Comparison of G6PDH Activity and LT50 Between P.tomentosa and P.suaveolens During Freezing Acclimation

The differences of glucose-6P dehydrogenase(G6PDH) activity and freezing resistance induced by freezing acclima-tion between cuttings of freezing-sensitive P.tomentosa and freezing-resistant P.suaveolens were compared for exploring the role of G6PDH on the enhancement of freezing resistance induced by freezing acclimation.After 5d of freezing acclimation at -3℃ ,the LT50 of P.tomentosa has deereased from -6.2℃ incontrol cuttings to -14.3℃ in freezing acclimated ones,and the increase of LT50 of P.tomentosa has decreased from -6.2℃ in control cutting to -14.3℃ in freezing acclimated ones,and the increase of G6PDH activity was observed in freezing acclimated cuttings as compared with control ones.Whereas,when P.suaveolens was freezing acclimated at -20℃ for 5d,the LT50 has decreased from -27.1℃ in control cuttings to -43.5℃ in freezing acclimated ones,and the activity of G6PDH increased considerably.In addition,the increase of LT50 and the decrease of G6PDH activity resulting from 2d of deacclimation at 25℃ were found in two kinds of freezing acclimated cuttings.It is concluded that the increase in the activity of G6PDH may associate with the inherited freezing resistance of species and the enhancemen of freezing resistance of cuttings,and may play an important role in the antifreeze process under freezing temperature,which would provide the basis for the study on the molecular mechanism of freezing resistance in P.suaveolens and the cloning of gene associated with freezing resistance.     

Characterization and role of glucose-6-phosphate dehydrogenase of Populus suaveolens in induction of freezing resistance

Glucose-6-phosphate dehydrogenase (G6PDH, EC 1.1.1.49) was purified from the leaves of 8-week-old Populus suaveolens cuttings. The enzyme activity in the absence and presence of reduced dithiothreitol (DTT_red) was determined. The results show that the G6PDH activity is not inactivated by pre-incubation with DTT_red, indicating that the purified enzyme probably presented in cytosol of P. suaveolens. The catalytic characteristics and kinetic parameters of cytosolic G6PDH purified from P. suaveolens cuttings were also studied. The results show that G6PDH is characterized by K _m value of 360 μmol·L⁻¹ for G6P and 16 μmol·L⁻¹ for NADP, a pH range of 7.3-8.9, and the maximum activity around pH 8.2. The enzyme activity is inhibited by various metabolites such as NADPH, NADH, GTP, UTP, ATP, AMP, ADP, CoA, acetyl CoA, fructose-6-phosphate (F6P), erythrose-4-phosphate (E4P), ribose-5-phosphate (R5P) and 3-phosphoglycerate (3-PG) (all at 1 mmol·L⁻¹ except for NADPH and NADH) to different extents. NADPH is the most effective inhibitor of enzyme activity, with an inhibition of 72.0%. The addition of metal ions such as MgCl₂, CaCl₂ and KCl (all 1.0 mmol·L⁻¹) to the standard reaction mixture has no remarkable influence on the cytosolic G6PDH activity. However, CdCl₂ (1.0 mmol·L⁻¹) causes high inhibitory effect on the enzyme activity. To explore the role of G6PDH on the enhancement of freezing resistance induced by freezing acclimation, the changes in the cytosolic G6PDH activity and freezing resistance (expressed as LT₅₀) of P. suaveolens cuttings during freezing acclimation at −20 °C were investigated. The results reveal that freezing acclimation decreases LT₅₀ of cuttings, and increases the activity of cytosolic G6PDH compared with control ones, while 2 d of de-acclimation at 25 °C result in a decrease in cytosolic G6PDH activity, and caused an increase in LT₅₀. Furthermore, the change in cytosolic G6PDH activity is found to be closely correlated to the degree of freezing resistance of cuttings during freezing acclimation. It is suggested that cytosolic G6PDH may be involved in the induction of freezing resistance of cuttings. [Supported by the Foundation of State-designated Base for Biology Researching and Teaching in Beijing Forestry University]     

