Expression analysis suggests novel roles for members of the Pht1 family of phosphate transporters in Arabidopsis

The completion of the Arabidopsis thaliana genome has revealed that there are nine members of the Pht1 family of phosphate transporters in this species. As a step towards identifying the role of this gene family in phosphorus nutrition, we have isolated the promoter regions from each of these genes, and fused them to the reporter genes beta-glucuronidase and/or green fluorescent protein. These chimeric genes have been introduced into A. thaliana, and reporter gene expression has been assayed in plants grown in soil containing high and low concentrations of inorganic phosphate (Pi). Four of these promoters were found to direct reporter gene expression in the root epidermis, and were induced under conditions of phosphate deprivation in a manner similar to previously characterised Pht1 genes. Other members of this family, however, showed expression in a range of shoot tissues and in pollen grains, which was confirmed by RT-PCR. We also provide evidence that the root epidermally expressed genes are expressed most strongly in trichoblasts, the primary sites for uptake of Pi. These results suggest that this gene family plays a wider role in phosphate uptake and remobilisation throughout the plant than was previously believed.     

Dose effect of UV-B irradiation on pollen tube growth and seed-specific promoter activities in irradiated pollen grains of Nicotiana plumbaginifolia

Low doses of UV-B irradiation applied to mature Nicotiana plumbaginifolia pollen grains stimulated pollen tube growth. The most pronounced effect was achieved after 1.5 min of irradiation. Using transgenic N. plumbaginifolia plants expressing the GFP reporter gene under the control of the seed-specific promoters USP (unknown seed protein) or LegB4 (legumin B4) genes, it was shown that these promoters are also inducible by UV-B irradiation of the pollen grains. The improvement of pollen viability and germination by UV-light is discussed with respect to effects on plant flowering and reproduction. Received: 10 November 1999 / Revision accepted: 14 February 2000     

Pollen- and anther-specific chi promoters from petunia: tandem promoter regulation of the chiA gene.

We have analyzed the spatial and temporal activities of chalcone flavanone isomerase (chi) A and B gene promoters from petunia. To study the tandem promoter regulation of chiA, various chiA promoter fragments were fused with the beta-glucuronidase (GUS) reporter gene. Analysis of transgenic plants containing these chimeric genes provided definitive proof that the chiA coding region is regulated by two distinct promoters (designated PA1 and PA2). We also showed that both promoters can function independently and that the chiA PA1 promoter is expressed in limb (epidermal and parenchyma cells), tube (inner epidermal and parenchyma cells), seed (seed coat, endosperm, and embryo), sepal, leaf, and stem. The use of chiA and chiB promoters in the regulation of anther- and pollen-specific gene expression has been studied. By analyzing transgenic plants containing chimeric genes consisting of chiA and B promoter fragments and the GUS reporter gene, we were able to identify a 0.44-kilobase chiA PA2 promoter fragment that drives pollen-specific gene expression and a 1.75-kilobase chiB PB promoter fragment that confers anther-specific (pollen and tapetum cells) expression to the GUS gene.     

Molecular analysis of Arabidopsis endosperm and embryo promoter trap lines: reporter-gene expression can result from T-DNA insertions in antisense orientation, in introns and in intergenic regions, in addition to sense insertion at the 5' end of genes

Random insertions of promoterless reporter genes in genomes are a common tool for identifying marker lines with tissue-specific expression patterns. Such lines are assumed to reflect the activity of endogenous promoters and should facilitate the cloning of genes expressed in the corresponding tissues. To identify genes active in seed organs, plant DNA flanking T-DNA insertions (T-DNAs) have been cloned in 16 Arabidopsis thaliana GUS-reporter lines. T-DNAs were found in proximal promoter regions, 5' UTR or intron with GUS in the same (sense) orientation as the tagged gene, but contrary to expectations also in inverted orientation in the 5' end of genes or in intergenic regions. RT-PCR, northern analysis, and data on expression patterns of tagged genes, compared with the expression pattern of the reporter lines, suggest that the expression pattern of a reporter gene will reflect the pattern of a tagged gene when inserted in sense orientation in the 5' UTR or intron. When inserted in the promoter region, the reporter-gene expression patterns may be restricted compared with the endogenous gene. Among the trapped genes, the previously described nitrate transporter gene AtNRT1.1, the cyclophilin gene ROC3, and the histone deacetylase gene AtHD2C were found. Reporter-gene expression when positioned in antisense orientation, for example, in the SLEEPY1 gene, is indicative of antisense expression of the tagged gene. For T-DNAs found in intergenic regions, it is suggested that the reporter gene is transcribed from cryptic promoters or promoters of as yet unannotated genes.     

