Ovule Development Research Articles

Reactive oxygen species are signaling molecules that modulate plant reproduction.

Reactive oxygen species (ROS) can serve as signaling molecules that are essential for plant growth and development but abiotic stress can lead to ROS increases to supraoptimal levels resulting in cellular damage. To ensure efficient ROS signaling, cells have machinery to locally synthesize ROS to initiate cellular responses and to scavenge ROS to prevent it from reaching damaging levels. This review summarizes experimental evidence revealing the role of ROS during multiple stages of plant reproduction. Localized ROS synthesis controls the formation of pollen grains, pollen-stigma interactions, pollen tube growth, ovule development,and fertilization. Plants utilize ROS-producing enzymes such as respiratory burst oxidase homologsand organelle metabolic pathways to generate ROS, while the presence of scavenging mechanisms, including synthesis of antioxidant proteins and small molecules, serves to prevent its escalation to harmful levels. In this review, we summarized the function of ROS and its synthesisand scavenging mechanisms in all reproductive stages from gametophyte development until completion of fertilization. Additionally, we further address the impact of elevated temperatures induced ROS on impairing these reproductive processes and of flavonol antioxidants in maintaining ROS homeostasis to minimize temperature stress to combat the impact of global climate change on agriculture.

DEVELOPMENT OF FEMALE REPRODUCTIVE STRUCTURES IN <i>BOECHERA</i> (BRASSICACEAE) SPECIES UNDER SEXUAL AND APOMICTIC REPRODUCTIVE MODES

The results of comparative embryological study of the two species from the genus Boechera (B. falcata and B. stricta), which are characterized by a sexual mode of reproduction, and the apomictic M4B accession, an ancient natural hybrid of B. stricta × B. retrofracta, are presented. A detailed description of the ovule development of the studied plants is described. A significant similarity of the ovule development has been shown; a general plan of the ovules structure is ortho-campylotropic, bitegmal, medionucellate. The development of the embryo sac in B. falcata and B. stricta corresponds to the monosporic Polygonum type; in the ovules of the M4B accession, an apomictic unreduced female gametophyte is predominantly formed as a result of Taraxacum-type diplospory. However, in this accession apomixis is facultative, since a small amount of the ovules in the ovary forms a reduced embryo sac with participation of meiosis, but unlike B. falcata and B. stricta, after the second meiotic division, a triad of megaspores is formed, of which the chalazal one is functional. In the ovules of the apomictic M4B accession, underdevelopment of the inner integument was often observed, which, apparently, correlates with arrest of the megasporocyte and embryo sac development, since destruction was often observed in ovules with a short inner integument. This abnormality is likely due to the hybrid nature of the M4B accession rather than apomixis. In this accession, the embryo is formed parthenogenetically, but its development requires endosperm, which apparently develops by pseudogamy, only after triple fusion (fertilization of the fused polar nuclei by sperm). In the absence of endosperm, the embryo perishes, and seed development is arrested.

Low-concentration exogenous 3-indoleacetic acid improves fruit-setting rate of Marsdenia tenacissima by inhibiting the expression of embryo abortion-related genes

Marsdenia tenacissima is a medicinal plant characterized by many flowers, few fruits, and a low fruit-setting rate. Exogenous auxins can improve the fruit-setting rate of plants; however, their impacts on M. tenacissima and regulatory mechanisms remain unclear. In this study, we conducted a field experiment to determine the fruit-setting rate, seed-setting rate, fruit size, and changes in transcriptional expression of related genes by spraying 10 and 50 mg·L-1 of 3-indoleacetic acid (IAA). The control plants were sprayed with distilled water. Our results indicated that the fruit-setting rate was 0.15 when treated with 10 mg·L-1 of IAA, which was 2.76-fold higher than that of the control. Compared with that of the control, the number of differentially expressed genes (DEGs) regulated by 10 mg·L-1 of IAA was 28.6-fold higher than that regulated by 50 mg·L-1 of IAA. These DEGs were closely related to hormone metabolism and fruit development. By transcriptome analysis, spraying 10 mg·L-1 of IAA increased the expressions of STP6, MYB17, and LAX3 and reduced those of CXE18, ILR1-like 3, and SAUR50; this possibly affected the ovule, embryo, and fruit development, thereby elevating the fruit-setting rate of M. tenacissima. Our results indicated that low IAA concentration increased the fruit-setting rate of M. tenacissima, providing theoretical and practical support for promoting the seed yield of M. tenacissima.

