The expression of alternative oxidase and uncoupling protein during fruit ripening in mango

Abstract

The expression of alternative oxidase (Aox) and uncoupling proteins (Ucp) was investigated during ripening in mango (Mangifera indica) and compared with the expression of peroxisomal thiolase, a previously described ripening marker in mango. The multigene family for the Aox in mango was expressed differentially during ripening. Abundance of Aox message and protein both peaked at the ripe stage. Expression of the single gene for the Ucp peaked at the turning stage and the protein abundance peaked at the ripe stage. Proteins of the cytochrome chain peaked at the mature stage of ripening. The pattern of protein accumulation suggested that increases in cytochrome chain components played an important role in facilitating the climacteric burst of respiration and that the Aox and Ucp may play a role in post-climacteric senescent processes. Because both message and protein for the Aox and Ucp increased in a similar pattern, it suggests that their expression is not controlled in a reciprocal manner but may be active simultaneously.

Figure 1

Southern-blot analyses of mango genomic DNA hybridized with DIG-labeled DNA clones of MnAox1a, 1b, 1c, and 2 (A–D) or MnUcp1 (E). Genomic DNA isolated from mango leaves was digested (3 μg per lane but in a pooled sample for MnAox) with the restriction enzyme(s): 1, Eco RI; 2, Hind III; 3, Eco RV; 4, Bam HI; 5, Eco RV/Bam HI; and 6, Bam HI/Hind III. DNA was electrophoresed on 0.8% (w/v) agarose gel against a DIG-labeled molecular mass marker (M_r). After blotting to Nylon⁺ membrane and hybridization with a known amount of the relevant DIG-labeled clone, blots were washed at a high stringency (0.1× SSC and 0.1% [w/v] SDS at 68 °C), detected, and then visualized using an LAS-1000. Diagnostic restriction fragments are indicated with their apparent molecular mass (base pairs).

Figure 2

Validation and summary of results of the real-time PCR approach to assay developmental expression of select genes throughout ripening of mango. A, Lack of cross-reactivity between MnAox primers was first established and the data analyzed for significance (refer to the text). B, Total RNA (1 μg) from mango fruit of five stages of ripeness was then reverse transcribed two independent times. Standard curves of cDNA from the stage of ripeness with greatest expression for each transcript were used to calculate relative abundance at the other stages. Unedited data was analyzed by the appropriate analysis of variance to calculate variance ratios (vr) and t statistics (refer to Table IB). The data was presented individually for each transcript where the stage of ripening that displayed maximal expression was set to 1. C, Corrections were made for differences in amplification efficiency (refer to Table IA) between primer sets for the various transcripts and then data were expressed relative to the greatest corrected expression value.

Figure 3

Immunoblots of total protein from ripening mango fruit using antibodies raised against various mitochondrial proteins. Protein was loaded in lanes for SDS-PAGE on an equal mass (wet weight) basis. Proteins cross-reacting with the various antibodies were visualized using an LAS-1000 and quantitated digitally, where the highest intensity band of the profile was set to 1 and others calculated relative to that value.