CaCl2在毛白杨幼苗抗冻性低温诱导中的作用

不同浓度的CaCl2 处理对毛白杨幼苗抗冻性的提高效应是不同的 ,其中 10mmol·L-1CaCl2 处理更有利于幼苗抗冻性的提高 ,而且处理最适宜时间为 5天 .低温锻炼不仅提高了毛白杨幼苗叶片和枝条中钙调素含量 ,同时也提高了幼苗抗冻性 .CaCl2 处理对低温锻炼的作用有加强效果 ,而钙离子螯合剂EGTA、钙离子通道阻断剂LaCl3 或钙调素拮抗剂CPZ处理则减弱低温锻炼效应 ,表现出CaM含量和幼苗抗冻性明显降低 .这表明Ca2 + CaM可能参与了幼苗抗冻性的诱导     

在低温锻炼中毛白杨幼苗可溶性总蛋白、RNA、DNA、RNase及抗冻性的变化

低温锻炼不仅提高了毛白杨幼苗存活率和抗冻性以及RNA和可溶性总蛋白的含量 ,降低了RNase活性 ,而且能减轻低温胁迫引起的RNA和可溶性总蛋白含量的下降程度和RNase活性的提高 ,有利于幼苗在恢复过程中RNA和可溶性总蛋白水平的迅速回升以及RNase活性的降低 .进一步研究发现 ,DNA含量无论在低温锻炼中还是在低温胁迫下或是在随后的恢复生长期均保持相对稳定 ;低温锻炼所引起的RNA含量的增加 ,与RNase活性的降低呈明显的负相关 ,与可溶性总蛋白含量的增加以及幼苗抗冻性的提高成正相关 .这表明低温锻炼可能抑制RNase活性 ,有效地促进RNA含量的增加 ,而RNA可能参与了可溶性总蛋白的合成及抗冻性的诱导     

Cloning and Sequence Analysis of a Glucose-6-Phosphate Dehydrogenase Gene PsG6PDH from Freezing-tolerant Populus suaveolens

A 1 207 bp cDNA fragment (PsG6PDH) was amplified by RT-PCR from cold-induced total RNA of the freez- ing-tolerant P. Suaveolens, using primers based on the highly conserved region of published plant glucose-6-phosphate dehydro- genase (G6PDH) genes. The sequence analysis showed that PsG6PDH coding region had 1 101 bp and encoded 367 predicted amino acid residues. Moreover, the nucleotide sequence of PsG6PDH showed 83%, 82%, 79%, 79% and 78% identity, and the derived amino acid sequence shared 44.2%, 44.7%, 42.0%, 40.5% and 43.9% identity with those of the Solanum tuberosum, Nicotiana ta- bacum, Triticum aestivum, Oryza sativa and Arabidopsis thaliana, respectively. The results show that PsG6PDH is a new member of G6PDH gene family and belongs to the cytosolic G6PDH gene. This is the first report on cloning of the G6PDH gene from woody plants.     

The role of antioxidant system in freezing acclimation-induced freezing resistance of Populus suaveolens cuttings

We investigated the changes in the contents of H2O2, malonaldehyde (MDA) and endogenous antioxidants, the activities of protective enzymes and some critical enzymes involved in the ascorbate-glutathione (ASA-GSH) cycle as well as freezing resistance (expressed as LT50) and correlations mentioned above, in detail using Populus suaveolens cuttings. The purpose was to explore the physiological mechanism of the enhancement of freezing resistance induced by freezing acclimation at –20°C, and to elucidate the physiological mechanisms by which trees adapt to freezing. The results showed that freezing acclimation enhanced the activities of superoxide dismutase (SOD), peroxidase (POD), catalase (CAT), monodehydroascorbate reductase (MDAR), ascorbate peroxidase (APX), dehydroascorbate reductase (DHAR) and glutathione reductase (GR). And it increased the contents of reduced ascorbate (ASA), reduced glutathione (GSH), dehydroascorbate (DHA) and oxidized glutathione (GSSG). However, H2O2 and MDA contents and LT50 of cuttings were decreased. LT50 in cuttings was found to be closely correlated to the levels of SOD, POD, CAT, APX, DHAR, MDAR, GR, H2O2, MDA, ASA, GSH, DHA and GSSG during freezing acclimation. This suggested that the enhancement of freezing resistance of cuttings induced by freezing acclimation may relate to the distinct increase for the levels of SOD, POD, CAT, APX, DHAR, MDAR, GR, ASA, GSH, DHA, and GSSG. In addition, the observed levels of APX, DHAR, MDAR, GR, ASA, DHA, GSH and GSSG were higher than those of SOD, POD and CAT during freezing acclimation. It indicated that a higher capacity of the ASA-GSH cycle is required for H2O2 detoxification, and growth and development of cuttings. Based on the obtained results, it can be concluded that the ASA-GSH cycle plays an important role in enhancement of freezing resistance of P. suaveolens cuttings during freezing acclimation.     

