Vesicular transport route of horseradish C1a peroxidase is regulated by N- and C-terminal propeptides in tobacco cells.

Peroxidases (PRX, EC 1.11.1.7) are widely distributed across microorganisms, plants, and animals; and, in plants, they have been implicated in a variety of secondary metabolic reactions. In particular, horseradish (Armoracia rusticana) root represents the main source of commercial PRX production. The prxC1a gene, which encodes horseradish PRX (HRP) C, is expressed mainly in the roots and stems of the horseradish plant. HRP C1a protein is shown to be synthesized as a preprotein with both a N-terminal (NTPP) and a C-terminal propeptide (CTPP). These propeptides, which might be responsible for intracellular localization or secretion, are removed before or concomitant with production of the mature protein. We investigated the functional role of HRP C1a NTPP and CTPP in the determination of the vesicular transport route, using an analytical system of transgenically cultured tobacco cells (Nicotiana tabacum, BY2). Here, we report that NTPP and CTPP are necessary and sufficient for accurate localization of mature HRP C1a protein to vacuoles of the vesicular transport system. We also demonstrate that HRP C1a derived from a preprotein lacking CTPP is shunted into the secretory pathway.

Molecular biology and application of plant peroxidase genes.

Peroxidases are a family of isozymes found in all plants; they are heme-containing monomeric glycoproteins that utilize either H(2)O(2) or O(2) to oxidize a wide variety of molecules. These important enzymes are used in enzyme immunoassays, diagnostic assays and industrial enzymatic reactions. Peroxidase genes and their promoters can be used for molecular breeding of useful plants. Transgenic techniques have also been used to investigate the physiological and molecular functions of peroxidase genes in plants. Here, we review transgenic studies of peroxidase genes, including the functional analyses of the enzymes and their promoters. Regarding application of peroxidase genes, it has been reported that overexpression of the tomato TPX2 gene or the sweet potato swpa1 gene conferred increased salt-tolerance or oxidative-stress tolerance, respectively. The growth stimulation effect in transgenic tobacco and hybrid aspen upon overexpression of horseradish peroxidase gene is also discussed.

In vivo conversion of a glycan to human compatible type by transformed tobacco cells.

Horseradish peroxidase isozyme C (HRP; EC 1.11.1.7) was used as a model protein to evaluate the capacity of tobacco cells transformed with human beta 1,4-galactosyltransferase (GT6) to modify and galactosylate a foreign glycoprotein. Cells transformed with the HRP gene are designated as BY2-HRP and GT6-HRP, for wild type BY2 and GT6 transformed cells, respectively. Expression of HRP cells was confirmed by isoelectric focusing, peroxidase activity staining, Western blotting, and enzymatic assays. The presence of HRP galactosylated N-glycans in GT6-HRP cells was analyzed by lectin staining, affinity chromatography, and structural analyses of pyridylamino-labeled RCA(120)-bound sugar chains. The structure of Gal(1)GlcNAc(1)Man(5)GlcNAc(2) was proposed based from the results of exoglycosidase digestions and two-dimensional sugar chain mapping. Unlike the HRP produced in BY2-HRP cells, the HRP from GT6-HRP cells has galactosylated glycoproteins that did not bind to the xylose-specific antiserum, suggesting the absence of the beta 1,2-xylose residue in the sugar chain.

Two types of HKT transporters with different properties of Na+ and K+ transport in Oryza sativa.

It is thought that Na+ and K+ homeostasis is crucial for salt-tolerance in plants. To better understand the Na+ and K+ homeostasis in important crop rice (Oryza sativa L.), a cDNA homologous to the wheat HKT1 encoding K+-Na+ symporter was isolated from japonica rice, cv Nipponbare (Ni-OsHKT1). We also isolated two cDNAs homologous to Ni-OsHKT1 from salt-tolerant indica rice, cv Pokkali (Po-OsHKT1, Po-OsHKT2). The predicted amino acid sequence of Ni-OsHKT1 shares 100% identity with Po-OsHKT1 and 91% identity with Po-OsHKT2, and they are 66-67% identical to wheat HKT1. Low-K+ conditions (less than 3 mM) induced the expression of all three OsHKT genes in roots, but mRNA accumulation was inhibited by the presence of 30 mM Na+. We further characterized the ion-transport properties of OsHKT1 and OsHKT2 using an expression system in the heterologous cells, yeast and Xenopus oocytes. OsHKT2 was capable of completely rescuing a K+-uptake deficiency mutation in yeast, whereas OsHKT1 was not under K+-limiting conditions. When OsHKTs were expressed in Na+-sensitive yeast, OsHKT1 rendered the cells more Na+-sensitive than did OsHKT2 in high NaCl conditions. The electrophysiological experiments for OsHKT1 expressed in Xenopus oocytes revealed that external Na+, but not K+, shifted the reversal potential toward depolarization. In contrast, for OsHKT2 either Na+ or K+ in the external solution shifted the reversal potential toward depolarization under the mixed Na+ and K+ containing solutions. These results suggest that two isoforms of HKT transporters, a Na+ transporter (OsHKT1) and a Na+- and K+-coupled transporter (OsHKT2), may act harmoniously in the salt tolerant indica rice.

