Nuclear calcium regulation of plant development

Lead Research Organisation: John Innes Centre
Department Name: Cell and Develop Biology

Abstract

Calcium signalling is essential for growth and development, in both plants and animals. In animals nuclear calcium release is a potent regulator of neuronal gene expression and of cell proliferation. Nuclear calcium signalling is also known to be essential in legumes to promote associations with nitrogen fixing bacteria and phosphate delivering arbuscular mycorrhizal fungi. Legumes are among the world's most important agricultural food crops that are beneficial to billions of farmers and consumers worldwide and provide an essential aspect of natural soil enrichment of organic nitrogen compounds.

The mechanisms of plant nuclear calcium signalling were poorly understood. During my work I have used the symbiotic associations in legumes as a platform to dissect plant nuclear calcium signalling. Using a wide range of approaches, I discovered a number of ion channels located at the nuclear envelope that are responsible for symbiotic nuclear calcium release. Among them, I defined the first plant nuclear-associated calcium channels encoded by cyclic nucleotide gated channels (CNGC15s). The CNCG15s sit at the nuclear envelope in a complex with a potassium permeable channel (DMI1), also required for the generation of the symbiotic nuclear calcium signals. Interestingly, CNGC15s and DMI1 are conserved across all land plants, including non-symbiotic species, strongly suggesting that they have other functions during plant development. Consistent with this I have found a number of defects in Arabidopsis lines mutated in CNGC15 and DMI1, that include root developmental defects. I propose that my research in legumes has revealed the generic plant machinery involved in the regulation of nuclear calcium release. By studying the components that regulate nuclear calcium release I will be able to understand when and where nuclear calcium signalling is important.

My proposal will focus on the role of nuclear calcium signalling during root development, both root growth and associations with symbiotic microorganisms. Consistent with a function for CNGC15 and DMI1 in root development I have observed nuclear calcium responses in Arabidopsis root meristematic cells during their response to the phytohormones auxin and cytokinin. These calcium responses are mechanistically different from the nuclear calcium signals observed in legumes during symbiotic associations. My proposed research integrates molecular biology, genetics, cell biology, chemistry, electrophysiology and mathematical modelling to investigate how CNGC15-DMI1 regulates nuclear calcium release leading to plant developmental processes. It will use a large collection of Arabidopsis mutant and transgenic lines, that I have already generated, with a panel of nuclear calcium sensors allowing detection of nuclear calcium signals in an array of Arabidopsis mutants. My work will dissect the functions that nuclear calcium signalling plays in root developmental processes and how diverse nuclear calcium signals are encoded. Finally, through a combination of transcriptomics and mutant screens, I will be uniquely poised to decipher the downstream signalling components associated with nuclear calcium signalling during root development.

Technical Summary

Root legume symbioses are the only plant system with a well-defined nuclear calcium signal. I have used this system to dissect nuclear calcium signalling in plants. I demonstrated the first nuclear calcium channels in plants, encoded by cyclic nucleotide gated channels (CNGC)15s, which form a complex with the potassium permeable channel, DMI1, to create a large channel complex capable of generating symbiotic nuclear calcium oscillations. These channels are conserved across many plant species, including non-symbiotic plants, and from this I concluded that this channel complex must play additional functions beyond the establishment of symbiotic associations. To assess this, I generated Arabidopsis mutants in DMI1 and CNGC15 in Arabidopsis. Mutations in Arabidopsis CNGC15 show defects in root development and fertility, as well as other defects yet to be validated. Interestingly Arabidopsis dmi1 mutants also show root developmental defects, although the effects are opposite to those observed in cngc15. These preliminary analyses imply that the channel complex that I discovered in legumes plays broader roles in plant development. I propose that this channel complex controls all aspects of nuclear calcium signalling in plants and thus through these analyses, I will be able to define where and when nuclear calcium signalling is important during plant development and environmental responses. My fellowship proposal focuses on the role of CNGC15 and DMI1 during root development, both root growth and symbiotic associations in legumes. I have demonstrated that phytohormones activate nuclear calcium responses in Arabidopsis root meristematic cells. In my proposed fellowship I will dissect the detailed mechanism of action of the DMI1/CNGC15 channel complex and its broader function during root development.

