Coupling Virus-Induced Gene Silencing to Exogenous Green Fluorescence Protein Expression Provides a Highly Efficient System for Functional Genomics in Arabidopsis and across All Stages of Tomato Fruit Development

Since the advent of the postgenomic era, efforts have focused on the development of rapid strategies for annotating plant genes of unknown function. Given its simplicity and rapidity, virus-induced gene silencing (VIGS) has become one of the preeminent approaches for functional analyses. However, several problems remain intrinsic to the use of such a strategy in the study of both metabolic and developmental processes. The most prominent of these is the commonly observed phenomenon of ""sectoring"" the tissue regions that are not effectively targeted by VIGS. To better discriminate these sectors, an effective marker system displaying minimal secondary effects is a prerequisite. Utilizing a VIGS system based on the tobacco rattle virus vector, we here studied the effect of silencing the endogenous phytoene desaturase gene (pds) and the expression and subsequent silencing of the exogenous green fluorescence protein (gfp) on the metabolism of Arabidopsis (Arabidopsis thaliana) leaves and tomato (Solanum lycopersicum) fruits. In leaves, we observed dramatic effects on primary carbon and pigment metabolism associated with the photobleached phenotype following the silencing of the endogenous pds gene. However, relatively few pleiotropic effects on carbon metabolism were observed in tomato fruits when pds expression was inhibited. VIGS coupled to gfp constitutive expression revealed no significant metabolic alterations after triggering of silencing in Arabidopsis leaves and a mild effect in mature green tomato fruits. By contrast, a wider impact on metabolism was observed in ripe fruits. Silencing experiments with an endogenous target gene of interest clearly demonstrated the feasibility of cosilencing in this system; however, carefully constructed control experiments are a prerequisite to prevent erroneous interpretation.

Molecular characterization of Arabidopsis thaliana PUF proteins - binding specificity and target candidates

PUF proteins regulate both stability and translation through sequence-specific binding to the 3` UTR of target mRNA transcripts. Binding is mediated by a conserved PUF domain, which contains eight repeats of approximately 36 amino acids each. Found in all eukaryotes, they have been related to several developmental processes. Analysis of the 25 Arabidopsis Pumilio (APUM) proteins presenting PUF repeats reveals that 12 (APUM-1 to APUM-12) have a PUF domain with 50-75% similarity to the Drosophila PUF domain. Through three-hybrid assays, we show that APUM-1 to APUM-6 can bind specifically to the Nanos response element sequence recognized by Drosophila Pumilio. Using an Arabidopsis RNA library in a three-hybrid screening, we were able to identify an APUM-binding consensus sequence. Computational analysis allowed us to identify the APUM-binding element within the 3` UTR in many Arabidopsis transcripts, even in important mRNAs related to shoot stem cell maintenance. We demonstrate that APUM-1 to APUM-6 are able to bind specifically to APUM-binding elements in the 3` UTR of WUSCHEL, CLAVATA-1, PINHEAD/ZWILLE and FASCIATA-2 transcripts. The results obtained in the present study indicate that the APUM proteins may act as regulators in Arabidopsis through an evolutionarily conserved mechanism, which may open up a new approach for investigating mRNA regulation in plants.

The Arabidopsis bZIP Gene AtbZIP63 Is a Sensitive Integrator of Transient Abscisic Acid and Glucose Signals

