Purification, immunolocalization, cryoprotective, and antifreeze activity of PCA60: A dehydrin from peach (Prunus persica)

Dehydrins are glycine-rich, hydrophilic, heat-stable proteins and are generally induced in response to a wide array of environmental stresses. In previous research (Artlip et al. 1997, Plant Molecular Biology 33: 61–70), a full-length dehydrin gene, ppdhn1 , was isolated from peach, and its expression was associated with qualitative and quantitative differences in cold hardiness in sibling genotypes of evergreen and deciduous peach. Similar results were obtained for levels of the corresponding 60 kDa peach dehydrin protein (PCA60). The objective of the present study was to purify the PCA60, test the purified protein for cryoprotective and/or antifreeze activity, and to determine the cellular localization of PCA60 using immunomicroscopy. PCA60 was extracted from winter bark tissues of peach ( Prunus persica [L.] Batsch) and purified in a two-step process. Separation was based on free-solution isoelectric focusing followed by size exclusion. Purified PCA60, as well as crude protein extract, preserved the in vitro enzymatic activity of lactate dehydrogenase after several freeze-thaw cycles in liquid nitrogen. PCA also exhibited distinct antifreeze activity as evidenced by ice crystal morphology and thermal hysteresis. This is the first time antifreeze activity has been demonstrated for dehydrins. Immunomicroscopy, utilizing an affinity-purified, polyclonal antibody developed against a synthetic peptide of the lysine-rich consensus portion of dehydrins, indicated that PCA60 was freely distributed in the cytoplasm, plastids, and nucleus of bark cells and xylem parenchyma cells. Although the functional role of dehydrins remains speculative, the data support the hypothesis that it plays a role in preventing denaturation of proteins exposed to dehydrative stresses.     

Cold acclimation in genetically related (sibling) deciduous and evergreen peach (Prunus persica [L.] Batsch). II. A 60-kilodalton bark protein in cold-acclimated tissues of peach is heat stable and related to the dehydrin family of proteins.

In several plant species, certain cold-regulated proteins share unique properties. These proteins are (a) heat stable and (b) hydrophilic and are related to the Group 2 late embryogenesis abundant or dehydrin family of proteins. Our previous work with sibling deciduous and evergreen peach genotypes demonstrated a correlation between the level of accumulation of certain bark proteins and cold-acclimation potential of these tissues. Here we identify a 60-kD bark protein in peach (Prunus persica [L.] Batsch), PCA60 ("peach cold acclimation"), that is accumulated during cold acclimation and is heat stable. Immunological studies indicated that this protein is related to the dehydrin family of proteins and accumulates at much higher levels in the bark tissues of the deciduous genotype than in the evergreen. Amino acid composition indicated that the 60-kD protein has a compositional bias for glycine (24%), glutamic acid/glutamine (11.4%), aspartic acid/asparagine (10%), and threonine (9.6%), contains relatively low levels of aromatic amino acids (phenylalanine and tyrosine), and is rich in hydrophilic amino acids. A novel characteristic of the 60-kD cold-acclimation protein is the presence of a repeating nine-amino acid sequence. A five-amino acid stretch, which is included within this repeating motif, shares striking homology with other cold-regulated proteins and dehydrins.     

Chemical and Biophysical Changes in the Plasma Membrane during Cold Acclimation of Mulberry Bark Cells (Morus bombycis Koidz. cv Goroji)

The lipid and protein composition of the plasma membrane isolated from mulberry (Morus bombycis Koidz.) bark cells was analyzed throughout the cold acclimation period under natural and controlled environment conditions. There was a significant increase in phospholipids and unsaturation of their fatty acids during cold acclimation. The ratio of sterols to phospholipids decreased with hardiness, primarily due to the large increase in phospholipids. The fluidity of the plasma membrane, as determined by fluorescent polarization technique, increased with hardiness. Electrophoresis of plasma membrane proteins including glycoproteins revealed change in banding pattern during the early fall to winter period. Some of the protein changes could be related to growth cessation and defoliation. However, minor changes in proteins also occurred during the most active period of hardening. Changes in glycoproteins were coincident both with changes in growth stages and with the development of cold hardiness.     

