Generation and physiological characterization of genome-edited Nicotiana benthamiana plants containing zeaxanthin as the only leaf xanthophyll.

MAIN CONCLUSION: Simultaneous genome editing of the two homeologous LCYe and ZEP genes of Nicotiana benthamiana results in plants in which all xanthophylls are replaced by zeaxanthin. Plant carotenoids act both as photoreceptors and photoprotectants in photosynthesis and as precursors of apocarotenoids, which include signaling molecules such as abscisic acid (ABA). As dietary components, the xanthophylls lutein and zeaxanthin have photoprotective functions in the human macula. We developed transient and stable combinatorial genome editing methods, followed by direct LC-MS screening for zeaxanthin accumulation, for the simultaneous genome editing of the two homeologous Lycopene Epsilon Cyclase (LCYe) and the two Zeaxanthin Epoxidase (ZEP) genes present in the allopolyploid Nicotiana benthamiana genome. Editing of the four genes resulted in plants in which all leaf xanthophylls were substituted by zeaxanthin, but with different ABA levels and growth habits, depending on the severity of the ZEP1 mutation. In high-zeaxanthin lines, the abundance of the major photosystem II antenna LHCII was reduced with respect to wild-type plants and the LHCII trimeric state became unstable upon thylakoid solubilization. Consistent with the depletion in LHCII, edited plants underwent a compensatory increase in PSII/PSI ratios and a loss of the large-size PSII supercomplexes, while the level of PSI-LHCI supercomplex was unaffected. Reduced activity of the photoprotective mechanism NPQ was shown in high-zeaxanthin plants, while PSII photoinhibition was similar for all genotypes upon exposure to excess light, consistent with the antioxidant and photoprotective role of zeaxanthin in vivo.

Analysis of state 1-state 2 transitions by genome editing and complementation reveals a quenching component independent from the formation of PSI-LHCI-LHCII supercomplex in Arabidopsis thaliana.

BACKGROUND: The light-harvesting antennae of photosystem (PS) I and PSII are pigment-protein complexes responsible of the initial steps of sunlight conversion into chemical energy. In natural environments plants are constantly confronted with the variability of the photosynthetically active light spectrum. PSII and PSI operate in series but have different optimal excitation wavelengths. The prompt adjustment of light absorption by photosystems is thus crucial to ensure efficient electron flow needed to sustain downstream carbon fixing reactions. Fast structural rearrangements equilibrate the partition of excitation pressure between PSII and PSI following the enrichment in the red (PSII-favoring) or far-red (PSI-favoring) spectra. Redox imbalances trigger state transitions (ST), a photoacclimation mechanism which involves the reversible phosphorylation/dephosphorylation of light harvesting complex II (LHCII) proteins by the antagonistic activities of the State Transition 7 (STN7) kinase/TAP38 phosphatase enzyme pair. During ST, a mobile PSII antenna pool associates with PSI increasing its absorption cross section. LHCII consists of assorted trimeric assemblies of Lhcb1, Lhcb2 and Lhcb3 protein isoforms (LHCII), several being substrates of STN7. However, the precise roles of Lhcb phosphorylation during ST remain largely elusive. RESULTS: We inactivated the complete Lhcb1 and Lhcb2 gene clades in Arabidopsis thaliana and reintroduced either wild type Lhcb1.3 and Lhcb2.1 isoforms, respectively, or versions lacking N-terminal phosphorylatable residues proposed to mediate state transitions. While the substitution of Lhcb2.1 Thr-40 prevented the formation of the PSI-LHCI-LHCII complex, replacement of Lhcb1.3 Thr-38 did not affect the formation of this supercomplex, nor did influence the amplitude or kinetics of PSII fluorescence quenching upon state 1-state 2 transition. CONCLUSIONS: Phosphorylation of Lhcb2 Thr-40 by STN7 alone accounts for ≈ 60% of PSII fluorescence quenching during state transitions. Instead, the presence of Thr-38 phosphosite in Lhcb1.3 was not required for the formation of the PSI-LHCI-LHCII supercomplex nor for re-equilibration of the plastoquinone redox state. The Lhcb2 phosphomutant was still capable of ≈ 40% residual fluorescence quenching, implying that a yet uncharacterized, STN7-dependent, component of state transitions, which is unrelated to Lhcb2 Thr-40 phosphorylation and to the formation of the PSI-LHCI-LHCII supercomplex, contributes to the equilibration of the PSI/PSII excitation pressure upon plastoquinone over-reduction.

A paler shade of green: engineering cellular chlorophyll content to enhance photosynthesis in crowded environments.

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.

Assessing photoprotective functions of carotenoids in photosynthetic systems of plants and green algae.

