ORIGINAL RESEARCH published: 10 June 2022 doi: 10.3389/fpls.2022.878037 Knock-Out of CmNAC-NOR Affects Melon Climacteric Fruit Ripening Bin Liu 1, Miguel Santo Domingo 1, Carlos Mayobre 1, Ana Montserrat Martín-Hernández 1,2, Marta Pujol 1, 2* and Jordi Garcia-Mas 1, 2* 1 Centre for Research in Agricultural Genomics (CRAG), CSIC-IRTA-UAB-UB, Barcelona, Spain, 2 Institut de Recerca i Tecnologia Agroalimentàries (IRTA), Barcelona, Spain Fruit ripening is an important process that affects fruit quality. A QTL in melon, ETHQV6.3, involved in climacteric ripening regulation, has been found to be encoded by CmNAC-NOR, a homologue of the tomato NOR gene. To further investigate CmNAC-NOR function, we obtained two CRISPR/Cas9-mediated mutants (nor-3 and nor-1) in the climacteric Védrantais background. nor-3, containing a 3-bp deletion altering the NAC domain A, resulted in ~8 days delay in ripening without affecting fruit quality. In contrast, the 1-bp deletion in nor-1 resulted in a fully disrupted NAC domain, which completely blocked climacteric ripening. The nor-1 fruits did not produce ethylene, no abscission layer was Edited by: Ana Margarida Fortes, formed and there was no external color change. Additionally, volatile components were University of Lisbon, Portugal dramatically altered, seeds were not well developed and flesh firmness was also altered. Reviewed by: There was a delay in fruit ripening with the nor-1 allele in heterozygosis of ~20 days. Our Supaart Sirikantaramas, Chulalongkorn University, Thailand results provide new information regarding the function of CmNAC-NOR in melon fruit Gholamreza Khaksar, ripening, suggesting that it is a potential target for modulating shelf life in commercial Chulalongkorn University, Thailand climacteric melon varieties. Daqi Fu, China Agricultural University, China Keywords: fruit ripening, melon (Cucumis melo L.), NAC-NOR, CRISPR, shelf life *Correspondence: Marta Pujol marta.pujol@irta.cat Jordi Garcia-Mas INTRODUCTION jordi.garcia@irta.cat Fruit maturation is an important developmental stage because the set of biochemical pathways Specialty section: involved in ripening make the fruit attractive, perfumed, and edible (Bouzayen et al., 2010). This article was submitted to In addition, the ripening process also helps seed dispersal (Wang et al., 2020a). There is Plant Biotechnology, considerable ongoing research to help understand the complex regulation of this important a section of the journal process (Bouzayen et al., 2010). Based on their ripening behavior, fleshy fruits have been Frontiers in Plant Science divided into two groups: climacteric and non-climacteric (McMurchie et al., 1972). Climacteric Received: 17 February 2022 fruits such as tomato are characterized by an ethylene burst accompanied by an increase in Accepted: 23 May 2022 respiration at the onset of ripening. In contrast, non-climacteric fruits such as orange lack Published: 10 June 2022 this ethylene-associated respiratory peak (Paul et al., 2012). Usually, climacteric fruits have a Citation: shorter shelf life than non-climacteric fruits (Hiwasa-Tanase and Ezura, 2014), and breeding Liu B, Santo Domingo M, Mayobre C, programs for fruit crops are oriented to increase shelf life to minimize postharvest losses Martín-Hernández AM, Pujol M and (Payasi and Sanwal, 2010). Garcia-Mas J (2022) Knock-Out of CmNAC-NOR Affects Melon Ethylene plays a primary role in initiating climacteric fruit ripening (McMurchie et al., Climacteric Fruit Ripening. 1972). Its production is low at the pre-climacteric stage, while there is a massive auto- Front. Plant Sci. 13:878037. stimulated ethylene production at the onset of the ripening stage. Exogenous ethylene treatment doi: 10.3389/fpls.2022.878037 can also induce the ethylene burst at the pre-climacteric stage of climacteric fruits, thereby Frontiers in Plant Science | www.frontiersin.org 1 June 2022 | Volume 13 | Article 878037 Liu et al. Knock-Out CmNAC-NOR Affects Melon Ripening advancing the ripening process (Hiwasa-Tanase and Ezura, 2013), and FaRIF (Martín-Pizarro et al., 2021). These studies 2014). In contrast, treatment with the ethylene inhibitor have provided valuable information on gene function related 1-methylcyclopropene (1-MCP) delays fruit ripening to regulation of fruit ripening. (Blankenship and Dole, 2003; Watkins, 2006). Antisense- Melon (Cucumis melo L.) is a suitable model to study induced repression of ethylene synthesis enzymes in tomato fruit ripening, because there are both climacteric and also delays fruit maturation (Hamilton et al., 1990). These non-climacteric genotypes (Ezura and Owino, 2008). Genetic results demonstrate the key role of ethylene in regulating analysis of a biparental population of the cantaloupe type ripening in climacteric fruits. Interestingly, some reports have Védrantais (VED, climacteric) × PI 161375 (SC, non-climacteric) shown that ethylene can also play a role in non-climacteric inbred lines indicated that ethylene production and fruit fruit ripening (Katz et al., 2004; Bouzayen et al., 2010), abscission are controlled by two independent loci, Al-3 and although in non-climacteric fruit, a low level of ethylene Al-4 (Périn et al., 2002). In recent studies, a near isogenic during the whole developmental process is found (Paul et al., line SC3-5-1 derived from the non-climacteric parental lines 2012). In melon, some fruit ripening processes are independent SC and the inodorus type Piel de Sapo (PS) had a climacteric of ethylene, such as flesh softening, sugar accumulation, or ripening phenotype, and two QTLs, ETHQB3.5 and ETHQV6.3, flesh color, that do not change in ethylene-suppressed melon were found to be involved in the regulation of climacteric fruit (Flores et al., 2001; Pech et al., 2008), confirming that ripening in SC3-5-1 (Eduardo et al., 2005; Moreno et al., the control of fruit ripening is a complex trait. 