Humberto J.O. Ramos

Differentially expressed genes and proteins upon drought acclimation in tolerant and sensitive genotypes of Coffea canephora

The aim of this study was to investigate the molecular mechanisms underlying drought acclimation in coffee plants by the identification of candidate genes (CGs) using different approaches. The first approach used the data generated during the Brazilian Coffee expressed sequence tag (EST) project to select 13 CGs by an in silico analysis (electronic northern). The second approach was based on screening macroarrays spotted with plasmid DNA (coffee ESTs) with separate hybridizations using leaf cDNA probes from drought-tolerant and susceptible clones of Coffea canephora var. Conilon, grown under different water regimes. This allowed the isolation of seven additional CGs. The third approach used two-dimensional gel electrophoresis to identify proteins displaying differential accumulation in leaves of drought-tolerant and susceptible clones of C. canephora. Six of them were characterized by MALDI-TOF-MS/MS (matrix-assisted laser desorption-time of flight-tandem mass spectrometry) and the corresponding proteins were identified. Finally, additional CGs were selected from the literature, and quantitative real-time polymerase chain reaction (qPCR) was performed to analyse the expression of all identified CGs. Altogether, >40 genes presenting differential gene expression during drought acclimation were identified, some of them showing different expression profiles between drought-tolerant and susceptible clones. Based on the obtained results, it can be concluded that factors involved a complex network of responses probably involving the abscisic signalling pathway and nitric oxide are major molecular determinants that might explain the better efficiency in controlling stomata closure and transpiration displayed by drought-tolerant clones of C. canephora.

Conserved threonine residues within the A-loop of the receptor NIK differentially regulate the kinase function required for antiviral signaling.

NSP-interacting kinase (NIK1) is a receptor-like kinase identified as a virulence target of the begomovirus nuclear shuttle protein (NSP). We found that NIK1 undergoes a stepwise pattern of phosphorylation within its activation-loop domain (A-loop) with distinct roles for different threonine residues. Mutations at Thr-474 or Thr-468 impaired autophosphorylation and were defective for kinase activation. In contrast, a mutation at Thr-469 did not impact autophosphorylation and increased substrate phosphorylation, suggesting an inhibitory role for Thr-469 in kinase function. To dissect the functional significance of these results, we used NSP-expressing virus infection as a mechanism to interfere with wild type and mutant NIK1 action in plants. The NIK1 knockout mutant shows enhanced susceptibility to virus infections, a phenotype that could be complemented with ectopic expression of a 35S-NIK1 or 35S-T469A NIK1 transgenes. However, ectopic expression of an inactive kinase or the 35S-T474A NIK1 mutant did not reverse the enhanced susceptibility phenotype of knockout lines, demonstrating that Thr-474 autophosphorylation was needed to transduce a defense response to geminiviruses. Furthermore, mutations at Thr-474 and Thr-469 residues antagonistically affected NIK-mediated nuclear relocation of the downstream effector rpL10. These results establish that NIK1 functions as an authentic defense receptor as it requires activation to elicit a defense response. Our data also suggest a model whereby phosphorylation-dependent activation of a plant receptor-like kinase enables the A-loop to control differentially auto- and substrate phosphorylation.

Characteristics of free endoglucanase and glycosidases multienzyme complex from Fusarium verticillioides

A novel multienzyme complex, E1C, and a free endoglucanase, E2 (GH5), from Fusarium verticillioides were purified. The E1C contained two endoglucanases (GH6 and GH10), one cellobiohydrolase (GH7) and one xylanase (GH10). Maximum activity was observed at 80 °C for both enzymes and they were thermostable at 50 and 60 °C. The activation energies for E1C and E2 were 21.3 and 27.5 kJ/mol, respectively. The KM for E1C was 10.25 g/L while for E2 was 6.58 g/L. Both E1C and E2 were activated by Mn(2+) and CoCl2 while they were inhibited by SDS, CuSO4, FeCl3, AgNO4, ZnSO4 and HgCl2. E1C and E2 presented endo-β-1,3-1,4-glucanase activity. E1C presented crescent activity towards cellopentaose, cellotetraose and cellotriose. E2 hydrolyzed the substrates cellopentaose, cellotetraose and cellotriose with the same efficiency. E1C showed a higher stability and a better hydrolysis performance than E2, suggesting advantages resulting from the physical interaction between proteins.

Proteomic analysis of the venom of the predatory ant Pachycondyla striata (Hymenoptera: Formicidae)

The ants use their venom for predation, defense, and communication. The venom of these insects is rich in peptides and proteins, and compared with other animal venoms, ant venoms remain poorly explored. The objective of this study was to evaluate the protein content of the venom in the Ponerinae ant Pachycondyla striata. Venom samples were collected by manual gland reservoir dissection, and samples were submitted to two-dimensional gel electrophoresis and separation by ion-exchange and reverse-phase high-performance liquid chromatography followed by mass spectrometry using tanden matrix-assisted laser desorption/ionization with time-of-flight (MALDI-TOF/TOF) mass spectrometry and electrospray ionization-quadrupole with time-of-flight (ESI-Q/TOF) mass spectrometry for obtaining amino acid sequence. Spectra obtained were searched against the NCBInr and SwissProt database. Additional analysis was performed using PEAKS Studio 7.0 (Sequencing de novo). The venom of P. striata has a complex mixture of proteins from which 43 were identified. Within the identified proteins are classical venom proteins (phospholipase A, hyaluronidase, and aminopeptidase N), allergenic proteins (different venom allergens), and bioactive peptides (U10-ctenitoxin Pn1a). Venom allergens are among the most expressed proteins, suggesting that P. striata venom has high allergenic potential. This study discusses the possible functions of the proteins identified in the venom of P. striata.

