Cell-free translation system with artificial lipid-monolayer particles as a unique tool for characterizing lipid-monolayer binding proteins.

Methods for functional analysis of proteins specifically localizing to lipid monolayers such as rubber particles and lipid droplets are limited. We have succeeded in establishing a system in which artificially prepared lipid monolayer particles are added to a cell-free translation system to confirm the properties of proteins that specifically bind to lipid monolayers in a translation-coupled manner.

CRK2 and C-terminal Phosphorylation of NADPH Oxidase RBOHD Regulate Reactive Oxygen Species Production in Arabidopsis.

Reactive oxygen species (ROS) are important messengers in eukaryotic organisms, and their production is tightly controlled. Active extracellular ROS production by NADPH oxidases in plants is triggered by receptor-like protein kinase-dependent signaling networks. Here, we show that CYSTEINE-RICH RLK2 (CRK2) kinase activity is required for plant growth and CRK2 exists in a preformed complex with the NADPH oxidase RESPIRATORY BURST OXIDASE HOMOLOG D (RBOHD) in Arabidopsis (Arabidopsis thaliana). Functional CRK2 is required for the full elicitor-induced ROS burst, and consequently the crk2 mutant is impaired in defense against the bacterial pathogen Pseudomonas syringae pv tomato DC3000. Our work demonstrates that CRK2 regulates plant innate immunity. We identified in vitro CRK2-dependent phosphorylation sites in the C-terminal region of RBOHD. Phosphorylation of S703 RBOHD is enhanced upon flg22 treatment, and substitution of S703 with Ala reduced ROS production in Arabidopsis. Phylogenetic analysis suggests that phospho-sites in the C-terminal region of RBOHD are conserved throughout the plant lineage and between animals and plants. We propose that regulation of NADPH oxidase activity by phosphorylation of the C-terminal region might be an ancient mechanism and that CRK2 is an important element in regulating microbe-associated molecular pattern-triggered ROS production.

Tonoplast-localized Ca2+ pumps regulate Ca2+ signals during pattern-triggered immunity in Arabidopsis thaliana.

One of the major events of early plant immune responses is a rapid influx of Ca2+ into the cytosol following pathogen recognition. Indeed, changes in cytosolic Ca2+ are recognized as ubiquitous elements of cellular signaling networks and are thought to encode stimulus-specific information in their duration, amplitude, and frequency. Despite the wealth of observations showing that the bacterial elicitor peptide flg22 triggers Ca2+ transients, there remain limited data defining the molecular identities of Ca2+ transporters involved in shaping the cellular Ca2+ dynamics during the triggering of the defense response network. However, the autoinhibited Ca2+-ATPase (ACA) pumps that act to expel Ca2+ from the cytosol have been linked to these events, with knockouts in the vacuolar members of this family showing hypersensitive lesion-mimic phenotypes. We have therefore explored how the two tonoplast-localized pumps, ACA4 and ACA11, impact flg22-dependent Ca2+ signaling and related defense responses. The double-knockout aca4/11 exhibited increased basal Ca2+ levels and Ca2+ signals of higher amplitude than wild-type plants. Both the aberrant Ca2+ dynamics and associated defense-related phenotypes could be suppressed by growing the aca4/11 seedlings at elevated temperatures. Relocalization of ACA8 from its normal cellular locale of the plasma membrane to the tonoplast also suppressed the aca4/11 phenotypes but not when a catalytically inactive mutant was used. These observations indicate that regulation of vacuolar Ca2+ sequestration is an integral component of plant immune signaling, but also that the action of tonoplast-localized Ca2+ pumps does not require specific regulatory elements not found in plasma membrane-localized pumps.

In vivo Imaging Enables Understanding of Seamless Plant Defense Responses to Wounding and Pathogen Attack.

