Papaya CpbHLH1/2 regulate carotenoid biosynthesis-related genes during papaya fruit ripening

The ripening of papaya is a physiological and metabolic process associated with accumulation of carotenoids, alternation of flesh color and flavor, which depending on genotype and external factors such as light and hormone. Transcription factors regulating carotenoid biosynthesis have not been analyzed during papaya fruit ripening. RNA-Seq experiments were implemented using different ripening stages of papaya fruit from two papaya varieties. Cis-elements in lycopene β-cyclase genes (CpCYC-B and CpLCY-B) were identified, and followed by genome-wide analysis to identify transcription factors binding to these cis-elements, resulting in the identification of CpbHLH1 and CpbHLH2, two bHLH genes. The expressions of CpbHLH1/2 were changed during fruit development, coupled with transcript increase of carotenoid biosynthesis-related genes including CpCYC-B, CpLCY-B, CpPDS2, CpZDS, CpLCY-E, and CpCHY-B. Yeast one-hybrid (Y1H) and transient expression assay revealed that CpbHLH1/2 could bind to the promoters of CpCYC-B and CpLCY-B, and regulate their transcriptions. In response to strong light, the results of elevated expression of carotenoid biosynthesis-related genes and the changed expression of CpbHLH1/2 indicated that CpbHLH1/2 were involved in light-mediated mechanisms of regulating critical genes in the carotenoid biosynthesis pathway. Collectively, our findings demonstrated several TF family members participating in the regulation of carotenoid genes and proved that CpbHLH1 and CpbHLH2 individually regulated the transcription of lycopene β-cyclase genes (CpCYC-B and CpLCY-B). This study yielded novel findings on regulatory mechanism of carotenoid biosynthesis during papaya fruit ripening.


Introduction
Carotenoids are important structural and functional pigments existed in all plants, which are important for photosynthesis, plant growth and development. Many fleshy fruits, including papaya and tomato, have a dramatic flesh color change resulted from synthesized carotenoids accumulating in chromoplast during fruit ripening. The content of lycopene and β-carotene would have a significant increase from color-break stage to mature fruit, respectively, in red and yellow-fleshed papaya and tomato. As an adaptive characteristic, animals can be attracted by the changed pigments and disperse the matured seeds which are therefore able to germinate 1,2 .
During fruit ripening, carotenoid biosynthesis pathway is regulated by an genetically programmed mechanism that involves phytohormones and environmental factors such as, light, water, and temperature 3 . In papaya carotenoid biosynthesis pathway, with phytoene synthase (PSY) functioned, phytoene is synthesized by condensing two molecules of geranylgeranyl diphosphate (GGPP) in the plastids. Combining the phytoene desaturase (PDS) with ζ-carotene desaturase (ZDS), phytoene is further converted into lycopene which is the precursor of carotene. In nature, carotenoids usually exist in transstructure. Only trans-lycopene can be catalyzed by downstream cyclase 4 . ZISO and CRTSO are key isomerases catalyzing the transformation of carotenoid cisstructure to trans-structure in plants. And the catalytic process also requires the participation of auxiliary factor flavin adenine dinucleotide (FAD) 5,6 . Afterward, one pathway lead to α-carotene via lycopene εand β-cyclase and the other result in β-carotene though chromoplast lycopene beta cyclase (CYC-B) or chloroplast lycopene beta cyclase (LCY-B) and further to zeaxanthin and lutein (Supplement Fig. S1) 7,8 . Meanwhile, recent studies have defined some transcription mechanisms required for the carotenoid biosynthesis-related genes during plant development. MADS-RIN, SPL-CNR, and NAC-NOR have been identified as crucial transcription factors (TFs) for regulating tomato fruit ripening resulting from mutations in these TF genes which inhibit fruit ripening process, such as softening and color development 9,10 .
