Osmotic dehydration of wheat seedlings in -0.5 MPa polyethylene glycol ( PEG) solutions for 24, 48 and 72 h resulted in mild, moderate and severe water stress respectively in leaves, but only caused mild water stress in roots as reflected by the changes in relative water content (RWC) . In response to the above water stress conditions, leaf total respiratory rate (Vt,) decreased progressively, and the alternative pathway (AP) capacity ( Valt) and its actual operation activity (ρValt) decreased more severely. Water stress also led to continuous reduction in cytochrome pathway (CP) activity ( ( ρ Vcyt) and different changes in the contribution of ρValt and p' Vcyt to Vt in leaves, with ρValt/ Vt, decreasing and ρ' Vcyt,/ Vt, increasing. The change pattern of root Vt was similar to that of its RWC, while root Valt and ρValt were found to decrease during the first 24 h of stress and thereafter recover to a level close to that of the control (Oh). These data indicate that the alternative pathway is sensitive to water stress and can adapt mild water stress. The results of northern hybridization using an alternative oxidase gene ( Aox) as probe revealed that there was a good correlation between Valt (also ρValt) and Aox mRNA levels in both leaves and roots, suggesting that water stress affects the development and operation of AP respiration through affecting the expression of the alternative oxidase gene.
Twelve peptides, including eight conservative amino acid residues in the amino acid sequence of hydrophilic S helix of the alternative oxidase (AOX), were synthesized by solid-phase method. The polypeptide was coupled with α-chymotrypsinogen, and the antibodies were obtained through immunizing domestic rabbit by injecting this complex. By using these antibodies, which were raised to immunoreact with total proteins of purified mitochondria from different organs of mung bean seedlings, we find that there are two hybridizable AOX bands in mitochondria. Their molecular weights are about 35 and 38 ku, respectively. Moreover, the respiratory parameters of hypocotyl, true leaf and cotyledon of mung bean seedlings show that true leaf has the highest total respiration (Vt), alternative pathway (AP) capacity (Valt) and the activity of AP (ρValt) among the three organs. Vt and ρValt of cotyledon ranked the second. Hypocotyl has the lowest V, and ρValt, but its Valt is higher than that of cotyledon. These results are consistent with the analysis of Western blotting of expression of AOX. The highest Vt and ρValt in true leaf are accompanied two hybridizable polypeptides of AOX protein, 35 ku and 38 ku respectively. The next is cotyledon Vt and ρValt with only one 38 ku hybridizable polypeptide of AOX protein. Hypocotyl Vt and ρValt is the lowest and its immunoblotting band is similar to that of cotyledon, but the expressive amount of 38 ku protein is less than that of cotyledon. The results suggest that the 35 ku AOX may contribute mainly to true leaf ρValt.
During the course of mungbean (Phaseolus radiatus L.) germination, the rate of ethylene production and the activity of ACC synthase (1_aminocyclopropane_1_carboxylic acid synthase, EC4.4.1.4) began to increase in the 5th day of germination, and reached its peak in the 10th day and then decreased. The ethylene production and the activity of ACC synthase were obviously promoted by 10 μmol/L exogenous IAA (indole_3_acetic acid). The production of superoxide radical (O -· 2) and hydrogen peroxide (H 2O 2) were also promoted by exogenous IAA, suggesting that there was some relationship between active oxygen production and the activity of ACC synthase induced by exogenous IAA. The production of ethylene and the activity of ACC synthase increased dramatically when the seedlings were treated with exogenous O -· 2, whereas the exogenous H 2O 2 had no effects on the production of ethylene and the activity of ACC synthase. Exogenous SOD (superoxide dismutase, one scavenger of O -· 2) could inhibit the production of ethylene and the activity of ACC synthase, but exogenous CAT (catalase) could not. So it was possible that IAA would stimulate the activity of ACC synthase by inducing the production of O -· 2 in germinating mungbean seedlings, and this might be one of the regulating mechanism of ethylene synthesis in higher plants; the production of H 2O 2 induced by IAA was not the cause of the increase of the activity of ACC synthase and the production of ethylene.