Evolution and development of fruits of Erycina pusilla and other orchid species.

Fruits play a crucial role in seed dispersal. They open along dehiscence zones. Fruit dehiscence zone formation has been intensively studied in Arabidopsis thaliana. However, little is known about the mechanisms and genes involved in the formation of fruit dehiscence zones in species outside the Brassicaceae. The dehiscence zone of A. thaliana contains a lignified layer, while dehiscence zone tissues of the emerging orchid model Erycina pusilla include a lipid layer. Here we present an analysis of evolution and development of fruit dehiscence zones in orchids. We performed ancestral state reconstructions across the five orchid subfamilies to study the evolution of selected fruit traits and explored dehiscence zone developmental genes using RNA-seq and qPCR. We found that erect dehiscent fruits with non-lignified dehiscence zones and a short ripening period are ancestral characters in orchids. Lignified dehiscence zones in orchid fruits evolved multiple times from non-lignified zones. Furthermore, we carried out gene expression analysis of tissues from different developmental stages of E. pusilla fruits. We found that fruit dehiscence genes from the MADS-box gene family and other important regulators in E. pusilla differed in their expression pattern from their homologs in A. thaliana. This suggests that the current A. thaliana fruit dehiscence model requires adjustment for orchids. Additionally, we discovered that homologs of A. thaliana genes involved in the development of carpel, gynoecium and ovules, and genes involved in lipid biosynthesis were expressed in the fruit valves of E. pusilla, implying that these genes may play a novel role in formation of dehiscence zone tissues in orchids. Future functional analysis of developmental regulators, lipid identification and quantification can shed more light on lipid-layer based dehiscence of orchid fruits.

Glucan Synthase-like 2 is Required for Seed Initiation and Filling as Well as Pollen Fertility in Rice

BackgroundThe Glucan synthase-like (GSL) genes are indispensable for some important highly-specialized developmental and cellular processes involving callose synthesis and deposition in plants. At present, the best-characterized reproductive functions of GSL genes are those for pollen formation and ovary expansion, but their role in seed initiation remains unknown.ResultsWe identified a rice seed mutant, watery seed 1-1 (ws1-1), which contained a mutation in the OsGSL2 gene. The mutant produced seeds lacking embryo and endosperm but filled with transparent and sucrose-rich liquid. In a ws1-1 spikelet, the ovule development was normal, but the microsporogenesis and male gametophyte development were compromised, resulting in the reduction of fertile pollen. After fertilization, while the seed coat normally developed, the embryo failed to differentiate normally. In addition, the divided endosperm-free nuclei did not migrate to the periphery of the embryo sac but aggregated so that their proliferation and cellularization were arrested. Moreover, the degeneration of nucellus cells was delayed in ws1-1. OsGSL2 is highly expressed in reproductive organs and developing seeds. Disrupting OsGSL2 reduced callose deposition on the outer walls of the microspores and impaired the formation of the annular callose sheath in developing caryopsis, leading to pollen defect and seed abortion.ConclusionsOur findings revealed that OsGSL2 is essential for rice fertility and is required for embryo differentiation and endosperm-free nucleus positioning, indicating a distinct role of OsGSL2, a callose synthase gene, in seed initiation, which provides new insight into the regulation of seed development in cereals.

