Ovule Initiation Research Articles

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.

MADS8 is indispensable for female reproductive development at high ambient temperatures in cereal crops.

Temperature is a major factor that regulates plant growth and phenotypic diversity. To ensure reproductive success at a range of temperatures, plants must maintain developmental stability of their sexual organs when exposed to temperature fluctuations. However, the mechanisms integrating plant floral organ development and temperature responses are largely unknown. Here, we generated barley and rice loss-of-function mutants in the SEPALLATA-like MADS-box gene MADS8. The mutants in both species form multiple carpels that lack ovules at high ambient temperatures. Tissue-specific markers revealed that HvMADS8 is required to maintain floral meristem determinacy and ovule initiation at high temperatures, and transcriptome analyses confirmed that temperature-dependent differentially expressed genes in Hvmads8 mutants predominantly associate with floral organ and meristem regulation. HvMADS8 temperature-responsive activity relies on increased binding to promoters of downstream targets, as revealed by a cleavage under targets and tagmentation (CUT&Tag) analysis. We also demonstrate that HvMADS8 directly binds to 2 orthologs of D-class floral homeotic genes to activate their expression. Overall, our findings revealed a new, conserved role for MADS8 in maintaining pistil number and ovule initiation in cereal crops, extending the known function of plant MADS-box proteins in floral organ regulation.

Ovule initiation: the essential step controlling offspring number in Arabidopsis

AbstractSeed is the offspring of angiosperms. Plants produce large numbers of seeds to ensure effective reproduction and survival in varying environments. Ovule is a fundamentally important organ and is the precursor of the seed. In Arabidopsis and other plants characterized by multi‐ovulate ovaries, ovule initiation determines the maximal ovule number, thus greatly affecting seed number per fruit and seed yield. Investigating the regulatory mechanism of ovule initiation has both scientific and economic significance. However, the genetic and molecular basis underlying ovule initiation remains unclear due to technological limitations. Very recently, rules governing the multiple ovules initiation from one placenta have been identified, the individual functions and crosstalk of phytohormones in regulating ovule initiation have been further characterized, and new regulators of ovule boundary are reported, therefore expanding the understanding of this field. In this review, we present an overview of current knowledge in ovule initiation and summarize the significance of ovule initiation in regulating the number of plant offspring, as well as raise insights for the future study in this field that provide potential routes for the improvement of crop yield.

Highlighted articles for June 2022

Highlighted articles for June 2022

PIN3 positively regulates the late initiation of ovule primordia in Arabidopsis thaliana

Ovule initiation determines the maximum ovule number and has great impact on seed number and yield. However, the regulation of ovule initiation remains largely elusive. We previously reported that most of the ovule primordia initiate asynchronously at floral stage 9 and PINFORMED1 (PIN1) polarization and auxin distribution contributed to this process. Here, we further demonstrate that a small amount of ovule primordia initiate at floral stage 10 when the existing ovules initiated at floral stage 9 start to differentiate. Genetic analysis revealed that the absence of PIN3 function leads to the reduction in pistil size and the lack of late-initiated ovules, suggesting PIN3 promotes the late ovule initiation process and pistil growth. Physiological analysis illustrated that, unlike picloram, exogenous application of NAA can’t restore these defective phenotypes, implying that PIN3-mediated polar auxin transport is required for the late ovule initiation and pistil length. qRT-PCR results indicated that the expression of SEEDSTICK (STK) is up-regulated under auxin analogues treatment while is down-regulated in pin3 mutants. Meanwhile, overexpressing STK rescues pin3 phenotypes, suggesting STK participates in PIN3-mediated late ovule initiation possibly by promoting pistil growth. Furthermore, brassinosteroid influences the late ovule initiation through positively regulating PIN3 expression. Collectively, this study demonstrates that PIN3 promotes the late ovule initiation and contributes to the extra ovule number. Our results give important clues for increasing seed number and yield of cruciferous and leguminous crops.

Interaction of brassinosteroid and cytokinin promotes ovule initiation and increases seed number per silique in Arabidopsis.

