Defense Proteins from Vigna Unguiculata Seed Exudates: Characterization and Inhibitory Activity Against Fusarium Oxysporum

Plants exude a variety of substances through their roots, germinating seeds and aerial parts. Some of these released compounds seem to have an inhibitory effect against pathogens. The aim of this work was to investigate and identify antifungal proteins present in exudates from imbibed cowpea seeds (Vigna unguiculata (L.) Walp). The obtained exudation was analyzed in regard to specific protein activities by enzymatic or immunological assays for plant defense proteins, from 4 h to 48 h of seed imbibition. Our results show that cowpea seeds exudates present several defense related proteins characterized as β-1,3-glucanases, cystatins, vicilins and lipid transfer proteins (LTPs), as well as a storage vacuole membrane α-TIP protein, since the very first hours of imbibition. These exudates also have an “in vitro” inhibitory effect on the growth of the fungus Fusarium oxysporum f. sp. phaseoli. Our results suggest that seed exudates should promote seed protection from soil pathogens.     

Purification of lipase from Cunninghamella verticillata by stepwise precipitation and optimized conditions for crystallization

Lipase from the oil-mill waste isolate Cunninghamella verticillata was purified by stepwise precipitation using acetone, as a sequel to our earlier conventional column chromatographic method [Gopinath et al. (2002)World Journal of Microbiology and Biotechnology 18, 449–458]. The yield of purified lipase was approx. 4-fold higher than by the previous method and the purified lipase was obtained with 70–80% acetone saturations. The enzyme was resolved as a single band with homogeneity both by native and by SDS–PAGE. The optimum condition for the lipase to crystallize was 5 g of enzyme in 0.05 M sodium phosphate buffer (pH 6.5) with 5 mM FeCl₂ and 10% 2-methyl 2,4-pentanediol (MPD).These authors equally contributed to this work     

Myrosinases from root and leaves of Arabidopsis thaliana have different catalytic properties

Myrosinases (EC 3.2.1.147) are β-thioglucoside glucosidases present in Brassicaceae plants. These enzymes serve to protect plants against pathogens and insect pests by initiating breakdown of the secondary metabolites glucosinolates into toxic products. Several forms of myrosinases are present in plants but the properties and role of different isoenzymes are not well understood. The dicot plant model organism Arabidopsis thaliana seems to contain six myrosinase genes (TGG1–TGG6). In order to compare the different myrosinases, cDNAs corresponding to TGG1 from leaves and TGG4 and TGG5 from roots were cloned and overexpressed in Pichia pastoris. The His-tagged recombinant proteins were purified using affinity chromatography and the preparations were homogenous according to SDS–PAGE analysis. Myrosinase activity was confirmed for all forms and compared with respect to catalytic activity towards the allyl-glucosinolate sinigrin. There was a 22-fold difference in basal activity among the myrosinases. The enzymes were active in a broad pH range, are rather thermostable and active in a wide range of salt concentrations but sensitive to high salt concentrations. The myrosinases showed different activation–inhibition responses towards ascorbic acid with maximal activity around 0.7–1 mM. No activity was registered towards desulphosinigrin and this compound did not inhibit myrosinase activity towards sinigrin. All myrosinases also displayed O-β-glucosidase activity, although with lower efficiency compared to the myrosinase activity. The differences in catalytic properties among myrosinase isozymes for function in planta are discussed.     

Identification and partial purification of DnaK homologue from extremely halophilic archaebacteria, Halobacterium cutirubrum

The levels of synthesis of six proteins were increased at elevated growth temperature of the extremely halophilic archaebacterium Halobacterium cutirubrum. One of these proteins, with an apparent molecular mass of 97 kDa on sodium dodecylsulfate–polyacrylamide gel electrophoresis (SDS–PAGE), bound to an ATP-agarose column in the presence of 4 M NaCl, but not in the absence of salt, indicating that this protein retained its ATP-binding activity only at high salt concentration. The NH₂-terminal sequence of this protein and the internal sequences of the tryptic peptides covering 1/3 of the total number of residues coincided with that deduced from the nucleotide sequence of the dnaK gene isolated from H. cutirubrum. The results strongly suggest that this apparent 97-kDa protein is the gene product of dnaK, although the molecular mass calculated from the nucleotide sequence is only 68,495, much smaller than the value of this protein determined by SDS–PAGE. Ferguson plot analysis indicated that this protein showed anomalous mobility on SDS–PAGE. We have purified DnaK homologue to greater than 90% homogeneity with stepwise elution from an ATP-agarose column.     

