Francoise Quigley

From genome to function: the Arabidopsis aquaporins

BackgroundIn the post-genomic era newly sequenced genomes can be used to deduce organismal functions from our knowledge of other systems. Here we apply this approach to analyzing the aquaporin gene family in Arabidopsis thaliana. The aquaporins are intrinsic membrane proteins that have been characterized as facilitators of water flux. Originally termed major intrinsic proteins (MIPs), they are now also known as water channels, glycerol facilitators and aqua-glyceroporins, yet recent data suggest that they facilitate the movement of other low-molecular-weight metabolites as well.ResultsThe Arabidopsis genome contains 38 sequences with homology to aquaporin in four subfamilies, termed PIP, TIP, NIP and SIP. We have analyzed aquaporin family structure and expression using the A. thaliana genome sequence, and introduce a new NMR approach for the purpose of analyzing water movement in plant roots in vivo.ConclusionsOur preliminary data indicate a strongly transcellular component for the flux of water in roots.

Endosymbiotic origin and codon bias of the nuclear gene for chloroplast glyceraldehyde-3-phosphate dehydrogenase from maize.

SummaryThe nuclei of plant cells harbor genes for two types of glyceraldehyde-3-phosphate dehydrogenases (GAPDH) displaying a sequence divergence corresponding to the prokaryote/eukaryote separation. This strongly supports the endosymbiotic theory of chloroplast evolution and in particular the gene transfer hypothesis suggesting that the gene for the chloroplast enzyme, initially located in the genome of the endosymbiotic chloroplast progenitor, was transferred during the course of evolution into the nuclear genome of the endosymbiotic host. Codon usage in the gene for chloroplast GAPDH of maize is radically different from that employed by present-day chloroplasts and from that of the cytosolic (glycolytic) enzyme from the same cell. This reveals the presence of subcellular selective pressures which appear to be involved in the optimization of gene expression in the economically important graminaceous monocots.

Organization and sequence of five tRNA genes and of an unidentified reading frame in the wheat chloroplast genome: evidence for gene rearrangements during the evolution of chloroplast genomes

SummaryThe genes for the initiator tRNACAUMet. tRNAUCCGly, tRNAGGUThr, tRNAUUCGlu and tRNAGUATyr and an open reading frame of 62 codons have been identified by sequencing a 2,358 by BamHl and a 1,378 by BamHI-Sst2 DNA fragments from wheat chloroplasts. A comparison of the organization of these five tRNA genes and of the open reading frame on the wheat, tobacco and spinach chloroplast genomes suggests that at least three genomic inversions must have occurred during the evolution of the wheat chloroplast genome from a spinach-like ancestor genome. Furthermore, it seems that in wheat the 91 by intergenic region between the genes for the initiator tRNAMet and the gene for tRNAUCCGly is one end-point of the 20 kbp genomic inversion proposed by Palmer and Thompson in the case of maize (Palmer and Thompson 1982). A 119 bp duplication is located at this junction: the first copy comprises the 91 by of the intergenic region and the first 28 by of the tRNAMet gene, the second copy is found downstream of the tRNAMet gene.

Three thioredoxin targets in the inner envelope membrane of chloroplasts function in protein import and chlorophyll metabolism

Thioredoxins (Trxs) are ubiquitous small proteins with a redox-active disulfide bridge. In their reduced form, they constitute very efficient protein disulfide oxidoreductases. In chloroplasts, two types of Trxs (f and m) coexist and play central roles in the regulation of the Calvin cycle and other processes. Here, we identified a class of Trx targets in the inner plastid envelope membrane of chloroplasts that share a CxxC motif ≈73 aa from their carboxyl-terminal end. Members of this group belong to a superfamily of Rieske iron–sulfur proteins involved in protein translocation and chlorophyll metabolism. These proteins include the protein translocon protein TIC55, the precursor NADPH:protochlorophyllide oxidoreductase translocon protein PTC52, which operates as protochlorophyllide a-oxygenase, and the lethal leaf spot protein LLS1, which is identical with pheophorbide a oxygenase. The role of these proteins in dark/light regulation and oxidative control by the Trx system is discussed.

Cotranscription of the S10- and spc-like operons in spinach chloroplasts and identification of three of their gene products.

SummaryThe organisation and expression of the rpl22, rps3, rpl16 and rpl14 genes, which belong to the S10- and spc-like operons of spinach chloroplasts, have been studied. Northern experiments and nuclease S1 mapping show that the two operon-like groups of genes are cotranscribed. It is demonstrated that the intron-containing rpl16 gene is spliced in vivo. Based on amino acid composition and protein sequence data, the products of the rpl22, rpl16 and rpl14 genes are identified respectively as the spinach chloroplast ribosomal proteins CS-L13, CS-L24 and CS-L29. The rpl22 gene product is a 5S rRNA binding protein and therefore is distinguishable from the homologous Escherichia coli L22 ribosomal protein.

