Detection and seasonal expression pattern of a pathogenesis-related protein (PR-10) in Douglas-fir (Pseudotsuga menziesii) tissues

Previously, a PR-10 protein Pin m III was described in western white pine. In this study, primers based on cDNA of Pin m III were utilized to obtain the genomic sequence of a Pin m III homologue – Pse m I – in Douglas-fir. A comparative analysis of a deduced amino acid sequence of Pin m III and Pse m I genes indicated about 80% similarity between the two protein sequences, and a consensus 20 amino acid sequence located around the p-loop sequence was used to synthesize a peptide of 20 amino acids. An antibody to this synthetic peptide was able to detect the Pse m I protein in Douglas-fir. The anti- Pse m I antibody was used in a western immunoblot to monitor seasonal variation of the Pse m I in Douglas-fir needles and its level was shown to increase with overwintering of Douglas-fir seedlings. However, unlike the Pin m III, there is no indication that the Pse m I is associated with frost hardiness. Analysis of infected Douglas-fir roots showed a possible trend to up-regulation of Pse m I by pathogens such as the laminated root rot fungus, Phellinus weirii . The expression of Pse m I protein in Douglas-fir seedlings is very low compared to the expression of Pin m III protein in western white pine seedlings. In addition, a light-harvesting complex I protein, PSI-F, was identified in Douglas-fir by N-terminal amino acid sequencing.     

A Simulation Model for the Annual Frost Hardiness and Freeze Damage of Scots Pine

The changes in the frost hardiness of Scots pine were modelledby a dynamic model where the input variables were temperatureand photoperiod and the phase of annual development. The damagecaused by freezing was described by the sigmoidal relationshipbetween the relative needle damage and freezing temperature.The model simulations were carried out using temperature datafrom two sites in central Finland—Suonenjoki and Tampere.The validity of the frost hardiness model was tested with measuredfrost hardiness data from Suonenjoki. The effects of climaticwarming were also simulated by increasing temperature of thelong-term climatic data. Genotypic differences in chilling requirement,which determines the timing of the reduction of hardening competence,were included in the simulations. The simulated needle damageincreased as a result of climatic warming, and the differencesin the chilling requirement had a stronger effect on the amountof damage in the warmed climate than in the present climate.A large variation between years was found in the level of damage. Annual development; climatic change; dynamic model; freeze damage; frost hardiness,Pinus sylvestris ; Scots pine     

Changing Environmental Effects on Frost Hardiness of Scots Pine During Dehardening

The effects of raised temperature and extended photoperiod onthe dehardening of quiescent and winter-hardy Scots pine saplingswere examined in an open-top-chamber experiment. The saplingswere exposed during winter to natural, square-curve fluctuating(between 1 and 11 °C with a 14 d interval), and constant(6 °C) temperatures with a natural and an extended (17 h)photoperiod. Frost hardiness of needles was determined by controlledfreezing tests and visual damage scoring. The constant 6 °Ctemperature treatment caused a gradual dehardening of needleswhereas under fluctuating temperatures the level of frost hardinessfluctuated. Trees exposed to extended photoperiods were lesshardy than under natural photoperiods after the initiation ofshoot elongation, but before this there were no clear differencesin frost hardiness between different photoperiodic treatments.The results indicate that the frost hardening competence ofScots pine changes during quiescence. Climate change; frost hardiness; hardening competence; photoperiod; Pinus sylvestris, Scots pine; temperature     

Seasonal fluctuation of dehydrins is related to osmotic status in Scots pine needles

Seasonal variation in dehydrins and other soluble proteins of Scots pine (Pinus sylvestris L.) needles, buds and bark were analyzed monthly for 1 year from 1998 to 1999. Dehydrin-related proteins of 60 and 56 kDa were identified immunologically in all tissues. The concentration of the 60-kDa dehydrin was highest during the winter (October-February) in buds and bark but increased in early spring (March-May) in needles. Accumulation of the 60-kDa dehydrin in the needles in springtime was related to the decreasing osmotic potentials of the needles. The 56-kDa dehydrin was present only during the growing season, as was a 50-kDa dehydrin, which only appeared in bud and bark tissues. The soluble protein concentration of needles did not differ significantly between seasons, but in bark and bud tissues the protein concentrations were at their lowest level in newly grown tissues (June-August). The level of several polypeptides was higher during the winter-spring period than in the growing season, especially in bark and bud tissues. These proteins may be related to cold hardiness or dormancy in overwintering Scots pine. Dehydrin-related proteins in needles are linked to springtime changes in the osmotic status of needles rather than to their cold acclimation.     