Plants are exposed to varied biotic stresses, including sequential or simultaneous attack by insects and pathogens. To overcome these complex stresses, plants must perceive each of the stresses, then integrate and relay the information throughout the plant body and eventually activate local and systemic resistance responses. Previous molecular genetic studies identified jasmonic acid and salicylic acid as key plant hormones of wound and immune responses. These hormones, combined with their antagonistic interaction, play critical roles in the initiation and regulation of defense responses against insects and pathogens. Aside from molecular and genetic information, the latest in vivo imaging technology has revealed that plant defense responses are regulated spatially and temporally. In this review, we summarize the current knowledge of local and systemic defense responses against wounding and diseases with a focus on past and recent advances in imaging technologies. We discuss how imaging-based multiparametric analysis has improved our understanding of the spatiotemporal regulation of dynamic plant stress responses. We also emphasize the importance of compiling the knowledge generated from individual studies on plant wounding and immune responses for a more seamless understanding of plant defense responses in the natural environment.

CYCLIC NUCLEOTIDE-GATED ION CHANNEL 2 modulates auxin homeostasis and signaling.

Cyclic nucleotide-gated ion channels (CNGCs) have been firmly established as Ca2+-conducting ion channels that regulate a wide variety of physiological responses in plants. CNGC2 has been implicated in plant immunity and Ca2+ signaling due to the autoimmune phenotypes exhibited by null mutants of CNGC2 in Arabidopsis thaliana. However, cngc2 mutants display additional phenotypes that are unique among autoimmune mutants, suggesting that CNGC2 has functions beyond defense and generates distinct Ca2+ signals in response to different triggers. In this study, we found that cngc2 mutants showed reduced gravitropism, consistent with a defect in auxin signaling. This was mirrored in the diminished auxin response detected by the auxin reporters DR5::GUS and DII-VENUS and in a strongly impaired auxin-induced Ca2+ response. Moreover, the cngc2 mutant exhibits higher levels of the endogenous auxin indole-3-acetic acid, indicating that excess auxin in the cngc2 mutant causes its pleiotropic phenotypes. These auxin signaling defects and the autoimmunity syndrome of the cngc2 mutant could be suppressed by loss-of-function mutations in the auxin biosynthesis gene YUCCA6 (YUC6), as determined by identification of the cngc2 suppressor mutant repressor of cngc2 (rdd1) as an allele of YUC6. A loss-of-function mutation in the upstream auxin biosynthesis gene TRYPTOPHAN AMINOTRANSFERASE OF ARABIDOPSIS (TAA1, WEAK ETHYLENE INSENSITIVE8) also suppressed the cngc2 phenotypes, further supporting the tight relationship between CNGC2 and the TRYPTOPHAN AMINOTRANSFERASE OF ARABIDOPSIS-YUCCA -dependent auxin biosynthesis pathway. Taking these results together, we propose that the Ca2+ signal generated by CNGC2 is a part of the negative feedback regulation of auxin homeostasis in which CNGC2 balances cellular auxin perception by influencing auxin biosynthesis.

CRK2 Enhances Salt Tolerance by Regulating Callose Deposition in Connection with PLDα1.

High salinity is an increasingly prevalent source of stress to which plants must adapt. The receptor-like protein kinases, including members of the Cys-rich receptor-like kinase (CRK) subfamily, are a highly expanded family of transmembrane proteins in plants that are largely responsible for communication between cells and the extracellular environment. Various CRKs have been implicated in biotic and abiotic stress responses; however, their functions on a cellular level remain largely uncharacterized. Here we have shown that CRK2 enhances salt tolerance at the germination stage in Arabidopsis (Arabidopsis thaliana) and also modulates root length. We established that functional CRK2 is required for salt-induced callose deposition. In doing so, we revealed a role for callose deposition in response to increased salinity and demonstrated its importance for salt tolerance during germination. Using fluorescently tagged proteins, we observed specific changes in the subcellular localization of CRK2 in response to various stress treatments. Many of CRK2's cellular functions were dependent on phospholipase D activity, as were the subcellular localization changes. Thus, we propose that CRK2 acts downstream of phospholipase D during salt stress, promoting callose deposition and regulating plasmodesmal permeability, and that CRK2 adopts specific stress-dependent subcellular localization patterns that allow it to carry out its functions.