Glucose modulates plant metabolism, growth, and development. In Arabidopsis (Arabidopsis thaliana), Hexokinase1 (HXK1) is a glucose sensor that may trigger abscisic acid (ABA) synthesis and sensitivity to mediate glucose-induced inhibition of seedling development. Here, we show that the intensity of short-term responses to glucose can vary with ABA activity. We report that the transient (2 h/4 h) repression by 2% glucose of AtbZIP63, a gene encoding a basic-leucine zipper (bZIP) transcription factor partially involved in the Snf1-related kinase KIN10-induced responses to energy limitation, is independent of HXK1 and is not mediated by changes in ABA levels. However, high-concentration (6%) glucose-mediated repression appears to be modulated by ABA, since full repression of AtbZIP63 requires a functional ABA biosynthetic pathway. Furthermore, the combination of glucose and ABA was able to trigger a synergistic repression of AtbZIP63 and its homologue AtbZIP3, revealing a shared regulatory feature consisting of the modulation of glucose sensitivity by ABA. The synergistic regulation of AtbZIP63 was not reproduced by an AtbZIP63 promoter-5`-untranslated region:beta-glucuronidase fusion, thus suggesting possible posttranscriptional control. A transcriptional inhibition assay with cordycepin provided further evidence for the regulation of mRNA decay in response to glucose plus ABA. Overall, these results indicate that AtbZIP63 is an important node of the glucose-ABA interaction network. The mechanisms by which AtbZIP63 may participate in the fine-tuning of ABA-mediated abiotic stress responses according to sugar availability (i.e., energy status) are discussed.

Elevated CO(2) increases photosynthesis, biomass and productivity, and modifies gene expression in sugarcane

Because of the economical relevance of sugarcane and its high potential as a source of biofuel, it is important to understand how this crop will respond to the foreseen increase in atmospheric [CO(2)]. The effects of increased [CO(2)] on photosynthesis, development and carbohydrate metabolism were studied in sugarcane (Saccharum ssp.). Plants were grown at ambient (similar to 370 ppm) and elevated (similar to 720 ppm) [CO(2)] during 50 weeks in open-top chambers. The plants grown under elevated CO(2) showed, at the end of such period, an increase of about 30% in photosynthesis and 17% in height, and accumulated 40% more biomass in comparison with the plants grown at ambient [CO(2)]. These plants also had lower stomatal conductance and transpiration rates (-37 and -32%, respectively), and higher water-use efficiency (c.a. 62%). cDNA microarray analyses revealed a differential expression of 35 genes on the leaves (14 repressed and 22 induced) by elevated CO(2). The latter are mainly related to photosynthesis and development. Industrial productivity analysis showed an increase of about 29% in sucrose content. These data suggest that sugarcane crops increase productivity in higher [CO(2)], and that this might be related, as previously observed for maize and sorghum, to transient drought stress.

Endogenous abscisic acid and protein contents during seed development of Araucaria angustifolia

This paper describes a proteome analysis and changes in endogenous abscisic acid (ABA) contents during seed development of Araucaria angustifolia (Bert.) O. Ktze. Megagametophytes and embryonic axis tissues exhibited a similar ABA variation pattern during seed development, reaching maximum values at the pre-cotyledonary stage. The embryonic axis protein content increased until the cotyledonary stage with following stabilization at mature seed. The two-dimensional electrophoresis at the torpedo developmental stage showed approximately 230 polypeptides against 340 in the mature stage. Peptide mass fingerprinting analyses identified three polypeptides, corresponding to an AtSAC4, a late embryogenesis abundant (LEA) and a storage protein, respectively.

Differential proteome analysis of mature and germinated embryos of Araucaria angustifolia

Araucaria angustifolia is an endangered Brazilian native conifer tree. The aim of the present work was to identify differentially expressed proteins between mature and germinated embryos of A. angustifolia, using one and two dimensional gel electrophoresis approaches followed by protein identification by tandem mass spectrometry. The identities of 32 differentially expressed protein spots from two dimensional gel maps were successfully determined, including proteins and enzymes involved in storage mobilization such as the vicilin-like storage protein and proteases. A label free approach, based on spectral counts, resulted in detection of 10 and 14 mature and germinated enriched proteins, respectively. Identified proteins were mainly related to energetic metabolism pathways, translational processes. oxidative stress regulation and cellular signaling. The integrated use of both strategies permitted a comprehensive protein expression overview of changes in germinated embryos in relation to matures, providing insights into the this process in a recalcitrant seed species. Applications of the data generated on the monitoring and control of in vitro somatic embryos were discussed. Published by Elsevier Ltd.

