Phosphorus

APOPLASM

A. Phosphorus

1. Root-Induced Phosphorus Mobilization in Soils

Phosphorus (P) is one of the major limiting factors for plant growth in many soils. Plant availability of inorganic phosphorus (Pi) can be limited by formation of sparingly soluble Ca phosphates, particularly in alkaline and calcareous soils;

by adsorption to Fe- and Al-oxide surfaces in acid soils; and by formation of Fe/

AI-P complexes with humic acids (94). Phosphorus deficiency can significantly alter the composition of root exudates in a way that is, at least in some plant species, related to an increased ability for mobilization of sparingly soluble P sources (29,3 1,7 1 ).

Increased root exudation of carboxylates (e.g., citrate, malate, oxalate) is a P-deficiency response, particularly in dicotyledonous plant species. Mobiliza- tion of Pi by exogenous application of carboxylates to various soils with low P availability has been demonstrated in numerous studies (22,94-97) and seems to be mediated by mechanisms of ligand exchange, dissolution, and occupation of P sorption sites (e.g., Fe/AI-P and Ca-P) in the soil matrix (22). Citrate and oxalate were found to be the most efficient carboxylates with respect to P mobili- zation in many of these model experiments according to high stability constants for complex formation with Fe, AI, and Ca (7 1 ). To mediate significant desorption of Pi. however, carboxylate concentrations in the millimolar range are required i n the soil solution, associated with a carboxylate accumulation of > l 0 pmol g~

of rhizosphere soil (98,99). A similar concentration level in the rhizosphere has so far been reported for only a limited number of plant species such as Lupinus n l b u s L. and members of the Proteaceae (22,27,31,45,52). For I cm apical root zones, where the most intense root exudation is frequently observed (see Sect.

2.2), the volume of rhizosphere soil solution can be calculated at about 30 pL cm

'

root length at a distance of I mm from the root surface (100). Assuming a residence time of 5 h for the root apex i n a given soil zone (42), carboxylate exudation at a rate of 6 nmol h" cm" root length is required to reach a concentra- tion level of I mM. Although the impact of microbial degradation (42) and the mechanical impedance of the soil matrix on root exudation ( 4 3 ) was not taken into account, this is similar to the exudation rates of citrate reported for proteoid roots of P-deficient Luyir1lr.s crll~lrs L. (17.35), which is a plant species with a proven ability for citrate-mediated P mobilization in the rhizosphere (22,33,34).

In cluster-rooted plant species, carboxylate accumulation i n the proteoid rhizo- sphere is further promoted by the high density of carboxylate-excreting root tips, which exhibit no more growth activity (3 l ) . Intense exudation of carboxylic acids in response to P deficiency has been reported also for oil-seed rape ( 101 ), spinach (102), red clover (99). and chickpea (17,82). Striim et al. (10.3) have suggested that P (and Fe) mobilization, mediated by root exudation of citrate and oxalate, might be related to the ability of various calcicole plant species to grow on calcar- eous soils. I n many studies, however. interpretation of data is biased by a lack of information about spatial variation of exudation along the root system and by suboptimal conditions during exudate collection (e.g., long-term collection under nonsterile conditions using inappropriate collection media-collection from root systems removed from solid substrates (see Sect. 2).

Although in many soils with low P availability, significant desorption of sparingly soluble Pi fornls requires at least millimolar concentration levels of specific carboxylates (e.g., citrate, oxalate) in the soil solution, much lower con- centrations (0. I 1nM) were necessary to reduce soil adsorption of Pi. which was applied simultaneously with carboxylates [ 100). Thus, competition of carboxyl- ates with pi for P sorption sites in the soil matrix may be a mechanism that can,

to some extent, prevent soil-fixation of Pi after fertilizer application even in plant species with moderate exudation rates.

