Mesozoic cupules and the origin of the angiosperm second integument.

The second integument of the angiosperm ovule is unique among seed plants, with developmental genetics that are distinct from those of the inner integument1. Understanding how the second integument should be compared to structures in other seed plants is therefore crucial to resolving the long-standing question of the origin of angiosperms2-6. Attention has focused on several extinct plants with recurved cupules that are reminiscent of the anatropous organization of the basic bitegmic ovules of angiosperms1-6, but interpretations have been hampered by inadequate information on the relevant fossils. Here we describe abundant exceptionally well-preserved recurved cupules from a newly discovered silicified peat dating to the Early Cretaceous epoch (around 125.6 million years ago) in Inner Mongolia, China. The new material, combined with re-examination of potentially related fossils, indicates that the recurved cupules of several groups of Mesozoic plants are all fundamentally comparable, and that their structure is consistent with the recurved form and development of the second integument in the bitegmic anatropous ovules of angiosperms. Recognition of these angiosperm relatives (angiophytes) provides a partial answer to the question of angiosperm origins, will help to focus future work on seed plant phylogenetics and has important implications for ideas on the origin of the angiosperm carpel.

Formins control dynamics of F-actin in the central cell of Arabidopsis thaliana.

In the female gamete of flowering plants, sperm nuclear migration is controlled by a constant inward movement of actin filaments (F-actin) for successful fertilization. This dynamic F-actin movement is ARP2/3-independent, raising the question of how actin nucleation and polymerization is controlled in the female gamete. Using confocal microscopy live-cell imaging in combination with a pharmacological approach, we assessed the involvement of another group of actin nucleators, formins, in F-actin inward movement in the central cell of Arabidopsis thaliana. We identify that the inhibition of the formin function, by formin inhibitor SMIFH2, significantly reduced the dynamic inward movement of F-actin in the central cell, indicating that formins play a major role in actin nucleation required for F-actin inward movement in the central cell.

Ultrastructural analysis of Rhynchospora ovules: The first record of Cyperaceae megagametophyte on transmission electron microscope.

Microsporogenesis and microgametogenesis are unusual in sedges (Cyperaceae), the third largest monocotyledonous family, as three microspores are aborted in favor of a single functional microspore. However, studies using light microscopy show that megasporogenesis and megagametogenesis occur normally. Nevertheless, the lack of ultrastructural details limits our knowledge of female gametophyte development in this family. Given the importance of morphological studies of reproductive structures, ovules and megagametophytes of Rhynchospora pubera were analyzed under transmission electron microscopy for the first time. Overall, ovules presented features similar to those described for the family, but ultrastructural details revealed an absence of a clear boundary between the egg cell and the central cell cytoplasm. Most interestingly, antipodal and nucellar cells showed several signs of vacuolar cell death, which suggest that programmed autolysis in sporogenous and gametophytic tissue is common in gametophyte development in the Cyperaceae. This may be related to the reproductive success of this family.

Arabidopsis KETCH1 Is Critical for the Nuclear Accumulation of Ribosomal Proteins and Gametogenesis.

Male and female gametophytes are generated from micro- or megaspore mother cells through consecutive meiotic and mitotic cell divisions. Defects in these divisions often result in gametophytic lethality. Gametophytic lethality was also reported when genes encoding ribosome-related proteins were mutated. Although numerous ribosomal proteins (RPs) have been identified in plants based on homology with their yeast and metazoan counterparts, how RPs are regulated, e.g., through dynamic subcellular targeting, is unknown. We report here that an Arabidopsis (Arabidopsis thaliana) importin ß, KETCH1 (karyopherin enabling the transport of the cytoplasmic HYL1), is critical for gametogenesis. Karyopherins are molecular chaperones mediating nucleocytoplasmic protein transport. However, the role of KETCH1 during gametogenesis is independent of HYPONASTIC LEAVES 1 (HYL1), a previously reported KETCH1 cargo. Instead, KETCH1 interacts with several RPs and is critical for the nuclear accumulation of RPL27a, whose mutations caused similar gametophytic defects. We further showed that knocking down KETCH1 caused reduced ribosome biogenesis and translational capacity, which may trigger the arrest of mitotic cell cycle progression and lead to gametophytic lethality.

Comparative gene expression profile analysis of ovules provides insights into Jatropha curcas L. ovule development.

