Histochemical Localization of {beta}-Glycerophosphatase Activity in Young Root Tips

Glycerophosphatase activity has been studied in frozen sectionsof maize, barley, and broad-bean root tips by the Gomori leadsulphide precipitation procedure. The enzyme was largely localizedat particulate sites in the cytoplasm, and in the cell walls.In maize and barley the particles were similar to acid phosphatase-containingspherosomes found in other tissues and were most active in thecortical cells. The wall reaction was highest in the outer cells,in agreement with biochemical studies. When excised roots wereincubated in the Gomori medium, staining was restricted to thesurface cells. The possible function of this surface activityand its relevance to ultrastructural studies is discussed.     

Localization and Kinetic Properties of {beta}-Glycerophosphatase in Barley Roots

The study of ß-glycerophosphatase activity in cell-wallpreparations and in excised root tips from barley seedlingssupports the view that the former, which constitutes about 20per cent of the activity of the whole homogenate, representsthe fraction located at the surface of the roots in vivo. Theactivities of the cell-wall suspension and intact roots arevirtually identical, and further show identical relations topH, substrate concentration (K_m), and competitive inhibitionby molybdate and inorganic phosphate (K_i). The enzyme must thereforebe freely exposed to the external solution without any permeabilitybarrier separating it from either substrate or inhibitors. Theabsence of any lag phase in the hydrolysis in excised root tipssuggests that the surface enzyme may be limited to the outermostlayers of the root. The solubilization of some of the activityof the cell-wall preparation by treatment with sodium chlorideand ammonium sulphate suggest that surface activity may havebeen lost from these preparations rather than adsorbed duringhomogenization and extraction. The K_m and pH-activity curveof the supernatant activity remaining after centrifugation ofthe cell-wall fraction indicate that only this enzyme and noother detectable glycerophosphatase exists in the roots.     

Modulation of Elicitor-Induced Chitinase and {beta}-1,3-Glucanase Activity by Hormones in Phaseolus vulgaris

Chitinase and ß-1 ,3-glucanase induction in Phaseolusvulgaris by cell wall elicitor from Col-letotrichum lindemuthianumhas been studied together with the effects of the hormones IAAand ethylene. Chitinase and ß-1, 3-glucanase increasedin response to the elicitor in the resistant cultivar, Kievit,but not in the susceptible cultivar, Pinto. However, both activitiesincreased in both cultivars in response to hormones in the absenceof elicitor; elicitor did not augment this response in cv. Kievit.Aminoethoxyvinyl glycine (AVG) abolished all responses exceptthose obtained by the application of ethylene. Of other hydrolasestested, only ß -galactosidase was induced by elicitor;this was similar for both cultivars but hormones were withouteffect. Evidence suggests that both chitinase and ß-l,3-glucanase are located within the cell rather than in theintercellular space. It is concluded that chitinase and ß;-l,3-glucanaseare coordinately synthesized as a defence response since theyhydrolyse complementary linkages in pathogen derived polysac-charides.Regulation of the induction of the two enzymes is primarilydue to ethylene and the lack of response in the compatible reactionappears to arise from an inability to synthesize ‘ stress’ ethylene. ¹Present address: School of Chemistry, Molecular and Biological Sciences, University of Sussex, Brighton BN1 9QJ, U.K. (Received March 15, 1991; Accepted June 13, 1991)     

Adhesion of Colletotrichum lindemuthianum Spores to Phaseolus vulgaris Hypocotyls and to Polystyrene

Adhesion of Colletotrichum lindemuthianum spores to Phaseolus vulgaris hypocotyls and to polystyrene was inhibited by the respiratory inhibitors sodium azide and antimycin A, indicating a requirement for metabolic activity in adhesion. Various commercial proteins and Tween 80 also reduced adhesion to both surfaces. Binding was enhanced by the presence of salts: sodium, potassium, calcium, and magnesium chlorides were equally effective. The removal of surface wax from hypocotyls by chloroform treatment greatly reduced their subsequent ability to bind spores. The results suggest a similar mechanism for spore adhesion to the plant surface and to polystyrene, involving purely physical surface properties rather than group-specific binding sites.     

