Screening and Identification of Candidate GUN1-Interacting Proteins in Arabidopsis thaliana

Chloroplasts are semi-autonomous organelles governed by the precise coordination between the genomes of their own and the nucleus for functioning correctly in response to developmental and environmental cues. Under stressed conditions, various plastid-to-nucleus retrograde signals are generated to regulate the expression of a large number of nuclear genes for acclimation. Among these retrograde signaling pathways, the chloroplast protein GENOMES UNCOUPLED 1 (GUN1) is the first component identified. However, in addition to integrating aberrant physiological signals when chloroplasts are challenged by stresses such as photooxidative damage or the inhibition of plastid gene expression, GUN1 was also found to regulate other developmental processes such as flowering. Several partner proteins have been found to interact with GUN1 and facilitate its different regulatory functions. In this study, we report 15 possible interacting proteins identified through yeast two-hybrid (Y2H) screening, among which 11 showed positive interactions by pair-wise Y2H assay. Through the bimolecular fluorescence complementation assay in Arabidopsis protoplasts, two candidate proteins with chloroplast localization, DJC31 and HCF145, were confirmed to interact with GUN1 in planta. Genes for these GUN1-interacting proteins showed different fluctuations in the WT and gun1 mutant under norflurazon and lincomycin treatments. Our results provide novel clues for a better understanding of molecular mechanisms underlying GUN1-mediated regulations.

FERONIA Confers Resistance to Photooxidative Stress in Arabidopsis

Plants absorb light energy required for photosynthesis, but excess light can damage plant cells. To protect themselves, plants have developed diverse signaling pathways which are activated under high-intensity light. Plant photoprotection mechanisms have been mainly investigated under conditions of extremely high amount of light; thus, it is largely unknown how plants manage photooxidative damage under moderate light intensities. In the present study, we found that FERONIA (FER) is a key protein that confers resistance to photooxidative stress in plants under moderate light intensity. FER-deficient mutants were highly susceptible to increasing light intensity and exhibited photobleaching even under moderately elevated light intensity (ML). Light-induced expression of stress genes was largely diminished by the fer-4 mutation. In addition, excitation pressure on Photosystem II was significantly increased in fer-4 mutants under ML. Consistently, reactive oxygen species, particularly singlet oxygen, accumulated in fer-4 mutants grown under ML. FER protein abundance was found to be elevated after exposure to ML, which is indirectly affected by the ubiquitin-proteasome pathway. Altogether, our findings showed that plants require FER-mediated photoprotection to maintain their photosystems even under moderate light intensity.

Characterization of UVA-Induced Alterations to Transfer RNA Sequences

Ultraviolet radiation (UVR) adversely affects the integrity of DNA, RNA, and their nucleoside modifications. By employing liquid chromatography–tandem mass spectrometry (LC–MS/MS)-based RNA modification mapping approaches, we identified the transfer RNA (tRNA) regions most vulnerable to photooxidation. Photooxidative damage to the anticodon and variable loop regions was consistently observed in both modified and unmodified sequences of tRNA upon UVA (λ 370 nm) exposure. The extent of oxidative damage measured in terms of oxidized guanosine, however, was higher in unmodified RNA compared to its modified version, suggesting an auxiliary role for nucleoside modifications. The type of oxidation product formed in the anticodon stem–loop region varied with the modification type, status, and whether the tRNA was inside or outside the cell during exposure. Oligonucleotide-based characterization of tRNA following UVA exposure also revealed the presence of novel photoproducts and stable intermediates not observed by nucleoside analysis alone. This approach provides sequence-specific information revealing potential hotspots for UVA-induced damage in tRNAs.

Soybean (Glycine max) PHYTOCHROME-INTERACTION FACTOR 1 induced skotomorphogenesis and de-etiolation in Arabidopsis

