SlTCP24 and SlTCP29 synergistically regulate compound leaf development through interacting with SlAS2 and activating transcription of SlCKX2 in tomato.

The complexity of compound leaves results primarily from the leaflet initiation and arrangement during leaf development. However, the molecular mechanism underlying compound leaf development remains a central research question. SlTCP24 and SlTCP29, two plant-specific transcription factors with the conserved TCP motif, are shown here to synergistically regulate compound leaf development in tomato. When both of them were knocked out simultaneously, the number of leaflets significantly increased, and the shape of the leaves became more complex. SlTCP24 and SlTCP29 could form both homodimers and heterodimers, and such dimerization was impeded by the leaf polarity regulator SlAS2, which interacted with SlTCP24 and SlTCP29. SlTCP24 and SlTCP29 could bind to the TCP-binding cis-element of the SlCKX2 promoter and activate its transcription. Transgenic plants with SlTCP24 and SlTCP29 double-gene knockout had a lowered transcript level of SlCKX2 and an elevated level of cytokinin. This work led to the identification of two key regulators of tomato compound leaf development and their targeted genes involved in cytokinin metabolic pathway. A model of regulation of compound leaf development was proposed based on observations of this study.

Diversification of JAZ-MYC signaling function in immune metabolism.

Jasmonate (JA) re-programs metabolism to confer resistance to diverse environmental threats. Jasmonate stimulates the degradation of JASMONATE ZIM-DOMAIN (JAZ) proteins that repress the activity of MYC transcription factors. In Arabidopsis thaliana, MYC and JAZ are encoded by 4 and 13 genes, respectively. The extent to which expansion of the MYC and JAZ families has contributed to functional diversification of JA responses is not well understood. Here, we investigated the role of MYC and JAZ paralogs in controlling the production of defense compounds derived from aromatic amino acids (AAAs). Analysis of loss-of-function and dominant myc mutations identified MYC3 and MYC4 as the major regulators of JA-induced tryptophan metabolism. We developed a JAZ family-based, forward genetics approach to screen randomized jaz polymutants for allelic combinations that enhance tryptophan biosynthetic capacity. We found that mutants defective in all members (JAZ1/2/5/6) of JAZ group I over-accumulate AAA-derived defense compounds, constitutively express marker genes for the JA-ethylene branch of immunity and are more resistant to necrotrophic pathogens but not insect herbivores. In defining JAZ and MYC paralogs that regulate the production of amino-acid-derived defense compounds, our results provide insight into the specificity of JA signaling in immunity.

SlBBX20 attenuates JA signalling and regulates resistance to Botrytis cinerea by inhibiting SlMED25 in tomato.

Jasmonic acid (JA) plays an important role in regulating plant growth and defence responses. Here, we show that a transcription factor that belongs to the B-box (BBX) family named SlBBX20 regulates resistance to Botrytis cinerea in tomato by modulating JA signalling. The response to JA was significantly suppressed when SlBBX20 was overexpressed in tomato. By contrast, the JA response was enhanced in SlBBX20 knockout lines. RNA sequencing analysis provided more evidence that SlBBX20 modulates the expression of genes that are involved in JA signalling. We found that SlBBX20 interacts with SlMED25, a subunit of the Mediator transcriptional co-activator complex, and prevents the accumulation of the SlMED25 protein and transcription of JA-responsive genes. JA contributes to the defence response against necrotrophic pathogens. Knocking out SlBBX20 or overexpressing SlMED25 enhanced tomato resistance to B. cinerea. The resistance was impaired when SlBBX20 was overexpressed in plants that also overexpressed SlMED25. These data show that SlBBX20 attenuates JA signalling by regulating SlMED25. Interestingly, in addition to developing enhanced resistance to B. cinerea, SlBBX20-KO plants also produced higher fruit yields. SlBBX20 is a potential target gene for efforts that aim to develop elite crop varieties using gene editing technologies.

