环境胁迫和抗坏血酸的氧化还原状态

为了解环境胁迫对植物体中抗坏血酸含量及氧化还原状态的影响，以不同强度的冰冻和干旱两种胁迫为例，研究了它们对沈阳几种针叶树离体叶抗坏血酸、脱氢抗坏血酸含量以及抗坏血酸-谷胱甘肽循环中4种酶活性的影响。结果表明，两种胁迫达到一定强度后，都能使还原态抗坏血酸含量下降而使脱氢抗坏血酸含量上升。冰冻使抗坏血酸过氧化酶和单脱氢抗坏血酸还原酶活性下降。轻度失水使这两种酶活性上升，失水加重后转而趋于下降。脱氢抗坏血酸还原酶和谷胱甘肽还原酶活性对两种胁迫反应均不如前两种酶敏感。结合以前的研究结果，认为这一H2O2清除系统在导致驯化(acclimation)的轻度胁迫作用下可以得到加强，而当胁迫强度过大时则其清除能力下降并使组织受到伤害。文中还报告了沈阳几种针叶树抗寒性和针叶中抗坏血酸含量及上述4种酶活性之间的相关关系。     

环境胁迫和抗坏血酸的氧化还原状态

为了解环境胁迫对植物体中抗坏血酸含量及氧化还原状态的影响,以不同强度的冰冻和干旱两种胁迫为例,研究了它们对沈阳几种针叶树离体叶抗坏血酸、脱氢抗坏血酸含量以及抗坏血酸-谷胱甘肽循环中4种酶活性的影响.结果表明,两种胁迫达到一定强度后,都能使还原态抗坏血酸含量下降而使脱氢抗坏血酸含量上升.冰冻使抗坏血酸过氧化酶和单脱氢抗坏血酸还原酶活性下降.轻度失水使这两种酶活性上升,失水加重后转而趋于下降.脱氢抗坏血酸还原酶和谷胱甘肽还原酶活性对两种胁迫反应均不如前两种酶敏感.结合以前的研究结果,认为这一H2O2清除系统在导致驯化(acclimation)的轻度胁迫作用下可以得到加强,而当胁迫强度过大时则其清除能力下降并使组织受到伤害.文中还报告了沈阳几种针叶树抗寒性和针叶中抗坏血酸含量及上述4种酶活性之间的相关关系.     

东北地区城市针叶树冬季滞尘效应研究

对不同种针叶树同一降尘条件及同种针叶树不同降尘条件的滞尘能力进行研究,结果表明,针叶树在东北的冬季有很强的滞尘作用,不同的针叶树滞尘能力排序为沙松冷杉>沙地云杉>红皮云杉>东北红豆杉>白皮松>华山松>油松,不同针叶树叶表面结构不同,滞尘量较小的白皮松、华山松和油松表面平滑,细胞与气孔排列整齐,而滞尘量较大的红皮云杉、沙松冷杉、东北红豆杉的针叶表皮平滑程度较差,细胞与气孔排列较前3种植物差,在红皮云杉叶表面上有大小不等的瘤状物,不同针叶树叶断面形状与滞尘量相关,白皮松和油松叶片的上表面呈弧形,不易附着灰尘;华山松叶片呈三棱形,上表面较窄,附着灰尘的量较小;两种云杉的叶片呈四棱形,上表面较3种松属植物的叶片要宽与平展,因此相对滞尘量要大;沙松冷杉和东北红豆杉的叶断面形状都较扁、平,这种断面正是构成其滞尘量大于3种松属和两种云杉属植物的主要因素。     

Reactive species, antioxidants and cold tolerance during deacclimation of Picea abies populations

