Salicylic acid-independent induction of pathogenesis-related protein transcripts by sugars is dependent on leaf developmental stage.

Soluble sugars have been found to regulate a number of genes involved in functions associated with sink metabolism, defense reactions and photosynthesis. As viruses and pathogens induce the expression of pathogenesis-related (PR) protein genes and have also been reported to lead to localized sugar accumulation in leaves, it was investigated whether a salicylic acid-independent but sugar-dependent pathway for PR-protein gene induction may exist in plant cells. Leaf discs of tobacco plants were floated on different sugar solutions, transcript accumulation and salicylic acid (SA) levels were subsequently determined. PR-Q and PAR-1 transcripts were found to be inducible by glucose, fructose and sucrose. No significant change in SA content could be detected, following incubation. On the other hand, SAR8.2 transcripts were repressed by elevated levels of soluble sugars and sorbitol, respectively, suggesting sensitivity to turgor pressure. Since leaves undergo sink to source transition during growth, sugar responsiveness was investigated in leaves of different developmental stages. Interestingly, induction of PR-Q and PAR-1 by soluble sugars was essentially restricted to fully expanded leaves and was independent of plant age. Induction by salicylate was not confined to the source capacity of a leaf but was dependent on the age of the respective leaf. Repression of transcripts encoding photosynthetic genes (ribulose-1,5-bisphosphate carboxylase/oxygenase (rbcS) and chlorophyll a/b binding protein (cab) by soluble sugars were largely independent from the leaf developmental state. These findings hint to the possibility of salicylic acid-independent defense reactions of plants against pathogens by induction of a set of PR proteins in source leaves. Furthermore, the data suggest different mechanisms for the induction of PR-protein genes and the repression of photosynthetic genes by soluble sugars.

Systemic Acquired Resistance Mediated by the Ectopic Expression of Invertase: Possible Hexose Sensing in the Secretory Pathway.

Systemic acquired resistance (SAR) has been reported to be associated with lesion-mimic mutants. Tobacco plants expressing vacuolar and apoplastic yeast-derived invertase (vaclnv and cwlnv, respectively) develop spontaneous necrotic lesions similar to hypersensitive responses caused by avirulent pathogens. Therefore, SAR and metabolic alterations leading to the activation of defense-related responses were studied in these plants. Defense-related gene transcripts, callose content, peroxidase activities, and levels of salicylic acid were found to be elevated. The defense reactions were accompanied by increased resistance toward potato virus Y and were measured as decreased viral spreading and reduced multiplication in systemic leaves of the transgenic plants. Interestingly, the accumulation of pathogenesis-related (PR) protein transcripts (PR-Q) and repression of photosynthetic gene transcripts (chlorophyll a/b binding protein) were inversely correlated and required the same threshold level of hexoses for induction and repression. Expression of a cytosolic yeast-derived invertase in transgenic tobacco plants with equally increased levels of sugars neither displayed SAR responses nor showed decreased levels of photosynthetic genes. It is suggested that hexose sensing in the secretory pathway is essential for mediating the activation of defense-related genes as well as repression of photosynthetic genes in vaclnv and cwlnv plants.

Cholera toxin elevates pathogen resistance and induces pathogenesis-related gene expression in tobacco.

In animals, plants and fungi, cholera toxin (CTX) can activate signalling pathways dependent on heterotrimeric GTP binding proteins (G-proteins). We transformed tobacco plants with a chimeric gene encoding the A1 subunit of CTX regulated by a light-inducible wheat Cab-1 promoter. Tissues of transgenic plants expressing CTX showed greatly reduced susceptibility to the bacterial pathogen Pseudomonas tabaci, accumulated high levels of salicylic acid (SA) and constitutively expressed pathogenesis-related (PR) protein genes encoding PR-1 and the class II isoforms of PR-2 and PR-3. In contrast, the class I isoforms of PR-2 and PR-3 known to be induced in tobacco by stress, by ethylene treatment and as part of the hypersensitive response to infection, were not induced and displayed normal regulation. In good agreement with these results, microinjection experiments demonstrated that CTX or GTP-gamma-S induced the expression of a PR1-GUS reporter gene but not that of a GLB-GUS reporter gene containing the promoter region of a gene encoding the class I isoform of PR-2. Microinjection and grafting experiments strongly suggest that CTX-sensitive G-proteins are important in inducing the expression of a subset of PR genes and that these G-proteins act locally rather than systemically upstream of SA induction.

