Association of Formation and Release of Cyclic AMP with Glucose Depletion and Onset of Chlorophyll Synthesis in Poterioochromonas malhamensis

Depletion of glucose from the culture medium by Poterioochromonas malhamensis results in cessation of growth and accumulation of cyclic adenosine 3′:5′-monophosphate (cAMP), followed by formation of chlorophyll and an increase in extracellular cAMP. Readdition of glucose to the culture medium causes P. malhamensis to release its intracellular cAMP into the medium. These results suggest that formation of the photosynthetic apparatus in P. malhamensis may be repressed by glucose, and that high cAMP conveys the regulatory information that the glucose supply is inadequate. This pattern is reminiscent of cAMP-mediated escape from catabolite repression in bacteria.     

Somaclonal variation in tuber disc-derived populations of potato

Summary Approximately 1,600 potato (Solanum tuberosum L.) plants of the cultivar Superior were regeneratedin vitro from meristems adventitiously initiated on tuber disc expiants. Direct regeneration from tuber disc cells, by passing a callus intermediary, is efficient and results in low frequencies of plants with gross phenotypic aberrations. The somaclonal plant population was statistically characterized in field plots over five asexual generations and in three diverse locations. When compared in advanced generations to a large population of control plants propagated from stem cuttings, the means of the somaclonal population were significantly different, often shifted in the desirable direction, for 16 of 22 horticulturally important traits. Somaclonal population variances statistically exceeded those of the controls for 13 of the 22 traits. Regressions between consecutive tuber generations and between locations or replications (blocks) within a generation were significant in the somaclonal population for all traits analyzed. In a few instances, significant control population regressions occurred that are interpreted to be the result of non-random, non-genetic factors primarily affecting control plants of low vigor. Selected somaclones exhibiting desirable alterations for yield, tuber number and shape, and vigor were stable over more than two consecutive asexual generations.Research supported by a grant from NPI, 417 Wakara Way, Salt Lake City, UT 84108     

The elastin associated glycoprotein gp115. Synthesis and secretion by chick cells in culture

Synthesis of gp115 by aorta smooth muscle cells and tendon fibroblasts isolated from chick embryos was investigated. gp115 was specifically immunoprecipitated by both polyclonal and monoclonal antibodies from cell lysates and culture medium of matrix free cells metabolically labeled with [3H]leucine and [35S]methionine. The component of gp115 isolated from the cell lysate had an apparent Mr in reduced sodium dodecyl sulfate polyacrylamide gels lower (105,000) than the protein isolated from the culture medium (Mr = 115,000). In immunoblot experiments, the latter corresponded in apparent Mr to the form isolated from chick tissues. gp115 was glycosylated in vitro; it was labeled with [3H]fucose, and when cells were cultured and labeled in the presence of tunicamycin, a lower Mr form with an apparent Mr = 90,000 was immunoprecipitated in both the cell lysate and the culture medium. In pulse-chase experiments, the intracellular and the extracellular forms were clearly suggestive of a direct precursor-product relationship in the absence of intermediate forms. The kinetics of secretion appeared very slow compared with that of other proteins of the extracellular matrix investigated in the same system; about 50-70% of gp115 in the form of the Mr = 105,000 species was still cell-associated after 4 h, whereas the half-time for secretion of fibronectin, type VI collagen, and tropoelastin was about 60 min, 3 h, and 60 min, respectively. Newly synthesized and processed cell-associated gp115 migrated in both reduced and non-reduced gels as a monomer. On the contrary, the secreted protein was present in the culture medium as large aggregates that did not enter the gel in the absence of reducing agents.     

A Saccharomyces cerevisiae Assay System to Investigate Ligand/AdipoR1 Interactions That Lead to Cellular Signaling

