Gonçalo Apolinário de Souza Filho

Identification and characterization of Gluconacetobacter diazotrophicus mutants defective in the solubilization of phosphorus and zinc

Gluconacetobacter diazotrophicus is a plant-growth-promoting bacterium, which is able to colonize sugarcane and other plant species of economic importance. The potentially beneficial effects promoted by this bacterium on plants are nitrogen-fixation, production of phythormones, action against pathogens and mineral nutrient solubilization. In this study, the molecular mechanisms associated with phosphorus and zinc solubilization were analyzed. A transposon mutant library was constructed and screened to select for mutants defective for phosphorous [Ca5(PO4)3OH] and zinc (ZnO) solubilization. A total of five mutants were identified in each screen. Both screenings, performed independently, allowed to select the same mutants. The interrupted gene in each mutant was identified by sequencing and the results demonstrate that the production of gluconic acid is a required pathway for solubilization of such nutrients in G. diazotrophicus.

Specific control of Arabidopsis BAK1/SERK4-regulated cell death by protein glycosylation

Precise control of cell death is essential for the survival of all organisms. Arabidopsis thaliana BRASSINOSTEROID INSENSITIVE 1-associated receptor kinase 1 (BAK1) and somatic embryogenesis receptor kinase 4 (SERK4) redundantly and negatively regulate cell death through elusive mechanisms. By deploying a genetic screen for suppressors of cell death triggered by virus-induced gene silencing of BAK1/SERK4 on Arabidopsis knockout collections, we identified STT3a, a protein involved in N-glycosylation modification, as an important regulator of bak1/serk4 cell death. Systematic investigation of glycosylation pathway and endoplasmic reticulum (ER) quality control (ERQC) components revealed distinct and overlapping mechanisms of cell death regulated by BAK1/SERK4 and their interacting protein BIR1. Genome-wide transcriptional analysis revealed the activation of members of cysteine-rich receptor-like kinase (CRK) genes in the bak1/serk4 mutant. Ectopic expression of CRK4 induced STT3a/N-glycosylation-dependent cell death in Arabidopsis and Nicotiana benthamiana. Therefore, N-glycosylation and specific ERQC components are essential to activate bak1/serk4 cell death, and CRK4 is likely to be among client proteins of protein glycosylation involved in BAK1/SERK4-regulated cell death.

Protein Poly(ADP-ribosyl)ation Regulates Arabidopsis Immune Gene Expression and Defense Responses

Perception of microbe-associated molecular patterns (MAMPs) elicits transcriptional reprogramming in hosts and activates defense to pathogen attacks. The molecular mechanisms underlying plant pattern-triggered immunity remain elusive. A genetic screen identified Arabidopsis poly(ADP-ribose) glycohydrolase 1 (atparg1) mutant with elevated immune gene expression upon multiple MAMP and pathogen treatments. Poly(ADP-ribose) glycohydrolase (PARG) is predicted to remove poly(ADP-ribose) polymers on acceptor proteins modified by poly(ADP-ribose) polymerases (PARPs) with three PARPs and two PARGs in Arabidopsis genome. AtPARP1 and AtPARP2 possess poly(ADP-ribose) polymerase activity, and the activity of AtPARP2 was enhanced by MAMP treatment. AtPARG1, but not AtPARG2, carries glycohydrolase activity in vivo and in vitro. Importantly, mutation (G450R) in atparg1 blocks its activity and the corresponding residue is highly conserved and essential for human HsPARG activity. Consistently, mutant atparp1atparp2 plants exhibited compromised immune gene activation and enhanced susceptibility to pathogen infections. Our study indicates that protein poly(ADP-ribosyl)ation plays critical roles in plant immune gene expression and defense to pathogen attacks.

Cloning and Characterization of a cDNA Encoding a Cowpea Seed Defensin and Analysis of its Expression

We have previously isolated a cowpea seed defensin with potent antifungal activity. Here we report the cloning of its cDNA obtained from RT-PCR. The sequence of the deduced protein VUDEF exhibited homology to plant defensins. Northern analysis revealed that VUDEF mRNA is accumulated during cowpea seed development and its level increases in seedling tissues after exposure to fungal pathogen and cold.

Essential role of the czc determinant for cadmium, cobalt and zinc resistance in Gluconacetobacter diazotrophicus PAl 5.

The mechanisms of cadmium, cobalt and zinc resistance were characterized in the plant-growth-promoting bacterium Gluconacetobacter diazotrophicus PAl 5. The resistance level of the wild-type strain was evaluated through the establishment of minimum inhibitory concentrations (MIC) of the soluble compounds CdCl2·H2O, CoCl2·6H2O and ZnCl2. Gluconacetobacter diazotrophicus PAl 5 was resistant to high concentrations of Cd, Co and Zn, with MICs of 1.2, 20 and 20 mM, respectively. Screening of an insertion library from transposon EZ-Tn5 in the presence of ZnO revealed that the mutant GDP30H3 was unable to grow in the presence of the compound. This mutant was also highly sensitive to CdCl2·H2O, CoCl2·6H2O and ZnCl2. Molecular characterization established that the mutation affected the czcA gene, which encodes a protein involved in metal efflux. In silico analysis showed that czcA is a component of the czcCBARS operon together with four other genes. This work provides evidence of the high tolerance of G. diazotrophicus PAl 5 to heavy metals and that czc is a determinant for metal resistance in this bacterium.

