Intracellular Solutes,Photosynthesis and Respiration of the Green Alga Blidingia minima in Response to Salinity Stress

The effects of different salinities ranging from 7–68‰ on the internal inorganic and organic solute concentrations, and on the photosynthesis and respiration have been investigated in the green alga Bladingia minima (Näg. ex Kütz.) Kylin. The levels of the main osmotic solutes K⁺, sucrose and proline increased with increasing salinities and vice versa, while Na⁺, Mg²⁺, Cl^? and PO^3–₄ played a minor role in the osmotic acclimation. In contrast to related Enteromorpha species, B. minima exhibited high NO^?₃ concentrations, which decreased under hypo- and hypersaline conditions. B. minima differs also from Enteromorpha by accumulating the tertiary sulphonium compound DMSP in osmotically significant amounts under gentle hypersaline conditions. B. minima revealed typical characteristics of a “sun-plant” having a high light compensation point together with a saturation of photosynthesis at high photon flux densities. The alga showed a broad photosynthetic stability under osmotic stress; only with extreme hypersaline conditions was photosynthesis partly inhibited. The rate of respiration remained constant in hypersaline media, and was stimulated under hyposaline conditions.     

Mutations Stabilize Small Subunit Ribosomal RNA in Desiccation-Tolerant Cyanobacteria <Emphasis Type="Italic">Nostoc</Emphasis>

The ribosomal RNA molecule is an ideal model for evaluating the stability of a gene product under desiccation stress. We isolated 8 Nostoc strains that had the capacity to withstand desiccation in habitats and sequenced their 16S rRNA genes. The stabilities of 16S rRNAs secondary structures, indicated by free energy change of folding, were compared among Nostoc and other related species. The results suggested that 16S rRNA secondary structures of the desiccation-tolerant Nostoc strains were more stable than that of planktonic Nostocaceae species. The stabilizing mutations were divided into two categories: (1) those causing GC to replace other types of base pairs in stems and (2) those causing extension of stems. By mapping stabilizing mutations onto the Nostoc phylogenetic tree based on 16S rRNA gene, it was shown that most of stabilizing mutations had evolved during adaptive radiation among Nostoc spp. The evolution of 16S rRNA along the Nostoc lineage is suggested to be selectively advantageous under desiccation stress. Electronic Supplementary Material Electronic Supplementary material is available for this article at and accessible for authorised users.     

Gene Expression in the Cyanobacterium <Emphasis Type="Italic">Anabaena</Emphasis> sp. PCC7120 under Desiccation

The N₂-fixing cyanobacterium Anabaena sp. PCC7120 showed an inherent capacity for desiccation tolerance. A DNA microarray covering almost the entire genome of Anabaena was used to determine the genome-wide gene expression under desiccation. RNA was extracted from cells at intervals starting from early to late desiccation. The pattern of gene expression in DNA fragments was categorized into seven types, which include four types of up-regulated and three types of down-regulated fragments. Validation of the data was carried out by RT-PCR on selected up-regulated DNA fragments and was consistent with the changes in mRNA levels. Our conclusions regarding desiccation tolerance for Anabaena sp. PCC7120 are as follows: (i) Genes for osmoprotectant metabolisms and the K⁺ transporting system are up-regulated from early to mid-desiccation; (ii) genes induced by osmotic, salt, and low-temperature stress are up-regulated under desiccation; (iii) genes for heat shock proteins are up-regulated after mid-desiccation; (iv) genes for photosynthesis and the nitrogen-transporting system are down-regulated during early desiccation; and (v) genes for RNA polymerase and ribosomal protein are down-regulated between the early and the middle phase of desiccation. Profiles of gene expression are discussed in relation to desiccation acclimation.     

Hydrogen uptake in Nostoc sp. strain PCC 73102. Cloning and characterization of a hupSL homologue

