Polyethylene glycol (PEG)-mediated transient gene expression in a red alga, Cyanidioschyzon merolae 10D

DNA introduction into cells is an essential technique for molecular genetic analysis. Here, we show that DNA is easily introduced into cells of the unicellular red alga Cyanidioschyzon merolae by a polyethylene glycol (PEG)-mediated protocol. In this study, the beta-tubulin gene of C. merolae was cloned on a plasmid and a hemagglutinin (HA) tag then added at the C-terminus. This plasmid was then introduced into C. merolae cells by a PEG-mediated transformation protocol. At 24 h after PEG-mediated transformation, intracellular localization of the tagged protein was detected by anti-HA immunocytochemistry, indicating the utility of this transient expression system for molecular genetic analyses.     

Bowman-birk proteinase inhibitor confers heavy metal and multiple drug tolerance in yeast

Cultured Coptis japonica cells show tolerance to various toxic compounds. By yeast functional screening of cadmium (Cd) plates with its cDNA library, we isolated a gene encoding Bowman-Birk proteinase inhibitor (CjBBI). The yeast transformant of CjBBI showed multiple tolerance to various drugs adding to Cd, and revealed reduced Cd accumulation in cells. Preferential organs for Cjbbi expression were aerial parts of intact plants, and the subcellular localization of CjBBI was shown, using its green fluorescent protein fusion, to be the apoplast. Induction of Cjbbi expression by Cd treatment suggested that CjBBI was responsible for the tolerance to Cd observed in C. japonica cells.     

Ethylene promotes submergence-induced expression of OsABA8ox1, a gene that encodes ABA 8'-hydroxylase in rice

A rapid decrease of the plant hormone ABA under submergence is thought to be a prerequisite for the enhanced elongation of submerged shoots of rice (Oryza sativa L.). Here, we report that the level of phaseic acid (PA), an oxidized form of ABA, increased with decreasing ABA level during submergence. The oxidation of ABA to PA is catalyzed by ABA 8'-hydroxylase, which is possibly encoded by three genes (OsABA8ox1, -2 and -3) in rice. The ABA 8'-hydroxylase activity was confirmed in microsomes from yeast expressing OsABA8ox1. OsABA8ox1-green fluorescent protein (GFP) fusion protein in onion cells was localized to the endoplasmic reticulum. The mRNA level of OsABA8ox1, but not the mRNA levels of other OsABA8ox genes, increased dramatically within 1 h after submergence. On the other hand, the mRNA levels of genes involved in ABA biosynthesis (OsZEP and OsNCEDs) decreased after 1-2 h of submergence. Treatment of aerobic seedlings with ethylene and its precursor, 1-aminocyclopropane-1-carboxylate (ACC), rapidly induced the expression of OsABA8ox1, but the ethylene treatment did not strongly affect the expression of ABA biosynthetic genes. Moreover, pre-treatment with 1-methylcyclopropene (1-MCP), a potent inhibitor of ethylene action, partially suppressed induction of OsABA8ox1 expression under submergence. The ABA level was found to be negatively correlated with OsABA8ox1 expression under ACC or 1-MCP treatment. Together, these results indicate that the rapid decrease in ABA levels in submerged rice shoots is controlled partly by ethylene-induced expression of OsABA8ox1 and partly by ethylene-independent suppression of genes involved in the biosynthesis of ABA.     

Bacterial lipopolysaccharides induce defense responses associated with programmed cell death in rice cells

PAMP (pathogen-associated molecular pattern) recognition plays an important role during the innate immune response in both plants and animals. Lipopolysaccharides (LPS) derived from Gram-negative bacteria are representative of typical PAMP molecules and have been reported to induce defense-related responses, including the suppression of the hypersensitive response, the expression of defense genes and systemic resistance in plants. However, the details regarding the precise molecular mechanisms underlying these cellular responses, such as the molecular machinery involved in the perception and transduction of LPS molecules, remain largely unknown. Furthermore, the biological activities of LPS on plants have so far been reported only in dicots and no information is thus available regarding their functions in monocots. In our current study, we report that LPS preparations for various becteria, including plant pathogens and non-pathogens, can induce defense responses in rice cells, including reactive oxygen generation and defense gene expression. In addition, global analysis of gene expression induced by two PAMPs, LPS and chitin oligosaccharide, also reveals a close correlation between the gene responses induced by these factors. This indicates that there is a convergence of signaling cascades downstream of their corresponding receptors. Furthermore, we show that the defense responses induced by LPS in the rice cells are associated with programmed cell death (PCD), which is a finding that has not been previously reported for the functional role of these molecules in plant cells. Interestingly, PCD induction by the LPS was not detected in cultured Arabidopsis thaliana cells.     

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.     

