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American Journal of 87(11): 1547±1560. 2000. INVITED SPECIAL PAPER

PLANT IN THE NEW MILLENNIUM: NEW TOOLS AND NEW INSIGHTS1

ELISON B. BLANCAFLOR2 AND SIMON GILROY3,4

2Plant Biology Division, The Samuel Roberts Noble Foundation Inc., 2510 Sam Noble Parkway, Ardmore, Oklahoma 73401 USA; and 3Biology Department, 208 Mueller Laboratory, The Pennsylvania State University, University Park, Pennsylvania 16802 USA

The highly regulated structural components of the cell form the basis of its . It is becoming increasingly recognized that cellular components are ordered into regulatory units ranging from the multienzyme complexes that allow metabolic channeling during primary to the ``transducon'' complexes of signal transduction elements that allow for the highly ef®cient transfer of information within the cell. Against this structural background the highly dynamic processes regulating cell function are played out. Recent technological advances in three areas have driven our understanding of the complexities of the structural and functional dynamics of . First, microscope and digital camera technology has seen not only improvements in the resolution of the optics and sensitivity of detectors, but also the development of novel microscopy applications such as confocal and multiphoton microscopy. These technologies are allowing cell to image the dynamics of living cells with unparalleled three-dimensional resolution. The second advance has been in the availability of increasingly powerful and affordable computers. The computer control/ analysis required for many of the new microscopy techniques was simply unavailable until recently. Third, there have been dramatic advances in the available probes to use with these new microscopy approaches. Thus the plant cell now has available a vast array of ¯uorescent probes that will report cell parameters as diverse as the pH of the , the oxygen level in a , or the dynamics of the . The combination of these new approaches has led to an increasingly detailed picture of how plant cells regulate their activities.

Key words: caged probes; cell biology (); ; green ¯uorescent ; laser tweezers; light microscopy, ratio imaging; signal transduction.

OVERVIEW techniques that can be exploited to aid in further understanding the inner workings of plant cells. The past decade has seen an explosion in the development The power of modern light microscopy to view plant cel- of new techniques in cell biology, particularly in the ®eld of lular relies almost as much on improving ¯uorescent light microscopy and imaging. New microscopes are contin- probes as it does on faster computers, sensitive photodetectors, uously being developed that have either improved optics or and advanced lasers. The green ¯uorescent protein (GFP), a the ability to image new features of the cell. High performance new cellular marker cloned from the jelly®sh Aequoria vic- cameras with improved detection capabilities are also becom- toria, has allowed investigators to monitor cytoskeletal and ing a common feature of many cell biology laboratories. Since dynamics in living plant cells (Kohler, 1998; Kost, its conception by Marvin Minsky in the 1950s, confocal mi- Spielhofer, and Chua, 1998; Marc et al., 1998) as well as long- croscopy has now become a routine technique and indispens- distance transport of and (e.g., Imlau, able tool not only for cell biological studies but also for mo- Truernit, and Sauer, 1999; Oparka et al., 1999; reviewed by lecular investigations (Lichtman, 1994). In addition, although Thompson and Schulz, 1999; Lazarowitz, 1999). Several spec- used by only a handful of research laboratories worldwide tral variants of this protein have also enabled researchers to since its recent introduction, applications of multiphoton ¯uo- monitor the dynamics of two or more in the cell rescence microscopy to biological questions are likely to grow simultaneously (Ellenberg, Lippincott-Schwartz, and Presley, rapidly within the next few years. This technique is poised to 1999; Palm and Wlodawer, 1999; Haseloff, 1999). These spec- provide a very real alternative to confocal microscopy in re- tral variants of GFP have also led to the design of transgenic vealing the three-dimensional dynamics of the plant cell intracellular sensors that take advantage of ¯uorescence (Denk, Strickler, and Webb, 1990). Computers are also fast resonance transfer (FRET) between different spectral evolving to meet the demanding requirements for image cap- forms of GFP (Miyawaki et al., 1997, 1999; Allen et al., 1999; ture and analysis that have become essential to make full use of these new instruments. For example, approaches such as Gadella, van der Krogt, and Bisseling, 1999). real-time computational image deconvolution (Scalettar et al., The combination of sophisticated instrumentation with new 1996) were until recently only available to microscopsists with ¯uorescent probes has transformed light microscopy into a access to a supercomputer. All these developments have trans- powerful analytical tool that has allowed researchers to gain lated into a repertoire of advanced microscopy and imaging new insights on basic plant cellular structure and function. As new approaches emerge and current technologies undergo re- ®nement, we are constantly presented with a new suite of tools 1 Manuscript received 3 February 2000; revision accepted 5 May 2000. to tackle previously intractable questions in plant cell biology. Funding for this work was through the Samuel Roberts Noble Foundation Due to space limitations, this review will highlight just some Inc. (E.B.B.) and grants from the National Aeronautics and Space Adminis- tration and the National Science Foundation (S.G.). The authors thank Dr. of the developments in optical microscopy and imaging tech- Richard S. Nelson and Dr. Sarah Swanson for comments on the manuscript. nology outlined above and how they have revolutionized our 4 Author for reprint requests (e-mail: [email protected]). view of plant cell function. We will also suggest some of the 1547 1548 AMERICAN JOURNAL OF BOTANY [Vol. 87 directions these new tools could lead us as we take plant cell the surface of individual hyphae can then be captured at different planes biology into the new millennium. within the cell using the confocal microscope. Figure 1 shows that the con- focal imaging approach not only produces blur-free images, but also captures ADVANCED OPTICAL MICROSCOPY the three-dimensional architecture of the cell. Thus each plane in the colonized INSTRUMENTATION AND TECHNIQUES cell has a unique pattern of hyphae. A projection of sequential optical sections taken throughout the depth of the cell shows the complex pattern of hyphae For more than three centuries, the light microscope has provided biologists as they ramify throughout the single colonized cell (Fig. 2). Such an appre- with a powerful visual and analytical tool to study cells and tissues. With the ciation of the three-dimensional details and complexity that forms the basis introduction of the electron microscope in the 1930s, researchers were able of cellular structure is now routinely possible in large part because of wide- to take cell biology several steps further into understanding cell function by spread availability of confocal microscopes. resolving not previously visible with the light microscope. Although Confocal microscopy has been applied to answer questions as varied as electron microscopy continues to play an essential role in characterizing cel- determining micro®bril orientation (Verbelen and Stickens, 1995), lular function it can only yield static images of cells and tissues. Yet it has in situ localization of RNA and DNA probes (Wymer et al., 1999), determin- always been clear to microscopists that the plant cell possesses an intricately ing the spatial and temporal characteristics of pH during growth patterned, highly dynamic, three-dimensional structure, which is thought to (Taylor, Slatter, and, Leopold, 1996; Bibikova et al., 1998) and for monitoring form the basis of cellular function. Conventional light microscopy in com- ¯uorescent dye movement to study cell-to-cell in and bination with other contrast-enhancing approaches such as differential inter- (Zhu, Lucas, and Rost, 1998; Gisel et al., 1999). Three-dimensional ference contrast (DIC) microscopy and phase contrast have provided tools to morphological examination of surface structures used to be the realm of scan- monitor the structural dynamics of the cell (for a full discussion of these ning electron microscopy (SEM), but confocal microscopy is proving to be techniques, see Ruzin, 1999). These microscopy approaches have continued an effective tool in these type of studies as well (Lemon and Posluszny, 1998; to be instrumental in cataloging the structural basis of plant cellular processes from 's discovery in 1665 that cells formed a functional unit of Melville et al., 1998), with the important advantage of being applicable to to modern high-resolution DIC video-enhanced microscopy that allows living cells. However, it is important to note that the confocal microscope has individual plant to be imaged in solution (Moore et al., 1997). only reached its place as an indispensable tool for the plant biologist because, However, cell biologists had to await signi®cant technological advances to be in parallel, researchers have been developing an ever increasing range of able to capture and, more recently, manipulate the dynamic, three-dimensional ¯uorescent reagents to visualize de®ned cellular structures and processes (see of speci®c molecular components of the plant cell in vivo. below).