Proteins in the roots of the perennial weeds chicory (Cichorium intybus L.) and dandelion (Taraxacum officinale Weber) are associated with overwintering

Roots are the overwintering structures of herbaceous perennial weeds growing in temperate climates. During the fall they accumulated reserves which are remobilized when growth resumes in the spring. An 18kDa (kilodalton) protein increases in both chicory and dandelion roots during the fall months. The proteins in both species are antigenically similar, and are recognized also by an antibody to a storage-protein deposited in Jerusalem artichoke (Helianthus tuberosus) tubers. In chicory, the protein is root-specific, but in dandelion it is detectable in the flowers, vestigial stem and the seed. Electrophoretic characterization of the 18-kDa protein shows that it is a single polypeptide, without subunits, with charge isomers of pI values close to pH 6.5. The major protein present in chicory and dandelion roots is unlike the vegetative storage proteins recently found in soybean or the storage proteins in the bark of trees.     

Resistance to Dutch Elm Disease Reduces Presence of Xylem Endophytic Fungi in Elms (Ulmus spp.)

Efforts to introduce pathogen resistance into landscape tree species by breeding may have unintended consequences for fungal diversity. To address this issue, we compared the frequency and diversity of endophytic fungi and defensive phenolic metabolites in elm (Ulmus spp.) trees with genotypes known to differ in resistance to Dutch elm disease. Our results indicate that resistant U. minor and U. pumila genotypes exhibit a lower frequency and diversity of fungal endophytes in the xylem than susceptible U. minor genotypes. However, resistant and susceptible genotypes showed a similar frequency and diversity of endophytes in the leaves and bark. The resistant and susceptible genotypes could be discriminated on the basis of the phenolic profile of the xylem, but not on basis of phenolics in the leaves or bark. As the Dutch elm disease pathogen develops within xylem tissues, the defensive chemistry of resistant elm genotypes thus appears to be one of the factors that may limit colonization by both the pathogen and endophytes. We discuss a potential trade-off between the benefits of breeding resistance into tree species, versus concomitant losses of fungal endophytes and the ecosystem services they provide.     

Gradients and dynamics of inner bark and needle osmotic potentials in Scots pine (Pinus sylvestris L.) and Norway spruce (Picea abies L. Karst)

Preconditions of phloem transport in conifers are relatively unknown. We studied the variation of needle and inner bark axial osmotic gradients and xylem water potential in Scots pine and Norway spruce by measuring needle and inner bark osmolality in saplings and mature trees over several periods within a growing season. The needle and inner bark osmolality was strongly related to xylem water potential in all studied trees. Sugar concentrations were measured in Scots pine, and they had similar dynamics to inner bark osmolality. The sucrose quantity remained fairly constant over time and position, whereas the other sugars exhibited a larger change with time and position. A small osmotic gradient existed from branch to stem base under pre‐dawn conditions, and the osmotic gradient between upper stem and stem base was close to zero. The turgor in branches was significantly driven by xylem water potential, and the turgor loss point in branches was relatively close to daily minimum needle water potentials typically reported for Scots pine. Our results imply that xylem water potential considerably impacts the turgor pressure gradient driving phloem transport and that gravitation has a relatively large role in phloem transport in the stems of mature Scots pine trees.     

Seasonal and year-to-year differences in food selection by beavers

Summary Selection of trees for food by a colony of beavers in central Massachusetts was studied from September 1972 through April 1974. The beavers exhibited both seasonal and year-to-year differences in preference for certain genera. Pine was selected against during fall but not spring, and there was a switch in preference from birch during fall 1972 to oak and witch hazel in fall 1973. These differences may partly reflect greater seasonal stability in concentrations of stored nutrients in coniferous tree bark than in deciduous tree bark, and greater year-to-year stability in bark concentrations of stored nutrients in non-mast-seeding species than in mast-seeding species (1972 was a mast year for oaks in central Massachusetts).     

