A novel root gravitropism mutant of Arabidopsis thaliana exhibiting altered auxin physiology

A root gravitropism mutant was isolated from the DuPont Arabidopsis thaliana T-DNA insertional mutagenesis collection. This mutant has reduced root gravitropism, hence the name rgrl. Roots of rgrl are shorter than those of wild-type, and they have reduced lateral root formation. In addition, roots of rgrl coil clockwise on inclined agar plates, unlike wild-type roots which grow in a wavy pattern. The rgrl mutant has increased resistance, as measured by root elongation, to exogenously applied auxins (6-fold to indole-3-acetic acid, 3-fold to 2,4-dichlorophenoxyacetic acid, and 2-fold to napthyleneacetic acid). It is also resistant to polar auxin transport inhibitors (2-fold to triiodobenzoic acid and 3- to 5-fold lo napthyleneacetic acid). The rgrl mutant does not appear to be resistant to other plant hormone classes. When grown in the presence of 10^?2 M 2.4-dichlorophenoxyacetic acid, rgrl roots have fewer root hairs than wild type. All these rgrl phenotypes are Mendelian recessives. Complementation tests indicate that rgrl is not allelic to previously characterized agravitropic or auxin-resistant mutants. The rgrl locus was mapped using visible markers to 1.4 ± 0.6 map units from the CHI locus at 1–65.4. The rgrl mutation and the T-DNA cosegregate, suggesting that rgrl was caused by insertional gene inactivation.     

拟南芥室内培养技术

本文报道了室内培养拟南芥的一些简便易行的改进技术.采用我们改进的营养土、蛭石、素沙混合培养介质和直播方式培养拟南芥,并根据其生物学特性在温度、空气湿度、土壤水分和光照等方面给予适当管理,能培养出生长更健壮、更好地满足实验要求的拟南芥植株.此外还介绍了播种、浇水、生育期调节、种子保存、病虫害防治和防混杂等环节的一些技巧措施.与其他培养方法相比,此法不仅简便、效果好,而且适合较简易的培养条件.     

Costs and benefits of cold tolerance in transgenic Arabidopsis thaliana

Cold tolerance in plants is an ecologically important trait that has been under intensive study for basic and applied reasons. Determining the fitness benefits and costs of cold tolerance has previously been difficult because cold tolerance is normally an induced trait that is not expressed in warm environments. The recent creation of transgenic plants constitutively expressing cold tolerance genes enables the investigation of the fitness consequences of cold tolerance in multiple temperature environments. We studied three genes from the CBF (C-repeat/dehydration responsive element binding factor) cold tolerance pathway, CBF1, 2 and 3, in Arabidopsis thaliana to test for benefits and costs of constitutive cold tolerance. We used multiple insertion lines for each transgene and grew the lines in cold and control conditions. Costs of cold tolerance, as determined by fruit number, varied by individual transgene. CBF2 and 3 overexpressers showed costs of cold tolerance, and no fitness benefits, in both environments. CBF1 overexpressing plants showed no fitness cost of cold tolerance in the control environment and showed a marginal fitness benefit in the cold environment. These results suggest that constitutive expression of traits that are normally induced in response to environmental stress will not always lead to costs in the absence of that stress, and that the ecological risks of CBF transgene escape should be assessed prior to their use in commercial agriculture.     

A novel calmodulin-binding protein functions as a negative regulator of osmotic stress tolerance in Arabidopsis thaliana seedlings

A clone for a novel Arabidopsisthaliana calmodulin (CaM)-binding protein of 25 kDa (AtCaMBP25) has been isolated by using a radiolabelled CaM probe to screen a cDNA expression library derived from A. thaliana cell suspension cultures challenged with osmotic stress. The deduced amino acid sequence of AtCaMBP25 contains putative nuclear localization sequences and shares significant degree of similarity with hypothetical plant proteins only. Fusion of the AtCaMBP25 coding sequence to reporter genes targets the hybrid protein to the nucleus. Bacterially expressed AtCaMBP25 binds, in a calcium-dependent manner, to a canonical CaM but not to a less conserved isoform of the calcium sensor. AtCaMBP25 is encoded by a single-copy gene, whose expression is induced in Arabidopsis seedlings exposed to dehydration, low temperature or high salinity. Transgenic plants overexpressing AtCaMBP25 exhibits an increased sensitivity to both ionic (NaCl) and non-ionic (mannitol) osmotic stress during seed germination and seedling growth. By contrast, transgenic lines expressing antisense AtCaMBP25 are significantly more tolerant to mannitol and NaCl stresses than the wild type. Thus, the AtCaMBP25 gene functions as a negative effector of osmotic stress tolerance and likely participates in stress signal transduction pathways.     