钙-钙调素对中东杨扦插苗的诱导抗冻作用

以零下低温锻炼和结合效应剂（CaCl2、钙离子螯合剂EGTA、钙离子通道阻断剂LaCl3或钙调素拮抗剂CPE）处理的低温锻炼下的中东扬扦插苗为试材，对其体内丙二醛（MDA）及钙调素CaM含量，SOD、POD及线粒体Ca^2-＋ATPase活性分别进行测定。结果表明，低温锻炼不仅在一定程度下提高了幼苗CaM含量，提高了SOD、POD和Ca^2＋-ATPase活性，降低了MDA含量；而且减少了低温胁迫所引起的SOD、POD，Ca^2＋＋ATPase和CaM含量的下降程度以及MDA的增加幅度。在低温锻炼的同时。用CaCl2处理能加强低温锻炼的效果，但这种效应可被EGTA、LaCl3或CPZ处理抑制。经低温锻炼后，幼苗中的CaM含量的增加有助于SOD、POD和线粒体Ca^2＋-ATPase活性的提高，进而对幼苗抗冻性的提高有明显的促进作用。     

低温锻炼与CaCl2处理对毛白杨幼苗可溶性总蛋白、CaM及抗冻性的影响

低温锻炼提高了毛白杨幼苗叶片和枝条中钙调素 (CaM)和可溶性总蛋白含量 ,同时也提高了幼苗的抗冻性 .CaCl2 处理对上述低温锻炼的作用有加强的效果 ,体现在明显提高了CaM和可溶性总蛋白含量以及幼苗抗冻性 ;钙离子螯合剂EGTA、钙离子通道阻断剂LaCl3 或钙调素拮抗剂CPZ处理则减弱了低温锻炼的效应 ,表现出CaM和可溶性总蛋白含量以及幼苗抗冻性明显降低 .进一步研究发现 ,CaCl2 处理的低温锻炼不仅能减轻低温胁迫引起的CaM和可溶性总蛋白含量的下降程度 ,而且有利于幼苗在恢复过程中CaM和可溶性总蛋白水平的迅速回升 .这表明Ca2 + CaM可能参与了CaM和可溶性总蛋白的合成及抗冻性的诱导     

Cloning and sequence analysis of a glucose-6-phosphate dehydrogenase gene <Emphasis Type="Italic">PsG6PDH</Emphasis> from freezing-tolerant <Emphasis Type="Italic">Populus suaveolens</Emphasis>

A 1207 bp cDNA fragment (PsG6PDH) was amplified by RT-PCR from cold-induced total RNA of the freezing-tolerant P. Suaveolens, using primers based on the highly conserved region of published plant glucose-6-phosphate dehydrogenase (G6PDH) genes. The sequence analysis showed that PsG6PDH coding region had 1 101 bp and encoded 367 predicted amino acid residues. Moreover, the nucleotide sequence of PsG6PDH showed 83%, 82%, 79%, 79% and 78% identity, and the derived amino acid sequence shared 44.2%, 44.7%, 42.0%, 40.5% and 43.9% identity with those of the Solanum tuberosum, Nicotiana tabacum, Triticum aestivum, Oryza sativa and Arabidopsis thaliana, respectively. The results show that PsG6PDH is a new member of G6PDH gene family and belongs to the cytosolic G6PDH gene. This is the first report on cloning of the G6PDH gene from woody plants.     

