Caroline Valente dissertação de mestrado - Universidade Federal ...

ABSTRACT
Mitochondria, containing in the inner membrane the proton-pumping
respiratory chain that transfers electrons via cytochromes and synthesizes ATP, were
isolated from embryogenic cells of A angustifolia. The membrane also includes a
second pathway characteristically inhibited by salicylhydroxamic acid (SHAM),
insensitive to cyanide, non-proton pumping and called alternative oxidase (AOX).
Other characterized alternative components are the alternative NADH
dehydrogenases, that were sensitive to flavone and, the plant uncoupling
mitochondrial protein (PUMP) that was identified for the first time in gymnosperms.
Mitochondria from A. angustifolia respired by oxygen consumption through the
oxidation of the substrates malate/glutamate and succinate. They were highly
functional, showing high phosphorylation capacity, demonstrated by the respiratory
controls values. The external rotenone-insensitive NADH dehydrogenase was
characterized through oxidation of externally added NADH in the absence or
presence of rotenone. Internal NADH dehydrogenase was characterized by the
oxidation of malate/glutamate in the presence of rotenone. The external and internal
NADH dehydrogenases were inhibited at 60% by the addition of flavone to
mitochondria respiring in the presence of FCCP. Complex I and succinate oxidase
were inhibited at 10-15% and 20%, respectively. The inhibition of the alternative
NADH dehydrogenases by flavone was also suggested through its effect on NADH
dehydrogenase and NADH cytochrome c reductase in disrupted mitochondria.
Flavone also caused a significant depolarization of the inner membrane in
mitochondria energized by NADH ou malate/glutamate oxidation. Mitochondria were
isolated from embryogenic cells of A. angustiofolia that had been submitted to
temperature changes (cold or heat) and compared to untreated. The PUMP protein
was identified in mitochondria from A. angustifolia through Western blotting and using
this assay we also identified the differences on the protein expression respective to
the treated and untreated cells. Polyclonal antibodies for Arabidopsis thaliana
(AtPUMP) that already are used in the identification of PUMP from several plant
species such as potato and tomato, were used in this assay. PUMP protein was
visualized as a protein with a molecular mass higher than 33 kDa. Cells exposed at
4°C for 48h increased the expression of PUMP when compared to the unstressed.
When cells were exposed to 43°C for 4h the effect seemed to be too severe and
caused the disappearance of the protein band. The activity of PUMP protein was
evaluated through the addition of BSA to the isolation and/or reaction medium. The
values for the respiratory control ratio showed that BSA prevents uncoupling at all
stress conditions indicating its inhibitory action on PUMP. Studies concerning the
morphology of the embryogenic cells submitted or not to temperature changes
showed that cold or heat caused damage to the embryonic and suspensor cells
interfering in the cell multiplication with decrease in the quantity and causing
deformation of the suspensor cells.