Confocal microscopyÐConfocal microscopy is proving to be one of the Multiphoton ¯uorescence microscopyÐA new approach to live cell im- most exciting advances in optical microscopy of the last century. Although conventional wide-®eld epi¯uorescence microscopy has been a powerful tool aging that is likely to have as dramatic an impact as confocal microscopy is for locating speci®c molecular components of the cell, it suffers from the multiphoton microscopy. This technique uses a laser to deliver a burst of low- problem of out-of-focus ¯uorescence interfering with the contrast and reso- energy, long-wavelength photons in a short pulse. The photon ¯ux density in lution of the ®nal image (Webb, 1999). With the commercialization of con- the pulses is high enough that some of the photons hit a target ¯uorochrome focal microscopes in 1988, biologists began seeing cells in three dimensions simultaneously. When the target absorbs two photons at essentially the same and with much more clarity than previously possible. The impact that confocal time, they produce a similar effect as a single short-wavelength photon with microscopy has had on cell biology is evident in the number of reviews and twice the energy (i.e., half the wavelength). For example, two photons of books written on the subject (e.g., Pawley, 1995; Sheppard and Shotton, 1997; infrared radiation (e.g., 700 nm) when absorbed simultaneously can excite a Webb, 1999). The number of scienti®c papers employing confocal microscopy UV or blue light excitable dye (e.g., a dye normally excited by 350 nm light; in plant biology has also grown dramatically since it was ®rst introduced and Xu et al., 1995; Sako et al., 1995). The probability that two photons will is indicative of the importance of this technology to plant science research arrive at the ¯uorochrome simultaneously drops off dramatically away from (Hepler and Gunning, 1998; Wymer et al., 1999). the focal plane of the microscope. Thus ¯uorescence is only effectively ex- Confocal microscopes work by exciting ¯uorescence with a highly focused cited at the focal plane, and an optical section analogous to a confocal optical beam of laser light. The laser illuminates the sample, and the emitted ¯uo- section is generated. Unlike the confocal approach, however, the optical sec- rescent light is collected by the microscope objective. Light emitted from the tion is produced by the excitation of the ¯uorochrome in a single focal plane. focal plane of the microscope passes through a pinhole aperture positioned in In addition, the multiphoton microscope imposes much less demand on the front of a photomultiplier detector. The photomultiplier then passes the de- detector optics because, unlike the confocal microscope, the emitted light does tected light signal to a computer, which will use it to generate an image. The not have to be accurately focused through a pinhole to achieve the optical critical feature of the confocal approach is that light emitted from points above sectioning effect. This helps improve the ef®ciency of detection and the ability or below the plane of focus is blocked by the pinhole and so never reaches to image deep into tissues. the detector. Thus only an image of the ¯uorescence from the focal plane is Multiphoton microscopy has the potential to circumvent some of the prob- observed (i.e., the microscope has generated an ``optical section''). A single lems inherent in confocal imaging. The high-intensity, short-wavelength ir- two-dimensional view of the optical section is then obtained by scanning the radiation used to excite the samples in many confocal imaging situations can laser from point to point over the sample until the entire focal plane of the be damaging when imaged over long periods. Also, the shorter wavelengths sample is imaged (Lichtman, 1994). Because only the light at the focal plane of light tend to penetrate less far into a sample (Gilroy, 1997). Multiphoton of the sample passes through the pinhole onto the detector, the resulting image is free from out- of-focus ¯uorescence. By collecting a series of optical sec- microscopy, on the other hand, limits excitation to the focal plane of the tions at different focal planes, a full three-dimensional image of the sample sample, and, therefore, photobleaching of the ¯uorochrome outside the focal can be reconstructed (Webb, 1999). As sectioning is performed using optics plane is minimized. This feature, in addition to the fact that the longer wave- rather than the physical sectioning of the sample, living cells can be analyzed. length photons used in multiphoton microscopy have less energy, cuts down A series of optical sections through a root cell of Medicago truncatula on phototoxicity to the sample (Denk, Piston, and Webb, 1995). Longer wave- colonized with a symbiotic (Glomus versiforme) demonstrates the length photons are also less prone to scattering, making it possible to image quality of images that can be obtained using confocal microscopy (Figs. 1 deeper into the sample (Gilroy, 1997; Fricker and Oparka, 1999). Multiphoton and 2). A root was stained with wheat germ agglutinin (WGA) linked to the microscopy promises to generate confocal-like images while making longer ¯uorescent dye Texas-red. WGA selectively binds to the N-acetoglucosamine observation times possible and should provide an important complement to acid residues on the hyphal surface. Fluorescence from the Texas red labeling the confocal microscope (Sako et al., 1995). November 2000] BLANCAFLOR AND GILROYÐPLANT CELL BIOLOGY IN NEW MILLENNIUM 1549

Figs. 1±2. Confocal images of an inner cortical cell colonized by arbuscular-mycorrhizal fungi in a root of Medicago truncatula. 1. A series of optical sections (a±f) taken at 1-␮m intervals using a Bio-Rad MRC 1024 confocal microscope of a root section stained with Texas-Red conjugated to wheat germ agglutinin, which binds to the surface of the hyphae. Single optical sections are free from out-of-focus ¯uorescence, a typical problem encountered with conventional ¯uorescence microscopy. 2. Projection of 29 optical sections taken at 0. 2-␮m intervals from the same cell depicted in Fig. 1. Confocal microscopy allows the projection of several optical sections to reveal the intricate three-dimensional structure of a cell. Note the highly complex distribution of hypha in a colonized cell (E. B. Blanca¯or, L. Zhao, and M. J. Harrison, unpublished data). Scale bar ϭ 10 ␮m.

