G-site residue S67 is involved in the fungicide-degrading activity of a tau class glutathione S-transferase from Carica papaya.

Thiram is a toxic fungicide extensively used for the management of pathogens in fruits. Although it is known that thiram degrades in plant tissues, the key enzymes involved in this process remain unexplored. In this study, we report that a tau class glutathione S-transferase (GST) from Carica papaya can degrade thiram. This enzyme was easily obtained by heterologous expression in Escherichia coli, showed low promiscuity toward other thiuram disulfides, and catalyzed thiram degradation under physiological reaction conditions. Site-directed mutagenesis indicated that G-site residue S67 shows a key influence for the enzymatic activity toward thiram, while mutation of residue S13, which reduced the GSH oxidase activity, did not significantly affect the thiram-degrading activity. The formation of dimethyl dithiocarbamate, which was subsequently converted into carbon disulfide, and dimethyl dithiocarbamoylsulfenic acid as the thiram degradation products suggested that thiram undergoes an alkaline hydrolysis that involves the rupture of the disulfide bond. Application of the GST selective inhibitor 4-chloro-7-nitro-2,1,3-benzoxadiazole reduced papaya peel thiram-degrading activity by 95%, indicating that this is the main degradation route of thiram in papaya. GST from Carica papaya also catalyzed the degradation of the fungicides chlorothalonil and thiabendazole, with residue S67 showing again a key influence for the enzymatic activity. These results fill an important knowledge gap in understanding the catalytic promiscuity of plant GSTs and reveal new insights into the fate and degradation products of thiram in fruits.

Systems Biology Applied to the Study of Papaya Fruit Ripening: The Influence of Ethylene on Pulp Softening.

Papaya is a fleshy fruit that undergoes fast ethylene-induced modifications. The fruit becomes edible, but the fast pulp softening is the main factor that limits the post-harvest period. Papaya fast pulp softening occurs due to cell wall disassembling coordinated by ethylene triggering that massively expresses pectinases. In this work, RNA-seq analysis of ethylene-treated and non-treated papayas enabled a wide transcriptome overview that indicated the role of ethylene during ripening at the gene expression level. Several families of transcription factors (AP2/ERF, NAC, and MADS-box) were differentially expressed. ACO, ACS, and SAM-Mtase genes were upregulated, indicating a high rate of ethylene biosynthesis after ethylene treatment. The correlation among gene expression and physiological data demonstrated ethylene treatment can indeed simulate ripening, and regulation of changes in fruit color, aroma, and flavor could be attributed to the coordinated expression of several related genes. Especially about pulp firmness, the identification of 157 expressed genes related to cell wall metabolism demonstrated that pulp softening is accomplished by a coordinated action of several different cell wall-related enzymes. The mechanism is different from other commercially important fruits, such as strawberry, tomato, kiwifruit, and apple. The observed behavior of this new transcriptomic data confirms ethylene triggering is the main event that elicits fast pulp softening in papayas.

Immobilization of papain: A review.

Papain is a cysteine protease from papaya, with many applications due to its broad specificity. This paper reviews for first time the immobilization of papain on different supports (organic, inorganic or hybrid supports) presenting some of the features of the utilized immobilization strategies (e.g., epoxide, glutaraldehyde, genipin, glyoxyl for covalent immobilization). Special focus is placed on the preparation of magnetic biocatalysts, which will permit the simple recovery of the biocatalyst even if the medium is a suspension. Problems specific to the immobilization of proteases (e.g., steric problems when hydrolyzing large proteins) are also defined. The benefits of a proper immobilization (enzyme stabilization, widening of the operation window) are discussed, together with some artifacts that may suggest an enzyme stabilization that may be unrelated to enzyme rigidification.

Biochemical Properties and Anti-Biofilm Activity of Chitosan-Immobilized Papain.

