Auxin-binding proteins without KDEL sequence in the moss Funaria hygrometrica

Whereas the important plant growth regulator auxin has multiple effects in flowering plants, it induces a specific cell differentiation step in the filamentous moss protonema. Here, we analyse the presence of classical auxin-binding protein (ABP1) homologues in the moss Funaria hygrometrica. Microsomal membranes isolated from protonemata of F. hygrometrica have specific indole acetic acid-binding sites, estimated to be about 3–5 pmol/mg protein with an apparent dissociation constant (K _d) between 3 and 5 μM. Western analyses with anti-ABP1 antiserum detected the canonical endoplasmic reticulum (ER)-localised 22–24 kDa ABP1 in Zea mays, but not in F. hygrometrica. Instead, polypeptides of 31–33 and 46 kDa were labelled in the moss as well as in maize. In F. hygrometrica these proteins were found exclusively in microsomal membrane fractions and were confirmed as ABPs by photo-affinity labelling with 5-azido-[7-³H]-indole-3-acetic acid. Unlike the classical corn ABP1, these moss ABPs did not contain the KDEL ER retention sequence. Consistently, the fully sequenced genome of the moss Physcomitrella patens, a close relative of F. hygrometrica, encodes an ABP1-homologue without KDEL sequence. Our study suggests the presence of putative ABPs in F. hygrometrica that share immunological epitopes with ABP1 and bind auxin but are different from the classical corn ABP1.     

Molecular cloning and characterization of the glutathione reductase gene from Stipa purpurea

Reactive oxygen species (ROS) are a key factor in abiotic stresses; excess ROS is harmful to plants. Glutathione reductase (GR) plays an important role in scavenging ROS in plants. Here, a GR gene, named SpGR, was cloned from Stipa purpurea and characterized. The full-length open reading frame was 1497 bp, encoding 498 amino acids. Subcellular localization analysis indicated that SpGR was localized to both the plasma membrane and nucleus. The expression of SpGR was induced by cold, salt, and drought stresses. Functional analysis indicated that ectopic expression of SpGR in Arabidopsis thaliana resulted in greater tolerance to salt stress than that of wild-type plants, but no difference under cold or drought treatments. The results of GR activity and GSSG and GSH content analyses suggested that, under salt stress, transgenic plants produced more GR to reduce GSSG to GSH for scavenging ROS than wild-type plants. Therefore, SpGR may be a candidate gene for plants to resist abiotic stress.     

Molecular cloning, characterization, and expression analysis of a cytosolic HSP90 gene from Haematococcus pluvialis

Heat shock protein 90 (HSP90) is a highly conserved molecular chaperone that plays key roles in the folding, maintenance of structural integrity, and regulation of a subset of cytosolic proteins. In this study, the cDNA of Haematococcus pluvialis HSP90 (designated HpHSP90) was cloned by the combination of homology cloning and rapid amplification of cDNA ends approaches. The full-length cDNA of HpHSP90 was of 2,606 bp, including an open reading frame of 2,109 bp encoding a polypeptide of 702 amino acids with predicted molecular weight of 80.14 kDa and theoretical isoelectric point of 5.07. BLAST analysis revealed that HpHSP90 shared high similarity with other known HSP90s, and the five conserved amino acid blocks defined as HSP90 protein family signatures were also identified in HpHSP90, which indicated that HpHSP90 should be a cytosolic member of the HSP90 family. Under different stress conditions, messenger RNA (mRNA) expression levels of HpHSP90 were quantified by quantitative RT-PCR. To H. pluvialis kept at different temperatures for 1 h, maximum HpHSP90 expression was observed in the range 5 to 10°C and 35 to 40°C and the expression level of HpHSP90 at 40°C was the highest (threefold compared with that at 25°C). In H. pluvialis kept at 35°C for different times, the mRNA expression level of HpHSP90 reached a maximum level after 7 h and then dropped progressively. The results indicate that HpHSP90 responded to cold and heat stresses with a temperature-dependent expression pattern as well as exposure time effect and could be used as a molecular biomarker in adverse stress environment.     

Molecular cloning and sequence analysis of methionine adenosyltransferase from the economic seaweed Undaria pinnatifida

The methionine adenosyltransferase gene (MAT) had been isolated from an economic seaweed Undaria pinnatifida by PCR using degenerate primers. The cDNA was 1,491 bp in length with an open reading frame of 1,194 nucleotides, encoding a deduced protein of 397 amino acids. The protein had a predicted molecular weight of 43.2 kDa, and the isoelectric point was 5.244. The sequence contains a 92 bp 5′-untranslated region (UTR) and a 205 bp 3′-UTR. The methionine adenosyltransferase (MAT) sequence of U. pinnatifida (UpMAT) shared 68–92 % identities with the previous published MAT sequences of other species. Phylogenetic analysis indicated that the phylogenetic relationship of UpMAT with some other seaweeds was closer than with those of higher plants. Under different stress conditions, the relative mRNA expression levels of the MAT of U. pinnatifida (UpMAT) were measured by real-time quantitative PCR, and the results demonstrated that the UpMAT might help to protect the alga against various abiotic stresses.     

