Periodic Reporting for period 2 - TIPTOP (Temperature Integration via Phase change and Translation Of Proteins in plants)
Reporting period: 2023-07-01 to 2024-12-31
This project seeks to understand how plants sense and respond to heat stress. This is an important topic for society because virtually all the food we consume is ultimately dependent on plants. The productivity of plants is highly influenced by heat stress. For example, it has been predicted that for every additional 1 ºC temperature increase as a consequence of climate change, crop yields of staples such as wheat and rice will decrease by about 10 %. Global food security is therefore at a high risk from increasing heat waves that can have major impacts on agricultural yields. Climate change is causing hotter, drier, summers and these are becoming more frequent. Despite the major importance of this topic, we understand relatively little about how plants actually sense and respond to heat. Work in my group has lead to the discovery of several mechanisms by which plants sense and respond to temperature. One particularly interesting temperature sensor involves protein phase change. During protein phase change, proteins are able to change from being fully soluble in a watery solution to being in droplets, much like a salad dressing separates into oil and vinegar.
This ability of proteins to sense temperature by undergoing "phase change" reflects a major temperature sensing mechanism in plants. This grant seeks to capitalize on this discovery and expand our understanding of how plants sense temperature in this way. By understanding how molecules in plants sense temperature, we ultimately aim to breed new crops that are more resilient to climate change. This will help safeguard food security.
Before we are able to directly work on crop plants, for this project we must first make fundamental discoveries about how protein phase change in response to temperature occurs. This work is done in the model plant Arabidopsis thaliana, since this is the most thoroughly understood plant from a scientific perspective owing to its excellent tractability as a model system.
Specific objectives of this project include:
-Determining how the amino-acid sequence of proteins controls the protein phase change response to temperature
We are identifying proteins that contain particular sequence motifs that confer temperature dependent phase change. We are then analyzing these sequences to identify the features that trigger temperature responsiveness. The goal of this objective is to understand thermosensing with sufficient clarity that we are able directly engineer new temperature responsive behavior by altering the amino acid composition of temperature responsive proteins.
-Identifying the temperature responsive proteome
Major advances in studying how proteins work in the cell now make it possible to study how proteins respond to temperature in the cell directly. We are therefore analyzing the cellular proteome to identify the major temperature responsive proteins. By comparing these with proteins that contain protein phase change sequence motifs, we are able to identify the proteins that likely undergo protein phase change and are thermally responsive. This will allow us to create a comprehensive understanding of the key thermosensors of the cell.
-Understanding how protein production is altered by temperature
As well as protein activity, the synthesis of some proteins by translation is also influenced by temperature. For example, RNA can adapt different conformations at different temperatures. We will investigate to see if these mechanisms are widely used in plants to sense and respond to temperature.
-Engineering desired temperature response pathways in plants. By taking the combined knowledge of both how proteins as well as mRNA responds to temperature, we will seek to engineer desired temperature responsive networks in cells. We will try this in both yeast, since it is a very tractable model system, as well as Arabidopsis.
This ability of proteins to sense temperature by undergoing "phase change" reflects a major temperature sensing mechanism in plants. This grant seeks to capitalize on this discovery and expand our understanding of how plants sense temperature in this way. By understanding how molecules in plants sense temperature, we ultimately aim to breed new crops that are more resilient to climate change. This will help safeguard food security.
Before we are able to directly work on crop plants, for this project we must first make fundamental discoveries about how protein phase change in response to temperature occurs. This work is done in the model plant Arabidopsis thaliana, since this is the most thoroughly understood plant from a scientific perspective owing to its excellent tractability as a model system.
Specific objectives of this project include:
-Determining how the amino-acid sequence of proteins controls the protein phase change response to temperature
We are identifying proteins that contain particular sequence motifs that confer temperature dependent phase change. We are then analyzing these sequences to identify the features that trigger temperature responsiveness. The goal of this objective is to understand thermosensing with sufficient clarity that we are able directly engineer new temperature responsive behavior by altering the amino acid composition of temperature responsive proteins.
-Identifying the temperature responsive proteome
Major advances in studying how proteins work in the cell now make it possible to study how proteins respond to temperature in the cell directly. We are therefore analyzing the cellular proteome to identify the major temperature responsive proteins. By comparing these with proteins that contain protein phase change sequence motifs, we are able to identify the proteins that likely undergo protein phase change and are thermally responsive. This will allow us to create a comprehensive understanding of the key thermosensors of the cell.
-Understanding how protein production is altered by temperature
As well as protein activity, the synthesis of some proteins by translation is also influenced by temperature. For example, RNA can adapt different conformations at different temperatures. We will investigate to see if these mechanisms are widely used in plants to sense and respond to temperature.
-Engineering desired temperature response pathways in plants. By taking the combined knowledge of both how proteins as well as mRNA responds to temperature, we will seek to engineer desired temperature responsive networks in cells. We will try this in both yeast, since it is a very tractable model system, as well as Arabidopsis.
We have made major progress in understanding the basis of the heat stress response pathway in Arabidopsis. We have discovered that the key heatshock genes HSFA1a, HSFA1b, HSFA1d and HSFA1e all contain prion like domains (PLDs), and these appear responsible for sensing and responding to temperature. When we alter the PLDs we are able to change the thermal responsiveness of seedlings. PLD1 acts as a negative regulator of the temperature response. When PLD1 is deleted we have a constitutively higher activity of HSFA1a. We were able to determine that PLD1 directly binds to Heat Shock Protein 70 (HSP70). By contrast, PLD2 of HSFA1a acts as a positive regulator in response to heat.
