Agnieszka Katarzyna Banaś

Blue light signalling in chloroplast movements

Chloroplast movements are among the mechanisms allowing plants to cope with changes in their environment. Chloroplasts accumulate at illuminated cell areas under weak light while they avoid areas exposed to strong light. These directional responses may be controlled by blue and/or red light, depending on the plant group. In terrestrial angiosperms only the blue light perceived by phototropins is active. The last decade has seen a rapid development of studies on the mechanism of directional chloroplast movements, which started with an identification of the photoreceptors. A forward genetic approach has been used to identify the components which control chloroplast movements. This review summarizes the current state of research into the signalling pathways which lead to chloroplast responses. First, the molecular properties of phototropins are presented, followed by a characterization both of proteins which are active downstream of phototropins and of secondary messengers. Finally, cross-talk between light signalling involved in chloroplast movements and other signalling pathways is discussed.

Photoprotective function of chloroplast avoidance movement: in vivo chlorophyll fluorescence study.

Light-induced chloroplast avoidance movement has long been considered to be a photoprotective mechanism. Here, we present an experimental model in which this function can be shown for wild type Arabidopsis thaliana. We used blue light of different fluence rates for chloroplast positioning, and strong red light inactive in chloroplast positioning as a stressing light. The performance of photosystem II was measured by means of chlorophyll fluorescence. After stressing light treatment, a smaller decrease in photosystem II quantum yield was observed for leaves with chloroplasts in profile position as compared with leaves with chloroplasts in face position. Three Arabidopsis mutants, phot2 (no avoidance response), npq1 (impaired zeaxanhtin accumulation) and stn7 (no state transition), were examined for their chloroplast positioning and chlorophyll fluorescence parameters under identical experimental conditions. The results obtained for these mutants revealed additional stressing effects of blue light as compared with red light.

The expression of phototropins in Arabidopsis leaves: developmental and light regulation

Phototropins are blue light receptors, which play different roles during plant development. Two phototropins of Arabidopsis thaliana, phot1 and phot2, have strongly overlapping functions. In seedlings, both photoreceptors are responsible for phototropism. In mature leaves they redundantly regulate leaf shape, stomatal opening, and the accumulation of chloroplasts, whereas phototropin2 alone controls chloroplast avoidance response. Light not only activates phototropins, but also affects the level of their expression. In Arabidopsis seedlings, PHOT1 is downregulated and PHOT2 is upregulated by light. Since data on transcription levels of phototropins in mature Arabidopsis leaves is scarce, a comprehensive real-time PCR study of PHOT1 and PHOT2 expression during development was performed, from seedlings to senescing leaves. So far, neither the phototropin expression nor its modulation by light have been investigated during senescence. The results show that the general regulation pattern remains conserved during Arabidopsis lifecycle, whereas the level of transcripts fluctuates over time, pointing to the significance of the light control for functioning of phototropins. The second part of the study determined the influence of photosynthesis-derived signals and photoreceptor-activated transduction pathways on phototropin mRNA levels. The effects of blue and red light were examined using Arabidopsis mutant lines deficient in photoreceptors. The results reveal a complex network of interactions between these receptors in the regulation of phototropin transcription profiles. Cryptochrome1 and phytochromeB appear to be main photoreceptors involved in the regulation of PHOT1 transcript accumulation. The expression of PHOT2 is dependent on both cryptochromes and phytochromeA.

Blue-light-activated phototropin2 trafficking from the cytoplasm to Golgi/post-Golgi vesicles

Summary The blue-light-induced trafficking of the UVA/blue light receptor phototropin2 is shown. Evidence is provided for the presence of two pathways, one directing phototropin2 to the Golgi and post-Golgi vesicles, and the other to degradation.

Expression of Enzymes Involved in Chlorophyll Catabolism in Arabidopsis Is Light Controlled

We found that the levels of mRNA of two enzymes involved in chlorophyll catabolism in Arabidopsis (Arabidopsis thaliana), products of two chlorophyllase genes, AtCLH1 and AtCLH2, dramatically increase (by almost 100- and 10-fold, respectively) upon illumination with white light. The measurements of photosystem II quantum efficiency in 3-(3,4-dichlorophenyl)-1,1-dimethylurea-inhibited leaves show that their expression is not related to photosynthesis but mediated by photoreceptors. To identify the photoreceptors involved, we used various light treatments and Arabidopsis photoreceptor mutants (cry1, cry2, cry1cry2, phot1, phot2, phot1phot2, phyA phyB, phyAphyB). In wild-type Columbia, the amount of transcripts of both genes increase after white-light irradiation but their expression profile and the extent of regulation differ considerably. Blue and red light is active in the case of AtCLH1, whereas only blue light raises the AtCLH2 mRNA level. The fundamental difference is the extent of up-regulation, higher by one order of magnitude in AtCLH1. Both blue and red light is active in the induction of AtCLH1 expression in all mutants, pointing to a complex control network and redundancy between photoreceptors. The blue-specific up-regulation of the AtCLH2 transcript is mediated by cryptochromes and modulated by phototropin1 and phytochromes. Individually darkened leaves were used to test the effects of senescence on the expression of AtCLH1 and AtCLH2. The expression profile of AtCLH1 remains similar to that found in nonsenescing leaves up to 5 d after darkening. In contrast, the light induction of AtCLH2 mRNA declines during dark treatment. These results demonstrate that the expression of enzymes involved in chlorophyll catabolism is light controlled.

