Ovule Enlargement Research Articles

Pollen Tube Content Facilitates and Increases the Potential of Endosperm Proliferation Irrespective of Fertilization in <i>Arabidopsis thaliana</i>

Sexual reproduction in angiosperms is a complex and precise process of regulation, which includes the pollen tube guidance, double fertilization, and the seed development process. The previous report identified that for seed development, there is a period of preparation for fertilization (between pollen tube guidance and double fertilization) that is called pollen-tube-dependent ovule enlargement morphology (POEM). It has been shown that the pollen tube content (PTC) plays a crucial role in the enlargement of ovules and the initiation of seed coat formation. However, we did not investigate the potential of endosperm proliferation in autonomous mutants at a later stage. Here, we investigated this phenomenon using vanillin staining and transparent experiments to examine the manner, in which the PTC affects the potential of endosperm formation. Interestingly, the PTC increased the number of endosperm nuclei without fertilization equally and synchronously in mea and fis2 ovules. This finding might help improve the study of apomixes and our understanding of how the molecular mechanisms that regulate this phenomenon will contribute to plant reproductive science in the future.

Elongation of Siliques Without Pollination 3 Regulates Nutrient Flow Necessary for Embryogenesis.

Apomixis, defined as the transfer of maternal germplasm to offspring without fertilization, enables the fixation of F1-useful traits, providing advantages in crop breeding. However, most apomictic plants require pollination to produce the endosperm. The endosperm is essential for embryogenesis, and its development is suppressed until fertilization. We show that the expression of a chimeric repressor of the Elongation of Siliques without Pollination 3 (ESP3) gene (Pro35S:ESP3-SRDX) induces ovule enlargement without fertilization in Arabidopsis thaliana. The ESP3 gene encodes a protein similar to the flowering Wageningen homeodomain transcription factor containing a StAR-related lipid transfer domain. However, ESP3 lacks the homeobox-encoding region. Genes related to the cell cycle and sugar metabolism were upregulated in unfertilized Pro35S:ESP3-SRDX ovules similar to those in fertilized seeds, while those related to autophagy were downregulated similar to those in fertilized seeds. Unfertilized Pro35S:ESP3-SRDX ovules partially nourished embryos when only the egg was fertilized, accumulating hexoses without central cell proliferation. ESP3 may regulate nutrient flow during seed development, and ESP3-SRDX could be a useful tool for complete apomixis that does not require pseudo-fertilization.

The effect of different gelling agents as part of culture medium on the rate of cucumber (<i>Cucumis sativus</i> L.) unpollinated ovules gynogenic development

Relevance. The induction of gynogenic development of the culture of unpollinated ovules of Cucumis sativus L., as a part of solid nutrient media agar-agar or Phytagel™ are used as a gelling agent. The gelling agent determines the mobilization of substances in the composition of the nutrient medium and has various effects on the explants, which affects the quality of the resulting regenerates. However, there are no scientific works that explaines the effect of these gelling agents on the development of ovules and compare them with each other for C. sativus L. The aim: investigate the effect of various gelling agent in the nutrient medium on the induction of gynogenesis and the development of cucumber unpollinated ovules.Materials and methods. There are two promising collection specimens of cucumber №58 and №831 of the laboratory of cucurbits crop breeding and seed production of FSBSI FSVC were included into research. IMC nutrient medium (Induction Medium for Cucurbitaceae) with 30 g/l sucrose, 200 mg/l ampicillin, 0.2 mg/l thidiazuron (TDZ) was used for the induction of gynogenesis; agar-agar at a concentration of 7 g/l or Phytagel™ at a concentration of 3.5 g/l was used as a gel-forming agents. Ovules were isolated from ovaries in the phase of half-opened flower (FL-1) and fully opened flower (FL). Cultivation was carried out in plastic Petri dishes with a diameter of 60 mm with an air gap of 28.8 cm3 – "KS No. 1", and glass culture jars with an air gap of 140 cm3 – "KS No. 2".Results. On containing Phytagel™ or agar-agar nutrient medium, the area of ovules during 30 days of cultivation increases irregularly. Coefficients of ovule enlargement between gelling agents differed from 1.7 to 2.6 times depending on the cultivation time. The average growth rate of ovules on media with Phytagel™ was 0.08 mm2/day, while on media with agar-agar it was 0.02 mm2/day. Gelling agents type and cucumber phenotype are significant factors affecting the increase in area of entered into unpollinated cucumber ovules culture. Herewith the share of the gelling agent effect was 55.01%, and the share of genotype effect was 14.53%. The effect of flower development stage or culture vessel type has not found for both of genotypes. In the study, it was possible to achieve the induction of gynogenesis in 67% of the unpollinated ovules genotype №831 on nutrient medium with agar-agar.Conclusion. Ovules development were faster on a nutrient medium using Phytagel™ as a gelling agent than on agaragar. At the same time, the percentage of induced ovules was significally higher on nutrient medium with agar-agar for both phenotypes. Gelling agents type and cucumber phenotype were found as significant factors of the induction and the development of unpollinated ovules.

