Light intensity dependent photosynthetic electron transport in eelgrass (Zostera marina L.)

Accepted Manuscript Light intensity dependent photosynthetic electron transport in eelgrass (Zostera marina L.) Xiao Qi Yang, Quan Sheng Zhang, Di Zhang, Zi Tong Sheng PII:

S0981-9428(17)30061-X

DOI:

10.1016/j.plaphy.2017.02.011

Reference:

PLAPHY 4808

To appear in:

Plant Physiology and Biochemistry

Received Date: 18 October 2016 Revised Date:

7 February 2017

Accepted Date: 9 February 2017

Please cite this article as: X.Q. Yang, Q.S. Zhang, D. Zhang, Z.T. Sheng, Light intensity dependent photosynthetic electron transport in eelgrass (Zostera marina L.), Plant Physiology et Biochemistry (2017), doi: 10.1016/j.plaphy.2017.02.011. 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.

ACCEPTED MANUSCRIPT 1

Light intensity dependent photosynthetic electron transport in eelgrass (Zostera marina L.)

2 Xiao Qi Yang, Quan Sheng Zhang*, Di Zhang, Zi Tong Sheng

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Ocean School, Yantai University, Yantai 264005, PR China

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* Corresponding author. Tel.: +86 0535 6706011; fax: +86 0535 6706299.

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E-mail address: [email protected] (Q.S. Zhang).

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Abstract

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Responses of electron transport to three levels of irradiation (20, 200, and 1200µmol photons m−2 s−1

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PAR; exposures called LL, ML and HL, respectively) were investigated in eelgrass (Zostera marina L.)

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utilizing the chlorophyll a fluorescence technique. Exposure to ML and HL reduced the maximum

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quantum yield of photosystem II (PSII) (Fv/Fm) and the maximum slope decrease of MR/MRO (VPSI),

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indicating the occurrence of photoinhibition of both PSII and photosystem I (PSI). A comparatively

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slow recovery rate of Fv/Fm due to longer half-life recovery time of PSII and 40% lower descending

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amplitude compared to other higher plants implied the poor resilience of the PSII. Comparatively, PSI

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demonstrated high resilience and cyclic electron transport (CEF) around PSI maintained high activity.

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With sustained exposure, the amplitudes of the kinetic components (L1 and L2), the probability of

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electron transfer from PSII to plastoquinone pool (ψET2o), and the connectivity among PSII units

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decreased, accompanied by an enhancement of energy dissipation. Principle component analysis

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revealed that both VPSI and Fv/Fm contributed to the same component, which was consistent with high

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connectivity between PSII and PSI, suggesting close coordination between both photosystems. Such

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coordination was likely beneficial for the adaption of high light. Exposure to LL significantly increased

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the activity of both PSI and CEF, which could lead to increased light harvesting. Moreover, smooth

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electron transport as indicated by the enhancement of L1, L2, ψET2o and the probability of electron

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transport to the final PSI acceptor sides, could contribute to an increase in light utilization efficiency.

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Keywords: Chlorophyll a fluorescence; Electron transport; Light exposure; Resilience; Zostera marina

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Abbreviations: CEF, cyclic electron flow; DF, delayed fluorescence; MR820nm, 820 nm modulated

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reflection; NPQ, non-photochemical quenching; PCA, principal components analysis; PF, prompt 1

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fluorescence; PSI, II, photosystem I, II; RC, reaction center; RLC, rapid light response curve;

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1. Introduction

4 As one of the most important producers in the coastal ecosystem, seagrasses play a key role in

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sediment stabilization and provision of extensive habitats for benthic organisms (Waycott et al. 2009).

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However, due to anthropogenic disturbance, seagrasses are globally in decline (Orth et al. 2006;

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Skinner et al. 2014; Munkes et al. 2015). Anthropogenic nutrient loading induces the growth of

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phytoplankton and macroalgal blooms, and the resulting light limitation has been revealed as one of the

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common stress factors for seagrass (Ralph et al. 2007; Mazzuca et al. 2009; Ochieng et al. 2010).

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Studies have shown that energy balance in photosystems depends on the optimization of light capture

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achieved via an increase of light utilization efficiency and an adjustment of the photosynthetic and

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photoprotection pigment pool (Ochieng et al. 2010). Furthermore, seagrasses are susceptible to

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disturbance via excess irradiation. To reduce the photo-oxidation damage of excess irradiation,

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seagrasses operate a series of regulatory mechanisms, such as photoprotection, photoinhibition and

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dynamic down-regulation of photosystem II (PSII) activity (Ralph et al. 2002). When exposed to

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excess light, dealing with the equilibrium between effective utilization of a limited light and thermal

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dissipation is of vital importance for plants (Dai et al. 2009). Energy production and thermal dissipation

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closely correlate with electron transport in the photosynthetic apparatus (Rochaix 2011, Takahashi and

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Badger 2011). Photosynthetic electron transport is an effective tool to explore the mechanisms seagrass

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utilizes to achieve the necessary energy balance needed for survival (Enríquez and Borowitzka 2010).

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Consequently, the study of the photosynthetic electron transport response to light is an important

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subject in seagrass.

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Eelgrass (Zostera marina L.) exhibits a common response following exposure to visible light:

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down-regulation of PSII activity. Repair of the PSII reaction center (RC) is frequently necessary due to

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rapid turnover of D1 protein. It is generally accepted that PSII repair is an important photoprotection

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mechanism (Nixon et al. 2010). Additionally, non-photochemical quenching (NPQ) of excess

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excitation energy acts as a major photoprotective mechanism of PSII (Ivanov et al. 2008; Lambrev et al.

