Changes of plasma membrane H<Superscript>+</Superscript>-ATPase activities of <Emphasis Type="Italic">Glycine max</Emphasis> seeds by PEG treatment

The soybean （Glycine max） Heihe No. 23 is sensitive to imbibitional chilling injury. Polyethylene glycol （PEG） treatment can improve chilling tolerance of soybean seeds to a certain extent. The changes of hydrolytic ATPase in plasma membranes and H^＋-pumping responses in soybean seeds were investigated during PEG treatments. Effects of exogenous calcium and exogenous ABA on the hydrolytic ATPase were also examined in order to understand the mechanism of chilling resistance. Highly purified plasma membranes were isolated by 6.0% aqueous two-phase partitioning from soybean seeds, as judged by the sensitivity of hydrolytic ATPase to sodium vanadate. PEG treatment resulted in a slight increase of the hydrolytic ATPase activity in 12 h. Then the activity decreased gradually, but still higher than the control. The H^＋-pumping activity increased steadily during PEG treatment. Exogenous calcium had both activating and inhibiting effects on the hydrolytic ATPase, but the activity was inhibited in soybean seeds treated with exogenous ABA. Results suggested that PEG treatment, not the exogenous calcium and ABA, up-regulated H^＋-ATPase activities in soybean seeds.     

Effects of osmoconditioning on mitochondrial respiration and phosphorylation in soybean seeds

Cotyledon mitochondrion respiration and oxidative phosphorylation activity were studied in two groups of soybean seeds. One group was primed with polyethylene glycol (PEG) for different periods of time, and the other was unprimed (control), and both were then exposed to imbibition at low temperatures before their germination. The results indicated that when L-Malate (L-Mal)and α-Ketoglutarate (α-Kg) were used as substrates, the ADP-stimulated mitochondria respiration rates of control seeds were markedly higher than state Ill respiration rates of primed seeds. However, the osmoconditioning pretreatment significantly enhanced the oxidative phosphorylation activity of cotyledon mitochondrion in 12 h. The oxidative phosphorylation activity of the mitochondrion of primed seeds was normal and the ADP/O value was consistent with the theoretical one. When reduced nicotinamide adenine dinucleotide (NADH) was used as the substrate, the mitochondria of control seeds still had oxidative phosphorylation activity, while ADP/O value was obviously lower than that of mitochondria of primed seeds. When Succinate (Succ) was used as the substrate, the oxidative phosphorylation activity of the primed seeds was normal after priming for 24 h. When different substrates were used, the emerging order of the oxidative phosphorylation activity of the primed seeds was NADH, α-Kg, Succ and in the last place L-Mal.The mechanism of soybean imbibitionl chilling injury and protective effect of PEG priming were discussed.     

等渗胁迫及其钙调节下青钱柳幼苗叶肉细胞中ATP酶的细胞化学定位及亚细胞变化

研究了等渗的盐和水分胁迫及其钙调节下,青钱柳幼苗叶肉细胞中ATP酶活性在亚细胞中的分布及其超微结构变化。在人工气候室中采用水培法,将青钱柳幼苗进行5个不同浓度处理：对照,85mMNaCl,85mMNaCl＋12mMCa（NO3）2,PEG（渗透势等于85mMNaCl）及PEG（渗透势等于85mMNaCI）＋12mMCa（NO3）2。结果表明：正常生长条件下,ATP酶活性较低并主要定位在细胞核中 等渗胁迫12d以后,ATP酶活性增大并以液泡中居多。等渗水分胁迫下出现的嗜锇颗粒较等渗盐胁迫下的多。由于外源钙的调节作用,等渗胁迫下ATP酶活性增加并主要转移至细胞核上,且在等渗水分胁迫下的嗜锇颗粒明显减少。ATP酶定位在细胞核中表明幼苗遭受胁迫伤害程度较轻,而定位在液泡中则表明受伤害程度较重。等渗处理4和20d后,等渗胁迫及其钙调节下青钱柳幼苗叶片超微结构被破坏程度较无钙调节处理,尤其是较等渗水分胁迫下的轻。初步认为,等渗盐胁迫下青钱柳幼苗遭受的胁迫伤害程度较等渗水分胁迫下的轻,而钙调节作用则以等渗水分胁迫下的效果较佳。     

Changes in freezing tolerance, plasma membrane H+-ATPase activity and fatty acid composition in Pinus resinosa needles during cold acclimation and de-acclimation

