Borate-Rhamnogalacturonan II Bonding Reinforced by Ca2+ Retains Pectic Polysaccharides in Higher-Plant Cell Walls

The extent of in vitro formation of the borate-dimeric-rhamnogalacturonan II (RG-II) complex was stimulated by Ca²⁺. The complex formed in the presence of Ca²⁺ was more stable than that without Ca²⁺. A naturally occurring boron (B)-RG-II complex isolated from radish (Raphanus sativus L. cv Aokubi-daikon) root contained equimolar amounts of Ca²⁺ and B. Removal of the Ca²⁺ by trans-1,2-diaminocyclohexane-N,N,N′,N′-tetraacetic acid induced cleavage of the complex into monomeric RG-II. These data suggest that Ca²⁺ is a normal component of the B-RG-II complex. Washing the crude cell walls of radish roots with a 1.5% (w/v) sodium dodecyl sulfate solution, pH 6.5, released 98% of the tissue Ca²⁺ but only 13% of the B and 22% of the pectic polysaccharides. The remaining Ca²⁺ was associated with RG-II. Extraction of the sodium dodecyl sulfate-washed cell walls with 50 mm trans-1,2-diaminocyclohexane-N,N,N′,N′-tetraacetic acid, pH 6.5, removed the remaining Ca²⁺_, 78% of B, and 49% of pectic polysaccharides. These results suggest that not only Ca²⁺ but also borate and Ca²⁺ cross-linking in the RG-II region retain so-called chelator-soluble pectic polysaccharides in cell walls.Boron (B) is an essential element for higher plant growth, although its primary function is not known (Loomis and Durst, 1992). Determining the sites of B in cells is required to identify its function. In cultured tobacco cells more than 80% of cellular B is in the cell wall (Matoh et al., 1993), whereas the membrane fraction (Kobayashi et al., 1997) and protoplasts (Matoh et al., 1992) do not contain a significant amount of B. In radish (Raphanus sativus L. cv Aokubi-daikon) root cell walls, B cross-links two RG-II regions of pectic polysaccharides through a borate-diol ester (Kobayashi et al., 1995, 1996). The association of B with RG-II has been confirmed in sugar beet (Ishii and Matsunaga, 1996), bamboo (Kaneko et al., 1997), sycamore and pea (O''Neill et al., 1996), and red wine (Pellerin et al., 1996). In cultured tobacco cells the B associated with RG-II accounts for about 80% of the cell wall B (Kobayashi et al., 1997) and RG-II may be the exclusive carrier of B in higher plant cell walls (Matoh et al., 1996). Germanic acid, which partly substitutes for B in the growth of the B-deprived plants (Skok, 1957), also cross-links two RG-II chains (Kobayashi et al., 1997). These results suggest that the physiological role of B is to cross-link cell wall pectic polysaccharides in the RG-II region and thereby form a pectic network.It is believed that in the cell wall pectic polysaccharides are cross-linked with Ca²⁺, which binds to carboxyl groups of the polygalacturonic acid regions (Jarvis, 1984). Thus, the ability of B and Ca²⁺ to cross-link cell wall pectic polysaccharides needs to be evaluated. In this report we describe the B-RG-II complex of radish root and the role of B-RG-II and Ca²⁺ in the formation of a pectic network.     

Evidence for a Slow-Turnover Form of the Ca2+-Independent Phosphoenolpyruvate Carboxylase Kinase in the Aleurone-Endosperm Tissue of Germinating Barley Seeds