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2012). Considerable attention focuses on PSII (Ralph et al. 2007, Ochieng et al. 2010, Skinner et al.

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2014), but information concerning the photochemical characteristics of photosystem I (PSI) is still 2

ACCEPTED MANUSCRIPT limited for eelgrass. In terrestrial plants, PSI photoinhibition exhibits slow recovery characteristics and

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the potential for secondary damage (Sonoike 2011). The consequence of PSI photoinhibition seems to

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be more severe compared to PSII (Goh et al. 2012; Yan et al. 2013). When PSI photoinhibition is

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induced, the resulting block of electron transport causes the collapse of ATP synthesis. Energy

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deficiency reduces repair cycle rates, thus aggravating the degree of PSII photoinhibition (Zhang et al.

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2011, Tikkanen et al. 2014). Optimal performance of photosynthesis is dependent on functional and

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structural coordination between PSI and PSII (Nellaepalli et al. 2012). Consequently, the mechanisms

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of interaction between PSII and PSI in response to light warrants further exploration.

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Eelgrass is a representative of temperate seagrass and one of the few higher plants that live in the

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ocean. Related studies suggest that the photosystem of eelgrass shows a slight difference to other

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higher plants (Silva and Santos 2003). E.g., members of the light harvesting complex B family extend

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quantity to enhance their photosynthetic performance via combination with NPQ (Olsen et al. 2016).

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When light capture levels exceed the assimilation of available carbon in eelgrass, the role of

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photorespiration becomes vital, as a primary electron sink. However, the contribution of the Mehler

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reaction is minor compared to that of photorespiration, which is inconsistent with studies on other

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plants (Buapet and Björk 2016). Consequently, the photosynthetic electron transport in eelgrass may

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exhibit special characteristics in response to light. Chlorophyll a fluorescence is extensively utilized for

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detecting the plant health status and the influences of environmental stress influences on photosynthetic

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performance (Enríquez and Borowitzka 2010; Ptushenko et al. 2013). Especially, the simultaneous

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measurement of prompt fluorescence (PF) and delayed fluorescence (DF) as well as 820 nm modulated

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reflection (MR820nm) are able to reveal the process of photosynthetic electron transport and hence, the

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interplay between PSII and PSI (Strasser et al. 2010).

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In this study, we investigated the comprehensive physiological responses of eelgrass photosynthetic

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electron transport through the chlorophyll a fluorescence technique. Our main objectives were to: (i)

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examine the variations of PSII and PSI activities; (ii) explore the connectivity of the electron transport

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chain; (iii) evaluate the coordination between PSII and PSI.

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2. Materials and methods

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2.1. Plant materials 3

ACCEPTED MANUSCRIPT 1 Mature eelgrasses were collected from Changdao (37º 91´N, 120º 73´E) in the Yantai, Shandong

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province, China throughout May 2016. Selected samples were healthy in appearance with intact

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rhizomes (6-9 internodes). We removed surface epiphytes and sediments as well as the older leaves and

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internodes prior to transfer into the laboratory. Eelgrasses were allowed to acclimate in filtered

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seawater in aquaria at a constant temperature of 15 °C for 3 d prior to use in the experiments. A LED

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lamp (LI-6400-04; Li-Cor, Lincoln, NE, USA) provided the light source above the aquaria under a 10:

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14 h light: dark photoperiod with a minimum saturation light intensity of 100 µmol photons m−2 s−1.

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Seawater in the aquaria was aerated to ensure mixing and the seawater was completely changed daily

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until experimental use to avoid nutrient limitation and excessive growth of phytoplankton. During the

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course of the experiment, we attached stainless steel clips to all eelgrass plants, keeping them upright to

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simulate their growing state in the natural environment.

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2.2. Light treatment

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Pre-cultured eelgrasses were dark-adapted overnight prior to light treatment and then perpendicularly

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exposed to three different light intensities (20, 200, and 1200 µmol photons m−2 s−1 PAR; exposures

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were referred to as LL, ML and HL, respectively), informed by the minimum saturating light intensity

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measured in our preliminary trials. The applied light intensity above the leaf surface of the upright LL

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exposure group was low, but still above the light compensation point (10 µmol photons m−2 s−1) of

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eelgrass (Villazán et al. 2013). Subsequent to exposure to their respective light treatments for 3 h,

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eelgrasses were immediately transferred to darkness for recovery. All treatments were conducted in

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illumination incubators (GZP-250N, Shanghai senxin experimental instrument co., LTD, China) with a

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controlled seawater temperature of 15 °C, employing separate plots of different light intensities.

