Humic acids are of great interest in many fields; however, they are inhibitors of fermentative processes applied to hydrothermally treated sewage sludge. Hence, the structure and composition of soluble and bound humic acidlike fractions from raw and hydrothermally treated sewage sludge were studied. Lipid, polysaccharide, protein and aromatic fractions were identified, as well as a high nitrogen content (7-10 %) and low solubility in alkaline media. Thus, they do not strictly meet the chemical definition of 'humic acids'. The soluble humic acid-like compounds had more aromatic and less protein content. Thermal hydrolysis of sewage sludge increased their aromaticity to the detriment of protein and polysaccharide fractions, while wet oxidation caused an increase in all structural fractions. Regarding the bound compounds, lipid, polysaccharide and aromatic fractions increased markedly during both treatments, although oxygen produced higher degradation of the protein fraction and, from 1 h, the partial degradation of aromatic compounds and an increase in the C/N atomic ratio (from 5.0 to 18.7 after 2 h). Therefore, hydrothermal treatments have a positive impact on the hydrolysate biodegradability due to the organic matter solubilisation, but also a negative impact linked to the higher solubilisation of the humic acid-like compounds and their structural changes.

Previous studies have shown that natural heteromolecularcomplexesmight be an alternative to synthetic chelates to correct iron (Fe)deficiency. To investigate the mechanism of action of these complexes,we have studied their interaction with Ca2+ at alkalinepH, Fe-binding stability, Fe-root uptake in cucumber, and chemicalstructure using molecular modeling. The results show that a heteromolecularFe complex including citric acid and lignosulfonate as binding ligands(Ls-Cit) forms a supramolecular system in solution with iron citrateinteracting with the hydrophobic inner core of the lignosulfonatesystem. These structural features are associated with high stabilityagainst Ca2+ at basic pH. Likewise, unlike Fe-EDDHA, rootFe uptake from Ls-Cit implies the activation of the main root responsesunder Fe deficiency at the transcriptional level but not at the post-transcriptionallevel. These results are consistent with the involvement of some plantresponses to Fe deficiency in the plant assimilation of complexedFe in Ls-Cit under field conditions.

Many studies have shown the capacity of soil humic substances (HS) to improve plant growth in natural ecosystems. This effect involves the activation of different processes within the plant at different coordinated molecular, biochemical, and physiological levels. However, the first event triggered by plant root-HS interaction remains unclear. Some studies suggest the hypothesis that the interaction of HS with root exudates involves relevant modification of the molecular conformation of humic self-assembled aggregates, including disaggregation, which might be directly involved in the activation of root responses. To investigate this hypothesis, we have prepared two humic acids. A natural humic acid (HA) and a transformed humic acid obtained from the treatment of HA with fungal laccase (HA enz). We have tested the capacity of the two humic acids to affect plant growth (cucumber and Arabidopsis) and complex Cu. Laccase-treatment did not change the molecular size but increased hydrophobicity, molecular compactness and stability, and rigidity of HA enz. Laccase-treatment avoided the ability of HA to promote shoot- and root-growth in cucumber and Arabidopsis. However, it does not modify Cu complexation features. There is no molecular disaggregation upon the interaction of HA and HA enz with plant roots. The results indicate that the interaction with plant roots induced in both HA and laccase-treated HA (HA enz), changes in their structural features that showed higher compactness and rigidity. These events might result from the interaction of HA and HA enz with specific root exudates that can promote intermolecular crosslinking. In summary, the results indicate that the weakly bond stabilized aggregated conformation (supramolecular-like) of HA plays a crucial role in its ability to promote root and shoot growth. The results also indicate the presence of two main types of HS in the rhizosphere corresponding to those non-interacting with plant roots (forming aggregated molecular assemblies) and those produced after interacting with plant root exudates (forming stable macromolecules).

The global decrease in soil fertility leads to a new agricultural scenario where eco-friendly solutions play an important role. The plant growth promotion through the use of microbes, especially endophytes and rhizosphere microbiota, has been proposed as a useful solution. Several studies have shown that humic substances are suitable vehicles for the inoculation of plant growth promoting bacteria, and that this combination has an enhanced effect on the stimulation of plant development. In this work, cucumber plants grown hydroponically have been pre-treated with a sedimentary humic acid (SHA) with known plant growth-enhancing effects, and culturable bacterial endophytes have been isolated from these plants. The hypothesis was that this pre-treatment with SHA could lead to the isolation of certain endophytic taxa whose proliferation within the plant could have been promoted as a result of the effects of the treatment with SHA, and that could eventually reinforce a potential synergistic effect of a combined application of those endophytic bacteria and SHA. The culturable endophytes that have been isolated from humic acid-treated cucumber plants have been identified as members of four main phyla:Proteobacteria,Firmicutes,Actinobacteria, andBacteroidetes. Isolates were characterized according to the following plant growth-promoting traits: nitrogen fixation/scavenging, phosphate solubilization, siderophore production and plant hormone production. Most of the isolates were able to fix/scavenge nitrogen and to produce plant hormones (indole-3-acetic acid and several cytokinins), whereas few isolates were able to solubilize phosphate and/or produce siderophores. The most promising endophyte isolates for its use in futures investigations as plant growth-promoting bacterial inocula werePseudomonassp. strains (that showed all traits),Sphingomonassp.,Stenotrophomonassp. strains, or someArthrobactersp. andMicrobacteriumsp. isolates.

