INNER NO OUTER Research Articles

Brassinosteroid signaling represses ZINC FINGER PROTEIN11 to regulate ovule development in Arabidopsis.

Brassinosteroid (BR) signaling and the C-class MADS-box gene AGAMOUS (AG) play important roles in ovule development in Arabidopsis (Arabidopsis thaliana). However, how BR signaling integrates with AG functions to control the female reproductive process remains elusive. Here, we showed that the regulatory role of BR signaling in proper ovule development is mediated by the transcriptional repressor gene ZINC FINGER PROTEIN 11 (ZFP11), which is a direct target of AG. ZFP11 expression initiates from the placenta upon AG induction and becomes prominent in the funiculus of ovule primordia. Plants harboring zfp11 mutations showed reduced placental length with decreased ovule numbers and some aborted ovules. During ovule development, the transcription factor BRASSINAZOLE-RESISTANT 1 (BZR1), which functions downstream of BR signaling, inhibits ZFP11 expression in the chalaza and nucellus. Weakened BR signaling leads to stunted integuments in ovules, resulting from the direct repression of INNER NO OUTER (INO) and WUSCHEL (WUS) by extended ZFP11 expression in the chalaza and nucellus, respectively. In addition, the zfp11 mutant shows reduced sensitivity to BR biosynthesis inhibitors and can rescue outer integument defects in brassinosteroid insensitive 1 (bri1) mutants. Thus, the precise spatial regulation of ZFP11, which is activated by AG in the placenta and suppressed by BR signaling in the central and distal regions of ovules, is essential for ensuring sufficient ovule numbers and proper ovule formation.

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.

Global gene expression profile and functional analysis reveal the conservation of reproduction-associated gene networks in Gossypium hirsutum.

Lastly, the bZIP gene family encompasses genes that have been reported to play a role in flower development, such as bZIP14 (FD). Notably, bZIP14 is essential for Flowering Locus T (FT) initiation of floral development in Arabidopsis (Abe et al. 2005). Cotton (Gossypium hirsutum L.) is the world's most extensively cultivated fiber crop. However, its reproductive development is poorly characterized at the molecular level. Thus, this study presents a detailed transcriptomic analysis of G. hirsutum at three different reproductive stages. We provide evidence that more than 64,000 genes are active in G. hirsutum during flower development, among which 94.33% have been assigned to functional terms and specific pathways. Gene set enrichment analysis (GSEA) revealed that the biological process categories of floral organ development, pollen exine formation, and stamen development were enriched among the genes expressed during the floral development of G. hirsutum. Furthermore, we identified putative Arabidopsis homologs involved in the G. hirsutum gene regulatory network (GRN) of pollen and flower development, including transcription factors such as WUSCHEL (WUS), INNER NO OUTER (INO), AGAMOUS-LIKE 66 (AGL66), SPOROCYTELESS/NOZZLE (SPL/NZZ), DYSFUNCTIONAL TAPETUM 1 (DYT1), ABORTED MICROSPORES (AMS), and ASH1-RELATED 3 (ASHR3), which are known crucial genes for plant reproductive success. The cotton MADS-box protein-protein interaction pattern resembles the previously described patterns for AGAMOUS (AG), SEEDSTICK (STK), SHATTERPROOF (SHP), and SEPALLATA3 (SEP3) homolog proteins from Arabidopsis. In addition to serving as a resource for comparative flower development studies, this work highlights the changes in gene expression profiles and molecular networks underlying stages that are valuable for cotton breeding improvement.

Seedless fruit in Annona squamosa L. is monogenic and conferred by INO locus deletion in multiple accessions

Key messageInheritance of the presence/absence of seeds in Annona squamosa is mediated by a single fully recessive gene and is caused by a deletion of the INNER NO OUTER (INO) locus.For some fruits, seedless varieties are desirable for consumption and processing. In the sugar apple tree (Annona squamosa L.), the seedless trait in the Thai seedless (Ts) and Brazilian seedless (Bs) accessions was associated with defective ovules and an apparent deletion of the INNER NO OUTER (INO) ovule development gene locus. Segregation analysis of F2 and backcross descendants of crosses of Bs to fertile wild-type varieties in this species with a multi-year generation time showed that seedlessness was recessive and controlled by a single locus. Comparison of whole genome sequence of a wild-type plant and a third accession, Hawaiian seedless (Hs), identified a 16 kilobase deletion including INO in this line. Ts and Bs lines were shown to have an identical deletion, indicating a common origin from a single deletion event. Analysis of microsatellite markers could not preclude the possibility that all three seedless accessions are vegetatively propagated clones. The sequence of the deletion site enabled a codominant assay for the wild-type and mutant genes allowing observation of complete cosegregation of the seedless/defective ovule phenotype with the INO deletion, showing maximal separation of less than 3.5 cM. The observed deletion is the only significant difference between the wild-type and Hs line over 587 kilobases, likely encompassing much more than 3.5 cM, showing that the deletion is the cause of seedless trait. The codominant markers and obtained progenies will be useful for introgression of the seedless trait into elite sugar apple lines and into other Annonas through interspecific crossings.

