Molecular Cloning of Osmotin and Regulation of Its Expression by ABA and Adaptation to Low Water Potential

In response to adaptation to NaCl, cultured tobacco cells (Nicotiana tabacum L. cv Wisconsin 38) synthesize a major 26 kilodalton protein which has been named osmotin due to its induction by low water potentials. To help characterize the expression of osmotin in adapted cells, a cDNA clone for osmotin has been isolated. Abscisic acid induces messenger RNA encoding osmotin. Levels of this mRNA in adapted cells are approximately 15-fold higher than in unadapted cells. Message for osmotin is present at constant levels through the growth cycle of adapted cells, while in unadapted cells, the level decreases during exponential phase of growth and increases again when the cells approach stationary phase. While abscisic acid induces the message for osmotin, a low water potential environment appears to be required for accumulation of the protein. An osmotic shock to unadapted cells does not increase the amount of message or protein present most likely because this treatment does not induce immediately the accumulation of abscisic acid. The increased expression of osmotin in adapted cells is not correlated with an increase in osmotin gene copy number. Osmotin is homologous to a 24 kilodalton NaCl-induced protein in tomato, as well as thaumatin, maize α-amylase/trypsin inhibitor and a tobacco mosaic virus-induced pathogenesis-related protein.     

Stable NaCl Tolerance of Tobacco Cells Is Associated with Enhanced Accumulation of Osmotin

Osmotin is a major protein which accumulates in tobacco cells (Nicotiana tabacum L. var Wisconsin 38) adapted to low water potentials. Quantitation of osmotin levels by immunoblots indicated that cells adapted to 428 millimolar NaCl contained 4 to 30 times the level of osmotin found in unadapted cells, depending on the stage of growth. Unadapted cells accumulated low levels of osmotin with apparent isoelectric points, (pl) of 7.8 and >8.2. Upon transfer of NaCl-adapted cells to medium without NaCl and subsequent growth for many cell generations, the amount of osmotin declined gradually to a level intermediate between that found in adapted and unadapted cells. NaCl-adapted cells grown in the absence of NaCl accumulated both pl forms; however, the form accumulated by cells adapted to NaCl (pl > 8.2) was most abundant. Adapted cells grown in the absence of NaCl exhibited absolute growth rates and NaCl tolerance levels which were intermediate to those of NaCl-adapted and unadapted cells. The association between osmotin accumulation and stable NaCl tolerance indicates that cells with a stable genetic change affecting the accumulation of osmotin are selected during prolonged exposure to high levels of NaCl. This stable alteration in gene expression probably affects salt tolerance.     

Plant Defense Genes Are Synergistically Induced by Ethylene and Methyl Jasmonate

Combinations of ethylene and methyl jasmonate (E/MeJA) synergistically induced members of both groups 1 and 5 of the pathogenesis-related (PR) superfamily of defense genes. E/MeJA caused a synergistic induction of PR-1b and osmotin (PR-5) mRNA accumulation in tobacco seedlings. E/MeJA also synergistically activated the osmotin promoter fused to a [beta]-glucuronidase marker gene in a tissue-specific manner. The E/MeJA responsiveness of the osmotin promoter was localized on a -248 to +45 fragment that exhibited responsiveness to several other inducers. E/MeJA induction also resulted in osmotin protein accumulation to levels similar to those induced by osmotic stress. Of the several known inducers of the osmotin gene, including salicylic acid (SA), fungal infection is the only other condition known to cause substantial osmotin protein accumulation in Wisconsin 38, a tobacco cultivar that does not respond hypersensitively to tobacco mosaic virus. Based on the ability of the protein kinase C inhibitor 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine to block ethylene induction of PR-1b mRNA accumulation and its inability to block osmotin mRNA induction by ethylene, these two PR gene groups appeared to have at least partially separate signal transduction pathways. Stimulation of osmotin mRNA accumulation by okadaic acid indicated that another protein kinase system is involved in regulation of the osmotin gene. SA, which is known to induce pathogen resistance in tobacco, could not induce the osmotin gene as much as E/MeJA and neither could it induce PR-1b as much as SA and MeJA combined.     

