An in vivo root hair assay for determining rates of apoptotic-like programmed cell death in plants
- Bridget V Hogg†1, 4,
- Joanna Kacprzyk†1,
- Elizabeth M Molony†1,
- Conor O'Reilly2,
- Thomas F Gallagher1,
- Patrick Gallois3 and
- Paul F McCabe1Email author
© Hogg et al; licensee BioMed Central Ltd. 2011
Received: 6 July 2011
Accepted: 13 December 2011
Published: 13 December 2011
In Arabidopsis thaliana we demonstrate that dying root hairs provide an easy and rapid in vivo model for the morphological identification of apoptotic-like programmed cell death (AL-PCD) in plants. The model described here is transferable between species, can be used to investigate rates of AL-PCD in response to various treatments and to identify modulation of AL-PCD rates in mutant/transgenic plant lines facilitating rapid screening of mutant populations in order to identify genes involved in AL-PCD regulation.
Programmed cell death (PCD) can be described as the organised destruction of a cell [1, 2] and plays an important role in many plant developmental pathways including xylogenesis, embryogenesis, root and leaf development and senescence [3, 4]. During the hypersensitive response it provides a defence response against invading pathogens  and has been shown to be part of the plants response to environmental stresses .
While there are several different forms of plant PCD [reviewed [7, 8]] and differences between plant PCD and apoptosis, the type of PCD which we observe in cell suspension cultures, apoptotic-like PCD (AL-PCD), has some shared features with apoptosis . For example, in response to biotic and abiotic stress, suspension culture cells have been shown to undergo nuclear condensation and internucleosomal DNA cleavage , activate caspase-like activity  and release cytochrome c from the mitochondria . In addition, both animal and plant cells demonstrate condensation of the cytoplasm. In plant cells the retraction and condensation of the cytoplasm leaves a visible gap between the cell wall and the plasma membrane resulting in a specific corpse morphology , a hallmark feature that has been a useful tool in quantifying rates of AL-PCD in plant suspension cultures from a wide variety of species. For example, the effects of chemically induced cell death in sycamore cell cultures  and in soybean cells , the effect of HR elicitors on PCD rates in tobacco  and Arabidopsis thaliana and the role of hydrogen peroxide on the induction of PCD in Arabidopsis thaliana, Glycine max and Nicotiana plumbaginifolia have all been investigated in cell suspension cultures. More recently, in transgenic lines, Burbridge et al.  showed that AL-PCD morphology could be used to investigate the effects of elevated levels of peroxidase on the AL-PCD induction threshold in transgenic cell suspension cultures of Nicotiana tabacum.
The AL-PCD associated corpse morphology has also been described in cells of whole plants. Delorme et al.,  showed that senescing cucumber cotyledon cells exhibit retraction of the cytoplasm and this retraction was associated with internucleosomal cleavage. During the hypersensitive response cells surrounding the site of infection undergo PCD, with the retraction of the cytoplasm thought to be a method for controlling the spread of infection . In fact, there are numerous examples in plants in which cytoplasm condensation occurs during PCD. These include, leaf morphogenesis in Monstera and lace plant leaves , in the final stages of senescence [19, 22] or in tissues undergoing the hypersensitive response [23, 24]. The lace plant Aponogeton madagascariensis provides in vivo system for studying PCD in real time  however in other species the cells undergoing AL-PCD in whole plants are often buried within the tissue and large-scale scoring of individual cells to evaluate changes in cell death rates is not always feasible. While cell suspension cultures have provided a model to investigate AL-PCD in individual cells, they can be labour intensive to establish and time is required to achieve a mature stable cell line. As such they are not amenable to investigation of altered AL-PCD rates in mutant/transgenic lines in which large amounts of time have already been invested and many lines may need to be scored. There is a need therefore to develop an in vivo model which utilises the AL-PCD morphology to evaluate changes in rates of PCD in whole plants.
