The Arabidopsis INNER NO OUTER (INO) gene acts exclusively and quantitatively in regulation of ovule outer integument development.

The INNER NO OUTER (INO) gene is essential for formation of the outer integument of ovules in Arabidopsis thaliana. Initially described lesions in INO were missense mutations resulting in aberrant mRNA splicing. To determine the null mutant phenotype, we generated frameshift mutations and found, in confirmation of results on another recently identified frameshift mutation, that such mutants have a phenotype identical to the most severe splicing mutant (ino-1), with effects specific to outer integument development. We show that the altered protein of an ino mRNA splicing mutant with a less severe phenotype (ino-4) does not have INO activity, and the mutant is partial because it produces a small amount of correctly spliced INO mRNA. Screening for suppressors of ino-4 in a fast neutron-mutagenized population identified a translocated duplication of the ino-4 gene, leading to an increase in the amount of this mRNA. The increased expression led to a decrease in the severity of the mutant effects, indicating that the amount of INO activity quantitatively regulates outer integument growth. The results further confirm that the role of INO in Arabidopsis development is specific to the outer integument of ovules where it quantitatively affects the growth of this structure.

Functional conservation of the grapevine candidate gene INNER NO OUTER for ovule development and seed formation.

Seedlessness represents a highly appreciated trait in table grapes. Based on an interesting case of seedless fruit production described in the crop species Annona squamosa, we focused on the Vitis vinifera INNER NO OUTER (INO) gene as a candidate. This gene encodes a transcription factor belonging to the YABBY family involved in the determination of abaxial identity in several organs. In Arabidopsis thaliana, this gene was shown to be essential for the formation and asymmetric growth of the ovule outer integument and its mutation leads to a phenotypic defect of ovules and failure in seed formation. In this study, we identified in silico the V. vinifera orthologue and investigated its phylogenetic relationship to INO genes from other species and its expression in different organs in seeded and seedless varieties. Applying cross-species complementation, we have tested its functionality in the Arabidopsis ino-1 mutant. We show that the V. vinifera INO successfully rescues the ovule outer integument growth and seeds set and also partially complements the outer integument asymmetric growth in the Arabidopsis mutant, differently from orthologues from other species. These data demonstrate that VviINO retains similar activity and protein targets in grapevine as in Arabidopsis. Potential implications for grapevine breeding are discussed.

Development and evolution of the unique ovules of flowering plants.

Ovules are the precursors to seeds and as such are critical to plant propagation and food production. Mutant studies have led to the identification of numerous genes regulating ovule development. Genes encoding transcription factors have been shown to direct ovule spacing, ovule identity and integument formation. Particular co-regulators have now been associated with activities of some of these transcription factors, and other protein families including cell surface receptors have been shown to regulate ovule development. Hormone levels and transport, especially of auxin, have also been shown to play critical roles in ovule emergence and morphogenesis and to interact with the transcriptional regulators. Ovule diversification has been studied using orthologs of regulatory genes in divergent angiosperm groups. Combining modern genetic evidence with expanding knowledge of the fossil record illuminates the possible origin of the unique bitegmic ovules of angiosperms.

Recent advances in understanding female gametophyte development.

The haploid female gametophyte (embryo sac) is an essential reproductive unit of flowering plants, usually comprising four specialized cell types, including the female gametes (egg cell and central cell). The differentiation of these cells relies on spatial signals which pattern the gametophyte along a proximal-distal axis, but the molecular and genetic mechanisms by which cell identities are determined in the embryo sac have long been a mystery. Recent identification of key genes for cell fate specification and their relationship to hormonal signaling pathways that act on positional cues has provided new insights into these processes. A model for differentiation can be devised with egg cell fate as a default state of the female gametophyte and with other cell types specified by the action of spatially regulated factors. Cell-to-cell communication within the gametophyte is also important for maintaining cell identity as well as facilitating fertilization of the female gametes by the male gametes (sperm cells).

Integument Development in Arabidopsis Depends on Interaction of YABBY Protein INNER NO OUTER with Coactivators and Corepressors.

Arabidopsis thaliana INNER NO OUTER (INO) is a YABBY protein that is essential for the initiation and development of the outer integument of ovules. Other YABBY proteins have been shown to be involved in both negative and positive regulation of expression of putative target genes. YABBY proteins have also been shown to interact with the corepressor LEUNIG (LUG) in several systems. In support of a repressive role for INO, we confirm that INO interacts with LUG and also find that INO directly interacts with SEUSS (SEU), a known corepressive partner of LUG. Further, we find that INO can directly interact with ADA2b/PROPORZ1 (PRZ1), a transcriptional coactivator that is known to interact with the histone acetyltransferase GENERAL CONTROL NONREPRESSIBLE PROTEIN 5 (GCN5, also known as HAG1). Mutations in LUG, SEU, and ADA2b/PRZ1 all lead to pleiotropic effects including a deficiency in the extension of the outer integument. Additive and synergistic effects of ada2b/prz1 and lug mutations on outer integument formation indicate that these two genes function independently to promote outer integument growth. The ino mutation is epistatic to both lug and ada2b/prz1 in the outer integument, and all three proteins are present in the nuclei of a common set of outer integument cells. This is consistent with a model where INO utilizes these coregulator proteins to activate and repress separate sets of target genes. Other Arabidopsis YABBY proteins were shown to also form complexes with ADA2b/PRZ1, and have been previously shown to interact with SEU and LUG. Thus, interaction with these corepressors and coactivator may represent a general mechanism to explain the positive and negative activities of YABBY proteins in transcriptional regulation. The LUG, SEU, and ADA2b/PRZ1 proteins would also separately be recruited to targets of other transcription factors, consistent with their roles as general coregulators, explaining the pleiotropic effects not associated with YABBY function.

