Vascular-specific activity of the Arabidopsis carotenoid cleavage dioxygenase 7 gene promoter

Carotenoid cleavage dioxygenases (CCDs) are involved in the production of diverse apocarotenoids including phytohormones, the visual molecules and the aromatic volatile compounds derived from carotenoids. Here, we examined the spatial expression of four of the CCD genes (AtCcd1, 4, 7 and 8) among the nine members of this family in Arabidopsis by RT-PCR. We found that the AtCcd7 gene showed strong expression in seeds. However, the promoter activity of the 1,867-bp 5′-upstream region of this gene exhibited a vascular specificity at all developmental stages throughout the transgenic Arabidopsis plants tested. The strength of the AtCcd7 promoter was also found to be lower than that of the 35S promoter by about 60%. The whole body expression of the β-glucuronidase (GUS) reporter gene driven by the AtCcd7 promoter in Arabidopsis plants was confirmed in different organs by RT-PCR and GUS enzymatic assays. Histochemical GUS staining further revealed that the AtCcd7 promoter has utility in limiting the expression of target genes to the vascular tissues in all plant organs such as the leaf, stem, root, flower and seed.     

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.     

Upstream regulatory regions from the maize Sh1 promoter confer tissue-specific expression of the ,-glucuronidase gene in tomato

A promoter fusion (Sh35) combining upstream regulatory regions from the maize Sh1 promoter with a truncated 35S promoter, Δ9035 (–90 to +8) has been compared with the original Sh1 promoter for its capacity to promote expression of the β-glucuronidase (GUS) gene in stably transformed tomato plants. For both promoters, very faint GUS expression was detected in the vegetative tissues, and no expression was detected in the fruit pericarp tissues. However, in the seed, Sh1 promoted low GUS expression but Sh35 directed 25-fold higher GUS expression. For both constructs, the profile of GUS expression was similar to that of endogenous sucrose synthase activity, but maximal GUS activity was reached 15 days after the peak of sucrose synthase activity. Received: 20 October 1998 / Revision received: 1 December 1998 / Accepted: 14 December 1998     

Effect of an enhanced CaMV 35S promoter and a fruit-specific promoter on uida gene expression in transgenic tomato plants

Summary Two different promoters, a cauliflower mosaic virus (CaMV) 35S promoter with a 5′-untranslated leader sequence from alfalfa mosaic virus RNA4 (designated as CaMV 35S/AMV) and an E-8 fruit-ripening-specific promoter, were compared to evaluate their effects on expression of the uidA reporter gene in transgenic tomato plants. In order to generate sufficient numbers of transgenic tomato plants, both a reliable regeneration system and an efficient Agrobacterium transformation protocol were developed using 8-d-old cotyledons of tomato (Lycopersicon ecsulentum Mill. cv. Swifty Belle). Two sets of constructs, both derivatives of the binary vector pBI121, were used in transformation of tomato whereby the uidA gene was driven either by the CaMV 35S/AMV or the E-8 fruit-ripening-specific promoter. Southern blot hybridization confirmed the stable integration of the chimeric uidA gene into the tomato genome. Fruit and leaf tissues were collected from T₀ and T₁ plants, and assayed for β-glucuronidase (GUS) enzyme activity. As expected, both vegetative and fruit tissues of transgenic plants carrying the uidA gene under the control of CaMV 35S/AMV showed varying levels of GUS activity, while no expression was observed in vegetative tissues of transgenic plants carrying the uidA gene driven by the E-8 promoter. All fruits from transgenic plants produced with both sets of constructs displayed expression of the uidA gene. However, when this reporter gene was driven by the CaMV 35S/AMV, GUS activity levels were significantly higher than when it was driven by the E-8 fruit-specific promoter. The presence/absence of the uidA gene in T₁ plants segregated in a 3∶1 Mendelian ratio.     

