Dynamic Relation between Expansion and Cellular Turgor in Growing Grape (Vitis vinifera L.) Leaves

Measurements of the growth and water relations of expanding grape (Vitis vinifera L.) leaves have been used to determine the relationship between leaf expansion rate and leaf cell turgor. Direct measurement of turgor on the small (approximately 15 micrometer diameter) epidermal cells over the midvein of expanding grape leaves was made possible by improvements in the pressure probe technique. Leaf expansion rate and leaf water status were perturbed by environmentally induced changes in plant transpiration. After establishing a steady state growth rate, a step decrease in plant transpiration resulted in a rapid and large increase in leaf cell turgor (0.25 megapascal in 5 minutes), and leaf expansion rate. Subsequently, leaf expansion rate returned to the original steady state rate with no change in cell turgor. These results indicate that the expansion rate of leaves may not be strongly related to the turgor of the leaf cells, and that substantial control of leaf expansion rate, despite changes in turgor, may be part of normal plant function. It is suggested that a strictly physical interpretation of the parameters most commonly used to describe the relationship between turgor and growth in plant cells (cell wall extensibility and yield threshold) may be inappropriate when considering the process of plant cell expansion.     

植物生理学研究中的压力探针技术

压力探针技术是一种用来测定微系统中压力大小和变化的新技术。其最初被设计用于直接测定巨型藻类的细胞膨压。随着操作装置的进一步微型化和精密化, 后来被应用于测定普通高等植物细胞膨压及其它水分关系参数。该技术的发展建立在一系列相应的流体物理学理论基础上。通过这些物理学公式的计算, 该技术能测定跨细胞膜或器官的水分运输速度以及它们的水力学导度; 测定溶液中水分和溶质的相对运输速度以及它们之间的相互影响; 还可以测定细胞壁的刚性等。目前压力探针技术已成为植物生理学和生态学领域研究中的多用途技术。它可以在细胞水平上原位测定水分及溶质跨膜运输及分布情况, 这对于阐明水通道功能具有极其重要的意义。此外, 木质部压力探针技术是目前唯一可以直接测定导管或管胞中负压的工具。该技术还可以用于单细胞汁液的样品采集, 结合微电极技术测定导管或其它细胞中的pH值、离子浓度以及细胞膜电位。本文重点介绍该技术使用的基本原理和相应的理论基础, 并详细地描述了操作过程中的技术和技巧。     

植物钙结合蛋白与钙离子结合鉴定技术的研究进展

Ca2＋在植物生长发育和环境适应过程中发挥着中心调控作用,钙信号是植物生长发育和逆境响应的主要调控因子,钙结合蛋白是植物钙信号传导途径的最重要组分之一,然而植物钙结合蛋白在体内和体外与Ca2＋结合的技术体系还有待完善和发展。为了系统总结植物钙结合蛋白的鉴定方法与技术,本文从定性结合、定量结合和结合方式等角度,综述了植物钙结合蛋白在体内和体外条件下与Ca2＋结合的原理、方法、特点和应用前景,详细阐述了近年来的主要检测方法,并对其今后的发展趋势作了展望。本文将为植物钙结合蛋白的分离、功能鉴定和作用机制的研究提供技术支撑。     

Use of the pressure probe in studies of stomatal function

Over the past few decades the pressure probe has been used extensively in studies of the hydromechanical and osmotic properties of plant cells. However, although pressure probe techniques have been employed successfully in the study of stomatal function, there is no detailed account of this special application of the pressure probe technique. This paper describes the construction and use of the pressure probe in studies relating to stomatal function, and reviews the current state of knowledge of stomatal function in relation to guard cell and leaf hydromechanical properties.     

