Populus euphratica APYRASE2 Enhances Cold Tolerance by Modulating Vesicular Trafficking and Extracellular ATP in Arabidopsis Plants

Apyrase and extracellular ATP play crucial roles in mediating plant growth and defense responses. In the cold-tolerant poplar, Populus euphratica, low temperatures up-regulate APYRASE2 (PeAPY2) expression in callus cells. We investigated the biochemical characteristics of PeAPY2 and its role in cold tolerance. We found that PeAPY2 predominantly localized to the plasma membrane, but punctate signals also appeared in the endoplasmic reticulum and Golgi apparatus. PeAPY2 exhibited broad substrate specificity, but it most efficiently hydrolyzed purine nucleotides, particularly ATP. PeAPY2 preferred Mg²⁺ as a cofactor, and it was insensitive to various, specific ATPase inhibitors. When PeAPY2 was ectopically expressed in Arabidopsis (Arabidopsis thaliana), cold tolerance was enhanced, based on root growth measurements and survival rates. Moreover, under cold stress, PeAPY2-transgenic plants maintained plasma membrane integrity and showed reduced cold-elicited electrolyte leakage compared with wild-type plants. These responses probably resulted from efficient plasma membrane repair via vesicular trafficking. Indeed, transgenic plants showed accelerated endocytosis and exocytosis during cold stress and recovery. We found that low doses of extracellular ATP accelerated vesicular trafficking, but high extracellular ATP inhibited trafficking and reduced cell viability. Cold stress caused significant increases in root medium extracellular ATP. However, under these conditions, PeAPY2-transgenic lines showed greater control of extracellular ATP levels than wild-type plants. We conclude that Arabidopsis plants that overexpressed PeAPY2 could increase membrane repair by accelerating vesicular trafficking and hydrolyzing extracellular ATP to avoid excessive, cold-elicited ATP accumulation in the root medium and, thus, reduced ATP-induced inhibition of vesicular trafficking.Low temperature is a major environmental factor that restrains plant growth and crop productivity (Yamaguchi-Shinozaki and Shinozaki, 2006). When temperatures fall below 0°C, plant cells experience dehydration and mechanical injury caused by ice crystallization (Webb and Steponkus, 1993; Yamazaki et al., 2008). Cold stress reduces plasma membrane (PM) integrity, which leads to the leakage of intracellular solutes. ATP can be an important signaling molecule when released into the extracellular matrix (ECM). Extracellular ATP (eATP) was shown to regulate a wide range of cellular processes (Roux and Steinebrunner, 2007; Clark and Roux, 2009, 2011; Tanaka et al., 2010; Clark et al., 2014), but its functions are dose dependent. For example, in Arabidopsis (Arabidopsis thaliana), low concentrations of eATP triggered stomatal opening, but high concentrations caused stomatal closure (Clark et al., 2011). Application of eATP at 100 to 200 μm increased hypocotyl elongation in etiolated seedlings, but higher doses led to a reduction of hypocotyl elongation (Roux et al., 2006). In fibers of cotton (Gossypium hirsutum), application of ATPγS and ADPβS at 30 μm each induced an increase in average fiber length, whereas 150 μm ATPγS or ADPβS inhibited the growth of fibers (Clark et al., 2010a). In addition, low doses of eATP (less than 100 μm) induced an obvious Ca²⁺ influx into the elongation zone of Arabidopsis roots, and higher doses of eATP (1,000 μm) caused a significant increase in Ca²⁺ efflux (Demidchik et al., 2011). In Populus euphratica, exposure to high levels of ATP (more than 500 μm) was shown to trigger programmed cell death in callus cells, but at low doses (less than 200 μm), callus cells did not die over the observation period (Sun et al., 2012a). The cold-elicited release of ATP through disrupted membranes and the buildup of ATP in the ECM may cause a reduction in cell viability. eATP also plays a fundamental role in mediating plant responses to environmental stresses, such as pathogens (Chivasa et al., 2009), wounds (Cao et al., 2014), high salt (Sun et al., 2012b), and osmotic stress (Jeter et al., 2004; Kim et al., 2009); however, the link between eATP and cold tolerance has not been fully established.Extracellular