The molecular conformation, but not disaggregation, of humic acid in water solution plays a crucial role in promoting plant development in the natural environment.

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).

Thermodynamic and spectroscopic data suggesting hypothetical humic fractions evolution on carbon cycle in soils.

Soil organic matter is considered by soil scientists as the interlayer that connect alive with mineral sides of the soil. In addition, microorganisms have in soil organic matter a source of carbon as well as a source of energy. We can observe a duality that can be analyzed from a biological, physicochemical, or even thermodynamic sense. From this last point of view carbon cycle follows its evolution on burial soil, and under certain temperature and pression conditions, up to fossil fuels or coals through kerogen being humic substances the ending point of biologically linked structures. When biological aspects are minimized, physicochemical aspects are maximized and carbonaceous structures are a source of energy but resilient to microorganism actions. Under these premises, we have isolated, purified, and analyzed different humic fractions. Heat of combustion of these humic fractions here analyzed reflects this situation and fitted the list of evolution stage of carbonaceous materials that step by step accumulates energy. Theoretical value of this parameter calculated from studied humic fractions, and by combination of its biochemical macromolecules yielded an exaggerated value in comparison to the real and measured value indicating a complexity of these humic structures, more than simpler molecules. Heat of combustion and excitation-emission matrices by fluorescence spectroscopy of isolated and purified grey and brown humic materials revealed different values for each fraction. Grey fractions showed a higher heat of combustion values and shorter λexc/λem, whereas brown fractions showed a lower heat of combustion and a larger λexc/λem. These data together with previous chemical analysis indicated a deep structural differentiation that can be observed by the Pyrolysis MS-GC data of the studied samples. Authors hypothesized that this incipient distinction between aliphatic and aromatic cores could evolve independently up to fossil fuel on one hand and coals on the other hand but separately.

Humic Acid Alleviates Fe Chlorosis in Graminaceous Plants Through Coordinated Fe-Dependent and Fe-Independent Mechanisms.

Many studies have shown the close relationship between the beneficial action of soil and sedimentary humic acids on the growth of plants cultivated in calcareous soils and their ability to improve Fe plant nutrition. These results have been ascribed to the humic acid (HA) capability to improve Fe solubility and bioavailability. However, other effects more related to a humic acid action on the specific mechanisms activated in roots of plants under Fe deficiency cannot be ruled out. Although this question has been studied in dicotyledonous plants, in graminaceous plants there are no specific studies. Here we investigate the ability of a humic acid extracted from peat (HA) to improve Fe nutrition in wheat plants cultivated under Fe deficient and sufficient conditions. The results show that HA can improve the physiological status of Fe deficient wheat plants by alleviating some of the deleterious consequences of Fe deficiency on plant development and increasing the plant ability to secrete phytosiderophores to the nutrient solution. This action of HA is associated with increases in the Fe-active pool in leaves that might be related to the mobilization of the Fe complexed by HA resulting from the interaction of HA with the phytosiderophores in the nutrient solution. The Fe translocation from the root to the shoot may be favored by the action of trans-Zeatin Riboside (tZR) since the leaf concentration of this phytohormone was enhanced by HA in Fe deficient plants.

The Singular Molecular Conformation of Humic Acids in Solution Influences Their Ability to Enhance Root Hydraulic Conductivity and Plant Growth.

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.

Maturation in composting process, an incipient humification-like step as multivariate statistical analysis of spectroscopic data shows.

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, 13C- 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 determined by 13C- NMR and pyrolysis GS/MS. However, these indexes were negatively correlated with polysaccharides and amides determined by FTIR, and lipids determined by pyrolysis GS/MS. The Kolmogorov-Smirnov Test showed that E4/E6, ε600, EEt/EBz, ε280 from UV/Visible; A440 from synchronous fluorescence; 1040/1400 and 1515/1715 by FTIR and, LIP from pyrolysis GS/MS were able to discriminate the samples in two different groups. The group formed by the transformed organic substances (humic, fulvic and composted materials) on the one hand, and the raw (fresh) organic materials on the other. These results, considered along with those obtained from the PCA analysis of spectroscopic data, indicated that composting could share secondary reactions and processes with the first steps of natural humification occurring in soil. Likewise, the results show that the organic molecules present in humic and composted materials are chemically different from the biomolecules present in fresh, no-transformed- materials.

Alternative Polyadenylation and Salicylic Acid Modulate Root Responses to Low Nitrogen Availability.

