High-Resolution Measurements in Plant Biology


Einstein once wrote the somewhat puzzling words: ‘Technological progress is like an axe in the hands of a pathological criminal’. Perhaps he had a particular branch of science in mind when expressing this view, because certainly in our own field – the plant sciences – all the evidence suggests that, thanks to technological progress in the past few decades, we are living in one of the most exciting and progressive eras experienced to date. Technology has allowed so many long-standing barriers to be overcome, and to such an extent that virtually the only factor now needed to advance knowledge is curiosity itself.

Recent progress in multiple technologies have enabled plant biologists to query their favorite experimental systems at the highest levels of measurement resolution and reach unchartered frontiers in the knowledge of plant growth, metabolism, development, and response to environmental cues. These technological advances have led to the birth of new approaches, the adoption and adaptation of instruments from other fields, and the integration of added disciplines in plant research. They allow accurate measurements of cell content and activity at the whole plant, tissue and organ, cell layer, single cell, and even cell compartment level. In our own disciplines, such developments are very well combined with the assortment of novel profiling and detection techniques that monitor both coding and non-coding nucleic acids and their modifications, proteins, small molecules, organelles, and many more biological substances and structures. The combination of these factors is frequently leading to major scientific breakthroughs that subsequently guide and impact the quality of science we perform today and shapes the science of tomorrow. With this in mind, we have assembled a Special Issue comprising reviews of the most advanced technologies and approaches for high-resolution measurements in plants from the leaders in their respective fields.

The life of all living multicellular organisms depends on complex interactions among regulatory networks based on the interplay of genes, gene products, hormone pathways, metabolites and signaling pathways. Current technology has dramatically changed the quality and resolution of ‘omics’ analyses, which are now being adapted from the level of the whole plant and whole organ to that of single-cell profiling. Rogers et al. (2012) address how the integration of advancements in single-cell type isolation – from complex organs and technological progress – to produce cell type-specific metabolite and transcriptome profiles has allowed phenotyping at the cellular level to develop, leading to the assigning of functional annotation to genes with previously unknown function, as well as refinements to existing gene networks. Besides critically presenting the most advanced methods for isolating single populations of cells for transcriptional profiling, the authors also discuss the impact that whole organ transcriptional profiles has had on functional comparisons across species and how plants can respond to stress. However, they also highlight how transcript analysis at the cellular level has allowed important steps forward in elucidating many gene networks that were masked at the organ level due to restricted expression and how this approach is aiding not only the functional annotation of genes with unknown function, but also refining existing gene networks, especially under conditions of stress. This work expands upon the most recent approaches to obtain single cell transcriptome profiling data, which pinpoint cell type-specific metabolic profiles and avoid missing important information from intact organ metabolomics.

Schmidt et al. (2012) report on how high-resolution RNA profiling could be applied to the study of male and female germline lineages in the flower reproductive organs. This is indeed a challenge considering that target cells constituting the germline lineages, particularly the female ones, are typically enclosed by sporophytic tissues that render them inaccessible. Advancements in isolating the relevant cells – largely through laser-assisted microdissection, micromanipulation and FACS in combination with array- and RNA-seq methodologies (the latter outperforms the former) – have provided insights into the transcriptional mechanisms underlying the specification and development of the plant germline. Such information complements the well characterized cytological changes involved in these processes and raises new questions regarding the evolutionary mechanisms underlying germline fate. These concepts at the specific cell-type level are further expanded upon in terms of the single-cell and subcellular metabolite profiling approaches described by Oikawa and Saito (2012) and Kueger et al. (2012), respectively. Both sets of authors point out that measurement of metabolites must take into consideration their vast variability in the concentration and chemical properties of the metabolites, which increases the complexity of the analyses, especially for unknown compounds. In this context, the authors present a critical analysis of the state-of-the-art in mass-spectrometry and non-mass-spectrometry instrumentation, which are helping to overcome several of the hurdles in metabolomics studies at the single-cell and subcellular resolutions. One major difficulty in this regard is the isolation of single cells and specific cell compartments prior to the downstream assays that could affect the quality of data obtained in such experiments. Plant genomics has benefitted from the development of next-generation sequencing technologies, while metabolomics has reached its current high levels of resolution thanks to advanced processing software and greater degrees of sensitivity and accuracy in terms of analytical instrumentation. While we are learning much about the genomics and metabolomics of plant tissues and cells, we can now also query plants for specific classes of chemicals (e.g. lipids) at the highest levels of resolution. More in terms of single-cell type analysis, the review by Tissier (2012) describes work with glandular trichomes and explores how these structures have been used to study cell diversity, as well as define specialized metabolic pathways andplant interactions with the external environment. The author reports on the application of technologies widely used for whole tissues to study specifically the development of these structures at the genomic and metabolomic levels.

