Structural and histochemical approach to the fruit and seed diversity of Cyperaceae in an evolutionary context.

KEY MESSAGE: The Cyperaceae fruit consistency depends on the mesocarp. Seed structure is diverse and related to the evolutionary history of their species. A new storage tissue is described for Cyperaceae. Anatomy and histochemistry of Cyperaceae fruits (including seeds) are poorly known due to their hard, isolating tissues that prevent anatomical techniques. We performed the first, most comprehensive structural diversity characterisation of fruit and seed in Cyperaceae, accompanied by an unprecedented histochemical characterisation of seeds for this family. We analysed fruits of 29 species, included in 19 genera and 12 tribes within the subfamilies Cyperoideae and Mapanioideae, using light microscopy. Cyperaceae fruits have a pericarp with a one-cell-layered exocarp and endocarp, and a multi-cell-layered mesocarp. The mesocarp of the Mapanioideae has a spongy-fleshy outer region and a hard inner region. The mesocarp of the Cyperoideae has only a hard region. The pericarp is free from the seed coat. Cyperaceae seeds have a three-layered seed coat, an embryo with haustorial function of its scutellum, and two storage tissues: the endosperm and a putative perisperm. Nine seed morphotypes and four seed subtypes were observed among the studied species. Our results suggested that the fruit consistency is determined by the mesocarp. Both the terms "nut" and "achene" should be accepted to refer to the dry fruit of the Cyperaceae until a widely accepted fruit classification for angiosperms is proposed. The Cyperaceae seed structural diversity is high and related to the evolutionary history of the species. The "perisperm" is a new tissue proposed for sedge seeds, and is here characterized for the first time. The seed coat has a different structure than the one described so far for the family.

Stigma/Style Cell-Cycle Inhibitor 1, a Regulator of Cell Proliferation, Interacts With a Specific 14-3-3 Protein and Is Degraded During Cell Division.

The final shape and size of plant organs are determined by a network of genes that modulate cell proliferation and expansion. Among those, SCI1 (Stigma/style Cell-cycle Inhibitor 1) functions by inhibiting cell proliferation during pistil development. Alterations in SCI1 expression levels can lead to remarkable stigma/style size changes. Recently, we demonstrated that SCI1 starts to be expressed at the specification of the Nicotiana tabacum floral meristem and is expressed at all floral meristematic cells. To elucidate how SCI1 regulates cell proliferation, we screened a stigma/style cDNA library through the yeast two-hybrid (Y2H) system, using SCI1 as bait. Among the interaction partners, we identified the 14-3-3D protein of the Non-Epsilon group. The interaction between SCI1 and 14-3-3D was confirmed by pulldown and co-immunoprecipitation experiments. 14-3-3D forms homo- and heterodimers in the cytoplasm of plant cells and interacts with SCI1 in the nucleus, as demonstrated by Bimolecular Fluorescence Complementation (BiFC). Analyses of SCI1-GFP fluorescence through the cell-cycle progression revealed its presence in the nucleoli during interphase and prophase. At metaphase, SCI1-GFP fluorescence faded and was no longer detected at anaphase, reappearing at telophase. Upon treatment with the 26S proteasome inhibitor MG132, SCI1-GFP was stabilized during cell division. Site-directed mutagenesis of seven serines into alanines in the predicted 14-3-3 binding sites on the SCI1 sequence prevented its degradation during mitosis. Our results demonstrate that SCI1 degradation at the beginning of metaphase is dependent on the phosphorylation of serine residues and on the action of the 26S proteasome. We concluded that SCI1 stability/degradation is cell-cycle regulated, consistent with its role in fine-tuning cell proliferation.

Heterochrony and repurposing in the evolution of gymnosperm seed dispersal units.

BACKGROUND: Plant dispersal units, or diaspores, allow the colonization of new environments expanding geographic range and promoting gene flow. Two broad categories of diaspores found in seed plants are dry and fleshy, associated with abiotic and biotic dispersal agents, respectively. Anatomy and developmental genetics of fleshy angiosperm fruits is advanced in contrast to the knowledge gap for analogous fleshy structures in gymnosperm diaspores. Improved understanding of the structural basis of modified accessory organs that aid in seed dispersal will enable future work on the underlying genetics, contributing to hypotheses on the origin of angiosperm fruits. To generate a structural framework for the development and evolution of gymnosperm fleshy diaspores, we studied the anatomy and histochemistry of Ephedra (Gnetales) seed cone bracts, the modified leaves surrounding the reproductive organs. We took an ontogenetic approach, comparing and contrasting the anatomy and histology of fleshy and papery-winged seed cone bracts, and their respective pollen cone bracts and leaves in four species from the South American clade. RESULTS: Seed bract fleshiness in Ephedra derives from mucilage accumulated in chlorenchyma cells, also found in the reduced young leaves before they reach their mature, dry stage. Cellulosic fibers, an infrequent cell type in gymnosperms, were found in Ephedra, where they presumably function as a source of supplementary apoplastic water in fleshy seed cone bracts. Papery-winged bract development more closely resembles that of leaves, with chlorenchyma mucilage cells turning into tanniniferous cells early on, and hyaline margins further extending into "wings". CONCLUSIONS: We propose an evolutionary developmental model whereby fleshy and papery-winged bracts develop from an early-stage anatomy shared with leaves that differs at the pollination stage. The ancestral fleshy bract state may represent a novel differentiation program built upon young leaf anatomy, while the derived dry, papery-winged state is likely built upon an existing differentiation pattern found in mature vegetative leaves. This model for the evolution of cone bract morphology in South American Ephedra hence involves a novel differentiation program repurposed from leaves combined with changes in the timing of leaf differentiation, or heterochrony, that can further be tested in other gymnosperms with fleshy diaspores.

