The physico-chemical characterization of a boiling stable antifreeze protein from a perennial grass (Lolium perenne)

We have characterized a cold-induced, boiling stable antifreeze protein. This highly active ice recrystallization inhibition protein shows a much lower thermal hysteresis effect and displays binding behavior that is uncharacteristic of any AFP from fish or insects. Ice-binding studies show it binds to the (1 0 1 0) plane of ice and FTIR studies reveal that it has an unusual type of highly beta-sheeted secondary structure. Ice-binding studies of both glycosylated and nonglycosylated expressed forms indicate that it adsorbs to ice through the protein backbone. These results are discussed in light of the currently proposed mechanisms of AFP action.     

Characterization of a family of ice-active proteins from the Ryegrass, Lolium perenne

Five genes coding for ice-active proteins were identified from an expressed sequence tag database of Lolium perenne cDNA libraries. Each of the five genes were characterized by the presence of an N-terminal signal peptide, a region enriched in hydrophilic amino acids and a leucine-rich region in four of the five genes that is homologous with the receptor domain of receptor-like protein kinases of plants. The C-terminal region of all five genes contains sequence homologous with Lolium and Triticum ice-active proteins. Of the four ice-active proteins (IAP1, IAP2, IAP3 and IAP5) cloned, three could be expressed in Escherichia coli and recovered in a functional form in order to study their ice activity. All three ice-active proteins had recrystallization inhibition activity but showed no detectable antifreeze or ice nucleation activity at the concentration tested. IAP2 and IAP5 formed distinct hexagonal-shaped crystals in the nanolitre osmometer as compared to the weakly hexagonal crystals produced by IAP3.     

Purification and characterization of antifreeze proteins from larvae of the beetle Dendroides canadensis

Summary Four antifreeze proteins (AFPs) were purified from larvae of the beetle Dendroides canadensis. The AFPs are similar in amino acid compositions, having high contents of hydrophilic amino acids (45–55 mol%) and cysteine (16 mol% Cys). Approximately half of the Cys residues form disulfide bridges, and both the disulfide bridges and free sulfhydryls are essential for activity. The N-terminals of the AFPs are blocked. The pH optimum of the AFPs is 7.8, but major loss of activity occurred only at very high pH (12.0). The detergents SDS and Triton X-100 did not inactivate the AFPs. Circular dichroism spectra indicate the presence of both and secondary structures in the AFPs, in addition to a large random structure component.Abbreviations AFP antifreeze protein - CD circular dichroism - DTT dithiothreitol - HPLC high pressure liquid chromatography - PAGE polyacrylamide gel electrophoresis - PAS periodic acid Schiff - SDS sodium dodecyl sulfate - TFA trifluoroacetic acid     

Isolation and characterisation of a sucrose: sucrose 1-fructosyltransferase gene from perennial ryegrass (Lolium perenne)

A sucrose: sucrose 1-fructosyltransferase (1-SST) gene and cDNA (Lp 1-SST) from perennial ryegrass (Lolium perenne) were isolated. The Lp 1-SST gene was fully sequenced and shown to contain three exons and two introns. Nucleotide sequence analysis of the 4824 bp Lp 1-SST genomic sequence revealed 1618 bp of 5' UTR and an open reading frame of 1962 bp encoding a protein of 653 amino acids. Lp 1-SST is 95% identical to the tall fescue 1-SST and contains plant fructosyltransferase functional domains. Lp 1-SST corresponds to a single copy gene in perennial ryegrass, and is expressed in young leaf bases and mature leaf sheaths. The recombinant Lp 1-SST protein from corresponding cDNA expression in Pichia pastoris showed 1-SST activity.     

Expression, purification, and antifreeze activity of carrot antifreeze protein and its mutants

Antifreeze proteins (AFPs) enable organisms to survive under freezing or sub-freezing conditions. AFPs have a great potential in the low temperature storage of cells, tissues, organs, and foods. This process will require a large number of recombinant AFPs. In the present study, the recombinant carrot AFP was highly expressed in Escherichia coli strain BL21 (DE3). The activity of the purified and refolded recombinant proteins was analyzed by measurement of thermal hysteresis (TH) activity and detection of in vitro antifreeze activity by measuring enhanced cold resistance of bacteria. Two carrot AFP mutants generated by site-directed mutagenesis were also expressed and purified under these conditions for use in parallel experiments. Recombinant DcAFP displayed a TH activity equivalent to that of native DcAFP, while mutants DcAFP-N130Q and rDcAFP-N130V showed 32 and 43% decreases in TH activity, respectively. Both the recombinant DcAFP and its mutants were able to enhance the cold resistance of bacteria, to degrees consistent with their respective TH activities.     

