https://doi.org/10.1093/plphys/kiac097 PLANT PHYSIOLOGY 2022: 189: 1450–1465 Development and carotenoid synthesis in dark-grown carrot taproots require PHYTOCHROME RAPIDLY REGULATED1 Research Article Downloaded from https://academic.oup.com/plphys/article/189/3/1450/6546316 by guest on 26 May 2023 Daniela Arias ,1 Angelica Ortega ,1 Christian González-Calquin,1 Luis Felipe Quiroz ,1 Jordi Moreno-Romero ,2,3 Jaime F. Martınez-Garcıa2,3 and Claudia Stange 1,*,† 1 Centro de Biologıa Molecular Vegetal, Facultad de Ciencias, Universidad de Chile, Santiago, Chile 2 Centre for Research in Agricultural Genomics (CRAG), CSIC-IRTA-UAB-UB, Barcelona, Spain 3 Institute for Plant Molecular and Cell Biology (IBMCP), CSIC-UPV, Universitat Politècnica de València, València, Spain *Author for correspondence: cstange@uchile.cl † Senior author. C.S. conceived the research plans. C.S. and J.F.M.G. supervised the experiments. D.A. and A.O performed the experiments. J.M.R., C.G.C., and L.F.Q. performed some experiments with D.A. J.F.M.G. provided technical assistance to D.A. C.S. designed the experiments and analyzed the data. C.S. con- ceived the project and wrote the article with contributions of all the authors. C.S. agrees to serve as the author responsible for contact and ensures communication. The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (https://academic.oup.com/plphys/pages/general-instructions) is: Claudia Stange (cstange@uchile.cl). Abstract Light stimulates carotenoid synthesis in plants during photomorphogenesis through the expression of PHYTOENE SYNTHASE (PSY), a key gene in carotenoid biosynthesis. The orange carrot (Daucus carota) synthesizes and accumulates high amounts of carotenoids in the taproot that grows underground. Contrary to other organs, light impairs carrot taproot development and represses the expression of carotenogenic genes, such as DcPSY1 and DcPSY2, reducing carotenoid accu- mulation. By means of RNA sequencing, in a previous analysis, we observed that carrot PHYTOCHROME RAPIDLY REGULATED1 (DcPAR1) is more highly expressed in the underground grown taproot compared with those grown in light. PAR1 is a transcriptional cofactor with a negative role in shade avoidance syndrome regulation in Arabidopsis (Arabidopsis thaliana) through the dimerization with PHYTOCHROME-INTERACTING FACTORs (PIFs), allowing a moderate synthesis of carotenoids. Here, we show that overexpressing AtPAR1 in carrot increases carotenoid production in taproots grown un- derground as well as DcPSY1 expression. The high expression of AtPAR1 and DcPAR1 led us to hypothesize a functional role of DcPAR1 that was verified through in vivo binding to AtPIF7 and overexpression in Arabidopsis, where AtPSY expres- sion and carotenoid accumulation increased together with a photomorphogenic phenotype. Finally, DcPAR1 antisense carrot lines presented a dramatic decrease in carotenoid levels and in relative expression of key carotenogenic genes as well as impaired taproot development. These results suggest that DcPAR1 is a key factor for secondary root development and carotenoid synthesis in carrot taproot grown underground. Received January 05, 2022. Accepted February 04, 2022. Advance access publication March 10, 2022 C The Author(s) 2022. Published by Oxford University Press on behalf of American Society of Plant Biologists. V Open Access This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs licence (https://creativecommons.org/licenses/by-nc-nd/4.0/), which permits non-commercial reproduction and distribution of the work, in any medium, provided the original work is not altered or transformed in any way, and that the work is properly cited. For commercial re- use, please contact journals.permissions@oup.com DcPAR1 is a key factor in taproot development PLANT PHYSIOLOGY 2022: 189; 1450–1465 | 1451 Introduction bind to LREs (Bae and Choi, 2008; Shen et al., 2008; Shin Light is an essential cue for plant development and growth et al., 2009; Toledo-Ortiz et al., 2010). This results in a rapid since it is required for photosynthesis and photomorpho- de-repression of PSY gene expression and a burst in the pro- genesis (Tripathi et al., 2019). In both processes, the plastid- duction of carotenoids in coordination with chlorophyll bio- ial pigments chlorophylls and carotenoids fulfil crucial roles. synthesis and chloroplast development for an optimal Synthesis of chlorophylls and carotenoids is strongly pro- transition to photosynthetic metabolism (Toledo-Ortiz et al., moted by light (Stange and Flores, 2012). Carotenoids also 2010). In addition to repressing gene expression, PIFs also provide yellow, orange, and red colors to flowers, fruits, and promote the expression of dozens of genes in the dark that some roots. These pigments are not only powerful antioxi- are also rapidly downregulated after seedling de-etiolation, dant that protect plants from photooxidative damage but such as A. thaliana PHYTOCHROME RAPIDLY REGULATED 1 also precursors of important compounds such as the phyto- (AtPAR1), that encodes a transcriptional cofactor of the hormones strigolactones and abscisic acid (ABA) that affect bHLH family. AtPAR1 physically interacts with PIFs and Downloaded from https://academic.oup.com/plphys/article/189/3/1450/6546316 by guest on 26 May 2023 plant development (Hirschberg, 2001; Howitt and Pogson, thereby prevents PIFs from binding to LREs found in pro- 2006; Sandmann et al., 2006; Rodrıguez-Concepción, 2010; moters of photomorphogenic genes, thus producing an in- Ruyter-Spira et al., 2013; Sandmann, 2015; Simpson et al., crease in carotenoids compared to plants grown in the dark 2016a). The first enzyme in carotenoid synthesis and the (Bou-Torrent et al., 2015). Indeed, A. thaliana lines that most regulated at the transcriptional and posttranscriptional overexpress AtPAR1 present an increase in total carotenoids level is phytoene synthase (PSY; Maass et al., 2009; in photosynthetic tissues, an increase in AtPSY expression Rodrıguez-Villalón et al., 2009; Rosas-Saavedra and Stange, and an exacerbated photomorphogenic phenotype shown 2016). In Arabidopsis (Arabidopsis thaliana) this enzyme as a reduced hypocotyl length (Roig-Villanova et al., 2007; is encoded by the AtPSY gene, whereas in carrot (Daucus Hao et al., 2012; Zhou et al., 2014; Bou-Torrent et al., 2015). carota), it is encoded by two genes, DcPSY1 and DcPSY2 Unlike other plants, orange carrots accumulate large (Ruiz-Sola and Rodriguez-Concepcion, 2012; Rodriguez- amounts of carotenoids in underground and dark-grown tap- Concepcion and Stange, 2013). roots (Stange et al., 2008; Fuentes et al., 2012; Rodriguez- When seedlings germinate in the dark, they exhibit a pale Concepción and Stange, 2013). Surprisingly, root exposure to yellow or rather whitish color due to the lack of pigmenta- white light (W) causes a reduction in the expression of caro- tion (etiolated seedlings). In these conditions, tenogenic genes such as PSYs and LYCOPENE BETA-CYCLASES PHYTOCHROME-INTERACTING FACTORs (PIFs) accumu- (LCYBs) and a decrease in carotenoid levels (Fuentes et al., late and repress photomorphogenesis. PIFs are a subfamily 2012; Llorente et al., 2017; Arias et al., 2020). Indeed, the car- of basic helix–loop–helix (bHLH) transcription factors that rot root grown in W has a thinner and greener phenotype act as central mediators in a variety of light-mediated and presents an enrichment of chloroplasts instead of carot- responses, including transitions from etiolated to photomor- enoid accumulating chromoplasts (Fuentes et al., 2012). The phogenic (de-etiolated) seedlings and plant responses to expression profile between carrot taproot grown in W and vegetation proximity (Leivar et al., 2009; Pham et al., 2018). underground (dark-grown) was analyzed and compared As part of their repressive role of the photomorphogenic de- through an RNA sequencing (RNA-seq) approach (Arias velopment in the dark, PIFs bind to AtPSY light-responsive et al., 2020). This led us to identify several carrot genes related