Pivotal role of STIP in ovule pattern formation and female germline development in Arabidopsis thaliana.

In spermatophytes the sporophytic (diploid) and the gametophytic (haploid) generations co-exist in ovules, and the coordination of their developmental programs is of pivotal importance for plant reproduction. To achieve efficient fertilization, the haploid female gametophyte and the diploid ovule structures must coordinate their development to form a functional and correctly shaped ovule. WUSCHEL-RELATED HOMEOBOX (WOX) genes encode a family of transcription factors that share important roles in a wide range of processes throughout plant development. Here, we show that STIP is required for the correct patterning and curvature of the ovule in Arabidopsis thaliana. The knockout mutant stip-2 is characterized by a radialized ovule phenotype due to severe defects in outer integument development. In addition, alteration of STIP expression affects the correct differentiation and progression of the female germline. Finally, our results reveal that STIP is required to tightly regulate the key ovule factors INNER NO OUTER, PHABULOSA and WUSCHEL, and they define a novel genetic interplay in the regulatory networks determining ovule development.

Type II arabinogalactans initiated by hydroxyproline-O-galactosyltransferases play important roles in pollen-pistil interactions.

Arabinogalactan-proteins (AGPs) are hydroxyproline-rich glycoproteins containing a high sugar content and are widely distributed in the plant kingdom. AGPs have long been suggested to play important roles in sexual plant reproduction. The synthesis of their complex carbohydrates is initiated by a family of hydroxyproline galactosyltransferase (Hyp-GALT) enzymes which add the first galactose to Hyp residues in the protein backbone. Eight Hyp-GALT enzymes have been identified so far, and in the present work a mutant affecting five of these enzymes (galt2galt5galt7galt8galt9) was analyzed regarding the reproductive process. The galt25789 mutant presented a low seed set, and reciprocal crosses indicated a significant female gametophytic contribution to this mutant phenotype. Mutant ovules revealed abnormal callose accumulation inside the embryo sac and integument defects at the micropylar region culminating in defects in pollen tube reception. In addition, immunolocalization and biochemical analyses allowed the detection of a reduction in the amount of glucuronic acid in mutant ovary AGPs. Dramatically low amounts of high-molecular-weight Hyp-O-glycosides obtained following size exclusion chromatography of base-hydrolyzed mutant AGPs compared to the wild type indicated the presence of underglycosylated AGPs in the galt25789 mutant, while the monosaccharide composition of these Hyp-O-glycosides displayed no significant changes compared to the wild-type Hyp-O-glycosides. The present work demonstrates the functional importance of the carbohydrate moieties of AGPs in ovule development and pollen-pistil interactions.

Genome-Wide Characterization and Functional Validation of the ACS Gene Family in the Chestnut Reveals Its Regulatory Role in Ovule Development.

Ovule abortion significantly contributes to a reduction in chestnut yield. Therefore, an examination of the mechanisms underlying ovule abortion is crucial for increasing chestnut yield. In our previous study, we conducted a comprehensive multiomic analysis of fertile and abortive ovules and found that ACS genes in chestnuts (CmACS) play a crucial role in ovule development. Therefore, to further study the function of ACS genes, a total of seven CmACS members were identified, their gene structures, conserved structural domains, evolutionary trees, chromosomal localization, and promoter cis-acting elements were analyzed, and their subcellular localization was predicted and verified. The spatiotemporal specificity of the expression of the seven CmACS genes was confirmed via qRT-PCR analysis. Notably, CmACS7 was exclusively expressed in the floral organs, and its expression peaked during fertilization and decreased after fertilization. The ACC levels remained consistently greater in fertile ovules than in abortive ovules. The ACSase activity of CmACS7 was identified using the genetic transformation of chestnut healing tissue. Micro Solanum lycopersicum plants overexpressing CmACS7 had a significantly greater rate of seed failure than did wild-type plants. Our results suggest that ovule fertilization activates CmACS7 and increases ACC levels, whereas an overexpression of CmACS7 leads to an increase in ACC content in the ovule prior to fertilization, which can lead to abortion. In conclusion, the present study demonstrated that chestnut ovule abortion is caused by poor fertilization and not by nutritional competition. Optimization of the pollination and fertilization of female flowers is essential for increasing chestnut yield and reducing ovule abortion.

