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Fruit-localized phytochromes regulate plastid biogenesis, starch synthesis, and carotenoid metabolism in tomato

Fruit-localized phytochromes regulate plastid biogenesis, starch synthesis, and carotenoid... Light signaling has long been reported to influence fruit biology, although the regulatory impact of fruit-localized photoreceptors on fruit development and metabolism remains unclear. Studies performed in phytochrome (PHY)- deficient tomato (Solanum lycopersicum) mutants suggest that SlPHYA, SlPHYB2, and to a lesser extent SlPHYB1 influence fruit development and ripening. By employing fruit-specific RNAi-mediated silencing of SlPHY genes, we demonstrated that fruit-localized SlPHYA and SlPHYB2 play contrasting roles in regulating plastid biogenesis and maturation in tomato. Our data revealed that fruit-localized SlPHYA, rather than SlPHYB1 or SlPHYB2, positively influ- ences tomato plastid differentiation and division machinery via changes in both light and cytokinin signaling-related gene expression. Fruit-localized SlPHYA and SlPHYB2 were also shown to modulate sugar metabolism in early devel- oping fruits via overlapping, yet distinct, mechanisms involving the co-ordinated transcriptional regulation of genes related to sink strength and starch biosynthesis. Fruit-specific SlPHY silencing also drastically altered the transcrip- tional profile of genes encoding light-repressor proteins and carotenoid-biosynthesis regulators, leading to reduced carotenoid biosynthesis during fruit ripening. Together, our data reveal the existence of an intricate PHY–hormonal interplay during fruit development and ripening, and provide conclusive evidence on the regulation of tomato quality by fruit-localized phytochromes. Keywords: Auxin, carotenoid, cytokinin, fleshy fruit, phytochrome, plastid division, tomato, Solanum lycopersicum, starch. Introduction Fleshy fruit growth, maturation, and ripening are under strict major crop and important model species for fleshy fruits, several developmental, hormonal, and epigenetic regulation, which in lines of evidence indicate that changes in light perception and turn are fine-tuned by a plethora of environmental stimuli (Kumar signaling can lead to significant alterations in fruit development et al., 2014; Giovannoni et al., 2017). Among environmental cues, and quality traits (Giliberto et  al., 2005; Schofield and Paliyath, light plays a significant role in determining fruit growth, pigmen- 2005; Azari et al., 2010b; Bianchetti et al., 2017). tation, and timing of ripening (Carvalho et al., 2011; Gupta et al., One of the earliest pieces of evidence of the influence 2014; Llorente et al., 2016a). In tomato (Solanum lycopersicum), a of light on tomato fruit biology dates back to 1954, when © The Author(s) 2018. Published by Oxford University Press on behalf of the Society for Experimental Biology. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. Downloaded from https://academic.oup.com/jxb/article/69/15/3573/4975406 by DeepDyve user on 18 July 2022 3574 | Bianchetti et al. fruit pigmentation was shown to be regulated by red/far red (Gupta et  al., 2014; Bianchetti et  al., 2017). However, as the (R/FR) light in a reversible manner (Piringer and Heinze, existing evidence supporting these findings is exclusively based 1954). First isolated only a few years later, phytochromes on studies performed in phy mutants, whether these responses (PHYs) act as molecular switches in response to R and FR are dependent on fruit-localized PHYs or are merely conse- light, existing as homodimers of two independently revers- quences of the collateral negative effects of PHY deficiency on ible subunits. Once activated by R light, PHYs are trans- vegetative plant growth remains to be elucidated. ported from the cytosol to the nucleus, where they counteract By employing fruit-specific RNAi-mediated silencing light-signaling repressor proteins, such as CONSTITUTIVE of SlPHY genes, we shed light on the functional specificity PHOTOMORPHOGENESIS1 (COP1), CULLIN4 of fruit-localized SlPHYs in controlling developmental and (CUL4), DNA DAMAGE-BINDING PROTEIN 1 metabolic processes associated with sugar and carotenoid accu- (DDB1), DETIOLATED1 (DET1), and PHYTOCHROME mulation, two essential nutritional quality traits of this edible INTERACTION FACTOR (PIF) (Deng and Quail, fruit. Our data also reveal that an intricate light–hormonal 1992; Pepper et  al., 1994; Schroeder et  al., 2002; Duek and signaling network involving key components of both auxin Fankhauser, 2005; Thomann et  al., 2005). In line with their and cytokinin signal transduction pathways is implicated in the role as repressors of photomorphogenic responses, either the PHY-dependent regulation of fruit plastid biogenesis, sugar down-regulation or loss-of-function of tomato genes encod- metabolism, and carotenoid accumulation. ing COP1, CUL4, DDB1, DET1, and PIF1a profoundly alter tomato fruit physiology and nutritional composition Materials and methods (Cookson et  al., 2003; Liu et  al., 2004; Davuluri et  al., 2005; Plant material and growth conditions Kolotilin et  al., 2007; Wang et  al., 2008; Azari et  al., 2010b; Enfissi et al., 2010; Llorente et al., 2016b). Tomato (Solanum lycopersicum L.) plants cv. Micro-Tom, which harbors the wild-type SlGLK2 allele (Carvalho et  al., 2011), were grown under In tomato, five PHY-encoding genes have been identified, −2 −1 controlled conditions of 250 µmol m s , a 12-h photoperiod, and air namely SlPHYA, SlPHYB1, SlPHYB2, SlPHYE, and SlPHYF temperature of 27/22  °C day/ night. The fruit stages examined were (Alba et  al., 2000b). The paralogous SlPHYB1 and SlPHYB2, immature green, mature green, breaker, and red ripe, which were harvested which originated during the Solanum whole-genome trip- on average at 8, 25, 32, and 44 d post-anthesis. All fruits were harvested at lication event (Tomato Genome Consortium, 2012), display the same time of the day with four biological replicates (each replicate was composed of a pool of at least five fruits from different plants). Columella, distinct expression profiles within tomato organs, pointing placenta, and seeds were immediately removed, and the remaining tissues to functional diversification (Hauser et  al., 1997; Weller et  al., were frozen in liquid nitrogen and stored at –80 °C until use. 2000). SlPHYB1 is more prominently expressed in vegetative tissues, whereas the highest SlPHYB2 expression levels are Generation of transgenic tomato plants detected in fruits (Hauser et al., 1997; Bianchetti et al., 2017). Three fragments specific to the coding sequences of SlPHYA, SlPHYB2, Moreover, evidence also suggests a more direct involvement of and both SlPHYB1 and SlPHYB2 were selected using BLAST queries SlPHYB1, rather than SlPHYB2, during early seedling pho- against the Sol Genomics Network database (https://solgenomics.net/, tomorphogenic responses (van Tuinen et  al., 1995a, 1995b; ITAG release 2.40) and the web-based computational tool pssRNAit Weller et al., 2000). Very little is known about the influence of (Dai and Zhao, 2011) was employed to avoid off-target silencing. Each SlPHYE and SlPHYF on tomato vegetative and reproductive fragment was independently cloned into pENTR D-TOPO plasmids (Invitrogen) using the primers listed in Supplementary Table  S1 at JXB development (Schrager-Lavelle et al., 2016). online. Subsequently, each fragment was recombined into the plant trans- Attempts to define the influence of fruit-localized PHYs on formation vector pK8GWIWG (Fernandez et  al., 2009). Transgenic fruit development and ripening have been relatively limited. Micro-Tom plants were generated by Agrobacterium-mediated transform- Brief R-light treatments of detached mature-green tomato fruits ation according to Pino et  al. (2010), with minor changes: cotyledons promote lycopene accumulation, a response reversed by sub- from 5-d-old seedlings were used for the transformation, and the zeatin −1 and kanamycin concentration were 5 µM and 70 mg l , respectively. All sequent treatment with FR light (Alba et  al., 2000a), which is plants used in the study were from the T generation. consistent with the hypothesis that fruit-localized PHYs play a regulatory role in controlling tomato fruit carotenogenesis. The marked accumulation of SlPHYA transcripts during fruit ripen- Fruit color and pigment quantification ing (Alba et al., 2000a) associated with the reduced fruit lycopene Changes in fruit color (Hue angle) were determined using a Konica levels observed in phyA tomato mutants (Gupta et al., 2014) raise Minolta CR-400 colorimeter as described in Su et al. (2015). Chlorophyll extraction and quantification were carried out as described in Lira et al. the possibility that this PHY may be an important regulator of (2016) with some modifications. Pericarp samples were weighed (typ- tomato fruit carotenoid biosynthesis. However, regardless of the ically 100  mg fresh weight, FW), ground in liquid nitrogen, immersed development stage or tissue considered, SlPHYB2 is the most in a 10× excess volume of N, N-dimethylformamide, and incubated at highly expressed PHY in tomato fruits (Bianchetti et al., 2017). room temperature for 24 h in absolute darkness and constant agitation Moreover, the phyB2 mutant also displays considerable changes (200  rpm). After centrifugation (9000 g, 5  min, 4  °C), the supernatant absorbance was recorded at 647 and 664 nm, and the total chlorophyll in the fruit carotenoid profile (Gupta et al., 2014), suggesting that content was estimated using the equations given by Porra et al. (1989). multiple PHYs are involved in regulating this metabolic process. For carotenoid extraction, approximately 200 mg FW of pericarp sam- Besides carotenogenesis, PHYs have also been found to ples were ground in liquid nitrogen and sequentially homogenized with control other aspects of tomato fruit development and metab- a solution of 100  µl of saturated NaCl, then 200  µl of dichlorometh- olism, including chloroplast biogenesis, chlorophyll accumula- ane, and finally 1 ml of hexane:diethyl ether (1:1, v/v). The supernatant was collected after centrifugation (5000 g, 10 min, 4 °C). The remaining tion, sugar metabolism, sink activity, and hormonal signaling Downloaded from https://academic.oup.com/jxb/article/69/15/3573/4975406 by DeepDyve user on 18 July 2022 Fruit-localized phytochromes influence tomato quality traits | 3575 carotenoids in the pellet were extracted three more times with 500 µl of (GACGTA), CG-hybrid (GACGTG), canonical AuxRE (TGTGTC), hexane:diethyl ether (1:1, v/v). All supernatant fractions were combined, and degenerate AuxRE (TGTGNC) motifs (Martı́nez-Garcı́a et al., 2000; completely vacuum-dried, and suspended with 200  µl of acetonitrile. Song et al., 2008; Chaabouni et al., 2009). Lycopene, β-carotene, lutein, and neurosporene levels were determined by high-performance liquid chromatography (HPLC) with a photodiode Statistical analysis array detector (PDA) as described by Lira et al. (2017). ANOVA and Student’s t-test were performed using the JMP statistical software package (14th edition; http://jmp.com). Comparisons with P<0.05 were considered statistically significant. Data from wild-type and Starch and soluble sugar quantification all independent transgenic lines were also compared with principal com- Starch and soluble sugar extractions were performed as described in ponent analysis (PCA) using the InfoStat software (http://infostat.com.ar). Bianchetti et  al. (2017). Briefly, approximately 200 mg FW of pericarp samples was extracted with 1 ml of 80% (v/v) methanol for 10 min at 80 °C followed by the collection of the supernatants by centrifugation Results (13 000 g, 10  min, 4  °C). The remaining pellets were re-extracted five times, and all supernatants were combined, completely vacuum-dried, Fruit-specific PHY knockdown in transgenic and suspended in 200 µl distilled water. Soluble sugars (i.e. sucrose, fruc- tomato plants tose, and glucose) were measured using a HPLC system equipped with an amperometric detector (Dionex, Sunnyvale, USA) and a CarboPac PA1 To investigate the role played by distinct PHYs in tomato fruit (4 × 250 mm) column (Purgatto et al., 2002). Starch levels were deter- development and ripening, we generated fruit-specific silenced mined from dried pellet as described in Suguiyama et al. (2014). tomato plants with reduced mRNA levels of SlPHYA, SlPHYB2, or both SlPHYB1 and SlPHYB2. This was achieved using a Antioxidant capacity and total phenolics hairpin-mediated RNAi approach based on the expression of Hydrophilic and lipophilic Trolox equivalent antioxidant capacities specific fragment sequences of these genes under the control of (TEACs) were spectrophotometrically determined as described in Lira the fruit-specific PPC2 promoter (Fernandez et al., 2009). The et al. (2016). Total phenolic content was determined in hydrophilic extracts RNAi transgenic plants obtained, hereafter designated as SlPHYA , by using the Folin–Ciocalteu method (Singleton and Rossi, 1965). RNAi RNAi SlPHYB2 , and SlPHYB1/B2 (Fig. 1A), were generated in a Micro-Tom background homozygous for the wild-type Plastid ultrastructure and abundance GOLDEN2-LIKE-2 (SlGLK2) allele (Carvalho et  al., 2011), Pericarp fragments taken from the pedicel region (green shoulder) of which encodes a transcription factor critically important for immature fruits were fixed at 4 °C in 2.5% (v/v) glutaraldehyde and 2% chloroplast development in tomato fruits (Powell et al., 2012). (v/v) paraformaldehyde in 0.1 M sodium phosphate buffer (pH 7.2). Transcript abundance analysis revealed that SlPHYA, Subsequently, the samples were post-fixed in 1% osmium tetroxide in 0.1 M sodium phosphate buffer (pH 7.2), dehydrated in a graded acetone ser- SlPHYB2, and both SlPHYB1 and SlPHYB2 were down-regu- RNAi RNAi RNAi ies, and embedded in Spurr’s resin. Ultrathin sections were stained with sat- lated in the SlPHYA , SlPHYB2 , and SlPHYB1/B2 urated uranyl acetate and lead citrate (Melo et al., 2016) and observed using lines, respectively (Fig. 1B). A search for potential tomato off- a JEOL JEM1011 transmission electron microscope. Sections from three targets via BLAST queries against the Sol Genomics Network immature fruits picked from different plants were analysed per genotype. database or via the public web-based computational tool pss- Plastid abundance was determined as described in Bianchetti et  al. (2017). Briefly, small pieces (1 × 1 mm) of pericarp were fixed in 3.5% RNAit (Dai and Zhao, 2011) failed to identify regions in the (v/v) glutaraldehyde for 1 h. Samples were washed twice and transferred tomato coding that exhibited the 21-nucleotide perfect iden- to 0.1 M NaEDTA pH 9.5 solution for 4 h at 60 °C in complete dark- tity threshold reported to cause off-target silencing (Xu et  al., ness. Pieces were softly disrupted and transferred to microscope slides. 