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Mangaba (Hancornia speciosa Gomes) is a native fruit of Brazil that has high antioxidant activity and may benefit human health. The goal of this study was to discriminate between accessions in the ...

Sugar-rich environments represent an important challenge for microorganisms. The osmotic and molecular imbalances resulting from this condition severely limit microbial metabolism and growth. Gluconacetobacter diazotrophicus is one of the most sugar-tolerant prokaryotes, able to grow in the presence of sucrose concentrations up to 30%. However, the mechanisms that control its tolerance to such conditions remain poorly exploited. The present work investigated the key mechanisms of tolerance to high sugar in G. diazotrophicus. Comparative proteomics was applied to investigate the main functional pathways regulated in G. diazotrophicus when cultivated in the presence of high sucrose. Among 191 proteins regulated by high sucrose, regulatory pathways related to sugar metabolism, nutrient uptake, compatible solute synthesis, amino acid metabolism, and proteolytic system were highlighted. The role of these pathways on high-sucrose tolerance was investigated by mutagenesis analysis, which revealed that the knockout mutants zwf::Tn5 (sugar metabolism), tbdr::Tn5 (nutrient uptake), mtlK::Tn5 (compatible solute synthesis), pepN::Tn5 (proteolytic system), metH::Tn5 (amino acid metabolism), and ilvD::Tn5 (amino acid metabolism) became more sensitive to high sucrose. Together, our results identified mechanisms involved in response to high sugar in G. diazotrophicus, shedding light on the combination of osmotolerance and sugar-tolerance mechanisms. KEY POINTS: • G. diazotrophicus intensifies glycolysis to metabolize the excess of sugar. • G. diazotrophicus turns down the uptake of nutrients in response to high sugar. • G. diazotrophicus requires amino acid availability to resist high sugar.

This study aimed to evaluate the effects of cryoprotectant agents on Passiflora eichleriana, Passiflora crystallina and Passiflora nitida seeds undergoing cryopreservation. The experiment was condu...

O objetivo deste trabalho foi determinar a influência da procedência, tipo de estaca e da concentração de ácido indol-3-butírico (AIB) no enraizamento de estacas de mangabeira. As procedências testadas foram: Cáceres e Chapada dos Guimarães, Mato Grosso, das quais foram obtidas estacas herbáceas e lenhosas de árvores adultas. As estacas foram imersas em soluções de AIB nas concentrações de 0, 2.000, 4.000, 6.000, 8.000 e 10.000 mg.L-1 por 10 segundos e plantadas em bandejas de poliestireno expandido contendo areia lavada como substrato. O cultivo ocorreu em casa de vegetação por 60 dias. Análises anatômicas foram realizadas para investigação de possíveis impedimentos à iniciação radicial. O delineamento utilizado foi o inteiramente casualizado em arranjo fatorial 6 x 2 (seis concentrações de AIB e duas procedências), com 4 repetições de 10 estacas por unidade experimental. Não houve enraizamento das estacas de mangabeira, sendo constatada mortalidade total das mesmas. A dificuldade enraizamento da espécie não pode ser justificada pela estrutura anatômica do caule, o qual não possui qualquer impedimento à emissão de raízes.

Cadmium (Cd) is a heavy metal used as raw material for several fertilizers and pesticides. The increase of Cd concentration in soils has been observed in cultivated areas, affecting animals, plants, and microorganisms. Gluconacetobacter diazotrophicus is a plant growth-promoting bacterium able to survive under adverse environmental conditions. Here, we investigated key mechanisms involved with the resistance of G. diazotrophicus to Cd. Proteomic analyses revealed that the main pathways regulated in response to Cd are nutrient uptake, multidrug efflux pumps, response to oxidative stress, and protein quality control system. Extracytoplasmic proteins related to multidrug efflux pumps were up-accumulated, while several proteins related to nutrients uptake were down-accumulated. The relevance of these pathways for bacterial resistance to Cd was investigated by reverse genetic analysis using mutants defective for nutrient uptake (tdbr, ompW, and oprB), multidrug efflux (czcC), response to oxidative stress (ggt), and protein quality control system (clpX). Our data demonstrated the essential role of the tdbr and czcC genes for resistance to Cd in G. diazotrophicus. These results contribute to a better understanding of the resistance mechanisms to Cd in G. diazotrophicus, shedding light on responses associated with extracytoplasmic compartments.

The use of plant growth-promoting bacteria represents an alternative to the massive use of mineral fertilizers in agriculture. However, some abiotic stresses commonly found in the environment, like salinity, can affect the efficiency of this approach. Here, we investigated the key mechanisms involved in the response of the plant growth-promoting bacterium Gluconacetobacter diazotrophicus to salt stress by using morphological and cell viability analyses, comparative proteomics, and reverse genetics. Our results revealed that the bacteria produce filamentous cells in response to salt at 100 mM and 150 mM NaCl. However, such a response was not observed at higher concentrations, where cell viability was severely affected. Proteomic analysis showed that salt stress modulates proteins involved in several pathways, including iron uptake, outer membrane efflux, osmotic adjustment, cell division and elongation, and protein transport and quality control. Proteomic data also revealed the repression of several extracytoplasmic proteins, especially those located at periplasm and outer membrane. The role of such pathways in the tolerance to salt stress was analyzed by the use of mutant defectives for Δtbdr (iron uptake), ΔmtlK and ΔotsA (compatible solutes synthesis), and ΔdegP (quality control of nascent extracytoplasmic proteins). ΔdegP presented the highest sensitivity to salt stress, Δtbdr, andΔmtlK also showed increased sensitivity, but ΔotsA was not affected. This is the first demonstration that DegP protein, a protease with minor chaperone activity, is essential for tolerance to salt stress in G. diazotrophicus. Our data contribute to a better understanding of the molecular bases that control the bacterial response/tolerance to salt stress, shedding light on quality control of nascent extracytoplasmic proteins.

