Nitric oxide is involved in phosphorus deficiency-induced cluster-root development and citrate exudation in white lupin.

*White lupin (Lupinus albus) forms specialized cluster roots characterized by exudation of organic anions under phosphorus (P) deficiency. Here, the role of nitric oxide (NO) in P deficiency-induced cluster-root formation and citrate exudation was evaluated. *White lupin plants were treated with the NO donor sodium nitroprusside (SNP) and scavenger or inhibitor of NO synthase under conditions of P deficiency (0 muM) or P sufficiency (50 muM). *Phosphorus deficiency enhanced NO production in primary and lateral root tips, with a greater increase in cluster roots than in noncluster roots. NO concentrations decreased with cluster root development from the pre-emergent stage, through the juvenile stage, to the mature stage. The P deficiency-induced increase in NO production was inhibited by antagonists of NO synthase and xanthine oxidoreductase, suggesting the involvement of these enzymes in NO production. SNP markedly increased the number of cluster roots. Citrate exudation from different root segments in P-deficient roots was positively correlated with endogenous root NO concentrations. *These findings demonstrate differential patterns of NO production in white lupin, depending on root zone, developmental stage and P nutritional status. NO appears to play a regulatory role in the formation of cluster roots and citrate exudation in white lupin under conditions of P deficiency.

Overexpression of malate dehydrogenase in transgenic alfalfa enhances organic acid synthesis and confers tolerance to aluminum.

Al toxicity is a severe impediment to production of many crops in acid soil. Toxicity can be reduced through lime application to raise soil pH, however this amendment does not remedy subsoil acidity, and liming may not always be practical or cost-effective. Addition of organic acids to plant nutrient solutions alleviates phytotoxic Al effects, presumably by chelating Al and rendering it less toxic. In an effort to increase organic acid secretion and thereby enhance Al tolerance in alfalfa (Medicago sativa), we produced transgenic plants using nodule-enhanced forms of malate dehydrogenase and phosphoenolpyruvate carboxylase cDNAs under the control of the constitutive cauliflower mosaic virus 35S promoter. We report that a 1.6-fold increase in malate dehydrogenase enzyme specific activity in root tips of selected transgenic alfalfa led to a 4.2-fold increase in root concentration as well as a 7.1-fold increase in root exudation of citrate, oxalate, malate, succinate, and acetate compared with untransformed control alfalfa plants. Overexpression of phosphoenolpyruvate carboxylase enzyme specific activity in transgenic alfalfa did not result in increased root exudation of organic acids. The degree of Al tolerance by transformed plants in hydroponic solutions and in naturally acid soil corresponded with their patterns of organic acid exudation and supports the concept that enhancing organic acid synthesis in plants may be an effective strategy to cope with soil acidity and Al toxicity.

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.

Phosphorus deficiency in Lupinus albus. Altered lateral root development and enhanced expression of phosphoenolpyruvate carboxylase.

The development of clustered tertiary lateral roots (proteoid roots) and the expression of phosphoenolpyruvate carboxylase (PEPC, EC 4.1.1.31) in roots were studied in white lupin (Lupinus albus L.) grown with either 1 mM P (+P-treated) or without P (-P-treated). The +P-treated plants initiated fewer clustered tertiary meristems and the emergence of these meristems was delayed compared with - P-treated plants. Proteoid root zones could be identified 9 d after emergence in both P treatments. Amounts of PEPC mRNA, PEPC specific activity, and enzyme protein were greater in proteoid roots than in normal roots beginning at 10, 12, and 14 d after emergence, respectively. The increases in PEPC mRNA, PEPC enzyme, and PEPC specific activity suggest that this enzyme is in part under transcriptional regulation. Recovery of organic acids from root exudates coincided with the increases in PEPC specific activity. The -P-treated plants exuded 40-, 20-, and 5-fold more citrate, malate, and succinate, respectively, than did +P-treated plants. Data presented support the hypothesis that white lupin has concerted regulation of proteoid root development, transcriptional regulation of PEPC, and biosynthesis of organic acids for exudation in response to P deficiency.

Root Carbon Dioxide Fixation by Phosphorus-Deficient Lupinus albus (Contribution to Organic Acid Exudation by Proteoid Roots).

When white lupin (Lupinus albus L.) is subjected to P deficiency lateral root development is altered and densely clustered, tertiary lateral roots (proteoid roots) are initiated. These proteoid roots exude large amounts of citrate, which increases P solubilization. In the current study plants were grown with either 1 mM P (+P-treated) or without P (-P-treated). Shoots or roots of intact plants from both P treatments were labeled independently with 14CO2 to compare the relative contribution of C fixed in each with the C exuded from roots as citrate and other organic acids. About 25-fold more acid-stable 14C, primarily in citrate and malate, was recovered in exudates from the roots of -P-treated plants compared with +P-treated plants. The rate of in vivo C fixation in roots was about 4-fold higher in -P-treated plants than in +P-treated plants. Evidence from labeling intact shoots or roots indicates that synthesis of citrate exuded by -P-treated roots is directly related to nonphotosynthetic C fixation in roots. C fixed in roots of -P-treated plants contributed about 25 and 34% of the C exuded as citrate and malate, respectively. Nonphotosynthetic C fixation in white lupin roots is an integral component in the exudation of large amounts of citrate and malate, thus increasing the P available to the plant.