Structural genes encoding an uptake hydrogenase of Nostoc sp. strain PCC 73102 were isolated. From partial libraries of genomic DNA, two clones (pNfo01 and pNfo02) were selected and sequenced, revealing the complete sequence of both a hupS (960 bases) and a hupL (1,593 bases) homologue in Nostoc sp. strain PCC 73102. A comparison between the deduced amino acid sequences of HupS and HupL of Nostoc sp. strain PCC 73102 and Anabaena sp. strain PCC 7120 showed that the HupS proteins are 89% identical and the HupL proteins are 91% identical. However, the noncoding region between the genes in Nostoc sp. strain PCC 73102 (192 bases) is longer than that of Anabaena sp. strain PCC 7120 and of many other microorganisms. Southern hybridizations using DNA from both N₂-fixing and non-N₂-fixing cells of Nostoc sp. strain PCC 73102 and different probes from within hupL clearly demonstrated that, in contrast to Anabaena sp. strain PCC 7120, there is no rearrangement within hupL of Nostoc sp. strain PCC 73102. Indeed, 6 nucleotides out of 16 within the potential recombination site are different from those of Anabaena sp. strain PCC 7120. Furthermore, we have recently published evidence demonstrating the absence of the bidirectional/reversible hydrogenase in Nostoc sp. strain PCC 73102. The present knowledge, in combination with the unique characteristics, makes Nostoc sp. strain PCC 73102 an interesting candidate for the study of deletion mutants lacking the uptake-type enzyme. Received: 20 August 1997 / Accepted: 24 November 1997     

Sucrose metabolism in spring and winter wheat in response to high irradiance, cold stress and cold acclimation

Effects of low‐temperature stress, cold acclimation and growth at high irradiance in a spring (Triticum aestivum L. cv. Katepwa) and a winter wheat (Triticum aestivum L. cv. Monopol) were examined in leaves and crowns with respect to the sucrose utilisation and carbon allocation. Light‐saturated and carbon dioxide (CO₂)‐saturated rates of CO₂ assimilation were decreased by 50% in cold‐stressed spring and winter wheat cultivars. Cold‐ or high light‐acclimated Katepwa spring wheat maintained light‐saturated rates of CO₂ assimilation comparable to those of control spring wheat. In contrast, cold‐ or high light‐acclimated winter wheat maintained higher light and CO₂‐saturated rates of CO₂ assimilation than non‐acclimated controls. In leaves, during either cold stress, cold acclimation or acclimation to high irradiance, the sucrose/starch ratio increased by 5‐ to 10‐fold and neutral invertase activity increased by 2‐ to 2.5‐fold in both the spring and the winter wheat. In contrast, Monopol winter wheat, but not Katepwa spring wheat, exhibited a 3‐fold increase in leaf sucrose phosphate synthase (SPS) activity, a 4‐fold increase in sucrose:sucrose fructosyl transferase activity and a 6.6‐fold increase in acid invertase upon cold acclimation. Although leaves of cold‐stressed and high light‐grown spring and winter wheat showed 2.3‐ to 7‐fold higher sucrose levels than controls, these plants exhibited a limited capacity to adjust either sucrose phosphate synthase or sucrose synthase activity (SS[s]). In addition, the acclimation to high light resulted in a 23–31% lower starch abundance and no changes at the level of fructan accumulation in leaves of either winter or spring wheat when compared with controls. However, high light‐acclimated winter wheat exhibited a 1.8‐fold higher neutral invertase activity and high light‐acclimated spring wheat exhibited an induction of SS(d) activity when compared with controls. Crowns of Monopol showed higher fructan accumulation than Katepwa upon cold and high light acclimation. We suggest that the differential adjustment of CO₂‐saturated rates of CO₂ assimilation upon cold acclimation in Monopol winter wheat, as compared with Katepwa spring wheat, is associated with the increased capacity of Monopol for sucrose utilisation through the biosynthesis of fructans in the leaves and subsequent export to the crowns. In contrast, the differential adjustment of CO₂‐saturated rates of CO₂ assimilation upon high light acclimation of Monopol appears to be associated with both increased fructan and starch accumulation in the crowns.     

Accumulation of trehalose in response to desiccation and salt stress in the terrestrial cyanobacterium Nostoc commune

Changes in photosynthetic activity and trehalose levels in field‐isolated, natural colonies of the terrestrial cyanobacterium Nostoc commune responding to desiccation and salt stress were investigated. As the water content decreased in N. commune colonies during desiccation, photosynthetic O₂‐evolving activity decreased and no activity was detected in desiccated colonies. A high level of O₂ evolution was restored in the colonies as they absorbed atmospheric moisture, indicating that only a small amount of water is required for reactivation of photosynthesis. No detectable trehalose was found in fully hydrated N. commune colonies; however, trehalose accumulation occurred in response to water loss during desiccation and high levels of trehalose were detected in the air‐dried colonies. Moreover, a 0.2 M NaCl treatment also induced trehalose accumulation to a level equivalent to that by desiccation. Photosynthetic O₂ evolution was inhibited by 0.2 M NaCl, indicating that N. commune can tolerate only low levels of salt. These results suggest that cessation of photosynthesis and trehalose accumulation occur in response to both matric water stress (desiccation) and osmotic water stress (high salt concentration), and that while trehalose may be a less effective osmoprotective compound than others, it is important for the extreme tolerance to desiccation observed in terrestrial cyanobacterium.     