Humic substances (HS, fulvic and humic acids) are widely used as fertilizers or plant growth stimulants, although their mechanism of action still remains partially unknown. Humic substances may be applied either directly to the soil or as foliar sprays. Despite both kind of application are commonly used in agricultural practices, most of the studies regarding the elicited response in plants induced by HS are based on the root-application of these substances. The present work aimed at discriminating between the mechanisms of action of foliar application versus root application of a sedimentary humic acid (SHA) on plant development. For this purpose, six markers related to plant phenotype, plant morphology, hormonal balance and root-plasma membrane H+-ATPase were selected. Both application strategies improved the shoot and root growth. Foliar applied- and root applied-SHA shared the capacity to increase the concentration of indole-3-acetic acid in roots and cytokinins in shoots. However, foliar application did not lead to short-term increases in either abscisic acid root-concentration or root-plasma membrane H+-ATPase activity which are, however, two crucial effects triggered by SHA root-application. Both application modes increased the root concentrations of jasmonic acid and jasmonoyl-isoleucine. These hormonal changes caused by foliar application could be a stress-related symptom and connected to the loss of leaves trichomes and the diminution of chloroplasts size seen by scanning electron microscopy. These results support the hypothesis that the beneficial effects of SHA applied to roots or leaves may result from plant adaptation to a mild transient stress caused by SHA application.

Humification is a process that plant and microbiota residues experiment in natural or agronomic soils under microorganisms action and environmental conditions. Under this process natural biomolecules - such as protein, carbohydrates or lignin - experience secondary biochemical and chemical reactions yielding to the formation of new organic biomolecules normally known as soil humus or humic substances (HS). In parallel, composting of fresh organic residues may be seen as an artificial process that involves many microorganism-induced secondary biochemical reactions that are probably also included in the first steps of natural humification in soils. In this context, we have applied multivariate statistical analysis to diverse and complementary analytical techniques (UV-Visible, synchronous fluorescence, FTIR, C-13-NMR and pyrolysis GS/MS) to follow the structural evolution of three groups of organic material: (i) fresh organic matter materials, (ii) compost of the fresh organic matter materials, and (iii) humic and fulvic acids including standards and references from the International Humic Substances Society. In order to discriminate among the three groups of organic materials, the set of data obtained from each analytical technique was analyzed using complementary statistical techniques: Correlations, Kolmogorov-Smirnov Test and Principal Component Analysis (PCA). The results showed positive correlations between UV-visible and fluorescence indexes and aromatic structures ...

Availability of fresh water for crop irrigation is becoming scarce and rather expensive. In this context, the research about the potential reutilization of non-conventional water sources becomes highly relevant, principally in arid and semi-arid areas. On many occasions, these new water resources involve water with a moderate concentration of salt, making it necessary to improve plant growth under moderate saline conditions. Besides plant breeding techniques, the use of molecules able to improve plant adaptation to saline conditions has great interest. Between these molecules, humic substances (HS) have proven to be efficient as stress-protectors under specific conditions of stress intensity and moment of application. The HS are main components of the soil organic matter and dissolved organic matter resulting from the biotic and abiotic transformation of fresh organic matter in natural ecosystems. Although knowledge about their structure is still under open debate, HS contain aromatic and aliphatic domains presenting O-, N- and S- containing functional groups with high biological and chemical activities. The aim of this presentation is to summarize the main effects of humic acids (HA) applied either on the root or on the shoot, on the metabolism and hormonal balance of plants cultivated under normal and stressing conditions, from studies carried out for our group and collaborations during the last years.

