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

Fruit-localized phytochromes and their downstream signaling cascades not only modulate chloroplast biogenesis in immature tomato fruits but also regulate sugar and carotenoid accumulation, two essential features of tomato fruit quality.


Introduction
Fleshy fruit growth, maturation, and ripening are under strict developmental, hormonal, and epigenetic regulation, which in turn are fine-tuned by a plethora of environmental stimuli (Kumar et al., 2014;Giovannoni et al., 2017). Among environmental cues, light plays a significant role in determining fruit growth, pigmentation, and timing of ripening (Carvalho et al., 2011;Gupta et al., 2014;Llorente et al., 2016a). In tomato (Solanum lycopersicum), a major crop and important model species for fleshy fruits, several lines of evidence indicate that changes in light perception and signaling can lead to significant alterations in fruit development and quality traits (Giliberto et al., 2005;Schofield and Paliyath, 2005;Azari et al., 2010b;Bianchetti et al., 2017).
One of the earliest pieces of evidence of the influence of light on tomato fruit biology dates back to 1954, when fruit pigmentation was shown to be regulated by red/far red (R/FR) light in a reversible manner (Piringer and Heinze, 1954). First isolated only a few years later, phytochromes (PHYs) act as molecular switches in response to R and FR light, existing as homodimers of two independently reversible subunits. Once activated by R light, PHYs are transported from the cytosol to the nucleus, where they counteract light-signaling repressor proteins, such as CONSTITUTIVE PHOTOMORPHOGENESIS1 (COP1), CULLIN4 (CUL4), DNA DAMAGE-BINDING PROTEIN 1 (DDB1), DETIOLATED1 (DET1), and PHYTOCHROME INTERACTION FACTOR (PIF) (Deng and Quail, 1992;Pepper et al., 1994;Schroeder et al., 2002;Duek and Fankhauser, 2005;Thomann et al., 2005). In line with their role as repressors of photomorphogenic responses, either the down-regulation or loss-of-function of tomato genes encoding COP1, CUL4, DDB1, DET1, and PIF1a profoundly alter tomato fruit physiology and nutritional composition (Cookson et al., 2003;Liu et al., 2004;Davuluri et al., 2005;Kolotilin et al., 2007;Wang et al., 2008;Azari et al., 2010b;Enfissi et al., 2010;Llorente et al., 2016b).
Attempts to define the influence of fruit-localized PHYs on fruit development and ripening have been relatively limited. Brief R-light treatments of detached mature-green tomato fruits promote lycopene accumulation, a response reversed by subsequent treatment with FR light (Alba et al., 2000a), which is 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 ripening (Alba et al., 2000a) associated with the reduced fruit lycopene levels observed in phyA tomato mutants (Gupta et al., 2014) raise the possibility that this PHY may be an important regulator of tomato fruit carotenoid biosynthesis. However, regardless of the development stage or tissue considered, SlPHYB2 is the most highly expressed PHY in tomato fruits (Bianchetti et al., 2017). Moreover, the phyB2 mutant also displays considerable changes in the fruit carotenoid profile (Gupta et al., 2014), suggesting that multiple PHYs are involved in regulating this metabolic process.
Besides carotenogenesis, PHYs have also been found to control other aspects of tomato fruit development and metabolism, including chloroplast biogenesis, chlorophyll accumulation, sugar metabolism, sink activity, and hormonal signaling (Gupta et al., 2014;Bianchetti et al., 2017). However, as the existing evidence supporting these findings is exclusively based on studies performed in phy mutants, whether these responses are dependent on fruit-localized PHYs or are merely consequences of the collateral negative effects of PHY deficiency on vegetative plant growth remains to be elucidated.
By employing fruit-specific RNAi-mediated silencing of SlPHY genes, we shed light on the functional specificity of fruit-localized SlPHYs in controlling developmental and metabolic processes associated with sugar and carotenoid accumulation, two essential nutritional quality traits of this edible fruit. Our data also reveal that an intricate light-hormonal signaling network involving key components of both auxin and cytokinin signal transduction pathways is implicated in the PHY-dependent regulation of fruit plastid biogenesis, sugar metabolism, and carotenoid accumulation.

Plant material and growth conditions
Tomato (Solanum lycopersicum L.) plants cv. Micro-Tom, which harbors the wild-type SlGLK2 allele (Carvalho et al., 2011), were grown under controlled conditions of 250 µmol m −2 s −1 , a 12-h photoperiod, and air temperature of 27/22 °C day/ night. The fruit stages examined were immature green, mature green, breaker, and red ripe, which were harvested on average at 8, 25, 32, and 44 d post-anthesis. All fruits were harvested at 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, placenta, and seeds were immediately removed, and the remaining tissues were frozen in liquid nitrogen and stored at -80 °C until use.