Effects of abscisic acid, ethylene and sugars on the mobilization of storage proteins and carbohydrates in seeds of the tropical tree Sesbania virgata (Leguminosae)

Endospermic legumes are abundant in tropical forests and their establishment is closely related to the mobilization of cell-wall storage polysaccharides. Endosperm cells also store large numbers of protein bodies that play an important role as a nitrogen reserve in this seed. In this work, a systems approach was adopted to evaluate some of the changes in carbohydrates and hormones during the development of seedlings of the rain forest tree Sesbania virgata during the period of establishment. Seeds imbibed abscisic acid (ABA), glucose and sucrose in an atmosphere of ethylene, and the effects of these compounds on the protein contents, alpha-galactosidase activity and endogenous production of ABA and ethylene by the seeds were observed. The presence of exogenous ABA retarded the degradation of storage protein in the endosperm and decreased alpha-galactosidase activity in the same tissue during galactomannan degradation, suggesting that ABA represses enzyme action. On the other hand, exogenous ethylene increased alpha-galactosidase activity in both the endosperm and testa during galactomannan degradation, suggesting an inducing effect of this hormone on the hydrolytic enzymes. Furthermore, the detection of endogenous ABA and ethylene production during the period of storage mobilization and the changes observed in the production of these endogenous hormones in the presence of glucose and sucrose, suggested a correlation between the signalling pathway of these hormones and the sugars. These findings suggest that ABA, ethylene and sugars play a role in the control of the hydrolytic enzyme activities in seeds of S. virgata, controlling the process of storage degradation. This is thought to ensure a balanced flow of the carbon and nitrogen for seedling development.

Ethylene and polyamine interactions in morphogenesis of Passiflora cincinnata: effects of ethylene biosynthesis and action modulators, as well as ethylene scavengers

Ethylene is a plant hormone that is of fundamental importance to in vitro morphogenesis, but in many species, it has not been thoroughly studied. Its relationship with polyamines has been studied mainly because the two classes of hormones share a common biosynthetic precursor, S-adenosylmethionine (SAM). In order to clarify whether competition between polyamines and ethylene influences in vitro morphogenetic responses of Passiflora cincinnata Mast., a climacteric species, different compounds were used that act on ethylene biosynthesis and action, or as ethylene scavengers. Treatment with the ethylene inhibitor, aminoethoxyvinylglycine (AVG) caused a greater regeneration frequency in P. cincinnata, whereas treatment with the ethylene precursor, 1-aminocyclopropane-1-carboxylic-acid (ACC) lessened regeneration frequencies. The data suggested that levels of polyamines and ethylene are not correlated with morphogenic responses in P. cincinnata. It was ascertained that neither the absolute ethylene and polyamine levels, nor competition between the compounds, correlated to the obtained morphogenic responses. However, sensitivity to, and signaling by, ethylene appears to play an important role in differentiation. This study reinforces previous reports regarding the requirement of critical concentrations and temporal regulation of ethylene levels for morphogenic responses. Temporal regulation also appeared to be a key factor in competition between the two biosynthetic pathways, without having any effects on morphogenesis. Further studies investigating the silencing or overexpression of genes related to ethylene perception, under the influence of polyamines in cell differentiation are extremely important for the complete understanding of this process.

Cell wall polysaccharides from cell suspension cultures of the Atlantic Forest tree Rudgea jasminoides (Rubiaceae)

Rudgea jasminoides (Rubiaceae) is a tropical tree species native of the Atlantic Forest in the south of Brazil. Previous studies with leaf cell walls of R. jasminoides showed a different proportion of cross-linked glycans compared to what is usually reported for eudicots. However, due to the difficulties of working with whole plant organs, cell suspensions of R. jasminoides, consisting of predominantly undifferentiated cells with mainly primary cell walls, were used to examine cell walls and extracellular soluble polysaccharides (EP) released into the culture medium. Sugar composition and linkage analysis showed homogalacturonans, xylogalacturonans and arabinogalactans to be the predominant EP. In the cell wall, homogalacturonans and arabinogalactans are the major pectins, and xyloglucans and xylans are the major cross-linking glycans. The presence of xylogalacturonans in the R. jasminoides cell cultures seems to be related to the occurrence of a homogeneous cell suspension with loosely attached cells. Although all alkali extractions from the cell walls yielded amounts of xyloglucan that exceed those of the xylans, the latter was found in a proportion that is higher than what has been usually reported for primary cell walls of most eudicots. The xyloglucan from cell walls of cell suspension cultures of R. jasminoides has low fucosylation levels and high proportion of galactosyl residues, a branching pattern commonly found in storage cell-wall xyloglucans.