Chitosan, the product of chitin deacetylation, is an excellent candidate for enzyme immobilization purposes. Here we demonstrate that papain, an endolytic cysteine protease (EC: 3.4.22.2) from Carica papaya latex immobilized on the matrixes of medium molecular (200 kDa) and high molecular (350 kDa) weight chitosans exhibits anti-biofilm activity and increases the antimicrobials efficiency against biofilm-embedded bacteria. Immobilization in glycine buffer (pH 9.0) allowed adsorption up to 30% of the total protein (mg g chitosan-1) and specific activity (U mg protein-1), leading to the preservation of more than 90% of the initial total activity (U mL-1). While optimal pH and temperature of the immobilized papain did not change, the immobilized enzyme exhibited elevated thermal stability and 6-7-fold longer half-life time in comparison with the soluble papain. While one-half of the total enzyme dissociates from both carriers in 24 h, this property could be used for wound-dressing materials design with dosed release of the enzyme to overcome the relatively high cytotoxicity of soluble papain. Our results indicate that both soluble and immobilized papain efficiently destroy biofilms formed by Staphylococcus aureus and Staphylococcus epidermidis. As a consequence, papain, both soluble and immobilized on medium molecular weight chitosan, is capable of potentiating the efficacy of antimicrobials against biofilm-embedded Staphylococci. Thus, papain immobilized on medium molecular weight chitosan appears a presumably beneficial agent for outer wound treatment for biofilms destruction, increasing antimicrobial treatment effectiveness.

Novel biotechnological formulations of cysteine proteases, immobilized on chitosan. Structure, stability and activity.

Bromelain, papain, and ficin are studied the most for meat tenderization, but have limited application due to their short lifetime. The aim of this work is to identify the adsorption mechanisms of these cysteine proteases on chitosan to improve the enzymes' stability. It is known that immobilization can lead to a significant loss of enzyme activity, which we observed during the sorption of bromelain (protease activity compared to soluble enzyme is 49% for medium and 64% for high molecular weight chitosan), papain (34 and 28% respectively) and ficin (69 and 70% respectively). Immobilization on the chitosan matrix leads to a partial destruction of protein helical structure (from 5 to 19%). Using computer modelling, we have shown that the sorption of cysteine proteases on chitosan is carried out by molecule regions located on the border of domains L and R, including active cites of the enzymes, which explains the decrease in their catalytic activity upon immobilization. The immobilization on chitosan does not shift the optimal range of pH (7.5) and temperature values (60 °C for bromelain and papain, 37-60 °C for ficin), but significantly increases the stability of biocatalysts (from 5.8 times for bromelain to 7.6 times for papain).

Broad range of substrate specificities in papain and fig latex enzymes preparations improve enumeration of Listeria monocytogenes.

Numerous applications of proteolytic enzymes include dissociation of fermented meat products for the enumeration of `foodborne pathogenic bacteria. The use of trypsin for this cause is abandoned due to the high concentration of the enzyme affecting released bacteria. Papain, as a suggested replacement, and fig latex preparation with high extent of papain-like enzymes have the potential to be applied for bacteria enumeration. Both enzymatic preparations, originating from papaya and fig, showed a broader range of substrate specificities including gelatinolytic activity, especially prominent in the case of ficin and attributed to both, cysteine protease ficin and serine protease by the analysis of 2D zymography with specific inhibitors. The activity towards native collagen, mild in the case of papain, and extensive in the case of fig latex was proved by structural analysis of digested collagen by infrared spectroscopy. Further exploration of their potential for dissociation of fermented meat products showed that both papain and fig latex enzymes are stable in the presence of detergents Tween 20 and Triton X-100 and effective in the enumeration of Listeria monocytogenes. Gelatenolytic activity, and at least partial collagenolytic activity and stability in procedure conditions make papaya and fig latex proteases potent for this application in significantly lower concentrations than previously used enzymes. As a mixture of proteolytic enzymes with divergent characteristics, fig latex preparation shows higher efficiency in Listeria monocytogenes release than papain, conserved even in the presence of stronger non-ionic detergent Triton X-100.

Correlation among PME activity, viscoelastic, and structural parameters for Carica papaya edible tissue along ripening.

