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. 2022 Mar 21;189(2):527–540. doi: 10.1093/plphys/kiac133

Pivotal roles of ELONGATED HYPOCOTYL5 in regulation of plant development and fruit metabolism in tomato

Chunli Zhang 1,2,3, Yujie Wu 1, Xiaorui Liu 1, Jiayi Zhang 1, Xin Li 4, Li Lin 1,2,3, Ruohe Yin 1,2,3,✉,†
PMCID: PMC9157105  PMID: 35312008

Abstract

The transcription factor ELONGATED HYPOCOTYL5 (HY5) plays critical roles in plant photomorphogenesis. Previous studies on HY5 have mainly focused on the seedling stage in Arabidopsis (Arabidopsis thaliana), and its functions in other plant species have not been well characterized, particularly at adult stages of development. In this report, we investigated the functions of tomato (Solanum lycopersicum) HY5 (SlHY5) from seedlings to adult plants with a focus on fruits. Genome-edited slhy5 mutants exhibited typical compromised photomorphogenesis in response to various light conditions. The slhy5 mutants showed reduced primary root length and secondary root number, which is associated with altered auxin signaling. SlHY5 promoted chlorophyll biosynthesis from seedling to adult stages. Notably, the promotive role of SlHY5 on chlorophyll accumulation was more pronounced on the illuminated side of green fruits than on their shaded side. Consistent with this light-dependent effect, we determined that SlHY5 protein is stabilized by light. Transcriptome and metabolome analyses in fruits revealed that SlHY5 has major functions in the regulation of metabolism, including the biosynthesis of phenylpropanoids and steroidal glycoalkaloids. These data demonstrate that SlHY5 performs both shared and distinct functions in relation to its Arabidopsis counterpart. The manipulation of SlHY5 represents a powerful tool to influence the two vital agricultural traits of seedling fitness and fruit quality in tomato.

Tomato HY5 regulates diverse aspects of plant physiology, including seedling hypocotyl growth, primary root growth, chlorophyll biosynthesis, and fruit metabolism.

Introduction

Light is a key environmental factor regulating plant development. Land plants have evolved multiple photoreceptors, including red/far-red light photoreceptor phytochromes, blue light photoreceptor cryptochromes, phototropins, the ZEITLUPE family of proteins, and the UV-B photoreceptor UV RESISTANCE LOCUS 8 (UVR8), to sense and respond to light signals (Kami et al., 2010). Light signaling pathways regulate diverse aspects of plant development, including seedling de-etiolation, root development, thermormorphogenesis, entrainment of the circadian clock, gravitropism, flowering time, and phototropism (Yadav et al., 2020). ELONGATED HYPOCOTYL5 (HY5) plays key roles in diverse light signaling pathways in Arabidopsis (Arabidopsis thaliana) and acts downstream of photoreceptors (Gangappa and Botto, 2016).

In the dark, HY5 is targeted for degradation by the E3 ligase CONSTITUTIVE PHOTOMORPHOGENIC 1 (COP1; Osterlund et al., 2000). Light inhibits COP1 and promotes HY5 accumulation (Osterlund et al., 2000). Light also regulates the transcriptional output of about one-third of all genes in Arabidopsis seedlings (Ma et al., 2001). Multiple transcriptional regulators contribute to diverse light signaling pathways, including HY5, PHYTOCHROME-INTERACTING FACTORs (PIFs), LONG HYPOCOTYL IN FAR-RED 1, LONG AFTER FAR-RED LIGHT 1, BBOX family members, and many more (Ballesteros et al., 2001; Duek and Fankhauser, 2003, 2005; Khanna et al., 2009; Tavridou et al., 2020; Yadav et al., 2020).

HY5 regulates the expression of thousands of its target genes in Arabidopsis seedlings (Lee et al., 2007; Zhang et al., 2011). HY5 contains a basic leucine zipper (bZIP) DNA-binding domain but no activation domain; to regulate gene transcription, it recruits cofactors with activation domains (Oyama et al., 1997; Xu et al., 2016; Bursch et al., 2020). Fusion of a transcriptional activation domain (VP16) or a transcriptional repression motif (SRDX) to HY5 in Arabidopsis results in transcriptional changes and exaggerated seedling photomorphogenic responses, indicating that HY5 requires cofactors to regulate gene transcription (Burko et al., 2020). HY5 protein binds to its own gene promoter, and HY5 is required but not sufficient for its own gene transcriptional induction by light (Abbas et al., 2014; Binkert et al., 2014). Interestingly, tomato (Solanum lycopersicum) transcription factors BBX20 and BBX21 interact with HY5 and cooperatively activate the transcription of HY5, and the excessive HY5 protein inhibits its own gene transcription, forming an autoregulatory negative feedback loop to coordinate HY5 levels in plants (Yang et al., 2022). Notably, most of these studies have been conducted at the seedling stage in Arabidopsis, and the functions of HY5 at later stages of development have been less explored. Moreover, HY5 putative orthologs in other plant species are not well understood.