In vivo creep and stress relaxation experiments to determine the wall extensibility and yield threshold for the sporangiophores of phycomyces

The pressure probe was used to conduct in vivo creep and in vivo stress relaxation experiments on the sporangiophores of Phycomyces blakesleeanus. The in vivo creep and in vivo stress relaxation methods are compared with respect to their utility for determining the irreversible wall extensibility and the yield threshold. The results of the in vivo stress relaxation experiments demonstrate that the growth usually does not cease when the external water supply is removed, and the turgor pressure does not decay for hours afterwards. A successful stress relaxation experiment requires that the cell enlargement rate (growth rate) be zero during the turgor pressure decay. In a few experiments, the growth rate was zero during the turgor pressure decay. However, in general only the yield threshold could be determined.

In vivo creep experiments proved to be easier to conduct and more useful in determining values for both the irreversible wall extensibility and the yield threshold. The results of the in vivo creep experiments demonstrate that small steps-up in turgor pressure, generally <0.02 MPa, elicit increases in growth rate as predicted by the growth equations and the augmented growth equations. The irreversible wall extensibility and the yield threshold were determined from these results. The results also demonstrate that steps-up in turgor pressure larger than 0.02 MPa, produce a different response; a decrease in growth rate. The decreased growth rate behavior is related to the magnitude of the step-up, and in general, larger steps-up in turgor pressure produce larger decreases in growth rate and longer periods of decreased growth rate. Qualitatively, this growth behavior is very similar to the “stretch response” previously reported by Dennison and Roth (1967).

     

Pressure probe technique to study transpiration in phycomyces sporangiophores

The growth equation for the rate of water uptake is augmented with a transpiration term. The obtained augmented growth equations are used to develop methodology which employs the pressure probe to measure transpiration rates from single plant cells. Experiments are conducted on the sporangiophores of Phycomyces blakesleeanus to demonstrate this technique.     

Nuclear magnetic resonance and plant metabolic engineering.

Nuclear magnetic resonance (NMR) can be used to measure metabolite levels and metabolic fluxes, to probe the intracellular environment, and to follow transport and energetics nondestructively. NMR methods are therefore powerful aids to understanding plant metabolism and physiology. Both spectroscopy and imaging can help overcome the unique challenges that plants present to the metabolic engineer by detecting, identifying, quantifying, and localizing novel metabolites in vivo and in extracts; revealing the composition and physical state of cell wall and other polymers; allowing the identification of active pathways; providing quantitative measures of metabolic flux; and testing hypotheses about the effects of engineered traits on plant physiological function. The aim of this review is to highlight recent studies in which NMR has contributed to metabolic engineering of plants and to illustrate the unique characteristics of NMR measurements that give it the potential to make greater contributions in the future.     

Ultrasound: mechanical gene transfer into plant cells by sonoporation

Development of nonviral gene transfer methods would be a valuable alternative of gene therapy or transformation. Ultrasound can produce a variety of nonthermal bioeffects via acoustic cavitation. Cavitation bubbles can induce cell death or transient membrane permeabilization (sonoporation) on cells. Application of sonoporation for gene transfer into cells or tissues develops quickly in recent years. Many studies have been performed in vitro exposure systems to a variety of cell lines transfected successfully. In vivo, cavitation initiation and control are more difficult, but can be enhanced by ultrasound contrast agents (microbubbles). The use of ultrasound for nonviral gene delivery has been applied for mammalian systems, which provides a fundamental basis and strong promise for development of new gene therapy methods for clinical medicine. In this paper, ultrasound applied to plant cell transformation or gene transfer is reviewed. Recently, most researches are focused on sonication-assisted Agrobacterium-mediated transformation (SAAT) in plant cells or tissues. Microbubbles are also proposed to apply to gene transfer in plant cells and tissues.     

Application of pressure probe techniques in studies of plant water relations]

This review introduces the pressure probe technique that was originally designed to detect the turgor of a giant algal cell, then adapted to measure the turgor and other water-relations parameters of higher plants, and now has developed into a diverse tool on researches of plant physiology and eco-physiology. This technique can be used to measure in situ the permeability of cell membranes to water and solutes at the resolution of single cells, and hence is a useful tool to study function and regulation of water channels (aquaporins) of intact plant cells. The recently developed xylem-pressure probe technique is the only way to directly measure the negative pressure in xylem conduits. In this review we introduce the basic principles and the theoretical backgrounds underlying the pressure probe. Finally some important achievements and applications of the pressure probe in studies of plant water relations are reviewed and discussed.     