2. Physiology of Carboxylate Exudation

Only limited information is available on the physiological basis of P deficiency- induced root exudation of carboxylates. Increased carboxylate release is fre- quently observed in later stages of P deficiency ( I 1,82). Major exudate com- pounds are malate, citrate, and also oxalate, especially in plant species where oxalate replaces malate as the major internal carboxylate anion, e.g. members of the Chenopodiaceae (99,102,104). Solution culture experiments have revealed that plant species with intense P deficiency-induced carboxylate exudation-such as oil-seed rape (101). chickpea, and white lupin (82)--accumulated organic acids mainly in the root tissue and, moreover, i n the root zones where exudation was most intense (e.g., subapical root zones, proteoid roots). In contrast, root exudation of carboxylates even decreased in response to P deficiency in plant species such as SysimOr-ium cd$cirlalr ( I O I ) , wheat, and tomato (82) and was associated with predominant carboxylate accumulation in the shoots (Fig. 4). The results suggest that accumulation of organic acids in the root tissue is a prerequi- site for enhanced root exudation of carboxylates under P-deficient conditions and may be determined by shoot/root partitioning of carboxylates or of carbohydrates as related precursors. Differential ‘+‘CO2 pulse-chase labeling experiments with shoots and roots of white lupin revealed predominant biosynthesis of carboxylates in the root tissue and particularly in proteoid roots under P-deficient conditions ( 1 l ) . This is i n good agreement with enhanced expression and in vitro activities of a specific set of glycolytic enzymes, such as sucrose synthase, phosphogluco- mutase, fructokinase, PPi-dependent phosphofructokinase, and PEP carboxylase detected in P-deficient root tissues (Fig. 5 ) . These enzymes may operate as an alternative pathway of carbohydrate catabolism under P-deficient conditions, which facilitates a more economic Pi utilization by Pi recycling. minimization of Pi consumption, and utilization of alternative P pools such as PPi ( 1 OS- 107).

Phosohorus deprivation seems to be generally associated with increased shoot- to-root allocation of carbohydrates, at least in the initial stages of P deficiency (108-1 IO). The ability to maintain glycolytic carbohydrate catabolism under P- deficient conditions is a prerequisite for the utilization of carbohydrates and thus also for the biosynthesis of carboxylates in the root tissue. However, Hoffland et al. (101) stated that at least a part of the carboxylates that accumulated in roots of P-deficient oil-seed rape was synthesized i n the shoot.

Nonphotosynthetic C O z tixation via phosphoenolpyruvate carboxylase (PEPC) can contribute a substantial proportion of carbon (>30%) for the biosyn- thesis of carboxylates in roots of P-deficient plants (Fig. S) ( I 1,82,101,1 1 1 - 1 13).

Thus, PEPC-mediated COz fixation may be interpreted as an anaplerotic carbon

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accumulation in root tissue

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P defidency-induced root exudation of carboxylates

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Tomato Syslm- Wheat Chick- Rape WhiteLupin roots roots

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Figure 4 P deficiency-induced changes in tissue concentrations and root exudation of

carboxylates in different plant species. The zero line represents the P-sufficient control.

(Adapted from Refs. 82 and 102.)

- activation

= = m = =

inhibition

Figure 5 Model of phosphorus (P) deficiency-induced physiologlcal changes associ-

ated with the release of P-mobilizing root exudates in cluster roots of white lupin. Solid lines indicate stimulation and dotted lines inhibition of biochemical reaction sequences or metabolic pathways in response to P deficiency. For a detailed description see Sec. 4.1.

Abbreviations: SS = sucrose synthase; FK = fructokinase; PGM = phosphoglucomutase;

PEP = phosphoenolpyruvate; PEPC = PEP-carboxylase; MDH = malate dehydrogenase;

ME = malic enzyme; CS = citrate synthase; PDC = pyruvate decarboxylase; ALDH = alcohol dehydrogenase; E-4-P = erythrose-4-phosphate; D A W = dihydroxyacetonephos- phate; APase = acid phosphatase.

supply to compensate for carbon losses related with root exudation of carboxyl- ates, which can make up between 5 and 25% of photosynthetic net fixation of CO? ( 1 1,7 1 , l 12). Phosphorus deficiency-induced induction of PEPC is regulated at the transcriptional and also at the posttranslational level by protein phosphory- lation ( l 12- l 14). The cytosolic enzyme catalyzes the carboxylation of phospho- enolpyruvate (PEP) to oxaloacetate, which can be further converted to malate by enhanced expression of cytosolic malate dehydrogenase (MDH) (35,112,l 14).