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2.3. Chlorophyll a fluorescence measurement

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Pulse-Amplitude Modulated (PAM) Fluorometer (Mini-PAM; Walz, Germany) was used to measure

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NPQ as well as the rapid light response curve (RLC) at 3 h-intervals. Eelgrasses were placed in the leaf

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clip from the fiber optic probe at a 60° angle to avoid shading or darkening. RLC was determined on 4

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dark-adapted samples with a pre-installed software routine, with actinic illumination levels of 0, 72,

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111, 150, 260, 400, 752, 1176 and 1818 µmol photons m-2 s-1. The exposure time for each actinic light

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level was 10 s. To determine photosynthetic efficiency (α) and relative maximum electron transport

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rate (rETRmax), we fitted the RLC data to the double exponential decay function. Multi-Function Plant Efficiency Analyzer 2 (M-PEA-2; Hansatech, UK) was used to simultaneously

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record the kinetics of PF, DF and MR820 nm and thus to synchronously monitor the two photosystem

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photochemical activities and to deeply dissect the electron transport chain. The M-PEA-2 emitted a

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saturating light pulse with an intensity of 5000 µmol m-2 s-1 for 10 s. PF and DF were recorded in the

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light and dark intervals. PF kinetics reflected the different steps and phases of PSII and we analyzed

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these via the JIP-test based on Strasser et al. (2010). In this paper, the following original data were used:

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the minimum fluorescence intensity (FO) at 50 µs, the maximum fluorescence intensity (Fm) and the

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fluorescence at 0.3 ms (K-step, FK), 3ms (J-step, FJ) and 30 ms (I-step, FI). The relative variable

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fluorescence at the K-step to the amplitude FJ–FO was defined as WK = (FK-FO)/(FJ-FO) and the relative

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variable fluorescence intensity at both J-step (VJ) and I-step (VI) were defined as: Vt = (Ft-FO)/(Fm-FO).

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The probabilities for an electron moving further than the PSII primary electron transport acceptor (QA),

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for an electron moving from QA- to the PSII secondary electron transport acceptor (QB), and for an

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electron moving from the reduced intersystem electron acceptors to the final PSI electron acceptors

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were respectively defined as ψET2o = 1-VJ, ψRE1o = 1-VI, and δRo = (1-VI)/(1-VJ). The quantum yield of

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the primary photochemistry, for the electron transport and for the reduction of PSI end electron

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acceptors were defined as φPo = Fv/Fm = 1-FO/Fm, φEo = φPo × (1/VJ) and φRo = φPo × (1/VJ),

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respectively. The apparent antenna size of the active PSII RC (ABS/RC) was defined as ABS/RC = MO

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× (1/VJ) × [1/(Fv/Fm)], where MO represented the approximated initial slope of the fluorescent transient.

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The effective dissipation of energy in the active RC of PSII (DIO/RC) was defined as DIO/RC = MO ×

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(1/VJ) × [1/(FO/Fm)]. The probability of the connectivity among PSII units (ω) was calculated as ω = p

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× [(Fm-FO)/Fm], where p represented the calculated connectivity parameter according to Zivcak et al.

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(2014). MR820nm obtained by saturating red light elicited a fast oxidation phase and a following

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reduction phase. To eliminate the disturbance caused by geometrical differences, we normalized

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MR820nm as MR/MRO. MRO was the modulated reflection signal of the first reliable MR measurement

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(taken at 7 ms) and MR was the signal during the course of illumination (Gao et al. 2014). Cyclic

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electron flow (CEF) around PSI was determined following the effects of far-red illumination for 100 s

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ACCEPTED MANUSCRIPT (Wang et al. 2006). The initial rate (0-0.3 s) of P700+ re-reduction (Vre-red), maximum decrease of slope

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(VPSI) and maximum increase of slope (VPSII-PSI) of MR/MRO were calculated using Excel 2003. All

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parameters derived from M-PEA-2 were simultaneously measured on the same leaf. Prior to the

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measurement of the fluorescence dynamics, leaves of exposure to light were randomly selected at 30

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min-intervals to dark-adapt for 15 min.

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Eelgrass leaves grow from the sediment towards the peak of the canopy and the meristematic tissues

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are located basally. Accordingly, we conducted all fluorescence measurements 2-3 cm above the base,

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ensuring consistency of the sample age.

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All data were analyzed using SPSS 22.0 statistical software. Effects of light treatment on the

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measured parameters (JIP-test parameters, VPSI, VPSII-PSI, Vre-red, L1, L2, NPQ, α, and rETRmax) were

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analyzed with repeated measures ANOVA using treatment time as the within-group factor and light

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intensity as the categorical factor. The kinetic models of VPSI and Fv/Fm were fitted, using the single

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exponential function fitting procedure of Origin 8.0. Decay kinetics of DF data was fitted to the time

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function DF (t) = L1× exp (-t/τ1) + L2× exp (-t/τ2) + L3, where L1, L2, and L3 were the amplitudes of the

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kinetics component and τ1 and τ2 were their lifetimes (in ms). Tukey’s tests were utilized for the post

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hoc comparisons. Differences were considered statistically significant when p < 0.05. Principal

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components analysis (PCA) was used to reveal the variability and correlation between parameters. Nine

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parameters (L1, Fv/Fm, ψET2o, φEo ψRE1o, δRo, φRo, VPSI, and Vre-red) connected with electron transport

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were selected to conduct the PCA analysis in our treatments of LL, ML and HL, respectively.

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Components with eigenvalue above greater than 1 were extracted.

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3. Results

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3.1. Effect of light exposure on kinetics of PF, DF and MR820 nm

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PF induction kinetics of eelgrass exhibited a similar sigmoid characteristic response to all three light

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intensities. The decrease of fluorescence intensity with enhanced light levels was more accentuated in 6

ACCEPTED MANUSCRIPT both PF and DF (Fig. 1a-b). Decay curves at I1 (at about 7 ms) exhibited a multiphase decrease

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tendency (Fig. 1c). In addition, the minimal MR/MRO, when the oxidation and re-reduction rates of

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P700 and PC were equal, increased in response to the elevation of light intensity (Fig. 1d). Supported by

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our calculated fluorescence parameters, these changes of fluorescence dynamics confirmed that the

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photosynthetic responses of electron transport differed in different light intensity.