Cell cycle regulation of cyclin-dependent kinases in tobacco cultivar Bright Yellow-2 cells.

Plants possess two major classes of cyclin-dependent kinases (CDK) with cyclin-binding motifs PSTAIRE (CDK-a) and PPTA/TLRE (CDK-b). Tobacco (Nicotiana tabacum L. cv Bright Yellow-2) cells are the most highly synchronizable plant culture, but no detailed analysis of CDK activities has been reported in this system. Here we describe isolation of new PPTALRE CDKs (Nicta;CdkB1) from Bright Yellow-2 cells and present detailed analysis of the mRNA, protein and kinase activity levels of CdkB1, and the PSTAIRE CDKA during the growth and cell cycles. CdkA and CdkB1 transcripts are more abundant in exponential than in stationary phase cells, but the two genes show strikingly different regulation during the cell cycle. CdkA mRNA and protein accumulate during G1 in cells re-entering the cell cycle, and immunoprecipitated histone H1 kinase activity increases at the G1/S boundary. Aphidicolin synchronized cells show the highest CDKA-associated histone H1 kinase activity during S-G2 phases, although CdkA mRNA and protein levels are not significantly regulated. In contrast, CdkB1 transcripts are present at very low levels until S phase and CDKB1 protein and kinase activity is almost undetectable in G1. CdkB1 mRNA accumulates through S until M phase and its associated kinase activity peaks at the G2/M boundary, confirming that transcription of PPTALRE CDKs is cell cycle regulated. We suggest that CDKA kinase activity likely plays roles at the G1/S phase boundary, during S phase, and at the G2/M phase transition, and that CDKB1 kinase activity is present only at G2/M.

An insulator element from the sea urchin Hemicentrotus pulcherrimus suppresses variation in transgene expression in cultured tobacco cells.

Specialized DNA sequences known as insulators protect genes from both the positive and negative influences of nearby chromatin. Many insulators have been identified in various species; however, few function in multiple species. We have shown that an insulator from the Ars (arylsulfatase) gene of the sea urchin Hemicentrotus pulcherrimus functions in plant cells. Normally, expression of an introduced chimeric GUS gene is inactivated in approximately 30% of transformed tobacco BY2 clones. Transgenes containing the Ars insulator, however, were expressed in all transformed tobacco BY2 cells. The insulator did not affect the copy number, the chromosomal position of transgene integration or maximum expression levels. These results suggest that the insulator functions to suppress the variation normally associated with transgene expression in tobacco BY2 cells.

Ectoine, the compatible solute of Halomonas elongata, confers hyperosmotic tolerance in cultured tobacco cells.

1,4,5,6-Tetrahydro-2-methyl-4-pyrimidinecarboxylic acid (ectoine) functions as a compatible osmolyte in the moderate halophile Halomonas elongata OUT30018. Ectoine is biosynthesized by three successive enzyme reactions from aspartic beta-semialdehyde. The genes encoding the enzymes involved in the biosynthesis, ectA, ectB, and ectC, encoding L-2,4-diaminobutyric acid acetyltransferase, L-2, 4-diaminobutyric acid transaminase, and L-ectoine synthase, respectively, have been previously cloned. To investigate the function of ectoine as a compatible solute in plant cells, the three genes were individually placed under the control of the cauliflower mosaic virus 35S promoter and introduced together into cultured tobacco (Nicotiana tabacum L.) cv Bright Yellow 2 (BY2) cells. The transgenic BY2 cells accumulated a small quantity of ectoine (14-79 nmol g(-1) fresh weight) and showed increased tolerance to hyperosmotic shock (900 mOsm). Furthermore, the transgenic BY2 cells exhibited a normal growth pattern even under hyperosmotic conditions (up to 530 mOsm), in which the growth of the untransformed BY2 (wild type) cells was obviously delayed. These results suggest that genetically engineered synthesis of ectoine results in the increased hyperosmotic tolerance of cultured tobacco BY2 cells despite the low level of accumulation of the solute.

