Silicon ameliorates manganese toxicity in cucumber by decreasing hydroxyl radical accumulation in the leaf apoplast

This work was focused on the role of silicon (Si) in amelioration of manganese (Mn) toxicity caused by elevated production of hydroxyl radicals (·OH) in the leaf apoplast of cucumber (Cucumis sativus L.). The plants were grown in nutrient solutions with adequate (0.5 μM) or excessive (100 μM) Mn concentrations with or without Si being supplied. The symptoms of Mn toxicity were absent in the leaves of Si-treated plants subjected to excess Mn, although the leaf Mn concentration remained extremely high. The apoplastic concentration of free Mn(2+) and H(2)O(2) of high Mn-treated plants was significantly decreased by Si treatment. Si supply suppressed the Mn-induced increased abundance of peroxidase (POD) isoforms in the leaf apoplastic fluid, and led to a rapid suppression of guaiacol-POD activity under excess Mn. The spin-trapping reagent 5-(diethoxyphosphoryl)-5-methyl-1-pyrroline-N-oxide was used to detect ·OH by electron paramagnetic resonance spectroscopy. Although supplying Si markedly decreased the accumulation of ·OH in the leaf apoplast with excess Mn, adding monosilicic acid to the Mn(2+)/H(2)O(2) reaction mixture did not directly affect the Fenton reaction in vitro. The results indicate that Si contributes indirectly to a decrease in ·OH in the leaf apoplast by decreasing the free apoplastic Mn(2+), thus regulating the Fenton reaction. A direct inhibitory effect of Si on guaiacol-POD activity (demonstrated in vitro) may also contribute to decreasing the POD-mediated generation of ·OH.     

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.     

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 M 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 M 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 M 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 M Mn and not showing Mn toxicity symptoms was higher than that of the plants grown at 10 M 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.     

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.     

Compartmentation of Assimilate Fluxes in Leaves

Abstract: Sugar levels in the apoplast of assimilate exporting leaves were studied in two groups of plant species with contrasting structures of companion cells in minor veins. These species are termed either "symplastic" (with intermediary cells) or "apoplastic" (with transfer or ordinary cells). Sugars were measured in intercellular washing fluid after extracting the apoplast by an infiltration-centrifugation technique. During the course of a day, sugar contents in the apoplast were, in general, lower in species with intermediary cells than in species with transfer or ordinary cells. In "symplastic" species, apoplastic sucrose concentrations were between 0.3 and 1 mM. In "apoplastic" species with transfer cells, they ranged between 2 and 6 mM. Apoplastic hexose contents were between 0.3 and 1 mM irrespective of presumed transport mode. "Symplastic" and "apoplastic" plants differed markedly in their response to a'translocation block. In "symplastic" plants, inhibition of assimilate export left apoplastic concentrations of sucrose and hexoses unchanged, whereas in "apoplastic" plants sugar levels increased, the maximal increase being observed with sucrose. In these plants, concentrations of sucrose were two to six times higher in the apoplast under export inhibition than in control leaves. The data suggest a different role of the leaf apoplast in the compartmentation and export of assimilates in the two plant groups under study.     

Does high bicarbonate supply to roots change availability of iron in the leaf apoplast?

The role of the leaf apoplast in iron (Fe) uptake into the leaf symplast is insufficiently understood, particularly in relation to the supposed inactivation of Fe in leaves caused by elevated bicarbonate in calcareous soils. It has been supposed that high bicarbonate supply to roots increases the pH of the leaf apoplast which decreases the physiological availability of Fe in leaf tissues. The study reported here has been carried out with sunflower plants grown in nutrient solution and with grapevine plants grown on calcareous soil under field conditions. The data obtained clearly show that the pH of the leaf apoplastic fluid was not affected by high bicarbonate supply in the root medium (nutrient solution and field experiments). The concentrations of total, symplastic and apoplastic Fe were decreased in chlorotic leaves of both sunflower (nutrient solution experiment) and grapevine plants in which leaf expansion was slightly inhibited (field experiment). However, in grapevine showing severe inhibition of leaf growth, total Fe concentration in chlorotic leaves was the same or even higher than in green ones, indicative to the so-called `chlorosis paradox'. The findings do not support the hypothesis of Fe inactivation in the leaf apoplast as the cause of Fe deficiency chlorosis since no increase was found in the relative amount of apoplastic Fe (% of total leaf Fe) either in the leaves of sunflower or grapevine plants. It is concluded that high bicarbonate concentration in the soil solution does not decrease Fe availability in the leaf apoplast.     

