Measurement of programmed cell death

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Measurement of programmed cell death, and necrosis in Arabidopsis thaliana cell suspension culture after various levels of heat induced stress.

Programmed cell death(PCD) is a genetically encoded self-destruct mechanism enclosed within the nuclear DNA of plant cells that has become essential for growth, development, maturity and defence to infection from the environment.

Plant development involves the elimination of cell organelles, protoplasts, tissues and organs. Characteristic features of apoptosis, a form of programmed cell death, were found e.g. in leaf senescence, abscission of flower parts, reproduction processes, tracheary element formation, and responses to various biotic and abiotic stresses. The role of phytohormones in programmed cell death is becoming evident. Apoptosis in meristems influences longevity and overall development of plants.

1 Table of Contents

1.1 Table of Figures

1.2 Table of Tables

2 Aim

The aim of this experiment was to determine and compare levels of programmed cell death (PCD) and necrosis at various temperatures in Arabidopsis thaliana cell suspension cultures. This was achieved by exposing the cultures to various temperatures inside a Grant(r)waterbath, and counting cells under microscope.

3 Introduction

Programmed cell death (PCD) is a genetically organised mechanism which allows cells to self-destruct, whenstimulatedby anappropriatetrigger. The initiation for this trigger, may come about as a result of the cell being no longerrequired,or, thecellhas becomeathreattotheplants overall health within the evolution and maturation of the plant. This has been defined “as a sequence of events that leads to the controlled and organized destruction of the cell” (Lockshin and Zakeri, 2004), or as stated by Gadjev (2008) “programmed cell death (PCD) is a genetically controlled process leading to selective elimination of unwanted or damaged cells in eukaryotes, and is essential for growth and development of multicellular organisms as well as for proper response to environment (Gadjev et al., 2008). PCD occurs in all plants and animals and is a crucial in protecting against infection and disease by restricting the spread of pathogens. PCD can be observed continuously in plants; from the tissues of germinating seeds, and fruit drop, to deciduous plants shedding their leaves in the form of abscission, and the storing of energy with senescence. Also sepals and petals having accomplished their role “may senesce and either abscise or remain in situ, protecting the initial growth of the ovule” (Gadjev et al., 2008).

There are however differences between plants and animals, in that plants lack immune systems like animals, so that they can’t resist infection or remove dead cells, but they do consist of a cell wall where, the dying cells manufacture substances to break itself down as well as storing infection and toxins within avacuolethat ruptures as the cell dies “thus protecting the overall healthy development of the organism as a whole (Reape et al., 2008). The importance of this PCD in plants cannot be underestimated as it helps to shape the development of the plant not only by removing damaged, unwanted or infected cells but also in development, such as Xylem vessels which are dead cells at maturity, but are “the cells that are elements of the water, and nutrient conducting system live to make strong walls”(Greenberg, 1996). “During tracheary elements differentiation, the cell wall not only remains but also undergoes reinforcement and thickening that is coordinated with the vacuole swelling and rupture. Only the fragment of the primary cell walls located between adjacent tracheary elements is hydrolyzed to form a channel” (Gadjev et al., 2008).

4 Cell Death

Cell deathis classed as abiological occurrence, whereby a cellceases to carry out its principal purpose. This may be as a consequence of the natural process whereby old cells die and are replaced by fresh ones, or may be as a consequence of disease, localized injury, or the failure of which the cells are part of such as leaf or flowers. PCD in plants is a crucial component for both development and defence and consists of more than one type, such as; apoptosis, autophagy, and necrosis and these all occur during different stages of a plants lifecycle, and “occurs during development, such as during xylogenesis, embryogenesis, parenchyma formation, several plant reproductive processes, seed development and leaf senescence” (Gadjev et al., 2008).

4.1 Development PCD

Developmental PCD occurs throughout the plant cells at different times and in various tissues such as reproductive organs and in some instances, the organs themselves undergo abscission. By removing the organs and tissues that are no longer necessary, the plant maintains its energy resources, eliminates possible entry sites for pathogens.

4.2 Apoptosis

PCDAApoptosis is the process of PCD in which a regulated sequence of events leads to the death of the cell without releasing harmful substances contained within, into the surrounding area. “The strict definition for apoptosis states that the cell eventually breaks up into apoptotic bodies that can then be engulfed by phagocytes” (Reape et al., 2008). Features of apoptosis are evident during some very specific cellular morphology, such as a retraction of the protoplast away from the cell wall as can be seen in figure 1. To differentiate between animal and plant PCD characterised by this retraction of the protoplast away from the cell wall in plants, it has been termed as AL-PCD.

Figure 1: - A.thaliana cells undergoing PCD. Morphology evident, showing the retraction of protoplast from cell wall (Daly, 2014).