Two types of heat shock factors in cultured tobacco cells.

We have isolated two types of complementary DNA (cDNA) sequences showing high similarity with the DNA-binding domain in heat shock factors (HSFs) from a cDNA library of tobacco suspension-cultured cells (Nicotiana tabacum L. cv. BY2). These two genes, NtHSF1 and NtHSF2, showed low similarity (37%) with each other. Both contained the typical conserved regions of HSFs (DNA-binding domains, leucine zipper repeats, nuclear localization signals, and tryptophan repeats). Transcripts of NtHSF1 and NtHSF2 were detected even at the normal temperature. The recombinant NtHSF1 and NtHSF2 proteins expressed in Escherichia coli bound specifically to a 183-bp DNA fragment of the Arabidopsis thaliana HSP18.2 promoter containing three sets of head-to-head and tail-to-tail repeats of the conserved pentamer unit (nGAAnnTTCnnGAAn).

A cis-element containing PAL-box functions in the expression of the wound-inducible peroxidase gene of horseradish.

In an attempt to understand the molecular basis of plant responses to wounding, we have investigated the transcriptional regulation of the wound-inducible prxC2 gene, which encodes horseradish peroxidase. In the previous work, 5'-deletion analysis revealed that a cis-element involved in the expression of the prxC2 gene is located at a position between -296 and -283 bp from the translation start site and contains a G-box sequence. We have also reported that a trans-acting factor, TFHP-1, can bind to the G-box sequence in this cis-element. Although the antisense RNA of TFHP-1 suppressed the expression of a prxC2 promoter-GUS chimeric gene in tobacco protoplasts, the sense RNA of TFHP-1 could not enhance this expression. These results suggested that other elements function in the expression of the prxC2 gene, and therefore additional deletion analysis of the promoter was performed with a transient expression system in cultured tobacco cells. Consequently, the second cis-element was found at a position between -177 and -134 bp from the translation start site of prxC2. This second cis-element contains a sequence similar to a PAL-box, and a nuclear protein possibly binds to it through the PAL-box sequence.

Isolation of growth-phase-specific promoters from cultured tobacco cells.

Strong promoters are required under several culture conditions for effective transgene expression in tobacco BY2 cells. We have isolated the promoter fragments of 4 genes exhibiting high homology to those of Arabidopsis thaliana 108C1T7 (unknown function) and F1-ATPase-delta, alcohol dehydrogenase and pectin esterase genes from a genomic DNA library of BY2 cells. Two of the four genes were strongly expressed during every phase of growth of BY2 cells, and the other two were expressed only during the stationary phase. Each of the promoter fragments was ligated to the GUS reporter gene and introduced into the chromosome of BY2 cells by Agrobacterium-mediated transformation. Growth-phase-dependent expression of the GUS gene was reproduced under the control of all 4 promoters observed with the original genes. Significantly higher expression was observed under the control of Nt108p during every phase of cell growth and under the control of NtADHp and NtPESp during the stationary phase than that under the control of the CaMV35S promoter.

Transgene expression systems in plant, a natural bioreactor.

Plants are important resources that have been providing us food from the earliest times. The rapid advances that have taken place in plant genetic engineering have made it possible to modify plants to increase food production and contribute to environmental purification. Transgenic plants are gaining increasing attention from the industry as a natural bioreactor for the production of industrial and chemical products. Useful expression systems based on promoters to optimize transgene expression in plant cells, hold the key to maximizing the potential of this concept of molecular-farming or industrial plants. This review, which is devoted to the use of plants for heterologous protein production, is divided into three parts. First, we introduce the nature of plant promoters and strategies for the isolation of novel promoters. In the second part, various promoters showing high-level constitutive, organ-specific, or inducible expression, are summarized as useful tools for realizing the efficient transcription of transgenes. Finally, problems in the expression of foreign gene in plant cells and future prospects in plant biotechnology are discussed.

Isolation and characterization of the E2F-like gene in plants.

The transcription factor E2F regulates the expression of genes involved in the progression of G1/S transition and DNA replication in mammalian cells. We cloned and characterized a cDNA (NtE2F) corresponding to a E2F homolog of tobacco (Nicotiana tabacum). The transcription of NtE2F was induced as cells progressed from G1 to the S phase and expressed much earlier than that of the proliferating cell nuclear antigen (PCNA) gene. We demonstrated that NtE2F can interact with the tobacco retinoblastoma (Rb)-related protein in a yeast two-hybrid assay. To further characterize NtE2F, the trans-activation activity of NtE2F was examined by using a transient assay in the tobacco Bright Yellow-2 (BY-2) cells with NtE2F fused to the DNA-binding domain of the veast transcriptional activator GAL4. NtE2F activated the transcription of the beta-glucuronidase (GUS) reporter gene driven by a cauliflower mosaic virus (CaMV) 35S core promoter containing the GAL4-binding sequence. This is the first report of the identification of a functionally equivalent E2F-like gene in plants.

