INTRODUCTION - The Rhizosphere Part of Atmosphere

APOPLASM

1. INTRODUCTION

Roots of plants growing in their natural environment have evolved and live in close contact with the solid phase of the soil: this interaction can determine changes both in root physiology ( I ) and anatomy (2) and in the chemical, physical, and microbiological properties of the soil. These phenomena occur in a limited area surrounding the root-the rhizosphere-where nutrient, energy, and signal exchanges make this environment decisively different from bulk soil, both from a chemical-physical and microbiological point of view.

The production and release of organic molecules by the root systems of plants have been extensively studied under a wide range of soil conditions (nutrient and water availability, presence of pollutants. etc., see Chaps. 2 and 3). Fur- thermore it has been clearly demonstrated that soil microorganisms are able to produce molecules that can affect the physiology and architecture of roots (3);

evidence has been also provided that molecular signals between plants and microorganisms are exchanged (see Chap. 7).

On the other hand, certain soil components affect per se plant growth and nutrition. Humic substances in particular can influence plant metabolism by inter- acting with a variety of biochemical mechanisms and physiological processes, stimulating growth and increasing the total amount of nutrients taken up by the plant (4). It has therefore been suggested that these compounds may have a fundamental influence not only on the composition and activity of rhizosphere soil

microbiota but also on the physiology of plants.

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This chapter focuses on the effects of humic substances present at the rhizosphere on plant growth and nutrient uptake. The main structural features of humic substances, their nutritional function, and the capacity to interact with plant metabolism are also presented.

II. DEFINITIONS AND MAIN FEATURES OF

HUMIC SUBSTANCES

Humic substances account for approximately 60% of soil organic matter. These compounds are the result of biological and chemical transformations of plant, animal, and microbial residues carried out by soil microorganisms (5). The re- sulting chemical compounds are more stable than their precursors. Although the molecular structures have not yet been exactly defined, humic substances are known to have many aromatic rings that interact with each other and with ali- phatic chains, giving rise to molecules of dimensions ranging from a few hundred to a few hundred thousand Daltons (fulvic and humic acids, respectively). Certain distinctive features of these two classes have been defined, such as acidity, the

presence of oxygen-containing functional groups (6) (Table l ) , and solubility in water, which is greater for fulvic acids. In recent years structural information on humic molecules has also been acquired using specific software that processes data obtained by pyrolysis-gas chromatography, pyrolysis-field ionization mass spectrometry, and physical, chemical, biological, and spectroscopic methods (7).

Using these approaches, tridimensional models have been presented showing the interactions between humic substances and mineral matrix of soil. The model is consistent with the presence of cavities within the humic molecules that could house organic compounds such as carbohydrates, proteins, lipids, and biocides.

Table 1 Range of Distribution of Oxygen-Containing Functional Groups in Humic and Fulvic Acids Isolated from Soils of Widely Different Climatic Zones (in mEq/ 100 g)

Humic acids Fulvic acids

Total acidity 560-890 640- I420

COOH 150-570 520- 1 120

Acidic OH 2 10-570 30-570

Weakly acidic

+

alcoholic OH 20-490 260-950 Quinone and kctonic C = O 10-560 120-420

OCH 3 30-80 30- I20

Nowadays the distinction between humic and fulvic acids is no longer considered valid (g), since it is based on conventional extraction and purification methods. It has furthermore been proved that there are humic molecules of very different molecular mass and degrees of solubility in the soil. Concentrations of humified organic matter in the soil solution of 250 mg C org/L or even higher have been reported (9- I I ) . Humic compounds behave in a typical manner and have the typical properties of colloid associations, polyelectrolytic solutions, and colloid dispersions (12); the degree of aggregation of humic compounds is, in fact, known to depend not only on their molecular structure but also on the solvat- ing conditions of the system. Humic molecules separate in water at low pH Val- ues, when the ionic force of the solution increases or when polyvalent metals are added to the solution. On the other hand, the smaller fractions of humified matter can remain in solution even at high salt concentrations and at a wide range of pH (3-8). Therefore an interaction between these molecules and plant roots appears plausible, and studies should be done to investigate the structure and behavior of humic matter at the rhizosphere.

