Tissue specificity of the sugarcane bacilliform virus promoter in oat,barley and wheat

The tissue-specificity of the sugarcane bacilliform virus (SCBV) promoter was investigated in oat, barley, and wheat to determine whether its expression pattern in one species was predictive of promoter specificity in the other closely related Gramineae species. Progeny of transgenic plants produced using constructs containing the SCBV promoter driving gusA were sampled at different stages of plant development and stained for GUS activity using a histochemical assay. Overall, the GUS staining patterns were most similar between oat and barley. In all three species, similar GUS staining patterns were observed in mature endosperms, leaves, and floral bracts of developing infloresences. No GUS staining was detected in oat embryos whereas the entire barley embryo was stained, and GUS staining was confined to the scutellum of wheat embryos. Oat and barley stems exhibited GUS staining whereas no GUS staining was observed in stems of the transgenic wheat plants. The SCBV promoter conferred strong GUS staining intensity in most tissues of oat and barley but was generally weaker in wheat. These differences in SCBV promoter specificity indicate that promoter evaluation should be conducted in the target species of interest rather than by extrapolation from expression patterns in other species.     

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     

Analysis of the activity of a γ-gliadin promoter in transgenic wheat and characterization of gliadin synthesis in wheat by MALDI-TOF during grain development

The wheat grain is the most important organ for human food and therefore is the target for much research focused on modifying its composition to improve nutritional and functional components. Genetic transformation provides a precise tool to alter the composition of wheat grain by expressing new genes or by down-regulating groups of proteins encoded by multigene families such as gliadins, which contain clusters of epitopes that are active groups in triggering celiac disease. For such work, specific promoters are required to express such constructs in the wheat endosperm. In the present study we report the isolation and characterization of a γ-gliadin promoter from transgenic wheat, and the analysis of gliadin synthesis during grain development in bread wheat by Matrix-Assisted Laser Desorption/Ionization Time-Of-Flight Mass Spectrometry (MALDI/TOF MS). The γ-gliadin promoter fragment was isolated from bread wheat by genome walking and was re-introduced, driving the expression of the gusA gene, by particle bombardment, giving fifteen independent transgenic lines. Detailed analysis of the sequence of the 885 bp promoter fragment showed that it contains three prolamin boxes but only one is conserved according to the consensus sequence reported. The AACA/TA motif is present twice in published γ-gliadin promoter sequences. The RY element i.e., CATGCAT or CATGCAC, is also present twice in the published promoter. Transgenic lines were classified as high, medium, and low expressers. The expression of the gusA gene was found only in the seeds of the transgenic lines. GUS staining was first detected in the outer endosperm of the lobes, and then it extended to the whole outer endosperm. GUS staining was not found in the aleurone layer nor in the embryo. The qRT-PCR data confirmed the data obtained by GUS staining. The expression of the gusA gene determined by qRT-PCR for the high expresser line (B281) was 4 and 8 times higher than that of medium (B282) and low (B286) expresser lines, respectively. MALDI/TOF-MS showed that gliadins exhibited different patterns of synthesis during the course of seed maturation. Thus, gliadins with masses higher than 36,000 Da were synthesised within the first 12 days post anthesis while those with masses lower than 36,000 Da were synthesised later. Results of GUS staining, qRT-PCR and MALDI/TOF-MS showed that the γ-gliadin promoter reported in this work could be a good candidate to downregulate wheat gliadins.     

Detailed analysis of the expression of an alpha-gliadin promoter and the deposition of alpha-gliadin protein during wheat grain development

Background and Aims

Alpha-gliadin proteins are important for the industrial quality of bread wheat flour, but they also contain many epitopes that can trigger celiac (cœliac) disease (CD). The B-genome-encoded α-gliadin genes, however, contain very few epitopes. Controlling α-gliadin gene expression in wheat requires knowledge on the processes of expression and deposition of α-gliadin protein during wheat grain development.