Phosphoenolpyruvate carboxylase (PEPC) activity was detected in aleurone-endosperm extracts of barley (Hordeum vulgare) seeds during germination, and specific anti-sorghum (Sorghum bicolor) C₄ PEPC polyclonal antibodies immunodecorated constitutive 103-kD and inducible 108-kD PEPC polypeptides in western analysis. The 103- and 108-kD polypeptides were radiolabeled in situ after imbibition for up to 1.5 d in ³²P-labeled inorganic phosphate. In vitro phosphorylation by a Ca²⁺-independent PEPC protein kinase (PK) in crude extracts enhanced the enzyme''s velocity and decreased its sensitivity to l-malate at suboptimal pH and [PEP]. Isolated aleurone cell protoplasts contained both phosphorylated PEPC and a Ca²⁺-independent PEPC-PK that was partially purified by affinity chromatography on blue dextran-agarose. This PK activity was present in dry seeds, and PEPC phosphorylation in situ during imbibition was not affected by the cytosolic protein-synthesis inhibitor cycloheximide, by weak acids, or by various pharmacological reagents that had proven to be effective blockers of the light signal transduction chain and PEPC phosphorylation in C₄ mesophyll protoplasts. These collective data support the hypothesis that this Ca²⁺-independent PEPC-PK was formed during maturation of barley seeds and that its presumed underlying signaling elements were no longer operative during germination.Higher-plant PEPC (EC 4.1.1.31) is subject to in vivo phosphorylation of a regulatory Ser located in the N-terminal domain of the protein. In vitro phosphorylation by a Ca²⁺-independent, low-molecular-mass (30–39 kD) PEPC-PK modulates PEPC regulation interactively by opposing metabolite effectors (e.g. allosteric activation by Glc-6-P and feedback inhibition by l-malate; Andreo et al., 1987), decreasing significantly the extent of malate inhibition of the leaf enzyme (Carter et al., 1991; Chollet et al., 1996; Vidal et al., 1996; Vidal and Chollet, 1997). These metabolites control the rate of phosphorylation of PEPC via an indirect target-protein effect (Wang and Chollet, 1993; Echevarría et al., 1994; Vidal and Chollet, 1997).Several lines of evidence support the view that this protein-Ser/Thr kinase is the physiologically relevant PEPC-PK (Li and Chollet, 1993; Chollet et al., 1996; Vidal et al., 1996; Vidal and Chollet, 1997). The presence and inducible nature of leaf PEPC-PK have been established further in various C₃, C₄, and CAM plant species (Chollet et al., 1996). In all cases, CHX proved to be a potent inhibitor of this up-regulation process so that apparent changes in the turnover rate of PEPC-PK itself or another, as yet unknown, protein factor were invoked to account for this observation (Carter et al., 1991; Jiao et al., 1991; Chollet et al., 1996). Consistent with this proposal are recent findings about PEPC-PK from leaves of C₃, C₄, and CAM plants that determined activity levels of the enzyme to depend on changes in the level of the corresponding translatable mRNA (Hartwell et al., 1996).Using a cellular approach we previously showed in sorghum (Sorghum bicolor) and hairy crabgrass (Digitaria sanguinalis) that PEPC-PK is up-regulated in C₄ mesophyll cell protoplasts following illumination in the presence of a weak base (NH₄Cl or methylamine; Pierre et al., 1992; Giglioli-Guivarc''h et al., 1996), with a time course (1–2 h) similar to that of the intact, illuminated sorghum (Bakrim et al., 1992) or maize leaf (Echevarría et al., 1990). This light- and weak-base-dependent process via a complex transduction chain is likely to involve sequentially an increase in pHc, inositol trisphosphate-gated Ca²⁺ channels of the tonoplast, an increase in cytosolic Ca²⁺, a Ca²⁺-dependent PK, and PEPC-PK.Considerably less is known about the up-regulation of PEPC-PK and PEPC phosphorylation in nongreen tissues. A sorghum root PEPC-PK purified on BDA was shown to phosphorylate in vitro both recombinant C₄ PEPC and the root C₃-like isoform, thereby decreasing the enzyme''s malate sensitivity (Pacquit et al., 1993). PEPC from soybean root nodules was phosphorylated in vitro and in vivo by an endogenous PK (Schuller and Werner, 1993; Zhang et al., 1995; Zhang and Chollet, 1997). A Ca²⁺-independent nodule PEPC-PK containing two active polypeptides (32–37 kD) catalyzed the incorporation of phosphate on a Ser residue of the target enzyme and was modulated by photosynthate transported from the shoots (Zhang and Chollet, 1997). Regulatory seryl phosphorylation of a heterotetrameric (α₂β₂) banana fruit PEPC by a copurifying, Ca²⁺-independent PEPC-PK was shown to occur in vitro (Law and Plaxton, 1997). Although phosphorylation was also detected in vivo and found to concern primarily the α-subunit, PEPC exists mainly in the dephosphorylated form in preclimacteric, climacteric, and postclimacteric fruit.In a previous study we showed that PEPC undergoes regulatory phosphorylation in aleurone-endosperm tissue during germination of wheat seeds (Osuna et al., 1996). Here we report on PEPC and the requisite PEPC-PK in germinating barley (Hordeum vulgare) seeds. PEPC was highly phosphorylated by a Ca²⁺-independent Ser/Thr PEPC-PK similar to that found in other plant systems studied previously (Chollet et al., 1996); however, the PK was already present in the dry seed and its activity did not require protein synthesis during imbibition.     

Increase in the Quantum Yield of Photoinhibition Contributes to Copper Toxicity in Vivo

