Improved in vitro selection of nitrate reductase-deficient mutants of Nicotiana plumbaginifolia

Summary The use of increasing knowledge on regulation of nitrate reductase activity in Nicotiana cell cultures is the basis for the described optimization of in vitro selection for nitrate reductase-deficient mutants by screening for chlorate resistance. Selection was carried out on haploid mesophyll protoplast-derived cell cultures of Nicotiana plumbaginifolia. It is demonstrated that revised selection results in high variant detectability and increased variant confirmability in comparison with the hitherto used selection scheme.     

Isolation of mutants of Arabidopsis thaliana in which accumulation of tobacco mosaic virus coat protein is reduced to low levels.

Summary We have found that Arahidopsis thaliana is susceptible to infection with a crucifer strain of tobacco mosaic virus (TMV-Cg); the coat protein of TMV-Cg accumulated to a high level in uninoculated rosette leaves several days after inoculation. As a first step in the search for host-coded factors that are involved in virus multiplication, we isolated mutants of A. thaliana in which the accumulation of TMV-Cg coat protein was reduced to low levels. Of 6000 M₂ plants descended from ethyl methanesulfonate-treated seeds, two such lines (PD 114 and PD378) were isolated. Genetic analyses suggested that the PD 114 phenotype was caused by a single nuclear recessive mutation, and that PD114 and PD378 belonged to the same complementation group. The coat protein accumulation of a tomato strain of TMV (TMVL) was also reduced in PD 114 plants compared to that in the wild-type plants. In contrast, PD114 plants infected with turnip crinkle or turnip yellow mosaic viruses, which belong to taxonomic groups other than Tobamovirus, expressed similar levels of these coat proteins as did infected wild-type plants.In this paper, we use the term multiplication (of a virus in a plant) to mean a substantial increase in virus concentration in the uninoculated leaves of the infected plant. Therefore, the efficiency of each process of invasion of the plant by the virus, uncoating, replication and degradation of the virus genome, formation and degradation of the virus particles, and spreading of the virus in the plant will affect the degree of multiplication     

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.     

Isolation and characterization of nitrate reductase-deficient mutants in tomato (Lycopersicon esculentum Mill.).

Summary Five nitrate reductase-deficient mutants of tomato were isolated from an M2 population after ethyl-methanesulphonate (EMS) seed treatment by means of selection for chlorate resistance. All mutations were monogenic and recessive and complementation analysis revealed that they were non-allelic. Biochemical and molecular characterization of these mutants showed that four of them are cofactor mutants while one is an apoenzyme mutant.     

Isolation and characterization of an auxin-inducible glutathione S-transferase gene of Arabidopsis thaliana

Genes homologous to the auxin-inducible Nt103 glutathione S-transferase (GST) gene of tobacco, were isolated from a genomic library of Arabidopsis thaliana. We isolated a clone containing an auxin-inducible gene, At103-1a, and part of a constitutively expressed gene, At103-1b. The coding regions of the Arabidopsis genes were highly homologous to each other and to the coding region of the tobacco gene but distinct from the GST genes that have been isolated from arabidopsis thusfar. Overexpression of a cDNA clone in Escherichia coli revealed that the AT103-1A protein had GST activity.     

Functional characterization of the Arabidopsis thaliana nitrate transporter CHL1 in the yeast Hansenula polymorpha

CHL1 (AtNRT1.1) is a dual-affinity nitrate transporter of Arabidopsis thaliana, in which phosphorylation at Thr 101 switches CHL1 from low to high nitrate affinity. CHL1 expressed in a Hansenula polymorpha high-affinity nitrate-transporter deficient mutant (Deltaynt1) restores nitrate uptake and growth. These events take place at nitrate concentrations as low as 500 muM, suggesting that CHL1 has a high-affinity for nitrate in yeast. Accordingly, CHL1 expressed in H. polymorpha presents a K (m) for nitrate of about 125 muM. The absence of nitrate, the CHL1 gene inducer, showed the high turnover rate of CHL1 expressed in yeast, which is counteracted by nitrate CHL1 induction. Furthermore, H. polymorpha strains expressing CHL1 become sensitive to 250 muM chlorate, as expected for CHL1 high-affinity behaviour. Given that CHL1 presented high affinity by nitrate, we study the role of CHL1 Thr101 in yeast. Strains producing CHL1Thr101Ala, unable to undergo phosphorylation, and CHL1Thr101Asp, where CHL1 phosphorylation is constitutively mimicked, were used. Yeast strains expressing CHL1Thr101Ala, CHL1Thr101Asp and CHL1 at the same rate showed that Deltaynt1CHL1Thr101Ala is strikingly unable to transport nitrate and contains a very low amount of CHL1 protein; however, Deltaynt1CHL1Thr101Asp restores nitrate uptake and growth, although no significant changes in nitrate affinity were observed. Our results show that CHL1-Thr101 is involved in regulating the levels of CHL1 expressed in yeast and suggest that the phosphorylation of this residue could be involved in targeting this nitrate transporter to the plasma membrane. The functional expression of CHL1 in H. polymorpha reveals that this yeast is a suitable tool for evaluating the real nitrate transport capacity of plant putative nitrate transporters belonging to different families and study their regulation and structure function relationship.     

