Measuring spatial variation in natural selection using randomly-sown seeds of Arabidopsis thaliana

We describe a simple method of measuring spatial variation in fitness using randomly sown seeds. Given a random initial distribution of seeds, any spatial variation in the final distribution of genotypes must be the result of spatial variation in natural selection. Departures from spatial randomness are tested using a modification of join-count statistics, based on the probability that two randomly chosen plants separated by a given distance carry the same genetic marker. Monte Carlo simulations showed that for the randomly sown seeds design the join-count test had higher power to detect spatial variation in fitness than analysis of variance. The power was relatively independent of the number of genotypes used or the number of microsites sampled. The method was tested in the field using three standard lines of Arabidopsis thaliana. At the end of the life cycle, adjacent plants were significantly more likely to be the same line than randomly chosen pairs and the spatial dependence extended to distances of nearly 50 cm. Simple experiments like this may be especially useful for “comparative ecological genetics”, allowing systematic studies of natural selection among many species or habitats.     

Proteomic analysis of S-nitrosylated proteins in Arabidopsis thaliana undergoing hypersensitive response

Nitric oxide (NO) has a fundamental role in the plant hypersensitive disease resistance response (HR), and S-nitrosylation is emerging as an important mechanism for the transduction of its bioactivity. A key step toward elucidating the mechanisms by which NO functions during the HR is the identification of the proteins that are subjected to this PTM. By using a proteomic approach involving 2-DE and MS we characterized, for the first time, changes in S-nitrosylated proteins in Arabidopsis thaliana undergoing HR. The 16 S-nitrosylated proteins identified are mostly enzymes serving intermediary metabolism, signaling and antioxidant defense. The study of the effects of S-nitrosylation on the activity of the identified proteins and its role during the execution of the disease resistance response will help to understand S-nitrosylation function and significance in plants.     

拟南芥雄性不育突变体ms1142的遗传定位与功能分析

经EMS诱变野生型拟南芥(Arabidopsis thaliana)群体筛选得到一株雄性不育突变体ms1142, 突变体的果荚短小, 不含种子。细胞学观察和扫描电镜结果表明, 突变体花药发育过程中, 花药中小孢子外壁异常、破裂, 最后没有花粉形成。遗传分析表明, 该突变体为隐性单核基因突变所致; 利用图位克隆的方法将MS1142基因定位于第1条染色体的BAC克隆F16P17上44 kb区间内, 目前尚未见该区间内有雄性不育基因的报道。以上结果结合生物信息学分析表明, MS1142是一个新的调控花药发育的关键基因。该工作为花药发育关键基因MS1142的克隆及功能分析奠定了基础。     

拟南芥白化突变体心口的基因定位与分析

EMS30是拟南芥经甲基磺酸乙酯（EMS）诱变得到的白化突变体。该突变体的叶绿体结构存在严重缺陷,同时伴随叶绿素缺失。遗传分析显示EMS30突变体的突变表型受隐性单基因控制。采用图位克隆的方法对EMS30突变基因进行定位的结果显示,该基因位于拟南芥第一条染色体的分子标记F21M12和F14N23之间的96kb区间内,该区间包含25个基因。通过生物信息学分析发现,该区间内有3个基因定位在叶绿体或与叶绿体发育相关。这些结果有助于该基因的克隆,为阐释叶绿体发育提供线索。     

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.     

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。     

天山北部拟南芥自然居群表型变化的环境依赖特征

拟南芥(Arabidopsis thaliana)是植物生物学的模式植物, 在分子遗传学方面已经积累了丰富的研究成果, 但目前对拟南芥自然分布的生境特点、表型变化的环境依赖特征等研究很少, 极大地限制了对拟南芥进化动力和机制的理解。为了了解在微环境下拟南芥种群分布和表型性状的变化特点, 对天山北部分布于塔尔巴哈台山、阿尔泰山和天山的10个拟南芥种群的分布特征、表型的变化特点, 以及与综合环境因子的相互关系进行了分析。结果表明: 除分枝数外, 株高、株重、根重、单个果实重量、单株果数、单株果重、果长、果实开裂力度、单株果重/总重9个特征在种群间变化显著, 可塑性能力较强; 但方差分析和变异系数结果显示, 角果长度、果实开裂力度在种群内和种群间的变化相对较小。表型特征在山系间、经纬度和海拔间的变化规律不明显。拟南芥主要分布于pH值和HCO₃ ^-含量低, 有机质丰富, 且有一定坡度的沙土地块上。种群内拟南芥分布频度很低, 在1.56%-10.69%之间, 空间自相关距离在15.4-46.7 cm之间变化较大, 10个种群均呈现极显著集群分布, 分布的集群性受果实开裂力度的影响显著, 而果实开裂力度随环境胁迫而极显著增加。总结认为: 天山北部拟南芥生长和分布主要受微环境的影响, 在干旱环境下, 拟南芥主要通过增加繁殖分配比例, 产生难开裂的果实, 促使种子短距离扩散于母株周围, 确保子代利用原适宜生境来生存繁衍。     