Evaluation of tissue specificity and expression strength of rice seed component gene promoters in transgenic rice

Using stable transgenic rice plants, the promoters of 15 genes expressed in rice seed were analysed for their spatial and temporal expression pattern and their potential to promote the expression of recombinant proteins in seeds. The 15 genes included 10 seed storage protein genes and five genes for enzymes involved in carbohydrate and nitrogen metabolism. The promoters for the glutelins and the 13 kDa and 16 kDa prolamins directed endosperm-specific expression, especially in the outer portion (peripheral region) of the endosperm, whilst the embryo globulin and 18 kDa oleosin promoters directed expression in the embryo and aleurone layer. Fusion of the GUS gene to the 26 kDa globulin promoter resulted in expression in the inner starchy endosperm tissue. It should be noted that the 10 kDa prolamin gene was the only one tested that required both the 5' and 3' flanking regions for intrinsic endosperm-specific expression. The promoters from the pyruvate orthophosphate dikinase (PPDK) and ADP-glucose pyrophosphorylase (AGPase) small subunit genes were active not only in the seed, but also in the phloem of vegetative tissues. Within the seed, the expression from these two promoters differed in that the PPDK gene was only expressed in the endosperm, whereas the AGPase small subunit gene was expressed throughout the seed. The GUS reporter gene fused to the alanine aminotransferase (AlaAT) promoter was expressed in the inner portion of the starchy endosperm, whilst the starch branching enzyme (SBE1) and the glutamate synthase (GOGAT) genes were mainly expressed in the scutellum (between the endosperm and embryo). When promoter activities were examined during seed maturation, the glutelin GluB-4, 26 kDa globulin and 10 kDa and 16 kDa prolamin promoters exhibited much higher activities than the others. The seed promoters analysed here exhibited a wide variety of activities and expression patterns, thus providing many choices suitable for various applications in plant biotechnology.     

Deletion analysis of pollen-expressed promoters

Summary We have used microprojectile bombardment of tobacco pollen to study the DNA sequences involved in the expression of pollen-expressed genes. Promoter-reporter gene fusions constructed with the promoters of three different pollen-expressed genes from tomato (LAT52, LAT56, and LAT59) and either theβ-glucuronidase or luciferase reporter genes were assayed by bombarding hydrated tobacco pollen with the gene constructs precipitated onto tungsten microprojectiles. Reporter gene expression can be assayed within 30 min, with the maximal level of expression between 6 and 12 h after bombardment. By constructing and assaying promoter deletion derivatives, we have been able to delimit regions of the promoters that are necessary for high level expression in pollen. We also demonstrate that results with this transient expression system parallel the expression levels seen in pollen from stably transformed transgenic plants. The microprojectile bombardment assay can be used to rapidly test constructs for pollen expression beforeAgrobacterium-mediated plant transformation. Furthermore, it may be possible to adapt the microprojectile bombardment technique to achieve stable transformation of pollen. Presented in the Session-in-Depth Genetic Transformation and Genetic Analysis Using Microprojectile Bombardment at the 41st Annual Meeting of the Tissue Culture Association, Houston, Texas, June 10–13, 1990. This work was supported by the U.S. Department of Agriculture, Washington, DC, ARS CRIS 5335-22230-002-00D, and by the NSF Center for Plant Developmental Biology, UC-Berkeley, DIR-8719933.     