Abscisic acid and auxin accumulation in Catasetum fimbriatum roots growing in vitro with high sucrose and mannitol content

Endogenous contents of indolyl-3-acetic acid (IAA) and abscisic acid (ABA) were quantified in excised roots of Catasetum fimbriatum (Orchidaceae) cultured in vitro on solidified Vacin and Went medium with 1, 2, 4, 6, 8 and 10 % sucrose, as well as 2 % sucrose plus mannitol. Maximum root growth was observed in media with 4 % sucrose and 2 % sucrose plus 2.2 % mannitol, suggesting that a moderate water or osmotic stress promotes orchid root growth. Contents of both ABA and IAA increased in parallel to increasing sucrose concentration and a correlation between root elongation and the ABA/IAA ratio was observed. Incubating isolated C. fimbriatum roots with radiolabeled tryptophan, we showed an accumulation of IAA and its conjugates.

Polyamines, amino acids, IAA and ABA contents during Ocotea catharinensis seed germination

The aim of this work was to Study biochemical variations of IAA (indole-3-acetic acid), ABA (abscisic acid). PAs (polyamines) and amino acids at endogenous levels, during seed germination in Ocotea catharinensis. Seeds were germinated in a vermiculite substratum (100%), samples being collected after 15, 30 and 60 days. Total amino acid levels decreased during the first 15 days. Followed by all increment at the end of germination. Among amino acids, higher concentration was observed in asparagine, this being the predominant amino acid during the whole germination period. Total PAs (free + conjugated) content increased during the first 15 days, followed by a decrease and stabilization between 30 and 60 days of germination. Among the PAs, free putrescine levels rose during the first 15 days, followed by a drop and Stabilization up to 60 days of germination, while spermidine and spermine (spm) contents diminished during the period. Only spin was detected in a conjugated form, with increasing concentrations starting from 30 days on. IAA levels increased during tire first 15 days. followed by a decrease and stabilization until the end of germination (60 days), while ABA contents dwindled during the first 15 days, with similar Values until the end of germination.

Genetic dissection of vitamin E biosynthesis in tomato

Vegetables are critical for human health as they are a source of multiple vitamins including vitamin E (VTE). In plants, the synthesis of VTE compounds, tocopherol and tocotrienol, derives from precursors of the shikimate and methylerythritol phosphate pathways. Quantitative trait loci (QTL) for alpha-tocopherol content in ripe fruit have previously been determined in an Solanum pennellii tomato introgression line population. In this work, variations of tocopherol isoforms (alpha, beta, gamma, and delta) in ripe fruits of these lines were studied. In parallel all tomato genes structurally associated with VTE biosynthesis were identified and mapped. Previously identified VTE QTL on chromosomes 6 and 9 were confirmed whilst novel ones were identified on chromosomes 7 and 8. Integrated analysis at the metabolic, genetic and genomic levels allowed us to propose 16 candidate loci putatively affecting tocopherol content in tomato. A comparative analysis revealed polymorphisms at nucleotide and amino acid levels between Solanum lycopersicum and S. pennellii candidate alleles. Moreover, evolutionary analyses showed the presence of codons evolving under both neutral and positive selection, which may explain the phenotypic differences between species. These data represent an important step in understanding the genetic determinants of VTE natural variation in tomato fruit and as such in the ability to improve the content of this important nutriceutical.

