Expression of cell wall related genes in basal and ear internodes of silking <Emphasis Type="Italic">brown-midrib-3</Emphasis>, caffeic acid <Emphasis Type="Italic">O</Emphasis>-methyltransferase (COMT) down-regulated,and normal maize plants

Background  

Silage maize is a major forage and energy resource for cattle feeding, and several studies have shown that lignin content and structure are the determining factors in forage maize feeding value. In maize, four natural brown-midrib mutants have modified lignin content, lignin structure and cell wall digestibility. The greatest lignin reduction and the highest cell wall digestibility were observed in the brown-midrib-3 (bm3) mutant, which is disrupted in the caffeic acid O-methyltransferase (COMT) gene.     

<Emphasis Type="Italic">ASYMMETRIC LEAVES1</Emphasis>, an <Emphasis Type="Italic">Arabidopsis</Emphasis> gene that is involved in the control of cell differentiation in leaves

During leaf development, the formation of dorsal-ventral and proximal-distal axes is central to leaf morphogenesis. To investigate the genetic basis of dorsoventrality and proximodistality in the leaf, we screened for mutants of Arabidopsis thaliana (L.) Heynh. with defects in leaf morphogenesis. We describe here the phenotypic analysis of three mutant alleles that we have isolated. These mutants show varying degrees of abnormality including dwarfism, broad leaf lamina, and aberrant floral organs and fruits. Genetic analysis revealed that these mutations are alleles of the previously isolated mutant asymmetric leaves1 ( as1). In addition to the leaf phenotypes described previously, these alleles display other phenotypes that were not observed. These include: (i) some rosette leaves with petiole growth underneath the leaf lamina; (ii) leaf vein branching in the petiole; and (iii) a leaf lamina with an epidermis similar to that on the petiole. The mutant phenotypes suggest that the ASYMMETRIC LEAVES1 ( AS1) gene is involved in the control of cell differentiation in leaves. As the first step in determining a molecular function for AS1, we have identified the AS1 gene using map-based cloning. The AS1 gene encodes a MYB-domain protein that is homologous to the Antirrhinum PHANTASTICA ( PHAN) and maize ROUGH SHEATH2 ( RS2) genes. AS1 is expressed nearly ubiquitously, consistent with the pleiotropic mutant phenotypes. High levels of AS1 expression were found in tissues with highly proliferative cells, which further suggests a role in cell division and early cell differentiation.     

The mouse mutants <Emphasis Type="Italic">recoil wobbler</Emphasis> and <Emphasis Type="Italic">nmf373</Emphasis> represent a series of <Emphasis Type="Italic">Grm1</Emphasis> mutations

The identification of novel mutant alleles is important for understanding critical functional domains of a protein and establishing genotype:phenotype correlations. The recoil wobbler (rcw) allelic series of spontaneous ataxic mutants and the ENU-induced mutant nmf373 genetically mapped to a shared region of chromosome 10. Their mutant phenotypes are strikingly similar; all have an ataxic phenotype that is recessive, early-onset, and is not associated with neurodegeneration. In this study we used complementation tests to show that these series of mutants are allelic to a knockout mutant of Grm1. Subsequently, a duplication of exon 4 and three missense mutations were identified in Grm1: I160T, E292D, and G337E. All mutations occurred within the ligand-binding region and changed conserved amino acids. In the rcw mutant, the Grm1 gene is expressed and the protein product is properly localized to the molecular layer of the cerebellar cortex. Grm1 is responsible for the generation of inositol 1,4,5-trisphosphate (IP₃). The inositol second messenger system is the central mechanism for calcium release from intracellular stores in cerebellar Purkinje cells. Several of the genes involved in this pathway are mutated in mouse ataxic disorders. The novel rcw mutants represent a resource that will have utility for further studies of inositol second-messenger-system defects in neurogenetic disorders.     

RNAi‐suppression of barley caffeic acid O‐methyltransferase modifies lignin despite redundancy in the gene family

Caffeic acid O‐methyltransferase (COMT), the lignin biosynthesis gene modified in many brown‐midrib high‐digestibility mutants of maize and sorghum, was targeted for downregulation in the small grain temperate cereal, barley (Hordeum vulgare), to improve straw properties. Phylogenetic and expression analyses identified the barley COMT orthologue(s) expressed in stems, defining a larger gene family than in brachypodium or rice with three COMT genes expressed in lignifying tissues. RNAi significantly reduced stem COMT protein and enzyme activity, and modestly reduced stem lignin content while dramatically changing lignin structure. Lignin syringyl‐to‐guaiacyl ratio was reduced by ~50%, the 5‐hydroxyguaiacyl (5‐OH‐G) unit incorporated into lignin at 10‐–15‐fold higher levels than normal, and the amount of p‐coumaric acid ester‐linked to cell walls was reduced by ~50%. No brown‐midrib phenotype was observed in any RNAi line despite significant COMT suppression and altered lignin. The novel COMT gene family structure in barley highlights the dynamic nature of grass genomes. Redundancy in barley COMTs may explain the absence of brown‐midrib mutants in barley and wheat. The barley COMT RNAi lines nevertheless have the potential to be exploited for bioenergy applications and as animal feed.     

