Impairment in karrikin but not strigolactone sensing enhances root skewing in Arabidopsis thaliana

Roots form highly complex systems varying in growth direction and branching pattern to forage for nutrients efficiently. Here mutations in the KAI2 (KARRIKIN INSENSITIVE) α/β‐fold hydrolase and the MAX2 (MORE AXILLARY GROWTH 2) F‐box leucine‐rich protein, which together perceive karrikins (smoke‐derived butenolides), caused alteration in root skewing in Arabidopsis thaliana. This phenotype was independent of endogenous strigolactones perception by the D14 α/β‐fold hydrolase and MAX2. Thus, KAI2/MAX2 effect on root growth may be through the perception of endogenous KAI2‐ligands (KLs), which have yet to be identified. Upon perception of a ligand, a KAI2/MAX2 complex is formed together with additional target proteins before ubiquitination and degradation through the 26S proteasome. Using a genetic approach, we show that SMAX1 (SUPPRESSOR OF MAX2‐1)/SMXL2 and SMXL6,7,8 (SUPPRESSOR OF MAX2‐1‐LIKE) are also likely degradation targets for the KAI2/MAX2 complex in the context of root skewing. In A. thaliana therefore, KAI2 and MAX2 act to limit root skewing, while kai2's gravitropic and mechano‐sensing responses remained largely unaffected. Many proteins are involved in root skewing, and we investigated the link between MAX2 and two members of the SKS/SKU family. Though KLs are yet to be identified in plants, our data support the hypothesis that they are present and can affect root skewing.     

钙离子参与拟南芥的缺氧信号传导

报告了钙流通抑制剂钌红对缺氧条件下拟南芥中ADH基因表达的诱导和植株存活的影响。结果表明 ,缺氧早期ADH基因的激活和表达需要钙离子 ,钌红处理可以延长缺氧条件下拟南芥植株的存活期。据此推测 :拟南芥中缺氧诱导的细胞死亡是一个钙离子介导的主动过程 ,钌红通过阻止细胞内钙离子浓度的增加而抑制这一过程。延长缺氧处理的时间会导致拟南芥叶片细胞内发生核凝聚和染色体断裂的现象 ,也进一步验证了这种构想。表明缺氧处理引起的叶片细胞损伤直至植株死亡是一个程序化的过程     

An allelic series at the KARRIKIN INSENSITIVE 2 locus of Arabidopsis thaliana decouples ligand hydrolysis and receptor degradation from downstream signalling

Karrikins are butenolide compounds present in post‐fire environments that can stimulate seed germination in many species, including Arabidopsis thaliana. Plants also produce endogenous butenolide compounds that serve as hormones, namely strigolactones (SLs). The receptor for karrikins (KARRIKIN INSENSITIVE 2; KAI2) and the receptor for SLs (DWARF14; D14) are homologous proteins that share many similarities. The mode of action of D14 as a dual enzyme receptor protein is well established, but the nature of KAI2‐dependent signalling and its function as a receptor are not fully understood. To expand our knowledge of how KAI2 operates, we screened ethyl methanesulphonate (EMS)‐mutagenized populations of A. thaliana for mutants with kai2‐like phenotypes and isolated 13 new kai2 alleles. Among these alleles, kai2‐10 encoded a D184N protein variant that was stable in planta. Differential scanning fluorimetry assays indicated that the KAI2 D184N protein could interact normally with bioactive ligands. We developed a KAI2‐active version of the fluorescent strigolactone analogue Yoshimulactone Green to show that KAI2 D184N exhibits normal rates of ligand hydrolysis. KAI2 D184N degraded in response to treatment with exogenous ligands, suggesting that receptor degradation is a consequence of ligand binding and hydrolysis, but is insufficient for signalling activity. Remarkably, KAI2 D184N degradation was hypersensitive to karrikins, but showed a normal response to strigolactone analogues, implying that these butenolides may interact differently with KAI2. These results demonstrate that the enzymatic and signalling functions of KAI2 can be decoupled, and provide important insights into the mechanistic events that underpin butenolide signalling in plants.     

Redundant roles of four ZIP family members in zinc homeostasis and seed development in Arabidopsis thaliana

Zinc (Zn) is essential for normal plant growth and development. The Zn-regulated transporter, iron-regulated transporter (IRT)-like protein (ZIP) family members are involved in Zn transport and cellular Zn homeostasis throughout the domains of life. In this study, we have characterized four ZIP transporters from Arabidopsis thaliana (IRT3, ZIP4, ZIP6, and ZIP9) to better understand their functional roles. The four ZIP proteins can restore the growth defect of a yeast Zn uptake mutant and are upregulated under Zn deficiency. Single and double mutants show no phenotypes under Zn-sufficient or Zn-limited growth conditions. In contrast, triple and quadruple mutants show impaired growth irrespective of external Zn supply due to reduced Zn translocation from root to shoot. All four ZIP genes are highly expressed during seed development, and siliques from all single and higher-order mutants exhibited an increased number of abnormal seeds and decreased Zn levels in mature seeds relative to wild type. The seed phenotypes could be reversed by supplementing the soil with Zn. Our data demonstrate that IRT3, ZIP4, ZIP6, and ZIP9 function redundantly in maintaining Zn homeostasis and seed development in A. thaliana.     

