Sucrose-phosphate synthase activity and yield analysis of tomato plants transformed with maize sucrose-phosphate synthase

Sucrose synthesis is a major element of the interactions between photosynthesis and plant growth and development. Tomato (Lycopersicon esculentum Mill. cv. UC82B) plants transformed with maize sucrose-phosphate synthase (SPS; EC 2.3.1.14) expressed from either a ribulose-1,5-bisphosphate carboxylase-oxygenase (Rubisco) small subunit promoter (SSU) or the cauliflower mosaic virus 35S promoter (35S) were used to study effects of increased sucrose synthesis rates on plant growth. The plants were grown in growth chambers, field plots, and open-top chambers. The 35S plants had a 2 to 3-fold increase in young-leaf SPS activity, a 10 to 20-fold increase in young-root SPS activity and no increase in young-fruit SPS activity. The leaf SPS activity in one of the 35S lines fell to control levels by two months of age. The SSU plants had a 4 to 5-fold increase in leaf SPS activity and no significant increase in root or young-fruit SPS activity. One 35S line, which maintained high leaf SPS activity throughout development, yielded 70–80% more than controls at both normal and elevated CO₂ in open-top chambers in the field and 20–30% more than controls in two additional field trials. The other 35S line and the two SSU lines either yielded less or did not differ from controls under several growth conditions. Since only one of four transformed lines showed an increase in yield, we can not yet conclude that increased leaf SPS activity leads to increased yield. However, increased leaf SPS activity appears to result in increased fruit sugar content since all three lines with increased leaf SPS usually also had increased fruit sugars. Received: 18 November 1996 / Accepted: 22 January 1997     

Development of insect resistant transgenic cotton lines expressing cry1EC gene from an insect bite and wound inducible promoter

Transgenic cotton lines were developed for high-level expression of a synthetic cry1EC gene from a wound inducible promoter. The tobacco pathogenesis related promoter PR-1a was modified by placing CaMV35S promoter on its upstream in reverse orientation. The resultant chimeric promoter CaMV35S(r)PR-1a expressed constitutively and was further up-regulated at the site of feeding by insects. It was induced more rapidly by treatment with salicylic acid (SA). The CaMV35S(r)PR-1a cry1EC expressing transgenic lines of cotton showed 100% mortality of Spodoptera litura larvae. The tightly regulated low-level expression of PR-1a was modified to a highly expressing constitutive expression by CaMV35S placed in reverse orientation. Salicylic acid treatment and wounding enhanced the expression further by the chimeric promoter. The leaves expressed more δ-endotoxin around the sites of insect bites. The levels of expression and induction varied among different transgenic lines, suggesting position effect. Some of the transgenic lines that expressed Cry1EC from the chimeric promoter at a low level also showed 100% mortality when induced with salicylic acid. A highly expressing insect bite and wound inducible promoter is desirable for developing insect resistant transgenic plants.     

Effective delivery of a nematode‐repellent peptide using a root‐cap‐specific promoter

The potential of the MDK4‐20 promoter of Arabidopsis thaliana to direct effective transgenic expression of a secreted nematode‐repellent peptide was investigated. Its expression pattern was studied in both transgenic Arabidopsis and Solanum tuberosum (potato) plants. It directed root‐specific β‐glucuronidase expression in both species that was chiefly localized to cells of the root cap. Use of the fluorescent timer protein dsRED‐E5 established that the MDK4‐20 promoter remains active for longer than the commonly used constitutive promoter CaMV35S in separated potato root border cells. Transgenic Arabidopsis lines that expressed the nematode‐repellent peptide under the control of either AtMDK4‐20 or CaMV35S reduced the establishment of the beet cyst nematode Heterodera schachtii. The best line using the AtMDK4‐20 promoter displayed a level of resistance >80%, comparable to that of lines using the CaMV35S promoter. In transgenic potato plants, 94.9 ± 0.8% resistance to the potato cyst nematode Globodera pallida was achieved using the AtMDK4‐20 promoter, compared with 34.4 ± 8.4% resistance displayed by a line expressing the repellent peptide from the CaMV35S promoter. These results establish the potential of the AtMDK4‐20 promoter to limit expression of a repellent peptide whilst maintaining or even improving the efficacy of the cyst‐nematode defence.     

