A role for plant natriuretic peptide immuno-analogues in NaCl- and drought-stress responses

Higher plants contain biologically active molecules that are recognized by anti-human atrial natriuretic polypeptide rabbit serum (anti-ANP). These molecules are termed immunoreactant plant natriuretic peptides (irPNPs) and have previously been shown to be associated with conductive tissue and to affect ion fluxes, protoplast volume regulation and stomatal guard cell responses. Herein an irPNP from the brassicaceus weed Erucastrum strigosum is identified and it is demonstrated that the relative amounts of irPNP expressed as a percentage of total water : methanol (50 : 50) extracted proteins are increased when plants are exposed to 300 m M NaCl. Since 100 and 200 m M NaCl reduce dry and fresh mass as well as increase total tissue NaCl load, it is hypothesized that irPNP up-regulation is a late and possibly adaptive response. IrPNP is also significantly up-regulated in Arabidopsis thaliana suspension culture cells in response to 150 m M NaCl and even more so in response to iso-osmolar amounts of sorbitol. Finally, a recombinant A. thaliana irPNP (AtPNP-A) promotes net water-uptake into the protoplast and thus volume increases. This response is dependent on de novo protein synthesis and may suggest a complex and possibly regulatory function for irPNP-like molecules in plant homeostasis.     

Natriuretic peptides--a class of heterologous molecules in plants

Immunological and physiological evidence suggests the presence of biologically active natriuretic peptide hormones (NPs) in plants. Evidence includes specific binding of rat atrial NP, [rANP (99-126)] to plant membranes and the promotion of cyclic guanosine-3',5'-monophosphate (cGMP) mediated stomatal responses. Furthermore, anti-ANP affinity purifies biologically active plant immunoreactants (irPNPs) and a biologically active Arabidopsis thaliana irPNP (AtPNP-A) has been identified. AtPNP-A belongs to a novel class of molecules that share some similarity with the cell wall loosening expansins but do not contain the carbohydrate-binding wall anchor, thus suggesting that irPNPs and ANP are heterologues. We hypothesise that irPNP-like molecules have evolved from primitive glucanase-like molecules that have been recruited to become systemically mobile modulators of homeostasis acting via the plasma membrane. Such a function is compatible with localisation in the conductive tissue and the physiological and cellular modes of action of irPNPs reported to-date.     

In Situ and in Vitro Binding of Natriuretic Peptide Hormones in Tradescantia multiflora

Abstract: An increasing body of evidence suggests that in plants, as in vertebrates, biologically active natriuretic peptide (NP) hormones play an important role in the regulation of the osmotic and ionic balance. The evidence includes isolation and immunoaffinity purification of biologically active natriuretic peptide analogues (irPNP) from ivy that promoted stomatal opening and specifically, rapidly and transiently increased cGMP levels in root conductive tissue. In this study we demonstrate that I¹²⁵-rat atrial natriuretic peptide (rANP) binds to plasma membranes from leaf and stem tissue of Tradescantia multiflora and importantly, both unlabelled rANP and irPNP can competitively displace that binding. In addition, tissue section autoradiography reveals specific in situ binding of I¹²⁵-rANP to leaf and stem tissue. The findings are consistent with the presence of a biologically active NP system in plants and suggest that NPs signal through a dedicated receptor system.     

Natriuretic Peptides--A New Class of Plant Hormone?

