Expression of two heterologous promoters, Agrobacterium rhizogenes rolC and cauliflower mosaic virus 35S, in the stem of transgenic hybrid aspen plants during the annual cycle of growth and dormancy

We monitored, for the first time, the activity of two model heterologous promoters, the Agrobacterium rhizogenes rolC and the cauliflower mosaic virus (CaMV) 35S, throughout the annual cycle of growth and dormancy in a perennial species, hybrid aspen. Each promoter was fused to the uidA -glucuronidase (GUS) reporter gene and the constructs were introduced into the hybrid aspen genome by Agrobacterium-mediated transformation. Both wildtype and transgenic plants were cultivated under different regimes of photoperiod and temperature to induce passage through one growth-dormancy-reactivation cycle, and at intervals GUS staining was assessed in stem sections. In rolC::uidA transformants, GUS activity in rapidly growing current-year shoots was not only tissue-specific, being localized to the phloem, but also cell-specific at the shoot base, where it was present only in the companion cells. However, during the onset of dormancy induced by short photoperiod, GUS activity shifted laterally from the phloem to include the cortex and pith. After subsequent exposure to chilling temperatures to induce the transition between the dormancy stages of rest and quiescence, GUS activity almost disappeared from all stem tissues, but regained its original phloem specificity and intensity after the shoots were reactivated by exposing them to long photoperiod and high temperatures. In contrast, GUS activity in the stem of 35S::uidA transformants was strong in all tissues except for the vascular cambium and xylem, and did not vary in intensity during the growth-dormancy-reactivation cycle. The lateral shift and increased intensity of GUS activity in the stem of rolC::uidA transformants during dormancy induction was shown to be associated with the accumulation of starch, and to be mimicked by incubating stem sections in sucrose, as well as glucose and fructose, but not sorbitol, prior to the GUS assay. Our results demonstrate that the activities of the rolC and 35S promoters varied in very different, unpredictable ways during the annual cycle of growth and dormancy in a perennial species, and indicate that the spatial and temporal variation in rolC promoter activity that we observed in the stem of transgenic hybrid aspen plants is attributable to cellular and seasonal changes in sucrose content.     

The Brassica napus extA promoter: a novel alternative promoter to CaMV 35S for directing transgene expression to young stem tissues and load bearing regions of transgenic apple trees (Malus pumila Mill.)

The Brassica napus extensin A gene is highly expressed in root tissue of oilseed rape. In an attempt to identify an effective root-specific promoter for biotechnological applications, we have examined the ability of the –940 extA promoter to drive expression of the gusA reporter gene in the vegetative tissues of apple (Malus pumila Mill cv. Greensleeves). Transgenic apple lines were produced by Agrobacterium tumefaciens-mediated transformation and GUS activity was analysed both quantitatively and qualitatively. The extA promoter was active in all tissues of young plants in all 15 clones examined. However Southern blot data suggested that only a proportion of the population contained the entire promoter and that others had suffered deletions of unknown length. This may have contributed to the variation seen in the quantitative and qualitative expression of GUS. Specific GUS activity was highest in the stems where it approached, and in some clones, exceeded that using the constitutive CaMV 35S promoter. Histochemical analysis confirmed that GUS was localised to tissues involved in structural support of the stem. Staining was particularly intense at nodal junctions where high tensile stress is exerted on the tissues. Maturing phloem tissues showed localisation of expression to the phloem parenchyma cells and phloem fibres. Transverse sections of the root revealed staining of primary procambial tissues including the young endodermis but no staining was seen in the cortex. Although the –940 extA promoter is clearly not root-specific in apple, it is likely to have useful biotechnological applications in tree species.     

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.     

The Promoter of a Metallothionein-Like Gene from the Tropical Tree Casuarina Glauca is Active in Both Annual Dicotyledonous and Monocotyledonous Plants

A chimeric gene consisting of the -glucuronidase (gusA) reporter gene under the control of the metallothionein-like promoter cgMT1 from the tropical tree Casuarina glauca was introduced into Nicotiana tabacum via Agrobacterium tumefaciens and into Oryza sativa by particle bombardment. The strongest histochemical staining for GUS activity was observed in the root system of the transgenic plants, and especially in lateral roots. In contrast, a relatively low level of reporter gene expression was seen in the aerial tissues and GUS staining was located mainly in the plant vascular system. The average ratio of GUS activity between root and leaf was found to be 13:1 in tobacco and 1.5:1 in rice. The pattern of cgMT1 promoter activity in floral organs was found to be different in tobacco and rice. High levels of gusA gene expression were detected in the ovules, pollen grains and tapetum, whereas in rice PcgMT1 directs expression to the vascular system of the floral organs. These results suggest that PcgMT1 is potentially useful in molecular breeding to express genes of interest whose products are preferentially needed in roots.     

