水稻细胞质雄性不育相关基因orf290功能域的初步研究

[目的]探讨线粒体基因orf290主要功能结构域与水稻配子体细胞质雄性不育的关系。[方法]利用生物信息学方法,分析ORF290功能结构域,构建其不同功能结构域的表达载体,完成遗传转化,采用碘-碘化钾染色法观察阳性植株的花粉育性及小穗育性,统计分析ORF290功能结构域与育性的关系。[结果]与保持系相比,各功能结构域的转基因植株农艺性状的差异不明显,花粉育性及自然结实率均有所降低,35S::orf290(T_0代)、35S::Rf1b 5'-orf290(T_1代)、35S::Rf1b 5'-orf216(T_0代)和35S::orf216(T_0代)花粉育性(结实率)分别为:50. 7%(46. 5%)、33%(42. 15%)、38. 7%(37. 9%)和60. 8%(50%)。[结论]结果表明orf290是细胞质雄性不育相关基因,且ORF290功能结构域位于C端,N端具有线粒体定位信号。     

拟南芥Rab1家族基因的克隆和功能研究

Yptl蛋白是酵母唯一的Rab1 GTP酶,调控囊泡从内质网到高尔基体的运输.酵母温敏突变株 ASY01是一个Ypt1基因功能部分缺失菌株,在26℃可以正常生长,但在37℃不能生长.拟南芥有4个Rab1基因,分别是AtRab1A1、AtRab1B1、AtRab1B2、AtRab1C1.克隆了所有4个AtRab1基因,构建酵母表达载体,转化温敏突变型酵母ASY01.温度敏感性实验结果表明,所有转基因菌株在37℃都恢复正常生长.说明拟南芥4个Rab1基因都与酵母Ypt1基因功能互补,都具有调节囊泡从内质网到高尔基体运输的功能.     

水稻OsNramp4介导锰离子吸收和转运的功能分析

锰(Mn)是植物生长发育必需的矿质营养元素。水稻天然抗性相关巨噬细胞蛋白(natural resistance-associated macrophage protein, OsNramp)家族有7个成员,其中4个具有Mn²⁺转运活性。OsNramp4已被鉴定为一个铝离子转运蛋白,但尚未报道其是否具有Mn²⁺转运活性。本文利用Mn²⁺吸收突变株Δsmf1进行OsNramp4功能互补实验,分析OsNramp4对Mn²⁺的响应,研究环境Mn²⁺胁迫对敲除突变体nramp4的生长势以及Mn²⁺吸收和转运的影响。结果显示:OsNramp4能互补Δsmf1的Mn²⁺吸收缺陷表型;水稻根部OsNramp4的表达受Mn²⁺的诱导;高Mn²⁺胁迫下, nramp4的长势明显优于野生型对照,突变体地上部Mn²⁺含量显著低于野生型,且根-茎Mn²⁺转运率明显低于野生型...     

拟南芥CBL9的质膜定位对花粉管顶端生长的影响

旨在揭示拟南芥CBL9(Calcineurin B-like protein 9)在花粉管顶端生长中的作用。基于花粉管瞬时表达体系,通过基因枪技术将带有黄色荧光蛋白(YFP)标签的CBL9在烟草花粉中进行瞬时表达,观察其亚细胞定位及过表达表型。针对CBL9的N端酰基化的保守位点,通过点突变技术构建CBL9G2A突变体,对比研究其表型及定位的改变情况。结果显示CBL9-YFP定位于花粉管顶端质膜及颗粒状细胞器上,CBL9过量表达可以引起显著的花粉管去极化生长。而CBL9G2A-YFP则表现出非特异性的胞内弥散定位,同时CBL9G2A过量表达没有显著影响花粉管的顶端生长。CBL9的N端酰基化位点是CBL9质膜定位的重要因素,而正确定位的CBL9才能发挥调控花粉管生长的相关功能。     

