Normalizing to housekeeping gene (HKG) can make results from quantitative real-time PCR (qRT-PCR) more reliable. Recent studies have shown that no single HKG is universal for all experiments. Thus, a suitable HKG should be selected before its use. Only a few studies on HKGs have been done in plants, and none in soybean, an economically important crop. Therefore, the present study was conducted to identify suitable HKG(s) for normalization of gene expression in soybean.

The pivotal role of glucose-6-phosphate dehydrogenase (G-6-PDH)-mediated nitric oxide (NO) production in the tolerance to oxidative stress induced by 100 mM NaCl in red kidney bean ( Phaseolus vulgaris ) roots was investigated. The results show that the G-6-PDH activity was enhanced rapidly in the presence of NaCl and reached a maximum at 100 mM. Western blot analysis indicated that the increase of G-6-PDH activity in the red kidney bean roots under 100 mM NaCl was mainly due to the increased content of the G-6-PDH protein. NO production and nitrate reductase (NR) activity were also induced by 100 mM NaCl. The NO production was reduced by NaN ₃ (an NR inhibitor), but not affected by N^ω -nitro- l -arginine ( l -NNA) (an NOS inhibitor). Application of 2.5 mM Na ₃ PO ₄ , an inhibitor of G-6-PDH, blocked the increase of G-6-PDH and NR activity, as well as NO production in red kidney bean roots under 100 mM NaCl. The activities of antioxidant enzymes in red kidney bean roots increased in the presence of 100 mM NaCl or sodium nitroprusside (SNP), an NO donor. The increased activities of all antioxidant enzymes tested at 100 mM NaCl were completely inhibited by 2.5 mM Na ₃ PO ₄ . Based on these results, we conclude that G-6-PDH plays a pivotal role in NR-dependent NO production, and in establishing tolerance of red kidney bean roots to salt stress.

The role of ethylene and hydrogen peroxide (H₂O₂) in the induction of the alternative respiratory pathway (AP) in calluses from wild-type (WT) Arabidopsis and ethylene-insensitive mutant etr1-3 under salt stress was investigated. The capacity and the contribution of the AP to the total respiration were significantly induced by 100 mM sodium chloride (NaCl) in WT calluses but only slightly induced in etr1-3 calluses. Ethylene emission was enhanced in WT calluses under salt stress. Application of 1-aminocyclopropane-1-carboxylic acid (an ethylene precursor) further increased the AP capacity in WT calluses but not in etr1-3 calluses under salt stress. Reduction of ethylene production by aminooxyacetic acid (AOA, an ethylene biosynthesis inhibitor) in WT calluses eliminated the NaCl-induced increase of ethylene emission and inhibited AP induction under salt stress, suggesting that ethylene is required for AP induction. H₂O₂ enhanced ethylene production while ethylene reduced H₂O₂ generation in WT calluses under salt stress. In addition, ethylene and H₂O₂ modulated NaCl-induced alternative oxidase gene (AOX1a) expression and the increase in pyruvate content in WT calluses. Inhibition of the AP by salicylhydroxamic acid in WT calluses under salt stress resulted in severe cellular damage as indicated by the high content of H₂O₂, malondialdehyde and more electrolyte leakage. Taken together, ethylene and H₂O₂ are involved in the salt-induced increase of the AP, which plays an important role in salt tolerance in WT calluses, and ethylene may be acting downstream of H₂O₂.