Methods

A 592-bp fragment of the promotor of a B-genome-encoded α-gliadin gene driving the expression of a GUS reporter gene was transformed into wheat. A large number of transgenic lines were used for data collection. GUS staining was used to determine GUS expression during wheat kernel development, and immunogold labelling and tissue printing followed by staining with an α-gliadin-specific antibody was used to detect α-gliadin protein deposited in developing wheat kernels. The promoter sequence was screened for regulatory motifs and compared to other available α-gliadin promoter sequences.

Key Results

GUS expression was detected primarily in the cells of the starchy endosperm, notably in the subaleurone layer but also in the aleurone layer. The α-gliadin promoter was active from 11 days after anthesis (DAA) until maturity, with an expression similar to that of a 326-bp low molecular weight (LMW) subunit gene promoter reported previously. An α-gliadin-specific antibody detected α-gliadin protein in protein bodies in the starchy endosperm and in the subaleurone layer but, in contrast to the promoter activity, no α-gliadin was detected in the aleurone cell layer. Sequence comparison showed differences in regulatory elements between the promoters of α-gliadin genes originating from different genomes (A and B) of bread wheat both in the region used here and upstream.

Conclusions

The results suggest that additional regulator elements upstream of the promoter region used may specifically repress expression in the aleurone cell layer. Observed differences in expression regulator motifs between the α-gliadin genes on the different genomes (A and B) of bread wheat leads to a better understanding how α-gliadin expression can be controlled.Key words: Alpha-gliadin, promoter, expression, deposition, wheat, Triticum aestivum, grain development     

Silicon carbide fiber-mediated DNA delivery into cells of wheat (Triticum acstivum L.) mature embryos

We have demonstrated that foreign DNA can be delivered into cells of mature embryos of wheat (Triticum aestivum L.) using silicon carbide fibers (SCF). The highest transient expression of thegusA (GUS) gene was detected when dry embryos were vortexed for 10–30 min in a SCF-DNA solution containing 90–120 g/l of sucrose. Up to 100 (on average 20–40) blue expression units per embryo were observed. Scutellum side and epiblast of the intact wheat embryos are preferentially transformed. When embryos with the coleoptilar tip removed were treated and allowed to germinate, GUS staining was observed in emerging leaf tissues. The potential of this new approach for stable transformation of wheat is under investigation. It has been found that callus tissues induced from the SCF treated embryos contain GUS-expressing sectors one month after treatment.     

Transformation and expression of a stilbene synthase gene of Vitis vinifera L. in barley and wheat for increased fungal resistance

 Transformation of barley and wheat via particle bombardment with a gene derived from Vitis vinifera L. (Vst1 gene) resulted in the expression of the foreign phytoalexin, resveratrol, in the transformed plants. Transgenic barley plants were regenerated from microspores and transgenic wheat plants from immature embryos were both selected on Basta. Stable integration of the gene in the genomes of transgenic barley and wheat plants, as well as their progeny, was analysed by Southern-blot analysis. The induction of the stilbene synthase promoter and the transient expression of stilbene synthase-specific mRNA after induction by wounding and infection were proofed in T₁ and T₂ progeny plants. An enhanced expression of the Vst1 gene under control of the stilbene synthase promoter was observed with enhancer sequences from the cauliflower mosaic virus 35s (CaMV 35s) promoter. The enzyme activity of the stilbene synthase was analysed in T₁ progeny plants. The first pathological results indicated an increased resistance of transgenic barley plants to Botrytis cinerea used as a model experimental system. Received: 5 November 1997 / Accepted: 11 November 1997     

Transgenic barley expressing a fungal xylanase gene in the endosperm of the developing grains