Ovule initiation is a key step that strongly influences ovule number and seed yield. Notably, mutants with enhanced brassinosteroid (BR) and cytokinin (CK) signaling produce more ovules and have a higher seed number per silique (SNS) than wild-type plants. Here, we crossed BR- and CK-related mutants to test whether these phytohormones function together in ovule initiation. We determined that simultaneously enhancing BR and CK contents led to higher ovule and seed numbers than enhancing BR or CK separately, and BR and CK enhanced each other. Further, the BR-response transcription factor BZR1 directly interacted with the CK-response transcription factor ARABIDOPSIS RESPONSE REGULATOR1 (ARR1). Treatments with BR or BR plus CK strengthened this interaction and subsequent ARR1 targeting and induction of downstream genes to promote ovule initiation. Enhanced CK signaling partially rescued the reduced SNS phenotype of BR-deficient/insensitive mutants whereas enhanced BR signaling failed to rescue the low SNS of CK-deficient mutants, suggesting that BR regulates ovule initiation and SNS through CK-mediated and -independent pathways. Our study thus reveals that interaction between BR and CK promotes ovule initiation and increases seed number, providing important clues for increasing the seed yield of dicot crops.

Gibberellins regulate ovule number through a DELLA-CUC2 complex in Arabidopsis.

Ovule development is a key process for plant reproduction, helping to ensure correct seed production. Several molecular factors and plant hormones such as gibberellins are involved in ovule initiation and development. Gibberellins control ovule development by the destabilization of DELLA proteins, whereas DELLA activity has been shown to act as a positive factor for ovule primordia emergence. But the molecular mechanism by which DELLA acts in ovule primordia initiation remained unknown. In this study we report that DELLA proteins participate in ovule initiation by the formation of a protein complex with the CUC2 transcription factor. The DELLA protein GAI requires CUC2 to promote ovule primordia formation, through the direct GAI-CUC2 interaction in placental cells that would determine the boundary regions between ovules during pistil development. Analysis of GAI-CUC2 interaction and co-localization in the placenta supports this hypothesis. Moreover, molecular analysis identified a subset of the loci for which the GAI protein may act as a transcriptional co-regulator in a CUC2-dependent manner. The DELLA-CUC2 complex is a component of the gene regulatory network controlling ovule primordia initiation in Arabidopsis.

Flower meristem maintenance by TILLERS ABSENT 1 is essential for ovule development in rice.

Plant development depends on the activity of pluripotent stem cells in meristems, such as the shoot apical meristem and the flower meristem. In Arabidopsis thaliana, WUSCHEL (WUS) is essential for stem cell homeostasis in meristems and integument differentiation in ovule development. In rice (Oryza sativa), the WUS ortholog TILLERS ABSENT 1 (TAB1) promotes stem cell fate in axillary meristem development, but its function is unrelated to shoot apical meristem maintenance in vegetative development. In this study, we examined the role of TAB1 in flower development. The ovule, which originates directly from the flower meristem, failed to differentiate in tab1 mutants, suggesting that TAB1 is required for ovule formation. Expression of a stem cell marker was completely absent in the flower meristem at the ovule initiation stage, indicating that TAB1 is essential for stem cell maintenance in the 'final' flower meristem. The ovule defect in tab1 was partially rescued by floral organ number 2 mutation, which causes overproliferation of stem cells. Collectively, it is likely that TAB1 promotes ovule formation by maintaining stem cells at a later stage of flower development.

Molecular Network for Regulation of Ovule Number in Plants.