Tomato is a model system for the study of photomorphogenesis, defense responses, and the development of fleshy fruits (Ballare et al., 1995; Kendrick et al., 1997; Cortes et al., 2016; Fantini et al., 2019; Rosado et al., 2019; Liu et al., 2020; Courbier et al., 2021). Silencing of tomato HY5 (abbreviated as SlHY5 herein) reduces anthocyanin and carotenoid accumulation (Liu et al., 2004). In addition, SlHY5 promotes tomato cold tolerance (Wang et al., 2019; Zhang et al., 2020), fruit ripening (Wang et al., 2021), and iron uptake (Guo et al., 2021). In this work, we aimed to investigate the major functions of SlHY5 in tomato development, from the seedling stage to ripening fruits. To this end, we generated slhy5 mutants by CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9-mediated gene editing. Our data show that SlHY5 regulates seedling fitness, including primary root length and hypocotyl length, which are of importance for agricultural practices. During adult stages, SlHY5 affected plant height but not flowering time. In the mature leaves and green fruits of adult plants, SlHY5 promoted chlorophyll accumulation in a light-intensity-dependent manner. Importantly, transcriptome analyses with ripening fruits indicated that a major function of SlHY5 is to regulate fruit metabolism, which we confirmed through metabolome analyses. Collectively, this work provides an overview of the functions of SlHY5 during tomato development and reveals that SlHY5 plays key roles in the regulation of tomato fruit metabolism.

Results

SlHY5 promotes root growth in tomato

To analyze the function of SlHY5, we generated slhy5 mutants in the tomato cultivar “Ailsa Craig” (AC) with a CRISPR/Cas9 system (Supplemental Figure S1A). We obtained fifteen independent putative slhy5 mutant lines. We selected for further analyses three independent lines carrying frameshift mutations in SlHY5 that introduce a premature stop codon (Supplemental Figure S1B). All three slhy5 alleles showed very low SlHY5 transcript levels (Figure 1A), which is consistent with previous findings that HY5 protein is required for its own gene transcription (Abbas et al., 2014; Binkert et al., 2014). Relative SlHY5 transcript levels were highest in roots, followed by fruits and flowers (Figure 1B). The slhy5 mutants developed substantially shorter primary roots than the wild-type (WT) 5 d after germination (DAG); the primary roots were also slower growing in the slhy5 mutants (Figure 2, A–D). Lateral root number of slhy5 mutants was fewer than WT (Figure 2E). Arabidopsis hy5 hyh double mutants also develop shorter primary roots and fewer lateral roots than the WT, a phenotype that is associated with auxin signaling (Sibout et al., 2006). We performed transcript analysis for several genes that are potentially involved in auxin signaling, including Auxin/Indole-3-Acetic Acid (Aux/IAA) and small auxin upregulated RNA (SAUR) genes in both slhy5 mutant and WT roots. Transcript levels of SlIAA11, SlIAA19, SlSAUR50, and SlSAUR50like were not obviously altered in slhy5 mutants (Supplemental Figure S2); however, expression of SlIAA3, SlIAA9, SlIAA17, SlIAA21, SlIAA23, and SlSAUR21like was lower in slhy5 mutants compared with the WT (Figure 2F). Thus, it appears that SlHY5 regulates tomato root growth by altering auxin signaling.

Figure 1.

Figure 1

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Transcript levels of SlHY5 in tomato. A, Expression of SlHY5 is strongly reduced in the slhy5 frame-shift mutants. Total RNA was prepared from tomato seedlings cultured on 1/2 MS solid medium under white light for 5 DAG. Means ± se are shown (n = 3). Asterisks indicate significant differences between slhy5 and WT (AC) with one-way ANOVA analysis, **P < 0.01. B, Transcript levels of SlHY5 in tomato tissues. IM, immature green fruits; MG, mature green fruits; BR, breaker ripening fruits. All the tissues were collected from 120 d plants excepted seedlings, which were 6 d old. Means ± se are shown (n = 3).

Figure 2.

Figure 2

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SlHY5 promotes root growth of tomato seedlings. A and B, The slhy5 mutants developed shorter primary root in relation to WT (AC). Representative image (A) and quantification of primary root length (B) of slhy5 mutants at seedling stage (5 DAG). Means ± sd are shown (n = 15) Scale bar = 2 cm. C, Primary root growth rates of the seedlings from third to fifth DAG. Means ± sd are shown (n = 15). D, Representative image for cell length of root tip in WT and slhy5 with 5 DAG seedlings under the Olympus BX43 microscope. The blue rectangular box represented a cell. E, Lateral root number of seedlings 4 DAG. Mean ± sd are shown (n = 15). F, The expression of auxin responsive genes in the root of slhy5 mutants. Total RNA was prepared from roots of seedling cultured on 1/2 MS solid medium for 5 DAG. Means ± se are shown (n = 3). Asterisks indicate significant differences between slhy5 and WT with one-way ANOVA analysis, *P < 0.05 and **P < 0.01 (**).

To further investigate the function of SlHY5 in root development, we generated FLAG-tagged SlHY5 overexpression lines driven by the 35S promoter in the WT background (SlHY5-OX). FLAG-SlHY5 fusion protein was detected in the SlHY5-OX lines (Supplemental Figure S3). However, the primary root length, primary root growth rate, and secondary root number were all similar among the three independent SlHY5-OX lines and the WT tomato (Supplemental Figure S4, A–D).

slhy5 mutants exhibit elongated hypocotyls under various light conditions at the seedling stage

We next systemically analyzed seedling photomorphogenesis of slhy5 mutant lines. In continuous darkness, both WT and slhy5 mutant seedlings developed the typical elongated hypocotyls, closed apical hook, and closed cotyledons characteristic of etiolated seedlings (Figure 3A). Under monochromatic blue, red, or far-red light, slhy5 mutants developed significantly longer hypocotyls than WT (Figure 3, B–D). Similarly, hypocotyl length was longer in slhy5 mutants relative to WT in seedlings grown in white light or white light supplemented with UV-B (Figure 3, E and F). UV-B inhibits seedling hypocotyl elongation through the UV-B photoreceptor SlUVR8 (Liu et al., 2020). Interestingly, UV-B inhibited seedling hypocotyl growth only partially through SlHY5, as UV-B still effectively repressed hypocotyl elongation in slhy5, albeit to a slightly lesser extent than in WT (Figure 3G).