High-quality sugar production by osgcs1 rice

Carbohydrates (sugars) are an essential energy-source for all life forms. They take a significant share of our daily consumption and are used for biofuel production as well. However, sugarcane and sugar beet are the only two crop plants which are used to produce sugar in significant amounts. Here, we have discovered and fine-tuned a phenomenon in rice which leads them to produce sugary-grain. We knocked-out GCS1 genes in rice by using CRISPR technology, which led to fertilization failure and pollen tube-dependent ovule enlargement morphology (POEM) phenomenon. Apparently, the POEMed-like rice ovule (‘endosperm-focused’) can grow near-normal seed-size unlike earlier observations in Arabidopsis in which gcs1 ovules (‘embryo-focused’) were aborted quite early. The POEMed-like rice ovules contained 10–20% sugar, with extremely high sucrose content (98%). Trancriptomic analysis revealed that the osgcs1 ovules had downregulation of starch biosynthetic genes, which would otherwise have converted sucrose to starch. Overall, this study shows that pollen tube content release is sufficient to trigger sucrose unloading at rice ovules. However, successful fertilization is indispensable to trigger sucrose-starch conversion. These findings are expected to pave the way for developing novel sugar producing crops suited for diverse climatic regions.

Comparative transcriptome analysis of the effect of different heat shock periods on the unfertilized ovule in watermelon (Citrullus lanatus)

In vitro gynogenesis is an important tool used in haploid or homozygous double-haploid plant breeding. However, because of low repeatability, embryoid induction rate and quality, the molecular mechanisms remain poorly understood. Heat shock treatment can promote the transformation of the gametophytic pathway into the sporophyte pathway, which induces the occurrence of haploid. In this study, unfertilized ovaries were heat shocked for 0 h (A0) before flowering and for 0 h (A1), 4 h (A3), 8 h (A5), 12 h (A7), and 24 h (A8), respectively, at 37°C at the first day of the flowering stage. The ovule enlargement rate was increased from 0% at 25°C to 96.8% at 37°C (24 h treatment). Thus, we aimed to investigate the gene expression patterns in unfertilized ovules of watermelon after different periods of heat shock by using RNA-Seq technology. The results showed that compared with A3, A5, A7, and A8, the biosynthesis of amino acid, glycine, serine and threonine metabolic pathways in A1 has changed significantly. This indicated that heat shock treatment affected the synthesis and transformation of amino acids during ovule expansion. The transcriptome data suggested gene expressions of ovule growth were significantly changed by heat-specific influences. The results provide new information on the complex relationship between in vitro gynogenesis and temperature. This provides a basis for further study of the mechanism of heat shock affecting the expansion of watermelon ovule.

Establishment of a novel method for the identification of fertilization defective mutants in Arabidopsis thaliana

Plant reproduction is an extremely important phenomenon, as it is strongly associated with plant genetics and early development. Additionally, foundations of the reproductive system have direct implications on plant breeding and agriculture. Investigation of the functions of male and female gametophytes is critical since their fusion is required for seed formation. Although a large number of mutants have been generated to understand the functions of male and female gametophytes, only a small number of genes required for plant fertilization have been identified to date. This is because the screening method used previously required the dissection of siliques, and fertilization-specific mutants exhibiting semi-fertility (or ∼50% fertility) were difficult to identify. Here, we report a new efficient screening method for the identification of fertilization defective mutants in Arabidopsis thaliana using vanillin staining. This method is based on the pollen tube-dependent ovule enlargement morphology (POEM) phenomenon, which generates a partial seed coat within the ovule without fertilization. Using this method, we successfully identified 23 putative fertilization defective mutants in Arabidopsis.

Pollen tube contents from failed fertilization contribute to seed coat initiation in Arabidopsis.

