ABSTRACT: Ovules are female reproductive organs of angiosperms, containing sporophytic integuments and gametophytic embryo sacs. After fertilization, embryo sacs develop into embryos and endosperm whereas integuments into seed coat. Ovule development is regulated by transcription factors (TF) whose expression is often controlled by microRNAs. Mutations of Arabidopsis DICER-LIKE 1 (DCL1), a microRNA processing protein, caused defective ovule development and reduced female fertility. However, it was not clear whether other microRNA processing proteins participate in this process and how defective ovule development influenced female fertility. We report that mutations of HUA ENHANCER1 (HEN1) and HYPONASTIC LEAVES 1 (HYL1) interfered with integument growth. The sporophytic defect caused abnormal embryo sac development and inability of mutant ovules to attract pollen tubes, leading to reduced female fertility. We show that the role of HEN1 in integument growth is cell-autonomous. Although AUXIN RESPONSE FACTOR 6 (ARF6) and ARF8 were ectopically expressed in mutant ovules, consistent with the reduction of microRNA167 in hen1, introducing arf6;arf8 did not suppress ovule defects of hen1, suggesting the involvement of more microRNAs in this process. Results presented indicate that the microRNA processing machinery is critical for ovule development and seed production through multiple microRNAs and their targets.
Project description:KANADI (KAN) transcription factors promote abaxial cell fate throughout plant development and are required for organ formation during embryo, leaf, carpel and ovule development. ABERRANT TESTA SHAPE (ATS, or KAN4) is necessary during ovule development to maintain the boundary between the two ovule integuments and to promote inner integument growth. Yeast two-hybrid assays identified ETTIN (ETT, or AUXIN RESPONSE FACTOR 3) as a transcription factor that could physically interact with ATS. ATS and ETT were shown to physically interact in vivo in transiently transformed tobacco epidermal cells using bimolecular fluorescence complementation. ATS and ETT were found to share an overlapping expression pattern during Arabidopsis ovule development and loss of either gene resulted in congenital fusion of the integuments and altered seed morphology. We hypothesize that in wild-type ovules a physical interaction between ATS and ETT allows these proteins to act in concert to define the boundary between integument primordia. We further show protein-protein interaction in yeast between ETT and KAN1, a paralog of ATS. Thus, a direct physical association between ETT and KAN proteins underpins their previously described common role in polarity establishment and organogenesis. We propose that ETT-KAN protein complex(es) constitute part of an auxin-dependent regulatory module that plays a conserved role in a variety of developmental contexts.
Project description:The angiosperm seed is composed of three genetically distinct tissues: the diploid embryo that originates from the fertilized egg cell, the triploid endosperm that is produced from the fertilized central cell, and the maternal sporophytic integuments that develop into the seed coat1. At the onset of embryo development in Arabidopsis thaliana, the zygote divides asymmetrically, producing a small apical embryonic cell and a larger basal cell that connects the embryo to the maternal tissue2. The coordinated and synchronous development of the embryo and the surrounding integuments, and the alignment of their growth axes, suggest communication between maternal tissues and the embryo. In contrast to animals, however, where a network of maternal factors that direct embryo patterning have been identified3,4, only a few maternal mutations have been described to affect embryo development in plants5-7. Early embryo patterning in Arabidopsis requires accumulation of the phytohormone auxin in the apical cell by directed transport from the suspensor8-10. However, the origin of this auxin has remained obscure. Here we investigate the source of auxin for early embryogenesis and provide evidence that the mother plant coordinates seed development by supplying auxin to the early embryo from the integuments of the ovule. We show that auxin response increases in ovules after fertilization, due to upregulated auxin biosynthesis in the integuments, and this maternally produced auxin is required for correct embryo development.