Role of grapevine SEPALLATA-related MADS-box gene VvMADS39 in flower and ovule development.

Seedlessness is one of the most important breeding goals for table grapes; thus, understanding the molecular genetic regulation of seed development and abortion is critical for the development of seedless cultivars. In the present study, we characterized VvMADS39, a class E MADS-box gene of grapevine (Vitis vinifera) orthologous to Arabidopsis SEP2. Heterologous overexpression of VvMADS39 in tomato reduced the fruit and seed size and seed number. Targeted mutagenesis of the homologous SlMADS39 in tomato induced various floral and fruit defects. It could reasonable to suppose that active VvMADS39 expression in "Thompson Seedless" may restrict cellular expansion, resulting in the development of smaller fruits and seeds, VvMADS39 may play a role in the regulation of ovule development in grapevine and contributes to seedless fruit formation. In contrast, VvMADS39 suppression in "Red Globe" was associated with enhanced histone H3 lysine 27 trimethylation in the promoter region of VvMADS39, allowing normal ovule and fruit development; Meanwhile, VvMADS39 interacts with VvAGAMOUS, and the activity of the VvMADS39-VvAGAMOUS dimer to induce integument development requires the activation and maintenance of VvINO expression. The synergistic cooperation between VvMADS39 and related proteins plays an important role in maintaining floral meristem characteristics, and fruit and ovule development.

Improvement of RNA In Situ Hybridisation for Grapevine Fruits and Ovules.

The European grapevine (Vitis vinifera L.) is one of the world's most widely cultivated and economically important fruit crops. Seedless fruits are particularly desired for table grapes, with seedlessness resulting from stenospermocarpy being an important goal for cultivar improvement. The establishment of an RNA in situ hybridisation (ISH) system for grape berries and ovules is, therefore, important for understanding the molecular mechanisms of ovule abortion in stenospermocarpic seedless cultivars. We improved RNA in situ hybridisation procedures for developing berries and ovules by targeting two transcription factor genes, VvHB63 and VvTAU, using two seeded varieties, 'Red Globe' and 'Pinot Noir', and two seedless cultivars, 'Flame Seedless' and 'Thompson Seedless'. Optimisation focused on the time of proteinase K treatment, probe length, probe concentration, hybridisation temperature and post-hybridisation washing conditions. The objectives were to maximise hybridisation signals and minimise background interference, while still preserving tissue integrity. For the target genes and samples tested, the best results were obtained with a pre-hybridisation proteinase K treatment of 30 min, probe length of 150 bp and concentration of 100 ng/mL, hybridisation temperature of 50 °C, three washes with 0.2× saline sodium citrate (SSC) solution and blocking with 1% blocking reagent for 45 min during the subsequent hybridisation. The improved ISH system was used to study the spatiotemporal expression patterns of genes related to ovule development at a microscopic level.

Transcriptomic Analysis of Hormone Signal Transduction, Carbohydrate Metabolism, Heat Shock Proteins, and SCF Complexes before and after Fertilization of Korean Pine Ovules.

The fertilization process is a critical step in plant reproduction. However, the mechanism of action and mode of regulation of the fertilization process in gymnosperms remain unclear. In this study, we investigated the molecular regulatory networks involved in the fertilization process in Korean pine ovules through anatomical observation, physiological and biochemical assays, and transcriptome sequencing technology. The morphological and physiological results indicated that fertilization proceeds through the demise of the proteinaceous vacuole, egg cell division, and pollen tube elongation. Auxin, cytokinin, soluble sugar, and soluble starch contents begin to decline upon fertilization. Transcriptomic data analysis revealed a large number of differentially expressed genes at different times before and after fertilization. These genes were primarily involved in pathways associated with plant hormone signal transduction, protein processing in the endoplasmic reticulum, fructose metabolism, and mannose metabolism. The expression levels of several key genes were further confirmed by qRT-PCR. These findings represent an important step towards understanding the mechanisms underlying morphological changes in the Korean pine ovule during fertilization, and the physiological and transcriptional analyses lay a foundation for in-depth studies of the molecular regulatory network of the Korean pine fertilization process.

Identification of key regulatory genes involved in the sporophyte and gametophyte development in Ginkgo biloba ovules revealed by in situ expression analyses.