Purification and characterization of a novel ribosome-inactivating protein from seeds of Trichosanthes kirilowii Maxim

A novel ribosome-inactivating protein, designated Trichosanthrip, was purified from mature seeds of Trichosanthes kirilowii Maxim by cation-exchange and gel-filtration chromatography. Trichosanthrip migrated as a single band in SDS–PAGE, with an apparent molecular mass of 13 kDa. The molecular mass of Trichosanthrip was 10,964.617 Da as determined by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Trichosanthrip showed N-glycosidase activity on 28 S rRNA and strongly inhibited cell-free protein synthesis, with an IC₅₀ of 1.6 ng/ml. Liquid chromatography–tandem mass spectrometry showed that Trichosanthrip was a novel protein with similar sequence to other proteins present in members of the Cucurbitaceae.     

Ecophysiological characterization of carnivorous plant roots: Oxygen fluxes,respiration, and water exudation

Various ecophysiological investigations on carnivorous plants in wet soils are presented. Radial oxygen loss from roots of Droseraceae to an anoxic medium was relatively low 0.02 – 0.07 mol(O₂) m^{– 2} s^–1 in the apical zone, while values of about one order of magnitude greater were found in both Sarracenia rubra roots and Genlisea violacea traps. Aerobic respiration rates were in the range of 1.6 – 5.6 mol kg^–1 (f.m.) s^–1 for apical root segments of seven carnivorous plant species and 0.4 – 1.1 mol kg^–1 (f.m.) s^–1 for Genlisea traps. The rate of anaerobic fermentation in roots of two Drosera species was only 5 – 14 % of the aerobic respiration. Neither 0.2 mM NaN₃ nor 0.5 mM KCN influenced respiration rate of roots and traps. In all species, the proportion of cyanide-resistant respiration was high and amounted to 65 – 89 % of the total value. Mean rates of water exudation from excised roots of 12 species ranged between 0.4 – 336 mm 3 kg^–1 (f.m.) s^–1 with the highest values being found in the Droseraceae. Exudation from roots was insensitive to respiration inhibitors. No significant difference was found between exudation rates from roots growing in situ in anoxic soil and those kept in an aerated aquatic medium. Carnivorous plant roots appear to be physiologically very active and well adapted to endure permanent soil anoxia.     

Identification of an ascorbate-dependent cytochrome<Emphasis Type="Italic"> b</Emphasis> of the tonoplast membrane sharing biochemical features with members of the cytochrome<Emphasis Type="Italic"> b</Emphasis>561 family