Plant seeds are essential for human beings, constituting 70% of carbohydrate resources worldwide; examples include rice, wheat, and corn. In angiosperms, fertilization of the egg by a sperm cell is required for seed formation; therefore, fertilization failure results in no seed formation, except in the special case of apomixis. Initially, plants produce many pollen grains inside the anthers; once the pollen grain is deposited onto the top of the pistil, the pollen tube elongates until it reaches the ovule. Generally, only one pollen tube is inserted into the ovule; however, we previously found that if fertilization by the first pollen tube fails, a second pollen tube could rescue fertilization via the so-called fertilization recovery system (FRS). Our previous reports also demonstrated that failed fertilization results in pollen tube-dependent ovule enlargement morphology (POEM), enlarged seeds, and partial seed coat formation if the pollen tube releases the pollen tube contents into the ovule. However, we have not determined whether all the ovules enlarge or produce seed coats if an ovule accepts the pollen tube contents. Therefore, we conducted a partial seed coat formation experiment taking into account both the FRS and POEM phenomena. Notably, the ratios of failed fertilization and the ovules with partial seed coats matched, indicating that all ovules initiate seed coat formation if the fertilization fails but the pollen tube contents enter the ovule. In addition, we confirmed that the agl62 mutant , defective in early endosperm formation, showed seed coat initiation with and without fertilization, indicating that for a normal seed coat initiation, fertilization is not required; however, for the completion of normal seed coat formation, both normal fertilization and endosperm formation are required. Further molecular evidence is required to understand these phenomena because very few factors related to FRS and POEM have been identified.

Pollen tube contents from failed fertilization contribute to seed coat initiation in Arabidopsis

Plant seeds are essential for human beings, constituting 70% of carbohydrate resources worldwide; examples include rice, wheat, and corn. In angiosperms, fertilization of the egg by a sperm cell is required for seed formation; therefore, fertilization failure results in no seed formation, except in the special case of apomixis. Initially, plants produce many pollen grains inside the anthers; once the pollen grain is deposited onto the top of the pistil, the pollen tube elongates until it reaches the ovule. Generally, only one pollen tube is inserted into the ovule; however, we previously found that if fertilization by the first pollen tube fails, a second pollen tube could rescue fertilization via the so-called fertilization recovery system (FRS). Our previous reports also demonstrated that failed fertilization results in pollen tube-dependent ovule enlargement morphology (POEM), enlarged seeds, and partial seed coat formation if the pollen tube releases the pollen tube contents into the ovule. However, we have not determined whether all the ovules enlarge or produce seed coats if an ovule accepts the pollen tube contents. Therefore, we conducted a partial seed coat formation experiment taking into account both the FRS and POEM phenomena. Notably, the ratios of failed fertilization and the ovules with partial seed coats matched, indicating that all ovules initiate seed coat formation if the fertilization fails but the pollen tube contents enter the ovule. In addition, we confirmed that the agl62 mutant , defective in early endosperm formation, showed seed coat initiation with and without fertilization, indicating that for a normal seed coat initiation, fertilization is not required; however, for the completion of normal seed coat formation, both normal fertilization and endosperm formation are required. Further molecular evidence is required to understand these phenomena because very few factors related to FRS and POEM have been identified.

Pollen tube contents from failed fertilization contribute to seed coat initiation in Arabidopsis

Plant seeds are essential for human beings, constituting 70% of carbohydrate resources worldwide; examples include rice, wheat, and corn. In angiosperms, fertilization of the egg by a sperm cell is required for seed formation; therefore, fertilization failure results in no seed formation, except in the special case of apomixis. Initially, plants produce many pollen grains inside the anthers; once the pollen grain is deposited onto the top of the pistil, the pollen tube elongates until it reaches the ovule. Generally, only one pollen tube is inserted into the ovule; however, we previously found that if fertilization by the first pollen tube fails, a second pollen tube could rescue fertilization via the so-called fertilization recovery system (FRS). Our previous reports also demonstrated that failed fertilization results in pollen tube-dependent ovule enlargement morphology (POEM), enlarged seeds, and partial seed coat formation if the pollen tube releases the pollen tube contents into the ovule. However, we have not determined whether all the ovules enlarge or produce seed coats if an ovule accepts the pollen tube contents. Therefore, we conducted a partial seed coat formation experiment taking into account both the FRS and POEM phenomena. Notably, the ratios of failed fertilization and the ovules with partial seed coats matched, indicating that all ovules initiate seed coat formation if the fertilization fails but the pollen tube contents enter the ovule. In addition, we confirmed that the agl62 mutant , defective in early endosperm formation, showed seed coat initiation with and without fertilization, indicating that for a normal seed coat initiation, fertilization is not required; however, for the completion of normal seed coat formation, both normal fertilization and endosperm formation are required. Further molecular evidence is required to understand these phenomena because very few factors related to FRS and POEM have been identified.