Local and Systemic Biosynthesis of Salicylic Acid in Infected Cucumber Plants.

Radiolabeling studies showed that salicylic acid (SA), an essential component in the signal transduction pathway leading to systemic acquired resistance, is synthesized from phenylalanine (Phe) and benzoic acid in cucumber (Cucumis sativus L.) plants inoculated with pathogens. Leaf discs from plants inoculated with either tobacco necrosis virus or Pseudomonas lachrymans incorporated more [14C]Phe into [14C]SA than mock-inoculated controls. The identity of SA was confirmed by gas chromatography-mass spectrometry. No reduction in specific activity of [14C]SA was observed for either free or bound SA between control and infected plants after feeding [14C]Phe. A specific inhibitor of Phe ammonia-lyase, 2-aminoindan-2-phosphonic acid, completely inhibited the incorporation of [14C]Phe into [14C]SA, although plants treated with 2-aminoindan-2-phosphonic acid could still produce [14C]SA from [14C]benzoic acid. Biosynthesis of SA in tissue inoculated with tobacco necrosis virus followed a transient pattern with the highest induction occurring 72 h postinoculation. Uninfected tissues from an infected plant synthesized de novo more SA than did controls. This suggests the involvement of a systemic signal triggering SA synthesis in tissue distant from the site of infection that display systemic acquired resistance.

Retention of cadmium in roots of maize seedlings. Role of complexation by phytochelatins and related thiol peptides.

Cd from roots of maize was partitioned in seedlings exposed to 3 microM CdSO4 for 1 to 7 d. Most of the root Cd (92-94%) was buffer soluble and provided the classical metal-induced cysteine-rich, high-molecular-weight Cd-binding complex. This complex, however, bound only part of the Cd within the roots, from 19% after 1 d of exposure to 59% by d 7. Three families of peptides formed the Cd-binding complex: (gamma-glutamic acid-cysteine)n-glycine [(gamma-Glu-Cys)n-Gly], or phytochelatins, (gamma-Glu-Cys)n, and (gamma-Glu-Cys)n-Glu. The monothiols gamma-Glu-Cys-Gly (glutathione), gamma-Glu-Cys, and gamma-Glu-Cys-Glu were absent from the complex. The n2 oligomers of any peptide were the least concentrated, whereas the n3 and n4 oligomers increased in the complex with exposure to Cd. By d 7, 75% of (gamma-Glu-Cys)4-Gly, 80% of (gamma-Glu-Cys)4, and 73% of (gamma-Glu-Cys)3-Glu were complexed with Cd. The peptide thiol:Cd molar ratio for the complexes was 1.01 +/- 0.07, as if the minimal amount of thiol was used to bind Cd. Acid-labile sulfide occurred in the complexes from d 1 onward at the low S2-;Cd molar ratio of 0.18 +/- 0.02.

Three families of thiol peptides are induced by cadmium in maize.

Phytochelatins ((gamma GluCys)nGly) are synthesized from glutathione by plants exposed to metals like Cd2+, Cu2+ and Zn2+. An intracellular complex formed by phytochelatins with Cd2+ and sulfide is thought to detoxify the metal possibly by sequestration in the vacuole. It was found that maize seedlings exposed to Cd2+ produced phytochelatins and two additional families of cysteine-containing peptides, (gamma GluCys)n and (gamma GluCys)nGlu. All thiol peptides with n = 2 and 3 were purified and their structure characterized by tandem mass spectrometry. For maize plants exposed to Cd2+ for 7 days, phytochelatins were synthesized preferentially in the first 24 h whereas the amounts of (gamma GluCys)n and (gamma GluCys)nGlu were the highest thereafter. This was probably due to an initial large pool of glutathione available in control plants compared with a dearth of gamma GluCys and no detectable gamma GluCysGlu. The (gamma GluCys)nGlu peptides were induced exclusively by Cd2+ as they were below the detection limit in control seedlings that contained low amounts of phytochelatins and (gamma GluCys)n. Since the Cys moiety of the peptides is essential for binding Cd2+, a role for accumulated (gamma GluCys)n and (gamma GluCys)nGlu in detoxifying Cd2+ in plants must be considered.