Two membrane-bound, ascorbate-dependent b-type cytochromes were identified in etiolated bean (Phaseolus vulgaris L.) hypocotyls. Following solubilization of microsomal membranes and anion-exchange chromatography at pH 8.0, two major cytochrome peaks (P-I and P-II) were separated. Both cytochromes were reduced by ascorbate and re-oxidized by monodehydroascorbate, but P-I reduction by ascorbate was higher and saturated at far lower concentrations of ascorbate with respect to P-II. The -band was symmetrically centered at 561 nm in P-I, but it was asymmetric in P-II with a maximum at 562 nm and shoulder at 557 nm. Ascorbate reduction of P-II, but not P-I, was inhibited by diethyl pyrocarbonate. Reduced P-II but not P-I was readily oxidized by certain ferric chelates, including FeEDTA and Fe-nitrilotriacetic acid. Purified P-I, associated with the plasma membrane, showed up as a 63-kDa glycosylated protein during sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) and behaved as a monomer of about 70 kDa during size-exclusion chromatography. P-I identified with a previously purified ascorbate-dependent b-type cytochrome of bean hypocotyl plasma membranes [P. Trost et al. (2000) Biochim Biophys Acta 1468:1–5]. Partially purified P-II, on the other hand, correlated with a heme-protein of 27 kDa in SDS–PAGE gels, was dimeric (60 kDa) during size-exclusion chromatography, and was associated with the tonoplast marker V-ATPase in sucrose gradients. The sequence of a peptide of 11 residues obtained by tryptic digestion of P-II was found to be identical to a segment of a putative cytochrome b561 of Zea mays and highly conserved in other related plant sequences, including that of Arabidopsis thaliana cytochrome b561-1 (CAA18169). The biochemical features fully support the assignment of P-II cytochrome to the family of cytochrome b561, ascorbate-dependent (CYBASC) cytochromes, which also includes cytochrome b561 of animal chromaffin granules. The presence of a cytochrome reducing ferric chelates on the tonoplast is consistent with the role of plant vacuoles in iron homeostasis.     

Expression of frutalin, an alpha-D-galactose-binding jacalin-related lectin, in the yeast Pichia pastoris

Frutalin is an α-d-galactose-binding lectin expressed in breadfruit seeds. Its isolation from plant is time-consuming and results in a heterogeneous mixture of different lectin isoforms. In order to improve and facilitate the availability of the breadfruit lectin, we cloned an optimised codifying frutalin mature sequence into the pPICZαA expression vector. This expression vector, designed for protein expression in the methylotrophic yeast Pichia pastoris, contains the Saccharomyces α-factor preprosequence to direct recombinant proteins into the secretory pathway. Soluble recombinant frutalin was detected in the culture supernatants and recognised by native frutalin antibody. Approximately 18–20 mg of recombinant lectin per litre medium was obtained from a typical small scale methanol-induced culture purified by size-exclusion chromatography. SDS–PAGE and Edman degradation analysis revealed that frutalin was expressed as a single chain protein since the four amino-acid linker peptide “T-S-S-N”, which connects α and β chains, was not cleaved. In addition, incomplete processing of the signal sequence resulted in recombinant frutalin with one Glu-Ala N-terminal repeat derived from the α-factor prosequence. Endoglycosidase treatment and SDS–PAGE analysis revealed that the recombinant frutalin was partly N-glycosylated. Further characterisation of the recombinant lectin revealed that it specifically binds to the monosaccharide Me-α-galactose presenting, nevertheless, lesser affinity than the native frutalin. Recombinant frutalin eluted from a size-exclusion chromatography column with a molecular mass of about 62–64 kDa, suggesting a tetrameric structure, however it did not agglutinate rabbit erythrocytes as native frutalin does. This work shows that the galactose-binding jacalin-related lectins four amino-acid linker peptide “T-S-S-N” does not undergo any proteolytic cleavage in the yeast P. pastoris and also that linker cleavage might not be essential for lectin sugar specificity.     

Isolation and characterization of a trypsin fraction from the pyloric ceca of chinook salmon (Oncorhynchus tshawytscha)

A trypsin fraction was isolated from the pyloric ceca of New Zealand farmed chinook salmon (Oncorhynchus tshawytscha) by ammonium sulfate fractionation, acetone precipitation and affinity chromatography. The chinook salmon enzyme hydrolyzed the trypsin-specific synthetic substrate benzoyl-dl-arginine-p-nitroanilide (dl-BAPNA), and was inhibited by the general serine protease inhibitor phenyl methyl sulfonyl fluoride (PMSF), and also by the specific trypsin inhibitors — soybean trypsin inhibitor (SBTI) and benzamidine. The enzyme was active over a broad pH range (from 7.5 to at least pH 10.0) at 25 °C and was stable from pH 4.0 to pH 10.0 when incubated at 20 °C, with a maximum at pH 8.0. The optimum temperature for the hydrolysis of dl-BAPNA by the chinook salmon enzyme was 60 °C, however, the enzyme was unstable at temperatures above 40 °C. The molecular mass of the chinook salmon trypsin was estimated as 28 kDa by SDS–PAGE.     

