Structural Biochemistry/Cell Organelles/Plant Cell/Heat Stress Response

Introduction
The heat stress response (HSR) in plants involves a complex network of different pathways which occurs in different cellular compartments. Scientists have been trying to figure out which is the main thermosensor that initiate the transcription of heat stress response gene in response to the heat stress.Up till now, scientists predicted that there are at least four major thermosensors in plants that activate a similar set of HSR gene which helps improving thermotolerance in plants, include a plasma membrane channel, an unfolded protein sensor in endoplasmatic recticulum, an unfolded protein sensor in cytosol, and a histone sensor in the nucleus. However, the relationship between these four thermosensors remains unknown.

The Significances of Heat Stress in Plants
1. Heat plays tremendous roles, especially those of adverse, in plant development, growth, reproduction and fertilization. Plant tissues involved in reproduction are especially vulnerable to the exposure of heat.

2. Plants are sessile organisms that cannot escape heat. Their metabolism is unique as they adapt their metabolism to counterbalance this disadvantage.

3. Cells and organelles in plants are designed in a way to combat and prevent damages caused by heat.

4. Studies for heat stress in plants help agriculture. Necessary measures for heat damage may be prepared from these studies. Under heat stress, programmed cell death can be activated in plants and this can lead to the shredding of leaves, flowers not blooming, little to no production of fruits, or simply the death of plants. In addition, the reproductive tissues in plants are especially sensitive to heat. A difference of a few degrees in temperature can lead to no crops for the season. This can have an enormous and negative impact on the economy. It is estimated that the 1980 and 1988 US heat waves resulted in overall damages of approximately 55 and 71 billion dollars

The Effects of Heat Stress in Plants
Heat stress has certain effects on different components in plant cells such as the stability of membranes, proteins, RNA and skeleton structure in cells.It also can change the way chemical reactions are carried out such as enzymatic reactions in the metabolic system. Due to the changes in these components, metabolic process of plant cells becomes imbalance. The disrupted state of metabolites can result in unwanted by-products such as reactive oxygen species (ROS).

In response to the changes of surrounding temperature, plants re-program their transcriptome, protome, metabolome and lipidome. This action basically induces changes in the composition of certain transcripts, metabolites and lipids which help the plants reset a new  metabolic balance so that the cells can survive and function as normal even at very high temperature. Moreover, when the temperature comes back to normal, plant cells can reverse the reprogram process and get back to the original metabolic balance that fit in with the current temperature. In addition, plants can also program cell death in response to the heat stress such as resulting in leaves shedding.

Types of Heat Response in Plants
Several heat treatments have been applied on plants to study the heat response in plants. There are three major set of heat treatments as mentioned in the article "How do plants feel the heat" by Ron Mittler* : no priming, stepwise priming, and gradual priming.

In the "no priming" treatment, the plants are introduced to a series of severe heat stress. For example, a plant that normally grows at 21oC is placed in an environment at 42-45oC for 0.5 to 1 hour. Plant survival is then measured 5-7 days after the treatment to determine the effect of heat stress. Due to this series of severe heat applied, the plant dies very quickly (exponential decay). The plants ability to survive after such treatment is referred to as basal thermotolerance, the ability of plants to sense and adapt to extreme heat without any priming.

"Stepwise priming" subjects the plants to an environment of moderate heat stress. This is set up by first letting the plants grow at 21oC, and then subjecting them to a temperature of 36-38oC for a short period time of 1.5 hours. They are then recovered to the normal temperature of 21oC for 2 hours after to which they are subjected to a severe temperature of 45oC. Their survivability under such condition (after priming) is known as acquired thermotolerance.

"Gradual priming" is the process which the temperature was increased gradually and steadily until it reaches the temperature equal to extreme heat of 45oC. This is supposed to imitate the conditions that is seen in nature. Plants under these conditions also exhibit acquired thermotolorence. For Arabidopsis thaliana, gradual priming increase the survival rate by more than 10%. It is preferred over both no priming heat stress and stepwise priming temperature changes.

The last type of heat treatment is "warming", which is not considered as one of the heat stress treatments. During warming treatment, plants are originally at a temperature lower than standard growing temperature of 12oC, instead of 21oC. They are then exposed to a temperature that is slightly higher than their ordinary growth temperature so instead of subjecting them to 21oC, they are grown at 27oC. This slight increase in temperature change from the norm is, however, not considered heat stress since plants actually do not express HSR markers under warming. In general, warming allows longer term adaptation and reprogramming of the development, which includes early flowering or shedding leaves.

The important message to take home from this experimental setup is that different heating treatments elicit different transcriptome responses. This suggests that there may be separate heat sensors and signaling pathways activating specific responses to rise in temperature.

Sensing heat in plants
When an A. thaliana experiences an increase in external temperature, its large surface-to-volume ratio makes sure that almost all macromolecules such as protein complexes, membranes and nucleic acid polymers in the plant cells to acknowledge the heat immediately. As it is true for every matter in the universe, the macromolecules' kinetic energy is raised by the introduction of heat into the system. This, consequently, results in reversible physical changes of the macromolecules. There are certain macromolecules that not only just "perceive" heat but also differentially trigger a unique signaling path that can specifically upregulate hundreds of HSR genes

There are 2 basic routes plants use to mediate heat stress 1. Direct effect in a specific sensor: The specific macromolecule that behaves as a sensor molecule could be directly affected by heat. It is observed when temperature-induced changes in its quaternary and tertiary structures occur. 2. Indirect effect in a specific sensor: In this route, the specific macromolecule sensor is indirectly influenced by heat due to the effects of heat on other components of the cell. For example, temperature-induced changes in membrane fluidity could affect a membrane protein which is embedded in. It is indirect because the membrane protein is actually affected by heat but is only subject to change when the fluidity of plasma membrane varies.