2008; Vegas et al., 2013). Previously, ETHQV6.3 was found Genes involved in fruit ripening have been largely studied to be encoded by a NAC transcription factor CmNAC-NOR in either climacteric or non-climacteric species (Gapper et al., (MELO3C016540.2), phylogenetically related to the tomato 2013; Osorio et al., 2013; Lü et al., 2018) and tomato has SlNAC-NOR (Ríos et al., 2017). TILLING mutants containing emerged as a prime model of climacteric fruit ripening (Alexander non-synonymous mutations in the coding region of and Grierson, 2002). Genetic characterization of several ripening- CmNAC-NOR had a delayed ripening phenotype, suggesting related mutants in tomato has advanced our knowledge of the that CmNAC-NOR is an important regulator of climacteric mechanisms that regulate fruit ripening (Giovannoni, 2007). ripening in melon. To further investigate the CmNAC-NOR The ripening-inhibitor (rin), non-ripening (nor), and Colorless function, in this study, we generated and phenotyped CRISPR/ non-ripening (Cnr) mutations have been useful to understand Cas9 mutants with different disruption levels. the transcriptional regulation of fruit ripening (Vrebalov et al., 2002; Manning et al., 2006; Giovannoni, 2007; Wang et al., 2020b). However, the milder ripening phenotypes observed MATERIALS AND METHODS for the CRISPR/Cas9 knockouts of these three genes have resulted in a re-evaluation of their original proposed role as Plant Material and Generation of master regulators of fruit ripening, suggesting that a network Constructs of partially redundant components exists to regulate this The cantaloupe inbred line VED (climacteric) was used in important biological process (Ito et al., 2017; Gao et al., 2019, this study. For editing CmNAC-NOR in VED, three gRNAs 2020; Wang et al., 2019, 2020a). Additional transcription factors (gRNA1, gRNA2, and gRNA3; Supplementary Table S1) were involved in the regulation of fruit ripening have also been designed, based on the genomic sequence of CmNAC-NOR, identified in tomato. The MADS-box transcription factor using Breaking-Cas (Oliveros et al., 2016). The gRNA1 and TOMATO AGAMOUS-LIKE1 (TAGL1; Itkin et al., 2009; Vrebalov gRNA2 sequences were inserted into the vector pBS_KS_Bsa_ et al., 2009) is highly expressed during fruit ripening. TAGL1- Bbs_tandem with the BbsI and BsaI sites, respectively, cut using silenced fruit did not ripe normally, with reduced levels of the SpeI and KpnI restriction enzymes, and then inserted into carotenoids and ethylene. FRUITFULL homologues (TDR4/ the final pB7-CAS9-TPC vector to obtain the gRNA1- FUL1 and MBP7/FUL2) are also MADS-box transcription factors gRNA2-CAS9 construct. The same protocol was used to generate involved in fruit ripening in an ethylene-independent manner, the gRNA2-gRNA1-CAS9 and gRNA3-gRNA1-CAS9 constructs. having redundant functions in cell wall modification (Bemer These constructs were transformed into Agrobacterium (AGL-0) et al., 2012). FUL1/2 and TAGL1 may regulate different subsets and identified by cloning PCR. Cloning vectors were kindly of the known RIN targets. APETALA2a (AP2a) is a negative provided by Professor Puchta (KIT, Germany). regulator of tomato fruit ripening, with its silencing causing elevated ethylene production and early fruit ripening (Chung Melon Transformation et al., 2010; Karlova et al., 2011). Other NAC proteins have Cotyledon transformation was used for melon transformation also been found to be involved in regulating ripening, among using Agrobacterium strain AGL-0 as described by Castelblanque them SlNAC1, SlNAC3, SlNAC4, and SlNAM1, suggesting that et al. (2008) except that the cotyledons were cut as in García- a complex regulatory network of fruit ripening exists (reviewed Almodóvar et al. (2017). In brief, half of the proximal parts in Liu et al., 2022). For non-climacteric fruit, strawberry is of the cotyledons from 1-day-old seeds were cut and co-cultured one of the most studied plants (Osorio et al., 2013) and several with transformed Agrobacterium for 20 min in the presence genes involved in strawberry fruit ripening have recently been of 200 μM acetosyringone. The inoculated explants with identified, including FaPYR1 (Chai et al., 2011), FaExp2 (Civello Agrobacterium were co-cultured for 3 days at 28°C on et al., 1999), FaASR (Chen et al., 2011), FaABI1 (Jia et al., regeneration medium supplemented with 0.5 mg/l Frontiers in Plant Science | www.frontiersin.org 2 June 2022 | Volume 13 | Article 878037 Liu et al. Knock-Out CmNAC-NOR Affects Melon Ripening 6-bencylaminopurine (BA), 0.1 mg/l Indole-3-acetic acid (IAA), at 250 rpm. The SPME fiber (50/30 μm DVB/CAR/PDMS, and 200 mM acetosyringone. Every 3–4 weeks, the green cluster Merck®, Darmstadt, Germany) was exposed to the vial headspace buds were cut and explants were moved to fresh selection for 30 