Project description:BACKGROUND: Arabidopsis ovules comprise four morphologically distinct parts: the nucellus, which contains the embryo sac, two integuments that become the seed coat, and the funiculus that anchors the ovule within the carpel. Analysis of developmental mutants has shown that ovule morphogenesis relies on tightly regulated genetic interactions that can serve as a model for developmental regulation. Redundancy, pleiotropic effects and subtle phenotypes may preclude identification of mutants affecting some processes in screens for phenotypic changes. Expression-based gene discovery can be used access such obscured genes. RESULTS: Affymetrix microarrays were used for expression-based gene discovery to identify sets of genes expressed in either or both integuments. The genes were identified by comparison of pistil mRNA from wild type with mRNA from two mutants; inner no outer (ino, which lacks the outer integument), and aintegumenta (ant, which lacks both integuments). Pools of pistils representing early and late stages of ovule development were evaluated and data from the three genotypes were used to designate genes that were predominantly expressed in the integuments using pair-wise and cluster analyses. Approximately two hundred genes were found to have a high probability of preferential expression in these structures, and the predictive nature of the expression classes was confirmed with reverse transcriptase polymerase chain reaction and in situ hybridization. CONCLUSION: The results showed that it was possible to use a mutant, ant, with broad effects on plant phenotype to identify genes expressed specifically in ovules, when coupled with predictions from known gene expression patterns, or in combination with a more specific mutant, ino. Robust microarray averaging (RMA) analysis of array data provided the most reliable comparisons, especially for weakly expressed genes. The studies yielded an over-abundance of transcriptional regulators in the identified genes, and these form a set of candidate genes for evaluation of roles in ovule development using reverse genetics.
Project description:Ovules are the female reproductive structures that develop into seeds. Angiosperm ovules include one, or more commonly two, integuments that cover the nucellus and female gametophyte. Mutations in the Arabidopsis KANADI (KAN) and YABBY polarity genes result in amorphous or arrested integument growth, suggesting that polarity determinants play key roles in ovule development. We show that the class III homeodomain leucine zipper (HD-ZIPIII) genes CORONA (CNA), PHABULOSA (PHB) and PHAVOLUTA (PHV) are expressed adaxially in the inner integument during ovule development, independent of ABERRANT TESTA SHAPE (ATS, also known as KANADI4) activity. Loss of function of these genes leads to aberrant integument growth. Additionally, over-expression of PHB or PHV in ovules is not sufficient to repress ATS expression, and can produce phenotypes similar to those of the HD-ZIPIII loss-of-function lines. The absence of evidence of mutual negative regulation by KAN and HD-ZIPIII transcription factors is in contrast to known mechanisms in leaves. Loss of HD-ZIPIII activity can partially compensate for loss of ATS activity in the ats cna phb phv quadruple mutant, showing that CNA/PHB/PHV act in concert with ATS to control integument morphogenesis. In a parallel pathway, ATS acts with REVOLUTA (REV) to restrict expression of INNER NO OUTER (INO) and outer integument growth. Based on these expression and genetic studies, we propose a model in which a balance between the relative levels of adaxial/abaxial activities, rather than maintenance of boundaries of expression domains, is necessary to support laminar growth of the two integuments.
Project description:Many homeotic MADS-box genes have been identified as controllers of the floral transition and floral development. However, information regarding Bsister (Bs)-function genes in monocots is still limited. Here, we describe the functional characterization of a Bs-group MADS-box gene FEMALE-STERILE (FST), whose frame-shift mutation (fst) results in abnormal ovules and the complete abortion of zygotic embryos and endosperms in rice. Anatomical analysis showed that the defective development in the fst mutant exclusively occurred in sporophytic tissues including integuments, fertilized proembryos and endosperms. Analyses of the spatio-temporal expression pattern revealed that the prominent FST gene products accumulated in the inner integument, nucellar cell of the micropylar side, apical and base of the proembryos and free endosperm nuclei. Microarray and gene ontology analysis unraveled substantial changes in the expression level of many genes in the fst mutant ovules and seeds, with a subset of genes involved in several developmental and hormonal pathways appearing to be down-regulated. Using both forward and reverse genetics approaches, we demonstrated that rice FST plays indispensable roles and multiple functions during ovule and early seed development. These findings support a novel function for the Bs-group MADS-box genes in plants.
Project description:BACKGROUND: The Arabidopsis outer ovule integument is a simple two-cell layered structure that grows around the developing embryo and develops into the outer layer of the seed coat. As one of the functions of the seed coat is the protection of the plant embryo, the outer ovule integument is an example for a plant organ whose morphogenesis has to be precisely regulated. RESULTS: To better characterise outer ovule integument morphogenesis, we have isolated some marker lines that show GFP expression in this organ. We have used those lines to identify distinct cell types in the outer integument and to demonstrate similarities between leaves and the outer integument. Using confocal microscopy, we showed that cell sizes and shapes differ between the two cell layers of the outer integument. Expression of KNAT1 in the integuments leads to extra cell divisions specifically in the outer layer of the outer integument. This is being compensated for by a decrease of cell volume in this layer, thus showing that mechanisms exist to control proper ovule integument morphogenesis. CONCLUSION: The Arabidopsis outer ovule integument can be used as a good model system to study the basic principles of plant organ morphogenesis. This work provides new insights into its development and opens new possibilities for the identification of factors involved in the regulation of cell division and elongation during plant organ growth.
