Application of Non-invasive Microsensing System to Simultaneously Measure Both H^＋ and O2 Fluxes Around the Pollen Tube

Various Ionic and molecular activities in the extraceUular environment are vital to plant cell physiological processes. A noninvasive microsensing system （NMS） based on either the scanning ion-selective electrode technique （SIET） or the scanning polarographlc electrode technique （SPET） is able to obtain information regarding the transportation of various Ions/molecules in Intact samples under normal physiological conditions. The two-probe simultaneous test system （2STS） Is an Integrated system composed of SIET, SPET, and a Xu-Kunkel sampling protocol. In the present study, 2STS was able to simultaneously measure fluxes of H^＋ and O2 of the Uly （Lillum Iongiflorum Thunb. cv. Ace） pollen tube while avoiding interference between the two probes. The results Indicate that the proton fluxes were effluxes, whereas the oxygen fluxes were Influxes, and they were closely correlated to each other surrounding the constitutive alkaline band region. Specifically, when the proton effluxes increased, the oxygen Influxes also increased. Therefore, the hypothesis of condensed active mitochondria existing in the alkalized area of the pollen tube proposed by Hepler＇s group is supported.     

用非损伤微测技术研究肿瘤细胞的耐药性与其胞外H+流变性的相关性

介绍了人乳腺癌细胞(药物敏感株MCF-7/S及耐药株MCF-7/R)在化疗药物阿霉素(adriamycin,ADR)处理下,细胞外pH和H 流动方向和速率的变化.为此,建立了一种基于非损伤微测技术(non-invasive micro-test technique,NMT)的药物抗性研究方法(drug resistance study method,DRSM),该方法可用于研究器官/组织/细胞外离子/分子活性与肿瘤细胞耐药性之间的相互关系.结果显示存在一个持续的并以固有振荡形式出现的胞外H 流现象.此外,耐药株净H 流在加ADR前趋近于零,而敏感株净H 流呈明显内流.敏感株和耐药株加ADR后净H 均呈外流,但耐药株的净H 外流速率要高于敏感株5倍.与净H 流动速率结果相一致的是胞外的pH也产生了相应的变化.因此,实验为胞外H 活性与肿瘤耐药性的相互关联提供了直接证据.     

Exclusion of a proton ATPase from the apical membrane is associated with cell polarity and tip growth in Nicotiana tabacum pollen tubes

Polarized growth in pollen tubes results from exocytosis at the tip and is associated with conspicuous polarization of Ca(2+), H(+), K(+), and Cl(-) -fluxes. Here, we show that cell polarity in Nicotiana tabacum pollen is associated with the exclusion of a novel pollen-specific H(+)-ATPase, Nt AHA, from the growing apex. Nt AHA colocalizes with extracellular H(+) effluxes, which revert to influxes where Nt AHA is absent. Fluorescence recovery after photobleaching analysis showed that Nt AHA moves toward the apex of growing pollen tubes, suggesting that the major mechanism of insertion is not through apical exocytosis. Nt AHA mRNA is also excluded from the tip, suggesting a mechanism of polarization acting at the level of translation. Localized applications of the cation ionophore gramicidin A had no effect where Nt AHA was present but acidified the cytosol and induced reorientation of the pollen tube where Nt AHA was absent. Transgenic pollen overexpressing Nt AHA-GFP developed abnormal callose plugs accompanied by abnormal H(+) flux profiles. Furthermore, there is no net flux of H(+) in defined patches of membrane where callose plugs are to be formed. Taken together, our results suggest that proton dynamics may underlie basic mechanisms of polarity and spatial regulation in growing pollen tubes.     

Carrier-mediated residual K++ and Na++ transport of human red blood cells

Summary Residual, i.e., (ouabain, bumetanide, and EGTA)-insensitive K⁺ and Na⁺ influxes as well as effluxes of human red blood cells are enhanced in isotonic solutions of low (Na-Cl+KCl) concentration using sucrose to maintain constant osmolarity. Various carrier models were tested to fit the experimental data of these fluxes simultaneously. The residual K⁺ and Na⁺ fluxes can be described on the basis of a carrier mechanism of competing substrates with modifier sites.     