Papaya fruit, widely consumed around the world, is mechanically and structurally affected by several enzymatic processes during ripening, where pectin methylesterase plays a key role. Hence, the aim of this work was to evaluate possible correlations among physicochemical changes, mechanical parameters, viscoelastic behavior, and enzyme activity of pectin methylesterase to provide information about the softening phenomenon by applying the Maxwell and Peleg models. Mechanical parameters were estimated by texture profile analysis, enzyme activity by Michaelis-Menten parameters, and viscoelastic behavior by relaxation test responses fitted to these models. The Maxwell model described properly mechanical changes during ripening, displaying a better adjustment (R2 > 0.97) than the Peleg model (0.80 < R2 < 0.84). Pearson correlation analysis (P ≤ 0.01) indicated an inversely proportional relation among firmness, total soluble solids, and the first elastic element of the Maxwell model. Besides, the PME Michaelis-Menten affinity constant showed a correlation between the first elastic element and the first viscoelastic element of the Maxwell model. Findings of this work pointed out that the first Maxwell elastic element could explain structural changes as papaya ripening advance, associated with pectin methylesterase activity, cell wall disruption, and cell assembling into the tissue. PRACTICAL APPLICATION: Mechanical and viscoelastic behavior of papaya fruit tissue were described by the Maxwell model associating both viscous and elastic elements to the softening process. The results provide background and practical knowledge to describe structural changes during the ripening process of papaya depending on its enzymatic activity. Outcomes could be further applied to understand changes in other fruits or food matrixes that soften during postharvest, storage, and food chain supply processes.

One-step recovery of latex papain from Carica papaya using three phase partitioning and its use as milk-clotting and meat-tenderizing agent.

Three Phase Partitioning (TPP) system as an elegant non-chromatographic and bulk separation method was successfully applied for the extraction and recovery of papain from the latex of Carica papaya. The optimized parameters of TPP allowed achieving a purification fold of 11.45 and activity recovery of 134% with 40% (NH4)2SO4, 1.0:0.75 ratio of crude extract: t-BuOH at pH and temperature of 6.0 and 25 °C, respectively. The recovered papain had a molecular weight of 23.2 kDa and revealed maximum activity at pH 6.0 and temperature of 50 °C. The maximum values of Km and Vmax parameters were 10.83 mg mL-1 and 33.33 U mL-1, respectively. The protease with 4 isoforms was stable at 40-80 °C and a pH range of 6.0-7.5 against numerous metal ions and none of them inactivated the recovered protease. Moreover, 10 mM Ca2+ improved 2-folds the activity and half-life of the protease at temperatures from 30 to 50 °C. The milk-clotting activity tests revealed high stability of latex papain at storage, namely at -20 °C compared to 4 °C and 25 °C for up than 5 weeks. As a meat tenderizing agent, it showed promising role under different treatments by improving the texture of tough meat. The findings indicated that one-step TPP system is a simple, quick, economical and very attractive process for fast recovery of latex papain compared to other proposed protocols.

Allergenicity reduction of cow's milk proteins using latex peptidases.

The present study evaluated four laticifer fluids as a novel source of peptidases capable of hydrolyzing proteins in cow's milk. The latex peptidases from Calotropis procera (CpLP), Cryptostegia grandiflora (CgLP), and Carica papaya (CapLP) were able to perform total hydrolysis of caseins after 30 min at pH 6.5, as confirmed by a significant reduction in the residual antigenicity. Casein hydrolysis by Plumeria rubra latex peptidases (PrLP) was negligible. Moreover, whey proteins were more resistant to proteolysis by latex peptidases; however, heat pretreatment of the whey proteins enhanced the degree of hydrolysis and reduced the residual antigenicity of the hydrolysates. The in vivo assays show that the cow's milk proteins hydrolysed by CgLP and CapLP exhibited no immune reactions in mice allergic to cow's milk, similar to a commercial partially hydrolysed formula. Thus, these peptidases are promising enzymes for the development of novel hypoallergenic formulas for children with a milk allergy.

Combination of Gluten-Digesting Enzymes Improved Symptoms of Non-Celiac Gluten Sensitivity: A Randomized Single-blind, Placebo-controlled Crossover Study.

INTRODUCTION: Recently, the population of individuals with non-celiac gluten sensitivity (NCGS) who do not have celiac disease but show improved symptoms with a gluten-free diet, has increased. Enzyme replacement therapy using digestive enzymes is expected to improve the symptoms of NCGS and be sustainable, since gluten-related proteins that are indigestible by the digestive system have been considered triggers of NCGS. METHODS: We selected patients with NCGS by screening demographic interviews, as well as performing medical evaluations, anti-gluten antibody tests, and gluten challenge tests. We performed a single-blind and crossover clinical trial with these subjects using a gluten challenge with the enzyme mixture or a placebo. Our designed enzyme mixture contained peptidase, semi alkaline protease, deuterolysin, and cysteine protease derived from Aspergillus oryzae, Aspergillus melleus, Penicillium citrinum, and Carica papaya L., respectively. RESULTS: Administration of the enzyme mixture significantly decreased the change in the score of the symptom questionnaire before and after the gluten challenge compared with administration of the placebo in patients with NCGS without adverse events. In particular, the changes in the score of the gluten-induced incomplete evacuation feeling and headaches were significantly improved. The serum levels of interleukin (IL)-8, tumor necrosis factor (TNF)-α, andregulated on activation, normal T cell expressed and secreted (RANTES) in subjects were not significantly changed by gluten, as expected from previous studies, and the enzyme mixture did not affect these inflammatory markers. CONCLUSION: In this human clinical study, we demonstrated the efficacy of the enzyme mixture derived from microorganisms and papaya in improving the symptoms of NCGS.