Generation of transgenic tomato plants
Three fragments specific to the coding sequences of SlPHYA, SlPHYB2, and both SlPHYB1 and SlPHYB2 were selected using BLAST queries against the Sol Genomics Network database (https://solgenomics.net/, ITAG release 2.40) and the web-based computational tool pssRNAit (Dai and Zhao, 2011) was employed to avoid off-target silencing. Each fragment was independently cloned into pENTR D-TOPO plasmids (Invitrogen) using the primers listed in Supplementary Table S1 at JXB online. Subsequently, each fragment was recombined into the plant transformation vector pK8GWIWG (Fernandez et al., 2009). Transgenic Micro-Tom plants were generated by Agrobacterium-mediated transformation according to Pino et al. (2010), with minor changes: cotyledons from 5-d-old seedlings were used for the transformation, and the zeatin and kanamycin concentration were 5 µM and 70 mg l −1 , respectively. All plants used in the study were from the T 2 generation.

Fruit color and pigment quantification
Changes in fruit color (Hue angle) were determined using a Konica Minolta CR-400 colorimeter as described in Su et al. (2015). Chlorophyll extraction and quantification were carried out as described in Lira et al. (2016) with some modifications. Pericarp samples were weighed (typically 100 mg fresh weight, FW), ground in liquid nitrogen, immersed in a 10× excess volume of N, N-dimethylformamide, and incubated at room temperature for 24 h in absolute darkness and constant agitation (200 rpm). After centrifugation (9000 g, 5 min, 4 °C), the supernatant absorbance was recorded at 647 and 664 nm, and the total chlorophyll content was estimated using the equations given by Porra et al. (1989).
For carotenoid extraction, approximately 200 mg FW of pericarp samples were ground in liquid nitrogen and sequentially homogenized with a solution of 100 µl of saturated NaCl, then 200 µl of dichloromethane, 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 carotenoids in the pellet were extracted three more times with 500 µl of hexane:diethyl ether (1:1, v/v). All supernatant fractions were combined, completely vacuum-dried, and suspended with 200 µl of acetonitrile. Lycopene, β-carotene, lutein, and neurosporene levels were determined by high-performance liquid chromatography (HPLC) with a photodiode array detector (PDA) as described by Lira et al. (2017).

Starch and soluble sugar quantification
Starch and soluble sugar extractions were performed as described in 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 (13 000 g, 10 min, 4 °C). The remaining pellets were re-extracted five times, and all supernatants were combined, completely vacuum-dried, and suspended in 200 µl distilled water. Soluble sugars (i.e. sucrose, fructose, and glucose) were measured using a HPLC system equipped with an amperometric detector (Dionex, Sunnyvale, USA) and a CarboPac PA1 (4 × 250 mm) column (Purgatto et al., 2002). Starch levels were determined from dried pellet as described in Suguiyama et al. (2014).

Antioxidant capacity and total phenolics
Hydrophilic and lipophilic Trolox equivalent antioxidant capacities (TEACs) were spectrophotometrically determined as described in Lira et al. (2016). Total phenolic content was determined in hydrophilic extracts by using the Folin-Ciocalteu method (Singleton and Rossi, 1965).

Plastid ultrastructure and abundance
Pericarp fragments taken from the pedicel region (green shoulder) of immature fruits were fixed at 4 °C in 2.5% (v/v) glutaraldehyde and 2% (v/v) paraformaldehyde in 0.1 M sodium phosphate buffer (pH 7.2). 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 series, and embedded in Spurr's resin. Ultrathin sections were stained with saturated uranyl acetate and lead citrate (Melo et al., 2016) and observed using a JEOL JEM1011 transmission electron microscope. Sections from three immature fruits picked from different plants were analysed per genotype.
Plastid abundance was determined as described in Bianchetti et al. (2017). Briefly, small pieces (1 × 1 mm) of pericarp were fixed in 3.5% (v/v) glutaraldehyde for 1 h. Samples were washed twice and transferred to 0.1 M NaEDTA pH 9.5 solution for 4 h at 60 °C in complete darkness. Pieces were softly disrupted and transferred to microscope slides. Isolated cells were visualized using a Leica microscope. Plastid densities in individual cells were estimated using the ImageJ program (https:// imagej.nih.gov/ij/). At least 40 individual cells were analysed per sample.

Transcriptional profile
Total RNA extraction, cDNA synthesis, primer design, and qPCR assays were performed as described by Quadrana et al. (2013). Primer sequences used are detailed in Supplementary Table S1. Quantitative real-time (qRT-)PCR reactions were performed in a StepOnePlus PCR Real-Time thermocycler (Applied Biosystems) in a final volume of 10 µl using 2× SYBR Green Master Mix reagent (Thermo Fisher Scientific). Melting curves were checked for unspecific amplifications and primer dimerization. Absolute fluorescence data were analysed using the LinRegPCR software package (Ruijter et al., 2009) to obtain quantitation cycle (C q ) values and to calculate primer efficiency. Transcript abundances were normalized against the geometric mean of two reference genes, CAC and EXPRESSED (Expósito-Rodriguez et al., 2008).

Statistical analysis
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 all independent transgenic lines were also compared with principal component analysis (PCA) using the InfoStat software (http://infostat.com.ar).

