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).

Some studies have reported that the capacity of humic substances to improve plant growth is dependent on their ability to increase root hydraulic conductivity. It was proposed that this effect is directly related to the structural conformation in solution of these substances. To study this hypothesis, the effects on root hydraulic conductivity and growth of cucumber plants of a sedimentary humic acid and two polymers-polyacrylic acid and polyethylene glycol-presenting a molecular conformation in water solution different from that of the humic acid have been studied. The results show that whereas the humic acid caused an increase in root hydraulic conductivity and plant growth, both the polyacrylic acid and the polyethylene glycol did not modify plant growth and caused a decrease in root hydraulic conductivity. These results can be explained by the different molecular conformation in water solution of the three molecular systems. The relationships between these biological effects and the molecular conformation of the three molecular systems in water solution are discussed.

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.

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)).

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 physiological and metabolic mechanisms behind the humic acid-mediated plant growth enhancement are discussed in detail. Experiments using cucumber (Cucumis sativus) plants show that the shoot growth enhancement caused by a structurally well-characterized humic acid with sedimentary origin is functionally associated with significant increases in abscisic acid (ABA) root concentration and root hydraulic conductivity. Complementary experiments involving a blocking agent of cell wall pores and water root transport (polyethylenglycol) show that increases in root hydraulic conductivity are essential in the shoot growth-promoting action of the model humic acid. Further experiments involving an inhibitor of ABA biosynthesis in root and shoot (fluridone) show that the humic acid-mediated enhancement of both root hydraulic conductivity and shoot growth depended on ABA signaling pathways. These experiments also show that a significant increase in the gene expression of the main root plasma membrane aquaporins is associated with the increase of root hydraulic conductivity caused by the model humic acid. Finally, experimental data suggest that all of these actions of model humic acid on root functionality, which are linked to its beneficial action on plant shoot growth, are likely related to the conformational structure of humic acid in solution and its interaction with the cell wall at the root surface.

The main objective of this study is to investigate the more relevant binding sites (functional groups) involved in the complexation of Fe(III) and Cu(II) by humic-based structures. To this aim we have generated a set of seven humic-based samples (MHA) by the application of a hemi-synthetic process consisting in the oxidative polymerization of different combinations of three sources of natural organic molecules: two humic acids obtained from peat and leonardite, and natural tannin extracted from the quebracho tree (Schinopsis sp.) bark. These MHA were extensively characterized by using complementary analytical techniques (13C NMR, size-exclusion chromatography, electron paramagnetic resonance, and total phenol-reductant content), and their ability to complex Fe(III) and Cu(II) was also calculated by fluorescence quenching method. The data were studied by using general correlation matrix and principal component analysis. The results obtained, taken together, indicated that in a set of humic-based structures presenting carboxylic, phenol and O-alkyl groups, and diverse aliphatic/aromatic character, the complexation of Fe(III) mainly involved specific structural arrangements including carboxylic groups distributed in aliphatic domains. However, Cu(II) complexation involved singular structural arrangements including phenols (and probably substituted phenols) and O-alkyl groups in side chains of aromatic domains.

A number of studies have shown the ability of natural organic matter (NOM) in general and humic substances (HS) in particular, to affect the development of plants and microorganisms in many different natural ecosystems and agroecosystems. Regarding plants, these NOM and HS effects were expressed in both root growth and architecture, and shoot growth. However, these effects were different in intensity and quality depending on several intrinsic and extrinsic factors associated with HS structure and concentration, plant species and soil properties. Two main mechanisms have been proposed to explain the beneficial action of NOM and HS on plant growth. An indirect effect expressed through the improvement of plant nutrition by increasing soil nutrient availability, principally some micronutrients (mostly P and Fe); and a possible direct action affecting the transcriptional and post-transcriptional regulation of several enzymes and molecular transporters in the root. These biological effects within the plant seem to be associated with both nutrient root uptake ability and the efficient use of the nutrient in plant leaves. In this communication, the relationships between the effects of HS on root development, shoot development, plant nutrition, and soil properties; are discussed. This study is developed in the context of the links existing between the signal role of some nutrients and the hormonal balance in both root and shoot.

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.