Arnoldo Rocha Façanha

Humic Acids Isolated from Earthworm Compost Enhance Root Elongation, Lateral Root Emergence, and Plasma Membrane H+-ATPase Activity in Maize Roots

Earthworms (Eisenia foetida) produce humic substances that can influence plant growth by mechanisms that are not yet clear. In this work, we investigated the effects of humic acids (HAs) isolated from cattle manure earthworm compost on the earliest stages of lateral root development and on the plasma membrane H+-ATPase activity. These HAs enhance the root growth of maize (Zea mays) seedlings in conjunction with a marked proliferation of sites of lateral root emergence. They also stimulate the plasma membrane H+-ATPase activity, apparently associated with an ability to promote expression of this enzyme. In addition, structural analysis reveals the presence of exchangeable auxin groups in the macrostructure of the earthworm compost HA. These results may shed light on the hormonal activity that has been postulated for these humic substances.

Chemical composition and bioactivity properties of size-fractions separated from a vermicompost humic acid.

Preparative high performance size-exclusion chromatography (HPSEC) was applied to humic acids (HA) extracted from vermicompost in order to separate humic matter of different molecular dimension and evaluate the relationship between chemical properties of size-fractions (SF) and their effects on plant root growth. Molecular dimensions of components in humic SF was further achieved by diffusion-ordered nuclear magnetic resonance spectroscopy (DOSY-NMR) based on diffusion coefficients (D), while carbon distribution was evaluated by solid state (CP/MAS) (13)C NMR. Seedlings of maize and Arabidopsis were treated with different concentrations of SF to evaluate root growth. Six different SF were obtained and their carbohydrate-like content and alkyl chain length decreased with decreasing molecular size. Progressive reduction of aromatic carbon was also observed with decreasing molecular size of separated fractions. Diffusion-ordered spectroscopy (DOSY) spectra showed that SF were composed of complex mixtures of aliphatic, aromatic and carbohydrates constituents that could be separated on the basis of their diffusion. All SF promoted root growth in Arabidopsis and maize seedlings but the effects differed according to molecular size and plant species. In Arabidopsis seedlings, the bulk HA and its SF revealed a classical large auxin-like exogenous response, i.e.: shortened the principal root axis and induced lateral roots, while the effects in maize corresponded to low auxin-like levels, as suggested by enhanced principal axis length and induction of lateral roots. The reduction of humic heterogeneity obtained in HPSEC separated size-fractions suggested that their physiological influence on root growth and architecture was less an effect of their size than their content of specific bioactive molecules. However, these molecules may be dynamically released from humic superstructures and exert their bioactivity when weaker is the humic conformational stability as that obtained in the separated size-fractions.

Bioactivity of chemically transformed humic matter from vermicompost on plant root growth.

Chemical reactions (hydrolysis, oxidation, reduction, methylation, alkyl compounds detachment) were applied to modify the structure of humic substances (HS) isolated from vermicompost. Structural and conformational changes of these humic derivatives were assessed by elemental analyses, size exclusion chromatography (HPSEC), solid-state nuclear magnetic resonance ((13)C CPMAS-NMR), and diffusion ordered spectroscopy (DOSY-NMR), whereas their bioactivity was evaluated by changes in root architecture and proton pump activation of tomato and maize. All humic derivatives exhibited a large bioactivity compared to original HS, both KMnO(4)-oxidized and methylated materials being the most effective. Whereas no general relationship was found between bioactivity and humic molecular sizes, the hydrophobicity index was significantly related with proton pump stimulation. It is suggested that the hydrophobic domain can preserve bioactive molecules such as auxins in the humic matter. In contact with root-exuded organic acids the hydrophobic weak forces could be disrupted, releasing bioactive compounds from humic aggregates. These findings were further supported by the fact that HS and all derivatives used in this study activated the auxin synthetic reporter DR5::GUS.