It has previously been suggested that plasma membrane ATPase (PM H+-ATPase, EC 3.6.1.3.) is a site of incipient freezing injury because activity increases following cold acclimation and there are published data indicating that activity of PM H+-ATPase is modulated by changes in lipids associated with the enzyme. To test and extend these findings in a tree species, we analyzed PM H+-ATPase activity and the fatty acid (FA) composition of glycerolipids in purified plasma membranes (PMs) prepared by the two-phase partition method from current-year needles of adult red pine (Pinus resinosa Ait.) trees. Freezing tolerance of the needles decreased from -56 degrees C in March to -9 degrees C in May, and increased from -15 degrees C in September to -148 degrees C in January. Specific activity of vanadate-sensitive PM H+-ATPase increased more than two-fold following cold acclimation, despite a concurrent increase in protein concentration. During de-acclimation, decreases in PM H+-ATPase activity and freezing tolerance were accompanied by decreases in the proportions of oleic (18:1) and linoleic (18:2) acids and increases in the proportions of palmitic (16:0) and linolenic (18:3) acids in total glycerolipids extracted from the plasma membrane fraction. This pattern of changes in PM H+-ATPase activity and the 18:1, 18:2 and 18:3 fatty acids was reversed during cold acclimation. In the PM fractions, changes in FA unsaturation, expressed as the double bond index (1 x 18:1 + 2 x 18:2 + 3 x 18:3), were closely correlated with changes in H+-ATPase specific activity (r2 = 0.995). Changes in freezing tolerance were well correlated with DBI (r2 = 0.877) and ATPase specific activity (r2 = 0.833) in the PM fraction. Total ATPase activity in microsomal fractions also closely followed changes in freezing tolerance (r2 = 0.969). We conclude that, as in herbaceous plants, simultaneous seasonal changes in PM H+-ATPase activity and fatty acid composition occur during cold acclimation and de-acclimation in an extremely winter hardy tree species under natural conditions, lending support to the hypothesis that FA-regulated PM H+-ATPase activity is involved in the cellular response underlying cold acclimation and de-acclimation.     

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。     

水胁迫条件下引物处理对巴西盾柱木种子萌发的影响

实验所用巴西盾柱木种子由圣保罗州Anhembi公司提供,种子经由98%的浓硫酸预处理15 min, 以打破物理休眠。分别在0.2%克菌丹溶液10 oC和27 oC下,-1.0 MPa聚乙二醇6000 10 oC和27 oC下,以及, 0.5 mol,0.75 mol,1.0 mol 硝酸钾溶液进行引物处理. 包括对照组种子,共8个实验处理,每一个处理5个重复,每一个处理实验用100粒种子,置于滤纸浸透引物聚乙二醇6000溶液的15厘米培养皿中,置于冰箱保持27oC恒温。实验引物渗透势为0.0, -0.2, -0.4, -0.6, -0.8, -1.0-MPa, -1.4 MPa. 实验同时将种子置于浸有引物聚乙二醇的卷纸中,观察水胁迫下的萌发情况。实验结果证明,随着聚乙二醇6000浓度的升高,巴西盾柱木种子萌发率降低,对照组萌发率比实验引物处理组高。聚乙二醇达到PEG -1.4 MPa,种子不能萌发。图2表3参10。     

Effect of harvest date,drying, short-term storage and freezing after chilling on the germination of rowan seeds

The effects of seed pretreatment and harvest date on the germination of European rowan (Sorbus aucuparia L) seeds were examined. In one experiment, seeds were subjected to drying, storage, soaking, warm and chilling treatments after harvesting in mid- and late August. In another experiment, fully imbibed (FI) seeds were given warm treatment for six weeks and then various durations of chilling (4?±?1°C) for up to 24 weeks. Thereafter, the seeds were adjusted to target moisture content of 35% and 30% or remained in the FI state and were then subjected to either subsequent chilling or freezing (?3°C) for up to 32 weeks. The treated seeds were allowed to germinate at a constant 15°C with 8 hours of lighting per day. Treatment effects were generally consistent for each harvest date. Seeds did not germinate unless they were chilled and germination rates were low unless the seeds were soaked. Warm treatment applied before chilling appeared to induce dormancy. The effect of storage varied with harvest date and storage treatment, with germination being highest for seeds harvested in late August. Mild freezing of ≥16 weeks duration following 16–24 weeks initial chilling resulted in >80% germination, more than could be achieved using chilling alone.     

