Intracellular localization of integrin-like protein and its roles in osmotic stress-induced abscisic acid biosynthesis in Zea mays

Summary. Plants have evolved many mechanisms to cope with adverse environmental stresses. Abscisic acid (ABA) accumulates significantly in plant cells in response to drought conditions, and this is believed to be a major mechanism through which plants enhance drought tolerance. In this study, we explore the possible mechanisms of osmotic stress perception by plant cells and the consequent induction of ABA biosynthesis. Immunoblotting and immunofluorescence localization experiments, using a polyclonal antibody against human integrin β1, revealed the presence of a protein in Zea mays roots that is similar to the integrin proteins of animals and mainly localized in the plasma membrane. Treatment with GRGDS, a synthetic pentapeptide containing an RGD domain, which interacted specifically with the integrin protein and thus blocked the cell wall–plasma membrane interaction, significantly inhibited osmotic stress-induced ABA biosynthesis in cells, and the GRGDS analog which does not contain the RGD domain had no effect. Our results show that a strong interaction exists between the cell wall and plasma membrane and that this interaction is largely mediated by integrin-like proteins. They also imply that the cell wall and/or cell wall–plasma membrane interaction plays important roles in the perception of osmotic stress. Accordingly, we conclude that the cell wall and/or cell wall–plasma membrane interaction mediated by the integrin-like protein plays important roles in osmotic stress-induced ABA biosynthesis in Zea mays. Correspondence: J. S. Liang, College of Bioscience and Biotechnology, Yangzhou University, Yangzhou 225009, People’s Republic of China.     

Cell biological mechanism for triggering of ABA accumulation under water stress inVicia faba leaves

Water stress-induced ABA accumulation is a cellular signaling process from water stress perception to activation of genes encoding key enzymes of ABA biosynthesis, of which the water stress-signal perception by cells or triggering mechanism of the ABA accumulation is the center in the whole process of ABA related-stress signaling in plants. The cell biological mechanism for triggering of ABA accumulation under water stress was studied in leaves ofVicia faba. Mannitol at 890 mmol ·kg^-1 osmotic concentration induced an increase of more than 5 times in ABA concentration in detached leaf tissues, but the same concentration of mannitol only induced an increase of less than 40 % in ABA concentration in protoplasts. Like in detached leaf tissues, ABA concentration in isolated cells increased more than 10 times under the treatment of mannitol at 890 mmol · kg^-1 concentration, suggesting that the interaction between plasmalemma and cell wall was essential to triggering of the water stress-induced ABA accumulation. Neither Ca²⁺-chelating agent EGTA nor Ca²⁺channel activator A23187 nor the two cytoskeleton inhibitors, colchicine and cytochalasin B, had any effect on water stress-induced ABA accumulation. Interestingly water stress-induced ABA accumulation was effectively inhibited by a non-plasmalemma-permeable sulfhydryl-modifier PCMBS (p-chloromercuriphenyl-sulfonic acid), suggesting that plasmalemma protein(s) may be involved in the triggering of water stress-induced ABA accumulation, and the protein may contain sulfhydryl group at its function domain.     

Cell biological mechanism for triggering of ABA accumulation under water stress inVicia faba leaves

Water stress-induced ABA accumulation is a cellular signaling process from water stress perception to activation of genes encoding key enzymes of ABA biosynthesis, of which the water stress-signal perception by cells or triggering mechanism of the ABA accumulation is the center in the whole process of ABA related-stress signaling in plants. The cell biological mechanism for triggering of ABA accumulation under water stress was studied in leaves ofVicia faba. Mannitol at 890 mmol ·kg^-1 osmotic concentration induced an increase of more than 5 times in ABA concentration in detached leaf tissues, but the same concentration of mannitol only induced an increase of less than 40 % in ABA concentration in protoplasts. Like in detached leaf tissues, ABA concentration in isolated cells increased more than 10 times under the treatment of mannitol at 890 mmol · kg^-1 concentration, suggesting that the interaction between plasmalemma and cell wall was essential to triggering of the water stress-induced ABA accumulation. Neither Ca²⁺-chelating agent EGTA nor Ca²⁺channel activator A23187 nor the two cytoskeleton inhibitors, colchicine and cytochalasin B, had any effect on water stress-induced ABA accumulation. Interestingly water stress-induced ABA accumulation was effectively inhibited by a non-plasmalemma-permeable sulfhydryl-modifier PCMBS (p-chloromercuriphenyl-sulfonic acid), suggesting that plasmalemma protein(s) may be involved in the triggering of water stress-induced ABA accumulation, and the protein may contain sulfhydryl group at its function domain.     