FLUORESCENT DYES Hϩ,Kϩ,Naϩ,Cl-,Zn2ϩ; Haugland, 1999). These indicators have driven much of the advances in our understanding of The new imaging technologies have provided unparalleled cellular control as they provide an image of the dynamics of opportunities to image the plant cell. However, the insights a well-de®ned cell component. Their usage is perhaps most they have revealed have resulted in large part because of the prevalent in the study of ionic signals such as Ca2ϩ and pH. explosion in the availability of optical probes that can be used For example, Ca2 ϩ-selective dyes such as Fluo-3 and Calcium with these techniques. are continuously developing Green, whose ¯uorescence increases in response to Ca2ϩ lev- new ¯uorescent labels to allow imaging of , nucleic els, have been instrumental in demonstrating a previously un- acids, , and just about any speci®c cell component. In suspected role for Ca2ϩ release from intracellular stores and addition, targeted probes for and even speci®c intracellular waves of Ca2ϩ in tube growth (reviewed are now commercially available (Haugland, 1999). Many of in Franklin-Tong, 1999). However, there have always been these reagents have yet to be successfully applied to plant limitations in interpreting ¯uorescence images from such dyes. cells, and we can expect some plant-speci®c problems with The signal from the dye may increase due to increased Ca2ϩ, the use of some probes. For example, although ¯uorescent but also due to dye relocalization, differences in dye concen- probes to image are widely used in tration, photobleaching, and differences in optics between re- cells (e.g., Montana, Farkas, and Loew, 1989; Haugland, 1999), plant biologists have had little success with equivalent gions of a tissue or even subregions of a cell. This limitation approaches. Similarly, the ubiquitous ``ester-loading'' method was elegantly overcome by applying the ratio analysis ap- employed to load ¯uorescent dyes into the animal cell cyto- proach (Tanasugarn et al., 1984; Tsien and Poenie, 1986). In plasm has had very limited success in plants where dyes may this approach a spectral shift in ¯uorescence is monitored rath- completely fail to penetrate the cell (Gilroy, 1997; Fricker et er than simply monitoring ¯uorescence intensity. A wave- al., 1999) or accumulate in the rather than the cyto- length of ¯uorescence that increases with ion concentration is plasm (Swanson and Jones, 1996). However, there is a vast compared with one that is invariant or decreases with ion level. array of untried ¯uorescent probes available to the plant bi- The ratio of these intensities is independent of dye concentra- ologist, and their potential to reveal speci®c insights into how tion and many of the other optical artifacts that make the signal the plant cell functions is enormous. This is especially true from single-wavelength dyes hard to quantify accurately. when they are combined with image analysis approaches, such The ratio analysis approach has allowed reliable, quantita- as ratio imaging, which allows ¯uorescence images to yield tive, spatial, and temporal analysis of images from ¯uorescent quantitative information about the dynamics of the cell. probes. growth and signaling in guard cells serve to highlight the power of this technology and the biological Fluorescence ratio imagingÐThe ¯uorescence of many surprises it has revealed. Ratio imaging has shown a tip-fo- ¯uorochromes is sensitive to their molecular environment. For cused gradient in Ca2ϩ centered on the growing tip of the pol- example, the ¯uorescence of perhaps the most widely used len tube (Fig. 3). The gradient is always associated with the ¯uorochrome, ¯uorescein, increases as the pH rises. This has growing tip and altering the gradient redirects growth (Malho led to the design of a whole spectrum of dyes that are selective and Trewavas, 1996; Bibikova, Zhigilei, and Gilroy, 1997). indicators of speci®c cell parameters or molecules. Thus there When tip growth ceases, the gradient is lost. Such data led to are dyes whose ¯uorescence reports parameters ranging from the idea that localized Ca2ϩ in¯ux across the plasma membrane mobility to speci®c ion concentrations (e.g., Ca2ϩ,Mg2ϩ, generated a tip-focused Ca2ϩ gradient that promoted 1550 AMERICAN JOURNAL OF BOTANY [Vol. 87

Figs. 3±6. Optical probes used in modern light microscopy to visualize cellular dynamics. 3. Ratio imaging of cytoplasmic Ca2ϩ levels in a pollen tube of Trandescantia. The pollen tube was microinjected with Indo-1 and Ca2ϩ levels determined from the emission ratio 400±430 nm/460±480 nm using a Zeiss LSM 410 UV confocal microscope. Ca2ϩ levels were pseudocolored according to the inset scale. Fluorescence ratio imaging reveals the highly localized calcium gradient at the pollen tube tip. Scale bar ϭ 10 ␮m. 4. Transient expression of GFP in an epidermal cell of Nicotiana tabacum. The coding region of enhanced GFP (eGFP) was linked to the cauli¯ower mosaic 35S promoter, biolistically bombarded, and the imaged using a Bio-Rad 1024 confocal microscope. Free GFP is distributed throughout the cell and can also be seen to enter the nucleus (n) but not the (c) (S. Yin, E. B. Blanca¯or, and N. L. Paiva, unpublished data). Scale bar ϭ 20 ␮m. 5. A leaf of Nicotiana benthamiana inoculated with mosaic virus that was genetically modi®ed to express GFP. The leaf shows green ¯uorescence associated with the veins when photographed under UV light. The advent of GFP technology has allowed the visualization of the pathways by which viruses move in living plants (N. Cheng and R. S. Nelson, unpublished data). Bar ϭ 1 cm. 6. Reorientation of Trandescantia pollen tube growth after local photoactivation of caged Ca2ϩ ionophore (A23187). Uncaging of the ionophore was accomplished by UV irradiating the region denoted by the white box using a 0.7-s scan of a Zeiss UV confocal microscope. Local activation of the ionophore allows Ca2ϩ in¯ux into the cell at that point. The pollen tube reorients toward the new site of elevated calcium induced by the localized Ca2ϩ in¯ux. Scale bar ϭ 10 ␮m. Inset shows ¯uorescence ratio imaging of Ca2ϩ levels in Trandescantia pollen tubes during localized photoactivation of caged Ca2ϩ ionophore (Bibikova et al., 1997). The pollen tube was microinjected with calcium green/rhodamine and Ca2ϩ levels pseudocolored according to the scale in Fig. 3. Note that Ca2ϩ is elevated at the site of ionophore uncaging. Scale bar ϭ 10 ␮m. November 2000] BLANCAFLOR AND GILROYÐPLANT CELL BIOLOGY IN NEW MILLENNIUM 1551 and therefore drives growth. The link between the Ca2ϩ gra- apoprotein (apoaequorin) and coelentrazine, its small cofactor. dient and growth was even more strongly made when it was Light emission from aequorin is triggered by Ca2ϩ with the noted by several researchers that pollen tubes grow in pulses rate of luminescence being proportional to the Ca2ϩ concen- and that the gradient also oscillated (reviewed in Franklin- tration. The for apoaequorin has been cloned, and when Tong, 1999). However, a closer examination revealed that the cells expressing this protein are treated with coelentrazine, story is not so simple. The increase in the Ca2ϩ gradient co- functional aequorin is reconstituted. The luminescence rate can incides with growth but comparing this to measurements made be calibrated to an absolute Ca2ϩ concentration making it pos- using the self-referencing (vibrating) microelectrode showed sible to use transgenically expressed aequorin as a Ca2ϩ sensor. Ca2ϩ in¯ux into the tip lagged the elevation of the cytosolic Plants such as tobacco and Arabidopsis have been stably trans- -gradient by 10 s (Pierson et al., 1996; Holdaway-Clarke et al., formed with aequorin