Zhen-Ming Pei

Calcium channels activated by hydrogen peroxide mediate abscisic acid signalling in guard cells.

Drought is a major threat to agricultural production. Plants synthesize the hormone abscisic acid (ABA) in response to drought, triggering a signalling cascade in guard cells that results in stomatal closure, thus reducing water loss. ABA triggers an increase in cytosolic calcium in guard cells ([Ca2+]cyt) that has been proposed to include Ca2+ influx across the plasma membrane. However, direct recordings of Ca 2+ currents have been limited and the upstream activation mechanisms of plasma membrane Ca2+ channels remain unknown. Here we report activation of Ca2+-permeable channels in the plasma membrane of Arabidopsis guard cells by hydrogen peroxide. The H2O2-activated Ca2+ channels mediate both influx of Ca2+ in protoplasts and increases in [Ca 2+]cyt in intact guard cells. ABA induces the production of H2O2 in guard cells. If H2O2 production is blocked, ABA-induced closure of stomata is inhibited. Moreover, activation of Ca2+ channels by H2O2 and ABA- and H2O2-induced stomatal closing are disrupted in the recessive ABA-insensitive mutant gca2. These data indicate that ABA-induced H2O2 production and the H2O 2-activated Ca2+ channels are important mechanisms for ABA-induced stomatal closing.

NADPH oxidase AtrbohD and AtrbohF genes function in ROS-dependent ABA signaling in arabidopsis

Reactive oxygen species (ROS) have been proposed to function as second messengers in abscisic acid (ABA) signaling in guard cells. However, the question whether ROS production is indeed required for ABA signal transduction in vivo has not yet been addressed, and the molecular mechanisms mediating ROS production during ABA signaling remain unknown. Here, we report identification of two partially redundant Arabidopsis guard cell‐expressed NADPH oxidase catalytic subunit genes, AtrbohD and AtrbohF, in which gene disruption impairs ABA signaling. atrbohD/F double mutations impair ABA‐induced stomatal closing, ABA promotion of ROS production, ABA‐induced cytosolic Ca2+ increases and ABA‐ activation of plasma membrane Ca2+‐permeable channels in guard cells. Exogenous H2O2 rescues both Ca2+ channel activation and stomatal closing in atrbohD/F. ABA inhibition of seed germination and root elongation are impaired in atrbohD/F, suggesting more general roles for ROS and NADPH oxidases in ABA signaling. These data provide direct molecular genetic and cell biological evidence that ROS are rate‐limiting second messengers in ABA signaling, and that the AtrbohD and AtrbohF NADPH oxidases function in guard cell ABA signal transduction.

Differential abscisic acid regulation of guard cell slow anion channels in Arabidopsis wild-type and abi1 and abi2 mutants.

Abscisic acid (ABA) regulates vital physiological responses, and a number of events in the ABA signaling cascade remain to be identified. To allow quantitative analysis of genetic signaling mutants, patch-clamp experiments were developed and performed with the previously inaccessible Arabidopsis guard cells from the wild type and ABA-insensitive (abi) mutants. Slow anion channels have been proposed to play a rate-limiting role in ABA-induced stomatal closing. We now directly demonstrate that ABA strongly activates slow anion channels in wild-type guard cells. Furthermore, ABA-induced anion channel activation and stomatal closing were suppressed by protein phosphatase inhibitors. In abi1-1 and abi2-1 mutant guard cells, ABA activation of slow anion channels and ABA-induced stomatal closing were abolished. These impairments in ABA signaling were partially rescued by kinase inhibitors in abi1 but not in abi2 guard cells. These data provide cell biological evidence that the abi2 locus disrupts early ABA signaling, that abi1 and abi2 affect ABA signaling at different steps in the cascade, and that protein kinases act as negative regulators of ABA signaling in Arabidopsis. New models for ABA signaling pathways and roles for abi1, abi2, and protein kinases and phosphatases are discussed.

Roles of Ion Channels in Initiation of Signal Transduction in Higher Plants.

All biological organisms perceive environmental and chemical signals via specific receptors or perception mechanisms. When stimulated, these receptors induce an intracellular cascade of events leading to modification of cellular activity or to regulation of specific genes, which in turn produces the biological response. Recently, progress has been made in identifying initial signal reception mechanisms and early events in signaling cascades in higher plants. lon channels, along with intracellular signaling proteins and second messengers, are critical components mediating early events in higher plant signal transduction. lon channel-mediated signal transduction in higher plants has notable differences from signaling mechanisms in animal systems. Of the many types of ion channels found in higher plants, recent findings have indicated that anion channels, along with Ca2+ channels, play critical and rate-limiting roles in the mediation of early events of signal transduction. We have now begun to obtain the first insights into the modes of regulation, membrane localization, and, in the case of K+ channels, molecular structure of higher plant ion channels. In this article, we focus mainly on nove1 findings concerning the function and regulation of anion and Caz+ channels and outline testable models of their involvement in signal transduction. Our objective is not only to summarize these findings but also to point out the many open questions involving early events in plant signal transduction. To illustrate the functions of higher plant ion channels in the initiation of signaling cascades, in the first section we discuss the molecular mechanisms of abscisic acid (ABA)-induced stomatal closing, with a special focus on new and emerging concepts. In the second section, we address Ca2+-dependent and Ca2+-independent signaling processes in plants and analyze certain putative parallels between initial guard cell signaling and both the initiation of defense responses and phytochrome-induced signaling.

