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.Mature fruit of nor mutant produces no ethylene burst, undergoes very little change in carotenoid content, and almost completely fails to ripen . Based on these results, NAC-NOR has long been considered one of the core TFs regulating the initiation and progression of tomato fruit ripening . 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 . 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. Ethylene and carotenoids were still synthesized, and fruit softening was initiated in CR-NOR fruit . 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 . 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, plant pot with drainage is a gain-of-function TF that regulates tomato fruit ripening by transcriptional inhibition . 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 .
The question arises as to whether there is a similar explanation for the mechanism of action of the nor 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 . 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 . Wang et al. 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 is a dominant-negative protein. They also speculated that the truncated protein in the nor mutant still has the ability to interact with other NAC proteins and to bind DNA without transcriptionally activating its targets . 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 . 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 . Furthermore, over expression 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 . 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 .
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 . In WT tomato, WT NOR protein interacts with other fruit ripening associated 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 . 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 . However, the specific mechanism of nor functional transformation in the mutants is unclear, and needs further research.The ripening process in tomato fruits is regulated by many ripening-related TFs, some of which play a positive role, such as RIN , TDR4 , and TAGL1 , whereas others play a negative role, such as AP2a and SlMADS1 . 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 . 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 . Sequencing results showed that the expression levels of genes related to these three pathways also changed accordingly .
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 . 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 . 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 . 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 . Ethylene production occurred, but at a reduced rate, and softening also occurred, but the mature fruit phenotype was orange-red rather than red . In contrast, compared with the control fruit, CR-NOR-like1 fruit had a delay in color break of at least 14 d . 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 over expression of NAC-NOR does affect fruit development and the timing of ripening.Plants produce a wide variety of human health-benefiting compounds. Numerous studies have shown that plants contain rich and complex profiles of phytochemicals, including anthocyanins and other polyphenols. The bio-active functionalities of these compounds, such as antioxidants, anti-inflammatory, anti-carcinogen, and maltase inhibition, may deter or prevent chronic diseases such as cancer, cardiovascular diseases, and diabetes. Therefore, there is an increasing demand for the integration of these bio-active compounds with food. The challenges, however, pots with drainage holes lie in the complex chemical profile of the plant-based materials and the lack of stability under processing and storage conditions of bio-active compounds. Several extraction techniques have been developed for separating these polyphenolic compounds from plant materials, such as absorption and ion-exchange technologies with microporous resins, liquid–liquid extraction, and membrane filtration. Some of the key limitations of these approaches include inefficient extraction of a large diversity of polyphenolic compounds, labor extensive processes using high volumes of organic solvent, and limited protections for the sensitive compounds against degradation after extraction. Thus, there is a significant need to develop environmentally and economically friendly approaches to efficiently separate and stabilize the high-value bio-active compounds from plant sources and deliver their health-promoting functionalities. Furthermore, these solutions may also need to address the sensory challenges with some of the plant bio-actives.
Microencapsulation processes have been applied to concentrate, protect, and facilitate the incorporation of the polyphenolic compounds from plant extracts into food and pharmaceutical matrices. By definition, microencapsulation refers to technologies of formulating solids, liquids, or gaseous materials into microparticles or dispersion, with diameters typically ranging between 0.1 and 1000 µm. The industrial applications of the microencapsulation process offer a wide array of advantages in delivering polyphenolics, such as: protecting encapsulated phyto-active compounds from degradation during processing and storage; controlling and targeting the release of the encapsulated compounds; and tailoring the undesirable physical characteristics of the polyphenolic compounds such as solubility, smell, and taste, etc. The most common coating materials are polymers, which include natural polymers and synthetic polymers , poly , and copolymers. The shell for these microparticles is often formed using both physical and chemical processes such as spray drying and coacervation. Encapsulation systems can provide protection for the bio-active compounds using a combination of exogenous preservatives and coating materials. However, conventional encapsulation systems lack mechanisms to selectively bind phytochemicals from plant extracts and to protect these health-promoting phytochemicals, often without exogenous preservatives. Biological microscale structures, such as microbial cells, have emerged as promising encapsulation carriers for bio-active compounds. The results of these prior studies illustrate that microbial cells, such as yeast cells, can bind and encapsulate purified phytochemicals and protect them from oxidative and thermal stresses. Thus, the key advantages of microbial cell-based encapsulation systems are these pre-existing cell based microstructures eliminate the need for expensive processes used for creating these microstructures from biopolymers; eliminate the exposure of phytochemicals to heat, oxygen, and other physical factors that may deteriorate the encapsulated compounds, and reduce/eliminate the need of exogenous preservatives and antioxidants. The current studies using microbial cells have focused on purified plant-derived compounds, and to the best of our knowledge, no study has evaluated the role of microbial carriers for binding and encapsulation of diverse phytochemicals from plant juices or concentrations. Furthermore, most of the studies using microbial cells have focused on yeast cells as a model system with limited emphasis on bacterial cells for the binding and encapsulation of complex polyphenolic compounds. Lactic acid bacteria are widely recognized for their role in food fermentation and are increasingly evaluated for their probiotic functionality, thus having a significant potential to impact food systems and human health. Despite this potential, there is limited evaluation of the potential of lactic acid bacteria to encapsulate diverse bio-actives. In addition, the unique structural and compositional features of lactic acid bacteria, including cell wall bio-polymer composition and structure, significantly high single-cell protein content, bio-affinity to interact with the gut, and high levels of antioxidant activity due to various small molecules, peptides, proteins, or enzymes makes these bacterial cells a preferred encapsulation matrix for plant-based bio-actives. Thus, the focus of this study was to evaluate the binding and encapsulation of phytochemicals from a model fruit juice using inactivated probiotic bacteria Lactobacillus casei. Muscadine grapes juice was selected in this study since these are popular and highly valued fruits with rich phytochemical profiles and antioxidant properties. Inactivated probiotic bacteria L. casei was selected as it is a widely used probiotic strain from a Lactobacillus family and thus is widely accepted as a beneficial ingredient in food systems. Heat-inactivated cells were selected for the encapsulation to limit the metabolism of the encapsulated compounds as well as to increase the permeability of the cells for the fruit phytochemicals. Furthermore, inactivated probiotic cells retain some of the beneficial probiotic functions, as illustrated by recent studies. To develop a simple approach that can be adapted by other researchers, a simple incubation process was utilized to bind and encapsulate phytochemicals from the juice matrix of MG using bacterial cells in this study. The anthocyanin content and antioxidant properties of the juice matrix before and after incubation was selected as an overall measure of encapsulation efficiency of complex phytochemicals from juice matrix. To further characterize the binding and localization of phytochemicals to bacterial cell matrix, multispectral fluorescence confocal imaging data was acquired. The binding and encapsulation yield of key selected phytochemical compounds from MG was also quantified using a high-performance liquid chromatography .