Studies on frost hardiness in Chlorella ellipsoidea II. Effects of inhibitors of RNA and protein synthesis and surfactants on the process of hardening

Chlorella ellipsoidea cells at an intermediate stage in theripening phase of the cell cycle were brought into contact withinhibitors of RNA and protein synthesis, and surfactants duringor after hardening at 3°C. Cycloheximide, actinomycin D and 5-fluorouracil inhibited thedevelopment of frost hardiness in the algal cells. In contrastwith cycloheximide, chloramphenicol did not cause complete inhibitionof hardiness increase, even at a high concentration. These resultssuggest that protein synthesis on cytoplasmic ribosomes is indispensablefor the development of frost hardiness, but protein synthesison chloroplast ribosomes has little or no involvement in thehardening process. Triton X-100 and sodium dodecyl sulfate inhibited the developmentof frost hardiness, but sodium cholate and sodium deoxycholatedid not. Hardened cells were more susceptible to sodium dodecylsulfate, in contrast to sodium cholate, sodium deoxycholateand Triton X-100, than unhardened cells. From the results, considerablealterations in both lipids and proteins constituting cellularmembranes appear to be involved in the process of hardening. (Received December 3, 1975; )     

A Second-order Dynamic Model for the Frost Hardiness of Trees

The development of frost hardiness in forest trees is describedby a dynamic model in which the input variables are the prevailingenvironmental conditions and the developmental stage of trees.The assumption of the model is that for each temperature andphotoperiod there is a discrete stationary level of frost hardiness,which is attained if these environmental factors remain constant.The dependence of the stationary level on temperature and photoperiodis assumed to be piece-wise linear and additive. The rate ofacclimation, i.e. frost hardening or dehardening, is describedas a second-order dynamic process with two time constants, thesecond of which changes depending on the stage of the annualdevelopment of the trees. The frost hardiness model was calibratedand tested using experimental data from Douglas fir [Pseudotsugamenziesii var. glauca (Beissn.) Franco] seedlings. The resultssuggest that the second-order model describes the changes infrost hardiness better than the first-order model with onlyone time constant.Copyright 1995, 1999 Academic Press Acclimation, developmental stage, Douglas fir, dynamic model, frost hardiness, photoperiod, Pseudotsuga menziesii, temperature     

Frost hardening and photosynthetic performance of Scots pine (Pinus sylvestris L.) needles. I. Seasonal changes in the photosynthetic apparatus and its function

Photosynthetic CO₂ uptake, the photochemical efficiency of photosystem II, the contents of chlorophyll and chlorophyll-binding proteins, and the degree of frost hardiness were determined in three-year-old Scots pine (Pinus sylvestris L.) trees growing in the open air but under controlled daylength. The following conditions were compared: 9-h light period (short day), 16-h light period (long day), and natural daylength. Irrespective of induction by short-day photoperiods or by subfreezing temperatures, frost hardening of the trees was accompanied by a long-lasting pronounced decrease in the photosynthetic rates of one-year-old needles. Under moderate winter conditions, trees adapted to a long-day photoperiod, assimilated CO₂ with higher rates than the short-day-treated trees. In the absence of strong frost, photochemical efficiency was lower under short-day conditions than under a long-day photoperiod. Under the impact of strong frost, photochemical efficiency was strongly inhibited in both sets of plants. The reduction in photosynthetic performance during winter was accompanied by a pronounced decrease in the content of chlorophyll and of several chlorophyll-binding proteins [light-harvesting complex (LHC)IIb, LHC Ib, and a chlorophyll-binding protein with MW 43 kDa (CP 43)]. This observed seasonal decrease in photosynthetic pigments and in pigment-binding proteins was irrespective of the degree of frost hardiness and was apparantly under the control of the length of the daily photoperiod. Under a constant 9-h daily photoperiod the chlorophyll content of the needles was considerably lower than under long-day conditions. Transfer of the trees from short-day to long-day conditions resulted in a significantly increased chlorophyll content, whereas the chlorophyll content decreased when trees were transferred from a long-day to a short-day photoperiod. The observed changes in photosynthetic pigments and pigment-binding proteins in Scots pine needles are interpreted as a reduction in the number of photosynthetic units induced by shortening of the daily light period during autumn. This results in a reduction in the absorbing capacity during the frost-hardened state. Received: 3 March 1997 / Accepted: 16 July 1997     

