Tissue-specific and developmental-specific expression of an Arabidopsis thaliana gene encoding the lipoamide dehydrogenase component of the plastid pyruvate dehydrogenase complex

We describe an Arabidopsis thaliana gene, ptlpd2, which codes for a protein with high amino acid similarity to lipoamide dehydrogenases (LPDs) from diverse species. Ptlpd2 codes for a precursor protein possessing an N-terminal extension predicted to be a plastid-targeting signal. Expression of the ptlpd2 cDNA in Escherichia coli showed the encoded protein possessed the predicted LPD activity. PTLPD2 protein, synthesized in vitro, was efficiently imported into isolated chloroplasts of Pisum sativum and shown to be located in the stroma. In addition, fusion proteins containing the predicted transit peptide of PTLPD2 or the entire protein fused at the N-terminus with the green fluorescent protein (GFP), showed accumulation in vivo in chloroplasts but not in mitochondria of A. thaliana. Expression of ptlpd2 was investigated by introducing ptlpd2 promoter--glucuronidase (GUS) gene fusions into Nicotiana tabacum. GUS expression was observed in seeds, flowers, root tips and young leaves. GUS activity was highest in mature seeds, decreased on germination and increased again in young leaves. Expression was also found to be temporally regulated in pollen grains where it was highest in mature grains at dehiscence. Database searches on ptlpd2 sequences identified a second A. thaliana gene encoding a putative plastidial LPD and two genes encoding proteins with high similarity to the mitochondrial LPD of P. sativum.     

Isolation and analysis of a genomic clone encoding a pokeweed antiviral protein

Partial cDNAs encoding a pokeweed antiviral protein were obtained by polymerase chain reaction from the poly(A)⁺ RNA of seeds, leaves, and roots using two specific primers based on the amino acid sequence of a pokeweed antiviral protein from the seeds (PAP-S). Using the cDNAs as a radioactive probe, 17 and 39 positive plaques were isolated from libraries containing the genomic DNA of Phytolacca americana digested with Bam HI partially and completely, respectively. The plaques were grouped into nine types by Southern hybridization. The type genomic clone encodes a protein of 294 amino acids. Its amino acid sequence is similar but not identical to that of PAP-S. A comparison of the two amino acid sequences suggested that the deduced protein contains extrapeptides of 24 and 9 amino acids at the NH₂ and the COOH terminals, respectively. The putative protein was expressed in Escherichia coli and shown to depurinate the specific adenine of wheat 25S rRNA, indicating that the protein encoded by a type genomic clone is a functional protein exhibiting RNA N-glycosidase activity.     

Systemic induction of proteinase-inhibitor-II gene expression in potato plants by wounding

The systemic induction of expression of the gene for proteinase inhibitor II after wounding different parts of potato (Solanum tuberosum L.) plants was analysed at the RNA level. Wounding of either leaves or tubers led to an induction of expression of this gene in non-wounded upper and lower leaves as well as in the upper stem segment, whereas no expression was observed in nonwounded roots or in the lower stem segment. The signal mediating the systemic induction in nonwounded tissue must therefore be able to move both acropetally and basipetally. The systemic wound response is specific for the expression of the proteinase-inhibitor-II gene as no influence was observed for the expression of genes encoding the small subunit of ribulose-1,5-bisphosphate carboxylase and the tuber storage protein patatin which were examined in parallel with the proteinase-inhibitor-II gene.Abbreviation ssRubisco small subunit of ribulose-1,5-bis-phosphate carboxylase     

Molecular cloning and characterization of novel isoforms of potato ADP-glucose pyrophosphorylase