The plant cuticle is required for osmotic stress regulation of abscisic acid biosynthesis and osmotic stress tolerance in Arabidopsis

Osmotic stress activates the biosynthesis of abscisic acid (ABA). One major step in ABA biosynthesis is the carotenoid cleavage catalyzed by a 9-cis epoxycarotenoid dioxygenase (NCED). To understand the mechanism for osmotic stress activation of ABA biosynthesis, we screened for Arabidopsis thaliana mutants that failed to induce the NCED3 gene expression in response to osmotic stress treatments. The ced1 (for 9-cis epoxycarotenoid dioxygenase defective 1) mutant isolated in this study showed markedly reduced expression of NCED3 in response to osmotic stress (polyethylene glycol) treatments compared with the wild type. Other ABA biosynthesis genes are also greatly reduced in ced1 under osmotic stress. ced1 mutant plants are very sensitive to even mild osmotic stress. Map-based cloning revealed unexpectedly that CED1 encodes a putative α/β hydrolase domain-containing protein and is allelic to the BODYGUARD gene that was recently shown to be essential for cuticle biogenesis. Further studies discovered that other cutin biosynthesis mutants are also impaired in osmotic stress induction of ABA biosynthesis genes and are sensitive to osmotic stress. Our work demonstrates that the cuticle functions not merely as a physical barrier to minimize water loss but also mediates osmotic stress signaling and tolerance by regulating ABA biosynthesis and signaling.     

AT14A mediates the cell wall-plasma membrane-cytoskeleton continuum in Arabidopsis thaliana cells

AT14A has a small domain that has sequence similarities to integrins from animals. Integrins serve as a transmembrane linker between the extracellular matrix and the cytoskeleton, which play critical roles in a variety of biological processes. Because the function of AT14A is unknown, Arabidopsis thaliana AT14A, which is a transmembrane receptor for cell adhesion molecules and a middle member of the cell wall-plasma membrane-cytoskeleton continuum in plants, has been described. AT14A, co-expressed with green fluorescent protein (GFP), was found to localize mainly to the plasma membrane. The mutant Arabidopsis at14a-1 cells exhibit various phenotypes with cell shape, cell cluster size, thickness, and cellulose content of cell wall, the adhesion between cells, and the adhesion of plasma membrane to cell wall varied by plasmolysis. Using direct staining of filamentous actin and indirect immunofluorescence staining of microtubules, cortical actin filaments and microtubules arrays were significantly altered in cells, either where AT14A was absent or over-expressed. It is concluded that AT14A may be a substantial middle member of the cell wall-plasma membrane-cytoskeleton continuum and play an important role in the continuum by regulating cell wall and cortical cytoskeleton organization.     

Lectin receptor kinases participate in protein-protein interactions to mediate plasma membrane-cell wall adhesions in Arabidopsis

Interactions between plant cell walls and plasma membranes are essential for cells to function properly, but the molecules that mediate the structural continuity between wall and membrane are unknown. Some of these interactions, which are visualized upon tissue plasmolysis in Arabidopsis (Arabidopsis thaliana), are disrupted by the RGD (arginine-glycine-aspartic acid) tripeptide sequence, a characteristic cell adhesion motif in mammals. In planta induced-O (IPI-O) is an RGD-containing protein from the plant pathogen Phytophthora infestans that can disrupt cell wall-plasma membrane adhesions through its RGD motif. To identify peptide sequences that specifically bind the RGD motif of the IPI-O protein and potentially play a role in receptor recognition, we screened a heptamer peptide library displayed in a filamentous phage and selected two peptides acting as inhibitors of the plasma membrane RGD-binding activity of Arabidopsis. Moreover, the two peptides also disrupted cell wall-plasma membrane adhesions. Sequence comparison of the RGD-binding peptides with the Arabidopsis proteome revealed 12 proteins containing amino acid sequences in their extracellular domains common with the two RGD-binding peptides. Eight belong to the receptor-like kinase family, four of which have a lectin-like extracellular domain. The lectin domain of one of these, At5g60300, recognized the RGD motif both in peptides and proteins. These results imply that lectin receptor kinases are involved in protein-protein interactions with RGD-containing proteins as potential ligands, and play a structural and signaling role at the plant cell surfaces.     

