pH-sensitivity of YFP provides an intracellular indicator of programmed cell death
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Programmed cell death (PCD) is an essential process for the life cycle of all multicellular organisms. In higher plants however, relatively little is known about the cascade of genes and signalling molecules responsible for the initiation and execution of PCD. To aid with the discovery and analysis of plant PCD regulators, we have designed a novel cell death assay based on low cytosolic pH as a marker of PCD.
The acidification that occurs in the cytosol during plant PCD was monitored by way of the extinction of YFP fluorescence at low pH. This fluorescence was recovered experimentally when bringing the intracellular pH back to 7, demonstrating that there was no protein degradation of YFP. Because it uses YFP, the assay is none-destructive, does not interfere with the PCD process and allows time-lapse studies to be carried out. In addition, changes of sub-cellular localisation can be visualised during PCD using the protein of interest fused to RFP. Coupled to a transient expression system, this pH-based assay can be used to functionally analyse genes involved in PCD, using point mutations or co-expressing PCD regulators. Transfecting mBAX and AtBI-1 in onion epidermal cells showed that the pH shift is downstream of PCD suppression by AtBI-1. In addition, this method can be used to score PCD in tissues of stably transformed transgenic lines. As proof of principle, we show the example of YFP extinction during xylogenesis in Arabidopsis. This demonstrates that the assay is applicable to PCD studies in a variety of tissues.
The observation that YFP fluorescence is lost during the plant PCD process provides a new tool to study the genetic regulation and cell biology of the process. In addition, plant cell biologists should make a note of this effect of PCD on YFP fluorescence to avoid misinterpretation of their data and to select a pH insensitive reporter if appropriate. This method represents an efficient and streamlined tool expected to bring insights on the process leading to the pH shift occurring during PCD.
KeywordsProgramme Cell Death Yellow Fluorescent Protein Onion Epidermal Cell Programme Cell Death Process Programme Cell Death Marker
PCD is a universal process across multicellular organisms that is highly regulated and tightly controlled by many genes. These genes are expected to act together to form organised cascades culminating in cell death. There are a few assays, which can be used to monitor PCD in plants in order to analyse the function and interaction of specific genes. These assays score PCD at various steps in the PCD process and each has its limitations. Some are destructive such as TUNEL assays for detecting the DNA fragmentation induced during PCD . This assay involves fixing and permeabilising cells before labelling DNA fragment 3' ends using a terminal deoxynucleotidyl transferase and labelled dUTP (fluorescein, biotin, digoxigenin). Enzymatic assays for caspase-like proteases have become relatively prevalent in the plant literature as many synthetic caspase substrates are now commercially available . Most chromogenic or fluorogenic substrates are based on a four amino acid peptide with has a higher affinity for a subset of animal caspases e.g. DEVD for caspase3 and 7. Typically plant protein extracts are buffered at pH 7 or 5.5 and incubated with one of the substrates at 50 to 100 μM. In addition, none destructive in situ caspase assay can be carried out using permeable caspase inhibitors coupled to the fluorescent molecule carboxyfluorescein . The reagent easily permeates the cells and can irreversibly bind to caspase-like proteases inside the cell. Unbound inhibitor molecules are washed away to eliminate background. In addition, two types of cell permeable substrates have been used in pollen for in vivo studies of caspase activation . BiotumTD has developed a cell permeable substrate (Nucview 488) which when cleaved by caspase-like proteases releases a DNA dye which migrates to the cell nucleus and stains nuclear DNA . The other substrate, CR(DEVD)2, is composed of two DEVD peptides coupled to the fluorophore cresyl violet (CR). Upon cleavage, the fluorescent CR marker is released as a red fluorescent product . Recently, Zhang et al. 2009  developed an in vivo PCD assay based on expressing a recombinant protein in cells that is a DEVD_FRET substrate cleaved by caspase3-like proteases. In this case, the fluorescence is lost when the substrate is cleaved. It requires costly confocal equipment. Finally, the dye mitotracker red can be used to detect a loss of mitochondrial membrane potential (ψmit) in cells undergoing PCD. A loss of ψmit has been reported e.g. in tobacco cells during heat-shock induced PCD  and during tracheary element formation . This dye is a cationic lipophilic fluorochrome, which acts by accumulating in the negatively charged matrix of the mitochondria. The accumulation of this probe in the mitochondria is dependent upon the strength of the ψmit, the loss of which results in a proportional loss of mitotracker fluorescence .
