Silencing of NtMPK4 impairs CO-induced stomatal closure, activation of anion channels and cytosolic Casignals in Nicotiana tabacum guard cells

Light-induced stomatal opening in C3 and C4 plants is mediated by two signalling pathways. One pathway is specific for blue light and involves phototropins, while the second pathway depends on photosyntheticaly active radiation (PAR). Here, the role of Nt MPK4 in light-induced stomatal opening was studied, as silencing of this MAP kinase stimulates stomatal opening. Stomata of Nt MPK4-silenced plants do not close in elevated atmospheric CO₂, and show a reduced response to PAR. However, stomatal closure can still be induced by abscisic acid. Measurements using multi-barrelled intracellular micro-electrodes showed that CO₂ activates plasma membrane anion channels in wild-type Nicotiana tabacum guard cells, but not in Nt MPK4-silenced cells. Anion channels were also activated in wild-type guard cells after switching off PAR. In approximately half of these cells, activation of anion channels was accompanied by an increase in the cytosolic free Ca²⁺ concentration. The activity of anion channels was higher in cells showing a parallel increase in cytosolic Ca²⁺ than in those with steady Ca²⁺ levels. Both the darkness-induced anion channel activation and Ca²⁺ signals were repressed in Nt MPK4-silenced guard cells. These data show that CO₂ and darkness can activate anion channels in a Ca²⁺-independent manner, but the anion channel activity is enhanced by parallel increases in the cytosolic Ca²⁺ concentration. Nt MPK4 plays an essential role in CO₂- and darkness-induced activation of guard-cell anion channels, through Ca²⁺-independent as well as Ca²⁺-dependent signalling pathways.     

Carbon dioxide induces increases in guard cell cytosolic free calcium

The hypothesis that increases in cytosolic free calcium ([Ca²⁺]_i) are a component of the CO₂ signal transduction pathway in stomatal guard cells of Commelina communis has been investigated. This hypothesis was tested using fura-2 fluorescence ratio photometry to measure changes in guard cell [Ca²⁺]_i in response to challenge with 700 µl l⁻¹ CO₂. Elevated CO₂ induced increases in guard cell [Ca²⁺]_i which were similar to those previously reported in response to abscisic acid. [Ca²⁺]_i returned to resting values following removal of the CO₂ and further application of CO₂ resulted in a second increase in [Ca²⁺]_i. This demonstrated that the CO₂-induced increases in [Ca²⁺]_i were stimulus dependent. Removal of extracellular calcium both prevented the CO₂-induced increase in [Ca²⁺]_i and inhibited the associated reduction in stomatal aperture. These data suggest that Ca²⁺ acts as a second messenger in the CO₂ signal transduction pathway and that an increase in [Ca²⁺]_i may be a requirement for the stomatal response to CO₂.     

Actin Dynamics Regulates Voltage-Dependent Calcium-Permeable Channels of the Vicia faba Guard Cell Plasma Membrane

Free cytosolic Ca²⁺ ([Ca²⁺]_cyt) is an ubiquitous second messenger in plant cell signaling, and [Ca²⁺]_cyt elevation is associated with Ca²⁺-permeable channels in the plasma membrane and endomembranes regulated by a wide range of stimuli. However, knowledge regarding Ca²⁺ channels and their regulation remains limited in planta . A type of voltage-dependent Ca²⁺-permeable channel was identified and characterized for the Vicia faba L. guard cell plasma membrane by using patch-clamp techniques. These channels are permeable to both Ba²⁺ and Ca²⁺, and their activities can be inhibited by micromolar Gd³⁺. The unitary conductance and the reversal potential of the channels depend on the Ca²⁺ or Ba²⁺ gradients across the plasma membrane. The inward whole-cell Ca²⁺ (Ba²⁺) current, as well as the unitary current amplitude and NP_o of the single Ca²⁺ channel, increase along with the membrane hyperpolarization. Pharmacological experiments suggest that actin dynamics may serve as an upstream regulator of this type of calcium channel of the guard cell plasma membrane. Cytochalasin D, an actin polymerization blocker, activated the NPo of these channels at the single channel level and increased the current amplitude at the whole-cell level. But these channel activations and current increments could be restrained by pretreatment with an F-actin stabilizer, phalloidin. The potential physiological significance of this regulatory mechanism is also discussed.     