Experimental approaches used to quantify physical parameters at cellular and subcellular levels

From a mechanical point of view, plant and hyphal cells are more complex than their animal counterparts because the variety of structural components determining cellular architecture is broader. In addition to cytoskeletal elements and the plasma membrane, the cell wall and turgor pressure equip plant and hyphal cells with structures analogous to an exoskeleton and a hydroskeleton, respectively. To quantify the physical properties of plant and hyphal cells, researchers have developed a plethora of experimental methods. This review provides an overview of experimental approaches that have been used to measure turgor pressure and to determine the mechanical properties of the plant cell wall at the subcellular level. It is completed by a glimpse into the arsenal of techniques that has been used to characterize the physical properties of cytoskeletal elements. These have mostly been used on animal cells, but we hope they will find their way into plant cell research. Finally, assays and tests to measure the generation of forces by cells and subcellular structures are discussed.     

Towards mechanistic models of plant organ growth

Modelling and simulation are increasingly used as tools in the study of plant growth and developmental processes. By formulating experimentally obtained knowledge as a system of interacting mathematical equations, it becomes feasible for biologists to gain a mechanistic understanding of the complex behaviour of biological systems. In this review, the modelling tools that are currently available and the progress that has been made to model plant development, based on experimental knowledge, are described. In terms of implementation, it is argued that, for the modelling of plant organ growth, the cellular level should form the cornerstone. It integrates the output of molecular regulatory networks to two processes, cell division and cell expansion, that drive growth and development of the organ. In turn, these cellular processes are controlled at the molecular level by hormone signalling. Therefore, combining a cellular modelling framework with regulatory modules for the regulation of cell division, expansion, and hormone signalling could form the basis of a functional organ growth simulation model. The current state of progress towards this aim is that the regulation of the cell cycle and hormone transport have been modelled extensively and these modules could be integrated. However, much less progress has been made on the modelling of cell expansion, which urgently needs to be addressed. A limitation of the current generation models is that they are largely qualitative. The possibilities to characterize existing and future models more quantitatively will be discussed. Together with experimental methods to measure crucial model parameters, these modelling techniques provide a basis to develop a Systems Biology approach to gain a fundamental insight into the relationship between gene function and whole organ behaviour.     

A model of tumor growth based on cell cycle kinetics

This work describes a mathematical model of growth based on the kinetics of the cell cycle. A traditional model of the cell cycle has been used, with the addition of a resting (G₀) state from which cells could reenter the reproductive cycle. The model assumes that a growth regulatory substance regulates the transition of cells to and from the resting state. Other transitions between the phases of the cycle were modeled as a first order process. Cell loss is an important feature of growth kinetics, and has been represented by a general but tractable mathematical form. The resulting model forms a system of ordinary nonlinear differential equations. Analytic methods are employed first in the study of this system. Simplifying assumptions regarding cell loss give rise to special cases for which equilibrium solutions can be found. One special case, which assumes first order loss from all cell cycle phases at equal rates, is presented here. For small time values, approximations corresponding to exponential growth were developed. The equations describing an intrinsic growth rate were derived. Simulation methods were used to further characterize the behavior of this model. Parameter values were chosen based on animal tumor cell cycle kinetic data, resulting in a set of 45 model simulations. Several tumor treatment protocols were simulated which illustrated the importance of the intrinsic growth rate and cell loss concepts. Although the qualitative behavior regarding absolute and relative growth is reasonable, this model awaits data for model fitting, parameter estimation, or revision of the equations.     