A polarographic, oxygen-selective, vibrating-microelectrode system for the spatial and temporal characterisation of transmembrane oxygen fluxes in plants

 A simple procedure is described for the fabrication of micrometer to nanometer-scale platinum electrodes to be used in a vibrating oxygen-selective system. The electrode was prepared by etching a fine platinum wire and insulating it with an electrophoretic paint. The dimensions allowed this electrode to be used with the “vibrating probe technique” in exploratory studies aimed at mapping and measuring the patterns of net influxes as well as effluxes of oxygen in Olea europaea L. leaves and roots with spatial and temporal resolutions of a few microns and a few seconds, respectively. The magnitude and spatial localisation of O₂ influxes in roots was characterised by two distinct peaks. The first, in the division zone, averaged 38 ± 5 nmol m⁻² s⁻¹; the second, in the elongation region, averaged 68 ± 6 nmol m⁻² s⁻¹. Long-term records of oxygen influx in the elongation region of the root showed an oscillatory regime characterised by a fast oscillation with periods of about 8–9 min. In leaves, the system allowed the measurement of real-time changes in O₂ evolution following changes in light. Furthermore, it was possible to obtain “topographical” images of the photosynthetically generated oxygen diffusing through different stomata from a region of the leaf of 120 μm × 120 μm. The combination of topographic and electrochemical information at the micrometer scale makes the system an efficient tool for studying biological phenomena involving oxygen diffusion. Received: 12 November 1999 / Accepted: 1 February 2000     

Effects of membrane potential on calcium fluxes of Pelvetia eggs

⁴⁵Ca²⁺ fluxes across the plasma membrane of zygotes of the fucoid alga, Pelvetia fastagiata (J. Ag.) De Toni, were studied in artificial sea waters of various potassium concentrations. Except for two cases, hyperpolarization of the cell membrane (with low [K⁺]) increases, and depolarization (with high [K⁺]) decreases the influx of Ca²⁺ over the range of [K⁺] studied (1–100 mM). The fractional increases of influx during hyperpolarization are close to the fractional increases in membrane potential but the decreases during depolarization are much smaller than those in membrane potential. In two anomalous cases, the influxes of ⁴⁵Ca²⁺ at a potassium concentration of 30 mM were about 20% higher than the control value instead of being 10% lower.The effluxes of ⁴⁵Ca²⁺ are increased by both hyperpolarization and by depolarization. On balance (and excepting the two anomalous cases) the net result of hyperpolarization should be to increase and that of depolarization to decrease intracellular [Ca²⁺].     

Elevated [Cl-]i, and [Na+]i inhibit Na+, K+, Cl- cotransport by different mechanisms in squid giant axons

Bumetanide-sensitive (BS) unidirectional fluxes of (36)Cl- or (22)Na+ were measured in internally dialyzed squid giant axons while varying the intra- or extracellular concentrations of Na+ and/or Cl-. Raising either [Cl-]i or [Na+]i resulted in a concentration-dependent reduction of the BS influx of both (36)Cl- and (22)Na+. Raising [Cl-]i above 200 mM completely blocked BS influxes. However, raising [Na+]i to 290 mM resulted in saturable but incomplete inhibition of both BS Na+ influx and BS Cl- influx. The consequences of varying intracellular Cl- on cotransporter effluxes were complex. At lower [Cl-]i values (below 100 mM) intracellular Cl- activated cotransporter effluxes. Surprisingly, however, raising [Cl-]i levels > 125 mM resulted in a [Cl-]i-dependent inhibition of BS effluxes of both Na+ and Cl-. On the other hand, raising [Na+]i resulted only in the activation of the BS Na+ efflux; intracellular Na+ did not inhibit BS efflux even at 290 mM. The inhibitory effects of intracellular Na+ on cotransporter-mediated influxes, and lack of inhibitory effects on BS effluxes, are consistent with the trans-side inhibition expected for an ordered binding/release model of cotransporter operation. However, the inhibitory effects of intracellular Cl- on both influxes and effluxes are not explained by such a model. These data suggest that Cl may interact with an intracellular site (or sites), which does not mediate Cl transport, but does modulate the transport activity of the Na+, K+, Cl- cotransporter.     