Genetic and Morpho-Physiological Differences among Transgenic and No-Transgenic Cotton Cultivars.

Three carbon-chain extension genes associated with fatty acid synthesis in upland cotton (Gossypium hirsutum), namely GhKAR, GhHAD, and GhENR, play important roles in oil accumulation in cotton seeds. In the present study, these three genes were cloned and characterized. The expression patterns of GhKAR, GhHAD, and GhENR in the high seed oil content cultivars 10H1014 and 10H1041 differed somewhat compared with those of 10H1007 and 2074B with low seed oil content at different stages of seed development. GhKAR showed all three cultivars showed higher transcript levels than that of 2074B at 10-, 40-, and 45-days post anthesis (DPA). The expression pattern of GhHAD showed a lower transcript level than that of 2074B at both 10 and 30 DPA but a higher transcript level than that of 2074B at 40 DPA. GhENR showed a lower transcript level than that of 2074B at both 15 and 30 DPA. The highest transcript levels of GhKAR and GhENR were detected at 15 DPA in 10H1007, 10H1014, and 10H1041 compared with 2074B. From 5 to 45 DPA cotton seed, the oil content accumulated continuously in the developing seed. Oil accumulation reached a peak between 40 DPA and 45 DPA and slightly decreased in mature seed. In addition, GhKAR and GhENR showed different expression patterns in fiber and ovule development processes, in which they showed high expression levels at 20 DPA during the fiber elongation stage, but their expression level peaked at 15 DPA during ovule development processes. These two genes showed the lowest expression levels at the late seed maturation stage, while GhHAD showed a peak of 10 DPA in fiber development. Compared to 2074B, the oil contents of GhKAR and GhENR overexpression lines increased 1.05~1.08 folds. These results indicated that GhHAD, GhENR, and GhKAR were involved in both seed oil synthesis and fiber elongation with dual biological functions in cotton.

2-NBDG Uptake in Gossypium hirsutum in vitro ovules: exploring tissue-specific accumulation and its impact on hexokinase-mediated glycolysis regulation.

Fluorescent glucose derivatives are valuable tools as glucose analogs in plant research to explore metabolic pathways, study enzyme activity, and investigate cellular processes related to glucose metabolism and sugar transport. They allow visualization and tracking of glucose uptake, its utilization, and distribution within plant cells and tissues. This study investigates the phenotypic and metabolic impact of the exogenously fed glucose derivative, 2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-2-deoxyglucose) (2-NBDG) on the fibers of Gossypium hirsutum (Upland cotton) ovule in vitro cultures. The presence of 2-NBDG in the culture medium did not lead to macroscopic morphological alterations in ovule and fiber development or to the acquisition of fluorescence or yellow coloration. Confocal laser scanning microscope imaging and chromatographic analysis of cotton ovules' outer rim cross-sections showed that the 2-NBDG is transported from the extracellular space and accumulated inside some outer integument cells, epidermal cells, and fertilized epidermal cells (fibers), but is not incorporated into the cell walls. Untargeted metabolic profiling of the fibers revealed significant changes in the relative levels of metabolites involved in glycolysis and upregulation of alternative energy-related pathways. To provide biochemical and structural evidence for the observed downregulation of glycolysis pathways in the fibers containing 2-NBDG, kinetics analysis and docking simulations were performed on hexokinase from G. hirsutum (GhHxk). Notably, the catalytic activity of heterologously expressed recombinant active GhHxk exhibited a five-fold decrease in reaction rates compared to D-glucose. Furthermore, GhHxk exhibited a linear kinetic behavior in the presence of 2-NBDG instead of the Michaelis-Menten kinetics found for D-glucose. Docking simulations suggested that 2-NBDG interacts with a distinct binding site of GhHxk9, possibly inducing a conformational change. These results highlight the importance of considering fluorescent glucose derivatives as ready-to-use analogs for tracking glucose-related biological processes. However, a direct comparison between their mode of action and its extrapolation into biochemical considerations should go beyond microscopic inspection and include complementary analytical techniques.