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3.2. Effect of light exposure on the photochemical activities of PSII and PSI

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Maximum quantum yield of PSII (Fv/Fm) decreased gradually with exposure duration (Fig.2a).

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However, the kinetics model proposed by Hanelt (1998) did not perfectly fit the time-course changes of

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Fv/Fm. Fv/Fm reduced in relation to light exposure by 2.1% (LL), 3.8% (ML) and 10% (HL). We

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found the slowest half-life time (t1/2) for Fv/Fm in eelgrass after exposure to HL. Although the

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descending amplitude of Fv/Fm was limited, the recovery rate was slower as indicated by the extended

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t1/2 of Fv/Fm (Fig.2a). Fv/Fm during LL and ML exposures fully recovered within 3 h of darkness,

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whereas Fv/Fm during HL exposure did not return to the initial value until the next morning. The

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maximum slope decrease of MR/MRO (VPSI), which was considered to be indicative of photochemical

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activity of PSI, increased by 21% following LL exposure and reduced by 13% and 23% following

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exposure to ML and HL, respectively (Fig. 2b). In case of HL, the calculated values of t1/2 in VPSI was

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1.39 h, exhibiting faster recovery rates as compared to Fv/Fm (Fig. 2b). Subsequent recovery of VPSI

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was similar to Fv/Fm.

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Exposure to all three light intensities resulted in faster rates of P700+ re-reduction. Eelgrass exposed to

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LL showed the highest initial rate (0–0.3 s) of P700+ re-reduction (Vre-red), whereas the lowest value of

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Vre-red occurred during HL exposure (Fig. 3). We observed complete recovery of Vre-red within 3 h of

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darkness.

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3.3. Effect of light exposure on electron transport

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The JIP-test parameters were studied according to the sequence of linear electron transfer in

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photosystems. Furthermore, the amplitudes of the kinetic components of DF decay (L1 and L2) were

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also discussed to verify the JIP-test results. Relative variable fluorescence at the K-step to the 7

ACCEPTED MANUSCRIPT amplitude FJ-FO (WK) decreased during LL exposure and elevated gradually during HL exposure. L1

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exhibited a reverse trend with WK increasing following LL and HL exposures (Fig. 4a-b). However, WK

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remained constant while L1 decreased significantly during ML exposure (Post-hoc tests, P < 0.05). The

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variations of L1 and WK showed a more sensitive response of DF compared to PF. The probability of

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electron transfer from PSII to the plastoquinone pool (ψET2o) gradually decreased for the duration of

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ML and HL exposures, whereas it increased for LL. We observed a similar tendency in the quantum

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yield for electron transport (φEo), L2 and the connectivity among PSII units (ω) (Fig. 4c-f). These

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parameters fully recovered within 3 h of darkness following exposure to LL and HL, whereas the

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parameters did not return to the initial value until the next morning following HL exposure. The

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probability of electron transport from QA- to QB (ψRE1o) increased with the duration of exposure, which

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we considered as a semi-quantitative indicator for relative changes in PSI content. The values of ψRE1o

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ranged from 0.18 to 0.40. Both δRo and φRo, which were related to the electron transport to the final PSI

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electron acceptors, exhibited a similar variation with ψRE1o (Fig. 5a-c). ψRE1o, δRo and φRo fully

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recovered within 3 h of darkness following HL exposure, whereas the parameters did not return to their

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initial values until the next morning, following exposure to ML and LL. Connectivity between PSII and

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PSI (VPSII-PSI) decreased after LL and increased after ML and HL exposure (Fig. 5d). VPSII-PSI

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completely recovered within 3 h of darkness.

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3.4. Effect of light exposure on energy distribution of PSII

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Both the efficiency of light utilization of PSII (α) and the relative maximum electron transport rate

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(rETRmax) increased following three exposures. We observed maximum increase of rETRmax and α

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following ML exposure. Values of α and rETRmax fully recovered within 3 h of darkness. NPQ

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increased with the elevation of light intensity and the elevation of NPQ following HL exposure was

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approximately twice the amount of that following ML exposure. JIP-test showed an apparent antenna

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size of active PSII RC (ABS/RC) and effective dissipation of energy in active RC (DIO/RC) with

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similar enhancement following ML and HL exposures (Table 2). However, the reduction of ABS/RC

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was not significant (Post-hoc tests, P = 0.683), whereas DIO/RC increased significantly after LL

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exposure (Post-hoc tests, P < 0.05). ABS/RC and DIO/RC after ML and LL exposure completely

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recovered within 3 h of darkness, whereas they did not recover to the initial values until the next

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morning for HL.

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3.5. Principle component analysis

4 LL PCA revealed that the modifications in the first Principle Component (Comp 1) determined

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about 52.2% of the total changes and was characterized by positive loadings of ψET2o, φEo, ψRE1o, φRo

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and δRo. Comp 2 reflected 16.9% of the total changes and was positively loaded by Fv/Fm and L1.