gamma-Glutamylcysteinylglutamic acid--a new homologue of glutathione in maize seedlings exposed to cadmium.

Exposure of plants to Cd induces the appearance of several thiols based on glutathione and known as class III metallothioneins (or phytochelatins). A new tripeptide with the structure gamma-GluCysGlu accumulated in roots and shoots of Cd-exposed maize seedlings. This thiol was purified and identified by tandem mass spectrometry. The fragmentation pattern of the maize tripeptide was identical to that of the synthetic compound. Like glutathione, this new tripeptide may serve as a precursor for longer-chain peptides involved in metal detoxification through the formation of Cd-binding complexes.

Ortho-anisic acid as internal standard for the simultaneous quantitation of salicylic acid and its putative biosynthetic precursors in cucumber leaves.

Salicylic acid (SA) acts as an endogenous signal for the induction of systemic acquired resistance (SAR) in plants infected by pathogens. In order to study SA biosynthesis in plants, a quantitative method using an internal standard was developed for the simultaneous measurement of SA and three of its putative bioprecursors, namely trans-cinnamic acid (CA), trans-ortho-hydroxycinnamic acid (oHCA), and benzoic acid (BA). ortho-Anisic acid (oANI) was found to be a suitable internal standard for all four compounds. It was not present as endogenous compound in cucumber leaves, no substance interfered with its detection, and it followed identical losses as SA, CA, oHCA, and BA during the extraction procedure. Baseline separation of these four compounds and of eight other plant phenolics was achieved in 35 min using deactivated reversed-phase HPLC. On-line detection was performed fluorimetrically for SA, oANI, and oHCA (with the excitation and emission wavelengths optimized for each compound) and with uv detection for CA and BA. Correlation equations for SA, CA, oHCA, and BA using oANI as internal standard were linear for a broad range of concentrations. The limits of detection (in ng/g fresh weight, FW) obtained during routine analyses were as follows: SA, 4; oHCA, 100; CA, 150; and BA, 500. The method was used to quantify levels of free and acid-hydrolyzed bound SA in cucumber plants either 5 days postinoculation with Pseudomonas lachrymans or after mock-inoculation with water on their first leaf.(ABSTRACT TRUNCATED AT 250 WORDS)

Alteration of thiol pools in roots and shoots of maize seedlings exposed to cadmium : adaptation and developmental cost.

