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Plant-made oral vaccines can be a cost-effective method to control infectious diseases of humans and farm animals. Pig edema is a bacterial disease caused by enterohemorrhagic Escherichia coli producing the toxin Shiga toxin 2e (Stx2e). In our previous report, we chose the non-toxic B subunit of Stx2e (Stx2eB) as a vaccine antigen, and Stx2eB was expressed in lettuce (Lactuca sativa L., cv. Green wave). We found that a double repeated Stx2eB (2×Stx2eB) accumulates to higher levels than a single Stx2eB. In this study, we analyzed progeny plants introduced with 2×Stx2eB in which the gene was expressed under the control of conventional cauliflower mosaic virus 35S RNA (CaMV 35S) promoter, and found that the lettuce underwent transgene silencing and bore few seeds. We resolved these problems by using a transgene cassette which harbored a transcriptional promoter derived from the lettuce ubiquitin gene and a longer version of HSPT. The lettuce harboring this expression construct will be valuable in establishing the seed lot system on the basis that thousands of seeds can be obtained from one plant body and the resulting progeny plants accumulate 2×Stx2eB at high levels without the transgene silencing.

High expression of a transgene is often necessary to produce useful substances in plants. The efficiency of mRNA translation is an important determinant of the level of transgene expression. In dicotyledonous plants, the 5′UTR of certain mRNAs act as translational enhancers, dramatically improving transgene expression levels. On the other hand, translation enhancers derived from dicotyledonous plants are not so much effective in monocotyledonous plants, which are important as industrial crops and as hosts for production of useful substances. In this study, we evaluated the polysome association on a large scale with high resolution for each 5′UTR variant from multiple transcription start site in normal and heat-stressed Oryza sativa suspension cultures. Translational enhancer candidates were selected from the resultant large-scale data set, and their enhancer activities were evaluated by transient expression assay. In this manner, we obtained several translational enhancers with significantly higher activities than previously reported enhancers. Their activities were confirmed in a different monocotyledonous plant, Secale cereale, and using a different reporter gene. In addition, enhancer activities of tested 5′UTRs were different between monocotyledonous and dicotyledonous plants, suggesting that the enhancer activities were not compatible between them. Overall, we demonstrate these useful 5′UTRs as enhancer sequence for transgene expression in monocotyledonous plants.

Plant high-affinity K+ (HAK) transporters are divided into four major clusters. Cluster I transporters, in particular, are thought to have high-affinity for K+. Of the 27 HAK genes in rice, eight HAK transporters belong to cluster I. In this study, we investigated the temporal expression patterns during K+ deficiency and K+ transport activity of these eight HAK transporters. The expression of seven HAK genes except OsHAK20 was detected. Expression of OsHAK1, OsHAK5 and OsHAK21 was induced in response to K+ deficiency; however, that of other genes was not. Six of the eight HAK transporters-OsHAK1, OsHAK5, OsHAK19, OsHAK20, OsHAK21, and OsHAK27-complemented the K+-transporter-deficient yeast or bacterial strain. Further, the yeast cells expressing OsHAK1 were more sensitive to Na+ than those expressing OsHAK5. Mutant analysis showed that the high-affinity K+ uptake activity was almost undetectable in oshak1 mutants in a low-K+ medium (0.02 mM). In addition, the high-affinity K+ uptake activity of wild-type plants was inhibited by mild salt stress (20 mM NaCl); however, Na+ permeability of OsHAK1 was not detected in Escherichia coli cells. The high-affinity K+ uptake activity by leaf blades was detected in wild-type plants, while it was not detected in oshak1 mutants. Our results suggest that OsHAK1 and OsHAK5 are the two important components of cluster I corresponding to low-K+ conditions, and that the transport activity of OsHAK1, unlike that of OsHAK5, is sensitive to Na+. Further, OsHAK1 is suggested to involve in foliar K+ uptake.

