Apoplastic Washing Fluid Research Articles

Roles of apoplastic peroxidases, laccases, and lignification in the manganese tolerance of hyperaccumulator Phytolacca americana

We investigated the response of Mn-hyperaccumulator Phytolacca americana L. to manganese excess as well as the relationships between lignin deposition in the plant’s leaves, peroxidase and laccase activities in the leaf apoplast, and Mn toxicity. The exceptionally high tolerance of P. americana to Mn, both in solution and in tissue, was confirmed. No visible brown spot was observed in the leaves of plants treated with ≤10,000 μM Mn for 10 days. Mn treatment significantly increased lignin content and laccase activity in the apoplastic washing fluid (AWF) of P. americana leaves. In contrast, an increase in the Mn supply was paralleled by a significant decrease in the concentration of total phenolic compounds (TPCs) and in water-soluble guaiacol peroxidase (SPOD) activity in leaf AWF. This result suggested that an increase in lignin deposition decreased the concentration of apoplastic TPCs that are available to generate potentially toxic intermediates by acting as peroxidase substrates. Thus, data of the present study indicate that lignin formation by laccase activities reduces Mn toxicity and increases Mn tolerance of P. americana by depressing SPOD-mediated formation of toxic intermediates from TPCs.

Bud development in corydalis (Corydalis bracteata) requires low temperature: a study of developmental and carbohydrate changes

Spring geophytes require a period of low temperature for proper flower development but the mechanism that underlies the relationship between cold treatment and flowering remains unknown. The present study aims to compare the developmental anatomy and carbohydrate content of the tuberous geophyte Corydalis bracteata growing under natural winter conditions from 10 to -10 degrees C (field-grown) and under a mild temperature regime of 18 degrees C (indoor-grown plants). Samples were studied under light and electron microscopy. A histochemical test (periodic acid--Schiff's) was employed to identify starch in sectioned material. Sugars were analysed by capillary gas chromatography. Apoplastic wash fluid was prepared. Under natural conditions, shoots were elongated, and buds gained in dry mass and developed normally. For indoor-grown plants, these parameters were lower in value and, from December, a progressive necrosis of flower buds was observed. The tuber consisted of the new developing one, which was connected to the bud, and the old tuber with its starch reserve. Due to the absence of plasmodesmata between new and old tuber cells, sugar transport cannot be through the symplast. Thus, a potential apoplastic route is proposed from old tuber phloem parenchyma cells to the adjacent new tuber cells. Sugar content in buds during the autumn months (September-November) was lower for indoor-grown plants than control plants, whereas the sugar content in tubers during the same period was similar for plants from both temperature treatments. However, the amount of apoplastic sugars in tubers of field-grown plants was almost 15-fold higher than in indoor-grown tubers. The results suggest that low temperature activates the apoplastic route of sugar transport in C. bracteata tubers and a consequent carbohydrate delivery to the bud. In the absence of cold treatment, the carbohydrate reserve is locked in old tuber cells so the nutrient supply to the buds is suppressed, possibly leading to bud abortion.

Physiological and proteomic characterization of manganese sensitivity and tolerance in rice (Oryza sativa) in comparison with barley (Hordeum vulgare).

