Dissection of upstream regulatory components of the Rho1p effector, 1,3-beta-glucan synthase,in Saccharomyces cerevisiae

In the budding yeast Saccharomyces cerevisiae, one of the main structural components of the cell wall is 1,3-beta-glucan produced by 1,3-beta-glucan synthase (GS). Yeast GS is composed of a putative catalytic subunit encoded by FKS1 and FKS2 and a regulatory subunit encoded by RHO1. A combination of amino acid alterations in the putative catalytic domain of Fks1p was found to result in a loss of the catalytic activity. To identify upstream regulators of 1,3-beta-glucan synthesis, we isolated multicopy suppressors of the GS mutation. We demonstrate that all of the multicopy suppressors obtained (WSC1, WSC3, MTL1, ROM2, LRE1, ZDS1, and MSB1) and the constitutively active RHO1 mutations tested restore 1,3-beta-glucan synthesis in the GS mutant. A deletion of either ROM2 or WSC1 leads to a significant defect of 1,3-beta-glucan synthesis. Analyses of the degree of Mpk1p phosphorylation revealed that among the multicopy suppressors, WSC1, ROM2, LRE1, MSB1, and MTL1 act positively on the Pkc1p-MAPK pathway, another signaling pathway regulated by Rho1p, while WSC3 and ZDS1 do not. We have also found that MID2 acts positively on Pkc1p without affecting 1,3-beta-glucan synthesis. These results suggest that distinct networks regulate the two effector proteins of Rho1p, Fks1p and Pkc1p.     

Genetic interactions of yeast eukaryotic translation initiation factor 5A (eIF5A) reveal connections to poly(A)-binding protein and protein kinase C signaling

The highly conserved eukaryotic translation initiation factor eIF5A has been proposed to have various roles in the cell, from translation to mRNA decay to nuclear protein export. To further our understanding of this essential protein, three temperature-sensitive alleles of the yeast TIF51A gene have been characterized. Two mutant eIF5A proteins contain mutations in a proline residue at the junction between the two eIF5A domains and the third, strongest allele encodes a protein with a single mutation in each domain, both of which are required for the growth defect. The stronger tif51A alleles cause defects in degradation of short-lived mRNAs, supporting a role for this protein in mRNA decay. A multicopy suppressor screen revealed six genes, the overexpression of which allows growth of a tif51A-1 strain at high temperature; these genes include PAB1, PKC1, and PKC1 regulators WSC1, WSC2, and WSC3. Further results suggest that eIF5A may also be involved in ribosomal synthesis and the WSC/PKC1 signaling pathway for cell wall integrity or related processes.     

Selection for water-soluble carbohydrate accumulation and investigation of genetic × environment interactions in an elite wheat breeding population

Key message

Water-soluble carbohydrate accumulation can be selected in wheat breeding programs with consideration of genetic × environmental interactions and relationships with other important characteristics such as relative maturity and nitrogen concentration, although the correlation between WSC traits and grain yield is low and inconsistent.

Abstract

The potential to increase the genetic capacity for water-soluble carbohydrate (WSC) accumulation is an opportunity to improve the drought tolerance capability of rainfed wheat varieties, particularly in environments where terminal drought is a significant constraint to wheat production. A population of elite breeding germplasm was characterized to investigate the potential for selection of improved WSC concentration and total amount in water deficit and well-watered environments. Accumulation of WSC involves complex interactions with other traits and the environment. For both WSC concentration (WSCC) and total WSC per area (WSCA), strong genotype × environment interactions were reflected in the clear grouping of experiments into well-watered and water deficit environment clusters. Genetic correlations between experiments were high within clusters. Heritability for WSCC was larger than for WSCA, and significant associations were observed in both well-watered and water deficit experiment clusters between the WSC traits and nitrogen concentration, tillering, grains per m², and grain size. However, correlations between grain yield and WSCC or WSCA were weak and variable, suggesting that selection for these traits is not a better strategy for improving yield under drought than direct selection for yield.