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Comp 3 explained 15.4% of the total changes and was marked by a high positive correlation of VPSI and

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Vre-red. Comp 1 of ML was equal to LL PCA, but Comp 2 incorporated the Comp 2 and 3 of LL PCA

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(Fig. 6a-b). The modifications in Comp 1 determined about 49.2% of the total changes, while Comp 2

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reflected 23.0% and was characterized by positive loadings of VPSI, Fv/Fm and L1 and, to a larger

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extent, by negative loadings of Vre-red. In contrast with the ML PCA, two parameters (ψET2o and Vre-red)

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transferred to the next component in HL PCA. We quantified the contribution of ψET2o as 0.68 in Comp

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2 and 0.59 in Comp 1. Moreover, Comp 1 reflected 39.9% of total variations, Comp 2 determined

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33.4%, and Comp 3 determined 11.8% (Fig. 6c-d).

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4. Discussion

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In our present study, the values of Fv/Fm decreased while exposed to HL, demonstrating the

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universal down-regulation of PSII activity as other higher plants. However, the amplitude of

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down-regulation of Fv/Fm is limited in comparison to higher plants, whose Fv/Fm almost decreased

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by 50% when exposed to high light (Ramalho et al, 2000; Huang et al. 2012; Li and Ma 2012). In

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addition to the low descending amplitude of Fv/Fm, the slow recovery rate implied a limited resilience

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of PSII. Time-course changes of Fv/Fm did not match the model of photoinhibition published by

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Hanelt (1998), indicating that the variation of PSII activity in eelgrass was incapable to exhibit the

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differences of fast (Pfast) and slow (Pslow) component reaction dynamics. Studies have shown that Pfast to

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be correlated with the rapid reversibility of PSII down-regulation triggered by high light

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(García-Mendoza and Colombo-Pallotta, 2007). Therefore, the Pfast deficiency could account for the

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low resilience of PSII in eelgrass. Despite the resilience of Fv/Fm and slight spatial-temporal

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fluctuation in different seasons and growth periods (Enríquez et al. 2002), we suggest this poor

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ACCEPTED MANUSCRIPT 1

resilience to be special. Poor PSII resilience may be a deficiency specific to eelgrass, rendering it

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incapable to adequate levels of photosynthetic electron transport to varying light environments. It has been often considered that high light induces selective photodamage to PSII in nature

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(Tikkanen et al. 2014). However, our results revealed a VPSI reduction of 13% and 23% in ML and HL,

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respectively, indicating the occurrence of PSI photoinhibition. PSI photoinhibition generally

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correlates with electron transport from PSII (Sonoike 2011). The limited down regulation amplitude

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of eelgrass, which could not functions as the control of photosynthetic electron transport allowed the

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maintenance of oxidized P700 under excess light, might resulted in PSI photoinhibition. Additionally,

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the PSI completely recovered within a brief period of time and the recovery rate was faster in

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comparison to Fv/Fm as evidenced by faster t1/2 of VPSI, indicating a higher relative resilience of PSI.

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We speculate about the existence of a trade-off between the repair of PSII and PSI following exposure

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to excess light, i.e. eelgrasses accelerate the repair of PSI subunits at the cost of slowing the turnover

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rate of the D1 protein.

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When exposed to LL, VPSI significantly increased, indicating the up-regulation of PSI activity,

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which might attribute to the elevation of a light harvesting capacity in PSI. It is well documented that

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the operation of state transition contributed to the adjustment of energy distribution in the two

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photosystems for optimal photosynthesis (Suzuki et al. 2012). PSI was preferentially excited in weak

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light, enhancing the cross-section of PSI, thus leading to a shift from state I to state II (Antal et al.

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2011). Moreover, state II further increased the promotion of CEF around PSI as evidenced by the

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enhancement of Vre-red during LL exposure. Such enhancement could participate in the adjustment of

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energy equilibrium between PSII and PSI since the primary function of CEF around PSI was to

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balance the ATP/NADPH ratio (Yamori et al. 2015).

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Detailed information about that electron transport carrier work to accommodate the demand of PSII

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and PSI during light exposure was also provided in the present study. In the case of HL, the reduction

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of L1, ψET2o, φEo and L2 indicated the over-reduction of the electron transport chain, thus blocking the

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electron transport. Oxidative stress is one of the common light-induced damages that derive from the

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electron transport block (Rochaix 2011). The decrease of connectivity among PSII units could act as

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an effective strategy to alleviate the oxidative stress via the reduction of effective electron carriers and

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the production of reactive oxygen species (Zivcak et al. 2014). Moreover, the observed significant

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increase of DIO/RC and NPQ indicated that more excitation energy could be dissipated as heat. With 10

ACCEPTED MANUSCRIPT the duration of exposure, the ABS/RC also tended to increase. It might attribute to that the

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dismantling of antenna proceeded at a rate slower in comparison to the reaction center dismantling

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rate (Solovchenko et al. 2013). This progress effectively reduced the number of reaction centers and

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correspondingly increased the apparent antenna size. In the case of ML, the response of WK was not

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consistent with that of L1 because DF was more sensitive compared to PF. Additionally, the lower

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reduction degrees of L1, ψET2o, φEo and L2 indicated that the inhibition degree of over-reduction of the

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electron transport chain was less than that of HL. Eelgrass exposed to ML has the highest light

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utilization efficiency and relative maximum electron transport rates and therefore exhibits optimal

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photosynthetic performance. For LL exposure, the values of ABS/RC did not change significantly,

10

indicating that the light harvesting capacity of PSII remained unchanged. However, the relative

11

maximum electron transport rates increased due to the light-induced activation of enzymes involved

12

in carbon metabolism and the opening of stomata (Maxwell and Johnson 2000). Moreover, the

13

observed increase of L1, ψET2o, φEo, L2, ψRE1o, δRo and φRo and the decrease of WK suggested smooth

14

operation of electron transport in photosystems. In spite of a constantly maintained PSII light

15

harvesting ability, this smooth electron transport could contribute to the promotion of light utilization

16

efficiency via an increase of α.

