Biology subjects

Symonds, K.

Publications and source records attributed to Symonds, K..

6 recordsLinked to original sources

bioRxiv2024

The Calmodulin-like proteins, CML13 and CML14 Function as Myosin Light Chains for the Class XI Myosins in Arabidopsis

Myosins are a crucial motor protein associated with the actin cytoskeleton in eukaryotic cells. Structurally, myosins form heteromeric complexes, with smaller light chains such as calmodulin (CaM) bound to isoleucine-glutamine (IQ) domains in the neck region. These interactions facilitate mechano-enzymatic activity. Recently, we identified Arabidopsis CaM-like (CML) proteins CML13 and CML14 as interactors with proteins containing multiple IQ domains, that function as the myosin VIII light chains. This study demonstrates that CaM, CML13, and CML14 specifically bind to the neck region of all 13 Arabidopsis myosin XI isoforms, with some preference among the CaM/CML-IQ domains. Additionally, we observed distinct residue preferences within the IQ domains for CML13, CML14, and CaM. In vitro experiments revealed that recombinant CaM, CML13, and CML14 exhibit calcium-independent binding to the IQ domains of myosin XIs. Furthermore, when co-expressed with MAP65-1-myosin fusion proteins containing the IQ domains of myosin XIs, CaM, CML13, and CML14 co-localize to microtubules. In vitro actin motility assays demonstrated that recombinant CML13, CML14, and CaM function as myosin XI light chains. A cml13 T-DNA mutant exhibited a shortened primary root phenotype that was complemented by the wild-type CML13 and was similar to that observed in a triple myosin XI mutant (xi3KO). Overall, our data indicate that Arabidopsis CML13 and CML14 are novel myosin XI light chains that likely participate in a breadth of myosin XI functions. HighlightMyosin XI proteins play a crucial role in the plant cytoskeleton, but their associated light chains have remained unidentified. Here, we show that calmodulin-like proteins, CML13 and CML14, serve as light chains for myosin XI, similar to their role for myosin VIII proteins

plant biology↗

bioRxiv2024

Characterization of the calmodulin-like protein family in Chara braunii and their conserved interaction with the calmodulin-binding transcription activator family

Calcium sensor proteins play important roles by detecting changes in intracellular calcium and relaying that information onto downstream targets through protein-protein interaction. Very little is known about calcium sensors from plant species that predate land colonization and the evolution of embryophytes. Here, we examined the genome of the multicellular algae, Chara braunii, for orthologs to the evolutionarily-conserved calcium sensor calmodulin (CaM), and for CaM-like proteins (CMLs). We identified one CaM and eight CML isoforms which rang in size from 16.4 to 21.3 kDa and are predicted to have between two to four calcium-binding (EF-hand) domains. Using recombinant protein, we tested whether CbCaM and CbCMLs1-7 possess biochemical properties of typical calcium sensors. CbCaM and the CbCMLs all displayed high-affinity calcium binding with estimated global KD values in the physiological {micro}M range. In response to calcium binding, CbCaM and the CbCMLs exhibited varying degrees of increase in exposed hydrophobicity, suggesting different calcium-induced conformational changes occur among isoforms. We found many examples of putative CaM targets encoded in the C. braunii genome and explored the ability of CbCaM and CbCMLs to interact in planta with a representative putative target, a C. braunii CaM-binding transcription factor (CbCAMTA1). CbCaM, CbCML2, and CbCML4 associated with the C-terminal region of CbCAMTA1. Collectively, our data support the hypothesis that complex calcium signaling and sensing networks involving CaM and CMLs evolved early in the green lineage. Similarly, it seems likely that calcium-mediated regulation of transcription occurs in C. braunii via CAMTAs and is an ancient trait predating embryophytic emergence. HighlightsAlthough calmodulin (CaM) and calmodulin-like (CML) proteins are well studied in vascular plants, little is known about their orthologs in ancient lineages. We characterized CaM and CMLs from Chara braunii, and assessed their ability to bind a representative target protein, a calmodulin-binding transcription factor, CbCAMTA1.

plant biology↗

bioRxiv2024

Characterization of Arabidopsis aldolases AtFBA4 and AtFBA5; inhibition by morin and interaction with calmodulin

Fructose bisphosphate aldolases (FBAs) catalyze the reversible cleavage of fructose 1,6-bisphosphate into dihydroxyacetone phosphate and glyceraldehyde 3-phosphate. We analyzed two previously uncharacterized cytosolic Arabidopsis FBAs, AtFBA4 and AtFBA5. Based on a recent report, we examined the interaction of AtFBA4 with calmodulin (CaM)-like protein 11 (AtCML11). AtFBA4 did not bind AtCML11, however, we found that CaM bound AtFBA5 in a Ca2+-dependent manner with high specificity and affinity (KD [~] 190 nM) and enhanced its stability. AtFBA4 and AtFBA5 exhibited Michaelis-Menten kinetics with Km and Vmax values of 180 {micro}M and 4.9 U/mg for AtFBA4, and 6.0 {micro}M and 0.30 U/mg for AtFBA5, respectively. The flavonoid morin inhibited both isozymes. Our study suggests that Ca2+ signalling and flavanols may influence plant glycolysis/gluconeogenesis.

biochemistry↗

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↗