In seed-bearing plants, the ovule (“small egg”) is the organ within the gynoecium that develops into a seed after fertilization. The gynoecium located in the inner compartment of the flower turns into a fruit. The number of ovules in the ovary determines the upper limit or the potential of seed number per fruit in plants, greatly affecting the final seed yield. Ovule number is an important adaptive characteristic for plant evolution and an agronomic trait for crop improvement. Therefore, understanding the mechanism and pathways of ovule number regulation becomes a significant research aspect in plant science. This review summarizes the ovule number regulators and their regulatory mechanisms and pathways. Specially, an integrated molecular network for ovule number regulation is constructed, in which phytohormones played a central role, followed by transcription factors, enzymes, other protein and micro-RNA. Of them, AUX, BR and CK are positive regulator of ovule number, whereas GA acts negatively on it. Interestingly, many ovule number regulators have conserved functions across several plant taxa, which should be the targets of genetic improvement via breeding or gene editing. Many ovule number regulators identified to date are involved in the diverse biological process, such as ovule primordia formation, ovule initiation, patterning, and morphogenesis. The relations between ovule number and related characteristics/traits especially of gynoecium/fruit size, ovule fertility, and final seed number, as well as upcoming research questions, are also discussed. In summary, this review provides a general overview of the present finding in ovule number regulation, which represents a more comprehensive and in-depth cognition on it.

Fine mapping and candidate gene analysis of a major locus controlling ovule abortion and seed number per silique in Brassica napus L.

A major QTL controlling ovule abortion and SN was fine-mapped to a 80.1-kb region on A8 in rapeseed, and BnaA08g07940D and BnaA08g07950D are the most likely candidate genes. The seed number per silique (SN), an important yield determining trait of rapeseed, is the final consequence of a complex developmental process including ovule initiation and the subsequent ovule/seed development. To explore the genetic mechanism regulating the natural variation of SN and its related components, quantitative trait locus (QTL) mapping was conducted using a doubled haploid (DH) population derived from the cross between C4-146 and C4-58B, which showed significant differences in SN and aborted ovule number (AON), but no obvious differences in ovule number (ON). QTL analysis identified 19 consensus QTLs for six SN-related traits across three environments. A novel QTL on chromosome A8, un.A8, which associates with multiple traits, except for ON, was stably detected across the three environments. This QTL explained more than 50% of the SN, AON and percentage of aborted ovules (PAO) variations as well as a moderate contribution on silique length (SL) and thousand seed weight (TSW). The C4-146 allele at the locus increases SN and SL but decreases AON, PAO and TSW. Further fine mapping narrowed down this locus into an 80.1-kb interval flanked by markers BM1668 and BM1672, and six predicted genes were annotated in the delimited region. Expression analyses and DNA sequencing showed that two homologs of Arabidopsis photosystem I subunit F (BnaA08g07940D) and zinc transporter 10 precursor (BnaA08g07950D) were the most promising candidate genes underlying this locus. These results provide a solid basis for cloning un.A8 to reduce the ovule abortion and increase SN in the yield improvement of rapeseed.

Asynchrony of ovule primordia initiation in Arabidopsis

ABSTRACTPlant ovule initiation determines the maximum of ovule number and has a great impact on the seed number per fruit. The detailed processes of ovule initiation have not been accurately described, although two connected processes, gynoecium and ovule development, have been investigated. Here, we report that ovules initiate asynchronously. The first group of ovule primordia grows out, the placenta elongates, the boundaries of existing ovules enlarge and a new group of primordia initiates from the boundaries. The expression pattern of different marker genes during ovule development illustrates that this asynchronicity continues throughout whole ovule development. PIN-FORMED1 polar distribution and auxin response maxima correlate with ovule primordia asynchronous initiation. We have established computational modeling to show how auxin dynamics influence ovule primordia initiation. Brassinosteroid signaling positively regulates ovule number by promoting placentae size and ovule primordia initiation through strengthening auxin response. Transcriptomic analysis demonstrates numerous known regulators of ovule development and hormone signaling, and many new genes are identified that are involved in ovule development. Taken together, our results illustrate that the ovule primordia initiate asynchronously and the hormone signals are involved in the asynchrony.

