Arabidopsis Synaptotagmin SYT1, a Type I Signal-anchor Protein,Requires Tandem C2 Domains for Delivery to the Plasma Membrane

The correct localization of integral membrane proteins to subcellular compartments is important for their functions. Synaptotagmin contains a single transmembrane domain that functions as a type I signal-anchor sequence in its N terminus and two calcium-binding domains (C₂A and C₂B) in its C terminus. Here, we demonstrate that the localization of an Arabidopsis synaptotagmin homolog, SYT1, to the plasma membrane (PM) is modulated by tandem C2 domains. An analysis of the roots of a transformant-expressing green fluorescent protein-tagged SYT1 driven by native SYT1 promoter suggested that SYT1 is synthesized in the endoplasmic reticulum, and then delivered to the PM via the exocytotic pathway. We transiently expressed a series of truncated proteins in protoplasts, and determined that tandem C₂A-C₂B domains were necessary for the localization of SYT1 to the PM. The PM localization of SYT1 was greatly reduced following mutation of the calcium-binding motifs of the C₂B domain, based on sequence comparisons with other homologs, such as endomembrane-localized SYT5. The localization of SYT1 to the PM may have been required for the functional divergence that occurred in the molecular evolution of plant synaptotagmins.     

Cold-Induced Freezing Tolerance in Arabidopsis

Changes in the physiology of plant leaves are correlated with enhanced freezing tolerance and include accumulation of compatible solutes, changes in membrane composition and behavior, and altered gene expression. Some of these changes are required for enhanced freezing tolerance, whereas others are merely consequences of low temperature. In this study we demonstrated that a combination of cold and light is required for enhanced freezing tolerance in Arabidopsis leaves, and this combination is associated with the accumulation of soluble sugars and proline. Sugar accumulation was evident within 2 h after a shift to low temperature, which preceded measured changes in freezing tolerance. In contrast, significant freezing tolerance was attained before the accumulation of proline or major changes in the percentage of dry weight were detected. Many mRNAs also rapidly accumulated in response to low temperature. All of the cold-induced mRNAs that we examined accumulated at low temperature even in the absence of light, when there was no enhancement of freezing tolerance. Thus, the accumulation of these mRNAs is insufficient for cold-induced freezing tolerance.     

The Calcium-Dependent and Calcium-Independent Membrane Binding of Synaptotagmin 1: Two Modes of C2B Binding

The Ca²⁺-independent membrane interactions of the soluble C2 domains from synaptotagmin 1 (syt1) were characterized using a combination of site-directed spin labeling and vesicle sedimentation. The second C2 domain of syt1, C2B, binds to membranes containing phosphatidylserine and phosphatidylcholine in a Ca²⁺-independent manner with a lipid partition coefficient of approximately 3.0 × 10² M^− 1. A soluble fragment containing the first and second C2 domains of syt1, C2A and C2B, has a similar affinity, but C2A alone has no detectable affinity to phosphatidylcholine/phosphatidylserine bilayers in the absence of Ca²⁺. Although the Ca²⁺-independent membrane affinity of C2B is modest, it indicates that this domain will never be free in solution within the cell. Site-directed spin labeling was used to obtain bilayer depth restraints, and a simulated annealing routine was used to generate a model for the membrane docking of C2B in the absence of Ca²⁺. In this model, the polybasic strand of C2B forms the membrane binding surface for the domain; however, this face of C2B does not penetrate the bilayer but is localized within the aqueous double layer when C2B is bound. This double-layer location indicates that C2B interacts in a purely electrostatic manner with the bilayer interface. In the presence of Ca²⁺, the membrane affinity of C2B is increased approximately 20-fold, and the domain rotates so that the Ca²⁺-binding loops of C2B insert into the bilayer. This Ca²⁺-triggered conformational change may act as a switch to modulate the accessibility of the polybasic face of C2B and control interactions of syt1 with other components of the fusion machinery.     