酿酒酵母GPCR蛋白Ste2亚细胞定位信号探索

G蛋白偶联受体(G protein-coupled receptor,GPCR)家族蛋白在细胞感受各种胞外信号过程中发挥重要作用。Ste2是酵母细胞中GPCR蛋白之一。大量文献报道了Ste2蛋白突变体对其功能和表达的影响,但关于Ste2亚细胞定位的研究相对较少。这项工作的目的在于确定Ste2亚细胞定位,探究Ste2不同跨膜域、胞内外环状结构域和N端、C端对其亚细胞定位的影响。构建了一系列结构域删除或替换突变体,通过荧光显微镜观察判断不同结构区域对Ste2亚细胞定位的影响,并通过与已知的细胞器标记蛋白共定位观察验证亚细胞定位判读结果。结果显示:野生型Ste2荧光信号出现在质膜和液泡内腔; C端缺失突变体荧光信号出现在质膜和内质网。在N端、C端、各环状结构域序列采用动物GPCR蛋白ORI7、OR17-40相应结构域替换的突变体中,C端替换导致液泡内腔信号消失,质膜信号强于野生型; N端和部分环状结构域替换不同程度减弱或消除了质膜定位,液泡腔内信号类似于野生型;部分突变体在胞内出现点状分布的荧光信号。由此推断:Ste2 N端,第一、第二胞外环状结构域和第三胞内环状结构域可能具有影响Ste2运输定位到质膜的功能;而C端则可能在Ste2离开细胞膜进入液泡的过程中发挥作用。初步确定了Ste2的不同结构区域对其定位的影响,为深入研究GPCR蛋白的亚细胞定位机制奠定基础。     

家蚕吡哆醛激酶基因定点突变及突变体功能

【目的】吡哆醛激酶（pyridoxal kinase,PLK）是维生素B₆关键代谢酶。前期研究克隆出家蚕Bombyx mori吡哆醛激酶cDNA,经序列比对发现几个重要且保守的氨基酸残基在此蛋白中被替换。为明确家蚕吡哆醛激酶分子若干特定位置上氨基酸残基在酶功能上的作用进行本研究。【方法】采用重叠延伸法对家蚕吡哆醛激酶Thr⁴⁷,Asn¹²¹, Ile⁵⁴, Arg⁸⁸和Trp²³⁰氨基酸残基进行定点突变, 构建表达载体pET-22b（+）-PLK并转入大肠杆菌Escherichia coli Rosetta中进行诱导表达,经亲和层析对重组蛋白进行纯化, 通过酶活性检测进行功能鉴定。【结果】家蚕吡哆醛激酶Thr⁴⁷,Ile⁵⁴和Arg⁸⁸氨基酸突变后酶的催化活力分别下降82%, 58%和85%;Asn¹²¹突变对酶的催化活力几乎没有影响;而Trp²³⁰突变导致酶丧失催化活性。【结论】本研究明确了选定氨基酸侧链基团在家蚕吡哆醛激酶催化功能上的意义。     

London/Swedish双突变APP转基因小鼠的鉴定

鉴定及评价APP双突变阿尔茨海默病的转基因小鼠模型。方法将London/Swedish双突变APP基因插入到PDGF启动子下游,构建转基因表达载体,通过显微注射法建立APP695^V652I/K596N/M597L双突变转基因C57BL/6J小鼠。PCR鉴定APP695双突变转基因小鼠的基因表型,RT-PCR和Western blotting检测APP突变基因表达,免疫组化检测APP695双突变转基因小鼠大脑病理改变。水迷宫检测APP695^V652I/K596N/M597L转基因小鼠的行为学改变。结果建立了2个品系的人APP695^V652I/K596N/M597L转基因小鼠。抗Aβ1-17免疫组织化学显示APP695双突变转基因小鼠海马区阳性细胞数较APP695^V652I单突变转基因小鼠,及野生小鼠阳性细胞数明显增多,胞膜着色明显加深。双突变转基因小鼠在5月龄时可检测到老年斑。行为学检测显示APP695^V652I/K596N/M597L双突变转基因小鼠学习记忆能力比APP695^V652I单突变转基因小鼠有明显下降。结论APP695^V652I/K596N/M597L转基因小鼠较APP695^V652I转基因小鼠更早出现老年斑及学习认知能力障碍。成功建立了人APP695^V652I/K596N/M597L转基因小鼠阿尔茨海默病模型,为研究阿尔茨海默病发病机制和药物研发提供了有价值的动物模型。     