elements (LREs, such as G-box), preventing PSY expression to photomorphogenesis, such as DcPHYA, DcPHYB, and and impeding the synthesis and accumulation of carote- DcPAR1, whose transcripts accumulated in the underground noids in these conditions (Toledo-Ortiz et al., 2010). Once dark-grown taproot but rapidly decreased in the root when the emerging seedlings perceive the light, photomorphogen- exposed to W (Arias et al., 2020). esis is triggered, which results in inhibition of stem elonga- In this work, we aimed to characterize the role of DcPAR1 tion (e.g. hypocotyls or epicotyls), promotion of cotyledon in carotenoid synthesis and development in carrot taproot. or/and leaf expansion, and promotion of chlorophyll and First, we showed that overexpression of AtPAR1 in trans- carotenoids synthesis, which result in the acquisition of the genic carrot leads to higher levels of carotenoids in taproots characteristic green color of plants (Quail, 2002; Kami et al., as well as higher DcPSY1 expression suggesting a key role of 2010; De Wit et al., 2016). Light perception is carried out by PAR1 in carrot carotenoid synthesis. The similarity of photoreceptors such as phytochromes (phys), crypto- AtPAR1 and DcPAR1 led us to suggest a similar molecular chromes, and phototropins (Kami et al., 2010; Stange and role of DcPAR1 as a bHLH member that was verified Flores, 2012; De Wit et al., 2016; Quian-Ulloa and Stange, through the in vivo binding to the bHLH AtPIF7 protein. In 2021). In particular, phys participate in the induction of the addition, in order to analyze if these two genes share func- expression of key carotenogenic genes such as PSY (Saijo tionality, that is, if they are orthologues, we determined the et al., 2008; Stange and Flores, 2012; Chen et al., 2014; DcPAR1 role in photomorphogenesis and in carotenoid syn- Bianchelli et al., 2018; Quian-Ulloa and Stange, 2021). During thesis by overexpressing it in Arabidopsis. We showed that de-etiolation, the light activation of phys triggers PIF phos- overexpression of AtPAR1 in transgenic carrot present higher phorylation, which are subsequently degraded by the protea- levels of carotenoids in taproots as well as higher DcPSY1 ex- some and/or inactivated, therefore making them unable to pression suggesting a key role of the PAR1 in carrot 1452 | PLANT PHYSIOLOGY 2022: 189; 1450–1465 Arias et al. carotenoid synthesis. Most importantly, DcPAR1 antisense adult plants (grown 4 months in the greenhouse), that over- carrot plants showed an impaired taproot development and expressed AtPAR1, were selected, established as independent a dramatic decrease in both carotenoid levels and in the rel- transgenic lines and subjected to molecular and biochemical ative expression of key carotenogenic genes. These results analysis (Figure 1, B and C). Among the four lines, two of suggest that DcPAR1 is a transcriptional cofactor with a key them presented higher levels of transgene relative expression role in the regulation of taproot development and caroten- in the taproot (OE1 and OE2; Figure 1B), which also showed oid synthesis in the carrot taproot grown underground. an increase in DcPSY1 expression relative to WT taproots (Figure 1B). On the contrary, DcPSY2 showed a decrease in Results the relative expression (Figure 1B), whereas the endogenous Carrot plants overexpressing AtPAR1 present higher DcPAR1 was expressed at similar or reduced levels than WT carotenoid levels and an increase in DcPSY1 plants (Supplemental Figure S1). Importantly, transgenic expression in the taproot grown underground OE1 and OE2 lines, which have the highest expression levels Downloaded from https://academic.oup.com/plphys/article/189/3/1450/6546316 by guest on 26 May 2023 As a first approximation to determine PAR1 function in ca- of AtPAR1 and DcPSY1, presented an average of 2.5 times rotenoid synthesis in the carrot taproot, we overexpressed more total carotenoids in their taproots (919.6 mg g–1 dry AtPAR1 in carrots. During carrot transformation it caught weight [DW] and 796.1 mg g–1 DW of carotenoids, respec- our attention that AtPAR1 embryos presented an orange tively) compared to WT plants (410.2 mg g–1 DW) phenotype instead of the normal whitish coloration of wild- (Figure 1C). Moreover, both OE1 and OE2 lines also present type (WT) embryos (Figure 1A). This early observation sug- higher b-carotene level (2.3 and 2.4 times more than WT gested that AtPAR1 could promote the synthesis of carote- roots) and only the OE1 line has significantly higher levels of noids in carrots in early stages of development. Next, four a-carotene in their taproots (Figure 1C). These results Figure 1 Molecular and biochemical analysis of AtPAR1 transgenic carrots. A, In vitro regeneration of carrot transformant plants. Embryo regener- ation stage at 18–20 weeks after explants transformation is shown. The arrow indicates orange embryos that later give rise to whole carrot plants in AtPAR1 (AtPAR1:GFP). WT, and DcPAR1as cream-colored embryos are also shown. Scale bar: 5 mm. B, Expression of AtPAR1, DcPSY1, and DcPSY2 in transgenic (OE1, OE2, OE5, and OE6) and WT carrot taproots. Ubiquitin was used as housekeeping gene. The RT-qPCR was performed with three biological replicates (three different samples from the same transgenic lines) and two technical repeats each. C, Total carotenoids, b- carotene and a-carotene levels in transgenic (OE1, OE2, OE5, and OE6) and WT carrot taproots. The analysis was repeated 3 times using three dif- ferent samples from each transgenic line and three pools from three WT plants. Carotenoids are indicated in microgram per gram of DW. In (B) and (C) values are means ± standard error (SE). Letters and asterisks indicate significant differences between WT and transgenic lines (one-way ANOVA analysis with Dunnett’s post-hoc test; ****P 5 0.0001, ***P 5 0.001, **P 5 0.01, *P 5 0.1). DcPAR1 is a key factor in taproot development PLANT PHYSIOLOGY 2022: 189; 1450–1465 | 1453 suggest that AtPAR1 positively regulates carotenoid synthesis transcriptional cofactor, as described for AtPAR1 (Galstyan by promoting the expression of the DcPSY1, but not et al., 2011). DcPSY2. AtPIF7 has been shown to bind to PHYB (Leivar et al., 2008) and to have a role in promoting Arabidopsis hypoco- DcPAR1 interacts with AtPIF7 in the nucleus tyls as a result of shade and warm temperature treatments The effect of AtPAR1 overexpression on carrot taproot upon (Li et al., 2012; Fiorucci et al., 2020). Considering that in A. DcPSY1 expression and carotenoid levels suggests that the thaliana a functional AtPAR1 binds in vivo to PIFs and other product of a carrot PAR1 homolog might have the same bHLH proteins in the nucleus (Hao et al., 2012; Cifuentes- role as AtPAR1 in A. thaliana. Therefore, we identified contig Esquivel et al., 2013; Paulisic et al., 2021), we aimed to deter- 42,760 (292 bp) obtained from the carrot transcriptome mine whether the DcPAR1 protein is nuclear and capable of (Arias et al., 2020) which presented a 98% identity with heterodimerizing with other bHLH proteins. First, we ob- AtPAR1 (At2g42870, Access No NM_129848.3) and obtained served that DcPAR1-GFP presents nuclear localization in the CDS sequence of DcPAR1 (Access No XM_017390696.1) cells of Nicotiana benthamiana leaves (Supplemental Figure Downloaded from https://academic.oup.com/plphys/article/189/3/1450/6546316 by guest on 26 May 2023 by searching in NCBI (Supplemental Figure S2). The com- S3). Next, we tested interaction of DcPAR1 to AtPIF7 in vivo plete CDS of DcPAR1 (363 bp) presents a 57.7% nucleotide in N. benthamiana cells by bimolecular fluorescence comple- identity with the complete AtPAR1 and 44% identity at the mentation (BiFC). In Figure 3, the reconstitution of YELLOW amino acid level (Figure 2A). We paid special attention to FLUORESCENCE PROTEIN (YFP) fluorescence is observed the bHLH domain considering that the AtPAR1 protein into the cell nucleus of leaves co-agroinfiltrated with the N- presents a bHLH domain that allows it to interact with PIFs