4.3 Autophagy

Autophagy is a type of slow cell death, generally thought to be a survival mechanism, as it involves a process of PCD which is important for balancing energy sources at crucial times of development in response to nutrient stresses, by promoting cellular senescence, whilst protecting against and preventing necrosis. Autophagy is also essential in removing damaged organelles, such as mitochondria, ribosomes and endoplasmic reticulum, as well as removing pathogens, and can be selective or non-selective in this removal. Autophagy iscytoplasmic, characterized by the formation of largevacuolesthat eat awayorganellesin a specific sequence prior to the destruction of thenucleus, as stated by Contento (2005) “Autophagy is a slow type of cell death whereby cells display a decreased cytoplasmic width and enlarged vacuole, processes which are thought to be related to the recycling of nutrients within the plant before the onset of death” (Contento et al., 2005)”.

4.4 Necrosis

Necrosis is a non-physiological process and is caused by external factors, such as infection, stress or injury. With necrosis being the terminal point for the cell, PCD isn’t a terminal stage for plant cells. As with some cases, the dead cells end up playing specific functions within the plant structure such as tracheids and vessel elements, found in xylem in vascular plants essential for support and water transportation. “In other cases, cells must die to form organs with proper functions” or cells die because they have “accomplished their function and/or are no longer required (e.g., petals in some flowers after pollination, leaf senescence)” (Gadjev et al., 2008).

5 Cells under view of microscope

marklive1eFor this experiment Arabidopsis thaliana suspension culture was used as described in the Methods sections. To view under microscope one must first be able to distinguish between cells alive and healthy, undergoing PCD or necrotic. Differences between stages can be seen in Figures 2, 3, and 4. Figure 2, shows healthy cells, showing the vacuole close to cell wall along with the nucleus, being visible. Figure 3 shows stages of PCD with the retraction of the protoplast away from the cell wall leaving a visible gap, whereas figure 4 shows necrotic cells, which appear to show no distinction of a vacuole, or nucleus however, creases are often viewed within the cell.

Figure 2:- Healthy A.thaliana cells showing vacuole and organelles tight to cell wall (Daly, 2014).

Once samples had been extracted and pipetted on to slides, being able to distinguish between the cells is crucial to be able to distinguish between the forms of cell death in a specified time frame if meaningful results were to be obtained.

6 Heat Induced Stress (Temperature)

PCD may also be initiated by many abiotic factors such as extreme temperatures, salinity, or pollutants. Extreme fluctuations in temperature, or rapid increase or decrease or a sustained prolonged period of temperature not suitable for the cells will have a harmful effect, which can lead to the commencement of PCD. “Metabolic activity is affected by heat stress and the cell cytoskeleton becomes destabilised which affects membrane fluidity and enzyme function is impaired due to the breakdown of proteins” and “consequently cold stress reduces “membrane fluidity and enzymatic activity”(Kacprzyk et al., 2011).

7 Methods

For this experiment, two 100ml 7 day oldArabidopsis thalianacell suspension cultures were used along with three, 100mL flasks of freshly prepared MS autoclaved Arabidopsis thalianamedia. These were poured into a singular beaker and mixed to ensure equal density, ensuring all replicates were identical before being aliquoted in 48 flasks containing 10ml each.

Tin foil lids were placed on the tops of the flasks to ensure no bacteria entered the flasks before being randomly placed into 6 groups each containing 8 flasks. The 6 groups were then labelled accordingly; C, (for control), 35°C, 47°C, 51°C, 57°C and 70°C. All flasks except the control, were then placed in an internal shaker mechanism, inside a Grant(r)waterbath which had been previously set at the required temperature, (as above) and heat treated for 10 minutes whilst been continuously shaken at 90 rpm. The heat stressed flasks including the control flasks were then placed back into a shaker incubator overnight, at 120 rpm and 23°C. Ensuring the cell suspension culture didn’t settle resulting the death of the cell due to lack of oxygen. Morphological changes can normally be detected between 1 and 3 hours after the heat treatment, but it is easier to determine PCD after maximum protoplast retraction. For this reason waiting approximately 24 hours gives best results. The following day (approx. 24hrs later) the flasks were divided amongst the students, with each student taking a flask from each group and pipetting 100µl of culture from each flask onto microscope slides. The slides was then covered and the slide placed under the microscope lens. Each slide was analysed for cell viability, PCD and Necrosis, by scoring the cells up to a value of 200 with all data being collaborated. As seen in the results table 1.

8 Results

Table 1: - Raw Data Collaborated

Table 1 shows all recorded data. Replicate number of 8 (n=8).

Figure 5:-Levels of cell viability and cell death in A.thaliana cell suspension cultures after varying degrees of temperature.

Error bars indicate higher degree of discrepancy at 51°C and 57°C. Fluorescein diacetate (FDA) may have been used to produce more accurate results.

