Polyphenol oxidase-mediated protection against oxidative stress is not associated with enhanced photosynthetic efficiency

Plant material

Wild-type Aa 4381 and low PPO mutant ABY-Aa 4521 red clover plants (Trifolium pratense L. 1753 ‘Milvus’; Lee et al., 2004) were grown from seeds in John Innes No. 3 compost in controlled environment conditions and were kept well-watered. For the cold/high light stress study, ten replicate plants were grown until 74 d old in a growth cabinet [20/16 °C day/night, 16-h photoperiod and a photosynthetic photon flux density (PPFD) of ∼300 µmol m^–2s^–1 at canopy level]. For each plant, two mature trifoliate leaves were randomly selected to monitor the carbon assimilation rate A and chlorophyll fluorescence (F_v/F_m, ΦPSII, q_P, q_N, NPQ; see below) with GFS-3000FL and PAM-2000 instruments, respectively (Heinz Walz GmbH, Effeltrich, Germany) as described below. These leaves were then excised, flash frozen in liquid nitrogen and stored at –80 °C. At the end of day 0, five randomly selected replicates were placed in an identical controlled environment cabinet except that temperature conditions were 2 °C day/night, 580 µmol m^–2s^–1 and a 16-h photoperiod. Control and stress-treated plants were monitored as before for a further 4 d. The entire experiment was repeated three times using new plant material on each occasion. Also, plants (three random replicates of each genotype as control and five stress-treated) were treated as above and their recovery monitored over 3 d following their return to control growth conditions at the end of day 4.

Where the effect of methyl viologen (MV) was to be tested, mature glasshouse-grown wild-type and mutant red clover plants were defoliated to ∼ 9 cm above soil surface, clonally replicated by division of entire plants and grown for at least 1 month under controlled environment conditions (20/16 °C day/night at 400 µmol m^–2s^–1 PPFD with a 12-h photoperiod). Clonal replicates were pre-assessed to minimize biological variation and improve the likelihood of identifying a PPO-related response to MV. Four clones of both mutant and wild-type were selected for similarity (allowing 10 % error) in terms of chlorophyll fluorescence (F_v/F_m, ΦPSII, q_P, NPQ; see below), total leaf protein and chlorophyll content, and total PPO activity. On the day of treatment, 1 h after the start of the photoperiod, each plant received both control and MV treatments by treating individual trifoliates which were selected at random, ensuring that there was no leaf–leaf contact. Treatments included an untreated control (no spray solution was applied) as well as 0·05 % Tween 20 containing 0, 1, 2·5, 5, 7·5 and 10 µm MV. The solutions were freshly prepared and applied as a saturating dosage of approx. 0·04 mL cm^–2 of trifoliate leaf surface area using an airbrush model 250 (Badger Air-Brush Co., http://www.badgerairbrush.com). Alternating between genotypes, the plants were sequentially removed from the growth cabinet, sprayed in a contained area and immediately returned to the illuminated cabinet. Chlorophyll fluorescence measurements (F_v/F_m, light-adapted F_v′/F_m′, q_N, NPQ) were taken with the Handy PEA fluorimeter (Hansatech Instruments, Norfolk, UK) as soon as the leaves had dried (2·5 h after treatment application) and again at 4·5 and 7·5 h after treatment application. Trifoliates from each genotype × treatment group were harvested, flash frozen in liquid nitrogen and stored at –80 °C until analysis.

