Isolation and Activity Analysis of Phytoene Synthase (ClPsy1) Gene Promoter of Canary-Yellow and Golden Flesh-Color Watermelon

Abstract

Watermelon (Citrullus lanatus) is an economically important cucurbit crop. Its pulp is rich in antioxidant carotenoids, which confer a variety of flesh colors. ClPsy1 (Phytoene Synthase) is the rate-limiting enzyme for carotenoid synthesis; however, the promoter activity of ClPsy1 is still unknown. In the present study, promoter sequences were isolated from four watermelon accessions: Cream of Saskatchewan pale yellow (COS), canary yellow flesh (PI 635597), golden flesh (PI 192938), and red flesh (LSW-177), all of which express ClPsy1 at extremely high levels. Sequence alignment and cis-element analysis disclosed six SNPs between the four lines all in COS, two of which (at the 598th and 1257th positions) caused MYC and MYB cis-element binding sequence variations, respectively. To confirm ClPsy1 gene promoter activity, full-length and deletion fragments of the promoter were constructed and connected to a β-D-glucosidase (GUS) vector and transferred into tomato fruits. GUS staining was performed to analyze the key segment of the promoter. The activity of the PI 192938 ClPsy1 full-length promoter exceeded that of COS. The deletion fragment from −1521 bp to −1043 bp exhibited strong promoter activity, and contained a MYB transcription factor-binding site mutation. We combined RNA-seq with qRT-PCR to analyze the gene expression pattern between the MYB transcription factor Cla97C10G196920 and ClPsy1 gene and found that Cla97C10G196920 (ClMYB21) showed the same expression trend with ClPsy1, which positively regulates carotenoid synthesis and metabolism.

1. Introduction

Carotenoids are natural pigments that exist widely in nature [1]; most are C40 terpenoids and their derivatives. They are usually orange, red, and yellow pigments produced by isoprenoid synthesis pathways in pigmented and photosynthetic tissues such as endosperm, leaves, roots, flowers, and the fruit flesh of plants [2]. Carotenoids are precursors of vitamin A [3], phytohormones [4,5], and various aromatic compounds [6], and are also indispensable micronutrients for human health. They facilitate anti-oxidation, immune regulation, anti-aging, and other processes essential to well-being. Carotenoids can reduce the risk of cancer and also improve the nutritional quality of horticultural crops [7,8].

Reference genomes were constructed by whole-genome sequencing of wild and cultivated watermelon varieties. Genetic loci associated with important agronomic traits were determined further through quantitative genetic analysis. Genome, transcriptome, metabolome, and other multi-omics technologies were integrated to mine key genes [9]. PSY is a key gene upstream of the carotenoid metabolic pathway. Three to five PSY family members are present in most plant genomes except that of Arabidopsis, which has only one. The PSY transcript consumes two geranylgeranyl pyrophosphate molecules in the methylerythritol 4-phosphate pathway to form the first carotenoid, phytoene, a substrate of downstream carotenoid synthesis [10]. PSY family members exhibit varying expression patterns in different plants. For example, in banana, MaPSY1 is expressed primarily in the peel and pulp, and at varying levels according to growth stage [11]. In addition, SlPSY1 promotes carotenoid accumulation in tomato fruits, while SlPSY2 acts primarily on green tissues and SlPSY3 acts on roots [12]. Similarly, in maize, ZmPSY1 plays a dominant role in endosperm carotenoid accumulation, whereas ZmPSY2 is related to photosynthesis and undergoes differential expression under photoinduction [13].