Recent immunological and functional evidence suggests the presenceof a biologically active natriuretic peptide hormone (NP) systemin plants. The evidence includes specific binding of rat atrialNP [rANP (99–126)] to isolated plant membranes and thepromotion of stomatal opening that is concentration and conformationdependent. The native circular molecule is active whereas thelinearized molecule shows no biological activity. Stomatal openingmediated by rANP (99–126) is inhibited by LY 83583, anantagonist of guanylate cyclase, while 8-Br-cGMP, a cell permeantcyclic guanosine-3'-5'-monophosphate (cGMP) analogue, mimicsrANP (99–126) effects. Most importantly, isolation andimmunoaffinity purification of biologically active plant NP(irPNP) fromHedera helixhas been achieved and immunoaffinitypurified peptide has been shown to induce rapid and specificincreases in cGMP levels inZea maysroot stele. Furthermore,rANP (99–126), irPNP and cGMP promote radial water movementsfrom the xylem ofTradescantia multifloraand these NP-inducedand cGMP-dependent increases are prevented by a water channelinhibitor. Taken together, the data are consistent with thepresence of a biologically active NP system that, as in vertebrates,signals via cGMP dependent pathways. The evidence suggests thatthe NP system has a role in maintaining water and salt homeostasisin plants.Copyright 1999 Annals of Botany Company Plant hormones, natriuretic peptides, stomata, cGMP, salt and water homeostasis.     

The Arabidopsis small G protein ROP2 is activated by light in guard cells and inhibits light-induced stomatal opening

ROP small G proteins function as molecular switches in diverse signaling processes. Here, we investigated signals that activate ROP2 in guard cells. In guard cells of Vicia faba expressing Arabidopsis thaliana constitutively active (CA) ROP2 fused to red fluorescent protein (RFP-CA-ROP2), fluorescence localized exclusively at the plasma membrane, whereas a dominant negative version of RFP-ROP2 (DN-ROP2) localized in the cytoplasm. In guard cells expressing green fluorescent protein-ROP2, the relative fluorescence intensity at the plasma membrane increased upon illumination, suggesting that light activates ROP2. Unlike previously reported light-activated factors, light-activated ROP2 inhibits rather than accelerates light-induced stomatal opening; stomata bordered by guard cells transformed with CA-rop2 opened less than controls upon light irradiation. When introduced into guard cells together with CA-ROP2, At RhoGDI1, which encodes a guanine nucleotide dissociation inhibitor, inhibited plasma membrane localization of CA-ROP2 and abolished the inhibitory effect of CA-ROP2 on light-induced stomatal opening, supporting the negative effect of active ROP2 on stomatal opening. Mutant rop2 Arabidopsis guard cells showed phenotypes similar to those of transformed V. faba guard cells; CA-rop2 stomata opened more slowly and to a lesser extent, and DN-rop2 stomata opened faster than wild-type stomata in response to light. Moreover, in rop2 knockout plants, stomata opened faster and to a greater extent than wild-type stomata in response to light. Thus, ROP2 is a light-activated negative factor that attenuates the extent of light-induced changes in stomatal aperture. The inhibition of light-induced stomatal opening by light-activated ROP2 suggests the existence of feedback regulatory mechanisms through which stomatal apertures may be finely controlled.     

Light perception in guard cells

Abstract. Guard cells perceive light via two photoreceptor systems: a blue-light-dependent photosystem and the guard cell chloroplast. Chloroplasts stimulate stomatal opening by transducing photosynthetic active radiation into proton pumping at the guard cell plasma membrane. In addition, guard cell chloroplasts fix CO₂ photosynthetically. Sugar from guard cell photosynthesis can contribute to the osmotic build-up required for opening. The blue-light-dependent photosystem activates proton pumping at the guard cell plasma membrane and stimulates starch hydrolysis. Available information on the photobiological properties of guard cells makes it possible to describe stomatal function in terms of the cellular components regulating stomatal movements. The blue light response is involved in stomatal opening in the early morning and stomatal responses to sunflecks. The guard cell chloroplast is likely to be involved in stomatal adaptations to sun, shade and to temperature. Interactions between these photosystems, a third photoreceptor in guard cells, phytochrome, and other mechanisms transducing stomatal responses such as VPD and carbon dioxide, provide the cellular basis for stomatal regulation.     

