Recovery of photosynthesis after exposure of intertidal algae to osmotic and temperature stresses: comparative studies of species with differing distributional limits

Summary This study examines possible relationships between stress tolerance by marine algae and distributions of these species. The ability to recover photosynthetic activity following dehydration or temperature treatments was the assay used to evaluate stress tolerance, and Porphyra perforata, Rhodoglossum affine, Gelidium coulteri, and Smithora naiadum differed in thresholds of tolerance, even though plants were collected from low tidal sites.Limits of dehydration tolerance were well correlated with limits of tidal distribution for these species. Additionally, other high tidal species tolerated severe dehydration while subtidal and low tidal species were sensitive to dehydration. High tidal individuals of P. perforata were also more tolerant of dehydration than were low tidal thalli of P. perforata.Limits of high or low temperature tolerance were not well correlated with tidal elevation for any groups of algae studied. However, cold-tolerant species had more northerly extensions, and warm-tolerant species had more southerly distributions. Thus, differential tolerance to temperature extremes may be an important influence for latitudinal ranges of species.By comparing the experimentally determined thresholds of stress with distributions of species, we test the role of stress in influencing photosynthesis and ultimately distributions of marine algae.     

高盐下条斑紫菜光合特性和S-腺苷甲硫氨酸合成酶基因表达的变化

为了研究条斑紫菜耐盐机理,对条斑紫菜叶状体进行了高盐胁迫处理,继而采用氧电极法测量了光合放氧速率和呼吸耗氧速率的变化,采用实时荧光定量PCR技术测量了S-腺苷甲硫氨酸合成酶(命名为PySAMS)基因的表达变化。结果显示藻体的光合与呼吸作用均受到高盐度海水的显著影响,随着盐度的增加,光合放氧率逐渐降低,呼吸耗氧率也逐渐降低。高盐度海水对PySAMS基因表达量也产生了显著影响,40和50盐度的海水诱导了PySAMS表达,但60至80盐度的海水却不同程度地抑制了PySAMS表达。据此推测,在面对较高盐度胁迫时条斑紫菜叶状体将逐步降低体内新陈代谢以度过不良环境。     

IDENTIFICATION OF THE HIGH‐TEMPERATURE RESPONSE GENES FROM PORPHYRA SERIATA (RHODOPHYTA) EXPRESSION SEQUENCE TAGS AND ENHANCEMENT OF HEAT TOLERANCE OF CHLAMYDOMONAS (CHLOROPHYTA) BY EXPRESSION OF THE PORPHYRA HTR2 GENE1

Temperature is one of the major environmental factors that affect the distribution, growth rate, and life cycle of intertidal organisms, including red algae. In an effort to identify the genes involved in the high‐temperature tolerance of Porphyra, we generated 3,979 expression sequence tags (ESTs) from gametophyte thalli of P. seriata Kjellm. under normal growth conditions and high‐temperature conditions. A comparison of the ESTs from two cDNA libraries allowed us to identify the high temperature response (HTR) genes, which are induced or up‐regulated as the result of high‐temperature treatment. Among the HTRs, HTR2 encodes for a small polypeptide consisting of 144 amino acids, which is a noble nuclear protein. Chlamydomonas expressing the Porphyra HTR2 gene shows higher survival and growth rates than the wild‐type strain after high‐temperature treatment. These results suggest that HTR2 may be relevant to the tolerance of high‐temperature stress conditions, and this Porphyra EST data set will provide important genetic information for studies of the molecular basis of high‐temperature tolerance in marine algae, as well as in Porphyra.     

Quantifying the three main components of salinity tolerance in cereals

Salinity stress is a major factor inhibiting cereal yield throughout the world. Tolerance to salinity stress can be considered to contain three main components: Na⁺ exclusion, tolerance to Na⁺ in the tissues and osmotic tolerance. To date, most experimental work on salinity tolerance in cereals has focused on Na⁺ exclusion due in part to its ease of measurement. It has become apparent, however, that Na⁺ exclusion is not the sole mechanism for salinity tolerance in cereals, and research needs to expand to study osmotic tolerance and tissue tolerance. Here, we develop assays for high throughput quantification of Na⁺ exclusion, Na⁺ tissue tolerance and osmotic tolerance in 12 Triticum monococcum accessions, mainly using commercially available image capture and analysis equipment. We show that different lines use different combinations of the three tolerance mechanisms to increase their total salinity tolerance, with a positive correlation observed between a plant's total salinity tolerance and the sum of its proficiency in Na⁺ exclusion, osmotic tolerance and tissue tolerance. The assays developed in this study can be easily adapted for other cereals and used in high throughput, forward genetic experiments to elucidate the molecular basis of these components of salinity tolerance.     

