Two Proton Pumps Operate in Parallel Across the Tonoplast of Vacuoles Isolated from Suspension Cells of Chenopodium rubrum L.*

The kinetics of vacuolar acidification upon addition of ATP and/or pyrophosphate (PPi) has been assayed on single immobilized vacuoles by computer-aided microfluorimetry of 9-aminoacridine, and by acridine orange absorption photometry on vacuole suspensions isolated from green suspension cells of Chenopodium rubrum L. Two proton pumps at the tonoplast, an ATPase and a pyrophosphatase (PPase), operate in parallel to acidify the vacuole with different contributions adding up to a transtonoplast Δ pH of 2.6 pH units at external pH 7.2. The saturable components of proton pumping reach half maximal velocity with 0.32 ± 0.06 mM ATP and 23 ± 2.5 μM PPi, respectively. At saturating substrate concentrations, ATPase and PPase hydrolyse ATP and PPi, respectively, at a ratio of 2.3. The same ratio holds for the corresponding proton fluxes maintaining a given steady-state vacuolar pH. We conclude that both pumps operate at the same stoichiometry.     

减压、低温条件下制样的几种植物液泡图象

样品制作中选用的固定剂不同,在电子显微镜下所得的图象差异甚大。高锰酸钾固定的样品中(Luft 1956,Mollenhauer1959),液泡是一个空泡,核也是一个空泡。用四氧化锇(Palade 1952)和醛类-四氧化锇双固定的样品(Sabatini等1963)核内有物质,但液泡仍是一空泡。所有电子显微技术样品制作都离不开固定、脱水、包埋等过程中化学试剂的反复处理,化学试剂处理细胞会抽去部分细胞内含物;或与细胞某些内含物结合形成络合物。从而     

Retention in Situ and Spectral Analysis of Fluorescent Vacuole Components in Sections of Plant Tissues

The contents of plant vacuoles vary in different organs and with the health of the plant, but little is known of the cell-to-cell distribution of soluble organic compounds within plant tissues. Soluble fluorescent phenolic compounds can be immobilized in plant tissues using an anhydrous freeze-substitution and resin embedment process. The vacuolar fluorescence can be characterized in fluorescence photomicrographs for variations in color and intensity, or more quantitatively with spectra obtained using a microspectrofluorometer. This is demonstrated here in freeze-substituted roots and leaves of soybean. Excitation and emission spectra of individual vacuoles can be compared with spectra of pure compounds to form profiles of the varied phenolic contents of plant vacuoles. Such analyses will add an important anatomical dimension to the study of plant defense and stress responses.     

Molecular Evidence for the Occurrence of H+-Transporting V-ATPase Subunit D and Two Different Forms of Subunit E in Leaves of the Obligate CAM Species Kalanchoë daigremontiana

Membrane proteins of purified tonoplast vesicles from leaves of Kalanchoë daigremontiana Hamet et Perrier were solubilized by the non-ionic detergent Triton X-114 and subsequently separated by MonoQ® anion-exchange chromatography. Special attention was given to the range of molecular masses around 30 kDa comprising the central stalk subunit peptides of the H⁺-transporting V-ATPase. Three polypeptides of apparent molecular masses of 32, 33 and 34 kDa were separated. Proteolytic fragments were obtained by trypsin digestion. Analysis by matrix-assisted laser desorption ionization (MALDI) mass spectrometry of tryptic fragments of the 32 and 33 kDa peptides and protein data- bank comparisons showed that they are two different forms of subunit E. N-terminal amino acid sequencing of tryptic fragments of the 34 kDa peptide showed that it is subunit D. This work provides for the first time unequivocal molecular evidence that the central stalk of the V-ATPase of the obligate CAM plant K. daigremontiana includes subunit D and different forms of subunit E.     

New Insight into the Structure and Regulation of the Plant Vacuolar H+-ATPase

Plant cells are characterized by a highly active secretory system that includes the large central vacuole found in most differentiated tissues. The plant vacuolar H⁺-ATPase plays an essential role in maintaining the ionic and metabolic gradients across endomembranes, in activating transport processes and vesicle dynamics, and, hence, is indispensable for plant growth, development, and adaptation to changing environmental conditions. The review summarizes recent advances in elucidating the structure, subunit composition, localization, and regulation of plant V-ATPase. Emerging knowledge on subunit isogenes from Arabidopsis and rice genomic sequences as well as from Mesembryanthemum illustrates another level of complexity, the regulation of isogene expression and function of subunit isoforms. To this end, the review attempts to define directions of future research on plant V-ATPase.     