Arabidopsis Synaptotagmin 1 Is Required for the Maintenance of Plasma Membrane Integrity and Cell Viability

Plasma membrane repair in animal cells uses synaptotagmin 7, a Ca²⁺-activated membrane fusion protein that mediates delivery of intracellular membranes to wound sites by a mechanism resembling neuronal Ca²⁺-regulated exocytosis. Here, we show that loss of function of the homologous Arabidopsis thaliana Synaptotagmin 1 protein (SYT1) reduces the viability of cells as a consequence of a decrease in the integrity of the plasma membrane. This reduced integrity is enhanced in the syt1-2 null mutant in conditions of osmotic stress likely caused by a defective plasma membrane repair. Consistent with a role in plasma membrane repair, SYT1 is ubiquitously expressed, is located at the plasma membrane, and shares all domains characteristic of animal synaptotagmins (i.e., an N terminus-transmembrane domain and a cytoplasmic region containing two C2 domains with phospholipid binding activities). Our analyses support that membrane trafficking mediated by SYT1 is important for plasma membrane integrity and plant fitness.     

The Arabidopsis Synaptotagmin1 Is Enriched in Endoplasmic Reticulum-Plasma Membrane Contact Sites and Confers Cellular Resistance to Mechanical Stresses

Eukaryotic endoplasmic reticulum (ER)-plasma membrane (PM) contact sites are evolutionarily conserved microdomains that have important roles in specialized metabolic functions such as ER-PM communication, lipid homeostasis, and Ca²⁺ influx. Despite recent advances in knowledge about ER-PM contact site components and functions in yeast (Saccharomyces cerevisiae) and mammals, relatively little is known about the functional significance of these structures in plants. In this report, we characterize the Arabidopsis (Arabidopsis thaliana) phospholipid binding Synaptotagmin1 (SYT1) as a plant ortholog of the mammal extended synaptotagmins and yeast tricalbins families of ER-PM anchors. We propose that SYT1 functions at ER-PM contact sites because it displays a dual ER-PM localization, it is enriched in microtubule-depleted regions at the cell cortex, and it colocalizes with Vesicle-Associated Protein27-1, a known ER-PM marker. Furthermore, biochemical and physiological analyses indicate that SYT1 might function as an electrostatic phospholipid anchor conferring mechanical stability in plant cells. Together, the subcellular localization and functional characterization of SYT1 highlights a putative role of plant ER-PM contact site components in the cellular adaptation to environmental stresses.Land plants are sessile organisms that are persistently challenged by physicomechanical forces that arise from the external environment. Some of the most common mechanical stimuli perceived by plants, collectively termed thigmostimuli (the Greek prefix thigmo means touch), are those induced by gradients in pressure (e.g. wind or tidal flows), by the gravity vector (e.g. ice and snow accumulation), or by direct impact with inanimate objects and/or living organisms (e.g. raindrops, hailstones, insects, canopy rubbing; Telewski, 2006). Mechanical stresses are not only exerted by the environment but are also intrinsic to the biophysics of plant growth (Landrein and Hamant, 2013). For instance, plants experience progressive mechanical self-loading as they increase in size or bear fruit (Almeras et al., 2004), intricate stress patterns are generated by different expansion rates between particular plant tissues (Mirabet et al., 2011; Sampathkumar et al., 2014), and plant cells, which are physically restrained by a rigid cell wall, generate turgor pressure characterized by circumferential tensile forces and radial compressive forces toward the plasma membrane (Telewski, 2006).Under mechanical stresses, different cell types, including meristematic, expanding, and fully differentiated cells, undergo physiological changes based on the sensing and integration of various mechanical signals (Monshausen and Haswell, 2013). Although it is well established that plants sense and respond to mechanical cues, our understanding of the various molecular mechanisms by which this is accomplished is limited, and most of our knowledge relies on comparisons with mechanosensors and transduction pathways identified in Escherichia coli and mammalian cells (Arnadóttir and Chalfie, 2010). Currently, two nonmutually exclusive mechanosensing models, namely, the ion channel and the tensegrity models, coexist in the plant literature. In the ion channel model, plant homologs of the bacterial mechanosensitive channel of small