Jatropha curcas, an economically important biofuel feedstock with oil-rich seeds, has attracted considerable attention among researchers in recent years. Nevertheless, valuable information on the yield component of this plant, particularly regarding ovule development, remains scarce. In this study, transcriptome profiles of anther and ovule development were established to investigate the ovule development mechanism of J. curcas. In total, 64,325 unigenes with annotation were obtained, and 1723 differentially expressed genes (DEGs) were identified between different stages. The DEG analysis showed the participation of five transcription factor families (bHLH, WRKY, MYB, NAC and ERF), five hormone signaling pathways (auxin, gibberellic acid (GA), cytokinin, brassinosteroids (BR) and jasmonic acid (JA)), five MADS-box genes (AGAMOUS-2, AGAMOUS-1, AGL1, AGL11, and AGL14), SUP and SLK3 in ovule development. The role of GA and JA in ovule development was evident with increases in flower buds during ovule development: GA was increased approximately twofold, and JA was increased approximately sevenfold. In addition, the expression pattern analysis using qRT-PCR revealed that CRABS CLAW and AGAMOUS-2 were also involved in ovule development. The upregulation of BR signaling genes during ovule development might have been regulated by other phytohormone signaling pathways through crosstalk. This study provides a valuable framework for investigating the regulatory networks of ovule development in J. curcas.

Identification and profiling of upland cotton microRNAs at fiber initiation stage under exogenous IAA application.

BACKGROUND: Cotton is the most essential textile crop worldwide, and phytohormones are critical for cotton fiber development. One example is the role of auxin in fiber initiation, but we know little molecular basis. MicroRNAs (miRNAs) have a significant function in cotton development; nevertheless their role in fiber initiation remains unclear. Here, exogenous IAA was applied to cotton plant before anthesis. Utilizing small RNA sequencing, the mechanism underlying miRNA-mediated regulation of fiber initiation under exogenous IAA treatment was investigated. RESULTS: With exogenous IAA application, the endogenous IAA and GA contents of IAA treated (IT) ovules were higher than control (CK) ovules at the fiber initiation stage, while endogenous ABA content was lower in IT than CK. Using scanning electron microscopy, we found the fiber number and size were significantly promoted in IT at 0 DPA. Fiber quality analysis showed that fiber length, uniformity, strength, elongation, and micronaire of IT were higher than CK, though not statistically significant, while lint percent was significantly higher in IT. We generated six small RNA libraries using - 3, 0, and 3 DPA ovules of IT and CK, and identified 58 known miRNAs and 83 novel miRNAs together with the target genes. The differential expressed miRNAs number between IT and CK at - 3, 0, 3 DPA was 34, 16 and 24, respectively. Gene ontology and KEGG pathway enrichment analyses for the target genes of the miRNAs expressed in a differential manner showed that they were significantly enriched in 30 terms and 8 pathways. QRT-PCR for those identified miRNAs and the target genes related to phytohormones and fiber development was performed, and results suggested a potential role of these miRNAs in fiber initiation. CONCLUSIONS: The exogenous IAA application affected the relative phytohormone contents in ovule and promoted fiber initiation in cotton. Identification and profiling of miRNAs and their targets at the fiber initiation stage provided insights for miRNAs' regulation function of fiber initiation. These findings not only shed light on the regulatory network of fiber growth but also offer clues for cotton fiber amelioration strategies in cotton.

Sex change in kiwifruit (Actinidia chinensis Planch.): a developmental framework for the bisexual to unisexual floral transition.

KEY MESSAGE: The developmental morphology of male and female kiwifruit flowers is tracked to delimit a framework of events to aid the study of divergence in floral gene expression. The transition from hermaphrodite to unisexual development of kiwifruit (Actinidia chinensis Planch) flowers has been reported previously, but differences in gene expression controlling sexual development for this species have not been associated with the major developmental changes occurring within pistils. We investigated the key stages in male and female flower development to define the point at which meristematic activities diverge in the two sexes. A combination of scanning electron microscopy and light microscopy was used to investigate pistil development from the earliest stages. We identified seven distinct stages characterized by differences in ovary size and shape, macrosporogenesis, ovule primordium development, anther locule lengthening, microspore wall thickening, and pollen degeneration. Sex differences were evident from the initial stage of development, with a laterally compacted gynoecium in male flowers. However, the key developmental stage, at which tissue differentiation clearly deviated between the two sexes, was stage 3, when flowers were 3.5 to 4.5 mm in length at approximately 10 d from initiation of stamen development. At this stage, male flowers lacked evident carpel meristem development as denoted by a lack of ovule primordium formation. Pollen degeneration in female flowers, probably driven by programmed cell death, occurred at the late stage 6, while the final stage 7 was represented by pollen release. As the seven developmental stages are associated with specific morphological differences, including flower size, the scheme suggested here can provide the required framework for the future study of gene expression during the regulation of flower development in this crop species.

