Re-evaluation of the nor mutation and the role of the NAC-NOR transcription factor in tomato fruit ripening

The 186 aa truncated protein produced by the tomato spontaneous non-ripening (nor) mutant enters the nucleus and combines with the promoters of its target genes, resulting in a gain of function.


Introduction
Plant fruits protect developing seeds and aid in their dissemination (Forlani et al., 2019). They are also an important food source for humans and animals and are rich in nutrients such as carbohydrates, fats, proteins, vitamins, and trace elements (Giovannoni, 2004;Klee and Giovannoni, 2011). Fleshy fruit ripening and the generation of quality attributes occur towards Downloaded from https://academic.oup.com/jxb/article-abstract/71/12/3560/5825550 by University of Zagreb user on 23 June 2020 the end of seed development and render fruit attractive to animal and human consumers, further aiding seed dispersal (Klee and Giovannoni, 2011). Understanding fruit ripening provides an important theoretical and practical basis for manipulating the ripening process, improving fruit quality, and prolonging fruit shelf life (Gómez et al., 2014;Pesaresi et al., 2014). Tomato is a model plant for studying the ripening of climacteric fruit because of its simple diploid genetics, small genome size (approximately 950 Mb), short life cycle, ease of transient and stable transformation, distinct ripening phenotypes, and abundant bioinformatics resources (Alexander and Grierson, 2002;Giovannoni, 2004;Klee and Giovannoni, 2011). Molecular genetics studies have shown that tomato fruit ripening is governed by a transcription regulation network that is coordinated by a series of ripening-related transcription factors (TFs) (Karlova et al., 2014;Liu et al., 2015;Giovannoni et al., 2017;Li et al., 2019) and ethylene (Alexander and Grierson, 2002;Li et al., 2019). Exploring the roles of these ripening-related TFs is an effective tool for understanding the mechanisms involved in fruit ripening. Tomato has abundant natural mutants (Giovannoni, 2007), some of which have obvious ripeninginhibited phenotypes, such as ripening-inhibitor (rin) (Vrebalov et al., 2002), non-ripening (nor) (Mizrahi et al., 1976;White, 2002;Kumar et al., 2018), Colorless non-ripening (Cnr) (Eriksson et al., 2004;Manning et al., 2006), Never-ripe (Nr) (Lanahan et al., 1994;Wilkinson et al., 1995;Yen et al., 1995), and Green-ripe (Gr) (Barry et al., 2005;Barry and Giovannoni, 2006). Nr and Gr are related to ethylene signal transduction, while rin, Cnr, and nor TFs are involved in the transcription regulation network controlling the expression of tomato fruit ripening-related genes that determines quality attributes. However, several detailed studies of rin, Cnr, and nor mutants involving CRISPR/ Cas9 gene editing have caused the roles of these mutants to be re-evaluated (Ito et al., 2017;Li et al., 2018;Wang et al., 2019;Gao et al., 2019;Wang et al., 2020).
In rin mutant, almost all ripening-related phenotypes, including ethylene biosynthesis, carotenoid accumulation, fruit softening, and flavor synthesis, were significantly inhibited. In addition, the sepal size of rin mutant is increased, and the inflorescence is less ordered (Vrebalov et al., 2002). Studies have shown that rin is formed by the deletion of the 3′ end of the MADS-RIN gene and the 5′ end of the MADS-MC gene, resulting in the formation of a RIN-MC fusion gene. MADS-RIN is considered to regulate tomato fruit ripening, while MADS-MC is considered to affect sepal development and inflorescence, and rin was considered a loss-of-function mutant of RIN (Vrebalov et al., 2002). The phenotype of the rin mutant is a near complete inhibition of ripening, and based on this evidence, RIN was considered the core TF required for the tomato fruit ripening process, including ethylene biosynthesis and signal transduction, carotenoid synthesis, cell wall metabolism, aroma synthesis, sucrose metabolism, and other biological pathways (Qin et al., 2012(Qin et al., , 2016Fujisawa et al., 2013;Wang et al., 2014;Kumar et al., 2016;Li et al., 2017). Recent studies, however, have shown that the fusion protein RIN-MC in the rin mutant retains biological functions, and the role of RIN has been re-evaluated in light of this evidence (Ito et al., 2017;Li et al., 2018). The RIN protein segment of the RIN-MC fusion protein functions in binding DNA, while the adjacent MC region possesses a transcription repression function. This chimeric protein, RIN-MC, produced by the rin mutant is thus a gain-of-function mutant and active TF (Ito et al., 2017;Li et al., 2018) responsible for the inhibition of expression of ripening genes. It was concluded from this evidence that RIN was not required for the initiation of ripening but was essential for the completion of normal ripening (Ito et al., 2017;Li et al., 2018Li et al., , 2019. Compared with wild-type (WT), the Cnr mutant has reduced ethylene synthesis, fruit softening, and carotenoid synthesis in pericarp tissue (Eriksson et al., 2004). Mapping and identification of Cnr by Manning et al. (2006) showed that SPL-CNR belongs to the SBP family of TFs. There was no alteration in the SPL-CNR DNA sequence, but its promoter region was hypermethylated, and the transcription of the SPL-CNR gene was inhibited, giving rise to the Cnr ripening mutant phenotype (Manning et al., 2006). This was the first report of methylation affecting the expression of fruit ripening genes, but the exact cause of the methylation of SPL-CNR in the Cnr mutant remains unclear. Using CRISPR/Cas9 to edit SPL-CNR in WT fruit, Gao et al. (2019) found that the ripening of CR-CNR fruits was similar to that of WT tomatoes, and CR-CNR fruits fail to show a Cnr mutant phenotype. Therefore, the mechanism of action of the Cnr mutant and the function of SPL-CNR requires explanation and further study. Studies on the nor mutant and the function of NAC-NOR have lagged behind those of RIN and SPL-CNR and there is little information available regarding the mechanism of action of nor and the function of NAC-NOR. The synthesis of ethylene and carotenoids in the fruit of the nor mutant is significantly inhibited, and the fruit does not ripen. Giovannoni et al. (United States Patent, No. US 6 762 347 B1) discovered by map-based cloning that the nor mutant was caused by the deletion of two adenines in the third exon of the NAC-NOR gene, which belongs to the NAC gene family. Due to this frameshift mutation, the NAC-NOR protein in the nor mutant encodes a truncated NOR protein of 186 amino acids (aa) (NOR186), which disrupts the transcriptional activation region but preserves the complete DNA-binding region. Based on this evidence, the nor mutant phenotype was considered to be due to loss of function of the NAC-NOR gene, and NAC-NOR was considered to be a core TF regulating the initiation of tomato fruit ripening. Most NAC-NOR-related studies are based on the use of the nor mutant as experimental material. Yuan et al. (2016) compared the proteome differences between the nor mutant and WT tomato fruit by isobaric tags for relative and absolute quantification (iTRAQ) and found that the accumulation of many ripening-related and disease-resistance proteins was altered in the nor mutant. Additionally, the NAC-NOR mutation in Penjar tomato inhibited various metabolic processes and prolonged the shelf life of fruit (Kumar et al., 2018), whereas the overexpression of NAC-NOR accelerated the senescence of tomato leaves (Ma et al., 2019).
In addition to NAC-NOR, several other NAC TFs have been reported to be involved in regulating tomato fruit ripening. For example, the overexpression of SlNAC1 in tomato resulted in a decrease in ethylene synthesis and the early softening of fruit, producing a yellow to orange phenotype Meng et al., 2016). In addition, the silencing of SlNAC4 in tomato fruit resulted in a 2-3 d delay in fruit ripening and significantly inhibited ethylene biosynthesis, chlorophyll degradation, and carotenoid accumulation (Zhu et al., 2014). The ripening process in tomato fruit with CRISPR/Cas9 gene editing of NOR-like1 (CR-NOR-like1) was significantly delayed for more than 2 weeks, and ethylene, carotenoid synthesis, and fruit softening were inhibited in CR-NOR-like1 fruit compared with WT (Gao et al., 2018). Surprisingly, however, we have recently been unable to obtain a nor mutant phenotype in NAC-NOR-edited fruit using CRISPR/Cas9 (Gao et al., 2019), which was published simultaneously by the de Maagd laboratory (Wang et al., 2019), who demonstrated that NOR186 has an inhibitory function affecting ripening. Thus, the nor mutant may be a gain-of-function mutant, similar to rin, although the specific mechanism of action is unclear. If nor is a gain-of-function mutant, the role of NAC-NOR in the normal development and ripening of tomato and the function of the normal NAC-NOR gene in tomato fruit development and ripening need to be re-evaluated.
In this study, we investigated the results of CR-NOR and OE-NOR at the physiological, cellular, and molecular levels. The results showed that the residual protein NOR186 of the nor mutant could not only enter the nucleus but also bind to the promoters of NAC-NOR target genes, but could not activate them. While mixing the WT NOR protein and the nor mutation NOR186 protein, the activation effect of NOR target promoters was inhibited compared with the WT NOR protein present alone. In addition, overexpression of the NAC-NOR gene in the nor mutant did not restore the normal ripening phenotype of tomato, providing evidence for the gain-of-function of NOR186 in the nor mutant. Transcript accumulation studies indicate that NAC-NOR still plays an important role as a positive regulator in tomato fruit ripening. These results re-evaluated the role of NAC-NOR in tomato fruit ripening and help place it in the context of the transcriptional regulatory network regulating tomato fruit ripening.

