Evolutionary recycling of light signaling components in fleshy fruits : new insights on the role of pigments to monitor ripening

Besides an essential source of energy, light provides environmental information to plants. Photosensory pathways are thought to have occurred early in plant evolution, probably at the time of the Archaeplastida ancestor, or perhaps even earlier. Manipulation of individual components of light perception and signaling networks in tomato (Solanum lycopersicum) affects the metabolism of ripening fruit at several levels. Most strikingly, recent experiments have shown that some of the molecular mechanisms originally devoted to sense and respond to environmental light cues have been re-adapted during evolution to provide plants with useful information on fruit ripening progression. In particular, the presence of chlorophylls in green fruit can strongly influence the spectral composition of the light filtered through the fruit pericarp. The concomitant changes in light quality can be perceived and transduced by phytochromes (PHYs) and PHY-interacting factors, respectively, to regulate gene expression and in turn modulate the production of carotenoids, a family of metabolites that are relevant for the final pigmentation of ripe fruits. We raise the hypothesis that the evolutionary recycling of light-signaling components to finely adjust pigmentation to the actual ripening stage of the fruit may have represented a selective advantage for primeval fleshy-fruited plants even before the extinction of dinosaur

Corrigendum : Evolutionary Recycling of Light Signaling Components in Fleshy Fruits: New Insights on the Role of Pigments to Monitor Ripening

In the original article, we neglected to include some funder information. The corrected ACKNOWLEDGMENTS section should be the following: ACKNOWLEDGMENTS We acknowledge the support of grants from EC (CarotenActors, 300862), CYTED (Ibercarot, 112RT0445), FEDER/MINECO (FPDI-2013-018882, BIO2011-23680, BIO2014-59092-P), MEC (AP2012-0189), and AGAUR (2014SGR-1434). The authors apologize for this error and state that this does not change the scientific conclusions of the article in any way. The original article has been updated

The shade avoidance syndrome in Arabidopsis : the antagonistic role of phytochrome A and B differentiates vegetation proximity and canopy shade

Light limitation caused by dense vegetation is one of the greatest threats to plant survival in natural environments. Plants detect such neighboring vegetation as a reduction in the red to far-red ratio (R:FR) of the incoming light. The low R:FR signal, perceived by phytochromes, initiates a set of responses collectively known as the shade avoidance syndrome, intended to reduce the degree of current or future shade from neighbors by overtopping such competitors or inducing flowering to ensure seed production. At the seedling stage these responses include increased hypocotyl elongation. We have systematically analyzed the Arabidopsis seedling response and the contribution of phyA and phyB to perception of decreased R:FR, at three different levels of photosynthetically active radiation. Our results show that the shade avoidance syndrome, induced by phyB deactivation, is gradually antagonized by phyA, operating through the so-called FR-High Irradiance Response, in response to high FR levels in a range that simulates plant canopy shade. The data indicate that the R:FR signal distinguishes between the presence of proximal, but non-shading, neighbors and direct foliar shade, via a intrafamily photosensory attenuation mechanism that acts to suppress excessive reversion toward skotomorphogenic development under prolonged direct vegetation shade

Tomato fruit carotenoid biosynthesis is adjusted to actual ripening progression by a light-dependent mechanism

Carotenoids are isoprenoid compounds that are essential for plants to protect the photosynthetic apparatus against excess light. They also function as health-promoting natural pigments that provide colors to ripe fruit, promoting seed dispersal by animals. Work in Arabidopsis thaliana unveiled that transcription factors of the phytochrome-interacting factor (PIF) family regulate carotenoid gene expression in response to environmental signals (i.e. light and temperature), including those created when sunlight reflects from or passes though nearby vegetation or canopy (referred to as shade). Here we show that PIFs use a virtually identical mechanism to modulate carotenoid biosynthesis during fruit ripening in tomato (Solanum lycopersicum). However, instead of integrating environmental information, PIF-mediated signaling pathways appear to fulfill a completely new function in the fruit. As tomatoes ripen, they turn from green to red due to chlorophyll breakdown and carotenoid accumulation. When sunlight passes through the flesh of green fruit, a self-shading effect within the tissue maintains high levels of PIFs that directly repress the master gene of the fruit carotenoid pathway, preventing undue production of carotenoids. This effect is attenuated as chlorophyll degrades, causing degradation of PIF proteins and boosting carotenoid biosynthesis as ripening progresses. Thus, shade signaling components may have been co-opted in tomato fruit to provide information on the actual stage of ripening (based on the pigment profile of the fruit at each moment) and thus finely coordinate fruit color change. We show how this mechanism may be manipulated to obtain carotenoid-enriched fruits.Peer ReviewedPostprint (published version

