Differential expression of genes potentially related to the callogenesis and in situ hybridization of SERK gene in macaw palm (Acrocomia aculeata Jacq.) Lodd. ex Mart.

For the purpose of understanding the molecular processes triggered during callus formation in macaw palm, the expression of seven genes potentially involved in this process, identified in previous studies and from the literature, was investigated by RT-qPCR. In addition, in situ hybridization of the SERK gene was performed. Leaf tissues from adult plants from two macaw palm accession were inoculated in a medium combined with Picloram at a concentration of 450 µM to induce callus. The expression analysis was performed from leaf samples from two accessions of different origins (Municipalities of Tiros, MG, and Buriti Vermelho, DF, Brazil), which are characterized as non-responsive (NR) and responsive (R), respectively. The material was collected before callus induction (0 DAI, initial day) and 120 days after callus induction (120 DAI). Genes related to development (SERK, OASA, EF1, ANN1) and stress (LEA, CAT2, and MDAR5) were evaluated. The results obtained showed that all the genes involved with the development had their expressions downregulated at 0 DAI when the accession R was compared with the accession NR. On the other hand, it was possible to observe that these genes were upregulated at 120 DAI. The LEA stress gene showed a tendency to increase expression in the NR accession, while the R accession showed decreased expression and the CAT2 and MDAR5 genes showed upregulation in both accessions. In situ hybridization showed SERK transcripts in the vascular bundles, indicating the expression of SERK in this region, in addition to its expression in calluses. The results obtained in this study support our hypothesis that the regulation of genes involved in the control of oxidative stress and development is crucial for the formation of calluses in macaw palm.

Involvement of structurally distinct cupuassu chitinases and osmotin in plant resistance to the fungus Moniliophthora perniciosa.

The cupuassu tree (Theobroma grandiflorum) is a crop of great economic importance to Brazil, mainly for its pulp and seeds, which are used in food industry. However, cupuassu fruit production is threatened by witches' broom disease caused by the fungus Moniliophthora perniciosa. As elements of its defense mechanisms, the plant can produce and accumulate pathogenesis-related (PR) proteins such as chitinases and osmotins. Here, we identified three cupuassu PR proteins (TgPR3, TgPR5 and TgPR8) from cupuassu-M. perniciosa interaction RNA-seq data. TgPR3 and TgPR8 corresponded to chitinases, and TgPR5 to osmotin; they are phylogenetically related to cacao and to Arabidopsis PR sequences involved in biotic and abiotic stress. The TgPR proteins' tridimensional structure was obtained through homology modeling, and molecular docking with chitin and chitosan showed that the TgPR proteins can interact with both cell wall molecules and presented a higher affinity for chitosan. TgPR gene expression was analyzed by RT-qPCR on resistant and susceptible cupuassu genotypes infected by M. perniciosa at 8, 24, 48 and 72 h after infection (hai). The TgPR genes showed higher expression in resistant plants compared to the susceptible ones, mainly for TgPR5 at 8 and 24 hai, while the expression was lower in the susceptible cupuassu plants. To our knowledge, this is the first in silico and in vitro reports of cupuassu PR protein. The data suggested that TgPRs could be involved in recognizing mechanisms of the plant's innate immune system through chitin receptors. Our results also suggest a putative role of chitinase/chitosanase for the TgPR5/osmotin.

Genome size, cytogenetic data and transferability of EST-SSRs markers in wild and cultivated species of the genus Theobroma L. (Byttnerioideae, Malvaceae).

The genus Theobroma comprises several trees species native to the Amazon. Theobroma cacao L. plays a key economic role mainly in the chocolate industry. Both cultivated and wild forms are described within the genus. Variations in genome size and chromosome number have been used for prediction purposes including the frequency of interspecific hybridization or inference about evolutionary relationships. In this study, the nuclear DNA content, karyotype and genetic diversity using functional microsatellites (EST-SSR) of seven Theobroma species were characterized. The nuclear content of DNA for all analyzed Theobroma species was 1C = ~ 0.46 pg. These species presented 2n = 20 with small chromosomes and only one pair of terminal heterochromatic bands positively stained (CMA+/DAPI- bands). The small size of Theobroma ssp. genomes was equivalent to other Byttnerioideae species, suggesting that the basal lineage of Malvaceae have smaller genomes and that there was an expansion of 2C values in the more specialized family clades. A set of 20 EST-SSR primers were characterized for related species of Theobroma, in which 12 loci were polymorphic. The polymorphism information content (PIC) ranged from 0.23 to 0.65, indicating a high level of information per locus. Combined results of flow cytometry, cytogenetic data and EST-SSRs markers will contribute to better describe the species and infer about the evolutionary relationships among Theobroma species. In addition, the importance of a core collection for conservation purposes is highlighted.

