Slow induction of chlorophyll a fluorescence excited by blue and red light in Tradescantia leaves acclimated to high and low light

Abstract

Tradescantia is a good model for assaying induction events in higher plant leaves. Chlorophyll (Chl) fluorescence serves as a sensitive reporter of the functional state of photosynthetic apparatus in chloroplasts. The fluorescence time-course depends on the leaf growth conditions and actinic light quality. In this work, we investigated slow induction of Chl a fluorescence (SIF) excited by blue light (BL, λ_max = 455 nm) or red light (RL, λ_max = 630 nm) in dark-adapted leaves of Tradescantia fluminensis acclimated to high light (~ 1000 µmol photons m⁻² s⁻¹; HL) or low light (~ 100 µmol photons m⁻² s⁻¹; LL). Our special interest was focused on the contribution of the avoidance response to SIF kinetics. Bearing in mind that BL and RL have different impacts on photoreceptors that initiate chloroplast movements within the cell (accumulation/avoidance responses), we have compared the SIF patterns during the action of BL and RL. The time-courses of SIF and kinetics of non-photochemical quenching (NPQ) of Chl a fluorescence revealed a certain difference when leaves were illuminated by BL or RL. In both cases, the yield of fluorescence rose to the maximal level P and then, after the lag-phase P–S–M₁, the fluorescence level decreased toward the steady state T (via the intermediate phases M₁–M₂ and M₂–T). In LL-acclimated leaves, the duration of the P–S–M₁ phase was almost two times longer that in HL-grown plants. In the case of BL, the fluorescence decay included the transient phase M₁–M₂. This phase was obscure during the RL illumination. Non-photochemical quenching of Chl a fluorescence has been quantified as ${\text{NPQ}} = F_{\text{m}}^{ 0} /F^{\prime}_{\text{m}} - 1$, where $F_{\text{m}}^{ 0}$ and $F^{\prime}_{\text{m}}$ stand for the fluorescence response to saturating pulses of light applied to dark-adapted and illuminated samples, respectively. The time-courses of such a formally determined NPQ value were markedly different during the action of RL and BL. In LL-grown leaves, BL induced higher NPQ as compared to the action of RL. In HL-grown plants, the difference between the NPQ responses to BL and RL illumination was insignificant. Comparing the peculiarities of Chl a fluorescence induced by BL and RL, we conclude that the avoidance response can provide a marked contribution to SIF and NPQ generation. The dependence of NPQ on the quality of actinic light suggests that chloroplast movements within the cell have a noticeable impact on the formally determined NPQ value. Analyzing kinetics of post-illumination decay of NPQ in the context of solar stress resistance, we have found that LL-acclimated Tradescantia leaves are more vulnerable to strong light than the HL-grown leaves.

Appendix

Chlorophyll fluorescence and anatomy peculiarities of Tradescantia leaves

Analysis of induction events in high plant leaves may be complicated by anatomical peculiarities of leaves. Cui et al. (1991), who measured chlorophyll and light gradients in sun and shade leaves of Spinacia oleracea, found that propagation of BL and RL within leaves appeared to be determined largely by light absorption. Light at 450, 550, and 680 nm (blue, green and red light) was greatly attenuated within the palisade layer by initial 150 μm of palisade tissue. In the context of BL and RL propagation into the depth of the Tradescantia leaf, we consider below how the initial rise of fluorescence to maximal value (parameter F_P) depends on the photon flux density and light quality. Here, we have to mention one interesting and unusual anatomical property of Tradescantia leaves (T. fluminensis and T. sillamontana) described by Ptushenko and collaborators (Ptushenko et al. 2016; Ptushenko and Ptushenko 2019). It is well-established fact that in traditional model plants, Arabidopsis and Spinacia oleracia, chloroplast-containing cells are spread over the palisade and spongy mesophyll. The light intensity appears to be rapidly reduced and spectrally dispersed with the leaf depth in the direction perpendicular to the leaf surface (Vogelmann and Evans 2002; Oguchi et al. 2011a, b). In contrast to Arabidopsis and Spinacia oleracia, the mesophyll layer in T. fluminensis leaves is relatively thin (~ 40 μm and ~ 70 μm in LL and HL plants, respectively). In T. fluminensis, chloroplast-containing cells are concentrated in mesophyll, which comprises only ~ 10–13% (HL leaves) or ~ 24% (LL leaves) of the whole leaf thickness (Ptushenko et al. 2016). The mesophyll layer consists on average of 2–3 layers of mesophyll cells. The tissues adjacent to mesophyll (thick layers of adaxial and abaxial epidermis; see cartoon in Fig. 14) are almost free of chloroplasts. Thus, in T. fluminensis the vast majority of chloroplast-containing cells are clustered in the relatively thin layer inside the leaf. This circumstance (relatively thin mesophyll) might be important from the optical point of view: since mesophyll cells are located within a relatively thin layer, dispersion of photosynthetically active light absorbed by mesophyll should be reduced.