Ethylene related TFs (EIN3/EILs and ERFs) regulate fruit development via implementing a transcriptional domino effect associated with ethylene-responsive genes. Reduced expression of SIAP2a and SIERF6 results in carotenoid accumulation and ethylene biosynthesis in tomato, suggesting that SIAP2a and SIERF6 act as negative regulators for carotenogenesis [11][12][13] . However, in Arabidopsis, these ethylene-responsive factors such as ERFs and RAP2.2 promote carotenoid biosynthesis by binding to PSY promoter 14 . In NAC family, carotenoid accumulation and ethylene synthesis can be positively regulated since SlNAC4 actively regulate expression of the ripening regulator RIN and cannot be induced by ethylene 15 . In Citrus, through directly binding to promoters, CsMADS6 upregulates the expression of PSY, PDS, and CCD1, implying CsMADS6 regulate the multi-targeted carotenogenesis 16 . Loss-of-function mutations in RCP1 belonging to MYB family led to downregulation of all carotenoid biosynthetic genes, suggesting RCP1 boosts carotenoid biosynthesis during flower development in Mimulus lewisii 17 . Many other important TF families, including HD-ZIP HOMEOBOX PROTEIN-1 (HB-1) and TAGL1, have been proven to play important roles in fruit development in many plant species 18,19 .
bHLH is a transcription factor family in fruit ripening because they can interact with environmental cues driving multiple aspects of downstream morphogenesis 20 . In high-pigment tomato mutants, such as hp1 and hp2, exposing to stronger light, anthocyanin and carotenoid will be accumulated. Meanwhile, in Arabidopsis, homologs of DDB1 and DET1 genes of hp1 and hp2 mutants showed negative regulation in light-mediated signaling pathway 21,22 . During tomato development, constitutive downregulation of PIF1 levels resulted in increased accumulation of carotenoids in the fruit because PIF1 directly repressed PSY gene expression 23 . Other results also showed that PIF1 and other PIFs promote PSY gene expression and carotenoid accumulation through responding to light signals during daily cycles of light and dark in mature Arabidopsis 24 .
Acting as transcriptional cofactors, bHLHs regulate target genes with other transcription regulators. Since bHLHs proteins are absent from an appropriate DNAbinding domain, they usually function as bHLH heterodimer 25 . PAR1 and HFR1 interact with PIFs and keep them from matching the target genes' promoters [26][27][28] . In recent decades, papaya fruit flesh has gained increased attention from breeders and consumers. Due to carotenoids content and composition, flesh color and nutritional quality have become increasingly important for fruit crop improvment 29 . CpCYC-B and CpLCY-B are crucial genes controlling flesh color and carotenoids profile in papaya. The red color of papaya flesh is from accumulating lycopene, while the yellow color is attributed to lycopene conversion to β-carotene and β-cryptoxanthin. Studying the regulatory mechanisms of CpCYC-B and CpLCY-B could lead to potential applications for improving fruit color and quality.
To unravel the molecular mechanism of papaya flesh color, we examined how bHLHs regulate CpCYC-B and CpLCY-B during papaya fruit development. To delineate the function of bHLHs on CpCYC-B and CpLCY-B, we conducted genome-wide identification of TFs targeting carotenogenesis by RNA-seq assay coupled with cis-element analysis and isolation for narrowing the numbers of TF families regulating CpCYC-B and CpLCY-B genes. More than 15 TF families were identified and 8 TFs were selected that may have positive or negative role in regulating CpCYC-B and CpLCY-B during fruit ripening. Yeast one-hybrid experiments and dual-luciferase transient expression assays demonstrated CpbHLH1 and CpbHLH2 can directly bind to the promoter upstream regions of CpCYC-B and CpLCY-B and individually inhibit or promote their transcription. Furthermore, we demonstrated that light might also involve in the regulation of CpbHLH1 and CpbHLH2 during fruits ripening.

