Dual-Located WHIRLY1 Interacting with LHCA1 Alters Photochemical Activities of Photosystem I and Is Involved in Light Adaptation in Arabidopsis

Abstract

Plastid-nucleus-located WHIRLY1 protein plays a role in regulating leaf senescence and is believed to associate with the increase of reactive oxygen species delivered from redox state of the photosynthetic electron transport chain. In order to make sure whether WHIRLY1 plays a role in photosynthesis, in this study, the performances of photosynthesis were detected in Arabidopsis whirly1 knockout (kowhy1) and plastid localized WHIRLY1 overexpression (oepWHY1) plants. Loss of WHIRLY1 leads to a higher photochemical quantum yield of photosystem I Y(I) and electron transport rate (ETR) and a lower non-photochemical quenching (NPQ) involved in the thermal dissipation of excitation energy of chlorophyll fluorescence than the wild type. Further analyses showed that WHIRLY1 interacts with Light-harvesting protein complex I (LHCA1) and affects the expression of genes encoding photosystem I (PSI) and light harvest complexes (LHCI). Moreover, loss of WHIRLY1 decreases chloroplast NAD(P)H dehydrogenase-like complex (NDH) activity and the accumulation of NDH supercomplex. Several genes encoding the PSI-NDH complexes are also up-regulated in kowhy1 and the whirly1whirly3 double mutant (ko1/3) but steady in oepWHY1 plants. However, under high light conditions (800 μmol m^-2 s^-1), both kowhy1 and ko1/3 plants show lower ETR than wild-type which are contrary to that under normal light condition. Moreover, the expression of several PSI-NDH encoding genes and ERF109 which is related to jasmonate (JA) response varied in kowhy1 under different light conditions. These results indicate that WHIRLY1 is involved in the alteration of ETR by affecting the activities of PSI and supercomplex formation of PSI with LHCI or NDH and may acting as a communicator between the plastids and the nucleus.

Keywords: WHIRLY1; electron transport rate (ETR); light; photochemical activities; photosystem I; plastid gene.

Figure 1

Analyses of the photosynthetic performance in leaves of the WHY1, lhca1 mutants and the wild-type plants by a Dual PAM (Pulse-amplitude modulation) device. Dual PAM measurements of photochemical yield of photosystem I Y(I) (A), yield of photosystem II Y(II) (B), electron transport rate (ETR) (C), and photosystem II fluorescence (NPQ) (D), respectively. Data presented are mean ± SE (n = 6–8). Significance analysis was performed by Tukey–Kramer method (p < 0.05) and the data were dividing into different significance groups.

Figure 2

Expression of photosystem I (PSI)-light harvest complexes (LHCI) encoded genes in WHY1 mutants and wild-type plants (A) Expression analysis of LHCI complex encoded genes in WHY1 mutants and wild-type plants by qPCR; (B) Expression analysis of PSI core complex encoded genes in WHY1 mutants and wild-type plants. The transcript level in each case was normalized to that of GAPC2 as a reference gene, and the expression level of WT was set as 1. Three biological replications were used to analyze. Asterisk indicate significant differences in gene expression compared to WT (Tukey–Kramer method, * p < 0.05, ** p < 0.01, *** p < 0.001).

Figure 3

The interaction confirmation of WHY1 and LHCA1. (A) The interaction confirmation of WHY1 and LHCA1 by yeast two hybrid. Yeast two hybrid interaction detection growth selections and X-Gal agarose overlay assay indicate an interaction between WHY1 and LHCA1; (B) CoIP of WHY1 and LHCA1 from photosystem I containing thylakoid membrane complexes. As input to the thylakoid membrane fractions 1, 2, and 3 (see Supplementary Figure S1, 1, free pigments; 2, LHCII and photosystem II complexes; 3, photosystem I complexes) were used. Coimmunoprecipitation (CoIP) was performed with the antibody specific for the HA peptide. Precipitated proteins were separated by SDS-PAGE. Immunoreactions were performed with the antibody specific for LHCA1 (22 kD), followed by incubation with the antibody directed towards LHCA2 (23 kD), LHCA3 (25 kD), LHCA4 (22 kD), and LHCB1 (20 kD) as control; (C) Confirmation of the interaction of WHY1 and LHCA1 by bimolecular fluorescence complementation assays (BiFC). The onion epidermal cells were transiently transformed with the full length WHY1 and the full length LHCA1. Constructs were fused to either c-myc-GFPn173 or HA-GFPc155 and vice versa. The WHY1 and LHCA1 fused to full length GFP and empty vector were used as controls. All constructs were under the control of the 35S promoter. Fluorescence images are shown on the upside and bright field images are shown on the underside, respectively. C1: WHY1-GFPn173 + LHCA1-GFPc155; C2: LHCA1-GFPn173 + WHY1-GFPc155; C3: WHY1-GFP; C4: LHCA1-GFP; C5: WHY1-GFPn173 + LHCA4-GFPc155; C1-2: scale bar 21 μm; C3-5: scale bar 100 μm.