Discovery of pollen tube-dependent ovule enlargement morphology phenomenon, a new step in plant reproduction

ABSTRACTIn animals, when semen is discharged into the uterus, the seminal plasma carries the sperm to the egg. In plants, the function of pollen tube contents (PTC) is analogous to that of the seminal plasma in animals, i.e., carrying sperm cells to the ovules for fertilization. Because the function of the seminal plasma is essential for fertilization in animals, we propose that the function of PTC must be important for plant fertilization. To understand the function of PTC, we examined the transcriptional variation after the release of PTC into the embryo sac. The phenotypic analysis revealed that ovules were enlarged without fertilization when the PTC was released into the ovule, entirely consistent with the transcriptome analysis. We identified a new plant phenomenon, pollen tube-dependent ovule enlargement morphology (POEM) phenomenon that occurs only when the ovule accepts PTC, irrespective of fertilization. POEM is a new phase between the pollen tube guidance and fertilization phases as a reproductive step. Here we established the in vitro POEM assay, which effectively measures POEM activity. Using this assay, we identified that a simple dose of plant hormone(s) cannot induce POEM. We also showed that this assay could be a powerful tool for identifying POEM factor(s).

Pollen tube contents initiate ovule enlargement and enhance seed coat development without fertilization.

In angiosperms, pollen tubes carry two sperm cells toward the egg and central cells to complete double fertilization. In animals, not only sperm but also seminal plasma is required for proper fertilization. However, little is known regarding the function of pollen tube content (PTC), which is analogous to seminal plasma. We report that the PTC plays a vital role in the prefertilization state and causes an enlargement of ovules without fertilization. We termed this phenomenon as pollen tube-dependent ovule enlargement morphology and placed it between pollen tube guidance and double fertilization. Additionally, PTC increases endosperm nuclei without fertilization when combined with autonomous endosperm mutants. This finding could be applied in agriculture, particularly in enhancing seed formation without fertilization in important crops.

A comparison of pollinator fig wasp development in figs of Ficus montana and its hybrids with Ficus asperifolia

AbstractFigs (Moraceae) and pollinator fig wasps (Hymenoptera: Agaonidae) have a highly specific mutualistic relationship but fig wasps occasionally enter atypical hosts, and this can lead to hybrid fig trees and the potential for gene flow between species. Many fig trees are dioecious, with fig wasp offspring developing in galled ovules inside figs on male trees, whereas seeds develop only in figs on female trees. We generated experimental hybrids between the Asian Ficus montana Blume and a closely related African species Ficus asperifolia Miquel. Male F1s were sterile if entered by Kradibia tentacularis (Grandi) (Agaonidae), the pollinator of F. montana, because its offspring always failed to develop, without ovule enlargement. As with the F1s, figs on most male backcross plants [F. montana × (F. montana × F. asperifolia)] also aborted shortly after pollinator entry, resulting in a higher turnover of figs than with F. montana, although the times taken for the figs to reach receptivity were similar. Pollinator larvae nonetheless consistently managed to develop inside the figs of one backcross plant and also occasionally in a few figs from another backcross individual. In these figs, galled ovules developed as normal, whereas in figs that aborted the galled ovules failed to enlarge. The sex ratio of K. tentacularis progeny in the backcross figs was female biased and did not differ from that in F. montana figs. Sycoscapter spec. (Hymenoptera: Pteromalidae), a parasitoid of K. tentacularis, was able to lay eggs and developed normally inside male backcross figs where its host was present.

