Nitric oxide emission from tobacco leaves and cell suspensions: rate limiting factors and evidence for the involvement of mitochondrial electron transport

Abstract

SummaryQuantitative data on nitric oxide (NO) production by plants, and knowledge of participating reactions and rate limiting factors are still rare. We quantified NO emission from tobacco (Nicotiana tabacum) wild-type leaves, from nitrate reductase (NR)-or nitrite reductase (NiR)-deficient leaves, from WT-or from NR-deficient cell suspensions and from mitochondria purified from leaves or cells, by following NO emission through chemiluminescence detection. In all systems, NO emission was exclusively due to the reduction of nitrite to NO, and the nitrite concentration was an important rate limiting factor. Using inhibitors and purified mitochondria, mitochondrial electron transport was identified as a major source for reduction of nitrite to NO, in addition to NR. NiR and xanthine dehydrogenase appeared to be not involved. At equal respiratory activity, mitochondria from suspension cells had a much higher capacity to produce NO than leaf mitochondria. NO emission in vivo by NiR-mutant leaves (which was not nitrite limited) was proportional to photosynthesis (high in light þCO 2 , low in light )CO 2 , or in the dark). With most systems including mitochondrial preparations, NO emission was low in air (and darkness for leaves), but high under anoxia (nitrogen). In contrast, NO emission by purified NR was not much different in air and nitrogen. The low aerobic NO emission of darkened leaves and cell suspensions was not due to low cytosolic NADH, and appeared only partly affected by oxygendependent NO scavenging. The relative contribution of NR and mitochondria to nitrite-dependent NO production is estimated.

Hunting for Plant Nitric Oxide Synthase Provides New Evidence of a Central Role for Plastids in Nitric Oxide Metabolism

“…These results indicate that additional mechanisms to reduce nitrite into NO exist in plant cells and that the decreased capability for NO synthesis of mutant plants with defective NR activity might result from their reduced nitrite levels (Modolo et al, 2005). Other enzymatic sources for nitrite-dependent NO synthesis exist in the plasma membrane (Stohr et al, 2001) and mitochondria (Planchet et al, 2005), whereas nonenzymatic production of NO from nitrite has been shown to occur in acidic and reducing environments, such as the apoplasm (Bethke et al, 2004) and plastids (Cooney et al, 1994). The highly reduced levels of L-Arg in the nia1 nia2 mutant (Modolo et al, 2006) might also compromise its ability to produce NO.…”

Nitrite Acts as a Transcriptome Signal at Micromolar Concentrations in Arabidopsis Roots

“…In contrast, NiR-deficient plants, which accumulate high nitrite levels, display chlorosis and dramatically reduced growth in tobacco (Nicotiana tabacum; Vaucheret et al, 1992) and rapid death in barley (Hordeum vulgare; Duncanson et al, 1993) if grown with nitrate. Such plants also produce high levels of nitric oxide (NO; Morot- Gaudry-Talarmain et al, 2002) because nitrite serves as a substrate for the formation of NO (Yamasaki et al, 1999;Rockel et al, 2002;Lea et al, 2004;Meyer et al, 2005;Planchet et al, 2005). It should be noted that nitrite can have a beneficial effect because it protects maize (Zea mays) roots from anoxia by reducing cytoplasmic acidosis (Libourel et al, 2006).…”

Sulfur deprivation-induced expression of THB1, a Chlamydomonas reinhardtii truncated hemoglobin, is mediated by nitrate reductase-dependent NO production

“…Intracellular nitrite is a limiting factor for NO production (Planchet et al, 2005). Strikingly, we found THB1 upregulation in S-starved cells that were incubated in ammonium-containing medium (Minaeva et al, 2017; Figs 1 and 2).…”