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Heterobaric leaves have bundle sheath extensions (BSEs) that compartmentalise the parenchyma, whereas homobaric leaves do not. The presence of BSEs affects leaf hydraulics and photosynthetic rate. The tomato (Solanum lycopersicum) obscuravenosa (obv) mutant lacks BSEs. Here we identify the obv gene and the causative mutation, a non-synonymous amino acid change that disrupts a C2H2 zinc finger motif in a putative transcription factor. This mutation exists as a rare polymorphism in the natural range of wild tomatoes, but has increased in frequency in domesticated tomatoes, suggesting that the latter diversified into heterobaric and homobaric leaf types. The obv mutant displays reduced vein density, leaf hydraulic conductance and photosynthetic assimilation rate. We show that these and other effects on plant development, including changes in leaf insertion angle, leaf margin serration, minor vein density and fruit shape, are controlled by OBV via changes in auxin signalling. Loss of function of the transcriptional regulator AUXIN RESPONSE FACTOR (ARF4) also results in defective BSE development, revealing an additional component of a novel genetic module controlling aspects of leaf development important for ecological adaptation and subject to breeding selection.One sentence summarydistribution of tomato heterobaric and homobaric leaves is controlled by a single-nucleotide polymorphism in an auxin-related transcription factor

Witches’ broom disease of cacao is caused by the pathogenic fungus Moniliophthora perniciosa. By using tomato (Solanum lycopersicum) cultivar Micro-Tom (MT) as a model system, we investigated the physiological and metabolic consequences of M. perniciosa infection to determine whether symptoms result from sink establishment during infection. Infection of MT by M. perniciosa caused reductions in root biomass and fruit yield, a decrease in leaf gas exchange, and down-regulation of photosynthesis-related genes. The total leaf area and water potential decreased, while ABA levels, water conductance/conductivity, and ABA-related gene expression increased. Genes related to sugar metabolism and those involved in secondary cell wall deposition were up-regulated upon infection, and the concentrations of sugars, fumarate, and amino acids increased. 14C-glucose was mobilized towards infected MT stems, but not in inoculated stems of the MT line overexpressing CYTOKININ OXIDASE-2 (35S::AtCKX2), suggesting a role for cytokinin in establishing a sugar sink. The up-regulation of genes involved in cell wall deposition and phenylpropanoid metabolism in infected MT, but not in 35S::AtCKX2 plants, suggests establishment of a cytokinin-mediated sink that promotes tissue overgrowth with an increase in lignin. Possibly, M. perniciosa could benefit from the accumulation of secondary cell walls during its saprotrophic phase of infection.

Moniliophthora perniciosa causes witches' broom disease of cacao and inflicts symptoms suggestive of hormonal imbalance. We investigated whether infection of the tomato (Solanum lycopersicum) model system Micro-Tom (MT) by the Solanaceae (S)-biotype of Moniliophthora perniciosa, which causes stem swelling and hypertrophic growth of axillary shoots, results from changes in host cytokinin metabolism. Inoculation of an MT-transgenic line that overexpresses the Arabidopsis CYTOKININ OXIDASE-2 gene (35S::AtCKX2) resulted in a reduction in disease incidence and stem diameter. RNA-sequencing analysis of infected MT and 35S::AtCKX2 revealed the activation of cytokinin-responsive marker genes when symptoms were conspicuous. The expression of an Moniliophthora perniciosa tRNA-ISOPENTENYL-TRANSFERASE suggests the production of isopentenyladenine (iP), detected in mycelia grown in vitro. Inoculated MT stems showed higher levels of dihydrozeatin and trans-zeatin but not iP. The application of benzyladenine induced symptoms similar to infection, whereas applying the cytokinin receptor inhibitors LGR-991 and PI55 decreased symptoms. Moniliophthora perniciosa produces iP that might contribute to cytokinin synthesis by the host, which results in vascular and cortex enlargement, axillary shoot outgrowth, reduction in root biomass and an increase in fruit locule number. This strategy may be associated with the manipulation of sink establishment to favour infection by the fungus.

Background 2,4-dihydroxy-7-methoxy-1,4-benzoxazin-3-one (DIMBOA), the main benzoxazinoid found in corn, elicits variable larval responses from different pest moths. For the widespread and highly polyphagous Spodoptera frugiperda (Lepidoptera: Noctuidae), the fall-armyworm (FAW), DIMBOA acts as a feeding stimulant and improves larval growth at low concentrations. The FAW present two host plant-related strains, corn and rice strains, related to host preference on corn and other Graminae or rice. Based on both host preference and strain divergence of the FAW on corn, a cereal containing DIMBOA, and rice, lacking this compound, we question if corn and rice strains larvae respond equally toward DIMBOA. We evaluated differential expression in the transcriptome of both midgut and fat body larval tissues of the two strains reared on either DIMBOA-enriched artificial diet or control diet and inferred Bayesian networks. Results We found differences in performance between corn and rice strain larvae reared on DIMBOA, as well as several differentially regulated contigs annotated as esterases, peptidases, transferases and reductases, all of them known for being related to responses of lepidopterans and other insects to DIMBOA. We also found a UDP-glucuronosyltransferase very similar to others found in many lepidopterans occupying a central hub within a transferase Bayesian network, suggesting that it is essential to an effective response to DIMBOA in FAW. Conclusion Our results suggest that there is an intrinsic cost for FAW rice strain larvae to metabolize corn-originated hydroxamic acids, which could have resulted in the partial host-associated genetic isolation found at FAW field populations.

Phytocystatins are tight-binding cysteine protease inhibitors produced by plants. The first phytocystatin described was isolated from Oryza sativa and, since then, cystatins from several plant species were reported, including from sugarcane. Sugarcane cystatins were unraveled in Sugarcane EST project database, after sequencing of cDNA libraries from various sugarcane tissues at different developmental stages and six sugarcane cystatins were cloned, expressed and characterized (CaneCPI-1 to CaneCPI-6). These recombinant proteins were produced in different expression systems and inhibited several cysteine proteases, including human cathepsins B and L, which can be involved in pathologies, such as cancer. In this review, we summarize a comprehensive history of all sugarcane cystatins, presenting an updated phylogenetic analysis; chromosomal localization, and genomic organization. We also present protein docking of CaneCPI-5 in the active site of human cathepsin B, insights about canecystatins structures; recombinant expression in different systems, comparison of their inhibitory activities against human cysteine cathepsins B, K, L, S, V, falcipains from Plasmodium falciparum and a cathepsin L-like from the sugarcane weevil Sphenophorus levis; and enlighten their potential and current applications in agriculture and health.

Background Our main purpose was to evaluate the expression of plastic and evolved genes involved in ecological speciation in the noctuid moth Spodoptera frugiperda, the fall armyworm (FAW); and to demonstrate how host plants might influence lineage differentiation in this polyphagous insect. FAW is an important pest of several crops worldwide, and it is differentiated into host plant-related strains, corn (CS) and rice strains (RS). RNA-Seq and transcriptome characterization were applied to evaluate unbiased genetic expression differences in larvae from the two strains, fed on primary (corn) and alternative (rice) host plants. We consider that genes that are differently regulated by the same FAW strain, as a response to different hosts, are “plastic”. Otherwise, differences in gene expression between the two strains fed on the same host are considered constitutive differences. Results Individual performance parameters (larval and pupal weight) varied among conditions (strains vs. hosts). A total of 3657 contigs was related to plastic response, and 2395 contigs were differentially regulated in the two strains feeding on preferential and alternative hosts (constitutive contigs). Three molecular functions were present in all comparisons, both down- and up-regulated: oxidoreductase activity, metal-ion binding, and hydrolase activity. Conclusions Metabolization of foreign chemicals is among the key functions involved in the phenotypic variation of FAW strains. From an agricultural perspective, high plasticity in families of detoxifying genes indicates the capacity for a rapid response to control compounds such as insecticides.

Forest fragmentation may affect mating and pollen dispersal patterns through conversion of continuous forests into small, spatially isolated remnant patches and individual trees in an anthropogenic landscape. We investigated reproductive investment and success, pollen dispersal, mating system, and genetic diversity and spatial structure of Qualea grandiflora trees in two environmental contexts: a continuous natural Cerrado area and isolated individuals on roadsides. Roadside trees produced more flowers and more fruit than Cerrado trees. Pollen dispersal kernels were fat-tailed in both contexts, indicating long-distance dispersal, but in Cerrado the mean pollen dispersal distance (524.7 m) and the effective number of pollen donors per mother-tree (Nep = 12.7) were higher than for roadside trees (60.9 m, Nep = 4.6). The levels and structure of genetic diversity, outcrossing rates ( $$t_{m}$$ > 0.98), and mating among relatives ( $$t_{m} - t_{s}$$ < 0.1) were similar in both environmental contexts. Allelic richness and number of private alleles were similar between the two environments. The fixation index was significantly lower in adults (minimum of 0.08) than in offspring (minimum of 0.23) in both contexts, suggesting selection against inbred individuals between offspring and adult stage. Our results indicate that the spatial isolation of roadside trees, by increasing the number of flowers produced, decreased pollinator movements, thereby reducing effective pollen flow and the number of pollen donors. All these results suggest that roadside trees can be used for harvesting seeds for recovery plans, and that these trees are a biological legacy, and reservoir of Q. grandiflora genetic diversity, from the original Cerrado forest.