The promoter of rbcS in a C3 plant (rice) directs organ-specific,light-dependent expression in a C4 plant (maize), but does not confer bundle sheath cell-specific expression

The small subunit of ribulose-bisphosphate carboxylase (Rubisco), encoded by rbcS, is essential for photosynthesis in both C3 and C4 plants, even though the cell specificity of rbcS expression is different between C3 and C4 plants. The C3 rbcS is specifically expressed in mesophyll cells, while the C4 rbcS is expressed in bundle sheath cells, and not mesophyll cells. Two chimeric genes were constructed consisting of the structural gene encoding -glucuronidase (GUS) controlled by the two promoters from maize (C4) and rice (C3) rbcS genes. These constructs were introduced into a C4 plant, maize. Both chimeric genes were specifically expressed in photosynthetic organs, such as leaf blade, but not in non-photosynthetic organs. The expressions of the genes were also regulated by light. However, the rice promoter drove the GUS activity mainly in mesophyll cells and relatively low in bundle sheath cells, while the maize rbcS promoter induced the activity specifically in bundle sheath cells. These results suggest that the rice promoter contains some cis-acting elements responding in an organ-pecific and light-inducible regulation manner in maize but does not contain element(s) for bundle sheath cell-specific expression, while the maize promoter does contain such element(s). Based on this result, we discuss the similarities and differences between the rice (C3) and maize (C4) rbcS promoter in terms of the evolution of the C4 photosynthetic gene.     

Novel and useful properties of a chimeric plant promoter combining CaMV 35S and MAS elements

The CaMV 35S and Ti plasmid mannopine synthetase (mas) promoters are commonly used by plant genetic engineers. To combine their useful properties, we constructed hybrid promoters incorporating elements from both. These promoters were spliced to the beta-glucuronidase reporter gene and introduced into tobacco and tomato plants by Agrobacterium cocultivation. T1 and T2 transgenic plant populations transformed with different constructs were assayed for the marker enzyme. Comparisons were made based on the range of expression levels found for each promoter construct. We found that a hybrid promoter incorporating the mas region from +65 to -301 and the 35S enhancer region from -90 to -941 had new and interesting properties. This promoter, called Mac, expressed gus at a level three to five times that expressed by a double 35S promoter in the leaves, and 10 to 15 times in hypocotyls and roots. The Mac promoter, however, showed only marginal wound inducibility. Five- to seven-fold wound induction required the presence of the region from -301 to -613 of mas. Reiteration of the 35S enhancer region, from -90 to -430, behind the 35S TATA box region or the mas +65 to -301 region had a smaller effect on expression, ranging from equal to twice the level of the single enhancer control.     

Evaluation in tobacco of the organ specificity and strength of therolD promoter,domain A of the 35S promoter and the 35S2 promoter

In order to study the expression in plants of therolD promoter ofAgrobacterium rhizogenes, we have constructed chimaeric genes placing the coding region of thegusA (uidA) marker gene under control of tworolD promoter fragments of different length. Similar results were obtained with both genes. Expression studies were carried out in transformed R₁ progeny plants. In mature transformed tobacco plants, therolD-gus genes were expressed strongly in roots, and to much lower levels in stems and leaves. This pattern of expression was transmitted to progeny, though the ratio of the level of expression in roots relative to that in leaves was much lower in young seedlings. The degree of root specificity inrolD-gus transformants was less than that of a gene constructed with domain A of the CaMV 35S promoter,domA-gus, but the level of root expression was much higher than with the latter gene. However, the level of expression of therolD-gus genes was less than that of agus gene with a 35S promoter with doubled domain B, 35S²-gus. TherolD-gus genes had a distinctive pattern of expression in roots, compared to that of the two other genes, with the strongest GUS activity observed in the root elongation zone and in vascular tissue, and much less in the root apex.     

