Physcomitrella patens mutants affected on heat dissipation clarify the evolution of photoprotection mechanisms upon land colonization

Abstract

Light is the source of energy for photosynthetic organisms; when in excess, however, it also drives the formation of reactive oxygen species and, consequently, photoinhibition. Plants and algae have evolved mechanisms to regulate light harvesting efficiency in response to variable light intensity so as to avoid oxidative damage. Nonphotochemical quenching (NPQ) consists of the rapid dissipation of excess excitation energy as heat. Although widespread among oxygenic photosynthetic organisms, NPQ shows important differences in its machinery. In land plants, such as Arabidopsis thaliana, NPQ depends on the presence of PSBS, whereas in the green alga Chlamydomonas reinhardtii it requires a different protein called LHCSR. In this work, we show that both proteins are present in the moss Physcomitrella patens. By generating KO mutants lacking PSBS and/or LHCSR, we also demonstrate that both gene products are active in NPQ. Plants lacking both proteins are more susceptible to high light stress than WT, implying that they are active in photoprotection. These results suggest that NPQ is a fundamental mechanism for survival in excess light and that upon land colonization, photosynthetic organisms evolved a unique mechanism for excess energy dissipation before losing the ancestral one found in algae.

Fig. 1.

Comparison of NPQ A. thaliana, P. patens, and C. reinhardtii. (A) Western blotting against NPQ involved polypeptides, PSBS, and LHCSR in Arabidopsis (At), Physcomitrella (Pp), and Chlamydomonas (Cr). Thylakoids (1 μg of Chl) were loaded in all cases. In the case of Cr, thylakoids were isolated from high light-grown cells; here, two LHCSR bands are recognized as in the study by Peers et al. (17). Bands are squared because LHCSR has a larger molecular mass in Cr with respect to Pp (29 vs. 23 kDa). Anti-LHCSR antibody recognizes other proteins with lower specificity (likely Lhc proteins) at different molecular masses in both At and Pp. NPQ (B) and fluorescence (C) kinetics were measured for Pp (black) compared with At (red). Actinic light intensities were shown to saturate photosynthetic capacity in both organisms, as shown by the fact that pulses induce a very small peak in fluorescence (Fm′), and thus activate maximal NPQ.

Fig. 2.

KO mutant generation and characterization. Scheme of constructs used for KO generation. Genomic region of PSBS (A) and LHCSR (B and C) genes is schematized, with exons shown in black. Gray boxes represent the genomic regions exploited for homologous recombination. Below are shown the constructs for homologous recombination: genes for antibiotic resistance are located between regions homologous to the genome (all primers used for mutagenesis are reported in Table S1). (D) Example of verification of DNA insertion in the genome by amplification of the right and left borders. An example of four independent lines of Psbs KO is shown. (E) Amplification of genomic DNA using primers external to the target recombination region. Mutants carrying a single insertion are identified by the different size of the amplified band with respect to WT. Amplificates were also sequenced to verify the insertion in the correct position. (F) Evaluation of PSBS and LHCSR gene expression assessed by RT-PCR in WT and different mutant lines. Elongation Factor-1 alpha (EF1α) is also reported as a control.

Fig. 3.

P. patens psbs and lhcsr KO phenotypes. (A) Western blotting using antibodies against LHCSR and PSBS. Western blotting against CP43 is also shown as a loading control. Thylakoids were loaded in all cases, 0.5 μg of Chl for anti-PSBS and 0.3 μg of Chl for all the others. (B) NPQ kinetics of selected lines. Curves for WT and psbs KO, lhcsr1 KO, and lhcsr2 KO are shown in black, blue, green, and red, respectively. Averages and SD are calculated from at least five independent measures.

Fig. 4.

Phenotypes of P. patens psbs and lhcsr KO double and triple mutants. (A) Western blotting using antibody against LHCSR and PSBS. (B) NPQ kinetics of selected lines. Curves for WT, lhcsr1 KO, psbs lhcsr1 KO, and psbs lhcsr1 lhcsr2 KO are shown in black, red, green, and blue, respectively. Averages and SD are calculated from at least five independent measures.

Fig. 5.

Photoprotection capacity of NPQ-affected mutants. The photoprotection capacity of the previously described mutants has been compared with WT by treating 5-day-old plants with an additional 3 days of high light. PSII quantum yield, expressed as Fv/Fm, was monitored daily. WT, psbs KO, and lhcsr1 KO are shown in black, red, and blue, respectively, whereas psbs lhcsr1 and psbs lhcsr1 lhcsr2 KO double and triple mutants are shown in green and cyan. Values for a WT culture that was kept to control light conditions are also reported in black dots. Data from mutants grown in control conditions were omitted for clarity because they were indistinguishable from data on WT culture.