拟南芥雄性不育突变体ms214的遗传与功能分析

经过EMS诱变、背景纯化以及遗传分析,得到一株隐性核基因控制的拟南芥雄性不育突变体ms214,用图位克隆的方法将突变基因MS214定位于拟南芥第一条染色体上顶端700kb的区间内。生物信息学分析发现,该区间内有一个与蜡质合成有关的基因CERl;测序分析表明在突变体ms214中,CERl基因第一个外显子上碱基由C^509变成了u^509的突变,导致CER蛋白在该处的氨基酸由脯氨酸^170变成了亮氨酸^170;等位实验结果表明ms214和cerl是等位突变体。ms214突变体的茎和果荚呈现出与野生型不同的亮绿色;组织切片观察结果表明,突变体花药发育各个时期无异常变化;扫描电镜观察发现ms214的茎和果荚的表皮没有蜡质的形成,但是突变体成熟花粉粒表面含油层异常,具有许多小的脂肪小滴。这些结果揭示了MS214蛋白质参与蜡质合成过程,而且脯氨酸^170是该蛋白质行使功能所必需的。     

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.     

Nucleotide variation at the myrosinase-encoding locus, TGG1, and quantitative myrosinase enzyme activity variation in Arabidopsis thaliana

The Arabidopsis thaliana TGG1 gene encodes thioglucoside glucohydrolase (myrosinase), an enzyme catalysing the hydrolysis of glucosinolate compounds. The enzyme is involved in plant defence against some insect herbivores, and is present in species of the order Capparales (Brassicales). Nucleotide variation was surveyed by sequencing c. 2.4 kb of the TGG1 locus in a sample of 28 worldwide A. thaliana accessions, and one Arabidopsis lyrata ssp. lyrata individual. Myrosinase activity was quantified for 27 of these same A. thaliana accessions, plus five additional others. Overall, estimated nucleotide diversity in A. thaliana was low compared to other published A. thaliana surveys, and the frequency distribution was skewed toward an excess of low-frequency variants. Furthermore, comparison to the outgroup species A. lyrata demonstrated that A. thaliana exhibited an excess of high-frequency derived variants relative to a neutral equilibrium model, suggesting a selective sweep. A. thaliana accessions differed significantly in total myrosinase activity, but analysis of variance detected no statistical evidence for an association between quantitative enzyme activity and alleles at the TGG1 myrosinase-encoding locus. We thus conclude that other, unsurveyed factors primarily affect the observed myrosinase activity levels in this species. The pattern of nucleotide variation was consistent with a model of positive selection but might also be compatible with a completely neutral model that takes into account the metapopulation behaviour of this highly inbreeding species which experienced a relatively recent worldwide expansion.     

拟南芥低磷胁迫反应分子机理研究的最新进展

本文综述了拟南芥低磷(Pi)胁迫反应分子机理的最新研究进展,重点介绍了低磷胁迫反应中的SUMOylation途径、转录因子在低磷反应中的功能、Pi平衡调节机制以及磷脂酶在Pi的循环利用过程中的作用,总结了已经鉴定的参与低磷胁迫反应的基因及其可能存在的相互关系。     

Standing genetic variation in FRIGIDA mediates experimental evolution of flowering time in Arabidopsis

