Female gametophyte development is required for nucellar-tip degeneration during Arabidopsis ovule development.

MAIN CONCLUSION: Genetic ablation of the female gametophyte provides direct evidence for the existence of interregional communication during Arabidopsis ovule development and the importance of the female gametophyte in nucellar-tip degeneration. The angiosperm ovule consists of three regions: the female gametophyte, the nucellus, and the integuments, all of which develop synchronously and coordinately. Previously, interregional communication enabling cooperative ovule development had been proposed; however, the evidence for these communications mostly relies on the analysis of mutant phenotypes. To provide direct evidence, we specifically ablated the Arabidopsis female gametophyte by expressing the diphtheria toxin fragment A (DTA) under the female gametophyte-specific DD13 promoter and analyzed its effects on the development of the nucellus and the integuments. We found that the female gametophyte is not required for integument development or for the orientation and curvature of the ovule body, but is necessary for nucellar-tip degeneration. The results presented here provide direct evidence for communication from the female gametophyte to the nucellus and demonstrate that Arabidopsis ovules require interregional communication for cooperative development.

Eight hydroxyproline-O-galactosyltransferases play essential roles in female reproductive development.

In angiosperms, ovules give rise to seeds upon fertilization. Thus, seed formation is dependent on both successful ovule development and tightly controlled communication between female and male gametophytes. During establishment of these interactions, cell walls play a pivotal role, especially arabinogalactan-proteins (AGPs). AGPs are highly glycosylated proteins decorated by arabinogalactan side chains, representing 90 % of the AGP molecule. AGP glycosylation is initiated by a reaction catalysed by hydroxyproline-O-galactosyltransferases (Hyp-GALTs), specifically eight of them (GALT2-9), which add the first galactose to Hyp residues. Five Hyp-GALTs (GALT2, 5, 7, 8 and 9) were previously described as essential for AGP functions in pollen and ovule development, pollen-pistil interactions, and seed morphology. In the present work, a higher order Hyp-GALT mutant (23456789) was studied, with a high degree of under-glycosylated AGPs, to gain deeper insight into the crucial roles of these eight enzymes in female reproductive tissues. Notably, the 23456789 mutant demonstrated a high quantity of unfertilized ovules, displaying abnormal callose accumulation both at the micropylar region and, sometimes, throughout the entire embryo sac. Additionally, this mutant displayed ovules with abnormal embryo sacs, had a disrupted spatiotemporal distribution of AGPs in female reproductive tissues, and showed abnormal seed and embryo development, concomitant with a reduction in AGP-GlcA levels. This study revealed that at least three more enzymes exhibit Hyp-O-GALT activity in Arabidopsis (GALT3, 4 and 6), and reinforces the crucial importance of AGP carbohydrates in carrying out the biological functions of AGPs during plant reproduction.

Germline ß-1,3-glucan deposits are required for female gametogenesis in Arabidopsis thaliana.

Correct regulation of intercellular communication is a fundamental requirement for cell differentiation. In Arabidopsis thaliana, the female germline differentiates from a single somatic ovule cell that becomes encased in ß-1,3-glucan, a water insoluble polysaccharide implicated in limiting pathogen invasion, regulating intercellular trafficking in roots, and promoting pollen development. Whether ß-1,3-glucan facilitates germline isolation and development has remained contentious, since limited evidence is available to support a functional role. Here, transcriptional profiling of adjoining germline and somatic cells revealed differences in gene expression related to ß-1,3-glucan metabolism and signalling through intercellular channels (plasmodesmata). Dominant expression of a ß-1,3-glucanase in the female germline transiently perturbed ß-1,3-glucan deposits, allowed intercellular movement of tracer molecules, and led to changes in germline gene expression and histone marks, eventually leading to termination of germline development. Our findings indicate that germline ß-1,3-glucan fulfils a functional role in the ovule by insulating the primary germline cell, and thereby determines the success of downstream female gametogenesis.

RNA HELICASE 32 is essential for female gametophyte development in Arabidopsis.