A Peptide Pair Coordinates Regular Ovule Initiation Patterns with Seed Number and Fruit Size

Ovule development in Arabidopsis thaliana involves pattern formation, which ensures that ovules are regularly arranged in the pistils to reduce competition for nutrients and space. Mechanisms underlying pattern formation in plants, such as phyllotaxis, flower morphogenesis, or lateral root initiation, have been extensively studied, and genes controlling the initiation of ovules have been identified. However, the fundamental patterning mechanism that determines the spacing of ovule anlagen within the placenta remained unexplored. Using natural variation analysis combined with quantitative trait locus analysis, we found that the spacing of ovules in the developing gynoecium and fruits is controlled by two secreted peptides, EPFL2 and EPFL9 (also known as Stomagen), and their receptors from the ERECTA (ER) family that act from the carpel wall and the placental tissue. We found that a signaling pathway controlled by EPFL9 acting from the carpel wall through the LRR-receptor kinases ER, ERL1, and ERL2 promotes fruit growth. Regular spacing of ovules depends on EPFL2 expression in the carpel wall and in the inter-ovule spaces, where it acts through ERL1 and ERL2. Loss of EPFL2 signaling results in shorter gynoecia and fruits and irregular spacing of ovules or even ovule twinning. We propose that the EPFL2 signaling module evolved to control the initiation and regular, equidistant spacing of ovule primordia, which may serve to minimize competition between seeds or facilitate equal resource allocation. Together, EPFL2 and EPFL9 help to coordinate ovule patterning and thereby seed number with gynoecium and fruit growth through a set of shared receptors.

On the Way to Ovules: The Hormonal Regulation of Ovule Development

This review focuses on the hormonal regulation of ovule development, especially on ovule initiation, patterning, and morphogenesis. Understanding of the genetic and molecular basis of ovule development is essential from both the scientific and economic perspective. The ovule represents an attractive system to study lateral organ development in plants, and, since ovules are the precursors of seeds, full comprehension of this process can be the key to the improvement of crops, especially those depending on high production of seeds and grains. Ovule initiation, patterning, and morphogenesis are governed by complex genetic and hormonal networks involving auxins, cytokinins, brassinosteroids, and gibberellins. These coordinate the determination of the ovule number, size, and shape through the regulation of the number of ovule primordia that arise from the placenta and/or ensuring their correct development into mature functional ovules. Here we summarize the current knowledge of how ovules are formed, paying special attention to the roles of these four plant hormones.

Genome-Wide Association Analysis Identifies Candidate Genes Regulating Seed Number Per Silique in Arabidopsis thaliana.

Seed weight and number ultimately determine seed yield. Arabidopsis seed number comprised of silique number and seed number per silique (SNS). Comparing seed development and weight, determinants of seed number remain largely uncharacterized. In this study, taking advantage of 107 available Arabidopsis accessions, genome-wide association analysis (GWAS) was employed to identify the candidate genes regulating SNS. GWAS-based genotype and phenotype association analysis identified 38 most significant SNPs marker sites that were mapped to specific chromosomal positions and allowed us to screen for dozens of candidate genes. One of them (PIN3) was selected for functional validation based on gene expression analysis. It is a positive regulator of Arabidopsis SNS. Although silique length of PIN3 loss of function mutant was not significantly changed, its SNS and seed density (SD) were significantly reduced as compared with the wild type. Notably, PIN3 overexpression lines driven by a placenta-specific promoter STK exhibited significantly shorter siliques, slightly reduced SNS, but significant increased SD compared with wild type, suggesting that PIN3 positively regulates SD through inducing ovule primordia initiation regardless of the placenta size. Ovule initiation determines the maximal possibility of SNS, and new genes and mechanism regulating SNS through modulating ovule initiation is worth further investigated.

Cell Wall Invertase Is Essential for Ovule Development through Sugar Signaling Rather Than Provision of Carbon Nutrients.

