signaling in MMCs induce multiple MMC-like cells. In summary, our findings elucidate a gene regulatory network that shows how the hormone BR generated in sporophytic ovule primordia cells restricts the origin of the female germline to a single cell.

Slow development allows redundant genes to restore the fertility of rpg1, a TGMS line in Arabidopsis.

Slow development has been shown to be a general mechanism to restore the fertility of thermo-sensitive and photoperiod-sensitive genic male sterile (TGMS and PGMS) lines in Arabidopsis. rpg1 is a TGMS line defective in primexine, which is essential for pollen wall pattern formation. Here, we showed that RPG1-GFP was highly expressed in microsporocytes, microspores, and pollen grains but not in the tapetum in the complemented transgenic line, suggesting that microsporocytes are the main sporophytic cells for primexine formation. Further cytological observations showed that primexine formation in rpg1 was partially restored under slow growth conditions, leading to its fertility restoration. RPG2 is the homolog of RPG1 in Arabidopsis. We revealed that the fertility recovery of rpg1 rpg2 was significantly reduced compared with that of rpg1 under low temperature. The RPG2-GFP protein was also expressed in microsporocytes in the RPG2-GFP (WT) transgenic line. These results suggest that RPG2 plays a redundant role in rpg1 fertility restoration. rpg1 plants were male sterile at the early growth stage, while their fertility was partially restored at the late developmental stage. The fertility of the rpg1 lateral branches was also partially restored. Further growth analysis showed that slow growth at the late reproductive stage or on the lateral branches led to fertility restoration. This work reveals the importance of gene redundancy in fertility restoration for TGMS lines and provides further insight into pollen wall pattern formation.

Ultrastructural observations of mitochondrial morphology through the life cycle of the brown alga, Mutiomo cylindricus (Cutleriaceae, Tilopteridales).

Mitochondrial morphology varies according to development and the physiological conditions of the cell. Here, we performed electron tomography using serial sections to analyze the number, individual volume, and morphological complexity of mitochondria in the cells across two generations in the life cycle of the brown alga Mutimo cylindricus. This species shows a heteromorphic alternation of generations between the macroscopic gametophyte and the crustose sporophyte during its life cycle and displays anisogamous sexual reproduction. We observed the mitochondria in the vegetative cells of gametophytes and sporophytes to mainly show tubular or discoidal shapes with high morphological complexity. The morphology of the mitochondria in the male and female gametes changed to a nearly spherical or oval shape from a tubular or discoidal shape before release. In this species, degradation of the paternal mitochondria was observed in the zygote 2 h after fertilization. Morphological changes in the mitochondria were not observed until 6 h after fertilization. Twenty-four-hour-old zygotes before and after cytokinesis showed a similar number of mitochondria as 6-h-old zygotes; however, the volume and morphological complexity increased. The results indicated that the maternal mitochondria did not undergo fission or fusion until this stage. Based on the analysis results of the number and total volume of mitochondria before and after the release of the gametes, it is possible that the mitochondria in the female gametes fuse immediately before release.

Cytoplasmic inheritance of mitochondria and chloroplasts in the anisogamous brown alga Mutimo cylindricus (Phaeophyceae).