min. Splitless injection was used in a 7890A gas medium in the presence of L-Phosphinothricin (PPT). When chromatograph (GC) equipped with a Sapiens-X5MS capillary the regenerated shoots were tall, they were cut, separated from column (30 m/0.25 mm/0.25 μm, Teknokroma®, Sant Cugat del the explants, and put individually into rooting media in large Vallès, Spain), with 10 min of thermal desorption at 250°C. The test tubes. When the rooted plantlets were large enough, a oven was set to 50°C for 1 min, then increasing by 5°C/min leaf section was cut to identify edited T0 plants. to 280°C and holding for 5 min. The carrier gas was helium at a head pressure of 13.37 psi. A mass spectrometer (MS) Genotyping and Ploidy Test 5975 C (Agilent Technologies®, Santa Clara, CA, United States) Genomic DNA was extracted from young leaves of melon was coupled to the GC, with a source temperature of 230°C plants by an improved CTAB method (Pereira et al., 2018). and the quadrupole temperature was set to 150°C. With an To genotype the candidate plants, the CAS9 gene was amplified untargeted analysis, volatiles were identified by comparison of to confirm that plants were transgenic; then, the target region their mass spectra with the NIST 11 library (NIST/EPA/NIH) of the three gRNAs was amplified and sequenced. Primers and by their Kovats retention index, calculated using a mix used in this study are listed in Supplementary Table S1. At of alkanes (C7-C40 in hexane, Merck®, Darmstadt, Germany) the same time, young leaves were harvested and sent to Iribov under the same chromatographic conditions. The relative content (Heerhugowaard, Netherlands) for the ploidy test using flow of each volatile was estimated by normalizing the peak area cytometry (FCM). to the internal standard peak. A Shapiro–Wilk normality test (α = 0.05) and a multiple variable t-test were carried out. A Wilcoxon test was used to compare edited plants against Identification of CAS9 Free T1 Plants wild type. Diploid T0 plants that carried the CAS9 gene were selected, grown, and self-pollinated to obtain the T1 seeds. The T1 seeds were germinated and genotyped, and the CAS9 free plants Gene Expression Analysis with or without CmNAC-NOR editions were selected for Total RNA was extracted from flesh tissue at harvest from further experiments. three biological replicates per genotype (Control, non-edited (NE), nor-3 and nor-1) using the Spectrum Plant Total RNA Fruit Phenotyping Kit (Sigma, Burlington, MA, United States). A DNase treatment Fruit quality traits, especially those associated to climacteric was performed using Turbo DNase kit (Invitrogen, Waltham, ripening behavior, were assessed after harvest as previously MA, United States). cDNA was synthesized using PrimeScript described (Ríos et al., 2017; Pereira et al., 2018). In brief, the kit (Takara, Kyoto, Japan). Quantitative RT-PCR was performed production of ethylene in the fruits was measured from 25 days in a LightCycler® 480 with LightCycler® 480 SYBR Green after pollination (DAP) to when fruit dropped, or 65 DAP I Master (Roche, Basel, Switzerland). Expression data are when it did not drop, using a non-invasive ethylene quantification presented as fold-change (2-ΔΔCT; Livak and Schmittgen, 2001), method (Pereira et al., 2017). The same method for measuring using CmCYP7 (MELO3C025848.2) as a reference gene for ethylene (Pereira et al., 2017) was used for ethylene treatment, normalization, which was validated in Saladié et al. (2015). where 250 ml of 50 ppm ethylene was injected in the bag with Primers used for RT-qPCR are listed in Supplementary Table S1. the fruit, which was then phenotyped after the bag had been kept closed for 1 week. The production of aroma was detected Data Analysis by olfactory evaluation of fruits from 25 DAP until harvest. DNA and protein sequence alignments were obtained with The number of days for abscission layer formation were also DNAMAN version 7. For the statistical analyses, the R (v3.5.3) recorded. External color change during fruit ripening was software (R Core Team, 2020) was used. ANOVAs and pairwise phenotyped visually. Fruits were weighed at harvest. Soluble t-test were performed using R package “rstats.” In general, solid content was analyzed with a digital hand refractometer significance was fixed at value of p <0.05. (Atago Co. Ltd., Tokyo, Japan). Flesh firmness was measured using a penetrometer (Fruit TestTM, Wagner Instruments). RESULTS Volatiles Analysis The aroma profiling of the flesh tissue of melon fruits was Generation of CmNAC-NOR Disrupted analyzed with GC–MS as previously described (Mayobre et al., Mutant Lines by CRISPR/Cas9 2021). Briefly, 2 g of frozen flesh was ground, weighed, and CmNAC-NOR contains three exons and encodes a protein of added to 20 ml chromatography vials with 1 g of NaCl and 353 amino acids (Ríos et al., 2017). In this study, three guide 7 ml of saturated NaCl solution containing 15 ppm of 3-hexanone RNAs (gRNAs) that specifically target the first or second exon as internal standard. Samples were stored at 4°C a maximum of CmNAC-NOR were designed (Figure 1A). After melon of 7 days. Solid-Phase Micro-Extraction (SPME) was carried transformation, we obtained 83 T0 plants containing the gRNA2- out by pre-heating samples for 15 min at 50°C and centrifuging gRNA1-CAS9 and six containing the gRNA1-gRNA2-CAS9 Frontiers in