Reduction of Cr(VI) by Desulfovibrio vulgaris and Microbacterium sp.

Many industrial wastes contain Cr(VI), a carcinogen and mutagen, the toxicity of which can be ameliorated by reduction to Cr(III). Microbacterium sp. NCIMB 13776 andDesulfovibrio vulgaris NCIMB 8303 reduced Cr(VI) to Cr(III) anoxically using 25 mM sodium citrate buffer (pH 7), with 25 mM sodium acetate and 25 mM sodium formate as electron donors at 30 °C, under which conditions the rates of reduction of 500 M sodium chromate were 77 and 6 nmol h^–1 mg dry cell wt for D. vulgaris and Microbacterium sp., respectively, these being increased to 127 and 17 nmol h^–1 mg dry cell wt in the presence of 20 mM MOPS/NaOH buffer.     

Comparative analyses of three legume species reveals conserved and unique root extracellular proteins

Increasing evidence suggests that root extracellular proteins are involved in interactions between roots and their soil environment. In the present study, exudates released by 6‐day‐old roots of the three legume species white lupin (Lupinus albus), soybean (Glycine max), and cowpea (Vigna sinensis) were collected under axenic conditions, and their constitutively secreted proteomes were analyzed. Between 42 and 93 unique root extracellular proteins with 2 or more different peptide fragments per protein were identified by LC‐MS/MS. Functional annotation of these proteins classified them into 14–16 different functional categories. Among those 14 homologous proteins were identified in at least two legume species. Among the unique proteins, 58 in white lupin, 85 in soybean, and 31 in cowpea were specific for each plant species, and many of them were classified in the same functional categories. Interestingly, in contrast to soybean and cowpea, two protein bands of approximately 16 and 30 kDa were present on the SDS‐PAGE gel of white lupin. The identification of these bands revealed a class III chitinase and a thaumatin‐like protein. Both belong to the class of pathogenesis‐related proteins. The results imply that root extracellular proteins play important roles in the cross‐talk between plant roots and the rhizosphere.     

Turnover and distribution of root exudates of Zea mays

Decomposition and distribution of root exudates of Zea mays L. were studied by means of ¹⁴CO₂ pulse labeling of shoots on a loamy Haplic Luvisol. Plants were grown in two-compartment pots, where the lower part was separated from the roots by monofilament gauze. Root hairs, but not roots, penetrated through the gauze into the lower part of the soil. The root-free soil in the lower compartment was either sterilized with cycloheximide and streptomycin or remained non-sterile. In order to investigate exudate distribution, 3 days after the ¹⁴C labeling, the lower soil part was frozen and sliced into 15, one-mm thick layers using a microtome. Cumulative ¹⁴CO₂ efflux from the soil during the first 3 days after ¹⁴C pulse labeling did not change during plant growth and amounted to about 13–20% of the total recovered ¹⁴C (41–55% of the carbon translocated below ground). Nighttime rate of total CO₂ efflux was 1.5 times lower than during daytime because of tight coupling of exudation with photosynthesis intensity. The average CO₂ efflux from the soil with Zea mays was about 74 g C g^–1 day^–1 (22 g C m^–2 day^–1), although, the contribution of plant roots to the total CO₂ efflux from the soil was about 78%, and only 22% was respired from the soil organic matter. Zea mays transferred about 4 g m^–2 of carbon under ground during 26 days of growth. Three zones of exudate concentrations were identified from the distribution of the ¹⁴C-activity in rhizosphere profiles after two labeling periods: (1) 1–2 (3) mm (maximal concentration of exudates) 2) 3–5 mm (presence of exudates is caused by their diffusion from the zone 1); (3) 6–10 mm (very insignificant amounts of exudates diffused from the previous zones). At the distance further than 10 mm no exudates were found. The calculated coefficient of exudate diffusion in the soil was 1.9 × 10^–7 cm² s^–1.     

