Figure 1.
Rapid light curves of basic photosynthetic parameters for wild type (closed symbols) and overexpressors of TGZ (open symbols). Data are presented as the means ± SE (n = 3). (A) Non photochemical quenching (NPQ) is higher for TGZ overexpressors over a range of light intensities. (B) Linear electron transport (LEF) is lower for TGZ overexpressors and saturates at around 600 µmol photons m−2s−1. (C) Yield of the overexpressors is only half of that of WT over the range of light intensities tested.
Figure 2.
Steady state photosynthesis in leaves.
In vivo fluorescence spectroscopy for intact WT (closed symbols) and TGZ overexpressors (open symbols). Data are presented as the means ± SE (n = 8). (A) Effective quantum yield at steady state (Fv/Fm’). (B) linear electron flow at steady state (LEF). (C) photoinhibition quenching (qI). (D) rate of Fv recovery in the dark.
Figure 3.
Estimates of the protonic circuit of photosynthesis.
In vivo monitoning of the protonic circuit of photosynthesis in WT (closed symbols) and TGZ OE (open symbols). Data are presented as the means ± SE (n = 8). (A) Light-induced pmf as estimated by the ECSt parameter. (B) Apparent conductivity of the ATPase of thylakoids (gH+). (C) The electric component of pmf (Δψ/pmf) as estimated by three wavelength deconvolution (insert shows typical traces). (D) Estimates of the ΔpH component of pmf in thylakoids as estimated by the ECSinv parameter.
Figure 4.
Regulation of the protonic circuit of photosynthesis.
WT (closed symbols) and TGZ OE (open symbols). Data are presented as the means ± SE (n = 8). (A) Sensitivity of antenna downregulation (qE) to LEF. It is apparent that for the same LEF TGZ OE shows a higher qE response. (B) Sensitivity of qE to the ΔpH component of pmf. This increased sensitivity indicates that antenna properties are significantly different from WT. (C) Sensitivity of qE to light induced pmf follows in transformed tobacco the same trend as in WT. (D) Proton efflux in thylakoids of TGZ OE is getting more sensitive to LEF in comparison to WT.
Figure 5.
Non invasive probing of tobacco antenna by rapid fluorescence induction curves.
(A) TGZ overexpression induces an increase Ka of PSIIa centers. (B) TGZ overexpession significantly affects the shape of the OJIP curve (fluorescence induction curve).
Figure 6.
Changes in antenna properties between WT and TGZ and the polyamines bound in LHCII, CP29, CP26 and CP24.
(A) Western blot immunolocalization indicating that the fraction used for polyamine determination contains TGZ in the OE, and Lhcb1–6 antenna proteins in WT and in TGZ OE. AbH (1∶1000) and Abpep (1∶500), antibodies against generic TGase and against TGZ, were respectively used. 1 to 6, anti Lhcb1–6 antibodies (1∶5000), were respectively used. (B) HPLC determination of bound-polyamines of the same fraction as above, indicating that TGZ over-expression increases bound Spd and Spm in the antenna of PSII.
Figure 7.
Chloroplast ultrastructure of WT and TGZ over-expressing Nicotiana tabacum leaf cells.
The TGZ-transformed chloroplasts (A,C) show an increased grana appresion and a reduced stroma thylakoid network versus the WT (B,D). In the TGZ over-expressing chloroplasts, large grana with an increased number of appresed thylakoids and a decline in thylakoidal interconections are observed. Flattened thylakoids and starch grains can be also detected sometimes in the TGZ-transformed chloroplasts (C). According to the TGZ chloroplast age, inclusion bodies of different size were visible (A). In the WT chloroplast (B,D), the thylakoid architecture is normal. Insert Fig. 7A: aspect of an in vitro-grown TGZ-transformed plant showing the youngest green leaves and the oldest white leaves. Insert Fig. 7B: aspect of an in vitro-grown WT plant. ib inclusion body, g grana, nt non-apressed thylakoid, p plastoglobuli, S starch grains, tv top views of grana.
Figure 8.
Absorbance spectroscopy in leaves.
Comparison of the raw signals between WT (black) and TGZ OE (gray). (A) The signal at 535 nm that is rich is information related to electrochromic shift and to the qE effect (B) The signal at 520 that is rich mainly in electrochromic shift (C) The raw 505 nm is rich in information related to electrochromic shift but is less contaminated by the qE effect.
Figure 9.
Activation of xanthophyll cycle in WT and TGZ OE.
First order decay kinetics were fitted to data from 3 independent experiments and corresponding tau values are illustrated in the graphs. Plants were illuminated with 500 µmol photons m−2s−1 for about 30 min. (A) Activation of xanthophyll cycle in WT. (B) Activation of xanthophyll cycle in TGZ OE. In TGZ OE xanthophyll cycle is induced about 3 times faster. Upon shuttering actinic the signal at 505 nm was marginally changed after 10 min indicating that contamination of the 505 nm from the qE response peaking at 535–540 nm is low.
Figure 10.
Polyamine titer in whole leaves.
TGZ OE have three times less polyamines than WT. In TGZ OE, Put is 40%, Spd 26% and Spm 31% of the WT values. Data are presented as the means ± SE (n = 3).
Figure 11.
Model of antenna proteins from dicots.
(A) Model of LHCII monomer from pea (from residue 10 to 232). Side view of LHCII showing the three transmembrane helixes (gray) and the glutamine residues (black) that could be potential substrate sites for the plastidal transglutaminase. Entry used for the model (PDB ID: 2BWH). (B) Model of CP29 from spinach (from residue 88 to 243). Side view of CP29 showing the three transmembrane helixes (gray) and the glutamine residues (black) that could be potential substrate sites for the plastidal transglutaminase. Entry used for the model (PDB ID: 3PL9). Please note that it is highly unlikely all these marked residues to be substrates for TGases. More likely substrates are those residues that are stroma exposed such as Q90 (panel B).
Figure 12.
An oversimplified scheme showing a possible mode of action of TGZ in tobacco thylakoids.
Higher TGase activity in chloroplast results in a higher polyamination of LHCII and CPs (this work), which in turn increase the absorption cross section of PSII. Under these conditions, antenna downregulation via qE response is getting more sensitive to LEF.