Functional Expression of Plant Lipases: The Case of CpLip1 from Carica papaya.

Carica papaya latex is one of the most studied sources of plant lipases. However, the complexity of the matrix composition makes it difficult to isolate and purify the lipolytic enzymes present in Carica papaya latex. Therefore, diverse strategies have been developed to study the catalytic properties of these enzymes.Recently the first lipase from Carica papaya latex (CpLip1) has been successfully cloned and expressed in order to study their catalytic properties. In order to improve the catalytic properties and increase the potential for its use at industrial scale.In this chapter, a practical protocol to recombinant CpLip1 lipase is given.

Chemoenzymatic synthesis of new derivatives of glycyrrhetinic acid with antiviral activity. Molecular docking study.

We present an efficient approach to the synthesis of a series of glycyrrhetinic acid derivatives. Six derivatives, five of them new compounds, were obtained through chemoenzymatic reactions in very good to excellent yield. In order to find the optimal reaction conditions, the influence of various parameters such as enzyme source, nucleophile:substrate ratio, enzyme:substrate ratio, solvent and temperature was studied. The excellent results obtained by lipase catalysis made the procedure very efficient considering their advantages such as mild reaction conditions and low environmental impact. Moreover, in order to explain the reactivity of glycyrrhetinic acid and the acetylated derivative to different nucleophiles in the enzymatic reactions, molecular docking studies were carried out. In addition, one of the synthesized compounds exhibited remarkable antiviral activity against TK + and TK- strains of Herpes simplex virus type 1 (HSV-1), sensitive and resistant to acyclovir (ACV) treatment.

Papain-like cysteine proteases in Carica papaya: lineage-specific gene duplication and expansion.

BACKGROUND: Papain-like cysteine proteases (PLCPs), a large group of cysteine proteases structurally related to papain, play important roles in plant development, senescence, and defense responses. Papain, the first cysteine protease whose structure was determined by X-ray crystallography, plays a crucial role in protecting papaya from herbivorous insects. Except the four major PLCPs purified and characterized in papaya latex, the rest of the PLCPs in papaya genome are largely unknown. RESULTS: We identified 33 PLCP genes in papaya genome. Phylogenetic analysis clearly separated plant PLCP genes into nine subfamilies. PLCP genes are not equally distributed among the nine subfamilies and the number of PLCPs in each subfamily does not increase or decrease proportionally among the seven selected plant species. Papaya showed clear lineage-specific gene expansion in the subfamily III. Interestingly, all four major PLCPs purified from papaya latex, including papain, chymopapain, glycyl endopeptidase and caricain, were grouped into the lineage-specific expansion branch in the subfamily III. Mapping PLCP genes on chromosomes of five plant species revealed that lineage-specific expansions of PLCP genes were mostly derived from tandem duplications. We estimated divergence time of papaya PLCP genes of subfamily III. The major duplication events leading to lineage-specific expansion of papaya PLCP genes in subfamily III were estimated at 48 MYA, 34 MYA, and 16 MYA. The gene expression patterns of the papaya PLCP genes in different tissues were assessed by transcriptome sequencing and qRT-PCR. Most of the papaya PLCP genes of subfamily III expressed at high levels in leaf and green fruit tissues. CONCLUSIONS: Tandem duplications played the dominant role in affecting copy number of PLCPs in plants. Significant variations in size of the PLCP subfamilies among species may reflect genetic adaptation of plant species to different environments. The lineage-specific expansion of papaya PLCPs of subfamily III might have been promoted by the continuous reciprocal selective effects of herbivore attack and plant defense.

Soluble expression of biologically active methionine sulfoxide reductase B1 (PaMsrB1) from Carica papaya in Escherichia coli and isolation of its protein targets.