Ultrastructural and Extracellular Protein Changes in Cell Suspension Cultures of Populus euphratica Associated with Low Temperature-induced Cold Acclimation

Populus euphratica Olive is the only tree species that can grow in the saline land and also survive cold winters in northwest China, and it plays a very important role in stabilizing the vulnerable ecosystem there. A cell suspension culture was initiated from callus derived from plantlets of Populus euphratica. Cold acclimation was induced (LT50 of-17.5 ℃) in cell suspension at 4-5 ℃ in the dark for 30 days and the freezing tolerance increased from LT50 of-12.5 ℃ in nonacclimated cells to LT50 of-17.5 ℃ in cold-acclimated cells. Microvacuolation, cytoplasmic augmentation and accumulation of starch granules were observed in cells that were cold-acclimated by exposure to low temperatures. Several qualitative and quantitative changes in proteins were noted during cold acclimation. Antibodies to carrot extracellular (apoplastic) 36 kD antifreeze protein did not cross react on immunoelectroblots with extracellular proteins in cell suspension culture medium of Populus euphratica, indicating no common epitopes in the carrot 36 kD antifreeze protein and P euphratica extracellular proteins. The relationship of these changes to cold acclimation in Populus euphratica cell cultures was discussed.     

秦岭细粘束孢产漆酶的培养条件

漆酶(Laccase,EC1.10.3.2)属于蓝色氧化酶家族的一种多酚氧化酶,是重要的木质纤维(lingocellulose)降解酶之一(Bertrand et al.,2002)。漆酶能催化降解多种芳香族化合物特别是酚类物质(Solomon et al.,1996)。由于漆酶具有430～790mV氧化还原电位(Shleev et al.,2003),能够将分子氧直接还原为水,在没有H2O2和其他次级代谢产物存在下,只要存在溶解氧,就可直接氧化底物(韩     

Effects of cold-hardening on compatible solutes and antioxidant enzyme activities related to freezing tolerance in Ammopiptanthus mongolicus seedlings

Cold acclimation is associated with many metabolic changes that lead to an increase of freezing tolerance. In order to investigate the biochemical process of cold acclimation in Ammopiptanthus mongolicus, seedlings were acclimated at 2℃ under 16-h photoperiod （150 μmol·m^-2·s^-1 photosynthetically active radiation） for 14 d. Freezing tolerance in seedlings increased after 14 d of cold-hardening. Contents of protein, proline and solute carbohydrate in cotyledon increased after cold acclimation. Patterns of isozymes of superoxide dismutase （SOD）, peroxidase, catalase and polyphenol oxidase （PPO） were investigated. The activities of SOD, peroxidase and PPO in cold acclimated plants were increased during cold-hardening. We deduced that compatible solutes and antioxidant enzymes play important roles in development of freezing tolerance during cold acclimation in this evergreen woody plant.     

钙对酸化处理平邑甜茶根系抗氧化酶活性及线粒体功能的影响

为探讨酸化处理及钙对酸化处理下平邑甜茶根系抗氧化酶活性及线粒体功能的影响,将平邑甜茶幼苗分2组分别置于pH3,4,5,6(对照)的1/2Hoagland营养液中及3个钙离子浓度梯度(10,20,30mmol·L-1)的pH值为3的1/2Hoagland营养液中,于第0,2,3,4天分别测定抗氧化酶活性及线粒体指标。结果表明:与对照相比,各处理的SOD,CAT酶活性在处理的第2天达到最大值,POD酶活性变化不一,pH3与pH4处理在第2天达到最大值,pH5处理在第3天达到最大值;根系线粒体膜通透性、膜电位及细胞色素c/a随着酸度增加和时间的延长逐渐降低;H2O2含量先降低后升高。低浓度外源钙(10,20mmol·L-1)处理能提高SOD,CAT,POD酶活性并维持一定时间,缓解线粒体各项功能的伤害,抑制H2O2的积累,主要表现在与单独酸处理相比H2O2含量降低了6.78%,13.43%,且20mmol·L-1钙离子缓解效果最好;30mmol·L-1钙离子处理却加剧了酸化伤害,H2O2含量上升了8.96%。酸化处理会对平邑甜茶根系造成伤害而营养液适量加钙可以缓解其伤害。     

