Chlorophyll a is a favorable substrate for Chlamydomonas Mg-dechelatase encoded by STAY-GREEN

Mg removal from chlorophyll by Mg-dechelatase is the first step of chlorophyll degradation. Recent studies showed that in Arabidopsis, Stay Green (SGR) encodes Mg-dechelatase. Though the Escherichia coli expression system is advantageous for investigating the properties of Mg-dechelatase, Arabidopsis Mg-dechelatase is not successfully expressed in E. coli. Chlamydomonas reinhardtii SGR (CrSGR) has a long, hydrophilic tail, suggesting that active CrSGR can be expressed in E. coli. After the incubation of chlorophyll a with CrSGR expressed in E. coil, pheophytin a accumulated, indicating that active CrSGR was expressed in E. coli. Substrate specificity of CrSGR against chlorophyll b and an intermediate molecule of the chlorophyll b degradation pathway was examined. CrSGR exhibited no activity against chlorophyll b and low activity against 7-hydroxymethyl chlorophyll a, consistent with the fact that chlorophyll b is degraded only after conversion to chlorophyll a. CrSGR exhibited low activity against divinyl chlorophyll a and chlorophyll a', and no activity against chlorophyllide a, protochlorophyll a, chlorophyll c(2), and Znchlorophyll a. These observations indicate that chlorophyll a is the most favorable substrate for CrSGR. When CrSGR was expressed in Arabidopsis cells, the chlorophyll content decreased, further confirming that SGR has Mg-dechelating activity in chloroplasts. (C) 2016 Elsevier Masson SAS. All rights reserved

Mg-dechelatase is involved in the formation of photosystem II but not in chlorophyll degradation in Chlamydomonas reinhardtii

The STAY-GREEN (SGR) gene encodes Mg-dechelatase which catalyzes the conversion of chlorophyll (Chl) a to pheophytin (Pheo) a. This reaction is the first and most important regulatory step in the Chl degradation pathway. Conversely, Pheo a is an indispensable molecule in photosystem (PS) II, suggesting the involvement of SGR in the formation of PSII. To investigate the physiological functions of SGR, we isolated Chlamydomonas sgr mutants by screening an insertion-mutant library. The sgr mutants had reduced maximum quantum efficiency of PSII (F-v/F-m) and reduced Pheo a levels. These phenotypes were complemented by the introduction of the Chlamydomonas SGR gene. Blue Native polyacrylamide gel electrophoresis and immunoblotting analysis showed that although PSII levels were reduced in the sgr mutants, PSI and light-harvesting Chl a/b complex levels were unaffected. Under nitrogen starvation conditions, Chl degradation proceeded in the sgr mutants as in the wild type, indicating that Chlamydomonas SGR is not required for Chl degradation and primarily contributes to the formation of PSII. In contrast, in the Arabidopsis sgr triple mutant (sgrl sgr2 sgrL), which completely lacks SGR activity, PSII was synthesized normally. These results suggest that the Arabidopsis SGR participates in Chl degradation while the Chlamydomonas SGR participates in PSII formation despite having the same catalytic property

Mg-dechelation of chlorophyll a by Stay-Green activates chlorophyll b degradation through expressing Non-Yellow Coloring 1 in Arabidopsis thaliana

The first step in chlorophyll a degradation is the extraction of the central Mg. This reaction is catalyzed by Mg-dechelatase encoded by Stay-Green (SGR) in land plants. SGR extracts Mg from chlorophyll a but not from chlorophyll b, and chlorophyll b must be converted to chlorophyll a before degradation. The first reaction of the chlorophyll b to chlorophyll a conversion is catalyzed by chlorophyll b reductase. Non-Yellow Coloring 1 (NYC1) and NYC1 like (NOL) are isozymes of chlorophyll b reductase. When SGR was transiently overexpressed in Arabidopsis, both chlorophyll a and b were degraded, suggesting that the chlorophyll b to chlorophyll a conversion is activated by SGR overexpression. To examine the involvement of chlorophyll b reductases in SGR-induced chlorophyll b degradation, SGR was transiently overexpressed in nyc1, nol, and nyc1 nol double mutants by dexamethasone treatment. It was found that in the wild type and nol mutant, chlorophyll a and b were degraded and all the chlorophyll-binding proteins decreased. Meanwhile, in nyc1 and nyc1 nol mutants, chlorophyll b degradation was suppressed and the light-harvesting complex of photosystem II remained. The mRNA and protein levels of NYC1 increased after SGR overexpression in wild type plants. These results suggest that Mg-dechelation of chlorophyll a by SGR activates chlorophyll b degradation by inducing the expression of NYC1. This is an effective regulation of a metabolic pathway

