- Email: [email protected]
Plasticity of photosynthesis after the ‘red light syndrome’ in cucumber
Accepted Manuscript Title: Plasticity of photosynthesis after the ‘red light syndrome’ in Cucumber Author: Govert Trouwborst Sander W. Hogewoning Olaf van Kooten Jeremy Harbinson Wim van Ieperen PII: DOI: Reference:
S0098-8472(15)00085-4 http://dx.doi.org/doi:10.1016/j.envexpbot.2015.05.002 EEB 2931
To appear in:
Environmental and Experimental Botany
Received date: Revised date: Accepted date:
4-2-2015 28-4-2015 11-5-2015
Please cite this article as: Trouwborst, Govert, Hogewoning, Sander W., Kooten, Olaf van, Harbinson, Jeremy, Ieperen, Wim van, Plasticity of photosynthesis after the ‘red light syndrome’ in Cucumber.Environmental and Experimental Botany http://dx.doi.org/10.1016/j.envexpbot.2015.05.002 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Plasticity of photosynthesis after the ‘red light syndrome’ in Cucumber Govert Trouwborst1,2, Sander W. Hogewoning1,2, Olaf van Kooten1, Jeremy Harbinson1, Wim van Ieperen1* 1
Wageningen University, Horticulture and Product Physiology Group, PO box 630,
6700AP Wageningen, the Netherlands 2
Present Address: Plant Lighting B.V., Veilingweg 46, 3981PC, Bunnik, The
Netherlands
*Corresponding author e-mail: [email protected]; Tel: +31 317-413281. Visiting adress: Droevendaalsesteeg 1, 6708PD, Wageningen, The Netherlands. Running title: Plasticity of photosynthesis after the ‘red light syndrome’ Date of submission: number of figures: number of tables: total word count:
Key words: chlorophyll fluorescence, Cucumis sativus, energy dissipation model for PSII, Light emitting diodes (LEDs), leaf acclimation, light quality, photosynthetic acclimation Abstract The quantum efficiency of photosynthesis of leaves is wavelength dependent and peaks in red (620-670 nm). However when Cucumber plants are raised under pure red light, leaf photosynthesis becomes severely impaired. This “red light syndrome” has been characterized before by a low Fv/Fm, unresponsive stomatal conductance (gs), a low photosynthetic capacity (Amax). It is not known if the syndrome also occurs in fully developed leaves that are exposed to pure red light after reaching maturity and if initially injured leaves can recover from the syndrome. This study investigates the plasticity leaf photosynthetic apparatus after inducing or releasing the “red light syndrome” in leaves of young Cucumber plants. The plants were grown under pure red (R) or mixed red/blue (RB; 70%R) LED light and subsequently exposed to RB (R/RB) and R (RB/R) light (100 µmol PPFD.m-2.s-1,
16h photoperiod) or kept at their initial growth spectrum (R/R) and (RB/RB). Acclimation of fully developed leaves was monitored with gas exchange and chlorophyll fluorescence (CF) over a period of 8 to 10 days after the shift. After switching to RB, R injured leaves recovered from photodamage within 4 days. Photosynthetic capacity (Amax) and gs partly recovered, but did not restrict the net CO2 assimilation rate at growth irradiance (A100), which increased to the same level as in healthy (RB/RB) leaves. After imposing injurious R to healthy mature leaves, they transiently developed signs of the red light-syndrome: a slightly decreased Fv/Fm and more severely reduced Amax and gs. However, A100 did not significantly decrease. CF quenching analysis revealed an potentially harmful increased quantum yield of non-regulated non-photochemical energy loss in PSII under R, which was higher in leaves that developed under R than in leaves that were exposed to R after reaching maturity. We conclude that exposure to pure red light is harmful to photosynthetic systems in both developing and developed leaves of cucumber, but the effect on CO2 assimilation rate and Fv/Fm is much more severe in developing leaves than in mature leaves at low growth irradiance. Chloroplasts of previously R light injured leaves can recover within a few days after releasing from R light, while stomatal conductance and other (partial) morphologically determined leaf factors do not completely acclimate. List of abbreviations A100: net photosynthetic rate at growth irradiance; An: net photosynthetic rate; Amg: maximum gross photosynthetic rate; Amax: maximum net photosynthetic rate; CF: chlorophyll fluorescence; Ci: internal CO2 concentration; ETR: electron transport rate; Fv/Fm: maximum quantum efficiency of PSII photochemistry in the dark; Fv’/Fm’: maximum quantum efficiency of PSII photochemistry in the light; gs: stomatal conductance; Jmax: maximum electron transport rate; LMA: leaf mass per area; Norg: organic nitrogen within the leaf (g m-2);
NPQ: non-photochemical quenching; PAR: photosynthetic active radiation; PNUE: photosynthetic nitrogen use efficiency QA: primary quinone acceptor of PSII; qP: PSII efficiency factor; RD: dark respiration rate; VCmax: Maximum carboxylation rate; α: initial quantum efficiency; θ: parameter for curvature; ΦPSII: PSII operating efficiency; ΦNPQ: regulated energy dissipation ΦNO: non regulated energy dissipation including fluorescence emission Introduction Light is an indispensable energy source for plant growth and is usually supplied by the Sun. Artificial light, however, is used as an energy source for plants in certain situations, such as in greenhouses at high latitudes where natural sunlight severely limits plant growth in the late autumn – early spring period (Trouwborst et al., 2010), or in growth cabinets for research purposes or in plant factories for urban farming. Many different lamp types are used to generate artificial light for plant growth and most of them produce a broad emission spectrum within the PAR range, although this is often superimposed upon distinct emission lines such as in the case of gas discharge lamps or fluorescent tubes (McCree, 1972b). All lamps have in common that they are spectrally very different from sun light, and this considerably influences plant morphology (Hogewoning et al., 2010a). Today, also light emitting diodes (LEDs) are considered as viable sources of plant growth light (Hogewoning et al., 2007; Massa et al., 2006; Massa et al., 2008; Trouwborst et al., 2010). In contrast to the broad spectrum light sources LEDs emit light in a narrow wavelength band (typically 25-50 nm half-power bandwidth). Of particular interest is the influence of wavelength on leaf photosynthesis. Early work on the spectral quantum efficiency for leaf photosynthesis has shown it is highest in the red region of the spectrum (Evans, 1987; Inada, 1976; McCree, 1972a). However, highest instantaneous photosynthesis does not necessarily result in optimal photosynthesis and growth in the long term. Leaves of cucumber plants that were grown under pure red LED-light (100 µmol m-2 s-1; 640 nm; R-grown leaves)
developed a low dark-adapted
Fv/Fm (Hogewoning et al., 2010b), which is
indicative for photodamage (Baker, 2008). This ‘red light syndrome’ is further characterized by unresponsive stomata, a low photosynthetic capacity, low photosynthetic nitrogen use efficiency, a low leaf mass per area and impaired growth (Hogewoning et al., 2010b). Similar results were observed with tomato (unpublished results), but none of these effects occurred in leaves that were grown under mixed red (640 nm) and blue (450 nm) light (RB-grown leaves). It appears therefore that exposure to red light alone during leaf development influences photosynthesis at different functional levels extending from the thylakoid to the whole leaf level. It is unknown if the adverse effects of red light during leaf development are structural and persist after a change in light spectrum or can be partially or completely overcome. Based on how photosynthesis acclimates to changes in light intensity, different extents of acclimation due to changes in light spectrum might be expected at thylakoid and whole leaf level. The objective of this study was to investigate the plasticity of photosynthesis at different functional levels (thylakoid to whole leaf) in response to the induction and release of the ‘red light syndrome’ in cucumber. This was done by changing the spectrum of incident light from pure red (640nm) to mixed red and blue light (640 & 450nm) on leaves with the ‘red light syndrome’, and vice versa on healthy leaves (without the ‘red light syndrome’).
At the thylakoid level we investigated changes in fate of excitation energy in PSII using chlorophyll fluorescence (Cailly et al., 1996; Genty et al., 1996; Hendrickson et al., 2004) before, during and after changes in light spectrum to assess changes in energy dissipation between photosynthetic electron transport (ΦPSII) and regulated (ΦNPQ) and constitutive energy dissipation processes (ΦNO). At the leaf level we used gas exchange measurements to determine changes in photosynthetic light- and CO2 response curves before, during and after changes in light spectrum, and we measured several leaf anatomical parameters. Materials & methods Plant material and growth conditions One week old seedlings (Cucumis sativus “Hoffmann’s Giganta”) were transplanted to a hydroponic growth system in a climate chamber as described in Hogewoning et al. (2010b) and further grown horizontally to avoid shading of older by younger
leaves. Immediately after transplanting, the plants were subjected to the following light treatments: 100% red LED (638 nm dominant wavelength) light (R) or a mixture of 70% red and 30% blue LED (450 nm dominant wavelength) light (RB) to allow full leaf development under distinct different light spectra. After three weeks, when the second leaves were fully expanded, half of the plants per light treatment were changed to the other light spectrum, resulting in 4 light treatments: 2 with a distinct change in spectral composition (RB/R and R/RB) and two controls (R/R or RB/RB). Photosynthetic photon flux density (PPFD) and duration of photoperiod were the same for all light treatments: 100±5 μmol m-2s-1 for 16h a day (Li-190, LiCor inc., Lincoln, NE, USA). Temperature and relative humidity inside the climate room were respectively 25°C and 70%.
Gas exchange and chlorophyll fluorescence measurements Measurements on the second fully expanded leaf started on the day at which half of the plants were subjected to the change in light spectrum (day zero). Photosynthetic irradiance-response curves were repeatedly measured over a time course of 10 days with a portable gas analyzer (LI-6400 with fluorescence head and standard LEDirradiance light source; Li-Cor Inc., Lincoln, NE, USA). Photosynthetic CO2response (A-Ci) curves were measured on the first and the 7th or 8th day of this period. The blue to red ratio of the actinic light in the clamp-on leaf chamber was always set equal to the growth irradiance of the particular leaf subjected to measurements. However above 900 μmol m-2 s-1 the blue light fraction in the Li-Cor CF measuring head gradually decreased from 30% to 18% at 1600 μmol m-2 s-1 due to limitations of the blue light source in the leaf chamber. Leaf chamber temperature, air flow speed and the CO2-concentration were set at 25 oC, 250 μmol s-1, and 380 μmol mol-1 respectively. Air humidity in the leaf chamber was kept similar as during growth. Photosynthetic rates (including correction for diffusion leaks) and chlorophyll fluorescence measurements were determined as described in Trouwborst et al., 2011.
Determinations of leaf parameters At the start and the 7th and 8th day of the experiment, leaf samples were taken to measure chlorophyll, Leaf mass per area (LMA) and organic nitrogen (Norg) as described in Trouwborst et al. (2010). The leaf absorptance spectrum was measured
in single nanometer steps according to Hogewoning et al. (2010b), and the quantum flux absorbed by the leaf was calculated by multiplying the absorptance spectrum by the growth-light spectrum.
Calculations and statistics Maximum PSII efficiency in light (Fv’/Fm’), PSII operating efficiency (ΦPSII), PSII efficiency factor (qP) (Baker, 2008) and the electron transport rate (ETR) at growth light level were calculated according to Baker et al. (2007). Fo’ was calculated according to Oxborough et al. (1997). For the calculation of ETR, we assumed an excitation balance of 0.5 and corrected for the measured leaf absorption and ΦPSII. The energy dissipation in PSII was calculated according to Genty et al. (1996) and Cailly et al., (1996). The quantum yield of PSII electron transport is ΦPSII, the quantum yield of regulated thermal dissipation, ΦNPQ, is calculated as Fs/Fm’Fs/Fm and the quantum yield of non-regulated energy dissipation, ΦNO, is calculated as Fs/Fm (for a more complete consideration of these quantum yields see Kramer (2004),
Hendrickson (2004) and Murchie and Harbinson (2014)).