Characterization of a large inversion in the spinach chloroplast genome relative to Marchantia: a possible transposon-mediated origin

SummaryA 7,022 by BamHI-EcoRI fragment, located in the inverted repeat of spinach chloroplast, has been sequenced. It contains a 2131 codon open reading frame (ORF) homologous to both tobacco ORFs 581 and 1708, and to Marchantia ORF 2136. Relative to the Marchantia chloroplast genome, spinach ORF 2131 is located at the end of a large inversion; the other end point is close to trnL, the position of which is the same in Marchantia, tobacco and spinach. In Marchantia, two 8 by direct repeats flanking two 10 by indirect repeats are present near the end points of the inversion. These repeats may result from a transposon-mediated insertion which would have facilitated the subsequent inversion. From a comparison of the gene organization of the spinach, tobacco, and Marchantia genomes in this region, we propose a step-wise process to explain the expansion of the inverted repeat from a Marchantia-like genome to the spinach/tobacco genome.

Strong functional GC pressure in a light-regulated maize gene encoding subunit GAPA of chloroplast glyceraldehyde-3-phosphate dehydrogenase: Implications for the evolution of GAPA pseudogenes

SummaryThe light-regulated nuclear gene encoding subunit A of chloroplast glyceraldehyde-3-phosphate dehydrogenase (subunit GAPA, geneGpa1) from maize is extremely G+C rich (67% in codons). The genomic surroundings of this gene have been characterized together with the sequences of two strongly conservedGpa pseudogenes isolated from a genomic maize library by differential cDNA hybridization. The comparisons show that the high G+C content of the maize gene is maintained independently of the surrounding noncoding sequences, which are G+C poor (42%), and only as long as the gene encodes a functional protein. After nonfunctionalization,Gpa pseudogenes rapidly loose G+C mainly due to enhanced turnover of CpG and CpXpG methylation sites. These results suggest that the maizeGpa1 gene is under strong functional GC pressure, due to constraints (CpG island) probably exerted at the transcriptional level. They also indicate thatGpa pseudogenes are methylated and that methylation was either the cause or the immediate consequence of their nonfunctionalization. It can be concluded further that the progenitor of pseudogenes 1 and 2 was a second functionalGpa gene (Gpa′), which, after duplication, accelerated in evolutionary rate due to relaxation of selective constraints. This is in agreement with the neutral theory of evolution. Comparison ofGpa intron sequences reveals a gradient of divergence: the more 3′ the position of an intron the more its sequence has diverged between the threeGpa genes. A speculative model is presented explaining these observations in terms of a homologous recombination of genes with their reverse-transcribed pre-mRNAs.

Nucleotide sequence and expression of a novel glycine-rich protein gene from Arabidopsis thaliana

A clone containing a gene coding for a novel glycine-rich protein has been identified in an Arabidopsis thaliana genomic library. The gene codes for a 339 amino acid protein and is interrupted by a 686 bp intervening sequence. The gene is present in one copy only. Transcripts accumulate mainly in hypocotyls and stems and the highest level is observed in rosettes before the start of stem elongation. The protein contains 71% of glycine residues and is highly hydrophobic.

In vitro-mutagenesis of NADPH:protochlorophyllide oxidoreductase B: two distinctive protochlorophyllide binding sites participate in enzyme catalysis and assembly

NADPH:protochlorophyllide oxidoreductase (POR) B is a key enzyme for the light-induced greening of etiolated angiosperm plants. It is nucleus-encoded, imported into the plastids posttranslationally, and assembled into larger light-harvesting POR:protochlorophyllide complexes termed LHPP (Reinbothe et al., Nature 397:80–84, 1999). An in vitro-mutagenesis approach was taken to study the role of the evolutionarily conserved Cys residues in pigment binding. Four Cys residues are present in the PORB of which two, Cys276 and Cys303, established distinct pigment binding sites, as shown by biochemical tests, protein import studies, and in vitro-reconstitution experiments. While Cys276 constituted the Pchlide binding site in the active site of the enzyme, Cys303 established a second, low affinity pigment binding site that was involved in the assembly and stabilization of imported PORB enzyme inside etioplasts.

The six genes of the Rubisco small subunit multigene family from Mesembryanthemum crystallinum, a facultative CAM plant

The nucleotide sequences of the entire gene family, comprising six genes, that encodes the Rubisco small subunit (rbcS) multigene family in Mesembryanthemum crystallinum (common ice plant), were determined. Five of the genes are arranged in a tandem array spanning 20 kb, while the sixth gene is not closely linked to this array. The mature small subunit coding regions are highly conserved and encode four distinct polypeptides of equal lengths with up to five amino acid differences distinguishing individual genes. The transit peptide coding regions are more divergent in both amino acid sequence and length, encoding five distinct peptide sequences that range from 55 to 61 amino acids in length. Each of the genes has two introns located at conserved sites within the mature peptide-coding regions. The first introns are diverse in sequence and length ranging from 122 by to 1092 bp. Five of the six second introns are highly conserved in sequence and length. Two genes, rbcS-4 and rbcS-5, are identical at the nucleotide level starting from 121 by upstream of the ATG initiation codon to 9 by downstream of the stop codon including the sequences of both introns, indicating recent gene duplication and/or gene conversion. Functionally important regulatory elements identified in rbcS promoters of other species are absent from the upstream regions of all but one of the ice plant rbcS genes. Relative expression levels were determined for the rbcS genes and indicate that they are differentially expressed in leaves.