ADP-glucose pyrophosphorylase (AGPase) is one of the major enzymes involved in starch biosynthesis in higher plants. We report here the molecular cloning of two cDNAs encoding so far uncharacterized isoforms (AGP S2 and AGP S3) of the potato enzyme. Sequence analysis shows that the two polypeptides are more homologous to previously identified large subunit polypeptides from potato and other plant species than to small subunit isoforms. This observation suggests that AGP S2 and AGP S3 represent novel large subunit polypeptides. agpS2 is expressed in several tissues of the potato plant, including leaves and tubers. Expression was stronger in sink leaves than in source leaves, indicating developmental regulation. In leaves, agpS2 expression was induced 2- to 3-fold by exogenous sucrose; therefore, agpS2 represents a new sucrose-responsive gene of starch metabolism. Expression of agpS3 was restricted to tubers: no agpS3 expression could be seen in leaves of different developmental stages, or when leaves were incubated in sucrose. Therefore, agpS3 represents the only AGPase gene so far characterized from potato, which is not expressed in leaves. Conversely, all four AGPase isoforms known from potato are expressed in tubers.     

The Bt gene <Emphasis Type="Italic">cry2Aa2</Emphasis> driven by a tissue specific ST-LS1 promoter from potato effectively controls <Emphasis Type="Italic">Heliothis virescens</Emphasis>

Expression of the Cry2Aa2 protein was targeted specifically to the green tissues of transgenic tobacco Nicotiana tabacum cv. Xanthi plants. This deployment was achieved by using the promoter region of the gene encoding the Solanum tuberosum leaf and stem specific (ST-LS1) protein. The accumulated levels of toxin in the leaves were found to be effective in achieving 100 mortality of Heliothis virescens larvae. The levels of Cry2Aa2 expression in the leaves of these transgenic plants were up to 0.21 of the total soluble proteins. Bioassays with R₁ transgenic plants indicated the inheritance of cry2Aa2 in the progeny plants. Tissue-specific expression of the Bt toxin in transgenic plants may help in controlling the potential occurrence of insect resistance by limiting the amount of toxin to only predated tissues. The results reported here validate the use of the ST-LS1 gene promoter for a targeted expression of Bt toxins in green tissues of plants.     

Molecular characterization of tobacco cDNAs encoding two small GTP-binding proteins

We have isolated two cDNAs encoding small GTP-binding proteins from leaf cDNA libraries. These cDNAs encode distinct proteins which show considerable homology to members of the ras superfamily. Np-ypt3, a 1044 bp long Nicotiana plumbaginifolia cDNA, encodes a 24.4 kDa protein which shows 65% amino acid sequence similarity to the Schizosaccharomyces pombe ypt3 protein. The N-ypt3 gene is differentially expressed in mature flowering plants. Expression of this gene is weak in leaves, higher in stems and roots, but highest in petals, stigmas and stamens. Nt-rab5, a 712 bp long Nicotiana tabacum SR1 cDNA, encodes a 21.9 kDa protein which displays 65% amino acid sequence similarity to mammalian rab5 proteins. The expression pattern of the Nt-rab5 gene is very similar to that of the Np-ypt3 gene. The Nt-rab5 gene is virtually not expressed in leaves, higher in stems and roots, and highest in flowers. Both the Nt-rab5 and Np-ypt3 proteins were expressed in Escherichia coli and shown to bind GTP.     

cDNA cloning and expression analysis of a mannose-binding lectin from <Emphasis Type="Italic">Pinellia pedatisecta</Emphasis>

Pinellia pedatisecta agglutinin (PPA) is a very basic protein that accumulates in the tuber of P. pedatisecta. PPA is a hetero-tetramer protein of 40 kDa, composed of two polypeptide chains A (about 12 kDa) and two polypeptides chains B (about 12 kDa). The full-length cDNA of PPA was cloned from P. pedatisecta using SMART RACE-PCR technology; it was 1146 bp and contained a 771 bp open reading frame (ORF) encoding a lectin precursor of 256 amino acid residues with a 24 amino acid signal peptide. The PPA precursor contained 3 mannose-binding sites (QXDXNXVXY) and two conserved domains of 43% identity, PPA-DOM₁ (polypeptides A) and PPA-DOM₂ (polypeptides B). PPA shared varying identities, ranging from 40% to 85%, with mannose-binding lectins from other species of plant families such as Araceae, Alliaceae, Iridaceae, Liliaceae, Amaryllidaceae and Bromeliaceae. Southern blot analysis indicated that ppa belonged to a multi-copy gene family. Expression pattern analysis revealed that ppa expressed in most tested tissues, with high expression being found in spadix, spathe and tuber. Cloning of the ppa gene not only provides a basis for further investigation of its structure, expression and regulatory mechanism, but also enables us to test its potential role in controlling pests and fungal diseases by transferring the gene into plants in the future.     