Isolation and Characterization of Hardening-Induced Proteins in Chlorella vulgaris C-27: Identification of Late Embryogenesis Abundant Proteins

Hardening-induced soluble proteins of Chlorella vulgaris BeijerinkIAM C-27 (formerly Chlorella ellipsoidea Gerneck IAM C-27) wereisolated and purified by two-dimensional high-performance liquidchromatography (2D-HPLC) on an anion-exchange column, with subsequentreversed-phase chromatography. Some of the proteins were resolvedby SDS-PAGE, characterized by amino-terminal sequencing andidentified by searching for homologies in databases. Separationof the soluble proteins during the hardening of Chlorella bya combination of 2D-HPLC and SDS-PAGE revealed that at least31 proteins were induced or increased in abundance. Of particularinterest was the induction after 12 h of a 10-kDa protein withthe amino-terminal amino acid sequence AGNKPITEQISDAVGAAGQKVGand the induction after 6 h of a 14-kDa protein with the amino-terminalsequence ALGEESLGDKAKNAFEDAKDAVKDAAGNVKEAV. The amino-terminalsequences of these proteins indicated that they were homologousto late embryogenesis abundant (LEA) proteins. Furthermore,the level of a 22-kDa protein also increased after 12 h. Theamino-terminal sequence of this protein, AAPLVGGPAPDFTAAAVFD,indicated that it was homologous to thioredoxin peroxidase. (Received June 9, 1995; Accepted September 12, 1995)     

Overexpression of a western white pine PR10 protein enhances cold tolerance in transgenic Arabidopsis

The PmPR10-1.10 protein from western white pine is known to be associated with frost hardiness, and up-regulated by seasonal cold acclimation and biotic and abiotic stresses. To gain insight into the molecular basis of cold hardiness, we investigated the potential physiological role of PmPR10-1.10 by gene overexpression in transgenic Arabidopsis plants. A binary vector was constructed for PmPR10-1.10 synthesis in higher plants and transgenic Arabidopsis lines were generated by Agrobacterium-mediated transformation. Following Western protein blot analysis confirming target protein production, transgenic Arabidopsis lines were tested for cold tolerance by electrolyte leakage analysis post treatment of different freezing temperatures. Our results demonstrate that accumulation of PmPR10-1.10 protein resulted in significantly greater freezing tolerance in transgenic plants than in wild type plants. This indicates that the transfer and selection of cold acclimation proteins like PmPR10-1.10 may be a breeding strategy for the development of freezing tolerance in conifers.     

Accumulation of Dehydrins and ABA-Inducible Proteins in Wheat Seedlings during Low-Temperature Acclimation

Using one-dimensional SDS-PAGE and immunochemical methods, we detected the presence and estimated the content of dehydrins and ABA-inducible (RAB) polypeptides in etiolated seedlings of four spring and three winter wheat (Triticum aestivum L.) cultivars differing in frost hardiness. We hardened three-day-old seedlings at 4°C for nine days or grew them at 22°C for a day (control seedlings). We established that heat-stable cold-regulated (COR) polypeptides with mol wts of 209, 196, 169, 66, 50, and 41 kD, which are characteristic of hardened wheat seedlings, were homologous to polypeptides from a dehydrin family and polypeptides with mol wts of 209, 196, 66, 50, and 41 kD were immunologically related to RAB-proteins. We supposed that these COR polypeptides were involved in the prevention of local protein dehydration and denaturation during hypothermia. Analysis of the relative content of COR proteins revealed a close correlation between the cultivar frost hardiness and the concentration of these proteins. It seems evident that different accumulation of dehydrins and RAB polypeptides in different cultivars of a single species is one of the causes for different plant frost hardiness.     