Planned Impact

The outcomes of the proposed research will be of significant benefit to farmers and plant breeders as it has the potential to impact on crop yield and thereby to the UK public in general by contributing to UK's economic competitiveness.
Over 800 million people lack adequate access to safe and nutritious food. The world faces an even greater crisis in food security as expected global population growth to over 9 billion by 2050, is coupled with global climate change. On the issue of global warming, we have underestimated extreme climate fluctuations at the decadal time scale, which will influence UK and worldwide food security by creating unpredictable food shocks. It is urgent to develop new strategies to sustainably enhance worldwide agricultural production. Thus, understanding plant development, and how plants can integrate diverse environmental stimuli into developmental responses, is essential to improve resilience in crop production. My work will establish a molecular underpinning to the understanding of root development, a strategic important research area.
Calcium (Ca2+) is a universal regulatory molecule and intimately couples primary biotic and abiotic signals to many cellular processes allowing plant development and adaptation to environmental changes. My proposed research is at the leading edge of studies in plant development and nuclear Ca2+ signalling, providing exciting, new insights into the mechanisms of nuclear Ca2+ signal transduction during plant development. I will unravel the regulatory mechanisms of the central regulator of nuclear Ca2+ signal, which is at the core of multiple developmental processes such as root growth and fertility, targets for plant breeders to improve crop production.
By elucidating the mechanism of plant nuclear Ca2+ generation, this proposed research will position the UK as the leader in a new research area. Beyond the excellence, the outcome of this proposed research, combined with the new technology of genome editing, has the potential to be directly transferred to the main crops constituting the world's breadbasket (eg. wheat, maize) and major crops in developing countries (eg. rice, millet, sorghum). This could impact the production at multiple levels, from the biggest farming regions in UK, USA and Russia to the small farmers in sub-Saharan Africa.
In summary, the proposed project opens a new research area in plant sciences by determining how nuclear Ca2+ signals are regulated to influence plant growth. This research will have strong social, economic impacts involving UK competitiveness and global food security.

Publications

10 25 50
 
Title Dual Color Sensors for Simultaneous Analysis of Calcium Signal Dynamics in the Nuclear and Cytoplasmic Compartments of Plant Cells 
Description Spatiotemporal changes in cellular calcium (Ca2+) concentrations are essential for signal transduction in a wide range of plant cellular processes. In legumes, nuclear and perinuclear-localized Ca2+ oscillations have emerged as key signatures preceding downstream symbiotic signaling responses. Förster resonance energy transfer (FRET) yellow-based Ca2+ cameleon probes have been successfully exploited to measure the spatiotemporal dynamics of symbiotic Ca2+ signaling in legumes. Although providing cellular resolution, these sensors were restricted to measuring Ca2+ changes in single subcellular compartments. In this study, we have explored the potential of single fluorescent protein-based Ca2+ sensors, the GECOs, for multicolor and simultaneous imaging of the spatiotemporal dynamics of cytoplasmic and nuclear Ca2+ signaling in root cells. Single and dual fluorescence nuclear and cytoplasmic-localized GECOs expressed in transgenic Medicago truncatula roots and Arabidopsis thaliana were used to successfully monitor Ca2+ responses to microbial biotic and abiotic elicitors. In M. truncatula, we demonstrate that GECOs detect symbiosis-related Ca2+ spiking variations with higher sensitivity than the yellow FRET-based sensors previously used. Additionally, in both M. truncatula and A. thaliana, the dual sensor is now able to resolve in a single root cell the coordinated spatiotemporal dynamics of nuclear and cytoplasmic Ca2+ signaling in vivo. The GECO-based sensors presented here therefore represent powerful tools to monitor Ca2+ signaling dynamics in vivo in response to different stimuli in multi-subcellular compartments of plant cells. 
Type Of Material Physiological assessment or outcome measure 
Year Produced 2018 
Provided To Others? Yes  
Impact the cytoplasmic and nucleoplasmic dual sensors developed in this work are powerful tools to analyze the subcellular Ca2+ dynamics between the cytoplasm and nucleus in planta. These sensors will unequivocally be of interest in exploring Ca2+-mediated signaling in response to diverse stimuli in different plant species. 
URL https://www.frontiersin.org/articles/10.3389/fpls.2018.00245/full
 
Description Medicago LINC complexes function in nuclear morphology, nuclear movement, and root nodule symbiosis 
Organisation Ohio State University
Country United States 
Sector Academic/University 
PI Contribution Supervision of the study Symbioses experiments
Collaborator Contribution Study of the LINC components
Impact Newman-Griffis AH, Del Cerro P, Charpentier M, Meier I. Medicago LINC complexes function in nuclear morphology, nuclear movement, and root nodule symbiosis. Plant Physiology. 2019. 179(2):491-506
Start Year 2016
 