Osmotin gene expression is controlled by elicitor synergism

Arachidonic acid (AA), a fatty-acid fungal elicitor, and a cellulase preparation from Aspergillus niger , a protein-type fungal elicitor, induced osmotin gene expression. Both elicitors activated the osmotin promoter fused to a β-glucuronidase (GUS) reporter gene in a tissue-specific manner in tobacco seedlings ( Nicotiana tabacum L. cv. Wisconsin 38). The cellulase preparation was more effective than AA at the concentrations tested and, unlike AA, also induced the accumulation of osmotin mRNA and protein. Combinations of AA and the cellulase preparation had a greater than additive effect on the activation of the osmotin promoter and the accumulation of osmotin mRNA and protein. Both AA and the cellulase preparation, when applied separately, were virtually ineffective in the induction of the osmotin promoter in cotyledon tissues. However, together they were able to induce synergistically GUS fused to the osmotin promoter. Increases in osmotin-promoter-driven GUS activity and accumulation of osmotin mRNA induced by AA, the cellulase preparation or their combination were reversed by norbornadiene, an ethylene action inhibitor, indicating that ethylene is involved in the induction of the osmotin gene by these elicitors.     

Enhanced expression of the human CD14 protein in tobacco using a 22-kDa alpha-zein signal peptide

The human CD14, a high affinity receptor for lipopolysaccharides (LPS), is involved in the innate immunity system and the inflammatory response. There is increasing interest in using recombinant approaches to produce purified CD14 protein for therapeutic uses. Plants provide ideal expression systems for the production of recombinant proteins, but the levels of expression of recombinant proteins produced in planta are still not high. To improve expression levels of CD14 the 22-kDa alpha-zein signal peptide (ZSP) from maize was fused to the human CD14 cDNA so that recombinant CD14 could stably accumulate in plant cells. The human CD14 gene and the modified human CD14 cDNA with the 22-kDa ZSP were respectively transformed into tobacco to produce transgenic plants. Western blot analysis confirmed human CD14 accumulation in the transgenic tobacco. The concentration of the recombinant protein in the tobacco leaves was measured by ELISA, and the results suggested that fusion with the 22-kDa alpha-ZSP effectively increased the accumulation of the recombinant protein (rCD14). The concentration of rCD14 in some of the transgenic lines was 19.54???g?g^?1 tobacco leaf (fw), which was about 0.6?% of the total soluble protein. The rCD14 protein showed natural LPS-binding bioactivity by using U937 cells mensuration. Our results suggested that the maize 22-kDa alpha-zein signal peptide could be used to increase the accumulation of recombinant protein in tobacco leaves so that proteins can be produced in abundant biomass.     

Characterization of osmotin : a thaumatin-like protein associated with osmotic adaptation in plant cells

Cultured tobacco (Nicotiana tabacum var Wisconsin 38) cells adapted to grow under osmotic stress synthesize and accumulate a 26 kilodalton protein (osmotin) which can constitute as much as 12% of total cellular protein. In cells adapted to NaCl, osmotin occurs in two forms: an aqueous soluble form (osmotin-I) and a detergent soluble form (osmotin II) in the approximate ratio of 2:3. Osmotin-I has been purified to electrophoretic homogeneity, and osmotin-II has been purified to 90% electrophoretic homogeneity. The N-terminal amino acid sequences of osmotins I and II are identical through position 22. Osmotin-II appears to be much more resistant to proteolysis than osmotin-I. However, it cross-reacts with polyclonal antibodies raised in rabbits against osmotin-I. Osmotin strongly resembles the sweet protein thaumatin in its molecular weight, amino acid composition, N-terminal sequence, and the presence of a signal peptide on the precursor protein. Thaumatin does not cross-react with antiosmotin. An osmotin solution could not be detected as sweet at a concentration at least 100 times that of thaumatin which could be detected as sweet. Immunocytochemical detection of osmotin revealed that osmotin is concentrated in dense inclusion bodies within the vacuole. Although antiosmotin did not label organelles, cell walls, or membranes, osmotin appeared sparsely distributed in the cytoplasm.     

Expression of the heparan sulfate proteoglycan, perlecan, during mouse embryogenesis and perlecan chondrogenic activity in vitro.

