GUS Staining Patterns Research Articles

Confirmation of 'Pollen- and Seed-Specific Gene Deletor' System Efficiency for Transgene Excision from Transgenic Nicotiana tabacum under Field Conditions.

The commercial application of genetically modified plants has been seriously impeded by public concern surrounding the potential risks posed by such plants to the ecosystem and human health. Previously, we have developed a 'pollen- and seed-specific Gene Deletor' system that automatically excised all transgenes from the pollen and seeds of greenhouse-grown transgenic Nicotiana tabacum. In this study, we conducted seven field experiments over three consecutive years to evaluate the stability of transgene excision under field conditions. Our results showed that transgenes were stably excised from transgenic Nicotiana tabacum under field conditions with 100% efficiency. The stability of transgene excision was confirmed based on PCR, as well as the GUS staining patterns of various organs (roots, leaves, petiole, stem, flower, fruit, and seeds) from transgenic N. tabacum. In six transgenic lines (D4, D10, D31, D56, and D43), the transgenes were stably deleted in the T0 and T1 generations. Thus, the 'Gene Deletor' system is an efficient and reliable method to reduce pollen- and seed-mediated unintentional gene flow. This system might help to alleviate the food safety concerns associated with transgenic crops.

A petal-specific InMYB1 promoter from Japanese morning glory: a useful tool for molecular breeding of floricultural crops.

Production of novel transgenic floricultural crops with altered petal properties requires transgenes that confer a useful trait and petal-specific promoters. Several promoters have been shown to control transgenes in petals. However, all suffer from inherent drawbacks such as low petal specificity and restricted activity during the flowering stage. In addition, the promoters were not examined for their ability to confer petal-specific expression in a wide range of plant species. Here, we report the promoter of InMYB1 from Japanese morning glory as a novel petal-specific promoter for molecular breeding of floricultural crops. First, we produced stable InMYB1_1kb::GUS transgenic Arabidopsis and Eustoma plants and characterized spatial and temporal expression patterns under the control of the InMYB1 promoter by histochemical β-glucuronidase (GUS) staining. GUS staining patterns were observed only in petals. This result showed that the InMYB1 promoter functions as a petal-specific promoter. Second, we transiently introduced the InMYB1_1 kb::GUS construct into Eustoma, chrysanthemum, carnation, Japanese gentian, stock, rose, dendrobium and lily petals by particle bombardment. GUS staining spots were observed in Eustoma, chrysanthemum, carnation, Japanese gentian and stock. These results showed that the InMYB1 promoter functions in most dicots. Third, to show the InMYB1 promoter utility in molecular breeding, a MIXTA-like gene function was suppressed or enhanced under the control of InMYB1 promoter in Arabidopsis. The transgenic plant showed a conspicuous morphological change only in the form of wrinkled petals. Based on these results, the InMYB1 promoter can be used as a petal-specific promoter in molecular breeding of floricultural crops.

High-Efficiency Stable Transformation of the Model Fern Species Ceratopteris richardii via Microparticle Bombardment

Ferns represent the most closely related extant lineage to seed plants. The aquatic fern Ceratopteris richardii has been subject to research for a considerable period of time, but analyses of the genetic programs underpinning developmental processes have been hampered by a large genome size, a lack of available mutants, and an inability to create stable transgenic lines. In this paper, we report a protocol for efficient stable genetic transformation of C. richardii and a closely related species Ceratopteris thalictroides using microparticle bombardment. Indeterminate callus was generated and maintained from the sporophytes of both species using cytokinin treatment. In proof-of-principle experiments, a 35S::β-glucuronidase (GUS) expression cassette was introduced into callus cells via tungsten microparticles, and stable transformants were selected via a linked hygromycin B resistance marker. The presence of the transgene in regenerated plants and in subsequent generations was validated using DNA-blot analysis, reverse transcription-polymerase chain reaction, and GUS staining. GUS staining patterns in most vegetative tissues corresponded with constitutive gene expression. The protocol described in this paper yields transformation efficiencies far greater than those previously published and represents a significant step toward the establishment of a tractable fern genetic model.

