Oxidative Phosphorylation by Mitochondria Extracted from Dry Sunflower Seeds

Abstract

The role of mitochondria in the phosphorylation of ADP to ATP in the early steps of seed germination has been studied. Mitochondria were extracted from dry sunflower (Helianthus annuus) seeds. Adenylate kinase-dependent ATP synthesis was inhibited by p ',p5-di(adenosine-5') results were obtained in similar experiments with mung bean and cucumber seeds (14). It was concluded from all these results that, as soon as water enters into the cells, preexisting mitochondria synthesize ATP by oxidative phosphorylation. More recently, active ATPase and Cyt oxidase could be extracted from quiescent maize embryos (6). Thus protective mechanisms must occur in the dry seed which allow the membranes to withstand dryness or rehydration without disorganization (2, 9, 25). In the present paper, we show that mitochondria extracted from dry sunflower seeds (Helianthus annuus) are able to produce ATP by the oxidation of various organic acids or NADH, and this production is abolished by inhibitors of the partial reactions involved in the process of oxidative phosphorylation. The morphology of these mitochondria is also described and discussed in relation to their function. MATERIALS AND METHODS Plant MaterialThe mechanisms by which ATP is produced upon imbibition of seeds and during the early phases of their germination has been a matter of debate for about three decades (for reviews see refs. 1, 17, 19). Although seed respiration has been established for a long time, its link with the phosphorylation of ADP to ATP is in doubt mainly for two reasons. Many authors observed no inhibition of germination by cyanide (19). Also, in spite of many attempts in various laboratories, phosphorylating mitochondria ofgerminating seeds have been obtained in only two cases, from peanut cotyledons imbibed for 40 min (27), and from cucumber cotyledons imbibed for 6 h (14). Moreover, the leakage of solutes during seed imbibition suggested some degree of membrane disorganization either in the dry state or following rehydration (5,23,26). It was believed, in particular, that mitochondria were damaged and had to be repaired during the germination period to become functional at the time of radicle emergence (13). Because fermentation has a low activity in most germinating seeds under aerated conditions (20), new pathways for ATP synthesis have been sought ( 17).However, in vivo studies on lettuce seeds showed that the ATP synthesis which occurs upon imbibition of the seeds is 02-dependent and KCN-sensitive, suggesting that the mechanism involved is oxidative phosphorylation (8). The same Sunflower seeds (Helianthus annuus cv Rodeo) obtained from CETIOM (France) were stored at 4°C in closed vessels containing silica gel. The seeds were dehusked by hand 1 or 2 d before utilization and stored at 4°C in flasks containing silica gel. Their water content was 4.5% (w/w) as determined after drying 24 h at 95°C. ChemicalsAll organic compounds were from Sigma and Merck, except Percoll (Pharmacia), Triton X-100 (Packard), f3-mercaptoethanol (Serva), and glyce...

Expression profiles of respiratory components associated with mitochondrial biogenesis during germination and seedling growth under normal and restricted conditions in wheat

“…Because no apparent increases occurred in the amount of most protein complexes examined during the initial hours of germination ( Fig. 2A), the observed respiration burst during this period can be ascribed to the presence of active protein complexes and/or to the rapid assembly of the preserved components as suggested by Attucci et al (1991). As shown in the previous study by mitochondrial macroarray (Khanam et al, 2007) and the present study by qRT-PCR, dry quiescent embryos contain transcripts of all mitochondria-and nuclear-encoded genes needed for mitochondrial biogenesis (Fig.…”

“…There are reports showing that oxygen consumption starts and proceeds via the cytchrome pathway (cyt path) and this main electron transport path supplies enough energy for sustaining embryo germination and early growth of developing seedlings (Ehrenshaft and Brambl, 1990;Attucci et al, 1991). On the other hand, there are a number of reports describing that plant embryo can germinate normally in the presence of cyt path inhibitors like KCN and antimycin A (AA), but germination and subsequent seedling growth are severely suppressed by inhibitors of the alternative (alt) path including salicylhydroxamic acid (SHAM) and n-propyl gallate (nPG) (Yentur and Leopold, 1976).…”

The Responses of Cytochrome Redox State and Energy Metabolism to Dehydration Support a Role for Cytoplasmic Viscosity in Desiccation Tolerance

“…Extraction of mitochondria from dry tissues often leads to loss of structural and functional integrity (Hoekstra and van Roekel, 1983) or, at best, gives a low yield of intact material (Attucci et al, 1991). Moreover, the necessary use of aqueous solvents during isolation from drying tissues implies that the changes in redox states of the Cyts observed in vitro may not reflect the actual in vivo redox state before the extraction.…”

Mitochondrial biogenesis as revealed by mitochondrial transcript profiles during germination and early seedling growth in wheat

“…It has been reported that poorly differentiated but fully Edited by Toru Terachi * Corresponding author. E-mail: nakamura@kobe-u.ac.jp † these authors contributed equally to this work functional mitochondria are present in dry sunflower embryos (Attucci et al, 1991). Such protomitochondria, upon imbibition, enlarge and elaborate a more complex inner membrane structure in peanut (Morohashi et al, 1981) and in rice (Howell et al, 2006).…”