terminal part of the YFP, NY fused to AtPIF7 (NY-AtPIF7) and with itself but not to DNA (Galstyan et al., 2011, 2012). and the C-terminal part of the YFP (CY) fused to DcPAR1 A conserved domain search analysis was performed predict- (CY-DcPAR1), thus demonstrating that both proteins inter- ing a complete bHLH sequence between residues 29 and act in vivo. Fluorescence is also detected in NY-DcPAR1 and 107 (Figure 2A). Comparing this predicted sequence with CY-DcPAR1 co-agroinfiltrations (Figure 3), indicating that AtPAR1 bHLH, DcPAR1 presented the key conserved Leu23 DcPAR1 can also homodimerize, as it was described for residue in Helix 1 (Figure 2B) and the conserved hydropho- AtPAR1 (Bou-Torrent et al., 2015). Together, these results bic residues such as Val, Ala, Leu, and Ile in Helix 2, all of suggested that DcPAR1 has the ability to dimerize with itself them important for dimer formation. In the basic domain, it and with PIFs, as it has been shown for AtPAR1. presents an Asp, Glu, and Lys at positions 5, 9, and 13, re- spectively, that, similar to AtPAR1 (Figure 2B), also differ Arabidopsis plants that express DcPAR1 present an from those (His5-Glu9-Arg13) required for DNA binding increase in carotenoids with a higher AtPSY (Heim et al., 2003). This sequence analysis suggests that expression and AtPSY protein abundance DcPAR1 could be able to dimerize with other bHLH proteins Taken in mind that overexpression of AtPAR1 results in but unable to bind DNA, functionally acting as a dwarf plants and increased AtPSY expression and carotenoid Figure 2 DcPAR1 and AtPAR1 protein alignment. A, The predicted bHLH domains are highlighted in two shades of gray. The left box delimits the basic domain, while the next boxes mark the helix 1, the loop, and the helix 2 subdomains, respectively. B, Alignment of AtPAR1 and DcPAR1 bHLH domains. The subdomain extensions and conserved amino acids were described by Heim et al. (2003) and Roig-Villanova et al. (2007). Circles highlight residues 5, 9, and 13 (Asp, Ala, and Glu in AtPAR1 and Asp, Glu, and Lys in DcPAR1). Star highlights the conserved leucine residue at position 23 in helix 1. Helix 2 shows hydrophobic residues, most of which are conserved in both proteins. Different and identical amino acids are highlighted in dark and white, respectively in each subdomain. All alignments were performed with Clustal Omega. 1454 | PLANT PHYSIOLOGY 2022: 189; 1450–1465 Arias et al. Downloaded from https://academic.oup.com/plphys/article/189/3/1450/6546316 by guest on 26 May 2023 Figure 3 Interaction in vivo between DcPAR1 and AtPIF7. BiFC assay between DcPAR1 and AtPIF7 is shown. The NY-AtPIF7/DcPAR1 corresponds to AtPIF7 or DcPAR1 genes cloned in fusion to N-terminal region of YFP (NY)gene and CY-AtPIF7/DcPAR1 corresponds to the same genes cloned in fusion to the CY. NY and CY are empty vectors. Nicotiana benthamiana leaves were co-infiltrated with the NY- and CY-derived plasmids to ex- press the indicated fusion proteins. The top row shows the fluorescence of reconstituted YFP (indicative of a positive interaction of YN and YC fusions), the middle row shows the bright field of the same area and the bottom row shows the merge of previous conditions. Scale bar: 50 mm. level in Arabidopsis (Roig-Villanova et al., 2007; Bou-Torrent present a dwarf phenotype (Figure 5A) with a significant re- et al., 2015), we overexpressed DcPAR1 in Arabidopsis to duced stem height and silique length (Figure 5B) at the ma- evaluate if it provides similar phenotypes as AtPAR1. We ture stage. These results suggest that the DcPAR1 is obtained more than 20 independent transgenic lines and se- functional in vivo in promoting carotenoid synthesis and lected four T3 transgenic lines that express DcPAR1 inhibiting growth in A. thaliana. (Figure 4A) for further analysis that was done in 2-week-old plants grown under W (long-day photoperiod). The relative Repression of DcPAR1 expression affects carrot expression of the endogenous AtPAR1 was not significantly taproots growth, carotenoid composition, and affected in the transgenic lines except for L1, which pre- carotenogenic gene expression sented a higher expression level than the WT line (Col-0; The next step was to evaluate the role of DcPAR1 in carrot. Figure 4A). Importantly, all transgenic lines presented a sig- For this, transgenic antisense carrot plants for DcPAR1 were nificant increase in AtPSY relative expression (Figure 4A), generated. During in vitro regeneration of DcPAR1 antisense similar to AtPAR1 overexpressing lines (Bou-Torrent et al., (DcPAR1as) plants, no phenotypic differences were observed 2015). The increase in the relative expression of AtPSY was compared to WT embryogenesis (Figure 1A). Eleven mature accompanied by an increase in the abundance of PSY pro- plants were obtained, and the molecular analysis confirmed tein in all transgenic lines (Figure 4B; Supplemental Figure that ten of them were transgenic (Supplemental Figure S6). S4 shows Coomassie blue staining as control) and increased Four transgenic lines were selected for further molecular total carotenoids compared to Col-0 (Figure 4C), suggesting and biochemical analysis. As shown in Figure 6A, all trans- that DcPAR1 positively regulates the expression of AtPSY genic lines presented an important reduction in DcPAR1 ex- and thus increasing carotenoids content in Arabidopsis. pression (between 80% and 99%) and a drastic reduction in To determine if DcPAR1 also affects elongation, we per- the taproot secondary growth at the mature stage, as shown formed phenotypic analysis in 2-week- and 1.5-month-old for line AS10 (Figure 6B). The taproots of a 4-month-old transgenic lines. In 2-week-old seedlings, transgenic lines pre- representative line that grew underground were considerably sented a reduced length in hypocotyl, cotyledons, and pri- smaller and thinner than WT taproots of the same age and mary leaves (Supplemental Figure S5) similar to that grown under the same conditions. The average mass of the reported for Arabidopsis AtPAR1 overexpressing lines (Roig- transgenic roots did not exceed 400 mg while the average Villanova et al., 2007; Hao et al., 2012; Zhou et al., 2014; mass of a 4-month-old WT carrot taproot can weigh up to Bou-Torrent et al., 2015). Moreover, all transgenic lines 7 g (Figure 6B). In addition, the roots were pale suggesting DcPAR1 is a key factor in taproot development PLANT PHYSIOLOGY 2022: 189; 1450–1465 | 1455 Downloaded from https://academic.oup.com/plphys/article/189/3/1450/6546316 by guest on 26 May 2023 Figure 4 Expression and carotenoid quantification of transgenic Arabidopsis lines overexpressing DcPAR1. A, Relative expression level of DcPAR1, AtPAR1, and AtPSY in 2-week-old 35S:DcPAR1-GFP (L1, L2, L3, and L4) transgenic lines. The relative expression levels were carried out with three biological replicates (n = 3) and two technical repeats each and normalized to housekeeping gene EFf1A4. Values are means ± SE (B) Immunoblot of DcPAR1-GFP, AtPSY, and actin in the transgenic Arabidopsis lines indicated in (A) using antiGFP, antiPSY, and anti-ACTIN antibodies. About 100 lg of total protein corresponding to three plants was loaded in each lane. Graphs present the semi-quantification of DcPAR1-GFP and AtPSY from Immunoblot using actin abundance as normalizer. Each graph shows the average of three Immunoblots and values are means ± SE. ImageJ program was used for WB semi quantification. MW, molecular weight; Col-0: WT plants; L1–L4, transgenic plants. C, Total carotenoid content in 2-week-old Col-0 and transgenic plants. Carotenoid quantification was carried out with two biological replicates (n = 3) for each line. Values are means ± SE. Concentration is presented in microgram per gram of DW. In (A–C): asterisks indicate significant differences between WT and trans- genic plants performed by one-way ANOVA analysis with Dunnett’s post-hoc test; ****P 5 0.0001, ***P 5 0.001, **P 5 0.01, *P 5 0.1. that a substantial reduction in carotenoid levels might have 8 months of