MAPKs regulate root growth by influencing auxin signaling and cell cycle-related gene expression in cadmium-stressed rice

This work aims to analyze the relationship between root growth, mitogen-activated protein kinase (MAPK), auxin signaling, and cell cycle-related gene expression in cadmium (Cd)-stressed rice. The role of MAPKs in auxin signal modification and cell cycle-related gene expression during root growth was investigated by disrupting MAPK signaling using the MAPKK inhibitor PD98059 (PD). Treatment with Cd caused a significant accumulation of Cd in the roots. A Cd-specific probe showed that Cd is mainly localized in the meristematic zone and vascular tissues. Perturbation of MAPK signaling using PD significantly suppressed root system growth under Cd stress. The transcription of six MAPK genes was inhibited by Cd compared to the control. Detection using DR5-GUS transgenic rice showed that the intensity and distribution pattern of GUS staining was similar in roots treated with PD or Cd, whereas in Cd plus PD-treated roots, the GUS staining pattern was similar to that of the control, which indicates a close association of MAPK signaling with auxin homeostasis under control and Cd stress conditions. The expression of most key genes of auxin signaling, including OsYUCCA, OsPIN, OsARF, and OsIAA, and of most cell cycle-related genes, was negatively regulated by MAPKs under Cd stress. These results suggest that the MAPK pathway plays specific roles in auxin signal transduction and in the control of the cell cycle in response to Cd stress. Altogether, MAPKs take part in the regulation of root growth via auxin signal variation and the modified expression of cell cycle-related genes in Cd-stressed rice. A working model for the function of MAPKs in rice root systems grown under Cd stress is proposed.

Analysis of cellulose synthase (CesA) promoter function in trees using Induced Somatic Sector Analysis (ISSA)

Detailed knowledge of the tissue specificity of gene expression is of central importance not only for our understanding of developmental processes during wood formation, but also is a prerequisite for the deliberate manipulation of xylogenesis candidate genes. Today, much of our knowledge about specific gene expression is based on annual model plants in part because perennial tree systems are often seen as too cumbersome for detailed promoter studies. Here we address this issue in a novel way with the investigation of a number of well characterised cellulose synthase (CesA) genes in the stems of perennial tree species using the in vivo Induced Somatic Sector Analysis (ISSA) system. Three primary and four secondary cell wall associated CesA gene promoters from Eucalyptus grandis and Arabidopsis thaliana were previously isolated and cloned in front of the GUS reporter gene then, using Agrobacterium-mediated transformation, transformed into cambial initials in stems of eucalypt and poplar plants. A CAMV35S promoter::GUS vector was also used as a control. After a period of growth, stems were harvested and GUS-staining patterns were analysed and interpreted. Results from this work show that in eucalypts the staining patterns are consistent with observations in other species whereas in poplar conflicting results were observed. These findings and the general utility of ISSA for these studies are discussed.

The scutellar vascular bundle–specific promoter of the wheat HD‐Zip IV transcription factor shows similar spatial and temporal activity in transgenic wheat, barley and rice

An HD-Zip IV gene from wheat, TaGL9, was isolated using a Y1H screen of a cDNA library prepared from developing wheat grain. TaGL9 has an amino acid sequence distinct from other reported members of the HD-Zip IV family. The 3' untranslated region of TaGL9 was used as a probe to isolate a genomic clone of the TaGL9 homologue from a BAC library prepared from Triticum durum L. cv. Langdon. The full-length gene containing a 3-kb-long promoter region was designated TdGL9H1. Spatial and temporal activity of TdGL9H1 was examined using promoter-GUS fusion constructs in transgenic wheat, barley and rice plants. Whole-mount and histochemical GUS staining patterns revealed grain-specific expression of TdGL9H1. GUS expression was initially observed between 3 and 8 days after pollination (DAP) in embryos at the globular stage and adjacent to the embryo fraction of the endosperm. Expression was strongest in the outer cell layer of the embryo. In developed wheat and barley embryos, strong activity of the promoter was only detected in the main vascular bundle of the scutellum, which is known to be responsible for the uptake of nutrients from the endosperm during germination and the endosperm-dependent phase of seedling development. Furthermore, this pattern of GUS staining was observed in dry seeds several weeks after harvesting but quickly disappeared during imbibition. The promoter of this gene could be a useful tool for engineering of early seedling vigour and protecting the endosperm to embryo axis pathway from pathogens during grain desiccation and storage.

