Glucuronidase Reporter Gene Research Articles

Cell specific, cross-species expression of myrosinases in Brassica napus, Arabidopsis thaliana and Nicotiana tabacum.

A prototypical characteristic of the Brassicaceae is the presence of the myrosinase-glucosinolate system. Myrosinase, the only known S-glycosidase in plants, degrades glucosinolates, thereby initiating the formation of isothiocyanates, nitriles and other reactive products with biological activities. We have used myrosinase gene promoters from Brassica napus and Arabidopsis thaliana fused to the beta -glucuronidase (GUS) reporter gene and introduced into Arabidopsis thaliana, Brassica napus and/or Nicotiana tabacum plants to compare and determine the cell types expressing the myrosinase genes and the GUS expression regulated by these promoters. The A. thaliana TGG1 promoter directs expression to guard cells and phloem myrosin cell idioblasts of transgenic A. thaliana plants. Expression from the same promoter construct in transgenic tobacco plants lacking the myrosinase enzyme system also directs expression to guard cells. The B. napus Myr1.Bn1 promoter directs a cell specific expression to idioblast myrosin cells of immature and mature seeds and myrosin cells of phloem of B. napus. In A. thaliana the B. napus promoter directs expression to guard cells similar to the expression pattern of TGG1. The Myr1.Bn1 signal peptide targets the gene product to the reticular myrosin grains of myrosin cells. Our results indicate that myrosinase gene promoters from Brassicaceae direct cell, organ and developmental specific expression in B. napus, A. thaliana and N. tabacum.

Tissue-specific expression of the ?-glucuronidase reporter gene in transgenic strawberry (Fragaria � ananassa) plants

The strawberry (Fragaria spp) is regarded as a false fruit because it originates from the receptacle, which is a non-ovarian tissue. For this reason, fruit-specific promoters isolated from plant species in which the fruit is derived from the ovary wall might not be suited to control gene expression in a fruit-specific way in strawberry. In order to achieve (false) fruit-specific expression in strawberry, we tested the petunia FBP7 (floral binding protein7) promoter, which proved to be active in the receptacles of petunia flowers, in transgenic strawberry fruits. In strawberry plants containing the FBP7 promoter fused to the β-glucuronidase (GUS) reporter gene (gus), GUS activity was found in floral and fruit tissues of all developmental stages tested but not in leaf, petiole and root tissue. Surprisingly, Northern blot analysis showed the presence of gus-derived mRNAs in root (strong) and petiole (weak) tissue of fbp7-gus plants in addition to the floral and fruit tissues. Therefore, it is concluded that the histological GUS phenotype does not necessarily correspond with expression at the mRNA level. mRNA quantification using the TaqMan polymerase chain reaction technology confirmed the Northern results and showed that in red strawberry tissue the cauliflower mosaic virus 35S promoter is at least sixfold stronger than the FBP7 promoter.

The expression of an extensin-like protein correlates with cellular tip growth in tomato.

Extensins are abundant proteins presumed to determine physical characteristics of the plant cell wall. We have cloned a cDNA encoding LeExt1 from a tomato (Lycopersicon esculentum Mill.) root hair cDNA library. The deduced sequence of the LeExt1 polypeptide defined a novel type of extensin-like proteins in tomato. Patterns of mRNA distribution indicated that expression of the LeExt1 gene was initiated in the root hair differentiation zone of the tomato rhizodermis. Cloning of the corresponding promoter and fusion to the -glucuronidase (GUS) reporter gene allowed detailed examination of LeExt1 expression in transgenic tomato plants. Evidence is presented for a direct correlation between LeExt1 expression and cellular tip growth. LeExt1/GUS expression was detectable in trichoblasts (=root hair-bearing cells), but not in atrichoblasts of the tomato rhizodermis. Both hair formation and LeExt1 expression was inducible by the plant hormone ethylene. Comparative analysis of the LeExt1/GUS expression was performed in transgenic tomato, potato (Solanum tuberosum), tobacco (Nicotiana tabacum), and Arabidopsis plants. In the apical/basal dimension, GUS staining was absent from the root cap and undifferentiated cells at the root tip in all species investigated. It was induced at the distal end of the differentiation zone and remained high proximally to the root/hypocotyl boundary. In the radial dimension, GUS expression was root hair specific in the solanaceous species. Whereas LeExt1 mRNA was exclusively detectable in the rhizodermis, root hair-specific expression correlated with GUS expression in germinating pollen tubes. This is correlative evidence for a role of LeExt1 in root hair tip growth [corrected].

