Green Leaves

Research 

The overarching question in my research program is: How do plants use calcium signals to process information about their environment?

We use the tools of protein biochemistry, cell biology, molecular genetics, and physiology to better understand how plants detect and respond to information from their environment.

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The Role of Calcium in Signal Transduction

As sessile organisms that cannot flee adverse growth conditions, plants have evolved a complex array of adaptive responses to environmental perturbations such as drought, temperature stress, salinity, and pathogen attack. Generally speaking, when a stimulus is perceived, signal transduction cascades activate and coordinate appropriate cellular responses that typically include changes in gene/protein expression and activity and lead to adjustments in metabolism and development. Although the specific cellular and physiological changes induced by stress vary according to the nature of the stimulus, one universal aspect is the use of calcium ions as important second messengers. One of the earliest cellular responses to stimuli involves the opening of calcium channels and the subsequent influx of calcium into the cytosol. Increases in [calcium]cyt above a threshold level activate a variety of calcium sensors (calcium-binding proteins) which, in turn, regulate the activity of myriad downstream targets such as kinases, transcription factors, metabolic enzymes, cytoskeletal components, etc. that ultimately orchestrate the cellular response required to adapt to a given stimulus.

Interestingly, cell imaging studies using calcium-sensitive dyes has revealed complex spatio-temporal patterns (termed “signatures”) of calcium influx/efflux in response to various stimuli. Evidence is emerging that distinct calcium signatures are evoked by different stimuli and thus encode information about the nature of the stimulus, like a Morse code. Calcium sensors are thought to detect and “decode” these signatures and thus contribute to the specificity of response. Although this model is an oversimplification and other 2nd messengers also participate in these signaling pathways, there is no question that calcium plays a central role in information processing by plants. This point is underscored by the remarkable array of calcium sensors that plants possess compared to other eukaryotes of similar genomic complexity.  Most of these sensors are likely unique to plants and remain unstudied.

The CaM and CaM-like (CML) family of Calcium sensors

Our lab is interested in understanding how calcium signals are used by plants to help coordinate cellular responses to environmental stress and during development. We are primarily focused on elucidating the roles of the various calcium sensors found specifically in plants. The two largest phylogenetic groups of calcium sensors in plants are the calcium-binding protein kinases (CPKs; ~35 members in Arabidopsis) and the calmodulin (CaMs; ~7 in Arabidopsis) family which includes the CaM-like proteins (CMLs; ~50 in Arabidopsis). CaM is a small (~148 residues), evolutionarily conserved, eukaryotic protein that possess calcium-binding domains, termed 4 EF-hand motifs. Upon binding calcium, CaM undergoes a conformational change that allows it to bind to (and typically regulate) various downstream target proteins. Numerous CaM-targets have been identified in plants (e.g. ion channels and pumps, kinases, phosphatases, metabolic proteins, transcription factors, etc.) and it is clear that CaM plays an important regulatory role in many cellular processes in plants. In contrast, much less is known about the plant-specific family of CMLs. Gene expression analysis using the model plant Arabidopsis indicates significant increases in transcript levels for many CMLs after stress treatments, suggesting a role in calcium-mediated stimulus response. Given the complexity of the CML family, it is likely they serve a variety of cellular functions.

Determining the functions of CMLs

In order to fully understand the role of calcium as a messenger in plant cells it is necessary to study the calcium sensors which propagate calcium signals. Why do plants need such a sophisticated array of calcium sensors? We use the genetic model plant Arabidopsis to address questions about the CML family. We have adopted a multi-disciplinary approach in our investigations of plant CMLs by using the tools of genetics (e.g. knockout mutants, overexpression of CMLs), molecular biology (e.g. gene expression, promoter analysis), biochemistry (e.g protein structural and functional analysis, protein-protein interactions), and whole-plant physiology (e.g. analysis of mutants under various growth regimes). CMLs vary in their sequence similarity to CaM (from about 20% to greater 80%) suggesting they occupy cellular roles distinct from CaM. Our working hypothesis is that CMLs operate as transducers of calcium signals: upon binding calcium, CMLs undergo a conformational change that allows them to bind to specific targets. To test this hypothesis we have been looking at the biochemical properties of CMLs and attempting to identify the protein targets that CMLs interact with. Our biochemical analyses (e.g NMR, hydrophobicity studies using ANS, calorimetry, etc) using recombinant CMLs indicate that most CMLs behave like bona fide calcium sensors but have unique properties compared to CaM. We have also identified putative novel targets for several CMLs and are actively characterizing these proteins and examining their cellular roles. In addition, genetic analysis of knockout plants has revealed aberrant developmental phenotypes that are shedding light on the physiological roles of these interesting proteins.

The complexity of calcium signalling in plants is impressive but also challenging to study. We are learning new and exciting things about how plants differ from other eukaryotes when it comes to sensing and responding to environmental cues. As we continue to explore the properties and roles of CMLs we expect many surprises and interesting revelations. Plants do things in unique ways and it is fun and rewarding to discover how they have evolved specialized proteins and systems to cope with life’s challenges!

The expression of many CMLs is induced by stress. Here, CML37 (left) and CML38 (centre), but not CML39 (right), are upregulated by a wound treatment that mimics insect feeding.

Close up of CML16 expression in guard cells.

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CML15 is mainly expressed in floral tissue.

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CML16 displays a broad expression pattern.

cml39 knockout mutants display developmental arrest a few days after germinating.

Most CMLs display large changes in tertiary structure upon calcium binding, exposing regions of hydrophobicity that interact with downstream targets.

Many, though not all, CMLs possess high affinity for calcium as a ligand. Such binding likely regulates their interaction with protein targets.

cml42 knockout plants have unusual trichome-branching patterns