Variation in the fruit development gene POINTED TIP regulates protuberance of tomato fruit tip.

The domestication of tomato has led to striking variations in fruit morphology. Here, we show a genome-wide association study (GWAS) to understand the development of the fruit tip and describe a POINTED TIP (PT) gene that encodes a C2H2-type zinc finger transcription factor. A single nucleotide polymorphism is found to change a histidine (H) to an arginine (R) in the C2H2 domain of PT and the two alleles are referred to as PTH and PTR. Knocking out PTH leads to development of pointed tip fruit. PTH functions to suppress pointed tip formation by downregulating the transcription of FRUTFULL 2 (FUL2), which alters the auxin transport. Our evolutionary analysis and previous studies by others suggest that the PTR allele likely hitch-hiked along with other selected loci during the domestication process. This study uncovers variation in PT and molecular mechanism underlying fruit tip development in tomato.

A novel regulatory complex mediated by Lanata (Ln) controls multicellular trichome formation in tomato.

Trichomes that originate from plant aerial epidermis act as mechanical and chemical barriers against herbivores. Although several regulators have recently been identified, the regulatory pathway underlying multicellular trichome formation remains largely unknown in tomato. Here, we report a novel HD-ZIP IV transcription factor, Lanata (Ln), a missense mutation which caused the hairy phenotype. Biochemical analyses demonstrate that Ln separately interacts with two trichome regulators, Woolly (Wo) and Hair (H). Genetic and molecular evidence demonstrates that Ln directly regulates the expression of H. The interaction between Ln and Wo can increase trichome density by enhancing the expression of SlCycB2 and SlCycB3, which we previously showed are involved in tomato trichome formation. Furthermore, SlCycB2 represses the transactivation of the SlCycB3 gene by Ln and vice versa. Our findings provide new insights into the novel regulatory network controlling multicellular trichome formation in tomato.

Hair interacts with SlZFP8-like to regulate the initiation and elongation of trichomes by modulating SlZFP6 expression in tomato.

Trichomes are specialized glandular or non-glandular structures that provide physical or chemical protection against insect and pathogen attack. Trichomes in Arabidopsis have been extensively studied as typical non-glandular structures. By contrast, the molecular mechanism underlying glandular trichome formation and elongation remains largely unknown. We previously demonstrated that Hair is essential for the formation of type I and type VI trichomes. Here, we found that overexpression of Hair increased the density and length of tomato trichomes. Biochemical assays revealed that Hair physically interacts with its close homolog SlZFP8-like (SlZFP8L), and SlZFP8L also directly interacts with Woolly. SlZFP8L-overexpressing plants showed increased trichome density and length. We further found that the expression of SlZFP6, which encodes a C2H2 zinc finger protein, is positively regulated by Hair. Using chromatin immunoprecipitation, yeast one-hybrid, and dual-luciferase assays we identified that SlZFP6 is a direct target of Hair. Similar to Hair and SlZFP8L, the overexpression of SlZFP6 also increased the density and length of tomato trichomes. Taken together, our results suggest that Hair interacts with SlZFP8-like to regulate the initiation and elongation of trichomes by modulating SlZFP6 expression in tomato.

miRNAs and lncRNAs in tomato: Roles in biotic and abiotic stress responses.

Plants are continuously exposed to various biotic and abiotic stresses in the natural environment. To cope with these stresses, they have evolved a multitude of defenses mechanisms. With the rapid development of genome sequencing technologies, a large number of non-coding RNA (ncRNAs) have been identified in tomato, like microRNAs (miRNAs) and long non-coding RNAs (lncRNAs). Recently, more and more evidence indicates that many ncRNAs are involved in plant response to biotic and abiotic stresses in tomato. In this review, we summarize recent updates on the regulatory roles of ncRNAs in tomato abiotic/biotic responses, including abiotic (high temperature, drought, cold, salinization, etc.) and biotic (bacteria, fungi, viruses, insects, etc.) stresses. Understanding the molecular mechanisms mediated by ncRNAs in response to these stresses will help us to clarify the future directions for ncRNA research and resistance breeding in tomato.

