PERSPECTIVE published: 05 September 2019 doi: 10.3389/fpls.2019.01071 Manipulation of Plastidial Protein Quality Control Components as a New Strategy to Improve Carotenoid Contents in Tomato Fruit Lucio D’Andrea 1† and Manuel Rodriguez-Concepcion 1,2* 1Centre for Research in Agricultural Genomics (CRAG) CSIC-IRTA-UAB-UB, Barcelona, Spain, 2 Consejo Superior de Investigaciones Científicas (CSIC), Barcelona, Spain Edited by: Kevin Davies, The New Zealand Institute for Plant & Carotenoids such as β-carotene (pro-vitamin A) and lycopene accumulate at high levels Food Research Ltd, during tomato (Solanum lycopersicum L.) fruit ripening, contributing to the characteristic New Zealand color and nutritional quality of ripe tomatoes. Besides their role as pigments in chromoplast- Reviewed by: Irini Pateraki, harboring tissues such as ripe fruits, carotenoids are important for photosynthesis University of Copenhagen, and photoprotection in the chloroplasts of photosynthetic tissues. Interestingly, recent Denmark work in Arabidopsis thaliana (L.) Heynh. has unveiled a critical role of chloroplast Marcello Salvatore Lenucci, University of Salento, Italy protein quality control components in the regulation of carotenoid biosynthesis. The Changjie Xu, accumulation (i.e. degradation rate) and activity (i.e. folding status) of phytoene synthase Zhejiang University, China Hiroshi Ezura, (PSY) and other Arabidopsis biosynthetic enzymes is modulated by chaperones such University of Tsukuba, Japan as Orange (OR) and Hsp70 in coordination with the stromal Clp protease complex. OR *Correspondence: and Clp protease were recently shown to also influence PSY stability and carotenoid Manuel Rodriguez-Concepcion accumulation in tomato. Here we show how manipulating the levels of plastid-localized manuel.rodriguez@cragenomica.es Hsp70 in transgenic tomato plants can also impact the accumulation of carotenoids Present address: † Max-Planck-Institut für Molekulare in ripe fruit. The resulting carotenoid profile and chromoplast ultrastructure, however, Pflanzenphysiologie, are different from those obtained in tomatoes from transgenic lines with increased OR Potsdam-Golm, Germany activity. These results suggest that different chaperone families target different processes Specialty section: related to carotenoid metabolism and accumulation during tomato ripening. We further This article was submitted to discuss other possible targets for future manipulation in tomato based on the knowledge Plant Metabolism and acquired in Arabidopsis. Chemodiversity, a section of the journal Keywords: carotenoids, Clp protease, chaperones, Hsp70, protein quality control, tomato Frontiers in Plant Science Received: 18 May 2019 Accepted: 07 August 2019 INTRODUCTION Published: 05 September 2019 Citation: Carotenoids are a group of isoprenoid molecules produced by all photosynthetic organisms and D’Andrea L and some non-photosynthetic bacteria and fungi (Rodriguez-Concepcion et al., 2018). In plants they Rodriguez-Concepcion M (2019) are essential as photoprotective pigments for photosynthesis and as precursors for the production Manipulation of Plastidial Protein of hormones (abscisic acid, strigolactones) and other signaling molecules. They also provide colors Quality Control Components as a New Strategy to Improve Carotenoid and aromas to many flowers and ripe fruits. Dietary carotenoids and their cleavage products Contents in Tomato Fruit. contribute to human and animal nutrition at many different levels. These features translate into Front. Plant Sci. 10:1071. huge economic value for the industry (e.g. as natural pigments) and major nutritional relevance doi: 10.3389/fpls.2019.01071 for consumers (Rodriguez-Concepcion et al., 2018; Ilahy et al., 2019). In plants, carotenoids are Frontiers in Plant Science | www.frontiersin.org 1 September 2019 | Volume 10 | Article 1071 D’Andrea and Rodríguez-Concepción Tomato Chaperones, Proteases and Carotenoids synthesized and accumulated in plastids. They derive from efficiently promoted by OR-His, was associated with increased geranylgeranyl diphosphate (GGPP), generated from prenyl expression of other chaperone-encoding genes (Yazdani et al., diphosphate substrates synthetized by the methylerythritol 2019). Levels of plastidial