PREMISE: In Arabidopsis thaliana, the role of the most important key genes that regulate ovule development is widely known. In nonmodel species, and especially in gymnosperms, the ovule developmental processes are still quite obscure. In this study, we describe the putative roles of Ginkgo biloba orthologs of regulatory genes during ovule development. Specifically, we studied AGAMOUS (AG), AGAMOUS-like 6 (AGL6), AINTEGUMENTA (ANT), BELL1 (BEL1), Class III HD-Zip, and YABBY Ginkgo genes. METHODS: We analyzed their expression domains through in situ hybridizations on two stages of ovule development: the very early stage that corresponds to the ovule primordium, still within wintering buds, and the late stage at pollination time. RESULTS: GBM5 (Ginkgo ortholog of AG), GbMADS8 (ortholog of AGL6) and GbC3HDZ1-2-3 were expressed in both the stages of ovule development, while GbMADS1, GbAGL6-like genes (orthologs of AGL6), GbBEL1-2 and YABBY Ginkgo orthologs (GbiYAB1B and GbiYABC) seem mostly involved at pollination time. GbANTL1 was not expressed in the studied stages and was different from GbANTL2 and GbBEL1, which seem to be involved at both stages of ovule development. In Ginkgo, the investigated genes display patterns of expression only partially comparable to those of other studied seed plants. CONCLUSIONS: The expression of most of these regulatory genes in the female gametophyte region at pollination time leads to suggest a communication between the sporophytic maternal tissue and the developing female gametophyte, as demonstrated for well-studied model angiosperms.

The role of pollination in controlling Ginkgo biloba ovule development.

Generally, in gymnosperms, pollination and fertilization events are temporally separated and the developmental processes leading the switch from ovule integument into seed coat are still unknown. The single ovule integument of Ginkgo biloba acquires the typical characteristics of the seed coat long before the fertilization event. In this study, we investigated whether pollination triggers the transformation of the ovule integument into the seed coat. Transcriptomics and metabolomics analyses performed on ovules just prior and after pollination lead to the identification of changes occurring in Ginkgo ovules during this specific time. A morphological atlas describing the developmental stages of ovule development is presented. The metabolic pathways involved in the lignin biosynthesis and in the production of fatty acids are activated upon pollination, suggesting that the ovule integument starts its differentiation into a seed coat before the fertilization. Omics analyses allowed an accurate description of the main changes that occur in Ginkgo ovules during the pollination time frame, suggesting the crucial role of the pollen arrival on the progression of ovule development.

The neglected other half - role of the pistil in plant heat stress responses.

Heat stress coinciding with reproductive stage leads to a significant loss in reproductive organs viability, resulting in lower seed-set and crop productivity. Successful fertilization and seed formation are determined by the viability of male and female reproductive organs. The impact of heat stress on the male reproductive organ (pollen) is studied more often compared to the female reproductive organ (pistil). This is attributed to easier accessibility of the pollen coupled with the notion that the pistil's role in fertilization and seed-set under heat stress is negligible. However, depending on species and developmental stages, recent studies reveal varying degrees of sensitivity of the pistil to heat stress. Remarkably, in some cases, the vulnerability of the pistil is even greater than the pollen. This article summarizes the current knowledge of the impact of heat stress on three critical stages of pistil for successful seed-set, that is, female reproductive organ development (gametogenesis), pollen-pistil interactions including pollen capture on stigma and pollen tube growth in style, as well as fertilization and early embryogenesis. Further, future research directions are suggested to unravel molecular basis of heat stress tolerance in pistil, which is critical for sustaining crop yields under predicted warming scenarios.

Arabidopsis Chloroplast protein for Growth and Fertility1 (CGF1) and CGF2 are essential for chloroplast development and female gametogenesis.

