Isolation and Characterization of a Starchless Mutant of Arabidopsis thaliana (L.) Heynh Lacking ADPglucose Pyrophosphorylase Activity

A mutant of Arabidopsis thaliana lacking ADPglucose pyrophosphorylase activity (EC 2.7.7.27) was isolated (from a mutagenized population of plants) by screening for the absence of leaf starch. The mutant grows as vigorously as the wild type in continuous light but more slowly than the wild type in a 12 hours light/12 hours dark photoperiod. Genetic analysis showed that the deficiency of both starch and ADPglucose pyrophosphorylase activity were attributable to a single, nuclear, recessive mutation at a locus designated adg1. The absence of starch in the mutant demonstrates that starch synthesis in the chloroplast is entirely dependent on a pathway involving ADPglucose pyrophosphorylase. Analysis of leaf extracts by two-dimensional polyacrylamide gel electrophoresis followed by Western blotting experiments using antibodies specific for spinach ADPglucose pyrophosphorylase showed that two proteins, present in the wild type, were absent from the mutant. The heterozygous F₁ progeny of a cross between the mutant and wild type had a specific activity of ADPglucose pyrophosphorylase indistinguishable from the wild type. These observations suggest that the mutation in the adg1 gene in TL25 might affect a regulatory locus.     

A th-1 Mutant of Arabidopsis thaliana Is Defective for a Thiamin-Phosphate-Synthesizing Enzyme: Thiamin Phosphate Pyrophosphorylase

We have examined the activity of the thiamin phosphate pyrophosphorylase in Arabidopsis thaliana wild type and in a mutant (th-1) which requires exogenous thiamin for growth. Mutant and wild-type plants grown in 1 × 10⁻⁷ molar thiamin were used for the examination of the production of thiamin and thiamin monophosphate (TMP) using 4-methyl-5-hydroxyethylthiazole phosphate and 2-methyl-4-amino-5-hydroxymethylpyrimidine pyrophosphate as substrates. While the wild-type strain formed both thiamin and TMP, the th-1 mutant did not. When TMP was added to the extracts, the th-1 mutant, as well as wild type, produced thiamin. Accordingly, it was concluded that the th-1 mutant was defective in the activity of TMP pyrophosphorylase. Some of the characteristics of the enzyme from the wild-type plant were examined. The optimum temperature for the reaction is 45°C, and the K_m values for the substrates are 2.7 × 10⁻⁶ molar for 4-methyl-5-hydroxyethylthiazole phosphate and 1.8 × 10⁻⁶ molar for 2-methyl-4-amino-5-hydroxymethylpyrimidine pyrophosphate.     

A Mutant of Arabidopsis thaliana Which Lacks Activation of RuBP Carboxylase In Vivo

A mutant of Arabidopsis thaliana has been isolated in which ribulose-1,5-bisphosphate carboxylase is present in a nonactivatable form in vivo. The mutation appears to affect carboxylase activation specifically, and not any other enzyme of the photosynthesis or photorespiratory cycles. The effect of the mutation on carboxylase activation is indirect, inasmuch as the properties of ribulose-1,5-bisphosphate carboxylase purified from the mutant are not distinguishable from those of the wild type enzyme. The mutant requires high levels of atmospheric CO₂ for growth because photosynthesis is severely impaired in atmospheres containing normal levels of CO₂, irrespective of the atmospheric O₂ concentration. In this respect, the mutant is distinguished from previously described high-CO₂ requiring mutants of Arabidopsis which have defects in photorespiratory carbon or nitrogen metabolism.     

A Mutant of Arabidopsis Deficient in the Elongation of Palmitic Acid

The overall fatty acid composition of leaf lipids in a mutant of Arabidopsis thaliana was characterized by an increased level of 16:0 and a concomitant decrease of 18-carbon fatty acids as a consequence of a single recessive nuclear mutation at the fab1 locus. Quantitative analysis of the fatty acid composition of individual lipids established that lipids synthesized by both the prokaryotic and eukaryotic pathways were affected by the mutation. Direct enzyme assays demonstrated that the mutant plants were deficient in the activity of 3-ketoacyl-acyl carrier protein synthase II; therefore, it is inferred that fab1 may encode this enzyme. Labeling experiments with [14C]acetate and lipase positional analysis indicated that the mutation results in a small shift in the partitioning of lipid synthesis between the prokaryotic and eukaryotic pathways. Synthesis of chloroplast lipids by the prokaryotic pathway was increased with a corresponding reduction in the eukaryotic pathway.     