chaperones (often referred to as heat- 4-phosphate (MEP) pathway (Figure 1A). The metabolic flux shock proteins) actually increase during natural fruit ripening through the MEP pathway and hence the supply of isoprenoid in tomato (Figures 1B, C) and several other plant systems, precursors for carotenoid biosynthesis is mainly limited by the presumably to deal with proteome changes and protein folding activity of the first enzyme, deoxyxylulose 5-phosphate synthase stress resulting from the chromoplast differentiation process (DXS). Another major regulatory checkpoint is the production (Bonk et al., 1996; Neta-Sharir et al., 2005; Barsan et al., 2012; of phytoene from GGPP catalyzed by phytoene synthase (PSY) Suzuki et al., 2015; Shukla et al., 2017; D’Andrea et al., 2018). in the first committed step of the carotenoid pathway (Camagna Interestingly, constitutive overexpression of one of such et al., 2019). The subsequent action of desaturases and isomerases chaperones, small heat shock protein 21 (Hsp21), appeared to transforms uncolored phytoene into red-colored lycopene. promote the conversion of chloroplasts to chromoplasts during Cyclization of lycopene ends branches the pathway to produce tomato fruit ripening (Neta-Sharir et al., 2005). β-carotene (pro-vitamin A) and derived β,β-xanthophylls Besides contributing to proper protein folding, assembly, and (including zeaxanthin and violaxanthin) in one branch and β,ε- subcellular targeting, chaperones, together with proteases, are xanthophylls (e.g. lutein) in the other (Figure 1A). central components of the protein quality control (PQC) systems Besides biosynthesis (and degradation) rates, carotenoid that ensure the stabilization, repair, or degradation of proteins storage capacity is a major determinant of carotenoid contents in that lose their native conformation and activity after stress plant tissues (Llorente et al., 2017; Sun et al., 2018). Chromoplasts episodes such as excess light, temperature peaks, oxidative stress are plastids specialized in the accumulation of high carotenoid or nutrient starvation (Trösch et al,. 2015; Majsec et al., 2017). levels. Ultrastructure of chromoplasts is mainly determined They hence promote correct protein folding, remove irreversibly by their carotenoid profiles, which widely differ among plant damaged proteins, and ensure plastid proteostasis. In agreement species, organs, and developmental stages (Sun et al., 2018). with the conclusion that PQC components are major regulators During tomato (Solanum lycopersicum L.) fruit ripening, of fruit carotenoid accumulation and chromoplast differentiation chromoplasts differentiate from chloroplasts as chlorophylls in tomato, down-regulation of the activity of the Clp proteolytic degrade and carotenoids accumulate to change the fruit color complex (the main stromal protease) resulted in defective from green to orange and red when ripe. The carotenoid differentiation of fruit chromoplasts and an altered carotenoid profile also changes from typical chloroplast carotenoids profile during tomato ripening (D’Andrea et al., 2018). Here we (lutein, β-carotene and β,β-xanthophylls) to mainly lycopene revise recent advances in our understanding of the connections and some β-carotene in chromoplasts (D’Andrea et al., 2018; between PQC and carotenoid accumulation and discuss how Yazdani et al., 2019). Extensive genetic evidence indicates that interference with PQC components can improve the carotenoid increasing carotenoid sink capacity leads to enhanced levels of profile of tomato fruits, one of the most highly consumed these health-promoting pigments in tomato fruit and other plant vegetables worldwide. systems (Sun et al., 2018). Yet our limited knowledge of how chromoplast differentiation and carotenoid storage are regulated has prevented to fully exploit this “pull” strategy as an alternative Learning From Arabidopsis or a complement to the “push” approach based on stimulating A direct connection between PQC and carotenoid biosynthesis is carotenoid biosynthesis. well established in Arabidopsis (Figure 1A). DXS is prone to lose its Orange (OR) is one of the very few proteins known to native structure, resulting in protein misfolding and aggregation in influence chromoplast differentiation (Rodriguez-Concepcion chloroplasts (Pulido et al., 2013). Delivery of such inactive forms et al., 2018; Sun et al., 2018). OR is a DNAJ-related protein that of DXS to the Hsp70 chaperone for unfolding is facilitated by shows chaperone activity (Park et al., 2016). Different OR versions their direct binding to J20, a DNAJ co-chaperone that functions promote PSY accumulation and activity and some prevent as a Hsp70 adaptor (Pulido et al., 2013). The fate of Hsp70-bound carotenoid (particularly β-carotene) metabolism (Bai et al., 2014; enzymes depends on the relative abundance of two plastidial Tzuri et al., 2015; Yuan et al., 2015; Zhou et al., 2015; Bai et al., Hsp100 chaperones: ClpB3 and ClpC1 (Pulido et al., 2016). Under 2016; Park et al., 2016; Chayut et al., 2017; Kim et al., 2018). In conditions promoting protein folding stress (e.g. heat), ClpB3 melon (Cucumis melo L.), a form of OR harboring an amino chaperones accumulate and assist Hsp70 to unfold DXS and release acid change from Arg to His at a position identified as a “golden it to the chloroplast stroma, allowing spontaneous refolding to its SNP” is responsible for the high β-carotene content of some active form. The default pathway, however, involves degradation cultivars (Chayut et al., 2017). This variant (OR-His) was found mediated by ClpC1, a Clp protease component (Pulido et al., to bind and promote PSY stability similarly to the OR-Arg form 2016). ClpC1 is part of the chaperone ring that unfolds protein while being much more efficient in preventing the conversion clients prior to degradation by the proteolytic core of the complex. of β-carotene into downstream products. Overexpression of Both pathways (refolding and degradation) are interconnected by Arabidopsis thaliana (L.) Heynh. OR-Arg and OR-His versions a mechanism involving the heat shock transcription factor HsfA2, in tomato was recently reported to result in increased levels which is induced by an unknown signal released by chloroplasts of fruit carotenoids (Yazdani et al., 2019). The chloroplast to when Clp protease activity is compromised (Llamas et al., 2017). chromoplast transition in transgenic fruit, which was most Higher HsfA2 levels induce the expression of genes encoding Frontiers in Plant Science | www.frontiersin.org 2 September 2019 | Volume 10 | Article 1071 D’Andrea and Rodríguez-Concepción Tomato Chaperones, Proteases and Carotenoids FIGURE 1 | PQC components involved in carotenoid biosynthesis. (A) Model of the regulation of biosynthetic enzymes by PQC components. Metabolite acronyms: GAP, glyceraldehyde 3-phosphate; DXP, deoxyxylulose 5-phosphate; MEP, methylerythritol 4-phosphate; IPP, isopentenyl diphosphate; DMAPP, dimethylallyl diphosphate; GGPP, geranylgeranyl diphosphate; SLs, strigolactones; ABA, abscisic acid. Dashed arrows represent several steps. The MEP pathway (boxed in green) provides substrates for the carotenoid pathway (boxed in orange). DXS and PSY catalyze the main flux-controlling steps of these pathways (large arrows). When active enzymes (oval) misfold or/and aggregate (spiked), specific chaperones participate in their unfolding for either reactivation or removal. DXS and PSY are degraded by the stromal Clp protease, a complex that also appears to control the levels of the indicated enzymes (bold): DXR, DXP reductoisomerase; HDS, hydroxymethylbutenyl diphosphate (HMBPP) synthase; HDR, HMBPP reductase; PDS, phytoene desaturase; ZDS, zeta-carotene desaturase; LUT5, cytochrome P450 beta-hydroxylase; BCH, non-heme di-iron beta-hydroxylase; ZEP, zeaxanthin epoxidase. (B) Expression levels of the indicated chaperone genes during tomato fruit development. Data from the Tomato Expression Atlas (http://tea.solgenomics.net/) were plotted to show the levels of transcripts encoding ClpB3 (Solyc02g088610), Hsp21 (Solyc03g082420), OR (Solyc03g093830), OR-like (Solyc09g010110) and J20 (Solyc05g053760) in fruit at several stages after anthesis. MG, mature green (equatorial pericarp); BR, breaker (equatorial pericarp); LR, light red (total pericarp); RR, red ripe (total pericarp). (C) Plastidial Hsp70 family members and their expression levels during tomato fruit development. The left panel shows a Maximum Likelihood unrooted tree constructed with MEGA6 using Arabidopsis