Making extra room for carotenoids in plant cells: new opportunities for biofortification

[EN] Plant carotenoids are essential for photosynthesis and photoprotection and provide colors in the yellow to red range to non-photosynthetic organs such as petals and ripe fruits. They are also the precursors of biologically active molecules not only in plants (including hormones and retrograde signals) but also in animals (including retinoids such as vitamin A). A carotenoid-rich diet has been associated with improved health and cognitive capacity in humans, whereas the use of carotenoids as natural pigments is widespread in the agrofood and cosmetic industries. The nutritional and economic relevance of carotenoids has spurred a large number of biotechnological strategies to enrich plant tissues with carotenoids. Most of such approaches to alter carotenoid contents in plants have been focused on manipulating their biosynthesis or degradation, whereas improving carotenoid sink capacity in plant tissues has received much less attention. Our knowledge on the molecular mechanisms influencing carotenoid storage in plants has substantially grown in the last years, opening new opportunities for carotenoid biofortification. Here we will review these advances with a particular focus on those creating extra room for carotenoids in plant cells either by promoting the differentiation of carotenoid-sequestering structures within plastids or by transferring carotenoid production to the cytosol.We greatly thank Carmen and Pilar Torres-Montilla for their collaboration and help in the design and execution of the figures. We also thank Luca Morelli and BioRender.com for some of the images. Work in our lab is funded by Spanish grants BIO2017-84041-P and PID2020-115810GB-I00 from the Agencia Estatal de Investigacion (AEI) and 202040E299 from Consejo Superior de Investigaciones Cientificas (CSIC) to MRC. STM was supported by PhD fellowship FPU16/04054 from the Spanish Ministerio de Educacion y CulturaTorres-Montilla, S.; Rodriguez-Concepcion, M. (2021). Making extra room for carotenoids in plant cells: new opportunities for biofortification. Progress in Lipid Research. 84:1-9. https://doi.org/10.1016/j.plipres.2021.101128S198

Environmentally driven transcriptomic and metabolic changes leading to color differences in “Golden Reinders” apples

Apple is characterized by its high adaptation to diverse growing environments. However, little is still known about how different environments can regulate at the metabolic or molecular level specific apple quality traits such as the yellow fruit peel color. In this study, changes in carotenoids and chlorophylls, antioxidants as well as differences in the transcriptome were investigated by comparing the peel of “Golden Reinders” apples grown at different valley and mountain orchards. Mountain environment favored the development of yellow color, which was not caused by an enhanced accumulation of carotenoids but rather by a decrease in the chlorophyll content. The yellow phenotype was also associated to higher expression of genes related to chloroplast functions and oxidative stress. Time-course analysis over the last stages of apple development and ripening, in fruit from both locations, further revealed that the environment differentially modulated isoprenoids and phenylpropanoid metabolism and pointed out a key role for H2O2 in triggering apple peel degreening. Overall, the results presented herein provide new insights into how different environmental conditions regulate pigment and antioxidant metabolism in apple leading to noticeable differences in the apple peel colorinfo:eu-repo/semantics/publishedVersio

The intrinsic chaperone network of Arabidopsis stem cells confers protection against proteotoxic stress

The biological purpose of plant stem cells is to maintain themselves while providing new pools of differentiated cells that form organs and rejuvenate or replace damaged tissues. Protein homeostasis or proteostasis is required for cell function and viability. However, the link between proteostasis and plant stem cell identity remains unknown. In contrast to their differentiated counterparts, we find that root stem cells can prevent the accumulation of aggregated proteins even under proteotoxic stress conditions such as heat stress or proteasome inhibition. Notably, root stem cells exhibit enhanced expression of distinct chaperones that maintain proteome integrity. Particularly, intrinsic high levels of the T-complex protein-1 ring complex/chaperonin containing TCP1 (TRiC/CCT) complex determine stem cell maintenance and their remarkable ability to suppress protein aggregation. Overexpression of CCT8, a key activator of TRiC/CCT assembly, is sufficient to ameliorate protein aggregation in differentiated cells and confer resistance to proteotoxic stress in plants. Taken together, our results indicate that enhanced proteostasis mechanisms in stem cells could be an important requirement for plants to persist under extreme environmental conditions and reach extreme long ages. Thus, proteostasis of stem cells can provide insights to design and breed plants tolerant to environmental challenges caused by the climate change