Research on manganese (Mn) toxicity and tolerance indicates that Mn toxicity develops apoplastically through increased peroxidase activities mediated by phenolics and Mn, and Mn tolerance could be conferred by sequestration of Mn in inert cell compartments. This comparative study focuses on Mn-sensitive barley (Hordeum vulgare) and Mn-tolerant rice (Oryza sativa) as model organisms to unravel the mechanisms of Mn toxicity and/or tolerance in monocots. Bulk leaf Mn concentrations as well as peroxidase activities and protein concentrations were analysed in apoplastic washing fluid (AWF) in both species. In rice, Mn distribution between leaf compartments and the leaf proteome using 2D isoelectric focusing IEF/SDS-PAGE and 2D Blue native BN/SDS-PAGE was studied. The Mn sensitivity of barley was confirmed since the formation of brown spots on older leaves was induced by low bulk leaf and AWF Mn concentrations and exhibited strongly enhanced H2O2-producing and consuming peroxidase activities. In contrast, by a factor of 50, higher Mn concentrations did not produce Mn toxicity symptoms on older leaves in rice. Peroxidase activities, lower by a factor of about 100 in the rice leaf AWF compared with barley, support the view of a central role for these peroxidases in the apoplastic expression of Mn toxicity. The high Mn tolerance of old rice leaves could be related to a high Mn binding capacity of the cell walls. Proteomic studies suggest that the lower Mn tolerance of young rice leaves could be related to Mn excess-induced displacement of Mg and Fe from essential metabolic functions. The results provide evidence that Mn toxicity in barley involves apoplastic lesions mediated by peroxidases. The high Mn tolerance of old leaves of rice involves a high Mn binding capacity of the cell walls, whereas Mn toxicity in less Mn-tolerant young leaves is related to Mn-induced Mg and Fe deficiencies.

Characterization of leaf apoplastic peroxidases and metabolites in Vigna unguiculata in response to toxic manganese supply and silicon

Previous work suggested that the apoplastic phenol composition and its interaction with apoplastic class III peroxidases (PODs) are decisive in the development or avoidance of manganese (Mn) toxicity in cowpea (Vigna unguiculata L.). This study characterizes apoplastic PODs with particular emphasis on the activities of specific isoenzymes and their modulation by phenols in the Mn-sensitive cowpea cultivar TVu 91 as affected by Mn and silicon (Si) supply. Si reduced Mn-induced toxicity symptoms without affecting the Mn uptake. Blue Native-PAGE combined with Nano-LC-MS/MS allowed identification of a range of POD isoenzymes in the apoplastic washing fluid (AWF). In Si-treated plants Mn-mediated induction of POD activity was delayed. Four POD isoenzymes eluted from the BN gels catalysed both H2O2-consuming and H2O2-producing activity with pH optima at 6.5 and 5.5, respectively. Four phenols enhanced NADH-peroxidase activity of these isoenzymes in the presence of Mn2+ (p-coumaric=vanillic>>benzoic>ferulic acid). p-Coumaric acid-enhanced NADH-peroxidase activity was inhibited by ferulic acid (50%) and five other phenols (50–90%). An independent component analysis (ICA) of the total and apoplastic GC-MS-based metabolome profile showed that Mn, Si supply, and the AWF fraction (AWFH2O, AWFNaCl) significantly changed the metabolite composition. Extracting non-polar metabolites from the AWF allowed the identification of phenols. Predominantly NADH-peroxidase activity-inhibiting ferulic acid appeared to be down-regulated in Mn-sensitive (+Mn, –Si) and up-regulated in Mn-tolerant (+Si) leaf tissue. The results presented here support the previously hypothesized role of apoplastic NADH-peroxidase and its activity-modulating phenols in Mn toxicity and Si-enhanced Mn tolerance.

Peroxidase activity and phenolic compounds content in maize root and leaf apoplast, and their association with growth

Apoplastic fluid originated from maize ( Zea mays L.) leaf and root growing and non-growing regions were analyzed for peroxidase (POD) activities and phenols content. The samples were obtained by two different methods, as apoplastic washing fluid (AWF) from excised leaf and root segments, and as root apoplastic fluid (RAF) collected from the surface of intact roots by filter papers. AWF and RAF were tested for peroxidative and oxidative activity of POD, being lowest in the growing zones and gradually increasing toward root base and leaf tip. Isoelectric focusing of AWF and RAF revealed the differences pronounced in cationic group of peroxidases. Quantitative and qualitative analyzes of phenolic compounds from root and leaf apoplastic fluid was performed by HPLC. The increase of peroxidase activities was parallel to the increase of coniferyl alcohol, as well as other phenolic compound concentrations with root and leaf age, indicating that increased phenolic concentrations reflect increased lignin production during maturation. The data presented in this paper indicate that the method for getting RAF using filter papers is a more reliable procedure than infiltration/centrifugation method for studying processes which take place in root and leaf apoplastic fluid.