Based on the morphology of gametes, sexual reproduction in brown algae is usually classified into three types: isogamy, anisogamy, and oogamy. In isogamy, chloroplasts and chloroplast DNA (chlDNA) in the sporophyte cells are inherited biparentally, while mitochondria (or mitochondrial DNA, mtDNA) is inherited maternally. In oogamy, chloroplasts and mitochondria are inherited maternally. However, the patterns of mitochondrial and chloroplast inheritance in anisogamy have not been clarified. Here, we examined derivation of mtDNA and chlDNA in the zygotes through strain-specific PCR analysis using primers based on single nucleotide polymorphism in the anisogamous brown alga Mutimo cylindricus. In 20-day-old sporophytes after fertilization, mtDNA and chlDNA derived from female gametes were detected, thus confirming the maternal inheritance of both organelles. Additionally, the behavior of mitochondria and chloroplasts in the zygotes was analyzed by examining the consecutive serial sections using transmission electron microscopy. Male mitochondria were isolated or compartmentalized by a double-membrane and then completely digested into a multivesicular structure 2h after fertilization. Meanwhile, male chloroplasts with eyespots were observed even in 4-day-old, seven-celled sporophytes. The final fate of male chloroplasts could not be traced. Organelle DNA copy number was also examined in female and male gametes. The DNA copy number per chloroplast and mitochondria in male gametes was lower compared with female organelles. The degree of difference is bigger in mtDNA. Thus, changes in different morphology and DNA amount indicate that maternal inheritance of mitochondria and chloroplasts in this species may be based on different processes and timing after fertilization.

Haploid spontaneous diploidization during apogamy of male gametophytes in Pyropia haitanensis (Bangiales, Rhodophyta)

Cytological observations were systematically employed for detecting cellular morphogenesis and chromosomal behavior of apogamous development of new sporophytes from individually cultured male gametophytes of Pyropia haitanensis. The results showed that the apical parts of premature male gametophytes initially underwent obvious apogamy, which primarily appeared in the functionally specialized spermatangial mother cells (SSMCs) that would not develop into highly differentiated spermatangia but change their developmental fate and become brownish red in color. These brownish red spermatangial mother cells (SMCs) were released accompanying the spermatia dissociated from mature spermatangia and then rarely formed circular carpospore-like cells (CLCs) and carpospore-like germlings (CLGs) that eventually developed into normal apogamous sporophytic conchocelis. Chromosome counts indicated that spermatangia and spermatia were haploid (n = 5), whereas SSMCs, CLCs, and CLGs were diploid (2n = 10). Furthermore, filamentous conchocelis and conchosporangial branch cells of apogamous sporophytes were diploid and no haploid, aneuploid, and polyploid were observed, suggesting that spontaneous chromosome doubling occurred in the process of apogamy and initiated rarely SSMCs rather than released spermatia. Moreover, pit connections were observed in the whole development of apogamous sporophytes like in heterozygous sporophytes. Mitotic chromosomal behaviors appeared during the first two divisions of conchospores that normally grew into haploid tetrads and developed into unisexual male gametophytes with the characteristics of apogamy in the aging cultures.

Nuclear Chaperone ASF1 is Required for Gametogenesis in Arabidopsis thaliana

Sexual reproduction in flowering plants is distinct from that in animals since gametogenesis requires production of haploid spores, which divide and differentiate into specialised gametophyte structures. Anti-Silencing Function 1 (ASF1) is a histone H3/H4 chaperone involved in chromatin remodeling during cell division, which we have found plays a critical role in gametophyte development in Arabidopsis thaliana. Using mutant alleles for the two ASF1 homologs, asf1a and asf1b, we show that ASF1 is required for successful development of gametophytes and acquisition of fertilisation competency. On the female side, reproductive failure is caused by aberrant development of ovules, leading to gamete degeneration. On the male side, we show both in vitro and in vivo that asf1 mutant pollen tube growth is stunted, limiting fertilisation to ovules nearest the stigma. Consistent with ASF1 importance in gametogenesis, we show that ASF1A and ASF1B are expressed throughout female and male gametogenesis. We show that the gametogenesis defects can be corrected by ASF1A and ASF1B transgenes, and that ASF1A and ASF1B act redundantly. Thus, in contrast to the role of ASF1 in sporophytic cell cycle progression, our data indicate that during reproduction, ASF1 is required for the precise nuclei differentiation necessary for gametophyte maturation and fertilisation.

AUXIN RESPONSE FACTOR17 Directly Regulates MYB108 for Anther Dehiscence.

