Role for Apyrases in Polar Auxin Transport in Arabidopsis

Recent evidence indicates that extracellular nucleotides regulate plant growth. Exogenous ATP has been shown to block auxin transport and gravitropic growth in primary roots of Arabidopsis (Arabidopsis thaliana). Cells limit the concentration of extracellular ATP in part through the activity of ectoapyrases (ectonucleoside triphosphate diphosphohydrolases), and two nearly identical Arabidopsis apyrases, APY1 and APY2, appear to share this function. These findings, plus the fact that suppression of APY1 and APY2 blocks growth in Arabidopsis, suggested that the expression of these apyrases could influence auxin transport. This report tests that hypothesis. The polar movement of [³H]indole-3-acetic acid in both hypocotyl sections and primary roots of Arabidopsis seedlings was measured. In both tissues, polar auxin transport was significantly reduced in apy2 null mutants when they were induced by estradiol to suppress the expression of APY1 by RNA interference. In the hypocotyl assays, the basal halves of APY-suppressed hypocotyls contained considerably lower free indole-3-acetic acid levels when compared with wild-type plants, and disrupted auxin transport in the APY-suppressed roots was reflected by their significant morphological abnormalities. When a green fluorescent protein fluorescence signal encoded by a DR5:green fluorescent protein construct was measured in primary roots whose apyrase expression was suppressed either genetically or chemically, the roots showed no signal asymmetry following gravistimulation, and both their growth and gravitropic curvature were inhibited. Chemicals that suppress apyrase activity also inhibit gravitropic curvature and, to a lesser extent, growth. Taken together, these results indicate that a critical step connecting apyrase suppression to growth suppression is the inhibition of polar auxin transport.In both animals and plants, cells release nucleotides into their extracellular matrix, where they function as signaling agents, inducing rapid increases in the concentration of cytosolic calcium that are transduced into downstream changes in cell physiology (Kim et al., 2006; Burnstock, 2007; Roux and Steinebrunner, 2007; Tanaka et al., 2010a, 2010b; Demidchik et al., 2011). Prominent among these downstream changes in plants are changes in the growth of cells, including the growth of pollen tubes (Steinebrunner et al., 2003), root hairs (Clark et al., 2010b), and cotton (Gossypium hirsutum) fibers (Clark et al., 2010a). These results suggest the possibility that the signaling changes induced by extracellular nucleotides intersect with signaling changes induced by one or more of the hormones that regulate plant cell growth. Consistent with this possibility, Tang et al. (2003) showed that a concentration of applied nucleotides that inhibited the gravitropic growth of roots could block the transport of the growth hormone auxin and that this effect could not be attributed to either pH changes or chelation of divalent cations. Correspondingly, Clark et al. (2010a) showed that when the application of nucleotides to cotton ovules growing in culture altered the rate of cotton fiber growth, it also induced the production of ethylene, a hormone known to regulate the growth of cotton fibers.Given the potency of extracellular nucleotides to regulate cellular activities, it would be important for cells to control the concentration of these nucleotides. In both animals and plants, the principal enzymes that limit the buildup of extracellular ATP (eATP) and extracellular ADP are ectoapyrases (apyrase; EC 3.6.1.5). These enzymes, which are nucleoside triphosphate diphosphohydrolases, are characterized by apyrase-conserved regions whose peptide sequences are highly similar throughout the plant and animal kingdoms (Clark and Roux, 2009). Based on this structural criterion, there are seven apyrases in Arabidopsis (Arabidopsis thaliana; APY1–APY7), and two of these, APY1 and APY2, share 87% protein sequence identity but are less than 30% similar to the other five apyrases. These two apyrases partially complement each other’s function and play central roles in growth control in Arabidopsis, as judged both by genetic and biochemical criteria (Wolf et al., 2007; Wu et al., 2007). Polyclonal antibodies raised to APY1 (Steinebrunner et al., 2000) inhibit the apyrase activity released into the medium of growing pollen tubes, and when these antibodies were added to the culture medium of germinated pollen, they both blocked the growth of the pollen and raised the concentration of ATP in the medium (Wu et al., 2007). Similarly, treatment of cultured cotton ovules with antibodies that recognize cotton fiber apyrase both inhibits the growth of the fibers and increases the concentration of ATP in the medium, further establishing the link between apyrase activity and regulation of the extracellular ATP concentration ([eATP]) in growing tissues (Clark et al., 2010a).Because wild-type pollen tubes expressing active APY1 or APY2 and cultured cotton fibers with wild-type apyrase activity grow at a normal rate, and because the antibodies inhibit apyrase activity (Wu et al., 2007), the growth inhibition induced by the antibodies further implicated apyrase activity as critical for the growth of these tissues. The antibodies were unlikely to enter the pollen tubes or cotton fibers, so these results also suggested that the pollen and cotton apyrases were ectoapyrases. However, these data do not rule out a possible Golgi function for APY1 and APY2 and for the cotton APY(s), as discussed by Wu et al. (2007) and Clark and Roux (2011). In fact, there is strong evidence that APY1 and APY2 are localized in the Golgi and may function there to regulate protein glycosylation and/or affect polysaccharide synthesis (Chiu et al., 2012; Schiller et al., 2012).Although the suppression of APY1/APY2 or of apyrase activity has a dramatic effect on growth, overexpression of APY1 or APY2 has much less of an effect. Constitutive expression of APY1 induces a small but statistically significant increase in the growth of etiolated hypocotyls, while overexpressing APY2 has no effect on this growth (Wu et al., 2007). This is probably because the wild-type levels of apyrase expression are near optimal for growth (Roux and Steinebrunner, 2007).The double knockout apy1apy2 is sterile, because the pollen of this mutant does not germinate (Steinebrunner et al., 2003). However, when APY1 is suppressed only approximately 60% by an inducible RNA interference (RNAi) construct in apy2 null mutants, pollen of these mutants will germinate, permitting fertilization and subsequent normal development, although the adult plants of these mutants are dwarf (Wu et al., 2007). Suppression of ectoapyrase activity would be expected to raise the equilibrium concentration of eATP (Wu et al., 2007), and since higher levels of eATP can inhibit auxin transport in roots (Tang et al., 2003), it was reasonable to hypothesize that the suppression of apyrase by RNAi could suppress auxin transport. The experiments described in this report test this hypothesis. The results indicate that suppression of APY1/APY2 expression in an inducible RNAi line, R2-4A (Wu et al., 2007), results in a significant inhibition of polar auxin transport in Arabidopsis hypocotyls and roots, with a concomitant altered distribution of endogenous auxin. Consistent with this result and with the results of Tang et al. (2003), suppression of APY1/APY2 also blocks the asymmetric distribution of a GFP reporter encoded by a DR5:GFP construct in gravistimulated primary roots of Arabidopsis seedlings and diminishes the extent of the elongation zone in these roots. These results are consistent with the novel conclusion that inhibition of auxin transport is a key step in the signaling pathway that links the inhibition of apyrase expression to growth inhibition.     

