The activity of superoxide dismutases (SODs) at the early stages of wheat deetiolation

Abstract

Unbound tetrapyrroles, i.e. protochlorophyllide (Pchlide), chlorophyllide and chlorophylls, bring the risk of reactive oxygen species (ROS) being generated in the initial stages of angiosperm deetiolation due to inefficient usage of the excitation energy for photosynthetic photochemistry. We analyzed the activity of superoxide dismutases (SODs) in etiolated wheat (Triticum aestivum) leaves and at the beginning of their deetiolation. Mn-SOD and three isoforms of Cu/Zn-SODs were identified both in etiolated and greening leaves of T. aestivum. Two Cu/Zn-SODs, denoted as II and III, were found in plastids. The activity of plastidic Cu/Zn-SOD isoforms as well as that of Mn-SOD correlated with cell aging along a monocot leaf, being the highest at leaf tips. Moreover, a high Pchlide content at leaf tips was observed. No correlation between SOD activity and the accumulation of photoactive Pchlide, i.e. Pchlide bound into ternary Pchlide:Pchlide oxidoreductase:NADPH complexes was found. Cu/Zn-SOD I showed the highest activity at the leaf base. A flash of light induced photoreduction of the photoactive Pchlide to chlorophyllide as well as an increase in all the SODs activity which occurred in a minute time-scale. In the case of seedlings that were deetiolated under continuous light of moderate intensity (100 μmol photons m-2 s-1), only some fluctuations in plastidic Cu/Zn-SODs and Mn-SOD within the first four hours of greening were noticed. The activity of SODs is discussed with respect to the assembly of tetrapyrroles within pigment-protein complexes, monitored by fluorescence spectroscopy at 77 K.

Fig 1. Identification of SOD forms in wheat leaves.

Native polyacrylamide gel electrophoresis (PAGE) of SODs in 6-day-old wheat leaves: Lane 1A –etiolated, Lane 1B –etiolated and then deetiolated under white light (100 μmol photons m^-2 s^-1) for 4 hours, Lane 1C –grown under a 14 hour photoperiod (100 μmol photons m^-2 s^-1). The inhibitors were: NaCN–the inhibitor of Cu/Zn-SOD (Lanes 2A-2C) and H₂O₂ –the inhibitor of Fe-SOD and of Cu/Zn-SOD (Lanes 3A-3C). Each well was loaded with 25 μg of protein.

Fig 2. Distribution of SOD isoforms and Pchlide fluorescence along etiolated wheat leaves.

A) Experimental model: tip (T), middle (M) and basal (B) leaf fragments were collected for the experiments; B) Native PAGE for the detection of SOD in T, M and B leaf fragments; the pooled sample was used for electrophoresis. Each well was loaded with 25 μg of protein; C) The distribution of the relative activity of SOD isoforms in B, M and T etiolated leaf fragments; the data represents the mean ± SD, n = 3, * p < 0.05, ** p < 0.01; D) 77 K fluorescence emission spectra of T, M and B etiolated leaf fragments; excitation: 440 nm; E) The average ratio of fluorescence intensity at 654 nm to the intensity at 633 nm (F654/F633) read from fluorescence spectra measured for single leaf fragments, the data represents mean ± SD, n > 30, *** p < 0.001.

Fig 3. Flash-induced Pchlide photoreduction in 6-day-old etiolated wheat leaves.

A) Native PAGE of SOD isoforms in M fragments of leaves: E—etiolated, EFD—etiolated and then illuminated with a single flash followed by incubation in darkness for 30 minutes; The pooled sample was used for electrophoresis; each well was loaded with 25 μg of protein B) The distribution of the relative activity of SOD isoforms in M leaf fragments; the data represents the mean ± SD, n = 3 (** p < 0.01; *** p < 0.001); C) Fluorescence emission spectra measured at 77 K for M fragments of laves; Excitation 440 nm.

Fig 4. Deetiolation of 6-day-old etiolated wheat leaves under continuous white light (100 μmol photons m^-2 s^-1).

A) Chlorophyll content; B) Carotenoid content; Statistically insignificant differences in A and B (p > 0.01) were marked with the same letters (n = 3); C) Representative 77 K fluorescence emission spectra for M leaf fragments; Excitation at 440 nm; D) Scatter of the position of fluorescence emission maximum (indicated with a green grid line in C) in the course of greening, as representative to the pooled sample taken for SOD native PAGE analysis; E) Average relative activity of SODs for M leaf fragments calculated with respect to the total SOD activity in a single lane of gel; 3–5 gels were compared for each greening time; the data represents the mean ± SD, n = 3–5.

Fig 5. SOD in isolated plastids.

A) Fluorescence emission spectra measured at 77 K for plastids isolated from leaves treated in the same way as for SOD identification; excitation: 440 nm; greening time–indicated in the legend; EFD–plastids isolated from leaves that were etiolated for 6 days and then illuminated with a flash of white light; plastid isolation was begun directly after the flash or indicated greening times and lasted c.a. 30 min; they were isolated under a dim green light; B) The distribution of the relative activity of SOD isoforms in plastids isolated from M leaf fragments; the data represents the mean ± SD (n = 3, * p < 0.05; ** p < 0.01).