Anomalous electron transport activity in a Photosystem I-deficient maize mutant

Photosynthesis mutations were induced in maize lines bearing the transposable DNA element system, Mutator. Two Photosystem I mutants (hcf101 and hcf104) which were isolated are described here. Maize plants homozygous for the hcf104 mutation are seedling lethal and exhibit a high in vivo chlorophyll fluorescence yield. They lack 60% of CP1, P700 and PSI-specific electron transport activity relative to normal sibling plants. The comparable depletion of these three measures of PS I content conforms to the pattern reported for many other PS I-deficient mutants. Maize plants homozygous for hcf101 are seedling lethal and also exhibit high in vivo chlorophyll fluorescence yield. They lack 80–90% of CP1 and P700 but sustain steady state levels of PS I-specific electron transport activity at 70% of normal. Previous reports of similar apparent PS I hyperactivity are discussed and an explanation for the elevated steady state level of PS I electron transport activity in hcf101 is proposed.Abbreviations CP1 chlorophyll-protein complex 1 - hcf high chlorophyll fluorescent - LHCI Light harvesting chlorophyll-protein complex I - PAGE polyacrylamide gel electrophoresis - P700 reaction center pigment of PS I - PQ plastoquinone     

Energy transfer for low temperature fluorescence in PS II mutant thylakoids

The Chl-protein complexes of three maize (Zea mays L.) mutants and one barley (Hordeum vulgare L.) mutant were analyzed using low temperature Chl fluorescence emissions spectroscopy and LDS-polyacrylamide gel electrophoresis. The maize mutants hcf-3, hcf-19, and hcf-114 all exhibited a high Chl fluorescence (hcf) phenotype indicating a disruption of the energy transfer within the photosynthetic apparatus. The mutations in each of these maize mutants affects Photosystem II. The barley mutant analyzed was the well characterized Chl b-less mutant chlorina-f2, which did not exhibit the hcf phenotype. Chlorina-f2 was used because no complete Chl b-less mutant of maize is available. Analysis of hcf-3, hcf-19, and hcf-114 revealed that in the absence of CP43, LHC II can still transfer excitation energy to CP47. These results suggest that in mutant membranes LHC II can interact with CP47 as well as CP43. This functional interaction of LHC II with CP47 may only occur in the absence of CP43, however, it is possible that LHC II is positioned in the thylakoid membranes in a manner which allows association with both CP43 and CP47.Abbreviations hcf high chlorophyll fluorescence - LDS lithium dodecyl sulfate - LHC II light-harvesting complex of Photosystem II - LHC I light-harvesting complex of Photosystem I - CPIa chlorophyll-protein complex consisting of LHC I and the PS I core complex - CPI chlorophyll-protein complex consisting of the PS I core complex - CP47 47 kDa chlorophyll-protein of the Photosystem II core - CP43 43 kDa chlorophyll-protein of the Photosystem II core - CP29 29 kDa chlorophyll-protein of Photosystem II - CP26 26 kDa chlorophyll-protein of Photosystem II - CP24 24 kDa chlorophyll-protein of Photosystem II - fp free pigments     

Site-selected transposon mutagenesis at the hcf106 locus in maize.

