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1 University of Groningen Photosynthetic Quantum Yield Dynamics Hogewoning, Sander W.; Wientjes, Emilie; Douwstra, Peter; Trouwborst, Govert; van Ieperen, Wim; Croce, Roberta; Harbinson, Jeremy Published in: Plant Cell DOI:.5/tpc IMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite from it. Please check the document version below. Document Version Publisher's PDF, also known as Version of record Publication date: Link to publication in University of Groningen/UMCG research database Citation for published version (APA): Hogewoning, S. W., Wientjes, E., Douwstra, P., Trouwborst, G., van Ieperen, W., Croce, R., & Harbinson, J. (). Photosynthetic Quantum Yield Dynamics: From Photosystems to Leaves. Plant Cell, (5), DOI:.5/tpc Copyright Other than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of the author(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons). Take-down policy If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim. Downloaded from the University of Groningen/UMCG research database (Pure): For technical reasons the number of authors shown on this cover page is limited to maximum. Download date: --8

2 Net assimilation ( mol CO m - s - ) A B C 8 nm 6 nm 7 nm 8 nm 6 nm 7 nm 8 nm 6 nm 7 nm Absorbed irradiance ( mol m - s - ) Supplemental Figure. Typical responses of net assimilation to irradiance within the light-limited range for leaves grown under a sunlight spectrum (A), a shadelight spectrum (B) and blue light (C). The slope of each curve expresses the quantum yield for CO fixation for the corresponding wavelength.

3 Signal (a.u.) A B 8 6 LHCII *CP9 C µg chlorophyll 6 9 LHCII/PSII D 6 9 g Chlorophyll loaded Supplemental Figure. Analysis of LHCII content in thylakoids of leaves grown under a sunlight spectrum, a shadelight spectrum, and blue light by SDS PAGE. (A) Example of SDS-PAGE for thylakoids (sunlight) loaded at different concentrations. (B) Linearity verification of Coomassie bound to CP9 and LHCII. Values of integrated optical density of the bands are plotted against the quantity of material loaded. (C) LHCII trimers/psii based on the four different concentrations of thylakoids (example for shadelight spectrum grown leaves). (D) Analysis of LHCII content. SDS-PAGE was followed by Coomassie staining, and densitometric analysis of the polypeptide was used to quantify LHCII (Lhcb-) levels relative to CP9 (Lhcb). Preparations containing the monomeric Lhcb antenna and PSI complexes were used as references. For the thylakoids of the leaves μg of chlorophylls were loaded. The Lhcb region of the gel is shown. Note that the PSI proteins do not overlap with CP9 or Lhcb-. The PSI/PSII ratio (Table main text) was calculated from the Chl a:b ratio of the membranes using the Chl content values of PSI-LHCI, PSII core, LHCII trimer and minor Lhcb (see Table main text) and the LHCII:PSII core ratio obtained for each sample from the analysis of the SDS page gel. The Chl a:b ratio is given by: PSI and PSII are on RC (reaction centre) basis and Taking PSII as, this simplifies to: represents the number of LHCII trimers per PSII RC. From which can be derived that PSI/PSII, as a function of the Chl a:b ratio and the number of LHCII trimers per PSII RC, is calculated as:

4 Signal antibody (a.u.) Relative PSI/PSII ratio A B 5 Shade C CP PsaD SUN SHADE BLUE Supplemental Figure. Quantification of PSI/PSII ratio by Protein immunoblotting. (A) Example of one Western blot of thylakoids from leaves grown under a blue light, shadelight and sunlight spectrum. Samples were loaded at four different concentrations and developed with antibodies against CP (a PSII core protein) and PsaD (a PSI core protein). Antibodies (Agrisera, Sweden) were diluted x and 7x for CP and PsaD, respectively. The level of antibody binding was monitored with secondary goat antirabbit IgG alkaline phosphatase antibody in combination with NBT/BCIP. (B) Example of signal linearity verification. Occasionally the last point was out of the linear range due to saturation. (C) Relative PSI/PSII ratios based on PsaD/CP ratios. The ratios for the blue light and shadelight spectrum samples were normalized to the sunlight spectrum sample (set to ). Error bars represent the s.e.m (N 5).

