Identification of pH-sensing Sites in the Light Harvesting Complex Stress-related 3 Protein Essential for Triggering Non-photochemical Quenching in Chlamydomonas reinhardtii

Abstract

Light harvesting complex stress-related 3 (LHCSR3) is the protein essential for photoprotective excess energy dissipation (non-photochemical quenching, NPQ) in the model green algaChlamydomonas reinhardtii Activation of NPQ requires low pH in the thylakoid lumen, which is induced in excess light conditions and sensed by lumen-exposed acidic residues. In this work we have used site-specific mutagenesisin vivoandin vitrofor identification of the residues in LHCSR3 that are responsible for sensing lumen pH. Lumen-exposed protonatable residues, aspartate and glutamate, were mutated to asparagine and glutamine, respectively. By expression in a mutant lacking all LHCSR isoforms, residues Asp(117), Glu(221), and Glu(224)were shown to be essential for LHCSR3-dependent NPQ induction inC. reinhardtii Analysis of recombinant proteins carrying the same mutations refoldedin vitrowith pigments showed that the capacity of responding to low pH by decreasing the fluorescence lifetime, present in the wild-type protein, was lost. Consistent with a role in pH sensing, the mutations led to a substantial reduction in binding the NPQ inhibitor dicyclohexylcarbodiimide.

Keywords: fluorescence; non-photochemical quenching; photoprotection; photosynthesis; photosynthetic pigment; photosystem II; plant biochemistry.

FIGURE 1.

Three-dimensional model of LHCSR3. Panel A, LHCSR3 structure modeled on LHCII and CP29 crystallographic structures. Putative protonatable sites are indicated. Panel B, zoom view on Asp¹¹⁷, Glu²²¹, and Glu²²⁴ residues; the distance between the different residues is indicated in yellow (Å).

FIGURE 2.

Alignment of LHCSR-like protein sequences.

FIGURE 3.

NPQ measurements and immunoblot analysis of LHCSR3 protein levels in npq4 lines expressing site-specific mutant versions of LHCSR3 affecting protonatable sites. Panel A, NPQ measurements on WT, npq4 mutant, and transgenic lines with LHCSR3 proteins carrying a single mutation on putative protonatable sites D2, E1–5. Panel B, immunoblot analysis of LHCSR3 accumulation on genotypes analyzed in panel A; immunoblot analysis of the D1 subunit of PSII is shown as a control for loading. Panel C, NPQ measurements on WT, npq4 lhcsr1 mutant, and transgenic lines with LHCSR3 proteins with double mutations on putative protonatable sites D2E2 and E1E2. Panel D, immunoblot analysis of LHCSR3 accumulation on genotypes analyzed in panel C. In all cases three independent biological replicates were analyzed. The experiments were reproduced two times.

FIGURE 4.

NPQ measurements and immunoblot analysis of LHCSR3 protein levels in npq4 lhcsr1 lines expressing site-specific mutant versions of LHCSR3 affecting protonatable sites. NPQ measurements on WT, npq4 lhcsr1 mutant, and transgenic lines with LHCSR3 proteins mutated on D2, E1, and E2 protonatable sites (panel A). Panel B, immunoblot analysis of LHCSR3 accumulation; immunoblot analysis of the D1 subunit of PSII is shown as a control for loading. In all cases three independent biological replicates were analyzed. The experiments were reproduced three times.

FIGURE 5.

Absorption and circular dichroism spectra of LHCSR recombinant proteins. Absorption spectra (panel A) and circular dichroism (panel B) in the visible region of LHCSR3 WT and D2E1E2 mutant refolded in vitro in the presence of chlorophylls and carotenoids. The experiments were reproduced three times, each time with two independent biological replicates.

FIGURE 6.

¹⁴DCCD binding in LHCSR3 recombinant WT and D2E1E2 mutant. Panel A, autoradiography of recombinant LHCSR3 WT and the D2E1E2 mutant treated with ¹⁴DCCD; microliters of sample (0.2 μg/μl of chlorophylls) loaded on SDS-PAGE are reported (15, 7.5, and 2.5 μl). Panel B, Coomassie staining of SDS-PAGE used for autoradiography. Panel C, ratio of the level of ¹⁴C observed by autoradiography signals and protein quantity obtained by densitometric analysis of Coomassie-stained gels. The experiments were performed two times; each time two independent biological replicates were analyzed with three technical replicates with different loading volume as indicated.

FIGURE 7.

Fluorescence decay kinetics. Fluorescence decay kinetics of recombinant LHCSR3 WT (panel A) and D2E1E2 mutant (panel B) at pH 7.5 or 5.0 in the presence of high (0.03%) or low (0.003%) detergent (α-DM) concentrations. The experiment was performed two times, each time with two independent biological replicates.

FIGURE 8.

77 K fluorescence emission spectra. Fluorescence emission spectra of recombinant LHCSR3 WT (panel A) and the D2E1E2 mutant (panel B) at 77 K measured at pH 5 or 7.5 in the presence of 0.03% α-DM or 0.003% α-DM. The experiments were reproduced three times, each time with two independent biological replicates.