Periodic Reporting for period 1 - CLAVHUB (Regulation of plasmodesmata signalling and transport by the CLAVATA1 and ARABIDOPSIS CRINKLY 4 receptor-like kinases)
Okres sprawozdawczy: 2021-12-27 do 2023-12-26
With global population growth and the threats of climate change, producing hardy, pest-resistant crops with larger yields is the holy grail of plant science. Historically, selectively breeding crops may have taken thousands of years and there was no knowledge of the genetic mechanisms underlying advantageous traits. In recent decades, researchers have linked phenotypic traits to specific genes and have found that many desirable traits are determined by a set of genes involved in regulating plant meristems.
Meristems are regions of the plant that maintain pools of undifferentiated stem cells and enable it to generate new structures, like roots, branches or flowers, throughout its entire lifetime. It is the regulation of these meristems that controls the plant’s overall architecture, allowing it to respond to environmental inputs and adapt its growth accordingly. By taking advantage of naturally-arising differences in meristem regulation, scientists have been able to modify crop architecture to increase yields, for example by selecting tomato plants with larger floral meristems that produce more and lager fruits, or dwarf rice plants that divert more resources to seed production rather than stem tissue.
Despite their agricultural significance, we still don’t fully understand the fundamental ways in which meristems are controlled. To investigate this, the CLAVHUB project used the model plant Arabidopsis thaliana, a member of the brassica family whose small size, short lifecycle and relatively simple genetics makes it particularly suitable for study and propagation in the lab. CLAVHUB concentrated on two Arabidopsis proteins involved in meristem regulation: ARABIDOPSIS CRINKLY 4 (ACR4) and CLAVATA 1 (CLV1).
ACR4 and CLV1 are both receptor kinases, a class of proteins straddling the cell membrane that can receive messages from outside the cell and relay them to effectors inside the cell. One interesting feature of ACR4 and CLV1 is that, as well as being distributed throughout the cell membrane, they also concentrate at specific points along the membrane. These points correspond to plasmodesmata, membrane-lined channels that enable nutrients and informational molecules to move from cell to cell through the cell wall. While small molecules can freely diffuse through plasmodesmata, the movement of larger molecules, such as RNAs or transcription factors, is selective. This is particularly important in plant development, which relies on regulatory molecules being able to move between specific regions to establish cell identity. However, how this selectivity is achieved it not well understood.
This brings us back to ACR4 and CLV1 because, in addition to clustering at plasmodesmata, the two proteins form complexes there. Furthermore, these complexes differ from the complexes they form elsewhere in the plasma membrane. This suggests that ACR4 and CLV1 have a plasmodesmata-specific function. The aim of CLAVHUB was to try to find out what this function was: a) whether plasmodesmata simply provide a platform on which to assemble signalling hubs, and/or b) if ACR4 and CLV1 are somehow influencing the function of plasmodesmata by modulating their permeability, and c) what other proteins are involved.
Meristems are regions of the plant that maintain pools of undifferentiated stem cells and enable it to generate new structures, like roots, branches or flowers, throughout its entire lifetime. It is the regulation of these meristems that controls the plant’s overall architecture, allowing it to respond to environmental inputs and adapt its growth accordingly. By taking advantage of naturally-arising differences in meristem regulation, scientists have been able to modify crop architecture to increase yields, for example by selecting tomato plants with larger floral meristems that produce more and lager fruits, or dwarf rice plants that divert more resources to seed production rather than stem tissue.
Despite their agricultural significance, we still don’t fully understand the fundamental ways in which meristems are controlled. To investigate this, the CLAVHUB project used the model plant Arabidopsis thaliana, a member of the brassica family whose small size, short lifecycle and relatively simple genetics makes it particularly suitable for study and propagation in the lab. CLAVHUB concentrated on two Arabidopsis proteins involved in meristem regulation: ARABIDOPSIS CRINKLY 4 (ACR4) and CLAVATA 1 (CLV1).
ACR4 and CLV1 are both receptor kinases, a class of proteins straddling the cell membrane that can receive messages from outside the cell and relay them to effectors inside the cell. One interesting feature of ACR4 and CLV1 is that, as well as being distributed throughout the cell membrane, they also concentrate at specific points along the membrane. These points correspond to plasmodesmata, membrane-lined channels that enable nutrients and informational molecules to move from cell to cell through the cell wall. While small molecules can freely diffuse through plasmodesmata, the movement of larger molecules, such as RNAs or transcription factors, is selective. This is particularly important in plant development, which relies on regulatory molecules being able to move between specific regions to establish cell identity. However, how this selectivity is achieved it not well understood.