and their Ca2؉ dynamics monitored ei 1997; Messerli and Robinson, 1997). We are still trying to ther by putting whole plants in a luminometer, or by visual- understand how these observations ®t into a model of the reg- izing light emission using a highly sensitive camera. ulation of tip growth, but Ca2ϩ incorporation into newly syn- Aequorin has revealed some surprising insights into plant thesized wall polymers, stretch-activated Ca2ϩ channels, and signaling and regulation. It has been used to show that there Ca2ϩ release from internal stores all seem plausible features are proliferating waves of Ca2ϩ and -speci®c responses that could be interacting to give these ion dynamics. Recent when challenged by a host of environmental signals such as imaging data suggest the Ca2ϩ gradient is controlled in part salinity, drought, cold, heat shock, blue light, elicitors, touch, by the Rop class of monomeric GTPases (Li et al., 1999) and or anoxia (reviewed in Trewavas, 1999). With aequorin it was possibly in¯uenced by the cytoskeleton (Bibiko- also shown that there are oscillations in Ca2ϩ associated with va, Blanca¯or, and Gilroy, 1999). Thus the ratio imaging data the circadian rhythms of the plant (Johnson et al., 1995). Ae- have not only indicated that Ca2ϩ is a critical player in the quorin has been targeted to various organelles and has shown, growth machinery but has also shown us that we have only for example, that not only is Ca2ϩ regulated inde- just begun to characterize its action. pendently of the cytosol (Johnson et al., 1995), but also, that In guard cells, the subtlety of potential signaling systems is despite the extensive pore system in the nuclear membrane, dramatically revealed by the ratio analysis approach. Guard nuclear Ca2ϩ is also regulated independently of cytosolic levels cells respond to multiple environmental signals including red (Van der Luit et al., 1999). 2ϩ and blue light, CO2, humidity, and the stress hormone ABA Arti®cial coelentrazines of varying Ca af®nities, aequorins (abscisic acid; Assmann and Shimazaki, 1999). This ability to with various spectral properties, and even a ratioable aequorin integrate numerous signals has led to intensive studies on sig- are available (Knight et al., 1993). However, imaging the rapid naling in these cells. Studies with inhibitors of Ca2ϩ signaling dynamics of Ca2ϩ at the cellular level using aequorin is very hinted at a Ca2ϩ-dependent system of signaling. However, only dif®cult, and imaging with subcellular resolution has proven after ratio imaging allowed the spatial and temporal dynamics impossible (unless the protein is speci®cally targeted to a sub- of the system to be seen and manipulated was the full extent cellular site) due to the inherently low signal generated by this of the complex signaling network in this single cell revealed luminescent protein. These applications are more suited to (reviewed in Hetherington et al., 1998). These studies showed analysis by ¯uorescent dyes and GFP-based sensors such as the operation of Ca2ϩ-dependent and -independent signaling the cameleon Ca2ϩ probe (see below). However, aequorin has systems in response to ABA that is in¯uenced by the environ- a far superior dynamic range than these ¯uorescent sensors mental history of the plant (Allan et al., 1994). Evidence has and can therefore easily detect rapid Ca2ϩ transients in whole also accumulated for information being encoded in the spatial plants. Aequorin's high signal-to-background also makes it us- patterns, amplitude, and frequency of transient elevations in able in plant cells where auto¯uorescence makes ¯uorescent Ca2ϩ levels occurring within the cytoplasm (Gilroy, sensors inapplicable. These advantages and disadvantages of Read, and Trewavas, 1990; Grabov and Blatt, 1998; Staxen et aequorin highlight that although there is a remarkable array of al., 1999). For example, Staxen et al. (1999) presented evi- probes and technologies available to the plant biologist to dence that the frequency of Ca2ϩ transients might encode the monitor cell behavior, each is appropriate to answer a speci®c ambient ABA concentration. Recent work with calmodulin- kind of question and it is in their combination and comple- dependent protein kinase II from mammalian cells shows a mentary usage that the real power of these new approaches single can decode Ca2ϩ transients into speci®c fre- lies. quency-related intermediate enzymatic activities (Dekoninck and Schulman, 1998), providing a model of how plant cells GREEN FLUORESCENT PROTEINS: NOVEL INSIGHTS might use such frequency-related information. It is clear that ON CELLULAR STRUCTURE, DYNAMICS, AND most cells respond to myriad signals with a remarkable ability SIGNALING IN LIVING PLANT CELLS to integrate and respond appropriately. We can anticipate that the guard cell story of a complex and dynamic regulatory net- The discovery of GFP and several of its spectral variants work will be reprised in many other systems as more plant from Aequorea victoria (Ellenberg, Lippincott-Schwartz, and cells receive equivalent, intensive analysis by cell physiolo- Presley, 1999; Palm and Wlodawer, 1999; Haseloff, 1999) and gists. more recently a red ¯uorescent protein from another anthozoan species (Matz et al., 1999) has provided a unique complement AequorinÐNo review of imaging and analysis of signaling to the battery of imaging devices that have been developed systems in plants would be complete without mentioning the during the latter part of the 20th century. GFP is a 27-kDa advances made in the use of aequorin as a luminescent Ca2ϩ protein that ¯uoresces bright green when excited by UV or indicator. Aequorin, like GFP, is a protein originally from the blue light (Fig. 4). GFP ¯uorescence depends on an internal jelly®sh A. victoria that has proven of immense use to cell chromophore and so, unlike other biological ¯uorochromes, it biologists. Aequorin is a luminescent protein consisting of an does not need the addition of a cofactor to ¯uoresce (Cubbit, 1552 AMERICAN JOURNAL OF BOTANY [Vol. 87

Wollenweber, and Heim, 1999; Prendegast, 1999). In addition, actin binding of talin, a mammalian actin-binding pro- mutated forms of GFP have been generated with altered spec- tein (designated GFP-mTn). GFP-mTn constructs decorated tral characteristics (e.g., Blue-FP, Cyan-FP, and Yellow-FP), as actin ®laments in tobacco pollen tubes and cultured BY2 cells, well as altered stabilities and solubilities (Heim and Tsien, which allowed the in vivo observation of actin dynamics in 1996). The gene for GFP can be fused to other target these cells. More recently, stable transformation of Arabidop- and the resulting construct genetically engineered into a cell. sis with the GFP-mTn and GFP-MAP4 fusions was used to The behavior of the resulting GFP-fusion protein can then be monitor actin ®lament and microtubule dynamics during tri- followed in vivo. The entire GFP protein appears necessary to chome development (Mathur et al., 1999; Mathur and Chua, generate the ¯uorochrome (Cubbit, Wollenweber, and Heim, 2000). 