基于液泡膜质子泵的硝态氮再利用研究进展

全面掌握洛川果园的土壤水分环境特征,不仅可为苹果的园址选择、砧穗组合和改进土壤水分管理措施提供理论依据,而且对我国苹果产区果园提质增效具有借鉴价值.采用定点土壤水分连续监测法,对洛川苹果园的总体土壤水分环境以及不同生长年限、不同立地类型和乔、矮化果园的土壤水分分异特征进行分析.结果表明： 苹果树根际区 (0～200 cm)土壤水分普遍亏欠,且0～60 cm土层的水分亏欠小于60～200 cm土层;生长季0～60 cm土层贮水量与降水量的变化一致,土壤相对含水量大多<60%,季节性旱象严重;果园剖面土壤含水量变异系数随土壤深度加深而递减;随果园生长年限的增大,土壤剖面贮水量下降;在栽培密度一致的条件下,矮化果园5 m土层土壤含水量均高于乔化果园,而栽培密度大的矮化果园的土壤贮水量低于栽培密度小的乔化果园;塬地成龄果园的土壤水分含量最高,川地次之,台地相对较低.密度对果园土壤水分含量有很大影响,在栽培密度一致的条件下,采用矮化栽培能减少土壤水分消耗,显著提高果园土壤含水量;挖株降低栽培密度是维持苹果园土壤水分平衡、实现可持续发展的有效途径.     

Salt-induced chloroplast protrusion is the process of exclusion of ribulose-1,5-bisphosphate carboxylase/oxygenase from chloroplasts into cytoplasm in leaves of rice

Chloroplast protrusions (CPs) are often observed under environmental stresses, but their role has not been elucidated. The formation of CPs was observed in the leaf of rice plants treated with 75 mm NaCl for 14 d. Some CPs were almost separated from the main chloroplast body. In some CPs, inner membrane structures and crystalline inclusions were included. Similar structures surrounded by double membranes were observed in the cytoplasm and vacuole. Ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) was detected in CPs and the similar structures in the cytoplasm and vacuole. These results suggest that CP is one of the pathways of Rubisco exclusion from chloroplasts into the cytoplasm under salinity, and the exclusions could be transported to vacuole for their degradation.     

DEVELOPMENT OF PERIPHERAL VACUOLES IN PLANT CELLS

The vacuolar apparatus of various plant cells consists of two distinct features: the large central vacuole and peripheral vacuoles which are derived from invaginations of the plasma membrane. Peripheral vacuoles are conspicuous structures in both living and fixed hair or filament cells of Tradescantia virginiana. They occur as spherical structures along the inner boundary of the peripheral cytoplasm and can be recognized as projections into the central vacuole. These structures are variable in size and number within a cell and can represent a significant proportion of the volume of the vacuole. Peripheral vacuoles most frequently are observed in motion with the streaming cytoplasm although their velocity is usually somewhat slower that that of the cytoplasmic organelles. Ultrastructural studies show two closely approximated membranes, one for each vacuole, in areas where a peripheral vacuole projects into the central vacuole. These are separated by an intermembrane zone continuous with the peripheral cytoplasm. The movement of organelles over the perimeter of the peripheral vacuole is presumed to occur along this intermembrane zone. The internal area of the peripheral vacuoles may appear empty although some contain a vesicular content of unknown origin and function.     

Compartment analysis of plant cells by means of turgor pressure relaxation: I. Theoretical considerations

Summary Turgor pressure relaxation curves for individual plant cells represent an important source of information for the plant physiologist. However, the accurate interpretation of these curves is strongly dependent on the model chosen to describe the plant cell. If the compartmentation of the cell into vacuole and cytoplasm is taken into account, a theoretical analysis shows that pressure relaxation curves can be represented by the sum of two exponential functions. Givena priori assumptions about the exchange area of the tonoplast and its reflection coefficient, the hydraulic conductivities of the plasmalemma and tonoplast can be determined and the proportion of the total cell volume occupied by the cytoplasm is also obtained. Numerical solutions to the flow equations have shown that the biphasic nature of pressure relaxations is maintained even when a permeable tonoplast is assumed. Depending on the magnitude of the reflection coefficient and the permeability of the vacuolar membrane, large errors can arise in the determination of the hydraulic conductivity of the tonoplast. However, under certain conditions, even a highly permeable tonoplast may behave like a nonpermeable membrane during pressure relaxation.     

