We believe that the major three challenges for humankind in the 21st century are food, energy, and the environment, including climate change and environmental degradation due to pollution and habitat loss. In a word: sustainability. Plant life plays an essential role in all three of these sustainability challenges. All of our food and the majority of our energy are produced by photosynthetic plants. Plants are major players in determining our climate, and agricultural expansion is a major factor in habitat encroachment and pollution of waterways by fertilizer application and runoff. Furthermore, these issues are not independent; as the climate changes, additional challenges are placed on plant performance and, thus, food supply and habitat. Research on plants is needed to provide solutions to these major challenges.
The fundamental biology of plants is similar to our own; they use the same genetic code, share many homologous genes, and even many regulatory mechanisms, basic biochemical pathways, and fundamental processes in cell biology. However, their form and lifestyle are fundamentally different. Plants can reach individual life spans of up to 5000 years; they can obtain adequate nutrition from the air and soil and survive adverse environmental conditions and attacks from pests, pathogens, and herbivores, despite remaining rooted in one spot for their lifetime. Plants are master chemists and can defend themselves with an incredible arsenal of chemicals. Many plants do not have a determinate body plan; a single genome is capable of producing an enormous range of size and form. Thus, plants are also valuable basic research objects since we can learn fundamental principles that are shared with humans and at the same time learn how different wiring can create such fundamental differences in form, biochemistry, and function.
While some of the richest ideas in the life sciences have been developed without application of specialized technology (the theory of natural selection and evolution stands out as a prime example), technology is more often a primary driver of new understanding. Two essential roles for new technology can be identified. First, advances in technology provide the means to test hypotheses formulated from less complete or less precise information. Second, technology provides new information to generate fresh hypotheses. Certainly the most influential advance in modern biology has been uncovering the molecular basis of inheritance and biological information transfer, namely, the discovery and structure of DNA and how this molecular information is transcribed into RNA and translated into proteins. This advance depended critically on technologies such as x-ray diffraction to test hypotheses generated from chemical analysis and, in turn, uncovered vast new fields of information that continue to revolutionize the course of biological research. The “central dogma” is also a prime example of how advances made possible by technology itself engenders powerful new technologies, accelerating the cycle of discovery. It is noteworthy that important new technologies can arise from pure curiosity-driven research, and these technologies can revolutionize areas far outside the original area of inquiry. Thermally stable DNA polymerases stand out as a prominent example.
In looking forward, we see the essential questions concerning technology and plant research to be threefold: (1) What existing technology do we need that is not currently being applied (Table 1)? (2) What new technology can be developed that we can readily envision (Table 2)? (3) What technology would we like to have but do not know how to create?
The third question is important because it helps us to set goals and helps us to recognize more easily the potential application of new developments in more distant fields of research. This exercise is also fun (we like to think of it as the Star Trek question): What would be really useful if it indeed existed? The creators of Star Trek and other science fiction imagined a variety of useful future technologies, one of which, the handheld communicator, has come to fruition as the cell phone, much as originally imagined. While warp drive and the transporter are not likely around the corner, the idea of these technologies continues to inspire the imagination.
Over the past decade, plant biology has matured and is homing in on many of the classic great questions: mechanisms of cell and tissue development, the interplay of hormones, mechanisms of cell wall construction and growth, the structure and function of signaling and metabolic networks, and mechanisms of environmental perception and adaptation. Highlights include the insight that protein degradation is central to hormone signaling and perception, the discovery of regulatory small RNAs, the identification of ion transporters as receptors, the discovery of flowering signals, the delineation of regulatory networks for hormones, the molecular basis for guard cell movement, discovery of genetic control networks for development, and breakthroughs in applications such as submergence tolerant rice (Oryza sativa; Xu et al., 2006; Leung, 2008). A series of editorials in the new journal Frontiers in Plant Physiology provide some recent perspectives regarding grand challenges for different fields of plant science (Frommer, 2010; Jorgensen, 2010; Deal, 2011; Gilroy, 2011; Heazlewood, 2011; Huber, 2011; Körner, 2011; Murphy, 2011; Sinha, 2011; Von Wirén, 2011; Zwieniecki and Dumais, 2011), and a group in the UK recently published a catalog of 100 questions that are considered major challenges for the future of plant sciences (Grierson et al., 2011). Application of the best of current technologies and development of new technologies are needed to meet the grand challenges in plant science. In this Perspective, we consider the utility of novel molecular and imaging technologies, two areas of recent and significant technology innovation. In addition to the discussion here, the reader is referred to a document developed by the National Academy of Sciences of the US entitled “A New Biology for the 21st Century” (Committee on a New Biology for the 21st Century, 2009). A central feature of this document is a thorough analysis of the Grand Challenges of Plant Science to develop ideas regarding advances through technology.
THE NEW OMICS TECHNOLOGIES: CATALOGING TECHNOLOGIES AND BEYOND
The early years of molecular genetics made rapid strides, especially in revealing genes and pathways important for plant development; essential cellular functions, such as transporters; and components of important biochemical pathways, such as photosynthesis and lipid biosynthesis. Progress accelerated severalfold when advances in sequencing technology, aided soon thereafter by advances in computational technology, opened the door to whole-genome sequencing forever changed the possibilities for discovery.