apyrases (or ectoapyrases) are the principal enzymes that limit eATP accumulation in both animals and plants (Todorov et al., 1997; Marcus et al., 2003; Wu et al., 2007). In Arabidopsis, suppression of apyrases (AtAPY1 and AtAPY2) led to a slight increase in eATP levels, indicating that APY1 and APY2 controlled the concentration of eATP (Lim et al., 2014). Apyrases are suggested to be involved in some of the signaling steps in plant growth. Apyrases play a crucial role in pollen germination (Steinebrunner et al., 2003; Wu et al., 2007), cotton fiber elongation (Clark et al., 2010a), and root hair growth (Liu et al., 2012). In addition, ectoapyrases contribute to regulating stomatal functions; chemical and immunological inhibition of apyrase activity induced stomatal closure (Clark et al., 2011). Recently, apyrases were shown to play important roles in the signaling steps in plant defense responses (Lim et al., 2014). Suppression of apyrase significantly altered the expression of genes involved in biotic stress responses (Lim et al., 2014). Additionally, our previous finding suggested that apyrase contributed to salt tolerance in P. euphratica (Sun et al., 2012b). NaCl shock elicited a significant rise in ATP in the ECM, but the eATP levels returned to basal levels after 20 min of salt treatment (Sun et al., 2012b). This was presumably due to ATP hydrolysis by ectoapyrase, which enabled P. euphratica to maintain low levels of eATP in a prolonged duration of salinity and, thus, prevent eATP-induced cell death (Sun et al., 2012a). Apyrase was also postulated to serve as a signal in stress responses. However, no studies have investigated in higher order plants whether apyrase promotes the hydrolysis of ATP at low temperatures and whether this activity is correlated to cold tolerance.In general, in higher order plants, low temperature causes a reduction in PM integrity. It is necessary for plant cells to reseal the PM disruption to prevent a decrease in cell viability (Yamazaki et al., 2008, 2010). PM resealing requires vesicular trafficking that includes both endocytosis and exocytosis (Togo et al., 1999; McNeil et al., 2003; Tam et al., 2010; Los et al., 2011). Ca²⁺-dependent exocytosis provides a membrane patch to the wound site, which relieves PM tension for resealing (Togo et al., 2000; Sonnemann and Bement, 2011). In animals, lysosomes are the major organelles that contribute to exocytosis-mediated membrane repair (Gerasimenko et al., 2001; Reddy et al., 2001; McNeil, 2002). Endocytosis also contributes to membrane repair by retrieving the wound site from the PM in a Ca²⁺-dependent manner (Idone et al., 2008). Shibasaki et al. (2009) suggested that low temperature inhibited the intracellular trafficking of auxin efflux carriers after the initiation of cold stress (9–12 h). However, it remains unclear whether vesicular trafficking is mediated by apyrase and eATP and contributes to cold tolerance during long-term cold stress and the subsequent recovery period.This study evaluated the roles of apyrase and eATP in cold stress signaling in woody plants. We focused on P. euphratica, because this species plays very important roles in stabilizing sand dunes and in sheltering agricultural regions in northwest China (Wei, 1993). In addition, P. euphratica trees can adapt to harsh temperature conditions in saline and alkaline desert sites (Wei, 1993). In this study, we showed that cold stress up-regulated APY2 expression in P. euphratica callus cells, but it did not induce the expression of APY1, another apyrase (Supplemental Fig. S1). Thus, APY2 may contribute to cold adaptation in P. euphratica. We tested this hypothesis by cloning the PeAPY2 gene from P. euphratica callus cells and transferring it into a model species, Arabidopsis. We then investigated the roles of PeAPY2 in eATP control and cold tolerance. Our data showed that PeAPY2 overexpression increased root membrane integrity and cold tolerance. This was likely due to effective PM repair, because endocytosis and exocytosis were up-regulated in transgenic plants. We concluded that PeAPY2 modulated eATP levels and enhanced vesicular trafficking and that these activities may have contributed to membrane resealing in cold-stressed PeAPY2-transgenic plants.     