Protein extraction for proteome analysis from cacao leaves and meristems, organs infected by Moniliophthora perniciosa, the causal agent of the witches' broom disease

Preparation of high-quality proteins from cacao vegetative organs is difficult due to very high endogenous levels of polysaccharides and polyphenols. In order to establish a routine procedure for the application of proteomic and biochemical analysis to cacao tissues, three new protocols were developed; one for apoplastic washing fluid (AWF) extraction, and two for protein extraction--under denaturing and nondenaturing conditions. The first described method allows a quick and easy collection of AWF--using infiltration-centrifugation procedure--that is representative of its composition in intact leaves according to the smaller symplastic contamination detected by the use of the hexose phosphate isomerase marker. Protein extraction under denaturing conditions for 2-DE was remarkably improved by the combination of chemically and physically modified processes including phenol, SDS dense buffer and sonication steps. With this protocol, high-quality proteins from cacao leaves and meristems were isolated, and for the first time well-resolved 1-DE and 2-DE protein patterns of cacao vegetative organs are shown. It also appears that sonication associated with polysaccharide precipitation using tert-butanol was a crucial step for the nondenaturing protein extraction and subsequent enzymatic activity detection. It is expected that the protocols described here could help to develop high-level proteomic and biochemical studies in cacao also being applicable to other recalcitrant plant tissues.

Seasonal differences and within-canopy variations of antioxidants in mature spruce ( Picea abies) trees under elevated ozone in a free-air exposure system

The effect of free-air ozone fumigation and crown position on antioxidants were determined in old-growth spruce ( Picea abies) trees in the seasonal course of two consecutive years (2003 and 2004). Levels of total ascorbate and its redox state in the apoplastic washing fluid (AWF) were increased under double ambient ozone concentrations (2 × O 3), whilst ascorbate concentrations in needle extracts were unchanged. Concentrations of apoplastic and symplastic ascorbate were significantly higher in 2003 compared to 2004 indicating a combined effect of the drought conditions in 2003 with enhanced ozone exposure. Elevated ozone had only weak effects on total glutathione levels in needle extracts, phloem exudates and xylem saps. Total and oxidised glutathione concentrations were higher in 2004 compared to 2003 and seemed to be more affected by enhanced ozone influx in the more humid year 2004 compared to the combined effect of elevated ozone and drought in 2003 as observed for ascorbate.

Overexpression of sweetpotato swpa4 peroxidase results in increased hydrogen peroxide production and enhances stress tolerance in tobacco

Plant peroxidases (POD) reduce hydrogen peroxide (H(2)O(2)) in the presence of an electron donor. Extracellular POD can also induce H(2)O(2) production and may perform a significant function in responses to environmental stresses via the regulation of H(2)O(2) in plants. We previously described the isolation of 10 POD cDNA clones from cell cultures of sweetpotato (Ipomoea batatas). Among them, the expression of the swpa4 gene was profoundly induced by a variety of abiotic stresses and pathogenic infections (Park et al. in Mol Gen Genome 269:542-552 2003; Jang et al. in Plant Physiol Biochem 42:451-455 2004). In the present study, transgenic tobacco (Nicotiana tabacum) plants overexpressing the swpa4 gene under the control of the CaMV 35S promoter were generated in order to assess the function of swpa4 in planta. The transgenic plants exhibited an approximately 50-fold higher POD specific activity than was observed in control plants. Both transient expression analysis with the swpa4-GFP fusion protein and POD activity assays in the apoplastic washing fluid revealed that the swpa4 protein is secreted into the apoplastic space. In addition, a significantly enhanced tolerance to a variety of abiotic and biotic stresses occurred in the transgenic plants. These plants harbored increased lignin and phenolic content, and H(2)O(2 )was also generated under normal conditions. Furthermore, they showed an increased expression level of a variety of apoplastic acidic pathogenesis-related (PR) genes following enhanced H(2)O(2) production. These results suggest that the expression of swpa4 in the apoplastic space may function as a positive defense signal in the H(2)O(2)-regulated stress response signaling pathway.