Activation of the Pathogen-Inducible Gst1 Promoter of Potato after Elicitation by Venturia inaequalis and Erwinia amylovora in Transgenic Apple (Malus × Domestica)

Rather than using a constitutive promoter to drive transgenes for resistance against fungal and bacterial diseases in genetic engineering of apple (Malus × domestica) cultivars, a promoter induced only after infection was preferred. The ability of the Pgst1 promoter from potato (Solanum tuberosum L.) to drive expression of the gusA reporter gene was determined in two genotypes of apple: the fruit cultivar Royal Gala and the M.26 rootstock. β-glucuronidase activity in the transgenic lines grown in a growth chamber was determined quantitatively using fluorometric assays and compared to the activity in Cauliflower Mosaic Virus (CaMV) 35S promoter-driven transgenic lines. In both apple genotypes, the Pgst1 promoter exhibited a low level of expression after bacterial and fungal inoculation compared to the level obtained with the PCaMV35S promoter (15% and 8% respectively). The Pgst1 promoter was systematically activated in apple at the site of infection with a fungal pathogen. It was also activated after treatment with salicylic acid, but not after wounding. Taken together, these data show that, although the Pgst1 promoter is less active than the PCaMV35S promoter in apple, its pathogen responsiveness could be useful in driving the expression of transgenes to promote bacterial and fungal disease resistance.     

The cauliflower mosaic virus (CaMV) 35S promoter sequence alters the level and patterns of activity of adjacent tissue- and organ-specific gene promoters

Here we report the effect of the 35S promoter sequence on activities of the tissue- and organ-specific gene promoters in tobacco plants. In the absence of the 35S promoter sequence the AAP2 promoter is active only in vascular tissues as indicated by expression of the AAP2:GUS gene. With the 35S promoter sequence in the same T-plasmid, transgenic plants exhibit twofold to fivefold increase in AAP2 promoter activity and the promoter becomes active in all tissue types. Transgenic plants hosting the ovary-specific AGL5:iaaM gene (iaaM coding an auxin biosynthetic gene) showed a wild-type phenotype except production of seedless fruits, whereas plants hosting the AGL5:iaaM gene along with the 35S promoter sequence showed drastic morphological alterations. RT-PCR analysis confirms that the phenotype was caused by activation of the AGL5:iaaM gene in non-ovary organs including roots, stems and flowers. When the pollen-, ovule- and early embryo-specific PAB5:barnase gene (barnase coding a RNase gene) was transformed, the presence of 35S promoter sequence drastically reduced transformation efficiencies. However, the transformation efficiencies were restored in the absence of 35S promoter, indicating that the 35S promoter might activate the expression of PAB5:barnase in non-reproductive organs such as calli and shoot primordia. Furthermore, if the 35S promoter sequence was replaced with the NOS promoter sequence, no alteration in AAP2, AGL5 or PAB5 promoter activities was observed. Our results demonstrate that the 35S promoter sequence can convert an adjacent tissue- and organ-specific gene promoter into a globally active promoter. Xuelian Zheng and Wei Deng contributed equally to this work and are considered co-first authors.     

Functional properties of the anaerobic responsive element of the maize Adh1 gene

The functional properties of the anaerobic responsive element (ARE) of the maize Adh1 gene have been analysed using a transient expression assay in electroporated maize protoplasts. The ARE functions in both orientations although inversion of the ARE sequence relative to the TATA box element produces slightly weaker promoter activity under anaerobic conditions and elevated expression under aerobic conditions. Promoter activity under anaerobic conditions is proportional to the number of complete ARE sequences in the Adh1 promotor. The ARE contains two sub-regions and dimers of sub-region II are as efficient as the wild-type sequence in activating gene expression under anaerobic conditions. However, sub-region I dimers do not appear capable of inducing gene expression in response to anaerobic stress. We conclude that sub-region II is essential for anaerobic induction of gene expression. Reporter gene expression remains constant when the spacing between sub-regions of the ARE is increased up to at least 64 bp, but increased spacing of 136 bp or greater abolishes expression in both aerobic and anaerobic conditions, indicating that a close association of the two sub-regions is required both for anaerobic responsiveness and for maximal levels of aerobic gene expression. When the ARE is placed upstream of position –90 of the CaMV 35S promoter, the ARE produces a high level of expression in both aerobic and anaerobic conditions. The general enhancement of gene expression driven by the hybrid ARE/35S promoter in aerobic conditions requires an intact sub-region II motif since mutation or deletion of sub-region II from the hybrid promoter reduces the level of expression to that observed for the truncated 35S promoter alone. In addition, mutation of the sub-region I sequences in the ARE/35S hybrid promoter does not significantly reduce expression in aerobic conditions, relative to pARE/35S(-90), suggesting that sub-region I does not contribute to this general enhancer function.     