Plant material and treatment
Red-fleshed papaya (Carica papaya L., cv. Hongling, SunUp, AU9) fruits at green, color break, and ripening stages were collected from experimental stations in Anxi and Yangzhong in Fujian, China. Two post-harvest treatments have been subjected: dark and light. During dark and light treatment, fruits were kept at 28°C for 2 days. Fruits with the same morphology were selected, such as shape, maturity, weight, and without virus defects. All experiments will be biologically replicated with three samples after being frozen with liquid nitrogen or −80°C.
RNA extraction, library construction, gene isolation, and sequence analysis Through grinding frozen papaya flesh samples, total RNA was extracted from fruits according to RNA-prep pure Plant Kit (Huayueyang) protocol. The quality and concentration of total RNAs were checked on an Agilent 2100 Bioanalyzer. After matching the qualification, mRNA samples were synthesized as cDNA and further constructed into libraries according to NEBNext Ultra RNA library Pre Kit for Illumine (NEB, E7530). The cDNA libraries were sequenced using Illumina NovaSeq with paired-end 150nt read length. By analyzing the RNAseq data, eight differentially expressed genes, named CpNAC3; CpbZIP-1/2/3; CpERF1/2; CpbHLH-1/2 were identified from the database for different papaya-ripening stages.

Quantitative real-time PCR analysis
The experiments of qRT-PCR were performed with above RNA libraries. The primers applying to qRT-PCR analysis were designed as shown in Supplementary Table  S2. The resulting qRT-PCR data were computed and analyzed using the formula 2 −ΔΔCt 30 . CpTBP1 adopted as an internal standard in papaya 31 . All experiments were implemented with three biological replications. The final values were presented with the mean of three biological replications.

Construction of vectors and plant transformation
Deletion constructs of CpCYC-B and CpLCY-B 5′ promotor sequences were amplied based on the annotated papaya genome at positions −0.5, −1.0, −1.5 kb (including −0.2/−0.3/−0.4/−0.5-absent element) and cloned into pDNOR207 vector using Gateway technology (Invitrogen). The targeting promoter fragments were then sub-cloned into pMDC162 and pGWB633 vectors using the Gateway conversion system. The Agrobacterium tumefaciensmediated transformation system was implemented to transform pMDC162 and pGWB633-deletion promoters' constructs into Arabidopsis Columbia (Col-0) ecotype.

Dual-luciferase transient expression assay
The transcription activity of CpbHLH1 and CpbHLH2 was analyzed through the double luciferase reporter gene system in tobacco (N. benthamiana). As effectors the CDS sequences of CpbHLH1 and CpbHLH2 were inserted into the pGreenII 62-SK vectors, including a CaMV35S-Rluc and internal reference REN regulated by the 35S promoters (pG62-CpbHLH1 and pG62-CpbHLH2). Meanwhile, the promoters of CpCYC-B and CpLCY-B genes were cloned into the pGreenII 0800-LUC double-reporter vectors 32 . Through co-transforming the Agrobacterium, respectively, containing effector and reporter constructs into tobacco leaves, the luciferase activities of LUC and REN were obtained using luciferase assay kit (Promega). At least eight biological replications were performed. Luminoskan Ascent Microplate Luminometer (Thermo Fisher Scientific) was used to collect data.
Yeast one-hybrid (Y1H) and yeast two-hybrid (Y2H) assays Y1H experiments were completed according to the Matchmaker Gold Y1H System protocol (Clontech). The CDS sequence of CpbHLH1 and CpbHLH2 were cloned into pGADT7 to fuse with the activation domain in the vector. The promoter fragments and short repeat promoter elements of CpCYC-B and CpLCY-B were cloned into pHIS2 vector. Co-transforming PGADT7 vectors containing transcription factors and pHIS2 vectors embodying promoters into yeast strain Y187. Yeasts were cultivated on SD basic medium without Leu and Trp for 3 days. After successfully growing on double dropout plate, the yeast colonies were picked to plate onto triple dropout minimal medium (SD/-Leu/-Trp/-His). Using positive and negative controls, the possible interactions between CpbHLH1/2 and CpCYC-B/CpLCY-B were defined by their growth status. Y2H experiment protocol was followed. Yeast cells co-transformed PGADT7-CpbHLH1 + PGBKT7-CpbHLH2 and PGADT7-CpbHLH2 + PGBKT7-CpbHLH1 were grown on triple-dropout and four-dropout minimal medium that basic medium lacks Leu, Trp, His, and Ade.

