Summary Calcium signalling is central to many plant processes, with families of calcium decoder proteins having expanded across the green lineage and redundancy existing between decoders. The liverwort Marchantia polymorpha has fast become a new model plant, but the calcium decoders that exist in this species remain unclear. We performed phylogenetic analyses to identify the calcineurin B‐like (CBL) and CBL‐interacting protein kinase (CIPK) network of M. polymorpha. We analysed CBL‐CIPK expression during salt stress, and determined protein–protein interactions using yeast two‐hybrid and bimolecular fluorescence complementation. We also created genetic knockouts using CRISPR/Cas9. We confirm that M. polymorpha has two CIPKs and three CBLs. Both CIPKs and one CBL show pronounced salt‐responsive transcriptional changes. All M. polymorpha CBL‐CIPKs interact with each other in planta. Knocking out CIPK‐B causes increased sensitivity to salt, suggesting that this CIPK is involved in salt signalling. We have identified CBL‐CIPKs that form part of a salt tolerance pathway in M. polymorpha. Phylogeny and interaction studies imply that these CBL‐CIPKs form an evolutionarily conserved salt overly sensitive pathway. Hence, salt responses may be some of the early functions of CBL‐CIPK networks and increased abiotic stress tolerance required for land plant emergence.

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Loss-of-function mutations in the CYP24A1 protein-coding region causing reduced 25 hydroxyvitamin D (25OHD) and 1,25 dihydroxyvitamin D (1,25(OH) 2 D) catabolism have been observed in some cases of infantile hypercalcemia type 1 (HCINF1), which can manifest as nephrocalcinosis, hypercalcemia and adult-onset hypercalciuria, and renal stone formation. Some cases present with apparent CYP24A1 phenotypes but do not exhibit pathogenic mutations. Here, we assessed the molecular mechanisms driving apparent HCINF1 where there was a lack of CYP24A1 mutation. We obtained blood samples from 47 patients with either a single abnormality of no obvious cause or a combination of hypercalcemia, hypercalciuria, and nephrolithiasis as part of our metabolic and stone clinics. We used liquid chromatography tandem mass spectrometry (LC-MS/MS) to determine serum vitamin D metabolites and direct sequencing to confirm CYP24A1 genotype. Six patients presented with profiles characteristic of altered CYP24A1 function but lacked protein-coding mutations in CYP24A1. Analysis upstream and downstream of the coding sequence showed single nucleotide variants (SNVs) in the CYP24A1 3 0 untranslated region (UTR). Bioinformatics approaches revealed that these 3 0 UTR abnormalities did not result in microRNA silencing but altered the CYP24A1 messenger RNA (mRNA) secondary structure, which negatively impacted translation. Our experiments showed that mRNA misfolding driven by these 3 0 UTR sequence-dependent structural elements was associated with normal 25OHD but abnormal 1,25(OH) 2 D catabolism. Using CRISPR-Cas9 gene editing, we developed an in vitro mutant model for future CYP24A1 studies. Our results form a basis for future studies investigating structure-function relationships and novel CYP24A1 mutations producing a semifunctional protein.