Improved metabolic action of a bacterial lysine decarboxylase gene in tobacco hairy root cultures by its fusion to arbcS transit peptide coding sequence

The gene of a bacterial lysine decarboxylase (ldc) fused to arbcS transit peptide coding sequence (tp), and under the control of the CaMV 35S promoter, was expressed in hairy root cultures ofNicotiana tabacum. The fusion of theldc to the targeting signal sequence improved the performance of the bacterial gene in the plant cells in many respects. Nearly all transgenic hairy root cultures harbouring the35S-tp-ldc gene contained distinctly higher lysine decarboxylase activity (from 1.5 to 30 pkat LDC per mg protein) than those which had been transformed with constructs in which the gene had been directly cloned behind the CaMV 35S promoter. The higher enzyme activity led to the accumulation of up to 0.7% cadaverine on a dry mass basis. In addition, part of the cadaverine pool was used for increased biosynthesis of anabasine, an alkaloid which was hardly detectable in control cultures. The best line contained anabasine levels of 0.5% dry mass, which could further be enhanced by feeding of lysine.     

Isolation and characterization of a novel plant promoter that directs strong constitutive expression of transgenes in plants

A novel, constitutively expressed gene, designated MtHP, was isolated from the model legume species Medicago truncatula. Sequence analysis indicates that MtHP most likely belongs to the PR10 multi-gene family. The MtHP promoter was fused to a -glucuronidase gene to characterize its expression in different plant species. Transient assay by microprojectile bombardment and hairy root transformation by Agrobacterium rhizogenes revealed GUS expression in leaf, stem, radicle and root in M. truncatula. Detailed analysis in transgenic Arabidopsis plants demonstrated that the promoter could direct transgene expression in different tissues and organs at various developmental stages; its expression pattern was similar to that of CaMV35S promoter, and the level of expression was higher than the reporter gene driven by CaMV35S promoter. Deletion analysis revealed that even a 107 bp fragment of the promoter could still lead to a moderate level of expression. The promoter was further characterized in white clover (Trifolium repens), a widely grown forage legume species. Strong constitutive expression was observed in transgenic white clover plants. Compared with CaMV35S promoter, the level of GUS activity in transgenic white clover was higher when the transgene was driven by MtHP promoter. Thus, the promoter provides a useful alternative to the CaMV35S promoter in plant transformation for high levels of constitutive expression.     

TRANSIENT TRANSFORMATION OF A CHLORARACHNIOPHYTE ALGA,LOTHARELLA AMOEBIFORMIS (CHLORARACHNIOPHYCEAE), WITH uidA AND egfp REPORTER GENES1

A transient genetic transformation system was established for a chlorarachniophyte alga, Lotharella amoebiformis K. Ishida et Y. Hara. We first isolated sequences that contain a putative promoter for a RUBISCO SSU (rbcS) gene and a terminator for another copy of rbcS gene from L. amoebiformis. With those promoter and terminator sequences, we developed two expression vectors, pLaRGus and pLaRGfp, which code uidA and egfp genes, respectively. The cells were then transformed with each vector using a microparticle bombardment system. When the cells were transformed with the pLaRGus, β‐glucuronidase (GUS) staining dyed several cells blue. Green fluorescent protein (GFP) fluorescence was observed in the cells transformed with pLaRGfp. The highest transient transformation efficiency, 35 per 2 × 10⁷ cells, was detected from the GUS staining. This study demonstrates that two reporter genes are expressed in L. amoebiformis cells when rbcS promoter and terminator are used. The conditions of transformation were also optimized. This is the first report of successful genetic transformation in chlorarachniophyte algae.     