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。     

Conserved structure and function of the Arabidopsis flowering time gene CONSTANS in Brassica napus

The Arabidopsis thaliana CONSTANS (CO) gene which promotes flowering in long days was recently isolated by chromosome walking. The mapping of QTLs controlling flowering time in Brassica species has identified genomic regions that contain homologues of the CO gene. Four genes homologous to the Arabidopsis CO gene were isolated from a pair of homoeologous loci in each of two doubled-haploid Brassica napus lines displaying different flowering times, N-o-1 and N-o-9. The four genes, BnCOa1, BnCOa9, BnCOb1 and BnCOb9, are located on linkage groups N10 and N19, and are highly similar to each other and to the Arabidopsis CO gene. Two regions of the proteins are particularly well conserved, a N-terminal region with two putative zinc fingers and a C-terminal region which may contain a nuclear localization signal. All four genes appear to be expressed in B. napus. The BnCOa1 allele was shown to complement the co-2 mutation in Arabidopsis in a dosage-dependent manner causing earlier flowering than in wild type under both long- and short-day conditions.     

拟南芥未知功能基因At4g22890 编码蛋白的叶绿体定位

蛋白质的亚细胞定位信息对于深入了解该蛋白质的功能具有重要意义。本文对一个预测的拟南芥叶绿体未知功能基因At4g22890 编码蛋白进行了叶绿体定位研究。我们克隆了该基因5′端长208 bp 的DNA 片段, 与绿色荧光蛋白(GFP) 基因构建重组表达载体pMON530-cTP-GFP, 经农杆菌介导转化拟南芥。转基因植株经激光共聚焦显微镜观察, GFP 荧光仅在叶绿体中观察到, 表明所克隆的DNA 序列编码的多肽能够将At4g22890 编码蛋白质引导进入叶绿体, 由此推测该蛋白质为叶绿体蛋白质。     

利用流式细胞仪分选拟南芥根尖发育早期非根毛细胞

建立了应用流式细胞仪分选植物特定类型细胞的方法。以拟南芥(Arabidopsis thaliana)Wer::GFP转基因株系为材料,用激光共聚焦显微镜鉴定GFP的表达位置,采用酶解法制备拟南芥根尖原生质体,应用流式细胞仪荧光激活细胞分选技术(FACS)分选收集GFP阳性细胞,并提取细胞的RNA。结果表明,Wer::GFP转基因株系仅在根表皮发育早期的非根毛细胞中表达GFP;利用酶解法制备的根尖原生质体数目较多;从FACS分选收集的细胞中提取的RNA质量较好,可用于研究特定类型细胞的基因表达谱。应用流式细胞仪分选拟南芥非根毛细胞的方法为研究植物特定类型细胞的基因表达谱及基因功能奠定了技术基础。     

Regulation of root hair density by phosphorus availability in Arabidopsis thaliana

We characterized the response of root hair density to phosphorus (P) availability in Arabidopsis thaliana. Arabidopsis plants were grown aseptically in growth media with varied phosphorus concentrations, ranging from 1 mmol m⁻³ to 2000 mmol m⁻³ phosphorus. Root hair density (number of root hairs per mm of root length) was analysed starting at 7 d of growth. Root hair density was highly regulated by phosphorus availability, increasing significantly in roots exposed to low-phosphorus availability. The initial root hairs produced by the radicle were not sensitive to phosphorus availability, but began to respond after 9 d of growth. Root hair density was about five times greater in low phosphorus (1 mmol m⁻³) than in high phosphorus (1000 mmol m⁻³) media. Root hair density decreased logarithmically in response to increasing phosphorus concentrations within that range. Root hair density also increased in response to deficiencies of several other nutrients, but not as strongly as to low phosphorus. Indoleacetic acid (IAA), the auxin transport inhibitor 2-(p-chlorophenoxy)-2-methylpropionic acid (CMPA), the ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC), and the ethylene synthesis inhibitor amino-oxyacetic acid (AOA) all increased root hair density under high phosphorus but had very little effect under low phosphorus. Low phosphorus significantly changed root anatomy, causing a 9% increase in root diameter, a 31% decrease in the cross-sectional area of individual trichoblasts, a 40% decrease in the cross-sectional area of individual atrichoblasts, and 45% more cortical cells in cross-section. The larger number of cortical cells and smaller epidermal cell size in low phosphorus roots increased the number of trichoblast files from eight to 12. Two-thirds of increased root hair density in low phosphorus roots was caused by increased likelihood of trichoblasts to form hairs, and 33% of the increase was accounted for by changes in low phosphorus root anatomy resulting in an increased number of trichoblast files. These results show that phosphorus availability can fundamentally alter root anatomy, leading to changes in root hair density, which are presumably important for phosphorus acquisition.     