Authentic seed-specific activity of the <Emphasis Type="Italic">Perilla</Emphasis> oleosin 19 gene promoter in transgenic <Emphasis Type="Italic">Arabidopsis</Emphasis>

The Perilla (Perilla frutescens L. cv. Okdong) oleosin gene, PfOle19, produces a 19-kDa protein that is highly expressed only in seeds. The activity of the −2,015 bp 5′-upstream promoter region of this gene was investigated in transgenic Arabidopsis plants using the fusion reporter constructs of enhanced green fluorescent protein (EGFP) and β-glucuronidase (GUS). The PfOle19 promoter directs Egfp expression in developing siliques, but not in leaves, stems or roots. In the transgenic Arabidopsis, EGFP fluorescence and histochemical GUS staining were restricted to early seedlings, indehiscent siliques and mature seeds. Progressive 5′-deletions up to the −963 bp position of the PfOle19 promoter increases the spatial control of the gene expression in seeds, but reduces its quantitative levels of expression. Moreover, the activity of the PfOle19 promoter in mature seeds is 4- and 5-fold greater than that of the cauliflower mosaic virus 35S promoter in terms of both EGFP intensity and fluorometric GUS activity, respectively.     

Stability of β-glucuronidase gene expression in transgenic Tricyrtis hirta plants after two years of cultivation

Transgenic plants of Tricyrtis hirta carrying the intron-containing β-glucuronidase (GUS) gene under the control of the CaMV35S promoter have been cultivated for two years. Four independent transgenic plants produced flowers 1–2 years after acclimatization, and all of them contained one copy of the transgene as indicated by inverse polymerase chain reaction (PCR) analysis. All the four transgenic plants showed stable expression of the gus gene in leaves, stems, roots, tepals, stamens and pistils as indicated by histochemical and fluorometric GUS assays, although differences in the GUS activity were observed among different organs of each transgenic plant. No apparent gus gene silencing was observed in transgenic T. hirta plants even after two years of cultivation.     

A strong constitutive gene expression system derived from <Emphasis Type="Italic">ibAGP1</Emphasis> promoter and its transit peptide

To develop a strong constitutive gene expression system, the activities of ibAGP1 promoter and its transit peptide were investigated using transgenic Arabidopsis and a GUS reporter gene. The ibAGP1 promoter directed GUS expression in almost entire tissues including rosette leaf, inflorescence stem, inflorescence, cauline leaf and root, suggesting that the ibAGP1 promoter is a constitutive promoter. GUS expression mediated by ibAGP1 promoter was weaker than that by CaMV35S promoter in all tissue types, but when GUS protein was targeted to plastids with the aid of the ibAGP1 transit peptide, GUS levels increased to higher levels in lamina, petiole and cauline leaf compared to those produced by CaMV35S promoter. The enhancing effect of ibAGP1 transit peptide on the accumulation of foreign protein was tissue-specific; accumulation was high in lamina and inflorescence, but low in root and primary inflorescence stem. The transit peptide effect in the leaves was maintained highly regardless of developmental stages of plants. The ibAGP1 promoter and its transit peptide also directed strong GUS gene expression in transiently expressed tobacco leaves. These results suggest that the ibAGP1 promoter and its transit peptide are a strong constitutive foreign gene expression system for transgenesis of dicot plants.     

Pineapple translation factor SUI1 and ribosomal protein L36 promoters drive constitutive transgene expression patterns in Arabidopsis thaliana

The availability of a variety of promoter sequences is necessary for the genetic engineering of plants, in basic research studies and for the development of transgenic crops. In this study, the promoter and 5′ untranslated regions of the evolutionally conserved protein translation factor SUI1 gene and ribosomal protein L36 gene were isolated from pineapple and sequenced. Each promoter was translationally fused to the GUS reporter gene and transformed into the heterologous plant system Arabidopsis thaliana. Both the pineapple SUI1 and L36 promoters drove GUS expression in all tissues of Arabidopsis at levels comparable to the CaMV35S promoter. Transient assays determined that the pineapple SUI1 promoter also drove GUS expression in a variety of climacteric and non-climacteric fruit species. Thus the pineapple SUI1 and L36 promoters demonstrate the potential for using translation factor and ribosomal protein genes as a source of promoter sequences that can drive constitutive transgene expression patterns.     

