Purification and Properties of Penicillin Acylase from Kluyvera citrophila

Penicillin acylase (EC 3.5.1.11) of Kluyvera citrophila KY7844 was purified approximately 120-fold by DEAE-cellulose chromatography, hydroxyapatite chromatography and isoelectro-focusing fractionation. The purified enzyme, with an approximate molecular weight of 63,000, appeared to be homogeneous in disc electrophoretic analysis, and showed isoelectric point (Ip) 8.12 and 13.0 units/mg of specific activity for cephalexin hydrolysis. The Michaelis constant (Km) for cephalexin and for 7-[1-(1H)-tetrazolylacetamido]-desacetoxycephalosporanic acid ((1H) T-7ADCA) was 1.4 mM and 3.6 mM, respectively. This enzyme was capable of producing (1H) T-7ADCA in 80% yield from 1-(1H)-tetrazolylacetate methylester and 7-aminodesacetoxycephalosporanic acid.     

A 150 kDa plasma membrane complex of AtNRT2.5 and AtNAR2.1 is the major contributor to constitutive high‐affinity nitrate influx in Arabidopsis thaliana

In plants that have been deprived of nitrate for a significant length of time, a constitutive high‐affinity nitrate transport system (cHATS) is responsible for initial nitrate uptake. This absorbed nitrate leads to the induction of the major nitrate transporters and enzymes involved in nitrate assimilation. By use of ¹³NO₃^– influx measurements and Blue Native polyacrylamide gel electrophoresis we examined the role of AtNRT2.5 in cHATS in wild type (WT) and various T‐DNA mutants of Arabidopsis thaliana. We demonstrate that AtNRT2.5 is predominantly expressed in roots of nitrate‐deprived WT plants as a 150 kDa molecular complex with AtNAR2.1. This complex represents the major contributor to cHATS influx, which is reduced by 63% compared with WT in roots of Atnrt2.5 mutants. The remaining cHATS nitrate influx in these mutants is due to a residual contribution by the inducible high‐affinity transporter encoded by AtNRT2.1/AtNAR2.1. Estimates of the kinetic properties of the NRT2.5 transporter reveal that its low K_m for nitrate makes this transporter ideally suited to detect and respond to trace quantities of nitrate in the root environment.     

Germination and seedling growth of bog plants in relation to the recolonization of milled peatlands

Two controlled experiments were conducted to evaluate the potential for vascular plants to germinate and establish in milled peatlands and to assess whether easily measured plant traits can be used to predict their probable success. Study species included twenty species of perennial herbs, shrubs and trees occurring frequently in undisturbed bogs or abandoned milled bogs in Québec, Canada. First, a glasshouse experiment was performed to test the effect of burial under peat on germination and seedling emergence. Second, a growth chamber experiment was conducted to measure relative growth rate and other growth parameters of seedlings between 1 and 3 weeks of age. In the burial experiment, seedling emergence decreased exponentially with peat depth for most species examined. The slope of the exponential decline varied between species and was strongly correlated to seed mass. Seeds less than 0.1 mg in mass were most sensitive to burial. In the seedling growth experiments, Betula species had the highest absolute and relative growth rates, which may help to explain their prevalence in milled bogs. Relative growth rate (RGR) was not correlated with seed mass, however it was strongly correlated with leaf area ratio (LAR) and especially specific leaf area (SLA) of seedlings, except for species with seed mass less than 0.01 mg. Screening of species for seed mass and SLA should help predict their germination and establishment success or failure in milled peatlands and allow more directed interventions to favour the establishment of desirable species in milled bogs. This revised version was published online in August 2006 with corrections to the Cover Date.     

Expression profiling of the estrogen responsive genes in response to phytoestrogens using a customized DNA microarray

Here, we examined phytoestrogens, isoflavones (genistein, daidzein, glycitein, biochanin A and ipriflavone), flavones (chrysin, luteolin and apigenin), flavonols (kaempferol and quercetin), and a coumestan, a flavanone and a chalcone (coumestrol, naringenin and phloretin, respectively) by means of a DNA microarray assay. A total of 172 estrogen responsive genes were monitored with a customized DNA microarray and their expression profiles for the above phytoestrogens were compared with that for 17beta-estradiol (E2) using correlation coefficients, or R values, after a correlation analysis by linear regression. While R values indicate the similarity of the response by the genes, we also examined the genes by cluster analysis and by their specificity to phytoestrogens (specific to genistein, daidzein or glycitein) or gene functions. Several genes were selected from p53-related genes (CDKN1A, TP53I11 and CDC14), Akt2-related genes (PRKCD, BRCA1, TRIB3 and APPL), mitogen-activated protein kinase-related genes (RSK and SH3BP5), Ras superfamily genes (RAP1GA1, RHOC and ARHGDIA) and AP-1 family and related genes (RIP140, FOS, ATF3, JUN and FRA2). We further examined the extracts from two local crops of soy beans (Kuro-daizu or Mochi-daizu) by comparing the gene expression profiles with those of E2 or phytoestrogens as a first step in utilizing the expression profiles for various applications.     