Genome-wide identification of the MADS-box gene family and SEP1 target genes regulating young embryo development in hazel (Corylus spp.) via ChIP sequencing

Hazel (Corylus spp.) is a nut-producing species with important cultivation value. The purpose of this study was to identify important MADS genes involved in the ovule and embryo development of hazel and explore the possible mechanism of MADS regulation on embryo development. After identifying all MADS-box gene members and differentially expressed genes in the hazel genome, we further verified the expression characteristics of important differentially expressed genes (DEGs) using in situ hybridization, and verified the interaction between important proteins using yeast two-hybrid analyses. Finally, ChIP-Seq analysis of ChSEP1 was conducted to identify its target genes. In total, 59 ChMADSs were identified across all 11 chromosomes, and protein length was predicted to vary between 89 and 524 amino acids. All ChMADSs were divided into Type I and Type II MADS-box genes with 14 subgroups. Most ChMADSs showed low expression, and 11 Type II ChMADSs were highly and differentially expressed in the ovule. Of these, floral quartet model genes, both ChMADS11 (AG-like) and ChMADS37 (SEP1) were highly expressed in young embryos at the rapid ovule growth stage and interacted with each other, indicating that they might coordinate embryo development through protein-protein interactions. In total, 4,143 ChSEP1 differential target genes were identified through ChIP-Seq. Important ChSEP1 target genes included ChSEP1/3, ChAG, ChSHP, and ChSTK as well as ChAGL6, ChAP1, ChFUL1, and ChTT16–2, indicating that ChSEP1 might regulate ovule development by cooperating with and mediating ChMADSs. Overall, our study provides new insights into the molecular mechanisms of hazel ovule development.

ETHNOSCIENCE STUDY OF YUR LOCAL WISDOM OF KANUM TRIBE IN MERAUKE

Kanum tribe is one of indigenous tribes in Merauke which depend their live on nature. One of their local wisdoms is yur, which was done on plant at harvest time. The principle of yur is prohibits human to take that plant at the certain time. The aim of this research is to describe yur local wisdom with ethno-science study. This research conducted in Tomer village, habitat of Kanum tribe in Naukenjerai District, Merauke. Data collected direct observation to Tomer village, interview with some people of Kanum tribe, and also literature study. The result of this study show that yur local wisdom can analyze with biology, physic, and chemistry science. This local wisdom can protect every step of plant growing, namely pollination, fertilization, development of ovule into seed, ovary into fruit, and until it becomes a perfectly ripe fruit. Furthermore, yur can take care of imbibition, photosynthesis, and transpiration process. Finally, from all of that, yur can conserve the growing process of plant until harvest time.

Genome-wide chromatin accessibility landscape and dynamics of transcription factor networks during ovule and fiber development in cotton

BackgroundThe development of cotton fiber is regulated by the orchestrated binding of regulatory proteins to cis-regulatory elements associated with developmental genes. The cis–trans regulatory dynamics occurred throughout the course of cotton fiber development are elusive. Here we generated genome-wide high-resolution DNase I hypersensitive sites (DHSs) maps to understand the regulatory mechanisms of cotton ovule and fiber development.ResultsWe generated DNase I hypersensitive site (DHS) profiles from cotton ovules at 0 and 3 days post anthesis (DPA) and fibers at 8, 12, 15, and 18 DPA. We obtained a total of 1185 million reads and identified a total of 199,351 DHSs through ~ 30% unique mapping reads. It should be noted that more than half of DNase-seq reads mapped multiple genome locations and were not analyzed in order to achieve a high specificity of peak profile and to avoid bias from repetitive genomic regions. Distinct chromatin accessibilities were observed in the ovules (0 and 3 DPA) compared to the fiber elongation stages (8, 12, 15, and 18 DPA). Besides, the chromatin accessibility during ovules was particularly elevated in genomic regions enriched with transposable elements (TEs) and genes in TE-enriched regions were involved in ovule cell division. We analyzed cis-regulatory modules and revealed the influence of hormones on fiber development from the regulatory divergence of transcription factor (TF) motifs. Finally, we constructed a reliable regulatory network of TFs related to ovule and fiber development based on chromatin accessibility and gene co-expression network. From this network, we discovered a novel TF, WRKY46, which may shape fiber development by regulating the lignin content.ConclusionsOur results not only reveal the contribution of TEs in fiber development, but also predict and validate the TFs related to fiber development, which will benefit the research of cotton fiber molecular breeding.