耐低温厌氧蛋白酶产生菌B-25的选育及产酶条件研究

从公园池塘污泥中分离到一株性状优良、耐低温的厌氧蛋白酶产生菌B-25,经形态、生理生化特性、16SrDNA序列分析鉴定为双酶梭状芽孢杆菌（Clostridium bifermentans）。对其最适产酶条件进行了初步研究,该菌培养96h时酶活力达到高峰,最适初始pH为7．5,在16℃、22℃、30℃培养酶活较高,该酶不耐高温,60℃以上酶活全部丧失。     

Evaluating a three dimensional model of diffuse photosynthetically active radiation in maize canopies

Diffuse photosynthetically active radiation (DPAR) is important during overcast days and for plant parts shaded from the direct beam radiation. Simulation of DPAR interception by individual plant parts of a canopy, separately from direct beam photosynthetically active radiation (PAR), may give important insights into plant ecology. This paper presents a model to simulate the interception of DPAR in plant canopies. A sub-model of a virtual maize canopy was reconstructed. Plant surfaces were represented as small triangular facets positioned according to three-dimensionally (3D) digitized data collected in the field. Then a second sub-model to simulate the 3D DPAR distribution in the canopy was developed by dividing the sky hemisphere into a grid of fine cells that allowed for the anisotropic distribution of DPAR over the sky hemisphere. This model, DSHP (Dividing Sky Hemisphere with Projecting), simulates which DSH (Divided Sky Hemisphere) cells are directly visible from a facet in the virtual canopy, i.e. not obscured by other facets. The DPAR reaching the center of a facet was calculated by summing the amounts of DPAR present in every DSH cell. The distribution of DPAR in a canopy was obtained from the calculated DPARs intercepted by all facets in the canopy. This DSHP model was validated against DPAR measurements made in an actual maize (Zea mays L.) canopy over selected days during the early filling stage. The simulated and measured DPAR at different canopy depths showed a good agreement with a R ² equaling 0.78 (n=120).     

