Differential expression of the lethal gene Luteus-Pa in cacao of the Parinari series.

The recessive lethal character Luteus-Pa is found in cacao (Theobroma cacao) genotypes of the Parinari series (Pa) and is characterized by expression of leaf chlorosis and seedling death. Several genotypes of the Pa series are bearers of the gene responsible for the expression of the Luteus-Pa character, which can be used as a tool for determining relationships between genotypes of this group. To evaluate this phenomenon, we analyzed the differential expression of genes between mutant seedlings and wild-type hybrid Pa 30 x 169 seedlings, with the aim of elucidating the possible lethal mechanisms of the homozygous recessive character Luteus-Pa. Plant material was harvested from leaves of wild and mutant seedlings at different periods to construct a subtractive library and perform quantitative analysis using real-time PCR. The 649 sequences obtained from the subtractive library had an average length of 500 bp, forming 409 contigs. The probable proteins encoded were grouped into 10 functional categories. Data from ESTs identified genes associated with Rubisco, peroxidases, and other proteins and enzymes related to carbon assimilation, respiration, and photosystem 2. Mutant seedlings were characterized by synthesizing defective PsbO and PsbA proteins, which were overexpressed from 15 to 20 days after seedling emergence.

Efficient method of protein extraction from Theobroma cacao L. roots for two-dimensional gel electrophoresis and mass spectrometry analyses.

Theobroma cacao is a woody and recalcitrant plant with a very high level of interfering compounds. Standard protocols for protein extraction were proposed for various types of samples, but the presence of interfering compounds in many samples prevented the isolation of proteins suitable for two-dimensional gel electrophoresis (2-DE). An efficient method to extract root proteins for 2-DE was established to overcome these problems. The main features of this protocol are: i) precipitation with trichloroacetic acid/acetone overnight to prepare the acetone dry powder (ADP), ii) several additional steps of sonication in the ADP preparation and extractions with dense sodium dodecyl sulfate and phenol, and iii) adding two stages of phenol extractions. Proteins were extracted from roots using this new protocol (Method B) and a protocol described in the literature for T. cacao leaves and meristems (Method A). Using these methods, we obtained a protein yield of about 0.7 and 2.5 mg per 1.0 g lyophilized root, and a total of 60 and 400 spots could be separated, respectively. Through Method B, it was possible to isolate high-quality protein and a high yield of roots from T. cacao for high-quality 2-DE gels. To demonstrate the quality of the extracted proteins from roots of T. cacao using Method B, several protein spots were cut from the 2-DE gels, analyzed by tandem mass spectrometry, and identified. Method B was further tested on Citrus roots, with a protein yield of about 2.7 mg per 1.0 g lyophilized root and 800 detected spots.

Changes in protein profile detected in seedlings of Caesalpinia peltophoroides (Fabaceae) after exposure to high concentration of cadmium.

Sibipiruna (Caesalpinia peltophoroides Benth) is a tree of the Brazilian Atlantic Forest. It is a flowering ornamental tree widely planted throughout Brazil and indicated for restoration of degraded areas. We examined protein profile changes in leaves of seedlings of C. peltophoroides grown in nutrient solution under greenhouse conditions, after exposure to cadmium (Cd; 32 mg/L). A two-dimensional gel was used to analyze proteins expressed in response to stress 24 and 72 h after initiation of treatment with Cd. Various protein bands were identified that were related to stress response and/or metabolic adjustments, including proteins involved with resistance to stress, including detoxification, degradation, antioxidant, transport, signal transduction, photosynthesis, electron transport, biosynthesis reactions, and transcription regulation. After 24 h of Cd exposure, the genes of most of these proteins were upregulated. These putative proteins were associated with resistance to stress, including heat shock proteins, heat stress transcriptional factor and other transcriptional factors, aquaporins, glutathione transferase and choline monooxygenase. Most of the putative proteins observed after 72 h of exposure to Cd were downregulated. They were mainly photosynthetic process proteins, such as NAD(P)H-quinone oxidoreductase, photosystem I assembly, and photosystem II CP47 chlorophyll apoprotein. There were also proteins involved with degradation, biosynthesis and antioxidant activity, such as ATP-dependent Clp protease, methylthioribose-1-phosphate and glutathione peroxidase 2. Based on preliminary proteomic analysis, we conclude that proteins related to photosynthetic activity are inhibited, decreasing plant performance under stress conditions and that several proteins related to defense mechanisms are activated, inducing the plant defense response.

Multivariate analysis of morphological characteristics of two species of passion flower with ornamental potential and of hybrids between them.

We estimated genetic parameters through multivariate analysis of two species of Passiflora and their hybrids, considered of ornamental potential, based on the morphological characteristics: flower diameter, corona diameter, corona filament size, flower peduncle length, petal length and width, sepal length and width, internode length, stem diameter, leaf length, leaf width (mm), and leaf area (cm(2)). Five specimens of Passiflora sublanceolata [ex P. palmeri var. sublanceolata], five of P. foetida var. foetida and 20 F(1) hybrids between the two were evaluated. A randomized block design with four replications was used. The data were submitted to variance analysis and multivariate procedures, principal components analysis and unweighted pair group method with arithmetic mean grouping. We found significant differences between genotypes for all these morphological parameters. The hybrid plants had the highest variability, making them the most indicated for future improvement programs. The various multivariate techniques gave similar results, allowing separation of the plants into three distinct groups, these being the two paternal species and the hybrids. The hybrids were closer to the male genitors, revealing a paternal effect on the inheritance of vegetative and floral characters. Based on estimates of genetic parameters, the floral characteristics are the most indicated for the selection of plants for ornamental purposes, since these characteristics displayed greatest variability, a variation index of more than one, and high genotypic determination coefficients.