5 Absorption (a.u.) Absorption (a.u.). A B sunlight spectrum C shadelight spectrum D blue light Supplemental Figure. (A) Absorbance spectra of LHCII (upper dotted line) and PSI-LHCI (lower dotted line) purified from chloroplasts of the sunlight spectrum grown cucumber leaves (spectra for blue light and shadelight spectrum grown leaves were identical), and that of the PSII C S M supercomplex of Arabidopsis thaliana (connected line). Spectra were normalized to the maximum in the Q y region. (B, C, D) Absorbance spectra of PSI (connected line) and PSII (dotted line) from leaves grown under the three different light spectra, normalized to the number of Chls a and b in each complex, as described in the Methods section of the main text. Note that at wavelengths >7nm PSII absorbance is close to zero, whereas in vivo quantum yields substantially greater than zero were measured at 77 nm and 7 nm (Supplemental Table ). This is at least partially due to the fact that the narrow-band pass filters used for the in vivo measurements (see Methods section of the main text), of which the centre wavelengths are reported, transmitted a small proportion of smaller wavelengths (>69 nm). These smaller wavelengths were partially absorbed by PSII, allowing some linear electron flow and therefore CO fixation.

6 PSII/(PSI + PSII) in vivo PSII/(PSII+PSI) in vitro Supplemental Figure 5. Relationship between the excitation balance of the two photosystems calculated as light fraction absorbed by PSII divided by the absorbed fraction by both PSI and PSII using an in vitro and in vivo approach (sunlight spectrum: open circles; shadelight spectrum: closed circles; blue light: squares). Only those data points corresponding with wavelengths preferentially exciting PSII are presented in the graph. The six crossed data points correspond with the absorption peaks of carotenoids (6 nm and 5 nm). If these data points are not taken into account, the linearity of the relationship between the in vitro and in vivo approach improves significantly. 5

7 Absorption (a.u.). A B Supplemental Figure 6. Absorbance spectra of the purified LHCII trimer (A) and PSI-LHCI (B) of sunlight spectrum grown cucumber leaves (red connected lines; spectra for blue light and shadelight spectrum grown leaves were similar), and Arabidopsis thaliana (black dotted lines). Due to the negligible differences in the absorbance spectra of the photosystem components of Cucumis sativus and Arabidopsis thaliana it is difficult to distinguish between the two lines in each graph. 6

8 Supplemental Table. The wavelength dependence of the quantum yield for CO fixation on an incident light basis ( action spectrum ) for Cucumis sativus leaves grown under an artificial sunlight spectrum (SUN), an artificial shadelight spectrum (SHADE) and blue light (BLUE). The action spectra are presented on an absolute (i.e. CO molecules fixed per incident photon; left table) and relative basis (right table). Concerning the absolute values (left), the LSD of the significant (P<.5) interaction between growth-light spectrum and measuring wavelength is. (P<.5). The spectra of the three different growth-light sources are shown in Figure in the print version. SUN SHADE BLUE SUN SHADE BLUE

9 Supplemental Table. The wavelength dependence of the quantum yield for CO fixation on an absorbed light basis for Cucumis sativus leaves grown under an artificial sunlight spectrum (SUN), an artificial shadelight spectrum (SHADE) and blue light (BLUE). The quantum yields are presented on an absolute (i.e. CO molecules fixed per absorbed photon; left table) and relative basis (right table). Concerning the absolute quantum yields (left), the LSD of the significant (P<.5) interaction between growth-light spectrum and measuring wavelength is.59 (P<.5). The spectra of the three different growth-light sources are shown in Figure in the print version. SUN SHADE BLUE SUN SHADE BLUE