Selection of callus cultures of sugarcane (Saccharum sp.) tolerant to NaCl and their response to salt stress

Stable callus cultures tolerant to NaCl (68 mM) were developed from salt-sensitive sugarcane cultivar CP65-357 by in vitro selection process. The accumulation of both inorganic (Na⁺, Cl⁻ and K⁺) and organic (proline and soluble sugars) solutes was determined in selected and non-selected calli after a NaCl shock in order to evaluate their implication in in vitro salt tolerance of the selected lines. Both salt-tolerant and non-selected calli showed similar relative fresh weight growth in the absence of NaCl. No growth reduction was observed in salt-tolerant calli while a significant reduction about 32% was observed in nonselected ones when both were cultivated on 68 mM NaCl. Accumulation of Na⁺ was similar in both salt-tolerant and non-selected calli in the presence of NaCl. Accumulation of Cl⁻ was lower in NaCl-tolerant than in non-selected calli while proline and soluble sugars were more accumulated in salt-tolerant than in non-selected calli when both were exposed to salt. K⁺ level decreased more severely in non-selected calli than in NaCl-tolerant ones after NaCl shock. The results indicated that K⁺ and Cl⁻ may play a key role in in vitro salt-tolerance in sugarcance cell lines obtained by in vitro selection and that organic solutes could contribute mainly to counteract the negative water potential of the outside medium.     

In vitro selection and characterization of polyethylene glycol (PEG) tolerant callus lines and regeneration of plantlets from the selected callus lines in sugarcane (Saccharum officinarum L.)

A system for in vitro selection of drought tolerant callus lines in sugarcane was developed. High molecular weight PEG was used as selective agent. Selected callus line grew better than non-selected callus when grown on different concentrations of PEG. The activity of antioxidant enzymes like CAT, POX, APX and SOD were high in selected callus than in non-selected callus. Osmolytes like proline and ascorbic acid were at higher levels in selected callus than in non-selected callus, however at higher concentrations (20–30 %) of PEG, levels of proline and ascorbic acid decreased. The frequency of organogenesis and number of plantlets decreased in selected callus than in non-selected callus. The results can be used for in vitro screening and manipulations of sugarcane for improvement of drought tolerance     

Proteomics analysis of sensitive and tolerant barley genotypes under drought stress

Drought is a severe environmental constraint to plant productivity and an important factor limiting barley yield. To investigate the initial response of barley to drought stress, changes in protein profile were analyzed using a proteomics technique. Three-day-old barley seedlings of sensitive genotype 004186 and tolerant genotype 004223 were given two treatments, one with 20 % polyethylene glycol and the second with drought induced by withholding water. After 3 days of treatments, proteins were extracted from shoots and separated by 2-dimensional polyacrylamide gel electrophoresis. Metabolism related proteins were decreased in the sensitive genotype under drought; however, they were increased in the tolerant genotype. Photosynthetic related proteins were decreased and increased among the three sensitive and three tolerant genotypes, respectively. In addition, amino acid synthesis and degradation related proteins were increased and decreased among the three tolerant genotypes. These results suggest that chloroplastic metabolism and energy related proteins might play a significant role in the adaptation process of barley seedlings under drought stress.     

Tissue-specific expression and functional role of dehydrins in heat tolerance of sugarcane (Saccharum officinarum)