conductance and putative stretch-activated Ca²⁺-permeable channels are gated in response to mechanical forces and trigger a signaling cascade through the rapid influx of extracellular Ca²⁺ toward the cytosol (Arnadóttir and Chalfie, 2010; Jensen and Haswell, 2012; Sukharev and Sachs, 2012; Kurusu et al., 2013). In the tensegrity model, plant cells operate as self-supporting structures stabilized by a dynamic prestress state in which all elements are in isometric tension (Fuller, 1961). In such a structure, the mechanical disturbance of any individual element allows stress signals to propagate and be transduced at relatively distant locations (Ingber, 2008). Thus, the mechanically stable cell walls provide structural support, and the constant remodeling of the underlying cytoskeleton in response to mechanical disturbances acts as a tensegrity sensor (Komis et al., 2002; Berghöfer et al., 2009; Nick, 2013).In this article, we characterize the Arabidopsis (Arabidopsis thaliana) type I anchor Synaptotagmin1 (SYT1/SYTA/NTMC2T1.1 [hereafter SYT1]; Craxton, 2010; Yamazaki et al., 2010; Lewis and Lazarowitz, 2010) as an important component required to withstand mechanical stress in plant cells. SYT1 belongs to a five-member family in Arabidopsis and shares a common modular structure with different members of the mammalian extended synaptotagmins (E-Syts) and yeast (Saccharomyces cerevisiae) tricalbins families of organelle tethers (Manford et al., 2012; Fig. 1A; Supplemental Fig. S1). These proteins act as molecular bridges between the ER and the PM at sites where both cellular membranes are in close proximity, called ER-PM contact sites. These specialized microdomains carry out important roles in organelle communication, lipid and Ca²⁺ homeostasis, and intracellular signaling in animal and yeast cells (Toulmay and Prinz, 2011; Helle et al., 2013; Prinz, 2014). In plants, these ER-PM contact sites have been morphologically described for decades (Staehelin, 1997), but their physiological roles have not been thoroughly characterized. Furthermore, the identity of molecular components at these sites has remained elusive until the recent characterization of the ER-PM localized Vesicle-Associated Protein27-1 (VAP27) and NETWORKED 3C (NET3C) markers in Arabidopsis (Wang et al., 2014).Open in a separate windowFigure 1.SYT1 displays dual endoplasmic reticulum (ER)-plasma membrane (PM) localization. A, Schematic representation of the SYT1 structure and functional domains. TM, Transmembrane domain; SMP, synaptotagmin-like-mitochondrial-lipid binding domain; C2A and C2B, Ca²⁺ and phospholipid binding domains. B and C, Coimmunolocalization of the endogenous SYT1 and the ER marker HDEL-GFP (B) or the PM marker PIN2-GFP (C) in 5-d-old root epidermal cells. Scale bars = 10 μm. D and E, Subcellular localization of the SYT1proSYT1-GFP marker (SYT1-GFP) in leaves. Images were acquired at the cortical (D) or equatorial regions (E) of 8-d-old leaf epidermal cells. Costaining with propidium iodide (PI) was used to facilitate the visualization of the cortical regions. Scale bars = 20 μm.In previous reports, SYT1 has been described as an essential component for PM integrity maintenance, especially under conditions of high potential for membrane disruption such as freezing or salt stresses (Schapire et al., 2008, 2009; Yamazaki et al., 2008). In these reports, SYT1 was proposed to act as a Ca²⁺-dependent regulator of membrane fusion, in analogy to the classical animal SYTs that mediate Ca²⁺-triggered vesicle fusion during neurotransmission (Carr and Munson, 2007). Another report highlighted a role for SYT1 in viral spreading from cell to cell (Lewis and Lazarowitz, 2010). The recent characterization of mammalian E-Syts in stress tolerance (Herdman et al., 2014) and the phylogenetic relationships of tricalbins and E-Syts with SYT1 (Craxton, 2010; Yamazaki et al., 2010) led us to hypothesize that SYT1 could be a functional ortholog of E-Syts and tricalbins in plants. Consistently, we found that it is localized in specific ER-PM subdomains in cortical cytoskeleton-depleted regions, it colocalizes with the VAP27 marker, and it anchors negatively charged phospholipids through its PM-targeted C2 domains. Additionally, we show that SYT1 loss of function causes mechanical instability at the tissue and cellular level without altering the gross ER morphology. Based on these findings, we conclude that SYT1 acts at ER-PM contact sites as part of structural platforms adjacent to the cortical cytoskeleton that are required for mechanical stress tolerance in Arabidopsis.     