In order to provide a simple, rapid and in vivo model for the investigation of AL-PCD rates we assessed a range of cell types, for their suitability to facilitate large-scale scoring of AL-PCD in whole plants. An important criterion in assessing their suitability being that they exhibit the AL-PCD associated corpse morphology when subjected to a known inducer of AL-PCD such as heat, and that these cells are easily visualized in large numbers. Root hairs are single cells, readily observed by light microscopy and the response of each individual root hair subjected to specific treatments can be determined easily. Using the corpse morphology as a visual indicator of AL-PCD, they provide a quick in vivo model for quantifying rates of AL-PCD. In addition, many root hairs can be scored per plant and exogenous compounds can be added to the root system and their effect on AL-PCD rates investigated. Plants can be grown quickly and easily and results obtained in a matter of days. Root hairs can also be used to investigate AL-PCD rates in mutant/transgenic plant lines. Lines whose root hairs have an altered AL-PCD response can then be selected for the production of cell suspension cultures if so desired.
We describe in this paper a simple model for evaluating AL-PCD in vivo. Using Arabidopsis thaliana we show that results can be obtained (from seed to scoring) in 6 days and that induction of AL-PCD in root hairs can be achieved both by heat (abiotic stress) and chemical stress (ethanol, acetylsalicylic acid, hydrogen peroxide and sodium chloride treatment). The AL-PCD phenotype is comparable to that observed in cell suspension cultures and the response to heat stress almost identical. We also demonstrate that AL-PCD of Arabidopsis thaliana root hairs is suppressed by Ac-DEVD-CHO, a caspase-3 inhibitor. To test whether this system can be used to screen for lines with an altered AL-PCD response we present results from At-DAD1 (Defender Against Apoptotic Cell Death) over-expressor line of Arabidopsis thaliana DAD-1 .
Results and Discussion
Caspase inhibitor study
Response of root hairs to a range of temperatures
Response of root hairs to ethanol
Response of root hairs to salt stress (NaCl), acetylsalicylic acid (ASA) and hydrogen peroxide
Modulation of AL-PCD levels in the At-DAD1 over-expressing line
AL-PCD in other plant systems
The applicability of the this novel in vivo root hair technique to plants other than Arabidopsis thaliana was confirmed by the induction of AL-PCD morphology in root hair cells of Medicago truncatula, Zea mays and Quercus robur and provides an effective model for the investigation of AL-PCD in, we believe, any plant species. It will facilitate the investigation of PCD in the adaptation, or lack thereof, of crop responses to abiotic stress such as cold, drought and salinity. For example, it could be envisaged that the induction of AL-PCD in root hairs of Zea mays undergoing cold stress may be an indicator of cold susceptibility/tolerance and provide a rapid initial screen for breeding lines of Zea mays that are adapted to a more temperate climate. The assessment of AL-PCD in root hairs can easily be conducted at different stages of plant growth and development. It is possible certain species have a greater tolerance to different abiotic stresses and the threshold for the induction AL-PCD by heat stress may be at a lower or higher temperature than that seen in this study. Our investigations to date suggest the AL-PCD morphology is a feature of most, if not all, plant species and can therefore be used to investigate regulation of AL-PCD in plants.
We describe a rapid and adaptable in vivo root hair model for accurately assessing rates of AL-PCD in plants. We show that, as in cell suspension cultures, abiotic stress (heat) or chemical stress (ethanol, ASA, NaCl, H2O2) induce cell death in root hairs that results in the AL-PCD corpse morphology. This morphology is associated with caspase-like activity. Using an At-DAD1 over-expressing line we demonstrate that this system can be used for screening lines altered in the expression of genes which are thought to be involved in PCD for perturbations in rates of AL-PCD. The transferability of this system to other plants, including an important crop species (Zea mays), is also demonstrated.