Chlorophyllase Activity and Chlorophyll Content in Wild and Mutant Plants of Arabidopsis thaliana

The activity of chlorophyllase in wild type (WT) was higher than in ethylene insensitive mutant (eti 5) of Arabidopsis thaliana (L.) Heynh plants during the vegetative period. Chlorophyll content in eti 5 leaves was higher than in WT but the difference decreased by the end of the experimental period.     

Alterations in Growth, Photosynthesis, and Respiration in a Starchless Mutant of Arabidopsis thaliana (L.) Deficient in Chloroplast Phosphoglucomutase Activity

A mutant of Arabidopsis thaliana (L.) Heynh. which lacks leaf starch was isolated by screening for plants which did not stain with iodine. The starchless phenotype, confirmed by quantitative enzymic analysis, is caused by a single recessive nuclear mutation which results in a deficiency of the chloroplast isozyme of phosphoglucomutase. When grown in a 12-h photoperiod, leaves of the wild-type accumulated substantial amounts of starch but lower levels of soluble sugars. Under these conditions, the mutant accumulated relatively high levels of soluble sugars. Rates of growth and net photosynthesis of the mutant and wild-type were indistinguishable when the plants were grown in constant illumination. However, in a short photoperiod, the growth of the mutant was severely impaired, the rate of photosynthesis was depressed relative to the wild-type, and the rate of dark respiration, which was high following the onset of darkness, exhibited an uncharacteristic decay throughout the dark period. The altered control of respiration by the mutant, which may be related to the relatively high levels of soluble carbohydrate that accumulate in the leaf and stem tissue, is believed to be partially responsible for the low growth rate of the mutant in short days. The depressed photosynthetic capacity of the mutant may also reflect a metabolic adaptation to the accumulation of high levels of soluble carbohydrate which mimics the effects of alterations in source/sink ratio. The activities of sucrose phosphate synthase and acid invertase are significantly higher in the mutant than in the wild-type whereas ADP-glucose pyrophosphorylase activity is lower. This suggests that the activities of these enzymes may be modulated in response to metabolite concentrations or flux through the pathways.     

A Mutant of Arabidopsis thaliana with a Reduced Response to Fusicoccin. I

Because fusicoccin (FC) has the the capacity to promote solute uptake, a selective procedure for isolating mutants of Arabidopsis thaliana with a reduced response to the toxin has been developed. The procedure is based on the incubation of A. thaliana seedlings in a solution containing the cation Paraquat (Pq) at a concentration that per se does not produce bleaching of the leaves upon illumination but does in the presence of FC because of the increased uptake of the toxic cation. Using this procedure, we identified, among the progenies of 2010 M1 ethyl methanesulfonate-mutagenized plants, two mutants that stay green after exposure to FC and Pq. Some properties and inheritance of one of the two mutants (5-2) are described. Morphology of mutant plants is almost indistinguishable from that of the wild type. However, 5-2 seeds germinate and produce viable seedlings in the presence of FC plus the aminoglycoside antibiotic hygromycin B: plants of the mutant do not wilt when exposed to FC and stomata do not open or open only partially. In the presence of FC, the mutant appears less responsive than the wild type as far as the increment in fresh weight, the enlargement of leaf disc area, or the stimulation of H+ extrusion is concerned. Inheritance of the trait is monogenic dominant or semidominant, depending on the test used.     

Chlorophyllase Activity and Chlorophyll Content in Wild Type and eti 5 Mutant of Arabidopsis thaliana Subjected to Low and High Temperatures

Chlorophyll a (Chl a) content and chlorophyllase (Chlase) activity from leaves of wild type (WT) and the ethylene-insensitive mutant (eti 5) of Arabidopsis thaliana (L.) Heynh during temperature stress and plant recovery have been studied. The plants were subjected to temperatures of 4 °C (LT) and 38 °C (HT) for 24 h. Chl a gradually decreased somewhat during stress and in the first day of recovery, especially in HT-treated plants. At the end of the experimental period (1 d stress and 10 d recovery) Chl a content was lower in eti 5 plants than in WT ones. The Chlase in WT was more affected than in eti 5 plants during the temperature treatment and the recovery period. This revised version was published online in July 2006 with corrections to the Cover Date.     

