Expression of Ixodes scapularis antifreeze glycoprotein enhances cold tolerance in Drosophila melanogaster

Drosophila melanogaster experience cold shock injury and die when exposed to low non-freezing temperatures. In this study, we generated transgenic D. melanogaster that express putative Ixodes scapularis antifreeze glycoprotein (IAFGP) and show that the presence of IAFGP increases the ability of flies to survive in the cold. Male and female adult iafgp-expressing D. melanogaster exhibited higher survival rates compared with controls when placed at non-freezing temperatures. Increased hatching rates were evident in embryos expressing IAFGP when exposed to the cold. The TUNEL assay showed that flight muscles from iafgp-expressing female adult flies exhibited less apoptotic damage upon exposure to non-freezing temperatures in comparison to control flies. Collectively, these data suggest that expression of iafgp increases cold tolerance in flies by preventing apoptosis. This study defines a molecular basis for the role of an antifreeze protein in cryoprotection of flies.     

Induction of freezing tolerance in spinach is associated with the synthesis of cold acclimation induced proteins

Spinach (Spinacia oleracea L. cv Bloomsdale) seedlings cultured in vitro were used to study changes in protein synthesis during cold acclimation. Seedlings grown for 3 weeks postsowing on an inorganic-nutrient-agar medium were able to increase their freezing tolerance when grown at 5°C. During cold acclimation at 5°C and deacclimation at 25°C, the kinetics of freezing tolerance induction and loss were similar to that of soil-grown plants. Freezing tolerance increased after 1 day of cold acclimation and reached a maximum within 7 days. Upon deacclimation at 25°C, freezing tolerance declined within 1 day and was largely lost by the 7th day. Leaf proteins of intact plants grown at 5 and 25°C were in vivo radiolabeled, without wounding or injury, to high specific activities with [³⁵S]methionine. Leaf proteins were radiolabeled at 0, 1, 2, 3, 4, 7, and 14 days of cold acclimation and at 1, 3, and 7 days of deacclimation. Up to 500 labeled proteins were separated by two-dimensional gel electrophoresis and visualized by fluorography. A rapid and stable change in the protein synthesis pattern was observed when seedlings were transferred to the low temperature environment. Cold-acclimated leaves contained 22 polypeptides not found in nonacclimated leaves. Exposure to 5°C induced the synthesis of three high molecular weight cold acclimation proteins (CAPs) (M_r of about 160,000, 117,000, and 85,000) and greatly increased the synthesis of a fourth high molecular weight protein (M_r 79,000). These proteins were synthesized during day 1 and throughout the 14 day exposure to 5°C. During deacclimation, the synthesis of CAPs 160, 117, and 85 was greatly reduced by the first day of exposure to 25°C. However, CAP 79 was synthesized throughout the 7 day deacclimation treatment. Thus, the induction at low temperature and termination at warm temperature of the synthesis of CAPs 160, 117, and 85 was highly correlated with the induction and loss of freezing tolerance. Cold acclimation did not result in a general posttranslational modification of leaf proteins. Most of the observed changes in the two-dimensional gel patterns could be attributed to the de novo synthesis of proteins induced by low temperature. In spinach leaf tissue, heat shock altered the pattern of protein synthesis and induced the synthesis of several heat shock proteins (HSPs). One polypeptide synthesized in cold-acclimated leaves had a molecular weight and net charge (M_r 79,000, pI 4.8) similar to that of a HSP (M_r 83,000, pI 4.8). However, heat shock did not increase the freezing tolerance, and cold acclimation did not increase heat tolerance over that of nonacclimated plants, but heat-shocked leaf tissue was more tolerant to high temperatures than nonacclimated or cold-acclimated leaf tissue. When protein extracts from heat-shocked and cold-acclimated leaves were mixed and separated in the same two-dimensional gel, the CAP and HSP were shown to be two separate polypeptides with slightly different isoelectric points and molecular weights.     