The Arabidopsis LAZY1 Family Plays a Key Role in Gravity Signaling within Statocytes and in Branch Angle Control of Roots and Shoots.

During gravitropism, the directional signal of gravity is perceived by gravity-sensing cells called statocytes, leading to asymmetric distribution of auxin in the responding organs. To identify the genes involved in gravity signaling in statocytes, we performed transcriptome analyses of statocyte-deficient Arabidopsis thaliana mutants and found two candidates from the LAZY1 family, AtLAZY1/LAZY1-LIKE1 (LZY1) and AtDRO3/AtNGR1/LZY2 We showed that LZY1, LZY2, and a paralog AtDRO1/AtNGR2/LZY3 are redundantly involved in gravitropism of the inflorescence stem, hypocotyl, and root. Mutations of LZY genes affected early processes in gravity signal transduction without affecting amyloplast sedimentation. Statocyte-specific expression of LZY genes rescued the mutant phenotype, suggesting that LZY genes mediate gravity signaling in statocytes downstream of amyloplast displacement, leading to the generation of asymmetric auxin distribution in gravity-responding organs. We also found that lzy mutations reversed the growth angle of lateral branches and roots. Moreover, expression of the conserved C-terminal region of LZY proteins also reversed the growth direction of primary roots in the lzy mutant background. In lateral root tips of lzy multiple mutants, asymmetric distribution of PIN3 and auxin response were reversed, suggesting that LZY genes regulate the direction of polar auxin transport in response to gravity through the control of asymmetric PIN3 expression in the root cap columella.

Interplay of Plasma Membrane and Vacuolar Ion Channels, Together with BAK1, Elicits Rapid Cytosolic Calcium Elevations in Arabidopsis during Aphid Feeding.

A transient rise in cytosolic calcium ion concentration is one of the main signals used by plants in perception of their environment. The role of calcium in the detection of abiotic stress is well documented; however, its role during biotic interactions remains unclear. Here, we use a fluorescent calcium biosensor (GCaMP3) in combination with the green peach aphid (Myzus persicae) as a tool to study Arabidopsis thaliana calcium dynamics in vivo and in real time during a live biotic interaction. We demonstrate rapid and highly localized plant calcium elevations around the feeding sites of M. persicae, and by monitoring aphid feeding behavior electrophysiologically, we demonstrate that these elevations correlate with aphid probing of epidermal and mesophyll cells. Furthermore, we dissect the molecular mechanisms involved, showing that interplay between the plant defense coreceptor BRASSINOSTEROID INSENSITIVE-ASSOCIATED KINASE1 (BAK1), the plasma membrane ion channels GLUTAMATE RECEPTOR-LIKE 3.3 and 3.6 (GLR3.3 and GLR3.6), and the vacuolar ion channel TWO-PORE CHANNEL1 (TPC1) mediate these calcium elevations. Consequently, we identify a link between plant perception of biotic threats by BAK1, cellular calcium entry mediated by GLRs, and intracellular calcium release by TPC1 during a biologically relevant interaction.

Using GCaMP3 to Study Ca2+ Signaling in Nicotiana Species.

Ca2+ signaling is a central component of plant biology; however, direct analysis of in vivo Ca2+ levels is experimentally challenging. In recent years, the use of genetically encoded Ca2+ indicators has revolutionized the study of plant Ca2+ signaling, although such studies have been largely restricted to the model plant Arabidopsis. We have developed stable transgenic Nicotiana benthamiana and Nicotiana tabacum lines expressing the single-wavelength fluorescent Ca2+ indicator, GCaMP3. Ca2+ levels in these plants can be imaged in situ using fluorescence microscopy, and these plants can be used qualitatively and semi-quantitatively to evaluate Ca2+ signals in response to a broad array of abiotic or biotic stimuli, such as cold shock or pathogen-associated molecular patterns (PAMPs). Furthermore, these tools can be used in conjunction with well-established N. benthamiana techniques such as virus-induced gene silencing (VIGS) or transient heterologous expression to assay the effects of loss or gain of function on Ca2+ signaling, an approach which we validated via silencing or transient expression of the PAMP receptors FLS2 (Flagellin Sensing 2) or EFR (EF-Tu receptor), respectively. Using these techniques, along with chemical inhibitor treatments, we demonstrate how these plants can be used to elucidate the molecular components governing Ca2+ signaling in response to specific stimuli.

