Influence of plant growth-promoting bacteria on leaf carbon and nitrogen metabolism of two drought-stressed neotropical tree species: a metabolomic approach.

Deforestation of Atlantic Forest has caused prolonged drought events in the last decades. The need for reforestation is growing, and the development of native seedlings that are more tolerant to drought stress is necessary. A biotechnological tool that improves plant tolerance is the use of plant growth-promoting bacteria (PGPB) as inoculants. Two species of PGPB were inoculated in drought-stressed seedlings of two neotropical tree species that have been used in environmental restoration programs: Cecropia pachystachya and Cariniana estrellensis. Biometrical, physiological, and metabolomic parameters from carbon and nitrogen pathways were evaluated. We found that the PGPB positively influenced photosynthesis and growth parameters in both trees under drought. The enzymes activities, the tricarboxylic acid cycle intermediates, the amino acids, and protein contents were also influenced by the PGPB treatments. The results allowed us to find the specific composition of secondary metabolites of each plant species. This study provides evidence that there is not a single mechanism involved in drought tolerance and that the inoculation with PGPB promotes a broad-spectrum tolerance response in Neotropical trees. The inoculation with PGPB appears as an important strategy to improve drought tolerance in Atlantic Forest native trees and enhance environmental restoration programs' success. MAIN CONCLUSION: The association with plant growth-promoting bacteria improved the tolerance to drought in Neotropical trees through biochemical, physiological, and biometrical parameters. This can enhance the success of forest restoration programs.

Integrated Analysis of the Transcriptome and Metabolome of Cecropia obtusifolia: A Plant with High Chlorogenic Acid Content Traditionally Used to Treat Diabetes Mellitus.

This investigation cultured Cecropia obtusifolia cells in suspension to evaluate the effect of nitrate deficiency on the growth and production of chlorogenic acid (CGA), a secondary metabolite with hypoglycemic and hypolipidemic activity that acts directly on type 2 diabetes mellitus. Using cell cultures in suspension, a kinetics time course was established with six time points and four total nitrate concentrations. The metabolites of interest were quantified by high-performance liquid chromatography (HPLC), and the metabolome was analyzed using directed and nondirected approaches. Finally, using RNA-seq methodology, the first transcript collection for C. obtusifolia was generated. HPLC analysis detected CGA at all sampling points, while metabolomic analysis confirmed the identity of CGA and of precursors involved in its biosynthesis. Transcriptome analysis identified differentially expressed genes and enzymes involved in the biosynthetic pathway of CGA. C. obtusifolia probably expresses a key enzyme with bifunctional activity, the hydroxycinnamoyl-CoA quinate hydroxycinnamoyl transferase and hydroxycinnamoyl-CoA shikimate/quinate hydroxycinnamoyl transferase (HQT/HCT), which recognizes shikimic acid or quinic acid as a substrate and incorporates either into one of the two routes responsible for CGA biosynthesis.

Plant growth-promoting bacteria improve leaf antioxidant metabolism of drought-stressed Neotropical trees.

MAIN CONCLUSION: Plant growth-promoting bacteria association improved the enzymatic and non-enzymatic antioxidant pathways in Neotropical trees under drought, which led to lower oxidative damage and enhanced drought tolerance in these trees. Water deficit is associated with oxidative stress in plant cells and may, thus, negatively affect the establishment of tree seedlings in reforestation areas. The association with plant growth-promoting bacteria (PGPB) is known to enhance the antioxidant response of crops, but this strategy has not been tested in seedlings of Neotropical trees. We evaluated the effects of inoculation with two PGPB (Azospirillum brasilense and Bacillus sp.) on the antioxidant metabolism of Cecropia pachystachya and Cariniana estrellensis seedlings submitted to drought. We measured the activity of antioxidant enzymes and the content of non-enzymatic antioxidants in leaves, and biometrical parameters of the seedlings. In both tree species, drought decreased the activity of antioxidant enzymes and the content of non-enzymatic antioxidant compounds. For C. pachystachya, the enzymatic and non-enzymatic pathways were mostly influenced by A. brasilense inoculation, which enhanced ascorbate peroxidase (APX) and superoxide dismutase activities and positively affected the level of non-enzymatic antioxidant compounds. In C. estrellensis, A. brasilense inoculation enhanced APX activity. However, A. brasilense and Bacillus sp. inoculation had more influence on the non-enzymatic pathway, as both bacteria induced a greater accumulation of secondary compounds (such as chlorogenic acid, gallic acid, rutin and synapic acid) compared to that in non-inoculated plants under drought. For both species, PGPB improved biometrical parameters related to drought tolerance, as specific leaf area and leaf-area ratio. Our results demonstrate that PGPB induced antioxidant mechanisms in drought-stressed Neotropical trees, increasing drought tolerance. Thus, PGPB inoculation provides a biotechnological alternative to improve the success of reforestation programmes.

Ultrasound-assisted extraction optimization and validation of an HPLC-DAD method for the quantification of polyphenols in leaf extracts of Cecropia species.