A hyperactive, Ca2+-dependent antifreeze protein in an Antarctic bacterium

In cold climates, some plants and bacteria that cannot avoid freezing use antifreeze proteins (AFPs) to lessen the destructive effects of ice recrystallization. These AFPs have weak freezing point depression activity, perhaps to avoid sudden, uncontrolled growth of ice. Here, we report on an uncharacteristically powerful bacterial AFP found in an Antarctic strain of the bacterium, Marinomonas primoryensis. It is Ca(2+)-dependent, shows evidence of cooperativity, and can produce over 2 degrees C of freezing point depression. Unlike most AFPs, it does not produce obvious crystal faceting during thermal hysteresis. This AFP might be capable of imparting freezing avoidance to M. primoryensis in ice-covered Antarctic lakes. A hyperactive bacterial AFP has not previously been reported.     

Associations between enzyme genotypes and dark respiration in perennial ryegrass,Lolium perenne L.

Summary In this study, we determined whether relationships existed between dark respiration and genotype at five enzyme polymorphisms in perennial ryegrass, Lolium perenne L. Positive correlations were found between Q ₁₀ of dark respiration and genotype at the phosphoglucomutase (PGM) and 6-phosphogluconate dehydrogenase (6PGD) loci. Plants doubly homozygous for the common allele at these loci were found to have Q ₁₀ values 20% higher than those for double heterozygotes. In plants that were heat stressed for five consecutive days, Q ₁₀ was found to be negatively correlated with apparent vigor after stressing. Individuals homozygous for PGM and 6PGD (with higher Q ₁₀ values) exhibited more apparent damage following the stress than heterozygous individuals. Both PGM and 6PGD occupy positions in metabolism with regulatory potential. Although caution must be used in assigning causal relationships, the results suggest that specific forms of these enzymes are directly related to, or are correlated with, the determinants of respiratory efficiency in L. perenne.     

Gene expression and genetic mapping analyses of a perennial ryegrass glycine-rich RNA-binding protein gene suggest a role in cold adaptation

A perennial ryegrass cDNA clone encoding a putative glycine-rich RNA binding protein (LpGRP1) was isolated from a cDNA library constructed from crown tissues of cold-treated plants. The deduced polypeptide sequence consists of 107 amino acids with a single N-terminal RNA recognition motif (RRM) and a single C-terminal glycine-rich domain. The sequence showed extensive homology to glycine-rich RNA binding proteins previously identified in other plant species. LpGRP1-specific genomic DNA sequence was isolated by an inverse PCR amplification. A single intron which shows conserved locations in plant genes was detected between the sequence motifs encoding RNP-1 and RNP-2 consensus protein domains. A significant increase in the mRNA level of LpGRP1 was detected in root, crown and leaf tissues during the treatment of plants at 4°C, through which freezing tolerance is attained. The increase in the mRNA level was prominent at least 2 h after the commencement of the cold treatment, and persisted for at least 1 week. Changes in mRNA level induced by cold treatment were more obvious than those due to treatments with abscisic acid (ABA) and drought. The LpGRP1 protein was found to localise in the nucleus in onion epidermal cells, suggesting that it may be involved in pre-mRNA processing. The LpGRP1 gene locus was mapped to linkage group 2. Possible roles for the LpGRP1 protein in adaptation to cold environments are discussed.     

Molecular characterization and sequencing of antifreeze proteins from larvae of the beetle Dendroides canadensis

The deduced amino acid sequences of antifreeze proteins (AFPs) from larvae of the beetle Dendroides canadensis were determined from both complementary DNAs (cDNAs) and from peptide sequencing. These consisted of proteins with a 25-residue signal peptide and mature proteins 83 (Dendroides antifreeze protein; DAFP-1) or 84 (DAFP-2) amino acids in length which differed at only two positions. Peptide sequencing yielded sequences which overlapped exactly with those of the deduced cDNA sequences of DAFP-1 and DAFP-2, while the partial sequence of another AFP (DAFP-3) matched 21 of 28 residues. Seven 12- or 13-mer repeating units are present in these antifreeze proteins with a consensus sequence consisting of: Cys-Thr-X₃-Ser-X₅-X₆-Cys-X₈-X₉-Ala-X₁₁-Thr-X₁₃, where X₃ and X₁₁ tend toward charged residues, X₅ tends toward threonine or serine, X₆ toward asparagine or aspartate, X₉ toward asparagine or lysine, and X₁₃ toward alanine in the 13-mers. The most interesting feature of these proteins is that throughout the length of the mature antifreeze proteins every sixth residue is a cysteine. These sequences are not similar to any of the known fish AFPs, but they are similar to AFPs from the beetle Tenebrio molitor. Accepted: 14 November 1997     