Involvement of intracellular Ca2+ in blue light-dependent proton pumping in guard cell protoplasts from Vicia faba

Recent studies have suggested that Ca²⁺/calmodulin (CaM) or CaM-like proteins may be involved in blue light (BL)-dependent proton pumping in guard cells. As the increase in cytosolic concentration of Ca²⁺ is required for the activation of CaM and CaM-like proteins, the origin of the Ca²⁺ was investigated by measuring BL-dependent proton pumping with various treatments using guard cell protoplasts (GCPs) from Vicia faba . BL-dependent proton pumping was affected neither by Ca²⁺ channel blockers nor by changes of Ca²⁺ concentration in the medium used for the GCPs. Addition of Ca²⁺ ionophores and an agonist to GCPs did not induce proton pumping. However, BL-dependent proton pumping was inhibited by 10 m M caffeine, which releases Ca²⁺ from the intracellular stores, and by 10 μ M 2,5-di-( tert -butyl)-1,4-benzohydroquinone (BHQ) and 10 μ M cyclopiazonic acid (CPA), inhibitors of Ca²⁺-ATPase in the sarcoplasmic and endoplasmic reticulum (ER). By contrast, the inhibitions were not observed by 10 μ M thapsigargin, an inhibitor of animal ER-type Ca²⁺-ATPase. The inhibitions by caffeine and BHQ were reversible. Light-dependent stomatal opening in the epidermis of Vicia was inhibited by caffeine, BHQ, and CPA. From these results, we conclude that the Ca²⁺ thought to be required for BL-dependent proton pumping may originate from intracellular Ca²⁺ stores, most likely from ER in guard cells, and that this origin of Ca²⁺ may generate a stimulus-specific Ca²⁺ signal for stomatal opening.     

DNA binding of citrus dehydrin promoted by zinc ion

Dehydrins are hydrophilic proteins that accumulate during embryogenesis and osmotic stress responses in plants. Here, we report an interaction between citrus dehydrin Citrus unshiu cold-regulated 15 kDa protein (CuCOR15) and DNA. Binding of CuCOR15 to DNA was detected by an electrophoretic mobility shift assay, a filter-binding assay and Southwestern blotting. The binding was stimulated by physiological concentrations of Zn²⁺, but little stimulation occurred when other divalent cations, such as Mg²⁺, Ca²⁺, Mn²⁺, Ni²⁺ and Cu²⁺, were substituted for Zn²⁺. Ethylenediaminetetraacetic acid cancelled the Zn²⁺-stimulated binding. A binding curve and competitor experiments suggested that the DNA binding of CuCOR15 exhibited low affinity and non-specificity. Moreover, tRNA competed with the DNA binding. Histidine-rich domains and a polylysine segment-containing domain participated in the DNA binding. These results suggest that CuCOR15 can interact with DNA, and also RNA, in the presence of Zn²⁺. Dehydrin may protect nucleic acids in plant cells during seed maturation and stress responses.     

From common signalling components to cell specific responses: insights from the cereal aleurone

Studies into the molecules underlying plant signal transduction events continue to reveal the involvement of highly conserved factors such as Ca²⁺, calmodulin, cyclic GMP and phospholipases in a remarkably diverse array of physiological processes. The hormonal response systems in the aleurone cells of the cereal grain and in the stomatal guard cell are beginning to reveal how diversity of response can be hard wired into these cells despite the use of these common signalling intermediates. In both the aleurone and the guard cell ABA signalling operates through the action of phospholipase D and alterations in a Ca²⁺-dependent signalling system. The role of phospholipase D is highly analogous in these two divergent cell types, perhaps reflecting the closeness of this enzyme to a conserved ABA receptor. However, specificity in response becomes evident in elements downstream from PLD, such as in the Ca²⁺ signalling system. For example, ABA has opposite effects on cytoplasmic Ca²⁺ in the aleurone and guard cell. Combining the Ca²⁺-dependent signalling activities in networks with parallel regulatory activities such as cyclic GMP appears to underlie the flexible regulatory systems that are the hallmark of plant cell function.     

Two plant Ca2+ pumps expressed in stomatal guard cells show opposite expression patterns during cold stress

Guard cells respond to cold and other stimuli with an oscillating Ca²⁺ signal, causing an opening or closing of the stomatal pore. However, the molecular basis for the regulation of Ca²⁺ transients in guard cells has so far not been elucidated. We have localized the expression of two closely related Ca²⁺-ATPases, ACA8 and ACA10 , to guard cells in Arabidopsis . Whereas ACA8 expression was limited to guard cells and vascular tissues, ACA10 was found to be ubiquitously expressed. Upon cold treatment, the expression of ACA8 was found to be upregulated while that of ACA10 was downregulated. This finding was supported by the presence of the cold responsive C-repeat/dehydration-responsive element motif in the ACA8 promoter sequence.     