Hsp70.1 (At4g24280) and Hsp70.2 (At5g49910) and tomato Hps70.1 (Solyc01g103450), Hsp70.2 (Solyc11g020040), Hsp70.3 (Solyc01g106260), and Hsp70.4 (Solyc01g106210) protein sequences lacking their predicted plastid-targeting sequences (http://www.cbs.dtu.dk/services/TargetP/). The graph on the right was made as described in (B). Frontiers in Plant Science | www.frontiersin.org 3 September 2019 | Volume 10 | Article 1071 D’Andrea and Rodríguez-Concepción Tomato Chaperones, Proteases and Carotenoids plastid-targeted chaperones such as ClpB3 and Hsp21, hence genes encoding plastid-targeted chaperones such as ClpB3 in boosting the unfolding and disaggregation capacity of chloroplasts transgenic tomatoes (D’Andrea et al., 2018). The two tomato genes to cope with protein folding stress (Llamas et al., 2017). This sort encoding OR homologues were also upregulated in transgenic of coordination between proteases and chaperones is a widespread fruits with reduced Clp protease activity (D’Andrea et al., 2018). mechanism that ensures protein homeostasis in several plant cell Because transgene-mediated downregulation of fruit Clp protease compartments. activity triggers an induction of nuclear genes encoding plastid- Besides DXS, the Clp protease regulates the levels of other targeted chaperones, it is possible that naturally occurring defects MEP pathway enzymes as well as many of the carotenoid pathway in the Clp protease-dependent degradation pathway during the enzymes, including PSY (Rodriguez-Concepcion et al., 2019). The differentiation of tomato fruit chromoplasts might be similarly common regulation of both pathways by the Clp protease likely signaled to the nucleus to eventually ensure correct folding (i.e. adjusts the supply of metabolic precursors for the production activity) of key enzymes such as DXS (via ClpB3) and PSY (via of carotenoids (Figure 1A). As described for DXS, degradation OR). Genes encoding ClpB3 and OR chaperones are induced of Arabidopsis PSY by the Clp proteolytic complex involves during normal fruit ripening (Figure 1B), supporting a role of interaction with ClpC1 (Welsch et al., 2018). The delivery pathway, these chaperones in keeping carotenoid biosynthetic enzymes however, appears to differ between these enzymes. PSY interacts active as chloroplast naturally differentiate into chromoplasts. with OR and Hsp70 chaperones (Zhou et al., 2015; Park et al., It is unknown whether other ripening-induced chaperones that 2016; Welsch et al., 2018). Unlike J20 (a canonical DNAJ protein), protect against protein misfolding and aggregation such as Hsp21 OR is a DNAJE1-type protein that lacks the J-domain and hence it (Figure 1B) or Hsp70 (Figure 1C) are also up-regulated in tomato is unable to interact with Hsp70 (Lu et al., 2006; Pulido and Leister, as they are in Arabidopsis when Clp protease activity is blocked 2018; Welsch et al., 2018). Consistent with the conclusion that (Llamas et al., 2017). J20 and OR play distinct roles in regulating the stability of their As indicated above, both Hsp21 and OR chaperones were protein clients, higher DXS protein but lower activity levels were found to promote the conversion of chloroplasts to chromoplasts observed in J20-defective mutants (Pulido et al., 2013; Pulido et al., and the accumulation of carotenoids when overproduced in 2016) whereas higher levels of both PSY protein and activity were tomato fruit (Neta-Sharir et al., 2005; Yazdani et al., 2019). detected in OR-overexpressing lines (Zhou et al., 2015; Park et al., From the rest of chaperones identified in Arabidopsis to play a 2016; Welsch et al., 2018). The current model proposes that OR role in the carotenoid-related PQC network (Figure 1A), only might promote refolding of misfolded or aggregated PSY proteins the tomato homologues of J20 and ClpB3 have been investigated which otherwise would be degraded by the Clp protease by a by altering their levels in transgenic plants. Their role on fruit pathway involving ClpC1 and, perhaps, Hsp70 (Figure 1A). In carotenoid biosynthesis, however, was not addressed. Tomato agreement with this model, PSY protein levels are up-regulated in antisense plants with reduced J20 or ClpB3 transcript levels mutants lacking Hsp70.2, one of the two Hsp70 isoforms found in showed reduced thermotolerance (Yang et al., 2006; Wang et al., Arabidopsis chloroplasts (Welsch et al., 2018). 