Seasonal Changes of the Metabolites in the Leaves, Bark and Xylem Tissues of Olive Tree (Olea europaea. L) II. Carbohydrates

Seasonal changes of starch and soluble carbohydrates in leaves,bark and xylem tissues of olive tree were examined during acomplete annual cycle. Leaf starch and soluble carbohydrateswere detected at high levels during the spring and autumn metabolicallyactive periods. The low level of leaf starch in combinationwith the drastic reduction of soluble carbohydrates and mannitol,defined the summer period of the low metabolic state of thetree. The low leaf starch level in conjunction with the risensoluble carbohydrate levels in leaves in winter were associatedwith cold acclimation processes. The bark and xylem tissueswere performing as starch deposition sites, and differencesin the extent of starch accumulation in these tissues were detectedduring the seasons. The starch fluctuations in bark and xylemwere discussed in relation to the translocation of metabolitesand other physiological processes. Mannitol, the most abundantleaf carbohydrate, was examined in relation to the reducingsugars exported to the bark. The bark mannitol was examinedin conjunction with the sucrose, glucose and starch levels inthe maturing bark tissues and was correlated to the low wintertemperatures. During the winter there was a drastic reductionof mannitol circulation from the bark to xylem. Olive tree, Olea europaea, L, carbohydrates     

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.     

Periodicity of cambial activity in Abies balsamea. I. Effects of temperature and photoperiod on cambial dormancy and frost hardiness

The relationship between from hardiness and growth potential, and their dependence on temperature and photoperiod, was investigated in the one-year-old cambium of balsam fir [Abies balsamea (L.) Mill.]. Six-year-old trees were exposed for 9 weeks to either the natural environment or one of 4 controlled environments in the fall (18 September-18 November), spring (12 April–14 June) and summer (19 July – 19 September). The 4 controlled environments were (1) WS, warm temperature (24/20°C in day/night) + short day (8 h). (2) WL. warm temperature (24/20°C) + long day (8 h + 1 h night break), (3) CS. cold temperature (9/5°C) + short day (8 h) and (4) CL, cold temperature (9/5°C) + long day (8 h + 1 h night break). At the beginning and end of each exposure, cambial activity was measured by recording the number of xylem, cambium and phloem cells, frost hardiness was estimated from the cambium's ability to survive freezing to –40°C, and cambial growth potential was deduced from the duration of the cell cycle and the production of xylem, cambium and phloem cells in cuttings cultured for 4 weeks with exogenous indole-3-acetic acid (IAA) under environmental conditions favourable for cambial activity. In the natural environment, frost hardening began in September and was completed in November, while dehardening occurred when the cambium reactivated. CL, CS, and to a lesser extent WS, promoted hardening in the summer and fall, but did not prevent dehardening in the spring. The cambial growth potential in the natural environment declined from a maximum in April to a low level in June, reached a minimum in September, then increased to a high level in November. This potential was promoted by CL and CS on all dates by WL in the summer and fall. The ratio of xylem to phloem induced by IAA treatment was greatest in June and least in September in cuttings from trees exposed to the natural environment, and was increased by CL and CS in the fall. The cambium in intact branches of trees protected from chilling during the fall and winter resumed cell cycling after less than 9 weeks of dormancy, but produced mostly or only phloem in the subsequent growing period. It is concluded that the frost hardiness of the cambium, the IAA-induced cycling of cambial cells, and IAA-induced xylem to phloem ratio vary independently with season, temperature and photoperiod, and that the periodicity of these processes is regulated endogenously.     

Accumulation of Free Proline in Citrus Leaves during Cold Hardening of Young Trees in Controlled Temperature Regimes

Free proline increased in leaves of orange (Citrus sinensis [L.] Osb. cv. Valencia) and grapefruit (Citrus paradisi Macfad. cv. Star Ruby) trees on a wide range of citrus rootstocks during cold hardening. Increases in sugars accompanied proline accumulation. During cold hardening, the rate of proline accumulation was greater in old than in young leaves. In leaves of grapefruit trees kept in the dark during cold hardening, neither proline nor sugars increased and the degree of cold hardiness was less than in trees exposed to light. Like sugar accumulations, proline accumulation does not reflect specific degrees of cold hardiness in citrus cultivars.     