SlBBX20 interacts with the COP9 signalosome subunit SlCSN5-2 to regulate anthocyanin biosynthesis by activating SlDFR expression in tomato.

Anthocyanins play vital roles in plant stress tolerance and growth regulation. Previously, we reported that the photomorphogenesis-related transcription factor SlBBX20 regulates anthocyanin accumulation in tomato. However, the underlying mechanism remains unclear. Here, we showed that SlBBX20 promotes anthocyanin biosynthesis by binding the promoter of the anthocyanin biosynthesis gene SlDFR, suggesting that SlBBX20 directly activates anthocyanin biosynthesis genes. Furthermore, we found by yeast two-hybrid screening that SlBBX20 interacts with the COP9 signalosome subunit SlCSN5-2, and the interaction was confirmed by bimolecular fluorescence complementation and coimmunoprecipitation assays. SlCSN5 gene silencing led to anthocyanin hyperaccumulation in the transgenic tomato calli and shoots, and SlCSN5-2 overexpression decreased anthocyanin accumulation, suggesting thSlCSN5-2 enhanced the ubiquitination of SlBBX20 and promoted the degradation of SlBBX20 in vivo. Consistently, silencing the SlCSN5-2 homolog in tobacco significantly increased the accumulation of the SlBBX20 protein. Since SlBBX20 is a vital regulator of photomorphogenesis, the SlBBX20-SlCSN5-2 module may represent a novel regulatory pathway in light-induced anthocyanin biosynthesis.

Transcriptome profiling of Capsicum annuum using Illumina- and PacBio SMRT-based RNA-Seq for in-depth understanding of genes involved in trichome formation.

Trichomes, specialized epidermal cells located in aerial parts of plants, play indispensable roles in resisting abiotic and biotic stresses. However, the regulatory genes essential for multicellular trichrome development in Capsicum annuum L. (pepper) remain unclear. In this study, the transcript profiles of peppers GZZY-23 (hairy) and PI246331 (hairless) were investigated to gain insights into the genes responsible for the formation of multicellular trichomes. A total of 40,079 genes, including 4743 novel genes and 13,568 differentially expressed genes (DEGs), were obtained. Functional enrichment analysis revealed that the most noticeable pathways were transcription factor activity, sequence-specific DNA binding, and plant hormone signal transduction, which might be critical for multicellular trichome formation in hairy plants. We screened 11 DEGs related to trichome development; 151 DEGs involved in plant hormone signal transduction; 312 DEGs belonging to the MYB, bHLH, HD-Zip, and zinc finger transcription factor families; and 1629 DEGs predicted as plant resistance genes (PRGs). Most of these DEGs were highly expressed in GZZY-23 or trichomes. Several homologs of trichome regulators, such as SlCycB2, SlCycB3, and H, were considerably upregulated in GZZY-23, especially in the trichomes. The transcriptomic data generated in this study provide a basis for future characterization of trichome formation in pepper.

HOMEODOMAIN PROTEIN8 mediates jasmonate-triggered trichome elongation in tomato.

Plant hormones can adjust the physiology and development of plants to enhance their adaptation to biotic and abiotic challenges. Jasmonic acid (JA), one of the immunity hormones in plants, triggers genome-wide transcriptional changes in response to insect attack and wounding. Although JA is known to affect the development of trichomes, epidermal appendages that form a protective barrier against various stresses, it remains unclear how JA interacts with developmental programs that regulate trichome development. In this study, we show that JA affects trichome length in tomato by releasing the transcriptional repression mediated by Jasmonate ZIM (JAZ) proteins. We identified SlJAZ4, a negative regulator preferentially expressed in trichomes, as the critical component in JA signaling in tomato trichomes. We also identified a homeodomain-leucine zipper gene, SlHD8, as the downstream regulator of JA signaling that promotes trichome elongation. SlHD8 is also highly expressed in trichomes and physically interacts with SlJAZ4. Loss-of-function mutations in SlHD8 caused shorter trichomes, a phenotype that was only partially rescued by methyl jasmonate treatment. Our dual-luciferase and chromatin immunoprecipitation-quantitative PCR assays revealed that SlHD8 regulates trichome elongation by directly binding to the promoters of a set of cell-wall-loosening protein genes and activating their transcription. Together, our findings define SlHD8-SlJAZ4 as a key module mediating JA-induced trichome elongation in tomato.