The effects of long-term elevation of air temperature and CO on the frost hardiness of Scots pine

The frost hardiness of 20 to 25-year-old Scots pine (Pinus sylvestris L.) saplings was followed for 2 years in an experiment that attempted to simulate the predicted climatic conditions of the future, i.e. increased atmospheric CO₂ concentration and/or elevated air temperature. Frost hardiness was determined by an electrolyte leakage method and visual damage scoring on needles. Elevated temperatures caused needles to harden later and deharden earlier than the controls. In the first year, elevated CO₂ enhanced hardening at elevated temperatures, but this effect disappeared the next year. Dehardening was hastened by elevating CO₂ in both springs. The frost hardiness was high (相似文献   

Isolation of the D1 Protein and a 28-kDa Fragment of the D2 Protein from Spinach Photosystem II Membranes

The 32-kDa D1 protein, which contained no lysine in spinachchloroplasts, as deduced from its DNA code, was isolated byhigh-performance gel permeation chromatography in the presenceof 0.1% SDS and 4 M urea. Three proteins of the photosystemII reaction center complex have a molecular mass of 30–35kDa, and two, the D2 protein and the peripheral 33-kDa protein,were severed into peptide fragments by Achromobacter lysyl endopeptidase(EC 3.4.14.50 [EC] ) before the chromatography. The isolated D1 proteindid not contain chlorophylls and pheophytins but had an absorptionmaximum at 265 nm probably due to bound plastoquinone. A peptidefragment of 28 kDa from the D2 protein was also isolated fromspinach photosystem II membranes and the wheat photosystem IIreaction center. Antibodies raised against the 28-kDa peptidefrom wheat bound to the 34-kDa D2 protein, which suggested thatthis peptide was the largest sequence of Aspl4-Lys265. The fragmentof wheat D2 protein showed absorption maxima at 413 and 682nm attributable to bound pheophytin that probably had been convertedfrom chlorophyll a during the isolation process. (Received June 29, 1987; Accepted October 21, 1987)     

Effects of Hormones on Morphogenesis and Cold Resistance in Berseem Clover (Trifolium alexandrinum L.)

Plants of berseem clover (Trifolium alexandrinum L.) cv. Taborwere raised under conditions inhibiting the acquisition of coldhardiness (non-hardened) or inducing cold hardiness (hardened).All non-hardened plants developed an elongated shoot and exhibitedconsiderable frost sensitivity, as measured by the extent ofthe reduction in yield of variable chlorophyll fluorescenceafter exposure to sub-zero temperature. Hardened plants developeda shorter shoot, with fewer leaves and a greater percentageof dry matter in the root system. These parameters were associatedwith a marked increase in frost resistance. Exogenous applicationof ABA to plants effected similar morphological modificationsin both hardening and non-hardening temperature regimes; plantsdeveloped a shorter primary shoot axis and leaves exhibiteda marked increase in frost hardiness. In berseem clover ABAcan thus substitute, at least partially, for the low temperaturetreatment required to induce cold hardiness. Spraying plantsraised under hardening conditions with gibberellic acid reversedthe effects of the hardening treatment, since they developedan elongated shoot and exhibited frost sensitivity comparableto non-treated plants grown under non-hardening conditions.It is concluded that these endogenous hormones are directlyinvolved in triggering changes in morphogenesis which accompanyphysiological and metabolic events associated with the inductionof plant cold hardiness. Key words: Frost resistance, morphogenesis, abscisic acid, giberellic acid, Trifolium alexandrinum     

Application of Impedance Spectroscopy for Selecting Frost Hardy Varieties of English Ryegrass