The calcium sensor calcineurin B-like 9 modulates abscisic acid sensitivity and biosynthesis in Arabidopsis

Calcium plays a pivotal role in plant responses to several stimuli, including pathogens, abiotic stresses, and hormones. However, the molecular mechanisms underlying calcium functions are poorly understood. It is hypothesized that calcium serves as second messenger and, in many cases, requires intracellular protein sensors to transduce the signal further downstream in the pathways. The calcineurin B-like proteins (CBLs) represent a unique family of calcium sensors in plant cells. Here, we report our analysis of the CBL9 member of this gene family. Expression of CBL9 was inducible by multiple stress signals and abscisic acid (ABA) in young seedlings. When CBL9 gene function was disrupted in Arabidopsis thaliana plants, the responses to ABA were drastically altered. The mutant plants became hypersensitive to ABA in the early developmental stages, including seed germination and post-germination seedling growth. In addition, seed germination in the mutant also showed increased sensitivity to inhibition by osmotic stress conditions produced by high concentrations of salt and mannitol. Further analyses indicated that increased stress sensitivity in the mutant may be a result of both ABA hypersensitivity and increased accumulation of ABA under the stress conditions. The cbl9 mutant plants showed enhanced expression of genes involved in ABA signaling, such as ABA-INSENSITIVE 4 and 5. This study has identified a calcium sensor as a common element in the ABA signaling and stress-induced ABA biosynthesis pathways.     

In vivo FRET–FLIM reveals ER-specific increases in the ABA level upon environmental stresses

Plant hormone abscisic acid (ABA) is essential for regulating plant growth and various stress responses. ABA-mediated signaling depends on local ABA levels rather than the overall cellular ABA concentration. While cellular concentration of ABA can be detected using Förster resonance energy transfer (FRET)-based ABA probes, direct imaging of subcellular ABA levels remains unsolved. Here, we modified the previously reported ABAleon2.1 and generated a new ABA sensor, named ABAleon2.1_Tao3. Via transient expression in tobacco (Nicotiana tabacum) protoplasts, we targeted ABAleon2.1_Tao3s to the endoplasmic reticulum (ER) membrane with the ABA sensing unit facing the cytosol and the ER, respectively, through a nanobody–epitope-mediated protein interaction. Combining FRET with fluorescence lifetime imaging microscopy, ABA-triggered-specific increases in the fluorescence lifetime of the donor mTurquoise in the ABAleon2.1_Tao3 were detected in both transient assays and stably transformed Arabidopsis plants. In tobacco protoplasts, ER membrane-targeted ABAleon2.1_Tao3s showed a generally higher basal level of ABA in the ER than that in the cytosol and ER-specific alterations in the level of ABA upon environmental cues. In ABAleon2.1_Tao3-transformed Arabidopsis roots, mannitol triggered increases in cytosolic ABA in the division zone and increases in ER ABA in the elongation and maturation zone within 1 h after treatment, both of which were abolished in the bg1-2 mutant, suggesting the requirement for BG1 in osmotic stress-triggered early ABA induction in Arabidopsis roots. These data demonstrate that ABAleon2.1_Tao3s can be used to monitor ABA levels in the cytosol and the ER, providing key information on stress-induced changes in the level of ABA in different subcellular compartments.

FRET-based ABA sensors targeted to the ER membrane through a nanobody–epitope-mediated protein assembly reveal specific alterations and dynamics of ABA in the ER upon various environmental stimuli.     

