Transient expression in Nicotiana benthamiana for rapid functional analysis of genes involved in non-photochemical quenching and carotenoid biosynthesis.

Plants must switch rapidly between light harvesting and photoprotection in response to environmental fluctuations in light intensity. This switch can lead to losses in absorbed energy usage, as photoprotective energy dissipation mechanisms can take minutes to hours to fully relax. One possible way to improve photosynthesis is to engineer these energy dissipation mechanisms (measured as non-photochemical quenching of chlorophyll a fluorescence, NPQ) to induce and relax more quickly, resulting in smaller losses under dynamic light conditions. Previous studies aimed at understanding the enzymes involved in the regulation of NPQ have relied primarily on labor-intensive and time-consuming generation of stable transgenic lines and mutant populations - approaches limited to organisms amenable to genetic manipulation and mapping. To enable rapid functional testing of NPQ-related genes from diverse organisms, we performed Agrobacterium tumefaciens-mediated transient expression assays in Nicotiana benthamiana to test if NPQ kinetics could be modified in fully expanded leaves. By expressing Arabidopsis thaliana genes known to be involved in NPQ, we confirmed the viability of this method for studying dynamic photosynthetic processes. Subsequently, we used naturally occurring variation in photosystem II subunit S, a modulator of NPQ in plants, to explore how differences in amino acid sequence affect NPQ capacity and kinetics. Finally, we functionally characterized four predicted carotenoid biosynthesis genes from the marine algae Nannochloropsis oceanica and Thalassiosira pseudonana and examined the effect of their expression on NPQ in N. benthamiana. This method offers a powerful alternative to traditional gene characterization methods by providing a fast and easy platform for assessing gene function in planta

pAUL: a gateway-based vector system for adaptive expression and flexible tagging of proteins in Arabidopsis.

Determination of protein function requires tools that allow its detection and/or purification. As generation of specific antibodies often is laborious and insufficient, protein tagging using epitopes that are recognized by commercially available antibodies and matrices appears more promising. Also, proper spatial and temporal expression of tagged proteins is required to prevent falsification of results. We developed a new series of binary Gateway cloning vectors named pAUL1-20 for C- and N-terminal in-frame fusion of proteins to four different tags: a single (i) HA epitope and (ii) Strep-tagIII, (iii) both epitopes combined to a double tag, and (iv) a triple tag consisting of the double tag extended by a Protein A tag possessing a 3C protease cleavage site. Expression can be driven by either the 35 S CaMV promoter or, for C-terminal fusions, promoters from genes encoding the chloroplast biogenesis factors HCF107, HCF136, or HCF173. Fusions of the four promoters to the GUS gene showed that endogenous promoter sequences are functional and drive expression more moderately and consistently throughout different transgenic lines when compared to the 35 S CaMV promoter. By testing complementation of mutations affected in chloroplast biogenesis factors HCF107 and HCF208, we found that the effect of different promoters and tags on protein function strongly depends on the protein itself. Single-step and tandem affinity purification of HCF208 via different tags confirmed the integrity of the cloned tags

One step and tandem-purification of HCF208.

<p>100 (A) or 200 µg (B, C) chlorophyll aliquots of solubilized membrane proteins were applied for purification. Aliquots of 20 µg chlorophyll from extracts and total amounts of eluates were separated by SDS-PAGE, transferred to a nitrocellulose membrane and immunodecorated with antibodies against the HA tag (Anti-HA-Peroxidase) and ATP-Synthase as a control. (A) One step purification of proteins from wild type and HCF208pAUL1 via the HA epitope and competitive elution. (B) Tandem purification of proteins from wild type and HCF208pAUL2 via Strep-tag<i>III</i> and 3xHA (C) Tandem purification of proteins from wild type and HCF208pAUL3 via ProtA tag +3C protease cleavage and Strep-tag<i>III</i>.</p

Schematic illustration of the Gateway compatible pAUL destination vector series, showing expression cassettes.

