High-resolution analysis of cell type-specific gene expression can provide valuable information about the complexity of transcriptional programs (). Comparison of the transcriptional profile of an organ to the cellular expression profile of that same organ has highlighted the limitations of performing the analysis at the organ level (Brady et al., 2007
). High-resolution profiles of specific cell types using both FACS and LCM have revealed additional information about organs that was masked when examining the entire organ. For example, cell type-specific expression analyses using FACS compared to expression in the whole root of Arabidopsis identified several hundred additional genes that were absent from whole root profiles and demonstrated the importance of considering gene expression at the cellular level (Schmid et al., 2005
; Iyer-Pascuzzi et al., 2011
). This discrepancy between whole organ and cell type expression is not limited to the root. In the leaf, separation of guard cells from mesophyll cells revealed the expression of guard cell specific ABA responsive genes (Leonhardt et al., 2004
). In combination with reverse genetics, this cell type-specific expression data allowed researchers to identify the function of a previously unknown guard cell-specific ABA responsive gene. In a third example, cells that were obtained by LCM from the epidermis and vascular tissues of coleoptiles from maize seedlings identified genes differentially expressed in epidermis and vascular tissues. Genes involved in secondary metabolism were epidermis-specific, whereas, genes encoding transporters and metal binding proteins were vascular-specific (Nakazono et al., 2003
). These cell type-specific profiles and the examples that follow have identified physiological mechanisms that are cell type-specific within an organ, which were not observed by whole organ transcriptomics confirming the need for high resolution cellular profiles.
Root stress response is cell type-specific
Plant growth and development do not strictly adhere to a predefined program and are directly influenced by the environment. As a result, plants have developed multiple mechanisms to deal with external stimuli. Early studies of this phenomenon were focused on the macro level; however, the sequencing of the Arabidopsis genome made it possible to analyze changes at the molecular level. The initial experiments dealt with the phenotypic characterization of mutants having an abnormal response to a specific stress, followed by a laborious mapping process to identify the gene or genes responsible for the phenotype. This approach successfully identified multiple environmental response genes; however, it was limited to analyzing single genes and made it difficult to define gene networks involved in the stress response. Advances in transcriptomics and metabolomics have made possible the generation of genome-wide transcript and metabolite profiles in response to many abiotic stresses and have uncovered multiple stress-specific regulatory networks (for reviews of abiotic stress response networks see (Hirayama and Shinozaki, 2010
; Urano et al., 2010
The Arabidopsis root provides an informative model system for the discovery of stress response networks because of its simple architecture and cellular organization. Hence, many efforts have focused on understanding how the Arabidopsis root perceives and responds to the soil environment using whole roots (Misson et al., 2005
; Kilian et al., 2007
; Van Hoewyk et al., 2008
; Zeller et al., 2009
). These observations have demonstrated that nutrient responsive genes impact gene expression and metabolite production in all parts of the plant as a mechanism to deal with oxidative damage and nutrient deprivation. By examining whole root responses, many important discoveries were made regarding metabolite biosynthesis, stress specific transcriptional responses and demonstrated many general stress response pathways; however, more recent experiments have shown the value of cell type-specific profiling.
Transcriptional profiling of individual cell types within the root demonstrated that many of the stress responses occur at the cellular level, which was obscured when examining the whole root (Dinneny et al., 2008
; Gifford et al., 2008
). To gain a comprehensive view as to how individual tissues respond to environmental stimuli, Dinneny and colleagues (2008)
profiled 6 different cell types across 4 longitudinal zones for changes in gene expression in response to salt exposure and iron deficiency. Of the nearly 4000 cell type-specific genes that were differentially expressed after salt exposure and 1300 genes after iron deficiency, the majority of these were changed in only one cell type, suggesting that a large part of stress regulation occurs at the cell or tissue level. Analysis of developmental zone-specific gene sets for each stress indicated that many genes were enriched in only one zone. Comparison of the cell type-specific and longitudinal data demonstrated that most of the enriched genes are longitudinal zone-specific, suggesting that salt and iron stress regulate processes on the basis of developmental context, in addition to cell type. A comparison of the cell type-specific gene sets showed only 20% overlap between salt and iron responsive genes, indicating that the gene response is stress specific but also likely contains a common stress response. Additional studies have shown similar cell type-specific responses to N, phosphorous, pH and sulfur (S) stress (Gifford et al., 2008
; Iyer-Pascuzzi et al., 2011
). A more recent comparison between the transcriptional profiles generated from whole roots and individual cell types during stress identified a common stress response in the whole root; however, evidence was lacking for a universal stress response (Iyer-Pascuzzi et al., 2011
). Although individual stresses uniquely impact the transcriptional profile, a cell type-specific stress response exists independent from the type of stress. This analysis also revealed that ABA signaling, which Dinneny and colleagues (2008)
previously regarded as a general stress response, regulates a different set of genes depending on the stress and cell type. Collectively, these results suggest that the gene response observed in whole roots is comprised largely of multiple individual cellular responses. The exact function of the stress response genes remains largely unknown, but mutant analyses suggest that they are directly linked to gene regulatory networks controlling growth and development.
