Physiological adaptations to phosphorus deficiency during proteoid root development in white lupin

Release of large amounts of citric acid from specialized root clusters (proteoid roots) of phosphorus (P)-deficient white lupin (Lupinus albus L.) is an efficient strategy for chemical mobilization of sparingly available P sources in the rhizosphere. The present study demonstrates that increased accumulation and exudation of citric acid and a concomitant release of protons were predominantly restricted to mature root clusters in the later stages of P deficiency. Inhibition of citrate exudation by exogenous application of anion-channel blockers such as ethacrynic- and anthracene-9-carboxylic acids may indicate involvement of an anion channel. Phosphorus-deficiency-induced accumulation and subsequent exudation of citric acid seem to be a consequence of both increased biosynthesis and reduced metabolization of citric acid in the proteoid root tissue, indicated by increased in-vitro activity and enzyme protein levels of phosphoenolpyruvate carboxylase (EC 4.1.1.31), and reduced activity of aconitase (EC 4.2.1.3) and root respiration. Similar to citric acid, acid phosphatase, which is secreted by roots and involved in the mobilization of the organic soil P fraction, was released predominantly from proteoid roots of P-deficient plants. Also ³³Pi uptake per unit root fresh-weight was increased by approximately 50% in juvenile and mature proteoid root clusters compared to apical segments of non-proteoid roots. Kinetic studies revealed a K _m of 30.7 μM for Pi uptake of non-proteoid root apices in P-sufficient plants, versus K _m values of 8.5–8.6 μM for non-proteoid and juvenile proteoid roots under P-deficient conditions, suggesting the induction of a high-affinity Pi-uptake system. Obviously, P-deficiency-induced adaptations of white lupin, involved in P acquisition and mobilization of sparingly available P sources, are predominantly confined to proteoid roots, and moreover to distinct stages during proteoid root development. Received: 10 September 1998 / Accepted: 22 December 1998     

Kinetic properties of phosphoenolpyruvate carboxylase from lupin nodules and roots

The kinetic properties of two forms of phosphoenolpyruvate carboxylase (PEPC I and PEPC II, EC 4.1, 1.31) from lupin ( Lupinus luteus L. cv. Ventus) nodules and one enzyme form (PEPC III) from roots were studied. The Michaelis constant (K_m) values for PEP, Mg²⁺ and especially HCO₃⁻were lower for PEPC I. Kinetic studies showed that aspartate is a competitive inhibitor at pH 7.2 and inhibitor constant (K_i) values are different for the three forms of PEPC. Malate is a competitive inhibitor for PEPC I and PEPC III and shows mixed-type inhibition for PEPC II. Malate inhibition is dependent upon the pH of the assay. Different effect of several metabolites was also observed. The temperature optimum was near 39°C for PEPC I and around 43°C for PEPC II and PEPC III. PEPC I appeared to be the most thermolabile. It is suggested that PEPC I from lupin nodules is closely associated with N₂ fixation.     

Adaptation of H+-pumping and plasma membrane H+ ATPase activity in proteoid roots of white lupin under phosphate deficiency

White lupin (Lupinus albus) is able to adapt to phosphorus deficiency by producing proteoid roots that release a huge amount of organic acids, resulting in mobilization of sparingly soluble soil phosphate in rhizosphere. The mechanisms responsible for the release of organic acids by proteoid root cells, especially the trans-membrane transport processes, have not been elucidated. Because of high cytosolic pH, the release of undissociated organic acids is not probable. In the present study, we focused on H+ export by plasma membrane H+ ATPase in active proteoid roots. In vivo, rhizosphere acidification of active proteoid roots was vanadate sensitive. Plasma membranes were isolated from proteoid roots and lateral roots from P-deficient and -sufficient plants. In vitro, in comparison with two types of lateral roots and proteoid roots of P-sufficient plants, the following increase of the various parameters was induced in active proteoid roots of P-deficient plants: (a) hydrolytic ATPase activity, (b) Vmax and Km, (c) H+ ATPase enzyme concentration of plasma membrane, (d) H+-pumping activity, (e) pH gradient across the membrane of plasmalemma vesicles, and (f) passive H+ permeability of plasma membrane. In addition, lower vanadate sensitivity and more acidic pH optimum were determined for plasma membrane ATPase of active proteoid roots. Our data support the hypothesis that in active proteoid root cells, H+ and organic anions are exported separately, and that modification of plasma membrane H+ ATPase is essential for enhanced rhizosphere acidification by active proteoid roots.     