The first intron of rice EPSP synthase enhances expression of foreign gene

The first intron (EPI) of rice 5-enolpyruvylshikimate 3-phosphate synthase gene was isolated by PCR from one clone with genomic EPSP synthase gene. Sequence analysis showed that the first intron is 704 bp in length with 36.2% G+C content. To investigate its effect on expression of foreign gene, we inserted the first intron between CaMV35S promoter and β-glucuronidase (GUS) gene. The transient expression results showed that GUS could be expressed effectively with EPI. The GUS activity in transgenic tobacco shows that the EPI can greatly enhance the expression level of β-glucuronidase (P < 0.01) compared with transgenic tobacco without the first intron, and 3-to 6-fold increase in GUS activity in some transgenic tobaccos. Northern blot indicated the first intron was spliced from GUS pre-mRNA, and the steady-state mRNA levels of GUS with EPI in transgenic tobaccos were higher than that in transgenic tobacco without EPI, which suggested that the first intron of EPSP was a non-translated intron.     

Comparative expression of β-glucuronidase with five different promoters in transgenic carrot (<Emphasis Type="Italic">Daucus carota</Emphasis> L.) root and leaf tissues

Tissue-specific patterns and levels of protein expression were characterized in transgenic carrot plants transformed with the β-glucuronidase (GUS) gene driven by one of five promoters: Cauliflower mosaic virus 35S (35S) and double 35S (D35S), Arabidopsis ubiquitin (UBQ3), mannopine synthase (mas2) from Agrobacterium tumefaciens or the rooting loci promoter (rolD) from A. rhizogenes. Five independently transformed carrot lines of each promoter construct were assessed for GUS activity. In leaves, activity was highest in plants with the D35S, 35S and UBQ3 promoters, while staining was weak in plants with the mas2 promoter, and only slight visual staining was present in the leaf veins of plants containing rolD promoter . Strong staining was seen in the lateral roots, including root tips, hairs and the vascular tissues of plants expressing the 35S, D35S and UBQ3. Lateral roots of plants containing the rolD construct also showed staining in these tissues while the mas2 promoter exhibited heightened staining in the root tips. Relatively strong GUS staining was seen throughout the tap root with all the promoters tested.. When GUS expression was quantified, the UBQ3 promoter provided the highest activity in roots of mature plants, while plants with the D35S and 35S promoter constructs had higher activity in the leaves. Although plants containing the mas2 promoter had higher levels of activity compared to the rolD plants, these two promoters were significantly weaker than D35S, 35S and UBQ3. The potential for utilization of specific promoters to target expression of desired transgenes in carrot tissues is demonstrated.     

A sweetpotato SRD1 promoter confers strong root-, taproot-, and tuber-specific expression in Arabidopsis, carrot, and potato

Harvestable, starch-storing organs of plants, such as fleshy taproots and tubers, are important agronomic products that are also suitable target organs for use in the molecular farming of recombinant proteins due to their strong sink strength. To exploit a promoter directing strong expression restricted to these storage organs, we isolated the promoter region (3.0 kb) of SRD1 from sweetpotato (Ipomoea batatas cv. ‘White Star’) and characterized its activity in transgenic Arabidopsis, carrot, and potato using the β-glucuronidase (GUS) gene (uidA) as a reporter gene. The SRD1 promoter conferred root-specific expression in transgenic Arabidopsis, with SRD1 promoter activity increasing in response to exogenous IAA. A time-course study of the effect of IAA (50 μM) revealed a maximum increase in SRD1 promoter activity at 24 h post-treatment initiation. A serial 5′ deletion analysis of the SRD1 promoter identified regions related to IAA-inducible expression as well as regions containing positive and negative elements, respectively, controlling the expression level. In transgenic carrot, the SRD1 promoter mediated strong taproot-specific expression, as evidenced by GUS staining being strong in almost the entire taproot, including secondary phloem, secondary xylem and vascular cambium. The activity of the SRD1 promoter gradually increased with increasing diameter of the taproot in the transgenic carrot and was 10.71-fold higher than that of the CaMV35S promoter. The SRD1 promoter also directed strong tuber-specific expression in transgenic potato. Taken together, these results demonstrate that the SRD1 promoter directs strong expression restricted to the underground storage organs, such as fleshy taproots and tubers, as well as fibrous root tissues.     