Control of basal jasmonate signalling and defence through modulation of intracellular cation flux capacity.

Unknown mechanisms tightly regulate the basal activity of the wound-inducible defence mediator jasmonate (JA) in undamaged tissues. However, the Arabidopsis fatty acid oxygenation upregulated2 (fou2) mutant in vacuolar two-pore channel 1 (TPC1D454N ) displays high JA pathway activity in undamaged leaves. This mutant was used to explore mechanisms controlling basal JA pathway regulation. fou2 was re-mutated to generate novel 'ouf' suppressor mutants. Patch-clamping was used to examine TPC1 cation channel characteristics in the ouf suppressor mutants and in fou2. Calcium (Ca2+ ) imaging was used to study the effects fou2 on cytosolic Ca2+ concentrations. Six intragenic ouf suppressors with near wild-type (WT) JA pathway activity were recovered and one mutant, ouf8, affected the channel pore. At low luminal calcium concentrations, ouf8 had little detectable effect on fou2. However, increased vacuolar Ca2+ concentrations caused channel occlusion, selectively blocking K+ fluxes towards the cytoplasm. Cytosolic Ca2+ concentrations in unwounded fou2 were found to be lower than in the unwounded WT, but they increased in a similar manner in both genotypes following wounding. Basal JA pathway activity can be controlled solely by manipulating endomembrane cation flux capacities. We suggest that changes in endomembrane potential affect JA pathway activity.

Salt stress-induced Ca2+ waves are associated with rapid, long-distance root-to-shoot signaling in plants.

Their sessile lifestyle means that plants have to be exquisitely sensitive to their environment, integrating many signals to appropriate developmental and physiological responses. Stimuli ranging from wounding and pathogen attack to the distribution of water and nutrients in the soil are frequently presented in a localized manner but responses are often elicited throughout the plant. Such systemic signaling is thought to operate through the redistribution of a host of chemical regulators including peptides, RNAs, ions, metabolites, and hormones. However, there are hints of a much more rapid communication network that has been proposed to involve signals ranging from action and system potentials to reactive oxygen species. We now show that plants also possess a rapid stress signaling system based on Ca(2+) waves that propagate through the plant at rates of up to ∼ 400 µm/s. In the case of local salt stress to the Arabidopsis thaliana root, Ca(2+) wave propagation is channeled through the cortex and endodermal cell layers and this movement is dependent on the vacuolar ion channel TPC1. We also provide evidence that the Ca(2+) wave/TPC1 system likely elicits systemic molecular responses in target organs and may contribute to whole-plant stress tolerance. These results suggest that, although plants do not have a nervous system, they do possess a sensory network that uses ion fluxes moving through defined cell types to rapidly transmit information between distant sites within the organism.

Wortmannin-induced vacuole fusion enhances amyloplast dynamics in Arabidopsis zigzag1 hypocotyls.

Gravitropism in Arabidopsis shoots depends on the sedimentation of amyloplasts in the endodermis, and a complex interplay between the vacuole and F-actin. Gravity response is inhibited in zigzag-1 (zig-1), a mutant allele of VTI11, which encodes a SNARE protein involved in vacuole fusion. zig-1 seedlings have fragmented vacuoles that fuse after treatment with wortmannin, an inhibitor of phosphatidylinositol 3-kinase, and underscore a role of phosphoinositides in vacuole fusion. Using live-cell imaging with a vertical stage microscope, we determined that young endodermal cells below the apical hook that are smaller than 70 µm in length are the graviperceptive cells in dark-grown hypocotyls. This result was confirmed by local wortmannin application to the top of zig-1 hypocotyls, which enhanced shoot gravitropism in zig-1 mutants. Live-cell imaging of zig-1 hypocotyl endodermal cells indicated that amyloplasts are trapped between juxtaposed vacuoles and their movement is severely restricted. Wortmannin-induced fusion of vacuoles in zig-1 seedlings increased the formation of transvacuolar strands, enhanced amyloplast sedimentation and partially suppressed the agravitropic phenotype of zig-1 seedlings. Hypergravity conditions at 10 g were not sufficient to displace amyloplasts in zig-1, suggesting the existence of a physical tether between the vacuole and amyloplasts. Our results overall suggest that vacuole membrane remodeling may be involved in regulating the association of vacuoles and amyloplasts during graviperception.