2006). The percentage of identity of the silencing fragments was Isolated cells were visualized using a Leica microscope. Plastid densities below 60% with non-target tomato PHY genes (Supplementary in individual cells were estimated using the ImageJ program (https:// imagej.nih.gov/ij/). At least 40 individual cells were analysed per sample. Table S2). Moreover, the length of stretches with perfect iden- tity between the RNAi fragments and non-target tomato PHY genes was ≤15 nucleotides (Supplementary Table  S2). In line Transcriptional profile with this, no off-target SlPHY silencing was detected in the Total RNA extraction, cDNA synthesis, primer design, and qPCR assays transgenic lines generated (Supplementary Fig. S1). were performed as described by Quadrana et al. (2013). Primer sequences used are detailed in Supplementary Table  S1. Quantitative real-time In all the transgenic lines, PHY knockdown was restricted to (qRT-)PCR reactions were performed in a StepOnePlus PCR Real- the fruit tissues as no significant PHY silencing was observed in Time thermocycler (Applied Biosystems) in a final volume of 10 µl using leaf samples (Fig. 1B). Transgenic lines exhibited normal plant 2× SYBR Green Master Mix reagent (Thermo Fisher Scientific). Melting growth and visual phenotypic features similar to those found in curves were checked for unspecific amplifications and primer dimeriza- wild-type (WT) plants (Supplementary Fig. S2). Overall, fruit- tion. Absolute fluorescence data were analysed using the LinRegPCR software package (Ruijter et al., 2009) to obtain quantitation cycle (C ) specific PHY knockdown caused no marked changes in fruit values and to calculate primer efficiency. Transcript abundances were size and ripening progression (Supplementary Fig. S3). normalized against the geometric mean of two reference genes, CAC and EXPRESSED (Expósito-Rodriguez et al., 2008). Fruit-localized SlPHYA and SlPHYB2 differentially impact chloroplast biogenesis and differentiation Gene promoter analysis during early fruit development Gene promoter analysis was performed using the promotor sequences available at the Sol Genomics Network. Typically, 3 kb upstream of the The PHY-dependent regulation of chloroplast development initial ATG codon of each sequence was analysed using the PlantPAN 2.0 has been extensively reported in leaf tissues of several spe- platform (http://plantpan2.itps.ncku.edu.tw/) (Chow et  al., 2016) for cies (Stephenson et  al., 2009; Inagaki et  al., 2015). Moreover, the presence of PBE-box (CACATG), G-box (CACGTG), CA-hybrid Downloaded from https://academic.oup.com/jxb/article/69/15/3573/4975406 by DeepDyve user on 18 July 2022 3576 | Bianchetti et al. RNAi RNAi Fig. 1. Fruit-specific PHY knockdown in transgenic tomato plants. (A) Constructs designed for generation of the SlPHYA , SlPHYB1/B2 , and RNAi SlPHYB2 transgenic lines. ‘A’ indicates the SlPHYA-specific fragment of the mRNA 5´ untranslated region (UTR). ‘B1/B2’ indicates the SlPHYB1/B2- specific fragment of the mRNA 5´ UTR. ‘B2’ indicates the SlPHYB2-specific fragment of the mRNA 5´ UTR. (B) Relative SlPHY mRNA levels in leaves, RNAi RNAi RNAi and immature green (IG), mature green (MG), and breaker (Bk) stages of fruits of the SlPHYA , SlPHYB2 , and SlPHYB1/B2 lines. The first and second fully expanded leaves from the top of 2-month-old plants were harvested. Transcript abundance was normalized against the wild-type (WT) sample. Statistically significant differences compared with the WT genotype were determined using Student’s t-test: *P<0.05. Data are means (±SE) of at least three biological replicates. (This figure is available in color at JXB online.) some recent reports have also indicated altered chlorophyll WT fruits (Fig. 2B). However, the higher chlorophyll content RNAi accumulation and chloroplast biogenesis in immature fruits of observed in SlPHYB2 immature fruits was not accompa- PHY-deficient tomato mutants (Gupta et al., 2014; Bianchetti nied by changes in plastid abundance but instead was linked et al., 2017). Compared to the WT, fruit-specific SlPHYA and to the up-regulation of the master regulator of chloroplast SlPHYB2 knockdown reduced and increased the chlorophyll development and maintenance, SlGLK2 (Fig. 2C). SlPHYB1/ content in immature fruits, respectively (Fig.  2A). However, B2 knockdown lines showed an intermediate impact on fruit RNAi chlorophyll levels in immature fruits from SlPHYB1/B2 chlorophyll content, plastid density, and SlGLK2 mRNA lev- plants were similar to WT counterparts. els, exhibiting unaltered chlorophyll levels and chloroplast Microscopy analysis of pericarp cells revealed that the abundance in pericarp cells and slightly higher expression of RNAi reduced chlorophyll content detected in SlPHYA imma- SlGLK2 compared to the WT (Fig. 2). RNAi RNAi ture fruits was associated with a reduction of up to 40% in Plastids of WT, SlPHYB2 , and SlPHYB1/B2 immature the number of chloroplasts per pericarp cell compared to fruits exhibited remarkably similar internal membranous structures, Downloaded from https://academic.oup.com/jxb/article/69/15/3573/4975406 by DeepDyve user on 18 July 2022 Fruit-localized phytochromes influence tomato quality traits | 3577 Fig. 2. Fruit-localized SlPHYA and SlPHYB2 differentially impact on chloroplast biogenesis and differentiation during early fruit development. (A) Total chlorophyll content in immature fruits. (B) Plastid abundance per pericarp cell of immature fruits. (C) Relative mRNA levels of GOLDEN2-LIKE-2 (SlGLK2) normalized against the wild-type (WT) sample. Statistically significant differences compared with the WT sample were determined using Student’s t-test: *P<0.05. Chlorophyll content and transcript abundance data are means (±SE) of at least three biological replicates. For plastid density, three fruits of each genotype were randomly picked, and two technical replicates were taken at the pedicel region of each fruit. Plastid density was determined in at least 40 individual cells per sample. (D) Representative TEM images of plastids in the pedicel region of immature fruits. Arrows indicate plastoglobuli. G, granal thylakoid. displaying well-developed grana and stroma thylakoids as well as fruit tissues (Shi et  al., 2012), SlCRF1, SlCRF2, and SlCRF5 RNAi numerous plastoglobuli (Fig. 2D, Supplementary Fig. S4). In con- were markedly down-regulated in SlPHYA lines, whereas trast, fruit-specific SlPHYA knockdown resulted in the formation SlCRF3 and SlCRF9 mRNA levels remained unchanged of chloroplasts with highly reduced grana, suggesting a promo- (Fig.  3C). As AtCRF2 is responsible for inducing AtPDV2, tive role of PHYA-mediated light perception on fruit plastid granal subsequently increasing plastid division rates in Arabidopsis development. Plastoglobuli and starch grains were observed equally (Okazaki et  al., 2009), the drastic down-regulation of both in fruit chloroplasts of the WT and all transgenic lines. SlCRF2 and SlPDV2 in SlPHYA-silenced fruits suggests that As neither SlPHYB2 nor the SlPHYB1/B2 knockdown a similar regulatory mechanism also takes place early in the altered chloroplast density per cell or plastid ultrastructure development of tomato fruits. (Fig.  2), fruit-localized SlPHYA seems to play a preponder- Alongside the down-regulation of cytokinin signaling genes, ant role in controlling chloroplast biogenesis and differenti- fruit-specific SlPHYA-silencing resulted in the up-regulation ation in early developing fruits. Transcript abundance analysis of tomato genes encoding light-signaling repressor proteins revealed that the reduced plastid abundance observed in such as COP1, CUL4, DDB1, and DET1 (Fig.  3D), which SlPHYA-silenced fruits was most probably explained are negative regulators of plastid division and maturation in by a drastic reduction in mRNA levels of genes encod- tomato and other species (Chory and Peto, 1990; Kolotilin ing key components of the plastid division machinery, such et al., 2007; Wang et al., 2008; Azari et al., 2010b). as FILAMENTOUS TEMPERATURE SENSITIVE-Z Collectively, these data suggest that fruit-localized PHYA (FtsZs), ACCUMULATION AND REPLICATION OF positively influences tomato plastid division machinery via CHLOROPLASTS (ARCs), and PLASTID DIVISION 2 changes in the transcript abundance of both light- and cyto- (PDV2), compared to the WT genotype (Fig. 3A). kinin-signaling genes, whereas PHYB2 negatively regulates Given the key role played by cytokinins in regulating plas- chlorophyll accumulation by controlling the expression of the tid division and maturation in plants and the widely reported master transcription factor of chloroplast development and crosstalk between this hormonal class and PHY signaling maintenance, SlGLK2. (Okazaki et  al., 2009; Cortleven and Schmülling, 2015), a transcriptional profiling of type-A TOMATO RESPONSE Fruit-localized PHYs regulate starch metabolism during REGULATOR (TRR) was performed. Four out of the five early fruit development type-A TRRs analysed were significantly down-regulated in RNAi Fruit-specific SlPHYA and SlPHYB2 knockdown promoted immature fruits of SlPHYA compared to the WT geno- starch accumulation during early fruit development (Fig. 4A). type (Fig.  3B). Moreover, among the five CYTOKININ In both the WT and transgenic lines, the highest starch content RESPONSE FACTOR genes most highly expressed in tomato Downloaded from https://academic.oup.com/jxb/article/69/15/3573/4975406 by DeepDyve user on 18 July 2022 3578 | Bianchetti et al. Fig. 3. SlPHYA-mediated regulation of chloroplast division machinery is associated with changes in the transcript abundance of light- and cytokinin- signaling genes. (A) Relative mRNA levels of genes encoding components of the plastid division machinery in immature fruits. (B) Relative mRNA levels of type-A TOMATO RESPONSE REGULATOR (TRR) genes in immature fruits. (C) Relative mRNA levels of CYTOKININ RESPONSE FACTOR (SlCRF) genes in immature fruits. (D) Relative mRNA levels of genes encoding light-signaling repressor proteins. Data are means (±SE) of at least three biological replicates. Transcript abundance was normalized against the wild-type (WT) sample. Statistically significant differences compared with the WT were determined using Student’s t-test: *P<0.05). FtsZ, filamentous temperature sensitive-Z; ARC, accumulation and replication of chloroplasts; PDV2, plastid division 2; COP1, constitutive photomorphogenic 1; CUL4, cullin 4; DDB1, UV-damaged DNA binding protein 1; DET1, de-etiolated1. RNAi was observed in immature green (IG) fruits, followed by development and ripening in both the SlPHYA and RNAi slightly more reduced levels at the mature green (MG) stage, SlPHYB2 lines. However, despite the negative impact of and undetectable levels from the breaker (Bk) stage onwards either SlPHYA- or SlPHYB2-silencing on SlAGPaseS1 expres- (Supplementary Fig. S5). sion, this gene exhibited higher expression levels than those Compared to the WT genotype, marked differences in the encoding AGPase large subunits (Supplementary Table  S3), transcript profiles of starch biosynthesis genes were observed suggesting that the catalytic AGPase subunit was not limiting in both SlPHYA- and SlPHYB2-silenced fruits (Fig.  4A, for starch biosynthesis in tomato fruits. RNAi RNAi Supplementary Fig. S6). Catalysing the first committed step in In both SlPHYA and SlPHYB2 immature fruits, the starch biosynthesis, ADP-glucose pyrophosphorylase (AGPase) starch synthase (STS)-encoding genes SlSTS1 and SlSTS2 is a heterotetramer comprising a pair of small/catalytic and a were markedly up-regulated compared to WT fruits, whereas pair of large/regulatory subunits (Kim et  al., 2007; Figueroa SlSTS3 was slightly down-regulated. For SlSTS6, higher tran- RNAi et  al., 2013). Among the tomato genes encoding the large script accumulation was observed in SlPHYA than in the AGPase subunits, both SlAGPaseL1 and SlAGPaseL3 were WT throughout fruit development and ripening (i.e. IG to up-regulated whereas SlAGPaseL2 mRNA levels remained RR stage) (Supplementary Fig.  