The RootScope: a simple high-throughput screening system for quantitating gene expression dynamics in plant roots
- Erin J Kast†1,
- Minh-Duyen T Nguyen†1,
- Rosalie E Lawrence†1,
- Christina Rabeler1 and
- Nicholas J Kaplinsky1Email author
© Kast et al.; licensee BioMed Central Ltd. 2013
Received: 22 May 2013
Accepted: 9 October 2013
Published: 12 October 2013
High temperature stress responses are vital for plant survival. The mechanisms that plants use to sense high temperatures are only partially understood and involve multiple sensing and signaling pathways. Here we describe the development of the RootScope, an automated microscopy system for quantitating heat shock responses in plant roots.
The promoter of Hsp17.6 was used to build a Hsp17.6p:GFP transcriptional reporter that is induced by heat shock in Arabidopsis. An automated fluorescence microscopy system which enables multiple roots to be imaged in rapid succession was used to quantitate Hsp17.6p:GFP response dynamics. Hsp17.6p:GFP signal increased with temperature increases from 28°C to 37°C. At 40°C the kinetics and localization of the response are markedly different from those at 37°C. This suggests that different mechanisms mediate heat shock responses above and below 37°C. Finally, we demonstrate that Hsp17.6p:GFP expression exhibits wave like dynamics in growing roots.
The RootScope system is a simple and powerful platform for investigating the heat shock response in plants.
KeywordsHeat shock Thermometer Thermostat Automated imaging Quantitative imaging Temperature Arabidopsis
A systems level understanding of plant responses to the environment requires quantitative gene expression data with high spatial and temporal resolution from multiple individuals [1, 2]. This type of data cannot be readily obtained using conventional gene expression assays. RT-PCR, microarray, and RNAseq approaches are limited by low spatial resolution unless tissues are either dissected manually or sorted using FACS. While in situ and GUS reporter based approaches can provide high spatial resolution, they require sacrificing the sampled tissue and thus cannot be used to easily monitor gene expression dynamics in individual plants over time. Both Luciferase and fluorescent protein (FP) based reporters expressed in living tissues can provide dynamic expression information, but only FP based reporters allow for both high spatial and temporal resolution.
Several systems for assaying gene expression dynamics in plant roots using FP reporters have previously been developed. All imaging approaches involve tradeoffs between spatial resolution, temporal resolution, and the number of plants that can be imaged at a time. Cost and ease of use are also important considerations. Light sheet microscopy has been used to generate high resolution 3D expression data in an Arabidopsis root at 10 minute intervals, but only one root can be imaged at a time . Two systems have been developed for imaging FPs in multiple roots in rapid succession. The RootChip is a microfluidics based system which allows extended imaging of eight (and potentially more) roots and allows rapid exchanges of the growth media [4, 5]. A second microfluidics device, the RootArray, uses confocal microscopy to image up to 64 roots in three dimensions and at high resolution. However, long acquisition times in this system limit the temporal resolution; imaging even a subset of the roots at high resolution takes several hours . While extremely powerful, both of these systems require plants to be grown in the microfluidic device so that the roots grow close to a cover slip which enables high resolution imaging. This is a labor-intensive procedure and requires specialized and relatively expensive microfluidics devices. We are interested in using gene expression dynamics as a phenotype in a genetic screen for high temperature stress 'thermostat’ genes. Because the screen involves characterizing large numbers of individuals we wanted to develop a system which maximizes the number of plants imaged simultaneously while retaining high spatial and temporal resolution and providing accurate control of plant temperature.
The motivation for this screen is to understand the genetic network(s) involved in heat shock (HS) sensing and signal transduction. Plants are sessile organisms and must endure widely varying environmental conditions, many of which result in stress and reductions in yield in agricultural systems. Heat is an abiotic stress that disrupts protein and membrane homeostasis at a cellular level. Heat stress is often accompanied by drought stress which exacerbates its deleterious effects. Global climate change is predicted to result in more frequent and severe episodes of both of these stresses [7, 8]. Plants have evolved a complex set of mechanisms for dealing with both stressful as well as non-stressful high temperatures [9–12]. At stressful temperatures the heat shock response (HSR) results in global changes in gene regulation. An important component of the HSR is the synthesis of Heat Shock Proteins (HSPs) which protect normal protein and membrane function. HSP function has been extensively characterized biochemically and genetically and is reasonably well understood [11, 13]. Much less is known about the high temperature sensing and signal transduction mechanisms, the 'thermostat’, which regulates the HSR .