Roots of intact 5-day-old maize (Zea mays L.) seedlings were exposed to 3 micromolar Cd during a 7-day period. Cysteine, gamma-glutamylcysteine, glutathione (GSH), and Cd-induced acid-soluble thiols (ASTs), including phytochelatins, were quantified in roots and shoots. Adaptation to Cd and its cost to seedling development were evaluated by measuring Cd content, tissue fresh weight, and rate of root elongation. Roots contained 60 to 67% of the Cd in the seedlings between 4 and 7 days of exposure. Exposure to Cd decreased the fresh weight gain in roots from day 4 onward without affecting the shoots. Between days 1.5 and 3.5 of Cd treatment, roots elongated more slowly than controls; however, their growth rate recovered thereafter and exceeded that of controls. Exposure to Cd did not appreciably affect the concentration of cysteine in the seedlings. However, the initial low concentration of gamma-glutamylcysteine increased (after a lag of 6 hours in roots and 2 days in shoots), reaching a plateau by day 6 at 28.5 nanomoles per gram of fresh weight in roots and by day 5 at 19.1 nanomoles per gram of fresh weight in shoots. During the first 9 hours of Cd exposure, the concentration of GSH in roots decreased dramatically (at 31.6 nanomoles per gram of fresh weight per hour) and thereafter decreased more slowly than in controls. The depletion of GSH in the roots (366 nanomoles per gram of fresh weight) matched the synthesis of ASTs (349 nanomoles per gram of fresh weight) during the first 48 hours. The concentration of ASTs in roots increased steadily thereafter to reach 662.2 nanomoles per gram of fresh weight by 6 days of Cd exposure. In shoots, Cd had little influence on the concentration of GSH, but ASTs still accumulated to 173.3 nanomoles per gram fresh weight after 5 days. The molar ratio of thiols in ASTs to Cd increased to a maximum of 10.24 in roots after 4 hours and of 4.25 in shoots after 2 days of Cd exposure. After 4 days, the ratio reached a plateau of approximately 2 in roots and between 2 and 3 in shoots, as if a steady state of Cd chelation had been achieved in both organs. The plateau coincided with recovered root elongation or an adaptation to Cd. The reduced fresh weight gain of the roots during this time, however, indicated that the synthesis of Cd-induced thiols was at a cost to root development.

Rapid high-performance liquid chromatographic determination with fluorescence detection of furosemide in human body fluids and its confirmation by gas chromatography-mass spectrometry.

Furosemide (FD: Lasix) is a loop diuretic which strongly increases both urine flow and electrolyte urinary excretion. Healthy volunteers were administered 40 mg orally (dissolved in water) and concentrations of FD were determined in serum and urine for up to 6 h for eight subjects, who absorbed water at a rate of 400 ml/h. Quantification was performed by HPLC with fluorescence detection (excitation at 233 nm, emission at 389 nm) with a limit of detection of 5 ng/ml for a 300-microliters sample. The elution of FD was completed within 4 min using a gradient of acetonitrile concentration rising from 30 to 50% in 0.08 M phosphoric acid. The delay to the peak serum concentration ranged from 60 to 120 min. FD was still easily measurable in the sera from all subjects 6 h after administration. In urine, the excretion rates reached their maximum between 1 and 3 h. The total amount of FD excreted in the urine averaged 11.2 mg (range 7.6-14.0 mg), with a mean urine volume of 3024 ml (range 2620-3596 ml). Moreover, the urine density was lower than 1.010 (recommended as an upper limit in doping analysis to screen diuretics) only for 2 h. An additional volunteer was administered 40 mg of FD and his urine was collected over a longer period. FD was still detectable 48 h after intake. Gas chromatography-mass spectrometry with different types of ionization was used to confirm the occurrence of FD after permethylation of the extract. Negative-ion chemical ionization, with ammonia as reactant gas, was found to be the most sensitive method of detection.

Simultaneous gas chromatography-mass spectrometry quantification of endogenous [C]- and applied [C]indole-3yl-acetic Acid levels in growing maize roots.

The use of stable indole-3yl-acetic acid (IAA) labeled by 6 atoms of (13)C allowed, after [(13)C]IAA treatment, simultaneous gas chromatography-mass spectrometry quantifications of both endogenous [(12)C]IAA and applied [(13)C]IAA levels in Zea mays L. roots. Root material was immersed for 1 hour in a buffered (pH 6.0) solution without or with [(13)C]IAA at 10(-7) molar. Both applied and endogenous IAA were thus measured for three zones of the roots (apical, elongating, differentiating) directly after treatment and also 2 hours later. Growth was followed over a 4 hour period. Roots not immersed elongated more than control roots (immersed in buffer), which grew more than IAA-treated roots. Immersion in buffer induced a large decrease (-68%) of [(12)C]IAA in the apical part of control roots, whereas immersion in [(13)C]IAA prevented most of it. No significant difference between control and treated roots occurred in the two other zones. Two hours after treatment, [(13)C]IAA had completely disappeared from the elongating zone even though [(12)C]IAA level was essentially stable. A direct relationship occurred between the level of IAA in the elongating zone and the growth of the root. This relationship was strongly disturbed if unmetabolized [(13)C]IAA was present. However, the relationship returned to its initial state when significant amounts of free [(13)C]IAA were no longer detectable. These results are discussed in terms of the stability of both types of compounds and the utility of the method of using stable isotopes of hormones, for the understanding of hormonal regulation of plant growth.