A Revolution of DNA Sequencing Technologies
Thirty years ago we were thrilled when we could analyze DNA sequence 75 bp at a time using Maxam and Gilbert reactions (Maxam and Gilbert, 1977), or we could study the expression of a gene using RNA gel blots (Alwine et al., 1977). At the same time, the first solid-phase synthesis of oligonucleotides became available. DNA sequencing technology is undergoing continual change, with next-generation sequencing (NGS) achieving ever-increasing throughput and concomitant reductions in cost (Metzker, 2010). NGS started with massively parallel signature sequencing, Polony, and 454 platforms and now comprises Illumina (Solexa), SOLiD, ion conductor, DNA nanoball, Helioscope single-molecule, single-molecule real time, single-molecule real-time RNA polymerase, nanopore, and Visigen sequencing platforms. It is estimated that the cost of DNA sequencing drops at a faster rate than the cost of computer data processing (Figure 1). The decoding of the human genome, completed by a public consortium of universities in 2003, cost in excess of $500 million. What was a grand challenge a few years ago, namely, the “$1000 human genome,” is now in sight (Mardis, 2006). Today, both oligonucleotide synthesis and DNA sequencing are performed by companies or large facilities (e.g., at the Beijing Genome Institute, which today has some 137 Illumina HiSeq2000 and 27 Applied Biosystems SOLiD 4.0 systems; www.genomics.cn/). Whole genomes can be sequenced in a day, and ambitious projects are making rapid progress in deciphering the genome sequence of thousands of Arabidopsis thaliana accessions (Cao et al., 2011; Ledford, 2011). We expect that the genomes of many crops will be deciphered in the next few years. In addition, NGS developed to survey microorganisms associated with plants, the microbiome (Siezen and Kleerebezem, 2011), will help us to understand the structure and dynamics of microbial communities as well as their role in plant biology (Bisseling et al., 2009).
Trajectory of Cost Reductions in DNA Sequencing.
Data replotted from: Wetterstrand K.A. For DNA sequencing costs, data are from the National Human Genome Research Institute Initiative Large-Scale Genome Sequencing Program available at www.genome.gov/sequencingcosts (accessed February 4, 2011).
Despite breakthrough advances, such as PCR, and cloning systems, such as Gateway (Hartley et al., 2000) and USER (Nour-Eldin et al., 2010), cloning is still cumbersome. Ambitious graduate students are often surprised that the way to a major breakthrough runs through frustrating valleys of cloning experiences (e.g., PCR errors or difficulties in generating complex constructs). Therefore, one of the big challenges is a quantum leap in methods to facilitate cloning and molecular construction. Significant cost reductions in DNA synthesis, currently in the range of ~$0.30/bp, will facilitate developments to overcome this roadblock. We hope that this technology will be complemented by comprehensive clone depositories for plants, like those available for yeast, Caenorhabditis elegans, and mouse. Examples include genome-wide small interfering RNA libraries for mouse and human and antibody libraries for almost all human proteins. While comprehensive protein data repositories are publicly available for some organisms, such as the subcellular localization of all yeast proteins, similar data sets for plant proteins are missing.
Another major challenge is the transfer of genes into plant genomes. While transformation of Arabidopsis is fast and simple, transformation of other species frequently requires special expertise and involves extended tissue culture phases that lead to somaclonal variation. Moreover, transformation technology based on Agrobacterium tumefaciens is limited by the size of the T-DNA and allows transfer of a limited number of genes at a time. At present, stacking of traits in plants is limited to a handful of constructs, while synthetic biology in bacteria is now at a stage in which complete chromosomes can be synthesized and introduced into bacterial hosts to replace the genetic information (Gibson et al., 2008, 2010). The generation of plant minichromosomes (Birchler et al., 2008) combined with the ability to synthesize artificial chromosomes promises to overcome many of the limitations, providing us with the potential to create breakthroughs in synthetic biology for the plant field in the future. One of the remaining big challenges for the plant field is the development of efficient transformation technologies for introducing these large synthetic DNAs into plant genomes and, more importantly, the knowledge of the biology needed to assemble useful information and circuitry on these chromosomes.
At the same time, adding genes in random positions of the genome is far less powerful compared with gene replacement strategies, which are common in yeast and mouse. Nonhomologous recombination has limited our ability to use recombination-based gene replacement strategies; thus a major challenge in the plant filed is targeted gene replacement (i.e., the ability to replace an endogenous gene with a mutated version).
Large-scale sequence analysis technology has opened a wide range of opportunities beyond genome sequencing, including genome-wide analysis of the transcriptome (the set of RNA transcripts present in an organism, organ, tissue, or even cell type at a certain stage of development or in response to environmental conditions), the methylome (DNA methlyation landscape; Pelizzola and Ecker, 2011), the translatome (RNAs bound to polysomes; Mustroph et al., 2009), and transcription factor binding sites on specific chromosomes using chromatin immunoprecipitation (ChIPSeq; Ferrier et al., 2011). It is now even possible to map the position of ribosomes to mRNA at the genome scale using NGS (Ingolia, 2010; Ingolia et al., 2011). Transcript analysis is further facilitated by high-throughput quantitative RT-PCR and Fluidigm’s microfluidic expression analysis (Moltzahn et al., 2011). The breakthrough in identifying small RNAs as key regulators has led to cataloging of all small RNAs using NGS approaches. A major gap in this area is that all methods focus on measuring steady state levels of RNAs. In most organisms, including plants, RNA turnover is still poorly understood. Also here, the new sequencing technologies will help us systematically to identify the RNA degradome (Endres et al., 2011; Zheng et al., 2011).