Figure 3.

Figure 3

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The slhy5 mutants exhibit elongated hypocotyls under various light conditions. A–F, Representative (upper) and quantification of hypocotyls (lower) for WT (WT, AC) and slhy5 mutants grown in dark (A), blue light (B), red light (C), far-red light (D), white (E), and white light supplemental with UV-B (F) for 4 d. Scale bar = 1 cm. Mean ± sd are shown (n = 20). G, hypocotyl length ratio of UV-B (F)/white light (E). Asterisks indicate significant differences between slhy5 and WT with one-way ANOVA analysis, *P < 0.05 and **P < 0.01. H and I, Western blot of SlHY5 accumulation in 35S:FLAG-SlHY5 seedlings. Seedlings grown in dark and then transferred to white light (H); and seedlings were transferred from white light to white light supplemented with UV-B (I).

Similarly, we also analyzed seedling photomorphogenesis of SlHY5-OX lines. In continuous darkness, SlHY5-OX lines exhibited typical skotomorphogenesis, suggesting that additional factors are required to promote photomorphogenesis (Supplemental Figure S5A). In general, under both white light and monochromatic light conditions, SlHY5-OX lines developed a hypocotyl phenotype opposite to that of slhy5 mutants; however, under far-red light, only one overexpression line had a slightly shorter hypocotyl than the WT (Supplemental Figure S5, B–E). Under UV-B, hypocotyl lengths of the SlHY5-OX lines were significantly shorter than those of the WT (Supplemental Figure S5F).

We then assessed whether SlHY5 protein abundance is regulated by light using SlHY5-OX lines. Seedlings were first grown in the dark and then transferred into white light for various periods. We found that white light promoted the accumulation of FLAG-SlHY5 fusion protein (Figure 3H), as did UV-B irradiation after initial growth in white light (Figure 3I). Thus, we conclude that light promotes the accumulation of SlHY5, which stimulates tomato seedling photomorphogenesis under various light conditions.

SlHY5 influences plant height and chlorophyll accumulation at adult stages

The tomato cultivar AC has an indeterminate growth pattern and can reach more than two meters in height, if not topped. Before flowering, slhy5 and SlHY5-OX lines were significantly taller and shorter than WT, respectively (Figure 4, A and B; Supplemental Figure S6, A and B). Indeterminate tomato cultivars are typically topped after several inflorescences are produced by the shoot apical meristem. We measured the length of the primary stem between the second and third inflorescence stem as a proxy for plant height after they had been topped. Again, slhy5 and SlHY5-OX lines were taller and shorter than WT, respectively (Figure 4C;Supplemental Figure S6, A–C). The flowering time of both slhy5 and SlHY5-OX lines was similar to that of WT, according to the leaf number until first inflorescence (Supplemental Figure S6D).

Figure 4.

Figure 4

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SlHY5 influences plant height and chlorophyll accumulation at adult stages. A and B, The slhy5 mutants are taller than WT (AC) at adult stage. Representative image (A) and quantification of plant height (B) of 30 DAG plants. Means ± sd are shown (n = 15). Scale bar = 5 cm. C, The length from second to third inflorescence to represent the plant height after tomato plants are topped. Means ± sd are shown (n = 15). D, Representative image of mature leaves and mature green fruits. Scale bar = 5 cm. E, Chlorophyll (Chl) content is lower in the slhy5 mutants than WT in mature leaves and mature green fruits. Means ± sd are shown (n = 3). Total chlorophyll content was used for statistical analysis. F, The representative image of tomato fruits cultured under natural light. The estimated boundary between illuminated and shaded sides was indicated by white dotted lines. Scale bar = 5 cm. G, The reduction of chlorophyll content in slhy5 fruit is more pronounced in the light side than the shaded side. Means ± se are shown (n = 3). Total chlorophyll content was used for statistical analysis. H, Expression of the key genes in the chlorophyll biosynthesis pathway in slhy5 seedlings. Means ± se are shown (n = 3). Asterisks indicate significant differences between slhy5 and WT with one-way ANOVA analysis. *P < 0.05 and **P < 0.01.

We also noticed that the WT appeared greener than the slhy5 mutants, as seen in seedlings, mature leaves, and mature green fruits (Figure 4, A and D; Supplemental Figure S6E). In fact, slhy5 mutants accumulated less chlorophyll than WT in these organs (Figure 4E;Supplemental Figure S6F). However, chlorophyll contents in SlHY5-OX lines were similar to that in WT (Supplemental Figure S6G). In the field, one side of tomato fruits faces light while the opposite side remains in the shade. Notably, we determined that the reduction in chlorophyll contents seen in slhy5 mutants was more severe in the pericarp of the illuminated side relative to the shaded side (Figure 4, F and G). We conclude that SlHY5 promotes chlorophyll accumulation in tomato fruits. The expression of several chlorophyll biosynthesis genes, such as SlCHLH (encoding the H SUBUNIT OF MG-CHELATASE), SlHEMA1 (encoding GLUTAMYL-TRNA REDUCTASE), and PROTOCHLOROPHYLLIDE OXIDOREDUCTASE (SlPOR), was lower in slhy5 mutants than in WT, as evidenced by RT-qPCR (Figure 4H). Therefore, we conclude that SlHY5 promotes chlorophyll biosynthesis and accumulation in different developmental stages in tomato.

