Requirement of a CCGAC cis-acting element for cold induction of the BN115 gene from winter Brassica napus

Mutation of the core pentamer, CCGAC, of two putative low temperature responsive elements (LTREs) in the 5-proximal region of the winter Brassica napus cold-induced gene BN115 was carried out. Analyses of transient expression of the resultant mutated BN115 promoter-GUS fusions revealed the loss of low-temperature regulation by the promoter. This indicates that the CCGAC sequence is critical to the low-temperature response in the BN115 gene. In contrast, mutation of two G-boxes, CACGTG, staggered between the LTREs in the same region of the promoter did not alter cold-inducible gene expression. Replacement of a possible enhancer region of the BN115 promoter with the enhancer from the CaMV 35S promoter resulted in a several-fold increase in low temperature-induced GUS activity.     

Construction and functional characteristics of tuber-specific and cold-inducible chimeric promoters in potato

The improvement of processing quality of potato products (fries and chips) demands less accumulation of reducing sugars (glucose and fructose) in cold-stored potato (Solanum tuberosum) tubers. Control of gene expression to achieve this requires promoters with specificity to tubers as well as inducible activity under low temperatures. Here we use overlapping extension PCR to construct two chimeric promoters, pCL and pLC, to control gene expression in a tuber-specific and cold-inducible pattern. This combined different combinations of the LTRE (low-temperature responsive element) from Arabidopsis thaliana cor15a promoter and the TSSR (tuber-specific and sucrose-responsive sequence) from potato class I patatin promoter. The cold-inducible and tuber-specific activities of the chimeric promoters were investigated by quantitative analysis of GUS activity in transgenic potato cultivar E3 plants. The results showed that the cis-elements, LTRE and TSSR, played responsive roles individually or in combination. pCL with the TSSR closer to the TATA-box showed substantially higher promoter activity than pLC with the LTRE closer to the TATA-box at either normal (20°C) or low temperature (2°C), suggesting that the promoter activity was closely associated with the position of the two elements. The chimeric promoter pCL with tuber-specific and cold-inducible features may provide valuable tool for controlling the expression of gene constructs designed to lower the formation of reducing sugars in tubers stored at low temperature and to improve the processing quality of potato products. The nucleotide sequence data reported will appear in the GenBank database under the accession numbers DQ494557 (pCL) and DQ494558 (pLC ).     

Regulation of BN115, a low-temperature-responsive gene from winter Brassica napus.

The genomic clone for BN115, a low-temperature-responsive gene, was isolated from winter Brassica napus and its sequence was determined. A 1.2-kb fragment of the 5' regulatory region (from bp -1107 to +100) was fused to the beta-glucuronidase (GUS) reporter gene and BN115-promoted GUS expression was observed in green tissues of transgenic B. napus plants only after incubation at 2 degrees C. No expression was observed after incubation at 22 degrees C, either in the presence or the absence of ABA. Microprojectile bombardment of winter B. napus leaves with a BN115 promoter/GUS construct yielded similar results and was used to analyze a series of deletions from the 5' end of the promoter. Results obtained from transient expression studies showed that the low-temperature regulation of BN115 expression involves a possible enhancer region between bp -1107 and -802 and a second positive regulatory region located between bp -302 and -274. Deletion analyses and results from replacement with a truncated cauliflower mosaic virus 35S promoter suggest that the minimal size required for any maintenance of low-temperature GUS expression is a -300-bp fragment. Within this fragment are two 8-bp elements with the sequence TGGCCGAC, which are identical to those present in the positive regulatory region of the promoter of the homologous Arabidopsis cor15a gene and to a 5-bp core sequence in the low-temperature- and dehydration-responsive elements identified in the promoter regions of several cold-responsive Arabidopsis thaliana genes.     

Molecular cloning and characterization of a CBF gene from Capsella bursa-pastoris.

A new CBF gene was cloned from Capsella bursa-pastoris(shepherd's purse) by rapid amplification of cDNA ends (RACE). The full-length cDNA of C. bursa-pastoris CBF gene (designated as Cbcbf) was 1034 bp long and contained a 657 bp open reading frame (ORF) encoding a putative DRE/CRT (LTRE)-binding protein of 219 amino acids. The predicted CbCBF protein was found to have a potential nuclear localization signal (NLS) in its N-terminal region followed by an AP2 DNA-binding motif and an acidic C-terminal half that might act as an activator domain. Bioinformatic analysis revealed that Cbcbf strongly resembled other CBF genes from Arabidopsis thaliana (cbf1, cbf2, cbf3) and Brassica napus (Bncbf5, Bncbf 7, Bncbf16 and Bncbf17). Subsequent cold acclimation assay showed that Cbcbf was relevant to cold acclimation. Our study implies that Cbcbf might have similar functions possessed by other CBF genes such as inducing the expression of some cold-regulated genes and increasing plants' freezing tolerance.     

Isolation and molecular characterization of a new CRT binding factor gene from Capsella bursa-pastoris

A new CRT binding factor (CBF) gene designated Cbcbf25 was cloned from Capsella bursa-pastoris, a wild grass, by the rapid amplification of cDNA ends (RACE). The full-length cDNA of Cbcbf25 was 898 bp with a 669 bp open reading frame (ORF) encoding a putative DRE/CRT (LTRE)-binding protein of 223 amino acids. The predicted CbCBF25 protein contained a potential nuclear localization signal (NLS) in its N-terminal region followed by an AP2 DNA-binding motif and a possible acidic activation domain in the C-terminal region. Bioinformatic analysis revealed that Cbcbf25 has a high level of similarity with other CBF genes like cbf1, cbf2, and cbf3 from Arabidopsis thaliana, and Bncbf5, Bncbf7, Bncbf16, and Bncbf17 from Brassica napus. A cold acclimation assay showed that Cbcbf25 was expressed immediately after cold triggering, but this expression was transient, suggesting that it concerns cold acclimation. Our study implies that Cbcbf25 is an analogue of other CBF genes and may participate in cold-response, by for example, controlling the expression of cold-regulated genes or increasing the freezing tolerance of plants.     