Isolation and Characterization of a Pseudomonas sp. That Mineralizes the s-Triazine Herbicide Atrazine.

A bacterium that was capable of metabolizing atrazine at very high concentrations (>1,000 ppm) was isolated from a herbicide spill site. The organism was differentiated by observing clearing zones on indicator agar plates containing 1,000 ppm atrazine. Detailed taxonomic studies identified the organism as a Pseudomonas sp., designated ADP, that was dissimilar to currently known species. Pseudomonas sp. strain ADP metabolized atrazine as its sole nitrogen source. Nongrowing suspended cells also metabolized atrazine rapidly; for example, 9 x 10(sup9) cells per ml degraded 100 ppm of atrazine in 90 min. Atrazine was metabolized to hydroxyatrazine, polar metabolites, and carbon dioxide. When uniformly ring-labeled [(sup14)C]atrazine was used, 80% of the radioactivity was liberated as (sup14)CO(inf2). These data indicated the triazine ring was completely mineralized. The isolation and characterization of Pseudomonas sp. strain ADP may contribute to efforts on atrazine bioremediation, particularly in environments containing very high pesticide levels.

Phosphorus Stress-Induced Proteoid Roots Show Altered Metabolism in Lupinus albus.

Proteoid roots develop in Lupinus albus L. in response to nutrient stress, especially P. Proteoid roots excrete citrate and thus increase the availability of P, Fe, and Mn in the rhizosphere. In an effort to understand citrate synthesis and organic acid metabolism in proteoid roots of lupin, we have evaluated in vitro enzyme activities of citrate synthase (CS), malate dehydrogenase (MDH), and phosphoenolpyruvate carboxylase (PEPC) in proteoid and normal roots of plants grown with or without P. Organic acid concentrations, respiration rates, and dark 14CO2-labeling patterns were also determined. The in vitro specific activities of CS, MDH, and PEPC and in vivo dark 14CO2 fixation were higher in proteoid roots compared to normal roots, particularly under P stress. Western blot analysis showed that PEPC enzyme protein was more highly expressed in -P proteoid roots compared to other tissues. The majority of the fixed 14C was found in organic acids, predominantly malate and citrate. A larger fraction of citrate was labeled in P- stressed proteoid roots compared to other root tissue. Respiration rates of proteoid roots were 31% less than those of normal roots. The data provide evidence for increased synthesis of citrate in proteoid roots compared to normal roots, particularly under P stress. A portion of the carbon for citrate synthesis is derived from nonautotrophic CO2 fixation via PEPC in proteoid roots.

Mineralization of the s-triazine ring of atrazine by stable bacterial mixed cultures.

Enrichment cultures containing atrazine (2-chloro-4-ethylamino-6-isopropylamino-1,3,5-triazine) at a concentration of 100 ppm (0.46 mM) as a sole nitrogen source were obtained from soils exposed to repeated spills of atrazine, alachlor, and metolachlor. Bacterial growth occurred concomitantly with formation of metabolites from atrazine and subsequent biosynthesis of protein. When ring-labeled [14C]atrazine was used, 80% or more of the s-triazine ring carbon atoms were liberated as 14CO2. Hydroxyatrazine may be an intermediate in the atrazine mineralization pathway. More than 200 pure cultures isolated from the enrichment cultures failed to utilize atrazine as a nitrogen source. Mixing pure cultures restored atrazine-mineralizing activity. Repeated transfer of the mixed cultures led to increased rates of atrazine metabolism. The rate of atrazine degradation, even at the elevated concentrations used, far exceeded the rates previously reported in soils, waters, and mixed and pure cultures of bacteria.

Proton and copper adsorption to maize and soybean root cell walls.

A surface complexation model which has been used to describe inner-sphere complexation on metal oxide surfaces was applied to the adsorption of Cu by isolated cell walls of 4-day and 28-day-old maize (Zea mays L. cv WF9 x Mo17) and 21-day-old soybean (Glycine max [L.] Merr. cv Dare) roots. Concentration dependence of the titration data prevented the determination of unique pK and capacitance values for the 4-day maize cell walls, though mean values obtained for the intrinsic pK of the titratable carboxyl groups were 3.0 (4-day maize), 3.6 (28-day maize), and 3.0 (21-day soybean) as determined by potentiometric titration with either NaOH or HCl in 20 millimolar NaCl. The constant capacitance model was applied to Cu sorption data from rapid batch equilibrium experiments in an ionic medium of 20 millimolar NaClO(4). Speciation calculations indicated that the formation of a bidentate surface complex was sufficient to describe the experimental data for all three types of plant material, with only one value for the pK and capacitance density. The intrinsic constants of Cu complexation by a neutral site are: log K = -0.3 +/- 0.1, -0.2 +/- 0.3, and 0.9 +/- 0.1 for 4-day and 28-day maize, and 21-day soybean, respectively. The integral capacitance density parameter, which describes the relationship between surface charge density and electrical potential, is several times larger than for crystalline mineral surfaces. This result indicates that the surface electrical potential remains low even when the surface charge density is high. Such behavior is characteristic of gels and porous oxides.