SlHY5 helps regulate the metabolism of ripening tomato fruit

To further characterize the function of SlHY5 in fruits, we performed transcriptome deep sequencing (RNA-seq) analysis with pericarp from ripening fruits (7 d after breaker ripening, BR + 7) from WT and slhy5 plants. Principal component analysis (PCA) revealed that the three replicates from each genotype grouped together, indicative of high reproducibility between biological replicates (Supplemental Figure S7A). We identified 2,065 downregulated genes and 1,014 upregulated genes in slhy5 as compared to WT (Supplemental Figure S7B; Supplemental Table S1). Of these genes, 775 of 1,007 (or 77%) differentially expressed genes (DEGs) were associated with a Kyoto Encyclopedia of Genes and Genomes (KEGG) classification term related to metabolism, including phenylpropanoid biosynthesis, flavonoid biosynthesis, amino acid metabolism, starch, and sucrose metabolism, and fructose and mannose metabolism (Supplemental Figure S7C). In addition, several DEGs had predicted functions in phytohormone signaling and environmental responses, consistent with the known targets of Arabidopsis HY5 at the seedling stage (Lee et al., 2007; Zhang et al., 2011).

Since transcriptome analysis indicated that the majority of DEGs were associated with metabolism, we performed a complementary metabolome analysis with a broadly targeted liquid chromatography–tandem mass spectrometry (LC–MS/MS) method on similar tissues. We identified a total of 723 annotated metabolites in WT and slhy5 (Supplemental Table S2). PCA showed that the metabolites detected grouped by genotype, suggesting that there was a difference in the composition of metabolites extracted from WT and the slhy5 mutants (Supplemental Figure S8A). Clustering analysis indicated that the 723 metabolites comprised 115 flavonoids, 113 phenolic acids, 88 amino acids and derivatives, 81 alkaloids, 78 lipids, 70 organic acids, 48 nucleotide and derivatives, 43 saccharides and alcohols, 19 lignans and coumarins, 14 terpenoids, 13 vitamin, and 41 additional compounds that do not fit into these main classes (Supplemental Figure S8B; Supplemental Table S2). We classified as differentially regulated metabolites those with variable importance in projection (VIP) ≥ 1 and a fold change (FC) ≤0.5 or ≥2. Relative to WT, 137 metabolites accumulated to lower levels in slhy5, while 46 metabolites were more abundant in slhy5 (Figure 5A;Supplemental Table S3). Clustering analysis indicated that these differentially abundant metabolites consisted of 76 flavonoids, 29 phenolic acids, 21 alkaloids, 8 amino acids and derivatives, 8 nucleotides and derivatives, 8 organic acids, and 8 lipids, as well as additional compounds (Figure 5B). We performed a KEGG pathway enrichment analysis using the identified differentially abundant metabolites, which revealed associations with flavonoid biosynthesis, flavone and flavonol biosynthesis, isoflavonoid biosynthesis, phenylpropanoid biosynthesis, and amino acid metabolism (Supplemental Figure S8C). Thus, our metabolome data were largely consistent with the transcriptome data.

Figure 5.

Figure 5

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Metabolome analysis of WT and slhy5 mutants in ripening fruits. A, The number of metabolites with significantly altered levels in slhy5. B, Clustering heatmap of 183 significant differentially accumulating metabolites in slhy5. The details of these differentially accumulating metabolites are shown in Supplemental Table S3. C, A simplified phenylpropanoid biosynthesis pathway. PAL, phenylalanine ammonia lyase; 4CL, 4-hydroxycinnamoyl CoA ligase; C4H, cinnamate 4-hydroxylase; HCT, cinnamoyl CoA shikimate/quinate transferase; C3H, p-coumaroyl ester 3-hydroxylase; HQT, hydroxycinnamoyl CoA quinate transferase; CHS, chalcone synthase; CHI, chalcone isomerase; F3H, flavanone-3-Hydroxylase; FLS, flavonol synthase. D, Relative content of caffeoyl quinic acids and flavonoids in pericarp. Mean ± sd are shown (n = 3). E, Fruit peel color at BR+7 stage. WT (AC) fruits have yellow peel and slhy5 mutants have colorless peel. F, Transcript levels of the key genes in caffeoyl quinic acids and flavonoids biosynthesis pathway. Means ± se are shown (n = 3). Asterisks indicate significant differences between slhy5 and WT *P < 0.05 and **P < 0.01 resulting from a one-way ANOVA analysis.

To validate the metabolome and transcriptome data, we focused on the phenylpropanoid and steroidal glycoalkaloid (SGA) biosynthesis pathways. The phenylpropanoid pathway branches out to support the biosynthesis of flavonoids and caffeoylquinic acids (Figure 5C). Moreover, the contents of major flavonoids, including naringenin chalcone, were lower in slhy5 (Figure 5D). The skin of slhy5 fruits appeared pink rather than the normal orange-red color of WT fruits (Figure 5E), consistent with the proposed function of naringenin chalcone in fruit color (Adato et al., 2009). In addition, the contents of several caffeoylquinic acids, including chlorogenic acid, were lower in slhy5 than in WT (Figure 5D). The transcript levels of many key genes in the phenylpropanoid pathway leading to the biosynthesis of flavonoids and caffeoylquinic acids were significantly downregulated in slhy5 relative to WT as determined by RT-qPCR analysis (Figure 5F). Thus, SlHY5 promotes the biosynthesis and accumulation of flavonoids and caffeoylquinic acids in fruits.