Studies on the functional roles of dehydrins (DHNs) in heat tolerance of plants are scarce. This study was conducted to immunohistolocalize DHNs in leaves of heat-tolerant (CP-4333) and heat-sensitive (HSF-240) sugarcane (Saccharum officinarum L.) clones at three phenological stages in order to elucidate their putative roles under heat stress. CP-4333 indicated greater amounts of heat-stable proteins than HSF-240 under heat stress. Western blotting revealed the expression of three DHNs in CP-4333 (13- and 15-kDa peptides at 48 h and an additional 18-kDa band at 72 h) and two (13 and 15 kDa at 48 h) in HSF-240 at formative stage; two DHNs in CP-4333 (20 and 25 kDa) and one in HSF-240 (20 kDa) at grand growth stage, while two DHNs in CP-4333 (20 and 22 kDa) and one in HSF-240 (20 kDa) at maturity stage. Tissue-specific immunohistolocalization showed that DHNs were expressed in stele particularly the phloem and the cells intervening bundle sheath and vascular bundles. Furthermore, DHNs were also found scattered along the epidermal and parenchymatous cells. Recovery of sugarcane from heat stress manifested a gradual disappearance of DHNs in both the clones, being quicker in sensitive clone (HSF-240). Results suggested specific implications for DHNs synthesis. Their synthesis in epidermis appears to protect the mesophyll tissues from heat injury. When associated to vascular tissue, they tend to ensure the normal photoassimilate loading into the sieve element–companion cell complex. DHNs diminution during recovery suggested that their expression was transitory. However, prolonged retention of DHNs by tolerant clone appears to be an adaptive advantage of sugarcane to withstand heat stress.     

Modulation of stress proteins and apoptotic regulators in the anoxia tolerant turtle brain

Freshwater turtles survive prolonged anoxia and reoxygenation without overt brain damage by well-described physiological processes, but little work has been done to investigate the molecular changes associated with anoxic survival. We examined stress proteins and apoptotic regulators in the turtle during early (1 h) and long-term anoxia (4, 24 h) and reoxygenation. Western blot analyses showed changes within the first hour of anoxia; multiple stress proteins (Hsp72, Grp94, Hsp60, Hsp27, and HO-1) increased while apoptotic regulators (Bcl-2 and Bax) decreased. Levels of the ER stress protein Grp78 were unchanged. Stress proteins remained elevated in long-term anoxia while the Bcl-2/Bax ratio was unaltered. No changes in cleaved caspase 3 levels were observed during anoxia while apoptosis inducing factor increased significantly. Furthermore, we found no evidence for the anoxic translocation of Bax from the cytosol to mitochondria, nor movement of apoptosis inducing factor between the mitochondria and nucleus. Reoxygenation did not lead to further increases in stress proteins or apoptotic regulators except for HO-1. The apparent protection against cell damage was corroborated with immunohistochemistry, which indicated no overt damage in the turtle brain subjected to anoxia and reoxygenation. The results suggest that molecular adaptations enhance pro-survival mechanisms and suppress apoptotic pathways to confer anoxia tolerance in freshwater turtles.     

Metabolite adjustments in drought tolerant and sensitive soybean genotypes in response to water stress

Soybean (Glycine max L.) is an important source of protein for human and animal nutrition, as well as a major source of vegetable oil. The soybean crop requires adequate water all through its growth period to attain its yield potential, and the lack of soil moisture at critical stages of growth profoundly impacts the productivity. In this study, utilizing (1)H NMR-based metabolite analysis combined with the physiological studies we assessed the effects of short-term water stress on overall growth, nitrogen fixation, ureide and proline dynamics, as well as metabolic changes in drought tolerant (NA5009RG) and sensitive (DM50048) genotypes of soybean in order to elucidate metabolite adjustments in relation to the physiological responses in the nitrogen-fixing plants towards water limitation. The results of our analysis demonstrated critical differences in physiological responses between these two genotypes, and identified the metabolic pathways that are affected by short-term water limitation in soybean plants. Metabolic changes in response to drought conditions highlighted pools of metabolites that play a role in the adjustment of metabolism and physiology of the soybean varieties to meet drought effects.     

Lipid composition and turnover in heterotrophic cell suspension cultures of Saccharum officinarum

Utilizing radioactively labelled precursors we found that fully heterotrophic sugarcane cells without carotenoids and chlorophyll and absolutely dependent on sucrose for growth were capable of incorporating galactose into both mono- and di-galactosyldiacylglycerol (MGDG and DGDG), sulfate and galactose into sulfolipid (SL), and phosphate into phosphatidylglycerol (PG). All above-mentioned lipids showed turnover allowing the calculation of their half-lifes: τ_0.5 = 28 h for MGDG. There is an initial increment in the labelling of DGDG (at the expense of MGDG) and then a decrement with τ_0.5 = 27 h. The SL shows a τ_0.5 = 30 h and τ_0.5 = 28 h for PG. It is evident that during differentiation changes in lipid metabolism occur allowing the cell not only to increase, but to localize preferentially the biosynthetic machinery of the above-mentioned lipids in the chloroplasts and to make the process light-dependent.     