The role of the novel adenosine 5′-phosphosulfate reductase in regulation of sulfate assimilation of <Emphasis Type="Italic">Physcomitrella patens</Emphasis>

Sulfate assimilation provides reduced sulfur for the synthesis of the amino acids cysteine and methionine and for a range of other metabolites. The key step in control of plant sulfate assimilation is the reduction of adenosine 5′-phosphosulfate to sulfite. The enzyme catalyzing this reaction, adenosine 5′phosphosulfate reductase (APR), is found as an iron sulfur protein in plants, algae, and many bacteria. In the moss Physcomitrella patens, however, a novel isoform of the enzyme, APR-B, has recently been discovered lacking the co-factor. To assess the function of the novel APR-B we used homologous recombination to disrupt the corresponding gene in P. patens. The knock-out plants were able to grow on sulfate as a sole sulfur source and the content of low molecular weight thiols was not different from wild type plants or plants where APR was disrupted. However, when treated with low concentrations of cadmium the APR-B knockout plants were more sensitive than both wild type and APR knockouts. In wild type P. patens, the two APR isoforms were not affected by treatments that strongly regulate this enzyme in flowering plants. The data thus suggest that in P. patens APS reduction is not the major control step of sulfate assimilation.     

Targeted knock-out of a gene encoding sulfite reductase in the moss Physcomitrella patens affects gametophytic and sporophytic development

A key step in sulfate assimilation into cysteine is the reduction of sulfite to sulfide by sulfite reductase (SiR). This enzyme is encoded by three genes in the moss Physcomitrella patens. To obtain a first insight into the roles of the individual isoforms, we deleted the gene encoding the SiR1 isoform in P. patens by homologous recombination and subsequently analysed the ΔSiR1 mutants. While ΔSiR1 mutants showed no obvious alteration in sulfur metabolism, their regeneration from protoplasts and their ability to produce mature spores was significantly affected, highlighting an unexpected link between moss sulfate assimilation and development, that is yet to be characterized.     

Geothermal bryophyte habitats in the South Sandwich Islands,maritime Antarctic

Question: How does geothermal activity influence terrestrial plant colonization, species composition and community development in the Antarctic? Location: South Sandwich Islands, maritime Antarctic. Methods: Bryophytes were documented during a biological survey of the archipelago in January and February 1997. Particular attention was given to sites under current or recent influence of geothermal activity. Temperature profiles obtained across defined areas of activity on several islands were linked with the presence of specific bryophytes. Results: Greatest bryophyte richness was associated with geothermally influenced ground. Of 35 moss and nine liverwort species recorded, only four mosses were never associated with heated ground, while eight of the liverworts and 50% of the mosses were found only on actively or recently heated ground. Some species occur in unheated sites elsewhere in the maritime Antarctic, but were absent from such habitats on the South Sandwich Islands. Several species occurred in distinct zones around fumaroles. Maximum temperatures recorded within the upper 0.5 cm of the vegetation surface were 40 ‐ 47 °C, with only Campylopus introflexus tolerating such temperatures. Maximum temperatures 2.5 or 5 cm below the vegetation surface of this moss reached 75 °C. Other bryophytes regularly present in zoned vegetation included the mosses Dicranella hookeri, Sanionia georgico‐uncinata, Pohlia nutans and Notoligotrichum trichodon, and the liverworts Cryptochila grandiflora and Marchantia berteroana. Surface temperatures of 25 ‐ 35 °C and subsurface temperatures of 50 ‐ 60 °C were recorded in these species. Conclusions: These exceptional plant communities illustrate the transport of viable propagules into the Antarctic. Individually ephemeral in nature, the longer term existence of geothermal habitats on islands along the Scotia Arc may have provided refugia during periods of glacial expansion, facilitating subsequent recolonization of Antarctic terrestrial habitats.     