The effect of copper on photoinhibition of photosystem II in vivo was studied in bean (Phaseolus vulgaris L. cv Dufrix). The plants were grown hydroponically in the presence of various concentrations of Cu²⁺ ranging from the optimum 0.3 μm (control) to 15 μm. The copper concentration of leaves varied according to the nutrient medium from a control value of 13 mg kg⁻¹ dry weight to 76 mg kg⁻¹ dry weight. Leaf samples were illuminated in the presence and absence of lincomycin at different light intensities (500–1500 μmol photons m⁻² s⁻¹). Lincomycin prevents the concurrent repair of photoinhibitory damage by blocking chloroplast protein synthesis. The photoinhibitory decrease in the light-saturated rate of O₂ evolution measured from thylakoids isolated from treated leaves correlated well with the decrease in the ratio of variable to maximum fluorescence measured from the leaf discs; therefore, the fluorescence ratio was used as a routine measurement of photoinhibition in vivo. Excess copper was found to affect the equilibrium between photoinhibition and repair, resulting in a decrease in the steady-state concentration of active photosystem II centers of illuminated leaves. This shift in equilibrium apparently resulted from an increase in the quantum yield of photoinhibition (Φ_PI) induced by excess copper. The kinetic pattern of photoinhibition and the independence of Φ_PI on photon flux density were not affected by excess copper. An increase in Φ_PI may contribute substantially to Cu²⁺ toxicity in certain plant species.Cu²⁺ is an essential micronutrient but in excess is toxic for plants. It is a redox-active metal that functions as an enzyme activator and is an important part of prosthetic groups of many enzymes (for review, see Sandmann and Böger, 1983). Copper concentrations in healthy plant tissues range from 5 to 20 mg kg⁻¹ dry weight. In Cu²⁺-rich environments, accumulation of Cu²⁺ in plant tissues depends on the species and cultivar. Cu²⁺ seems to have several sites of action, which vary among plant species. Toxic concentrations of Cu²⁺ inhibit metabolic activity, which leads to suppressed growth and slow development. Most Cu²⁺ ions are immobilized to the cell walls of roots or of mycorrhizal fungi (Kahle, 1993).When the tolerance mechanisms in the root zone become overloaded, Cu²⁺ is translocated by both the xylem and phloem up to the leaves. Excess Cu²⁺ may replace other metals in metalloproteins or may interact directly with SH groups of proteins (Uribe and Stark, 1982). Cu²⁺-induced free-radical formation may also cause protein damage (for review, see Fernandes and Henriques, 1991; Weckx and Clijsters, 1996). High concentrations of Cu²⁺ may catalyze the formation of the hydroxyl radical from O₂ and H₂O₂. This Cu²⁺-catalyzed Fenton-type reaction takes place mainly in chloroplasts (Sandmann and Böger, 1980). The hydroxyl radical may start the peroxidation of unsaturated membrane lipids and chlorophyll (Sandmann and Böger, 1980), and these inhibitory mechanisms might contribute to the observed inhibition of photosynthetic electron transport by excess Cu²⁺ (Clijsters and Van Assche, 1985).The role of Cu²⁺ as an inhibitor of photosynthetic electron transport has been studied in vitro. Both the donor (Cedeno-Maldonado and Swader, 1972; Samuelsson and Öquist, 1980; Schröder et al., 1994) and acceptor (Mohanty et al., 1989; Yruela et al., 1992, 1993, 1996a, 1996b; Jegerschöld et al., 1995) sides of PSII have been proposed to be the most sensitive site for Cu²⁺ action. On the donor side, Cu²⁺ is thought to inhibit electron transport to P680, the primary donor of PSII (Schröder et al., 1994). On the acceptor side, Cu²⁺ interactions with the pheophytin-Q_A-Fe²⁺-domain or Cu²⁺-induced modifications in the amino acid or lipid structure close to the Q_A- and Q_B-binding sites have been suggested to cause the inhibition of electron transport (Jegerschöld et al., 1995; Yruela et al., 1996a, 1996b).Celeno-Maldonado and Swader (1972) noticed that preincubation of chloroplasts in the light enhanced the Cu²⁺-induced inhibition of electron transport, and that PSII was more susceptible to this kind of inhibition than was PSI. The hypothetical acceptor- and donor-side mechanisms of the light-induced inhibition of electron transport, photoinhibition, involve the same domains of attack as Cu²⁺. Both acceptor- and donor-side photoinhibition trigger the D1 polypeptide of the PSII reaction center for degradation (for review, see Aro et al., 1993). The damaged D1 protein is degraded, and the recovery of PSII activity needs de novo synthesis of D1 protein. Photoinhibition occurs at all light intensities (Tyystjärvi and Aro, 1996); therefore, the cycle of PSII photoinhibition, which is followed by degradation, and, finally, resynthesis of the D1 protein, runs constantly in plant cells in the light. If the photoinhibition-repair cycle is allowed to run for some time at a constant light intensity, equilibrium is reached. At equilibrium (steady state), all three reaction rates (photoinhibition, D1 degradation, and D1 synthesis) are equal. Healthy plants are often able to maintain a high steady-state concentration of active PSII under widely varying light intensities. Even if the concentration of active PSII is lowered by high light, the concentration of D1 protein tends to stay fairly constant (Cleland et al., 1990; Kettunen et al., 1991). In the bean (Phaseolus vulgaris L.) plants used in this study the steady-state D1 protein content remained almost constant even in the presence of excess Cu²⁺.The effect of Cu²⁺ on photoinhibition in vivo has been studied very little. Vavilin et al. (1995) suggest that excess Cu²⁺ may slow the PSII repair cycle in the green alga Chlorella pyrenoidosa, and Ouzounidou et al. (1997) suggest that Cu²⁺ inhibits adaptation to light in maize. In the current study we show that excess Cu²⁺ induces a large increase in the rate constant of photoinhibition in vivo in a higher plant.     

