Biology subjects

Symonds, K.

Publications and source records attributed to Symonds, K..

3 recordsLinked to original sources

bioRxiv2023

Arabidopsis CML13 and CML14 Have Essential And Overlapping Roles In Plant Development

Calmodulin-like proteins (CaM-like; CML) are the largest family of calcium-binding proteins in plants, yet the functions of most CMLs are unknown. Arabidopsis CML13 and CML14 are closely related paralogs that interact with the isoleucine-glutamine (IQ) domains of myosins, IQ-domain (IQD) proteins, and CaM-activated transcription factors (CAMTAs). Here, we explored the physiological roles of CML13 and CML14 during development by using dexamethasone-inducible RNA silencing to suppress either CML13 or CML14 transcript levels. In the absence of inducible suppression, CML13- and CML14-RNA-interference lines were indistinguishable from WT plants throughout development. In contrast, induction of silencing treatment led to rapid increases in RNA-hairpin production that correlated with a targeted reduction in CML13 or CML14 transcript levels and a range of developmental and morphological effects. RNA suppression treatment did not impair the germination of CML13- or 14-RNA-interference lines, but these seedlings were chlorotic, displayed high mortality, and failed to achieve seedling establishment. Induced RNA suppression of mature plants led to reduced silique length, shorter roots, and rapid leaf senescence in CML13- and 14-RNA-interference plants. Plants induced for RNA suppression at 2 weeks post-germination exhibited a much stronger phenotype than treatment of 3-, 4-, or 5-week-old plants. Collectively, our data indicate that both CML13 and CML14 are essential for normal development and function across a broad range of tissues and developmental stages. HighlightCML13 and CML14 are biochemically unique among the CML family and interact with proteins that possess IQ domains. Here, we show that both paralogs are essential for normal plant growth and development.

plant biology↗

bioRxiv2023

Functional Characterization of Calmodulin-like Proteins, CML13 and CML14, as Novel Light Chains of Arabidopsis Class VIII Myosins

Myosins are important motor proteins that associate with the actin cytoskeleton. Structurally, myosins function as heteromeric complexes where smaller light chains, such as calmodulin (CaM), bind to isoleucine-glutamine (IQ) domains in the neck regions to facilitate mechano-enzymatic activity. We recently identified Arabidopsis CaM-like (CML) proteins, CML13 and CML14 as interactors of proteins containing multiple IQ domains, including a member of the myosin VIII class. Here, using in vivo and in vitro assays we demonstrate that CaM, CML13, and CML14 bind the neck region of all four Arabidopsis myosin VIII isoforms. Among ten CML isoforms tested for in planta binding to myosins VIIIs, CaM, CML13, and CML14 gave the strongest signals using in planta split-luciferase protein-interaction assays. In vitro, recombinant CaM, CML13, and CML14 showed specific, high-affinity, calcium-independent binding to the IQ domains of myosin VIIIs. Subcellular localization analysis indicated that CaM, CML13, and CML14 co-localized to plasma membrane-bound puncta when co-expressed with RFP-myosin fusion proteins containing IQ- and tail-domains of myosin VIIIs. In addition, in vitro actin-motility assays using recombinant myosin holoenzymes demonstrated that CaM, CML13, and CML14 function as light chains for myosin VIIIs. Collectively, our data indicate that Arabidopsis CML13 and CML14 are novel myosin VIII light chains. HighlightMyosins are key proteins in the plant cytoskeleton, but the identity of their light chain components is unknown. Here, we show that calmodulin-like proteins function as novel myosin light chains.

plant biology↗

bioRxiv2023

Arabidopsis calmodulin-like proteins CML13 and CML14 interact with proteins that have IQ domains

In response to Ca2+ signals, the evolutionarily-conserved Ca2+ sensor calmodulin (CaM) regulates protein targets via direct interaction. Plants possess many CaM-like (CML) proteins, but their binding partners and functions are mostly unknown. Here, using Arabidopsis CML13 as bait in a yeast two-hybrid screen, we isolated putative targets from three, unrelated protein families, namely, IQD proteins, calmodulin-binding transcriptional activators (CAMTAs), and myosins, all of which possess tandem isoleucine-glutamine (IQ) structural domains. Using the split-luciferase complementation assay in planta and the yeast 2-hybrid system, CML13 and CML14 showed a preference for interaction with tandem over single IQ domains. Relative to CaM, CML13 and CML14 displayed weaker signals when tested with the non-IQ, CaM-binding domain of glutamate decarboxylase or the single IQ domains of CNGC20 (cyclic-nucleotide gated channel-20) or IQM1 (IQ motif protein1). We examined IQD14 as a representative tandem IQ-protein and found that only CaM, CML13, and CML14 interacted with IQD14 among 12 CaM/CMLs tested. CaM, CML13, and CML14 bound in vitro to IQD14 in the presence or absence of Ca2+. Binding affinities were in the nM range and were higher when two tandem IQ domains from IQD14 were present. Green fluorescent protein-tagged versions of CaM, CML13, and CML14 localized to both the cytosol and nucleus in plant cells but were partially relocalized to the microtubules when co-expressed with IQD14 tagged with mCherry. These and other data are discussed in the context of possible roles for these CMLs in gene regulation via CAMTAs and cytoskeletal activity via myosins and IQD proteins.

plant biology↗