The timely release of mature pollen following anther dehiscence is essential for reproduction in flowering plants. AUXIN RESPONSE FACTOR17 (ARF17) plays a crucial role in pollen wall pattern formation, tapetum development, and auxin signal transduction in anthers. Here, we showed that ARF17 is also involved in anther dehiscence. The Arabidopsis (Arabidopsis thaliana) arf17 mutant exhibits defective endothecium lignification, which leads to defects in anther dehiscence. The expression of MYB108, which encodes a transcription factor important for anther dehiscence, was dramatically down-regulated in the flower buds of arf17 Chromatin immunoprecipitation assays and electrophoretic mobility shift assays showed ARF17 directly binds to the MYB108 promoter. In an ARF17-GFP transgenic line, in which ARF17-GFP fully complements the arf17 phenotype, ARF17-GFP was observed in the endothecia at anther stage 11. The GUS signal driven by the MYB108 promoter was also detected in endothecia at late anther stages in transgenic plants expressing promoterMYB108::GUS Thus, the expression pattern of both ARF17 and MYB108 is consistent with the function of these genes in anther dehiscence. Furthermore, the expression of MYB108 driven by the ARF17 promoter successfully restored the defects in anther dehiscence of arf17 These results demonstrated that ARF17 regulates the expression of MYB108 for anther dehiscence. Together with its function in microcytes and tapeta, ARF17 likely coordinates the development of different sporophytic cell layers in anthers. The ARF17-MYB108 pathway involved in regulating anther dehiscence is also discussed.

Auxin production in diploid microsporocytes is necessary and sufficient for early stages of pollen development.

Gametophytic development in Arabidopsis depends on nutrients and cell wall materials from sporophytic cells. However, it is not clear whether hormones and signaling molecules from sporophytic tissues are also required for gametophytic development. Herein, we show that auxin produced by the flavin monooxygenases YUC2 and YUC6 in the sporophytic microsporocytes is essential for early stages of pollen development. The first asymmetric mitotic division (PMI) of haploid microspores is the earliest event in male gametophyte development. Microspore development in yuc2yuc6 double mutants arrests before PMI and consequently yuc2yuc6 fail to produce viable pollens. Our genetic analyses reveal that YUC2 and YUC6 act as sporophytic genes for pollen formation. We further show that ectopic production of auxin in tapetum, which provides nutrients for pollen development, fails to rescue the sterile phenotypes of yuc2yuc6. In contrast, production of auxin in either microsporocytes or microspores rescued the defects of pollen development in yuc2yuc6 double mutants. Our results demonstrate that local auxin biosynthesis in sporophytic microsporocytic cells and microspore controls male gametophyte development during the generation transition from sporophyte to male gametophyte.

Genomic structural variation-mediated allelic suppression causes hybrid male sterility in rice

Hybrids between divergent populations commonly show hybrid sterility; this reproductive barrier hinders hybrid breeding of the japonica and indica rice (Oryza sativa L.) subspecies. Here we show that structural changes and copy number variation at the Sc locus confer japonica–indica hybrid male sterility. The japonica allele, Sc-j, contains a pollen-essential gene encoding a DUF1618-domain protein; the indica allele, Sc-i, contains two or three tandem-duplicated ~ 28-kb segments, each carrying an Sc-j-homolog with a distinct promoter. In Sc-j/Sc-i hybrids, the high-expression of Sc-i in sporophytic cells causes suppression of Sc-j expression in pollen and selective abortion of Sc-j-pollen, leading to transmission ratio distortion. Knocking out one or two of the three Sc-i copies by CRISPR/Cas9 rescues Sc-j expression and male fertility. Our results reveal the gene dosage-dependent allelic suppression as a mechanism of hybrid incompatibility, and provide an effective approach to overcome the reproductive barrier for hybrid breeding.

