Anil H. Shirsat

Sequences responsible for the tissue specific promoter activity of a pea legumin gene in tobacco

SummaryMaturing pea cotyledons accumulate large quantities of storage proteins at a specific time in seed development. To examine the sequences responsible for this regulated expression, a series of deletion mutants of the legA major seed storage protein gene were made and transferred to tobacco using the Bin19 disarmed Agrobacterium vector system. A promoter sequence of 97 bp including the CAAT and TATA boxes was insufficient for expression. Expression was first detected in a construct with 549 bp of upstream flanking sequence which contained the the leg box element, a 28 bp conserved sequence found in the legumintype genes of several legume species. Constructs containing-833 and-1203 bp of promoter sequence significantly increased levels of expression. All expressing constructs preserved seed specificity and temporal regulation. The results indicate that promoter sequences between positions-97 and-549 bp are responsible for promoter activity, seed specificity, and temporal regulation of the pea legA gene. Sequences between positions-549 and-1203 bp appear to function as enhancer-like elements, to increase expression.

The Arabidopsis extensin gene is developmentally regulated, is induced by wounding, methyl jasmonate, abscisic and salicylic acid, and codes for a protein with unusual motifs.

Abstract. A single-copy extensin gene (atExt1) has been isolated from Arabidopsis thaliana (L.) Heynh. The deduced amino acid sequence consists of 374 amino acids which are organised into highly ordered repeating blocks in which Ser(Pro)4 and Ser(Pro)3 motifs alternate. Two copies of the Tyr-X-Tyr-Lys motif and 13 copies of the Val-Tyr-Lys motif are present, showing that this extensin may be highly cross-linked, possessing the capacity for both intra and inter-molecular bond formation. The gene atExt1 is normally expressed in the root and is silent in the leaf; wounding reverses this pattern, turning on the gene in the leaf and repressing it in the root. The promoter contains motifs which have been found to activate plant defence genes in response to salicylic acid, abscisic acid and methyl jasmonate; when these compounds are applied to the roots, the atExt1 gene is activated in the leaf.

Tissue-specific expression of a pea legumin gene in seeds of Nicotiana plumbaginifolia

A 3.4-kilobase genomic DNA fragment from Pisum sativum L. containing the LegA gene, which encodes a major legumin storage protein, was transferred to Nicotiana plumbaginifolia using an Agrobacterium tumefaciens strain containing the Bin 19 binary vector system. Northern hybridisation analysis of legA-transformed plants demonstrated that legumin-specific RNA was present in developing seeds but not in developing leaves. Legumin protein was immunologically detected in the mature seeds of legA-transformed plants, and was present as the correct-size protein composed of disulphide-bonded polypeptides. It is concluded that the transferred pea genomic fragment contains all the information necessary for seed-specific expression of the legA gene, and for correct processing of the primary transcript and the precursor legumin protein.

Transformation of Brassica napus L. (oilseed rape) using Agrobacterium tumefaciens and Agrobacterium rhizogenes - a comparison.

Abstract We have produced transgenic oilseed rape plants by two different transformation strategies. The first method utilised an A. rhizogenes binary system comprising of p Ri1855 and p Bin19 plasmids, to induce hairy root proliferation at the cotyledonary nodes. Clonal hairy root explants selected on kanamycin were then used to regenerate whole transgenic rape plants which though exhibiting the hairy root phenotype to varying degrees, were fertile. These plants were outcrossed with “wild type” rape lines and the F1 progeny analysed to show segregation of hairy root and antibiotic resistance genes. In the second method, an A. tumefaciens binary system was used to transform inflorescence stalks of different varieties of rape, using modifications of existing protocols which resulted in more efficient transformation. These modifications included the use of acetosyringone, the use of N. plumbaginifolia feeder cells, the inclusion of Seaplaque agarose in the media, and the postponement of kanamycin selection in the shooting medium for 2–3 weeks. Regenerated shoots were rooted and whole rape plants produced. Putative transgenic plants were subjected to phenotypic and Southern analyses to confirm the presence or absence of the introduced genes. The methods are effective with both Spring and Winter varieties of rape. Details of the two methods are presented and the advantages and disadvantages of each method are discussed.

The unusual Arabidopsis extensin gene atExt1 is expressed throughout plant development and is induced by a variety of biotic and abiotic stresses

We detail the expression of the Arabidopsis thaliana (L.) Heynh. atExt1 extensin gene. atExt1 is normally expressed in roots and inflorescences, and is induced by wounding, exogenously supplied salicylic acid, methyl jasmonate, auxins and brassinosteroids. Northern assays and histochemical analysis of transgenics expressing an atExt1::gus fusion show that this gene is also induced by the brassica pathogen Xanthomonas campestris pv. campestris and that this induction is restricted to tissues close to the site of infection. Expression at regions of abscission and senescence also implicates atExt1 in these important developmental processes.