Photosynthetic measurements

Unless otherwise stated, photosynthetic parameters were measured 5 h after the onset of the photoperiod, alternating between treatment and genotype. Leaf CO₂ assimilation rates were measured in vivo using a GFS-3000FL system (Heinz Walz, Effeltrich, Germany) incorporating a red–blue LED light source (Walz 3055-FL). Reference [CO₂] was set at 380 µmol mol^–1, the leaf temperature was maintained at 20 °C and vapour pressure deficit was maintained at 6·6 kPa (Long and Bernacchi, 2003; Purdy et al., 2013). Chlorophyll fluorescence parameters were measured using a PAM-2000 portable fluorometer (Heinz Walz) which was operated via the Data Acquisition software DA-2000 or PamWin to estimate the maximum quantum yield of PSII photochemistry (F_v/F_m), the quantum efficiency of PSII electron transport (ФPSII), the degree of photochemical (q_P) and non-photochemical quenching of chlorophyll fluorescence [estimated both as q_N (F_m–F_m′)/(F_m–F_o′) and NPQ (F_m–F_m′)/F_m′; Genty et al., 1989; Baker, 2008; Murchie and Lawson, 2013]. Randomly selected, mature and fully expanded leaves were dark adapted under treatment conditions for at least 20 min before measuring first the dark-adapted (F_v/F_m) and subsequently the light-adapted (A, ФPSII, q_P q_N and NPQ) parameters. Measurements of net carbon assimilation rate and light-adapted electron transport were undertaken, first at the growth irradiance of 300 µmol m^–2s^–1 after which PPFD was increased to 1300 µmol m^–2s^–1 (determined in preliminary experiments to be saturating for CO₂ assimilation). In each case measurements of light-adapted electron transport were collected once a steady-state carbon assimilation rate had been established (Wang et al., 2012). When using the Handy PEA fluorimeter, measurements of light-adapted and dark-adapted fluorescence were made to obtain F_v′/F_m′, F_v/F_m, NPQ and q_N. Plants were kept inside the growth cabinet and light-adapted minimal (F_o′) and maximal chlorophyll fluorescence yield parameters (F_m′) were measured first. The leaf clip shutter was then closed for dark adaptation while light-adapted measurements of the remaining trifoliates continued. Once all were measured and the leaf measuring areas had sufficiently dark adapted, F_o and F_m were measured. Dark-adapted F_v/F_m, light-adapted F_v′/F_m′, NPQ and q_N were calculated as before.

Protein extraction and quantification

Frozen leaf samples were ground to a fine powder in liquid nitrogen. Protein was extracted by addition of 5 mL g^–1f. wt of extraction buffer (0·2 m Na₂HPO₄, pH 7 and 71 mm ascorbic acid, containing 0·1 % Triton X-100 and 0·5 % of protease inhibitor cocktail; Sigma protease cocktail plant, Sigma Aldrich, Poole, UK), which was further ground to a smooth homogenate. If the extract was to be used for measurements of PPO activity the Triton X-100 and protease inhibitor were omitted from the extraction buffer. Where required, aliquots of the homogenate were taken for chlorophyll quantification (Arnon, 1949). The homogenate was then centrifuged at 10 000 g for 10 min at 4 °C. The protein content of the clarified homogenates was estimated spectrophotometrically (Bradford, 1976) with reference to a bovine serum albumin standard curve.

Determination of PPO activity

Prior to each experiment, the PPO phenotype of the wild-type and mutant plants was confirmed either by a rapid PPO phenotype screening assay adapted from Winters et al. (2008) or by measuring total (active + latent) PPO enzyme pool of mature leaves under control growth conditions. For the rapid PPO phenotype screening assay, protein was extracted in a liquid nitrogen pre-cooled pestle and mortar in McIlvaine buffer (pH 7) at a ratio of 5 mL g^–1f. wt. After centrifugation at 10 000 g for 10 min at room temperature, the colour of the supernatant was scored; a brown colour indicated a wild-type phenotype, while retention of the initial green colour indicated a low PPO mutant phenotype.

Alternatively, PPO activity in protein extracts which had been desalted to McIlvaine buffer, pH 7, on pre-conditioned columns of Bio-Gel P6DG (Bio-Rad Laboratories, Hemel Hempstead, UK) was measured spectrophotometrically according to Winters et al. (2008), with minor modifications. Standard reactions were carried out in 1·53 mL containing 20 µL of desalted extract, 0·15 mm copper sulphate and 9·87 mm 4-methylcatechol in McIlvaine buffer (pH 7) alone or including 0·26 % (w/v) SDS. The reaction was initiated by addition of 4-methylcatechol and the change in absorbance at 420 nm was monitored during the initial 25 s. Rates were calculated from the linear phase of the curve as ΔA min^–1 with SWIFT II software (Biochrom, Cambridge, UK). One unit of PPO enzyme (1 U) was defined as the amount of enzyme that produced 1 µmol of quinone per minute and is expressed per mg protein in the desalted PPO enzyme extract. A conversion factor of U = 2·717 × Δ optical density was used for the calculations (Alder and Magnussen, 1959; Lee et al., 2008).

