Targeted quantitative profiling of metabolites and gene transcripts associated with 4-aminobutyrate (GABA) in apple fruit stored under multiple abiotic stresses

4-Aminobutyrate accumulates in plants under abiotic stress. Here, targeted quantitative profiling of metabolites and transcripts was conducted to monitor glutamate- and polyamine-derived 4-aminobutyrate production and its subsequent catabolism to succinate or 4-hydroxybutyrate in apple (Malus x domestica Borkh.) fruit stored at 0 °C with 2.5 kPa O2 and 0.03 or 5 kPa CO2 for 16 weeks. Low-temperature-induced protein hydrolysis appeared to be responsible for the enhanced availability of amino acids during early storage, and the resulting higher glutamate level stimulated 4-aminobutyrate levels more than polyamines. Elevated CO2 increased the levels of polyamines, as well as succinate and 4-hydroxybutyrate, during early storage, and 4-aminobutyrate and 4-hydroxybutyrate over the longer term. Expression of all of the genes likely involved in 4-aminobutyrate metabolism from glutamate/polyamines to succinate/4-hydroxybutyrate was induced in a co-ordinated manner. CO2-regulated expression of apple GLUTAMATE DECARBOXYLASE 2, AMINE OXIDASE 1, ALDEHYDE DEHYDROGENASE 10A8 and POLYAMINE OXIDASE 2 was evident with longer term storage. Evidence suggested that respiratory activities were restricted by the elevated CO2/O2 environment, and that decreasing NAD+ availability and increasing NADPH and NADPH/NADP+, respectively, played key roles in the regulation of succinate and 4-hydroxybutyate accumulation. Together, these findings suggest that both transcriptional and biochemical mechanisms are associated with 4-aminobutyrate and 4-hydroxybutyrate metabolism in apple fruit stored under multiple abiotic stresses.


Supplementary Materials and Methods S1-GHB, Succinate and Pyridine Dinucleotide Analyses
To determine GHB in each treatment replicate, 37.5 mg of frozen apple powder for each of the four subsamples for a treatment replicate was pooled. The pooled tissue was spiked with 148 pmol of the internal standard GHB-d6 (C/D/N Isotopes), as well as 155 pmol GHB (Sigma) to improve the sensitivity of the method, and combined with 500 µL of 80 % (v/v) ethanol and homogenized with a small pestle for 5 min. The homogenate was clarified by centrifugation at 21,000 x g for 20 min and the pellet was discarded. The supernatant was combined with 200 µL of chloroform, vortexed for 1 min, followed by addition of 300 µL of Milli-Q water and an additional 2 min of vortexing. The mixture was centrifuged as described above, and the polar layer was collected and passed through a 0.45-μm nylon syringe filter. A 120-µL aliquot of the filtered polar layer was aliquoted into glass inserts and dried down by rotary concentrator (Savant SPD 2010, Thermo Scientific, Asheville, NC, U.S.A.) for 15 h. The dried residue was resuspended in 40 μL of 20 g L -1 methoxyamine hydrochloride in pyridine by vortexing for 10 s, and then incubated for 90 min at 37 ºC. Subsequently, 80 μL N-Methyl-N-(trimethylsilyl) trifluoroacetamide was added, and the sample was incubated for 45 min at 37 ºC. The solution was allowed to stand overnight at room temperature, and then a 1-µL aliquot of the derivatized plant extract was split-injected onto the gas chromatograph-mass spectrometer/mass spectrometer (Scion 436-GC, Bruker Daltonics Inc., Fremont, CA, U.S.A.) through a 30 m, 0.25 mm ID, 0.25 mm df column (BR-5ms, Bruker) carried by helium gas. Initially, the column oven temperature was held at 50 ºC for 5 min, followed by an increase of 5 ºC min -1 up to 140 ºC, and then a 20 ºC min -1 increase to 300 ºC, which was held for 1 min. Three solvent washes of the injector syringe were performed prior to each sample injection. The transfer line was kept at 250 ºC, and the MS-MS source was maintained at 200 ºC. GHB-d6 was monitored by precursor and product ions of m/z 239 and m/z 146, respectively, while GHB was monitored by precursor and product ions of m/z 233 and m/z 146, respectively. Stock solutions (1 g L -1 ) of GHB (Sigma) and GHB-d6 (C/D/N Isotopes) were each generated by dissolving the appropriate compound in HPLC-grade water. A 25-µL aliquot of the stock solution was mixed with 975 µL of HPLCgrade water, followed by a number of 500 µL serial dilutions in HPLC-grade water to produce a set of GHB solutions in the µg L -1 range (~6 to 195 pg µL -1 range used here). Thereafter, 50 µL of each GHB solution was pipetted into glass inserts (Fisher) accompanied by 15 µL of a 195 pg µL -1 GHB-d6 solution. To account for matrix effects, 120 µL of apple extracts was added to each glass insert. These were dried down, derivatized, and injected onto the GC-MS-MS alongside apple samples. To create the calibration curve, the ratio of the peak area of GHB to the peak area of GHB-d6 was plotted against the amount of GHB injected for each standard. To determine the amount of GHB present in the tissue, the ratio of the peak area of GHB to the peak area of GHB-d6 was determined and substituted in the equation of the calibration curve to calculate the amount of GHB injected. For apple samples, 0.19 pmol of GHB was subtracted from this value to account for the exogenous GHB added during extraction. One standard and one blank were run every five to six samples. Use of an isotopic internal standard allows for corrections stemming from extraction efficiency, degree of derivatization, and any small changes in chromatography or detection due to its structural resemblance and small difference in mass 1 .