Fruit-specific PHY knockdown in transgenic tomato plants
To investigate the role played by distinct PHYs in tomato fruit development and ripening, we generated fruit-specific silenced tomato plants with reduced mRNA levels of SlPHYA, SlPHYB2, or both SlPHYB1 and SlPHYB2. This was achieved using a hairpin-mediated RNAi approach based on the expression of specific fragment sequences of these genes under the control of the fruit-specific PPC2 promoter (Fernandez et al., 2009). The transgenic plants obtained, hereafter designated as SlPHYA RNAi , SlPHYB2 RNAi , and SlPHYB1/B2 RNAi (Fig. 1A), were generated in a Micro-Tom background homozygous for the wild-type GOLDEN2-LIKE-2 (SlGLK2) allele (Carvalho et al., 2011), which encodes a transcription factor critically important for chloroplast development in tomato fruits (Powell et al., 2012).
Transcript abundance analysis revealed that SlPHYA, SlPHYB2, and both SlPHYB1 and SlPHYB2 were down-regulated in the SlPHYA RNAi , SlPHYB2 RNAi , and SlPHYB1/B2 RNAi lines, respectively (Fig. 1B). A search for potential tomato offtargets via BLAST queries against the Sol Genomics Network database or via the public web-based computational tool pss-RNAit (Dai and Zhao, 2011) failed to identify regions in the tomato coding that exhibited the 21-nucleotide perfect identity threshold reported to cause off-target silencing (Xu et al., 2006). The percentage of identity of the silencing fragments was below 60% with non-target tomato PHY genes (Supplementary  Table S2). Moreover, the length of stretches with perfect identity between the RNAi fragments and non-target tomato PHY genes was ≤15 nucleotides (Supplementary Table S2). In line with this, no off-target SlPHY silencing was detected in the transgenic lines generated ( Supplementary Fig. S1).
In all the transgenic lines, PHY knockdown was restricted to the fruit tissues as no significant PHY silencing was observed in leaf samples (Fig. 1B). Transgenic lines exhibited normal plant growth and visual phenotypic features similar to those found in wild-type (WT) plants ( Supplementary Fig. S2). Overall, fruitspecific PHY knockdown caused no marked changes in fruit size and ripening progression ( Supplementary Fig. S3).

Fruit-localized SlPHYA and SlPHYB2 differentially impact chloroplast biogenesis and differentiation during early fruit development
The PHY-dependent regulation of chloroplast development has been extensively reported in leaf tissues of several species (Stephenson et al., 2009;Inagaki et al., 2015). Moreover, some recent reports have also indicated altered chlorophyll accumulation and chloroplast biogenesis in immature fruits of PHY-deficient tomato mutants (Gupta et al., 2014;Bianchetti et al., 2017). Compared to the WT, fruit-specific SlPHYA and SlPHYB2 knockdown reduced and increased the chlorophyll content in immature fruits, respectively ( Fig. 2A). However, chlorophyll levels in immature fruits from SlPHYB1/B2 RNAi plants were similar to WT counterparts.
Microscopy analysis of pericarp cells revealed that the reduced chlorophyll content detected in SlPHYA RNAi immature fruits was associated with a reduction of up to 40% in the number of chloroplasts per pericarp cell compared to WT fruits (Fig. 2B). However, the higher chlorophyll content observed in SlPHYB2 RNAi immature fruits was not accompanied by changes in plastid abundance but instead was linked to the up-regulation of the master regulator of chloroplast development and maintenance, SlGLK2 (Fig. 2C). SlPHYB1/ B2 knockdown lines showed an intermediate impact on fruit chlorophyll content, plastid density, and SlGLK2 mRNA levels, exhibiting unaltered chlorophyll levels and chloroplast abundance in pericarp cells and slightly higher expression of SlGLK2 compared to the WT (Fig. 2).
Plastids of WT, SlPHYB2 RNAi , and SlPHYB1/B2 RNAi immature fruits exhibited remarkably similar internal membranous structures, displaying well-developed grana and stroma thylakoids as well as numerous plastoglobuli (Fig. 2D, Supplementary Fig. S4). In contrast, fruit-specific SlPHYA knockdown resulted in the formation of chloroplasts with highly reduced grana, suggesting a promotive role of PHYA-mediated light perception on fruit plastid granal development. Plastoglobuli and starch grains were observed equally in fruit chloroplasts of the WT and all transgenic lines.
As neither SlPHYB2 nor the SlPHYB1/B2 knockdown altered chloroplast density per cell or plastid ultrastructure (Fig. 2), fruit-localized SlPHYA seems to play a preponderant role in controlling chloroplast biogenesis and differentiation in early developing fruits. Transcript abundance analysis revealed that the reduced plastid abundance observed in SlPHYA-silenced fruits was most probably explained by a drastic reduction in mRNA levels of genes encoding key components of the plastid division machinery, such as FILAMENTOUS TEMPERATURE SENSITIVE-Z (FtsZs), ACCUMULATION AND REPLICATION OF CHLOROPLASTS (ARCs), and PLASTID DIVISION 2 (PDV2), compared to the WT genotype (Fig. 3A).
Given the key role played by cytokinins in regulating plastid division and maturation in plants and the widely reported crosstalk between this hormonal class and PHY signaling (Okazaki et al., 2009;Cortleven and Schmülling, 2015), a transcriptional profiling of type-A TOMATO RESPONSE REGULATOR (TRR) was performed. Four out of the five type-A TRRs analysed were significantly down-regulated in immature fruits of SlPHYA RNAi compared to the WT genotype (Fig. 3B). Moreover, among the five CYTOKININ RESPONSE FACTOR genes most highly expressed in tomato fruit tissues (Shi et al., 2012), SlCRF1, SlCRF2, and SlCRF5 were markedly down-regulated in SlPHYA RNAi lines, whereas SlCRF3 and SlCRF9 mRNA levels remained unchanged (Fig. 3C). As AtCRF2 is responsible for inducing AtPDV2, subsequently increasing plastid division rates in Arabidopsis (Okazaki et al., 2009), the drastic down-regulation of both SlCRF2 and SlPDV2 in SlPHYA-silenced fruits suggests that a similar regulatory mechanism also takes place early in the development of tomato fruits.
Collectively, these data suggest that fruit-localized PHYA positively influences tomato plastid division machinery via changes in the transcript abundance of both light-and cytokinin-signaling genes, whereas PHYB2 negatively regulates chlorophyll accumulation by controlling the expression of the master transcription factor of chloroplast development and maintenance, SlGLK2.