Adiponectin is a mammalian hormone that exerts anti-diabetic, anti-cancer and cardioprotective effects through interaction with its major ubiquitously expressed plasma membrane localized receptors, AdipoR1 and AdipoR2. Here, we report a Saccharomyces cerevisiae based method for investigating agonist-AdipoR interactions that is amenable for high-throughput scale-up and can be used to study both AdipoRs separately. Agonist-AdipoR1 interactions are detected using a split firefly luciferase assay based on reconstitution of firefly luciferase (Luc) activity due to juxtaposition of its N- and C-terminal fragments, NLuc and CLuc, by ligand induced interaction of the chimeric proteins CLuc-AdipoR1 and APPL1-NLuc (adaptor protein containing pleckstrin homology domain, phosphotyrosine binding domain and leucine zipper motif 1-NLuc) in a S. cerevisiae strain lacking the yeast homolog of AdipoRs (Izh2p). The assay monitors the earliest known step in the adiponectin-AdipoR anti-diabetic signaling cascade. We demonstrate that reconstituted Luc activity can be detected in colonies or cells using a CCD camera and quantified in cell suspensions using a microplate reader. AdipoR1-APPL1 interaction occurs in absence of ligand but can be stimulated specifically by agonists such as adiponectin and the tobacco protein osmotin that was shown to have AdipoR-dependent adiponectin-like biological activity in mammalian cells. To further validate this assay, we have modeled the three dimensional structures of receptor-ligand complexes of membrane-embedded AdipoR1 with cyclic peptides derived from osmotin or osmotin-like plant proteins. We demonstrate that the calculated AdipoR1-peptide binding energies correlate with the peptides’ ability to behave as AdipoR1 agonists in the split luciferase assay. Further, we demonstrate agonist-AdipoR dependent activation of protein kinase A (PKA) signaling and AMP activated protein kinase (AMPK) phosphorylation in S. cerevisiae, which are homologous to important mammalian adiponectin-AdipoR1 signaling pathways. This system should facilitate the development of therapeutic inventions targeting adiponectin and/or AdipoR physiology.     