Molecular dissection of the alpha-dystroglycan- and integrin-binding sites within the globular domain of human laminin-10

The adhesive interactions of cells with laminins are mediated by integrins and non-integrin-type receptors such as alpha-dystroglycan and syndecans. Laminins bind to these receptors at the C-terminal globular domain of their alpha chains, but the regions recognized by these receptors have not been mapped precisely. In this study, we sought to locate the binding sites of laminin-10 (alpha5beta1gamma1) for alpha(3)beta(1) and alpha(6)beta(1) integrins and alpha-dystroglycan through the production of a series of recombinant laminin-10 proteins with deletions of the LG (laminin G-like) modules within the globular domain. We found that deletion of the LG4-5 modules did not compromise the binding of laminin-10 to alpha(3)beta(1) and alpha(6)beta(1) integrins but completely abrogated its binding to alpha-dystroglycan. Further deletion up to the LG3 module resulted in loss of its binding to the integrins, underlining the importance of LG3 for integrin binding by laminin-10. When expressed individually as fusion proteins with glutathione S-transferase or the N-terminal 70-kDa region of fibronectin, only LG4 was capable of binding to alpha-dystroglycan, whereas neither LG3 nor any of the other LG modules retained the ability to bind to the integrins. Site-directed mutagenesis of the LG3 and LG4 modules indicated that Asp-3198 in the LG3 module is involved in the integrin binding by laminin-10, whereas multiple basic amino acid residues in the putative loop regions are involved synergistically in the alpha-dystroglycan binding by the LG4 module.     

胡杨谷胱甘肽过氧化物酶PeGPX基因的克隆及转化植株耐盐性分析

胡杨(Populus euphratica Oliv.)具有极强抗盐碱能力。本实验室前期胡杨微阵列芯片数据结果显示:盐胁迫下,胡杨谷胱甘肽过氧化物酶基因(PeGPX)的转录上调,暗示该基因可能对胡杨耐盐性具有一定的作用。为分析 GPX 对植物耐盐性的贡献,本研究以胡杨为材料,利用 RT-PCR 方法克隆了胡杨谷胱甘肽过氧化物酶PeGPX基因,并在烟草中过量表达该基因,以分析谷胱甘肽过氧化物酶活性与植物耐盐性的关系。研究结果显示,实验中克隆的 cDNA (PeGPX)编码谷胱甘肽过氧化物酶,其 ORF 为 693 bp,其蛋白由 231 个氨基酸编码。过量表达 PeGPX 基因的烟草与野生型烟草的耐盐性实验结果显示,野生型烟草植株在加 NaCl(200 mmol/L)的 MS 培养基中生长 15 d 后,无明显的长高,且不长根;而转基因烟草在同样的加盐培养基上,生长基本没有受到抑制,植株生长状态良好,并且能够长根。光合数据显示,在盐胁迫下过量表达 PeGPX 基因烟草的净光合速率受到影响明显小于野生型烟草的净光合速率。酶活数据显示,转基因株系 GPX 酶活与野生型的相比在盐胁迫下活性有非常显著的提高。我们的研究结果说明:过表达 PeGPX 基因使得烟草的耐盐性得到显著提高,这对深入研究PeGPX基因在胡杨耐盐机制中的作用具有重要的意义。高,这对深入研究PeGPX基因在胡杨耐盐机制中的作用具有重要的意义。     

Arabidopsis fatty acid desaturase FAD2 is required for salt tolerance during seed germination and early seedling growth

Fatty acid desaturases play important role in plant responses to abiotic stresses. However, their exact function in plant resistance to salt stress is unknown. In this work, we provide the evidence that FAD2, an endoplasmic reticulum localized ω-6 desaturase, is required for salt tolerance in Arabidopsis. Using vacuolar and plasma membrane vesicles prepared from the leaves of wild-type (Col-0) and the loss-of-function Arabidopsis mutant, fad2, which lacks the functional FAD2, we examined the fatty acid composition and Na+-dependent H+ movements of the isolated vesicles. We observed that, when compared to Col-0, the level of vacuolar and plasma membrane polyunsaturation was lower, and the Na+/H+ exchange activity was reduced in vacuolar and plasma membrane vesicles isolated from fad2 mutant. Consistent with the reduced Na+/H+ exchange activity, fad2 accumulated more Na+ in the cytoplasm of root cells, and was more sensitive to salt stress during seed germination and early seedling growth, as indicated by CoroNa-Green staining, net Na+ efflux and salt tolerance analyses. Our results suggest that FAD2 mediated high-level vacuolar and plasma membrane fatty acid desaturation is essential for the proper function of membrane attached Na+/H+ exchangers, and thereby to maintain a low cytosolic Na+ concentration for salt tolerance during seed germination and early seedling growth in Arabidopsis.     