2019). By contrast, transgenic tomato lines overexpressing the endogenous J20 gene showed enhanced thermotolerance, in part because the expression of HsfA2 and other genes encoding heat Translating to Tomato shock transcription factors was higher in J20-overexpressing Tomato has risen to the podium of model plants in carotenoid lines under heat stress (Wang et al., 2019). Interestingly, the biotechnology due to the high abundance of carotenoids in the J20 homologue was found to interact with Hsp70.2 (Wang et al., fruit together with the astounding genetic resources available and 2019), one of the four plastid-targeted isoforms of Hsp70 present the recent publication of its genome sequence. From early reverse in tomato (Figure 1C). Hsp70.2 (but not J20 or other Hsp70- genetic approaches to manipulate the carotenoid content of tomato encoding genes) is strongly upregulated during fruit ripening fruits it was deduced that post-transcriptional mechanisms are (Figure 1C). Hsp70.2 also interacts with the J20-related DNAJ key to regulate carotenoid accumulation during fruit ripening proteins CDJ1 and CDJ2, which maintain photosystem II under (Giuliano et al., 2008; Fraser et al., 2009; Rodriguez-Concepcion chilling stress (Kong et al., 2014) and protect Rubisco from et al., 2018). However, the specific nature of such mechanisms degradation under heat stress (Wang et al., 2015). No functional has remained little explored. Work in Arabidopsis has recently information is available for the other three tomato plastid- provided important clues on the relevance of PQC for this matter targeted Hsp70 isoforms. as well as molecular tools to manipulate the differentiation of chromoplasts and the carotenoid profile of tomato fruits. A general conclusion is that the basic mechanisms unveiled in Arabidopsis Filling the Gaps chloroplasts appear to be conserved in tomato chromoplasts. Hsp70 chaperones appear as a central node in the Arabidopsis For example, decrease of Clp protease activity during tomato carotenoid-related PQC network (Figure 1A) but their role in ripening using an artificial microRNA (amiRNA) against one of tomato ripening has not been addressed. To fill this gap, we the catalytic core subunits resulted in increased DXS and PSY generated transgenic tomato plants with decreased expression protein levels in ripe fruit (D’Andrea et al., 2018), suggesting that of the ripening-induced Hsp70.2 gene (Figure 2). Constructs both enzymes are Clp protease targets in Arabidopsis and tomato. were generated as described (D’Andrea et al., 2018) to express Also similar to that observed in Arabidopsis (Llamas et al., 2017), a Hsp70.2-specific amiRNA under the control of the ripening- reduced Clp protease activity led to upregulated expression of induced PLI promoter (Estornell et al., 2009). After transformation Frontiers in Plant Science | www.frontiersin.org 4 September 2019 | Volume 10 | Article 1071 D’Andrea and Rodríguez-Concepción Tomato Chaperones, Proteases and Carotenoids of tomato cv. MicroTom (MT) plants, T1 lines with decreased phytoene desaturase (PDS in Figure 1A), a possible Clp protease levels of Hsp70.2 transcripts were selected for further studies. client (Welsch et al., 2018; Rodriguez-Concepcion et al., 2019), The fruits of these lines showed no significant changes in the have actually been found in chromoplasts (Al-Babili et al., 1996; expression of tomato genes encoding the fruit DXS1 and PSY1 Bonk et al., 1996). Loss of Hsp70.2 might make inactive enzymes isoforms (Figure 2A). Ripe fruits from T2 lines showed increased more available for reactivation upon unfolding by chaperones levels of lycopene and, to a lower extent, other fruit carotenoids such as OR in the case of PSY1. While it is possible that Hsp70.2 (Figure 2B). Consistent with their high lycopene enrichment, might regulate other processes related to carotenoid biosynthesis transgenic fruit of the H70.2-10 line showed a stronger red or storage, both ripe fruit color and chromoplast ultrastructure color. These results suggest that Hsp70.2 negatively regulates were similar in MT and Hsp70.2-defective lines (Figure 2C). carotenoid biosynthesis. This