OsPINOID Regulates Stigma and Ovule Initiation through Maintenance of the Floral Meristem by Auxin Signaling.

Stigma and ovule initiation is essential for sexual reproduction in flowering plants. However, the mechanism underlying the initiation of stigma and ovule primordia remains elusive. We identified a stigma-less mutant of rice (Oryza sativa) and revealed that it was caused by the mutation in the PINOID (OsPID) gene. Unlike the pid mutant that shows typical pin-like inflorescences in maize (Zea mays) and Arabidopsis (Arabidopsis thaliana), the ospid mutant does not display any defects in inflorescence development and flower initiation, and fails to develop normal ovules in most spikelets. The auxin activity in the young pistil of ospid was lower than that in the wild-type pistil. Furthermore, the expression of most auxin response factor genes was down-regulated, and OsETTIN1, OsETTIN2, and OsMONOPTEROS lost their rearrangements of expression patterns during pistil and stamen primordia development in ospid Moreover, the transcription of the floral meristem marker gene, OSH1, was down-regulated and FLORAL ORGAN NUMBER4, the putative ortholog of Arabidopsis CLAVATA3, was up-regulated in the pistil primordium of ospid These results suggested that the meristem proliferation in the pistil primordium might be arrested prematurely in ospid Based on these results, we propose that the OsPID-mediated auxin signaling pathway plays a crucial role in the regulation of rice stigma and ovule initiation by maintaining the floral meristem.

The role of the integuments in pollen tube guidance in flowering plants.

In angiosperms, pollen tube entry into the ovule generally takes place through the micropyle, but the exact role of the micropyle in pollen tube guidance remains unclear. A limited number of studies have examined eudicots with bitegmic micropyles, but information is lacking in ovules of basal/early-divergent angiosperms with unitegmic micropyles. We have evaluated the role of the micropyle in pollen tube guidance in an early-divergent angiosperm (Annona cherimola) and the evolutionarily derived Arabidopsis thaliana by studying γ-aminobutyric acid (GABA) and arabinogalactan proteins (AGPs) in wild-type plants and integument-defective mutants. A conserved inhibitory role of GABA in pollen tube growth was shown in A. cherimola, in which AGPs surround the egg apparatus. In Arabidopsis, the micropyle formed only by the outer integument in wuschel-7 mutants caused a partial defect in pollen tube guidance. Moreover, pollen tubes were not observed in the micropyle of an inner no outer (ino) mutant in Arabidopsis, but were observed in homologous ino mutants in Annona. The similar distribution of GABA and AGPs observed in the micropyle of Arabidopsis and Annona, together with the anomalies from specific integument mutants, support the role of the inner integument in preventing multiple tube entrance (polytubey) in these two phylogenetically distant genera.

Arabidopsis PIRL6 Is Essential for Male and Female Gametogenesis and Is Regulated by Alternative Splicing.

Plant intracellular Ras-group leucine-rich repeat (LRR) proteins (PIRLs) are related to Ras-interacting animal LRR proteins that participate in developmental cell signaling. Systematic knockout analysis has implicated some members of the Arabidopsis (Arabidopsis thaliana) PIRL family in pollen development. However, for PIRL6, no bona fide knockout alleles have been recovered, suggesting that it may have an essential function in both male and female gametophytes. To test this hypothesis, we investigated PIRL6 expression and induced knockdown by RNA interference. Knockdown triggered defects in gametogenesis, resulting in abnormal pollen and early developmental arrest in the embryo sac. Consistent with this, PIRL6 was expressed in gametophytes: functional transcripts were detected in wild-type flowers but not in sporocyteless (spl) mutant flowers, which do not produce gametophytes. A genomic PIRL6-GFP fusion construct confirmed expression in both pollen and the embryo sac. Interestingly, PIRL6 is part of a convergent overlapping gene pair, a scenario associated with an increased likelihood of alternative splicing. We detected multiple alternative PIRL6 mRNAs in vegetative organs and spl mutant flowers, tissues that lacked the functionally spliced transcript. cDNA sequencing revealed that all contained intron sequences and premature termination codons. These alternative mRNAs accumulated in the nonsense-mediated decay mutant upf3, indicating that they are normally subjected to degradation. Together, these results demonstrate that PIRL6 is required in both male and female gametogenesis and suggest that sporophytic expression is negatively regulated by unproductive alternative splicing. This posttranscriptional mechanism may function to minimize PIRL6 protein expression in sporophyte tissues while allowing the overlapping adjacent gene to remain widely transcribed.