Visualization of autophagy in Arabidopsis using the fluorescent dye monodansylcadaverine and a GFP-AtATG8e fusion protein

Autophagy is a process that is thought to occur in all eukaryotes in which cells recycle cytoplasmic contents when subjected to environmental stress conditions or during certain stages of development. Upon induction of autophagy, double membrane-bound structures called autophagosomes engulf portions of the cytoplasm and transfer them to the vacuole or lysosome for degradation. In this study, we have characterized two potential markers for autophagy in plants, the fluorescent dye monodansylcadaverine (MDC) and a green fluorescent protein (GFP)-AtATG8e fusion protein, and propose that they both label autophagosomes in Arabidopsis. Both markers label the same small, apparently membrane-bound structures found in cells under conditions that are known to induce autophagy such as starvation and senescence. They are usually seen in the cytoplasm, but occasionally can be observed within the vacuole, consistent with a function in the transfer of cytoplasmic material into the vacuole for degradation. MDC-staining and the GFP-AtATG8e fusion protein can now be used as very effective tools to complement biochemical and genetic approaches to the study of autophagy in plant systems.     

Mobile arrays of vacuole ripples are common in plant cells

In tobacco (Nicotiana tabacum L.), a structure frequently interpreted as endoplasmic reticulum, was clearly identified as a set of ripple-shaped protrusions of the vacuole into the surrounding cytoplasm. The occurrence in other species suggests that these ripples might be common in vacuolated plant cells. The apparent mobility of the ripples depends on the integrity of the F-actin network. This raises questions concerning the precise composition and architecture of the cytoplasm/vacuole border. The stereotypic concept of the central vacuole as a kind of inflated balloon with a smooth surface has to be debated. Received: 7 November 1997 / Revision received: 27 April 1998 / Accepted: 28 April 1998     

In vivo imaging and quantitative monitoring of autophagic flux in tobacco BY-2 cells

Autophagy has been shown to play essential roles in the growth, development and survival of eukaryotic cells. However, simple methods for quantification and visualization of autophagic flux remain to be developed in living plant cells. Here, we analyzed the autophagic flux in transgenic tobacco BY-2 cell lines expressing fluorescence-tagged NtATG8a as a marker for autophagosome formation. Under sucrose-starved conditions, the number of punctate signals of YFP-NtATG8a increased, and the fluorescence intensity of the cytoplasm and nucleoplasm decreased. Conversely, these changes were not observed in BY-2 cells expressing a C-terminal glycine deletion mutant of the NtATG8a protein (NtATG8aΔG). To monitor the autophagic flux more easily, we generated a transgenic BY-2 cell line expressing NtATG8a fused to a pH-sensitive fluorescent tag, a tandem fusion of the acid-insensitive RFP and the acid-sensitive YFP. In sucrose-rich conditions, both fluorescent signals were detected in the cytoplasm and only weakly in the vacuole. In contrast, under sucrose-starved conditions, the fluorescence intensity of the cytoplasm decreased, and the RFP signal clearly increased in the vacuole, corresponding to the fusion of the autophagosome to the vacuole and translocation of ATG8 from the cytoplasm to the vacuole. Moreover, we introduce a novel simple easy way to monitor the autophagic flux non-invasively by only measuring the ratio of fluorescence of RFP and YFP in the cell suspension using a fluorescent image analyzer without microscopy. The present in vivo quantitative monitoring system for the autophagic flux offers a powerful tool for determining the physiological functions and molecular mechanisms of plant autophagy induced by environmental stimuli.     

FURTHER OBSERVATIONS ON THE PHENOMENON OF SECONDARY VACUOLATION IN LIVING CELLS

Invagination of the plasma membrane in plant cells forms peripheral or endocytic structures which often contain a complement of membrane-bound vesicles. These structures, or secondary vacuoles, move with the streaming cytoplasm although their velocities are somewhat slower than that for the various organelles within the cytoplasm. They glide over the nucleus or flow from the peripheral cytoplasm onto a transvacuolar strand and continue unabated along the length of a strand. These structures may detach from the plasma membrane as sacs to become positioned in the cytoplasm directly under the tonoplast and project into the primary vacuole. Some endocytic vacuoles may separate from the peripheral cytoplasm and remain free within the primary vacuole; subsequently they can re-associate with the cytoplasm. While the content and function of these vacuoles are yet to be determined, indirect evidence indicates that they are pinocytic in character since the content of an invagination is confined to the sac upon its detachment from the plasma membrane and is subsequently transported throughout the cell by cyclosis.     

