Context-dependent antisense transcription from a neighboring gene interferes with the expression of mNeonGreen as a functional in vivo fluorescent reporter in the chloroplast of Chlamydomonas reinhardtii.

Advancing chloroplast genetic engineering in Chlamydomonas reinhardtii remains challenging, decades after its first successful transformation. This study introduces the development of a chloroplast-optimized mNeonGreen fluorescent reporter, enabling in vivo observation through a sixfold increase in fluorescence via context-aware construct engineering. Our research highlights the influence of transcriptional readthrough and antisense mRNA pairing on post-transcriptional regulation, pointing to novel strategies for optimizing heterologous gene expression. We further demonstrate the applicability of these insights using an accessible experimentation system using glass-bead transformation and reestablishment of photosynthesis using psbH mutants, focusing on the mitigation of transcriptional readthrough effects. By characterizing heterologous expression using regulatory elements such as PrrnS, 5'atpA, and 3' rbcL in a sense-transcriptional context, we further documented up to twofold improvement in fluorescence levels. Our findings contribute new tools for molecular biology research in the chloroplast and evidence fundamental gene regulation processes that could enable the development of more effective chloroplast engineering strategies. This work not only paves the way for more efficient genetic engineering of chloroplasts but also deepens our understanding of the regulatory mechanisms at play.

Copper excess detoxification is mediated by a coordinated and complementary induction of glutathione, phytochelatins and metallothioneins in the green seaweed Ulva compressa.

In order to analyze the involvement of intracellular thiol-chelators in the accumulation and detoxification of copper, the marine alga Ulva compressa was cultivated with increasing concentrations of copper such as 2.5, 5, 7.5 and 10 µM for up to 12 d, and the amount of intracellular copper, glutathione (GSH), phytochelatins (PCs) and transcripts encoding three metallothioneins (MTs) were determined. Over this exposure period and concentration range there was a linear correlation between intracellular copper and the copper concentration in the culture medium. Increases in GSH concentrations occurred mainly between days 1 and 3 and at lower concentrations of copper (2.5 and 5 µM). The level of PCs, and particularly PC2, increased from day 1 of exposure mainly at higher concentrations of copper (7.5 and 10 µM). The levels of transcripts encoding MT7 increased at day 3, whereas those of MT3 and MT6 increased between days 9-12, mainly at higher concentrations of copper. Thus in U. compressa, the initial responses to increasing intracellular copper concentrations are increases in GSH and PCs that are followed by higher levels of MTs expression, suggesting that thiol-containing peptides and proteins may participate in copper accumulation and detoxification responding in a coordinated and complementary manner. In addition, the alga was cultivated with 10 µM copper for 5 d and transferred to synthetic seawater with no copper and cultivated for 3 d. The release of copper from cells to culture medium was observed and accompanied by a similar nanomolar amount of GSH; no PCs or small proteins were detected. These results could suggest that a component of the detoxification mechanism also involves the release of copper and GSH to the extracellular medium.

Copper-induced overexpression of genes encoding antioxidant system enzymes and metallothioneins involve the activation of CaMs, CDPKs and MEK1/2 in the marine alga Ulva compressa.

Transcriptomic analyses were performed in the green macroalga Ulva compressa cultivated with 10µM copper for 24h. Nucleotide sequences encoding antioxidant enzymes, ascorbate peroxidase (ap), dehydroascorbate reductase (dhar) and glutathione reductase (gr), enzymes involved in ascorbate (ASC) synthesis l-galactose dehydrogenase (l-gdh) and l-galactono lactone dehydrogenase (l-gldh), in glutathione (GSH) synthesis, γ-glutamate-cysteine ligase (γ-gcl) and glutathione synthase (gs), and metal-chelating proteins metallothioneins (mt) were identified. Amino acid sequences encoded by transcripts identified in U. compressa corresponding to antioxidant system enzymes showed homology mainly to plant and green alga enzymes but those corresponding to MTs displayed homology to animal and plant MTs. Level of transcripts encoding the latter proteins were quantified in the alga cultivated with 10µM copper for 0-12 days. Transcripts encoding enzymes of the antioxidant system increased with maximal levels at day 7, 9 or 12, and for MTs at day 3, 7 or 12. In addition, the involvement of calmodulins (CaMs), calcium-dependent protein kinases (CDPKs), and the mitogen-activated protein kinase kinase (MEK1/2) in the increase of the level of the latter transcripts was analyzed using inhibitors. Transcript levels decreased with inhibitors of CaMs, CDPKs and MEK1/2. Thus, copper induces overexpression of genes encoding antioxidant enzymes, enzymes involved in ASC and GSH syntheses and MTs. The increase in transcript levels may involve the activation of CaMs, CDPKs and MEK1/2 in U. compressa.