Fractional and structural characterization of hemicelluloses from perennial ryegrass (Lolium perenne) and cocksfoot grass (Dactylis glomerata)

Sequential three-stage treatments with 80% EtOH containing 0.2% NaOH, 2.5% H2O2-0.2% EDTA containing 1.5% NaOH and 2.5% H2O2-0.2% TAED containing 1.0% NaOH at 75 degrees C for 3h released 8.0% and 10.4%, 79.1% and 77.0% and 12.9% and 12.5% of the original hemicelluloses from perennial grass and cocksfoot grass, respectively. It was found that the four alkaline peroxide-soluble hemicellulosic fractions contained higher amounts of xylose (33.4-38.2%), uronic acids (9.3-15.3%) and rhamnose (3.0-3.9%), but were lower in glucose (25.1-28.3%), galactose (13.3-15.3%) and mannose (0.4-1.5%) than those of the two alkaline EtOH-soluble hemicellulosic fractions in which glucose (32.9-36.0%), xylose (20.1-22.6%), arabinose (14.1-21.4%), galactose (16.6-19.9%), mannose (4.1-9.9%) and uronic acids (3.4-7.4%) were the major sugar components. 13C NMR spectroscopy confirmed that all the six hemicellulosic fractions were composed of galactoarabinoxylans, 4-O-methylglucuronoarabinoxylans and beta-glucan. In addition, the studies showed that the four alkaline peroxide-soluble hemicellulosic fractions were more linear and acidic and had larger molecular weights (Mw, 28,400-38,650 g mol(-1)) than those of the two alkaline EtOH-soluble hemicellulosic fractions (Mw, 16,460-17,420 g mol(-1)).     

Characterization of an antifreeze protein from the polar diatom Fragilariopsis cylindrus and its relevance in sea ice

Antifreeze proteins (AFPs), characterized by their ability to separate the melting and growth temperatures of ice and to inhibit ice recrystallization, play an important role in cold adaptation of several polar and cold-tolerant organisms. Recently, a multigene family of AFP genes was found in the diatom Fragilariopsis cylindrus, a dominant species within polar sea ice assemblages. This study presents the AFP from F. cylindrus set in a molecular and crystallographic frame. Differential protein expression after exposure of the diatoms to environmentally relevant conditions underlined the importance of certain AFP isoforms in response to cold. Analyses of the recombinant AFP showed freezing point depression comparable to the activity of other moderate AFPs and further enhanced by salt (up to 0.9 °C in low salinity buffer, 2.5 °C at high salinity). However, unlike other moderate AFPs, its fastest growth direction is perpendicular to the c-axis. The protein also caused strong inhibition of recrystallization at concentrations of 1.2 and 0.12 μM at low and high salinity, respectively. Observations of crystal habit modifications and pitting activity suggested binding of AFPs to multiple faces of the ice crystals. Further analyses showed striations caused by AFPs, interpreted as inclusion in the ice. We suggest that the influence on ice microstructure is the main characteristic of these AFPs in sea ice.     

Identification of the ice-binding face of a plant antifreeze protein

The antifreeze protein of Lolium perenne, a perennial ryegrass, was previously modeled as a beta-roll with two extensive flat beta-sheets on opposite sides of the molecule. Here we have validated the model with a series of nine site-directed steric mutations in which outward-pointing short side-chain residues were replaced by tyrosine. None of these disrupted the fold. Mutations on one of the beta-sheets and on the sides of the protein retained 70% or greater activity. Three mutations that clustered on the other flat surface lost up to 90% of their antifreeze activity and identify this beta-sheet as the ice-binding face.     

Isolation and characterization of cold-regulated transcriptional activator <Emphasis Type="Italic">LpCBF3</Emphasis> gene from perennial ryegrass (<Emphasis Type="Italic">Lolium perenne</Emphasis> L.)