For all of these genome-scale lines of inquiry, NGS technologies are replacing array-based technologies, such as microarrays or tiling arrays, not only due to lower cost and higher throughput, but also because the technology allows for quantitative analyses (e.g., counting of transcripts). Studies suggest that genome-wide RNA-seq is more sensitive and has a much larger quantitative range than microarray analysis and is at least comparable to quantitative RT-PCR. Nature Genetics has dedicated a website to collect the recent publications that continue to revolutionize this field (www.nature.com/nrg/series/nextgeneration/index.html).
With these new technologies comes a new set of challenges, such as optimizing library construction, amplification strategies, read length bias, and, importantly, data analysis and storage (Pop and Salzberg, 2008; Ozsolak and Milos, 2011). However, these technologies provide low resolution with respect to cells, and expression typically has been analyzed principally at the whole-plant or organ level. In recent years, considerable progress has been made in the field of cell-specific gene expression (Wee and Dinneny, 2010), a critical set of information for multicellular organisms. Progress was initiated by fluorescence-activated plant cell sorting (Brady et al., 2007), and this is being supplemented by methods that rely on tagging and affinity-purifying nuclear ribosome-associated RNAs using INTACT (Deal and Henikoff, 2011) or translatome-based methods (Mustroph et al., 2009). The latter two approaches achieve cell specificity by driving the affinity tag from cell-specific promoters.
Robotic technologies also play an increasing role in cataloging technologies, most prominently for the use of yeast two-hybrid technologies to map the interactome of plants (Lalonde et al., 2010; Dreze et al., 2011). NGS-based methods may soon replace the need for robotics here as well. Microfluidics may also play a role in molecular cataloging, as, for example, the microfluidic chip for high-throughput analysis of in vitro kinetics of protein interactions (Bates and Quake, 2009). Molecular interactions can also be measured in high-throughput platforms using label-free plasmon resonance energy imaging (Lausted et al., 2011). This technology may prove crucial since it will take us from a qualitative perspective of protein interactions to quantitative data sets.
While cataloging technologies expand our knowledge dramatically, a major gap remains regarding understanding the function of most cataloged genes, their regulation, and their interactions in networks. For example, a huge number of proteins in the best-studied plant, Arabidopsis, have no assigned biochemical activity. Moreover, while some proteins have putative functions assigned to them, these are based only on sequence homology (i.e., we do not know what their substrates or ligands are nor what specific pathways they mediate). Examples include the hundreds of receptor-like kinases and ~1000 F-box proteins. Importantly, we still lack an understanding of most cellular and subcellular processes in the multicellular plant at high temporal and spatial resolution. We have the tools to synthesize and engineer whole chromosomes in microorganisms, and soon we will have this capability in plants and can begin to construct minichromosomes. The biggest challenge will be determining how to engineer these chromosomes and build their regulatory circuits efficiently. If we consider plants as complex machines, similar to automobile engineering, we need a complete understanding of the underlying principles and the interplay of parts to engineer plants at will. This will require careful dissection of gene and protein functions combined with cellular and organismal physiology, systems biology, and computational modeling.
Progress in Mass Spectrometry Technology
Over the past few decades, massive progress has been made in mass spectrometry instrumentation and analysis tools, and wide distribution of this instrumentation has provided access to the technology to most plant scientists (McLafferty, 2011). Specific instruments have been developed for many different applications, ranging from small molecules to large protein complexes. Currently, the sensitivity of select instruments reaches the zeptomolar range (nanoelectrospray ionization; Shen et al., 2004). Some instruments provide extraordinary mass accuracy; Fourier transform ion cyclotron resonance mass spectrometry is an important and powerful tool in direct mass spectrometry analyses due to its ultrahigh resolution (>1,000,000) and mass accuracy (<1 ppm). High mass resolution is useful in empirical formula calculations and compound identification (Lei et al., 2011). These advances have fueled the development of genome-wide analysis of ion profiles (ionomics; Salt et al., 2008), metabolite profiles (metabolomics; Giavalisco et al., 2009), protein composition and relative abundance (proteomics; Heazlewood, 2011), the analysis of posttranslational protein modifications (phosphoproteomics; Ytterberg and Jensen, 2010), as well as analysis of other types of modifications, such as methylation, O-glycosylation, etc., and even protein interactions (interactomics; Gavin et al., 2011).These data sets will help us evaluate the relative contribution of transcriptional and posttranslational networks to the regulation of gene function. Ionomics and metabolomics today are largely defined by mass spectrometric analyses and can provide information down to the cellular level (Mach, 2008; Benfey, 2011; Conn et al., 2011). While these technologies are limited regarding subcellular resolution, techniques such as nonaqueous fractionation, in which cellular processes are arrested by rapid freezing and addition of organic solvents and organellar fractions are subsequently purified on density gradients, provide insights into the subcellular metabolome, ionome, or proteome (Krüger et al., 2011).
These mass spectrometry technologies are providing us with a molecular inventory and allow us systematically to identify important levels of posttranslational regulatory networks. Improvements in quantification will be critical for comparative analyses. Data sets derived using this rapidly progressing technology will be key to systems biology approaches that attempt to develop quantitative models of cells and organisms (Lee et al., 2010). With the increased availability of genome sequences, proteomics will be feasible and efficient in species other than Arabidopsis.
