Heterologous expression of moss light-harvesting complex stress-related 1 (LHCSR1), the chlorophyll a-xanthophyll pigment-protein complex catalyzing non-photochemical quenching, in Nicotiana sp

Abstract

Oxygenic photosynthetic organisms evolved mechanisms for thermal dissipation of energy absorbed in excess to prevent formation of reactive oxygen species. The major and fastest component, called non-photochemical quenching, occurs within the photosystem II antenna system by the action of two essential light-harvesting complex (LHC)-like proteins, photosystem II subunit S (PSBS) in plants and light-harvesting complex stress-related (LHCSR) in green algae and diatoms. In the evolutionary intermediate Physcomitrella patens, a moss, both gene products are active. These proteins, which are present in low amounts, are difficult to purify, preventing structural and functional analysis. Here, we report on the overexpression of the LHCSR1 protein from P. patens in the heterologous systems Nicotiana benthamiana and Nicotiana tabacum using transient and stable nuclear transformation. We show that the protein accumulated in both heterologous systems is in its mature form, localizes in the chloroplast thylakoid membranes, and is correctly folded with chlorophyll a and xanthophylls but without chlorophyll b, an essential chromophore for plants and algal LHC proteins. Finally, we show that recombinant LHCSR1 is active in quenching in vivo, implying that the recombinant protein obtained is a good material for future structural and functional studies.

Keywords: LHCSR; NPQ; Physcomitrella; light-harvesting complex (antenna complex); photobiology; photosynthesis; photosynthetic pigment; recombinant protein expression; tobacco.

FIGURE 1.

Expression analysis of N. benthamiana and N. tabacum transformation using two different PpLHCSR1 constructs and localization of PpLHCSR1 in thylakoid membranes of tobacco. A–D, time course PpLHCSR1 accumulation trial in N. benthamiana upon transient expression. Leaf disks collected daily postagroinfiltration were ground in loading buffer, fractionated by SDS-PAGE, and transferred to a PVDF membrane. Immunoblot analyses using a homemade α-PpLHCSR antibody of oeLHCSR1 (A) or oeLHCSR1 His tag (B) are reported. The time course of LHCSR1 for oeLHCSR1 (C) or oeLHCSR1 His tag (D) is shown. Data are presented as means ± S.D. (error bars) of three independent experiments. E and F, immunoblot analysis of oeLHCSR1 (E) or oeLHCSR1 His tag (B) plants of stably transformed N. tabacum. Antibody α-PpLHCSR was used. An equal volume for each leaf disk was loaded. Immunodetection using α-CP43 antibody is shown as a control of equal loading. P. patens and N. tabacum WT thylakoids (1 μg of Chl) were loaded as positive and negative controls, respectively. Thyl., thylakoids; Nb, N. benthamiana; Nt, N. tabacum. G, immunoblot analysis of thylakoid membranes purified from WT, oeLHCSR1 (oeSR1), and oeLHCSR1 His tag (oeSR1 HisTag).

FIGURE 2.

Fractionation of WT and oeLHCSR1 solubilized thylakoids of N. benthamiana and N. tabacum. A, sucrose gradient of thylakoids from WT and oeLHCSR1 of N. benthamiana and N. tabacum plants solubilized with 0.8% α-DM. B, Coomassie Blue-stained SDS-PAGE analysis of green bands collected from the sucrose gradient. An equal volume (50 μl) of each band was loaded. A polypeptide corresponding to the molecular weight of LHCSR1, underlined in red, was clearly visible in B1 and B2 (corresponding to free pigments and monomeric LHCs, respectively). C, immunoblot analysis with α-PpLHCSR antibody. Thylakoids (Thyl) from oeLHCSR1 and WT were loaded as positive and negative controls, respectively. B1, B2, and B3 from WT thylakoids were loaded as additional negative controls.

FIGURE 3.

Absorption (Abs) spectra of fractions obtained from sucrose gradient. Spectra of B1 fraction from WT (black line) versus oeLHCSR1 (gray line) of N. benthamiana (A) and N. tabacum (B) are shown. Absorption spectra of B2 fraction from WT (black line) and oeLHCSR1 (gray line) of N. benthamiana (C) and N. tabacum (D) are shown. Peak wavelengths are indicated. a.u., arbitrary units.

FIGURE 4.

PpLHCSR1 His tag isolation and biochemical characterization. A, absorption (Abs) spectra of fraction eluted with 250 mm imidazole (gray line) after solubilization of oeLHCSR1 His tag thylakoids with α-DM. The absorption spectrum of solubilized thylakoids (black line) is also reported. a.u., arbitrary units. B, Coomassie Blue-stained SDS-PAGE gel analysis of fractions eluted with different imidazole concentrations. Samples containing 2 μg of Chl were loaded in each slot. Thylakoids (3 μg of Chl) of WT and oeLHCSR1 His tag (HT) of N. tabacum and WT of P. patens were also loaded as controls. Immunoblot analysis using antibody α-PpLHCSR is shown in the panel below the gel. Sol, solubilized thylakoids; FT, flow-through; Thyl., thylakoids; Nt, N. tabacum. C, sucrose gradient fractionation obtained by loading the fractions eluted from the Ni²⁺ column with 250 mm imidazole (on the left). Coomassie stain of SDS-PAGE of B2 and P fractions is shown on the right. D, absorption spectra of B1 (red line) and B2 (gray line) fractions from the sucrose gradient. LHCII (black line) is reported for comparison.