Seasonal changes in levels of reactive oxygen species (ROS), low-molecular weight antioxidants and activities of antioxidant enzymes were analyzed in relation to the freezing tolerance of 1-year-old needles from four populations of Norway spruce. Throughout the study period (from January until May), no significant changes were observed in the superoxide anion radical (O ₂ ^·? ) or hydrogen peroxide (H₂O₂) levels in the needles. By contrast, a marked reduction was observed in concentrations of low-molecular weight antioxidants, including flavonoids (FL), ascorbic acid (AsA) and slight glutathione (GSH), during deacclimation. The activities of superoxide dismutase (SOD) (EC. 1.15.1.1.) and guaiacol peroxidase (PO) (EC. 1.11.1.7.) also decreased significantly. The activity of catalase (CAT) (EC. 1.11.1.6.) did not change significantly. Levels of low-molecular weight antioxidants (AsA, FL and GSH) and SOD activity were correlated significantly with freezing tolerance in the studied populations. The reactions were similar in all populations. This suggests that the response of the antioxidant system depends more strongly on climatic conditions than on population origin. The ability of spruce trees to cope with active oxygen species is discussed as an aspect of defense and a factor associated with freezing tolerance.     

Changes in chlorophyll and carotenoid contents in radish (Raphanus sativus) cotyledons show different time courses during senescence

Changes of chlorophylls and carotenoids from green to yellow cotyledons of the radish ( Raphanus sativus ) were simultaneously and systematically analysed by high-performance liquid chromatography (HPLC) with photodiode array detection. Twenty-one components, seven chlorophylls and 14 carotenoids, were detectable. Seven chlorophylls and five carotenoids were identified from the results of HPLC analyses. Most chlorophyll species degraded during senescence, whereas carotenoids showed different behaviour in their metabolism depending on pigment species. For instance, during senescence, the contents of lutein and violaxanthin changed only slightly, β-carotene in 5-day-senescent cotyledons became 2.7 times higher than non-senescent leaves. Carotenoids of radish cotyledons were classified into three groups by their changes in concentration during senescence (increased, degraded and constant) and their roles discussed.     

Cold-regulated proteins with potent antifreeze and cryoprotective activities in spruces (Picea spp.)

Antifreeze proteins (AFPs) were obtained from intercellular spaces of spruce needles Picea abies (L.) Karst. and Picea pungens Engelm. by vacuum infiltration with ascorbic acid, followed by centrifugation to recover the infiltrate. As shown by sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE), apoplastic proteins are accumulated in these spruce species as a group of 5–9 polypeptide bands. These proteins have a molecular mass of 7–80 kDa. The spruce AFPs have the ability to modify the growth of ice and thermal hysteresis, TH, caused by these AFPs was close to 2.0 °C at a concentration of 400 μg/ml. The antifreeze activity of proteins from these winter-hardy coniferous species showed a positive correlation with the concentration of proteins after cold acclimation of needle tissues. Apoplastic proteins from winter spruce needles exhibited antifreeze activity, whereas no such activity was observed in extracts from summer needles. When we examined the possible role of spruce AFPs in cryoprotection, we found that lactate dehydrogenase, LDH, activity was higher after freezing in the presence of AFPs compared with bovine serum albumin. Amino-terminal sequence comparisons indicated that a 27-kDa protein from both P. abies and P. pungens was similar to some pathogenesis-related proteins namely chitinases, also from conifer species. These results show that spruces produce AFPs that are secreted into the apoplast of needles. The accumulation of AFPs in extracellular spaces caused by seasonal cold acclimation during winter indicates that these proteins may play a role in the acquisition of freezing tolerance of needle cells in coniferous species.     

Recovery kinetics of photochemical efficiency in winter stressed conifers: the effects of growth light environment,extent of the season and species

Evergreens undergo reductions in maximal photochemical efficiency (F_v/F_m) during winter due to increases in sustained thermal energy dissipation. Upon removing winter stressed leaves to room temperature and low light, F_v/F_m recovers and can include both a rapid and a slow phase. The goal of this study was to determine whether the rapid component to recovery exists in winter stressed conifers at any point during the season in a seasonally extreme environment. Additional goals were to compare the effects of species, growth light environment and the extent of the winter season on recovery kinetics in conifers. Four species (sun and shade needle) were monitored during the winter of 2007/2008: eastern white pine (Pinus strobus), balsam fir (Abies balsamea), Taxus cuspidata and white spruce (Picea glauca). F_v/F_m was measured in the field, and then monitored indoors at room temperature and low light for 6 days. The results showed that all species showed a rapid component to recovery in early winter that disappeared later in the season in sun needles but was present in shade needles on most days monitored during winter. There were differences among species in the recovery kinetics across the season, with pine recovering the most slowly and spruce the most quickly. The results suggest an important role for the rapidly reversible form of energy dissipation in early winter, as well as important differences between species in their rate of recovery in late winter/early spring which may have implications for spring onset of photosynthesis.     