Expression of the basement membrane heparan sulfate proteoglycan (HSPG), perlecan (Pln), mRNA, and protein has been examined during murine development. Both Pln mRNA and protein are highly expressed in cartilaginous regions of developing mouse embryos, but not in areas of membranous bone formation. Initially detected at low levels in precartilaginous areas of d 12.5 embryos, Pln protein accumulates in these regions through d 15.5 at which time high levels are detected in the cartilage primordia. Laminin and collagen type IV, other basal lamina proteins commonly found colocalized with Pln, are absent from the cartilage primordia. Accumulation of Pln mRNA, detected by in situ hybridization, was increased in d 14.5 embryos. Cartilage primordia expression decreased to levels similar to that of the surrounding tissue at d 15.5. Pln accumulation in developing cartilage is preceded by that of collagen type II. To gain insight into Pln function in chondrogenesis, an assay was developed to assess the potential inductive activity of Pln using multipotential 10T1/2 murine embryonic fibroblast cells. Culture on Pln, but not on a variety of other matrices, stimulated extensive formation of dense nodules reminiscent of embryonic cartilaginous condensations. These nodules stained intensely with Alcian blue and collagen type II antibodies. mRNA encoding chondrocyte markers including collagen type II, aggrecan, and Pln was elevated in 10T1/2 cells cultured on Pln. Human chondrocytes that otherwise rapidly dedifferentiate during in vitro culture also formed nodules and expressed high levels of chondrocytic marker proteins when cultured on Pln. Collectively, these studies demonstrate that Pln is not only a marker of chondrogenesis, but also strongly potentiates chondrogenic differentiation in vitro.     

Cytokinin stress changes the developmental regulation of several defence-related genes in tobacco

Tobacco shoots exposed to elevated endogenous or exogenous cytokinin levels are unable to develop roots and lack apical dominance. We have isolated cDNA copies of five mRNA species that accumulate to elevated levels in such cytokinin-stressed shoots via differential screening of a cDNA library of transgenic shoots which contain an active T-DNA cytokinin gene (T-cyt gene) from Agrobacterium tumefaciens. Four of the cDNA clones were found to correspond to plant defence-related mRNAs, encoding extensin, chitinase, PR-1 and a PR-1-like protein, respectively. In normal tobacco plants PR-1 mRNA is relatively rare in all organs. The other four mRNAs occur at relatively low levels in shoots, especially in leaves, but are very prevalent in roots. Extensin mRNA, for example, is not detectable in leaves, while it is an abundant mRNA in roots and stems. In normal shoots cultured on cytokinin-containing medium all five mRNAs accumulate to elevated levels, similar to those found in transgenic T-cyt shoots. We conclude that the imposed cytokinin stress causes changes in the tissue-specific control of the levels of several defence-related mRNA species in tobacco.     

Proteins Produced during Salt Stress in Tobacco Cell Culture

The protein pattern of cultured tobacco (Nicotiana tabacum L. var Wisconsin 38) cells that have become adapted to a medium containing 10 grams NaCl per liter was compared to that of unadapted cells on one-dimensional sodium dodecyl sulfate gels. Two protein bands (32,000 and 20,000 daltons) were much more abundant in the salt-adapted cells, and one protein (26,000 daltons) was unique to the salt cells. This protein pattern did not change during the growth cycle of the cells. When salt-adapted cells are transferred to control medium, their ability to grow in the salt-containing medium returns to that of control cells after one passage in the control medium (Hasegawa, Bressan, Handa 1980 Plant Cell Physiol 21: 1347). Within this time the levels of the 32,000 and 20,000 dalton proteins also return to that of the control cells, but the 26,000 dalton protein does not disappear until after at least two passages in control medium. Amino acid analyses of these three proteins revealed that they all contain some hydroxyproline.     

Computer, Simulated Evaluation of Possible Mechanisms for Sequestering Metal Ion Activity in Plant Vacuoles: II. Zinc

Various mechanisms have been suggested for sequestering Zn ion activity in vacuoles of Zn-tolerant plants. One of these mechanisms, complexation in the vacuole with organic acids, has received some support in the recent literature. However, the lack of experimental evidence for anticipated vacuolar compartmentation and concerning the nature of metal-ligand species occurring in the vacuole has been criticized. In this study we have used computer modeling of chemical equilibria to predict the metalligand species in vacuoles of tobacco (Nicotiana tabacum) cultured cells. Results of this thermodynamic evaluation support the conclusion that citrate in the concentration range encountered in tobacco cultured cells exposed to 300 or 2000 μm Zn has high potential for forming soluble complexes with Zn, over the entire probable range of vacuolar pH 4 to 7. Complexation of Zn with oxalate is also predicted, especially in cells exposed to high Zn levels. Malate, though the most abundant acid present, showed little potential for competing with other ligands for Zn. Overall, results suggest that vacuolar sequestration of Zn by high levels of vacuolar citrate may be a central mechanism in the accumulation of Zn in plants exposed to either low or high levels of this metal.     