Tissue and cell-specific localization of rice aquaporins and their water transport activities

Water transport in plants is greatly dependent on the expression and activity of water transport channels, called aquaporins. Here, we have clarified the tissue- and cell-specific localization of aquaporins in rice plants by immunoblotting and immunocytochemistry using seven isoform-specific aquaporin antibodies. We also examined water transport activities of typical aquaporin family members using a yeast expression system in combination with a stopped-flow spectrophotometry assay. OsPIP1 members, OsPIP2;1, OsTIP1;1 and OsTIP2;2 were expressed in both leaf blades and roots, while OsPIP2;3, OsPIP2;5 and OsTIP2;1 were expressed only in roots. In roots, large amounts of aquaporins accumulated in the region adjacent to the root tip (around 1.5-4 mm from the root tip). In this region, cell-specific localization of the various aquaporin members was observed. OsPIP1 members and OsTIP2;2 accumulated predominantly in the endodermis and the central cylinder, respectively. OsTIP1;1 showed specific localization in the rhizodermis and exodermis. OsPIP2;1, OsPIP2;3 and OsPIP2;5 accumulated in all root cells, but they showed higher levels of accumulation in endodermis than other cells. In the region at 35 mm from the root tip, where aerenchyma develops, aquaporins accumulated at low levels. In leaf blades, OsPIP1 members and OsPIP2;1 were localized mainly in mesophyll cells. OsPIP2;1, OsPIP2;3, OsPIP2;5 and OsTIP2;2 expressed in yeast showed high water transport activities. These results suggest that rice aquaporins with various water transport activities may play distinct roles in facilitating water flux and maintaining the water potential in different tissues and cells.     

The plant growth-promoting fungus Penicillium simplicissimum GP17-2 induces resistance in Arabidopsis thaliana by activation of multiple defense signals

Arabidopsis thaliana grown in soil amended with barley grain inocula of Penicillium simplicissimum GP17-2 or receiving root treatment with its culture filtrate (CF) exhibited clear resistance to Pseudomonas syringae pv. tomato DC3000 (Pst). To assess the contribution of different defense pathways, Arabidopsis genotypes implicated in salicylic acid (SA) signaling expressing the NahG transgene or carrying disruption in NPR1 (npr1), jasmonic acid (JA) signaling (jar1) and ethylene (ET) signaling (ein2) were tested. All genotypes screened were protected by GP17-2 or its CF. However, the level of protection was significantly lower in NahG and npr1 plants than it was in similarly treated wild-type plants, indicating that the SA signaling pathway makes a minor contribution to the GP17-2-mediated resistance and is insufficient for a full response. Examination of local and systemic gene expression revealed that GP17-2 and its CF modulate the expression of genes involved in both the SA and JA/ET signaling pathways. Subsequent challenge of GP17-2-colonized plants with Pst was accompanied by direct activation of SA-inducible PR-2 and PR-5 genes as well as potentiated expression of the JA-inducible Vsp gene. In contrast, CF-treated plants infected with Pst exhibited elevated expression of most defense-related genes (PR-1, PR-2, PR-5, PDF1.2 and Hel) studied. Moreover, an initial elevation of SA responses was followed by late induction of JA responses during Pst infection of induced systemic resistance (ISR)-expressing plants. In conclusion, we hypothesize the involvement of multiple defense mechanisms leading to an ISR of Arabidopsis by GP17-2.     

Transcriptional Induction of Two Genes for CCaPs, Novel Cytosolic Proteins, in Arabidopsis thaliana in the Dark

Ca2+-signaling in downstream effectors is supported by many kinds of Ca2+-binding proteins, which function as a signal mediator and a Ca2+-buffering protein. We found in Arabidopsis thaliana a new type of Ca2+-binding protein, CCaP1, which consists of 152 amino acid residues, and binds (45)Ca2+ even in the presence of a high concentration of Mg2+. We found two other proteins with similar motifs, CCaP2 and CCaP3. These three proteins had no organelle localization signal and their green fluorescent protein (GFP) fusions were detected in the cytosol. Real-time PCR and histochemical analysis of promoter-beta-glucuronidase fusions revealed that CCaP1 was predominantly expressed in petioles while CCaP2 was expressed in roots. CCaP3 was hardly expressed. Expression of CCaP1 and CCaP2 was enhanced in darkness and became maximal after 24 h. Immunoblotting revealed petiole-specific accumulation of CCaP1. Expression of CCaP1 and CCaP2 was suppressed by a high concentration of Ca2+ and other metal ions. Deletion of sucrose from the medium markedly increased the mRNA levels of CCaP1 and CCaP2 within 2 h. Gibberellic acid enhanced the expression of CCaP1 and CCaP2 by 5- and 2.5-fold, respectively, after 6 h. CCaP1 and CCaP2 were suppressed in the petiole and the root, respectively, by light and the product of photosynthesis (sucrose) or both. These results suggest that CCaP1 functions as a mediator in response to continuous dark or gibberellic acid.     

