Engineering the phototropin photocycle improves photoreceptor performance and plant biomass production.

The ability to enhance photosynthetic capacity remains a recognized bottleneck to improving plant productivity. Phototropin blue light receptors (phot1 and phot2) optimize photosynthetic efficiency in Arabidopsis thaliana by coordinating multiple light-capturing processes. In this study, we explore the potential of using protein engineering to improve photoreceptor performance and thereby plant growth. We demonstrate that targeted mutagenesis can decrease or increase the photocycle lifetime of Arabidopsis phototropins in vitro and show that these variants can be used to reduce or extend the duration of photoreceptor activation in planta Our findings show that slowing the phototropin photocycle enhanced several light-capturing responses, while accelerating it reduced phototropin's sensitivity for chloroplast accumulation movement. Moreover, plants engineered to have a slow-photocycling variant of phot1 or phot2 displayed increased biomass production under low-light conditions as a consequence of their improved sensitivity. Together, these findings demonstrate the feasibility of engineering photoreceptors to manipulate plant growth and offer additional opportunities to enhance photosynthetic competence, particularly under suboptimal light regimes.

Native mass spectrometry reveals the conformational diversity of the UVR8 photoreceptor.

UVR8 is a plant photoreceptor protein that regulates photomorphogenic and protective responses to UV light. The inactive, homodimeric state absorbs UV-B light, resulting in dissociation into monomers, which are considered to be the active state and comprise a ß-propeller core domain and intrinsically disordered N- and C-terminal tails. The C terminus is required for functional binding to signaling partner COP1. To date, however, structural studies have only been conducted with the core domain where the terminal tails have been truncated. Here, we report structural investigations of full-length UVR8 using native ion mobility mass spectrometry adapted for photoactivation. We show that, while truncated UVR8 photoconverts from a single conformation of dimers to a single monomer conformation, the full-length protein exists in numerous conformational families. The full-length dimer adopts both a compact state and an extended state where the C terminus is primed for activation. In the monomer the extended C terminus destabilizes the core domain to produce highly extended yet stable conformations, which we propose are the fully active states that bind COP1. Our results reveal the conformational diversity of full-length UVR8. We also demonstrate the potential power of native mass spectrometry to probe functionally important structural dynamics of photoreceptor proteins throughout nature.

Regulation of Arabidopsis gene expression by low fluence rate UV-B independently of UVR8 and stress signaling.

UV-B exposure of plants regulates expression of numerous genes concerned with various responses. Sudden exposure of non-acclimated plants to high fluence rate, short wavelength UV-B induces expression via stress-related signaling pathways that are not specific to the UV-B stimulus, whereas low fluence rates of UV-B can regulate expression via the UV-B photoreceptor UV RESISTANCE LOCUS 8 (UVR8). However, there is little information about whether non-stressful, low fluence rate UV-B treatments can activate gene expression independently of UVR8. Here, transcriptomic analysis of wild-type and uvr8 mutant Arabidopsis exposed to low fluence rate UV-B showed that numerous genes were regulated independently of UVR8. Moreover, nearly all of these genes were distinct to those induced by stress treatments. A small number of genes were expressed at all UV-B fluence rates employed and may be concerned with activation of eustress responses that facilitate acclimation to changing conditions. Expression of the gene encoding the transcription factor ARABIDOPSIS NAC DOMAIN CONTAINING PROTEIN 13 (ANAC13) was studied to characterise a low fluence rate, UVR8-independent response. ANAC13 is induced by as little as 0.1 µmol m-2 s-1 UV-B and its regulation is independent of components of the canonical UVR8 signaling pathway COP1 and HY5/HYH. Furthermore, UV-B induced expression of ANAC13 is independent of the photoreceptors CRY1, CRY2, PHOT1 and PHOT2 and phytochromes A, B, D and E. ANAC13 expression is induced over a range of UV-B wavelengths at low doses, with maximum response at 310 nm. This study provides a basis for further investigation of UVR8 and stress independent, low fluence rate UV-B signaling pathway(s).

Difference in the action spectra for UVR8 monomerisation and HY5 transcript accumulation in Arabidopsis.

The photoreceptor UV RESISTANCE LOCUS 8 (UVR8) activates photomorphogenic responses when plants are exposed to ultraviolet-B (UV-B) light. However, whereas the absorption spectrum of UVR8 peaks at 280 nm, action spectra for several photomorphogenic UV-B responses show maximal photon effectiveness at 290-300 nm. To investigate this apparent discrepancy we measured the effectiveness of UV wavelengths in initiating two responses in Arabidopsis: photoconversion of homodimeric UVR8 into the monomeric form, which is active in signaling, and accumulation of transcripts of the ELONGATED HYPOCOTYL 5 (HY5) transcription factor, which has a key role in UVR8-mediated responses. When purified UVR8 or Arabidopsis leaf extracts were exposed to UV light monomerisation was maximal at approximately 280 nm, which correlates with the UVR8 absorption spectrum. When intact plants were exposed to UV, monomerisation was most strongly initiated at approximately 290 nm, and this shift in maximal effectiveness could be explained by strong absorption or reflectance at 280 nm by leaf tissue. Notably, the action spectrum for accumulation of HY5 transcripts in the same leaf tissue samples used to assay UVR8 dimer/monomer status peaked at approximately 300 nm. Possible reasons for the difference in maximal photon effectiveness of UVR8 monomerisation and HY5 transcript accumulation in leaf tissue are discussed.