The Rhizosphere
VI. CONCLUSIONS
It is likely that of the vast array of compounds released by plant roots, very few have a direct effect on the growth of soil-grown plants. One must ask whether they serve any useful purpose at all. The fears and uncertainty about what is physiologically normal were expressed by Ayers and Thornton in 1968 (97); their
concerns are as valid today as they were then. The absolute and relative quantities released are at present no more than approximations based largely on a very
narrow selection of short-lived agricultural plants.
In attempting to classify types of secretions on the basis of what might happen in the rhizosphere, the foregoing discussion has taken a fairly distrusting view of data derived from i n vitro experiments such as solution cultures, those using sterilized soil, and those using highly contrived situations where the reality of normal soil-grown plants is disregarded. The discussion has also highlighted the difficulties faced by some secretions and how these difficulties will decrease their likelihood of bringing about the process they are purported to bring about.
However, arguments in cases such as tolerance of A1 toxicity and the acquisition of Fe and Mn can be strengthened by invoking the right set of circumstances.
Root products represent a vast array of predominantly organic compounds.
Of these, secretions represent a small proportion, but they are deemed the most likely of all root products to have a direct effect on the growth of the plant that produced them. When a secretion is released by a root, all the following are likely to affect its behavior.
1. Site of secretion appropriate or inappropriate 2. Microbial assimilation/degradation
3. Chemical alteration/degradation (e.g., oxidation)
4. Sorption and persistence with or without activity (e.g., ectoenzymes) 5 . Diffusion and reaction with the target (e.g., complex with AI3+ or Fe”) 6. Mechanism of uptake (e.g., reabsorption by the root)
Very few root secretions can be expected to be effective unless “the right set of circumstances” arises sufficiently often. Further, research involving soil- grown plants is required to establish whether the right set of circumstances, as discussed above, does make a real contribution to the well-being of field-grown plants. Similarly, close scrutiny of all research must be made, particularly when results obtained in solution cultures and other contrived situations are believed to be relevant for plants growing in soil.
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3
The Release of Root Exudates as Affected by the Plant’s
Physiological Status
Gunter Neumann and Volker Romheld Universitat Hohenheim, Stuttgart, Germany
1. INTRODUCTION
Apart from the function of plant roots as organs for nutrient uptake, roots are also able to release a wide range of organic and inorganic compounds into the root environment. Soil-chemical changes related to the presence of these com- pounds and products of their microbial turnover are important factors affecting microbial populations, availability of nutrients, solubility of toxic elements in the rhizosphere, and thereby the ability of plants to cope with adverse soil-chemical conditions (1). Organic rhizodeposition includes lysates, liberated by autolysis of sloughed-off cells and tissues, as well as root exudates, released passively (diffusates) or actively (secretions) from intact root cells (Table 1; see also Chap. 2). In annual plant species, 30-60% of the photosynthetically fixed carbon is translocated to the roots, and a considerable proportion of this carbon (up to 70%) can be released into the rhizosphere (2,3), as pointed out in Chaps. 4,6, and 12. This rhizodeposition is affected by multiple factors, such as light intensity, temperature, nutritional status of the plants, activity of retrieval mechanisms, vari- ous stress factors, mechanical impedance, sorption characteristics of the growth medium, and microbial activity in the rhizosphere. This chapter focuses on the release of water-soluble organic root exudates and highlights effects of the physi- ological status on root exudation and its significance for adaptations to adverse soil conditions and nutrient efficiency. Since the methods employed for collection and analysis of root exudates play an important role in the qualitative and quanti-
41
Table 1 Root Exudates Detected in Higher Plants Class of compounds Single components
~~
Sugars
Amino acids and amides
Aliphatic acids
Aromatic acids
Miscellaneous phenolics Fatty acids
Sterols Enzymes
Micellaneous
Arabinose, glucose, fructose, galactose, maltose, raffinose, rhamnose, ribose, sucrose, xylose
All 20 proteinogenic amino acids, aminobutyric acid, homo- serine, cysrathionine, mugineic acid phytosiderophores (mugineic acid, deoxymugineic acid, hydroxymugineic acid, epi-hydroxymugineic acid, avenic acid, distichonic acid A)
Formic, acetic, butyric, popionic, malic. citric, isocitric. ox- alic, fumaric. malonic, succinic, maleic, tartaric, oxalo- acetic, pyruvic, oxoglutaric, maleic, glycolic, shikimic, cis-aconitic, trans-aconitic, valeric, gluconic
p-Hydroxybenzoic, caffeic, p-coumaric, ferulic, gallic, gen- tisic, protocatechuic, salicylic, sinapic, syringic Flavonols, flavones, flavanones, anthocyanins, isoflavonoids Linoleic, linolenic, oleic, palmitic, stearic
Campestrol, cholesterol, sitosterol, stigmasterol
Amylase, invertase, cellobiase. desoxyribonuclease, ribonu- clease, acid phosphatase, phytase, pyrophosphatase apy- rase, peroxidase, protease
Vitamins, plant growth regulators (auxins, cytokinins, gib- berellins), alkyl sultides, ethanol, H'. K ' nitrate, phos- phate, HCOl
Soltrcr: Adapted from Ref. 274.
tative interpretation of measured exudate data, methodological aspects are also discussed in an introductory section.