The carotenoid metabolic pathway is complex, and features the expression of multiple genes and regulation by multiple factors including growth, development, and environmental conditions [2,14]. Although some genes involved in carotenoid synthesis and degradation have been analyzed, the regulation of these processes is still poorly understood. The importance of transcription factors in regulating gene expression is well-known. Multiple transcription factors interact directly with carotenoid gene promoters and regulate their expression. For example, ethylene response factors and RAP2.2 can activate PSY to increase carotenoid accumulation in Arabidopsis leaves [15]. SlBBX20 can bind directly to the G-box on the promoter and activate PSY1 expression, thus raising carotenoid levels in SlBBX20 overexpression lines [16]. Tomato SlMYB72 binds directly to phytoene synthase, ζ-carotene isomerase and lycopene β-cyclase genes to regulate carotenoid biosynthesis [17]. The light-sensitive bHLH transcription factor PIF1 downregulates the Arabidopsis PSY gene by binding directly to a G-box acting element in the PSY promoter [18]. Chromatin immunoprecipitation analysis showed that tomato PIF1a binds specifically to the PBE box of the PSY1 promoter, inhibits its expression, and ultimately reduces carotenoid biosynthesis [19]. MADS-box transcription factors such as TAGL1 [20], RIN [21] and FUL11 [22] synthesize and accumulate carotenoids during tomato fruit ripening by directly or indirectly activating SlPSY1. Other MADS-box proteins such as SlMADS1 [23] and SlFYEL [24] inhibit SlPSY1, while SlCMB1 exhibits a positive regulatory role during fruit ripening [25]. In citrus fruits, the transcript of CsMADS6 can bind to the promoters of CsPSY and CsPDS to increase their expression levels [26]. The citrus CsLCYb1 promoter has numerous hormone-responsive elements, such as a gibberellin responsive element GARE motif, a salicylic acid responsive element TCA motif, and an auxin responsive element TGA motif, to enable hormone-induced high-level gene expression [27]. The PDS promoter initiates high GUS expression in chromophore-forming organs and gene transcription in green tissues to synthesize final products [28]. Inhibition of SlNAC4 will lead to the decrease of PSY1 expression, and the increase of LCYB, LCYE, and CYCB expression, resulting in the decrease of total carotenoid synthesis and producing orange fruit [29].

In this study, we isolated the ClPsy1 promoter sequences of four different watermelon lines: Cream of Saskatchewan pale-yellow flesh (COS), canary-yellow flesh (PI 635597), golden flesh (PI 192938), and red flesh (LSW-177). We then performed a comparative analysis of their cis-elements, constructed plant expression vectors, and completed a transient-transformed expression analysis. We found a MYB transcription factor (TF) Cla97C10G196920, and analyzed its gene expression pattern in different watermelons. Our findings may provide a foundation for further clarification of the role of the ClPsy1 gene in carotenoid metabolism and its regulatory mechanism in PI 192938 and COS.

2. Materials and Methods

2.1. Plant Materials

Pale yellow-flesh COS, canary yellow-flesh PI 635597, golden-flesh PI 192938, and red-flesh LSW-177 seeds were preserved and provided by the Melon Molecular Breeding Laboratory, Northeast Agricultural University, and were grown at the Xiang Yang Farm of Northeast Agricultural University, Harbin, China. Tomato (Lycopersicon esculentum cv Ailsa Craig) seeds were provided by the Tomato Molecular Breeding Laboratory of Northeast Agricultural University and grown in the greenhouse at the Horticulture Station of Northeast Agricultural University.

2.2. Promoter Isolation and Sequence Analysis

Ten days after planting, young leaves were sampled and frozen for DNA extraction using the improved hexadecyl trimethyl ammonium bromide method. According to the genome resequencing results, specific primers were designed to isolate the full promoter sequences from the ATG start codon in four different accessions (Supplementary Table S1). PCR reactions were performed in our previous study [30]. Amplified PCR products were constructed into the pMD18-T vector (Takara, Dalian, China), and then transformed into Escherichia coli DH5α for sequencing. Multiple sequence alignments were performed by using the DNAMAN program. A cis-element analysis of promoter sequences was performed by PlantCARE (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/, accessed on 12 October 2022).

2.3. Promoter Deletion Fragments’ Vector Construction

PCR amplification of the full promoter fragment of ClPsy1 and three deletion fragments (gradually truncated every 500 bp from the promoter) was performed, and primers with homology arms were designed (Supplementary Table S1). The amplified fragment was inserted into the corresponding site upstream of the GUS reporter gene of pCAMBIA3301 (replacing the CaMV35S promoter) and digested with BamHI and NcoI. The eight vectors were designated as P_PI 192938_1987, P_PI 192938_1521, P_PI 192938_1043, P_PI 192938_550, P_COS_1987, P_COS_1521, P_COS_1043, and P_COS_550. The CaMV35S:GUS fusion in pCAMBIA3301 was used as a positive expression control designated as 35S. Untransformed Agrobacterium GV3101 was used as a negative control designated as -CK. After sequence verification, the vectors were transformed into Agrobacterium tumefaciens strain GV3101 by the freeze-thaw method, after which the complete constructs were transformed into tomato fruits to test promoter activity.