Jasmonate response of the Nicotiana tabacum agglutinin promoter in Arabidopsis thaliana

NICTABA is a carbohydrate-binding protein (also called lectin) that is expressed in several Nicotiana species after treatment with jasmonates and insect herbivory. Analyses with tobacco lines overexpressing the NICTABA gene as well as lines with reduced lectin expression have shown the entomotoxic effect of NICTABA against Lepidopteran larvae, suggesting a role of the lectin in plant defense. Until now, little is known with respect to the upstream regulatory mechanisms that are controlling the expression of inducible plant lectins. Using Arabidopsis thaliana plants stably expressing a promoter-β-glucuronidase (GUS) fusion construct, it was shown that jasmonate treatment influenced the NICTABA promoter activity. A strong GUS staining pattern was detected in very young tissues (the apical and root meristems, the cotyledons and the first true leaves), but the promoter activity decreased when plants were getting older. NICTABA was also expressed at low concentrations in tobacco roots and expression levels increased after cold treatment. The data presented confirm a jasmonate-dependent response of the promoter sequence of the tobacco lectin gene in Arabidopsis. These new jasmonate-responsive tobacco promoter sequences can be used as new tools in the study of jasmonate signalling related to plant development and defense.

Quantitative expression analysis of selected transcription factors in pavement, basal and trichome cells of mature leaves from Arabidopsis thaliana

Gene expression levels of several transcription factors from Arabidopsis thaliana that were described previously to be involved in leaf development and trichome formation were analysed in trichome, basal and pavement cells of mature leaves. Single cell samples of these three cells types were collected by glass micro-capillaries. Real-time reverse transcription (RT)-PCR was used to analyse expression patterns of the following transcription factors: MYB23, MYB55, AtHB1, FILAMENTOUS FLOWER (FIL)/YABBY1 (YAB1), TRIPTYCHON (TRY) and CAPRICE (CPC). A difference in the expression patterns of TRY and CPC was revealed. Contrary to the CPC expression pattern, no transcripts of TRY could be detected in pavement cells. FIL/YAB1 was exclusively expressed in trichome cells. AtHB1 was highly expressed throughout all three cell types. MYB55 was higher expressed in basal cells than in trichome and pavement cells. MYB23 showed a pattern of low expression in pavement cells, medium in basal cells and high expression in trichomes. Expression patterns obtained by single cell sampling and real-time RT-PCR were compared to promoter GUS fusions of the selected transcription factors. Therefore, we regenerated two transgenic Arabidopsis lines that expressed the GUS reporter gene under control of the promoters of MYB55 and YAB1. In conclusion, despite their function in leaf morphogenesis, all six transcription factors were detected in mature leaves. Furthermore, single cell sampling and promoter GUS staining patterns demonstrated the predominant presence of MYB55 in basal cells as compared to pavement cells and trichomes.

Investigating glutamate receptor-like gene co-expression in Arabidopsis thaliana.

There is increasing evidence of the important roles of glutamate receptors (GLRs) in plant development and in adaptation to stresses. However, the studies of these putative ion channels, both in planta and in Xenopus oocytes, may have been limited by our lack of knowledge of possible GLR heteromer formation in plants. We have developed a modification of the single-cell sampling technique to investigate GLR co-expression, and thus potential heteromer formation, in single cells of Arabidopsis thaliana leaves. Micro-EXpression amplification (MEX) has allowed us to amplify gene transcripts from a single cell, enabling expression of up to 100 gene transcripts to be assayed. We measured, on average, the transcripts of five to six different AtGLRs in a single cell. However, no consistent patterns of co-expression or cell-type-specific expression were detected, except that cells sampled from the same plant showed similar expression profiles. The only discernible feature was the detection of AtGLR3.7 in every cell examined, an observation supported by GUS staining patterns in plants stably expressing promoter::uidA fusions. In addition, we found AtGLR3.7 expression in oocytes induces a Ba2+-, Ca2+- and Na+-permeable plasma membrane conductance.