Description Simultaneous Analysis of Calcium Signal Dynamics in the Nuclear and Cytoplasmic Compartments of Plant Cells 
Organisation French National Institute of Agricultural Research
Department INRA Toulouse
Country France 
Sector Public 
PI Contribution We generated the Dual calcium sensor and the stably transformed plants (Arabidopsis thaliana and Medicago truncatula) We performed the research in Arabidopsis thaliana
Collaborator Contribution The Carvalho-Niebel lab generated DUAL sensor and stably transformed Medicago truncatula Performed the experiment in Medicago truncatula
Impact Kelner A, Leitao N, Chabaud M, Charpentier M*, De Carvalho-Niebel F*. Dual Color Sensors for Simultaneous Analysis of Calcium Signal Dynamics in the Nuclear and Cytoplasmic Compartments of Plant Cells. Frontiers in Plant Science. 2018. 9:245.
Start Year 2017
 
Description Co-organiser of the satellite meeting on Signal Transduction Pathways in Nitrogen-Fixing Symbioses 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact The meeting focused on molecular, genetic and in vivo dynamic aspects of plant symbiotic signaling, and offered the possibility to highlight the recent progress in Nitrogen-fixing signal transduction. This satellite meeting provided a privileged frame for presentations, discussions and exchanges around the latest scientific discoveries in plant signal transduction related to Nitrogen-fixing symbiosis, that were not covered during the 13rd European Nitrogen Fixation Conference (ENFC) in Stockholm, Sweden.
Year(s) Of Engagement Activity 2018
URL http://enfc2018.org/
 
Description Invited Speaker: Host Microbe Interactions- Norwich Research Park Group 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Professional Practitioners
Results and Impact Discussion within the Norwich research park scientists working on Host-Microbe Interactions in plant and animal.
Year(s) Of Engagement Activity 2018
 
Description Invited plenary speaker: 13th European Nitrogen Fixation Conference 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Prolific discussions and interest in nitrogen fixation signaling, as well as in promoting equality and diversity in science
Year(s) Of Engagement Activity 2018
 
Description Invited seminar at the 3rd Adam Kondorosi Symposium: Frontiers in Beneficial Plant Microbe Interactions 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact The objective of this Symposium was to create a scientific event that is at the forefront of fundamental research in beneficial plant-microbe interactions.
The Symposium brought together about 150 participants in a rather informal atmosphere, facilitating exchanges. This promoted exciting discussion with researchers - in particular those at the early stage of their career.
Year(s) Of Engagement Activity 2017
URL https://symposium.inra.fr/ak-ips2-2017_eng/
 
Description Invited speaker at the XIX International Botanical Congress, Shenzhen, China 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Undergraduate students
Results and Impact Invited speaker at on Calcium transport and signaling.
Year(s) Of Engagement Activity 2017
 
Description Invited speaker: Gordon Research Conference on Organellar Channels and Transporters, Mount Snow, USA 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact This dedicated conference provided a unique opportunity for animal and plant scientists working on different organelles to share data and ideas to help addressing current challenges of organelle biology, foster collaborations, and identify new ways to treat common and rare diseases based on better understanding of mechanisms of ion/metabolite transport in various organelles.
Year(s) Of Engagement Activity 2017
 
Description Invited speaker: International Plant Molecular Biology Conference, Montpellier, France 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact stimulated discussion promoting new collaboration.
Year(s) Of Engagement Activity 2018
 
Description Invited speaker: Max Planck Institute for Plant Breeding Research, Cologne, Germany 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Professional Practitioners
Results and Impact Exciting discussion promoting new collaboration
Year(s) Of Engagement Activity 2019
 
Description Invited speaker: Max Planck Institute of Molecular Plant Physiology, Golm, Germany 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Professional Practitioners
Results and Impact Discussion with scientists to increase collaborative effort aiming at improving crop production
Year(s) Of Engagement Activity 2018
 
Description Invited speaker: SEB Functional organisation of the nuclear periphery, Florence, Italy 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact This session included presentations on recent advances in knowledge and understanding molecular functions, dynamics and interactions of proteins of the nuclear envelope across kingdoms; topics will include the proteins of the linker of cytoskeleton and nucleoskeleton complex; the nuclear pore complex; the nucleoskeleton and lamina and the interactions of the nuclear envelope with the cytoskeleton. It included applications ranging from the role of nuclear envelope proteins in plant stress and disease responses to developmental disorders caused by laminopathies. The session generated very stimulated discussions.
Year(s) Of Engagement Activity 2018
 
Description Organization of the International Plant calcium signalling conference 2017 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact This conference brought all the plant calcium community together. It triggered very stimulating and interesting discussions and new opportunities of collaboration for many attendees.
Year(s) Of Engagement Activity 2017
URL http://plantcalciumsignalling.com/