A model of stomatal conductance to quantify the relationship between leaf transpiration, microclimate and soil water stress

A model of stomatal conductance was developed to relate plant transpiration rate to photosynthetic active radiation (PAR), vapour pressure deficit and soil water potential. Parameters of the model include sensitivity of osmotic potential of guard cells to photosynthetic active radiation, elastic modulus of guard cell structure, soil‐to‐leaf conductance and osmotic potential of guard cells at zero PAR. The model was applied to field observations on three functional types that include 11 species in subtropical southern China. Non‐linear statistical regression was used to obtain parameters of the model. The result indicated that the model was capable of predicting stomatal conductance of all the 11 species and three functional types under wide ranges of environmental conditions. Major conclusions included that coniferous trees and shrubs were more tolerant for and resistant to soil water stress than broad‐leaf trees due to their lower osmotic potential, lignified guard cell walls, and sunken and suspended guard cell structure under subsidiary epidermal cells. Mid‐day depression in transpiration and photosynthesis of pines may be explained by decreased stomatal conductance under a large vapour pressure deficit. Stomatal conductance of pine trees was more strongly affected by vapour pressure deficit than that of other species because of their small soil‐to‐leaf conductance, which is explainable in terms of xylem tracheids in conifer trees. Tracheids transport water by means of small pit‐pairs in their side walls, and are much less efficient than the end‐perforated vessel members in broad‐leaf xylem systems. These conclusions remain hypothetical until direct measurements of these parameters are available.     

蚕豆下表皮细胞外钙调素的存在及其对气孔运动的调节

细胞外钙调素可能作为多肽第一信使,调节细胞增殖,花粉萌发,特定基因表达等生理过程,气孔能灵敏地对外界刺激作出反应,快速开闭,本文用免疫电镜和免疫荧光显微镜技术证明保卫细胞及其它表皮细胞胞外都存在钙调素;外源纯化钙调素能促进气孔关闭,抑制气孔开放,最适浓度为10^-8mol/L;不能透过质膜的大分子钙调素拮抗剂W—-agarose和钙调素抗血清都能抑制气孔关闭,促进开放,说明保卫细胞的内源胞外钙调素确实能促进气孔关闭,抑制开放。而且只能在细胞外起作用,推测在自然情况下,保卫细胞内源胞外钙调素可能作为胞外第一信使和其它信号分子一起调节气孔的开关运动,而且可能在环境刺激与细胞响应之间起重要作用。     

蚕豆下表皮细胞外钙调素的存在及其对气孔运动的调节

细胞外钙调素可能作为多肽第一信使,调节细胞增殖、花粉萌发、特定基因表达等生理过程.气孔能灵敏地对外界刺激作出反应,快速开闭.本文用免疫电镜和免疫荧光显微镜技术证明保卫细胞及其它表皮细胞胞外都存在钙调素.外源纯化钙调素能促进气孔关闭、抑制气孔开放,最适浓度为10-8mol/L;不能透过质膜的大分子钙调素拮抗剂W7-agarose和钙调素抗血清都能抑制气孔关闭、促进开放,说明保卫细胞的内源胞外钙调素确实能促进气孔关闭、抑制开放,而且只能在细胞外起作用.推测在自然情况下,保卫细胞内源胞外钙调素可能作为胞外第一信使和其它信号分子一起调节气孔的开关运动,而且可能在环境刺激与细胞响应之间起重要作用.     

Stochastic dynamics of actin filaments in guard cells regulating chloroplast localization during stomatal movement

Actin filaments and chloroplasts in guard cells play roles in stomatal function. However, detailed actin dynamics vary, and the roles that they play in chloroplast localization during stomatal movement remain to be determined. We examined the dynamics of actin filaments and chloroplast localization in transgenic tobacco expressing green fluorescent protein (GFP)-mouse talin in guard cells by time-lapse imaging. Actin filaments showed sliding, bundling and branching dynamics in moving guard cells. During stomatal movement, long filaments can be severed into small fragments, which can form longer filaments by end-joining activities. With chloroplast movement, actin filaments near chloroplasts showed severing and elongation activity in guard cells during stomatal movement. Cytochalasin B treatment abolished elongation, bundling and branching activities of actin filaments in guard cells, and these changes of actin filaments, and as a result, more chloroplasts were localized at the centre of guard cells. However, chloroplast turning to avoid high light, and sliding of actin fragments near the chloroplast, was unaffected following cytochalasin B treatment in guard cells. We suggest that the sliding dynamics of actin may play roles in chloroplast turning in guard cells. Our results indicate that the stochastic dynamics of actin filaments in guard cells regulate chloroplast localization during stomatal movement.     

