Structural Insights into the Substrate Specificity of (S)-Ureidoglycolate Amidohydrolase and Its Comparison with Allantoate Amidohydrolase

In plants, the ureide pathway is a metabolic route that converts the ring nitrogen atoms of purine into ammonia via sequential enzymatic reactions, playing an important role in nitrogen recovery. In the final step of the pathway, (S)-ureidoglycolate amidohydrolase (UAH) catalyzes the conversion of (S)-ureidoglycolate into glyoxylate and releases two molecules of ammonia as by-products. UAH is homologous in structure and sequence with allantoate amidohydrolase (AAH), an upstream enzyme in the pathway with a similar function as that of an amidase but with a different substrate. Both enzymes exhibit strict substrate specificity and catalyze reactions in a concerted manner, resulting in purine degradation. Here, we report three crystal structures of Arabidopsis thaliana UAH (bound with substrate, reaction intermediate, and product) and a structure of Escherichia coli AAH complexed with allantoate. Structural analyses of UAH revealed a distinct binding mode for each ligand in a bimetal reaction center with the active site in a closed conformation. The ligand directly participates in the coordination shell of two metal ions and is stabilized by the surrounding residues. In contrast, AAH, which exhibits a substrate-binding site similar to that of UAH, requires a larger active site due to the additional ureido group in allantoate. Structural analyses and mutagenesis revealed that both enzymes undergo an open-to-closed conformational transition in response to ligand binding and that the active-site size and the interaction environment in UAH and AAH are determinants of the substrate specificities of these two structurally homologous enzymes.     

Novel<Emphasis Type="Italic"> eceriferum</Emphasis> mutants in<Emphasis Type="Italic"> Arabidopsis thaliana</Emphasis>

We conducted a novel non-visual screen for cuticular wax mutants in Arabidopsis thaliana (L.) Heynh. Using gas chromatography we screened over 1,200 ethyl methane sulfonate (EMS)-mutagenized lines for alterations in the major A. thaliana wild-type stem cuticular chemicals. Five lines showed distinct differences from the wild type and were further analyzed by gas chromatography and scanning electron microscopy. The five mutants were mapped to specific chromosome locations and tested for allelism with other wax mutant loci mapping to the same region. Toward this end, the mapping of the cuticular wax (cer) mutants cer10 to cer20 was conducted to allow more efficient allelism tests with newly identified lines. From these five lines, we have identified three mutants defining novel genes that have been designated CER22, CER23, and CER24. Detailed stem and leaf chemistry has allowed us to place these novel mutants in specific steps of the cuticular wax biosynthetic pathway and to make hypotheses about the function of their gene products.Abbreviations EMS Ethyl methane sulfonate - SEM Scanning electron microscopy - SSLP Simple sequence length polymorphism - WT Wild type     

Shoot apical meristem structure and STM expression in has mutant of Arabidopsis thaliana

Wild-type and the handshake (has) mutants in Arabidopsis thaliana were analyzed. Compared to the wild-type, has mutants display a number of morphological alterations, which can largely be traced back to altered meristem function. Analyses of apical meristem of mutant plants showed that mutation affected meristem structure and patterns of STM expression.     

Structure of Flower in Arabidopsis thaliana: Spatial Pattern Formation

Morphological analysis of flowers was carried out in Arabidopsis thaliana wild type plants and agamous and apetala2 mutants. No direct substitution of organs takes place in the mutants, since the number and position of organs in them do not correspond to the structure of wild type flower. In order to explain these data, a notion of spatial pattern formation in the meristem was introduced, which preceded the processes of appearance of organ primordia and formation of organs. Zones of acropetal and basipetal spatial pattern formation in the flower of wild type plants were postulated. It was shown that the acropetal spatial pattern formation alone took place in agamous mutants and basipetal spatial pattern formation alone, in apetala2 mutants. Different variants of flower structure are interpreted as a result of changes in the volume of meristem (space) and order of spatial pattern formation (time).     

How do plant shoots bend up? — The initial step to elucidate the molecular mechanisms of shoot gravitropism usingArabidopsis thaliana

In higher plants, shoots show a negative gravitropic response. To elucidate the molecular mechanisms of this phenomenon, mutational analyses usingArabidopsis thaliana are in progress. This minireview aims to present recent developments in the genetic analysis of shoot gravitropism in this organism. We focus mainly on our studies on the novelshootgravitropic (sgr) mutants inArabidopsis thaliana that have dramatic defects in shoot gravitropism.     