S6). Finally, distinct expres- RNAi unchanged in immature fruits of SlPHYA plants. It is sion patterns were observed for the starch branching enzyme worth mentioning that SlAGPaseL1 was the large AGPase sub- (SBE)-encoding genes, as SlSBE1 was up-regulated in all the unit most expressed in immature tomato fruits (Supplementary transgenic lines from MG to Bk stage whereas SlSBE2 was RNAi RNAi Table S3; Petreikov et al., 2006); therefore, the 3-fold increment down-regulated in both SlPHYA and SlPHYB2 from in its mRNA levels correlates well with the higher starch lev- IG to RR stage (Supplementary Fig. S6). RNAi RNAi els and reduced soluble sugar levels detected in SlPHYA The increased accumulation of starch in SlPHYA fruits immature fruits compared to the WT counterparts (Fig.  4, correlated well with higher mRNA levels of SlLIN5 and Supplementary Figs S5, S6). SlLIN6 (Fig.  4D), which encode cell-wall invertases critic- SlAGPaseS1, which encodes the small/catalytic AGPase ally important for sink activity in tomato (Fridman and Zamir, subunit, was consistently down-regulated throughout fruit 2003; Kocal et al., 2008). By applying an unsupervised method Downloaded from https://academic.oup.com/jxb/article/69/15/3573/4975406 by DeepDyve user on 18 July 2022 Fruit-localized phytochromes influence tomato quality traits | 3579 Fig. 4. Fruit-localized phytochromes regulate sugar metabolism during early fruit development. (A) Schematic representation of the major steps of starch biosynthesis and graphs showing starch content and transcript abundance of starch biosynthesis-related genes in immature fruits. (B) Soluble sugar contents in immature fruits. (C) Summed values of the three soluble sugars analysed (i.e. sucrose + glucose + fructose). (D) Relative mRNA levels of tomato genes encoding invertases (SlLIN) in immature fruits. (E) Relative mRNA levels of AUXIN RESPONSE FACTOR 4 (SlARF4) in immature fruits. For simplicity, the mean of the three values for the transgenic lines is shown. Values for each transgenic line are presented in Supplementary Figs S5, S6. Data are means (±SE) of at least three biological replicates. Statistically significant differences compared with the wild-type (WT) sample were determined using Student’s t-test: *P<0.05. IG, immature green; MG, mature green; Bk, breaker; RR, red ripe; Glc-1-P, glucose 1-phosphate; ADPG, adenosine diphosphate glucose; AGPase, ADP-glucose pyrophosphorylase; STS, starch synthase; SBE, starch branching enzyme. (i.e. principal component analysis, PCA) to search for pat- promoter sequences of SlLIN5 and SlLIN6 genes (Bianchetti terns in the expression profiles of genes related to sink- and et al., 2017). starch-biosynthesis, we demonstrated a clear separation of the RNAi RNAi WT, SlPHYA , and SlPHYB2 groups (Supplementary PHY-dependent regulation of fruit carotenoid Fig. S7). biosynthesis is associated with transcriptional changes Previous findings have indicated that AUXIN RESPONSE in light- and auxin-signaling genes FACTOR4 (SlARF4) is a major negative regulator of starch The very well-characterized PHY-mediated signaling networks biosynthesis in early developing tomato fruits (Sagar et  al., controlling carotenogenesis in vegetative tissues (Toledo-Ortiz 2013; Bianchetti et  al., 2017). Recent evidence also indicates et  al., 2010) contrasts with the considerably more limited that SlARF4 plays a repressor role in controlling the tran- information regarding the fruit-localized PHY-dependent script abundance of sink-related genes, including SlLIN5 and signaling cascades regulating carotenoid biosynthesis in fleshy SlLIN6 (Bianchetti et al., 2017). In accordance with this, fruit- fruits (Llorente et  al., 2016b, 2017). Carotenoid profiling specific SlPHYA and SlPHYB2 knockdown drastically reduced revealed a significant reduction in lycopene content in red ripe SlARF4 mRNA abundance in early developing tomato fruits RNAi RNAi (RR) fruits of both the SlPHYA and SlPHYB2 lines (Fig.  4E). Although the direct transcriptional regulation of compared to the WT (Fig.  5A, Supplementary Table  S4). In tomato AGPase, STS, and SBE genes by transcription factors contrast, the content of all other carotenoids analysed (i.e. phy- associated with auxin- or light-signaling remains to be deter- toene, phytofluene, β-carotene, and lutein) remained virtually mined, the presence of PBE-box, G-box, CA-hybrid, and/or unchanged in ripe fruits of the transgenic lines compared to CG-hybrid motifs (Martı́nez-Garcı́a et  al., 2000; Song et  al., WT counterparts. As lycopene is the main carotenoid accu- 2008) as well as canonical and/or degenerated ARF-binding mulated in ripe tomato, fruit-specific SlPHYA- or SlPHYB2- Auxin Response Element (AuxRE) motifs within the 3-kb knockdown led to a slight, yet significant, reduction in total promoter sequence of these genes (Supplementary Fig. S8) is carotenoid content compared to the WT genotype (Fig.  5A, consistent with the hypothesis that light- and/or auxin-related Supplementary Table  S4). In accordance with this, signifi- transcription factors might directly control the expression of cantly lower mRNA levels of genes encoding carotenoid starch biosynthesis-related genes. Similarly, PIF, HY5, and/ biosynthesis-related enzymes such as GERANYLGERANYL or ARF-binding motifs have also been identified within the Downloaded from https://academic.oup.com/jxb/article/69/15/3573/4975406 by DeepDyve user on 18 July 2022 3580 | Bianchetti et al. Fig. 5. Fruit-specific SlPHYA or SlPHYB2 knockdown represses carotenoid biosynthesis during tomato fruit ripening. (A) Lycopene, phytoene, phytofluene, β-carotene, lutein, and total carotenoid content in red ripe fruits. (B) Schematic representation of carotenoid biosynthetic pathway and graphs showing the transcript abundance of carotenoid biosynthesis genes in ripening fruits. Intermediate reactions are omitted. For simplicity, the mean of the three values for the transgenic lines is shown. Values for each transgenic line are presented in Supplementary Fig. S9, Supplementary Table S4. Data are means (±SE) of at least three biological replicates. Statistically significant differences compared with the wild-type (WT) sample were determined using Student’s t-test: *P<0.05. MG, mature green; Bk, breaker; RR, red ripe; MEP, methylerythritol 4-phosphate; GGDP, geranylgeranyl diphosphate; GGPS, GGDP synthase; PSY, phytoene synthase; PDS, phytoene desaturase; LCYβ, chloroplast-specific β-lycopene cyclase; CYCβ, chromoplast- specific β-lycopene cyclase. (This figure is available in color at JXB online.) DIPHOSPHATE SYNTHASE (GGPS), PHYTOENE fruits (Azari et  al., 2010b; Llorente et  al., 2016b) whereas SYNTHASE 1 (PSY1), and PHYTOENE DESATURASE auxin response factors such as SlARF2a and SlARF2b play (PDS) were observed in ripe fruits of SlPHYA and SlPHYB2- the opposite role (Hao et  al., 2015). To gain insight into the silenced lines than in WT counterparts (Fig. 5B, Supplementary potential role played by these signaling components during Fig.  S9). In line with the relatively limited reduction in total the PHY-dependent regulation of carotenoid biosynthesis in carotenoids, no significant differences in lipophilic antioxidant tomato fruits, the transcript abundance of their encoding genes RNAi RNAi activity were observed between ripe WT and transgenic fruits was profiled in both SlPHYA and SlPHYB2 ripening (Supplementary Table  S5). Interestingly, however, red ripe fruits (Fig. 6, Supplementary Fig. S10). Among the four SlPIF SlPHYB2-down-regulated fruits exhibited increased hydro- genes most highly expressed in fruits (Rosado et  al., 2016), philic antioxidant activity compared to the WT, which may SlPIF1a, SlPIF1b, and SlPIF4/5 mRNA levels were signifi- be associated with the higher content of total phenolics also cantly higher in SlPHYB2-down-regulated fruits compared RNAi detected in SlPHYB2 ripe fruits (Supplementar y Table S5). to the WT counterparts during fruit ripening (MG, Bk, and Accumulating evidence indicates that light-signaling RR stages), whereas the opposite was observed for SlPIF3 repressors such as SlPIF1a, SlCOP1, SlCUL4, SlDDB1, and transcripts. Although less pronounced, the overall impacts of SlDET1 negatively regulate carotenoid biosynthesis in tomato fruit-specific SlPHYA knockdown on tomato PIF expression Downloaded from https://academic.oup.com/jxb/article/69/15/3573/4975406 by DeepDyve user on 18 July 2022 Fruit-localized phytochromes influence tomato quality traits | 3581 Fig. 6. PHY-dependent regulation of fruit carotenogenesis is associated with transcriptional changes in auxin- and light-signaling genes. (A) Transcript abundance of tomato genes encoding PHYTOCHROME INTERACTING FACTORs (SlPIFs). (B) Transcript abundance of tomato genes encoding the light-signaling repressors CONSTITUTIVE PHOTOMORPHOGENIC 1 (SlCOP1), CULLIN 4 (SlCUL4), UV-DAMAGED DNA BINDING PROTEIN 1 (SlDDB1), and DE-ETIOLATED1 (SlDET1). (C) Transcript abundance of the tomato AUXIN RESPONSIVE FACTOR 2a and 2b (SlARF2a and SlARF2b) genes. For simplicity, the mean of the three values for the transgenic lines is shown. Values for each transgenic line are presented in Supplementary Fig. S10. Data are means (±SE) of at least three biological replicates. Statistically significant differences compared with the wild-type (WT) sample were determined using Student’s t-test: *P<0.05. MG, mature green; Bk, breaker; RR, red ripe. RNAi profiles were similar to those observed in the SlPHYB2 and quality traits. Differently from the pleiotropic phenotyp- lines (Fig. 6, Supplementary Fig. S10). ical alterations observed in phy mutants (Gupta et  al., 2014; Among the genes encoding light-signaling repressors, Bianchetti et al., 2017), the fruit-specific silencing of the target SlCUL4, SlDDB1, and SlDET1 exhibited significantly SlPHY genes resulted in no obvious impacts on plant vegeta- higher mRNA levels in SlPHYA-silenced fruits in com- tive growth and overall yield. This suggests that the perturb- parison to the WT at all fruit development stages analysed ation in fruit metabolism caused by the fruit-specific SlPHY (Fig. 6B, Supplementary Fig. S10). Moreover, strikingly higher manipulation does not propagate from fruits to the rest of the SlDET1 transcript abundance was also detected in SlPHYB2- plant, which agrees with the limited transference of substances knockdown compared to WT fruits at all ripening stages (i.e. out of this predominantly sink organ. MG, Bk, and RR) whereas SlCOP1 and SlDDB1 mRNA lev- In a previous work, we demonstrated that a global defi- RNAi els were also up-regulated in SlPHYB2 fruits exclusively ciency in functional PHYs drastically reduces chlorophyll con- at the MG stage. Transcript levels of the positive regulators tent and chloroplast abundance in tomato fruits (Bianchetti of tomato fruit carotenogenesis SlARF2a and SlARF2b were et al., 2017). Therefore, the PHY-mediated regulation of plas- RNAi RNAi considerably lower in SlPHYA and SlPHYB2 fruits, tid biogenesis and maturation widely reported for leaf tissues particularly at the Bk and RR stages (Fig. 6C, Supplementary (Stephenson et  al., 2009; Oh and Montgomery, 2014; Melo Fig. S10). A PCA plot in which the expression profile of carot- et al., 2016) seems to be conserved early in the development of enoid biosynthesis-related genes as well as SlPIFs, SlCOP1, tomato fruits. In this current work, it is further demonstrated SlCUL4, SlDDB1, SlDET1, SlARF2a, and SlARF2b were rep- that fruit-localized SlPHYA and SlPHYB2 play distinct roles RNAi RNAi resented revealed that the WT, SlPHYA , and SlPHYB2 in controlling chloroplast biogenesis and activity during early groups clearly separated from each other at the red ripe stage stages of tomato fruit development. (Supplementary Fig. S11). The results indicate that SlPHYA-mediated light percep- Altogether, these data suggest that both SlPHYA and tion promotes fruit chloroplast biogenesis and differenti- SlPHYB2 play overlapping roles in promoting the paralogues ation, as inferred from the reduced chlorophyll content, lower SlARF2a and SlARF2b and repressing light-signaling repres- chloroplast abundance, and poorly-developed grana stacking RNAi sors such as SlPIF1a, SlPIF1b, SlPIF4/5, SlCOP1, SlCUL4, detected in SlPHYA immature fruits (Fig. 2). In line with SlDDB1, and SlDET1, which in turn mediate the PHY- this observation, an analysis of single and multiple phy mutants dependent regulation of carotenoid biosynthesis in ripening also suggested that SlPHYA is a major regulator of chloro- tomato fruits. phyll accumulation in tomato fruits (Gupta et  al., 2014). In land plants, chloroplast division depends on nucleus-encoded proteins that form ring structures at the division site (Jarvis Discussion and López-Juez, 2013). Our findings clearly demonstrate that Studies performed on PHY-deficient mutants have suggested fruit-localized SlPHYA influences the transcript levels of genes that PHY-dependent light perception participates in the regu- derived from the ancestral prokaryotic cell-division machinery, lation of several aspects of tomato fruit biology (Gupta et  al., such as SlFtsZ (i.e. SlFtsZ1, SlFtsZ2) and SlARCs (i.e. SlARC3 2014; Bianchetti et  al., 2017). Here, we applied a RNAi- and SlARC6), as well as those encoding chloroplast division- mediated organ-specific silencing approach to investigate the related proteins specific to land plants, such as SlPDV2. In impact of fruit-localized SlPHYs on tomato fruit physiology Arabidopsis, PDV2 determines the rate of chloroplast division Downloaded from https://academic.oup.com/jxb/article/69/15/3573/4975406 by DeepDyve user on 18 July 2022 3582 | Bianchetti et al. and is positively regulated by cytokinins, being strongly pro- the transcriptional profile of starch biosynthesis-related genes moted in transgenic plants overexpressing the cytokinin sign- at the immature green stage (Fig.  4). AGPase, which cataly- aling-related transcription factor CRF2 (Okazaki et al., 2009; ses the rate-limiting reaction in the starch synthesis pathway, is Cortleven and Schmülling, 2015). SlCRF2, along with other both transcriptionally and post-translational regulated by light SlCRF and TRR genes, were drastically repressed in PHYA- (Harn et al., 2000; Geigenberger, 2011), although the role played down-regulated fruits, implying that changes in