The effects of phenotypic plasticity on photosynthetic performance in winter rye, winter wheat and Brassica napus

The contributions of phenotypic plasticity to photosynthetic performance in winter (cv Musketeer, cv Norstar) and spring (cv SR4A, cv Katepwa) rye (Secale cereale) and wheat (Triticum aestivum) cultivars grown at either 20°C [non‐acclimated (NA)] or 5°C [cold acclimated (CA)] were assessed. The 22–40% increase in light‐saturated rates of CO₂ assimilation in CA vs NA winter cereals were accounted for by phenotypic plasticity as indicated by the dwarf phenotype and increased specific leaf weight. However, phenotypic plasticity could not account for (1) the differential temperature sensitivity of CO₂ assimilation and photosynthetic electron transport, (2) the increased efficiency and light‐saturated rates of photosynthetic electron transport or (3) the decreased light sensitivity of excitation pressure and non‐photochemical quenching between NA and NA winter cultivars. Cold acclimation decreased photosynthetic performance of spring relative to winter cultivars. However, the differences in photosynthetic performances between CA winter and spring cultivars were dependent upon the basis on which photosynthetic performance was expressed. Overexpression of BNCBF17 in Brassica napus generally decreased the low temperature sensitivity (Q₁₀) of CO₂ assimilation and photosynthetic electron transport even though the latter had not been exposed to low temperature. Photosynthetic performance in wild type compared to the BNCBF17‐overexpressing transgenic B. napus indicated that CBFs/DREBs regulate not only freezing tolerance but also govern plant architecture, leaf anatomy and photosynthetic performance. The apparent positive and negative effects of cold acclimation on photosynthetic performance are discussed in terms of the apparent costs and benefits of phenotypic plasticity, winter survival and reproductive fitness.     

Two related,low-temperature-induced genes from Brassica napus are homologous to the human tumour bbc1 (breast basic conserved) gene

In order to identify genes involved in cold acclimation, we have constructed a cDNA library from Brassica napus (cv. Samouraï) cold-acclimated etiolated seedlings. By differential screening, a cDNA clone named pBnC24 (Brassica napus Cold), corresponding to a new cold-inducible plant gene, was isolated. Northern blot hybridizations using total RNA from acclimated and unacclimated seedlings confirmed that BnC24 represents a cold-regulated gene. In contrast with a number of cold-inducible plant genes, BnC24 does not seem to be responsive to abscisic acid (ABA). In addition, further screening of the cold-acclimated cDNA library using pBnC24 cDNA as a probe, allowed the isolation of a second type of homologous cDNA. Sequence analysis showed that the two BnC24 genes encode basic 24 kDa proteins, which are highly hydrophilic and rich in alanine, lysine and arginine. The nucleotide and deduced amino acid sequences of these clones do not show any homology with other previously described cold-induced plants genes. However they have strong homology with a recently discovered human tumour gene, bbc1 (breast basic conserved), which seems to be highly conserved in eukaryotes.     

Purification, characterization, and structural analysis of a plant low-temperature-induced protein.

We have purified to near homogeneity a recombinant form of the protein BN28 (rBN28), expressed in response to low temperature in Brassica napus plants, and we have determined its solution structure. Antibodies raised against rBN28 were used to characterize the recombinant and native proteins. Similar to many other low-temperature-induced proteins, BN28 is extremely hydrophilic, such that it remains soluble following boiling. Immunoblot analysis of subcellular fractions indicated that BN28 was not strongly associated with cellular membranes and was localized exclusively within the soluble fraction of the cell. Contrary to predicted secondary structure that suggested significant helical content, circular dichroism analysis revealed that rBN28 existed in aqueous solution largely as a random coil. However, the helical propensity of the protein could be demonstrated in the presence of trifluoroethanol. Nuclear magnetic resonance analysis further showed that rBN28 was in fact completely unstructured (100% coil) in aqueous solution. Although it had earlier been speculated that BN28-like proteins from Arabidopsis thaliana might possess antifreeze protein activity (S. Kurkela and M. Franck [1990] Plant Mol Biol 15: 137-144), no such activity could be detected in ice recrystallization assays with rBN28.     

Inheritance and expression patterns of BN28, a low temperature induced gene in Brassica napus, throughout the Brassicaceae.

Molecular genetics is becoming an important tool in the breeding and selection of agronomically important traits. BN28 is a low temperature induced gene in Brassicaceae species. PCR and Southern blot analysis indicate that BN28 is polymorphic in the three diploid genomes: Brassica rapa (AA), Brassica nigra (BB), and Brassica oleracea (CC). Of the allotetraploids, Brassica napus (AACC) is the only species to have inherited homologous genes from both parental genomes. Brassica juncea (AABB) and Brassica carinata (BBCC) have inherited homologues from the AA and CC genomes, respectively, while Sinapsis arvensis (SS) contains a single homologue from the BB genome and Sinapsis alba (dd) appears to be different from all the diploid parents. All species show message induction when exposed to low temperature. However, differences in expression were noticed at the protein level, with silencing occurring in the BB genome at the level of translation. Results suggest that silencing is occurring in diploid species where duplication may not have occurred. Molecular characterization and inheritance of BN28 homologues in the Brassicaceae may play an important role in determining their quantitative function during exposure to low temperature. Key words : Brassicaceae, BN28, inheritance, polymorphism.