Genome-wide mutagenesis of <Emphasis Type="Italic">Zea mays</Emphasis> L. using <Emphasis Type="Italic">RescueMu</Emphasis> transposons

Derived from the maize Mu1 transposon, RescueMu provides strategies for maize gene discovery and mutant phenotypic analysis. 9.92 Mb of gene-enriched sequences next to RescueMu insertion sites were co-assembled with expressed sequence tags and analyzed. Multiple plasmid recoveries identified probable germinal insertions and screening of RescueMu plasmid libraries identified plants containing probable germinal insertions. Although frequently recovered parental insertions and insertion hotspots reduce the efficiency of gene discovery per plasmid, RescueMu targets a large variety of genes and produces knockout mutants.     

Chloroplast genome aberration in micropropagation-derived albino <Emphasis Type="Italic">Bambusa edulis</Emphasis> mutants, <Emphasis Type="Italic">ab1</Emphasis> and <Emphasis Type="Italic">ab2</Emphasis>

Two albino mutants (ab1 and ab2) have been derived from long-term shoot proliferation of Bambusa edulis. Based on transmission electronic microscopy data, the chloroplasts of these mutants were abnormal. To study the mutation of gene regulation in the aberrant chloroplasts, we designed 19 pairs of chloroplast-encoded gene primers for genomic and RT-PCR. Only putative NAD(P)H-quinone oxidoreductase chain 4L (ndhE; DQ908943) and ribosomal protein S7 (rps7; DQ908931) were conserved in both the mutant and wild-type plants. The deletions in the chloroplast genome of these two mutants were different: nine genes were deleted in the chloroplast genomic aberration in ab1 and 11 genes in ab2. The chloroplast genes, NAD(P)H-quinone oxidoreductase chain 4 (ndhD; DQ908944), chloroplast 50S ribosomal protein L14 (rpl14; DQ908934), and ATP synthase beta chain (atpB; DQ908948) were abnormal in both mutants. The gene expressions of 18 of these 20 genes were correlated with their DNA copy number. The two exceptions were: ATP synthase CF0 A chain (atpI; DQ908946), whose expression in both mutants was not reduced even though the copy number was reduced; ribosomal protein S19 (rps19; DQ908949), whose expression was reduced or it was not expressed at all even though there was no difference in genomic copy number between the wild-type and mutant plants. The genomic PCR results showed that chloroplast genome aberrations do occur in multiple shoot proliferation, and this phenomenon may be involved in the generation of albino mutants.     

<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.     

Isolation and Characterization of Transposon-Insertional Mutants from <Emphasis Type="Italic">Paenibacillus </Emphasis><Emphasis Type="Italic">polymyxa</Emphasis> E681 Altering the Biosynthesis of Indole-3-Acetic Acid

We screened a mini-Tn10 insertional mutant library of the spore-forming bacterium Paenibacillus polymyxa E681 with variable indole-3-acetic acid (IAA) productivity. Four mutants, of which two showed a decrease in IAA production and the other two showed an increase in IAA production, were finally selected. Further analyses demonstrated different levels of IAA intermediates from culture supernatant of wild-type strain and mutants. In addition, mutants showed different promotions on the early growth of 10-day-old maize in terms of the increase in shoot and root weights. DNA fragments flanking the transposon insertion in four mutants were cloned and sequenced. The target sites of insertion were gene gpr1, disrupted at two sites, 49 bp downstream of the spo0F gene, and relA/spoT homologue, which codes for GPR1/FUN34/YaaH family protein, stage 0 sporulation protein F, and RelA/SpoT domain protein, respectively. This evidence suggests that there may be a number of genes involved in the regulation of IAA biosynthesis of P. polymyxa.     

