The Effects Of Different Temperature Stresses And Salicylic Acid Treatment On Cyanide-Resistant Respiration And The Molecular Biological Studies On Expression Of Alternative Oxidase Gene In Tobacco Callus

In this paper, we studied effects of different temperature stresses and salicylic acid on cyanide-resistant respiration in tobacco callus, expression patterns of alternative oxidase (AOX) protein and its encoding gene (Aoxl) during these physiological courses were also studied.In the first part, the relationship between cyanide-resistant respiration and expression of alternative oxidase (AOX) in tobacco callus treated by different temperatures was studied. The results showed that the alternative pathway(AP) capacity(Valt) and the activity of AP(pValt) could be significantly induced by different temperatures(14? and 4?), and the increased amplitude of Valt was concordant with the decreased amplitude of temperature, but the difference of pValt between the calli treated by 14 ? and 4? was not significant as that of Valt. The Western blot results showed AOX in tobacco callus was present with the major form of homodimers, and the state could not be changed by the treatment of different low temperatures. The changes of total AOX protein induced by low temperatures were concordant with those of Valt and pValt, indicating the enhancement of cyanide-resistant respiration was in relation to theincrease of total AOX. The abundances of AOX monomers in calli treated by 14? and 4? were approximately equal, but the quantity of AOX homodimers increased with the decline in temperature, suggesting the low temperatures induced the increase in AOX homodimers primarily.In the second part, We studied the effects of high temperature stress on the operation of cyanide-resistant respiration and the expression of the Aoxl gene. The results showed the total respiration(Vt), Valt and pValt all decreased during the high temperature treatment. The contribution of AP to Vt(pValt /Vt) declined within the 0-12h of stress, but there was partial recovery afterwards. However, the contribution of CP to Vt(p' Vcyt /Vt) was in opposition to pVa|t/Vt, and was higher than pValt/Vt during the treatment, suggesting the CP was still the major pathway of electron transport under the high temperature stress. The Northern blot results demonstrated the level of AOX mRNA was in good line with the changes of Valt and pValt, indicating high temperature stress decreased the operation of cyanide-resistant respiration by inhibiting the expression of Aoxl gene.In the third part, the effects of Salicylic acid(SA) treatment on Valt, pValt and the expression of AOX in tobacco callus were studied. We found the Valt and pValt were dramatically induced in early stage(0-8h) of SA treatment, and decreased subsequently(8-16h) to a stable status(16-24h). However, they changed unsynchronously, and the effects of SA treatment on pValt were greater than those on Valt. In addition, SA treatment also led to a relocation of respiratory electron flux between the two pathways. During the first 8h of SA treatment, electron flowed through the AP increased in contrast to the decrease in the CP, and led AP become the major pathway of electron transport instead of CP. Along with the elongation of SA treatment, electron flux diverted partially back to CP and resulted in a balance between AP and CP after 16h of SA treatment. The Western blot showed the changes of total AOX and the AOX monomer but not of AOX homodimer were in good line with thechanges of Valt and pValt. These results indicated the increases of Valt and pValt induced by SA in tobacco callus were depended on the synthesis of AOX , and the AOX monomer has the greater action on Valt and pValt than the homodimer.