Protein characterization

SDS–PAGE was performed according to Laemmli (1970) with either hand-cast 12·5 % single percentage gels or linear gradient pre-cast gels (4–20 % Mini-PROTEAN TGX gels or 10–20 % Ready Gel Tris–HCl gels; Bio-Rad). Protein was electrophoretically transferred to nitrocellulose filters and PPO was detected immunologically with anti-red clover (T. pratense) PPO1 primary antiserum (a kind gift from M. L. Sullivan; Sullivan et al., 2004) used at 1 : 1000 dilution and alkaline phosphatase-conjugated swine anti-rabbit secondary antiserum (Dako, Glostrup, Denmark) used at 1 : 2000 dilution. Cross-reactions were detected as dark blue formazan bands formed by reduction of nitrobluetetrazolium and were recorded digitally with a GS-800 calibrated densitometer (Bio-Rad).

Alternatively, samples containing 4 µg protein were diluted to a final volume of 200 µL with extraction buffer (0·2 m Na₂HPO₄, 71 mm ascorbic acid, pH 7) and were transferred to nitrocellulose with the Bio-Dot SF Microfiltration Apparatus (Bio-Rad).

Oxidized proteins were detected using the Oxyblot Protein Oxidation Detection Kit according to the manufacturer’s instructions (Millipore, Watford, UK). Cross-reactions were detected by enhanced chemiluminescence and recorded by exposure to blue-light-sensitive film (GE Healthcare Life Sciences, Little Chalfont, UK).

Statistical analysis

Statistical analyses of photosynthetic and biochemical data were performed with GenStat 13.2 (VSN International, Hemel Hempstead, UK) using restricted maximum likelihood (REML) or ANOVA functions. Where necessary data were transformed and normal distribution confirmed by testing for homogeneity (Bartlett’s test) before statistical analysis. The significance of the observations was determined using a Bonferroni multiple comparison test (adjusted P < 0·05/n with n number of comparisons).

Effect of presence of PPO activity on photosynthesis under cold stress

The effect of exposure to and recovery from a cold/high light stress was determined in leaves of wild-type and mutant plants. No significant differences were observed between dark-adapted F_v/F_m of wild-type and mutant red clover at any time under control growth conditions (Fig. 1A). In contrast, a progressive decrease was observed for both lines immediately after initiation of the cold/high light stress treatment at the end of day 0. The F_v/F_m values of wild-types and mutants during exposure to the stress were not significantly different except for day 4 (Fig. 1A). When measured under control growth conditions, the CO₂ assimilation rate A was typically greater in mutants than in wild-types although this was only significant on day 2 (Fig. 1B). As a result of the stress treatment, A declined rapidly and was complete within 1 d of treatment, but there were no significant differences between the lines at any point. This effect was observed under both growth and saturating irradiances (Fig. 1B, Supplementary Data Fig. S1A). Similarly, the ΦPSII of both lines was comparable under control growth conditions and declined in response to the cold and high light stress treatment (Fig. 1C). The decline in ΦPSII was attributed largely to an early increase in q_N (day 1; Fig. 2A) and a later (day 3) decrease in q_p under growth irradiance (Fig. 2B). Differences in q_N between control and treated mutants were greater than between control and treated wild-types (Fig. 2A). Treatment differences in q_N were less obvious when measurements were made under saturating irradiance (Supplementary Data Fig. S2). Measurements of ΦPSII, q_N and q_P under saturating irradiance did not amplify any potential genotypic differences regardless of treatment (Figs S1B, S2). Regardless of measurement irradiance, no significant differences between lines or treatments were observed when non-photochemical quenching was calculated as NPQ (data not shown).

Recovery of dark-adapted F_v/F_m and ΦPSII to levels observed in untreated control was rapid upon return to control growth conditions and was established within 24 h after return to optimal conditions. Differences between the wild-type and low PPO mutant line were not observed (Fig. 1A, C). The CO₂ assimilation rate recovered equally rapidly for both genotypes but only reached 73·9 % of the control values (Fig. 1B).