A novel functional gene associated with cold tolerance at the seedling stage in rice

Identification and cloning of cold‐tolerant genes that can stably express under different cold environments are crucial for molecular rice breeding for cold tolerance. In the previous study, we identified a cold‐tolerant QTL at the seedling stage, qCTS‐9 which could be detected under different cold environments using a recombinant inbred line (RIL) population derived from a cold‐tolerant variety Lijiangxintuanheigu (LTH) and a cold‐sensitive variety Shanhuangzhan 2 (SHZ‐2). In this study, eight candidate genes within the qCTS‐9 interval were identified through integrated analysis of QTL mapping with genomewide differential expression profiling of LTH. The qRT‐PCR assay showed that only Os09g0410300 exhibited different expression patterns between LTH and SHZ‐2 during cold stress, and significantly positive correlation was found between cold induction of Os09g0410300 and seedling cold tolerance in the RI lines. Five SNPs and one InDel in the promoters of Os09g0410300 were detected between LTH and SHZ‐2, and the InDel marker ID410300 designed based on the insertion–deletion polymorphism in the promoter was significantly associated with seedling cold tolerance in RIL population. Further, Os09g0410300 over‐expression plants exhibited enhanced cold tolerance at the seedling stage compared with the wild‐type plants. Thus, our results suggest that Os09g0410300 is the functional gene underlying qCTS‐9. To our knowledge, it is a novel gene contributed to enhance cold tolerance at the seedling stage in rice. Identification of the functional gene underlying qCTS‐9 and development of the gene‐specific marker will facilitate molecular breeding for cold tolerance at the seedling stage in rice through transgenic approach and marker‐assisted selection (MAS).     

Hyperactive spruce budworm antifreeze protein expression in transgenic Drosophila does not confer cold shock tolerance

Drosophila melanogaster, a freeze intolerant and cold shock sensitive insect, was transformed with the hyperactive insect antifreeze protein gene (AFP) from the spruce budworm, Choristoneura fumiferana. Transformation P-element constructs (pCasper) were made with CfAFP 337 isoform DNA using a strong constitutive promoter, Actin 5c. This is the first report of insect AFP used to transform another insect. Properly folded active insect AFP was only detected when signal sequences were used to target proteins to the endoplasmic reticulum for secretion into the hemolymph. The 18 residue Drosophila binding protein signal sequence (BiP) constructs resulted in transformed fly lines with significantly higher AFP expression in hemolymph than when the native C. fumiferana AFP signal sequence was used. The resultant transgene fly lines have the highest levels of thermal hysteresis, 0.8 degrees C, seen for any engineered Drosophila. Despite the high level of expression, even higher than some overwintering fish with natural levels of endogenous AFP, the transformants did not display any cold shock resistance compared to controls or low AFP expressing lines. These results indicate that insect AFP alone cannot protect Drosophila from cold shock and may not be useful for Drosophila cryopreservation.     

Isozyme marker loci associated with cold tolerance and maturity in maize

Summary Two maize (Zea mays L.) populations, AS1(S) and ECR-A, were evaluated for allozyme frequency changes associated with selection for improved seedling emergence, early season vigor and early maturity. Eleven marker loci were examined and four loci were used for indirect selection in an attempt to modify cold tolerance and maturity. Allozyme-selected divergent subpopulations were produced by compositing selected S₁ progeny from cycle one (C1) of AS1(S) and from C2 of ECR-A. These subpopulations and S₁ generations from all cycles resulting from phenotypic selection, ECR-A C1 through C7 and AS1(S) CO through C6, were tested in cold tolerance and agronomic performance trials over five environments in 1986. Seedling emergence and seedling dry weight did not improve with phenotypic selection in ECR-A, while plant height, ear height, grain yield, grain moisture, days to mid-silk and days to mid-pollen were reduced significantly. Contrasts between divergent allozyme-selected subpopulations from ECR-A were significant for grain moisture and mid-pollen date. For AS1(S), seeding emergence increased, while plant and ear height decreased with phenotypic selection. Contrasts between allozyme-selected subpopulations were significant for plant and ear height. Changes associated with marker-based selection for AS1(S) were not in the same direction as with phenotypic selection. Selection for favorable allozyme genotypes may be effective in changing certain traits in populations that have been modified by direct selection, however results may not be predictable.Contribution from the Department of Agronomy, Wisconsin Agric. Exp. Stn., Madison, WI. Part of a thesis submitted by the senior author in partial fulfillment of the requirements for a Ph. D. received June, 1987. Research supported by the College of Agric. and Life Sci., University of Wisconsin-Madison, Dekalb-Pfizer Genetics, Garst Seed Company, and Pioneer Hi-Bred.     