Other generic live/dead assays used in PCD studies are non-destructive such as when using fluorescein diacetate (FDA) in an in vivo enzymatic assay. The assay relies on cellular esterase activity as a marker of live cells; the enzyme converts the non-fluorescent FDA to fluorescein . Because FDA is used to score live cells, a time course of life to death cannot be generated in the same cell as FDA fluorescence persists in dead cells. Further assays are based on the loss of membrane permeability that occurs during PCD. The molecules Evans blue, sytox green and sytox orange are excluded from live cells and only diffuse into the cells when membrane permeability is compromised. Evans blue labels the whole cell blue while sytox green and sytox orange light up the nucleus after binding to DNA [10, 11].
As plant vacuoles are acidic with a pH < 6 , it is logical to expect that vacuole rupture during PCD could result in cytosolic acidification. For example, authors of xylem differentiation studies had suggested that a cytosolic pH drop may occur when the vacuole ruptured during xylem PCD [13, 14]. In addition, vacuole disruption was proposed as a defining characteristic for bona fide PCD as vacuole collapse was reported not to occur in necrotic cell death . During PCD activated by self-incompatibility (SI) in the pollen tubes of Papaver rhoeas, Bosch et al. 2007  were able to measure a dramatic drop of intracellular pH. Using a pH sensitive probe, they found that the intracellular pH of pollen tubes undergoing SI dropped from pH 7 to pH 5.5. A pH of 5.5 is remarkably close to the pH optima measured in vitro for most of the caspase-like activities detected during plant PCD , suggesting that lowering the pH may be part of the activation process for caspase-like activities in plants.
Evidence in the literature [4, 14] and our own observation that YFP fluorescence was lost during the PCD process led us to consider that a large pH drop was a general feature of plant PCD and therefore a good candidate to develop a novel marker for PCD in plant cells.
BAX induces PCD and intracellular acidification in onion cells
To investigate the intracellular pH during PCD of onion cells, Ptilosarcus GFP (PtGFP) was chosen as a pH probe because of a broader pH-responsiveness and a greater acidic-stability than other pH probes such as pHluorins . First, onion epidermal cells were bombarded with ptGFP under the 35S promoter, permeabilised and equilibrated using buffers at pH ranging from 5 to 8. Fluorescent ratiometric measurements (F475/F390) were taken. The calibration curve obtained (Figure 1C) fitted the first part of the sigmoidal curve described over a wider range of pH using recombinant ptGFP. Next, 35S::ptGFP and 35S::BAX were co-bombarded to induce PCD and measure intracellular pH. After 30 hours of expression, all control cells expressing ptGFP only had an average intracellular pH of 7.3, a value typical for plant cell cytoplasm. By contrast at the same time point, cells co-expressing BAX and ptGFP had an acidic pH of 5.7 (Figure 1C). This pH value is consistent with the value of 5.5 reported by Bosch et al. 2007  during PCD in pollen and is in support of using a buffer at pH 5.5 to develop a marker of the PCD process in plant cells.
pH 5.5 attenuates the fluorescent signal of YFP in vivo
PCD attenuates YFP but not RFP fluorescence in vivo
In order to test whether YFP fluorescence attenuation by low pH could be used to score PCD in vivo, we co-transfected cells with RFP td-tomato and BAX fused to YFP (BAX::YFP) (Figure 2D, E). The number of YFP and RFP expressing cells were counted at 12 h, 24 h and 48 h after bombardment (Figure 2E). At 12 h, every transfected cell was expressing both constructs and the number of BAX::YFP and RFP fluorescent cells was the same. At 24 h post-bombardment, the cells were re-counted and there was a small reduction (< 20%) in the number of cells fluorescent for both BAX::YFP and RFP. By the 48 h time point, there was an 80% reduction in the number of BAX::YFP fluorescent cells. For RFP, the number of fluorescent cells remained unchanged across all time points. To investigate the correlation between loss of YFP fluorescence and PCD, caspase-like protease activity was followed in cells expressing BAX. For this purpose, cells transfected with 35S::BAX were incubated with FAM-VAD-FMK. The number of cells exhibiting caspase-like protease activity across the time points was found to correlate with the number of cells with an attenuated YFP signal (Figure 2E).