Intracellular Na and K distribution in Debaryomyces hansenii. Cloning and expression in Saccharomyces cerevisiae of DhNHX1

Debaryomyces hansenii is a salt-tolerant yeast that contains high amounts of internal Na(+). Debaryomyces hansenii kept more sodium than Saccharomyces cerevisiae in both the cytoplasm and vacuole when grown under a variety of NaCl concentrations. These results indicate a higher tolerance of Debaryomyces to high internal Na(+), and, in addition, suggest the existence of a transporter driving Na(+) into the vacuole. Moreover, a gene encoding a Na(+) (K(+))/H(+) antiporter from D. hansenii was cloned and sequenced. The gene, designated DhNHX1, exhibited significant homology with genes of the NHE/NHX family. DhNHX1 expression was induced neither at low pH nor by extracellular NaCl. A mutant of S. cerevisiae lacking its own Na(+) transporters (ena1-4Delta nha1 Delta nhx1 Delta), when transformed with DhNHX1, partially recovered cation tolerance as well as the ability to accumulate Na(+) and K(+) into the vacuole. Our analysis provides evidence that DhNhx1p transports Na(+) (and K(+)) into the vacuole and that it can play an important role in ion homeostasis and salt tolerance.     

Impact of hypoosmotic challenges on spongy architecture of the cytoplasm of the giant marine alga Valonia utricularis

Summary. The ultrastructure of the several micrometers thick cytoplasmic layer of the giant marine alga Valonia utricularis displays characteristics which are apparently linked with the capability of this alga to regulate turgor pressure. Transmission and scanning electron microscopy of cells prefixed in different ways, including a protocol that allows prefixation of the alga in a turgescent state, revealed a highly dendritic network of cytoplasmic strands connecting and enveloping the chloroplasts and the nuclei. Innumerable vacuolar entities are embedded in the network, giving the cytoplasm a spongy appearance. Vacuolar perfusion of turgor-pressure-clamped cells with prefixation solution containing tannic acid presented evidence that these vacuolar entities together with the huge central vacuole form a large unstirred continuum. In contrast to the tonoplast, the plasmalemma followed smoothly the lining of the cell wall, even at the numerous cell wall ingrowths. Sucrose, but not polyethylene glycol 6000, induced chloroplast clustering. Acute hypoosmotic treatment (established by reduction of external NaCl or by replacement of part of the external NaCl by equivalent osmotic concentrations of sucrose or polyethylene glycol 6000) resulted in a local relocation of the chloroplasts and cytoplasm towards the central vacuole. This effect did not occur when the relatively low reflection coefficients of these two osmolytes were taken into account. The increase in spacing between the spongy cytoplasm and the plasmalemma by chloroplast relocation (viewed by confocal laser scanning microscopy) was associated with a speckled appearance of the affected surface area under the light microscope. As indicated by electron microscopy, hypoosmotically induced chloroplast relocation resulted from disproportionate swelling of the vacuolar entities located close to the plasmalemma. The cytoskeleton in the cytoplasm and the mucopolysaccharide network in the central vacuole apparently resisted swelling of these compartments. This finding has the important consequence that relevant hydrostatic pressure gradients can be built up throughout the entire multifolded vacuolar space. This gradient could represent the trigger for turgor pressure regulation which is manifested electrically first in the tonoplast.Correspondence and reprints: Lehrstuhl für Biotechnologie, Biozentrum, Am Hubland, 97074 Würzburg, Federal Republic of Germany.     