Moreover, the PEPC reaction results in liberation of Pi from PEP and may be therefore regarded as an alternative pathway for PEP catabolization via pyruvate kinase, which depends on the presence of ADP and Pi (106).

Especially in dicotyledonous plant species such as tomato, chickpea, and white lupin (82,l 1 l), with a high cation/anion uptake ratio, PEPC-mediated bio- synthesis of carboxylates may also be linked to excessive net uptake of cations due to inhibition of uptake and assimilation of nitrate under P-deficient conditions (Fig. 5) (17.1 1 1.1 15). Excess uptake of cations is balanced by enhanced net re- lease of protons (82,l I 1,116), provided by increased biosynthesis of organic acids via PEPC as a constituent of the intracellular pH-stat mechanism ( 1 17). In these plants, P deficiency-mediated proton extrusion leads to rhizosphere acidi- fication, which can contribute to the solubilization of acid soluble Ca phosphates in calcareous soils (Fig. 5 ) (34,l 18,l 19). In some species (e.g., chickpea, white lupin, oil-seed rape, buckwheat), the enhanced net release of protons is associated with increased exudation of carboxylates, whereas in tomato, carboxylate exuda- tion was negligible despite intense proton extrusion (82,120).

In many plants, citrate and malate are the major carboxylate anions that

accumulate i n the root tissue in response to P deprivation (82,101, I 12,l 13). In- creased citrate accumulation is frequently associated not only with enhanced ac- tivity of PEPC but also with a reduction in aconitase activity (82), which is in- volved in the turnover of citrate within the tricarboxylic acid (TCA) cycle. Thus, citrate accumulation is probably a consequence of both increased biosynthesis and reduced turnover ofcitrate under P-deficient conditions (Fig. 5). Citrate accu- mulation during proteoid root development in white lupin was also associated with reduced respiration and a concomitant decrease of soluble intracellular Pi (17,l 12). It was suggested that Pi limitation of the respiratory chain may induce a feedback inhibition of citrate turnover in the TCA cycle, in order to prevent excessive production of reducing equivalents (Fig. S) (17). Accordingly. in roots of Phaseol~rs vdgrrris L., P deprivation decreased the activity of the respiratory cytochrome pathway but increased the ratio of NADH/NAD and cyanide-resis- tant respiration ( 12 1 ). The P deficiency-induced inhibition of nitrate assimilation (see above) may also contribute to increased citrate accumulation in the root tissue by downregulation of citrate conversion to 2-oxoglutarate (Fig. 5 ) . which is an important acceptor for amino N as a product of NO3- reduction (122). From these findings, it may be concluded that increased accumulation of organic acids

and particularly of citrate in the root tissue is a P deficiency-induced metabolic disorder, and the release of high amounts of citrate and protons into the rhizo- sphere in some plant species might serve as a detoxification mechanism to prevent cytoplasmic acidosis. A similar mechanism has been discussed for the detoxifica- tion of lactic acid, which accumulates in root tips of maize under hypoxic condi- tions ( 1 23) with a comparable intracellular carboxylate concentration (20-30 pmol g" root fresh weight) and similar exudation rates (2000-5700 nmol h"

g" root fresh weight) as observed in proteoid roots of P-deficient white lupin ( 17,7 I , 123). Besides of increased root exudation of carboxylates and protons, some othcr strategies may also prevent excessive accumulation of organic acids in the root tissue of P-deficient plants. Increased contribution of the Pi-independent cyanide-resistant pathway to root respiration under P deprivation, which was de- tected i n roots of Phtrsrolus w l g n r i s L. ( 124), although not associated with ATP production, may enable the operation of the TCA cycle, and thus the turnover of citrate under P-deficient conditions. Similarly, an ATP-dependent citrate lyase, which is predominantly expressed in juvenile proteoid roots of white lupin during early stages of P deficiency-induced citrate accumulation, may be involved in the cleavage of citrate into oxaloacetate and acetyl-coA. In later stages of pro- teoid root development, the activity of citrate lyase decreases, probably due to ATP limitation, associated with increased accumulation and finally increased ex- udation of citrate (17) (Massonneau et al., unpublished). Another function of citrate lyase i n P-deficient root tissue may be the redistribution of acetyl-coA for the biosynthesis of lipids and phenolics (see Sect. 4.1.3.) under metabolic conditions favoring the accumulation of di- and tricarboxylates. Increased root to shoot translocation of carboxylates in P-deficient Ricinus conzmmi.v L. has been reported by Jeschke et al. ( 1 2S), and may be related to the higher storage capacity for carboxylates in the leaf vacuoles (17).