Metabolism of Benzyladenine is Impaired in a Mutant of Arabidopsis thaliana Lacking Adenine Phosphoribosyltransferase Activity

Formation of the riboside-5′-monophosphate is a general feature of the metabolism of cytokinins in plants. As part of a study of the biological significance of the nucleotide form of cytokinins, we analyzed a mutant of Arabidopsis thaliana deficient in adenine phosphoribosyltransferase (APRT) activity for its ability to metabolize N⁶-benzyladenine (BA). Formation of N⁶-benzyladenosine-5′-monophosphate (BAMP) was assayed in vivo, by feeding tritiated BA to wild-type and mutant plantlets, and in crude plantlet extracts. Metabolites were separated by high performance liquid chromatography and quantitated by on-line liquid scintillation spectrometry. BA was rapidly absorbed by A. thaliana plantlets and primarily converted to BAMP and to BA 7- and 9-glucosides. BA was also rapidly absorbed by APRT-deficient plantlets, but its conversion to BAMP was strongly reduced. Formation of BAMP from N⁶-benzyladenosine was not affected in the mutant plantlets. In vitro conversion of BA to its nucleoside-5′-monophosphate was detected in crude extracts of wild-type plantlets, but not in extracts of APRT-deficient plantlets. Therefore, results of both assays indicate that APRT-deficient tissue does not convert BA to BAMP to a significant extent. Further, nondenaturing isoelectric focusing analysis of APRT activity in leaf extracts indicated that the enzyme activities which metabolize adenine and BA into their corresponding riboside-5′-monophosphate in extracts of wild-type plantlets have the same apparent isoelectric point. These activities were not detected in extracts prepared from APRT-deficient plantlets. Thus, these results demonstrate that APRT is the main enzyme which converts BA to its nucleotide form in young A. thaliana plants and that the ribophosphorylation of BA is not a prerequisite of its absorption by the plantlets.     

Isolation and Identification of an Arabidopsis thaliana Mutant with Serration Leaf Margin

Leaf polarity determines leaf flatness development directly, and abnormal polarity usually results in many abnormal leaves, which subsequently affects many physiological functions of plants. So the normal leaf development is important to plants. Here, an abnormal serration leaf margin mutant with abnormal leaf trichome development, named pCB1294, was isolated from an activation tagging Arabidopsis mutant pool through reverse genetics. By Tail PCR, the mutant gene loci At5g41663 encoding miR319b was successfully identified. Real time PCR shows the relative expression level of miR319b gene in the pCB1294 mutant is eleven times of higher than that of the wild (col). Our study lay the foundation for further studying the genetic mechanism of leaf polarity and investigating the interaction between miR319b and leaf morphology.     

拟南芥叶边缘锯齿状突变体的分离与鉴定

叶的极性建立直接决定叶的平展性发育,极性改变导致叶形态异常,影响植物体的各种正常生理活动。利用反向遗传学方法,从拟南芥基因激活标签突变体库中分离到一个叶片边缘锯齿状表型的突变体（命名为pCB1294）,该突变体同时表现出叶表皮腺毛形态发育异常。通过TailPCR方法成功定位突变基因为At5g41663,该基因编码miR319b基因。Real time PCR显示,pCB1294突变体植株中miR319b基因的表达量是野生型（col）植株的11倍多。所得结果为进一步研究miRNA调控叶极性的分子机制和进一步分析miR319b与叶形态发生的关系奠定了基础。     

Phylogenetic Analysis of ADP-Glucose Pyrophosphorylase Subunits Reveals a Role of Subunit Interfaces in the Allosteric Properties of the Enzyme