cytokinin by PHYs in this regulatory process remains elusive. During signaling mediate the PHYA-dependent regulation of plas- early fruit development, SlPHYA-suppressed fruits exhibited tid division during early stages of tomato fruit  development. increased mRNA levels of both SlAGPaseL1 and SlAGPaseL3, In agreement with this, accumulating evidence indicates that which encode AGPase large subunits, and SlSTS1, SlSTS2, and there is an intensive crosstalk between the PHY and cytokinin SlSTS6, which encode starch synthase enzymes, along with an signaling cascades, with particular involvement of CRF and increase in starch accumulation and reduced soluble sugar con- type-A ARR proteins (Salomé et al., 2006; Oh et al., 2009). tent, thus indicating a repressor role for fruit-localized SlPHYA Fruit-specific SlPHYA-silencing also promoted the mRNA on the first steps of starch synthesis in tomato fruits. Whether accumulation of genes encoding all the major light-signaling the up-regulation of starch biosinthesis-related genes is a com- repressor proteins already described to negatively regulate chloro- pensatory mechanism to cope with the fewer and poorly devel- RNAi plast biogenesis in tomato fruits, i.e. SlCOP1, SlCUL4, SlDDB1, oped chloroplasts observed in SlPHYA immature fruits and SlDET1 (Liu et  al., 2004; Kolotilin et  al., 2007; Wang et  al., remains to be investigated. In contrast, the increased starch 2008; Azari et  al., 2010a). Defective mutants or transgenic lines accumulation detected in SlPHYB2-silenced immature fruits with reduced levels of each of these genes are known to develop was not associated with increments in transcript abundance more chloroplasts containing more grana/thylakoids in both of AGPase-encoding genes nor with prominent reductions in leaves and immature fruits (Cookson et al., 2003; Liu et al., 2004; soluble sugars, but instead were accompanied by increments in Kolotilin et al., 2007; Wang et al., 2008; Azari et al., 2010a), which SlSTS1 and SlSTS2 mRNA levels. Furthermore, as no signifi- in some cases, such as in the SlDET1-knockout mutant, is asso- cant alterations in plastid abundance or internal structure were RNAi ciated with the up-regulation of plastid biogenesis-related genes observed in SlPHYB2 immature fruits, it seems likely that (Kolotilin et  al., 2007). Therefore, the presence of fewer chloro- this genetic manipulation caused less prominent changes than plasts with poorly developed or almost no grana in immature fruits SlPHYA-silencing on reactions taking place within fruit chlo- of the SlPHYA-suppressed lines agrees with the higher transcript roplasts, including starch biosynthesis. Altogether, these find- abundance of SlCOP1, SlDDB1, and particularly SlCUL4 and ings suggest that SlPHYA and SlPHYB2 negatively regulate SlDET1 in these transgenic lines compared to the WT genotype. starch synthesis via overlapping, yet distinct, mechanisms. In contrast, fruit-localized SlPHYB2 was shown to play a The influence of auxin on fruit sugar metabolism has been negative role in chlorophyll accumulation, as evidenced by increasingly reported (Purgatto et al., 2002; Yuan and Carbaugh, the increment in chlorophyll content in immature fruits of 2007; Bianchetti et  al., 2017). In tomato, SlARF4 has been RNAi SlPHYB2 plants with no impact in chloroplast number described as a key negative regulator of starch synthesis dur- in pericarp cells. As SlPHYB2 fruit-specific silencing led to ing early fruit development via the transcriptional and post- higher SlGLK2 mRNA levels compared to the WT genotype, transcriptional down-regulation of AGPase (Sagar et  al., 2013). it seems plausible to suggest that the effect of SlPHYB2 on Recent findings have also indicated that PHYs strictly regulate fruit chloroplasts is mediated by SlGLK2, the master regula- the transcript abundance of this particular auxin response factor tor of chloroplast development in tomato fruits (Powell et al., in both vegetative (Melo et al., 2016) and fruit tissues (Bianchetti 2012). Further suggesting that the SlPHYB2-mediated regu- et al., 2017). In line with this, the increased starch accumulation in lation of SlGLK2 expression is essential for the consequent pre-ripening SlPHYA- and SlPHYB2-silenced fruits correlated changes in fruit chlorophyll accumulation, no obvious changes well with the down-regulation of SlARF4 in these transgenic RNAi RNAi in chlorophyll content were observed in phyb2 mutants from lines (Fig.  4). In fact, SlPHYA rather than SlPHYB2 tomato varieties that lacked functional SlGLK2 proteins (Gupta exhibited the most expressive decrease in SlARF4, and only the et al., 2014). In agreement with these findings, PHY-dependent former displayed increased mRNA levels of AGPase-encoding transcriptional regulation of GLK genes has been increasingly genes in immature fruits. Together, these data strongly suggest reported in vegetative tissues of other plant species (Oh and that fruit-localized PHYA, and to some extent SlPHYB2, posi- Montgomery, 2014; Song et al., 2014). tively modulates SlARF4, which in turn represses starch biosyn- Alterations in chloroplast number, internal structure, and thetic enzymes, such as AGPase and STS, consequently limiting size during the early development of tomato fruits signifi- starch synthesis in pre-ripening tomato fruits. cantly impact the abundance of metabolites associated with Previous findings indicated that a global deficiency in organoleptic and nutritional quality at the ripe stage (Galpaz functional phytochromes transcriptionally represses both et  al., 2008; Cocaliadis et  al., 2014). Intense starch synthe- sink-related and starch biosynthesis-related enzymes in early sis and degradation take place in tomato fruit chloroplasts developing tomato fruits, suggesting a promotive role of PHYs at the unripe and breaker stages, respectively (Schaffer and on the regulation of these processes (Bianchetti et  al., 2017). Petreikov, 1997). Whereas the global deficiency in PHYs sig- However, it remained unclear whether these responses were nificantly reduces the starch content in immature tomato fruits dependent on fruit-localized PHYs or were the consequence (Bianchetti et  al., 2017), fruit-localized SlPHYA or SlPHYB2 of collateral negative effects of the global PHY deficiency suppression increased fruit starch levels and markedly altered on vegetative plant growth. Here, we shed light on this topic Downloaded from https://academic.oup.com/jxb/article/69/15/3573/4975406 by DeepDyve user on 18 July 2022 Fruit-localized phytochromes influence tomato quality traits | 3583 by showing that fruit-localized SlPHYA, and to some extent PHY-dependent signaling cascades in the fruit carotenoid bio- SlPHYB2, repress both starch metabolism and key determi- synthetic pathway. Consistent with this, our findings indicate nants of tomato fruit sink strength, including SlLIN5 transcript that fruit-localized SlPHYA and SlPHYB2 positively influ- accumulation (Fridman and Zamir, 2003; Kocal et  al., 2008). ence the transcript accumulation of all the major carotenoid Consequently, the down-regulation in starch synthesis and biosynthesis-related genes, including SlGGPS, SlPSY1, SlPDS, sink activity previously observed in fruits of the PHY-deficient SlCYCβ, and SlLYCβ, consequently modifying the lycopene mutant aurea (Bianchetti et al., 2017) may be due either to lim- and total carotenoid content in this fleshy fruit. Light-signaling itations in vegetative growth and metabolism or to the com- repressor proteins such as SlDET1, SlDDB1, SlCOP1, SlCUL4, binatory effect of the deficiency in all phytochromes instead of and more recently SlPIF1a have been identified as key nega- only in SlPHYA or SlPHYB2. Moreover, it also seems tempt- tive regulators of tomato fruit carotenoid synthesis (Liu et al., ing to suggest that the fewer and poorly-developed chloroplasts 2004; Kolotilin et al., 2007; Wang et al., 2008; Azari et al., 2010a; RNAi detected in SlPHYA immature fruits restrict photoassimi- Llorente et  al., 2016b). Among these, the transcription factor late production via fruit photosynthesis; therefore, the observed SlPIF1a was shown to directly bind to the promoter of SlPSY1 up-regulation of sink-related genes in transgenic fruits may to repress fruit carotenogenesis (Llorente et  al., 2016b), thus represent a compensatory mechanism to maintain fruit growth resembling the action of its ortholog in Arabidopsis (AtPIF1) and intense starch accumulation despite potential limitations in in controlling carotenoid biosynthesis in leaf tissues (Toledo- fruit-localized photoassimilation. Ortiz et  al., 2010). Therefore, the marked up-regulation of The link between PHY-dependent light perception and SlDET1, SlDDB1, SlCOP1, SlCUL4, SlPIF1a, and SlPIF1b carotenoid metabolism in both vegetative and fruit tissues has together with the overall repression of carotenoid biosynthesis been highlighted by a number of studies (Alba et  al., 2000a; observed in both SlPHYA- and SlPHYB2-silenced fruits imply Llorente et  al., 2016b). Exposure of wild-type tomato fruits that light-signaling repressor proteins participate in SlPHYA- to red light (Alba et  al., 2000a) or constitutively silencing of and SlPHYB2-mediated regulation of fruit carotenogenesis. SlPIF1a (Llorente et  al., 2016b) promotes tomato fruit lyco- In addition, it is becoming increasingly well established that pene accumulation, thereby implying a positive role of auxin represses tomato ripening and down-regulates lycopene Fig. 7. Proposed model for phytochrome-mediated signaling events controlling chloroplast biogenesis, and sugar and carotenoid metabolism in tomato fruits. (A) SlPHYA- and SlPHYB2-dependent light perception regulate fruit plastid division and maturation, respectively. By promoting key members of the cytokinin signaling-related CRF and TRR gene family, SlPHYA up-regulates SlPDV2, a rate-limiting component of the plastid division machinery. Moreover, the SlPHYA-mediated down-regulation of light-signaling repressors, such as SlCOP1, SlDET1, SlDDB1, and SlCUL4, induces other major components of the chloroplast division machinery, such as SlFTsZs and SlARCs. In contrast, Sl-PHYB2 represses the chloroplast differentiation transcription factor SlGLK2, consequently limiting chloroplast differentiation during early fruit development. (B) Fruit-localized SlPHYA and SlPHYB2 play overlapping roles in repressing and promoting starch and carotenoid biosynthesis, respectively. Both SlPHYA and SlPHYB2 induce SlARF4, a negative regulator of AGPase and starch accumulation in tomato fruits. In contrast, these same photoreceptors promote both SlARF2 paralogues and inhibit all the major genes encoding light-signaling repressor proteins, consequently up-regulating most components of the tomato carotenoid biosynthetic route. Arrows at the ends of lines indicate stimulatory effects, whereas bars indicate inhibitory effects. AGPase, ADP-glucose pyrophosphorylase; ARC, accumulation and replication of chloroplasts; ARF, auxin response factor; COP1, constitutive photomorphogenic 1; CRF, cytokinin response factor; CUL4, cullin 4; DDB1, UV-damaged DNA binding protein 1; DET1, de-etiolated1; FtsZ, filamentous temperature sensitive-Z; GGPS, geranylgeranyl pyrophosphate synthase; GLK2, golden2-like-2; PDS, phytoene desaturase; PDV2, plastid division 2; PIF, phytochrome interacting factor; PSY, phytoene synthase; TRR, tomato response regulator. Downloaded from https://academic.oup.com/jxb/article/69/15/3573/4975406 by DeepDyve user on 18 July 2022 3584 | Bianchetti et al. biosynthetic genes (Su et  al., 2015). Among tomato ARF Table S3. Relative transcript ratios of SlAGPase in immature genes, two paralogs, SlARF2a and SlARF2b, have emerged as fruits. key positive regulators of tomato fruit ripening and lycopene Table S4. Carotenoid profiles in red ripe fruits. accumulation (Hao et al., 2015). Either SlPHYA or SlPHYB2 Table S5. Antioxidant activity and total phenolics in red ripe fruit-specific silencing profoundly reduced both SlARF2a and fruits. SlARF2b, suggesting the involvement of these auxin signaling elements in the PHY-dependent regulation of carotenoid bio- Acknowledgements synthesis in tomato fruits. Overall, our results shed light on the specific role played The authors sincerely thank Prof. Lazaro E.  P. Peres for providing the Micro-Tom GLK2 seeds. This work was supported by the CNPq by fruit-localized phytochromes and their downstream signal- (Conselho Nacional de Desenvolvimento Científico e Tecnológico, ing cascades, showing that plastid division, as well as sugar and grant no. 442045/2014-0) and the FAPESP (Fundação de Amparo carotenoid metabolism, are profoundly regulated by SlPHYA- à Pesquisa do Estado de São Paulo, grant nos. 2013/18056-2 and and SlPHYB2-mediated light perception. A model summariz- 2016/01128-9). ing the influence of fruit-localized SlPHYs on tomato fruit physiology is presented in Fig.  7. According to this model, References SlPHYA and SlPHYB2 play overlapping roles in regulating starch and carotenoid biosynthesis, whereas they differentially Alba R, Cordonnier-Pratt MM, Pratt LH. 2000a. Fruit-localized phytochromes regulate lycopene accumulation independently of ethylene regulate distinct aspects of fruit plastid biogenesis and mat- production in tomato. Plant Physiology 123, 363–370. uration. 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Effects of NAA, AVG, and 1-MCP on ethylene Su L, Diretto G, Purgatto E, Danoun S, Zouine M, Li Z, Roustan JP, biosynthesis, preharvest fruit drop, fruit maturity, and quality of ‘Golden Bouzayen M, Giuliano G, Chervin C. 2015. Carotenoid accumulation Supreme’ and ‘Golden Delicious’ apples. HortScience 42, 101–105. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal of Experimental Botany Oxford University Press