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

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

Molecular mechanisms of plastidial differentiation

Los carotenoides son isoprenoides que tanto los organismos fotosintéticos como algunos no fotosintéticos producen. Tienen funciones relacionadas con la fotosínte sis, la fotoprotección, la señalización y la pigmentación. A pesar de que los humanos no podemos sintetizarlos, los carotenoides proporcionan propiedades nutricionales muy favorables para nuestra salud, principalmente por su papel como precursores de vitamina A. Las plantas sintetizan carotenoides en los plastos a partir del geranil geranil difosfato (GGPP) que se sintetiza a partir de la ruta del metileritritol 4-fosfato (MEP). Los cromoplastos son plastos especializados en la producción y acumulación de carotenoides que normalmente se originan por la diferenciación a partir de cloro plastos, pero el mecanismo de diferenciación no ha sido identificado. La transición cloroplasto-cromoplasto solo ocurre en algunos órganos de algunas especies, nor malmente en paralelo con otros muchos procesos como todos aquellos relacionados con la maduración del fruto. El objetivo de esta tesis doctoral ha sido la caracteri zación de un sistema artificial de diferenciación cloroplasto-cromoplasto en hojas de Nicotiana benthamiana, desencadenado por la expresión transitoria del gen bacteri ano crtB, que codifica una fitoeno sintasa, el primer paso de la ruta biosintética de carotenoides. Para una mejor caracterización de nuestro sistema artificial, inicialmente nos enfocamos en hojas de N. benthamiana cuatro días después de la agroinfiltración con crtB (una vez que un fenotipo amarillo estaba plenamente establecido), y las comparamos con regiones de las mismas hojas expresando GFP como control de agroinfiltración. Las regiones que expresaban crtB acumulaban fitoeno, aumentaban el contenido de carotenoides totales, detenían su actividad fotosintética y cambia ban la morfología de sus plastos hacia una similar a cromoplastos. De hecho, este fenotipo solamente se inducía cuando crtB se localizaba en los plastos, produciendo fitoeno a partir del GGPP derivado de la ruta del MEP. El análisis de RNA-seq a las 96 horas post-infiltración (hpi) mostró unos perfiles de transcripción globales con similaridades con el sistema de maduración de fruto en tomate (en el que los cloro plastos se diferencian en cromoplastos de forma natural) pero no con el proceso de senescencia en hojas de Arabidopsis thaliana (donde los cromoplastos degeneran a gerontoplastos). Para respaldar nuestros resultados, anotamos un nuevo genoma de N. benthamiana, utilizando genes de Arabidopsis como referencia e identificando homólogos para diferentes familias génicas. ix La segunda pare de la tesis estuvo enfocada en describir los eventos de ex presión génica durante la transición cloroplasto-cromoplasto. Un segundo exper imento de RNA-seq se llevó a cabo, abarcando ocho puntos desde las 22 a las 56 hpi. Los análisis de estos datos usando el nuevo genoma anotado de N. benthamiana y la comparación de los resultados con experimentos de RNA-seq enfocados en la diferenciación cloroplasto-cromoplasto en frutos mostró se trata de un proceso muy heterogéneo entre distintas especies vegetales, compartiendo solamente un pequeño porcentaje de sus perfiles de expresión génica. En N. benthamiana, se encontraron dos picos de cambios de expresión que correlacionan con las caídas en la actividad foto sintética: uno al principio y otro al final del proceso, con una etapa de relajación entre medias. Se observó un primer evento de represión en la expresión que afectaba a la expresión génica relacionada con el metabolismo primario y las chaperonas, proba blemente causado por el consumo de GGPP o por una respuesta ante la presencia de crtB. Esta represión fue seguida de una inducción en la expresión correlacionada con la acumulación de fitoeno y el descenso de la fotosíntesis, que afectaba a la expresión de genes de la ruta del MEP y genes relacionados con el ácido jasmónico (JA). El pico final de cambio en la expresión génica ocurrió simultáneamente con el segundo y definitivo evento de reducción fotosintética y el incremento de carotenoides totales. Los niveles hormonales de JA incrementaron en este último evento, mientras que los genes de la biosíntesis de carotenoides no estuvieron afectados durante el exper imento. Además de contribuir a un mejor entendimiento de la cromoplastogénesis, estos resultados proveen de anotaciones realizadas in-silico para futuros estudios en N. benthamiana.Carotenoids are isoprenoids produced by all photosynthetic and some non photosynthetic organisms. They have functions related to photosynthesis, photo protection, pigmentation and signaling. Despite humans cannot synthesize them, carotenoids provide important nutritional and health-promoting properties, mainly as vitamin A precursors. Plants synthesize carotenoids in plastids from geranylger anyl diphosphate (GGPP) synthesized by the methylerythritol 4-phosphate (MEP) pathway. Chromoplasts are plastids specialized in carotenoid production and ac cumulation which are usually differentiated from pre-existing chloroplasts, but the differentiation mechanism remains unknown. Chloroplast-to-chromoplast transi tion only occurs in some organs of some plant species, normally in parallel to many other processes such as those related to fruit ripening. The goal of this thesis work has been to characterize an artificial system of chloroplast-to-chromoplast differen tiation in leaves of Nicotiana benthamiana, triggered by the transient expression of the bacterial crtB gene which encode a phytoene synthase, the first step of the carotenoid biosynthetic pathway. To better characterize our artificial system, we initially focused on N. benthami ana leaves four days after the agroinfiltration with crtB (once a yellow phenotype was fully established), and compared them to regions of the same leaves express ing GFP as a control. crtB-expressing leaves accumulated phytoene, increased total carotenoid content, stopped their photosynthetic activity and changed the morphol ogy of their chloroplats to chromoplast-like plastids. Strikingly, this phenotype was only induced when crtB localized in plastids, producing phytoene from GGPP de rived from the MEP pathway. RNA-seq at 96 hours post-infiltration (hpi) showed global transcription profiles with similarities to the tomato fruit ripening system (where chloroplasts naturally differentiate into chromoplasts) but not to the leaf senescence process in Arabidopsis thaliana (where chloroplasts degenerate to become gerontoplasts). To support our results, a new genome of N. benthamiana was anno tated, using Arabidopsis genes as reference and identifying homologs for different gene families. The second part of the thesis was focused on describing gene expression events during chloroplast-to-chromoplast transition. A second RNA-seq experiment was carried out encompassing eight time-points, from 22 to 56 hpi. Analysis of these data using the newly annotated N. benthamiana genome and comparison of the results with RNA-seq experiments covering chloroplast-to-chromoplast differentiation in vii fruits showed that this process is very heterogenous among different plant systems, sharing just a small proportion of their gene expression profiles. In N. benthamiana, two peaks were found in gene expression changes that correlated with drops in pho tosynthetic activity: one at the beginning and another at the end of the time course, with a relaxation in the middle. A first event of down-regulation affected primary metabolism and chaperone gene expression, probably caused by GGPP consump tion or as a response to crtB presence. This down-regulation was followed by an up-regulation correlated to the first event of phytoene accumulation and photosyn thesis decrease that affected MEP pathway and jasmonic acid (JA) related gene ex pression. The final peak of gene expression changes occurred simultaneously with the second and definite event of photosynthesis reduction and total carotenoid in crease. JA hormone levels increased at this last event, while carotenoid biosynthesis genes were not affected during the investigated time-course. Besides contributing to a better understanding of chromoplastogenesis, these results provide in-silico cu rated annotations for future studies in N. benthamiana.