Mapping of QTLs associated with cold tolerance during the vegetative stage in rice

Low-temperature stress is an important factor affecting the growth and development of rice (Oryza sativa L.) in temperate and high-elevation areas. Cold stress may cause various seedling injuries, delayed heading and yield reduction due to spikelet sterility. In this study, 181 microsatellite marker loci were used to identify quantitative trait loci (QTLs) associated with cold tolerance at the vegetative stage in 191 recombinant inbred lines (RILs) derived from a cross of a cold-tolerant temperate japonica cultivar (M-202) with a cold-sensitive indica cultivar (IR50). Different temperature regimes were applied in growth chambers on 191 RILs. The temperature regimes imposed in the growth chamber simulated cold-stress injuries at the seedling and late vegetative stages. In this study a major QTL was identified on chromosome 12, designated as qCTS12a, that was closely associated with cold-induced necrosis and wilting tolerance, and accounted for 41% of the phenotypic variation. A number of QTLs with smaller effects were also detected on eight rice chromosomes.     

MUSTANG is a novel family of domesticated transposase genes found in diverse angiosperms

While transposons have traditionally been viewed as genomic parasites or "junk DNA," the discovery of transposon-derived host genes has fueled an ongoing debate over the evolutionary role of transposons. In particular, while mobility-related open reading frames have been known to acquire host functions, the contribution of these types of events to the evolution of genes is not well understood. Here we report that genome-wide searches for Mutator transposase-derived host genes in Arabidopsis thaliana (Columbia-0) and Oryza sativa ssp. japonica (cv. Nipponbare) (domesticated rice) identified 121 sequences, including the taxonomically conserved MUSTANG1. Syntenic MUSTANG1 orthologs in such varied plant species as rice, poplar, Arabidopsis, and Medicago truncatula appear to be under purifying selection. However, despite the evidence of this pathway of gene evolution, MUSTANG1 belongs to one of only two Mutator-like gene families with members in both monocotyledonous and dicotyledonous plants, suggesting that Mutator-like elements seldom evolve into taxonomically widespread host genes.     

Testing for equal rates of cladogenesis in diverse taxa

Taxa differ widely in numbers of species, which may be due either to chance alone or to factors that cause differences in speciation and extinction rates between taxa. To test whether an observed distribution of species over taxa differs from the distribution expected from chance alone, one must take into account that neither speciation nor extinction rates are known. This paper introduces a way to estimate speciation and extinction probabilities from the distribution of extant species over families and to test whether the observed distribution is different from expected. Application of this procedure to the distributions of bird, hexapod, primate, and angiosperm species over taxa provides statistical evidence of differences in rates of cladogenesis between taxa.     

A natural variant of type I antifreeze protein with four ice-binding repeats is a particularly potent antifreeze.

A 4.3-kDa variant of Type I antifreeze protein (AFP9) was purified from winter flounder serum by size exclusion chromatography and reversed-phase HPLC. By the criteria of mass, amino acid composition, and N-terminal sequences of tryptic peptides, this variant is the posttranslationally modified product of the previously characterized AFP gene 21a. It has 52 amino acids and contains four 11-amino acid repeats, one more than the major serum AFP components. The larger protein is completely alpha-helical at 0 degree C, with a melting temperature of 18 degrees C. It is considerably more active as an antifreeze than the three-repeat winter flounder AFP and the four-repeat yellowtail flounder AFP, both on a molar and a mg/mL basis. Several structural features of the four-repeat winter flounder AFP, including its larger size, additional ice-binding residues, and differences in ice-binding motifs might contribute to its greater activity. Its abundance in flounder serum, together with its potency as an antifreeze, suggest that AFP9 makes a significant contribution to the overall freezing point depression of the host.     