Photosynthesis and Photoprotection in Overwintering Plants

Abstract: Seasonal differences in the capacity of photosynthetic electron transport, leaf pigment composition, xanthophyll cycle characteristics and chlorophyll fluorescence emission were investigated in two biennial mesophytes ( Malva neglecta and Verbascum thapsus ) that grow in full sunlight, and in leaves/needles of sun and shade populations of several broad-leafed evergreens and conifers (Vinca minor, Euonymus kiautschovicus, Mahonia repens, Pseudotsuga menziesii [Douglas fir], and Pinus ponderosa). Both mesophytic species maintained or upregulated photosynthetic capacity in the winter and exhibited no upregulation of photoprotection. In contrast, photosynthetic capacity was downregulated in sun leaves/needles of V. minor, Douglas fir, and Ponderosa pine, and even in shade needles of Douglas fir. Interestingly, photosynthetic capacity was upregulated during the winter in shade leaves/needles of V. minor, Ponderosa pine and Euonymus kiautschovicus. Nocturnal retention of zeaxanthin and antheraxanthin, and their sustained engagement in a state primed for energy dissipation, were observed largely in the leaves/needles of sun-exposed evergreen species during winter. Factors that may contribute to these differing responses to winter stress, including chloroplast redox state, the relative levels of source and sink activity at the whole plant level, and apoplastic versus symplastic phloem loading, are discussed.     

长白山红松臭冷杉光谱反射随海拔的变化

为了研究红松、臭冷杉光谱反射率随海拔梯度的变化,本研究使用光谱仪测量了长白山两种主要的针叶树种红松、臭冷杉不同海拔针叶的光谱反射率,分别对比了两个树种不同海拔间光谱反射率、光谱指数的差异,这些特征可以反映植物叶片色素组成和色素含量等信息。结果表明：两种针叶树在不同海拔的光谱反射率变化明显,光谱指数也有显著差异,证明相关生理状况发生明显变化。对比各海拔光谱反射率发现,1279m处红松、臭冷杉针叶的色素总含量较高,叶绿素指数反映出748m处红松、臭冷杉针叶叶绿素含量显著高于1040m和1279m处,Car：Chl指数的差异说明红松、臭冷杉均在748处受到较弱的胁迫,臭冷杉在1040m受到较强的胁迫,对比各波段反射率随海拔的变化,发现红松、臭冷杉针叶色素含量都随海拔变化有明显的变化,而且臭冷杉的叶片水分含量、叶片结构也可能容易受到海拔影响。     

远紫外辐射对紫杉幼苗针叶膜脂过氧化及内源保护系统的影响

实验室条件下用远紫外线(UV-BC)光源照射紫杉幼苗,随照射时间延长,针叶的离子渗出率、膜脂过氧化水平、组织自动氧化速率及H₂O₂含量显著增加,可溶性蛋白、抗坏血酸、类胡萝卜素和叶绿素含量下降,叶绿体光系统II电子传递活性显著下降,外源活性氧清除剂苯甲酸钠和抗坏血酸对针叶膜脂过氧化有抑制作用;甲基紫精和DDC对针叶膜脂过氧化有促进效果,远紫外线引起的紫杉伤害可能和针叶树的越冬光氧化伤害有类似之处.紫杉苗对紫外辐射的抗性远高于一般农作物.     

SEASONALITY AND THERMAL ACCLIMATION OF REACTIVE OXYGEN METABOLISM IN FUCUS VESICULOSUS (PHAEOPHYCEAE)