A modified
version of the Farquhar, Von Caemmerer and Berry (FvCB) model (Farquhar et al., 1980) was fitted to the A-Ci response data. We estimated Jmax and Vcmax normalized to 25°C using the non-linear fitting procedure NLIN in SAS (release 9.1.3; SAS institute, Cary, NC, USA) described in detail according to Trouwborst et al. (2011). A non-rectangular hyperbola (Thornley, 1976) was fitted to the photosynthesis irradiance-response data using the non-linear fitting procedure NLIN in SAS to determine dark respiration (RD), maximum photosynthetic rate (Amax), light-limited quantum efficiency of CO2 assimilation (α) and the scaling constant for the curvature (θ) of the leaves in the different treatments. All treatments were repeated four times (2-4 plants per replicate). Fisher’s LSD was used to make post-hoc multiple comparisons among spectral treatment means from significant one-way analysis of variance (ANOVA) tests (P <0.05). Results Photosynthetic responses at the leaf level As expected all leaves, which developed under pure R light, vastly suffered the redlight syndrome at maturity. They showed a reduced CO2 assimilation capacity (Amax), reduced CO2 assimilation at growth irradiance (A100), a reduced Fv/Fm (Fig. 1)
and a decreased gs (Table 1) compared to leaves that were grown under a combination of red and blue (RB) LED light. Immediately after changing the growth light spectra (from R to RB and vice versa) leaves started to acclimate. This acclimation lasted approximately 7-8 days after which Amax, A100, gs and Fv/Fm were stabilized at new levels (Fig. 1). Releasing the injured leaves from R light (R/RB-leaves) resulted in an approximately 2-fold increase of Amax, while exposure of non-injured leaves to the R-spectrum (RB/Rleaves) caused almost halving of Amax (see also Fig. 2A). Similar responses were observed for gs at growth irradiance, but the effect on Ci was very limited (Table 1). Changes in Fv/Fm were much stronger after alleviating from R light (R/RB) than vice versa (RB/R): full recovery of Fv/Fm occurred in R/RB leaves while only a very small reduction was shown in RB/R leaves (Fig. 1C). Interestingly, the effect of spectrum changes on A100 differed between the release from and the change to R light: A100 in R/RB leaves raised to the same level as in RB/RB leaves within a time course of 4 days, while A100 in RB/R leaves did not change at all after the spectrum change (Fig. 1B). At the end of the experiment, both the PSII operating efficiency (ΦPSII) and estimated ETR at growth irradiance were reduced in R/R compared to RB/RB grown leaves (Table 1). After the spectrum change from R to RB both parameters increased to the level displayed by RB/RB leaves, while only small reductions in ΦPSII and ETR were observed after a spectrum change to injurious R-light (Table 1).The lightlimited quantum efficiency of CO2 assimilation (α) was reduced in R-grown compared to RB-grown leaves, but completely recovered after a change from an R to an RB-spectrum. No reduction in α was observed after the opposite spectrum change from RB to R light (Table 1). Respiration in darkness (RD) was slightly higher in RB/RB and R/RB-leaves than in R/R and RB/R-leaves (Table 1). The maximum carboxylation rate allowed by Rubisco (VCmax) and maximum rate of linear electron transport, i.e. that through PSII, (Jmax) showed similar patterns across the light treatments as Amax. CF quenching analysis and the capacity of leaves to cope with excess energy At growth irradiance and at the end of the acclimation period, the estimated quantum yield of regulated non-photochemical energy loss in PSII (ΦNPQ) was very small (≤0.051), though significant differences were observed between all treatments (Table
1). The quantum yield of non-regulated non-photochemical energy loss in PSII (ΦNO) was much larger (≤0.35) and differed significantly between the spectral treatments: ΦNO was ~50% higher in R/R leaves compared to RB/RB leaves at growth irradiance, and full adjustment of ΦNO to the level in healthy RB/RB leaves was observed in R/RB leaves after the change in spectrum. Exposing healthy leaves to pure R-light, on the other hand, resulted in only a weak increase of ΦNO at growth irradiance at the end of the acclimation period (Table 1). Analysis of CF quenching parameters was extended beyond growth irradiance to investigate further differences in the capacity of leaves to cope with excess energy between the spectral treatments. Differences in PSII efficiency between treatments increased with increasing irradiance (Fig. 2B) with the least steep decrease in RB/RB and the steepest decrease in R/R-leaves. The parameters Fv’/Fm’ (the maximum quantum yield of PSII in light; Fig 2C) and qP (the PSII efficiency factor (Baker, 2008); Fig. 2D), which are often used to assess to what extent changes in PSII operating efficiency are attributable to changes in non-photochemical quenching (NPQ) or to changes in the openness of PSII reaction centers (Baker, 2008), showed some conspicuous differences between the spectral treatments. The decrease of Fv’/Fm’ with irradiance was low, and smaller and less discriminating between spectral treatments than the accompanying decrease in qP with irradiance. Decreases in ΦPSII with increasing irradiance were increasingly due to differences in qP and hardly due to changes in Fv’/Fm’ (Fig. 3A and B; n.b. ΦPSII = Fv’/Fm’ × qP). The increase in quantum yield of regulated non-photochemical energy loss in PSII (ΦNPQ) with irradiance paralleled the decrease in Fv’/Fm’ with irradiance (Fig 2C, E) and small differences were observed between the spectral treatments: ΦNPQ converged to similar values at high irradiances in RB/RB and R/RB leaves, while substantially lower values for ΦNPQ were measured at high irradiance in leaves that ended in R light, with R/R leaves having a lower maximum value of ΦNPQ than the RB/R leaves (Fig. 2CE). The quantum yield of non-regulated non-photochemical energy loss in PSII (ΦNO) was highest in R/R leaves at all irradiances. All leaves, which ended in RB light (R/RB and RB/RB), showed an approximately similar, limited increase in ΦNO at low irradiance, which was saturated above 300 µmol PPFD.m-2.s-1, while ΦNO in R/RB leaves did not differ from RB/RB and R/BR leaves at low irradiance but increasingly deviated with increasing irradiance (Fig. 2F).
Acclimation of leaf parameters LMA, Norg, chlorophyll content and leaf absorption were lowest for R/R-leaves and highest for RB/RB-leaves. Acclimation to the other spectrum resulted in intermediate values (Table 2). The chlorophyll a/b ratio was significantly higher in RB/RB-leaves than in R/R- and RB/R-leaves. The %Norg did not differ between treatments. Photosynthetic Nitrogen Use Efficiency (PNUE) and Amax/LMA were the lowest for R/R- and RB/R-leaves. Amax/chl was lowest for R/R leaves and highest for RB/RBand R/RB-leaves, while RB/R-leaves showed intermediate values. Discussion From previous research on blue-light dose-responses it has been concluded that blue light is qualitatively required for the development of normal photosynthetic functioning leaves in cucumber (Hogewoning et al., 2010b). Also in this study the absence of blue light in the growth light spectrum caused significant reductions of Amax gs and Fv/Fm, indicating damage to PSII. Present results are in full agreement with previous research and in this study we expand towards a more detailed analysis of the ‘red light syndrome’ and its dynamics after changes in the growth light spectrum. This research has been conducted under light limiting conditions (relative low irradiance) where plants are in their light limited range of photosynthesis and excess light energy is low. These circumstances are common for climate chambers used in plant research and in growth systems for vertical farming (plant factories) in which LEDs increasingly are implemented as light sources. We here present results and CF quenching analysis at growth irradiance, which is of explanatory value for the changes that occurred in photosynthesis in the leaves in this study. We also present the CF quenching analysis at further increasing irradiance, which displays changes in the photosynthetic systems that are not fully expressed at growth irradiance.
Effects of R light at growth irradiance A reduced Fv/Fm is usually associated with photodamage of PSII (photoinhibition) or with slowly reversible down-regulation of PSII, which both occur in response to a wide range of stresses (Baker, 2008; Demmig-Adams and Adams, 2006). However, here the usual causes for a reduced Fv/Fm did not apply: growth irradiance was low (100 µmol m-2 s-1) and the plants were neither nutrient, temperature nor water stressed. Sink limitation related down regulation of PSII was previously discounted
for (Hogewoning et al., 2010b). At growth irradiance, only leaves that developed and were kept under pure R light (R/R) showed reduced photosynthesis (A100;Table 1). This reduction in A100 can be largely attributed to changes at the chloroplast level, because in all spectral treatments growth irradiance was within the light-limited range of photosynthesis (Fig. 2A), and because the observed differences in stomatal conductance (up to ~60% lower in R/R compared to RB/RB) did not result in large differences in Ci (Table 1). Observed changes in A100 between R/R and RB/RB grown leaves were in close agreement with estimated reductions in estimated ETR: both were approximately 26% lower in R/R (Table 1). The smaller estimated ETR is mainly due to a decreased ΦPSII (23% lower) as the decrease in leaf absorption was very limited (4% lower; Table 1&2). It is well known that gs can be reduced under pure R light compared to RB (Sharkey and Raschke, 1981; Goins et al., 1997), probably because blue light stimulates additional opening of stomata, mediated by the blue-light absorbing carotenoid zeaxanthin (Zeiger et al., 2002). Recently we reported that pure R light during leaf development significantly decreased stomatal density in cucumber leaves, largely due to a positive effect on epidermal cell size (Savvides et al., 2012). The combination of these two effects account for the strongly reduced gs in leaves that were grown under R/R compared to RB/RB. The intermediate values for gs in the R/RB treatment and the RB/R treatment after full acclimation were most likely caused by aperture responses to the final light spectrum, restricted by the stomatal density that was set during leaf development. Despite these changes in gs, the effect on CO2 assimilation was limited due to the negligible changes in Ci at the low light intensity (Table 1). The differences between R/R- and RB/RB-leaves in LMA, Norg, chlorophyll a/b ratio, Vcmax, Jmax and PNUE (Table 2) have already been discussed in detail by Hogewoning et al. (2010b). The changes in these parameters after changes in the light spectrum (i.e. in
RB/R and R/RB leaves) displayed changes that were
consistent with those usually observed during acclimation to low (RB to R) or to high light (R to RB) conditions (e.g. Pons and De Jong-van Berkel, 2004; Pons and Pearcy, 1994; Table 1&2). After releasing injured leaves from R or exposing healthy developed leaves to R leaves started to adjust their photosynthetic characteristics, displaying a great degree of plasticity. Changing the light spectrum from R to RB light caused α, A100
and ETR to fully recover to the level of RB/RB-leaves, whereas leaf absorption, LMA, Norg, gs and Amax also increased but remained lower than in RB/RB-leaves. The latter reveals limitations on the plasticity for acclimation, which could be partly due to unchangeable differences in leaf morphology (a lower LMA at R/R than at RB/RB) which were induced during previous leaf development under R light. Although not directly measured, leaf thickness was probably reduced in leaves that developed under R light, consistent with the lower LMA and the lower stomatal density (at similar stomatal index) and larger epidermal cell size observed in R grown compared to RB grown cucumber leaves in a previous experiment (Savvides et al., 2012). Normalizing Amax for LMA (i.e. expressing it at dry weight basis) resulted in a calculated full adjustment of Amax at dry weight basis, which was also found for Amax normalized for chlorophyll content and PNUE (Table 2), supporting the suggestion that leaf structure is limiting for Amax. Oguchi and co-workers (2003) concluded earlier that Amax of low light developed leaves, which are later exposed to high light, are physically restricted by the cell size of the leaves and the unoccupied horizontal cell surface along which the chloroplasts can expand. As boundaries in cell size and maximal stomatal conductance are set during the leaf developmental phase (Schoch et al., 1980; Sims and Pearcy, 1992), the implication for later plasticity in acclimation is evident. We conclude that the limitation of assimilation rate at growth irradiance in R/R-leaves is mostly due to a disturbed light reaction or photosynthetic metabolism. Limitations due to stomatal effects or leaf light absorption clearly are of minor importance.