Chemical, biochemical, and microbiological conditions at the rhizosphere differ widely from those of bulk soil; this can lead to changes in the dynamics and structure of humified organic matter. On the other hand, little is known about the molecular structure and degree of aggregation of humic molecules at the rhizosphere. A few attempts have been made to determine whether organic acids released by plant roots may change the structure of humic molecules ( 13). Analy- ses by size-exclusion chromatography (SEC) have revealed that high-molecular- weight molecules treated with acidic solutions or organic acids release humic

molecules of lower molecular weight that possess higher biological activities than those of the former molecules. Similar results were also obtained using maize root exudates (14). The disaggregation process can be explained by the micellar behavior of humic substances in solution (15). On the other hand, one must also take into account that the use of SEC to estimate changes in the molecular mass of acid-treated humic compounds has certain limitations that justify caution in the interpretation of the results (16).

111. SOURCE OF NUTRIENTS

A. Constitutive Nutrients

Fresh organic matter plays a fundamental role in plant nutrition by supplying nutrients released through degradation processes; however, humified organic substances also become a source of nutrients when subjected to mineralization processes. The main aspects of the cycle of organic matter at the rhizosphere soil are reported in Chap. 6.

Generally more than 95% of the total nitrogen in the soil is present in

the organic fraction (17); humic substances in particular (which contain 3 4 % nitrogen) act as a storehouse and supplier of nitrogen for plant roots and microorganisms ( 1 8). A relatively large amount of aminic nitrogen present in the soil is incorporated in humified matter, up to 50% of which is supposed to be present as peptides and proteins ( 1 9). Recent studies have proved the presence of protease enzymatic activity at the rhizosphere (20).

Other than a nutritional role linked to mineralization processes, humic compounds have been hypothesized to directly affect plant nutrition, since it has been suggested that roots may take up low-molecular-weight humic molecules (21).

Interestingly, plants have been observed to express carriers for amino acids (22) and small peptides (23) at the root level. Certain components of the humic fraction have been found inside root cells and were, moreover, translocated to the shoots (24,25). Recent experiments performed on rice cells in suspension culture seem to suggest that they may use carbon skeletons from humic molecules to synthesize proteins and DNA (26).

As well as peptides, nitrogen can be found in humic substances as heterocyclic molecules. These can account for up to 50% of total nitrogen present in humified organic matter and are mainly purines, pyrimidines, indols, quinolines, isoquinolines, aminobenzofuranes, and piperidine and pyrrolidine derivatives, which are presumably integrated into the structure of humified substances ( 1 7).

Information relative to the role of the heterocyclic component of humic nitrogen in plant nutrition is very scarce. These structures are subjected to a variety of trans- formation processes in the soil; although they appear to be quite stable, it has been proved (27,28) that this fraction of nitrogen is not inert and can be microbiologi- cally and chemically converted into inorganic compounds. The contribution of this fraction to nitrogen nutrition in plants is still unknown; on the other hand, taking into account the microbial activity, the higher energetic availability, and the chemical-biological conditions found at the rhizosphere, humic nitrogen is likely to be subjected to transformations differing from those in the bulk soil.

Phosphorus in its inorganic form is a nutrient showing low solubility and mobility in the soil, as it easily reacts with the soil mineral components (clay,

iron, and aluminum oxides, and carbonates) (17). The content of phosphorus in humic substances ranges between 0.1 and 1.0%; it is particularly abundant in humic acids. By using j'P nuclear magnetic resonance (NMR), Bedrok et al. (29) showed that different forms of phosphorus can be associated with humic fractions of different molecular size. In fractions of high molecular weight, phosphorus is usually found as esaphosphate inositol, whereas in those of lower molecular weight, it is usually present as inorganic orthophosphate. Both types of phosphorus have an important nutritional value: the former compound is a substrate for phosphatases, which are particularly abundant at the rhizosphere (30), whereas the latter, bound to humic compounds by aluminium and iron bridges, becomes available in the presence of high concentrations of organic and tricarboxylic

acids, which are usually present in the rhizosphere soil (see Chaps. 2 and 3).