Plant Science | www.frontiersin.org 3 June 2022 | Volume 13 | Article 878037 Liu et al. Knock-Out CmNAC-NOR Affects Melon Ripening A C D B E FIGURE 1 | Generation of CmNAC-NOR disrupted mutant lines by CRISPR/Cas9. (A) Illustration of the CmNAC-NOR gene structure and the three gRNAs in exons 1 and 3. (B) Six independent mutations at gRNA3 targeting sites. (C) Ploidy test of transgenic lines. Left, diploid line NOR-g3-22; Middle, tetraploid line NOR-g3-20; and Right, total number of diploid and tetraploid plants obtained. (D) Validated edited lines by Sanger sequencing. The yellow boxes and dotted lines indicate the deleted nucleotides and their position. (E) Amino acid alignment of the CmNAC-NOR gRNA3-target region in VED (WT), nor-3, and nor-1. NAC subdomain A is shown by a dashed green line above the protein sequences. construct, but none of them were edited. In contrast, we obtained of ethylene in nor-3 when compared to VED and NE, but 39 T0 lines that contained the gRNA3-gRNA1-CAS9 construct, without a significant difference in the amount of ethylene and six mutations at the gRNA3 target site were detected produced (Figure 2A). For the nor-1 mutant in homozygosis, (Figure 1B). As melon tissue culture often induces the generation we did not detect any ethylene production even at 65 DAP of tetraploid plants (Ezura et al., 1992), we looked at the (Figure 2A). Interestingly, there was a ~ 20-day delay in ethylene ploidy of 15 individuals and found three diploid plants production for the nor-1 allele in heterozygosis, and the amount (Figure 1C). These diploid plants contained two different of ethylene produced was much lower than with the controls mutations, a-3 bp and a-1 bp deletion, which were named nor-3 (Figure 2A). and nor-1, respectively (Figure 1D). The −3 bp deletion in VED is a cantalupensis melon, which has intense aroma nor-3 results in the loss of the proline in position 17 (Figure 1E), during climacteric fruit ripening due to the high production which is predicted as a deleterious change (score:-14.874) by of esters (Obando-Ulloa et al., 2008; Mayobre et al., 2021). PROVEAN (Protein Variation Effect Analyzer; Choi and Chan, Therefore, we also compared the aroma production between 2015). The −1 bp deletion in nor-1 results in major changes the controls and both nor mutants. In nor-3 fruits, the aroma from amino acid 16 and the generation of a truncated protein production was significantly delayed compared to the controls of 37 aa (Figure 1E). The functional regions in the NAC at the ripe stage (Figure 2B). However, we did not detect subdomain A of the CmNAC-NOR protein were totally disrupted aroma production in nor-1. To investigate which volatiles were in nor-1 (Figure 1E), suggesting that nor-1 is a loss-of-function altered in the mutants, we used GC–MS to study the fruit mutant. In the following studies, we used T1 plants of the flesh volatile profile. As shown in Figures 2C,D, we found two lines and VED and T1 non-edited (NE) plants as controls. no significant differences in the volatile profile between nor-3 and controls, but nor-1 mutants had a completely different profile with a major decrease in total VOCs produced, which Ethylene Production, Volatile Profile, and explains the lack of aroma by olfactory evaluation. In the Gene Expression in nor Mutants nor-1 mutant, we detected far fewer ester compounds and an Given the key role of ethylene in initiating climacteric fruit increase in aldehydes, furans, and terpenes compared to the ripening (McMurchie et al., 1972), we first compared the wild-type VED (Supplementary Table S2). ethylene production between the controls and both nor mutants. To understand the regulation of ripening by CmNAC-NOR, The ethylene production was recorded from 25 DAP, and the we analyzed the expression of genes related to key ripening results showed a significant delay (~8 days) in the production pathways: ethylene biosynthesis, ester production, flesh softening, Frontiers in Plant Science | www.frontiersin.org 4 June 2022 | Volume 13 | Article 878037 Liu et al. Knock-Out CmNAC-NOR Affects Melon Ripening that CmACS1 and CmACO1 were expressed in both control A lines, VED and NE, at similar levels as the nor-3 mutant, while their expression was dramatically reduced in the nor-1 mutant (Supplementary Figure S1). To elucidate the regulation of aroma production by CmNAC-NOR, we evaluated two genes involved in ester production, one alcohol dehydrogenase CmADH2 (MELO3C014897.2; Manríquez et al., 2006) and one alcohol acyl-transferase CmAAT1 (MELO3C024771.2; B El-Sharkawy et al., 2005). The expression of both genes was repressed in the nor-1 mutant, whereas they were highly expressed in nor-3 and control lines, which is in agreement with the decrease of esters in the nor-1 mutant (Supplementary Figure S1; Figures 2B–D). Two polygalacturonases (CmPGs MELO3C013129.2 and MELO3C016494.2) involved in melon fruit softening during ripening (Hadfield et al., 1998; Nishiyama et al., 2007; Saladié et al., 2015) were also evaluated and we observed that they C were repressed in the nor-1 mutant compared to nor-3 and control lines (Supplementary Figure S1). To estimate the effect of CmNAC-NOR in carotenoid synthesis, we evaluated the expression of CmOr (MELO3C005449.2), a gene involved in beta-carotene accumulation in melon flesh (Tzuri et al., 2015). The results showed