Diversity and Developmental Expression of L-type Calcium Channel ��2 Proteins and Their Influence on Calcium Current in Murine Heart

By now, little is known on L-type calcium channel (LTCC) subunits expressed in mouse heart. We show that CaVβ2 proteins are the major CaVβ components of the LTCC in embryonic and adult mouse heart, but that in embryonic heart CaVβ3 proteins are also detectable. At least two CaVβ2 variants of ∼68 and ∼72 kDa are expressed. To identify the underlying CaVβ2 variants, cDNA libraries were constructed from poly(A)⁺ RNA isolated from hearts of 7-day-old and adult mice. Screening identified 60 independent CaVβ2 cDNA clones coding for four types of CaVβ2 proteins only differing in their 5′ sequences. CaVβ2-N1, -N4, and -N5 but not -N3 were identified in isolated cardiomyocytes by RT-PCR and were sufficient to reconstitute the CaVβ2 protein pattern in vitro. Significant L-type Ca²⁺ currents (I_Ca) were recorded in HEK293 cells after co-expression of CaV1.2 and CaVβ2. Current kinetics were determined by the type of CaVβ2 protein, with the ∼72-kDa CaVβ2a-N1 shifting the activation of I_Ca significantly to depolarizing potentials compared with the other CaVβ2 variants. Inactivation of I_Ca was accelerated by CaVβ2a-N1 and -N4, which also lead to slower activation compared with CaVβ2a-N3 and -N5. In summary, this study reveals the molecular LTCC composition in mouse heart and indicates that expression of various CaVβ2 proteins may be used to adapt the properties of LTCCs to changing myocardial requirements during development and that CaVβ2a-N1-induced changes of I_Ca kinetics might be essential in embryonic heart.Cardiac contractions require Ca²⁺ influx in cardiomyocytes from the extracellular fluid, which leads to Ca²⁺ release from the sarcoplasmic reticulum via ryanodine receptors (1).This Ca²⁺-induced Ca²⁺ release (CICR)⁴ causes a marked increase in intracellular Ca²⁺ concentration for short periods of time and underlies cardiac contraction (2, 3). The Ca²⁺ influx into cardiac myocytes is mediated by high voltage-activated L-type Ca²⁺ channels (LTCCs), which are heteromultimeric complexes comprised predominantly of the pore-forming CaVα₁ subunit and the auxiliary CaVβ subunit (4). In heart, the principal CaVα₁ subunit, CaVα₁c (CaV1.2), is encoded by the Cacna1C gene (5). Four genes (Cacnb1-4) encoding CaVβ subunits have been identified that are expressed in the heart of different species including human, rabbit, and rat (6, 7, 8).CaVβ proteins are ∼500 amino acid cytoplasmic proteins that bind to the CaVα₁ I-II intracellular loop (9) and affect channel gating properties (4), trafficking (10, 11), regulation by neurotransmitter receptors through G-protein βγ subunit activation (12), and sensitivity to drugs (13). The CaVβ primary sequence encodes five domains, arranged V1-C1-V2-C2-V3. V1, V2, and V3 are variable domains, whereas C1 and C2 are conserved (14). Structural studies reveal that C1 and C2 form a SH3 domain (Src homology 3 domain) and a NK domain (nucleotide kinase domain), respectively (15). Although C1-V2-C2 makes the CaVβ core, in heart the V1 region appears critical for the kinetics of I_Ca and heart function. Accordingly a mutation in the V1 region of the Cacnb2 gene was recently identified as an underlying cause of Brugada syndrome (16).In mice-targeted deletion of the Cacnb2 gene (17) but not of Cacnb1 (18), Cacnb3 (19, 20), or Cacnb4 (21) leads to a morphologically and functionally compromised heart, which causes severe defective remodeling of intra- and extra-embryonic blood vessels and death at early embryonic stages both when the Cacnb2 gene was targeted globally or in a cardiac myocyte-specific way (17). Although these results point to an essential role of CaVβ2 for I_Ca and cardiac function, the existence of various CaVβ2 splice variants and heterogeneity of the expressed CaVβ2 proteins require further studies on the subunit composition of LTCCs in the mouse heart. In addition and in view of the growing number of preclinical studies using mouse models carrying definite Ca²⁺ channel subunits as transgenes in heart tissue, the identification of the relevant gene products underlying the endogenous mouse cardiac L-type channel is essential. Recent mouse models (e.g. 22, 23, 24) carrying a rat CaVβ2 splice variant (“rat CaVβ2a”) (25) expressed in rat and rabbit brain (26), but not in rabbit heart (26), have only escalated this requirement, because it has never been shown that the mouse orthologue of this variant is endogenously expressed in the mouse heart.So far, five CaVβ2 variants varying only in the V1 domain have been identified from different species (25, 27, 28) and in human heart these variants have been obtained mainly by RT-PCR approaches (29, 30). In contrast, there is little information on the CaVβ proteins present in mouse heart, their respective splice variants, and expression ratios. We therefore started to study CaVβ expression in the murine heart using Western blots and cDNA cloning and to reveal their functional impact on LTCCs formed by the murine CaV1.2 protein.     

