Optimization and validation of bacterial assay with AtDAO1
To optimize and validate the proposed method, we used E. coli cultures producing recombinant AtDAO1 to test the effect of different experimental conditions on oxIAA production. AtDAO1 catalyzes the oxidation of IAA (Fig. 1b) and this reaction represents the most important mechanism for IAA inactivation in plants [19, 22, 30, 31]. Recombinant AtDAO1 was produced in bacterial cultures as an N-terminally tagged hexahistidine fusion protein and its production was confirmed by Western blot analysis (Additional file 2: Figure S1). In the first experiment, the enzymatic activity of recombinant AtDAO1 was tested by supplementing bacterial cultures producing AtDAO1 with 0.001 mM IAA and measuring the levels of oxIAA in the supernatant and in the pellet fractions separately by UHPLC–ESI-MS/MS. Previous work has shown that the in vitro catalytic activity of recombinant DAO proteins [20, 33] requires, in addition to the substrate IAA, the co-substrate 2-oxoglutarate, and the cofactors (NH4)2Fe(SO4)2 and ascorbate in the reaction mixture. We therefore carried out, the assay by adding IAA to the bacterial cultures with or without a mixture containing co-substrate and cofactors (DAO cofactor mixture). Bacterial cultures producing the GFP protein supplemented with IAA in the presence or absence of the DAO cofactor mixture were used as negative controls. Untreated bacterial cultures were used as mock samples. Bacterial cultures were sampled prior to the treatment with IAA (T0) and after over-night incubation at 20 °C with the substrate, and with or without the DAO cofactor mixture. Under these conditions, the supernatant fraction exhibited a strong accumulation of oxIAA in all the samples compared with the pellet (Fig. 2a). Our analysis shows the concentration of oxIAA to be higher in the culture media of bacterial cells transformed with AtDAO1 that were simultaneously incubated with IAA and DAO cofactor mixture than in the samples taken from the same bacterial culture that was treated with IAA alone (Fig. 2a). However, background levels of oxIAA were observed in the culture media of AtDAO1-producing bacteria that were not treated with IAA (mock), prior treatment (T0) and in all GFP samples (Fig. 2a). Low levels of IAA were detected in culture media before treatment with exogenous IAA (Additional file 4: Figure S2). Moreover, the basal accumulation of oxIAA that we observed in bacterial cultures could have been derived from the spontaneous non-enzymatic conversion of IAA into oxIAA, as has previously been reported in control samples from in vitro AtDAO assays [20]. Our results showed that AtDAO1 activity could be monitored by direct analysis of the levels of oxIAA that accumulate in the supernatant fraction of the AtDAO1-producing bacterial cultures that had been supplemented simultaneously with low levels of exogenous IAA and DAO cofactor mixture. Therefore, in all further tests only the supernatant fractions were analyzed. It should be noted that in case this method will be adopted to test other substrates, rather than IAA, both supernatant and pellet fractions should be first examined, in order to establish where the enzymatic products accumulate the most. In contrast with IAA-producing phytopatogens (e.g., Pseudomonas syringae, Agrobacterium tumefaciens, etc.), E. coli is not capable of synthesizing IAA and does not possess an endogenous IAA-response machinery, making it ideal for hosting and testing the activity of IAA-related enzymes. However, if other molecules will be used as substrate and tested with this method, the presence of an endogenous substrate-associated machinery in E. coli should be verified first. In order to prevent possible bias due to the experimental conditions, as well as those arising from host genetics, negative controls (e.g., GFP-producing bacteria) should always be included in the experiments.