Molecular cloning and characterization of the enzyme UDP-glucose: protein transglucosylase from potato

UDP-Glc:protein transglucosylase (UPTG) (EC 2.4.1.112) is an autocatalytic glycosyl-transferase previously postulated as a protein that primes starch biosynthesis. Polyclonal antibodies raised against UPTG purified from potato (Solanum tuberosum L.) tubers were used to screen a potato swelling stolon tip cDNA expression library. The isolation, cloning and sequencing of two cDNAs corresponding to UPTG are described. Recombinant UPTG was labelled after incubation with UDP-[¹⁴C]-Glc and Mn²⁺, indicating that it was enzymatically active. It was determined that purified as well as recombinant UPTG can be reversibly glycosylated by UDP-Glc, UDP-Xyl or UDP-Gal. RNA hybridization studies and western blot analysis indicate that UPTG mRNA and protein are expressed in all potato tissues. Databank searches revealed a high degree of identity between UPTG and several plant sequences that encode for proteins with apparent localization at the cytoplasmic face of the Golgi apparatus and at plasmodesmata. The biochemical properties of UPTG and the apparent lack of a signal peptide that could allow its entrance into plastids argue against the postulated role of UPTG in starch synthesis and point towards a possible role of the protein in the synthesis of cell wall polysaccharides.     

Cloning and electrophysiological analysis of KST1, an inward rectifying K+ channel expressed in potato guard cells.

Potassium uptake by guard cells represents part of the osmotic motor which drives stomatal opening. Patch-clamp measurements have identified inward rectifying K+ channels capable of mediating K+ uptake in guard cells and various other plant cell types. Here we report the molecular cloning and characterization of a voltage-dependent K+ channel (KST1) from potato (Solanum tuberosum L.) guard cells. In situ hybridization shows expression of kst1 in guard cells. Two-electrode voltage-clamp and patch-clamp studies of the gene product after cRNA injection into Xenopus oocytes identified KST1 as a slowly activating, voltage-dependent, inward rectifying K+ channel. The single channel current voltage curve was linear in the range -160 to +20 mV, with a deduced single channel conductance of 7 pS in symmetrical 100 mM K+. This channel type, modulated by pH changes within the physiological range, required ATP for activation. In line with the properties of a K(+)-selective channel, KST1 was permeable to K+, Rb+ and NH4+ and excluded Na+ and Li+. Cs+ at submillimolar concentrations blocked the channel in a voltage-dependent manner. Related studies on potato guard cell protoplasts confirmed the biophysical characteristics of the kst1 gene product (KST1) in the heterologous expression system. Therefore, KST1 represents a major K+ uptake channel in potato guard cells.     

Anion channels as central mechanisms for signal transduction in guard cells and putative functions in roots for plant-soil interactions

In higher plants anion channels have recently been suggested to play key roles in controlling cellular functions, including turgor- and osmoregulation, stomatal movements, anion transport, signal transduction and possibly also signal propagation. In guard cells and roots, physiological functions of anion channels have been proposed which will be discussed here. In initial investigations it was proposed that anion channels in the plasma membrane of guard cells provide a prominent control mechanism for stomatal closing. The proposed model suggests that anion channel activation and the resulting anion efflux from guard cells cause membrane depolarization, thereby driving K⁺ efflux through outward-rectifying K⁺ channels required for stomatal closing. This article provides a brief review of new and recent insights into the molecular properties and cell biological functions of anion channels in guard cells. Furthermore, recently implicated putative functions of anion channels in roots during salt stress, xylem loading and Al³⁺ tolerance are addressed.     