A novel methodology to develop a foot and mouth disease virus (FMDV) peptide-based vaccine in transgenic plants

The expression of antigens in transgenic plants has been increasingly used as an alternative to the classical methodologies for antigen expression in the development of experimental vaccines. However, an important limitation in most cases is the low concentration of the recombinant antigens in the plant tissues, which reduces the possibilities of practical applications. Because the site of insertion of the transferred DNA into the cellular chromosomal DNA is at random, different levels of foreign protein expression in independent transformants is expected. Strategies to allow the evaluation of a high number of the transgenic individuals, usually an expensive and very time consuming process, would permit the selection of those plants presenting the highest levels of recombinant protein expression. Here, we present the development of an experimental immunogen based in the expression of a highly immunogenic epitope from foot and mouth disease virus (FMDV) fused to the glucuronidase ( gus A) reporter gene, which allows selection of the transgenic plants by the ß-glucuronidase (ßGUS) enzymatic activity. We produced transgenic plants of alfalfa expressing the immunogenic site between amino acid residues 135–160 of structural protein VP1 (VP135–160), fused to the ßGUS protein. Plants expressing the highest levels of the immunogenic epitope VP135–160, analyzed by Western blot, were efficiently selected based on their levels of ßGUS enzymatic activity. The FMDV epitope expressed in plants was highly immunogenic in mice which developed, after immunization, a strong anti-FMDV antibody response against a synthetic peptide representing the region VP135–160, to native virus VP1, and to purified FMDV particles. Additionally, these mice were completely protected against experimental challenge with the virulent virus. To our knowledge, this constitutes the first report of a peptide-based vaccine produced in transgenic plants that induces a protective immune response when used in experimental hosts. Also, these results demonstrated the possibility of using a novel and simple methodology for obtaining transgenic plants expressing high levels of foreign immunogenic epitopes, which could be directly applied in the development of plant-based vaccines.

Promoters derived from banana bunchy top virus-associated components S1 and S2 drive transgene expression in both tobacco and banana

Potential promoter regions of the Banana bunchy top virus (BBTV)-associated DNA components S1 and S2 were fused to the #-glucuronidase reporter gene and assessed for activity in both tobacco (Nicotiana tabacum cv. Xanthi) and banana (Musa spp. cv. Bluggoe). Transient assays indicated that all the S1- and S2-derived promoters were active and had greater expression in tobacco than banana. In stably transformed tobacco and banana, the S1- and S2-derived promoters directed expression in root meristems and trichomes. The S1 promoter was also expressed in the vascular tissue of leaves and roots, while both the S1 and S2 promoters were active in tobacco leaf trichomes and pollen. In banana, expression was significantly enhanced by the inclusion of the maize polyubiquitin intron 3' to the promoter. Interestingly, there was some evidence to indicate that S1 promoter fragments containing part of the open reading frame at the 5' end of the promoter had enhanced activity, suggesting that promoter elements may not be confined to the non-coding region.

Temperature-responsive genetic loci in the plant pathogen Pseudomonas syringae pv. glycinea The GenBank accession numbers for the nucleotide sequences of mutants 560, 561, 562, 563, 564, 568, 570, 574, 590, 591, 593, 596, 599, 601, 605, 608, 613, 617, 618, 626 and 632 determined in this work are AF274322–AF274342, respectively.