木本植物花药培养的进展

近十年来,已从木本植物的8个科、9个属、约23 个种中获得了单倍体植株,它们是：茄科的滨黎叶拘祀 (Lycium halimifolzum Mill.)^[33],宁夏拘祀(L. barbarum L.)t'3拘祀(L. chinense Mill.)^[2]。杨柳科的 黑杨（Populus nigra L. )[33,小叶杨X黑杨（P. simonit Carr. X P. nigra L.),大青杨（P. ussuriensis Komar. )^[32],加拿大白杨X香杨（P. canadensis Moench X P. koreana Rehd.),哈青杨X钻天杨(P. harbinensis Wang et Skv. X P. pyramidalis Roz.)^[4],银白杨X小 叶杨（P. albs L. X P. simonii Carr.)^[5],中东杨（P. berolinensis Dipp. ),中东杨X钻夭杨(P. berolinensis Dipp. X P. pyramidalis Roz. ),小青杨（P. pseudo-simonii Kitagawa.)^[6],小青杨X钻天杨（P. pseudo-stmonzi Kitagawa. X P. pyramidalis Roz.)^[5],小叶杨（P. simonii Carr.) M,胡杨（P. euphratica Oliv. )^[7][8]。芸香科的权 壳（Poncirus trifolata (L.) Raf.)^[25], 柑桔（Citrus microcarpa Bge.)^[9] 葡萄科的葡萄(Vitis vinifera L.)^[10]蔷薇科苹果属的揪子（Malus pruni f olia (Wi- 11d.) Borkh.)^[11]以及苹果（Malus pumilea Mill.)^[12]。七叶树科的欧洲七叶树（Aesculus hippocastanum L.)^[30] 无患子科的荔枝（Litchi chinensis Sonn.)^[13]。此外,在 我国已从杉木花药培养获得小植株^[14],油茶也已从花 药愈伤组织分化出绿芽点^[15]。     

CO2浓度升高和不同氮肥水平下源库处理对粳稻茎鞘非结构性碳水化合物积累和转运的影响

为探究CO₂浓度升高和不同氮肥水平下源库处理对粳稻茎鞘非结构性碳水化合物(NSC)积累和转运的影响,利用开顶式气室(OTC),设置2个CO₂浓度([CO₂]):对照(背景大气,a[CO₂])和在背景大气[CO₂]基础上升高200μmol·mol^-1(e[CO₂])。以常规粳稻"南粳9108"为试验材料,在OTC内采用盆栽方式,设置低N(N1,10 g N·m^-2)、中N(N2,20 g N·m^-2)和高N(N3,30 g N·m^-2)3个施N水平。抽穗期源库改变设剪叶(LC)和疏花(SR)处理,以不处理为对照。测定并计算了抽穗期和成熟期叶片N含量、茎鞘NSC积累量(TM_NSC)、NSC表观转运量(ATMNSC)及其对籽粒产量的表观贡献率(AC_NSC)。采用方差分析、相关分析和逐步回归方法对上述观测数据进行分析。结果表明,[CO₂]升高显著降低抽穗期叶片N含量,显著促进中N水平的NSC积累。在不同[CO₂]和N水平下,SR处理均导致成熟期茎鞘TM_NSC显著升高,ATM_NSC和AC_NSC显著降低;在背景大气和不同N水平下,LC处理均显著降低成熟期TM_NSC,显著提高ATM_NSC,但[CO₂]升高下LC处理对成熟期TM_NSC和ATM_NSC均无显著影响。LC处理对籽粒产量及其构成未产生显著影响。粒叶比越高,成熟期TM_NSC和千粒重越低,ATM_NSC、AC_NSC、籽粒产量和收获指数越高。综合影响AC_NSC的因素为粒叶比、抽穗期和成熟期TM_NSC;综合影响籽粒产量的因素为粒叶比、成熟期叶片N含量和TM_NSC,这些综合影响均可用多元回归模型定量表述。     