Characterization of a chlorate-hypersensitive,high nitrate reductase Arabidopsis thaliana mutant

Summary A population of A. thaliana, produced by self-fertilization of ethylmethane sulfonate treated plants, was exposed to chlorate in the watering solution, and plants showing early susceptibility symptoms were rescued. Among the progeny lines of these plants five were shown to be repeatably chlorate-hypersusceptible. One of these lines (designated C-4) possessed elevated activity of nitrate reductase (NR). The NR activity of mutant C-4 was higher than that of normal plants throughout the life cycle. Nitrite reductase and glutamine synthetase activities of C-4 were normal, as were chlorate uptake rate and tissue nitrate content. The elevated NR activity apparently was responsible for the chlorate hypersusceptibility of C-4. Inheritance studies of NR indicated that the elevated activity of C-4 was probably controlled by a single recessive allele.     

Isolation and characterization of an Arabidopsis thaliana gene for the 54 kDa subunit of the signal recognition particle

The first step in the routing of newly synthesized proteins into the secretory pathway is the binding of the nascent signal sequence to the signal recognition particle. The mammalian signal recognition particle is a complex consisting of 6 proteins and a single 7S RNA molecule. Signal recognition particle-like complexes have been described from wheat and maize but none of the protein components have yet been described from any plant species. Here we report the cloning and characterization of an Arabidopsis thaliana gene encoding the 54 kDa protein subunit of the signal recognition particle. This is the first report of a SRP-54 sequence for any plant species and the first genomic sequence for any multicellular organism.Abbreviations ER endoplasmic reticulum - PCR polymerase chain reaction - SRP signal recognition particle     

Identification and characterization of a chlorate-resistant mutant of Arabidopsis thaliana with mutations in both nitrate reductase structural genes NIA1 and NIA2

Mutant plants defective in the assimilation of nitrate can be selected by their resistance to the herbicide chlorate. In Arabidopsis thaliana, mutations at any one of nine distinct loci confer chlorate resistance. Only one of the CHL genes, CHL3, has been shown genetically to be a nitrate reductase (NR) structural gene (NIA2) even though two NR genes (NIA1 and NIA2) have been cloned from the Arabidopsis genome. Plants in which the NIA2 gene has been deleted retain only 10% of the wildtype shoot NR activity and grow normally with nitrate as the sole nitrogen source. Using mutagenized seeds from the NIA2 deletion mutant and a modified chlorate selection protocol, we have identified the first mutation in the NIA1 NR structural gene. nia1, nia2 double mutants have only 0.5% of wild-type shoot NR activity and display very poor growth on media with nitrate as the only form of nitrogen. The nial-1 mutation is a single nucleotide substitution that converts an alanine to a threonine in a highly conserved region of the molybdenum cofactor-binding domain of the NR protein. These results show that the NIA1 gene encodes a functional NR protein that contributes to the assimilation of nitrate in Arabidopsis.     

Molecular characterization of multiple cDNA clones for ADP-glucose pyrophosphorylase from Arabidopsis thaliana

PCR amplification of cDNA prepared from poly(A)⁺ RNA from aerial parts of Arabidopsis thaliana, using degenerate nucleotide primers based on conserved regions between the large and small subunits of ADP-glucose pyrophosphorylase (AGP), yielded four different cDNAs of ca. 550 nucleotides each. Based on derived amino acid sequences, the identities between the clones varied from 49 to 69%. Sequence comparison to previously published cDNAs for AGP from various species and tissues has revealed that three of the amplified cDNAs (ApL1, ApL2 and ApL3) correspond to the large subunit of AGP, and one cDNA (ApS) encodes the small subunit of AGP. Both ApL1 and ApS were subsequently found to be present in a cDNA library made from Arabidopsis leaves. All four PCR products are encoded by single genes, as found by genomic Southern analysis.     

Occurrence of xanthine dehydrogenase in Chlamydomonas reinhardii: A common cofactor shared by xanthine dehydrogenase and nitrate reductase

Wild-type Chlamydomonas reinhardii cells have xanthine dehydrogenase activity when grown with nitrate, nitrite, urea, or amino acid media. Mutant strains 102, 104, and 307 of Chlamydomonas, lacking both xanthine dehydrogenase and nitrate reductase activities, were incapable of restoring the NADPH-nitrate reductase activity of the mutant nit-1 of Neurospora crassa, whereas wild type cells and mutants 203 and 305 had xanthine dehydrogenase and were able to reconstitute the nitrate reductase activity of nit-1 of Neurospora. Therefore, it is concluded that in Chlamydomonas a common cofactor is shared by xanthine dehydrogenase and nitrate reductase. Xanthine dehydrogenase is repressed by ammonia and seems to be inessential for growth of Chlamydomonas.     