Signaling Processes for Initiating Smooth Muscle Contraction upon Neural Stimulation

Relationships among biochemical signaling processes involved in Ca²⁺/calmodulin (CaM)-dependent phosphorylation of smooth muscle myosin regulatory light chain (RLC) by myosin light chain kinase (MLCK) were determined. A genetically-encoded biosensor MLCK for measuring Ca²⁺-dependent CaM binding and activation was expressed in smooth muscles of transgenic mice. We performed real-time evaluations of the relationships among [Ca²⁺]_i, MLCK activation, and contraction in urinary bladder smooth muscle strips neurally stimulated for 3 s. Latencies for the onset of [Ca²⁺]_i and kinase activation were 55 ± 8 and 65 ± 6 ms, respectively. Both increased with RLC phosphorylation at 100 ms, whereas force latency was 109 ± 3 ms. [Ca²⁺]_i, kinase activation, and RLC phosphorylation responses were maximal by 1.2 s, whereas force increased more slowly to a maximal value at 3 s. A delayed temporal response between RLC phosphorylation and force is probably due to mechanical effects associated with elastic elements in the tissue. MLCK activation partially declined at 3 s of stimulation with no change in [Ca²⁺]_i and also declined more rapidly than [Ca²⁺]_i during relaxation. The apparent desensitization of MLCK to Ca²⁺ activation appears to be due to phosphorylation in its calmodulin binding segment. Phosphorylation of two myosin light chain phosphatase regulatory proteins (MYPT1 and CPI-17) or a protein implicated in strengthening membrane adhesion complexes for force transmission (paxillin) did not change during force development. Thus, neural stimulation leads to rapid increases in [Ca²⁺]_i, MLCK activation, and RLC phosphorylation in phasic smooth muscle, showing a tightly coupled Ca²⁺ signaling complex as an elementary mechanism initiating contraction.Increases in [Ca²⁺]_i3 in smooth muscle cells lead to Ca²⁺/CaM-dependent MLCK activation and RLC phosphorylation. Phosphorylation of RLC increases actin-activated myosin MgATPase activity leading to myosin cross-bridge cycling with force development (1–3).The activation of smooth muscle contraction may be affected by multiple cellular processes. Previous investigations show that free Ca²⁺/CaM is limiting for kinase activation despite the abundance of total CaM (4–6). The extent of RLC phosphorylation is balanced by the actions of MLCK and myosin light chain phosphatase, which is composed of three distinct protein subunits (7). The myosin phosphatase targeting subunit, MYPT1, in smooth muscle binds to myosin filaments, thus targeting the 37-kDa catalytic subunit (type 1 serine/threonine phosphatase, PP1c) to phosphorylated RLC. RLC phosphorylation and muscle force may be regulated by additional signaling pathways involving phosphorylation of RLC by Ca²⁺-independent kinase(s) and inhibition of myosin light chain phosphatase, processes that increase the contraction response at fixed [Ca²⁺]_i (Ca²⁺-sensitization) (8–14). Many studies indicate that agonist-mediated Ca²⁺-sensitization most often reflects decreased myosin light chain phosphatase activity involving two major pathways including MYPT1 phosphorylation by a Rho kinase pathway and phosphorylation of CPI-17 by PKC (8, 14–16). Additionally, phosphorylation of MLCK in its calmodulin-binding sequence by a Ca²⁺/calmodulin-dependent kinase pathway has been implicated in Ca²⁺ desensitization of RLC phosphorylation (17–19). How these signaling pathways intersect the responses of the primary Ca²⁺/CaM pathway during physiological neural stimulation is not known.There is also evidence that smooth muscle contraction requires the polymerization of submembranous cytoskeletal actin filaments to strengthen membrane adhesion complexes involved in transmitting force between actin-myosin filaments and external force-transmitting structures (20–23). In tracheal smooth muscle, paxillin at membrane adhesions undergoes tyrosine phosphorylation in response to contractile stimulation by an agonist, and this phosphorylation increases concurrently with force development in response to agonist. Expression of nonphosphorylatable paxillin mutants in tracheal muscle suppresses acetylcholine-induced tyrosine phosphorylation of paxillin, tension development, and actin polymerization without affecting RLC phosphorylation (24, 25). Thus, paxillin phosphorylation may play an important role in tension development in smooth muscle independently of RLC phosphorylation and cross-bridge cycling.Specific models relating signaling mechanisms in the smooth muscle cell to contraction dynamics are limited when cells in tissues are stimulated slowly and asynchronously by agonist diffusing into the preparation. Field stimulation leading to the rapid release of neurotransmitters from nerves embedded in the tissue avoids these problems associated with agonist diffusion (26, 27). In urinary bladder smooth muscle, phasic contractions are brought about by the parasympathetic nervous system. Upon activation, parasympathetic nerve varicosities release the two neurotransmitters, acetylcholine and ATP, that bind to muscarinic and purinergic receptors, respectively. They cause smooth muscle contraction by inducing Ca²⁺ transients as elementary signals in the process of nerve-smooth muscle communication (28–30). We recently reported the development of a genetically encoded, CaM-sensor for activation of MLCK. The CaM-sensor MLCK contains short smooth muscle MLCK fused to two fluorophores, enhanced cyan fluorescent protein and enhanced yellow fluorescent protein, linked by the MLCK calmodulin binding sequence (6, 14, 31). Upon dimerization, there is significant FRET from the donor enhanced cyan fluorescent protein to the acceptor enhanced yellow fluorescent protein. Ca²⁺/CaM binding dissociates the dimer resulting in a decrease in FRET intensity coincident with activation of kinase activity (31). Thus, CaM-sensor MLCK is capable of directly monitoring Ca²⁺/CaM binding and activation of the kinase in smooth muscle tissues where it is expressed specifically in smooth muscle cells of transgenic mice. We therefore combined neural stimulation with real-time measurements of [Ca²⁺]_i, MLCK activation, and force development in smooth muscle tissue from these mice. Additionally, RLC phosphorylation was measured precisely at specific times following neural stimulation in tissues frozen by a rapid-release electronic freezing device (26, 27). Results from these studies reveal that physiological stimulation of smooth muscle cells by neurotransmitter release leads to rapid increases in [Ca²⁺]_i, MLCK activation, and RLC phosphorylation at similar rates without the apparent activities of Ca²⁺-independent kinases, inhibition of myosin light chain phosphatase, or paxillin phosphorylation. Thus, the elemental processes for phasic smooth muscle contraction are represented by this tightly coupled Ca²⁺ signaling complex.     

