Physiological significance of the structure and components of the apoplast canal system for water absorption in plants

The non-linear differential equation that describes the coupling between water transport and solute transport in the apoplast canal system in plants was proposed by Katou and Furumoto in 1986. In the present paper, we analytically solved the equation in order to find the law describing the canal system. In the canal system, water transport is regulated linearly by solute transport under physiological conditions. The approximate solution of the differential equations defines the conditions of the structure and components of the apoplast canal for optimal water absorption. Water absorption during cell elongation in plants requires that the apoplast canal be composed of a cell wall with an appropriate diffusion coefficient for solute.     

pH, abscisic acid and the integration of metabolism in plants under stressed and non-stressed conditions. II. Modifications in modes of metabolism induced by variation in the tension on the water column and by stress.

The hydrolysis of ATP(4-) by the plasmalemma and tonoplast H(+)/ATPases and by the tonoplast pyrophosphatase results in the export of a proton to the apoplast or vacuole with remaining in the cytoplasm. As the enzymes that synthesize ATP(4-) require as a substrate it is proposed that protons are an essential substrate for ATP(4-) synthesis. Thus, the entry of protons to the cytoplasm by sym- and antiports will control the rate of ATP(4-) synthesis. Evidence is adduced that plants control the tension on the water column by removing water to or from the 'cellular reservoir' and guard cells by generating osmotic gradients. Schemes are presented that propose a series of metabolic changes that result in a seamless transition through the following states: (1) the import of K(+), Cl(-) and water from the apoplast to the vacuole, the K(+) being admitted to the cytoplasm via a Ca(2+)-activated K(+)-H(+) symport and the water via a Ca(2+)-activated aquaporin; (2) the continued import of K(+) and water from the apoplast to the vacuole with the concomitant export of protons and the synthesis of malate from glucose in the cytoplasm for importation into the vacuole; (3) when the tension on the water column is optimal, respiration and photosynthesis is maximal resulting in biosynthetic reactions and growth; (4) when tension on the water column increases, K(+), Cl(-) and water are exported from the vacuole to the apoplast; (5) the continued export of K(+) and water from the vacuole to the apoplast with malate for export being synthesized in the cytoplasm; the export of K(+) resulting in the acidification of the vacuole; and (6) a further increase in tension results in the deactivation of the plasmalemma H(+)/ATPase by a further increase in cytoplasmic Ca(2+) which also indirectly activates the alternative oxidase. It is suggested that mitochondrial pyruvate is partly oxidized by the TCA cycle and is partly exported to the cytoplasm where it is carboxylated to form malate(1-) for continued export to the apoplast. K(+) is transferred from the vacuole to the apoplast, the K(+) being replaced by protons from the export of mitochondrial pyruvate. The maintenance of the tonoplast electrochemical gradient is thought to result in an increase in the pH of the apoplast which may cause the hydrolysis of abscisic acid precursors with the resulting abscisic acid opening Ca(2+) channels so that the above events are reinforced. (7) This mode is proposed to continue by the metabolism of glucose to four phosphoenolpyruvate, three of which are carboxylated to malate(1-) for continued export to the apoplast with K(+) from the vacuole, the 'stress-tolerant quiescent state'.     

Water Flow in Beta vulgaris Storage Tissue

The relative magnitudes of the hydraulic resistances, water capacities, and water potential equilibration time constants for the single cell, for the apoplast, and for the symplast in higher plant tissue are assessed. Swelling of beetroot (Beta vulgaris, var. `Detroit Red') storage tissue sections in pure water is measured using a displacement transducer. This method of measurement avoids the difficulty of solute diffusion in the apoplast. Theoretical analysis of the experimental results shows that the main path of water flow into the tissue is the apoplast rather than the symplast, that the main resistance to water flow into the cells is usually the cell membrane rather than the apoplast, but that in some cases the apoplast resistance and water capacity can contribute significantly to the water potential equilibration time constant of the tissue.     

