Role of thylakoid Ndh complex and peroxidase in the protection against photo‐oxidative stress: fluorescence and enzyme activities in wild‐type and ndhF‐deficient tobacco

Abstract

An Ndh‐deficient mutant of tobacco (Nicotiana tabacum cv. Petit Havana) was prepared by disrupting the ndhF gene in a transplastomic approach. The mutant (ΔndhF) showed 10% of the Ndh complex activity (EC 1.6.5.3) and 8% of the NDH‐F polypeptide of that of non‐transformed plants. However, in ΔndhF, NDH‐A, another Ndh polypeptide, was still present at 50% of the level in non‐transformed plants. ΔndhF tobacco showed higher sensitivity than non‐transformed plants to photo‐oxidative stress (as judged by chlorophyll bleaching) caused by increased light intensity and paraquat applications. These photo‐oxidative treatments increased the amount and activity of the Ndh complex, thylakoid peroxidase, post‐illumination chlorophyll fluorescence and non‐photochemical quenching (NPQ) of chlorophyll fluorescence in non‐transformed but not in ΔndhF tobacco. Highly stressed non‐transformed plants showed a rapid post‐rise decline of chlorophyll fluorescence, probably indicating a re‐oxidation of reduced plastoquinone. The results indicate that, in normal plants, the Ndh complex and thylakoid peroxidase (EC 1.11.1.7) provide and remove electrons, respectively, to balance the redox level of the intermediates of cyclic electron transport. In this way, they optimize the generation of the transmembrane H+ gradient of thylakoids and, as a consequence, increase the NPQ and the protection against photo‐oxidative stress.

Chloroplasts regulate leaf senescence: delayed senescence in transgenic ndhF-defective tobacco

“…On the other hand, paraquat treatment, which increases the production of ROS, enhances senescence symptoms in barley 23 and tobacco leaves. 20 It has been proposed that the mitochondrial complex I increases to compensate the energy deficit in aging animal tissues. 27 In this way, aging tissues would respond like young ones to a lowering of ATP levels.…”

“…The Ndh complex is responsible for the increase in chlorophyll fluorescence (dependent on the level of reduced plastoquinone) after the interruption of actinic illumination. 20 Accordingly, fluorescence rose in wt but not DndhF incubated leaf disks (Figure 3c). This is another indication that the Ndh complex remained at a low level during incubations of DndhF but not of wt leaf disks.…”

“…The DndhF and wt tobacco genotypes were distinguishable because the amplification with F2/F4 primers ( Figure 1a) produced a 515 bp band with wt DNA and a 1928 bp band with DndhF DNA. 20 A faint 515 bp band was amplified, in addition to the stronger 1928 bp band, with DndhF tobacco DNA (Figure 1b). Also faint bands of NDH-F, J and K polypeptides and of Ndh activity were detected in DndhF tobacco (Figure 1c and d) indicating that it was not completely homoplastomic.…”

“…Again, the MDA level was approximately 135 and 85% higher in the Zymographic detection of the NADH dehydrogenase activity of the Ndh complex after native electrophoresis of 120 mg protein solubilized from thylakoid preparations of the fourth leaves of wt and DndhF tobacco 40 days after germination. Details of PCR amplification, the source of the specific antisera, preparation of extracts, electrophoretic and Western-blot methods and zymographic identification/determination are described in previous publications 15,16,20,25 and in Online Supplementary Information second and fourth leaves of DndhF than in the corresponding wt leaves. Even the eighth leaf, that had similar MDA levels in 72-day-old DndhF and wt, showed a small increase of MDA in 79-day-old flowering wt.…”

“…[14][15][16] In all, 11 genes (ndh) of plastid DNA encode [17][18][19] components of the Ndh complex which participates in a chlororespiratory electron transport chain 15,20,21 that regulates the redox state of transporters to optimize 22 the rate of cyclic electron transport. In chlororespiration, the Ndh complex provides electrons to plastoquinone, and the Mehler reaction (producing superoxide anion radical, Á O 2 À , which is converted to H 2 O 2 by SOD) and peroxidase/oxidase reactions remove electrons from reduced iron sulfur protein and reduced plastoquinone, respectively.…”