Fruit-localized phytochromes regulate plastid biogenesis, starch synthesis, and carotenoid metabolism in tomato

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Oxford University Press
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Copyright © 2022 Society for Experimental Biology
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0022-0957
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1460-2431
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10.1093/jxb/ery145
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Abstract

Light signaling has long been reported to influence fruit biology, although the regulatory impact of fruit-localized photoreceptors on fruit development and metabolism remains unclear. Studies performed in phytochrome (PHY)- deficient tomato (Solanum lycopersicum) mutants suggest that SlPHYA, SlPHYB2, and to a lesser extent SlPHYB1 influence fruit development and ripening. By employing fruit-specific RNAi-mediated silencing of SlPHY genes, we demonstrated that fruit-localized SlPHYA and SlPHYB2 play contrasting roles in regulating plastid biogenesis and maturation in tomato. Our data revealed that fruit-localized SlPHYA, rather than SlPHYB1 or SlPHYB2, positively influ- ences tomato plastid differentiation and division machinery via changes in both light and cytokinin signaling-related gene expression. Fruit-localized SlPHYA and SlPHYB2 were also shown to modulate sugar metabolism in early devel- oping fruits via overlapping, yet distinct, mechanisms involving the co-ordinated transcriptional regulation of genes related to sink strength and starch biosynthesis. Fruit-specific SlPHY silencing also drastically altered the transcrip- tional profile of genes encoding light-repressor proteins and carotenoid-biosynthesis regulators, leading to reduced carotenoid biosynthesis during fruit ripening. Together, our data reveal the existence of an intricate PHY–hormonal interplay during fruit development and ripening, and provide conclusive evidence on the regulation of tomato quality by fruit-localized phytochromes. Keywords: Auxin, carotenoid, cytokinin, fleshy fruit, phytochrome, plastid division, tomato, Solanum lycopersicum, starch. Introduction Fleshy fruit growth, maturation, and ripening are under strict major crop and important model species for fleshy fruits, several developmental, hormonal, and epigenetic regulation, which in lines of evidence indicate that changes in light perception and turn are fine-tuned by a plethora of environmental stimuli (Kumar signaling can lead to significant alterations in fruit development et al., 2014; Giovannoni et al., 2017). Among environmental cues, and quality traits (Giliberto et  al., 2005; Schofield and Paliyath, light plays a significant role in determining fruit growth, pigmen- 2005; Azari et al., 2010b; Bianchetti et al., 2017). tation, and timing of ripening (Carvalho et al., 2011; Gupta et al., One of the earliest pieces of evidence of the influence 2014; Llorente et al., 2016a). In tomato (Solanum lycopersicum), a of light on tomato fruit biology dates back to 1954, when © The Author(s) 2018. Published by Oxford University Press on behalf of the Society for Experimental Biology. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. Downloaded from https://academic.oup.com/jxb/article/69/15/3573/4975406 by DeepDyve user on 18 July 2022 3574 | Bianchetti et al. fruit pigmentation was shown to be regulated by red/far red (Gupta et  al., 2014; Bianchetti et  al., 2017). However, as the (R/FR) light in a reversible manner (Piringer and Heinze, existing evidence supporting these findings is exclusively based 1954). First isolated only a few years later, phytochromes on studies performed in phy mutants, whether these responses (PHYs) act as molecular switches in response to R and FR are dependent on fruit-localized PHYs or are merely conse- light, existing as homodimers of two independently revers- quences of the collateral negative effects of PHY deficiency on ible subunits. Once activated by R light, PHYs are trans- vegetative plant growth remains to be elucidated. ported from the cytosol to the nucleus, where they counteract By employing fruit-specific RNAi-mediated silencing light-signaling repressor proteins, such as CONSTITUTIVE of SlPHY genes, we shed light on the functional specificity PHOTOMORPHOGENESIS1 (COP1), CULLIN4 of fruit-localized SlPHYs in controlling developmental and (CUL4), DNA DAMAGE-BINDING PROTEIN 1 metabolic processes associated with sugar and carotenoid accu- (DDB1), DETIOLATED1 (DET1), and PHYTOCHROME mulation, two essential nutritional quality traits of this edible INTERACTION FACTOR (PIF) (Deng and Quail, fruit. Our data also reveal that an intricate light–hormonal 1992; Pepper et  al., 1994; Schroeder et  al., 2002; Duek and signaling network involving key components of both auxin Fankhauser, 2005; Thomann et  al., 2005). In line with their and cytokinin signal transduction pathways is implicated in the role as repressors of photomorphogenic responses, either the PHY-dependent regulation of fruit plastid biogenesis, sugar down-regulation or loss-of-function of tomato genes encod- metabolism, and carotenoid accumulation. ing COP1, CUL4, DDB1, DET1, and PIF1a profoundly alter tomato fruit physiology and nutritional composition Materials and methods (Cookson et  al., 2003; Liu et  al., 2004; Davuluri et  al., 2005; Plant material and growth conditions Kolotilin et  al., 2007; Wang et  al., 2008; Azari et  al., 2010b; Enfissi et al., 2010; Llorente et al., 2016b). Tomato (Solanum lycopersicum L.) plants cv. Micro-Tom, which harbors the wild-type SlGLK2 allele (Carvalho et  al., 2011), were grown under In tomato, five PHY-encoding genes have been identified, −2 −1 controlled conditions of 250 µmol m s , a 12-h photoperiod, and air namely SlPHYA, SlPHYB1, SlPHYB2, SlPHYE, and SlPHYF temperature of 27/22  °C day/ night. The fruit stages examined were (Alba et  al., 2000b). The paralogous SlPHYB1 and SlPHYB2, immature green, mature green, breaker, and red ripe, which were harvested which originated during the Solanum whole-genome trip- on average at 8, 25, 32, and 44 d post-anthesis. All fruits were harvested at lication event (Tomato Genome Consortium, 2012), display the same time of the day with four biological replicates (each replicate was composed of a pool of at least five fruits from different plants). Columella, distinct expression profiles within tomato organs, pointing placenta, and seeds were immediately removed, and the remaining tissues to functional diversification (Hauser et  al., 1997; Weller et  al., were frozen in liquid nitrogen and stored at –80 °C until use. 2000). SlPHYB1 is more prominently expressed in vegetative tissues, whereas the highest SlPHYB2 expression levels are Generation of transgenic tomato plants detected in fruits (Hauser et al., 1997; Bianchetti et al., 2017). Three fragments specific to the coding sequences of SlPHYA, SlPHYB2, Moreover, evidence also suggests a more direct involvement of and both SlPHYB1 and SlPHYB2 were selected using BLAST queries SlPHYB1, rather than SlPHYB2, during early seedling pho- against the Sol Genomics Network database (https://solgenomics.net/, tomorphogenic responses (van Tuinen et  al., 1995a, 1995b; ITAG release 2.40) and the web-based computational tool pssRNAit Weller et al., 2000). Very little is known about the influence of (Dai and Zhao, 2011) was employed to avoid off-target silencing. Each SlPHYE and SlPHYF on tomato vegetative and reproductive fragment was independently cloned into pENTR D-TOPO plasmids (Invitrogen) using the primers listed in Supplementary Table  S1 at JXB development (Schrager-Lavelle et al., 2016). online. Subsequently, each fragment was recombined into the plant trans- Attempts to define the influence of fruit-localized PHYs on formation vector pK8GWIWG (Fernandez et  al., 2009). Transgenic fruit development and ripening have been relatively limited. Micro-Tom plants were generated by Agrobacterium-mediated transform- Brief R-light treatments of detached mature-green tomato fruits ation according to Pino et  al. (2010), with minor changes: cotyledons promote lycopene accumulation, a response reversed by sub- from 5-d-old seedlings were used for the transformation, and the zeatin −1 and kanamycin concentration were 5 µM and 70 mg l , respectively. All sequent treatment with FR light (Alba et  al., 2000a), which is plants used in the study were from the T generation. consistent with the hypothesis that fruit-localized PHYs play a regulatory role in controlling tomato fruit carotenogenesis. The marked accumulation of SlPHYA transcripts during fruit ripen- Fruit color and pigment quantification ing (Alba et al., 2000a) associated with the reduced fruit lycopene Changes in fruit color (Hue angle) were determined using a Konica levels observed in phyA tomato mutants (Gupta et al., 2014) raise Minolta CR-400 colorimeter as described in Su et al. (2015). Chlorophyll extraction and quantification were carried out as described in Lira et al. the possibility that this PHY may be an important regulator of (2016) with some modifications. Pericarp samples were weighed (typ- tomato fruit carotenoid biosynthesis. However, regardless of the ically 100  mg fresh weight, FW), ground in liquid nitrogen, immersed development stage or tissue considered, SlPHYB2 is the most in a 10× excess volume of N, N-dimethylformamide, and incubated at highly expressed PHY in tomato fruits (Bianchetti et al., 2017). room temperature for 24 h in absolute darkness and constant agitation Moreover, the phyB2 mutant also displays considerable changes (200  rpm). After centrifugation (9000 g, 5  min, 4  °C), the supernatant absorbance was recorded at 647 and 664 nm, and the total chlorophyll in the fruit carotenoid profile (Gupta et al., 2014), suggesting that content was estimated using the equations given by Porra et al. (1989). multiple PHYs are involved in regulating this metabolic process. For carotenoid extraction, approximately 200 mg FW of pericarp sam- Besides carotenogenesis, PHYs have also been found to ples were ground in liquid nitrogen and sequentially homogenized with control other aspects of tomato fruit development and metab- a solution of 100  µl of saturated NaCl, then 200  µl of dichlorometh- olism, including chloroplast biogenesis, chlorophyll accumula- ane, and finally 1 ml of hexane:diethyl ether (1:1, v/v). The supernatant was collected after centrifugation (5000 g, 10 min, 4 °C). The remaining tion, sugar metabolism, sink activity, and hormonal signaling Downloaded from https://academic.oup.com/jxb/article/69/15/3573/4975406 by DeepDyve user on 18 July 2022 Fruit-localized phytochromes influence tomato quality traits | 3575 carotenoids in the pellet were extracted three more times with 500 µl of (GACGTA), CG-hybrid (GACGTG), canonical AuxRE (TGTGTC), hexane:diethyl ether (1:1, v/v). All supernatant fractions were combined, and degenerate AuxRE (TGTGNC) motifs (Martı́nez-Garcı́a et al., 2000; completely vacuum-dried, and suspended with 200  µl of acetonitrile. Song et al., 2008; Chaabouni et al., 2009). Lycopene, β-carotene, lutein, and neurosporene levels were determined by high-performance liquid chromatography (HPLC) with a photodiode Statistical analysis array detector (PDA) as described by Lira et al. (2017). ANOVA and Student’s t-test were performed using the JMP statistical software package (14th edition; http://jmp.com). Comparisons with P<0.05 were considered statistically significant. Data from wild-type and Starch and soluble sugar quantification all independent transgenic lines were also compared with principal com- Starch and soluble sugar extractions were performed as described in ponent analysis (PCA) using the InfoStat software (http://infostat.com.ar). Bianchetti et  al. (2017). Briefly, approximately 200 mg FW of pericarp samples was extracted with 1 ml of 80% (v/v) methanol for 10 min at 80 °C followed by the collection of the supernatants by centrifugation Results (13 000 g, 10  min, 4  °C). The remaining pellets were re-extracted five times, and all supernatants were combined, completely vacuum-dried, Fruit-specific PHY knockdown in transgenic and suspended in 200 µl distilled water. Soluble sugars (i.e. sucrose, fruc- tomato plants tose, and glucose) were measured using a HPLC system equipped with an amperometric detector (Dionex, Sunnyvale, USA) and a CarboPac PA1 To investigate the role played by distinct PHYs in tomato fruit (4 × 250 mm) column (Purgatto et al., 2002). Starch levels were deter- development and ripening, we generated fruit-specific silenced mined from dried pellet as described in Suguiyama et al. (2014). tomato plants with reduced mRNA levels of SlPHYA, SlPHYB2, or both SlPHYB1 and SlPHYB2. This was achieved using a Antioxidant capacity and total phenolics hairpin-mediated RNAi approach based on the expression of Hydrophilic and lipophilic Trolox equivalent antioxidant capacities specific fragment sequences of these genes under the control of (TEACs) were spectrophotometrically determined as described in Lira the fruit-specific PPC2 promoter (Fernandez et al., 2009). The et al. (2016). Total phenolic content was determined in hydrophilic extracts RNAi transgenic plants obtained, hereafter designated as SlPHYA , by using the Folin–Ciocalteu method (Singleton and Rossi, 1965). RNAi RNAi SlPHYB2 , and SlPHYB1/B2 (Fig. 1A), were generated in a Micro-Tom background homozygous for the wild-type Plastid ultrastructure and abundance GOLDEN2-LIKE-2 (SlGLK2) allele (Carvalho et  al., 2011), Pericarp fragments taken from the pedicel region (green shoulder) of which encodes a transcription factor critically important for immature fruits were fixed at 4 °C in 2.5% (v/v) glutaraldehyde and 2% chloroplast development in tomato fruits (Powell et al., 2012). (v/v) paraformaldehyde in 0.1 M sodium phosphate buffer (pH 7.2). Transcript abundance analysis revealed that SlPHYA, Subsequently, the samples were post-fixed in 1% osmium tetroxide in 0.1 M sodium phosphate buffer (pH 7.2), dehydrated in a graded acetone ser- SlPHYB2, and both SlPHYB1 and SlPHYB2 were down-regu- RNAi RNAi RNAi ies, and embedded in Spurr’s resin. Ultrathin sections were stained with sat- lated in the SlPHYA , SlPHYB2 , and SlPHYB1/B2 urated uranyl acetate and lead citrate (Melo et al., 2016) and observed using lines, respectively (Fig. 1B). A search for potential tomato off- a JEOL JEM1011 transmission electron microscope. Sections from three targets via BLAST queries against the Sol Genomics Network immature fruits picked from different plants were analysed per genotype. database or via the public web-based computational tool pss- Plastid abundance was determined as described in Bianchetti et  al. (2017). Briefly, small pieces (1 × 1 mm) of pericarp were fixed in 3.5% RNAit (Dai and Zhao, 2011) failed to identify regions in the (v/v) glutaraldehyde for 1 h. Samples were washed twice and transferred tomato coding that exhibited the 21-nucleotide perfect iden- to 0.1 M NaEDTA pH 9.5 solution for 4 h at 60 °C in complete dark- tity threshold reported to cause off-target silencing (Xu et  al., ness. Pieces were softly disrupted and transferred to microscope slides. 