The Role of Hydrogen Peroxide-Producing and Hydrogen Peroxide-Consuming Peroxidases in the Leaf Apoplast of Cowpea in Manganese Tolerance

The apoplast is considered the leaf compartment decisive for manganese (Mn) toxicity and tolerance in cowpea (Vigna unguiculata). Particularly apoplastic peroxidases (PODs) were proposed to be key enzymes in Mn toxicity-induced processes. The presented work focuses on the characterization of the role of hydrogen peroxide (H2O2)-producing (NADH peroxidase) and H2O2-consuming peroxidase (guaiacol POD) in the apoplastic washing fluid (AWF) of leaves for early stages of Mn toxicity and genotypic differences in Mn tolerance of cowpea. Leaf AWF of the Mn-sensitive cultivar (cv) TVu 91 but not of the Mn-tolerant cv 1987 showed an increase of guaiacol-POD and NADH-peroxidase activities at elevated AWF Mn concentrations. two-dimensional resolutions of AWF proteins revealed that cv TVu 91 expressed more and additional proteins at high Mn treatment, whereas Mn-tolerant cv TVu 1987 remained nearly unaffected. In both cultivars, NADH-peroxidase activity and accompanied H2O2 formation rate in vitro were significantly affected by Mn2+, p-coumaric acid, and metabolites occurring in the AWF. The total phenol concentration in the AWF was indicative of advanced stages of Mn toxicity but was rather unrelated to early stages of Mn toxicity and genotypic differences in Mn tolerance. The NADH oxidation by AWF PODs was significantly delayed or enhanced in the presence of the protein-free AWF from cv TVu 1987 or cv TVu 91, respectively. High-performance liquid chromatography analysis of AWF indicates the presence of phenols in cv TVu 1987 not observed in cv TVu 91. We conclude from our studies that the H2O2-producing NADH peroxidase and its modulation by stimulating or inhibiting phenolic compounds in the leaf apoplast play a major role for Mn toxicity and Mn tolerance in cowpea.

Does apoplastic ascorbic acid enhance manganese tolerance of Vigna unguiculata and Phaseolus vulgaris?

AbstractIn cowpea (Vigna unguiculata), the development of manganese (Mn) toxicity is considered to be accompanied by the formation of reactive oxygen species, oxidized Mn, and phenoxy radicals in the leaf apoplast. Ascorbic acid (AA) is a common antioxidant in plants, and the oxidation of AA, particularly in the leaf apoplast, contributes to the first line of defence against several biotic and abiotic stress factors. The objective of the present study was to contribute to a better understanding of the role of AA in Mn leaf‐tissue tolerance of cowpea and common bean (Phaseolus vulgaris). Five cowpea cultivars (cvs.) differed greatly in Mn tolerance, which was expressed in differences in numbers of brown spots on leaves and in peroxidase (POD) activity in the apoplastic washing fluid (AWF). In a Mn‐sensitive cv., after 3 d of Mn treatment, brown spots were formed, and POD activities were increased, accompanied by a release of proteins into the apoplast. In the AWF, the concentration of AA and the ratio of AA : (AA+DHA) decreased already after 1 d, and to only 2% after 3 d. In the leaf tissue, the ratio was nearly unaffected, and the total AA+DHA content in the leaf tissue was even increased with advanced expression of Mn toxicity. The application of AA solutions in the range of 5–10 μM via the petiole slightly enhanced Mn tolerance as indicated by the reduction of brown spots (however inconsistently) and POD activity (consistently) in the AWF. Common bean cultivars differing in ozone tolerance, which has been reported to be due to a high AA availability in the leaf apoplast, were studied for their Mn tolerance. Clear differences in Mn tolerance between the cultivars existed, however, these differences were not related to their ozone tolerance. From these results, we conclude that the maintenance of sufficient AA levels in the leaf apoplast contribute to Mn tolerance, but does not fully explain genotypic differences in Mn tolerance in cowpea and common bean.

