Outer Integument Research Articles

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.

Ontogenia de la semilla de Yucca periculosa (Agavaceae)

Las semillas de algunas especies de Yucca han sido estudiadas por Arnott y Horner. Ellos se refieren principalmente al estado maduro y afirman que el tejido de reserva extraembrionario es un perispermo. En el presente trabajo se muestran evidencias ontogénicas de que se trata de un endospermo con desarrollo de tipo nuclear. La forma de la semilla se aproxima a un prisma triangular de poco menos de 1 cm de longitud, es de color negro y el surco de la rafe se observa fácilmente. La cubierta seminal deriva únicamente del tegumento externo. La pared periclinal externa de la exotesta aparece heterogéneamente engrosada. La mesotesta y enclotesta proliferan desigualmente hacia el interior de la semilla formando un ruminado en el endospermo. Hacia la madurez, desaparecen los derivados del tegumento interno ocasionando la fusión de la cutícula intertegumentaria con la tegmen-nucelar; éstas aportan información ontogénica valiosa, debido a su persistencia aún cuando el tejido que las originó hubiere desaparecido. El embrión acelera su desarrollo a las 10 semanas después de antesis, es cilíndrico, plegado en forma de 'S' y sus dos tercios calaza les corresponden al cotiledón.

Morphogenesis and global analysis of transcriptional profiles of Celastrus orbiculatus aril: unravelling potential genes related to aril development

As a specialized seed appendage, the aril is a noteworthy and taxonomically important feature of Celastrus orbiculatus; the aril also has important ornamental value and biological functions. To better understand the mechanism of aril development, paraffin sections were used to examine its morphogenesis, and RNA-sequencing (RNA-seq) technology was employed for transcriptional profiling of the developing aril. Our results revealed that the aril of C. orbiculatus was formed by the cells of the exostomal region of the outer integument. By analyzing global changes in the transcriptome, 41,560,806, 42,789,340, and 44,496,748 clean reads from three cDNA libraries were generated and assembled into 87,600 unigenes with N50 of 1328 bp, in which 31,971 (36.49%) were annotated to Nr database. Furthermore, a total of 25,914 (29.79%), 14,394 (16.4%) and 11,957 (13.65%) genes were successfully annotated with hierarchical 48 GO terms, 26 KOGs and 32 KEGG pathways. Totally, 104, 1887 and 1967 differently expressed genes were identified in CO-F2 versus CO-F3, CO-F2 versus CO-YF1 and CO-F3 versus CO-YF1, respectively. GO and KEGG pathway analyses of DEGs involved in cell–cell communication and plant hormone signal transduction contributed to aril development were identified. Furthermore, we found putative key target genes of WRKY, Aux/IAA, ARF and MADS-box family’s transcription factors related to aril development. Real-time qPCR was performed on eight genes randomly selected from important transcription factors to validate the expression profiles obtained by RNA-seq. This work provides a platform for future genetic and functional genomics research on the molecular mechanisms of aril structure, and expands our understanding of the molecular mechanism and evolutionary analysis of aril development in other members of the Celastraceae.

Ovule, Fruit, and Seed Development ofOrectanthe sceptrumand Its Systematic Relevance to Xyridaceae (Poales)

Premise of research. Orectanthe consists of only two species with a distribution limited to the Guayana Highlands, in locations where sample collection is challenging. The genus belongs to Abolbodoideae, one of the two subfamilies of Xyridaceae. The development of the ovule, fruit, and seed of Orectanthe sceptrum was studied using a comparative approach to provide useful information for the taxonomy and phylogeny of the family.Methodology. Flowers and fruits at different developmental stages were analyzed by light and scanning electron microscopy. Optimization of select characters on a previously published phylogeny was conducted to evaluate the evolution of some embryological characters in Poales. The seed weights of O. sceptrum and species of Abolboda and Xyris were measured for comparison.Pivotal results. Orectanthe sceptrum presents anatropous, tenuinucellate, and bitegmic ovules with a micropyle bounded by both integuments. The multilayered outer integument forms the seed wing, which is a unique feature of this genus. The development of megagametophyte is of the Polygonum type. The seed has a tanniferous hypostase, a starchy endosperm, and a reduced and undifferentiated embryo. The seed coat is composed of a tanniferous tegmen (endotegmen and exotegmen) and testa (endotesta and exotesta). The mechanical layer of the seed coat is the exotesta. The fruit has a sclerenchymatous exocarp. The ancestral character-state reconstructions show that anatropous, crassinucellate ovules, megagametophyte development of the Polygonum type, and nuclear endosperm are the ancestral conditions in Poales. Orthotropous, tenuinucellate ovules appear in the most recent common ancestor of the xyrid, restiid, and graminid clades. The seed weight of O. sceptrum is one to two orders of magnitude greater than the seed weight of Abolboda and Xyris species.Conclusions. Anatropous ovules and a seed coat formed by endotegmen, exotegmen, endotesta, and exotesta link Orectanthe to Abolboda (Abolbodoideae). Such characteristics are not found in Xyris (Xyridoideae), which is consistent with the subfamilial division. The presence of anatropous ovules in Abolbodoideae most likely represents a character reversal. The greater seed weight of O. sceptrum may explain the presence of wings in the seeds.

