VIPP1, a nuclear gene of Arabidopsis thaliana essential for thylakoid membrane formation

The conversion of light to chemical energy by the process of photosynthesis is localized to the thylakoid membrane network in plant chloroplasts. Although several pathways have been described that target proteins into and across the thylakoids, little is known about the origin of this membrane system or how the lipid backbone of the thylakoids is transported and fused with the target membrane. Thylakoid biogenesis and maintenance seem to involve the flow of membrane elements via vesicular transport. Here we show by mutational analysis that deletion of a single gene called VIPP1 (vesicle-inducing protein in plastids 1) is deleterious to thylakoid membrane formation. Although VIPP1 is a hydrophilic protein it is found in both the inner envelope and the thylakoid membranes. In VIPP1 deletion mutants vesicle formation is abolished. We propose that VIPP1 is essential for the maintenance of thylakoids by a transport pathway not previously recognized.     

Transposable elements and small RNAs contribute to gene expression divergence between Arabidopsis thaliana and Arabidopsis lyrata

Transposable elements (TEs) are often the primary determinant of genome size differences among eukaryotes. In plants, the proliferation of TEs is countered through epigenetic silencing mechanisms that prevent mobility. Recent studies using the model plant Arabidopsis thaliana have revealed that methylated TE insertions are often associated with reduced expression of nearby genes, and these insertions may be subject to purifying selection due to this effect. Less is known about the genome-wide patterns of epigenetic silencing of TEs in other plant species. Here, we compare the 24-nt siRNA complement from A. thaliana and a closely related congener with a two- to threefold higher TE copy number, Arabidopsis lyrata. We show that TEs--particularly siRNA-targeted TEs--are associated with reduced gene expression within both species and also with gene expression differences between orthologs. In addition, A. lyrata TEs are targeted by a lower fraction of uniquely matching siRNAs, which are associated with more effective silencing of TE expression. Our results suggest that the efficacy of RNA-directed DNA methylation silencing is lower in A. lyrata, a finding that may shed light on the causes of differential TE proliferation among species.     

Efficient gene tagging in Arabidopsis thaliana using a gene trap approach

Large quantities of DNA sequence information about plant genes are rapidly accumulating in public databases, but to progress from DNA sequence to biological function a mutant allele for each of the genes ideally should be available. Here we describe a gene trap construct that allowed us to disrupt transcribed genes with a high efficiency in Arabidopsis thaliana. In the T-DNA vector used, the expression of a bacterial reporter gene coding for neomycin phosphotransferase II (nptII) depends on the in vivo generation of a translation fusion upon the T-DNA integration into the Arabidopsis genome. Analysis of 20 selected transgenic lines showed that 12 lines are T-DNA insertion mutants. The disrupted genes analyzed encoded ribosomal proteins (three lines), aspartate tRNA synthase, DNA ligase, basic-domain leucine zipper DNA binding protein, ATP-binding cassette transporter, and five proteins of unknown function. Four tagged genes were new for Arabidopsis. The results presented here suggest that gene trapping, using nptII as a reporter gene, can be as high as 80% and opens novel perspectives for systematic gene tagging in A. thaliana.     

Diversity and molecular evolution of the RPS2 resistance gene in Arabidopsis thaliana

The RPS2 gene in Arabidopsis thaliana governs resistance to strains of the bacterial pathogen, Pseudomonas syringae pv. tomato, that express the avrRpt2 gene. The two loci are involved in a gene-for-gene interaction. Seventeen accessions of A. thaliana were sequenced to explore the diversity present in the coding region of the RPS2 locus. An unusually high level of nucleotide polymorphisms was found (1.26%), with nearly half of the observed polymorphisms resulting in amino acid changes in the RPS2 protein. Seven haplotypes (alleles) were identified and their evolutionary relationships deduced. Several of the alleles conferring resistance were found to be closely related, whereas susceptibility to disease was conferred by widely divergent alleles. The possibility of selection at the RPS2 locus is discussed.     

