| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
|
First published online March 21, 2008; 10.1104/pp.106.081497 Plant Physiology 147:391-401 (2008) © 2008 American Society of Plant Biologists OPEN ACCESS ARTICLE
Characterization of Cold-Responsive Extracellular Chitinase in Bromegrass Cell Cultures and Its Relationship to Antifreeze Activity1,[OA]Environmental Stress Research Unit, Division of Plant Sciences, National Institute of Agrobiological Sciences, Tsukuba, Ibaraki 305–8602, Japan
A cold-responsive chitinase gene, BiCHT1, was isolated from bromegrass (Bromus inermis) ‘Manchar’ suspension cells. BiCHT1 messenger RNA was detected at low levels in nonstressed bromegrass cells, whereas its accumulation was induced by incubation at 10°C and 4°C as detected by northern- and western-blot analyses. BiCHT1 was highly homologous to rye CHT9, known to encode an antifreeze protein. BiCHT1 was overexpressed in Escherichia coli and bromegrass cells using genetic transformation procedures. BiCHT1 products expressed in both systems had chitinase activity, but the expressed proteins did not affect the growth of ice crystals in any conditions tested. Besides cold stress, the expression of the BiCHT1 gene was up-regulated by exposure to 35°C, but not by salt or osmotic stress, abscisic acid, or ethephon. BiCHT1 messenger RNA did not accumulate in response to methyl jasmonate and salicylic acid, but was slightly increased by prolonged culture at 25°C and only transiently by chitin. Antifreeze activity detected in the culture medium was induced at 4°C but only slightly at 10°C. It was also induced by ethephon treatment, but not by abscisic acid, chitin, or prolonged incubation at 25°C. The results of transgenics and expression analyses suggest that the BiCHT1 product is a major protein with chitinase activity secreted in the medium of cold-treated cells and is unlikely to be responsible for the antifreeze activity detected in the culture medium.
Low temperature is a critical environmental factor that significantly influences plant growth, development, and crop yield. Most plants from temperate regions can acclimate to cold and develop freezing tolerance (Guy, 1990  ). Cold acclimation has been characterized by physiological and biochemical changes that result from the selective increases or decreases in the biosynthesis of a large number of distinct proteins (Bohnert et al., 1995). These changes in protein patterns are due in part to changes in the transcriptional activity of the corresponding genes. Many cold-inducible genes have been cloned from various plants (Thomashow, 1999). Some of these products are involved in the stabilization of membranes and macromolecules, metabolic shifts, scavenging of oxygen radicals, and signal transduction pathways. The function of numerous other cold-inducible products is still unknown. Analysis of genes whose expression is induced during cold acclimation is important to understand the mechanism of freezing tolerance and for use in breeding freezing-tolerant plants.
Many overwintering cereals accumulate antifreeze proteins (AFPs) during cold acclimation (Antikainen and Griffith, 1997
Chitinases (EC 3.2.1.14) hydrolyze chitin (N-acetyl-D-glucosamine polymers) and are present in many plant species. Plant chitinases are induced by pathogens or elicitors and are important enzymes in defense mechanisms. However, chitinases are also involved in diverse biological functions, such as somatic embryogenesis (De Jong et al., 1992
In our previous study, BiCHT1 encoding a putative chitinase was isolated from bromegrass (Bromus inermis) ‘Manchar’ based on microarray analyses (T. Nakamura, J. Yazaki, N. Kishimoto, S. Kikuchi, and M. Ishikawa, unpublished data [hereafter referred to as Nakamura et al., unpublished data]). Expression of BiCHT1 mRNA was increased by low temperature, suggesting the product may be involved in cold acclimation. BiCHT1 was highly homologous to winter rye CHT9, known to encode an AFP (Yeh et al., 2000
Deduced Amino Acid Sequence of BiCHT1 BiCHT1 cDNA was cloned from mRNA isolated from cold-treated bromegrass cells as previously described (Nakamura et al., unpublished data). The cDNA is 1,168 bp long with an open reading frame of 957 bp, encoding a peptide of 319 amino acids (predicted molecular mass is 33.2 kD). TargetP and PSORT analyses indicated with a certainty of 82% to 96.2% that there is a signal sequence of 20 amino acids that targets the protein to outside the cell (predicted molecular mass without the signal sequence is 31.3 kD). Comparison of the BiCHT1 amino acid sequence with known proteins demonstrated a homology with class I chitinase (Fig. 1 ). BiCHT1 protein had two consensus motifs (a chitin-binding domain and a catalytic domain) normally found in members of this family. BiCHT1 was highly homologous to winter rye CHT9, known as an AFP gene. The deduced amino acid sequence of BiCHT1 showed 89% identity with that of rye CHT9.