The normal progression of mitotic cycles and synchronized development within female reproductive organs are pivotal for sexual reproduction in plants. Nevertheless, our understanding of the genetic regulation governing mitotic cycles during the haploid phase of higher plants remains limited. In this study, we characterized RNA HELICASE 32 (RH32), which plays an essential role in female gametogenesis in Arabidopsis. The rh32 heterozygous mutant was semi-sterile, whereas the homozygous mutant was nonviable. The rh32 mutant allele could be transmitted through the male gametophyte, but not the female gametophyte. Phenotypic analysis revealed impaired mitotic progression, synchronization, and cell specification in rh32 female gametophytes, causing the arrest of embryo sacs. In the delayed pollination test, none of the retarded embryo sacs developed into functional female gametophytes, and the vast majority of rh32 female gametophytes were defective in the formation of the large central vacuole. RH32 is strongly expressed in the embryo sac. Knock-down of RH32 resulted in the accumulation of unprocessed 18 S pre-rRNA, implying that RH32 is involved in ribosome synthesis. Based on these findings, we propose that RH32 plays a role in ribosome synthesis, which is critical for multiple processes in female gametophyte development.

Principles of amyloplast replication in the ovule integuments of Arabidopsis thaliana.

Plastids in vascular plants have various differentiated forms, among which amyloplasts are crucial for starch storage and plant productivity. Despite the vast knowledge of the binary-fission mode of chloroplast division, our understanding of the replication of non-photosynthetic plastids, including amyloplasts, remains limited. Recent studies have suggested the involvement of stromules (stroma-filled tubules) in plastid replication when the division apparatus is faulty. However, details of the underlying mechanism(s) and their relevance to normal processes have yet to be elucidated. Here, we developed a live analysis system for studying amyloplast replication using Arabidopsis (Arabidopsis thaliana) ovule integuments. We showed the full sequence of amyloplast development and demonstrated that wild-type amyloplasts adopt three modes of replication, binary fission, multiple fission, and stromule-mediated fission, via multi-way placement of the FtsZ ring. The minE mutant, with severely inhibited chloroplast division, showed marked heterogeneity in amyloplast size, caused by size-dependent but wild-type modes of plastid fission. The dynamic properties of stromules distinguish the wild-type and minE phenotypes. In minE cells, extended stromules from giant amyloplasts acquired stability, allowing FtsZ ring assembly and constriction, as well as the growth of starch grains therein. Despite hyper-stromule formation, amyloplasts did not proliferate in the ftsZ null mutant. These data clarify the differences between amyloplast and chloroplast replication and demonstrate that the structural plasticity of amyloplasts underlies the multiplicity of their replication processes. Furthermore, this study shows that stromules can generate daughter plastids via the assembly of the FtsZ ring.

An ATP-binding cassette transporter, OsABCB24, is involved in female gametophyte development and early seed growth in rice.

Female gametogenesis has been rarely studied due to gametophyte lethality and the unavailability of related genetic resources. In this study, we identified a rice ATP-binding cassette transporter, OsABCB24, whose null function displayed a significantly reduced seed setting rate by as much as 94%-100% compared with that of the wild type (WT). The reciprocal cross of WT and mutant plants demonstrated that the female reproductive organs in mutants were functionally impaired. Confocal microscopy observations revealed that, although megasporogenesis remained unaffected in CRISPR/Cas9 osabcb24 mutants, the formation of female gametophytes was interrupted. Additionally, the structure of the syncytial nucleus was impaired during the initial stages of endosperm formation. Histochemical analysis showed that OsABCB24 was preferentially expressed at the conjunction of receptacle and ovary, spanning from the functional megaspore stage to the two-nucleate embryo sac stage. Further, OsABCB24 was identified as an endoplasmic reticulum membrane-localized protein. Notably, the overexpression of OsABCB24 triggered a 1.5- to 2-fold increase in grain production compared to the WT. Our findings showed that OsABCB24 plays a key role in both female gametophyte development and the early development of seeds.