Chlorophyll fluorescence quenching and energy dissipation analysis None of the leaves investigated in present study displayed a serious light-induced decrease in Fv’/Fm’ with increasing irradiance (Fig. 2C) or with decreasing PSII (Fig. 3B). This implies that all these leaves had a relatively small ability for NPQ (Bilger and Björkman, 1990; Genty et al., 1990; Demmig-Adams et al., 1996), which is normal for leaves of fast growing species that developed under low light conditions (Demmig-Adams and Adams, 2006) such as the cucumber leaves in present study. Of the variation in ΦPSII at growth irradiance between spectral treatments (R/R vs. RB/RB) ~60% was attributable to differences in qP, a measure of impact that PSII trap closure has on the light-use efficiency for PSII electron transport (Murchie and
Harbinson, 2014) and ~40% to differences in Fv’/Fm’ (changes in non-photochemical quenching (NPQ)) (Table 1). However, with increasing measuring light intensity the difference in qP between spectral treatments became much more pronounced than the differences observed in NPQ (Fv’/Fm’, Fig. 2C and D). Though the actual effects in our experiments were small due to the low growth irradiance, it implies that leaves developing in pure R light became more vulnerable to photodamage, which seems consistent with a greater extent of ‘shade acclimation’ (Hogewoning et al., 2010b; Lichtenthaler et al., 1980; Matsuda et al., 2004; Matsuda et al., 2008; Voskresenskaya, 1979). With irradiance increasing far beyond growth irradiance and further increasing excess light energy, ΦNO increased in the R/R leaves to a level which was 60% higher than in RB/RB-leaves (Fig. 2F). This increasing fraction of ΦNO implies an increasing susceptibility for photodamage of leaves under R light when they are exposed to higher growth irradiances than in current experiment. A correlation between the rate of photodamage and excitation dissipation by basal dissipation mechanisms (ΦNO) has been shown (Kato et al., 2003) though in this case ΦNO was calculated using a model (Demmig-Adams et al., 1996), which is less robust than the model used here. More generally, however, the photoprotective effect of the qE component of NPQ (which gives rise to ΦNPQ) has been amply shown (eg Bilger and Bjorkman, 1990; Krause and Behrend, 1986; Niyogi et al., 1998; Muller et al., 2001; Li et al., 2002; Ruban et al., 2012). If, together with photochemistry (ΦPSII) dissipation by the qE mechanism (ΦNPQ) is protective, then a deficiency of both of those, which will result in dissipation by the less competitive, slower mechanisms that give rise to ΦNO, will result in more photodamage. Photodamage is, however, complex. While some photodamage is due to damaging side reactions occurring within photosystem II (the kind that NPQ can protect against; eg Vass, 2011), photodamage can also be caused by direct effects of light on the Mn cluster of the oxygen evolving cluster of PSII (eg Takahashi and Badger, 2011; Hakala et al., 2006). Such direct effects of light are not protected against by NPQ and therefore an increasing dissipation by ΦNO would not result in any more (or less) photodamage by that mechanism. Interestingly R/RB leaves showed a similar pattern as RB/RB leaves with no additional increases in ΦNO with increasing irradiance above 300 µmol.m-2.s1
.These recovered leaves seem to be perfectly capable to handle excess light energy
in a regulated way.
Until ΦPSII had decreased to about 0.65 the increase in ΦNO was similar R/RB, RB/RB and RB/R leaves (Fig. 3C), but below this value the responses of the leaves differed due to the different extents of development of ΦNPQ (Fig. 3D). The weaker development of dissipation by the regulated NPQ in the R/R and RB/R leaves (a consequence of the small decrease in Fv’/Fm’ in this leaf (Fig. 3B)) results in a greater non-regulated dissipation – a greater ΦNO – when ΦPSII was less than 0.65. In contrast, in the RB/RB leaves ΦNO only increased slightly once ΦPSII has fallen below 0.65 as a result of the stronger development of inducible NPQ (a reflection of the lower Fv’/Fm’) at ΦPSII below 0.65. At saturating irradiances assimilation (Amax) differed for all four treatments (Fig. 2A). However, at the higher irradiances, the Fv’/Fm’ versus ΦPSII (Fig. 3B) relationship revealed only two responses for the four treatments. The Fv’/Fm’ versus ΦPSII of RB/R leaves followed the same pattern as R/R-leaves. This pattern was distinctly lower for the leaves grown under, or acclimated to RB-light and is paralleled by the nearly identical relationships of ΦNPQ and ΦNO to decreasing ΦPSII shown by these leaves (Fig. 3C and 3D). The responses of ΦNPQ and ΦNO to decreasing ΦPSII shown by leaves grown under, or acclimated to, red light reveals differences between the R/R and RB/R leaves, with the R/R leaves having a greater development of ΦNO and less development of ΦNPQ than the RB/R leaves. As a result, ΦNO is greater at lower ΦPSII values in the R/R leaves than in RB/R leaves, and ΦNPQ is correspondingly lower. This reveals on thylakoid level that RB/R-leaves only partially developed the full red-light syndrome with the duration of the experiment. Thus apart from shade acclimation the ‘red light syndrome’ also occurs after normal leaf development, though not at its full extent. So far the ‘red light syndrome’ has only be studied in detail in cucumber. It would therefore be very speculative to assume at this stage that the described responses are generic.
Conclusions We conclude that exposure to pure red light at low growth irradiance is harmful to chloroplasts in both developing and developed leaves of cucumber. The effect on CO2 assimilation rate is more severe in developing leaves than in leaves, which developed under a combination of red and blue light at low growth irradiance. Chloroplasts in leaves, which previously developed under red light can fully recover
within a few days after releasing them from R light, while leaf photosynthesis at increasing irradiances was limited at the leaf level possibly due to constraints imposed by morphology. CF quenching analysis revealed detailed symptoms of the ‘red light syndrome’, of which the higher quantum yield for non-regulated nonphotochemical energy loss in PSII (ΦNO) in leaves, which are exposed to R light, is most pronounced. Acknowledgements This work was financially supported by the Dutch technology foundation STW (WPB.6662), Philips, and Plant Dynamics. We are grateful to Joost Ruijsch and Theo Damen for assembling the LED arrays, to Hennie Halm and Jan van Walsem for N measurements.
References Baker NR. 2008. Chlorophyll fluorescence: a probe of photosynthesis in vivo. Annual Review of Plant Biology 59, 89-113. Baker NR, Harbinson J, Kramer DM. 2007. Determining the limitations and regulation of photosynthetic energy transduction in leaves. Plant Cell And Environment 30, 1107-1125. Bilger W, Björkman O. 1990. Role of the xanthophyll cycle in photoprotection elucidated by measurements of light-induced absorbance changes, fluorescence and photosynthesis in leaves of Hedera canariensis. Photosynthesis Research 25, 173185. Cailly A, Rizza F, Genty B, Harbinson J. 1996. Fate of excitation at PSII in leaves. The non-photochemical side. 10th FESPP meeting, Vol. 86. Florence, Italy: Plant Physiology and Biochemistry, special issue, p.86. Demmig-Adams B, III WWA, Barker DH, Logan BA, Bowling DR, Verhoeven AS. 1996. Using chlorophyll fluorescence to assess the fraction of absorbed light allocated to thermal dissipation of excess excitation. Physiologia Plantarum 98, 253264.
Demmig-Adams B, Adams III WW. 2006. Photoprotection in an ecological context: the remarkable complexity of thermal energy dissipation. New Phytologist 172, 11-21. Evans JR. 1987. The dependence of quantum yield on wavelength and growth irradiance. Australian Journal Of Plant Physiology 14, 69-79. Farquhar GD, Caemmerer SV, Berry JA. 1980. A biochemical-model of photosynthetic CO2 assimilation in leaves of C-3 species. Planta 149, 78-90. Genty B, Harbinson J, Briantais J, Baker NR. 1990. The relationship between non-photochemical quenching of chlorophyll fluorescence and the rate of photosystem 2 photochemistry in leaves. Photosynthesis Research 25, 249-257. Genty B, Harbinson J, Cailly A, Rizza F. 1996. Fate of excitation at PS II in leaves. The nonphotochemical side. The Third BBRSC Robert Hill Symposium on photosynthesis, Vol. 31. Sheffield, UK: University of Sheffield, department of molecular Biology and biotechnology, abstract no. p28. Goins GD, Yorio NC, Sanwo MM, Brown CS. 1997. Photomorphogenesis, photosynthesis, and seed yield of wheat plants grown under red light-emitting diodes (LEDs) with and without supplemental blue lighting. Journal Of Experimental Botany 48, 1407-1413. Hakala M, Rantamäki S, Puputti EM, Tyystjärvi T, Tyystjärvi E. 2006. Photoinhibition of manganese enzymes: Insights into the mechanism of photosystem II photoinhibition. Journal of Experimental Botany 57, 1809–1816. Hendrickson L, Furbank RT, Chow WS. 2004. A simple alternative approach to assessing the fate of absorbed light energy using chlorophyll fluorescence. Photosynthesis Research 82, 73-81. Hogewoning SW, Trouwborst G, Engbers GJ, Harbinson J, Van Ieperen W, Ruijsch J, Schapendonk AHCM, Pot SC, Van Kooten O. 2007. Plant physiological acclimation to irradiation by light emitting diodes (LEDs). Acta Horticulturae (ISHS) 761, 183-191. Hogewoning SW, Douwstra P, Trouwborst G, van Ieperen W, Harbinson J. 2010a. An artificial solar spectrum substantially alters plant development compared
with usual climate room irradiance spectra. Journal Of Experimental Botany 61, 1267-1276. Hogewoning SW, Trouwborst G, Maljaars H, Poorter H, van Ieperen W, Harbinson J. 2010b. Blue light dose-responses of leaf photosynthesis, morphology, and chemical composition of Cucumis sativus grown under different combinations of red and blue light. Journal Of Experimental Botany 61, 3107-3117. Inada K. 1976. Action Spectra for Photosynthesis in Higher-Plants. Plant And Cell Physiology 17, 355-365. Kato MC, Hikosaka K, Hirotsu N, Makino A, Hirose T. 2003. The excess light energy that is neither utilized in photosynthesis nor dissipated by photoprotective mechanisms determines the rate of photoinactivation in photosystem II. Plant and Cell Physiology 44, 318–325. Klughammer C, Schreiber U. 2008. Complementary PSII quantum yields calculated from simple fluorescense parameters measured by PAM fluorometry and the saturation pulse method. PAM application notes 1, 27-35. Kramer D, Johnson G, Kiirats O, Edwards G. 2004. New Fluorescence Parameters for the Determination of QA Redox State and Excitation Energy Fluxes. Photosynthesis Research 79, 209-218. Krause GH, Behrend U. 1986. ΔpH-dependent chlorophyll fluorescence quenching indicating a mechanism of protection against photoinhibition of chloroplasts. FEBS Letters 200, 298–302. Li XP, Muller-Moule P, Gilmore AM, Niyogi KK. 2002. PsbS-dependent enhancement of feedback de-excitation protects photosystem II from photoinhibition. Proceedings of the National Academy of Sciences of the United States of America 99, 15222–15227. Lichtenthaler HK, Buschmann C, Rahmsdorf U. 1980. The importance of blue light for the development of sun-type chloroplasts. In: Senger H, ed. The blue light syndrome. Berlin [etc.]: Springer, 485-494. Massa GD, Emmerich JC, Morrow RC, Bourget CM, Mitchell CA. 2006. Plantgrowth lighting for space life support: a review. Gravitational and space biology 19, 19-29.