SGAs are cholesterol-derived specialized metabolites produced by solanaceous plant species (Figure 6A; Milner et al., 2011). We detected a total of 11 SGAs by LC–MS/MS, of which 8 accumulated to lower levels in slhy5 in comparison to WT (Figure 6B; Supplemental Table S3). GLYCOALKALOID METABOLISM (GAME) genes play key roles in SGA biosynthesis (Itkin et al., 2013). Transcript levels of several SlGAME genes (including SlGAME1, SlGAME2, SlGAME6, SlGAME11, SlGAME18, and SlGAME25) were significantly downregulated in slhy5 relative to WT as determined by RT-qPCR analysis (Figure 6C). Thus, SlHY5 appears to play important roles in tomato fruit metabolism by regulating the expression of genes in different biosynthesis pathways.

Figure 6.

Figure 6

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SlHY5 regulates SGAs biosynthesis. A, Biosynthetic pathway of steroidal alkaloids in Solanaceae plants. B, Relative content of SGAs in WT (AC) and slhy5. Mean ± sd are shown (n = 3). C, Transcript levels of the key genes in SGAs biosynthesis pathway. Means ± se are shown (n = 3). Asterisks indicate significant differences between slhy5 and WT *P < 0.05 and **P < 0.01 resulting from a one-way ANOVA analysis.

Discussion

In Arabidopsis, HY5 plays critical roles during seedling growth and development (Oyama et al., 1997; Gangappa and Botto, 2016). It is important to extend the knowledge acquired concerning HY5 function from studies conducted in Arabidopsis to crop plants. The key domains harbored by HY5, such as the Val-Pro motif, which is important for its interaction with COP1, and the bZIP domain, are highly conserved (Supplemental Figure S9), suggesting conserved functions. Yet, the characterization of HY5 putative orthologs in rice (Oryza sativa), birdsfoot trefoil (Lotus japonicus), and garden pea (Pisum sativum) revealed that they have both shared and distinct roles (Nishimura et al., 2002; Weller et al., 2009; Burman et al., 2018). We determined here that SlHY5 is critical for photomorphogenesis under monochromatic light conditions at the seedling stage, like its Arabidopsis counterpart. Notably, slhy5 mutants were hyposensitive to all light treatments, including red, blue, and far-red light, and UV-B in the hypocotyl growth assays (Figure 3, B–G). Overall, SlHY5-OX lines exhibited exaggerated photomorphogenic responses in seedling hypocotyl length under white, red, and blue light, but not under far-red light. Only one overexpression line showed a slightly stronger response than the WT under far-red light, an observation that requires further detailed analysis.

At the adult stage, SlHY5 affected plant morphology mainly in two regards: plant height and pigment accumulation. Transcriptome and metabolome analyses supported the notion that SlHY5 regulates metabolism of ripening fruits via transcriptional regulation. Our work thus revealed that, besides seedling photomorphogenesis, SlHY5 also plays critical roles in plant development and metabolism at the adult stage.

Expression of SlHY5 was relatively high in roots, which is consistent with its key function in root development. The root system is essential for the uptake of water and nutrients, and thus for crop yield (Gangappa and Botto, 2016). Shoot-derived HY5 can move to the root to regulate nutrient uptake in Arabidopsis (Chen et al., 2016) and iron uptake in tomato (Guo et al., 2021). Interestingly, HY5 appears to exert plant species-specific roles in root development: for example, it inhibits nodulation in L. japonicus, while no obvious function for the protein in root development was identified in garden pea, another legume (Oyama et al., 1997; Nishimura et al., 2002; Burman et al., 2018). In Arabidopsis, hy5 mutant seedlings exhibit shorter primary root lengths but show increased initiation and elongation of lateral roots (Oyama et al., 1997; Chen et al., 2016). Interestingly, Arabidopsis hy5 hyh double mutants maintained shorter primary root lengths but had similar lateral root growth than the WT (Sibout et al., 2006). Tomato has only one copy of SlHY5. We showed that slhy5 independent mutant lines develop shorter primary roots and fewer lateral roots, which is similar to Arabidopsis hy5 hyh double mutants. Arabidopsis hy5 hyh double mutants exhibit several shoot phenotypes, including fused cotyledons indicative of altered auxin signaling. In contrast, tomato slhy5 mutant lines develop normal cotyledons. Transcript analyses indicate that auxin signaling is enhanced in Arabidopsis hy5 hyh double mutants; however, it is reduced in tomato slhy5 mutants (Sibout et al., 2006). This discrepancy is possibly due to the different tissues used for transcript analyses, that is, whole seedlings for hy5 hyh versus roots for slhy5 (Sibout et al., 2006). Although SlHY5 overexpression lines showed enhanced responses to light in hypocotyl length assays, these lines exhibited WT root phenotypes. It is possible that additional factors are needed for the overexpressed SlHY5 to alter root growth. The detailed mechanism by which SlHY5 regulates tomato root development awaits further investigations.

Arabidopsis hy5 mutants have reduced chlorophyll contents in seedlings (Oyama et al., 1997). We show here that tomato SlHY5 also promotes chlorophyll accumulation at the adult stage, probably via transcriptional regulation of key genes along the chlorophyll biosynthesis pathway. Interestingly, the effect of SlHY5 on chlorophyll accumulation was more prominent on the illuminated side of fruits relative to their shaded side (Figure 4, F and G). We observed that both white light and UV-B can promote SlHY5 protein accumulation. Moreover, the effect of SlHY5 appeared to be more pronounced in the outer layers of the tomato pericarp than in the inner layers (Figure 4F). These observations suggest a model in which light inhibition of SlCOP1 results in the accumulation of SlHY5 itself, leading to photomorphogenic responses, including the biosynthesis and accumulation of chlorophylls. More than one functional copy of SlCOP1 is thought to be encoded by the tomato genome, as available slcop1 mutant alleles do not show typical photomorphogenic phenotypes as Arabidopsis cop1 mutants do (Jones et al., 2012).