Horn and Chapman (2012) report on recent advances in lipidomics linked to related developments in mass spectrometry (MS) that are enabling the simultaneous identification and quantification of lipid species from complex structures at the tissue, cellular and organelle resolution levels, as well as an exploration of the functional and structural role of lipids in plants. Thanks to such advancements – plus the breadth of techniques increasingly available to researchers to dissect the lipid composition of extract, as well as the possibility to visualize lipids in a subcellular context – it is obvious that new and exciting screens can be designed to identify and characterize mutants of lipid functions and signaling. Of particular note in this respect are those approaches at the macro scale, such as ‘shotgun lipidomics’ (described in the review), which enable the almost simultaneous analysis, directly from extracts, of hundreds of lipids. At the nano scale, ‘direct organelle MS’ (DOMS) appears to be an emerging and powerful approach to profile lipids at the organelle level by extracting lipids from organelles in isolation, or from intact cells, within a capillary tip, followed by their identification and quantification using direct-infusion nanospray MS. Recent developments in MS-based technologies provide new spatial information regarding metabolite accumulation.

Lee et al. (2012) describe MS Imaging (MSI) experiments that include tissue preparation, matrix application [in Matrix Assisted Laser Desorption Ionization (MALDI) assays], MSI data acquisition, and finally data analysis and metabolite image assembly. MSI data is obtained by desorbing analyte molecules from the tissue (including depth profiling) and ionization to create charged ions. Following ionization performed by, for example, MALDI, Desorption Electrospray Inonization (DESI), or Secondary Ion MS (SIMS), the ions are introduced to the mass analyzer. Chemical images are subsequently formed by compiling spectra of ions of interest, at present mostly up to the single-cell resolution level, because increasing the spatial resolution reduces the sensitivity of chemical analyses; although, a resolution of a few μm is currently achievable. The authors review recent applications of MSI technologies in plants, as for example in surface lipids and secondary metabolites. Overcoming major drawbacks in MSI methods, including temporal resolution, large acquisition times, sensitivity, and chemical identification issues, will unquestionably make these technologies of great value in the near future.

MSI is largely complementary to methods of optical imaging. It is fascinating to witness how technological advances in the optical imaging of cellular structures are facilitating the dissection of the activities of subcellular compartments to levels that were not even imaginable just over a decade ago. Sparkes and Brandizzi (2012) report how plant scientists have adopted fluorescent protein technology to image subcellular dynamics of plant cell organelles at a spatial and temporal resolution, and to manipulate the distribution of fluorescent markers to identify the genes responsible for the inner activities of plant cells by coupling light microscopy with genetics and genomics. The authors also report on innovative approaches based on laser trap microscopy to manipulate the position of organelles and probe fundamental and exciting questions about inter-organelle relationships in live cells. In addition, the reviews by Choi et al. (2012) and Okumoto (2012) report on clever adaptations of fluorochrome properties to query the composition and the activity of subcellular compartments, so that it is now possible to monitor cell-signaling molecules such as Ca2+ and H2O2 with specific probes in live cells in real time using standard microscopy instrumentation. The authors of these reviews present an extensive coverage of the sensors available to plant researchers and critically analyze their suitability for addressing the challenges that plant cell imaging offers. At the same time, they also provide a critical overview of the controls required in these kinds of analyses. In the last decade or so, a number of new genetically encoded fluorochromes for the measurement of signaling molecules have been improved from earlier versions. For example, in plants, a newer Ca2+ indicator, YC3.6, in which the YFP of YC2.1 was replaced with a circularly permutated version of this fluorescent protein, can now be used to reduce the FRET range limits of YC2.1. Furthermore, the authors bring to our attention not only the applications of sensors derived from the green fluorescent protein for measurements of redox levels, but also the implementation of genetically encoded sensors derived from unusual sources. This is the example of ‘Hyper’, an H2O2 sensor, which is based on the regulatory domain of an E. coli transcription factor OxyR (OxyR-RD), which is used naturally by the bacterium to monitor H2O2 levels. Progress in using genetically encoded probes in plant biology was initially slow compared to other fields; however, this has changed in recent years, and more than 70 different ligands are now available (as of 2011).