降解1,3-二甲基-2-咪唑烷酮细菌的分离及功能评价

[背景] 1,3-二甲基-2-咪唑烷酮（1,3-Dimethyl-2-Imidazolidinone,DMI）作为一种强极性非质子溶剂,在生产和应用过程的环境中有稳定残留问题,存在安全隐患。[目的] 分离筛选具有降解DMI能力的微生物菌株,为清除环境中残留的DMI提供优良的微生物菌种资源。[方法] 从DMI生产区域土壤采集样品分离DMI抗性微生物,采用形态学及分子生物学鉴定确定其分类地位,并对DMI降解能力进行测定。[结果] 分离到最高能够耐受5%（体积分数） DMI的微生物菌株,形态学及分子生物学鉴定初步表明获得的菌株DT-1和DT-2为贝莱斯芽孢杆菌（Bacillus velezensis）;全细胞及细胞提取液均具有降解DMI的能力;其中菌株DT-1及其细胞提取液对1%（体积分数） DMI的降解率分别达到48%和68%。[结论] 从DMI生产区域土壤中分离到具有DMI降解能力的芽孢杆菌,不但可为DMI污染的微生物治理提供优良微生物资源,而且扩展了人们对芽孢杆菌生物学功能的认识。     

Effect of NaCl on leaf H+-ATPase and the relevance to salt tolerance in two contrasting poplar species

During a 30-day period of increasing salinity, we examined the effects of NaCl on leaf H⁺-ATPase and salinity tolerance in 1-year-old plants of Populus euphratica Oliv. (salt resistant) and P. popularis 35–44 (P. popularis) (salt sensitive). Electron probe X-ray microanalysis of leaf mesophyll revealed that P. euphratica had a higher ability to retain lower NaCl concentrations in the cytoplasm, as compared to P. popularis. The sustained activity of H⁺ pumps (by cytochemical staining) in salinised P. euphratica suggests a role in energising salt transport through the plasma membrane (PM) and tonoplast. Salt-induced alterations of leaf respiration, ATP content and expression of PM H⁺-ATPase were compared between the two species. Results show that P. euphratica retained a constant respiratory rate, ATP production and protein abundance of PM H⁺-ATPase (by Western blotting) in salt-stressed plants. P. euphratica was able to maintain a comparatively high capacity of ATP hydrolysis and H⁺ pumping during prolonged salt exposure. By contrast, the activity and expression of PM H⁺-ATPase were markedly decreased in P. popularis leaves in response to salt stress. Furthermore, NaCl-stressed P. popularis plants showed a marked decline of respiration (70%) and ATP production (66%) on day 30. We conclude that the inability of P. popularis to transport salt to the apoplast and vacuole was partly due to the decreased activity of H⁺ pumps. As a consequence, cytosolic ion concentrations were observed to be comparatively high for an extended period of time, so that cell metabolism, in particular respiration, was disrupted in P. popularis leaves.     

Extracellular ATP Signaling Is Mediated by H2O2 and Cytosolic Ca2+ in the Salt Response of Populus euphratica Cells

Extracellular ATP (eATP) has been implicated in mediating plant growth and antioxidant defense; however, it is largely unknown whether eATP might mediate salinity tolerance. We used confocal microscopy, a non-invasive vibrating ion-selective microelectrode, and quantitative real time PCR analysis to evaluate the physiological significance of eATP in the salt resistance of cell cultures derived from a salt-tolerant woody species, Populus euphratica. Application of NaCl (200 mM) shock induced a transient elevation in [eATP]. We investigated the effects of eATP by blocking P2 receptors with suramin and PPADS and applying an ATP trap system of hexokinase-glucose. We found that eATP regulated a wide range of cellular processes required for salt adaptation, including vacuolar Na⁺ compartmentation, Na⁺/H⁺ exchange across the plasma membrane (PM), K⁺ homeostasis, reactive oxygen species regulation, and salt-responsive expression of genes related to K⁺/Na⁺ homeostasis and PM repair. Furthermore, we found that the eATP signaling was mediated by H₂O₂ and cytosolic Ca²⁺ released in response to high salt in P. euphratica cells. We concluded that salt-induced eATP was sensed by purinoceptors in the PM, and this led to the induction of downstream signals, like H₂O₂ and cytosolic Ca²⁺, which are required for the up-regulation of genes linked to K⁺/Na⁺ homeostasis and PM repair. Consequently, the viability of P. euphratica cells was maintained during a prolonged period of salt stress.