An isoelectric focusing study of the effect of methyl-esterified pectic substances on the production of extracellular pectin isoenzymes by soft rot Erwinia spp.

The production of extracellular pectic isoenzymes by seven strains of soft rot bacteria, Erwinia carotovora subsp. carotovora, E.c. atroseptica and E. chrysanthemi , when grown in media containing four different pectic substances with different degrees of methylation or with potato tuber cell-wall extract was examined by isoelectric focusing activity staining. In addition to the isoenzymes of pectate lyase, polygalacturonase and pectin methyl esterase produced constitutively or following induction by polygalacturonic acid (PGA) and coded by known genes, between two and seven novel isoenzymes of the three enzymes with a wider pI range were apparently induced by the pectins and cell-wall extract. Pectin lyase, which is induced in vitro by DNA-damaging agents, was not produced in the absence of mitomycin C in a medium containing PGA but up to two isoenzymes were found with pectin or cell-wall extract. In contrast, cellulase isoenzyme production was not affected by pectin or cell-wall extract. A greater number of novel isoenzymes of all pectic enzymes except pectin lyase tended to be produced in media containing Link pectin, which is PGA methylated to 98%, than the other pectic substances and cell-wall extract. Pectate lyase and polygalacturonase were induced by pectin lyase-degraded products of highly methylated pectin but not by PGA in an E. chrysanthemi strain with all its known pei and peh genes mutated. The results suggest that the production of novel pectic isoenzymes could be related to the presence of CH⁺₃ groups and that their induction differs from that for isomers induced by PGA-degraded products and DNA-damaging agents or produced constitutively.     

Isolation of Pectolytic clostridia from Potatoes

S ummary : A method for selective counting and isolation of pectolytic clostridia in the presence of Erwinia carotovora is described; using this method pectolytic clostridia have been found in numbers of 8 × 10⁵–1 × 10⁸/g of rotting potato tissue in the presence of 1–4 × 10⁸ E. carotovora /g.     

Cytokinin biochemistry in relation to leaf senescence. VIII. Translocation, metabolism and biosynthesis of cytokinins in relation to sequential leaf senescence of tobacco

Cytokinin bases (zeatin and dihydrozeatin) and ribosides (zeatin riboside and dihydrozeatin riboside) were identified as major cytokinins in tobacco xylem sap by radioimmunoassay. When ³H-labelled zeatin riboside or dihydrozeatin riboside were supplied to tobacco plants via the xylem, leaves of differing maturity did not differ appreciably in level of radioactivity or in metabolism of the cytokinin. The major metabolites of zeatin riboside in leaves were adenine, adenosine and adenine nucleotides, whereas that of dihydrozeatin riboside was dihydrozeatin 7-glucoside. Incorporation of [¹⁴C]adenine into zeatin was evident in upper green leaves. indicating that young leaves have the capacity to synthesize cytokinins in situ. In contrast, fully expanded green leaves and senescing tobacco leaves exhibited little or no incorporation of [¹⁴C]adenine into cytokinins. This difference in cytokinin biosynthetic capacity may contribute to the differing cytokinin levels in leaves of different matirity, and may participate in control of sequential leaf senescence in tobacco.     

Hydrogen peroxide does not function downstream of salicylic acid in the induction of PR protein expression

The roles of salicylic acid (SA) and H₂O₂ in the induction of PR proteins in tobacco have been examined. Studies were conducted on wild-type tobacco and plants engineered to express a bacterial salicylate hydroxylase capable of metabolizing SA to catechol (SH-L plants). Wild-type and PR-1a—GUS-transformed plants express PR-1a following challenge with Pseudomonas syringae pathovar syringae , SA or 2,6-dichloro-isonicotinic acid (INA). In contrast, SH-L plants failed to respond to SA but did express PR-1a following INA treatment. H₂O₂ and the irreversible catalase inhibitor 3-amino-1,2,4-triazole (3-AT) were found to be weak inducers of PR-1a expression (relative to SA) in wild-type tobacco but were unable to induce PR-1a in SH-L plants, suggesting that the action of these compounds depends upon the accumulation of SA. A model has been proposed suggesting that SA binds to and inhibits a catalase inducing an increase in H₂O₂ leading to PR protein expression. Catalase activity has been measured in tobacco and no significant changes in activity following infection with P. syringae pv. syringae were detected. Furthermore, inhibition of catalase activity in vitro in plant extracts requires pre-incubation and only occurs at SA concentrations above 250 µM. Leaf disks pre-incubated with 1 mM SA do accumulate SA to these levels and PR-1a is efficiently induced but there is no apparent inhibition of catalase activity. It is also shown that a SA-responsive gene, PR-1a, and a H₂O₂-sensitive gene, AoPR-1, are both relatively insensitive to 3-AT suggesting that induction of these genes is unlikely to be due entirely to inhibition of an endogenous catalase.     