玉米根细胞中类整合素蛋白与α-微管蛋白的共定位及其可能的相互作用

采用间接免疫荧光标记法对玉米根细胞中的类整合素蛋白和细胞骨架主要组分之一的α-微管蛋白进行了荧光定位。结果表明：类整合素蛋白主要分布在质膜上。与对照相比,用与类整合素蛋白特异结合的5肽GRGDS处理后,质膜上类整合素的分布更为均匀,微管的排列密度降低,而用不与类整合素蛋白特异结合的GRGDS类似物SDGRG处理则对类整合素蛋白分布和微管蛋白的排列均无明显影响。微管蛋白解聚剂或稳定剂处理改变类整合素在质膜上的分布。这些结果表明类整合素蛋白与微管蛋白间有复杂的相互作用。     

ABA synthesis in protoplasts of different origin in response to osmotic stress

All the protoplasts analysed in this study whatever the original plant material were able to accumulate ABA under osmotic stress. The time course of ABA accumulation strongly differed according to the plant material. In both rose petal or Amaranthus leaf protoplasts, the increase in ABA level was significant but transient. Protoplasts prepared from Amaranthus cell suspensions behaved differently, showing a late and durable accumulation of ABA. Similar patterns of changes in ABA accumulation were observed in the original plant material under osmotic stress. A pretreatment of plant material by fluridone induced a strong inhibition of ABA accumulation whatever the origin of protoplasts was. This result suggests that ABA could be synthesised via the carotenoid pathway in the absence of the cell wall.     

A vacuolar β-glucosidase homolog that possesses glucose-conjugated abscisic acid hydrolyzing activity plays an important role in osmotic stress responses in Arabidopsis

The phytohormone abscisic acid (ABA) plays a critical role in various physiological processes, including adaptation to abiotic stresses. In Arabidopsis thaliana, ABA levels are increased both through de novo biosynthesis and via β-glucosidase homolog1 (BG1)-mediated hydrolysis of Glc-conjugated ABA (ABA-GE). However, it is not known how many different β-glucosidase proteins produce ABA from ABA-GE and how the multiple ABA production pathways are coordinated to increase ABA levels. Here, we report that a previously undiscovered β-glucosidase homolog, BG2, produced ABA by hydrolyzing ABA-GE and plays a role in osmotic stress response. BG2 localized to the vacuole as a high molecular weight complex and accumulated to high levels under dehydration stress. BG2 hydrolyzed ABA-GE to ABA in vitro. In addition, BG2 increased ABA levels in protoplasts upon application of exogenous ABA-GE. Overexpression of BG2 rescued the bg1 mutant phenotype, as observed for the overexpression of NCED3 in bg1 mutants. Multiple Arabidopsis bg2 alleles with a T-DNA insertion in BG2 were more sensitive to dehydration and NaCl stress, whereas BG2 overexpression resulted in enhanced resistance to dehydration and NaCl stress. Based on these observations, we propose that, in addition to the de novo biosynthesis, ABA is produced in multiple organelles by organelle-specific β-glucosidases in response to abiotic stresses.     

The coronatine-insensitive 1 mutation reveals the hormonal signaling interaction between abscisic acid and methyl jasmonate in Arabidopsis guard cells. Specific impairment of ion channel activation and second messenger production