Involvement of lipid transfer proteins in resistance against a non-host powdery mildew in Arabidopsis thaliana

Non-specific lipid transfer proteins (LTPs) are involved in the transport of lipophilic compounds to the cuticular surface in epidermal cells and in the defence against pathogens. The role of glycophosphatidylinositol (GPI)-anchored LTPs (LTPGs) in resistance against non-host mildews in Arabidopsis thaliana was investigated using reverse genetics. Loss of either LTPG1, LTPG2, LTPG5 or LTPG6 increased the susceptibility to penetration of the epidermal cell wall by Blumeria graminis f. sp. hordei (Bgh). However, no impact on pre-penetration defence against another non-host mildew, Erysiphe pisi (Ep), was observed. LTPG1 was localized to papillae at the sites of Bgh penetration. This study shows that, in addition to the previously known functions, LTPGs contribute to pre-invasive defence against certain non-host powdery mildew pathogens.     

小拟南芥ApZFP基因异源超表达促进拟南芥开花并提高耐逆性

锌指蛋白(ZFP)是一类重要的转录因子, 广泛参与植物的生长发育和非生物胁迫应答。新疆小拟南芥(Arabidopsispumila)又名无苞芥, 是十字花科短命植物, 具有高光效、繁殖力强和适应干旱等生物学特征, 而且比模式植物拟南芥(A.thaliana)更耐高盐胁迫。将前期克隆的小拟南芥锌指蛋白基因ApZFP通过花滴法转化到哥伦比亚生态型拟南芥(Col-0)中,获得了独立表达的转基因株系。表型观察发现, 过量表达ApZFP基因可促使拟南芥在长短日照下均提前开花。实时荧光定量PCR结果显示, 转基因拟南芥株系中, 光周期途径中的CO基因和年龄途径中的SPL基因表达上调; 春化、环境温度和自主途径中的FLC基因表达下调; 编码成花素的基因FT及下游开花相关基因AP1和LFY的表达量均升高。进一步通过盐、干旱和ABA胁迫处理ApZFP转基因株系的种子和幼苗, 发现在胁迫处理下, 与对照相比, 转基因拟南芥种子萌发率较高, 幼苗主根较长。因此推测, ApZFP在植物发育过程中具有多种功能, 可能既参与植物的开花转变过程, 又同其它植物的锌指蛋白基因一样, 参与植物的耐逆过程。     

Effects of gene expression on molecular evolution in Arabidopsis thaliana and Arabidopsis lyrata

We analyzed the complete genome sequence of Arabidopsis thaliana and sequence data from 83 genes in the outcrossing A. lyrata, to better understand the role of gene expression on the strength of natural selection on synonymous and replacement sites in Arabidopsis. From data on tRNA gene abundance, we find a good concordance between codon preferences and the relative abundance of isoaccepting tRNAs in the complete A. thaliana genome, consistent with models of translational selection. Both EST-based and new quantitative measures of gene expression (MPSS) suggest that codon preferences derived from information on tRNA abundance are more strongly associated with gene expression than those obtained from multivariate analysis, which provides further support for the hypothesis that codon bias in Arabidopsis is under selection mediated by tRNA abundance. Consistent with previous results, analysis of protein evolution reveals a significant correlation between gene expression level and amino acid substitution rate. Analysis by MPSS estimates of gene expression suggests that this effect is primarily the result of a correlation between the number of tissues in which a gene is expressed and the rate of amino acid substitution, which indicates that the degree of tissue specialization may be an important determinant of the rate of protein evolution in Arabidopsis.     

染色体展片法观察拟南芥雄性减数分裂过程中的染色体形态

减数分裂指DNA复制1次, 细胞核分裂2次, 产生染色体数目减半的单倍体配子, 是真核生物有性生殖所必需的环节。拟南芥(Arabidopsis thaliana)是分子遗传学研究的传统模式生物。近年来, 随着显微镜技术的快速发展, 利用细胞学方法观察拟南芥减数分裂过程中的染色体形态和同源染色体互作事件, 将有助于深入认识减数分裂的分子遗传机制。该文详细描述了染色体展片法观察拟南芥雄性减数分裂细胞中的染色体形态。