Relationship between promoter strength and transformation frequencies using mannose selection for the production of transgenic sugar beet

The relationship between the expression level of the selectable marker gene and transformation frequency was investigated in transgenic sugar beets with five different promoters, a modified cauliflower mosaic virus 35S RNA promoter (E35S), an enhanced nopaline synthase promoter (ENOS), a modified mannopine synthase promoter (SMAS), a heat shock protein promoter (HSP80) and a chlorophyll a/b-binding protein promoter (CAB3), to drive the expression of the selectable marker gene. The selection system employed was based on the Escherichia coli phosphomannose isomerase (PMI) gene as selectable marker gene and mannose as selective agent. The selected transgenic shoots were analysed for PMI activity and the average activity for each promoter was found to be 5.9 (HSP80), 31 (SMAS), 38 (E35S), 49 (ENOS) and 61 (CAB3) mU/mg. The weakest promoter, HSP80, resulted in the lowest transformation frequency (0.30%), suggesting that this promoter was too weak to confer sufficient resistance to mannose. On the other hand, the strongest promoters, ENOS and CAB3, only gave intermediate transformation frequencies, 0.44% and 0.47% respectively, while the somewhat weaker SMAS promoter produced the highest transformation frequency, 0.89%. Thus, these data suggest that the activity of the selectable PMI gene should be above a certain threshold level; however, above this level, no simple correlation between the PMI activities, calculated as averages, and transformation frequencies could be deduced. However, extended data analysis by dividing the transgenic shoots into 4 groups according to their PMI activities (low(<10 mU/mg), medium (10–50 mU/mg), high (50–100 mU/mg) and very high (>100 mU/mg) expressers) revealed a significant positive correlation between the relative number of shoots having medium levels of expression and transformation frequency. This indicated that promoters that predominanthly give rise to intermediate expression levels of the selectable PMI gene result in high transformation frequencies.     

Meristem-specific gene expression directed by the promoter of the S-phase-specific gene,cyc07, in transgenic Arabidopsis

A genomic clone for the cyc07 gene, which is expressed specifically at the S phase during the cell cycle in synchronous cultures of periwinkle (Catharanthus roseus) cells, was isolated. Determination of the nucleotide sequence of the clone revealed that the cyc07 gene consists of seven exons separated by six introns. Genomic Southern analysis indicated that the cyc07 gene is present as a single copy per haploid genome in periwinkle. Expression of related genes was detected in a wide range of other plants. Transgenic Arabidopsis plants were generated that expressed the gene for -glucuronidase (GUS) under the control of the promoter of the cyc07 gene. The tissue-specific pattern of expression directed by the promoter was investigated by analysis of GUS activity. Histochemical tests demonstrated that 589 bp of the 5-upstream sequence of the cyc07 gene could direct specifical expression of the GUS reporter gene in meristematic tissues in transgenic plants. The spatial pattern of expression directed by the promoter was closely correlated with meristematic activity and cell proliferation, suggesting an association between the function of the cyc07 gene and cell proliferation.     

Expression of CaMV35S-GUS gene in transgenic rice plants

Summary To understand the properties of the cauliflower mosaic virus (CaMV) 35S promoter in a monocotyledonous plant, rice (Oryza sativa L.), a transgenic plant and its progeny expressing the CaMV35S-GUS gene were examined by histochemical and fluorometric assays. The histochemical study showed that -glucuronidase (GUS) activity was primarily localized at or around the vascular tissue in leaf, root and flower organs. The activity was also detected in the embryo and endosperm of dormant and germinating seeds. The fluorometric assay of various organs showed that GUS activity in transgenic rice plants was comparable to the reported GUS activity in transgenic tobacco plants expressing the CaMV35S-GUS gene. The results indicate that the level of expression of the CaMV 35S promoter in rice is similar to that in tobacco, a dicotyledonous plant, suggesting that it is useful for expression of a variety of foreign genes in rice plants.