Alanine aminotransferase catalyses the breakdown of alanine after hypoxia in Arabidopsis thaliana

Alanine aminotransferase (AlaAT) catalyses the reversible transfer of an amino group from glutamate to pyruvate to form 2-oxoglutarate and alanine. The regulation of AlaAT in several plant species has been studied in response to low-oxygen stress, light and nitrogen application. In this study, induction of Arabidopsis AlaAT1 and AlaAT2 during hypoxia was observed at the transcriptional level, and an increase in enzyme activity was detected in hypoxically treated roots. In addition, the tissue-specific expression of AlaAT1 and AlaAT2 was analysed using promoter:GUS fusions. The GUS staining patterns indicated that both AlaAT genes are expressed predominantly in vascular tissues. We manipulated AlaAT expression to determine the relative importance of this enzyme in low-oxygen stress tolerance and nitrogen metabolism. This was done by analysing T-DNA mutants and over-expressing barley AlaAT in Arabidopsis. The AlaAT1 knockout mutant (alaat1-1) showed a dramatic reduction in AlaAT activity, suggesting that AlaAT1 is the major AlaAT isozyme in Arabidopsis. Over-expression of barley AlaAT significantly increased the AlaAT activity in the transgenic plants. These plants were analysed for metabolic changes over a period of hypoxic stress and during their subsequent recovery. The results showed that alaat1-1 plants accumulate more alanine than wild-type plants during the early phase of hypoxia, and the decline in accumulated alanine was delayed in the alaat1-1 line during the post-hypoxia recovery period. When alanine was supplied as the nitrogen source, alaat1-1 plants utilized alanine less efficiently than wild-type plants did. These results indicate that the primary role of AlaAT1 is to break down alanine when it is in excess. Therefore, AlaAT appears to be crucial for the rapid conversion of alanine to pyruvate during recovery from low-oxygen stress.

Gene Trapping in Arabidopsis Reveals Genes Involved in Vascular Development

The procambium is made up of stem cells that give rise to various vascular cells in plants. To understand the molecular nature of procambium cells, we tried to identify genes that characterize procambium cells using Arabidopsis gene trap lines. Among 26,000 gene trap lines, we found 67 lines in which beta-glucuronidase (GUS) staining occurred along vascular tissues in cotyledons and/or adult leaves. Although four gene trap lines showed procambium-preferential GUS expression, their expression patterns differed from each other during procambium development in root tips and young rosette leaves. Genomic regions flanking the gene trap insertion points in 25 of the 67 lines were determined, including three lines showing preferential GUS staining of the procambium. The three procambium-related genes encoded PINHEAD, katanin and an unknown DUF740 domain-containing protein. We discuss procambium development based on the functions and the differential GUS staining patterns of the procambium-related genes.

Distinct and overlapping roles of two gibberellin 3‐oxidases in Arabidopsis development

Gibberellin (GA) 3-oxidase, a class of 2-oxoglutarate-dependent dioxygenases, catalyzes the conversion of precursor GAs to their bioactive forms, thereby playing a direct role in determining the levels of bioactive GAs in plants. Gibberellin 3-oxidase in Arabidopsis is encoded by a multigene family consisting of at least four members, designated AtGA3ox1 to AtGA3ox4. It has yet to be investigated how each AtGA3ox gene contributes to optimizing bioactive GA levels during growth and development. Using quantitative real-time PCR analysis, we have shown that each AtGA3ox gene exhibits a unique organ-specific expression pattern, suggesting distinct developmental roles played by individual AtGA3ox members. To investigate the sites of synthesis of bioactive GA in plants, we generated transgenic Arabidopsis that carried AtGA3ox1-GUS and AtGA3ox2-GUS fusions. Comparisons of the GUS staining patterns of these plants with that of AtCPS-GUS from previous studies revealed the possible physical separation of the early and late stages of the GA pathway in roots. Phenotypic characterization and quantitative analysis of the endogenous GA content of ga3ox1 and ga3ox2 single and ga3ox1/ga3ox2 double mutants revealed distinct as well as overlapping roles of AtGA3ox1 and AtGA3ox2 in Arabidopsis development. Our results show that AtGA3ox1 and AtGA3ox2 are responsible for the synthesis of bioactive GAs during vegetative growth, but that they are dispensable for reproductive development. The stage-specific severe GA-deficient phenotypes of the ga3ox1/ga3ox2 mutant suggest that AtGA3ox3 and AtGA3ox4 are tightly regulated by developmental cues; AtGA3ox3 and AtGA3ox4 are not upregulated to compensate for GA deficiency during vegetative growth of the double mutant.