Cross-talk between salicylic acid and NaCl-generated reactive oxygen species and nitric oxide in tomato during acclimation to high salinity

Hydrogen peroxide (H₂O₂) and nitric oxide (NO) generated by salicylic acid (SA) are considered to be functional links of cross‐tolerance to various stressors. SA‐stimulated pre‐adaptation state was beneficial in the acclimation to subsequent salt stress in tomato (Solanum lycopersicum cv. Rio Fuego). At the whole‐plant level, SA‐induced massive H₂O₂ accumulation only at high concentrations (10^?3–10^?2M), which later caused the death of plants. The excess accumulation of H₂O₂ as compared with plants exposed to 100 mM NaCl was not associated with salt stress response after SA pre‐treatments. In the root tips, 10^?3–10^?2M SA triggered the production of reactive oxygen species (ROS) and NO with a concomitant decline in the cell viability. Sublethal concentrations of SA, however, decreased the effect of salt stress on ROS and NO production in the root apex. The attenuation of oxidative stress because of high salinity occurred not only in pre‐adapted plants but also at cell level. When protoplasts prepared from control leaves were exposed to SA in the presence of 100 mM NaCl, the production of NO and ROS was much lower and the viability of the cells was higher than in salt‐treated samples. This suggests that, the cross‐talk of signalling pathways induced by SA and high salinity may occur at the level of ROS and NO production. Abscisic acid (ABA), polyamines and 1‐aminocyclopropane‐1‐carboxylic acid, the compounds accumulating in pre‐treated plants, enhanced the diphenylene iodonium‐sensitive ROS and NO levels, but, in contrast to others, ABA and putrescine preserved the viability of protoplasts.     

The Effect of In Vitro Culture Conditions on the Pattern of Photoinhibition during Acclimation of Gardenia Plantlets to Ex Vitro Conditions

We tested the effect of growing conditions during micropropagation on the fast kinetics of chlorophyll (Chl) fluorescence of Gardenia jasminoides Ellis plantlets during a 4-week acclimation to ex vitro. We studied whether photoautotrophic growing in vitro produced plantlets with less photoinhibition impairment during acclimation. Of the growing conditions stimulating photoautotrophy in vitro, only loose tube caps had a positive effect, whereas low sucrose or sucrose-free content in the medium and high PPFD showed a negative effect. Thus, plantlets cultured with 3 % (m/v) of sucrose were subsequently less photoinhibited throughout acclimation than those cultured with low sucrose (0.5 %) or sucrose-free media. Moreover, at the end of acclimation the former plantlets showed F_v/F_m and F_v/F₀ ratios typical of unstressed ex vitro plants as well as a higher Chl content and ratio of Chls to carotenoids. Plantlets cultured at a photosynthetic photon fluence density (PPFD) of 50 µmol m^–2 s^–1 also showed a better performance at the end of acclimation than those cultured at a higher (110 µmol m^–2 s^–1) PPFD. Thus except in the case of loose-tube closure, gardenia plantlets cultured in vitro under conventional sucrose concentration and PPFD are the least photoinhibited during acclimation. Nevertheless, significant interactions between the in vitro growing factors were observed at the end of acclimation.     

Isolation and characterization of a novel strain of genus <Emphasis Type="Italic">Dietzia</Emphasis> capable of multiple-extreme resistance

An ultraviolet (UV) radiation resistant gram-positive bacterium, Dietzia sp. MG4 strain, was isolated from the Sirch Hot Spring (Kerman, Iran), then it was identified on the basis of morphological and biochemical characteristics, and 16S rRNA gene sequencing. The effects of temperature, pH, desiccation, different percentage of NaCl, hydrogen peroxide (H₂O₂), mitomycin C (MMC) and high levels of radiation on viability or growth rate of MG4 strain were investigated. Also heavy metal tolerance of MG4 strain was assayed. 16S rDNA sequence of the isolate exhibited 99.69% similarity with Dietzia sp. and this result was confirmed by phylogenetic analysis. Viability of this strain was obtained D₉₁ according to D index after exposure to 25 J/cm² UV radiation dose, and D₃₀ after desiccation stress (for 28 days) using flow cytometery. The D₁₀ value for a microorganism is defined as the stress dose necessary to provide 10% survivors. Therefore, this strain showed high resistance to UV-C radiation and moderate resistance to desiccation. Optimal growth of MG4 strain was observed at pH 9, temperature of 30°C and 5% (w/v) NaCl. Isolated Dietzia was resisted up to 3 mM of nickel and 0.2 mM of mercury ions. Also this strain could tolerate 1–4% (v/v) H₂O₂ and 8 µg/mL of MMC as oxidant agents. To the best of our knowledge, this is the first study on multiple extreme resistant Dietzia sp. MG4 strain.     