Seasonal monitoring of Drosophila suzukii (Diptera: Drosophilidae) in a mixed fruit production system

Drosophila suzukii Matsumura (Diptera: Drosophilidae), an invasive pest native to Southeast Asia, is now reported throughout North America and Europe. We used traps baited with apple cider vinegar to monitor D. suzukii adult presence in multiple crops and associated fruiting plants at the Wolfskill USDA Germplasm Repository in Winters, CA, USA from 2011 to 2013. Traps were placed in small (~ 160 m × 40 m on average) almond, apricot, cherry, fig, grape, mulberry, peach, persimmon, plum, and pomegranate deciduous fruit orchard blocks as well as a citrus block and evergreen trees located near a house at the repository. D. suzukii was present in all blocks with the greatest monthly deciduous fruit captures in the cherry and fig blocks. Few D. suzukii were captured in almond, apricot, pomegranate and grape blocks. Deciduous fruit blocks had two distinct periods of trap capture: spring through midsummer and again in fall. Most deciduous fruit blocks had low trap captures during the hottest summer months (August to September) and the coldest winter months (December to April). However, from late December through mid-January, high trap captures were associated with the citrus and house sites. This study provides seasonal trapping data of D. suzukii adults in an unsprayed multi-crop mosaic, and may serve as a model of adult capture patterns across smaller mixed-crop commercial orchards and associated urban landscapes.     

Seasonal changes in stem diameter and leaf development in a tropical montane forest

Abstract. Seasonal patterns of stem diameter changes in evergreen and deciduous species of a tropical montane forest in the Central Himalayas (300–2250 m a.s.l.) were investigated in relation to leaf development. Ca. 75 % of the annual rainfall in this region occurs in a short period, from mid-June to mid-September and the remaining months are dry. It was assumed that changes in stem diameter are correlated with changes in water stress. Each evergreen species could be characterized by leaf longevity of about one year; each species showed pronounced summer leaf drop and simultaneous new leaf formation. Winter stem shrinkage was more pronounced in deciduous species than in evergreen ones. The deciduous species also showed a greater proportional loss of leaf mass (before abscission) than the evergreen species. Winter leaf fall in deciduous species was related to the pronounced stem shrinkage. The leaf fall enabled these species to control further water loss. Being more resistant to desiccation, the evergreen species retained their leaves throughout the winter but showed gradual loss of leaf mass, presumably in order to control water loss. In all species, leaf expansion was completed before the onset of the rainy season, when water stress was high. This strategy has definite advantages in a climate with a monsoon pattern of rainfall. Evergreen species, showing pronounced leaf drop in summer, have advantages over deciduous species; hence their preponderance in the region.     

Overwintering sites and winter mortality of Euseius finlandicus (Acari: Phytoseiidae) in a peach orchard in northern Greece

In peach orchards of northern Greece Euseius finlandicus Oudemans overwinters in various sites on the trees, usually in groups of 5–15 females. Overwintering females were mostly found in bark crevices near the hibernation cocoons of the peach moth Adoxophyes orana (Fischer von Rosslerstamm), and also in small crevices of the bark and in pedicels left after fruit harvesting. Empty scales of dead diaspidids and dead coccids, cocoons of lacewings and mummies of parasitized aphids sporadically found on trees, were less common overwintering sites for females of the mite. In two successive years, mortality of overwintering females was very low, approximately 4 and 4.5% which could be due to the mild climate of northern Greece and the high cold tolerance of the mite. This revised version was published online in July 2006 with corrections to the Cover Date.     