Light-independent developmental regulation of cab gene expression in Arabidopsis thaliana seedlings.

We found a transient increase in the amount of mRNA for four nuclear genes encoding chloroplast proteins during early development of Arabidopsis thaliana. This increase began soon after germination as cotyledons emerged from the seed coat; it occurred in total darkness and was not affected by external factors, such as gibberellins or light treatments used to stimulate germination. Three members of the cab gene family and the rbcS-1A gene exhibited this expression pattern. Because timing of the increase coincided with cotyledon emergence and because it occurred independently of external stimuli, we suggest that this increase represents developmental regulation of these genes. Further, 1.34 kilobases of the cab1 promoter was sufficient to confer this expression pattern on a reporter gene in transgenic Arabidopsis seedlings. The ability of the cab genes to respond to phytochrome preceded this developmental increase, showing that these two types of regulation are independent.     

Background-dependent effects of polyglutamine variation in the Arabidopsis thaliana gene ELF3

Tandem repeats (TRs) have extremely high mutation rates and are often considered to be neutrally evolving DNA. However, in coding regions, TR copy number mutations can significantly affect phenotype and may facilitate rapid adaptation to new environments. In several human genes, TR copy number mutations that expand polyglutamine (polyQ) tracts beyond a certain threshold cause incurable neurodegenerative diseases. PolyQ-containing proteins exist at a considerable frequency in eukaryotes, yet the phenotypic consequences of natural variation in polyQ tracts that are not associated with disease remain largely unknown. Here, we use Arabidopsis thaliana to dissect the phenotypic consequences of natural variation in the polyQ tract encoded by EARLY FLOWERING 3 (ELF3), a key developmental gene. Changing ELF3 polyQ tract length affected complex ELF3-dependent phenotypes in a striking and nonlinear manner. Some natural ELF3 polyQ variants phenocopied elf3 loss-of-function mutants in a common reference background, although they are functional in their native genetic backgrounds. To test the existence of background-specific modifiers, we compared the phenotypic effects of ELF3 polyQ variants between two divergent backgrounds, Col and Ws, and found dramatic differences. In fact, the Col-ELF3 allele, encoding the shortest known ELF3 polyQ tract, was haploinsufficient in Ws × Col F₁ hybrids. Our data support a model in which variable polyQ tracts drive adaptation to internal genetic environments.     

Differential regulation of an auxin-producing nitrilase gene family in Arabidopsis thaliana.

Nitrilases (nitrile aminohydrolase, EC 3.5.5.1) convert nitriles to carboxylic acids. We report the cloning, characterization, and expression patterns of four Arabidopsis thaliana nitrilase genes (NIT1-4), one of which was previously described [Bartling, D., Seedorf, M., Mithöfer, A. & Weiler, E. W. (1992) Eur. J. Biochem. 205, 417-424]. The nitrilase genes encode very similar proteins that hydrolyze indole-3-acetonitrile to the phytohormone indole-3-acetic acid in vitro, and three of the four genes are tandemly arranged on chromosome III. Northern analysis using gene-specific probes and analysis of transgenic plants containing promoter-reporter gene fusions indicate that the four genes are differentially regulated. NIT2 expression is specifically induced around lesions caused by bacterial pathogen infiltration. The sites of nitrilase expression may represent sites of auxin biosynthesis in A. thaliana.     

Molecular cloning and DNA sequence of the Arabidopsis thaliana alcohol dehydrogenase gene.