Positional cloning of <Emphasis Type="Italic">ds1</Emphasis>, the target leaf spot resistance gene against <Emphasis Type="Italic">Bipolaris sorghicola</Emphasis> in sorghum

Target leaf spot is one of the major sorghum diseases in southern Japan and caused by a necrotrophic fungus, Bipolaris sorghicola. Sorghum resistance to target leaf spot is controlled by a single recessive gene (ds1). A high-density genetic map of the ds1 locus was constructed with simple sequence repeat markers using progeny from crosses between a sensitive variety, bmr-6, and a resistant one, SIL-05, which allowed the ds1 gene to be genetically located within a 26-kb region on the short arm of sorghum chromosome 5. The sorghum genome annotation database for BTx623, for which the whole genome sequence was recently published, indicated a candidate gene from the Leucine-Rich Repeat Receptor Kinase family in this region. The candidate protein kinase gene was expressed in susceptible plants but was not expressed or was severely reduced in resistant plants. The expression patterns of ds1 gene and the phenotype of target leaf spot resistance were clearly correlated. Genomic sequences of this region in parental varieties showed a deletion in the promoter region of SIL-05 that could cause reduction of gene expression. We also found two ds1 alleles for resistant phenotypes with a stop codon in the coding region. The results shown here strongly suggest that the loss of function or suppression of the ds1 protein kinase gene leads to resistance to target leaf spot in sorghum.     

The brown midrib3 (bm3) mutation in maize occurs in the gene encoding caffeic acid O-methyltransferase.

The brown midrib mutations are among the earliest described in maize. Plants containing a brown midrib mutation exhibit a reddish brown pigmentation of the leaf midrib starting when there are four to six leaves. These mutations are known to alter lignin composition and digestibility of plants and therefore constitute prime candidates in the breeding of silage maize. Here, we show that two independent brown midrib3 (bm3) mutations have resulted from structural changes in the COMT gene, which encodes the enzyme O-methyltransferase (COMT; EC 2.1.1.6), involved in lignin biosynthesis. Our results indicate that the bm3-1 allele (the reference mutant allele) has arisen from an insertional event producing a COMT mRNA altered in both size and amount. By sequencing a COMT cDNA clone obtained from bm3-1 maize, a retrotransposon with homology to the B5 element has been found to be inserted near the junction of the 3' coding region of the COMT gene intron. The second bm3 allele, bm3-2, has resulted from a deletion of part of the COMT gene. These alterations of the COMT gene were confirmed by DNA gel blot and polymerase chain reaction amplification analyses. These results clearly demonstrate that mutations at the COMT gene give a brown midrib3 phenotype. Thus, the gene genetically recognized as bm3 is the same as the one coding for COMT.     

Cloning and characterization of the histidine kinase gene<Emphasis Type="Italic"> Dic1</Emphasis> from<Emphasis Type="Italic"> Cochliobolus heterostrophus</Emphasis> that confers dicarboximide resistance and osmotic adaptation

A gene for a putative two-component histidine kinase, which is homologous to os-1 from Neurospora crassa, was cloned and sequenced from the plant-pathogenic fungus Cochliobolus heterostrophus. The predicted protein possessed the conserved histidine kinase domain, the response regulator domain, and six tandem repeats of 92-amino-acids at the N-terminal end that are found in histidine kinases from other filamentous fungi. Introduction of the histidine kinase gene complemented the deficiency of the C. heterostrophus dic1 mutant, suggesting that the Dic1 gene product is a histidine kinase. Dic1 mutants are resistant to dicarboximide and phenylpyrrole fungicides, and they are sensitive to osmotic stress. We previously classified dic1 alleles into three types, based on their phenotypes. To explain the phenotypic differences among the dic1 mutant alleles, we cloned and sequenced the mutant dic1 genes and compared their sequences with that of the wild-type strain. Null mutants for Dic1, and mutants with a deletion or point mutation in the N-terminal repeat region, were highly sensitive to osmotic stress and highly resistant to both fungicides. A single amino acid change within the kinase domain or the regulator domain altered the sensitivity to osmotic stress and conferred moderate resistance to the fungicides. These results suggest that this predicted protein, especially its repeat region, has an important function in osmotic adaptation and fungicide resistance.Communicated by C. A. M. J. J. van den Hondel     

Overproduction of Delta-Endotoxins by Sporeless <Emphasis Type="Italic">Bacillus</Emphasis><Emphasis Type="Italic">thuringiensis</Emphasis> Mutants Obtained by Nitrous Acid Mutagenesis

Asporogenic and oligosporogenic Bacillus thuringiensis mutants having the ability to overproduce insecticidal crystal protein were generated by using nitrous acid (50 mg/ml), as chemical mutagenic agent. Insecticidal crystal proteins produced by asporogenic mutants remained encapsulated within the cells. Delta-endotoxin production by most of mutants was improved compared to the corresponding wild strains BNS3 and a mutant M26. The overproduction by asporogenic and oligosporogenic mutants was attributed to defect in genes involved in sporulation and to random mutations affecting cell metabolism at different pathways and delta-endotoxin synthesis. Sporeless bioinsecticides could be developed based on stable and environmentally safe Bacillus thuringiensis mutants.     