Made available in DSpace on 2019-10-06T17:04:47Z (GMT). No. of bitstreams: 0 Previous issue date: 2019-02-13 National Institute of Agrobiological Sciences A sugarcane gene encoding a dirigent-jacalin, ShDJ, was induced under drought stress. To elucidate its biological function, we integrated a ShDJ-overexpression construction into the rice Nipponbare genome via Agrobacterium-mediated transformation. Two transgenic lines with a single copy gene in T 0 were selected and evaluated in both the T 1 and T 4 generations. Transgenic lines had drastically improved survival rate under water deficit conditions, at rates close to 100%, while WT did not survive. Besides, transgenic lines had improved biomass production and higher tillering under water deficit conditions compared with WT plants. Reduced pectin and hemicellulose contents were observed in transgenic lines compared with wild-type plants under both well-watered and water deficit conditions, whereas cellulose content was unchanged in line #17 and reduced in line #29 under conditions of low water availability. Changes in lignin content under water deficit were only observed in line #17. However, improvements in saccharification were found in both transgenic lines along with changes in the expression of OsNTS1/2 and OsMYB58/63 secondary cell wall biosynthesis genes. ShDJ-overexpression up-regulated the expression of the OsbZIP23, OsGRAS23, OsP5CS, and OsLea3 genes in rice stems under well-watered conditions. Taken together, our data suggest that ShDJ has the potential for improving drought tolerance, plant biomass accumulation, and saccharification efficiency. Instituto Agronômico (IAC) Centro de Cana PPG-Genética Faculdade de Medicina de Ribeirão Preto Universidade de São Paulo Department of Plant Biology Institute of Biology University of Campinas Instituto de Ciência e Tecnologia Universidade Federal de São Paulo Instituto Agronômico (IAC) Centro de Ecofisiologia e Biofísica Departamento de Biologia Universidade Federal de Lavras Departamento de Biologia Faculdade de Filosofia Ciências e Letras de Ribeirão Preto Universidade de São Paulo Faculdade de Ciências Agrárias e Veterinárias Universidade Estadual Paulista Júlio de Mesquita Filho (UNESP) Centro de Energia Nuclear na Agricultura (CENA) University of São Paulo Texas A&M Agrilife Research & Extension Center Faculdade de Ciências Agrárias e Veterinárias Universidade Estadual Paulista Júlio de Mesquita Filho (UNESP)

A sugarcane gene encoding a dirigent-jacalin, ShDJ, was induced under drought stress. To elucidate its biological function, we integrated a ShDJ-overexpression construction into the rice Nipponbare genome via Agrobacterium-mediated transformation. Two transgenic lines with a single copy gene in T0 were selected and evaluated in both the T1 and T4 generations. Transgenic lines had drastically improved survival rate under water deficit conditions, at rates close to 100%, while WT did not survive. Besides, transgenic lines had improved biomass production and higher tillering under water deficit conditions compared with WT plants. Reduced pectin and hemicellulose contents were observed in transgenic lines compared with wild-type plants under both well-watered and water deficit conditions, whereas cellulose content was unchanged in line #17 and reduced in line #29 under conditions of low water availability. Changes in lignin content under water deficit were only observed in line #17. However, improvements in saccharification were found in both transgenic lines along with changes in the expression of OsNTS1/2 and OsMYB58/63 secondary cell wall biosynthesis genes. ShDJ-overexpression up-regulated the expression of the OsbZIP23, OsGRAS23, OsP5CS, and OsLea3 genes in rice stems under well-watered conditions. Taken together, our data suggest that ShDJ has the potential for improving drought tolerance, plant biomass accumulation, and saccharification efficiency.

Complementation of the “Micro-Tom” tomato tangerine mutant with a Citrus CRTISO allele restores the wild-type fruit carotenoid profile, indicating that the Citrus allele encodes an authentic functional carotenoid isomerase. Citrus fruits are rich in carotenoids; the genus offers a large diversity in composition, yet to be fully explored to improve fruit nutritional quality. As perennial tree species, Citrus lack the resources for functional genetic studies, requiring the use of model plant systems. Here, we used the “Micro-Tom” (MT) tomato carrying the tangerine mutation (t), deficient for the carotenoid isomerase (CRTISO) gene, to functionally characterize the homologous C. sinensis genes. We identified four putative loci in the C. sinensis genome, named CsCRTISO, CsCRTISO-Like 1, CsCRTISO-Like 2, and CsCRTISO-Like 2B, with the latter as a presumed duplication of CRTISO-Like 2. In general, all the Citrus paralogs showed less expression specialization than the tomato ones, with CsCRTISO being the most expressed gene in all tissues analyzed. MT-t plants were successfully complemented with the CsCRTISO, and fruits showed a carotenoid profile similar to the control, indicating that the Citrus allele indeed encodes an authentic functional carotenoid isomerase and that the signal peptide is functional in tomato. MT was silenced using an inverted repeat of a fragment from the Citrus CRTISO resulting in a stronger phenotype than MT-t. MT-t and MT silenced for CRTISO presented an overall decrease in transcript accumulation of all genes from the biosynthesis pathway. The expression of the Citrus CRTISO gene is able to restore the biosynthesis of carotenoids with the appropriate regulation in MT-t. The decrease in transcript accumulation in MT-t and MT-CRTISO-suppressed lines reinforces previous suggestions that transcriptional regulation of the carotenoid biosynthesis involves regulatory loops by intermediate products.

Drought is the most significant environmental stress for agricultural production worldwide, and tremendous efforts have been made to improve crop yield under the increasing water scarcity. Transcription factors are major players in the regulation of water stress-related genes in plants. Recently, different MYB transcription factors were characterized for their involvement in drought response. A sugarcane R2R3-MYB gene (ScMYBAS1) and its four alternative forms of transcript (ScMYAS1-2, ScMYBAS1-3, ScMYBAS1-4 and ScMYBAS1-5) were identified in this study. The subcellular localization, in Nicotiniana benthamiana, of the TFs fused in frame with GFP revealed that ScMYBAS1-2-GFP and ScMYBAS1-3-GFP were observed in the nucleus. The overexpression of ScMYBAS1-2 and ScMYBAS1-3 spliced transcripts in rice promoted change in plant growth under both well-watered and drought conditions. The ScMYBAS1-2 and ScMYBAS1-3 transgenic lines revealed a higher relative water content (RWC) compared to the wild type before maximum stress under drought conditions. The ScMYBAS1-2 transgenic lines showed a reduction in biomass (total dry weight). Conversely, ScMYBAS1-3 showed an increased biomass (total dry weight) relative to the wild-type. The overexpression of ScMYBAS1-3 in rice transgenic lines showed involvement with drought tolerance and biomass and, for this reason, was considered a good target for plant transformation, particularly for use in developing genotypes with drought tolerance and biomass accumulation.

Red-fleshed oranges (Citrus sinensis) contain high levels of carotenoids and lycopene. The growing consumer demand for products with health benefits has increased interest in these types of Citrus cultivars as a potential source of nutraceuticals. However, little is known about the physiology of these cultivars under Brazilian conditions. Transcriptome and gene expression analyses are important tools in the breeding and management of red-fleshed sweet orange cultivars. Reverse transcription quantitative polymerase chain reaction is a method of quantifying gene expression, but various standardizations are required to obtain precise, accurate, and specific results. Among the standardizations required, the choice of suitable stable reference genes is fundamental. The objective of this study was to evaluate the stability of 11 candidate genes using various tissue and organ samples from healthy plants or leaves from citrus greening disease (Huanglongbing)-symptomatic plants of a Brazilian red-fleshed cultivar ('Sanguínea de Mombuca'), in order to select the most suitable reference gene for investigating gene expression under these conditions. geNorm and NormFinder identified genes that encoded translation initiation factor 3, ribosomal protein L35, and translation initiation factor 5A as the most stable genes under the biological conditions tested, and genes coding actin (ACT) and the subunit of the PSI reaction center subunit III were the least stable. Phosphatase, malate dehydrogenase, and ACT were the most stable genes in the leaf samples of infected plants.