SlHY5 was recently shown to regulate ethylene biosynthesis in fruits (Wang et al., 2021). Our transcriptome analysis revealed that it may also affect other phytohormone signaling pathways in fruits (Supplemental Figure S7C). Transcriptome analysis at the seedling stage showed that only a small portion of HY5-regulated genes have roles in metabolism (Lee et al., 2007), while our transcriptome analyses in fruits revealed that most genes regulated by SlHY5 affect fruit metabolism. Previous targeted metabolite analysis studies have demonstrated the key roles of SlHY5 in regulating anthocyanin biosynthesis in tomato (Liu et al., 2018; Qiu et al., 2019). Transient silencing of SlHY5 by virus-induced gene silencing revealed that SlHY5 regulates the biosynthesis of SGAs (Wang et al., 2018). Moreover, there is evidence that SlHY5 binds to the promoters of several genes encoding enzymes involved in SGA biosynthesis (Wang et al., 2018). We further tested these effects with a metabolome approach and discovered that SlHY5 indeed regulates the accumulation of many metabolites with stable slhy5 mutant. The metabolome analyses identified some known metabolites regulated by SlHY5, including flavonoids and SGAs, as well as additional SlHY5 targets including amino acids and nucleotides (Figure 5, B and D and 6B). We show that SlHY5 regulates several key genes in the biosynthesis pathway of SGAs with stable slhy5 mutant lines (Figure 6C).

Among the significantly differentially accumulated metabolites in slhy5, phenylpropanoids and alkaloids were overrepresented (Figure 5B;Supplemental Table S3). Therefore, we focused on these two large families of metabolites for detailed analysis. Sugar metabolism is important for fruit development. Our transcriptome analysis identified DEGs related to sugar metabolism (Supplemental Figure S7C). We selected four detected DEGs related to sugar metabolism including the genes encoding sucrose phosphate synthase (SlSPS), sucrose synthase (SlSS3), neutral invertase (SlNI), and fructokinase (SlFK2) for RT-qPCR analysis. Expression patterns of three out of the four selected genes in transcriptome can be confirmed by RT-qPCR with an exception of SlNI. The expression of SlNI was shown to be enhanced in slhy5 in the transcriptome analysis, which could not be confirmed with RT-qPCR analysis (Supplemental Figure S10). Furthermore, we also analyzed some other sugar metabolism-related genes including those encoding fructose 1,6-bisphosphatase (SlFBP), fructose 1,6-bisphosphatase (SlG6PDH), and sucrose transporter (SlSUT4), that were not obviously altered in slhy5 with transcriptome analysis. Interestingly, expression of SlFBP was shown significantly higher in slhy5 than in the WT in the RT-qPCR analysis (Supplemental Figure S10). However, our metabolome analysis did not detect many sugars or related metabolites with altered levels in slhy5. The level of D-Fructose-1,6-biphosphate in slhy5 was ˂50% of that in the WT (Supplemental Table S3). It is possible that more comprehensive approach is needed for sugar analysis. Several other metabolites, including carotenoids, were not detected in our metabolome analyses. More specific and comprehensive metabolomic approaches will be needed to further explore the metabolic pathways regulated by SlHY5 in the future.

In summary, besides regulation of seedling photomorphogenesis, our results present additional key functions of SlHY5 at adult stages of tomato development, including effects on plant height, pigment accumulation, and fruit metabolism. Overall, we show not only that SlHY5 is important for seedling photomorphogenesis, but that manipulation of this protein is a promising strategy to improve tomato plant fitness and fruit quality.

Materials and methods

Plant materials and growth conditions

The tomato (S. lycopersicum) cultivar AC was used in this work. The slhy5 mutants were generated with a CRISPR/CAS9 approach as reported previously (Zhang et al., 2021). The SlHY5 overexpression lines (35S:SlHY5-FLAG) were generated in the AC background similar as reported previously (Zhang et al., 2021). The primers used are included in Supplemental Table S4. Seedlings were grown for 4 d on half-strength Murashige and Skoog (1/2 MS) medium under different light conditions after germination. For hypocotyl assays, light conditions consisted of: white light (3.6 μmol m−2 s−1), blue light (5 μmol m−2 s−1), red light (60 μmol m−2 s−1), far-red light (5 μmol m−2 s−1), and white light supplemented with 1.5 μmol m−2 s−1 UV-B 1. Hypocotyl length was quantified with ImageJ software (http://rsbweb.nih.gov/ij/). For root development, seeds were transferred to 1/2 MS medium after germination. Plates were grown vertically under a 16-h light/8-h dark photoperiod at 25°C (100 μmol m−2 s−1 white light). The lower half of each petri dish was covered with aluminum foil and black cloth with the upper part exposed to light. To keep the roots in continuous darkness while exposing the shoots to light, the aluminum foil and black cloth were positioned to cover the seeds, with the top edge of the foil and cloth about 2 mm above the seeds. For sampling of fruits, flowers were tagged on the day of anthesis to evaluate fruit ripening stages. Adult tomato plants were cultivated under natural sunlight in a simple greenhouse with a plastic cover. One side of each tomato fruit was naturally in the shade, as the other side received most of the incoming sunlight (Figure 4, F–G).