Polycomb proteins RING1A/B promote H2A monoubiquitination to regulate female gametophyte development in Arabidopsis.

A functional female gametophyte is the basis of successful sexual reproduction in flowering plants. During female gametophyte development, the megaspore mother cell (MMC), which differentiates from a single subepidermal somatic cell in the nucellus, undergoes meiosis to produce four megaspores; only the one at the chalazal end, referred to as the functional megaspore (FM), then undergoes three rounds of mitosis and develops into a mature embryo sac. Here, we report that RING1A and RING1B (RING1A/B), two functionally redundant Polycomb proteins in Arabidopsis, are critical for female gametophyte development. Mutations of RING1A/B resulted in defects in the specification of the MMC and the FM, and in the subsequent mitosis of the FM, thereby leading to aborted ovules. Detailed analysis revealed that several genes essential for female gametophyte development were ectopically expressed in the ring1a ring1b mutant, including Argonaute (AGO) family genes and critical transcription factors. Furthermore, RING1A/B bound to some of these genes to promote H2A monoubiquitination (H2Aub). Taken together, our study shows that RING1A/B promote H2Aub modification at key genes for female gametophyte development, suppressing their expression to ensure that the development progresses correctly.

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.

Understanding megasporogenesis through model plants: contemporary evidence and future insights.

The megasporangium serves as a model system for understanding the concept of individual cell identity, and cell-to-cell communication in angiosperms. As development of the ovule progresses, three distinct layers, the epidermal (L1), the subepidermal or the hypodermal (L2) and the innermost layers (L3) are formed along the MMC (megaspore mother cell). The MMC, which is the primary female germline cell, is initiated as a single subepidermal cell amongst several somatic cells. MMC development is governed by various regulatory pathways involving intercellular signaling, small RNAs and DNA methylation. The programming and reprograming of a single nucellar cell to enter meiosis is governed by 'permissive' interacting processes and factors. Concomitantly, several nucellar sister cells are prevented from germline fate also by a set of 'repressive' factors. However, in certain angiosperms, anomalies in development of the female gametophyte have been observed. The sporophytic tissue surrounding the female gametophyte affects the gametophyte in multiple ways. The role of genes and transcription factors in the development of the MMC and in the regulation of various processes studied in selected model plants such as Arabidopsis is explained in detail in this paper. However, as angiosperms display enormous diversity, it is important to investigate early stages of megasporogenesis in other plant systems as well. Such studies provide valuable insights in understanding the regulation of megasporogenesis and the evolution of the female gametophyte from gymnosperms to flowering plants.

SlCRCa is a key D-class gene controlling ovule fate determination in tomato.

Cell fate determination and primordium initiation on the placental surface are two key events for ovule formation in seed plants, which directly affect ovule density and seed yield. Despite ovules form in the marginal meristematic tissues of the carpels, angiosperm carpels evolved after the ovules. It is not clear how the development of the ovules and carpels is coordinated in angiosperms. In this study, we identify the S. lycopersicum CRABS CLAW (CRC) homologue SlCRCa as an essential determinant of ovule fate. We find that SlCRCa is not only expressed in the placental surface and ovule primordia but also functions as a D-class gene to block carpel fate and promote ovule fate in the placental surface. Loss of function of SlCRCa causes homeotic transformation of the ovules to carpels. In addition, we find low levels of the S. lycopersicum AINTEGUMENTA (ANT) homologue (SlANT2) favour the ovule initiation, whereas high levels of SlANT2 promote placental carpelization. SlCRCa forms heterodimer with tomato INNER NO OUTER (INO) and AGAMOUS (AG) orthologues, SlINO and TOMATO AGAMOUS1 (TAG1), to repress SlANT2 expression during the ovule initiation. Our study confirms that angiosperm basal ovule cells indeed retain certain carpel properties and provides mechanistic insights into the ovule initiation.

OsRH52A, a DEAD-box protein, regulates functional megaspore specification and is required for embryo sac development in rice.