1999) and so the entire 27-kDa GFP coding region must be Although these are the only reports to date on visualizing fused to the gene of interest. Remarkably, attaching the 27- the cytoskeleton in living plant cells using GFP fusions, we kDa GFP to many proteins does not alter their activity, al- can anticipate that studies on the plant cytoskeleton will ad- though this cannot be taken for granted and must be rigorously vance rapidly in the next few years because of this technol- proven in vitro and in vivo before results from the GFP tagged ogy. Additional advances will come by applying multiphoton protein can be easily interpreted. However, GFP fusions have microscopy to intact plants expressing the GFP-cytoskeleton been applied with great success to follow the dynamics of the binding protein fusions to image cytoskeletal dynamics in plant cytoskeleton, endomembrane traf®cking, organelle dy- cells located deeper in an intact plant organ (Potter et al., namics, and macromolecular transport. GFP has also been 1996; Schwille et al., 1999). Studies with ¯uorescent analog used to mark speci®c cell types and to follow cytochemistry already suggest that microtubules on different events. These topics are discussed below. faces of a cell may have vastly different dynamics and or- ganization (Yuan et al., 1992), and studies on ®xed material The plant cytoskeletonÐThrough the use of highly speci®c have shown that the cytoskeleton in the innermost cells of antibodies against cytoskeletal proteins, researchers were able roots reorganizes rapidly when exposed to a variety of hor- to infer from ®xed, static images how the plant cytoskeleton mones and other stimuli (Shibaoka, 1994; Blanca¯or and reorganizes during various developmental stages and in re- Hasenstein, 1995a, b; Blanca¯or, Jones, and Gilroy, 1998; sponse to a variety of environmental stimuli (Lloyd, 1987; Cyr, Nick, 1998; Hasenstein, Blanca¯or, and Lee, 1999). Imaging 1994; Hush and Overall, 1996; Nick, 1998). A more dynamic the cytoskeleton deep into living tissues using multiphoton element was added to this snapshot view of the cell through and GFP technology should reveal the dynamic nature of the use of ¯uorescent analog cytochemistry, whereby a ¯uo- these responses. Furthermore, the availability of several spec- rescently labeled protein (e.g., rhodamine-tubulin) is intro- tral variants of GFP will be of enormous value in allowing duced into the cell by microinjection. The labeled protein multiple tagging experiments to probe the interaction be- could then be incorporated into the pool of normal cellular tween different components of the plant cytoskeleton in liv- proteins and their behavior monitored by ¯uorescence or con- ing cells. For example, studies of the dynamics of the inter- focal microscopy. Using this technique, the highly dynamic action between microtubules and actin should be high on the nature of both the cortical and spindle microtubule cytoskel- priority list of plant cytoskeletal researchers (Tominaga et al., eton of living plant cells were directly observed and quanti®ed 1997; Collings et al., 1998). (Wasteneys, Gunning, and Hepler, 1993; Hush et al., 1994; Yuan et al., 1994; Wymer et al., 1997; Himmelspach et al., Visualizing intracellular organelle dynamics and 1999). Similar progress has been made with the actin cyto- endomembrane traf®ckingÐThe use of GFP has also led to skeleton. By microinjecting small amounts of ¯uorescently la- new insights on the dynamics and functions of various cellular beled phalloidin, a low molecular weight fungal metabolite organelles and the endomembrane system of plants. The cre- that binds preferentially to F-actin, it was possible to observe ation of GFP fusions that contain speci®c localization sequenc- the actin cytoskeleton in living plant cells undergoing division es results in the retention of GFP in speci®c organelles and (Schmit and Lambert, 1990; Cleary, 1995), in pollen tubes membrane systems and allows their direct observation in vivo (Miller, Lancelle, and Hepler, 1996), and in Transdescantia (Kohler, 1998). Although membrane-permeable ¯uorescent hairs (Ren et al., 1997). dyes have been available to observe the dynamics of some The procedures to load ¯uorescently labeled proteins into cellular components such as the (ER) cells, however, can be a technically demanding and slow pro- and mitochondria in living plant cells, questions as to their cess, especially for walled plant cells. Microinjection, being speci®city have been a major concern (Hepler and Gunning, an invasive technique, can also perturb cellular function. Fur- 1998). Furthermore, rapid photobleaching and the possible thermore, the technique suffers from limited observation times toxic effects of these ¯uorescent dyes have made long-term and is only applicable to isolated or exposed single cells (Kost, observation of these organelles in living plant cells dif®cult Mathur, and Chua, 1999). These limitations have been partially (Scott et al., 1999). overcome with the use of GFP. By fusing the microtubule- So far, the most common and readily available GFP-based binding domain of the microtubule-associated protein 4 subcellular markers are constructs that have the ER peptide (MAP4) with GFP and transiently expressing the recombinant retention signal, KDEL. By fusing the KDEL sequence to the protein in plant cells, Marc et al. (1998) were able to label coding sequence of GFP and expressing this construct in cortical microtubules in living epidermal cells. A time course plants, the dynamic cortical network of membranous tubules analysis of the labeled microtubules revealed intricate dynam- representing the ER could be visualized (Boevink et al., 1996, ics including localized reorientations, lengthening, shortening, 1998). This new vital ER marker has already provided new and translocation of microtubules. information on structure/function of the endomembrane sys- Kost, Spielhofer, and Chua (1998) have taken a similar ap- tem as well as vesicle traf®cking in plants (Hawes, Brandizzi, proach for actin by transiently expressing GFP fused to the and Andreeva, 1999). For example, GFP fused to the ER re- November 2000] BLANCAFLOR AND GILROYÐPLANT CELL BIOLOGY IN NEW MILLENNIUM 1553 tention signal has not only demonstrated the highly dynamic and mechanisms by which viruses moved in plants were poor- nature of the plant endomembrane system, but has also re- ly understood primarily because of an inability to observe the vealed the existence of rapidly moving organelles (Haseloff infection process in live tissue over time (Nelson and van Bel, and Siemering, 1998). Although these organelles have been 1998; Santa Cruz, 1999). However, through the observation of suggested to be proplastids, an alternative explanation as to GFP ¯uorescence from chimeric viruses, researchers can ef- the identity of these organelles has been proposed (Gunning, fectively monitor the vein classes by which viruses move after 1998). Using a GFP fusion targeted to the infection in near-real time conditions (Fig. 5; Roberts et al., (made by linking GFP to the transmembrane domain of a rat 1997; Cheng et al., 2000). sialyl transferase) in combination with a second construct con- New insights as to the manner in which viruses move from sisting of GFP fused to the Arabidopsis H/KDEL homologue cell to cell are also beginning to come to light. Although the of the HDEL receptor, aERD2, the simultaneous visu- association of viral components with some of the host cell alization of the Golgi and ER was possible. This provided the components has been reported previously, these associations ®rst evidence that the ER and Golgi in plants are closely as- were demonstrated mainly through electron and light micros- sociated (Boevink et al., 1998). copy of ®xed plant tissues (Nelson and van Bel, 1998). With In addition to the ER and Golgi, GFP has also been targeted modi®ed viruses expressing GFP fusions to viral proteins, spe- to the mitochondria (Kohler et al., 1997b), vacuole (Sanse- ci®c localization of viral proteins to plasmodesmata (Itaya et bastiano et al., 1998), (Kohler et al., 1997a; Tirlapur al., 1997; Oparka et al., 1997) and other subcellular compo- et al., 1999), and the nucleus (Chytilova, Macas, and Gal- nents of the plant host have been elegantly demonstrated. For braith, 1999). Among other ®ndings, this work has