Cell suspension cultures growth
Cell suspension cultures of Arabidopsis thaliana were grown essentially as described in . Briefly cultures were grown in 100 ml of liquid Murashige and Skoog (basal salts 4.3 g l-1 ) medium containing 0.5 mg l-1 NAA, 0.05 mg l-1 kinetin and 3% (w/v) sucrose, pH 5.8 with agitation on an orbital shaker (100 rev/min) under low light conditions (6 μmol m-2 s-1 ) in a controlled environment room, 23°C, 24 hr light. Cultures were subcultured every 7 days by taking 10 ml and adding to 100 ml of fresh medium.
Heat treatment of cell suspension cultures
Ten ml of seven day old cultures was placed in sterile 100 ml flasks. The flasks were then placed in a shaking (85 oscillations/minute) waterbath (Grant OLS200) set to the desired temperature for ten minutes. Following heat treatment flasks were returned to the controlled environment room, with shaking, until scoring 24 hr later.
Seeds of Arabidopsis thaliana, Medicago truncatula and Zea mays were sterilised in 20% (V/V) commercial bleach (final concentration of NaOCl approximately 1%) followed by washing four times with sterile distilled water (SDW). Seeds of Medicago truncatula were scarified using fine sandpaper before sterilisation for 3 min in 20% (V/V) commercial bleach. Zea mays and Arabidopsis thaliana seeds where sterilized for twenty minutes. Following sterilisation all seeds where plated in a single line on their respective growing medium to allow the roots to grow down the surface. Arabidopsis thaliana seeds were plated on semi-solid half-strength Murashige and Skoog (basal salts, 2.15 g l-1 ) medium, 1% sucrose and 1.5% agar in 9 cm Petri dishes and stratified at 4°C for 24 hr, before being placed vertically at 22°C, 16 hr light, 8 hr dark. Zea mays seeds were germinated on Whatman filter paper No.1 that had been soaked in half-strength Hoagland's medium (1.6 g l-1 , Sigma) in square Petri dishes (12 cm × 12 cm), five seeds per Petri dish. The Petri dishes where then sealed with parafilm wrapped in aluminium foil and placed vertically at 22°C. Following sterilisation seeds of Medicago truncatula were left to imbibe for 24 hr at 4°C and then plated on nitrogen-free modified Fahraeus medium  (1 mM CaCl2, 0.5 mM MgSO4, 0.7 mM KH2PO4, 0.8 mM Na2HPO4, 50 μM FeEDTA (pH 6.5) including the following microelements, CuSO4, MnSO4, ZnSO4, H3BO3, and Na2MoO4 at a final concentration of 0.1 mg l-1 ) with 1.5% w/v agar in square (12 cm × 12 cm) Petri dishes, six seeds per plate. Following three days at 4°C the plates were placed vertically at 24°C day (16 hr), 18°C night (8 hr).
For the germination of Quercus robur, acorns were soaked at 4°C for 5 days in water. Following soaking acorns were half buried in vermiculite and covered with damp tissue. Acorns were then placed at 22°C, 16 hr light, 8 hr dark, and watered generously every second day. Three to four weeks later seedlings were selected where significant root growth had occurred. Following removal of the vermiculite from the roots by gentle washing in SDW, seedlings were then subjected to the heat treatment as described below.
Heat treatment of root hairs
All heat treatments were carried out in SDW using a Grant OLS200 waterbath set the desired temperature, without shaking, for ten minutes. Five day old seedlings of Arabidopsis thaliana were carefully transferred with the forceps from the growth medium to 15 ml Falcon tubes containing 10 ml of sterile distilled water (SDW). Seed germination on the vertically positioned Petri dishes prevented developing roots from penetrating the surface of the medium and therefore reduced the background root hair damage caused by transfer from the growth plate. Following heat treatment seedlings were then returned to the constant temperature room at 23°C in the light for 6 hr, after which AL-PCD was scored. Both Medicago truncatula and Zea mays were heat treated in 6 cm Petri dishes containing 5 ml of SDW. Five day old seedlings were used for heat treatment Medicago truncatula and 3 day old seedlings were used for Zea mays. Following heat treatment, seedlings of Medicago truncatula were returned to the controlled environment room 24°C day (16 hr), 18°C night (8 hr) and scored for AL-PCD 24 hr later. Seedlings of Zea mays were incubated in the dark at room temperature following heat treatment for 6 hr until scored for AL-PCD. Three to four week old Quercus robur seedlings were heat treated in square Petri dishes (10 cm × 10 cm) filled with approximately 50 ml of SDW. Following heat treatment, seedlings were incubated in the light at room temperature for 6 hr before scoring for AL-PCD.