Expression pattern of carotenoid biosynthesis genes during papaya fruit development
The fruit color of papaya is determined by accumulation of different types of carotenoid compositions and content, and the expression level of the carotenoid biosynthesis genes play important roles in papaya flesh color, fruit firmness, and nutritional profile during papaya fruit development. To understand carotenoid biosynthesis genes' expression pattern during papaya fruit ripening, RNA-Seq analysis was performed to detect carotenoidrelated genes' expression level during five stages of fruit development (Supplement Fig. S2). CpCYC-B, CpCHY-B, and CpLCY-B were significantly upregulated during fruit ripening; CpZDS, CpPDS, and CpLCY-E were also upregulated from stage 3 (50% ripening stage) to stage 5 (100% ripening stage) (Fig. 1).

Identification and characterization of putative transcription factors involved in CpCYC-B and CpLCY-B regulation
To further analyze the potential transcription factors regulating carotenoid biosynthesis genes involved in papaya fruit ripening, RNA-Seq analysis was implemented using libraries constructed from papaya varieties SunUp (from S1 to S5) and AU9 (GRN, CB, and RP) fruit at different developmental stages. To calculate reads per kilo bases per million reads (RPKM), significant expressed tags in different samples were identified. The differentially expressed transcription factors in SunUp and AU9 samples were identified based on the following criteria: P < 0.01, FDR < 0.01, and fold change >2. Expression profiles of different TFs were classified as bHLH, GRAS, SBP, and bZIP between the first stage and the final stage in SunUp and AU9 cultivars. In total, 23 and 27 different expressed TFs were identified in SunUp and AU9, respectively. Most TFs were downregulated in two cultivars. Of the 23 TFs in Sunup, 17 TFs, including bHLHs, C2H2s, and GRFs, were downregulated, and 5 TFs, including bZIP and B3, were upregulated. Of the 27 TFs in AU9, 21 TFs, including BES1, WRKY, and TCP, were downregulated, and 6 TFs, including bZIP and AP2, were upregulated (Fig. 2). Some of transcription factors were selected for further analysis.
To test whether these transcription factors can bind promoters of CpCYC-B and CpLCY-B during papaya fruit development, we analyzed the promoter sequences in silico and constructed a series of chimeric genes containing truncated fragments of CpCYC-B and CpLCY-B promoters and GUS reporter gene. Promoters were analyzed for potential TFs binding sites using the PlantCare database. The potential regulatory elements in upstream −1.5 kb promoters of CpCYC-B and CpLCY-B have been summarized in Table 1. Most of them are responsive to hormones and light. CGTCA-motif and ERF, bHLH boxs were the consensus cis-elements of CpCYC-B and Fig. 1 qRT-PCR expression patterns of six carotenoid genes were shown during papaya fruit ripening. S1, 10% ripening; S2, 30% ripening; S3, 50% ripening; S4, 70% ripening; S5, 100% ripening. Value presented as mean + SE of three replicates CpLCY-B promoters. For example, bHLH binding site (-CANNTG-), was found at −113 and −164, respectively, in CpCYC-B and CpLCY-B promoters. The distribution of different regulatory elements in different region of promoters implied that some special elements played special function at different developmental stages.