The exact mechanism of gene induction during stress is also unclear, but likely involves a signal transduction pathway. Calcium is an important second messenger that has been associated with multiple stress responses. For example, salt stress elicits an increase in calcium that activates an anti-porter to promote export of sodium. When the calcium response was examined in different cells, researchers were unable to determine which cells contribute to this response (Knight et al., 1997
). Further work that examined calcium dynamics at the cellular level using a combination of YFP-aequorin (calcium reporter protein) gene fusion under control of UAS and cell type-specific GAL4 enhancer trap lines to direct cell type-specific expression, demonstrated a peak of calcium response in the epidermis and endodermis of the root in response to salt stress (Kiegle et al., 2000
). The authors also noted that the response varied depending upon the stress; drought stress was epidermis specific while cold stress was uniform in all cell-types. Another common signaling mechanism is redox regulation involving reactive oxygen species (ROS), which are key factors in many cellular activities and signal transduction pathways, making them good candidates as modifiers of the stress response. In agreement with this, ROS are associated with the activation of several genes and proteins including defense related genes, mitogen activated protein kinases (MAPKs) that transmit cellular responses to external stimuli, multiple stress-related genes and genes that control plant development (Desikan et al., 2001
; Shin and Schachtman, 2004
; Tsukagoshi et al., 2010
). Under conditions of potassium, N and phosphorous stress, ROS levels are increased in a cell type-specific manner, that is stress dependent (Shin et al., 2005
). Under potassium and N starvation, ROS accumulates primarily in the epidermis; under phosphorous stress, ROS accumulates in the cortex. In most studies, however, it is currently unclear whether ROS is driving cell type-specific gene expression or cell type-specific expression results in ROS accumulation.
The epidermis layer: trichomes and metabolite production
The leaf epidermis is composed of pavement cells, border cells, guard cells, trichomes (leaf hairs) and trichome socket cells, each with its own specialized function. Because the epidermis is exposed to the surrounding environment, it is the first line of defense against environmental stress, including herbivores and pathogens. The epidermis layer synthesizes the building blocks for the construction of the cuticle, an extracellular layer largely composed of the polymer cutin and waxes. To identify proteins involved in the biosynthesis of waxes and cutin, Suh et al. (2005)
manually dissected epidermal peels from Arabidopsis and determined transcript profiles in both rapidly expanding and nonexpanding cells at different positions along the inflorescence stem (Suh et al., 2005
). Known epidermis-specific genes were correctly classified and 15% of the transcripts preferentially expressed in the epidermis were enriched in genes encoding proteins predicted to be membrane associated and involved in lipid metabolism. Several recent studies followed the transcriptome, proteome and metabolome of the epidermis layer in fleshy fruit, particularly in tomato. For example, manually dissected exocarp (i.e. the outer layer including the epidermis) and mesocarp (i.e. flesh, the inner layer) tissues were used for both transcriptome and metabolite profiling and hundreds of genes and metabolites were identified that were enriched in the outer layer (Lemaire-Chamley et al., 2005
; Mintz-Oron et al., 2008
). In melon, an extraordinary range of complementary analytical technologies were used to profile the metabolome, volatiles and mineral elements in fruit at a number of time points during the final ripening process and tissues collected across the fruit flesh from rind to the seed cavity (Moing et al., 2011
). In this extensive study, approximately 2000 metabolite signatures and 15 mineral elements were determined in an assessment of temporal and spatial melon fruit development. Very recently, LCM was combined with NGS to characterize the transcriptomes of the five principal tissues of tomato fruit including the outer and inner epidermal layers, collenchyma, parenchyma, and vascular tissues. More than 20,000 expressed unigenes were identified, including a large number that displayed cell type specific or distinct expression patterns in specific tissues (Matas et al., 2011
The trichomes are large single cells extending from the epidermis which are capable of synthesis, storage and secretion of multiple secondary metabolites that aid in defense, including, terpenes, acyl sugars, phenylpropanoids and flavonoids. These metabolites are of interest to humans because they are often used as food flavors, perfumes and as pharmaceuticals. There are two major types of trichomes, nonglandular and glandular. Arabidopsis contains only nonglandular trichomes where as other species such as Solanum (tomato) possess both glandular and nonglandular trichomes. Among the glandular trichomes, there are several predominate types dependent upon the plant species. Trichomes are extensively used as a model system to address questions regarding developmental patterning and to study metabolite biosynthesis because of their location on the external surface of the plant. Because they represent single cells, they have been used by many researchers to generate cell type-specific transcriptional and metabolite profiles. The major conclusions from some of these studies are discussed below.