The development of the embryo sac is an important factor that affects seed setting in rice. Numerous genes associated with embryo sac (ES) development have been identified in plants; however, the function of the DEAD-box RNA helicase family genes is poorly known in rice. Here, we characterized a rice DEAD-box protein, RH52A, which is localized in the nucleus and cytoplasm and highly expressed in the floral organs. The knockout mutant rh52a displayed partial ES sterility, including degeneration of the ES (21%) and the presence of a double-female-gametophyte (DFG) structure (11.8%). The DFG developed from two functional megaspores near the chalazal end in one ovule, and 3.4% of DFGs were able to fertilize via the sac near the micropylar pole in rh52a. RH52A was found to interact with MFS1 and ZIP4, both of which play a role in homologous recombination in rice meiosis. RNA-sequencing identified 234 down-regulated differentially expressed genes associated with reproductive development, including two, MSP1 and HSA1b, required for female germline cell specification. Taken together, our study demonstrates that RH52A is essential for the development of the rice embryo sac and provides cytological details regarding the formation of DFGs.

Strigolactones Might Regulate Ovule Development after Fertilization in Xanthoceras sorbifolium.

Strigolactones (SLs) were recently defined as a novel class of plant hormones that act as key regulators of diverse developmental processes and environmental responses. Much research has focused on SL biosynthesis and signaling in roots and shoots, but little is known about whether SLs are produced in early developing seeds and about their roles in ovule development after fertilization. This study revealed that the fertilized ovules and early developing pericarp in Xanthoceras sorbifolium produced minute amounts of two strigolactones: 5-deoxystrigol and strigol. Their content decreased in the plants with the addition of exogenous phosphate (Pi) compared to those without the Pi treatment. The exogenous application of an SL analog (GR24) and a specific inhibitor of SL biosynthesis (TIS108) affected early seed development and fruit set. In the Xanthoceras genome, we identified 69 potential homologs of genes involved in SL biological synthesis and signaling. Using RNA-seq to characterize the expression of these genes in the fertilized ovules, 37 genes were found to express differently in the fertilized ovules that were aborting compared to the normally developing ovules. A transcriptome analysis also revealed that in normally developing ovules after fertilization, 12 potential invertase genes were actively expressed. Hexoses (glucose and fructose) accumulated at high concentrations in normally developing ovules during syncytial endosperm development. In contrast, a low ratio of hexose and sucrose levels was detected in aborting ovules with a high strigolactone content. XsD14 virus-induced gene silencing (VIGS) increased the hexose content in fertilized ovules and induced the proliferation of endosperm free nuclei, thereby promoting early seed development and fruit set. We propose that the crosstalk between sugar and strigolactone signals may be an important part of a system that accurately regulates the abortion of ovules after fertilization. This study is useful for understanding the mechanisms underlying ovule abortion, which will serve as a guide for genetic or chemical approaches to promote seed yield in Xanthoceras.

Development of pollinated and unpollinated ovules in Ginkgo biloba: unravelling the role of pollen in ovule tissue maturation.

In gymnosperms such as Ginkgo biloba, the arrival of pollen plays a key role in ovule development, before fertilization occurs. Accordingly, G. biloba female plants geographically isolated from male plants abort all their ovules after the pollination drop emission, which is the event that allows the ovule to capture pollen grains. To decipher the mechanism induced by pollination required to avoid ovule senescence and then abortion, we compared the transcriptomes of pollinated and unpollinated ovules at three time points after the end of the emission of pollination drop. Transcriptomic and in situ expression analyses revealed that several key genes involved in programmed cell death such as senescence and apoptosis, DNA replication, and cell cycle regulation were differentially expressed in unpollinated ovules compared to pollinated ovules. We provide evidence that the pollen captured by the pollination drop affects auxin local accumulation and might cause deregulation of key genes required for the ovule's programmed cell death, activating both the cell cycle regulation and DNA replication genes.

Maternal nitric oxide homeostasis impacts female gametophyte development under optimal and stress conditions.