Reproductive Mechanisms in Ginkgo and Cycas: Sisters but not Twins

The study of reproductive mechanisms is of particular interest for a real understanding of seed plant evolution. Spermatophytes comprise angiosperms and four orders of gymnosperms (Cycadales, Ginkgoales, Coniferales, and Gnetales) whose main characteristic is the reproduction via seeds. Ginkgo and cycads form a sister clade to the other gymnosperms and occupy a key phylogenetic position in-between the extinct Paleozoic seed ferns and the other extant gymnosperms. This review focuses on the similarities and differences between the reproductive mechanisms of Ginkgo and Cycas, from the morphogenesis of the male and female organs to the pollination and fertilization events. Together with the morphological and cytological description, the latest available molecular data on the reproductive organ development of the two plant genera are discussed. This will, hopefully, pave the path for new studies aiming at filling the gaps in our understanding of the hormonal and genetic regulation of their reproductive mechanisms. The whole reproductive process is presented in detail, providing a comprehensive and organic picture together with complete illustrations and photographic material. Each phase of the reproductive process is dissected, pointing out the main similarities and differences found among the two genera. The comparison comprises the male and female reproductive organs development, with a focus on pollen ontogeny, shape, ultrastructure, and germination as well as ovule development and patterning, female gametophyte formation, and ovule integument differentiation. Particular attention is given to the pollination and fertilization events focusing on the role of reproductive fluids as well as zoogamy.

Identification and expression analysis of MADS-box gene family in Docynia delavayi (Franch.) Schneid.

MADS-box gene family is a significant transcription factor family that plays a crucial role in regulating plant growth, development, signal transduction, and other processes. In order to study the characteristics of MADS-box gene family in Docynia delavayi (Franch.) Schneid. and its expression during different stages of seed germination, this study used seedlings at different stages of germination as materials and screened MADS-box transcription factors from the transcriptome database of D. delavayi using bioinformatics methods based on transcriptome sequencing. The physical and chemical properties, protein conservative motifs, phylogenetic evolution, and expression patterns of the MADS-box transcription factors were analyzed. Quantitative real-time PCR (qRT-PCR) was used to verify the expression of MADS-box gene family members during different stages of seed germination in D. delavayi. The results showed that 81 genes of MADS-box gene family were identified from the transcriptome data of D. delavayi, with the molecular weight distribution ranged of 6 211.34-173 512.77 Da and the theoretical isoelectric point ranged from 5.21 to 10.97. Phylogenetic analysis showed that the 81 genes could be divided into 15 subgroups, among which DdMADS27, DdMADS42, DdMADS45, DdMADS46, DdMADS53, DdMADS61, DdMADS76, DdMADS77 and DdMADS79 might be involved in the regulation of ovule development in D. delavayi. The combination of the transcriptome data and the qRT-PCR analysis results of D. delavayi seeds indicated that DdMADS25 and DdMADS42 might be involved in the regulation of seed development, and that DdMADS37 and DdMADS38 might have negative regulation effects on seed dormancy. Previous studies have reported that the MIKC* subgroup is mainly involved in regulating flower organ development. For the first time, we found that the transcription factors of the MIKC* subgroup exhibited a high expression level at the early stage of seed germination, so we speculated that the MIKC* subgroup played a regulatory role in the process of seed germination. To verify the accuracy of this speculation, we selected DdMADS60 and DdMADS75 from the MIKC* subgroup for qRT-PCR experiments, and the experimental results were consistent with the expression trend of transcriptome sequencing. This study provides a reference for further research on the biological function of D. delavayi MADS-box gene family from the perspective of molecular evolution.