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The multi-signal fluorescence parameters were grouped in PCA on the basis of their role in the light

18

response, thus revealing the common physiological principle in our data. The parameters of ψET2o, φEo,

19

ψRE1o, φRo and δRo were related to the PSII acceptor electron transport and contributed to Comp 1,

20

suggesting a coordinated functionality at the PSII donor side. Correspondingly, L1 and Fv/Fm

21

contributed to Comp 2, suggesting closely correlated PSII activity to the PSII donor side (OEC). Such

22

coordination between PSII and PSI was particularly close during exposure to ML and HL as indicated

23

by both the distribution of Fv/Fm and VPSI into Comp 2 and the relatively higher connectivity between

24

both photosystems. However, Fv/Fm and VPSI were distributed into different components in LL PCA,

25

respectively, indicating a weak coordination between both photosystems, which may be detrimental

26

for the operation of optimal photosynthetic performance as evidenced by the lower values of

27

rETRmax and α during LL exposure.

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In contrast to higher plants, the main differences of eelgrass photochemical characteristics are as

29

following: (1) it is generally recognized that PSII is very susceptible to light and has efficient and

30

dynamically regulated repair machinery (Tikkanen et al. 2014); however, our results revealed that the 11

ACCEPTED MANUSCRIPT decrease amplitude of Fv/Fm was limited and the recovery of PSII was slow; in addition, the

2

deficiency of Pfast further accounted for the poor resilience of PSII in eelgrass, which may be a

3

deficiency specific to eelgrass; (2) it is generally recognized that the PSI are efficiently protected

4

against photodamage, and in a rare case of damage when comparing to PSII; once the PSI

5

photoinhibition occurs, the subsequent recovery of PSI is extremely slow (Sonoike 2011); however,

6

our results revealed the occurrence of PSI photoinhibition; moreover, PSI completely recovered

7

within a brief period and the recovery rate was faster in comparison to PSII, suggesting a higher

8

relative resilience of PSI. In summary, our results demonstrated that the regulating capacity of PSII

9

was limited, while the regulating capacity of PSI remained strong. The close coordination between

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both photosystems endowed the eelgrass with high potential for adaption to high light.

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Contributions

13

XY and QZ conceived and designed the experiments, XY and DZ conducted experiments and carried

14

out the data analysis, XY and QZ wrote the manuscript. All authors participated in the preparation and

15

review of the manuscript.

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16 Acknowledgements

18

This work was financially supported by the National Natural Science Foundation of China (No.

19

41376154) and the Graduate Innovation Foundation of Yantai University (No. YDZD1714).

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17

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5 Figure Captions

7

Fig. 1 Variations in chlorophyll a fluorescence intensity of OJIP transient (a), 20µs delayed

8

fluorescence (b) and modulated reflection at 820 nm (d) exposed to 20µmol photons m−2 s−1 (LL),

9

200µmol photons m−2 s−1 (ML) and 1200µmol photons m−2 s−1 (HL), respectively. (c) Decay kinetics of

10

delayed fluorescence at the characteristic point I1 (7ms) in eelgrass leaves at different light treatments.

11

Each curve represents the average of 5 replicates

12

Fig. 2 Time courses of the changes in (a) PSII activity (Fv/Fm) and (b) PSI activity (VPSI) exposed to

13

LL, ML and HL, respectively. The numbers inside the figure are the half-life time of Fv/Fm and VPSI

14

during recovery period. Each curve represents the fit to the exponential function (all R2 values > 0.96).

15

White and light grey represent the period of exposure and recovery, respectively. The means ± SD are

16

calculated from 5 independent samples

17

Fig. 3 Time course of the variations in the activity of cyclic electron transport around PSI (Vre-red) in

18

eelgrass leave exposed to LL, ML and HL. White and light grey represent the period of exposure and

19

recovery, respectively. The means ± SD are calculated from 5 independent samples

20

Fig. 4 Time courses of changes in related parameters of electron transport from donor side of PSII to

21

QA exposed to LL. ML and HL, respectively. (a) The relative variable fluorescence at the K-step to the

22

amplitude (FJ-FO). (b) The amplitude of submillisecond component calculated by fitting the decay

23

kinetics of delayed fluorescence data to the time function DF (t) = L1× exp (-t/τ1) + L2× exp (-t/τ2) + L3.