role might involve delivering Thus, chromoplasts from transgenic ripe fruits contained similar fruit PSY1 and likely other carotenoid pathway enzymes to vesicles, plastoglobules, and undulating membranes (i.e. the degradation by the Clp protease complex (Figure 1A). Protein remnants of envelopes formed around lycopene crystals) as those complexes containing Hsp70 and enzymatically inactive forms of observed in untransformed controls (Figure 2C). It is possible FIGURE 2 | Hsp70 and OR chaperones differentially control carotenoid biosynthesis in tomato. (A) Transcript levels of the indicated genes in the pericarp of red ripe fruits. Tomato MicroTom (MT) plants were transformed with an amiRNA construct to silence the Hsp70.2 gene during fruit ripening. Ripe fruits from greenhouse- grown untransformed and transgenic T1 plants were collected at 52 days post-anthesis (dpa) and used for real-time quantitative PCR (qPCR) analysis of Hsp70.2, DXS1 (Solyc01g067890) and PSY1 (Solyc03g031860) expression using the ACT gene (Solyc04g011500) as normalizer. Mean and SD values (n ≥ 2) are shown relative to those in untransformed MT fruit. Asterisks mark statistically significant differences relative to MT samples (T-test, P < 0.05). (B) Levels of carotenoids in the pericarp of red ripe fruits from the indicated lines. MT plants were transformed with a construct previously used to constitutively overexpress Arabidopsis OR-His under the control of the 35S promoter in tomato plants of the M82 cultivar (Yazdani et al., 2019). A representative T1 line was selected based on the distinctive orange color of their fruit. T2 plants of this line (oxOR-121) were grown in the greenhouse together with untransformed controls and T2 plants of Hsp70.2-silenced lines analyzed in (A), and ripe fruits were collected at 52 dpa for pericarp carotenoid extraction and analysis by HPLC. Mean and SD values (n ≥ 2) of individual and total carotenoid levels are shown relative to those in MT fruit. Asterisks mark statistically significant differences relative to MT samples (T-test, P < 0.05). (C) Representative images of chromoplasts. Fruits were collected from the indicated lines at 52 dpa and used for TEM analysis of chromoplast ultrastructure. Bar, 1 µm. Previously described methods (D’Andrea et al., 2018) were used for amiRNA synthesis, plant transformation, RNA and carotenoid extraction, qPCR, HPLC, TEM, and statistical analyses. Frontiers in Plant Science | www.frontiersin.org 5 September 2019 | Volume 10 | Article 1071 D’Andrea and Rodríguez-Concepción Tomato Chaperones, Proteases and Carotenoids that other plastidial Hsp70 chaperones besides Hsp70.2 might other plastidial proteins and complexes (Lee et al., 2007; Mishra ensure protein homeostasis during chromoplast differentiation and Grover, 2016), its overproduction might provide additional in Hsp70.2-defective transgenic lines. At least some of them benefits to fruit ripening, including an enhanced resistance to might also ensure that the tomato DXS1 enzyme also remains multiple stresses affecting protein folding. A similar but maybe active to provide more substrates for PSY1 to channel into the enhanced phenotype might be achieved by the ripening-specific carotenoid pathway during fruit ripening. overexpression of the transcription factor HsfA2, found to Consistent with the conclusion that Hsp70 and OR have upregulate the expression of genes encoding chaperones such opposite roles, tomato fruit carotenoids can be increased either as ClpB3 and Hsp21 in Arabidopsis. Future research should by down-regulating Hsp70 (Figure 2) or by up-regulating also identify the motifs in carotenoid biosynthetic enzymes that OR (Yazdani et al., 2019). But unlike Hsp70.2-defective lines, determine their misfolding propensity or stability as well as the constitutive overexpression of the Arabidopsis OR-His protein basic mechanisms underlying chromoplast differentiation. Such in tomato MT plants produced fruits that were most enriched information would provide new powerful tools to improve the in phytoene (likely the result of enhanced PSY activity) and nutritional quality of tomatoes and other fruits. β-carotene instead of lycopene (Figure 2B). The enrichment in β-carotene, which resulted in ripe fruits with a distinctive orange hue in some lines is similar to that reported in other plant systems DATA AVAILABILITY when OR-His proteins are overproduced. It has been suggested that OR might repress β-carotene metabolism or/and degradation The datasets generated for this study are available on request to by interacting with cleaving enzymes such as CCD4 (Chayut et al., the corresponding author. 