Transgenic proteoid roots of white lupin: a vehicle for characterizing and silencing root genes involved in adaptation to P stress

White lupin (Lupinus albus L.) has become an illuminating model for the study of plant adaptation to phosphorus (P) deficiency. It adapts to -P stress with a highly coordinated modification of root development and biochemistry resulting in short, densely clustered secondary roots called proteoid (or cluster) roots. In order to characterize genes involved in proteoid root formation and function in a homologous system, we have developed an Agrobacterium rhizogenes-based transformation system for white lupin roots that allows rapid analysis of reporter genes as well as RNA interference (RNA(i))-based gene silencing. We used this system to characterize a lupin multidrug and toxin efflux (Lupinus albus MULTIDRUG AND TOXIN EFFLUX, LaMATE) gene previously shown to have enhanced expression under -P stress. Here, we show that LaMATE had high expression in proteoid roots not only under -P, but also under -Fe, -N, -Mn and +Al stress. A portion containing the putative LaMATE promoter was fused to GUS and enhanced green fluorescence protein (EGFP) reporter genes, and a translational LaMATE::EGFP fusion was constructed under control of the LaMATE promoter. The LaMATE promoter directed P-dependent GUS and EGFP expression to proteoid roots. Confocal microscopy in white lupin and Arabidopsis point to the plasma membrane as the likely location of the LaMATE protein. LaMATE displayed homology to FRD3 in Arabidopsis, but did not complement an Arabidopsis ferric reductase defective 3 (FRD3) mutant. RNA(i)-based gene silencing was shown to effectively reduce LaMATE expression in transformed white lupin roots. LaMATE RNAi-silenced plants displayed an about 20% reduction in dry weight.     

Metabolic regulation and gene expression of root phosphoenolpyruvate carboxylase by different nitrogen sources

Alfalfa (Medicago sativa L.) N-sufficient plants were fed 1·5 mM N in the form of NO₃⁻, NH₄⁺ or NO₃⁻ in conjunction with NH₄⁺, or were N-deprived for 2 weeks. The specific activity of phosphoenolpyruvate carboxylase (PEPC) from the non-nodulated roots of N-sufficient plants was increased in comparison with that of N-deprived plants. The PEPC value was highest with NO₃⁻ nutrition, lowest with NH₄⁺ and intermediate in plants that were fed mixed salts. The protein was more abundant in NO₃⁻-fed plants than in either NH₄⁺- or N mixed-fed plants. Nitrogen starvation decreased the level of PEPC mRNA, and nitrate was the N form that most stimulated PEPC gene expression. The malate content was significantly lower in NO₃⁻-deprived than in NO₃⁻-sufficient plants. Root malate accumulation was high in NO₃⁻-fed plants, but decreased significantly in plants that were fed with NH₄⁺. The effect of malate on the desalted enzyme was also investigated. Root PEPC was not very sensitive to malate and PEPC activity was inhibited only by very high concentrations of malate. Asparagine and glutamine enhanced PEPC activity markedly in NO₃⁻-fed plants, but failed to affect plants that were either treated with other N types or N starved. Glutamate and citrate inhibited PEPC activity only at optimal pH. N-nutrition also influenced root nitrate and ammonium accumulation. Nitrate accumulated in the roots of NO₃⁻- and (NO₃⁻ + NH₄⁺)-fed plants, but was undetectable in those administered NH₄⁺. Both the nitrate and the ammonium contents were significantly reduced in NO₃⁻- and (NO₃⁻ + NH₄⁺)-starved plants. Root accumulation of free amino acids was strongly influenced by the type of N administered. It was highest in NH₄⁺-fed plants and the most abundant amides were asparagine and glutamine. It was concluded that root PEPC from alfalfa plants is N regulated and that nitrate exerts a strong influence on the PEPC enzyme by enhancing both PEPC gene expression and activity.     