A cell surface receptor mediates extracellular Ca 2+ sensing in guard cells

Extracellular Ca2+ (Ca2+o) is required for various physiological and developmental processes in animals and plants. In response to varied Ca2+o levels, plants maintain relatively constant internal Ca2+ content, suggesting a precise regulatory mechanism for Ca2+ homeostasis. However, little is known about how plants monitor Ca2+o status and whether Ca2+o-sensing receptors exist. The effects of Ca2+o on guard cells in promoting stomatal closure by inducing increases in the concentration of cytosolic Ca2+ ([Ca2+]i) provide a clue to Ca2+o sensing. Here we have used a functional screening assay in mammalian cells to isolate an Arabidopsis complementary DNA clone encoding a Ca2+-sensing receptor, CAS. CAS is localized to the plasma membrane, exhibits low-affinity/high-capacity Ca2+ binding, and mediates Ca2+o-induced [Ca2+]i increases. CAS is expressed predominantly in the shoot, including guard cells. Repression of CAS disrupts Ca2+o signalling in guard cells, and impairs bolting (swift upward growth at the transition to seed production) in response to Ca2+ deficiency, so we conclude that CAS may be a primary transducer of Ca2+o in plants.

A novel chloride channel in Vicia faba guard cell vacuoles activated by the serine/threonine kinase, CDPK.

Calcium‐Dependent Protein Kinases (CDPKs) in higher plants contain a C‐terminal calmodulin‐like regulatory domain. Little is known regarding physiological CDPK targets. Both kinase activity and multiple Ca2+‐dependent signaling pathways have been implicated in the control of stomatal guard cell movements. To determine whether CDPK or other protein kinases could have a role in guard cell signaling, purified and recombinant kinases were applied to Vicia faba guard cell vacuoles during patch‐clamp experiments. CDPK activated novel vacuolar chloride (VCL) and malate conductances in guard cells. Activation was dependent on both Ca2+ and ATP. Furthermore, VCL activation occurred in the absence of Ca2+ using a Ca2+‐independent, constitutively active, CDPK* mutant. Protein kinase A showed weaker activation (22% as compared with CDPK). Current reversals in whole vacuole recordings shifted with the Nernst potential for Cl‐and vanished in glutamate. Single channel recordings showed a CDPK‐activated 34 +/− 5 pS Cl‐ channel. VCL channels were activated at physiological potentials enabling Cl‐ uptake into vacuoles. VCL channels may provide a previously unidentified, but necessary, pathway for anion uptake into vacuoles required for stomatal opening. CDPK‐activated VCL currents were also observed in red beet vacuoles suggesting that these channels may provide a more general mechanism for kinase‐dependent anion uptake.

OSCA1 mediates osmotic-stress-evoked Ca2+ increases vital for osmosensing in Arabidopsis

Water is crucial to plant growth and development. Environmental water deficiency triggers an osmotic stress signalling cascade, which induces short-term cellular responses to reduce water loss and long-term responses to remodel the transcriptional network and physiological and developmental processes. Several signalling components that have been identified by extensive genetic screens for altered sensitivities to osmotic stress seem to function downstream of the perception of osmotic stress. It is known that hyperosmolality and various other stimuli trigger increases in cytosolic free calcium concentration ([Ca2+]i). Considering that in bacteria and animals osmosensing Ca2+ channels serve as osmosensors, hyperosmolality-induced [Ca2+]i increases have been widely speculated to be involved in osmosensing in plants. However, the molecular nature of corresponding Ca2+ channels remain unclear. Here we describe a hyperosmolality-gated calcium-permeable channel and its function in osmosensing in plants. Using calcium-imaging-based unbiased forward genetic screens we isolated Arabidopsis mutants that exhibit low hyperosmolality-induced [Ca2+]i increases. These mutants were rescreened for their cellular, physiological and developmental responses to osmotic stress, and those with clear combined phenotypes were selected for further physical mapping. One of the mutants, reduced hyperosmolality-induced [Ca2+]i increase 1 (osca1), displays impaired osmotic Ca2+ signalling in guard cells and root cells, and attenuated water transpiration regulation and root growth in response to osmotic stress. OSCA1 is identified as a previously unknown plasma membrane protein and forms hyperosmolality-gated calcium-permeable channels, revealing that OSCA1 may be an osmosensor. OSCA1 represents a channel responsible for [Ca2+]i increases induced by a stimulus in plants, opening up new avenues for studying Ca2+ machineries for other stimuli and providing potential molecular genetic targets for engineering drought-resistant crops.