Borisjuk et al. (2012) specifically illustrate the applicability of magnetic resonance imaging (MRI), a technique often used in animal diagnostics, for the non-invasive analysis of several aspects of plant physiology and development, plant/environment interactions, biodiversity, gene function, and metabolism. This interesting report describes how MRI did not find immediate success in plant analyses due to tissue-related technical difficulties. This, however, now seems to have been largely overcome thanks to recent developments in hardware, the realization of ultra-high magnetic fields, and the development of new imaging techniques, therefore suggesting that technological advances may continue to bring fields together and increase imaging resolution at multiple levels. MRI technology has also been applied in plants to measure – in a non-invasive manner – the flow of solutes in the vascular tissues. Knoblauch and Oparka (2012) describe how an array of bio-imaging tools have advanced our capabilities to investigate long-distance assimilates in the phloem sieve-tube system. High resolution and precise structural data are crucial in understanding the driving forces behind translocation in the phloem, a major issue in the vascular biology field that has remained not fully understood for many years. It seems likely that while visualization technologies are currently limited to immobilized samples, they will soon permit dynamic live-sieve element imaging at resolutions lower than 20 nm.

The integration of several disciplines, including high-resolution microscopy, genetics, genomics and physics, is leading to a better understanding of how plant-specific components and structures develop and function. For example, Nevo et al. (2012) demonstrate how such an approach has led to the understanding of dynamic macroscopic organization of the photosynthetic apparatus of plants in response to the light environment and chromatic adaptation. Among other important approaches, the review summarizes exciting findings on the study of the organization of the thylakoid membranes from electron tomography, which allows – to a resolution of several nanometers – the in silico 3D reconstruction of subcellular structuresfrom a series of projection images obtained through an axis perpendicular to the electron beam. The work critically analyzes what have at times been controversial results in the history of the study of the thylakoid membranes, and does so not only in light of the technical challenges offered by specific plant tissues, but also the implementation of photosynthetic mutants and alternative yet powerful techniques (e.g. atomic force microscopy, used to study the organization, composition and architecture of thylakoid membranes).

Finally, all the great advancements in high-resolution measurements benefit greatly from today’s very high standards and degree of competence in genome sequencing. Hamilton and Buell (2012) summarize this by describing the latest available technologies, which have now progressed to such a level that genome sequencing is possible in the majority of institutes and universities for whose research programs these facilities are needed, and will also most likely extend to individual labs in the near future. This widespread ability to access the genomes and genotypes of multiple plant species no doubt expedites the pace of plant biology research in its different disciplines. Nevertheless, the authors also describe the major challenges that we face in the years to come in terms of genomic data handling and mining owing to the ever-increasing output of genome sequencing.

The array of approaches to high-resolution measurements in plants described in this Special Issue of The Plant Journal was made possible because of major technological advancements. The utilization of such tools entails a combination of methodologies originating from multiple disciplines, such as chemistry and physics, and their sub-disciplines (e.g. mass spectrometry, optical engineering, electronics and nanotechnology). As a large number of these new approaches require varied levels of different types of expertise in a single lab or institute, it is expected that they will not be available to the entire research community. Nevertheless, we hope that this Special Issue will help to raise awareness among our readers of such state-of-the-art technologies, and greatly promote their use in studying plant biology.