In plants, low temperatures can activate the CBF cold response pathway playing a prominent role in cold acclimation by triggering a set of cold-related gene expressions. CBF homologous gene, designated as LpCBF3, from a cold-tolerant perennial ryegrass (Lolium perenne L.) accession was identified. It carries the sequences for nuclear localization signal (NLS), AP2 DNA-binding domains and an acidic activation present in most of the plant CBF proteins. Southern analysis indicated the presence of three homologs of LpCBF3 gene in perennial ryegrass genome, and only one amino acid variation in LpCBF3 protein between cold-tolerant and -sensitive perennial ryegrass accessions. In their putative promoter regions, some differential regions were found. Northern blotting and RT-PCR analysis found that LpCBF3 reached the highest expression after 1.5 h of cold treatment (4 degrees C). The COR homologous gene, a downstream gene of CBF, can be expressed in the plant stem of cold-tolerant perennial ryegrass accessions without cold treatment. Without cold treatment, the COR gene cannot be activated in cold-sensitive perennial ryegrass accessions. Cold treatment can prompt expression levels of COR homologous genes in both perennial ryegrass accessions. In transgenic Arabidopsis, the overexpression of LpCBF3 with the 35S promoter resulted in dwarf-like plants, later flowering and greater freezing tolerance.     

Expression of a beetle, Dendroides canadensis, antifreeze protein in Drosophila melanogaster

Antifreeze protein 1 (DAFP-1), from the beetle Dendroides canadensis, was expressed in Drosophila melanogaster. Mean thermal hysteresis values (the difference between freezing and melting points), indicative of antifreeze protein activity, in the hemolymph of transgenic flies were found to be as high as 6.23+/-0.10 degrees C (using the nanoliter osmometer). Direct comparisons of the capillary and nanoliter osmometer techniques for measuring THA were made, illustrating the much higher values obtained by the latter. Transgenic Drosophila had supercooling points, both in contact with ice and not, that were slightly, but significantly, lower than wild-type controls (1.5-2.0 degrees C and 2.0-4.0 degrees C, respectively). The results indicate functionality of DAFP-1 in Drosophila melanogaster (the ability of DAFP-1 to inhibit both inoculative freezing across the cuticle and freezing initiated by endogenous ice nucleators). The much larger effects of DAFPs in inhibiting inoculative freezing and ice nucleation in Dendroides canadensis relative to the transgenic Drosophila may partially result from the lower DAFP concentrations and activities in Drosophila, however the absence of multiple types of DAFPs and absence of tissue specific expression may also contribute. Transgenic Drosophila were also able to live significantly longer than controls at 0 degrees C and 4 degrees C, indicating that DAFP-1 is able to increase cold tolerance at above freezing temperatures.     

Rapid cycling of nitrogen and phosphorus from dying roots of Lolium perenne

Summary Previous experiments, using ³²P pulse labelling, showed that when roots of Lolium perenne were detached from the shoot, a substantial proportion of the phosphorus in the roots could within a few weeks be released and be captured by another, living plant. This paper describes experiments designed to confirm and further investigate this rapid nutrient transfer. Roots from plants grown with ample N and P were detached and placed in litter bags in soil. They lost up to 60% of their initial N and up to 70% of their P in three weeks. Even when roots were grown with deficient P supply, resulting in C:P ratios of 300–400, they lost 20–30% of their initial P. Time-courses of ³²P loss from roots suspended in solution gave results which agreed with these figures. The initially rapid rate of ³²P loss had declined greatly within three weeks. In a pot experiment small L. perenne plants showed a marked increase in their N and P content during 30 days after a neighbouring large plant's shoot was removed, supporting rapid capture of nutrients lost from the detached roots. To investigate P loss from roots while attached to the shoot, L. perenne shoots were clipped every four days and ³²P loss from the roots measured. After the third clip the rate of loss increased, eventually to more than four times that from the control plants.     

Application of silicon sources increases silicon accumulation in perennial ryegrass turf on two soil types

This study investigated the ability of perennial ryegrass to accumulate silicon and the factors that may influence plant silicon accumulation. Plants were grown in the greenhouse in two soil types, peat:sand mix and Hagerstown-silt-loam, amended with two commercially available sources of silicon, calcium silicate slag and wollastonite at 0, 0.5, 1, 2, 5 and 10 t/ha. Shoot tissue of nine-week-old perennial ryegrass plants was analyzed for silicon content (%) and found to reach a dry matter concentration of up to 4% in this study. Silicon accumulation in perennial ryegrass was influenced by the soil type and source, and was higher in plants grown in low-silicon peat:sand mix compared to Hagerstown-silt-loam. Silicon content (%) in the plants consistently increased with increasing rates of silicon in all four soil and source combinations. Acetic acid (HAc) extractable silicon and Ca increased in both soil types when amended with either of the silicon sources. Effects of silicon sources on soil pH varied with soil type. This study indicates that soil type, source of silicon, and rate of silicon application are important factors influencing the uptake of silicon by perennial ryegrass which is a widely used turfgrass species in golf courses, sports fields, and residential lawns in the United States.