2006). The percentage of identity of the silencing fragments was Isolated cells were visualized using a Leica microscope. Plastid densities below 60% with non-target tomato PHY genes (Supplementary in individual cells were estimated using the ImageJ program (https:// imagej.nih.gov/ij/). At least 40 individual cells were analysed per sample. Table S2). Moreover, the length of stretches with perfect iden- tity between the RNAi fragments and non-target tomato PHY genes was ≤15 nucleotides (Supplementary Table  S2). In line Transcriptional profile with this, no off-target SlPHY silencing was detected in the Total RNA extraction, cDNA synthesis, primer design, and qPCR assays transgenic lines generated (Supplementary Fig. S1). were performed as described by Quadrana et al. (2013). Primer sequences used are detailed in Supplementary Table  S1. Quantitative real-time In all the transgenic lines, PHY knockdown was restricted to (qRT-)PCR reactions were performed in a StepOnePlus PCR Real- the fruit tissues as no significant PHY silencing was observed in Time thermocycler (Applied Biosystems) in a final volume of 10 µl using leaf samples (Fig. 1B). Transgenic lines exhibited normal plant 2× SYBR Green Master Mix reagent (Thermo Fisher Scientific). Melting growth and visual phenotypic features similar to those found in curves were checked for unspecific amplifications and primer dimeriza- wild-type (WT) plants (Supplementary Fig. S2). Overall, fruit- tion. Absolute fluorescence data were analysed using the LinRegPCR software package (Ruijter et al., 2009) to obtain quantitation cycle (C ) specific PHY knockdown caused no marked changes in fruit values and to calculate primer efficiency. Transcript abundances were size and ripening progression (Supplementary Fig. S3). normalized against the geometric mean of two reference genes, CAC and EXPRESSED (Expósito-Rodriguez et al., 2008). Fruit-localized SlPHYA and SlPHYB2 differentially impact chloroplast biogenesis and differentiation Gene promoter analysis during early fruit development Gene promoter analysis was performed using the promotor sequences available at the Sol Genomics Network. Typically, 3 kb upstream of the The PHY-dependent regulation of chloroplast development initial ATG codon of each sequence was analysed using the PlantPAN 2.0 has been extensively reported in leaf tissues of several spe- platform (http://plantpan2.itps.ncku.edu.tw/) (Chow et  al., 2016) for cies (Stephenson et  al., 2009; Inagaki et  al., 2015). Moreover, the presence of PBE-box (CACATG), G-box (CACGTG), CA-hybrid Downloaded from https://academic.oup.com/jxb/article/69/15/3573/4975406 by DeepDyve user on 18 July 2022 3576 | Bianchetti et al. RNAi RNAi Fig. 1. Fruit-specific PHY knockdown in transgenic tomato plants. (A) Constructs designed for generation of the SlPHYA , SlPHYB1/B2 , and RNAi SlPHYB2 transgenic lines. ‘A’ indicates the SlPHYA-specific fragment of the mRNA 5´ untranslated region (UTR). ‘B1/B2’ indicates the SlPHYB1/B2- specific fragment of the mRNA 5´ UTR. ‘B2’ indicates the SlPHYB2-specific fragment of the mRNA 5´ UTR. (B) Relative SlPHY mRNA levels in leaves, RNAi RNAi RNAi and immature green (IG), mature green (MG), and breaker (Bk) stages of fruits of the SlPHYA , SlPHYB2 , and SlPHYB1/B2 lines. The first and second fully expanded leaves from the top of 2-month-old plants were harvested. Transcript abundance was normalized against the wild-type (WT) sample. Statistically significant differences compared with the WT genotype were determined using Student’s t-test: *P<0.05. Data are means (±SE) of at least three biological replicates. (This figure is available in color at JXB online.) some recent reports have also indicated altered chlorophyll WT fruits (Fig. 2B). However, the higher chlorophyll content RNAi accumulation and chloroplast biogenesis in immature fruits of observed in SlPHYB2 immature fruits was not accompa- PHY-deficient tomato mutants (Gupta et al., 2014; Bianchetti nied by changes in plastid abundance but instead was linked et al., 2017). Compared to the WT, fruit-specific SlPHYA and to the up-regulation of the master regulator of chloroplast SlPHYB2 knockdown reduced and increased the chlorophyll development and maintenance, SlGLK2 (Fig. 2C). SlPHYB1/ content in immature fruits, respectively (Fig.  2A). However, B2 knockdown lines showed an intermediate impact on fruit RNAi chlorophyll levels in immature fruits from SlPHYB1/B2 chlorophyll content, plastid density, and SlGLK2 mRNA lev- plants were similar to WT counterparts. els, exhibiting unaltered chlorophyll levels and chloroplast Microscopy analysis of pericarp cells revealed that the abundance in pericarp cells and slightly higher expression of RNAi reduced chlorophyll content detected in SlPHYA imma- SlGLK2 compared to the WT (Fig. 2). RNAi RNAi ture fruits was associated with a reduction of up to 40% in Plastids of WT, SlPHYB2 , and SlPHYB1/B2 immature the number of chloroplasts per pericarp cell compared to fruits exhibited remarkably similar internal membranous structures, Downloaded from https://academic.oup.com/jxb/article/69/15/3573/4975406 by DeepDyve user on 18 July 2022 Fruit-localized phytochromes influence tomato quality traits | 3577 Fig. 2. Fruit-localized SlPHYA and SlPHYB2 differentially impact on chloroplast biogenesis and differentiation during early fruit development. (A) Total chlorophyll content in immature fruits. (B) Plastid abundance per pericarp cell of immature fruits. (C) Relative mRNA levels of GOLDEN2-LIKE-2 (SlGLK2) normalized against the wild-type (WT) sample. Statistically significant differences compared with the WT sample were determined using Student’s t-test: *P<0.05. Chlorophyll content and transcript abundance data are means (±SE) of at least three biological replicates. For plastid density, three fruits of each genotype were randomly picked, and two technical replicates were taken at the pedicel region of each fruit. Plastid density was determined in at least 40 individual cells per sample. (D) Representative TEM images of plastids in the pedicel region of immature fruits. Arrows indicate plastoglobuli. G, granal thylakoid. displaying well-developed grana and stroma thylakoids as well as fruit tissues (Shi et  al., 2012), SlCRF1, SlCRF2, and SlCRF5 RNAi numerous plastoglobuli (Fig. 2D, Supplementary Fig. S4). In con- were markedly down-regulated in SlPHYA lines, whereas trast, fruit-specific SlPHYA knockdown resulted in the formation SlCRF3 and SlCRF9 mRNA levels remained unchanged of chloroplasts with highly reduced grana, suggesting a promo- (Fig.  3C). As AtCRF2 is responsible for inducing AtPDV2, tive role of PHYA-mediated light perception on fruit plastid granal subsequently increasing plastid division rates in Arabidopsis development. Plastoglobuli and starch grains were observed equally (Okazaki et  al., 2009), the drastic down-regulation of both in fruit chloroplasts of the WT and all transgenic lines. SlCRF2 and SlPDV2 in SlPHYA-silenced fruits suggests that As neither SlPHYB2 nor the SlPHYB1/B2 knockdown a similar regulatory mechanism also takes place early in the altered chloroplast density per cell or plastid ultrastructure development of tomato fruits. (Fig.  2), fruit-localized SlPHYA seems to play a preponder- Alongside the down-regulation of cytokinin signaling genes, ant role in controlling chloroplast biogenesis and differenti- fruit-specific SlPHYA-silencing resulted in the up-regulation ation in early developing fruits. Transcript abundance analysis of tomato genes encoding light-signaling repressor proteins revealed that the reduced plastid abundance observed in such as COP1, CUL4, DDB1, and DET1 (Fig.  3D), which SlPHYA-silenced fruits was most probably explained are negative regulators of plastid division and maturation in by a drastic reduction in mRNA levels of genes encod- tomato and other species (Chory and Peto, 1990; Kolotilin ing key components of the plastid division machinery, such et al., 2007; Wang et al., 2008; Azari et al., 2010b). as FILAMENTOUS TEMPERATURE SENSITIVE-Z Collectively, these data suggest that fruit-localized PHYA (FtsZs), ACCUMULATION AND REPLICATION OF positively influences tomato plastid division machinery via CHLOROPLASTS (ARCs), and PLASTID DIVISION 2 changes in the transcript abundance of both light- and cyto- (PDV2), compared to the WT genotype (Fig. 3A). kinin-signaling genes, whereas PHYB2 negatively regulates Given the key role played by cytokinins in regulating plas- chlorophyll accumulation by controlling the expression of the tid division and maturation in plants and the widely reported master transcription factor of chloroplast development and crosstalk between this hormonal class and PHY signaling maintenance, SlGLK2. (Okazaki et  al., 2009; Cortleven and Schmülling, 2015), a transcriptional profiling of type-A TOMATO RESPONSE Fruit-localized PHYs regulate starch metabolism during REGULATOR (TRR) was performed. Four out of the five early fruit development type-A TRRs analysed were significantly down-regulated in RNAi Fruit-specific SlPHYA and SlPHYB2 knockdown promoted immature fruits of SlPHYA compared to the WT geno- starch accumulation during early fruit development (Fig. 4A). type (Fig.  3B). Moreover, among the five CYTOKININ In both the WT and transgenic lines, the highest starch content RESPONSE FACTOR genes most highly expressed in tomato Downloaded from https://academic.oup.com/jxb/article/69/15/3573/4975406 by DeepDyve user on 18 July 2022 3578 | Bianchetti et al. Fig. 3. SlPHYA-mediated regulation of chloroplast division machinery is associated with changes in the transcript abundance of light- and cytokinin- signaling genes. (A) Relative mRNA levels of genes encoding components of the plastid division machinery in immature fruits. (B) Relative mRNA levels of type-A TOMATO RESPONSE REGULATOR (TRR) genes in immature fruits. (C) Relative mRNA levels of CYTOKININ RESPONSE FACTOR (SlCRF) genes in immature fruits. (D) Relative mRNA levels of genes encoding light-signaling repressor proteins. Data are means (±SE) of at least three biological replicates. Transcript abundance was normalized against the wild-type (WT) sample. Statistically significant differences compared with the WT were determined using Student’s t-test: *P<0.05). FtsZ, filamentous temperature sensitive-Z; ARC, accumulation and replication of chloroplasts; PDV2, plastid division 2; COP1, constitutive photomorphogenic 1; CUL4, cullin 4; DDB1, UV-damaged DNA binding protein 1; DET1, de-etiolated1. RNAi was observed in immature green (IG) fruits, followed by development and ripening in both the SlPHYA and RNAi slightly more reduced levels at the mature green (MG) stage, SlPHYB2 lines. However, despite the negative impact of and undetectable levels from the breaker (Bk) stage onwards either SlPHYA- or SlPHYB2-silencing on SlAGPaseS1 expres- (Supplementary Fig. S5). sion, this gene exhibited higher expression levels than those Compared to the WT genotype, marked differences in the encoding AGPase large subunits (Supplementary Table  S3), transcript profiles of starch biosynthesis genes were observed suggesting that the catalytic AGPase subunit was not limiting in both SlPHYA- and SlPHYB2-silenced fruits (Fig.  4A, for starch biosynthesis in tomato fruits. RNAi RNAi Supplementary Fig. S6). Catalysing the first committed step in In both SlPHYA and SlPHYB2 immature fruits, the starch biosynthesis, ADP-glucose pyrophosphorylase (AGPase) starch synthase (STS)-encoding genes SlSTS1 and SlSTS2 is a heterotetramer comprising a pair of small/catalytic and a were markedly up-regulated compared to WT fruits, whereas pair of large/regulatory subunits (Kim et  al., 2007; Figueroa SlSTS3 was slightly down-regulated. For SlSTS6, higher tran- RNAi et  al., 2013). Among the tomato genes encoding the large script accumulation was observed in SlPHYA than in the AGPase subunits, both SlAGPaseL1 and SlAGPaseL3 were WT throughout fruit development and ripening (i.e. IG to up-regulated whereas SlAGPaseL2 mRNA levels remained RR stage) (Supplementary Fig.  