Fig. 4
Metabolism of exogenous GABA in plants. The decay (metabolism) of D6-GABA in citrus leaves presented in concentration and log of the concentration versus time (a). Leaves were incubated in 10 mM D6-GABA for 3 h, washed with water, and then transferred to distilled water for 0, 1, 2, 3, 4, and 5 h. Effect of the exogenous D6-GABA application on the relative gene expression of several citrus genes involved in GABA biosynthesis and catabolism (b). Leaf samples, for RNA extraction, were collected at 6 h post treatment (dpt) with or without GABA [mock (0 mM vs.10 mM GABA]. Bars represents the relative increase in the gene expression level in GABA-treated plants relative to the control, while the error bars represent standard deviation (SDs). Gene expressions were normalized using five housekeeping genes including; elongation factor 1-alpha (EF1), F-box/kelch-repeat protein (F-box), glyceraldehyde-3-phosphate dehydrogenase GAPC1, cytosolic (GAPC1, also known as GAPDH), and SAND family protein (SAND), which previously showed high stability for transcript normalization in citrus under biotic stress [19,20,21]. The changes in the gene expression levels were analyzed with the 2−ΔΔCT method. Samples were analyzed in triplicate for each biological replicate (n = 5). The full list of expressed genes, names, accession numbers, and primers are available in Additional file 1: Table S1. Schematic diagram of the metabolism of exogenous D6-GABA in citrus plants. Metabolism of D6-GABA to D4-succinic acid and derivatization of D4-succinic with methyl chloroformate (c)