In order to achieve higher expression of a transgene, it is important to optimize not only the promoter, 5′-untranslated region (UTR), and localization signal, but also the transcriptional terminator. We previously reported that the terminator derived from the Arabidopsis thaliana heat shock protein 18.2 gene (HSPT) increases gene expression in various plant species. In this study, with the goal of further increasing the expression level of transgenes, we evaluated a longer version of HSPT, corresponding to the 878 bp downstream of the stop codon, in cultured tobacco cells. The longer version of HSPT increased the expression levels of various genes including Renilla reniformis luciferase, Photinus pyralis luciferase, horseradish (Armoracia rusticana) peroxidase, and the non-toxic B subunit of Stx2e, a candidate vaccine protein for pig edema disease. This effect was not observed in a transient expression system. This element represents a useful tool for expression of transgenes integrated into the nuclear genome in plant cells.

Suspension-cultured plant cells are feasible bioreactors for the production of pharmaceutical proteins. Posttranslational modification, such as glycosylation, is important for in vivo biological and physiological roles of proteins. However, little is known about the effect of suspension-culture conditions on protein N-glycosylation. In this study, we studied the N-glycan structures of soluble and endogenous glycoproteins at different growth stages of suspension-cultured Arabidopsis thaliana T87 cells. At the late growth stage, the amount of N-glycans with terminal N-acetylglucosamine (GlcNAc) decreased, whereas that of N-glycans with fucose and xylose increased. This indicates that N-glycans have more plant-specific sugar residues as the cultivation reaches the late or end stage of plant growth.

Portulaca (Portulaca oleracea cv.), a garden plant, e ciently removes endocrine-disrupting chemicals (EDCs) including bisphenol A (BPA) from hydroponic solution. We hypothesized that polyphenol oxidase (PPO) was involved in the initial steps of detoxifying EDCs in portulaca roots. In order to elucidate the molecular basis of portulaca’s ability to metabolize EDCs, we rst isolated ve PPO genes (PoPPO1–5) that were expressed mainly in portulaca roots. Among these genes, PoPPO2, PoPPO4 and PoPPO5 were introduced into cultured tobacco cells and expressed as active forms. We found that crude extracts from the cells expressing PoPPO2, PoPPO5, and to a lesser extent PoPPO4, could metabolize BPA. In addition, we found that the BPA metabolites from crude extracts of cells expressing PoPPO2, PoPPO4 and PoPPO5 were identical to those of portulaca. Moreover, PoPPO2 and PoPPO5 also caused hydroxylation of octylphenol, nonylphenol and 17β-estradiol. erefore, these results strongly suggest that PoPPOs signi cantly contribute to the superior ability of portulaca to metabolize EDCs.

Chimeric repressor gene-silencing technology (CRES-T) is a powerful tool that has recently been developed for the functional analysis of plant transcription factors and for the genetic manipulation of plant traits. For CRES-T, a transcription factor is converted to a strong repressor by fusion with an SRDX repression domain, which is then expressed in plants to induce a loss-of-function phenotype. However, the traditional CRES-T vectors are inconvenient for gene cloning and promoter exchange. In this study, we developed new CRES-T vectors that are efficient and convenient to use by employing the Gateway system, a new vector backbone and a terminator derived from the heat shock protein 18.2 (HSP) gene. Our test experiments revealed that the CRES-T vector containing the Gateway linker sequence within the transcribed region showed reduced efficiency of CRES-T when compared with the traditional CRES-T vector. However, the HSP terminator compensated for the negative effect of the Gateway sequence and improved the efficiency of CRES-T in all cases tested and resulted in the highest efficiency achieved to date. We found that the HSP terminator increased transcription efficiency or transcript stability; in contrast, these factors were negatively affected by the Gateway linker sequence in our vector system. We, therefore, propose that the appropriate CRES-T vector should be chosen depending on situations and purposes.

