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.

Proteomic response of Moniliophthora perniciosa exposed to pathogenesis-related protein-10 from Theobroma cacao.

TcPR-10, a member of the pathogenesis-related protein 10 family, was identified in EST library of interactions between Theobroma cacao and Moniliophthora perniciosa. TcPR-10 has been shown to have antifungal and ribonuclease activities in vitro. This study aimed to identify proteins that are differentially expressed in M. perniciosa in response to TcPR-10 through a proteomic analysis. The fungal hyphae were subjected to one of four treatments: control treatment or 30-, 60- or 120-min treatment with the TcPR-10 protein. Two-dimensional maps revealed 191 differentially expressed proteins, 55 of which were identified by mass spectrometry. The proteins identified in all treatments were divided into the following classes: cell metabolism, stress response, zinc binding, phosphorylation mechanism, transport, autophagy, DNA repair, and oxidoreductases. The predominant class was stress-response proteins (29%), such as heat shock proteins; these proteins exhibited the highest expression levels relative to the control treatment and are known to trigger defense mechanisms against cytotoxic drugs as well as TcPR-10. Oxidoreductases (25%) were overexpressed in the control and in 30-min treatments but exhibited reduced expression at 120 min. These proteins are involved in the repair of damage caused by oxidative stress due to the contact with TcPR- 10. Consistent with the antifungal activity of TcPR-10, several proteins identified were related to detoxification, autophagy or were involved in mechanisms for maintaining fungal homeostasis, such as ergosterol biosynthesis. These results show that the sensitivity of the fungus to TcPR-10 involves several biochemical routes, clarifying the possible modes of action of this antifungal protein.

Variability analysis of 'Persian' acid lime tree selections using agronomic and molecular markers.

'Persian' acid lime (PAL) is the most important triploid commercial citrus crop planted in the world. Little is known about the genetic variability of the selections used in Brazil. Therefore, 25 genotypes originating from the PAL, and three control species, Citrus sunki, C. limon, and C. aurantiifolia, were assessed using inter-simple sequence repeat (ISSR) and inter-retrotransposon amplified polymorphism (IRAP) molecular markers and agronomic traits of the fruit. The dendrograms were designed using the mean Euclidean distance for the physicochemical attributes of the fruit (weight, length, diameter, peel color, peel thickness, number of seeds, juice yield, titratable acidity, soluble solids, and ratio) and the Jaccard distances using the data from the ISSR and IRAP molecular markers. In the physicochemical analysis, the genotypes were grouped according to species. The trait that contributed most to the diversity among accessions was the number of seeds. The 17 ISSR primers produced 69 polymorphic bands in the molecular analysis, and the seven IRAP primers generated 30 polymorphic bands. The markers detected polymorphisms within and among the PALs; two groups were formed within the PALs.

Comparative study of putative 9-cis-epoxycarotenoid dioxygenase and abscisic acid accumulation in the responses of Sunki mandarin and Rangpur lime to water deficit.

Abscisic acid is a plant hormone that participates in essential plant physiological processes, especially during adaptation to many environmental stresses, such as water deficit. The relationship between ABA accumulation and the expression of putative carotenoid cleavage dioxygenase (CCD) genes was investigated in the pot-cultivated leaves and roots of the 'Rangpur' lime and 'Sunki Maravilha' mandarin plants. Transpiration, stomatal resistance and leaf growth were evaluated when these genotypes were subjected to continuous water deficit. Under water deficit conditions, the 'Rangpur' lime extracts used greater amounts of water when compared to the 'Sunki Maravilha' plants, which reached the greatest stomatal resistance 5 days before 'Rangpur' lime. When subjected to water deficit, the roots and leaves of 'Sunki Maravilha' showed a progressive increase in ABA accumulation; however, in 'Rangpur' lime, alternations between high and low ABA concentrations were observed. These results suggest a retroactive feeding regulation by ABA. In 'Rangpur' lime the NCED2, NCED3 and CCD4a genes were expressed at the highest levels in the roots, and NCED5 was highly expressed in the leaves; in 'Sunki Maravilha', the NCED2 and NCED5 genes were most highly expressed in the roots, and NCED2 was most highly expressed in the leaves. However, for both genotypes, the transcription of these genes only correlated with ABA accumulation during the most severe water deficit conditions. The 'Rangpur' lime behaved as a vigorous rootstock; the leaf growth remained unaltered even when water was scarce. However, 'Sunki Maravilha' adaptation was based on the equilibrium of the response between the root and the aerial tissues due to water restriction. The use of the Sunki mandarin in combination with a scion with similar characteristics as its own, which responds to water deficit stress by accumulating ABA in the leaves, may display good drought tolerance under field conditions.