A Putative Calcium-Permeable Cyclic Nucleotide-Gated Channel, CNGC18, Regulates Polarized Pollen Tube Growth

A tip-focused Ca^2＋ gradient is tightly coupled to polarized pollen tube growth, and tip-localized influxes of extracellular Ca^2＋ are required for this process. However the molecular identity and regulation of the potential Ca^2＋ channels remains elusive. The present study has implicated CNGC18 （cyclic nucleotide-gated channel 18） in polarized pollen tube growth, because its overexpression induced wider and shorter pollen tubes. Moreover, CNGC18 overexpression induced depolarization of pollen tube growth was suppressed by lower extracellular calcium （[Ca^2＋]ex）. CNGC18-yellow fluorescence protein （YFP） was preferentially localized to the apparent post-Golgi vesicles and the plasma membrane （PM） in the apex of pollen tubes. The PM localization was affected by tip-localized ROP1 signaling. Expression of wild type ROP1 or an active form of ROP1 enhanced CNGC18-YFP localization to the apical region of the PM, whereas expression of RopGAP1 （a ROP1 deactivator） blocked the PM localization. These results support a role for PM-Iocalized CNGC18 in the regulation of polarized pollen tube growth through its potential function in the modulation of calcium influxes.     

Single-cell, real-time measurements of extracellular oxygen and proton fluxes fromSpirogyra grevilleana

Summary We have adapted the self-referencing microelectrode technique to allow sensitive and noninvasive measurement of oxygen fluxes around single cells. The self-referencing technique is based on the translational movement of a selective microelectrode through the gradient next to the cell wall or membrane. The electrode is moved at a known frequency and between known points. The differential electrode output values are converted into a directional measurement of flux by the Fick equation. By coupling the newly developed oxygen-selective self-referencing electrochemical microelectrode (SREM-O₂) system with self-referencing ionselective proton measurements (SRIS-H⁺) we have characterized oxygen and proton fluxes from a single cell of the filamentous green algaSpirogyra gre illeana (Hass.). Oxygen showed a net efflux and protons showed a net influx when the cell was illuminated. These photosynthesis-dependent fluxes were found to be spatially associated with the chloroplasts and were sensitive to treatment with dichlorophenyldimethylurea. In the dark the directions of oxygen and proton fluxes were reversed. This oxygen influx was associated with mitochondrial respiration and was reduced by 78% when the cells was treated with 0.5 mM KCN. The residual cyanide-resistant respiration was inhibited by the application of 5 mM salicylhydroxamic acid, an inhibitor of the alternative oxidase. Similarly the cytochrome pathway was also inhibited by the presence of 20 M NO, while the cyanide-resistant alternative oxidase was not. These results demonstrate the use of the newly developed SREM-O₂ system to measure and characterize metabolic fluxes at a level of sensitivity that allows for subcellular resolution. These measurements, in conjunction with SERIS-H⁺ measurements, have led to new insights in our understanding of basic cellular physiology in plant cells.Abbreviations SRIS self-referencing ion selective - SREM self-referencing electrochemical microelectrode - ICP inductive coupled plasma spectroscopy     

The influence of pH and membrane potential on passive Na+ and K+ fluxes in human red blood cells

Passive (ouabain-insensitive) Na+ and K+ effluxes from human red blood cells were measured over the range pHo 6.2-8.5. On raising pHo, Na+ efflux increased and this was mainly attributable to the piretanide-sensitive component: K+ efflux likewise but attributable to both piretanide-sensitive and piretanide-insensitive components. On replacing Cl- with non-penetrating anions (mainly gluconate), Na+ and K+ effluxes increased, mostly attributable to the piretanide-insensitive components. On restoring pHi either by reducing pHo or by applying DIDS, the influence of pHo on Na+ and K+ effluxes was diminished. These results suggest that pHi rather than Em is the dominant influence. Passive Na+ and K+ effluxes and influxes in the presence of bumetanide were tested fro conformity to the Ussing independence relationship. For K+, the calculated and observed ratios agreed, indicating that the sodium pump, 'cotransport' and leak wholly account for K+ fluxes in human red blood cells. For Na+, the ratios did not agree and a 1:1 Na+/Na+ exchange did not account for the discrepancy. Pathways for Na+ appear to be more numerous than for K+.     