Selective pressure against horizontally acquired prokaryotic genes as a driving force of plastid evolution

Altres ajuts: del Consejo Nacional de Investigaciones Científicas y Técnicas- Argentina (CONICET) i del Programa Iberoamericano de Ciencia y Tecnología para el Desarrollo (IBERCAROT).The plastid organelle comprises a high proportion of nucleus-encoded proteins that were acquired from different prokaryotic donors via independent horizontal gene transfers following its primary endosymbiotic origin. What forces drove the targeting of these alien proteins to the plastid remains an unresolved evolutionary question. To better understand this process we screened for suitable candidate proteins to recapitulate their prokaryote-to-eukaryote transition. Here we identify the ancient horizontal transfer of a bacterial polyphenol oxidase (PPO) gene to the nuclear genome of an early land plant ancestor and infer the possible mechanism behind the plastidial localization of the encoded enzyme. Arabidopsis plants expressing PPO versions either lacking or harbouring a plastid-targeting signal allowed examining fitness consequences associated with its subcellular localization. Markedly, a deleterious effect on plant growth was highly correlated with PPO activity only when producing the non-targeted enzyme, suggesting that selection favoured the fixation of plastid-targeted protein versions. Our results reveal a possible evolutionary mechanism of how selection against heterologous genes encoding cytosolic proteins contributed in incrementing plastid proteome complexity from non-endosymbiotic gene sources, a process that may also impact mitochondrial evolution

Specific Hsp100 chaperones determine the fate of the first enzyme of the plastidial isoprenoid pathway for either refolding or degradation by the stromal Clp protease in Arabidopsis

The lifespan and activity of proteins depend on protein quality control systems formed by chaperones and proteases that ensure correct protein folding and prevent the formation of toxic aggregates. We previously found that the Arabidopsis thaliana J-protein J20 delivers inactive (misfolded) forms of the plastidial enzyme deoxyxylulose 5-phosphate synthase (DXS) to the Hsp70 chaperone for either proper folding or degradation. Here we show that the fate of Hsp70-bound DXS depends on pathways involving specific Hsp100 chaperones. Analysis of individual mutants for the four Hsp100 chaperones present in Arabidopsis chloroplasts showed increased levels of DXS proteins (but not transcripts) only in those defec- tive in ClpC1 or ClpB3. However, the accumulated enzyme was active in the clpc1 mutant but inactive in clpb3 plants. Genetic evidence indicated that ClpC chaperones might be required for the unfolding of J20-delivered DXS protein coupled to degradation by the Clp protease. By contrast, biochemical and genetic approaches confirmed that Hsp70 and ClpB3 chaperones interact to collaborate in the refolding and activation of DXS. We conclude that specific J-proteins and Hsp100 chaperones act together with Hsp70 to recognize and deliver DXS to either reactivation (via ClpB3) or removal (via ClpC1) depending on the physiological status of the plastid

Interference with Clp protease impairs carotenoid accumulation during tomato fruit ripening

Profound metabolic and structural changes are required for fleshy green fruits to ripen and become colorful and tasty. In tomato (Solanum lycopersicum), fruit ripening involves the differentiation of chromoplasts, specialized plastids that accumulate carotenoid pigments such as β-carotene (pro-vitamin A) and lycopene. Here, we explored the role of the plastidial Clp protease in chromoplast development and carotenoid accumulation. Ripening-specific silencing of one of the subunits of the Clp proteolytic complex resulted in β-carotene-enriched fruits that appeared orange instead of red when ripe. Clp-defective fruit displayed aberrant chromoplasts and up-regulated expression of nuclear genes encoding the tomato homologs of Orange (OR) and ClpB3 chaperones, most probably to deal with misfolded and aggregated proteins that could not be degraded by the Clp protease. ClpB3 and OR chaperones protect the carotenoid biosynthetic enzymes deoxyxylulose 5-phosphate synthase and phytoene synthase, respectively, from degradation, whereas OR chaperones additionally promote chromoplast differentiation by preventing the degradation of carotenoids such as β-carotene. We conclude that the Clp protease contributes to the differentiation of chloroplasts into chromoplasts during tomato fruit ripening, acting in co-ordination with specific chaperones that alleviate protein folding stress, promote enzyme stability and accumulation, and prevent carotenoid degradation

Synthetic conversion of leaf chloroplasts into carotenoid-rich plastids reveals mechanistic basis of natural chromoplast development