Amyloplast displacement is necessary for gravisensing in Arabidopsis shoots as revealed by a centrifuge microscope.

The starch-statolith hypothesis proposes that starch-filled amyloplasts act as statoliths in plant gravisensing, moving in response to the gravity vector and signaling its direction. However, recent studies suggest that amyloplasts show continuous, complex movements in Arabidopsis shoots, contradicting the idea of a so-called 'static' or 'settled' statolith. Here, we show that amyloplast movement underlies shoot gravisensing by using a custom-designed centrifuge microscope in combination with analysis of gravitropic mutants. The centrifuge microscope revealed that sedimentary movements of amyloplasts under hypergravity conditions are linearly correlated with gravitropic curvature in wild-type stems. We next analyzed the hypergravity response in the shoot gravitropism 2 (sgr2) mutant, which exhibits neither a shoot gravitropic response nor amyloplast sedimentation at 1 g. sgr2 mutants were able to sense and respond to gravity under 30 g conditions, during which the amyloplasts sedimented. These findings are consistent with amyloplast redistribution resulting from gravity-driven movements triggering shoot gravisensing. To further support this idea, we examined two additional gravitropic mutants, phosphoglucomutase (pgm) and sgr9, which show abnormal amyloplast distribution and reduced gravitropism at 1 g. We found that the correlation between hypergravity-induced amyloplast sedimentation and gravitropic curvature of these mutants was identical to that of wild-type plants. These observations suggest that Arabidopsis shoots have a gravisensing mechanism that linearly converts the number of amyloplasts that settle to the 'bottom' of the cell into gravitropic signals. Further, the restoration of the gravitropic response by hypergravity in the gravitropic mutants that we tested indicates that these lines probably have a functional gravisensing mechanism that is not triggered at 1 g.

Analyses of a gravistimulation-specific Ca2+ signature in Arabidopsis using parabolic flights.

Gravity is a critical environmental factor affecting the morphology and functions of organisms on the Earth. Plants sense changes in the gravity vector (gravistimulation) and regulate their growth direction accordingly. In Arabidopsis (Arabidopsis thaliana) seedlings, gravistimulation, achieved by rotating the specimens under the ambient 1g of the Earth, is known to induce a biphasic (transient and sustained) increase in cytoplasmic calcium concentration ([Ca(2+)]c). However, the [Ca(2+)]c increase genuinely caused by gravistimulation has not been identified because gravistimulation is generally accompanied by rotation of specimens on the ground (1g), adding an additional mechanical signal to the treatment. Here, we demonstrate a gravistimulation-specific Ca(2+) response in Arabidopsis seedlings by separating rotation from gravistimulation by using the microgravity (less than 10(-4)g) conditions provided by parabolic flights. Gravistimulation without rotating the specimen caused a sustained [Ca(2+)]c increase, which corresponds closely to the second sustained [Ca(2+)]c increase observed in ground experiments. The [Ca(2+)]c increases were analyzed under a variety of gravity intensities (e.g. 0.5g, 1.5g, or 2g) combined with rapid switching between hypergravity and microgravity, demonstrating that Arabidopsis seedlings possess a very rapid gravity-sensing mechanism linearly transducing a wide range of gravitational changes (0.5g-2g) into Ca(2+) signals on a subsecond time scale.

An Arabidopsis E3 ligase, SHOOT GRAVITROPISM9, modulates the interaction between statoliths and F-actin in gravity sensing.