Background Citrus breeding programs have many limitations associated with the species biology and physiology, requiring the incorporation of new biotechnological tools to provide new breeding possibilities. Diversity Arrays Technology (DArT) markers, combined with next-generation sequencing, have wide applicability in the construction of high-resolution genetic maps and in quantitative trait locus (QTL) mapping. This study aimed to construct an integrated genetic map using full-sib progeny derived from Murcott tangor and Pera sweet orange and DArTseq™ molecular markers and to perform QTL mapping of twelve fruit quality traits. A controlled Murcott x Pera crossing was conducted at the Citrus Germplasm Repository at the Sylvio Moreira Citrus Centre of the Agronomic Institute (IAC) located in Cordeirópolis, SP, in 1997. In 2012, 278 F1 individuals out of a family of 312 confirmed hybrid individuals were analyzed for fruit traits and genotyped using the DArTseq markers. Using OneMap software to obtain the integrated genetic map, we considered only the DArT loci that showed no segregation deviation. The likelihood ratio and the genomic information from the available Citrus sinensis L. Osbeck genome were used to determine the linkage groups (LGs). Results The resulting integrated map contained 661 markers in 13 LGs, with a genomic coverage of 2,774 cM and a mean density of 0.23 markers/cM. The groups were assigned to the nine Citrus haploid chromosomes; however, some of the chromosomes were represented by two LGs due the lack of information for a single integration, as in cases where markers segregated in a 3:1 fashion. A total of 19 QTLs were identified through composite interval mapping (CIM) of the 12 analyzed fruit characteristics: fruit diameter (cm), height (cm), height/diameter ratio, weight (g), rind thickness (cm), segments per fruit, total soluble solids (TSS, %), total titratable acidity (TTA, %), juice content (%), number of seeds, TSS/TTA ratio and number of fruits per box. The genomic sequence (pseudochromosomes) of C. sinensis was compared to the genetic map, and synteny was clearly identified. Further analysis of the map regions with the highest LOD scores enabled the identification of putative genes that could be associated with the fruit quality characteristics. Conclusion An integrated linkage map of Murcott tangor and Pera sweet orange using DArTseq™ molecular markers was established and it was useful to perform QTL mapping of twelve fruit quality traits. The next generation sequences data allowed the comparison between the linkage map and the genomic sequence (pseudochromosomes) of C. sinensis and the identification of genes that may be responsible for phenotypic traits in Citrus. The obtained linkage map was used to assign sequences that had not been previously assigned to a position in the reference genome. Electronic supplementary material The online version of this article (doi:10.1186/s12864-017-3629-2) contains supplementary material, which is available to authorized users.

The miniature tomato (Solanum lycopersicum) cultivar Micro-Tom (MT) has become an important platform to investigate plant–pathogen interactions. In the case of the witches’ broom disease of Theobroma cacao (cacao), the existence of Moniliophthora perniciosa isolates pathogenic to Solanaceae (S-biotype) may enable the use of MT to circumvent limitations of the cacao host, whereas the availability of a non-infective cacao C-biotype allows the evaluation of contrasting responses of MT. Infection of MT by the S-biotype led to stem swelling and axillary shoot growth to form broom-like symptoms similar to the biotrophic phase in cacao, but the infected tissues did not progress to necrosis. Conversely, inoculation with the C-biotype did not cause typical symptoms, but reduced plant height, appearing as a non-host interaction. Histopathological characterization of the S-biotype infection of MT by light and electron microscopy indicated limited germ tube penetration, preferentially through wounds at the base of trichomes or actively through the epidermis. No intracellular mycelium was observed, corroborating the lack of the necrotrophic stage of the pathogen. The analysis of gene expression during the interaction between the S- or C-biotype with MT indicated that expression of plant defence-associated genes differs for kinetics and intensity between a compatible or incompatible M. perniciosa–MT interaction. The pattern of spore germination and low rate of mycelia penetration suggests that the S-biotype is not a fully adapted tomato pathogen, but possibly a case of broken non-host resistance, and evidence suggests the occurrence of a non-host MT response against the C-biotype.

Several plants use secondary metabolites against insects and pathogens attack. Among such metabolites known glycoalkaloids are the most studied. Plants from Solanaceae Family are the most abundant on those alkaloids. Despite alfa-tomatine is a known glycoalkaloid, its specific action against Moniliophthora perniciosa, fungi responsible for witches broom disease in cocoa plantations was obtained in this work. Alfa-tomatine was infusion-extracted from Solanum lycopersicum leaves, and its action against the fungi growth was shown on the bioassays. In these tests, M. perniciosa growth was totally inhibited but other fungus tested, the inhibition was not totally, or did not present effect on growth, showing in this way, the specificity of alfa-tomatine to M. perniciosa. The advantages of work with a natural molecule for the fungi control is the stability of the molecule, its resistance to high temperature and pressure and for its use, the simple method of extraction used. The confirmation of the alfa-tomatine molecule was done by High Performance Liquid Chromatography and Mass Spectrometry in comparison to the Sigma standard.

Garlic cultivars are sexually sterile under standard growth conditions, with direct implications for commercial production costs as well as breeding programs. Garlic is propagated commercially via bulblets, which facilitates disease transmission and virus load accumulation over vegetative generations. Tissue culture produces virus-free clones that are more productive, while keeping the desired traits of the cultivar. Consequently, this technique allows studies of garlic genetics as well as guarantees genetic conservation of varieties. We aimed at analyzing the in vitro regeneration of eight marketable cultivars of garlic using root segments as explants. For each genotype, bulblet-derived explants were isolated and introduced into MS medium supplemented with 2,4-D and 2-iP. Calli were transferred to MS medium supplemented with 8.8 mM BAP and 0.1 mM NAA (regeneration medium A), or with 4.6 mM kinetin alone (regeneration medium B). The calli were then evaluated for regeneration frequency after sixty days of in vitro cultivation. The noble cultivar 'Jonas' presented the highest rates of plant regeneration among the cultivars tested. The medium A, which contained auxin and cytokinin, induced the highest regeneration rates of all cultivars. The process described herein is simple, reproducible and can potentially be used as a tool in molecular breeding strategies for other marketable cultivars and genotypes of garlic.

RNA interference (RNAi), a gene-silencing mechanism that involves providing double-stranded RNA molecules that match a specific target gene sequence, is now widely used in functional genetic studies. The potential application of RNAi-mediated control of agricultural insect pests has rapidly become evident. The production of transgenic plants expressing dsRNA molecules that target essential insect genes could provide a means of specific gene silencing in larvae that feed on these plants, resulting in larval phenotypes that range from loss of appetite to death. In this report, we show that the tomato leafminer (Tuta absoluta), a major threat to commercial tomato production, can be targeted by RNAi. We selected two target genes (Vacuolar ATPase-AandArginine kinase) based on the RNAi response reported for these genes in other pest species. In view of the lack of an artificial diet forT. absoluta, we used two approaches to deliver dsRNA into tomato leaflets. The first approach was based on the uptake of dsRNA by leaflets and the second was based on “in planta-induced transient gene silencing” (PITGS), a well-established method for silencing plant genes, used here for the first time to deliverin planta-transcribed dsRNA to target insect genes.Tuta absolutalarvae that fed on leaves containing dsRNA of the target genes showed an ∼60% reduction in target gene transcript accumulation, an increase in larval mortality and less leaf damage. We then generated transgenic ‘Micro-Tom’ tomato plants that expressed hairpin sequences for both genes and observed a reduction in foliar damage byT. absolutain these plants. Our results demonstrate the feasibility of RNAi as an alternative method for controlling this critical tomato pest.

RNA interference (RNAi), a gene-silencing mechanism that involves providing double-stranded RNA molecules that match a specific target gene sequence, is now widely used in functional genetic studies. The potential application of RNAi-mediated control of agricultural insect pests has rapidly become evident. The production of transgenic plants expressing dsRNA molecules that target essential insect genes could provide a means of specific gene silencing in larvae that feed on these plants, resulting in larval phenotypes that range from loss of appetite to death. In this report, we show that the tomato leafminer (Tuta absoluta), a major threat to commercial tomato production, can be targeted by RNAi. We selected two target genes (Vacuolar ATPase-A and Arginine kinase) based on the RNAi response reported for these genes in other pest species. In view of the lack of an artificial diet for T. absoluta, we used two approaches to deliver dsRNA into tomato leaflets. The first approach was based on the uptake of dsRNA by leaflets and the second was based on “in planta-induced transient gene silencing” (PITGS), a well-established method for silencing plant genes, used here for the first time to deliver in planta-transcribed dsRNA to target insect genes. Tuta absoluta larvae that fed on leaves containing dsRNA of the target genes showed an ∼60% reduction in target gene transcript accumulation, an increase in larval mortality and less leaf damage. We then generated transgenic ‘Micro-Tom’ tomato plants that expressed hairpin sequences for both genes and observed a reduction in foliar damage by T. absoluta in these plants. Our results demonstrate the feasibility of RNAi as an alternative method for controlling this critical tomato pest.

We investigated the potential of peat additions to improve plant growth and fertility and to reduce plant metal uptake in waste foundry sands (WFS) landfills. The WFS contains 78211 mg kg(-1) and 371 mg kg(-1) concentrations of Cr and Ni, respectively, and varied metal concentrations. The experiment investigated the growth of Brassica juncea plants on fertilized WFS mixed with peat at concentrations of 0, 2.5, 5, and 10% (w/w). The highest peat treatment allowed substantial plant growth and increased Ni mass in shoots, which was positively correlated to shoot biomass increments. On a concentration basis, peat additions did not increase shoot Ni values, thus suggesting that plants grown on peat-treated WFS may not increase risks to human and ecological receptors. Chromium was below detection levels in shoots for all peat treatments. Peat-treated substrates also promoted increased CEC values and higher water holding capacity, therefore improving the WFS agronomical properties. These results indicate that peat can be used as an amendment to assist in the phytoremediation of WFS landfill areas. However, there was evidence for increased mobilization of Cr and Ni in the substrate solution which can pose a threat to local groundwater.