Auxin Regulates Cotton Fiber Initiation via GhPIN-Mediated Auxin Transport.

Cotton fibers are seed trichomes that make cotton unique compared with other plants. At anthesis, IAA, a major auxin in plants, accumulates in the fiber cell to promote cell initiation. However, many important aspects of this process are not clear. Here, auxin distribution patterns indicated by auxin-dependent DR5::GUS (β-glucuronidase) expression in cotton ovules were studied during fiber cell differentiation and cell initiation [-2 to 2 DPA (days post-anthesis)]. The nucellus and fiber cell were two major sites where auxin accumulates. The accumulation in the nucellus started from -1 DPA, and that in fiber cells from 0 DPA. Immunolocalization analysis further suggests that the IAA accumulation in fiber initials began before flower opening. Furthermore, we demonstrate that accumulated IAA in fiber initials was mainly from efflux transport and not from in situ synthesis. Eleven auxin efflux carrier (GhPIN) genes were identified, and their expression during ovule and fiber development was investigated. Ovule-specific suppression of multiple GhPIN genes in transgenic cotton inhibited both fiber initiation and elongation. In 0 DPA ovules, GhPIN3a, unlike other GhPIN genes, showed additional localization of the transcript in the outer integument. Collectively, these results demonstrate the important role of GhPIN-mediated auxin transport in fiber-specific auxin accumulation for fiber initiation.

The MYB107 Transcription Factor Positively Regulates Suberin Biosynthesis.

Suberin, a lipophilic polymer deposited in the outer integument of the Arabidopsis (Arabidopsis thaliana) seed coat, represents an essential sealing component controlling water and solute movement and protecting seed from pathogenic infection. Although many genes responsible for suberin synthesis are identified, the regulatory components controlling its biosynthesis have not been definitively determined. Here, we show that the Arabidopsis MYB107 transcription factor acts as a positive regulator controlling suberin biosynthetic gene expression in the seed coat. MYB107 coexpresses with suberin biosynthetic genes in a temporal manner during seed development. Disrupting MYB107 particularly suppresses the expression of genes involved in suberin but not cutin biosynthesis, lowers seed coat suberin accumulation, alters suberin lamellar structure, and consequently renders higher seed coat permeability and susceptibility to abiotic stresses. Furthermore, MYB107 directly binds to the promoters of suberin biosynthetic genes, verifying its primary role in regulating their expression. Identifying MYB107 as a positive regulator for seed coat suberin synthesis offers a basis for discovering the potential transcriptional network behind one of the most abundant lipid-based polymers in nature.

Laser Microdissection and Spatiotemporal Pinoresinol-Lariciresinol Reductase Gene Expression Assign the Cell Layer-Specific Accumulation of Secoisolariciresinol Diglucoside in Flaxseed Coats

The concentration of secoisolariciresinol diglucoside (SDG) found in flaxseed (Linum usitatissimum L.) is higher than that found in any other plant. It exists in flaxseed coats as an SDG-3-hydroxy-3-methylglutaric acid oligomer complex. A laser microdissection method was applied to harvest material from different cell layers of seed coats of mature and developing flaxseed to detect the cell-layer specific localization of SDG in flaxseed; NMR and HPLC were used to identify and quantify SDG in dissected cell layers after alkaline hydrolysis. The obtained results were further confirmed by a standard molecular method. The promoter of one pinoresinol-lariciresinol reductase gene of L. usitatissimum (LuPLR1), which is a key gene involved in SDG biosynthesis, was fused to a β-glucuronidase (GUS) reporter gene, and the spatio-temporal regulation of LuPLR1 gene expression in flaxseed was determined by histochemical and activity assays of GUS. The result showed that SDG was synthesized and accumulated in the parenchymatous cell layer of the outer integument of flaxseed coats.