Plant growth-promoting bacteria are a promising alternative to improve agricultural sustainability. Gluconacetobacter diazotrophicus is an osmotolerant bacterium able to colonize several plant species, including sugarcane, coffee, and rice. Despite its biotechnological potential, the mechanisms controlling such osmotolerance remain unclear. The present study investigated the key mechanisms of resistance to osmotic stress in G. diazotrophicus. The molecular pathways regulated by the stress were investigated by comparative proteomics, and proteins essential for resistance were identified by knock-out mutagenesis. Proteomics analysis led to identify regulatory pathways for osmotic adjustment, de novo saturated fatty acids biosynthesis, and uptake of nutrients. The mutagenesis analysis showed that the lack of AccC protein, an essential component of de novo fatty acid biosynthesis, severely affected G. diazotrophicus resistance to osmotic stress. Additionally, knock-out mutants for nutrients uptake (Δtbdr and ΔoprB) and compatible solutes synthesis (ΔmtlK and ΔotsA) became more sensitive to osmotic stress. Together, our results identified specific genes and mechanisms regulated by osmotic stress in an osmotolerant bacterium, shedding light on the essential role of cell envelope and extracytoplasmic proteins for osmotolerance.

Background Plants interact with a variety of microorganisms during their life cycle, among which beneficial bacteria deserve special attention. Gluconacetobacter diazotrophicus is a beneficial bacterium able to fix nitrogen and promote plant growth. Despite its biotechnological potential, the mechanisms regulating the interaction between G. diazotrophicus and host plants remain unclear. Methods We analyzed the response of G. diazotrophicus to cocultivation with Arabidopsis thaliana seedlings. Bacterial growth in response to cocultivation and plant exudates was analyzed. Through comparative proteomic analysis, G. diazotrophicus proteins regulated during cocultivation were investigated. Finally, the role of some up-accumulated proteins in the response G. diazotrophicus to cocultivation was analyzed by reverse genetics, using insertion mutants. Results Our results revealed the induction of bacterial growth in response to cocultivation. Comparative proteomic analysis identified 450 bacterial proteins, with 39 up-accumulated, and 12 down-accumulated in response to cocultivation. Among the up-accumulated pathways, the metabolism of pentoses and protein synthesis were highlighted. Proteins potentially relevant to bacterial growth response such as ABC-F-Etta, ClpX, Zwf, MetE, AcnA, IlvC, and AccC were also increased. Reverse genetics analysis, using insertion mutants, revealed that the lack of ABC-F-Etta and AccC proteins severely affects G. diazotrophicus response to cocultivation. Our data demonstrated that specific mechanisms are activated in the bacterial response to plant exudates, indicating the essential role of “ribosomal activity” and “fatty acid biosynthesis” in such a process. This is the first study to demonstrate the participation of EttA and AccC proteins in plant-bacteria interactions, and open new perspectives for understanding the initial steps of such associations.

Brazil is one of the centers of diversity of the genus Passiflora L . and therefore has great potential for the utilization of the genetic resources in agronomy. Passiflora mucronata Lam. is a wild species that presents great characteristics for ornamental use. This work aimed to evaluate the effect of different concentrations of cryoprotectants dimethylsulfoxide (DMSO) and sucrose (0, 7, 14, 21 and 28%) on the cryopreservation of Passiflora mucronata seeds. After the application of the cryoprotectants, the seeds were stored in liquid nitrogen for 5 days, thawed, and later, seeded in vermiculite and maintained at a temperature of 27.7 ± 1.9°C. The cryoprotectant type and the application of increasing doses affect the physiological quality of seeds after storage in liquid nitrogen. Cryopreservation may be used for the long-term conservation of seeds, without the use of cryoprotectants.

Hancornia speciosa Gomes, popularly known as mangabeira, is a fruit tree belonging to the Apocynaceae family, native to the Brazilian Cerrado. The fruit is widely used by the local population as an alternative source of income. Limited information is available about this species, which increases the difficulty of conserving its genetic resources and exploiting mangabeira as an economic resource. The objective of this research was to evaluate the genetic diversity and genetic structure of H. speciosa from Chapada dos Guimarães. Twenty-four trees and ten inter simple sequence repeats (ISSR) primers were evaluated. Of the 57 bands obtained, 33 (57.89%) presented polymorphism. The analysis using Structure defined two different clusters (K = 2), which were consistent with the unweighted pair group method with arithmetic mean (UPGMA) clustering analysis. The number of observed alleles (Na = 1.58), number of effective alleles (Ne = 1.29), Nei’s genetic distance (He = 0.18) and Shannon index (I = 0.27) were considered low among the population. The analysis of molecular variance (AMOVA) revealed that the variability within the cluster (83.39%) was higher than among clusters (16.61%). ISSR primers proved effective for detection of genetic polymorphism in H. speciosa, and could be utilized for strategies that aim at conservation, plant breeding programs, and commercial use.