Calreticulin localizes to plant intra/extracellular peripheries of highly specialized cells involved in pollen-pistil interactions

Calcium (Ca2+) plays essential roles in generative reproduction of angiosperms, but the sites and mechanisms of Ca2+ storage and mobilization during pollen-pistil interactions have not been fully defined. Both external and internal Ca2+ stores are likely important during male gametophyte communication with the sporophytic and gametophytic cells within the pistil. Given that calreticulin (CRT), a Ca2+-buffering protein, is able to bind Ca2+ reversibly, it can serve as a mobile store of easily releasable Ca2+ (so called an exchangeable Ca2+) in eukaryotic cells. CRT has typical endoplasmic reticulum (ER) targeting and retention signals and resides primarily in the ER. However, localization of this protein outside the ER has also been revealed in both animal and plant cells, including Golgi/dictyosomes, nucleus, plasma membrane/cell surface, plasmodesmata, and even extracellular matrix. These findings indicate that CRT may function in a variety of different cell compartments and specialized structures. We have recently shown that CRT is highly expressed and accumulated in the ER of plant cells involved in pollen-pistil interactions in Petunia, and we proposed an essential role for CRT in intracellular Ca2+ storage and mobilization during the key reproductive events. Here, we demonstrate that both CRT and exchangeable Ca2+ are localized in the intra/extracellular peripheries of highly specialized plant cells, such as the pistil transmitting tract cells, pollen tubes, nucellus cells surrounding the embryo sac, and synergids. Based on our present results, we propose that extracellularly located CRT is also involved in Ca2+ storage and mobilization during sexual reproduction of angiosperms.

Marchantia MpRKD Regulates the Gametophyte-Sporophyte Transition by Keeping Egg Cells Quiescent in the Absence of Fertilization

Unlike in animals, the life cycle of land plants alternates between two multicellular generations, the haploid gametophyte and the diploid sporophyte [1]. Gamete differentiation initiates the transition from the gametophyte to the sporophyte generation and, upon maturation, the egg cell establishes a quiescent state that is maintained until fertilization. This quiescence represents a hallmark of the gametophyte-sporophyte transition. The underlying molecular mechanisms are complex and best characterized in the flowering plant Arabidopsis thaliana [2-4]. However, only few genes with egg cell-specific expression or defects have been identified [5-10]. Intriguingly, ectopic expression of members of a clade of RWP-RK domain (RKD)-containing transcription factors, which are absent from animal genomes [11-13], can induce an egg cell-like transcriptome in sporophytic cells of A.thaliana. Yet, to date, loss-of-function experiments have not produced phenotypes affecting the egg cell, likely due to genetic redundancy and/or cross-regulation among the five RKD genes of A.thaliana [10]. To reduce genetic complexity, we explored the genome of Marchantia polymorpha, a liverwort belonging to the basal lineage of extant land plants [14-17]. Based on sequence homology, we identified a single M.polymorpha RKD gene, MpRKD, which is orthologous to all five A.thaliana RKD genes. Analysis of the MpRKD expression pattern and characterization of lines with reduced MpRKD activity indicate that it functions as a regulator of gametophyte development and the gametophyte-sporophyte transition. In particular, MpRKD is required to establish and/or maintain the quiescent state of the egg cell in the absence of fertilization.

Pollen proteomics: from stress physiology to developmental priming.

Key messagePollen development and stress.In angiosperms, pollen or pollen grain (male gametophyte) is a highly reduced two- or three-cell structure which plays a decisive role in plant reproduction. Male gametophyte development takes place in anther locules where diploid sporophytic cells undergo meiotic division followed by two consecutive mitotic processes. A desiccated and metabolically quiescent form of mature pollen is released from the anther which lands on the stigma. Pollen tube growth takes place followed by double fertilization. Apart from its importance in sexual reproduction, pollen is also an interesting model system which integrates fundamental cellular processes like cell division, differentiation, fate determination, polar establishment, cell to cell recognition and communication. Recently, pollen functionality has been studied by multidisciplinary approaches which also include OMICS analyses like transcriptomics, proteomics and metabolomics. Here, we review recent advances in proteomics of pollen development and propose the process of developmental priming playing a key role to guard highly sensitive developmental processes.