Insight into “serpentine syndrome” of Albanian, endemic violets (Viola L., Melanium Ging. section) – Looking for unique, adaptive microstructural floral, and embryological characters

The genus Viola, particularly Melanium section, rich in metallophytes, is an excellent taxon for study of microevolutionary and adaptation processes. Pollen, ovule, and microstructural floral characters were investigated by LM, SEM, and CLSM in seven endemic Albanian violets, five serpentinophytes (Viola albanica, V. dukadjinica, V. albanica × V. dukadjinica, V. raunsiensis, and V. macedonica), two from chalk soil (V. aetolica and V. schariensis), and in their closest relatives (V. lutea ssp. sudetica, V. tricolor ssp. tricolor, and V. arvensis) for their taxonomic usefulness and adaptive value. Three among analyzed characters were common in all Albanian violets however not unique. Serpentinophytes, V. aetolica and V. schariensis possessed hairs deep inside the spur, developed pollen heteromorphism, both increase the chance of pollination in unpredictable conditions and had strongly developed tannin rich layer in the outer integument of the young ovules with a protective role. They also all exhibited high pollen viability (86.9 ± 10.2%), high frequency of normally developed, enlarged (fertilized) ovules in ovary (65.0 ± 24.0%), but also high frequency of degenerations in developing ovules (40.4 ± 9.8%). Several flower characters may be adaptive in the unfavorable, high altitude environment, including serpentine soils. High pollen viability and normally developed fertilized ovules are sufficient for Albanian species maintenance.

MYB107 and MYB9 Homologs Regulate Suberin Deposition in Angiosperms.

Suberin, a polymer composed of both aliphatic and aromatic domains, is deposited as a rough matrix upon plant surface damage and during normal growth in the root endodermis, bark, specialized organs (e.g., potato [Solanum tuberosum] tubers), and seed coats. To identify genes associated with the developmental control of suberin deposition, we investigated the chemical composition and transcriptomes of suberized tomato (Solanum lycopersicum) and russet apple (Malus x domestica) fruit surfaces. Consequently, a gene expression signature for suberin polymer assembly was revealed that is highly conserved in angiosperms. Seed permeability assays of knockout mutants corresponding to signature genes revealed regulatory proteins (i.e., AtMYB9 and AtMYB107) required for suberin assembly in the Arabidopsis thaliana seed coat. Seeds of myb107 and myb9 Arabidopsis mutants displayed a significant reduction in suberin monomers and altered levels of other seed coat-associated metabolites. They also exhibited increased permeability, and lower germination capacities under osmotic and salt stress. AtMYB9 and AtMYB107 appear to synchronize the transcriptional induction of aliphatic and aromatic monomer biosynthesis and transport and suberin polymerization in the seed outer integument layer. Collectively, our findings establish a regulatory system controlling developmentally deposited suberin, which likely differs from the one of stress-induced polymer assembly recognized to date.

HAPLESS13-Mediated Trafficking of STRUBBELIG Is Critical for Ovule Development in Arabidopsis.

Planar morphogenesis, a distinct feature of multicellular organisms, is crucial for the development of ovule, progenitor of seeds. Both receptor-like kinases (RLKs) such as STRUBBELIG (SUB) and auxin gradient mediated by PIN-FORMED1 (PIN1) play instructive roles in this process. Fine-tuned intercellular communications between different cell layers during ovule development demands dynamic membrane distribution of these cell-surface proteins, presumably through vesicle-mediated sorting. However, the way it’s achieved and the trafficking routes involved are obscure. We report that HAPLESS13 (HAP13)-mediated trafficking of SUB is critical for ovule development. HAP13 encodes the μ subunit of adaptor protein 1 (AP1) that mediates protein sorting at the trans-Golgi network/early endosome (TGN/EE). The HAP13 mutant, hap13-1, is defective in outer integument growth, resulting in exposed nucellus accompanied with impaired pollen tube guidance and reception. SUB is mis-targeted in hap13-1. However, unlike that of PIN2, the distribution of PIN1 is independent of HAP13. Genetic interference of exocytic trafficking at the TGN/EE by specifically downregulating HAP13 phenocopied the defects of hap13-1 in SUB targeting and ovule development, supporting a key role of sporophytically expressed SUB in instructing 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.

