McDaniel and Sarkissian (Genetics 57:843,1967) reported the manifestation of hybrid vigor in O2 consumption and P/O ratios by isolated seedlings (4-5 d etiolated) from hybrids of maize. We are further investigating this phenomenon by monitoring O2 consumption of highly purified mitochondria from pedigree stocks of 5 inbred and 8 reciprocal hybrid lines. We have also subjected purified seedling (4-5 d etiolated) mitochondria from the 13 lines to a system generating in organello protein synthesis to detect both quantitative and qualitative differences in protein synthesis by mitochondria.
Mitochondria were isolated from 4-5 d etiolated seedling tissue of 13 pedigreed lines (Oh43, W23, M14, Mo17, B73, Oh43 X W23, W23 X Oh43, Oh43 X M14, M14 X Oh43, W23 X M14, M14 X W23, Mo17 X B73, B73 X Mo17) and purified on discontinuous sucrose gradients by the method of Forde (PNAS 75:3841, 1978). For measurement of O2 consumption under State I conditions (no exogenous Krebs cycle intermediates or ADP) depletion of O2 was monitored on a Clarke electrode apparatus.
Concentration of protein was determined (Bradford,
Anal. Biochem. 72:248, 1976) and mitochondria concentration correlated
with amount of protein. Mitochondria of the inbreds Oh43 and W23 and their
reciprocal hybrids show positive heterosis in the slopes of the regression
lines plotting %O2/ug protein versus time
|Line||Mean slope (B) x 10-4|
|Oh43 X W23||9.14|
|W23 X Oh43||7.75|
These differences are significant between the inbreds as a group and the hybrids as a group but not significant differences between inbreds or between hybrids (p<0.05). The remaining 9 lines are currently under investigation.
Mitochondria were isolated for in organello protein synthesis studies by a similar procedure modified to accommodate small samples (0.5 g fresh weight tissue) (Boutry, Eur. J. Biochem. 127:129, 1982). Purified mitochondria were incubated on a shaker in the dark for 90 min in media including 35S-methionine and succinate/ADP as an energy-generating system to measure incorporation of label into acid-insoluble material according to Forde (ibid.). Bacterial contamination was ruled out by plating aliquots on complete media and performing replicate experiments with acetate as the only carbon source (Forde and Leaver, PNAS 7 7:418, 1980). Three to 4 replicates were carried out for each line.
Mitochondria purified from 0.5 g seedling tissue incorporate a significant amount of 35S-methionine into TCA-insoluble material. Incorporation is chloramphenicol-sensitive and cycloheximide-insensitive. Inbred mitochondria incorporate significantly more than hybrid mitochondria of the same tissue sample:
We are further investigating this observation. Some hypotheses we are testing include: 1) Hybrid cells are larger than inbred cells in seedlings of 4-5 d incubation, thereby reducing the number of mitochondria in 0.5 g fresh weight. 2) The number of mitochondria per cell, regardless of cell size, is modified in hybrids thereby altering number of mitochondria in 0.5 g fresh weight. 3) Hybrid mitochondria differ in uptake of 35S-methionine and/or succinate/ADP from their inbred parents, thereby altering specific activity of newly synthesized proteins.
Mitochondria labelled with 35S-methionine for 90 min as described above were subjected to SDS-PAGE according to Laemmli (Nature 227:680, 1970), impregnated with PPO/DMSO and fluorographed. Typical N cytoplasm protein profiles were observed with consistent presence of several larger MW proteins (> 92 kD). Genotypic differences were apparent; these patterns seem to be strictly passed on in hybrids in a maternal fashion. No apparent differences were found attributable to hybridity per se.
Mitochondria were also isolated and purified from 0.5 g of 20 d plants as described above. Purification was complete monitored by the lack of green color in the final mitochondrial pellet. Incorporation of 35S-methionine was very low (200-500 cpm) and no significant differences were detectable among lines. Protein profiles after SDS/PAGE/fluorography were virtually identical for all lines regardless of genotype or inbred/hybrid status.
We continue to investigate the manifestation of hybrid vigor by seedling mitochondria in both respiratory activity and quantitative and qualitative protein synthesis differences.
Christine M. Nebiolo, D. B. Walden and Kevin D.
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