Heavy water inhibition of alkali cation transport across the muscle membrane. II. A comparison of the action of D20 and ouabain on the sodium efflux and rubidium influx in magnesium media

The action of heavy water and ouabain on sodium effluxes and rubidium influxes has been measured and compared in frog muscles (m. sartorius, R. temporaria). Approximately half of muscle sodium was substituted by lithium by preliminary incubation in mixed sodium-lithium media. The ratio of the ouabain-sensitive parts of rubidium influx and sodium efflux is 7.3:10.5, and that of D2O-sensitive parts of corresponding parameters is 7.5:11.3. A conclusion is made that D2O-effect on the Na, K-ATPase system of muscles under investigation resembles ouabain-effect on sodium effluxes as well as on rubidium influxes.     

Covariance of ion flux measurements allows new interpretation of Xenopus laevis oocyte physiology.

An animal-vegetal net ionic current identified previously using voltage probe techniques in maturing Xenopus laevis oocytes has now been investigated using noninvasive ion-selective microelectrodes. Three-dimensional fluxes of hydrogen (H(+)), potassium (K(+)), and bicarbonate (HCO(3)(-)) were characterized with respect to the developmental stage and hemisphere of the oocyte and presence of surrounding follicular tissue. Variable effluxes of H(+) and HCO(3)(-) were recorded from both the animal and vegetal hemispheres. Variable influxes and effluxes of K(+) were also observed. The equatorial region, silent by voltage probe, exhibited fluxes of H(+) and K(+). Simultaneous measurement of pairs of ions allowed correlation analysis of two ion types. Notably for H(+) and K(+) data, positive and negative correlation at animal and vegetal poles respectively offer an explanation of the unpredictable results obtained when individual ions were observed independently.     

Vertical nutrient transport and its effects on epilimnetic phosphorus in four calcareous lakes

The influence of vertical nutrient transport on epilimnetic phosphorus was studied comparatively in four calcareous Wisconsin lakes during 1972. In the two lakes with steep metalimnetic nutrient gradients (Mendota, Delavan), upward fluxes by entrainment and eddy diffusion exceeded all other influxes combined; here epilimnetic total-P concentrations increased during period of high windpower and thermocline migration, and decreased during comparatively stagnant intervals. In the two lakes lacking upper metalimnetic P gradients (Green, Fish), higher windpower had little or no effect on epilimnetic phosphorus.In each of the four lakes epilimnetic P declined in early summer until a quasi steady-state was achieved between P influxes and P sedimentation. In Mendota and Delavan steady-state featured higher concentrations of total-P, and much higher epilimnetic concentrations of particulate-P and chlorophyll, than in Green and Fish Lakes — mainly on account of the high fluxes of molybdate-reactive, biologically-available P through the seasonal thermocline. The flux analysis illustrates why mean lake TP concentration after ice-out is an inconsistent measure (not sufficient statistic) for estimating nutrient potential and chlorophyll standing crops during the following summer in stratified lakes.     

Boron deficiency alters cytosolic Ca2+ concentration and affects the cell wall components of pollen tubes in Malus domestica

• Boron (B) is essential for normal plant growth, including pollen tube growth. B deficiency influences various physiological and metabolic processes in plants. However, the underlying mechanism of B deficiency in pollen tube growth is not sufficiently understood. In the present research, the influence of B deficiency on apple (Malus domestica) pollen tube growth was studied and the possible regulatory mechanism evaluated.
• Apple pollen grains were cultured under different concentrations of B. Scanning ion‐selective electrode technique, fluorescence labelling and Fourier‐transform infrared (FTIR) analysis were used to detect calcium ion flux, cytosolic Ca²⁺ concentration ([Ca²⁺]cyt), actin filaments and cell wall components of pollen tubes.
• B deficiency inhibited apple pollen germination and induced retardation of tube growth. B deficiency increased extracellular Ca²⁺ influx and thus led to increased [Ca²⁺]cyt in the pollen tube tip. In addition, B deficiency modified actin filament arrangement at the pollen tube apex. B deficiency also altered the deposition of pollen tube wall components. Clear differences were not observed in the distribution patterns of cellulose and callose between control and B deficiency treated pollen tubes. However, B deficiency affected distribution patterns of pectin and arabinogalactan proteins (AGP). Clear ring‐like signals of pectins and AGP on control pollen tubes varied according to B deficiency. B deficiency further decreased acid pectins, esterified pectins and AGP content at the tip of the pollen tube, which were supported by changes in chemical composition of the tube walls.
• B appears to have an active role in pollen tube growth by affecting [Ca²⁺]cyt, actin filament assembly and pectin and AGP deposition in the pollen tube. These findings provide valuable information that enhances our current understanding of the mechanism regulating pollen tube growth.