Identification of apple genes
Putative appleSSADH1 and MdSSADH2 genes were identified using a BLAST search of the Arabidopsis SSADH sequence (GenBank Acc No. NM_106592) against the apple genome (www.rosaceae.org). Two sequences were highly similar to the Arabidopsis sequence:  Table S1 for a list of primers). A translation of the fragment revealed that it was 79 to 89% identical to the sequences used to design the degenerate primers. The sequence was used to design primers for 5' and 3' for RACE.
The primers CT-F30 and CT-F31 were used for 5′ RACE and nested PCR, respectively; CT-R30 and CT-R31 were used for 3′ RACE and nested PCR, respectively. Sequences obtained from the RACE reactions were combined to assemble a contig representing an apple ALANINE TRANSAMINASE (ALA-T). The translated apple ALA-T is 490 amino acids in length and 77 to 90% identical to the sequences used to design the degenerate primers. Apple ALA-T is 97% identical to the last 507 amino acids of a 560 amino acid predicted peptide from the apple genome, MDP0000168683. The apple ALA-T sequence was used to design primers for quantitative reverse transcriptase polymerase chain reaction (qPCR).
The apple genome database (www.rosaceae.org) was searched utilizing known Arabidopsis PAOs (PAO1, At5g13700; PAO2, At2g43020; PAO3, At3g59050; PAO4, At1g65840; PAO5, At4g29720) sequences as queries at the nucleotide and amino acid levels. Six putative apple PAO genes were identified with following accession numbers: MDP0000261625; MDP0000321972; MDP00001888553; MDP0000702799; and, MDP0000941459. Singlestranded cDNA from 'Empire' apple was generated as described above. cDNA containing open reading frames of the apple PAO homologs were amplified using gene specific primers (see Supplementary Information Table S2 for a list of primers). All six putative apple PAO genes appeared to be expressed in leaves and were cloned into pCR2.1-TOPO (Invitrogen) vector using the manufacturer's protocol. At least three clones from independent PCR origin were sequenced for each PAO gene. The apple PAO gene sequences were designated as PAO1, PAO2, PAO3, PAO4, PAO5 and PAO6 and deposited in the GenBank (Acc. No. KT184496 -KT184501, respectively). Table S1. Synthetic oligonucleotides utilized for identification and cloning of apple ALA-T.

Primer
Sequence ( Table S4. Impact of elevated CO 2 on the amino acid composition of 'Empire' apple fruit during 16 weeks of postharvest storage under low temperaure and low oxygen conditions. Data represent the mean ± SE (nmol per gram fresh mass) of four storage treatment replicates.