culture. With the purpose of verifying that the occurred (Figure 6B). In addition, expression of key genes re- taproot phenotype in silenced plants persisted in time, quired for carotenoid synthesis, DcPSY1, DcPSY2, DcLCYE, plants were grown for four additional months (except the DcLCYB2, DcCHXB1, and DcCHXB2, was significantly lower in AS10 line that was harvested before and could not be recov- all silenced lines relative to the WT, showing a reduction be- ered). We observed that, although the size of transgenic tween 40% and 99% (Figure 6C). Moreover, a 50%–80% de- roots increased during the 4 months, they were still smaller crease in total carotenoid levels in all silenced lines was also and thinner than the WT taproots and presented a greenish obtained (Figure 6D). Likewise, all the transgenic lines coloration in the external part of the taproot and a yellow- showed a 4- to 60-fold decrease in a-carotene and 4- to 58- ish coloration in the taproot center (Figure 7A). The relative fold in b-carotene (Figure 6E). However, an unexpected 6- expression level of DcPAR1 in 8 months transgenic lines to 15-fold increase in lutein levels was obtained (Figure 6E). remained reduced between 60% and 75% (Figure 7A). This pigment accumulates in photosynthetic tissues and in Likewise, the relative expression of key carotenogenic genes the WT carrot roots that grow exposed to light but not in in transgenic lines was still lower than in WT plants the orange carrot taproot that grows underground (Fuentes (Figure 7B), confirming that DcPAR1 regulates the expression et al., 2012). of carotenogenic genes in carrot root. In our experience, WT plants present a similar taproot Carotenoids and chlorophylls quantification showed that phenotype and carotenoid content between three to DcPAR1as lines presented 2.4-fold fewer total carotenoids 1456 | PLANT PHYSIOLOGY 2022: 189; 1450–1465 Arias et al. Downloaded from https://academic.oup.com/plphys/article/189/3/1450/6546316 by guest on 26 May 2023 Figure 5 Phenotype of mature transgenic Arabidopsis lines overexpressing DcPAR1. A, Picture showing the length and phenotype of representa- tive 6-week-old plants of Col-0 and T3 transgenic lines. B, Stem and (C) Silique length of 6-week-old Col-0 and T3 transgenic plants. Each value is the mean ± SE/SD of five plants each and using ImageJ program. Asterisks indicate significant differences between WT and transgenic plants per- formed by one-way ANOVA analysis with Dunnett’s post-hoc test; ****P 5 0.0001, ***P 5 0.001, **P 5 0.01, *P 5 0.1. (Figure 7C) and an increase of three- to five-fold to eight- as in the root that grew in light (Arias et al., 2020). PAR1 is fold in chlorophyll a and b reaching 100–250 mg g–1 DW a recognized negative regulator of the shade avoidance syn- (Figure 7D), suggesting that the absence of PAR1 leads to drome (SAS) and a positive factor during seedling de- chloroplast instead of chromoplasts differentiation. Likewise, etiolation in Arabidopsis (Roig-Villanova et al., 2007; Hao transgenic antisense plants presented a 3.5- to 14-fold re- et al., 2012; Zhou et al., 2014). In shade, the expression of duction in a-carotene and three- to nine-fold reduction in PAR1, PAR2, and the LONG HYPOCOTYL IN FAR RED 1 is in- b-carotene levels with respect to WT plants. As in younger duced, factors that encode transcriptional bHLH cofactors taproots, a significant increase in lutein was also observed, that participate in the compensation of excessive hypocotyl which reached up to 200–250 mg g–1 DW (compared to elongation during SAS (Roig-Villanova et al., 2007; Bou- 30 mg g–1 DW in WT) (Figure 7E), thus explaining the yel- Torrent et al., 2008; Hao et al., 2012; Zhou et al., 2014). lowish coloration of antisense transgenic taproots with re- From these factors, PAR1 interacts with several bHLH pro- spect to the orange pigmentation of WT (Figure 7A). Taking teins that act as positive regulators of SAS such as BR- all the results together, we suggest that DcPAR1 is a func- ENHANCED EXPRESSION (BEE), BES1-INTERACTING MYC- tional cofactor that has an important regulatory role not LIKE (BIM), and PIFs by partially preventing the binding of only in carotenoid synthesis in carrot taproot that grows un- these transcription factors to LREs of photomorphogenic derground, but also in secondary taproot development. genes (Roig-Villanova et al., 2006, 2007; Hao et al., 2012; Cifuentes-Esquivel et al., 2013; Bou-Torrent et al., 2015; Discussion Ballare and Pierik, 2017; Fernández-Milmanda and Ballare, DcPAR1 boosts carotenoid synthesis and promotes a 2021; Quian-Ulloa and Stange, 2021). Importantly, PAR1 pro- photomorphogenic phenotype in Arabidopsis motes PSY expression in leaves under shade (Bou-Torrent Carotenoid biosynthesis regulation in plants has been exten- et al., 2015) and, of the positive SAS regulators BEEs, BIMs, sively studied. Although there is much information about and PIFs, only PIFs participate in repressing PSY expression how this process is regulated by several factors, there is still and carotenoid synthesis in the shade (Toledo-Ortiz et al., much to explore with respect to how light regulates carot- 2010). PIFs remain stable in the shade condition and bind to enoid biosynthesis in plant organs that normally grow with the LRE motifs (Lorrain et al., 2008; Hornitschek et al., 2012). no direct W, such as roots (Stanley and Yuan, 2019; Quian- Arabidopsis RNAi-PAR1 and par2 mutant lines have longer Ulloa and Stange, 2021). With the purpose to understand roots than WTs. This suggest that AtPAR1, and its homolog how carotenoids are synthesized in orange carrot taproot, AtPAR2 (Roig-Villanova et al., 2006, 2007), are active in roots we compared through RNA-seq the expression profile of and necessary for their correct development (Supplemental carrot roots that grew in W with respect to those that grew Figure S7). underground (Arias et al., 2020). We identified several pho- AtPAR1 is a nuclear and atypical bHLH, it presents a func- tomorphogenic genes that were upregulated in the under- tional HLH protein domain, but it lacks a functional DNA- ground condition and among them, DcPAR1, DcPHYA, and binding domain, so its regulatory function is performed DcPIF4 presented twice as much expression level in darkness through the dimerization with PIFs transcription factors, DcPAR1 is a key factor in taproot development PLANT PHYSIOLOGY 2022: 189; 1450–1465 | 1457 Downloaded from https://academic.oup.com/plphys/article/189/3/1450/6546316 by guest on 26 May 2023 Figure 6 Phenotype, expression analysis, and carotenoid content in 4-month-old DcPAR1as transgenic carrot taproots. A, Relative expression level of DcPAR1 in AS1, AS5, AS6, and AS10 transgenic lines. The analysis was performed with three biological replicates (three different samples from the same transgenic lines) and two technical repeats each and normalized to the housekeeping Ubiquitin gene. WT was used as calibrator. B, Representative phenotype of a WT and a DcPAR1as transgenic carrot root (line AS10) grown 4 months underground after transplanting. Bar: 1 cm for left and center. Bar: 0.5 cm for the magnified version on the right. C, Relative expression level of DcPSY1, DcPSY2, DcLCYE, DcLCYB2, DcCHXB1, and DcCHXB2. The relative expression was carried out with three biological replicates (three different samples from the same transgenic lines) and two technical repeats each and normalized to the housekeeping Ubiquitin gene. WT was used as calibrator. D, Total carotenoids were quantified in a spectrophotometer at 474 nm and (E) lutein, a-carotene, and b-carotene levels were determined by HPLC-DAD. Pigment content is indicated in microgram per gram of DW and was measured with three biological replicates (three different samples from the same transgenic lines). In (A) and (C–E): Values are means ± SE. Asterisks indicate significant differences between transgenic lines and WT plant determined by one-way ANOVA analysis with Dunnett’s post-hoc test; ****P 5 0.0001, ***P 5 0.001, **P 5 0.01, *P 5 0.1. such