pH, abscisic acid and the integration of metabolism in plants under stressed and non-stressed conditions: cellular responses to stress and their implication for plant water relations

A paradigm for the response of plants to stress is presented which suggests that plants move towards a state of minimal metabolic activity as a stress intensifies and remain in that state until that stress is relieved. The paradigm is based on the proposition that cells that interface with the transpiration stream employ variations on the following theme to move towards that state. Tension on the apoplastic water opens a mechanosensitive Ca2+ channel, a response that is augmented by apoplastic ABA. The resulting elevated cytoplasmic Ca2+ deactivates a plasmalemma H+/ATPase and also activates a K(+)-H+ symport. The inflow of K+ and H+ depolarizes the membrane and renders the apoplast less acidic, the protons being removed to the vacuole and the K+ ions being re-exported via the K+ outward rectifying channel. The onset of darkness in guard and mesophyll cells deactivates the plasmalemma H+/ATPase and then the events outlined above ensue except that these cells do not appear to utilize either Ca2+ or ABA during these changes. In stressed cells it is proposed that elevated cytoplasmic Ca2+ activates the release of an ABA precursor from a stored form. ABA is then released in the apoplast after export of the precursor if the activity of the K(+)-H+ symport has brought the apoplastic pH close to 7.0. It is proposed that aquaporins in the xylem parenchyma and mesophyll cells are opened by elevated cytoplasmic Ca2+ when the water potential of the transpiration stream is high so that water can be stored in the 'xylem parenchyma reservoir'. The water in this reservoir is then used to increase the water potential in the transpiration stream when the water column is under tension and to help repair embolisms by a mechanism that resembles stomatal closure.     

Effect of apoplastic solutes on water potential in elongating sugarcane leaves

Solute concentration in the apoplast of growing sugarcane (Saccharum spp. hybrid) leaves was measured using one direct and several indirect methods. The osmotic potential of apoplast solution collected directly by centrifugation of noninfiltrated tissue segments ranged from −0.25 megapascal in mature tissue to −0.35 megapascal in tissue just outside the elongation zone. The presence of these solutes in the apoplast manifested itself as a tissue water potential equal to the apoplast osmotic potential. Since the tissue was not elongating, the measurements were not influenced by growth-induced water uptake and no significant tension was detected with the pressure chamber. Further evidence for a significant apoplast solute concentration was obtained from pressure exudation experiments and comparison of methods for estimating tissue apoplast water fraction. For elongating leaf tissue the centrifugation method could not be used to obtain direct measurements of apoplast solute concentration. However, several other observations suggested that the apoplast water potential of −0.35 to −0.45 megapascal in elongating tissue had a significant osmotic component and small, but significant tension component. Results of experiments in which exudate was collected from pressurized tissue segments of different ages suggested that a tissue age-dependent dynamic equilibrium existed between intra- and extracellular solutes.     

不同提取液提取水稻幼苗质外体蛋白效果的比较

提取植物组织质外体蛋白质的主要困难是提取效率低且易被细胞质蛋白污染。为解决上述问题,以12天93-11水稻幼苗为试验材料,使用3种含不同浓度钾和钙离子的缓冲液作为提取液进行提取效果比较。3种提取液的相同成分都是0.1mol/L Tris-HCl pH 7.6,1mmol/L PMSF,区别点在于:Buffer A含0.2mol/L KCl;Buffer B含0.2mol/L CaCl₂;Buffer C含0.1mol/L KCl和0.1mol/L CaCl₂。结果表明,Buffer A的蛋白产率达到了(0.49±0.07)mg/g FW(叶片)和(0.83±0.06)mg/g FW(根部),比Buffer B和Buffer C分别提高了122.7%和53.1%(叶片)以及102.4%和59.6% (根部)。六磷酸葡萄糖脱氢酶活性检测的结果表明在这些蛋白质提取物中细胞质蛋白的污染率很低,可以控制在1%以下。这些实验结果说明通过优化提取液,建立了有效提取水稻幼苗质外体蛋白质的方法,可应用于植物质外体蛋白质组学研究。     