     

Diencephalo-telencephalic changes of tyrosine hydroxylase in rats and grass frogs after sleep deprivation

Based on sleep deprivation-produced changes of electrographic parameters of the wakefulness-sleep cycle (WSC) in rats and frogs (Rana temporaria), dynamics of activity of tyrosine hydroxylase, the key enzyme of dopamine synthesis, was studied immunohistochemically in substantia nigra and nigrostriatal pathway in rats and in striatum, paraventricular organ, and extrahypothalamic pathways in frogs. Changes in dynamics of tyrosine hydroxylase in rats and in frogs are revealed after the 6-h sleep deprivation and after 2 h of postdeprivation sleep. This allows determining the degree of participation of corticostriatal neuroregulatory and hypothalamo-pituitary neurosecretory systems and their role in regulation of WSC. Possible evolutionary peculiarities of morphofunctional differences in homoiothermal and poikilothermal animals are discussed.     

Effects of water-soluble carbodiimide on "fast" sodium channel gating in the rat sensory neuron membrane

Currents through "fast" (tetrodotoxin-sensitive) channels before and after external application of solutions containing 100 mM 1-ethyl-3-dimethylaminopropyl)carbodiimide-HCl (WSC) were measured during volage clamping at the membrane of dialyzed neurons of rat spinal ganglia. Treating the membrane with WSC (pH 4.8–4.9) led to a 5 to 20-fold reduction in sodium conductance, a 1.5–2.5-fold deceleration in the dynamics of current increase, and less abrupt voltage-dependent sodium channel activation curves. The shifted effective charge of activation was normally halved. The WSC produced no effect on activation parameters at normal pH (7.6). It was deduced that the changes observed resulted from WSC reacting with carboxyl groups located on the outer surface of the membrane. These groups are thought to be involved in the system of charge movements of the sodium channel gating mechanism.Institute of Cytology, Academy of Sciences of the USSR, Leningrad. Translated from Neirofiziologiya, Vol. 19, No. 1, pp. 46–53, January–February, 1987.     

Morphofunctional Analysis of Effect of Short-Term Sleep Deprivation on the Wakefulness–Sleep Cycle in Young and Adult Rats

The work presents comparative data on changes of neurophysiological, time characteristics of the wakefulness–sleep cycle (WSC) and morphofunctional state of neurosecretory cells in supraoptic nucleus of the hypothalamus, which develop under influence of a 6-h long sleep deprivation in adult and one-month old rats. It is shown that the rebound of sleep develops in adult animals with a delay, after the 3rd hour and is characterized by a moderate increase of portions of slow-wave (SSP) and fast-wave (FSP) sleep phases in WSC and by a decrease of the wakefulness portion. Morphological analysis of the hypothalamus nonapeptidergic system has revealed a rise of content of neurosecretory material in fibers of supraoptic nucleus cells an in area of supraoptic-pituitary tract, as well as marked hyperemia that indicates activation of processes of secretion of neurohormones into the general blood flow; these reactions are similar to reactions of this system to stress. In rat pups the sleep rebound develops in 0.5 h after the end of the deprivation procedure and is characterized by more pronounced, statistically significant changes in WSC. Individual WSC become very short and almost all of them are completed with episodes of FSP. A statistically significant rise of power of the -wave band in electrogram spectra of hippocampus and somatosensory cortex in SSP, whereas peak of the activity in FSP is shifted to -waves. Ratios of SSP and FSP to wakefulness in individual WSC in mature animals increase after the deprivation 1.53 and 1.85 times, while they are elevated in one-month old animals 5.25 and 6.75 times, respectively. The obtained morphofunctional data allow believing that deprivation is the stress factor of low intensity for adult animals, whereas it may be considered as the stress action of intermediate and even high intensity for rat pups, which changes essentially the interrelations in WSC. Participation of central mechanisms of regulation of sleep and vigilance, which provide processes of compensation of damaging action of deprivation on WSC in the maturing animals, is discussed.     

A novel role for the mating type (MAT) locus in the maintenance of cell wall integrity in Saccharomyces cerevisiae