Further Properties of Adenosine Triphosphatase and {beta}-Glycerophosphatase from Barley Roots

Cell wall preparations from barley roots contain ATP-ase activitythat is stimulated by monovalent cations at alkaline pH values,above that obtained with calcium or magnesium ions. Sodium isthe most effective cation followed by potassium, lithium, andrubidium. Similar activation is obtained with a soluble enzymefraction and with excised root tips. ß-Glycerophosphataseshows no stimulation by calcium and sodium or potassium haveonly a small stimulatory effect. Disc electrophoresis demonstratesthe group character of ATP-ase and ß-glycerophosphataseactivities which consist of multiple forms either specific toone or other substrate or hydrolysing both.     

The Distribution of {beta}-Glycerophosphatase in Young Roots of Pisum sativum L.

The distribution of ß-glycerophosphatase activityin young roots of Pisum sativum, cultivar Alaska, has been examinedby biochemical and histochemical methods. Results obtained bythe two approaches are broadly similar, and indicate that highenzyme activity is associated with cells of the root cap, outerlayers of the cortex, differentiating xylem elements and phloemfibres, and cortical cells surrounding emerging lateral roots.The significance of this distribution in relation to a possiblefunction of ß-glycerophosphatase is discussed.     

A specific inhibitor of Colletotrichum lindemuthianum protease from kidney bean (Phaseolus vulgaris) seeds

A specific protein—an inhibitor of Colletotrichum lindemuthianum protease—was isolated from kidney bean seeds in a homogeneous form. The purification procedure included gel filtration, isoelectric focusing and affinity chromatography on trypsin-Sepharose column. The latter was introduced to separate the fungal protease inhibitor from closely related trypsin and chymotrypsin inhibitors present in kidney bean seeds.     

Pterocarpan Accumulation in Pod Infection-droplets and Pod Tissues of Phaseolus vulgaris Inoculated with Colletotrichum lindemuthianum1

Following inoculation of pods of several cultivars of French bean (Red Kidney Selections W 243, 244, 245), with Colletotrichum lindemuthianum (Sacc. & Magn.) Bri. and Cav. ANZ races 1 and 2, phaseollin, phaseollidin and 6-α-hydroxyphaseollin were estimated in infection-droplets and tissue extracts by u.v. spectrophotometry and HPLC. The examples represented both compatible and incompatible cultivar-race interactions and confirmed a positive correlation between degree of incompatibility andphaseollin concentration or content. The data supported the concept that a phytoalexin index may be of value in selecting for disease resistance or studying the quantitative inheritance of phaseollin in studies of anthracnose resistance in French beans.Concentrations of phaseollidin and 6-α-hydroxyphaseollin were low and were not correlated with degree of compatibility of cultivar-race interactions. The evidence did not support the putative role of 6-α-hydroxyphaseollin as an in planta degra, dative phaseollin metabolite associated with French bean –C. lindemuthianum compatible interactions.     

Resistance to Colletotrichum lindemuthianum in Phaseolus vulgaris: a case study for mapping two independent genes

Anthracnose, caused by the hemibiotrophic fungal pathogen Colletotrichum lindemuthianum is a devastating disease of common bean. Resistant cultivars are economical means for defense against this pathogen. In the present study, we mapped resistance specificities against 7 C. lindemuthianum strains of various geographical origins revealing differential reactions on BAT93 and JaloEEP558, two parents of a recombinant inbred lines (RILs) population, of Meso-american and Andean origin, respectively. Six strains revealed the segregation of two independent resistance genes. A specific numerical code calculating the LOD score in the case of two independent segregating genes (i.e. genes with duplicate effects) in a RILs population was developed in order to provide a recombination value (r) between each of the two resistance genes and the tested marker. We mapped two closely linked Andean resistance genes (Co-x, Co-w) at the end of linkage group (LG) B1 and mapped one Meso-american resistance genes (Co-u) at the end of LG B2. We also confirmed the complexity of the previously identified B4 resistance gene cluster, because four of the seven tested strains revealed a resistance specificity near Co-y from JaloEEP558 and two strains identified a resistance specificity near Co-9 from BAT93. Resistance genes found within the same cluster confer resistance to different strains of a single pathogen such as the two anthracnose specificities Co-x and Co-w clustered at the end of LG B1. Clustering of resistance specificities to multiple pathogens such as fungi (Co-u) and viruses (I) was also observed at the end of LG B2.     