Overexpression of Multiple Dehydrin Genes Enhances Tolerance to Freezing Stress in Arabidopsis

To elucidate the contribution of dehydrins (DHNs) to freezing stress tolerance in Arabidopsis, transgenic plants overexpressing multiple DHN genes were generated. Chimeric double constructs for expression of RAB18 and COR47 (pTP9) or LTI29 and LTI30 (pTP10) were made by fusing the coding sequences of the respective DHN genes to the cauliflower mosaic virus 35S promoter. Overexpression of the chimeric genes in Arabidopsis resulted in accumulation of the corresponding dehydrins to levels similar or higher than in cold-acclimated wild-type plants. Transgenic plants exhibited lower LT50 values and improved survival when exposed to freezing stress compared to the control plants. Post-embedding immuno electron microscopy of high-pressure frozen, freeze-substituted samples revealed partial intracellular translocation from cytosol to the vicinity of the membranes of the acidic dehydrin LTI29 during cold acclimation in transgenic plants. This study provides evidence that dehydrins contribute to freezing stress tolerance in plants and suggests that this could be partly due to their protective effect on membranes.     

Overexpression of TaFBA-A10 from Winter Wheat Enhances Freezing Tolerance in Arabidopsis thaliana

Journal of Plant Growth Regulation - Freezing stress is the principal abiotic stress that is not conducive to plant growth and yield. Fructose-1, 6-bisphosphate aldolase (FBA; EC 4.1.2.13) is a key...     

Maintenance of Membrane Fluidity during Development of Freezing Tolerance of Winter Wheat Seedlings

Fluidity of membrane lipids of shoot and root tissue and of chloroplasts from young wheat seedlings of contrasting freezing tolerance was investigated by measuring the motion and order parameters after spin labeling. A striking similarity was observed in membrane lipid fluidity of the five cultivars grown at 22 C. After cold hardening by growth at 2 C, a small change in membrane lipid fluidity was observed, but this was not correlated with the development of freezing tolerance, and there was no alteration in the transition temperature of membrane lipids. The results show that neither changes in membrane lipid fluidity nor transition temperature are a necessary feature of cold acclimation in wheat.     

The Signaling Mechanism of Arabidopsis CRY1 Involves Direct Interaction with COP1

Dark-grown transgenic Arabidopsis seedlings expressing the C-terminal domains (CCT) of the cryptochrome (CRY) blue light photoreceptors exhibit features that are normally associated only with light-grown seedlings, indicating that the signaling mechanism of Arabidopsis CRY is mediated through CCT. The phenotypic properties mediated by CCT are remarkably similar to those of the constitutive photomorphogenic1 (cop1) mutants. Here we show that Arabidopsis cryptochrome 1 (CRY1) and its C-terminal domain (CCT1) interacted strongly with the COP1 protein. Coimmunoprecipitation studies showed that CRY1 was bound to COP1 in extracts from both dark- and light-grown Arabidopsis. An interaction also was observed between the C-terminal domain of Arabidopsis phytochrome B and COP1, suggesting that phytochrome signaling also proceeds, at least in part, through direct interaction with COP1. These findings give new insight into the initial step in light signaling in Arabidopsis, providing a molecular link between the blue light receptor, CRY1, and COP1, a negative regulator of photomorphogenesis.     

冷诱导转录因子CBF1转化草莓及其抗寒性鉴定

以草莓栽培品种哈尼"为材料,利用根癌农杆菌介导的叶盘法将拟南芥冷诱导转录激活因子CBF1(C-re-peat binding factor)导入草莓中.转化植株经25 mg/L Km筛选后进行PCR和Southern杂交鉴定,结果显示CBF1基因整合进草莓基因组中.采用电解质渗漏法和生长恢复法对转基因株系进行抗寒生理鉴定,结果显示,转基因株系的电导率普遍低于对照;将转基因株系和对照于-2-0℃放置7 d,在转基因株系83中有65%的植株出现了萎蔫,而对照植株有91%出现了萎蔫,经22-25℃下进行恢复生长,转基因株系83有74%完全恢复,而对照株系只有18%恢复.结果表明CBF1基因导入草莓中可以提高草莓对低温胁迫的抵抗力.     