The main objective of the present work was to study nutritive strategies for lessening the CH(4) formation associated to ruminant tropical diets. In vitro gas production technique was used for evaluating the effect of tannin-rich plants, essential oils, and biodiesel co-products on CH(4) formation in three individual studies and a small chamber system to measure CH(4) released by sheep for in vivo studies was developed. Microbial rumen population diversity from in vitro assays was studied using qPCR. In vitro studies with tanniniferous plants, herbal plant essential oils derived from thyme, fennel, ginger, black seed, and Eucalyptus oil (EuO) added to the basal diet and cakes of oleaginous plants (cotton, palm, castor plant, turnip, and lupine), which were included in the basal diet to replace soybean meal, presented significant differences regarding fermentation gas production and CH(4) formation. In vivo assays were performed according to the results of the in vitro assays. Mimosa caesalpineaefolia, when supplemented to a basal diet (Tifton-85 hay Cynodon sp, corn grain, soybean meal, cotton seed meal, and mineral mixture) fed to adult Santa Ines sheep reduced enteric CH(4) emission but the supplementation of the basal diet with EuO did not affect (P0.05) methane released. Regarding the microbial studies of rumen population diversity using qPCR with DNA samples collected from the in vitro trials, the results showed shifts in microbial communities of the tannin-rich plants in relation to control plant. This research demonstrated that tannin-rich M. caesepineapholia, essential oil from eucalyptus, and biodiesel co-products either in vitro or in vivo assays showed potential to mitigate CH(4) emission in ruminants. The microbial community study suggested that the reduction in CH(4) production may be attributed to a decrease in fermentable substrate rather than to a direct effect on methanogenesis.

The seeds of Theobroma cacao (cacao) are the source of cocoa, the raw material for the multi-billion dollar chocolate industry. Cacao’s two most important traits are its unique seed storage triglyceride (cocoa butter) and the flavor of its fermented beans (chocolate). The genome of T. cacao is being sequenced, and to expand the utility of the genome sequence to the improvement of cacao, we are evaluating Theobroma grandiflorum, the closest economically important species of Theobroma for its potential use in a comparative genomic study. T. grandiflorum differs from cacao in important agronomic traits such as flavor of the fermented beans, disease resistance to witches’ broom and abscission of mature fruits. By comparing genomic sequences and analyzing viable inter-specific hybrids, we hope to identify the key genes that regulate cacao’s most important traits. We have investigated the utility in T. grandiflorum of three types of markers (microsatellite markers, single-strand conformational polymorphism markers and single nucleotide polymorphism (SNP) markers) developed in cacao. Through sequencing of amplicons of 12 diverse individuals of both cacao and T. grandiflorum, we have identified new intra- and inter-specific SNPs. Two markers which had no overlap of alleles between the species were used to genotype putative inter-specific hybrid seedlings. Sequence conservation was significant and species-specific differences numerous enough to suggest that comparative genomics of T. grandiflorum and T. cacao will be useful in elucidating the genetic differences that lead to a variety of important agronomic trait differences.

Plant hormones are likely key regulators of arbuscular mycorrhizae (AM) development. However, their roles in AM are not well known. Here mutants in five hormone classes introgressed in a single tomato (Lycopersicon esculentum Mill. Syn Solanum lycopersicum L.) background (cv. Micro-Tom) were used to determine their effects on AM development and the expression of defense-related genes (chitinases and b-1,3-glucanases) in roots. Under low P conditions, mutant epinastic (epi) and Never ripe (Nr), ethylene overproducer and low sensitivity, respectively, had the intraradical colonization by Glomus clarum highly inhibited, as compared to the control Micro-Tom (MT). No significant alterations in fungal colonization were observed in mutants affecting other hormone classes. Under low P conditions, the steady state levels of transcripts encoding a class I basic chitinase (chi9) were higher in mycorrhizal epi and Nr mutant roots as compared to MT controls. In contrast the steady state levels of a class III acidic b-1,3-glucanase (TomPR-Q'a) transcripts in mycorrhizal epi mutant roots were significantly lower than in mycorrhizal MT roots. Root colonization in epi mutants was accompanied by several alterations in fungal morphology, as compared to root colonization in MT controls. The data suggest that ethylene may play an important role in controlling intraradical arbuscular mycorrhizal fungal growth. Os hormônios vegetais são possíveis reguladores chave do desenvolvimento de micorrizas arbusculares (MAS). Contudo, seus papéis em MA são pouco conhecidos. No presente estudo, foram utilizados mutantes em cinco classes hormonais introgredidos em uma única cultivar (cv. Micro-Tom) de tomateiro (Lycopersicon esculentum Mill. Syn Solanum lycopersicum L.) para determinar seus efeitos no desenvolvimento de MA e expressão de genes relacionados à defesa (quitinases e b-1,3-glucanases) em raízes. Sob condição de baixo P, os mutantes epinastic (epi) e Never ripe (Nr), os quais são super produtores e pouco sensíveis a etileno, respectivamente, tiveram a colonização intra-radicular por Glomus clarum inibida quando comparada com o controle Micro-Tom (MT). Não se observou alterações significativas na colonização fúngica nos mutantes afetando outras classes hormonais. Sob condição de baixo P, o nível de transcritos codificando uma quitinase básica de classe I (chi9) foi mais elevado em raízes micorrizadas dos mutantes epi e Nr, quando comparado com o controle MT. Em contraste, o nível de transcritos de uma b-1,3-glucanase ácida da classe III (TomPR-Q'a) em raízes micorrizadas do mutante epi foi significativamente menor que em raízes micorrizadas de MT. A colonização de raízes no mutante epi foi acompanhada por várias alterações na morfologia fúngica, quando comparada com o controle MT. Os resultados sugerem que o etileno pode desempenhar um importante papel controlando o crescimento fúngico intra-radicular nas MAS.

SUMMARY The basidiomycete Crinipellis perniciosa is the causal agent of witches' broom disease of Theobroma cacao (cocoa). Hypertrophic growth of infected buds ('brooms') is the most dramatic symptom, but the main economic losses derive from pod infection. To identify cocoa genes differentially expressed during the early stages of infection, two cDNA libraries were constructed using the suppression subtractive hybridization (SSH) approach. Subtraction hybridization was conducted between cDNAs from infected shoot-tips of the susceptible genotype 'ICS 39' and the resistant 'CAB 214', in both directions. A total of 187 unique sequences were obtained, with 83 from the library enriched for the susceptible 'ICS 39' sequences, and 104 for the resistant 'CAB 214'. By homology search and ontology analyses, the identified sequences were mainly putatively categorized as belonging to 'signal transduction', 'response to biotic and abiotic stress', 'metabolism', 'RNA and DNA metabolism', 'protein metabolism' and 'cellular maintenance' classes. Quantitative reverse transcription amplification (RT-qPCR) of 23 transcripts identified as differentially expressed between genotypes revealed distinct kinetics of gene up-regulation at the asymptomatic stage of the disease. Expression induction in the susceptible 'ICS 39' in response to C. perniciosa was delayed and limited, while in 'CAB 214' there was a quicker and more intense reaction, with two peaks of gene induction at 48 and 120 h after inoculation, corresponding to morphological and biochemical changes previously described during colonization. Similar differences in gene induction were validated for another resistant genotype ('CAB 208') in an independent experiment. Validation of these genes corroborated similar hypothetical mechanisms of resistance described in other pathosystems.

The most abundant form in which phosphorus occurs in seeds is phytate (myo-inositol hexakisphosphate), mostly known as an antinutrient for animals, given its ability to complex proteins and minerals, despite its antioxidant and anticarcinogenic properties. However, phytate synthesis is still poorly understood, both in terms of its regulation and metabolic route, and relatively few works have addressed the control mechanism of phytate accumulation during seed development. Aiming at understanding the control mechanism of phytate synthesis, we examined myo-inositol-3-phosphate synthase (MIPS) (EC 5.5.1.4) activity and gene expression during seed development of common bean. Phytate concentration was low at the initial stage of seed development, coinciding with a period of the most intense seed metabolism, but followed by a period of high enzymatic activity and gene expression of MIPS when a decrease in its specific activity and transcription was detected throughout seed development until 20 days after flowering; however, the specific activity of MIPS dropped more expressively than the gene expression, matching with higher phytate concentration. Hence, we show that there is evidence of one control point regulating phytate synthesis with MIPS enzyme.