Fruit-localized PHYs regulate starch metabolism during early fruit development
Fruit-specific SlPHYA and SlPHYB2 knockdown promoted starch accumulation during early fruit development (Fig. 4A). In both the WT and transgenic lines, the highest starch content was observed in immature green (IG) fruits, followed by slightly more reduced levels at the mature green (MG) stage, and undetectable levels from the breaker (Bk) stage onwards ( Supplementary Fig. S5).
Compared to the WT genotype, marked differences in the transcript profiles of starch biosynthesis genes were observed in both SlPHYA-and SlPHYB2-silenced fruits (Fig. 4A, Supplementary Fig. S6). Catalysing the first committed step in starch biosynthesis, ADP-glucose pyrophosphorylase (AGPase) is a heterotetramer comprising a pair of small/catalytic and a pair of large/regulatory subunits (Kim et al., 2007;Figueroa et al., 2013). Among the tomato genes encoding the large AGPase subunits, both SlAGPaseL1 and SlAGPaseL3 were up-regulated whereas SlAGPaseL2 mRNA levels remained unchanged in immature fruits of SlPHYA RNAi plants. It is worth mentioning that SlAGPaseL1 was the large AGPase subunit most expressed in immature tomato fruits (Supplementary  Table S3; Petreikov et al., 2006); therefore, the 3-fold increment in its mRNA levels correlates well with the higher starch levels and reduced soluble sugar levels detected in SlPHYA RNAi immature fruits compared to the WT counterparts (Fig. 4,  Supplementary Figs S5, S6).
SlAGPaseS1, which encodes the small/catalytic AGPase subunit, was consistently down-regulated throughout fruit development and ripening in both the SlPHYA RNAi and SlPHYB2 RNAi lines. However, despite the negative impact of either SlPHYA-or SlPHYB2-silencing on SlAGPaseS1 expression, this gene exhibited higher expression levels than those encoding AGPase large subunits (Supplementary Table S3), suggesting that the catalytic AGPase subunit was not limiting for starch biosynthesis in tomato fruits.
In both SlPHYA RNAi and SlPHYB2 RNAi immature fruits, the starch synthase (STS)-encoding genes SlSTS1 and SlSTS2 were markedly up-regulated compared to WT fruits, whereas SlSTS3 was slightly down-regulated. For SlSTS6, higher transcript accumulation was observed in SlPHYA RNAi than in the WT throughout fruit development and ripening (i.e. IG to RR stage) ( Supplementary Fig. S6). Finally, distinct expression patterns were observed for the starch branching enzyme (SBE)-encoding genes, as SlSBE1 was up-regulated in all the transgenic lines from MG to Bk stage whereas SlSBE2 was down-regulated in both SlPHYA RNAi and SlPHYB2 RNAi from IG to RR stage ( Supplementary Fig. S6).
The increased accumulation of starch in SlPHYA RNAi fruits correlated well with higher mRNA levels of SlLIN5 and SlLIN6 (Fig. 4D), which encode cell-wall invertases critically important for sink activity in tomato (Fridman and Zamir, 2003;Kocal et al., 2008). By applying an unsupervised method  Previous findings have indicated that AUXIN RESPONSE FACTOR4 (SlARF4) is a major negative regulator of starch biosynthesis in early developing tomato fruits (Sagar et al., 2013;Bianchetti et al., 2017). Recent evidence also indicates that SlARF4 plays a repressor role in controlling the transcript abundance of sink-related genes, including SlLIN5 and SlLIN6 (Bianchetti et al., 2017). In accordance with this, fruitspecific SlPHYA and SlPHYB2 knockdown drastically reduced SlARF4 mRNA abundance in early developing tomato fruits (Fig. 4E). Although the direct transcriptional regulation of tomato AGPase, STS, and SBE genes by transcription factors associated with auxin-or light-signaling remains to be determined, the presence of PBE-box, G-box, CA-hybrid, and/or CG-hybrid motifs (Martıńez-Garcıá et al., 2000;Song et al., 2008) as well as canonical and/or degenerated ARF-binding Auxin Response Element (AuxRE) motifs within the 3-kb promoter sequence of these genes ( Supplementary Fig. S8) is consistent with the hypothesis that light-and/or auxin-related transcription factors might directly control the expression of starch biosynthesis-related genes. Similarly, PIF, HY5, and/ or ARF-binding motifs have also been identified within the promoter sequences of SlLIN5 and SlLIN6 genes (Bianchetti et al., 2017).