Apoplastic peroxidases and ascorbate are involved in manganese toxicity and tolerance of Vigna unguiculata

Excessive manganese (Mn) supply induced the formation of brown spots on leaves as typical Mn toxicity symptoms in cowpea (Vigna unguiculata L. Walp.) grown in hydroponics. Differences in Mn resistance between cv. TVu 91 (Mn‐sensitive) and cv. TVu 1987 (Mn‐tolerant) expressed in the density of brown spots in older leaves were due to higher Mn tissue tolerance. Apoplastic water‐soluble peroxidase (POD) in the apoplastic washing fluid (AWF) was enhanced by increasing Mn leaf content and generally significantly higher in leaves of cv. TVu 91 than in cv. TVu 1987. Electrophoresis of AWF revealed the presence of several water‐soluble POD isoenzymes. At toxic Mn supply, the activities of these and additional POD isoenzymes increased more in the Mn‐sensitive cultivar. Levels of ascorbic acid in the apoplast and cytoplasm of the Mn‐sensitive cv. TVu 91 decreased with increasing leaf Mn contents, whereas Mn‐tolerant cv. TVu 1987 was not affected. Mn treatment lead to a stimulation of the enzymes of the ascorbic acid regeneration system (monodehydroascorbic acid reductase and glutathione reductase) in both cultivars, but the activation of glutathione reductase was clearly more enhanced in the Mn‐tolerant cultivar TVu 1987. The results provide circumstantial evidence that apoplastic ascorbate and peroxidases are involved in the expression of Mn toxicity and genotypic Mn tolerance.

Leaf apoplastic silicon enhances manganese tolerance of cowpea ( Vigna unguiculata)

The relationship between the manganese (Mn) and silicon (Si) concentrations in apoplastic washing fluids (AWF) and the severity of Mn toxicity symptoms were investigated in the leaves of the Mn-sensitive cowpea cultivar (Vigna unguiculata (L.) Walp.) in solution-culture experiments. The expression of Mn toxicity symptoms was prevented when 1.44 mmol/L Si was supplied together with 50 μmol/L Mn. However, distinct Mn toxicity symptoms were observed in plants pretreated with 1.44 mmol/L Si and then exposed to 50 μmol/L Mn without concurrent Si supply. The Mn concentrations in the AWF of leaves but not in the bulk-leaf tissue were decreased by both Si treatments. Independent from the Si treatment period, Si concentrations in the bulk-leaf tissue were increased. However, the Si concentrations in the AWF of the plants pretreated with Si were lower than those of plants supplied with Si and Mn concurrently. The severity of Mn toxicity symptoms and the guaiacol-peroxidase (POD) activity in AWF of these plants were not significantly correlated with the Mn concentrations in AWF but were highly significantly correlated with the Si concentrations in AWF. The results show that the decrease of Mn concentration in the apoplast caused by Si supply was insufficient to explain the enhanced Mn tolerance by Si supply. A possible role of apoplastic soluble Si was indicated.

Effects of silicon supply on apoplastic manganese concentrations in leaves and their relation to manganese tolerance in cowpea (Vigna unguiculata (L.) Walp.)

The effects of silicon (Si) supply on manganese (Mn) toxicity symptoms and Mn and Si concentrations in the leaf apoplast in a Mn-sensitive cowpea cultivar (Vigna unguiculata (L.) Walp. cv. TVu 91) were investigated in solution culture experiments. When 1.44 mM Si was supplied concurrently with 50 μM Mn, the Mn toxicity symptoms were clearly avoided without decreasing the total Mn concentration. On the other hand, the symptoms were not completely alleviated when the plants were pretreated with 1.44 mM Si and then exposed to 50 μM Mn without concurrent Si supply. Plants of both of these treatments exhibited lower Mn concentrations in the apoplastic washing fluids but higher amounts of adsorbed Mn on the cell walls than the plants treated with 50 μM Mn without Si supply. However, the difference in Mn concentration between plants with continuous and interrupted Si supply was not significant. Moreover, the Mn concentration in the apoplastic washing fluids of the plants with continuous supply of 1.44 mM Si and 50 μM Mn and not showing Mn toxicity symptoms was higher than that of the plants grown at 10 μM Mn without Si supply which showed distinct Mn toxicity symptoms. These results show that Si supply alleviates Mn toxicity not only by decreasing the concentration of soluble apoplastic Mn through the enhanced adsorption of Mn on the cell walls. A role of the soluble Si in the apoplast in the detoxicification of apoplastic Mn is indicated.

