烟草叶片液泡内氨基酸成份研究

液泡是植物细胞中特有的大型细胞器,具有重要的生理功能。本文报导了用双酶直接酶解法从烟草叶肉细胞中分离原生质体和完整液泡。在最适保存条件下,原生质体和液泡分别在３６和１２小时后,尚有一半保持活力。液泡内含有大量游离氨基酸,液泡膜ＡＴＰａｓｅ的最适ｐＨ为７．０,受Ｃｌ－激活,受ＮＯ３－抑制。     

聚多曲霉菌原生质体和液泡的制备及优化

原生质体的大量制备是研究原生质体转化、诱变、融合等技术的关键,液泡的制备对研究液泡中分解、转运有机物的特性具有重要意义,但目前分离技术还不够成熟.本研究从聚多曲霉菌菌丝中分离原生质体和液泡并对分离条件进行优化,以聚多曲霉菌DJ515-2菌丝为材料,探索不同因素对原生质体和液泡制备分离效果的影响.结果表明,聚多曲霉菌DJ515-2在菌龄42 h,以3％纤维素酶、1％蜗牛酶和3％的溶壁酶组成复合酶液,25℃下酶解4 h,原生质体达到最大产量,为5.167×105个/mL.同时,在此基础上裂解液泡的最优条件为pH7.5、1.0mol/L KCl、0.020％Triton X-10,其产量达2.63×105个/mL,产率为原生质体的50.8％,相比采用多元碱化合物诱导真菌原生质体裂解释放液泡的产率增加了 40％～45％.本研究为真菌和植物的各种原生质体技术及基于亚细胞层面的研究提供了材料基础.     

植物原生质体的纯化

本文叙述了用改良离心管漂洗纯化原生质体中的细胞和亚细胞碎片的方法。在这方法中使用了一个通底的带有小细管的改良离心管。经过这种纯化,原生质体的产量是较高的。玉米(Zea mays L.)茎每克鲜重平均收获6.2×10~5个原生质体,矮牵牛(Petunia hybrida Vilm.)叶每克鲜重平均收获2.7×10~6个原生质体。纯化后植物原生质体的活力是较高的,当培养时,观察到玉米茎原生质体的细胞分裂;矮牵牛叶片原生质体进行了细胞分裂,并发育成愈伤组织,愈伤组织转移到分化培养基上长出了根。     

植物原生质体培养方法

1　植物原生质体培养的简史植物细胞原生质体 ,在植物学上指植物细胞通过质壁分离后 ,可以和细胞壁分开的那部分细胞物质。原生质体分离纯化后 ,须在适当的培养基上应用适当的培养方法 ,才能再生细胞壁 ,并启动细胞持续分裂 ,直至形成细胞团、长成愈伤组织或胚状体、分化和发育成苗 ,最终再生完整植株。其中 ,选择合适的培养方法始终是原生质体培养中最基础也是最关键的一环。植物原生质体培养方法起源于植物单细胞培养方法。早在 190 2年 ,Haberlant通过实验就预言 :体外培养单个细胞可通过其分裂得到培养组织。直到 1954年 ,植物单细胞培…     

对原生质体纯化技术的评估

苏格兰作物研究所及Polytechnic的S.Millam等比较了纯化芸苔原生质体的3种技术的效果:(1)在13％甘露醇中低速(150g)离心沉淀;(2)用蔗糖溶液浮集;(3)在覆以等体积Percoll的甘露醇中的低速离心沉淀。 Millam等未发现3种方法中总原生质俸回收率有显著差异。方法(1)、(3)的同收率为35.6～37.6％,而方法(2)的回收率为26.6％。但是,液泡化与叶绿体型原生质体亚类间的相对回收率有显著差异。方法(3)的液泡化原生质体回收     

鱼腥藻Anabaena PCC7120原生质球和液泡的同步诱导

植物和真菌的液泡,都是通过原生质体途径被分离后才得以深入研究.要分离蓝藻液泡,首先要求制备蓝藻原生质体(球),而这一技术在蓝藻方面长期不过关.最近十年来,作者在这方面进行了不断的努力,先后在蓝藻原生质球制备1、培养再生和融合2等方面获得进展,这方面的研究导致了蓝藻液泡的重新发现3.所发现的液泡由无机盐诱导产生,经电镜检查为单位膜所包围,故确定为液泡.借助较为成功的原生质球制备技术,首先分离了无机盐诱导形成的液泡,在此基础上进一步发现和分离了非诱导液泡4,在分离非诱导液泡的试验过程中发现了原生质球和液泡的同步诱导作用.       