UV-B预处理诱导拟南芥耐旱性的提高

为了解UV-B提高拟南芥(Arabidopsis thaliana)耐旱性的生理机制,将2周龄的野生型拟南芥(WT)和sto突变体幼苗用不同剂量UV-B预处理1周,再用30%PEG模拟干旱处理24 h,对植株的表型进行统计,并测定类黄酮、脯氨酸和MDA含量。结果表明,低剂量UV-B预处理能够提高拟南芥的耐旱性,植株的类黄酮与脯氨酸含量分别提高了20%～40%和50%～65%,细胞膜受损程度降低,从而提高了保水性。低剂量UV-B提高拟南芥耐旱性的效应在sto突变体中消失,证明这种效应在分子机制上可能与STO蛋白相关。     

Isolation of Arabidopsis thaliana mutants hypersensitive to gamma radiation

A screening method for mutants of Arabidopsis thaliana hypersensitive to -radiation has been devised. Plants grown from ethyl methanesulfonate (EMS)-treated seeds were irradiated at the seedling stage, which is highly radiosensitive due to extensive cell division. Severe growth inhibition of mutant plants by a -ray dose which only slightly affects wild-type plants was the selective criterion. Twelve true-breeding hyper-sensitive lines were isolated from a total of 3394 screened plants. Genetic analysis of five of the lines revealed five new genes, designated RAD1-RAD5. These Arabidopsis RAD mutants are phenotypically similar to mutants in the RAD52 epistasis group of Saccharomyces cerevisiae, which are highly sensitive to ionizing radiation but not hypersensitive to UV light. One possibility is that the Arabidopsis mutants are defective in a nonhomologous or illegitimate recombination mechanism used by plants for repair of chromosome breaks.     

The influence of matrix attachment regions on transgene expression in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> wild type and gene silencing mutants

Many studies in both animal and plant systems have shown that matrix attachment regions (MARs) can increase the expression of flanking transgenes. However, our previous studies revealed no effect of the chicken lysozyme MARs (chiMARs) on transgene expression in the first generation transgenic Arabidopsis thaliana plants transformed with a β-glucuronidase gene (uidA) unless gene silencing mutants were used as genetic background for transformation. In the present study, we investigated why chiMARs do not influence transgene expression in transgenic wild-type Arabidopsis plants. We first studied the effect of chiMARs on transgene expression in the progeny of primary transformants harboring chiMAR-flanked T-DNAs. Our data indicate that chiMARs do not affect transgene expression in consecutive generations of wild-type A. thaliana plants. Next, we examined whether these observed results in A. thaliana transformants are influenced by the applied transformation method. The results from in vitro transformed A. thaliana plants are in accordance with those from in planta transformed A. thaliana plants and again reveal no influence of chiMARs on transgene expression in A. thaliana wild-type transformants. The effect of chiMARs on transgene expression is also examined in in vitro transformed Nicotiana tabacum plants, but as for A. thaliana, the transgene expression in tobacco transformants is not altered by the presence of chiMARs. Taken together, our results show that the applied method or the plant species used for transformation does not influence whether and how chiMARs have an effect on transgene expression. Finally, we studied the effect of MARs (tabMARs) of plant origin (tobacco) on the transgene expression in A. thaliana wild-type plants and suppressed gene silencing (sgs2) mutants. Our results clearly show that similar to chiMARs, the tobacco-derived MARs do not enhance transgene expression in a wild-type background but can be used to enhance transgene expression in a mutant impaired in gene silencing. Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible for authorized users. Miguel F.C. De Bolle, Katleen M.J. Butaye Contributed equally to this work     

Activities of auxin polar transport in inflorescence axes of flower mutants ofArabidopsis thaliana: Relevance to flower formation and growth