that CmOr was not differentially expressed when comparing both mutants nor-1, nor-3, and the control lines VED and NE (Supplementary Figure S1). Partially Disrupting CmNAC-NOR in nor-3 Delays Fruit Ripening but Does Not Affect Fruit Quality D The results from ethylene and aroma production suggest that nor-3 has a delayed ripening phenotype, and similar results were obtained with other ripening-related traits. The flesh color of nor-3 at 40 and 49 DAP was similar to that of NE at 32 and 40 DAP, respectively (Figure 3A), confirming the 8–9 days ripening delay in nor-3. In addition, abscission layer formation (Figure 3B) and rind color change (Figure 3C) of nor-3 fruit were also significantly delayed compared to the controls at the ripe stage. However, we found no significant difference in the amount of ethylene produced (Figure 2A), which is consistent with our previous findings in CmNAC-NOR TILLING mutants (Ríos et al., 2017). We also measured the soluble solids content FIGURE 2 | Ethylene production and volatile profile in nor mutants. (SSC, Figure 3D), fruit weight (Figure 3E), and flesh firmness (A) Ethylene production in VED, NE (non-edited control), nor-3, nor-1 (Figure 3F) at harvest, and no significant differences were homozygote, and nor-1 heterozygote according to days after pollination detected between NE, nor-3, and VED. (DAP). Means are plotted ±SD (n = 5) except nor-1 heterozygote (n = 1). (B) Phenotypic comparison of aroma production in VED, NE, and nor-3 at harvest. Lower case letters indicate significant differences (p < 0.05, n > 5). CmNAC-NOR Knock-Out in nor-1 Blocks (C,D) Relative volatile content in VED, NE, nor-3, and nor-1 at harvest by mass (C) and mass percentage (D). Lower case letters indicate significant Climacteric Ripening differences (p < 0.05, n = 3); large size black dots represent outliers. The behavior of the nor-1 mutant at the fruit ripening stage differed from that of the nor-3 mutant, with the progress of climacteric ripening totally blocked in nor-1 in contrast to and carotenoid production at harvest. Two key genes involved only being delayed in nor-3. Ethylene production, rind color in the biosynthesis of ethylene: 1-aminocyclopropane-1- change, aroma production, and abscission layer formation did carboxylate synthase (CmACS1 MELO3C021182.2) and not occur in nor-1 (Figure 4A). As shown in Figure 4B, the 1-aminocyclopropane-1-carboxylate oxidase (CmACO1 external color of VED changed to yellow at 38 DAP, while MELO3C014437.2) increased their expression during ripening the rind color of nor-1 remained green at 78 DAP. Comparing in climacteric genotypes (Saladié et al., 2015). We observed flesh color and carotenoid content, nor-1 flesh was slightly Frontiers in Plant Science | www.frontiersin.org 5 June 2022 | Volume 13 | Article 878037 Liu et al. Knock-Out CmNAC-NOR Affects Melon Ripening A D B E C F FIGURE 3 | Partially disrupting CmNAC-NOR in nor-3 delays fruit ripening. (A) Fruit ripening phenotype of nor-3 and NE (non-edited control) under natural ripening conditions at days after pollination (DAP). (B-F) Phenotypic comparisons according to abscission layer formation (B), color change of the rind (C), soluble solids content (D), fruit weight (E), and flesh firmness (F) among NE, nor-3, and VED. Lower case letters indicate significant differences (p < 0.05, n > 5). less orange than VED (Figure 4B); however, the total carotenoid (Figure 4H), but the abscission layer was induced 3 days after content was not significantly different (data not shown). ethylene treatment (Figure 4I). Surprisingly, nor-1 seeds were not well developed (Figure 4C), resulting in an extremely low germination rate (1.25%; Figures 4D,E), while seed development was not affected in DISCUSSION nor-3 (Figure 4C), which had an 82.86% germination rate (Figures 4D,E). In addition, the flesh of nor-1 was firmer NAC transcription factors are a large gene family involved in than that of VED and NE fruits (Figure 4F). We found no plant development and the environment stress response (Guo significant difference in SSC (Figure 4G) or fruit weight and Gan, 2006; Nuruzzaman et al., 2013; Hernández and Sanan- (Figure 4H) between VED, NE, and nor-1 fruits. Mishra, 2017). Among them, the SlNAC-NOR gene from tomato is well known as a key regulator in fruit ripening (Giovannoni, 2007). In previous studies, we have characterized melon Ethylene Treatment Did Not Recover CmNAC-NOR, a close homologue of SlNAC-NOR according Climacteric Ripening in nor-1 to a phylogenetic analysis with NAC genes from several species, Given that ethylene plays a major role in the ripening of and demonstrated its involvement in fruit ripening with the climacteric fruit (Ayub et al., 1996), we explored whether delayed ripening phenotype of two TILLING mutant lines (Ríos external ethylene treatment could induce ripening in nor-1 et al., 2017). RNA-seq expression analysis of CmNAC-NOR in mutants. The results showed that climacteric ripening was not Ved (climacteric) and PS (non-climacteric) at different ripening induced after 1 week of external ethylene treatment. Rind color stages showed that both lines have a similar expression profile did not change to yellow (Figure 4B), fruits did not produce during ripening (Supplementary Figure S2). In this study, aroma, and flesh firmness slightly decreased but was still we obtained two