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     

Optimization of biolistic bombardment parameters for <Emphasis Type="Italic">Dendrobium</Emphasis> Sonia 17 calluses using GFP and GUS as the reporter system

Genetic transformation system of Dendrobium Sonia 17 was optimized using green fluorescent protein (GFP) and -glucuronidase (GUS) gene as the reporter systems. The 35S-sgfp-TYG-nos (p35S) and pSMDFR, carrying sgfp and gusA gene, respectively, were co-bombarded into the calluses. Parameters optimized were acceleration pressure, target distance, gold particle size, pre-bombardment cultured time, plasmid DNA precipitation, total plasmid DNA and the ratio of the plasmids co-bombarded. Both reporter systems responded similarly to the bombardment parameters investigated. Based on the GUS/GFP spot counts, the GFP expression rate was higher than that for GUS under the control of the same promoter, CaMV 35S. GFP could be used as the reporter system for the co-bombardment as it was rapid and non-destructive system to monitor the transformed tissues. A combination of GFP and antibiotic resistance gene was used to select stable putative transformants.     

Inheritance of gusA and neo genes in transgenic rice

Inheritance of foreign genes neo and gusA in rice (Oryza sativa L. cv. IR54 and Radon) has been investigated in three different primary (T₀) transformants and their progeny plants. T₀ plants were obtained by co-transforming protoplasts from two different rice suspension cultures with the neomycin phosphotransferase II gene [neo or aph (3) II] and the -glucuronidase gene (uidA or gusA) residing on separate chimeric plasmid constructs. The suspension cultures were derived from callus of immature embryos of indica variety IR54 and japonica variety Radon. One transgenic line of Radon (AR2) contained neo driven by the CaMV 35S promoter and gusA driven by the rice actin promoter. A second Radon line (R3) contained neo driven by the CaMV 35S promoter and gusA driven by a promoter of the rice tungro bacilliform virus. The third transgenic line, IR54-1, contained neo driven by the CaMV 35S promoter and gusA driven by the CaMV 35S.Inheritance of the transgenes in progeny of the transgenic rice was investigated by Southern blot analysis and enzyme assays. Southern blot analysis of genomic DNA showed that, regardless of copy numbers of the transgenes in the plant genome and the fact that the two transgenes resided on two different plasmids before transformation, the introduced gusA and neo genes were stably transmitted from one generation to another and co-inherited together in transgenic rice progeny plants derived from self-pollination. Analysis of GUS and NPT II activities in T₁ to T₂ plants provided evidence that inheritance of the gusA and neo genes was in a Mendelian fashion in one plant line (AR2), and in an irregular fashion in the two other plant lines (R3 and IR54-1). Homozygous progeny plants expressing the gusA and neo genes were obtained in the T₂ generation of AR2, but the homozygous state was not found in the other two lines of transgenic rice.     

Promoters for pregenomic RNA of banana streak badnavirus are active for transgene expression in monocot and dicot plants

Two putative promoters from Australian banana streak badnavirus (BSV) isolates were analysed for activity in different plant species. In transient expression systems the My (2105 bp) and Cv (1322 bp) fragments were both shown to have promoter activity in a wide range of plant species including monocots (maize, barley, banana, millet, wheat, sorghum), dicots (tobacco, canola, sunflower, Nicotiana benthamiana, tipu tree), gymnosperm (Pinus radiata) and fern (Nephrolepis cordifolia). Evaluation of the My and Cv promoters in transgenic sugarcane, banana and tobacco plants demonstrated that these promoters could drive high-level expression of either the green fluorescent protein (GFP) or the -glucuronidase (GUS) reporter gene (uidA) in vegetative plant cells. In transgenic sugarcane plants harbouring the Cv promoter, GFP expression levels were comparable or higher (up to 1.06% of total soluble leaf protein as GFP) than those of plants containing the maize ubiquitin promoter (up to 0.34% of total soluble leaf protein). GUS activities in transgenic in vitro-grown banana plants containing the My promoter were up to seven-fold stronger in leaf tissue and up to four-fold stronger in root and corm tissue than in plants harbouring the maize ubiquitin promoter. The Cv promoter showed activities that were similar to the maize ubiquitin promoter in in vitro-grown banana plants, but was significantly reduced in larger glasshouse-grown plants. In transgenic in vitro-grown tobacco plants, the My promoter reached activities close to those of the 35S promoter of cauliflower mosaic virus (CaMV), while the Cv promoter was about half as active as the CaMV 35S promoter. The BSV promoters for pregenomic RNA represent useful tools for the high-level expression of foreign genes in transgenic monocots.     