The T-box transcription factor Brachyury regulates epithelial–mesenchymal transition in association with cancer stem-like cells in adenoid cystic carcinoma cells

Abstract Background The high frequencies of recurrence and distant metastasis of adenoid cystic carcinoma (AdCC) emphasize the need to better understand the biological factors associated with these outcomes. To analyze the mechanisms of AdCC metastasis, we established the green fluorescence protein (GFP)-transfected subline ACCS-GFP from the AdCC parental cell line and the metastatic ACCS-M GFP line from an in vivo metastasis model. Methods Using these cell lines, we investigated the involvement of the epithelial–mesenchymal transition (EMT) and cancer stem cell (CSCs) in AdCC metastasis by real-time RT-PCR for EMT related genes and stem cell markers. Characteristics of CSCs were also analyzed by sphere-forming ability and tumorigenicity. Short hairpin RNA (shRNA) silencing of target gene was also performed. Results ACCS-M GFP demonstrated characteristics of EMT and additionally displayed sphere-forming ability and high expression of EMT-related genes (Snail, Twist1, Twist2, Slug, zinc finger E-box binding homeobox 1 and 2 [Zeb1 and Zeb2], glycogen synthase kinase 3 beta [Gsk3β and transforming growth factor beta 2 [Tgf-β2]), stem cell markers (Nodal, Lefty, Oct-4, Pax6, Rex1, and Nanog), and differentiation markers (sex determining region Y [Sox2], Brachyury, and alpha fetoprotein [Afp]). These observations suggest that ACCS-M GFP shows the characteristics of CSCs and CSCs may be involved in the EMT of AdCC. Surprisingly, shRNA silencing of the T-box transcription factor Brachyury (also a differentiation marker) resulted in downregulation of the EMT and stem cell markers. In addition, sphere-forming ability, EMT characteristics, and tumorigenicity were simultaneously lost. Brachyury expression in clinical samples of AdCC was extremely high and closely related to EMT. This finding suggests that regulation of EMT by Brachyury in clinical AdCC may parallel that observed in vitro in this study. Conclusions The use of a single cell line is a limitation of this study. However, parallel data from in vitro and clinical samples suggest the possibility that EMT is directly linked to CSCs and that Brachyury is a regulator of EMT and CSCs.</p

Search for intermediate-mass black hole binaries in the third observing run of Advanced LIGO and Advanced Virgo

International audienceIntermediate-mass black holes (IMBHs) span the approximate mass range 100−105 M⊙, between black holes (BHs) that formed by stellar collapse and the supermassive BHs at the centers of galaxies. Mergers of IMBH binaries are the most energetic gravitational-wave sources accessible by the terrestrial detector network. Searches of the first two observing runs of Advanced LIGO and Advanced Virgo did not yield any significant IMBH binary signals. In the third observing run (O3), the increased network sensitivity enabled the detection of GW190521, a signal consistent with a binary merger of mass ∼150 M⊙ providing direct evidence of IMBH formation. Here, we report on a dedicated search of O3 data for further IMBH binary mergers, combining both modeled (matched filter) and model-independent search methods. We find some marginal candidates, but none are sufficiently significant to indicate detection of further IMBH mergers. We quantify the sensitivity of the individual search methods and of the combined search using a suite of IMBH binary signals obtained via numerical relativity, including the effects of spins misaligned with the binary orbital axis, and present the resulting upper limits on astrophysical merger rates. Our most stringent limit is for equal mass and aligned spin BH binary of total mass 200 M⊙ and effective aligned spin 0.8 at 0.056 Gpc−3 yr−1 (90% confidence), a factor of 3.5 more constraining than previous LIGO-Virgo limits. We also update the estimated rate of mergers similar to GW190521 to 0.08 Gpc−3 yr−1.Key words: gravitational waves / stars: black holes / black hole physicsCorresponding author: W. Del Pozzo, e-mail: [email protected]† Deceased, August 2020