Actin-regulated water permeability of two transport channels of plasmodesmata in roots of winter wheat cultivars varying in drought resistance

In roots of 5-6-day old seedlings of three cultivars of the winter wheat, varying in drought-resistance: Bezostaya 1 (low resistant), Mironovskaya 808 (resistant), and Albidum 114 (highly resistant) water permeability of two transport channels of plasmodesmata was studied at the action of cytochalasin B, which is known to inhibit polymerization of cytoskeleton actin filaments, by a pulse method of NMR, on the background of increasing water loss in the seedlings. It has been found that the registered coefficients of water self diffusion, two of which (D2 and D3) depend on the water permeability of different transport channels of plasmodesmata, differ in opposite directions. This may suggest that in roots of drought-resistant plants, after a moderate water loss, a diffusive water flow through the cytoplasmic symplast increases (demonstrated by an increase of D2), while that through the vacuolar symplast decreases (seen by an increase of D3). After a high water loss in seedlings, we noticed an even greater increase in water permeability of the cytoplasmic symplast, and a decrease in water permeability of the vacuolar symplast, however, in the roots of low resistant cultivars these changes were poorly expressed, if at all. Under stress-less conditions cytochalasin B would result in an increased water transport through the cytoplasmic channel of plasmodesmata due apparently to a destruction of their actin-myosin sphincters. Both weak and average degrees of water loss would strengthen the cytochalasin B exerted influence on plasmodesmal water conductance, that may testify to a synergetic action of these two factors. After a significant water loss this action was kept only partially, because the inhibitor, on blocking the cytoplasmic channel, did increase at the same time the effect of water stress, limiting water flows through the vacuolar symplast and, simultaneously, raising the water inflow to the apoplast.     

Proton flows across the plasma membrane in microperforated characean internodes: tonoplast injury and involvement of cytoplasmic streaming

Microperforation of characean cell wall with a glass micropipette in the absence of the tonoplast impalement was found to cause rapid alkalinization of the apoplast by 2–3 pH units, which may rigidify the cell wall structure, thus protecting the cell from further injury. A similar but a deeper insertion of a microneedle, associated with piercing the tonoplast and with an action potential generation, led to a considerable delay in the apoplast alkalinization without affecting the amplitude of the eventual increase in pH. The retardation by the mechanically elicited action potential of the incision-mediated pH transients in the apoplast contrasted sharply to the enhancement of these pH transients by the action potential triggered electrically before the microperforation. Hence, the delay of the apoplast alkalinization was not related to basic ionic mechanisms of plant action potentials. Measurements of the vacuolar pH after mechanical elicitation of an action potential indicate that the tonoplast piercing was accompanied by leakage of protons from the vacuole into the cytoplasm, which may strongly acidify the cytoplasm around the wounded area, thus collapsing the driving force for H⁺ influx from the medium into the cytoplasm. The lag period preceding the onset of external alkalinization was found linearly related to the duration of temporal cessation of cytoplasmic streaming. The results suggest that the delayed alkalinization of the apoplast in the region of tonoplast wounding reflects the localized recovery of the proton motive force across the plasmalemma during replacement of the acidic cytoplasm with fresh portions of unimpaired cytoplasm upon restoration of cytoplasmic streaming.     

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.     