6种拟南芥突变体热敏感性差异的比较分析

为了解拟南芥（Arabidopsis thaliana）热敏感突变体的热敏感性,对6个常用的拟南芥热敏感突变体hot1、apx2、fes1a、hsfa7a、hop1-2-3和hsp70-15进行了比较分析。结果表明,6个突变体的热敏感性均高于野生型,但他们之间的热敏感性有显著差异,45℃极度高温下90 min,hot1的白化死亡率最高,处理105 min后,fes1a也出现高比率的白化死亡,处理135 min后,apx2、hsfa7a和hop1-2-3表现出几乎相同的损伤现象,热损伤均比hsp70-15严重。因此,6种突变体的热敏感性依次为hot1 > fes1a > apx2、hsfa7a、hop1-2-3 > hsp70-15。     

Identification of Arabidopsis thaliana variants with differential glyphosate responses

To facilitate future investigations of glyphosate-resistance mechanisms, three approaches were taken to obtain Arabidopsis thaliana variants that differed in glyphosate response. Recurrent selection by spraying with sub-lethal glyphosate concentrations was performed with Columbia-0 seedlings. After seven cycles of treatment, no resistance was found. A population of 800,000 ethylmethanesulfonate-mutagenized M(2) seedlings was screened on agar containing 0.2mM glyphosate, a lower concentration than that previously used in other studies, and no resistant mutants were recovered. Seventy-two Arabidopsis ecotypes were screened with glyphosate and a range of responses was observed. In a follow-up experiment on a subset of these ecotypes, reduction of seed yield by 11.5 g/ha glyphosate (about 1% the typical field use rate) ranged among ecotypes from 0% to >90%, relative to untreated controls. However, even the least sensitive ecotypes were severely injured by relatively low glyphosate rates. Overall, attempts to select Arabidopsis seedlings that were significantly glyphosate-resistant were unsuccessful and consistent with previous reports. Arabidopsis ecotypes identified with differential glyphosate responses could be used for further studies though the inherently high sensitivity of Arabidopsis to glyphosate could limit their utility in studying glyphosate-resistance mechanisms.     

Nitrate reductase deficient cell lines from diploid cell cultures and lethal mutant M2 plants of Arabidopsis thaliana

Summary Cell suspensions of diploid Arabidopsis thaliana were screened for resistance to chlorate on a medium with ammonium nitrate as the nitrogen source, and after plating on filters to increase the plating efficiency. Thirty-nine lines were selected, four of which were still resistant after two years of subculturing on non-selective medium. Of the latter lines three were nitrate reductase deficient but exhibited some residual nitrate reductase activity; the fourth line showed a high level of enzyme activity. Screening M2-seeds for callus production on selective medium with amino acids as the nitrogen source and chlorate revealed resistant calli in 17 out of 483 M2-groups. Nine well-growing lines, all but one (G3) exhibiting no detectable in vivo nitrate reductase activity, were classified as defective in the cofactor. Two lines (G1 and G3) could be analysed genetically at the plant level. Chlorate resistance was monogenic and recessive. Sucrose gradient fractionation of callus extracts of G1 revealed that a complete enzyme molecule can be assembled. Nitrate reductase activity in G1 could partly be restored by excess molybdenum. It is suggested that G1 is disturbed in the catalytic properties of the cofactor. It appeared that G1 is neither allelic with another molybdenum repairable mutant (B73) nor with another cofactor mutant (B25). Wilting of intact G1 plants could be ascribed to non-closing stomata.     

Characterization of three male-sterile mutants of Arabidopsis thaliana exhibiting alterations in meiosis

Male-sterile mutants are being studied to deepen our understanding of the complex processes of microsporogenesis and microgametogenesis. Due to difficulties associated with isolating the mutated gene, there is currently very little molecular information on the defects responsible for male sterility. As a first step in utilizing male-sterile mutants to better understand the bio-chemical and molecular processes that control pollen development, we have characterized a number of Arabidopsis thaliana lines that were generated by seed transformation and exhibit male sterility. We report here the identification and characterization of three male-sterile A. thaliana lines, all of which are tagged with T-DNA and show aberrant meiosis. A detailed cytochemical study was conducted on these lines to better understand the timing and nature of each mutation and to investigate how these mutations affect subsequent steps of pollen development. All three mutants undergo apparently normal morphogenesis until the onset of meiosis. In one line (6492) the mutation is most notable at the tetrad stage when up to eight microspores can be seen in each callose-encased tetrad. The resulting mutant microspores are of variable sizes and contain different amounts of DNA. Two other mutants (7219 and 7593) possess many common features, including variable developmental pathways, failure to produce callose, production of vacuolate, coenocytic (multi-nucleate) cells that are surrounded by persistent microsporocyte walls, and asynchronous patterns of development. Unlike the situation in wild-type plants, where developmental stages are correlated with bud length, such correlations are almost impossible with these two mutants. The sporogenous tissue within all three of these mutant lines collapses prior to anthesis.     

