Rapid light curves: A powerful tool to assess photosynthetic activity

Abstract

Rapid light curves provide detailed information on the saturation characteristics of electron transport, as well as the overall photosynthetic performance of a plant. Rapid light curves were collected from samples of Zostera marina grown under low and high-light conditions (50 and 300 μmol photons m⁻² s⁻¹) and the distinctive patterns of RLC parameters are discussed, in terms of differential sink capacity and PSII reaction centre closure. Derived cardinal points of a rapid light curve (α, E_k and rETR_max) describe the photosynthetic capacity of a seagrass leaf, its light adaptation state and its capacity to tolerate short-term changes in light. The shapes of the corresponding F and ${F^{\prime }}_{\text{m}}$ curves also provide information on the development of the trans-thylakoid proton gradient and thermal energy dissipation. Low-light leaves showed limited photosynthetic capacity and reduced activity of non-photochemical quenching pathways, whereas photosynthesis of high light leaves were not limited and showed an elevated level of non-photochemical quenching, possibly associated with xanthophyll cycle activity. Light-dark kinetics are also discussed in relation to relaxation of non-photochemical quenching and its various components. A curve fitting model is recommended based on the double exponential decay function. In this paper, we explain the fundamental aspects of a RLC, describe how it reflects the response to light exposure of a leaf, how to interpret these curves, and how to quantitatively describe and compare RLCs.

Introduction

Seagrass leaves are able to maintain photosynthetic activity over a wide range of light conditions. They have various mechanisms to optimize light interception and utilization: these include canopy architecture, chloroplast distribution and thylakoid membrane organization (Dawson and Dennison, 1996, White and Critchley, 1999). Like corals, seagrasses are exposed to highly variable light fields due to wave action (wave focusing), shading by clouds, leaf blade oscillation, self-shading, as well as tidal and solar oscillations (Falkowski et al., 1990). Seagrasses can grow in a wide range of light climates, from the intertidal zone where they are exposed to full sunlight (ca. 2000 μmol photons m⁻² s⁻¹), to very deep meadows up to 50 m (Lee Long et al., 1993). Leaves adapted to a high-light (HL) environment have a range of physiological characteristics, such as lower chlorophyll content, high photosynthetic capacity and active photoprotective mechanisms (such as xanthophyll pigments), while low-light (LL) leaves generally show the reverse (Demmig-Adams et al., 1999). These characteristics can change photosynthetic activity according to the prevailing light conditions, as well as the seasonal light climate. Pigment content usually takes several days to weeks for acclimation, while the xanthophyll cycle can be regulated in minutes, and effective quantum yield can change in seconds (Ralph et al., 2002b). There are also cyclical changes in photosynthetic activity, such as diurnal fluctuations in photosynthetic efficiency (Ralph et al., 1998, Durako and Kunzelman, 2002, Enríquez et al., 2002).

Over the past 15 years, the measurement of the chlorophyll a fluorescence has proven to be a powerful method of assessing the properties of the photosynthetic apparatus (Schreiber, 2004). PAM fluorometers use three different lights to manipulate the photosynthetic apparatus (specifically the chlorophyll a molecule), which in turn emits different quantities of fluorescence. Firstly, weak measuring light (0.15 μmol photons m⁻² s⁻¹) induces a fluorescence emission without inducing photosynthesis, and this is used to determine the proportion of closed PSII reaction centres. Once a PSII reaction centre captures a photon, it must pass the energy to the electron transport chain before it opens again and captures another photon. The fluorescence emitted (fluorescence yield) as a result of the measuring light only, is called minimum fluorescence (F_o for dark-adapted and F for light-adapted samples as measured immediately before the saturating pulse). The second light source used to assess photosynthetic activity is a saturating pulse (>10,000 μmol photons m⁻² s⁻¹, 0.4–0.8 s), which is used to close all PSII reaction centres, resulting in a substantially greater fluorescence emission. This is called maximum fluorescence (F_m if dark-adapted or ${F^{\prime }}_{\text{m}}$ if light-adapted). The third light source is used to manipulate the photosynthetic apparatus and is known as actinic light. This is used to induce photosynthesis and can range up to 2000 μmol photons m⁻² s⁻¹. Dark-adaptation allows PSII reaction centres to open, electron transport chain to be oxidized, photoprotective mechanisms (xanthophyll cycle) to be relaxed and the trans-thylakoid (ΔpH) gradient to be depleted. Measurement of the photosynthetic efficiency can be derived from the minimum and maximum values: maximum quantum yield of PSII (F_v/F_m = [F_m − F_o]/F_m, requires dark-adapted leaves) and effective quantum yield of PSII (${\Phi }_{\text{PSII}}=\Delta F/{F^{\prime }}_{\text{m}}=[{F^{\prime }}_{\text{m}}-F]/{F^{\prime }}_{\text{m}}$ requires light-adapted leaves; Genty et al., 1989). Φ_PSII provides an indication of the amount of energy used in photochemistry. Effective quantum yield (Φ_PSII) measured with light-adapted leaves are lower than dark-adapted (F_v/F_m), due to the inherent impact of non-photochemical quenching reducing the light-adapted quantum yield (Beer et al., 2001).