In Arabidopsis (as in all organisms) HSP expression is strongly induced in response to heat stress. HSP regulation is mediated by the activity of a family of temperature and cAMP regulated Ca2+ channels (CNGC2,4, and 6), a calmodulin (CAM3), a kinase (CBK3) and a phosphatase (PP7) [14–19]. These genes are predicted to function upstream of a family of highly redundant Heat Shock transcription Factors (HSFs) which directly regulate much, but not all, HSP expression [20–23]. HSF activity is also thought to be regulated through direct physical interactions with the HSPs HSP70 and HSP90 and a HS induced HSF binding protein (HSBP), creating feedback loops which finely tune the HSR [24–27]. Notably, none of these thermostat genes were identified based on their mutant phenotypes, perhaps due to redundancy in the HSR network. Instead, these genes were identified based on homology with other systems, expression profiling, or biochemical interactions and they have all been characterized using reverse genetics approaches.
Many details of the HS thermostat’s function are complex and not fully understood. The CNGC and HSF gene families exemplify this complexity. Based on studies in mammalian systems, CNGCs are thought to function as hetero-tetramers and the HSFs as homo- and hetero-trimers [14, 27, 28]. The in vivo subunit composition of these complexes has not been described in plants. Since the function of these genes is likely influenced by their co-complex interaction partners the analysis of their mutant phenotypes is complicated. Both the CNGC and HSF families contain members which, when mutated, exhibit opposite phenotypes. cngc2 and 4 mutants increase Ca2+ influx into cells and result in elevated thermotolerance while cngc6 mutants decrease Ca2+ influx and exhibit decreased thermotolerance [14, 17]. Similarly, HSFA1a,b,d, and e are positive regulators of the HSR and are required for thermotolerance while HSFB1 and HSFB2b attenuate the HSR [20, 21, 23]. Not only are the physical interactions among Arabidopsis family members not well understood, but their interactions with and regulation by other molecules are also poorly described. For instance, the CNGCs are activated by the second messenger cAMP yet only a few genes involved in cAMP production have been identified in Arabidopsis. None have demonstrated biological functions in plants [14, 29, 30]. Given these ambiguities it is likely that multiple components of the HSR network remain unidentified.
Unbiased forward genetics approaches based on thermotolerance defects have identified only a few HSR genes including HSP101 (HOT1), a chitinase (HOT2), and an exportin (HIT2) [31–33]. None of these genes have obvious sensing or signaling functions and, to our knowledge, no thermostat genes have been identified by a high temperature thermotolerance screen. This suggests that screening for thermotolerance defects using conventional assays may be of limited value in identifying HS thermostat genes.
To overcome these limitations we have developed the RootScope which generates the quantitative data required to identify new and characterize existing components of the HSR thermostat. The RootScope consists of a robust HSR reporter and an automated imaging system for monitoring the reporter in multiple plants with high temporal and spatial resolution.
Construction of the HSP17.6p:GFP reporter line
The Hsp17.6 C-CI (AT1G53540) promoter was amplified from genomic DNA using primers AT1G53540-FWD (5′-CACCATTCAGGTAATCAGGTTGTCTGC-3′) and AT1G53540-REV (5′-CGTTTCACTTCCTCTTGTGATTGC-3′). These primers amplify 713 bp starting in the 3′ UTR of the upstream gene AT1G53530 and ending at the ATG start codon of AT1G53540. CACC was added to the 5′ end of AT1G53540-FWD to provide directionality for TOPO cloning into pENTR/D-TOPO (Invitrogen, Grand Island, NY). TOPO cloning of the PCR product into pENTR/D-TOPO was followed by Gateway mediated LR recombination (Invitrogen) into destination vector pFAST-R07 (Shimada et al., 2010) to create a C-terminal promoter:GFP fusion (HSP17.6p:GFP). This construct was transformed into Col-O plants using Agrobacterium tumefaciens strain GV3101 (Clough and Bent, 1998). Primary transformants were identified using the RFP seed marker contained in the pFAST-R07 construct and were allowed to self-fertilize to generate homozygous lines. Homozygous lines were screened for GFP expression at basal temperatures (22°C) and after a 1 hour 37°C heat shock as 3 day old seedlings. Fluorescence imaging was performed using a Leica MZ16F microscope equipped with a 2x Planapo lens, a GFP3 filter set, and an EL6000 light source (Leica Microsystems).
Construction of the automated imaging system
The heated stage consists of an Inheco CPAC Ultraflat HT microplate heater with a temperature range of 4°C to 110°C (Inheco). Microplates were placed on the CPAC and held in place by a custom 3D-printed chamber which incorporates the heated glass plate from a Tokai Hit INU-GSI stage top incubator. For horizontal root growth the heated stage was mounted on an ASI LS-50 linear stage which provided Z (focus) movement. The LS-50 linear stage was then mounted on an ASI MS-2000 XY stage to create a heated XYZ stage. For vertical root growth a heated XY stage was built by directly mounting the heated stage on the MS-2000 XY stage. All ASI stages were controlled by a LX-4000 control unit (ASI).