Effects of Xylem pH on Transpiration from Wild-Type and flacca Tomato Leaves : A Vital Role for Abscisic Acid in Preventing Excessive Water Loss Even from Well-Watered Plants

The pH of xylem sap from tomato (Lycopersicon esculentum) plants increased from pH 5.0 to 8.0 as the soil dried. Detached wild-type but not flacca leaves exhibited reduced transpiration rates when the artificial xylem sap (AS) pH was increased. When a well-watered concentration of abscisic acid (0.03 μm) was provided in the AS, the wild-type transpirational response to pH was restored to flacca leaves. Transpiration from flacca but not from wild-type leaves actually increased in some cases when the pH of the AS was increased from 6.75 to 7.75, demonstrating an absolute requirement for abscisic acid in preventing stomatal opening and excessive water loss from plants growing in many different environments.Jones (1980) and Cowan (1982) were the first to suggest that plants can “measure” soil water status independently of shoot water status via the transfer of chemical information from roots to shoots. Dehydrating roots in drying soil synthesize ABA more rapidly than fully turgid tissue, and resultant increases in the ABA concentration of xylem sap flowing toward the still-turgid shoot constitutes a chemical signal to the leaves (for review, see Davies and Zhang, 1991): the xylem vessels give up their contents to the leaf apoplast, thereby increasing the ABA concentration in this compartment. ABA receptors on the external surface of stomatal guard cells respond to the apoplastic ABA concentration (Hartung, 1983; Anderson et al., 1994; but see Schwartz et al., 1994). When bound, the receptors transduce a reduction in guard cell turgor, which leads to stomatal closure (Assmann, 1993). This maintains shoot water potential despite the reduction in soil water availability.Another chemical change related to soil drying in the absence of a reduction in shoot water status is an increase in the pH of the xylem sap flowing from the roots (Schurr et al., 1992). The pH of the xylem and/or apoplastic sap of plants can also change dramatically in response to soil flooding, diurnal or annual rhythms, and mineral nutrient supply (Table ​(TableI)I) in the absence of concomitant changes in either root or shoot water status. We already know that, like the increase in xylem ABA concentration described above, an increase in xylem pH can also act as a signal to leaves to close their stomata (Wilkinson and Davies, 1997). Since the conditions that affect xylem/apoplastic pH can also affect transpiration (light intensity [Cowan et al., 1982]; soil drying [Davies and Zhang, 1991]; nitrate supply [Clarkson and Touraine, 1994]; soil flooding [Else, 1996]), the possibility exists that the pH change that they induce could be the means by which they alter stomatal aperture. Table IpH changes that occur in plant xylem or apoplastic sap under various conditions It was originally suggested that an increase in xylem sap pH could putatively enhance stomatal closure by changing the distribution of the ABA that is present in all nonstressed plants at a low “background” concentration, without requiring de novo ABA synthesis (Schurr et al., 1992; Slovik and Hartung, 1992a, 1992b). This hypotheses is built on the well-known fact that weak acids such as ABA accumulate in more alkaline compartments (Kaiser and Hartung, 1981). More recently, Wilkinson and Davies (1997) and Thompson et al. (1997) directly demonstrated that increases in xylem sap pH reduced rates of water loss from Commelina communis and tomato (Lycopersicon esculentum) leaves detached from well-watered plants. This was found to be mediated by the relatively low endogenous concentration of ABA (about 0.01 mmol m⁻³) contained in the xylem vessels and apoplast of these leaves, a concentration of ABA that did not itself affect transpiration at a well-watered sap pH of 6.0. The mechanism by which the combination of high sap pH and such a low concentration of ABA was able to increase the apoplastic ABA concentration sufficiently to close stomata was also elucidated: the mesophyll and epidermis cells of these leaves had a greatly reduced ability to sequester ABA away from the apoplast when the pH of the latter was increased by the incoming xylem sap (Wilkinson and Davies, 1997).In contrast to the indirect ABA-mediated effect of pH on stomata, it was also demonstrated that increasing the pH of the external solution (from 5.0 to 7.0) bathing isolated abaxial epidermis tissue peeled from well-watered C. communis leaves actually increased stomatal aperture (Wilkinson and Davies, 1997). Mechanisms for this direct effect of pH on guard cells have been speculated on by Thompson et al. (1997). If this process were to occur in vivo, environments that increase xylem sap pH could potentially induce excessive water loss from the plants experiencing them, over and above rates of transpiration occurring in unstressed plants. The latter may contain stomata with apertures smaller than the maximum that is possible, even under favorable local conditions. It was assumed that high-pH-induced apoplastic ABA accumulation in C. communis in vivo was sufficient to override the direct stomatal opening effect seen in the isolated tissue (Wilkinson and Davies, 1997). To test these possibilities, effects of pH on transpiration rates from leaves of the flacca mutant of tomato were investigated. flacca does not synthesize ABA as efficiently as wild-type tomato (Parry et al., 1988; Taylor et al., 1988). It contains a very low endogenous ABA concentration (Tal and Nevo, 1973), although it retains the ability to respond to an application of this hormone (Imber and Tal, 1970). The results demonstrate not only that ABA mediates high xylem sap pH-induced stomatal closure but also that it is necessary to prevent high xylem sap pH-induced stomatal opening and dangerously excessive water loss.     