allowed example, by linking the tobacco mosaic virus movement pro- the monitoring of the mobility of thin tubular projections orig- tein (MP) to GFP, colocalization of MP-GFP to cellular actin inating from plastids (Kohler et al., 1997a; Tirlapur et al., was observed and MP was demonstrated to bind actin in vitro 1999) and the movement and shape changes of the nucleus (McLean, Zupan, and Zambryski, 1995). MP-GFP has also (Chytilova, Macas, and Galbraith, 1999). Furthermore, by gen- been shown to localize to ¯uorescent aggregates and ®lamen- erating a protein fusion between GFP and a spliceosomal pro- tous structures in infected BY2 cells. Treatment of infected tein, the dynamics of coiled bodies, nuclear organelles in- cells with microtubule-depolymerizing compounds disrupted volved in RNA processing, have been observed in higher the ®lamentous strands providing evidence for MP-microtu- plants for the ®rst time (Boudonck, Dolan, and Shaw, 1999). bule association (Heinlein et al., 1995, 1998). These studies By creating GFP fusions to known cellular proteins or their all indicate that the local spread of viruses could be facilitated putative targeting peptides, it has also been possible to deter- by interactions between a virus and these components of the mine the dynamics and localization patterns of these proteins host plant (Reichel, Mas, and Beachy, 1999). in living plant cells. For example, GFP linked to phragmo- The progress made from studies on movement plastin, a protein known to be associated with for- has had a signi®cant impact on plant cell biology and plant mation, has allowed the dynamics of early cell plate devel- . The ability of viruses to penetrate the physical opment to be observed in vivo (Gu and Verma, 1997). A barriers presented by the plant has opened new ideas on the unique family of calmodulin-dependent Ca2ϩ-ATPases were lo- fundamental mechanisms by which macromolecules are trans- calized to the ER and of Arabidopsis (Hong ported throughout the plant. These transport stories have re- et al., 1999) and a calmodulin-binding transporter protein was vealed perhaps one of the major discoveries of the past decade, shown to be associated with the plasma membrane of that plant can be transported from cell to cell. (Schuurink et al., 1998). Various proteins This was originally reported for the Knotted gene in (e.g., CRY2, COP1, phyB) implicated in light signal trans- (Lucas et al., 1995), but there are now an ever-expanding set duction were linked to GFP and shown to translocate to the of results pointing to a remarkable informational macromolec- nucleus (Kleiner et al., 1999; Stacey and von Arnim, 1999; ular exchange between plant cells that are likely to coordinate Yamaguchi et al., 1999; Stacey, Hicks, and von Arnim, 1999), many aspects of plant development and function (Lucas and while an isocitrate dehydrogenase isoenzyme fused to GFP Wolf, 1999; Oparka et al., 1999). The reader is referred to the was demonstrated to localize exclusively to the mitochondria several excellent reviews published recently that have thor- (Galvez et al., 1998). GFP fusions to transit peptides of oughly addressed these issues (Lazarowitz, 1999; Lazarowitz phage-type organellar RNA polymerases also allowed local- and Beachy, 1999; Santa Cruz, 1999; Thompson and Schulz, ization of this protein to the mitochondria and plastids. These 1999). data provided additional support for the involvement of these polymerases in the transcriptional machinery of plant mito- Plant cellular signalingÐAnother exciting use of GFP fu- chondria and plastids (Hedtke et al., 1999). Therefore, with sion proteins is to allow the visualization and quanti®cation of the availability of several spectral variants of GFP and the protein±protein interactions. This technique is called ¯uores- ability to target GFP to almost any cellular organelle desired, cence resonance energy transfer (FRET) and relies on the phe- we predict that the direct interactions between these different nomenon whereby a donor ¯uorescent , when excited organelles and/or protein fusions will be an area of intense with the appropriate wavelength of light, transfers some of its research. emission energy to an adjacent acceptor chromophore. Since FRET is dependent on the proximity of the donor to the ac- Macromolecular transport and virus movements in ceptor, measurement of the ef®ciency of FRET can be used to plantsÐVirus movement in plants is a complex process that estimate the distance between donor and acceptor ¯uoro- is generally considered to involve both cell- to-cell movement phores. Therefore if two different proteins or molecules are via the plasmodesmata (Reichel, Mas, and Beachy, 1999) and tagged with either a donor or acceptor ¯uorophore, FRET can systemic transport via the vascular system (Nelson and van determine whether these proteins are in close association (Gor- Bel, 1998). Despite being intensively studied, the pathways don et al., 1998). 1554 AMERICAN JOURNAL OF BOTANY [Vol. 87

The concept of FRET in combination with GFP technology video-rate scanning two-photon microscopy (Fan et al., 1999); offers new methods for elucidating signal transduction path- therefore it will be possible to quantify rapid changes in Ca2ϩ ways in plants. For example, spectral variants of GFP were deep in an intact plant organ. recently used to construct a novel intracellular Ca2ϩ sensor referred to as ``cameleon.'' This probe was designed so that GFP as a cell- and tissue-speci®c markerÐIn develop- two spectral variants of GFP with overlapping emission and mental biology, labeling of cells to follow cell fate has relied excitation spectra were fused at opposite ends of calmodulin mainly on the microinjection of dyes into speci®c cell types or immunocytochemical detection of speci®c cell types after (a Ca2ϩ-binding protein) and the M13 peptide, (the calmodu- lin-binding domain from myosin light chain kinase). The two tissue ®xation (Brand, 1999). With GFP, it is now possible to varieties of cameleon constructs are the yellow-cameleon, mark speci®c cells or tissues in living Arabidopsis plants and follow their fates directly during development (Fricker and which consists of cyan ¯uorescent protein (CFP) and the yel- Oparka, 1999; Haseloff, 1999). This was accomplished by us- low ¯uorescent protein (YFP), and the blue cameleon, which ing the targeted gene expression approach that uses the GAL4 consists of blue ¯uorescent protein (BFP) and GFP (Miyawaki yeast transcription activator. In this ``enhancer-trap'' strategy, et al., 1997). the GAL4 gene is randomly integrated into the Arabidopsis Cameleons report Ca2ϩ in the following manner. Binding of 2ϩ , bringing it under the control of different genomic Ca to the calmodulin within the cameleon induces a confor- enhancer sequences that can direct a wide range of expression mational change causing it to bind to the M13 peptide. The patterns in the plant. However, in order to obtain ef®cient ex- cameleon molecule is folded in the process such that the donor pression, it was necessary to alter codon usage by employing (e.g., CFP) and acceptor (e.g., YFP) are brought in to closer a derivative sequence (GAL4-VP19). With GAL4-VP19 proximity to each other, thereby increasing FRET ef®ciency. linked to mGFP5, which codes for ER-targeted GFP, the gen- The donor:acceptor ¯uorescence ratio can then be calibrated 2ϩ erated expression patterns of GFP in Arabidopsis roots could to the cytoplasmic Ca concentration (Miyawaki et al., 1997, be readily visualized with a confocal microscope (Haseloff, 1999). Although the BFP constructs suffer from low signal 1999). strength in plants, plant-expressed CFP constructs are bright Using the enhancer trap