梅花(Prunus mume Sieb et Zucc)远缘杂种及其亲本的差热分析与冻害关系的研究

用差热分析(Differential thermal analysis: DTA)研究了山桃(Prunus davidiana)、杏(P. armeniaca)、青岛“粉红梅”(P.mume cv.'Fenhong Mei,)、“小绿萼”(P.mume cv.'Small Green Calyx')及其种问杂种“小绿萼”梅×山桃、青岛“粉红梅”×杏和杏×青岛“粉红梅”的低温放热(Low temperature exotherm)与冻害关系,以及皮部和木质部的冰冻类型(Freezing pattern)。在差热分析中,观察到亲本和杂种的木质部都有二次放热现象。低温放热后,引起木质部和髓射线薄壁细胞死亡,原生质膜透性急剧增加。在杂种与亲本之间,存在着明显的差异。分离的皮部却只出现一次高温放热(High temperature exotherm)。高温放热是与冻害无关的。文末讨论了梅花及其杂种在北京越冬的主要障碍及有关栽培措施。     

Frequency of inversions affects senescence phenology of Acer pseudoplatanus and Fagus sylvatica

In mountainous regions, inversion situations with cold-air pools in the valleys occur frequently, especially in fall and winter. With the accumulation of inversion days, trees in lower elevations experience lower temperature sums than those in middle elevations. In a two-year observational study, deciduous trees, such as Acer pseudoplatanus and Fagus sylvatica, on altitudinal transects responded in their fall leaf senescence phenology. Phenological phases were advanced and senescence duration was shortened by the cold temperatures in the valley. This effect was more distinct for late phases than for early phases since they experienced more inversion days. The higher the inversion frequency, the stronger the signal was. Acer pseudoplatanus proved to be more sensitive to cold temperatures compared to Fagus sylvatica. We conclude that cold-air pools have a considerable impact on the vegetation period of deciduous trees. Considering this effect, trees in the mid hillside slopes gain advantages compared to lower elevations. Our findings will help to improve knowledge about ecological drivers and responses in mountainous forest ecosystems.     

Seasonal occurrence and distribution of myxomycetes on different types of leaf litter in a warm temperate forest of western Japan

The seasonal occurrence and distribution of myxomycetes on different types of newly defoliated leaf litter were examined in a secondary forest in a warm temperate region of western Japan. The two types of leaf litter (deciduous trees, Prunus verecunda and Quercus variabilis, and evergreen trees, Q. glauca and Cinnamomum camphora) were incubated in trays on the forest floor. A total of 45 myxomycete species were recorded from 3021 collected samples that occurred at the July peak during the warmest and humidest season from April to November. The occurrence of species was significantly related to the changes in mean temperature and minimum temperature on both leaf types under humid conditions. Myxomycete assemblages were divided into three seasonal phases. Most of the species occurred in June–September, while a few species demonstrated characteristic distributions; i.e., Didymium melanospermum appeared in April– May and Diderma umbilicatum appeared in October– November. The respective leaf types supported the reproduction of myxomycetes with high species richness and diversity, with 34 species and H’ = 2.59 on deciduous trees and similarly 30 species and H’ = 2.49 on evergreen trees. Several species, however, exhibited a preference for either the deciduous tree or evergreen tree leaves. Thus, a mixed forest that defoliates during different two seasons yields a greater species diversity of myxomycete assemblage.     

Comparative freezing patterns of bark and xylem of “Siberian C” and “Redhaven” peach twigs

An electrophoretic mobility technique was used to study freezing patterns in excised peach twigs. Moisture content was the only qualitative difference in initial freezing patterns of similar tissues of Redhaven and Siberian C. Siberian C contained up to 18% less moisture than Redhaven. Major differences in the shape of the transition pattern were detected between bark and xylem. Even though bark tissues had twice the water content of xylem, the bark exhibited equilibrium freezing while the xylem underwent nonequilibrium freezing. Bark water must be intimately associated with the living protoplast, while xylem water is less closely associated with cellular components. Comparison of bark and xylem freezing curves with sucrose and cellulose model systems suggested that bark freezing was similar to the sucrose model while xylem freezing was similar to the cellulose model.