GhLTPG1, a cotton GPI-anchored lipid transfer protein, regulates the transport of phosphatidylinositol monophosphates and cotton fiber elongation.

The cotton fibers are seed trichomes that elongate from the ovule epidermis. Polar lipids are required for the quick enlargement of cell membrane and fiber cell growth, however, how lipids are transported from the ovules into the developing fibers remains less known. Here, we reported the functional characterization of GhLTPG1, a GPI-anchored lipid transport protein, during cotton fiber elongation. GhLTPG1 was abundantly expressed in elongating cotton fibers and outer integument of the ovules, and GhLTPG1 protein was located on cell membrane. Biochemical analysis showed that GhLTPG1 specifically bound to phosphatidylinositol mono-phosphates (PtdIns3P, PtdIns4P and PtdIns5P) in vitro and transported PtdInsPs from the synthesis places to the plasma membranes in vivo. Expression of GhLTPG1 in Arabidopsis caused an increased number of trichomes, and fibers in GhLTPG1-knockdown cotton plants exhibited significantly reduced length, decreased polar lipid content, and repression of fiber elongation-related genes expression. These results suggested that GhLTPG1 protein regulates the cotton fiber elongation through mediating the transport of phosphatidylinositol monophosphates.

PROTEINS OF THE INTEGUMENTARY SYSTEM OF THE HONEYBEE, Apis mellifera.

The integument of insects and other arthropods is composed of an inner basal lamina coated by the epidermis, which secretes the bulk of the outer integument layer, the cuticle. The genome sequencing of several insect species has allowed predicting classes of proteins integrating the cuticle. However, only a small proportion of them, as well as other proteins in the integumentary system, have been validated. Using two-dimensional gel electrophoresis coupled with mass spectrometry, we identified 45 different proteins in a total of 112 selected gel spots derived from thoracic integument samples of developing honeybee workers, including 14 cuticular proteins (AmelCPR 3, AmelCPR 12, AmelCPR 16, AmelCPR 27, apidermin 2, apidermin 3, endocuticle structural glycoprotein SgAbd-8-like, LOC100577363, LOC408365, LOC413679, LOC725454, LOC100576916, LOC725838, and peritrophin 3-C analogous). Gene ontology functional analysis revealed that the higher proportions of the identified proteins have molecular functions related to catalytic and structural molecule activities, are involved in metabolic biological processes, and pertain to the protein class of structural or cytoskeletal proteins and hydrolases. It is noteworthy that 26.7% of the identified proteins, including five cuticular proteins, were revealed as protein species resulting from allelic isoforms or derived from posttranslational modifications. Also, 66.7% of the identified cuticular proteins were expressed in more than one developmental phase, thus indicating that they are part of the larval, pupal, and adult cuticle. Our data provide experimental support for predicted honeybee gene products and new information on proteins expressed in the developing integument.

Morphoanatomy of the ovary and ovule in Bromeliaceae subfamily Tillandsioideae and its systematic relevance

Tillandsioideae are composed of nine genera, most of these with unclear delimitation. The morphoanatomy of ovules and ovaries of 83 species from seven different genera was studied under light or scanning electron microscopy to contribute to the understanding of the relationships in the group. The nucellar epidermis and the parietal tissue showed a regular pattern for all Tillandsioideae. The presence of chalazal appendages, however, and their shape and size varied between the genera analysed. Most species had a single acuminate chalazal appendage. Catopsis was the only genus with multiple appendages. Guzmania and Racinaea lacked this structure. The integuments also presented a variable length and thickness among the different genera. The four to six layers of the outer integument were unique in Catopsis and the three cell layers condition in the inner integument of Tillandsia should be a retained feature as it was also observed in Catopsis. Many characteristics in ovule morphology presented here have great systematic value for distinguishing genera of the Tillandsioideae. In addition, these features can also help in the understanding of ovule evolution in Bromeliaceae as a whole.

