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Humic substances (HS) represent the organic material mainly widespread in nature. HS have positive effects on plant physiology by improving soil structure and fertility and by influencing nutrient uptake and root architecture. The biochemical and molecular mechanisms underlying these events are only partially known. HS have been shown to contain auxin and an “auxin-like” activity of humic substances has been proposed, but support to this hypothesis is fragmentary. In this review article, we are giving an overview of available data concerning molecular structures and biological activities of humic substances, with special emphasis on their hormone-like activities.
Humic substances (HS) are the largest constituent of soil organic matter (~60%) and are considered as a key component of the terrestrial ecosystem, being responsible for many complex chemical reactions in soil.1 They can not be decomposed readily because of their intimate interactions with soil mineral phases and are chemically too complex to be used by micro organisms. As far as soil is concerned, one of the most striking characteristics of HS is their ability to interact with metal ions, oxides, hydroxides, mineral and organic compounds,2 including toxic pollutants,3–5 to form water-soluble and water-insoluble complexes. Through the formation of these complexes, can dissolve, mobilize and transport metals and organics in soils and waters, or accumulate in soil horizons. Accumulation of such complexes can contribute to a reduction of toxicity, e.g., of aluminium (Al),6,7 or to remove Cr(VI) from aqueous solutions.8 Recently Wang and Mulligan (2009) have used HS in the arsenic and heavy metal remediation, indicating the HS as a possible remedial to reduce and avoid further contamination. Moreover HS can interact with xenobiotic organic molecules such as pesticides2,9–11 and influence nutrient availability (N, S, P), especially of those nutrients present at a very low concentration.12 Moreover, these are able to produce various morphological, physiological and biochemical effects on higher plants.13–15
Numerous studies have shown that HS enhance root, leaf and shoot growth but also stimulate the germination of various crop species.16 These positive effects are explained as an interaction between HS and physiological and metabolic processes.15,17 The addition of HS stimulate nutrient uptake,16,20 cell permeability21 and seems to regulate mechanisms involved in plant growth stimulation.22–26
It is not easy to distinguish between the direct and indirect effects of these substances. In fact, some of their positive effects may be ascribed to a general improvement of soil fertility, leading to a higher nutrient availability for plants. Whilst, in other cases, HS seem to positively influence metabolic and signalling pathways involved in the plant development, by acting directly on specific physiological targets.27,28 For this reason, understanding HS biological activity and the molecular mechanisms through which they exert their functions is becoming an important ecological task and a valid tool in facing environmental problems.
This review is aimed at clarifying the main signalling events governing the physiological effects of HS on plant metabolism, in order to shed more light on nature, properties, dynamics and functions of HS as part of soil and agricultural ecosystems.
A focal point of this topic is to verify the existence of a relationship between structure and biological properties of HS.29 So far the structure of HS is under debated. The main obstacle is the lack of a repetitive sequences and the variety of chemical and biological reactions involved in their genesis,30 that make HS very complex and multifaceted molecules, able to exert important signalling and nutritional functions in soil-plant system. The points of view more debated concern the polymeric nature and the supramolecular association of HS. Different authors have considered the HS a polymeric material with high molecular mass (100–300 kDa),1,29,31,32 originated from the lignin decomposition33 and from abiotic catalysts such as primary minerals and layer silicates.34
Alvarez-Puebla et al.35 proposed a macromolecular model of HS in which simple (though heterogeneous) monomeric units progressively build up into high molecular weight polymers by random condensation and oxidation processes (Fig. 1).35 In this model is demonstrated that linear or branched polymeric chain, assuming several conformational folding that would give more resistant to microbial degradation and lengthen their turnover in the soil.36
A supramolecular association of heterogeneous molecules held together by hydrophobic interactions (van der Waals, π-π, ion-dipole) and hydrogen bonds has been proposed by Piccolo,37,38 Simpson et al.39 and Schaumann.40 The whole of these forces stabilizes the structure of molecular aggregates. Moreover, the stability of HS aggregates in solution seems to be of a dynamic nature and influenced by the solution ionic strength and pH.41,42 At the alkaline pH the HS are in disperse form because intramolecular hydrogen bondings are completely disrupted.43 Acidification of HS with weak acids (i.e., acetic acid) solutions produces a significant decrease of molecular sizes or disaggregation for disruption of weak non covalent interactions, such as van der Waals, π-π, and CH-π.43,44 At lowest pH values (<3) the HS collapse into smaller aggregated. This phenomenon is mainly due to the protonation of carboxylate groups that favor an increase in the number of intra- and intermolecular hydrogen bonds inside the humic macromolecules.38,45
