An atypical member of the light-harvesting complex stress-related protein family modulates diatom responses to light

Abstract

Diatoms are prominent phytoplanktonic organisms that contribute around 40% of carbon assimilation in the oceans. They grow and perform optimally in variable environments, being able to cope with unpredictable changes in the amount and quality of light. The molecular mechanisms regulating diatom light responses are, however, still obscure. Using knockdown Phaeodactylum tricornutum transgenic lines, we reveal the key function of a member of the light-harvesting complex stress-related (LHCSR) protein family, denoted LHCX1, in modulation of excess light energy dissipation. In contrast to green algae, this gene is already maximally expressed in nonstressful light conditions and encodes a protein required for efficient light responses and growth. LHCX1 also influences natural variability in photoresponse, as evidenced in ecotypes isolated from different latitudes that display different LHCX1 protein levels. We conclude, therefore, that this gene plays a pivotal role in managing light responses in diatoms.

Fig. 1.

Diatom LHCX expression and NPQ characteristics in dark, low light and high light conditions. (A) De-epoxidation state (DES) determined as DT/(DD + DT), and relative level of the pool of xanthophyll (DT + DD), normalized to the fucoxanthin (Fx) content, obtained from HPLC analysis. (B) NPQ response from cells grown as described above. NPQ was calculated as (Fm-Fm′)/Fm′ (34), where Fm and Fm′ are the maximum fluorescence emission measured in dark (30-min adaptation before exposure) and cells exposed for 5 min to 700 μmol photons m⁻²·s⁻¹, respectively. (C) Accumulation of the four P. tricornutum LHCX transcripts determined by qRT-PCR from cells grown in a 12:12 h light:dark cycle and collected in the dark (dark), after 2 h of low light treatment (70 μmol photons m⁻²·s⁻¹, LL), and after 1 h of a subsequent low light to high light shift (600 μmol photons m⁻²·s⁻¹, HL). RPS was used as reference gene. Error bars are relative to three independent experiments. (D) Western blotting showing LHCX protein accumulation. Proteins were detected with antibodies against the LHCSR/LHCX and the PSII subunit D2 was used as loading control.

Fig. 2.

Diurnal changes in DES, NPQ, and LHCX1 transcript (A), and LHCX protein levels (B). Cells were exposed to a 12:12 h light:dark regime at 70 μmol photons m⁻²·s⁻¹, and samples were collected at the indicated times during the light phase for the different measurements. Error bars refer to duplicate measurements in three biological samples. NPQ, DES, and blot analyses were performed as specified in Fig. 1.

Fig. 3.

LHCX1 regulates NPQ and growth in P. tricornutum. (A) Analyses of LHCX1 mRNA by qRT-PCR (Left) and protein levels (Right), and (B) NPQ (Left) and DES (Right) values, in wild-type (Pt1) and three RNAi silenced lines, sampled 8 h after the onset of illumination. Analyses were performed as described in Fig. 1. Relative transcript levels were determined using RPS as a reference gene and values normalized to gene expression levels of the wild type (Pt1). (C) Growth curve of the Pt1 and lhcx1 cells (Left) under 12 h:12 h light:dark regime at 30 μmol photons m⁻²·s⁻¹ (SE refers to duplicate measurements from three biological samples), and growth tests on cells spotted on solid media (Right). A total of 5 μL of different cell dilutions (1, 0.5, 0.25, and 0.12·10⁶·mL cells) were spotted and pictures were taken after 5 d.

Fig. 4.

Pt4 is a natural NPQ variant displaying altered LHCX expression. (A) Analysis of 10 P. tricornutum ecotypes isolated from different locations (Left). Maximum NPQ as a function of growth temperature (Right). Cells were grown at the indicated temperatures for at least 3 wk. Samples were collected 2 h after the onset of illumination and NPQ was measured as in Fig. 1. (B) NPQ (Left and Center) in Pt1, Pt4, and two Pt4 transgenic lines overexpressing LHCX1. Right shows their DES values. (C) LHCX1 mRNA levels by qRT-PCR using RPS as reference gene (Left) and protein accumulation (Right). A total of 50 μg of total protein extracts was used in the Western blot analysis for Pt4 and Pt1. To better highlight differences, 20 μg has been loaded for the Pt4 and Pt4 overexpressing lines (OE1 and OE2). Proteins were detected with antibodies against the LHCSR/LHCX, and the photosystem II subunit D2 was used as a loading control.