Methyl jasmonate (MeJA) elicits stomatal closing similar to abscisic acid (ABA), but whether the two compounds use similar or different signaling mechanisms in guard cells remains to be clarified. We investigated the effects of MeJA and ABA on second messenger production and ion channel activation in guard cells of wild-type Arabidopsis (Arabidopsis thaliana) and MeJA-insensitive coronatine-insensitive 1 (coi1) mutants. The coi1 mutation impaired MeJA-induced stomatal closing but not ABA-induced stomatal closing. MeJA as well as ABA induced production of reactive oxygen species (ROS) and nitric oxide (NO) in wild-type guard cells, whereas MeJA did not induce production of ROS and NO in coi1 guard cells. The experiments using an inhibitor and scavengers demonstrated that both ROS and NO are involved in MeJA-induced stomatal closing as well as ABA-induced stomatal closing. Not only ABA but also MeJA activated slow anion channels and Ca(2+) permeable cation channels in the plasma membrane of wild-type guard cell protoplasts. However, in coi1 guard cell protoplasts, MeJA did not elicit either slow anion currents or Ca(2+) permeable cation currents, but ABA activated both types of ion channels. Furthermore, to elucidate signaling interaction between ABA and MeJA in guard cells, we examined MeJA signaling in ABA-insensitive mutant ABA-insensitive 2 (abi2-1), whose ABA signal transduction cascade has some disruption downstream of ROS production and NO production. MeJA also did not induce stomatal closing but stimulated production of ROS and NO in abi2-1. These results suggest that MeJA triggers stomatal closing via a receptor distinct from the ABA receptor and that the coi1 mutation disrupts MeJA signaling upstream of the blanch point of ABA signaling and MeJA signaling in Arabidopsis guard cells.     

Functional sieve element protoplasts

Sieve element (SE) protoplasts were liberated by exposing excised phloem strands of Vicia faba to cell wall-degrading enzyme mixtures. Two types of SE protoplasts were found: simple protoplasts with forisome inclusions and composite twin protoplasts-two protoplasts intermitted by a sieve plate-of which one protoplast often includes a forisome. Forisomes are giant protein inclusions of SEs in Fabaceae. Membrane integrity of SE protoplasts was tested by application of CFDA, which was sequestered in the form of carboxyfluorescein. Further evidence for membrane intactness was provided by swelling of SE protoplasts and forisome dispersion in reaction to abrupt lowering of medium osmolarity. The absence of cell wall remnants as demonstrated by negative Calcofluor White staining allowed patch-clamp studies. At negative membrane voltages, the current-voltage relations of the SE protoplasts were dominated by a weak inward-rectifying potassium channel that was active at physiological membrane voltages of the SE plasma membrane. This channel had electrical properties that are reminiscent of those of the AKT2/3 channel family, localized in phloem cells of Arabidopsis (Arabidopsis thaliana). All in all, SE protoplasts promise to be a powerful tool in studying the membrane biology of SEs with inherent implications for the understanding of long-distance transport and signaling.     

Phosphorylation of the inward-rectifying potassium channel KAT1 by ABR kinase in Vicia guard cells

A 48-kDa protein kinase was detected in Vicia faba guard cell protoplasts by an in-gel protein kinase assay using a recombinant peptide (KAT1C) of the carboxyl-terminus of an inward-rectifying voltage-dependent K+ channel cloned from Arabidopsis thaliana, KAT1. This protein kinase (ABR* kinase) was activated by pretreatment of guard cell protoplasts with ABA, but not by pretreatment with IAA, 2,4-D, kinetin or GA3. The activation of ABR* kinase was dependent on the time and concentration of ABA. The kinase activity was sensitive to staurosporine and K-252a, protein kinase inhibitors, and insensitive to Ca2+. No ABR* kinase activity was detected in mesophyll cell protoplasts. These characteristics of ABR* kinase are consistent with those of an ABA-responsive protein kinase (ABR kinase) reported previously [Mori and Muto (1997), Plant Physiol. 113: 833]. These results indicate that ABR* kinase phosphorylates the inward-rectifying K+ channel in response to treatment of stomatal guard cells with ABA. The data reported here provide evidence that this ABA-responsive protein kinase may promote ABA signaling by directly phosphorylating guard cell ion channels.     

Abscisic acid-regulated responses of aba2-1 under osmotic stress: the abscisic acid-inducible antioxidant defence system and reactive oxygen species production