<p>(A) C-terminal fusion vectors pAUL1-16. Expression is driven by either 2x p<i>35 </i><i>S CaMV</i> or endogenous promoter sequences from <i>A. thaliana</i> (p<i>HCF107</i>, p<i>HCF136</i>, p<i>HCF173</i>): pAUL1-3 and pAUL13 carry <i>p35S CaMV</i>; pAUL4-6 and pAUL14 carry p<i>HCF107</i>; pAUL7-9 and pAUL15 carry p<i>HCF136</i>; pAUL10-12 and pAUL16 carry p<i>HCF173</i>. Protein tags are: 3xHA single tag (pAUL1, 4, 7, 10); Strep-tag<i>III</i> single tag (pAUL13-16); 3xHA/Strep-tag<i>III</i> double tag (pAUL2, 5, 8, 11); and 3xHA/Strep-tag<i>III</i>/ProtA triple tag +3C protease cleavage site (pAUL3, 6, 9, 12). (B) N-terminal fusion vectors pAUL17-20. Vectors carry coding sequences for 3xHA single tag (pAUL17); 3xHA/Strep-tag<i>III</i> double tag (pAUL18); 3xHA/Strep-tag<i>III</i>/ProtA triple tag +3C protease cleavage site (pAUL19); and Strep-tag<i>III</i> single tag (pAUL20).</p

Complementation analysis of <i>hcf208</i>and <i>hcf107.2</i> with a representative pAUL vector set.

<p>(A) Schematic illustration of promoter/cDNA/tag combinations generated for transformation of <i>hcf107.2</i> and <i>hcf208</i>. (B) Fluorometric analysis of HCF208- and HCF107 complemented plants, wild type and <i>hcf208/hcf107.2</i> mutant plants. Pseudo-color images of maximum quantum efficiency of photosystem II (Fv/Fm) are displayed for HCF107 and of photochemical quenching efficiency (qP) are displayed for HCF208. 3 independent transformants were tested for each construct. Values for each line investigated are illustrated in diagrams. (C) Western blot analysis of complemented lines, wild type and mutant plants. 50 µg of crude protein extract were loaded. Membranes were decorated with Anti-HA-Peroxidase antibody for HCF208 and HCF107; HCF107 was also visualized by an HCF107-specific antibody.</p

Oligonuleotides used for cloning of target genes.

<p>Oligonuleotides used for cloning of target genes.</p

Modulation of xanthophyll cycle impacts biomass productivity in the marine microalga Nannochloropsis

Life on earth depends on photosynthetic primary producers that exploit sunlight to fix CO2 into biomass. Approximately half of global primary production is associated with microalgae living in aquatic environments. Microalgae also represent a promising source of biomass to complement crop cultivation, and they could contribute to the development of a more sustainable bioeconomy. Photosynthetic organisms evolved multiple mechanisms involved in the regulation of photosynthesis to respond to highly variable environmental conditions. While essential to avoid photodamage, regulation of photosynthesis results in dissipation of absorbed light energy, generating a complex trade-off between protection from stress and light-use efficiency. This work investigates the impact of the xanthophyll cycle, the light-induced reversible conversion of violaxanthin into zeaxanthin, on the protection from excess light and on biomass productivity in the marine microalgae of the genus Nannochloropsis. Zeaxanthin is shown to have an essential role in protection from excess light, contributing to the induction of nonphotochemical quenching and scavenging of reactive oxygen species. On the contrary, the overexpression of zeaxanthin epoxidase enables a faster reconversion of zeaxanthin to violaxanthin that is shown to be advantageous for biomass productivity in dense cultures in photobioreactors. These results demonstrate that zeaxanthin accumulation is critical to respond to strong illumination, but it may lead to unnecessary energy losses in light-limiting conditions and accelerating its reconversion to violaxanthin provides an advantage for biomass productivity in microalgae

Characterization of promoters 2x p<i>35S CaMV</i>, p<i>HCF107</i>, p<i>HCF136</i>, and p<i>HCF173</i> fused to the <i>GUS</i> reporter gene.

<p>(A) GUS staining of 5-day-old transgenic <i>A. thaliana</i> seedlings. (B) Histochemical localization of GUS activity in leaf sections of 3-week-old transgenic <i>A. thaliana</i> plants. UE, upper epidermis; PM, palisade mesophyll; SM, spongy mesophyll; LE, lower epidermis. (C) GUS activities in transgenic <i>A. thaliana</i> lines. In each case, 10 independent transgenic lines were tested 15 or 30 days after germination. Median values are shown as black bars and indicated at the top of each column. MU, 4-methylumbelliferone.</p