Promoter of the rolC gene of Agrobacterium rhizogenes can be strongly regulated in glandular cell of transgenic tobacco

A 577-bp promoter segment of Agrobacterium rhizogenes rolC, previously known as the phloem-specific gene expression promoter, was fused to the 5′ end of a reporter gene, β-glucuronidase (GUS), uidA. This rolC-promoter-driven expression of the GUS gene was found to be significantly strong in glandular cells in transgenic tobacco plants. Analysis of this segment of the promoter sequence revealed a myb response element.     

Transgene expression in strawberries driven by a heterologous phloem-specific promoter

Strawberry is susceptible to diseases caused by phytoplasmas, mycoplasma-like prokaryotes restricted to sieve elements in the phloem tissue of infected plants. One strategy to improve strawberry resistance to phytoplasmas involves transgenic expression of anti-microbial peptide genes in phloem. For targeted phloem-specific expression, we constructed a binary vector with an expression cassette bearing the -glucuronidase (GUS) reporter gene (uidA) under control of the Arabidopsis sucrose-H⁺ symporter gene (AtSUC2) promoter. Transgenic strawberry lines were generated with high efficiencies by a modified transformation protocol, which combines the adoption of a 3-day pre-selection period following transformation, and the addition of 10-M thidiazuron to the regeneration medium. Histological GUS activity indicated that the reporter gene was expressed specifically in phloem of leaves, petioles, and roots of transgenic plants. The results suggest that the transformation protocol and the AtSUC2 promoter may be useful for engineering phytoplasma-resistant transgenic strawberries.     

The potato Lhca3.St.1 promoter confers high and stable transgene expression in chrysanthemum,in contrast to CaMV-based promoters

Theenhanced cauliflower mosaic virus 35S (dCaMV) promoter and the potatoLhca3.St.1 promoter were evaluated for their expressionabilities in chrysanthemum. The promoters were fused to the-glucuronidase(GUS) reporter gene with and without flanking matrix-associated regions (MARs).They were transferred into chrysanthemum viaAgrobacterium-mediated transformation. The quantitativeevaluation of GUS activity in a total of 127 independently derivedtransformantsestablished that in chrysanthemum the Lhca3.St.1 promoterwas 175 fold more active in the leaves than the dCaMV promoter was. The latterwas as poor in expression as the single CaMV promoter. The use of suchCaMV-based promoters in the genetic engineering of chrysanthemum should bediscouraged when high levels of transgene expression are desired. No clearinfluence of the presence of MARs was observed on the variability of GUS geneexpression, in contrast to earlier studies in tobacco. This may indicate apossible plant species dependent activity of MAR elements.Lhca3.St.1 promoter-driven GUS activity was relativelyhigher in the stem of chrysanthemum and proved stable over extensive timeperiods. Therefore this potato promoter is attractive to obtain high expressionlevels in chrysanthemum.     

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.     

TheCaMV 35S promoter is highly active on floral organs and pollen of transgenic strawberry plants