The cell wall and stress response component (Wsc) protein family in the yeast Saccharomyces cerevisiae is encoded by at least three genes, WSC1, WSC2, and WSC3. The Wsc proteins are putative upstream activators of the RHO1-regulated PKC1-MAP kinase cascade, and are required for maintenance of cell wall integrity and the stress response. Deletion of WSC1 causes a cell lysis defect that is exacerbated by deleting WSC2 or WSC3. This cell lysis defect can be rescued by adding osmotic stabilizers, such as 1?M sorbitol, to the medium, and by overexpressing PKC1 or RHO1. To advance our understanding of the function of the WSC genes, we performed a genetic screen to identify other components of the pathways they regulate. Here we report our findings. MAT a 1 and MATα2 were identified as dosage-dependent suppressors of the lysis defect of a wscΔ mutant. Overexpression of MAT a 1 or MATα2 was found to suppress the heat shock sensitivity, in addition to the lysis defect, of the wscΔ mutant. Phenotypic suppression by these two genes, MAT a 1 and MATα2, is significantly stronger when they are overexpressed in cells of the opposite mating type. Deletion of MAT a 1 exacerbates the lysis defect of haploid and diploid wscΔ strains. Our results suggest that the MAT locus plays a role in responses similar to those regulated by WSC and provide evidence for a regulatory effect of the MAT locus outside the realm of cell type determination.     

Phytoplankton composition in the Weddell-Scotia Confluence area during austral spring in relation to hydrography

During the EPOS leg 2 cruise of the RV Polarstern, carried out in late austral spring of 1988–1989, the composition of phytoplankton in relation to the distribution of hydrographic parameters was studied in four successive transects carried out along 49°W and 47°W, across the Weddell-Scotia Confluence (WSC) and the marginal ice zone (which overlapped in part). In all transects, a maximum of phytoplankton biomass was found in the WSC, in surface waters stabilized by ice melting. Different phytoplankton assemblages could be distinguished. North of the Scotia Front (the northern limit of the WSC) diatoms with Chaetoceros neglectus, Nitzschia spp. and (Thalassiosira gravida) dominated the phytoplankton community. This assemblage appeared to have seeded a biomass maximum which occupied, during the first transect, an area of the WSC, south of the Scotia Front. The southernmost stations of the first transect and all the stations to the south of the Scotia Front in the other transects were populated by a flagellate assemblage (with a cryptomonad, Pyramimonas spp. and Phaeocystis sp.) and an assemblage of diatoms (Corethron criophilum and Tropidoneis vanheurkii among others) associated to the presence of ice. During the last three transects, the flagellate assemblage formed a bloom in the low salinity surface layers of the WSC zone. The bulk of the biomass maximum was formed by the cryptomonad which reached concentrations up to 4×10⁶ cells l^–1 towards the end of the cruise. Multivariate analysis is used to summarize phytoplankton composition variation. The relationships between the distribution of the different assemblages and the hydrographic conditions indicate that the change of dominance from diatoms to flagellates in the WSC zone was related to the presence of water masses from different origin.Data presented here were collected during the European Polarstern Study (EPOS) sponsored by the European Science Foundation     

Effects of Favorable Alleles for Water-Soluble Carbohydrates at Grain Filling on Grain Weight under Drought and Heat Stresses in Wheat

Drought, heat and other abiotic stresses during grain filling can result in reductions in grain weight. Conserved water-soluble carbohydrates (WSC) at early grain filling play an important role in partial compensation of reduced carbon supply. A diverse population of 262 historical winter wheat accessions was used in the present study. There were significant correlations between 1000-grain weight (TGW) and four types of WSC, viz. (1) total WSC at the mid-grain filling stage (14 days after flowering) produced by leaves and non-leaf organs; (2) WSC contributed by current leaf assimilation during the mid-grain filling; (3) WSC in non-leaf organs at the mid-grain filling, excluding the current leaf assimilation; and (4) WSC used for respiration and remobilization during the mid-grain filling. Association and favorable allele analyses of 209 genome-wide SSR markers and the four types of WSC were conducted using a mixed linear model. Seven novel favorable WSC alleles exhibited positive individual contributions to TGW, which were verified under 16 environments. Dosage effects of pyramided favorable WSC alleles and significantly linear correlations between the number of favorable WSC alleles and TGW were observed. Our results suggested that pyramiding more favorable WSC alleles was effective for improving both WSC and grain weight in future wheat breeding programs.     

High molecular weight water-soluble chitosan protects against apoptosis induced by serum starvation in human astrocytes

The effect of high molecular weight water-soluble chitosan (WSC) on serum starvation-induced apoptosis in human astrocytes (CCF-STTG1 Cells) was investigated. WSC, having an average molecular weight of 300 kDa and a degree of deacetylation over 90%, can be produced using a simple multi-step membrane separation process. Serum starvation led to growth arrest, rounding up of cells and appearance of p53 bands. Prolonged (48 h) incubation in serum starved medium led to cell detachment and death. WSC significantly protected the serum starvation-induced cellular rounding up and protected the serum starvation-induced cell death as tested by flow cytometry. WSC also protected serum starvation-induced p53 activation as determined by Western blot. These results suggest that WSC may prevent serum starvation-induced apoptosis of CCF-STTG1 cells via p53 inactivation.     

