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

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

The RNA-dependent DNA methylation pathway is required to restrict SPOROCYTELESS/NOZZLE expression to specify a single female germ cell precursor in Arabidopsis.

In higher plants, the female germline is formed from the megaspore mother cell (MMC), a single cell in the premeiotic ovule. Previously, it was reported that mutants in the RNA-dependent DNA methylation (RdDM) pathway might be involved in restricting the female germline to a single nucellus cell. We show that the DRM methyltransferase double mutant drm1drm2 also presents ectopic enlarged cells, consistent with supernumerary MMC-like cells. In wild-type ovules, MMC differentiation requires SPOROCYTELESS/NOZZLE (SPL/NZZ), as demonstrated by the spl/nzz mutant failing to develop an MMC. We address the poorly understood upstream regulation of SPL/NZZ in ovules, showing that the RdDM pathway is important to restrict SPL/NZZ expression. In ago9, rdr6 and drm1drm2 mutants, SPL/NZZ is expressed ectopically, suggesting that the multiple MMC-like cells observed might be attributable to the ectopic expression of SPL/NZZ. We show that the ovule identity gene, SEEDSTICK, directly regulates AGO9 and RDR6 expression in the ovule and therefore indirectly regulates SPL/NZZ expression. A model is presented describing the network required to restrict SPL/NZZ expression to specify a single MMC.

Asexual reproduction through seeds: the complex case of diplosporous apomixis.

Apomixis is considered a potentially revolutionary tool to generate high-quality food at a lower cost and shorter developmental time due to clonal seed production through apomeiosis and parthenogenesis. In the diplosporous type of apomixis, meiotic recombination and reduction are circumvented either by avoiding or failing meiosis or by a mitotic-like division. Here, we review the literature on diplospory, from early cytological studies dating back to the late 19th century to recent genetic findings. We discuss diplosporous developmental mechanisms, including their inheritance. Furthermore, we compare the strategies adopted to isolate the genes controlling diplospory with those to produce mutants forming unreduced gametes. Nowadays, the dramatically improved technologies of long-read sequencing and targeted CRISPR/Cas mutagenesis justify the expectation that natural diplospory genes will soon be identified. Their identification will answer questions such as how the apomictic phenotype can be superimposed upon the sexual pathway and how diplospory genes have evolved. This knowledge will contribute to the application of apomixis in agriculture.

Gynoecium size and ovule number are interconnected traits that impact seed yield.

Angiosperms form the largest group of land plants and display an astonishing diversity of floral structures. The development of flowers greatly contributed to the evolutionary success of the angiosperms as they guarantee efficient reproduction with the help of either biotic or abiotic vectors. The female reproductive part of the flower is the gynoecium (also called pistil). Ovules arise from meristematic tissue within the gynoecium. Upon fertilization, these ovules develop into seeds while the gynoecium turns into a fruit. Gene regulatory networks involving transcription factors and hormonal communication regulate ovule primordium initiation, spacing on the placenta, and development. Ovule number and gynoecium size are usually correlated and several genetic factors that impact these traits have been identified. Understanding and fine-tuning the gene regulatory networks influencing ovule number and pistil length open up strategies for crop yield improvement, which is pivotal in light of a rapidly growing world population. In this review, we present an overview of the current knowledge of the genes and hormones involved in determining ovule number and gynoecium size. We propose a model for the gene regulatory network that guides the developmental processes that determine seed yield.

The Importance of Cytokinins during Reproductive Development in Arabidopsis and Beyond.

Fertilization and seed formation are fundamental events in the life cycle of flowering plants. The seed is a functional unit whose main purpose is to propagate the plant. The first step in seed development is the formation of male and female gametophytes and subsequent steps culminate in successful fertilization. The detailed study of this process is highly relevant because it directly impacts human needs, such as protecting biodiversity and ensuring sustainable agriculture to feed the increasing world population. Cytokinins comprise a class of phytohormones that play many important roles during plant growth and development and in recent years, the role of this class of phytohormones during reproduction has become clear. Here, we review the role of cytokinins during ovule, pollen and seed formation at the genetic and molecular levels. The expansion of knowledge concerning the molecular mechanisms that control plant reproduction is extremely important to optimise seed production.

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.

CUP-SHAPED COTYLEDON1 (CUC1) and CUC2 regulate cytokinin homeostasis to determine ovule number in Arabidopsis.