Plant-pathogenic bacteria may sense variations in environmental factors, such as temperature, to adapt to plant-associated habitats during pathogenesis or epiphytic growth. The bacterial blight pathogen of soybean, Pseudomonas syringae pv. glycinea PG4180, preferentially produces the phytotoxin coronatine at 18 degrees C and infects the host plant under conditions of low temperature and high humidity. A miniTn5-based promoterless glucuronidase (uidA) reporter gene was used to identify genetic loci of PG4180 preferentially expressed at 18 or 28 degrees C. Out of 7500 transposon mutants, 61 showed thermoregulated uidA expression as determined by a three-step screening procedure. Two-thirds of these mutants showed an increased reporter gene expression at 18 degrees C whilst the remainder exhibited higher uidA expression at 28 degrees C. MiniTn5-uidA insertion loci from these mutants were subcloned and their nucleotide sequences were determined. Several of the mutants induced at 18 degrees C contained the miniTn5-uidA insertion within the 32.8 kb coronatine biosynthetic gene cluster. Among the other mutants with increased uidA expression at 18 degrees C, insertions were found in genes encoding formaldehyde dehydrogenase, short-chain dehydrogenase and mannuronan C-5-epimerase, in a plasmid-borne replication protein, and in the hrpT locus, involved in pathogenicity of P. syringae. Among the mutants induced at 28 degrees C, insertions disrupted loci with similarities to a repressor of conjugal plasmid transfer, UV resistance determinants, an isoflavanoid-degrading enzyme, a HU-like DNA-binding protein, two additional regulatory proteins, a homologue of bacterial adhesins, transport proteins, LPS synthesis enzymes and two proteases. Genetic loci from 13 mutants did not show significant similarities to any database entries. Results of plant inoculations showed that three of the mutants tested were inhibited in symptom development and in planta multiplication rates. Temperature-shift experiments suggested that all of the identified loci showed a rather slow induction of expression upon change of temperature.

Analysis of a promoter for the NADH - glutamate synthase gene in rice (Oryza sativa): cell type-specific expression in developing organs of transgenic rice plants

This paper originates from a presentation at the International Conference on Assimilate Transport and Partitioning, Newcastle, NSW, August 1999 The entire 3.7 kbp 5´-upstream region (–2840 to +886) from the translational start codon of NADH–glutamate synthase (NADH–GOGAT, EC 1.4.1.14) gene from rice (Oryza sativa L.) or the region sequentially deleted from the 5´-end was fused with the β−glucuronidase (GUS) reporter gene. The chimeric gene was introduced into calli derived from rice scutellum via Agrobacterium tumefaciens-mediated transformation and tissue-specific GUS activity determined in T0 generations. When the entire region was fused, GUS activity was detected in vascular bundles of the developing leaf blade and in dorsal and lateral vascular bundles of developing grains. This corresponds with our previous immunodetection of NADH–GOGAT protein (Hayakawa et al., Planta 193, 455–460, 1994). A series of deletion experiments showed that a 149-nucleotide region between –142 and +7 was essential for promoter activity in the NADH–GOGAT gene.

Biochemical characterization and expression of RLK4, a receptor-like kinase from Arabidopsis thaliana

The Arabidopsis RLK4 gene encodes a Ser/Thr receptor-like protein kinase. A lysine to glutamic acid substitution in an essential subdomain of the RLK4 catalytic domain, results in an inactive protein kinase. Transphosphorylation experiments, using the inactive form of RLK4 as a substrate for an active form, indicated that autophosphorylation could proceed via an intermolecular mechanism. Multiple sites are phosphorylated at the catalytic domain of RLK4, as indicated by two-dimensional (2D) thin-layer electrophoresis and chromatography. To analyze the expression pattern of RLK4, we used the RLK4 promoter to drive the expression of the β- glucuronidase ( GUS) reporter gene in transgenic plants. GUS activity was observed in the shoot apex and roots. In roots, the GUS staining pattern was limited to the lateral roots and at the root-hypocotyl transition zone. Analysis of an RLK4 mutant line did not show any difference in the phenotype with respect to the wild type. The expression of other related RLKs with similar expression patterns suggest there is functional redundancy for RLK4.