Pathogenic Variability in Colletotrichum lindemuthianum and Evaluation of Resistance in Phaseolus vulgaris in the North-Western Himalayan Region of India

Eightyfive isolates of Colletotrichum lindemuthianum (bean anthracnose) collected from different kidney bean growing areas of a hilly state (Himachal Pradesh) of India, were characterized on the basis of their reaction types on International and CIAT differentials. On international differentials, 12 races viz., Alpha-Brazil, Beta, Gamma and Ind I to Ind IX were characterized. The races designated as Ind I to Ind IX were different from those identified in Europe and USA, thus forming a new race group from the Indian subcontinent. On the CIAT differential set the 85 isolates have been grouped into 19 races. Of these, only races 65 and 73 resembled the North American races. Exotic accessions AB 136 and G 2333 were resistant to all the Indian races. However, race specific resistance has been found in a number of indigenous and exotic genotypes of Phaseolus vulgaris.     

Effect of light treatment and endogenous growth hormones on {alpha}-and {beta}-amylase activities in cotyledons of Phaseolus vulgaris L.

The influence of light and endogenous plant-growth regulatorson the evolution of - and ß-amylases in cotyledonsof Phaseolus vulgaris L. was investigated. Both enzymes, whichare not present in ungerminated seeds, appear during germinationof intact seedlings or incubation of excised cotyledons. -Amylaseactivity decreases upon exposure to light. This inhibition iscorrelated with a drastic increase in chlorophyll content anda change in the endogenous gibberellin-inhibitor balance. ß-Amylaseactivity was not affected by light treatment but was by thepresence of endogenous cytokinins. (Received February 3, 1977; )     

l-Phenylalanine Ammonia-Lyase Activity During Germination of Phaseolus vulgaris

l-Phenylalanine ammonia-lyase (PAL) activity develops in excised bean axes after approximately 5 hours of incubation and reaches a maximum level after 14 hours of incubation. Light does not affect the development of activity, but puromycin, cycloheximide, actinomycin D, and 5-fluorouracil inhibit.     

Partial Purification and Characterization of Gibberellin 3{beta}-hydroxylase from Immature Seeds of Phaseolus vulgaris L.

Gibberellin 3/ß-hydroxylase,a 2-oxoglutarate-dependentdioxygenase that catalyzes the hydroxylation of GA₂₀ to GA₁,was purified 313-fold from immature seeds of Phaseolus vulgarisL. The mol wt of the enzyme was estimated to be 42,000 by gelfiltration HPLC and SDS-polyacrylamide gel electrophoresis.The enzyme exhibited maximum activity at pH 7.7. The Km valuesfor [2,3-³H]GA20 and [2,3-³H]GA, were 0.29µu and 0.33µm, respectively. The enzyme requires 2-oxoglutarate asa cosubstrate; the K_m value for 2-oxoglutarate was 250µMusing [³H]- GA₂₀ as a substrate. Fe²⁺ and ascorbate significantlyactivated the enzyme at all purification steps, while catalaseand BSA activated the purified enzyme only. The enzyme was inhibitedby divalent cations Mn²⁺, Co²⁺, Ni²⁺, Cu²⁺, Zn²⁺, Cd²⁺ and Hg²⁺.3ß-Hydroxylation of [³H]- GA₂₀ was also inhibitedby non-radioactive GA₅, GA₉,GA₁₅, GA₂₀ and GA₄₄. The possiblesite of 3ß-hydroxylation in gibberellin biosynthesisis discussed in terms of the substrate specificity of partiallypurified gibberellin 3ß-hydroxylase. (Received February 29, 1988; Accepted June 3, 1988)     

The plant nitrogen mobilization promoted by Colletotrichum lindemuthianum in Phaseolus leaves depends on fungus pathogenicity