S6). Finally, distinct expres- RNAi unchanged in immature fruits of SlPHYA plants. It is sion patterns were observed for the starch branching enzyme worth mentioning that SlAGPaseL1 was the large AGPase sub- (SBE)-encoding genes, as SlSBE1 was up-regulated in all the unit most expressed in immature tomato fruits (Supplementary transgenic lines from MG to Bk stage whereas SlSBE2 was RNAi RNAi Table S3; Petreikov et al., 2006); therefore, the 3-fold increment down-regulated in both SlPHYA and SlPHYB2 from in its mRNA levels correlates well with the higher starch lev- IG to RR stage (Supplementary Fig. S6). RNAi RNAi els and reduced soluble sugar levels detected in SlPHYA The increased accumulation of starch in SlPHYA fruits immature fruits compared to the WT counterparts (Fig.  4, correlated well with higher mRNA levels of SlLIN5 and Supplementary Figs S5, S6). SlLIN6 (Fig.  4D), which encode cell-wall invertases critic- SlAGPaseS1, which encodes the small/catalytic AGPase ally important for sink activity in tomato (Fridman and Zamir, subunit, was consistently down-regulated throughout fruit 2003; Kocal et al., 2008). By applying an unsupervised method Downloaded from https://academic.oup.com/jxb/article/69/15/3573/4975406 by DeepDyve user on 18 July 2022 Fruit-localized phytochromes influence tomato quality traits | 3579 Fig. 4. Fruit-localized phytochromes regulate sugar metabolism during early fruit development. (A) Schematic representation of the major steps of starch biosynthesis and graphs showing starch content and transcript abundance of starch biosynthesis-related genes in immature fruits. (B) Soluble sugar contents in immature fruits. (C) Summed values of the three soluble sugars analysed (i.e. sucrose + glucose + fructose). (D) Relative mRNA levels of tomato genes encoding invertases (SlLIN) in immature fruits. (E) Relative mRNA levels of AUXIN RESPONSE FACTOR 4 (SlARF4) in immature fruits. For simplicity, the mean of the three values for the transgenic lines is shown. Values for each transgenic line are presented in Supplementary Figs S5, S6. Data are means (±SE) of at least three biological replicates. Statistically significant differences compared with the wild-type (WT) sample were determined using Student’s t-test: *P<0.05. IG, immature green; MG, mature green; Bk, breaker; RR, red ripe; Glc-1-P, glucose 1-phosphate; ADPG, adenosine diphosphate glucose; AGPase, ADP-glucose pyrophosphorylase; STS, starch synthase; SBE, starch branching enzyme. (i.e. principal component analysis, PCA) to search for pat- promoter sequences of SlLIN5 and SlLIN6 genes (Bianchetti terns in the expression profiles of genes related to sink- and et al., 2017). starch-biosynthesis, we demonstrated a clear separation of the RNAi RNAi WT, SlPHYA , and SlPHYB2 groups (Supplementary PHY-dependent regulation of fruit carotenoid Fig. S7). biosynthesis is associated with transcriptional changes Previous findings have indicated that AUXIN RESPONSE in light- and auxin-signaling genes FACTOR4 (SlARF4) is a major negative regulator of starch The very well-characterized PHY-mediated signaling networks biosynthesis in early developing tomato fruits (Sagar et  al., controlling carotenogenesis in vegetative tissues (Toledo-Ortiz 2013; Bianchetti et  al., 2017). Recent evidence also indicates et  al., 2010) contrasts with the considerably more limited that SlARF4 plays a repressor role in controlling the tran- information regarding the fruit-localized PHY-dependent script abundance of sink-related genes, including SlLIN5 and signaling cascades regulating carotenoid biosynthesis in fleshy SlLIN6 (Bianchetti et al., 2017). In accordance with this, fruit- fruits (Llorente et  al., 2016b, 2017). Carotenoid profiling specific SlPHYA and SlPHYB2 knockdown drastically reduced revealed a significant reduction in lycopene content in red ripe SlARF4 mRNA abundance in early developing tomato fruits RNAi RNAi (RR) fruits of both the SlPHYA and SlPHYB2 lines (Fig.  4E). Although the direct transcriptional regulation of compared to the WT (Fig.  5A, Supplementary Table  S4). In tomato AGPase, STS, and SBE genes by transcription factors contrast, the content of all other carotenoids analysed (i.e. phy- associated with auxin- or light-signaling remains to be deter- toene, phytofluene, β-carotene, and lutein) remained virtually mined, the presence of PBE-box, G-box, CA-hybrid, and/or unchanged in ripe fruits of the transgenic lines compared to CG-hybrid motifs (Martı́nez-Garcı́a et  al., 2000; Song et  al., WT counterparts. As lycopene is the main carotenoid accu- 2008) as well as canonical and/or degenerated ARF-binding mulated in ripe tomato, fruit-specific SlPHYA- or SlPHYB2- Auxin Response Element (AuxRE) motifs within the 3-kb knockdown led to a slight, yet significant, reduction in total promoter sequence of these genes (Supplementary Fig. S8) is carotenoid content compared to the WT genotype (Fig.  5A, consistent with the hypothesis that light- and/or auxin-related Supplementary Table  S4). In accordance with this, signifi- transcription factors might directly control the expression of cantly lower mRNA levels of genes encoding carotenoid starch biosynthesis-related genes. Similarly, PIF, HY5, and/ biosynthesis-related enzymes such as GERANYLGERANYL or ARF-binding motifs have also been identified within the Downloaded from https://academic.oup.com/jxb/article/69/15/3573/4975406 by DeepDyve user on 18 July 2022 3580 | Bianchetti et al. Fig. 5. Fruit-specific SlPHYA or SlPHYB2 knockdown represses carotenoid biosynthesis during tomato fruit ripening. (A) Lycopene, phytoene, phytofluene, β-carotene, lutein, and total carotenoid content in red ripe fruits. (B) Schematic representation of carotenoid biosynthetic pathway and graphs showing the transcript abundance of carotenoid biosynthesis genes in ripening fruits. Intermediate reactions are omitted. For simplicity, the mean of the three values for the transgenic lines is shown. Values for each transgenic line are presented in Supplementary Fig. S9, Supplementary Table S4. Data are means (±SE) of at least three biological replicates. Statistically significant differences compared with the wild-type (WT) sample were determined using Student’s t-test: *P<0.05. MG, mature green; Bk, breaker; RR, red ripe; MEP, methylerythritol 4-phosphate; GGDP, geranylgeranyl diphosphate; GGPS, GGDP synthase; PSY, phytoene synthase; PDS, phytoene desaturase; LCYβ, chloroplast-specific β-lycopene cyclase; CYCβ, chromoplast- specific β-lycopene cyclase. (This figure is available in color at JXB online.) DIPHOSPHATE SYNTHASE (GGPS), PHYTOENE fruits (Azari et  al., 2010b; Llorente et  al., 2016b) whereas SYNTHASE 1 (PSY1), and PHYTOENE DESATURASE auxin response factors such as SlARF2a and SlARF2b play (PDS) were observed in ripe fruits of SlPHYA and SlPHYB2- the opposite role (Hao et  al., 2015). To gain insight into the silenced lines than in WT counterparts (Fig. 5B, Supplementary potential role played by these signaling components during Fig.  S9). In line with the relatively limited reduction in total the PHY-dependent regulation of carotenoid biosynthesis in carotenoids, no significant differences in lipophilic antioxidant tomato fruits, the transcript abundance of their encoding genes RNAi RNAi activity were observed between ripe WT and transgenic fruits was profiled in both SlPHYA and SlPHYB2 ripening (Supplementary Table  S5). Interestingly, however, red ripe fruits (Fig. 6, Supplementary Fig. S10). Among the four SlPIF SlPHYB2-down-regulated fruits exhibited increased hydro- genes most highly expressed in fruits (Rosado et  al., 2016), philic antioxidant activity compared to the WT, which may SlPIF1a, SlPIF1b, and SlPIF4/5 mRNA levels were signifi- be associated with the higher content of total phenolics also cantly higher in SlPHYB2-down-regulated fruits compared RNAi detected in SlPHYB2 ripe fruits (Supplementar y Table S5). to the WT counterparts during fruit ripening (MG, Bk, and Accumulating evidence indicates that light-signaling RR stages), whereas the opposite was observed for SlPIF3 repressors such as SlPIF1a, SlCOP1, SlCUL4, SlDDB1, and transcripts. Although less pronounced, the overall impacts of SlDET1 negatively regulate carotenoid biosynthesis in tomato fruit-specific SlPHYA knockdown on tomato PIF expression Downloaded from https://academic.oup.com/jxb/article/69/15/3573/4975406 by DeepDyve user on 18 July 2022 Fruit-localized phytochromes influence tomato quality traits | 3581 Fig. 6. PHY-dependent regulation of fruit carotenogenesis is associated with transcriptional changes in auxin- and light-signaling genes. (A) Transcript abundance of tomato genes encoding PHYTOCHROME INTERACTING FACTORs (SlPIFs). (B) Transcript abundance of tomato genes encoding the light-signaling repressors CONSTITUTIVE PHOTOMORPHOGENIC 1 (SlCOP1), CULLIN 4 (SlCUL4), UV-DAMAGED DNA BINDING PROTEIN 1 (SlDDB1), and DE-ETIOLATED1 (SlDET1). (C) Transcript abundance of the tomato AUXIN RESPONSIVE FACTOR 2a and 2b (SlARF2a and SlARF2b) genes. For simplicity, the mean of the three values for the transgenic lines is shown. Values for each transgenic line are presented in Supplementary Fig. S10. Data are means (±SE) of at least three biological replicates. Statistically significant differences compared with the wild-type (WT) sample were determined using Student’s t-test: *P<0.05. MG, mature green; Bk, breaker; RR, red ripe. RNAi profiles were similar to those observed in the SlPHYB2 and quality traits. Differently from the pleiotropic phenotyp- lines (Fig. 6, Supplementary Fig. S10). ical alterations observed in phy mutants (Gupta et  al., 2014; Among the genes encoding light-signaling repressors, Bianchetti et al., 2017), the fruit-specific silencing of the target SlCUL4, SlDDB1, and SlDET1 exhibited significantly SlPHY genes resulted in no obvious impacts on plant vegeta- higher mRNA levels in SlPHYA-silenced fruits in com- tive growth and overall yield. This suggests that the perturb- parison to the WT at all fruit development stages analysed ation in fruit metabolism caused by the fruit-specific SlPHY (Fig. 6B, Supplementary Fig. S10). Moreover, strikingly higher manipulation does not propagate from fruits to the rest of the SlDET1 transcript abundance was also detected in SlPHYB2- plant, which agrees with the limited transference of substances knockdown compared to WT fruits at all ripening stages (i.e. out of this predominantly sink organ. MG, Bk, and RR) whereas SlCOP1 and SlDDB1 mRNA lev- In a previous work, we demonstrated that a global defi- RNAi els were also up-regulated in SlPHYB2 fruits exclusively ciency in functional PHYs drastically reduces chlorophyll con- at the MG stage. Transcript levels of the positive regulators tent and chloroplast abundance in tomato fruits (Bianchetti of tomato fruit carotenogenesis SlARF2a and SlARF2b were et al., 2017). Therefore, the PHY-mediated regulation of plas- RNAi RNAi considerably lower in SlPHYA and SlPHYB2 fruits, tid biogenesis and maturation widely reported for leaf tissues particularly at the Bk and RR stages (Fig. 6C, Supplementary (Stephenson et  al., 2009; Oh and Montgomery, 2014; Melo Fig. S10). A PCA plot in which the expression profile of carot- et al., 2016) seems to be conserved early in the development of enoid biosynthesis-related genes as well as SlPIFs, SlCOP1, tomato fruits. In this current work, it is further demonstrated SlCUL4, SlDDB1, SlDET1, SlARF2a, and SlARF2b were rep- that fruit-localized SlPHYA and SlPHYB2 play distinct roles RNAi RNAi resented revealed that the WT, SlPHYA , and SlPHYB2 in controlling chloroplast biogenesis and activity during early groups clearly separated from each other at the red ripe stage stages of tomato fruit development. (Supplementary Fig. S11). The results indicate that SlPHYA-mediated light percep- Altogether, these data suggest that both SlPHYA and tion promotes fruit chloroplast biogenesis and differenti- SlPHYB2 play overlapping roles in promoting the paralogues ation, as inferred from the reduced chlorophyll content, lower SlARF2a and SlARF2b and repressing light-signaling repres- chloroplast abundance, and poorly-developed grana stacking RNAi sors such as SlPIF1a, SlPIF1b, SlPIF4/5, SlCOP1, SlCUL4, detected in SlPHYA immature fruits (Fig. 2). In line with SlDDB1, and SlDET1, which in turn mediate the PHY- this observation, an analysis of single and multiple phy mutants dependent regulation of carotenoid biosynthesis in ripening also suggested that SlPHYA is a major regulator of chloro- tomato fruits. phyll accumulation in tomato fruits (Gupta et  al., 2014). In land plants, chloroplast division depends on nucleus-encoded proteins that form ring structures at the division site (Jarvis Discussion and López-Juez, 2013). Our findings clearly demonstrate that Studies performed on PHY-deficient mutants have suggested fruit-localized SlPHYA influences the transcript levels of genes that PHY-dependent light perception participates in the regu- derived from the ancestral prokaryotic cell-division machinery, lation of several aspects of tomato fruit biology (Gupta et  al., such as SlFtsZ (i.e. SlFtsZ1, SlFtsZ2) and SlARCs (i.e. SlARC3 2014; Bianchetti et  al., 2017). Here, we applied a RNAi- and SlARC6), as well as those encoding chloroplast division- mediated organ-specific silencing approach to investigate the related proteins specific to land plants, such as SlPDV2. In impact of fruit-localized SlPHYs on tomato fruit physiology Arabidopsis, PDV2 determines the rate of chloroplast division Downloaded from https://academic.oup.com/jxb/article/69/15/3573/4975406 by DeepDyve user on 18 July 2022 3582 | Bianchetti et al. and is positively regulated by cytokinins, being strongly pro- the transcriptional profile of starch biosynthesis-related genes moted in transgenic plants overexpressing the cytokinin sign- at the immature green stage (Fig.  4). AGPase, which cataly- aling-related transcription factor CRF2 (Okazaki et al., 2009; ses the rate-limiting reaction in the starch