We have evaluated the expression of the reporter -glucuronidase (GUS) gene driven by the cauliflower mosaic virus 35S (CaMV 35S) promoter in flowers and pollen from 14 independent transgenic strawberry lines. Of the 14 lines evaluated, 13 (92.8%) showed GUS activity—as estimated by the histochemical GUS assay—in some floral organs, with expression being most common in the flower stem, sepals, petals, ovary and stigma. Ten of these thirteen transgenic lines (77%) showed GUS activity in pollen, although the percentages of positive pollen per flower varied greatly among the different lines. A study of the GUS expression during pollen maturation showed that the (CaMV 35S) promoter showed low expression in pollen from flower buds before anthesis but was activated in mature pollen following anther dehiscence. The percentages of pollen grains that showed GUS activity ranged from 2.1% to 46.3%. These percentages were similar or even higher when mature pollen was stored dry at room temperature for 2 weeks. After 5 weeks of storage, the percentages of GUS-positive pollen decreased in two of the six lines analysed but remained at similar values in the other four lines. GUS activity was also measured in protein extracts of mature pollen by means of the fluorometric GUS assay, with the values obtained ranging from 3.8 mol MU mg protein^–1 h^–1 to 0.26 mol MU mg protein^–1 h^–1. Contrary to the generally held view that the CaMV 35S promoter is virtually silent in pollen, we conclude that it is highly expressed in transgenic strawberry pollen.Abbreviations CaMV 35S Cauliflower mosaic virus promoter - GUS -Glucuronidase (EC 3.2.1.31) - MU 4-Methyl umbelliferone - nos Nopaline synthase promoter - nptII Neomycin phosphotransferase - X-Gluc 5-Bromo-4-chloro-3-indolyl--d-glucuronic acid     

Evaluation of putative seed-specific promoters for Linum usitatissimum

The GUS reporter gene was used to test four different putativeseed-specific promoters in developing and mature seeds, leaves and roots fromlinseed flax (Linum usitatissimum). The promoters testedincluded the regulatory regions of the -ketoacyl-CoA synthase gene (KCS)and the napin protein gene from Brassica napus, thepromoter regions of the 'unknown seed protein' (USP), and a legumin proteingene(LeB4) from Vicia faba and the CaMV 35S promoter (positivecontrol). The promoter-GUS constructs were inserted into L.usitatissimum via Agrobacterium mediatedtransformation, and GUS activity evaluated using histochemical andfluorimetrical assays. All the promoters showed some activity, but only CaMV35S, LeB4 and USP exhibited an expression level high enough to be useful inlinseed flax. Plants with USP-GUS showed the earliest GUS activity at 5 to 6days after flowering (daf) and persisting until 40 daf. Expression of GUS underthe control of the LeB4 promoter was measurable 11 daf and was still detectableat 40 daf. The KCS-GUS construct showed a low level of GUS activity between 14daf and 40 daf. Plants transformed with USP-GUS or LeB4-GUS exhibited a lowlevel of GUS activity in leaves and roots of some of the transformants,indicating the need for generating large numbers of primary transformants,followed by careful evaluation and selection for ones with not only the desiredlevel of expression, but also the desired spatial and temporal expression.     

A repeated chromosomal DNA sequence is amplified as a circular extrachromosomal molecule in rice (Oryza sativa L.)

Summary The regulation in tobacco of the rolB and rolC promoters of Agrobacterium rhizogenes pRi 1855 T_L-DNA was studied by using the -glucuronidase (GUS) reporter system in transgenic plants. A 20- to 100-fold increase of GUS activity was selectively induced by auxin in rolB-GUS transformed mesophyll protoplasts, whereas this auxin-dependent increase was only 5-fold in rolC-GUS protoplasts. Moreover, both gene fusions exhibited similar tissue-specific expression in aerial parts but different patterns in roots. The spatial pattern of rolBGUS expression could be strongly modified by the addition of exogenous auxin, further suggesting that auxin plays a central role in the regulation of the rolB promoter in tobacco. The tissue-specific and auxin-dependent regulation of the rolB promoter is discussed in relation to the effects of the rolB gene on rhizogenesis and on cellular responses to auxin.Abbreviations BA benzoic acid - 6-BAP benzylaminopurine - 2,4-D 2,4-dichlorophenoxyacetic acid - GUS -glucuronidase - 2,4,5-T 2,4,5,-trichlorophenoxyacetic acid - 2,4,6-T 2,4,6-trichlorophenoxyacetic acid - IAA indoleacetic acid - NAA naphthaleneacetic acid - MU 4-methyl umbelliferone - 35S CaMV cauliflower mosaic virus 35S (promoter) - TCA trichloroacetic acid - X-Glu 5-bromo-4chloro-3-indolyl -d-glucuronic acid     