Ovule formation is essential for realizing crop yield because it determines seed number. The underlying molecular mechanism, however, remains elusive. Here, we show that cell wall invertase (CWIN) functions as a positive regulator of ovule initiation in Arabidopsis (Arabidopsis thaliana). In situ hybridization revealed that CWIN2 and CWIN4 were expressed at the placenta region where ovule primordia initiated. Specific silencing of CWIN2 and CWIN4 using targeted artificial microRNA driven by an ovule-specific SEEDSTICK promoter (pSTK) resulted in a substantial reduction of CWIN transcript and activity, which blocked ovule initiation and aggravated ovule abortion. There was no induction of carbon (C) starvation genes in the transgenic lines, and supplementing newly forming floral buds with extra C failed to recover the ovule phenotype. This indicates that suppression of CWIN did not lead to C starvation. A group of hexose transporters was downregulated in the transgenic plants. Among them, two representative ones were spatially coexpressed with CWIN2 and CWIN4, suggesting a coupling between CWIN and hexose transporters for ovule initiation. RNA-sequencing analysis identified differentially expressed genes encoding putative extracellular receptor-like kinases, MADS-box transcription factors, including STK, and early auxin response genes in response to CWIN-silencing. Our data demonstrate the essential role of CWIN in ovule initiation, which is most likely to occur through sugar signaling instead of C nutrient contribution. We propose that CWIN-mediated sugar signaling may be perceived by, and transmitted through, hexose transporters or receptor-like kinases to regulate ovule formation by modulating downstream auxin signaling and MADS-box transcription factors.

Phylogenetic analyses of key developmental genes provide insight into the complex evolution of seeds

Gene duplication plays a decisive role in organismal diversification and in the appearance of novel structures. In plants the megagametophyte covered by the integuments, which after fertilization becomes the seed constitutes a novel structure: the ovule. In Arabidopsis thaliana, genetic mechanisms regulating ovule development, including the genetics underlying ovule initiation, ovule patterning and integument development, have been identified. Among seed plants, integuments are not only a novelty in evolution, but integuments also present an enormous morphological variation. This study is focused on the evolution of gene families that play a role in the proper morphological development of the integuments, BELL1 (BEL1), KANADIs (KAN1, KAN2, and KAN4/ATS), UNICORN (UCN) and SHORT INTEGUMENTS1 (SIN1). In Arabidopsis, BEL1 establishes the initiation of integument development. KAN1 and 2 act in the proper development of the outer integument. While ABERRANT TESTA SHAPE (ATS), is involved in the correct separation of both integuments. UCN acts in planar growth of the outer integument repressing ATS. SIN1 is involved in cell elongation in the integuments. The results of our analyses show that each of these genes has a different evolutionary history and that while gymnosperms appear to have a simpler ovule morphology, they have more homologues of these candidate genes than angiosperms. In addition, we present the conserved and novel motifs for each of these genes among seed plants and their selection constraints, which may be related to functional changes and to the diversity of ovule morphologies.

Regulation of ovule initiation by gibberellins and brassinosteroids in tomato and Arabidopsis: two plant species, two molecular mechanisms.

Ovule primordia formation is a complex developmental process with a strong impact on the production of seeds. In Arabidopsis this process is controlled by a gene network, including components of the signalling pathways of auxin, brassinosteroids (BRs) and cytokinins. Recently, we have shown that gibberellins (GAs) also play an important role in ovule primordia initiation, inhibiting ovule formation in both Arabidopsis and tomato. Here we reveal that BRs also participate in the control of ovule initiation in tomato, by promoting an increase on ovule primordia formation. Moreover, molecular and genetic analyses of the co-regulation by GAs and BRs of the control of ovule initiation indicate that two different mechanisms occur in tomato and Arabidopsis. In tomato, GAs act downstream of BRs. BRs regulate ovule number through the downregulation of GA biosynthesis, which provokes stabilization of DELLA proteins that will finally promote ovule primordia initiation. In contrast, in Arabidopsis both GAs and BRs regulate ovule number independently of the activity levels of the other hormone. Taken together, our data strongly suggest that different molecular mechanisms could operate in different plant species to regulate identical developmental processes even, as for ovule primordia initiation, if the same set of hormones trigger similar responses, adding a new level of complexity.

