Table I. ALD1 catalyzes the transamination of various amino acids other than l-Lys in vitro but these ALD1-mediated reactions are not detected in planta.

The in vitro activities of the ALD1 aminotransferase toward the indicated amino acids were assessed by incubation of ALD1 protein with the amino acid substrate and pyruvate as an acceptor oxoacid for 30 min. The molar concentrations of the product amino acid Ala and the given substrate amino acid were determined by GC-MS, and the percentage of transamination was calculated by forming the quotient [Ala]/([substrate amino acid] + [Ala]). The percentage values represent means ± sd of the values from three replicate assays. Relative in vitro activities were calculated from the transamination percentages by relating the mean transamination values to the one determined for l-Lys. The in vitro formation of product oxoacids or oxoacid-derived compounds was determined by the indicated GC-MS procedure. Retention times, molecular ions, and specific ion fragments in the mass spectra of the derivatized products are indicated. Underlined m/z values represent dominant masses in mass spectra, and ion chromatograms for the m/z values indicated in boldface have been used for substance quantification in planta. The molecular structures of several of the nonderivatized and derivatized products and the mass spectra of the latter are given in Figures 1C, 3C, and 5, C and D, as well as in Supplemental Figure S8. The detectability of oxoacid products in leaf extracts from Psm-inoculated or mock-treated Col-0 and ald1 plants (48 hpi) is indicated. The level of a detected product in leaf tissue is given in ng g⁻¹ fresh weight (means ± sd; n = 3); nd, not detected. Asterisks denote statistically significant differences between Psm- and mock-treated samples of a genotype (P < 0.05, two-tailed Student’s t test). The results for additional tested amino acid substrates are summarized in Supplemental Table S1.

Amino Acid	ALD1 in Vitro Assay					Chemical Analyses					In Planta Analyses
	Relative in Vitro Activity	Percentage Transamination (30 min)	Possible Product Oxoacid (or Derived Product)	Compound	In Vitro Detection of Product	GC-MS Procedure	Compound	M+	Retention Time	Ion Fragments	In Planta Detection	Col-0 Mock	Col-0 Psm	ald1 Mock	ald1 Psm
	%							m/z	min	m/z
l-Lys	100	77.75 ± 6.58	2,3-DP	1	Yes	A	1a	141	11.6	141, 126, 108	Yes	41 ± 7	1,557 ± 240*	39 ± 4	47 ± 9
						B	1b	255	13.8	255, 169, 127	Yes	75 ± 25	1,253 ± 119*	79 ± 28	104 ± 12
d-Lys	0.1	0.05 ± 0.01	2,3-DP	1	No	A, B		–	–	–	–	–	–	–	–
l,l-DAP	16.3	12.64 ± 1.19	6-Carboxy-2,3-DP	2	Yes	A	2a	199	17.3	199, 140, 108	No	nd	nd	nd	nd
meso-DAP	12.9	10.01 ± 1.76			Yes	B	2b	341	17.3	239, 168, 108	No	nd	nd	nd	nd
l-Orn	52.9	36.31 ± 8.89	Δ2-Pyr2C	3	Yes	B	3b	241	13.0	241, 155, 113	No	nd	nd	nd	nd
2,4-DABA	2.0	1.58 ± 0.21	4-Amino-2-oxo-butyric acid		No	B		–	–	–	–	–	–	–	–
l-Met	78.5	61.04 ± 7.41	α-Keto-Met	4	Yes	A	4a	162	12.0	162, 103, 61	No	nd	nd	nd	nd
l-Leu	57.0	44.34 ± 1.53	α-Keto-Leu	5	Yes	A	5a	144	6.9	144,102, 85	No	nd	nd	nd	nd
l-Ile	0.5	0.36 ± 0.04	α-Keto-Ile		No	A		144	6.8	144, 85, 57	No	nd	nd	nd	nd
l-Val	0.3	0.23 ± 0.02	α-Keto-Val		Yes	A		130	4.9	130, 71, 59	No	nd	nd	nd	nd
l-Phe	10.6	8.25 ± 0.42	Phenylpyruvic acid	6	Yes	A	6a	178	14.2	178, 119, 91	Yes	30 ± 8	79 ± 22*	37 ± 6	87 ± 23*
l-Trp	7.6	6.09 ± 0.63	Indole-3-pyruvic acid	7	Yes	A	7a	217	23.3	217, 130, 77	No	nd	nd	nd	nd
l-Glu	12.4	9.61 ± 0.96	α-Ketoglutaric acid		Yes	A		174	12.2	143, 115, 87	Yes	5,981 ± 817	8,826 ± 1,064*	6,507 ± 725	7,806 ± 784
l-Aad	11.9	9.23 ± 1.68	α-Ketoadipic acid	8	Yes	A	8a	188	13.9	157, 129, 101	Yes	86 ± 15	147 ± 15*	91 ± 5	181 ± 39*
l-Asn	6.1	4.72 ± 0.21	2-Oxosuccinamic acid		Yes	A		145	15.0	144, 115, 69	No	nd	nd	nd	nd