The High chlorophyll fluorescence106 (Hcf106) gene in maize is required for chloroplast membrane biogenesis, and the hcf106-mum1 allele is caused by the insertion of a Robertson's Mutator Mu1 element into the promoter of the gene. Seedlings homozygous for hcf106-mum1 are pale green and die 3 weeks after germination, but only in the presence of Mutator activity conferred by active, autonomous Mu regulatory transposons elsewhere in the genome. When Mutator activity is lost, the mutant phenotype is suppressed, and homozygous plants have an almost wild-type phenotype. To isolate derivative alleles at the hcf106 locus that no longer require Mutator activity for phenotypic expression, we have developed a method for site-selected transposon mutagenesis in maize. This procedure, first described for Caenorhabditis elegans and Drosophila, involves using polymerase chain reaction (PCR) to screen pools of individuals for insertions and deletions in genes of known sequence. Pools of seedlings segregating for the progenitor allele hcf106-mum1 were screened by PCR for insertions and deletions associated with Robertson's Mutator. In a 360-bp target region, two new insertions and one deletion were identified in only 700 Mu-active gametes screened. One of the insertions was in the progenitor hcf106-mum1 allele and the other was in the wild-type allele, but all three new alleles were found to have break-points at the same nucleotide in the first intron. Unlike the hcf-106-mum1 progenitor allele, the deletion and one of the insertions conferred pale green seedling lethal phenotypes in the absence of mutator activity. However, the second insertion had a weak, viable phenotype under these conditions.(ABSTRACT TRUNCATED AT 250 WORDS)     

Molecular analysis of high-copy insertion sites in maize

High-copy transposon mutagenesis is an effective tool for creating gene disruptions in maize. In order to molecularly define transposon-induced disruptions on a genome-wide scale, we optimized TAIL-PCR to amplify genomic DNA flanking maize Robertson’s Mutator insertions. Sample sequencing from 43 Mutator stocks and the W22 inbred line identified 676 non-redundant insertions, and only a small fraction of the flanking sequences showed significant similarity to maize repetitive sequences. We further designed and tested 79 arbitrary primers to identify 12 primers that amplify all Mutator insertions within a DNA sample at 3.1-fold redundancy. Importantly, the products are of sufficient size to use as substrates or probes for hybridization-based identification of gene disruptions. Our adaptation simplifies previously published TAIL-PCR protocols and should be transferable to other high-copy insertional mutagens.     

Reactivation of a silenced minimal Mutator transposable element system following low-energy nitrogen ion implantation in maize

In maize, Mutator transposable elements are either active or silenced within the genome. In response to environmental stress, silenced Mutator elements could be reactivated, leading to changes in genome structure and gene function. However, there is no direct experimental evidence linking environmental stress and Mutator transposon reactivation. Using a maize line that contains a single inactive MuDR and a lone nonautonomous Mutator element, a Mu1 insertion in the recessive reporter allele a1-mum2 in an inactive Mutator background, we directly assessed Mutator reactivation following low-energy nitrogen ion implantation. We observed that N⁺ implantation decreased cytosine methylation in MuDR terminal inverted repeats and increased expression of mudrA and mudrB. Both changes were associated with increased transpositional activity of MuDR through reactivation of the inactive minimal Mutator transposable element system. This study provides direct evidence linking environmental stress agents and Mutator transposon mobilization in maize. In addition, the observed changes to DNA methylation suggest a new mechanism for mutations by low-energy ion implantation.     

gaMS-1: A gametophytic male sterile mutant in maize

Several pollen-specific genes from different species have been isolated and characterized at the molecular level, but the precise role of most of them is unknown. Mutant analysis represents a direct approach to uncovering gene function, but the paucity of available mutants affecting pollen development and/or function and the poor characterization of the known mutants have so far limited the exploitation of this approach. Here we present the cytological characterization ofgametophytic male sterile-1 (gaMS-1), a maize mutant that we identified in a program of transposon insertion mutagenesis for the production of mutations in gametophytically acting genes involved in microsporogenesis.gaMS-1 is expressed during or immediately after the first microspore division and leads to the production of immature, nonfunctional pollen grains. The mutation appears to affect the events leading to the developmental switch that follows the first microspore mitosis.     