植物铝胁迫响应基因的研究进展

铝毒是酸性土壤中植物生长和作物生产的主要限制因子.近年来的很多研究应用差异显示PCR、抑制差减cDNA文库和DNA微正列等技术,在一些铝耐受型和敏感型植物中鉴定了很多铝胁迫响应基因.本研究通过参阅国内外有关报道和结合本实验室的研究成果,从铝诱导的通道蛋白、代谢相关、胁迫和细胞死亡以及信号转导相关基因4个方面的研究进展进行了综述.     

Cell sampling and analysis (SiCSA): metabolites measured at single cell resolution

By using a fine oil-filled glass microcapillary mounted on a micromanipulator, the solutes of individual plant cells can be sampled. These samples can then be analysed using a range of physical and chemical methods. Hydrostatic pressure (cell pressure probe), osmotic pressure (picolitre osmometer), organic solutes (enzyme-linked fluorescence microscope spectrometry or capillary electrophoresis), inorganic solutes (X-ray microdroplet analysis or capillary electrophoresis), (14)C (mass spectrometry), proteins (microdroplet immunoblotting), and mRNA (rt PCR) have been measured. Collectively, the battery of techniques is called single cell sampling and analysis (SiCSA) and all of the techniques have relevance to the study of plant metabolism at the resolution of the individual cell. This review summarizes the techniques for SiCSA and presents examples of applications used in this laboratory, in particular those relating to cell metabolism.     

Extraction and analysis of sap from individual wheat leaf cells: the effect of sampling speed on the osmotic pressure of extracted sap

Abstract. A modification to the pressure probe is described which allows very rapid extraction of sap samples from single higher plant cells. The performance of this rapid-sampling probe was assessed and compared with the unmodified probe for cells of both wheat and Tradescantia. Under some conditions, the unmodified probe operated too slowly to avoid dilution of cell sap during the extraction process. This led to values for apparent sample osmotic pressures that were below the turgor pressures for the same cells. The problem was particularly acute in young wheatleaf epidermal cells which are small, elongate and have high turgor pressure. These exhibited rapid water influx when their turgor was depressed during the sampling of their contents (half-time for pressure recovery in wheat cells was less than 1 s while in Tradescantia cells it was 3–5 s). Dilution during sampling was apparently negligible when the rapid sampling probe was used. The study was complemented by a simple model of the way cells dilute during sampling. Quantitative predictions of the model were consistent with our observed findings. The model is used to assess the major factors which determine a cell's susceptibility to dilution during sampling.     

纳米材料在农作物领域的应用及展望

近年来,纳米材料成为农业领域的一个研究热点,受到了国内外学者的广泛关注。纳米材料基于其尺寸小的特点,可以在穿透植物细胞壁后,通过内吞作用被细胞吸收,进而对植物生长产生影响。纳米材料被广泛应用于植物遗传转化、作物生长发育和植物健康等农作物领域,尤其在遗传转化领域,其可作为转化载体与基因编辑技术综合运用,对作物进行遗传改良。基于此,对纳米材料在植物体内的吸收、转运机制及其在农作物领域的应用进展进行了综述,重点探讨了纳米材料在植物遗传转化方面的研究进展,并对其在农作物领域亟待解决的问题和后续发展方向进行了展望,以期为拓宽纳米材料的应用领域提供参考。     

Osmotic Biosensors. How to Use a Characean Internode for Measuring the Alcohol Content of Beer

Isolated internodes of Chara corallina and Nitella flexilis have been used to determine the concentration of one passively permeating solute in the presence of non-permeating solutes. The technique was based on the fact that the shape of the peaks of the biphasic responses of cell turgor (as measured in a conventional way using the cell pressure probe) depended on the concentration and composition of the solution and on the permeability and reflection coefficients of the solutes. Peak sizes were proportional to the concentration of the permeating solute applied to the cell. Thus, using the selective properties of the cell membrane as the sensing element and changes of turgor pressure as the physical signal, plant cells have been used as a new type of biosensor based on osmotic principles. Upon applying osmotic solutions, the responses of cell turgor (P) exactly followed the P(t) curves predicted from the theory based on the linear force/flow relations of irreversible thermodynamics. The complete agreement between theory and experiment was demonstrated by comparing measured curves with those obtained by either numerically solving the differential equations for volume (water) and solute flow or by using an explicit solution of the equations. The explicit solution neglected the solvent drag which was shown to be negligible to a very good approximation. Different kinds of local beers (regular and de-alcoholized) were used as test solutions to apply the system for measuring concentrations of ethanol. The results showed a very good agreement between alcohol concentrations measured by the sensor technique and those obtained from conventional techniques (enzymatic determination using alcohol dehydrogenase or from measurement of the density and refraction index of beer). However, with beer as the test solution, the characean internodes did show irreversible changes of the transport properties of the membranes leading to a shift in the responses when cells were treated for longer than 1 h with diluted beer. The accuracy and sensitivity of the osmotic biosensor technique as well as its possible applications are discussed.     