approach, root-cap-speci®c pro- (Blanca¯or, Dowd, and Gilroy, unpublished data) making the moters were recently identi®ed and used to genetically ablate CFP-YFP cameleon the appropriate choice for use with plant (i.e., selectively kill through the localized expression of a cy- tissues. totoxic protein) root caps of Arabidopsis to study the conse- Cameleons were ®rst used to measure intracellular calcium quences that such ablations have on root development (Hase- in mammalian cells (Miyawaki et al., 1997; Emmanouilidou loff, 1999). With the availability of speci®cally marked cell et al., 1999), but the yellow cameleon construct has now been lines, researchers were also able to follow cellular fate and successfully engineered into plant cells (Allen et al., 1999; infer positional information that contributes to a speci®c type Fricker and Oparka, 1999; Gadella, van der Krogt, and Bis- of patterning in roots and shoots (Berger et al., 1998a, b; Sa- seling, 1999). Allen et al. (1999) constitutively expressed this batini et al., 1999). Cell-speci®c marking using GFP has also yellow cameleon in Arabidopsis and used this to investigate been useful in the identi®cation of endodermal protoplasts for cytoplasmic Ca2ϩ changes in guard cells. Extracellular Ca2ϩ or patch-clamp experiments (Maathuis et al., 1998) and compan- ABA application induced cytoplasmic Ca2ϩ transients in guard ion cells for single-cell detection of gene transcripts (Brandt cells, which were similar to the changes observed using ¯uo- et al., 1999). rescent dyes. These results indicate that GFP-based Ca2ϩ in- One of the more exciting prospects of the enhancer trap dicators are suitable for plants and therefore will be of enor- approach is the ability to activate other genes of interest in the mous value in understanding the complex role of Ca2ϩ in plant GFP-marked cell lines. This can be accomplished by subclon- cell signal transduction. ing the desired gene behind a tandem array of ®ve optimized In addition to Ca2ϩ, pH has also been shown to tightly reg- GAL4 binding sites (the Upstream Activation Sequences) and ulate numerous cellular signaling events and physiological transforming plants with this construct. In the absence of the processes in plant cells (Guern et al., 1991). Like previous GAL4 transcriptional activator, however, the desired gene is studies with Ca2ϩ, measurement of cytoplasmic pH has relied not expressed. By crossing this plant with a line with a known on ¯uorescent ratiometric dyes (Bibikova et al., 1998; Fricker pattern of GAL4 expression, known because of the GFP ex- et al., 1999; Scott and Allen, 1999). GFP-based pH indicators pression patterns, the silenced gene is activated only in the have also been recently developed for monitoring cytoplasmic cells where GAL4 is expressed (Haseloff, 1999). Using this and organelle pH in mammalian cells (Kneen et al., 1998; approach, it should be possible, for example, to express the Llopis et al., 1998). Various laboratories are attempting to en- GFP-based cameleon or pH indicators in speci®c cells or tis- gineer these GFP-based pH indicators into plants, and it will sues in the plant and monitor signaling-related ion ¯uxes in be exciting to see whether these pH sensors function in living these cells. Since the ¯uorescence signal will be coming ex- plant cells. clusively from speci®c cells, the problem of interfering signals The use of GFP-based ion sensors to study plant signal from other tissues is eliminated. Furthermore, it should also transduction is still in its infancy, but is likely to generate be possible to express particular GFP-protein fusions such as information not previously obtainable with the traditional ¯uo- the GFP:MAP4 or GFP:talin fusions described above in spe- rescent dyes. This is due to the fact that these GFP-based ion ci®c cells and follow the dynamics of the cytoskeleton as these sensors can be easily targeted to organelles (see discussion cells progress developmentally. above); therefore the subtle changes in ions that appear to be part of the signaling cascades of some plant processes such as OPTICAL TECHNIQUES FOR MICROMANIPULATION gravitropism can now be determined (Legue et al., 1997; Ro- OF PLANT CELLS sen, Chen, and Masson, 1999; Scott and Allen, 1999). Fur- In addition to the improved ability to visualize dynamic thermore, the cameleon indicators can now be imaged with structures and quantify various physiological processes in November 2000] BLANCAFLOR AND GILROYÐPLANT CELL BIOLOGY IN NEW MILLENNIUM 1555

Fig. 7. Displacement of mineral-containing vesicles (statoliths) using optical tweezers. The arrow indicates the initial trapping of statoliths on one side of the . Similar to the graviresponse, bend toward the side of statolith accumulation (E. B. Blanca¯or, and S. Gilroy, unpublished data). Scale bar ϭ 20 ␮m. cells, optical microscopy is now being used as a precise tool Fallon, Shacklock, and Trewavas, 1993), the signaling path- to manipulate both the physical and chemical properties of the way of stomatal closure (Gilroy, Read, and Trewavas, 1990) cell. The ®elds of and physics have contributed sub- and in the localization of growth in pollen tubes (Fig. 6; Malho stantially to the development of these new optical approaches and Trewavas, 1996; Bibikova, Zhigilei, and Gilroy, 1997). and a representative from each ®eld is discussed below. Caged inositol triphosphate (IP3) has revealed a role for an IP3 mobilizable internal Ca2ϩ pool in the stomatal closure response Caged probe technologyÐCaged or photoactivatable (Blatt, Thiel, and Trentham, 1990; Gilroy, Read, and Trewa- probes provide the opportunity to manipulate cell vas, 1990) and pollen tube growth (Franklin-Tong, 1999) and or signaling activities in situ using light. All caged probes caged ABA has been used to localize one site of ABA per- operate on a similar theme. A biologically active molecule is ception to an intracellular site in the guard cell (Allan et al., derivatized by a caging group, which then sterically blocks the 1994). Given the ability to quantitatively regulate the spatial activity of the molecule. The caging group is chosen carefully and temporal release of a caged molecule with subcellular pre- to be photoactivatable. Thus, when the caging group absorbs cision, we can predict an increasing application of such ap- light (usually only UV light is energetic enough for these ap- proaches to probe the spatial and temporal requirements of plications), it photolyzes, releasing the biologically active mol- regulation in the plant cell. ecule. Some photolysis by-products are also released, and their potential biological activity has to be carefully checked. How- Laser micromanipulationÐThe use of lasers is not only ever, the caged molecules used in plant cells to date have re- limited to providing a coherent light source for microscopic vealed no indication of biological activity or cytotoxicity of imaging of cells, but has also been used for the micromanip- these by products. ulation of whole cells and organelles. Laser micromanipulation When the caged molecule is loaded into the cell (e.g., by can be divided into two types. The ®rst type, called laser twee- microinjection), then illumination using a standard ¯uores- zers or optical trapping, makes use of a continuous-wave, low- cence microscope is all it takes to release the molecule in the power infrared laser beam. The laser beam is focused on an cell. More sophisticated setups use a ¯ash UV illuminator or object at the focal plane of the microscope, which results in localized UV laser irradiation to produce highly controlled the refraction of the incident laser beam. The refraction of the temporal or spatial uncaging. In addition, the more