2 g in 100 mL of 2 N HCl). The mixtures were incubated for 30 min at 30 °C, after which 2 mL of 2 N NaOH was added. The A540 nm was measured.
ACCD activity was evaluated quantitatively by measuring the amount of α-ketobutyrate produced by the deamination of ACC. ACCD activity was expressed in μmol of α-ketobutyrate mg−1 protein h−1 . Protein Selleckchem LGK974 concentrations were determined using the BioRad (Promega) reagent. Three independent replicate flasks were analyzed. The experiment was repeated three times. To evaluate ACCD activity and expression in mycelia induced by plant interaction, 1 g sterile cucumber root tissue was added to fungus cultures (previously grown in rich SM) in SM with no ammonium or carbon sources. Canola (Brassica napus cv. SARI) seeds (1 g=200 seeds) were surface sterilized (10 min in 1.5% sodium hypochlorite) and incubated for 1 h at room temperature either in sterile 0.03 M MgSO4 as a blank control, in Trichoderma fungal spores (109 g per seeds) or in a bacterial suspension of P. putida UW4 or E. coli tac∷Tas-acdS, at OD600 nm=0.5. The E. coli ACCD overexpressors were tested with and without isopropyl-β-d-1-thiogalactopyranoside (IPTG) induction of the transcription of the tac promoter. Selleck Alectinib Six seeds were placed in each seed-pack growth pouch (125 × 157 mm; Mega International) filled with 12 mL of distilled water. Ten replicate pouches were used
for each treatment. The assay was repeated three independent times. The pouches were incubated upright in a plastic tray partially filled with water at 25 °C in a growth chamber with a 12-h photoperiod and a light intensity of 12.9 μmol m−2 s−1. After 4–5 days, the seedling root length was measured. Root colonization assays were performed according to Viterbo et al.(2005). Briefly, at the end of pouch assay experiments, canola roots were sterilized in 1% NaOCl for 2 min, washed with sterile-distilled water, weighed and homogenized using an ULTRA-TURRAX apparatus (Janke & Kunkel) in 20 mL of water for 1 min. Serial dilutions were plated for CFU counts on Trichoderma selective medium
(Vargas Gil et al., 2009) at 28 °C. jmp8 software (SAS Thalidomide Institute Inc., Cary, NC) was used for statistical analyses. Data were analyzed using one-way anova. Mean comparisons were made using the Tukey–Kramer honestly significant difference multiple range test at P<0.05. Degenerate primers designed according to conserved amino acid sequences (VQEHWVDW and AFITDPVYEG) of fungal ACCDs (see Material and methods) enabled isolation of a 650-bp DNA fragment. Segments 750-bp and 250-bp long, of the upstream and downstream regions, respectively, were obtained by nested PCR amplification with specific primers according to the Genome Walker procedure (Viterbo et al., 2002). The Tas-acdS ORF encodes a 348-amino acid protein with an expected molecular mass of 37 kDa. blast search shows extensive homology to fungal and bacterial ACCD sequences. Figure 1 shows an alignment of ACCD from T.