Melatonin-related mitochondrial respiration responses are associated with growth promotion and cold tolerance in plants

Melatonin has the ability to improve plant growth and strengthened plant tolerance to environmental stresses; however, the effects of melatonin on mitochondrial respiration in plants and the underlying biochemical and molecular mechanisms are still unclear. The objective of the study is to determine possible effects of melatonin on mitochondrial respiration and energy efficiency in maize leaves grown under optimum temperature and cold stress and to reveal the relationship between melatonin-induced possible alterations in mitochondrial respiration and cold tolerance. Melatonin and cold stress, alone and in combination, caused significant increases in activities and gene expressions of pyruvate dehydrogenase, citrate synthase, and malate dehydrogenase, indicating an acceleration in the rate of tricarboxylic acid cycle. Total mitochondrial respiration rate, cytochrome pathway rate, and alternative respiration rate were increased by the application of melatonin and/or cold stress. Similarly, gene expression and protein levels of cytochrome oxidase and alternative oxidase were also enhanced by melatonin and/or cold stress. The highest values for all these parameters were obtained from the seedlings treated with the combined application of melatonin and cold stress. The activity and gene expression of ATP synthase and ATP concentration were augmented by melatonin under control and cold stress. On the other hand, cold stress reduced markedly plant growth parameters, including root length, plant height, leaf surface area, and chlorophyll content and increased the content of reactive oxygen species (ROS), including superoxide anion and hydrogen peroxide and oxidative damage, including malondialdehyde content and electrolyte leakage level; however, melatonin significantly promoted the plant growth parameters and reduced ROS content and oxidative damage under control and cold stress. These data revealed that melatonin-induced growth promotion and cold tolerance in maize is associated with its modulating effect on mitochondrial respiration.     

Individual variation in whole-animal hypoxia tolerance is associated with cardiac hypoxia tolerance in a marine teleost

Hypoxia is a pervasive problem in coastal environments and is predicted to have enduring impacts on aquatic ecosystems. Intraspecific variation in hypoxia tolerance is well documented in fish; however, the factors underlying this variation remain unknown. Here, we investigate the role of the heart in individual hypoxia tolerance of the European sea bass (Dicentrarchus labrax). We found individual whole-animal hypoxia tolerance is a stable trait in sea bass for more than 18 months (duration of study). We next examined in vitro cardiac performance and found myocardial muscle from hypoxia-tolerant individuals generated greater force, with higher rates of contraction and relaxation, than hypoxic-sensitive individuals during hypoxic exposure. Thus, whole-animal hypoxia tolerance is associated with cardiac hypoxia tolerance. As the occurrence of aquatic hypoxia is expected to increase in marine ecosystems, our experimental data suggest that cardiac performance may influence fish survival and distribution.     

甘蔗耐寒相关miRNA的生物信息学分析及其靶基因预测

为了解不同基因型甘蔗(Saccharum officinarum)响应低温胁迫的分子机制,该研究以低温胁迫4 ℃处理 24 h 后的3个不同耐寒性甘蔗品种的叶片为材料进行Illumina HiSeqTM 2000 高通量测序,构建了18个低温胁迫前后sRNA文库。结果表明:(1)共获得分属于84个家族的322个已知miRNA及预测得到110个新miRNA,并在已知miRNA中筛选出100个差异表达miRNA(61个上调,39个下调),新miRNA中筛选出37个差异表达 miRNA(15个上调, 22个下调)。(2)利用psRNATarget、TargetFinder、Tapirhybrid软件对所获得的差异表达miRNA进行靶基因预测,得到1 844个靶基因并进行GO分析揭示其主要功能类别,即分子功能、细胞组分与生物过程。(3)为验证高通量测序数据的可靠性,筛选14个miRNA及其靶基因进行qRT-PCR验证,结果显示这些miRNA均被检测发现且大多表达结果与测序结果一致。(4)鉴定出部分差异表达miRNA的靶基因,这些基因参与植物生长、发育及低温胁迫反应。综上认为,耐寒型甘蔗体内miRNA直接或间接作用靶基因实现表达调控相关代谢途径,对其重要农艺性状均起着关键的调控作用。     

Endotoxin tolerance is associated with altered GTP-binding protein function.