24

(c) Probability of electron moves further than QA. (d) Quantum yield for electron transport. (e) The

25

amplitude of millisecond kinetic component derived from above function. (f) Probability of the

26

connectivity among PSII units. White and light grey represent the period of exposure and recovery,

27

respectively. The means ± SD are calculated from 5 independent samples

28

Fig. 5 Changes in related parameters of electron transport from QA- to the acceptor side of PSI exposed

29

to LL. ML and HL, respectively. (a) Probability with which a PSII trapped electron is transferred from

30

QA- beyond PSI. (b) Probability with which a PSII trapped electron is transferred from reduced

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16

ACCEPTED MANUSCRIPT intersystem electron acceptors to the final electron acceptors of PSI. (c) Quantum yields for reduction

2

of the end electron acceptors at the PSI acceptor side. (d) Connectivity between PSII and PSI. White

3

and light grey represent the period of exposure and recovery, respectively. The means ± SD are

4

calculated from 5 independent samples

5

Fig. 6 Principle component analysis of parameters related to photosynthetic electron transport in

6

eelgrass of exposure to LL (a), ML (b) and HL (c and d), respectively. Parameters attached to the same

7

component were encircled by the same color cycle. Component 1 was surrounded by gray cycle and

8

Component 2 was dark cycle as well as Component 3 was light grey cycles

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9 Table Captions

11

Table 1 Measured and calculated parameters derived from OJIP transient

12

Table 2 Variations of selected parameters derived from chlorophyll fluorescence in eelgrass leaves

13

after exposure to LL, ML and HL, respectively. Each light treatment started from 8:00 am and

14

continuously lasted for 6 hours. Initial, Exposure and Recovery respectively represented measuring

15

time prior to 8: 00 (pre-cultured plants were dark-adapted overnight), after 11: 00 (3 h of exposure) and

16

13: 00 (3 h of recovery). Letters indicate the significant differences at p < 0.05. The means ± SD are

17

calculated from 5 independent samples

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ACCEPTED MANUSCRIPT Table 1 Parameters

Physiological interpretation

Basic JIP-test parameters Fluorescence at time t after onset of actinic illumination

FO = F50µs

Minimum fluorescence, all PSII reaction centers (RC) are open

FK = F300µs

Fluorescence intensity at the K-step (300µs) of OJIP

FJ = F2ms

Fluorescence intensity at the J-step (2ms) of OJIP

FI = F30ms

Fluorescence intensity at the I-step (30ms) of OJIP

Fm = Fp

Maximal fluorescence, at the peak P of OJIP

Vt = (Ft - FO)/( Fm -FO)

Relative variable fluorescence at time t

VK = (FK- FO)/( Fm -FO)

Relative variable fluorescence at the K-step

VJ = (FJ-FO)/(Fm-FO)

Relative variable fluorescence at the J-step

W100µs = (F100µs-FO)/(FJ-FO)

Relative variable fluorescence at 100µs to the amplitude FJ-FO

WK = (FK- FO)/(FJ - FO)

Relative variable fluorescence at the K-step to the amplitude FJ-FO

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Quantum efficiencies or flux ratios

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Ft

φPo = 1-FO/Fm=FV/Fm

Maximum quantum yield for primary photochemistry

ψET2o = 1-VJ

Probability that an electron moves further than QA

φEo = (1-FO/Fm)/(1-VJ)

Quantum yield for electron transport

ψRE1o = 1-VI

Probability that an electron moves from reduced QA beyond PSI

φRo = (1-FO/Fm)/(1-VI)

Quantum yield for reduction of the end electron acceptors at the PSI acceptor side

Probability that an electron is transported from the reduced intersystem

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δRo = (1-VI)/(1-VJ)

electron acceptors to the final electron acceptors of PSI

Phenomenological energy fluxes

Apparent antenna size of active PSII RC

DIo/RC = MO × (1/VJ) ×[1/(FO/Fm)]

Effective dissipation of energy in active RC

EP

ABS/RC = MO × (1/VJ)(1/(Fv/Fm))

Connectivity among PSII units

AC C

WE =1-[(F2ms-F300µs)/(F2ms-F50µs)]1/5

Model-derived value of relative variable fluorescence at 100µs calculated for unconnected PSII units

C =(WE-W100µs)/[VJ×W100µs×(1-WE) ]

Curvature constant of initial phase of the O-J curve

P2G = C×[FO/(FJ-FO)]

Overall grouping probability

P=[P2G×(Fm/FO-1)]/[1+P2G×(Fm/FO-1)]

Connectivity parameter

ω=P×[(Fm-FO)/Fm]

Probability of the connectivity among PSII units

1

ACCEPTED MANUSCRIPT Table 2 Parameter

Initial

Exposure LL d

0.232±0.015

Recovery

ML c

0.318±0.030

HL a

LL

0.260±0.012

b

0.186±0.012

ML d

0.187±0.040

HL d

0.190±0.011d

0.186±0.012

rETRmax

12.11±1.03d

22.89±2.20c

46.20±2.89 a

37.82±3.40b

11.31±1.92d

12.76±1.19d

11.13±0.25d

ABS/RC

1.089±0.0396c

1.071±0.049c

1.263±0.066b

1.580±0.082a

1.079±0.002c

1.083±0.018c

1.328±0.103b

DIO/RC

0.254±0.010d

0.275±0.004c

0.288±0.013c

0.455±0.028a

0.242±0.005d

0.246±0.014d

0.310±0.014b

NPQ

0e

0.291±0.027c

0.387±0.031b

0.849±0.030a

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α

AC C

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SC

0.103±0.015d

2

0.168±0.077d

0.282±0.016c

ACCEPTED MANUSCRIPT Fig. 1

75

3500

a

3000

b

P

I

2000

J 1500

k

1000

LL ML HL

O 500

45

30

15

0

0 -2

-1

10

10

0

1

10

2

10

10

3

-1

4

10

0

10

10

10

Time (ms) d

60

1.000

MR/MRo

DF intensity at I1

2

10

3

10

3

10

10

4

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0.998

SC

1.002

c

20

1

10

JIP-time (ms)

80

40

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2500

DF20ms intensity (i.e.)