From a chemical point of view the HS are molecular aggregates consisting of sugar, fatty acids, polypeptides, aliphatic chains and aromatic rings.39 HS are operationally classified into humin and humic and fulvic acids in relation to different solubility at acid and alkaline pH.1 This classification was based only on superficial criteria and no indication on chemical behavior or structures was deduced.1
Different authors hypothesized that HS may be adsorbed by plant roots, even if high molecular weight (HMW) and low molecular weight (LMW) fractions seem to behave differently.15,18,21,46,71 Up to today it has not been yet completely clarified the mechanisms through which HS interact with the root cells and may subsequently influence plant physiology and growth. Among the modifications induced by HS on treated plants, changes in size and development were the first to be studied analytically. Under particular conditions, HS can stimulate plant growth in terms of increase in plant length and dry or fresh weight.47,48 These effects seem to depend on the concentration49 and source of the substance,50 on the plant species47,48 and age, as well as on the culture conditions of the trial. Recently, many studies have confirmed the hypothesis of a direct effect of HS on plant physiology, in particular concerning root hair formation51 and lateral root development (Fig. 2).23,24,52
The effects of HS on plant metabolic processes have been extensively reviewed.15,44 For instance, many reports showed that HS influence respiration,53 protein synthesis54 and enzyme activity in higher plants.53,55–57 As far as the photosynthesis process is concerned, few reports, focusing on the chlorophyll content and electrons transport, are available.58–61
In light of the importance of mineral nutrition for overall plant productivity, the effects of HS on ion uptake represent one of the topics which received more attention by scientists. They appear to be variable and selective, depending on the HS typology and concentration, on plant species, and on composition and pH of the medium.14,15,25,62–65 A positive effect of HS on nutrient uptake has been reported for the major inorganic elements, such as nitrogen, phosphorus, potassium66 and sulphur,48 but different HS fractions seem to differently affect their uptake.67,68 A recent study focused on iron uptake and assimilation in cucumber plants treated with purified leonardite humic acid (PHA), evidenced an induction of the expression of CsFRO1 and CsIRT1, encoding a Fe(III) chelate-reductase and a Fe(II) root transporter respectively.69 These results strongly support the hypothesis that beneficial effects of HS on plant development may, at least in part, depend on their capacity to improve Fe availability for plant uptake under Fe-deficient conditions.65 Pinton et al.70 evidenced the capacity of a specific fraction of HS (water extracted fraction WEHS) to stimulate some of the typical plant responses to Fe deficiency, such as the induction of Fe(III) chelate-reductase activity. This action of WEHS was also associated to significant increase in rhizosphere acidification.
Furthermore, as a consequence of the environmental impact that the nitrogen fertilization has assumed during the last century, several studies were conduced to figure out how the presence of HS may interfere with the nitrate uptake and assimilation by plants.14,25,27,63,70–72 Results obtained demonstrated a strong positive effect of the LMW fractions on nitrate uptake and assimilation, whereas HMW fractions induced only weakly the same pathways,15 in agreement with previous data.71
More recently, three independent studies aimed to better clarify the mechanisms through which HS stimulate the nitrate uptake in maize,23,27,70 demonstrated that the increment of nitrate influx measured in response to HS depends at least partially on a transcriptional activation of a gene encoding a major H+-ATPase of maize (Mha2), likely leading to the generation of a more favorable electrochemical gradient. In fact, nitrate influx across the plasmalemma of root cells is coupled to the favorable H+ electrochemical gradient created by the plasma membrane H+-ATPases.73–76
A recent study used a wide transcriptomic approach to study the biological processes involved in the Arabidopsis thaliana response to HS.77 This study represents the first report concerning the global molecular mechanisms governing the HS-plant interaction. From our preliminary data it may be hypothesized that HS influence plant development by interfering with the transcription of genes involved in meristem formation and organization, cell cycle, microtubule organization and cytokinesis. Moreover, a great amount of the transcripts isolated has been shown to belong to several classes of transcription factors and DNA binding proteins, confirming. Even if the exact mechanisms through which HS exert their effects on plant physiology are still partially unclear, many evidences suggest that they may involve at least in part a hormone-like activity.