Process of tomato root colonization by a pathogenic strain of Fusarium oxysporum f. sp. lycopersici in comparison with a non‐pathogenic strain

A pathogenic strain of Fusarium oxysporum f. sp. lycopersici transformed with the glucuronidase (GUS) reporter gene was used to study the colonization process of tomato (Lycopersicon esculentum) roots in hydroponic culture. The plants were treated exactly as those used in a different study with a non‐pathogenic strain of F. oxysporum in order to compare the two types of colonization process. The pathogenic strain rapidly colonized the root surface, forming a dense network of hyphae – shown by the GUS staining assay – as early as 48 h after inoculation. The first images of the pathogen penetrating into the epidermis of the root were observed 24 h after inoculation. The plant showed defence reactions mainly in the hypodermis, but also in the cortex. Generally these barriers failed to prevent the centripetal growth of the pathogen towards the stele. The GUS activity moved with the actively growing hyphae into the tissues; the stele appeared intensely stained 7 d after inoculation, whereas by this time the hyphae at the root surface did not show any more staining. The hyphae formed a dense network at the apex of the root, but direct contact between hyphae and living cells was prevented by several layers of sloughed cap cells. Although rarely observed, penetration of the pathogen into the apex was possible and led to a rapid destruction of apical cells. The only differences between this pattern of root colonization and that observed for a non‐pathogenic strain of F. oxysporum appear to concern the frequency of dead apices and the intensity of fungal colonization in the cortex. When the pathogen passed around the barrier formed in the hypodermis it always reached the xylem, although this barrier and other defence reactions induced at different levels in the cortex always prevented the non‐pathogenic strain from reaching the stele. These observations suggest that the main differences between the two types of interaction – between the plant and either the pathogenic or the non‐pathogenic strain – are quantitative rather than qualitative.

Characterization of an auxin-inducible 1-aminocyclopropane-1-carboxylate synthase gene, VR-ACS6, of mungbean (Vigna radiata (L.) Wilczek) and hormonal interactions on the promoter activity in transgenic tobacco.

A genomic clone for VR-ACS6, an isozyme of auxin-inducible ACC synthase of mungbean, was isolated, and its promoter activity was examined in transgenic tobacco. The clone contained 1,612 bp long 5' untranscribed region and its coding sequence consisted of three exons and two introns. Genomic Southern hybridization indicated that VR-ACS6 is a single copy gene. The transcription initiation site was a cytosine present at 231-base upstream the translation start codon. The VR-ACS6 promoter contained DNA sequences homologous to various functionally identified auxin-responsive elements. To demonstrate hormonal response of the promoter region, transgenic tobacco plants carrying the 1,719 bp VR-ACS6 promoter/-glucuronidase (GUS) fusion gene were generated. Strong GUS expression occurred by auxin treatment of leaves of T0 transformants and hypocotyls of T1 etiolated seedlings. Magnitude of the response to auxin was dose-dependent, and the increased GUS activity was detected at 0.1 microM and higher concentrations of IAA. Other plant hormones did not induce GUS activity, but greatly modified the response to auxin. Cytokinin enhanced the IAA-induced expression of GUS reporter gene, whereas ABA and ethylene suppressed the expression. These characteristics of VR-ACS6 promoter activity in transgenic tobacco are in good accordance with the expression patterns of the gene in mungbean hypocotyls. Histochemical staining showed that GUS activity was evident in both etiolated and light grown seedings treated with IAA. Cytokinin enhanced the intensity of auxin-induced GUS stain and also expanded the stained area, whereas ABA and ethylene reduced both intensity and area of the stain.

Compatibility of different pollination techniques with microprojectile bombardment of Norway spruce and Scots pine pollen

Transformed pollen could be used in crossings to provide a new approach for producing transgenic seedlings of gymnosperms recalcitrant to tissue culture. The aim of this study was to investigate different pollination techniques in Norway spruce (Picea abies (L.) Karst.) and in Scots pine (Pinus sylvestris L.) using both natural pollen and pollen bombarded with DNA-coated microprojectiles. Cone and seed set after liquid pollinations with natural and bombarded pollen was compared with conventional crossings in which dried natural pollen was used. Liquid pollination proved inferior to conventional crossings. In Norway spruce, the percentages of mature cones and full seeds were lower with liquid pollination than with the conventional method; and in Scots pine, the number of conelets was lower than in pollinations in which dried pollen was used. With bombarded pollen, the results were even lower. To improve possibilities to obtain transgenic progenies, a method was developed that allows the use of bombarded pollen in conventional crossings. This technique is based on dehydration of pollen suspensions and storage at -20°C, which results in a slight decrease in the germination ability of pollen but leads to a high level of the beta -glucuronidase reporter gene expression. This is the first report on transient foreign gene expression in plant material stored in a metabolically inactive form for 1 month before gene expression was assayed.