Images were acquired using a Hamamatsu ORCA II BT-512 camera cooled to -55°C in both horizontal and vertical arrangements. Exposure times ranged from 75–200 msec depending on the plant line being imaged. For horizontal root growth the heated XYZ stage and camera were mounted under a Leica MZ16F microscope equipped with a 2x Planapo lens, a GFP3 filter set, and an EL6000 light source (Leica Microsystems). For vertical root growth the heated XY stage was mounted vertically on a 1.5″ mounting post (Thorlabs). A Leica Z16 APO macroscope with a 2x Planapo lens and a 1x tube lens (Leica) was coupled to a DFM dichroic filter cube with a MDF-GFP filter set (Thorlabs) and the ORCA II camera using custom adapters. Illumination was provided by a M470-L2 high power LED collimated using an ACL2520-A lens (Thorlabs). These components were mounted on a 8″ x 24″ x 1/2″ aluminum breadboard which in turn was mounted on a Parker Daedal CR4900-8 crossed roller bearing stage (Parker Daedal). The LS-50 linear stage was connected to the breadboard to provide focus control by moving the entire microscope relative to the XY stage. All of these components were built up on a 12″ x 36″ optical breadboard (Vere).
Both the LED and EL6000 light sources were shuttered using a USB connected Arduino UNO driving a SainSmart 2-Channel relay (SainSmart). For the EL6000 light source the relay was directly connected to the light source shutter inputs. For the high power LED light source the relay was connected to the DIM input of a Luxdrive 1400 mA Buckblock (LUXdrive) which was used to drive the LED.
μManager microscopy automation software  running on a 64 bit Windows 7 (Microsoft) system was used to control the Arduino based shutter, the LX-4000 stage control unit, and the ORCA II camera. Root images were quantitated and, in some cases straightened, using custom ImageJ  and Perl scripts. Alignment of straightened roots was performed using the Template Matching and Slice Alignment ImageJ plugin .
Arabidopsis seeds were surface sterilized with 30% bleach and 0.1% SDS for 15 minutes, rinsed three times in sterile water, and plated on OmniTray single well microplates (Thermo Scientific product #242811) containing 60 ml of 0.5x MS media. The plated seeds were stratified for three days at 4°C and then transferred to Percival E-30B growth chambers (Percival Scientific) set at 22°C with 24 hours of light for three days.
Generation of a Hsp17.6p:GFP reporter for the heat shock response
Assaying the HSR in multiple plants with high spatial and temporal resolution requires a live cell reporter with a high dynamic range and a strong signal when induced. We chose Hsp17.6 (AT1G53540) because this small HSP is consistently among the most highly induced HSPs in both our own and published HS microarray experiments [21, 37, 38]. We cloned the AT1G53540 promoter upstream of GFP in the pFAST-R07 vector  and transformed the resulting Hsp17.6p:GFP construct into Arabidopsis. We used this vector because it contains a RFP seed marker which allows for easy identification of plants which contain the reporter. This feature will be useful for identifying plants homozygous for the reporter in the future. We selected 46 independent transformants and allowed each to self-fertilize to generate plants homozygous for the reporter. Homozygous lines were screened for GFP expression in 3 day old roots at basal temperatures and an hour after a one hour 37°C heat shock. None of the lines exhibited GFP expression at basal temperatures, consistent with a lack of Hsp17.6 expression in the absence of cellular stress. The intensity of the GFP signal after the heat shock varied among lines and we chose three of the brightest lines (8, 11, and 26) for further characterization (data not shown).
A simple high-throughput screening system for quantitating gene expression dynamics in plant roots
The advantage of this setup compared to a microfluidic device is that we can easily and quickly plate 100 (or more) seeds on MS media on a single, cheap, and commercially available microplate. Seed placement is not critical as the roots do not have to grow in defined micro-channels. Root positions are identified manually using the μManager slide explorer plugin once the plate has been transferred to the microscope for imaging. The seeds are germinated in an incubator, grown for 3 or 4 days, and then transferred to the microscope where they can be simultaneously heat shocked and imaged. The system allows us to quantitate the HSR at high temporal (every 4 minutes with 100 plants) and spatial (~2.4μm with our current camera and optics) resolution with a 12 bit dynamic range.