Plant materials and growth conditions
WT tomato (Solanum lycopersicum) cultivar Ailsa Craig (abbreviated as AC) and NAC-NOR gene transgenic lines were grown under controlled greenhouse condition with natural light. Standard greenhouse culture conditions with regular fertilizer application were used. Comparing the ripening process of tomato fruit by calculating the time from flowering to fruit discoloration, flowers were tagged at anthesis to record the ripening stages accurately through fruit development. Fruits of WT and NAC-NOR transgenic lines were harvested and collected at different ripening stages: mature green (MG), breaker (Br), and 3, 6, and 9 d after breaker (B+3, B+6, and B+9, respectively). Pericarp tissues of the harvested fruits were collected and frozen in liquid nitrogen immediately and stored at −80 °C until use.
Total RNA isolation and quantitative real-time PCR analysis RNA isolation from fruits or all other tissues was performed with the RNeasy Mini Kit (Qiagen, Germany) according to the manufacturer's protocol. DNaseI (Qiagen, Germany) digestion was performed to remove genomic DNA. Transcript One-Step gDNA Removal and cDNA Synthesis SuperMix (TransGen, China) were used to synthesize cDNA from 2 μg total DNaseI-treated RNA. Quantitative real-time PCR (qRT-PCR) was conducted using SYBR Green PCR Master Mix (TransGen, China) with a CFX96 Real-Time PCR System (Bio-Rad, USA). The tomato ACTIN gene (Solyc03g078400) was used as the internal control. Relative gene expression values were calculated according to the 2 −△△Ct method (Livak & Schmittgen, 2001). For each sample, three biological replicates were included. All primers used for qRT-PCR are listed in Supplementary Table S1.

