[Show abstract][Hide abstract] ABSTRACT: The internal ribosome entry site (IRES) enables polycistronic expression of multiple genes from a single mRNA controlled by one promoter. In general, only the most 5′ coding sequence is abundantly translated from polycistronic mRNAs in eukaryotic cells; IRES-mediated translation allows additional coding sequences at other positions to be translated at detectable levels. However, IRES-mediated translation often results in much lower protein expression than translation of single-gene mRNAs. We first aimed to improve IRES-mediated gene expression with a transient expression system based on Nicotiana benthamiana. We demonstrated that the presence of two cassettes comprised of an IRES-mediated Venus coding sequence results in higher Venus expression than one cassette. The double IRES cassette system is expected to be useful for expressing a gene of interest polycistronically in plants. Using the double IRES cassette system, we found that polycistronic expression of a reporter gene and two copies of an IRES-mediated RNA silencing suppressor gene protected the transcribed mRNA from RNA silencing-mediated degradation. This is the first report of a mRNA self-protection system from RNA silencing by IRES-mediated expression of a viral suppressor in plants.
Preview · Article · Mar 2015 · Plant Biotechnology
[Show abstract][Hide abstract] ABSTRACT: Eukaryotes possess several RNA surveillance mechanisms that prevent undesirable aberrant RNAs from accumulating. Arabidopsis XRN2, XRN3, and XRN4 are three orthologs of the yeast 5'-to-3' exoribonuclease, Rat1/Xrn2, that function in multiple RNA decay pathways. XRN activity is maintained by FIERY1 (FRY1), which converts the XRN inhibitor, adenosine 3', 5'-bisphosphate (PAP), into 5'AMP. To identify the roles of XRNs and FRY1 in suppression of non-coding RNAs, strand-specific genome-wide tiling arrays and deep strand-specific RNA-Seq analyses were carried out in fry1 and xrn single and double mutants. In fry1-6, about 2000 new transcripts were identified that extended the 3' end of specific mRNAs; many of these were also observed in genotypes that possess the xrn3-3 mutation, a partial loss-of-function allele. Mutations in XRN2 and XRN4 in combination with xrn3-3 revealed only a minor effect on 3' extensions, indicating that these genes may be partially redundant with XRN3. We also observed the accumulation of 3' remnants of many DCL1-processed microRNA (miRNA) precursors in fry1-6 and xrn3-3. These findings suggest that XRN3, in combination with FRY1, is required to prevent the accumulation of 3' extensions that arise from thousands of mRNA and miRNA precursor transcripts.
[Show abstract][Hide abstract] ABSTRACT: Sucrose plays an important role in several cellular processes since it is a general source of metabolic energy, serves as a precursor for starch and cellulose synthesis, and is a metabolic starting point for carboxylate- and amino acid synthesis. While plant vacuole is the main cellular storage pool, where sucrose accumulates to high concentrations, only a small number of vacuolar sugar transporters have been identified and characterized to date. We initially identified a vacuolar sucrose transporter (NtSUT4) from tobacco BY-2 cells and established transgenic tobacco BY-2 cell lines that overexpress NtSUT4-GFP (BY-SUTG cells). Using a model system for synchronous cell elongation in miniprotoplasts (evacuolated cells) prepared from tobacco BY-2 cells, we found that NtSUT4-GFP overexpression inhibited cell growth towards the cell major axis. Moreover, under the same conditions, we found that the cell walls were well stained by calcofluor in BY-SUTG cells than in wild type BY-2 cells. These results suggest that NtSUT4 is involved in cell shape via sucrose homeostasis in plant cells.
Full-text · Article · Oct 2010 · Journal of Plant Research
[Show abstract][Hide abstract] ABSTRACT: Aquaporin is a water channel that increases water permeability through membranous structures. In plants, vacuoles are essential organelles that undergo dynamic volume changes during cell growth. To understand the contribution of aquaporins to plant cell growth, we developed a transgenic tobacco BY-2 cell line overexpressing the tonoplast intrinsic protein (TIP), gammaTIP. Vacuolar membranes of isolated vacuoles from gammaTIP-overexpressing cells showed higher water permeation activities than those from wild-type cells. We then examined the role of gammaTIP in vacuolar regeneration of evacuolated tobacco BY-2 protoplasts (miniprotoplasts). Vacuolar regeneration from thin to thick tube-network vacuoles and subsequent development of large vacuoles was accelerated in miniprotoplasts of this cell line. A parallel increase in the rate of cell expansion indicated a tight relationship between vacuolar development and cellular volume increases. Interestingly, overexpression of tobacco gammaTIP also enhanced cell division. Thus, increased vacuolar aquaporin activity may accelerate both cell expansion and cell division by increasing water permeability through the vacuolar membrane.
No preview · Article · Dec 2008 · Plant and Cell Physiology