Cyclophilin Participates in Responding to Stress Situations in Porphyra haitanensis (Bangiales,Rhodophyta)

Porphyra haitanensis (T. J. Chang & B. F. Zheng) is an important economic alga found off the southern coast of China. It has evolved a strong tolerance against stress, which is an important survival characteristic. Cyclophilin has been shown to be involved in the stress response of plants and algae. To investigate the tolerance against stress in Porphyra, we isolated the cyclophilin PhCYP18 gene (Accession number JQ413239 ) and measured its expression over different generations and stress conditions. In P. haitanensis, cyclophilin PhCYP18 accumulated more in the filamentous sporophyte generation than in the blade gametophyte generation. This difference was thought to be due to harsh environments and a gene dosage effect. It has been found, however, that PhCYP18 expression was dysregulated in blades under high salt stress, strong irradiance stress and multifactorial stress compared to blades under normal conditions. Moreover, the changes were not linearly related to the degree of stress. It was therefore thought that PhCYP18 actively responded to stress situations and induced strong stress tolerance, which is evident in P. haitanensis.     

Heat challenges can enhance population tolerance to thermal stress in mussels: a potential mechanism by which ship transport can increase species invasiveness

It is unclear whether transport by human vectors can increase the robustness of translocated populations and thereby enhance their invasiveness. To test this concept, we investigated the effect of heat stress on the tolerance of mussel populations towards a second stress event of the same kind. The heat challenges we mimicked can be faced by marine invertebrates that are transported through regions with high sea surface temperatures on ship hulls or in ballast water tanks. The study included 5 mussel species that were collected at sites in Brazil, Chile, Finland, Germany (Baltic Sea) and Portugal. In parallel laboratory experiments, monospecific groups of individuals were exposed to heat challenges that caused 60–83% mortality in the experimental groups within 15–28 days. The surviving individuals were exposed to a second stress event of the same kind, while their survival was then compared to the robustness of conspecifics that had not been exposed to elevated temperatures before. We observed that thermal tolerance was significantly enhanced by previous heat stress experience in case of Semimytilus algosus from Chile and in case of Mytilus edulis from Germany. Our results suggest that heat challenges, which marine invertebrates experience during transport, can enhance stress tolerance in founder populations of these species in their non-native range by potentially increasing the frequency of genetically adapted genotypes. This points at the necessity to learn more about selection acting on organisms during human-mediated transport—in the aquatic but also in the terrestrial environment.     

Photosystem I-Mediated Regulation of Water Splitting in the Red Alga, Porphyra sanjuanensis

The marine red alga, Porphyra sanjuanensis is found mainly in the high intertidal zone and at low tide subject to frequent and extreme water stress, often accompanied by high temperatures and light intensities. Such exposures can lead to severe desiccation which is accompanied by the progressive loss of photosynthetic activity. Even following the loss of more than 90% of the thallus water content the alga recovers rapidly when returned to seawater. This stress-induced, reversible inactivation of photosynthesis is believed to be a protective adaptation which prevents photodamage to the exposed alga. Effects of light, inhibitors of water splitting, and electron donors to PSI on variable fluorescence and water splitting suggest that activity of the oxygen evolving complex is regulated by the PSI-driven reduction of a component of intersystem electron transport.     

Variant forms of a group I intron in nuclear small-subunit rRNA genes of the marine red alga Porphyra spiralis var. amplifolia

A group IC1 intron occurs in nuclear small-subunit (18S) ribosomal RNA (SSU rRNA) genes of the marine red alga Porphyra spiralis var. amplifolia. This intron occurs at the same position as the self- splicing group IC1 introns in nuclear SSU rDNAs of the fungus Pneumocystis carinii and in the green alga Chlorella ellipsoidea and shares sequence identity with the Pneumocystis carinii intron in domains L1, P1, P2, and L2, outside the conserved core. Three size variants, differing in amount of sequence in L1, exist and are differentially distributed in geographically distinct populations. Preliminary data suggest that the largest variant can self-splice in vitro. Short open reading frames are present but do not correspond to known genes. Repeated nucleotide motifs, reminiscent of duplicated target sites of transposons or Alu elements, are associated with the intron and with one of the variant forms of L1. Insertions are present in nuclear SSU rDNAs of several other Porphyra species and of the red alga Bangia atropurpurea; insertionless rDNA variants also occur in several Porphyra species. Our observations are most readily explained by intron mobility, although it remains unclear how transfer could have been mediated between genomes of organisms as ecologically diverse as marine red algae, freshwater green algae, and a mammalian-pathogenic fungus.      