ADP-glucose pyrophosphorylase (AGPase) catalyzes a rate-limiting step in glycogen and starch synthesis in bacteria and plants, respectively. Plant AGPase consists of two large and two small subunits that were derived by gene duplication. AGPase large subunits have functionally diverged, leading to different kinetic and allosteric properties. Amino acid changes that could account for these differences were identified previously by evolutionary analysis. In this study, these large subunit residues were mapped onto a modeled structure of the maize (Zea mays) endosperm enzyme. Surprisingly, of 29 amino acids identified via evolutionary considerations, 17 were located at subunit interfaces. Fourteen of the 29 amino acids were mutagenized in the maize endosperm large subunit (SHRUNKEN-2 [SH2]), and resulting variants were expressed in Escherichia coli with the maize endosperm small subunit (BT2). Comparisons of the amount of glycogen produced in E. coli, and the kinetic and allosteric properties of the variants with wild-type SH2/BT2, indicate that 11 variants differ from the wild type in enzyme properties or in vivo glycogen level. More interestingly, six of nine residues located at subunit interfaces exhibit altered allosteric properties. These results indicate that the interfaces between the large and small subunits are important for the allosteric properties of AGPase, and changes at these interfaces contribute to AGPase functional specialization. Our results also demonstrate that evolutionary analysis can greatly facilitate enzyme structure-function analyses.ADP-glucose pyrophosphorylase (AGPase) catalyzes the conversion of Glc-1-P (G-1-P) and ATP to ADP-Glc and pyrophosphate. This reaction represents a rate-limiting step in starch synthesis (Hannah, 2005). AGPase is an allosteric enzyme whose activity is regulated by small effector molecules. In plants, AGPase is activated by 3-phosphoglyceraldehyde (3-PGA) and deactivated by inorganic phosphate (Pi).Plant AGPase is a heterotetramer consisting of two identical large and two identical small subunits. The large and small subunits of AGPase were generated by a gene duplication. Subsequent sequence divergence has given rise to complementary rather than interchangeable subunits. Indeed, both subunits are needed for AGPase activity (Hannah and Nelson, 1976, Burger et al., 2003). Biochemical studies have indicated that both subunits are important for catalytic and allosteric properties (Hannah and Nelson, 1976; Greene et al., 1996a, 1996b; Ballicora et al., 1998; Laughlin et al., 1998; Frueauf et al., 2001; Kavakli et al., 2001a, 2001b; Cross et al., 2004, 2005; Hwang et al., 2005, 2006, 2007; Kim et al., 2007; Ventriglia et al., 2008). Surprisingly, Georgelis et al. (2007, 2008) showed that, in angiosperms, the small subunit is under greater evolutionary pressure compared with the large subunit. Detailed analyses have shown that the greater constraint on the small subunit is due to its broader tissue expression patterns compared with the large subunit and the fact that the small subunit must interact with multiple large subunits.Large subunits have undergone more duplication events than have small subunits (Georgelis et al., 2008). This has led to the creation of five groups of large subunits that differ in their patterns of tissue of expression (Akihiro et al., 2005; Crevillen et al., 2005; Ohdan et al., 2005). Crevillen et al. (2003) studied the biochemical properties of four Arabidopsis (Arabidopsis thaliana) AGPases consisting of the four different large subunits and the only functional small subunit in Arabidopsis. The different AGPases had different kinetic and allosteric properties. More specifically, the AGPases differed in their affinity for the allosteric regulator 3-PGA and the substrates G-1-P and ATP. This possibly reflects the different 3-PGA, G-1-P, and ATP levels in the various tissues. This evidence indicates that not only did the different large subunit groups subfunctionalize in terms of expression, but also these groups may have specialized in terms of protein function. While the study of Crevillen et al. (2003) pointed to functional specialization of the large subunit, the identity of the amino acid sites in the large subunit that account for these kinetic and allosteric differences was not pursued.Georgelis et al. (2008) presented supporting evidence for AGPase large subunit specialization by identifying positively selected amino acid sites in the phylogenetic branches following gene duplication events. We also identified amino acid residues that were conserved in one large subunit group but not conserved in another large subunit group (type I functional divergence; Gu, 1999) and amino acid residues that are conserved within large subunit groups but are variable among large subunit groups (type II functional divergence; Gu, 2006). Positively selected type I and type II sites could have contributed to specialization of the different large subunit groups. Indeed, positively selected type II sites in several proteins have been proven via site-directed mutagenesis (Bishop, 2005; Norrgård et al., 2006; Cavatorta et al., 2008; Courville et al., 2008) to be important for protein function and functional specialization. Additionally, several positively selected type I and type II amino acid sites in the large AGPase subunit identified in our previous evolutionary analysis (Georgelis et al., 2008) have been implicated in the kinetic and allosteric properties and heat stability of AGPase. The role of these sites was demonstrated by site-directed mutagenesis experiments of large subunits from Arabidopsis, maize endosperm, and potato (Solanum tuberosum) tuber (Ballicora et al., 1998, 2005; Kavakli et al., 2001a; Jin et al., 2005; Linebarger et al., 2005; Ventriglia et al., 2008). These analyses indicate that the rest of the amino acid sites identified as positive type I and type II sites in our previous evolutionary analysis (Georgelis et al., 2008) represent promising candidate targets for mutagenesis.To identify large subunit amino acids that are possibly important in controlling enzyme properties and that may have contributed to large subunit specialization, we conducted site-directed mutagenesis of the maize endosperm large subunit encoded by Shrunken-2 (Sh2). We specifically identified amino acids of SH2 that correspond to amino acid sites that were detected as positive type I and type II sites during the large subunit evolution (Georgelis et al., 2008). We then replaced the SH2 residues with amino acids of a group different from the SH2 family. Several amino acid sites important for the kinetic and allosteric properties and heat stability of AGPase were identified. Our results indicate that the subunit interfaces between the large and small subunits are important for the allosteric properties of AGPase. They also indicate that amino acid changes at subunit interfaces have been important for AGPase specialization in terms of allosteric properties. These experiments also support the idea that the majority of positively selected sites as detected by codon substitution models (Nielsen and Yang, 1998; Yang et al., 2000) and type II sites are not false positives. Site-directed mutagenesis of such sites can greatly facilitate enzyme structure-function analyses.     