diploid CmNAC-NOR-disrupted melon plants significantly more than VED and NE fruits (Figure 4F). We did with different edited sites (nor-1 and nor-3) by using the not see a significant change in SSC (Figure 4G) or fruit weight CRISPR/Cas9 system (Figure 1). nor-1 is a complete knock-out Frontiers in Plant Science | www.frontiersin.org 6 June 2022 | Volume 13 | Article 878037 Liu et al. Knock-Out CmNAC-NOR Affects Melon Ripening CmNAC-NOR. Moreover, in the heterozygous nor-1, there was A also a 20 day delay in ethylene production (Figure 2A), suggesting that nor-1 might have potential for extending the shelf life of fruit in melon breeding programs. In tomato, fruit ripening is affected in both natural and CRISPR/Cas9 knock out mutants of SlNAC-NOR (Gao et al., B F 2020). However, the phenotype of fruit of SlNAC-NOR mutations (CR-NOR) induced by CRISPR/Cas9 has been found to be much less severe than the natural mutant slnac-nor (Wang et al., 2019; Gao et al., 2020). Mature fruit of the slnac-nor mutant does not produce an ethylene burst (Giovannoni, 2007; Adaskaveg et al., 2021) and has little carotenoid content (Giovannoni et al., 1995; Kumar et al., 2018), but CR-NOR fruits can produce ethylene and synthesize much more carotenoids than slnac-nor at the ripe stage (Gao et al., 2020). Therefore, slnac-nor was C G reported as a gain-of-function mutation (Gao et al., 2020) suggesting that SlNAC-NOR does not act as a master regulator but as a major gene controlling the ripening process (Wang D et al., 2020a). In the VED climacteric melon, fruit ripening occurred around 35 ~ 40 DAP (Figure 2A) and was associated with a transient increase in autocatalytic ethylene production, accompanied by changes in rind and flesh color, flesh firmness, sugar content, and aroma production (Pereira et al., 2018, E 2020; Mayobre et al., 2021). In the nor-3 knock down mutant, H the mutation was located within the DNA/protein binding region of the NAC-NOR transcription factor, so we could expect a reduction of its binding affinity, affecting its regulation of ripening-related genes. As it was expected, we observed a delay in ripening (ethylene and aroma production), suggesting I that the mutated protein was still functional but less efficient (Figure 2). This phenotype agreed with Ríos et al. (2017), where non-synonymous mutations in the conserved NAC domain region caused a delay in fruit ripening. Concerning fruit quality, we demonstrated that even though the fruit nor-3 ripened later, it was able to produce a peak of ethylene similar to FIGURE 4 | CmNAC-NOR knock out in nor-1 blocks climacteric ripening. VED, which probably allowed the nor-3 fruit attain the same (A) Difference of ripening behavior among NE, VED, nor-3, and nor-1. (B) Fruit rind and flesh of VED and nor-1 under natural ripening conditions. quality parameters as VED such as VOCs profile or flesh (C) Phenotypic comparison of seeds of nor-3 and nor-1 at 38 and 78 DAP, firmness (Figure 3), that are ethylene-dependent or partially respectively. (D,E) Germination efficiency of nor-3 and nor-1 seeds. (F-H) dependent (Pech et al., 2008). This was confirmed by the Phenotypic comparison according to flesh firmness (F), soluble solids content transcription analysis of genes involved in ethylene biosynthesis, (G), and fruit weight (H) between NE, VED, and nor-1. nor-1 + Eth: nor-1 fruit ester production, and fruit softening, where nor-3 showed after ethylene treatment. (I) Abscission layer of nor-1 appears after ethylene treatment. Lower case letters indicate significant differences (p < 0.05, n > 5). similar expression data as VED at harvest. In the complete knock-out mutant nor-1, ethylene production was blocked (Figure 2A), the expression of CmACS1 and CmACO1 was mutant where climacteric ripening behavior is almost absent, repressed (Supplementary Figure S1), the aroma component without ethylene or aroma production, a volatile profile similar changed (Figure 2C), CmAAT1 and CmADH2 were to unripe fruit, absence of abscission layer, with no external downregulated (Supplementary Figure S1), the rind color did color change (Figures 2, 4) and with low expression of several not change from green to yellow (Figure 4B), no abscission ripening-related genes (Supplementary Figure S1), suggesting layer formed, the flesh was firmer than VED (Figure 4F), and that CmNAC-NOR plays a significant role in regulating fruit two CmPGs were downregulated (Supplementary Figure S1). ripening in melon. Nor-3 is a knock-down mutant with one The flesh color of nor-1 seemed visually less orange than VED amino acid deletion at the NAC (NAM, ATAF1,2, CUC2) (Figure 4B), although we did not detect significant differences domain, which causes around 8 days delay in ripening, but in carotenoid content, nor in CmOr expression without affecting either fruit quality (Figure 3), nor gene (Supplementary Figure S1). It remains to be tested if the expression of several ripening-related genes at harvest composition of individual carotenoid compounds is altered in (Supplementary Figure S1), suggesting a potential way to the mutant, without modifying the total carotenoid content. control fruit ripening in melon by disrupting Our findings suggest that nor-1 resembles the tomato phenotypes Frontiers in Plant Science | www.frontiersin.org 7 June 2022 | Volume 13 | Article 878037 Liu et al. Knock-Out CmNAC-NOR