PF intensity (a.u.)

60

0.996

0 0.0

0.2

0.4

0.6

0.8

1.0

0.994

-1

10

AC C

EP

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Decay time (ms)

1

0

10

10

1

2

10

JIP-time (ms)

10

4

ACCEPTED MANUSCRIPT Fig. 2 Exposure

0.90

Recovery

a 0.85

0.75

0.70

t1/2(LLrec)=2.1h

LL ML HL

0.65

t1/2(MLrec)=1.9h t1/2(HLrec)=2.4h

0.60 1

2

3

4

6

Recovery

Exposure

1.4

5

SC

0

b t (LLrec)=1.31h 1/2

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VPSI

1.2

t (MLrec)=0.68h

1.0

1/2

0.8

t (HLrec)=1.39h 1/2

0.6 1

2

3

4

5

6

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0

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Fv/Fm

0.80

AC C

EP

Time (h)

2

ACCEPTED MANUSCRIPT Fig.3 Recovery

Exposure

1.8

LL ML HL

1.6

1.2

1.0

0.8

0.6

0.4 0

1

2

3

4

5

6

AC C

EP

TE D

M AN U

SC

Time (h)

RI PT

Vre-red

1.4

3

ACCEPTED MANUSCRIPT Fig. 4 Recovery

Exposure

0.40

Recovery

Exposure

160

0.35 120

L1

Wk

0.30 80

40

LL ML HL

0.20

a

b

0.15

0 0

1

2

3

4

0.8

5

6

0

Recovery

Exposure

1

0.7

4

5

6

Recovery

ϕEo 0.4

M AN U

ψ ET2o

0.5

0.5

3

SC

0.6 0.7

0.6

2

Exposure

RI PT

0.25

0.3

d

c 0.4

0.2

0

1

2

3

4

Exposure

30

5

6

0

Recovery

27

1

2

3

4

0.7

5

6

Recovery

Exposure

0.6

24

18

15

e

12 1

2

3

4

5

6

EP

0

AC C

Time (h)

4

ω

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L2

0.5 21

0.4

0.3

f 0.2 0

1

2

3

Time (h)

4

5

6

ACCEPTED MANUSCRIPT Fig. 5 Exposure

0.5

Recovery

Exposure

0.6

b

a 0.4

0.5

δRo

0.3

0.4

0.2 0.3

LL ML HL

0.1

0.2

0.0 0

1

2

3

4

0

6

Recovery

Exposure

0.4

5

2

1.6

d

0.3

VPSII-PSII

1.2

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1.0

0.1

4

5

6

SC

1.4

0.2

3

Recovery

Exposure

c

ϕRo

1

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ψRE10

Recovery

0.8

0.0

0.6

0

1

2

3

4

6

0

1

2

3

Time (h)

AC C

EP

TE D

Time (h)

5

5

4

5

6

ACCEPTED MANUSCRIPT Fig. 6 1.0

Vre-red VPSI

0.5

L1

δRo ψ

0.0

ϕ

-1.5 -1.0

ψ

-0.5

Co mp 0.0 one 0.5 nt 1

0.5

ET2o

0.0

-0.5

1.5

L1

b

1.0

Eo

1.0

1.0

2 nt ne po m Co

Fv/Fm

VPSI

0.5

Component 2

RE1o

ϕ

Ro

ϕ

ψ 0.0

Eo ET2o

ϕ

Ro

M AN U

ψ

RE1o

δRo

-0.5

Vre-red -1.0 -1.0

-0.5

0.0

0.5

1.0

Component 1

1.0

L1 Fv/Fm

c

ϕ

Eo

VPSI

Component 2

0.5

ψ

ET2o

ϕ

Ro

TE D

δRo

0.0

ψ

Vre-red

-0.5

-1.0 -1.0

-0.5

0.0

RE1o

0.5

1.0

d

Vre-red

Component 3

AC C

0.5

EP

Component 1

1.0

ϕ

L1

0.0

ψ Eo

ET2o

ψ

RE1o

ϕ

Fv/Fm

Ro

δRo

VPSI

-0.5

-1.0 -1.0

-0.5

0.0

RI PT

Fv/Fm

SC

Component 3

a

0.5

1.0

Component 1

6

ACCEPTED MANUSCRIPT Highlights •Poor PSII resilience may be a deficiency specific to eelgrass, rendering it incapable to adequate levels of photosynthetic electron transport to varying light environments. •Compared to PSII, PSI has a higher relative resilience.

RI PT

•Coordination between PSII and PSI is particularly close during exposure to excess light as indicated by principle component analysis.

•Eelgrass exposed to excess light has the higher light utilization efficiency and relative maximum electron transport rates and therefore exhibits optimal photosynthetic performance, indicating the high

AC C

EP

TE D

M AN U

SC

light requirement.

ACCEPTED MANUSCRIPT Contributions Xiao Qi Yang and Quan Sheng Zhang conceived and designed the experiments, Xiao Qi Yang and Di Zhang conducted experiments and carried out the data analysis, Xiao Qi Yang and Quan Sheng Zhang

AC C

EP

TE D

M AN U

SC

RI PT

wrote the manuscript. All authors participated in the preparation and review of the manuscript.