Nitrogen (N) is probably the most important macronutrient and its scarcity limits plant growth, development and fitness. N starvation response has been largely studied by transcriptomic analyses, but little is known about the role of alternative polyadenylation (APA) in such response. In this work, we show that N starvation modifies poly(A) usage in a large number of transcripts, some of them mediated by FIP1, a component of the polyadenylation machinery. Interestingly, the number of mRNAs isoforms with poly(A) tags located in protein-coding regions or 5'-UTRs significantly increases in response to N starvation. The set of genes affected by APA in response to N deficiency is enriched in N-metabolism, oxidation-reduction processes, response to stresses, and hormone responses, among others. A hormone profile analysis shows that the levels of salicylic acid (SA), a phytohormone that reduces nitrate accumulation and root growth, increase significantly upon N starvation. Meta-analyses of APA-affected and fip1-2-deregulated genes indicate a connection between the nitrogen starvation response and salicylic acid (SA) signaling. Genetic analyses show that SA may be important for preventing the overgrowth of the root system in low N environments. This work provides new insights on how plants interconnect different pathways, such as defense-related hormonal signaling and the regulation of genomic information by APA, to fine-tune the response to low N availability.

Root ABA and H+-ATPase are key players in the root and shoot growth-promoting action of humic acids.

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 (Lpr).

Role of cis-zeatin in root responses to phosphate starvation.

Phosphate (Pi) is an essential nutrient for all organisms. Roots are underground organs, but the majority of the root biology studies have been done on root systems growing in the presence of light. Root illumination alters the Pi starvation response (PSR) at different intensities. Thus, we have analyzed morphological, transcriptional and physiological responses to Pi starvation in dark-grown roots. We have identified new genes and pathways regulated by Pi starvation that were not described previously. We also show that Pi-starved plants increase the cis-zeatin (cZ) : trans-zeatin (tZ) ratio. Transcriptomic analyses show that tZ preferentially represses cell cycle and PSR genes, whereas cZ induces genes involved in cell and root hair elongation and differentiation. In fact, cZ-treated seedlings show longer root system as well as longer root hairs compared with tZ-treated seedlings, increasing the total absorbing surface. Mutants with low cZ concentrations do not allocate free Pi in roots during Pi starvation. We propose that Pi-starved plants increase the cZ : tZ ratio to maintain basal cytokinin responses and allocate Pi in the root system to sustain its growth. Therefore, cZ acts as a PSR hormone that stimulates root and root hair elongation to enlarge the root absorbing surface and to increase Pi concentrations in roots.

The polyadenylation factor FIP1 is important for plant development and root responses to abiotic stresses.

Root development and its response to environmental changes is crucial for whole plant adaptation. These responses include changes in transcript levels. Here, we show that the alternative polyadenylation (APA) of mRNA is important for root development and responses. Mutations in FIP1, a component of polyadenylation machinery, affects plant development, cell division and elongation, and response to different abiotic stresses. Salt treatment increases the amount of poly(A) site usage within the coding region and 5' untranslated regions (5'-UTRs), and the lack of FIP1 activity reduces the poly(A) site usage within these non-canonical sites. Gene ontology analyses of transcripts displaying APA in response to salt show an enrichment in ABA signaling, and in the response to stresses such as salt or cadmium (Cd), among others. Root growth assays show that fip1-2 is more tolerant to salt but is hypersensitive to ABA or Cd. Our data indicate that FIP1-mediated alternative polyadenylation is important for plant development and stress responses.

Both chemical and crystalline phase configuration influence the molecular features of humic acids in humic-calcium-phosphates fertilizers.

Phosphate-metal-humic complexes are very relevant in nature due to their crucial role in phosphate availability for plants and microorganisms. Synthetic phosphate-calcium-humic acid (HA) complexes have proven to be efficient sources of available phosphorus for crops. However, the current knowledge about their structure and molecular features is very poor. The structural implications of phosphate interaction with humic binding sites through calcium bridges, in both monocalcium phosphate and dicalcium phosphate is investigated by using molecular modeling, 31P-NMR, 1H-NMR and X-ray diffractometry. The conformational changes in the molecular configuration of the humic acid involved in the interaction resulting from the synthetic process is also studied by using HPSEC and synchronous fluorescence. The results obtained allow us to identify the phosphate type in the crystalline phase that is involved in the interaction of humic acid binding sites and the different forms of calcium phosphate. Synchronous fluorescence also shows that whereas the conformational configuration of the HA binding site is only partially affected in the monocalcium phosphate interaction, it changes in the case of dicalcium phosphate showing simpler molecular arrangements. These changes in the molecular conformation of the binding site in HA in solution may influence the biological activity of the humic acid. On the other hand, HPSEC studies show that the humic-calcium-phosphate interaction is accompanied by increases in the humic acid apparent size distribution. This effect is more intense in the case of monocalcium phosphate system probably due the influence of pH.