In the following experiment, we investigated the effect of temperature and exposure time to exogenous IAA and the DAO cofactor mixture on AtDAO1-mediated oxIAA production. In previous studies, in vitro DAO assays were performed by incubating the reaction mixture containing the purified recombinant DAOs at 30 °C for 1 h [20, 33]. We therefore carried out an experiment in which AtDAO1- and GFP-producing bacterial cultures were incubated at 20 °C or 30 °C with IAA and in the presence or absence of the DAO cofactor mixture. Samples were taken after 1 or 6 h of treatment with IAA. To reduce the impact of the natural non-enzymatic oxidation of IAA on the overall oxIAA production, bacterial cultures were treated with a higher concentration of IAA (0.01 mM) than that adopted in the initial experiment (0.001 mM). Untreated bacterial cultures were used as mock samples and culture media that had not been inoculated with bacteria (designed ‘no bacteria’) were sampled and analyzed as an additional control to monitor the background oxIAA formation that derives from the naturally occurring oxidation of IAA. We observed a very strong accumulation of oxIAA in AtDAO1 samples taken from cultures that were treated with IAA and the DAO cofactor mixture, compared with AtDAO1 from samples treated with IAA only (Fig. 2b), indicating that the simultaneous supplementation of the DAO cofactor mixture and a higher concentration of IAA significantly enhanced AtDAO1-mediated oxIAA production. This finding was also confirmed when AtDAO1-producing bacterial cultures were incubated with 0.1 mM IAA (Fig. 3a). The concentration of oxIAA was higher in AtDAO1 samples after prolonged treatment (6 h) with substrate and the DAO cofactor mixture, than in samples that had a shorter exposure time (1 h) (Fig. 2b). Furthermore, oxIAA levels were slightly higher in the samples that were incubated at 20 °C compared with samples from cultures kept at 30 °C (Fig. 2b). No differences in oxIAA levels were detected among any GFP samples, any ‘no bacteria’ samples, nor any mock AtDAO1 samples (Fig. 2b), suggesting that low levels of non-enzymatic oxidation of IAA occurs in the culture media. Together, these results suggest that incubation with the substrate and the DAO cofactor mixture for 6 h at 20 °C provides the optimal conditions for accumulating enzymatic product. These experimental settings were therefore adopted to develop the method further in later experiments with other enzymes involved in IAA inactivation; only GFP samples were included in the next experiments as a negative control.
IAA-conjugating activity of AtGH3.6, AtGH3.17, AtUGT84B1 and AtUGT74D1 in bacterial assay
Earlier work has demonstrated that Group II members of the GH3 family convert IAA into IAA-amino acids [9]. Some of the IAA-amino acid conjugates, such as IAAsp and IAGlu, are the most abundant in Arabidopsis [18] and are believed to be irreversible conjugates that cannot be hydrolyzed to form free IAA [12]. Staswick et al. [9] tested the in vitro activity of several recombinant Arabidopsis GH3 proteins and carried out a screening of amino acid preferences using a TLC-based assay by which it was shown that AtGH3.17 prefers Glu over other amino acids, whereas AtGH3.6 exhibits activity with Asp. We therefore examined whether AtGH3.6 and AtGH3.17 could also catalyze the same reaction (Fig. 1b) directly in bacterial cultures that produce these recombinant proteins. AtGH3.6 and AtGH3.17 genes were produced in E. coli individually and the production of recombinant fusion proteins was confirmed by Western blot analysis (Additional file 2: Figure S1). Cultures of AtGH3.6- and AtGH3.17-producing bacteria were tested in a reaction with or without 0.1 mM IAA in combination with the GH3 cofactor mixture (containing as co-substrates, aspartic and glutamic acid, and as cofactors, ATP and MgCl2) to determine whether IAAsp and/or IAGlu were formed. Figure 3b shows that the reaction mediated by AtGH3.6 yielded the accumulation of both IAA-amino acid conjugates, IAAsp and IAGlu, which is consistent with previous results reported by Staswick et al. [9]. Interestingly, AtGH3.6 preferred Glu over Asp since IAGlu concentration was higher than IAAsp under our experimental conditions (Fig. 3b). Bacterial cultures producing AtGH3.17 that were treated with IAA and the GH3 cofactor mixture accumulated very high levels of IAGlu while almost no IAAsp was detected (Fig. 3c), confirming that AtGH3.17 favors Glu over Asp as reported by Staswick et al. [9]. Accumulation of IAAsp and IAGlu was always below the limits of detection in mock samples taken from AtGH3.6- and AtGH3.17-producing bacterial cultures (Fig. 3b, c).
