Characteristics Of Mitochondrial Alternative Electron Transfer Pathways In Chorispora Bungeana Under Chilling Stress

Chorispora Bungeana Fisch. & C.A. Mey. (C. bungeana) is a representative alpine subnival plant. It mainly distributes in an ice free cirque (with a 3800-3900m height above sea level) beside the Glacier No. 1 in the source area of Urumqi River in Tianshan mountains, Xinjiang province, China, where the average temperature is below subzero during the growth period from June to September. It can survive under many environmental stresses like freezing, enhanced UV-B irradiation and lower oxygen partial pressure. Great attention had been paid to the exploration of its special mechanism to resist external frequent temperature fluctuations and freezing temperatures. Plant mitochondria possess multiple energy dissipating pathways like alternative oxidase pathway, uncoupling pathway and alternative NAD(P)H dehydrogenase pathway, but the function of these pathways in C. bungeana is still not clear. The present research is focus on the anti-stress characteristic of plant mitochondria and cold resistance mechanism of alternative pathways in C. bungeana. Furthermore, we study the relationship between cold resistance and protein-protein interaction of alternative NAD(P)H pathway, which help us to further understand the antifreezing mechanism of this pathway. The results as following:Results of HPLC found that redox state of cellular ubiquinone is very active in early chilling stress. Comparing with 25°C, the level of reductive ubiquinone kept decreasing under different chilling temperature. Meanwhile, chilling induction can not impair cell activity since MMP kept stabilizing under early chilling and the accumulation of ROS production did not fluctuate at the same time. These findings suggested that the special mechanism of chilling resistance in the alpine subnival plant is tightly linked with redox balance of its cellular redox molecule and, in early chilling, the redox transition of ubiquinone can not only ensure the fluency of electron transfer in mitochondria but also facilitate the regulation of the whole-cell redox states leading to adaptation of cellular regulations. Accumulation of 14-3-3 protein in plasma membrane was very pronounced during chilling but it's quicker under 0℃than that under 4℃, this regulation under 4℃was persistent but was limited by long-term 0℃chilling and 14-3-3 proteins decreased to the normal level after one-day 0℃chilling. Mitochondrial alternative NADH oxidation supplied more efficient ability to resist severe circumstance. The enhancement of rotenone-insensitive NADH dehydrogenase under 4℃also showed an accumulative effect and the response under 0℃was quiker than that 4℃, however, this increase could not be impaired by prolonged 0℃chilling. Neither antibody screen nor oxygen consume assay could find the existence of alternative oxidase in C. bungeana mitochondria. Uncoupling protein could be found by antibody screen but it didn't respond to chilling. It was concluded that plasma membrane and mitochondrial alternative NADH dehydrogenase actively functioned with cooperative or complementary way to resist cold stress in C. bungeana.Based on NADH-tetrazalium activity and histological stain in gel, we isolated LPD in native state with undenatured PAGE from the mitochondria of C.bungeana. The following size-exclusion chromatography and native/SDS-PAGE analysis showed that LPD formed a complex with other proteins. This isolated LPD showed a NAD(P)H quinone catalysis like its mammalian analog, and CoQ₀ could be used as electron acceptor with high selectivity in the catalysis of NAD(P)H oxidation. Except for binding with FAD, FMN also was found in this complex, but it did not participate in the LPD catalysis process because loss of FMN after further purification on BN-PAGE did not impair the enzyme activity. Also, antibody screen showed that the co-purified 26kD subunits belonged to small heat-shock protein (sHsp), which was found to be able to interact with LPD by co-immunoprecipitation assay, but it didn't involve in the catalysis since the dissociation on BN-PAGE restored the activity of NAD(P)H oxidation. So LPD in C. bungeana mitochondria could function a NAD(P)H quinone oxidoreduction in new formation with sHsp participated in it, and this process may contribute to the recovery of the oxidized cellular ubiquinone and mediate stress resistance. Furthermore, Based on native isolation on undenatured PAGE, we got one protein complex from Arabidopsis mitochondria which mediated a NAD(P)H quinone oxidoreduction and many subunits were involved in it. After identification with MALDI-ToF and enzyme assays, LPD and N-acetyl-γ-glutamyl-phosphate reductase (NAGPR) were found to catalyze the quinone-dependent NAD(P)H dehydrogenation. Meanwhile, malate dehydrogenase (MDH), Aspartate aminotransterase (AAT) and Chaperonin 20 (Cpn20) combined with them to form complex. Through immunofluorescence assay we found that LPD, NAGPR and Cpn20 partially co-located in the cells. Furthermore, all of these proteins interacted and assembled together in vitro, LPD and Cpn20 could co-immunoprecipitate (co-IP) with NAGPR from the lysate of isolated mitochondria, MDH activity also was assayed in the elution of co-IP. So it can be suggested that these known proteins in mitochondrial matrix could combine with each other, which resulted in coupling of NAD(P)H quinone oxidation with NADH influx under physiological condition, LPD and NAGPR acted as catalyzing subunits, they could respectively oxidize NADH and NADPH, MDH and AAT mediated NADH input through malate/aspartate shuttle. These findings efficiently facilitated us to understand the exact mechanism of mitochondrial NAD(P)H oxidation in plant.