Laurian S. Robert

Conserved structure and function of the Arabidopsis flowering time gene CONSTANS in Brassica napus.

The Arabidopsis thaliana CONSTANS (CO) gene which promotes flowering in long days was recently isolated by chromosome walking. The mapping of QTLs controlling flowering time in Brassica species has identified genomic regions that contain homologues of the CO gene. Four genes homologous to the Arabidopsis CO gene were isolated from a pair of homoeologous loci in each of two doubled-haploid Brassica napus lines displaying different flowering times, N-o-1 and N-o-9. The four genes, BnCOa1, BnCOa9, BnCOb1 and BnCOb9, are located on linkage groups N10 and N19, and are highly similar to each other and to the Arabidopsis CO gene. Two regions of the proteins are particularly well conserved, a N-terminal region with two putative zinc fingers and a C-terminal region which may contain a nuclear localization signal. All four genes appear to be expressed in B. napus. The BnCOa1 allele was shown to complement the co-2 mutation in Arabidopsis in a dosage-dependent manner causing earlier flowering than in wild type under both long- and short-day conditions.

Isolation and characterization of a polygalacturonase gene highly expressed in Brassica napus pollen

A cDNA clone, Sta 44-4, corresponding to a mRNA highly expressed in Brassica napus cv. Westar stamens, was isolated by differential screening and characterized. Northern blot and in situ analyses demonstrated that Sta 44-4 is synthesized in pollen beginning at the late uninucleate stage and reaches a maximum in trinucleate microspores. Sta 44-4 displayed significant sequence similarity to known pollen polygalacturonase genes. The B. napus pollen polygalacturonase gene was shown to be part of a small gene family and to display some polymorphism among different cultivars.

Molecular Characterization of Two Brassica napus Pollen-Expressed Genes Encoding Putative Arabinogalactan Proteins

Two highly homologous cDNA clones, Sta 39’3 and Sta 39’4, corresponding to mRNAs highly expressed in Brassica napus cv Wester stamens were isolated by differential screening and characterized. Northern blot and in situ analyses demonstrated that Sta 39’3 and Sta 39’4 transcripts accumulate in pollen following the first pollen mitosis and are abundant at pollen maturity, thus identifying them as “late” pollen genes. Sta 39’3 and Sta 39’4 belong to a small gene family. Their predicted proteins share similarities with those deduced from recently isolated cDNAs encoding arabinogalactan proteins, which include a similar molecular mass (approximately 13 kD); high levels of alanine, proline, serine, and threonine, which are interspersed throughout the protein with no obvious repetitive motif; low levels of cysteine, histidine, tryptophan, and tyrosine; and hydrophobic N- and C-terminal ends. The Sta 39’3 and Sta 39’4 proteins may play a role in pollen germination and tube growth.

Control of seed germination in transgenic plants based on the segregation of a two-component genetic system

We have developed a repressible seed-lethal (SL) system aimed at reducing the probability of transgene introgression into a population of sexually compatible plants. To evaluate the potential of this method, tobacco plants were transformed with an SL construct comprising gene 1 and gene 2 from Agrobacterium tumefaciens whereby gene 1 was controlled by the seed-specific phaseolin promoter modified to contain a binding site for the Escherichia coli TET repressor (R). The expression of this construct allows normal plant and seed development but inhibits seed germination. Plants containing the SL construct were crossed with plants containing the tet R gene to derive plant lines where the expression of the SL construct is repressed. Plant lines that contained both constructs allowed normal seed formation and germination, whereas seeds in which the SL construct was separated from the R gene through segregation did not germinate. The requirements of such a method to efficiently control the flow of novel traits among sexually compatible plants are discussed.

Cloning and characterization of a Brassica napus gene encoding a homologue of the B subunit of a heteromeric CCAAT-binding factor

The CCAAT motif present in the promoter of several genes is recognized in yeast and animals by a highly specific heteromeric factor (variously called HAP, CBF, CP1 or NF-Y) which is composed of a minimum of three subunits. A plant homologue of the CBF-B/HAP2 subunit is described for the first time in this report. Sequence comparison of the Brassica napus (Bn) CCAAT-binding factor (CBF) B subunit with the homologous yeast and animal proteins revealed that the critical amino-acid domains involved in DNA binding and subunit assembly are also conserved in plants. Interestingly, the Gln-rich regions found in the animal and yeast proteins, which may be involved in transcriptional activation, are absent in the Bn CBF-B subunit. The analysis of various cDNAs and of a genomic clone revealed the presence of alternatively spliced transcripts which could originate from different promoters.

Low temperature induced defence gene expression in winter wheat in relation to resistance to snow moulds and other wheat diseases.

