Molecular and physiological studies of phytochrome function in light direction sensing in Physcomitrium patens

Abstract

The main objective of this thesis was to generate libraries of mutants of the phytochrome and phototropin gene family and to analyse them physiologically in order to gain a better insight into the steering processes involved in phytochrome-mediated light directional sensing in Physcomitrium patens. Since Physcomitrella has no less than seven phytochrome genes, with both specific and overlapping functions, and five phototropin genes, the CRISPR/Cas9 technology was used to overcome this obstacle. A highly-efficient multiplex CRISPR/Cas9 based on the co-delivery of an improved Cas9 plasmid with multiple sgRNA plasmids was established and used to target multiple phytochrome genes simultaneously in different combinations. This improved multiplexing approach, together with an efficient screening procedure based on high-resolution PAGE gels to identify high-order multiple mutants prior to sequencing, allowed the isolation of phy7- lines along with lower-order mutants and phot5- mutants. Phototropic responses, initially assayed in unilateral, collimated R, confirmed that phytochrome is essential for R phototropism in Physcomitrella as observed in the aphototropic phenotype of the phy7- mutant. This conclusion was corroborated by the R/FR reversibility observed with light pulses, which also showed that the phytochrome molecules responsible are relatively immobile in the cell. Physiological analysis of lower-order mutants revealed a certain degree of functional redundancy for all phytochromes except for phy1 and phy3 which appear to not be involved in phototropism. Interestingly, the strongest phenotype was observed in the phy5a phy5c- double mutants where the absence of both phy5a and phy5c abolished the phototropic response as effectively as in the phy7- line. This result clearly indicated a dominant role of both phy5a and phy5c in the phototropic response. Furthermore, phy5c- mutants showed negative phototropism, suggesting a very specific role of this phytochrome in mediating the response, as well as providing a crucial information to further elucidate the mechanism of directional light sensing. These results showed that phy5a and phy5c act antagonistically, one generating positive and the other negative phototropic responses, revealing an additional level of complexity in the mechanism previously hypothesised in the Jaffe/Etzold/Haupt model of phytochrome action in direction sensing. WT and phy mutants were also analysed in their polarotropic responses. phy5a phy5c- double mutant showed no reaction to polarized light, providing the robust genetic evidence that photo- and polarotropic responses to R involve the same photoreceptors, namely phy5a and phy5c. Given the encouraging results obtained in the first phototropism assay, phototropic reactions of the WT and mutated lines were therefore measured quantitatively. The fluence-rate response analysis provided interesting insights into the optical nature of the phototropic response in Physcomitrella filaments, suggesting that a lens effect is the predominant effect in detecting light direction. Lens effect would explain the presence of negative phototropism at low fluence rates, positive phototropism at medium fluence rates, and in particular, aphototropism observed at the highest fluence rate tested. This notion was also supported by the refractive index experiments carried out in WT caulonemata. The positive curvature observed in the WT under simultaneous irradiation with R and FR and the weak positive phototropism observed in the phy5c- mutant suggested the formation of a Pfr gradient, possibly due to photocycling between Pr and Pfr, reflecting the light gradient established within the cell and ultimately inducing the phototropic response. Correct sensing of light direction in Physcomitrella would be ensured by antagonistic effects of phy5a and phy5c, allowing fine modulation of light sensitivity, especially at low fluence rates. In order to achieve a better understanding of the phy-phot interaction at the plasma membrane, phototropism was investigated in phot5- mutants. The results revealed an unexpected phototropic phenotype, although with a weakened bending curvature compared to WT. The generation of phy5c photA2- double mutants via CRISPR/Cas9 and their physiological analysis further confirmed the importance phy-phot interaction complex in directional sensing. Although these results suggest that phototropin is involved in mediating phytochrome induced phototropism, they contradict the idea of phototropin as an essential component of the signalling complex. One possibility is that phytochrome may also bind to an unknown additional protein that is associated with the phy-phot complex and provides an anchoring function at the plasma membrane. Phototropic responses were also assessed under unilateral, collimated B light. Previously, a low fluence rate had failed to induce a phototropic response. Higher B fluence rates used in this work were effective in the WT. This response was lost in both phy7- and phy5a phy5c- mutants. On the other hand, phot5- lines and phy5c photA2- mutants also showed positive phototropism. Thus, although the response is rather insensitive, B-induced phototropism occurs in caulonemata and is mediated predominantly by phy5a and phy5c as in R. The data generated from the CRISPR/Cas9 experiments, together with the optimisation of the mutant screening procedure, have increased our understanding of the CRISPR/Cas9 system in plants and in Physcomitrella in particular. The multiplex mutants and light experiments provided deep insights into the photoperception system and allowed the creation of a model that, combining previous work and the present results, could explain phytochrome-mediated directional sensing in Physcomitrella filament tip cells.