Photometric Assays for Chloroplast Movement Responses to Blue Light

Gabryś, Halina; Banaś, Agnieszka Katarzyna; Hermanowicz, Paweł; Krzeszowiec, Weronika; Leśniewski, Sebastian; Łabuz, Justyna; Sztatelman, Olga

doi:10.21769/bioprotoc.2310

Cited by 6 publications

(10 citation statements)

References 14 publications

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“…Chloroplast movements were assessed with the photometric method, as described in Gabryś et al (2017) . In this approach, chloroplast movements are followed by measurements of changes in leaf transmittance.…”

Section: Methodsmentioning

confidence: 99%

Phototropin Interactions with SUMO Proteins

Łabuz

Sztatelman

Jagiełło-Flasińska

et al. 2021

Plant and Cell Physiology

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The disruption of the sumoylation pathway affects processes controlled by the two phototropins of Arabidopsis thaliana, phot1 and phot2. Phototropins, plant UVA/blue light photoreceptors, regulate growth responses and fast movements aimed at optimizing photosynthesis, such as phototropism, chloroplast relocations and stomatal opening. Sumoylation is a posttranslational modification, consisting in the addition of a SUMO (SMALL UBIQUITIN-RELATED MODIFIER) protein to a lysine residue in the target protein. Besides affecting stability of proteins, it regulates their activity, interactions and subcellular localization. We examined physiological responses controlled by phototropins, phototropism and chloroplast movements, in sumoylation pathway mutants. Chloroplast accumulation in response to both continuous and pulse light was enhanced in the E3 ligase siz1 mutant, in a manner dependent on phot2. A significant decrease in phototropin2 protein abundance was observed in this mutant after blue light treatment both in seedlings and mature leaves. Using plant transient expression and yeast two hybrid assays, we found that phototropins interacted with SUMO proteins mainly through their N-terminal parts, which contain the photosensory LOV domains. The covalent modification of phototropins by SUMO was verified using an Arabidopsis sumoylation system reconstituted in bacteria followed by the MS analysis. Lys 297 was identified as the main target of SUMO3 in the phot2 molecule. Finally, sumoylation of phot2 was detected in Arabidopsis mature leaves upon light or heat stress treatment.

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Section: Methodsmentioning

confidence: 99%

Phototropin Interactions with SUMO Proteins

Łabuz

Sztatelman

Jagiełło-Flasińska

et al. 2021

Plant and Cell Physiology

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“…After recording the initial transmittance level, leaf segments were illuminated with weak blue light (1.6 μmol m −2 s −1 ) for 45 min followed by strong blue light (108 μmol m −2 s −1 ) for the same time. The following parameters were measured/calculated for both responses: (1) amplitude—the transmittance change after 45 min, (2) velocity—the first derivative of the initial linear fragment of the transmittance curve (for details see Gabryś et al 2017 ). To measure the fluence rate response curves chloroplast relocations were induced by a continuous blue light of fluence rates increasing stepwise, each step lasting 45 min.…”

Section: Methodsmentioning

confidence: 99%

“…The abundance of transcripts was analyzed with the relative standard curve method (Delta-delta Ct method), normalizing to the reference gene Ubi4. The reference gene was chosen according to the technical approach used in previous Brachypodium studies (Hong et al 2008 ; Chambers et al 2012 ). The primers used for amplification are shown in Table 1 .…”

Section: Methodsmentioning

confidence: 99%

“…Quantitative measurements of chloroplast movements were performed on ca. 0.8 cm long leaf segments using a double-beam photometer (Gabryś et al 2017). A red light of 660 nm, 0.1 μmol m −2 s −1 , modulated with a frequency of 1 3 800 Hz was used to monitor changes in the transmittance through the leaf.…”

Section: Photometric Measurements Of Chloroplast Movementsmentioning

confidence: 99%

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Chloroplasts in C3 grasses move in response to blue-light