[EN] Plastids, the defining organelles of plant cells, undergo physiological and morphological changes to fulfill distinct biological functions. In particular, the differentiation of chloroplasts into chromoplasts results in an enhanced storage capacity for carotenoids with industrial and nutritional value such as beta-carotene (provitamin A). Here, we show that synthetically inducing a burst in the production of phytoene, the first committed intermediate of the carotenoid pathway, elicits an artificial chloroplast-to-chromoplast differentiation in leaves. Phytoene overproduction initially interferes with photosynthesis, acting as a metabolic threshold switch mechanism that weakens chloroplast identity. In a second stage, phytoene conversion into downstream carotenoids is required for the differentiation of chromoplasts, a process that involves a concurrent reprogramming of nuclear gene expression and plastid morphology for improved carotenoid storage. We hence demonstrate that loss of photosynthetic competence and enhanced production of carotenoids are not just consequences but requirements for chloroplasts to differentiate into chromoplasts.We greatly thank Jaume F. Martinez-Garcia and Ralf Welsch for fruitful discussions on the manuscript; Ralf Welsch and Li Li for providing seeds of the Arabidopsis ccd1 ccd4 and ator atorl mutants, respectively; Juan Jose Lopez-Moya and Maria Luisa Domingo-Calap for the gift of the HcProWMV-pGWB702 vector; and M. Rosa Rodriguez-Goberna for excellent technical support. The help of Marti Bernardo, Fidel Lozano, Lidia Jimenez, and members of the CRAG core facilities is also appreciated. This work was funded by the European Regional Development Fund and Spanish Agencia Estatal de Investigacion Grants BIO2017-84041-P, BIO2017-83184-R, BIO2017-90877-REDT, BES-2017-080652, and AGL2017-85563-C2-1-R; Ministry of Education, Culture and Sports Grants AP2012-3751 and FPU16/04054; and Generalitat de Catalunya Grant 2017SGR-710. We also thank the financial support of the European Union's Horizon 2020 (EU-H2020) COST Action CA15136 (EuroCaroten) and Marie S. Curie Action (MSCA) 753301 (Arcatom), the Severo Ochoa Programme for Centres of Excellence in R&D 2016-2019 Grant SEV-2015-0533 and the Generalitat de Catalunya CERCA Programme (to CRAG). B.L. is supported by grants from the CSIRO Synthetic Biology Future Science Platform and Macquarie University. L.M. is supported by La Caixa Foundation PhD INPhINIT Fellowship LCF/BQ/IN18/11660004, which received funding from the EU-H2020 through MSCA Grant 713673. A.R.F. is supported by Deutsche Forschungsgemeinschaft Grant DFG TRR 175.Llorente, B.; Torres-Montilla, S.; Morelli, L.; Florez-Sarasa, I.; Matus, JT.; Ezquerro, M.; D'andrea, L.... (2020). Synthetic conversion of leaf chloroplasts into carotenoid-rich plastids reveals mechanistic basis of natural chromoplast development. Proceedings of the National Academy of Sciences of the United States of America (Online). 117(35):21796-21803. https://doi.org/10.1073/pnas.2004405117S21796218031173

An Engineered extraplastidial pathway for carotenoid biofortification of leaves

Carotenoids are lipophilic plastidial isoprenoids highly valued as nutrients and natural pigments. A correct balance of chlorophylls and carotenoids is required for photosynthesis and therefore highly regulated, making carotenoid enrichment of green tissues challenging. Here we show that leaf carotenoid levels can be boosted through engineering their biosynthesis outside the chloroplast. Transient expression experiments in Nicotiana benthamiana leaves indicated that high extraplastidial production of carotenoids requires an enhanced supply of their isoprenoid precursors in the cytosol, which was achieved using a deregulated form of the main ratedetermining enzyme of the mevalonic acid (MVA) pathway. Constructs encoding bacterial enzymes were used to convert these MVA-derived precursors into carotenoid biosynthetic intermediates that do not normally accumulate in leaves, such as phytoene and lycopene. Cytosolic versions of these enzymes produced extraplastidial carotenoids at levels similar to those of total endogenous (i.e. chloroplast) carotenoids. Strategies to enhance the development of endomembrane structures and lipid bodies as potential extraplastidial carotenoid storage systems were not successful to further increase carotenoid contents. Phytoene was found to be more bioaccessible when accumulated outside plastids, whereas lycopene formed cytosolic crystalloids very similar to those found in the chromoplasts of ripe tomatoes. This extraplastidial production of phytoene and lycopene led to an increased antioxidant capacity of leaves. Finally, we demonstrate that our system can be adapted for the biofortification of leafy vegetables such as lettuce