Raimundo Villarroel

Analysis of 1.9 Mb of contiguous sequence from chromosome 4 of Arabidopsis thaliana

The plant Arabidopsis thaliana (Arabidopsis) has become an important model species for the study of many aspects of plant biology. The relatively small size of the nuclear genome and the availability of extensive physical maps of the five chromosomes provide a feasible basis for initiating sequencing of the five chromosomes. The YAC (yeast artificial chromosome)-based physical map of chromosome 4 was used to construct a sequence-ready map of cosmid and BAC (bacterial artificial chromosome) clones covering a 1.9-megabase (Mb) contiguous region, and the sequence of this region is reported here. Analysis of the sequence revealed an average gene density of one gene every 4.8 kilobases (kb), and 54% of the predicted genes had significant similarity to known genes. Other interesting features were found, such as the sequence of a disease-resistance gene locus, the distribution of retroelements, the frequent occurrence of clustered gene families, and the sequence of several classes of genes not previously encountered in plants.

The functional organization of the nopaline A. tumefaciens plasmid pTiC58

Abstract We have employed the P type plasmid RP4 and the transposons Tn1 and Tn7 to isolate insertion and deletion mutations in the nopaline Ti-plasmid pTiC58. Mutations that inactivate all known Ti phenotypes have been located on the physical map. Most importantly, we have positioned several regions involved in the determination of oncogenicity. They correspond to regions of homology between octopine and nopaline plasmids. One of these regions is part of the T-DNA, the Ti-plasmid DNA present in transformed plant cells. There are also segments of the T-DNA that are not essential for oncogenicity. One of these determines the biosynthesis of nopaline in tumors. The latter regions might allow insertion of foreign DNA that can then be introduced into plant cells.

Characterization of a rice gene showing organ-specific expression in response to salt stress and drought.

Protein changes induced by salinity stress were investigated in the roots of the salt-sensitive rice cultivar Taichung native 1. We found eight proteins to be induced and obtained partial sequences of one with a molecular mass of 15 kilodaltons and an isoelectric point of 5.5. Using an oligonucleotide probe based on this information, a cDNA clone, salT, was selected and found to contain an open reading frame coding for a protein of 145 amino acid residues. salT mRNA accumulates very rapidly in sheaths and roots from mature plants and seedlings upon treatment with Murashige and Skoog salts (1%), air drying, abscisic acid (20 microM), polyethylene glycol (5%), sodium chloride (1%), and potassium chloride (1%). Generally, no induction was seen in the leaf lamina even when the stress should affect all parts of the plant uniformly. The organ-specific response of salT is correlatable with the pattern of Na+ accumulation during salt stress.

An S18 ribosomal protein gene copy at the Arabidopsis PFL locus affects plant development by its specific expression in meristems.

In Arabidopsis, mutation at PFL causes pointed first leaves, reduced fresh weight and growth retardation. We have cloned the wild‐type PFL gene by T‐DNA tagging, and demonstrate that it complements the mutant phenotype. PFL codes for ribosomal protein S18, based on the high homology with rat S18 and on purification of S18‐equivalent peptides from plant ribosomes. pfl represents the first mutation in eukaryotic S18 proteins or their S13 prokaryotic counterparts, involved in translation initiation. Arabidopsis contains three S18 gene copies dispersed in the genetic map; they are all transcribed and code for completely identical proteins. No transcript is detected from the mutated gene, S18A. The activity of the S18A promoter is restricted to meristems, with a markedly high expression at the embryonic heart stage, and to wounding sites. This means that plants activate an extra copy of this ribosomal protein gene in tissues with cell division activity. We postulate that in meristematic tissues plants use transcriptional control to synthesize extra ribosomes to increase translational efficiency. In analogy with this, an additional, developmentally regulated gene copy might be expected for all ribosomal proteins.

Comprehensive analysis of gene expression in Nicotiana tabacum leaves acclimated to oxidative stress

The molecular mechanisms by which plants acclimate to oxidative stress are poorly understood. To identify the processes involved in acclimation, we performed a comprehensive analysis of gene expression in Nicotiana tabacum leaves acclimated to oxidative stress. Combining mRNA differential display and cDNA array analysis, we estimated that at least 95 genes alter their expression in tobacco leaves acclimated to oxidative stress, of which 83% are induced and 17% repressed. Sequence analysis of 53 sequence tags revealed that, in addition to antioxidant genes, genes implicated in abiotic and biotic stress defenses, cellular protection and detoxification, energy and carbohydrate metabolism, de novo protein synthesis, and signal transduction showed altered expression. Expression of most of the genes was enhanced, except for genes associated with photosynthesis and light-regulated processes that were repressed. During acclimation, two distinct groups of coregulated genes (“early-” and “late-response” gene regulons) were observed, indicating the presence of at least two different gene induction pathways. These two gene regulons also showed differential expression patterns on an oxidative stress challenge. Expression of “late-response” genes was augmented in the acclimated leaf tissues, whereas expression of “early-response” genes was not. Together, our data suggest that acclimation to oxidative stress is a highly complex process associated with broad gene expression adjustments. Moreover, our data indicate that in addition to defense gene induction, sensitization of plants for potentiated gene expression might be an important factor in oxidative stress acclimation.