We investigated the interaction among abscisic acid (ABA), reactive oxygen species (ROS) and antioxidant defence system in the transduction of osmotic stress signalling using Arabidopsis thaliana WT (Columbia ecotype, WT) and an ABA-deficient mutant (aba2-1). For this, 50 μm ABA and osmotic stress, induced with 40% (w/v) polyethylene glycol (PEG8000; -0.7 MPa), were applied to WT and aba2-1 for 6, 12 or 24 h. Time course analysis was undertaken for determination of total/isoenzyme activity of the antioxidant enzymes, superoxide dismutase (SOD; EC 1.15.1.1), catalase (CAT; EC 1.11.1.6), ascorbate peroxidase (APX; EC 1.11.1.11), NADPH oxidase (NOX; EC 1.6.3.1) activity; scavenging activity of the hydroxyl radical (OH˙), hydrogen peroxide (H(2) O(2) ); endogenous ABA and malondialdehyde (MDA). The highest H(2) O(2) and MDA content was found in PEG-treated groups of both genotypes, but with more in aba2-1. ABA treatment under stress reduced the accumulation of H(2) O(2) and MDA, while it promoted activity of SOD, CAT and APX. APX activity was higher than CAT activity in ABA-treated WT and aba2-1, indicating a protective role of APX rather than CAT during osmotic stress-induced oxidative damage. Treatment with ABA also significantly induced increased NOX activity. Oxidative damage was lower in ABA-treated seedlings of both genotypes, which was associated with greater activity of SOD (Mn-SOD1 and 2 and Fe-SOD isoenzymes), CAT and APX in these seedlings after 24 h of stress. These results suggest that osmotic stress effects were overcome by ABA treatment because of increased SOD, CAT, APX and NOX.     

Regulation of Arabidopsis thaliana Ku genes at different developmental stages under heat stress

Ku, a heterodimeric protein consisting of 70- and 80-kDa subunits, is involved in many cellular processes, such as DNA replication, cell cycle regulation and heat shock response. Moreover, the expression of Arabidopsis thaliana Ku genes (AtKu) is modulated by certain plant hormones through several signal transduction pathways. This study investigated how AtKu are regulated by heat stress. AtKu expression in 3-week-old young seedlings was down-regulated by heat stress in a time-dependent manner, as examined using real-time quantitative PCR, GUS reporter systems, and western blotting analysis. Additionally, the heat-induced repression of AtKu was mediated through the abscisic acid (ABA) biosynthetic pathway, as shown by the reversal of AtKu suppression in the ABA biosynthesis mutant, aba3, and by an increase in the ABA level as analyzed by reverse-phase high performance liquid chromatography. Heat stress-induced regulation of AtKu repression also involved ethylene signaling, DNA repair pathways, and fatty acid synthesis. Furthermore, AtKu expression was repressed in stems, rosette leaves, and cauline leaves in 4-5-week-old plants under heat stress, whereas it remained unchanged in roots and primary inflorescence, indicating that heat differentially modulated AtKu expression in distinct tissues of Arabidopsis.     

Expansins are involved in cell growth mediated by abscisic acid and indole-3-acetic acid under drought stress in wheat

Expansin protein is a component of the cell wall generally accepted to be the key regulator of cell wall extension during plant growth. Plant hormones regulate expansin gene expression as well as plant growth during drought stress. However, the relationship between expansin and plant hormone is far from clear. Here, we studied the involvement of expansin in plant cell growth mediated by the hormones indole-3-acetic acid (IAA) and abscisic acid (ABA) under osmotic stress which was induced by polyethylene glycol (PEG)-6000. Wheat coleoptiles from a drought-resistant cultivar HF9703 and a drought-sensitive cultivar 921842 were used to evaluate cell growth and expansin activity. Osmotic stress induced the accumulation of ABA. ABA induced expansin activity mainly by enhancing expansin expression, since ABA induced cell wall basification via decreasing plasma membrane H⁺-ATPase activity, which was unfavorable for expansin activity. Although ABA induced expansin activity and cell wall extension, treatment with exogenous ABA and/or fluridone (FLU, an ABA inhibitor) suggested that ABA was involved in the coleoptile growth inhibition during osmotic stress. IAA application to detached coleoptiles also enhanced coleoptile growth and increased expansin activity, but unlike ABA, IAA-induced expansin activity was mainly due to the decrease of cell wall pH by increasing plasma membrane H⁺-ATPase activity. Compared with drought-sensitive cultivar, the drought-resistant cultivar could maintain greater expansin activity and cell wall extension, which was contributive to its resultant faster growth under water stress.