Promoters of nuclear‐encoded respiratory chain Complex I genes from Arabidopsis thaliana contain a region essential for anther/pollen‐specific expression

Regulatory promoter regions responsible for the enhanced expression in anthers and pollen are defined in detail for three nuclear encoded mitochondrial Complex I (nCI) genes from Arabidopsis thaliana. Specific regulatory elements were found conserved in the 5′ upstream regions between three different genes encoding the 22 kDa (PSST), 55 kDa NADH binding (55 kDa) and 28 kDa (TYKY) subunits, respectively. Northern blot analysis and transgenic Arabidopsis plants carrying progressive deletions of the promoters fused to the β-glucuronidase (GUS) reporter gene by histochemical and fluorimetric methods showed that all three promoters drive enhanced expression of GUS specifically in anther tissues and in pollen grains. In at least two of these promoters the –200/–100 regions actively convey the pollen/anther-specific expression in gain of function experiments using CaMV 35S as a minimal promoter. These nCI promoters thus contain a specific regulatory region responding to the physiological demands on mitochondrial function during pollen maturation. Pollen-specific motifs located in these regions appear to consist of as little as seven nucleotides in the respective promoter context.     

Expression pattern and activity of six glutelin gene promoters in transgenic rice

The shortage of strong endosperm-specific expression promoters for driving the expression of recombinant protein genes in cereal endosperm is a major limitation in obtaining the required level and pattern of expression. Six promoters of seed storage glutelin genes (GluA-1, GluA-2, GluA-3, GluB-3, GluB-5, and GluC) were isolated from rice (Oryza sativa L.) genomic DNA by PCR. Their spatial and temporal expression patterns and expression potential in stable transgenic rice plants were examined with beta-glucuronidase (GUS) used as a reporter gene. All the promoters showed the expected spatial expression within the endosperm. The GluA-1, GluA-2, and GluA-3 promoters directed GUS expression mainly in the outer portion (peripheral region) of the endosperm. The GluB-5 and GluC promoters directed GUS expression in the whole endosperm, with the latter expressed almost evenly throughout the whole endosperm, a feature different from that of other rice glutelin gene promoters. The GluB-3 promoter directed GUS expression solely in aleurone and subaleurone layers. Promoter activities examined during seed maturation showed that the GluC promoter had much higher activity than the other promoters. These promoters are ideal candidates for achieving gene expression for multiple purposes in monocot endosperm but avoid promoter homology-based gene silencing. The GluC promoter did not contain the endosperm specificity-determining motifs GCN4, AACA, and the prolamin-box, which suggests the existence of additional regulatory mechanism in determining endosperm specificity.     

The 5[prime] Flanking Regions of Vicilin and Napin Storage Protein Genes Are Down-Regulated by Desiccation in Transgenic Tobacco

Drying of seeds, when imposed prematurely, elicits a switch in metabolism; events unique to development, such as synthesis of storage protein, are terminated, whereas syntheses associated with germination and growth are initiated. To determine the role of desiccation in down-regulating the expression of genes for storage proteins, the desiccation responsiveness of the 5[prime] and 3[prime] regulatory regions of the genes encoding the pea storage protein vicilin and the Brassica napus storage protein napin was tested in transgenic tobacco seed. Chimeric genes were introduced into tobacco; these genes consisted of the coding region of the reporter gene for [beta]-glucuronidase (GUS) and 5[prime] and/or 3[prime] regions from the vicilin or napin genes or, as controls, the same regions derived from constitutively expressed genes, presumed to be desiccation insensitive. In transgenic seed expressing the gene constructs containing the vicilin or napin promoters, GUS activities declined during late seed development, and more dramatically, after imbibition of mature dry seed or prematurely dried seed. In contrast, GUS activities increased after seed rehydration when the constitutive viral promoter replaced the storage-protein gene 5[prime] region. Transient expression assays support the hypothesis that premature drying down-regulates the expression of the storage-protein gene promoter. Following desiccation, this region may become insensitive to positive controlling factors; alternatively, changes to trans-acting factors may occur.     

