Rapid analysis of an osmotic stress responsive promoter by transient expression in intact wheat embryos

Expression of the wheat ‘Em’ genes in embryos isstrongly induced both by abscisic acid and by subjection toosmotic stress. We have examined the basis of this inductionin an homologous system by con structing fusions between thepromoter sequences of a cloned ‘Em’ gene and theGUS reporter gene. The ABA-and stress-mediated expression ofthese constructs has been assayed following delivery to intactwheat embryos by particle bombardment. Staining of bombardedembryos with the chromogenic substrate X-gluc enabled a simpleand rapid visual identification of promoter activity by scoringthe numbers of stained spots. Although not rigorously quantitative,a general correspondence between number of expression events(spots) and promoter activity could be inferred when the resultsobtained with bombarded embryos were compared with those obtainedby the fluorlmetnc measurement of GUS activity in cereal aleuroneprotoplasts transfected with the same constructs. Analysis ofa series of 5'-deletions of the ‘Em’ promoters indicatedthat the stress-responsive cis-acting regulatory elements comprisethe same sequences as those responsible for ABA-mediated geneexpression. Key words: Abscisic acid, osmotic stress, Em genes, wheat embryos, transient expression     

Molecular analysis of the ependymin gene and functional test of its promoter region by transient expression in Brachydanio rerio.

Ependymins are secretory products of meningeal cells and represent the predominant glycoproteins in the cerebrospinal fluid from various orders of teleost fish. In the zebrafish, their expression starts between 48 and 72 h post-fertilization. Generally, they share characteristics with proteins involved in cell-contact phenomena. Here, we characterize the ependymin gene from Brachydanio rerio and its flanking regions. The sequence was obtained from clones generated using the polymerase chain reaction (PCR), including a variation of an "anchored" PCR. Also, clones from a conventional phage library were analyzed. We found that the transcribed portion is arranged in six exons. Transient expression of an ependymin-promoter-lacZ gene fusion in zebrafish embryos revealed that the 2.0-kb upstream regulatory region used is sufficient to direct the ependymin-specific correct temporal and spatial expression pattern of the lacZ reporter gene.     

The promoter of barley trypsin-inhibitor BTI-CMe,discriminates between wheat and barley endosperm protoplasts in transient expression assays

Several promoter fragments from the barley gene coding for trypsin inhibitor, BTI-CMe, have been fused to the -glucuronidase (GUS) reporter gene and these chimeric constructs used for transient expression in protoplasts. Transfection of developing endosperm protoplasts from barley (cv Bomi) show a maximum GUS expression of about 50% of that driven by the cauliflower mosaic virus 35S promoter, while in wheat endosperm protoplasts expression is less than 10%. No significant expression is found in transfected leaf protoplasts from barley, wheat or tobacco (<2% of the 35S control). All the information required for endosperm and barley specificity is present in the 343 bp proximal to the translation initiation site.Abbreviations MS Murashige and Skoog medium - PEG polyethyleneglycol - GUS -glucuronidase - MU methylumbelliferone - MUG 4-methylumbelliferyl--D glucuronide - pp protoplasts     

Factors influencing Agrobacterium-mediated transient expression of uidA in wheat inflorescence tissue.

A critical step in the development of Agrobacterium tumifaciens-mediated transformation is the establishment of optimal conditions for T-DNA delivery into tissue from which whole plants can be regenerated. The efficient transformation of inflorescence tissue from 'Baldus', a commercial wheat variety, using the Agrobacterium strain AGLI harbouring the binary vector pAL156 is reported here. The effects of various factors on delivery and the transient expression of the uidA gene were studied including the duration of preculture, vacuum infiltration, the effect of sonication treatments, and Agrobacterium cell density. Optimal T-DNA delivery (as measured by uidA activity) was obtained from inflorescence tissues precultured for 21 d and sonicated. Increasing Agrobacterium cell density, the duration of inoculation/co-cultivation, and vacuum pressure, up to a threshold, increased uidA expression. The investigation of factors that influence T-DNA delivery is an important first step in the utilization of Agrobacterium in the transformation of immature wheat inflorescence tissue.     

Functional analysis of homologous and heterologous promoters in strawberry fruits using transient expression

The isolation and characterization of fruit-specific promoters are critical for the manipulation of the nutritional value and quality of fruits by genetic engineering. The analysis of regulatory sequences of many ripening-related genes has remained elusive for many species due to their low transformation efficiency and/or lengthy regeneration of a small number of transgenic plants. Strawberry is an important crop and represents one of the most widely studied non-climacteric model systems. However, until recently, its difficult regeneration has limited the functional study of promoters by stable transformation. A protocol based on biolistic transient transformation has been developed in order to study the function of promoters in a fast and efficient manner in strawberry fruits. The protocol has been applied to the study of the GalUR promoter, a gene involved in the biosynthesis of vitamin C in this fruit. The activity of the GalUR promoter is restricted to the fruit, being strictly dependent on light. The analysis of deletion series revealed the presence of a minimum activation region 397 bp upstream of the gene with a putative G-box motif, and a negative regulatory region between -397 and -518 bp, where an I-box was identified. The transient assay has been used to study the activity of the tomato polygalacturonase and the pepper fibrillin promoters in strawberry fruits. Whereas slight activity was observed with the fibrillin promoter, no significant activity was found with the polygalacturonase promoter. The GalUR promoter in transiently transformed ripe tomato fruits showed no activity, indicating the presence of regulatory sequences specific for its function in strawberry fruit.     