The electron transport rate (ETR) was found to be closely related to the photosynthetic activity when measured by oxygen evolution or CO₂ uptake (Beer et al., 1998). Relative ETR is an approximation of the rate of electrons pumped through the photosynthetic chain (Beer et al., 2001):

$$\text{relative}\text{electron}\text{transport}\text{rate}(\text{rETR})={\Phi }_{\text{PSII}}\times \text{PAR}$$

Photon energy captured by a chlorophyll a molecule can either drive photosynthesis (photochemical quenching, qP), be emitted as fluorescence, or be converted to heat (non-photochemical quenching, qN and NPQ). Heat dissipation is linked to the xanthophyll cycle, which protects the photosynthetic apparatus from high-light damage. Quenching analysis monitors the development of qP and qN (or NPQ) (Schreiber, 2004), while NPQ can be used to infer activity of the xanthophyll cycle (Demmig-Adams et al., 1999, Ralph et al., 2002b). Quenching analysis compares the fluorescence yield during a saturating pulse under actinic light conditions (${F^{\prime }}_{\text{m}}$ and F), with the dark-adapted values (F_m and F_o). There are two coefficients of non-photochemical quenching; qN and Stern–Volmer quenching (NPQ). Stern–Volmer quenching (NPQ) is more sensitive to energy dissipation within the antennae matrix (containing xanthophylls, where energy dependent quenching occurs), while it is relatively insensitive to lower levels of qN (0–0.4) which are mainly associated with thylakoid membrane energization (Schreiber, 2004). We recommended using the Stern–Volmer coefficient (NPQ), as it is a more robust assessment of non-photochemical quenching, since it is not dependent upon ${F^{\prime }}_{\text{o}}$ and is not affected by F_o quenching (Schreiber, 2004). Non-photochemical quenching and Φ_PSII are correlated, where Φ_PSII decreases with increasing irradiance, as more electrons accumulate at the PSII acceptor side and there is a relative increase in non-photochemical quenching (heat energy dissipation). For further details see Schreiber (2004).

Light curves can assess not only the present photosynthetic capacity, but the plant's potential activity over a wide range of ambient light intensities. This type of measurement is known as a photosynthesis-irradiance curve (P–E, formerly P–I; Falkowski and Raven, 1997). Photosynthesis–irradiance curves are commonly used to measure and describe the acclimation of the photosynthetic apparatus to a range of light intensities, using O₂ or CO₂ gas exchange measurements. P–E curves have been measured for many years and numerous mathematical models have been used to describe the photosynthetic rate, as well as several characteristics of the curves (Platt et al., 1980, Harrison and Platt, 1986).

In comparison to traditional light curves, a rapid light curve (RLC) measures the effective quantum yield (Φ_PSII) as a function of irradiance. If rETR is plotted against PAR, a RLC looks similar to a traditional oxygen-based P–E curve; however it is not a P–E curve and should not be interpreted as if it is one (Hawes et al., 2003). In situations where the light field is rapidly fluctuating, a RLC can provide a reliable assessment of photosynthetic activity, by integrating the leaf's ability to tolerate light fluctuation, as well as reflecting its immediate short-term light history (Schreiber et al., 1997, White and Critchley, 1999). Whereas, a single Φ_PSII determination would be strongly dependent upon the light climate immediately prior to saturating pulse (Rascher et al., 2000, Schreiber, 2004). A RLC does the same as a P–E curve; it measures the photosynthetic performance as a function of irradiance, however a RLC does not achieve steady state conditions during each light step (Schreiber et al., 1997), whereas a P–E does. Normally, a RLC uses only 10 s of actinic light at each of eight light steps, the Φ_PSII and the rETR indicate the actual state of photosynthesis, not the optimal state as shown in a steady state P–E curve which is independent of light pre-history. RLCs have been called rapid P–I curves (Beer et al., 1998) light response curves (Schreiber, 2004) and instant light-response curves (Rascher et al., 2000). We recommend using the term RLC, as it shows the light-acclimation state over the past few minutes, but is also strongly influenced by its long-term pre-history (such as HL or LL adapted plants). RLCs have been used in a range of seagrass ecophysiological investigations. Since the photophysiology of a seagrass leaf changes throughout the daily cycle of irradiance (Durako and Kunzelman, 2002), RLCs reflect the relative condition throughout diel and tidal cycles (Beer et al., 1998, Ralph and Gademann, 1999, Ralph et al., 2002b).

Both RLC and traditional P–E curves have three distinct regions: the light limited, the light-saturated and the photoinhibited region (supra-optimal irradiance). With low irradiance, photosynthesis is limited by the irradiance. The rise of the curve in the light-limiting region (α) is proportional to efficiency of light capture (effective quantum yield; Schreiber, 2004). Minimum saturating irradiance (E_k) is determined by finding the interception of α with the maximum photosynthetic rate (Sakshaug et al., 1997). E_k is related to quenching, where photochemical quenching dominates below E_k, while non-photochemical quenching dominates the fluorescence quenching above E_k (Henley, 1993). Since irradiance is continuously fluctuating and acclimation takes some time, E_k is constantly changing and rarely matches the instantaneous irradiance (Sakshaug et al., 1997). Under moderate irradiance, the capacity of the electron transport chain limits photosynthesis and the curve reaches a plateau, where maximum photosynthetic capacity occurs (rETR_max) (Schreiber, 2004). With even higher irradiance (supra-saturating), the curve often tends to decline. With traditional P–E curves, this decline is usually associated with photoinhibition (Henley, 1993); however with RLCs this decline could be linked to dynamic down-regulation of PSII (White and Critchley, 1999), not photoinhibition as there is insufficient time for photodamage to occur.

Given that the Diving-PAM is now a well-integrated tool of seagrass research, we felt that it was timely to provide an explanation on how best to use RLC. The majority of published fluorescence in the seagrass literature deals with quantum yield estimates, while this powerful techniques has been largely ignored, possibly due to insufficient understanding of what a RLC is capable of providing. In this paper, we provide an overview of the use and interpretation of RLCs and discuss some techniques for the quantitative interpretation of these curves. We present data describing changes in photophysiology of a population of Zostera marina acclimated to two light climates. With the use of RLCs, we demonstrate how a range of cardinal points describing photosynthesis were modified during light climate acclimation.