An RNA helicase (AtSUV3) is present in Arabidopsis thaliana mitochondria.
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The proteins involved in mitochondrial mRNA processing and degradation in higher plants have yet to be identified. As a first step towards this aim, we report here the characterisation of a nuclear-encoded DExH box RNA helicase (AtSUV3) localised in Arabidopsis thaliana mitochondria. The AtSUV3 mRNA is assembled from the 16 exons of a weakly expressed unique gene and the predicted protein has a calculated molecular weight of 63.6 kDa. Subcellular fractionation of transgenic plants expressing AtSUV3/GUS fusion proteins localises this protein in mitochondria. The N-terminal domain of AtSUV3 containing the motifs characteristic of DExH box RNA helicases exhibits a low endogenous ATPase activity in vitro which can be stimulated by the presence of mitochondrial RNA, confirming that AtSUV3 is an RNA helicase. (+info)
Complete sequence of RNA 1 and the presence of tRNA-like structures in all RNAs of Potato mop-top virus, genus Pomovirus.
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The complete nucleotide sequence (6043 nt) of RNA 1 from Potato mop-top virus (PMTV-Sw), the type member of the genus Pomovirus, was determined. The first (5'-terminal) open reading frame (ORF 1) encodes a predicted protein of 148 kDa. ORF 2 extends through the opal stop codon of ORF 1 producing a predicted readthrough protein of 206 kDa which resembles the RNA-dependent RNA polymerases (RdRp) of other fungal-transmitted viruses. It includes a methyltransferase, a helicase and a GDD RdRp motif, respectively. Phylogenetic analyses of RdRps indicated that PMTV is most closely related to Beet soil-borne virus (genus Pomovirus), Broad bean necrosis virus (genus Pomovirus) and Soil-borne wheat mosaic virus (genus Furovirus), and is more distantly related to the other viruses of the former furovirus group. The 5' and 3' termini of RNA 1 in PMTV contained untranslated regions (UTR) of 114 nt and 489 nt, respectively. The 3'-UTR of RNA 1 contained a tRNA-like structure, which has previously been reported in the 3'-UTR of RNA 2 but not RNA 3. However, in this study, the tRNA-like structure was also found in the 3'-UTR of RNA 3, which confirms its presence in the 3'-UTRs of all three RNAs of PMTV. (+info)
Transformation of tobacco and potato with cDNA encoding the full-length genome of potato leafroll virus: evidence for a novel virus distribution and host effects on virus multiplication.
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A full-length cDNA copy of the genome of Potato leafroll virus (PLRV) was introduced into the genome of tobacco and potato plants by Agrobacterium tumefaciens-mediated transformation. Transgenic lines were obtained in which the transgene was readily detected by PCR with DNA extracted from T(1) tobacco seedlings and clonally multiplied potato plants. PLRV-specific genomic and sub- genomic RNAs, coat protein antigen and virus particles were detected in transgenic plants. Aphids fed on the transgenic tobacco plants readily transmitted PLRV to test plants. Infected transgenic tobacco plants, like non-transgenic (WT) PLRV-infected plants, displayed no symptoms of the infection but transgenic plants of potato were severely stunted. In parallel tests, the mean PLRV titres in WT tobacco plants and transgenic tobacco plants were 600 and 630 ng virus/g leaf, respectively, although differences in PLRV titres among transgenic plants were much greater than those among infected WT plants. In similar tests with potato, the mean PLRV titre of WT plants was 50 ng virus/g leaf whereas higher concentrations (up to 3400 ng virus/g leaf) accumulated in transgenic potato plants. In tissue prints of stems, PLRV was detected in similar proportions of phloem cells in transgenic and infected WT plants. In transgenic tobacco and potato plants, but not in infected WT plants, a few stem epidermal cells also contained virus. From tissue prints of transgenic tobacco leaves, it was estimated that about one in 40000 mesophyll cells contained virus, but in transgenic potato, a greater proportion of mesophyll cells was infected. (+info)
Tomato chlorotic dwarf viroid: an evolutionary link in the origin of pospiviroids.
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Over 40 isolates of potato spindle tuber viroid (PSTVd) have been reported from potato, other Solanum species and greenhouse tomato. These isolates have sequence similarities in the range 95-99%. A viroid which caused chlorotic leaves and severe dwarfing of plants in greenhouse tomato crops was detected. The viroid was found to hybridize readily with PSTVd probes. It migrated faster than PSTVd in return-polyacrylamide gel electrophoresis and was not amplified in RT-PCR by a primer pair based on the lower strand of the central conserved region of PSTVd. Nucleotide sequencing of the viroid indicated that it is a circular RNA of 360 nt, with less than 90% sequence similarities with PSTVd isolates. The Variable domain (V) has less than 60% and the Terminal Right domain less than 90% sequence similarity, while the remainder of the molecule has greater than 97% similarity with PSTVd. Because of its less-than 90% sequence similarities, unique V domain, lack of seed-transmission and lack of cross-protection by PSTVd, the viroid from tomato is proposed to be a distinct viroid species (tomato chlorotic dwarf viroid; TCDVd) which also differs from two viroids infecting tomato in nature. TCDVd may be an evolutionary link in the development of crop viroids, with Mexican papita viroid as the ancestral viroid. (+info)
Specificity of starch synthase isoforms from potato.