Activity of β-glucuronidase in Root Tips of Different Types of Transgenic Sugar Beet Plants

Expression of the β-glucuronidase (GUS) reporter gene driven by the CaMV 35S, rolC, nos and mas promoters was assessed in the tips of 12 independent clones of transgenic sugar beet (Beta vulgaris) roots. Three questions were addressed: 1) expression pattern specific for a given promoter, 2) expression pattern variability, and 3) relationship between gene expression and cell differentiation. Characteristic patterns of tissue-specific expression were distinguished for each promoter. Striking differences, however, were found between some clones, bearing the same construct. Statistical analysis of the pattern variability proved that the variability is significantly lower within the construct than between constructs. rolC-GUS clones exhibited the lowest and CaMV 35S clones the highest pattern variability. Comparisons between the four promoters showed consistent GUS activity in areas playing a key role in tissue determination (the elongation zone) where cells switch from frequent mitosis and mostly isodiametrical growth, typical for the promeristem, to rapid elongation and differentiation. All of the clones were highly GUS-positive in the elongation zone of stele. Activity was commonly localised in the stele of the maturation zone for CaMV 35S, rolC and mas-GUS clones. CaMV 35S-GUS clones were highly active in the promeristem.

Complementation of a potato virus X mutant mediated by bombardment of plant tissues with cloned viral movement protein genes.

Microprojectile bombardment was used to examine the transport function of the 25 kDa movement protein (MP) encoded in the triple gene block of potato virus X (PVX). A 25 kDa MP-defective full-length cloned PVX genome carrying a beta-glucuronidase (GUS) reporter gene was co-bombarded with 35S promoter constructs containing either the 25 kDa MP gene of wild-type PVX, the MP gene of either of two tobamoviruses (tomato mosaic virus or crucifer tobamovirus), red clover necrotic mosaic dianthovirus (RCNMV) or brome mosaic bromovirus (BMV). When inoculated alone, the MP-defective PVX was unable to move out of the inoculated cell, as visualized by in situ staining for GUS activity. However, cell-to-cell movement of the mutant PVX genome was restored by co-inoculation with 35S constructs containing the MP cDNA of PVX, either tobamovirus or RCNMV. The BMV MP construct did not complement movement of the defective PVX. These results show that co-bombardment of cDNA of an MP-defective virus with plasmids designed to express MP of other viruses could be used as a fast and simple method for transcomplementation experiments.

The Promoter of the Gene Encoding the C4 Form of Phosphoenolpyruvate Carboxylase Directs Mesophyll-Specific Expression in Transgenic C4 Flaveria spp.

The function of the C4 mechanism of photosynthesis depends on the strict compartmentation of the enzymes involved. Here, we investigate the regulatory mechanisms that ensure the mesophyll-specific expression of the C4 isoform of phosphoenolpyruvate carboxylase. We show that 2 kb of the 5[prime] flanking region of the Flaveria trinervia C4 PpcA1 gene is sufficient to direct mesophyll-specific expression of the [beta]-glucuronidase reporter gene in transgenic F. bidentis (C4) plants. In young leaves of seedlings, the activity of this promoter is dependent on the developmental stage of the mesophyll cells. It is induced in a basipetal fashion (leaf tip to base) during leaf development. The promoter region of the orthologous nonphotosynthetic Ppc gene of F. pringlei (C3) induces reporter gene expression mainly in the vascular tissue of leaves and stems as well as in mesophyll cells of transgenic F. bidentis plants. Our experiments demonstrate that during the evolution of the C4 Flaveria species, cis-acting elements of the C4 Ppc gene must have been altered to achieve mesophyll-specific expression.