     

Inorganic nutrient and oxygen fluxes across the sediment–water interface in the inshore macrophyte areas of a shallow estuary (Lake Illawarra,Australia)

Rates of inorganic nutrient and oxygen fluxes, and gross community primary productivity were investigated using incubated cores in July, August and September 2001, in a seagrass meadow of Lake Illawarra, a barrier estuary in New South Wales, Australia. The results indicated that rates of gross primary productivity were high, varying from C = 0.62 to 1.89 g m^–2 d^–1; low P/R ratios of 0.28–0.48 define the system as heterotrophic and indicate that more carbon is respired than is produced. In order to determine the effect of macroalgae on O₂ and nutrient fluxes, measurements were also conducted on cores from which the macroalgae had been removed. The results showed that the O₂ fluxes during light incubations were significantly lower in the cores without macroalgae (P<0.01), indicating that macroalgae could be a significant contributor to the primary production in the lake. In general, nutrient fluxes showed a typical diurnal variation with an efflux from sediments in the dark and a reduced efflux (or uptake) in the light. Dissolved inorganic nitrogen (NO₂ ^–+ NO₃ ^–+NH₄ ⁺) net fluxes were directed from the sediments towards the water column and dominated by the NH₄ ⁺ fluxes (>80%). NO₂ ^–+ NO₃ ^– and o-P fluxes were always very low during the sampling period. The increasing tendency of net nutrient effluxes, especially NH₄ ⁺ from July to September, is consistent with the increase of the water temperature and seagrass biomasses. However, in September, significantly lower light, dark and net NH₄ ⁺ effluxes were found in the cores with macroalgae (SA-sediments) compared with the cores without macroalgae (S-sediments). These results support the hypothesis that actively-growing dense macroalgal mats (i.e., algal blooms in September) may act as a filter reducing the flux of nutrients to the water column.     

Passive and active transport properties of a gill model, the cultured branchial epithelium of the freshwater rainbow trout (Oncorhynchus mykiss)

Branchial epithelia of freshwater rainbow trout were cultured on permeable supports, polyethylene terephthalate membranes ("filter inserts"), starting from dispersed gill epithelial cells in primary culture. Leibowitz L-15 media plus foetal bovine serum and glutamine, with an ionic composition similar to trout extracellular fluid, was used. After 6 days of growth on the filter insert with L-15 present on both apical and basolateral surfaces, the cultured preparations exhibited stable transepithelial resistances (generally 1000-5000 ohms cm2) typical of an electrically tight epithelium. Under these symmetrical conditions, transepithelial potential was zero, and unidirectional fluxes of Na+ and Cl- across the epithelium and permeability to the paracellular marker polyethylene glycol-4000 (PEG) were equal in both directions. Na+ and Cl- fluxes were similar to one another and linearly related to conductance (inversely related to resistance) in a manner indicative of fully conductive passive transport. Upon exposure to apical fresh water, transepithelial resistance increased greatly and a basolateral-negative transepithelial potential developed. At the same time, however, PEG permeability and unidirectional effluxes of Na+ and Cl- increased. Thus, total conductance fell, and ionic fluxes and paracellular permeability per unit conductance all increased greatly, consistent with a scenario whereby transcellular conductance decreases but paracellular permeability increases upon dilution of the apical medium. In apical fresh water, there was a net loss of ions from the basolateral to apical surfaces as effluxes greatly exceeded influxes. However, application of the Ussing flux ratio criterion, in two separate series involving different methods for measuring unidirectional fluxes, revealed active influx of Cl- against the electrochemical gradient but passive movement of Na+. The finding is surprising because the cultured epithelium appears to consist entirely of pavement-type cells.     