Seasonal gene expression signatures of delayed fertilization in Fagaceae.

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

An exonuclease V homologue is expressed predominantly during early megasporogenesis in apomictic Brachiaria brizantha.

An exonuclease V homologue from apomictic Brachiaria brizantha is expressed and localized in nucellar cells at key moments when these cells differentiate to give rise to unreduced gametophytes. Brachiaria is a genus of forage grasses with economical and agricultural importance to Brazil. Brachiaria reproduces by aposporic apomixis, in which unreduced embryo sacs, derived from nucellar cells, other than the megaspore mother cell (MMC), are formed. The unreduced embryo sacs produce an embryo without fertilization resulting in clones of the mother plant. Comparative gene expression analysis in ovaries of sexual and apomictic Brachiaria spp. revealed a sequence from B. brizantha that showed a distinct pattern of expression in ovaries of sexual and apomictic plants. In this work, we describe a gene named BbrizExoV with strong identity to exonuclease V (Exo V) genes from other grasses. Sequence analysis in signal prediction tools showed that BbrizExoV might have dual localization, depending on the translation point. A longer form to the nucleus and a shorter form which would be directed to the chloroplast. This is also the case for monocot sequences analyzed from other species. The long form of BbrizExoV protein localizes to the nucleus of onion epidermal cells. Analysis of ExoV proteins from dicot species, with exception of Arabidopsis thaliana ExoVL protein, showed only one localization. Using a template-based AlphaFold 2 modelling approach the structure of BbrizExoV in complex with metal and ssDNA was predicted based on the holo structure of the human counterpart. Features predicted to define ssDNA binding but a lack of sequence specificity are shared between the human enzyme and BbrizExoV. Expression analyses indicated the precise site and timing of transcript accumulation during ovule development, which coincides with the differentiation of nucelar cells to form the typical aposporic four-celled unreduced gametophyte. A putative function for this protein is proposed based on its homology and expression pattern.

Seedless fruit in Annona squamosa L. is monogenic and conferred by INO locus deletion in multiple accessions

Key messageInheritance of the presence/absence of seeds in Annona squamosa is mediated by a single fully recessive gene and is caused by a deletion of the INNER NO OUTER (INO) locus.For some fruits, seedless varieties are desirable for consumption and processing. In the sugar apple tree (Annona squamosa L.), the seedless trait in the Thai seedless (Ts) and Brazilian seedless (Bs) accessions was associated with defective ovules and an apparent deletion of the INNER NO OUTER (INO) ovule development gene locus. Segregation analysis of F2 and backcross descendants of crosses of Bs to fertile wild-type varieties in this species with a multi-year generation time showed that seedlessness was recessive and controlled by a single locus. Comparison of whole genome sequence of a wild-type plant and a third accession, Hawaiian seedless (Hs), identified a 16 kilobase deletion including INO in this line. Ts and Bs lines were shown to have an identical deletion, indicating a common origin from a single deletion event. Analysis of microsatellite markers could not preclude the possibility that all three seedless accessions are vegetatively propagated clones. The sequence of the deletion site enabled a codominant assay for the wild-type and mutant genes allowing observation of complete cosegregation of the seedless/defective ovule phenotype with the INO deletion, showing maximal separation of less than 3.5 cM. The observed deletion is the only significant difference between the wild-type and Hs line over 587 kilobases, likely encompassing much more than 3.5 cM, showing that the deletion is the cause of seedless trait. The codominant markers and obtained progenies will be useful for introgression of the seedless trait into elite sugar apple lines and into other Annonas through interspecific crossings.