野生大豆盐碱胁迫相关GsTIFY11b的克隆与功能分析

以耐盐碱野生大豆(Glycine soja L.G07256)为材料, 采用同源克隆方法和RT-PCR技术获得一个TIFY 类基因的全长cDNA(命名为GsTIFY11b)。进化树分析表明, 与其他物种相比, GsTIFY11b与拟南芥的AtTIFY11a基因相似性最高, 达到56%; 序列分析表明GsTIFY11b蛋白除具有 TIFY保守结构域外, 还具有一个N端保守结构域和一个C端保守的Jas结构域; 实时荧光定量PCR结果显示该基因受盐和碱胁迫诱导表达; 将GsTIFY11b转化拟南芥来验证其耐盐碱功能, 获得两个转基因纯合体株系, 盐碱胁迫分析结果表明, GsTIFY11b的超量表达没能提高拟南芥对盐碱胁迫的耐性, 并且与野生型相比, 转基因植株在种子萌发期和苗期表现出对盐胁迫更加敏感。盐胁迫信号通路相关marker基因在转基因拟南芥中的表达特性分析表明, GsTIFY11b可以调控RD29B、KIN1、DREB等基因的转录。在洋葱表皮细胞中瞬时表达GsTIFY11b-GFP融合蛋白的结果表明, GsTIFY11b定位于细胞核中。上述结果表明, 该基因在细胞核中起着转录调节子的作用, 可能是通过调控盐胁迫信号通路中关键基因的表达来改变植物对盐胁迫的耐受性。     

AtRabD2b, a Functional Ortholog of the Yeast Ypt1, Controls Various Growth and Developmental Processes in Arabidopsis

Rab1-related guanosine-5′-triphosphatases (GTPases) regulate the intracellular endoplasmic reticulum (ER)-to-Golgi vesicle transport in yeast, mammalian and plant systems. Arabidopsis contains four Rab1 GTPases which are further divided into two functionally distinct subclasses, RabD1 and RabD2. In this study, we investigated the function of an Arabidopsis RabD2b gene. Yeast complementary assay demonstrated that AtRabD2b is a functional counterpart of the yeast Ypt1. The active GTP-bound mutant (AtRabD2b [Q67L]) maintained the complementary ability of ypt1 mutant as wild-type AtRabD2b. Both AtRabD2b and AtRabD2b [Q67L] localize on the Golgi stacks and a second subpopulation of punctate structures in tobacco leaf cells. RT-PCR assay showed that AtRabD2b is ubiquitously expressed in different plant tissues. Transgenic plants overexpressing yellow fluorescent protein (YFP)-AtRabD2b [Q67L] fusion gene exhibited dwarfism, altered morphology of rosette leaves, and upward-pointing siliques in the stems. The downregulation of AtRabD1, AtRabD2b, and AtRabD2c, due to cosuppression of the AtRabD2b gene in the YFP-AtRabD2b [Q67L] transgenic plants, resulted in stunted bushy growth phenotype, very low fertility, and the necrosis at the apical region of the stem. Together, these data indicated that AtRabD2b gene regulates the ER-to-Golgi membrane trafficking as Ypt1 and plays a role in several aspects of plant growth and development.     

Distinct localization of two closely related Ypt3/Rab11 proteins on the trafficking pathway in higher plants.

Ypt/Rab proteins are Ras-related small GTPases that act on the intracellular membrane through the trafficking pathway, and their function depends on their localization. Approximately 25 genes encoding Ypt3/Rab11-related proteins exist in Arabidopsis, but the reason for the presence of many genes in plants remains unclear. Pea Pra2 and Pra3, members of Ypt3/Rab11, are closely related proteins. Because possible orthologs are conserved among dicots, they can be studied to determine their possible localization. Biochemical analysis revealed that these proteins were localized on distinct membranes in pea. Furthermore, using green fluorescent protein-Pra2 and green fluorescent protein-Pra3 fusion proteins, we demonstrated that these proteins are distinctively localized on the trafficking pathway in tobacco Bright Yellow 2 cells. Pra2 was predominantly localized on Golgi stacks and endosomes, which did not support the localization of Pra2 on the endoplasmic reticulum (Kang, J. G., Yun, J., Kim, D. H., Chung, K. S., Fujioka, S., Kim, J. I., Dae, H. W., Yoshida, S., Takatsuto, S., Song, P. S., and Park, C. M. (2001) Cell 105, 625--636). In contrast, Pra3 was likely to be localized on the trans-Golgi network and/or the prevacuolar compartment. We concluded that Pra2 and Pra3 proteins are distinctively localized on the trafficking pathway. This finding suggests that functional diversification takes place in the plant Ypt3/Rab11 family.     