A hydromechanical and biochemical model of stomatal conductance

A mathematical model of stomatal conductance is presented. It is based on whole‐plant and epidermal hydromechanics, and on two hypotheses: (1) the osmotic gradient across guard cell membranes is proportional to the concentration of ATP in the guard cells; and (2) the osmotic gradient that can be sustained per unit of ATP is proportional to the turgor pressure of adjacent epidermal cells. In the present study, guard cell [ATP] is calculated using a previously published model that is based on a widely used biochemical model of C₃ mesophyll photosynthesis. The conductance model for Vicia faba L. is parameterized and tested As with most other stomatal models, the present model correctly predicts the stomatal responses to variations in transpiration rate, irradiance and intercellular CO₂. Unlike most other models, however, this model can predict the transient stomatal opening often observed before conductance declines in response to decreases in humidity, soil water potential, or xylem conductance. The model also explicitly accommodates the mechanical advantage of the epidermis and correctly predicts that stomata are relatively insensitive to the ambient partial pressure of oxygen, as a result of the assumed dependence on ATP concentration.     

Changes in stomatal function and water use efficiency in potato plants with altered sucrolytic activity

As water availability for agriculture decreases, breeding or engineering of crops with improved water use efficiency (WUE) will be necessary. As stomata are responsible for controlling gas exchange across the plant epidermis, metabolic processes influencing solute accumulation in guard cells are potential targets for engineering. In addition to its role as an osmoticum, sucrose breakdown may be required for synthesis of other osmotica or generation of the ATP needed for solute uptake. Thus, alterations in partitioning of sucrose between storage and breakdown may affect stomatal function. In agreement with this hypothesis, potato (Solanum tuberosum) plants expressing an antisense construct targeted against sucrose synthase 3 (SuSy3) exhibited decreased stomatal conductance, a slight reduction in CO(2) fixation and increased WUE. Conversely, plants with increased guard cell acid invertase activity caused by the introduction of the SUC2 gene from yeast had increased stomatal conductance, increased CO(2) fixation and decreased WUE. (14)CO(2) feeding experiments indicated that these effects cannot be attributed to alterations in photosynthetic capacity, and most likely reflect alterations in stomatal function. These results highlight the important role that sucrose breakdown may play in guard cell function and indicate the feasibility of manipulating plant WUE through engineering of guard cell sucrose metabolism.     

The nitrate transporter AtNRT1.1 (CHL1) functions in stomatal opening and contributes to drought susceptibility in Arabidopsis

The movement of guard cells in stomatal complexes controls water loss and CO(2) uptake in plants. Examination of the dual-affinity nitrate transporter gene AtNRT1.1 (CHL1) revealed that it is expressed and functions in Arabidopsis guard cells. CHL1 promoter-beta-glucuronidase and CHL1 promoter-green fluorescent protein constructs showed strong expression in guard cells, and immunolocalization experiments with anti-CHL1 antibody confirmed these results. To assess CHL1 function, chl1 mutant plants grown in the presence of nitrate were examined. Compared with wild-type plants, chl1 mutants had reduced stomatal opening and reduced transpiration rates in the light or when deprived of CO(2) in the dark. These effects result in enhanced drought tolerance in chl1 mutants. At the cellular level, chl1 mutants showed reduced nitrate accumulation in guard cells during stomatal opening and failed to show nitrate-induced depolarization of guard cells. In wild-type guard cells, nitrate induced depolarization, and nitrate concentrations increased threefold during stomatal opening. These results identify an anion transporter that functions in stomatal opening and demonstrate that CHL1 supports stomatal function in the presence of nitrate.     

Subcellular localization of β-1,3-glucanase in Puccinia recondita f.sp. tritici-infected wheat leaves