Affects Melon Ripening of slnac-nor and CR-NOR mutants. Unlike the tomato CRISPR fruit ripening; CRISPR/Cas9 mutants of NOR-like1 delayed mutant CR-NOR, the main climacteric ripening components fruit ripening and seriously affected seed development, were almost blocked in nor-1 melon. In addition, nor-1 was reducing the number and weight of seeds, which showed insensitive to external ethylene treatment (Figure 4), except poor germination (Gao et al., 2018). However, the target for abscission layer formation, suggesting that CmNAC-NOR genes associated with seed development are still unknown. gene is a major key regulator of fruit ripening in melon. Only a recent study in grape showed that the NAC domain The different phenotypes of tomato and melon CRISPR gene VvNAC26, which positively regulates ethylene and NAC-NOR mutants might be explained by their different editing ABA-related genes to influence seed and fruit development, patterns. Although they lose the transcriptional regulation interacts with the transcription factor VvMADS9 (Zhang region, their NAC domain is altered at different levels. The et al., 2021). NAC domain contains five subdomains (A-E) that play an Non-climacteric melon cultivars as Piel de Sapo (PS), a important role in DNA-binding (Kikuchi et al., 2000; Ernst variety belonging to the inodorous group in the melo et al., 2004). The slnac-nor mutant has been found to contain subspecies, produce low amount of ethylene, insufficient a complete NAC domain, resulting in a gain-of-function mutation, to trigger the climacteric response and do not abscise when while the CR-NOR mutant produced a truncated protein of ripe. Climacteric varieties as VED, a variety from the 47 aa, which lost NAC subdomains B-E, but still had the cantaloupensis group in the melo subspecies, show a typical NAC subdomain A (Gao et al., 2020). Here, the editing of climacteric fruit ripening behavior, with a sharp ethylene nor-1 started from the NAC subdomain A (Figure 1E), so peak and noticeable related climacteric traits as abscission the whole NAC domain was affected in nor-1, resulting in a layer formation at around 35 DAP. There are no significant loss-of-function mutation, whereas nor-3 lost a single amino differences between both types in soluble solid content nor acid at NAC subdomain A, resulting in a delay of ripening. firmness of the flesh (Pereira et al., 2020), but the fruit Fruit flavor is an important trait as it affects consumer volatiles produced by both types are different (Mayobre preferences. Volatile esters are major contributors to fruit flavor et al., 2021). Three climacteric QTLs involved in fruit giving the fruity aroma to climacteric melons (El Hadi et al., ripening have been characterized in melon, ETHQB3.5, 2013). Compared to the controls, the esters content was ETHQV6.3, and ETHQV8.1 (Vegas et al., 2013; Pereira et al., dramatically reduced in nor-1 and the content of aldehydes 2020). So far, only the causal gene for ETHQV6.3 has been was increased, which explain the green, fresh aroma of these identified (CmNAC-NOR, Ríos et al., 2017). Introgression fruits. The nor-1 VOCs profile was more similar to unripe lines carrying the climacteric allele of each of the three melons or to non-climacteric melons such as inodorus types QTL in the non-climacteric PS background are able to (Mayobre et al., 2021) than to the VED profile. These results induce a very mild climacteric response (Vegas et al., 2013; suggest that CmNAC-NOR could be involved in the regulation Pereira et al., 2020). However, when combined in pairs or of the AAT genes, which are known to be ethylene-dependent the three of them together, they interact epistatically, and are responsible for volatile ester formation (El-Sharkawy producing a dramatic climacteric effect in the non-climacteric et al., 2005; Cao et al., 2021). As expected, when we measured background (unpublished). These data suggest that the the expression of CmAAT1, we observed that it was downregulated non-climacteric PS may be impaired in ethylene production in the nor-1 mutant, compared to the control lines and the due to variations in more than one gene, and that the nor-3 mutant. This is also consistent with a recent study that combination of two or more genes is necessary to rescue reported that the NAC transcription factor PpNAC1 (with the typical climacteric response. In addition, the homology to SlNAC-NOR) regulates fruit flavor ester biosynthesis non-climacteric allele of ETHQV8.1 in the VED background in peach by activating PpAAT1 expression (Cao et al., 2021). delays ripening but does not result in a strong non-climacteric However, to demonstrate that CmNAC-NOR directly binds phenotype (Pereira et al., 2020). A plausible hypothesis is CmAAT1, further experiments are needed. that at least these three genes/QTL are responsible of the An unexpected phenotype of nor-1 was that seeds were ripening differences between non-climacteric melons from not well developed (Figure 4C). This phenotype has not the inodorus group and the climacteric cantaloupe type. previously been reported in the tomato NOR mutant or in However, the complex molecular mechanisms cannot be yet other NAC genes in species such as peach (Pirona et al., understood until the causal genes of the other two QTL 2013), apple (Yeats et al., 2019), and strawberry (Martín- are identified. Still, we