In adverse environments, the number of fertilizable female gametophytes (FGs) in plants is reduced, leading to increased survival of the remaining offspring. How the maternal plant perceives internal growth cues and external stress conditions to alter FG development remains largely unknown. We report that homeostasis of the stress signaling molecule nitric oxide (NO) plays a key role in controlling FG development under both optimal and stress conditions. NO homeostasis is precisely regulated by S-nitrosoglutathione reductase (GSNOR). Prior to fertilization, GSNOR protein is exclusively accumulated in sporophytic tissues and indirectly controls FG development in Arabidopsis (Arabidopsis thaliana). In GSNOR null mutants, NO species accumulated in the degenerating sporophytic nucellus, and auxin efflux into the developing FG was restricted, which inhibited FG development, resulting in reduced fertility. Importantly, restoring GSNOR expression in maternal, but not gametophytic tissues, or increasing auxin efflux substrate significantly increased the proportion of normal FGs and fertility. Furthermore, GSNOR overexpression or added auxin efflux substrate increased fertility under drought and salt stress. These data indicate that NO homeostasis is critical to normal auxin transport and maternal control of FG development, which in turn determine seed yield. Understanding this aspect of fertility control could contribute to mediating yield loss under adverse conditions.

JAGGER localization and function are dependent on GPI anchor addition.

KEY MESSAGE: GPI anchor addition is important for JAGGER localization and in vivo function. Loss of correct GPI anchor addition in JAGGER, negatively affects its localization and function. In flowering plants, successful double fertilization requires the correct delivery of two sperm cells to the female gametophyte inside the ovule. The delivery of a single pair of sperm cells is achieved by the entrance of a single pollen tube into one female gametophyte. To prevent polyspermy, Arabidopsis ovules avoid the attraction of multiple pollen tubes to one ovule-polytubey block. In Arabidopsis jagger mutants, a significant number of ovules attract more than one pollen tube to an ovule due to an impairment in synergid degeneration. JAGGER encodes a putative arabinogalactan protein which is predicted to be anchored to the plasma membrane by a glycosylphosphatidylinositol (GPI) anchor. Here, we show that JAGGER fused to citrine yellow fluorescent protein (JAGGER-cYFP) is functional and localizes mostly to the periphery of ovule integuments and transmitting tract cells. We further investigated the importance of GPI-anchor addition domains for JAGGER localization and function. Different JAGGER proteins with deletions in predicted ω-site regions and GPI attachment signal domain, expected to compromise the addition of the GPI anchor, led to disruption of JAGGER localization in the cell periphery. All JAGGER proteins with disrupted localization were also not able to rescue the polytubey phenotype, pointing to the importance of GPI-anchor addition to in vivo function of the JAGGER protein.

HISTONE DEACETYLASE19 Controls Ovule Number Determination and Transmitting Tract Differentiation.

The gynoecium is critical for the reproduction of flowering plants as it contains the ovules and the tissues that foster pollen germination, growth, and guidance. These tissues, known as the reproductive tract (ReT), comprise the stigma, style, and transmitting tract (TT). The ReT and ovules originate from the carpel margin meristem (CMM) within the pistil. SHOOT MERISTEMLESS (STM) is a key transcription factor for meristem formation and maintenance. In all above-ground meristems, including the CMM, local STM downregulation is required for organ formation. However, how this downregulation is achieved in the CMM is unknown. Here, we have studied the role of HISTONE DEACETYLASE 19 (HDA19) in Arabidopsis (Arabidopsis thaliana) during ovule and ReT differentiation based on the observation that the hda19-3 mutant displays a reduced ovule number and fails to differentiate the TT properly. Fluorescence-activated cell sorting coupled with RNA-sequencing revealed that in the CMM of hda19-3 mutants, genes promoting organ development are downregulated while meristematic markers, including STM, are upregulated. HDA19 was essential to downregulate STM in the CMM, thereby allowing ovule formation and TT differentiation. STM is ectopically expressed in hda19-3 at intermediate stages of pistil development, and its downregulation by RNA interference alleviated the hda19-3 phenotype. Chromatin immunoprecipitation assays indicated that STM is a direct target of HDA19 during pistil development and that the transcription factor SEEDSTICK is also required to regulate STM via histone acetylation. Thus, we identified factors required for the downregulation of STM in the CMM, which is necessary for organogenesis and tissue differentiation.

