Functional characterization of mitochondrial and cytosolic aldehyde dehydrogenases in maize (Zea mays L.).

摘要

Aldehyde dehydrogenases (ALDHs) are a group of enzymes that catalyze oxidation of aldehydes in the presence of coenzyme NAD+ or NADP+. Few plant ALDHs have been studied at protein level, although many ALDH genes have been cloned. To date no functional characterization of plant ALDHs has been reported. This dissertation is focused on functional characterization of four ALDHs from maize, RF2A, RF2B, RF2C and RF2D. RF2A and RF2B are targeted to mitochondria, while RF2C and RF2D are located in cytosol. RF2A is required for normal pollen development: the anthers in lower florets from spikelets of homozygous mutant rf2a plants in normal cytoplasm stop developing at certain stage and no pollen or little pollen in these anthers is produced; in Texas cytoplasm plants, the homozygous rf2a mutant plants are completely male sterile, i.e., no pollen is produced in either upper or lower florets of spikelets. Cell fractionation experiments and mitochondrial ALDH assays have revealed that RF2A is an active ALDH enzyme located in mitochondrial matrix. Purified recombinant RF2A oxidizes a broad spectrum of aldehydes, including 3- to 9-carbon aliphatic aldehydes, aromatic aldehydes and some other aldehydes, suggesting it plays multiple roles in vivo. RF2B is also located in mitochondria, as revealed by in vitro import experiments. Purified recombinant RF2B only oxidizes short chain aliphatic aldehydes, suggesting its major role may be involved in oxidizing acetaldehyde. The expression patterns of rf2 and rf2b are partially overlapping. In conclusion, although both RF2A and RF2B are mitochondrial ALDHs, their biological functions are apparently differentiated.; Maize rf2c and rf2d have about 70% and 81% amino acid similarity with rf2a/rf2b and between each other, respectively. Kinetic analyses performed on purified RF2C and RF2D proteins revealed that both proteins can oxidize α,β-unsaturated aldehydes generated via lipid peroxidation. The K_ms for these aldehydes for RF2C and RF2D are in low micromolar range. Accumulation of transcripts and protein of both genes was induced by application of ABA, wounding and hypoxia, all of which result in lipid peroxidation. Hence, RF2C and RF2D can be considered as part of the plant's defense system against stresses.