Genetic segregation of microsatellite markers in Saccharum officinarum and S. spontaneum

Genetic mapping techniques can be used to study the interaction between two different genomes after hybridization. This study investigated a Saccharum officinarum (Green German or GG, 2n approximately 11x approximately 110) x S. spontaneum (IND 81-146 or IND, 2n approximately 7x approximately 56) interspecific cross. Segregation of 193 microsatellite (SSR) loci was evaluated in the F(1) progeny of 169 full-sibs of the cross. Following the two-way pseudo-testcross strategy and 'cross pollination' population type, linkage groups (LG) and phases were established for each parent map, using the criteria of LOD score > or = 3.0 and a maximum recombination frequency of 0.35. Of the 193 markers analyzed, 61 were IND-specific, 106 were GG-specific, and 26 were heterozygous in both parents. About 78% of the markers segregated in a Mendelian fashion and 22% were distorted (as evaluated by chi(2)-tests, P < or = 0.01). The GG map included 91 marker loci arranged into 25 LG covering 1180 cM of the officinarum genome. The IND map consisted of 46 marker loci assembled into 10 LG, which spanned 614 cM of the spontaneum genome. A specific chromosome associated with segregation distortion was detected in the female (GG) genome only, probably as a result of double reduction. The segregation patterns of the marker loci indicated a centromere-driven distortion process with the shared allelic markers (as putative centromeres) regulating the placement and association of markers with opposite phase (coupling vs repulsion) and dosage on either side. Although incomplete, the framework maps were informative with respect to segregation distortion, chromosome fusion, rearrangements, and translocations, observed in both parental genomes as a result of their merger.     

Vancomycin stress response in a sensitive and a tolerant strain of Streptococcus pneumoniae

The vancomycin stress response was studied in Streptococcus pneumoniae strains T4 (TIGR4) and Tupelo. Vancomycin affected the expression of 175 genes, including genes encoding transport functions and enzymes involved in aminosugar metabolism. The two-component systems TCS03, TCS11, and CiaRH also responded to antibiotic treatment. We hypothesize that the three regulons are an important part of the bacterium's response to vancomycin stress.     

Manipulating cytoplasmic pH under anoxia: A critical test of the role of pH in the switch from aerobic to anaerobic metabolism

Ethanol production by maize (Zea mays L.) root tips, measured by an enzymic assay of the suspending medium, was correlated with changes in the cytoplasmic pH, determined by in-vivo ³¹P nuclear magnetic resonance (NMR) spectroscopy, following the onset of anoxia. Strong evidence for the role of the cytoplasmic pH in triggering the switch to ethanol production under anoxia was obtained by: (i) varying the pH of the suspending medium between pH 4 and pH 10; and (ii) using the permeant weak base methylamine to combat the acidification of the cytoplasm induced by the anoxic conditions. Experimentally, it proved to be much easier to manipulate the cytoplasmic pH under anoxia after the pH had stabilised, rather than during the initial rapid acidification that occurred following the onset of anoxia, and in the presence of methylamine, it was possible to impose a normal aerobic cytoplasmic pH value on tissue that was metabolising anaerobically. By this means it was possible to demonstrate the reversibility of the pH effect on ethanol production under anoxia and thus to provide good evidence in support of the biochemical pH-stat model of the anoxic response. The NMR measurement of the cytoplasmic pH in the presence of methylamine was achieved by using a manganese pretreatment technique to eliminate interference between the cytoplasmic and vacuolar P_i signals, and it seems likely that the experimental approach used here will have further applications in studies of the metabolic response to anoxia.Abbreviations Caps 3-(cyclohexylamino)-1-propane sulphonic acid - Mes 2-(N-morpholino)-ethane sulphonic acid - NMR nuclear magnetic resonance - Pi inorganic phosphate We acknowledge the financial support of the Agricultural and Food Research Council and G.G.F. acknowledges the receipt of a Research Fellowship from the Royal Commission for the Exhibition of 1851.     