Local treatment with indole-3-acetic acid induces differential growth responses in Zea mays L. roots.

Differential growth responses of primary Zea mays L. roots were induced after asymmetrical applications of IAA-loaded beads along their elongating zone (1.0-4.0 mm from tip) and were modulated according to the initial growth rates of the roots. The amplitude and location (midpoint) of curvature, the elongation of the roots and the uptake of IAA were measured after 4 h. The amount of curvature depended on the location of the bead and upon the amount of IAA taken up. Curvature occurred towards the site of IAA application and the zone (midpoint) of curvature was always found to be basipetal to the IAA-loaded bead, indicating the transport of a growth signal in a basipetal direction from the zone of IAA application towards the zone of curvature. Two regions within the elongation zone were thus characterized: an acropetal region (between 1.0-3.5 mm from the tip) in which IAA induced a growth signal and a basipetal region (3.5-6.5 mm from the tip) which displayed the growth response; neither of these regions were able to carry out both physiological functions. The acropetal region was extended in fast-growing roots or when the amount of applied IAA was increased. No significant growth stimulation of fast-growing roots was induced regardless of the amount of IAA supplied, but the growth of slow-growing roots was stimulated with very low amounts of IAA ([Symbol: see text]100 pg/root after 4 h). Inhibition of growth of both classes of roots increased with amounts of IAA between 100-600 pg/root after 4 h, but no further inhibition of growth appeared after treatment with greater amounts of IAA. A model based on these data shows a possible relationship between the uptake of IAA and root growth and indicates a clear relationship between an optimal level of IAA and root growth. The responses of the elongating cells of primary maize roots to applied IAA depend on their developmental stage and on the initial growth rates of the root.

Maize root growth and localized indol-3yl-acetic Acid treatment: a new methodological approach.

Resin beads loaded with indol-3yl-acetic acid (IAA) were used as asymmetrical donors along the elongation zone of intact primary Zea mays L. roots. A strong curvature, towards and above the bead, occurred when IAA was applied at a mean distance of 2.20 mm from the tip. No curvature was detected after applications at 3.89 and 5.71 mm from the tip. Correspondence analysis, a new methodological approach in plant hormone studies, permitted the evaluation of the relative influence of several factors on the curvature observed for each root. The parameters considered were the initial growth rate, the exact location of the bead (1.64-2.73 millimeters from tip) and the quantity of IAA absorbed. Roots which grew rapidly bent earlier than slowly growing ones and the more basal the treatment was, the less curvature occurred. Surprisingly, the amount of IAA taken up (between 1.2 and 2.2 times the endogenous IAA content) was found to have no influence on either the time-course or the magnitude of this growth inhibition (curvature). The usefulness of this multivariate analysis is also discussed.

Local application of indole-3-acetic acid, by resin beads to intact growing maize roots.

Five types of anion-exchanger resin beads which had adsorbed indole-3-acetic acid (IAA) were tested as IAA donors. The rate of IAA-uptake by beads was a function of time and pH. The release was relatively steady during 6 h application on vertical maize roots. No IAA degradation occurred in the beads (Amberlite IRA 400 type) but 45.8% was metabolised in the roots during treatment. Beads loaded with IAA and placed on one side of the root (at 2.20±0.03 mm from the tip) induced a curvature towards and above the bead (23.3±1.1 degrees after 5.25 h application). In contrast, control beads (without IAA) did not change the axial growth rate. Applied IAA seemed to move differently from endogenous IAA. The use of resin beads loaded with IAA offers a technique to study the effects of local IAA application on intact growing roots.