Proton (H+) flux signature for the presymbiotic development of the arbuscular mycorrhizal fungi

Ion dynamics are important for cell nutrition and growth in fungi and plants. Here, the focus is on the relationship between the hyphal H(+) fluxes and the control of presymbiotic growth and host recognition by arbuscular mycorrhizal (AM) fungi. Fluxes of H(+) around azygopores and along lateral hyphae of Gigaspora margarita during presymbiotic growth, and their regulation by phosphate (P) and sucrose (Suc), were analyzed with an H(+)-specific vibrating probe. Changes in hyphal H(+) fluxes were followed after induction by root exudates (RE) or by the presence Trifolium repens roots. Differential sensitivity to P-type ATPase inhibitors (orthovanadate or erythrosin B) suggests an asymmetric distribution or activation of H(+)-pump isoforms along the hyphae of the AM fungi. Concentration of P and Suc affected the hyphal H(+) fluxes and growth rate. However, further increases in H+ efflux and growth rate were observed when the fungus was growing close to clover roots or pretreated with RE. The H(+) flux data correlate with those from polarized hyphal growth analyses, suggesting that spatial and temporal alterations of the hyphal H(+)fluxes are regulated by nutrient availability and might underlie a pH signaling elicitation by host RE during the early events of the AM symbiosis.

Enhanced Proton Translocating Pyrophosphatase Activity Improves Nitrogen Use Efficiency in Romaine Lettuce

A simple genetic manipulation triggers enhanced nitrogen use efficiency in lettuce. Plant nitrate (NO3−) acquisition depends on the combined activities of root high- and low-affinity NO3− transporters and the proton gradient generated by the plasma membrane H+-ATPase. These processes are coordinated with photosynthesis and the carbon status of the plant. Here, we present the characterization of romaine lettuce (Lactuca sativa ‘Conquistador’) plants engineered to overexpress an intragenic gain-of-function allele of the type I proton translocating pyrophosphatase (H+-PPase) of Arabidopsis (Arabidopsis thaliana). The proton-pumping and inorganic pyrophosphate hydrolytic activities of these plants are augmented compared with control plants. Immunohistochemical data show a conspicuous increase in H+-PPase protein abundance at the vasculature of the transgenic plants. Transgenic plants displayed an enhanced rhizosphere acidification capacity consistent with the augmented plasma membrane H+-ATPase proton transport values, and ATP hydrolytic capacities evaluated in vitro. These transgenic lines outperform control plants when challenged with NO3− limitations in laboratory, greenhouse, and field scenarios. Furthermore, we report the characterization of a lettuce LsNRT2.1 gene that is constitutive up-regulated in the transgenic plants. Of note, the expression of the LsNRT2.1 gene in control plants is regulated by NO3− and sugars. Enhanced accumulation of 15N-labeled fertilizer by transgenic lettuce compared with control plants was observed in greenhouse experiments. A negative correlation between the level of root soluble sugars and biomass is consistent with the strong root growth that characterizes these transgenic plants.

Chemical nature of soil humified fractions and their bioactivity

The aim of this work was to evaluate the humus composition from an Ultisol from Campos dos Goytacazes, RJ, Brazil. Soil samples of four depths (0-0.05, 0.05-0.10, 0.10-0.20 and 0.20-0.40 m) and its chemical nature were analysed by elemental composition, E4/E6 ratios and Fourier transformed infrared spectroscopy. The bioactivity of these humified substances was evaluated through their action on maize root growth and H + -ATPase activity of roots microsomes. In topsoil, the content of high condensed alkaline soluble humic substances is greater than that found in the subsuperficial layers. The chemical nature of humic and fulvic acids also varied with the soil depth. The humic acids isolated from the soil samples exhibited higher bioactivity compared with the fulvic acids. Moreover, the results suggest that more condensed humic substances can promote highest stimulation of the microsomal H + -ATPases from maize roots. These data reinforce the concept that the activity of the H + pumps can be used as a biochemical marker for evaluation of humic substances

