Cloning, Sequencing and Expression Analysis of a cDNA Encoding Glutamate Dehydrogenase Gene in Broccoli During Postharvest Senescence

Quality of broccoli deteriorates rapidly after harvest due to major physio-biochemical changes. In this study, the changes in ammonia content and activity and gene expression of glutamate dehydrogenase (GDH; EC 1.4.1.2) during storage at 20°C for five days were investigated. The branchlets were separated from the florets at the end of each storage period. Ammonia assay showed that the level in the branchlet portion was almost constant while in the floret portion increased rapidly after three days of storage to about seven times the content at harvest. Enzymatic analysis, on the other hand, revealed that GDH, in both amination and deamination activities, decreased in the branchlets while a gradual increase was observed in the floret portion as storage progressed. To better understand these biochemical changes, a cDNA encoding GDH in broccoli was isolated, cloned and sequenced. The partial cDNA clone referred to as BoGDH (Brassica oleracea glutamate dehydrogenase gene; AB212934) encodes an mRNA of 781 bp. The deduced amino acid sequence showed highest similarity with the GDH gene from Arabidopsis associated with stress. Although the transcript was not consistent with enzyme activity, RNA gel blot analysis revealed that BoGDH was present in both branchlet and floret portions throughout the storage duration. The results suggest that GDH plays an essential role during postharvest senescence. Furthermore, it is likely that its expression is controlled by multigenes and regulated either transcriptionally or posttranscriptionally.