2020

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Key message Brachypodium distachyonis a good model for studying chloropla st movements in the crop plants, wheat, rye and barley. The movements are activated only by blue light, similar to Arabidopsis. Abstract Chloroplast translocations are ubiquitous in photosynthetic organisms. On the one hand, they serve to optimize energy capture under limiting light, on the other hand, they minimize potential photodamage to the photosynthetic apparatus in excess light. In higher plants chloroplast movements are mediated by phototropins (phots), blue light receptors that also control other light acclimation responses. So far, Arabidopsis thaliana has been the main model for studying the mechanism of blue light signaling to chloroplast translocations in terrestrial plants. Here, we propose Brachypodium distachyon as a model in research into chloroplast movements in C3 cereals. Brachypodium chloroplasts respond to light in a similar way to those in Arabidopsis. The amino acid sequence of Brachypodium PHOT1 is 79.3% identical, and that of PHOT2 is 73.6% identical to the sequence of the corresponding phototropin in Arabidopsis. Both phototropin1 and 2 are expressed in Brachypodium, as shown using quantitative real-time PCR. Intriguingly, the light-expression pattern of BradiPHOT1 and BradiPHOT2 is the opposite of that for Arabidopsis phototropins, suggesting potential unique light signaling in C3 grasses. To investigate if Brachypodium is a good model for studying grass chloroplast movements we analyzed these movements in the leaves of three C3 crop grasses, namely wheat, rye and barley. Similarly to Brachypodium, chloroplasts only respond to blue light in all these species.

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“…Chloroplast movements triggered by blue light were assessed using the photometric method according to Gabryś et al (2017), which relies on measurements of changes in leaf transmittance. Chloroplast responses to continuous blue light (Luxeon Star Royal Blue LXHL-FR5C LED, 460 nm) of 1.6 or 120 μmol·m −2 ·s −1 were measured in detached leaves of overnight dark-adapted 4- to 5-week-old plants.…”

Section: Methodsmentioning

confidence: 99%

UV-B Induces Chloroplast Movements in a Phototropin-Dependent Manner

et al. 2019

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We examined the impact of UV-B irradiation on chloroplast movements in Arabidopsis leaves. Directional chloroplast movements induced by blue light have been described in multiple plant species. In weak light, chloroplasts accumulate at periclinal cell walls to increase light capture. In strong light, chloroplasts exhibit the avoidance response, as they move towards anticlinal walls to protect the photosynthetic apparatus from light-induced damage. In Arabidopsis, chloroplast movements are triggered by phototropins, phot1 and phot2, which are known as blue/UV-A photoreceptors. We found that irradiation with UV-B of 3.3 µmol·m−2·s−1 induced chloroplast accumulation in wild-type plants. UV-B-triggered accumulation was dependent on the presence of phototropins, especially phot1, but not on UVR8 (the canonical UV-B photoreceptor). Irradiation with strong UV-B of 20 µmol·m−2·s−1 did not induce substantial chloroplast relocations in wild-type leaves. However, in the jac1 mutant, which is defective in chloroplast accumulation, strong UV-B elicited chloroplast avoidance. This indicated that UV-B can also activate signaling to the avoidance response. To assess the possibility of indirect effects of UV-B on chloroplast movements, we examined the impact of UV-B on the actin cytoskeleton, which serves as the motile system for chloroplast movements. While irradiation with UV-B of 3.3 µmol·m−2·s−1 did not affect the actin cytoskeleton, strong UV-B disrupted its structure as shown using an Arabidopsis line expressing Lifeact-green fluorescent protein (GFP). In wild-type plants, pretreatment with strong UV-B attenuated chloroplast responses triggered by subsequent blue light irradiation, further indicating that this UV-B intensity also indirectly affects chloroplast movements. Taken together, our results suggest that the effect of UV-B on chloroplast movement is twofold: it directly induces phototropin-mediated movements; however, at higher intensities, it attenuates the movements in a nonspecific manner.

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Photometric Assays for Chloroplast Movement Responses to Blue Light

Abstract: Assessment of chloroplast movements by measuring changes in leaf transmittance is discussed, with special reference to the conditions necessary for reliable estimation of blue light-activated chloroplast responses.

Cited by 6 publications

References 14 publications

Phototropin Interactions with SUMO Proteins

Phototropin Interactions with SUMO Proteins

Chloroplasts in C3 grasses move in response to blue-light

UV-B Induces Chloroplast Movements in a Phototropin-Dependent Manner

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