Agrobacterium-mediated transformation of dormant lateral buds in poplar trees reveals developmental patterns in secondary stem tissues.

In an attempt to devise a method for the rapid creation of somatic transgenic wood sectors of sufficient size that would allow us to detect and analyse altered wood characteristics within them, we have explored the manual wounding and subsequent infection with Agrobacterium of dormant lateral buds in poplar. Following treatment and transformation with a 35S-GUS construct, frequent stable transformation was found in the form of distinct and specific GUS staining patterns in the outer cortex, cambial region (including primary and secondary xylem and phloem) and pith. Sector frequency and size were consistent with anatomical features of dormant lateral buds at the time of manual wounding and Agrobacterium-infection. The suitability of somatic sector analysis for functional genomic studies as well as for studies investigating pattern formation and the developmental fate of various cell-types within poplar stems is discussed.

Transformation of cambial tissue in vivo provides an efficient means for induced somatic sector analysis and gene testing in stems of woody plant species.

Large-scale functional analysis of genes and transgenes suspected to be involved in wood development in trees is hindered by long generation times, low transformation and regeneration efficiencies and difficulties with phenotypic assessment of traits, especially those that appear late in a tree's development. To avoid such obstacles many researchers have turned to model plants such as Arabidopsis thaliana (L.) Heynh., Zinnia elegans Jacq. and Nicotiana ssp., or have focused their attention on in vitro wood formation systems or in vivo approaches targeting primary meristems for transformation. Complementing such efforts, we report the use of Agrobacterium to introduce transgenes directly into cambial cells of glasshouse-grown trees in order to create transgenic somatic tissue sectors. These sectors are suitable for phenotypic evaluation and analysis of target gene function. In our experiments the wood formation zone containing the cambium of Eucalyptus, Populus and Pinus species of varying age was inoculated with Agrobacterium containing a CaMV 35S::GUS construct. Following an initial wound response, frequent and stable transformation was observed in the form of distinct GUS-staining patterns (sectors) in newly formed secondary tissues. Sector size and extent depended on the cell type transformed, the species and the length of time treated plants were allowed to grow (more than two years in some cases). Induced somatic sector analysis (ISSA) can now be efficiently used to study cell fate and gene function during secondary growth in stems of forest tree species.

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.

Spatial and temporal expression of the orchid floral homeotic gene DOMADS1 is mediated by its upstream regulatory regions.

The orchid floral homeotic gene, DOMADSI, is a marker gene specifically expressed in the transitional shoot apical meristem during floral transition in Dendrobium Madame Thong-In. DOMADSI is not detectable in vegetative tissues except a weak expression in the stem. Its transcript is uniformly localized in both of the inflorescence meristem and floral primordia, and later expressed in almost all of the floral organs. We isolated and sequenced a 3.5 kb DOMADSI promoter fragment upstream of the transcription start site, demonstrating the location of several putative DNA-binding sites, through which MADS-box and class I knox genes may modulate the DOMADSI expression. To gain insight into the molecular basis of the regulation of DOMADS1, deletion analysis of the DOMADSI::beta-glucuronidase (GUS) gene fusions was performed by means of the stable orchid transformation systems. The study shows that the full-length upstream promoter sequence confers the same spatial and temporal GUS staining pattern as that of the distribution of DOMADSI RNA during orchid development. We also identified the distinct cis-acting regulatory regions required for the control of DOMADS1 expression in vegetative and reproductive tissues, as well as the shoot apical meristem during floral transition.

Analysis of a dehiscence zone endo-polygalacturonase in oilseed rape (Brassica napus) and Arabidopsis thaliana: evidence for roles in cell separation in dehiscence and abscission zones, and in stylar tissues during pollen tube growth.

