Preamble: Koyaanisqatsi is a Hopi word that means “unbalanced life”. This word not only astutely describes the state of physiological stress, which organisms experience when environmental conditions rapidly change, but also describes the current state of our planet. Transformational change in our global research enterprise is needed to focus efforts towards establishing a more sustainable society. While research efforts across many fields will make important contributions here, the study of plants presents a unique opportunity to advance sustainability through research that is also biomedically relevant. Plants are a source of sustainable human nutrition and novel pharmaceuticals, plant cells synthesize biodegradable materials, and plant bodies capture and sequester carbon from the atmosphere at massive scales. Currently, however, our ability to use plants towards sustainable outcomes is in peril due to the extreme sensitivity of plant growth and development to environmental change. To understand the effects of climate change on ecosystems and to advance engineering efforts to tune the resilience of crops through biotechnology, advances in our basic understanding of how plants sense and acclimate to physiological stress is needed to fulfill the promise that plants may play in our future.
There may be no better place to focus our efforts than on water, which constitutes the most limiting resource for plant survival across the planet. Over the past 12 years, research in my lab has led to the discovery of novel modes of acclimation to water-associated stress that have broad physiological and agricultural relevance. We have defined the developmental and molecular mechanisms that control these responses and established new paradigms for how to experimentally approach the study of plant-environment interactions. The key difference between the work that my lab has done and what I want my group to achieve in the future lies in the depth and breadth at which we need to pursue our biological questions, to make the next biggest discoveries, and have the broadest impact on sustainability. Our current and future work will break current paradigms by: 1) Uncovering new cellular mechanisms plants use to resist water loss, 2) Utilizing the extraordinary diversity of plant species to discover novel tricks for extreme living and 3) Inventing the future of phenomics through robotics and in-field sensing of root systems. These areas of research will foster focused, mechanistic inquiry that reveals innovative adaptations to stress, which have a strong potential for application due to the physiologically realistic and field-informed experimental approaches we invent and deploy.
FOCUS AREA 1: Uncovering new cellular mechanisms that allow plants to resist water loss. Water affects the physical stresses acting on and within plant cells [3,4]. These mechanical cues are sensed by the plant and trigger osmotic-signal transduction events that ultimately regulate gene expression. While progress has been limited in defining the basic osmotic tolerance mechanism in plants, we have recently made a leap forward in characterizing this pathway using the single-celled model alga Chlamydomonas reinhardtii as a genetic system. We utilized a bar-coded, sequence-indexed mutant library in Chlamydomonas, developed by Martin Jonikas (Princeton University), which allowed us to query the functions of ~80% of the genes in the genome across 121 environmental conditions and a dozen osmotic stresses (Vilarrasa-Blasi et al. in preparation). We have characterized mutants of orthologous genes in the multicellular land plant Arabidopsis to identify broadly conserved osmo-tolerance pathways including genes controlling cytoskeletal organization, ion transport, plastid signaling and protein palmitoylation.
In organisms that lack cell walls, the actin cytoskeleton allows cells to mechanically interact with their environments (e.g. cell migration) through the formation of actin bundles and cables that exert force against the plasma membrane and affect the shape of the cell. In plant cells, however, the tremendous hydrostatic pressures that build up make such actin-mediated forces insignificant. As plant cells lose water to the environment, turgor pressure subsides, and the plasma membrane separates from the cell wall in a process called plasmolysis (Fig. 1A). Under these conditions, plant cells may be subject to the same range of forces as animal cells. Our mutant screen in Chlamydomonas identified PROFILIN as an essential gene under osmotic stress, and in Arabidopsis, profilin mutants undergo stress-induced cell lysis, which suggests broad conservation in function across green plants. Profilin binds actin monomers to regulate polymerization and cable formation. Indeed, we have revealed that in both species the actin cytoskeleton undergoes a massive remodelling under osmotic stress, such that the initially finely branched actin network condenses into large cables that wrap around the cell, just under the plasma membrane (Fig. 1B). We hypothesize that these actin cables may provide structural stability to the cell and prevent water loss under conditions where turgor is no longer at play. Through a collaboration with Chiara Daraio (Caltech) who studies the mechanical properties of biological materials, we utilized nano-indentation to probe tissue-level mechanics and determined that an actin-dependent rigidification of cells occurs under osmotic stress. These results support the existence of an actin-based mechanism to prevent water loss under osmotic stress and open up future opportunities to utilize the vast knowledge of actin dynamics to study this new mechanism for stress tolerance.
The signaling events that lead to changes in cytoskeletal structure are likely initiated at the plasma membrane (PM), which forms attachments to the cell wall. We are currently determining the polymers in the cell wall that facilitate these attachment sites. Recent work by other groups has suggested that many different stimuli lead to the recruitment of signaling proteins into nanoclusters with lower mobility . It is hypothesized that wall-plasma-membrane interactions may serve as recruitment points for these signaling proteins and we are now identifying such complexes using the proximity labeling technique Turbo-ID. Our goal is to define the proteins within these complexes and understand the specific linkages they make to the cell wall to enable the perception of osmotic stress and plasmolysis. Together our work in Focus Area 1 will establish the cellular mechanisms that govern perception and acclimation to water limiting conditions and identify the signaling pathways that trigger and shape the acclimation response.