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 generatedframeshift 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.

BELL1 interacts with CRABS CLAW and INNER NO OUTER to regulate ovule and seed development in pomegranate.

Pomegranate (Punica granatum) flowers are classified as bisexual flowers and functional male flowers. Functional male flowers have sterile pistils that show abnormal ovule development. In previous studies, we identified INNER NO OUTER (INO), CRABS CLAW (CRC) and BELL1 (BEL1), which were specifically expressed in bisexual and functional male flowers. However, the functions of ovule identity genes and the mechanism underlying ovule sterility in pomegranate remain unknown. Here, we found that the integument primordia formed and then ceased developing in the ovules of functional male flowers with a vertical diameter of 8.1-13.0 mm. Megaspore mother cells were observed in bisexual flowers when the vertical diameters of flowers were 10.1-13.0 mm, but not in functional male flowers. We analyzed the expression patterns of ovule-related genes in pomegranate ovule sterility and found that PgCRC mRNA was highly expressed at a critical stage of ovule development in bisexual flowers. Ectopic expression of PgCRC and PgINO was sufficient to increase seed number in transgenic lines. PgCRC partially complemented the Arabidopsis (Arabidopsis thaliana) crc mutant, and PgINO successfully rescued the seeds set in the Arabidopsis ino mutant. The results of Y2H assays, BiFC assays and genetic data analyses showed that PgCRC and PgINO directly interact with PgBEL1. Our results also showed that PgCRC and PgINO could not interact directly with MADS-box proteins and that PgBEL1 interacted with SEPALLATA (PgSEP) proteins. We report the function of PgCRC and PgINO in ovule and seed development and show that PgCRC and PgINO interact with PgBEL1. Thus, our results provide understanding of the genetic regulatory networks underlying ovule development in pomegranate.

Transcription factors KNAT3 and KNAT4 are essential for integument and ovule formation in Arabidopsis.

Integuments form important protective cell layers surrounding the developing ovules in gymno- and angiosperms. Although several genes have been shown to influence the development of integuments, the transcriptional regulatory mechanism is still poorly understood. In this work, we report that the Class II KNOTTED1-LIKE HOMEOBOX (KNOX II) transcription factors KNOTTED1-LIKE HOMEBOX GENE 3 (KNAT3) and KNAT4 regulate integument development in Arabidopsis (Arabidopsis thaliana). KNAT3 and KNAT4 were co-expressed in inflorescences and especially in young developing ovules. The loss-of-function double mutant knat3 knat4 showed an infertility phenotype, in which both inner and outer integuments of the ovule are arrested at an early stage and form an amorphous structure as in the bell1 (bel1) mutant. The expression of chimeric KNAT3- and KNAT4-EAR motif repression domain (SRDX repressors) resulted in severe seed abortion. Protein-protein interaction assays demonstrated that KNAT3 and KNAT4 interact with each other and also with INNER NO OUTER (INO), a key transcription factor required for the outer integument formation. Transcriptome analysis showed that the expression of genes related with integument development is influenced in the knat3 knat4 mutant. The knat3 knat4 mutant also had a lower indole-3-acetic acid (IAA) content, and some auxin signaling pathway genes were downregulated. Moreover, transactivation analysis indicated that KNAT3/4 and INO activate the auxin signaling gene IAA INDUCIBLE 14 (IAA14). Taken together, our study identified KNAT3 and KNAT4 as key factors in integument development in Arabidopsis.

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.

Restriction of iron loading into developing seeds by a YABBY transcription factor safeguards successful reproduction in Arabidopsis

Iron (Fe) storage in plant seeds is not only necessary for seedling establishment following germination but is also a major source of dietary Fe for humans and other animals. Accumulation of Fe in seeds is known to be low during early seed development. However, the underlying mechanism and biological significance remain elusive. Here, we show that reduced expression of Arabidopsis YABBY transcription factor INNER NO OUTER (INO) increases embryonic Fe accumulation, while transgenic overexpression of INO results in the opposite effect. INO is highly expressed during early seed development, and decreased INO expression increases the expression of NATURAL RESISTANCE-ASSOCIATED MACROPHAGE PROTEIN 1 (NRAMP1), which encodes a transporter that contributes to seed Fe loading. The relatively high embryonic Fe accumulation conferred by decreased INO expression is rescued by the nramp1 loss-of-function mutation. We further demonstrated that INO represses NRAMP1 expression by binding to NRAMP1-specific promoter region. Interestingly, we found that excessive Fe loading into developing seeds of ino mutants results in greater oxidative damage, leading to increased cell death and seed abortion, a phenotype that can be rescued by the nramp1 mutation. Taken together, these results indicate that INO plays an important role in safeguarding reproduction by reducing Fe loading into developing seeds by repressing NRAMP1 expression.

