拟南芥WRKY61转录因子的转录活性与互作蛋白分析

该研究采用双荧光素酶报告系统、酵母双杂交和双分子荧光互补实验,对拟南芥AtWRKY61的转录活性及与AtWRKY61转录因子的互作蛋白进行了分析,并用qRT-PCR方法分析AtWRKY61对多种非生物逆境的响应特征,为进一步揭示AtWRKY61的功能与分子调控机制奠定基础。绿色荧光蛋白介导的亚细胞定位分析显示,AtWRKY61定位于细胞核内;基于原生质体的双荧光素酶报告系统和酵母实验发现,AtWRKY61具有转录抑制活性。qRT-PCR分析表明,AtWRKY61对多种非生物逆境的处理具有明显的响应,可能是在多条信号通路中发挥作用。酵母双杂交与双分子荧光互补分析表明,AtWRKY61与自身以及同组的AtWRKY9和AtWRKY72存在互作关系,暗示可能通过形成WRKY复合物来行使特定的转录抑制功能。     

盐胁迫对拟南芥AtPUB18基因的诱导表达及其启动子分析

用300mmol/L NaCl处理拟南芥幼苗,分别于处理后0、1、2、4、8、16、24、48h通过Northern Blot检测其AtPUB18基因的表达量。结果显示:拟南芥AtPUB18基因的表达量受高盐胁迫的诱导而升高,于处理后4h表达量达到最高,处理后16h表达量最低。采用PCR技术克隆AtPUB18的启动子,序列为1 974bp;序列分析发现启动子内含有大量与非生物胁迫相关的顺式作用元件,如HSE、LTR、MBS及ABRE;将启动子克隆到表达载体pCambia1300-221-GUS中,驱动报告基因GUS表达。组织化学染色结果表明,未经过高盐处理的幼苗中GUS基因表达水平很低;300mmol/L NaCl处理后GUS基因表达量显著升高。研究表明,AtPUB18的表达受高盐胁迫诱导,且AtPUB18基因的启动子是一个盐胁迫诱导型启动子。     

盐胁迫对二穗短柄草幼苗生长及不同器官中盐离子稳态的影响

采用4种浓度的NaCl溶液(50、100、150、200 mmol/L)处理二穗短柄草和拟南芥(对照)幼苗,测定不同浓度盐胁迫下2种植物幼苗的生长指标和离子分布,以探讨二穗短柄草在盐胁迫下主要阳离子平衡机制.结果表明:(1)盐胁迫显著抑制二穗短柄草根叶的生物量积累.(2)根冠比数据显示,在盐胁迫条件下二穗短柄草能够更好地维系地下部分的生物量积累.(3)在4种浓度盐胁迫下,二穗短柄草叶中Na+含量低于根系,而且K+、Cl-含量和K+/Na+比值始终高于根系,说明在二穗短柄草中Na+从地下到地上的转运受到抑制,但对Cl-的转运缺乏有效的调控.(4)回归分析发现,盐胁迫下二穗短柄草和拟南芥根部Na+与K+含量变化呈正相关关系,而在叶部则不相关,说明二穗短柄草和拟南芥Na+与K+在根部具有相同的离子通道,而在叶部却具有各自独立的转运途径.     

The HKM gene, which is identical to the MS1 gene of Arabidopsis thaliana, is essential for primexine formation and exine pattern formation

A male-sterile mutant of Arabidopsis thaliana was isolated by T-DNA tagging screening. Using transmission electron microscopy analysis, we revealed that the microspores of this mutant did not have normal thick primexine on the microspore at the tetrad stage. Instead, a moderately electron-dense layer formed around the microspores. Although microspores without normal primexine failed to form a proper reticulate exine pattern at later stages, sporopollenin was deposited and an exine-like hackly structure was observed on the microspores during the microspore stage. Thus, this mutant was named hackly microspore (hkm). It is speculated that the moderately electron-dense layer was primexine, which partially played its role in sporopollenin deposition onto the microspore. Cytological analysis revealed that the tapetum of the hkm mutant was significantly vacuolated, and that vacuolated tapetal cells crushed the microspores, resulting in the absence of pollen grains within the anther at anthesis. Single nucleotide polymorphism analysis demonstrated that the hkm mutation exists within the MS1 gene, which has been reportedly expressed within the tapetum. Our results suggest that the critical process of primexine formation is under sporophytic control .     