Tobacco retinoblastoma-related protein phosphorylated by a distinct cyclin-dependent kinase complex with Cdc2/cyclin D in vitro.

The retinoblastoma (Rb) protein was originally identified as a product of a tumour suppressor gene that plays a pivotal role in regulating both the cell cycle and differentiation in mammals. The growth-suppressive activity of Rb is regulated by phosphorylation with cyclin-dependent kinase (CDK), and inactivation of the Rb function is one of the critical steps for transition from the G1 to the S phase. We report here the cloning of a cDNA (NtRb1) from Nicotiana tabacum which encodes a Rb-related protein, and show that this gene is expressed in all the organs examined at the mRNA level. We have demonstrated that NtRb1 interacts with tobacco cyclin D by using yeast two-hybrid and in vitro binding assays. In mammals, cyclin D can assemble with CDK4 and CDK6, but not with Cdc2, to form active complexes. Surprisingly, tobacco cyclin D and Cdc2 proteins can form a complex in insect cells, which is able to phosphorylate tobacco Rb-related protein in vitro. Using immunoprecipitation with the anti-cyclin D anti-body, cyclin D can be found in a complex with Cdc2 in suspension-cultured tobacco BY-2 cells. These results suggest that the cdc2 gene modulates the cell cycle through the phosphorylation of Rb-related protein by forming an active complex with cyclin D in plants.

Characterization of biosynthetic enzymes for ectoine as a compatible solute in a moderately halophilic eubacterium, Halomonas elongata.

1,4,5,6-Tetrahydro-2-methyl-4-pyrimidinecarboxylic acid (ectoine) is an excellent osmoprotectant. The biosynthetic pathway of ectoine from aspartic beta-semialdehyde (ASA), in Halomonas elongata, was elucidated by purification and characterization of each enzyme involved. 2,4-Diaminobutyrate (DABA) aminotransferase catalyzed reversively the first step of the pathway, conversion of ASA to DABA by transamination with L-glutamate. This enzyme required pyridoxal 5'-phosphate and potassium ions for its activity and stability. The gel filtration estimated an apparent molecular mass of 260 kDa, whereas molecular mass measured by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was 44 kDa. This enzyme exhibited an optimum pH of 8.6 and an optimum temperature of 25 degreesC and had Kms of 9.1 mM for L-glutamate and 4.5 mM for DL-ASA. DABA acetyltransferase catalyzed acetylation of DABA to gamma-N-acetyl-alpha,gamma-diaminobutyric acid (ADABA) with acetyl coenzyme A and exhibited an optimum pH of 8.2 and an optimum temperature of 20 degreesC in the presence of 0.4 M NaCl. The molecular mass was 45 kDa by gel filtration. Ectoine synthase catalyzed circularization of ADABA to ectoine and exhibited an optimum pH of 8.5 to 9.0 and an optimum temperature of 15 degreesC in the presence of 0.5 M NaCl. This enzyme had an apparent molecular mass of 19 kDa by SDS-PAGE and a Km of 8.4 mM in the presence of 0. 77 M NaCl. DABA acetyltransferase and ectoine synthase were stabilized in the presence of NaCl (>2 M) and DABA (100 mM) at temperatures below 30 degreesC.

Expression of a foreign gene in Chlamydomonas reinhardtii chloroplast.

Chimeric genes for expression of a foreign gene in the Chlamydomonas reinhardtii chloroplast were constructed. These chimeric genes are composed of the promoter from chloroplast genes, rbcL, psbA, and atpA, 5'- and 3'-untranslated regions, and the Escherichia coli beta-glucuronidase (GUS) structural gene (uidA) as a foreign gene. Three types of chloroplast transformants (RG, PG, and AG), which contained the rbcL-uidA, psbA-uidA, and atpA-uidA chimeric genes integrated in the chloroplast genome, were generated by particle bombardment. The AG transformant grown under photoautotrophic conditions showed the highest GUS activity (130 nmol/min/mg protein) so far reported in C. reinhardtii, and the accumulated GUS protein accounted for 0.08% of the total soluble proteins. GUS activity in RG was 12% of that in AG, and no activity was detected in PG. We also measured the GUS activity from transformants grown under heterotrophic conditions, but the culture conditions made little difference in activity levels. The difference in the amount of accumulated GUS protein in the transformants was paralleled by the difference in the level of transcripts, and the pattern of gene expression was not the same as that of the endogenous genes in the chloroplast.