Requirement of nucleotide exchange factor for Ypt1 GTPase mediated protein transport

Small GTPases of the rab family are involved in the regulation of vesicular transport. It is believed that cycling between the GTP- and GDP-bound forms, and accessory factors regulating this cycling are crucial for rab function. However, an essential role for rab nucleotide exchange factors has not yet been demonstrated. In this report we show the requirement of nucleotide exchange factor activity for Ypt1 GTPase mediated protein transport. The Ypt1 protein, a member of the rab family, plays a role in targeting vesicles to the acceptor compartment and is essential for the first two steps of the yeast secretory pathway. We use two YPT1 dominant mutations that contain alterations in a highly conserved GTP-binding domain, N121I and D124N. YPT1-D124N is a novel mutation that encodes a protein with nucleotide specificity modified from guanine to xanthine. This provides a tool for the study of an individual rab GTPase in crude extracts: a xanthosine triphosphate (XTP)-dependent conditional dominant mutation. Both mutations confer growth inhibition and a block in protein secretion when expressed in vivo. The purified mutant proteins do not bind either GDP or GTP. Moreover, they completely inhibit the ability of the exchange factor to stimulate nucleotide exchange for wild type Ypt1 protein, and are potent inhibitors of ER to Golgi transport in vitro at the vesicle targeting step. The inhibitory effects of the Ypt1-D124N mutant protein on both nucleotide exchange activity and protein transport in vitro can be relieved by XTP, indicating that it is the nucleotide-free form of the mutant protein that is inhibitory. These results suggest that the dominant mutant proteins inhibit protein transport by sequestering the exchange factor from the wild type Ypt1 protein, and that this factor has an essential role in vesicular transport.     

The Golgi-localized Arabidopsis endomembrane protein12 contains both endoplasmic reticulum export and Golgi retention signals at its C terminus

Endomembrane proteins (EMPs), belonging to the evolutionarily conserved transmembrane nine superfamily in yeast and mammalian cells, are characterized by the presence of a large lumenal N terminus, nine transmembrane domains, and a short cytoplasmic tail. The Arabidopsis thaliana genome contains 12 EMP members (EMP1 to EMP12), but little is known about their protein subcellular localization and function. Here, we studied the subcellular localization and targeting mechanism of EMP12 in Arabidopsis and demonstrated that (1) both endogenous EMP12 (detected by EMP12 antibodies) and green fluorescent protein (GFP)-EMP12 fusion localized to the Golgi apparatus in transgenic Arabidopsis plants; (2) GFP fusion at the C terminus of EMP12 caused mislocalization of EMP12-GFP to reach post-Golgi compartments and vacuoles for degradation in Arabidopsis cells; (3) the EMP12 cytoplasmic tail contained dual sorting signals (i.e., an endoplasmic reticulum export motif and a Golgi retention signal that interacted with COPII and COPI subunits, respectively); and (4) the Golgi retention motif of EMP12 retained several post-Golgi membrane proteins within the Golgi apparatus in gain-of-function analysis. These sorting signals are highly conserved in all plant EMP isoforms and, thus, likely represent a general mechanism for EMP targeting in plant cells.     

A rab1 GTPase is required for transport between the endoplasmic reticulum and golgi apparatus and for normal golgi movement in plants

We describe a green fluorescent protein (GFP)-based assay for investigating membrane traffic on the secretory pathway in plants. Expression of AtRab1b(N121I), predicted to be a dominant inhibitory mutant of the Arabidopsis Rab GTPase AtRab1b, resulted in accumulation of a secreted GFP marker in an intracellular reticulate compartment reminiscent of the endoplasmic reticulum. This accumulation was alleviated by coexpressing wild-type AtRab1b but not AtRab8c. When a Golgi-targeted and N-glycosylated variant of GFP was coexpressed with AtRab1b(N121I), the variant also accumulated in a reticulate network and an endoglycosidase H-sensitive population appeared. Unexpectedly, expression of AtRab1b(N121I), but not of the wild-type AtRab1b, resulted in a reduction or cessation of vectorial Golgi movement, an effect that was reversed by coexpression of the wild type. We conclude that AtRab1b function is required for transport from the endoplasmic reticulum to the Golgi apparatus and suggest that this process may be coupled to the control of Golgi movement.     