Arabidopsis thaliana Atvsp is homologous to soybean VspA and VspB,genes encoding vegetative storage protein acid phosphatases,and is regulated similarly by methyl jasmonate,wounding, sugars,light and phosphate

The soybean vegetative storage proteins, VSP and VSP, are acid phosphatases that accumulate to very high levels in hypocotyls, young leaves and flowers and pods. The genes encoding the soybean VSP are activated by jasmonate, wounding, sugars and light and down regulated by phosphate and auxin. In this study, expression of an Arabidopsis thaliana gene (Atvsp) encoding a protein homologous to soybean Vsp and Vsp, was examined and compared to expression of the soybean Vsp genes. Atvsp mRNA was present at high levels in flowers and buds and at low levels in roots, stems, leaves and siliques. Expression of Atvsp in leaves could be induced by wounding or by treatment of illuminated plants with methyl jasmonate and sucrose. Roots of plants with wounded leaves also accumulated Atvsp mRNA indicating that this gene can be regulated by a transmissible wound signal. Phosphate partially inhibited expression of Atvsp. Arabidopsis proteins of 29 and 30 kDa crossreacted with antibodies against soybean VSP. These proteins were very abundant in flowers and the proteins accumulated in leaves and roots of plants treated with methyl jasmonate. The level of these proteins in flowers was similar to the levels of soybean VSP in young soybean leaves. Overall, these data indicate that Arabidopsis Atvsp and soybean VspA/B genes are regulated similarly and that in both plants, the gene products can accumulate to high levels. This suggests that genes homologous to VspA/B may be of greater general significance than previously recognized.     

Molecular characterization of a phloem-specific gene encoding the filament protein, Phloem Protein 1 (PP1), from Cucurbita maxima

Sieve elements in the phloem of most angiosperms contain proteinaceous filaments and aggregates called P-protein. In the genus Cucurbita, these filaments are composed of two major proteins: PP1, the phloem filament protein, and PP2, the phloem lectin. The gene encoding the phloem filament protein in pumpkin (Cucurbita maxima Duch.) has been isolated and characterized. Nucleotide sequence analysis of the reconstructed gene gPP1 revealed a continuous 2430 bp protein coding sequence, with no introns, encoding an 809 amino acid polypeptide. The deduced polypeptide had characteristics of PP1 and contained a 15 amino acid sequence determined by N-terminal peptide sequence analysis of PP1. The sequence of PP1 was highly repetitive with four 200 amino acid sequence domains containing structural motifs in common with cysteine proteinase inhibitors. Expression of the PP1 gene was detected in roots, hypocotyls, cotyledons, stems, and leaves of pumpkin plants. PP1 and its mRNA accumulated in pumpkin hypocotyls during the period of rapid hypocotyl elongation after which mRNA levels declined, while protein levels remained elevated. PP1 was immunolocalized in slime plugs and P-protein bodies in sieve elements of the phloem. Occasionally, PP1 was detected in companion cells. PP1 mRNA was localized by in situ hybridization in companion cells at early stages of vascular differentiation. The developmental accumulation and localization of PP1 and its mRNA paralleled the phloem lectin, further suggesting an interaction between these phloem-specific proteins.     