Simultaneous sulfide and nitrate removal in anaerobic reactor under shock loading

The performance of anaerobic reactor for simultaneous sulfide and nitrate removal under substrate shock loading was studied. The response to the shock loading could be divided into three stages i.e. disturbance, inertial and recovery periods. The effect of the shock loading was directly proportional to the intensity of the shock loads. The reactor performance was stable at a relatively lower intensity (1.5 times shock load), while it was considerably affected by higher intensity (higher than 2.0 times shock load). Nevertheless, the reactor performance recovered from disturbances at all the tested shock loads. The effluent sulfide-sulfur concentration was found as sensitive parameter, which increased up to 18 times of that at steady state; it could be used as an indicator of the reactor’s performance.     

Dissimilatory nitrate reduction in fungi under conditions of hypoxia and anoxia: a review

Recent progress in studies of anaerobic nitrate reduction and nitrous oxide formation in fungi has been reviewed. Current understanding of the biochemistry of nitrate and nitrite reduction to nitrous oxide and ammonium under oxygen limitation is presented, with emphasis on patterns of fungal co-denitrification, properties of the enzymes involved, and prevalence of nitrate respiration among fungal species.     

Physiological,biochemical, and metabolic responses of a Taxus baccata L. callus culture under drought stress

In Vitro Cellular & Developmental Biology - Plant - The metabolic stimulation induced by abiotic stress is an efficient strategy for the production of secondary metabolites in sterile and...     

Dissimilatory nitrate reduction in fungi under conditions of hypoxia and anoxia: A review

Recent progress in studies of anaerobic nitrate reduction and nitrous oxide formation in fungi has been reviewed. Current understanding of the biochemistry of nitrate and nitrite reduction to nitrous oxide and ammonium under oxygen limitation is presented, with emphasis on patterns of fungal co-denitrification, properties of the enzymes involved, and prevalence of nitrate respiration among fungal species.     

Effects of NaCl stress on proline and cation accumulation in salt sensitive and tolerant turfgrasses

Summary Concentrations of proline, sodium and potassium in shoot tissues of five turfgrass species were measured following exposure to 170 mM NaCl salinity stress. Salt tolerant ‘Fults’ alkaligrass and ‘Dawson’ red fescue restricted the accumulation of Na-ions to significatnly low levels compared to the salt sensitive Kentucky bluegrasses (‘Adelphi’ and ‘Ram I’) and ‘Jamestown’ red fescue. Accumulation of proline began in all species within 24 h of initiation of salt stress but at a more rapid rate and higher overall concentration for ‘Fults’ alkaligrass. Proline levels were variable and too low in relation to sodium accumulations to have any significant osmoregulatory role in salt tolerance among all cultivars tested with the possible exception of alkaligrass.     

Differences in fennel seed responses to drought stress at the seed formation stage in sensitive and tolerant genotypes

To understand the effects of drought on fennel seed production and determine the underlying molecular processes, various fennel genotypes were exposed to drought stress. The yield and quality, including aromatic oil content, of fennel seeds were reduced by drought during seed development. To explore drought-induced biological processes in fennel, a label-free/gel-free proteomic analysis was performed. In Gaziantep and Tatmaj cultivars, which are sensitive and tolerant fennel genotypes, respectively, 106 and 92 drought-responsive proteins were identified. Comparison of protein-functional profiles indicated that proteins classified in stress, cell, and protein synthesis/degradation categories consisted important responsive mechanisms against drought stress. Pathway analysis visualized that the tricarboxylic acid cycle is important for both cultivars. In Tatmaj, moderate activation of proteins related to oxidative pentose phosphate pathway was detected along with an increase in photosynthesis-related proteins. Furthermore, cluster analysis of drought-responsive proteins using protein abundance at milky, dough, and mature stages identified protein homeostasis as a mechanism of drought tolerance in fennel. These results suggest that coordinated energy consumption and supply might be a drought-tolerance mechanism in fennel plants.