2.4. Transient Transformation of Ripe Green Tomatoes

Transient transformation of tomato fruit was performed using Orzaez’s method [31], with minor modifications. A. tumefaciens strain GV3101 monoclonal isolates containing the target vector were isolated and cultured in 500 μL of LB (Luria–Bertani) liquid medium supplemented with kanamycin (50 mg/L) and rifampicin (20 mg/L) at 28 °C, 150 r/min for 24 h. All culture medium was then transferred to a 50 mL induction medium (LB medium containing 200 mM acetosyringone and 10 mM MES (2-morpholinoethanesulfonic acid), pH = 5.6, supplemented with appropriate antibiotics) for further culture. The next day, the liquid culture was centrifuged and re-suspended in the infection medium (10 mM MgCl₂, 10 mM MES, 200 mM acetosyringone, pH = 5.6) to adjust OD₆₀₀ to about 1.0. The liquid culture was then mixed gently at room temperature away from light (20 r/min) for about 2 h.

Ripe green tomatoes with intact stems were harvested for subsequent transient transformation. A 1 mL aliquot of liquid culture was extracted by syringe and injected into the tomato fruit through the pedicle. After cultivation in the dark for 3 days, a cross-section of the fruit was used for GUS (β-D-glucosidase) histochemical staining. All samples were frozen in liquid nitrogen for subsequent GUS quantification. Three biological replicates were performed for each group (P_PI 192938_550, P_PI 192938_1043, P_PI 192938_1521, P_PI 192938_1987, P_COS_550, P_COS_1043, P_COS_1521, P_COS_1987, and 35S, respectively).

2.5. β-D-Glucuronidase Assays

GUS staining solution was prepared in a quantity that covered the materials completely, and was incubated at 37 °C for 24 h in the dark until blue gradually appeared. After complete staining, chlorophyll was removed with 75% ethanol for 12–24 h until the negative control was completely decolorized. Specimens of approximately 1 g from each group were placed in liquid nitrogen to freeze and extract the protein sample solution. Protein concentration was determined by the Bradford method (Coolaber, Beijing, China) and standard curves were drawn. GUS activity was determined by 4-MUG fluorescence under 455 nm emission light and 365 nm excitation light by using a Multiskan FC microplate reader (Thermo Fisher Scientific Inc., Waltham, MA, USA)

2.6. Gene-Expression Pattern Analysis

Flesh samples of four different accessions were collected from fruit centers at 10, 18, 26, 34 and 42 days after pollination (DAP). Three fruits grown under similar conditions were collected per sample. Flesh tissues were immediately frozen in liquid nitrogen and stored at −80 °C for RNA extraction. A Plant Total RNA Isolation Kit (Sangon, Shanghai, China) and ReverTra Ace qPCR RT Kit (TOYOBO, Osaka, Japan) were used for total RNA extraction and cDNA synthesis, respectively. Specific primers for MYB21 TF Cla97C10G196920 were designed for gene expression analysis through quantitative real-time polymerase chain reaction (qRT-PCR) (Supplementary Table S1). An AceQ qPCR SYBR Green Master Mix (Vazyme, Nanjing, China) was used to perform qRT-PCRs in the QTOWER Real-Time PCR System (Analytik Jena, Jena, Germany) according to the manufacturer’s instructions. qRT-PCR amplification and mixing were performed as previously described [30]. Each experiment was performed with three biological repetitions and three technical repetitions. Relative gene expression levels were determined using the 2^−∆∆Ct method [32].

2.7. Statistical Analysis

The data of GUS enzymatic activity and qRT-PCR were expressed as the mean ±SD of three independent replicates. One-way analysis of variance (ANOVA) was performed in Microsoft Excel (Microsoft Office 2021, Microsoft, Albuquerque, NM, USA) to analyze the significance of data differences. A p-value < 0.05 indicated a significant difference.