Glucosylation is another important mechanism of IAA conjugation that is implicated in the inactivation of IAA in plants. Members of the UDP-glucosyl transferase (UGT) super family can catalyze glucosylation of plant hormones, including IAA, using UDP-glucose as a co-substrate. Previous studies have demonstrated that two AtUGTs, AtUGT84B1 and AtUGT74D1, can convert IAA to IAGlc in vitro (Fig. 1b) [14, 15]. To investigate if AtUGT84B1 and AtUGT74D1 could catalyze the glucosylation of IAA in our experimental conditions, we expressed these two genes in E. coli individually and confirmed the production of recombinant fusion proteins by Western blot analysis (Additional file 2: Figure S1). Next, we treated bacterial cultures with or without 0.1 mM IAA in combination with the UGT cofactor mixture (containing as co-substrate, UDPG, and as cofactors, MgSO4 and KCl) and measured IAGlc levels. IAGlc accumulated in samples taken from cultures of bacteria transformed with either AtUGT84B1 or AtUGT74D1 that were treated with 0.1 mM IAA and UGT cofactor mixture, while IAGlc levels were below the limits of detection in samples taken from the same bacterial cultures that were not treated with IAA and UGT cofactor mixture (Fig. 3d, e). IAAsp, IAGlu and IAGlc levels were below the limits of detection in all GFP samples treated with IAA and cofactor mixtures, and in all mock GFP samples (Fig. 3f). Taken together, these results demonstrate that AtGH3 and AtUGT proteins heterologously produced in E. coli cells can efficiently conjugate IAA with amino acids and sugar, respectively, by performing the enzymatic assay with IAA and specific cofactor mixtures directly in bacterial cultures.
OxIAA-conjugating activity of AtUGT84B1 and AtUGT74D1 and competition assay
Conjugation of oxIAA with glucose to form oxIAGlc is an additional and relevant metabolic step in the oxIAA pathway. oxIAGlc, together with oxIAA, are the major degradation products of IAA in Arabidopsis [12, 18, 19], suggesting that this reaction also contributes to IAA homeostasis. Previous studies have demonstrated that recombinant AtUGT74D1 protein converts not only IAA to IAGlc [15], but also oxIAA to oxIAGlc (Fig. 1b) [16]. To verify whether recombinant AtUGT proteins exhibit glucosyl transferase activity towards oxIAA under our experimental conditions, we treated AtUGT84B1- and AtUGT74D1-producing bacterial cultures with 0.1 mM oxIAA in combination with the UGT cofactor mixture. oxIAGlc was detected in cultures that produced AtUGT84B1 and AtUGT74D1, after treatment with oxIAA (Fig. 4a, b), indicating that these two AtUGTs can catalyze the glucosylation of oxIAA. To determine the substrate preference of these two AtUGTs, we performed a competition experiment in which cultures of AtUGT84B1- and AtUGT74D1-producing bacteria were treated simultaneously with the same concentration of IAA and oxIAA and with the UGT cofactor mixture. When IAA and oxIAA competed with each other, IAA was found to be a better substrate as IAGlc levels were much higher than oxIAGlc in samples from both bacterial cultures expressing either of the two AtUGTs (Fig. 4a, b). IAGlc and oxIAGlc were always below the limits of detection in GFP samples as well as in both AtUGTs mock samples (Fig. 4). Our finding is in contrast to that reported by Tanaka et al. [16]; they showed that recombinant AtUGT74D1 had a higher specificity towards oxIAA than IAA in vitro. The discrepancy between these findings could be due to the different experimental conditions that were adopted to study the enzymatic activity.
Taken together, these results provide evidence that our method is suitable for testing different substrates by supplementing the compounds of interest, individually or simultaneously, directly in the bacterial cultures producing the recombinant protein. In the present study, we were also able to show that AtUGT84B1 can catalyze not only the glucosylation of IAA but also of oxIAA to their corresponding glucosides. To the best of our knowledge, this is the first report in which AtUGT84B1 glucosyl transferase activity has been tested and demonstrated toward oxIAA.