Reduced Development of Grey Mould (Botrytis cinerea) in Bean and Tomato Plants by Calcium Nutrition

Fertilization of bean plants grown in perlite with 1 and 3 mM CaCl₂ or Ca(NO₃)₂ reduced severity of grey mould as compared with control plants or plants fertilized with 5 mM of the compounds. Fertilization with Ca(NO₃)₂ reduced severity leaf grey mould and fruit ghost spots of tomato plants grown in perlite by 70 and 45%, respectively. The rate of decrease varied with the position of the fruits on the plants. Leaves from plants treated with calcium or otherwise [KNO₃, (NH₄)₂SO₄] produced less ethylene than leaves of nontreated plants. Rate of growth of B. cinerea was lower on growth medium prepared from washings from leaves of calcium fertilized plants than from leaves from other treatments. The fertilizer combination Ca(H₂PO₄)₂+ CaSO₄ (1 and 3 g/kg soil) applied once to tomato plants grown in soil reduced severity of leaf grey mould by 80 % (significant at P = 0.05) but 1–3 g CaSO₄/kg soil only tended to reduce disease severity (30–40 %, not significant) as compared with the control. The compounds CaCl₂ and Ca(NO₃)₂ increased significantly ( P = 0.05) the growth of B. cinerea on synthetic medium when applied at rates of 1 0–10.0 mM whereas reduction of growth was observed with 0.1 mM of the compounds and of CaSO₄.     

Development of necrosis and activation of disease resistance in transgenic tobacco plants with severely reduced catalase levels

Numerous studies argue that salicylic acid (SA) is an important component of the plant signal transduction pathway(s) leading to disease resistance. The discovery that the SA-binding protein is a catalase, whose activity is blocked by SA, led to the proposal that one of SA's modes of action is to inhibit this H₂O₂-degrading enzyme and thus elevate H₂O₂ levels. To test this model, an attempt was made to mimic the action of SA by reducing the synthesis of catalase using antisense RNA technology. Analyses of transgenic tobacco plants that expressed the tobacco catalase 1 ( cat1 ) or catalase 2 ( cat2 ) gene in an antisense orientation indicate that there is no correlation between modest to high levels of reduction in catalase activity and activation of plant defenses such as pathogenesis-related (PR)-1 protein synthesis. However, three independent antisense catalase transgenic plants (ASCAT1 Nos 16, 17, and 28), which exhibited the most severe reduction in catalase activity (∼90% or more), developed chlorosis or necrosis on some of their lower leaves. These same leaves accumulated very high levels of PR-1 proteins and showed enhanced resistance to tobacco mosaic virus. Necrosis and elevated SA, which appear to result from severe depression of catalase levels, may be responsible for the induction of these defense responses.     

Serological and biochemical variation among potato strains of Erwinia carotovora subsp. atroseptica and their taxonomic relationship to other E. carotovora strains

The serological and biochemical characteristics of 32 Erwinia carotovora subsp. atroseptica strains from potato were compared with 48 other pectolytic Erwinia strains. Biochemical characteristics were examined by the API 20E and API 50CHE systems. Numerical analysis using the Euclidean distance coefficients and clustering by the unweighted average pair group method indicated that these E. carotovora subsp. atroseptica strains formed a distinct cluster (subphenon A₁) that could be differentiated from other E. carotovora strains. Three non-potato strains also belonged to this group; two of these were from tomato and the other from Chinese cabbage. Named E. carotovora subsp. atroseptica strains from other hosts clustered into other phenons. Sixty-three per cent of subphenon A₁ strains tested in this study typed into serogroup I. One potato strain in another phenon also typed into this serogroup. The subphenon A₁ strains that did not type into serogroup I typed into serogroups XVIII, XX, or XXII. Many of these strains, however, expressed several different O antigens which were also expressed by E. carotovora strains in other phenons.     