Spatial and temporal expression of the response regulators ARR22 and ARR24 in Arabidopsis thaliana

ARR22 (At3g04280) is a novel Type A response regulator whose function in Arabidopsis is unknown. RT-PCR analysis has shown that expression of the gene takes place in flowers and developing pods with the tissues accumulating different proportions of splice variants. Spatial analysis of expression, using ARR22::GUS plants as a marker, has revealed that the reporter protein accumulates specifically at the junction between the funiculus and the chalazal tissue. Expression can be up-regulated at this location by wounding the developing seed. A detailed analysis has failed to detect ARR22 expression at any other sites and, to support this assertion, the only evidence for tissue ablation in ARR22::Barnase plants is during seed development, with the consequence that embryo growth is attenuated. Ectopic expression of ARR22, driven by either the CaMV 35S or the pea plastocyanin (PPC) promoters, resulted in the generation of plants exhibiting extremely stunted root and shoot growth. No viable progeny could be isolated from the PPC::ARR22 transgenic lines. An RT-PCR analysis of a recently annotated gene (ARR24-At5g26594), that exhibits 66% amino acid similarity to ARR22, has shown that expression is also predominantly in floral and silique tissues. Examination of ARR24::GUS plants has revealed that the activity of the promoter is primarily restricted to pollen grains indicating that this gene is unlikely to display an overlapping function with ARR22. Analyses of individual KO lines of either ARR22 or ARR24 have failed to identify a mutant phenotype under the growth conditions employed and the double knockout ARR22/ARR24 line is also indistinguishable from wild-type plants. These results are discussed in the light of the proposed role of response regulators in plant growth and development.     

Induction and expression of seed-specific promoters in Arabidopsis embryo-defective mutants

In order to assess the importance of morphogenesis on the induction of promoter markers for storage and Lea programmes, advantage was taken of the emb mutations producing embryos arrested at a wide range of developmental stages in Arabidopsis. These embryos are viable during their stage of developmental arrest and continue to divide further, but apparently without further differentiation into the main organs and tissues of the normal embryos. Eight independent emb mutants arrested in their development prior to the cotyledon stage were selected. These emb embryos lack the normal morphology of the wild-type embryos when the synthesis of storage and Lea proteins are normally initiated. The 2S1-uidA chimeric gene, representative of the maturation programme and the Em 1-uidA chimeric gene, representative of the desiccation programme were introduced by crosses into the emb background. In the eight emb lines, the expression of the GUS reporter gene directed by the 2S1 and Em 1 promoters was observed in the aborted seeds irrespective of their stage of developmental arrest. The time of induction of the expression of both promoters was the same in the arrested embryos as compared with the normal embryos within the same silique. Thus, the activation of these two promoters is triggered by the same signal and can occur in the absence of morphogenesis. However, in the absence of normal organ formation, the expression of the reporter gene under the control of the 2S1 and Em 1 promoters was evident throughout the whole seed tissues. In normal seed development, the hormone abscisic acid (ABA) activates the promoters of the 2S1 and Em 1 genes. One of the important members of the signal transduction pathway of ABA is the ABI3 protein. It has been shown previously that this protein is a prerequisite for the induction of Em 1 by ABA in seeds. A good correlation with the expression of the ABI3 promoter and the 2S1 and Em 1 promoters was found in emb seeds tissues. This observation suggests that the promoters of the 2S1 and the Em 1 genes are expressed in the mutant seeds not at a basal level, but are probably induced by ABA, as in normal seed development.     