Blue light‐induced apoplastic acidification of Arabidopsis thaliana guard cells: Inhibition by ABA is mediated through protein phosphatases

The phytohormone abscisic acid (ABA) inhibits blue light‐induced apoplastic acidification of guard cells. The signal transduction pathway of ABA, mediating this response, was studied using ABA‐insensitive ( abi ) mutants of Arabidopsis thaliana . Apoplastic acidification was monitored with a flat tipped pH‐electrode placed on epidermal strips, in which only guard cells were viable. Blue light‐induced apoplastic acidification was reduced by vanadate and diethylstilbestrol (DES), indicating involvement of plasma membrane‐bound H⁺‐ATPases. In wild type epidermal strips, ABA reduced blue light‐induced acidification to 63%. The inhibition did not result from an increased cytoplasmic free Ca²⁺ concentration in guard cells, since factors that increase the Ca²⁺ concentration stimulated apoplastic acidification. Apoplastic acidification was not inhibited by ABA in abi1 and abi2 mutants. In abi1 epidermal strips ABA had no effect on the acidification rate, while it stimulated apoplastic acidification in abi2 . The ABA response in both mutants could be partially restored with protein kinase and phosphatase inhibitors. The abi1 guard cells became ABA responsive in the presence of okadaic acid, a protein phosphatase inhibitor. In abi2 guard cells the wild type ABA response was partially restored by K‐252a, a protein kinase inhibitor. Apoplastic inhibition is thus mediated through the protein phosphatases encoded by ABI1 and ABI2 . The results with protein kinase and protein phosphatase inhibitors indicate that ABI1 and ABI2 are involved in separate signal transduction pathways.     

Turgor- dependent membrane permeability in relation to calcium level

The relationship between the inhibiting effect of Ca²⁺ and of low turgor pressure on K⁺ release from fresh-cut discs of carrot ( Daucus carota var. Nantes) storage tissue was studied. A range of Ca²⁺ concentrations in the tissue was obtained by adding 0.5 m M EDTA or CaSO₄ at different concentrations to the medium. Calcium inhibited K⁺ release in fully turgid cells (2.5 μmol K⁺ g⁻¹ h⁻¹ in 0.5 m M EDTA vs 0.4 μmol K⁺ g⁻¹ h⁻¹ in 10 m M CaSO₄). Less turgid cells, obtained by equilibration with 0.2 M mannitol, released K⁺ at only 30% of the rate of the turgid cells, yet the pattern of K⁺ release as a function of Ca²⁺ level was similar in both turgid and non-turgid cells. Removal of calcium by EDTA occasionally injured cell membranes in the fully turgid discs but never in the less turgid ones. In view of the additive effect of Ca²⁺ and low turgor on K⁺ release regardless of the treatment order, it is suggested that the two factors exert their effect on membrane permeability independently of each other.     

The stomatal response to CO2 is linked to changes in guard cell zeaxanthin*

The mechanisms mediating CO₂ sensing and light–CO₂ interactions in guard cells are unknown. In growth chamber-grown Vicia faba leaves kept under constant light (500 μ mol m^–2 s^–1) and temperature, guard cell zeaxanthin content tracked ambient [CO₂] and stomatal apertures. Increases in [CO₂] from 400 to 1200 cm³ m^–3 decreased zeaxanthin content from 180 to 80 mmol mol^–1 Chl and decreased stomatal apertures by 7·0 μ m. Changes in zeaxanthin and aperture were reversed when [CO₂] was lowered. Guard cell zeaxanthin content was linearly correlated with stomatal apertures. In the dark, the CO₂-induced changes in stomatal aperture were much smaller, and guard cell zeaxanthin content did not change with chamber [CO₂]. Guard cell zeaxanthin also tracked [CO₂] and stomatal aperture in illuminated stomata from epidermal peels. Dithiothreitol (DTT), an inhibitor of zeaxanthin formation, eliminated CO₂-induced zeaxanthin changes in guard cells from illuminated epidermal peels and reduced the stomatal CO₂ response to the level observed in the dark. These data suggest that CO₂-dependent changes in the zeaxanthin content of guard cells could modulate CO₂-dependent changes of stomatal apertures in the light while a zeaxanthin-independent CO₂ sensing mechanism would modulate the CO₂ response in the dark.     

