The Salt Overly Sensitive (SOS) pathway plays an important role in the regulation of Na + /K + ion homeostasis and salt tolerance in Arabidopsis thaliana. Previously, we reported that the calcium binding proteins SOS3 and SOS3-LIKE CALCIUM BINDING PROTEIN8 (SCaBP8) nonredundantly activate the protein kinase SOS2. Here, we show that SOS2 phosphorylates SCaBP8 at its C terminus but does not phosphorylate SOS3. In vitro, SOS2 phosphorylation of SCaBP8 was enhanced by the bimolecular interaction of SOS2 and SCaBP8 and did not require calcium ions. In vivo, this phosphorylation was induced by salt stress, occurred at the membrane, stabilized the SCaBP8-SOS2 interaction, and enhanced plasma membrane Na + /H + exchange activity. When a Ser at position 237 in the SCaBP8 protein (the SOS2 phosphorylation target) was mutated to Ala, SCaBP8 was no longer phosphorylated by SOS2 and the mutant protein could not fully rescue the salt-sensitive phenotype of the scabp8 mutant. By contrast, when Ser-237 was mutated to Asp to mimic the charge of a phosphorylated Ser residue, the mutant protein rescued the scabp8 salt sensitivity. These data demonstrate that calcium sensor phosphorylation is a critical component of SOS pathway regulation of salt tolerance in Arabidopsis.

Background: Biosynthetic gene clusters are unusual in plants, yet may provide insight into the associated evolution of secondary metabolism. Results: The cytochromes P450 CYP76M5-8, found in one such cluster, have multiple functions in rice diterpenoid metabolism. Conclusion: Plant biosynthetic gene clusters can encode metabolic plasticity. Significance: Such plasticity may enable further evolution, and be a broader feature of plant secondary metabolism.

All higher plants contain an ent-kaurene oxidase (KO), as such a cytochrome P450 (CYP) 701 family member is required for gibberellin (GA) phytohormone biosynthesis. While gene expansion and functional diversification of GA-biosynthesis-derived diterpene synthases into more specialized metabolism has been demonstrated, no functionally divergent KO/CYP701 homologs have been previously identified. Rice (Oryza sativa) contains five CYP701A subfamily members in its genome, despite the fact that only one (OsKO2/CYP701A6) is required for GA biosynthesis. Here we demonstrate that one of the other rice CYP701A subfamily members, OsKOL4/CYP701A8, does not catalyze the prototypical conversion of the ent-kaurene C4a-methyl to a carboxylic acid, but instead carries out hydroxylation at the nearby C3a position in a number of related diterpenes. In particular, under conditions where OsKO2 catalyzes the expected conversion of ent-kaurene to ent-kaurenoic acid required for GA biosynthesis, OsKOL4 instead efficiently reacts with ent-sandaracopimaradiene and ent-cassadiene to produce the corresponding C3a-hydroxylated diterpenoids. These compounds are expected intermediates in biosynthesis of the oryzalexin and phytocassane families of rice antifungal phytoalexins, respectively, and can be detected in rice plants under the appropriate conditions. Thus, it appears that OsKOL4 plays a role in the more specialized diterpenoid metabolism of rice, and our results provide evidence for divergence of a KO/CYP701 family member from GA biosynthesis. This further expands the range of enzymes recruited from the ancestral GA primary pathway to the more complex and specialized labdane-related diterpenoid metabolic network found in rice.

The Arabidopsis (Arabidopsis thaliana) genome encodes nine Salt Overly Sensitive3 (SOS3)-like calcium-binding proteins (SCaBPs; also named calcineurin B-like protein [CBL]) and 24 SOS2-like protein kinases (PKSs; also named as CBL-interacting protein kinases [CIPKs]). A general regulatory mechanism between these two families is that SCaBP calcium sensors activate PKS kinases by interacting with their FISL motif. In this study, we demonstrated that phosphorylation of SCaBPs by their functional interacting PKSs is another common regulatory mechanism. The phosphorylation site serine-216 at the C terminus of SCaBP1 by PKS24 was identified by liquid chromatography-quadrupole mass spectrometry analysis. This serine residue is conserved within the PFPF motif at the C terminus of SCaBP proteins. Phosphorylation of this site of SCaBP8 by SOS2 has been determined previously. We further showed that CIPK23/PKS17 phosphorylated CBL1/SCaBP5 and CBL9/SCaBP7 and PKS5 phosphorylated SCaBP1 at the same site in vitro and in vivo. Furthermore, the phosphorylation stabilized the interaction between SCaBP and PKS proteins. This tight interaction neutralized the inhibitory effect of PKS5 on plasma membrane H + -ATPase activity. These data indicate that SCaBP phosphorylation by their interacting PKS kinases is a critical component of the SCaBP-PKS regulatory pathway in Arabidopsis.