Transport and action of ascorbate at the plant plasma membrane

The plasmalemma is both a bridge and a barrier between the cytoplasm and the outside world. It is a dynamic interface that perceives and transmits information concerning changes in the environment to the nucleus to modify gene expression. In plants, ascorbate is an essential part of this dialogue. The concentration and ratio of reduced to oxidized ascorbate in the apoplast, for example, possibly modulates cell division and growth. The leaf apoplast contains millimolar amounts of ascorbate that protect the plasmalemma against oxidative damage. The apoplastic ascorbate-dehydroascorbate redox couple is linked to the cytoplasmic ascorbate-dehydroascorbate redox couple by specific transporters for either or both metabolites. Although evidence about the mechanisms driving ascorbate or dehydroascorbate transport remains inconclusive, these carrier proteins potentially regulate the level and redox status of ascorbate in the apoplast. The redox coupling between compartments facilitated by these transport systems allows coordinated control of key physiological responses to environmental cues.     

Transient alkalinization in the leaf apoplast of Vicia faba L. depends on NaCl stress intensity: an in situ ratio imaging study

The apoplast is suggested to be involved not only in the response, but also in the perception and transduction of various environmental signals. In this context, apoplastic alkalinization has previously been discussed as a general stress factor caused by abiotic and biotic stress events. In this study, an ion-sensitive fluorescence probe in combination with inverted fluorescence microscopy has been used for in planta monitoring of apoplastic shoot pH during challenging of Vicia faba L. plants by NaCl stress encountered at the roots. We demonstrate that transient increases in leaf apoplastic pH are dependent on the NaCl stress intensity. Moreover, we have visualized spatial pH gradients within the leaf apoplast. Our results indicate that these pH responses are propagated from root to leaf and that this occurs along the apoplast.     

The apoplastic antioxidant system in Prunus: response to long-term plum pox virus infection

This work describes, for the first time, the changes taking place in the antioxidative system of the leaf apoplast in response to plum pox virus (PPV) in different Prunus species showing different susceptibilities to PPV. The presence of p-hydroxymercuribenzoic acid (pHMB)-sensitive ascorbate peroxidase (APX) (class I APX) and pHMB-insensitive APX (class III APX), superoxide dismutase (SOD), peroxidase (POX), NADH-POX, and polyphenoloxidase (PPO) was described in the apoplast from both peach and apricot leaves. PPV infection produced different changes in the antioxidant system of the leaf apoplast from the Prunus species, depending on their susceptibility to the virus. In leaves of the very susceptible peach cultivar GF305, PPV brought about an increase in class I APX, POX, NADH-POX, and PPO activities. In the susceptible apricot cultivar Real Fino, PPV infection produced a decrease in apoplastic POX and SOD activities, whereas a strong increase in PPO was observed. However, in the resistant apricot cultivar Stark Early Orange, a rise in class I APX as well as a strong increase in POX and SOD activities was noticed in the apoplastic compartment. Long-term PPV infection produced an oxidative stress in the apoplastic space from apricot and peach plants, as observed by the increase in H2O2 contents in this compartment. However, this increase was much higher in the PPV-susceptible plants than in the resistant apricot cultivar. Only in the PPV-susceptible apricot and peach plants was the increase in apoplastic H2O2 levels accompanied by an increase in electrolyte leakage. No changes in the electrolyte leakage were observed in the PPV-inoculated resistant apricot leaves, although a 42% increase in the apoplastic H2O2 levels was produced. Two-dimensional electrophoresis analyses revealed that the majority of the polypeptides in the apoplastic fluid had isoelectric points in the range of pI 4-6. The identification of proteins using MALDI-TOF (matrix-assisted laser desorption/ionization-time of flight) and peptide mass fingerprinting analyses showed the induction of a thaumatin-like protein as well as the decrease of mandelonitrile lyase in peach apoplast due to PPV infection. However, most of the selected polypeptides showed no homology with known proteins. This fact emphasizes that, at least in Prunus, most of the functions of the apoplastic space remain unknown. It is concluded that long-term PPV infection produced an oxidative stress in the leaf apoplast, contributing to the deleterious effects produced by PPV infection in leaves of inoculated, susceptible Prunus plants.     