3. Results

3.1. ClPsy1 Promoter Sequence and cis-Acting Element Analysis

The ClPsy1 promoter fragment located upstream of the ATG start codon was obtained by PCR, and preliminarily identified as the full-length ClPsy1 promoter. Cis-acting element analysis showed that the ClPsy1 promoter contained many enhancers such as TATA-box, CAAT-box, and G-box, as well as other cis-acting elements listed in (Supplementary Table S2). We compared the promoter sequences of the four accessions. Our findings were consistent with those of Liu et al. (2022) [30], in that only COS had a total of six SNP mutations (Figure 1). Further analysis of COS resequencing data showed that the SNPs at the 598th and 1257th positions were two unique mutations [30]. We subsequently analyzed the cis-acting elements of four different accessions by PlantCARE. In canary-yellow-flesh COS, the SNPs at the 598th and 1257th positions featured T→C and T→C substitutions, resulting in mutant MYC and MYB transcription factor binding sites, respectively. We also found two other mutations, one at 6th position in PI 192938 that featured T→C, and the other at the 1424th position in PI 635597 that featured T→C. Meanwhile, our analysis of cis-acting elements revealed that the two distinct mutations did not cause changes in transcription factor binding sites.

3.2. Analysis of ClPsy1 Promoter Activity in Transformed Tomato

The activity of the ClPsy1 promoter was evaluated by transient transformed expression in tomato fruits. The promoter GUS vector is shown in Figure 2. GUS staining was deepest on the transverse section injected with the original 35S promoter (designated as 35S), while the section injected with A. tumefaciens without the vector did not produce a blue stain (designated as -CK). Although both the ventricles and seeds of the transformed tomatoes appeared blue, the color intensities varied (Figure 3a). Glucuronidase enzymatic activities in the transformed tomatoes are shown in Figure 3b.

Horizontal comparison revealed that P_PI 192938_1987 had deeper staining than P_PI 192938_1521, and that P_PI 192938_1521 was more deeply stained than P_PI 192938_1043. The staining of P_PI 192938_1043 and P_PI 192938_550 was equivalent. The staining results were consistent in COS, namely, the staining of P_COS_1987 was deeper than that of P_COS_1521, the staining of P_COS_1521 was deeper than that of P_COS_1043, and that of P_COS_1043 was similar to that of P_COS_550 (Figure 3a). The quantitative determination of GUS enzymatic activity was consistent with all of the staining results (Figure 3b). The GUS activity of P_PI 192938_1987 was higher than P_PI 192938_1521, while that of P_PI 192938_1521 exceeded that of P_PI 192938_1043, and that of P_PI 192938_550 approximated that of P_PI 192938_1043. The trend of decreasing GUS activity was the same as in COS, namely, P_COS_1987 exhibited higher GUS activity than P_COS_1521, P_COS_1521 GUS activity was greater than that of P_COS_1043, and P_COS_550 GUS activity approached P_COS_1043. GUS enzymatic activity was higher in P_COS_1987 than in P_COS_1521, greater in P_COS_1521 than in P_COS_1043, and similar between P_COS_550 and P_COS_1043. The staining result and GUS enzyme activity of different promoter fragment lengths in the same line showed that the promoter sequence could drive GUS gene expression in tomato fruit Lycopersicon esculentum cv Ailsa Craig, demonstrating that promoter sequences with different lengths had promoter functions, but their promoter activities were different.

Longitudinal comparison among different strains showed that the staining of P_PI 192938_1987 was deeper than that of P_COS_1987, that of P_PI 192938_1521 was deeper than P_COS_1521, that of P_PI 192938_1043 was similar to P_COS_1043, and that of P_PI 192938_550 was similar to that of P_COS_550 (Figure 3a). GUS enzyme activity was higher in P_PI 192938_1987 than in P_COS_1987, greater in P_PI 192938_1521 than in P_COS_1521, and similar between P_PI 192938_1043 and P_COS_1043 and between P_PI 192938_550 and P_COS_550 (Figure 3b). The results of GUS staining with the same promoter length in different lines showed that the activity of the ClPsy1 gene promoter was stronger in PI 192938 than in COS. Promoter activity decreased with fragment deletion; the main range was from −1521 bp to −1043 bp. Combined with the mutation of the cis-acting element of the promoter, it can be further speculated that the MYB transcription factor may be a key regulator of ClPsy1 gene expression.