Characterization of Zinc Uptake, Binding, and Translocation in Intact Seedlings of Bread and Durum Wheat Cultivars

Durum wheat (Triticum turgidum L. var durum) cultivars exhibit lower Zn efficiency than comparable bread wheat (Triticum aestivum L.) cultivars. To understand the physiological mechanism(s) that confers Zn efficiency, this study used ⁶⁵Zn to investigate ionic Zn²⁺ root uptake, binding, and translocation to shoots in seedlings of bread and durum wheat cultivars. Time-dependent Zn²⁺ accumulation during 90 min was greater in roots of the bread wheat cultivar. Zn²⁺ cell wall binding was not different in the two cultivars. In each cultivar, concentration-dependent Zn²⁺ influx was characterized by a smooth, saturating curve, suggesting a carrier-mediated uptake system. At very low solution Zn²⁺ activities, Zn²⁺ uptake rates were higher in the bread wheat cultivar. As a result, the Michaelis constant for Zn²⁺ uptake was lower in the bread wheat cultivar (2.3 μm) than in the durum wheat cultivar (3.9 μm). Low temperature decreased the rate of Zn²⁺ influx, suggesting that metabolism plays a role in Zn²⁺ uptake. Ca inhibited Zn²⁺ uptake equally in both cultivars. Translocation of Zn to shoots was greater in the bread wheat cultivar, reflecting the higher root uptake rates. The study suggests that lower root Zn²⁺ uptake rates may contribute to reduced Zn efficiency in durum wheat varieties under Zn-limiting conditions.Soils that contain insufficient levels of the essential plant micronutrient Zn are common throughout the world. As a result, Zn deficiency is a widespread problem in crop plants, especially cereals (Graham et al., 1992). The importance of plant foods as sources of Zn, particularly in the marginal diets of developing countries, is well established (Welch, 1993). The development of crop plants that are efficient Zn accumulators is therefore a potentially important endeavor. In addition to its effects on nutrition, Zn deficiency in crops is relevant to other areas of human health. Another consequence of Zn-deficient soils is the tendency for plants grown in such soils to accumulate heavy metals. For example, in the Great Plains region of North America, where soil Zn levels are low and naturally occurring Cd is present, durum wheat (Triticum turgidum L. var durum) grains accumulate Cd to relatively high concentrations (Wolnik et al., 1983). The presence of Cd in food represents a potential human health hazard and, in response, international trade standards have been proposed to limit the levels of Cd in exported grain (Codex Alimentarius Commission, 1993). Thus, there is a need to understand the physiological processes that control acquisition of Zn from soil solution by roots and mobilization of Zn within plants.It has been demonstrated in recent years that crop plants vary in their ability to take up Zn, particularly when its availability to roots is limited. Zn efficiency, defined as the ability of a plant to grow and yield well in Zn-deficient soils, varies among wheat cultivars (Graham and Rengel, 1993). In field trials, durum wheat cultivars have been shown to be consistently less Zn efficient than bread wheat (Triticum aestivum L.) cultivars (Graham et al., 1992). Similarly, durum wheat varieties were reported to be less Zn efficient than bread wheat varieties when grown in chelate-buffered hydroponic nutrient culture (Rengel and Graham, 1995a).The physiological mechanism(s) that confers Zn efficiency has not been identified. Processes that could influence the ability of a plant to tolerate limited amounts of available Zn include higher root uptake, more efficient utilization of Zn, and enhanced Zn translocation within the plant. Cakmak et al. (1994) showed that a Zn-inefficient durum wheat cultivar exhibited Zn-deficiency symptoms earlier and more intensely than a Zn-efficient bread wheat cultivar even though the Zn tissue concentrations were similar in both lines, suggesting differential utilization of Zn in the two cultivars. Rates of Zn translocation to shoots were shown to vary among sorghum cultivars, although correlations with Zn efficiency were not established (Ramani and Kannan, 1985). Root uptake kinetics have been reported to vary between rice cultivars having different Zn requirements, with high-Zn-requiring cultivars exhibiting consistently higher root uptake rates (Bowen, 1986). In contrast, a correlation between Zn efficiency and rates of root Zn uptake in bread and durum wheat cultivars could not be demonstrated (Rengel and Graham, 1995b).In grasses Zn influx into the root symplasm has been hypothesized to occur as the free Zn²⁺ ion (Halvorson and Lindsay, 1977), as well as in the form of Zn complexes with nonprotein amino acids known as phytosiderophores (Tagaki et al., 1984) or phytometallophores (Welch, 1993). Concentration-dependent uptake of free Zn²⁺ ions has been shown to be saturable in several species, including maize (Mullins and Sommers, 1986), barley (Veltrup, 1978), and wheat (Chaudhry and Loneragan, 1972), suggesting that ionic uptake in grasses occurs via a carrier-mediated system. However, several of these studies have been criticized on the basis that excessively high (and physiologically unrealistic) Zn²⁺ concentrations were used (Kochian, 1993).This study was undertaken to examine unidirectional Zn²⁺ influx and translocation to shoots in Zn-efficient bread wheat lines and Zn-inefficient durum wheat lines. Experiments were performed in the absence of added phytometallophores and results are presumed to represent influx of ionic Zn²⁺. Zn activities in the nanomolar range were used to more closely mimic free Zn²⁺ levels occurring naturally in soil solution. The results presented here indicate that a Zn-efficient bread wheat cultivar maintained higher rates of Zn uptake than a Zn-inefficient durum wheat cultivar, particularly at low (and physiologically relevant) solution Zn²⁺ activities.     