The oilseed rape (Brassica napus) endo-polygalacturonase (endo-PG) RDPG1 is involved in middle lamella breakdown during silique opening. We investigated tissue-specific expression of RDPG1 in transgenic Arabidopsis thaliana. Cellular localization of endo-PG protein in Arabidopsis siliques was determined by immuno-electron microscopy. An Arabidopsis orthologue, ADPG1, was isolated and aligned with the sequence of RDPG1. The proximal 5' sequences as well as introns are largely conserved. Analysis of the histological GUS-staining pattern of two RDPG1 promoter-GUS (beta-glucuronidase) constructs in transgenic Arabidopsis revealed that the conserved proximal part of the 5'-flanking region directs expression in dehiscence zones of siliques and anthers, floral abscission zones and stylar tissues during pollen tube growth, branch points between stems and pedicel and expression associated with the apical meristem of seedlings, while the distal part of the RDPG1 5'-flanking region contains elements involved in vascular-associated expression in petals, cotyledons and roots. Subsequent RT-PCR analysis, on RNA from the corresponding rape tissues, confirms the staining pattern revealed in transgenic Arabidopsis, thereby justifying the use of Arabidopsis as a reliable model system for analysis of oilseed rape regulatory sequences.

Identification and localization of transformed cells in agrobacterium tumefaciens-induced plant tumors

Agrobacterium tumefaciens-induced tumors of dicotyledonous plants consist of well-defined vascular bundle-like structures originating from transformed cells. The current view that 25% of the tumor cells are transformed has been re-investigated by using beta-glucuronidase (gus)-gene-containing wild-type bacteria (A281 p35S gus-int). Regularly growing stem and leaf tumors showed irregular GUS-staining patterns in the different plant species, Ricinus communis L., Cucurbita maxima L., Vicia faba L. and Kalanchoe daigremontiana Hamet et Perrier. Variable staining and inconsistency between staining and tumor growth suggested an inhibition of gus expression. By polymerase chain reaction (PCR) and reverse transcriptase-PCR analyses it became evident that gus is also integrated into the DNA of unstainable tumor parts but not expressed. These results and area calculations of tissues unable to contain the bacterial transferred-DNA with gus provide strong evidence that in A. tumefaciens-induced tumors most cells, or even all, are transformed, i.e. ca. 100%.

Molecular dissection of the AGAMOUS control region shows that cis elements for spatial regulation are located intragenically.

AGAMOUS (AG) is an Arabidopsis MADS box gene required for the normal development of the internal two whorls of the flower. AG RNA accumulates in distinct patterns early and late in flower development, and several genes have been identified as regulators of AG gene expression based on altered AG RNA accumulation in mutants. To understand AG regulatory circuits, we are now identifying cis regulatory domains by characterizing AG::beta-glucuronidase (GUS) gene fusions. These studies show that a normal AG::GUS staining pattern is conferred by a 9.8-kb region encompassing 6 kb of upstream sequences and 3.8 kb of intragenic sequences. Constructs lacking the 3.8-kb intragenic sequences confer a GUS staining pattern that deviates both spatially and temporally from normal AG expression. The GUS staining patterns in the mutants for the three negative regulators of AG, apetala2, leunig, and curly leaf, showed the predicted change of expression for the construct containing the intragenic sequences, but no significant change was observed for the constructs lacking this intragenic region. These results suggest that intragenic sequences are essential for AG regulation and that these intragenic sequences contain the ultimate target sites for at least some of the known regulatory molecules.

Isolation and expression in transgenic tobacco and rice plants, of the cassava vein mosaic virus (CVMV) promoter.

The cassava vein mosaic virus (CVMV) is a double stranded DNA virus which infects cassava plants (Manihot esculenta Crantz) and has been characterized as a plant pararetrovirus belonging to the caulimovirus subgroup. Two DNA fragments, CVP1 of 388 nucleotides from position -368 to +20 and CVP2 of 511 nucleotides from position -443 to +72, were isolated from the viral genome and fused to the uidA reporter gene to test promoter expression. The transcription start site of the viral promoter was determined using RNA isolated from transgenic plants containing the CVMV promoter:uidA fusion gene. Both promoter fragments were able to cause high levels of gene expression in protoplasts isolated from cassava and tobacco cell suspensions. The expression pattern of the CVMV promoters was analyzed in transgenic tobacco and rice plants, and revealed that the GUS staining pattern was similar for each construct and in both plants. The two promoter fragments were active in all plant organs tested and in a variety of cell types, suggesting a near constitutive pattern of expression. In both tobacco and rice plants, GUS activity was highest in vascular elements, in leaf mesophyll cells, and in root tips.