Expression of an Aspartate Kinase Homoserine Dehydrogenase Gene Is Subject to Specific Spatial and Temporal Regulation in Vegetative Tissues, Flowers, and Developing Seeds.

Although the regulation of amino acid synthesis has been studied extensively at the biochemical level, it is still not known how genes encoding amino acid biosynthesis enzymes are regulated during plant development. In the present report, we have used the [beta]-glucuronidase (GUS) reporter gene to study the regulation of expression of an Arabidopsis thaliana aspartate kinase-homoserine dehydrogenase (AK/HSD) gene in transgenic tobacco plants. The polypeptide encoded by the AK/HSD gene comprises two linked key enzymes in the biosynthesis of aspartate-family amino acids. AK/HSD-GUS gene expression was highly stimulated in apical and lateral meristems, lateral buds, young leaves, trichomes, vascular and cortical tissues of growing stems, tapetum and other tissues of anthers, pollen grains, various parts of the developing gynoecium, developing seeds, and, in some transgenic plants, also in stem and leaf epidermal trichomes. AK/HSD-GUS gene expression gradually dimished upon maturation of leaves, stems, floral tissues, and embryos. GUS expression was relatively low in roots. During seed development, expression of the AK/HSD gene in the embryo was coordinated with the initiation and onset of storage protein synthesis, whereas in the endosperm it was coordinated with the onset of seed desiccation. Upon germination, AK/HSD-GUS gene expression in the hypocotyl and the cotyledons was significantly affected by light. The expression pattern of the A. thaliana AK/HSD-GUS reporter gene positively correlated with the levels of aspartate-family amino acids and was also very similar to the expression pattern of the endogenous tobacco AK/HSD mRNA as determined by in situ hybridization.

The Tissue-Specific Expression of a Tobacco Phytochrome B Gene.

We have isolated a genomic clone from Nicotiana tabacum, designated Nt-PHYB-1, encoding a type-II, "green tissue" phytochrome apoprotein. Recombinant genes, consisting of the 3319-bp promoter of the Nt-PHYB-1 gene (including the entire 5[prime] untranslated sequence but not the ATG) or its deletion derivatives and the bacterial [beta]-glucuronidase reporter gene, were constructed and transferred into tobacco. The expression patterns and levels of the endogenous Nt-PHYB-1, as well as those of the transgenes, were determined by RNase protection assays and by [beta]-glucuronidase histochemical staining. We show that (a) the PHYB-1 gene has three transcription start sites, (b) the abundance of the three PHYB-1-specific mRNAs is different, and that (c) it is not regulated by light. However, we do demonstrate that transcription of the endogenous PHYB-1 gene and that of the recombinant genes exhibit a well-defined organ and tissue specificity. This tobacco PHYB gene is relatively highly expressed in leaf, stem, and different floral organs but not in root. Deletion analysis of the Nt-PHYB-1 promoter indicates that a 382-bp region, located between -1472 and -1089, is required for high-level expression of this gene.

The Activation of the Potato PR-10a Gene Requires the Phosphorylation of the Nuclear Factor PBF-1.

The pathogenesis-related gene PR-10a (formerly STH[middot]2) is induced in various organs of potato after wounding, elicitor treatment, or infection by Phytophthora infestans. Deletion analysis of the promoter of the PR-10a gene enabled us to identify a 50-bp region, located between positions -155 and -105, necessary for the elicitor responsiveness of the [beta]-glucuronidase reporter gene in transgenic potato plants. Within this region, a 30-bp sequence, located between positions -135 and -105, was necessary for the activation of the promoter by the elicitor. However, strong promoter activity after elicitor treatment required the presence of a 20-bp sequence located between positions -155 and -135. The region between -135 and -105 was specifically recognized by two nuclear factors, PBF-1 (PR-10a Binding Factor 1) and PBF-2, and binding of PBF-1 was coordinated with the accumulation of the PR-10a mRNA. Gel shift assays using nuclear extracts pretreated with sodium deoxycholate or alkaline phosphatase suggested that PBF-1 is a multimeric factor in which at least one of the constituent proteins can be phosphorylated. Treatment with alkaline phosphatase also indicated that binding of PBF-1 is positively regulated by phosphorylation and that it is phosphorylated only in tissues in which PR-10a is expressed. The use of protein phosphatase and kinase inhibitors in vivo provided additional evidence that wounding and elicitor treatment induce the phosphorylation of PBF-1 and that this phosphorylation is associated with gene activation.