Response of Antarctic, temperate, and tropical microalgae to temperature stress

The global temperature increase has significant implications on the survival of microalgae which form the basis of all aquatic food webs. The aim of this study was to compare the response of similar taxa of microalgae from the Antarctic (Chlamydomonas UMACC 229, Chlorella UMACC 237, and Navicula glaciei UMACC 231), temperate (Chlamydomonas augustae UMACC 247, Chlorella vulgaris UMACC 248, and Navicula incerta UMACC 249), and tropical (C. augustae UMACC 246, C. vulgaris UMACC 001, and Amphiprora UMACC 239) regions to changing temperature. The Antarctic, temperate, and tropical strains were grown over specific temperature ranges of 4 °C to 30 °C, 4 °C to 32 °C, and 13 °C to 38 °C, respectively. The three Antarctic strains survived at temperatures much higher than their ambient regime. In comparison, the tropical strains are already growing at their upper temperature limits. The three Chlorella strains from different regions are eurythermal, with a large overlap on tolerance ranging from 4 °C to 38 °C. The specific growth rate (μ) of the Antarctic Navicula decreased (<0.34 day^?1) at temperatures above 4 °C, showing it to be sensitive to temperature increase. If further warming of Earth occurs, N. glaciei UMACC 231 is likely to have the most deleterious consequences than the other two Antarctic microalgae studied. The percentage of polyunsaturated fatty acids (PUFA) decreased with increasing temperature in the Antarctic Navicula. As temperature increases, the growth and nutritional value of this commonly occurring diatom in the Antarctic may decrease, with consequences for the aquatic food web. Of the three Chlamydomonas strains, only the Antarctic strain produced predominantly PUFA, especially 16:3 (48.4–57.2 % total fatty acids).     

Effect of shade on leaf photosynthetic capacity,light-intercepting,electron transfer and energy distribution of soybeans

Lectin receptor-like kinases (LecRLK) are widespread in higher plants and their effects on abiotic stress tolerance are gradually being reported. However, little information is available on LecRLK functions in bryophytes. Here, an L-type LecRLK gene (PnLecRLK1) was characterized from the Antarctic moss Pohlia nutans. Subcellular localization analysis revealed that PnLecRLK1 was a plasma membrane protein. The expression of PnLecRLK1 was rapidly induced by simulated cold, salt, and drought stresses as well as by exogenously applied abscisic acid (ABA) and methyl jasmonate. Transgenic Arabidopsis plants of overexpressing PnLecRLK1 exhibited enhanced tolerance to chilling-stress and increased ABA sensitivity. Additionally, the expression levels of genes in the C-repeat binding factor (CBF) signaling pathway such as AtCBF1, AtCBF2, AtCBF3 and AtCOR47 were markedly increased in transgenic Arabidopsis. Furthermore, the expression levels of ABA-responsive genes, such as AtABI4, AtABI5, AtMYB2 and AtDREB2A, were also significantly up-regulated in transgenic Arabidopsis. Therefore, our results suggested that PnLecRLK1 functions as a membrane-bound regulator that increases chilling stress tolerance and ABA sensitivity to enable P. nutans to adapt to polar climates.     

Combined action of antioxidant defense system and osmolytes in chilling shock-induced chilling tolerance in Jatropha curcas seedlings

Low non-freezing temperature is one of the major environmental factors affecting growth, development and geographical distribution of chilling-sensitive plants, Jatropha curcas is considered as a sustainable energy plants with great potential for biodiesel production. In this study, chilling shock at 5 °C followed by recovery at 26 °C for 4 h significantly improved survival percentage of J. curcas seedlings under chilling stress at 1 °C. In addition, chilling shock could obviously enhance the activities of antioxidant enzymes superoxide dismutase (SOD), ascorbate peroxidase (APX), catalase (CAT) and glutathione reductase (GR), and the levels of antioxidants ascorbic acid (AsA) and glutathione (GSH), as well as the contents of osmolytes proline and betaine in leaves of seedlings of J. curcas compared with the control without chilling shock. During the process of recovery, GR activity, AsA, GSH, proline and betaine contents sequentially increased, whereas SOD, APX and CAT activities gradually decreased, but they markedly maintained higher activities than those of control. Under chilling stress, activities of SOD, APX, CAT, GR and GPX, and contents of AsA, GSH, proline and betaine, as well as the ratio of the reduced antioxidants to total antioxidants [AsA/(AsA + DHA) and GSH/(GSH + GSSG)] in the shocked and non-shock seedlings all dropped, but shocked seedlings sustained significantly higher antioxidant enzyme activity, antioxidant and osmolyte contents, as well as ratio of reduced antioxidants to total antioxidants from beginning to end compared with control. These results indicated that the chilling shock followed by recovery could improve chilling tolerance of seedlings in J. curcas, and antioxidant enzymes and osmolytes play important role in the acquisition of chilling tolerance.     