Seeds derive from ovules upon fertilization and therefore the total number of ovules determines the final seed yield, a fundamental trait in crop plants. Among the factors that co-ordinate the process of ovule formation, the transcription factors CUP-SHAPED COTYLEDON 1 (CUC1) and CUC2 and the hormone cytokinin (CK) have a particularly prominent role. Indeed, the absence of both CUC1 and CUC2 causes a severe reduction in ovule number, a phenotype that can be rescued by CK treatment. In this study, we combined CK quantification with an integrative genome-wide target identification approach to select Arabidopsis genes regulated by CUCs that are also involved in CK metabolism. We focused our attention on the functional characterization of UDP-GLUCOSYL TRANSFERASE 85A3 (UGT85A3) and UGT73C1, which are up-regulated in the absence of CUC1 and CUC2 and encode enzymes able to catalyse CK inactivation by O-glucosylation. Our results demonstrate a role for these UGTs as a link between CUCs and CK homeostasis, and highlight the importance of CUCs and CKs in the determination of seed yield.

Attenuation of pattern recognition receptor signaling is mediated by a MAP kinase kinase kinase.

Pattern recognition receptors (PRRs) play a key role in plant and animal innate immunity. PRR binding of their cognate ligand triggers a signaling network and activates an immune response. Activation of PRR signaling must be controlled prior to ligand binding to prevent spurious signaling and immune activation. Flagellin perception in Arabidopsis through FLAGELLIN-SENSITIVE 2 (FLS2) induces the activation of mitogen-activated protein kinases (MAPKs) and immunity. However, the precise molecular mechanism that connects activated FLS2 to downstream MAPK cascades remains unknown. Here, we report the identification of a differentially phosphorylated MAP kinase kinase kinase that also interacts with FLS2. Using targeted proteomics and functional analysis, we show that MKKK7 negatively regulates flagellin-triggered signaling and basal immunity and this requires phosphorylation of MKKK7 on specific serine residues. MKKK7 attenuates MPK6 activity and defense gene expression. Moreover, MKKK7 suppresses the reactive oxygen species burst downstream of FLS2, suggesting that MKKK7-mediated attenuation of FLS2 signaling occurs through direct modulation of the FLS2 complex.

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.

Cytochrome P450 CYP78A9 is involved in Arabidopsis reproductive development.

Synchronized communication between gametophytic and sporophytic tissue is crucial for successful reproduction, and hormones seem to have a prominent role in it. Here, we studied the role of the Arabidopsis (Arabidopsis thaliana) cytochrome P450 CYP78A9 enzyme during reproductive development. First, controlled pollination experiments indicate that CYP78A9 responds to fertilization. Second, while CYP78A9 overexpression can uncouple fruit development from fertilization, the cyp78a8 cyp78a9 loss-of-function mutant has reduced seed set due to outer ovule integument development arrest, leading to female sterility. Moreover, CYP78A9 has a specific expression pattern in inner integuments in early steps of ovule development as well as in the funiculus, embryo, and integuments of developing seeds. CYP78A9 overexpression did not change the response to the known hormones involved in flower development and fruit set, and it did not seem to have much effect on the major known hormonal pathways. Furthermore, according to previous predictions, perturbations in the flavonol biosynthesis pathway were detected in cyp78a9, cyp78a8 cyp78a9, and empty siliques (es1-D) mutants. However, it appeared that they do not cause the observed phenotypes. In summary, these results add new insights into the role of CYP78A9 in plant reproduction and present, to our knowledge, the first characterization of metabolite differences between mutants in this gene family.

The impact of heat stress in plant reproduction.

The increment in global temperature reduces crop productivity, which in turn threatens food security. Currently, most of our food supply is produced by plants and the human population is estimated to reach 9 billion by 2050. Gaining insights into how plants navigate heat stress in their reproductive phase is essential for effectively overseeing the future of agricultural productivity. The reproductive success of numerous plant species can be jeopardized by just one exceptionally hot day. While the effects of heat stress on seedlings germination and root development have been extensively investigated, studies on reproduction are limited. The intricate processes of gamete development and fertilization unfold within a brief timeframe, largely concealed within the flower. Nonetheless, heat stress is known to have important effects on reproduction. Considering that heat stress typically affects both male and female reproductive structures concurrently, it remains crucial to identify cultivars with thermotolerance. In such cultivars, ovules and pollen can successfully undergo development despite the challenges posed by heat stress, enabling the completion of the fertilization process and resulting in a robust seed yield. Hereby, we review the current understanding of the molecular mechanisms underlying plant resistance to abiotic heat stress, focusing on the reproductive process in the model systems of Arabidopsis and Oryza sativa.

Auxin and Flower Development: A Blossoming Field.