Disease resistance to bacterial pathogens affected by the amount of ferredoxin-I protein in plants

Ferredoxin-I (Fd-I) is a fundamental protein that is involved in several metabolic pathways. The amount of Fd-I found in plants is generally regulated by environmental stress, including biotic and abiotic events. In this study, the correlation between quantity of Fd-I and plant disease resistance was investigated. Fd-I levels were increased by inoculation with Pseudomonas syringae pv. syringae but were reduced by Erwinia carotovora ssp. carotovora . Transgenic tobacco over-expressing Fd-I with the sense sweet pepper Fd-I gene ( pflp ) was resistant to E. carotovora ssp. carotovora and the saprophytic bacterium P. fluorescens. By contrast, transgenic tobacco with reduced total Fd-I and the antisense pflp gene was susceptible to E. carotovora ssp. carotovora and P. fluorescens . Both of these transgenic tobaccos were resistant to P. syringae pv. syringae . By contrast, the mutated E. carotovora ssp. carotovora , with a defective harpin protein, was able to invade the sense- pflp transgenic tobacco as well as the non-transgenic tobacco. An in vitro kinase assay revealed that harpin could activate unidentified kinases to phosphorylate PFLP. These results demonstrate that Fd-I plays an important role in the disease defence mechanism.     

Transgenic tobacco with a reduced catalase activity develops necrotic lesions and induces pathogenesis-related expression under high light

Transgenic tobacco deficient in either Cat1 (Cat1AS), Cat2 (Cat2AS), or both (CatGH) was generated through sense and antisense technology. Cat1AS, Cat2AS, and CatGH plants showed no visible phenotype when grown at low light (100 µmol m⁻² sec⁻¹. Under these conditions, deficiency in Cat1 and/or Cat2 did not lead to constitutive pathogenesis-related (PR-1) expression and did not potentiate PR-1 induction by exogenous salicylic acid. This demonstrates that catalase suppression per se is not a sufficient signal for PR-1 induction. In Cat1-deficient plants exposed to higher light intensities (250–1000 µmol m⁻² sec⁻¹), PR-1 expression was induced without pathogenic challenge and multiplication of Pseudomonas syringae pv. syringae was repressed. Yet, it is unlikely that Cat1 deficiency is mimicking the mode of action of salicylic acid in tobacco, because, concurrent with PR-1 induction, Cat1 deficiency at high light provoked severe leaf damage, characterized by white necrotic lesions. Taken together, these results do not support the model that catalase inactivation is the key route by which salicylic acid induces PR defense responses in healthy tissue. However, because catalase deficiency is potentially lethal to leaves, catalase inactivation by salicylic acid could be of importance in the establishment of hypersensitive responses.     

Oxidative burst elicited by Bacillus mycoides isolate Bac J, a biological control agent, occurs independently of hypersensitive cell death in sugar beet

Response of sugar beet cultivars C40 and USH11 to syringe infiltration of live and dead Bacillus mycoides isolate Bac J, a biological control agent, and virulent and avirulent isolates of Erwinia carotovora pv. betavasculorum was measured by monitoring systemic acquired resistance control of Cercospora beticola, specific activity of chitinase and beta-glucanase, the oxidative burst, and hypersensitive cell death at the infiltration site. Priming sugar beet with B. mycoides Bac J (1 x 10(8) cells/ml) and avirulent isolates of E. carotovora pv. betavasculorum (1 x 10(6) cells/ml) reduced C. beticola symptoms by nearly 70% on distal, untreated leaves. Systemic resistance responses elicited by live B. mycoides Bac J and avirulent E. carotovora pv. betavasculorum isolates, measured by assays for chitinase and beta-glucanase, were statistically equivalent, and biphasic hydrogen peroxide production was observed. Although similar in timing, the second hydrogen peroxide burst was twofold lower for B. mycoides Bac J than for avirulent E. carotovora pv. betavasculorum. Hypersensitive cell death was elicited by avirulent E. carotovora pv. betavasculorum but not B. mycoides Bac J. An oxidative burst was elicited by spray-applied B. mycoides Bac J under both light and green light conditions, indicating that the signal produced by B. mycoides Bac J was not reliant on the stomata for entry into sugar beet. A working model for signal delivery and systemic resistance induction by B. mycoides Bac J in sugar beet is proposed.     