Small RNA profiling reveals that an ovule-specific microRNA, cja-miR5179, targets a B-class MADS-box gene in Camellia japonica.

BACKGROUND AND AIMS: The functional specialization of microRNA and its target genes is often an important factor in the establishment of spatiotemporal patterns of gene expression that are essential to plant development and growth. In different plant lineages, understanding the functional conservation and divergence of microRNAs remains to be explored. METHODS: To identify small regulatory RNAs underlying floral patterning, we performed a tissue-specific profiling of small RNAs in various floral organs from single and double flower varieties (flowers characterized by multiple layers of petals) in Camellia japonica. We identified cja-miR5179, which belongs to a deeply conserved microRNA family that is conserved between angiosperms and basal plants but frequently lost in eudicots. We characterized the molecular function of cja-miR5179 and its target - a B-function MADS-box gene - through gene expression analysis and transient expression assays. KEY RESULTS: We showed that cja-miR5179 is exclusively expressed in ovule tissues at the early stage of floral development. We found that cja-miR5179 targets the coding sequences of a DEFICIENS-like B-class gene (CjDEF) mRNA, which is located in the K motif of the MADS-box domain; and the target sites of miR5179/MADS-box were consistent in Camellia and orchids. Furthermore, through a petal transient-expression assay, we showed that the BASIC PENTACYSTEINE proteins bind to the GA-rich motifs in the cja-miR5179 promoter region and suppresses its expression. CONCLUSIONS: We propose that the regulation between miR5179 and a B-class MADS-box gene in C. japonica has a deep evolutionary origin before the separation of monocots and dicots. During floral development of C. japonica, cja-miR5179 is specifically expressed in the ovule, which may be required for the inhibition of CjDEF function. This work highlights the evolutionary conservation as well as functional divergence of small RNAs in floral development.

Arabidopsis TCP4 transcription factor inhibits high temperature-induced homeotic conversion of ovules.

Abnormal high temperature (HT) caused by global warming threatens plant survival and food security, but the effects of HT on plant organ identity are elusive. Here, we show that Class II TEOSINTE BRANCHED 1/CYCLOIDEA/ PCF (TCP) transcription factors redundantly protect ovule identity under HT. The duodecuple tcp2/3/4/5/10/13/17/24/1/12/18/16 (tcpDUO) mutant displays HT-induced ovule conversion into carpelloid structures. Expression of TCP4 in tcpDUO complements the ovule identity conversion. TCP4 interacts with AGAMOUS (AG), SEPALLATA3 (SEP3), and the homeodomain transcription factor BELL1 (BEL1) to strengthen the association of BEL1 with AG-SEP3. The tcpDUO mutant synergistically interacts with bel1 and the ovule identity gene seedstick (STK) mutant stk in tcpDUO bel1 and tcpDUO stk. Our findings reveal the critical roles of Class II TCPs in maintaining ovule identity under HT and shed light on the molecular mechanisms by which ovule identity is determined by the integration of internal factors and environmental temperature.

Genetic and Phenotypic Analysis of Ovule Development in Arabidopsis.

The plant seed is a remarkable structure that represents the single most important energy source in global diets. The stages of reproductive growth preceding seed formation are particularly important since they influence the number, size, and quality of seed produced. The progenitor of the seed is the ovule, a multicellular organ that produces a female gametophyte while maintaining a range of somatic ovule cells to protect the seed and ensure it receives maternal nourishment. Ovule development has been well characterized in Arabidopsis using a range of molecular, genetic, and cytological assays. These can provide insight into the mechanistic basis for ovule development, and opportunities to explore its evolutionary conservation. In this chapter, we describe some of these methods and tools that can be used to investigate early ovule development and cell differentiation.