Selection of Reference Genes for Expression Study in Pulp and Seeds of Theobroma grandiflorum (Willd. ex Spreng.) Schum.

Cupuassu (Theobroma grandiflorum [Willd. ex Spreng.] Schum) is a species of high economic importance in Brazil with great potential at international level due to the multiple uses of both its seeds and pulp in the industry of sweets and cosmetics. For this reason, the cupuassu breeding program focused on the selection of genotypes with high pulp and seed quality-selection associated with the understanding of the mechanisms involved in fruit formation. Gene expression is one of the most used approaches related to such understanding. In this sense, quantitative real-time PCR (qPCR) is a powerful tool, since it rapidly and reliably quantifies gene expression levels across different experimental conditions. The analysis by qPCR and the correct interpretation of data depend on signal normalization using reference genes, i.e. genes presenting a uniform pattern of expression in the analyzed samples. Here, we selected and analyzed the expression of five genes from cupuassu (ACP, ACT, GAPDH, MDH, TUB) to be used as candidates for reference genes on pulp and seed of young, maturing and mature cupuassu fruits. The evaluation of the gene expression stability was obtained using the NormFinder, geNorm and BestKeeper programs. In general, our results indicated that the GAPDH and MDH genes constituted the best combination as reference genes to analyze the expression of cupuassu samples. To our knowledge, this is the first report of reference gene definition in cupuassu, and these results will support subsequent analysis related to gene expression studies in cupuassu plants subjected to different biotic or abiotic conditions as well as serve as a tool for diversity analysis based on pulp and seed quality.

First Microsatellite Markers Developed from Cupuassu ESTs: Application in Diversity Analysis and Cross-Species Transferability to Cacao.

The cupuassu tree (Theobroma grandiflorum) (Willd. ex Spreng.) Schum. is a fruitful species from the Amazon with great economical potential, due to the multiple uses of its fruit´s pulp and seeds in the food and cosmetic industries, including the production of cupulate, an alternative to chocolate. In order to support the cupuassu breeding program and to select plants presenting both pulp/seed quality and fungal disease resistance, SSRs from Next Generation Sequencing ESTs were obtained and used in diversity analysis. From 8,330 ESTs, 1,517 contained one or more SSRs (1,899 SSRs identified). The most abundant motifs identified in the EST-SSRs were hepta- and trinucleotides, and they were found with a minimum and maximum of 2 and 19 repeats, respectively. From the 1,517 ESTs containing SSRs, 70 ESTs were selected based on their functional annotation, focusing on pulp and seed quality, as well as resistance to pathogens. The 70 ESTs selected contained 77 SSRs, and among which, 11 were polymorphic in cupuassu genotypes. These EST-SSRs were able to discriminate the cupuassu genotype in relation to resistance/susceptibility to witches' broom disease, as well as to pulp quality (SST/ATT values). Finally, we showed that these markers were transferable to cacao genotypes, and that genome availability might be used as a predictive tool for polymorphism detection and primer design useful for both Theobroma species. To our knowledge, this is the first report involving EST-SSRs from cupuassu and is also a pioneer in the analysis of marker transferability from cupuassu to cacao. Moreover, these markers might contribute to develop or saturate the cupuassu and cacao genetic maps, respectively.

An opaque-2-like transcription factor from pearl millet.

Pearl millet (Pennisetum glaucum L.), a species of the Poaceae family, is an important food crop in Africa, Asia and South America. Its nutritional value is due to storage prolamins accumulated in the seeds. In other species of the same family, the expression of the genes coding for storage prolamins is mediated by the regulatory protein opaque-2. In this paper we show that an opaque-2 -like protein is present in pearl millet too and is expressed during the early stages of seed development. The organization of the gene coding for this protein is similar to that of orthologous genes in other Poaceae species, i.e. six exons separated by five introns. A comparison of amino acid homologies with other described opaque-2 proteins is presented.

Characterization of pearl millet prolamins.

We report the physical-chemical characterization of the major alcohol-soluble proteins present in seeds of pearl millet (Pennisetum glaucum) by SDS-PAGE, bidimensional gel electrophoresis, MALDI-TOF/MS and RP-HPLC. We demonstrate the presence of three major prolamins, called A-, B- and C-pennisetin with mass values around 27, 22 and 12 kDa, respectively. We present partial amino acid sequences of these major proteins, which should allow the posterior isolation of the respective genes.