First frost: Effects of single and repeated freezing events on acclimation in Picea abies and other boreal and temperate conifers

In experiments with needles of Picea abies, we tested the specific hypothesis that a single night of freezing acts as a signal that triggers a rapid increase in low temperature (LT) tolerance, and the more general hypothesis that repeated or prolonged freezing stimulates increased LT acclimation. In three growth chamber experiments involving acclimation under early- to mid-autumn light and temperature conditions followed by one or more freezing treatments, we found no significant effect of a single night of freezing on LT tolerance, and only limited and inconsistent effects of repeated and prolonged freezing. We also tested the effect of prolonged storage at −5 °C on LT tolerance on samples of three boreal and three temperate conifer species during acclimation under field conditions, and again found no consistent enhancement of LT tolerance attributable to freezing in either group. In agreement with our own and others’ anecdotal observations that some species can attain nearly maximal LT tolerance in the absence of freezing under field conditions, we conclude that freezing is neither required nor a major influence in LT acclimation, at least in well-studied boreal conifer species, while the effects of freezing on temperate conifers are not as well-documented. We conclude that freezing treatment of conifer seedlings to ensure sufficient hardiness for late planting seems to offer little practical advantage.     

Aluminum-induced decrease in CO2 assimilation in citrus seedlings is unaccompanied by decreased activities of key enzymes involved in CO2 assimilation

'Cleopatra' tangerine (Citrus reshni Hort. ex Tanaka) seedlings were irrigated daily for 8 weeks with 1/4 strength Hoagland's nutrient solution containing 0 (control) or 2 mM aluminum (Al). Leaves from Al-treated plants had decreased CO2 assimilation and stomatal conductance, but increased intercellular CO2 concentrations compared with control leaves. On a leaf area basis, 2 mM Al increased activities of key enzymes in the Calvin cycle, including ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco), NADP-glyceraldehyde-3-phosphate dehydrogenase (GAPDH), phosphoribulokinase (PRK), stromal fructose-1,6-bisphosphatase (FBPase), and a key enzyme in starch synthesis, ADP-glucose pyrophosphorylase (AGPase), compared with control leaves. Aluminum had no effect on cytosolic FBPase activity, but it decreased sucrose phosphate synthase (SPS) activity. Aluminum had no effect on area-based concentrations of carbohydrates, glucose-6-phosphate (G6P) and fructose 6-phosphate (F6P) or the G6P:F6P ratio, but it decreased the area-based concentration of 3-phosphoglycerate (PGA). Photochemical quenching coefficient (qP) and electron transport rate through PSII were greatly reduced by Al. Non-photochemical quenching coefficient (NPQ) was less affected by Al than qP and electron transport rate through PSII. We conclude that the reduced rate of CO2 assimilation in Al-treated leaves was probably caused by a combination of factors such as reduced electron transport rate through PSII, increased closure of PSII reaction centers and increased photorespiration.     

不同类型的GGR及其浓度对紫叶风箱果硬枝扦插的影响

为探究不同种类、浓度的GGR对紫叶风箱果硬枝扦插的影响,用4种类型的GGR设置不同浓度(50~350 mg·L-1)处理插穗后进行扦插试验。结果表明:4种GGR处理在50~350 mg·L-1时对风箱果插穗生根均有一定的促进作用,以150 mg·L-1的效果最好,存活率达92.66%。GGR-10在促进插穗生根及根系生长方面效果最好;GGR-6和GGR-7一般;GGR-8的效果较差。     

