Isolation of protoplast from callus of Populus euphratica and H fluxes across plasma membrane under NaCl stress

We used callus of Populus euphratica Olive to isolate protoplasts, and H fluxes across plasma membrane were investi-gated. The concentration of enzymes for protoplast isolation, e.g. cellulase, pectolyase, macerozyme, hemicellulase, and sorbitol content, incubation time were systemically studied. High yield and viability of protoplast was achieved after 6-8 hours incubation of P. euphratica callus in enzyme solution containing 1.5% (w:v) cellulase R-10, 0.1% (w:v) pectolyase Y-23, 0.2% (w:v) macerozyme R-10, 0.05% (w:v) hemicellulase and 0.75-0.80 mol·L-1 sorbitol. Non-invasively ion selective microelectrode technique was used to access proton fluxes in the absence and presence of NaCl (20 mmol·L-1). Salt-induced transient net H efflux was observed in the plasma membrane of P. euphratica cells. The shift of H flux response to NaCl shock and the relevance to salt tolerance were discussed.     

Anti-ulcer constituents of Annona squamosa twigs

Phytochemical investigation of Annona squamosa twigs, resulted in isolation and identification of twelve known (1-12) compounds among them one 1-(4-β-D-glucopyranosyloxyphenyl)-2-(β-D-glucopyranosyloxy)-ethane (11) is synthetically known but first time isolated from natural sources. Their structures were elucidated using 1D and 2D NMR spectroscopic analysis. The isolated compounds (2-8, 11) were evaluated for H⁺ K⁺-ATPase activity. Three of these compounds (+)-O-methylarmepavine (2), N-methylcorydaldine (3), isocorydine (6) showed promising anti-secretory activity. Activity of these compounds, comparable to the standard drug omeprazole is novel to our finding. Moreover, there is no information accessible regarding the pharmacological effect of A. squamosa on the gastrointestinal system. This study is the first of its kind to show the significant anti-ulcer effect of A. squamosa. The present study aimed to evaluate the gastroprotective effect of A. squamosa (AS) and to identify its active constituents. Anti-ulcer activity was evaluated against cold restraint (CRU), pyloric ligation (PL), aspirin (ASP), alcohol (AL) induced gastric ulcer and histamine (HA) induced duodenal ulcer model and further confirmed through in vitro assay of H⁺ K⁺-ATPase activity and plasma gastrin level. AS and its chloroform and hexane fraction attenuated ulcer formation in CRU, PL, HA model and displayed anti-secretory activity in vivo through reduced free, total acidity and pepsin in PL, confirmed by in vitro inhibition of H⁺ K⁺-ATPase activity with corresponding decrease in plasma gastrin level. Cytoprotection of AS was apparent with protection in AL, ASP models and enhanced mucin level in PL.     

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.     

Regeneration and gene transformation systems of Robinia pseudoacacia ‘Idaho’ mediated by Agrobacterium tumefaciens

Robinia pseudoacacia ‘Idaho’ is one of several multi-purpose trees used in ornamental, soil and water conservation, fodder and nectar sources. Plant abiotic stress tolerance transformed by genes could meet the requirements for reclamation of arid or alkalid lands and vegetation restoration. For this paper, we studied the effects of auxin and cytokine on Idaho locust in vitro regeneration and the establishment of gene transformation systems for plants mediated by Agrobacterium tumefaciens. Results showed that the ratios of cytokinin and auxin were the major factors affecting adventitious bud differentiation on a MS medium; the concentration of 0.5 mg&#8226;L^–1 6-BA benefitted callus proliferation and 0.25 mg&#8226;L^–1 IBA promoted shoot rooting; however, a higher IBA concentration will inhibit rooting. The most effective antitoxin for screening transgenic Idaho locust shoots was G418 and the most sensitive concentra-tion of it was 8 mg&#8226;L^–1.     