Proteins and Antifreeze Activity in the Culture Medium of Bromegrass during Cold Acclimation A 32-kD protein increased in the culture media of cold-acclimated wild-type bromegrass cells as detected by SDS-PAGE (Fig. 2A ). The protein cross reacted with an antibody raised against rice (Oryza sativa) class I chitinase, CHT3. This allows us to conclude that the major protein increased in cold-acclimated bromegrass culture medium is a chitinase. The results coincided well with our microarray and RNA-blot data that showed that the expression of an endochitinase gene (BiCHT1) was up-regulated by cold and the mRNA accumulated with increasing exposure to cold (Nakamura et al., unpublished data).
No antifreeze activity or thermal hysteresis was detected in the culture medium before cold treatment, as a flat round ice crystal grew rapidly with decreasing temperature in the antifreeze assay (Fig. 2B). Antifreeze activity in the culture medium increased during cold acclimation as seen in the formation of a specific ice shape (hexagonal) and retardation of ice crystal growth after 5 d of treatment at 4°C (Fig. 2D). After 3 weeks of cold treatment, the ice crystal formed hexagonal column and bipyramidal shapes, indicating further increases in the antifreeze activity (Fig. 2E).
Expression and purification of recombinant BiCHT1 proteins were performed as described in "Materials and Methods." As shown in Figure 3A , a tag-fused protein of 48 kD was detected by Coomassie Brilliant Blue staining after induction by isopropyl-β-D-thiogalactopyranoside (IPTG). Most recombinant BiCHT1 proteins were insoluble (recovered in the centrifugation pellet), but there were some soluble (recovered in the supernatant) recombinant proteins (Fig. 3B). The soluble, tag-fused protein bound to S-protein agarose and was successfully eluted after specific digestion with a protease to form a 33-kD recombinant protein (Fig. 3B). Both the tag-fused (48 kD) and tag-removed (33 kD) recombinant proteins cross reacted with anti-CHT3 antibody (data not shown). Chitinase activity was detected in the total soluble protein fraction containing the tag-fused protein and with a high specific activity in the tag-removed recombinant BiCHT1 protein fraction (Table I ). However, the tag-removed recombinant BiCHT1 protein fraction had no antifreeze activity even at 2 mg/mL concentration (Fig. 6H).
Overexpression of BiCHT1 Protein in Culture Media of Transgenic Bromegrass Cells and Antifreeze Activity of the Media To investigate the native form of BiCHT1 protein, the BiCHT1 gene was introduced into bromegrass suspension-cultured cells under the control of a cauliflower mosaic virus (CaMV) 35S promoter (Fig. 4A ). We obtained 19 geneticin-resistant bromegrass cell lines, in 12 lines of which transgene-specific PCR product was detected (data not shown). Southern-blot analysis (Fig. 4B) revealed that the introduced BiCHT1 gene was integrated into the genome in at least six of these transgenic bromegrass cell lines. The expression of BiCHT1 mRNA in the transformants was determined by northern-blot analysis (Fig. 4C). Two cell lines, one expressing relatively low levels of BiCHT1 mRNA (T2) and the other expressing high levels of BiCHT1 mRNA (T6), were selected for further experiments.
The culture medium of these transgenic lines showed increased chitinase activities compared to that of wild-type cells (Table I). SDS-PAGE of the culture medium revealed that two independent transgenic cell lines (T2 and T6) overexpressed the BiCHT1 products with the molecular mass identical to the one induced by exposure to 4°C and 10°C (Fig. 5A ). The BiCHT1 products were confirmed to be purified from culture media by adsorption to chitin affinity beads (Fig. 5A). The molecular mass of the expressed protein (32 kD) was consistent with the calculated one (31.3 kD, without the signal sequence) of the BiCHT1 protein based on the deduced amino acid sequence. Western analysis showed cross reaction of the expressed proteins with anti-CHT3 antiserum (Fig. 5B). When SDS-PAGE of culture media was performed in the absence of β-mercaptoethanol (β-ME), the expressed BiCHT1 protein showed a decreased mobility (35 kD; Fig. 5C, top arrow) as compared to the mobility in the presence of β-ME (Fig. 5C, bottom arrow). Gel-based enzyme assays detected chitinase activities in both the 35-kD (–β-ME) and 32-kD (+β-ME) bands (Fig. 5D). This indicated that chitinase denatured in the presence of β-ME could refold in the gel to regain an active form during incubation with the buffer following the SDS-PAGE. These results allow us to conclude that the BiCHT1 product is a chitinase secreted into the culture medium.