FIGURE 5.

Abundance of the recombinant PpLHCSR1 protein in the different expression systems. A, LHCSR1 immunotitration of oeLHCSR1 thylakoids of N. benthamiana and N. tabacum. 0.125, 0.25, 0.5, and 0.75 μg of Chl were loaded for each sample. In the case of LHCSR1 His tag purified protein, 0.012, 0.025, and 0.05 μg of Chl were loaded. B, LHCSR1 immunotitration of oeLHCSR1 His tag thylakoids of N. benthamiana and N. tabacum. Filters were probed with α-PpLHCSR antibody.

FIGURE 6.

PpLHCSR1 localization among pigment-binding complexes of N. benthamiana and N. tabacum. A and B, non-denaturing gel electrophoresis of pigment-binding complexes from thylakoids of oeLHCSR1 and WT of N. benthamiana (A) and N. tabacum (B) after solubilization with 0.8% α-DM. 500 μg of Chl were loaded for each sample. C and D, Coomassie blue staining and immunoblotting analysis of the fractions eluted from the non-denaturing gel slices shown in A and B and separated in a second dimension under denaturing conditions. The numbering of fractions reported on the top of each filter corresponds to that of gel slices cut from the non-denaturing gel. A polypeptide corresponding to the molecular weight of LHCSR1, underlined in red, was clearly visible in slices 5, 6, and 7 of oeLHCSR1 from both plant systems. Chlorophyll a/b ratios are indicated underneath each filter.

FIGURE 7.

Spectroscopic analysis of recombinant PpLHCSR1 protein purified from Deriphat-PAGE. A, absorption (Abs) spectrum of slice 6 eluted from the gel. The sample containing PpLHCSR1 protein from oeLHCSR1 thylakoids (gray line) is compared with the corresponding fraction purified from N. benthamiana WT solubilized thylakoids (black line). B, absorption spectrum of slice 6 eluted from the gel of N. tabacum thylakoids. The sample containing PpLHCSR1 protein from oeLHCSR1 thylakoids (gray line) compared with the corresponding fraction purified from WT (black line). C, comparison among absorption spectra of fraction 6 containing LHCSR1 protein from N. benthamiana (black line) and N. tabacum (light gray line) and native LHCSR from P. patens (dark gray line) isolated in Pinnola et al. (22). a.u., arbitrary units.

FIGURE 8.

Untagged PpLHCSR1 isolation and biochemical characterization. A, immunoblot of fractions obtained by solubilizing thylakoids by different concentration of α-DM probed with α-PpLHCSR antibody. An equal amount of Chl (0.25 μg) was loaded for each fraction. Thylakoids of oeLHCSR1 of N. benthamiana and N. tabacum are shown as reference. Thyl, thylakoids; Nb, N. benthamiana; Nt, N. tabacum. B, sucrose gradient of supernatant obtained by solubilizing thylakoids from WT and oeLHCSR1 (oeSR1) of both plant systems with 0.8% α-DM. C, polypeptide composition of fractions harvested from the sucrose gradient of oeLHCSR1 plants. 0.5 μg of Chl was loaded for B2, and 0.3 μg of Chl was loaded for other green bands. In the lower panel, the immunoblot with α-PpLHCSR antibody is shown. D, absorption (Abs) spectra of monomeric LHCs (pink line) and B1–2 (black line) from the sucrose gradient of N. tabacum and from B1–2 (red line) and B2–3 (blue line) from the sucrose gradient of N. benthamiana. a.u., arbitrary units.

FIGURE 9.

Purification of LHCSR1 without His tag. A, sucrose gradient of PpLHCSR1-enriched fraction. Enrichment was obtained by pooling the LHCSR-containing fractions from the first sucrose gradient (not shown). This second gradient allowed for separation of free pigments. B, Coomassie Blue-stained SDS-PAGE gel analysis of fraction B2. LHCSR His tag (LHCSR HT) was also loaded for reference. Loading was 0.3 μg of Chl/lane. C, absorption (Abs) spectra of LHCSR1 with and without His tag (gray and black lines, respectively). a.u., arbitrary units.

FIGURE 10.

Determination of Chl to polypeptide ratio in LHCSR1. A and B, Coomassie-stained gels of various dilutions (0.6, 0.3, 0.15, 0.075, and 0.035 μg of Chl) of LHCSR, LHCSR His tag, and LHCII (0.6, 0.3, 0.15, and 0.075 μg of Chl). C, plot of Coomassie stain versus chlorophyll amount for LHCII (upper panel) and LHCSR (lower panel) showing linearity of the Coomassie binding (R² ≥ 0.99). D, plot of Coomassie stain versus chlorophyll amount for LHCII (upper panel) and LHCSR His tag (lower panel) (R² ≥ 0.99). The figure shows the result of a typical experiment.

FIGURE 11.

Relationship between NPQ activity and accumulation of PpLHCSR1 protein. A, NPQ kinetic measurements on N. tabacum leaf disks. N. tabacum samples expressing different levels of PpLHCSR1 were compared with WT. B, correlation between NPQ activity at the last light point (maximum NPQ) of N. tabacum plants and LHCSR accumulation (R² ≥ 0.96). Data are presented as a means ± S.D. (error bars) (n ≥ 3).