The establishment of the species-specific floral organ body plan involves many coordinated spatiotemporal processes, which include the perception of positional information that specifies floral meristem and floral organ founder cells, coordinated organ outgrowth coupled with the generation and maintenance of inter-organ and inter-whorl boundaries, and the termination of meristem activity. Auxin is integrated within the gene regulatory networks that control these processes and plays instructive roles at the level of tissue-specific biosynthesis and polar transport to generate local maxima, perception, and signaling. Key features of auxin function in several floral contexts include cell nonautonomy, interaction with cytokinin gradients, and the central role of MONOPTEROS and ETTIN to regulate canonical and noncanonical auxin response pathways, respectively. Arabidopsis flowers are not representative of the enormous angiosperm floral diversity; therefore, comparative studies are required to understand how auxin underlies these developmental differences. It will be of great interest to compare the conservation of auxin pathways among flowering plants and to discuss the evolutionary role of auxin in floral development.

The emerging role of small RNAs in ovule development, a kind of magic.

In plants, small RNAs have been recognized as key genetic and epigenetic regulators of development. Small RNAs are usually 20 to 30 nucleotides in length and they control, in a sequence specific manner, the transcriptional or post-transcriptional expression of genes. In this review, we present a comprehensive overview of the most recent findings about the function of small RNAs in ovule development, including megasporogenesis and megagametogenesis, both in sexual and apomictic plants. We discuss recent studies on the role of miRNAs, siRNAs and trans-acting RNAs (ta-siRNAs) in early female germline differentiation. The mechanistic complexity and unique regulatory features are reviewed, and possible directions for future research are provided.

Alternative Splicing Generates a MONOPTEROS Isoform Required for Ovule Development.

The plant hormone auxin is a fundamental regulator of organ patterning and development that regulates gene expression via the canonical AUXIN RESPONSE FACTOR (ARF) and AUXIN/INDOLE-3-ACETIC ACID (Aux/IAA) combinatorial system. ARF and Aux/IAA factors interact, but at high auxin concentrations, the Aux/IAA transcriptional repressor is degraded, allowing ARF-containing complexes to activate gene expression. ARF5/MONOPTEROS (MP) is an important integrator of auxin signaling in Arabidopsis development and activates gene transcription in cells with elevated auxin levels. Here, we show that in ovules, MP is expressed in cells with low levels of auxin and can activate the expression of direct target genes. We identified and characterized a splice variant of MP that encodes a biologically functional isoform that lacks the Aux/IAA interaction domain. This MP11ir isoform was able to complement inflorescence, floral, and ovule developmental defects in mp mutants, suggesting that it was fully functional. Our findings describe a novel scenario in which ARF post-transcriptional regulation controls the formation of an isoform that can function as a transcriptional activator in regions of subthreshold auxin concentration.

Ovule development, a new model for lateral organ formation.

In spermatophytes the ovules upon fertilization give rise to the seeds. It is essential to understand the mechanisms that control ovule number and development as they ultimately determine the final number of seeds and, thereby, the yield in crop plants. In Arabidopsis thaliana, ovules arise laterally from a meristematic tissue within the carpel referred to as placenta. For a correct determination of the number of ovules, a precise establishment of the positions where ovule primordia emerge is needed, and a tight definition of the boundaries between ovules is therefore also required. In the last decades, few factors have been identified to be involved in the determination of ovule number. Recently, plant hormones have also been revealed as fundamental players in the control of the initiation of ovule formation. In this review we summarize the current knowledge about both the molecular and hormonal mechanisms that control ovule formation in Arabidopsis thaliana.

Toward understanding the role of CYP78A9 during Arabidopsis reproduction.

After fertilization in Arabidopsis, auxin response in ovules triggers fruit development through the stimulation of gibberellin metabolism. In a recent work, we showed that this model could not explain why CYP78A9 overexpression can uncouple these processes. The specific expression pattern of CYP78A9 suggests its involvement during reproductive development. Moreover, controlled pollination showed that CYP78A9 responds to fertilization. The genetic evidence supports the idea that CYP78A9 and its closest paralogs participate in a pathway that control floral organ size and ovule integuments development as denoted by the phenotypes of es1-D overexpression and cyp78a8 cyp78a9 double mutants. Furthermore, according to previous predictions, perturbations in the flavonol biosynthesis pathway were detected in cyp78a9, cyp78a8 cyp78a9 and es1-D mutants. However, they do not cause the observed phenotypes. Our results add new insights into the role of CYP78A9 in plant reproduction and present the first characterization of metabolite differences between mutants in this gene family.