as AtPIF4 and AtPIF1 (Roig-Villanova et al., 2007; Hao Figure 2B). Likewise, the essential amino acids that deter- et al., 2012; Bou-Torrent et al., 2015). mine the protein–protein interactions are still conserved in Our bioinformatic analysis indicated that DcPAR1 has a DcPAR1 (Heim et al., 2003; Figure 2B) which in functionality similar structural pattern to AtPAR1, especially at the bHLH terms allows DcPAR1 to interact with other bHLH proteins, domain, suggesting that it could fulfill similar functions. such as itself and AtPIF7 (Figure 3), an essential characteris- Importantly, the key amino acids at the basic domain that tic for a functional PAR1. determines the binding to DNA are also replaced by similar AtPAR1 interacts with AtPIF1, and consequently induces amino acids that are present in AtPAR1 (Heim et al., 2003; the expression of AtPSY, thus increasing the content of 1458 | PLANT PHYSIOLOGY 2022: 189; 1450–1465 Arias et al. Downloaded from https://academic.oup.com/plphys/article/189/3/1450/6546316 by guest on 26 May 2023 Figure 7 Phenotype, expression analysis, and pigment content of 8-month-old DcPAR1as transgenic carrot taproots. A, Representative phenotype of carrot taproots of a WT and AS1, AS5, and AS6 DcPAR1as transgenic plants grown 8 months underground after transplanting (line AS10 was harvested at 4 months and not shown here). Bar: 1 cm. Graph in the right shows the relative expression level of DcPAR1 in the indicated lines. Analysis was performed with three biological replicates (three different samples from the same transgenic lines) and two technical repeats each and normalized to the housekeeping Ubiquitin gene. B, Relative expression level of DcPSY1, DcPSY2, and DcLCYB2. Relative expression was carried out with three biological replicates (three different samples from the same transgenic lines) and two technical repeats each and normalized to the housekeeping Ubiquitin gene. C, Total carotenoids levels and (D) cholorophylls a and b in WT, AS1, AS5, and AS6 DcPAR1as carrot taproots. E, Lutein, a-carotene, and b-carotene levels was determined by HPLC-DAD. Pigment content is indicated in microgram per gram of DW and was measured in triplicate, with three biological replicates (three different samples from the same transgenic lines). In (A–E): values are means ± SE. Asterisks indicate significant differences between transgenic lines and WT determined by one-way ANOVA analysis with Sidak post-hoc test; ****P 5 0.0001, ***P 5 0.001, **P 5 0.01, *P 5 0.1. carotenoids together with the promotion of a photomor- AtPAR1 was not affected suggesting that PAR1 does not in- phogenic phenotype (Roig-Villanova et al., 2007; Bou- duce its own expression in Arabidopsis. It remains to be elu- Torrent et al., 2015). Interestingly, Arabidopsis plants overex- cidated whether the DcPAR1 protein would be promoting pressing DcPAR1-GFP and grown in W, presented a stable AtPSY transcription through binding exclusively to PIF-type accumulation of DcPAR1-GFP, suggesting that it is stable in transcription factors or if there are other transcription fac- W, similarly to AtPAR1 that is stable in low and high R:FR, tors that may be involved in this process, such as BEEs and but not in the dark (Zhou et al., 2014). The higher DcPAR1- BIMs (Cifuentes-Esquivel et al., 2013). GFP accumulation correlates statistically with an increase in Moreover, according to the dwarf phenotype of AtPSY relative expression levels and in PSY protein with a fi- Arabidopsis lines overexpressing AtPAR1 (Roig-Villanova nal rise in total carotenoids (Figure 4C), which strongly sug- et al., 2007), 2-week-old DcPAR1 transgenic lines presented gest a positive role of DcPAR1 on carotenoids synthesis in several characteristics of a pronounced photomorphogenic Arabidopsis. Indeed, the expression of the endogenous phenotype (Supplemental Figure S5) that remains until DcPAR1 is a key factor in taproot development PLANT PHYSIOLOGY 2022: 189; 1450–1465 | 1459 1.5 months (Figure 5). Given these results, we strongly sug- The posttranscriptional gene silencing of DcPAR1 in car- gest that DcPAR1 and AtPAR1 are orthologs. rots is a better strategy to determine the role of the endoge- nous gene in carrots. DcPAR1 antisense had important DcPAR1 positively regulates carotenoid synthesis in consequences on the carotenoid levels (Figures 6 and 7), carrot taproot which decrease significantly in correlation to a decrease in Considering that AtPAR1 positively regulates the carotenoids the expression of all carotenogenic genes analyzed, including synthesis in Arabidopsis photosynthetic tissue (Roig- both, DcPSY1 and DcPSY2 (Figures 6 and 7). Similar results Villanova et al., 2007; Bou-Torrent et al., 2015) we aimed to were reported in DcLCYB1 antisense carrots (Moreno et al., determine the in vivo role of AtPAR1 in carotenoid synthesis 2013), where a decrease of 40%–80% in total carotenoid lev- in the carrot taproot. els together with a decrease in the expression levels of Carrot AtPAR1-GFP lines with the highest transgene ex- DcPSY1 and DcPSY2 was observed (Moreno et al., 2013). pression (OE1 and OE2) presented a significant increase in Moreover, DcPAR1as lines showed a dramatic reduction in Downloaded from https://academic.oup.com/plphys/article/189/3/1450/6546316 by guest on 26 May 2023 the relative expression levels of DcPSY1, but not in DcPSY2 the expression on all carotenogenic genes evaluated. and DcPAR1, in a significant correlation with an increase in Possibly, DcPAR1 has the ability to bind to PIFs or other un- carotenoid levels in the taproot of mature carrots (Figure 1) known transcription factors some of which would directly but not in total carotenoid level in leaves (Supplemental downregulate or upregulate DcPSY1, DcPSY2, DcLCYB2, Figure S8), suggesting that AtPAR1:GFP is sufficient and spe- DcLCYE, DcCBHX1, and/or DcCBHX2. It is also possible that cific to increase carotenoid production in taproots. DcPAR1–DcPIFs complex exerts a direct regulation upon Interestingly, the transgenic lines with the lower transgene DcPSYs and their activation has a direct impact on the other expression level (OE5 and OE6), produced a significant re- genes in the pathway. To establish a more in-depth explana- duction in DcPSY1 (except for OE5), DcPSY2, and DcPAR1 tion in this regard, it is necessary to determine which other expression but without affecting the level of carotenoids, factors, in addition to PIFs, are binding to PAR1 in carrots which remains similar to the WT. The decrease in the rela- during development and which transcription factors bind to tive expression of DcPAR1 may be an endogenous compen- promoters of carotenogenic genes, regulating their expres- sation mechanism leading to speculate that AtPAR1 may sion. In Arabidopsis, ELONGATED HYPOCOTYL 5 (HY5) regulate the expression of its ortholog gene in carrot. Given binds to PIFs and also competes with PIFs for binding to these results, we suggest a positive regulation of AtPAR1 on LRE, promoting PSY expression and inducing the differentia- DcPSY1 but not on DcPSY2, and that the effect generated is tion of etioplasts into chloroplasts with increased carote- due to the expression of AtPAR1 and not because of the en- noids and chlorophylls in plants (Von Lintig et al., 1997; dogenous gene. Ronen et al., 1999; Woitsch and Römer, 2003; Toledo-Ortiz The regulation of AtPAR1 on the different DcPSY homo- et al., 2010). It has been proposed that there would be a ge- logs may depend on the transcription factors that bind to netic interaction between AtPAR1 and AtHY5 because dou- their promoters that can be regulated by AtPAR1. It could ble antisense-PARs/hy5 mutants present a more elongated be possible that AtPAR1 dimerizes to some endogenous car- hypocotyl than simple mutants (Zhou et al., 2014). rot PIFs that bind preferably to DcPSY1 rather than to Therefore, HY5 role in PAR1 antisense lines could be ex- DcPSY2 promoter, and the higher AtPAR1 abundance may plored. Nevertheless, these results support the hypothesis heterodimerize