Gerda Cnops

The Arabidopsis thaliana Homolog of Yeast BRE1 Has a Function in Cell Cycle Regulation during Early Leaf and Root Growth

Chromatin modification and transcriptional activation are novel roles for E3 ubiquitin ligase proteins that have been mainly associated with ubiquitin-dependent proteolysis. We identified HISTONE MONOUBIQUITINATION1 (HUB1) (and its homolog HUB2) in Arabidopsis thaliana as RING E3 ligase proteins with a function in organ growth. We show that HUB1 is a functional homolog of the human and yeast BRE1 proteins because it monoubiquitinated histone H2B in an in vitro assay. Hub knockdown mutants had pale leaf coloration, modified leaf shape, reduced rosette biomass, and inhibited primary root growth. One of the alleles had been designated previously as ang4-1. Kinematic analysis of leaf and root growth together with flow cytometry revealed defects in cell cycle activities. The hub1-1 (ang4-1) mutation increased cell cycle duration in young leaves and caused an early entry into the endocycles. Transcript profiling of shoot apical tissues of hub1-1 (ang4-1) indicated that key regulators of the G2-to-M transition were misexpressed. Based on the mutant characterization, we postulate that HUB1 mediates gene activation and cell cycle regulation probably through chromatin modifications.

The TORNADO1 and TORNADO2 Genes Function in Several Patterning Processes during Early Leaf Development in Arabidopsis thaliana

In multicellular organisms, patterning is a process that generates axes in the primary body plan, creates domains upon organ formation, and finally leads to differentiation into tissues and cell types. We identified the Arabidopsis thaliana TORNADO1 (TRN1) and TRN2 genes and their role in leaf patterning processes such as lamina venation, symmetry, and lateral growth. In trn mutants, the leaf venation network had a severely reduced complexity: incomplete loops, no tertiary or quaternary veins, and vascular islands. The leaf laminas were asymmetric and narrow because of a severely reduced cell number. We postulate that the imbalance between cell proliferation and cell differentiation and the altered auxin distribution in both trn mutants cause asymmetric leaf growth and aberrant venation patterning. TRN1 and TRN2 were epistatic to ASYMMETRIC LEAVES1 with respect to leaf asymmetry, consistent with their expression in the shoot apical meristem and leaf primordia. TRN1 codes for a large plant-specific protein with conserved domains also found in a variety of signaling proteins, whereas TRN2 encodes a transmembrane protein of the tetraspanin family whose phylogenetic tree is presented. Double mutant analysis showed that TRN1 and TRN2 act in the same pathway.

The RON1/FRY1/SAL1 gene is required for leaf morphogenesis and venation patterning in Arabidopsis

To identify genes involved in vascular patterning in Arabidopsis (Arabidopsis thaliana), we screened for abnormal venation patterns in a large collection of leaf shape mutants isolated in our laboratory. The rotunda1-1 (ron1-1) mutant, initially isolated because of its rounded leaves, exhibited an open venation pattern, which resulted from an increased number of free-ending veins. We positionally cloned the RON1 gene and found it to be identical to FRY1/SAL1, which encodes an enzyme with inositol polyphosphate 1-phosphatase and 3′ (2′),5′-bisphosphate nucleotidase activities and has not, to our knowledge, previously been related to venation patterning. The ron1-1 mutant and mutants affected in auxin homeostasis share perturbations in venation patterning, lateral root formation, root hair length, shoot branching, and apical dominance. These similarities prompted us to monitor the auxin response using a DR5-GUS auxin-responsive reporter transgene, the expression levels of which were increased in roots and reduced in leaves in the ron1-1 background. To gain insight into the function of RON1/FRY1/SAL1 during vascular development, we generated double mutants for genes involved in vein patterning and found that ron1 synergistically interacts with auxin resistant1 and hemivenata-1 but not with cotyledon vascular pattern1 (cvp1) and cvp2. These results suggest a role for inositol metabolism in the regulation of auxin responses. Microarray analysis of gene expression revealed that several hundred genes are misexpressed in ron1-1, which may explain the pleiotropic phenotype of this mutant. Metabolomic profiling of the ron1-1 mutant revealed changes in the levels of 38 metabolites, including myoinositol and indole-3-acetonitrile, a precursor of auxin.

Chromosome landing at the Arabidopsis TORNADO1 locus using an AFLP-based strategy

Abstract The Arabidopsis tornado1 (trn1) mutation causes severe dwarfism combined with twisted growth of all organs. We present a chromosome landing strategy, using amplified restriction fragment length polymorphism (AFLP) marker technology, for the isolation of the TRN1 gene. The recessive trn1 mutation was identified in a C24 transgenic line and is located 5 cM from a T-DNA insertion. We mapped the TRN1 locus to the bottom half of chromosome 5 relative to visible and restriction fragment length polymorphism (RFLP) markers. Recombinant classes within a 3-cM region around TRN1 were used to build a high-resolution map in this region, using the AFLP technique. Approximately 300 primer combinations have been used to test about 26 000 fragments for polymorphisms. Seventeen of these AFLP markers were identified in the 3-cM region around TRN1. These markers were mapped within this region using individual recombinants. Four of these AFLP markers co-segregate with TRN1 whereas one maps at one recombinant below TRN1. We isolated and cloned three of these AFLP markers. These markers identified two yeast artificial chromosome (YAC) clones, containing the RFLP marker above and the AFLP marker below TRN1, demonstrating that these YACs span the TRN1 locus and that chromosome landing has been achieved, using an AFLP-based strategy.