HvPIP1;6, a barley (Hordeum vulgare L.) plasma membrane water channel particularly expressed in growing compared with non-growing leaf tissues

The aim of the present study was to identify water channel(s) which are expressed specifically in the growth zone of grass leaves and may facilitate growth-associated water uptake into cells. Previously, a gene had been described (HvEmip) which encodes a membrane intrinsic protein (MIP) and which is particularly expressed in the base 1 cm of barley primary leaves. The functionality of the encoding protein was not known. In the present study on leaf 3 of barley (Hordeum vulgare L.), a clone was isolated, termed HvPIP1;6, which has 99% amino acid sequence identity to HvEmip and belongs to the family of plasma membrane intrinsic proteins (PIPs). Expression of HvPIP1;6 was highest in the elongation zone, where it accounted for >85% of expression of known barley PIP1s. Within the elongation zone, faster grower regions showed higher expression than slower growing regions. Expression of HvPIP1;6 was confined to the epidermis, with some expression in neighboring mesophyll cells. Expression of HvPIP1;6 in Xenopus laevis oocytes increased osmotic water permeability 4- to 6-fold. Water channel activity was inhibited by pre-incubation of oocytes with 50 microM HgCl(2) and increased following incubation with the phosphatase inhibitor okadaic acid or the plant hormone ABA. Plasma membrane preparations were analyzed by Western blots using an antibody that recognized PIP1s. Levels of PIP1s were highest in the elongation and adjacent non-elongation zone. The developmental expression profile of HvPIP2;1, the only known barley water channel belonging to the PIP2 subgroup, was opposite to that of HvPIP1;6.     

Targeting and glycosylation of patatin the major potato tuber protein in leaves of transgenic tobacco

Patatin, the most abundant protein in the storage parenchyma cells of potato (Solanum tuberosum L.) tubers, is a vacuolar glycoprotein that consists of a number of closely related polypeptides and is encoded by a large gene family. To analyse the glycosylation pattern and the nature of the glycans on a single patatin polypeptide in a heterologous tissue we introduced a single chimaeric patatin gene into tobacco (Nicotiana tabacum L.) and studied its product in leaves. Patatin isolated from the leaves of transgenic tobacco plants is glycosylated at asparagine (Asn)⁶⁰, and Asn⁹⁰, but the third glycosylation site (Asn²⁰²) has no glycan. The two glycans are typical small complex glycans with xylose, fucose, mannose and N-acetylglucosamine in a ratio 1:1:3:2, the same ratio as found on patatin isolated from potato tubers. Expression of patatin in tobacco leaves was accompanied by the correct processing of the signal peptide, and the proper targeting of the glyco-protein to the vacuoles of mesophyll cells.Abbreviations Asn asparagine - ConA concanavalin A - EndoH endoglycosidase H - Fuc fucose - GlcNAc N-acetylglucosamine - HPLC high-performance liquid chromatography - Man mannose - PAGE polyacrylamide gel electrophoresis - SDS sodium dodecyl-sulfate - Ser serine - TFMS trifluoromethanesulfonic acid - Thr threonine - Xyl xylose     

Isolation and Characterization of cDNAs Encoding Ribosome Inactivating Protein from Dianthus sinensis L.

To isolate a ribosome inactivating protein (RIP) gene, six plant species were surveyed for antiviral activity. Crude proteins extracted from these plants were tested for the antiviral activity against tobacco mosaic virus (TMV) in Nicotiana glutinosa. All the plants, Spinacia oleracea, Amaranthus lividus, Dianthus superbus, Dianthus sinensis and Celosia cristata, with an exception of Oenanthe stolonifera, presented 70–90% inhibition of viral infectivity. In an attempt to search for the RIP gene from D. sinensis, partial cDNA was obtained by polymerase chain reaction (PCR) of the poly(A)⁺ RNA from D. sinensis leaves. DNA gel blot analysis showed that D. sinensis has multi-copy RIP genes. The expression of RIP gene was investigated in the flower, leaf, root and stem of D. sinensis, and was found to be most abundant in the leaf. Using the partial cDNA as a probe, seven full-length cDNAs were isolated from a library prepared from D. sinensis leaves. They were divided into three groups on the basis of their nucleotide sequence homology. The three representative clones, cDsRIP1, cDsRIP2 and cDsRIP3 were completely sequenced. They all had an open reading frame of 882 bp. The cDsRIP2 showed 79% homology with dianthin 30 and saporin genes; 59% with PAP and Mirabilis antiviral protein MAP genes. From the analysis of deduced amino acid sequences, it was predicted that D. sinensis RIP cDNAs might have a putative signal peptide of 23 amino acid residues at their N-terminus. When the cDNA was expressed in E. coli, the bacteria was unable to grow upon IPTG induction, suggesting that expression of the gene renders toxicity to E. coli cells.     