植物原生质体的制备与活力检测研究进展

原生质体是进行植物遗传改良和细胞各种生理生化特性研究的平台.本文对近些年制备原生质体的材料选择、预处理、游离、纯化和活力检测等方面的研究进展进行了综述,分析了影响原生质体的分离和纯化的有关因素,并根据相关文献讨论了今后原生质体重点研究方向.     

空间实验室提高了植物原生质体的融合

目前,电融合正用作植物原生质体融合以产生体细胞杂种和组织培养物的常规方法。然而,还存在着几个问题。为了保持细胞融合位置、防止细胞旋转,施加电流的时间必须比获得融合所需要的可逆性膜分解时间更长,电场强度更大。这就导致融合细胞活性的降低。此外,也很难进行不同比重的(如液泡化和非液泡化的)原生质体的细胞融合,因为,它们不能悬浮在同样的融合培养基中。作为西德非载人探空火箭计划的一部分。W. Mehrle及其同事进行了一个无线电控制     

丹参悬浮培养细胞原生质体的制备和活力检测

对丹参悬浮培养细胞原生质体制备条件进行了研究,并利用FDA染色和钙离子荧光探针Fluo-3/AM装载对制备得到的原生质体的活力和功能进行了检测。丹参悬浮培养细胞原生质体的制备条件为:悬浮培养细胞酶解的适宜酶液组合为纤维素酶1.5%、果胶酶0.3%和离析酶0.5%;适宜的甘露醇浓度为0.4 mol/L;酶解时间为12 h;在600 r/min转速下离心5 min收集,纯化得到原生质体,其产量为1.1×106/g FW,FDA检测显示其活力为95%以上,荧光探针Fluo-3/AM可成功装载到原生质体中。     

植物原生质体在分子细胞生物学研究中的应用

植物原生质体是去除了细胞壁的裸露细胞,其具有细胞全能性,现广泛应用于植物分子细胞生物学的研究中,可以大大缩减实验周期,并有助于得到体内实验的实时检测数据。该文除了介绍植物原生质体的提取和纯化方法外,还对国内外利用各种植物的原生质体进行细胞瞬时转化、亚细胞定位、细胞融合和大分子复合物相互作用等试验进行了总结和讨论。植物原生质体还可用于基因表达模式的实时检测,并作为生物反应器的受体细胞进行代谢物的体外生产。此外,还对当前该技术所面临的瓶颈进行了分析,为植物原生质体在分子细胞生物学领域的应用提供帮助,为技术的优化和推广提供参考。     

A procedure for isolating vacuoles from leaves of the halophyte Suaeda maritima

Abstract. A method is described here for isolating protoplasts and vacuoles from leaves of the halophyte Suacda maritima. Integrity of the protoplasts and vacuoles was tested by staining and shown to be more than 75%, while use of biochemical markers, staining and light microscopy suggested a high degree of purity of the vacuoles. Phosphatase and NADH cytochrome- c -reductase were associated with vacuoles; phosphatase showed an eight-fold enrichment and NADH cytochrome- c -reductase a 3.5-fold enrichment relative to protoplasts. The vacuoles contained only 15% of the protein in protoplasts.     

Barley aleurone cells contain two types of vacuoles. Characterization Of lytic organelles by use of fluorescent probes

Light microscopy was used to study the structure and function of vacuoles in living protoplasts of barley (Hordeum vulgare cv Himalaya) aleurone. Light microscopy showed that aleurone protoplasts contain two distinct types of vacuole: the protein storage vacuole and a lysosome-like organelle, which we have called the secondary vacuole. Fluorescence microscopy using pH-sensitive fluorescent probes and a fluorogenic substrate for cysteine proteases showed that both protein storage vacuoles and secondary vacuoles are acidic, lytic organelles. Ratio imaging showed that the pH of secondary vacuoles was lower in aleurone protoplasts incubated in gibberellic acid than in those incubated in abscisic acid. Uptake of fluorescent probes into intact, isolated protein storage vacuoles and secondary vacuoles required ATP and occurred via at least two types of vanadate-sensitive, ATP-dependent tonoplast transporters. One transporter catalyzed the accumulation of glutathione-conjugated probes, and another transported probes not conjugated to glutathione.     