小菊花粉生活力及贮藏力的研究

采用液体培养基法对几种小菊花粉生活力及贮藏力进行了研究。结果表明,小菊花粉在常规的硼、钙培养基上很难萌发,添加PEG后可显著提高花粉萌发率,在15%蔗糖+20mg/L硼酸+10mg/L CaCl2+15%PEG6000培养基上萌发率最高,可达38.4%。小菊花粉贮藏时不宜干燥,贮藏时间随着温度的降低而延长,在-80℃不干燥条件下,花粉活力可以保持60d以上。     

预处理对巴西盾柱木种子吸涨、活力测试及其加速老化的影响

对巴西盾柱木(Peltophorum dubium)种子进行了4种处理的吸胀实验,即将种子分别置于10℃和27℃下0.2% Captan溶液中和-1.0MPa的PEG6000溶液中,每个处理有四个重复,40粒种子,置于培养皿中浸满实验溶液的双层过滤纸上,覆以透气良好的薄膜.种子在实验前均用98%的浓硫酸处理15分钟以打破机械休眠.吸胀曲线显示,种子在吸胀过程中最终重量的增加值为70%-150%,以后进入一个停滞期.在5种处理下(对照组,PEG6000溶液中10℃和27℃下预处理,Captan溶液中10℃和27℃下预处理),对种子萌发所受到的影响进行了实验.每一个处理分别进行3个次级处理,即在实验前,将种子用蒸馏水分别浸泡12、24和36小时.萌发率最高的是经过蒸馏水浸泡12小时对照组种子和PEG 27℃组的种子,萌发率达到100%;萌发率最低的是蒸馏水中浸泡36小时的PEG 27℃组的种子,萌发率为52%;经过蒸馏水浸泡24小时的PEG 10℃组种子的平均萌发时间为1.08天.蒸馏水中浸泡12小时的PEG 27℃组种子平均萌发时间为2.42天,其它处理的萌发时间值介于两者之间.对预处理和未经预处理种子通过加速老化实验,对其活力和生存力进行了测试.经72小时加速老化,种子萌发率低或没有萌发力.对照组种子在培养皿(27℃)和室温条件下蛭石中的萌发率都比处理组高,表现了对老化更强的抗性.     

外源脱落酸对干旱胁迫下美国红枫抗旱性的影响

以当年生盆栽美国红枫扦插苗为试验材料,采用不同浓度的脱落酸(ABA)溶液喷施美国红枫扦插苗,测定干旱胁迫下植株的相对电导率、脯氨酸(Pro)、丙二醛(MDA)及可溶性糖含量等各项生理指标,研究外源喷施ABA对美国红枫抗旱性的影响。结果表明,在干旱条件下,外源喷施ABA可以缓解其伤害程度,促进美国红枫叶片中脯氨酸和可溶性糖含量的增加,抑制MDA的积累,延缓叶片相对电导率上升,其中以10 mg/L浓度ABA处理效果最明显。因此,干旱胁迫下,喷施一定浓度的脱落酸可有效提高美国红枫的抗旱能力,维持其正常生理代谢。     

色木槭和拧筋槭种子脱水耐性分析——基于含水量、发芽率和ABA含量变化的结果

通过采集不同发育阶段种子并对其进行自然干燥处理,以含水量、发芽率和ABA含量变化为指标,研究了色木槭和拧筋槭种子在自然发育和干燥过程中的脱水耐性变化.结果表明,色木槭和拧筋槭种子虽经历了成熟脱水,但种子仍不耐脱水;其发芽率和活力指数随发育进程而逐渐增加,但随自然脱水进程而逐渐降低;色木槭和拧筋槭种子最大脱水耐性分别出现在花后100 d和120 d;在整个发育过程中,两树种种子的ABA(脱落酸)含量均较低;拧筋槭种子中ABA含量在脱水过程中呈下降趋势,而色木槭种子中ABA含量在脱水过程中虽有升高但变化不显著.推测ABA含量低可能为色木槭和拧筋槭种子不耐脱水的主要原因之一.     

假色槭种子层积过程中内源激素含量的动态变化

通过对假色槭种子层积过程中内源激素含量的动态变化进行的研究,结果表明:在自然层积过程中,IAA表现为先上升再下降,再上升再下降的趋势,ABA基本表现为下降趋势,GA呈现先下降,再上升,再下降,再上升的趋势。IAA/ABA,GA/ABA在整个过程中基本表现为上升趋势。说明IAA、GA在种子休眠解除过程中起到一定促进作用,ABA起到抑制作用。     

Effect of Seed Maturity and Pretreatment on Dormancy and Germination of Sorbus mougeotii Seeds

The effect of harvest time on seed germination, desiccation tolerance and level of dormancy is not known in Sorbus mougeotii (Soyer-Will. & Godron). Dry weight (DW), moisture content (MC) and leakage conductivity (LC) were tested on seeds from six harvest times. Chilling requirement and germinability were tested on seeds without drying and after drying and rehydration. The effect of postharvest ripening of seeds with or without drying was also tested. The DW was stable during the last 6 weeks of maturation, whereas MC initially decreased but later increased simultaneously with an increase in LC. Seeds were desiccation tolerant, deeply dormant and after a chilling treatment germinated to a high percentage at all harvest times. The chilling requirement decreased significantly with later harvest, suggesting a decrease in level of dormancy. Drying and postharvest ripening reduced and synchronized the chilling requirement.     