Influence of Sugars on Blue Light-Induced Chloroplast Relocations

The aim of this study was to investigate the influence of sugars on blue light-induced chloroplast movements. Sucrose and glucose inhibited chloroplast responses in the detached leaves of Arabidopsis thaliana and in Lemna trisulca fronds in a concentration and time-dependent manner. The prolonged exposure necessary for inhibition indicates that sugars may act via altered gene expression. Overexpression of phototropin2, a photoreceptor responsible for the strong blue light response of chloroplasts, counteracted the sugar effect. This may suggest that sugars modify some component(s) of the phototropin2-mediated signal transduction pathway. The expression of PHOT2 was not suppressed by sugars in wild type plants, it was even up-regulated by glucose. Impaired chloroplast movements were observed only in mature Arabidopsis plants. The mRNA of SAG12, a late senescence marker, was not detectable in the sugar-incubated leaves. The SAG13 mRNA level and its regulation by sugars were similar in wild type and PHOT2 overexpressor. Thus, the sugar insensitivity of 35S:PHOT2 chloroplast responses was not due to delayed senescence. The sugar-induced transduction pathway involved remains unclear. 3-O-methylglucose did not affect chloroplast movements suggesting the participation of a hexokinase-dependent pathway. Only the amplitude of avoidance response was reduced in gin2-1, a hexokinase1 null mutant. Probably other hexokinases, or glycolysis-associated signals play a role in the suppression of chloroplast responses.

Fine tuning chloroplast movements through physical interactions between phototropins.

Highlight Physical interactions between phototropin molecules can alter their signaling outcomes, providing the means for the plant to fine-tune its blue light responses.

Abscisic acid and blue light signaling pathways in chloroplast movements in Arabidopsis mesophyll

Abscisic acid (ABA) and phototropins act antagonistically to control stomatal movements. Here, we investigated the role of ABA in phototropin-directed chloroplast movements in mesophyll cells of Arabidopsis thaliana. We analyzed the expression of phototropins at mRNA and protein level under the influence of ABA. PHOT1 mRNA level was decreased by ABA in the dark while it was insensitive to ABA in light. PHOT2 mRNA level was independent of the hormone treatment. The levels of phototropin proteins were down-regulated by ABA, both in darkness and light. No impact of exogenous ABA on amplitudes and kinetics of chloroplast movements was detected. Chloroplast responses in wild type Arabidopsis and three mutants, abi4, abi2 (abscisic acid insensitive4, 2) and aba1 (abscisic acid1), were measured to account for endogenous ABA signaling. The chloroplast responses were slightly reduced in abi2 and aba1 mutants in strong light. To further investigate the effect, abi2 and aba1 mutants were supplemented with exogenous ABA. In the aba1 mutant, the reaction was rescued but in abi2 it was unaffected. Our results show that ABA is not directly involved in phototropin-controlled chloroplast responses in mature leaves of Arabidopsis. However, the disturbance of ABA biosynthesis and signaling in mutants affects some elements of the chloroplast movement mechanism. In line with its role as a stress hormone, ABA appears to enhance plant sensitivity to light and promote the chloroplast avoidance response.

6,4–PP Photolyase Encoded by AtUVR3 is Localized in Nuclei, Chloroplasts and Mitochondria and its Expression is Down-Regulated by Light in a Photosynthesis-Dependent Manner

Pyrimidine dimers are the most important DNA lesions induced by UVB irradiation. They can be repaired directly by photoreactivation or indirectly by the excision repair pathways. Photoreactivation is carried out by photolyases, enzymes which bind to the dimers and use the energy of blue light or UVA to split bonds between adjacent pyrimidines. Arabidopsis thaliana has three known photolyases: AtPHR1, AtCRY3 and AtUVR3. Little is known about the cellular localization and regulation of AtUVR3 expression. We have found that its transcript level is down-regulated by light (red, blue or white) in a photosynthesis-dependent manner. The down-regulatory effect of red light is absent in mature leaves of the phyB mutant, but present in leaves of phyAphyB. UVB irradiation does not increase AtUVR3 expression in leaves. Transiently expressed AtUVR3-green fluorescent protein (GFP) is found in the nuclei, chloroplasts and mitochondria of Nicotiana benthamiana epidermal cells. In the nucleoplasm, AtUVR3-GFP is distributed uniformly, while in the nucleolus it forms speckles. Truncated AtUVR3 and muteins were used to identify the sequences responsible for its subcellular localization. Mitochondrial and chloroplast localization of AtUVR3 is independent of its N-terminal sequence. Amino acids located at the C-terminal loop of the protein are involved in its transport into chloroplasts and its retention inside the nucleolus.

Corrigendum: Fine tuning chloroplast movements through physical interactions between phototropins

Olga Sztatelman*, Justyna Łabuz, Paweł Hermanowicz, Agnieszka Katarzyna Banaś, Aneta Bażant, Piotr Zgłobicki, Chhavi Aggarwal, Marcin Nadzieja, Weronika Krzeszowiec, Wojciech Strzałka and Halina Gabryś 1 Department of Plant Biotechnology, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Gronostajowa 7, 30-387 Krakow, Poland 2 Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Pawińskiego 5a, 02-106 Warsaw, Poland 3 Malopolska Centre of Biotechnology, Jagiellonian University, Gronostajowa 7A, 30-387 Krakow, Poland