The New Genetics
For Arabidopsis, the production of extensive T-DNA insertion line collections has dramatically facilitated reverse genetics approaches. Multiple insertions are available for a large proportion of protein-encoding genes, and the specific mutation that is causative for an altered phenotype identified by forward genetic screens often can be identified within weeks. In Arabidopsis, it is now possible to use NGS to identify mutations in an F2 mutant population through next-generation mapping (Schneeberger et al., 2009; Austin et al., 2011). Similarly, the application of a combination of deletion generation by fast neutron bombardment with NGS promises to expand reverse genetics to many other plant species, thus significantly enhancing functional analyses in crops and other species (M.K. Barton, personal communication). Targeted induced local lesions in genomes (TILLING) has been another valuable tool for mutagenesis, and refinements such as ecotilling and deletion-TILLING provide novel means for high-throughput genotyping (Kurowska et al., 2011).
In parallel, advances in quantitative trait locus mapping, specifically association mapping, promise major advances for model systems and crop plants (Poland et al., 2011). The new genetics includes the methodology of chemical genetics, which expands our ability to detect phenotypes by directly targeting protein function through the use of small molecules that bind directly to proteins and alter protein function, thus circumventing problems with redundancy and lethality. Identification of the targeted protein can be pursued by interaction assays or sensitized genetic screens. This approach has been deployed with great success to gain major new insights into hormone signaling (Melcher et al., 2009; Nishimura et al., 2009; Park et al., 2009).
The New Structural Biology
Progress in expression systems, throughput, and analytical tools has lead to a dramatic increase in structural solutions of proteins. This holds true for membrane and soluble proteins. Structures that have been obtained recently for hormone receptors and recent progress in solving structures of transporters in the presence and absence of ligands have begun to provide us with snapshots of the molecular processes of enzyme and transporter activities (Krishnamurthy et al., 2009; Weyand et al., 2011). As yet, there is not a project to determine structures for plant proteins on a large scale, but such an endeavor would have great value in advancing our understanding of biochemical mechanisms and in looking at diversity and similarity in the proteome through the perspective of protein structure.
THE NEW CELL BIOLOGY AND IN VIVO BIOCHEMISTRY: IMAGING AND CELL BIOLOGICAL TECHNOLOGIES
Imaging is a principal means of assessing phenotype and function and an essential part of the modern biological tool kit. Operating over a range of spatial scales, spanning from tissues to single molecules, imaging provides the advantages of being able to observe and measure phenotype and function at cellular and even molecular resolution and to reveal biological dynamics in living tissue. These are capabilities that biochemical techniques rarely are able to achieve. And while physiological techniques such as microelectrode-based measurement have single-cell and even single-channel resolution, they are limited in their range of application and do not allow for efficient observation of variance over cellular scales.
Visible Light Imaging: Probe Development
In recent years, parallel advances in fluorescence-based microscopy methods and probes have revolutionized cell biology. The demonstration that intrinsically fluorescent proteins from jellyfish could be fused to other sequences and expressed and imaged in other species opened the floodgates to a new generation of experimentation and tool building. The fact that these probes are genetically encoded provides the possibility to tag target proteins with genetic specificity, a level not previously possible with antibodies. In addition, genetic encoding enables noninvasive introduction of probes at similar levels and spatio-temporal control as the native molecules. Noninvasive introduction is especially valuable in plant cells, whose rigid cell wall makes microinjection difficult and often presents challenges for the uptake and specific localization of probes introduced from the external solution (Fehr et al., 2004). It is now common practice to tag proteins of interest with fluorescent proteins to probe their localization, behavior, and possible interactions with other molecules in the cell.
Genetic encoding has also provided significant advantages for the development of new probes and has enabled the development of novel screens and tools. Improved and new probes have been developed by screening for naturally evolved variants and using molecular genetic techniques and high-throughput screening to evolve probes in the lab. Some key parameters that have been targeted are brightness, spectral output, pH sensitivity, oligomerization, and sophisticated properties such as photoconversion (Shaner et al., 2004, 2005; McKinney et al., 2009; Wu et al., 2011). Examples of novel screens and tools made possible by the genetic encoding of fluorescent probes include large-scale screens for protein localization (Cutler et al., 2000), which has been done effectively in yeast but remains to be done on a useful scale in plants, cell-type expression analysis based on fluorescence-activated cell sorting of disrupted and green fluorescent protein (GFP)–expressing cells (Birnbaum et al., 2003, 2005; Benfey, 2011), the ability to split probes into two components to use reconstitution of fluorescence as an assay for interaction of their tagged partners (Kerppola, 2008), and the creation of an ever growing array of novel cellular sensors (Shaner et al., 2005). Most of these sensors are genetically encoded and can thus be introduced into any cell or organism that is genetically transformable. These sensors can be based on changes in the expression or localization of a tagged and responsive protein (PH domain [Halet, 2005] or auxin probes [Ottenschlager et al., 2003]) or on a physical phenomenon called Förster resonance energy transfer (FRET) to probe changes in the spatial relationship of two compatible probes. FRET sensors are exemplified by small molecule sensors based on binding proteins that have different conformational states in the bound and unbound condition (ions, sugars, ATP, and signaling molecules, such as calcium or cyclic nucleotides) (Frommer et al., 2009). These genetically encoded sensors afford us with new possibilities to access new dimensions, such as millisecond time resolution and, importantly, cellular and subcellular resolution down to limited areas of the cell membrane (Okumoto et al., 2008). FRET can also be used to follow the activities of enzymes (such as proteases) in vivo, determine the phosphorylation state of a protein domain, measure membrane potential of subcellular membranes, and quantify tension between cytoskeletal proteins during growth (Frommer et al., 2009; Grashoff et al., 2010; Meng and Sachs, 2011; Zhou et al., 2011). Microfluidics has revolutionized single-cell analysis in yeast and animal systems (Bermejo et al., 2010, 2011b, 2011a). The recent development of a microfluidic multichannel device for root growth and imaging, the RootChip, now enables moderate throughput screening of mutants using the large spectrum of available FRET sensors in plants (Grossmann et al., 2011).