A putative HS hormone-like activity is not surprising as it is known that soils vary in their native auxin content78 and fertile soils contain greater amounts of auxin then less fertile ones.79,80 Auxin and gibberellin levels are usually higher in the rhizosphere than in the bulk soil, probably as a consequence of increased microbial populations or of an accelerated metabolism owing to the presence of root exudates. Although numerous soil and rhizosphere microorganisms, as well as the root systems of higher plants, have been reported as producing auxin81 and gibberellins,82 there is little information about their stability and only indirect conclusions have been made about their presence in amounts high enough to be biologically active.83
At the beginning of the 20th century, Bottomley84 hypothesized that the growth promoting activity of the HS could be due to a hormone-like activity. Such argument was then deeply faced by a number of studies and later definitely corroborated by results demonstrating the immunological or spectrometric identification of indol acetic acid (IAA) inside several HS.17,23,27,85 In addition, the hypothesis of a HS auxin-like activity was also supported by reports showing a positive effect of such substances on specific targets of auxin action. Mha2, a major maize isoform of H+-ATPase that is preferentially expressed in guard cells, phloem and root epidermal cells and that appears to be strongly stimulated at the transcriptional level in response to auxin,86 evidenced a significant upregulation of its mRNA abundance in roots of maize seedlings treated for 48 hours with earthworm low molecular size HS.27 Furthermore, Russel and collaborators,87 by studying the effects of two different molecular weight fractions of HS on pea, evidenced an auxin-like effect of both fractions on stomatal opening as influenced by phospholipase A2, that is considered to be involved in auxin signalling.88,89
In former experiments, Muscolo et al.90,91 showed a morphogenetic effect of HS on Nicotiana plumbaginifolia leaf explants, probably triggered by a modification of peroxidase and esterase activities. These effects, peculiar to humic fraction with a low relative molecular mass (<3,500 Da), were similar to those produced by IAA. A subsequent study on homogeneous carrot (Daucus carota) cell cultures compared the effects of the low relative molecular mass humic fraction to different auxins.17 This humic fraction caused an increase in carrot cell growth similar to that induced by 2,4-dichlorophenoxyacetic acid (2,4-D) and promoted morphological changes similar to those induced by IAA. In addition, Muscolo et al.17 demonstrated that IAA and LMW fractions richer in carboxylic groups bind in the same way with carrot cell membranes. Zandonadi et al.52 comparatively evaluate the effects of indole-3-acetic acid and humic acids (HA) isolated from different soils substances on maize root development and on activities of plasmalemma and tonoplast H+ pumps. They observed that HA as well as low IAA concentrations (10−10 and 10−15 M) stimulated root growth by inducing the proliferation of lateral roots (Fig. 3) along with a differential activation not only of the plasma membrane, but also of vacuolar H+-ATPases and H+-pyro-phosphatase.
A different theory about the auxin-like activity of the HS has been assumed by Schmidt et al.92 (2007). To further investigate a possible hormone-like effect of water-soluble humic molecules (WEHS), they grown Arabidopsis plants in sterile medium containing WEHS in concentrations ranging from 1–20 mg C org. Application of WEHS were found to significantly increase the number and length of root hairs. Further experiments reported that the phenotypes of Arabidopsis auxin-related mutants, all exhibiting a reduced number of root hairs, were not rescued by the application of WEHS. In addition, mutants defective in root hair initiation such as rhd6, known to develop normal hairs in the presence of ethylene or auxin, were not affected by a wide range of applied concentration of WEHS. The authors concluded that HS cannot substitute for these hormones in promoting root hair growth, and suggested that HS can alter root development without significantly affecting the plant's auxin homeostasis. This assumption was also supported by the lack of responsiveness of the auxin-responsive GH3 gene. Transcripts of genes from the GH3 family accumulate following auxin exposure, probably to dampen auxin signaling by inactivating IAA via conjugation to amino acids.93 Application of high concentration of HS (more than 5 mg C L−1) for two hours evidenced only a slight increase in transcript abundance in roots and caused no significant change in mRNA accumulation in leaves, further confirming the hypothesis that the changes in root morphology are not mediated via an auxin-signalling pathway.
It is important to note that this theory is not complemented with a detailed characterization of the HS analyzed. Because of the different features and the complex chemical composition of HS, an efficient and complete characterization, from both a chemical and a spectroscopic point of view, is an essential requirement to match data obtained from different studies. In this case the extraction procedure and the lacking in characterization of the humic substances analyzed make the results not completely suitable for further comparison with any other information present in literature. Moreover, the authors expressed the theory that WEHS do not exert their effects in an auxin-like manner without investigate its inner content of indole-3 acetic acid. According to all these lacking in description, the conclusion of the authors could be speculative rather than theoretical and it would need a more detailed investigation to be considered.