Identification, genomic organization, and expression profiles of single C2H2 zinc finger transcription factors in tomato (Solanum lycopersicum).

C2H2 zinc finger proteins (ZFPs) play essential roles in leaf morphogenesis and floral development, as well as heat stress response and trichome formation, which activate or inhibit gene transcription mainly through interactions with nucleic acids, such as single-strand DNA, RNA binding or RNA/DNA bidirectional binding, and protein interaction. Single C2H2 ZFPs is the subfamily of ZFPs, but little of single C2H2 ZFP family is known in tomato. In this study, we identified 30 single ZFP genes in tomato using bioinformatics-based methods. Gene structures, phylogeny, conserved motifs, cis-element of promoter, chromosomal localization, gene duplication, and expression patterns of these single C2H2 ZFP genes were analyzed. Sequence analysis showed that most single C2H2 ZFP genes possessed only one exon, except for SlC1-liZFP1 and SlC1-liZFP2. These single C2H2 ZFP genes were asymmetrically distributed on 10 chromosomes, excluding 2 and 12 chromosomes. In addition, 24 of these genes were predicated to have experienced segmental duplication. Cis-element prediction indicated that many important elements were located in the putative promoter regions, like light and gibberellic acid (GA)-responsive elements. The expression profiles of these genes in different tissues and various hormones and stress treatment were further analyzed. Many genes were lowly expressed in all tissues, whereas some were specifically expressed in certain tissues, like SlC1-liZFP2 in young leaves, and SlC1-liZFP15 in fruits. Furthermore, these genes could also be induced by several hormones and stresses, including IAA, ETH, GA, cold, and drought. This study sets a good foundation for further characterizing the biological roles of single C2H2 ZFP genes in tomato.

The loss of function of HEL, which encodes a cellulose synthase interactive protein, causes helical and vine-like growth of tomato.

Helical growth is an economical way for plant to obtain resources. The classic microtubule-microfibril alignment model of Arabidopsis helical growth involves restriction of the appropriate orientation of cellulose microfibrils appropriately in the cell walls. However, the molecular mechanism underlying tomato helical growth remains unknown. Here, we identified a spontaneous tomato helical (hel) mutant with right-handed helical cotyledons and petals but left-handed helical stems and true leaves. Genetic analysis revealed that the hel phenotype was controlled by a single recessive gene. Using map-based cloning, we cloned the HEL gene, which encodes a cellulose interacting protein homologous to CSI1 of Arabidopsis. We identified a 27 bp fragment replacement that generated a premature stop codon. Transgenic experiments showed that the helical growth phenotype could be restored by the allele of this gene from wild-type Pyriforme. In contrast, the knockout mutation of HEL in Pyriforme via CRISPR/Cas9 resulted in helical growth. These findings shed light on the molecular control of the helical growth of tomato.

NDW, encoding a receptor-like protein kinase, regulates plant growth, cold tolerance and susceptibility to Botrytis cinerea in tomato.