Genotypic Differences in Antioxidative Stress and Salt Tolerance of Three Poplars Under Salt Stress

To evaluate genotypic difference in antioxidative ability and salt tolerance in poplars, the authors investigated the effects of increasing content of soil NaCl on salt concentration in leaves, superoxide dismutase (SOD) and peroxidase (POD) activities, malondialdehyde (MDA) content, and membrane permeability (MP) in Populus euphratica Oliv., P. popularis “35–44,” and P. × euramericana cv. I-214 (hereafter abbreviated as P. cv. I-214). Na⁺ and Cl⁻ concentrations in leaves of P. popularis increased markedly over the increase of the duration of exposure to salinity, and culminated after 28 days of salt stress. SOD and POD activities declined correspondingly, followed by significant increases of MDA and MP, and leaf injury was finally observed. Compared with P. popularis, leaf Na⁺ and Cl⁻ in P. cv. I-214 exhibited a trend similar to P. popularis, but a lower salt-induced increase of MDA and permeability was observed and lighter leaf necrosis occurred. In contrast to P. popularis and P. cv. I-214, SOD and POD activities in P. euphratica leaves increased rapidly at the beginning of salt stress with a light soil NaCl concentration of 58.5 mmol/L. Furthermore, salt ion concentration, MDA content, and MP in P. euphratica leaves did not increase significantly during 28 days of increasing salt stress. Therefore, the increase in MP in P. popularis and P. cv. I-214 had a close relationship with a salt buildup in leaves under increasing salt stress. Salt-induced declines of SOD and POD activities might accelerate lipid peroxide and consequently resulted in ion leakage. P. euphratica rapidly activated antioxidant enzymes after the onset of salt stress, which might reduce the accumulation of reactive oxygen species and the subsequent acceleration of lipid peroxide. P. euphratica leaves exhibited a higher capacity to exclude salt in a longer period of increasing salinity, thus limited salt-induced lipid peroxide and MP, which contributed to membrane integrity maintenance and salt tolerance of P. euphratica. Translated from Journal of Beijing Forestry University, 2005, 27(3) (in Chinese)     

Anti-ulcer potential of Haldinia cordifolia

Haldinia cordifolia stem has been evaluated for its antiulcer potential. Enzyme assay-guided fractionation of the chloroform extract yielded 7-hydroxycoumarin as the active constituent which showed interesting H⁺/K⁺ ATPase inhibitory activity.     

Effects of Afforestation with Pinus contorta on Nutrient Content, Acidity and Exchangeable Cations in Peat

Nutrient contents, acidity and exchangeable cations in the upper300 mm of peat beneath lodgepole pine have been compared withthose in peat from contiguous unplanted areas at each of sixsites. Nitrogen, phosphorus and potassium contents, which aresignificantly higher in flushed blanket bog peat than in peatfrom either raised bog or unflushed blanket bog, are not significantlyaltered by the presence of trees. Calcium and magnesium contents,on the other hand, although similar in peat from different bogtypes, are lower in peat beneath the tree crop. ExchangeableCa²⁺ Mg²⁺ and K²⁺ are replaced by Na⁺ and H⁺ in planted peatbut the increase in H⁺ content exceeds the loss of base cations,reflecting a higher number of exchange sites. This increaseaccounts for lower values of both base saturation and pH inplanted peat and is attributed to enhanced decomposition promotedby aerobic conditions beneath the tree crop.     

Cross Breeding of Populus and Its Hybrids for Cold Resistance

Populus tomento a was crossed with P. tremuloidis, P grandiden ata, P. lba × P. grandidentata and P. alba × Ulmus s . ta pumila in order to maintain its rapid growth and high wood quality and improve its resistance to cold. Two methods were used to increase the germination rate from 1.5% to 41.1% and the remaining rate from 1.7% to 44.2%. Forty crossing combinations were conducted and 2 744 hybrid seedlings were obtained. MX. t4 × P grandiden ata (G-1-58), MX3 × P. tremuloidis (T-44-60), MX2 × P. tremuloidis (l-13-87-37) and MX2 × (P. alba × P. grandidentata) were regarded as superior combinations after analysis and selection. Thirty seedlings of these combinations and 11 triploid seedlings identified by counting their chromosomes were selected as super plants.     