Pollen-Specific Expression of <Emphasis Type="Italic">Oryza sativa Indica</Emphasis> Pollen Allergen Gene (<Emphasis Type="Italic">OSIPA</Emphasis>) Promoter in Rice and <Emphasis Type="Italic">Arabidopsis</Emphasis> Transgenic Systems

Earlier, a pollen-specific Oryza sativa indica pollen allergen gene (OSIPA), coding for expansins/pollen allergens, was isolated from rice, and its promoter—upon expression in tobacco and Arabidopsis—was found active during the late stages of pollen development. In this investigation, to analyze the effects of different putative regulatory motifs of OSIPA promoter, a series of 5′ deletions were fused to β-glucuronidase gene (GUS) which were stably introduced into rice and Arabidopsis. Histochemical GUS analysis of the transgenic plants revealed that a 1631 bp promoter fragment mediates maximum GUS expression at different stages of anther/pollen development. Promoter deletions to −1272, −966, −617, and −199 bp did not change the expression profile of the pollen specificity. However, the activity of promoter was reduced as the length of promoter decreased. The region between −1567 and −199 bp was found adequate to confer pollen-specific expression in both rice and Arabidopsis systems. An approximate 4-fold increase in the GUS activity was observed in the pollen of rice when compared to that of Arabidopsis. As such, the OSIPA promoter seems promising for generation of stable male-sterile lines required for the production of hybrids in rice and other crop plants.     

Transgene expression driven by heterologous ribulose-1,5-bisphosphate carboxylase/oxygenase small-subunit gene promoters in the vegetative tissues of apple (Malus pumila Mill.)

It is desirable that the expression of transgenes in genetically modified crops is restricted to the tissues requiring the encoded activity. To this end, we have studied the ability of the heterologous ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) small-subunit (SSU) gene promoters, RBCS3CP (0.8 kbp) from tomato (hycopersion esculentum Mill.) and SRS1P (1.5 kbp) from soybean (Glycine max [h.] Mers.), to drive expression of the β-glucuronidase (gusA) marker gene in apple (Malus pumila Mill.). Transgenic lines of cultivar Greensleeves were produced by Agrobacterium-mediated transformation and the level of gusA expression in the vegetative tissues of young plants was compared with that produced using the cauliflower mosaic virus (CaMV) 35S promoter. These quantitative GUS data were assessed for their relationship to the copy number of transgene loci. The precise location of GUS activity in leaves was identified histochemically. The heterologous SSU promoters were active primarily in the green vegetative tissues of apple, although activity in the roots was noticeably higher with the RBCS3C promoter than with the SRS1 promoter. The mean GUS activity in leaf tissue of the SSU promoter transgenics was approximately half that of plants containing the CaMV 35S promoter. Histochemical analysis demonstrated that GUS activity was localised to the mesophyll and palisade cells of the leaf. The influence of light on expression was also determined. The activity of the SRS1 promoter was strictly dependent on light, whereas that of the RBCS3C promoter appeared not to be. Both SSU promoters would be suitable for the expression of transgenes in green photosynthetic tissues of apple. Received: 15 June 1999 / Accepted: 12 August 1999     

A sterol biosynthetic geneAtCYP51A2 promoter for constitutive and ectopic expression of a transgene in plants