A mini-Tn5-derived transposon with reportable and selectable markers enables rapid generation and screening of insertional mutants in Gram-negative bacteria

We re-engineered a classic tool for mutagenesis and gene expression studies in Gram-negative bacteria. Our modified Tn5-based transposon contains multiple features that allow rapid selection for mutants, direct quantification of gene expression and straightforward cloning of the inactivated gene. The promoter-less gfp-km cassette provides selection and reporter assay depending on the activity of the promoter upstream of the transposon insertion site. The cat gene facilitates positive antibiotic selection for mutants, while the narrow R6Kγ replication origin forces transposition in recipient strains lacking the pir gene and enables cloning of the transposon flanked with the disrupted gene from the chromosome. The suicide vector pCKD100, a plasmid that could be delivered into recipient cells through biparental mating or electroporation, harbours the modified transposon. We used the transposon to mutagenize Pectobacterium versatile KD100, Pseudumonas coronafaciens PC27R and Escherichia coli 35150N. The fluorescence intensities of mutants expressing high GFP could be quantified and detected qualitatively. Transformation efficiency from conjugation ranged from 1600 to 1900 CFU per ml. We sequenced the upstream flanking regions, identified the putative truncated genes and demonstrated the restoration of the GFP phenotype through marker exchange. The mini-Tn5 transposon was also utilized to construct mutant a library of P. versatile for forward genetic screens.     

Targeted mutagenesis of a conserved anther‐expressed P450 gene confers male sterility in monocots

Targeted mutagenesis using programmable DNA endonucleases has broad applications for studying gene function in planta and developing approaches to improve crop yields. Recently, a genetic method that eliminates the need to emasculate the female inbred during hybrid seed production, referred to as Seed Production Technology, has been described. The foundation of this genetic system relied on classical methods to identify genes critical to anther and pollen development. One of these genes is a P450 gene which is expressed in the tapetum of anthers. Homozygous recessive mutants in this gene render maize and rice plants male sterile. While this P450 in maize corresponds to the male fertility gene Ms26, male fertility mutants have not been isolated in other monocots such as sorghum and wheat. In this report, a custom designed homing endonuclease, Ems26+, was used to generate in planta mutations in the rice, sorghum and wheat orthologs of maize Ms26. Similar to maize, homozygous mutations in this P450 gene in rice and sorghum prevent pollen formation resulting in male sterile plants and fertility was restored in sorghum using a transformed copy of maize Ms26. In contrast, allohexaploid wheat plants that carry similar homozygous nuclear mutations in only one, but not all three, of their single genomes were male fertile. Targeted mutagenesis and subsequent characterization of male fertility genes in sorghum and wheat is an important step for capturing heterosis and improving crop yields through hybrid seed.     

Exochelin genes in Mycobacterium smegmatis: identification of an ABC transporter and two non-ribosomal peptide synthetase genes

Many strains of mycobacteria produce two ferric chelating substances that are termed exochelin (an excreted product) and mycobactin (a cell-associated product). These agents may function as iron acquisition siderophores. To examine the genetics of the iron acquisition system in mycobacteria, ultraviolet (UV) and transposon (Tn611 ) mutagenesis techniques were used to generate exochelin-deficient mutants of Mycobacterium smegmatis strains ATCC 607 and LR222 respectively. Mutants were identified on CAS siderophore detection agar plates. Comparisons of the amounts of CAS-reactive material excreted by the possible mutant strains with that of the wild-type strains verified that seven UV mutant strains and two confirmed transposition mutant strains were deficient in exochelin production. Cell-associated mycobactin production in the mutants appeared to be normal. From the two transposon mutants, the mutated gene regions were cloned and identified by colony hybridization with an IS6100 probe, and the DNA regions flanking the transposon insertion sites were then used as probes to clone the wild-type loci from M. smegmatis LR222 genomic DNA. Complementation assays showed that an 8 kb Pst I fragment and a 4.8 kb Pst I/SacI subclone of this fragment complemented one transposon mutant (LUN2) and one UV mutant (R92). A 10.1 kb SacI fragment restored exochelin production to the other transposon mutant (LUN1). The nucleotide sequence of the 15.3 kb DNA region that spanned the two transposon insertion sites overlapped the 5′ region of the previously reported exochelin biosynthetic gene fxbA and contained three open reading frames that were transcribed in the opposite orientation to fxbA. The corresponding genes were designated exiT, fxbB and fxbC. The deduced amino acid sequence of ExiT suggested that it was a member of the ABC transporter superfamily, while FxbB and FxbC displayed significant homology with many enzymes (including pristinamycin I synthetase) that catalyse non-ribosomal peptide synthesis. We propose that the peptide backbone of the siderophore exochelin is synthesized in part by enzymes resembling non-ribosomal peptide synthetases and that the ABC transporter ExiT is responsible for exochelin excretion.     