Figure 6: - Line graph indicating the decline of PCD and increase of necrosis as temperature increases

Results revealed that PCD increased as temperature increased up until a point where the cells did not get the time to induce PCD and became necrotic instantly. As illustrated by the graph in figure 6 it can be seen that moderate heat stress caused most of the cells to die via PCD whilst higher temperatures caused the cells to die via necrosis without any cytoplasmic withdrawal as seen on figure 4. This demonstrates what Reape (2008) stated “that the withdrawal of the cytoplasm is an active result of PCD rather than a mere collapse of the cell in on itself.

Figure 7: - Arabidopsis after 10 minutes of Heat Treatment

After 10 minutes of heat treatment at 47°C, and below, most cells seem to survive, whereas above this temperature the rate of PCD increases. Once the cells are exposed to temperatures above 57°C, the cells die, but as a result of necrosis.

9 Discussion

There is no doubt as to the importance of PCD to plant growth, survival, development and structural strength. Plants eliminate cells, and organs, in response to stress and environmental conditions to assist in growth and survival. Broad leaf trees discard their leaves and flowers in autumn, whereas fertilised cells in ovules on the same plant are retained. PCD is also vital to a plant’s defence system, by way of a hypersensitive response that occurs when a plant cell recognises, directly or indirectly, of invading pathogens and prevents the systemic spread by voluntary sacrificing an infected cell in order to prevent the spread and limits the growth of the pathogen.

“It looks like every single plant cell has a special mechanism that it can activate to kill itself, and plants use it as they develop and also to protect themselves: if a plant cell recognises a disease-causing pathogen it switches on the death programme. It basically sacrifices itself as well as killing the pathogen to protect the rest of the plant” (Kacprzyk et al., 2011).

So can PCD be controlled, and used adventitiously to benefit? In some cases leaves become yellow on plants due to lack of nutrients or aging known as senescence. Senescence is a genetically controlled process which is internally programmed, but if this process has not gone too far, the leaves can become green again, reversing the PCD, so yellowing does not lead to death. In this instance “chloroplasts have initially lost considerable quantities of proteins and chlorophyll, but are somehow able to reverse the degradation process and synthesize various compounds again”(Krul, 1974). “In plant biology the term programmed cell death is widely used to describe genetically controlled forms of cell death” (Gadjev et al., 2008) With greater understanding, genetically controlled PCD could be exploited to control plants growth cycles, Size, longevity, breed resistant varieties, greater crop production and yield. For example activating PCD in cells involved in tissue development could be selected to eliminate their nucleated protoplasts, and thus enabling them to perform structural and translocatory functions, giving rise to more larger productive crops with greater yields. In addition plants that only survive for short periods of time, such as some herbaceous genus that develop and die in weeks, or crops that can’t survive in extreme conditions could be manipulated to suppress the genes that trigger the PCD thus maintaining their development

10 Conclusion

As discussed earlier PCD is a controlled organised process that is essential for plants which results in cellular destruction, activated throughout every part of the plant right through its entire life cycle. PCD is crucial for vegetative and reproductive developmental processes, senescence programmes, pathogen defence mechanisms and stress responses.

As discussed earlier “programmed cell death (PCD) describes a processes that results in a highly controlled, and organised, form of cellular destruction, activated through every part of the plant throughout its entire life cycle. PCD is a critical component of many vegetative and reproductive developmental processes, senescence programmes, pathogen defence mechanisms and stress responses. Cell destruction can manifest as apoptotic-like, necrotic or autophagic cell death and these processes are likely to overlap extensively, sharing several regulatory mechanisms” (Kacprzyk et al., 2011).

As seen throughout the results plant cells under stress

11 References

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GADJEV, I., STONE, J. M. & GECHEV, T. S. 2008. Programmed cell death in plants: new insights into redox regulation and the role of hydrogen peroxide. International review of cell and molecular biology, 270, 87-144.

GREENBERG, J. T. 1996. Programmed cell death: a way of life for plants. Proceedings of the National Academy of Sciences, 93, 12094-12097.

KACPRZYK, J., DALY, C. T. & MCCABE, P. F. 2011. The botanical dance of death: programmed cell death in plants. Advances in botanical research, 60, 170.

KRUL, W. R. 1974. Nucleic acid and protein metabolism of senescing and regenerating soybean cotyledons. Plant physiology, 54, 36-40.

LOCKSHIN, R. A. & ZAKERI, Z. 2004. Apoptosis, autophagy, and more. The international journal of biochemistry & cell biology, 36, 2405-2419.

REAPE, T. J., MOLONY, E. M. & MCCABE, P. F. 2008. Programmed cell death in plants: distinguishing between different modes. Journal of experimental botany, 59, 435-444.