PHY-dependent regulation of fruit carotenoid biosynthesis is associated with transcriptional changes in light-and auxin-signaling genes
The very well-characterized PHY-mediated signaling networks controlling carotenogenesis in vegetative tissues (Toledo-Ortiz et al., 2010) contrasts with the considerably more limited information regarding the fruit-localized PHY-dependent signaling cascades regulating carotenoid biosynthesis in fleshy fruits (Llorente et al., 2016b(Llorente et al., , 2017. Carotenoid profiling revealed a significant reduction in lycopene content in red ripe (RR) fruits of both the SlPHYA RNAi and SlPHYB2 RNAi lines compared to the WT (Fig. 5A, Supplementary Table S4). In contrast, the content of all other carotenoids analysed (i.e. phytoene, phytofluene, β-carotene, and lutein) remained virtually unchanged in ripe fruits of the transgenic lines compared to WT counterparts. As lycopene is the main carotenoid accumulated in ripe tomato, fruit-specific SlPHYA-or SlPHYB2knockdown led to a slight, yet significant, reduction in total carotenoid content compared to the WT genotype (Fig. 5A,  Supplementary Table S4). In accordance with this, significantly lower mRNA levels of genes encoding carotenoid biosynthesis-related enzymes such as GERANYLGERANYL 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. DIPHOSPHATE SYNTHASE (GGPS), PHYTOENE SYNTHASE 1 (PSY1), and PHYTOENE DESATURASE (PDS) were observed in ripe fruits of SlPHYA and SlPHYB2silenced lines than in WT counterparts (Fig. 5B, Supplementary  Fig. S9). In line with the relatively limited reduction in total carotenoids, no significant differences in lipophilic antioxidant activity were observed between ripe WT and transgenic fruits (Supplementary Table S5). Interestingly, however, red ripe SlPHYB2-down-regulated fruits exhibited increased hydrophilic antioxidant activity compared to the WT, which may be associated with the higher content of total phenolics also detected in SlPHYB2 RNAi ripe fruits (Supplementary Table S5).
Accumulating evidence indicates that light-signaling repressors such as SlPIF1a, SlCOP1, SlCUL4, SlDDB1, and SlDET1 negatively regulate carotenoid biosynthesis in tomato fruits (Azari et al., 2010b;Llorente et al., 2016b) whereas auxin response factors such as SlARF2a and SlARF2b play the opposite role (Hao et al., 2015). To gain insight into the potential role played by these signaling components during the PHY-dependent regulation of carotenoid biosynthesis in tomato fruits, the transcript abundance of their encoding genes was profiled in both SlPHYA RNAi and SlPHYB2 RNAi ripening fruits (Fig. 6, Supplementary Fig. S10). Among the four SlPIF genes most highly expressed in fruits , SlPIF1a, SlPIF1b, and SlPIF4/5 mRNA levels were significantly higher in SlPHYB2-down-regulated fruits compared to the WT counterparts during fruit ripening (MG, Bk, and RR stages), whereas the opposite was observed for SlPIF3 transcripts. Although less pronounced, the overall impacts of fruit-specific SlPHYA knockdown on tomato PIF expression  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, chromoplastspecific β-lycopene cyclase. (This figure is available in color at JXB online.) profiles were similar to those observed in the SlPHYB2 RNAi lines (Fig. 6, Supplementary Fig. S10).
Among the genes encoding light-signaling repressors, SlCUL4, SlDDB1, and SlDET1 exhibited significantly higher mRNA levels in SlPHYA-silenced fruits in comparison to the WT at all fruit development stages analysed (Fig. 6B, Supplementary Fig. S10). Moreover, strikingly higher SlDET1 transcript abundance was also detected in SlPHYB2knockdown compared to WT fruits at all ripening stages (i.e. MG, Bk, and RR) whereas SlCOP1 and SlDDB1 mRNA levels were also up-regulated in SlPHYB2 RNAi fruits exclusively at the MG stage. Transcript levels of the positive regulators of tomato fruit carotenogenesis SlARF2a and SlARF2b were considerably lower in SlPHYA RNAi and SlPHYB2 RNAi fruits, particularly at the Bk and RR stages (Fig. 6C, Supplementary  Fig. S10). A PCA plot in which the expression profile of carotenoid biosynthesis-related genes as well as SlPIFs, SlCOP1, SlCUL4, SlDDB1, SlDET1, SlARF2a, and SlARF2b were represented revealed that the WT, SlPHYA RNAi , and SlPHYB2 RNAi groups clearly separated from each other at the red ripe stage (Supplementary Fig. S11).
Altogether, these data suggest that both SlPHYA and SlPHYB2 play overlapping roles in promoting the paralogues SlARF2a and SlARF2b and repressing light-signaling repressors such as SlPIF1a, SlPIF1b,SlPIF4/5,SlCOP1,SlCUL4,SlDDB1,and SlDET1, which in turn mediate the PHYdependent regulation of carotenoid biosynthesis in ripening tomato fruits.