Temperature and growth-induced water potential

When the steins of dark-grown soybean [Glycine max (L.) Merr.] seedlings grew rapidly at favorable temperatures in saturating humidities, a water potential of about 0·2 MPa was induced by growth ($pS_o-$pS_w, where $pS_o is the water potential of the basal nonelongating tissue and $pS_w is the water potential of the elongating tissue). If this water potential was caused by high concentrations of solute in the apoplast, as has been proposed, lowering the temperature should have little effect on the potential. On the other hand, if the water potential was caused by apoplast tensions generated by growth, then the tensions should disappear as growth is inhibited by low temperatures. We observed that the growth-induced water potential became too small to detect when growth was inhibited by temperatures as low as 13—5 °C. The disappearance was observed as a rise in apoplast water potential using a thermocouple psychrometer for intact plants, a rise in cell turgor using a miniature pressure probe and a decrease in apoplast tensions using a pressure chamber. The disappearance was not caused by a loss of solute from the apoplast because the tensions fully accounted for the growth-induced water potential at all temperatures. The results are consistent with the lack of solute measured directly in the apoplast solutions at high temperatures (Nonami & Boyer 1987). Therefore, it was concluded that little solute was present in the apoplast at any temperature, and the growth-induced water potential was associated mostly with a tension that moved water from the xylem and into the surrounding cells to meet the demand of cell enlargement.     

Apoplastic mobility of sucrose in storage parenchyma of sugar beet

The apoplastic movement of sucrose through storage parenchyma discs (2.4 mm thick) from roots of sugar beet ( Beta vulgaris var. altissima ) was investigated in order to evaluate the suitability of the apoplast for transcellular sugar transport. The sucrose permeability of the discs (P = 5.7 × 10⁻⁸ cm s⁻¹ at 25°C) was more than two orders of magnitude lower than that of an equally thick layer of unstirred water. This is due to the small volume fraction of free space (3.1%) and the decreased diffusion coefficient D of sucrose in the cell walls. The effective diffusion coefficient of the apoplast (6 to 9 × 10⁻⁷ cm² s⁻¹ at 25°C) was determined independently of the cross sectional area of free space by treating the time course of fluxes according to Fick's second law. The high diffusion resistance of the apoplast has to be considered in models of native parenchyma transport.     

Compartmentation of solutes and water in developing sugarcane stalk tissue

Previous studies have suggested that the apoplast solution of sugarcane stalk tissue contains high concentrations of sucrose, but the accuracy of these reports has been questioned because sucrose leakage from damaged cells may have influenced the results. In this study, the solute potential of the apoplast and symplast of the second (immature), tenth, twentieth, thirtieth, and fortieth internodes of field-grown sugarcane (Saccharum spp. hybrid) stalk tissue was determined by two independent methods. Solute potential of the apoplast was measured either directly by osmometry from solution collected by centrifugation, or inferred from the initial water potential of fully hydrated tissue determined by thermocouple psychrometry before the tissue was progressively dehydrated for generation of water potential isotherms. Both methods produced nearly identical values ranging from −0.6 to −1.8 megapascals for immature and mature tissue, respectively. The solute potential of the symplast determined by either method ranged from −1.0 to approximately −2.2 megapascals for immature and mature internodes, respectively. Solute quantitation by HPLC agreed with concentrations inferred from osmometry. Washing thirtieth internode tissue in deionized water increased pressure potential from 0.29 to 1.96 megapascals. The apoplast of mature sugarcane stalk tissue is a significant storage compartment for sucrose containing as much as 25% of the total tissue water volume and as much as 21% of the stored sucrose.     

Measurement of diffusion within the cell wall in living roots of Arabidopsis thaliana