竹节花黄斑驳病毒启动子的缺失分析及功能

竹节花黄斑驳病毒(CoYMV)是侵染单子叶植物竹节花的一 种双链环状DNA病毒,它的启动子可介导外源基因在烟草韧皮部特异表达。为了研究其组织 特异性表达的最佳启动子区域,对CoYMV启动子进行了5′端五种不同长度的缺失分析,用不同长度的启动子片段与GUS基因及NOS3′端转录中止序列构建了全长启动子及5 个缺失启动子序列的六个嵌合GUS基因植物表达载体。利用农杆菌将上述嵌合基因转化烟草 外植体后,每种表达载体都获得了一批转基因烟草植株。转化再生烟草植株的PCR分析、GUS 酶活测定及GUS组织染色的结果表明六种类型的嵌合基因已整合到烟草染色体中,并有五种 表达出GUS活性。缺失到870bp的启动子比全长启动子(1040bp)的活性约高78%,870bp比585bp启动子介导的GUS活性略高但差别不明显,缺失到447和232时GUS活性有明显下 降,但仍具有韧皮部特异表达的特性。当缺失到TATA box附近的44bp时启动子丧失组织特 异性,GUS活性也降低到测不出来的水平。以上结果表明CoYMV启动子从转录起始位点上游 870bp～230bp及232bp下游区分别与启动子的活性和韧皮部组织特异性密切相关,870bp上游可能存在一个负调控序列,所以该启动子的活性和组织特异性的最佳调控区应在87 0bp或585bp的下游区。CoYMV启动子与35S启动子驱动GUS基因在烟草中表达的活性相比, 前者为后者的70%左右,考虑到前者仅在韧皮部细胞表达而后者为组成型表达,所以CoYMV启 动子在韧皮部的活性可能与35S启动子相当或更高。CoYMV启动子在其它转基因植物中驱动外 源基因表达的特点正在研究中。     

Genetic transformation through the use of hyperhydric tobacco meristems

Exposed shoot meristems from normal and hyperhydric (vitrified) tobacco, Nicotiana tabacum, were bombarded with gold particles either coated with plasmid DNA containing neomycin phosphotransferase (NPTII), rolC and -glucuronidase (GUS) genes (plasmid pGA-GUSGFrolC) or left uncoated. Meristems bombarded with uncoated particles were co-cultivated with Agrobacterium tumefaciens strain EHA 101 harboring the binary vector pGA-GUSGFrolC. Whole-plant transformants were produced from 4 of 40 hyperhydric meristems bombarded with uncoated particles followed by co-cultivation with A. tumefaciens. One transgenic plant was obtained from 40 normal, non-hyperhydric meristems treated. Transformation was verified by growth on kanamycin-containing medium, GUS assays, PCR, and Southern analysis. The plants tested through Southern analysis appeared to have 2 or more copies of the transgene insert. Seeds obtained from self-pollination of these transgenic plants segregated 3:1 or 15:1 (kanamycin resistant:sensitive) when germinated on medium containing 100 mg/l kanamycin, indicating transfer of foreign genes through the sexual cycle. Whole-plant transformants were not produced from 50 normal tobacco meristems bombarded with plasmid-coated gold particles and not exposed to engineered A. tumefaciens, but 1 plant of 60 bombarded hyperhydric meristems produced transgenic roots, the result of a chimera. We suggest that hyperhydric meristems are more readily transformed.     

Vascular expression driven by the promoter of a gene encoding a high-affinity potassium transporter HAK5 from <Emphasis Type="Italic">Eucalyptus grandis</Emphasis>

The construction of expression cassettes harboring tissue-specific promoters is a viable alternative to limit transgene expression to specific organs and cell types. In this study, we have functionally characterized the promoter of a Eucalyptus grandis gene encoding a putative high-affinity HAK5-like potassium (K⁺) transporter (designated EgHAK5) showing root-specific expression. The ability of the EgHAK 5′-flanking region (~1.3 kb) to drive root-specific expression of a reporter gene (β-glucuronidase; GUS) was examined using transgenic tobacco plants. Histochemical analysis revealed enhanced GUS staining in the vasculature of leaves, hypocotyls and roots, which was also confirmed in histological cross-sections. Moreover, the relative expression of GUS in the roots of the generated transgenic lines was increased in response to K⁺ starvation. Overall, our results indicate that, in a heterologous system, the EgHAK5 promoter shows expression in vascular tissues, mainly within the phloem, and is up-regulated upon potassium deprivation.