Of course, the jellyfish fluorescent proteins and relatives have not been the only active area for genetically based optical probe development. Binary probes have been created based on specific peptide motifs, and, recently, RNA motifs that interact with chemical fluors that are introduced to the cell have been developed. For example, the tetracysteine tags used for biarsenical labeling, termed FlAsH and ReAsH by Martin et al. (2005), offer the advantage of being considerably smaller than fluorescent proteins, whose large size can interfere with target protein function or assembly into complexes. However, they also can have high backgrounds due to off-target labeling. A recent report of specific RNA labeling using an evolved 60-base aptamer and a purpose-synthesized label is very exciting, with a variety of potential applications in plant biology (Paige et al., 2011).
Next-Generation Probes and Optogenetics
Plants have a wealth of light absorbing proteins, and these have been exploited to create useful new cell biological tools. For example, fluorescent tags have been created based the photoreceptors phytochrome and phototropin. As with tetracysteine tags, their primary advantage to date is their small size, but unlike tetracysteine tags, they do not create problems with off-target background. These proteins have been used not only to create probes for protein position, but also tools to alter protein function using light as a trigger (Leung et al., 2008; Levskaya et al., 2009; Wu et al., 2009) (Figure 2). These join a handful of other optically sensitive tools based on bacterial proteins, which have been used in exciting experiments to manipulate neuronal function through light sensate control of ion channels, a technique that has been dubbed optogenetics (Deisseroth, 2011). These light-based tools allow control of protein function with exquisite resolution in time and space and represent a bold and potentially revolutionary frontier in cell biology. While many of these tools are created with protein modules evolved in plants, we are not aware of their application in plant cells to date.
Reversible Manipulation of Protein Function Using Targeted Illumination.
Fusion proteins featuring Phytochrome and its effector phytochrome interacting factor3 (PIF) allow for reversible control of protein recruitment to the plasma membrane at the spatial precision of optical resolution and on a time scale of seconds. Levskaya et al. (2009) showed that these tools could be used to manipulate cell shape by locally activating Rho-GTPase signaling through light-mediated recruitment of Rho effector proteins to the plasma membrane of mammalian cells. A fusion protein of cyan fluorescent protein (CFP) and Phytochrome B (PHY) is tethered to the plasma membrane, where activation by red light recruits phytochrome interacting factor3 fused to yellow fluorescent protein (YFP) and the catalytic domain of the RacGEF Tiam (ITSN DHPH). The concentration of the RacGEF domain at the membrane activates its target Rho GTPase Cdc42, which in turn recruits a sensor consisting of the GBD binding domain of the actin polymerization factor WASP fused to the mCherry fluorescent protein. (Reprinted by permission from Macmillan Publishers Ltd.: Nature [Levskaya et al., 2009;Figure 4], copyright 2009.)
We anticipate that the development of new tools and methods based on genetically encoded probes and photosensitive proteins will be a very active area of research in the next few years. As organisms that have evolved to use and respond sensitively to light cues, plants represent a potentially rich reservoir of raw material for the creation of new photosensitive tools to manipulate protein localization and function.
Visible Light Imaging: Instrumentation
Extraction of information from live-cell imaging is limited by several instrument-dependent factors, including the ability to detect the labeled structures above specimen background and instrument noise, the spatial resolution of the microscope, the time resolution of image acquisition, the stability of the label under sustained imaging, and damage to the specimen caused by free radical generation.
The development of highly quantum efficient back-thinned detectors with improved electronics and on-chip amplification in electron multiplying charge coupled devices (EMCCDs) and, more recently, scientific-grade complementary metal-oxide-semiconductor (CMOS) cameras have made significant advances in addressing most of the above limitations. With >90% quantum efficiency and reduction of dark current and read noise to levels approaching photon noise-limited performance into very low light regimes, EMCCD cameras have made single-molecule detection widely available while also allowing images to be captured at tens of millisecond intervals without significant read noise penalties. These rapid frame rates enable observation of molecular dynamics at shorter time scales and permit three-dimensional (3D) imaging at rates that were formerly the domain of two-dimensional (2D) imaging (Figure 3). In addition, more sensitive detection allows significantly lower excitation energies to be used, extending the life of probes and reducing cellular damage by free radical generation. High-end CMOS cameras are now being introduced that improve on EMCCD performance for a number of applications. Specifically, they provide large fields of view and can acquire images with high dynamic range at extremely fast frame rates with minimal read noise penalty. EMCCDs continue to have better quantum efficiency, but CMOS technology is catching up in signal-to-noise performance.