Egg activation-triggered shape change in the Dictyota dichotoma (Phaeophyceae) zygote is actin-myosin and secretion dependent.

Background and Aims: Cellular morphogenesis in land plants and brown algae is typically a slow process involving growth established by an interplay of turgor pressure and cell wall rigidity. However, a recent study showed that zygotes of the brown alga Dictyota dichotoma undergo a rapid shape change from a sphere to an elongated spheroid in about 90 s, establishing the first body axis. Methods: Using a combination of pharmacology, staining techniques, membrane depolarization and microscopy techniques (brightfield, transmission electron microscopy and confocal laser scanning microscopy), egg activation and the shape change of the egg cell of D. dichotoma was studied. Key Results: It was established that elongation of the zygote does not involve growth, i.e. a positive change in size. The elongation is dependent on F-actin and myosin but independent of microtubules. Secretion was also found to be necessary for elongation after addition of brefeldin A. Moreover, a temporal correlation between extracellular matrix secretion and elongation was observed. Ionomycin and high potassium seawater are capable of triggering the onset of elongation, suggesting a role for membrane depolarization and calcium influx in the signalling mechanism. The elongated cells are shorter in the presence of ionomycin, suggesting a role for calcium in elongation. Conclusions: A model is proposed in which the fast elongation of the fertilized egg in Dictyota is accomplished by a force generated by F-actin and myosin, regulated by cytoplasmic calcium concentrations and by secretion during elongation lowering the antagonistic force. The finding of early extracellular matrix secretion, membrane depolarization and ionophore-triggered egg activation suggest significant differences in the mechanism of egg activation signalling between D. dichotoma and the oogamous brown algal model system Fucus .

Immunodetection of some pectic, arabinogalactan proteins and hemicellulose epitopes in the micropylar transmitting tissue of apomictic dandelions (Taraxacum, Asteraceae, Lactuceae).

In apomictic Taraxacum species, the development of both the embryo and the endosperm does not require double fertilisation. However, a structural reduction of ovular transmitting tissue was not observed in apomictic dandelions. The aim of this study was to analyse the chemical composition of the cell walls to describe the presence of arabinogalactan proteins (AGPs), hemicellulose and some pectic epitopes in the micropylar transmitting tissue of apomictic Taraxacum. The results point to (1) the similar distribution of AGPs in different developmental stages, (2) the absence of highly methyl-esterified homogalacturonan (HG) in transmitting tissue of ovule containing a mature embryo sac and the appearance of this pectin domain in the young seed containing the embryo and endosperm, (3) the similar pattern of low methyl-esterified pectin occurrence in both an ovule and a young seed with an embryo and endosperm in apomictic Taraxacum and (4) the presence of hemicelluloses recognised by LM25 and LM21 antibodies in the reproductive structure of Taraxacum.

Arabidopsis phosphatidylinositol-phospholipase C2 (PLC2) is required for female gametogenesis and embryo development.

MAIN CONCLUSION: AtPLC2 is an essential gene in Arabidopsis, since it is required for female gametogenesis and embryo development. AtPLC2 might play a role in cell division during embryo-sac development and early embryogenesis. Phosphoinositide-specific phospholipase C (PI-PLC) plays an important role in signal transduction during plant development and in the response to various biotic- and abiotic stresses. The Arabidopsis PI-PLC gene family is composed of nine members, named PLC1 to PLC9. Here, we report that PLC2 is involved in female gametophyte development and early embryogenesis. Using two Arabidopsis allelic T-DNA insertion lines with different phenotypic penetrations, we observed both female gametophytic defects and aberrant embryos. For the plc2-1 mutant (Ws background), no homozygous plants could be recovered in the offspring from self-pollinated plants. Nonetheless, plc2-1 hemizygous mutants are affected in female gametogenesis, showing embryo sacs arrested at early developmental stages. Allelic hemizygous plc2-2 mutant plants (Col-0 background) present reduced seed set and embryos arrested at the pre-globular stage with abnormal patterns of cell division. A low proportion (0.8%) of plc2-2 homozygous mutants was found to escape lethality and showed morphological defects and disrupted megagametogenesis. PLC2-promoter activity was observed during early megagametogenesis, and after fertilization in the embryo proper. Immunolocalization studies in early stage embryos revealed that PLC2 is restricted to the plasma membrane. Altogether, these results establish a role for PLC2 in both reproductive- and embryo development, presumably by controlling mitosis and/or the formation of cell-division planes.

