Rodolphe Boivin

Structural and Evolutionary Relationships Among Chitinases of Flowering Plants

Abstract. The analysis of nuclear-encoded chitinase sequences from various angiosperms has allowed the categorization of the chitinases into discrete classes. Nucleotide sequences of their catalytic domains were compared in this study to investigate the evolutionary relationships between chitinase classes. The functionally distinct class III chitinases appear to be more closely related to fungal enzymes involved in morphogenesis than to other plant chitinases. The ordering of other plant chitinases into additional classes mainly relied on the presence of auxiliary domains—namely, a chitin-binding domain and a carboxy-terminal extension—flanking the main catalytic domain. The results of our phylogenetic analyses showed that classes I and IV form discrete and well-supported monophyletic groups derived from a common ancestral sequence that predates the divergence of dicots and monocots. In contrast, other sequences included in classes I* and II, lacking one or both types of auxiliary domains, were nested within class I sequences, indicating that they have a polyphyletic origin. According to phylogenetic analyses and the calculation of evolutionary rates, these chitinases probably arose from different class I lineages by relatively recent deletion events. The occurrence of such evolutionary trends in cultivated plants and their potential involvement in host–pathogen interactions are discussed.

Construction of a Tn5 derivative encoding bioluminescence and its introduction in Pseudomonas, Agrobacterium and Rhizobium

SummaryA simple method based upon the use of a Tn5 derivative, Tn5-Lux, has been devised for the introduction and stable expression of the character of bioluminescence in a variety of gram-negative bacteria. In Tn5-Lux, the luxAB genes of Vibrio harveyi encoding luciferase are inserted on a SalI-BglII fragment between the kanamycin resistance (Kmr) gene and the right insertion sequence. The transposon derivative was placed on a transposition suicide vehicle by in situ recombination with the Tn5 suicide vector pGS9, to yield pDB30. Mating between Escherichia coli WA803 (pDB30) and a strain from our laboratory, Pseudomonas sp. RB100C, gave a Kmr transfer frequency of 10-6 per recipient, a value 10 times lower than that obtained with the original suicide vehicle pGS9. Tn5-Lux was also introduced by insertion mutagenesis in other strains of gram-negative soil bacteria. The bioluminescence marker was expressed in the presence of n-decanal, and was monitored as chemiluminescence in a liquid scintillation counter. The recorded light intensities were fairly comparable among the strains, and ranged between 0.2 to 1.8x106 cpm for a cell density of 103 colony forming units/ml. Nodules initiated by bioluminescent strains of Rhizobium leguminosarum on two different hosts were compared for intensity of the bioluminescence they produced.

Promoter for a Brassica napus ribulose bisphosphate carboxylase/oxygenase small subunit gene binds multiple nuclear factors and contains a negative-strand open reading frame encoding a putative transmembrane protein

Using a fractionated genomic bank, we have cloned and characterized a Brassica napus gene (rbcSF1) encoding the small sub-unit of ribulose 1,5-bisphosphate carboxylase. The promoter of this gene contains a 29 bp direct repeat capable of forming a single or a double hairpin loop, and three elements that are recognized by leaf nuclear proteins in vitro. The most upstream are the S-box, a small A/T-rich sequence between −516 and −512, and the F-box between −492 and −475. Finally, we have also observed binding to the G-box, a regulatory element common to numerous plant promoters. The promoter of rbcSF1 also has a 113 amino acids open reading frame (ORF113) in the non-coding strand. When used to probe a northern blot of leaf RNA, this ORF hybridizes to a 1.5 kb transcript. The protein encoded by ORF113 contains a transmembrane domain.

Stage-specific transcription of the homeobox gene Bnhd1 in young tissues and flowers of Brassica napus

The Bnhd1 cDNA, only distantly related to published homebox gene sequences, was isolated from Brassica napus by the polymerase chain reaction. The Bnhd1 transcript was detected in all organs of young seedlings, but only in the youngest leaves and flowers of mature plants. A 4- and 7-fold increase in transcription levels was observed after wounding of young roots and leaves, respectively.

Root-specific expression of a glycine-rich protein gene in Brassica napus

We have constructed complementary DNA and genomic libraries from Brassica napus and isolated clones corresponding to three closely related sequences encoding glycine-rich proteins. A probe from BnRG22, the most expressed sequence in 7-day old seedlings, identified a major transcription signal at 1.2-kb, highly abundant in roots and detected also in stems but not in other organs of the plant. The synthesis of these transcripts is regulated during development up to 30 days after germination. Moreover, flooding and drought have antagonistic effects on its expression. BnRG22 encodes a protein containing as much as 62% of glycine residues and an amino-terminal signal peptide. This protein shows three repeats of 42 residues each and includes the (Gly-X)n and many Gly-Tyr-Gly motifs. We propose for this protein a structural model featuring a β-pleated sheet domain made of anti-parallel strands.

Evidence of Somaclonal Variation in Somatic Embryo-Derived Plantlets of White Spruce (Picea Glauca (Moench) Voss

Unlike angiosperms (especially crop plant species), conifers are considered to be genetically stable following somatic embryogenesis. However, we have been able to identify four different variegata phenotypes among 2270 somatic embryo-derived white spruces. The four types of variegated plants differ from each other with respect to the extent and distribution of their chlorophyll-deficient needles. Microscopy shows that certain leaves of a selected variant are formed of a chimeral mixture of green and white cells. Cells in completely white needles of this variegated plant are characterized by large nuclei with predominant euchromatin, absence of large cytoplasmic vacuoles, and vacuolized plastids with aberrant morphologies. Various observations suggest that the recovered variegata phenotypes reflect some kind of genetic instability of either chloroplastic or nuclear genomes. Molecular approaches, including the use of RAPD markers, are currently employed to find out whether DNA rearrangements are involved in conferring these variegata phenotypes.

A novel approach to the rapid isolation and nucleotide sequencing of genomic clones.

In this study, a genomic library subdivided into fractions was rapidly screened by a Southern detection technique. Deletion libraries were obtained from recovered genomic clones by single random cuts with nuclease S1. These deletion libraries proved useful for localizing genes in the inserts and yielded, after size fractionation, nested deletions suitable for nucleotide sequencing. An heterologous vector (pDB21) carried the insert used as probe for all hybridizations involved in the process of genomic clones isolation and characterization.