EFFECTS OF SALINITY AND LIGHT INTENSITY ON THE RESUMPTION OF PHOTOSYNTHESIS IN REHYDRATED CYANOBACTERIAL MATS FROM BAJA CALIFORNIA SUR,MEXICO1

Lyngbya mats in the intertidal of the Laguna Ojo de Liebre are metabolically active for only a few days every 2 weeks during spring tides, with environmental conditions varying greatly during these periods of hydration. Pulse amplitude modulated fluorometry (PAM) and oxygen measurements were used to measure photosynthetic activity during the first few hours after rehydration under various light intensities and salinities. Upon rehydration, a transitory maximum in respiratory activity (10–30 min) occurred before the resumption of photosynthesis, which could recover in about 2 h. Salinities outside the mats' natural range (35–50 psu) were detrimental to photosynthetic recovery. Both high (100 psu) and low (0–10 psu) salinities slowed recovery as well as lowered the overall photosynthetic yield. Photosynthesis was initiated earlier and recovered more rapidly with increasing light intensity. In addition, the positive effect of light on rates of recovery was disproportionately greater at lower salinities (10–25 psu) where high light (500 W·m^?2) counteracted the negative effects of low‐salt stress early in recovery. However, higher light intensities became photoinhibitory later in recovery (>2 h). Photosynthesis did not recover uniformly within the mat. Filaments deeper in the mat most likely recovered later than those near the surface due to high light attenuation. The ability of the mats to tolerate desiccation and take advantage of hydration periods even when conditions are suboptimal enables these mats to predominate in the intertidal environment.     

Salinity tolerance of the copepod Apocyclops dengizicus (Lepeschkin, 1900), a key food chain organism in the Salton Sea,California

The copepod Apocyclops dengizicus is a key item in the food chain of the Salton Sea where the salinity is currently 45 g 1^–1. The salinity of the Salton Sea may reach 90 g 1 ^–1 within the next 20 years. This study examined the salinity tolerance of this copepod.Large copepodite and adult A. dengizicus were introduced into various salinities with and without acclimation. The 96 h LC₅₀ without acclimation was 101 g 1^–1. Mortality (at 96 h) without acclimation was low at salinities of 90 g 1 ^–1 or less.Copepod cultures were maintained, with successful reproduction of at least one new generation, at salinities of from 0.5 to 68 g 1 ^–1 for at least 120 days. Copepods maintained at higher salinities, up to 79 g 1 ^–1, remained alive up to 90 days, but a new generation was not produced. In laboratory studies of larval production and survivorship, few nauplii were released at salinities of 68 g 1 ^–1 or higher, and none survived to the copepodite stage.     

Incorporation of 14C in the cyanobacterium Synechococcus PCC 6301 following salt stress

Synechococcus PCC 6301 synthesized sucrose as a compatible solute following hyperosmotic shock induced by NaCl. Initial rates of photosynthetic ¹⁴C incorporation were reduced following salt shock. Photosynthetic rates were comparable in cells enriched for glycogen (by growth in NO ₃ ^- -deficient medium) and cells grown in NO ₃ ^- -sufficient medium in the absence of osmotic shock. Incorporation of ¹⁴C was predominantly into the NaOH fraction and the residual acidic fraction in cells grown in NO ₃ ^- -sufficient medium, whereas incorporation was predominantly into the residual acidic fraction in cells grown in NO ₃ ^- -deficient medium. Following salt stress, ¹⁴C incorporation was initially into the ethanol-soluble fraction and the majority of tracer was recovered in sucrose. Carbon-14 was detected in sucrose in cells which had been enriched for [¹⁴C]glycogen prior to salt stress, inferring that glycogen can act as a carbon source for sucrose synthesis following salt stress. Changes in the specific activity of sucrose are consistent with an initial synthesis of sucrose from glycogen followed by synthesis of sucrose using newly fixed carbon, in response to salt stress.This work was supported by the Agricultural and Food Research Council.     