However, antifreeze activity was not detected in the culture medium (50-fold concentrated) of transgenic bromegrass cell lines (T6 and T2) grown for 7 d at 25°C (Fig. 6, D and G). These samples contained approximately 0.25 to 0.35 mg/mL of the BiCHT1 product. Further concentration of the culture media to 200-fold failed to show antifreeze activity (data not shown). Antifreeze activity was not detected even after the addition of 5 to 20 mM CaCl2, KCl, NaCl, or EDTA (data not shown). Preincubation of the harvested culture medium (50-fold concentrated, molecular weight [MW] > 5 kD) at 4°C for 1 to 7 d in the presence and absence of medium filtrates (MW cutoff <5 kD) from wild-type cell cultures grown at 4°C for 3 weeks did not induce antifreeze activity (data not shown). In the meantime, higher antifreeze activity was detected in the 50-fold concentrated culture medium from wild-type cells grown at 4°C (ice crystals with sharper hexagonal shapes formed) than from those grown at 10°C (Fig. 6 , C compared to B). The levels of the BiCHT1 protein in these samples were approximately 0.04 mg/mL (4°C) and 0.14 mg/mL (10°C). Antifreeze activity levels seemed unrelated with BiCHT1 protein concentrations. When the transgenic bromegrass cell line (T6) was grown at 4°C and 10°C for 7 d, the culture medium (50-fold concentrated) showed antifreeze activity, but the levels were similar to those of wild-type cells: wild type at 4°C = T6 at 4°C > wild type at 10°C = T6 at 10°C (Fig. 6, F compared to C, and E compared to B). Serial dilution of these samples showed similar declines in the activity (data not shown). SDS-PAGE and western-blot analyses revealed that the levels of BiCHT1 product in these samples were: T6 at 10°C > T6 at 4°C >> wild type at 10°C > wild type at 4°C (Fig. 7 ). BiCHT1 protein levels of these samples seemed unrelated with antifreeze activity levels.
Expression of BiCHT1 Proteins during a Normal Culture Condition
The expression of BiCHT1 chitinase was investigated in the media of wild-type bromegrass suspension cells at a normal (no stress/no treatment) growth condition by SDS-PAGE and western blots (Fig. 8
). The chitinase protein in the culture media gradually increased during growth at 25°C, with the highest accumulation on 10- to 14-d incubation periods, which correspond to the stationary phase in the culture growth stage (Ishikawa et al., 2006
Effect of Various Stress, Hormone, and Chemical Treatments on BiCHT1 Expression and Antifreeze Activity
Northern-blot analyses were performed to determine how the expression of the BiCHT1 gene is regulated in response to environmental stresses. The data in Figure 9
shows that the levels of BiCHT1 mRNA were slightly increased at the stationary phase (14 d after inoculation). The expression of BiCHT1 was most increased by incubation at 4°C and 10°C and moderately at 35°C. BiCHT1 mRNA was not increased by salt or water stress. Abscisic acid (ABA), anisomycin, and sphingosine have been shown to induce freezing tolerance in bromegrass cultured cells at nonhardening temperatures (Ishikawa et al., 1990
In Figure 10 , methyl jasmonate (MeJA), salicylic acid (SA), and ethylene (given in the form of ethephon), which are known to be involved in pathogenesis-related responses, did not increase the expression of BiCHT1 mRNA as compared to the respective controls (ethanol only). The expression of BiCHT1 mRNA was rapidly but only transiently increased 7- to 9-fold by the addition of soluble chitin (ethylene glycol chitin [EGC]) and insoluble chitin. Incubating cells with 1 mM hydrogen peroxide (H2O2) did not affect the expression. Prolonged exposure (14 d) to 10 mM H2O2 and chitin (soluble or insoluble) increased the mRNA level in a manner that intensified the stationary phase-specific increase of the expression.