Development of data for the identification and characterization of proteins found in Rhodnius prolixus, Triatoma lecticularia and Panstrongylus herreri

The data presented here were obtained from the saliva of three triatominae, Rhodnius prolixus, Triatoma lecticularia and Panstrongylus herreri from Montandon et al. study, doi:10.1016/j.ibmb.2016.02.009 [3]. These data were obtained from spectra generated by the mass spectrometry of proteins observed through the analysis of 2-D electrophoretic profiles. The data were analyzed according to the UniProt code, protein name, protein group, isoelectric point and molecular weight, electrophoretic profile, molecular mass referring to UniProt, volume percentage referring to the spot of the electrophoretic profile, number of peptides and percent coverage found by mass spectrometry related to the particular proteins. In addition, there characterizations made the most significant protein per spot, and also characterizations made for biological processes and molecular functions for all identified proteins.

Two-dimensional electrophoresis-based proteomic analysis of Phaseolus vulgaris in response to Colletotrichum lindemuthianum.

Brazil is the largest producer of the common bean (Phaseolus vulgaris L.), a staple food for a large proportion of the Brazilian population and its main protein source. Bean anthracnose is a common and broadly distributed bean disease and can cause yield losses of up to 100%. Alterations in the proteome of common bean leaves as a response to challenge by Colletotrichum lindemuthianum were analyzed using two-dimensional gel electrophoresis and mass spectrometry. The cultivar AND 277 was inoculated with an incompatible race of C. lindemuthianum. In comparison with non-inoculated plants, we observed 34 proteins differentially accumulated in leaves collected 12, 24, and 48 h post inoculation. The protein spots obtained from 2DE gels were then analyzed by MALDITOF/TOF mass spectrometry. Seventeen proteins were identified by MS/MS fragmentation. Most of them corresponded to enzymes belonging to photosynthesis and carbon metabolism, antioxidant systems, genetic information processing, defense and stress response, chaperones and folding catalysts, and phenylpropanoid or flavonoid biosynthesis. Our findings indicate that these proteins are related to the common bean response to C. lindemuthianum.

Broad range flavonoid profiling by LC/MS of soybean genotypes contrasting for resistance to Anticarsia gemmatalis (Lepidoptera: Noctuidae)

Attack by herbivores is a major biotic stress limiting the soybean crop production. Plant defenses against caterpillars include the production of secondary metabolites such as flavonoids, which constitute a diverse group of plant secondary metabolites. Thus, a more discriminate metabolic profiling between genotypes are important for a more comprehensive and reliable characterization of soybean resistance. Therefore, in this study a non-targeted LC/MS-based for analysis of flavonoid profiles of soybean genotypes contrasting to the resistance to A. gemmatalis was applied. Clustering analysis revealed profiles highly distinct between the susceptible UFV 105 AP and the resistant IAC 17 genotypes. This comparative approach enables to identify directly from leaf extract some new compounds related to resistance, some of which were present in higher abundance specifically in the IAC 17 genotype: four Quercetin conjugates, Rutin (Quercetin 3-O-Rutinoside), Quercetin-3,7-O- di-glucoside, Quercetin-3-O-rhamnosylglycoside-7-O-glucoside and Quercetin-3-O-rhamnopyranosyl-glucopyranoside-rhamnopyranoside; two Genistein conjugates, Genistein-7-O-diglucoside-dimalonylated and Genistein-7-O-6-O-malonylglucoside; and one Daidzein conjugate, Daidzein-7-O-Glucoside-malonate. The most abundant flavonoid glycoconjugates in soybean leaves belongs to Quercetin and Kaempferol classes. However, only one from the identified compounds was classified as a Kaempferol. The Kaempferol-3-O-L-rhamnopyranosyl-glucopyranoside showed high abundance in the resistant genotype IAC 17. The metabolic profiles generated by LC/MS allowed the reconstruction of the flavonoid biosynthetic pathways, which revealed a constitutive character for herbivory resistance in the resistant genotype IAC-17 and a metabolic regulation for the rechanneling of Quercetin, Kaempferol and Genistein conjugates in soybean. Highest relative abundances were detected for glyconjugates, such as Rutin, Quercetin 3-O-rhamnosylglycoside-7-O-glucoside and Quercitin-3-O-rhamnopyranosyl-glucopyranoside-rhamnopyranoside in the leaves of the resistant genotype.