Crucial Role of Extracellular Polysaccharides in Desiccation and Freezing Tolerance in the Terrestrial Cyanobacterium Nostoc commune

The cyanobacterium Nostoc commune is adapted to the terrestrial environment and has a cosmopolitan distribution. In this study, the role of extracellular polysaccharides (EPS) in the desiccation tolerance of photosynthesis in N. commune was examined. Although photosynthetic O₂ evolution was not detected in desiccated colonies, the ability of the cells to evolve O₂ rapidly recovered after rehydration. The air-dried colonies contained approximately 10% (wt/wt) water, and field-isolated, natural colonies with EPS were highly water absorbent and were rapidly hydrated by atmospheric moisture. The cells embedded in EPS in Nostoc colonies were highly desiccation tolerant, and O₂ evolution was not damaged by air drying. Although N. commune was determined to be a mesophilic cyanobacterium, the cells with EPS were heat tolerant in a desiccated state. EPS could be removed from cells by homogenizing colonies with a blender and filtering with coarse filter paper. This treatment to remove EPS did not damage Nostoc cells or their ability to evolve O₂, but O₂ evolution was significantly damaged by desiccation treatment of the EPS-depleted cells. Similar to the EPS-depleted cells, the laboratory culture strain KU002 had only small amount of EPS and was highly sensitive to desiccation. In the EPS-depleted cells, O₂ evolution was also sensitive to freeze-thaw treatment. These results strongly suggest that EPS of N. commune is crucial for the stress tolerance of photosynthesis during desiccation and during freezing and thawing.     

Development of desiccation tolerance and vitrification by preculture treatment in suspension-cultured cells of the liverwort Marchantia polymorpha

Some cultured plant cells are able to acquire tolerance to various stresses when they are cultured under suitably controlled conditions. Induction of a high level of desiccation tolerance in suspension-cultured cells of the liverwort Marchantia polymorpha was examined for studying the mechanisms of desiccation tolerance and vitrification at the cellular level. Desiccation tolerance level of cells was very low and the survival rate was less than 10% after exposure to drying below 0.1 g H₂O g⁻¹ dry weight (DW). Preculture treatment in 0.5 M sucrose medium was the most effective method for inducing a high level of desiccation tolerance in cells and the survival rate was 87% even after being desiccated to below 0.1 g H₂O g⁻¹ DW. Preculture treatment caused alteration of cell structures and accumulation of a large amount of sucrose and newly synthesized proteins in cells. Abundant sucrose and preculture-induced proteins were necessary for full development of desiccation tolerance in the cells. When water content decreased to below 0.1 g H₂O g⁻¹ DW, desiccation-tolerant cells that had been precultured were vitrified above 0°C and maintained stable viability. We have succeeded in the induction of desiccation tolerance that allows formation of intracellular glass with cell viability at ambient temperatures by controlling culture conditions, and our results suggest that suspension-cultured cells of M. polymorpha are useful for studying cellular mechanisms for the development of desiccation tolerance and the stabilization of vitrified cells.     

The role of extracellular polysaccharides produced by the terrestrial cyanobacterium Nostoc sp. strain HK-01 in NaCl tolerance

The terrestrial cyanobacterium Nostoc sp. HK-01 was more tolerant to NaCl stress than the aquatic cyanobacterium Anabaena sp. PCC 7120 (also called Nostoc sp. PCC 7120) which is similar to Nostoc sp. HK-01 in phylogeny. We determined the amount of extracellular polysaccharides (capsular and released polysaccharides) from the cells of both strains cultured with or without 200 mM NaCl. The amount of capsular polysaccharides from Nostoc HK-01 reached approximately 65% of the dry weight whereas that from Anabaena PCC 7120 only occupied approximately 18% of the dry weight under NaCl stress. Anabaena PCC 7120 grew well under NaCl stress when both polysaccharides from Nostoc HK-01 were added to the culture. However, Anabaena PCC 7120 barely grew under NaCl stress when both of its polysaccharides were added. Extracellular polysaccharides from Nostoc HK-01 contained abundant fucose and glucuronic acid in comparison with those from Anabaena PCC 7120. Under NaCl stress, the composition ratios of sugars in the extracellular polysaccharides from Anabaena PCC 7120 hardly changed in comparison with those in ordinary culture conditions. By contrast, the composition ratios of sugars in the extracellular polysaccharides from Nostoc HK-01 changed under NaCl stress. These results suggest that the effect of extracellular polysaccharides from Nostoc HK-01 on NaCl tolerance comes from the increased amount of capsular polysaccharides, the sugar composition, and the change of the sugar composition ratio under NaCl stress.