Immunoblot analysis of SlHY5

Seedlings were ground to a fine powder in liquid nitrogen. Total proteins were extracted in buffer containing 100-mM Tris–HCl pH 8.0, 50-mM EDTA, 250 mM NaCl, 0.7% (w/v) SDS (Sodium dodecyl sulfate), 10-mM NaF, 15-mM β-glycerolphosphate, 15-mM p-nitrophenyl phosphate, 1-mM dithiothreitol, and 1× EDTA-free Protease Inhibitor. Anti-FLAG (Sigma-aldrich, St. Louis, MO 63103, USA; A8592) and Anti-GAPDH (Proteintech, Rosemont, IL, USA; 60004-1) antibodies were used for immunoblotting.

RT-qPCR

Total RNA was isolated from tomato tissues with a Plant RNeasy kit (OMEGA, Bienne, Switzerland). First-strand cDNA was synthesized with the TransScript SuperMix (TransGen Biotech Beijing, China). Actin (Solyc03g078400) served as the reference gene. RT-qPCR reactions were performed with Fast SYBR Green Master Mix on a CFX96 real-time fluorescence quantitative RT-PCR system (Bio-Rad, Hercules, CA, USA). Except in Figure 1B, expression of each gene was normalized against that in the WT, which was set to 1. For Figure 1B, the expression of HY5 in different tissues was normalized against that in seedlings, which was set to 1. The primers used here are included in Supplemental Table S4.

Chlorophyll extraction and detection

Chlorophylls were extracted from fruits and leaves by soaking in 80% (v/v) acetone for 3 h in darkness. After centrifugation at 13,000 g for 10 min at room temperature, chlorophyll content was determined using cleared supernatants based on absorption at 663 and 646 nm and a previously published formula (Burman et al., 2018).

Metabolome analysis

Sample preparation and extraction

The pericarps of fruits at ripening stages were pooled from at least four individual plants as one biological replicate. Three biological replicates were used for both metabolomes which were carried out by Wuhan Metware Biotechnology Co., Ltd (http://www.metware.cn/). The pericarp was lyophilized and crushed into powder using a mixer mill (MM 400, Retsch) with a zirconium bead for 1.5 min at 30 Hz. About 100 mg powder was weighted and extracted overnight at 4°C with 1.2 mL 70% (v/v) aqueous methanol. Following centrifugation at 12,000 g for 10 min, the extracts were filtered (SCAA-104, 0.22-μm pore size; ANPEL) before ultra-performance liquid chromatography–tandem mass spectrometry (UPLC–MS/MS) analysis.

UPLC conditions

The sample extracts were analyzed using an UPLC–ESI–MS/MS system (UPLC, SHIMADZU Nexera X2, www.shimadzu.com.cn/; MS, Applied Biosystems 4500 Q TRAP, www.appliedbiosystems.com.cn/). The analytical conditions were as follows, UPLC: column, Agilent SB-C18 (1.8 µm, 2.1 mm*100 mm); The mobile phase consisted of solvent A, pure water with 0.1% formic acid (v/v), and solvent B, acetonitrile with 0.1% formic acid (v/v). Sample measurements were performed with a gradient program that employed the starting conditions of 95% A, 5% B. Within 9 min, a linear gradient to 5% A, 95% B was programmed, and a composition of 5% A, 95% B was kept for 1 min. Subsequently, a composition of 95% A, 5.0% B was adjusted within 1.10 min and kept for 2.9 min. The column oven was set to 40°C; The injection volume was 4 μL. The effluent was alternatively connected to an ESI-triple quadrupole-linear ion trap (QTRAP)-MS.

ESI-QTRAP–MS/MS

LIT and triple quadrupole (QQQ) scans were acquired on a triple quadrupole-linear ion trap mass spectrometer (QTRAP), AB4500 Q TRAP UPLC/MS/MS System, equipped with an ESI Turbo Ion-Spray interface, operating in positive and negative ion mode and controlled by Analyst version 1.6.3 software (AB Sciex, Framingham, MA, USA). The ESI source operation parameters were as follows: ion source, turbo spray; source temperature 550°C; ion spray voltage 5,500 V (positive ion mode)/−4,500 V (negative ion mode); ion source gas I (GSI), GSII, and curtain gas were set at 50, 60, and 25.0 psi, respectively; the collision gas was high. Instrument tuning and mass calibration were performed with 10 and 100 μmol/L polypropylene glycol solutions in QQQ and LIT modes, respectively. QQQ scans were acquired as multiple reaction monitoring (MRM) experiments with collision gas (nitrogen) set to medium. DP and CE for individual MRM transitions were done with further DP and CE optimization. A specific set of MRM transitions were monitored for each period according to the metabolites eluted within this period.

Analytical method

Material characterization was carried out according to the secondary spectrum information based on self-built database (metware database). Metabolite quantification was performed by MRM of triple quadrupole scans. After obtaining the mass spectrometry analysis data, the peak area integration of mass spectrometry was carried out for all metabolites. Unsupervised PCA was performed by statistics function prop within R (www.r-project.org). The data were unit variance scaled before unsupervised PCA. The hierarchical cluster analysis (HCA) results of samples and metabolites were presented as heatmaps with dendrograms. HCA was carried out by R package heatmap. For HCA, normalized signal intensities of metabolites (unit variance scaling) are visualized as a color spectrum. Significantly regulated metabolites between groups were determined by variable VIP ≥ 1 and absolute Log2FC ≥ 1. VIP values were extracted from OPLS-DA (orthogonal partial least squares-discrimination analysis) result, which also contain score plots and permutation plots, was generated using R package “MetaboAnalystR.” The data was log transform (log2) and mean centering before OPLS-DA. In order to avoid overfitting, a permutation test (200 permutations) was performed. Identified metabolites were annotated using KEGG Compound database (http://www.kegg.jp/kegg/compound/), annotated metabolites were then mapped to KEGG Pathway database (http://www.kegg.jp/kegg/pathway.html). Pathways with significantly regulated metabolites mapped to were then fed into metabolite sets enrichment analysis, their significance was determined by hypergeometric test’s P-values.