2017; Kim et al., 2018). Also similar to other plant systems (Yuan et al., 2015; Park et al., 2016), OR-His promoted the development of membranous chromoplasts in tomato ripe fruit (Figure 2C). AUTHOR CONTRIBUTIONS The available data allow to conclude that Hsp70 and OR play LD’A performed experiments and collected data and information. distinct but complementary roles for both carotenoid biosynthesis LD’A and MR-C analyzed the data, discussed the results, prepared (i.e. enzyme activity) and storage (i.e. chromoplast differentiation) the figures, and wrote the paper. during normal fruit ripening in tomato. Looking Forward FUNDING The described results demonstrate that manipulation of plastidial This work was supported by grants from the Spanish Ministry PQC components related to carotenoid metabolism and of Economy and Competitiveness (BIO2017-84041-P and storage works to modify the carotenoid profile of tomato fruits. BIO2017-90877-REDT), the Ministry of Education, Culture and Manipulation of Hsp21, OR or/and Clp protease activity has Sports (AP2012-0189), the Generalitat de Catalunya (2017SGR- additionally been found to increase tolerance to environmental 710), and the European COST Action EuroCaroten (CA15136). stress (Neta-Sharir et al., 2005; D’Andrea et al., 2018; Kim et al., We also thank the financial support of the MINECO Severo 2018). PQC components identified in Arabidopsis to regulate Ochoa Programme for Centres of Excellence in R&D 2016-2019 carotenoid biosynthesis and found in tomato to improve (SEV-2015-0533) and the Generalitat de Catalunya CERCA thermotolerance, such as J20 (Wang et al., 2019), ClpB3 (Yang Programme to CRAG. et al., 2006) or HsfA2 (Fragkostefanakis et al., 2016), remain unexplored in terms of their impact on tomato fruit carotenoid accumulation. We hypothesize that the overexpression of ClpB3 ACKNOWLEDGMENTS might lead to higher levels of carotenoids but also other MEP- derived plastidial isoprenoids such as tocopherols (vitamin E) We thank Dr. Li Li (Cornell University) for the construct due to a predicted higher DXS activity (Llamas et al., 2017). As to express the Arabidopsis OR-His in tomato and M. Rosa ClpB3 also promotes the disaggregation, assembly and stability of Rodriguez-Goberna (CRAG) for excellent technical support. REFERENCES Bai, C., Rivera, S. M., Medina, V., Alves, R., Vilaprinyo, E., Sorribas, A., et al. (2014). An in vitro system for the rapid functional characterization of genes Al-Babili, S., von Lintig, J., Haubruck, H., and Beyer, P. (1996). A novel, involved in carotenoid biosynthesis and accumulation. 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Dual role for tomato conducted in the absence of any commercial or financial relationships that could heat shock protein 21: protecting photosystem II from oxidative stress and be construed as a potential conflict of interest. promoting color changes during fruit maturation. Plant Cell 17, 1829–1838. doi: 10.1105/tpc.105.031914 Copyright © 2019 D’Andrea and Rodriguez-Concepcion. This is an open-access article Park, S., Kim, H. S., Jung, Y. J., Kim, S. H., Ji, C. Y., Wang, Z., et al. (2016). Orange distributed under the terms of the Creative Commons Attribution License (CC BY). protein has a role in phytoene synthase stabilization in sweetpotato. Sci. Rep. 6, The use, distribution or reproduction in other forums is permitted, provided the original 33563. doi: 10.1038/srep33563 author(s) and the copyright owner(s) are credited and that the original publication in Pulido, P., and Leister, D. (2018). Novel DNAJ-related proteins in Arabidopsis this journal is cited, in accordance with accepted academic practice. No use, distribution thaliana. New Phytol. 217, 480–490. doi: 10.1111/nph.14827 or reproduction is permitted which does not comply with these terms. Frontiers in Plant Science | www.frontiersin.org 7 September 2019 | Volume 10 | Article 1071