Discussion
Studies performed on PHY-deficient mutants have suggested that PHY-dependent light perception participates in the regulation of several aspects of tomato fruit biology (Gupta et al., 2014;Bianchetti et al., 2017). Here, we applied a RNAimediated organ-specific silencing approach to investigate the impact of fruit-localized SlPHYs on tomato fruit physiology and quality traits. Differently from the pleiotropic phenotypical alterations observed in phy mutants (Gupta et al., 2014;Bianchetti et al., 2017), the fruit-specific silencing of the target SlPHY genes resulted in no obvious impacts on plant vegetative growth and overall yield. This suggests that the perturbation in fruit metabolism caused by the fruit-specific SlPHY manipulation does not propagate from fruits to the rest of the plant, which agrees with the limited transference of substances out of this predominantly sink organ.
In a previous work, we demonstrated that a global deficiency in functional PHYs drastically reduces chlorophyll content and chloroplast abundance in tomato fruits (Bianchetti et al., 2017). Therefore, the PHY-mediated regulation of plastid biogenesis and maturation widely reported for leaf tissues (Stephenson et al., 2009;Oh and Montgomery, 2014;Melo et al., 2016) seems to be conserved early in the development of tomato fruits. In this current work, it is further demonstrated that fruit-localized SlPHYA and SlPHYB2 play distinct roles in controlling chloroplast biogenesis and activity during early stages of tomato fruit development.
The results indicate that SlPHYA-mediated light perception promotes fruit chloroplast biogenesis and differentiation, as inferred from the reduced chlorophyll content, lower chloroplast abundance, and poorly-developed grana stacking detected in SlPHYA RNAi immature fruits (Fig. 2). In line with this observation, an analysis of single and multiple phy mutants also suggested that SlPHYA is a major regulator of chlorophyll 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 and López-Juez, 2013). Our findings clearly demonstrate that fruit-localized SlPHYA influences the transcript levels of genes derived from the ancestral prokaryotic cell-division machinery, such as SlFtsZ (i.e. SlFtsZ1, SlFtsZ2) and SlARCs (i.e. SlARC3 and SlARC6), as well as those encoding chloroplast divisionrelated proteins specific to land plants, such as SlPDV2. In Arabidopsis, PDV2 determines the rate of chloroplast division  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. and is positively regulated by cytokinins, being strongly promoted in transgenic plants overexpressing the cytokinin signaling-related transcription factor CRF2 (Okazaki et al., 2009;Cortleven and Schmülling, 2015). SlCRF2, along with other SlCRF and TRR genes, were drastically repressed in PHYAdown-regulated fruits, implying that changes in cytokinin signaling mediate the PHYA-dependent regulation of plastid division during early stages of tomato fruit development. In agreement with this, accumulating evidence indicates that there is an intensive crosstalk between the PHY and cytokinin signaling cascades, with particular involvement of CRF and type-A ARR proteins (Salomé et al., 2006;. Fruit-specific SlPHYA-silencing also promoted the mRNA accumulation of genes encoding all the major light-signaling repressor proteins already described to negatively regulate chloroplast biogenesis in tomato fruits, i.e. SlCOP1, SlCUL4, SlDDB1, and SlDET1 (Liu et al., 2004;Kolotilin et al., 2007;Wang et al., 2008;Azari et al., 2010a). Defective mutants or transgenic lines with reduced levels of each of these genes are known to develop more chloroplasts containing more grana/thylakoids in both leaves and immature fruits (Cookson et al., 2003;Liu et al., 2004;Kolotilin et al., 2007;Wang et al., 2008;Azari et al., 2010a), which in some cases, such as in the SlDET1-knockout mutant, is associated with the up-regulation of plastid biogenesis-related genes (Kolotilin et al., 2007). Therefore, the presence of fewer chloroplasts with poorly developed or almost no grana in immature fruits of the SlPHYA-suppressed lines agrees with the higher transcript abundance of SlCOP1, SlDDB1, and particularly SlCUL4 and SlDET1 in these transgenic lines compared to the WT genotype.
In contrast, fruit-localized SlPHYB2 was shown to play a negative role in chlorophyll accumulation, as evidenced by the increment in chlorophyll content in immature fruits of SlPHYB2 RNAi plants with no impact in chloroplast number in pericarp cells. As SlPHYB2 fruit-specific silencing led to higher SlGLK2 mRNA levels compared to the WT genotype, it seems plausible to suggest that the effect of SlPHYB2 on fruit chloroplasts is mediated by SlGLK2, the master regulator of chloroplast development in tomato fruits (Powell et al., 2012). Further suggesting that the SlPHYB2-mediated regulation of SlGLK2 expression is essential for the consequent changes in fruit chlorophyll accumulation, no obvious changes in chlorophyll content were observed in phyb2 mutants from tomato varieties that lacked functional SlGLK2 proteins (Gupta et al., 2014). In agreement with these findings, PHY-dependent transcriptional regulation of GLK genes has been increasingly reported in vegetative tissues of other plant species (Oh and Montgomery, 2014;Song et al., 2014).
Alterations in chloroplast number, internal structure, and size during the early development of tomato fruits significantly impact the abundance of metabolites associated with organoleptic and nutritional quality at the ripe stage (Galpaz et al., 2008;Cocaliadis et al., 2014). Intense starch synthesis and degradation take place in tomato fruit chloroplasts at the unripe and breaker stages, respectively (Schaffer and Petreikov, 1997). Whereas the global deficiency in PHYs significantly reduces the starch content in immature tomato fruits (Bianchetti et al., 2017), fruit-localized SlPHYA or SlPHYB2 suppression increased fruit starch levels and markedly altered the transcriptional profile of starch biosynthesis-related genes at the immature green stage (Fig. 4). AGPase, which catalyses the rate-limiting reaction in the starch synthesis pathway, is both transcriptionally and post-translational regulated by light (Harn et al., 2000;Geigenberger, 2011), although the role played by PHYs in this regulatory process remains elusive. During early fruit development, SlPHYA-suppressed fruits exhibited increased mRNA levels of both SlAGPaseL1 and SlAGPaseL3, which encode AGPase large subunits, and SlSTS1, SlSTS2, and SlSTS6, which encode starch synthase enzymes, along with an increase in starch accumulation and reduced soluble sugar content, thus indicating a repressor role for fruit-localized SlPHYA on the first steps of starch synthesis in tomato fruits. Whether the up-regulation of starch biosinthesis-related genes is a compensatory mechanism to cope with the fewer and poorly developed chloroplasts observed in SlPHYA RNAi immature fruits remains to be investigated. In contrast, the increased starch accumulation detected in SlPHYB2-silenced immature fruits was not associated with increments in transcript abundance of AGPase-encoding genes nor with prominent reductions in soluble sugars, but instead were accompanied by increments in SlSTS1 and SlSTS2 mRNA levels. Furthermore, as no significant alterations in plastid abundance or internal structure were observed in SlPHYB2 RNAi immature fruits, it seems likely that this genetic manipulation caused less prominent changes than SlPHYA-silencing on reactions taking place within fruit chloroplasts, including starch biosynthesis. Altogether, these findings suggest that SlPHYA and SlPHYB2 negatively regulate starch synthesis via overlapping, yet distinct, mechanisms.