Arabidopsis Roots and Shoots Have Different Mechanisms for Hypoxic Stress Tolerance

Arabidopsis has inducible responses for tolerance of O₂ deficiency. Plants previously exposed to 5% O₂ were more tolerant than the controls to hypoxic stress (0.1% O₂ for 48 h) in both roots and shoots, but hypoxic acclimation did not improve tolerance to anoxia (0% O₂). The acclimation of shoots was not dependent on the roots: increased shoot tolerance was observed when the roots of the plants were removed. An adh (alcohol dehydrogenase) null mutant did not show acclimation of the roots but retained the shoot survival response. Abscisic acid treatment also differentiated the root and shoot responses; pretreatment induced root survival in hypoxic stress conditions (0.1% O₂) but did not induce any increase in the survival of shoots. Cycloheximide blocked both root and shoot acclimation, indicating that both acclimation mechanisms are dependent on protein synthesis.The supply of O₂ to plant tissues may be restricted under certain environmental conditions (Hook and Crawford, 1978). When air spaces normally present in the soil become saturated with water, the root environment becomes hypoxic or anoxic as a result of O₂ consumption by respiring roots and microorganisms and the insufficient diffusion of O₂ through water (Armstrong, 1979). O₂ deficiency is thought to be a major determinant in the adverse effects of waterlogging on crops and other plant species (Jackson et al., 1991). Plants have evolved inducible metabolic mechanisms to cope with these ephemeral, low-O₂-stress conditions. When exposed to low-O₂ conditions, plants switch to the expression of “anaerobic” polypeptides (Sachs et al., 1980, 1996). The induction of these proteins may be responsible for the tolerance to O₂ deficiency that would otherwise be lethal. A number of anaerobic polypeptides have been identified as enzymes involved in glycolysis and ethanol fermentation (for a recent review, see Vartapetian and Jackson, 1997), and this supports the view that when O₂ is limiting, oxidative catabolism of sugars is hindered and ethanolic fermentation acts as an alternative energy-producing pathway.Ethanol is the main end product of anaerobic metabolism in plants (Smith and ap Rees, 1979; Good and Muench, 1993). Unlike lactate, which is also generated under O₂ deficiency, ethanol is a relatively nontoxic end product (Jackson et al., 1982) and does not lead to the acidification of the cytoplasm, a major determinant in intolerance to O₂ deficiency (Roberts et al., 1984, 1985). The induction of glycolytic enzymes probably reflects the need for increased glycolysis to compensate for the lower ATP yield of ethanol fermentation.The importance of ethanol fermentation is supported by studies of adh (alcohol dehydrogenase) null mutants in a number of species (Schwartz, 1966; Harberd and Edwards, 1982; Jacobs et al., 1988; Matsumura et al., 1995), which report reduced tolerance to O₂ deficiency in these plants.Some plant tissues exposed to a period of mild hypoxia show more tolerance to subsequent hypoxic or anoxic stress than plants kept in fully aerated conditions before the stress (for review, see Drew, 1997; see also more recent work on tomato [Germain et al., 1997] and rice [Ellis and Setter, 1999]).In this study we examined the survival of Arabidopsis plants after exposure to anoxic or hypoxic stress. Our results demonstrate that hypoxic pretreatment protects against hypoxic stress and that different mechanisms of acclimation to hypoxic stress are operative in root and shoot tissues.