To confirm that the attenuation of YFP fluorescence observed in cells expressing BAX::YFP and RFP td-tomato (Figure 2D) was due to a pH drop and not to protein degradation, we buffered these cells to pH 7 for 1 hour. The lost BAX::YFP fluorescence was recovered in 95% of cells (Figure 2F). This confirmed that the pH drop measured during PCD is responsible for the loss of the YFP signal, while having no effect on RFP fluorescence.
YFP can form the basis of a pH cell death assay to study the interaction of genes regulating PCD
48 h after bombardment, cells with YFP fluorescence were counted. After scoring, the cells were incubated with X-GLUC to count cells with GUS enzymatic activity. The result is given in Figure 3C. Bombarding YFP as a negative control gave a 100% of YFP fluorescent cells. When expressing the fusion BAX::YFP, only 20% of GUS positive cells were YFP positive. This implied that 80% of transfected cells had lost YFP fluorescence 48 h after bombardment. This value obtained with GUS as a reference gene, is the same as the value obtained in previous experiments using the RFP td-tomato gene as a reference (see Figure 2E). BAX::YFP and Bcl-2 co-transfection gave 100% of YFP positive cells, whereas BAX::YFP and AtBI-1 co-transfection, gave 60% of YFP positive cells. The clear suppression effect of these two anti-PCD genes on the loss of YFP fluorescence in BAX::YFP expressing cells further demonstrated that the loss of YFP signal can be taken as a measure of PCD.
Use of YFP to visualise PCD in whole root tissue
We present here a method that uses YFP fluorescence as an indicator of the intracellular acidification that occurs during plant PCD. BAX expression in onion cells induced a loss of mitochondrial membrane potential, caspase-like activity and plasma membrane retraction, three hallmarks of plant PCD. We confirmed that the cytoplasmic pH drop from 7.3 to 5.5 described to occur during PCD in pollen tubes , occurred too in BAX-induced PCD in onion cells. We measured this pH change using the pH probe ptGFP, however this technique is too slow and labour intensive to score routinely PCD in plant cells. By contrast, we demonstrate here that the pH shift can be visualised much more simply as a loss of YFP fluorescence. The loss of YFP fluorescence correlated with induced caspase-like activity and could be inhibited by the PCD suppressors AtBI-1 and Bcl2. PCD can therefore be scored as absence of YFP fluorescence in cell expressing YFP using a fluorescence microscope and appropriate detection settings.
YFP can be expressed in the cell of choice either on its own or as a fusion to a protein of interest to confirm expression, providing the fusion does not affect protein function. The ability to measure the consequence of gene expression in a none-destructive manner is an important tool to study gene function in any particular process. The use of YFP fluorescence as a PCD marker means that data can be captured in real time including information on sub-cellular localisation and kinetics. Conveniently, RFP fluorescence is not affected by changes in intracellular pH and remains detectable until the very last stages of PCD. This makes RFP proteins convenient references for transfection or gene expression. For example, sub-cellular localisation or expression can be monitored after the pH shift by fusing the gene of interest to the RFP sequence. The results section above shows examples using RFP td-tomato, RFP mCherry and GUS as transfection reference. Any reporter protein found to exhibit the required stability at low-pH would constitute a suitable alternative to these three proteins for use in our pH cell death assay.
As a demonstration of the usefulness of YFP as a PCD marker, we transiently expressed the mouse BAX gene to induce PCD in onion epidermal cells. Animal BAX has already been shown to induce PCD in plant cell . Apoptosis induction in animal cells is linked with the localisation of BAX at the mitochondria, and the same BAX localisation is associated with PCD induction in plants . BAX- induced PCD can be prevented by over expressing the plant PCD suppressor gene AtBI-1[20, 34]. We found BAX to behave in the onion assay exactly as predicted from the published studies above, reinforcing the proposition that data obtained using pH shift as a marker of PCD correlate with the results obtained with other PCD markers. In addition, we found the animal anti-apoptotic gene Bcl2 to block BAX-induced cell death in plants, possibly through direct physical interaction between the two proteins as described in animal cells [35, 36]. Incidentally, these experiments showed that AtBI-1 and human Bcl-2 both suppressed PCD upstream of the pH shift, providing some insight in the cell death cascade in plants. Finally, we show here that onion cells are suitable for PCD studies. These cells come as a single layer of flat cells, facilitating microscopic observation. Onion epidermal cells are easy to handle and transfect using biolistics. We found that in addition to a pH shift, other PCD markers can be detected in onion cells such as caspase-like activities, loss of mitochondrial membrane potential and plasma membrane retraction.