Loss of Halophytism by Interference with SOS1 Expression

The contribution of SOS1 (for Salt Overly Sensitive 1), encoding a sodium/proton antiporter, to plant salinity tolerance was analyzed in wild-type and RNA interference (RNAi) lines of the halophytic Arabidopsis (Arabidopsis thaliana)-relative Thellungiella salsuginea. Under all conditions, SOS1 mRNA abundance was higher in Thellungiella than in Arabidopsis. Ectopic expression of the Thellungiella homolog ThSOS1 suppressed the salt-sensitive phenotype of a Saccharomyces cerevisiae strain lacking sodium ion (Na⁺) efflux transporters and increased salt tolerance of wild-type Arabidopsis. thsos1-RNAi lines of Thellungiella were highly salt sensitive. A representative line, thsos1-4, showed faster Na⁺ accumulation, more severe water loss in shoots under salt stress, and slower removal of Na⁺ from the root after removal of stress compared with the wild type. thsos1-4 showed drastically higher sodium-specific fluorescence visualized by CoroNa-Green, a sodium-specific fluorophore, than the wild type, inhibition of endocytosis in root tip cells, and cell death in the adjacent elongation zone. After prolonged stress, Na⁺ accumulated inside the pericycle in thsos1-4, while sodium was confined in vacuoles of epidermis and cortex cells in the wild type. RNAi-based interference of SOS1 caused cell death in the root elongation zone, accompanied by fragmentation of vacuoles, inhibition of endocytosis, and apoplastic sodium influx into the stele and hence the shoot. Reduction in SOS1 expression changed Thellungiella that normally can grow in seawater-strength sodium chloride solutions into a plant as sensitive to Na⁺ as Arabidopsis.Accompanying the production and accumulation of osmolytes and other protective molecules, an important aspect of plant responses leading to salt stress tolerance is the regulation of uptake, reexport, and control over the distribution of sodium ions (Na⁺; Hasegawa et al., 2000; Tester and Davenport, 2003). Na⁺ appear to enter the root by several pathways (Essah et al., 2003; Pardo et al., 2006), although the nature of participating genes and their interaction in pathways require further investigation. Once Na⁺ has entered the root endodermis, a tissue that represents a barrier to ions (Peng et al., 2004), it is generally assumed that the ion enters the xylem following the movement of water to aerial parts of the plant. Despite substantial efflux of Na⁺ across the plasma membrane of root cells, the net flux of Na⁺ is unidirectional from soil to roots and then to the shoot, except for possible recirculation via the phloem (Tester and Davenport, 2003). In a range of species, the severity of damaging symptoms is positively correlated with the content of Na⁺ reaching photosynthetic tissues (Davenport et al., 2005; Ren et al., 2005; Munns et al., 2006). However, halophytic species can accumulate very high amounts of Na⁺ in vacuoles, such that Na⁺ may account for most of the total cellular osmotic potential (Tester and Davenport, 2003), and the presence of Na⁺ accelerates growth in euhalophytes to some degree (Adams et al., 1998). Emerging as the major advantage of halophytes appears to be their exceptional control over Na⁺ influx combined with export mechanisms, the ability to coordinate its distribution to various tissues, and efficient sequestration of Na⁺ into vacuoles. These characteristics are of particular advantage when plants are subjected to a sudden increase of Na⁺ salts in their environment (Hasegawa et al., 2000), whereas gradual increases in Na⁺ may be tolerated even by plants that are not halophytic in nature.Na⁺-ATPases, major Na⁺ export systems in organisms such as fungi and the moss Physcomitrella patens, have not been found in higher plants (Lunde et al., 2007). In Arabidopsis (Arabidopsis thaliana), transporters of monovalent (alkali) cations, such as HKT1 (Berthomieu et al., 2003; Rus et al., 2004), members of the NHX family (Yamaguchi et al., 2005; Pardo et al., 2006), and SOS1 (for Salt Overly Sensitive 1; Shi et al., 2000, 2002, 2003), have been shown to play roles in the movement and distribution of Na⁺ ions. Studies have shown the involvement of nonselective ion channels with roles in the transport of Na⁺ ions, but the genes encoding such function(s) have not been identified (Demidchik and Maathuis, 2007). SOS1, whose deletion resulted in a strong salt-sensitivity phenotype in Arabidopsis, encodes a plasma membrane Na⁺/H⁺ antiporter involved in removing Na⁺ ions from cells (Shi et al., 2000). This efflux strategy, which may be sufficient for the survival of unicellular organisms, must be accompanied by other means of Na⁺ confinement to avoid carryover of Na⁺ between cells in futile cycles. Hence, the physiological role of a plasma membrane Na⁺/H⁺ antiporter must be embedded in the context of tissue, organ, and whole plant distribution of ions and their transporters. A recent discovery on cell layer-specific differential responses to the salt stress of root cells supported this notion (Dinneny et al., 2008).In Arabidopsis, the SOS1 gene is most strongly expressed in the epidermis of the root tip region and in cells adjacent to vascular tissues (Shi et al., 2002). Based on the salt concentration in shoot, root, and xylem sap of wild-type Arabidopsis and its sos1 knockout mutants, the SOS1 antiporter is assumed to function in Na⁺ export under severe salt stress conditions (Shi et al., 2002). However, detailed knowledge about how a Na⁺ excluder achieves salt tolerance in a multicellular eukaryote is still missing. Significantly also, even though SOS1 has been an intensely studied component of the ion homeostasis mechanism, its involvement in the exceptional salt tolerance of halophytes is not known.Thellungiella salsuginea (salt cress), which had before been called T. halophila by us, is a close relative of Arabidopsis, which has become a model to study the genetic basis of this plant''s extreme tolerance to a variety of abiotic stress factors, including salinity (Inan et al., 2004; Gong et al., 2005; Vera-Estrella et al., 2005; Volkov and Amtmann, 2006; Amtmann, 2009). Thellungiella lacks specialized morphological structures, such as salt glands or large sodium storage cells found in other halophytes, making it a useful model for studying stress tolerance mechanisms that could be applicable to further understanding or to embark on engineering of conventional crops (Inan et al., 2004). Recently, it has been reported that Thellungiella had lower net Na⁺ uptake compared with Arabidopsis. The unidirectional influx of Na⁺ ions to roots appeared to be more restricted and/or tightly controlled in Thellungiella than in Arabidopsis. To compensate for greater influx, Arabidopsis roots showed higher Na⁺ efflux (Wang et al., 2006).Here, we wished to explore the role(s) by which ThSOS1, the SOS1 homolog in Thellungiella, could be involved in shaping the halophytic character of the species using ectopic expression of the gene in yeast and in Arabidopsis and Thellungiella SOS1-RNA interference (RNAi) lines. The results identified ThSOS1 as a genetic element whose activity limits Na⁺ accumulation and affects the distribution of Na⁺ ions at high concentration, thus acting as a major tolerance determinant.