A Signaling Pathway Linking Nitric Oxide Production to Heterotrimeric G Protein and Hydrogen Peroxide Regulates Extracellular Calmodulin Induction of Stomatal Closure in Arabidopsis

Extracellular calmodulin (ExtCaM) regulates stomatal movement by eliciting a cascade of intracellular signaling events including heterotrimeric G protein, hydrogen peroxide (H₂O₂), and Ca²⁺. However, the ExtCaM-mediated guard cell signaling pathway remains poorly understood. In this report, we show that Arabidopsis (Arabidopsis thaliana) NITRIC OXIDE ASSOCIATED1 (AtNOA1)-dependent nitric oxide (NO) accumulation plays a crucial role in ExtCaM-induced stomatal closure. ExtCaM triggered a significant increase in NO levels associated with stomatal closure in the wild type, but both effects were abolished in the Atnoa1 mutant. Furthermore, we found that ExtCaM-mediated NO generation is regulated by GPA1, the Gα-subunit of heterotrimeric G protein. The ExtCaM-dependent NO accumulation was nullified in gpa1 knockout mutants but enhanced by overexpression of a constitutively active form of GPA1 (cGα). In addition, cGα Atnoa1 and gpa1-2 Atnoa1 double mutants exhibited a similar response as did Atnoa1. The defect in gpa1 was rescued by overexpression of AtNOA1. Finally, we demonstrated that G protein activation of NO production depends on H₂O₂. Reduced H₂O₂ levels in guard cells blocked the stomatal response of cGα lines, whereas exogenously applied H₂O₂ rescued the defect in ExtCaM-mediated stomatal closure in gpa1 mutants. Moreover, the atrbohD/F mutant, which lacks the NADPH oxidase activity in guard cells, had impaired NO generation in response to ExtCaM, and H₂O₂-induced stomatal closure and NO accumulation were greatly impaired in Atnoa1. These findings have established a signaling pathway leading to ExtCaM-induced stomatal closure, which involves GPA1-dependent activation of H₂O₂ production and subsequent AtNOA1-dependent NO accumulation.Plant guard cells control opening and closure of the stomata in response to phytohormones (e.g. abscisic acid [ABA]) and various environmental signals such as light and temperature, thereby regulating gas exchange for photosynthesis and water status via transpiration (Schroeder et al., 2001). Cytosolic calcium ([Ca²⁺]_i) has been shown to be a key second messenger that changes in response to multiple stimuli in guard cells (McAinsh et al., 1995; Grabov and Blatt, 1998; Wood et al., 2000). A large proportion of Ca²⁺ is localized in extracellular space. It has been shown that external Ca²⁺ concentration ([Ca²⁺]_o) promotes stomatal closure and induces oscillation in [Ca²⁺]_i in guard cells (MacRobbie, 1992; McAinsh et al., 1995; Allen et al., 2001). However, how the guard cells perceive [Ca²⁺]_o concentration and convert [Ca²⁺]_o changes into [Ca²⁺]_i changes was not understood until a calcium-sensing receptor (CAS) in the plasma membrane of guard cells in Arabidopsis (Arabidopsis thaliana) was identified (Han et al., 2003). The external Ca²⁺ (Ca²⁺_o)-induced [Ca²⁺]_i increase is abolished in CAS antisense lines (Han et al., 2003). Both [Ca²⁺]_o and [Ca²⁺]_i show diurnal oscillation that is determined by stomatal conductance, whereas the amplitude of [Ca²⁺]_i oscillation is reduced in CAS antisense lines (Tang et al., 2007). The reduced amplitude of [Ca²⁺]_i diurnal oscillation in response to Ca²⁺_o treatment suggests the potential existence of other [Ca²⁺]_o sensor(s) that may transmit [Ca²⁺]_o information into the [Ca²⁺]_i response in coordination with CAS. Extracellular calmodulin (ExtCaM) could be such an additional [Ca²⁺]_o sensor.Calmodulin is a well-known Ca²⁺ sensor that is activated upon binding of Ca²⁺. It has been shown that calmodulin exists not only intracellularly but also extracellularly in many plant species (Biro et al., 1984; Sun et al., 1994, 1995; Cui et al., 2005). ExtCaM has been implicated in several important biological functions, such as the promotion of cell proliferation, pollen germination, and tube growth (Sun et al., 1994, 1995; Ma and Sun, 1997; Ma et al., 1999; Cui et al., 2005; Shang et al., 2005). ExtCaM is found in the cell wall of guard cells in Vicia faba and in the epidermis of Arabidopsis by immunogold labeling/electron microscopy and western-blot analyses, respectively, and the endogenous CaM in the extracellular space has been shown to regulate stomatal movements (Chen et al., 2003; Xiao et al., 2004). Under natural conditions, once the activity of ExtCaM has been inhibited by its membrane-impermeable antagonist W7-agrose or CaM