高等植物花色苷在液泡中的存在状态及其着色效应

综述了花色苷被摄入液泡的原因、花色苷在液泡中的存在状态及其对植物细胞的着色效应。花色苷在植物细胞质中合成后转运到液泡里是为了解除其对蛋白质和DNA等细胞功能分子的毒性。花色苷的液泡区隔化是花色苷在植物细胞中发挥正常功能的前提。在大多数植物中,花色苷在绝大多数情况下完全溶解在液泡里。但是,花色苷也能在液泡里形成颗粒,这些颗粒可以划分为花色苷体和花色苷液泡包涵体两类。花色苷体由膜包裹,其形成是液泡中小的有色囊泡逐渐合并的结果,发育完全的花色苷体为典型的球状、具比液泡更深的红色;液泡里的花色苷体具高密度,呈现为含高浓度花色苷的不溶性小球;花色苷体的存在可导致液泡的强烈色彩。花色苷液泡包涵体可能具备蛋白质基质,既无膜包裹又无内部结构,其形成是转运进液泡的花色苷与蛋白质基质结合的结果;液泡里的花色苷液泡包涵体形状不规则,象果冻;在花色苷液泡包涵体中,花色苷可能通过氢键连接于蛋白质基质的一个有限空间位点;花色苷液泡包涵体被认为是液泡中花色苷的"陷阱",优先摄取花色素3,5-二糖苷或酰化的花色苷;花色苷液泡包涵体的存在可增加液泡色彩的强度并导致"蓝化"。     

Enhanced resistance to salt,cold and wound stresses by overproduction of animal cell death suppressors Bcl-xL and Ced-9 in tobacco cells - their possible contribution through improved function of organella

Overproduction of animal cell death suppressors Bcl-xL and Ced-9 conferred enhanced resistance to UV-B and paraquat treatment in tobacco plants [Mitsuhara et al. (1999) CURR: Biol. 9: 775]. We report here that the progeny could germinate in 0.2 M NaCl or at 10 degrees C under light, while control plants could not. Suspension-cultured Bcl cells resisted NaCl treatment maintaining an active mitochondrial membrane potential for longer than control cells. When intracellular pH was determined by in vivo (31)P-NMR, immediate cytoplasmic acidification by 0.3 M NaCl treatment in control cells was found to be suppressed in transgenic cells. Monitoring of cytoplasmic and vacuolar pHs in control cells indicated the vacuole was disrupted 40 min after 0.5 M NaCl treatment, while the compartment between the cytoplasm and vacuole was likely to remain intact in Bcl cells for 100 min. Enhanced shoot regeneration from cut leaf pieces and more vigorous rooting from cut stem ends were found in transgenic plants. The Bcl protein was abundant in all subcellular fractions. Based on the results in transgenic plants carrying a mutant bcl-xL gene, Bcl-xL is thought to suppress cell death and enhance the viability of plants in stressful environments by contributing to the maintenance of the homeostasis of organella.     

Na2CO3胁迫下星星草(Puccinellia tenuiflora)幼苗叶表皮和叶肉细胞中K、Na的相对含量

对不同强度Na2CO3胁迫处理下星星草幼苗叶片表皮和叶肉细胞中K、Na的透射电镜X-射线电子探针显微分析和叶片表面扫描电镜X-射线电子探针显微分析,结果表明：在相同胁迫强度下,无论是表皮细胞还是叶肉细胞的细胞壁和液泡中的Na相对含量均明显高于细胞质中的Na相对含量,并且K的相对含量均明显比相应部位Na的相对含量高,细胞壁与液泡中的Na相对含量变化范围非常接近。在Na2CO3胁迫浓度低于0．1molL^-1时,在相同胁迫强度下,K的相对含量高于Na的相对含量,使细胞质保持相对高的K／Na比。而尽管向细胞壁和液泡分流了大量的Na,但是细胞质中的Na相对含量仍然随着Na2CO,胁迫强度的增加而增加,一方面证明星星草在Na2CO3胁迫下维持相对高的K／Na比的能力是有一定限度的,另一方面暗示星星草作为盐生植物在盐碱环境中一定程度上Na可以部分地代替K而行使部分K的生理功能。     

