A striking paradox is that genes with conserved protein sequence, function and expression pattern over deep time often exhibit extremely divergent cis-regulatory sequences. It remains unclear how ...such drastic cis-regulatory evolution across species allows preservation of gene function, and to what extent these differences influence how cis-regulatory variation arising within species impacts phenotypic change. Here, we investigated these questions using a plant stem cell regulator conserved in expression pattern and function over ~125 million years. Using in-vivo genome editing in two distantly related models, Arabidopsis thaliana (Arabidopsis) and Solanum lycopersicum (tomato), we generated over 70 deletion alleles in the upstream and downstream regions of the stem cell repressor gene CLAVATA3 (CLV3) and compared their individual and combined effects on a shared phenotype, the number of carpels that make fruits. We found that sequences upstream of tomato CLV3 are highly sensitive to even small perturbations compared to its downstream region. In contrast, Arabidopsis CLV3 function is tolerant to severe disruptions both upstream and downstream of the coding sequence. Combining upstream and downstream deletions also revealed a different regulatory outcome. Whereas phenotypic enhancement from adding downstream mutations was predominantly weak and additive in tomato, mutating both regions of Arabidopsis CLV3 caused substantial and synergistic effects, demonstrating distinct distribution and redundancy of functional cis-regulatory sequences. Our results demonstrate remarkable malleability in cis-regulatory structural organization of a deeply conserved plant stem cell regulator and suggest that major reconfiguration of cis-regulatory sequence space is a common yet cryptic evolutionary force altering genotype-to-phenotype relationships from regulatory variation in conserved genes. Finally, our findings underscore the need for lineage-specific dissection of the spatial architecture of cis-regulation to effectively engineer trait variation from conserved productivity genes in crops.
Calcium (Ca
)-dependent protein kinases (CDPKs or CPKs) are a unique family of Ca
sensor/kinase-effector proteins with diverse functions in plants. In
, CPK28 contributes to immune homeostasis by ...promoting degradation of the key immune signaling receptor-like cytoplasmic kinase BOTRYTIS-INDUCED KINASE 1 (BIK1) and additionally functions in vegetative-to-reproductive stage transition. How CPK28 controls these seemingly disparate pathways is unknown. Here, we identify a single phosphorylation site in the kinase domain of CPK28 (Ser318) that is differentially required for its function in immune homeostasis and stem elongation. We show that CPK28 undergoes intermolecular autophosphorylation on Ser318 and can additionally be transphosphorylated on this residue by BIK1. Analysis of several other phosphorylation sites demonstrates that Ser318 phosphorylation is uniquely required to prime CPK28 for Ca
activation at physiological concentrations of Ca
, possibly through stabilization of the Ca
-bound active state as indicated by intrinsic fluorescence experiments. Together, our data indicate that phosphorylation of Ser318 is required for the activation of CPK28 at low intracellular Ca
to prevent initiation of an immune response in the absence of infection. By comparison, phosphorylation of Ser318 is not required for stem elongation, indicating pathway-specific requirements for phosphorylation-based Ca
-sensitivity priming. We additionally provide evidence for a conserved function for Ser318 phosphorylation in related group IV CDPKs, which holds promise for biotechnological applications by generating CDPK alleles that enhance resistance to microbial pathogens without consequences to yield.
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