Effect of cold stress on PPO activity, protein and chlorophyll content

Measurements of total PPO activity levels (active + latent) confirmed the PPO phenotype of the two red clover genotypes, with significantly higher activities being recorded for wild-types (48·98 U of PPO enzyme mg^–1 protein) than mutants (0·25 U of PPO enzyme mg^–1 protein) regardless of the treatment conditions (adjusted P < 0·0083). No change in the PPO activities of either mutants or wild-types was observed regardless of treatment, such that these differences between the lines persisted throughout the stress treatment period, with no evidence for either loss of activity from wild-type or induction of activity in mutants (Fig. 3). In the wild-type, total activity typically exceeded the active pool by two-fold (Fig. 3).

Immunodetection of PPO confirmed that the lack of activity in the mutants was due to absence of the PPO protein, detected at molecular weights of 59·9 ± 1·3 and 46·7 ± 1·0 kDa (Fig. 4A). No changes in PPO protein abundance were observed in response to treatment or recovery (Fig. 4A) and the total protein profile was similar between the two red clover lines regardless of treatment (Fig. 4B). Similarly, apart from day 7, there were no significant differences in chlorophyll content between the lines under optimal growth conditions as a result of the stress treatment and subsequent recovery (Supplementary Data Fig. S3). Over the duration of the experiment, the chlorophyll content averaged 1·89 ± 0·03 mg chlorophyll (Chl_a + Chl_b) g^–1f. wt regardless of line or treatment (Fig. S3) and the Chl_a/Chl_b ratio was similar for wild-type and low PPO mutants under control and stress conditions and in the subsequent recovery phase (data not shown).

Effect of PPO on photosynthetic response to oxidative stress induced by MV

Rapid phenotype screening of the two mother plants prior to cloning was used to confirm their wild-type and low PPO mutant phenotype. Individual trifoliate leaves from the clonal replicates were exposed to MV and the effect on photosynthesis was assessed. In the wild-type an effect of MV on photosynthetic parameters was observed at doses between 2·5 and 10 µm and was most apparent 7·5 h after treatment (Fig. 5) (data for 2·5 and 4·5 h not shown). In contrast, the mutant was largely unaffected by MV treatment (Fig. 5). At MV doses of 2·5 µm and above, wild-types exhibited lower light-adapted F_v′/F_m′ and higher NPQ than the mutant. The high variability seen in the data could have been a result of high genetic diversity due to complexity of the PPO gene family and the fact that red clover is an outbreeder. MV treatment had no effect on measurements of active PPO (mean of 3·18 ± 0·63 and 0·24 ± 0·03 U mg^–1 protein for the wild-type and mutant, respectively) despite a linear increase in total (active + latent) PPO activity levels in leaves of wild-type plants (Fig. 6A). The total PPO activity of the wild-type leaf used for 0 µm treatment (spray treatment control) appears to be an outlier, but it was included in the interest of clarity. It is possible that in this case despite selection of leaves at a similar developmental stage (mature fully expanded) for treatment, the measurement made on the trifoliate leaves treated with 0 µm MV could have been of a different chronological age to the others and hence contained PPO activity inconsistent with the other samples (Lanker et al., 1987; Goud and Kachole, 2012).

The presence of PPO was associated with protein quality, as a higher degree of protein oxidation was consistently observed in leaf extracts from mutants than was present in samples from wild-type leaves (Fig. 7A). A comparison of the protein oxidation profiles in wild-type and mutant leaves indicated that the increased intensity of protein oxidation in mutants was not the result of additionally oxidized protein species, but rather because of an increase in oxidation of those proteins targeted in the wild-type. These proteins had estimated molecular weights of 124 ± 2·03, 115 ± 2·83, 106 ± 1·72, 58·5 ± 1·12, 51·3 ± 0·94, 46·8 ± 0·84 and 27·5 ± 1·07 kDa (Fig. 7B). Specificity of the detection was confirmed by lack of signal from comparable negative controls (data not shown).