EMBRYO SAC DEVELOPMENT 1 affects seed setting rate in rice by controlling embryo sac development.

Seed setting rate is one of the critical factors that determine rice yield. Grain formation is a complex biological process, whose molecular mechanism is yet to be improved. Here we investigated the function of an OVATE family protein, Embryo Sac Development 1 (ESD1), in the regulation of seed setting rate in rice (Oryza sativa) by examining its loss-of-function mutants generated via clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated9 (Cas9) technology. ESD1 was predominantly expressed at Stage 6 of panicle development, especially in the ovules. esd1 mutants displayed reduced seed setting rates with normal stamen development and pollen tube growth but abnormal pistil group. Investigation of embryo sacs revealed that during the mitosis of functional megaspores, some egg cells degraded during differentiation in esd1 mutants, thereby hindering subsequent fertilization process and reducing seed setting rate. In addition, the transcriptional level of O. sativa anaphase-promoting complex 6, a reported embryo sac developing gene, was significantly reduced in esd1 mutants. These results support that ESD1 is an important modulator of ESD and seed setting rate in rice. Together, this finding demonstrates that ESD1 positively regulates the seed setting rate by controlling ESD in rice and has implications for the improvement of rice yield.

Gibberellins negatively modulate ovule number in plants.

Ovule formation is a complex developmental process in plants, with a strong impact on the production of seeds. Ovule primordia initiation is controlled by a gene network, including components of the signaling pathways of auxin, brassinosteroids and cytokinins. By contrast, gibberellins (GAs) and DELLA proteins, the negative regulators of GA signaling, have never been shown to be involved in ovule initiation. Here, we provide molecular and genetic evidence that points to DELLA proteins as novel players in the determination of ovule number in Arabidopsis and in species of agronomic interest, such as tomato and rapeseed, adding a new layer of complexity to this important developmental process. DELLA activity correlates positively with ovule number, acting as a positive factor for ovule initiation. In addition, ectopic expression of a dominant DELLA in the placenta is sufficient to increase ovule number. The role of DELLA proteins in ovule number does not appear to be related to auxin transport or signaling in the ovule primordia. Possible crosstalk between DELLA proteins and the molecular and hormonal network controlling ovule initiation is also discussed.

Ovule identity mediated by pre-mRNA processing in Arabidopsis.

Ovules are fundamental for plant reproduction and crop yield as they are the precursors of seeds. Therefore, ovule specification is a critical developmental program. In Arabidopsis thaliana, ovule identity is redundantly conferred by the homeotic D-class genes SHATTERPROOF1 (SHP1), SHP2 and SEEDSTICK (STK), phylogenetically related to the MADS-domain regulatory gene AGAMOUS (AG), essential in floral organ specification. Previous studies have shown that the HUA-PEP activity, comprised of a suite of RNA-binding protein (RBP) encoding genes, regulates AG pre-mRNA processing and thus flower patterning and organ identity. Here, we report that the HUA-PEP activity additionally governs ovule morphogenesis. Accordingly, in severe hua-pep backgrounds ovules transform into flower organ-like structures. These homeotic transformations are most likely due to the dramatic reduction in SHP1, SHP2 and STK activity. Our molecular and genome-wide profiling strategies revealed the accumulation of prematurely terminated transcripts of D-class genes in hua-pep mutants and reduced amounts of their respective functional messengers, which points to pre-mRNA processing misregulation as the origin of the ovule developmental defects in such backgrounds. RNA processing and transcription are coordinated by the RNA polymerase II (RNAPII) carboxyl-terminal domain (CTD). Our results show that HUA-PEP activity members can interact with the CTD regulator C-TERMINAL DOMAIN PHOSPHATASE-LIKE1 (CPL1), supporting a co-transcriptional mode of action for the HUA-PEP activity. Our findings expand the portfolio of reproductive developmental programs in which HUA-PEP activity participates, and further substantiates the importance of RNA regulatory mechanisms (pre-mRNA co-transcriptional regulation) for correct gene expression during plant morphogenesis.

Cytokinin response factors integrate auxin and cytokinin pathways for female reproductive organ development.