Pathogenesis-related protein b1' in plants and in cell suspension cultures of Nicotiana glutinosa Nicotiana debneyi and an amphidiploid cross (N. glutinosa×N. debneyi)

The expression of PR-protein b₁' in plants and cell suspension cultures of Nicotiana glutinosa L., Nicotiana debneyi Domin, and an amphidiploid cross of these two species, a hybrid, has been investigated. An enzyme linked immunosorbent assay has been employed to determine the concentration of PR-protein b₁' in extracts. The PR-Protein b₁' was constitutively produced in intact plants of the hybrid (around 25 μg g⁻¹ leaf tissue), while only trace amounts of the protein (< 50 ng g⁻¹ leaf tissue) were found in plants of the two parents. In suspension culture, the concentrations of PR-protein b₁' were 8, 0.4 and less than 0.1 mg l⁻¹ medium for the hybrid. N. debneyi and N. glutinosa , respectively. Only trace amounts of the protein were found in extracts from cells. Seven days after infection by tobacco mosaic virus (TMV) the concentration of PR-protein b₁' in leaves of N. glutinosa was 22.5 μg g⁻¹ leaf tissue. In N. debneyi and the hybrid a relatively limited induction of PR-protein b₁' by TMV was observed. The influence of various phenoxyacetic acids on the expression of PR-protein b₁' in the 3 cell cultures has been investigated. Cultures of N. glutinosa responded to treatments with 2,4-D and 2,4,5-T while cultures of N. debneyi and the hybrid were essentially unaffected. In the former case a concentration of 5–10 mg l⁻¹ 2,4,5-T was optimal and cells were most responsive to the treatment 4 days after subcultivation. The concentration of PR-protein b₁' in elicited cell cultures of N. glutinosa was 2 to 4 mg l⁻¹ medium.     

Metabolism of cell wall polysaccharides in cell suspension cultures of Populus alba in relation to cell growth

Changes in the composition of cell walls and extracellular polysaccharides (ECP) were studied during the growth of suspension-cultured Populus alba cells. Three growth phases, namely the cell division phase, cell elongation phase and stationary phase, were distinguished. The active deposition of polysaccharides in cell wall fractions (50 m M Na₂CO₃-, 1 M KOH-, 4 M KOH-soluble and 4 M KOH-insoluble) was observed during the elongation phase. A 50 m M Na₂CO₃-soluble pectic fraction mainly composed of 1,4-linked galactan and arabinan except acidic sugars. The 1,4-linked galactan decreased markedly during elongation. In 1 and 4 M KOH-soluble hemicellulosic fractions, non-cellulosic 1,4-glucan and xyloglucan were observed as major components, respectively. These polysaccharides also decreased during elongation. A large amount of polysaccharides was secreted into the medium as ECP. Neutral sugars were detected predominantly by sugar composition analysis. Acidic sugars, such as galacturonic acid, were less than 12% of total. In this study, active metabolism of pectic polysaccharides in addition to hemicellulosic polysaccharides, especially neutral side chains of pectin, during cell growth, was clarified.     

Erwinia carotovora subsp. carotovora and Erwinia-derived elicitors HrpN and PehA trigger distinct but interacting defense responses and cell death in Arabidopsis

We have used an hrp-positive strain of the soft rot pathogen Erwinia carotovora subsp. carotovora to elucidate plant responses to this bacterial necrotroph. Purified virulence determinants, harpin (HrpN) and polygalacturonase (PehA), were used as tools to facilitate this analysis. We show that HrpN elicits lesion formation in Arabidopsis and tobacco and triggers systemic resistance in Arabidopsis. Establishment of resistance is accompanied by the expression of salicylic acid (SA)-dependent, but also jasmonate/ethylene (JA/ET)-dependent, marker genes PR1 and PDF1.2, respectively, suggesting that both SA-dependent and JA/ET-dependent defense pathways are activated. Use of pathway-specific mutants and transgenic NahG plants show that both pathways are required for the induction of resistance. Arabidopsis plants treated simultaneously with both elictors PehA, known to trigger only JA/ET-dependent defense signaling, and HrpN react with accelerated and enhanced induction of the marker genes PR1 and PDF1.2 both locally and systemically. This mutual amplification of defense gene expression involves both SA-dependent and JA/ET-dependent defense signaling. The two elicitors produced by E. carotovora subsp. carotovora also cooperate in triggering increased production of superoxide and lesion formation.     