Antifreeze activity in culture media (50-fold concentrated) of cells grown with some of these treatments was determined (data not presented). No antifreeze activity was detected in the medium of cells grown at 25°C in the presence of 75 µM ABA for 7 to 14 d or in the presence of chitin for 7 d. Prolonged incubation (14 d) of cells at 25°C without any treatment did not induce antifreeze activity in the medium. Antifreeze activity was detected in the medium concentrates of cells grown with ethephon (0.7–7 mM) at 25°C for 2 and 4 d.
Freezing is one of the most critical environmental stresses, and freezing tolerance is important for plants to survive severe cold winter periods. Plants show diverse levels of adaptation to cold (Thomashow, 1998 ), and adaptations involve regulation of enzyme activities and changes in gene expression. Smooth bromegrass is one of the most cold hardy grass species (Sakai and Larcher, 1987) and may serve as an important source of freezing tolerance genes.
Freezing tolerance is induced in bromegrass suspension cells by exposure to low temperatures (Robertson et al., 1987 Expression of BiCHT1 products in E. coli was difficult. Recombinant BiCHT1 proteins expressed in E. coli BL21 (DE3) pLysS were completely aggregated and insoluble (data not shown). On the other hand, E. coli Origami (DE3) pLysS, which has oxidative cytoplasm, could partially produce recombinant BiCHT1 proteins in a soluble form (Fig. 3B). Therefore, formation of sufficient disulfide bonds may be required for solubilization of recombinant BiCHT1 proteins. ScanProsite analysis predicted the presence of four disulfide bonds in the chitin-binding domain and its vicinity, which may be important for the chitin-binding activity. The soluble BiCHT1 products obtained (tag-fused, tag-removed) had chitinase activity (Table I), but no antifreeze activity was detected in these protein fractions (BiCHT1 product concentration in the tag-removed fraction was 2 mg/mL; Fig. 6H).
Protein structure analysis showed that BiCHT1 has a chitin-binding domain and a catalytic domain and was expected to be secreted. The BiCHT1 gene was successfully introduced by Agrobacterium-mediated transformation into bromegrass cells and overexpressed using the CaMV 35S promoter. There was an increase in a 32-kD protein band in SDS-PAGE gels of total protein fractions from the culture medium of overexpressing transgenic lines (Fig. 5). This band cross reacted with anti-CHT3 antibody and also bound to chitin beads. The culture medium of overexpressing lines had increased chitinase activities (Table I). The enzymic activities were subjected to zymogram analyses. Zymograms are not quantitative; however, they do offer a rough estimate of the approximate molecular masses of active enzymes. The chitinase zymogram (Fig. 5D) revealed one band with chitinolytic activity with an approximate molecular mass of 32 kD. These data confirmed the production and secretion of BiCHT1 proteins with the proper chitinase function (most probably with the proper structure) by the transformed bromegrass cells. However, the culture medium concentrates of BiCHT1 overexpressing cell lines did not modify the growth of ice in the antifreeze assays (BiCHT1 product concentration, 0.25–0.35 mg/mL; Fig. 6, D and G). Antifreeze activities were not detected even at the concentration of 1.0 to 1.4 mg/mL. At the concentration (0.25–2 mg/mL) used for assaying the products from transgenic E. coli and bromegrass, apoplast chitinases isolated from cold-acclimated winter rye showed high antifreeze activities (bipyramid ice crystals) according to Hon et al. (1994
Antifreeze activity was detected in the apoplast of freezing-tolerant cereals (Antikainen and Griffith, 1997 When the transgenic bromegrass cell lines were grown at 4°C, the culture medium concentrates showed antifreeze activity levels similar to that of wild-type cells grown at 4°C (Fig. 6) in spite of greater accumulation of BiCHT1 products in the medium (Fig. 7). This allows us to discount the possibility that some cofactors synthesized during cold acclimation at 4°C may be required for the latent antifreeze activity of the BiCHT1 product to become detectable. We also found that antifreeze activity in the medium concentrates of cells grown at 4°C was not affected by the addition of anti-CHT3 antibody (data not shown). This implies that the protein domain responsible for the antifreeze activity was not affected by binding of anti-CHT3 antibody.