Reporter gene GUS expression was analyzed to check the activity of these cis-elements in transgenic  Fig. 3c-h). The GUS activity with −0.5 kb region of CpCYC-B and CpLCY-B promoter was, respectively, stronger and weaker than another two types of transgenic plants with −1.0 and −1.5 promoters. These results were identical to the relative expression results of different length promoters (Fig. 3a, b), implying that −0.5 kb promoters of CpCYC-B and CpLCY-B were corresponding to chromoplast-and chloroplast-specific expression patterns, respectively. We further deleted the −0.5 kb promoters of CpCYC-B and CpLCY-B into −0.2, −0.3, and −0.4 kb fragments. We discovered that Pro-CpCYC-B::GUS had a relatively higher expression level with −0.2 kb promoter than other forms of transgenic Arabidopsis. However, Pro-CpLCY-B:: GUS showed similar relative expression levels driven by different promoters fragments (Supplement Fig. S3). In addition, we mutated two elements, -GAAAGAA-(311 bp) and -ATTTCAAA-(ERF-responsive element) in −0.5 kb of CpLCY-B and CpCYC-B promoters, respectively, and the results indicated that −0.5 kb of Pro-CpLCY-B::GUS without -GAAAGAA-element exhibited a relatively higher expression level compared with those containing this element, suggesting -GAAAGAA-element might act as a suppressor in CpLCY-B promoter. Without ERF element in −0.5 kb Pro-CpCYC-B::GUS T1 transgenic plants, there was no obvious expression difference with other transgenic promoter fragment in Arabidopsis (Supplement Fig. S3A and B). However, when we treated the transgenic Arabidopsis containing −1.0 kb CpCYC-B and −1.0 kb CpLCY-B promoter with 80 mg/L ethephon, GUS activity appeared to be weaker (Supplement Fig.  S3C-H). This result implied that ERF element might act as a negative cis-element.
By in silico analysis and experimental validation, we identified that −0.5 kb promoter of CpCYC-B and CpLCY-B played important regulatory roles and included some fruit-specific elements, e.g., ERF-responsive element, bHLH-box, in addition to the novel negative regulatory element (-GAAAGAA-) in CpLCY-B promoter.
Combining the result of the expression of TF family members during different fruit ripening stages and TF family motif in −0.5 kb promoters of CpCYC-B and CpLCY-B, we obtained four types of transcription factor families as shown in Fig. 4a. According to their distribution in promoters of CpCYC-B and CpLCY-B, all of their binding sites existed in −0.5 kb CpCYC-B promoter, while, bHLH and ERF binding sites existed in −0.5 kb CpLCY-B promoter. The characterizations of four TF families were shown in Table 2. Eight TFs belonging to four types of TF families were selected from database (Fig. 2) .supercontig_244.3). The transcription of the eight TFs was validated by RNA-Seq and qRT-PCR. The expression levels of CpbHLH1 and CpERF1/2 were decreased from the initial stage. As shown in Fig. 4b, the expression level of CpNAC3 had been decreased from stage 2; the expression patterns of CpbZIP1/2/3 were random, and for example, the expression level of CpbZIP1 first increased, then decreased and finally increased again. For CpbHLH2, expression level first increased at color break stage, decreased from stage 2, and then increased significantly from stage 4. These results were further demonstrated by RNA-Seq data (Supplement Fig. S4).