Analysis of an osmotically regulated pathogenesis-related osmotin gene promoter

Osmotin is a small (24 kDa), basic, pathogenesis-related protein, that accumulates during adaptation of tobacco (Nicotiana tabacum) cells to osmotic stress. There are more than 10 inducers that activate the osmotin gene in various plant tissues. The osmotin promoter contains several sequences bearing a high degree of similarity to ABRE, as-1 and E-8 cis element sequences. Gel retardation studies indicated the presence of at least two regions in the osmotin promoter that show specific interactions with nuclear factors isolated from cultured cells or leaves. The abundance of these binding factors increased in response to salt, ABA and ethylene. Nuclear factors protected a 35 bp sequence of the promoter from DNase I digestion. Different 5 deletions of the osmotin promoter cloned into a promoter-less GUS-NOS plasmid (pBI 201) were used in transient expression studies with a Biolistic gun. The transient expression studies revealed the presence of three distinct regions in the osmotin promoter. The promoter sequence from –108 to –248 bp is absolutely required for reporter gene activity, followed by a long stretch (up to –1052) of enhancer-like sequence and then a sequence upstream of –1052, which appears to contain negative elements. The responses to ABA, ethylene, salt, desiccation and wounding appear to be associated with the –248 bp sequence of the promoter. This region also contains a putative ABRE (CACTGTG) core element. Activation of the osmotin gene by various inducers is discussed in view of antifungal activity of the osmotin protein.     

Differential synthesis in vitro of barley aleurone and starchy endosperm proteins

To widen the selection of proteins for gene expression studies in barley seeds, experiments were performed to identify proteins whose synthesis is differentially regulated in developing and germinating seed tissues. The in vitro synthesis of nine distinct barley proteins was compared using mRNAs from isolated endosperm and aleurone tissues (developing and mature grain) and from cultured (germinating) aleurone layers treated with abscisic acid (ABA) and GA₃. B and C hordein polypeptides and the salt-soluble proteins β-amylase, protein Z, protein C, the chymotrypsin inhibitors (CI-1 and 2), the α-amylase/subtilisin inhibitor (ASI) and the inhibitor of animal cell-free protein synthesis systems (PSI) were synthesized with mRNA from developing starchy endosperm tissue. Of these proteins, β-amylase, protein Z, and CI- 1 and 2 were also synthesized with mRNA from developing aleurone cells, but ASI, PSI, and protein C were not. CI-1 and also a probable amylase/protease inhibitor (PAPI) were synthesized at high levels with mRNAs from late developing and mature aleurone. These results show that mRNAs encoding PAPI and CI-1 survive seed dessication and are long-lived in aleurone cells. Thus, expression of genes encoding ASI, PSI, protein C, and PAPI is tissue and stage-specific during seed development. Only ASI, CI-1, and PAPI were synthesized in significant amounts with mRNA from cultured aleurone layers. The levels of synthesis of PAPI and CI-1 were independent of hormone treatment. In contrast, synthesis of α-amylase (included as control) and of ASI showed antagonistic hormonal control: while GA promotes and ABA reduces accumulation of mRNA for α-amylase, these hormones have the opposite effect on ASI mRNA levels.     

Overexpression of a tobacco osmotin gene in carrot (Daucus carota L.) enhances drought tolerance

Osmotin and osmotin-like proteins belong to the PR-5 pathogenesis-related group of proteins and are induced in response to various types of biotic and abiotic stresses in several plant species. Carrot was transformed with a tobacco osmotin gene that encodes a protein lacking the vacuolar-sorting motif that is composed of a 20-amino-acid sequence at the C-terminal end, under the control of the cauliflower mosaic virus 35S promoter, using Agrobacterium-mediated transformation. Transgene integration and expression were confirmed by Southern and western blot analyses, and three selected transgenic lines were evaluated for their ability to tolerate drought stress. Under drought stress conditions, all transformants exhibited slower rates of wilting compared with the wild-type plants and recovered faster when the drought stress was alleviated. Transformants showed lower levels of hydrogen peroxide accumulation, reduced lipid peroxidation and electrolyte leakage, and higher leaf water content under drought stress. Our results provide additional evidence for the protective ability of the osmotin protein against drought stress conditions and suggest a possible means to achieve tolerance against this abiotic stress in plants.