The influence of auxin on fruit sugar metabolism has been increasingly reported (Purgatto et al., 2002;Yuan and Carbaugh, 2007;Bianchetti et al., 2017). In tomato, SlARF4 has been described as a key negative regulator of starch synthesis during early fruit development via the transcriptional and posttranscriptional down-regulation of AGPase (Sagar et al., 2013). Recent findings have also indicated that PHYs strictly regulate the transcript abundance of this particular auxin response factor in both vegetative (Melo et al., 2016) and fruit tissues (Bianchetti et al., 2017). In line with this, the increased starch accumulation in pre-ripening SlPHYA-and SlPHYB2-silenced fruits correlated well with the down-regulation of SlARF4 in these transgenic lines (Fig. 4). In fact, SlPHYA RNAi rather than SlPHYB2 RNAi exhibited the most expressive decrease in SlARF4, and only the former displayed increased mRNA levels of AGPase-encoding genes in immature fruits. Together, these data strongly suggest that fruit-localized PHYA, and to some extent SlPHYB2, positively modulates SlARF4, which in turn represses starch biosynthetic enzymes, such as AGPase and STS, consequently limiting starch synthesis in pre-ripening tomato fruits.
Previous findings indicated that a global deficiency in functional phytochromes transcriptionally represses both sink-related and starch biosynthesis-related enzymes in early developing tomato fruits, suggesting a promotive role of PHYs on the regulation of these processes (Bianchetti et al., 2017). However, it remained unclear whether these responses were dependent on fruit-localized PHYs or were the consequence of collateral negative effects of the global PHY deficiency on vegetative plant growth. Here, we shed light on this topic by showing that fruit-localized SlPHYA, and to some extent SlPHYB2, repress both starch metabolism and key determinants of tomato fruit sink strength, including SlLIN5 transcript accumulation (Fridman and Zamir, 2003;Kocal et al., 2008). Consequently, the down-regulation in starch synthesis and sink activity previously observed in fruits of the PHY-deficient mutant aurea (Bianchetti et al., 2017) may be due either to limitations in vegetative growth and metabolism or to the combinatory effect of the deficiency in all phytochromes instead of only in SlPHYA or SlPHYB2. Moreover, it also seems tempting to suggest that the fewer and poorly-developed chloroplasts detected in SlPHYA RNAi immature fruits restrict photoassimilate production via fruit photosynthesis; therefore, the observed up-regulation of sink-related genes in transgenic fruits may represent a compensatory mechanism to maintain fruit growth and intense starch accumulation despite potential limitations in fruit-localized photoassimilation.
The link between PHY-dependent light perception and carotenoid metabolism in both vegetative and fruit tissues has been highlighted by a number of studies (Alba et al., 2000a;Llorente et al., 2016b). Exposure of wild-type tomato fruits to red light (Alba et al., 2000a) or constitutively silencing of SlPIF1a (Llorente et al., 2016b) promotes tomato fruit lycopene accumulation, thereby implying a positive role of PHY-dependent signaling cascades in the fruit carotenoid biosynthetic pathway. Consistent with this, our findings indicate that fruit-localized SlPHYA and SlPHYB2 positively influence the transcript accumulation of all the major carotenoid biosynthesis-related genes, including SlGGPS, SlPSY1, SlPDS, SlCYCβ, and SlLYCβ, consequently modifying the lycopene and total carotenoid content in this fleshy fruit. Light-signaling repressor proteins such as SlDET1, SlDDB1, SlCOP1, SlCUL4, and more recently SlPIF1a have been identified as key negative regulators of tomato fruit carotenoid synthesis (Liu et al., 2004;Kolotilin et al., 2007;Wang et al., 2008;Azari et al., 2010a;Llorente et al., 2016b). Among these, the transcription factor SlPIF1a was shown to directly bind to the promoter of SlPSY1 to repress fruit carotenogenesis (Llorente et al., 2016b), thus resembling the action of its ortholog in Arabidopsis (AtPIF1) in controlling carotenoid biosynthesis in leaf tissues (Toledo-Ortiz et al., 2010). Therefore, the marked up-regulation of SlDET1, SlDDB1, SlCOP1, SlCUL4, SlPIF1a, and SlPIF1b together with the overall repression of carotenoid biosynthesis observed in both SlPHYA-and SlPHYB2-silenced fruits imply that light-signaling repressor proteins participate in SlPHYAand SlPHYB2-mediated regulation of fruit carotenogenesis.
In addition, it is becoming increasingly well established that 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. biosynthetic genes (Su et al., 2015). Among tomato ARF genes, two paralogs, SlARF2a and SlARF2b, have emerged as key positive regulators of tomato fruit ripening and lycopene accumulation (Hao et al., 2015). Either SlPHYA or SlPHYB2 fruit-specific silencing profoundly reduced both SlARF2a and SlARF2b, suggesting the involvement of these auxin signaling elements in the PHY-dependent regulation of carotenoid biosynthesis in tomato fruits.
Overall, our results shed light on the specific role played by fruit-localized phytochromes and their downstream signaling cascades, showing that plastid division, as well as sugar and carotenoid metabolism, are profoundly regulated by SlPHYAand SlPHYB2-mediated light perception. A model summarizing the influence of fruit-localized SlPHYs on tomato fruit physiology is presented in Fig. 7. According to this model, SlPHYA and SlPHYB2 play overlapping roles in regulating starch and carotenoid biosynthesis, whereas they differentially regulate distinct aspects of fruit plastid biogenesis and maturation. Compared to SlPHYB2, SlPHYA-dependent light perception seems to play a major role in promoting plastid division and differentiation as well as in controlling sink-related transcripts in tomato fruits. The data implicate cytokinin signaling-related proteins as mediators of the SlPHYA-dependent regulation of the plastid division machinery, and specific ARF genes as potential intermediates in the PHY-mediated regulation of fruit sugar and carotenoid metabolism. Altogether, these findings show that fruit-specific manipulation of PHY genes represents a promising approach to differentially regulate multiple biosynthetic pathways and, consequently, to modify the nutritional value of edible fleshy fruits.