红毛五加扦插繁殖技术试验研究

通过对红毛五加穗条的不同药剂、扦插时间和扦插基质对红毛五加存活率影响的试验研究,结果表明：（1）红毛五加在河沙基质中扦插存活率最高,达78.75%;（2）ABT药剂50 mg.L-1、6 h处理穗条,2 a生枝条扦插存活率最高,达70%;（3）ABT、NAA、IAA、IBA药剂50 mg.L-1、6 h处理,比其他处理方式能显著提高扦插苗存活率。且ABT、NAA、IAA药剂50 mg.L-1、6 h处理可用于生产推广。     

不同基质和IBA浓度对野蔷薇及木香扦插生根的影响

采用不同基质、不同IBA浓度处理野蔷薇、木香插穗,研究扦插基质、激素浓度对扦插生根的影响。结果表明野蔷薇在A2B2(100 mg·L-1IBA+蛭石)、木香在A3B1(200 mg·L-1IBA+珍珠岩)组合中成活率、生根数、平均根长最好。     

4-己基间苯二酚和曲酸对舞毒蛾酚氧化酶的抑制作用

以舞毒蛾为材料,分离提取酚氧化酶,研究4-己基间苯二酚(4HR)和曲酸对舞毒蛾酚氧化酶活力的影响.结果表明:4HR对该虫的单酚酶和二酚酶活性均表现很强的抑制作用,其IC_(50)分别为0.000 41和0.000 35mmol·L~(-1).其中,4HR对单酚酶活力表达的迟滞时间有明显的延长效应,浓度为0.000 2 mmol·L~(-1)时可使单酚酶活力表达迟滞时间从181 s延长到253 s;而当浓度为0.000 5 mmol·L~(-1)时,其迟滞时间则延长至372 s.以邻苯二酚为底物时,4HR对二酚酶的抑制作用表现为典型的竞争型抑制类型,抑制常数K_1为0.000 15 mmol·L~(-1).曲酸对舞毒蛾的单酚酶和二酚酶活性均表现较强的抑制作用,其IC_(50)分别为0.06和0.92 mmol·L~(-1).曲酸对单酚酶活力表达的迟滞时间也有明显的延长效应,浓度为0.1 mmol·L~(-1)时可使单酚酶活力表达迟滞时间从306 s延长到732 s;而当浓度为0.15 mmol·L~(-1)时,其迟滞时间则延长至900 s.以邻苯二酚为底物时,曲酸对二酚酶的抑制作用表现为典型的竞争型抑制类型,抑制常数K_i为0.51 mmol·L~(-1).     

Dynamics of low-temperature acclimation in temperate and boreal conifer foliage in a mild winter climate

To provide baseline data for physiological studies of extreme low-temperature (LT) tolerance in boreal conifers, we profiled LT stress responses, liquid nitrogen (LN(2))-quench tolerance, and sugar concentrations in foliage of boreal-temperate species pairs in the genera Abies, Picea and Pinus, growing in an arboretum in a temperate oceanic climate from August 2006 through April 2007. The boreal species acclimated more rapidly and deeply than the temperate species, acquiring LN(2)-quench tolerance by late November, despite unusually warm conditions throughout the autumn and early winter. Maximum LT tolerance in the temperate species was in the -25 to -35 degrees C range, and was reached only after a period of freezing temperatures in late January and February. During LT acclimation in the temperate species, sigmoid temperature-relative electrolyte leakage (REL) curves shifted toward lower temperatures, whereas in boreal species there was both a temperature shift and a lowering of the maximum REL until it fell below a threshold associated with irreversible injury. These differences may reflect differences in mechanisms of LT acclimation and LT tolerance. The concentrations of total and individual sugars did not show a clear pattern that could differentiate the boreal and temperate groups. Raffinose and, in three of the six species, stachyose showed the closest association with LT tolerance. Sugar concentrations, principally sucrose, decreased during mild weather, perhaps because of respiratory losses or phloem export, and increased after periods of freezing temperatures. Low-temperature acclimation in boreal species appears to follow a rigid program that may affect their ability to avoid excessive respiratory losses in the event of continued climate warming in boreal regions.