Project description:Apomixis in Hieracium subgenus Pilosella initiates in ovules when sporophytic cells termed aposporous initial (AI) cells enlarge near sexual cells undergoing meiosis. AI cells displace the sexual structures and divide by mitosis to form unreduced embryo sac(s) without meiosis (apomeiosis) that initiate fertilization-independent embryo and endosperm development. In some Hieracium subgenus Pilosella species, these events are controlled by the dominant LOSS OF APOMEIOSIS (LOA) and LOSS OF PARTHENOGENESIS (LOP) loci. In H. praealtum and H. piloselloides, which both contain the same core LOA locus, the timing and frequency of AI cell formation is altered in derived mutants exhibiting abnormal funiculus growth and in transgenic plants expressing rolB which alters cellular sensitivity to auxin. The impact on apomictic and sexual reproduction was examined here when a chimeric RNAse gene was targeted to the funiculus and basal portions of the ovule, and also when polar auxin transport was inhibited during ovule development following N-1-naphthylphthalamic acid (NPA) application. Both treatments led to ovule deformity in the funiculus and distal parts of the ovule and LOA-dependent alterations in the timing, position, and frequency of AI cell formation. In the case of NPA treatment, this correlated with increased expression of DR5:GFP in the ovule, which marks the accumulation of the plant hormone auxin. Our results show that sporophytic information potentiated by funiculus growth and polar auxin transport influences ovule development, the initiation of apomixis, and the progression of embryo sac development in Hieracium. Signals associated with ovule pattern formation and auxin distribution or perception may influence the capacity of sporophytic ovule cells to respond to LOA.
Project description:This experiment was designed to identify genes expressed preferentially in the two integuments of the Arabidopsis ovule. Pistils from wild type and two ovule mutants were compared against each aintegumenta-4 (ant-4) which lacks both integuments and inner no outer (ino-1) which lacks the outer integument. Genes that are highly expressed only in the integuments were expected to be reduced in expression in the mutants, as compared with wild type. Pistils containing ovules through all stages of ovule development prior to pollination were pooled for one experiment (FULL arrays), and for two separate experiments, a set of early differentiation stages (EARLY arrays) and a set of later differentiation stages (LATE ARRAYS) were pooled. Wild type and mutant lines are in the ecotype Landsberg erecta.
Project description:The ovule plays a critical role in cereal yield as it is the site of fertilization and the progenitor of the grain. The ovule primordium is generally comprised of three domains, the funiculus, chalaza, and nucellus, which give rise to distinct tissues including the integuments, nucellar projection, and embryo sac. The size and arrangement of these domains varies significantly between model eudicots, such as Arabidopsis thaliana, and agriculturally important monocotyledonous cereal species, such as Hordeum vulgare (barley). However, the amount of variation in ovule development among genotypes of a single species, and its functional significance, remains unclear. To address this, wholemount clearing was used to examine the details of ovule development in barley. Nine sporophytic and gametophytic features were examined at ovule maturity in a panel of 150 European two-row spring barley genotypes, and compared with grain traits from the preceding and same generation. Correlations were identified between ovule traits and features of grain they produced, which in general highlighted a negative correlation between nucellus area, ovule area, and grain weight. We speculate that the amount of ovule tissue, particularly the size of the nucellus, may affect the timing of maternal resource allocation to the fertilized embryo sac, thereby influencing subsequent grain development.
Project description:The number of integuments found in angiosperm ovules is variable. In orchids, most species show bitegmic ovules, except for some mycoheterotrophic species that show ovules with only one integument. Analysis of ovules and the development of the seed coat provide important information regarding functional aspects such as dispersal and seed germination. This study aimed to analyze the origin and development of the seed coat of the mycoheterotrophic orchid Pogoniopsis schenckii and to compare this development with that of other photosynthetic species of the family. Flowers and fruits at different stages of development were collected, and the usual methodology for performing anatomical studies, scanning microscopy, and transmission microscopy following established protocols. P. schenckii have ategmic ovules, while the other species are bitegmic. No evidence of integument formation at any stage of development was found through anatomical studies. The reduction of integuments found in the ovules could facilitate fertilization in this species. The seeds of P. schenckii, Vanilla planifolia, and V. palmarum have hard seed coats, while the other species have seed coats formed by the testa alone, making them thin and transparent. P. schenckii, in contrast to the other species analyzed, has a seed coat that originates from the nucellar epidermis, while in other species, the seed coat originates from the outer integument.