Molecular Responses to Abscisic Acid and Stress Are Conserved between Moss and Cereals.

Promoter elements from the wheat Em gene have been characterized. These elements are inducible by abscisic acid (ABA) and by osmotic stress. In this study, we demonstrated that the same promoter elements function in a distantly related plant species, the moss Physcomitrella patens. Transient and stable expression of the [beta]-glucuronidase reporter gene was used to determine that the heterologous wheat promoter also responds to osmotic stress and ABA in moss. Mutational analysis of the promoter indicated that the mechanism of gene regulation is conserved in both species. Gel retardation and DNase I footprint analyses were conducted to characterize further the interaction of moss transcription factors with the Em promoter. In addition, the synthesis of stress-related polypeptides in moss was observed. The evolutionary significance of these data and the potential for studying the entire ABA perception-response pathway in moss are discussed.

Low Temperature Induces the Accumulation of Phenylalanine Ammonia-Lyase and Chalcone Synthase mRNAs of Arabidopsis thaliana in a Light-Dependent Manner.

Anthocyanins, which accumulate in leaves and stems in response to low temperature and changes in light intensity, are synthesized through the phenylpropanoid pathway that is controlled by key enzymes that include phenylalanine ammonia-lyase (PAL) and chalcone synthase (CHS). In this work we demonstrate that PAL and CHS mRNAs accumulate in leaves of Arabidopsis thaliana (L.) Heynh. upon exposure to low temperature in a light-dependent manner. The regulation of the PAL1 gene expression by low temperature and light was examined by analyzing the expression of the [beta]-glucuronidase (uidA) reporter gene in transgenic Arabidopsis plants containing the uidA gene of Escherichia coli under the control of the PAL1 promoter. The results indicate that the accumulation of PAL1 mRNA is transcriptionally regulated. Histochemical staining for [beta]-glucuronidase activity showed that the PAL1 promoter is preferentially activated in photosynthetically active cells, paralleling anthocyanin accumulation. Moreover, we show that light may also be implicated in the regulation of the CHS gene in response to bacterial infiltration. Finally, using two transparent testa Arabidopsis mutants that are unable to accumulate anthocyanins, we demonstrate that these pigments are not required for successful development of freezing tolerance in this species.

Species-dependent expression of the hyoscyamine 6 beta-hydroxylase gene in the pericycle.

The tropane alkaloid scopolamine is synthesized in the pericycle of branch roots in certain species of the Solanaceae. The enzyme responsible for the synthesis of scopolamine from hyoscyamine is hyoscyamine 6 beta-hydroxylase (H6H). The gene for H6H was isolated from Hyoscyamus niger. It has an exon/intron organization very similar to those for ethylene-forming enzymes, suggesting a common evolutionary origin. The 827-bp 5' flanking region of the H6H gene was fused to the beta-glucuronidase (GUS) reporter gene and transferred to three solanaceous species by Agrobacterium-mediated transformation systems: H. niger and belladonna (Atropa belladonna), which have high and low levels, respectively, of H6H mRNA in the root, and tobacco (Nicotiana tabacum), which has no endogenous H6H gene. Histochemical analysis showed that GUS expression occurred in the pericycle and at the root meristem of transgenic H. niger hairy roots, but only at the root meristem of transgenic H. niger hairy roots, but only at the root meristem of hairy roots and plants of transgenic tobacco. In transgenic hairy roots and regenerated plants of belladonna, the root meristem was stained with GUS activity, except for a few transformants in which the vascular cylinder was also stained. These studies indicate that the cell-specific expression of the H6H gene is controlled by some genetic regulation specific to scopolamine-producing plants.