We have established important protocols for assaying temperature responsiveness of the plant cell proteome. These methods (Limited Proteolysis Mass Spectrometry, LiP-MS) enable us to measure the response of the whole proteome to changes in temperature dynamically. We are combining these approaches with new advances in machine learning for the prediction of protein structures and protein complexes to investigate key temperature responsive proteins in plants.
We have made very strong progress understanding how particular transcription factors directly sense and respond to temperature, and we are writing these results up for a publication currently. This mechanistic insight is enabling us to directly alter the sensitivity of temperature responsive transcription factors through rationale changes to their sequence. This is a major goal of the project.
We have used bioinformatic and computational biology approaches to understand how particular proteins containing disordered domains respond to temperature. This has enabled us to identify key residues and sequence motifs in the proteins of interest that confer temperature responsiveness. We can connect these residues to plants from different climates, strongly indicating that these domains are thermosensory and adaptive. These results are very important because they will help us engineer proteins with altered thermal responsiveness. These results also help us understand the fundamental biophysical basis of temperature perception.
We are performing medium high throughput assays to identify thermal responsiveness in candidate proteins by investigating phase change responses to temperature. These experiments are performed in the Nicotiana benthamiana system, and are enabling us to identify many temperature responsive proteins. These proteins are going to be investigated in more detail with follow up assays. For example, we will perform CRISPR-Cas9 mutagenesis of key targets to identify temperature related phenotypes.
We have established important protocols for assaying temperature responsiveness of the plant cell proteome. These methods (Limited Proteolysis Mass Spectrometry, LiP-MS) enable us to measure the response of the whole proteome to changes in temperature dynamically. We are combining these approaches with new advances in machine learning for the prediction of protein structures and protein complexes to investigate key temperature responsive proteins in plants.
We have made very strong progress understanding how particular transcription factors directly sense and respond to temperature, and we are writing these results up for a publication currently. This mechanistic insight is enabling us to directly alter the sensitivity of temperature responsive transcription factors through rationale changes to their sequence. This is a major goal of the project.
We have used bioinformatic and computational biology approaches to understand how particular proteins containing disordered domains respond to temperature. This has enabled us to identify key residues and sequence motifs in the proteins of interest that confer temperature responsiveness. We can connect these residues to plants from different climates, strongly indicating that these domains are thermosensory and adaptive. These results are very important because they will help us engineer proteins with altered thermal responsiveness. These results also help us understand the fundamental biophysical basis of temperature perception.
We are performing medium high throughput assays to identify thermal responsiveness in candidate proteins by investigating phase change responses to temperature. These experiments are performed in the Nicotiana benthamiana system, and are enabling us to identify many temperature responsive proteins. These proteins are going to be investigated in more detail with follow up assays. For example, we will perform CRISPR-Cas9 mutagenesis of key targets to identify temperature related phenotypes.
We have made progress beyond the state of the art in understanding key mechanisms of temperature perception:
(1) We have identified the role of PLD domains in HSFs and how these respond to temperature in Arabidopsis. This was previously unknown.
(2) We have identified how a bHLH transcription factor is regulated by temperature at the protein level. Even when overexpressed, this transcription factor has no significant phenotypes. This is because it contains thermosensory domains. When we carefully re-engineer these domains, we are able to completely alter the temperature response behavior of the plant. This was not previously described.
(3) We have discovered an entirely new thermosensory mechanism involving intramolecular inhibitory domains on flexible protein tethers. This discovery shows a new temperature sensing mechanism not previously known.
(4) We have identified sequence motifs and amino acid compositions that play a role in making intrinsically disordered proteins temperature responsive. This has not been described before
Key expected results by by the end of the project:
The major overarching goal of the project is to develop sufficient understanding of the mechanisms of temperature perception that we are able to directly engineer thermal responsiveness in plant signaling networks and alter temperature responses. We are on track to achieve this major goal, since we have made major progress in discovering temperature responsive mechanisms in plants. We are able to already alter temperature responses in directed ways in a number of key cases, and these will be greatly expanded by the end of the project.
(1) We have identified the role of PLD domains in HSFs and how these respond to temperature in Arabidopsis. This was previously unknown.
(2) We have identified how a bHLH transcription factor is regulated by temperature at the protein level. Even when overexpressed, this transcription factor has no significant phenotypes. This is because it contains thermosensory domains. When we carefully re-engineer these domains, we are able to completely alter the temperature response behavior of the plant. This was not previously described.
(3) We have discovered an entirely new thermosensory mechanism involving intramolecular inhibitory domains on flexible protein tethers. This discovery shows a new temperature sensing mechanism not previously known.
(4) We have identified sequence motifs and amino acid compositions that play a role in making intrinsically disordered proteins temperature responsive. This has not been described before
Key expected results by by the end of the project:
The major overarching goal of the project is to develop sufficient understanding of the mechanisms of temperature perception that we are able to directly engineer thermal responsiveness in plant signaling networks and alter temperature responses. We are on track to achieve this major goal, since we have made major progress in discovering temperature responsive mechanisms in plants. We are able to already alter temperature responses in directed ways in a number of key cases, and these will be greatly expanded by the end of the project.