Gene structure and expression of the high-affinity nitrate transport system in rice roots

Rice has a preference for uptake of ammonium over nitrate and can use ammonium-N efficiently. Consequently, transporters mediating ammonium uptake have been extensively studied, but nitrate transporters have been largely ignored. Recently,some reports have shown that rice also has high capacity to acquire nitrate from growth medium, so understanding the nitrate transport system in rice roots is very important for improving N use efficiency in rice. The present study identified four putative NRT2 and two putative NAR2 genes that encode components of the high-affinity nitrate transport system (HATS) in the rice (Oryza sativa L. subsp, japonica cv. Nipponbare) genome. OsNRT2.1 and OsNRT2.2 share an identical coding region sequence, and their deduced proteins are closely related to those from monocotyledonous plants. The two NAR2 proteins are closely related to those from mono-cotyledonous plants as well. However, OsNRT2.3 and OsNRT2.4 are more closely related to Arabidopsis NRT2 proteins. Relative quantitative reverse tranecdption-polymerase chain reaction analysis showed that all of the six genes were rapidly upregulated and then downregulated in the roots of N-starved rice plants after they were re-supplied with 0.2 mM nitrate, but the response to nitrate differed among gene members.The results from phylogenetic tree, gene structure and expression analysis implied the divergent roles for the individual members of the rice NRT2 and NAR2 families. High-affinity nitrate influx rates associated with nitrate induction in rice roots were investigated and were found to be regulated by external pH. Compared with the nitrate influx rates at pH 6.5, alkaline pH (pH 8.0) inhibited nitrate Influx, and acidic pH (pH 5.0) enhanced the nitrate influx In I h nitrate induced roots, but did not significantly affect that in 4 to 8 h nitrate induced roots.     

高粱高亲和硝酸盐转运蛋白NRT2/3基因家族鉴定、表达与DNA变异分析

硝态氮是作物吸收无机氮素的主要形态,硝酸盐转运蛋白2(nitrate transporter 2,NRT2)作为高亲和性的转运蛋白,以硝酸盐作为特异性底物,在可利用的硝酸盐受限时,高亲和性转运系统被激活,在硝酸盐吸收、转运过程中发挥着重要作用。大多数NRT2不能单独转运硝酸盐,需在硝酸盐同化相关蛋白2(nitrate assimilation related protein 2,NAR2)的协助下才能完成硝酸盐的吸收或转运。作物氮利用效率受环境条件影响,品种间存在差异,因此培育高氮素利用效率品种有重大意义。高粱(Sorghum bicolor)具有耐贫瘠特性,对土壤中的氮素吸收和利用效率较高。本研究结合高粱基因组数据库对NRT2/3基因家族成员基因结构、染色体定位、理化性质、二级结构与跨膜结构域、信号肽与亚细胞定位、启动子区顺式作用元件、系统进化、单核苷酸多态性(single nucleotide polymorphism,SNP)的识别与注释及选择压力进行了全面分析。通过生物信息学分析,筛选出5个NRT2s(命名为SbNRT2-1a、2-1b、SbNRT2-2–4)基因和2个NAR2s(SbNRT3-1–2)基因,较谷子略少。分布在3条染色体上,分为4个亚家族,同一亚族中基因结构高度相似;高粱NRT2/3亲水性平均值均为正值,表明均为疏水性蛋白;α-螺旋和无规则卷曲占二级结构总量的比例大于70%;亚细胞定位均在质膜上,其中NRT2s蛋白不含信号肽,NRT3s蛋白含信号肽;进一步对其跨膜结构域进行分析,发现NRT2s家族成员跨膜结构域个数均大于10个,而NRT3s家族成员跨膜结构域个数为2个;高粱与玉米(Zea mays)NRT2/3s的共线性较好;蛋白结构域显示存在MFS_1和NAR2蛋白结构域,可执行高亲和力硝酸盐转运;系统进化树分析可知,高粱与玉米和谷子的NRT2/3基因亲缘关系更近;基因启动子顺式作用元件分析发现,SbNRT2/3基因的启动子区均具有数个植物激素和逆境应答元件,可以响应高粱生长和环境变化;基因表达热图显示低氮条件下在根诱导表达的是SbNRT2-1a、SbNRT2-1b和SbNRT3-1,推测可在高粱根部表达并调控对硝酸盐的吸收或转运过程。在SbNRT2-4和SbNRT2-1a等发现多个非同义SNP变异;选择压力分析表明,高粱NRT2/3基因家族在进化过程中受纯化选择作用。SbNRT2/3基因表达及蚜虫侵染影响与基因在不同组织中的表达分析结果一致,SbNRT2-1b和SbNRT3-1在感染蚜虫品系5-27sug根部表达显著,高粱蚜虫侵染叶片显著降低了SbNRT2-3、SbNRT2-4和SbNRT3-2的表达水平。本研究初步对高粱全基因组NRT2/3基因家族进行鉴定、表达与DNA变异分析,为高粱氮高效研究提供了基础。