用激光扫描共聚焦显微镜观察雪松花粉和花粉管

为更直观地观察和显示花粉和花粉管中细胞结构及其细胞核的状态与行为。雪松花粉和花粉管经卡诺液固定,分别以埃氏苏木精、曙红、Hoechst 33243单染和曙红-Hoechst 33342双染后,用冬青油整体透明,在激光扫描共聚焦显微镜下观察。4种染色法观察效果不同;以曙红-Hoechst 33342双染的样品观察效果最佳,在紫外光激发下清晰地显示出细胞核,在488 nm激光激发下不仅能清晰看到花粉和花粉管壁结构,且能分辨管细胞、柄细胞及体细胞的结构特点和空间位置关系。建立了一种快速简便的适于在激光扫描共聚焦显微镜下观察花粉和花粉管中成员细胞结构及其细胞核的状态、行为的制片技术;激光扫描共聚焦显微镜具有独特的共轭成像装置、连续光学扫描、图像三维重组和多通道检测等功能,极好地展示了雪松花粉和花粉管的结构特点,相比于传统的光学显微镜和荧光显微镜,其观察到的图像更清晰、更直观、更具立体感。     

Ca2+-permeable channels in the plasma membrane of Arabidopsis pollen are regulated by actin microfilaments

Cytosolic free Ca2+ and actin microfilaments play crucial roles in regulation of pollen germination and tube growth. The focus of this study is to test the hypothesis that Ca2+ channels, as well as channel-mediated Ca2+ influxes across the plasma membrane (PM) of pollen and pollen tubes, are regulated by actin microfilaments and that cytoplasmic Ca2+ in pollen and pollen tubes is consequently regulated. In vitro Arabidopsis (Arabidopsis thaliana) pollen germination and tube growth were significantly inhibited by Ca2+ channel blockers La3+ or Gd3+ and F-actin depolymerization regents. The inhibitory effect of cytochalasin D (CD) or cytochalasin B (CB) on pollen germination and tube growth was enhanced by increasing external Ca2+. Ca2+ fluorescence imaging showed that addition of actin depolymerization reagents significantly increased cytoplasmic Ca2+ levels in pollen protoplasts and pollen tubes, and that cytoplasmic Ca2+ increase induced by CD or CB was abolished by addition of Ca2+ channel blockers. By using patch-clamp techniques, we identified the hyperpolarization-activated inward Ca2+ currents across the PM of Arabidopsis pollen protoplasts. The activity of Ca2+-permeable channels was stimulated by CB or CD, but not by phalloidin. However, preincubation of the pollen protoplasts with phalloidin abolished the effects of CD or CB on the channel activity. The presented results demonstrate that the Ca2+-permeable channels exist in Arabidopsis pollen and pollen tube PMs, and that dynamic actin microfilaments regulate Ca2+ channel activity and may consequently regulate cytoplasmic Ca2+.     

Nonsteady-State Three Compartment Tracer Kinetics: II. Sodium Flux Transients in the Toad Urinary Bladder in Response to Short Circuit

The theoretical approach presented in the previous paper provides an analytical method for determining the unidirectional, nonsteady-state fluxes in a three compartment system. Based on this a study was made of the sodium flux transients in the toad urinary bladder. A transient time-dependent state was generated by suddenly short-circuiting a bladder previously maintained in an open-circuited steady state. The sequence of experiments suggested by the theory provided the data required for the analysis. The results of these tracer experiments were consistent with the complex non-three compartmental structure of this tissue. As a result both of the inadequacy of the three compartment model in representing the tissue and of certain experimental difficulties, attempts at a quantitative solution were not entirely successful. Useful information was nevertheless obtained through a careful use of this model, and a qualitative analysis implied that the sodium influxes into the tissue at both of its surfaces are sensitive to changes in electrical potential while both effluxes are insensitive to this change. This suggests that both of the effluxes result from active processes while both influxes are associated with passive processes. The net transepithelial transport of sodium would then necessarily result from a more complex polarization than that proposed by Koefoed-Johnsen and Ussing.     

选择性微电极在植物生理学研究中的应用

选择性微电极技术是一种不仅能直接测定活的生物细胞或细胞器内的离子或分子活度,而且能对活的生物相邻的位置、功能和代谢速率可能不同的特定微区细胞表面的离子或分子流（flux）分别测定的电生理方法。具有操作简便、实时、非损伤性（测定离子或分子流）、灵敏度高（可达10^-12moles cm^-2s^-1）等优点。因为它是用微型化（尖端直径为0.5-5μm）的离子或分子选择性电极直接对准样品测定,不同于其他化学测定需取样品,所以能连续测定和自动监测,具有广阔的应用前景。该文阐述了选择性微电极测定原理,总结了选择性微电极技术在植物生理学研究中的应用进展,并展望了其发展前景。