Effect of salt stress on growth and cation compartmentation in leaves of two plant species differing in salt tolerance

Salinity can cause toxic symptoms, especially in mature leaves after long-term exposure. Thus, Na+ accumulation in leaves could be responsible for salt toxicity. The infiltration-centrifugation technique was employed for the isolation of apoplastic washing fluids (AWF) from leaves and the detection of Na+, K+ and Ca2+ was carried out by ion chromatography. The Na+ concentrations in the leaf apoplast of salt-sensitive corn and salt-tolerant cotton plants significantly increased with higher Na+ supply. Nevertheless, the apoplastic Na+ concentration did not exceed 10 and 30 mmol/L, respectively, after salt exposure up to 150 mmol/L in the medium in short- and long-term salt treatments. Higher Na+ concentrations were found in the leaf apoplast of salt-tolerant cotton in comparison to those of salt-sensitive corn, particularly in fully expanded leaves. No bound Na+ was found in the leaf apoplast. The Na+ concentration in the leaf apoplast did not reach high enough concentrations to be responsible for the decline in leaf growth under salinity. Supplemental Ca2+ did not affect Na+ concentration in the leaf apoplast under salinity. Apoplastic Ca2+ concentration remained constant, while K+ concentrations increased in the leaf apoplast under salinity. Our results do not support the hypothesis by Oertli (1968) who proposed that salt accumulation in the leaf apoplast could be responsible for the death of leaves in plants exposed to salinity.

Is the infiltration-centrifugation technique appropriate for the isolation of apoplastic fluid? A critical evaluation with different plant species.

The suitability of the infiltration-centrifugation method for collection of apoplastic fluid from intact leaves was evaluated for different plant species. Large differences with respect to infiltrability of the leaves, which correlated inversely with stomatal and mesophyll resistance, became apparent. Osmolality of infiltration medium (deionised water, 0.2 mM CaCl2, 10 mM KCl, 180 mM 2-[N-morpholino]ethane-sulphonic acid) and incubation time, time passed between onset of infiltration and end of centrifugation, revealed relatively little influence on the composition of the apoplastic washing fluid (AWF). In contrast, the pH of the infiltrated solution highly influenced the concentration of sucrose and hexoses. With increasing centrifugation force, hexosephosphate isomerase (HPI) activity in the AWF, which was taken as an indication for cytoplasmic contamination, increased. At the same time, Ca2+ concentration in the AWF increased even more. Since Ca2+ cannot originate from the cytoplasm, the suitability of HPI as marker for cytoplasmic contamination is questioned. From the composition of the AWF, it is concluded that, if centrifugation force does not exceed 1 000 g, cytoplasmic contamination is negligible and that the infiltration-centrifugation technique reveals an easy and inexpensive way to study apoplastic solutes. The infiltration-centrifugation method was also suitable to determine apoplastic air volume (Vair) and apoplastic water volume (Vwater), which are necessary for the calculation of the ion concentration in the leaf apoplast. It could be shown that the leaves of different species and the apical and basal leaves of single plants differ in Vair and Vwater.