Skotomorphogenesis occurs after germination and before excavation in plants. It inhibits excessive absorbed energy in cells and can prevent the lethal photooxidative damage caused by transitioning from skotomorphogenesis to photomorphogenesis for light energy utilization. To investigate the mechanisms underlying photoreactions in soybean [Glycine max (L.) Merr.], we identified and isolated soybean phytochrome-interaction factor 1 (GmPIF1). A yeast two-hybrid (Y2H) assay showed that GmPIF1 interacted with photoactive PHYTOCHROME A (PHYA) and B (PHYB) in both soybean and Arabidopsis (GmPHYA, GmPHYB, AtPHYA, and AtPHYB). To analyze its function, we ectopically over-expressed GmPIF1 in wild type and pif1 mutant Arabidopsis. In etiolated seedlings, GmPIF1 caused hypocotyl elongation, cotyledon closed, apical hooks folded, and less accumulation of protochlorophyllide. In Y2H, GmPIF1 interacted with AtHDA15 that inhibited chlorophyll synthesis under dark conditions. After transition from darkness to white light, GmPIF1 promotes the reduction of photobleaching and induced de-etiolation. Moreover, GmPIF1 inhibited PHYA- and PHYB-mediated seed germination. Our findings increase our understanding of the regulatory network of light response in soybean and provide useful gene resources for soybean breeding in programs and genetics engineering.

Distinct photooxidation-induced cell death pathways lead to selective killing of human breast cancer cells

Lack of effective treatments for aggressive breast cancer is still a major global health problem. We previously reported that Photodynamic Therapy using Methylene Blue as photosensitizer (MB-PDT) massively kills metastatic human breast cancer, marginally affecting healthy cells. In this study we aimed to unveil the molecular mechanisms behind MB-PDT effectiveness. Through lipidomic and biochemical approaches we demonstrated that MB-PDT efficiency and specificity relies on polyunsaturated fatty acids-enriched membranes and on the better capacity to deal with photooxidative damage displayed by non-tumorigenic cells. We found out that, in tumorigenic cells, lysosome membrane permeabilization is accompanied by ferroptosis and/or necroptosis. Our results broadened the understanding of MB-PDT-induced photooxidation mechanisms and specificity in breast cancer cells. Therefore, we demonstrated that efficient approaches could be designed on the basis of lipid composition and metabolic features for hard-to-treat cancers. The results further reinforce MB-PDT as a therapeutic strategy for highly aggressive human breast cancer cells.

Two dominant boreal conifers use contrasting mechanisms to reactivate photosynthesis in the spring

Boreal forests are dominated by evergreen conifers that show strongly regulated seasonal photosynthetic activity. Understanding the mechanisms behind seasonal modulation of photosynthesis is crucial for predicting how these forests will respond to changes in seasonal patterns and how this will affect their role in the terrestrial carbon cycle. We demonstrate that the two co-occurring dominant boreal conifers, Scots pine (Pinus sylvestris L.) and Norway spruce (Picea abies), use contrasting mechanisms to reactivate photosynthesis in the spring. Scots pine downregulates its capacity for CO2 assimilation during winter and activates alternative electron sinks through accumulation of PGR5 and PGRL1 during early spring until the capacity for CO2 assimilation is recovered. In contrast, Norway spruce lacks this ability to actively switch between different electron sinks over the year and as a consequence suffers severe photooxidative damage during the critical spring period.

A thylakoid membrane-bound and redox-active rubredoxin (RBD1) functions in de novo assembly and repair of photosystem II

Photosystem II (PSII) undergoes frequent photooxidative damage that, if not repaired, impairs photosynthetic activity and growth. How photosynthetic organisms protect vulnerable PSII intermediate complexes during de novo assembly and repair remains poorly understood. Here, we report the genetic and biochemical characterization of chloroplast-located rubredoxin 1 (RBD1), a PSII assembly factor containing a redox-active rubredoxin domain and a single C-terminal transmembrane α-helix (TMH) domain. RBD1 is an integral thylakoid membrane protein that is enriched in stroma lamellae fractions with the rubredoxin domain exposed on the stromal side. RBD1 also interacts with PSII intermediate complexes containing cytochrome b559. Complementation of the Chlamydomonas reinhardtii (hereafter Chlamydomonas) RBD1-deficient 2pac mutant with constructs encoding RBD1 protein truncations and site-directed mutations demonstrated that the TMH domain is essential for de novo PSII assembly, whereas the rubredoxin domain is involved in PSII repair. The rubredoxin domain exhibits a redox midpoint potential of +114 mV and is proficient in 1-electron transfers to a surrogate cytochrome c in vitro. Reduction of oxidized RBD1 is NADPH dependent and can be mediated by ferredoxin-NADP+ reductase (FNR) in vitro. We propose that RBD1 participates, together with the cytochrome b559, in the protection of PSII intermediate complexes from photooxidative damage during de novo assembly and repair. This role of RBD1 is consistent with its evolutionary conservation among photosynthetic organisms and the fact that it is essential in photosynthetic eukaryotes.