Ethrel and 1-methylcyclopropene treatment
Wide-type tomato fruits at the mature green stage were treated with 0.4% ethrel or double distilled water (DDW) as control for 10 min (Breitel et al., 2016), and then dried carefully and placed at room temperature for 12 h. Wild-type tomato fruits at the breaker stage were treated with 1.0 mg l −1 the ethylene signaling inhibitor 1-methylcyclopropene (1-MCP) or air as control for 16 h (Hao et al., 2015). The pericarp tissues were sliced and frozen in liquid nitrogen after treatment for RNA isolation and qRT-PCR. For each treatment, three biological replicates from independent samples were included.

Subcellular localization
The coding sequence (CDS) fragment of NAC-NOR and NOR#19 without the stop codon were amplified respectively by PCR (primers used are listed in Supplementary Table S2) and then inserted into the pEAQ-GFP vector to produce the fusion construct NOR-GFP/NOR#19-GFP using the ClonExpress II One-Step Cloning Kit (Vazyme, China). The CDS region of NOR186 and RIN (without the stop codon) was amplified respectively by PCR and inserted into the pEAQ-mCherry vector to produce the fusion protein NOR186-mCherry/RIN-mCherry using ClonExpress II One Step Cloning Kit (Vazyme, China). Then, pEAQ-NOR-GFP, pEAQ-NOR186-mCherry, pEAQ-NOR#19-GFP, pEAQ-RIN-mCherry, and the control vectors (pEAQ-GFP and pEAQ-mCherry) were transferred into Agrobacterium tumefaciens strain GV3101 and injected into 4-week-old tobacco leaves (Luo et al., 2017). The green fluorescence of green fluorescent protein (GFP) and the red fluorescence of mCherry were observed and captured by a laser confocal microscope (Leica, Germany) after 48 h of infiltration.

pYLCRISPR/Cas9Pubi-H-NOR and pCAMBIA-1300-221-NOR-3×HA vector construction and tomato genetic transformation
CRISPR-P (http://cbi.hzau.edu.cn/crispr/) was used to select four sgRNAs that targeted NAC-NOR; the four sgRNAs were cloned into the pYLCRISPR/Cas9Pubi-H binary plasmid using Golden Gate ligation (Ma et al., 2015). The CDS sequence of NAC-NOR (without the stop codon) and a 3×HA-tag (at the C-terminus) were amplified respectively and cloned into the pCAMBIA-1300-221 vector to generate the 35S:NOR-3×HA construct for overexpression of the NAC-NOR gene (primers used are listed in Supplementary Table S2). The two vectors sequenced correctly were transformed into A. tumefaciens strain GV3101. Then pYLCRISPR/Cas9Pubi-H was transformed into AC tomato, while pCAMBIA-1300-221-NOR-3×HA was transformed into the WT tomato (AC) and the nor mutant as described previously (Van Eck et al., 2006). The transgenic tomato lines were selected through their hygromycin resistance.

DNA extraction and mutation analysis
Total genomic DNA was extracted from tomato leaves using the DNA Secure Plant Kit (Tiangen, China) according to the manufacturer's instructions. The DNA was used as a template for amplifying the desired gene fragments using primers flanking the target sites or the possible offtarget sites. For each target, two of the most likely off-target sites were tested. The oligonucleotide primers used for off-target analysis are listed in Supplementary Tables S3, S4.

Gene overexpression analysis
Tomato fruits of the NAC-NOR gene overexpression in WT (OE-NOR in WT#2 and OE-NOR in WT#8) and in nor mutant (OE-NOR in nor#1 and OE-NOR in nor#16) at the stage of 20 d post-anthesis (dpa) were used for detection of the expression level of the NAC-NOR gene. Wild-type AC tomato fruits of 20 dpa were used as the control. Three biological replicates were performed.

RNA sequencing and bioinformatics analysis
Total RNA was extracted from tomato fruits at the B+3 stage of WT, nor#11, OE-NOR in WT#2, and the nor mutant using the RNeasy Mini Kit (Qiagen, Germany); the genomic DNA was removed by DNase I (Qiagen, Germany) digestion. For each sample, three biological replicates were performed. mRNA enrichment, RNA-seq library construction, and sequencing were performed by Novogene (China). The clean data were mapped to the tomato reference genome (version SL2.50) using TopHat software (version 2.0.14). Fragments were assigned to genes by feature counts and count programs, and gene expression abundance was represented by the reads per kilobase of transcript per million mapped reads (RPKM) value. Differential expression analysis between nor#11 and WT, OE-NOR in WT#2 and WT, and nor mutant and WT were identified by DESeq2 Library (Anders and Huber 2010). The fold change of gene expression was calculated by RPKM nor#11 /RPKM WT , RPKM OE-NOR-in-WT#2 /RPKM WT , and RPKM nor /RPKM WT . Genes were considered as differentially expressed genes (DEGs) if |log 2 (fold change)|>1 and modified P adj <0.05. The common DEGs in nor#11/WT and OE-NOR in WT#2/WT with opposite regulatory patterns were screened for further analysis of the function of NAC-NOR.

GO enrichment analysis
GO enrichment analysis was performed using the GO seq R package (Young et al., 2010) based on DEGs that were common in nor#11/WT and OE-NOR in WT#2/WT with opposite regulatory patterns, with a threshold of P<0.05. Proteins were filtered based on their grouping into cellular components, molecular functions, and biological functions.

Ethylene measurement
Tomato fruits of WT, nor#11, OE-NOR in WT#2, and nor mutant at different ripening stages (MG, Br, B+3, B+6, and B+9) were harvested, weighed, and placed at room temperature for 2 h to avoid measuring 'wound ethylene'. Subsequently, each fruit was transferred into 300 ml gas-tight jars, tabbed, sealed, and incubated at room temperature. After 2 h, 1 ml gas samples were withdrawn immediately and then analysed by GC-2014 gas chromatography (Shimadzu, Japan). At least three biological replicates, each with three technical replicates, were performed for each sample. Ethylene concentrations were calculated by comparing sample peak areas with ethylene standards of known concentration, ethylene production (nl g −1 h −1 ) was calculated by the following formula: Ethylene production (nl g −1 h −1 )=[C×(300−M)/M]/2, where C is ethylene concentration and M is the mass of the fruit.