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.

A digital 3D reference atlas reveals cellular growth patterns shaping the Arabidopsis ovule.

A fundamental question in biology is how morphogenesis integrates the multitude of processes that act at different scales, ranging from the molecular control of gene expression to cellular coordination in a tissue. Using machine-learning-based digital image analysis, we generated a three-dimensional atlas of ovule development in Arabidopsis thaliana, enabling the quantitative spatio-temporal analysis of cellular and gene expression patterns with cell and tissue resolution. We discovered novel morphological manifestations of ovule polarity, a new mode of cell layer formation, and previously unrecognized subepidermal cell populations that initiate ovule curvature. The data suggest an irregular cellular build-up of WUSCHEL expression in the primordium and new functions for INNER NO OUTER in restricting nucellar cell proliferation and the organization of the interior chalaza. Our work demonstrates the analytical power of a three-dimensional digital representation when studying the morphogenesis of an organ of complex architecture that eventually consists of 1900 cells.

Brassinosteroids regulate outer ovule integument growth in part via the control of INNER NO OUTER by BRASSINOZOLE-RESISTANT family transcription factors.

Brassinosteroids (BRs) play important roles in regulating plant reproductive processes. BR signaling or BR biosynthesis null mutants do not produce seeds under natural conditions, but the molecular mechanism underlying this infertility is poorly understood. In this study, we report that outer integument growth and embryo sac development were impaired in the ovules of the Arabidopsis thaliana BR receptor null mutant bri1-116. Gene expression and RNA-seq analyses showed that the expression of INNER NO OUTER (INO), an essential regulator of outer integument growth, was significantly reduced in the bri1-116 mutant. Increased INO expression due to overexpression or increased transcriptional activity of BRASSINAZOLE-RESISTANT 1 (BZR1) in the mutant alleviated the outer integument growth defect in bri1-116 ovules, suggesting that BRs regulate outer integument growth partially via BZR1-mediated transcriptional regulation of INO. Meanwhile, INO expression in bzr-h, a null mutant for all BZR1 family genes, was barely detectable; and the outer integument of bzr-h ovules had much more severe growth defects than those of the bri1-116 mutant. Together, our findings establish a new role for BRs in regulating ovule development and suggest that BZR1 family transcription factors might regulate outer integument growth through both BRI1-dependent and BRI1-independent pathways.

Characteristics of INNER NO OUTER Homologous Genes in Wild Tomato Species

INNER NO OUTER belongs to the YABBY transcription factor family and determines the abaxial identity of the outer ovule integument. In this study, complete genomic INO sequences were identified and characterized in 12 accessions of 11 tomato species (Solanum section Lycopersicon). It was shown that the identified INO genes encode proteins with the conserved zinc finger and YABBY domains. The INO sequences of all analyzed tomato species contain 243 (12.9% of the aligned length) variable sites, 43 of which are exon-specific and include 22 nonsynonymous SNPs leading to amino acid residue substitutions in zinc finger domain (10), YABBY domain (2), and interdomain regions (10). A deletion of 10 aa at the C-terminal interdomain sequence is characteristic of the S. arcanum INO.

From Plant Ontology to Gene Ontology and back

Our recent goal for the Plant Ontology (PO) is to have it integrated with the Gene Ontology (GO). The simplest aspect of this is to link morphological and anatomical images of structures for PO terms with genes involved in the development of those structures (GO terms). This, also, would include images of expression analyses by in situ hybridization. By using a process of reciprocal illumination, we will be able to clarify and/or redefine PO terms. In particular, an example of this is the integument development during ovule maturation in seed plants. Gymnosperms have one integument; whereas, Angiosperms (flowering plants) have two integuments, i.e., an inner integument surrounded by an outer integument. The question that arises is which of the two integuments in the Angiosperms is the equivalent of the single integument in the gymnosperms? In Angiosperms, the gene INNER NO OUTER (INO) is involved in the proper development of the outer integument but not in the inner integument and in ino mutants there is no outer integument. INO genes are angiosperm specific as no orthologs have been found in gymnosperms. Thus, it appears that the inner integument of the Angiosperms is equivalent –homologous- to the single integument of the gymnosperms and the PO terms can be revised accordingly.

Structure Analysis of INNER NO OUTER (INO) Homologs in Capsicum Species

The plant transcription factor INNER NO OUTER (INO) plays a major role in development of the outer integument of bitegmic ovules. INO sequences of ten Capsicum species were identified. Levels of nucleotide and amino acid variability in Capsicum INO were determined, and 18 amino acid residue substitutions localized in the YABBY domain and in the interdomain region were revealed. The ZnF domain was invariant in all the analyzed Capsicum species. In the constructed dendrogram, all the Capsicum species form a single cluster in the INO clade, the basal branches to which are formed by the INO proteins of the Solanum and Nicotiana species.