3.3. MYB TF Cla97C10G196920 Positively Regulates Carotenoid Metabolism in Watermelon Pulp

In our previous studies, we speculated that two MYB transcription factors and one MYC2 might influence ClPsy1 expression. Combined with published RNA-seq, it was found that Cla97C10G196920 was highly expressed and specifically expressed in red fruit flesh [30]. We verified the MYB TF Cla97C10G196920 expression pattern in four different accessions. The conserved domains of Cla97C10G196920 (148 aa) were extracted and compared to the Arabidopsis Information Resource: AtMYB21 was the homolog. We obtained the qRT-PCR data of Cla97C10G196920 in four different watermelon accessions during five DAPs (Figure 4). The total carotenoid content accumulated continuously and Cla97C10G196920 gene expression increased with ripening. Gene expression of Cla97C10G196920 in pale-yellow-flesh COS was lower than in canary yellow flesh PI 635597, while that of PI 635597 was lower than in golden flesh PI 192938. LSW-177 was a red flesh-colored accession with the same promoter region as PI 192938. It was found that Cla97C10G196920 expression level in red flesh LSW-177 was almost the same as that in golden flesh PI 192938 at the ripening stage. The gene expression level of LSW-177 was slightly higher than in PI 192938, due to the different degree of carotenoid accumulation in the early stage of fruit development. Cla97C10G196920 positively regulated carotenoid metabolism in watermelon flesh with fruit ripening. We speculated that ClMYB21 TF Cla97C10G196920 was positively correlated with carotenoid accumulation, and subsequently performed experiments to verify the interaction of ClMYB21 and the ClPsy1 gene and the function of the key transcription factor.

4. Discussion

Members of the PSY gene family have been discovered in cucurbit crops such as watermelon, papaya, and muskmelon, as well as tomato and other fruit crops. Watermelon pulp accumulates carotenoids, giving rise to a variety of flesh colors. The composition, content, and related genes of carotenoids in four different watermelon pulps has been reported in our previous studies [33,34]. PI 192938 (golden flesh) and COS (canary-yellow flesh) differ significantly in β-carotene and lutein content [33]. In previous works, we chose a population of red-flesh and pale-yellow-flesh watermelons to find that ClLCYB was the key gene to regulate red pulp [35,36,37]. Liu et al. investigated inbred lines of pale-yellow-flesh and canary-yellow-flesh watermelons to verify that ClCyf might encode the canary-yellow flesh-color and carotenoid accumulation levels [38]. Branham et al. used a population of red-flesh and orange-flesh watermelons. They determined that the orange color was encoded by a recessive gene, and found a 2.4Mb region linked on chromosome 1 that included ClPsy1 [39]. Through forward genetics, ClPsy1 was identified as the main gene for controlling the golden flesh color of PI 192938 [30]. However, little is known about how the ClPsy1 gene contributes to the carotene metabolic pathway in watermelons.

The coding and promoter-region sequences of 26 watermelon subspecies were analyzed by Liu et al. [30]. In the current study, upstream −1987 bp promoters of four watermelon lines were isolated and analyzed by sequence alignment and cis-element analysis. Compared with the other three accessions, COS had special mutation sites and changed the cis-acting elements. A total of six SNP mutations, and only two at cis-acting element sites, were found in COS. SNPs at the 598th and 1257th positions caused mutations in the MYC and MYB transcription factor binding sites, respectively. The qRT-PCR results, combined with the findings of Liu et al. [30], suggest that the two SNPs may explain the reduced ClPsy1 expression in COS compared to PI 192938. We hypothesize that the two TFs’ binding-site mutations may have caused the low expression of ClPsy1, and will conduct subsequent analysis to verify the promoter activity.