Nuclear mutations affecting plastoquinone accumulation in maize

We have isolated and characterized two nuclear mutations which affect plastoquinone accumulation in maize. The mutations, hcf103 and hcf114, modify the same genetic locus. Plants homozygous for either mutant allele exhibit reduced PS II electron transport activity, reduced variable chlorophyll fluorescence and reduced delayed fluorescence yield. In these ways, hcf103 and hcf114 resemble previously described PS II mutants which lack stably assembled PS II reaction center complexes. However, unlike most previously described PS II mutants, hcf103 and hcf114 possess stable membrane-associated PS II complexes. Plastoquinone (PQ-9), which performs a variety of redox functions essential to normal non-cyclic electron transport, is severely depleted in the mutants. The lack of PS II electron transport activity is attributed to the absence of PQ-9. This is the first report of mutants deficient in PQ which do not also suffer serious pleiotropic defects.Abbreviations PS II Photosystem II - PQ plastoquinone - Q_A and Q_B primary and secondary stable electron acceptors of PS II - HPLC high pressure liquid chromatography - LDS-PAGE lithium dodecyl sulfate polyacrylamide gel electrophoresis - TLC thin layer chromatography     

Rhizobium etli cytochrome mutants with derepressed expression of cytochrome terminal oxidases and enhanced symbiotic nitrogen accumulation

 A method to isolate mutants with derepressed expression of cytochrome oxidases and better symbiotic performance is presented. A mutant of Rhizobium etli, CFN030, isolated by its azide-resistant phenotype, was obtained by transposon Tn5-mob mutagenesis. This mutant has a derepressed expression of cytochrome aa₃, higher respiratory activities when cultured microaerobically and an improved symbiotic nitrogen fixation capacity. This phenotype was similar to the previously described mutant CFN037, which was isolated by its increased capacity to oxidize N,N,N′,N′-tetramethyl-p-phenylenediamine (TMPD) [Soberón M et al. (1990) J Bacteriol 172:1676–1680]. We show here that although both mutants have a similar symbiotic phenotype, they are affected in different genes. Strain CFN030 has the Tn5 inserted in the chromosome while in strain CFN037 the transposon was located in plasmid b. Cytochrome spectral analysis of both mutant strains in the post-exponential phase of growth, showed the expression of an additional terminal oxidase (cbb₃) that is not expressed in the wild-type strain. Received: 10 April 1995/Received revision: 21 August 1995/Accepted: 7 September1995     

<Emphasis Type="Italic">Mutator</Emphasis>-like elements identified in melon,Arabidopsis and rice contain ULP1 protease domains

The transposon Mutator was first identified in maize, and is one of the most active mobile elements in plants. The Arabidopsis thaliana genome contains at least 200 Mutator-like elements (MULEs), which contain the Mutator-like transposase gene, and often additional genes. We have detected a novel type of MULEs in melon (CUMULE), which, besides the transposase, contains two ubiquitin-like specific protease-like sequences (ULP1). This element is not present in the observed location in some melon cultivars. Multiple copies of this element exist in the Cucumis melo genome, and it has been detected in other Cucurbitaceae species. Analysis of the A. thaliana genome revealed more than 90 CUMULE-like elements, containing one or two Ulp1-like sequences, although no evidence of mobility exists for these elements. We detected various putative transposable elements containing ULP1-like sequences in rice. The discovery of these MULEs in melon and Arabidopsis, and the existence of similar elements in rice and maize, suggest that a proteolytic function may be important for this subset of the MULE transposable elements. Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible for authorized users. Nucleotide sequence data reported are available in the GenBank database under the accession number AY524004.     