Pleiotropic changes in Arabidopsis <Emphasis Type="Italic">f5h</Emphasis> and <Emphasis Type="Italic">sct</Emphasis> mutants revealed by large-scale gene expression and metabolite analysis

Hydrocinnamic acid esters, lignin, flavonoids, glucosinolates, and salicylic acid protect plants against UV exposure, oxidative stress, diseases, and herbivores. Through the phenylpropanoid pathway, certain Brassicaceae family members, including Arabidopsis thaliana and Brassica napus, accumulate large amounts of the anti-nutritive sinapoylcholine (sinapine) in the seed. We successfully down-regulated activities of key enzymes in the pathway including F5H and SCT and achieved reduction of sinapine and lignin in B. napus seeds. Despite this success, it was unclear how multiple agronomic traits were affected in the transgenic plants. Here, we report altered large-scale gene expression of new alleles of f5h and sct mutants of A. thaliana and resultant accumulation of sinapoylglucose, disinapoylglucose, quercetin-3-O-rhamnoside, salicylic acid glucoside, and total indolyl glucosinolates in the two mutants. Expression of several flowering genes was altered in these mutants when grown under drought and NaCl treatments. Furthermore, both mutants were more susceptible to fungal infection than the wild type. Microarray experiments identified distinctive spatial and temporal expression patterns of gene clusters involved in silique/seed developmental processes and metabolite biosynthesis in these mutants. Taken together, these findings suggest that both f5h and sct mutants exhibit major differences in accumulation of diverse metabolites in the seed and profound changes in global large-scale gene expression, resulting in differential pleiotropic responses to environmental cues.     

Interaction of gene <Emphasis Type="Italic">HSM3</Emphasis> with genes of the epistatic <Emphasis Type="Italic">RAD6</Emphasis> group in yeast <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

In eukaryotes, damage tolerance of matrix DNA is mainly determined by the repair pathway under the control of the RAD6 epistatic group of genes. This pathway is also a main source of mutations generated by mutagenic factors. The results of our recent studies show that gene HSM3 participating in the control of adaptive mutagenesis increases the frequency of mutations induced by different mutagens. Mutations rad18, rev3, and mms2 controlling various stages of the RAD6 pathway are epistatic with mutation hsm3 that decreases UV-induced mutagenesis to the level typical for single radiation-sensitive mutants. The level of mutagenesis in the double mutant srs2 hsm3 was lower than in both single mutants. Note that a decrease in the level of mutagenesis relative to the single mutant srs2 depends on the mismatch repair, since this level in the triple mutant srs2 hsm3 pms1 corresponds to that in the single mutant srs2. These data show that the mutator phenotype hsm3 is probably determined by processes occurring in a D loop. In a number of current works, the protein Hsm3 was shown to participate in the assembly of the proteasome complex S26. The assembly of proteasomes is governed by the N-terminal domain. Our results demonstrated that the Hsm3 protein contains at least two domains; the N-terminal part of the domain is responsible for the proteasome assembly, whereas the C-terminal portion of the protein is responsible for mutagenesis.     

Molecular analysis of mutants of the <Emphasis Type="Italic">Neurospora</Emphasis> adenylosuccinate synthetase locus

The ad-8 gene of Neurospora crassa, in addition to being used for the study of purine biology, has been extensively studied as a model for gene structure, mutagenesis and intralocus recombination. Because of this there is an extensive collection of well-characterized N. crassa ad-8 mutants in the Fungal Genetics Stock Center collection. Among these are spontaneous mutants and mutants induced with X-ray, UV or chemical mutagens. The specific lesions in these mutants have been genetically mapped at high resolution. We have sequenced the ad-8 locus from 13 of these mutants and identified the molecular nature of the mutation in each strain. We compare the historical fine-structure map to the DNA and amino acid sequence of each allele. The placement of the individual lesions in the fine-structure map was more accurate at the 5' end of the gene and no mutants were identified in the 3' untranslated region of this gene. We additionally analysed ad-8(+) alleles in 18 N. crassa strains subjected to whole-genome sequence analysis and describe the variability among Neurospora strains and among fungi and other organisms.