The FLX gene of Arabidopsis is required for FRI-dependent activation of FLC expression

The Arabidopsis FLOWERING LOCUS C (FLC) gene encodes a MADS box protein that acts as a dose-dependent repressor of flowering. Mutants and ecotypes with elevated expression of FLC are late flowering and vernalization responsive. In this study we describe an early flowering mutant in the C24 ecotype, flc expressor (flx), that has reduced expression of FLC. FLX encodes a protein of unknown function with putative leucine zipper domains. FLX is required for FRIGIDA (FRI)-mediated activation of FLC but not for activation of FLC in autonomous pathway mutants. FLX is also required for expression of the FLC paralogs MADS AFFECTING FLOWERING 1 (MAF1) and MAF2.     

Flowering in tobacco needs gibberellins but is not promoted by the levels of active GA1 and GA4 in the apical shoot

Flowering of Nicotiana tabacum cv Xhanti depends on gibberellins because gibberellin-deficient plants, due to overexpression of a gibberellin 2-oxidase gene (35S:NoGA2ox3) or to treatment with the gibberellin biosynthesis inhibitor paclobutrazol, flowered later than wild type. These plants also showed inhibition of the expression of molecular markers related to floral transition (NtMADS-4 and NtMADS-11). To investigate further the role of gibberellin in flowering, we quantified its content in tobacco plants during development. We found a progressive reduction in the levels of GA1 and GA4 in the apical shoot during vegetative growth, reaching very low levels at floral transition and beyond. This excludes these two gibberellins as flowering-promoting factors in the apex. The evolution of active gibberellin content in apical shoots agrees with the expression patterns of gibberellin metabolism genes: two encoding gibberellin 20-oxidases (NtGA20ox1 = Ntc12, NtGA20ox2 = Ntc16), one encoding a gibberellin 3-oxidase (NtGA3ox1 = Nty) and one encoding a gibberellin 2-oxidase (NtGA2ox1), suggesting that active gibberellins are locally synthesized. In young apical leaves, GA1 and GA4 content and the expression of gibberellin metabolism genes were rather constant. Our results support that floral transition in tobacco, in contrast to that in Arabidopsis, is not regulated by the levels of GA1 and GA4 in apical shoots, although reaching a threshold in gibberellin levels may be necessary to allow meristem competence for flowering.     

AtAMT1;4, a Pollen-Specific High-Affinity Ammonium Transporter of the Plasma Membrane in Arabidopsis

Pollen represents an important nitrogen sink in flowers to ensurepollen viability. Since pollen cells are symplasmically isolatedduring maturation and germination, membrane transporters arerequired for nitrogen import across the pollen plasma membrane.This study describes the characterization of the ammonium transporterAtAMT1;4, a so far uncharacterized member of the ArabidopsisAMT1 family, which is suggested to be involved in transportingammonium into pollen. The AtAMT1;4 gene encodes a functionalammonium transporter when heterologously expressed in yeastor when overexpressed in Arabidopsis roots. Concentration-dependentanalysis of ¹⁵N-labeled ammonium influx into roots of AtAMT1;4-transformedplants allowed characterization of AtAMT1;4 as a high-affinitytransporter with a K_m of 17 µM. RNA and protein gel blotanalysis showed expression of AtAMT1;4 in flowers, and promoter–genefusions to the green fluorescent protein (GFP) further definedits exclusive expression in pollen grains and pollen tubes.The AtAMT1;4 protein appeared to be localized to the plasmamembrane as indicated by protein gel blot analysis of plasmamembrane-enriched membrane fractions and by visualization ofGFP-tagged AtAMT1;4 protein in pollen grains and pollen tubes.However, no phenotype related to pollen function could be observedin a transposon-tagged line, in which AtAMT1;4 expression isdisrupted. These results suggest that AtAMT1;4 mediates ammoniumuptake across the plasma membrane of pollen to contribute tonitrogen nutrition of pollen via ammonium uptake or retrieval.     

Expression and RNA interference-induced silencing of the dammarenediol synthase gene in Panax ginseng

Panax ginseng is one of the most highly valued herbal medicines in the Orient, where it has gained an almost magical reputation for being able to maintain the quality of life. The root of ginseng contains noble tetracyclic triterpenenoid saponins, which are thought to be the major effective ingredients in P. ginseng. The first committed step in ginsenoside synthesis is the cyclization of 2,3-oxidosqualene to dammarenediol II by oxidosqualene cyclase, dammarenediol synthase (DDS). The gene encoding DDS has been characterized. Here, we investigated the expression of the DDS gene together with the genes involved in ginsenoside biosynthesis (SS, SE, PNX, PNY, PNY2 and PNZ). Expression of DDS mRNA was higher in flower buds compared with root, leaf and petiole of ginseng plants. Elicitor (methyl jasmonate) treatment up-regulated the expression of DDS mRNA. Ectopic expression of DDS in a yeast mutant (erg7) lacking lanosterol synthase resulted in the production of dammarenediol and hydroxydammarenone which were confirmed by liquid chromatography-atmospheric pressure chemical ionization mass spectrometry (LC/APCIMS). RNA interference (RNAi) of DDS in transgenic P. ginseng resulted in silencing of DDS expression which leads to a reduction of ginsenoside production to 84.5% in roots. These results indicate that expression of DDS played a vital role in the biosynthesis of ginsenosides in P. ginseng.