with and prevent this type of PIFs binding to that DcPAR1 would be a positive regulator of carotenoid DcPSY1 promoter. Indeed, LREs are located in the DcPSY1 synthesis in carrot taproot. and DcPSY2 promoters (Simpson et al., 2018). It may be in- Carrot DcPAR1as lines produce a significant increase in lu- teresting to determine which transcription factors directly tein (in 4- and 8-month-old carrot taproots) similar to WT bind to the promoters of DcPSY1 and DcPSY2 in carrots. carrot roots grown in light, but absent in taproots grown Since DcPSY2 is mostly induced by salt stress and ABA and underground (Fuentes et al., 2012; Arias et al., 2020). Most that AREB transcription factors are able to bind to DcPSY2 confusing is that DcCHXB1 and DcCHXB2 involved in lutein promoter (Simpson et al., 2018), we suggest that DcPSY2, synthesis are deeply downregulated (Figure 6C), suggesting and not DcPSY1, is induced under abiotic stress (Simpson that the enzymes may be stabilized by an unknown et al., 2018). It remains to be determined whether DcPSY1 mechanism. could be most associated to carotenoid synthesis during In addition, 8-month-old DcPAR1as lines accumulate or- taproot development. On the other hand, in transgenic car- ange pigmented carotenoids in the phloem, while cortex rots that overexpress the Arabidopsis 1-deoxyxy-lulose-5- and xylem display yellow/green pigmentation, a differential phosphate synthase (a gene that participates in the synthe- effect depending on the root tissue. Moreover, an increase sis of the metabolic precursors for carotenoids) an increase in chlorophylls is also observed that are normally absent in in carotenoid levels was observed also with a significant cor- WT taproots. These results suggest that DcPAR1 is required relation with an increase in the expression of DcPSY1 and for correct carotenoid synthesis. Indeed, DcPAR1as lines pre- DcPSY2 (Simpson et al., 2016b) suggesting that both genes sent chlorophylls similar than the WT roots that were determine the levels of carotenoids in carrot taproots. grown in W (Fuentes et al., 2012) in which DcPAR1 1460 | PLANT PHYSIOLOGY 2022: 189; 1450–1465 Arias et al. expression is lower than that in the taproot grown under- et al., 2001). Considering that DcPAR1as lines present a ground (Arias et al., 2020). short and thin taproot with more lateral root abundance, we propose that DcPAR1 may promote auxin metabolism DcPAR1 positively regulates carrot taproot de-repression. Therefore, it remains unknown if DcPAR1 development could have a role in hormone signaling during taproot It has been shown that the accumulation of carotenoids in development. carrot roots is genetically associated with a homolog of the Based on our results, we propose a simple model regard- Arabidopsis PSEUDO-ETIOLATION IN LIGHT (PEL) gene ing DcPAR1 role in carotenoid synthesis and taproot devel- (Iorizzo et al., 2016). PEL presumably acts as a repressor of opment (Figure 8). Exposure of the orange carrot roots to photomorphogenesis. Carrot varieties with a loss-of-function light (R/L) decreases DcPAR1 transcripts levels in the nu- allele of the PEL gene accumulate carotenoids in the root, cleus, where PIFs are recruited and inactivated by PHYs, and suggesting that high pigment contents might result from a competing with HY5 for binding to LRE, causing a low ex- pression of carotenogenic genes such as DcPSYs. This leads Downloaded from https://academic.oup.com/plphys/article/189/3/1450/6546316 by guest on 26 May 2023 derepressed development of carotenoid-accumulating plas- tids (i.e. chloroplasts in the light but chromoplasts in the to a reduced amount of PSY protein accumulation in plas- dark; Llorente et al., 2017). Our results support this possibil- tids, which in turn generated a drop in total carotenoid ac- ity and go further in proposing DcPAR1 as an antagonistic cumulation with reduced a-carotene and b-carotene levels factor of PEL1. Indeed, the expression of AtPAR1 in carrots but an increase in lutein and chlorophylls. This might result produces orange embryo showing an early and positive ef- in chloroplast (rather than chromoplasts) differentiation and fect of PAR1 on carotenoid synthesis (Figure 1) but it did impairment in taproot development. On the contrary, in not generate an evident change in photomorphogenic phe- roots grown underground (e.g. covered with soil; R/D), the notype at the mature stage. higher transcripts levels of DcPAR1 may conduct to a higher Although AtPAR1 antisense produced elongated and recruitment and inactivation of PIFs, promoting an increased hyper-etiolated plants (Zhou et al., 2014), carrot DcPAR1as DcPSYs expression given by HY5. lines presented similar embryogenic and photomorphogenic As a consequence, high levels of PSY accumulate in plas- development than control plants, although they presented tids that lead to an increase in carotenoids synthesis (espe- up to 95% of DcPAR1 reduced expression. However, the cially a-carotene and b-carotene) that would promote consequences in the taproot of DcPAR1as adult plants were chromoplast differentiation and secondary root develop- drastic, showing a dramatic reduction in the size (thickness ment. Indeed, it was shown recently that synthetically in- and length) and weight of the taproot in 4-month-old ducing a burst in the production of phytoene (the product plants, a tendency that was maintained at 8 months of de- of the PSY enzymes and the first committed intermediate of velopment. The phenotype obtained is partly reminiscent of the carotenoid pathway) elicits an artificial chloroplast-to- that observed when silencing DcLCYB1, where the roots gen- chromoplast differentiation in leaves (Llorente et al., 2020). erated were thinner than the controls (Moreno et al., 2013) Altogether, these results suggest that DcPAR1 is a key factor but less drastic than DcPAR1as, showing a relevant role for for secondary root development and carotenoid synthesis in DcPAR1 in carrot taproot development. carrot taproot grown underground. Carrot root development has been previously reported to be closely related to carotenoid synthesis (Suslow et al., Conclusions 1999; Clotault et al., 2008). Thus, it is possible that both pro- DcPAR1, like its Arabidopsis counterpart AtPAR1, induces cesses are being regulated by similar factors at the transcrip- AtPSY expression, boosts carotenoid synthesis, and produces tional or posttranscriptional level. PAR1 seems to be a good a dwarf phenotype when overexpressed in A. thaliana while candidate to participate in both processes. These results and in carrot, DcPAR1 positively regulates carotenoid synthesis those reported in Arabidopsis suggest that PAR1 has an im- and secondary root development in carrot taproot grown portant regulatory role on carotenoid synthesis and develop- underground. ment. Considering that PAR1 is upstream of the pigment synthesis pathway, it has a broader functional role on the Materials and methods carotenoid synthesis in parallel with the regulation of root Bioinformatic analysis development in the absence of W. AtPAR1 reduces hypo- National Center for Biotechnology Information (NCBI) plat- cotyl elongation in shade through the repression of auxin- form was used to obtain the complete CDS of DcPAR1 using induced genes such as the SMALL AUXIN UP RNA15 the contig 42,760 (202 bp) that corresponds to DcPAR1 and (SAUR15) and SAUR68 likely through their ability to inhibit was obtained from the RNA-seq analysis (Arias et al., 2020). DNA binding of PIFs or other bHLH to its binding motifs in Conserved domain search analysis was performed using the SAUR15 and SAUR68 promoters (Roig-Villanova et al., the InterPro Scan (https://www.ebi.ac.uk/interpro/search/se 2006, 2007; Bou-Torrent et al., 2008, 2015; Hao et al., 2012; quence-search) and SMART (http://smart.embl-heidelberg. Zhou et al., 2014). Moreover, some of the phenotypes of de/) in order to identify the bHLH functional regions in AtPAR1 overexpressing lines are similar to those reported in DcPAR1. Sequence alignments were