Previous studies have suggested that guanine nucleotide regulatory (G) proteins modulate endotoxin-stimulated peritoneal macrophage arachidonic acid (AA) metabolism. Endotoxin-stimulated metabolism of AA by peritoneal macrophages is decreased in endotoxin tolerance (Rogers et al. Prostaglandins 31: 639-650, 1986). These observations led to a study of G protein function and AA metabolism by peritoneal macrophages in endotoxin tolerance. Endotoxin tolerance was induced by the administration of sublethal doses of endotoxin. AA metabolism was assessed by measurement of thromboxane B2 (TxB2), a cyclooxygenase metabolite. NaF (5 mM), an activator of G proteins, significantly stimulated TxB2 synthesis in control macrophages from 7.7 +/- 0.2 to 19.1 +/- 0.6 (SE) ng/ml (P less than 0.05) at 2 h and was partially inhibited by pertussis toxin, suggesting a G protein-dependent mechanism. Salmonella enteritidis endotoxin (50 micrograms/ml) stimulated a similar increase in TxB2 levels (23 +/- 0.4 ng/ml, P less than 0.05). In contrast to control macrophages, macrophages from endotoxin-tolerant rats stimulated with either NaF or S. enteritidis endotoxin had TxB2 levels that were only 30 and 2% of the respective stimulated control cells. Basal guanosine-triphosphatase (GTPase) activity (33 +/- 6 pmol.mg-1.min-1) in endotoxin-tolerant macrophage membranes was significantly lower (P less than 0.05) than control basal activity (158 +/- 5 pmol.mg-1.min-1). This suppression of macrophage GTPase activity was apparent 48 h after the first in vivo sublethal endotoxin injection (100 micrograms/kg ip). The reduced GTPase activity paralleled in vitro cellular hyporesponsiveness to endotoxin-stimulated TxB2 production.(ABSTRACT TRUNCATED AT 250 WORDS)     

我国东部底栖无脊椎动物主要分类单元耐污值

根据采自安徽南部山区溪流、江苏南京秦淮河流域和紫金山地区小水体的92个大型底栖无脊椎动物样本,以及文献记载的北京、天津、安徽、湖南和辽宁的135个大型底栖无脊椎动物样本数据,用调整后的多样指数分级标准H'＞4极清洁,3～4清洁,2～3轻污,1～2中污,＜1重污,确定了227个样本所在样点水质类型.采用Lenat(1993)耐污值计算法,共获得81个分类单元的耐污值,其中昆虫纲鞘翅目3科2属,广翅目1科2属,蜉蝣目7科14属,(礻责)翅目2科2属,毛翅目6科7属,蜻蜓目3科1属,双翅目3科3亚科12属,软体动物2属5种,环节动物1属4种,扁形动物1属,甲壳动物1属.根据采集经验和文献记载,核定了73个分类单元(科、属)的耐污值,并初步探讨了上述各分类单元耐污值所适用的地区.     

Accumulation of terpenoid phytoalexins in maize roots is associated with drought tolerance

Maize (Zea mays) production, which is of global agro‐economic importance, is largely limited by herbivore pests, pathogens and environmental conditions, such as drought. Zealexins and kauralexins belong to two recently identified families of acidic terpenoid phytoalexins in maize that mediate defence against both pathogen and insect attacks in aboveground tissues. However, little is known about their function in belowground organs and their potential to counter abiotic stress. In this study, we show that zealexins and kauralexins accumulate in roots in response to both biotic and abiotic stress including, Diabrotica balteata herbivory, Fusarium verticillioides infection, drought and high salinity. We find that the quantity of drought‐induced phytoalexins is positively correlated with the root‐to‐shoot ratio of different maize varieties, and further demonstrate that mutant an2 plants deficient in kauralexin production are more sensitive to drought. The induction of phytoalexins in response to drought is root specific and does not influence phytoalexin levels aboveground; however, the accumulation of phytoalexins in one tissue may influence the induction capacity of other tissues.