Mapping of Quantitative Trait Loci for Butter Content and Hardness in Cocoa Beans (Theobroma cacao L.)

Cocoa butter is an important raw material for the chocolate, pharmaceutical, and cosmetic industries. The butter content and quality in cocoa beans are genetically controlled characteristics, and affect its commercial value and industrial applicability. In the present work, an F2 population derived from the cross between the ICS-1 and Scavina-6 cocoa clones was used for molecular mapping. A linkage map was constructed based on amplified fragment length polymorphism, random amplified polymorphic DNA, and simple sequence repeat markers, resulting in a total of 273 markers, distributed in 14 linkage groups (LGs). Phenotyping of butter content was performed after ether extraction and butter hardness was determined by sweeping differential calorimetry. One quantitative trait locus (QTL) associated to butter content was mapped at linkage group 9 (LG9) and two QTLs for butter hardness were identified at linkage groups 9 and 7 (LG9 and LG7). The two QTLs mapped at the LG9 explained 51.0% and 28.8% of the phenotypic variation for butter content and hardness, respectively. These QTLs were concentrated in the same map region, suggesting a close genetic linkage or pleiotropic effect. The QTLs identified may be useful in further marker-assisted selection breeding programs aimed at cocoa butter quality improvement.

Development and characterization of novel tetra-, tri- and di-nucleotide microsatellite markers in cacao (Theobroma cacao L.)

The cacao plant, Theobroma cacao L., produces white seeds (beans) that form the major ingredient of processed chocolate. A great deal of research effort has been expended to the development of new genetically modified cacao plants with improved productivity and resistance and beans of good industrial quality. The availability of suitable genetic markers is an important aspect of the efficient selection and breeding of this perennial species. We describe the development of 123 microsatellite loci of cacao. An optimized protocol was used to construct and screen a microsatellite-enriched genomic library from which we isolated 64 di-nucleotide, 45 tri-nucleotide and 14 tetra-nucleotide microsatellite loci. The primers were tested on samples from five different T. cacao accessions, one accession from T. grandiflorum and one accession from Herranea sp. Among the 123 loci, 54 were polymorphic, 61 were monomorphic and eight did not present an amplification product. These new markers will be useful in future studies by increasing the accuracy of genotypic assessments in diverse cocoa tree populations as well as in other species of the Theobroma genus.

A paper-based electroelution system for protein recovery from stained sodium dodecyl sulfate-polyacrylamide gels.

Electroelution is a widely used methodology for protein purification. In this study, a practical and low-cost system for protein electroelution from stained polyacrylamide gels was developed. For this, a horizontal protein electroelution cuve was constructed with glass plates, 1.5-ml capacity microcentrifuge tubes, and dialysis membrane. Analyses of the system efficiency showed high protein recovery from nonfixed and fixed sodium dodecyl sulfate (SDS)-polyacrylamide gels.

Self-incompatibility in passion fruit: cellular responses in incompatible pollinations

Self-incompatibility (SI) is a genetic mechanism in angiosperms that prevents selfing. The SI system in passion fruit (Passiflora edulis Sims) was investigated using hand pollinations. Pollen tube growth was inspected by microscopy, and sequence analysis of potential regulators of this process was carried out. The results revealed that the pollen tubes grew slowly and were often completely arrested in the stigma in an incompatible combination. Under these circumstances the pollen tube was rapidly and significantly rearranged, followed by the rapid deposition of callose in the stigma during the SI response. The structural changes in the pollen grain after an incompatible pollination were investigated using scanning electron microscopy. Furthermore, ultrastructural observations during incompatible interactions showed that the membrane system of the pollen tube was damaged, and fertilisation was not observed or was considerably delayed when compared to compatible interactions. The analysis presented here provides evidence that the passion fruit genome presents similar sequences to those encoding factors involved in SI in different species. These results suggest that, in the SI system of passion fruit, the rejection of an incompatible pollen grain is characterised by drastic structural changes in both pollen and pollen tube.

Characterization of a saccharide-binding protein from Talisia esculenta seeds with trypsin inhibitory activity.

Some proteins exhibit characteristics that suggest they have a primary, if not an exclusive role in nutrient reserve storage. The best studied examples are the storage proteins that accumulate specifically in developing seeds. Some of these protein demonstrate biological activities that could contribute to resistance to pest, pathogens or abiotic stresses. In this study we present the biochemical characterization and cloning of the major protein from seeds of T. esculenta (Talisin), a member of the Sapindaceae family. The N-terminal sequence of the protein isolated was used to produce a degenerated primer. This primer allowed the amplification of the Talisin cDNA by RTPCR from mRNA of the T. esculenta seeds protein. The sequence analysis of the cloned cDNA, demonstrated a 756 bp sequence encoding a peptide of 198 amino acids. The deduced peptide presented high similarity to a typical VSP, the 22-kDa protein in lychee (73 %) and 50.0 % identity to Theobroma bicolor reserve protein. Identities of 52.0 % and 44.0 % to trypsin inhibitors from Treobroma mammosum and Populus tremula respectively. In conclusion, we may suggest that Talisin could be a seed storage protein with affinity properties, i.e. interacts with carbohydrates and trypsin enzyme.