The Juxtamembrane Linker of Full-length Synaptotagmin 1 Controls Oligomerization and Calcium-dependent Membrane Binding

Synaptotagmin 1 (Syt1) is the calcium sensor for synchronous neurotransmitter release. The two C2 domains of Syt1, which may mediate fusion by bridging the vesicle and plasma membranes, are connected to the vesicle membrane by a 60-residue linker. Here, we use site-directed spin labeling and a novel total internal reflection fluorescence vesicle binding assay to characterize the juxtamembrane linker and to test the ability of reconstituted full-length Syt1 to interact with opposing membrane surfaces. EPR spectroscopy demonstrates that the majority of the linker interacts with the membrane interface, thereby limiting the extension of the C2A and C2B domains into the cytoplasm. Pulse dipolar EPR spectroscopy provides evidence that purified full-length Syt1 is oligomerized in the membrane, and mutagenesis indicates that a glycine zipper/GXXXG motif within the linker helps mediate oligomerization. The total internal reflection fluorescence-based vesicle binding assay demonstrates that full-length Syt1 that is reconstituted into supported lipid bilayers will capture vesicles containing negatively charged lipid in a Ca²⁺-dependent manner. Moreover, the rate of vesicle capture increases with Syt1 density, and mutations in the GXXXG motif that inhibit oligomerization of Syt1 reduce the rate of vesicle capture. This work demonstrates that modifications within the 60-residue linker modulate both the oligomerization of Syt1 and its ability to interact with opposing bilayers. In addition to controlling its activity, the oligomerization of Syt1 may play a role in organizing proteins within the active zone of membrane fusion.     

ABA and Low Temperature Induce Freezing Tolerance via Distinct Regulatory Pathways in Wheat

The role of ABA in the induction of freezing tolerance was investigatedin two wheat (T. aestivum L.) cultivars, Glenlea (spring var)and Fredrick (winter var). Exogenous application of ABA (5x10^–5M for 5 days at 24°C) increased the freezing tolerance ofintact plants by only 3°C (LT₅₀) in both cultivars. Maximalfreezing tolerance (LT₅₀ of –9°C for Glenlea and –17°Cfor Fredrick) could only be obtained with a low temperaturetreatment (6/2°C; day/night) for 40 days. These resultsshow that exogenously applied ABA cannot substitute for lowtemperature requirementto induce freezing tolerance in intactwheat plants. Furthermore, there was no increase in the endogenousABA level of wheat plants during low temperature acclimation,suggesting the absence of an essential role for ABA in the developmentof freezing tolerance in intact plants. On the other hand, ABAapplication (5x10^–5 M for 5 days at 24°C) to embryogenicwheat calli resulted in an increase of freezing tolerance similarto that achieved by low temperature. However, as in intact plants,there was no increase in the endogenous ABA level during lowtemperature acclimation of calli. These results indicate thatthe induction of freezing tolerance by low temperature is notassociated with an increase in ABA content. Using an antibodyspecific to a protein family associated with the developmentof freezing tolerance, we demonstrated that the induction offreezing tolerance by ABA in embryogenic wheat calli was correlatedwith the accumulation of a new 32 kDa protein. This proteinis specifically induced by ABA but shares a common antigenicitywith those induced by low temperature. These results suggestthat ABA induces freezing tolerance in wheat calli via a regulatorymechanism different from that of low temperature. (Received June 15, 1993; Accepted September 16, 1993)     

Betaine Improves Freezing Tolerance in Wheat

The accumulation of the osmolyte betaine was found to be correlatedwith the development of freezing tolerance (FT) of two wheatcultivars where it increases by about three fold during thecold acclimation period. Exogenous betaine application resultedin a large increase in total osmolality mostly due to betaineaccumulation. Plants that accumulated betaine are more tolerantto freezing stress since a four day exposure to 250 mM betaineresulted in a LT₅₀ of –8°C (in spring wheat Glenlea)and –9°C (in winter wheat Fredrick) compared to –3°C(Glenlea) and –4°C (Fredrick) for control non-exposedplants. Betaine treatment (250 mM) during cold acclimation increasedFT in an additive manner since the LT₅₀ reached –14°C(Glenlea) and –22°C (Fredrick) compared to –8°C(Glenlea) and –16°C (Fredrick) for plants that arecold acclimated in the absence of betaine. These results showthat betaine treatment can improve FT by more than 5°C inboth non-acclimated and cold-acclimated plants. The betainetreatment resulted in the induction of a subset of low temperatureresponsive genes, such as the wcor410, and wcor413, that arealso induced by salinity or drought stresses. In addition tothese genetic responses, betaine treatment was also able toimprove the tolerance to photoin-hibition of PSII and the steady-stateyield of electron transport over PSII in a manner that mimickedcold-acclimated plants. These data also suggest that betaineimproves FT by eliciting some of the genetic and physiologicalresponses associated with cold acclimation. (Received April 23, 1998; Accepted September 4, 1998)