To verify the activity of the ClPsy1 promoter in the two lines and its regulatory effect, we used transient transformation of tomato fruit to detect GUS activity. GUS immunohistochemical staining of tomato cross sections has been widely used because of its accuracy and convenience [40,41]. For example, the activity of the MaDREB promoter in regulating banana ripening was verified by GUS staining of tomato fruit [42]. GUS staining of tomato fruit and Arabidopsis thaliana revealed the complex regulatory mechanism of the LCYb1 promoter in citrus [27]. Transient transformation of tomato fruit is a simple, clear, intuitive, and convenient method to analyze promoter activity [27,41]. Therefore, the selection of this technique for operation is reasonable. Our association of varied GUS enzyme activities with vectors encoding different promoter deletion fragments, and our finding of increased ClPsy1 promoter activity in PI 192938 compared to COS, combined with the SNP-associated mutant binding sites of MYC and MYB transcription factors, suggest that the two mutations may explain the decreased ClPsy1 expression. Based on our association of variable GUS activities to different deletion fragments from both PI 192938 and COS, we hypothesize that the key functional segment of the ClPsy1 promoter ranged from −1521 bp to −1043 bp. The mutation binding sites of MYC and MYB transcription factors suggest that MYB is more likely to be an important transcription factor affecting ClPsy1 promoter activity.

Plant promoters play a crucial role in the regulation of gene expression. Cis-acting elements serve as the building blocks of the overall regulatory network of plants, and control the degree of gene expression. Genes that encode MYB transcription factors constitute the largest and most important gene family in plants, and contribute to development, metabolism, defense, differentiation, and stress response [43]. The canonical classification and nomenclature of this gene family was first established in A. thaliana, via 25 groups based on the MYB domain and amino acid motifs in C-terminal regions [44]. MYB transcription factors are distributed widely in higher plants, and are among the largest transcription factors. They are characterized by a highly conserved N-terminal domain. This transcription factor participates in many physiological and biochemical processes and has been widely studied [45]. The first plant R2R3-MYB gene, COLORED1 (C1), identified in Zea mays, is essential for anthocyanin biosynthesis in aleurone tissues [46]. MYB transcription factors regulate the synthesis of biomolecules such as anthocyanins and flavonoids in plants, which in turn control changes in pigmentation and aromatic substances. Examples include the accumulation of anthocyanin in the peel of the Hongyang kiwifruit [47] and the deposition of anthocyanin in tomato fruit [48]. Transient transformation of kiwifruit AdMYB7 overexpressed in tobacco showed that it could activate the LCY-β gene of kiwifruit and regulate the carotenoid metabolic pathway [49]. Overexpression of PaMYB10 enhanced red coloration of apricot skin during ripening [50]. FcMYB21 and FcMYB123 have important regulatory roles in Ficus carica peel pigmentation [51]. Based on the knowledge of the critical function of MYB TFs, we used RNA-seq to predict a MYB TF Cla97C10G196920, which regulated carotenoid metabolism. We found that Cla97C10G196920 was homologous to AtMYB21, and verified gene expression patterns in four different accessions. qRT-PCR showed that Cla97C10G196920 positively regulated carotenoid synthesis and accumulation with fruit ripening. Cla97C10G196920 expression was lowest in COS and the highest in LSW-177, which had the same promoter sequence as PI 192938. We combined the published RNA-seq (BioProject number SRP012849) found that Cla97C10G196920 expression in fruit flesh was specific, and speculated that it may be a key transcription factor to regulate carotenoid mainly in watermelon fruit. We hypothesized that ClMYB21 may be a key transcription factor regulating carotenoid synthesis in watermelon, and conducted follow-up experiments to verify its role.

5. Conclusions

Compared with four different accession promoter sequences, only canary-yellow-flesh COS had six SNPs, of which two changed MYC and MYB transcription factors’ binding sites. We confirmed the activity of the ClPsy1 promoter by using transient transformation of tomato fruit, which provided a quick and simple method for rapid gene function verification. The analysis of PI 192938 and COS promoter activities and cis-elements suggested that MYB may be a key regulator of ClPsy1 gene expression. We demonstrated MYB TF (ClMYB21) Cla97C10G196920 gene expression which exhibited expression trends similar as ClPsy1, and predicted that it may be positively involved in carotenoid synthesis.