The developmental programme of the pistil is under the control of both auxin and cytokinin. Crosstalk between these factors converges on regulation of the auxin carrier PIN-FORMED 1 (PIN1). Here, we show that in the triple transcription factor mutant cytokinin response factor 2 (crf2) crf3 crf6 both pistil length and ovule number were reduced. PIN1 expression was also lower in the triple mutant and the phenotypes could not be rescued by exogenous cytokinin application. pin1 complementation studies using genomic PIN1 constructs showed that the pistil phenotypes were only rescued when the PCRE1 domain, to which CRFs bind, was present. Without this domain, pin mutants resemble the crf2 crf3 crf6 triple mutant, indicating the pivotal role of CRFs in auxin-cytokinin crosstalk.

Effects of High Temperature on Embryological Development and Hormone Profile in Flowers and Leaves of Common Buckwheat (Fagopyrum esculentum Moench).

Common buckwheat is a valuable crop, mainly due to the beneficial chemical composition of its seeds. However, buckwheat cultivation is limited because of unstable seed yield. The most important reasons for the low yield include embryo and flower abortion. The aim of this work is to verify whether high temperature affects embryological development in this plant species. The experiment was conducted on plants of a Polish cultivar 'Panda' and strain PA15, in which the percentage of degenerating embryo sacs was previously determined and amounted to 32% and 10%, respectively. The plants were cultivated in phytotronic conditions at 20 °C (control), and 30 °C (thermal stress). The embryological processes and hormonal profiles in flowers at various developmental stages (buds, open flowers, and wilted flowers) and in donor leaves were analyzed in two-month-old plants. Significant effects of thermal stress on the defective development of female gametophytes and hormone content in flowers and leaves were observed. Ovules were much more sensitive to high temperature than pollen grains in both genotypes. Pollen viability remained unaffected at 30 °C in both genotypes. The effect of temperature on female gametophyte development was visible in cv. Panda but not in PA15 buds. A drastic reduction in the number of properly developed embryo sacs was clear in open flowers at 30 °C in both genotypes. A considerable increase in abscisic acid in open flowers ready for fertilization may serve as a signal inducing flower senescence observed in the next few days. Based on embryological analyses and hormone profiles in flowers, we conclude that cv. 'Panda' is more sensitive to thermal stress than strain PA15, mainly due to a much earlier response to thermal stress involving impairment of embryological processes already in the flower buds.

An Arabidopsis divergent pumilio protein, APUM24, is essential for embryogenesis and required for faithful pre-rRNA processing.

Pumilio RNA-binding proteins are largely involved in mRNA degradation and translation repression. However, a few evolutionarily divergent Pumilios are also responsible for proper pre-rRNA processing in human and yeast. Here, we describe an essential Arabidopsis nucleolar Pumilio, APUM24, that is expressed in tissues undergoing rapid proliferation and cell division. A T-DNA insertion for APUM24 did not affect the male and female gametogenesis, but instead resulted in a negative female gametophytic effect on zygotic cell division immediately after fertilization. Additionally, the mutant embryos displayed defects in cell patterning from pro-embryo through globular stages. The mutant embryos were marked by altered auxin maxima, which were substantiated by the mislocalization of PIN1 and PIN7 transporters in the defective embryos. Homozygous apum24 callus accumulates rRNA processing intermediates, including uridylated and adenylated 5.8S and 25S rRNA precursors. An RNA-protein interaction assay showed that the histidine-tagged recombinant APUM24 binds RNAin vitro with no apparent specificity. Overall, our results demonstrated that APUM24 is required for rRNA processing and early embryogenesis in Arabidopsis.

Transgenerational phenotype aggravation in CAF-1 mutants reveals parent-of-origin specific epigenetic inheritance.

