Summary: In natural ecosystems, plants compete for space, nutrients and light. The optically dense canopies limit the penetration of photosynthetically active radiation and light often becomes a growth‐limiting factor for the understory. The reduced availability of photons in the lower leaf layers is also a major constraint for yield potential in canopies of crop monocultures. Traditionally, crop breeding has selected traits related to plant architecture and nutrient assimilation rather than light use efficiency. Leaf optical density is primarily determined by tissue morphology and by the foliar concentration of photosynthetic pigments (chlorophylls and carotenoids). Most pigment molecules are bound to light‐harvesting antenna proteins in the chloroplast thylakoid membranes, where they serve photon capture and excitation energy transfer toward reaction centers of photosystems. Engineering the abundance and composition of antenna proteins has been suggested as a strategy to improve light distribution within canopies and reduce the gap between theoretical and field productivity. Since the assembly of the photosynthetic antennas relies on several coordinated biological processes, many genetic targets are available for modulating cellular chlorophyll levels. In this review, we outline the rationale behind the advantages of developing pale green phenotypes and describe possible approaches toward engineering light‐harvesting systems.

Background: Microalgae are efficient producers of lipid-rich biomass, making them a key component in developing a sustainable energy source, and an alternative to fossil fuels. Chlorella species are of special interest because of their fast growth rate in photobioreactors. However, biological constraints still cast a significant gap between the high cost of biofuel and cheap oil, thus hampering perspective of producing CO₂-neutral biofuels. A key issue is the inefficient use of light caused by its uneven distribution in the culture that generates photoinhibition of the surface-exposed cells and darkening of the inner layers. Efficient biofuel production, thus, requires domestication, including traits which reduce optical density of cultures and enhance photoprotection. Results: We applied two steps of mutagenesis and phenotypic selection to the microalga Chlorella vulgaris. First, a pale-green mutant (PG-14) was selected, with a 50% reduction of both chlorophyll content per cell and LHCII complement per PSII, with respect to WT. PG-14 showed a 30% increased photon conversion into biomass efficiency vs. WT. A second step of mutagenesis of PG-14, followed by selection for higher tolerance to Rose Bengal, led to the isolation of pale-green genotypes, exhibiting higher resistance to singlet oxygen (strains SOR). Growth in photobioreactors under high light conditions showed an enhanced biomass production of SOR strains with respect to PG-14. When compared to WT strain, biomass yield of the pale green + sor genotype was enhanced by 68%. Conclusions: Domestication of microalgae like Chlorella vulgaris, by optimizing both light distribution and ROS resistance, yielded an enhanced carbon assimilation rate in photobioreactor.

Microalgae represent a carbon-neutral source of bulk biomass, for extraction of high-value compounds and production of renewable fuels. Due to their high metabolic activity and reproduction rates, species of the genus Chlorella are highly productive when cultivated in photobioreactors. However, wild-type strains show biological limitations making algal bioproducts expensive compared to those extracted from other feedstocks. Such constraints include inhomogeneous light distribution due to high optical density of the culture, and photoinhibition of the surface-exposed cells. Thus, the domestication of algal strains for industry makes it increasingly important to select traits aimed at enhancing light-use efficiency while withstanding excess light stress. Carotenoids have a crucial role in protecting against photooxidative damage and, thus, represent a promising target for algal domestication. We applied chemical mutagenesis to Chlorella vulgaris and selected for enhanced tolerance to the carotenoid biosynthesis inhibitor norflurazon. The NFR (norflurazon-resistant) strains showed an increased carotenoid pool size and enhanced tolerance towards photooxidative stress. Growth under excess light revealed an improved carbon assimilation rate of NFR strains with respect to WT. We conclude that domestication of Chlorella vulgaris, by optimizing both carotenoid/chlorophyll ratio and resistance to photooxidative stress, boosted light-to-biomass conversion efficiency under high light conditions typical of photobioreactors. Comparison with strains previously reported for enhanced tolerance to singlet oxygen, reveals that ROS resistance in Chlorella is promoted by at least two independent mechanisms, only one of which is carotenoid-dependent.

Summary: High‐temperature bioconversion of lignocellulose into fermentable sugars has drawn attention for efficient production of renewable chemicals and biofuels, because competing microbial activities are inhibited at elevated temperatures and thermostable cell wall degrading enzymes are superior to mesophilic enzymes. Here, we report on the development of a platform to produce four different thermostable cell wall degrading enzymes in the chloroplast of Chlamydomonas reinhardtii. The enzyme blend was composed of the cellobiohydrolase CBM3GH5 from C. saccharolyticus, the β‐glucosidase celB from P. furiosus, the endoglucanase B and the endoxylanase XynA from T. neapolitana. In addition, transplastomic microalgae were engineered for the expression of phosphite dehydrogenase D from Pseudomonas stutzeri, allowing for growth in non‐axenic media by selective phosphite nutrition. The cellulolytic blend composed of the glycoside hydrolase (GH) domain GH12/GH5/GH1 allowed the conversion of alkaline‐treated lignocellulose into glucose with efficiencies ranging from 14% to 17% upon 48h of reaction and an enzyme loading of 0.05% (w/w). Hydrolysates from treated cellulosic materials with extracts of transgenic microalgae boosted both the biogas production by methanogenic bacteria and the mixotrophic growth of the oleaginous microalga Chlorella vulgaris. Notably, microalgal treatment suppressed the detrimental effect of inhibitory by‐products released from the alkaline treatment of biomass, thus allowing for efficient assimilation of lignocellulose‐derived sugars by C. vulgaris under mixotrophic growth.

Summary: Higher plants Photosystem I absorbs far‐red light, enriched under vegetation canopies, through long‐wavelength Chls to enhance photon capture. Far‐red absorption originates from Chl pairs within the Lhca3 and Lhca4 subunits of the LHCI antenna, known as the ‘red cluster’, including Chls a 603 and a 609. We used reverse genetics to produce an Arabidopsis mutant devoid of red‐shifted absorption, and we obtained high‐resolution cryogenic electron microscopy structures of PSI‐LHCI complexes from both wild‐type and mutant plants. Computed excitonic coupling values suggested contributions from additional nearby pigment molecules, namely Chl a 615 and violaxanthin in the L2 site, to far‐red absorption. We investigated the structural determinants of far‐red absorption by producing further Arabidopsis transgenic lines and analyzed the spectroscopic effects of mutations targeting these chromophores. The two structures solved were used for quantum mechanics calculations, revealing that excitonic interactions alone cannot explain far‐red absorption, while charge transfer states were needed for accurate spectral simulations. Our findings demonstrate that the molecular mechanisms of light‐harvesting under shaded conditions rely on very precise tuning of chromophore interactions, whose understanding is crucial for designing light‐harvesting complexes with engineered absorption spectra.