The feasibility of producing plant cell wall polysaccharide-hydrolysing feed enzymes in the endosperm of barley grain was investigated. The coding region of a modified xylanase gene (xynA) from the rumen fungus, Neocallimastix patriciarum, linked with an endosperm-specific promoter from cereal storage protein genes was introduced into barley by Agrobacterium-mediated transformation. Twenty-four independently transformed barley lines with the xylanase gene were produced and analysed. The fungal xylanase was produced in the developing endosperm under the control of either the rice glutelin B-1 (GluB-1) or barley B1 hordein (Hor2-4) promoter. The rice GluB-1 promoter provided an apparently higher expression level of recombinant proteins in barley grain than the barley Hor2-4 promoter in both transient and stable expression experiments. In particular, the mean value for the fungal xylanase activity driven by the GluB-1 promoter in the mature grains of transgenic barley was more than twice that with the Hor2-4 promoter. Expression of the xylanase transgene under these endosperm-specific promoters was not observed in the leaf, stem and root tissues. Accumulation of the fungal xylanase in the developing grains of transgenic barley followed the pattern of storage protein deposition. The xylanase was stably maintained in the grain during grain maturation and desiccation and post-harvest storage. These results indicate that the cereal grain expression system may provide an economic means for large scale production of feed enzymes in the future.     

Method for hull‐less barley transformation and manipulation of grain mixed‐linkage beta‐glucan

Hull‐less barley is increasingly offering scope for breeding grains with improved characteristics for human nutrition; however, recalcitrance of hull‐less cultivars to transformation has limited the use of these varieties. To overcome this limitation, we sought to develop an effective transformation system for hull‐less barley using the cultivar Torrens. Torrens yielded a transformation efficiency of 1.8%, using a modified Agrobacterium transformation method. This method was used to over‐express genes encoding synthases for the important dietary fiber component, (1,3;1,4)‐β‐glucan (mixed‐linkage glucan), primarily present in starchy endosperm cell walls. Over‐expression of the HvCslF6 gene, driven by an endosperm‐specific promoter, produced lines where mixed‐linkage glucan content increased on average by 45%, peaking at 70% in some lines, with smaller increases in transgenic HvCslH1 grain. Transgenic HvCslF6 lines displayed alterations where grain had a darker color, were more easily crushed than wild type and were smaller. This was associated with an enlarged cavity in the central endosperm and changes in cell morphology, including aleurone and sub‐aleurone cells. This work provides proof‐of‐concept evidence that mixed‐linkage glucan content in hull‐less barley grain can be increased by over‐expression of the HvCslF6 gene, but also indicates that hull‐less cultivars may be more sensitive to attempts to modify cell wall composition.     

fist: an Arabidopsis mutant with altered cell division planes and radial pattern disruption during embryogenesis

A T-DNA-tagged, embryo-defective Arabidopsis thaliana mutant, fist, was identified and shown to exhibit defects in nuclear positioning and cell division orientation beginning at the four-cell stage of the embryo proper. Cell division orientation was randomised, with each embryo exhibiting a different pattern. Periclinal divisions did not occur after the eight-cell embryo proper stage and fist embryos lacked a histologically distinct protoderm layer. Terminal embryos resembled globular-stage embryos, but were a disorganised mass containing 30–100 cells. Some terminal embryos (5%) developed xylem-like elements in outer surface cells, indicating that the fist mutation affects radial pattern. A soybean β-conglycinin seed storage protein gene promoter, active in wild-type embryos from heart stage to maturity, was also active in terminal fist embryos despite their disorganised globular state. This indicated that some pathways of cellular differentiation in fist embryos proceed independently of both organised division plane orientation and normal morphogenesis. Endosperm morphogenesis in seeds containing terminal fist embryos was arrested at one of three distinct developmental stages and appeared unlinked to fist embryo morphogenesis. The β-conglycinin seed storage protein gene promoter, normally active in cellularised wild-type endosperm, was inactive in fist endosperm, indicating abnormal development of fist endosperm at the biochemical level. These data indicate that the fist mutation, either directly or indirectly, results in defects in cell division orientation during the early stages of Arabidopsis embryo development. Other aspects of the fist phenotype, such as defects in endosperm development and radial pattern formation, may be related to abnormal cell division orientation or may occur as pleiotropic effects of the fist mutation. Received: 15 July 1997 / Accepted: 9 September 1997     