To investigate the effects of CpbHLH1/2 on CpCYC-B and CpLCY-B expression, dual-luciferase transient expression assays were performed. The LUC/REN ratio was lower than the positive control and negative control (CpbHLH1/Empty) when either the CpCYC-B or CpLCY-B pro-LUC reporter construct was co-transfected with the CaMV35S-CpbHLH1 effectors, implying that CpbHLH1 repressed CpCYC-B and CpLCY-B expression activities (Fig. 6c, d). In contrast, the LUC/REN ration of CpbHLH2-CpCYC-B/CpLCY-B was higher than the negative control (CpbHLH1/Empty) when previous promoters-LUC reporter constructs was co-transfected with the CaMV35S-CpbHLH2 effectors, indicating that CpbHLH2 may involve the activated regulation of CpCYC-B and CpLCY-B (Fig. 6e, f). Besides, LUC/REN ratios of four types of experimental groups were found to   To investigate if CpbHLH1/2 could regulate gene expression on carotenoid biosynthesis pathway (including CpCYC-B and CpLCY-B) in response to light, we collected SunUp fruit from S1 to S3 stage (green, color break, and ripe). Under 28°C, papaya fruits of each stage were divided into two parts, one part was exposed to strong white light for 2 days, another part was kept in dark. After 2 days of treatment, expression levels of CpbHLH1/2, CpCYC-B, and CpLCY-B were examined by qRT-PCR. In Fig. 7, the expression levels of CpCYC-B and CpLCY-B in light were higher than in dark. In contrast, the expression levels of CpbHLH1/2 were much lower in light than in dark.
Especially, at stages S1 and S2 when the carotenoids have not been accumulated, the expression levels of CpCYC-B and CpbHLH2 were twice in light and in dark, respectively. At ripe stage S3, there was no obvious expression difference for CpbHLH1/2, CpCYC-B, and CpLCY-B. In conclusion, CpbHLH1/2 may be among the factors regulating CpCYC-B and CpLCY-B in response to light.

Discussion
Carotenoids are very important pigments for plant growth and development, contributing to fruit flesh color and nutritional properties, and protecting the photosynthetic apparatus. In fruit, carotenoid accumulation could be promoted by light, but also could be harmed by The way their production is regulated by light is common 21,[33][34][35] . Lycopene conversion into carotenoids in ripening papaya flesh was regulated by CpbHLH1/2 based on a molecular mechanism that is similar in Arabidopsis leaves or other plants in response to light 24 . In red grapefruit, lycopene content in shaded fruits is 49-fold than fruit exposing to light, implying shading-promoted carotenoid accumulation 36 .
During papaya fruits ripening, transcription factors play a significant role in carotenoid biosynthesis and enhance carotenoid accumulation (Fig. 1). In order to identify TFs related to papaya carotenoids biosynthesis, we analyzed RNA-Seq data of different papaya fruit in SunUp and AU9, especially the period during color break stage when the content of carotenoids changes sharply. More than 40 TF genes were identified that are positively or negatively regulated during papaya fruit development (Fig. 2). These TF genes belong to NAC, bHLH, ERF, bZIP, Dof, MYB, SBP, and C2H2. To find the possible TF families binding to the CpCYC-B and CpLCY-B promoters, these promoters were identified and isolated. By analysis of promoter fragments, −0.5 kb promoter sequences of CpCYC-B and CpLCY-B were considered as the effective region in response to fruit ripening (Fig. 3). Binding sites of NAC, bHLH, bZIP, and ERF were found in −0.5 kb promoter of CpCYC-B, while binding sites of bHLH and bZIP were found in −0.5 kb in CpLCY-B (Table 2). In previous studies, all of these TF families are involved in light response. In Arabidopsis, responding to light, bZIP16 both negatively inhibit the cell elongation and positively promote seed germination 37 . In tomato transgenic plants containing the main ORFs of SlbZIP1 and SlbZIP2, the sugar content (sucrose/glucose/fructose) was 1.5-fold higher than non-transgenic fruits because SlbZIP transactivates the asparagine synthase and proline dehydrogenase genes 38 . In Citrullus colocynthis, higher transcripts of CcNAC1 and CcNAC2 following red light imply that NAC genes might function in light signaling pathway 39 . Besides, CpNAC1 was found to play a positive regulatory role in carotenoid accumulation through boosting CpPDS2/4 transcription by direct binding to their promoters 34 . When transferring Arabidopsis thaliana triose phosphate/phosphate translocator (tpt) mutants from low light to high light, transcripts expression level of four AP2/ERF genes, ERF6, RRTF1, ERF104, and ERF105 will absolutely and quickly decreased within 10 min, implying that AP2/ERF-TFs were responsive to strong light 40 . Through Y1H experiments, CpbHLH1/2 could bind to CpCYC-B and CpLCY-B promoters (Fig. 5). This suggested that CpbHLH1/2 are required to regulate the expression of CpCYC-B and CpLCY-B.