To quantify the diffusion constant of small molecules in the plant cell wall, fluorescence from carboxyfluorescein (CF) in the intact roots of Arabidopsis thaliana was recorded. Roots were immersed in a solution of the fluorescent dye and viewed through a confocal fluorescence microscope. These roots are sufficiently transparent that much of the apoplast can be imaged. The diffusion coefficient, D(cw), of CF in the cell wall was probed using two protocols: fluorescence recovery after photobleaching and fluorescence loss following perfusion with dye-free solution. Diffusion coefficients were obtained from the kinetics of the fluorescent transients and modelling apoplast geometry. Apoplastic diffusion constants varied spatially in the root. In the elongation zone and mature cortex, D(cw)=(3.2+/-1.4)x10(-11) m(2) s(-1), whereas in mature epidermis, D(cw)=(2.5+/-0.7)x10(-12) m(2) s(-1), at least an order of magnitude lower. Relative to the diffusion coefficient of CF in water, these represent reductions by approximately 1/15 and 1/195, respectively. The low value for mature epidermis is correlated with a suberin-like permeability barrier that was detected with either autofluorescence or berberine staining. This study provides a quantitative estimate of the permeability of plant cell walls to small organic acids-a class of compounds that includes auxin and other plant hormones. These measurements constrain models of solute transport, and are important for quantitative models of hormone signalling during plant growth and development.     

Accumulation of sugars in the xylem apoplast observed under water stress conditions is controlled by xylem pH

Severe water stress constrains, or even stops, water transport in the xylem due to embolism formation. Previously, the xylem of poplar trees was shown to respond to embolism formation by accumulating carbohydrates in the xylem apoplast and dropping xylem sap pH. We hypothesize that these two processes may be functionally linked as lower pH activates acidic invertases degrading sucrose and inducing accumulation of monosaccharides in xylem apoplast. Using a novel in vivo method to measure xylem apoplast pH, we show that pH drops from ~6.2 to ~5.6 in stems of severely stressed plants and rises following recovery of stem water status. We also show that in a lower pH environment, sugars are continuously accumulating in the xylem apoplast. Apoplastic carbohydrate accumulation was reduced significantly in the presence of a proton pump blocker (orthovanadate). These observations suggest that a balance in sugar concentrations exists between the xylem apoplast and symplast that can be controlled by xylem pH and sugar concentration. We conclude that lower pH is related to loss of xylem transport function, eventually resulting in accumulation of sugars that primes stems for recovery from embolism when water stress is relieved.     

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.     

Origin of growth-induced water potential : solute concentration is low in apoplast of enlarging tissues

We developed a new method to measure the solute concentration in the apoplast of stem tissue involving pressurizing the roots of intact seedlings (Glycine max [L.] Merr. or Pisum sativum L.), collecting a small amount of exudate from the surface of the stem under saturating humidities, and determining the osmotic potential of the solution with a micro-osmometer capable of measuring small volumes (0.5 microliter). In the elongating region, the apoplast concentrations were very low (equivalent to osmotic potentials of −0.03 to −0.04 megapascal) and negligible compared to the water potential of the apoplast (−0.15 to −0.30 megapascal) measured directly by isopiestic psychrometry in intact plants. Most of the apoplast water potential consisted of a negative pressure that could be measured with a pressure chamber (−0.15 to −0.28 megapascal). Tests showed that earlier methods involving infiltration of intercellular spaces or pressurizing cut segments caused solute to be released to the apoplast and resulted in spuriously high concentrations. These results indicate that, although a small amount of solute is present in the apoplast, the major component is a tension that is part of a growth-induced gradient in water potential in the enlarging tissue. The gradient originates from the extension of the cell walls, which prevents turgor from reaching its maximum and creates a growth-induced water potential that causes water to move from the xylem at a rate that satisfies the rate of enlargement. The magnitude of the gradient implies that growing tissue contains a large resistance to water movement.     