An antiserum raised against the purified 33-kDa β-1,3-glucanase of wheat (Triticum aestivum L.) was employed to investigate the ultrastructural localization of the enzyme in wheat leaves infected with Puccinia recondita Rob. ex Desm. f.sp. tritici Eriks. and Henn. using a post-embedding immunogold labelling technique. In both compatible and incompatible interactions, β-1,3-glucanase was detected in the host plasmalemma and in the domain of the host cell wall near the plasmalemma of the mesophyll cells, but higher concentrations of the enzyme were detected in infected resistant wheat leaves than in infected susceptible ones. β-1,3-Glucanase was also found in the secondary thickening of xylem vessels and in the walls of guard cells, epidermal cells and phloem elements, while no labelling was observed in host organelles, viz. vacuoles, mitochondria, endoplasmic reticulum, Golgi bodies, nuclei and chloroplasts. A low concentration of the enzyme was detected on the intercellular hyphal wall and in the hyphal cytoplasm. In the compatible interaction, β-1,3-glucanase was demonstrated to accumulate predominantly in the haustorial wall and extrahaustorial matrix. In the incompatible interaction, strong labelling for β-1,3-glucanase was found in host cell wall appositions, in the extracellular matrix in the intercellular space, and in electron-dense structures of host origin which occurred in the incompatible interaction only. Received: 22 July 1997 / Accepted: 16 August 1997     

Active ROP2 GTPase inhibits ABA- and CO2-induced stomatal closure

ROP GTPases function as molecular switches in diverse cellular processes. Previously, we showed that ROP2 GTPase is activated upon light irradiation, and thereby negatively regulates light-induced stomatal opening. Here we studied the role of ROP2 during stomatal closure. The expression of a constitutively active form of ROP2 (CA-rop2) in Arabidopsis thaliana and Vicia faba resulted in slower and reduced stomatal closure in response to abscisic acid (ABA) and CO(2) . In contrast, the expression of a dominant-negative form of ROP2 (DN-rop2) and the knockout mutation of ROP2 (rop2 KO) promoted ABA-induced stomatal closure in Arabidopsis. As early as 10 min after ABA treatment, ROP2 was inactivated and translocated to the cytoplasm of the stomatal guard cells. To elucidate the mechanism by which active ROP2 suppresses stomatal closure, we monitored endocytotic membrane trafficking, which is regulated by Rho GTPases in animal cells. We found that the endocytosis of plasma membrane (PM), as tracked by FM4-64, was lower in CA-rop2-expressing guard cells than in those of wild-type plants, which suggests that active ROP2 suppresses the endocytotic internalization of PM, a process required for stomatal closure. Together, our results suggest that ROP2 is inactivated by ABA, and that this inactivation is required for the timely stomatal closure.     

Guard cell photosynthesis is critical for stomatal turgor production,yet does not directly mediate CO2‐ and ABA‐induced stomatal closing

Stomata mediate gas exchange between the inter‐cellular spaces of leaves and the atmosphere. CO₂ levels in leaves (Ci) are determined by respiration, photosynthesis, stomatal conductance and atmospheric [CO₂]. [CO₂] in leaves mediates stomatal movements. The role of guard cell photosynthesis in stomatal conductance responses is a matter of debate, and genetic approaches are needed. We have generated transgenic Arabidopsis plants that are chlorophyll‐deficient in guard cells only, expressing a constitutively active chlorophyllase in a guard cell specific enhancer trap line. Our data show that more than 90% of guard cells were chlorophyll‐deficient. Interestingly, approximately 45% of stomata had an unusual, previously not‐described, morphology of thin‐shaped chlorophyll‐less stomata. Nevertheless, stomatal size, stomatal index, plant morphology, and whole‐leaf photosynthetic parameters (PSII, qP, qN, F_V′/F_M′) were comparable with wild‐type plants. Time‐resolved intact leaf gas‐exchange analyses showed a reduction in stomatal conductance and CO₂‐assimilation rates of the transgenic plants. Normalization of CO₂ responses showed that stomata of transgenic plants respond to [CO₂] shifts. Detailed stomatal aperture measurements of normal kidney‐shaped stomata, which lack chlorophyll, showed stomatal closing responses to [CO₂] elevation and abscisic acid (ABA), while thin‐shaped stomata were continuously closed. Our present findings show that stomatal movement responses to [CO₂] and ABA are functional in guard cells that lack chlorophyll. These data suggest that guard cell CO₂ and ABA signal transduction are not directly modulated by guard cell photosynthesis/electron transport. Moreover, the finding that chlorophyll‐less stomata cause a ‘deflated’ thin‐shaped phenotype, suggests that photosynthesis in guard cells is critical for energization and guard cell turgor production.