Arabidopsis CML13 and CML14 Have Essential and Overlapping Roles in Plant Development

Abstract

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. Under dexamethasone treatment, seeds of CML13- and CML14-RNA-interference lines exhibited differential sensitivity to exogenous ABA compared to WT seeds. Induced RNA suppression of mature plants led to reduced silique length, shorter roots, and rapid leaf senescence in CML13- and 14-RNA-interference plants which correlated with increased gene expression of the senescence marker SAG13. 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.

Functional characterization of calmodulin-like proteins, CML13 and CML14, as novel light chains of Arabidopsis class VIII myosins

“…We previously showed that suppression of CML13 or CML14 expression results in strong, aberrant phenotypes throughout development ( Symonds et al , 2023 ). An earlier report examined the phenotypes of myosin mutants and found that a myosin VIII mutant, atm1 , and myosin XI triple mutants (X1-1, -2, -K), xi3KO , exhibited reduced hypocotyl elongation relative to wild-type plants ( Olantunji and Kelly, 2020 ).…”

“…In Arabidopsis, these putative targets represent 56 different proteins, each with multiple IQ domains, and thus, like CaM, the number and variety of cellular processes that CML13/14 may participate in is expansive. Both CML13 and CML14 are important in plant growth throughout development, as suppression of either leads to a pleiotropic phenotype ( Symonds et al , 2023 ). Moreover, CML13 and CML14 function in abiotic stress responses, and a recent report found that cml13 mutants exhibit enhanced germination under salinity stress ( Hau et al , 2023 ).…”

“…For fluorescence microscopy assays, N. benthmaniana growth conditions were as previously described ( Belausov et al , 2023 ). For hypocotyl assays, Arabidopsis seeds were sown onto 0.5× Murashige and Skoog (MS) agar plates with or without 5 µM dexamethasone (Dex), as previously described, before cold stratifying in the dark at 4 °C for 48 h ( Symonds et al , 2023 ). Plates were then moved to the growth chamber in the dark and hypocotyls were allowed to elongate for 5 d before measuring the hypocotyl lengths with ImageJ ( Rueden et al , 2017 ).…”

“…Plates were then moved to the growth chamber in the dark and hypocotyls were allowed to elongate for 5 d before measuring the hypocotyl lengths with ImageJ ( Rueden et al , 2017 ). The Dex-inducible hpRNAi lines were produced previously ( Symonds et al , 2023 ). The Myosin VIII 4KO line, hereafter referred to as myosin viii4KO , was previously described ( Talts et al , 2016 ).…”

“…We recently reported that CML13 and CML14 interact with proteins that possess multiple IQ domains, including members of the IQ67-domain family (IQDs), CaM-binding transcriptional activators (CAMTAs), and class VIII myosins ( Teresinski et al , 2023 ). Moreover, we found that CML13 and CML14 are important for normal plant growth and development ( Symonds et al , 2023 ) and participate in the salinity stress response ( Hau et al , 2023 ). Here, we expand on these previous findings to investigate whether CML13 and CML14 are functional MLCs.…”