农杆菌属介导的植物细胞遗传转化研究现状

自20实际70年代末提出农杆菌属介导产生转基因植物以来,已相继有很多农作物和园艺植物被农杆菌成功转化。在一些发达国家已有大量种植的转基因经济作物如玉米、大豆、棉花、马铃薯、番茄等。但越来越多的经济作物以及园林树木和园艺植物对粒子轰击等转化方法无效或效果不佳,因而农杆菌因其介导的遗传转化率较高就越来越受到重视。但是,农杆菌介导的转基因在表达稳定性和可预见性方面仍然存在诸多问题。本文介绍了有关农杆菌分子生物学的基础研究及提高农杆菌介导的遗传转化效率的研究进展。     

The Contractile Vacuole of Amoeba proteus. II. Numerical Analysis of the Steady-State Hypothesis*

SYNOPSIS. At temperatures below 15 C, the contractile vacuole cycle of Amoeba proteus includes a presystolic plateau. The hypothesis attributing this plateau to a steady-state equilibrium between active filling processes and osmotic losses of water from the vacuole into the cytoplasm has been expressed in an equation predicting vacuolar diameter as a function of time for the later part of the cycle. Computer-generated model cycles have been compared with actual recorded cycles at 15 C, 10 C and 5 C and conditions of best fit were determined. Statistical analysis shows that recorded cycles are quite compatible with the steady-state hypothesis.     

Lysosomal Activities of the Vacuole in Damaged and Recovering Plant Cells

ON the evidence from cytochemical studies it seems that a wide range of lysosomal enzymes is present in the vacuole of plant cells^1,2. Cytological studies have also suggested that biochemically identified lysosomal particles are equivalent to small vacuoles and because membranous material has been seen within these vacuoles it was suggested that the plant cell vacuole is functionally equivalent to the secondary lysosome of the animal cell³. In this role it would be engaged in the. breakdown of cytoplasmic constituents and, using light microscope cinematography, it has been shown that after certain experimental environmental changes, structures appeared and then disappeared in the vacuoles of yeast cells⁴. Generally, electron micrographs of plant cells have remnants of disorganized membranes within their vacuoles, which have consequently come to be regarded as repositories of waste materials.     

Expression of a cauliflower tonoplast aquaporin tagged with GFP in tobacco suspension cells correlates with an increase in cell size

In plants, vacuoles are essential organelles that undergo dynamic volume changes during cell growth due to rapid and high flow of water through tonoplast water-carrying channels composed of integral proteins (tonoplast aquaporins). The tonoplast BobTIP26-1 from cauliflower has previously been shown to be an efficient active aquaporin in Xenopus leavis oocytes. In this study we used tobacco (Nicotiana tabacum cv. Wisconsin 38) suspension cells to examine the effect of BobTIP26-1 expression. In order to follow the intracellular localisation of the protein in real time, the gfp sequence was fused downstream to the BobTIP26-1 coding region. The fusion protein BobTIP26-1::GFP is less active than BobTIP26-1 by itself when expressed in Xenopus oocytes. Nevertheless, this fusion protein is well targeted to the tonoplast of the plant suspension cell when expressed via Agrobacterium co-cultivation. A complex tonoplast labelling is shown when young vacuolated cells are observed. The expression of the fusion protein does not affect the growth rate of the cells but increases their volume. We postulate that the increase in cell volume is triggered by the fusion protein allowing vacuolar volume increase.     

Fine Structure of the Contractile Vacuole Pore in Paramecium*

The pore through which a Paramecium contractile vacuole communicates with the external environment is a 1.2 μm long and 1 μm diameter cylindrical orifice in the pellicle. During diastole, the vacuole:pore junction is closed by a substantial diaphragm which parts to the side at systole. The diaphragm is composed of inner and outer membranes continuous with the vacuole and pore membranes, respectively, and an intervening cytoplasmic layer containing filaments and irregular membranous tubules and vesicles. Microtubules, organized into 2 sets, are an important component of the pore apparatus. One set of ～ 16 microtubules forms an annulus around the pore. These microtubules are organized into a right-handed helix with a pitch of 0.5-0.6 μm, and thus complete slightly more than 2 turns in their course from the level of the diaphragm to the pore outer lip. They appear to be embedded in a layer of dense material immediately adjacent to the pore membrane. The other set consists of 5 or more bands of 10–20 microtubules which radiate in a slight left-handed helix from an insertion at the pore out over the vacuole surface to the ampullae.     

