Alain Takvorian

Characterization of two members of the Arabidopsis thaliana gene family, Atβfruct3 and Atβfruct4, coding for vacuolar invertases

Abstract We have isolated and characterized two Arabidopsis thaliana cDNAs and their cognate genes, Atβfruct3 and Atβfruct4 , encoding vacuolar forms of invertase. Our sequencing results showed that the gene Atβfruct3 is located downstream of the 3-ketoacyl-acyl carrier protein synthase III gene ( AtKasIII ). Atβfruct3 and 4 are functional and organized into seven exons and six introns with an identical organization. The Atβfruct3 and Atβfruct4 genes encode, respectively, polypeptides of 648 and 664 residues that contain all the characteristic hallmarks of vacuolar invertases. A. thaliana is the first plant of which both cell-wall ( Atβfruct1 and Atβfruct2 ) and vacuolar ( Atβfruct3 and Atβfruct4 ) genes are characterized. The same number of exons and introns is seen in the genes Atβfruct1 , Atβfruct3 and Atβfruct4 as well as in all other invertase genes described to date. However, the position of the third intron is different in Atβfruct3 and Atβfruct4 . Atβfruct2 shows a different organization. A neighbour-joining distance tree shows that the A. thaliana vacuolar invertases described here are, as expected, more closely related to vacuolar invertases from other plant species (e.g., carrot) than to the A. thaliana cell-wall invertases. The evolution of plant invertase genes from a common ancestral gene is discussed. Our results demonstrate that in A. thaliana , at least two genes encoding vacuolar invertases are expressed during the development of the plant. Southern blot hybridization experiments suggest the presence of one copy of, respectively, Atβfruct3 and Atβfruct4 per haploid genome, and Northern blot analysis demonstrates that vacuolar invertase genes are highly expressed in stems, roots, flowers and at very low levels in mature leaves.

An evolutionary conserved group of plant GSK-3/shaggy-like protein kinase genes preferentially expressed in developing pollen

Genes and cDNAs encoding plant protein kinases highly homologous to the animal GSK-3/shaggy subfamily were isolated from Arabidopsis thaliana, Brassica napus, Petunia hybrida and Nicotiana tabacum using the P. hybrida PSK6 GSK-3/shaggy related cDNA as a probe. All the derived protein sequences contained the characteristic catalytic domain of GSK-3/shaggy protein kinases. Sequence comparisons within the catalytic domain with other plant GSK-3/shaggy like kinases clearly indicate that the novel sequences form an isolated group of genes termed the PSK6 group. All the proteins within this group possess an amino-terminal extension which contains short amino acid motifs highly conserved between species and possibly implicated in mitochondrial targeting. Northern hybridisation experiments and reverse transcriptase PCR analysis demonstrated that these novel cDNAs are predominantly expressed in developing pollen. The three genes isolated from P. hybrida and A. thaliana show the same genomic organisation into 12 introns and 13 exons. Although the size of the introns varies, their positions are conserved between genes and species. The comparison of these gene structures and the analysis of deduced protein sequences belonging to different plants hold important information to understand the function of individual members. They suggest that some of the characterised sequences represent most likely true orthologues whereas others must be paralogues. They also allow us to discuss the evolution of the plant GSK-3/shaggy like gene family with regard to plant speciation.

STRUCTURE, ORGANIZATION AND PUTATIVE FUNCTION OF THE GENES IDENTIFIED WITHIN A 23.9-KB FRAGMENT FROM ARABIDOPSIS THALIANA CHROMOSOME IV

In the framework of the complete genome sequencing programme of the crucifer Arabidopsis thaliana, a 23.9-kb fragment from the long arm of chromosome IV has been analysed. This paper presents a methodological approach, integrating computerized predictions, database screening, the sequencing of cognate cDNAs and a PCR-based detection of expression that allows the accumulation of an important amount of information from an anonymous sequence. This work revealed the organization of novel genes and the vestige of a copia-like retrotransposon. The gene AtRH1 encodes the first member of a new subfamily of the plant DEAD box RNA helicases. A recurrent and complete search of dbEST has been used to evaluate the number of different RNA helicases expressed in A. thaliana. On the 18 discriminated members of the family, only a small number seems to be expressed at a relatively high level. The putative gene AtTS1 encodes a novel terpene synthase in A. thaliana, and the genes G14587-5 and G14587-6 encode unknown proteins. This study illustrates most of the situations that could be encountered during the analysis of an anonymous sequence from A. thaliana.

The wheat mitochondrial rps13 gene: RNA editing and co-transcription with the atp6 gene

Abstract Northern analyses and reverse transcription-polymerase chain-reaction (RT-PCR) experiments, followed by PCR amplification product sequencing, were performed on total mitochondrial (mt) RNAs from wheat seedlings and tissue cultures. It was shown that the rps13 gene, which encodes ribosomal protein S13, and the atp6 gene, which encodes subunit 6 of the ATP synthase complex, were co-transcribed. However, rps13 transcripts were virtually undetectable in seedlings under conditions where atp6 transcripts appeared abundant. In addition, markedly higher steady state transcript levels were observed in tissue culture. Expression of the mitochondrial rps13 gene was confirmed by showing that its transcripts were edited. Slight differences between editing patterns of tissue-culture and whole-plant transcripts were found. Taken together, these results suggest that in vitro culture could disturb the post-transcriptional regulation of gene expression.

Genes encoding subunit 6 of NADH dehydrogenase and subunit 6 of ATP synthase are co-transcribed in maize mitochondria

Abstract The mitochondrial nad6 gene of maize was identified and mapped 1 kb downstream from the atp6 gene. It encodes a 220 amino-acid polypeptide. Using Northern hybridization experiments and RT-PCR analysis, we showed that both nad6 and atp6 are co-transcribed in maize mitochondria. RNA editing of the mitochondrial nad6 transcript was studied by cDNA sequencing. Twelve edited sites were identified at the same positions as those already identified in the wheat mitochondria nad6 transcript. Alignments of nucleotide and amino-acid sequences of the mitochondrial nad6 genes of maize, wheat, and Brassica campestris, show that the wheat gene encodes a shorter polypeptide (229 amino acids) than was previously thought.