Carotenoid extraction and liquid chromatography-quadrupole time of flight mass spectrometry analysis
Carotenoids of tomato fruits at the B+9 stage from WT, nor mutant, nor#11, and OE-NOR in WT#2 were extracted as described previously (Fantini et al., 2013) with minor modifications. The whole extraction process avoided light and was carried out at low temperature because carotenoids are volatile and oxidize in organic solvents. For each sample, four independent extractions were performed. Carotenoids were detected and identified, and the relative contents were determined by liquid chromatography-quadrupole time of flight mass spectrometry as previously described (Fantini et al., 2013).

Firmness measurement
In order to study the effect of NAC-NOR on tomato fruit softening, we measured the firmness of tomato fruits from WT, nor mutant, nor#11, and OE-NOR in WT#2 at different ripening stages (MG, Br, B+3, B+6, B+9) using a TA.XT Plus texture analyser (Stable Micro Systems, UK) with a 5 cm diameter cylindrical stainless probe. Fruit firmness examination was performed as described previously with minor revision (Wu and Abbott, 2002). The junction of outer and radial pericarp was compressed 2 mm at a test speed of 1 mm s −1 ; visible vascular bundles, fissures, and locular tissue were avoided. Each fruit was measured at two or three sites, and the average of the maximum force developed during the test was used as one biological replicate. At least three fruits per genotype and stage were measured.

Protein expression and electrophoretic mobility shift assay
The partially coding sequence of NAC-NOR and the whole coding sequence of NOR186 and NOR#19 were amplified from the cDNA of AC, nor mutant, and nor#19, respectively. Then the products were inserted into the linearized pGEX-GST vector, which was digested with BamHI and XhoI to produce recombinant NOR/NOR186/NOR#19 protein with a glutathione S-transferase (GST) tag using ClonExpress II One Step Cloning Kit (Vazyme, China). The N-GST-NOR/NOR186/ NOR#19 fusion protein was expressed in Escherichia coli strain Rosetta (DE3) and induced with 1 mM isopropyl β-D-1-thiogalactopyranoside at 37 °C for 6 h, at 37 °C for 6 h, and at 20 °C for 12 h, respectively, and further purified with Glutathione Sepharose 4B (GE Healthcare, Sweden) following the manufacturer's protocol. The oligonucleotide probes (shown in Supplementary  et al., 2016) regions derived from the promoters of SlACS2, SlGgpps2, and SlPL were synthesized (Sangon Biotech, China) and biotin-labeled using the Biotin 3′ End DNA Labeling Kit (Thermo Scientific, USA). An electrophoretic mobility shift assay (EMSA) was carried out as previously described (Han et al., 2016;Tan et al., 2018). Both the unlabeled probes and the mutant probes were used for competition; GST protein alone was used as negative control.

Dual-luciferase reporter assay
A dual-luciferase reporter assay was performed to analyse the transcriptional activity of NAC-NOR, NOR186, and NOR#19 with the promoters of SlACS2, SlGgpps2, and SlPL. The CDS fragment of NAC-NOR, NOR186, and NOR#19 was amplified and cloned into the 35S promoter-driven pEAQ vector (Sainsbury et al., 2009) to construct the effector vectors pEAQ-NOR, pEAQ-NOR186, and pEAQ-NOR#19. The 1-2 kb promoter regions of SlACS2, SlGgpps2, and SlPL that were reported in our previous study (Gao et al., 2018) were amplified and inserted into the linearized double-reporter vector pGreenII 0800-LUC (Hellens et al., 2005) to construct pGreenII 0800-SlACS2-LUC/pGreenII 0800-SlGgpps2-LUC/pGreenII 0800-SlPL-LUC using ClonExpress II One Step Cloning Kit (Vazyme, China). Subsequently, the recombinant effector was transferred into A. tumefaciens strain EHA105, and the reporters were transferred into EHA105 (pSoup). The A. tumefaciens containing effector/reporter was inoculated, collected, and suspended in infiltration buffer (10 mM MES, 10 mM MgCl2, 150 mM acetosyringone). The effector and reporter were mixed (9:1) and then co-infiltrated into 4-week-old tobacco (Nicotiana benthamiana) leaves as described previously (Fan et al., 2016;Tan et al., 2018). To investigate whether NOR186 has a competitive effect on the NOR protein, NOR186 was added as the same concentration with NOR, and then mixed with the reporter at 9:1 to perform the infiltration. After 48 h of infiltration, LUC and REN luciferase activities were measured respectively using the dual-luciferase assay kit (Promega, USA) with the Luminoskan Ascent Microplate Luminometer (Thermo Fisher Scientific, USA). The results were calculated as the ratio of LUC to REN. At least five biological replicates were performed for each combination.

Statistical analysis
Significance analysis of corresponding experimental data was conducted using IBM SPSS Statistics 20 software. Pairwise comparisons were computed using Student's t-test (*P<0.05 and **P<0.01), while multiple comparisons were subjected to ANOVA using Duncan's test. Statistically significant differences (P<0.05) are indicated by different lowercase letters.