Numerous types of AFP have been isolated from different organisms. A common structural feature of many animal AFPs is the presence of repeated amino acid sequences. Repeat sequences have been found in fish antifreeze glycoproteins, fish type I and type IV AFPs, and insect AFPs, while fish type II and III AFPs have no repetitive sequences (Cheng, 1998
BiCHT1 is a single-copy gene in bromegrass (data not shown). The antibody used in the western-blot studies specifically cross reacts with the Cht-3 product and not with Cht-2 in the class I chitinase family of rice (Nishizawa et al., 1999
Expression of BICHT1 was up-regulated and its product accumulated in long-term exposure to low temperatures (Figs. 2, 5, 7, and 9). The level of BiCHT1 mRNA expression at 10°C was similar to that at 4°C (Fig. 9) in contrast to antifreeze activity levels described below. It was also expressed at 35°C. Many genes induced by temperature stresses are also known to be induced by salt or drought stress (Seki et al., 2003
Antifreeze activity detected in the culture medium was induced by incubation of bromegrass cells at 4°C but only slightly at 10°C (Fig. 6, C and B). It was also induced by ethephon (0.7–7 mM) treatment (data not shown). But it was not induced by ABA (50–75 µM) treatment nor by chitin or long-term incubation (14 d) at 25°C (data not shown). Antifreeze activity detected in the apoplasts of winter rye plants has been known to be induced by cold treatment, drought (Yu and Griffith, 2001 The results of transgenic E. coli and bromegrass cells and expression analyses of BiCHT1, taken together, allow us to conclude that the BiCHT1 product is a major protein with chitinase activity secreted in the medium from cold-treated bromegrass cells but is unlikely to be responsible for cold-induced antifreeze activity in the culture medium by itself, although BiCHT1 is highly homologous to winter rye CHT9, known as an AFP gene. Isolation of alternative AFP candidates from bromegrass is currently under way in our laboratory.
We are currently investigating possible functions of BiCHT1 and its product using the bromegrass cell lines overexpressing BiCHT1. Cold-inducible wheat chitinases, one of which has high homology to BiCHT1 and rye CHT9, have recently been found to be involved in snow mold resistance (Kawakami and Terami, 2005
Plant Material and Culture Conditions
A nonembryogenic suspension culture of smooth bromegrass (Bromus inermis) ‘Manchar’ was used in this work, and growth conditions were as described previously (Nakamura and Ishikawa, 2006
Bromegrass cells were subjected to salt stress by adding NaCl to the culture medium at a final concentration of 50 mM or 100 mM. Water stress was provided by adding 100 g/L or 200 g/L of polyethylene glycol to the culture medium. Cold treatment was conducted by transferring cultures grown for 3 d at 25°C either directly to 10°C or 4°C (Figs. 5–7 For ABA treatment, (±) ABA (Sigma-Aldrich) was added to the ER medium at a final concentration of 50 µM. Anisomycin (in DMSO) was added to cell cultures at a final concentration of 10 µM or 20 µM. D-Erythro-sphingosine (in DMSO) was added to cell cultures at a final concentration of 5 µM or 10 µM. A 10 mM MeJA stock solution (in ethanol) was added to cell cultures at a final concentration of 1 µM or 10 µM. A 100 mM SA stock solution (in ethanol) was added to cell cultures at a final concentration of 10 µM or 100 µM. A 100 mg/mL ethephon stock solution (in ethanol) was added to cell cultures at a final concentration of 0.1 mg/mL (0.7 mM) or 1 mg/mL (7 mM). However, prolonged incubation with 1 mg/mL ethephon was sometimes lethal to bromegrass cells. H2O2 solution was added to cell cultures at a final concentration of 1 mM or 10 mM. For chitin treatment, 5 mg of EGC (soluble) or 30 mg of chitin (insoluble) was added to 50 mL cell culture medium. All these chemical, hormone, and stress treatments were performed at 25°C. The cells were harvested at designated time periods during stress treatments or incubation with the chemicals and used for total RNA extraction.
Bromegrass cell cultures were filtered with an 80-µm nylon mesh, and the obtained liquid media were filtered to remove cell debris using a 0.22-µm Millex-GP syringe-driven filter (Millipore). The media were concentrated using Apollo 20 mL (QMWL, 5 kD; Orbital Biosciences) for a 50-fold concentration of extracellular proteins. The concentrated media was used for SDS-PAGE, western-blot analyses, and antifreeze activity assays.