OAF is a DAF-like gene that controls ovule development in plants

We previously found that the RING-type E3 ligase DEFECTIVE IN ANTHER DEHISCENCE1- (DAD1-) Activating Factor (DAF) controls anther dehiscence by activating the jasmonate biosynthetic pathway in Arabidopsis. Here, we show that in Arabidopsis, the DAF ancestor was duplicated into three genes (DAF, Ovule Activating Factor (OAF), DAFL2), which evolved divergent partial functions from their ancestor through subfunctionalization. In this case, DAF-DAD1-JA signaling regulates anther dehiscence, whereas OAF controls ovule development by negatively regulating cinnamyl alcohol dehydrogenase 9 (CAD9) activity and being negatively regulated by miR847 itself in Arabidopsis. Downregulation of OAF or upregulation of CAD9 and miR847 caused similar abortion of ovule formation due to precocious ovule lignification in transgenic Arabidopsis. Interestingly, only one DAF-like gene, PaOAF, exists in the monocot orchids, which has likely evolved through nonfunctionalization and maintains a conserved function as Arabidopsis OAF in regulating ovule development since defective ovules were observed in the virus-induced gene silencing (VIGS) PaOAF Phalaenopsis orchids. The absence of the DAF ortholog and its function in orchids is likely due to the evolution of stamens to a unique pollinium structure that lacks the feature of anther dehiscence. These findings expand the current knowledge underlying the multifunctional evolution and diverse functionalization of duplicate gene pairs within/among plants.

Conservation of the restricted expression of Brassicaceae Bsister-like genes in seeds requires a transposable element in Arabidopsis thaliana.

Changes in transcription factor binding sites (TFBSs) can alter the spatio-temporal expression pattern and transcript abundance of genes. Loss and gain of TFBSs were shown to cause shifts in expression patterns in numerous cases. However, we know little about the evolution of extended regulatory sequences incorporating many TFBSs. We compare, across the crucifers (Brassicaceae, cabbage family), the sequences between the translated regions of Arabidopsis Bsister- (ABS-) like MADS-box genes (including paralogous GOA-like genes) and the next gene upstream, as an example of family wide evolution of putative regulatory regions (PURRs). ABS-like genes are essential for integument development of ovules and endothelium formation in seeds of Arabidopsis thaliana. A combination of motif based GO-enrichment and reporter gene analysis using A. thaliana as common trans-regulatory environment allows analysis of selected Brassicaceae Bsister gene PURRs. Comparison of TFBS of transcriptionally active ABS-like genes with those of transcriptionally largely inactive GOA-like genes shows that the number of in silico predicted TF binding sites (TFBSs) is similar between paralogs, emphasizing the importance of experimental verification for in silico characterization of TFBS activity and analysis of their evolution. Further, our data show highly conserved expression of Brassicaceae ABS-like genes almost exclusively in the chalazal region of ovules. The Arabidopsis-specific insertion of a transposable element (TE) into the ABS PURRs is required for stabilizing this spatially restricted expression, while other Brassicaceae achieve chalaza-specific expression without TE insertion. We hypothesize that the chalaza-specific expression of ABS is regulated by CREs provided by the TE.

Identification of IPT9 in Brachiaria brizantha (syn. Urochloa brizantha) and expression analyses during ovule development in sexual and apomictic plants