Relationships between the activity of auxin polar transport and flower formation were studied using several flower mutants ofArabidopsis thaliana. The activity of auxin polar transport in the upper portion of inflorescence axis of wildtype plants ofArabidopsis thaliana was significantly lower than that of the basal part. The activities of auxin polar transport in the upper portion of inflorescence axes ofap1 andclv1 mutants were significantly higher than that of wild-type plant. However, those of other flower mutants tested,ap3-1, ag, pi, Fl-40, Fl-54, Fl-89 andpin-formed, were extremely low as compared with that of wild one. We got some evidence that the reduction of the activity of auxin polar transport is concerned with the growth and development of plants. We could mimic it by the removal of all flowers and pods including mature or immature seeds. Moreover, artificial pollination inap3-1 andpi mutants, in which no seeds are found naturally, resulted in the partial recovery of the activity of auxin polar transport in inflorescence axis. Considering these results in this study together with the fact that inhibitors of auxin polar transport generated almost same disruptions ofpin-formed orpinoid mutants which normally had no flowers in inflorescence axis (Okadaet al. 1991, Uedaet al. 1992, Bennettet al. 1995), the systern of auxin polar transport and its activity in inflorescence axis seems to be essential for the development of flower bud in early stage ofArabidopsis thaliana, and the activity of auxin polar transport is also regulated by the formation of flowers and seeds in inflorescence axis.     

Development of inflorescences inArabidopsis thaliana

Those mutants were studied whose defects resulted in the morphological changes of inflorescences inArabidopsis thaliana. We characterized newly isolatedcorymbosa mutants andacaulis5 mutants. Thecorymbosa1-1 mutation was caused by the defects in the elongation of pedicels and the previously identifiederecta mutation belonged to this class. Thecorymbosa2-1 mutation was caused mainly by the increase of the number of the floral buds in the inflorescence. The expression of theERECTA gene whose defect resulted to the corymbose inflorescence was analyzed. TheERECTA gene was expressed in subsets of cells in both the peripheral zone and central zone and was thought to have important role for the development of inflorescences. The phenotypes of theacaulis5 mutation was pronounced just after the transition from the vegetative to reproductive growth phase. We found that the expressions of the genes for EXGT-A1 and γ-TIP were drastically reduced in theacaulis5 mutants. The extended abstract of a paper presented at the 13th International Symposium in Conjugation with Award of the International Prize for Biology “Frontier of Plant Biology”     

Inhibition of CAT enzyme activity in Arabidopsis thaliana

Previously it was shown that transient chloramphenicol acetyltransferase (CAT) marker gene expression in Arabidopsis thaliana and Nicotiana tabacum resulted in significant differences in the accumulation of the CAT reaction products in radioactive CAT assays. Compared to Nicotiana tabacum, conversion of chloramphenicol to the acetylated products in Arabidopsis thaliana extracts was rather low. Here we report that the low CAT enzyme activity can be attributed in part to a heat sensitive CAT inhibitory effect in extracts of Arabidopsis thaliana. CAT enzyme activity in transgenic tobacco is inhibited by extracts from Arabidopsis. This inhibitory effect diminishes when Arabidopsis extracts were heat incubated. CAT activity in transgenic Arabidopsis lines was very low and was only detected in heat incubated extracts. Alternatively, enzyme-linked immunosorbent assays (ELISAs) can be used to detect the CAT protein in transgenic Arabidopsis.Abbreviations CAT chloramphenicol acetyltransferase - CAM chloramphenicol - ELISA enzyme linked immunosorbent assay     

拟南芥基因密码子偏爱性分析

密码子偏爱性对外源基因的表达强度有一定影响,特别是编码蛋白质N端7～8个氨基酸残基的密码子.通过对拟南芥染色体中26 827个蛋白质对应的基因密码子进行分析,得到了编码氨基酸的61种密码子在拟南芥中的使用频率,并与大肠杆菌和哺乳动物进行了比较,结果表明三者间的密码子偏爱性有较大差异.这一分析结果对于动物基因在植物中的表达,及植物基因在微生物中的表达具有一定指导意义.同时提供了一种直接以XML文档为数据源解析巨型XML格式染色体数据的方法.     

Phenotypic alterations in<Emphasis Type="Italic"> Arabidopsis thaliana</Emphasis> plants caused by<Emphasis Type="Italic"> Rhodococcus fascians</Emphasis> infection

Arabidopsis thaliana (L.) Heynh. plants were challenged with Rhodococcus fascians at several developmental stages and using different inoculation procedures. A variety of morphological alterations was scored on the infected plants; some of them resembled phenotypes of A. thaliana mutants in their shoot apical meristem (SAM) organization. Infection with R. fascians did not affect SAM organization in wild type nor in SAM mutants. Anatomical studies on the new organs formed after infection with R. fascians demonstrated extensive bacterial colonization. Colonization and concomitant production of specific signals are the likely cause of malformations.     