BACKGROUND: Chloroplasts are essential organelles of plant cells for not only being the energy factory but also making plant cells adaptable to different environmental stimuli. The nuclear genome encodes most of the chloroplast proteins, among which a large percentage of membrane proteins have yet to be functionally characterized. RESULTS: We report here functional characterization of two nuclear-encoded chloroplast proteins, Chloroplast protein for Growth and Fertility (CGF1) and CGF2. CGF1 and CGF2 are expressed in diverse tissues and developmental stages. Proteins they encode are associated with chloroplasts through a N-terminal chloroplast-targeting signal in green tissues but also located at plastids in roots and seeds. Mutants of CGF1 and CGF2 generated by CRISPR/Cas9 exhibited vegetative defects, including reduced leaf size, dwarfism, and abnormal cell death. CGF1 and CGF2 redundantly mediate female gametogenesis, likely by securing local energy supply. Indeed, mutations of both genes impaired chloroplast integrity whereas exogenous sucrose rescued the growth defects of the CGF double mutant. CONCLUSION: This study reports that two nuclear-encoded chloroplast proteins, Chloroplast protein for Growth and Fertility (CGF1) and CGF2, play important roles in vegetative growth, in female gametogenesis, and in embryogenesis likely by mediating chloroplast integrity and development.

Characterization of floral morphoanatomy and identification of marker genes preferentially expressed during specific stages of cotton flower development.

MAIN CONCLUSION: Characterization of anther and ovule developmental programs and expression analyses of stage-specific floral marker genes in Gossypium hirsutum allowed to build a comprehensive portrait of cotton flower development before fiber initiation. Gossypium hirsutum is the most important cotton species that is cultivated worldwide. Although cotton reproductive development is important for fiber production, since fiber is formed on the epidermis of mature ovules, cotton floral development remains poorly understood. Therefore, this work aims to characterize the cotton floral morphoanatomy by performing a detailed description of anther and ovule developmental programs and identifying stage-specific floral marker genes in G. hirsutum. Using light microscopy and scanning electron microscopy, we analyzed anther and ovule development during 11 stages of flower development. To better characterize the ovule development in cotton, we performed histochemical analyses to evaluate the accumulation of phenolic compounds, pectin, and sugar in ovule tissues. After identification of major hallmarks of floral development, three key stages were established in G. hirsutum floral development: in stage 1 (early-EF), sepal, petal, and stamen primordia were observed; in stage 2 (intermediate-IF), primordial ovules and anthers are present, and the differentiating archesporial cells were observed, marking the beginning of microsporogenesis; and in stage 6 (late-LF), flower buds presented initial anther tapetum degeneration and microspore were released from the tetrad, and nucellus and both inner and outer integuments are developing. We used transcriptome data of cotton EF, IF and LF stages to identify floral marker genes and evaluated their expression by real-time quantitative PCR (qPCR). Twelve marker genes were preferentially expressed in a stage-specific manner, including the putative homologs for AtLEAFY, AtAPETALA 3, AtAGAMOUS-LIKE 19 and AtMALE STERILITY 1, which are crucial for several aspects of reproductive development, such as flower organogenesis and anther and petal development. We also evaluated the expression profile of B-class MADS-box genes in G. hirsutum floral transcriptome (EF, IF, and LF). In addition, we performed a comparative analysis of developmental programs between Arabidopsis thaliana and G. hirsutum that considered major morphoanatomical and molecular processes of flower, anther, and ovule development. Our findings provide the first detailed analysis of cotton flower development.

Ovule Development and in Planta Transformation of Paphiopedilum Maudiae by Agrobacterium-Mediated Ovary-Injection.

In this paper, the development of the Paphiopedilum Maudiae embryo sac at different developmental stages after pollination was assessed by confocal laser scanning microscopy. The mature seeds of P. Maudiae consisted of an exopleura and a spherical embryo, but without an endosperm, while the inner integument cells were absorbed by the developing embryo. The P. Maudiae embryo sac exhibited an Allium type of development. The time taken for the embryo to develop to a mature sac was 45-50 days after pollination (DAP) and most mature embryo sacs had completed fertilization and formed zygotes by about 50-54 DAP. In planta transformation was achieved by injection of the ovaries by Agrobacterium, resulting in 38 protocorms or seedlings after several rounds of hygromycin selection, corresponding to 2, 7, 5, 1, 3, 4, 9, and 7 plantlets from Agrobacterium-mediated ovary-injection at 30, 35, 42, 43, 45, 48, 50, and 53 DAP, respectively. Transformation efficiency was highest at 50 DAP (2.54%), followed by 2.48% at 53 DAP and 2.45% at 48 DAP. Four randomly selected hygromycin-resistant plants were GUS-positive after PCR analysis. Semi-quantitative PCR and quantitative real-time PCR analysis revealed the expression of the hpt gene in the leaves of eight hygromycin-resistant seedlings following Agrobacterium-mediated ovary-injection at 30, 35, 42, 43, 45, 48, 50, and 53 DAP, while hpt expression was not detected in the control. The best time to inject P. Maudiae ovaries in planta with Agrobacterium is 48-53 DAP, which corresponds to the period of fertilization. This protocol represents the first genetic transformation protocol for any Paphiopedilum species and will allow for expanded molecular breeding programs to introduce useful and interesting genes that can expand its ornamental and horticulturally important characteristics.