Promoter regions of the ExtA extensin gene from Brassica napus control activation in response to wounding and tensile stress

To identify controlling cis acting promoter regions in the B. napus extA extensin gene, expression in transgenic tobacco of 5′ −159, −433, −664, −789 and −940 bp promoter truncations linked to the uidA (B-glucuronidase) reporter coding sequence were analysed. The −159 and −433 bp truncations directed non specific expression in all cell types within the plant. An activator region which increased expression levels 10 fold in all cell types was located between −159 to −433 bp. A repressor region was found between −664 to −789 bp; removal of this region resulted in a 15 fold increase in expression. Histochemical analysis showed that transgenics containing the −664, −789 and −940 bp truncations directed expression of the fusion gene only in the phloem. A negative regulatory region located between −433 to −664 bp repressed expression in non-phloem cell types. In areas of the plant subject to tensile stress, the repression exerted by the negative regulatory region was overcome, allowing expression in all cell types. The quantitative repressor and activator regions which controlled absolute expression levels in all cell types were seperate from the negative regulatory region which controlled cell type specific expression in response to tensile stress. A wound responsive region was found to be located between −940 to −3500 bp. Thus, the extA gene is under complex control, being regulated by 4 sets of positively and negatively acting cis regions, which control wound inducibility, activation in response to tensile stress, and quantitative expression levels.

Extensin over‐expression in Arabidopsis limits pathogen invasiveness

SUMMARY The function of the cell wall protein extensin has been the subject of much speculation since it was first isolated over 40 years ago. In order to investigate the role of extensins in plant defence, we used the gain-of-function strategy to generate transgenic Arabidopsis plants over-expressing the EXT1 extensin gene. These were infected with the virulent bacterial pathogen Pseudomonas syringae DC3000 and symptom development was monitored. Lesions on the transgenics were on average five-fold smaller than those on the wild-type, did not increase in area over the time period of infection, accumulated a small bacterial load and showed very little chlorosis outside the lesion boundary. By contrast, lesions on the wild-type were large, spread to over 50% of the leaf area, continued to increase in size over the time course of the infection, accumulated a bacterial load 100-fold higher than that found in the transgenics, and showed a large chlorotic area outside the lesion boundary. SEM of lesions showed no evidence of bacteria at the lesion boundary in the extensin-over-expressing transgenics, whereas bacteria were always seen at the lesion boundary on the wild-type. Analysis of transgenics carrying an EXT1-GUS promoter-reporter fusion showed expression of GUS in a ring around the boundary of the lesion. Basal defences and signal transduction pathways involved in plant defence were not perturbed in the transgenics, as shown by the analysis of the expression of PR1 and PDF1.2 genes. These results show that extensin over-expression limits pathogen invasiveness.

Gene copy number and levels of expression in transgenic plants of a seed specific gene

Abstract Two variants of the seed specific pea legA gene encoding legumin, the major storage protein of pea seeds were transferred to Nicotiana plumbaginifolia plants using the Agrobacterium tumefaciens binary vector Bin 19. A wide variation was observed both in the gene copy number of the introduced genes and in the level of pea legumin synthesised in the seeds of several individual transgenic N. plumbaginifolia plants. The number of gene copies of the introduced legA genes could not be correlated with expression levels in the same plants, suggesting that the variation in expression resulted from other factors, such as chromosomal position effects of the integrated genes in the Nicotiana genome.

A transposon-like structure in the 5′ flanking sequence of a legumin gene from Pisum sativum

SummaryThe legumin storage proteins of Pisum sativum are coded for by a multigene family. An insertion element (Pis1) has been found integrated into the 5′ flanking sequence of the legC legumin seed storage protein gene. This element contains all the sequence features of the CACTA family of insertion elements, has perfect 12 bp inverted repeats at its termini, and generates a target host site duplication upon integration. An 8 bp sequence within the left arm of the insertion element shows perfect homology to a sequence in the legC flanking region. Three stem-loop structures which can be formed within the element have the same stem sequence.

The Brassica napus extA extensin gene is expressed in regions of the plant subject to tensile stresses

The expression of extA, an extensin gene from Brassica napus L. (oilseed rape) was examined in transgenic Nicotiana tabacum L. (tobacco) and untransformed Brassica juncea L. and B. napus tissues. Northern analysis showed that this gene maintained its normal pattern of expression when transferred to tobacco. In transgenic tobacco plants containing an extA promoter/β-glucuronidase coding sequence fusion, expression of extA was detected in the external and internal phloem of the main stem. High expression levels were seen in cortical parenchyma cells at the point where the axillary flowering branch joined the main stem. Expression was greatest in regions where the maximum tensile stress would seem to be exerted on the main stem by the weight of the axillary branch. It was confirmed that this expression pattern was due to tensile stress by using weights to induce expression of the fusion gene in axillary flowering stalks. In B. juncea pods, in-situ hybridisation studies showed that the extensin gene was strongly expressed in cells of the carpel walls within which considerable tensile stresses develop.