Transcriptome analysis

Sample preparation

The pericarps of fruits at ripening stages were pooled from at least four individual plants as one biological replicate. Three biological replicates were used for both transcriptome analyses, which were carried out by Wuhan Metware Biotechnology Co., Ltd (http://www.metware.cn/). The transcriptome raw data were deposited in the Sequence Read Archive (SRA) of NCBI, with accession number PRJNA796431 (https://www.ncbi.nlm.nih.gov/sra/PRJNA796431).

Library preparation for transcriptome sequencing

A total amount of 1-µg RNA per sample was used as input material for the RNA sample preparations. Sequencing libraries were generated using NEB Next Ultra RNA Library Prep Kit for Illumina (NEB, Ipswich, MA, USA) following manufacturer’s recommendations and index codes were added to attribute sequences to each sample. Library quality was assessed on the Agilent Bioanalyzer 2100 system.

Clustering and sequencing

The clustering of the index-coded samples was performed on a cBot Cluster Generation System using TruSeq PE Cluster Kit v3-cBot-HS (Illumia San Diego, CA, USA) according to the manufacturer’s instructions. After cluster generation, the library preparations were sequenced on an Illumina Novaseq 6000 platform and 150-bp paired-end reads were generated.

Data analysis

Raw data (raw reads) of fastq format were first processed through in-house perl scripts. In this step, clean data (clean reads) were obtained by removing reads containing adapter, reads containing ploy-N, and low-quality reads from raw data. At the same time, Q20, Q30, and GC content on the clean data were calculated. All the downstream analyses were based on the clean data with high quality. Differential expression analysis of two groups was performed using the DESeq2 package, and DESeq2 provides statistical routines for determining differential expression in digital gene expression data using a model based on the negative binomial distribution through indices of FC (log2 ratio) and P-value (false discovery rate [FDR]). DEGs were selected based on the criteria |log2(FC) | ≥1 and FDR < 0.05.

Statistical analyses

Significant differences between slhy5 (or SlHY5-OX) and WT were tested by one-way analysis of variance tests.

Accession numbers

Sequence data from this article can be found in the Sol Genomics Network databases (https://solgenomics.net/) under accession numbers: SlHY5 (Solyc08g061130), SlUVR8 (Solyc05g018615), SlBBX20 (Solyc01g110180), SlBBX21 (Solyc12g089240), and SlACTIN (Solyc03g078400).

Supplemental data

The following materials are available in the online version of this article.

Supplemental Figure S1. Generation of SlHY5 frame-shift mutant lines with CRISPR/CAS9 approach.

Supplemental Figure S2. Transcript levels of several potentially auxin responsive genes.

Supplemental Figure S3. Detection of FLAG-SlHY5 fusion protein in SlHY5 overexpression lines.

Supplemental Figure S4. Root phenotypes of the SlHY5-OX lines.

Supplemental Figure S5. The SlHY5-OX seedlings exhibit shorter hypocotyls under various light conditions.

Supplemental Figure S6. SlHY5 influences adult plant height and chlorophyll accumulation in seedlings.

Supplemental Figure S7. Transcriptome analysis of WT and the slhy5 mutant.

Supplemental Figure S8. Metabolome analysis of WT and the slhy5 mutant.

Supplemental Figure S9. Sequence alignment of HY5 from different species.

Supplemental Figure S10. Transcript analysis for the levels of selected sugar metabolism-elated genes in ripening tomato fruit by RT-qPCR.

Supplemental Table S1. The DEGs in slhy5 ripening fruit.

Supplemental Table S2. A list of all the detected metabolites.

Supplemental Table S3. Differentially accumulated metabolites in slhy5 in relation to WT (≥2 FC).

Supplemental Table S4. The primers used in this study.

Supplementary Material

kiac133_Supplementary_Data
Click here for additional data file. (1.9MB, zip)

Acknowledgments

We thank the Plant Editors for editing of this manuscript, all the other members in our laboratory for kind support of this work and helpful discussions.

Funding

This study was supported by the National Key Research and Development Program of China (2018YFD1000800), by the National Natural Science Foundation of China (32070261, 32170246, and 31870261), by Natural Science Foundation of Shanghai (22ZR1431300), by a Startup Fund for Young Faculty at SJTU (21X010500765), by Zhiyuan scholar program at SJTU and by the Medicine and Engineering interdisciplinary Research Fund of SJTU (YG2021ZD07).

Conflict of interest statement. None declared.

 

C.Z. performed most of the experiments, analyzed data, and prepared the initial draft of the manuscript. X.R.L. generated the SlHY5 over expression lines. Y.W. and J.Z. contributed to Figure 2, H and I. X.L. and L.L. contributed to the metabolome analysis. R.Y. designed the experiments, analyzed the data, and wrote the manuscript.

The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (https://academic.oup.com/plphys/pages/general-instructions) is Ruohe Yin (ruohe.yin@sjtu.edu.cn).

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

kiac133_Supplementary_Data
Click here for additional data file. (1.9MB, zip)

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