Higher plants use the sedimentation of amyloplasts in statocytes as statolith to sense the direction of gravity during gravitropism. In Arabidopsis thaliana inflorescence stem statocyte, amyloplasts are in complex movement; some show jumping-like saltatory movement and some tend to sediment toward the gravity direction. Here, we report that a RING-type E3 ligase SHOOT GRAVITROPISM9 (SGR9) localized to amyloplasts modulates amyloplast dynamics. In the sgr9 mutant, which exhibits reduced gravitropism, amyloplasts did not sediment but exhibited increased saltatory movement. Amyloplasts sometimes formed a cluster that is abnormally entangled with actin filaments (AFs) in sgr9. By contrast, in the fiz1 mutant, an ACT8 semidominant mutant that induces fragmentation of AFs, amyloplasts, lost saltatory movement and sedimented with nearly statically. Both treatment with Latrunculin B, an inhibitor of AF polymerization, and the fiz1 mutation rescued the gravitropic defect of sgr9. In addition, fiz1 decreased saltatory movement and induced amyloplast sedimentation even in sgr9. Our results suggest that amyloplasts are in equilibrium between sedimentation and saltatory movement in wild-type endodermal cells. Furthermore, this equilibrium is the result of the interaction between amyloplasts and AFs modulated by the SGR9. SGR9 may promote detachment of amyloplasts from AFs, allowing the amyloplasts to sediment in the AFs-dependent equilibrium of amyloplast dynamics.

Gravitropism and mechanical signaling in plants.

Mechanical stress is a critical signal affecting morphogenesis and growth and is caused by a large variety of environmental stimuli such as touch, wind, and gravity in addition to endogenous forces generated by growth. On the basis of studies dating from the early 19th century, the plant mechanical sensors and response components related to gravity can be divided into two types in terms of their temporal character: sensors of the transient stress of reorientation (phasic signaling) and sensors capable of monitoring and responding to the extended, continuous gravitropic signal for the duration of the tropic growth response (tonic signaling). In the case of transient stress, changes in the concentrations of ions in the cytoplasm play a central role in mechanosensing and are likely a key component of initial gravisensing. Potential candidates for mechanosensitive channels have been identified in Arabidopsis thaliana and may provide clues to these rapid, ionic gravisensing mechanisms. Continuous mechanical stress, on the other hand, may be sensed by other mechanisms in addition to the rapidly adapting mechnaosensitive channels of the phasic system. Sustaining such long-term responses may be through a network of biochemical signaling cascades that would therefore need to be maintained for the many hours of the growth response once they are triggered. However, classical physiological analyses and recent simulation studies also suggest involvement of the cytoskeleton in sensing/responding to long-term mechanoresponse independently of the biochemical signaling cascades triggered by initial graviperception events.

Green leaf volatile sensory calcium transduction in Arabidopsis.

Plants perceive volatile organic compounds (VOCs) released by mechanically- or herbivore-damaged neighboring plants and induce various defense responses. Such interplant communication protects plants from environmental threats. However, the spatiotemporal dynamics of VOC sensory transduction in plants remain largely unknown. Using a wide-field real-time imaging method, we visualize an increase in cytosolic Ca2+ concentration ([Ca2+]cyt) in Arabidopsis leaves following exposure to VOCs emitted by injured plants. We identify two green leaf volatiles (GLVs), (Z)-3-hexenal (Z-3-HAL) and (E)-2-hexenal (E-2-HAL), which increase [Ca2+]cyt in Arabidopsis. These volatiles trigger the expression of biotic and abiotic stress-responsive genes in a Ca2+-dependent manner. Tissue-specific high-resolution Ca2+ imaging and stomatal mutant analysis reveal that [Ca2+]cyt increases instantly in guard cells and subsequently in mesophyll cells upon Z-3-HAL exposure. These results suggest that GLVs in the atmosphere are rapidly taken up by the inner tissues via stomata, leading to [Ca2+]cyt increases and subsequent defense responses in Arabidopsis leaves.