Anatomy of the reproductive structures of Stenanona flagelliflora (Annonaceae)

The genus Stenanona comprises 15 species distributed from Mexico to Colombia. Stenanona flagelliflora is the only species in which the reproductive structures are found on the flagellum, a unique phenomenon in the flora of Mexico. Our objective is to recognize and describe anatomical features of flowers and fruits that contribute to the systematics of the species. We collected floral buds in three different stages of development (buds I, II, and III), flowers at anthesis, and immature and mature fruits in the ecological reserve of the ejido Adolfo Lopez Mateos, Catemaco, Veracruz, Mexico; the samples were fixed in formalin–acetic acid–alcohol (FAA), cut, stained, and mounted using conventional histological techniques. The receptacle has numerous vascular bundles distributed in two cylinders: the perianth is trimerous; it has 32 stamens arranged in four whorls; the anthers have a deltoid-shaped extension of connective tissue; the gynoecium has 5–7 free carpels. Bud II showed a bitegmic and crassinucellate ovule with four layers of the outer integument and two of the inner integument, an ovary with trichomes on the outer epidermis and reduced style, and anthers in different stages of development. Bud III showed epidermis with bulliform cells in the style and stigma. The fruit is of berry type; the ripe fruit shows longitudinal series of fibers across the mesocarp. The endosperm is ruminate; the embryo is small and undifferentiated. These features are common in the Annonaceae family; however, the number of integumentary layers in the ovule and the presence of papillae on the style and stigma are novel characters.

Ovule and megagametophyte development in selected species of Apeibeae and Grewieae (Malvaceae–Grewioideae) from South America and its systematic implications

This is the first embryological report of the Grewioideae subfamily, which is meant to contribute to the characterisation of the genera Corchorus, Luehea and Triumfetta. Ovule and female gametophyte development in C. argutus, L. divaricata and T. semitriloba was analysed. The ovules of all species are anatropous, bitegmic and of crassinucellate mixed type. The micropyle of the studied species is formed by the outer integument (exostome). The ovule of L. divaricata differs from those of the other two species because the chalazal tissue expands forming a cap, which gives rise to a wing in the seed. All species present one hypostase. The megaspore mother cell gives rise to a linear megaspore triad in C. argutus and L. divaricata, whereas in T. semitriloba, triads and diads can be observed in the same ovule. The chalazal megaspore develops a seven-celled and eight-nucleate female gametophyte corresponding to the Polygonum type. The synergids of L. divaricata have hooks and a conspicuous filiform apparatus. The antipodal cells in C. argutus are persistent, whereas in the other species, they are small and ephemeral. The embryological characters are compared with those of other taxa within the family and the megagametophyte formation in these species is discussed.

Rice caryopsis development I: Dynamic changes in different cell layers.

Rice caryopsis as one of the most important food sources for humans has a complex structure that is composed of maternal tissues including the pericarp and testa and filial tissues including the endosperm and embryo. Although rice caryopsis studies have been conducted previously, a systematic characterization throughout the entire developmental process is still lacking. In this study, detailed morphological examinations of caryopses were made during the entire 30‐day developmental process. We observed some rapid changes in cell differentiation events and cataloged how cellular degeneration processes occurred in maternal tissues. The differentiations of tube cells and cross cells were achieved by 9 days after pollination (DAP). In the testa, the outer integument was degenerated by 3 DAP, while the outer layer of the inner integument degenerated by 7 DAP. In the nucellus, all tissues with the exception of the nucellar projection and the nucellar epidermis degenerated in the first 5 DAP. By 21 DAP, all maternal tissues, including vascular bundles, the nucellar projection and the nucellar epidermal cells were degenerated. In summary, this study provides a complete atlas of the dynamic changes in cell differentiation and degeneration for individual maternal cell layers of rice caryopsis.