Shoot iron status and auxin are involved in iron deficiency-induced phytosiderophores release in wheat.

BACKGROUND: The release of phytosiderephores (PS) to the rhizosphere is the main root response to iron (Fe) deficiency in graminaceous plants. We have investigated the role of the Fe status in the shoot as well as of the signaling pathways controlled by three relevant phytoregulators - indolacetic acid (IAA), ethylene and nitric oxide (NO) - in the regulation of this root response in Fe-starved wheat plants. To this end, the PS accumulation in the nutrient solution and the root expression of the genes encoding the nicotianamine aminotransferase (TaNAAT) and ferritin (TaFER) have been evaluated in plants subjected to different treatments. RESULTS: The application of Fe to leaves of Fe-deficient plants prevented the increase in both PS root release and TaNAAT gene expression thus showing the relevant role of the shoot to root communication in the regulation of PS root release and some steps of PS biosynthesis. Experiments with specific hormone inhibitors showed that while ethylene and NO did not positively regulate Fe-deficiency induced PS root release, auxin plays an essential role in the regulation of this process. Moreover, the application of IAA to Fe-sufficient plants promoted both PS root release and TaNAAT gene expression thus indicating that auxin might be involved in the shoot to root signaling network regulating Fe-deficiency root responses in wheat. CONCLUSIONS: These results therefore indicate that PS root release in Fe-deficient wheat plants is directly modulated by the shoot Fe status through signaling pathways involving, among other possible effectors, auxin.

New Amphiphilic Composite for Preparing Efficient Coated Potassium-Fertilizers for Top-Dressing Fertilization of Annual Crops.

This study describes the efficiency of a new coating material for preparing granulated potassium-fertilizers with a potassium release to the soil solution sensitive to rainfall intensity. The composite is prepared by reaction of an alkyd-resin with cement in the absence of water. The complementary use of diverse analytical techniques showed that the presence of the cement fraction induced alkyd resin reticulation and gradual cement-resin hardening. Scanning electron microscopy revealed the formation of micro and nanopores within cement-clusters, whose water permeability is affected by the resin reticulation and amphiphilic character. Potassium release was evaluated in water, soil-columns, and in soil-plant trials in pots and open-field. Agronomic results were consistent with potassium release rates obtained in water solution and soil columns. The composite-coated potassium fertilizer was more efficient than the noncoated one in providing plant available potassium, with this effect being dependent on water presence in soil.

A light-sensitive mutation in Arabidopsis LEW3 reveals the important role of N-glycosylation in root growth and development.

Plant roots have the potential capacity to grow almost indefinitely if meristematic and lateral branching is sustained. In a genetic screen we identified an Arabidopsis mutant showing limited root growth (lrg1) due to defects in cell division and elongation in the root meristem. Positional cloning determined that lrg1 affects an alpha-1,2-mannosyltransferase gene, LEW3, involved in protein N-glycosylation. The lrg1 mutation causes a synonymous substitution that alters the correct splicing of the fourth intron in LEW3, causing a mix of wild-type and truncated protein. LRG1 RNA missplicing in roots and short root phenotypes in lrg1 are light-intensity dependent. This mutation disrupts a GC-base pair in a three-base-pair stem with a four-nucleotide loop, which seems to be necessary for correct LEW3 RNA splicing. We found that the lrg1 short root phenotype correlates with high levels of reactive oxygen species and low pH in the apoplast. Proteomic analyses of N-glycosylated proteins identified GLU23/PYK10 and PRX34 as N-glycosylation targets of LRG1 activity. The lrg1 mutation reduces the positive interaction between Arabidopsis and Serendipita indica. A prx34 mutant showed a significant reduction in root growth, which is additive to lrg1. Taken together our work highlights the important role of N-glycosylation in root growth and development.

Involvement of Hormone- and ROS-Signaling Pathways in the Beneficial Action of Humic Substances on Plants Growing under Normal and Stressing Conditions.