Effect of high external NaCl concentrations on ion transport within the shoot of Lupinus albus. I. Ions in xylem sap

Abstract. Xylem sap was collected from individual leaves of intact transpiring lupin plants exposed to increasing concentrations of NaCl by applying pneumatic pressure to the roots. Concentrations of Na⁺ and Cl⁻ in the xylem sap increased linearly with increases in the external NaCl concentration, averaging about 10% of the external concentration. Concentrations of K⁺ and NO₃⁻, the other major inorganic ions in the sap, were constant at about 2.5 and 1.5 mol m⁻³, respectively. There was no preferential direction of Na ⁺ or Cl⁻ to either young or old leaves: leaves of all ages received xylem sap having similar concentrations of Na⁺ and Cl⁻, and transpiration rates (per unit leaf area) were also similar for all leaves. Plants exposed to 120–160 mol m⁻³ NaCl rapidly developed injury of oldest leaves; when this occurred, the Na⁺ concentration in the leaflet midrib sap had increased to about 40 mol m⁻³ and the total solute concentration to 130 osmol m⁻³. This suggests that uptake of salts from the transpiration stream had fallen behind the rate of delivery to the leaf and that salts were building up in the apoplast.     

Hypocotyl growth of Pinus pinaster seedlings. Changes in α-cellulose, and in pectic and hemicellulosic polysaccharides

Lorences, E. P., Suárez, L. and Zarra, I. 1987. Hypocotyl growth of Pinus pinaster seedlings. Changes in a-cellulose, and in pectic and hemicellulosic polysaccharides.
The changes in pectic and hemicellulosic polysaccharides of hypocotyl cell walls during the growth of intact seedlings of Pinus pinaster Aiton were investigated, α -Cellulose and the water-soluble hemicellulose presented the most conspicuous changes during hypocotyl growth. The relative amount of the water-soluble hemicellulose decreased from day 7 to day 13 when hypocotyls were in the rapid growth phase, and stabilized when hypocotyl growth ceased. In this fraction, the relative amount of non-cellulosic glucose decreased dramatically during hypocotyl growth, while the relative amount of xylose increased. We suggest that these changes may be due to partial degradation of xyloglucan present in the water_:soluble hemicellulose fraction, accompanied by the synthesis of a xylan.     

Comparison of pectic enzymes produced by Erwinia chrysanthemi, Erwinia carotovora subsp. carotovora, and Erwinia carotovora subsp. atroseptica

Erwinia spp. that cause soft-rot diseases in plants produce a variety of extracellular pectic enzymes. To assess the correlation between patterns of pectic enzyme production and taxonomic classification, we compared the enzymes from representative strains. Supernatants obtained from polygalacturonate-grown cultures of nine strains of Erwinia chrysanthemi, three strains of E. carotovora subsp. carotovora, and three strains of E. carotovora subsp. atroseptica were concentrated and subjected to ultrathin-layer polyacrylamide gel isoelectric focusing. Pectate lyase, polygalacturonase, and exo-poly-alpha-D-galacturonosidase activities were visualized by staining diagnostically buffered pectate-agarose overlays with ruthenium red after incubation of the overlays with the isoelectric focusing gels. The isoelectric focusing profiles of pectate lyase and polygalacturonase were nearly identical for strains of E. carotovora subsp. carotovora and E. carotovora subsp. atroseptica, showing three pectate lyase isozymes with isoelectric points higher than 8.7 and a polygalacturonase with pI of ca. 10.2. Isoelectric focusing profiles of the E. chrysanthemi pectic enzymes were substantially different. Although there was considerable intraspecific heterogeneity, all strains produced at least four isozymes of pectate lyase, which could be divided into three groups: basic (pI, ca. 9.0 to 10.0), slightly basic (pI, ca. 7.0 to 8.5), and acidic (pI, ca. 4.0 to 5.0). Several strains of E. chrysanthemi also produced a single form of exo-poly-alpha-D-galacturonosidase (pI, ca. 8.0).(ABSTRACT TRUNCATED AT 250 WORDS)     