On the contrary, recently Dobbss and collaborators,26 using Arabidopsis and tomato seedlings, demonstrated that various characterized humic acids need the auxin transduction pathway to be active. The increase in number of lateral root exhibited in Arabidopsis and tomato wild-type seedlings treated with different HS led authors to hypothesize the presence of auxin-like compounds in these organic matter. Nevertheless the same substances did not induce lateral root formation in a tomato mutant (dgt) characterized by a defective gene for auxin response (Fig. 4). They concluded that probably HS may act as a “buffer”, either absorbing or releasing signalling molecules, according to modifications in the rizosphere, such as the acidification brought about by the activity of plasma membrane H+-ATPase23,52 or exudation of organic acids,24,94 thus behaving as a regulator of hormonal balance with respect to lateral root emergence.
More recently, the auxinic activity of HS in the initiation of lateral roots has been deeply studied in the model plant Arabidopsis thaliana,28 by utilizing a combination of genetic and molecular approaches (Figs. 5 and and66). The widely used auxin reporter DR5::GUS95 was employed to visualize auxin responses in roots96,97 and to characterize the distribution of LRP stages in both wild type (Col-0) and aux1 mutant background. In addition, the transcription of the known early auxin responsive genes IAA5 and IAA19,98–101 in parallel treatments with HS and comparable IAA concentrations was evaluated.
The authors concluded that HS exert their action on lateral root development mostly through their auxinic activity and clearly demonstrated the presence of a small amount of IAA in the fraction analyzed, corresponding to a concentration of 34 nM. At the same time, the presence of additional factors independent from auxin was evident based on IAA5 and IAA19 expression suggesting that more systematic approaches are needed to unravel the molecular mode of action of HS. These aspects are currently under investigation in our laboratory.
Because of their complicated and changeable nature, a debate on HS auxin-like activity is still open. However, the observed hormonal effects did not always correlate to the amount of IAA detected in the humic acids. For this reason, the presence of different compounds of the auxin family or of molecules that might either mimic the action or stimulate the plant endogenous metabolism of auxin cannot be ruled out. Functional genomics, transcriptomics or proteomics may represent a good strategy in shedding light on HS biological activity.
HS, as the major component of soil organic matter, have been widely studied in various areas of agriculture, such as soil chemistry, fertility and plant physiology. HS plays an important role in controlling the behavior and mobility of polluters in the environment and contribute substantially in improving the global soil fertility status. These features together with a major demand of safe food and sustainable agricultural have contributed to enlarge the environmental significance of HS, which have been recently recognized as a possible tool in facing environmental problems.
Many of their positive effects on soil and plant growth have been demonstrated to rely on their chemical composition, but progress in research on HS has been considerably hampered by the lack of characterization of the humic fractions being used.
The auxinic activity of HS, demonstrated in recent studies, is probably the main biological factor responsible for the positive effects exerted by HS on plant physiology. The stimulatory effect on Arabidopsis lateral root development observed in response to HS, has been found mainly in the first stages, when cells start to divide, suggesting that HS response may involve mechanisms as the stimulation of cell division and differentiation, which it is know to be under the control of auxin. Moreover, physiological and molecular data suggest brassinosteroids as a putative additional factor throught which HS could exert their effects on plant development. This finding has been further supported by recent transcriptomic results. A great amount of the genes isolated by means of a cDNA-AFLP approach have been demonstrated to be auxin regulated and related to developmental process, as differentiation and organization of meristems, embriogenesys, citokynesis and microtubules organization (Trevisian, personal communication).
All together, these results provide evidence that HS need auxin transduction pathway to establish their action on plant physiology, but evidenced also the existence of different signalling cascades involved in the global physiological response of plants to these substances (Fig. 7). This could be considered a starting point in elucidating mechanisms that occur in plant at molecular level in response to HS. Further studies are needed to assess the molecular targets and signalling pathways involved in the cross-talk between HS and plant cells. These features together with a major demand of safe food and sustainable agricultural have contributed to enlarge the environmental significance of HS.
Fertilizer factories are now redirecting their production to biostimulants, based on humic substances and other organic compounds and recently, in Italy, biostimulants were inserted in the Legislative Decree n. 217/2006 (“New regulation about fertilizer” MiPAF).
This is an important result which supports the fundamental project of recycling partially humified organic wastes, derived from plant, wood, food and other human activities, as beneficial soil amendments. For this reason, understanding HS biological activity and the molecular mechanisms through which they exert their functions is becoming an important ecological task and a valid tool in facing environmental problems.
Previously published online: www.landesbioscience.com/journals/psb/article/11211