Comparative Study on Antioxidative System in Normal and Vitrified Shoots of Populus suaveolens in Tissue Culture

To explore the physiological and biochemical mechanism of the occurrence of vitrified shoots of Populus suaveolens in tissue culture, the changes in water, chlorphyll, lignin, H202, phenylalanine ammonialyase (PAL), malonaldehyde (MDA), protective enzymatic systems, and some key enzymes involved in the ascorbate- glutathione cycle were comparatively studied in both normal and vitrified shoots of P suaveolens. The results show that the lower activities of peroxidase (POD), catalase (CAT), ascorbate peroxidase (APX), dehydroascorbate reductase (DHAR), glutathione reductase (GR) and PAL, and the less contents of chlorphyll, lignin, ascorbate (ASA) and reduced glutathione (GSH) as well as the lower ratios of ASA / DHA and GSH / GSSG are observed in vitrified shoots than in normal ones during the whole culture period, While in comparison with normal shoots, the higher activity of superoxide dismutase (SOD) and the more concentrations of water, H2O2, MDA, dehydroascorbate (DHA) and oxidized glutathione (GSSG) are found in vitrified shoots. Statistical analysis indicates that the enhanced activity of SOD and the decreased activities of CAT and POD as well as some enzymes involved in the ascorbate-glutathione cycle might be closely correlated to the accumulation of H202. The less regeneration of ASA and GSH and the lower capacity of the ascorbate-glutathione cycle observed in vitrified shoots might be due to a significant decrease in APX, MDAR, DHAR and GR activities and a decline in redox status of ASA and GSH. The decreases in chlorphyll content might result in a decline in photosynthesis. The lower activities of POD and PAL could result in the decrease of lignin synthesis and cell wall ligination, which might be the key factor leading to the increase in water content. It is concluded that the deficiency of detoxification capacity caused by the lower capacity of the ascorbate-glutathione pathway and the decreased activity of protective enzymatic system might lead to the large accumulation of H2O2 and the enhancement of membrane lipid peroxidation, which might be the main cause leading to the occurrence of vitrifying shoots of P.suaveolens in tissue culture.     

Deposition patterns in bulk precipitation and throughfall in a subtropical mixed forest in central-south China

The chemistry and deposition pattern in bulk precipitation andthroughfall (TF) were examined and evaluated based on the 3-yearobservations in Shaoshan subtropical deciduous-conifer mixedforest in central-south China. The TF chemistry was notablychanged when passing through canopies, which probably was attributedto the dry deposition (DD) on leaf surface and the canopy exchanges.Base cations' (Ca²⁺, Mg²⁺ and K⁺) fluxes were significantlyenriched in TF, in particular for K⁺. The annual K⁺ canopy exchangewas 12 times larger than DD, and canopy exchange of Ca²⁺, Mg²⁺and K⁺ was four times as high as the DD. The canopy exchangeof base cations in association with weak acid accounted for28.4 per cent of total leached base cations, which was one ofthe important factors to modify the TF chemistry.     

Ectomycorrhizal fungus (Paxillus involutus) and hydrogels affect performance of Populus euphratica exposed to drought stress

? Mycorrhizal fungi and hydrogels (water-absorbing polymers) can improve water availability for trees. The combination of both factors for plant performance under water limitation has not yet been studied.

? To investigate the influence of the ectomycorrhizal fungus Paxillus involutus, hydrogel and the combination of both factors, a drought-sensitive poplar, Populus euphratica, was examined in this study.

? After 16 weeks of inoculation, no ectomycorrhizas were found. Nevertheless, P. involutus-inoculated poplars displayed increased concentrations of soluble sugars and osmolality, leading to an improved water status. Growth was diminished compared with non-inoculated P. euphratica. The presence of hydrogels in the rooting medium resulted in increased biomass and higher plant water content and decreased the osmolality of plant tissues. Drought markedly decreased water contents in rooting medium and plants, and leaf chlorophyll fluorescence, and stimulated the root growth, concentrations of soluble sugars and osmolality in plants. Under drought conditions, P. euphratica exhibited osmoregulation by accumulation of low-molecular-weight carbohydrates.

? These data indicate that adding hydrogels to soils may improve the plant performance. The reasons for improved osmoregulation by fungi and hydrogels were probably related to their stimulating influence on the formation of soluble carbohydrates under drought conditions.