Several studies have used transcriptional profiles to try and elucidate the biochemical pathways involved in secondary metabolite production. Although enzyme transcript levels do not always correlate with metabolite levels, they are suggestive of metabolite production. Using Arabidopsis, the comparison of transcriptional profiles generated from trichomes and whole leaves from two trichomeless mutants yielded 3231 genes that appeared to be specific to trichomes (Jakoby et al., 2008
). Among the up-regulated genes were multiple genes involved in biosynthesis pathways that generate secondary metabolites. Trichomes and root hairs were previously known to share a transcriptional network; however the similarity of downstream targets was largely unknown. Comparison of the trichome and root hair expression profiles showed largely overlapping genes, strongly suggesting they share downstream targets. Interestingly, the pathways involved in secondary metabolite synthesis were specific to each cell type. Metabolite profiles in glandular trichomes isolated from four distinct sweet basil lines revealed an array of phenylpropanoids and terpenoid derived compounds that were line-dependent (Xie et al., 2008
). Comparison of the enzyme expression in the phenylpropanoid and terpenoid biosynthesis pathways also demonstrated line-dependent expression profiles. However, the enzyme profile was not always indicative of the metabolite profile. A transcriptional profile in Catharanthus roseus
was generated from the cells of the epidermis using carborundum abrasion in an attempt to identify components of the monoterpenoid indole alkaloid (MIA) biosynthesis pathway, which are key components of several anticancer drugs (Murata et al., 2008
). This profile, termed the epidermome, identified several novel MIA pathway genes in the epidermis that were not present in the public Catharanthus
EST database, in addition to genes involved in triterpene and flavonoid biosynthesis. The leaf epidermome demonstrates that a single layer of epidermal tissue produces multiple types of metabolites. However, it does not distinguish which cell type is responsible for production of these metabolites.
Based upon function, Solanum
glandular trichomes are classified as either secreting (type I and IV) or storage (type VI). Recent experiments profiling glandular trichomes isolated from Solanum habrochaites
leaves demonstrated that all three trichome types contain multiple methylated forms of myricetin, with the tetramethylated form predominating (Schmidt et al., 2011
). Myricetin flavonoid is highly methylated and has been reported in a variety of plants but the O
-methyltransferases (OMTs) responsible for their biosynthesis were unknown until recently. Using transcriptional profiling, Schmidt and colleagues (2011)
identified two genes enriched in the trichomes that encode enzymes capable of methylating myricetin, with the highest level being found in the type I and IV trichomes. Experiments are underway to confirm that these genes function as OMTs. Comparison of transcriptional and metabolite profiles generated from glandular secretory trichomes (type I, IV, VI and VII) in the leaves of five species of Solanum
, found that type I and type IV glandular trichomes are very similar at the transcript level (McDowell et al., 2011
). The authors also noted that the metabolite levels, but not the metabolite profiles, were different between type I and IV and type VI, and that type VII has limited metabolite synthesis and storage compared to other types. They also noted groups of compounds that were specific to certain trichome types and that were not evenly distributed across the various species. In agreement with previous observations (Harada et al., 2010
), several trichome types expressed carbon fixation genes, suggesting trichomes may serve as a site of carbon fixation to be used in secondary metabolism.
The sesquiterpene class of terpenoids are synthesized via modification of a prenyl diphosphate intermediate by terpene synthases (TPSs). A combination of transcriptome and metabolome data has been successful in identifying a sesquiterpene synthase that results in production of β
-caryophyllene and α-humulene specifically in the type VI glands of the leaf but not in the stem of tomato (Schilmiller et al., 2010
). In a separate study, a comparison of transcriptional profiles generated from Solanum lycopersicum
and Solanum habrochaites
stem trichomes was performed to identify TPSs, specifically those TPSs involved in production of sesquiterpenes (Bleeker et al., 2011
). The authors found 7 synthases expressed in Solanum lycopersicum
and 6 in Solanum habrochaites
, one of which was induced by jasmonic acid, suggesting its involvement in herbivore-stress response. The majority of the TPSs that were enriched were sesquiterpene synthases and many of these had been demonstrated to produce multiple sesquiterpenes. TPSs for many of the predominant sesquiterpenes were not identified from these data, further supporting the idea that a single TPS is likely responsible for producing more than one sesquiterpene.