antibody, stomatal opening under light is enhanced and stomatal closure in darkness is inhibited in V. faba and Arabidopsis (Chen et al., 2003; Xiao et al., 2004). [Ca²⁺]_i and cytosolic hydrogen peroxide (H₂O₂) changes, two events involved in ExtCaM-regulated stomatal movement (Chen et al., 2004), are likely regulated by light/darkness (Chen and Gallie, 2004; Tang et al., 2007), suggesting that ExtCaM plays an important physiological role in the regulation of stomatal diurnal rhythm. Calmodulin-binding proteins have been found in the protoplast of suspension-cultured Arabidopsis cells, supporting the idea that ExtCaM functions as a peptide-signaling molecule (Cui et al., 2005). Furthermore, ExtCaM triggers [Ca²⁺]_i elevation in guard cells of V. faba and Arabidopsis and in lily (Lilium daviddi) pollen (Chen et al., 2004; Xiao et al., 2004; Shang et al., 2005). These observations support the notion that ExtCaM could be a potential [Ca²⁺]_o sensor for external calcium, and this external calcium sensing could subsequently regulate the [Ca²⁺]_i level through a signaling cascade.It is interesting that ExtCaM and ABA induce some parallel changes in second messengers in guard cell signaling. Our previous studies show that ExtCaM induces [Ca²⁺]_i increase and H₂O₂ generation through the Gα-subunit (GPA1) of a heterotrimeric G protein, and increased H₂O₂ further elevates [Ca²⁺]_i (Chen et al., 2004). G protein, Ca²⁺, and H₂O₂ are well-known second messengers in ABA-induced guard cell signaling (McAinsh et al., 1995; Grabov and Blatt, 1998; Pei et al., 2000; Wang et al., 2001; Zhang et al., 2001; Liu et al., 2007). However, the signaling cascade triggered by ExtCaM in guard cells is poorly understood. New ABA signaling components in guard cells could provide a clue in the study of the molecular mechanism of ExtCaM guard cell signaling.Recently, nitric oxide (NO) has been shown to serve as an important signal molecule involved in many aspects of developmental processes, including floral transition, root growth, root gravitropism, adventitious root formation, xylogenesis, seed germination, and orientation of pollen tube growth (Beligni and Lamattina, 2000; Pagnussat et al., 2002; He et al., 2004; Prado et al., 2004; Gabaldón et al., 2005; Stohr and Stremlau, 2006). Increasing evidence points to a role for NO as an essential component in ABA signaling in guard cells (Garcia-Mata and Lamattina, 2001, 2002; Neill et al., 2002). It has been shown that nitrate reductase (NR) reduces nitrite to NO, and the nia1, nia2 NR-deficient mutant in Arabidopsis showed reduced ABA induction of stomatal closure (Desikan et al., 2002; Bright et al., 2006). Although animal nitric oxide synthase (NOS) activity has been detected in plants and inhibitors of mammalian NOS impair NO production in plants (Barroso et al., 1999; Corpas et al., 2001), the gene(s) encoding NOS in plants is still not clear. AtNOS1 in Arabidopsis was initially reported to encode a protein containing NOS activity (Guo et al., 2003). However, recent studies have raised critical questions regarding the nature of AtNOS1 and suggested that AtNOS1 appears not to encode a NOS (Crawford et al., 2006; Zemojtel et al., 2006). However, the originally described Atnos1 mutant is deficient in NO accumulation (Crawford et al., 2006). Consequently, AtNOS1 was renamed AtNOA1 (for NITRIC OXIDE ASSOCIATED1; Crawford et al., 2006). Therefore, the Atnoa1 mutant provides a useful tool for dissecting the function of NO in plants. At present, the molecules that regulate NO generation in ABA-mediated guard cell signaling are not clear. Evidence suggests that H₂O₂, a second messenger important for the regulation of many developmental processes and stomatal movement (Pei et al., 2000; Zhang et al., 2001; Coelho et al., 2002; Demidchik et al., 2003; Kwak et al., 2003), regulates NO generation in guard cells (Lum et al., 2002; He et al., 2005; Bright et al., 2006).Given the parallel signaling events induced by ABA and ExtCaM, we investigated whether NO is involved in the regulation of ExtCaM-induced stomatal closure in Arabidopsis and whether it is linked to G protein and H₂O₂, two key regulators of both ExtCaM and ABA regulation of stomatal movements. Using Arabidopsis mutants (e.g. GPA1 null mutants, the NO-producing mutant Atnoa1, and the guard cell H₂O₂ synthetic enzymatic mutant atrbohD/F) combined with pharmacological analysis, we present compelling evidence to establish a linear functional relationship between Gα, H₂O₂, and NO in ExtCaM guard cell signaling.     

