Light harvesting proteins regulate non-photochemical fluorescence quenching in the marine diatom Thalassiosira pseudonana

Highlights

• NPQ does not solely depend on the presence of the XC in T. pseudonana.

• NPQ is regulated by light harvesting proteins, especially Lhcx6.

• Other than NPQ, other metabolic pathways participate in photoprotection.

Abstract

Diatoms utilize various mechanisms to enhance heat dissipation when they are subjected to drastic fluctuations in the light intensity. The activation of the xanthophyll cycle (XC) leading to non-photochemical fluorescence quenching (NPQ) is one of the important mechanisms. We used the model diatom Thalassiosira pseudonana to investigate the factors controlling the kinetics of NPQ and XC. By adding chemicals to cells exposed to excess light, we found that the increase in NPQ during high light (HL) exposure could not be inhibited by NH₄Cl and 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), while the increase in diatoxanthin (Dt) could be prevented by DCMU and not by NH₄Cl, suggesting that the characteristics of NPQ do not solely depend on the presence of the XC. During HL exposure, the up-regulation of the light harvesting complex protein × 6 (Lhcx6) and Lhcx genes suggests that they are involved in photoprotection. The addition of DCMU and NH₄Cl significantly elevated the transcript levels of three of the Lhcx genes during HL treatment, especially Lhcx6. The results suggest that reactive oxygen species (ROS) generated by DCMU and transthylakoid ΔpH changes elicited by NH₄Cl may contribute to the development of NPQ during HL exposure by inducing the gene expression of Lhcx instead of controlling the XC. Among these, Lhcx6 protein may play a key role in managing light responses in T. pseudonana. Proteomic data demonstrated that the elevated Calvin cycle and increased synthesis of antioxidants, pseudouridines and plastoglobulins may raise the capability of diatoms to cope with light stress.

Introduction

Diatoms, eukaryotic unicellular microalgae, are widespread in oceans and rivers and are estimated to account for 20% of the primary productivity in the world [1], [2]. Why are diatoms so successful? One important reason is that diatoms possess an excellent capability to cope with rapid changes in light intensity resulting from fast agitation of surface waters in the ocean.

NPQ is a vital photoprotective process in plants and algae that dissipates surplus light energy in the form of heat. The mechanism of NPQ is well characterized in higher plants and is associated with the XC. The XC in higher plants embraces the conversion from the violaxanthin to zeaxanthin in high light and the counter reaction in low light [3]. However, The XC in diatoms is different from that in higher plants. It involves the de-epoxidation of diadinoxanthin (Dd) to Dt in high light treatment and the back reaction in low light irradiance or in darkness [4], [5]. Although the XC is essential for thermal dissipation, the formation of transthylakoid ΔpH [6] and PsbS protein in the PSII antenna are also necessary for NPQ induction [7]. In Phaeodactylum tricornutum, the application of certain uncouplers such as NH₄Cl, the ADRY reagent and the protonophore 2,4-dinitrophenol, which can effectively dissipate the transthylakoid ΔpH, inhibited NPQ and the conversion of Dd to Dt [8]. This suggests that NPQ in P. tricornutum relies on the transthylakoid ΔpH. DCMU, which restrains photosynthetic electron transfer, was used to study relationship between photosynthetic electron transport and NPQ in P. tricornutum. The addition of DCMU to P. tricornutum cells exposed to surplus light hardly affected NPQ and the functioning of the XC [8]. The authors proposed that the XC may be activated by cyclic electron transport in PSI in the presence of DCMU. T. pseudonana is a species of marine centric diatom and is the first eukaryotic marine phytoplankton used for whole genome sequencing. The relationship between NPQ and the XC in T. pseudonana has not been reported to date.

For higher plants under high light stress, conformational changes of light harvesting complexes (Lhc) promote the thermal dissipation of surplus light energy, which is triggered by protonation of PsbS protein [9]. In T. pseudonana, the Lhcx6 protein in the Lhc was found to participate in dissipation of surplus light energy during high light stress [10]. This suggests that the Lhcx6 protein in diatoms has a similar function to PsbS in higher plants. Phylogenetic analysis found five LI818-like genes (Lhcx) in the genome of T. pseudonana, including Lhcx1, Lhcx2, Lhcx4, Lhcx5 and Lhcx6 [11]. To date, roles of Lhcx genes in the regulation of NPQ remain largely unknown for T. pseudonana.

In this study, the marine model diatom T. pseudonana was used to examine the responses of NPQ, XC and Lhcx genes to excess light treatment in the presence of various chemicals. Several responses to inhibitors in T. pseudonana were found which were different from those in P. tricornutum. Furthermore, a comparative proteomics method was employed to examine the proteomic changes in T. pseudonana under excess light stress. The purpose of this proteomic study was to figure out which metabolic pathways in diatom cells are involved in the response to excess light stress and attempt to provide evidence for changes in the XC and NPQ at the protein level. These results will provide new insights into functioning of the XC and NPQ formation in marine diatoms during excess light acclimation.