Two metallocarboxypeptidase inhibitors are implicated in tomato fruit development and regulated by the Inner No Outer transcription factor

The TCMP-1 and TCMP-2 genes of tomato code for metallocarboxypeptidase inhibitors and show sequential, tightly regulated expression patterns during flower and fruit development. In particular, TCMP-1 is highly expressed in flower buds before anthesis, while TCMP-2 in ripe fruits. Their expression pattern suggests that they might play a role in fruit development. Here, to investigate their function, we altered their endogenous levels by generating transgenic plants harbouring a chimeric gene expressing the TCMP-1 coding sequence under the control of the TCMP-2 promoter. The expression of the transgene caused an earlier fruit setting with no visible phenotypic effects on plant and fruit growth. The altered TCMP-1 regulation determines an increased level of TCMP-1 in the fruit and unexpected changes in the levels of both TCMPs in flower buds before anthesis, suggesting a mechanism of transcriptional cross-regulation. We in silico analysed TCMPs promoter regions for the presence of common cis acting elements related to ovary/fruit development and we found that both promoters contain putative binding sites for INNER NO OUTER (INO), a transcription factor implicated in ovule development. By chromatin immunoprecipitation, we proved that INO binds to TCMP-1 and TCMP-2 promoters, thereby representing a candidate regulatory factor for coordinated control of TCMPs.

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.

The pomegranate (Punica granatum L.) genome and the genomics of punicalagin biosynthesis.

Pomegranate (Punica granatum L.) is a perennial fruit crop grown since ancient times that has been planted worldwide and is known for its functional metabolites, particularly punicalagins. Wehave sequenced and assembled the pomegranate genome with 328Mb anchoredintoninepseudo-chromosomes and annotated 29229 gene models. A Myrtales lineage-specific whole-genome duplication event was detected that occurred in the common ancestor before the divergence of pomegranate and Eucalyptus. Repetitive sequences accounted for 46.1% of the assembled genome. We found that the integument development gene INNER NO OUTER (INO) was under positive selection and potentially contributed to the development of the fleshy outer layer of the seed coat, an edible part of pomegranate fruit. The genes encoding the enzymes for synthesis and degradation of lignin, hemicelluloses and cellulose were also differentially expressed between soft- and hard-seeded varieties, reflecting differences in their accumulation in cultivars differing in seed hardness. Candidate genes for punicalagin biosynthesis were identified and their expression patterns indicated that gallic acid synthesis in tissues could follow different biochemical pathways. The genome sequence of pomegranate provides a valuable resource for the dissection of many biological and biochemical traits and also provides important insights for the acceleration of breeding. Elucidation of the biochemical pathway(s) involved in punicalagin biosynthesis could assist breeding efforts to increase production of this bioactive compound.

SEXUAL STERILITY is Essential for Both Male and Female Gametogenesis in Tomato.

Gametogenesis is a key step in the production of ovules or pollen in higher plants. The molecular aspects of gametogenesis are well characterized in the model plant Arabidopsis; however, little information is known in tomato, which is a model plant for fleshy fruit development. In this study, we characterized a tomato (Solanum lycopersicum L.) γ-ray mutant, sexual sterility (Slses), that exhibited both male and female sterility. Morphological analysis revealed that the Slses mutant forms incomplete ovules and wilted anthers devoid of pollen grains at the anthesis stage. Genetic and next-generation sequencing analyses revealed that the Slses mutant carried a 13 bp deletion within the first exon of a homolog of SPOROCYTELESS/NOZZLE (SPL/NZZ), which plays an important role in gametogenesis in Arabidopsis. Complementation analysis in which the complete SlSES genomic region was introduced into the Slses mutant fully restored normal phenotypes, demonstrating that Solyc07g063670 is responsible for the Slses mutation. SlSES probably act as a transcriptional repressor because of an EAR motif at the C-terminal region. Gene expression levels of WUSCHEL (SlWUS) and INNER NO OUTER (SlINO), both of which are required for ovule development, were dramatically reduced in the early stages of pistil development in the Slses mutant, suggesting a positive regulatory role for SlSES in the transcription of gametogenesis genes and differences in the regulation of INO (SlINO) and integument development by SPL/NZZ (SLSES) between Arabidopsis and tomato. Taken together, our results indicate that SlSES is a novel tomato gametogenesis gene essential for both male and female gametogenesis.

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

BackgroundThe 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.ResultsINO 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.ConclusionsThe 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.Electronic supplementary materialThe online version of this article (doi:10.1186/s12870-016-0835-z) contains supplementary material, which is available to authorized users.