Mutations in the TORNADO2 gene affect cellular decisions in the peripheral zone of the shoot apical meristem of Arabidopsis thaliana

SummaryAn EMS (ethyl methanesulfonate) mutagenesis effector screen performed with the STM:GUS marker line in Arabidopsis thaliana identified a loss-of-function allele of the TORNADO2 gene. The histological and genetic analyses described here implicate TRN2 in SAM function, where the peripheral zone in trn2 mutants is enlarged relative to the central stem cell zone. The trn2 mutant allele partially rescues the phenotype of shoot meristemless mutants but behaves additively to wuschel and clavata3 alleles during the vegetative phase and in the outer floral whorls. The development of carpels in trn2wus-1 double mutant flowers indicates that pluripotent cells persist in floral meristems in the absence of TRN2 function and can be recruited for carpel anlagen. The data implicate a membrane-bound plant tetraspanin protein in cellular decisions in the peripheral zone of the SAM.

Processes underlying branching differences in fodder crops

Plant architectural characteristics are under strong genetic regulation. Economically important traits for forage crops such as biomass yield, ground cover and persistence can be improved by selecting for particular aerial architectural characteristics. Here, we present an easily applicable method for the spatiotemporal description of branching patterns in red clover (Trifolium pratense) and perennial ryegrass (Lolium perenne), two of the most important forage crops in Europe. A detailed analysis of genotypes with contrasting branching phenotypes demonstrates that in these species different factors are the main determinants of shoot branching characteristics. In red clover, bud outgrowth and to a lesser extent bud formation explain inter-genotype branching differences. In perennial ryegrass, differences in the capacity to form new buds determined largely the differences between forage and turf types. However, when a set of four forage types was compared in a separate experiment, variation in quantity and pace of bud formation and bud outgrowth explained the differences in aerial architecture. In both crops, branching patterns are likely determined by several processes, and highly branched phenotypes can result from the formation of more buds, an increased probability of bud outgrowth, or a combination of these processes. Furthermore, the presence of more buds is partly caused by more bud outgrowth.

TRANSGENIC ARABIDOPSIS TESTER LINES WITH DOMINANT MARKER GENES

The map positions of a set of eight T-DNA insertions in theArabidopsis genome have been determined by using closely linked visible markers. The insertions are dispersed over four of the five chromosomes. Each T-DNA insert contains one or more of the chimeric marker genes neomycin phosphotransferase (neo), hygromycin phosphotransferase (hpt), phosphinothricin acetyltransferase (bar),β-glucuronidase (gusA) and indole-3-acetamide hydrolase (iaaH). Theneo, hpt andbar marker genes are dominant in a selective germination assay or when used as DNA markers in a polymerase chain reaction. These dominant markers will allow recombinants to be discerned in a germinating F2 population, one generation earlier than with a conventional recessive marker. The transgenic marker lines will speed up and simplify the isolation of recombinants in small genetic intervals, a rate-limiting step in positional cloning strategies. The transgenic lines containing thehpt marker will also be of interest for the isolation of deletion mutants at the T-DNA integration sites.

Phenotypic Assessment of Variability in Tillering and Early Development in Ryegrass (Lolium spp.)

Tillering is enormously variable in the genus Lolium. Exploitation of tillering characteristics in breeding programs requires a systematic characterization of this trait at the between-species, within-species, and within-cultivar levels. We have analyzed tillering in forage and turf cultivars of L. perenne, forage cultivars of L. multiflorum and annual L. temulentum genotypes. The collection was also enriched with wild L. perenne populations.

Morphological and Molecular Diversity of Branching in Red Clover (Trifolium pratense)

Mixed grass-clover grasslands are an essential element of sustainable farming systems. The presence of clover in the mixture contributes significantly to the reduction of nitrogen fertilizer application needs, and results in improved nutritional value. In red clover, architecture is under genetic and environmental control. Similarly to what has been found in other plant species, we anticipate that architectural changes in red clover will strongly influence traits such as forage yield, re-growth capacity, seed yield and persistence. The genetic aspect of branching has been widely studied in model plants but did not obtain much attention in the past in red clover. Our aim is to translate knowledge from model plants on genes involved in meristem initiation, bud formation, and the activity and determination of the apical meristems to red clover.

Morphological and Molecular Characterization of Branching in Red Clover (Trifolium pratense)

Clover is an essential element of sustainable grasslands. Clover reduces the need for nitrogen fertilizer and results in improved nutritional value of grasslands. Plant architecture, which is under genetic and environmental control, may have a strong influence on traits such as forage yield, re-growth capacity, seed yield and persistence in red clover. The genetic aspect of branching has been widely studied in model plants but has received little attention in red clover. Our present aim is to translate the knowledge regarding genes involved in bud outgrowth from model plants to red clover. Branching was studied in two environments during one growing season in clonal replicates of two genotypes with contrasting architecture, a highly branched and prostrate genotype (Crossway_2), and a poorly branched and erect genotype (Diplomat_8). The number of nodes and the quantity as well as the position of bud outgrowth into branches differed greatly between genotypes and were similar across both environments. The influence of auxin and strigolactone on bud outgrowth was investigated by applying these hormones to isolated single node segments. Furthermore, genes from the strigolactone pathway were isolated from red clover and their expression was studied in various tissues of the two genotypes.