Cecropia species are traditionally used in Latin American folk medicine and are available as food supplements with little information warranting their quality. The optimum conditions for the extraction of chlorogenic acid (CA), total flavonoids (TF) and flavonolignans (FL) from leaves of Cecropia species were determined using a fractional factorial design (FFD) and a central composite design (CCD). A reversed-phase high-performance liquid chromatographic method coupled to a diode array detector (HPLC-DAD) was validated for the quantification of CA, TF and FL, following the ICH guidelines. Quantitative and Principal Component Analysis (PCA) was also performed. The extraction-optimization methodology enabled us developing an appropriate extraction process with a time-efficient execution of experiments. The experimental values agreed with those predicted, thus indicating suitability of the proposed model. The validation parameters for all chemical markers of the quantification method were satisfactory. The results revealed that the method had excellent selectivity, linearity, precision (repeatability and intermediate precision were below than 2 and 5%, respectively) and accuracy (98-102%). The limits of detection and quantification were at nanogram per milliliter (ng/mL) level. In conclusion, the simultaneous quantification of chemical markers using the proposed method is an appropriate approach for species discrimination and quality evaluation of Cecropia sp.

Fluctuating Asymmetry as a Bioindicator of Environmental Stress Caused by Pollution in a Pioneer Plant Species.

Fluctuating asymmetry (FA) is a widely used tool to detect developmental instability and plants under stressful conditions are expected to exhibit increased values of asymmetry, as well as higher levels of herbivory. This study evaluated whether dust from roads can cause major deviations in the axis of symmetry of leaves of the pioneer plant species Cecropia pachystachya Trécul (Urticaceae). It was also investigated whether plants exposed to dust have greater nitrogen content and higher levels of herbivory levels. Ten leaves of 20 individuals of C. pachystachya were collected on two roads with different levels of dust deposition and a control area. FA was calculated as the size-scaled difference between the right (RW) and left (LW) leaf widths and leaf area removed was determined by the ratio between leaf area removed and total leaf area. C. pachystachya plants in areas under strong influence of dust pollution exhibited the highest FA values (0.279 cm), whereas plants in the control area exhibited the lowest. A positive relationship between levels of leaf area removed by insects and a gradient of dust pollution was also observed. Differences in foliar nitrogen concentration among sampling areas indicated differences in leaf quality and influenced herbivory levels of Cecropia. This study indicated that FA can be used as an indicator of developmental instability of plants and those individuals under the impact of road dust and pollution might be more susceptible to insect attack.

Distribution of the herbicide atrazine in a microcosm with riparian forest plants.

Pesticides applied on sugarcane reach the subsoil of riparian forests and probably contaminate the river water. This work was conducted to learn about the phytoremediation of atrazine and subsoil contamination using the common riparian forest species of Cecropia hololeuca Miq. and Trema micranta (L.) Blum. These plants were grown in soil microcosms where (14)C-atrazine at 1/10 of the field-recommended dose was applied at the bottom of the microcosm simulating the movement from contaminated ground water to the upper soil layers and into plants. Residues of (14)C-atrazine were detected in all parts of the microcosm including soil, rhizosphere and the roots in different layers of the microcosm, stem and leaves. Atrazine mineralization was higher (10.2%) in the microcosms with plants than the control microcosms without plants (1.2%). The upward movement of this pesticide from deeper to more superficial soil layers occurred in all the microcosms with plants, powered by evapotranspiration process. From the atrazine applied in this study about 45% was taken up by C. hololeuca and 35% by T. micrantha. The highest amount of radioactivity (%) was found in the fine roots and the specific radioactivity (% g(-1)) showed that thick, fine roots and leaves bioaccumulate atrazine. The enhanced mineralization of atrazine as well the phytostabilization effect of the tree biomass will reduce the bioavailability of these residues and consequently decrease the hazardous effects on the environment.

Experimental design as a tool to evaluate chlorogenic and caffeic acids extracted from Cecropia glaziovii Sneth.

The effects of different parameters, including ethanol concentration, time of drug:solvent contact, temperature and the presence of a preservative, on chlorogenic acid (CGA) and caffeic acid (CFA) yields in Cecropia glaziovii Sneth extracts were investigated using an experimental design. In order to quantify the phenolic acids in these extracts a high-performance liquid chromatography-diode array detection (HPLC-DAD) method was developed and validated. Extracts with 80% ethanol presented a higher CGA content, but low amounts of CFA. Extracts with 20% ethanol showed a higher CFA concentration, but a sharp reduction in CGA extraction yield. The presence of a preservative, under the same extraction conditions, resulted in a slight difference or no difference in the CGA and CFA extraction yields. When the temperature was controlled at refrigerator or room temperature, a slight alteration in the concentrations of the phenolics studied was observed. The present approach can be applied in order to determine the optimum conditions for the preparation of C. glaziovii Sneth extracts based on CGA or CFA extraction yield as a chemical marker.