Heat sensing at Plasma Membrane
The fluidity of the plasma membrane can increase when there is an increase in temperature. Scientists have found out that the increased fluidity of the plasma membrane will activate calcium channels, which will lead to an influx of Calcium ions into the cell. This is the primary heat sensing event in the moss Physcomitrella patens. This is the mechanism of detecting heat. However, the actual heat stress sensor in plants cells in still unknown. The A. thalianagenome encodes over 40 calcium channels and many of them are known to be located in the plasma membrane. These presences of calcium channels strongly suggest that calcium ions are the ones, which are responsible for sensing heat.

Beside activating the Calcium channels, membrane fluidity change might also trigger lipid signaling due to the change of temperature.

How does a heat stress-induced inward calcium flux can control regulate multiple signaling pathways in plants?

In A. thaliana, the following 4 pathways are known to happen:

1. The calmodulin AtCaM3 signals heat stress and it itself is involved in the activation of different transcription factors such as WRKY39 and HSFs

2. Chain reactions may be triggered by heat signaling. It is known that an inward flux of calcium activates unique calcium-dependent protein kinases or the ROS-producing enzyme NADPH oxidase. A protein kinases is a kinase enzyme that modifies other proteins by chemically adding phosphate groups to them.

3. AtCaM3 activated calcium/calmodulin-binding protein kinase. This protein kinase phosphorylates HSFA1a.

4. An HSP90/FKBP-dependent kinanse can also mediate HSF phosphorylation causued from calcium binding to calmodulin

Lipid Signaling
On top of ion channel activation in the plasma membrane, membrane fluidity changes caused by increase heat can also trigger lipid signaling. During heat stress, phospholipase D (PLD) and phosphatidylinositol-4-phosphate 5-kinase (PIPK) are activated. Lipid signaling molecules such as phosphatidic acid, D-myo-inositol-1,4,5-trisphosphate (IP3), and phosphatidylinositol-4-phosphate (PIP2) start to accumulate from heat stress and tigger the calcium channels open, resulting in calcium influx. On a side note, it should be noted that a reduction in phospholipase C9 activity is correlated with lower concentration of IP3, sHSP downregulation, and lower thermotolerance.

The mechanistic relationship between lipid signaling response to heat stress and plasma membrane channels (whether it is direct or indirect) is still unclear. The chronological order of events in heat stress sensing and signaling response also remains as a question to be answered. It is likely that the signaling pathways function downstream to what's responsible for sensing heat stress in plasma membrane.

Change in Histone occupancy
Histones are proteins that help package DNA. As seen in wild-type plants, the increase in temperature causes a significant decrease in concentration of certain histones such as H2A.Z which contains the nucleosomes that might trigger the changes in transcriptome so that HSR genes are initiated. Scientists also found that the decrease in H2A.Z histone concentration might also cause the change in the expression of a specific transcription factor or other regulatory proteins which can initiate the response of transcriptome. After screening for mutants impaired in heat sensing, it was discovered that the ARP6 gene is possibly responsible for mediation of responses to temperature changes. ARP6 codes for a SWR1 subunit that is required in order for H2A.Z to be inserted into nucleosomes, instead of H2A. It was shown that warming in wild-type plants result in significant decrease in H2A.Z occupancy in nucleosomes where the transcription for warming-induced genes starts. Since less histones are in the way, this allows for more transcriptions of these warming-induced genes. Less H2A.Z occupancy in certain HSP promoters can also affect transcription factor and other regulatory proteins' expression and DNA binding ability, ultimately inducing transcriptome response. It is, however, unsure whether the occupancy of the histones is the cause for heat sensing related to acquired thermotolerance. [5].

Unfolded Protein Response
Heat stress may activate the unfolded protein response (UPR) in both ER and Cytosol which can initiate heat stress response(HSR). The UPR is the weakening of protein stability. In the ER pathway, unfolded proteins can enter the nuclei and therefore affect the transcription of certain genes. This will lead to an accumulation of ER chaperone transcripts which will alter the metabolism of the cell in order to adapt to the heat. Beside heat stress, UPR can also be activated by specific chemicals that cause UPR or changes in certain abiotic aspects. Because few unfolded proteins and HSR chaperones are likely to exist under mild heat conditions, the UPR is considered to be not as sensitive as the PM heat response.

ROS Signaling
Different metabolic pathways are likely to depend on enzymes with different responsiveness to unnecessary heat. From this reason. it has been proposed that heat stress might uncouple some metabolic pathways and cause the accumulation of unwanted by-products. An example of such by-product is reactive oxygen species (ROS). Studies have shown that the accumulation of ROS is mostly likely due to the change in fluidity of the plasma membrane. This event is most likely a positive feedback loop since the accumulation of ROS will also open up more calcium channels in the plasma membrane and therefore lead to more calcium influx into the cell. Therefore, it seems that the plasma membrane heat sensing is highly interlinked with ROS signaling. A large accumulation of ROS may lead to program cell death.

Conclusion
Although scientists have proposed possible ways in which plants can sense heat, there is still much we don’t understand about the specific mechanisms and the order of events. How the signals from the speciated sensors integrate with each other within the network of signal transduction is also still of an important question that is yet to be answered. There could be additional pathways not yet discovered but crucial to heat stress response.