The ectopic overexpression of a citrus gibberellin 20-oxidase enhances the non-13-hydroxylation pathway of gibberellin biosynthesis and induces an extremely elongated phenotype in tobacco

Transgenic plants of Nicotiana tabacum overexpressing a gibberellin (GA) 20-oxidase cDNA ( CcGA20ox1 ) from citrus, under the control of the 35S promoter, were taller (up to twice) and had larger inflorescences and longer flower peduncles than those of control plants. Hypocotyls of transgenic seedlings were also longer (up to 4 times), and neither the seedlings nor the growing plants elongated further after application of GA₃. Hypocotyl and stem lengths were reduced by application of paclobutrazol, and this inhibition was reversed by exogenous GA₃. The ectopic overexpression of CcGA20ox1 enhanced the non-13-hydroxylation pathway of GA biosynthesis leading to GA₄, apparently at the expense of the early-13-hydroxylation pathway. The level of GA₄ (the active GA from the non-13-hydroxylation pathway) in the shoot of transgenic plants was 3–4 times higher than in control plants, whereas that of GA₁, formed via the early-13-hydroxylation pathway (the main GA biosynthesis pathway in tobacco), decreased or was not affected. GA₄ applied to the culture medium or to the expanding leaves was found to be at least equally active as GA₁ on stimulating hypocotyl and stem elongation of tobacco plants. The results suggest that the tall phenotype of tobacco transgenic plants was due to their higher content of GA₄, and that the GA response was saturated by the presence of the transgene.     

HydroxyproUne-rich Glycoprotein Accumulation in TMV-infected Tobacco Showing Systemic Acquired Resistance to Powdery Mildew

Tobacco cv. Havana 425, with systemic-acquired resistance (SAR) to an otherwise compatible Erysiphe cicho-racearum DC. race after TMV infection, was infected with TMV basaily (4^th and 5^th leaves) and the hydroxyproline-rich glycoproteins (HRGPs) were determined in two experiments (experiment 1 and experiment 2) by analysing cell wall hydroxyproline (Hyp) in the 6^th leaf. In basal TMV infected plants Hyp content (μmol^-1 FW) was greater than in controls in both experiments: the increase was significant at all times except 16 days after TMV inoculation in experiment 1; the pool of data of experiment 2 was also significantly increased. In TMV protected + E. cichoracearum challenged leaves compared to untreated controls significant increases in Hyp were also noted between the pools of data in both experiments. No differences were found between Hyp content in protected compared to protected + challenged leaves in both experiments. These results show accumulation of HRGPs in Havana tobacco associated with TMV infection and SAR activation against E, cichoracearum. The accumulation appears to be due to the inducer, since no further increase was detected in protected leaves after challenging.     

Geibberllin and temperature influence carbohydrate content and flowering in Phalaenopsis

When Phalaenopsis amabilis is grown under high temperature (30/25°C, day/night), flowering is blocked, and this can be reversed by gibberellin A₃ (GA₃) treatment. Associated with GA₃ treatment under high temperature are increases in sucrose, glucose and fructose as compared with warm-treated plants. Spraying with sucrose solution alone caused leaf epinasty in plants grown under high temperature. Epinasty was released by about 9 days of GA₃ treatment. In GA₃-treated plants under high temperatures, sucrose application to the source leaves led to an increase in sugar content in both leaves and inflorescence. In contrast, although in warm-treated plants sucrose application to the source leaves increased sugar content in the leaves, it did not increase sucrose content in the inflorescence. These results corroborate our hypothesis that in Phalaenopsis GA₃ stimulates sink activity in the apical meristem and promotes the translocation of sucrose from source leaves to the apex of the inflorescence, where it accumulates. GA₃ treatment led to an increase in sucrose synthase activity and had no effect on invertase activity.