In addition to experiments carried out using transient expression, we show the application of our method to the observation of differentiating xylem cells in Arabidopsis roots. PCD in differentiating xylem can be readily studied in zinnia cell culture  but not inside intact tissue such as root, as xylem cells are surrounded by several layers of cells. Our preliminary experiment suggests that YFP could be used to visualise PCD during the normal development of xylem. To our knowledge this is a first report using a PCD marker in live root and further work is required to characterised PCD in that experimental system. Nevertheless, our experiment demonstrates the power of cell-specific expression of YFP to visualise in real time and in planta, the pH shift marker of PCD. This approach could be extended to other developmental cell death systems.
In conclusion, we show here that the fluorescence of cells expressing YFP is greatly reduced at a specific stage of PCD in plants. This observation is the basis of the PCD assay described here. Combined with gene co-expression systems, this assay provides a convenient tool to study both the genetic regulation and the cell biology of PCD. Additionally, because this loss of YFP fluorescence is a specific marker of pH during PCD, it can be used to bring insights in the pH shift process itself. Finally, cell biologists may be unaware of this effect of PCD upon YFP fluorescence, which could lead to misinterpretation of expression data. To avoid this, a pH insensitive reporter should be selected.
pART7 with CaMV 35S and Ptilosarcus GFP (PtGFP) was bought from Nanolight, pinetop, USA. Mouse BAX-alpha fused to the a yellow fluorescent protein (YFP) and YFP (pEYFP-N1 #6006-1 CLONTECH Laboratories, Inc.) clones were provided by A. Gilmore, Manchester UK, and cloned as Eco47-Xho1 fragments into pDH51  cut with Sma1-Xho1. pDH51 provides a CaMV 35S promoter and terminator. A human Bcl2 cDNA clone was provided by T. Nishimoto, Kyushu Japan, and cloned as a BamH1-Sac1 fragment from pcDEB into pcGUS cut with BamH1-Sac1, providing a CaMV 35S promoter and NOS terminator. AtBAX-Inhibitor1 (At5g47120) was obtained from EST ATTS1836 via NASC, the European Arabidopsis Stock Centre, with the full coding sequence obtained by adding the sequence coding for MDAFSSF at the 5' end. The ORF was excised out of pBluescript SK+ into pDH51 using BamH1-Sal1. pRTL2-GUS expressing the beta-glucuronidase (GUS) gene is a gift from J. Carrington, College Station, USA. pAN57 containing mannosidase-tdtomato (RFP td-tomato) was obtained from Andreas Nebenführ . All plasmid preps were performed using 'Nucleospin plasmid ' or 'Nucleobond Xtra midi plus' from Macherey-Nagel, Duren, BD.
Each onion slice was transfected with a total of 10 μg of plasmid DNA. YFP and RFP plasmids were used in a 1:1 molar ratio. However, because GUS histochemical assays are more sensitive than YFP fluorescence detection, a dilution series of GUS plasmid concentration relative to YFP plasmid was carried out first to determine that a 1:1 ratio of cells expressing both GUS and YFP corresponded to a plasmid molar ratio of GUS 1:2.8 YFP. DNA was first precipitated onto 60 mg aliquots of 1.6 μm sterilised gold particles. Aliquots of gold particles were first vortexed for 30 seconds and then sonicated for 3 minutes. The DNA was then added in a volume under 80 μL, followed by 100 μL of 2.5 M CaCl2 and 20 μL of spermine 0.1 M with vortexing in-between each addition. The mix was further vortexed for 3 minutes and then left on ice for 15 minutes. The supernatant was removed and the particles were washed in 500 μL of ethanol. After 15 minutes on ice, the supernatant was removed and the particles were then washed again in ethanol this time using 200 μL. After one final 15-minute incubation on ice the supernatant was removed and the particles were re-suspended in 40 μL of ethanol. Bombardments were carried out on squares of onion fleshy scales of about 4 cm2 and 3 mm thick, each onion square was mounted on 1% agar plates to hold it in position to be fired upon and to prevent drying. A 10 μL volume of particles was deposited onto the micro-projectile and left to dry before being used in the gun. Samples were fired using 1100 psi rupture discs in a PDS-1000/HE particle gun (BioRad) at a distance of 9 cm under a vacuum of -27 inches Hg (0.925 bar). Bombarded onion pieces were then kept epidermal side down on the agars plates, which were sealed with parafilm and kept at 23°C until use.