Relative importance of Na+, Cl−, and abscisic acid in NaCl induced inhibition of root growth of rice seedlings

The relative importance of endogenous abscisic acid (ABA), as well as Na⁺ and Cl^– in NaCl-induced responses related to growth in roots of rice seedlings were investigated. The increase in ammonium, proline and H₂O₂ levels, and cell wall peroxidase (POD) activity has been shown to be related to NaCl-inhibited root growth of rice seedlings. Increasing concentrations of NaCl from 50 to 150 mM progressively decreased root growth and increased both Na⁺ and Cl^–. Treatment with NaCl in the presence of 4,4-diisothiocyano-2,2-disulfonic acid (DIDS, a nonpermeating amino-reactive disulfonic acid known to inhibit the uptake of Cl^–) had less Cl^– level in roots than that in the absence of DIDS, but did not affect the levels of Na⁺, and responses related to growth in roots. Treatment with 50 mM Na-gluconate (the anion of which is not permeable to membrane) had similar Na⁺ level in roots as that with 100 mM NaCl. It was found that treatment with 50 mM Na-gluconate effected growth reduction and growth-related responses in roots in the same way as 100 mM NaCl. All these results suggest that Cl^– is not required for NaCl-induced responses in root of rice seedlings. Endogenous ABA level showed no increase in roots of rice seedlings exposed to 150 mM NaCl. It is unlikely that ABA is associated with NaCl-inhibited root growth of rice seedlings.     

Lipoxygenase is an abundant protein in cucumber exudates

The presence of lipoxygenase (LOX) has been reported in many plant organs. High LOX activity (1–2 katal/mg protein) was detected in exudates from cut cucumber (Cucumis sativus L.) stems and petioles. Exudate LOX had a pH optimum of 5.0, an estimated molecular weight of 95 kDa and cross-reacted on sodium-dodecyl-sulfate gels with anti-LOX antibodies raised against soybean leaf LOX isoenzymes. Lipoxygenase activity was detected on native gels stained with o-dianisidine using linoleic acid as a substrate. Enzyme activity was similar with linoleic and linolenic acid and 2 times greater with arachidonic acid as substrate. At pH 6.8, LOX metabolized linoleic acid into 13- and 9-hydroperoxides at a ratio of 12. Linolenic acid was preferentially oxidized at carbon 13. Lipoxygenase activity was inhibited by n-propyl gallate (IC₅₀ 300 nM) and nordihydroguaiaretic acid (IC₅₀ 25 nM), but not by nonsteroidal anti-inflammatory drugs. LOX activity was enhanced 4.5-fold by 300 mM Ca²⁺. Spermine at 1 mM, and putrescine and spermidine at 2 mM completely inhibited LOX activity, but at low concentrations spermine (100 mM) and spermidine (100–500 mM) significantly stimulated LOX activity: 8- and 4.5-fold, respectively. Tissue printing of stem, petiole and hypocotyl sections with subsequent incubation with the antiserum raised against soybean leaf LOX revealed the presence of LOX in the internal and external phloem and in the sieve tubes.Abbreviations DTT dithiothreitol - EDTA ethylenediaminetetraacetic acid - EGTA ethyleneglycol-bis(-aminoethyl ether) N,N,N,N-tetraacetic acid - 9(S)-HpOD 9-(S)-hydroperoxy-(E,Z)10,12-octadecadienoic acid - 13(S)-HpOD 13-(S)-hydroperoxy(Z,E)-9,11-octadecadienoic acid - IC inhibition constant - IEF isoelectrofocusing - LOX lipoxygenase - NDGA nordihydroguaiaretic acid - PBS phosphate buffered saline - PAGE polyacrylamide gel electrophoresis - SDS sodium dodecyl sulfate We would like to thank Ulla Jarlfors for exellent technical assistance with the histological analysis. The research reported in this paper was supported in part by grants to J.K. from the R.J. Reynolds Tobacco Company and Cooperative Agreement 43YK-5-0030 of the USDA-ARS. Journal paper 93-11-12 of the Kentucky Agricultural Experiment Station, Lexington.     