Osmoregulation of a Pyrroline-5-Carboxylate Reductase Gene in Arabidopsis thaliana

In Arabidopsis thaliana (L.) Heynh. proline can account for up to 20% of the free amino acid pool after salt stress. Proline accumulation occurs in plants mainly by de novo synthesis from glutamate. The last step of the proline biosynthetic pathway is catalyzed by pyrroline-5-carboxylate (P5C) reductase. A gene (AT-P5C1) encoding this enzyme in A. thaliana has been cloned and sequenced. Expression of AT-P5C1 in Escherichia coli resulted in the complementation of a proC mutant to prototrophy. A comparison of the AT-P5C1 primary and secondary structures with those of six P5C reductases of other organisms is presented. With the exception of several functionally important amino acid residues, little conservation in the primary structure is seen; much greater similarity exists in the putative secondary structure. The AT-P5C1 protein is probably cytosolic. Under normal growth conditions, the P5C reductase mRNA level was significantly higher in roots and ripening seeds than in green tissue. A salt treatment of A. thaliana plants resulted in a 5-fold induction of the AT-P5C1 transcript, suggesting osmoregulation of the AT-P5C1 promoter region. Moreover, a time-course experiment indicated that this induction precedes proline accumulation.

Physical mapping of DNA base sequence homologies between an octopine and a nopaline Ti plasmid of Agrobacterium tumefaciens.

A detailed physical map of the homologous and non-homologous regions between an octopine (pTiAch5) and a nopaline (pTiC58) Ti plasmid was determined by Southern type hybridization and by electron microscope heteroduplex analysis. This map was correlated with the functional maps of both plasmids. For the Southern type hybridizations, total labelled pTiAch5 DNA was hybridized to Southern blots of restriction fragments from a series of hybrid plasmids containing overlapping segments of the whole TiC58 plasmid. Reciprocal experiments were also carried out. The common sequences between the two plasmids (±30%) are restricted to four major stretches of homology. Analysis of heteroduplexes between pTiAch5 and several hybrid plasmids containing specific regions of pTiC58, and of heteroduplexes between hybrid plasmids derived from pTiC58 and pTiAch5 provided a detailed map of the fine structure of the four major homology regions. Two regions are distributed in the same relative order as compared to a common reference point, and two are inversed. Three regions contain a number of small, mostly asymmetrical substitution loops. Several regions distributed over the common DNA sequences were found to be partially homologous.

Genome-scale Arabidopsis promoter array identifies targets of the histone acetyltransferase GCN5

We have assembled approximately 20 000 Arabidopsis thaliana promoter regions, compatible with functional studies that require cloning and with microarray applications. The promoter fragments can be captured as modular entry clones (MultiSite Gateway format) via site-specific recombinational cloning, and transferred into vectors of choice to investigate transcriptional networks. The fragments can also be amplified by PCR and printed on glass arrays. In combination with immunoprecipitation of protein-DNA complexes (ChIP-chip), these arrays enable characterization of binding sites for chromatin-associated proteins or the extent of chromatin modifications at genome scale. The Arabidopsis histone acetyltransferase GCN5 associated with 40% of the tested promoters. At most sites, binding did not depend on the integrity of the GCN5 bromodomain. However, the presence of the bromodomain was necessary for binding to 11% of the promoter regions, and correlated with acetylation of lysine 14 of histone H3 in these promoters. Combined analysis of ChIP-chip and transcriptomic data indicated that binding of GCN5 does not strictly correlate with gene activation. GCN5 has previously been shown to be required for light-regulated gene expression and growth, and we found that GCN5 targets were enriched in early light-responsive genes. Thus, in addition to its transcriptional activation function, GCN5 may play an important role in priming activation of inducible genes under non-induced conditions.

Molecular identification of catalases from Nicotiana plumbaginifolia (L.)

We have isolated three different catalase cDNAs from Nicotiana plumbaginifolia (cat1, cat2, and cat3) and a partial sequence of a fourth catalase gene (cat4) that shows no discernible expression based on Northern analysis. The catalase sequences were used to determine the similarity with other plant catalases and to study the transcriptional response to paraquat, 3‐aminotriazole, and salicylic acid. 3‐Aminotriazole induces mRNA levels of cat1, cat2 and cat3, indicating that a reduction in catalase activity positively affects catalase mRNA abundance. Salicylic acid that binds catalase in vitro, had no effect on catalase transcript levels at physiological concentrations. Paraquat resulted in the induction of cat1.

An analysis of the boundaries of the octopine TL-DNA in tumors induced by Agrobacterium tumefaciens

SummaryThe octopine Ti plasmid of Agrobacterium tumefaciens strain Ach5 contains a 13.5 kb TL-region and a 6 kb TR-region which can independently be transferred to plant nuclear DNA. A direct repeat of 25 bp flanks the TL-region, and is related to the direct repeat flanking the nopaline T-region (Zambryski et al. 1982; Yadav et al. 1982). Two right TL-DNA borders, recloned from transformed plant cells, are located in or very near to the right copy of the direct repeat. One left TL-DNA endpoint lies within the left repeat copy, another one is located 57 bp inside of this sequence. These observations are analogous to and generalize the ones made with the nopaline system. A further analogy is the observation that one tumor clone contained a tandem junction of two TL-DNA copies. The junction sequence of 389 bp contains, to the left, a 15 bp sequence representing a direct repeat of a sequence in the right end of TL. The rest of the junction consists mainly of a unit of 40 bp of plant origin directly repeated 6 times. This structure indicates that the generation of the tandem repeat of two TL-DNA copies in this particular tumor line took place during or after the insertion of an original copy in the plant genome.