The frost hardening and frost damage of 12 varieties of Englishryegrass (Lolium perenne) was studied by electrical impedancespectroscopy. For the measurement of the impedance spectrum(80 Hz to 1 MHz) a 10 mm length sample was cut from the stemabove the growing point, but the growing point was included.The impedance spectra were analysed by an asymmetric distributedcircuit model. The impedance spectra were measured at two phasesof hardiness and after freezing, i.e. (a) before hardening,(b) after hardening in controlled conditions, and (c) aftercontrolled frost exposure at -16 °C after hardening. Twomodel parameters, i.e. intra- and extracellular resistance,increased with hardening. The intracellular resistance and theskewness factor before hardening, and the ratio between thosetwo parameters before and after hardening, strongly correlatedwith hardening of different varieties of English ryegrass. Theextracellular resistance and the relaxation time decreased asa result of the frost exposure at -16 °C. Cold acclimation; electrical impedance; English ryegrass; frost hardiness; impedance spectroscopy; Lolium perenne     

Geographic pattern of genetic variation in photosynthetic capacity and growth in two hardwood species from British Columbia

Geographic patterns of intraspecific variations in traits related to photosynthesis and biomass were examined in two separate common garden experiments using seed collected from 26 Sitka alder (Alnus sinuata Rydb.) and 18 paper birch (Betula papyrifera Marsh.) populations from climatically diverse locations in British Columbia, Canada. Exchange rates of carbon dioxide and water vapour were measured on 2-year-old seedlings to determine the maximum net instantaneous photosynthetic rate, mesophyll conductance, stomatal conductance, and photosynthetic water use efficiency. Height, stem diameter, root and shoot dry mass and fall frost hardiness data were also obtained. Mean population maximum photosynthetic rate ranged from 10.35 to 14.57 μmol CO₂ m^–2 s^–1 in Sitka alder and from 14.76 to 17.55 μmol CO₂ m^–2 s^–1 in paper birch. Based on canonical correlation analyses, populations from locations with colder winters and shorter (but not necessarily cooler) summers had higher maximum photosynthetic rates implying the existence of an inverse relationship between leaf longevity and photosynthetic capacity. Significant canonical variates based on climatic variables derived for the seed collection sites explained 58% and 41% of variation in the rate of photosynthesis in Sitka alder and paper birch, respectively. Since growing season length is reflected in date of frost hardiness development, an intrinsic relationship was found between photosynthetic capacity and the level of fall frost hardiness. The correlation was particularly strong for paper birch (r=–0.77) and less strong for Sitka alder (r=–0.60). Mean population biomass accumulation decreased with increased climate coldness. These patterns may be consequential for evaluation of the impact of climate change and extension of the growing season on plant communities. Received: 12 July 1999 / Accepted: 24 November 1999     

Carbohydrate Metabolism and Frost Hardiness in Pine and Spruce Seedlings Grown at Different Photoperiods and Thermoperiods

Large changes occur in carbohydrate contents of pine (Pinus silvestris L.) and spruce (Picea abies (L.) Karst.) seedlings cold-hardened by photoperiod or by combined photo- and thermo-period. The largest change is in sucrose content, which is almost doubled after six weeks short-day (6/18 h) treatment; and more than doubled (spruce) or more than tripled (pine), when also temperature is lowered (10/5°C). Development of frost hardiness is strongly correlated with the change in carbohydrate contents. At dehardening, the carbohydrate content decreases rapidly, especially in pine, and the raffinose formed during the rest period disappears within 2–4 weeks. Frost hardiness decreases in parallel. The content of soluble carbohydrates may thus play a role in frost hardiness, although it is not the only factor. Bud formation at cold acclimation is not directly correlated with the changes in carbohydrate content and hardiness.     

Nursery Culture Impacts Cold Hardiness in Longleaf Pine (Pinus palustris) Seedlings

Success in restoring longleaf pine ecosystems depends on outplanting high‐quality longleaf pine seedlings. One important and relatively understudied attribute of seedling quality is cold hardiness. A suite of trials was conducted to investigate the influence of common nursery cultural practices on longleaf pine cold hardiness. Cold hardiness was increased with higher rates of nitrogen, unaffected by copper coating containers, greater for foliage than for root‐collar tissue, and tended to increase with increases in container size.     