BackgroundIn Brachiaria sexual reproduction, during ovule development, a nucellar cell differentiates into a megaspore mother cell (MMC) that, through meiosis and mitosis, gives rise to a reduced embryo sac. In aposporic apomictic Brachiaria, next to the MMC, other nucellar cells differentiate into aposporic initials that enter mitosis directly forming an unreduced embryo sac. The IPT (isopentenyltransferase) family comprises key genes in the cytokinin (CK) pathway which are expressed in Arabidopsis during ovule development. BbrizIPT9, a B. brizantha (syn. Urochloa brizantha) IPT9 gene, highly similar to genes of other Poaceae plants, also shows similarity with Arabidopsis IPT9, AtIPT9. In this work, we aimed to investigate association of BbrizIPT9 with ovule development in sexual and apomictic plants.Methods and resultsRT-qPCR showed higher BbrizIPT9 expression in the ovaries of sexual than in the apomictic B. brizantha. Results of in-situ hybridization showed strong signal of BbrizIPT9 in the MMC of both plants, at the onset of megasporogenesis. By analyzing AtIPT9 knockdown mutants, we verified enlarged nucellar cell, next to the MMC, in a percentage significantly higher than in the wild type, suggesting that knockout of AtIPT9 gene triggered the differentiation of extra MMC-like cells.ConclusionsOur results indicate that AtIPT9 might be involved in the proper differentiation of a single MMC during ovule development. The expression of a BbrizIPT9, localized in male and female sporocytes, and lower in apomicts than in sexuals, and effect of IPT9 knockout in Arabidopsis, suggest involvement of IPT9 in early ovule development.

Control of ovule development in Vitis vinifera by VvMADS28 and interacting genes.

Seedless grapes are increasingly popular throughout the world, and the development of seedless varieties is a major breeding goal. In this study, we demonstrate an essential role for the grapevine MADS-box gene VvMADS28 in morphogenesis of the ovule. We found that VvMADS28 mRNA accumulated in the ovules of a seeded cultivar, 'Red Globe', throughout the course of ovule and seed development, especially within the integument/seed coat. In contrast, in the seedless cultivar 'Thompson Seedless', VvMADS28 was expressed only weakly in ovules, and this was associated with increased levels of histone H3 lysine 27 trimethylation (H3K27me3) within the VvMADS28 promoter region. RNAi-mediated transient suppression of VvMADS28 expression in 'Red Globe' led to reduced seed size associated with inhibition of episperm and endosperm cell development. Heterologous overexpression of VvMADS28 in transgenic tomatoes interfered with sepal development and resulted in smaller fruit but did not obviously affect seed size. Assays in yeast cells showed that VvMADS28 is subject to regulation by the transcription factor VvERF98, and that VvMADS28 could interact with the Type I/ Mβ MADS-domain protein VvMADS5. Moreover, through DNA-affinity purification-sequencing (DAP-seq), we found that VvMADS28 protein specifically binds to the promoter of the grapevine WUSCHEL (VvWUS) gene, suggesting that maintenance of the VvMADS28-VvMADS5 dimer and VvWUS expression homeostasis influences seed development. Taken together, our results provide insight into regulatory mechanisms of ovule and seed development associated with VvMADS28.

Ovule Number and Flower Size in Pea (Pisum sativum L.): Variation, Heritability, and Correlation with Some Components of Productivity

The seed yield in pea (Pisum sativum L.) depends on numerous environmental and genotypic factors. The ongoing climate changes draw one’s attention to genetics and variation of underexplored reproductive traits. This study focuses on ovule number (ON) and flower size expressed as a length of flag petal (FL), in terms of their heritability, variability, and correlation with 1000 seed mass (TSM), seed number per pod, and seed/ovule ratio. A set of pea accessions was planted over several years in field conditions. Some of these accessions were also grown in a glasshouse. The chosen values were scored on living plants or while harvesting seeds. Nonparametric statistical methods were applied. Heritability of ON and FL was studied in five hybrid F1/F2 combinations. We found a relatively low (ca. 0.5) broad sense heritability of both ON and FL. Among other traits, TSM and ON reproduced best over the years. FL exhibited no reproducible correlations with other traits and cannot be used as a predictor of productivity. Water deficit and heat stress reduce seed yield in pea due to both seed abortion and development of fewer ovules. The differential and heritable ability to retain ON may become a basis for breeding pea cultivars adapted to changing climate.