Heterologous expression and regulation of the lysA genes of Pseudomonas aeruginosa and Escherichia coli

Summary Chlorsulfuron-resistant mutants of Arabidopsis thaliana were isolated by screening for growth of seedlings in the presence of the herbicide. Both whole plants and derived tissue cultures were resistant to concentrations of the herbicide approximately 300-fold higher than that required to prevent growth of the wild-type. The resistance is due to a single dominant nuclear mutation at a locus designated csr which has been genetically mapped to chromosome-3. Acetohydroxy acid synthase activity in extracts from chlorsulfuron-resistant plants was much less-susceptible to inhibition by chlorsulfuron and a structurally related inhibitor than the activity in wild-type extracts. This suggests that the csr locus is the structural gene for acetohydroxy acid synthase.     

拟南芥GH3.9基因的过表达及其表型分析

为研究GH3.9基因在植物生长发育过程中的作用,利用RT-PCR成功克隆到GH3.9基因,该基因全长为1 750bp。通过构建pEGAD-GH3.9过表达载体转化拟南芥,获得过表达GH3.9基因纯系转基因株系GH3.9ox-3和GH3.9ox-7。对拟南芥野生型(WT)和转基因株系(GH3.9ox-3和GH3.9ox-7)幼苗用不同光强和光质进行处理,结果显示:在蓝光、红光、远红光等不同光照强度下培养,过表达株系幼苗下胚轴的生长均明显受到抑制,且较野生型明显;采用不同光周期处理拟南芥幼苗,过表达幼苗下胚轴的伸长明显低于野生型;对成年植株表型进行观察,发现过表达株系植株矮小、雄蕊变短、果荚短小。研究表明:GH3.9基因参与了拟南芥生长发育调控,过表达GH3.9基因对拟南芥生长有抑制作用。     

<Emphasis Type="Italic">Anthocyaninless1</Emphasis> gene of <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> encodes a UDP-glucose:flavonoid-3-<Emphasis Type="Italic">O</Emphasis>-glucosyltransferase

We isolated several mutants of Arabidopsis thaliana (L.) Heynh. that accumulated less anthocyanin in the plant tissues, but had seeds with a brown color similar to the wild-type. These mutants were allelic with the anthocyaninless1 (anl1) mutant that has been mapped at 15.0 cM of chromosome 5. We performed fine mapping of the anl1 locus and determined that ANL1 is located between the nga106 marker and a marker corresponding to the MKP11 clone. About 70 genes are located between these two markers, including three UDP-glucose:flavonoid-3-O-glucosyltransferase-like genes and a glutathione transferase gene (TT19). A mutant of one of the glucosyltransferase genes (At5g17050) was unable to complement the anl1 phenotype, showing that the ANL1 gene encodes UDP-glucose:flavonoid-3-O-glucosyltransferase. ANL1 was expressed in all tissues examined, including rosette leaves, stems, flower buds and roots. ANL1 was not regulated by TTG1.     

Analysis of a repetitive DNA family from Arabidopsis arenosa and relationships between Arabidopsis species

We have analysed a family of highly repetitive DNA from Arabidopsis arenosa (L.) Lawalrée [syn. Cardaminopsis arenosa (L.) Hayck] composed of AT-rich tandem repeats of 166–179 bp in head to tail organization. Sequence comparison between several repeat units revealed a high level of divergence of 4.5% to 25%. The sequence family shows more than 58% homology to satellite sequences described in Arabidopsis thallana (L.) Heynh. but no homology to other satellite repeats in the Cruciferae. Within the genus Arabidopsis the satellite sequence was found to be present in A. thaliana and Arabidopsis suecica (Fries) Norrlin, but not in Arabidopsis griffithiana (Boiss.) N. Busch and Arabidopsis pumila (Stephan) N. Busch. In situ hybridization to metaphase chromosomes of A. arenosa (2n=4x=32) showed the sequence to be localized at the centromeres of all 32 chromosomes with substantial hybridization along the chromosome arms. Using Southern hybridization and in situ hybridization, we give evidence that A. suecica is a hybrid of A. thaliana and A. arenosa. A considerable reorganization of the A. thaliana satellite sequence pAL1 occurred in the hybrid genome while no molecular change of the A. arenosa repeat was observed in the hybrid. Analysis of related repeats enabled differentiation between closely related genomes and are useful for the investigation of hybrid genomes.