Na2CO3胁迫下星星草（Puccinellia tenuiflora）幼苗叶表皮和叶肉细胞中K、Na的相对含量

对不同强度Na2CO3胁迫处理下星星草幼苗叶片表皮和叶肉细胞中K、Na的透射电镜X-射线电子探针显微分析和叶片表面扫描电镜X-射线电子探针显微分析,结果表明：在相同胁迫强度下,无论是表皮细胞还是叶肉细胞的细胞壁和液泡中的Na相对含量均明显高于细胞质中的Na相对含量,并且K的相对含量均明显比相应部位Na的相对含量高,细胞壁与液泡中的Na相对含量变化范围非常接近。在Na2CO3胁迫浓度低于0.1molL-1时,在相同胁迫强度下,K的相对含量高于Na的相对含量,使细胞质保持相对高的K/ Na比。而尽管向细胞壁和液泡分流了大量的Na,但是细胞质中的Na相对含量仍然随着Na2CO3胁迫强度的增加而增加,一方面证明星星草在Na2CO3胁迫下维持相对高的K/ Na比的能力是有一定限度的,另一方面暗示星星草作为盐生植物在盐碱环境中一定程度上Na可以部分地代替K而行使部分K的生理功能。     

The Membrane Potential of Nitella translucens

The effects of changing the external concentrations of Na, K,Ca, and Cl on the potentials of the cytoplasm and the vacuolewith respect to the bathing medium of the internodal cells ofNitella translucens have been investigated. The potential differencebetween the vacuole and the cytoplasm is practically unaffectedby the concentration changes. The observed changes of potentialdifference are therefore attributed to the boundary separatingthe cytoplasm from the medium; this boundary is possibly a plasmalemma–cellwall complex. The difference of potential between the cell walland the medium has also been measured and, in the presence ofCa, shown to be markedly sensitive only to the external Ca concentration.The results are divided into two sections: (a) for cells pretreatedin 5 mM NaCl, the subsequent experiments being carried out inCa-free media, and (b) for cells initially immersed in a standardartificial pond water containing the chlorides of Na, K, Ca.With the pretreated cells the external Na/K ratio was variedwith the total NaCl+KCl concentration kept constant at 1.1 mM.The results suggest that over a limited range of concentrationsthe cytoplasm-medium potential difference can be described byan equation similar in form to a Goldman equation but containingonly terms for Na and K, the average value of the permeabilityratio (= P_Na/P_K) being 0.27. In the presence of Ca the effectsof Na and K on the cytoplasm-medium potential difference aregreatly reduced, while the effect of Ca is relatively large.The results cannot be fitted to any form of Goldman equationcontaining terms for the major ions. The possibility of a contributionto the plasmalemma potential from electrogenic pumps is brieflydiscussed. Measurements of the Na and K content of the cytoplasmand the vacuole have been made for the pretreated cells. TheNa concentration in the cytoplasm is 37 mM and in the vacuole73 mM; the K concentration is 93 mM in the cytoplasm and 67mM in the vacuole. The Nernst potentials for both ions are comparedwith the cytoplasm-medium and cytoplasm-vacuole potential differences.This analysis shows that Na is actively transported from thecytoplasm into the medium as well as into the Vacuole; K ispumped into the cytoplasm from the medium but appears to beclose to electrochemical equilibrium across the tonoplast. ThisConfirms previously published work.     

Degradation of lipid vesicles in the yeast vacuole requires function of Cvt17, a putative lipase

The vacuole/lysosome serves an essential role in allowing cellular components to be degraded and recycled under starvation conditions. Vacuolar hydrolases are key proteins in this process. In Saccharyomces cerevisiae, some resident vacuolar hydrolases are delivered by the cytoplasm to vacuole targeting (Cvt) pathway, which shares mechanistic features with autophagy. Autophagy is a degradative pathway that is used to degrade and recycle cellular components under starvation conditions. Both the Cvt pathway and autophagy employ double-membrane cytosolic vesicles to deliver cargo to the vacuole. As a result, these pathways share a common terminal step, the degradation of subvacuolar vesicles. We have identified a protein, Cvt17, which is essential for this membrane lytic event. Cvt17 is a membrane glycoprotein that contains a motif conserved in esterases and lipases. The active-site serine of this motif is required for subvacuolar vesicle lysis. This is the first characterization of a putative lipase implicated in vacuolar function in yeast.     