Behavior of the Contractile Vacuole of Tetrahymena pyriformis W: A Redescription with Comments on the Terminology

SYNOPSIS. The behavior of the contractile vacuole of Tetrahymena pyriformis W has been recorded and analyzed quantitatively by cinephotography. The vacuole fills in a stepwise fashion by the confluence of ampullae which appear regularly at the beginning of systole and whose membranes are continuous with that of the contractile vacuole throughout the cycle. The vacuole may subsequently fill slowly by a means not discernible by light microscopy. The vacuole rounds up at the beginning of systole and shortly thereafter the ampullae reappear around the perimeter of the vacuole. They are expanded by fluid forced into them from the vacuole. Round-up and the mode of growth of the ampullae indicate that the contractile vacuole is truly contractile. Expulsion occurs soon after the appearance of the ampullae and terminates the cycle. Contraction is initiated at regular intervals by a timing mechanism which is independent of the size of the vacuole. Suitable terminology to describe the structure and behavior of the contractile vacuole is discussed.     

高等植物根边缘细胞的发育调控及其生物学功能

植物根边缘细胞是从根冠表皮游离出来并聚集在根尖周围的一群特殊细胞 ,以前曾称为根冠脱落细胞。最近的证据表明 ,绝大多数物种边缘细胞具有生物学活性 ,其发育是受内外信号调控。边缘细胞一旦从根表皮游离后 ,其代谢活性大大上升、基因表达明显不同于根冠细胞。最近 ,与边缘细胞发育早期和晚期相关的两个基因PsUGT1和RCPME1分别被克隆和鉴定。边缘细胞能特异性地合成、分泌一系列的化学物质 ,包括花色素苷、抗生素、特异性酶类及其他化学物质能抑制或促进根际周围的细菌、真菌、病毒、线虫等的生长以及中和根际周围一些有毒化学物质如铝毒。因此 ,边缘细胞在植物生长发育过程中起着多种生物学功能     

PPiase-Activated ATP-Dependent H+ Transport at the Tonoplast of Mesophyll Cells of the CAM Plant Kalanchoë daigremontiana*

The co-ordinated action of the two proton-transporting enzymes at the tonoplast of the CAM plants. daigremontiana, viz. the ATPase and the PP_iase, was studied by measuring fluorescent dye quenching. The initial rates of ATP and PP_i-dependent H⁺ transport into tonoplast vesicles were additive, i.e. the sum of the rates obtained with each substrate alone was in the range obtained with both substrates added together at the same time. Conversely, the activities of the two H⁺ pumps were non-additive in establishing the steady-state level, indicating that the final steady state was under thermodynamic control of a maximal attainable proton gradient. The initial rates of ATP-dependent H⁺ transport were stimulated enormously if ATP was added a few minutes after pre-energization of the vesicles with PP_i. This stimulation was observed only when the PP_iase was active. A similar effect was not found for PP_i-dependent H⁺ transport after pre-energization with ATP. Hence, a PP_iase-activated ATP-dependent H⁺ transport can be distinguished from the basic ATP- and the basic PP_i-dependent H⁺ transport. In parallel a PP_i-dependent stimulation of ATP hydrolysis in the absence of ionophores was measured, which can only be attributed to the activity of the PP_iase. PP_iase-activated ATP-dependent H⁺ transport depends on the presence of permeant anions. It shows properties of both H⁺ transport activities, i.e. the chloride and malate stimulation and the DCCD inhibition of the ATP-dependent H⁺ transport activity, the nitrate stimulation and the KF inhibition of the PP_i-dependent H⁺ transport activity. Only MgPP_i and MgATP were effective as the respective substrates. The PP_iase-activated ATP-dependent H⁺ transport had a half life of about 5–9 minutes. It is concluded that the PP_iase may play an important role in kinetic regulation of the ATPase, and implications for CAM metabolism are discussed.     