The expression of NAC-NOR is ripening-related and induced by ethylene
To investigate the activity of the NAC-NOR gene during fruit development, the accumulation of NAC-NOR transcripts in various WT tomato plant organs and during fruit development and ripening was measured by qRT-PCR. The results showed that the expression of the NAC-NOR gene in vegetative organs such as root, stem, and leaf of tomato was low, while it was high in reproductive organs such as flower and fruit (Fig. 1A), which suggested that it may play an important role in tomato fruit ripening. Ethylene is a key hormone in the ripening of climacteric tomato, and many ripening-related genes are induced by ethylene during fruit ripening (Barry and Giovannoni, 2007;Kumar et al., 2014). To study the relationship between NAC-NOR expression and ethylene, we used treatment with an ethylene-generating compound (ethrel) and an ethylene perception inhibitor (1-methylcyclopropene; 1-MCP) to treat WT tomato fruits at mature green and breaker stages of fruit ripening, respectively. The results showed that the expression of the NAC-NOR gene in tomato fruit was induced by ethylene but inhibited by 1-MCP (Fig. 1B). Based on the above results, it could be hypothesized that the NAC-NOR gene is specifically expressed during tomato ripening and induced by exogenous ethylene treatment. This is consistent with a role for NAC-NOR in tomato fruit ripening, but this hypothesis requires further functional verification.  Re-evaluation of the role of NAC-NOR in tomato fruit ripening | 3565 CRISPR/Cas9-mediated mutagenesis of the NAC-NOR gene inhibited fruit ripening but resulted in a phenotype that was different from that of the nor mutant To further study the role of NAC-NOR in tomato fruit ripening, we carried out CRISPR/Cas9-mediated mutagenesis of the NAC-NOR gene by transgenic methods. Two homozygous CR-NOR plants named nor#11 and nor#19 were obtained, which encode truncated NAC-NOR proteins with 47 and 61 aa, respectively ( Fig. 2A; Gao et al., 2019). The DNA-binding and transcriptional regulatory region (TRR) of the NAC-NOR protein were both destroyed in nor#11 and nor#19 (Fig. 2A). These results suggest that nor#11 and nor#19 are loss-of-function mutants suitable for the analysis of NAC-NOR gene function. The CR-NOR phenotype is not consistent with the mature phenotype of the nor mutant (Wang et al., 2019;Gao et al., 2019); however, the authors of these studies did not provide sufficient information to describe the ripening process of CR-NOR fruit. To study the effect of NAC-NOR on the fruit development and ripening process in tomato, the days from flowering to fruit color break were recorded. Color break in nor#11 and nor#19 fruits was delayed only 3 d compared with that in WT fruits, which is accordance with the CRISPR-NOR phenotype described by Wang et al. (2019), but the subsequent accumulation of carotenoids in nor#11 and nor#19 fruits was significantly reduced compared with that in the control tomato fruit (Fig. 2D). The WT fruit turned completely red 9 d after breaker (B+9), while the fruits of nor#11 and nor#19 maintained an orange-yellow phenotype. The breaker stage of the nor mutant occurred approximately 50 d after anthesis, significantly later than that of CR-NOR, and the accumulation of pigments in the fruit of the nor mutant was significantly inhibited compared with that of WT (Fig. 2D). Even 30 d after color break, the fruit of the nor mutant still remained light yellow, while the color of nor#11 and nor#19 fruits was orange-red and more similar to that of the WT fruit (Gao et al., 2019). The color of CR-NOR fruit was obviously different from that of the nor mutant fruit at the final ripening stage (B+30) (Gao et al., 2019). These results indicated that CR-NOR inhibited tomato fruit ripening, but this inhibition was much less severe than the inhibition of fruit ripening in the nor mutant fruit; these results confirmed Wang et al.'s results and our earlier observations (Gao et al. 2019;Wang et al., 2019) and provide new evidence to explain them.
Overexpression of NAC-NOR significantly accelerates the fruit ripening process in tomato but cannot completely restore the ripening phenotype of the nor mutant fruit To provide additional biological evidence for the role of the NAC-NOR gene in fruit ripening, we overexpressed NAC-NOR in WT tomato. A total of 10 T 0 transgenic tomatoes were obtained. Two lines with elevated NAC-NOR gene expression were selected as OE-NOR in WT#2 and OE-NOR in WT#8 for further research. The expression of NAC-NOR in OE-NOR in WT#2 and OE-NOR in WT#8 was higher than that in WT tomato fruits (Fig. 2B). At the same time, we observed and recorded the ripening characteristics of OE-NOR fruits. The results showed that color break in WT tomatoes occurred 38 d after flowering, but in OE-NOR in WT#2 and OE-NOR in WT#8, color break occurred approximately 26 d after flowering (Fig. 2C). We also found that OE-NOR in WT#2 and OE-NOR in WT#8 fruits accumulated pigments more rapidly after color break and finally showed a deep red phenotype (Fig. 2C). We also overexpressed the NAC-NOR gene in the nor mutant (Fig. 2B). The results showed that although OE-NOR in the nor mutant significantly accelerated fruit ripening compared with the nor mutant, it could not completely restore the ripening phenotype of the nor mutant to the WT pattern (Fig. 2D). Color break in the nor mutant with OE-NOR occurred earlier than that in nor mutant fruit, but later than that in WT (Fig. 2D). These results indicate that the phenotype of the nor mutant is not explained solely by the loss function of NAC-NOR. OE-NOR in WT tomato fruit, however, accelerated tomato fruit ripening, implying that NAC-NOR does play an important role in regulating the tomato fruit development and ripening process.