cannot rule out that additional genes Pizarro et al., 2021). However, there are some reports are responsible for conferring a non-climacteric response suggesting that NAC transcription factors regulate seed in other non-climacteric melon types phylogenetically distant development and play a role in seed germination (Kim from PS. et al., 2008; Park et al., 2011; Wang et al., 2021; Liu et al., In this study, we provide evidence that supports 2022). In a recent study, knock out of the ClNAC68 gene CmNAC-NOR as a key player in regulating climacteric fruit in watermelon delayed seed maturation and germination, ripening in melon. As a master regulator, CmNAC-NOR but the germination rate was not affected (Wang et al., independently mediates many ripening-associated traits. Our 2021), suggesting that there are additional NAC genes with findings also suggest that CmNAC-NOR can be a potential diverse functions that regulate seed development. In tomato, target in breeding programs to modulate fruit maturation NOR-like1 has been shown to be a positive regulator of and shelf life in melon. Frontiers in Plant Science | www.frontiersin.org 8 June 2022 | Volume 13 | Article 878037 Liu et al. Knock-Out CmNAC-NOR Affects Melon Ripening DATA AVAILABILITY STATEMENT de Catalunya and 2017 SGR 1319 grant from the Generalitat de Catalunya to JG-M. BL was also supported by grants from The original contributions presented in the study are included Youth Project of National Natural Science Foundation of China in the article/Supplementary Material, further inquiries can (31902035), The International Postdoctoral Exchange Fellowship be directed to the corresponding authors. Program of China (20170053), and a postdoctoral grant from the Severo Ochoa Programme for Centres of Excellence in R&D 2016–2010 (SEV-2015-0533). MS was supported by a AUTHOR CONTRIBUTIONS grant BES-2017-079956 funded by MCIN/AEI/ 10.13039/501100011033 and by “ESF Investing in your future”. BL did the experimental work and data analysis and wrote CM was supported by FI grant from the Secretaria d’Universitats the original draft of the manuscript. MS grew the plants, i Recerca del Departament d’Empresa i Coneixement de la phenotyped the ripening behavior, and measured the ethylene Generalitat de Catalunya and the co-funding of the European content. CM performed the expression and volatile experiments. Social Fund (ESF)—“ESF is investing in your future.” AM-H supervised the CRISPR-Cas9 and genetic transformation experiments. MP and JG-M designed and supervised the work and reviewed and edited the manuscript. All authors contributed ACKNOWLEDGMENTS to the article and approved the submitted version. The authors thank Fuensanta García and Elena del Blanco for technical assistance in field and lab operations and Laura FUNDING Valverde for carotenoid measurements. This work was supported by grants AGL2015–64625-C2–1-R and RTI2018-097665-B-C2 funded by MCIN/ SUPPLEMENTARY MATERIAL AEI/10.13039/501100011033 and by “ERDF A way of making Europe,” the Severo Ochoa Programme for Centres of Excellence The Supplementary Material for this article can be found online in R&D 2016–2010 (SEV-2015-0533) funded by MCIN/ at: https://www.frontiersin.org/articles/10.3389/fpls.2022.878037/ AEI/10.13039/501100011033, the CERCA Programme/Generalitat full#supplementary-material REFERENCES Chen, J., Liu, D., Jiang, Y., Zhao, M., Shan, W., Kuang, J., et al. (2011). Molecular characterization of a strawberry FaASR gene in relation to fruit ripening. Adaskaveg, J. A., Silva, C. J., Huang, P., and Blanco-Ulate, B. (2021). 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BMC Genomics 16:440. doi: 10.1186/ Publisher’s Note: All claims expressed in this article are solely those of the s12864-015-1649-3 authors and do not necessarily represent those of their affiliated organizations, Vegas, J., Garcia-Mas, J., and Monforte, A. J. (2013). Interaction between QTLs or those of the publisher, the editors and the reviewers. Any product that may induces an advance in ethylene biosynthesis during melon fruit ripening. be evaluated in this article, or claim that may be made by its manufacturer, is Theor. Appl. Genet. 126, 1531–1554. doi: 10.1007/s00122-013-2071-3 not guaranteed or endorsed by the publisher. Vrebalov, J., Pan, I. L., Arroyo, A. J. M., McQuinn, R., Chung, M., Poole, M., et al. (2009). Fleshy fruit expansion and ripening are regulated by the Copyright © 2022 Liu, Santo Domingo, Mayobre, Martín-Hernández, Pujol and tomato SHATTERPROOF gene TAGL1. Plant Cell 21, 3041–3062. doi: Garcia-Mas. This is an open-access article distributed under the terms of the Creative 10.1105/tpc.109.066936 Commons Attribution License (CC BY). The use, distribution or reproduction in Vrebalov, J., Ruezinsky, D., Padmanabhan, V., White, R., Medrano, D., Drake, R., other forums is permitted, provided the original author(s) and the copyright owner(s) et al. (2002). A MADS-box gene necessary for fruit ripening at the tomato are credited and that the original publication in this journal is cited, in accordance ripening-inhibitor (rin) locus. Science 296, 343–346. doi: 10.1126/ with accepted academic practice. No use, distribution or reproduction is permitted science.1068181 which does not comply with these terms. Frontiers in Plant Science | www.frontiersin.org 11 June 2022 | Volume 13 | Article 878037