In papaya, the transcriptional regulation mechanism of carotenogenesis mediated by bHLHs is unclear. In current research, three models of transcriptional regulation by bHLHs were proposed. First, in dark or etiolated seedlings, responding to shade or night, PIFs mainly act as constitutive transcriptional activators. Under diurnal conditions, reversely, light will weaken or remove the Fig. 7 Under the treatment of 2-day dark and white light, the qRT-PCR expressions of four genes were examined from stages 1 to 3. The abscissa represents different developmental stages of papaya fruits, and the ordinate means qRT-PCR expression level. Stage 1: green stage; stage 2: color break; stage 3: ripe activation of PIFs. A considerable portion of PIFassociated genes are upregulated in pif mutants, particularly during etiolating process, implying that PIFs repress the expression of those genes 41,42 . Second, during deetiolation, PIFs also repress the constitutive transcription of a comparatively small subset of light-induced genes, which indicated that PIFs might have two function depending on the promoter sequences and environment cues. For example, PIF1 repressed the expression of PSY and CBF2 in dark or diurnal conditions 24,43,44 , and PIF1 and PIF3 also could inactivate the genes associated with chlorophyll biosynthesis and photosynthesis during deetiolation 45,46 . In the third model, PIFs constitutively coactivate momently light-induced genes such as ELIP2 during de-etiolation, suggesting other undiscovered transcriptional co-activator may participate in this process. In the dark, PIFs do not repress early light-induced genes like ELIP1 and ELIP2, but PIFs are required for their rapid light induction 47,48 . Acting as a co-activator, PIFs respond to light positively and lead to decrease transient expression of ELIP2. This model may also conduct for a broader spectrum of genes, perhaps some light-responsive genes, SIGE and ELIP genes, which are not regulated by PIFs in dark or light. In our results, the regulatory mechanism of CpbHLH-1/2 was similar to previous models. From results in Fig. 7, we peopose two bHLH mediated-models: (1) light activate Pr phytochrome into Pfr, resulting in the degradation of CpbHLH1, thus there was no active CpbHLH1 to inhibit the expression of CpCYC-B and CpLCY-B during strong white light as in the first model; (2) CpbHLH2 might co-activate CpCYC-B and CpLCY-B expression with other activators. Protein-protein interaction assays suggested that CpbHLH1 could not physically interact with CpbHLH2, that they could not co-operatively regulate CpCYC-B and CpLCY-B (Supplement Fig. S7).
In previous studies, PSY was reported to be the only one gene that was controlled by PIF transcription factors involving light-mediated regulation in carotenoid pathway, because it is considered as the main-rate limiting enzyme of carotenoid pathway 23 . In current study, bHLH family members, CpbHLH1 and CpbHLH2 bind promoter regions of CpCYC-B and CpLCY-B and regulate their expression during papaya fruit development. Correlating with the decreased expression of CpbHLH1/2, expression level of the CpCYC-B and CpLCY-B was elevated during fruit ripening. We analyzed transcriptional regulation network and identified some potential family members regulating carotenoid biosynthesis genes in response to light or other factors during papaya fruit development in a genome scale. Our results first reported bHLH family members involved in the regulated network of carotenoid biosynthesis-related genes in papaya, which revealed two novel TFs of bHLHs and their regulation mechanism related to carotenoid biosynthesis genes in response to light. These findings contribute to uncover regulatory cascades, which could be directly or indirectly regulate fruit ripening in papaya responding to light.