Chromatin is assembled by histone chaperones such as chromatin assembly factor CAF-1. We had noticed that vigor of Arabidopsis thaliana CAF-1 mutants decreased over several generations. Because changes in mutant phenotype severity over generations are unusual, we asked how repeated selfing of Arabidopsis CAF-1 mutants affects phenotype severity. CAF-1 mutant plants of various generations were grown, and developmental phenotypes, transcriptomes and DNA cytosine-methylation profiles were compared quantitatively. Shoot- and root-related growth phenotypes were progressively more affected in successive generations of CAF-1 mutants. Early and late generations of the fasciata (fas)2-4 CAF-1 mutant displayed only limited changes in gene expression, of which increasing upregulation of plant defense-related genes reflects the transgenerational phenotype aggravation. Likewise, global DNA methylation in the sequence context CHG but not CG or CHH (where H = A, T or C) changed over generations in fas2-4. Crossing early and late generation fas2-4 plants established that the maternal contribution to the phenotype severity exceeds the paternal contribution. Together, epigenetic rather than genetic mechanisms underlie the progressive developmental phenotype aggravation in the Arabidopsis CAF-1 mutants and preferred maternal transmission reveals a more efficient reprogramming of epigenetic information in the male than the female germline.

A parthenogenesis gene of apomict origin elicits embryo formation from unfertilized eggs in a sexual plant.

Apomixis is a naturally occurring mode of asexual reproduction in flowering plants that results in seed formation without the involvement of meiosis or fertilization of the egg. Seeds formed on an apomictic plant contain offspring genetically identical to the maternal plant. Apomixis has significant potential for preserving hybrid vigor from one generation to the next in highly productive crop plant genotypes. Apomictic Pennisetum/Cenchrus species, members of the Poaceae (grass) family, reproduce by apospory. Apospory is characterized by apomeiosis, the formation of unreduced embryo sacs derived from nucellar cells of the ovary and, by parthenogenesis, the development of the unreduced egg into an embryo without fertilization. In Pennisetum squamulatum (L.) R.Br., apospory segregates as a single dominant locus, the apospory-specific genomic region (ASGR). In this study, we demonstrate that the PsASGR-BABY BOOM-like (PsASGR-BBML) gene is expressed in egg cells before fertilization and can induce parthenogenesis and the production of haploid offspring in transgenic sexual pearl millet. A reduction of PsASGR-BBML expression in apomictic F1 RNAi transgenic plants results in fewer visible parthenogenetic embryos and a reduction of embryo cell number compared with controls. Our results endorse a key role for PsASGR-BBML in parthenogenesis and a newly discovered role for a member of the BBM-like clade of APETALA 2 transcription factors. Induction of parthenogenesis by PsASGR-BBML will be valuable for installing parthenogenesis to synthesize apomixis in crops and will have further application for haploid induction to rapidly obtain homozygous lines for breeding.

Gibberellins Regulate Ovule Integument Development by Interfering with the Transcription Factor ATS.

Gibberellins (GAs) are plant hormones that regulate most plant life cycle aspects, including flowering and fruit development. Here, we demonstrate the implication of GAs in ovule development. DELLA proteins, negative GA response regulators, act as positive factors for ovule integument development in a mechanism that involves transcription factor ABERRANT TESTA SHAPE (ATS). The seeds of the della global mutant, a complete loss-of-function of DELLA, and the ats-1 mutant are remarkably similar, with a round shape, a disorganized testa, and viviparism. These defects are the result of an alteration in integuments that fail to fully develop and are shorter than in wild-type plants. ats-1 also shows some GA-related phenotypes, for example, higher germination rates and early flowering. In fact, ats-1 has elevated GA levels due to the activation of GA biosynthesis genes, which indicates that ATS inhibits GA biosynthesis. Moreover, DELLAs and ATS proteins interact, which suggests the formation of a transcriptional complex that regulates the expression of genes involved in integument growth. Therefore, the repression of GA biosynthesis by ATS would result in the stabilization of DELLAs to ensure correct ATS-DELLA complex formation. The requirement of both activities to coordinate proper ovule development strongly argues that the ATS-DELLA complex acts as a key molecular factor. This work provides the first evidence for a role of GAs in ovule and seed development.

Activation of CYCD7;1 in the central cell and early endosperm overcomes cell-cycle arrest in the Arabidopsis female gametophyte, and promotes early endosperm and embryo development.