Although the ability of humic (HA) and fulvic acids (FA) to improve plant growth has been demonstrated, knowledge about the mechanisms responsible for the direct effects of HA and FA on the promotion of plant growth is scarce and fragmentary. Our study investigated the causal role of both root PM H+-ATPase activity and ABA in the SHA-promoting action on both root and shoot growth. The involvement of these processes in the regulation of shoot cytokinin concentration and activity was also studied. Our aim was to integrate such plant responses for providing new insights to the current model on the mode of action of HA for promoting root and shoot growth. Experiments employing specific inhibitors and using Cucumis sativus L. plants show that both the root PM H+-ATPase activity and root ABA play a crucial role in the root growth-promoting action of SHA. With regard to the HA-promoting effects on shoot growth, two pathways of events triggered by the interaction of SHA with plant roots are essential for the increase in root PM H+-ATPase activity-which also mediates an increase in cytokinin concentration and action in the shoot-and the ABA-mediated increase in hydraulic conductivity (Lp(r)).

Iron deficiency in plants is caused by a low availability of iron in the soil, and its main visual symptom is leaf yellowing due to a decrease in chlorophyll content, along with a reduction in plant growth and fruit quality. Foliar sprays with Fe compounds are an economic alternative to the treatment with expensive synthetic Fe-chelates applied to the soil, although the efficacy of foliar treatments is rather limited. Generally, plant response to Fe-foliar treatments is monitored by measuring chlorophyll content (or related parameters as SPAD index). However, different studies have shown that foliar Fe sprays cause a local regreening and that translocation of the applied Fe within the plant is quite low. In this context, the aim of this study was to assess the effects of foliar applications of different Fe compounds [FeSO4, Fe(III)-EDTA, and Fe(III)-heptagluconate] on Fe-deficient cucumber plants, by studying the main physiological plant root responses to Fe deficiency [root Fe(III) chelate reductase (FCR) activity; acidification of the nutrient solution; and expression of the Fe deficiency responsive genes encoding FCR, CsFRO1, Fe(II) root transporter CsIRT1, and two plasma membrane H+-ATPases, CsHA1 and CsHA2], along with SPAD index, plant growth and Fe content. The results showed that the overall assessment of Fe-deficiency root responses improved the evaluation of the efficacy of the Fe-foliar treatments compared to just monitoring SPAD indexes. Thus, FCR activity and expression of Fe-deficiency response genes, especially CsFRO1 and CsHA1, preceded the trend of SPAD index and acted as indicators of whether the plant was sensing or not metabolically active Fe due to the treatments. Principal component analysis of the data also provided a graphical tool to evaluate the evolution of plant responses to foliar Fe treatments with time.

The ability of rhizospheric humic substances to improve plant growth has been well established by many studies carried out using diverse plant species cultivated under many different conditions. These beneficial effects of humic substances on plant development are expressed in both root and shoot. However, the mechanisms responsible for this action of humic substances are only partially known and poorly integrated. In fact, although the studies focused on plant root development are numerous, those dealing with plant shoot development are scarce. Likewise, studies integrating humic effects on root and shoot are also few. In this context, the main goal of this work is to summarize some of the results regarding the effects of humic substances on plant development within a hypothetical holistic framework that will allow us to interconnect these findings and disclose some features of the functional crosstalk between the effects on soil, root and shoot. Furthermore, the significance of all these mechanisms in plants growing in the field is also discussed.

The role of soil humus in soil fertility and crop production has been well established by many studies and practical farming experience. This role is related to the presence of a family of organic substances, known as humic substances, with the capacity to increase the pool of plant-available nutrients in soil for root uptake. The improvement in plant growth and mineral nutrition is directly linked to the physicochemical features of humic substances, and more specifically, to their capacity to form stable chemical complexes with metals. This chapter reviews the main mechanisms behind the action of humic substances in improving plant mineral nutrition. It also discusses the main signalling pathways that might be involved in the regulation of these humic substances.

A number of studies have demonstrated the key role of nitric oxide in the regulation of many fundamental physiological processes that includes plant responses to abiotic and biotic stresses. On the other hand, beneficial action of humic substances on plant growth has been well corroborated, particularly when plants are subjected to abiotic stresses. Furthermore, several recent works have reported the functional links between the plant growth promoting action of humic substances and nitric oxide production and function in plants. In this article, we try to briefly review and discuss the main results showing the relationships between nitric oxide function and humic substances action on plants, also stressing the nitric oxide-dependent involvement of other plant growth regulators, such as auxin, ethylene, abscisic acid, and cytokinins.

The main aim of this communication is to discuss the current knowledge about the potential direct mechanisms that are involved in the beneficial action of humic substances on plant development. To this end, we present and discuss here recent results obtained in our laboratory, along with other findings published by other authors. Finally, we propose a hypothetical whole mechanism for explaining the action of humic substances on plant development. In this schema, we point out those steps that remain unclear.