Inhibition of Phosphate Uptake in Corn Roots by Aluminum-Fluoride Complexes

F forms stable complexes with Al at conditions found in the soil. Fluoroaluminate complexes (AlFx) have been widely described as effective analogs of inorganic phosphate (Pi) in Pi-binding sites of several proteins. In this work, we explored the possibility that the phytotoxicity of AlFx reflects their activity as Pi analogs. For this purpose, 32P-labeled phosphate uptake by excised roots and plasma membrane H+-ATPase activity were investigated in an Al-tolerant variety of maize (Zea mays L. var. dwarf hybrid), either treated or not with AlFx. In vitro, AlFx competitively inhibited the rate of root phosphate uptake as well as the H+-ATPase activity. Conversely, pretreatment of seedlings with AlFxin vivo promoted no effect on the H+-ATPase activity, whereas a biphasic effect on Pi uptake by roots was observed. Although the initial rate of phosphate uptake by roots was inhibited by AlFx pretreatment, this situation changed over the following minutes as the rate of uptake increased and a pronounced stimulation in subsequent 32Pi uptake was observed. This kinetic behavior suggests a reversible and competitive inhibition of the phosphate transporter by fluoroaluminates. The stimulation of root32Pi uptake induced by AlFx pretreatment was tentatively interpreted as a phosphate starvation response. This report places AlF3 and AlF4 − among Al-phytotoxic species and suggests a mechanism of action where the accumulation of Pi-mimicking fluoroaluminates in the soil may affect the phosphate absorption by plants. The biochemical, physiological, and environmental significance of these findings is discussed.

Arabidopsis sodium dependent and independent phenotypes triggered by H+-PPase up-regulation are SOS1 dependent

Coordinate regulation of transporters at both the plasma membrane and vacuole contribute to plant cells ability to adapt to a changing environment and play a key role in the maintenance of the chemiosmotic circuits required for cellular growth. The plasma membrane (PM) Na⁺/H⁺ antiporter (SOS1) is involved in salt tolerance, presumably in sodium extrusion; the vacuolar type I H⁺-PPase AVP1 is involved in vacuolar sodium sequestration, but its overexpression has also been shown to alter the abundance and activity of the PM H⁺-ATPase. Here we investigate the relationship between these transporters utilizing loss-of-function mutants of SOS1 (sos1) and increased expression of AVP1 (AVP1OX). Heightened expression of AVP1 enhances pyrophosphate-dependent proton pump activity, salt tolerance, ion vacuolar sequestration, K⁺ uptake capacity, root hair development, osmotic responses, and PM ATPase hydrolytic and proton pumping activities. In sos1 lines overexpressing AVP1, these phenotypes are negatively affected demonstrating that sos1 is epistatic to AVP1. Enhanced AVP1 protein levels require SOS1 and this regulation appears to be post-translational.

P5A-Type ATPase Cta4p Is Essential for Ca2+ Transport in the Endoplasmic Reticulum of Schizosaccharomyces pombe

This study establishes the role of P5A-type Cta4 ATPase in Ca2+ sequestration in the endoplasmic reticulum by detecting an ATP-dependent, vanadate-sensitive and FCCP insensitive 45Ca2+-transport in fission yeast membranes isolated by cellular fractionation. Specifically, the Ca2+-ATPase transport activity was decreased in ER membranes isolated from cells lacking a cta4+ gene. Furthermore, a disruption of cta4+ resulted in 6-fold increase of intracellular Ca2+ levels, sensitivity towards accumulation of misfolded proteins in ER and ER stress, stimulation of the calcineurin phosphatase activity and vacuolar Ca2+ pumping. These data provide compelling biochemical evidence for a P5A-type Cta4 ATPase as an essential component of Ca2+ transport system and signaling network which regulate, in conjunction with calcineurin, the ER functionality in fission yeast.

An outlook on ion signaling and ionome of mycorrhizal symbiosis

The 450-million-year-old interaction between the majority of land plants and mycorrhizal fungi is one of the most ancient, abundant, and ecologically important symbiosis on earth. The early events in the evolution of mycorrhizal symbioses seem to have involved reciprocal genetic changes in ancestral plants and free-living fungi. new data on the mechanism of action of specific signaling molecules and how it influence and is influenced by the membrane ions fluxes and cytoplasm ion oscillations which integrate the symbiotic ionome are improving our understanding of the molecular bases of the mycorrhization process. This mini-review will highlight topics regarding what is known about the ionome and ionic communication in the arbuscular mycorrhizal symbiosis focusing on the signals involved in the development of symbioses. Here we present an overview integrating the available data with the prospects of the research in the field.