Figure 4

Expression of NDH-encoded genes in different WHY1 mutants and wild-type plants. (A) Expression analysis of NDH membrane complex encoded genes in WHY1 mutants and wild-type plants by qPCR; (B) Expression analysis of NDH subcomplex-A encoded genes in WHY1 mutants and wild-type plants. The transcript level in each case was normalized to that of GAPC2 as a reference gene, and the expression level of WT was set as 1. Three biological replications were used in the analysis. Asterisk indicate significant differences in gene expression compared to WT (Tukey–Kramer method, * p < 0.05, ** p < 0.01, *** p < 0.001).

Figure 4

Expression of NDH-encoded genes in different WHY1 mutants and wild-type plants. (A) Expression analysis of NDH membrane complex encoded genes in WHY1 mutants and wild-type plants by qPCR; (B) Expression analysis of NDH subcomplex-A encoded genes in WHY1 mutants and wild-type plants. The transcript level in each case was normalized to that of GAPC2 as a reference gene, and the expression level of WT was set as 1. Three biological replications were used in the analysis. Asterisk indicate significant differences in gene expression compared to WT (Tukey–Kramer method, * p < 0.05, ** p < 0.01, *** p < 0.001).

Figure 5

The stability of NDH-PSI complexes of thylakoid membranes and NDH activities in the different WHY1 and lhca1 mutants. (A) Determination of NDH activity by chlorophyll fluorescence analysis. Five-week-old plants including different WHY1 and lhca1 mutants, why1lhca1 double mutant and wild-type (WT) were illuminated for 5 min with AL (200 μmol m⁻² s⁻¹). After illumination, the subsequent transient increase in chlorophyll fluorescence were recorded as an indicator of NDH activity (a.u.; arbitrary units). The fluorescence levels were standardized by the Fm level. Curves shows trace of chlorophyll fluorescence in mutants and WT plants. ndho line was used as positive control; (B) Quantification of NDH activity measured in (A). The transient increase in chlorophyll fluorescence in mutants was quantified by comparing the peak area with that of WT. The peak area of WT was set as 1. Data presented are the mean of ± SE (n = 5–6); (C) Blue Native PAGE of thylakoid membrane, Band I is the NDH-PSI supercomplex detected in PWHY-HA line; (D) 2D SDS-PAGE separation and immunodetection of NDH-PSI supercomplex containing NDH18 protein (I) and monomer NDH18 (II); (E) Immunodetection of thylakoid membrane, the antibody against to HA peptide, LHCA1, LHCA4, NDH18 (provided by Shikanai group, Japan) and PsaA in kowhy1 (a), oepWHY1-HA (b), PWHY1-HA (c), and PWHY1-HA in lhca1 background, lhca1/PWHY1-HA (d).

Figure 6

Measurement of photosynthetic parameters in WHY mutants and wild-type plants after high light treatment. (A) Electron transport rate (ETR) in WHY mutants and wild-type plants under high light; (B) Non-photochemical quenching (NPQ) in WHY mutants and wild-type plants under high light. Means ± SE of at least 10 independent measurements are shown. Asterisk indicate significant differences compared to WT (* p < 0.05, Student’s t test).

Figure 7

Heat map of gene expression in kowhy1 and ko1/3 mutants after high light treatment. Gene expression in kowhy1, ko1/3 mutants after high light treatment was analyzed by qPCR. The transcript level in each case was normalized to that of GAPC2 as a reference gene, and the expression level of WT was set as 1. Three biological replications were used to analyze. Heat map were drawn by Heml software used log2 of expression.