Antifreeze activity was detected according to Hon et al. (1994)
Northern- and Southern-blot analyses were performed as described previously (Nakamura et al., 1997
For construction of the chitinase expression vector, PCR was performed using BiCHT1 cDNA as a template with the forward primer 5'-GCCACCATGGGAGGACTTGTGGTGGCG-3' and reverse primer 5'-TCATTACTATGCGAACGGCCTCTG-3'. The PCR products were cloned into the pGEM-T Easy vector (Promega). After confirmation of the DNA sequence, the DNA fragment encoding BiCHT1 was excised by digestion with NcoI and EcoRI, and ligated into the expression vector pET-32a (Novagen), previously digested by the same enzymes. The recombinant plasmid, pET-BiCHT1, was introduced into E. coli Origami (DE3) pLysS (Novagen). For induction of recombinant BiCHT1 protein, 50-mL suspension (cell density, A600 = 1.0) of E. coli harboring pET-BiCHT1 was incubated with 1 mM IPTG for 24 h at 20°C. The bacteria harvested by centrifugation were sonicated in 5 mL of buffer comprising 50 mM Tris-HCl, pH 8.0, 50 mM NaCl, 1 mM EDTA, and centrifuged to obtain the supernatant (soluble protein fraction). Recombinant BiCHT1 was purified from the soluble protein fraction (5 mL) using an S·Tag rEK Purification kit (Novagen) according to the method specified by the supplier.
SDS-PAGE was carried out by the method of Laemmli (1970)
Chitinase activity was assayed colorimetrically with glycol chitin as a substrate. The sample solution (100 µL) was added to 900 µL of 100 mM sodium acetate buffer, pH 7.0, containing 0.1% (w/v) EGC (Seikagaku). After incubation of the reaction mixture at 37°C for 5 h, the amount of reducing sugars produced was determined by reading A420 using the modified Schales method (Imoto and Yagishita, 1971 To examine chitin-binding ability, a slurry of 500 µL of chitin beads (New England Biolabs) was added to 50 mL of culture medium. After gentle agitation at 4°C for 2 h, the beads were pelleted and washed extensively with 100 mM sodium phosphate buffer, pH 7.0. The chitin beads were resuspended in 200 µL of SDS-PAGE loading buffer and boiled for 5 min. The samples were directly used for SDS-PAGE analysis.
For zymogram assays of chitinase activity, samples were analyzed on 12.5% SDS-polyacrylamide gels containing 0.01% (w/v) EGC according to Trudel and Asselin (1989)
BiCHT1 cDNA was cloned into a binary vector, pBI121, under the control of a CaMV 35S promoter. The resultant plasmid, pBI-BiCHT1 (Fig. 4A), was introduced into Agrobacterium tumefaciens EHA105 using a freeze-thaw method (Holsters et al., 1978 Sequence data from this article can be found in the GenBank/EMBL data libraries under accession number AB428423 (BiCHT1).
We thank Dr. Y. Nishizawa (NIAS) for the gift of anti-chitinase antibody and Ms Y. Koike and R. Iwanami (NIAS) for maintenance of the cultures. We thank Dr. A.J. Robertson (University of Saskatchewan) for critical reading of the manuscript. We dedicate this article to the late Prof. Marilyn Griffith who made great contributions to antifreeze studies in plants with her pioneering works and also kindly showed us antifreeze assays. Received April 11, 2006; accepted March 11, 2008; published March 21, 2008.
1 This work was supported by the Rice Genome Project (grant nos. MA–2121 and PR1207); by the Bio Design Program from Ministry of Agriculture, Forestry and Fisheries, Japan; by Bio-oriented Technology Research Advancement Institution, Japan (a PRBRAIB grant); and by Japan Society for the Promotion of Science (grants-in-aid for scientific research nos. 17658011 and 16380030 to M.I.). 
2 These authors equally contributed to the article. The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Masaya Ishikawa (isikawam{at}affrc.go.jp).
[OA] Open Access articles can be viewed online without a subscription. www.plantphysiol.org/cgi/doi/10.1104/pp.106.081497 * Corresponding author; e-mail isikawam{at}affrc.go.jp.