Ultraviolet light and ozone stimulate accumulation of salicylic acid,pathogenesis-related proteins and virus resistance in tobacco

In tobacco (Nicotiana tabacum L. cv. Xanthinc), salicylic acid (SA) levels increase in leaves inoculated by necrotizing pathogens and in healthy leaves located above the inoculated site. Systemic SA increase may trigger disease resistance and synthesis of pathogenesis-related proteins (PR proteins). Here we report that ultraviolet (UV)-C light or ozone induced biochemical responses similar to those induced by necrotizing pathogens. Exposure of leaves to UV-C light or ozone resulted in a transient ninefold increase in SA compared to controls. In addition, in UV-light-irradiated plants, SA increased nearly fourfold to 0.77 g·g^–1 fresh weight in leaves that were shielded from UV light. Increased SA levels were accompanied by accumulation of an SA conjugate and by an increase in the activity of benzoic acid 2-hydroxylase which catalyzes SA biosynthesis. In irradiated and in unirradiated leaves of plants treated with UV light, as well as in plants fumigated with ozone, PR proteins 1a and 1b accumulated. This was paralleled by the appearance of induced resistance to a subsequent challenge with tobacco mosaic virus. The results suggest that UV light, ozone fumigation and tobacco mosaic virus can activate a common signal-transduction pathway that leads to SA and PR-protein accumulation and increased disease resistance.Abbreviations PR protein pathogenesis-related protein - SA salicylic acid - TMV tobacco mosaic virus - UV ultraviolet This work was financed by grants from the U.S. Department of Agriculture (Competitive Research Grants Office), Division of Energy Biosciences of U.S. Department of Energy, the Rockefeller Foundation, the New Jersey Commission for Science and Technology, and the New Jersey Agricultural Experiment Station.     

The type-1 and type-2 ribosome-inactivating proteins from<Emphasis Type="Italic"> Iris</Emphasis> confer transgenic tobacco plants local but not systemic protection against viruses

The antiviral activity of the type-2 ribosome-inactivating protein (RIP) IRAb from Iris was analyzed by expressing IRAb in tobacco (Nicotiana tabacum L. cv. Samsun NN) plants and challenging the transgenic plants with tobacco mosaic virus (TMV). Although constitutive expression of IRAb resulted in an aberrant phenotype, the plants were fertile. Transgenic tobacco lines expressing IRAb showed a dose-dependent enhanced resistance against TMV infection but the level of protection was markedly lower than in plants expressing IRIP, the type-1 RIP from Iris that closely resembles the A-chain of IRAb. To verify whether IRIP or IRAb can also confer systemic protection against viruses, transgenic RIP-expressing scions were grafted onto control rootstocks and leaves of the rootstocks challenged with tobacco etch virus (TEV). In spite of the strong local antiviral effect of IRIP and IRAb the RIPs could not provide systemic protection against TEV. Hence our results demonstrate that expression of the type-1 and type-2 RIPs from Iris confers tobacco plants local protection against two unrelated viruses. The antiviral activity of both RIPs was not accompanied by an induction of pathogenesis-related proteins. It is suggested that the observed antiviral activity of both Iris RIPs relies on their RNA N-glycohydrolase activity towards TMV RNA and plant rRNA.Abbreviations GUS -Glucuronidase - IRAb Iris agglutinin b - IRIP Iris type-1 RIP - PAG Polynucleotide:adenosine glycosylase - PAP Phytolacca americana antiviral protein - PR Pathogenesis-related - RIP Ribosome-inactivating protein - TCS Trichosanthin - TEV Tobacco etch virus - TMV Tobacco mosaic virus     