Gradual vs. abrupt reduction of carpels in syncarpous gynoecia: A case study from Polyscias subg. Arthrophyllum (Araliaceae: Apiales).

PREMISE OF THE STUDY: Revealing the relative roles of gradual and abrupt transformations of morphological characters is an important topic of evolutionary biology. Gynoecia apparently consisting of one carpel have evolved from pluricarpellate syncarpous gynoecia in several angiosperm clades. The process of reduction can involve intermediate stages, with one fertile and one or more sterile carpels (pseudomonomery). The possible origin of monomery directly via an abrupt change of gynoecium merism has been a matter of dispute. We explore the nature of gynoecium reduction in a clade of Araliaceae. METHODS: The anatomy and development of unilocular gynoecia are investigated using light and scanning electron microscopy in two members of Polyscias subg. Arthrophyllum. Gynoecium diversity in the genus is discussed in a phylogenetic framework. KEY RESULTS: Unilocular gynoecia with one fertile ovule have evolved at least four times in Polyscias, including one newly discovered case. The two unilocular taxa investigated are unicarpellate, without any traces of reduced sterile carpels. Carpel orientation is unstable, and the ovary roof and style contain numerous vascular bundles without clearly recognizable dorsals or ventrals. In contrast to pluricarpellate Araliaceae and Apiaceae, the cross zone is apparently oblique in the unicarpellate species. CONCLUSIONS: No support was found for gradual gynoecium reduction via pseudomonomery. The abrupt origin of monomery via direct change of gynoecium merism and the unstable carpel orientation observed are related to the general lability of the flower groundplan in Polyscias. The apparent occurrence of the unusual oblique cross zone in unicarpellate Araliaceae can be explained by developmental constraints.

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.

GhLTPG1, a cotton GPI-anchored lipid transfer protein, regulates the transport of phosphatidylinositol monophosphates and cotton fiber elongation.

The cotton fibers are seed trichomes that elongate from the ovule epidermis. Polar lipids are required for the quick enlargement of cell membrane and fiber cell growth, however, how lipids are transported from the ovules into the developing fibers remains less known. Here, we reported the functional characterization of GhLTPG1, a GPI-anchored lipid transport protein, during cotton fiber elongation. GhLTPG1 was abundantly expressed in elongating cotton fibers and outer integument of the ovules, and GhLTPG1 protein was located on cell membrane. Biochemical analysis showed that GhLTPG1 specifically bound to phosphatidylinositol mono-phosphates (PtdIns3P, PtdIns4P and PtdIns5P) in vitro and transported PtdInsPs from the synthesis places to the plasma membranes in vivo. Expression of GhLTPG1 in Arabidopsis caused an increased number of trichomes, and fibers in GhLTPG1-knockdown cotton plants exhibited significantly reduced length, decreased polar lipid content, and repression of fiber elongation-related genes expression. These results suggested that GhLTPG1 protein regulates the cotton fiber elongation through mediating the transport of phosphatidylinositol monophosphates.

Fluorescent whole-mount RNA in situ hybridization (F-WISH) in plant germ cells and the fertilized ovule.

First evidence on gene function and regulation is provided by the cellular expression pattern in complex tissues. However, to understand the activity of a specific gene, it is essential to analyze the regulatory network, which controls the spatio-temporal translation pattern during the entire life span of the transcribed mRNA. To explore mechanisms which control mRNA abundance and localization in space and time, it is necessary to visualize mRNAs quantitatively with a subcellular resolution, without sectioning the tissues. We have adapted and optimized a protocol for colorimetric whole-mount RNA in situ hybridization (WISH) using egg cell-specific digoxigenin (DIG) labeled probes (Hejátko et al., 2006) [1] on ovules and early seeds of Arabidopsis. Furthermore, we established a fluorescent whole-mount RNA in situ hybridization (F-WISH) protocol, which allows mRNA visualization on a subcellular level. The polar localized mRNA of SBT4.13, encoding a subtilase, was identified using this protocol. Both methods are described and discussed in detail. Additionally a (F)-WISH flow-chart is provided along with a troubleshooting table.

The role of OsMSH4 in male and female gamete development in rice meiosis.