Plants utilize different mechanisms to respond and adapt to continuously changing environmental factors. Receptor-like protein kinases (RLKs) comprise one of the largest families of plant transmembrane signaling proteins, which play critical and diverse roles in plant growth, development, and stress response. Here, we identified the necrotic dwarf (ndw) mutant introgression line (IL) 6-2, which demonstrated stunting, leaf curl, and progressive necrosis at low temperatures. Based on map-based cloning and transgenic analysis, we determined that the phenotype of ndw mutant is caused by decreased expression of NDW, which encodes an RLK. NDW is a plasma membrane and cytoplasmic located protein. Overexpression of NDW can restore both of the semi-dwarf and necrotic phenotype in IL6-2 at low temperatures, further we found that NDW could significantly reduce susceptibility to Botrytis cinerea. On the contrary, knockdown NDW in M82 plants could increase the sensitivity to B. cinerea. Furthermore, transcriptional expression analysis showed that NDW affects the expression of genes related to the abscisic acid (ABA) signaling pathway. Taken together, these results indicate that NDW plays an important role in regulating plant growth, cold tolerance and mitigating susceptibility to Botrytis cinerea.

The HD-Zip IV transcription factor SlHDZIV8 controls multicellular trichome morphology by regulating the expression of Hairless-2.

Trichomes are specialized epidermal appendages that serve as excellent models to study cell morphogenesis. Although the molecular mechanism underlying trichome morphogenesis in Arabidopsis has been well characterized, most of the regulators essential for multicellular trichome morphology remain unknown in tomato. In this study, we determined that the recessive hairless-2 (hl-2) mutation in tomato causes severe distortion of all trichome types, along with increased stem fragility. Using map-based cloning, we found that the hl-2 phenotype was associated with a 100 bp insertion in the coding region of Nck-associated protein 1, a component of the SCAR/WAVE complex. Direct protein-protein interaction was detected between Hl-2 and Hl (SRA1, specifically Rac1-associated protein) using yeast two-hybrid and co-immunoprecipitation assays, suggesting that these proteins may work together during trichome formation. In addition, knock-down of a HD-Zip IV transcription factor, HDZIPIV8, distorted trichomes similar to the hl-2 mutant. HDZIPIV8 regulates the expression of Hl-2 by binding to the L1-box in the Hl-2 promoter region, and is involved in organizing actin filaments. The brittleness of hl-2 stems was found to result from decreased cellulose content. Taken together, these findings suggest that the Hl-2 gene plays an important role in controlling multicellular trichome morphogenesis and mechanical properties of stems in tomato plants.

Transcriptomic and functional analyses uncover the regulatory role of lncRNA000170 in tomato multicellular trichome formation.

Trichomes are universal specific structures originating from nearly all terrestrial plants. Although quantities of long non-coding RNAs (lncRNAs) have been identified in many plant species, the role of lncRNAs in trichome formation still remains unknown. Here, we identified a total of 1303 lncRNAs in the young stems of woolly mutant LA3560 (Wo) and its non-woolly segregants (WT). Out of these lncRNAs, 86 lncRNAs were obviously upregulated in Wo and 110 lncRNAs were downregulated. We determined that seven lncRNAs were highly expressed in stem trichomes compared to trichome-free stems and several other tissues of LA3560 by a quantitative reverse transcriptase-polymerase chain reaction, including lncRNA000746, lncRNA000170, lncRNA000277, lncRNA000774, lncRNA000756, lncRNA000100, and lncRNA000898. Transgenic experiments revealed that overexpression of lncRNA000170 inhibited type I trichome formation on the lower stems of the adult transgenic plants. We further determined that lncRNA000170 was transcribed from the complementary strand of Solyc10g006360, for which expression can be induced by lncRNA000170 in its overexpression lines and woolly mutants. Solyc10g006360 overexpression also caused type I trichome decrease. In addition, several trichome regulators, such as Wo, H, SlCycB2, and SlCycB3, were markedly downregulated in lncRNA000170 overexpression lines. These findings demonstrate that lncRNA000170 may be involved in the regulatory pathway mediated by these trichome regulators.

Genome-wide identification, genomic organization and expression profiles of SlARR-B gene family in tomato.