Arabidopsis thaliana provides an excellent experimental plant system for molecular genetics because of its remarkably small genome size, near absence of dispersed middle repetitive DNA, and short life cycle. We have cloned and determined the nucleotide sequence of a single-copy gene from A. thaliana likely to be the gene encoding alcohol dehydrogenase (ADH; alcohol:NAD+ oxidoreductase, EC 1.1.1.1). The gene was isolated from a random recombinant library by cross-hybridization with a maize Adh1 gene probe. The DNA sequence contains an open reading frame capable of encoding a polypeptide the same length as maize ADH1 and ADH2 (379 amino acids) and having approximately equal to 80% homology with both maize enzymes. This open reading frame is interrupted by six introns whose positions are conserved with six of the nine intron positions present in both maize genes. The 5' and 3' untranslated regions are, respectively, 58 and 204 base pairs long. Sequences important for eukaryotic gene expression such as the TATA box, polyadenylylation signal, and intron splicesite sequences are found in the expected locations. The gene hybridizes to a specific anaerobically induced RNA in Arabidopsis whose appearance correlates with the anaerobic induction of Arabidopsis ADH protein.     

Global and local genome mapping in Arabidopsis thaliana by using recombinant inbred lines and random amplified polymorphic DNAs.

A population of Arabidopsis thaliana recombinant inbred lines was constructed and used to develop a high-density genetic linkage map containing 252 random amplified polymorphic DNA markers and 60 previously mapped restriction fragment length polymorphisms. Linkage groups were correlated to the classical genetic map by inclusion of nine phenotypic markers in the mapping cross. We also applied a technique for local mapping that allows targeting of markers to a selected genome region by pooling DNA from recombinant inbred lines based on their genotype. We conclude that random amplified polymorphic DNAs, used in conjunction with a recombinant inbred population, can facilitate the genetic and physical characterization of the Arabidopsis genome and that this method is generally applicable to other organisms for which appropriate populations either are available or can be developed.     

Male-biased transmission of deleterious mutations to the progeny in Arabidopsis thaliana

The extent and cause of male-biased mutation rates, the higher number of mutations in sperm than in eggs, is currently an active and controversial subject. Recent evidence indicates that this male (sperm) bias not only occurs in animals but also in plants. The higher mutation rate in plant sperm was inferred from rates of evolution of neutral DNA regions, and the results were confined to the mitochondria and chloroplasts of gymnosperms. However, the relative transmission rates of deleterious mutations, which have substantial evolutionary consequences, have rarely been studied. Here, an investigation is described by using the hermaphroditic self-compatible flowering plant Arabidopsis thaliana, in which we artificially increased the rate of mutation in pollen (i.e., sperm donor) and maternal (i.e., egg donor) parents, by using two kinds of UV irradiation in parallel and separate experiments, and assessed the deleterious effects on fitness of the F(2) generation. The results show that more deleterious induced mutations are transmitted to the progeny by a sperm than by an egg. These findings provide the first experimental evidence that more deleterious mutations are inherited from sperm than from an egg in any organism. Possible causes underlying this male bias are discussed.     

Genome organization in dicots: genome duplication in Arabidopsis and synteny between soybean and Arabidopsis

Synteny between soybean and Arabidopsis was studied by using conceptual translations of DNA sequences from loci that map to soybean linkage groups A2, J, and L. Synteny was found between these linkage groups and all four of the Arabidopsis chromosomes, where GenBank contained enough sequence for synteny to be identified confidently. Soybean linkage group A2 (soyA2) and Arabidopsis chromosome I showed significant synteny over almost their entire lengths, with only 2-3 chromosomal rearrangements required to bring the maps into substantial agreement. Smaller blocks of synteny were identified between soyA2 and Arabidopsis chromosomes IV and V (near the RPP5 and RPP8 genes) and between soyA2 and Arabidopsis chromosomes I and V (near the PhyA and PhyC genes). These subchromosomal syntenic regions were themselves homeologous, suggesting that Arabidopsis has undergone a number of segmental duplications or possibly a complete genome duplication during its evolution. Homologies between the homeologous soybean linkage groups J and L and Arabidopsis chromosomes II and IV also revealed evidence of segmental duplication in Arabidopsis. Further support for this hypothesis was provided by the observation of very close linkage in Arabidopsis of homologs of soybean Vsp27 and Bng181 (three locations) and purple acid phosphatase-like sequences and homologs of soybean A256 (five locations). Simulations show that the synteny and duplications we report are unlikely to have arisen by chance during our analysis of the homology reports.