Comparative studies on ovule development in Lardizabalaceae (Ranunculales)

Ovules development and structure of 6 species of 5 genera in Lardizabalaceae were studied under SEM and LM. Ovule morphology show diversity in the family: anatropous (Sinofranchetia and Decaisnea) or campylotropous (Sargentodoxa, Akebia and Holboellia); ovule number is 1 (Sargentodoxa) to 20 (even up to 180 in Akebia and Holboellia); mature ovule size is from 150μm (Holboellia) to 350μm (Sinofranchetia and Sargentodoxa) in length; placenta marginal or laminar-diffuse; bistomic or endostomic, in Decaisnea, the micropyle is formed by inner integument only; and the thickness of outer integument is vary from 2 to 3 to 5–6 cell layers (Decaisnea). Akebia and Holboellia had secretory hairs surrounding ovules. While, morphological features common to all species included (1) bitegmic integument, cup-shaped inner integument (consists of 2–3 cell layers) derived from an annular ring, and hood-shaped outer integument developed from semi-annular initiation (except Sargentodoxa, the cup-shaped outer integument with semi-annular initiation); (2) inner integument initiated at the stage of sporogenous cell or megaspore mother cell, while outer integument formed later; (3) crassinucellar, the megaspore mother cell 3–6 cell layer deep within the nucellus; (4) the short funiculus, a vascular bundle reached to the chalazal pole region connecting ovule to placenta, and no funiculus appendage was found.

The Transcriptional Repressor MYB2 Regulates Both Spatial and Temporal Patterns of Proanthocyandin and Anthocyanin Pigmentation in Medicago truncatula.

Accumulation of anthocyanins and proanthocyanidins (PAs) is limited to specific cell types and developmental stages, but little is known about how antagonistically acting transcriptional regulators work together to determine temporal and spatial patterning of pigmentation at the cellular level, especially for PAs. Here, we characterize MYB2, a transcriptional repressor regulating both anthocyanin and PA biosynthesis in the model legume Medicago truncatula. MYB2 was strongly upregulated by MYB5, a major regulator of PA biosynthesis in M. truncatula and a component of MYB-basic helix loop helix-WD40 (MBW) activator complexes. Overexpression of MYB2 abolished anthocyanin and PA accumulation in M. truncatula hairy roots and Arabidopsis thaliana seeds, respectively. Anthocyanin deposition was expanded in myb2 mutant seedlings and flowers accompanied by increased anthocyanin content. PA mainly accumulated in the epidermal layer derived from the outer integument in the M. truncatula seed coat, starting from the hilum area. The area of PA accumulation and ANTHOCYANIDIN REDUCTASE expression was expanded into the seed body at the early stage of seed development in the myb2 mutant. Genetic, biochemical, and cell biological evidence suggests that MYB2 functions as part of a multidimensional regulatory network to define the temporal and spatial pattern of anthocyanin and PA accumulation linked to developmental processes.

Immunohistochemical Localization of Endogenous IAA in Peach (Prunus persica L.) Fruit during Development

Peach (Prunus persica L.) is a model species for stone fruit studies within the Rosaceae family. Auxin plays an important role in the development of peach fruit. To reveal the distribution of auxin in the tissues of peach fruit, immunohistochemical localization of IAA was carried out in the seed, mesocarp, and endocarp in developing peach fruit using an anti-indole-3-acetic acid (anti-IAA) monoclonal antibody. A strong IAA signal was observed throughout the outer and inner integument during peach fruit development, and the distribution was zonal. The IAA signal was mainly focused in mucilage layers in the outer integument. The outer integument may function to produce or store IAA in the seed; a strong IAA signal was detected in the cells around the vascular tissue, whereas a weak IAA signal was located in the vascular tissues. In the mesocarp, the cells around the vascular bundle tissue gave rise to an IAA signal that increased in the late phase of fruit growth, which coincided with a significant increase in fruit growth. The distribution of IAA, however, was changed when fruit was treated with auxin transport inhibitors NPA (1-N-naphthylphthalamic acid) or TIBA (2, 3, 5-triiodobenzoic acid); in mesocarp tissues, an IAA signal was detected mainly in vessels of the treated fruit. During the critical period of endocarp lignification, the vessel lignification process was negatively correlated with IAA signal. The present results confirmed that the distribution of IAA was different in various tissues of peach fruit according to the developmental stage. This research provides cytological data for further study of the regulatory mechanism of auxin in peach fruit.