Overexpression of the rice Osmyb4 gene increases chilling and freezing tolerance of Arabidopsis thaliana plants

The expression of the gene Osmyb4, detected at low level in rice (Oryza sativa) coleoptiles grown for 3 days at 29 degrees C, is strongly induced by treatments at 4 degrees C. At sublethal temperatures of 10 and 15 degrees C, its expression in rice seedlings is already evident, but this effect cannot be vicariated by other stresses or ABA treatment. We demonstrate by transient expression that Myb4 transactivates the PAL2, ScD9 SAD and COR15a cold-inducible promoters. The Osmyb4 function in vivo is demonstrated overexpressing its cDNA in Arabidopsis thaliana plants (ecotype Wassilewskija) under the control of the constitutive CaMV 35S promoter. Myb4 overexpressing plants show a significant increased cold and freezing tolerance, measured as membrane or Photosystem II (PSII) stability and as whole plant tolerance. Finally, in Osmyb4 transgenic plants, the expression of genes participating in different cold-induced pathways is affected, suggesting that Myb4 represents a master switch in cold tolerance.     

A non-destructive selection method for resistance to fusicoccin in Arabidopsis thaliana

A mutagenised population of seeds of Arabidopsis thaliana was allowed to germinate in the presence of the positively charged aminoglycoside hygromycin (4 μg/ml) and the fungal toxin fusicoccin (5×10^–6 m). This hygromycin concentration, which is non-toxic by itself, becomes toxic when used together with fusicoccin, which stimulates cation uptake. Seeds that had germinated after 3–5 days and produced seedlings with green cotyledons were potentially resistant to fusicoccin and were therefore transferred into sterile Magenta vessels containing 1/2-strength Murashige and Skoog medium. This selection procedure is non-destructive, i.e. it allows the recovery of viable seedlings and their growth into adult plants thus permitting direct physiological characterisation. Received: 16 February 1998 / Revision received: 11 August 1998 / Accepted: 13 August 1998     

Heterosis in the freezing tolerance of crosses between two Arabidopsis thaliana accessions (Columbia-0 and C24) that show differences in non-acclimated and acclimated freezing tolerance

Heterosis is broadly defined as the increased vigour of hybrids in comparison to their parents. In the model plant Arabidopsis thaliana, a significant heterosis effect on leaf-freezing tolerance was observed in the F(1) generation of a cross between the accessions Columbia-0 (Col) and C24. Parental Col plants were significantly more freezing-tolerant than C24 plants in both the acclimated and non-acclimated (NA) states. Mid-parent heterosis was observed in the F(1) plants, both in the basic tolerance of non-adapted plants and in freezing tolerance after cold acclimation. Best-parent heterosis, on the other hand, was only found after cold acclimation. The heterosis effect was reduced in the F(2) populations such that only mid-parent heterosis was evident. The leaf content of soluble sugars (fructose (Fru), glucose (Glc), sucrose (Suc) and raffinose (Raf)) increased dramatically in the F(1) plants after cold acclimation as compared to the parental lines. The content of proline (Pro), however, was only moderately increased in the F(1) plants under the same conditions. Correlation analyses showed that only Raf content was consistently related to leaf-freezing tolerance in both the acclimated and NA states. A quantification of mRNA levels in leaves of parental and F(1) lines using quantitative real-time RT-PCR showed no clear indication for an involvement of the investigated genes (CBF (C-repeat binding factor)1, CBF2, (cold-regulated protein (COR) 6.6, COR15a, COR15b, COR47 and COR78) in the heterosis effect.     