Inactivation of Rhizoctonia solani toxin by a putative α-glucosidase from coconut leaves for control of sheath blight disease in rice

Inactivation of a host-specific toxin, RS-toxin, induced by Rhizoctonia solani, the cause of rice sheath blight disease was investigated. A putative -glucosidase identified based on enzyme assay and Western blot analysis was purified from coconut (Cocos nucifera; the only known non-host of R. solani) leaves and tested for its efficacy in degrading RS-toxin. SDS–PAGE analysis showed the appearance of a 97 kDa protein, which appeared in proteins extracted from coconut leaf bits during 48 and 96 h after RS-toxin-treatment and the protein eventually disappeared. A comparison of the u.v. spectra read at 150–300 nm revealed conspicuous disturbances in the absorbance at 24 h of incubation of RS-toxin with the coconut leaf protein extracts as compared to that at 12 h, indicating the possible degradation of RS-toxin by coconut leaf -glucosidase during incubation. Incubation of rice leaf sheath bits with coconut leaf protein extracts significantly reduced electrolyte leakage due to RS-toxin 30 min after the toxin treatment. Simultaneously, there was a significant reduction in sheath blight symptoms when the incubation of rice leaf sheaths with the coconut leaf protein extracts was extended up to 96 or 120 h. This appears to be the first report of purification and characterization of a putative plant -glucosidase.     

Expression of a cry2Aa gene in an acrystalliferous Bacillus thuringiensis strain and toxicity of Cry2Aa against Helicoverpa armigera

In the recent past research has been mainly focused on the expression of cry1 genes of Bacillus thuringiensis (Bt) to engineer lepidopteran insect resistance in plants. Search for structurally different toxins is necessary for the management of resistance development in insects. The intact cry2Aa operon (3.95 kb) of a new isolate of Bt, 47-8, was subcloned into a Bt shuttle vector, pHT3101 (6.7 kb). Recombinant pHT3101 containing the cry2Aa operon of Bt strain 47-8 was named as pTN2Aa and used to transform acrystalliferous Bt strain 4Q7 by electroporation. Phase contrast microscopic observation revealed the presence of crystalline inclusions in the transformants of Bt strain 4Q7 harbouring pTN2Aa. SDS–PAGE of a spore–crystal mixture prepared from transformants of acrystalliferous Bt strain 4Q7 harbouring pTN2Aa showed a single band of about 65 kDa alone confirming the expression of the cloned cry2Aa. Bioassay with Helicoverpa armigera showed 71.4% mortality caused by the proteins encoded by the newly cloned cry2Aa gene (at the concentration of 2.3 g/l) on the seventh day and all the survivors that escaped from Cry2Aa toxicity showed severe (81–99%) inhibition in larval growth.     

Mutation of the regulatory phosphorylation site of tobacco nitrate reductase results in high nitrite excretion and NO emission from leaf and root tissue

In wild-type Nicotiana plumbaginifolia Viv. and other higher plants, nitrate reductase (NR) is regulated at the post-translational level and is rapidly inactivated in response to, for example, a light-to-dark transition. This inactivation is caused by phosphorylation of a conserved regulatory serine residue, Ser 521 in tobacco, and interaction with divalent cations or polyamines, and 14-3-3 proteins. The physiological importance of the post-translational NR modulation is presently under investigation using a transgenic N. plumbaginifolia line. This line expresses a mutated tobacco NR where Ser 521 has been changed into aspartic acid (Asp) by site-directed mutagenesis, resulting in a permanently active NR enzyme [C. Lillo et al. (2003) Plant J 35:566–573]. When cut leaves or roots of this line (S₅₂₁) were placed in darkness in a buffer containing 50 mM KNO₃, nitrite was excreted from the tissue at rates of 0.08–0.2 mol (g FW)^–1 h^–1 for at least 5 h. For the control transgenic plant (C1), which had the regulatory serine of NR intact, nitrite excretion was low and halted completely after 1–3 h. Without nitrate in the buffer in which the tissue was immersed, nitrite excretion was also low for S₅₂₁, although 20–40 mol (g FW)^–1 nitrate was present inside the tissue. Apparently, stored nitrate was not readily available for reduction in darkness. Leaf tissue and root segments of S₅₂₁ also emitted much more nitric oxide (NO) than the control. Importantly, NO emission from leaf tissue of S₅₂₁ was higher in the dark than in the light, opposite to what was usually observed when post-translational NR modulation was operating.Abbreviations NR Nitrate reductase - NO Nitric oxide - Ser Serine - WT Wild type     