中间锦鸡儿WRKY75基因对拟南芥耐受盐和ABA能力的影响

该研究从旱生灌木中间锦鸡儿中克隆得到1个CiWRKY75基因。序列分析显示,CiWRKY75开放阅读框长570bp,编码189个氨基酸,含有1个WRKYGQK基序和1个C2H2型锌指结构,属于第二类WRKY转录因子。亚细胞定位显示,CiWRKY75定位于细胞核。实时荧光定量PCR检测表明,CiWRKY75基因的表达受盐胁迫和ABA诱导。在拟南芥中过量表达CiWRKY75后,与野生型拟南芥相比,转基因株系种子的萌发率在盐胁迫下降低,并且对盐胁迫的耐受能力明显减弱;ABA处理下,2个转基因株系的种子萌发率(10.3%、9.6%)较野生型(25.9%)明显降低。研究表明,CiWRKY75是中间锦鸡儿对盐和ABA响应的重要调控因子。     

Identification of Arabidopsis thaliana variants with differential glyphosate responses

To facilitate future investigations of glyphosate-resistance mechanisms, three approaches were taken to obtain Arabidopsis thaliana variants that differed in glyphosate response. Recurrent selection by spraying with sub-lethal glyphosate concentrations was performed with Columbia-0 seedlings. After seven cycles of treatment, no resistance was found. A population of 800,000 ethylmethanesulfonate-mutagenized M(2) seedlings was screened on agar containing 0.2mM glyphosate, a lower concentration than that previously used in other studies, and no resistant mutants were recovered. Seventy-two Arabidopsis ecotypes were screened with glyphosate and a range of responses was observed. In a follow-up experiment on a subset of these ecotypes, reduction of seed yield by 11.5 g/ha glyphosate (about 1% the typical field use rate) ranged among ecotypes from 0% to >90%, relative to untreated controls. However, even the least sensitive ecotypes were severely injured by relatively low glyphosate rates. Overall, attempts to select Arabidopsis seedlings that were significantly glyphosate-resistant were unsuccessful and consistent with previous reports. Arabidopsis ecotypes identified with differential glyphosate responses could be used for further studies though the inherently high sensitivity of Arabidopsis to glyphosate could limit their utility in studying glyphosate-resistance mechanisms.     

Processed Pseudogenes, Processed Genes, and Spontaneous Mutations in the Arabidopsis Genome

We identified 411 processed sequences in the Arabidopsis thaliana genome based on the fact that they have lost their intron(s) and have a length that is at least 95% of the length of the gene that gave rise to them. These sequences were generated by 230 different genes and clearly originated from retrotranspositons events because most of them (91%) have a poly(A)-tail. They are composed of 376 sequences with frame shifts and/or premature stop codons (processed pseudogenes) and 35 sequences without disablements (processed genes). Eleven of these processed genes are likely functional retrotransposed genes because they have low Ka/Ks ratios and high Ks values, and their sequences match numerous Arabidopsis ESTs. Processed sequences are mostly randomly distributed in the Arabidopsis genome and their rate of accumulation has steadily been decreasing since it peaked some 50 MYA. In contrast with the situation observed in mammals, the processed sequences found in the Arabidopsis genome originate from genes with high copy numbers and not from highly expressed genes. The patterns of spontaneous mutations in Arabidopsis are slightly different than those of mammals but are similar to those observed in Drosophila. This suggests that methylated cytosine deamination is less frequent in Arabidopsis than in mammals. Electronic Supplementary Material Electronic Supplementary material is available for this article at and accessible for authorised users. [Reviewing Editor: Dr. Juergen Brosius]     