     

Spice oil cinnamaldehyde exhibits potent anticandidal activity against fluconazole resistant clinical isolates

Fluconazole resistance is becoming an important clinical concern. We studied the in vitro effects of cinnamaldehyde against 18 fluconazole-resistant Candida isolates. MIC₉₀ of cinnamaldehyde against different Candida isolates ranged 100–500 μg/ml. Growth and sensitivity of the organisms were significantly affected by cinnamaldehyde at different concentrations. The rapid irreversible action of this compound on fungal cells suggested membrane-located targets for its action. Insight studies to mechanism suggested that cinnamaldehyde exerts its antifungal activity by targeting sterol biosynthesis and plasma membrane ATPase activity. Inhibition of H⁺-ATPase leads to intracellular acidification and cell death. Toxicity against H9c2 rat cardiac myoblasts was studied to exclude the possibility of further associated cytotoxicity. The observed selectively fungicidal characteristics against fluconazole-resistant Candida isolates signify a promising candidature of this essential oil as an antifungal agent in treatments for candidosis.     

毛竹PeAP2基因及其启动子的克隆与表达初步分析

APETALA2(AP2)基因在植物生长发育过程中发挥着重要作用.利用RT-PCR和RACE方法,从毛竹中克隆到1个AP2同源基因的全长cDNA序列,命名为PeAP2.序列分析表明:PeAP2基因全长1 750 bp,其中,5′端非编码区106bp,3′端非编码区174 bp,开放阅读框1 470 bp,编码1个489 aa的蛋白,该蛋白含有2个AP2结构域,属于AP2/EREBP家族的AP2亚家族.PeAP2蛋白与来自其它单子叶植物的AP2蛋白均有着较高同源性,其中,与二穗短柄草的AP2蛋白同源性最高,达74.85％.实时定量PCR分析显示:PeAP2基因在毛竹的根、茎、叶、鞘和节5种器官中均有表达,其中,叶片中的表达丰度最高,鞘中次之,而在根、茎、节中的表达丰度接近,均较低.利用hiTAIL-PCR方法克隆获得了PeAP2上游启动子区序列1 359 bp,分析显示其含有光、激素等多种信号应答相关的作用元件.     

Cloning and sequence analysis of gene encoding plasma aquaporin of Tamarix albiflonum

Plant aquaporins are water-selected-channels in plants and are involved in seed germination, cell elongation, stoma movement, fertilization and so on. Some plant aquaporins also play an important role in drought stress response. In this paper, the gene encoding the Tamarix albiflonum Aquaporin (AQP) was amplified by 5′rapid amplification of cDNA end (RACE) on the basis of the sequence information obtained from the expressed sequence tag of the subtractive hybridization library constructed under PEG6000 stress. The cDNA of the T. albiflonum AQP gene is 1,043 bp long, encoding a protein of 287 amino acids with a predicted molecular mass of 30.9 kDa, has 6 transmembrane regions, and possessing the major intrinsic protein (MIP) family signal consensus sequence SGXHXNPAVT and the higher plant plasma membrane intrinsic protein (PIP) highly conservative sequence GGGANXXXXGY and TGI/TNPARSL /FGAA I/VI/VF/YN. A comparative molecular analysis of the nucleotide sequence in National Center for Biotechnology Information (NCBI) databases showed that it shared 95% homology with the gene of Arabidopsis thaliana (MIP-C), with a theoretical isoelectric point 8.84. __________ Translated from Bulletin of Botanical Research, 2006, 26(4): 475–479 [译自: 植物研究]     

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.     

盐胁迫对16种幼苗渗透调节物质的影响

采用盆栽的方法,对16种2年生幼苗盐(NaCl)胁迫40 d,并进行耐盐性研究。结果表明:随盐浓度的升高,各树种的脯氨酸、可溶性糖和甜菜碱含量均呈上升趋势,在400 mmol·L-1盐浓度下,沙枣和水蜡的脯氨酸含量比对照分别增加了662.0%和562.8%,而可溶性蛋白含量在各树种间差异较大;Na+和Cl-含量在不同部位(根、茎和叶)均随盐浓度增加而增加,鞑靼忍冬、药鼠李、风箱树和醉鱼草叶片中Na+含量增加了5 20倍。K+和Ca2+含量随树种不同表现各异。综合各树种盐害症状和渗透调节物质变化特性,认为柽柳、唐古特白刺、西伯利亚白刺、卫矛和沙柳具有高度耐盐性,水蜡、沙枣、糖朴、美国皂荚和药鼠李具有中高度耐盐性,杜梨、竹柳、鞑靼忍冬、醉鱼草、风箱树和美国白蜡具有中度耐盐性。脯氨酸、可溶性糖和矿质元素含量(尤其是Na+、K+和Cl-)的变化对植物耐盐能力的影响较普遍也较大。     