In angiosperms, double fertilization of the egg and central cell of the megagametophyte leads to the development of the embryo and endosperm, respectively. Control of cell cycle progression in the megagametophyte is essential for successful fertilization and development. Central cell-targeted expression of the D-type cyclin CYCD7;1 (end CYCD7;1) using the imprinted FWA promoter overcomes cycle arrest of the central cell in the Arabidopsis female gametophyte in the unfertilized ovule, leading to multinucleate central cells at high frequency. Unlike FERTILIZATION-INDEPENDENT SEED (fis) mutants, but similar to lethal RETINOBLASTOMA-RELATED (rbr) mutants, no seed coat development is triggered. Unlike the case with loss of rbr, post-fertilization end CYCD7;1 in the endosperm enhances the number of nuclei during syncytial endosperm development and induces the partial abortion of developing seeds, associated with the enhanced size of the surviving seeds. The frequency of lethality was less than the frequency of multinucleate central cells, indicating that these aspects are not causally linked. These larger seeds contain larger embryos composed of more cells of wild-type size, surrounded by a seed coat composed of more cells. Seedlings arising from these larger seeds displayed faster seedling establishment and early growth. Similarly, two different embryo-lethal mutants also conferred enlarged seed size in surviving siblings, consistent with seed size increase being a general response to sibling lethality, although the cellular mechanisms were found to be distinct. Our data suggest that tight control of CYCD activity in the central cell and in the developing endosperm is required for optimal seed formation.

Embryology of Cardiopteris (Cardiopteridaceae, Aquifoliales), with emphasis on unusual ovule and seed development.

Cardiopteris (Cardiopteridaceae), a twining herb of two or three species distributed from Southeast Asia to Northern Australia, requires an embryological study for better understanding of its reproductive features. The present study of C. quinqueloba showed that the ovule and seed development involves a number of unusual structures, most of which are unknown elsewhere in angiosperms. The ovule pendant from the apical placenta is straight (not orthotropous), ategmic, and tenuinucellate, developing a monosporic seven-celled/eight-nucleate female gametophyte with an egg apparatus on the funicular side. Fertilization occurs by a pollen tube entering from the funicular side, resulting in a zygote on the funicular side. The endosperm is formed by the cell on the funicular side in the two endosperm cell stage. While retaining a (pro)embryo/endosperm as it is, the raphe (differentiating late in pre-fertilization stages) elongates toward the antiraphal side during post-fertilization stages, resulting in an anatropous seed. The two-cell-layered nucellar epidermis (belatedly forming by periclinal divisions), along with the raphe, envelops the embryo/endosperm entirely as the seed coat. The possibility was discussed that the arrested integument development triggers a series of the subsequent unusual structures of ovule and seed development. The fertilization mode in Cardiopteris underpins the hypothesis that the Polygonum‒type female gametophyte comprises two four-celled archegonia.

An integrative model of the control of ovule primordia formation.

Upon hormonal signaling, ovules develop as lateral organs from the placenta. Ovule numbers ultimately determine the number of seeds that develop, and thereby contribute to the final seed yield in crop plants. We demonstrate here that CUP-SHAPED COTYLEDON 1 (CUC1), CUC2 and AINTEGUMENTA (ANT) have additive effects on ovule primordia formation. We show that expression of the CUC1 and CUC2 genes is required to redundantly regulate expression of PINFORMED1 (PIN1), which in turn is required for ovule primordia formation. Furthermore, our results suggest that the auxin response factor MONOPTEROS (MP/ARF5) may directly bind ANT, CUC1 and CUC2 and promote their transcription. Based on our findings, we propose an integrative model to describe the molecular mechanisms of the early stages of ovule development.

Patterning of the angiosperm female gametophyte through the prism of theoretical paradigms.

The FG (female gametophyte) of flowering plants (angiosperms) is a simple highly polar structure composed of only a few cell types. The FG develops from a single cell through mitotic divisions to generate, depending on the species, four to 16 nuclei in a syncytium. These nuclei are then partitioned into three or four distinct cell types. The mechanisms underlying the specification of the nuclei in the FG has been a focus of research over the last decade. Nevertheless, we are far from understanding the patterning mechanisms that govern cell specification. Although some results were previously interpreted in terms of static positional information, several lines of evidence now show that local interactions are important. In the present article, we revisit the available data on developmental mutants and cell fate markers in the light of theoretical frameworks for biological patterning. We argue that a further dissection of the mechanisms may be impeded by the combinatorial and dynamical nature of developmental cues. However, accounting for these properties of developing systems is necessary to disentangle the diversity of the phenotypic manifestations of the underlying molecular interactions.