Electric field-induced fusion of isolated vacuoles and protoplasts of different developmental and metabolic provenience

Using an electric field pulse technique, we induced fusion between vacuoles and protoplasts of Kalanchoë daigremontiana , between protoplasts from etiolated and green leaf mesophyll, and between mesophyll protoplasts from plants of different physiological properties ( Avena sativa : C₃ mechanism of photosynthesis, Kalanchoë daigremontiana : crassulacean acid metabolism). Close membrane contact amongst protoplasts or between protoplasts and vacuoles (as required for fusion) was achieved by the application of an alternating, non-uniform electric field to the suspension. Due to the dielectrophoresis effect the cells attach to each other along the field lines. The fusion process is initiated by the injection of an electric field pulse of high intensity and short duration (μs range). The field intensity has to be sufficiently high to induce reversible breakdown in the area of close membrane contact. After the application of the field pulse, the fusion process is initiated and completed within seconds to a few minutes, depending on the material investigated.
Fusion occurs between protoplasts and vacuoles as well as between protoplasts of different species. Both tonoplast and plasma membranes completely intermingled, indicating that in contrast to suggestions in the literature these membranes are compatible. Furthermore the cytoplasms of etiolated and green protoplasts obviously do not mix after fusion is completed, as etioplasts and chloroplasts kept separated from each other. In all experiments the volume of the fusion product equalled the sum of the compartments that underwent fusion. The wide spectrum of possible applications resulting from these fusion experiments in relation to metabolic problems is discussed.     

Compartmentation of aluminium in leaves of an Al-accumulator,Fagopyrum esculentum Moench

Buckwheat (Fagopyrum esculentum Moench.) is an Al-accumulating plant, but the internal mechanism(s) of detoxification of Al is not fully understood. We investigated the subcellular localization of Al in the leaves of this plant (cv. Jianxi) by directly isolating protoplasts and vacuoles. Pure protoplasts and vacuoles from the leaves of buckwheat, grown hydroponically in Al solution, were obtained based on light-microscopic observation and the activities of marker enzymes of cytosol and vacuoles. More than 80% of total Al in the leaves was present in the protoplasts, and was identified as an Al-oxalate complex (1:3 ratio) by (27)Al-nuclear magnetic resonance. Oxalate and Al in the protoplasts was localized in the vacuoles. These results suggest that internal detoxification of Al in the buckwheat leaves is achieved by both complexation with oxalate and sequestration into vacuoles.     

Simple method to isolate vacuoles and protoplasts for patch-clamp experiments

It was not possible to obtain protoplasts or vacuoles from the thallus of the liverwortConocephalum conicum by applying cell-wall-degrading enzymes. Therefore, a surgical method was developed to isolate protoplasts and vacuoles. A thallus was plasmolyzed and cut. The few protoplasts along the cutting edge that were not destroyed emerged from the edge under deplasmolysis and became thus accessible for a patch pipette. Whereas under slightly hypoosmolar conditions the emerging protoplast remained largely intact, more hypoosmolar conditions gave rise to isolated vacuoles. This method to isolate protoplasts and vacuoles could also be applied to other plant tissues like leaves ofArabidopsis thaliana. Patch-clamp measurements were performed with isolated vacuoles and excised tonoplast patches. A slowly activating vacuolar channel inC. conicum displayed the characteristic features of higher-plant slowly activating vacuolar channels.Abbreviations AP action potential - SV channel slowly activating vacuolar channel     

Presence of the cyanogenic glucoside dhurrin in isolated vacuoles from sorghum

Large numbers of vacuoles (10⁶-10⁷) have been isolated from Sorghum bicolor protoplasts and analyzed for the cyanogenic glucoside dhurrin. Leaves from light-grown seedlings were incubated for 4 hours in 1.5% cellulysin and 0.5% macerase to yield mesophyll protoplasts which then were recovered by centrifugation, quantitated by a hemocytometer, and assayed for cyanogenic glucosides. Mature vacuoles, released from the protoplasts by osmotic shock, were purified on a discontinuous Ficoll gradient and monitored for intactness by their ability to maintain a slightly acid interior while suspended in an alkaline buffer as indicated by neutral red stain. Cyanide analysis of the protoplasts and the vacuoles obtained there from yielded equivalent values of 11 μmoles of cyanogenic glucoside dhurrin per 10⁷ protoplasts or 10⁷ vacuoles. This work supports an earlier study from this laboratory which demonstrated that the vacuole is the site of accumulation of the cyanogenic glucoside in Sorghum.     