金花茶查尔酮异构酶基因全长克隆与表达的初步研究

利用RT-PCR和RACE技术,从我国珍稀濒危植物金花茶花瓣中获得了查尔酮异构酶(Chalcone isomerase,CHI)基因的cDNA全长,命名为Cn-CHI,GenBank登录号HQ269805.1。碱基序列分析表明,Cn-CHI基因全长953 bp,包含56 bp的5’非翻译区、204 bp的3’ 非翻译区和一个长为693 bp编码230个氨基酸的开放阅读框。氨基酸序列比对分析表明,Cn-CHI基因编码蛋白与蔷薇科、杜鹃花科、茄科等植物的CHI蛋白同源性都在75%以上,与山茶科山茶属茶树CHI蛋白同源性高达99%。相对荧光定量PCR分析表明,Cn-CHI基因在金花茶花蕾发育过程中呈现先急剧上升后平缓下降的趋势;在花器官的不同部位中,Cn-CHI基因表达量最高的是雌蕊,其次是雄蕊,在萼片和花瓣中的表达量较低且相差不大。     

广州市酸雨对不同森林冠层淋溶规律的研究

１９９８年４月至１９９９年３月对广州市白云山马尾松林和常绿阔叶林、广州市龙眼洞马尾松林两试验点进行了酸雨的监测,并测定和分析了林内穿透雨物理量及化学量,旨在探讨酸雨对不同森林冠层养分淋溶规律的影响。结果表明：（１）广州市酸雨占次数的７９．７％或占降雨量的９５．１％。（２）酸雨通过林冠层后,ｐＨ值明显增加。（３）在马尾松林和常绿阔叶林中,某些单次降雨出现ＳＯ４＾２－、ＮＯ３＾－、ＮＨ４＾＋Ａｌ＾３＋、Ｎａ＾＋的负淋溶现象,说明森林对这些离子（特别是ＮＯ３＾－、Ａａ＾３＋）具有吸收作用;阔叶林全年的ＮＯ３＾－和Ａｌ＾３＋净淋溶为负值,说明阔叶林比马尾松林对这两种离子具有更强的吸收能力。（４）雨水酸度增加（即ｐＨ值减小）,明显提高阳离子Ｃａ＾２＋、Ｍｇ＾２＋、Ｋ＾＋和Ｎａ＾＋冠层淋溶面分率。（５）ＮＨ４＾＋、ＳＯ４＾     

Verbascoside isolated from Tectona grandis mediates gastric protection in rats via inhibiting proton pump activity

Evidences have suggested that Tectona grandis (TG) attenuates gastric mucosal injury; however its mechanism has not yet been established. The aim of present study was to evaluate the gastroprotective mechanism of ethanolic extract of TG (E-EtOH), butanolic fraction (Fr-Bu) and to identify its active constituents. Anti-ulcer activities were evaluated against cold restraint (CRU) and pyloric ligation (PL) induced gastric ulcer models and further confirmed through H⁺ K⁺-ATPase inhibitory activity. Cytoprotective activity was evaluated in alcohol (AL) induced gastric ulcer model and further through PGE₂ level. E-EtOH and Fr-Bu attenuated ulcer formation in CRU. Moreover E-EtOH and Fr-Bu displayed potent anti-secretory activity as evident through reduced free acidity and pepsin activity in PL, confirmed further by in vitro inhibition of H⁺ K⁺-ATPase activity. In addition cytoprotective potential of E-EtOH and Fr-Bu were apparent with protection in AL model, increased PGE₂ content and enhanced mucin level in PL. Phytochemical investigations of Fr-Bu yielded terpenoides and a phenolic glycoside, verbascoside. The anti-secretory mechanism of verbascoside mediated apparently through inhibition of H⁺ K⁺-ATPase with corresponding decrease in plasma gastrin level, is novel to our finding. Gastroprotection elicited by TG might be through proton pump inhibition and consequent augmentation of the defensive mechanism.