Rapid 3D Time-Lapse Imaging.
Maximum projections made from spinning disk confocal stacks acquired from Arabidopsis leaf epidermal cells expressing GFP:a-tubulin 6 to label cortical microtubules. The images show the junction between two pavement cells. Twenty-five confocal sections were acquired every 5 s to generate 3D volumes ~7 μm in depth. (Image courtesy of D.W. Ehrhardt and Y. Fu, unpublished data.)
In thicker cells and specimens, background from the specimen needs to be reduced, so these cameras are often coupled with a means of optical sectioning, such as parallel scanning confocal imaging (spinning disk and its relatives). This combination has made possible extended dynamic imaging of single-protein complexes in plant cells (Paredez et al., 2006; Nakamura et al., 2010). Total internal reflection fluorescence (TIRF) is also commonly used in combination with EMCCD cameras. TIRF works by reflecting excitation light off of the interface between the cover glass and the specimen, creating an evanescent field above the interface that can usefully excite fluorescence in a plane ~150 nm thin, far thinner than optical sections created by confocal technologies. However, the volume that can be imaged is therefore confined to a single location in space: the thin plane just above the cover glass. While the depth of TIRF excitation is shallower than the typical plant cell wall, TIRF microscopes have nonetheless been used successfully to obtain high-quality images in the plant cell cortex (Konopka and Bednarek, 2008; Staiger et al., 2009). It remains unclear whether these images come from an evanescent field generated at the cell wall membrane boundary or from reducing the background fluorescence by low-angle illumination. TIRF and array scanning systems can be outfitted with devices to perform targeted photobleaching and photoactivation of probes, thus combining sensitive detection and extended fluorescence life with the ability to perform photomanipulation experiments.
While TIRF and spinning disk systems are coming into increasing use for rapid and sensitive detection of fluorescent probes with background rejection, significant improvements have also been made in point scanning microscopes. Conventional point scanning microscopes remain workhorses for quantitative 3D imaging and offer advantages when larger fields of view are required and greater out of focus light rejection is needed when working past the cover slip interface. These instruments are improved with every cycle of development, for example, by offering more sensitive detectors and modalities, such as spectral imaging, that are not commonly available on spinning disk and TIRF systems. Many imaging facilities find both array-scanning disk and point-scanning instruments to be valuable. Perhaps the most significant advance in point-scanning instruments (other advances will be discussed below) is the advent of multiphoton excitation, especially when combined with nondescanned detection. Multiphoton imaging has three potential advantages over single-photon imaging. First, the photon density required for excitation occurs only precisely at the scanned focal point; thus, no significant excitation occurs in the cell outside this very small volume. This greatly reduces photobleaching and cytotoxicity due to excitation of fluors outside the interrogated focal plane. Second, the long wavelengths used for excitation are scattered less as they pass through the specimen, permitting improved imaging in deeper tissue. Finally, since the location of emitted photons is determined by the position of the focal spot, spatial filtering with an aperture in the image path is not needed. In fact, all that matters is when photons are collected, not where they are collected. Thus, even highly scattered photons can be detected and used to create the image, significantly increasing the sensitivity of imaging. Multiphoton imaging has revolutionized in vivo imaging in brain tissue in animals and is being applied increasingly to plant tissues (Feijó and Moreno, 2004; Hamamura et al., 2011; Roppolo et al., 2011).
Optical sectioning can also be achieved computationally by systems that acquire images from multiple focal planes (deconvolution microscopes) or with multiple illumination patterns (structured illumination [SI]), coupled with processing by sophisticated algorithms. These systems are more sensitive than confocal microscopes because no light is thrown out by a physical filter, but unless coupled with arrays of processors, they do not provide resolved images in real time, limiting their use in applications that require immediate feedback to the observer. As computational power historically grows exponentially, it is anticipated that soon these systems will achieve real-time performance at reasonable cost. It is the author’s (D.W.E.) experience, however, that deconvolution of structures at or near highly refractile cell walls is particularly challenging. Furthermore, when imaging thick specimens, the captured volume can include substantial signal from outside of the volume, significantly reducing the signal to background.
Light sheet technology provides one of the newest means of obtaining optical sections using fluorescent probes. In light sheet imaging, excitation energy is provided at right angles to the imaging axis, typically by scanning a laser beam through an objective to produce a sheet of light at a defined plane through the specimen. Fluorophores above and below the plane of the light sheet are not excited, producing an optical section. Light sheet microscopy has been used successfully to create 3D images of developing mammalian, insect, and fish embryos while exposing them to levels light of two to three orders of magnitude below that of conventional and confocal imaging (Keller et al., 2008, 2010; Reynaud et al., 2008). It has been applied recently to Arabidopsis tissue to image both root development and subcellular events (Maizel et al., 2011; Sena et al., 2011) (Figure 4). While more limited in lateral resolution than confocal imaging, light sheet imaging has produced useful images of cytoskeletal organization and endosome dynamics at light levels that are near physiological (Maizel et al., 2011).
Light Sheet–Based Imaging of Arabidopsis Seedlings.