Evolutionary patterns in neotropical Helieae (Gentianaceae): evidence from morphology, chloroplast and nuclear DNA sequences

Parsimony-based phylogenetic analyses of the neotropical tribe Helieae (Gentianaceae) are presented, including 22 of the 23 genera and 60 species. This study is based on data from morphology, palynology, and seed micromorphology (127 structural characters), and DNA sequences (matK, trnL intron, ITS). Phylogenetic reconstructions based on ITS and morphology provided the greatest resolution, morphological data further helping to tentatively place several taxa for which DNA was not available (Celiantha, Lagenanthus, Rogersonanthus, Roraimaea, Senaea, Sipapoantha, Zonanthus). Celiantha, Prepusa and Senaea together appear as the sister clade to the rest of Helieae. The remainder of Helieae is largely divided into two large subclades, the Macrocarpaea subclade and the Symbolanthus subclade. The first subclade includes Macrocarpaea, sister to Chorisepalum, Tochia, and Zonanthus. Irlbachia and Neblinantha are placed as sisters to the Symbolanthus subclade, which includes Aripuana, Calolisianthus, Chelonanthus, Helia, Lagenanthus, Lehmanniella, Purdieanthus, Rogersonanthus, Roraimaea, Sipapoantha, and symbolanthus. Generic-level polyphyly is detected in Chelonanthus and Irlbachia. Evolution of morphological characters is discussed, and new pollen and seed characters are evaluated for the first time in a combined morphological-molecular phylogenetic analysis.

Stigma/style cell cycle inhibitor 1 (SCI1), a tissue-specific cell cycle regulator that controls upper pistil development

P>A cDNA encoding a small lysine-rich protein of unknown function was identified in a tobacco (Nicotiana tabacum) stigma/style suppression subtractive hybridization cDNA library. After its characterization, the corresponding gene was designated stigma/style cell cycle inhibitor 1 (SCI1). Fluorescence microscopy with an SCI1-GFP protein fusion demonstrated its nuclear localization, which was confined to the interchromatic region. Real-time RT-PCR and in situ hybridization experiments showed that SCI1 is stigma/style-specific and developmentally regulated. SCI1 RNAi knockdown and overexpression plants had stigmas/styles with remarkably enlarged and reduced areas, respectively, which was attributable to differences in cell numbers. These results indicate that SCI1 is a tissue-specific negative cell cycle regulator. The differences in cell division had an effect on the timing of the differentiation of the stigmatic papillar cells, suggesting that their differentiation is coupled to stigma cell divisions. This is consistent with a role for SCI1 in triggering differentiation through cell proliferation control. Our results revealed that SCI1 is a novel tissue-specific gene that controls cell proliferation/differentiation, probably as a component of a developmental signal transduction pathway.

Polyamines, IAA and ABA during germination in two recalcitrant seeds: Araucaria angustifolia (Gymnosperm) and Ocotea odorifera (Angiosperm)

Background and Aims Plant growth regulators play an important role in seed germination. However, much of the current knowledge about their function during seed germination was obtained using orthodox seeds as model systems, and there is a paucity of information about the role of plant growth regulators during germination of recalcitrant seeds. In the present work, two endangered woody species with recalcitrant seeds, Araucaria angustifolia (Gymnosperm) and Ocotea odorifera (Angiosperm), native to the Atlantic Rain Forest, Brazil, were used to study the mobilization of polyamines (PAs), indole-acetic acid (IAA) and abscisic acid (ABA) during seed germination. Methods Data were sampled from embryos of O. odorifera and embryos and megagametophytes of A. angustifolia throughout the germination process. Biochemical analyses were carried out in HPLC. Key Results During seed germination, an increase in the (Spd + Spm) : Put ratio was recorded in embryos in both species. An increase in IAA and PA levels was also observed during seed germination in both embryos, while ABA levels showed a decrease in O. odorifera and an increase in A. angustifolia embryos throughout the period studied. Conclusions The (Spd + Spm) : Put ratio could be used as a marker for germination completion. The increase in IAA levels, prior to germination, could be associated with variations in PA content. The ABA mobilization observed in the embryos could represent a greater resistance to this hormone in recalcitrant seeds, in comparison to orthodox seeds, opening a new perspective for studies on the effects of this regulator in recalcitrant seeds. The gymnosperm seed, though without a connective tissue between megagametophyte and embryo, seems to be able to maintain communication between the tissues, based on the likely transport of plant growth regulators.