Plastid-related sequences, derived from putative nuclear or plastome genes, were searched in a large collection of expressed sequence tags (ESTs) and genomic sequences from the Citrus Biotechnology initiative in Brazil. The identified putative Citrus chloroplast gene sequences were compared to those from Arabidopsis, Eucalyptus and Pinus. Differential expression profiling for plastid-directed nuclear-encoded proteins and photosynthesis-related gene expression variation between Citrus sinensis and Citrus reticulata, when inoculated or not with Xylella fastidiosa, were also analyzed. Presumed Citrus plastome regions were more similar to Eucalyptus. Some putative genes appeared to be preferentially expressed in vegetative tissues (leaves and bark) or in reproductive organs (flowers and fruits). Genes preferentially expressed in fruit and flower may be associated with hypothetical physiological functions. Expression pattern clustering analysis suggested that photosynthesis- and carbon fixation-related genes appeared to be up- or down-regulated in a resistant or susceptible Citrus species after Xylella inoculation in comparison to non-infected controls, generating novel information which may be helpful to develop novel genetic manipulation strategies to control Citrus variegated chlorosis (CVC).

Plant hormones play a crucial role in integrating endogenous and exogenous signals and in determining developmental responses to form the plant body throughout its life cycle. In citrus species, several economically important processes are controlled by phytohormones, including seed germination, secondary growth, fruit abscission and ripening. Integrative genomics is a powerful tool for linking newly researched organisms, such as tropical woody species, to functional studies already carried out on established model organisms. Based on gene orthology analyses and expression patterns, we searched the Citrus Genome Sequencing Consortium (CitEST) database for Expressed Sequence Tags (EST) consensus sequences sharing similarity to known components of hormone metabolism and signaling pathways in model species. More than 600 homologs of functionally characterized hormone metabolism and signal transduction members from model species were identified in citrus, allowing us to propose a framework for phytohormone signaling mechanisms in citrus. A number of components from hormone-related metabolic pathways were absent in citrus, suggesting the presence of distinct metabolic pathways. Our results demonstrated the power of comparative genomics between model systems and economically important crop species to elucidate several aspects of plant physiology and metabolism.

Plants not only evolve but also reduce oxygen in photosynthesis. An inevitable consequence of this normal process is the production of reactive oxygen species (ROS). Plants are adequately protected by the presence of multiple antioxidative enzymes in the cytosol and also in the different cell organelles such as chloroplasts, mitochondria, and peroxisomes. Traditionally, ROS were considered to be only a toxic byproduct of aerobic metabolism. However, recently it has become apparent that plants actively produce these molecules which may control many different physiological processes such as abiotic and biotic stress response, pathogen defense and systemic signaling. The search results using the Citrus Genome Program in Brazil (CitEST) for oxidative stress and the antioxidant enzyme system in Citrus Sinensis variety ‘Pera IAC’ indicated that the multiple ROS-scavenging enzymes were expressed throughout all citrus tissues. The analyses demonstrated the ubiquitous expression of metallothioneins, probably indicating a constitutive expression pattern. Oxalate oxidase has been identified as the most abundant expressed gene in developing fruits, which suggests a specific function in the ripening of citrus fruit. Moreover, infected leaves with Xylella fastidiosa and Leprosis citri showed a massive change in their ROS gene expression profile which may indicate that the suppression of ROS detoxifying mechanisms may be involved in the induction of the diseases.

Background Providing double-stranded RNA (dsRNA) to insects has been proven to silence target genes, and this approach has emerged as a potential method to control agricultural pests by engineering plants to express insect dsRNAs. A critical step of this technology is the screening of effective target genes essential for insect development and/or survival. The tomato leafminer (Tuta absoluta Meyrick) is a major Solanum lycopersicum (tomato) pest that causes significant yield losses and has recently invaded Europe, from where it is spreading at an alarming rate. To explore RNA interference (RNAi) against T. absoluta, sequence information on potential target genes is necessary, but only a few sequences are available in public databases. Results We sequenced six libraries from RNA samples from eggs, adults, and larvae at four stages, obtaining an overall total of around 245 million reads. The assembled T. absoluta transcriptome contained 93,477 contigs with an average size of 1,574 bp, 59.8 % of which presented positive Blast hits, with 19,995 (21.4 %) annotated by gene ontology. From the transcriptome, most of the core genes of the RNAi mechanism of Lepidoptera were identified indicating the potential suitability of T. absoluta for gene silencing. No contigs displayed significant similarity with a RNA-dependent RNA Polymerase. Genes from the juvenile hormone and ecdysteroid biosynthetic pathways were identified, representing potential target genes for systemic silencing. Comparisons of transcript profiles among stages revealed 1,577 genes differentially expressed at earlier larval stages, from which potential gene targets were identified. Five of these genes were evaluated using in vitro transcribed dsRNA absorbed by tomato leaflets, which were fed to 1st instar T. absoluta larvae, resulting in significant reduction of larval body weight while exhibiting significant knockdown for three of the genes. Conclusions The transcriptome we generated represents a valuable genomic resource for screening potential gene targets that affect the development or survival of T. absoluta larvae. Five novel genes that showed greater expression at the 1st larval stage were demonstrated to be effective potential RNAi targets by reducing larval weight and can be considered good candidates for use in RNAi-mediated crop protection. Electronic supplementary material The online version of this article (doi:10.1186/s12864-015-1841-5) contains supplementary material, which is available to authorized users.

A sample of 94 accessions of Theobroma cacao L. (cacao), representing four populations from the Brazilian Amazon (Acre, Rondonia, lower Amazon and upper Amazon) were analyzed using microsatellite markers to assess the genetic diversity and the natural population structure. From the 19 microsatellite loci tested, 11 amplified scorable products, revealing a total of 49 alleles, including two monomorphic loci. The Brazilian upper Amazon population contained the largest genetic diversity, with the most polymorphic loci, the highest observed heterozygosity; and the majority of rare alleles, thereby this region might be con- sidered part of the center of diversity of the species. The observed heterozygosity for all the Brazilian populations (Ho=0.347) was comparable with values reported for other similar upper Amazon Forastero cacao populations, with the Acre and Rondonia displaying the lowest values. The lower Amazon popu- lation, traditionally defined as highly homozygous, presented an unexpectedly high observed heterozygosity (Ho=0.372), disclosing rare and distinct alleles, with large identity with the upper Amazon population. It was hypothesized that part of the lower Amazon population might derive from successive natural or intentional introduction of planting material from other provenances, mainly upper Amazon. Most of the loci exhibited a lower observed heterozygosity than expected, suggesting that self-pollination might be more common than usually assumed in cacao, but excess of homozygotes might also derive from sub-grouping (Wahlund effect) or from sampling related individuals. Most of the gene diversity was found to occur within groups, with small differentiation between the four Brazilian Amazon populations, typical of species with high gene flow. Abbreviations: AFLP - amplified fragment length polymorphism; bp - base pairs; CAB - Cacao Amazon Brazil; cDNA - complementary DNA; CIRAD - ''Centre de Cooperation Internationale en Recherche Agronomique pour le Developpement''; CTAB - hexadecytrimethyl ammonium bromide; dNTPs - de- oxyribonucleotides; EDTA - ethylenediaminetetraacetic acid; ERJOH - ''Estacao de Recursos Geneticos do Cacau JoseHaroldo''; RAPD - random amplified polymorphic DNA; rDNA - ribosomal DNA; RFLP - restriction fragment length polymorphism

Banana (Musa spp.) is one of the most consumed fruits worldwide. Production is based mainly on triploid cultivars, and most genetic improvement programs aim to generate tetraploid hybrids obtained from the crossing of established triploid cultivars with a diploid parent genotype, improved or wild, exhibiting the trait of interest, normally resistance to biotic factors. Microsatellites were used to investigate the genetic variability and relationships between 58 Musa genotypes, including 49 diploids and nine triploid cultivars maintained at the Musa germplasm collection of the Brazilian dessert banana breeding program. Thirty-three primer pairs developed for banana were tested, and nine amplified reproducible and discrete fragments, producing a total of 115 alleles. The average number of alleles amplified per primer was 12.8, ranging from 10 to 15. The diploid genotypes presented the largest genetic variability, demonstrated by the large number of alleles detected, and the low similarity between the clones. The phenetic analysis clustered the triploid cultivars in a separated group, with the exception of the ‘Nanica’ and ‘Gros Michel’ cultivars, which showed high similarity with the diploid cultivar ‘Mambee Thu’. It was not possible to separate the wild diploid genotypes from the cultivated ones, indicating a common origin of these genotypes. A high proportion of duplicated alleles and/or loci was observed for diploid and triploid genotypes. The information gathered about the similarity between diploid and triploid accessions will help to define potential crosses to maximize the recovery of the typical fruit qualities required in Brazil (AAB, Pome and Silk dessert banana).