(A) View of the central components of a digital scanned laser light sheet fluorescence microscope. The illumination system excites the fluorophores in a thin planar volume by rapidly scanning a micrometer-wide Gaussian laser beam inside the specimen. Fluorescence is collected at right angles to the illuminated plane by the detection system. The planar excitation volume and the focal plane of the detection system overlap. The intensity of the laser beam can be modulated in synchrony with the scanning process (SI). (Left figure by P. Theer.)
(B) Close-up of the sample chamber (boxed region in [A]). The root of the plant is growing on the surface of a Phytagel cylinder immersed in culture medium (half-strength Murashige and Skoog medium), while its leaves are in the air. The chamber is equipped with a perfusion system exchanging the whole chamber volume every 15 min and a sun-like lighting system covering the plant leaves from above.
(C) Side view of the two types of sample holder used for imaging. (Left) The root of the plant grows into a 0.5% Phytagel cylinder. For the image acquisition process, the Phytagel cylinder is extruded from the capillary, which is rigidified by an embedded carbon rod. (Right) The root grows through a plastic cone filled with 0.5% Phytagel maintained by a ring holder. In both designs, 2-d-old seedlings were transferred to the holders and further cultured in a tilted position such that the root grew toward the glass until the onset of imaging into the chambers indicated below. (
Research in Developmental Biology and Plant Physiology - (1964-1985)
From 1964 to 1974, I worked in the Department of Biochemistry at the University of Cambridge, at first as a graduate student, then as a Research Fellow of Clare College Cambridge and as a Research Fellow of the Royal Society. In 1968, and again in 1971, I did research on tropical plants in Malaysia, based in the Botany Department of the University of Malaya, Kuala Lumpur, and also at the Rubber Research Institute of Malaya. From 1974 to 1985, I worked at the International Crops Research Institute for the Semi-Arid Tropics (ICRISAT) in Hyderabad, India, on the physiology of tropical legume crops, first as Principal Plant Physiologist, and later as Consultant Physiologist.
Hormone Production in Plants
The first problem I investigated in my research career was how the hormone auxin (indole-3-acetic acid) is produced in plants. See Hormone Production in Plants below. When I started research on this subject, nobody knew how it was made, and years of efforts had failed to clarify the situation. As I reflected on the biochemical mechanisms by which auxin can be produced I realised that it could be a non-specific breakdown product of the amino-acid tryptophan and that it was likely to be produced in dying cells, as proteins break down, releasing tryptophan. When I started this research, in the 1960's, little attention was paid to dying cells in plants or animals. Nevertheless programmed cell death - also known as apoptosis - is now a very fashionable topic of research in cell biology.
I showed dying cells could produce auxin as a by-product of their autolysis or self-digestion. See The Production of Auxin by Autolysing Tissues
There was nothing specific about the way it was produced in plants. Auxin was also produced, for example, by autolysing yeast cells, and also by autolysing rat liver. Many of the places in which auxin is known to be produced in plants are places where cells die, for example in germinating seeds, as storage tissues breakdown. Auxin is known to be produced in developing leaves and buds, and its formation is roughly proportionate to the development of veins within the leaf. Veins contain xylem or wood cells, and when wood cells develop, the walls thicken up and the cell contains break down and are dissolved away, so cell death occurs in all young growing tissues. Perhaps the differentiating xylem cells were a source of auxin, released as they died.
I studied this question in stems in which new xylem cells were being formed as a result of cambial activity, and found that these thickening stems did indeed produce auxin. See The Production of Auxin by Tobacco Internode Tissues
I also looked at auxin production in senescent leaves. As they go yellow, the cells breakdown and sure enough I found the auxin levels increased dramatically. See Production of Auxin by Detached Leaves. There was only one supposed site of auxin production in plants in which dying cells were not present, namely the tips of coleoptiles in cereal seedlings. These sheathing structures around the seedling shoot were some of the first organs in which auxin was studied and were of particular importance in the classical literature on auxin production. However, although auxin was present in coleoptile tips, I found there was no persuasive evidence that it was made there, and found that in fact it was probably accumulating there having been carried up from the seed in the sap. See Do Coleoptile Tips Produce Auxin?