Effect of light on the NADPH-protochlorophyllide oxidoreductase of Arabidopsis thaliana

A cDNA encoding the NADPH-protochlorophyllide oxidoreductase (Pchlide reductase) of Arabidopsis thaliana has been isolated and sequenced. The cDNA contains the complete reading frame for the precursor of the Pchlide reductase. The deduced amino acid sequence of the Arabidopsis enzyme closely resembles the corresponding sequences of barley and oat. The cDNA has been used as a template for the synthesis of the enzyme protein in Escherichia coli. An antiserum was raised against this enzyme protein and both the antiserum and the cDNA were used as experimental tools to study the effects of light on the Pchlide reductase in A. thaliana.When etiolated seedlings of Arabidopsis were exposed to light the enzyme activity and the concentration of the enzyme protein rapidly declined. Similar light effects have been described previously for other angiosperms. In contrast to most of these species, however, in Arabidopsis only minor changes in Pchlide reductase mRNA content could be observed when etiolated seedlings were exposed to light.     

Genetic mapping of the barley nitrate reductase-deficient nar1 and nar2 loci

Summary The nar2 locus that codes for a protein involved in molybdenum cofactor function in nitrate reductase and other molybdoenzymes was mapped to barley chromosome 7. F₂ genotypic data from F₃ head rows indicated nar2 is located 8.4±2.1 and 23.0± 4.6 cm from the narrow leaf dwarf (nld) and mottled seedling (mt2) loci, respectively. This locates the nar2 locus at 54.7±3.1 cm from the short-haired rachilla (s) locus near the centromere of chromosome 7. Close linkage of nar2 with DDT resistance (ddt) and high lysine (lys3) loci was detected but could not be quantified due to deviations from the individual expected 121 segregations for the ddt and lys3 genes. Southern blots of wheat-barley addition lines probed with a nitrate reductase cDNA located the NADH : nitrate reductase structural gene, nar1, to chromosome 6.Scientific Paper No. 7762. College of Agriculture and Home Economics Research Center, Washington State University, Project No. 0745. This investigation was supported in part by United States Department of Agriculture Grant No. 86-CRCR-1-2004     

In vitro morphogenesis of arrested embryos from lethal mutants of Arabidopsis thaliana

Summary Arrested embryos from lethal (emb) mutants of Arabidopsis thaliana were rescued on a nutrient medium designed to promote plant regeneration from immature wild-type cotyledons. The best response was observed with mutant embryos arrested at the heart to cotyledon stages of development. Embryos arrested at a globular stage produced callus but failed to turn green or form normal shoots in culture. Many of the mutant plants produced in culture were unusually pale with abnormal leaves, rosettes, and patterns of reproductive development. Other plants were phenotypically normal except for the presence of siliques containing 100% aborted seeds following self-pollination. These results demonstrate that genes with essential functions during plant embryo development differ in their pattern of expression at later stages of the life cycle. Most of the 15 genes examined in this study were essential for embryogenesis but were required again for subsequent stages of development. Only EMB24 appeared to be limited in function to embryo development. These differences in the response of mutant embryos in culture may facilitate the classification of embryonic lethals and the identification of genes with developmental rather than housekeeping functions.     

Gene targeting in Arabidopsis thaliana.

Summary Gene targeting of a chromosomally integrated transgene in Arabidopsis thaliana is reported. A chimeric gene consisting of the promoter of the 35S RNA of CaMV, the polyadenylation signal of the octopine synthase gene and the coding region of the bacterial hygromycin phosphotransferase gene (hpt), which was rendered non-functional by deletion of 19 bp, was introduced into the genome of A. thaliana using Agrobacterium-mediated gene transfer. A total of 3.46 x 10⁸ protoplasts isolated from 17 independent transgenic Arabidopsis lines harbouring the defective chimeric hpt gene were transformed via direct gene transfer using various DNA forms containing only the intact coding region of the hpt gene. Out of 150 hygromycin-resistant colonies appearing in the course of these experiments, four were the result of targeted recombination of the incoming DNA with the defective chromosomal locus as revealed by PCR and Southern blot analysis. Comparison with the number of transformants obtained when an hpt gene controlled by a promoter and terminator from the nopaline synthase gene was employed results in a maximal ratio of homologous to non-homologous transformation in A. thaliana of 1 x 10^–4.