Supplementary data
Supplementary data are available at JXB online. Fig. S1. Transcriptional profile of tomato PHY genes in PHY-silenced fruits. Fig. S2. Vegetative phenotypes of the transgenic plants. Fig. S3. Visual phenotypes and color changes of PHYsilenced fruits. Fig. S4. Plastid structure in PHY-silenced fruits. Fig. S5. Carbohydrate profile in PHY-silenced fruits. Fig. S6. Transcript abundance of starch biosynthetic genes in PHY-silenced fruits. Fig. S7. PCA of the expression profile of sink-related and starch biosynthesis-related genes. Fig. S8. HY5-, PIF-, and ARF-binding motifs identified in the promoter regions of starch biosynthesis-related tomato genes. Fig. S9. Carotenoid metabolism during ripening in PHYsilenced fruits. Fig. S10. Transcript abundance of photomorphogenesis-and auxin-related genes in PHY-silenced fruits. Fig. S11. PCA of the expression profiles of photomorphogenesis-related, auxin-related, and carotenoid biosynthesis-related genes. Table S1. Primer sequences. Table S2. Homology of the RNAi fragments. Table S3. Relative transcript ratios of SlAGPase in immature fruits. Table S4. Carotenoid profiles in red ripe fruits. Table S5. Antioxidant activity and total phenolics in red ripe fruits.