It was suggested that root exudation of high amounts of carboxylates in some P-deficient plant species is mediated by anion channels with a concomittant release of protons via plasmalemma H'-ATPase (Fig. 5 ) to maintain charge bal- ance ( l 1,17,34). However, enhanced leakiness of membranes in response to P deprivation may also contribute to enhanced release of sugars, amino acids, and organic acids (84; see also Chap. 2). Schilling et al. (29) reported a shift i n the qualitative composition of sugars i n root exudates of maize and pea, leading to a higher proportion of pentoses at the expense of glucose and sucrose under P- deficient conditions. P deficiency-induced root exudation of sugars and amino acids has been related to increased mycorrhizal colonization (126,127).

3. Exudation of Phenolic Compounds

I n many plants, P deficiency also enhances production and root exudation of phenolic compounds (Fig. S) (27,3 1,128- 130). Increased biosynthesis of pheno-

lics under P-deficient conditions was suggested as another metabolic bypass reac- tion involved in liberation and recycling of Pi in P-starved cells (106). Antibiotic properties of certain phenolic compounds (e.g., isoflavonoids) in root exudates (69) may not only counteract infection by root pathogens but also prevent the microbial degradation of exudate compounds involved in P mobilization (31).

Certain root flavonoids have been identified as signal molecules for spore germi- nation and hyphal growth of arbuscular mycorrhizae, and flavonoids are likely to be important also as signaling compounds for the establishment of ectomy- corrhizae ( 1 3 I , 132) (this subject is reviewed in Chap. 7). Phenolics may further contribute to P mobilization by reduction of sparingly soluble Fell1 phosphates (Fig. S) (3 l ) . The specific release of piscidic acid (p-hydroxyphenyl tartaric acid) from roots of P-deficient pigeon pea (CNjcrn~rs cc+uz L.), which is a strong chela- tor for FeIII, has been related to enhanced mobilization of Fe-phosphates in Alfi- sols ( 133). However, considering the comparatively low exudation rates, piscidic acid may be more relevant as a signaling compound for the establishment of microbial associations (e.g.. arbuscular mycorrhiza (AM), rhizobia).

4. Root-Secretory Phosphohydrolases

Enhanced secretion of acid phosphatases (APase) (24,52,134) and phytases ( 1 35) by plant roots and also by rhizosphere microorganisms (136) under P-deficient conditions may contribute to Pi acquisition by hydrolysis of organic P esters in the rhizosphere (Fig. S), which can comprise up to 30-80% of the total soil phosphorus. In many soils, however, the availability of organic phosphorus seems to be limited mainly by the low solubility of certain P forms, such as Ca- and Fe/Al-phytates, which can make up a major propotion of the soil-organic P ( 137- 139). Beissner ( I 04) reported that oxalic acid in root exudates can contribute to some extent to phytate mobilization in soils. Similarly, in ^;I P-deficient sandy soil, more Pi was liberated by simultaneous application of acid phosphatase and organic acids identified in rhizosphere soil solution of Hclkm ltrzcllrlrrrcr than by separate application of organic acids or acid phosphatase, respectively (Fig. 6).

Another limiting factor for phosphatase-mediated P mobilization is the low mo- bility of the hydrolytic enzymes (APase, phytase), mainly associated with the root cell wall and with mucilage in apical root zones (140). An alternative function of root secretory acid phosphatases may be the rapid retrieval of phosphorus by hydrolysis of organic P, which is permanently lost by diffusion or from sloughed off and damaged root cells (Fig. S ) (141).

B. Nitrogen and Potassium