A Mutant of Arabidopsis thaliana That Defines a New Locus for Glycine Decarboxylation

A novel photorespiratory mutant of Arabidopsis thaliana, designatedgld2, was isolated based on a growth requirement for abnormallyhigh levels of atmospheric CO₂. Photosynthetic CO₂ fixationwas inhibited in the mutant following illumination in air butnot in atmosphere containing 2% O₂. Photosynthetic assimilationof ¹⁴CO₂ in an atmosphere containing 50% O₂ resulted in accumulationof 48% of the soluble label in glycine in the mutant comparedto 9% in the wild type. The rate of glycine decarboxylationby isolated mitochondria from the mutant was reduced to 6% ofthe wild type rate. In genetic crosses, the mutant complementedtwo previously described photorespiratory mutants of A. thalianathat accumulate glycine during photosynthesis in air due todefects in glycine decarboxylase (glyD, now designated gld1)and serine transhydroxymethylase (stm). Because glycine decarboxylaseis a complex of four enzymes, these results are consistent witha mutation in a glycine decarboxylase subunit other than thataffected in the gld1 mutant. The two gld loci were mapped tochromosomes 2 and 5, respectively. ³Present address: Department of Crop and Soil Sciences, MichiganState University, East Lansing, MI 48824, U.S.A. ⁴Present address: Department of Applied Bioscience, Facultyof Agriculture, Hokkaido University, Kita-Ku, Sapporo, 060 Japan ⁵Present address: Department of Biology, Carnegie Institutionof Washington, 290 Panama Street, Standford, CA 94305, U.S.A.     

黑曲霉纤维素酶突变子T-DNA插入位点的遗传分析及性质鉴定

采用羧甲基纤维素钠筛选培养基,对黑曲霉(Aspergillus niger)T-DNA突变子文库进行筛选,分离到一株纤维素酶分泌水平较低的菌株AN-108,为野生型菌株的83.3%。进一步测定该突变子固体发酵的纤维素酶活力,与野生型菌株相比没有明显差别,推测与固体发酵培养基中含有的天然糖类有关。在添加不同糖类的CMC-Na平板上培养该突变子,菌落周围均出现较明显的水解圈,结果显示糖类可能作为诱导物克服突变带来的影响。为了确定突变子AN-108中何种基因被阻断,采用反向PCR方法分析了T-DNA插入位点的序列,获得序列经过比对分析发现,该序列与黑曲霉An14g03730同源程度达90%,编码富含脯氨酸蛋白(proline-rich protein,PRP)。     

拟南芥网状突变体E-210基因精细定位

通过甲基磺酸乙酯(EMS)诱变与遗传分析,从拟南芥(Arabidopsis thaliana)中筛选到一株隐性单基因控制的网状突变体E-210.该突变体植株生长缓慢,叶脉呈绿色,叶肉呈黄色.通过透射电镜观察,发现野生型植株和突变植株在叶绿体结构上差异不大,猜测该突变体E-210基因与叶绿体的发育可能没有直接关系,而很可能同叶绿素或叶绿体的生物合成有关.通过图位克隆的方法,将该突变体的突变基因定位在第5条染色体上的MRBl7和MBG8-5的分子标记之间,精确到87.130 kb.对MRB17和MBG8-5的分子标记之间的22个基因进行了分析,预测突变体E-210基因可能是At5g54770,编码THI1,即噻唑合成酶.     

拟南芥prr5突变体对ABA的响应

以拟南芥为材料,统计PRRs （pseudo-response regulators）突变体 prr5及其野生型经ABA处理后的萌发率、根长和NaCl处理后的萌发率,并采用实时定量PCR方法,对不同浓度ABA处理的拟南芥幼苗中的PRR5基因表达进行分析.结果表明：prr5突变体对ABA弱敏感,其种子萌发率比野生型显著或极显著增高,主根比野生型长,且PRR5基因表达受ABA抑制.同时,NaCl处理后,prr5的萌发率比野生型极显著增高.因此,推测prr5可能为ABA信号通路相关基因.     

Deoxycytidine Triphosphate Deaminase: Characterization of an Escherichia coli Mutant Deficient in the Enzyme

A mutant of Escherichia coli, previously shown to contain abnormal nucleoside triphosphate pools, was found to be defective in its ability to synthesize thymidine nucleotides. The defect is not in the enzyme thymidylate synthetase but in deoxycytidine triphosphate deaminase, an enzyme that supplies deoxyuridine monophosphate, the substrate for thymidylate synthetase.