Conservation of the role of INNER NO OUTER in development of unitegmic ovules of the Solanaceae despite a divergence in protein function.

BACKGROUND: The INNER NO OUTER (INO) gene is expressed in the outermost cell layer of the outer integument of bitegmic ovules and is essential for this organ's growth. The role and cross-species functional conservation of INO orthologs were examined in members of the Solanaceae, which have unitegmic ovules. Unitegmy has evolved several times in disparate angiosperm lineages. INO expression has been observed in the outermost cell layers of all examined unitegmic ovules, but the functional role of INO in unitegmic ovules has not previously been evaluated. RESULTS: INO orthologs were unambiguously identified in tobacco and tomato by sequence homology. Expression of the tomato INO gene was limited to the outer cell layer of the single integument indicating that this single integument has properties of the outer integument. Expression occurred only after integument initiation, later than observed in ovules of other examined angiosperms. Virus-induced knock-down of expression of the INO ortholog in tobacco inhibited growth of the outer cell layer of the integument leading to a decrease in both integument extension and curvature of the ovule. The altered ovules closely resemble those of the aberrant testa shape (ats) ino mutant combination in Arabidopsis where we see the effect of the ino mutation on a single fused integument produced by the ats mutation. Despite significant sequence identity and similar expression patterns, the tomato INO coding region was not able to complement the Arabidopsis ino mutant. CONCLUSIONS: The similarity of effects of ino mutations on the unitegmic ovules of tobacco and the fused integuments of the Arabidopsis ats mutant show that: 1) INO orthologs play the same role in promoting integument growth in ovules of tobacco and Arabidopsis; and 2) the unitegmic ovules of tobacco (and hence other solanaceous species) are most likely the result of a congenital fusion of two ancestral integuments. Our results further indicate that INO has a conserved role in growth of the outermost cell layer of integuments. The curvature of solanaceous ovules is driven by unequal growth of the outer layers of the single integument that likely correspond to an ancestral outer integument.

Rare genetic variation at Zea mays crtRB1 increases beta-carotene in maize grain.

Breeding to increase beta-carotene levels in cereal grains, termed provitamin A biofortification, is an economical approach to address dietary vitamin A deficiency in the developing world. Experimental evidence from association and linkage populations in maize (Zea mays L.) demonstrate that the gene encoding beta-carotene hydroxylase 1 (crtRB1) underlies a principal quantitative trait locus associated with beta-carotene concentration and conversion in maize kernels. crtRB1 alleles associated with reduced transcript expression correlate with higher beta-carotene concentrations. Genetic variation at crtRB1 also affects hydroxylation efficiency among encoded allozymes, as observed by resultant carotenoid profiles in recombinant expression assays. The most favorable crtRB1 alleles, rare in frequency and unique to temperate germplasm, are being introgressed via inexpensive PCR marker-assisted selection into tropical maize germplasm adapted to developing countries, where it is most needed for human health.

Advances in maize genomics and their value for enhancing genetic gains from breeding.

Maize is an important crop for food, feed, forage, and fuel across tropical and temperate areas of the world. Diversity studies at genetic, molecular, and functional levels have revealed that, tropical maize germplasm, landraces, and wild relatives harbor a significantly wider range of genetic variation. Among all types of markers, SNP markers are increasingly the marker-of-choice for all genomics applications in maize breeding. Genetic mapping has been developed through conventional linkage mapping and more recently through linkage disequilibrium-based association analyses. Maize genome sequencing, initially focused on gene-rich regions, now aims for the availability of complete genome sequence. Conventional insertion mutation-based cloning has been complemented recently by EST- and map-based cloning. Transgenics and nutritional genomics are rapidly advancing fields targeting important agronomic traits including pest resistance and grain quality. Substantial advances have been made in methodologies for genomics-assisted breeding, enhancing progress in yield as well as abiotic and biotic stress resistances. Various genomic databases and informatics tools have been developed, among which MaizeGDB is the most developed and widely used by the maize research community. In the future, more emphasis should be given to the development of tools and strategic germplasm resources for more effective molecular breeding of tropical maize products.

Expression-based discovery of candidate ovule development regulators through transcriptional profiling of ovule mutants.

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.

Roles of polarity determinants in ovule development.

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.

ABERRANT TESTA SHAPE encodes a KANADI family member, linking polarity determination to separation and growth of Arabidopsis ovule integuments.

The Arabidopsis aberrant testa shape (ats) mutant produces a single integument instead of the two integuments seen in wild-type ovules. Cellular anatomy and patterns of marker gene expression indicate that the single integument results from congenital fusion of the two integuments of the wild type. Isolation of the ATS locus showed it to encode a member of the KANADI (KAN) family of putative transcription factors, previously referred to as KAN4. ATS was expressed at the border between the two integuments at the time of their initiation, with expression later confined to the abaxial layer of the inner integument. In an inner no outer (ino) mutant background, where an outer integument does not form, the ats mutation led to amorphous inner integument growth. The kan1kan2 double mutant exhibits a similar amorphous growth of the outer integument without affecting inner integument growth. We hypothesize that ATS and KAN1/KAN2 play similar roles in the specification of polarity in the inner and outer integuments, respectively, that parallel the known roles of KAN proteins in promoting abaxial identity during leaf development. INO and other members of the YABBY gene family have been hypothesized to have similar parallel roles in outer integument and leaf development. Together, these two hypotheses lead us to propose a model for normal integument growth that also explains the described mutant phenotypes.