The type-B authentic response regulators (ARR-Bs) function as positive regulators of cytokinin signal transduction and play important roles in abiotic stress resistance and plant development. However, little of ARR-B family is known in tomato. In this study, we performed a comprehensive analysis of ARR-B family factors in tomato. In total, 12 genes encoding ARR-B transcription factors (named as SlARR-B1-SlARR-B12) were identified from tomato. We analyzed the structures, chromosome locations, phylogeny, protein motifs, and expression profiles of these SlARR-B genes. Gene structure analysis showed that 5-12 exons and 4-11 introns existed in the SlARR-B genes. These SlARR-B genes were asymmetrically distributed on eight chromosomes in tomato. Phylogenetic tree of SlARR-B genes from tomato and other plant species revealed that SlARR-B genes were classified into 6 subfamilies. SlARR-B proteins had typical conserved domains, including Motif 1 and Motif 2. The investigation of the expression profiles of SlARR-B genes in all the examined tissues demonstrated that these genes were differentially expressed, including roots, stems, leaves, flowers, and fruits at developmental stages. Notably, the expression of SlARR-B11 and SlARR-B12 exhibited high expression levels in flowers. Each gene was induced by at least one of different phytohormones (SA, IAA, ABA, IBA, 6-BA, JA, GA, and ETH) and four abiotic stress treatments (heat, drought, salt, and cold). This study sets a good foundation for further characterization of the SlARR-B transcription factors in plant development and abiotic stress responses of tomato.

WOOLLY, interacting with MYB transcription factor MYB31, regulates cuticular wax biosynthesis by modulating CER6 expression in tomato.

Cuticular waxes play a crucial role not only in plant defense against biotic and abiotic stresses, but also in the quality and storability of fruits, such as the tomato (Solanum lycopersicum). Although the biosynthetic pathways of waxes have been extensively characterized, the regulatory mechanisms underlying wax biosynthesis in tomato remain largely unclear. Here, we show that Woolly (Wo), a multicellular trichome regulator, is involved in modulating wax biosynthesis in tomato. Wo enhances the expression of the wax biosynthetic genes SlCER6, SlKCR1, and SlPAS2, and the wax transporter gene SlLTP, and thereby promotes wax accumulation. Furthermore, Wo directly binds to the L1-box in the promoter of SlCER6, an essential element of the very-long-chain fatty acid elongase complex. Intriguingly, overexpression (OE) or knock-down of SlMYB31, an MYB transcription factor that physically interacts with Wo in vivo and in vitro, produces marked changes in wax composition, and whereas Wo knock-down inhibits wax accumulation in SlMYB31-OE lines, SlMYB31 knock-down inhibits wax accumulation in Wo-OE lines, implying that these two genes function in the same pathway. Lastly, SlCER6 expression is induced by abscisic acid in a manner that is partially dependent on Wo. These results demonstrate that Wo and SlMYB31 cooperatively control tomato cuticular wax biosynthesis by regulating the expression of SlCER6.

An allelic variant of GAME9 determines its binding capacity with the GAME17 promoter in the regulation of steroidal glycoalkaloid biosynthesis in tomato.

Steroidal glycoalkaloids (SGAs) are cholesterol-derived molecules found in the family Solanaceae. SGA content varies among different plant species and varieties. However, the genetic mechanisms regulating SGA content remain unclear. Here, we demonstrate that genetic variation in GLYCOALKALOID METABOLISM 9 (GAME9) is responsible for the variation in SGA content in tomato (Solanum lycopersicum). During a sequential analysis we found a 1 bp substitution in the AP2/ERF binding domain of GAME9. The 1 bp substitution in GAME9 was significantly associated with high SGA content and determined the binding capacity of GAME9 with the promoter of GAME17, a core SGA biosynthesis gene. The high-SGA GAME9 allele is mainly present in S. pimpinellifolium and S. lycopersicum var. cerasiforme populations and encodes a protein that can bind the GAME17 promoter. In contrast, the low-SGA GAME9 allele is mainly present in the big-fruited varieties of S. lycopersicum and encodes a protein that shows weak binding to the GAME17 promoter. Our findings provide new insight into the regulation of SGA biosynthesis and the factors that affect the accumulation of SGA in tomato.