Asn229 in the third helix of VPAC1 receptor is essential for receptor activation but not for receptor phosphorylation and internalization: comparison with Asn216 in VPAC2 receptor

After stimulation with agonist, G protein coupled receptors (GPCR) undergo conformational changes that allow activation of G proteins to transduce the signal, followed by phosphorylation by kinases and arrestin binding to promote receptor internalization. Actual paradigm, based on a study of GPCR-A/rhodopsin family, suggests that a network of interactions between conserved residues located in transmembrane (TM) domains (mainly TM3, TM6 and TM7) is involved in the molecular switch leading to GPCR activation.

We evaluated in CHO cells expressing the VPAC₁ receptor the role of the third transmembrane helix in agonist signalling by point mutation into Ala of the residues highly conserved in the secretin-family of receptors: Y²²⁴, N²²⁹, F²³⁰, W²³², E²³⁶, G²³⁷, Y²³⁹, L²⁴⁰. N²²⁹A VPAC₁ mutant was characterized by a decrease in both potency and efficacy of VIP stimulated adenylate cyclase activity, by the absence of agonist stimulated [Ca²⁺]_i increase, by a preserved receptor recognition of agonists and antagonist and by a preserved sensitivity to GTP suggesting the importance of that residue for efficient G protein activation. N²²⁹D mutant was not expressed at the membrane, and the N²²⁹Q with a conserved mutation was less affected than the A mutant. Agonist stimulated phosphorylation and internalization of N²²⁹A and N²²⁹Q VPAC₁ were unaffected. However, the re-expression of internalized mutant receptors, but not that of the wild type receptor, was rapidly reversed after VIP washing. Receptor phosphorylation, internalization and re-expression may be thus dissociated from G protein activation and linked to another active conformation that may influence its trafficking.

Mutation of that conserved amino acid in VPAC₂ could be investigated only by a conservative mutation (N²¹⁶Q) and led to a receptor with a low VIP stimulation of adenylate cyclase, receptor phosphorylation and internalization. This indicated the importance of the conserved N residue in the TM3 of that family of receptors.     

Trs20 is Required for TRAPP II Assembly

The modular TRAPP complexes act as nucleotide exchangers to activate the Golgi Ypt/Rab GTPases, Ypt1 and Ypt31/Ypt32. In yeast, TRAPP I acts at the cis‐Golgi and its assembly and structure are well characterized. In contrast, TRAPP II acts at the trans‐Golgi and is poorly understood. Especially puzzling is the role of Trs20, an essential TRAPP I/II subunit required neither for the assembly of TRAPP I nor for its Ypt1‐exchange activity. Mutations in Sedlin, the human functional ortholog of Trs20, cause the cartilage‐specific disorder SEDT. Here we show that Trs20 interacts with the TRAPP II‐specific subunit Trs120. Furthermore, the Trs20‐Trs120 interaction is required for assembly of TRAPP II and for its Ypt32‐exchange activity. Finally, Trs20‐D46Y, with a single‐residue substitution equivalent to a SEDT‐causing mutation in Sedlin, interacts with TRAPP I, but the resulting TRAPP complex cannot interact with Trs120 and TRAPP II cannot be assembled. These results indicate that Trs20 is crucial for assembly of TRAPP II, and the defective assembly caused by a SEDT‐linked mutation suggests that this role is conserved .     