拟南芥钙调素定点突变基因分离及其在钙不依赖钙调素结合蛋白检测中的应用

动植物系统研究表明,钙调素不仅在结合钙离子时调节多种靶酶或靶蛋白的活性,而且没有钙离子结合时,还可以通过结合钙不依赖的钙调素结合蛋白,发挥多种生物学作用．然而,目前却没有体内分析钙调素与钙不依赖钙调素结合蛋白相互作用的方法．首先,采用定点突变的方式,得到了拟南芥钙调素亚型2的多个突变基因mCaM2,随后,大肠杆菌重组表达突变蛋白的电泳迁移率及⁴⁵Ca²⁺覆盖分析表明,得到了编码失去钙结合能力的钙调素的突变基因mCaM2₁₂₃₄, mCaM2₁₂₃₄突变钙调素中所有4个钙结合EF-hand结构域中的关键氨基酸谷氨酸均突变为谷氨酰胺．在酵母双杂交体系中,作为诱饵蛋白的突变钙调素mCaM2₁₂₃₄与我们前期体外方法报道的钙不依赖性钙调素结合蛋白AtIQD26存在相互作用．这将为钙不依赖性钙调素结合蛋白提供有用的体内研究工具,有利于我们全面认识钙-钙调素-钙调素结合蛋白信号途径．     

一株具广谱抗真菌活性细菌菌株的分离鉴定及拮抗物的理化特性

从玉米小斑病叶片上分离到一株细菌(CGMCC No. 1982)。拮抗谱测定结果表明, 该菌株对常见致病真菌均具有不同程度的拮抗作用, 表现广谱抗真菌活性。对该菌株的菌落、菌体形态观察, 生理生化特性分析及16S rDNA序列测定结果表明, 该菌株为一株枯草芽孢杆菌。以玉米小斑病菌做为指示真菌, 对拮抗物的理化特性进行了研究, 结果表明, 在硫酸铵饱和度为60%时获得的拮抗物沉淀具较好的抗真菌活性, 并且该拮抗物对热、酸和碱较稳定; 对蛋白酶、氯仿敏感; 对紫外线部分敏感。该菌株表现出较好的微生物杀菌剂开     

蜱类分类系统的变更

随着分子生物学技术在蜱类研究中的应用,及对蜱类系统发生的深入研究,蜱的分类及命名发生了很大变更。而中国在这一领域的研究还停留在20世纪90年代,分类系统过时。文章对中国曾经采用的蜱类分类系统与现今世界上普遍认可的分类系统进行了详细比较,以期引起注意,从而促进蜱类系统学及其他研究领域的发展。     

荒漠生物结皮中藻类和苔藓植物研究进展

藻类和苔藓植物是荒漠植被演替过程中常见的两类先锋植物, 同时也是生物结皮中生物量最大的2个类群。该文综述了近年来干旱半干旱荒漠地区生物结皮中藻类和苔藓两大类植物区系及其生态作用的研究进展, 重点介绍藻类结皮、苔藓结皮的生态作用以及二者之间存在的生态学关系。在此基础上对荒漠生物结皮中藻类与苔藓植物的研究前景进行了展望, 指出荒漠生物结皮中藻类与苔藓共生机理的探讨是未来的研究重点, 这对进一步探明生物结皮中藻类和苔藓植物之间的相互作用, 揭示它们的生态学关系具有重要的理论意义和实践价值。