Advances in the molecular mechanism controlling chromoplast differentiation

Trabajo presentado al Seminario del CRAG (Internal Seminar), celebrado Online el 6 de noviembre de 2020.Peer reviewe

Artificial biogenesis of chromoplasts from leaf chloroplasts

Trabajo presentado a la II Reunión Nacional sobre Carotenoides en Microorganismos, Plantas, Alimentación y Salud, celebrada en la Estación Experimental del Zaidín (EEZ-CSIC), Granada (España) del 7 al 8 de Noviembre de 2019.Peer reviewe

Artificial chromoplast biogenesis

In the present invention, we show for the first time that the supply of phytoene to feed the carotenoid pathway functions as a primary determinant of chromoplast biogenesis in green tissues. We provide evidence that exceeding a threshold level of phytoene, the first committed intermediate of the carotenoid pathway, is sufficient to induce chloroplast-to-chromoplast differentiation in leaves. The present invention thus reveals a novel aspect of carotenoid biology and additionally presents a powerful tool for biotechnological applications of the modulation of plastid identity, that could be used in the development of new biofortified crops, food or as a source of extracts for a variety of industriesPeer reviewedCRAG Centre de Recerca en Agrigenòmica, Consejo Superior de Investigaciones Científicas (España)A1 Solicitud de patente con informe sobre el estado de la técnic

Artificial chromoplast biogenesis

[EN] In the present invention, we show for the first time that the supply of phytoene to feed the carotenoid pathway functions as a primary determinant of chromoplast biogenesis in green tissues. We provide evidence that exceeding a threshold level of phytoene, the first committed intermediate of the carotenoid pathway, is sufficient to induce chloroplast-to-chromoplast differentiation in leaves. The present invention thus reveals a novel aspect of carotenoid biology and additionally presents a powerful tool for biotechnological applications of the modulation of plastid identity, that could be used in the development of new biofortified crops, food or as a source of extracts for a variety of industries[FR] Dans la présente invention, nous avons démontré que la première fois que l'alimentation en phytoène pour alimenter la voie caroténoïde fonctionne comme un déterminant primaire de la biogenèse de chromoplaste les tissus verts. L'invention fournit la preuve qu'en dépassant un niveau seuil de phytoène, le premier intermédiaire engagé de la voie caroténoïde, est suffisant pour induire une différenciation des chloroplastes en chromoplastes dans les feuilles. La présente invention révèle ainsi un nouvel aspect de la biologie des caroténoïdes et présente en outre un outil puissant pour des applications biotechnologiques de la modulation de l'identité des plastes, qui pourraient être utilisés dans le développement de nouvelles cultures bioenrichies, d'aliments ou en tant que source d'extraits pour une variété d'industriesPeer reviewedCRAG Centre de Recerca en Agrigenòmica, Consejo Superior de Investigaciones Científicas (España)A1 Solicitud de patente con informe sobre el estado de la técnic