Nitrogen plays an essential role in the nutrient relationship between plants and pathogens. Some studies report that the nitrogen-mobilizing plant metabolism that occurs during abiotic and biotic stress could be a 'slash-and-burn' defence strategy. In order to study nitrogen recycling and mobilization in host plants during pathogen attack and invasion, the Colletotrichum lindemuthianum/Phaseolus vulgaris interaction was used as a model. C. lindemuthianum is a hemibiotroph that causes anthracnose disease on P. vulgaris. Non-pathogenic mutants and the pathogenic wild-type strain were used to compare their effects on plant metabolism. The deleterious effects of infection were monitored by measuring changes in chlorophyll, protein, and amino acid concentrations. It was shown that amino acid composition changed depending on the plant-fungus interaction and that glutamine accumulated mainly in the leaves infected by the pathogenic strain. Glutamine accumulation correlated with the accumulation of cytosolic glutamine synthetase (GS1 alpha) mRNA. The most striking result was that the GS1 alpha gene was induced in all the fungus-infected leaves, independent of the strain used for inoculation, and that GS1 alpha expression paralleled the PAL3 and CHS defence gene expression. It is concluded that a role of GS1 alpha in plant defence has to be considered.     

Changes in Cell Wall Polysaccharides During the Growth of Phaseolus vulgaris Leaves

The changes in pectic and hemicellulosic polysaccharides, and-cellulose during the expansion growth of the primary leavesof Phaseolus vulgaris L. var. Pinta have been studied. -Celluloseincreased continuously with age, while pectic and water-solublehemicellulose extracted with 4% KOH fractions slightly decreased.The water-soluble hemicelluloses extracted with 24% KOH showedthe most conspicuous changes, increasing until the 8th day,when the absolute growth rate was maximal, and thereafter decreasing.Furthermore, the study of the molecular mass distribution ofpectin, and water-soluble polysaccharides extracted with 4%and 24% KOH, showed an increase in the degree of polymerizationof polyuronic acid and xylan, and an important depolymerizationof galactan and xyloglucan. Accordingly, the mechanism of cellwall loosening in the leaf cell walls is similar to that describedfor plant axes. Key words: Cell wall, growth, leaf     

Identification of an ancestral resistance gene cluster involved in the coevolution process between Phaseolus vulgaris and its fungal pathogen Colletotrichum lindemuthianum.

The recent cloning of plant resistance (R) genes and the sequencing of resistance gene clusters have shed light on the molecular evolution of R genes. However, up to now, no attempt has been made to correlate this molecular evolution with the host-pathogen coevolution process at the population level. Cross-inoculations were carried out between 26 strains of the fungal pathogen Colletotrichum lindemuthianum and 48 Phaseolus vulgaris plants collected in the three centers of diversity of the host species. A high level of diversity for resistance against the pathogen was revealed. Most of the resistance specificities were overcome in sympatric situations, indicating an adaptation of the pathogen to the local host. In contrast, plants were generally resistant to allopatric strains, suggesting that R genes that were efficient against exotic strains but had been overcome locally were maintained in the plant genome. These results indicated that coevolution processes between the two protagonists led to a differentiation for resistance in the three centers of diversity of the host. To improve our understanding of the molecular evolution of these different specificities, a recombinant inbred (RI) population derived from two representative genotypes of the Andean (JaloEEP558) and Mesoamerican (BAT93) gene pools was used to map anthracnose specificities. A gene cluster comprising both Andean (Co-y; Co-z) and Mesoamerican (Co-9) host resistance specificities was identified, suggesting that this locus existed prior to the separation of the two major gene pools of P. vulgaris. Molecular analysis revealed a high level of complexity at this locus. It harbors 11 restriction fragment length polymorphisms when R gene analog (RGA) clones are used. The relationship between the coevolution process and diversification of resistance specificities at resistance gene clusters is discussed.     

Histochemical Localization of Alkaline Phosphatases and {beta}-Glucosidases in Cucurbitaceae

The distribution of alkaline phosphatases and ß-glucosidasesin the tissues of the stems of Colocynthls citrullus, Cucumissativus and Cucurbita pepo was studied. There was similarityin the pattern of distribution for these two groups of enzymesand that of acid phosphatases. The activities of these enzymesvaried not only from plant to plant but also from tissue totissue. However both enzymes showed increasing activity withplant age and localization was correspondingly more restricted.The significance of these localizations is discussed.