A Novel Non-wounding Transient Expression Assay for Cereals Mediated by Agrobacterium tumefaciens

A novel Agrobacterium tumefaciens-mediated transient expression assay (AmTEA) was developed for young plants of different cereal species and the model dicot Arabidopsis thaliana. AmTEA was evaluated using five promoters (six constructs) and two reporter genes, gus and egfp. The constitutive 35S promoter and the promoter of the rice glutaredoxin gene showed gus and egfp expression in the cereals analyzed in the present study. A promoter for the DEAD-box RNA helicase family protein gene from Arabidopsis showed similar expression patterns of reporter genes in stable transgenic lines as well as in transient expression lines of Arabidopsis. Agrobacterium tumefaciens co-cultivation and plant incubation times were optimized using 35S and the rice expressed protein gene promoter (R2-273). The possibility of non-specific expression of the reporter genes was ruled out by using the antibiotic carbenicillin and the comparison of expression of the reporter genes driven by full-length and truncated R2-273 promoters. AmTEA considerably reduced time, space, labor, and cost requirements. Ease of use with stress treatments is another major advantage of this method. AmTEA can be automated and used for large-scale studies to decipher promoter and gene functions with the ultimate goal to enhance the performance of cereal crops against biotic and abiotic stresses.     

Optimized expression of dual reporter genes in transient transfection of purified Toxoplasma gondii using different promoters

Fluorescent protein and luciferase genes are valuable reporter genes and have been widely used for noninvasive monitoring of gene expression in living tissues and cells. We tested expression of the dual reporter genes in transient transfection of purified Toxoplasma gondii tachyzoites. Two copies of the enhanced yellow fluorescent protein (EYFP) gene were put under the control of 3 representative T.?gondii promoters (GRA1, SAG1, and DHFR). Fluorescence from each EYFP reporter was significantly higher than that from a green fluorescent protein (GFP) reporter. The GRA1-EYFP reporter gave the highest fluorescence. Although both fluorescence and luciferase were expressed in the dual reporter system, the luciferase reporter was more efficient than either the EYFP or GFP reporters, and it required fewer parasites to be successfully used.     

Vicilin and Napin Storage-Protein Gene Promoters Are Responsive to Abscisic Acid in Developing Transgenic Tobacco Seed but Lose Sensitivity following Premature Desiccation

In transgenic tobacco (Nicotiana tabacum L.) seed, expression of chimeric [beta]-glucuronidase (GUS) genes containing the vicilin or napin storage-protein gene promoters is responsive to premature drying and declines upon rehydration (L. Jiang, W.L. Downing, C.L. Baszczynski, A.R. Kermode [1995] Plant Physiol 107: 1439-1449). Desiccation may cause changes in the content of or sensitivity to abscisic acid (ABA), partially or wholly removing the effects of this "modulator" of developmental gene expression. We studied the enhancement of GUS reporter enzyme activities in transgenic tobacco by exogenous ABA before and after drying. A racemic mixture of ABA at 10 [mu]M caused a 2- to 3-fold increase in GUS activity in developing transgenic seed expressing chimeric genes containing the vicilin or napin gene promoters. However, when these seeds were prematurely dried prior to their culture on ABA medium, enhancement of GUS activity was virtually abolished. Use of optically pure ABAs revealed that the enhancement in GUS activity in developing fresh seed was due largely to the natural (+) form of ABA. Chimeric constructs containing a viral 35S promoter did not respond to ABA whether or not premature drying was applied. Thus, vicilin and napin chimeric genes, initially sensitive to ABA, become relatively insensitive to the hormone following drying. A decline in ABA sensitivity may be an important factor in the cessation of storage-protein gene expression.     

Comparing constitutive promoters using CAT activity in transgenic tobacco plants

The effectiveness of different promoters for use in transgenic tobacco was compared using a reporter gene expressing chloramphenicol acetyl transferase (CAT). Plasmids with CAT gene controlled by cauliflower mosaic virus 35S (CaMV 35S), rice actin1 (Ract1) and tobacco polyubiquitin (Tubi.u4) promoters were delivered into tobacco plants by Agrobacterium-mediated transformation. The Ract1 promoter, previously shown to be a strong promoter in rice and other monocots, failed to promote strong expression in tobacco. CAT expression was greatest from the vector carrying Tubi.u4 with a 5'UTR and leader intron without a ubiquitin monomer. In transgenic plants harboring the Tubi.u4 promoter, CAT expression was approximately twice that of the CaMV 35S promoter. Our results suggest that foreign genes under the control of a ubiquitin promoter devoid of monomer will be useful for high-level gene expression in tobacco.