Ethanol (Merck) was added to 7 day old cells at a final concentration of 10% (v/v). Cells were then returned to the controlled environment room with shaking (100 rev/min) for 24 hr until scoring. Five day old Arabidopsis thaliana seedlings were placed in 6 cm Petri dishes containing 5 ml of 10% ethanol (v/v) (Merck). Petri dishes containing the seedlings were then incubated at room temperature for 6 hr until scoring.
Five day old Arabidopsis thaliana seedlings were placed in 6 cm Petri dishes containing 5 ml of 0.08 mM ASA solution (Sigma). The Petri dishes containing the seedlings were then incubated at room temperature in constant light for 24 hr until scoring.
Five day old Arabidopsis thaliana seedlings were incubated in 6 cm Petri dishes containing 5 ml of 100 mM NaCl solution (Sigma) for 5 min. Afterwards seedlings were transferred to 6 cm Petri dishes containing 5 ml of SDW and kept in constant light at room temperature for 24 hr until scoring.
Five day old Arabidopsis thaliana seedlings were incubated in 6 cm Petri dishes containing 5 ml of 25 mM H2O2 solution (Sigma) for 5 min. The Petri dishes containing the seedlings were then incubated at room temperature in constant light for 24 hr until scoring.
Caspase inhibitor study
Prior to heat-treatment, five day old Arabidopsis thaliana seedlings were incubated for 1 hour in 6 cm Petri dishes containing 5 ml of 1 μM Ac-DEVD-CHO (A.G. Scientific) in SDW. Subsequently Petri dishes were transferred to a waterbath set to 49°C for ten minutes. Following heat treatment seedlings were incubated at room temperature in constant light for 24 hr until scoring. Ac-DEVD-CHO stock was prepared in DMSO (10 mg/ml) and used within one month. DMSO solvent control had no influence on root hairs viability (data not shown).
Cells/roots were stained with fluorescein diacetate (FDA) a live/dead stain. Only viable cells/root hairs are able to cleave FDA to form fluorescein which, when excited by a wavelength of 485 nm, fluoresces green. Root hairs/cells were stained in a 1 μg/ml solution of FDA on the standard microscope slides and immediately examined under white light and fluorescent light. Whole Arabidopsis seedlings were placed on the slides; whereas only roots of Medicago trunculata, Zea mays and Quercus robur were used to facilitate placing the cover slip on the top of the specimen. As root hairs are single cells on the root surface, the morphology of individual root hairs along the main root can be readily observed. Root hairs/cells that were positive for FDA staining were scored as alive. Root hairs/cells negative for FDA staining were examined further and scored as either AL-PCD, having a retracted cytoplasm, or necrotic, having no retracted cytoplasm and therefore no distinguishable morphology compared to living cells under the light microscope. The percentage for each category was calculated as a percentage of the total number of roots hairs scored (typically ~ 100) averaged over at least three replicates. Images were taken using either an Olympus BX60 or Leica DML8 microscope with Leica DFC240C colour camera attached and captured using Leica application suite V3.1.0 software. Data is expressed in graphs as AL-PCD and Total Cell Death (TCD, where TCD = AL-PCD + necrosis).
EMM was funded by a UCD seedfunding grant awarded to COR. BVH was funded by an Irish DAFF Research Fund Programme awarded to TG and PMcC. JK is funded by The Embark Initiative, a Government of Ireland scholarship, operated by The Irish Research Council for Science, Engineering and Technology. The authors thank Dr. Theresa Reape for critical reading and revision of the manuscript.
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