synthesis pathway, is Cortleven and Schmülling, 2015). SlCRF2, along with other both transcriptionally and post-translational regulated by light SlCRF and TRR genes, were drastically repressed in PHYA- (Harn et al., 2000; Geigenberger, 2011), although the role played down-regulated fruits, implying that changes in cytokinin by PHYs in this regulatory process remains elusive. During signaling mediate the PHYA-dependent regulation of plas- early fruit development, SlPHYA-suppressed fruits exhibited tid division during early stages of tomato fruit  development. increased mRNA levels of both SlAGPaseL1 and SlAGPaseL3, In agreement with this, accumulating evidence indicates that which encode AGPase large subunits, and SlSTS1, SlSTS2, and there is an intensive crosstalk between the PHY and cytokinin SlSTS6, which encode starch synthase enzymes, along with an signaling cascades, with particular involvement of CRF and increase in starch accumulation and reduced soluble sugar con- type-A ARR proteins (Salomé et al., 2006; Oh et al., 2009). tent, thus indicating a repressor role for fruit-localized SlPHYA Fruit-specific SlPHYA-silencing also promoted the mRNA on the first steps of starch synthesis in tomato fruits. Whether accumulation of genes encoding all the major light-signaling the up-regulation of starch biosinthesis-related genes is a com- repressor proteins already described to negatively regulate chloro- pensatory mechanism to cope with the fewer and poorly devel- RNAi plast biogenesis in tomato fruits, i.e. SlCOP1, SlCUL4, SlDDB1, oped chloroplasts observed in SlPHYA immature fruits and SlDET1 (Liu et  al., 2004; Kolotilin et  al., 2007; Wang et  al., remains to be investigated. In contrast, the increased starch 2008; Azari et  al., 2010a). Defective mutants or transgenic lines accumulation detected in SlPHYB2-silenced immature fruits with reduced levels of each of these genes are known to develop was not associated with increments in transcript abundance more chloroplasts containing more grana/thylakoids in both of AGPase-encoding genes nor with prominent reductions in leaves and immature fruits (Cookson et al., 2003; Liu et al., 2004; soluble sugars, but instead were accompanied by increments in Kolotilin et al., 2007; Wang et al., 2008; Azari et al., 2010a), which SlSTS1 and SlSTS2 mRNA levels. Furthermore, as no signifi- in some cases, such as in the SlDET1-knockout mutant, is asso- cant alterations in plastid abundance or internal structure were RNAi ciated with the up-regulation of plastid biogenesis-related genes observed in SlPHYB2 immature fruits, it seems likely that (Kolotilin et  al., 2007). Therefore, the presence of fewer chloro- this genetic manipulation caused less prominent changes than plasts with poorly developed or almost no grana in immature fruits SlPHYA-silencing on reactions taking place within fruit chlo- of the SlPHYA-suppressed lines agrees with the higher transcript roplasts, including starch biosynthesis. Altogether, these find- abundance of SlCOP1, SlDDB1, and particularly SlCUL4 and ings suggest that SlPHYA and SlPHYB2 negatively regulate SlDET1 in these transgenic lines compared to the WT genotype. starch synthesis via overlapping, yet distinct, mechanisms. In contrast, fruit-localized SlPHYB2 was shown to play a The influence of auxin on fruit sugar metabolism has been negative role in chlorophyll accumulation, as evidenced by increasingly reported (Purgatto et al., 2002; Yuan and Carbaugh, the increment in chlorophyll content in immature fruits of 2007; Bianchetti et  al., 2017). In tomato, SlARF4 has been RNAi SlPHYB2 plants with no impact in chloroplast number described as a key negative regulator of starch synthesis dur- in pericarp cells. As SlPHYB2 fruit-specific silencing led to ing early fruit development via the transcriptional and post- higher SlGLK2 mRNA levels compared to the WT genotype, transcriptional down-regulation of AGPase (Sagar et  al., 2013). it seems plausible to suggest that the effect of SlPHYB2 on Recent findings have also indicated that PHYs strictly regulate fruit chloroplasts is mediated by SlGLK2, the master regula- the transcript abundance of this particular auxin response factor tor of chloroplast development in tomato fruits (Powell et al., in both vegetative (Melo et al., 2016) and fruit tissues (Bianchetti 2012). Further suggesting that the SlPHYB2-mediated regu- et al., 2017). In line with this, the increased starch accumulation in lation of SlGLK2 expression is essential for the consequent pre-ripening SlPHYA- and SlPHYB2-silenced fruits correlated changes in fruit chlorophyll accumulation, no obvious changes well with the down-regulation of SlARF4 in these transgenic RNAi RNAi in chlorophyll content were observed in phyb2 mutants from lines (Fig.  4). In fact, SlPHYA rather than SlPHYB2 tomato varieties that lacked functional SlGLK2 proteins (Gupta exhibited the most expressive decrease in SlARF4, and only the et al., 2014). In agreement with these findings, PHY-dependent former displayed increased mRNA levels of AGPase-encoding transcriptional regulation of GLK genes has been increasingly genes in immature fruits. Together, these data strongly suggest reported in vegetative tissues of other plant species (Oh and that fruit-localized PHYA, and to some extent SlPHYB2, posi- Montgomery, 2014; Song et al., 2014). tively modulates SlARF4, which in turn represses starch biosyn- Alterations in chloroplast number, internal structure, and thetic enzymes, such as AGPase and STS, consequently limiting size during the early development of tomato fruits signifi- starch synthesis in pre-ripening tomato fruits. cantly impact the abundance of metabolites associated with Previous findings indicated that a global deficiency in organoleptic and nutritional quality at the ripe stage (Galpaz functional phytochromes transcriptionally represses both et  al., 2008; Cocaliadis et  al., 2014). Intense starch synthe- sink-related and starch biosynthesis-related enzymes in early sis and degradation take place in tomato fruit chloroplasts developing tomato fruits, suggesting a promotive role of PHYs at the unripe and breaker stages, respectively (Schaffer and on the regulation of these processes (Bianchetti et  al., 2017). Petreikov, 1997). Whereas the global deficiency in PHYs sig- However, it remained unclear whether these responses were nificantly reduces the starch content in immature tomato fruits dependent on fruit-localized PHYs or were the consequence (Bianchetti et  al., 2017), fruit-localized SlPHYA or SlPHYB2 of collateral negative effects of the global PHY deficiency suppression increased fruit starch levels and markedly altered on vegetative plant growth. Here, we shed light on this topic Downloaded from https://academic.oup.com/jxb/article/69/15/3573/4975406 by DeepDyve user on 18 July 2022 Fruit-localized phytochromes influence tomato quality traits | 3583 by showing that fruit-localized SlPHYA, and to some extent PHY-dependent signaling cascades in the fruit carotenoid bio- SlPHYB2, repress both starch metabolism and key determi- synthetic pathway. Consistent with this, our findings indicate nants of tomato fruit sink strength, including SlLIN5 transcript that fruit-localized SlPHYA and SlPHYB2 positively influ- accumulation (Fridman and Zamir, 2003; Kocal et  al., 2008). ence the transcript accumulation of all the major carotenoid Consequently, the down-regulation in starch synthesis and biosynthesis-related genes, including SlGGPS, SlPSY1, SlPDS, sink activity previously observed in fruits of the PHY-deficient SlCYCβ, and SlLYCβ, consequently modifying the lycopene mutant aurea (Bianchetti et al., 2017) may be due either to lim- and total carotenoid content in this fleshy fruit. Light-signaling itations in vegetative growth and metabolism or to the com- repressor proteins such as SlDET1, SlDDB1, SlCOP1, SlCUL4, binatory effect of the deficiency in all phytochromes instead of and more recently SlPIF1a have been identified as key nega- only in SlPHYA or SlPHYB2. Moreover, it also seems tempt- tive regulators of tomato fruit carotenoid synthesis (Liu et al., ing to suggest that the fewer and poorly-developed chloroplasts 2004; Kolotilin et al., 2007; Wang et al., 2008; Azari et al., 2010a; RNAi detected in SlPHYA immature fruits restrict photoassimi- Llorente et  al., 2016b). Among these, the transcription factor late production via fruit photosynthesis; therefore, the observed SlPIF1a was shown to directly bind to the promoter of SlPSY1 up-regulation of sink-related genes in transgenic fruits may to repress fruit carotenogenesis (Llorente et  al., 2016b), thus represent a compensatory mechanism to maintain fruit growth resembling the action of its ortholog in Arabidopsis (AtPIF1) and intense starch accumulation despite potential limitations in in controlling carotenoid biosynthesis in leaf tissues (Toledo- fruit-localized photoassimilation. Ortiz et  al., 2010). Therefore, the marked up-regulation of The link between PHY-dependent light perception and SlDET1, SlDDB1, SlCOP1, SlCUL4, SlPIF1a, and SlPIF1b carotenoid metabolism in both vegetative and fruit tissues has together with the overall repression of carotenoid biosynthesis been highlighted by a number of studies (Alba et  al., 2000a; observed in both SlPHYA- and SlPHYB2-silenced fruits imply Llorente et  al., 2016b). Exposure of wild-type tomato fruits that light-signaling repressor proteins participate in SlPHYA- to red light (Alba et  al., 2000a) or constitutively silencing of and SlPHYB2-mediated regulation of fruit carotenogenesis. SlPIF1a (Llorente et  al., 2016b) promotes tomato fruit lyco- In addition, it is becoming increasingly well established that pene accumulation, thereby implying a positive role of auxin represses tomato ripening and down-regulates lycopene Fig. 7. Proposed model for phytochrome-mediated signaling events controlling chloroplast biogenesis, and sugar and carotenoid metabolism in tomato fruits. (A) SlPHYA- and SlPHYB2-dependent light perception regulate fruit plastid division and maturation, respectively. By promoting key members of the cytokinin signaling-related CRF and TRR gene family, SlPHYA up-regulates SlPDV2, a rate-limiting component of the plastid division machinery. Moreover, the SlPHYA-mediated down-regulation of light-signaling repressors, such as SlCOP1, SlDET1, SlDDB1, and SlCUL4, induces other major components of the chloroplast division machinery, such as SlFTsZs and SlARCs. In contrast, Sl-PHYB2 represses the chloroplast differentiation transcription factor SlGLK2, consequently limiting chloroplast differentiation during early fruit development. (B) Fruit-localized SlPHYA and SlPHYB2 play overlapping roles in repressing and promoting starch and carotenoid biosynthesis, respectively. Both SlPHYA and SlPHYB2 induce SlARF4, a negative regulator of AGPase and starch accumulation in tomato fruits. In contrast, these same photoreceptors promote both SlARF2 paralogues and inhibit all the major genes encoding light-signaling repressor proteins, consequently up-regulating most components of the tomato carotenoid biosynthetic route. Arrows at the ends of lines indicate stimulatory effects, whereas bars indicate inhibitory effects. AGPase, ADP-glucose pyrophosphorylase; ARC, accumulation and replication of chloroplasts; ARF, auxin response factor; COP1, constitutive photomorphogenic 1; CRF, cytokinin response factor; CUL4, cullin 4; DDB1, UV-damaged DNA binding protein 1; DET1, de-etiolated1; FtsZ, filamentous temperature sensitive-Z; GGPS, geranylgeranyl pyrophosphate synthase; GLK2, golden2-like-2; PDS, phytoene desaturase; PDV2, plastid division 2; PIF, phytochrome interacting factor; PSY, phytoene synthase; TRR, tomato response regulator. Downloaded from https://academic.oup.com/jxb/article/69/15/3573/4975406 by DeepDyve user on 18 July 2022 3584 | Bianchetti et al. biosynthetic genes (Su et  al., 2015). Among tomato ARF Table S3. Relative transcript ratios of SlAGPase in immature genes, two paralogs, SlARF2a and SlARF2b, have emerged as fruits. key positive regulators of tomato fruit ripening and lycopene Table S4. Carotenoid profiles in red ripe fruits. accumulation (Hao et al., 2015). Either SlPHYA or SlPHYB2 Table S5. Antioxidant activity and total phenolics in red ripe fruit-specific silencing profoundly reduced both SlARF2a and fruits. SlARF2b, suggesting the involvement of these auxin signaling elements in the PHY-dependent regulation of carotenoid bio- Acknowledgements synthesis in tomato fruits. Overall, our results shed light on the specific role played The authors sincerely thank Prof. Lazaro E.  P. Peres for providing the Micro-Tom GLK2 seeds. This work was supported by the CNPq by fruit-localized phytochromes and their downstream signal- (Conselho Nacional de Desenvolvimento Científico e Tecnológico, ing cascades, showing that plastid division, as well as sugar and grant no. 442045/2014-0) and the FAPESP (Fundação de Amparo carotenoid metabolism, are profoundly regulated by SlPHYA- à Pesquisa do Estado de São Paulo, grant nos. 2013/18056-2 and and SlPHYB2-mediated light perception. A model summariz- 2016/01128-9). ing the influence of fruit-localized SlPHYs on tomato fruit physiology is presented in Fig.  7. According to this model, References SlPHYA and SlPHYB2 play overlapping roles in regulating starch and carotenoid biosynthesis, whereas they differentially Alba R, Cordonnier-Pratt MM, Pratt LH. 2000a. Fruit-localized phytochromes regulate lycopene accumulation independently of ethylene regulate distinct aspects of fruit plastid biogenesis and mat- production in tomato. Plant Physiology 123, 363–370. uration. 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Published: Jun 27, 2018

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