Identification of lncRNAs involved in rice ovule development and female gametophyte abortion by genome-wide screening and functional analysis.

BACKGROUND: As important female reproductive tissues, the rice (Oryza sativa L.) ovule and female gametophyte is significant in terms of their fertility. Long noncoding RNAs (lncRNAs) play important and wide-ranging roles in the growth and development of plants and have become a major research focus in recent years. Therefore, we explored the characterization and expression change of lncRNAs during ovule development and female gametophytic abortion. RESULTS: In our study, whole-transcriptome strand-specific RNA sequencing (ssRNA-seq) was performed in the ovules of a high-frequency female-sterile rice line (fsv1) and a wild-type rice line (Gui99) at the megaspore mother cell meiosis stage (stage 1), functional megaspore mitosis stage (stage 2) and female gametophyte mature stage (stage 3). By comparing two rice lines, we identified 152, 233, and 197 differentially expressed lncRNAs at the three ovule developmental stages. Functional analysis of the coherent target genes of these differentially expressed lncRNAs indicated that many lncRNAs participate in multiple pathways such as hormone and cellular metabolism and signal transduction. Moreover, there were many differentially expressed lncRNAs acting as the precursors of some miRNAs that are involved in the development of ovules and female gametophytes. In addition, we have found that lncRNAs can act as decoys, competing with mRNAs for binding to miRNAs to maintain the normal expression of genes related to ovule and female gametophyte development. CONCLUSION: These results provide important clues for elucidating the female gametophyte abortion mechanism in rice. This study also expands our understanding about the biological functions of lncRNAs and the annotation of the rice genome.

Molecular cloning and functional characterization of a seed-specific VvßVPE gene promoter from Vitis vinifera.

MAIN CONCLUSION: The grapevine VvßVPE promoter is specifically expressed in the seed. The - 1306~- 1045 bp core region restricts expression in other tissues and organs. Vacuolar processing enzyme (VPE) is a cysteine proteinase regulating vacuolar protein maturation and executing programmed cell death (PCD) in plants. Vitis vinifera (Vv)ßVPE is a ß-type VPE showing seed-specific expression that processes seed proteins during ovule development. However, the regulation of the seed-specific gene expression is far from understood. In this study, we characterize VvßVPE promoter (pVvßVPE) from 12 seeded and seedless grape genotypes. 94.56% of the pVvßVPE coding sequence is consistent. Two ßVPE promoters were constructed and transformed into Arabidopsis thaliana via ß-glucuronidase (GUS) fused expression vectors, using cv. Pinot Noir and cv. Thompson as seed and seedless candidates. GUS staining in different tissues and organs revealed that VvßVPE expresses specifically in the embryo, including the cotyledon, hypocotyl and suspensor, but not in the leaf, stem, root or flowers of the seedling. Using promoter deletion analysis, we created four incomplete VvßVPE promoters and found each pVvßVPE deletion could drive GUS gene to express in seeds. Interestingly, seed specificity disappeared when the promoter missed the core - 1306~- 1045 bp region. All deletion promoters presenting various quantified GUS activities indicate that the region - 1704~- 1306 bp inhibits, and the region - 705~- 861 bp promotes gene expression of VvßVPE. Our results demonstrate that pVvßVPE is a seed-specific promoter in both seeded and seedless grapes. Moreover, the core region of pVvßVPE (- 1306~- 1045 bp) is the key one responsible for seed-specific expression.

PINOID is required for lateral organ morphogenesis and ovule development in cucumber.