Cold hardening of winter wheat at 2 °C for 1-6 wks increased resistance to the snow mould pathogens LTB, Typhula incarnata, and Microdochium nivale as well as to powdery mildew (Blumaria graminis f. sp. graminis) and stripe rust (Puccinia striiformis). Using microarrays and hardening of winter wheat for 0.25, 0.5, 1, 7, 21 and 49 d, an upregulation of a wide range of stress-response genes that include defence-related and abiotic stress-related genes, transcription factors including several lipoxygenases and ethylene responsive factors, and WRKY genes was observed. For the majority of these genes, the upregulation occurred later in the 21-49 d hardening treatments and coincided with the highest expression levels of snow mould resistance. Defence-related sequences were upregulated to a greater extent and were more numerous in the snow mould resistant line CI14106 compared to cold hardy DH+268. Transcript profiling of candidate defence and other stress-related genes under prolonged conditions at -3 °C with or without snow mould infection showed that there was a decline in transcripts of the defence-related genes PR1.1b and NPR3 during the 12wks incubation. Additionally, 14 d hardening was insufficient to permit full expression of the jasmonic acid synthesis gene, allene oxide synthase (AOS) and the fructan degrading enzyme β-fructofuranosidase compared the 42 d hardening treatment. The snow mould resistant line CI14106 was able to maintain higher transcript levels of AOS for longer conditions compared to the susceptible line Norstar under artificial snow mould conditions. These results explain the nature of cold-induced resistance to snow moulds and provide direction on establishing selection criteria for improving resistance and cold tolerance in winter wheat.

The promoter of a Brassica napus polygalacturonase gene directs pollen expression of β-glucuronidase in transgenic Brassica plants

A 647-bp 5′-flanking fragment obtained from genomic clone Sta 44G(2) belonging to a family of polygalacturonase genes expressed inBrassica napus pollen was fused to theβ-glucuronidase (GUS) marker gene. This fusion construct was introduced intoB. napus plants viaAgrobacterium tumefaciens transformation. Analysis of the transgenicB. napus plants revealed that this promoter fragment is sufficient to direct GUS expression specifically in the anther and that GUS activity increases in pollen during maturation.

Characterization of a low-temperature-induced cDNA from winter Brassica napus encoding the 70 kDa subunit of tonoplast ATPase

A cDNA clone, pBN59, was isolated by differential screening of a cDNA library of winter Brassica napus during cold acclimation. Nucleotide sequence of BN59 was found to be homologous to that encoding the 70 kDa subunit of the vacuolar H+-ATPase in plants. Transcripts hybridizing to BN59 accumulated during exposure to low temperatures and to the exogenous application of abscisic acid (ABA). Western blot analyses also indicated an increase in the 70 kDa subunit during cold acclimation. The accumulation of an endomembrane H+-ATPase is consistent with the observation of osmotic adjustment, increases in endogenous ABA and the proliferation of endomembranes during cold acclimation.

Sequence and expression of endogenous S-locus glycoprotein genes in self-compatible Brassica napus

Abstract.Brassica napus is an amphidiploid plant which is self-compatible even though it is derived from hybridisation of the self-incompatible species B. oleracea and B. campestris. Experiments were undertaken to establish if S-locus glycoprotein (SLG) genes exist in B. napus and whether these are expressed as in self-incompatible Brassica species. Two different stigma-specific cDNA sequences homologous to SLG genes were obtained from the B. napus cultivar Westar. One of these sequences, SLGWS1, displayed highest homology to class I SLG alleles, whereas the other, SLGWS2, showed greatest homology to class II SLG genes. Both were expressed at high levels in Westar stigmas following a developmental pattern typical of SLG genes in the self-incompatible diploids. We infer that they represent the endogenous SLG genes at the two homoeologous S-loci. The occurrence of normally expressed SLG genes and its relevance to the self-compatible phenotype of B. napus is discussed.

Promoter sequences from two different Brassica napus tapetal oleosin-like genes direct tapetal expression of β-glucuronidase in transgenic Brassica plants

To investigate the sequences responsible for the regulated expression of tapetal-specific oleosin-like genes, ca. 2 kb of the 5′-upstream regions from two divergent genes, OlnB;4 and OlnB;13, were isolated, sequenced and fused to the reporter gene β-glucuronidase for study in transgenic Brassica napus plants. Although the proteins encoded by these two genes are highly divergent, except for the conserved oleosin-like domain, the first 250 bp of their 5′-upstream regions was 86% identical, including a region of 150 bp upstream from the TATA box. Analysis of 42 independent transformants by histochemical and fluorometric methods showed that both promoters directed tapetal-specific expression that peaked at the 4 mm flower bud stage.