Coupling diurnal cytosolic Ca2+ oscillations to the CAS-IP3 pathway in Arabidopsis

Various signaling pathways rely on changes in cytosolic calcium ion concentration ([Ca2+]i). In plants, resting [Ca2+]i oscillates diurnally. We show that in Arabidopsis thaliana, [Ca2+]i oscillations are synchronized to extracellular Ca2+ concentration ([Ca2+]o) oscillations largely through the Ca2+-sensing receptor CAS. CAS regulates concentrations of inositol 1,4,5-trisphosphate (IP3), whichinturndirects release of Ca2+ from internal stores. The oscillating amplitudes of [Ca2+]o and [Ca2+]i are controlled by soil Ca2+ concentrations and transpiration rates. The phase and period of oscillations are likely determined by stomatal conductance. Thus, the internal concentration of Ca2+ in plant cells is constantly being actively revised.

Hydrogen sulphide enhances photosynthesis through promoting chloroplast biogenesis, photosynthetic enzyme expression, and thiol redox modification in Spinacia oleracea seedlings

Hydrogen sulphide (H(2)S) is emerging as a potential messenger molecule involved in modulation of physiological processes in animals and plants. In this report, the role of H(2)S in modulating photosynthesis of Spinacia oleracea seedlings was investigated. The main results are as follows. (i) NaHS, a donor of H(2)S, was found to increase the chlorophyll content in leaves. (ii) Seedlings treated with different concentrations of NaHS for 30 d exhibited a significant increase in seedling growth, soluble protein content, and photosynthesis in a dose-dependent manner, with 100 μM NaHS being the optimal concentration. (iii) The number of grana lamellae stacking into the functional chloroplasts was also markedly increased by treatment with the optimal NaHS concentration. (iv) The light saturation point (Lsp), maximum net photosynthetic rate (Pmax), carboxylation efficiency (CE), and maximal photochemical efficiency of photosystem II (F(v)/F(m)) reached their maximal values, whereas the light compensation point (Lcp) and dark respiration (Rd) decreased significantly under the optimal NaHS concentration. (v) The activity of ribulose-1,5-bisphosphate carboxylase (RuBISCO) and the protein expression of the RuBISCO large subunit (RuBISCO LSU) were also significantly enhanced by NaHS. (vi) The total thiol content, glutathione and cysteine levels, internal concentration of H(2)S, and O-acetylserine(thiol)lyase and L-cysteine desulphydrase activities were increased to some extent, suggesting that NaHS also induced the activity of thiol redox modification. (vii) Further studies using quantitative real-time PCR showed that the gene encoding the RuBISCO large subunit (RBCL), small subunit (RBCS), ferredoxin thioredoxin reductase (FTR), ferredoxin (FRX), thioredoxin m (TRX-m), thioredoxin f (TRX-f), NADP-malate dehydrogenase (NADP-MDH), and O-acetylserine(thiol)lyase (OAS) were up-regulated, but genes encoding serine acetyltransferase (SERAT), glycolate oxidase (GYX), and cytochrome oxidase (CCO) were down-regulated after exposure to the optimal concentration of H(2)S. These findings suggest that increases in RuBISCO activity and the function of thiol redox modification may underlie the amelioration of photosynthesis and that H(2)S plays an important role in plant photosynthesis regulation by modulating the expression of genes involved in photosynthesis and thiol redox modification.

Exploring the Mechanism of Physcomitrella patens Desiccation Tolerance through a Proteomic Strategy

The moss Physcomitrella patens has been shown to tolerate abiotic stresses, including salinity, cold, and desiccation. To better understand this plants mechanism of desiccation tolerance, we have applied cellular and proteomic analyses. Gametophores were desiccated over 1 month to 10% of their original fresh weight. We report that during the course of dehydration, several related processes are set in motion: plasmolysis, chloroplast remodeling, and microtubule depolymerization. Despite the severe desiccation, the membrane system maintains integrity. Through two-dimensional gel electrophoresis and image analysis, we identified 71 proteins as desiccation responsive. Following identification and functional categorization, we found that a majority of the desiccation-responsive proteins were involved in metabolism, cytoskeleton, defense, and signaling. Degradation of cytoskeletal proteins might result in cytoskeletal disassembly and consequent changes in the cell structure. Late embryogenesis abundant proteins and reactive oxygen species-scavenging enzymes are both prominently induced, and they might help to diminish the damage brought by desiccation.Bryophytes as the first land plants are believed to have colonized the land from a fresh water origin, requiring adaptive mechanisms that survival of dehydration. Physcomitrella patens is such a non-vascular bryophyte and shows rare desiccation tolerance in its vegetative tissues. Previous studies showed that during the course of dehydration, several related processes are set in motion: plasmolysis, chloroplast remodeling and microtubule depolymerization. And proteomic alteration supported the cellular structural changes in respond to desiccation stress. In this addendum, we report that Golgi bodies are absent and adaptor protein complex AP-1 large subunit is downregulated during the course of dehydration. Those phenomena may be adverse in protein posttranslational modification, protein sorting and cell walls synthesis under the desiccation condition.