Massa GD, Kim H-H, Wheeler RM, Mitchell CA. 2008. Plant productivity in response to LED lighting. Hortscience 43, 1951-1956. Matsuda R, Ohashi-Kaneko K, Fujiwara K, Goto E, Kurata K. 2004. Photosynthetic characteristics of rice leaves grown under red light with or without supplemental blue light. Plant And Cell Physiology 45, 1870-1874. Matsuda R, Ohashi-Kaneko K, Fujiwara K, Kurata K. 2008. Effects of blue light deficiency on acclimation of light energy partitioning in PSII and CO2 assimilation capacity to high irradiance in spinach leaves. Plant And Cell Physiology 49, 664-670. McCree KJ. 1972a. Action Spectrum, Absorptance And Quantum Yield Of Photosynthesis In Crop Plants. Agricultural Meteorology 9, 191-216. McCree KJ. 1972b. Test of Current Definitions of Photosynthetically Active Radiation against Leaf Photosynthesis Data. Agricultural Meteorology 10, 443-453. Müller P, Li XP, Niyogi KK. 2001. Non-photochemical quenching. A response to excess light energy. Plant Physiology 125, 1558–1566. Murchie EH, Harbinson J. 2014 Non-Photochemical Fluorescence Quenching Across Scales: From Chloroplasts to Plants to Communities. In: Demmig-Adams B, Garab G, Adams W, Govindjee. Non-Photochemical Quenching and Energy Dissipation in Plants, Algae and Cyanobacteria. Berlin [etc.]: Springer, 553-582. Niyogi KK, Grossman AR, Björkman O. 1998. Arabidopsis mutants define a central role for the xanthophyll cycle in the regulation of photosynthetic energy conversion. The Plant Cell 10, 1121–1134. Oguchi R, Hikosaka K, Hirose T. 2003. Does the photosynthetic light-acclimation need change in leaf anatomy? Plant Cell And Environment 26, 505-512. Oxborough K, Baker NR. 1997. Resolving chlorophyll a fluorescence images of photosynthetic efficiency into photochemical and non-photochemical components – calculation of qP and Fv-/Fm-; without measuring Fo. Photosynthesis Research 54, 135-142. Pons TL, De Jong-van Berkel YEM. 2004. Species-specific variation in the importance of the spectral quality gradient in canopies as a signal for photosynthetic resource partitioning. Annals Of Botany 94, 725-732.
Pons TL, Pearcy RW. 1994. Nitrogen reallocation and photosynthetic acclimation in response to partial shading in soybean plants. Physiologia Plantarum 92, 636-644. Ruban AV, Johnson MP, Duffy CDP. 2012. The photoprotective molecular switch in the photosystem II antenna. Biochimica et Biophysica Acta – Bioenergetics 1817, 167–181. Savvides A, Fanourakis D, van Ieperen W. 2012. Co-ordination of hydraulic and stomatal conductances across light qualities in cucumber leaves. Journal Of Experimental Botany 63, 1135-1143. Schoch PG, Zinsou C, Sibi M. 1980. Dependence of the stomatal index on environmental-factors during stomatal differentiation in leaves of Vigna-Sinensis L. 1. Effect of light-intensity. Journal Of Experimental Botany 31, 1211-1216. Sharkey TD, Raschke K. 1981. Effect of Light Quality on Stomatal Opening in Leaves of Xanthium strumarium L. Plant Physiology 68, 1170-1174. Sims DA, Pearcy RW. 1992. Response of leaf anatomy and photosynthetic capacity in Alocasia-Macrorrhiza (Araceae) to a transfer from low to high light. American Journal of Botany 79, 449-455. Takahashi S, Badger MR. 2011. Photoprotection in plants: A new light on photosystem II damage. Trends in Plant Science, 16, 53–60. Thornley JHM. 1976. Mathematical models in plant physiology : a quantitative approach to problems in plant and crop physiology London: Academic press. Trouwborst G, Oosterkamp J, Hogewoning SW, Harbinson J, W. Van Ieperen. 2010. The responses of light interception, photosynthesis and fruit yield of cucumber to LED-lighting within the canopy. Physiologia Plantarum 138, 289-300. Trouwborst G, Hogewoning SW, Harbinson J, van Ieperen W. 2011. Photosynthetic acclimation in relation to nitrogen allocation in cucumber leaves in response to changes in irradiance. Physiologia Plantarum 142, 157-169. Vass I. 2012. Molecular mechanisms of photodamage in the Photosystem II complex. Biochimica et Biophysica Acta (BBA) - Bioenergetics 1817, 209–217.
Voskresenskaya N. 1979. Effect of light quality on carbon metabolism. In: Gibbs M, Latzko E, eds. Encyclopedia of plant physiology, new series, Photosynthesis II, Vol. 6. Berlin: Springer Verlag, 174-180. Zeiger E, Talbott LD, Frechilla S, Srivastava A, Zhu J. 2002. The guard cell chloroplast: a perspective for the twenty-first century. New Phytologist 153, 415– 424.
Table 1. The effect of a change in growth light spectrum on photosynthetic parameters of fully expanded cucumber leaves developed under red (R) or a combination of red and blue light (RB) and exposed to a different spectrum for a period of 7-8 days (n=4; >2 plants per replicate). Different letters indicate statistical significant differences (P<0.05). Parameter1
R/R
RB/R
R/RB
RB/RB
Fv/Fm
0.759c
0.795b
0.808a
0.810a
RD (μmol m-2 s-1)
0.97c
1.00bc
1.25ab
1.31a
α
0.060b
0.066a
0.070a
0.066a
θ
0.78
0.78
0.7
0.77
Amax (μmol m-2 s-1)
8.47d
12.08c
17.11b
19.67a
Vcmax (μmol m-2 s-1)
32.2d
48.9c
66.2b
75.0a
Jmax (μmol m-2 s-1)
58.6d
90.6c
131.2b
153.1a
A100 (μmol m-2 s-1)
3.84b
4.69a
4.82a
4.73a
ΦPSII
0.60c
0.71b
0.73ab
0.74a
ΦNPQ
0.051a
0.032c
0.036b
0.027d
ΦNO
0.35a
0.26b
0.23c
0.23c
Fv’/Fm’
0.732d
0.776c
0.785b
0.793a
0.827d
0.911c
0.924b
0.932a
27.8c
33.2b
34.1ab
35.0a
0.129c
0.162c
0.241b
0.304a
320b
322b
339a
346a
Growth irradiance
qP -2 -1
ETR (μmol m s ) -2 -1
gs (mol m s ) -1
Ci (μmol mol ) 1
Fv/Fm provides a measure for damage to the photosynthetic apparatus and was measured after 30
minutes of darkness; Amax, RD, α and Θ are derived from the photosynthesis light response curves and VCmax and Jmax from the A-Ci curves. All other parameters in this table are measured at steady state growth irradiance (100 µmol.m-2.s-1).
Table 2. The effect of a change in growth light spectrum on leaf parameters of fully expanded cucumber leaves developed under red (R) or a combination of red and blue (RB) light and exposed to a different spectrum for a period of 7-8 days (n=4; >2 plants per replicate). Different letters indicate statistical significant differences (P<0.05). R/R
RB/R
R/RB
RB/RB
LMA (g m-2)
19.7c
23.9b
23.7b
28.1a
Norg (g m-2)
0.81c
0.99b
1.07ab
1.24a
4.11
4.15
4.51
4.42
Chlorophyll (mg m )
405.1b
481.7a
516.3a
550.2a
Chlorophyll a/b ratio
3.40b
3.43b
3.49ab
3.54a
PNUE
10.06b
12.34b
16.14a
16.15a
Pmax/LMA
0.40b
0.51b
0.72a
0.71a
Pmax/chlor
19.91c
25.40b
33.78a
35.69a
Leaf absorption (%)
91.7c
94.1b
94.1b
95.7a
Stomatal density
475b
723a
Epidermis cell density
1715b
2184a
Stomatal index
0.17
0.20
Stomatal apertures
0.82
0.93
%Norg -2
Figure legends Fig. 1. Time courses of the measured photosynthetic rate at saturating irradiance (Amax; panel A) and at growth irradiance (A100; panel B), and of Fv/Fm (panel C) of plants which were grown under either pure red irradiance or a combination of red and blue irradiance and were swapped to the other growth spectrum at day 0 (R/RB and RB/R). The controls are from plants that were not swapped between light spectra (R/R and RB/RB) at day 0. Each data point represents the mean of 4 repetitions (>2 plants per replicate) and vertical bars represent the SE.
Fig. 2. The effect of a change in growth light spectrum on the irradiance-response of CO2 exchange (2A), the maximum PSII efficiency in the light (Fv’/Fm’; 2B), the PSII efficiency factor (qP; 2C), the PSII operating efficiency (2D), the regulated (ΦNPQ; 2E) and the non-regulated (ΦNO; 2F) non-photochemical energy dissipation in cucumber leaves developed under red (R) or a combination of red and blue (RB)
irradiance after an acclimation period of 7-8 days. For symbols see legend. Lines through data points of the irradiance response curves represent the fit to the nonrectangular hyperbola (eq. 1). For each data point n=4 (>2 plants per replicate) and vertical bars represent the SE.
Fig. 3. The effect of a change in growth light spectrum on (A) the PSII efficiency factor (qP), and on (B) the regulated energy dissipation (ΦNPQ), and on (C) the nonregulated non-photochemical energy dissipation (ΦNO), and on (D) the nonphotochemical quenching (1 - Fv’/Fm’) versus the PSII operating efficiency (ΦPSII) in fully expanded cucumber leaves either developed under red (R) or a combination of red and blue (RB) irradiance after an acclimation period of 7-8 days. Each data point represents the mean of 4 repetitions (>2 plants per replicate) and vertical bars and horizontal bars represent the SE. For symbols see legend.
25 A -2 -1
Photosynthetic rate ( µmol m s )
20 15 10 5 0 6 B 5 4 3
Fv/Fm
0 0.82 C 0.80 0.78 0.76
RB/RB R/R RB/R R/RB
0.74 0.72 0.00
Fig. 1.
0
2
4 6 Days
8
10
15
0.60 10
0.6 0
50
100 0.4
RB/RB R/R RB/R R/RB
5 0 1.0
C
0.2 D 0.0
0.8
0.8
0.6
0.6
0.4
0.4
0.2
0.2 E
F 0.0
0.8
0.8
0.6
0.6
0.4
0.4
0.2
0.2
0.0
Fig. 2.
0
400 800 1200 1600 0 PPF (µmol m-2 s-1)
φPSII
0.8
0.70
0.0
φNPQ
0.80
qP
20
B 1.0
400 800 1200 1600 PPF (µmol m-2 s-1)
0.0
φNO
-2 -1
Assimilation rate ( µmol m s ) Fv '/Fm'
A
1.0
B 1.0
A
qP
0.6 0.4
0.6
0.7 0.9
Fv'/Fm'
0.8
0.8
0.4 0.6
0.8
0.2 0.7
0.6
0.4 0.3 0.2
0.8
RB/RB R/R RB/R R/RB
0.6 Φ NO
D 0.8
C
0.6
0.4
0.4
0.2
0.2
0.0 0.8 Fig. 3.