Use of Illumina sequencing to identify transposon insertions underlying mutant phenotypes in high‐copy Mutator lines of maize

High‐copy transposons have been effectively exploited as mutagens in a variety of organisms. However, their utility for phenotype‐driven forward genetics has been hampered by the difficulty of identifying the specific insertions responsible for phenotypes of interest. We describe a new method that can substantially increase the throughput of linking a disrupted gene to a known phenotype in high‐copy Mutator (Mu) transposon lines in maize. The approach uses the Illumina platform to obtain sequences flanking Mu elements in pooled, bar‐coded DNA samples. Insertion sites are compared among individuals of suitable genotype to identify those that are linked to the mutation of interest. DNA is prepared for sequencing by mechanical shearing, adapter ligation, and selection of DNA fragments harboring Mu flanking sequences by hybridization to a biotinylated oligonucleotide corresponding to the Mu terminal inverted repeat. This method yields dense clusters of sequence reads that tile approximately 400 bp flanking each side of each heritable insertion. The utility of the approach is demonstrated by identifying the causal insertions in four genes whose disruption blocks chloroplast biogenesis at various steps: thylakoid protein targeting (cpSecE), chloroplast gene expression (polynucleotide phosphorylase and PTAC12), and prosthetic group attachment (HCF208/CCB2). This method adds to the tools available for phenotype‐driven Mu tagging in maize, and could be adapted for use with other high‐copy transposons. A by‐product of the approach is the identification of numerous heritable insertions that are unrelated to the targeted phenotype, which can contribute to community insertion resources.     

Molecular characterization demonstrates that the <Emphasis Type="Italic">Zea mays</Emphasis> gene <Emphasis Type="Italic">sugary2</Emphasis> codes for the starch synthase isoform SSIIa

Mutations in the maize gene sugary2 (su2) affect starch structure and its resultant physiochemical properties in useful ways, although the gene has not been characterized previously at the molecular level. This study tested the hypothesis that su2 codes for starch synthase IIa (SSIIa). Two independent mutations of the su2 locus, su2-2279 and su2-5178, were identified in a Mutator-active maize population. The nucleotide sequence of the genomic locus that codes for SSIIa was compared between wild type plants and those homozygous for either novel mutation. Plants bearing su2-2279 invariably contained a Mutator transposon in exon 3 of the SSIIa gene, and su2-5178 mutants always contained a small retrotransposon-like insertion in exon 10. Six allelic su2 ^– mutations conditioned loss or reduction in abundance of the SSIIa protein detected by immunoblot. These data indicate that su2 codes for SSIIa and that deficiency in this isoform is ultimately responsible for the altered physiochemical properties of su2 ^– mutant starches. A specific starch synthase isoform among several identified in soluble endosperm extracts was absent in su2-2279 or su2-5178 mutants, indicating that SSIIa is active in the soluble phase during kernel development. The immediate structural effect of the su2 ^– mutations was shown to be increased abundance of short glucan chains in amylopectin and a proportional decrease in intermediate length chains, similar to the effects of SSII deficiency in other species.     

Construction of a Slime Negative Transposon Mutant in Staphylococcus epidermidis Using the Enterococcus faecalis Transposon Tn917

The slime-producing Staphylococcus epidermidis strain sensu strictu CNS23 was transformed by protoplast transformation with the plasmid pTV1 which carries transposon Tn917. Using this transposon mutagenesis system we obtained the Tn917-inserted mutant CT512, which has lost the ability to produce slime. A single insertion of the trasposon Tn917 into the chromosome of CT512 could be detected by Southern hybridization. This mutant showed a significantly higher stability concerning its slime-negative phenotype compared with spontaneous slime-negative mutants of S. epidermidis strain CNS23. In slime-ELISA no slime-associated antigen could be detected in extracts of the transposon mutant. Compared to slime-positive S. epidermidis strains, CT512 lacked in accumulative growth in microtiter tube test.     

