Table I. Amino acid exchanges in PPCK isoforms occurring among C₃ and C₄ Flaveria spp. compared with C₃ and C₄ species from Brassicales and Poaceae.

Amino acids are listed using standard single-letter code, with positions marked according to the F. pringlei PPCKA sequence. An alignment of all sequences is presented in Supplemental File S3. Boldface letters indicate amino acid exchanges found in some intermediate species and species with C₄; italic letters indicate amino acid exchanges found in all intermediate species and species with C₄; underlined letters indicate amino acid exchanges found only in species with C₄ photosynthesis. Species indicated with single asterisks perform C₃ photosynthesis; those indicated with double asterisks perform C₄ photosynthesis; all others are C₃-C₄ intermediates and the C₄-like F. brownii (Fbr). Dashes indicate that sequence information is missing or a residue is not present. Species abbreviations are as follows: Arabidopsis (At), C. gynandra (Cg), maize (Zm), rice (Os), S. italica (Si), and sorghum (Sb). The accession numbers for the sequences are listed in Supplemental File S2, except for C. gynandra; the sequence for C. gynandra is included at the end of Supplemental File S1.

Species		Residue Position Based on FpPPCKA Protein Sequence
	PPCK	4	49	80	87	109	123	135	147	160	163	165	185	211	217	233	257	270	273
Flaveria spp.
Fp*	A*	A	T	E	I	T	S	I	S	G	E	G	R	Y	E	L	L	S	E
Fro*	A*	A	T	E	I	A	S	I	S	G	E	G	R	Y	E	L	L	S	E
Fc	A	A	T	D	I	T	S	I	S	A	E	R	M	Y	E	L	F	S	E
Fa	A	A	T	D	I	T	S	I	S	A	D	R	M	Y	E	L	F	S	E
Fpu	A	A	T	D	I	T	S	I	S	A	E	R	M	Y	E	L	F	S	E
Fra	A	A	T	D	I	T	S	I	S	A	D	R	M	Y	E	L	F	N	E
Fbr	A	A	T	D	I	T	S	I	S	A	E	R	M	Y	E	L	F	S	E
Fb**	A**	T	T	D	I	T	S	L	G	A	D	R	M	H	D	L	F	N	K
Ft**	A**	T	T	D	I	T	S	L	G	A	D	R	M	H	D	L	F	N	K
Fp*	B*	V	T	E	I	S	S	I	S	A	E	G	R	Y	D	I	L	S	E
Fro*	B*	V	T	E	I	S	S	I	N	A	E	G	R	Y	D	I	L	S	E
Fc	B	–	T	E	I	S	S	I	S	A	E	G	R	Y	D	M	L	S	E
Fpu	B	–	T	E	I	S	S	I	S	A	E	G	R	Y	D	M	L	S	E
Fra	B	–	T	E	M	A	S	I	S	A	E	G	R	Y	D	I	L	S	E
Fbr	B	V	T	E	I	S	S	I	S	A	E	G	R	Y	D	M	L	S	E
Fb**	B**	V	I	E	M	A	G	I	S	G	E	G	R	Y	D	M	L	S	E
Ft**	B**	–	I	E	M	A	G	I	S	A	E	G	R	Y	D	M	L	S	E
Brassicales
At*	1*	T	L	D	M	P	S	I	T	G	–	E	Y	Y	E	I	F	R	E
Cg**		T	L	N	M	A	S	I	V	G	–	E	R	Y	E	I	F	N	A
At*	2*	E	L	E	M	S	A	V	G	G	–	E	R	N	D	K	F	N	N
Poaceae
Os*	1*	A	L	D	V	H	A	V	V	G	–	R	R	Y	E	A	F	S	G
Zm**	1**	A	L	A	L	P	A	V	A	G	–	R	R	S	E	A	L	T	G
Sb**	1**	A	L	A	L	P	A	V	A	G	–	R	R	S	D	A	L	T	G
Si**	1**	A	L	A	L	P	A	V	A	G	–	R	R	G	E	A	F	T	G
Bd*	1*	E	L	E	M	P	A	V	R	G	–	G	G	S	D	L	F	N	G
Bd*	2*	E	L	E	M	P	A	V	R	G	–	G	G	S	D	L	F	N	G
Os*	3*	E	L	D	M	P	A	V	R	G	–	I	G	G	D	L	F	S	G
Sb**	3**	–	–	D	M	V	A	V	G	G	G	R	G	–	D	–	–	S	G
Zm**	4**	Q	L	D	M	P	A	V	R	G	A	G	G	G	D	L	F	S	G
Si**	3**	Q	L	D	M	P	A	V	R	G	G	G	G	G	E	L	F	S	G
Os*	2*	E	L	D	M	P	A	V	R	G	–	G	G	G	E	L	F	S	G
Bd*		E	L	E	M	P	A	V	R	G	E	G	G	G	E	L	F	S	G
Sb**	2**	E	L	E	M	P	A	V	R	G	G	G	G	G	E	L	F	S	G
Zm**	3**	E	L	E	M	P	A	V	R	G	G	G	G	G	E	L	F	S	G
Zm**	2**	E	L	E	I	P	A	V	R	G	G	G	G	G	E	L	F	S	G
Si**	2**	E	L	E	M	P	A	V	R	G	G	G	G	G	E	L	F	S	G