Lateral organ development is essential for cucumber production. The protein kinase PINOID (PID) participates in distinct aspects of plant development by mediating polar auxin transport in different species. Here, we obtained a round leaf (rl) mutant that displayed extensive phenotypes including round leaf shape, inhibited tendril outgrowth, abnormal floral organs, and disrupted ovule genesis. MutMap+ analysis revealed that rl encodes a cucumber ortholog of PID (CsPID). A non-synonymous single nucleotide polymorphism in the second exon of CsPID resulted in an amino acid substitution from arginine to lysine in the rl mutant. Allelic testing using the mutant allele C356 with similar phenotypes verified that CsPID was the causal gene. CsPID was preferentially expressed in young leaf and flower buds and down-regulated in the rl mutant. Subcellular localization showed that the mutant form, Cspid, showed a dotted pattern of localization, in contrast to the continuous pattern of CsPID in the periphery of the cell and nucleus. Complementation analysis in Arabidopsis showed that CsPID, but not Cspid, can partially rescue the pid-14 mutant phenotype. Moreover, indole-3-acetic acid content was greatly reduced in the rl mutant. Transcriptome profiling revealed that transcription factors, ovule morphogenesis, and auxin transport-related genes were significantly down-regulated in the rl mutant. Biochemical analysis showed that CsPID physically interacted with a key polarity protein, CsREV (REVOLUTA). We developed a model in which CsPID regulates lateral organ morphogenesis and ovule development by stimulating genes related to auxin transport and ovule development.

The pomegranate (Punica granatum L.) genome provides insights into fruit quality and ovule developmental biology.

Pomegranate (Punica granatum L.) has an ancient cultivation history and has become an emerging profitable fruit crop due to its attractive features such as the bright red appearance and the high abundance of medicinally valuable ellagitannin-based compounds in its peel and aril. However, the limited genomic resources have restricted further elucidation of genetics and evolution of these interesting traits. Here, we report a 274-Mb high-quality draft pomegranate genome sequence, which covers approximately 81.5% of the estimated 336-Mb genome, consists of 2177 scaffolds with an N50 size of 1.7 Mb and contains 30 903 genes. Phylogenomic analysis supported that pomegranate belongs to the Lythraceae family rather than the monogeneric Punicaceae family, and comparative analyses showed that pomegranate and Eucalyptus grandis share the paleotetraploidy event. Integrated genomic and transcriptomic analyses provided insights into the molecular mechanisms underlying the biosynthesis of ellagitannin-based compounds, the colour formation in both peels and arils during pomegranate fruit development, and the unique ovule development processes that are characteristic of pomegranate. This genome sequence provides an important resource to expand our understanding of some unique biological processes and to facilitate both comparative biology studies and crop breeding.

GID1 expression is associated with ovule development of sexual and apomictic plants.

KEY MESSAGE: BbrizGID1 is expressed in the nucellus of apomictic Brachiaria brizantha, previous to aposporous initial differentiation. AtGID1a overexpression triggers differentiation of Arabidopsis thaliana MMC-like cells, suggesting its involvement in ovule development. GIBBERELLIN-INSENSITIVE DWARF1 (GID1) is a gibberellin receptor previously identified in plants and associated with reproductive development, including ovule formation. In this work, we characterized the Brachiaria brizantha GID1 gene (BbrizGID1). BbrizGID1 showed up to 92% similarity to GID1-like gibberellin receptors of other plants of the Poaceae family and around 58% to GID1-like gibberellin receptors of Arabidopsis thaliana. BbrizGID1 was more expressed in ovaries at megasporogenesis than in ovaries at megagametogenesis of both sexual and apomictic plants. In ovules, BbrizGID1 transcripts were detected in the megaspore mother cell (MMC) of sexual and apomictic B. brizantha. Only in the apomictic plants, expression was also observed in the surrounding nucellar cells, a region in which aposporous initial cells differentiate to form the aposporic embryo sac. AtGID1a ectopic expression in Arabidopsis determines the formation of MMC-like cells in the nucellus, close to the MMC, that did not own MMC identity. Our results suggest that GID1 might be involved in the proper differentiation of a single MMC during ovule development and provide valuable information on the role of GID1 in sexual and apomictic reproduction.

The superoxide dismutase genes might be required for appropriate development of the ovule after fertilization in Xanthoceras sorbifolium.