Metabolic engineering of cultured tobacco cells.

Construction of a gene expression system in tobacco cultured cells (BY2) was studied. A 925 bp promoter fragment of a heat-shock protein gene (HSP18.2) of Arabidopsis thaliana showed clear heat-shock response of expression of the beta-glucuronidase (GUS) reporter gene in BY2 cells. Similar results were observed in a 500 mL flask and 3-L jar fermentor. Isolation of strong promoters in BY2 cells was tried. cDNA clones, in which the mRNA level is high in log-phase cells and the copy number in the genome is low, were isolated. These clones showed high homology with F1-ATPase (mitochondria type), elongation factor 1-alpha, and a gene with an unknown function of A. thaliana (clone 27), respectively. A 5'-flanking region of clone 27 showed 6.2 times the promoter activity of the CaMV35S promoter in BY2 cells. Three cDNA clones, which are expressed in the stationary growth phase of BY2 cells, were isolated by a differential screening. These clones showed high sequence homologies to alcohol dehydrogenase, pectin esterase, and extensin. Promoters of these genes will be useful in gene expression in high cell-density culture.

Expression pattern of tobacco cyclin genes.

We have previously isolated three cDNA clones (Ntcyc25, Ntcyc27, Ntcyc29) encoding mitotic cyclins from tobacco (Nicotiana tabacum), and in this report we describe the expression patterns of these genes. RNA gelblot analysis showed that the mitotic cyclin genes were expressed predominantly in actively dividing meristematic tissues such as the shoot apex and young developing leaves. In situ hybridization analysis indicated that while theNtcyc29 gene was expressed consistently at low levels in shoot apex, but its transcripts were found at relatively high levels in the flower bud, indicating the possibility that theNtcyc29 product plays a role in flower development. TheNtcyc25 andNtcyc29 genes were expressed differentially in the root apex during the cell cycle, confirming the result obtained using synchronized-cultured tobacco cells. These results suggest that the transcriptionally regulated cyclin genes may be important in controlling cell division in tobacco.

cis-regulatory elements of the peroxidase gene in Arabidopsis thaliana involved in root-specific expression and responsiveness to high-salt stress.

The pattern of organ-specific expression of Arabidopsis thaliana peroxidase was similar to that in horseradish. Tobacco plants transformed with the gus gene fused to the -580 bp deletion (Ea-580) of A. thaliana exhibited high GUS expression in roots. Gel retardation and footprinting analyses showed that at least three domains of fragment between -172 and -1 bp have cis-acting element activities. Several physiological functions for plant peroxidases have been suggested; for example, a metabolic adaptation to salinity in the environment can be induced by certain specific elements of the peroxidase gene. The prxEa promoter fragments (Ea-580 and Ea-390) show multiple cis-acting elements in the control of expression in high-salt stress. These data suggest that the DNA-binding factor may be involved in the regulation of gene expression in specific organ and salt stress.

Construction of gene expression system in cultured tobacco cells.

To construct a gene expression system in cultured tobacco cells, useful regulatory elements of plant genes were studied. The promoter of the horseradish peroxidase gene, prxC2, showed high activity in tobacco cells, and it contained enhancer sequences and a cis element for wound induction. The heat shock promoter of the HSP18.2 gene from A. thaliana had strong activity of transcription when the incubation temperature of tobacco cells was shifted from 25 degrees C to 37 degrees C. These elements could be good candidates for foreign gene expression in tobacco cells.

Molecular cloning and characterization of a cDNA clone that encodes a Cdc2 homolog from Nicotiana tabacum.

We have isolated a cDNA clone (cdc2Nt1) that encodes a homolog of p34(cdc2/CDC28) kinase from tobacco (Nicotiana tabacum). The cdc2Nt1 protein showed extensive similarity to other homologs of Cdc2 from plants. Complementation studies showed that the cdc2Nt1 gene was able to overcome cell cycle arrest at both the G1/S and the G2/M transitions of cdc28ts mutants of budding yeast, demonstrating that the cdc2Nt1 protein was able to replace the Cdc28 kinase at both the G1/S and the G2/M transitions. Analysis of gene expression demonstrated that the cdc2Nt1 gene was transcribed constitutively throughout the cell cycle but that it was preferentially expressed in activity dividing tobacco BY-2 cells.