The top panel in Fig. 14 depicts the histogram, in which we compare the $F_{\text{P}} /F_{\text{m}}^{ 0}$ ratios (for definition, see Fig. 1) determined in LL- and HL-acclimated leaves upon the action of weak (40 μmol photons m⁻² s⁻¹) or strong (500 μmol photons m⁻² s⁻¹) actinic light. Here, we refer to maximal intensity of fluorescence $F_{\text{P}}$ measured at the very beginning of actinic light irradiation, when the regulatory effects associated with the NPQ and avoidance responses to be negligible. In LL-grown leaves illuminated by weak actinic light, the $F_{\text{P}} /F_{\text{m}}^{ 0}$ ratio determined for BL was lower than that in the case of RL illumination. We can explain this result by more significant attenuation of the BL flux compared to RL. In HL-grown plants, however, both BL and RL induced an increase in fluorescence to the same level F_P. The latter result could be explained by morphological peculiarities of Tradescantia leaves. Acclimation of Tradescantia to HL conditions causes the thickening of leaves and the formation of elongated column-type cells in adaxial epidermis (Ptushenko et al. 2016). Cui et al. (1991), who investigated light propagation within Spinacia oleracia leaves, put forward the curious hypothesis that thicker palisade tissue of sun leaves could facilitate the light propagation to spongy mesophyll, providing light penetration further into the leaf. They speculated that elongated cells might act as the lenses that promoted the transmission of light further into the mesophyll cells. In that event one could expect that in HL leaves both BL and RL would propagate more efficiently toward the mesophyll cells. Actually, for HL-grown T. fluminensis leaves, the $F_{\text{P}} /F_{\text{m}}^{ 0}$ ratio was the same both for weak BL and weak RL (Fig. 14). In the case of strong actinic light, the $F_{\text{P}} /F_{\text{m}}^{ 0}$ ratio was practically the same ($F_{\text{P}} /F_{\text{m}}^{ 0}$ = 0.98–0.99), regardless of the growth light intensity and the quality of actinic light. This suggests that the fluxes of strong BL and strong RL in the vicinity of mesophyll were enough to excite fluorescence of maximal intensity F_P.

Fig. 14

a Effects of intensity of blue (BL) and red (RL) actinic light on the fluorescence parameter $F_{\text{P}} /F_{\text{m}}^{ 0}$(for definition, see Fig. 1) in LL- and HL-acclimated T. fluminensis leaves, as indicated. Mean values (n = 4–6) ± SE. b Cartoon illustrating the mesophyll layer position and light propagation across the leaf

The comparison of photo-reducible PQ pool capacities in LL and HL plants

One can evaluate the relative sizes of the photo-reducible PQ pool (parameter Q₀) in LL- and HL-acclimated Tradescantia from the kinetics of fast induction of Chl a fluorescence (the OJIP curve) as described in (Suslichenko and Tikhonov 2019). Parameter Q₀ was calculated as the integral $Q_{0} = I_{2} \int_{{t_{1} }}^{{t_{2} }} {\alpha (t)dt = I_{2} \times W}$, where in I₂ the PFD for PSII and W is the so-called work integral. The variable α(t) = 1 − F(t)/F_m contains F(t) and F_m, the values of which denote the current and maximal intensities of Chl a fluorescence, respectively. The twofold difference between the Q₀ values for LL- and HL-acclimated Tradescantia leaves, Q₀(LL)/Q₀(HL) ≈ 2, suggests markedly increased capacity of the PQ pool in LL leaves compared to HL samples (for more details, see Suslichenko and Tikhonov 2019). A modified approach to the estimation of Q₀ proposes the integration of the variable β(t) = 1 − [F(t) − F₀]/[F_m − F₀], instead of α(t) (Tóth et al. 2007). It is not difficult to show that both approaches lead to close values of parameter Q₀. Elementary algebraic manipulations shows that α(t) =  β(t)*[(F_m − F₀)/F_m]. The correction factor, α(t)/β(t) = (F_m − F₀)/F_m, was of the same value in LL and HL leaves, (F_m − F₀)/F_m = 0.8 (see Fig. 1 in Suslichenko and Tikhonov 2019). This shows that both approaches would lead to the same proportionality between the PQ pool capacities in LL and HL samples, Q₀(LL)/Q₀(HL) ≈ 2.

Chlorophyll fluorescence and the avoidance response in Tradescantia leaves

In the context of the chloroplasts avoidance response, which can influence the time-course of SIF, we address to experimental data reported by Ptushenko et al. (2017), who investigated optical properties of T. fluminensis leaves in plants cultivated at LL and HL under experimental conditions close to those used in our work. Figure 15a demonstrates the spectrum of optical transmittance of dark-adapted T. fluminensis leaves grown under the HL conditions (800 μmol photons m⁻² s⁻¹). This spectrum was extracted and re-plotted from Fig. 1 presented in (Ptushenko et al. 2017). Figure 15b demonstrates how the leaf transmittance, determined at 455, 550, 630, and 680 nm, increased upon illumination of T. fluminensis leaves by continuous blue light (475 nm, 150 μmol photons m⁻² s⁻¹). Undoubtedly, the light-induced blooming of the leaf was caused by a decrease in the light absorptance caused by the chloroplast avoidance response. Actually, one can see that the most significant increase in the optical transmittance was observed at 455 and 680 nm (maximally absorbed by Chl molecules), rather than that in the green area of spectrum (~ 550 nm). Interestingly that the half-time of the light-induced increase in optical transparence in HL-grown leaves (t_1/2 ~ 5–7 min) was similar to the appearance of the intermediate state M₂ of SIF observed upon illumination by BL of LL- and HL-acclimated T. fluminensis leaves (Fig. 3).

Fig. 15

Optical transmittance spectrum for dark-adapted T. fluminensis leaves grown under the HL conditions (a) and kinetics of the light-induced changes in optical transmittance at four wavelength (455, 550, 630 and 680 nm) induced by continuous blue light (b). All the data shown here were extracted from Fig. 1 in (Ptushenko et al. 2017) and then transformed into the kinetic curves shown in b