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.Fil: Cruz, Joelma O.. Universidade de Sao Paulo; BrasilFil: Abramo Barrera San Martin, Juca. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Botánica Darwinion. Academia Nacional de Ciencias Exactas, Físicas y Naturales. Instituto de Botánica Darwinion; Argentina. Universidade de Sao Paulo; BrasilFil: Lubini, Greice. Universidade de Sao Paulo; BrasilFil: Strini, Edward J.. Universidade de Sao Paulo; BrasilFil: Sobral, Rómulo. Universidade do Minho; PortugalFil: Pinoti, Vitor F.. Universidade de Sao Paulo; BrasilFil: Ferreira, Pedro B.. Universidade de Sao Paulo; BrasilFil: Thomé, Vanessa. Universidade de Sao Paulo; BrasilFil: Quiapim, Andréa C.. Universidade de Sao Paulo; BrasilFil: Dornelas, Marcelo C.. Universidade Estadual de Campinas; BrasilFil: Pranchevicius, Maria Cristina S.. Universidade Federal do São Carlos; BrasilFil: Madueño, Francisco. Consejo Superior de Investigaciones Científicas; EspañaFil: Costa, M. Manuela R.. Universidade do Minho; PortugalFil: Goldman, Maria Helena S.. Universidade de Sao Paulo; Brasi

Characterization and optimization of ArtinM lectin expression in <it>Escherichia coli</it>

<p>Abstract</p> <p>Background</p> <p>ArtinM is a <smcaps>d</smcaps>-mannose-specific lectin from <it>Artocarpus integrifolia</it> seeds that induces neutrophil migration and activation, degranulation of mast cells, acceleration of wound healing, induction of interleukin-12 production by macrophages and dendritic cells, and protective T helper 1 immune response against <it>Leishmania major</it>, <it>Leishmania amazonensis</it> and <it>Paracoccidioides brasiliensis</it> infections. Considering the important biological properties of ArtinM and its therapeutic applicability, this study was designed to produce high-level expression of active recombinant ArtinM (rArtinM) in <it>Escherichia coli</it> system.</p> <p>Results</p> <p>The ArtinM coding region was inserted in pET29a(+) vector and expressed in <it>E. coli</it> BL21(DE3)-Codon Plus-RP. The conditions for overexpression of soluble ArtinM were optimized testing different parameters: temperatures (20, 25, 30 or 37°C) and shaking speeds (130, 200 or 220 rpm) during induction, concentrations of the induction agent IPTG (0.01-4 mM) and periods of induction (1-19 h). BL21-CodonPlus(DE3)-RP cells induced under the optimized conditions (incubation at 20°C, at a shaking speed of 130 rpm, induction with 0.4 mM IPTG for 19 h) resulted in the accumulation of large amounts of soluble rArtinM. The culture provided 22.4 mg/L of rArtinM, which activity was determined by its one-step purification through affinity chromatography on immobilized <smcaps>d</smcaps>-mannose and glycoarray analysis. Gel filtration showed that rArtinM is monomeric, contrasting with the tetrameric form of the plant native protein (jArtinM). The analysis of intact rArtinM by mass spectrometry revealed a 16,099.5 Da molecular mass, and the peptide mass fingerprint and esi-cid-ms/ms of amino acid sequences of peptides from a tryptic digest covered 41% of the total ArtinM amino acid sequence. In addition, circular dichroism and fluorescence spectroscopy of rArtinM indicated that its global fold comprises β-sheet structure.</p> <p>Conclusions</p> <p>Overall, the optimized process to express rArtinM in <it>E. coli</it> provided high amounts of soluble, correctly folded and active recombinant protein, compatible with large scale production of the lectin.</p

Pollination triggers female gametophyte development in immature Nicotiana tabacum flowers.

In Nicotiana tabacum, female gametophytes are not fully developed at anthesis, but flower buds pollinated 12 h before anthesis produce mature embryo sacs. We investigated several pollination-associated parameters in N. tabacum flower buds to determine the developmental timing of important events in preparation for successful fertilization. First, we performed hand pollinations in flowers from stages 4 to 11 to study at which developmental stage pollination would produce fruits. A Peroxtesmo test was performed to correlate peroxidase activity on the stigma surface, indicative of stigma receptivity, with fruit set. Pollen tube growth and female gametophyte development were microscopically analyzed in pistils of different developmental stages. Fruits were obtained only after pollinations of flower buds at late stage 7 and older; fruit weight and seed germination capacity increased as the developmental stage of the pollinated flower approached anthesis. Despite positive peroxidase activity and pollen tube growth, pistils at stages 5 and 6 were unable to produce fruits. At late stage 7, female gametophytes were undergoing first mitotic division. After 24 h, female gametophytes of unpollinated pistils were still in the end of the first division, whereas those of pollinated pistils showed egg cells. RT-qPCR assay showed that the expression of the NtEC1 gene, a marker of egg cell development, is considerably higher in pollinated late stage 7 ovaries compared with unpollinated ovaries. To test whether ethylene is the signal eliciting female gametophyte maturation, the expression of ACC synthase was examined in unpollinated and pollinated stage 6 and late stage 7 stigmas/styles. Pollination induced NtACS expression in stage 6 pistils, which are unable to produce fruits. Our results show that pollination is a stimulus capable of triggering female gametophyte development in immature tobacco flowers and suggests the existence of a yet undefined signal sensed by the pistil

Analysis of the Nicotiana tabacum Stigma/Style Transcriptome Reveals Gene Expression Differences between Wet and Dry Stigma Species1[W][OA]

The success of plant reproduction depends on pollen-pistil interactions occurring at the stigma/style. These interactions vary depending on the stigma type: wet or dry. Tobacco (Nicotiana tabacum) represents a model of wet stigma, and its stigmas/styles express genes to accomplish the appropriate functions. For a large-scale study of gene expression during tobacco pistil development and preparation for pollination, we generated 11,216 high-quality expressed sequence tags (ESTs) from stigmas/styles and created the TOBEST database. These ESTs were assembled in 6,177 clusters, from which 52.1% are pistil transcripts/genes of unknown function. The 21 clusters with the highest number of ESTs (putative higher expression levels) correspond to genes associated with defense mechanisms or pollen-pistil interactions. The database analysis unraveled tobacco sequences homologous to the Arabidopsis (Arabidopsis thaliana) genes involved in specifying pistil identity or determining normal pistil morphology and function. Additionally, 782 independent clusters were examined by macroarray, revealing 46 stigma/style preferentially expressed genes. Real-time reverse transcription-polymerase chain reaction experiments validated the pistil-preferential expression for nine out of 10 genes tested. A search for these 46 genes in the Arabidopsis pistil data sets demonstrated that only 11 sequences, with putative equivalent molecular functions, are expressed in this dry stigma species. The reverse search for the Arabidopsis pistil genes in the TOBEST exposed a partial overlap between these dry and wet stigma transcriptomes. The TOBEST represents the most extensive survey of gene expression in the stigmas/styles of wet stigma plants, and our results indicate that wet and dry stigmas/styles express common as well as distinct genes in preparation for the pollination process