The importance of soil humus in soil fertility has been well established many years ago. However, the knowledge about the whole mechanisms by which humic molecules in the rhizosphere improve plant growth remains partial and rather fragmentary. In this review we discuss the relationships between two main signaling pathway families that are affected by humic substances within the plant: one directly related to hormonal action and the other related to reactive oxygen species (ROS). In this sense, our aims are to try the integration of all these events in a more comprehensive model and underline some points in the model that remain unclear and deserve further research.

Root-Shoot Signaling crosstalk involved in the shoot growth promoting action of rhizospheric humic acids.

Numerous studies have shown the ability of humic substances to improve plant development. This action is normally reflected in an enhancement of crop yields and quality. However, the mechanisms responsible for this action of humic substances remain rather unknown. Our studies have shown that the shoot promoting action of sedimentary humic acids is dependent of its ability to increase root hydraulic conductivity through signaling pathways related to ABA, which in turn is affected in roots by humic acids in an IAA-NO dependent way. Furthermore, these studies also indicate that the primary action of humic acids in roots might also be physical, resulting from a transient mild stress caused by humic acids associated with a fouling-cleaning cycle of wall cell pores. Finally the role of alternative signal molecules, such as ROS, and corresponding signaling pathways are also discussed and modeled in the context of the above-mentioned framework.

Abscisic Acid Regulation of Root Hydraulic Conductivity and Aquaporin Gene Expression Is Crucial to the Plant Shoot Growth Enhancement Caused by Rhizosphere Humic Acids.

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.

D-Root: a system for cultivating plants with the roots in darkness or under different light conditions.

In nature roots grow in the dark and away from light (negative phototropism). However, most current research in root biology has been carried out with the root system grown in the presence of light. Here, we have engineered a device, called Dark-Root (D-Root), to grow plants in vitro with the aerial part exposed to the normal light/dark photoperiod while the roots are in the dark or exposed to specific wavelengths or light intensities. D-Root provides an efficient system for cultivating a large number of seedlings and easily characterizing root architecture in the dark. At the morphological level, root illumination shortens root length and promotes early emergence of lateral roots, therefore inducing expansion of the root system. Surprisingly, root illumination also affects shoot development, including flowering time. Our analyses also show that root illumination alters the proper response to hormones or abiotic stress (e.g. salt or osmotic stress) and nutrient starvation, enhancing inhibition of root growth. In conclusion, D-Root provides a growing system closer to the natural one for assaying Arabidopsis plants, and therefore its use will contribute to a better understanding of the mechanisms involved in root development, hormonal signaling and stress responses.

The relative abundance of oxygen alkyl-related groups in aliphatic domains is involved in the main pharmacological-pleiotropic effects of humic acids.

Despite the rather common presence of humic acid (HA), our full knowledge of its biological effect is still lacking. In this article, we first performed a physicochemical characterization of several HAs, and next, we evaluated their ability to affect interleukin-2 secretion, antibody secretion, wound healing (an in vitro model using HaCaT cells), cancer growth (the Lewis lung carcinoma model), and protection against hepatotoxicity. In all tested reactions, HA showed significant stimulation on immune reactions, including suppression of cancer growth and inhibition of lipopolysaccharide-induced hepatotoxicity. These effects were dependent on its chemical properties. The pleiotropic effects of HA observed in this article suggest the possible role of these compounds in human nutrition.

Structural characterization of anion-calcium-humate complexes in phosphate-based fertilizers.

Fertilizers based on phosphate-metal-humate complexes are a new family of compounds that represents a more sustainable and bioavailable phosphorus source. The characterization of this type of complex by using solid (31)P NMR in several fertilizers, based on single superphosphate (SSP) and triple superphosphate (TSP) matrices, yielded surprising and unexpected trends in the intensity and fine structure of the (31)P NMR peaks. Computational chemistry methods allowed the characterization of phosphate-calcium-humate complexes in both SSP and TSP matrices, but also predicted the formation of a stable sulfate-calcium-humate complex in the SSP fertilizers, which has not been described previously. The stability of this complex has been confirmed by using ultrafiltration techniques. Preference towards the humic substance for the sulfate-metal phase in SSP allowed the explanation of the opposing trends that were observed in the experimental (31)P NMR spectra of SSP and TSP samples. Additionally, computational chemistry has provided an assignment of the (31)P NMR signals to different phosphate ligands as well as valuable information about the relative strength of the phosphate-calcium interactions within the crystals.