Calcium deficiency in potato (Solanum tuberosum ssp. tuberosum) leaves and its effects on the pectic composition of the apoplastic fluid

Potato plants (Solanum tuberosum ssp. tuberosum cv. Adelheid), multiplied in vitro, were cultivated in growth chambers on nutrient solution at calcium regimes of 1000, 90, 60 or 30 μM Ca. An absolute Ca deficiency, particularly at the low Ca‐supply levels of 30 and 60 μM Ca, manifested itself initially in the form of marginal necrosis in younger, but not in the youngest, leaves of the potato plants. Further symptoms were rolling of the leaf lamina, browning of veins and roots, and finally necrosis also of the youngest leaves. Only in an advanced stage of Ca deficiency, the meristem of the shoots died. Ca‐deficiency symptoms could be expected at a Ca content in the leaves of less than 5 mg Ca (g dry weight)−1. However, there was no close negative correlation between the extent of leaf damage and the total Ca content of the leaves. In order to obtain information about the Ca concentration in the apoplast fluid of the leaves, apoplastic washing fluid was extracted by an infiltration‐centrifugation technique. A low Ca supply reduced the Ca concentration both in the apoplast fluid of the leaves and in the cell walls. Up to 60% more diffusible pectin fragments were then found in the apoplast of younger leaves, as compared to the control supplied with an optimum Ca level of 1000 μM. The amount of diffusible pectins accounted for 1–2% of the total pectin content of younger potato leaves. The size of the existing pectin fragments varied depending on the Ca supply. Compared with an optimum Ca supply of 1000 μM, fewer monomers and up to 7 times more diffusible pectin fragments with a degree of polymerization 9–20 were present at the low Ca‐supply level (30 μM). In addition, polygalacturonase activity in tissue homogenates increased remarkably with Ca deficiency. Thus it appears that one major effect of Ca deficiency was a stimulation of the activity of polygalacturonase, which could control the breakdown of pectic polysaccharides in the cell wall. Whether the release of potentially biologically active pectic fragments in cell walls might be involved in the occurrence of Ca‐deficiency symptoms is discussed.

Impacts of ozone on Plantago major: apoplastic and symplastic antioxidant status.

The aim of this work was to examine the correspondence between apoplastic/symplastic antioxidant status and previously reported plant age-related shifts in the ozone (O3 ) resistance of Plantago major L. Seed-grown plants were fumigated in duplicate controlled environment chambers with charcoal/Purafil?-filtered air (CFA) or CFA plus 70 nmol mol-1 O3 for 7 h d-1 over a 42 d period. Measurements of stomatal conductance and antioxidants were made after 14, 28 and 42 d fumigation, on leaves at an equivalent stage of development (youngest fully expanded leaf, measured c. 9 d after emergence). Ozone exposure resulted in a similar decline in stomatal conductance across plant ages, indicating that increases in O3 resistance with plant age were mediated through changes in the tolerance of leaf tissue rather than enhanced pollutant exclusion. Leaf apoplastic washing fluid was found to contain 'unspecific' peroxidase, ascorbate peroxidase, superoxide dismutase and ascorbate, but not glutathione and the enzymes required to facilitate the regeneration of ascorbate from its oxidized forms. A weak induction in the activity of certain symplastic antioxidants was found after 14 d O3 fumigation, despite a lack of visible symptoms of injury, but shifts in symplastic antioxidant enzyme activity were not consistent with previously observed increases in resistance to O3 with plant age. By contrast, changes in 'unspecific' peroxidase activity and in the small pool of ascorbate in the leaf apoplast were found to accompany age-related shifts in O3 resistance. It is concluded that constituents of the leaf apoplast may constitute a potentially important front line defence against O3 .

Apoplastic Peroxidase Generates Superoxide Anions in Cells of Cotton Cotyledons Undergoing the Hypersensitive Reaction to Xanthomonas campestris pv. malvacearum Race 18