Dynamic function and regulation of apoplast in the plant body

Apoplast is the internal environment of plant. Our body posses the internal environment that consists of blood, lympha, and tissue fluid. Plant cells are also cultivated and surrounded by a liquid medium in the apoplast. As well as various important functions of the internal environment in our body, apoplast function is also prerequisite for the plant life. There are so far seven distinct functions of apoplast. (1) Growth regulation with apoplastic enzymes by altering cell-wall properties through degradation, synthesis, orientation and cross-linking of supra-molecules of cell walls, such as cellulose, non-cellulosic polysaccharides, proteins, and lignin; (2) Skeleton sustained by cellulose microfibrils, lignin and various types of structural proteins with distinctively high content of hydroxyproline, proline or glycine; (3) Skin to defend symplast from desiccation, pathogens' attack and harmful environmental factors, such as ozone and sulfur dioxide; (4) Transportation route for not only well-known molecules of water, inorganic ions, and sugar, but also plant hormones, oligosaccharides and proteins; (5) Homeostasis of the internal environment by controlling ionic balance, pH and water content; (6) Adhesion of cell to cell; (7) Gas exchange space of leaf for photosynthesis. The present article reviews the recent advances in studies of several aspects of the dynamic function and regulation of apoplast.     

Regulation of Intercellular Water Exchange in Various Zones of Maize Root under Stresses

Apical root meristems and segments of root elongation zone were sampled from 4- to 5-day-old Zea mays L. seedlings. The vacuolar ATPase and pyrophosphatase, the tonoplast marker enzymes, and the tonoplast -, -, and -aquaporins were visualized by means of indirect immunofluorescent microscopy with the use of the respective antibodies. Following cell plasmolysis (700 mM mannitol, 2.5 h), the vacuolar ATPase and pyrophosphatase were detected in cell wall pores where plasmodesmata remained detached from the plasmolyzed protoplasts. This finding provides further evidence for existence of the vacuolar symplast in the elongation zone of maize root, which may ensure intercellular continuity of plant tissues. The pulsed NMR method was used to study the self-diffusion of water molecules. The diffusive decay in the root elongation zone was nonexponential, and it was transformed to three exponential terms with characteristic coefficients of self-diffusion; two of these coefficients (D ₂ and D ₃) characterize the water self-diffusion in the cytoplasmic and vacuolar symplasts of root, respectively. The root apical meristem was also investigated with NMR technique by virtue of paramagnetic doping of the apoplast. This approach allowed selective studying of water diffusion within the symplast compartments. Partial dehydration with PEG-6000, 12 and 20%, for 2.5 h and chemical stressors (ABA and salicylic acid, 0.1 mM, 24 h) were applied to modify water permeability of plasmodesmata and tonoplast aquaporins. The transcellular water permeability increased in the root meristem under the action of all stress factors. In the root elongation zone exposed to partial dehydration, the water exchange in the apoplast became the dominant component. Other stress factors affected water relations in different manners. ABA elevated the water permeability of the vacuolar symplast, in contrast to salicylic acid that decreased water conductance of both the cytoplasmic and vacuolar symplasts.     

The guard-cell apoplast as a site of abscisic acid accumulation in Vicia faba L.

Abscisic acid (ABA) integrates the water status of a plant and causes stomatal closure. Physiological mechanisms remain poorly understood, however, because guard cells flanking stomata are small and contain only attomol quantities of ABA. Here, pooled extracts of dissected guard cells of Vicia faba L. were immunoassayed for ABA at sub‐fmol sensitivity. A pulse of water stress was imposed by submerging the roots in a solution of PEG. The water potentials of root and leaf declined during 20 min of water stress but recovered after stress relief. During stress, the ABA concentration in the root apoplast increased, but that in the leaf apoplast remained low. The ABA concentration in the guard‐cell apoplast increased during stress, providing evidence for intra‐leaf ABA redistribution and leaf apoplastic heterogeneity. Subsequently, the ABA concentration of the leaf apoplast increased, consistent with ABA import via the xylem. Throughout, the ABA contents of the guard‐cell apoplast, but not the guard‐cell symplast, were convincingly correlated with stomatal aperture size, identifying an external locus for ABA perception under these conditions. Apparently, ABA accumulates in the guard‐cell apoplast by evaporation from the guard‐cell wall, so the ABA signal in the xylem is amplified maximally at high transpiration rates. Thus, stomata will display apparently higher sensitivity to leaf apoplastic ABA if stomata are widely open in a relatively dry atmosphere.