Oxidative stress proteins as an indicator of a low quality of eucalyptus clones for the pulp and paper industry.

Eucalyptus is a genus widely cultivated in many tropical and subtropical regions of the world as one of the main sources of raw materials for the pulp and paper industry. Identification of clones and selection of genotypes with desirable agronomic characteristics would be useful. We assessed eucalyptus full-sibs that varied in wood quality, using a combination of two-dimensional gel electrophoresis and mass spectrometry to identify differentially expressed proteins as candidates for quality markers. Thirty-one differently expressed proteins were identified, including three proteins of clone X1, four of clone X2, and 12 each of clones X3 and X4. These proteins are involved in various biological processes, including polyphosphate biosynthesis, catalytic activity, nucleotide excision repair, cellular metabolic processes, cell redox homeostasis, response to salt stress, response to temperature, oxidation and reduction processes, cellular water homeostasis, and protein phosphorylation. In the cambial region of each clone, the proteins ketol-acid reductoisomerase, uncharacterized protein MG428, receptor-like serine/threonine-protein kinase and a heat shock protein were found in larger quantities in clone X4 than in clone X1. These proteins are known to be related to protection against oxidative stress and biosynthesis of lignin. A high buildup of proteins involved in response to stress in the cambial region of eucalyptus would indicate clones with undesirable characteristics for use in the pulp and paper industry.

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.

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'.

Expression analysis of transcription factors from the interaction between cacao and Moniliophthora perniciosa (Tricholomataceae).

Cacao (Theobroma cacao) is one of the most important tropical crops; however, production is threatened by numerous pathogens, including the hemibiotrophic fungus Moniliophthora perniciosa, which causes witches' broom disease. To understand the mechanisms that lead to the development of this disease in cacao, we focused our attention on cacao transcription factors (TFs), which act as master regulators of cellular processes and are important for the fine-tuning of plant defense responses. We developed a macroarray with 88 TF cDNA from previously obtained cacao-M. perniciosa interaction libraries. Seventy-two TFs were found differentially expressed between the susceptible (Catongo) and resistant (TSH1188) genotypes and/or during the disease time course--from 24 h to 30 days after infection. Most of the differentially expressed TFs belonged to the bZIP, MYB and WRKY families and presented opposite expression patterns in susceptible and resistant cacao-M. perniciosa interactions (i.e., up-regulated in Catongo and down-regulated in TSH1188). The results of the macroarray were confirmed for bZIP and WRKY TFs by real-time PCR. These differentially expressed TFs are good candidates for subsequent functional analysis as well as for plant engineering. Some of these TFs could also be localized on the cacao reference map related to witches' broom resistance, facilitating the breeding and selection of resistant cacao trees.

Single nucleotide polymorphisms from Theobroma cacao expressed sequence tags associated with witches' broom disease in cacao.

In order to increase the efficiency of cacao tree resistance to witches' broom disease, which is caused by Moniliophthora perniciosa (Tricholomataceae), we looked for molecular markers that could help in the selection of resistant cacao genotypes. Among the different markers useful for developing marker-assisted selection, single nucleotide polymorphisms (SNPs) constitute the most common type of sequence difference between alleles and can be easily detected by in silico analysis from expressed sequence tag libraries. We report the first detection and analysis of SNPs from cacao-M. perniciosa interaction expressed sequence tags, using bioinformatics. Selection based on analysis of these SNPs should be useful for developing cacao varieties resistant to this devastating disease.

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.