The in vitro regeneration ability of tomato (Lycopersicon esculentumMill.) remains the main limiting factor for efficient genetic transformation. However, competence for in vitro regeneration can be transferred from wild Lycopersicon species to the cultivated tomato. The high regeneration capacity presented by the MsK genotype, derived from L. peruvianum, was attributed to the dominant loci Rg-1 and Rg-2 .I n the present work, we have transferred the high organogenetic competence from the MsK genotype to the cultivar Micro-Tom, which presents miniature size (8-cm tall) and a rapid life cycle (75 days from seed to fruit ripening). We obtained stable and non-segregating F6 ‘Micro-MsK’ plants, which were expressively superior to Micro-Tom in regeneration capacity, when hypocotyls but not cotyledons were used as explants. In addition to the regeneration ability, the obtained genotype presented a delayed leaf senescence and a reduced apical dominance. The development of ‘Micro-MsK’ genotype will enable the application of insertional mutagenesis in functional genomics of tomato. © 2004 Elsevier Ireland Ltd. All rights reserved.

Microsatellite markers were used to characterize 35 banana (Musa spp.)genotypes cultivated in Brazil, including triploid cultivars and tetraploid hybrids. A total of 33 Musa-specific primers were tested, and 11 produced clear ,reproducible and discrete bands. The average number of alleles amplified per primer was 6.1, ranging from 4 to 8, with a total of 67 alleles identified. Phenetic analysis based on Jaccard similarity index derived from presence or absence of the alleles agreed with the morphological classification. Bootstrap analysis divided the genotypes into four clusters, according to genomic group and subgroup classification. The first cluster contained the majority of cultivars which have ‘A’ genome alone; while the second contained all triploid cultivars of the subgroup Prata (Pome) and their tetraploid hybrids. The third cluster contained cultivar ‘Maca’ together with other genotypes considered for breeding purposes as similar to the Silk subgroup. These last two clusters formed a larger group including the majority of genotypes that resulted from hybridization between M. acuminata and M. balbisiana. The microsatellite loci were highly informative, with some pair of primers generating an unique finger printing for each genomic group and discriminating a genotype of doubtful classification, although somatic mutants from a subgroup were seldom distinguished from their original clone. Tetraploid hybrids exhibited distortion in the proportion of alleles donated by their triploid female parent. For a few primers, some genotypes exhibited a higher number of alleles than expected from their ploidy level, suggesting the occurrence of duplicated alleles or duplicated chromosomal regions.

Raw materials for cocoa butter substitutes, replacements or equivalents depend mostly on the unsteady supply from wild stands of plants, while there is no current supply of Neotropical origin. Seed fats from Theobroma species (T cacao, T bicolor, T grandiflorum, T obovatum, T subincanum, T speciosum, T sylvestre and T microcarpum, plus the closely related species Herrania mariae) were analysed for fatty acid and triacylglycerol composition by gas and liquid chromatography respectively, for iodine value, for melting point by open capillary tube and for solid fat content (SFC) by nuclear magnetic resonance. All Theobroma species had significantly lower palmitate levels than T cacao, except for T sylvestre and T speciosum, T microcarpum presented highly unsaturated fat (C18:2), while H mariae had high levels of arachidate. Fats from T sylvestre and T speciosum had a similar iodine value to T cacao and a higher melting point. No fat from the other species presented a similar melting profile to cocoa butter. T sylvestre and T bicolor were the most similar to T cacao but had a higher SFC at human body temperature. T sylvestre and T speciosum seed fats had more POP than cocoa butter. Fats from seeds of T speciosum, T sylvestre and T bicolor could be recommended as cocoa butter substitutes, while fats from species of the section Glossopetalum could be employed in products requiring fats with a lower melting point. © 2002 Society of Chemical Industry

Brown rust (causal agent Puccinia melanocephala) is an important sugarcane disease that is responsible for large losses in yield worldwide. Despite its importance, little is known regarding the genetic diversity of this pathogen in the main Brazilian sugarcane cultivation areas. In this study, we characterized the genetic diversity of 34 P. melanocephala isolates from 4 Brazilian states using loci identified from an enriched simple sequence repeat (SSR) library. The aggressiveness of 3 isolates from major sugarcane cultivation areas was evaluated by inoculating an intermediately resistant and a susceptible cultivar. From the enriched library, 16 SSR-specific primers were developed, which produced scorable alleles. Of these, 4 loci were polymorphic and 12 were monomorphic for all isolates evaluated. The molecular characterization of the 34 isolates of P. melanocephala conducted using 16 SSR loci revealed the existence of low genetic variability among the isolates. The average estimated genetic distance was 0.12. Phenetic analysis based on Nei's genetic distance clustered the isolates into 2 major groups. Groups I and II included 18 and 14 isolates, respectively, and both groups contained isolates from all 4 geographic regions studied. Two isolates did not cluster with these groups. It was not possible to obtain clusters according to location or state of origin. Analysis of disease severity data revealed that the isolates did not show significant differences in aggressiveness between regions.

Background Sugarcane is a major crop worldwide as raw material for sugar and ethanol production. Drought is one of the most limiting factor that affects sugarcane productivity. In order to understand the mechanisms of drought response, field and greenhouse assays were conducted with two droughtcontrasting sugarcane genotypes (IACSP94-2094/tolerant and IACSP97-7065/sensitive), and several genes up/downregulated under drought stress identified by microarrays and RNAseq analyses. Ten differential expressed genes in both assays were evaluated by qPCR [1], and three of them showed the transcriptional profile related drought tolerance: aLipoxygenase (ScLOX), acting in the biosynthesis of the jasmonic acid precursor, and recent studies showed their role in defense against drought stress [3]; bDehydrin, correlated to drought stress and associated to maintenance of turgor cells, [2]; cDirigent-jacalin, associated to resistance disease and abiotic stress tolerance [4] and also related to jasmonic acid, an important hormone on plant defense. These genes were chosen as target for functional analyses in rice and sugarcane transgenic plants.

Cocoa butter is the major commercial product from seeds of Theobroma cacao (cacao). However, most cacao breeding programs have only considered as selection criteria total seed yield per plant and disease resistance. Progress in cocoa butter yield might be possible if variability for fat content exists and its associations with the other production components are established. The average fat content in dry unfermented seeds for 490 accessions of the Centro de Pesquisa do Cacau germplasm collection in Ilheus, Bahia, Brazil was 53.2%, ranging from 45.4% in CC 57 to 60.3% in NA 312. Fat content averaged over series indicated that genotypes originally collected in the Upper Amazonian region had higher fat levels, while the Trinitario-Criollo and Bahian genotypes tended to have lower fat content. When considering total fat yield plant-1 an opposite trend arose with the series with low fat content having the highest total fat yield. The correlation between fat content and fat yield plant-1 was not significant, while there was a significant negative correlation between dry seed yield plant-1 and fat content. Despite the significant negative association between fat content and dry seed yield plant-1, the correlation coefficient was rather low, and genotypes could be identified with both high fat content and dry seed yield. A significant pollen effect on fat content was observed based on a complete diallel crossing scheme between three genotypes with high and three with low levels. The average fat content values in progenies were similar to the average of the parents, and the segregation range transgressed the distribution of the parents.

Background Proper conservation of plant samples, especially during remote field collection, is essential to assure quality of extracted DNA. Tropical plant species contain considerable amounts of secondary compounds, such as polysaccharides, phenols, and latex, which affect DNA quality during extraction. The suitability of ethanol (96% v/v) as a preservative solution prior to DNA extraction was evaluated using leaves of Jatropha curcas and other tropical species. Results Total DNA extracted from leaf samples stored in liquid nitrogen or ethanol from J. curcas and other tropical species (Theobroma cacao, Coffea arabica, Ricinus communis, Saccharum spp., and Solanum lycopersicon) was similar in quality, with high-molecular-weight DNA visualized by gel electrophoresis. DNA quality was confirmed by digestion with EcoRI or HindIII and by amplification of the ribosomal gene internal transcribed spacer region. Leaf tissue of J. curcas was analyzed by light and transmission electron microscopy before and after exposure to ethanol. Our results indicate that leaf samples can be successfully preserved in ethanol for long periods (30 days) as a viable method for fixation and conservation of DNA from leaves. The success of this technique is likely due to reduction or inactivation of secondary metabolites that could contaminate or degrade genomic DNA. Conclusions Tissue conservation in 96% ethanol represents an attractive low-cost alternative to commonly used methods for preservation of samples for DNA extraction. This technique yields DNA of equivalent quality to that obtained from fresh or frozen tissue.