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In higher plants several isoforms of starch synthase contribute to the extension of glucan chains in the synthesis of starch. Different isoforms are responsible for the synthesis of essentially linear amylose chains and branched, amylopectin chains. The activity of granule-bound starch synthase I from potato has been compared with that of starch synthase II from potato following expression of both isoforms in Escherichia coli. Significant differences in their activities are apparent which may be important in determining their specificities in vivo. These differences include affinities for ADPglucose and glucan substrates, activation by amylopectin, response to citrate, thermosensitivity and the processivity of glucan chain extension. To define regions of the isoforms determining these characteristic traits, chimeric proteins have been produced by expression in E. coli. These experiments reveal that the C-terminal region of granule-bound starch synthase I confers most of the specific properties of this isoform, except its processive elongation of glucan chains. This region of granule-bound starch synthase I is distinct from the C-terminal region of other starch synthases. The specific properties it confers may be important in defining the specificity of granule-bound starch synthase I in producing amylose in vivo. (+info)
Primary structure of a 21-kD protein from potato tubers.
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A 21-kD protein isolated earlier from potato tubers (Solanum tuberosum L.) has two isoforms, with pI 6.3 and 5.2, which were separated by fast protein ion-exchange chromatography on a Mono Q column. The primary structures of the two forms consisted of 187 and 186 amino acid residues. Both isoforms are composed of two polypeptide chains, designated A and B, linked by a single disulfide bond between Cys-146 of the A chain and Cys-7 of the B chain. The amino acid sequences of the A chains of the two forms, consisting of 150 residues each, differ in a single amino acid residue at position 52 (Val --> Ile), while the B chains, containing 37 and 36 residues, respectively, have substitutions at nine positions (Leu-8 --> Ser-8, Lys-25--Asp-26 --> Asn-25--Glu-26, Ile-31--Ser-32 --> Val-31--Leu-32, Lys-34--Gln-35--Val-36--Gln-37 --> Gln-34--Glu-35--Val-36). Both isoforms form stable inhibiting complexes with human leukocyte elastase and are less effective against chymotrypsin and trypsin. (+info)
A cysteine protease gene is expressed early in resistant potato interactions with Phytophthora infestans.
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A potato cysteine protease (cyp) cDNA expressed at an early stage of an incompatible interaction with Phytophthora infestans was isolated. Both the nucleotide and deduced amino acid sequences are highly homologous to those of a tomato cysteine protease, CYP1. Striking protein similarity to all known cathepsins in animals, particularly cathepsin K, was also observed. However, unlike cathepsins, a granulin binding domain is located near the carboxyl terminus of the putative CYP protein. In animals, granulins bind to receptors in the plasma membrane and signal cell growth and division. A ribonuclease protection assay demonstrated that the cyp gene is tightly regulated and is induced 15 h post inoculation with P. infestans in potato leaves either with high field resistance or in which a resistance (R) gene is activated. We conclude that a common signaling pathway is activated in each form of resistance. (+info)
Activation of the potato tuber ADP-glucose pyrophosphorylase by thioredoxin.
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The potato tuber (Solanum tuberosum L.) ADP-glucose pyrophosphorylase (ADP-GlcPPase) catalyzes the first committed step in starch biosynthesis. The main type of regulation of this enzyme is allosteric, and its activity is controlled by the ratio of activator, 3-phosphoglycerate to inhibitor, P(i). It was reported (Fu, Y., Ballicora, M. A., Leykam, J. F., and Preiss, J. (1998) J. Biol. Chem. 273, 25045-25052) that the enzyme was activated by reduction of the Cys(12) disulfide linkage present in the catalytic subunits. In this study, both reduced thioredoxin f and m from spinach (Spinacia oleracea) leaves reduced and activated the enzyme at low concentrations (10 microM) of activator (3-phosphoglycerate). Fifty percent activation was at 4.5 and 8.7 microM for reduced thioredoxin f and m, respectively, and 2 orders of magnitude lower than for dithiothreitol. The activation was reversed by oxidized thioredoxin. Cys(12) is conserved in the ADP-GlcPPases from plant leaves and other tissues except for the monocot endosperm enzymes. We postulate that in photosynthetic tissues, reduction could play a role in the fine regulation of the ADP-GlcPPase mediated by the ferredoxin-thioredoxin system. This is the first time that a covalent mechanism of regulation is postulated in the synthesis of starch. (+info)