A calcium influx precedes organogenesis in Graptopetalum

Abstract. An account is given of an investigation of net ionic currents and specific ion fluxes occuring during the initiation of organogenesis in detached leaves of Graptopetalum paraguayense E. Walther, in which a dramatic change in growth polarity is cytomorphologically evident 3–5 d after leaf detachment from the plant. Using the vibrating probe, it was possible to identify a peak of ionic current which is focused over the area of the leaf base where organogenesis is initiated. This net current is largest during the initial 12h after leaf detachment. With ion-selective microelectrodes capable of measuring H⁺, K⁺ and Ca²⁺ ion fluxes simultaneously in the same region of the leaf base, H⁺ and K⁺ fluxes remain relatively steady during the initial 24 h after detachment, while a large lanthanum-sensitive Ca²⁺ influx decreases by 50% from 2 to 12h. By 24h, Ca²⁺ transport is dominated by an efflux. We present evidence from a quantitative comparison of the ion current data collected using these two techniques, that Ca²⁺, H⁺ and K⁺ transport accounts for the major electrogenic ion fluxes during 2 and 12 but not 24 h after leaf detachment. The possibility is addressed that these ion currents, which precede organogenesis, and in particular the predominant Ca²⁺ flux, play a role in the establishment of growth polarity in higher plant tissues.     

Effects of saponin extracts from Solanum elaeagnifotium fruits on ion uptake by clover seedlings

Solanum elaeagnifolium Cav. fruits contain high concentrations of steroidal saponins. Treatment of 3-day-old clover seedlings with aqueous fruit extracts modified Ca²⁺ uptake without significantly altering K⁺ and H₂PO₄⁻ uptake. The extracts increased Ca²⁺ uptake in the concentration range of 0.2 to 20 m M Ca²⁺. Uptake curves could be represented by two phases. In the lower phase (0.2-1.0 m M Ca²⁺), this change could be related to an increase in V_max. Pretreatment of seedlings with saponin extracts significantly reduced ATP-dependent Ca²⁺ uptake and Ca²⁺-dependent ATPase activity in a fraction isolated from root homogenates by centrifugation at 1500 g for 15 min. Saponins purified from S. eleagnifolium extracts by thin-layer chromatography modified in vitro the Ca²⁺-ATPase activity of this fraction, indicating that the steroid may act directly on Ca²⁺ transport across membranes.     

Role of abscisic acid in a Ca2+ second messenger system

Owen, J. H. 1988. Role of abscisic acid in a Ca²⁺ second messenger system. - Physiol. Plant. 72:637–641.
Recent investigations have shown that Ca²⁺ acts as a second messenger in plant cell coupling of response to stimulus. Data now suggests that abscisic acid (ABA) plays a role as Ca²⁺'agonist' in this Ca²⁺ messenger system, although the molecular basis of such an interaction has not yet been fully investigated. ABA appears to possess a universal Ca²⁺'agonist' role because it elicits responses in systems as diverse as mammalian contractile tissue and cyanobacteria. ABA may therefore be of a wider biological significance than previously recognised.     

Development and accumulation of ABA in fluridone-treated and drought-stressed Vicia faba plants under different light conditions

The effects of fluridone on guard cell morphology, chloroplast ultrastructure and accumulation of drought stress-induced abscisic acid (ABA) were studied in Vicia faba L. plants grown under different light conditions. Drought stress was induced by allowing the leaves to lose 12% of their fresh weight. The appearance of defective and undeveloped stomata, and chloroplasts with a destroyed thylakoid membrane system was found in fluridone-treated plants grown at a photosynthetic photon flux (PPF) of 600 μmol m^-2 s^-1. Plants grown at a PPF of 40 μmol m^-2 s^-1 had diminished levels of ABA after imposition of dehydration. Fluridone treatment reduced the level of ABA in both unstressed and dehydrated leaves. Accumulation of ABA in the control plants was considerably reduced when they were exposed to dark periods of 24, 48 and 72 h just before imposition of the stress. Twenty-four hours after the dark treatment dehydration of the leaves resulted in a 3-fold decrease in the level of stress-induced ABA, and 72 h after dark treatment the amount of stress-induced ABA approximated the prestressed values. Fluridone-treated plants failed to accumulate ABA under water stress. In addition to functionally active chloroplasts, well-developed and functional stomata are required for drought stress to elicit a rise in ABA.     