Identification, expression, and evolutionary analyses of plant lipocalins

Lipocalins are a group of proteins that have been characterized in bacteria, invertebrate, and vertebrate animals. However, very little is known about plant lipocalins. We have previously reported the cloning of the first true plant lipocalins. Here we report the identification and characterization of plant lipocalins and lipocalin-like proteins using an integrated approach of data mining, expression studies, cellular localization, and phylogenetic analyses. Plant lipocalins can be classified into two groups, temperature-induced lipocalins (TILs) and chloroplastic lipocalins (CHLs). In addition, violaxanthin de-epoxidases (VDEs) and zeaxanthin epoxidases (ZEPs) can be classified as lipocalin-like proteins. CHLs, VDEs, and ZEPs possess transit peptides that target them to the chloroplast. On the other hand, TILs do not show any targeting peptide, but localization studies revealed that the proteins are found at the plasma membrane. Expression analyses by quantitative real-time PCR showed that expression of the wheat (Triticum aestivum) lipocalins and lipocalin-like proteins is associated with abiotic stress response and is correlated with the plant's capacity to develop freezing tolerance. In support of this correlation, data mining revealed that lipocalins are present in the desiccation-tolerant red algae Porphyra yezoensis and the cryotolerant marine yeast Debaryomyces hansenii, suggesting a possible association with stress-tolerant organisms. Considering the plant lipocalin properties, tissue specificity, response to temperature stress, and their association with chloroplasts and plasma membranes of green leaves, we hypothesize a protective function of the photosynthetic system against temperature stress. Phylogenetic analyses suggest that TIL lipocalin members in higher plants were probably inherited from a bacterial gene present in a primitive unicellular eukaryote. On the other hand, CHLs, VDEs, and ZEPs may have evolved from a cyanobacterial ancestral gene after the formation of the cyanobacterial endosymbiont from which the chloroplast originated.     

Photosynthetic efficiency and survival of Dactylis glomerata and Lolium perenne following low temperature stress

Resistance to low temperature is crucial for overwintering crops. In this work we compared the resistance to low temperature treatment of some varieties of two forage grass species Dactylis glomerata L. and Lolium perenne L. in order to elucidate the reason for the better resistance found in some species. The variety Amila of D. glomerata and Diament of L. perenne were more tolerant to low temperature stress during the emergence and tillering phases as compared to the varieties Amera and Gagat. The improved tolerance and ability for recovery after stress were associated with the better recovery of photosynthetic efficiency of these varieties and better survival of their shoots after low temperature stress.     

Natural variability in Drosophila larval and pupal NaCl tolerance

The regulation of NaCl is essential for the maintenance of cellular tonicity and functionality, and excessive salt exposure has many adverse effects. The fruit fly, Drosophila melanogaster, is a good osmoregulator and some strains can survive on media with very low or high NaCl content. Previous analyses of mutant alleles have implicated various stress signaling cascades in NaCl sensitivity or tolerance; however, the genes influencing natural variability of NaCl tolerance remain for the most part unknown. Here, we use two approaches to investigate natural variation in D. melanogaster NaCl tolerance. We describe four D. melanogaster lines that were selected for different degrees of NaCl tolerance, and present data on their survival, development, and pupation position when raised on varying NaCl concentrations. After finding evidence for natural variation in salt tolerance, we present the results of Quantitative Trait Loci (QTL) mapping of natural variation in larval and pupal NaCl tolerance, and identify different genomic regions associated with NaCl tolerance during larval and pupal development.     