0.6
0.4 ΦPSII
0.2
0.8
0.6
0.4 ΦPSII
0.2
0.0 0.0
Φ NPQ
0.2
S0098-8472(15)00085-4 http://dx.doi.org/doi:10.1016/j.envexpbot.2015.05.002 EEB 2931
To appear in:
Environmental and Experimental Botany
Received date: Revised date: Accepted date:
4-2-2015 28-4-2015 11-5-2015
Please cite this article as: Trouwborst, Govert, Hogewoning, Sander W., Kooten, Olaf van, Harbinson, Jeremy, Ieperen, Wim van, Plasticity of photosynthesis after the ‘red light syndrome’ in Cucumber.Environmental and Experimental Botany http://dx.doi.org/10.1016/j.envexpbot.2015.05.002 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Plasticity of photosynthesis after the ‘red light syndrome’ in Cucumber Govert Trouwborst1,2, Sander W. Hogewoning1,2, Olaf van Kooten1, Jeremy Harbinson1, Wim van Ieperen1* 1
Wageningen University, Horticulture and Product Physiology Group, PO box 630,
6700AP Wageningen, the Netherlands 2
Present Address: Plant Lighting B.V., Veilingweg 46, 3981PC, Bunnik, The
Netherlands
*Corresponding author e-mail: [email protected]; Tel: +31 317-413281. Visiting adress: Droevendaalsesteeg 1, 6708PD, Wageningen, The Netherlands. Running title: Plasticity of photosynthesis after the ‘red light syndrome’ Date of submission: number of figures: number of tables: total word count:
Key words: chlorophyll fluorescence, Cucumis sativus, energy dissipation model for PSII, Light emitting diodes (LEDs), leaf acclimation, light quality, photosynthetic acclimation Abstract The quantum efficiency of photosynthesis of leaves is wavelength dependent and peaks in red (620-670 nm). However when Cucumber plants are raised under pure red light, leaf photosynthesis becomes severely impaired. This “red light syndrome” has been characterized before by a low Fv/Fm, unresponsive stomatal conductance (gs), a low photosynthetic capacity (Amax). It is not known if the syndrome also occurs in fully developed leaves that are exposed to pure red light after reaching maturity and if initially injured leaves can recover from the syndrome. This study investigates the plasticity leaf photosynthetic apparatus after inducing or releasing the “red light syndrome” in leaves of young Cucumber plants. The plants were grown under pure red (R) or mixed red/blue (RB; 70%R) LED light and subsequently exposed to RB (R/RB) and R (RB/R) light (100 µmol PPFD.m-2.s-1,
16h photoperiod) or kept at their initial growth spectrum (R/R) and (RB/RB). Acclimation of fully developed leaves was monitored with gas exchange and chlorophyll fluorescence (CF) over a period of 8 to 10 days after the shift. After switching to RB, R injured leaves recovered from photodamage within 4 days. Photosynthetic capacity (Amax) and gs partly recovered, but did not restrict the net CO2 assimilation rate at growth irradiance (A100), which increased to the same level as in healthy (RB/RB) leaves. After imposing injurious R to healthy mature leaves, they transiently developed signs of the red light-syndrome: a slightly decreased Fv/Fm and more severely reduced Amax and gs. However, A100 did not significantly decrease. CF quenching analysis revealed an potentially harmful increased quantum yield of non-regulated non-photochemical energy loss in PSII under R, which was higher in leaves that developed under R than in leaves that were exposed to R after reaching maturity. We conclude that exposure to pure red light is harmful to photosynthetic systems in both developing and developed leaves of cucumber, but the effect on CO2 assimilation rate and Fv/Fm is much more severe in developing leaves than in mature leaves at low growth irradiance. Chloroplasts of previously R light injured leaves can recover within a few days after releasing from R light, while stomatal conductance and other (partial) morphologically determined leaf factors do not completely acclimate. List of abbreviations A100: net photosynthetic rate at growth irradiance; An: net photosynthetic rate; Amg: maximum gross photosynthetic rate; Amax: maximum net photosynthetic rate; CF: chlorophyll fluorescence; Ci: internal CO2 concentration; ETR: electron transport rate; Fv/Fm: maximum quantum efficiency of PSII photochemistry in the dark; Fv’/Fm’: maximum quantum efficiency of PSII photochemistry in the light; gs: stomatal conductance; Jmax: maximum electron transport rate; LMA: leaf mass per area; Norg: organic nitrogen within the leaf (g m-2);
NPQ: non-photochemical quenching; PAR: photosynthetic active radiation; PNUE: photosynthetic nitrogen use efficiency QA: primary quinone acceptor of PSII; qP: PSII efficiency factor; RD: dark respiration rate; VCmax: Maximum carboxylation rate; α: initial quantum efficiency; θ: parameter for curvature; ΦPSII: PSII operating efficiency; ΦNPQ: regulated energy dissipation ΦNO: non regulated energy dissipation including fluorescence emission Introduction Light is an indispensable energy source for plant growth and is usually supplied by the Sun. Artificial light, however, is used as an energy source for plants in certain situations, such as in greenhouses at high latitudes where natural sunlight severely limits plant growth in the late autumn – early spring period (Trouwborst et al., 2010), or in growth cabinets for research purposes or in plant factories for urban farming. Many different lamp types are used to generate artificial light for plant growth and most of them produce a broad emission spectrum within the PAR range, although this is often superimposed upon distinct emission lines such as in the case of gas discharge lamps or fluorescent tubes (McCree, 1972b). All lamps have in common that they are spectrally very different from sun light, and this considerably influences plant morphology (Hogewoning et al., 2010a). Today, also light emitting diodes (LEDs) are considered as viable sources of plant growth light (Hogewoning et al., 2007; Massa et al., 2006; Massa et al., 2008; Trouwborst et al., 2010). In contrast to the broad spectrum light sources LEDs emit light in a narrow wavelength band (typically 25-50 nm half-power bandwidth). Of particular interest is the influence of wavelength on leaf photosynthesis. Early work on the spectral quantum efficiency for leaf photosynthesis has shown it is highest in the red region of the spectrum (Evans, 1987; Inada, 1976; McCree, 1972a). However, highest instantaneous photosynthesis does not necessarily result in optimal photosynthesis and growth in the long term. Leaves of cucumber plants that were grown under pure red LED-light (100 µmol m-2 s-1; 640 nm; R-grown leaves)
developed a low dark-adapted
Fv/Fm (Hogewoning et al., 2010b), which is
indicative for photodamage (Baker, 2008). This ‘red light syndrome’ is further characterized by unresponsive stomata, a low photosynthetic capacity, low photosynthetic nitrogen use efficiency, a low leaf mass per area and impaired growth (Hogewoning et al., 2010b). Similar results were observed with tomato (unpublished results), but none of these effects occurred in leaves that were grown under mixed red (640 nm) and blue (450 nm) light (RB-grown leaves). It appears therefore that exposure to red light alone during leaf development influences photosynthesis at different functional levels extending from the thylakoid to the whole leaf level. It is unknown if the adverse effects of red light during leaf development are structural and persist after a change in light spectrum or can be partially or completely overcome. Based on how photosynthesis acclimates to changes in light intensity, different extents of acclimation due to changes in light spectrum might be expected at thylakoid and whole leaf level. The objective of this study was to investigate the plasticity of photosynthesis at different functional levels (thylakoid to whole leaf) in response to the induction and release of the ‘red light syndrome’ in cucumber. This was done by changing the spectrum of incident light from pure red (640nm) to mixed red and blue light (640 & 450nm) on leaves with the ‘red light syndrome’, and vice versa on healthy leaves (without the ‘red light syndrome’).
At the thylakoid level we investigated changes in fate of excitation energy in PSII using chlorophyll fluorescence (Cailly et al., 1996; Genty et al., 1996; Hendrickson et al., 2004) before, during and after changes in light spectrum to assess changes in energy dissipation between photosynthetic electron transport (ΦPSII) and regulated (ΦNPQ) and constitutive energy dissipation processes (ΦNO). At the leaf level we used gas exchange measurements to determine changes in photosynthetic light- and CO2 response curves before, during and after changes in light spectrum, and we measured several leaf anatomical parameters. Materials & methods Plant material and growth conditions One week old seedlings (Cucumis sativus “Hoffmann’s Giganta”) were transplanted to a hydroponic growth system in a climate chamber as described in Hogewoning et al. (2010b) and further grown horizontally to avoid shading of older by younger
leaves. Immediately after transplanting, the plants were subjected to the following light treatments: 100% red LED (638 nm dominant wavelength) light (R) or a mixture of 70% red and 30% blue LED (450 nm dominant wavelength) light (RB) to allow full leaf development under distinct different light spectra. After three weeks, when the second leaves were fully expanded, half of the plants per light treatment were changed to the other light spectrum, resulting in 4 light treatments: 2 with a distinct change in spectral composition (RB/R and R/RB) and two controls (R/R or RB/RB). Photosynthetic photon flux density (PPFD) and duration of photoperiod were the same for all light treatments: 100±5 μmol m-2s-1 for 16h a day (Li-190, LiCor inc., Lincoln, NE, USA). Temperature and relative humidity inside the climate room were respectively 25°C and 70%.
Gas exchange and chlorophyll fluorescence measurements Measurements on the second fully expanded leaf started on the day at which half of the plants were subjected to the change in light spectrum (day zero). Photosynthetic irradiance-response curves were repeatedly measured over a time course of 10 days with a portable gas analyzer (LI-6400 with fluorescence head and standard LEDirradiance light source; Li-Cor Inc., Lincoln, NE, USA). Photosynthetic CO2response (A-Ci) curves were measured on the first and the 7th or 8th day of this period. The blue to red ratio of the actinic light in the clamp-on leaf chamber was always set equal to the growth irradiance of the particular leaf subjected to measurements. However above 900 μmol m-2 s-1 the blue light fraction in the Li-Cor CF measuring head gradually decreased from 30% to 18% at 1600 μmol m-2 s-1 due to limitations of the blue light source in the leaf chamber. Leaf chamber temperature, air flow speed and the CO2-concentration were set at 25 oC, 250 μmol s-1, and 380 μmol mol-1 respectively. Air humidity in the leaf chamber was kept similar as during growth. Photosynthetic rates (including correction for diffusion leaks) and chlorophyll fluorescence measurements were determined as described in Trouwborst et al., 2011.
Determinations of leaf parameters At the start and the 7th and 8th day of the experiment, leaf samples were taken to measure chlorophyll, Leaf mass per area (LMA) and organic nitrogen (Norg) as described in Trouwborst et al. (2010). The leaf absorptance spectrum was measured
in single nanometer steps according to Hogewoning et al. (2010b), and the quantum flux absorbed by the leaf was calculated by multiplying the absorptance spectrum by the growth-light spectrum.
Calculations and statistics Maximum PSII efficiency in light (Fv’/Fm’), PSII operating efficiency (ΦPSII), PSII efficiency factor (qP) (Baker, 2008) and the electron transport rate (ETR) at growth light level were calculated according to Baker et al. (2007). Fo’ was calculated according to Oxborough et al. (1997). For the calculation of ETR, we assumed an excitation balance of 0.5 and corrected for the measured leaf absorption and ΦPSII. The energy dissipation in PSII was calculated according to Genty et al. (1996) and Cailly et al., (1996). The quantum yield of PSII electron transport is ΦPSII, the quantum yield of regulated thermal dissipation, ΦNPQ, is calculated as Fs/Fm’Fs/Fm and the quantum yield of non-regulated energy dissipation, ΦNO, is calculated as Fs/Fm (for a more complete consideration of these quantum yields see Kramer (2004),
Hendrickson (2004) and Murchie and Harbinson (2014)).
