STUDIES ON RESPIRATORY ENZYMES AND OXYGEN UPTAKE BY MITOCHONDRIA OF CMS SYSTEMS OF SORGHUM
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Date
2002
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MPKV, UNIVERSITY LIBRARY RAHURI
Abstract
The mam objective of the present investigation was to study some
biochemical parameters associated with CMS phenotype m sorghum using
isolated mitochondria Mitochondria were isolated from the etiolated
seedlings of the two CMS systems of sorghum viz, 1049A, 1049B, RSV33R,
SPH-1229 and 104A, 104B, RS-585, CSH-15R The assays of soluble
proteins and respiratory enzymes viz, cytochrome c oxidase and succinate
dehydrogenase were earned out from isolated mitochondria Further, the
total respiration, cyanide-sensitive, cyanide-insensitive and residual
respiration were measured from the isolated mitochondria and etiolated
seedlings of these lines The protocol for the isolation and punfication of
mitochondiral DNA was also standardized.
The isolated mitochondna from both the CMS lmes, 1049 A and
104A, contained higher soluble protems than their respective isonuclear
mamtamer, restorer and hybnd lmes
The cyanide-sensitive respiration was measured by using SHAM, a
potential inhibitor of alternative oxidase, while cyanide-insensitive
respiration was measured by using KCN, a potential inhibitor of cytochrome
c oxidase TUe cyanide-sensitive respiration was higher,
whereas cyanide-insensitive respiration was lower in isolated mitochondria
and etiolated seedlings of CMS lines than their corresponding mamtamer,
restorer and hybrid lines When both inhibitors were added simultaneously
in the reaction media, residual respiration was observed in isolated
mitochondria and etiolated seedlings of both the CMS systems
The cytochrome c oxidase and succinate dehydrogenase activities in
both the CMS lines were lower than their respective isonuclear mamtamer
lines and also restorer and hybrid lines The CMS system 104A, 104B,
RS-585 and CSH-15R showed comparatively higher cytochrome c oxidase
and succinate dehydrogenase activities than the other CMS system, 1049A,
1049B,RSV-33Rand SPH-1229
The higher protein content, cyanide-sensitive respiration and lower
cytochrome c oxidase activity in CMS lines than their isonuclear lines
indicates that mutation might have occurred in the mitochondrial DNA which
probably resulted in the synthesis of new proteinĀ® Specifically the
respiratory complex IV might have been mutated because some of the
subuntis of this complex are encoded by the mitochondrial genome,
reflecting the alteration in the cytochrome c oxidase activity and cyamdesensitive
respiration The lower cyanide-insensitive respiration and
succinate dehydrogenase activity in CMS lines than their corresponding
isonuclear maintamer lines suggested that it migh t be the result of the CMS
character an d not the cause of CMS, because alternativ e oxidas e and
succinate dehydrogenase are encode d by the nuclea r DNA The lower
alternate oxidase activity m CMS than its isonuclear lme may perhaps be
correlated to the higher yield or yield contributin g characters That's why
the hybrid CSH-15 R yields more than the hybrid SPH-1229
The protocols for the isolatio n and purification of mitochondria and
mitochondrial DNA were standardized. The alkalilysi s metho d yielded
higher mtDNA than freezing and thawing method The freezing and thawin g
method required les s time fo r the isolation o f mtDNA than the alkalilysi s
method, however the DNA isolated b y these two methods was found to be
pure By employmg these two methods, one can avoid the use of percoll
gradient and sucrose density gradient ultra-centrifugation for the isolation of
mitochondria and als o organic solvents for the purificatio n of mtDNA
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