Plant methionine sulfoxide reductase B1 (MsrB1) protects the photosynthetic apparatus from oxidative damage by scavenging reactive oxygen species to repair Met-oxidized proteins in response to abiotic stresses and biotic attack. Papaya MsrB1 (PaMsrB1) was identified previously to interact with papaya ringspot virus NIa-Pro, and this interaction inhibits the import of PaMsrB1 into the chloroplast. Further functional characterization of PaMsrB1 requires the production of a biologically active purified recombinant protein. In this report, PaMsrB1 as a fusion protein containing an N-terminal maltose-binding protein (MBP) was expressed in Escherichia coli Rosetta (DE3) cells and purified. Production of soluble fusion protein was greater when the cells were cultured at 16 °C than at 37 °C. The Factor Xa protease digested MBP-PaMsrB1 fusion protein and subsequently purified recombinant PaMsrB1 specifically reduced the R-diastereomer of methionine sulfoxide (MetSO) and Dabsyl-MetSO to Met in the presence of dithiothreitol. Eight chloroplast-localized and five non-chloroplast-localized candidate proteins that interact with PaMsrB1 were isolated by affinity chromatography and liquid chromatography coupled to tandem mass spectrometry. The results provide a platform to further understand the anti-oxidative defense mechanism of PaMsrB1.

Development of an Economical Method to Reduce the Extractable Latex Protein Levels in Finished Dipped Rubber Products.

Natural rubber latex (NRL) allergy is caused by the extractable latex proteins in dipped rubber products. It is a major concern for the consumers who are sensitive to the allergenic extractable proteins (EP) in products such as NRL gloves. Objective of this research was to develop an economical method to reduce the EP in finished dipped NRL products. In order to reduce the EP levels, two natural proteases, bromelain from pineapple and papain from papaya, were extracted and partially purified using (NH4)2SO4. According to the newly developed method, different glove samples were treated with a 5% solution of each partially purified enzyme, for 2 hours at 60°C. Residual amounts of in treated samples were quantified using the modified Lowry assay (ASTM D5712-10). Bromelain displayed a 54 (±11)% reduction of the EP from the dipped rubber products, whereas it was 58 (±8)% with papain. These results clearly indicate that the selected natural proteases, bromelain, and papain contribute significantly towards the reduction of the total EP in finished NRL products. Application of bromelain enzyme for the aforementioned purpose has not been reported up to date, whereas papain has been used to treat raw NRL towards reducing the EP.

Biosynthesis of (S)-naproxen starch ester by Carica papaya lipase in intermittent opening reaction mode.

Inorder to brought S-naproxen into small intestine, an optically pure (S)-naproxen starch ester was produced by lipase through enantio-selective trans-esterification of racemic naproxen methyl ester with pretreatment starch in solvent system. With carefully selection of the reaction medium (isooctane), lipase (Carica Papaya Lipase, CPL) and the reaction mode (intermittent opening), a high conversion rate (48.6%) and enantiomeric excess of product (99.6%) was obtained. The slow release macromolecular (S)-Naproxen had been synthesized to improve the efficacy of racemic naproxen and overcome its side effects. The enanitomeric ratio of CPL (E=52.5) was higher than CRL (E=22) and greatly influenced by the byproduct methyl alcohol. The intermittent opening reaction mode was the effective way to remove the inhibition of methyl alcohol and to improve the enantio-selectivity of CPL. S-naproxen starch was confirmed by HPLC and 1H NMR. This method may also apply to preparation the other optically pure 2-phenylpropionic acid derivatives. S-naproxen starch was a new optically pure derivatives possessing emulsifying and slow release properties would be widely applied to the food, pharmaceutical and biomedical industries.

A wild origin of the loss-of-function lycopene beta cyclase (CYC-b) allele in cultivated, red-fleshed papaya (Carica papaya).