Arabidopsis CYP51A2 (AtCYP51A2) mediates the sterol 14α-demethylation step inde novo sterol biosynthesis, and is constitutively and highly expressed in all plant tissues (Kim et al., 2005). We exploited the molecular features of its expression and the fundamental role of sterol biosynthesis in cells to develop a plant-derived promoter. Our GUS expression analysis between transgenicArabidopsis lines forAtCYP51A2::GUS and35S::GUS revealed that activity of theAtCYP51A2 promoter was comparable to that of the35S promoter, based on enzymatic activities and protein levels. TheAtCYP51A2 promoter was also constitutively active in transgenic tobacco, indicating that 5′ regulatory elements could be conserved amongCYP51 promoters in dicot plants. A homologue ofAtCYP51A2 was identified from rape seed, a crop species closely related toArabidopsis. Its constitutive tissue expression pattern implies that the application of thisAtCYP51A2 promoter is possible for that species. Based on these results, we present a new binary vector system with the plant-derivedAtCYP51A2 promoter, which is able to constitutively and ectopically drive a transgene in various dicotyledonous plants. These two authors are equally contributed to this work.     

Isolation of the Chinese rose sHSP gene promoter and its differential regulation analysis in transgenic Arabidopsis plants

In our previous study, we identified a Rosa chinensis heat shock protein (HSP) gene, RcHSP17.8, which was induced by abiotic stresses, such as high temperature and osmotic stress. To analyze the expression of RcHSP17.8 and the function of cis-acting elements in the promoter region, a 1,910 bp fragment of the upstream sequence of the RcHSP17.8 translation initiation codon and five promoter deletion fragments were fused to a β-glucuronidase (GUS) report gene. These plasmids were transferred to Arabidopsis thaliana via Agrobacterium. GUS staining was seen in all the organs, especially in the vascular tissues after heat treatment. In transgenic Arabidopsis, GUS expression driven by the full length promoter was significantly higher under heat shock, but no GUS activity was detected under other abiotic stresses. Deletion analysis indicated that the region from −178 to −771 was essential for the promoter’s response to high temperature.     

A Comparison of Constitutive Promoters for Expression of Transgenes in Alfalfa (Medicago Sativa)

The activity of constitutive promoters was compared in transgenic alfalfa plants using two marker genes. Three promoters, the 35S promoter from cauliflower mosaic virus (CaMV), the cassava vein mosaic virus (CsVMV) promoter, and the sugarcane bacilliform badnavirus (ScBV) promoter were each fused to the beta-glucuronidase (gusA) gene. The highest GUS enzyme activity was obtained using the CsVMV promoter and all alfalfa cells assayed by in situ staining had high levels of enzyme activity. The 35S promoter was expressed in leaves, roots, and stems at moderate levels, but the promoter was not active in stem pith cells, root cortical cells, or in the symbiotic zones of nodules. The ScBV promoter was active primarily in vascular tissues throughout the plant. In leaves, GUS activity driven by the CsVMV promoter was approximately 24-fold greater than the activity from the 35S promoter and 38-fold greater than the activity from the ScBV promoter. Five promoters, the double 35S promoter, figwort mosaic virus (FMV) promoter, CsVMV promoter, ScBV promoter, and alfalfa small subunit Rubisco (RbcS) promoter were used to control expression of a cDNA from Trichoderma atroviride encoding an endochitinase (ech42). Highest chitinase activity in leaves, roots, and root nodules was obtained in plants containing the CsVMV:ech42 transgene. Plants expressing the endochitinase were challenged with Phoma medicaginis var. medicaginis, the causal agent of spring black stem and leaf spot of alfalfa. Although endochitinase activity in leaves of transgenic plants was 50- to 2650-fold greater than activity in control plants, none of the transgenic plants showed a consistent increase in disease resistance compared to controls. The high constitutive levels of both GUS and endochitinase activity obtained demonstrate that the CsVMV promoter is useful for high-level transgene expression in alfalfa.