Made available in DSpace on 2013-08-12T18:46:36Z (GMT). No. of bitstreams: 0 Previous issue date: 2012-09-01 Made available in DSpace on 2013-09-30T18:37:46Z (GMT). No. of bitstreams: 0 Previous issue date: 2012-09-01 Submitted by Vitor Silverio Rodrigues (vitorsrodrigues@reitoria.unesp.br) on 2014-05-20T13:50:24Z No. of bitstreams: 0 Made available in DSpace on 2014-05-20T13:50:24Z (GMT). No. of bitstreams: 0 Previous issue date: 2012-09-01 Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) Despite the wide use of plant regeneration for biotechnological purposes, the signals that allow cells to become competent to assume different fates remain largely unknown. Here, it is demonstrated that the Regeneration1 (Rg1) allele, a natural genetic variation from the tomato wild relative Solanum peruvianum, increases the capacity to form both roots and shoots in vitro; and that the gibberellin constitutive mutant procera (pro) presented the opposite phenotype, reducing organogenesis on either root-inducing medium (RIM) or shoot-inducing medium (SIM). Mutants showing alterations in the formation of specific organs in vitro were the auxin low-sensitivity diageotropica (dgt), the lateral suppresser (ls), and the KNOX-overexpressing Mouse ears (Me). dgt failed to form roots on RIM, Me increased shoot formation on SIM, and the high capacity for in vitro shoot formation of ls contrasted with its recalcitrance to form axillary meristems. Interestingly, Rg1 rescued the in vitro organ formation capacity in proRg1 and dgtRg1 double mutants and the ex vitro low lateral shoot formation in pro and ls. Such epistatic interactions were also confirmed in gene expression and histological analyses conducted in the single and double mutants. Although Me phenocopied the high shoot formation of Rg1 on SIM, it failed to increase rooting on RIM and to rescue the non-branching phenotype of ls. Taken together, these results suggest REGENERATION1 and the DELLA mutant PROCERA as controlling a common competence to assume distinct cell fates, rather than the specific induction of adventitious roots or shoots, which is controlled by DIAGEOTROPICA and MOUSE EARS, respectively. Univ São Paulo, ESALQ, Dept Biol Sci LCB, Lab Hormonal Control Plant Dev, BR-13418900 Piracicaba, SP, Brazil Univ São Paulo, Lab Plant Breeding, CENA, BR-13400970 Piracicaba, SP, Brazil Univ Estadual Paulista, Inst Biol, Dept Genet, Lab Mol Genet Plant Dev, BR-18618970 Botucatu, SP, Brazil Univ Estadual Paulista, Inst Biol, Dept Genet, Lab Mol Genet Plant Dev, BR-18618970 Botucatu, SP, Brazil FAPESP: 07/07175-0

Understanding the mating patterns of populations of tree species is a key component of ex situ genetic conservation. In this study, we analysed the genetic diversity, spatial genetic structure (SGS) and mating system at the hierarchical levels of fruits and individuals as well as pollen dispersal patterns in a continuous population of Theobroma cacao in Pará State, Brazil. A total of 156 individuals in a 0.56 ha plot were mapped and genotyped for nine microsatellite loci. For the mating system analyses, 50 seeds were collected from nine seed trees by sampling five fruits per tree (10 seeds per fruit). Among the 156 individuals, 127 had unique multilocus genotypes, and the remaining were clones. The population was spatially aggregated; it demonstrated a significant SGS up to 15 m that could be attributed primarily to the presence of clones. However, the short seed dispersal distance also contributed to this pattern. Population matings occurred mainly via outcrossing, but selfing was observed in some seed trees, which indicated the presence of individual variation for self-incompatibility. The matings were also correlated, especially within (Ρ(p(m))=0.607) rather than among the fruits (Ρ(p(m))=0.099), which suggested that a small number of pollen donors fertilised each fruit. The paternity analysis suggested a high proportion of pollen migration (61.3%), although within the plot, most of the pollen dispersal encompassed short distances (28 m). The determination of these novel parameters provides the fundamental information required to establish long-term ex situ conservation strategies for this important tropical species.

Cacao (Theobroma cacao L.) is one of the main tropical crops worldwide. It is cultivated primarily to provide cacao liquor, butter, and powder for the chocolate industry, essentially due to its flavour properties. Unfortunately, destructive and newly encountered diseases have frequently been the major factors that limit cacao production in nearly all producing countries. The primary challenge is to develop improved cacao tree cultivars with durable and sustainable resistance to these diseases that at the same time have high bean quality for chocolate production. To achieve this goal, the use of functional genomics can be a key step to speed the development of such cultivars. During the last 10 years various functional genomics and some proteomic projects have been initiated, including expressed sequence tag and BAC libraries construction, cacao genome sequencing, expression studies of cacao tissues challenged with the main pathogens (Moniliophthora perniciosa, Phytophthora spp.) or subjected to other stress conditions (e.g. drought), and expression studies related to cacao quality flavour. Various others tools such as cacao in vitro culture, plant transformation or bioinformatics were developed to attend the necessity of cacao studies. Functional genomics research associated with genetics (molecular markers, maps and QTLs) will help to select new cacao varieties with agronomic characteristics demanded by farmers.

Contrary to the theory that geographic isolation is the main trigger for speciation, recent studies emphasize the continuous adaptation to different habitats as the driving force initiating diversification. In this way, adaptive divergence in response to contrasting selective pressures of populations of the same species in geographically or ecologically continuous environments may occur if long-lasting barriers are induced by biotic or abiotic events. Plants of the Amazon floodplains withstand annual periods of flooding which can last seven months. To verify if the regularity of the “flood pulse” of the Amazon River can induce speciation, we investigated populations of Himatanthus sucuuba (Apocynaceae) colonizing whitewater floodplains (varzea) and non-flooded uplands (terra-firme) in the region. In independent experiments, we simulated flooding conditions, to evaluate the germination and growth of seedlings from both environments. The two populations showed significant differences for most parameters evaluated. Thus, flooding is apparently a feature strong enough to promote phenotypic differentiation among varzea and terra firme populations. Indeed, molecular analysis showed genetic difference between populations, revealing that different ecological pressures may promote adaptive changes in Amazonian plants to insure establishment in different environments.

Given the susceptibility of tomato plants to pests, the aim of the present study was to understand how hormones are involved in the formation of tomato natural defences against insect herbivory. Tomato hormone mutants, previously introgressed into the same genetic background of reference, were screened for alterations in trichome densities and allelochemical content. Ethylene, gibberellin, and auxin mutants indirectly showed alteration in trichome density, through effects on epidermal cell area. However, brassinosteroids (BRs) and jasmonates (JAs) directly affected trichome density and allelochemical content, and in an opposite fashion. The BR-deficient mutant dpy showed enhanced pubescence, zingiberene biosynthesis, and proteinase inhibitor expression; the opposite was observed for the JA-insensitive jai1-1 mutant. The dpy x jai1-1 double mutant showed that jai1-1 is epistatic to dpy, indicating that BR acts upstream of the JA signalling pathway. Herbivory tests with the poliphagous insect Spodoptera frugiperda and the tomato pest Tuta absoluta clearly confirmed the importance of the JA-BR interaction in defence against herbivory. The study underscores the importance of hormonal interactions on relevant agricultural traits and raises a novel biological mechanism in tomato that may differ from the BR and JA interaction already suggested for Arabidopsis.

Herbicide selectivity is an agricultural technology largely exploited in chemical strategies of weed control. The joint action of several protection mechanisms avoids phytotoxicity from herbicide treatment, maintaining the level of agronomically accepted damage to a minimum, or even totally avoiding them. The major mechanism of herbicide selectivity derives from the differential metabolism between weed and crop plant species, with weeds presenting a limited ability to perform it under agronomically recommended conditions. In this case, phytotoxicity can be interpreted as an overcoming of the maximum protection capacity offered by the mechanisms of selectivity, or when considering metabolism as the main factor, the overcoming of the inherent plant ability to detoxify a particular molecule. Considering that herbicide metabolism requires energy disposal, symptoms of phytotoxicity characterize an additional waste of energy that should not be accepted as a natural physiologic response; therefore it might result in yield losses. To avoid or minimize crop losses or damages, it is required that herbicide application recommendations are based on results from rigorously conducted selectivity experiments, as well as that there is an increase in the awareness of growers about the best use of each product. A seletividade dos herbicidas é uma tecnologia agrícola que tem sido vastamente explorada nas estratégias de controle químico de plantas daninhas. É resultado da ação conjunta de diversos mecanismos que protegem a cultura da fitotoxicidade dos tratamentos herbicidas, mantendo-a com níveis de injúrias aceitáveis agronomicamente, ou mesmo na ausência destas. O principal mecanismo de seletividade dos herbicidas é o metabolismo diferencial desses produtos entre plantas daninhas e cultivadas, em que, nas situações de recomendação agronômica, as plantas daninhas são menos hábeis em realizá-lo. Neste caso, a fitotoxicidade pode ser entendida como a suplantação da capacidade máxima de proteção oferecida pelos mecanismos de seletividade ou, considerando o metabolismo como o principal mecanismo, como a superação da capacidade intrínseca da espécie em detoxificar determinada molécula. Considerando-se que o metabolismo de herbicidas envolve gasto de energia, os sintomas de fitotoxicidade caracterizam um segundo gasto energético que não deve ser aceito como uma resposta fisiológica natural, portanto pode resultar em perdas de rendimento das culturas. Para evitar ou minimizar as perdas ou injúrias às culturas, é necessário que as recomendações de herbicidas sejam baseadas em trabalhos de seletividade conduzidos com adequado rigor experimental; bem como é importante a conscientização dos agricultores quanto a melhor forma de utilizar cada produto.