Antifreeze protein accumulation in freezing-tolerant cereals

Freezing-tolerant plants withstand extracellular ice formation at subzero temperatures. Previous studies have shown that winter rye ( Secale cereale L.) accumulates proteins in the leaf apoplast during cold acclimation that have antifreeze properties and are similar to pathogenesis-related proteins. To determine whether the accumulation of these antifreeze proteins is common among herbaceous plants, we assayed antifreeze activity and total protein content in leaf apoplastic extracts from a number of species grown at low temperature, including both monocotyledons (winter and spring rye, winter and spring wheat, winter barley, spring oats, maize) and dicotyledons (spinach, winter and spring oilseed rape [canola], kale, tobacco). Apoplastic polypeptides were also separated by SDS-PAGE and immunoblotted to determine whether plants generally respond to low temperature by accumulating pathogenesis-related proteins. Our results showed that significant levels of antifreeze activity were present only in the apoplast of freezing-tolerant monocotyledons after cold acclimation at 5/2⁰C. Moreover, only a closely related group of plants, rye, wheat and barley, accumulated antifreeze proteins similar to pathogenesis-related proteins during cold acclimation. The results indicate that the accumulation of antifreeze proteins is a specific response that may be important in the freezing tolerance of some plants, rather than a general response of all plants to low temperature stress.     

Differential proteomic analysis of apoplastic proteins during initial phase of salt stress in rice

The plant cell apoplast, which consists of all the compartments beyond the plasma membrane, is implicated in a variety of functions during plant growth and development as well as in plant defence responses to stress conditions. To evaluate the role of apoplastic proteins in initial phase of salt stress, a 2-DE based differential proteomics approach has been used to identify apoplastic salt response proteins. Six salt response proteins have been identified, among them, an apoplastic protein OsRMC, which belongs to cysteine-rich repeat receptor like protein kinase subfamily but without the kinase domain, has shown drastically increased abundance in response to salt stress during the initial phase. Our results show, OsRMC negative regulates the salt tolerance of rice plants. These results indicated that plant apoplastic proteins may have important role in plant salt stress response signal pathway.Key words: rice, apoplast, proteomic, salt stress, receptor-like protein kinase, OsRMC     

Antioxidants in the apoplast and symplast of beech (Fagus sylvatica L.) leaves: seasonal variations and responses to changing ozone concentrations in air

Concentrations of the antioxidants ascorbate and glutathione were measured in the apoplast of beech (Fagus sylvatica L.) leaves and in leaf tissue. During early leaf development, reduced ascorbate (ASC) was almost absent from the apoplast, whereas levels of oxidized ascorbate (DHA) were high. Less than 20% of the apoplastic ascorbate was reduced. ASC increased towards midsummer, reaching top levels of about 4molm^?3 apoplast volume in July and August. Reduction increased to 60–75% in summer. Neither DHA reductase nor glutathione was detected in the apoplast of beech leaves. Levels of apoplastic ascorbate were compared with ambient concentrations of ozone in air. Statistical analysis indicated a significant interrelation between atmospheric ozone and apoplastic ascorbate. In midsummer of 1993, contents of DHA were increased in the apoplast when ozone concentrations were high. Apoplastic ASC was also positively correlated with ambient ozone concentrations, but with a delay of 3 to 7d. In leaf tissue, levels of ascorbate were between 17 and 21 μmolg^?1 FW in summer. Except for late April and November, more than 95% of the intracellular ascorbate was reduced. Glutathione contents were lowest during the summer. Oxidation was increased in spring and autumn, when apoplastic ascorbate was also largely oxidized. Usually, 80 to 90% of the glutathione was reduced. During the summer, intracellular concentrations of oxidized glutathione (GSSG) were increased, with a delay of about 1d following periods of high ambient ozone concentrations. The transitory accumulation of GSSG may be explained by slow enzymatic regeneration of glutathione.     

Nitrate does not result in iron inactivation in the apoplast of sunflower leaves

It has been hypothesized that nitrate (NO(3)(-)) nutrition might induce iron (Fe) deficiency chlorosis by inactivation of Fe in the leaf apoplast (H.U. Kosegarten, B. Hoffmann, K. Mengel [1999] Plant Physiol 121: 1069-1079). To test this hypothesis, sunflower (Helianthus annuus L. cv Farnkasol) plants were grown in nutrient solutions supplied with various nitrogen (N) forms (NO(3)(-), NH(4)(+) and NH(4)NO(3)), with or without pH control by using pH buffers [2-(N-morpholino)ethanesulfonic acid or 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid]. It was shown that high pH in the nutrient solution restricted uptake and shoot translocation of Fe independently of N form and, therefore, induced Fe deficiency chlorosis at low Fe supply [1 micro M ferric ethylenediaminedi(O-hydroxyphenylacetic acid)]. Root NO(3)(-) supply (up to 40 mM) did not affect the relative distribution of Fe between leaf apoplast and symplast at constant low external pH of the root medium. Although perfusion of high pH-buffered solution (7.0) into the leaf apoplast restricted (59)Fe uptake rate as compared with low apoplastic solution pH (5.0 and 6.0, respectively), loading of NO(3)(-) (6 mM) showed no effect on (59)Fe uptake by the symplast of leaf cells. However, high light intensity strongly increased (59)Fe uptake, independently of apoplastic pH or of the presence of NO(3)(-) in the apoplastic solution. Finally, there are no indications in the present study that NO(3)(-) supply to roots results in the postulated inactivation of Fe in the leaf apoplast. It is concluded that NO(3)(-) nutrition results in Fe deficiency chlorosis exclusively by inhibited Fe acquisition by roots due to high pH at the root surface.     