Temporal Distinction between Male and Female Floral Organ Development in Nicotiana tabacum cv. Xanthi (Solanaceae).

Early floral developmental investigations provide crucial evidence for phylogenetic and molecular studies of plants. The developmental and evolutionary mechanisms underlying the variations in floral organs are critical for a thorough understanding of the diversification of flowers. Ontogenetic comparisons between anthers and pistil within single flowers were characterized over time in Nicotiana tabacum cv. Xanthi. The ages of 42 tobacco flower or flower primordia were estimated using corolla growth analysis. Results showed that the protodermal layer in carpel primordia contributes to carpel development by both anticlinal and periclinal divisions. Periclinal divisions in the hypodermal layer of the placenta were observed around 4.8 ± 1.3 days after the formation of early carpel primordia (ECP) and ovule initiation occurred 10.0 ± 0.5 days after ECP. Meiosis in anthers and ovules began about 8.9 ± 1.1 days and 14.4 ± 1.3 days after ECP, respectively. Results showed an evident temporal distinction between megasporogenesis and microsporogenesis. Flower ages spanned a 17-day interval, starting with flower primordia containing the ECP and anther primordia to the tetrad stage of meiosis in megasporocytes and the bicellular stage in pollen grains. These results establish a solid foundation for future studies in order to identify the developmental and molecular mechanisms responsible for the mating system in tobacco.

Brassinosteroid and Cytokinin Synergistically Increase Ovule Initiation and Seed Number Per Silique in <i>Arabidopsis</i>

The main determinant of crop yield is seed yield. In Arabidopsi, many of the genes that regulate seed number are involved in gametophyte and zygote development, and their overexpression cannot increase seed number. However, several mutants with enhanced brassinosteroid (BR) and cytokinin (CK) signals show increased seed number per silique (SNS). Here, we studied the crosstalk between BR and CK signals in regulating SNS through crossing BR- and CK-related mutants. Compared with the mutants ckx3 ckx5 and bzr1-1D , the triple mutant ckx3 ckx5 bzr1-1D with enhanced BR and CK signals formed elongated placentae and showed increased ovule initiation and SNS, indicating that BR and CK synergistically up-regulate SNS. Although BR enhanced the CK signal and CK enhanced the BR signal, while CK partially rescued the phenotypes of BR deficient- and -insensitive mutants, BR could not rescue the phenotypes of CK-deficient mutants. This suggested that CK functions downstream of BR in regulating SNS and that BR regulates SNS through CK-mediated or CK-independent pathways. Comparative transcriptome analyses of ckx3 ckx5 , bzr1-1D , and ckx3 ckx5 bzr1-1D identified known and new genes involved in ovule initiation. The BR-induced transcription factor BZR1 was found to directly interact with the B-type regulator of CK, ARR1, and this interaction enhanced the ability of ARR1 to target and induce genes related to ovule development. Our results demonstrate that BR and CK synergistically increase SNS through co-regulating downstream genes involved in ovule identity and initiation. The genes involved in this process have potential applications in enhancing crop yield.

The Ovulatory Signal Precipitates LRH-1 Transcriptional Switching Mediated by Differential Chromatin Accessibility

In the ovary, follicular growth and maturation are complicated processes that involve a series of morphological and physiological changes in oocytes and somatic cells leading to ovulation and luteinization, essential processes for fertility. Given the complexity of ovulation, characterization of genome-wide regulatory elements is essential to understand the mechanisms governing the expression of specific genes in the rapidly differentiating follicle. We therefore employed a systems biology approach to determine global transcriptional mechanisms during the early stages of the ovulatory process. We demonstrate that, following the hormonal signal that initiates ovulation, granulosa cells undergo major modification of distal regulatory elements, which coincides with cistrome reprogramming of the indispensable orphan nuclear receptor liver receptor homolog-1 (LRH-1). This cistromic reorganization correlates with the extensive changes in gene expression in granulosa cells leading to ovulation. Together, our study yields a highly detailed transcriptional map delineating ovarian cell differentiation during the initiation of ovulation.