This brings us back to ACR4 and CLV1 because, in addition to clustering at plasmodesmata, the two proteins form complexes there. Furthermore, these complexes differ from the complexes they form elsewhere in the plasma membrane. This suggests that ACR4 and CLV1 have a plasmodesmata-specific function. The aim of CLAVHUB was to try to find out what this function was: a) whether plasmodesmata simply provide a platform on which to assemble signalling hubs, and/or b) if ACR4 and CLV1 are somehow influencing the function of plasmodesmata by modulating their permeability, and c) what other proteins are involved.
A major part of CLAVHUB was to identify proteins interacting with ACR4 and CLV1 to give further clues as to their role at plasmodesmata. One approach involved tagging proteins that came in close proximity to ACR4 or CLV1 using an enzyme called TurboID. Any proteins marked by ACR4-/CLV1-TurboID with a biotin tag could be fished out with beads that bind to the biotin and then identified by mass spectrometry. These experiments are still in progress, but we have identified some promising candidates, including proteins involved in plasmodesmata formation and regulation. Further experiments will determine whether ACR4 and CLV1 are indeed interacting with these critical plasmodesmata proteins and helping to regulate plasmodesmata function.
Another goal was to determine how proteins like AC4 and CLV1 are actually directed to plasmodesmata in the first place. As plasmodesmata are continuous with the plasma membrane, it is not obvious how some proteins come to be preferentially located at plasmodesmata. To identify which parts of ACR4 are required to deliver it to plasmodesmata, I deleted regions of ACR4 and observed whether these truncated proteins were still located at the plasmodesmata. Using this approach, I narrowed down the responsible region to part of the extracellular domain. As this region is exposed to the cell wall, it could be interacting with structural components of plasmodesmata, helping the anchor ACR4. Further work will determine if this is the case.
Finally, CLVAHUB aimed to communicate scientific findings to different audiences. As well as discussing this work with other scientists at conferences and meetings, my teaching activities brought me in contact with undergraduate students with whom I could discuss the ins and outs of being a researcher. In addition, I created a blog to share my insights into academia and research life. While the blog did reach some non-scientists, the primary feedback I received was from more junior scientists and other academic parents who appreciated me sharing my experiences.
Another goal was to determine how proteins like AC4 and CLV1 are actually directed to plasmodesmata in the first place. As plasmodesmata are continuous with the plasma membrane, it is not obvious how some proteins come to be preferentially located at plasmodesmata. To identify which parts of ACR4 are required to deliver it to plasmodesmata, I deleted regions of ACR4 and observed whether these truncated proteins were still located at the plasmodesmata. Using this approach, I narrowed down the responsible region to part of the extracellular domain. As this region is exposed to the cell wall, it could be interacting with structural components of plasmodesmata, helping the anchor ACR4. Further work will determine if this is the case.
Finally, CLVAHUB aimed to communicate scientific findings to different audiences. As well as discussing this work with other scientists at conferences and meetings, my teaching activities brought me in contact with undergraduate students with whom I could discuss the ins and outs of being a researcher. In addition, I created a blog to share my insights into academia and research life. While the blog did reach some non-scientists, the primary feedback I received was from more junior scientists and other academic parents who appreciated me sharing my experiences.
CLAVHUB has laid the groundwork for further research into the role of receptor kinases in plasmodesmata function. Data from TurboID experiments have provided new avenues for exploration and further research in our interdisciplinary team is expected to reveal more proteins involved in plasmodesmal signalling and transport. Findings in Arabidopsis will direct more focussed research in crop species, which are often much harder to work with.
Given their importance in plant development, growth and immunity, understanding how plasmodesmata are regulated could provide new targets for synthetic peptides or plant breeding strategies to produce crops better adapted to climate challenges and pests. Furthermore, by sharing my experiences as a young female researcher with kids, both through my contact with biology students and via my blog, I hope I have broadened the perspectives of the next generation of researchers.
Given their importance in plant development, growth and immunity, understanding how plasmodesmata are regulated could provide new targets for synthetic peptides or plant breeding strategies to produce crops better adapted to climate challenges and pests. Furthermore, by sharing my experiences as a young female researcher with kids, both through my contact with biology students and via my blog, I hope I have broadened the perspectives of the next generation of researchers.