We analyzed the impact of ethylene and auxin disturbances on callus, shoots and Agrobacterium rhizogenes-induced hairy root formation in tomato (Solanum lycopersicum L.). The auxin low-sensitivity dgt mutation showed little hairy root initiation, whereas the ethylene low-sensitivity Nr mutation did not differ from the control Micro-Tom cultivar. Micro-Tom and dgt hairy roots containing auxin sensitivity/biosynthesis rol and aux genes formed prominent callus onto media supplemented with cytokinin. Under the same conditions, Nr hairy roots did not form callus. Double mutants combining Rg1, a mutation conferring elevated shoot formation capacity, with either dgt or Nr produced explants that formed shoots with little callus proliferation. The presence of rol + aux genes in Rg1 hairy roots prevented shoot formation. Taken together, the results suggest that although ethylene does not affect hairy root induction, as auxin does, it may be necessary for auxin-induced callus formation in tomato. Moreover, excess auxin prevents shoot formation in Rg1.

The amount of cotyledon polyphenolic cells varies extensively within the Theobroma species. The polyphenolic compounds of these cells play a protective role and furthermore have an important function in the development of chocolate flavour. The morphology of the polyphenolic cells of the mesophyll is described and the development of these idioblasts in Theobroma cacao L., T. subincanum Mart., T. obovatum Klotzsch ex Bernoulli, T. grandiflorum (Willd. ex Spreng.) K. Schum., T. microcarpum Mart., T. bicolor Bonpl. and T. speciosum Willd. ex Spreng analysed. The total polyphenolic content in the seeds as determined by spectrophotometry showed a variation of about forty times. The alive, transparent polyphenolic cells are scattered throughout the cotiledonary mesophyll. However the polyphenolic cells of T. cacao and T. grandiflorum are also aligned perpendicularly with respect to the mesophyll borders and, in addition, both species display polyphenolic cells with a natural translucent purple colour. All the species analysed contained polyphenolic cells scattered throughout the parenchymal cells and also in a lengthwise association with vascular bundles. In T. bicolor and T. speciosum, the species with the lowest polyphenolic contents, these cells were mostly located around the vascular bundles. Using Scanning Electron Microscopy, the polyphenolic cells demonstrated a complex cytoarchitecture, and after fixing with glutaraldhyde, the polyphenolic secretion was shown to remain as a single unit or was organized into round droplets. Transmission Electron Microscopy displayed immature plastids from young mesophyll cells containing eletron-dense deposits similar to phenolic substances, suggesting that Theobroma plastids are involved in the synthesis of phenolics. A quantidade de células polifenólicas dos cotilédones das espécies de Theobroma variam extensivamente. Os compostos polifenólicos destas células além da função de proteção participam no desenvolvimento do aroma do chocolate. A morfologia das células polifenólicas do mesofilo é descrita e também é analisado o desenvolvimento dos idioblastos em T. cacao L., T. subincanum Mart., T. obovatum Klotzsch ex Bernoulli, T. grandiflorum (Willd. ex Spreng.) K. Schum., T. microcarpum Mart., T. bicolor Bonpl. e T. speciosum Willd. ex Spreng. A quantidade de polifenóis totais determinada por espectrofotometria apresentou uma grande variação, cerca de 40 vezes entre T. cacao (variedade forastero). As células polifenólicas apresentam-se transparentes e dispersas pelo mesofilo cotiledonar. Em T. cacao e T. grandiflorum grande número de células estão alinhadas perpendicularmente à borda do mesofilo e apresentam coloração púrpura translúcida. Entretanto, em todas as espécies as células polifenólicas também associam-se ao feixe vascular. Nas espécies T. bicolor e T. speciosum, com baixo conteúdo polifenólico, estão localizadas apenas na região do feixe vascular. À microscopia eletrônica de varredura, as células polifenólicas apresentam uma complexa citoarquitetura e após a fixação em glutaraldeído, a secreção polifenólica exibe um só conteúdo ou fragmenta-se em várias gotas esféricas. À microscopia eletrônica de transmissão aparecem nas células jovens do mesofilo, plastídeos imaturos eletrondensos, semelhantes a substâncias polifenólicas sugerindo que em Theobroma os plastídeos estão envolvidos na síntese de polifenóis.

Fermentation of Theobroma cacao (cacao) seeds is an absolute requirement for the full development of chocolate flavor precursors. An adequate aeration of the fermenting cacao seed mass is a fundamental prerequisite for a satisfactory fermentation. Here, we evaluated whether a controlled inoculation of cacao seed fermentation using a Kluyveromyces marxianus hybrid yeast strain, with an increased pectinolytic activity, would improve an earlier liquid drainage ('sweatings') from the fermentation mass, developing a superior final product quality. Inoculation with K. marxianus increased by one third the volume of drained liquid and affected the microorganism population structure during fermentation, which was detectable up to the end of the process. Introduction of the hybrid yeast affected the profile of total seed protein degradation evaluated by polyacrylamide gel electrophoresis, with improved seed protein degradation, and reduction of titrable acidity. Sensorial evaluation of the chocolate obtained from beans fermented with the K. marxianus inoculation was more accepted by analysts in comparison with the one from cocoa obtained through natural fermentation. The increase in mass aeration during the first 24 h seemed to be fundamental for the improvement of fermentation quality, demonstrating the potential application of this improved hybrid yeast strain with superior exogenous pectinolytic activity.

Localization of the cotyledon reserves of Theobroma grandiflorum (Willd. ex Spreng.) K. Schum., T. subincanum Mart., T. bicolor Bonpl. and their analogies with T. cacao L.). Cotyledon mesophyll cell morphology and lipid and protein synthesis of T. grandiflorum, T. subincanum and T. bicolor were analyzed and compared with T. cacao. These species possess foliar cotyledons folded around the hypocotyl radicle axis, typical of Sterculiaceae. Fruit size, morphology and weight are very distinct amongst the four species and so are the respective seeds. The main axis of the T. grandiflorum and T. bicolor seeds measured about 30 mm, while T. subincanum and T. cacao seeds measured 17 mm and 26 mm respectively. The seed weights of T. grandiflorum, T. bicolor, T. subincanum and T. cacao were 11.6 g, 9.4 g, 2.1 g and 3.0 g, respectively. The cotyledon mesophylls of the four species contained mainly polysaccharides and lipid-protein reserve cells. Theobroma cacao, T. grandiflorum and T. subincanum were composed of greater than 50% lipids. For the four species, lipid globules gradually accumulated adjacent to the cell wall, and these globules measured from 1 to 3 µm. TEM showed low-density proteins inside the central vacuole of the young mesophyll cells of T. cacao. The protein reserves of the mature cells were densely scattered amongst the lipid bodies, and a few starch granules occurred together with the cotyledon mesophyll of the four species. Polyphenolic cells were found throughout the mesophyll cells or aligned with the respective vascular bundles. Immature cells demonstrated the capacity to synthesize all these reserves, but gradually the pre-determined cells produced mainly lipid-protein reserves. Besides the unique characteristics of the T. cacao products, the lipid-protein synthesis capacities of T. grandiflorum, T. subincanum and T. bicolor suggest various possibilities for new industrialized food, pharmaceutical and cosmetic products.

Theobroma cacao, the chocolate tree, is an important tropical tree-crop that provides sustainable economic and environmental benefits to some of the poorest and most ecologically sensitive areas of the world. Recent progress in the development of genomics tools for cacao is reviewed. These include a reference molecular genetic map, simple sequence repeats and other molecular markers, two germplasm databases with microsatellite DNA fingerprints and other molecular data, many quantitative trait loci mapping projects which have identified disease resistance and yield component loci, several expressed sequence tag resources, a cacao microarray, bacterial artificial chromosome libraries, and a genetic transformation system. The evolutionary relatedness of cacao with other important crops and model plant systems positions cacao genomics to play a significant role in translational plant genomics. The future prospects for the contribution of cacao genomics to improvement of this crop for sustainable cacao production and as a tool for poverty alleviation and environmental stabilization are discussed.

RNA silencing mechanisms are conserved throughout eukaryotic evolution, possibly due to their importance in viral resistance and other aspects of cell biology. Here, we explored the Citrus EST (CitEST) database in search of sequences related to the most important known genes involved in RNA silencing. Transcripts strongly matching Argonaute (AGO), Dicer-like (DCL), Hua enhancer (HEN), and RNA-dependent RNA Polymerase (RdRP) were found in many of the citrus libraries. The reads were clustered and quantified. This shows that post-transcriptional gene silencing apparatus is active in citrus. It seems plausible that a better understanding of the players of RNA silencing in Citrus spp. and related genera may help create new tools to defeat the viral diseases that affect the citrus industry. Functional analyses of these citrus genes would enable the pursuit of this hypothesis. Made available in DSpace on 2022-08-25T13:19:50Z (GMT). No. of bitstreams: 1 Genetic-machinery-for-RNA-silencing-and-defense-against-viruses-in-Citrus..pdf: 72954 bytes, checksum: 1fa92887c8b6a1161571bacee6c34c59 (MD5) Previous issue date: 2007