performed using Clustal auxin, brassinosteroid, or gibberellin mutants (Nakazawa Omega platform (https://www.ebi.ac.uk/Tools/msa/clustalo/). DcPAR1 is a key factor in taproot development PLANT PHYSIOLOGY 2022: 189; 1450–1465 | 1461 Downloaded from https://academic.oup.com/plphys/article/189/3/1450/6546316 by guest on 26 May 2023 Figure 8 Proposed model depicting the role of DcPAR1 in carrot taproot development and carotenoid synthesis. Roots grown exposed to W (R/ L) present reduced expression of DcPAR1 in the nucleus. We propose that PIFs are inactivated by PHYs and that HY5 competes with PIFs for bind- ing to LRE, causing a low expression of carotenogenic genes such as DcPSYs. As a consequence, low level of PSY protein accumulates in plastids, producing reduced levels of total carotenoids, a-carotene, and b-carotene accumulation but higher levels of chlorophylls and lutein, correspond- ing with chloroplast differentiation. Taproot development is also impaired. On the contrary, in the nucleus of roots grown underground (R/D), a high expression levels of DcPAR1 takes place that permits a higher recruitment and inactivation of PIFs, promoting an increased DcPSYs expression given by HY5. Therefore, high levels of PSY accumulate in plastids leading to increased carotenoids synthesis corresponding with chromoplast dif- ferentiation and secondary taproot development. DcPAR1 gene amplification and vector construction sucrose and 0.7% w/v agar) and grown in a culture The complete CDS sequence of PAR1 of carrot (D. carota) chamber under 16-h long-day photoperiod at 23 C–25 C var. Nantaise (363 pb without stop codon) (NCBI access (W). Plants of 10- to 20-d postgermination were used for number XM_017390696.1) was amplified from carrot taproot Agrobacterium-mediated transformation as described in cDNA, using Herculase II Fusion DNA Polymerase (Agilent Gonzalez-Calquin and Stange (2020). Briefly, the epicotyls of Technologies, Santa Clara, CA, USA) and DcPAR1.F and WT plants grown in vitro, were cut and co-cultivated in DcPAR1.R primers (Supplemental Table S1). The sequence presence of A. tumefaciens strain GV3101 carrying the was cloned into the entry PCR8/GW/TOPO vector DcPAR1-GFP, DcPAR1as, or AtPAR1:GFP vectors and placed (Invitrogen, Waltham, MA, USA) according to manufacturer’s on solidified MS in darkness. After 2 d, the explants were instructions and sequenced (Macrogen Corp., Rockville, MD, transferred to MS culture medium supplemented with 1 mg USA). Clones of PCR8:DcPAR1 in sense and antisense orien- L–1 of 2,4-D, 50 mg L–1 of Hygromycin (for transgene selec- tation were recombined with the destination vector pGWB5, obtaining the binary vectors pG5_35S:PAR1:GFP (sense tion), and 200 mg L–1 of Timentin (for Agrobacterium con- DcPAR1-GFP) and pG5_35S:PAR1as (antisense DcPAR1as) trol) for embryo development. After 4–5 weeks in darkness, that were used to overexpress and silence DcPAR1, respec- the explants were transferred to MS culture medium supple- tively. The pBF1_35S:AtPAR1-GFP binary vector was selected mented with 0.5 mg L–1 of 2,4-D, 100 mg L–1 of Hygromycin to overexpress AtPAR1 as a fusion to the GFP reporter gene and 200 mg L–1 of Timentin and grown in W (16-h long-day (AtPAR1-GFP) and has been described elsewhere (Roig- photoperiod at 23 C–25 C) for 7 weeks. Finally, the Villanova et al., 2007). All binary plasmids were transformed hygromycin-resistant embryos were transferred to MS cul- into Agrobacterium tumefaciens strain GV3101 for stable ture medium without hormones to promote elongation of Arabidopsis (A. thaliana) and carrot transformation. carrot seedlings. Plants of 5–7 cm length were transferred to a mixture of soil: vermiculite (1:1) and maintained under the Agrobacterium-mediated transformation of same photoperiod and temperature conditions described D. carota and A. thaliana above. Transgenic plants were selected by amplifying the re- Commercial seeds of D. carota var. Nantaise were surface- spective transgene form genomic DNA:AtPAR1 gene in the sterilized and sown in culture medium Murashige and Skoog case of carrot plants transformed with pBF1:AtPAR1:GFP (MS; 4.4 g L–1 MS salts; Murashige and Skoog, 1962, 20 g L–1 (Bou-Torrent et al., 2015) with AtqPAR1.F and AtqPAR1.R 1462 | PLANT PHYSIOLOGY 2022: 189; 1450–1465 Arias et al. primers (Supplemental Table S1), DcPAR1 gene in the case with CTAB buffer (2% (w/v) CTAB, 2% (w/v) PVP40, 25-mM of Arabidopsis plants using primers DcqPAR1.F and ethylenediaminetetraacetic acid (EDTA), 2-M NaCl, 100-mM DcqPAR1.R (Supplemental Table S1) or including GFP ampli- Tris–HCl (pH 8.0), and 0.05% spermidine trihydrochloride) fication in the case of carrot plants transformed with and precipitated with LiCl (10 M) overnight. RNA was resus- pG5:PAR1as, using primers DcPAR1.R and qEGFP.R pended in nuclease-free water and the genomic DNA traces (Supplemental Table S1). were eliminated by a 40 min DNAse I treatment. For cDNA Arabidopsis thaliana WT plants (Col-0 ecotype) were synthesis, 3 mg of total RNA was incubated with 1 mM of transformed using floral dip (Zhang et al., 2006). Seeds were Oligo-AP primer (Supplemental Table S1) and Improm II re- surface sterilized in a solution of sodium hypochlorite (50% verse transcriptase (Promega) according to the manufac- v/v) for 6 min, washed 3 times with sterile water and sown turer’s recommendations. Real time expression (RT-qPCR) in solid MS medium (4.4 g L–1 MS salts, 0.44% vitamins, was performed as described in Fuentes et al. (2012) and 0.01% myo-inositol and 0.7% agar pH 5.8 with or without se- Moreno et al. (2013) in a Stratagene Mx3000P thermocycler, lection). They were kept in a growth chamber with a 16-h using SYBR Green double-strand DNA-binding dye. Downloaded from https://academic.oup.com/plphys/article/189/3/1450/6546316 by guest on 26 May 2023 long-day photoperiod illuminated with cool-white fluores- AtqPAR1.F and AtqPAR1.R primers were used to amplify a cent light (115 -lmol m2s–1) at 22 C for 2–6 weeks depend- specific fragment of the coding sequences of AtPAR1 ing on the experimental assay. Selected transgenic lines were (AT2G42870) and DcqPAR1.F and DcqPAR1.R for DcPAR1 transferred to a greenhouse in a mixture of soil:vermiculite (XM_017390696.1; Supplemental Table S1). DcqUbi.F and (1:1) for molecular analysis. The selection of T3 homozygous DcqUbi.R primers were used to amplify Ubiquitin and transgenic lines was carried out by cultivation of selected AtqPP2A.F and AtqPP2A.R to amplify PP2A housekeeping transgenic T1, T2 and then T3 seeds in MS medium with genes. Specific primers of carotenogenic genes DcPSY1, hygromycin (15-lg mL–1) and selected those T3 with up to DcPSY2, DcLCYB2, DcLCYE, DcCHXB1, DcCHXB2, and AtPSY 95% survival for further molecular, biochemical, and pheno- were the same as described in Fuentes et al. (2012) and typical analyses. summarized in Supplemental Table S1. The RT-qPCR analy- sis was performed with three biological replicates and two Pigments extraction and quantification technical repeats and all reaction specificities were tested In carrot and Arabidopsis, carotenoid and chlorophyll ex- with melting gradient dissociation curves. To test for signifi- traction was carried out as described previously (Simpson cant differences in gene expression, results were subjected et al., 2016b). Pigments were extracted from 20 to 100 mg of to a one-way analysis of variance (ANOVA) (P 5 0.05, confi- fresh carrot transgenic taproots and 100 mg of entire 2- dence interval 95%) with Dunnett’s post-hoc test according week-old Arabidopsis T3 lines with 4 mL of hexane:aceto- to the General Linear Models option in the statistical soft- ne:ethanol (2:1:1 v/v). After centrifugation, the upper phase ware package Graphpad Prism. The relative transcript levels was recovered and dried with N2. For total carotenoids and were obtained using the PfaffI equation (Pfaffl, 2001). chlorophylls quantification, pigments were resuspended in 1 mL of acetone and quantified in a spectrophotometer BiFC assay (Shimadzu) at 750, 662, 