新疆北部拟南芥自然居群表型变异与协变

拟南芥(Arabidopsis thaliana)自然居群的表型特征代表其在自然环境下的适应状况, 不同居群间特征的对比可以为了解拟南芥表型变化规律, 进而分析其形成过程和机制提供重要线索。本研究以分布于新疆北部天山、塔尔巴哈台山和阿尔泰山的10个种群的9个表型性状为基础, 对比分析了小尺度、局域尺度和区域尺度环境下原生境拟南芥种群表型性状的变化。结果发现, 不同性状对环境变化的反应不同, 其中株高、株重、根重、根长、单个果实重、果实开裂力度在3种环境尺度下种群间的差异均达到极显著水平, 而分枝数、果实长度的种群间变化不显著, 种群间的表型分化系数较低。不同环境尺度下株重、根重、单株果数均表现出一致的协变格局, 反映了生理功能性状之间整合对拟南芥适应环境的重要性。同时, 各种群间整体的性状协变差异性明显, 根长、单个果实重、分枝数、果实长度、果实开裂力度等特征与其他特征协变具有明显的局部性, 局域尺度和区域尺度环境之间的变化较大。聚类分析发现区域尺度上的不同种群聚合在一起的现象非常突出, 进一步表明拟南芥的表型特征受微环境的强烈影响。Mantel检验表明, 小尺度上10个种群株高、株重、根重、单个果实重、果实长度、果实开裂力度6个性状变化存在显著的空间相关性, 而分枝数、根长的相关性却不显著。因此, 我们认为拟南芥表型变化受小尺度环境的影响强烈, 但在表型层面并非所有性状都与原生境气候存在遗传关联。     

拟南芥根原生韧皮部筛管分子的超微结构

运用高压冷冻替代方法固定处理材料,在透射电镜下观察了拟南芥(Arabidopsis thaliana L.)根原生韧皮部筛管分子在发育过程中的超微结构变化.结果表明:在筛管分子发育过程中,细胞核具有细胞程序化死亡的典型特征,出现核膜内陷、核质聚集并边缘化、核膜破毁以及最后核消失.核膜在破毁前一直呈饱满状态,未出现核膜皱缩、核裂瓣和核周腔明显膨大等现象.在成熟筛管分子的细胞质内,具单层膜的淀粉状颗粒.这些淀粉状颗粒常与线粒体在一起,可能为线粒体的产能活动提供基质.小液泡发生于内质网,未见大液泡的形成.     

Cytosolic proline is required for basal freezing tolerance in Arabidopsis

The amino acid proline accumulates in many plant species under abiotic stress conditions, and various protective functions have been proposed. During cold stress, however, proline content in Arabidopsis thaliana does not correlate with freezing tolerance. Freezing sensitivity of a starchless plastidic phosphoglucomutase mutant (pgm) indicated that localization of proline in the cytosol might stabilize the plasma membrane during freeze–thaw events. Here, we show that re-allocation of proline from cytosol to vacuole was similar in the pyrroline-5-carboxylate synthase 2–1 (p5cs2–1) mutant and the pgm mutant and caused similar reduction of basal freezing tolerance. In contrast, the starch excess 1–1 mutant (sex1-1) had even lower freezing tolerance than pgm but did not affect sub-cellular localization of proline. Freezing sensitivity of sex1-1 mutants affected primarily the photosynthetic electron transport and was enhanced in a sex1-1::p5cs2–1 double mutant. These findings indicate that several independent factors determine basal freezing tolerance. In a pgm::p5cs2–1 double mutant, freezing sensitivity and proline allocation to the vacuole were the same as in the parental lines, indicating that the lack of cytosolic proline was the common cause of reduced basal freezing tolerance in both mutants. We conclude that cytosolic proline is an important factor in freezing tolerance of non-acclimated plants.     

Prenylcysteine methylesterase in Arabidopsis thaliana

Prenylated proteins undergo a series of post-translational modifications, including prenylation, proteolysis, and methylation. Collectively, these modifications generate a prenylcysteine methylester at the carboxyl terminus and modulate protein targeting and function. Prenylcysteine methylation is the only reversible step in this series of modifications. However, prenylcysteine -carboxyl methylesterase (PCME) activity has not been described in plants. We have detected a specific PCME activity in Arabidopsis thaliana membranes that discriminates between biologically relevant and irrelevant prenylcysteine methylester substrates. Furthermore, we have identified an Arabidopsis gene (At5g15860) that encodes measurable PCME activity in recombinant yeast cells with greater specificity for biologically relevant prenylcysteine methylesters than the activity found in Arabidopsis membranes. These results suggest that specific and non-specific esterases catalyze the demethylation of prenylcysteine methylesters in Arabidopsis membranes. Our findings are discussed in the context of prenylcysteine methylation/demethylation as a potential regulatory mechanism for membrane association and function of prenylated proteins in Arabidopsis.     