The intertidal brown macroalga Fucus vesiculosus L. acclimates its defense against reactive oxygen in response to both (1) growth at different temperatures in laboratory culture and (2) seasonal changes in environmental conditions. Fucus vesiculosus was grown in seawater at 0° C, 20° C, and at 0° C with a 3-h daily emersion at −10° C. Algae grown at low temperature, both with and without freezing, produced less reactive oxygen after severe freezing stress than those grown at 20° C. These differences were correlated with growth temperature-induced changes in activities of superoxide dismutase (SOD), glutathione reductase, and ascorbate peroxidase. The contents of tocopherols increased with increased cultivation temperature, whereas the activity of catalase and the content of glutathione and ascorbate did not change. Growth at 0° C increased the resistance of photosynthesis to freezing and reduced photoinhibition in high light at 5° C; the latter effect was further increased in algae subject to daily freezing. These data suggest that elevated activity of reactive oxygen scavenging enzymes, especially SOD, increases the resistance to photoinhibition, at least at low temperature, as well as being important for freezing tolerance. Seasonal changes in reactive oxygen metabolism showed a similar pattern to those elicited by temperature in laboratory culture. Summer samples had lower activities of most reactive oxygen scavenging enzymes than algae collected in autumn and winter when water temperatures were lower. In contrast to the laboratory experiments, ascorbate content did change and was lower during the winter than summer, whereas the content of glutathione was not influenced by season. Overall, the data not only indicate that temperature plays an important role in the regulation of stress tolerance and reactive oxygen metabolism but also suggest that other factors are also involved.     

Freezing tolerance/intolerance and cryoprotectant synthesis in terrestrially overwintering anurans in the Great Plains,USA

Mechanistic bases for freezing tolerance in anurans have been well-studied only in wood frogs, Rana sylvatica, so comprehensive explanations for the mechanisms and evolution of freezing tolerance in anurans are lacking. We measured crystallization temperatures, freezing tolerance/intolerance, and tissue glucose and glycogen phosphorylase activities in frozen and unfrozen winter-acclimated Pseudacris triseriata, Bufo cognatus and B. woodhousei. Freezing occurred at higher subzero temperatures on wet substrate than on dry substrate in all species, indicating susceptibility to inoculative freezing. P. triseriata was freeze-tolerant, but survival was dependent on the level of supercooling prior to freezing. All Bufo were freezing intolerant, regardless of crystallization temperature. Glucose was significantly elevated by freezing in both liver (35-fold) and leg muscle (22-fold) in winter P. triseriata, but only liver glucose was significantly elevated in B. cognatus. However, freezing did not alter glycogen phosphorylase activity in either species. Liver phosphorylase activity was significantly higher in P. triseriata than in B. cognatus, suggesting that capacity for mobilizing glucose from liver glycogen is associated with freezing tolerance. Summer measurements of liver phosphorylase activity, however, did not differ between species. Thus, P. triseriata, but not B. cognatus, exhibited winter increment of liver phosphorylase activity that is correlated with the development of freezing tolerance.Abbreviation T _b body temperature - T _c crystallization temperature - T _r rebound temperature - T _eq equilibrium temperature     

On the mechanisms of frost injury and frost hardening of spruce chloroplasts

Hill reaction and noncyclic photophosphorylation of isolated class C chloroplasts of spruce (Picea abies (L.) Karst.), as well as ¹⁴CO₂ fixation by whole needles at constant laboratory conditions proceeded at high rates during spring and early summer, declined during late summer and autumn by about 60%, remained at this level during winter, and recovered quickly in early spring. During summer, the whole needles proved to be frost labile, since after exposure to-20°C and careful thawing, fast chlorophyll degradation occurred. In addition, only photosynthetically inactive chloroplasts could be isolated from those precooled needles. On the contrary, during winter the photochemical activities of plastids from freshly harvested needles did not differ from those of artificially frozen-thawed needles. When isolated spruce chloroplasts were exposed to the same subfreezing temperatures as the whole needles, no influence of freezing on the photochemical activities was observed, irrespective of whether the plastids were isolated from frost sensitive or frost hardened needles. It is concluded that frost damage to spruce chloroplasts is due to an attack of membrane toxic compounds or lytic enzymes which were liberated upon freezing from more labile compartments. Frost hardening of the chloroplasts, as determined by the stability of chlorophyll after exposure of the needles to low temperatures, as well as by the isolation of photosynthetically active chloroplasts from such precooled needles, appeared to depend at least on 2 processes: (i) an alteration of the composition of the photosynthetically active membranes and (ii) and additional stabilization of these membranes by protecting substances. The first process was indicated by a large increase (decrease) of the capability of isolated chloroplasts for PMS-mediated photophosphorylation which accompanied natural or artificial frost hardening (dehardening). Production of cryoprotecting compounds was suggested by a significant higher stability against NaCl observed with class C chloroplasts isolated from frost hardened needles as compared to that of plastids from frost labile material. The decrease of the capability for both, the ferricyanide dependent photoreactions of the plastids and the CO₂ fixation by whole needles, which was observed during the frost hardening phase, cannot be due to freezing injuries; it rather appears to be a consequence of the frost hardening process.     