Cotton cotyledons displayed a hypersensitive reaction (HR) in the resistant cultivar Reba B50 after infiltration with the avirulent race 18 of Xanthomonas campestris pv. malvacearum (Xcm). Generation of active oxygen species during the HR was studied biochemically and cytochemically. O2·¯ was detected in cotyledon disks by the cytochrome c reduction assay 3 h after inoculation. This activity was inhibited by superoxide dismutase (SOD) and by the peroxidase inhibitors salicylhydroxamic acid (SHAM) and KCN but not by the NADPH oxidase inhibitor diphenyleneiodonium chloride (DPI). Strong NADH oxidation activity also was found 3 h after inoculation in crude extracts or in apoplastic washing fluid and was dramatically decreased after treatment with SHAM or KCN. NADH oxidation was activated by 2,4- dichlorophenol and MnCl2, indicating the involvement of a peroxidase. Activity of cationic peroxidase isoforms (pI 9 to 9.5) constitutively expressed in cotyledons was found to be enhanced 3 h after inoculation in the resistant cultivar. Activities of apoplastic peroxidase(s) and H2O2 accumulation were observed cytochemically, 3 and 4 h post inoculation, respectively. When digitonin, a O2·- elicitor, was infiltrated into cotyledons of resistant and susceptible cultivars, generation of O2·¯ radicals was shown to be reduced by SOD and inhibited by SHAM and KCN as observed after infection, and also by DPI. Our results strongly suggest that cotton cotyledons contain two O2·¯- generating systems and that cells undergoing the HR in response to an avirulent race of Xcm produce O2·¯ through the activation of an apoplastic peroxidase.

Apoplastic Ion Concentration of Intact Leaves of Field Bean ( Vicia faba) as Influenced by Ammonium and Nitrate Nutrition

Summary In order to differentiate between free and exchangeable apoplastic cations, intact leaves of Vicia faba were vacuum infiltrated with solutions differing in cation exchange strength (water and 50 mol · m -3 BaCl 2 ). Apoplastic washing fluids were subsequently obtained by careful centrifugation. While for the bivalent cations Ca 2+ and Mg 2+ it was possible to differentiate between a free and bound fraction this was not the case with K + . Concentration of free apoplastic Ca 2+ and K + varied between 0.3–0.8 mol· m -3 and 1.3–4.5 mol · m -3 , respectively. Total apoplastic Ca 2+ accounted for up to 40 % of total leaf Ca 2+ content while just 1–8 % of total leaf Mg 2+ and K + content were found in this compartment. Surprisingly, the N-form revealed little influence on apoplastic ion concentration. Independently from the form of N-nutrition, NH 4 + was not found in the leaf apoplast while NO 3 - concentration was rather low (0.01–0.03 mol · m -3 ). The pH of the intercellular washing fluid (PH 6.1) remained unaffected by the N-form.

Apoplastic Peroxidases and Lignification in Needles of Norway Spruce (Picea abies L.).

The objective of the present study was to investigate the correlation of soluble apoplastic peroxidase activity with lignification in needles of field-grown Norway spruce (Picea abies L.) trees. Apoplastic peroxidases (EC 1.11.1.7) were obtained by vacuum infiltration of needles. The lignin content of isolated cell walls was determined by the acetyl bromide method. Accumulation of lignin and seasonal variations of apoplastic peroxidase activities were studied in the first year of needle development. The major phase of lignification started after bud break and was terminated about 4 weeks later. This phase correlated with a transient increase in apoplastic guaiacol and coniferyl alcohol peroxidase activity. NADH oxidase activity, which is thought to sustain peroxidase activity by production of H2O2, peaked sharply after bud break and decreased during the lignification period. Histochemical localization of peroxidase with guaiacol indicated that high activities were present in lignifying cell walls. In mature needles, lignin was localized in walls of most needle tissues including mesophyll cells, and corresponded to 80 to 130 [mu]mol lignin monomers/g needle dry weight. Isoelectric focusing of apoplastic washing fluids and activity staining with guaiacol showed the presence of strongly alkaline peroxidases (isoelectric point [greater than or equal to] 9) in all developmental stages investigated. New isozymes with isoelectric points of 7.1 and 8.1 appeared during the major phase of lignification. These isozymes disappeared after lignification was terminated. A strong increase in peroxidase activity in autumn was associated with the appearance of acidic peroxidases (isoelectric point [less than or equal to] 3). These results suggest that soluble alkaline apoplastic peroxidases participate in lignin formation. Soluble acidic apoplastic peroxidases were apparently unrelated to developmentally regulated lignification in spruce needles.