Genetic mapping of Theobroma cacao (Malvaceae) seedlings of the Parinari series, carriers of the lethal gene Luteus-Pa.

The lethal gene 'Luteus-Pa' is found in cacao genotypes (Theobroma cacao) of the Parinari (Pa) series, from Peru. Seedlings affected by this gene have yellowing leaves and subsequently die. We mapped this gene based on microsatellite markers and RAPDs, in order to elucidate the inheritance of 'Luteus-Pa' and investigate possible lethal mechanisms. DNA samples of genitors were amplified with 87 SSR and 64 RAPD primers. The SSR primers amplified 65 RAPD primers, giving 179 polymorphic bands. After screening with SSR and RAPD markers, we selected 20 SSR primers, two SSR primers with ESTs and 22 RAPD primers that were polymorphic for genitors Pa 30 and Pa 169. Only two of the 22 RAPD primers and three of the 20 SSR primers were informative and polymorphic in the analysis of the bulk samples of progenies. Among these, primer RAPD E11 produced a band linked to the lethal gene (38.5 cM); none of the SSRs were associated with 'Luteus-Pa'.

Leaf carbon assimilation and molecular phylogeny in Cattleya species (Orchidaceae).

We examined leaf CO(2) assimilation and how it varied among species within the orchid genus Cattleya. Measurements of CO(2) assimilation and maximum quantum yield of PS II (Fv/Fm) were made for mature leaves of nine species using a portable system for photosynthesis measurement and a portable fluorometer. Leaf area was measured with an area meter, and the specific leaf mass was determined. DNA of nine Cattleya species and two species of Hadrolaelia was extracted using the CTAB protocol. Each sample was amplified and sequenced using primers for the trnL gene. The phylogenetic analyses, using neighbor-joining and maximum parsimony methods, retrieved a group that included Cattleya and Hadrolaelia species, in which the unifoliate species were separated from the bifoliates. The topologies of the two cladograms showed some similarities. However, C. guttata (bifoliate) was placed in the unifoliate clade in the neighbor-joining tree, while C. warneri (unifoliate) was not placed in this clade in the maximum parsimony tree. Most Cattleya species keep the leaf stomata closed from 6 am to 4 pm. We suggest that C. elongata, C. tigrina and C. tenuis have C(3)-crassulacean acid metabolism since they open their stomata around 12 am. The Fv/Fm values remained relatively constant during the measurements of CO(2) assimilation. The same was observed for the specific leaf mass values, although great variations were found in the leaf area values. When the species were grouped using molecular data in the neighbor-joining analysis, no relation was observed with CO(2) assimilation.

Ultrastructural and metabolic disorders induced by short-term cadmium exposure in Avicennia schaueriana plants and its excretion through leaf salt glands.

Environmental cadmium (Cd) sources have increased in mangrove sediments in recent decades, inducing cellular damage to many plants. Avicennia schaueriana is abundant in mangrove sites and has been subject to Cd contamination. The possible effects of Cd toxicity and the structural and physiological disturbances to this plant were studied. Can this plant express early cellular tolerance mechanisms to such metal contamination? Seedlings of A. schaueriana were collected from sites of their natural occurrence, placed in plastic pots containing nutrient solution for 60 days, and subsequently exposed to increasing Cd concentrations for 5 days under experimental conditions. The anatomical, ultrastructural and physiological changes induced by Cd were analysed. Cd accumulated mainly in the root system and in pneumatophores, stems and leaves, induced differential accumulation of mineral nutrients, but did not induce necrosis or changes in leaf anatomy. However, there was a decrease in starch grains and an increase in deposited electron-dense material in the cortex and vascular bundles. Cd induced both increases in calcium (Ca) content in shoots and Ca oxalate crystal precipitation in leaf mesophyll and was detected in crystals and in the secretion of salt glands. Our observations and experimental results provide evidence of Cd tolerance in A. schaueriana. As a new feature, despite the clear cellular physiological disorders, this plant is able to eliminate Cd through leaf salt glands and immobilise it in Ca crystals, representing fast mechanisms for Cd exclusion and complexation in leaves in heavy metal coastal polluted marine ecosystems.

Preliminary analysis of expressed sequences of genes in Genipa americana L. plant roots exposed to cadmium in nutrient solution.

Many cell functions are redundantly executed in cells, and the experimental approaches that analyze the group of proteins, whose expression is modified in a specific functional condition, enable the identification of the group of proteins that are expressed under stress conditions. The objective of the present study was the evaluation of the genetic expression induced by cadmium (Cd) in Genipa americana L. (Rubiaceae) plants cultivated in nutritive solution, in order to help further studies concerning its use as a plant phytoremediator of such a metallic element. Plants were exposed to increasing concentrations of Cd (0.5, 1, 2, 4, 8, and 16 mg/L), together with the control, in nutritive solution. After the application of the treatments, root tips were harvested for the construction of a cDNA library. Of the 165 expressed sequence tags (ESTs) generated with the construction of the cDNA library, 81 showed homology to genes deposited in the NCBI database, 67 did not show similarity to any available gene, and 17 ESTs demonstrated homology with unknown genes. Of the most abundant cDNAs, 16 ESTs were similar to sequences of metallothionein genes. The analysis of ESTs, obtained from the root of G. americana through the construction of a cDNA library, allowed the identification of genes probably associated with proteins and enzymes related to the defense mechanisms of plants when they undergo biotic and abiotic stresses.