Antikainen M, Griffith M (1997) Antifreeze protein accumulation in freezing-tolerant cereals. Physiol Plant 99: 423–432[CrossRef] Bohnert HJ, Nelson DE, Jensen RG (1995) Adaptation to environmental stress. Plant Cell 7: 1099–1111[CrossRef][Web of Science][Medline] Cheng C-HC (1998) Evolution of the diverse antifreeze proteins. Curr Opin Genet Dev 8: 715–720[CrossRef][Web of Science][Medline] Chun JU, Yu XM, Griffith M (1998) Genetic studies of antifreeze proteins and their correlation with winter survival in wheat. Euphytica 102: 219–226[CrossRef][Web of Science] De Jong AJ, Cordewener J, Lo Shiavo F, Terzi M, Vandekerckhove J, Van Kammen A, De Vries SC (1992) A carrot somatic embryo mutant is rescued by chitinase. Plant Cell 4: 425–433 Ewart KV, Lin Q, Hew CL (1999) Structure, function and evolution of antifreeze proteins. Cell Mol Life Sci 55: 271–283[CrossRef][Web of Science][Medline] Gamborg OL, Wetter LR (1975) Plant Tissue Culture Methods. National Research Council of Canada, Saskatoon, Canada Griffith M, Yaish MW (2004) Antifreeze proteins in overwintering plants: a tale of two activities. Trends Plant Sci 9: 399–405[CrossRef][Web of Science][Medline] Guy CL (1990) Cold acclimation and freezing stress tolerance: role of protein metabolism. Annu Rev Plant Physiol Plant Mol Biol 41: 187–223[Web of Science] Hiilovaara-Teijo M, Hannukkala A, Griffith M, Yu XM, Pihakaski-Maunsbach K (1999) Snow-mold-induced apoplastic proteins in winter rye leaves lack antifreeze activity. Plant Physiol 121: 665–674 Holsters M, De Waele D, Depicker A, Messens E, Van Montagu M, Schell J (1978) Transfection and transformation of Agrobacterium tumefaciens. Mol Gen Genet 163: 181–187[CrossRef][Web of Science][Medline] Hon WC, Griffith M, Chong P, Yang DSC (1994) Extraction and isolation of antifreeze proteins from winter rye (Secale cereale L.) leaves. Plant Physiol 104: 971–980[Abstract] Hon WC, Griffith M, Mlynarz A, Kwok YC, Yang DSC (1995) Antifreeze proteins in winter rye are similar to pathogenesis-related proteins. Plant Physiol 109: 879–889[Abstract] Huang T, Duman JG (2002) Cloning and characterization of a thermal hysteresis (antifreeze) protein with DNA-binding activity from winter bittersweet nightshade, Solanum dulcamara. Plant Mol Biol 48: 339–350[CrossRef][Web of Science][Medline] Imoto T, Yagishita K (1971) A simple activity measurement of lysozyme. Agric Biol Chem 35: 1154–1156[Web of Science] Ishikawa M, Robertson AJ, Gusta LV (1990) Effect of temperature, light, nutrients and dehardening on abscisic acid induced cold hardiness in Bromus inermis Leyss suspension cultured cells. Plant Cell Physiol 31: 51–59 Ishikawa M, Suzuki M, Nakamura T, Kishimoto T, Robertson AJ, Gusta LV (2006) Effect of growth phase on survival of bromegrass suspension cells following cryopreservation and abiotic stresses. Ann Bot (Lond) 97: 453–459 Kasprzewska A (2003) Plant chitinases: regulation and function. Cell Mol Biol Lett 8: 809–824[Web of Science][Medline] Kawakami A, Terami F, inventors. January 4, 2005. Low temperature expression chitinase cDNAs and method for isolating the same. U.S. Patent Application No. 6838271 Kuiper MJ, Davies PL, Walker VK (2001) A theoretical model of a plant antifreeze protein from Lolium perenne. Biophys J 81: 3560–3565[Web of Science][Medline] Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680–685[CrossRef][Medline] Liou YC, Tocilj A, Davies PL, Jia Z (2000) Mimicry of ice structure by surface hydroxyls and water of a β-helix antifreeze protein. Nature 406: 322–324[CrossRef][Medline] Meyer K, Keil M, Naldrett MJ (1999) A leucine rich repeat protein of carrot that exhibits antifreeze activity. FEBS Lett 447: 171–178[CrossRef][Web of Science][Medline] Mouille G, Robin S, Lecomte M, Pagant S, Höfte H (2003) Classification and identification of Arabidopsis cell wall mutants using Fourier Transform InfraRed (FT-IR) microspectroscopy. Plant J 35: 393–404[CrossRef][Web of Science][Medline] Nakamura T, Ishikawa M (2006) Transformation