NAC-NOR regulates several aspects of tomato fruit ripening
To study the effects of NAC-NOR on the important physiological characteristics of tomato fruit ripening, we measured ethylene production, carotenoid content, and fruit firmness of WT, nor, nor#11 and OE-NOR in WT#2 tomato fruits at different ripening stages (MG,Br,B+3,B+6,and B+9). The results showed that ethylene production (Fig. 3A), carotenoid content (Fig. 3B), and fruit softening (Fig. 3C) of the nor#11 fruit were significantly reduced compared with those of WT fruit at the same stage; ethylene production ( Fig. 3A), carotenoid content (Fig. 3B), and fruit softening (Fig. 3C) of the OE-NOR in WT#2 fruit were significantly higher than those of the WT control fruit at the same stage, whereas the nor mutant fruit produced a very small amount of ethylene (Fig. 3A) and carotenoids (Fig. 3B), and the fruit firmness (Fig. 3C) of nor mutant was significantly higher than that of WT and CR-NOR mutants from 3 d after the breaker (B+3) stage. These results indicated that the NAC-NOR gene was a positive regulator of tomato fruit ripening but the inhibition of ripening in the nor mutant was much more severe than that in CR-NOR mutants.
To further elucidate the molecular mechanism of NAC-NOR regulation of tomato fruit ripening, we performed transcriptome sequencing (RNA-sequencing) on tomato fruits of nor#11, OE-NOR in WT#2, nor mutant, and WT at 3 d after color break. We screened DEGs based on the threshold of |log 2 (fold change)|>1 and P adj <0.05. When all data were compared with the transcript accumulation in the WT fruits, the results showed that 1428 genes were significantly up-regulated and 997 genes were significantly down-regulated in nor#11. Fig. 3. NAC-NOR regulates many aspects of tomato fruit development and ripening, including ethylene production, carotenoid content, and fruit firmness. (A) Ethylene production. Fruits were harvested at different stages, and placed at room temperature for 2 h to allow 'wound ethylene' to subside. Subsequently, each fruit was transferred into 300 ml gas-tight jars, tabbed, sealed, and incubated at room temperature. After 2 h, 1 ml gas samples were withdrawn immediately and then analysed; significant differences (P<0.05) are indicated by lowercase letters. (B) Carotenoid content of the fruit of four different samples (WT, nor, and nor#11 and OE-NOR in WT#2) was quantified by HPLC at the B+9 stage. The upper panel shows the phenotype of tomato fruits at the B+9 stage, and the lower panel shows the carotenoid content. Significant differences (P<0.05) are indicated by lowercase letters. (C) The firmness of tomato fruits. Fruits were harvested at different stages, kept at room temperate, and used for each measurement of the force required for probe penetration (F max ). Significant differences (P<0.05) are indicated by lowercase letters. B+3, 3 d after breaker; B+6, 6 d after breaker; B+9, 9 d after breaker; Br, breaker; MG, mature green.
A total of 1185 genes were significantly up-regulated and 1406 genes were significantly down-regulated in OE-NOR in WT#2, while 2673 genes were up-regulated and 1827 genes were down-regulated in the nor mutant (Fig. 4A). We screened the key DEGs related to fruit ripening from nor#11 and compared the transcript levels to those in OE-NOR in WT#2 (Table 1). GO enrichment analysis of DEGs by GO seq showed that CR-NOR affected not only carotenoid biosynthesis and cell wall metabolism (zeaxanthin epoxidase activity, terpenoid biosynthesis process, xyloglucan: xyloglucosyl transferase activity) but also the synthesis or metabolic processes of sugar, carbohydrate, fatty acid, anthocyanin, flavanol, and other substances (sucrose synthase activity, S-adenosylmethioninedependent methyltransferase activity, flavonol synthase activity, anthocyanin 5-O-glucosyltransferase activity, glycolysis process, carbohydrate metabolic process, lipid transport, fatty acid metabolic process) (Fig. 4B). Combining the DEG data from the RNA-seq data of the above materials, we found that the extent of down-regulation of genes related to ethylene synthesis and signal transduction (SlACS2,SlACS4,SlACO1,SlACO3,SlETR4,SlE4,SlE8,and SlERF2),carotenoid metabolism (SlDXS,SlGgpps2,SlPSY1,SlPDS,SlZDS,and SlCRTISO), and fruit softening (SlPG2a,SlPME2,SlTBG4,SlPL,SlCEL2,SlCEL8,and SlEXP1) in the nor mutant was significantly higher than that of the CR-NOR mutant. Different transcriptional levels of these key ripening-related genes may be one reason for the differences in phenotype between the nor mutant and CR-NOR mutant (Fig. 5A). To verify the validity of the sequencing results as well as to confirm the regulatory effect of NAC-NOR on the key ripening-related genes, qRT-PCR was used to measure the expression of selected genes (SlACS2, SlACO3, SlE4, SlERF2, SlGgpps2, SlSGR1, SlPG2a, SlTBG4, SlEXP1, and SlCEL2) at the B+3 stage of WT, nor#11, nor#19, OE-NOR in WT#2, and OE-NOR in WT#8 (Fig. 5B), and the results were consistent with the sequencing results. Taken together, the above results indicated that NAC-NOR regulates several aspects of tomato fruit ripening and is beyond any doubt important, but the phenotype of the nor mutant is not only caused by the loss of NAC-NOR function.