Anaerobic respiration and antioxidant responses of Corythucha ciliata (Say) adults to heat-induced oxidative stress under laboratory and field conditions

High temperature often induces oxidative stress and antioxidant response in insects. This phenomenon has been well documented under controlled laboratory conditions, but whether it happens under fluctuating field conditions is largely unknown. In this study, we used an invasive lace bug (Corythucha ciliata) as a model species to compare the effects of controlled thermal treatments (2 h at 33–43 °C with 2 °C intervals in the laboratory) and naturally fluctuating thermal conditions (08:00–14:00 at 2-h intervals (29.7–37.2 °C) on a hot summer day in a field in Shanghai, China) on lipid peroxidation (malondialdehyde (MDA) was the marker) and anaerobic respiration (lactate dehydrogenase (LDH) was the marker), as well as superoxide dismutase (SOD), catalase (CAT), glutathione (GSH), and glutathione reductase (GR). The results show that MDA concentration increased significantly in response to heat stresses with similar trend in the laboratory and field. LDH activities did not significantly vary across temperatures in the laboratory-exposed individuals, but they significantly increased by rising temperature in the field. The activities or concentrations of SOD, CAT, GSH, and GR all significantly increased with increasing temperature in the two populations. These findings indicate that high temperature induces oxidative stress, resulting in high anaerobic respiration and antioxidant defenses in C. ciliata under both the laboratory and field conditions, which likely provide a defense mechanism against oxidative damage due to the accumulation of ROS.     

Exogenous hydrogen peroxide enhanced the thermotolerance of Festuca arundinacea and Lolium perenne by increasing the antioxidative capacity

Heat stress is one of the most detrimental environment stresses for plants. Hydrogen peroxide (H₂O₂) is produced quickly in response to various stresses and likely plays a positive role in transmitting stress signal in organisms. This investigation addressed whether an exogenous H₂O₂ application would affect the heat response of turfgrasses and induce acclimation. Tall fescue (Festuca arundinacea cv. Barlexas) and perennial ryegrass (Lolium perenne cv. Accent), two important cool-season turfgrasses and forages, were sprayed with 10 mM H₂O₂ before they were treated with heat stress (38/30 °C, day/night) and compared with plants maintained at control temperatures (26/15 °C, day/night). Prior to the initiation of heat stress, H₂O₂ pretreatment increased the activities of guaiacol peroxidase (POD), catalase (CAT), ascorbate peroxidase (APX), glutathione reductase (GR) and glutathione-dependent peroxidase (GPX) and the ascorbate and glutathione pool, and it decreased the GSH/GSSG ratio. During the heat stress process, pretreated plants from both grasses exhibited higher turfgrass quality and relative water content, and they experienced lower oxidative damage and H₂O₂ levels. Moreover, the activities of APX, GR, GPX and glutathione-S-transferase increased significantly in response to H₂O₂ pretreatment under heat stress. These results suggested that H₂O₂ most likely participated in the transduction of redox signaling and induced the antioxidative defense system, including various enzymatic and nonenzymatic H₂O₂ scavengers. The scavengers played important roles in improving the thermotolerance of tall fescue and perennial ryegrasses.     

Cold-Adapted RTX Lipase from Antarctic Pseudomonas sp. Strain AMS8: Isolation, Molecular Modeling and Heterologous Expression

A new strain of psychrophilic bacteria (designated strain AMS8) from Antarctic soil was screened for extracellular lipolytic activity and further analyzed using molecular approach. Analysis of 16S rDNA showed that strain AMS8 was similar to Pseudomonas sp. A lipase gene named lipAMS8 was successfully isolated from strain AMS8, cloned, sequenced and overexpressed in Escherichia coli. Sequence analysis revealed that lipAMS8 consist of 1,431 bp nucleotides that encoded a polypeptide consisting of 476 amino acids. It lacked an N-terminal signal peptide and contained a glycine- and aspartate-rich nonapeptide sequence at the C-terminus, which are known to be the characteristics of repeats-in-toxin bacterial lipases. Furthermore, the substrate binding site of lipAMS8 was identified as S²⁰⁷, D²⁵⁵ and H³¹³, based on homology modeling and multiple sequence alignment. Crude lipase exhibited maximum activity at 20 °C and retained almost 50 % of its activity at 10 °C. The molecular weight of lipAMS8 was estimated to be 50 kDa via sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The optimal expression level was attained using the recombinant plasmid pET32b/BL21(DE3) expressed at 15 °C for 8 h, induced by 0.1 mM isopropyl β-D thiogalactoside (IPTG) at E. coli growth optimal density of 0.5.