The emerging importance of type I MADS box transcription factors for plant reproduction.

Based on their evolutionary origin, MADS box transcription factor genes have been divided into two classes, namely, type I and II. The plant-specific type II MIKC MADS box genes have been most intensively studied and shown to be key regulators of developmental processes, such as meristem identity, flowering time, and fruit and seed development. By contrast, very little is known about type I MADS domain transcription factors, and they have not attracted interest for a long time. A number of recent studies have now indicated a key regulatory role for type I MADS box factors in plant reproduction, in particular in specifying female gametophyte, embryo, and endosperm development. These analyses have also suggested that type I MADS box factors are decisive for setting reproductive boundaries between species.

[Embryo initiation from Pinus sibirica megagametophytes in in vitro culture].

Megagametophytes of Siberian pine were cultured on an in vitro culture medium 1/2 LV supplemented with growth regulators 2,4-dichlorophenoxyacetic acid (2,4-D) and benzylaminopurine (6-BAP) to form embryos. The competency of somatic cell of explants to embryogenesis manifested itself in an organized growth and polarity. A coenocyte consisting of long vacuolated cells was formed in the megagametophyte culture. Then, the migration of the nuclei to one of the poles of the cell, their division, and formation of embryoids was observed. The megagametophyte culture of the Siberian pine differed from the zygotic embryo culture by the absence of asymmetric division in the vacuolated cell.

The Arabidopsis APC4 subunit of the anaphase-promoting complex/cyclosome (APC/C) is critical for both female gametogenesis and embryogenesis.

The anaphase-promoting complex/cyclosome (APC/C) is an E3 ubiquitin ligase that is involved in regulating cell-cycle progression. It has been widely studied in yeast and animal cells, but the function and regulation of the APC/C in plant cells are largely unknown. The Arabidopsis APC/C comprises at least 11 subunits, only a few of which have been studied in detail. APC4 is proposed to be a connector in the APC/C in yeast and animals. Here, we report the functional characterization of the Arabidopsis APC4 protein. We examined three heterozygous plant lines carrying apc4 alleles. These plants showed pleiotropic developmental defects in reproductive processes, including abnormal nuclear behavior in the developing embryo sac and aberrant cell division in embryos; these phenotypes differ from those reported for mutants of other subunits. Some ovules and embryos of apc4/+ plants also accumulated cyclin B protein, a known substrate of APC/C, suggesting a compromised function of APC/C. Arabidopsis APC4 was expressed in meristematic cells of seedlings, ovules in pistils and embryos in siliques, and was mainly localized in the nucleus. Additionally, the distribution of auxin was distorted in some embryos of apc4/+ plants. Our results indicate that Arabidopsis APC4 plays critical roles in female gametogenesis and embryogenesis, possibly as a connector in APC/C, and that regulation of auxin distribution may be involved in these processes.

Apomictic and sexual ovules of Boechera display heterochronic global gene expression patterns.

We have compared the transcriptomic profiles of microdissected live ovules at four developmental stages between a diploid sexual and diploid apomictic Boechera. We sequenced >2 million SuperSAGE tags and identified (1) heterochronic tags (n = 595) that demonstrated significantly different patterns of expression between sexual and apomictic ovules across all developmental stages, (2) stage-specific tags (n = 577) that were found in a single developmental stage and differentially expressed between the sexual and apomictic ovules, and (3) sex-specific (n = 237) and apomixis-specific (n = 1106) tags that were found in all four developmental stages but in only one reproductive mode. Most heterochronic and stage-specific tags were significantly downregulated during early apomictic ovule development, and 110 were associated with reproduction. By contrast, most late stage-specific tags were upregulated in the apomictic ovules, likely the result of increased gene copy number in apomictic (hexaploid) versus sexual (triploid) endosperm or of parthenogenesis. Finally, we show that apomixis-specific gene expression is characterized by a significant overrepresentation of transcription factor activity. We hypothesize that apomeiosis is associated with global downregulation at the megaspore mother cell stage. As the diploid apomict analyzed here is an ancient hybrid, these data are consistent with the postulated link between hybridization and asexuality and provide a hypothesis for multiple evolutionary origins of apomixis in the genus Boechera.