The "crystals" in the sugar beet (Beta vulgaris L.) leaves

Crystal containing cells widely distributed in plant tissues, though the origin of the crystals and their functions are still opened to question. Membrane vesicles in beet leaves are visible in electronic microscope. They originate in cytoplasm and penetrate into vacuole by pinocytosis with participation of tonoplast. In light microscope, vesicles are luminous likewise crystals in crystal cells. Such vesicles-"crystals" fulfill crystal cells also. The content of vesicles-"crystals" are electronic transparent at every path of leaf development. It was proposed that distinct vesicles-"crystals" in cytoplasm and vacuole and mass of them in crystal cells, vein bundles, and epidermal cells--all of them are lytic compartments. Later, obviously, true crystals are formed.     

Biochemical changes during sucrose deprivation in higher plant cells. Phosphorus-31 nuclear magnetic resonance studies

An experimental arrangement was described that enables nuclear magnetic resonance spectra of compressed plant cells to be recorded while circulating a medium through the sample. The system provided a convenient arrangement for monitoring by 31P NMR the behavior of plant cells over a long period of time under different conditions such as sucrose starvation. Perfusion of compressed sycamore cells with sucrose-free culture medium triggered a progressive decrease in the glucose 6-P and uridine-5'-diphosphate-alpha-D-glucose resonances over 30 h. When almost all the intracellular carbohydrate pool had disappeared the nucleotide triphosphate resonances decline progressively. These changes were accompanied by a Pi accumulation in the vacuole and a phosphorylcholine (P-choline) accumulation in the cytoplasm. The very long lag phase observed for ATP and P-choline evolution was comparable with that observed for the progressive intracellular digestion of cytoplasmic constituents (Journet, E., Bligny, R. and Douce, R. (1986) J. Biol. Chem. 261, 3193-3199). Addition of sucrose in the circulating system after a long period of sucrose starvation led to a disappearance of the cytoplasmic Pi resonance and a marked increase in that of glucose 6-P. Under these conditions the vacuolar Pi pool did not fluctuate to buffer the Pi in the cytoplasm. The results suggest that Pi which has been sequestered in the vacuole during the course of sucrose starvation is not restored to the cytoplasm for rapid metabolic processes. Furthermore, the presence of P-choline in plant cells in large excess should be considered as a good marker of membrane utilization after a long period of sucrose starvation and is very likely related to stress.     

Phosphorus-31 NMR Studies of Wild-Type and NaCl-Tolerant Citrus Cultured Cells

Ben–Hayyim, G. and Navon, G. 1985. Phosphorus–31NMR studies of wild–type and NaCl–tolerant Citruscultured cells.—J. exp. Bot. 36: 1877–1888. Theinternal pH of the cytoplasm and vacuole and the relative distributionof internal P_i concentrations between those two cell compartmentshave been determined by ³¹P nuclear magnetic resonance spectroscopyin wild–type and NaCl–tolerant cell lines of Shamoutiorange (Citrus sinensis L. Osbeck). Wild–type cells accumulatehigher amounts of P_i than the NaCl–tolerant cells whenexposed to equal external P_i concentrations. This additionalP_i is located mainly in the vacuole. When both types of cellsare exposed to increasing external P_i concentrations, the internalP_i concentrations increase. The cytoplasmic P_i concentrationreaches saturation at a rather low external P_i concentrationwhile the vacuolar P_i can be increased by a large factor. Transferof cells from aerobic to anaerobic conditions causes an immediateincrease of P_i in the cytoplasm and a slow acidification. Exposureof cells to NaCl during the period of their growth results inan increase in total P_i concentration with a large increasein the ratio of vacuolar to cytoplasmic P_i levels. When thesecells are exposed to NaCl for a short time, total internal P_iconcentration docs not change significantly but its proportionschange in favour of the vacuole. pH values of the cytoplasmand the vacuole under all these conditions are rather constant,the value being 5.8–6.0 for the vacuole and 7.4–7.6for the cytoplasm. Moreover, subjecting these cells to a widerange of external pH values does not change their intracellularpH. These results indicate that a strong regulation of internalpH is operating in both types of cells. The presence of a phosphorylatedmetabolite with an unusual pH titration curve, located in thevacuole, is also reported. Key words: Citrus, callus, ³¹P-NMR, NaCI tolerance, intracellular pH