Extrinsic proteins of photosystem II: an intermediate member of PsbQ protein family in red algal PS II.

The oxygen-evolving photosystem II (PS II) complex of red algae contains four extrinsic proteins of 12 kDa, 20 kDa, 33 kDa and cyt c-550, among which the 20 kDa protein is unique in that it is not found in other organisms. We cloned the gene for the 20-kDa protein from a red alga Cyanidium caldarium. The gene consists of a leader sequence which can be divided into two parts: one for transfer across the plastid envelope and the other for transfer into thylakoid lumen, indicating that the gene is encoded by the nuclear genome. The sequence of the mature 20-kDa protein has low but significant homology with the extrinsic 17-kDa (PsbQ) protein of PS II from green algae Volvox Carteri and Chlamydomonas reinhardtii, as well as the PsbQ protein of higher plants and PsbQ-like protein from cyanobacteria. Cross-reconstitution experiments with combinations of the extrinsic proteins and PS IIs from the red alga Cy. caldarium and green alga Ch. reinhardtii showed that the extrinsic 20-kDa protein was functional in place of the green algal 17-kDa protein on binding to the green algal PS II and restoration of oxygen evolution. From these results, we conclude that the 20-kDa protein is the ancestral form of the extrinsic 17-kDa protein in green algal and higher plant PS IIs. This provides an important clue to the evolution of the oxygen-evolving complex from prokaryotic cyanobacteria to eukaryotic higher plants. The gene coding for the extrinsic 20-kDa protein was named psbQ' (prime).     

The relation between growth cessation and frost hardening in Scots pines of different origins

The cessation of shoot elongation, diameter growth and needle elongation were compared with the initiation of frost hardening of the stems and needles in an 8-year-old provenance trial of Scots pine (Pinus sylvestris L.) established in central Finland. The saplings were of six different origins ranging from Estonia to northern Finland, forming a latitudinal gradient of ca. 10°N. The frost hardiness of the stems of current-year shoots was assessed by electrical impedance analysis and that of current-year needles by electrolyte leakage and visual scoring of damage. Artificial freezing tests were used in the assessments. The pattern of growth cessation (shoot and needle elongation, diameter growth) tended to follow the latitude of origin, i.e. growth ceased in the northernmost provenance first and in the southernmost one last. Both stems and needles of the northern provenances hardened earlier than the southern ones, but the differences in hardiness disappeared as hardening progressed. Growth cessation and initial hardening to -15°C were clearly correlated at the provenance level, indicating that growth must cease prior to hardening, and that earlier cessation of growth predicts earlier frost hardening of stems and needles. No differences in frost hardiness of stems were found at the provenance level at the end of the growing period in August. At that time, the frost hardiness of needles of the northernmost provenance was higher than that of other origins. Within the provenance, the stems were less hardy than the needles.     

Genetic analysis of frost hardiness traits in tuber-bearingSolanum species

The inheritance of frost hardiness and cold acclimation potential traits was studied in three segregating populations derived from a cross betweenSolanum commersonii Dun. PI 243503 (cmm) andSolanum cardiophyllum Lindl., PI 184762 (cph), two parental genotypes with contrasting frost hardiness and cold acclimation potential. The levels of frost hardiness and cold acclimation potential were expressed as the LT₅₀, the temperature at which 50% of the cells in leaf discs were killed, as measured by the ion leakage method, following a controlled freeze test There was considerable variation in both frost hardiness and cold acclimation potential in all three segregating populations (F₁ F₁ xcmm, and F₁ xcph). Frost hardiness and cold acclimation potential were not correlated, suggesting that these two traits are under independent genetic control. The analysis of generation means indicated that the variation for both traits could be best explained by an additive-dominance model, with additive gene effects the most important Broad-sense heritability was 0.73 and 0.74 in the F₁ population, for frost hardiness and cold acclimation potential, respectively, and was 0.85 for either trait in the F₁ xcmm population, indicating that these two traits are highly inheritable. Our results suggest that it should be possible to incorporate the frost hardiness and cold acclimation traits from S.commersonii into cultivated potato species.