Arabidopsis histidine kinase CKI1 acts upstream of histidine phosphotransfer proteins to regulate female gametophyte development and vegetative growth.

Cytokinin signaling is mediated by a multiple-step phosphorelay. Key components of the phosphorelay consist of the histidine kinase (HK)-type receptors, histidine phosphotransfer proteins (HP), and response regulators (RRs). Whereas overexpression of a nonreceptor-type HK gene CYTOKININ-INDEPENDENT1 (CKI1) activates cytokinin signaling by an unknown mechanism, mutations in CKI1 cause female gametophytic lethality. However, the function of CKI1 in cytokinin signaling remains unclear. Here, we characterize a mutant allele, cki1-8, that can be transmitted through female gametophytes with low frequency (approximately 0.17%). We have recovered viable homozygous cki1-8 mutant plants that grow larger than wild-type plants, show defective megagametogenesis and rarely set enlarged seeds. We found that CKI1 acts upstream of AHP (Arabidopsis HP) genes, independently of cytokinin receptor genes. Consistently, an ahp1,2-2,3,4,5 quintuple mutant, which contains an ahp2-2 null mutant allele, exhibits severe defects in megagametogenesis, with a transmission efficiency of <3.45% through female gametophytes. Rarely recovered ahp1,2-2,3,4,5 quintuple mutants are seedling lethal. Finally, the female gametophytic lethal phenotype of cki1-5 (a null mutant) can be partially rescued by IPT8 or ARR1 (a type-B Arabidopsis RR) driven by a CKI1 promoter. These results define a genetic pathway consisting of CKI1, AHPs, and type-B ARRs in the regulation of female gametophyte development and vegetative growth.

VERDANDI is a direct target of the MADS domain ovule identity complex and affects embryo sac differentiation in Arabidopsis.

In Arabidopsis thaliana, the three MADS box genes SEEDSTICK (STK), SHATTERPROOF1 (SHP1), and SHP2 redundantly regulate ovule development. Protein interaction studies have shown that a multimeric complex composed of the ovule identity proteins together with the SEPALLATA MADS domain proteins is necessary to determine ovule identity. Despite the extensive knowledge that has become available about these MADS domain transcription factors, little is known regarding the genes that they regulate. Here, we show that STK, SHP1, and SHP2 redundantly regulate VERDANDI (VDD), a putative transcription factor that belongs to the plant-specific B3 superfamily. The vdd mutant shows defects during the fertilization process resulting in semisterility. Analysis of the vdd mutant female gametophytes indicates that antipodal and synergid cell identity and/or differentiation are affected. Our results provide insights into the pathways regulated by the ovule identity factors and the role of the downstream target gene VDD in female gametophyte development.

Factors influencing fecundity in experimental crosses of water lotus (Nelumbo nucifera Gaertn.) cultivars.

BACKGROUND: Breeding programs for the water lotus (Nelumbo nucifera) are hampered by an inability to account for variation in seed set associated with crosses between different cultivars. We studied seed set in two reciprocal crosses between lotus cultivars ('Guili' × 'Aijiangnan' and 'Molingqiuse' × 'Qinhuaiyanzhi') to obtain insights into factors that govern fecundity in these experimental hybrids. Pollen viability, stigma receptivity and embryo development were compared for each hybrid and reciprocal cross. RESULTS: Pollen viability of the individual cultivars ranged from 4.1% to 20.2%, with the highest level (>11.9%) for all cultivars observed from the earliest collected grains (05:00-06:00 a.m.). Stigmatic pollen germination peaked at 4 h after pollination and varied from 4.8 to 60.6 grains per stigma among the crosses. Production of normal embryos ranged from 7.6% to 58.8% at 1 d after pollination and from 0 to 25% by 11 d after pollination. Seed set in crosses (0.2-23.3%) was generally lower than in open-pollinated plants (8.4-26.5%). Similar to the germination results, seed set was substantially reduced in both reciprocal crosses. CONCLUSIONS: These results suggested that poor pollen fertility, low stigma receptivity, and embryo abortion were responsible for the failure of the crosses 'Molingqiuse' × 'Qinhuaiyanzhi', 'Qinhuaiyanzhi' × 'Molingqiuse', and 'Aijiangnan' × 'Guili'.