FOCUS AREA 2: Defining an innovation map for gene regulation across plants that reveals novel tricks for extreme living. Genomes of a wide variety of organisms that are rare, unculturable, or even extinct, are becoming widely available. These species are often sequenced because of the unique developmental or physiological traits they exhibit. However, this new post-genomics era presents us with a challenge: How do we identify and characterize the function of genes when a genetic approach is not feasible? What new approaches are needed to discover the genetic innovations that underlie adaptive traits? Answering these questions will reveal the major milestones in the evolution of life on Earth, and the key components of regulatory pathways that can successfully be tinkered with to engineer the phenotype of an organism.
One of the pathways we have focused most on is abscisic acid (ABA) signaling, which is well characterized as a stress hormone acting downstream of drought and mediates changes in growth and water use. Surprisingly, we have found that species exhibiting extreme stress tolerance diverge in whether ABA represses or activates root growth and we are now working to determine how changes in the downstream targets of the ABA pathway may mediate such dramatically different physiological outcomes. ABA perception leads to the phosphorylation of ABRE-BINDING FACTOR (ABF) TFs, which regulate the bulk of ABA-dependent transcriptional responses. We have recently utilized DNA Affinity Purification Sequencing (DAP-seq) to define the genomic sites bound by all four members of the ABF family across four different species of the Brassicaceae family and are now working to complete a pan-species network for the entire Brassicaceae family. The Brassicaceae family serves as an excellent model to explore divergence in stress resilience as it contains plants that grow under extreme environmental stress as well as agriculturally and bioenergy relevant crops. We have generated global maps of direct TF binding that define the degree to which the function of a gene regulatory network is conserved or has diverged across species. Our family-wide survey will represent the most complete understanding of gene regulatory network evolution in plants and may reveal the pace and the targets of regulatory innovation. These genomic analyses are being complemented with comparative anatomy and single-cell RNAseq expression studies of roots across the Brassicaceae family to establish a comparative anatomical atlas that illuminates stress-resilient root functions.
FOCUS AREA 3: Inventing the future of phenomics through robotics and in-field sensing of root systems. In the lab we often simplify the environmental conditions organisms are grown under to reduce technical variation and increase the power of our visualization techniques. To study roots, plants are typically grown in sterile gel-based media that exposes the whole plant and root system to light. It is worth considering, however, that the native lives of these organisms might be quite different. Soil is a complex matrix of particles of different sizes and chemistries, pockets of air, heterogeneous distributions of water and nutrients and a micro-cosmos of bacteria, fungi and animals rivaling the biodiversity found in any rainforest canopy. I think it is useful to envision the types of biology that might exist in soil and to devise ways of investigating these aspects using thoughtfully designed experimental approaches.
We have developed an integrated system termed GLO-Roots (Growth and Luminescence Observatory for Roots), which is composed of custom growth vessels, reporter genes, imaging systems and image analysis software that allows root architecture, gene expression and microbial associations to be studied in soil grown plants (Fig. 2A)[12,13]. Luminescence-based reporters enable high-contrast visualization of roots and avoid the high-energy illumination required in fluorescence-based imaging. We have used GLO-Roots to reveal potentially adaptive responses of root systems to simulated drought treatments, which are difficult to study using gel-based media. We have paired this powerful method with robotics technology to enable automated time-lapse imaging of root systems. GLO-Bot I, designed in collaboration with local Bay Area startup Modular Science, was a low-cost “maker-bot” system that enabled the first set of 93 accessions to be imaged. Our newly installed GLO-Bot II plant handling system, designed in collaboration with Let’s Go Robotics, utilizes industry-standard robotics components and will provide a level of reliability and throughput needed to expand the scope of our experiments (Fig. 2B). We have now introduced our luminescence-based reporters into a panel of 183 Arabidopsis accessions to survey the diversity of root forms and environmental responses across this species. Through an active collaboration with Guillaume Lobet (Forschungszentrum Jülich and UC Louvain) we have built in silico models of Arabidopsis root systems and are parameterizing these to express variation captured in our phenotypic surveys. These models will be used to estimate physiological properties of root systems such as hydraulics and nutrient uptake efficiency. We plan to use these modeled structure-function relationships to compute traits that can then be used in GWA studies. Modeling physiological outcomes of root architectures may also be useful for identifying alternative strategies plants use to survive similar environmental pressures.
The frontier for understanding structure-function relationships in any organism is to study them in the natural environment. In-field phenotyping of plants will expand the diversity of species and environments that can be explored and, therefore, the number of adaptive strategies that can be discovered. We have collaborated with Stanford Electrical Engineers Amin Arbabian and Brutus Khuri-Yakub to build and test thermo-accoustic systems that may be useful for detecting roots and water in field conditions . Another collaboration with the Stanford undergraduate robotics club has led to in-field camera systems that will capture time-lapse movies of roots in the field together with soil-wetting patterns. We will continue to work with local colleagues and robotics companies to establish tools in the future that further break barriers regarding physiologically relevant imaging of plants. Together with the fundamental insight gathered at the cellular and whole plant level for Focus Areas 1 and 2, Area 3 will allow us to test the relevance of this knowledge to realistic environmental challenges, which will ultimately establish how these discoveries might one day impact plant-based innovation for sustainability.
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