The formation of auxin in developing xylem cells in the trunks of trees as they grow would mean that a gradient of auxin would be set up across the cambium, the region of dividing cells that separates the wood from the bark. I directly measured auxin levels in the xylem cambium and young phloem cells, from the inside of the bark and showed that there was indeed such a gradient. This was one of the first chemical gradients to be characterised in either animals or plants of a chemical known to have morphogenetic effects. See Auxin in the Cambium and its Differentiating Derivatives
Since dying cells produce auxin, and since dying cells occur within all higher plants as a result of xylem differentiation this raised an evolutionary question. Had the responsiveness of plants to this cell-breakdown product, acting as a chemical signal of cell death, evolved only after cell death became an integral part of plant grown with the evolution of a vascular system? Or have plants already become sensitive to auxin before the vascular system evolved? In fact it was already known that non-vascular land plants, like mosses and liverworts are sensitive to low concentrations of auxin in the environment. They react by producing root hairs, or rhizoids. If this sensitivity had developed in response to dying cells, it would enable mosses and liverworts to produce rhizoids which increase the surface area for absorption of nutrients, in places where there was decaying organic matter, in other words when nutrients were most likely to be abundant. Is auxin really present in such situations? I examined the humus on which mosses and liverworts were growing both in the tropics and in temperate countries and found that it did in fact contain auxin in quantities sufficient to produce rhizoid formation. This suggested an evolutionary origin for the auxin responses in higher plants. First, plants evolved sensitivity to auxin as a signal of organic decay in the external environment. Later, as cell death became an integral part of plant growth the evolution of the vascular system, this hormonal-response system became internalised and auxin evolved the wide range of signalling roles that it has today. See The Occurrence and Significance of Auxin in the Substrata of Bryophytes
At the end of my time at Cambridge, I published a comprehensive review of research on production of auxin and other hormones in plants, summarising the dying-cell hypothesis. See The Production of Hormones in Higher Plants
Auxin Transport in Plants
In plants auxin is transported from the shoot tips towards the root tips by the polar auxin transport system. I investigated which tissues were most involved in this transport process, and whether the polarity of stems could be reversed: I found it could not be. With my colleague Philip Rubery, I worked out the cellular basis of polar auxin transport. Our hypothesis, the so-called chemiosmotic hypothesis, was subsequently confirmed and is now generally accepted. We predicted the existence of auxin efflux carrier proteins preferentially located at the basal end of cells. These proteins were identified in the twenty-first century, and are now called PIN proteins; they are an important focus for contemporary research on plant development. [link to papers on auxin transport]
Cell Differentiation in Plants
As cells in plants turn into wood cells, called xylem cells, they thicken up their walls, and then the cell contents die and dissolve. The developing xylem cells also dissolve away their end walls, so that the dead, empty cells form tiny tubes through which the sap flow from the roots to the shoots. It seemed to me very likely that as new xylem cells formed and became part of the water-conducting system, the contents of the self-digested cells would be flushed away with the sap, and be carried upwards in it. I analysed the sap from several species of plants to see if it did contain breakdown products and enzymes of the kind likely to be involved in the autolysis, or self-digestion, of the differentiating xylem cells, and found that indeed it did. See Some Constituents of Xylem Sap and their Possible Relationship to Xylem Differentiation
Ageing, Growth And Death
In 1974, I published a paper in Nature on the ageing growth and death of cells in which I put forward a new hypothesis that accounts for many of the facts of cellular senescence and regeneration in plants and in animals. In essence, I proposed that harmful breakdown products build up in cells as they age, and that cells can be regenerated by asymmetric cell division so that one of the cells receives more of these harmful products. Thus one daughter cell will pay the price of mortality while the other is rejuvenated. This kind of asymmetric division takes place in the growing regions of plants, the meristems, and in stem cells in animals. It also occurs in the formation of egg cells in plants and animals. In both cases, the meiotic division of the egg mother cell results in one supremely regenerated cell, the egg cell, and three other cells which soon die. In animals these very mortal sisters of the egg cell are called polar bodies.
Scientific Papers on Plant and Cell Biology
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Hormone Production In Plants
Auxin Transport In Plants
The Ageing and Death of Cells
Philosophy of Biology
The Production of Hormones in Higher Plants
Do Coleoptile Tips Produce Auxin?
Auxin in the Cambium and its Differentiating Derivatives
The Occurrence and Significance of Auxin in the Substrata of Bryophytes
The Production of Auxin by Autolysing Tissues
Production of Auxin by Detached Leaves
The Production of Auxin by Tobacco Internode Tissues
Effects of Osmotic Stress on Polar Auxin Transport in Avena Mesocotyl Sections
Carrier-mediated Auxin Transport
The Polarity of Auxin Transport in Inverted Cuttings
Auxin Transport in Secondary Tissues
Effect of pH and Surface Charge on Cell Uptake of Auxin
Polar Auxin Transport in Leaves of Monocotyledons
Rupert's research reports as Rosenheim Research Fellow
Cellulase and Cell Differentiation in Acer pseudoplantanus
A Cellulase in Hevea Latex
Cellulase in Latex and its Possible Significance in Cell Differentiation
Some Constituents of Xylem Sap and their Possible : Relationship to Xylem Differentiation
The Ageing, Growth and Death of Cells
Effect of harvest methods on the second flush yield of : short-duration pigeonpea (Cajanus cajan)
Factors affecting growth and yield of short-duration pigeonpea and its potential
A perennial cropping system from pigeonpea grown in post-rainy season
Effect of seed-grading on the yields of chickpea and pigeonpea
Varietal Differences in Seed Size and Seedling Growth of Pigionpea and Chickpea
Effects of Pod Exposure on the Yield of Chickpeas
Iron Chlorosis in Chickpea Grown on High pH Calcareous Vertisol
Growth and Development of Chickpeas under Progressive Moisture Stress
Comparisons of Earlier- and Later-formed Pods of Chickpeas (Cicer arietinum)
Comparisons of Earlier- and Later-formed Pods of Pigeonpeas (Cajanus cajan)
The Effects of Flower Removal on the Seed Yield of Pigeonpeas (Cajanus cajan)
Growth, development and nutrient uptake in pigeonpeas (Cajanus cajan)
A Hydrodynamical Model of Pod-Set in Pigeonpea (Cajunus Cajan)
Pigeonpea as a Winter Crop in Peninsular India
The Expression and Influence on Yield of the 'Double-Podded' Character in Chickpeas
Some Effects of the Physiological State of Pigeonpeas : on the Incidence of the Wilt Disease
Book – The Anatomy of the Pigeonpea
Three Approaches to Biology