Proteome analysis of diploid,tetraploid and hexaploid wheat: towards understanding genome interaction in protein expression

Hexaploid wheat (Triticum aestivum L.) is derived from a complex hybridization procedure involving three diploid species carrying the A, B and D genomes. The proteome patterns of diploid, tetraploid and hexaploid wheat were analyzed to explore the genome interaction in protein expression. At least two species from each of the diploid and tetraploid were used to compare their proteome maps with a hexaploid wheat cv. Chinese Spring. The ancestral cultivars were selected based on their history of closeness with the cultivated wheat. Proteins were extracted from seed flour and separated by two-dimensional electrophoresis (2-DE) with isoelectric focusing of pH range from 4-10. 2-DE maps of cultivated and ancestral species were analyzed by computer assisted image analyzer. The region of high molecular weight glutenin subunits of hexaploid wheat showed similarity with those of the diploid donors, BB and DD genomes. The omega gliadin, which is controlled by B genome in common wheat, was assumed to have evolved as a result of interaction between AA and BB genomes. The low molecular weight glutenins and alpha and beta gliadin regions were contributed by the three genomes. This result suggests that the function of donor genomes particularly in the expression of proteins in hexaploid wheat is not totally independent; rather it is the product of interactions among the diploid genomes in the hexaploid nuclear constitutions. The expression of nonstorage proteins was affected substantially due to the removal of the D genome from hexaploid constitution. Location of the structural gene controlling one of the alpha amylase inhibitor proteins in the nonstorage protein region was identified in the short arm of chromosome 3D.     

Differential rRNA genes expression in bread wheat and its inheritance

The expression of the ribosomal RNA (rRNA) genes from rye, located within the nucleolus organizer regions (NORs), is repressed by cytosine methylation in wheat x rye hybrids and in triticale, as consequence of nucleolar dominance. Our previous study revealed that bread wheat cultivars with a maximum number of four Ag-NORs presented high level of rDNA cytosine methylation when compared to others with a maximum of six Ag-NORs. In order to evaluate the inheritance of the Ag-NORs number and NOR methylation patterns, we produced F₁ hybrids between bread wheat cultivars with four Ag-NORs and bread wheat cultivars with six Ag-NORs (in the direct and reciprocal senses). The F₂ progenies of these F₁ hybrids were also evaluated for the NOR number and methylation patterns. Parent bread wheat cultivars with a maximum of four Ag-NORs after treated with 5-azacytidine evidenced a maximum of six Ag-NORs per metaphase cell and a maximum of six nucleoli per interphase nucleus, confirming that the expression of the rRNA genes in bread wheat is related to cytosine methylation. Most of the F₁ hybrids showed a maximum number of four or six Ag-NORs, similarly to that of the female parent suggesting a non-mendelian inheritance, while other hybrids presented four or six Ag-NORs in both senses of the cross. The F₁ NOR methylation patterns showed some fragments common to their parents but also novel fragments suggesting genomic and/or chromosome rearrangements after hybridization. Despite the different NOR patterns among the parents, an invariable NOR pattern was found among the F₁ plants suggesting a tendency to stability, which was also transmitted to the F₂. The F₂ progenies showed plants with a maximum of four, five and/or six Ag-NORs. The ratio of plants with four, five and/or six Ag-NORs per F₂ progeny was variable and did not follow any specific mendelian proportion. These results allowed us to suggest that the inheritance of the number of Ag-NORs by the F₁ and F₂ plants did not follow any mendelian inheritance and were not correlated to NOR methylation patterns in contrast to what was verified for their parents.     

Marker gene expression driven by the maize ubiquitin promoter in transgenic wheat

Selecting a promoter for driving transgene expression is one of the most important factors to consider in a transformation project. Information about the native regulation of the promoter activity is important, but it is also necessary to consider how that activity will be affected when integrated into the genome of the transformed plants. Study of a promoter performance in individually transformed lines provides useful information in this area. The maize ubiquitin 1 (Ubi‐1) promoter has been widely used to drive constitutive transgene expression in monocotyledonous plants. However, lack of data on its activity in individual transformed wheat lines constitutes a gap in the understanding and predictability of this promoter's performance. In this paper, we began addressing this problem by examining the expression of the marker gene uidA, coding for β‐glucuronidase (GUS), under the control of the maize Ubi‐1 promoter in individual transgenic wheat (Triticum aestivum L.) lines from different wheat varieties. The expression of uidA driven by this promoter depended to a great extent on the specific transformation event. Whilst expression was strong and constitutive in all tissues in some of the lines analysed, there were also transgenic lines in which GUS activity was restricted to only a few tissues. In general the maize Ubi‐1 promoter had strong activity in young, metabolically active tissues and in pollen grains.