Legends for Supporting Information
Figure S1 Kin displays no -subunit functionality. Heterologous expression in the yeast
gal83 sip1 sip2 mutant of Arabidopsis KIN and yeast GAL83 as a positive control.
Cells were plated on minimal medium (-uracil) with glucose or glycerol/ethanol as the only
carbon source. Pictures were taken after 3 days.
Figure S2 Maximum likelihood phylogeny of SNF4/AMPKγ and plant KINβγ FCD-Ia genes.
The number next to each node corresponds to non-parametric bootstrap support values for the
corresponding clades.
Figure S3 Alignment of the FCD of hybrid  proteins from 5 different plant species .
Figure S4 Alignment the FCD of hybrid proteins (FCD-Ia) from 5 different plant species
together with the human AMPK and yeast SNF4 protein sequences, highlighting
conservation of the CBS motifs.
Figure S5 Alignment of the non-canonical -like FCD proteins with AMPK and SNF4
protein sequences.
Figure S6 Alignment of hybrid proteins (FCD-Ia) from 5 different plant species with the
non-canonical -like FCD proteins to identify conserved amino acids in the FCD-Ia proteins.
Figure S7 in silico analysis of nucleotide binding using docking of AMP in the crystallized
structure of AMPKγ1 and in optimized homology models of KINβγ and KINγ (A) (B) Glide
scores for AMP binding in the AMP1 and AMP2 sites of wild type and in silico mutated
AMPKγ1 and KINβγ. AMPK1 amino acids R69, D244, R268, Y240, R69, K169, S241
(AMP1) and T86, T88 and D89 (AMP2) were mutated in silico to Gly residues to disrupt
AMP binding. R69Q, R298G and mutations were reported to affect AMP binding; N255I is a
negative control (Xiao et al., 2007). L54, K57, P72, G83, S225 and Y240 are the 6 amino
acids highly conserved in KIN-like hybrid plant proteins and animal and fungal proteins
with reported -subunit functionality; they were mutated in silico to residues present in noncanonical -subunits. KINβγ amino acids S392, F432, G434, Q435, C437, P456 (AMP1) and
V209, L213, R262, V285, A286 and E374 (AMP2) were identified based on modeled binding
pockets and mutated in silico to Gly or Ala residues. L179, K182, P197, G208, S376 and
Y391 are the corresponding 6 amino acids highly conserved in KIN-like hybrid plant
proteins and animal and fungal proteins with reported -subunit functionality; they were
mutated in silico to residues present in non-canonical -subunits.
Figure S8 The mutant kin line SALK_074554. (A) T-DNA insertion site in exon 4 resulting
in complete knockout at the protein level. (B) No obvious growth defects can be observed.
Figure S9 Wild type response of transient KIN10 over-expression in kin mutant mesophyll
protoplasts. Upscaled experiment for target gene expression analysis by qRT-PCR. CT values
are given for WT (blue) and kin (red) control protoplasts and WT (green) and kin (purple)
protoplasts expressing KIN10.
Figure S10 Expression of Arabidopsis canonical and non-canonical -subunit FCD genes
throughout development using Genevestigator. (A) FCD-Ia (At1g09020/ KIN), (B) FCD-Ib
(At1g69800; At3g48530/KIN), (C) FCD-Ic (At1g80090; At1g15330/PV42a), (D) FCD-II
family genes (At3g52550; At5g63490; At5g50530).
Figure S11 Bayesian phylogenetic tree of the carbohydrate binding domains of β-subunits,
KINβγ, SEX4 and LSF1 homologs based on the sampling and phylogenetic reconstructions of
Janeček et al. (2011). Numbers indicate Bayesian posterior probability.