Ploidy Distribution of the Harmful Bloom Forming Macroalgae Ulva spp. in Narragansett Bay, Rhode Island, USA, Using Flow Cytometry Methods.

Macroalgal blooms occur worldwide and have the potential to cause severe ecological and economic damage. Narragansett Bay, RI is a eutrophic system that experiences summer macroalgal blooms composed mostly of Ulva compressa and Ulva rigida, which have biphasic life cycles with separate haploid and diploid phases. In this study, we used flow cytometry to assess ploidy levels of U. compressa and U. rigida populations from five sites in Narragansett Bay, RI, USA, to assess the relative contribution of both phases to bloom formation. Both haploid gametophytes and diploid sporophytes were present for both species. Sites ranged from a relative overabundance of gametophytes to a relative overabundance of sporophytes, compared to the null model prediction of √2 gametophytes: 1 sporophyte. We found significant differences in cell area between ploidy levels for each species, with sporophyte cells significantly larger than gametophyte cells in U. compressa and U. rigida. We found no differences in relative growth rate between ploidy levels for each species. Our results indicate the presence of both phases of each of the two dominant bloom forming species throughout the bloom season, and represent one of the first studies of in situ Ulva life cycle dynamics.

Pollen tubes introduce Raspberry bushy dwarf virus into embryo sacs during fertilization processes

We developed a fertilization method in which pollen tubes entered into embryo sacs without any need to contact surrounding female sporophytic cells by using Torenia fournieri (Torenia) plants under the condition of hindering movement of the virus from a stigma, which is the first infection site leading to systemic infection. When RBDV-infected Torenia pollen grains were used for the developed fertilization method, the virus was transmitted to the seeds by pollen tubes germinating from them. On the other hand, no seeds were infected with the virus when Torenia plants were pollinated with healthy Torenia pollen grains in combination with RBDV-infected raspberry pollen grains, which caused the virus infection in the stigma by penetration of their pollen tubes arrested in its style. Our results indicate that vertical transmission of RBDV by pollen occurs in the transport of the virus into embryo sacs by pollen tubes reaching the embryo sacs.

Epigenetic landscape of germline specific genes in the sporophyte cells of Arabidopsis thaliana.

In plants, the germline lineages arise in later stages of life cycle as opposed to animals where both male and female germlines are set aside early in development. This developmental divergence is associated with germline specific or preferential expression of a subset of genes that are normally repressed for the rest of plant life cycle. The gene regulatory mechanisms involved in such long-term suppression and short-term activation in plant germline remain vague. Thus, we explored the nature of epigenetic marks that are likely associated with long-term gene repression in the non-germline cells. We accessed available Arabidopsis genome-wide DNA methylation and histone modification data and queried it for epigenetic marks associated with germline genes: genes preferentially expressed in sperm cells, egg cells, synergid cells, central cells, antipodal cells or embryo sac or genes that are with enriched expression in two or more of female germline tissues. The vast majority of germline genes are associated with repression-related epigenetic histone modifications in one or more non-germline tissues, among which H3K9me2 and H3K27me3 are the most widespread repression-related marks. Interestingly, we show here that the repressive epigenetic mechanisms differ between male and female germline genes. We also highlight the diverse states of epigenetic marks in different non-germline tissues. Some germline genes also have activation-related marks in non-germline tissues, and the proportion of such genes is higher for female germline genes. Germline genes include 30 transposable element (TE) loci, to which a large number of 24-nt long small interfering RNAs were mapped, suggesting that these small RNAs take a role in suppressing them in non-germline tissues. The data presented here suggest that the majority of Arabidopsis gamete-preferentially/-enriched genes bear repressive epigenetic modifications or regulated by small RNAs.