Models to study the pathogenesis of thyroid autoimmunity.

In vitro and in vivo models to study the pathogenesis of thyroid autoimmunity are reviewed. Animal models with experimentally induced or spontaneously developed autoimmune thyroid disease as well as transplantation models have been used extensively in these studies, but also the use of thyroid cell cultures from both humans and animals has contributed to the present state of knowledge. Cytokines may play a role in the pathogenic mechanism in thyroid autoimmunity. The major in vitro and in vivo effects of for example interleukin-1, tumour necrosis factor and gamma-interferon on differentiated thyroid cell functions are inhibitory. The advantage of using cell cultures has been the possibility of studying an influence on thyrocytes from a single agent individually, such as cytokines, hormones or growth factors. The disadvantage is that an organism is under the influence of a multitude of factors that can only be investigated in vivo in intact organisms. Both types of models have therefore been important in the understanding of thyroid autoimmunity.     

Estimation of Polymer Rigidity in Cell Walls of Growing and Nongrowing Celery Collenchyma by Solid-State Nuclear Magnetic Resonance in Vivo

When the growth of a plant cell ceases, its walls become more rigid and lose the capacity to extend. Nuclear magnetic resonance relaxation methods were used to determine the molecular mobility of cell wall polymers in growing and nongrowing live celery (Apium graveolens L.) collenchyma. To our knowledge, this is the first time this approach has been used in vivo. Decreased polymer mobility in nongrowing cell walls was detected through the 13C-nuclear magnetic resonance spectrum by decreases in the proton spin-spin relaxation time constant and in the intensity of a sub-spectrum corresponding to highly mobile pectins, which was obtained by a spectral editing technique based on cross-polarization rates. Flexible, highly methyl-esterified pectins decreased in relative quantity when growth ceased. A parallel increase in the net longitudinal orientation of cellulose microfibrils was detected in isolated cell walls by polarized Fourier-transformed infrared spectrometry.     

Soil microbes and plant fertilization

With respect to the adverse effects of chemical fertilization on the environment and their related expenses, especially when overused, alternative methods of fertilization have been suggested and tested. For example, the combined use of chemical fertilization with organic fertilization and/or biological fertilization is among such methods. It has been indicated that the use of organic fertilization with chemical fertilization is a suitable method of providing crop plants with adequate amount of nutrients, while environmentally and economically appropriate. In this article, the importance of soil microbes to the ecosystem is reviewed, with particular emphasis on the role of plant growth-promoting rhizobacteria, arbuscular mycorrhizal fungi, and endophytic bacteria in providing necessary nutrients for plant growth and yield production. Such microbes are beneficial to plant growth through colonizing plant roots and inducing mechanisms by which plant growth increases. Although there has been extensive research work regarding the use of microbes as a method of fertilizing plants, it is yet a question how the efficiency of such microbial fertilization to the plant can be determined and increased. In other words, how the right combination of chemical and biological fertilization can be determined. In this article, the most recent advances regarding the effects of microbial fertilization on plant growth and yield production in their combined use with chemical fertilization are reviewed. There are also some details related to the molecular mechanisms affecting the microbial performance and how the use of biological techniques may affect the efficiency of biological fertilization.