Synthesis and physicochemical and dynamic mechanical properties of a water-soluble chitosan derivative as a biomaterial

The physicochemical and rheological properties of a water-soluble chitosan (WSC) derivative were characterized in order to facilitate its use as a novel material for biomedical applications. The WSC was prepared by conjugating glycidyltrimethylammonium chloride (GTMAC) onto chitosan chains. Varying the molar ratio of GTMAC to chitosan from 3:1 to 6:1 produced WSCs with a degree of substitution (DS) that ranged from 56% to 74%. The WSC with the highest DS was soluble in water up to concentrations of 25 g/dL at room temperature. An increase in the polymer concentration gradually increased both the pH and conductivity of the WSC solutions. The rheological properties of the WSC solutions were found to be dependent on the salt and polymer concentrations as well as the DS value. In the absence of salt, the rheological behavior of the WSC was found to be typical of that for a polyelectrolyte in the dilute solution regime. However, the addition of salt decreased the viscosity of the polymer solution due to the reduction of electrostatic repulsions by the positively charged trimethylated ammonium groups of the WSC. In the concentrated regime, the viscosity of the WSCs was found to follow a power-law expression. The lowest DS WSC had the more favorable viscoelastic properties that were attributed to its high molecular weight, as confirmed by the stress relaxation spectra and intrinsic viscosity measurements. The effect of DS on the degree of interaction between WSC and the lipid egg phosphatidylcholine was investigated by FTIR analysis. Overall, the lower DS WSC had enhanced rheological properties and was capable of engaging in stronger intermolecular physical interactions.     

Hyperosmotic stress response and regulation of cell wall integrity in Saccharomyces cerevisiae share common functional aspects

The osmosensitive phenotype of the hog1 strain is suppressed at elevated temperature. Here, we show that the same holds true for the other commonly used HOG pathway mutant strains pbs2 and sho1ssk2ssk22, but not for ste11ssk2ssk22. Instead, the ste11ssk2ssk2 strain displayed a hyperosmosensitive phenotype at 37 degrees C. This phenotype is suppressed by overexpression of LRE1, HLR1 and WSC3, all genes known to influence cell wall composition. The suppression of the temperature-induced hyperosmosensitivity by these genes prompted us to investigate the role of STE11 and other HOG pathway components in cellular integrity and, indeed, we were able show that HOG pathway mutants display sensitivity to cell wall-degrading enzymes. LRE1 and HLR1 were also shown to suppress the cell wall phenotypes associated with the HOG pathway mutants. In addition, the isolated multicopy suppressor genes suppress temperature-induced cell lysis phenotypes of PKC pathway mutants that could be an indication for shared targets of the PKC pathway and high-osmolarity response routes.     

Phylogenetic origin and transcriptional regulation at the post-diauxic phase of SPI1, in Saccharomyces cerevisiae

The gene SPI1, of Saccharomyces cerevisiae, encodes a cell wall protein that is induced in several stress conditions, particularly in the postdiauxic and stationary phases of growth. It has a paralogue, SED1, which shows some common features in expression regulation and in the null mutant phenotype. In this work we have identified homologues in other species of yeasts and filamentous fungi, and we have also elucidated some aspects of the origin of SPI1, by duplication and diversification of SED1. In terms of regulation, we have found that the expression in the post-diauxic phase is regulated by genes related to the PKA pathway and stress response (MSN2/4, YAK1, POP2, SOK2, PHD1, and PHO84) and by genes involved in the PKC pathway (WSC2, PKC1, and MPK1).     