Identification of two fructose transport and phosphorylation pathways in Xanthomonas campestris pv. campestris.

Summary Fructose was shown to be phosphorylated by a specific phosphoenolpyruvatc-dependent phosphotransferase system (PTS) in Xanthomonas campestris pv. campestris. Transposon mutagenesis of X. campestris was performed and two mutants affected in growth on fructose were isolated. Both mutants were deficient in PTS activity. Comparison of the rate of uptake and phosphorylation of fructose in the wild-type and in the mutant strains revealed the presence of a second fructose permeation and phosphorylation pathway in this bacterium: an unidentified permease coupled to an ATP-dependent fructokinase. One of the two mutants was also deficient in fructokinase activity. Chromosomal DNA fragments containing the regions flanking the transposon insertion site were cloned from both mutant strains. Their physical study revealed that the insertion sites were separated by 1.4 kb, allowing the reconstruction of a wild-type DNA fragment which complemented one of the two mutants. The region flanking the transposon insertion site was sequenced in one of the mutants, showing that the transposon had interrupted the gene encoding the fructose Ell. The mutant strains also failed to utilize mannose, sucrose and mannitol, suggesting the existence of a branch point between the metabolism of fructose and of these latter carbohydrates.     

Single-gene knockout of a novel regulatory element confers ethionine resistance and elevates methionine production in Corynebacterium glutamicum

Despite the availability of genome data and recent advances in methionine regulation in Corynebacterium glutamicum, sulfur metabolism and its underlying molecular mechanisms are still poorly characterized in this organism. Here, we describe the identification of an ORF coding for a putative regulatory protein that controls the expression of genes involved in sulfur reduction dependent on extracellular methionine levels. C. glutamicum was randomly mutagenized by transposon mutagenesis and 7,000 mutants were screened for rapid growth on agar plates containing the methionine antimetabolite d,l-ethionine. In all obtained mutants, the site of insertion was located in the ORF NCgl2640 of unknown function that has several homologues in other bacteria. All mutants exhibited similar ethionine resistance and this phenotype could be transferred to another strain by the defined deletion of the NCgl2640 gene. Moreover, inactivation of NCgl2640 resulted in significantly increased methionine production. Using promoter lacZ-fusions of genes involved in sulfur metabolism, we demonstrated the relief of l-methionine repression in the NCgl2640 mutant for cysteine synthase, o-acetylhomoserine sulfhydrolase (metY) and sulfite reductase. Complementation of the mutant strain with plasmid-borne NCgl2640 restored the wild-type phenotype for metY and sulfite reductase.     

Transposon Mutagenesis in the Study of Plant Development

Transposon mutagenesis has provided one of the first and most important routes to gene identification and characterization. In the 17 years since the bz1 gene was first tagged with Activator (Ac), more than 60 genes involved in plant development have been cloned using elements such as Supressor-mutator (Spm) and Mutator (Mu) from maize and Tag1 from Arabidopsis. The advantages of transposon-induced alleles in the study of developmental processes go beyond cloning to include sector analysis, generation of new alleles, and conditional expression based on suppression. The laborious technique of directed tagging that led to many of these successes is now being supplanted by systematic projects to produce large collections of transposon insertions that are precharacterized using PCR-based methods and publicly accessible for both forward and reverse genetics. Of the tens of thousands of new genes postulated to exist in Arabidopsis and other species, most are turning out to have no obvious phenotypic effect. The challenge for functional genomics is now to elucidate the apparently subtle actions of genes at a rate commensurate with their discovery. Referee: Dr. Paul Chomet, Monsanto Co., 62 Maritime Dr., Mystic, CT 06355