Endogenous inhibitors in seed ofFraxinus mandshurica Rupr and their effect on germination and regeneration

Bioassay and GC-MS determination indicated that inhibitors of seeds ofFraxinus mandshurica Rupr were distributed mainly in zones of β-inhibitors (including ABA) and of high and low R_f value. The inhibitory activity of outer seed coat of the dormant seed (control treatment) was higher than that of seeds with only the inner coat attached e.g. ABA was 114 and 54ng/g fresh weight respectively. The inhibitory effect of No. 12 zone was even higher than ABA. The inhibitory activity of all the zones decreased after stratification (St) and burying (A0) treatment, especially that of the outer seed coat, whose ABA content decreased to 7(St) and 11 (AO) ng/g(fw). However, the decrease in inhibition of root growth lagged behind the decrease in germination. It is concluded that burial is advantageous to regeneration. Inhibitors of the outer seed coat and seeds were mostly destroyed under the adverse conditions of natural dissemination (Di treatment) indicating that Di was not conducive to regeneration. MS analysis revealed that the largest peaks of No. 6, 7 and 12 zones were linolcic acid, olcic acid and 17-C, 18-C, and 20-C long chain fatty acids. Several coumarins were found in No. 6 and 7 zones, and cadinencs, farnesol and sesquiterpenes were found in No. 6 and 12 zones having common bioorigins with ABA.     

Effects of drought stresses induced by polyethylene glycol on germination of Pinus sylvestris var. mongolica seeds from natural and plantation forests on sandy land

Effects of drought stresses induced by polyethylene glycol (PEG) (0.0%, 10%, 15%, 20%, 25%, and 30%, with four replicates) on germination of Mongolian pine (Pinus sylvestris var. mongolica) seeds produced in plantations (southern Keerqin sandy land) and natural forests (Hulunbeier sandy plain) were observed. The results indicated that the seeds from both provenances did not germinate when PEG concentration was more than 25%. The time of initial germination and that of its completion of stressed seeds from both provenances were delayed when compared with the unstressed seeds. The germination capacity and germination rate of natural seeds were significantly higher than those of plantation seeds for all treatment levels (P < 0.05). The mean growth rates of radicle and hypocotyl from natural seeds were significantly higher than those from plantation seeds at all treatment levels below 20% PEG treatment (P < 0.05). The ratios of radicle to hypocotyl of 20% PEG treatment were significant higher than those of the corresponding controls for both provenances (P < 0.05). These results suggested that Mongolian pine seeds/seedlings had stronger resistance to PEG drought stresses; 10% PGE stress did not significantly influence germination. Natural seeds exhibited more resistance to PEG stress than plantation seeds. It was concluded that drought stress on seed germination might be one cause of obstructed natural regeneration of Mongolian pine plantations on sandy land. It is recommended that natural seeds be used for afforestation, and light drought stress (e.g., 10% PEG stress) may be useful in improving seed germination and the growth of radicles and hypocotyls.     

Effect of solid matrix priming during moist chilling on dormancy breakage and germination of seeds of four fir species

The ability of solid matrix priming (SMP) to overcome seed dormancy ofseeds of four fir species (Pacific silver fir, subalpine fir, grand fir andnoble fir), was examined. Seeds were subjected to treatments that combinedmoistchilling and SMP (SMP-chilling) and their germination compared to that elicitedby traditional moist chilling. For the SMP-chilling, six solid matrices withdifferent water holding capacities were tested (sphagnum, cat litter, peatmoss,Agro-Lig Greens Grade, Agro-Lig Micro Fine and sand). For each of the speciestested, germination performance was significantly improved by SMP-chilling; theresults were dependent on the type of solid matrix, the matrix water contentandthe treatment duration. SMP-chilling, using the best solid matrices for each ofthe species tested, resulted in germination percentages and rates that were24–35% and 8–21% higher, respectively, as compared with thatyieldedfrom control moist-chilling. High germination percentages (79% or greater) ofthree of the four species (Pacific silver fir, subalpine fir, and grand fir)were promoted after just four weeks (grand fir in sand) or eight weeks oftreatment (Pacific silver fir in peat moss and subalpine fir in Agro-Lig GreensGrade). The germination rate of grand fir seeds benefited from SMP-chillingperiods longer than four weeks, but this resulted in germination during thedormancy-breaking treatment. The water contents of the solid matrices that wereoptimal in terms of eliciting the best germination performance were 320% (w/w)for peat moss, 40% for Agro-Lig Greens Grade and 5–15% for sand.Conducting SMP at chilling temperatures (4 °C) is a practicalapproach to shorten the time required for effective dormancy termination ofAbies seeds; treatment times were shortened by 27 days forPacific silver fir and subalpine fir and more vigorous seedling growth wasexhibited by grand fir and noble fir.     