拟南芥抗盐突变体的RAPD分析

以筛选得到可以稳定遗传的抗盐单基因突变体2^#和15^#以及野生型拟南芥为材料进行RAPD分析,150条引物中有3条引物在突变体之间扩增出多态性,不仅证明了DNA水平突变的发生,而且表明它们之间遗传背景相似,是一系列抗盐性不同的近似等位基因系。1条引物在突变体的扩增产物比在野生型的扩增产物多出一个大小约为1200bp的片段,初步认为该片段与抗盐的主效基因有关。     

拟南芥根原生韧皮部筛管分子的超微结构

运用高压冷冻替代方法固定处理材料,在透射电镜下观察了拟南芥（Arabidopsis thaliana L.)根原生韧皮部筛管分子在发育过程中的超微结构变化。结果表明：在筛管分子发育过程中,细胞核具有细胞程序死亡的典型特征,出现核膜内陷、核质聚集并边缘化,核膜破毁以及最后核消失,核膜在破毁前一直呈饱满状态,未出现核膜皱缩,核裂瓣和核周腔明显膨大等现象。在成熟筛管分子的细胞质内,具单层膜的淀粉状颗粒,这些淀粉状态颗粒常与线粒体在一起,可能为线粒体的产能活动提供基质,小液泡发生于内质网,未见大液泡的形成。     

Clinal variation in freezing tolerance among natural accessions of Arabidopsis thaliana

Low temperature represents a form of abiotic stress that varies predictably with latitude and altitude and to which organisms have evolved multiple physiological responses. Plants provide an especially useful experimental system for investigating the ecological and evolutionary dynamics of tolerance to low temperature because of their sessile lifestyle and inability to escape ambient atmospheric conditions. Here, intraspecific variation in freezing tolerance was investigated in Arabidopsis thaliana by conducting freezing tolerance assays on 71 accessions collected from across the native range of the species. Assays were performed at multiple minimum temperatures and on both cold-acclimated and non-cold-acclimated individuals. Considerable variation in freezing tolerance was observed among accessions both with and without a prior cold-acclimation treatment, suggesting that differences among accessions in cold-acclimation capacity as well as differences in intrinsic physiology contribute to variation in this phenotype. A highly significant positive relationship was observed between freezing tolerance and latitude of origin of accessions, consistent with a major role for natural selection in shaping variation in this phenotype. Clinal variation in freezing tolerance in A. thaliana coupled with considerable knowledge of the underlying genetics and physiology of this phenotype should allow evolutionary genetic analysis at multiple levels.     

QTL architecture of resistance and tolerance traits in Arabidopsis thaliana in natural environments

Quantitative-genetic approaches have offered significant insights into phenotypic evolution. However, quantitative-genetic analyses fail to provide information about the evolutionary relevance of specific loci. One complex and ecologically relevant trait for plants is their resistance to herbivory because natural enemies can impose significant damage. To illustrate the insights of combined molecular and ecological research, we present the results of a field study mapping quantitative trait loci (QTL) for resistance and tolerance to natural rabbit herbivory in the genetic model, Arabidopsis thaliana. Replicates of the Ler x Col recombinant inbred lines were planted into field sites simulating natural autumn and spring seasonal germination cohorts. Shortly after flowering, herbivores removed the main flowering inflorescence (apical meristem). We found several main-effect QTL for resistance within each seasonal cohort and significant QTL-season interactions, demonstrating that the loci underlying resistance to a single herbivore differ across seasonal environments. The presence of QTL x environment also shows that variation at specific loci is only available to selection in some environments. Despite significant among-line variance components, no QTL for tolerance were detected. The combined results of the quantitative-genetic and QTL analyses demonstrate that many loci of small effect underlie tolerance to damage by rabbits, and counter the hypothesis of locus-specific tradeoffs between resistance and tolerance. The results also provide insights as to the locus-specific nature of evolutionary constraints, i.e. some loci influence flowering time and resistance in both seasonal cohorts. Our results show how linking molecular-genetic tools with field studies in ecologically relevant settings can clarify the role of specific loci in the evolution of quantitative traits.     

油菜素内酯不敏感型的拟南芥突变型筛选

自1979年Grove等首次从油菜（＆．wM－。。WL·）花粉中分离出油菜素内酯（brassinolide,BR）以来,人们已在该激素的生理反应和对植物生长发育等方面进行了许多研究（Kalinich等1985,Mandava1988,吴登如和赵硫橘1993）。但由于这类激素在10－'mol／L浓度水平就能诱导大豆、水稻等多种植物细胞的生长和分裂（Sasse1991）,而且在植物体内含量极低,因此用传统的方法研究它的作用方式非常困难。目前,利用激素突变体来研究激素代谢及其分子机制已有不少成功的例子,如生长素（Keily和Bradford1986,Lincoln等1990）、赤霉素（Singh…