The tomato WV gene encoding a thioredoxin protein is essential for chloroplast development at low temperature and high light intensity.

BACKGROUND: Chloroplast biogenesis, a complex process in higher plants, is the key to photoautotrophic growth in plants. White virescent (wv) mutants have been used to unfold the molecular mechanisms underlying the regulation of chloroplast development and chloroplast gene expression in plants. However, most of genes controlling white virescent phenotype still remain unknown. RESULTS: In this study, we identified a temperature- and light intensity-sensitive mutant, named as wv. The content of chlorophyll was dramatically decreased in the immature leaves of wv mutant under the conditions of low temperature and high-light intensity. TEM observation showed that the chloroplasts in the young leaves of wv mutant lacked an organized thylakoid membrane, whereas crescent-shaped chloroplasts with well-developed stromal and stacked grana thylakoids in the mature leaves were developed. Immunoblot analyses suggested that proteins of photosynthetic complexes were decreased substantially in wv mutants. Based on map-based cloning and transgenic analysis, we determined that the wv phenotype was caused by single base mutation in the first intron of WV gene, which encoded a thioredoxin protein with 365 amino acids. qRT-PCR analysis revealed that the expression of WV gene was significantly down-regulated in wv mutant. In addition, knockdown of WV gene through RNAi also resulted in white virescent young leaves, suggesting that the mutation possibly blocks the differentiation of chloroplasts through inhibiting the expression of WV gene. Furthermore, the expression of WV peaked in apical buds and gradually decreased along with the developmental stage, which was consistent with the wv mutant phenotype. Expression analysis of chloroplast-encoded genes by qRT-PCR showed that the wv mutation affected the expression pattern of chloroplast-encoded PEP dependent genes. CONCLUSION: Our results suggested that wv mutant was sensitive to low temperature and light intensity. WV gene was essential for chloroplast differentiation. A single base mutation in the first intron resulted in down-regulation of WV gene expression, which inhibited the expression of chloroplast-encoded genes, thereby blocking chloroplast formation and chlorophyll synthesis.

A tomato B-box protein SlBBX20 modulates carotenoid biosynthesis by directly activating PHYTOENE SYNTHASE 1, and is targeted for 26S proteasome-mediated degradation.

Carotenoids play important roles in many biological processes, such as light harvesting, photoprotection and visual attraction in plants. However, the regulation of carotenoid biosynthesis is still not fully understood. Here, we demonstrate that SlBBX20, a B-box (BBX) zinc-finger transcription factor, is a positive regulator of carotenoid accumulation in tomato (Solanum lycopersicum). Overexpression of SlBBX20 leads to dark green fruits and leaves and higher levels of carotenoids relative to the wild-type. Interactions between SlBBX20 and DE-ETIOLATED 1 (SlDET1) lead to the ubiquitination and 26S proteasome-mediated degradation of SlBBX20. Moreover, deficiencies in the components of the CUL4-DDB1-DET1 complex enhanced the stability of the SlBBX20 protein. Thus, we conclude that SlBBX20 is a substrate of the CUL4-DDB1-DET1 E3 ligase. SlBBX20 can activate the expression of PHYTOENE SYNTHASE 1, encoding a key enzyme in carotenoid biosynthesis, by directly binding to a G-box motif in its promoter, which results in the elevated levels of carotenoids in SlBBX20 overexpression lines. We identified a key regulator of carotenoid biosynthesis and demonstrated that the stability of SlBBX20 is regulated by ubiquitination. These findings provide us a new target for the genetic improvement of the nutritional quality of tomato fruit.