Characterization of the induction and attenuation kinetics of the Hsp17.6p:GFP reporter
Hsp17.6p:GFP expression correlates with the heat shock temperature
Hsp17.6p:GFP expression exhibits wave like dynamics
The RootScope is a simple and flexible system that makes it possible to determine the dynamics of gene expression in large numbers of plant roots with high temporal and spatial resolution. The time and cost required to set up plants for screening are minimized since seeds are plated on standard media in microplates and plating seeds for an experiment takes only minutes. The most time consuming step in a RootScope experiment is the manual identification of root positions once the plate is transferred to the microscope. This step takes less than 20 minutes per plate for an experienced undergraduate student. It should be possible to automate this task using image recognition algorithms; however, since automated localization may need to be confirmed and fine-tuned manually, the advantages of automating this step may not be significant in practice. In this paper we report using the RootScope as a fluorescence microscope for quantifying a GFP based transcriptional reporter. Similar to existing non-fluorescent phenotyping systems such as Phytomorph, the microscope is flexible and could also be used to generate high resolution incident light images to characterize growth dynamics in multiple plants simultaneously [42, 43]. Because the optics we have used are based on a zoom macroscope both larger and smaller fields of view can be selected (with concomitant changes in resolution). Finally, although we have chosen to image roots, there is no technical reason why the aerial portions of plants could not be imaged using this system.
The induction kinetics of Hsp17.6 in this system is slower than previous reports of HSP protein and transcript accumulation in which HSPs accumulate within half an hour of a heat shock [44, 45]. The sensitivity of our system and the maturation kinetics of GFP are both likely to contribute to this difference. Both RT-PCR and enzymatic Western blot detection techniques amplify small signals significantly making it possible to detect small numbers of molecules while our imaging approach does not. This limits the sensitivity of our system at very low Hsp17.6 expression levels. The folding, chromophore maturation, and degradation dynamics of GFP are also likely to influence the kinetics reported here, most likely by introducing delays in both the appearance and attenuation of the GFP signal. Hsp17.6p:GFP was built using pFAST-R07 which contains EGFP [39, 46]. EGFP is a GFP variant which exhibits approximately a four fold increase in the rate of chromophore maturation compared to wild type GFP. GFP variants with even faster maturation rates such as GFPmut2 or GFPm have been demonstrated to mature over twice as fast as EGFP in vitro although no in planta maturation rate data exist in the literature. While the use of a rapidly maturing GFP variant has the potential to decrease the delay between transcription and detection of the GFP signal using the RootScope, there is no reporter that we are aware of which can completely eliminate this delay. Since we are not concerned with the absolute timing of these events and given the relatively rapid expression dynamics we have observed, we do not anticipate that these artifacts will prohibit the use of this system for identifying HSR thermostat mutants. In addition to using a rapidly maturing GFP to decrease delays in the onset of the signal, the use of a translational as opposed to a transcriptional reporter might result in more accurate degradation kinetics.
The ability to monitor the kinetics of the HSR using Hsp17.6p:GFP should allow us to take a forward genetics approach to identifying HSR genes based on changes in Hsp17.6p:GFP induction and decay kinetics. We have EMS mutagenized 3,000 Hsp17.6p:GFP seeds and harvested pools of 4–5 self-pollinated plants. 80 seeds from each pool will be screened on plates with 20 non-mutagenized seeds as controls. This pooled approach should allow us to identify most of the mutants in each family and strikes a balance between throughput (each run takes 12 hours) and coverage. Assuming 200 days of screening in the first year we could screen 800–1000 families which, while it would not reach saturation, should uncover several HSR thermostat mutants . We will inspect the data both manually and computationally (based on extracting the times at which each plant’s HSR signal rises above a baseline and reaches a maximum and on the slope of the induction and attenuation phases) to identify mutants that have altered HSR kinetics, localization, or respond as if they are hotter or colder than they actually are. In addition to identifying HSR thermostat mutants we also anticipate discovering mutations which disrupt the wave like expression dynamics of Hsp17.6p:GFP. These mutants may provide insights into the co-ordination of developmental events and stress responses. This system will also be useful for uncovering the effects of previously identified HSR genes (many of which do not display single mutant phenotypes) on HSR dynamics.
We have demonstrated that the RootScope can be used to generate gene expression data with high temporal and spatial resolution in multiple plants simultaneously. We have used the system to uncover evidence that supports the hypothesis that multiple distinct high temperature responses exist in plants. Additionally, we have documented previously unknown waves of HS induced gene expression. Apart from characterizing the HSR, the RootScope should be of general use for quantitating other types of gene expression dynamics. Using other types of reporters [2, 49], it should also be useful for quantitating metabolite and small molecule dynamics in plant roots.
We would like to thank John Kelly and Steve Palmer for their support in building the RootScope and Bill Gresh for providing plant care. Catherine Crouch and Carl Grossman provided valuable advice about microscope optics. We would like to thank two anonymous reviewers for their comments and suggestions which helped us to improve this paper. The research reported in this publication was supported by the Friedman Fund for Physiology at Swarthmore College and by the National Institute of General Medical Sciences of the National Institutes of Health under award number 1R15GM093933. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health or of Swarthmore College.
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