Root exudates as mediators of mineral acquisition in low-nutrient environments

Plant developmental processes are controlled by internal signals that depend on the adequate supply of mineral nutrients by soil to roots. Thus, the availability of nutrient elements can be a major constraint to plant growth in many environments of the world, especially the tropics where soils are extremely low in nutrients. Plants take up most mineral nutrients through the rhizosphere where micro-organisms interact with plant products in root exudates. Plant root exudates consist of a complex mixture of organic acid anions, phytosiderophores, sugars, vitamins, amino acids, purines, nucleosides, inorganic ions (e.g. HCO₃ ^–, OH^–, H⁺), gaseous molecules (CO₂, H₂), enzymes and root border cells which have major direct or indirect effects on the acquisition of mineral nutrients required for plant growth. Phenolics and aldonic acids exuded directly by roots of N₂-fixing legumes serve as major signals to Rhizobiaceae bacteria which form root nodules where N₂ is reduced to ammonia. Some of the same compounds affect development of mycorrhizal fungi that are crucial for phosphate uptake. Plants growing in low-nutrient environments also employ root exudates in ways other than as symbiotic signals to soil microbes involved in nutrient procurement. Extracellular enzymes release P from organic compounds, and several types of molecules increase iron availability through chelation. Organic acids from root exudates can solubilize unavailable soil Ca, Fe and Al phosphates. Plants growing on nitrate generally maintain electronic neutrality by releasing an excess of anions, including hydroxyl ions. Legumes, which can grow well without nitrate through the benefits of N₂ reduction in the root nodules, must release a net excess of protons. These protons can markedly lower rhizosphere pH and decrease the availability of some mineral nutrients as well as the effective functioning of some soil bacteria, such as the rhizobial bacteria themselves. Thus, environments which are naturally very acidic can pose a challenge to nutrient acquisition by plant roots, and threaten the survival of many beneficial microbes including the roots themselves. A few plants such as Rooibos tea (Aspalathus linearis L.) actively modify their rhizosphere pH by extruding OH^– and HCO₃ ^– to facilitate growth in low pH soils (pH 3 – 5). Our current understanding of how plants use root exudates to modify rhizosphere pH and the potential benefits associated with such processes are assessed in this review.     

Effects of water deficit on N2(C2H2) fixation in cowpea and groundnut

The effect of water deficit on nodulation, N₂ fixation, photosynthesis, and total soluble sugars and leghemoglobin in nodules was investigated in cowpea and groundnut. Nitrogenase activity completely ceased in cowpea with a decrease in leaf water potential ( _leaf) from –0.4 MPa to –0.9 MPa, while in groundnut it continued down to –1.7 MPa. With increasing water stress, the acetylene reduction activity (ARA) declined very sharply in cowpea, but ARA gradually decreased in groundnut. Even with mild water stress ( _leaf of 0.2 MPa), nodule fresh weight declined 50% in cowpea partly due to a severe nodule shedding whereas nodule fresh weight declined in groundnut only when _leaf decreased by 1.0 MPa. No nodule shedding was noticed even at a higher stress level in groundnut. Photosynthesis and stomatal conductance were also more stable in groundnut than in cowpea under water stress. There was a sharp increase in total soluble sugars and leghemoglobin in the nodules of groundut with water stress, but no definite trend could be found in cowpea.