The C-Terminal Domains SnRK2 Box and ABA Box Have a Role in Sugarcane SnRK2s Auto-Activation and Activity

Resistance to drought stress is fundamental to plant survival and development. Abscisic acid (ABA) is one of the major hormones involved in different types of abiotic and biotic stress responses. ABA intracellular signaling has been extensively explored in Arabidopsis thaliana and occurs via a phosphorylation cascade mediated by three related protein kinases, denominated SnRK2s (SNF1-related protein kinases). However, the role of ABA signaling and the biochemistry of SnRK2 in crop plants remains underexplored. Considering the importance of the ABA hormone in abiotic stress tolerance, here we investigated the regulatory mechanism of sugarcane SnRK2s—known as stress/ABA-activated protein kinases (SAPKs). The crystal structure of ScSAPK10 revealed the characteristic SnRK2 family architecture, in which the regulatory SnRK2 box interacts with the kinase domain αC helix. To study sugarcane SnRK2 regulation, we produced a series of mutants for the protein regulatory domains SnRK2 box and ABA box. Mutations in ScSAPK8 SnRK2 box aimed at perturbing its interaction with the protein kinase domain reduced protein kinase activity in vitro. On the other hand, mutations to ScSAPK ABA box did not impact protein kinase activity but did alter the protein autophosphorylation pattern. Taken together, our results demonstrate that both SnRK2 and ABA boxes might play a role in sugarcane SnRK2 function.


Supplementary
: Unrooted phylogenetic tree of sugarcane, maize and Arabidopsis SnRK2s inferred by Maximum Likelihood. The tree is drawn to scale, with branch lengths representing the number of substitutions per site. Bootstrapping analysis was performed 1000 times, and the values (in %) are shown at each node. Values above 80% indicate branches are well supported by our phylogenetic reconstruction. Branches corresponding to the conserved monocotyledons SnRK2 proteins are colored in green. Figure S2: Schematic representation of the full-length ScSAPK8, ScSAPK9, and ScSAPK10. Each protein has an N-terminal kinase domain and a Cterminal region containing the regulatory domains SnRK2-box and ABA-box. Numbers represent the amino acid positions of each protein domain. Figure S3: Enzymatic activity of ScSAPK8 WT under variable enzyme concentration and after ATP pre-incubation. The data show the quantity of phosphorylated peptide produced after 1 hour, measured by the ratio of fluorescence intensity at 665 nm (streptavidin-XL665 emission excited by phospho-specific Eucryptate conjugated antibody) and 620 nm (Eu-cryptate emission). The ScSAPK8 activity was higher with ATP pre-incubation and also increased in an enzyme concentrationdependent manner. Figure S4: Coomassie-stained SDS-PAGE of ScSAPK8 WT and mutants. The image represents all the proteins after affinity purification and dilution to 20 μM final concentration. The protein concentration was estimated by the Bradford method (Sigma-Aldrich) before gel loading. Figure S5: ScSAPK8 WT autophosphorylation. A: Deconvoluted mass spectrum at time 0, 1 hour, 5 hours and overnight, generated by MAX ENT1 software (Waters). The blue arrow represents the intact kinase mass, and the blue stars represent the detected masses with one or more phosphorylations. The green arrows indicate the exact kinase mass with the loss of methionine that could occur during protein expression. The green stars represent masses of this kinase form with one or more phosphorylations. B: Graphical representation of protein autophosphorylation over time.

Supplementary
The percentage values were calculated using the ion counts extracted from the deconvoluted spectrum. Figure S6: ScSAPK8-M312A autophosphorylation. A: Deconvoluted mass spectrum at time 0, 1 hour, 5 hours and overnight, generated by MAX ENT1 software (Waters). The blue arrow represents the intact kinase mass, and the blue stars represent the detected masses with one or more phosphorylations. The green arrows indicate the exact kinase mass with the loss of methionine that could occur during protein expression. The green stars represent masses of this kinase form with one or more phosphorylations. B: Graphical representation of protein autophosphorylation over time.

Supplementary
The percentage values were calculated using the ion counts extracted from the deconvoluted spectrum. Figure S7: ScSAPK8-I315A autophosphorylation. A: Deconvoluted mass spectrum at time 0, 1 hour, 5 hours and overnight, generated by MAX ENT1 software (Waters). The blue arrow represents the intact kinase mass, and the blue stars represent the detected masses with one or more phosphorylations. The green arrows indicate the exact kinase mass with the loss of methionine that could occur during protein expression. The green stars represent masses of this kinase form with one or more phosphorylations. B: Graphical representation of protein autophosphorylation over time.

Supplementary
The percentage values were calculated using the ion counts extracted from the deconvoluted spectrum. Figure S8: ScSAPK8-L319A autophosphorylation. A: Deconvoluted mass spectrum at time 0, 1 hour, 5 hours and overnight, generated by MAX ENT1 software (Waters). The blue arrow represents the intact kinase mass, and the blue stars represent the detected masses with one or more phosphorylations. The green arrows indicate the exact kinase mass with the loss of methionine that could occur during protein expression. The green stars represent masses of this kinase form with one or more phosphorylations. B: Graphical representation of protein autophosphorylation over time.

Supplementary
The percentage values were calculated using the ion counts extracted from the deconvoluted spectrum.

Supplementary Figure S9: ScSAPK8-∆ABA-box autophosphorylation. A:
Deconvoluted mass spectrum at time 0, 1 hour, 5 hours and overnight, generated by MAX ENT1 software (Waters). The blue arrow represents the intact kinase mass, and the blue stars represent the detected masses with one or more phosphorylations. B: Graphical representation of protein autophosphorylation over time. The percentage values were calculated the ion counts extracted from the deconvoluted spectrum.

Supplementary Figure S10: ScSAPK8-ABAbox-group1 autophosphorylation. A:
Deconvoluted mass spectrum at time 0, 1 hour, 5 hours and overnight, generated by MAX ENT1 software (Waters). The blue arrow represents the intact kinase mass, and the blue stars represent the detected masses with one or more phosphorylations. B: Graphical representation of protein autophosphorylation over time. The percentage values were calculated the ion counts extracted from the deconvoluted spectrum.

Supplementary Figure S11: ScSAPK8-ABAbox-group2 autophosphorylation. A:
Deconvoluted mass spectrum at time 0, 1 hour, 5 hours and overnight, generated by MAX ENT1 software (Waters). The blue arrow represents the intact kinase mass, and the blue stars represent the detected masses with one or more phosphorylations. B: Graphical representation of protein autophosphorylation over time. The percentage values were calculated the ion counts extracted from the deconvoluted spectrum. Figure S12: ScSAPK8-ABAbox-group3 autophosphorylation. A: Deconvoluted mass spectrum at time 0, 1 hour, 5 hours and overnight, generated by MAX ENT1 software (Waters). The blue arrow represents the intact kinase mass, and the blue stars represent the detected masses with one or more phosphorylations. B: Graphical representation of protein autophosphorylation over time. The percentage values were calculated the ion counts extracted from the deconvoluted spectrum. Figure S14: ScSAPK10 WT and mutant ScSAPK10 ∆N-term ∆ABA-box autophosphorylation. Deconvoluted mass spectrum at time 1 hour generated by MAX ENT1 software (Waters). The blue arrow represents the intact kinase mass, and the blue stars represent the detected masses with one or more phosphorylations.