Meiosis is essential for gametogenesis in sexual reproduction in rice (Oryza sativa L.). We identified a MutS-homolog (MSH) family gene OsMSH4 in a trisomic plant. Cytological analysis showed that developments of both pollen and embryo sacs in an Osmsh4 mutant were blocked due to defective chromosome pairing. Compared with the wild type, the Osmsh4 mutant displayed a significant ~21.9% reduction in chiasma frequency, which followed a Poisson distribution, suggesting that class I crossover formation in the mutant was impaired. Temporal and spatial expression pattern analyses showed that OsMSH4 was preferentially expressed in meiocytes during their meiosis, indicating a critical role in gametogenesis. Subcellular localization showed that OsMSH4-green fluorescent protein was predominantly located in the nucleus. OsMSH4 could interact with another MSH member (OsMSH5) through the N-terminus and C-terminus, respectively. Direct physical interaction between OsMSH5, OsRPA1a, OsRPA2b, OsRPA1c, and OsRPA2c was identified by yeast two-hybrid assays and further validated by pull-down assays. Our results supported the conclusion that the OsMSH4/5 heterodimer plays a key role in regulation of crossover formation during rice meiosis by interaction with the RPA complex.

CORONA, PHABULOSA and PHAVOLUTA collaborate with BELL1 to confine WUSCHEL expression to the nucellus in Arabidopsis ovules.

Angiosperm ovules consist of three proximal-distal domains - the nucellus, chalaza and funiculus - demarcated by developmental fate and specific gene expression. Mutation in three paralogous class III homeodomain leucine zipper (HD-ZIPIII) genes leads to aberrations in ovule integument development. Expression of WUSCHEL (WUS) is normally confined to the nucellar domain, but in this triple mutant expression expands into the chalaza. MicroRNA-induced suppression of this expansion partially suppresses the effects of the HD-ZIPIII mutations on ovule development, implicating ectopic WUS expression as a component of the mutant phenotype. bell1 (bel1) mutants produce aberrant structures in place of the integuments and WUS is ectopically expressed in these structures. Combination of bel1 with the HD-ZIPIII triple mutant leads to a striking phenotype in which ectopic ovules emerge from nodes of ectopic WUS expression along the funiculi of the primary ovules. The synergistic phenotype indicates that BEL1 and the HD-ZIPIII genes act in at least partial independence in confining WUS expression to the nucellus and maintaining ovule morphology. The branching ovules of the mutant resemble those of some fossil gymnosperms, implicating BEL1 and HD-ZIPIII genes as players in the evolution of the unbranched ovule form in extant angiosperms.

Comparative De Novo Transcriptome Analysis of Fertilized Ovules in Xanthoceras sorbifolium Uncovered a Pool of Genes Expressed Specifically or Preferentially in the Selfed Ovule That Are Potentially Involved in Late-Acting Self-Incompatibility.

Xanthoceras sorbifolium, a tree species endemic to northern China, has high oil content in its seeds and is recognized as an important biodiesel crop. The plant is characterized by late-acting self-incompatibility (LSI). LSI was found to occur in many angiosperm species and plays an important role in reducing inbreeding and its harmful effects, as do gametophytic self-incompatibility (GSI) and sporophytic self-incompatibility (SSI). Molecular mechanisms of conventional GSI and SSI have been well characterized in several families, but no effort has been made to identify the genes involved in the LSI process. The present studies indicated that there were no significant differences in structural and histological features between the self- and cross-pollinated ovules during the early stages of ovule development until 5 days after pollination (DAP). This suggests that 5 DAP is likely to be a turning point for the development of the selfed ovules. Comparative de novo transcriptome analysis of the selfed and crossed ovules at 5 DAP identified 274 genes expressed specifically or preferentially in the selfed ovules. These genes contained a significant proportion of genes predicted to function in the biosynthesis of secondary metabolites, consistent with our histological observations in the fertilized ovules. The genes encoding signal transduction-related components, such as protein kinases and protein phosphatases, are overrepresented in the selfed ovules. X. sorbifolium selfed ovules also specifically or preferentially express many unique transcription factor (TF) genes that could potentially be involved in the novel mechanisms of LSI. We also identified 42 genes significantly up-regulated in the crossed ovules compared to the selfed ovules. The expression of all 16 genes selected from the RNA-seq data was validated using PCR in the selfed and crossed ovules. This study represents the first genome-wide identification of genes expressed in the fertilized ovules of an LSI species. The availability of a pool of specifically or preferentially expressed genes from selfed ovules for X. sorbifolium will be a valuable resource for future genetic analyses of candidate genes involved in the LSI response.