Iron deficiency induces cluster (proteoid) root formation in Casuarina glauca

The effect of iron deficiency, phosphorus, NaHCO₃, chelator supply and nitrogen source on the formation of cluster (proteoid) roots was investigated in Casuarina glauca growing in water culture. The addition of iron-binding chelators (e.g. EDDHA, DTPA, EDTA) or increase in nutrient solution pH with NaHCO₃ resulted in the formation of cluster roots when plants were grown in solution lacking iron. Phosphorus supply even at a concentration of 500 µM did not inhibit cluster root formation if EDDHA was added to the iron-deficient medium. Cluster root formation was influenced significantly by nitrogen source and occurred only in nitrate-fed plants.C. glauca seemed to be very sensitive to iron deficiency as shown by plant chlorosis when grown on alkaline soil. The symptoms of chlorosis decreased as the chlorophyll content in shoots and the number of cluster roots increased, suggesting that the alleviation of iron deficiency in plant tissues was correlated with cluster root formation. It appears that iron deficiency is more important than phosphorus deficiency in inducing the formation of cluster roots in C. glauca.     

Molecular control of acid phosphatase secretion into the rhizosphere of proteoid roots from phosphorus-stressed white lupin

White lupin (Lupinus albus) grown under P deficiency displays a suite of highly coordinated adaptive responses. Included among these is secretion of copious amounts of acid phosphatase (APase). Although numerous reports document that plants secrete APases in response to P deficiency, little is known of the biochemical and molecular events involved in this process. Here we characterize the secreted APase protein, cDNA, and gene from white lupin. The secreted APase enzyme is a glycoprotein with broad substrate specificity. It is synthesized as a preprotein with a deduced M(r) of 52,000 containing a 31-amino acid presequence. Analysis of the presequence predicts that the protein is targeted to outside the cell. The processed protein has a predicted M(r) of 49,000 but migrates as a protein with M(r) of 70,000 on sodium dodecyl sulfate gels. This is likely due to glycosylation. Enhanced expression is fairly specific to proteoid roots of P-stressed plants and involves enhanced synthesis of both enzyme protein and mRNA. Secreted APase appears to be encoded by a single gene containing seven exons interrupted by six introns. The 5'-upstream putative promoter of the white lupin-secreted APase contains a 50-base pair region having 72% identity to an Arabidopsis APase promoter that is responsive to P deficiency. The white lupin-secreted APase promoter and targeting sequence may be useful tools for genetically engineering important proteins from plant roots.     

Alfalfa root nodule phosphoenolpyruvate carboxylase: characterization of the cDNA and expression in effective and plant-controlled ineffective nodules

Phosphoenolpyruvate carboxylase (PEPC) plays a key role in N₂ fixation and ammonia assimilation in legume root nodules. The enzyme can comprise up to 2% of the soluble protein in root nodules. We report here the isolation and characterization of a cDNA encoding the nodule-enhanced form of PEPC. Initially, a 2945 bp partial-length cDNA was selected by screening an effective alfalfa nodule cDNA library with antibodies prepared against root nodule PEPC. The nucleotide sequence encoding the N-terminal region of the protein was obtained by primer-extension cDNA synthesis and PCR amplification. The complete amino acid sequence of alfalfa PEPC was deduced from these cDNA sequences and shown to bear striking similarity to other plant PEPCs. Southern blots of alfalfa genomic DNA indicate that nodule PEPC is a member of a small gene family. During the development of effective root nodules, nodule PEPC activity increases to a level that is 10- to 15-fold greater than that in root and leaf tissue. This increase appears to be the result of increases in amount of enzyme protein and PEPC mRNA. Ineffective nodules have substantially less PEPC mRNA, enzyme protein and activity than do effective nodules. Maximum expression of root nodule PEPC appears to be related to two signals. The first signal is associated with nodule initiation while the second signal is associated with nodule effectiveness. Regulation of root nodule PEPC activity may also involve post-translational processes affecting enzyme activity and/or degradation.     

Phosphate availability regulates root system architecture in Arabidopsis

Plant root systems are highly plastic in their development and can adapt their architecture in response to the prevailing environmental conditions. One important parameter is the availability of phosphate, which is highly immobile in soil such that the arrangement of roots within the soil will profoundly affect the ability of the plant to acquire this essential nutrient. Consistent with this, the availability of phosphate was found to have a marked effect on the root system architecture of Arabidopsis. Low phosphate availability favored lateral root growth over primary root growth, through increased lateral root density and length, and reduced primary root growth mediated by reduced cell elongation. The ability of the root system to respond to phosphate availability was found to be independent of sucrose supply and auxin signaling. In contrast, shoot phosphate status was found to influence the root system architecture response to phosphate availability.