The nor mutant truncated protein NOR186 is located in the nucleus and can bind to but not activate the promoters of ripening-related target genes
The nor mutant gene lacks two adenines, resulting in the early termination of translation and the production of the truncated 186-aa protein NOR186. Despite the lack of a TRR, the complete DNA-binding region is present in NOR186 ( Fig. 2A). Subcellular localization showed that NOR186 was located in the nucleus, while the NOR#19 truncated protein from nor#19, generated by CRISPR/Cas9, was not located in the nucleus (Fig. 6), which indicated that nor#19 is a true loss-offunction mutant. Promoter analysis and RNA-seq analysis indicated that SlACS2, SlGgpps2, and SlPL, which are involved in ethylene biosynthesis, carotenoid synthesis, and fruit softening, respectively, are the target genes of the NAC-NOR protein.
EMSA results showed that the NOR186 protein could bind to the conserved promoter regions of the three target genes, while the truncated protein NOR#19 could not bind to these promoter regions (Fig. 7A). Dual-luciferase reporter assay results showed that the relative LUC/REN ratio in tobacco leaves co-transformed with CaMV35S-NOR and CaMV35S-REN/pSlACS2-LUC, CaMV35S-REN/pSlGgpps2-LUC, or CaMV35S-REN/pSlPL-LUC was significantly higher than when co-transformed with CaMV35S-Empty and CaMV35S-REN/pSlACS2-LUC, CaMV35S-REN/pSlGgpps2-LUC, or CaMV35S-REN/pSlPL-LUC, but there was no significant difference between NOR186/NOR#19 and the control group. These results indicated that NAC-NOR could activate the promoter activity of SlACS2, SlGgpps2, and SlPL in tobacco, while NOR186 and NOR#19 could not (Fig. 7B). When CaMV35S-NOR was co-expressed with CaMV35S-NOR186 (mixing the WT NOR protein and the nor mutation NOR186 protein), the activation effect of SlACS2, SlGgpps2, and SlPL promoters was inhibited compared with the WT NOR protein occurring alone. This result demonstrated that NOR186 is a dominant-negative protein, which is consistent with the hypothesis of Wang et al. (2019). These results also explain why overexpressed NOR in the nor mutant cannot fully restore its immature phenotype. Based on the above results, it is concluded that NAC-NOR protein can enter the nucleus, bind to and activate the promoters of its target genes, while NOR186 also can enter the nucleus and bind to the promoter region of the target genes but cannot activate their promoters and play the role of a dominant-negative protein.
Thus, nor is a gain-of-function mutant.

NAC-NOR gene editing delays the initiation of tomato fruit ripening
Mature fruit of nor mutant produces no ethylene burst, undergoes very little change in carotenoid content, and almost completely fails to ripen (Mizrahi et al., 1976;Davies et al., 1991;White 2002;Kumar et al., 2018). Based on these results, NAC-NOR has long been considered one of the core TFs regulating the initiation and progression of tomato fruit ripening (Giovannoni, 2004(Giovannoni, , 2007. However, the fruit of homozygotes with NAC-NOR gene mutations induced by CRISPR/Cas9 technology shows a much less severe phenotype than the nor mutant, and the fruit undergoes significant ripening (Fig. 2C, D; Gao et al., 2019;Wang et al., 2019). The ripening of CR-NOR tomato fruit was partially inhibited, the breaker stage of CR-NOR fruit was delayed by only 3 d compared with that of WT fruit  2C). Ethylene and carotenoids were still synthesized, and fruit softening was initiated in CR-NOR fruit (Fig. 3). The color of CR-NOR tomato fruit was close to that of WT fruit 30 d after the color break stage, but the mature phenotype was obviously different (Gao et al., 2019). From these observations, it can be inferred that NAC-NOR gene editing delays the initiation of tomato fruit ripening. Recent studies have found that the RIN-MC fusion protein of the rin mutant, rather than being a loss of function mutation, is a gain-of-function TF that regulates tomato fruit ripening by transcriptional inhibition (Ito et al., 2017;Li et al., 2018). This new evidence shows that the RIN gene is not a necessary element for ripening initiation but is required for the development of full ripening attributes (Ito et al., 2017;Li et al., 2018). The question arises as to whether there is a similar explanation for the mechanism of action of the nor mutation.

NOR186 protein from the nor mutant inhibits accumulation of some ripening genes and nor is a gain-of-function mutation
The de Maagd laboratory at Wageningen University and our laboratory generated CR-NOR mutants in WT tomato by CRISPR/Cas9 technology and showed that the ripening of CR-NOR tomato fruit was partially inhibited (Gao et al., 2019;Wang et al., 2019). The color change in CR-NOR fruit after color break was slower than that in WT fruit, and CR-NOR fruit became orange, rather than red, 9 d after color break, which is obviously different from the almost completely inhibited ripening phenotype of nor mutant fruit ( Fig. 2C; Wang et al., 2019). Wang et al. (2019) further edited NOR186 in the nor mutant to obtain a phenotype similar to that of CR-NOR in the WT background. They concluded that the mutant nor protein (here referred to as NOR186) is a dominant-negative protein. They also speculated that the truncated protein (NOR186) in the nor mutant still has the ability to interact with other NAC proteins and to bind DNA without transcriptionally activating its targets (Wang et al., 2019). Here, we provide further experimental evidence for the hypothesis that NOR186 is localized in the nucleus and is capable of binding to the promoters of SlACS2, SlPL, and SlGgpps2 target genes, but is incapable of activating them (Fig. 7). This contrasts with the behavior of NOR#19, produced by CRISPR-Cas9, which does not enter the nucleus, does not bind to and cannot activate the promoters of SlACS2, SlPL, and SlGgpps2 (Fig. 7). Furthermore, overexpression of NAC-NOR in the nor background could not completely restore the ripening quality attributes to the level of WT fruits, as exemplified by their inability to turn completely red 15 d after color break (Fig. 2D). This phenotype was explained by co-expressing CaMV35S-NOR with CaMV35S-NOR186; the activation effect of SlACS2, SlGgpps2, and SlPL promoters was inhibited compared with the WT NOR protein present alone (Fig. 7B). Based on our above results, and combining the hypothesis of Wang et al., we constructed a model of the NOR and nor mutants' functions in tomato fruit ( Fig. 8; Wang et al., 2019Wang et al., , 2020. In WT tomato, WT NOR protein interacts with other fruit ripeningassociated TFs including other NAC TFs, binds to the NACRS, and activates ripening-associated target genes such as SlACS2, SlPL, and SlGgpps2 to regulate tomato fruit ripening. Besides, other NAC TFs that are not interact with NOR protein can also bind to the NACRS and activate the same genes at the following tomato ripening stages (Fig. 8A). In nor mutant fruit, NOR186 lacks the TRR, but retains the DNA binding region, and the protein complex of NOR is still present and can bind to the promoters of the target genes, but is unable to activate them. In addition, NOR186 can play a space-occupying role and stop other NAC TFs from binding the same NACRS site of the same target genes such as SlACS2, SlPL, and SlGgpps2 at the following tomato ripening stages (Fig. 8B). However, the specific mechanism of nor functional transformation in the mutants is unclear, and needs further research. Fig. 6. The truncated NOR186 protein is located in the nucleus. Subcellular localization of NAC-NOR, NOR186 (residual NOR truncated protein in the nor mutant), and NOR#19 (NOR truncated protein in NOR CRISPR line nor#19) was performed in 4-week-old tobacco (N. benthamiana) leaves and photographs were taken 48 h post-incubation under confocal microscopy. GFP and mCherry were co-expressed as the control. NOR-GFP and RIN-mCherry (marker protein for the nucleus) were co-expressed in the nucleus of tobacco leaves, NOR186-mCherry and NOR-GFP were also co-expressed in the nucleus, while NOR#19-GFP and the GFP protein alone were dispersed throughout the cell. Green and red fluorescence images were taken in a dark field, while the outline of the cell was photographed in a brightfield.