Rate of dehydration and cumulative desiccation stress interacted to modulate desiccation tolerance of recalcitrant cocoa and ginkgo embryonic tissues

Rate of dehydration greatly affects desiccation tolerance of recalcitrant seeds. This effect is presumably related to two different stress vectors: direct mechanical or physical stress because of the loss of water and physicochemical damage of tissues as a result of metabolic alterations during drying. The present study proposed a new theoretic approach to represent these two types of stresses and investigated how seed tissues responded differently to two stress vectors, using the models of isolated cocoa (Theobroma cacao) and ginkgo (Ginkgo biloba) embryonic tissues dehydrated under various drying conditions. This approach used the differential change in axis water potential (DeltaPsi/Deltat) to quantify rate of dehydration and the intensity of direct physical stress experienced by embryonic tissues during desiccation. Physicochemical effect of drying was expressed by cumulative desiccation stress [integralf(psi,t)], a function of both the rate and time of dehydration. Rapid dehydration increased the sensitivity of embryonic tissues to desiccation as indicated by high critical water contents, below which desiccation damage occurred. Cumulative desiccation stress increased sharply under slow drying conditions, which was also detrimental to embryonic tissues. This quantitative analysis of the stress-time-response relationship helps to understand the physiological basis for the existence of an optimal dehydration rate, with which maximum desiccation tolerance could be achieved. The established numerical analysis model will prove valuable for the design of experiments that aim to elucidate biochemical and physiological mechanisms of desiccation tolerance.     

Capacity for protein synthesis following heat stimulus of Drosophila associates with heat tolerance but does not underlie the latitudinal tolerance cline

Across populations of Drosophila melanogaster along the Australian eastern coastline latitudinal clines occur in both heat-knockdown tolerance and hardened heat-knockdown tolerance – low latitude tropical populations being more tolerant. A latitudinal cline also occurs for rates of total protein synthesis following a mild heat stress, with tropical populations having higher rates. Since the control of protein synthesis following heat stress is an important component of the cellular heat-shock response, we hypothesised that the higher rates of synthesis that follow a heat stimulus lead to higher knockdown tolerance and underpins the cline. However, levels of heat-stimulated total protein synthesis have been negatively related to heat-hardening capacity, a somewhat conflicting result. Here we examine the relationship between these physiological and adaptive traits in a set of 40 family lines derived from a hybrid laboratory population established by crossing populations from either end of the latitudinal transect. Among these lines high levels of heat-stimulated total protein synthesis were associated with both low basal and low heat-hardened adult knockdown time, confirming the importance of a negative relationship between protein synthesis and thermal tolerance. This result, when considered along with the directions of the latitudinal clines in protein synthesis and tolerance, suggests that variation in rates of heat-stimulated total protein synthesis is not a factor contributing to the latitudinal cline in heat tolerance. Given the robustness of this negative relationship we discuss possible explanations and future experiments to elucidate how the cellular heat stress response might facilitate increased knockdown tolerance.     

Growth,cell volume,and fine structure of Porphyra umbilicalis in relation to osmotic tolerance

Porphyra umbilicalis, a marine red alga occurring in the intertidal zone of the cold North Sea, tolerates a wide range of osmotic conditions from 0.2 x to 6 x artificial seawater medium ASP₁₂. In cells osmotically adapted for two weeks, photosynthesis and respiration are progressively inhibited in media more concentrated than 2 x. In both hypo- and hyperosmotic stress ranges, the most striking fine structural change is the development of vacuoles. In comparison to 1 x medium, where vacuoles are virtually lacking, the vacuolar part of the protoplasm increases 6-fold in 0.2 x and 10-fold in 3.5 x medium, respectively. However, at extreme hyperosmotic stress (6 x medium) the vacuolar part is extremely small. The largest cell volumes are found in 0.2 x and 3.5 x media, the smallest one in 6 x medium. In the osmotically regulated range (0.2–3.5 x medium), the regulated parameter is the volume of the protoplasm without the vacuolar system. It is suggested that at hyperosmotic stress the vacuoles may serve as osmotically active compartment, probably by accumulation of inorganic ions. The intracellular content of Floridean starch granules decreases with increasing osmotic pressure, possibly indicating the significance of soluble organic constituents as osmotically active solutes.Member of the Arbeitsgemeinschaft für Elektronenmikroskople un der Ticrärztlichen Hochschule Hannover     