Presence of indole-3-acetic acid in chloroplasts ofNicotiana tabacum andPinus sylvestris

The compartmentation and metabolism of indole-3-acetic acid (IAA) was examined in protoplasts derived from needles ofPinus sylvestris L., leaves of normal plants ofNicotiana tabacum L., leaves ofN. tabacum plants carrying the T-DNA gene 1 (rG1 plants) and leaves ofN. tabacum plants carrying the T-DNA gene 2 (rG2 plants) by using a rapid cell-fractionation method. In all tissues, 30%–40% of the IAA pool was located in the chloroplast, while the remainder was found in the cytosol. Quantitative analysis of indole-3-ethanol (IEt) showed that in bothPinus andNicotiana the IEt pool was located exclusively in the cytosol. The only plant that contained endogenous indoleacetamide (IAAm) was therG1-mutant ofN. tabacum, expressing theAgrobacterium tumefaciens T-DNA gene 1. Cellular fractionation of protoplasts from this transgenic plant showed that the entire IAAm pool was located in the cytosol. Feeding experiments utilizing [5-³H]tryptophan, [5-³H]IEt, [1′-¹⁴C] and [2′-¹⁴C]IAA demonstrated that the biosynthesis and catabolism of IAA occurred in the cytosol in bothPinus and in the wild type and the different mutants ofNicotiana. Furthermore, the biosynthesis of IAAm in therG1 plants was also shown to be localized in the cytosol.     

A plasma membrane zinc transporter from Medicago truncatula is up-regulated in roots by Zn fertilization,yet down-regulated by arbuscular mycorrhizal colonization

Here we present a Zn transporter cDNA named MtZIP2 from the model legume Medicago truncatula. MtZIP2 encodes a putative 37 kDa protein with 8-membrane spanning domains and has moderate amino acid identity with the Arabidopsis thaliana Zn transporter AtZIP2p. MtZIP2 complemented a Zn-uptake mutant of yeast implying that the protein encoded by this gene can transport Zn across the yeast's plasma membrane. The product of a MtZIP2-GFP fusion construct introduced into onion cells by particle bombardment likewise localized to the plasma membrane. The MtZIP2 gene was expressed in roots and stems, but not in leaves of M. truncatula and, in contrast to all other plant Zn transporters characterized thus far, MtZIP2 was up-regulated in roots by Zn fertilization. Expression was highest in roots exposed to a toxic level of Zn. MtZIP2 expression was also examined in the roots of M. truncatula when colonized by the obligate plant symbiont, arbuscular mycorrhizal (AM) fungi, since AM fungi are renowned for their ability to supply plants with mineral nutrients, including Zn. Expression was down-regulated in the roots of the mycorrhizal plants and was associated with a reduced level of Zn within the host plant tissues.     

Cloning,expression in yeast,and functional characterization of CYP71D14, a root-specific cytochrome P450 from <Emphasis Type="Italic">Petunia hybrida</Emphasis>

Alkaloids, which are naturally occurring amines, are biosynthesized and accumulated in plant tissues such as roots, leaves, and stems. Many alkaloids have pharmacological effects on humans and animals. Cytochrome P450 (P450 or CYP) monooxygenases are known to play key roles in the biosynthesis of alkaloids in higher plants. A cDNA clone encoding a P450 protein consisting of 502 amino acids was isolated from Petunia hybrida. The deduced amino acid sequence of the cDNA clone showed a high level of similarity with the other P450 species in the CYP71D family; hence, this novel P450 was named CYP71D14. Among plant P450 species, CYP71D14 had 45.7% similarity in its amino acid sequence to CYP71D12, which is involved in the biosynthesis of the indole alkaloids vinblastine and vincristine. Expression of CYP71D14 mRNA in Petunia plants was examined by Northern blot analysis by using a full-length cDNA of CYP71D14 as a probe. CYP71D14 mRNA was expressed most abundantly in the roots. The nucleotide sequence of CYP71D14 has been submitted to the DDBJ, EMBL, and GenBank nucleotide databases under the accession number AB028462. An erratum to this article can be found at