SCI1 Is a Direct Target of AGAMOUS and WUSCHEL and Is Specifically Expressed in the Floral Meristematic Cells.

The specified floral meristem will develop a pre-established number of floral organs and, thus, terminate the floral meristematic cells. The floral meristematic pool of cells is controlled, among some others, by WUSCHEL (WUS) and AGAMOUS (AG) transcription factors (TFs). Here, we demonstrate that the SCI1 (Stigma/style cell-cycle inhibitor 1) gene, a cell proliferation regulator, starts to be expressed since the floral meristem specification of Nicotiana tabacum and is expressed in all floral meristematic cells. Its expression is higher in the floral meristem and the organs being specified, and then it decreases from outside to inside whorls when the organs are differentiating. SCI1 is co-expressed with N. tabacum WUSCHEL (NtWUS) in the floral meristem and the whorl primordia at very early developmental stages. Later in development, SCI1 is co-expressed with NAG1 (N. tabacum AG) in the floral meristem and specialized tissues of the pistil. In silico analyses identified cis-regulatory elements for these TFs in the SCI1 genomic sequence. Yeast one-hybrid and electrophoresis mobility shift assay demonstrated that both TFs interact with the SCI1 promoter sequence. Additionally, the luciferase activity assay showed that NAG1 clearly activates SCI1 expression, while NtWUS could not do so. Taken together, our results suggest that during floral development, the spatiotemporal regulation of SCI1 by NtWUS and NAG1 may result in the maintenance or termination of proliferative cells in the floral meristem, respectively.

Stomatal Development and Conductance of a Tropical Forage Legume Are Regulated by Elevated [CO2] Under Moderate Warming.

The opening and closing of stomata are controlled by the integration of environmental and endogenous signals. Here, we show the effects of combining elevated atmospheric carbon dioxide concentration (eCO 2; 600 µmol mol-1) and warming (+2°C) on stomatal properties and their consequence to plant function in a Stylosanthes capitata Vogel (C3) tropical pasture. The eCO 2 treatment alone reduced stomatal density, stomatal index, and stomatal conductance (gs ), resulting in reduced transpiration, increased leaf temperature, and leading to maintenance of soil moisture during the growing season. Increased CO2 concentration inside leaves stimulated photosynthesis, starch content levels, water use efficiency, and PSII photochemistry. Under warming, plants developed leaves with smaller stomata on both leaf surfaces; however, we did not see effects of warming on stomatal conductance, transpiration, or leaf water status. Warming alone enhanced PSII photochemistry and photosynthesis, and likely starch exports from chloroplasts. Under the combination of warming and eCO 2, leaf temperature was higher than that of leaves from the warming or eCO 2 treatments. Thus, warming counterbalanced the effects of CO2 on transpiration and soil water content but not on stomatal functioning, which was independent of temperature treatment. Under warming, and in combination with eCO 2, leaves also produced more carotenoids and a more efficient heat and fluorescence dissipation. Our combined results suggest that control on stomatal opening under eCO 2 was not changed by a warmer environment; however, their combination significantly improved whole-plant functioning.

Bioactive Organocopper Compound from Pseudomonas aeruginosa Inhibits the Growth of Xanthomonas citri subsp. citri.

Citrus canker is a very destructive disease of citrus species. The challenge is to find new compounds that show strong antibiotic activity and low toxicity to plants and the environment. The objectives of the present study were (1) to extract, purify and evaluate the secondary metabolites with antibiotic activity produced by Pseudomonas aeruginosa LV strain in vitro against Xanthomonas citri subsp. citri (strain 306), (2) to determine the potential of semi-purified secondary metabolites in foliar application to control citrus canker under greenhouse conditions, and (3) to identify antibiotic activity in orange leaf mesophyll infected with strain 306, by electron microscopy. Two pure bioactive compounds were isolated, an organocopper antibiotic compound (OAC) and phenazine-1-carboxamide. Phenazine-1-carboxamide did not show any antibiotic activity under the experimental conditions used in this study. The OAC showed a high level of antibiotic activity with a minimum inhibitory concentration of 0.12 µg mL(-1). In greenhouse tests for control of citrus canker in orange trees, the semi-purified fraction F3d reduced lesion formation by about 97%. The concentration used was 500 times lower than that for the recommended commercial copper-based product. Electron microscopy showed that F3d altered the exopolysaccharide matrix and caused cell lysis of the pathogen inside the citrus canker lesions. These results suggest that secondary metabolites produced by inducing P. aeruginosa LV strain have a high potential to be used as a bioproduct to control citrus canker.