Accumulation of an apoplastic solute in the guard-cell wall is sufficient to exert a significant effect on transpiration in Vicia faba leaflets

Accumulation of recently photosynthesized sucrose in the guard‐cell wall is the empirical foundation for a hypothesis that links the rates of photosynthesis, translocation, and transpiration (Plant Physiology 114, 109–118). Critical assumptions of this hypothesis were tested by use of Vicia faba, an apoplastic phloem loader. Following measurements of the leaflet‐apoplastic‐water volume (by P–V isotherm analysis) and the guard‐cell wall volume (by 3‐D analysis), intact leaflets were fed dilute solutions of mannitol, an impermeant non‐toxic osmolyte. Even at bulk‐leaflet mannitol concentrations that would have only a negligible osmotic effect on stomata, transpiration at constant temperature, water‐vapour pressure, air movement and irradiance was diminished up to 25%, compared with controls. This effect on transpiration, a manifestation of smaller stomatal aperture size, was explained by accumulation of mannitol, up to 350 mol m ^{? 3}, in the estimated aqueous volume of the guard‐cell wall. The conclusion is that mannitol, a xenobiotic with structural similarity to sucrose, can move throughout the apoplast of a transpiring leaflet and accumulate in an osmotically significant concentration in the guard‐cell wall. These data therefore provide support for a new role for sucrose as a signal metabolite that integrates essential functions of the whole leaf. In addition, the results raise questions about the physiological or experimental accumulation of other guard‐cell‐targeted apoplastic solutes such as plant growth regulators, particularly abscisic acid, and ions.     

Multiple forms of phosphoenolpyruvate carboxylase in mesophyll, epidermal and guard cells of Vicia faba

The distribution of phosphoenolpyruvate carboxylase (PEPCase, EC 4.1.1.31) in different leaf‐cell‐types and tissues of Vicia faba L. cv. 3‐fach Weiße was studied. The highest specific PEPCase activity was found in guard cell protoplasts (16.3 µmol mg⁻¹ protein h⁻¹) whereas for epidermal and mesophyll protoplasts remarkably lower specific activities were found (1.6 and 1.0 µmol mg⁻¹ protein h⁻¹, respectively). On chlorophyll and protoplast basis, a similar distribution of enzyme activity was observed. Compared with epidermal extracts, the specific PEPCase activity of mesophyll tissue was 17‐fold lower. Immunological studies with polyclonal antibodies to PEPCase indicated 3 immunoreactive proteins in epidermal tissue and guard cell protoplasts with molecular masses of 107 000, 110 000, and 112 000. Only the M_r 107 000 protein was found in extracts of mesophyll and epidermis protoplasts. Western immunoblots after native electrophoresis of epidermal and mesophyll proteins showed a significant difference in PEPCase mobility. It is assumed, that the immunostained proteins of M_r 110 000 and 112 000 represent isoforms or subunits of the PEPCase and that they are involved in stomatal movements.     

Molecular Cloning of Osmotin and Regulation of Its Expression by ABA and Adaptation to Low Water Potential

In response to adaptation to NaCl, cultured tobacco cells (Nicotiana tabacum L. cv Wisconsin 38) synthesize a major 26 kilodalton protein which has been named osmotin due to its induction by low water potentials. To help characterize the expression of osmotin in adapted cells, a cDNA clone for osmotin has been isolated. Abscisic acid induces messenger RNA encoding osmotin. Levels of this mRNA in adapted cells are approximately 15-fold higher than in unadapted cells. Message for osmotin is present at constant levels through the growth cycle of adapted cells, while in unadapted cells, the level decreases during exponential phase of growth and increases again when the cells approach stationary phase. While abscisic acid induces the message for osmotin, a low water potential environment appears to be required for accumulation of the protein. An osmotic shock to unadapted cells does not increase the amount of message or protein present most likely because this treatment does not induce immediately the accumulation of abscisic acid. The increased expression of osmotin in adapted cells is not correlated with an increase in osmotin gene copy number. Osmotin is homologous to a 24 kilodalton NaCl-induced protein in tomato, as well as thaumatin, maize α-amylase/trypsin inhibitor and a tobacco mosaic virus-induced pathogenesis-related protein.     