Quantitative characterization of protoplasts and vacuoles from suspension-cultured cells of Catharanthus roseus

A simple and efficient procedure for isolation of protoplasts and then vacuoles from cultured cells of Catharanthus roseus (L.) G. Don is presented. Protoplasts were disrupted by an osmotic shock and the vacuoles vere purified by flotation on a single-step gradient. A comparison of the content and concentration of solutes (proteins, sugars, organic acids, alkaloids, mineral ions) in protoplasts and cells showed that massive and selective losses occur for most solutes during protoplast preparation. These are attributed to the osmotic adjustment and changes of membrane permeabilities occurring during plasmolysis. Data concerning the size, yield and purity of the isolated vacuoles are discussed. By analysis of isolated vacuoles, the vacuolar concentration and localization of solutes within protoplasts have been determined. The limits of this latter approach are stressed, however. Some evidence in favour of the selection of a special class of vacuoles during isolation is reported and discussed.     

1-Aminocyclopropane-1-carboxylic-acid-dependent ethylene production during re-formation of vacuoles in evacuolated protoplasts of Petunia hybrida

Summary Ethylene formation from 1-aminocycloprane-1-carboxylic acid (ACC) was studied in whole protoplasts, evaluolated protoplasts and isolated vacuoles from mesophyll cells of Petunia hybrida L. cv. Pink Magic. The re-formation of the large, central vacuole in evacuolated protoplasts and morphological characteristics of both types of protoplasts were examined by electron microscopy. Both the normal, whole protoplasts and vacuoles isolated from them produced ethylene from ACC at similar rates. Freshly-prepared evacuolated protoplasts had lost the capacity to produce ethylene. Re-formation of the central vacuole in these evacuolated protoplasts occurred between 14 to 17 h of incubation in the recovery medium and was followed by the development of ethyleneforming activity. Both these processes were inhibited by cycloheximide, indicating a requirement for new protein synthesis. Light stimulated the conversion of ACC to ethylene in both the regenerating, whole protoplasts and the evacuolated protoplasts that had re-formed the central vacuole.Abbreviations ACC 1-aminocyclopropane-1-carboxylic acid - AVG aminoethoxyvinylglycine - CHI cycloheximide     

Sodium, Potassium, Chloride, and Betaine Concentrations in Isolated Vacuoles from Salt-Grown Atriplex gmelini Leaves

Vacuoles were isolated via protoplasts from the leaves of a halophyte Atriplex gmelini C.A.Mey., grown in culture solution supplemented with 250 millimolar NaCl. Lysis of the protoplasts was induced by lowering the medium osmolarity (1.2 to 1.0 molar sorbitol) and adding a detergent, a synthesized cholate derivative, 3-([3-cholamidopropyl] dimethylammonio)-1-propanesulfonate at a concentration of 0.5 millimolar and the resulting vacuoles were purified by successive dilution and floatation. Isolated vacuoles contained almost the same concentration of sodium (569 millimolar) and chloride (260 millimolar) as recorded in protoplasts (582 and 254 millimolar, respectively), suggesting that the vacuoles are the major sequestration site of NaCl in leaves of halophytes. Betaine concentration in the protoplasts was about 16 millimolar, while that in vacuoles was only about 0.24 millimolar, indicating that betaine is accumulated in the cytoplasm as a compatible solute.     

Transport and subcellular localization of polyamines in carrot protoplasts and vacuoles

Putrescine and spermidine uptake in carrot (Daucus carota L., cv “Tip top”) protoplasts and isolated vacuoles was studied. Protoplasts and vacuoles accumulated polyamines very quickly, with maximum absorption within 1 to 2 minutes. The insertion of a washing layer containing 100 millimolar unlabeled putrescine or spermidine did not change this pattern, but strongly reduced the uptake of putrescine and spermidine in protoplasts and in vacuoles. The dependence of spermidine uptake on the external concentration was linear up to the highest concentrations tested in protoplasts, while that in vacuoles showed saturation kinetics below 1 millimolar (K_m = 61.8 micromolar) and a linear component from 1 to 50 millimolar. Spermidine uptake in protoplasts increased linearly between pH 5.5 and 7.0, while there was a distinct optimum at pH 7.0 for vacuoles. Preincubation of protoplasts with 1 millimolar Ca²⁺ affected only surface binding but not transport into the cells. Nonpermeant polycations such as La³⁺ and polylysine inhibited spermidine uptake into protoplasts. Compartmentation studies showed that putrescine and spermidine were partly vacuolar in location and that exogenously applied spermidine could be recovered inside the cells. The characteristics of the protoplast and vacuolar uptake system induce us to put forward the hypothesis of a passive influx of polyamines through the plasmalemma and of the presence of a carrier-mediated transport system localized in the tonoplast.