Evidence for nickel/proton antiport activity at the tonoplast of the hyperaccumulator plant Alyssum lesbiacum

The mechanism of nickel uptake into vacuoles isolated from leaf tissue of Alyssum lesbiacum was investigated to help understand the ability of this species to hyperaccumulate Ni. An imaging system was designed to monitor Ni uptake by single vacuoles using the metal-sensitive fluorescent dye, Newport Green. Nickel uptake into isolated vacuoles from leaf tissue of A. lesbiacum was enhanced by the presence of Mg/ATP, presumably via energisation of the vacuolar H(+)-ATPase (V-ATPase). This ATP-stimulated Ni uptake was abolished by bafilomycin (a diagnostic inhibitor of the V-ATPase) and by dissipation of the transmembrane pH difference with an uncoupler. These observations are consistent with Ni(2+)/nH(+) antiport activity at the tonoplast driven by a proton electrochemical gradient established by the V-ATPase, which would provide a mechanism for secondary active transport of Ni(2+) into the vacuole. This study provides insights into the molecular basis of Ni tolerance in Alyssum, and may aid in the identification of genes involved in Ni hyperaccumulation.     

Using Fluorescent Proteins to Visualize and Quantitate Chlamydia Vacuole Growth Dynamics in Living Cells

The obligate intracellular bacterium Chlamydia elicits a great burden on global public health. C. trachomatis is the leading bacterial cause of sexually transmitted infection and also the primary cause of preventable blindness in the world. An essential determinant for successful infection of host cells by Chlamydia is the bacterium''s ability to manipulate host cell signaling from within a novel, vacuolar compartment called the inclusion. From within the inclusion, Chlamydia acquire nutrients required for their 2-3 day developmental growth, and they additionally secrete a panel of effector proteins onto the cytosolic face of the vacuole membrane and into the host cytosol. Gaps in our understanding of Chlamydia biology, however, present significant challenges for visualizing and analyzing this intracellular compartment. Recently, a reverse-imaging strategy for visualizing the inclusion using GFP expressing host cells was described. This approach rationally exploits the intrinsic impermeability of the inclusion membrane to large molecules such as GFP. In this work, we describe how GFP- or mCherry-expressing host cells are generated for subsequent visualization of chlamydial inclusions. Furthermore, this method is shown to effectively substitute for costly antibody-based enumeration methods, can be used in tandem with other fluorescent labels, such as GFP-expressing Chlamydia, and can be exploited to derive key quantitative data about inclusion membrane growth from a range of Chlamydia species and strains.     

Electrical Events Correlated with Contractile Vacuole Activity in the Suctorian, Heliophrya

SYNOPSIS. The surface membrane potentials of suctorian genus Heliophrya were studied with intracellular electrodes. Resting membrane potentials averaged -32 mV, and spontaneous depolarizing potentials occurring at apparently random intervals and having a variety of waveforms were routinely observed. Such spontaneous potentials were correlated in time with visually monitored contractile vacuole activity. Individual contractile vacuoles had unique, although somewhat variable, electrical signatures. In the presence of an intracellular electrode all vacuoles contracted independently, but at approximately the same frequency. The amplitude of the electrical potentials increased when the membrane was hyperpolarized and decreased when it was depolarized. The sign of such potentials reversed at between -10 mV and the zero membrane potential. A 20% decrease in the membrane resistance was measured at the peak of the spontaneous depolarizing potentials.     

Multiple Vacuoles in Plant Cells: Rule or Exception?

It is generally accepted that plant cells can contain multiple vacuoles with different functions, for example lytic vacuoles with lysosome-like properties and protein storage vacuoles for reserve accumulation. Recent data call into question the generality of this theory. In this study, we review the published evidence for the existence of multiple vacuoles. We conclude that the multivacuole hypothesis is valid for a number of cases, but care should be taken before assuming that it applies universally.     

植物硫营养代谢、调控与生物学功能

植物作为无机硫的主要还原者,在全球的硫循环中起着关键作用。植物对土壤中硫酸盐的吸收运输和同化代谢,以及一系列具有重要生物学功能的含硫代谢产物的合成,不但与植物生长发育、耐逆和抗病虫害等密切相关,而且影响农作物产量与品质。硫营养的代谢和调控非常复杂,且生物学功能众多。本文综述了近年来植物硫营养代谢及调控及其在逆境胁迫中的生物学功能等方面的新进展,同时讨论了该领域悬而未决的重要生物学问题和研究动向,进而提出硫营养在农业生产上的重要性和所面临的新问题。