PREMISE OF THE STUDY: The red flesh of some papaya cultivars is caused by a recessive loss-of-function mutation in the coding region of the chromoplast-specific lycopene beta cyclase gene (CYC-b). We performed an evolutionary genetic analysis of the CYC-b locus in wild and cultivated papaya to uncover the origin of this loss-of-function allele in cultivated papaya. METHODS: We analyzed the levels and patterns of genetic diversity at the CYC-b locus and six loci in a 100-kb region flanking CYC-b and compared these to genetic diversity levels at neutral autosomal loci. The evolutionary relationships of CYC-b haplotypes were assessed using haplotype network analysis of the CYC-b locus and the 100-kb CYC-b region. KEY RESULTS: Genetic diversity at the recessive CYC-b allele (y) was much lower relative to the dominant Y allele found in yellow-fleshed wild and cultivated papaya due to a strong selective sweep. Haplotype network analyses suggest the y allele most likely arose in the wild and was introduced into domesticated varieties after the first papaya domestication event. The shared haplotype structure between some wild, feral, and cultivated haplotypes around the y allele supports subsequent escape of this allele from red cultivars back into wild populations through feral intermediates. CONCLUSIONS: Our study supports a protracted domestication process of papaya through the introgression of wild-derived traits and gene flow from cultivars to wild populations. Evidence of gene flow from cultivars to wild populations through feral intermediates has implications for the introduction of transgenic papaya into Central American countries.

A novel assay for the detection of anthelmintic activity mediated by cuticular damage to nematodes: validation on Caenorhabditis elegans exposed to cysteine proteinases.

Plant cysteine proteinases (CPs) from Carica papaya kill parasitic and free-living nematodes in vitro by hydrolysis of the worm cuticle, a mechanism that is different to all commercially available synthetic anthelmintics. We have developed a cheap and effective, rapid-throughput Caenorhabditis elegans-based assay for screening plant CP extracts for anthelmintic activity targeting cuticular integrity. The assay exploits colorimetric methodology for assessment of cuticular damage, and is based on the ability of viable cells to incorporate and bind Neutral red dye within lysosomes and to release the dye when damaged. Living worms are pre-stained with the dye, exposed to CPs and then leakage of the dye through the damaged cuticle is quantified by spectrophotometry. In contrast to motility assays and semi-subjective interpretation of microscopical images, this colorimetric assay is independent of observer bias. Our assay was applied to a series of C. elegans bus mutant strains with leaky cuticles and to cystatin knockout mutants. At ambient temperature and over 0.5-24 h, both bus mutants and the cystatin knockouts were highly susceptible to CPs, whereas wild-type Bristol N2 worms were essentially unstained by Neutral red and unaffected by CPs, providing validation for the utility of this assay.

Reverse micellar extraction of papain with cationic detergent based system: An optimization approach.

In this study, reverse micellar extraction of papain model system was performed using cetyltrimethylammonium bromide (CTAB)/iso-octane/hexanol/butanol system to optimize the forward and back extraction efficiency (BEE). A maximum forward extraction efficiency of 55.0, 61.0, and 54% was achieved with an aqueous phase pH of 11.0, 150 mM CTAB/iso-octane and 0.1 M NaCl, respectively. Taguchi's orthogonal array was applied to optimize the pH of stripping phase, concentration of isopropyl alcohol (IPA) and potassium chloride (KCl) for maximizing BEE. The optimal levels of stripping phase pH, concentration of IPA and KCl were found to be 6, 20% (v/v), and 0.8 M, respectively. Under these optimal levels, the BEE was found to be 88% after which enzyme activity was recovered with 2.5-fold purification. Further optimization was performed using artificial neural network-linked genetic algorithm, where the BEE was improved to 90.52% with pH 6, IPA (%) = 19.938, and KCl (M) = 0.729.

The Papaya Transcription Factor CpNAC1 Modulates Carotenoid Biosynthesis through Activating Phytoene Desaturase Genes CpPDS2/4 during Fruit Ripening.

Papaya fruits accumulate carotenoids during fruit ripening. Although many papaya carotenoid biosynthesis pathway genes have been identified, the transcriptional regulators of these genes have not been characterized. In this study, a NAC transcription factor, designated as CpNAC1, was characterized from papaya fruit. CpNAC1 was localized exclusively in nucleus and possessed transcriptional activation activity. Expression of carotenoid biosynthesis genes phytoene desaturases (CpPDSs) and CpNAC1 was increased during fruit ripening and by propylene treatment, which correlates well with the elevated carotenoid content in papaya. The gel mobility shift assays and transient expression analyses demonstrated that CpNAC1 directly binds to the NAC binding site (NACBS) motifs in CpPDS2/4 promoters and activates them. Collectively, these data suggest that CpNAC1 may act as a positive regulator of carotenoid biosynthesis during papaya fruit ripening possibly via transcriptional activation of CpPDSs such as CpPDS2/4.