Role of the Apoplast in the Control of Assimilate Transport,Photosynthesis, and Plant Productivity

A concept is suggested, which supposes that assimilates are transferred within the plant downward through phloem sieve tubes and, after entering the stem apoplast, are carried up with the ascending flow of transpiration water. After entering the apoplast of fully expanded leaves, these solutes are reexported through the phloem. Thus, a common pool of assimilates with uniform concentration is formed in the plant apoplast. According to this concept, the mechanism of assimilate demand represents a response of photosynthetic apparatus to changes in the apoplastic level of metabolites consumed by sink organs. The ratios of labeled photoassimilates differ between the apoplast and mesophyll cells. Most of the apoplastic labeled carbon is contained in sucrose, less in amino acids, and even less in hexoses. The ¹⁴C-labeling of amino acids increases and the sucrose/hexose labeling ratio decreased under conditions of enhanced nitrate supply. The well-known effect of relative inhibition of assimilate export from leaves under conditions of enhanced nitrogen supply is explained by an enhanced hydrolysis of apoplast-derived sucrose due to the increase in invertase activity, rather than by diversion of primary photosynthetic products from sucrose synthesis to other pathways required for activated growth processes in leaves. This notion is based on observations that the sucrose/hexose ratio is reduced to a greater extent in the apoplast than in the symplast. The last assumption was supported by data obtained after artificial changes in the apoplastic pH. In these experiments intact plants were placed in the atmosphere of NH₃ or HCl vapors, which induced opposite changes in relative content of labeled assimilates in the apoplast and in the photosynthetic rate.     

Proteins accumulate in the apoplast of winter rye leaves during cold acclimation

During cold acclimation, winter rye ( Secale cereale L.) plants develop the ability to tolerate freezing temperatures by forming ice in intercellular spaces and xylem vessels. In this study, proteins were extracted from the apoplast of rye leaves to determine their role in controlling extracellular ice formation. Several polypeptides in the 15 to 32 kDa range accumulated in the leaf apoplast during cold acclimation at 5°C and decreased during deacclimation at 20°C. A second group of polypeptides (63, 65 and 68 kDa) appeared only when the leaves were maximally frost tolerant. Ice nucleation activity, as well as the previously reported antifreeze activity, was higher in apoplastic extracts from cold-acclimated than from nonacclimated rye leaves. These results indicate that apoplastic proteins exert a direct influence on the growth of ice. In addition, freezing injury was greater in extracted cold-acclimated leaves than in unextracted cold-acclimated leaves, which suggests that the proteins present in the apoplast are an important component of the mechanism by which winter rye leaves tolerate ice formation     

Identification of NaCl stress-responsive apoplastic proteins in rice shoot stems by 2D-DIGE

Plants have evolved sophisticated systems to cope with adverse environmental conditions such as cold, drought, and salinity. Although a number of stress response networks have been proposed, the role of plant apoplast in plant stress response has been ignored. To investigate the role of apoplastic proteins in the salt stress response, 10-day old rice plants were treated with 200mM NaCl for 1, 6 or 12h, and the soluble apoplast proteins of rice shoot stems were extracted for differential analysis, compared with untreated controls, by 2-D DIGE saturation labeling techniques. One hundred twenty-two significantly changed spots were identified by LC-MS/MS, and 117 spots representing 69 proteins have been identified. Of these proteins, 37 are apoplastic proteins according to the bioinformatic analysis. These proteins are mainly involved in the processes of carbohydrate metabolism, oxido-reduction, and protein processing and degradation. According to their functional categories and cluster analysis, a stress response model of apoplastic proteins has been proposed. These data indicate that the apoplast is important in plant stress signal reception and response.     

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.