Carotenes and xanthophylls act as photoreceptors in the photosystems of plants and algae by absorbing light energy which drives photosynthetic electron transport. Moreover, these carotenoid pigments protect chloroplasts from excess light and from reactive species generated during oxygenic photosynthesis. These pigments share similar spectral properties, a feature which contrasts with the extreme level of conservation of their relative composition and abundance in leaves across taxa. Such a conservation through evolution suggested each carotenoid species had a peculiar role, which indeed has been investigated by different approaches. These studies included the purification of individual carotenoid-binding proteins and their characterization in vitro. In a complementary approach, plant and algal mutants devoid of selected carotenoid species have been produced. The physiological characterization of these mutants revealed that the integrated contributions of all carotenoid species provide the most efficient response to photooxidative stress. In this chapter, we provide step-by-step guides for characterizing the in vivo antioxidant activity of carotenoids in plants and green algae, and methods for quantifying the effect of photooxidative stress in genotypes with altered carotenoid composition or impaired defense mechanisms.

Loss of a single chlorophyll in CP29 triggers re-organization of the Photosystem II supramolecular assembly.

In land plants, both efficient light capture and photoprotective dissipation of chlorophyll excited states in excess require proper assembly of Photosystem II supercomplexes PSII-LHCs. These include a dimeric core moiety and a peripheral antenna system made of trimeric LHCII proteins connected to the core through monomeric LHC subunits. Regulation of light harvesting involves re-organization of the PSII supercomplex, including dissociation of its LHCII-CP24-CP29 domain under excess light. The Chl a603-a609-a616 chromophore cluster within CP29 was recently identified as responsible for the fast component of Non-Photochemical Quenching of chlorophyll fluorescence. Here, we pinpointed a chlorophyll-protein domain of CP29 involved in the macro-organization of PSII-LHCs. By complementing an Arabidopsis knock-out mutant with CP29 sequences deleted in the residue binding chlorophyll b614/b3-binding, we found that the site is promiscuous for chlorophyll a and b. By plotting NPQ amplitude vs. CP29 content we observed that quenching activity was significantly reduced in mutants compared to the wild type. Analysis of pigment-binding supercomplexes showed that the missing Chl did hamper the assembly of PSII-LHCs supercomplexes, while observation by electron microscopy of grana membranes highlighted the PSII particles were organized in two-dimensional arrays in mutant grana partitions. As an effect of such array formation electron transport rate between QA and QB reduced, likely due to restricted plastoquinone diffusion. We conclude that chlorophyll b614, rather being part of pigment cluster responsible for quenching, is needed to maintain full rate of electron flow in the thylakoids by controlling protein-protein interactions between PSII units in grana partitions.

High Carotenoid Mutants of Chlorella vulgaris Show Enhanced Biomass Yield under High Irradiance.

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.

A microalgal-based preparation with synergistic cellulolytic and detoxifying action towards chemical-treated lignocellulose.

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 ß-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.

Expression of a Hyperthermophilic Cellobiohydrolase in Transgenic Nicotiana tabacum by Protein Storage Vacuole Targeting.

Plant expression of microbial Cell Wall Degrading Enzymes (CWDEs) is a valuable strategy to produce industrial enzymes at affordable cost. Unfortunately, the constitutive expression of CWDEs may affect plant fitness to variable extents, including developmental alterations, sterility and even lethality. In order to explore novel strategies for expressing CWDEs in crops, the cellobiohydrolase CBM3GH5, from the hyperthermophilic bacterium Caldicellulosiruptor saccharolyticus, was constitutively expressed in N. tabacum by targeting the enzyme both to the apoplast and to the protein storage vacuole. The apoplast targeting failed to isolate plants expressing the recombinant enzyme despite a large number of transformants being screened. On the opposite side, the targeting of the cellobiohydrolase to the protein storage vacuole led to several transgenic lines expressing CBM3GH5, with an enzyme yield of up to 0.08 mg g DW-1 (1.67 Units g DW-1) in the mature leaf tissue. The analysis of CBM3GH5 activity revealed that the enzyme accumulated in different plant organs in a developmental-dependent manner, with the highest abundance in mature leaves and roots, followed by seeds, stems and leaf ribs. Notably, both leaves and stems from transgenic plants were characterized by an improved temperature-dependent saccharification profile.

Identification of a pigment cluster catalysing fast photoprotective quenching response in CP29.

Non-photochemical quenching is the photoprotective heat dissipation of chlorophyll-excited states. In higher plants, two quenching sites are located in trimeric LHCII and monomeric CP29 proteins. Catalysis of dissipative reactions requires interactions between chromophores, either carotenoid, chlorophyll or both. We identified CP29 protein domains involved in quenching by complementing an Arabidopsis deletion mutant with sequences deleted in pigment-binding or pH-sensitive sites. Acidic residues exposed to the thylakoid lumen were found not essential for activation of thermal dissipation in vivo. Chlorophylls a603 (a5) and a616 were identified as components of the catalytic pigment cluster responsible for quenching reaction(s), in addition to xanthophyll L2 and chlorophyll a609 (b5). We suggest that a conformational change induced by acidification in PsbS is transduced to CP29, thus bringing chlorophylls a603, a609 and a616 into close contact and activating a dissipative channel. Consistently, mutations on putative protonatable residues, exposed to the thylakoid lumen and previously suggested to regulate xanthophyll exchange at binding site L2, did not affect quenching efficiency.

Combined resistance to oxidative stress and reduced antenna size enhance light-to-biomass conversion efficiency in Chlorella vulgaris cultures.

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.