Adaptive changes in pigment complex of <Emphasis Type="Italic">Pinus sylvestris</Emphasis> needles upon cold acclimation

We studied seasonal changes in the content and ratio between photosynthetic pigments in one-yearold needles of Scotch pine (Pinus sylvestris L.) growing in Central Yakutia. Maximum accumulation of chlorophylls in developed young needles occurred in July when light and temperature conditions were favorable. In this period, the needles were notable for a relatively high level of β-carotene and neoxanthin and a reduced content of lutein and the pigments of violaxanthin cycle (VXC). In the course of autumn hardening, the content of chlorophylls decreased two times. Total content of carotenoids remained the same, but pigment composition considerably changed when plants progressed from a vegetating to frost-resistant state. We revealed time and temperature ranges of variation for individual carotenoids. In the beginning of hardening at reduced and low abovezero temperatures, the content of β-carotene in the needles decreased, the pigment-protein complexes (PPC) became enriched in lutein, the pigment pool of VXC gradually increased, and the content of neoxanthin transiently rose. When average daily air temperature further decreased to near- zero values, the content of zeaxanthin sharply rose. In winter, high levels of lutein and zeaxanthin were maintained. Main changes in pigment complex of the needles of P. sylvestris were completed before the coming of steady below-zero temperatures. The obtained data suggested that, upon seasonal decrease in temperature in early stages of hardening, a decrease in the level of chlorophyll promotes a reduction in the quantity of absorbed radiant energy. Apparently, this is accompanied by activation of the role of lutein and neoxanthin that perform specific photoprotective functions in antenna PPC associated with a gradual decrease in plants’ ability to quench singlet energy of excited chlorophyll. Accumulation of zeaxanthin as a result of inhibition of back reaction of epoxidation at near-zero temperatures creates necessary prerequisites for turning on the mechanisms of steady dissipation of absorbed light energy, which do not depend on transmembrane proton gradient of thylakoids. At the same time, zeaxanthin can perform antioxidant functions both in PPC and in the lipid phase of thylakoid membranes. The obtained data point to an adaptive nature of the observed reactions and a specific role of individual pigments in structural and functional reorganization of photosynthetic machinery in the course of development of frost-resistance in the needles.     

Cold acclimation induces freezing tolerance via antioxidative enzymes, proline metabolism and gene expression changes in two chrysanthemum species

Cold acclimation is necessary for chrysanthemum to achieve its genetically determined maximum freezing tolerance, but the underlying physiological and molecular mechanisms are unclear. The aim of this study was to discover whether changes in antioxidative enzymes, proline metabolism and frost-related gene expression induced by cold acclimation are related to freezing tolerance. Our results showed that the semi-lethal temperature (LT₅₀) decreased from ?7.3 to ?23.5 °C in Chrysanthemum dichrum and ?2.1 to ?7.1 °C in Chrysanthemum makinoi, respectively, after cold acclimation for 21 days. The activities of SOD, CAT and APX showed a rapid and transient increase in the two chrysanthemum species after 1 day of cold acclimation, followed by a gradual increase during the subsequent days and then stabilization. qRT-PCR analysis showed that the expression levels of some isozyme genes (Mn SOD, CAT and APX) were upregulated, which was consistent with the SOD, CAT and APX activities, while others remained relatively constant (Fe SOD and Cu/Zn SOD). P5CS and PDH expression were increased under cold acclimation and the level of P5CS presented similar trends as proline content, indicating proline accumulation was via P5CS and PDH cooperation. Cold acclimation also promoted DREB, COR413 and CSD gene expression. The activities of three enzymes and gene expression were higher in C. dichrum than in C. makinoi after cold acclimation. Our data suggested that cold-inducible freezing-tolerance could be attributed to higher activity of antioxidant enzymes, and increased proline content and frost-related gene expression during different periods.     