Developmental anatomy of cotyledons and leaves in has mutant of Arabidopsis thaliana

Summary. In this work, we analyzed the developmental anatomy of cotyledons and leaves in the has mutant of Arabidopsis thaliana. It is a recessive T-DNA insertion mutation that causes changes in the size, shape, and tissue organization of the cotyledons and leaves of has plants. Analysis of has cotyledons revealed a prominent decrease in the cell number and an increase in the area of cotyledon cells and intercellular spaces of has plants. At early stages of development, has leaves are fingerlike structures, but later they develop small, lobed blades with rare trichomes. An important characteristic of the mutant leaf anatomy is the absence of mesophyll tissue differentiation. In addition, both cotyledons and leaves display a disrupted pattern of vascular bundles. Furthermore, mutant plants are defective in root and shoot morphology, indicating that the has mutation affects a number of aspects in plant development. Correspondence and reprints: Institute of Botany and “Jevremovac” Botanical Garden, Faculty of Biology, Belgrade University, Takovska 43, 11 000 Belgrade, Serbia.     

An Arabidopsis gene with homology to glutathione S-transferases is regulated by ethylene

A cDNA clone obtained from Arabidopsis leaf RNA encodes a 24 kDa protein with homology to glutathione S-transferases (GST). It is most homologous with a tobacco GST (57% identity). In Arabidopsis, expression of GST mRNA is regulated by ethylene. Exposure of plants to ethylene increased the abundance of GST mRNA, while treatment with norbornadiene had the reverse effect. Ethylene had no effect on the mRNA level in ethylene-insensitive etr1 plants. The abundance of this mRNA increased with the age of plants. DNA hybridizations indicate that GSTs are encoded by a large multigene family in Arabidopsis.     

Regulation of cyclin-dependent kinases in Arabidopsis thaliana

In plants, different families of cyclin-dependent kinases (CDKs) and cyclins have been identified, indicating that also in plants the progression through the cell cycle is regulated by CDKs. In all eukaryotes, CDKs exert their activity through well-controlled phosphorylations of specific substrates on serine/threonine residues. Such post-translational modifications are universal mechanisms in signal transduction pathways. They allow the organism to differentiate, regulate growth and/or adapt to environmental changes, the latter being crucial for plants because of their sedentary life-style. This adaptation might explain the occurrence of a special CDK type with plant-specific features. This review focuses on the involvement of plant CDKs in different phases of the cell cycle in Arabidopsis thaliana and outlines their regulation by binding to other proteins, and by phosphorylation and dephosphorylation.     

拟南芥AtPUB18的亚细胞定位和酶活性及AtPUB18的表达

通过RT-PCR技术从拟南芥中克隆到AtPUB18全长ORF。利用VectorNTI和MEGA 5.0对蛋白序列进行生物信息学分析结果显示,AtPUB18和AtPUB19蛋白序列相似性为74.9%,一致性为63.5%;构建AtPUB18启动子(1 974bp)融合GUS报告基因载体并转化拟南芥,组织化学染色分析显示,低温和干旱诱导后转基因植株中AtPUB18表达水平显著提高;构建AtPUB18与绿色荧光蛋白基因(GFP)融合的瞬时表达载体并转化原生质体,激光共聚焦显微镜观察发现,AtPUB18-GFP融合蛋白分布在细胞核和细胞质中;构建AtPUB18与麦芽糖结合蛋白基因(MBP)融合表达载体并转化大肠杆菌表达融合蛋白,纯化后的蛋白进行泛素连接酶活性检测结果显示,在小麦泛素激活酶E1和人泛素结合酶E2存在时,AtPUB18具有泛素连接酶活性。研究表明,AtPUB18是一个有功能的泛素连接酶,定位在细胞膜和细胞质中,可能参与拟南芥对非生物胁迫的响应。     

Expression Patterns of Duplicate Genes in the Developing Root in Arabidopsis thaliana