TRAPPII subunits are required for the specificity switch of a Ypt-Rab GEF

Ypt-Rab GTPases are key regulators of the various steps of intracellular trafficking. Guanine nucleotide-exchange factors (GEFs) regulate the conversion of Ypt-Rabs to the GTP-bound state, in which they interact with effectors that mediate all the known aspects of vesicular transport. An interesting possibility is that Ypt-Rabs coordinate separate steps of the transport pathways. The conserved modular complex TRAPP is a GEF for the Golgi gatekeepers Ypt1 and Ypt31/32 (Refs 5-7). However, it is not known how Golgi entry and exit are coordinated. TRAPP comes in two configurations: the seven-subunit TRAPPI is required for endoplasmic reticulum-to-Golgi transport, whereas the ten-subunit TRAPPII functions in late Golgi. The two essential TRAPPII-specific subunits Trs120 and Trs130 have been identified as Ypt31/32 genetic interactors. Here, we show that they are required for switching the GEF specificity of TRAPP from Ypt1 to Ypt31. Moreover, a trs130ts mutation confers opposite effects on the intracellular localization of these GTPases. We suggest that the Trs120-Trs130 subcomplex joins TRAPP in the late Golgi to switch its GEF activity from Ypt1 to Ypt31/32. Such a 'switchable' GEF could ensure sequential activation of these Ypts, thereby coordinating Golgi entry and exit.     

Expression of rat beta(1,4)-N-acetylglucosaminyltransferase III in Nicotiana tabacum remodels the plant-specific N-glycosylation

Plant N -linked glycans differ substantially from their mammalian counterparts, mainly with respect to modifications of the core glycan, which typically contains a β(1,2)-xylose and an α(1,3)-fucose. The addition of a bisecting N -acetylglucosamine residue by β(1,4)- N -acetylglucosaminyltransferase III (GnTIII) is known to control the processing of N -linked glycans in mammals, for example by preventing α(1,6)-fucosylation of the core glycan. In order to outcompete plant-specific β(1,2)-xylose and α(1,3)-fucose modifications, rat GnTIII was expressed either with its native localization domain (GnTIII) or with the cytoplasmic tail, transmembrane domain and stem region (CTS) of Arabidopsis thaliana mannosidase II (ManII) (GnTIII^A.th.). Both CTSs targeted enhanced yellow fluorescent protein (eYFP) to a brefeldin A-sensitive compartment, indicative of Golgi localization. GnTIII expression increased the fraction of N -glycans devoid of xylose and fucose from 13% ± 7% in wild-type plants to 60% ± 8% in plants expressing GnTIII^A.th.. N -Glycans of plants expressing rat GnTIII contained three major glycan structures of complex bisected, complex, or hybrid bisected type, accounting for 70%–85% of the total N -glycans. On expression of GnTIII^A.th., N -glycans displayed a higher heterogeneity and were of hybrid type. Co-expression of A. thaliana ManII significantly increased the amount of complex bisected structures relative to the plants expressing GnTIII or GnTIII^A.th., whereas co-expression of human ManII did not redirect the pool of hybrid structures towards complex-type structures. The method described offers the advantage that it can be implemented in any desired plant system for effective removal of β(1,2)-xylose and α(1,3)-fucose from the N -glycan.     

NaCl-induced changes in cytosolic free Ca2+ in Arabidopsis thaliana are heterogeneous and modified by external ionic composition

Increases in cytosolic free Ca²⁺ ([Ca²⁺]_cyt) are common to many stress-activated signalling pathways, including the response to saline environments. We have investigated the nature of NaCl-induced [Ca²⁺]_cyt signals in whole Arabidopsis thaliana seedlings using aequorin. We found that NaCl-induced increases in [Ca²⁺]_cyt are heterogeneous and mainly restricted to the root. Both the concentration of NaCl and the composition of the solution bathing the root have profound effects on the magnitude and dynamics of NaCl-induced increases in [Ca²⁺]_cyt. Alteration of external K⁺ concentration caused changes in the temporal and spatial pattern of [Ca²⁺]_cyt increase, providing evidence for Na⁺-induced Ca²⁺ influx across the plasma membrane. The effects of various pharmacological agents on NaCl-induced increases in [Ca²⁺]_cyt indicate that NaCl may induce influx of Ca²⁺ through both plasma membrane and intracellular Ca²⁺-permeable channels. Analysis of spatiotemporal [Ca²⁺]_cyt dynamics using photon-counting imaging revealed additional levels of complexity in the [Ca²⁺]_cyt signal that may reflect the oscillatory nature of NaCl-induced changes in single cells.