The fou2 mutation in the major vacuolar cation channel TPC1 confers tolerance to inhibitory luminal calcium

The SV channel encoded by the TPC1 gene represents a Ca²⁺- and voltage-dependent vacuolar cation channel. Point mutation D454N within TPC1 , named fou2 for fatty acid oxygenation upregulated 2 , results in increased synthesis of the stress hormone jasmonate. As wounding causes Ca²⁺ signals and cytosolic Ca²⁺ is required for SV channel function, we here studied the Ca²⁺-dependent properties of this major vacuolar cation channel with Arabidopsis thaliana mesophyll vacuoles. In patch clamp measurements, wild-type and fou2 SV channels did not exhibit differences in cytosolic Ca²⁺ sensitivity and Ca²⁺ impermeability. K⁺ fluxes through wild-type TPC1 were reduced or even completely faded away when vacuolar Ca²⁺ reached the 0.1-m m level. The fou2 protein under these conditions, however, remained active. Thus, D454N seems to be part of a luminal Ca²⁺ recognition site. Thereby the SV channel mutant gains tolerance towards elevated luminal Ca²⁺. A three-fold higher vacuolar Ca/K ratio in the fou2 mutant relative to wild-type plants seems to indicate that fou2 can accumulate higher levels of vacuolar Ca²⁺ before SV channel activity vanishes and K⁺ homeostasis is impaired. In response to wounding fou2 plants might thus elicit strong vacuole-derived cytosolic Ca²⁺ signals resulting in overproduction of jasmonate.     

Sweet taste receptor interacting protein CIB1 is a general inhibitor of InsP3-dependent Ca2+ release in vivo

In a search for sweet taste receptor interacting proteins, we have identified the calcium- and integrin-binding protein 1 (CIB1) as specific binding partner of the intracellular carboxyterminal domain of the rat sweet taste receptor subunit Tas1r2. In heterologous human embryonic kidney 293 (HEK293) cells, the G protein chimeras Gα_16gust44 and Gα_15i3 link the sweet taste receptor dimer TAS1R2/TAS1R3 to an inositol 1,4,5-trisphosphate (InsP₃)-dependent Ca²⁺ release pathway. To demonstrate the influence of CIB1 on the cytosolic Ca²⁺ concentration, we used sweet and umami compounds as well as other InsP₃-generating ligands in FURA-2-based Ca²⁺ assays in wild-type HEK293 cells and HEK293 cells expressing functional human sweet and umami taste receptor dimers. Stable and transient depletion of CIB1 by short-hairpin RNA increased the Ca²⁺ response of HEK293 cells to the InsP₃-generating ligands ATP, UTP and carbachol. Over-expression of CIB1 had the opposite effect as shown for the sweet ligand saccharin, the umami receptor ligand monosodium glutamate and UTP. The CIB1 effect was dependent on the thapsigargin-sensitive Ca²⁺ store of the endoplasmic reticulum (ER) and independent of extracellular Ca²⁺. The function of CIB1 on InsP₃-evoked Ca²⁺ release from the ER is most likely mediated by its interaction with the InsP₃ receptor. Thus, CIB1 seems to be an inhibitor of InsP₃-dependent Ca²⁺ release in vivo .     

Localization of calcium ions in wounded characean internodal cells

Ca²⁺ was localized in chemically injured internodal cells of the characean alga Nitella flexilis (L.) Ag. using alizarin red and antimonate precipitation. The presence of Ca²⁺ in the antimonate precipitates was verified by X-ray analysis and EGTA chelation. Callose-containing amorphous wound walls were induced by 0·1 m m chlortetracycline (CTC) and cellulosic fibrillar wound walls were induced by 50 m m CaCl₂. Numerous precipitates were found in the amorphous wound walls and in the adjacent cytoplasm. Precipitates were mainly localized in single membrane-bound cisternae, probably of the endoplasmic reticulum, which accumulate at the wound and become a component of the amorphous wound wall via membrane fusion. In fibrillar wound walls, which do not contain membranous residues, precipitate density was significantly lower and similar to that in the secondary cell wall.
The data suggest that the high Ca²⁺ content of amorphous wound walls is due to incorporation of cytoplasmic Ca²⁺ stores. The possible function of amorphous wound walls in maintaining cellular Ca²⁺ homeostasis is discussed.