The promoter of the asi gene directs expression in the maternal tissues of the seed in transgenic barley

The bifunctional -amylase/subtilisin inhibitor (BASI) is an abundant protein in barley seeds, proposed to play multiple and apparently diverse roles in regulation of starch hydrolysis and in seed defence against pathogens. In the Triticeae, the protein has evolved the ability to specifically inhibit the main group of -amylases expressed during germination of barley and encoded by the amy1 gene family found only in the Triticeae. The expression of the asi gene that encodes BASI has been reported to be controlled by the hormones abscisic acid (ABA) and gibberellic acid (GA). Despite many studies at the gene and protein level, the function of this gene in the plant remains unclear. In this study, the 5-flanking region (1033 bp, 1033-asi promoter) and the 3-flanking region (655 bp) of the asi gene were isolated and characterised. The 1033-asi promoter sequence showed homology to a number of ciselements that play a role in ABA and GA regulated expression of other genes. With a green fluorescent protein gene (gfp) as reporter, the 1033-asi promoter was studied for spatial, temporal and hormonal control of gene expression. The 1033-asi promoter and its deletions direct transient gfp expression in the pericarp and at low levels in mature aleurone cells, and this expression is not regulated by ABA or GA. In transgenic barley plants, the 1033-asi promoter directed tissue-specific expression of the gfp gene in developing grain and germinating grain but not in roots or leaves. In developing grain, expression of gfp was observed specifically in the pericarp, the vascular tissue, the nucellar projection cells and the endosperm transfer cells and the hormones ABA or GA did not regulate this expression. In mature germinating grain gfp expression was observed in the embryo but not in aleurone or starchy endosperm. However, GA induced gfp expression in the aleurone of mature imbibed seeds from which the embryo had been removed. Expression in maternal rather than endosperm tissues of the grain suggests that earlier widespread assumptions that the protein is expressed largely in the endosperm may have been largely based on analysis of mixed grain tissues. This novel pattern of expression suggests that both activities of the protein may be primarily involved in seed defence in the peripheral tissues of the seed.     

The promoter of the wheat puroindoline-a gene (<Emphasis Type="Italic">PinA</Emphasis>) exhibits a more complex pattern of activity than that of the <Emphasis Type="Italic">PinB</Emphasis> gene and is induced by wounding and pathogen attack in rice

Puroindolines form the molecular basis of wheat grain hardness. However, little is known about puroindoline gene regulation. We previously reported that the Triticum aestivum puroindoline-b gene (PinB) promoter directs β-glucuronidase gene (uidA) seed-specific expression in transgenic rice. In this study, we isolated a puroindoline-a gene (PinA), analyzed PinA promoter activity by 5′ deletions and compared PinA and PinB promoters in transgenic rice. Seeds of PinA-1214 and PinB-1063 transgenic plants strongly expressed uidA in endosperm, in the aleurone layer and in epidermis cells in a developmentally regulated manner. The GUS activity was also observed in PinA-1214 embryos. Whereas the PinB promoter is seed specific, the PinA promoter also directed, but to a lower level, uidA expression in roots of seedlings and in the vascular tissues of palea and pollen grains of dehiscent anthers during flower development. In addition, the PinA promoter was induced by wounding and by Magnaporthe grisea. By deletion analysis, we showed that the “390-bp” PinA promoter drives the same expression pattern as the “1214-bp” promoter. Moreover, the “214-bp” PinA promoter drives uidA expression solely in pollen grains of dehiscent anthers. The presence of putative cis-regulatory elements that may be related to PinA expression is discussed from an evolutionary point of view. By electrophoretic mobility shift assay, we showed that putative cis-elements (WUN-box, TCA motifs and as-1-like binding sites) whose presence in the PinA promoter may be related to wounding and/or the pathogen response form complexes with nuclear extracts isolated from wounded wheat leaves.