Breeding for Bio-ethanol Production in Lolium perenne L.: Association of Allelic Variation with High Water-Soluble Carbohydrate Content

Increasing the extractable sugar yield from perennial crops is one strategy to generate renewable fuels such as bio-ethanol. Lolium perenne L. (perennial ryegrass) can contain significant (>30% dry matter) water-soluble sugars in the form of polymeric fructan which is readily extracted, broken down and fermented to bio-ethanol. A population of L. perenne generated from four parents which differed in water-soluble carbohydrate (WSC) content was subjected to multiple rounds of selection and recombination on the basis of early spring WSC content to produce a high WSC, and a low WSC population. A control population was generated by selecting the same number of plants at random. The alleles present at six candidate gene loci were analysed before and after selection and correlated to WSC content. Significant differences in the allele frequency of L. perenne soluble-acid invertase1:4 were observed between the three populations with one haplotype significantly associated with the high WSC C2^S+ population (after three rounds of selection and two rounds of recombination). Moreover, WSC content was also associated with biomass accumulation. Thus, in addition to a 2.84-fold increase in WSC yield, the C2^S+ population also had 1.48-fold more biomass per plant, resulting in 3.9-fold higher WSC yield per plant than the control population.     

Genome-Wide Association of Stem Water Soluble Carbohydrates in Bread Wheat

Water soluble carbohydrates (WSC) in stems play an important role in buffering grain yield in wheat against biotic and abiotic stresses; however, knowledge of genes controlling WSC is very limited. We conducted a genome-wide association study (GWAS) using a high-density 90K SNP array to better understand the genetic basis underlying WSC, and to explore marker-based breeding approaches. WSC was evaluated in an association panel comprising 166 Chinese bread wheat cultivars planted in four environments. Fifty two marker-trait associations (MTAs) distributed across 23 loci were identified for phenotypic best linear unbiased estimates (BLUEs), and 11 MTAs were identified in two or more environments. Liner regression showed a clear dependence of WSC BLUE scores on numbers of favorable (increasing WSC content) and unfavorable alleles (decreasing WSC), indicating that genotypes with higher numbers of favorable or lower numbers of unfavorable alleles had higher WSC content. In silico analysis of flanking sequences of trait-associated SNPs revealed eight candidate genes related to WSC content grouped into two categories based on the type of encoding proteins, namely, defense response proteins and proteins triggered by environmental stresses. The identified SNPs and candidate genes related to WSC provide opportunities for breeding higher WSC wheat cultivars.     

Variations of Water-Soluble Carbohydrate Contents in Different Age Class Modules of Leymus chinensis Populations in Sandy and Saline-Alkaline Soil on the Songnen Plains of China

Leymus chinensis （Trin.） Tzveh is a rhizomatous perennial herbage of Gramineae. Reproduction is mainly by vegetative reproduction. Tillering nodes and rhizomes of L. chinensis serve as organs for both vegetative reproduction and nutrient storage. Water-soluble carbohydrate （WSC） contents were measured in tillering nodes, nodes and Internodes of rhizomes of different age classes of L. chinensis populations at three development stages, namely the dough ripe stage, the vegetative growth stage after full ripeness, and the withering stage, in two habitats： sandy soil and saline-alkaline soil. The results showed that WSC content in tillering nodes of the three age classes of L. chinensis were all markedly decreased with increasing age in both sandy soil and saline-alkaline solh A similar trend of changes In WSC contents was observed in the nodes and internodes of rhizomes in different age classes In both habitats. The highest WSC contents were in 2-age-class nodes and internodes of rhizomes, followed by those In the 1 age class, with the lowest WSC contents found in 3-age-class nodes and internodes of rhizomes at the dough ripe and vegetative growth stages after full ripening. In turn, WSC contents decreased with Increasing age at the withering stage in both habitats. The WSC content in each age class of internode was higher than that in the node of rhizome at three development stages in both habitats.     

Diencephalo-telencephalic changes of tyrosine hydroxylase in rats and common frogs (Rana temporaria) after sleep deprivation

Based on sleep deprivation-produced changes of electrographic parameters of the wakefulness--sleep cycle (WSC) in rats and common frogs, dynamics of activity of tyrosine hydroxylase, the key enzyme of dopamine synthesis, was studied immunohistochemically in substantia nigra and nigrostriatal pathway in rats and in striatum, paraventricular organ, and extrahypothalamic pathways in frogs. There are revealed changes in dynamics of tyrosine hydroxylase in rats and in common frogs after the 6-h sleep deprivation and after 2 h of postdeprivation sleep. This allows determining the degree of participation of corticostriatal neuroregulatory and hypothalamo-pituitary neurosecretory systems and their role in regulation of WSC. Possible evolutionary peculiarities of morphofunctional differences in homoiothermal and poikilothermal animals are discussed.