645, and 474 nm in quartz cuvettes. The PCR8:DcPAR1 vector was recombined with BiFC vectors Absorbance at 662, 645, and 474 nm permitted to deter- (Gateway-based BiFC binary vectors, pYFC43 and pYFN43; mine the concentration of chlorophyll a, chlorophyll b, and Belda-Palazón et al., 2012), obtaining N-YFP:DcPAR1 and C- total carotenoids, respectively. The absorbance at 750 nm YFP:DcPAR1. The BiFC binary vectors (Belda-Palazón et al., determines the turbidity of the sample which may result in 2012) and N-YFP:AtPIF7 and C-YFP:AtPIF7 vectors were pro- underestimation of the pigment concentration. Then, con- vided by Dr Jaume Martınez-Garcıa (IBMCP CSIC-UPV, centration of chlorophyll a, chlorophyll b and total carote- Valencia, Spain). The four vectors were transformed in noids was determined as described previously (Lichtenthaler Agrobacterium GV3101 and used for N. benthamiana agroin- and Buschmann, 2001). Individual carotenoids were quanti- filtration (Simpson et al., 2018). Four days after injection, fied in a Shimadzu HPLC (LC-10AT) with a diode array de- YFP fluorescence was detected under a LEICA TCS SP5 con- tector using a RP-18 Lichrocart 125-4 reverse phase column focal microscope with a light excitation wavelength of (Merck, Kenilworth, NJ, USA) and a mobile phase composed 488 nm and a filter for YFP at 520–560 nm. The images were of acetonitrile:methanol:isopropanol (85:10:5 v/v). The sepa- obtained and processed with Leica LAS AF Lite software. ration of carotenoids and chlorophyll pigments was carried out with a 1.5 mL min–1 flow rate, at room temperature, Arabidopsis protein extraction and western blot and under isocratic conditions. The identification of the pig- The Arabidopsis protein extraction was performed according ments was performed as described by Simpson et al. to Saucedo et al. (2019) with modifications. Briefly, 400 mg (2016b). of fresh tissue of entire 2-week-old Arabidopsis plants were pulverized with liquid nitrogen and then 4 mL of extraction RNA extraction and RT-qPCR buffer [0.5 M Tris–HCl pH 8, 0.7 M Sucrose, 2 protease inhib- RNA was obtained from 20 to 100 mg of fresh carrot tap itor tablets (complete Mini, EDTA-free-Thermo Scientific Lot roots or leaves and entire 2-week-old Arabidopsis plants # UG27666820), 50- mM EDTA, KCl 0.1 M, b-mercaptoetha- that were pulverized with liquid nitrogen, homogenized nol 0.2%)] were added and homogenized until the sample DcPAR1 is a key factor in taproot development PLANT PHYSIOLOGY 2022: 189; 1450–1465 | 1463 was completely thawed. Then, 2 mL of basic saturated phe- required for carotenoid synthesis in the dark-grown carrot taproot. Mol Genet Genomics 295: 1379–1392 nol pH 8.0 (Winkler, Hackensack, NJ, USA; # 20192088) were Bae G, Choi G (2008) Decoding of light signals by plant phyto- added, shaken 10 min in ice, and centrifuged at 8,000 g for chromes and their interacting proteins. Annu Rev Plant Biol 59: 19 min at 4 C. The supernatant was recovered and four vol- 281–311 umes of 0.1-M ammonium acetate in methanol (Merck gra- Ballare CL, Pierik R (2017) The shade-avoidance syndrome: multiple dient for liquid chromatography) were added, and proteins signals and ecological consequences. Plant Cell Environ 40: 2530–2543 were precipitate overnight at –20 C. After centrifugation, Belda-Palazón B, Ruiz L, Martı E, Tárraga S, Tiburcio AF, Culiá~ nez F, the pellet was washed with 0.1-M ammonium acetate in Farràs R, Carrasco P, Ferrando A (2012) Aminopropyltransferases in- methanol, and with acetone. The remaining acetone was volved in polyamine biosynthesis localize preferentially in the nucleus evaporated at room temperature and the proteins (pellet) of plant cells. PLoS One 7: e46907 were stored at –20 C in resuspension buffer (100-mM Tris– Bianchetti ER, Silvestre Lira B, Santos Monteiro S, Demarco D, Purgatto E, Rothan C, Rossi M, Freschi L (2018) Fruit-localized HCl pH 7.0, 1% SDS). The extraction was carried out in trip- phytochromes regulate plastid biogenesis, starch synthesis, and ca- licate for each transgenic line and control. Downloaded from https://academic.oup.com/plphys/article/189/3/1450/6546316 by guest on 26 May 2023 rotenoid metabolism in tomato. J Exp Bot 69: 3573–3586 Bou-Torrent J, Roig-Villanova I, Galstyan A, Martınez-Garcıa JF Accession numbers (2008) PAR1 and PAR2 integrate shade and hormone transcrip- Sequence data from this article can be found in the tional networks. Plant Signal Behav 3: 453–454 GenBank/EMBL data libraries under the accession numbers Bou-Torrent J, Toledo-Ortiz G, Ortiz-Alcaide M, Cifuentes- indicated in Supplemental Figure S2. Esquivel N, Halliday KJ, Martinez-Garcıa JF, Rodriguez- Concepcion M (2015) Regulation of carotenoid biosynthesis by shade relies on specific subsets of antagonistic transcription factors Supplemental data and cofactors. Plant Physiol 169: 1584–1594 The following materials are available in the online version of Chen F, Li B, Li G, Charron JB, Dai M, Shi X, Deng XW (2014) this article. Arabidopsis phytochrome a directly targets numerous promoters Supplemental Table S1. Primers used in this work. for individualized modulation of genes in a wide range of path- ways. Plant Cell 26: 1949–1966 Supplemental Figure S1. Expression of DcPAR1 in carrot Cifuentes-Esquivel N, Bou-Torrent J, Galstyan A, Gallemı M, Sessa lines overexpressing AtPAR1. G, Salla Martret M, Roig-Villanova I, Ruberti I, Martınez-Garcıa Supplemental Figure S2. Sequences of contig 42.760 JF (2013) The bHLH proteins BEE and BIM positively modulate the (Arias et al., 2020) and complete CDS of the DcPAR1 gene shade avoidance syndrome in Arabidopsis seedlings. Plant J 75: (access number XM_017390696.1). 989–1002 Clotault J, Peltier D, Berruyer R, Thomas M, Briard M, Geoffriau Supplemental Figure S3. Subcellular localization of E (2008) Expression of carotenoid biosynthesis genes during carrot DcPAR1:GFP. root development. J Exp Bot 59: 3563–3573 Supplemental Figure S4. Sodium dodecyl sulfate–poly- De Wit M, Galv~ao VC, Fankhauser C (2016) Light-mediated hor- acrylamide gel electrophoresis (SDS–PAGE) of Arabidopsis monal regulation of plant growth and development. Annu Rev DcPAR1 transgenic lines. Plant Biol 67: 513–537 Supplemental Figure S5. Phenotypic analysis of 2-week- Fernández-Milmanda GL, Ballare CL (2021) Shade avoidance: expanding the color and hormone palette. Trends Plant Sci 26: old Arabidopsis transgenic lines that overexpress DcPAR1. 509–523 Supplemental Figure S6. Transgenic selection of Fiorucci AS, Galv~ao VC, Ince YÇ, Boccaccini A, Goyal A, DcPAR1as carrot lines. Allenbach Petrolati L, Trevisan M, Fankhauser C (2020) Supplemental Figure S7. Root length of Arabidopsis PHYTOCHROME INTERACTING FACTOR 7 is important for early seedlings with reduced levels in AtPAR1 and AtPAR2. responses to elevated temperature in Arabidopsis seedlings. New Phytol 226: 50–58 Supplemental Figure S8. Total carotenoids in leaves of Fuentes P, Pizarro L, Moreno JC, Handford M, Rodriguez- carrot lines overexpressing AtPAR1. Concepcion M, Stange C (2012) Light-dependent changes in plas- tid differentiation influence carotenoid gene expression and accu- Funding mulation in carrot roots. Plant Mol Biol 79: 47–59 This study was supported by Chilean ANID Fondecyt Galstyan A, Bou-Torrent J, Roig-Villanova I, Martınez-Garcıa JF (2012) A dual mechanism controls nuclear localization in the atyp- Grant 1180747 (C.S.), MCIN/AEI/10.13039/501100011033/ ical basic-helix-loop-helix protein PAR1 of Arabidopsis thaliana. and “ERDF A way of making Europe” grant BIO2017-85316- Mol Plant 5: 669–677 R and MCIN/AEI/10.13039/501100011033 grant PID2020- Galstyan A, Cifuentes-Esquivel N, Bou-Torrent J, Martinez-Garcia 115782GB-I00 to J.F.M.G., European Commission contract JF (2011) The shade avoidance syndrome in Arabidopsis: a funda- H2020-MCSAIF-2017 (proposal 797473) to J.M.R. mental role for atypical basic helix-loop-helix proteins as transcrip- tional cofactors. Plant J 66: 258–267 Conflict of interest statement. 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