YFP and RFP fluorescence
YFP positive cells and RFP positive cells were scored using a Leica DM5500 fitted with a Photometrics cascade II 512B EMCCD camera (Photometrics UK) and a dual filter YFP/dsRED (part 51019; Chroma Technology Corp.).
Mitotracker staining for mitochondria depolarisation
Onion cells were incubated for five minutes in 100 nM mitotracker red CMX ROS (Invitrogen) and observed immediately using a fluorescence microscope and a dual filter YFP/dsRED (part 51019; Chroma Technology Corp.).
In situ caspase activity
Activity was detected using the cell permeable and labelled Val-Ala-Asp peptide: FAM-VAD-FMK (APO LOGIX). Onion cells were incubated for 10 minutes in 4 μl of the 30× working dilution of the inhibitor diluted in 300 μl of water. Cells were washed twice in 5 ml of sterile distilled water before microscopic observation.
Fluorescence ratio imaging of ptGFP
Fluorescence ratios were calculated in accordance to Schulte et al. (2006) . Briefly, fluorescence images at excitation wavelengths of 475 nm and 390 nm were taken using a Nikon TE2000 PFS Widefield FRAP microscope fitted with a Cascade II EMCCD camera using a 10×/0.30 plan fluor objective. For this, cells expressing ptGFP were excited using a GFP filter (480/20) or a DAPI filter (402/15) with the emission wavelength centred on 540 nm. Images for each excitation were captured using Metamorph acquisition software from Molecular Devices. The ImageJ 1.42 free software was used to calculate a signal intensity value for every image. The ratio was subsequently calculated for the signal values obtained for an excitation at 475 nm and at 390 nm.
Calibrating ptGFP signal
A calibration curve was created by incubating ptGFP-expressing cells in buffers at pH ranging from 5 to 8. For this, F475/F390 ratiometric readings were taken in epidermal cells expressing PtGFP and incubated in solutions buffered at a pH ranging from 5 to 8. For this, 30 hours after bombardment, onion epidermal cell peels transfected with PtGFP were incubated in 6 wells plates, each well containing 3 mL of pH-buffered solution and 0.003% Triton-X-100. Triton X-100 was added in order to permeabilise cell membranes and to facilitate equilibration with the extra cellular buffer. Buffers used were: sodium acetate 50 mM, pH 5, MES (2-(N-morpholino) ethanesulfonic acid) 50 mM, pH 6 and 7, and Tris-HCl (Tris (hydroxymethyl) aminomethane hydrochloride) 50 mM, pH 8. Onion epidermal peels were incubated in these solutions for 1 hour, with slow shaking before fluorescence analysis.
GUS staining epidermal peels
Tissue samples were submerged in GUS staining solution (sodium phosphate buffer at pH 7, 10 mM EDTA, 0.1% Triton X-100, 2 mM potassium ferricyanide and 2 mM potassium ferrocyanide). For every one mL of this staining solution used, 1 μl of 5-Bromo-4-chloro-3-indolyl β-D-glucuronide (X-Gluc) (Melford, UK) was added from a stock at 50 mg/ml in dimethyl sulfoxide (DMSO). Samples submerged in the stain were vacuum infiltrated for 3 minutes, followed by 16 hours incubation at 37°C in the dark (plates were sealed with parafilm and then wrapped in tin foil to exclude light). The GUS stain solution was then removed and the tissue samples fixed in 70% ethanol.
Fluorescent microscopy of roots
The constructs and the line expressing both the YFP-CesA7 fusion (Cellulose synthase subunit A7) and the mCherry endoreticulum (ER) reporter have been described in Wightman et al. 2008 . Seedlings were grown in continuous light on vertical 0.5×MS salts (Duchefa), 1.5% agar plates. Root images were captured on a DMR microscope using an YFP/mCherry dual (51019) filter, a HCX PL APO CS ×63 water NA 1.2 objective and a SPOT Xplorer 4MP camera.
We thank C. Plieth for very helpful advice on the use of PtGFP and A. Day for kindly giving us access to his PDS-1000/HE particle gun. The Bioimaging Facility microscopes used in this study were purchased with grants from BBSRC, Wellcome Trust and the University of Manchester Strategic Fund. Special thanks go to Peter March and Robert Fernandez for their help with microscopy. BY holds a DTA BBSRC studentship.
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