A modified
version of the Farquhar, Von Caemmerer and Berry (FvCB) model (Farquhar et al., 1980) was fitted to the A-Ci response data. We estimated Jmax and Vcmax normalized to 25°C using the non-linear fitting procedure NLIN in SAS (release 9.1.3; SAS institute, Cary, NC, USA) described in detail according to Trouwborst et al. (2011). A non-rectangular hyperbola (Thornley, 1976) was fitted to the photosynthesis irradiance-response data using the non-linear fitting procedure NLIN in SAS to determine dark respiration (RD), maximum photosynthetic rate (Amax), light-limited quantum efficiency of CO2 assimilation (α) and the scaling constant for the curvature (θ) of the leaves in the different treatments. All treatments were repeated four times (2-4 plants per replicate). Fisher’s LSD was used to make post-hoc multiple comparisons among spectral treatment means from significant one-way analysis of variance (ANOVA) tests (P <0.05). Results Photosynthetic responses at the leaf level As expected all leaves, which developed under pure R light, vastly suffered the redlight syndrome at maturity. They showed a reduced CO2 assimilation capacity (Amax), reduced CO2 assimilation at growth irradiance (A100), a reduced Fv/Fm (Fig. 1)
and a decreased gs (Table 1) compared to leaves that were grown under a combination of red and blue (RB) LED light. Immediately after changing the growth light spectra (from R to RB and vice versa) leaves started to acclimate. This acclimation lasted approximately 7-8 days after which Amax, A100, gs and Fv/Fm were stabilized at new levels (Fig. 1). Releasing the injured leaves from R light (R/RB-leaves) resulted in an approximately 2-fold increase of Amax, while exposure of non-injured leaves to the R-spectrum (RB/Rleaves) caused almost halving of Amax (see also Fig. 2A). Similar responses were observed for gs at growth irradiance, but the effect on Ci was very limited (Table 1). Changes in Fv/Fm were much stronger after alleviating from R light (R/RB) than vice versa (RB/R): full recovery of Fv/Fm occurred in R/RB leaves while only a very small reduction was shown in RB/R leaves (Fig. 1C). Interestingly, the effect of spectrum changes on A100 differed between the release from and the change to R light: A100 in R/RB leaves raised to the same level as in RB/RB leaves within a time course of 4 days, while A100 in RB/R leaves did not change at all after the spectrum change (Fig. 1B). At the end of the experiment, both the PSII operating efficiency (ΦPSII) and estimated ETR at growth irradiance were reduced in R/R compared to RB/RB grown leaves (Table 1). After the spectrum change from R to RB both parameters increased to the level displayed by RB/RB leaves, while only small reductions in ΦPSII and ETR were observed after a spectrum change to injurious R-light (Table 1).The lightlimited quantum efficiency of CO2 assimilation (α) was reduced in R-grown compared to RB-grown leaves, but completely recovered after a change from an R to an RB-spectrum. No reduction in α was observed after the opposite spectrum change from RB to R light (Table 1). Respiration in darkness (RD) was slightly higher in RB/RB and R/RB-leaves than in R/R and RB/R-leaves (Table 1). The maximum carboxylation rate allowed by Rubisco (VCmax) and maximum rate of linear electron transport, i.e. that through PSII, (Jmax) showed similar patterns across the light treatments as Amax. CF quenching analysis and the capacity of leaves to cope with excess energy At growth irradiance and at the end of the acclimation period, the estimated quantum yield of regulated non-photochemical energy loss in PSII (ΦNPQ) was very small (≤0.051), though significant differences were observed between all treatments (Table
1). The quantum yield of non-regulated non-photochemical energy loss in PSII (ΦNO) was much larger (≤0.35) and differed significantly between the spectral treatments: ΦNO was ~50% higher in R/R leaves compared to RB/RB leaves at growth irradiance, and full adjustment of ΦNO to the level in healthy RB/RB leaves was observed in R/RB leaves after the change in spectrum. Exposing healthy leaves to pure R-light, on the other hand, resulted in only a weak increase of ΦNO at growth irradiance at the end of the acclimation period (Table 1). Analysis of CF quenching parameters was extended beyond growth irradiance to investigate further differences in the capacity of leaves to cope with excess energy between the spectral treatments. Differences in PSII efficiency between treatments increased with increasing irradiance (Fig. 2B) with the least steep decrease in RB/RB and the steepest decrease in R/R-leaves. The parameters Fv’/Fm’ (the maximum quantum yield of PSII in light; Fig 2C) and qP (the PSII efficiency factor (Baker, 2008); Fig. 2D), which are often used to assess to what extent changes in PSII operating efficiency are attributable to changes in non-photochemical quenching (NPQ) or to changes in the openness of PSII reaction centers (Baker, 2008), showed some conspicuous differences between the spectral treatments. The decrease of Fv’/Fm’ with irradiance was low, and smaller and less discriminating between spectral treatments than the accompanying decrease in qP with irradiance. Decreases in ΦPSII with increasing irradiance were increasingly due to differences in qP and hardly due to changes in Fv’/Fm’ (Fig. 3A and B; n.b. ΦPSII = Fv’/Fm’ × qP). The increase in quantum yield of regulated non-photochemical energy loss in PSII (ΦNPQ) with irradiance paralleled the decrease in Fv’/Fm’ with irradiance (Fig 2C, E) and small differences were observed between the spectral treatments: ΦNPQ converged to similar values at high irradiances in RB/RB and R/RB leaves, while substantially lower values for ΦNPQ were measured at high irradiance in leaves that ended in R light, with R/R leaves having a lower maximum value of ΦNPQ than the RB/R leaves (Fig. 2CE). The quantum yield of non-regulated non-photochemical energy loss in PSII (ΦNO) was highest in R/R leaves at all irradiances. All leaves, which ended in RB light (R/RB and RB/RB), showed an approximately similar, limited increase in ΦNO at low irradiance, which was saturated above 300 µmol PPFD.m-2.s-1, while ΦNO in R/RB leaves did not differ from RB/RB and R/BR leaves at low irradiance but increasingly deviated with increasing irradiance (Fig. 2F).
Acclimation of leaf parameters LMA, Norg, chlorophyll content and leaf absorption were lowest for R/R-leaves and highest for RB/RB-leaves. Acclimation to the other spectrum resulted in intermediate values (Table 2). The chlorophyll a/b ratio was significantly higher in RB/RB-leaves than in R/R- and RB/R-leaves. The %Norg did not differ between treatments. Photosynthetic Nitrogen Use Efficiency (PNUE) and Amax/LMA were the lowest for R/R- and RB/R-leaves. Amax/chl was lowest for R/R leaves and highest for RB/RBand R/RB-leaves, while RB/R-leaves showed intermediate values. Discussion From previous research on blue-light dose-responses it has been concluded that blue light is qualitatively required for the development of normal photosynthetic functioning leaves in cucumber (Hogewoning et al., 2010b). Also in this study the absence of blue light in the growth light spectrum caused significant reductions of Amax gs and Fv/Fm, indicating damage to PSII. Present results are in full agreement with previous research and in this study we expand towards a more detailed analysis of the ‘red light syndrome’ and its dynamics after changes in the growth light spectrum. This research has been conducted under light limiting conditions (relative low irradiance) where plants are in their light limited range of photosynthesis and excess light energy is low. These circumstances are common for climate chambers used in plant research and in growth systems for vertical farming (plant factories) in which LEDs increasingly are implemented as light sources. We here present results and CF quenching analysis at growth irradiance, which is of explanatory value for the changes that occurred in photosynthesis in the leaves in this study. We also present the CF quenching analysis at further increasing irradiance, which displays changes in the photosynthetic systems that are not fully expressed at growth irradiance.
Effects of R light at growth irradiance A reduced Fv/Fm is usually associated with photodamage of PSII (photoinhibition) or with slowly reversible down-regulation of PSII, which both occur in response to a wide range of stresses (Baker, 2008; Demmig-Adams and Adams, 2006). However, here the usual causes for a reduced Fv/Fm did not apply: growth irradiance was low (100 µmol m-2 s-1) and the plants were neither nutrient, temperature nor water stressed. Sink limitation related down regulation of PSII was previously discounted
for (Hogewoning et al., 2010b). At growth irradiance, only leaves that developed and were kept under pure R light (R/R) showed reduced photosynthesis (A100;Table 1). This reduction in A100 can be largely attributed to changes at the chloroplast level, because in all spectral treatments growth irradiance was within the light-limited range of photosynthesis (Fig. 2A), and because the observed differences in stomatal conductance (up to ~60% lower in R/R compared to RB/RB) did not result in large differences in Ci (Table 1). Observed changes in A100 between R/R and RB/RB grown leaves were in close agreement with estimated reductions in estimated ETR: both were approximately 26% lower in R/R (Table 1). The smaller estimated ETR is mainly due to a decreased ΦPSII (23% lower) as the decrease in leaf absorption was very limited (4% lower; Table 1&2). It is well known that gs can be reduced under pure R light compared to RB (Sharkey and Raschke, 1981; Goins et al., 1997), probably because blue light stimulates additional opening of stomata, mediated by the blue-light absorbing carotenoid zeaxanthin (Zeiger et al., 2002). Recently we reported that pure R light during leaf development significantly decreased stomatal density in cucumber leaves, largely due to a positive effect on epidermal cell size (Savvides et al., 2012). The combination of these two effects account for the strongly reduced gs in leaves that were grown under R/R compared to RB/RB. The intermediate values for gs in the R/RB treatment and the RB/R treatment after full acclimation were most likely caused by aperture responses to the final light spectrum, restricted by the stomatal density that was set during leaf development. Despite these changes in gs, the effect on CO2 assimilation was limited due to the negligible changes in Ci at the low light intensity (Table 1). The differences between R/R- and RB/RB-leaves in LMA, Norg, chlorophyll a/b ratio, Vcmax, Jmax and PNUE (Table 2) have already been discussed in detail by Hogewoning et al. (2010b). The changes in these parameters after changes in the light spectrum (i.e. in
RB/R and R/RB leaves) displayed changes that were
consistent with those usually observed during acclimation to low (RB to R) or to high light (R to RB) conditions (e.g. Pons and De Jong-van Berkel, 2004; Pons and Pearcy, 1994; Table 1&2). After releasing injured leaves from R or exposing healthy developed leaves to R leaves started to adjust their photosynthetic characteristics, displaying a great degree of plasticity. Changing the light spectrum from R to RB light caused α, A100
and ETR to fully recover to the level of RB/RB-leaves, whereas leaf absorption, LMA, Norg, gs and Amax also increased but remained lower than in RB/RB-leaves. The latter reveals limitations on the plasticity for acclimation, which could be partly due to unchangeable differences in leaf morphology (a lower LMA at R/R than at RB/RB) which were induced during previous leaf development under R light. Although not directly measured, leaf thickness was probably reduced in leaves that developed under R light, consistent with the lower LMA and the lower stomatal density (at similar stomatal index) and larger epidermal cell size observed in R grown compared to RB grown cucumber leaves in a previous experiment (Savvides et al., 2012). Normalizing Amax for LMA (i.e. expressing it at dry weight basis) resulted in a calculated full adjustment of Amax at dry weight basis, which was also found for Amax normalized for chlorophyll content and PNUE (Table 2), supporting the suggestion that leaf structure is limiting for Amax. Oguchi and co-workers (2003) concluded earlier that Amax of low light developed leaves, which are later exposed to high light, are physically restricted by the cell size of the leaves and the unoccupied horizontal cell surface along which the chloroplasts can expand. As boundaries in cell size and maximal stomatal conductance are set during the leaf developmental phase (Schoch et al., 1980; Sims and Pearcy, 1992), the implication for later plasticity in acclimation is evident. We conclude that the limitation of assimilation rate at growth irradiance in R/R-leaves is mostly due to a disturbed light reaction or photosynthetic metabolism. Limitations due to stomatal effects or leaf light absorption clearly are of minor importance.