UV light laser produces forces that pull the object toward the laser that is used, the more probe is activated. Thus the cell biologist beam, which can be used to hold the object in place. When can now control the spatial, temporal, and amplitude aspects applied to cells, moving the laser beam can pull organelles or of a molecule's activity in the living cell. As with ¯uorescent whole cells from one place to another (Berns, 1998; Greulich probes, the range of caged probes that are available has grown and Pilarczyk, 1998). dramatically in the last few years. There are now caged hor- One example of the use of laser tweezers in biology has mones, amino acids, peptides, drugs (such as caged taxol), been to address the problem of gravity sensing in the green nucleotides, ionophores, ¯uorochromes, and a range of caged- alga Chara. Chara is anchored to its substratum via single- chelators and even caged- (Haugland, 1999). Most cell, root-like structures called rhizoids. The tips of these rhi- are commercially available, although some of the more spe- zoids contain numerous vesicles that are ®lled with heavy min- cialized reagents must be obtained from the laboratory where eral deposits (Fig. 7). Upon tilting the rhizoids by 90Њ, these they were synthesized. vesicles sediment rapidly to the new lower ¯ank of the cell. Not surprisingly this technology has been applied to manip- Curvature commences shortly after sedimentation, and it is ulate a wide range of control features of animal cells (Adams believed that the sedimentation of these vesicles is responsible and Tsien, 1993). However, though used in several plant stud- for the initial perception of gravity leading to the redirection ies, its potential has yet to be fully explored by plant cell of rhizoid growth (Sievers, Buchen and Hodick, 1996). Laser biologists. For example, caged Ca2ϩ,Ca2ϩ chelators, and ion- tweezers have been used to mimic the gravitational displace- ophores have been used to show a requirement for Ca2ϩ in the ment of these vesicles and have been shown to alter the di- response to GA and ABA in aleurone cells (Gilroy, 1996), rection of rhizoid growth (Fig. 7; Leitz, Schnepf, and Greulich, phytochrome action (Shacklock, Read, and Trewavas, 1992; 1995). By combining laser trapping experiments and imaging 1556 AMERICAN JOURNAL OF BOTANY [Vol. 87 techniques such as ¯uorescence ratio analysis, it is now pos- CONCLUSIONS AND PROSPECTS sible to link displacement of the mineral containing vesicles to other cell regulatory elements (e.g., changes in Ca2ϩ distri- The rapid advances in optical probes and imaging technol- bution) that have been proposed to drive growth reorientation ogy make these exciting times for plant cell biology. The three during rhizoid gravitropism (Sinclair and Trewavas, 1997). lines of technological advances that have brought us to this Optical trapping techniques have also been applied to study unprecedented view of the dynamic plant cell show no sign the physical properties of the actin cytoskeleton in living plant of slowing. First we can predict a continuing impact of ever- cells. In this case, light from an argon-ion laser is focused onto increasing computing power. Image analysis is inherently a a transvacuolar strand and the force needed to displace strand- computer-based science and as computing power increases we associated vesicles is measured to give an indication of the can predict that techniques to improve resolution and extract viscoelastic properties of actin (Schindler, 1995). Using this even more complex information from raw ¯uorescence im- technique, it was demonstrated that various factors including ages, such as real-time image deconvolution, will become lipids, Ca2؉, pH, aluminum, and the plant hormones auxin and commonplace. The second thread of advance will be new and cytokinin alter the tension of the actin cytoskeleton in soybean exciting imaging technologies. We have highlighted multipho- suspension cells (Grabski et al., 1994; Grabski and Schindler, ton microscopy as a novel imaging approach with enormous 1995, 1996). Furthermore, Ca2ϩ-regulated kinases and phos- promise, but this is but one of the many technologies, such as phatases have been identi®ed as possible signaling molecules ¯uorescence lifetime imaging and correlation imaging, on the that modulate the changes in the viscoelastic properties of the horizon. The third line of advance will be in ¯uorescent and actin cytoskeleton (Grabski et al., 1998). However, whether luminescent probes. GFP technology is clearly an important the transvacuolar strands from which tension measurements step towards the ultimate goal of noninvasively monitoring the dynamics of the components of the plant cell, but there will were taken actually represent the actin cytoskeleton remains be an increasing need for ¯uorescent probes to monitor not questionable. With the availability of GFP to directly visualize only levels and distribution of regulators but also their activity. the cytoskeleton in living plant cells (see above; Kost, Spiel- For example, recently, calmodulin activity has been imaged hofer, and Chua, 1998; Marc et al., 1998), it would be inter- using novel ¯uorescent dyes (Hahn, Waggoner, and Taylor, esting to combine optical trapping techniques and ¯uorescence 1990; Torok et al., 1998) and a GFP-based probe (Persechini imaging to probe further into the physical properties of the and Cronk, 1999). These successes suggest that developing cytoskeleton. probes to image the activities of cellular regulators is an at- The second type of laser micromanipulation is called optical tainable goal. scissors or laser ablation. This technique relies on the precise The most signi®cant advances will be made by combining delivery of a high-powered laser beam to a target cell or or- these three areas of technology. For example, ¯uorescence cor- ganelle without affecting the areas surrounding the target (Po- relation microscopy uses powerful computational analysis of nelies et al., 1994; Berns, 1998; Greulich and Pilarczyk, 1998). ¯uorescence detected by very sensitive imaging sensors With this technique, whole plant cells have been ablated to (Haupts et al., 1998; Schwille et al., 1999). It has been used precisely de®ne the gravity-sensing cells in root caps of Ara- to look at the changes in parameters such as molecular dif- bidopsis (Blanca¯or, Fasano, and Gilroy, 1998, 1999). Signals fusional constants of GFP fusion proteins to image their in- that affect cell fate determination during root development teractions with other cell components (Brock et al., 1999; Trier have also been studied with the use of laser ablation (van den et al., 1999). Computation, imaging, and ¯uorescent probes Berg et al., 1997; Berger, 1998; Sabatini et al., 1999). have come together to reveal the dynamics of the environment Other applications of laser ablation include exposing the around a single molecule and have provided a very powerful plasma membrane of plant cells by ablating out a small region tool to monitor the activities of the living cell. The application of the cell wall to facilitate genetic transformation (Guo, of such technologies to the vast array of unanswered questions Liang, and Berns, 1995) or to allow easy access of micropi- in plant development, function, and regulation will be a major pettes for patch-clamp experiments (Henriksen and Assmann, goal for plant cell biology researchers in the new millennium. 1997; Bouget, Berger, and Brownlee, 1998). Laser ablation was also used to cut a hole in the wall of a single pollen grain LITERATURE CITED to isolate and eventually amplify its genomic DNA (Matsun- aga et al., 1999). 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