The role of standing genetic variation in adaptive evolution remains unclear. Although there has been much progress in identifying candidate genes that underlie adaptive traits, we still lack direct evidence that natural allelic variation in these genes can actually mediate adaptive evolution. In this study, we investigate the role of natural allelic variation in two candidate flowering time genes, in response to selection for early flowering in Arabidopsis thaliana : FRIGIDA ( FRI ) and FLOWERING LOCUS C ( FLC ). We performed artificial selection for early flowering under 'spring-' and 'winter-annual' growth conditions using an outbred population of A. thaliana produced by intermating 19 natural accessions. FRI and FLC are involved in A. thaliana 's response to winter conditions, and nonfunctional and weak alleles at these loci are know to reduce flowering time, particularly under spring-annual conditions. Our results provide direct evidence that natural allelic variation in FRI can provide rapid and predictable adaptive evolution in flowering time under spring-annual conditions. We observed a strong response to selection, in terms of reducing flowering time, in both growth conditions (~2 standard deviation reduction). Concomitantly, the frequency of functional FRI alleles under spring-annual conditions was reduced by 68%, in agreement with predicted changes. No significant changes in allele frequencies were observed in FRI in the winter-annual growth condition or in FLC for either growth conditions. These results indicate that changes in flowering time are mediated by different genetic factors under spring- and winter-annual growth conditions, and that other loci must also be contributing to the response to selection.     

Evolutionary genetics of resistance and tolerance to natural herbivory in Arabidopsis thaliana

Resistance and tolerance are widely viewed as two alternative adaptive responses to herbivory. However, the traits underlying resistance and tolerance remain largely unknown, as does the genetic architecture of herbivory responses and the prevalence of genetic trade-offs. To address these issues, we measured resistance and tolerance to natural apical meristem damage (AMD) by rabbits in a large field experiment with recombinant inbred lines (RILs) of Arabidopsis thaliana (developed from a cross between the Columbia x Landsberg erecta ecotypes). We also measured phenological and morphological traits hypothesized to underlie resistance and tolerance to AMD. Recombinant inbred lines differed significantly in resistance (the proportion of replicates within an RIL that resisted herbivory), and early flowering plants with tall apical inflorescences were more likely to experience damage. Tolerance (the difference in fitness between the damaged and undamaged states), also differed significantly among RILs, with some lines overcompensating for damage and producing more fruit in the damaged than undamaged state. Plastic increases in basal branch number, basal branch height, and senescence date in response to damage were all associated with greater tolerance. There was no evidence for a genetic trade-off between resistance and tolerance, an observation consistent with the underlying differences in associated morphological and phenological characters. Selection gradient analysis detected no evidence for direct selection on either resistance or tolerance in this experiment. However, a statistical model indicates that the pattern of selection on resistance depends strongly on the mean level of tolerance, and selection on tolerance depends strongly on the mean level of resistance. These observations are consistent with the hypothesis that selection may act to maintain resistance and tolerance at intermediate levels in spatially or temporally varying environments or those with varying herbivore populations.     

A novel role for oleosins in freezing tolerance of oilseeds in Arabidopsis thaliana

Oil bodies in seeds of higher plants are surrounded with oleosins. Here we demonstrate a novel role for oleosins in protecting oilseeds against freeze/thaw-induced damage of their cells. We detected four oleosins in oil bodies isolated from seeds of Arabidopsis thaliana , and designated them OLE1, OLE2, OLE3 and OLE4 in decreasing order of abundance in the seeds. For reverse genetics, we isolated oleosin-deficient mutants ( ole1 , ole2 , ole3 and ole4 ) and generated three double mutants ( ole1 ole2 , ole1 ole3 and ole2 ole3 ). Electron microscopy showed an inverse relationship between oil body sizes and total oleosin levels. The double mutant ole1 ole2 , which had the lowest levels of oleosins, had irregular enlarged oil-containing structures throughout the seed cells. Germination rates were positively associated with oleosin levels, suggesting that defects in germination are related to the expansion of oil bodies due to oleosin deficiency. We found that freezing followed by imbibition at 4°C abolished seed germination of single mutants ( ole1 , ole2 and ole3 ), which germinated normally without freezing treatment. The treatment accelerated the fusion of oil bodies and the abnormal-positioning and deformation of nuclei in ole1 seeds, which caused seed mortality. In contrast, ole1 seeds that had undergone freezing treatment germinated normally when incubated at 22°C instead of 4°C, because degradation of oils abolished the acceleration of fusion of oil bodies during imbibition. Taken together, our findings suggest that oleosins increase the viability of over-wintering oilseeds by preventing abnormal fusion of oil bodies during imbibition in the spring.     

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.