Chlorophyll fluorescence quenching and energy dissipation analysis None of the leaves investigated in present study displayed a serious light-induced decrease in Fv’/Fm’ with increasing irradiance (Fig. 2C) or with decreasing PSII (Fig. 3B). This implies that all these leaves had a relatively small ability for NPQ (Bilger and Björkman, 1990; Genty et al., 1990; Demmig-Adams et al., 1996), which is normal for leaves of fast growing species that developed under low light conditions (Demmig-Adams and Adams, 2006) such as the cucumber leaves in present study. Of the variation in ΦPSII at growth irradiance between spectral treatments (R/R vs. RB/RB) ~60% was attributable to differences in qP, a measure of impact that PSII trap closure has on the light-use efficiency for PSII electron transport (Murchie and
Harbinson, 2014) and ~40% to differences in Fv’/Fm’ (changes in non-photochemical quenching (NPQ)) (Table 1). However, with increasing measuring light intensity the difference in qP between spectral treatments became much more pronounced than the differences observed in NPQ (Fv’/Fm’, Fig. 2C and D). Though the actual effects in our experiments were small due to the low growth irradiance, it implies that leaves developing in pure R light became more vulnerable to photodamage, which seems consistent with a greater extent of ‘shade acclimation’ (Hogewoning et al., 2010b; Lichtenthaler et al., 1980; Matsuda et al., 2004; Matsuda et al., 2008; Voskresenskaya, 1979). With irradiance increasing far beyond growth irradiance and further increasing excess light energy, ΦNO increased in the R/R leaves to a level which was 60% higher than in RB/RB-leaves (Fig. 2F). This increasing fraction of ΦNO implies an increasing susceptibility for photodamage of leaves under R light when they are exposed to higher growth irradiances than in current experiment. A correlation between the rate of photodamage and excitation dissipation by basal dissipation mechanisms (ΦNO) has been shown (Kato et al., 2003) though in this case ΦNO was calculated using a model (Demmig-Adams et al., 1996), which is less robust than the model used here. More generally, however, the photoprotective effect of the qE component of NPQ (which gives rise to ΦNPQ) has been amply shown (eg Bilger and Bjorkman, 1990; Krause and Behrend, 1986; Niyogi et al., 1998; Muller et al., 2001; Li et al., 2002; Ruban et al., 2012). If, together with photochemistry (ΦPSII) dissipation by the qE mechanism (ΦNPQ) is protective, then a deficiency of both of those, which will result in dissipation by the less competitive, slower mechanisms that give rise to ΦNO, will result in more photodamage. Photodamage is, however, complex. While some photodamage is due to damaging side reactions occurring within photosystem II (the kind that NPQ can protect against; eg Vass, 2011), photodamage can also be caused by direct effects of light on the Mn cluster of the oxygen evolving cluster of PSII (eg Takahashi and Badger, 2011; Hakala et al., 2006). Such direct effects of light are not protected against by NPQ and therefore an increasing dissipation by ΦNO would not result in any more (or less) photodamage by that mechanism. Interestingly R/RB leaves showed a similar pattern as RB/RB leaves with no additional increases in ΦNO with increasing irradiance above 300 µmol.m-2.s1
.These recovered leaves seem to be perfectly capable to handle excess light energy
in a regulated way.
Until ΦPSII had decreased to about 0.65 the increase in ΦNO was similar R/RB, RB/RB and RB/R leaves (Fig. 3C), but below this value the responses of the leaves differed due to the different extents of development of ΦNPQ (Fig. 3D). The weaker development of dissipation by the regulated NPQ in the R/R and RB/R leaves (a consequence of the small decrease in Fv’/Fm’ in this leaf (Fig. 3B)) results in a greater non-regulated dissipation – a greater ΦNO – when ΦPSII was less than 0.65. In contrast, in the RB/RB leaves ΦNO only increased slightly once ΦPSII has fallen below 0.65 as a result of the stronger development of inducible NPQ (a reflection of the lower Fv’/Fm’) at ΦPSII below 0.65. At saturating irradiances assimilation (Amax) differed for all four treatments (Fig. 2A). However, at the higher irradiances, the Fv’/Fm’ versus ΦPSII (Fig. 3B) relationship revealed only two responses for the four treatments. The Fv’/Fm’ versus ΦPSII of RB/R leaves followed the same pattern as R/R-leaves. This pattern was distinctly lower for the leaves grown under, or acclimated to RB-light and is paralleled by the nearly identical relationships of ΦNPQ and ΦNO to decreasing ΦPSII shown by these leaves (Fig. 3C and 3D). The responses of ΦNPQ and ΦNO to decreasing ΦPSII shown by leaves grown under, or acclimated to, red light reveals differences between the R/R and RB/R leaves, with the R/R leaves having a greater development of ΦNO and less development of ΦNPQ than the RB/R leaves. As a result, ΦNO is greater at lower ΦPSII values in the R/R leaves than in RB/R leaves, and ΦNPQ is correspondingly lower. This reveals on thylakoid level that RB/R-leaves only partially developed the full red-light syndrome with the duration of the experiment. Thus apart from shade acclimation the ‘red light syndrome’ also occurs after normal leaf development, though not at its full extent. So far the ‘red light syndrome’ has only be studied in detail in cucumber. It would therefore be very speculative to assume at this stage that the described responses are generic.
Conclusions We conclude that exposure to pure red light at low growth irradiance is harmful to chloroplasts in both developing and developed leaves of cucumber. The effect on CO2 assimilation rate is more severe in developing leaves than in leaves, which developed under a combination of red and blue light at low growth irradiance. Chloroplasts in leaves, which previously developed under red light can fully recover
within a few days after releasing them from R light, while leaf photosynthesis at increasing irradiances was limited at the leaf level possibly due to constraints imposed by morphology. CF quenching analysis revealed detailed symptoms of the ‘red light syndrome’, of which the higher quantum yield for non-regulated nonphotochemical energy loss in PSII (ΦNO) in leaves, which are exposed to R light, is most pronounced. Acknowledgements This work was financially supported by the Dutch technology foundation STW (WPB.6662), Philips, and Plant Dynamics. We are grateful to Joost Ruijsch and Theo Damen for assembling the LED arrays, to Hennie Halm and Jan van Walsem for N measurements.
References Baker NR. 2008. Chlorophyll fluorescence: a probe of photosynthesis in vivo. Annual Review of Plant Biology 59, 89-113. Baker NR, Harbinson J, Kramer DM. 2007. Determining the limitations and regulation of photosynthetic energy transduction in leaves. Plant Cell And Environment 30, 1107-1125. Bilger W, Björkman O. 1990. Role of the xanthophyll cycle in photoprotection elucidated by measurements of light-induced absorbance changes, fluorescence and photosynthesis in leaves of Hedera canariensis. Photosynthesis Research 25, 173185. Cailly A, Rizza F, Genty B, Harbinson J. 1996. Fate of excitation at PSII in leaves. The non-photochemical side. 10th FESPP meeting, Vol. 86. Florence, Italy: Plant Physiology and Biochemistry, special issue, p.86. Demmig-Adams B, III WWA, Barker DH, Logan BA, Bowling DR, Verhoeven AS. 1996. Using chlorophyll fluorescence to assess the fraction of absorbed light allocated to thermal dissipation of excess excitation. Physiologia Plantarum 98, 253264.
Demmig-Adams B, Adams III WW. 2006. Photoprotection in an ecological context: the remarkable complexity of thermal energy dissipation. New Phytologist 172, 11-21. Evans JR. 1987. The dependence of quantum yield on wavelength and growth irradiance. Australian Journal Of Plant Physiology 14, 69-79. Farquhar GD, Caemmerer SV, Berry JA. 1980. A biochemical-model of photosynthetic CO2 assimilation in leaves of C-3 species. Planta 149, 78-90. Genty B, Harbinson J, Briantais J, Baker NR. 1990. The relationship between non-photochemical quenching of chlorophyll fluorescence and the rate of photosystem 2 photochemistry in leaves. Photosynthesis Research 25, 249-257. Genty B, Harbinson J, Cailly A, Rizza F. 1996. Fate of excitation at PS II in leaves. The nonphotochemical side. The Third BBRSC Robert Hill Symposium on photosynthesis, Vol. 31. Sheffield, UK: University of Sheffield, department of molecular Biology and biotechnology, abstract no. p28. Goins GD, Yorio NC, Sanwo MM, Brown CS. 1997. Photomorphogenesis, photosynthesis, and seed yield of wheat plants grown under red light-emitting diodes (LEDs) with and without supplemental blue lighting. Journal Of Experimental Botany 48, 1407-1413. Hakala M, Rantamäki S, Puputti EM, Tyystjärvi T, Tyystjärvi E. 2006. Photoinhibition of manganese enzymes: Insights into the mechanism of photosystem II photoinhibition. Journal of Experimental Botany 57, 1809–1816. Hendrickson L, Furbank RT, Chow WS. 2004. A simple alternative approach to assessing the fate of absorbed light energy using chlorophyll fluorescence. Photosynthesis Research 82, 73-81. Hogewoning SW, Trouwborst G, Engbers GJ, Harbinson J, Van Ieperen W, Ruijsch J, Schapendonk AHCM, Pot SC, Van Kooten O. 2007. Plant physiological acclimation to irradiation by light emitting diodes (LEDs). Acta Horticulturae (ISHS) 761, 183-191. Hogewoning SW, Douwstra P, Trouwborst G, van Ieperen W, Harbinson J. 2010a. An artificial solar spectrum substantially alters plant development compared
with usual climate room irradiance spectra. Journal Of Experimental Botany 61, 1267-1276. Hogewoning SW, Trouwborst G, Maljaars H, Poorter H, van Ieperen W, Harbinson J. 2010b. Blue light dose-responses of leaf photosynthesis, morphology, and chemical composition of Cucumis sativus grown under different combinations of red and blue light. Journal Of Experimental Botany 61, 3107-3117. Inada K. 1976. Action Spectra for Photosynthesis in Higher-Plants. Plant And Cell Physiology 17, 355-365. Kato MC, Hikosaka K, Hirotsu N, Makino A, Hirose T. 2003. The excess light energy that is neither utilized in photosynthesis nor dissipated by photoprotective mechanisms determines the rate of photoinactivation in photosystem II. Plant and Cell Physiology 44, 318–325. Klughammer C, Schreiber U. 2008. Complementary PSII quantum yields calculated from simple fluorescense parameters measured by PAM fluorometry and the saturation pulse method. PAM application notes 1, 27-35. Kramer D, Johnson G, Kiirats O, Edwards G. 2004. New Fluorescence Parameters for the Determination of QA Redox State and Excitation Energy Fluxes. Photosynthesis Research 79, 209-218. Krause GH, Behrend U. 1986. ΔpH-dependent chlorophyll fluorescence quenching indicating a mechanism of protection against photoinhibition of chloroplasts. FEBS Letters 200, 298–302. Li XP, Muller-Moule P, Gilmore AM, Niyogi KK. 2002. PsbS-dependent enhancement of feedback de-excitation protects photosystem II from photoinhibition. Proceedings of the National Academy of Sciences of the United States of America 99, 15222–15227. Lichtenthaler HK, Buschmann C, Rahmsdorf U. 1980. The importance of blue light for the development of sun-type chloroplasts. In: Senger H, ed. The blue light syndrome. Berlin [etc.]: Springer, 485-494. Massa GD, Emmerich JC, Morrow RC, Bourget CM, Mitchell CA. 2006. Plantgrowth lighting for space life support: a review. Gravitational and space biology 19, 19-29.