Indeed, these acids can form complexes with the iron bound to the surface of the humic matter, thus releasing the phosphate (3 1,32).

Notwithstanding the importance of sulfur from a nutritional point of view and its ascertained presence in humic matter, information relative to the availability of this nutrient is rather scarce (17). Humic molecules contain sulfur as proteins, amino acidic residues, sulfate esters, and possibly in the form of stable thiazine rings. Sulfur can be released from these organic compounds following the action of microorganisms using organic carbon as a source of energy. The chemical bond of the nutrients plays an important role in the type of organic matter mineralization. Hunt et al. (33) have distinguished five classes of chemical bonds (C-C, N-C, S-C, S-0-C, and P-0-C). Microorganisms oxi- dizing carbon provide energy to mineralize the compounds characterized by the first three types of bonds. On the other hand, compounds with sulfur and phosphorus atoms present as esters can be mineralized by the action of extracellular hy- drolases, according to the need of the element (34). Although sulfur is more mobile than phosphorus in the soil ( 3 5 ) , these processes at the rhizosphere may have great importance for plant nutrition; information in the literature concerning these aspects is, however, scarce.

B. Complexing Properties

Humic substances can form complexes with metals, including cationic micronutrients (36), thanks to the presence of electron-donor functional groups in these molecules. It therefore appears evident that due to these properties, humic substances can contribute to the regulation of the chemical balances of metals, thus influencing their solubility (5). With regard to plant availability, the molecular dimension and solubility of humic substances are very important.

Fractions of higher molecular mass, which are mostly insoluble, can with- hold large amounts of metals, especially in alkaline environments. Metals are thus subtracted from precipitation and subsequent crystallization, processes that would decrease their availability (37), and a reserve of micronutrients is created in equilibrium with complexing molecules. On the other hand, under conditions of high metal concentrations, complexation by humified organic matter may limit the amount of metal in solution; under these conditions, interchain bonds may form, causing humic molecules to precipitate. This process can be important for heavy metals, the activity of which can thus be reduced to nontoxic levels (9).

Soluble humified organic matter that may be present in the soil (38) can help to increase metal transport by diffusion to the roots (39) and favor micronutrient uptake by the plants. The contribution of these organic fractions to the dynamics of metals at the rhizosphere is not known; however, it is interesting to observe that, unlike other organic molecules present at the rhizosphere which can chelate

or complex metals (e.g., organic acids. phytosiderophores, microbial sidero-

phores), humic substances are much more stable in regard to microbial degradation.

IV. ROLE OF HUMIC SUBSTANCES AS

NATURALCHELATES

Humic substances have been extensively considered as natural chelates for cationic micronutrients. Thanks to their ability to form complexes with metal cations such as iron, it is generally accepted that they can mobilize then1 from soil parti- cles to the root surface. The quantitative aspects of this process have not yet been elucidated. It is reasonable to think that the importance of the metal-humic substance complexes depends on the metal-humic matter ratio. As proposed by Lindsay and Schwab (40) for the movement of the Fe-EDTA chelate from the solid phase of the soil to the roots, the following scenario can be hypothesized in the case of soluble humic molecules: at a low soluble humic molecule-Fe ratio, the molecules will tend to mobilize Fe from the solid phase to form stable complexes. If the amount of Fe is not sufficient to form humic macromolecules of lower solubility, the soluble complex will move by diffusion toward the roots.