II. COLLECTION OF ROOT EXUDATES-
METHODOLOGICAL ASPECTS
A. Collection Techniques with Trap Solutions
Water-soluble root exudates are most frequently collected by immersion of root systems into aerated trap solutions for a defined time period (Fig. 1A). The tech- nique is easy to perform and permits kinetic studies by repeated measurements over time using the same plants. While it is possible to get a first impression about qualitative exudation patterns and even quantitative changes in response to different preculture conditions, the technique also includes several restrictions that should be taken into account for the interpretation of experimental data.
- . ... . . .... . ,
0.2
C
Figure 1 Techniques for collection of root exudates. (A) Solution culture system (282);
root exudates collected from the whole root system by immersion into aerated trap solu- tions under sterile conditions (optional). (B) Plant culture in solid media (vermiculite, sand); root exudates collected from the whole root system by percolation of the culture vessels with trap solution (9,l l). (C) Localized root exudate sampling from plants grown in solution culture. Exudates collected into trap solution inside of sealed plastic rings straddling the root (28) or by application of sorption media (filter paper, agar, ion-exchange resins) onto the root surface (l7,45). (D) Localized collection of rhizosphere soil solution from plants grown in soil culture. Rhizoboxes with removable front lids for plant culture (root windows under field conditions). Collection by insertion of microsuction cups (made from HPLC capillaries, 1 mm in diameter) connected to a vacuum collection device (50) or by application of sorption media (filter paper, agar, ion-exchange resins) onto the root surface (27.45).
Application should be restricted to plants grown in nutrient solution, since re-
moval of root systems from solid media (soil, sand) is almost certainly associated with mechanical damage of root cells, resulting in overestimation of exudation
rates.
On
the other hand, it has frequently been demonstrated that the mechanical impedance of solid growth media leads to alterations in root morphology andstimulates root exudation (43). In liquid culture media, simulation of the mechan-
ical forces imposed on roots of soil-grown plants may be achieved by the addition of small glass beads (5-7). Alternatively, exudate collection from plants grown
in solid media (sand, vermiculite) may be performed by percolating the culture vessels with the trap solution for a defined time period (Fig. IS) after removal of rhizosphere products accumulated during the preceding culture period by re- peated washing steps (8-1 l ) . For this approach, however, recovery experiments and comparison with results obtained from experiments in liquid culture are es- sential, since sorption of certain exudate compounds to the matrix of solid culture media cannot be excluded. As a modification of the percolation technique, car- tridges filled with selective adsorption media (e.g., XAD resin for hydrophobic compounds, anion exchange resins for carboxylates). which are installed in the tube below the plant culture vessel, can be employed for the enrichment of distinct exudate constituents (12,13). After adsorption to a resin, exudate compounds are also protected against microbial degradation.
Trap solutions employed for the collection of water-soluble root exudates are nutrient solutions of the same composition as the culture media (8,10,1 I ) solutions of 0.5-2.5 mM C a S 0 4 or CaCI2 to provide Calf for membrane stabiliza- tion (14) or simply distilled water (9,lS- 17). Since the osmotic strength of nutri- ent solutions is generally low, short-term treatments ( 1 -2 h) even with distilled water are not likely to affect membrane permeability by osmotic stress. Accord- ingly, comparing exudation of amino acids from roots of Brcmiccl nnpus L. into nutrient solution, 20 mM KCI, or distilled water, respectively, revealed no differ- ences during collection periods between 0.5 and 6 h (18). In contrast, Cakamak and Marschner ( 1 9) reported increased exudation of sugars and amino acids from roots of wheat and cotton during a collection period of 6 h when distilled water instead of 1 mM CaSOJ was applied as the trap solution. Thus, for longer collec- tion periods or for repeated measurements, only complete or at least diluted nutri- ent solutions should be employed as trap solutions in order to avoid depletion of nutrients and excessive leaching of Ca?+. Long-term exposure of plant roots to external solutions of very low ionic strength is also likely to increase exudation rates due to an increased transmembrane concentration gradient of solutes (20,2 I ).
Exudate collection in trap solutions usually requires subsequent concentra- tion steps (vacuum evaporation, lyophilization) due to the low concentration of exudate compounds. Depending on the composition of the trap solution, the re- duction of sample volume can lead to high salt concentrations, which may inter- fere with subsequent analysis or may even cause irreversible precipitation of certain exudate compounds (e.g., Ca-citrate, Ca-oxalate, proteins). Therefore, if possible, removal of interfering salts by use of ion exchange resins prior to sample concentration is recommended. Alternatively, solid-phase extraction techniques may be employed for enrichment of exudate compounds from the diluted trap solution ( I 1,22). High-molecular-weight compounds may be concentrated by pre- cipitation with organic solvents [methanol, ethanol, acetone 80% (v/v) for poly- saccharides and proteins] or acidification [trichloroacetic acid 10% (w/v), per-