of suspension cultures of bromegrass (Bromus inermis Leyss) by Agrobacterium tumefaciens. Plant Cell Tissue Organ Cult 84: 293–299[CrossRef][Web of Science] Nakamura T, Yokota S, Muramoto Y, Tsutsui K, Oguri Y, Fukui K, Takabe T (1997) Expression of a betaine aldehyde dehydrogenase gene in rice, a glycinebetaine nonaccumulator, and possible localization of its protein in peroxisomes. Plant J 11: 1115–1120[CrossRef][Web of Science][Medline] Nishizawa Y, Hibi T (1991) Rice chitinase gene: cDNA cloning and stress-induced expression. Plant Sci 76: 211–218 Nishizawa Y, Kishimoto N, Saito A, Hibi T (1993) Sequence variation, differential expression and chromosomal location of rice chitinase genes. Mol Gen Genet 241: 1–10[CrossRef][Web of Science][Medline] Nishizawa Y, Nishio Z, Nakazono K, Soma M, Nakajima E, Ugaki M, Hibi T (1999) Enhanced resistance to blast (Magnaporthe grisea) in transgenic rice by constitutive expression of rice chitinase. Theor Appl Genet 99: 383–390[CrossRef][Web of Science] Robertson AJ, Gusta LV, Reaney MJT, Ishikawa M (1987) Protein synthesis in bromegrass (Bromus inermis Leyss.) cultured cells during the induction of frost tolerance by abscisic acid or low temperature. Plant Physiol 84: 1331–1336 Sakai A, Larcher W (1987) Frost Survival of Plants. Springer-Verlag, Berlin Seki M, Kamei A, Yamaguchi-Shinozaki K, Shinozaki K (2003) Molecular responses to drought, salinity and frost: common and different paths for plant protection. Curr Opin Biotechnol 14: 194–199[CrossRef][Web of Science][Medline] Sicheri F, Yang DS (1995) Ice-binding structure and mechanism of an antifreeze protein from winter flounder. Nature 375: 427–431[CrossRef][Medline] Stressmann M, Kitao S, Griffith M, Moresoli C, Bravo LA, Marangoni AG (2004) Calcium interacts with antifreeze proteins and chitinase from cold-acclimated winter rye. Plant Physiol 135: 364–376 Thomashow MF (1998) Role of cold-responsive genes in plant freezing tolerance. Plant Physiol 118: 1–7 Thomashow MF (1999) Plant cold acclimation, freezing tolerance genes and regulatory mechanisms. Annu Rev Plant Physiol Plant Mol Biol 50: 571–599[CrossRef][Web of Science] Trudel J, Asselin A (1989) Detection of chitinase activity after polyacrylamide gel electrophoresis. Anal Biochem 178: 362–366[CrossRef][Web of Science][Medline] Worrall D, Elias L, Ashford D, Smallwood M, Sidebottom C, Lillford P, Telford J, Hold C, Bowles D (1998) A carrot leucine-rich-repeat protein that inhibits ice recrystallization. Science 282: 115–117 Wu G, Robertson AJ, Liu X, Zheng P, Wilen RW, Nesbitt NT, Gusta LV (2004) A lipid transfer protein gene BG-14 is differentially regulated by abiotic stress, ABA, anisomycin, and sphingosine in bromegrass (Bromus inermis). J Plant Physiol 161: 449–458[CrossRef][Web of Science][Medline] Yeh S, Moffatt BA, Griffith M, Xiong F, Yang DS, Wiseman SB, Sarhan F, Danyluk J, Xue YQ, Hew CL, et al (2000) Chitinase genes responsive to cold encode antifreeze proteins in winter cereals. Plant Physiol 124: 1251–1264 Yu XM, Griffith M (2001) Winter rye antifreeze activity increases in response to cold and drought, but not abscisic acid. Physiol Plant 112: 78–86[CrossRef][Medline] Yu XM, Griffith M, Wiseman SB (2001) Ethylene induces antifreeze activity in winter rye leaves. Plant Physiol 126: 1232–1240 Zhang D, Hrmova M, Wan CH, Wu C, Balzen J, Cai W, Wang J, Densmore LD, Fincher GB, Zhang H, et al (2004) Members of a new group of chitinase-like genes are expressed preferentially in cotton cells with secondary walls. Plant Mol Biol 54: 353–372[CrossRef][Web of Science][Medline] Zhang D-Q, Liu B, Feng D-R, He Y-M, Wang S-Q, Wang H-B, Wang J-F (2004) Significance of conserved asparagine residues in the thermal hysteresis activity of carrot antifreeze protein. Biochem J 377: 589–595[CrossRef][Web of Science][Medline]
|
|||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| ASPB Publications | PLANT PHYSIOLOGY® | THE PLANT CELL | |
|---|---|---|---|
TOP
ABSTRACT
RESULTS