We investigated the efficiency of the 5� -untranslated region (UTR) of the tobacco alcohol dehydrogenase gene (NtADH-5� UTR) as a translational enhancer in chrysanthemum (Chrysanthemum morifolium) and torenia (Torenia fournieri). Three constructs were introduced: control, "ADH� ," cauliflower mosaic virus 35S RNA (CaMV 35S) promoter::b-glucuronidase (GUS); NtADH-5� UTR with a spacer, "ADHS," CaMV 35S promoter::NtADH-5� UTR::25-bp spacer::GUS; and NtADH-5� UTR with no spacer, "ADH� ," CaMV 35S promoter::NtADH-5� UTR::GUS. The highest GUS activity in ADHS and ADHfor chrysanthemum was about 45 and 190 times, respectively, and for torenia was 12 and 22 times, respectively, than that for the ADHplants. NtADH-5� UTR enhanced translational efficiency in both species. With the lowest translational efficiency set to 1, the relative translational efficiencies were 1 to 15 (ADH� ), 47 to 568 (ADHS), and 267 to 1360 (ADH� ) for chrysanthemum, and 1 to 3 (ADH� ), 47 to 114 (ADHS), and 85 to 226 (ADH� ) for torenia. NtADH-5� UTR would facilitate practical breeding and fundamental genetic research in chrysanthemum and torenia.

Specifically accessible chromatin sites, detected as DNase I hypersensitive sites (DNase I HSs), have been found at promoters of most genes in yeast, human as well as in Arabidopsis chromatin. The DNase I HSs are constitutively present at promoters of most genes both at inactive and activated states. On the other hand, appearance and disappearance of DNase I HSs have been observed at several developmentally regulated genes in different tissues of animals. Patterns of appearance and disappearance of DNase I HSs were correlated with transcriptional competency (potential of expression) of the associated genes, suggesting that DNase I HSs are absent from the promoters of transcriptionally incompetent (unexpressed nor inducible) genes. To test this hypothesis in plants, we analyzed DNase I hypersensitivity of several well- characterized genes showing distinct patterns of tissue-specific expression in Arabidopsis leaves, roots and suspension cells. Distinct DNase I HSs were present at transcriptionally competent gene promoters, but absent from incompetent gene promoters. The absence of DNase I HS suggests that nucleosomes are randomly positioned on incompetent promoters, and may contribute to the transcriptional incompetency by blocking the binding of activators in tissues where expression of these genes is unnecessary.

Plants contain a regulatory pathway similar to that involving retinoblastoma (RB) protein in animals. Here, we analyze an RB homolog, tobacco RETINOBLASTOMA-RELATED1 (NtRBR1), that encodes a protein possessing 13 potential phosphorylation sites for cyclin-dependent kinases (CDKs). We found that 11 synthetic peptides containing sequences from NtRBR1 were phosphorylated differentially by imunoprecipitates of cyclin/CDK complexes extracted from tobacco BY-2 cells. We raised antibodies that specifically recognize these sites of phosphorylation in NtRBR1. Distinct sites were phosphorylated during the cell cycle, suggesting that NtRBR1 is phosphorylated by different types of cyclin/CDK complexes.

Variable levels of transgene expression are frequently observed among independent transgenic plants. Although different chromatin condensation states surrounding randomly integrated transgenes have previously been thought to be one of the causes for this variability, condensation states of plant chromatin have not been examined systematically. In this study, to analyze the condensation states of Arabidopsis chromatin, we evaluated the degree of chromatin condensation by measuring overall accessibility to DNase I (DNase I sensitivity) at 500-base resolution. We analyzed 30 variably expressed genes in an 80-kb genomic region, a gene repressed by polycomb group and heterochromatin protein 1-like genes, two genes near the heterochromatin, and a retrotransposon within the genetically-defined centromere in Arabidopsis. The centromeric region was significantly DNase I insensitive, however, sensitivity of these genes was similar irrespective of the individual gene expression levels.

The E2F transcription factors play important roles in the regulation of gene expression at the G1/S transition in plants. Here, we show that the rice proliferating cell nuclear antigen (PCNA) promoter is activated by transient expression of tobacco NtE2F and Arabidopsis AtDPa in tobacco cells. This transcriptional activation is repressed by co-transfection with a plasmid encoding the tobacco Rb-related protein (NtRBR1), whereas further co-expression of cyclin D overcomes this repression. Importantly, the rice PCNA promoter is activated when cells are transfected with cyclin D alone, and this activation is enhanced by co-transfection with plasmids encoding NtE2F and AtDPa. These results suggest that the effect of cyclin D expression is mediated not only by its associated kinase, which allows it to phosphorylate NtRBR1 thereby releasing the NtE2F/NtDP complex to activate transcription, but also by a mechanism which does not involve transfected NtRBR1.