苹果蠹蛾热激蛋白Hsp90基因的克隆及热胁迫下的表达分析

世界检疫性害虫苹果蠹蛾Cydia pomonella是一种温度耐受可塑性很高的物种。本研究针对温度波动可能导致其耐热性增强的科学问题, 采用生测法鉴定了苹果蠹蛾实验种群的高温耐受阈值, 采用同源克隆、 RACE和实时荧光定量PCR (RT-qPCR)等方法研究了苹果蠹蛾热激蛋白Hsp90基因的应激表达对耐热性的重要作用。高温耐受阈值研究结果表明, 苹果蠹蛾实验种群的死亡率随温度的升高和时间的延长显著性升高, 1-5龄幼虫分别经50℃和52℃高温处理2, 5和10 min后, 3龄幼虫耐热性最差, 5龄幼虫最强。50℃和52℃分别处理10 min和5 min均可导致1-4龄幼虫全部死亡, 而5龄幼虫在这两种处理下仍有25.0%和11.1%的存活率。以35℃处理的5龄雌幼虫为材料克隆苹果蠹蛾Hsp90基因全长cDNA, 结果显示该基因全长为2 470 bp, 完整开放阅读框为2 148 bp, 共编码716个氨基酸, 预测分子量为82.07 kDa, 命名为Cphsp90 (GenBank登录号JN624775)。该基因编码的氨基酸序列与亚洲玉米螟Ostrinia furnacalis和甘蓝夜蛾Mamestra brassicae等昆虫的Hsp90的氨基酸序列一致性高达96%, 表明了Hsp90家族的保守特性。Cphsp90 mRNA的相对表达量在32~44℃高温胁迫下随温度的升高而显著增高, 证实Cphsp90是诱导型热激基因, 且mRNA相对表达量与胁迫程度正相关。Cphsp90基因的表达还具有组织特异性, 35℃处理幼虫的表皮中Cphsp90相对表达量显著高于血淋巴、 脂肪体和中肠, 应激响应最为活跃。与未经温热预处理的昆虫相比, 35℃温热预处理3 h后的5龄幼虫在40, 45和50℃更高的温度胁迫下, Cphsp90 mRNA达到最高表达量所需要的胁迫温度有所提升, 由未经预热处理的40℃处理10 min提高到45℃处理10 min, 这与温热预处理会增强5龄幼虫耐热性的现象相符, 表明Cphsp90基因的响应表达在苹果蠹蛾耐热性及其可塑性过程中发挥重要的作用。     

Tolerance to oxidative stress induced by desiccation in Porphyra columbina (Bangiales, Rhodophyta)

Unravelling the mechanisms underlying desiccation tolerance is crucial in order to understand the position of algal species in the intertidal zone. The alga Porphyra columbina lives in the uppermost part of the rocky intertidal zones around the world and was selected as a model for this study. Naturally desiccated plants were collected during low tide and studied for morphological changes, oxidative burst induction, biomolecule oxidation, antioxidant responses, and photosynthetic status. Naturally hydrated plants collected during high tides were used for comparative purposes. In addition, changes induced by desiccation were assessed in vitro and the capacity to recover from desiccation was determined by rehydrating the fronds in seawater. The global results show that desiccation induces morphological and cellular alterations accompanied by a loss of ～96% of the water content. Overproduction of reactive oxygen species (ROS) was induced by desiccation and two peaks of H(2)O(2) were detected at 1 and 3 h of desiccation. However, during in vitro rehydration post-desiccation, the ROS quickly returned to the basal levels. At the biomolecular level, only a low production of oxidized proteins was recorded during desiccation, whereas the activity of diverse antioxidant enzymes increased. However, this activity diminished to near basal levels during rehydration. The photosynthetic efficiency (F(v)/F(m)) during desiccation declined by 94-96% of the values recorded in hydrated plants. This reduction was generated by the low levels of trapped energy flux per cross-section (TRo/CS), electron transport flux per CS (ETo/CS), and density of reaction centres (RC/SCo) as well as the chlorophyll content. The inverse pattern was observed for the levels of phycocyanin and phycoerythrin content. F(v)/F(m) and the photosynthetic indicators were restored to normal levels after only 5 min of rehydration. The results indicate that desiccation in P. columbina causes overproduction of ROS that is efficiently attenuated. The morphological and photosynthetic changes could be operating as tolerance mechanisms due to the fact that these responses principally prevent biomolecular alteration and cellular collapse. Thus, the activation of different physiological mechanisms helps to explain the high tolerance to desiccation of P. columbina and, at least in part, the position of this species at the highest level in the intertidal zone.