Popular Rice (Oryza sativa L.) Cultivars Show Contrasting Responses to Heat Stress at Gametogenesis and Anthesis

ABSTRACTStudies on heat tolerance in rice (Oryza sativa L.) have focused on a range of accessions that are currently not grown or cultivated in small localized geographic regions, while popular rice cultivars occupying millions of hectares have not been tested for their response to heat stress. Hence, 18 popular rice cultivars grown across tropical and subtropical regions were subjected to 38°C for 6 h of heat stress at anthesis and five selected contrasting entries were exposed to six consecutive days (6 h each day) of stress coinciding with both gametogenesis and anthesis. Among the tested entries, Ciherang was the most tolerant, after both 6 h at anthesis and 6 d of stress across both gametogenesis and anthesis, whereas Moroberekan and KDML‐105 were the most susceptible. Using a quick and easy phenotyping protocol and by considering all the sterile spikelets across target panicles, the crucial physiological window “spikelets with exserted anthers but with no ovule enlargement” was identified as the most sensitive stage with exposure to heat stress at both critical reproductive stages. Geographic origin of rice varieties was not clearly related to the degree of tolerance or susceptibility to heat stress because both conditions were observed in cultivars from each target region. Our findings complement other ongoing efforts that are using heat‐tolerant landraces and wild rice as donors to further increase tolerance to sustain global rice production under future warmer climate.

Gynogenesis in the vine cacti Hylocereus and Selenicereus (Cactaceae)

Gynogenesis was investigated on the allotetraploid Selenicereus megalanthus and the diploid Hylocereus polyrhizus and Hylocereus undatus vine cactus species. Unpollinated ovules from developing flower buds containing microspores at middle uninucleate developmental stage were cultured on MS basal medium containing 2,4-D/TDZ with different sucrose concentrations. Ovule size increased under dark culture conditions in all the three species and the level of response was species and sucrose concentration dependent. The best responses were achieved in the two S. megalanthus accessions, E-123 and J-80, at 0.18 and 0.26 M sucrose. Only ovule enlargement was obtained in H. undatus and both ovule enlargement and callus were obtained in H. polyrhizus. Development in both species ceased and embryoids were not formed. Plant regeneration was directly and indirectly obtained in both S. megalanthus accessions. Ploidy level was determined for a total of 29 S. megalanthus gynogenic plants using flow cytometry: 15 were found to be dihaploid (plants with the gametophytic chromosome number) and the other 14 were found to have higher ploidy levels. This is the first report of successful gynogenesis in Cactaceae. The dihaploids of S. megalanthus successfully produced by ovule culture techniques opens new perspectives in vine cacti breeding.

Evidence for Stigmatic Self-incompatibility, Pollination Induced Ovule Enlargement and Transmitting Tissue Exudates in the Paleoherb, Saururus cernuus L. (Saururaceae)

Saururus cernuus, a species belonging to the primitive herbaceous angiosperm family Saururaceae, exhibits high rates of self-sterility. We investigated the structural and functional aspects of pollen-carpel interactions following cross and self pollination to assess the tissue specific site and timing of self-sterility and factors important for successful cross pollen tube growth. Self-sterility was due to inhibition of self pollen germination at a dry stigma. Self pollination was associated with anomalous foot formation, reduced cell wall expansion and secretory activity of stigmatic papillae, and callose production in stigmatic papillae. Following germination, cross compatible pollen tubes entered a solid core of transmitting tissue and grew to the base of a short style. Entry of cross pollen tubes into the ovary was coincident with ovule enlargement which placed the micropyle in the proximity of cross pollen tube tips. Ovule enlargement also occurred following self pollination. Cross pollen tubes either entered an exudate-filled micropyle directly from the style, or growth in the ovary was localized to the epidermis of the locule and outer integument prior to entry into the micropyle. Prior to pollination, the transmitting tract was void of secretions except for exudate in the micropyle. Growth of pollen tubes on the locule and integument was associated with exudate apparently arising from transmitting cells adjacent to growing pollen tubes. The present study provides the first evidence in a primitive herbaceous species of stigmatic self-incompatibility (SI) in association with a dry stigma, pollination-induced signalling events affecting development of carpellary tissues, and micropylar exudates.

On the structure of Cardiocarpus tritolopus, a cordaite seed from the lower—middle pennsylvanian of Kentucky

A new species of Cardiocarpus Brongniart is described from the Lower—Middle Pennsylvania of Kentucky. The integument is divided into four distinct layers: a sarcotesta, sclerotesta, a layer between the sclerotesta and endotesta, and the endotesta. The most characteristic layer is the endotesta which consists of large thin-walled cells. The vascular system consists of a single strand that enters the base of the seed and extends distally to form a massive chalazal platform. From this basal strand, two lateral traces arise that extend distally through the integument (sarcotesta) in the primary plane of the seed. Stages in the development of the megagametophyte are discussed together with a characterization of post-pollination ovule enlargement.