KEY MESSAGE: Superoxide dismutase genes were expressed differentially along with developmental stages of fertilized ovules in Xanthoceras sorbifolium, and the XsMSD gene silencing resulted in the arrest of fertilized ovule development. A very small percentage of mature fruits (ca. 5%) are produced relative to the number of bisexual flowers in Xanthoceras sorbifolium because seeds and fruits are aborted at early stages of development after pollination. Reactive oxygen species (ROS) in plants are implicated in an extensive range of biological processes, such as programmed cell death and senescence. Superoxide dismutase (SOD) activity might be required to regulate ROS homeostasis in the fertilized ovules of X. sorbifolium. The present study identified five SOD genes and one SOD copper chaperone gene in the tree. Their transcripts were differentially expressed along different stages of fertilized ovule development. These genes showed maximum expression in the ovules at 3 days after pollination (DAP), a time point in which free nuclear endosperm and nucleus tissues rapidly develop. The XsCSD1, XsFSD1 and XsMSD contained seven, eight, and five introns, respectively. Analysis of the 5'-flanking region of XsFSD1 and XsMSD revealed many cis-acting regulatory elements. Evaluation of XsMSD gene function based on virus-induced gene silencing (VIGS) indicated that the gene was closely related to early development of the fertilized ovules and fruits. This study suggested that SOD genes might be closely associated with the fate of ovule development (aborted or viable) after fertilization in X. sorbifolium.

Prolonged embryogenesis in Austrobaileya scandens (Austrobaileyaceae): its ecological and evolutionary significance.

The embryology of basal angiosperm lineages (Amborella, Nymphaeales and Austrobaileyales) is central to reconstructing the early evolution of flowering plants. Previous studies have shown that mature seeds in Austrobaileyales are albuminous, with a small embryo surrounded by a substantial diploid endosperm. However, little is known of seed ontogeny and seedling germination in Austrobaileya scandens, sister to all other extant Austrobaileyales. Standard histochemical techniques were used to study ovule/seed development and germination of Austrobaileya. Early development of the endosperm in Austrobaileya is ab initio cellular with pronounced cell proliferation. The nucellus transiently accumulates some starch, but is obliterated by expansion of a massive endosperm, where all embryo-nourishing reserves are ultimately stored. Twelve months elapse from fertilization to fruit abscission. Seeds are dispersed with a minute embryo, requiring 12 additional months for seedling establishment. The 2 yr required for seedling establishment is an apomorphic feature of Austrobaileya, probably related to germination in extremely dark understory conditions. Remarkably, although Austrobaileya seeds are nearly 50 times larger (by length) than the smallest seeds of extinct and extant members of early divergent angiosperm lineages, the embryo to seed ratio (E : S) falls squarely within the narrow range that characterizes the albuminous seeds of ancient flowering plant lineages.

Identification and expression analysis of microRNAs during ovule development in rice (Oryza sativa) by deep sequencing.

KEY MESSAGE: MicroRNA (miRNA) expression profiles during rice ovule development revealed the possible miRNA-mediated regulation between ovule sporophytic tissue and female gametophyte and the involvement of miRNAs in programmed cell death. MiRNAs are 20-24-nucleotide small RNAs that play key roles in the regulation of many growth and developmental processes in plants. Rice ovule development comprises a series of biological events, which are regulated by complex molecular mechanisms. To gain insight into miRNA-mediated regulation of rice ovule development, Illumina sequencing was used to examine the expression of miRNAs from the megaspore mother cell meiosis stage to the fertilized ovule stage. Based on the sequencing data, 486 known and 204 novel miRNAs were identified during rice ovule development. Moreover, 56, 65 and 11 differentially expressed miRNAs between adjacent developmental stages were identified. By analyzing transcriptome and degradome data, we identified 41, 65 and 12 coherent target genes for the differentially expressed miRNAs in ovule development. We found that changes in the expression of plant hormone-related miRNAs may play important roles in embryo sac development, providing evidence for cross-talk communication between sporophytic tissue and the female gametophyte. Additionally, we revealed that miRNAs may be involved in programmed cell death after fertilization. Finally, we constructed miRNA-mediated regulatory networks that are active during rice ovule development.