This same spinach leaf antiserum has been shown to recognize AGPase protein purified from tomato leaf and fruit , and both of the antisera have been shown to recognize the small subunit of AGPase from a variety of other monocotyledonous and dicotyledonous species . The gels from which blots were prepared were loaded in two ways. First, lanes were loaded with samples of pellet, homogenate, and supernatant fractions, each of which contained the same activity of the plastidial marker enzyme, alkaline pyrophosphatase . If the AGPase protein is plastidial, the intensity of the AGPase band on the blot should be the same for these fractions. However, if the AGPase protein is wholly or substantially extraplastidial, the intensity of the band should be much greater in the homogenate and supernatant fractions than in the pellet fraction. Second, lanes were loaded with supernatant and pellet fractions that contained the same activity of the cytosolic marker enzyme, alcohol dehydrogenase . If the AGPase protein is cytosolic, the intensity of the AGPase band on the blot should be the same for the two fractions. However, if the AGPase protein is wholly or substantially plastidial, the intensity of the band should be much greater in the pellet fraction than in the supernatant fraction. In samples from pericarp and columella, both antisera strongly recognized only one band of an appropriate molecular mass to be a subunit of AGPase . Where lanes were loaded with equal activities of plastidial marker enzyme, the band was of approximately equal intensity in homogenate, pellet, and supernatant fractions . However, where lanes contained equal activities of cytosolic marker enzyme, growing strawberries vertically the band was visible in the pellet and not the supernantant fraction . These data indicate that AGPase protein is primarily or exclusively plastidial in the pericarp and the columella.

Our study provides strong evidence that AGPase activity and protein is mainly or exclusively plastidial in the pericarp and the columella of the developing tomato fruit. This conclusion is consistent with our observation that the ratio of ADP-Glc to UDP-Glc in developing fruit is very low . We suggest that ADP-Glc in tomato fruit is synthesized via a plastidial AGPase from Glc phosphate imported from the cytosol. Consistent with this idea, envelopes of plastids from tomato fruit are reported to have a hexose-phosphate-phosphate exchange transporter . The pathway we suggestfor tomato appears to occur in all other organs for which reliable information is available, including the embryos of oilseed rape and pea and the tubers of potato . Our results are at variance with those of Chen and colleagues, who reported that the stroma and the cytosol were labeled in sections of developing pericarp challenged with an antiserum to tomato AGPase. Chen et al. suggested that the cytosolic protein they detected might be an untransported precursor of the plastidial AGPase. It is likely that the antisera we used recognized primarily or exclusively the small subunit of the tomato enzyme. The amino acid sequences of small subunits are highly conserved between species, whereas those of large subunits are divergent . In studies with purified AGPase from tomato fruit, Chen and Janes found that the spinach antiserum recognized only the small subunit. It is possible, therefore, that the cytosolic protein detected by Chen and colleagues was an inactive form of the large subunit, which we did not detect. Regardless of the nature of the cytosolic antigen detected by Chen et al. , our results provide strong evidence that little or no active AGPase is present outside the plastid in developing fruit.Plant fruits protect developing seeds and aid in their dissemination .

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 . Fleshy fruit ripening and the generation of quality attributes occur towards the end of seed development and render fruit attractive to animal and human consumers, further aiding seed dispersal . Understanding fruit ripening provides an important theoretical and practical basis for manipulating the ripening process, improving fruit quality, and prolonging fruit shelf life . Tomato is a model plant for studying the ripening of climacteric fruit because of its simple diploid genetics, small genome size , short life cycle, ease of transient and stable transformation, distinct ripening phenotypes, and abundant bio-informatics resources . 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  and ethylene . 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 , some of which have obvious ripening inhibited phenotypes, such as ripening-inhibitor , non-ripening , Colorless non-ripening , Never-ripe , and Green-ripe . 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 . 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 .

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. MADSRIN 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 . 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 . 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 . 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 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 . Compared with wild-type , the Cnr mutant has reduced ethylene synthesis, fruit softening, and carotenoid synthesis in pericarp tissue . Mapping and identification of Cnr by Manning et al. 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 SPLCNR gene was inhibited, giving rise to the Cnr ripening mutant phenotype . 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 SPLCNR in WT fruit, Gao et al. 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. 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 , 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 NACNOR 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. compared the proteome differences between the nor mutant and WT tomato fruit by isobaric tags for relative and absolute quantification and found that the accumulation of many ripening-related and disease-resistance proteins was altered in the nor mutant. Additionally, the NACNOR mutation in Penjar tomato inhibited various metabolic processes and prolonged the shelf life of fruit , drainage planter pot whereas the over expression of NAC-NOR accelerated the senescence of tomato leaves . In addition to NAC-NOR, several other NAC TFs have been reported to be involved in regulating tomato fruit ripening. For example, the over expression of SlNAC1 in tomato resulted in a decrease in ethylene synthesis and the early softening of fruit, producing a yellow to orange phenotype . 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 .

The ripening process in tomato fruit with CRISPR/Cas9 gene editing of 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 . Surprisingly, however, we have recently been unable to obtain a nor mutant phenotype in NAC-NOR-edited fruit using CRISPR/Cas9 , which was published simultaneously by the de Maagd laboratory , who demonstrated 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, over expression of the NACNOR 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.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 , 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 . To study the relationship between NAC-NOR expression and ethylene, we used treatment with an ethylene-generating compound and an ethylene perception inhibitor 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 .