Massa GD, Kim H-H, Wheeler RM, Mitchell CA. 2008. Plant productivity in response to LED lighting. Hortscience 43, 1951-1956. Matsuda R, Ohashi-Kaneko K, Fujiwara K, Goto E, Kurata K. 2004. Photosynthetic characteristics of rice leaves grown under red light with or without supplemental blue light. Plant And Cell Physiology 45, 1870-1874. Matsuda R, Ohashi-Kaneko K, Fujiwara K, Kurata K. 2008. Effects of blue light deficiency on acclimation of light energy partitioning in PSII and CO2 assimilation capacity to high irradiance in spinach leaves. Plant And Cell Physiology 49, 664-670. McCree KJ. 1972a. Action Spectrum, Absorptance And Quantum Yield Of Photosynthesis In Crop Plants. Agricultural Meteorology 9, 191-216. McCree KJ. 1972b. Test of Current Definitions of Photosynthetically Active Radiation against Leaf Photosynthesis Data. Agricultural Meteorology 10, 443-453. Müller P, Li XP, Niyogi KK. 2001. Non-photochemical quenching. A response to excess light energy. Plant Physiology 125, 1558–1566. Murchie EH, Harbinson J. 2014 Non-Photochemical Fluorescence Quenching Across Scales: From Chloroplasts to Plants to Communities. In: Demmig-Adams B, Garab G, Adams W, Govindjee. Non-Photochemical Quenching and Energy Dissipation in Plants, Algae and Cyanobacteria. Berlin [etc.]: Springer, 553-582. Niyogi KK, Grossman AR, Björkman O. 1998. Arabidopsis mutants define a central role for the xanthophyll cycle in the regulation of photosynthetic energy conversion. The Plant Cell 10, 1121–1134. Oguchi R, Hikosaka K, Hirose T. 2003. Does the photosynthetic light-acclimation need change in leaf anatomy? Plant Cell And Environment 26, 505-512. Oxborough K, Baker NR. 1997. Resolving chlorophyll a fluorescence images of photosynthetic efficiency into photochemical and non-photochemical components – calculation of qP and Fv-/Fm-; without measuring Fo. Photosynthesis Research 54, 135-142. Pons TL, De Jong-van Berkel YEM. 2004. Species-specific variation in the importance of the spectral quality gradient in canopies as a signal for photosynthetic resource partitioning. Annals Of Botany 94, 725-732.
Pons TL, Pearcy RW. 1994. Nitrogen reallocation and photosynthetic acclimation in response to partial shading in soybean plants. Physiologia Plantarum 92, 636-644. Ruban AV, Johnson MP, Duffy CDP. 2012. The photoprotective molecular switch in the photosystem II antenna. Biochimica et Biophysica Acta – Bioenergetics 1817, 167–181. Savvides A, Fanourakis D, van Ieperen W. 2012. Co-ordination of hydraulic and stomatal conductances across light qualities in cucumber leaves. Journal Of Experimental Botany 63, 1135-1143. Schoch PG, Zinsou C, Sibi M. 1980. Dependence of the stomatal index on environmental-factors during stomatal differentiation in leaves of Vigna-Sinensis L. 1. Effect of light-intensity. Journal Of Experimental Botany 31, 1211-1216. Sharkey TD, Raschke K. 1981. Effect of Light Quality on Stomatal Opening in Leaves of Xanthium strumarium L. Plant Physiology 68, 1170-1174. Sims DA, Pearcy RW. 1992. Response of leaf anatomy and photosynthetic capacity in Alocasia-Macrorrhiza (Araceae) to a transfer from low to high light. American Journal of Botany 79, 449-455. Takahashi S, Badger MR. 2011. Photoprotection in plants: A new light on photosystem II damage. Trends in Plant Science, 16, 53–60. Thornley JHM. 1976. Mathematical models in plant physiology : a quantitative approach to problems in plant and crop physiology London: Academic press. Trouwborst G, Oosterkamp J, Hogewoning SW, Harbinson J, W. Van Ieperen. 2010. The responses of light interception, photosynthesis and fruit yield of cucumber to LED-lighting within the canopy. Physiologia Plantarum 138, 289-300. Trouwborst G, Hogewoning SW, Harbinson J, van Ieperen W. 2011. Photosynthetic acclimation in relation to nitrogen allocation in cucumber leaves in response to changes in irradiance. Physiologia Plantarum 142, 157-169. Vass I. 2012. Molecular mechanisms of photodamage in the Photosystem II complex. Biochimica et Biophysica Acta (BBA) - Bioenergetics 1817, 209–217.
Voskresenskaya N. 1979. Effect of light quality on carbon metabolism. In: Gibbs M, Latzko E, eds. Encyclopedia of plant physiology, new series, Photosynthesis II, Vol. 6. Berlin: Springer Verlag, 174-180. Zeiger E, Talbott LD, Frechilla S, Srivastava A, Zhu J. 2002. The guard cell chloroplast: a perspective for the twenty-first century. New Phytologist 153, 415– 424.
Table 1. The effect of a change in growth light spectrum on photosynthetic parameters of fully expanded cucumber leaves developed under red (R) or a combination of red and blue light (RB) and exposed to a different spectrum for a period of 7-8 days (n=4; >2 plants per replicate). Different letters indicate statistical significant differences (P<0.05). Parameter1
R/R
RB/R
R/RB
RB/RB
Fv/Fm
0.759c
0.795b
0.808a
0.810a
RD (μmol m-2 s-1)
0.97c
1.00bc
1.25ab
1.31a
α
0.060b
0.066a
0.070a
0.066a
θ
0.78
0.78
0.7
0.77
Amax (μmol m-2 s-1)
8.47d
12.08c
17.11b
19.67a
Vcmax (μmol m-2 s-1)
32.2d
48.9c
66.2b
75.0a
Jmax (μmol m-2 s-1)
58.6d
90.6c
131.2b
153.1a
A100 (μmol m-2 s-1)
3.84b
4.69a
4.82a
4.73a
ΦPSII
0.60c
0.71b
0.73ab
0.74a
ΦNPQ
0.051a
0.032c
0.036b
0.027d
ΦNO
0.35a
0.26b
0.23c
0.23c
Fv’/Fm’
0.732d
0.776c
0.785b
0.793a
0.827d
0.911c
0.924b
0.932a
27.8c
33.2b
34.1ab
35.0a
0.129c
0.162c
0.241b
0.304a
320b
322b
339a
346a
Growth irradiance
qP -2 -1
ETR (μmol m s ) -2 -1
gs (mol m s ) -1
Ci (μmol mol ) 1
Fv/Fm provides a measure for damage to the photosynthetic apparatus and was measured after 30
minutes of darkness; Amax, RD, α and Θ are derived from the photosynthesis light response curves and VCmax and Jmax from the A-Ci curves. All other parameters in this table are measured at steady state growth irradiance (100 µmol.m-2.s-1).
Table 2. The effect of a change in growth light spectrum on leaf parameters of fully expanded cucumber leaves developed under red (R) or a combination of red and blue (RB) light and exposed to a different spectrum for a period of 7-8 days (n=4; >2 plants per replicate). Different letters indicate statistical significant differences (P<0.05). R/R
RB/R
R/RB
RB/RB
LMA (g m-2)
19.7c
23.9b
23.7b
28.1a
Norg (g m-2)
0.81c
0.99b
1.07ab
1.24a
4.11
4.15
4.51
4.42
Chlorophyll (mg m )
405.1b
481.7a
516.3a
550.2a
Chlorophyll a/b ratio
3.40b
3.43b
3.49ab
3.54a
PNUE
10.06b
12.34b
16.14a
16.15a
Pmax/LMA
0.40b
0.51b
0.72a
0.71a
Pmax/chlor
19.91c
25.40b
33.78a
35.69a
Leaf absorption (%)
91.7c
94.1b
94.1b
95.7a
Stomatal density
475b
723a
Epidermis cell density
1715b
2184a
Stomatal index
0.17
0.20
Stomatal apertures
0.82
0.93
%Norg -2
Figure legends Fig. 1. Time courses of the measured photosynthetic rate at saturating irradiance (Amax; panel A) and at growth irradiance (A100; panel B), and of Fv/Fm (panel C) of plants which were grown under either pure red irradiance or a combination of red and blue irradiance and were swapped to the other growth spectrum at day 0 (R/RB and RB/R). The controls are from plants that were not swapped between light spectra (R/R and RB/RB) at day 0. Each data point represents the mean of 4 repetitions (>2 plants per replicate) and vertical bars represent the SE.
Fig. 2. The effect of a change in growth light spectrum on the irradiance-response of CO2 exchange (2A), the maximum PSII efficiency in the light (Fv’/Fm’; 2B), the PSII efficiency factor (qP; 2C), the PSII operating efficiency (2D), the regulated (ΦNPQ; 2E) and the non-regulated (ΦNO; 2F) non-photochemical energy dissipation in cucumber leaves developed under red (R) or a combination of red and blue (RB)
irradiance after an acclimation period of 7-8 days. For symbols see legend. Lines through data points of the irradiance response curves represent the fit to the nonrectangular hyperbola (eq. 1). For each data point n=4 (>2 plants per replicate) and vertical bars represent the SE.
Fig. 3. The effect of a change in growth light spectrum on (A) the PSII efficiency factor (qP), and on (B) the regulated energy dissipation (ΦNPQ), and on (C) the nonregulated non-photochemical energy dissipation (ΦNO), and on (D) the nonphotochemical quenching (1 - Fv’/Fm’) versus the PSII operating efficiency (ΦPSII) in fully expanded cucumber leaves either developed under red (R) or a combination of red and blue (RB) irradiance after an acclimation period of 7-8 days. Each data point represents the mean of 4 repetitions (>2 plants per replicate) and vertical bars and horizontal bars represent the SE. For symbols see legend.
25 A -2 -1
Photosynthetic rate ( µmol m s )
20 15 10 5 0 6 B 5 4 3
Fv/Fm
0 0.82 C 0.80 0.78 0.76
RB/RB R/R RB/R R/RB
0.74 0.72 0.00
Fig. 1.
0
2
4 6 Days
8
10
15
0.60 10
0.6 0
50
100 0.4
RB/RB R/R RB/R R/RB
5 0 1.0
C
0.2 D 0.0
0.8
0.8
0.6
0.6
0.4
0.4
0.2
0.2 E
F 0.0
0.8
0.8
0.6
0.6
0.4
0.4
0.2
0.2
0.0
Fig. 2.
0
400 800 1200 1600 0 PPF (µmol m-2 s-1)
φPSII
0.8
0.70
0.0
φNPQ
0.80
qP
20
B 1.0
400 800 1200 1600 PPF (µmol m-2 s-1)
0.0
φNO
-2 -1
Assimilation rate ( µmol m s ) Fv '/Fm'
A
1.0
B 1.0
A
qP
0.6 0.4
0.6
0.7 0.9
Fv'/Fm'
0.8
0.8
0.4 0.6
0.8
0.2 0.7
0.6
0.4 0.3 0.2
0.8
RB/RB R/R RB/R R/RB
0.6 Φ NO
D 0.8
C
0.6
0.4
0.4
0.2
0.2
0.0 0.8 Fig. 3.
0.6
0.4 ΦPSII
0.2
0.8
0.6
0.4 ΦPSII
0.2
0.0 0.0
Φ NPQ
0.2