NAC-NOR is a positive regulator of tomato fruit ripening, and its function is different from that of NOR-like1
The ripening process in tomato fruits is regulated by many ripening-related TFs, some of which play a positive role, such as RIN (Ito et al., 2017;Li et al., 2019), TDR4 (Bemer et al., 2012;Fujisawa et al., 2014;Zhao et al., 2019), and TAGL1 (Itkin et al., 2009;Vrebalov et al., 2009), whereas others play a negative role, such as AP2a (Karlova et al., 2011;Wang et al., 2019) and SlMADS1 (Dong et al., 2013). Here, we found that the ripening process of CR-NOR fruit is delayed and inhibited, while the ripening process of OE-NOR fruit is significantly accelerated (Fig. 2C). Physiological analysis of the materials revealed a significant decrease in ethylene production, carotenoid accumulation, and fruit softening in CR-NOR fruit and a significant increase in OE-NOR fruit (Fig. 3). Sequencing results showed that the expression levels of genes related to these three pathways also changed accordingly (Fig. 5). All of the data indicated that NAC-NOR is a positive regulator of tomato fruit ripening. It has been reported that four NAC family members, SlNAC1, SlNAC4, NOR-like1, and NAC-NOR, participate in tomato fruit ripening Zhu et al., 2014;Meng et al., 2016;Gao et al., 2018Gao et al., , 2019Wang et al., 2019). There are examples where different members of the same gene family of TFs, such as RIN and TDR4, participate in the regulation of target genes by forming oligomers (Li et al., 2019). However, it is still unclear whether the four NAC gene-coding proteins interact with each other. NAC-NOR and our previously reported NOR-like1 belong to the same evolutionary clade. They have a close relationship and have 62.84% amino acid sequence identity (Gao et al., 2018). However, the ripening phenotypes of the fruit in which each of these genes has been edited are significantly different. Compared with WT, CR-NOR fruit had only a 3 d delay in color break and the accumulation of pigments was slower than in WT (Fig. 2C). Ethylene production occurred, but at a reduced rate, and softening also occurred, but the mature fruit phenotype was orange-red rather than red (Gao et al., 2019). In contrast, compared with the control fruit, CR-NOR-like1 fruit had a delay in color break of at least 14 d (Gao et al., 2018). After color break, the production of pigments, ethylene biosynthesis, and softening were also significantly inhibited. Thus, NOR and NOR-like1 have some similar functions but also some obvious differences in the development and maturation of tomato fruits.
NOR-like1 appears to be more important for fruit ripening initiation, whereas NAC-NOR has a stronger influence on carotenoid accumulation. There may not be an absolute separation of functions, however, since overexpression of NAC-NOR does affect fruit development and the timing of ripening. In the WT tomato, the complete NAC-NOR protein can enter the nucleus, interact with other ripening-related TFs, bind to the NACRS (which other NAC TFs also can bind to at different ripening stages) of ripening-related genes, such as SlACS2, SlGgpps2, and SlPL, and activate their expression, promoting ethylene production, carotenoid accumulation, and fruit softening. (B) In the nor mutant, the truncated NOR186 protein can enter the nucleus, interact with other ripening-related TFs, and bind to the NACRS of ripening-related genes, such as SlACS2, SlGgpps2, and SlPL, but cannot activate the transcription of these genes due to the lack of a transcriptional regulation region. On the other hand, the complex of NOR186-other TFs may occupy the NACRS promoter regions of its target genes when other NAC TFs need to bind at the following ripening stages, thereby inhibiting the binding of other NAC TFs to these regions. As a result, ethylene production, carotenoid accumulation, and fruit softening are depressed. Downloaded from https://academic.oup.com/jxb/article-abstract/71/12/3560/5825550 by University of Zagreb user on 23 June 2020

Supplementary data
Supplementary data are available at JXB online. Dataset S1. RNA-Seq data of nor#11/WT, OE-NOR in WT#2/WT, and nor/WT. Table S1. Primers used for qRT-PCR. Table S2. Primers used for vector construction. Table S3. Primers used for off-target site mutation analysis. Table S4. Detection of mutations on putative off-target sites. Table S5. Probes containing NACRS used in EMSA.