Data on gene expression in the development of the root in Arabidopsis thaliana were used to test for expression profile differences among multi-gene families and to examine the extent to which expression differences accompanied coding sequences divergence within families. Significant differences among families were observed on two principal axes, accounting for over 80% of the variance in the expression data. The number of synonymous nucleotide substitutions per synonymous site (d_S) and the number of nonsynonymous nucleotide substitutions per nonsynonymous site (d_N) were estimated between the members of two-member families (N=428) and between phylogenetically independent sister pairs (N=190) of sequences within larger families. Ribosomal proteins and a few other proteins were exceptional in showing highly divergent expression patterns in spite of very low levels of amino acid sequence divergence, as indicated by the low d_N relative to d_S. However, the majority of gene duplicates showed relatively high levels of amino acid sequence divergence without appreciable change in expression pattern in the cell types analyzed. Reviewing Editor:Dr. Manyuan Long     

GFP-FABD2 fusion construct allows in vivo visualization of the dynamic actin cytoskeleton in all cells of Arabidopsis seedlings

In vivo visualization of filamentous actin in all cells of Arabidopsis thaliana seedlings is essential for understanding the numerous roles of the actin cytoskeleton in diverse processes of cell differentiation. A previously introduced reporter construct based on the actin-binding domain of mouse talin proved to be useful for unravelling some of these aspects in cell layers close to the organ surface. However, cells more deeply embedded, especially stelar cells active in polar transport of auxin, show either diffuse or no fluorescence at all due to the lack of expression of the fusion protein. The same problem is encountered in the root meristem. Recently introduced actin reporters based on fusions between A. thaliana fimbrin 1 and GFP gave brilliant results in organs from the root differentiation zone upwards to the leaves, however failed to depict the filamentous actin cytoskeleton in the transition zone of the root, in the apical meristem and the root cap. To overcome these problems, we have prepared new transgenic lines for the visualization of F-actin in vivo. We report here that a construct consisting of GFP fused to the C-terminal half of A. thaliana fimbrin 1 reveals dynamic arrays of F-actin in all cells of stably transformed A. thaliana seedlings.     

An Arabidopsis mutant showing aberrations in male meiosis

A male-sterile mutant, mei-1, of Arabidopsis thaliana is described. In this mutant, instead of a tetrad of four microspores being formed after meiosis, a tetrad consisting of from five to eight microspores is formed. The microspores show a wide range of sizes and of DNA contents. The mutant is female-fertile. This mutant was produced by seed transformation with Agrobacterium and appears to be T-DNA tagged.     

Reduction of mineral nutrient availability accelerates flowering of Arabidopsis thaliana

The time of flowering is regulated by various environmental cues, and in some plant species, it is known to be affected by abiotic stresses. We investigated the effect of nutrient stress caused by an abrupt reduction of mineral nutrition on flowering of Arabidopsis thaliana. We used a hydroponic culture system that enabled us to precisely control nutrient levels. When plants were grown in full-strength nutrient solution for several weeks and then transferred to a diluted medium, the time from sowing to bud appearance was significantly shortened. This acceleration of flowering was more pronounced in short days than in long days, and stronger in the ecotype Landsberg erecta than in Columbia and San Feliu-2. The response was also affected by the age of plants at the beginning of nutrient stress and by the concentration of the diluted medium: earlier treatment and more diluted solutions strengthened the effect. Flowering was affected by nutrient stress, not by a change in the osmotic potential of the medium: addition of mannitol to a 1000-fold diluted solution had no effect on the promotion of flowering. When 3-week-old Landsberg erecta plants were exposed to 1000-fold diluted nutrient solution in an 8-h day length, flower bud appearance was strongly and reproducibly advanced by 10.8–12.8 d compared with control plants (which developed buds 41.1–46.2 d after sowing). This treatment can serve as an optimized protocol for future studies concerning physiological, molecular and ecological aspects of flower induction by nutrient stress in A. thaliana.