Effects of moist chilling and solid matrix priming on germination of loblolly pine (Pinus taeda L.) seeds

Loblolly pine (Pinus taeda L.) seeds from sources with a mild climate under maritime influence (North Carolina) required shorter moist chilling to achieve maximum germination vigor than seeds from sources with a harsher continental climate (Oklahoma). Solid matrix priming (SMP) for 6 d achieved as much as 60 d of moist chilling to improve rapidity, synchrony and completeness of germination for three of the four families studied. SMP after moist chilling increased the rapidity, synchrony and completeness of germination. The benefit of SMP was greatest for non-stratified seeds and the benefit decreased with length of moist chilling. In general, delaying planting for one week after SMP had minor effects on germination when seeds were kept in the SMP matrix at 4°C. Delayed planting after SMP can increase germination rapidity and synchrony of seeds that have received long moist chilling and reduce the benefit of SMP in non-moist-chilled seeds.     

Shoot water status and ABA responses of transgenic hybrid larch Larix kaempferi x L. decidua to ectomycorrhizal fungi and osmotic stress

It has been postulated that osmotic effects on plant tissue are mediated by abscisic acid (ABA). Hybrid larch (Larix kaempferi (Lambert) Carr. x L. decidua Mill.) plantlets, transformed with the ABA-inducible wheat Em promoter associated with the Gus reporter gene, were axenically inoculated with two ectomycorrhizal fungi: Cenococcum geophilum Fr., considered tolerant to water stress, and Laccaria bicolor (Marie) Orton, considered less tolerant to drought. The mycorrhizal and non-mycorrhizal transgenic plantlets were subjected to osmotic stress by adding polyethylene glycol (PEG) to the culture medium. In the presence of PEG, L. bicolor and C. geophilum reduced shoot water potential and turgor potential, but increased host osmotic potential. Treatment of plantlets with PEG induced a significant increase in endogenous ABA concentrations. Laccaria bicolor and C. geophilum behaved similarly and significantly decreased the ABA response of plantlets to PEG treatment. Moreover, inoculation with either fungus regulated the ABA response of the plantlets even when the fungus was separated from the host by a cellophane sheet that prevented mycorrhiza formation. Although the wheat Em promoter was inducible in larch plantlets, it was not regulated by endogenous ABA. Induction of the wheat Em promoter in larch plantlets depended on organ type, with maximum induction in the root apex. Induction of the Em promoter was significantly decreased by mycorrhizal inoculation.     

Breaking seed dormancy in European rowan seeds and its implications for regeneration

The effects of the presence of a medium, seed moisture content, warm and chilling treatments on the germination response of European rowan (Sorbus aucuparia L.) seeds of Irish origin were investigated with the aim of reducing precocious (or premature) germination and maximising germination after pretreatment. After adjusting seed moisture content, seeds of two lots were subjected to warm treatment for 0–6 weeks followed by 0–50 weeks of chilling. The seeds then were allowed to germinate at a constant 15 °C with 8 h lighting per day or 20 (dark)/30 °C (light). Some seeds were treated in the fully imbibed (FI) state in a medium (standard operational treatment). Seed lot effects were evident, but treatment effects were consistent in each lot. Warm treatment greatly increased germination and reduced precocious germination. The presence of a medium was not required to achieve high germination and seed moisture content close to the FI state was needed to maximise germination. Climate change may reduce the ability of this species to regenerate naturally, favouring other species that require less chilling.     

香果树种子发芽对水分胁迫的响应

通过用PEG（聚乙二醇）配制不同浓度溶液,模拟不同土壤水势梯度,达到不同水分胁迫水平,进行香果树种子发芽水分胁迫实验。研究结果表明;水势在0～-0．4MPa,随PEG浓度升高,香果树种子发芽率、发芽指数、苗长、活力指数均呈下降趋势。PEG浓度升至20%,种子发芽完全受抑制。表明香果树种子在轻度水分胁迫下,有一定的耐旱能力。