Genetic and environmental contributions to the winter hardiness of conifers

Summary

Both genetic and environmental components are involved in the processes of winter hardening and dehardening which permit needles of conifers like Norway spruce to survive very low temperatures (< - 30°C) over winter and to recover fully in time for the following and subsequent seasons. One of the major environmental effects of increasing concentrations of atmospheric ozone (O₃) in recent summers has been to affect detrimentally the ability of conifer needles to harden properly and at the correct rate the following autumn. Part of the mechanism by which this occurs in the cytoplasm of needle cells has been traced to detrimental effects on both the Δ¹² fatty acid desaturase and the unusual Δ⁵ desaturase which appears to be part of the low temperature survival mechanism of conifers. The genetic component of winter hardening also involves needle lipids. Studies of lipids in the needles of Norway spruce trees of different provenances growing for many years in the UK have shown that they change during winter hardening as though they were still on trees in the original sites throughout Europe from which the seeds of these trees were initially collected.     

Efficiency of the photosynthetic apparatus in developing needles of Norway spruce (Picea abies L. Karst.)

The photosynthetic performance of developing spruce (Picea abies L. Karst.) needles was investigated. As revealed by previous reports, the biosynthesis of chlorophylls and carotenoids was not following the characteristic chloroplast ultrastructure building up during needle elongation process. The aim of our study was to investigate photosynthetic capability (evaluated by oxygen evolution and chlorophyll a fluorescence kinetics measurements), the dynamics of chloroplast pigments biosynthesis and the expression of major photosynthetic proteins as well as to find out possible correlation between components of issue. Low amounts of chlorophylls and carotenoids, LHC II and Rubisco LSU were detected in the embryonic shoot of vegetative buds. Although PS II was functional, oxygen production was not sufficient to compensate for respiration in the same developmental stage. The light compensation point of respiration was successively lowered during the needle elongation. Nevertheless the significant increase in photosynthetic pigments as well as the high level of expression of LHC II and Rubisco LSU proteins was observed in the later stages of needle development. Our results suggest that, besides light, some other environmental factors could be critical for producing fully functional chloroplasts in rapidly growing young needles.     

Role of chlorophylls and carotenoids in seed tolerance to abiotic stressors

It is found that chlorophylls are not fully destructed during seed ripening and can be detected in appreciable quantities in physiologically mature seeds. The elevated content of residual chlorophylls reduces seed tolerance to abiotic stresses. The seed carotenoids were represented mainly by lutein and, in much smaller quantities, by β-carotene. Carotenoids were found to accumulate in seeds during accelerated aging and during seed germination at high temperatures. The ratio of carotenoid to chlorophyll content (Car/Chl) is proposed to be a measure of seed tolerance to stress factors. The seeds with elevated Car/Chl ratio were characterized by higher tolerance to stress treatments. It is supposed that the presence of chlorophylls in seeds enhances oxidative stress induced by abiotic stress factors. Carotenoids are considered as antioxidants protecting the seeds against oxidative stress.     

Farinose freezing （Primula flavonoids,are associated with high tolerance in fairy primrose malacoides） plants

The deposition of surface （farinose） flavonoids on aerial parts of some Primula species is a well-documented but poorly understood phenomenon. Here, we show that flavonoid deposition on the leaves and winter buds may contribute strongly to preventing freezing damage in these plants. The ice nucleation temperature of fairy primrose （Primula malacoides） leaves covered with natural flavone was approximately 6~C lower compared to those that had their flavone artificially removed. Additionally, farinose flavonoids on the leaves reduced subse- quent electrolyte leakage （EL） from the cells exposed to freezing temperatures. Interestingly, exogenous application of flavone at 4 mg/g fresh weight to P. malacoides leaves, which had the original flavone mechanically removed, restored freezing tolerance, and diminished EL from the cells to pretreatment values. Our results suggest that farinose flavonoids may function as mediators of freezing tolerance in P. malacoides, and exogenous application of flavone could be used to reduce freezing damage during sudden but predictable frost events in other plant species.