Cesco et al. (41) observed that a humic fraction-water-extractable humic substances (WEHS)-purified from a water extract of sphagnum peat using XAD- 8 amberlite resin can solubilize Fe present as insoluble hydroxide and mobilize it in a soil column, making it available for exchange with organic chelating agents released by the roots, like the phytosiderophores. It is interesting to note that the method used to extract WEHS does not involve the use of extractants, such as sodium hydroxide, which may modify the chemical-physical characteristics of humic matter (42). The dynamics of Fe mobilization by humic substances must, however, take into account conditions at the rhisosphere, such as pH and redox potential, and the presence of other types ofchelating agents of microbial (sidero- phores) or plant (organic acids and phytosiderophores) origin. Plants are known

to possess different mechanisms for responding to limited micronutrient availability. In the case of Fe, two strategies have been observed (43), for dicots and nongramineous monocots (strategy I) and for graminaceae (strategy II), respectively.

In the first case the mechanisms are based on an increased reducing capacity of Fe(lII)-chelates, a necessary step in the uptake process, with a COncUrrent increase in acidification and release of organic acids into the rhizosphere; in the latter case molecules having high affinity for Fe (phytosiderophores) are synthe- sized and released into the rhizosphere when Fe is lacking.

It is interesting to observe that response mechanisms to Fe deficiency have been studied ahnost exclusively using synthetic chelates such as EDTA and ED-

DHA or, in a few cases, organic acids released by the roots (such as citrate and malate). It is however reasonable to suppose that a mixture of natural chelates is present in the soil and in the rhizosphere (44). Among natural chelates, humic molecules may play an important role in the mechanisms involved in Fe uptake.

Lobartini and Orioli (45) have shown that iron deficient sunflower plants could use Fe-humate added to the nutrient solutions as a source of iron, leading to the disappearance of chlorosis symptoms. A response of plants to the treatment could be observed even if the quantity of iron absorbed was only one-tenth of that obtained using Fe-EDTA as a source of iron. The same authors also proved that barley plants were able to accumulate syFe from solutions containing Fe-humate with mechanisms involving micronutrient adsorption at the root apoplast. The ability of cucumber plants to recover from conditions of iron deficiency after supplying two different humic fractions containing “endogenous” Fe to the nutrient solution was also proved by Pinton et al. (46). The efficiency in the use of Fe present in the two fractions was related to their molecular mass and solubility. Iron present in the low-molecular-weight ( < l kDa) WEHS fractions was used almost completely; whereas only 25% of that contained in larger (characterized by a dimension >S kDa) humate, obtained by extraction with Na-pyrophos- phate, was used by the plants. Clear evidence that humic substances can be used as a source of iron has recently been provided by Pinton et al. (47). The Fe(II1)- WEHS complex was in fact reduced both by roots of iron-deficient cucumber plants and by the plasma membrane vesicles isolated from the roots. The Fe(II1)- WEHS complex was reduced in vivo more efficiently than Fe-EDTA; in addition, a more rapid or greater recovery from symptoms of chlorosis was observed in plants treated with the Fe-humate complex than in those treated with other Fe sources (Fe-EDTA, Fe-citrate, and FeC13). The greater efficiency seems to be related not only to Fe(1ll) reduction by roots but also to a stimulation of proton extrusion by the humic fraction. Recovery from deficiency also occurred when plants were grown in a nutrient solution at alkaline pH and in thc presence of CaCOl (48), a situation closer to that causing iron chlorosis in the soil.

Based on the above observation, it can conceivably be concluded that the presence of Fe-WEHS-type complexes can be of relevance to the Fe nutrition of both strategy I and strategy I1 plants (Fig. 1). In the case of strategy I plants, an effect on the mechanisms of active proton extrusion should also be considered (see Sect. V).

V. EFFECT ON MECHANISMS OF NUTRIENT UPTAKE

Humic substances have been proved to stimulate plant growth and nutrient accu- mulation (for review scc Refs. 4,49, and SO). Various studies performed on ex- cised roots or whole plants show that usually the uptake of cationic and anionic

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