NAC TFs have the conserved N-terminal NAC region with about 150 aa including both nuclear localization signal (NLS) and DNA binding regions, which are further divided into five subdomains designated from A to E (A, C, and D are more highly conserved compared to B and E) and a C-terminal region as a transcriptional regulatory/activation region (TRR and TAR, respectively) ( Figure 1 ). In particular, members of the NAC family have a conserved domain fold containing one β-sheet flanked by an α-helical element, and the C-subdomain has a DNA recognition conserved motif sequence (WKATGT/NDK) that specifically binds to the developmental core CGT(GA) and the stress core CGTG (Mathew and Agarwal, 2018) ( Figures 1B, E ). In addition, NACs include activators and repressors for gene expression and repression, respectively, resulting from the NAC repression domain (NARD) in the D subdomain, such as the rice NAC020 and NAC026, which show bifunctionality having both activation and repression properties (Mathew et al., 2016). In addition, NAC proteins have homodimers and heterodimers, and dimerization is required for stable DNA binding to post-transcriptional and translational modifications in target genes (Chen et al., 2016) ( Figure 1B ). In conclusion, the N-terminal region in NACs contains the repression domain, while the C-terminal region is the transactivation region to a large degree. These properties allow NACs to have multiple regulatory patterns at the transcriptional, post-transcriptional, and translational levels, serving as activators and repressors in response to developmental and environmental cues.

Most of the members of the NAC TF family are localized in the nucleus, although a few of them are situated in other organelles such as the plasma membrane (PM), the cytoplasm, and the endoplasmic reticulum (ER) (Olsen et al., 2005; Kim et al., 2006; Kim et al., 2007; Seo et al., 2008; Wang et al., 2016; Bhattacharjee et al., 2017). Notably, membrane-bound NACs may be anchored in membrane systems, including the PM, ER membrane, and nuclear membrane, in inactive forms. Stimulation by developmental or environmental cues promotes their release and trafficking from the membrane to the nucleus for the control of target gene expression in a common regulatory pattern (Kim et al., 2006; Kim et al., 2007; Seo et al., 2008; Wang et al., 2016; Bhattacharjee et al., 2017). Altogether, the multiple localizations and the translocation traits of NAC TFs implicate various biological functions and regulatory mechanisms in response to developmental and environmental cues.

Environmental factors have played a vital role in the diversification of the NAC family, to a great extent, enabling higher plant survival and prosperity on land. Construction of a phylogeny tree based on the NAC family from green algae to higher plants can help to better understand the origin and expansion of NACs from their aquatic to terrestrial evolution (Mathew and Agarwal, 2018). Higher plant-specific NACs are potentially from the ancestral WRKY protein, concomitant with the transition from an aquatic to a terrestrial environment through two landmark expansions—the evolution of bryophytes from streptophytes, followed by the development of flowering angiosperm plants from vascular plants—which highlight the vital role of NACs in plant evolution (Yamasaki et al., 2013; Maugarny-Calès et al., 2016; Mathew and Agarwal, 2018).

The subfamily of FaNAC035 (Moyano et al., 2018; Martín-Pizarro et al., 2021), a key regulator of strawberry fruit development and ripening, is a typical case. Phylogenetic analysis showed that this subfamily also followed the evolutionary relationships from aquatic to terrestrial, and some conserved motifs were discovered to be lost when comparing ferns, gymnosperms, and angiosperms ( Figures 1C–E ). We supposed that four subdomains (motif1–motif4) appeared after the first evolutionary expansion; subsequently, the angiosperm NACs evolved to the conserved motif7 and motif8, with motif7 being conserved only in dicotyledons rather than in monocotyledons ( Figures 1C, D ). The number of genes also varies, with monocotyledons having only one member while dicotyledons have more than two members ( Figure 1C ). In brief, the gene numbers and conserved motifs are species-specific and have developed diverse functions in adapting to developmental and stress processes.

Based on wide phylogenetic analysis, the NAC family of green plants is divided into six major groups (groups I–VI) as follows: 1) the basal group I functions in water conduction and cell support; 2) the developmental group II, which includes CUC1–CUC3 (AtNAC054, AtNAC031, and AtNAC098), function in shoot apical meristem (SAM) formation and cotyledon separation, associated with the ETH–auxin pathways; 3) group III has the transmembrane motif (TMM); 4) group IV functions in the timing control of several developmental processes, including flower evolution, such as AtNAC034 for flowering time, AtNAC009 for cell division, and AtNAC042 for longevity; 5) group V regulates the stress responses; and 6) group VI has neo- and/or sub-functionalization in different plant species. In summary, NACs regulate many developmental and stress processes through phytohormones (Ooka et al., 2003; Ernst et al., 2004).

Higher plants have developed multiple regulatory mechanisms to maintain a balance between the expression and turnover of NACs partly through NAC regulatory loops (Mathew and Agarwal, 2018). In the past years, significant progress has been made toward understanding the regulatory mechanisms of NACs in controlling their homeostasis at the DNA, RNA, and protein levels, including 1) transcriptional regulation by their upstream TFs to form messenger RNA precursors (pre-mRNAs) in the nucleus; 2) post-transcriptional regulation at the nucleotide level, including microRNA (miRNA)-mediated inhibition and/or splicing of pre-mRNAs and their transport from the synthesis site to the action sites; 3) post-translational regulation at the protein level, including the transfer of the cleave-mediated membrane-tethered proteins to the nucleus, which are associated with specific active groups, protein degradation by proteasome machinery, oligomerization, and protein interactions (Mathew and Agarwal, 2018).

Regarding the primary function of NACs in secondary cell wall thickening and xylem vessel element formation, the NAC secondary wall thickening promoting factor2, NST2/AtNAC066, and ORE1 (ORESARA1/AtNAC092) are regulated by WRKY12 and EIN2 (ethylene-insensitive 2) (Wild and Krutzfeldt, 2010; Kim et al., 2014), while the secondary wall-associated NAC domain protein1, SND1/NAC012, appears autoregulatory in nature (Wang et al., 2011). In sweet potato (Ipomoea batatas), the homeostasis of IbNAC1 is regulated by the competitive binding of two upstream TFs (the activator bHLH3 and the repressor bHLH4) to its promoter (Chen et al., 2016). Seven vascular-related NAC domains (AtVNDs) can induce the expression of AtVND7 by binding to its promoter (Endo et al., 2015; Nakano et al., 2015).

NAC TFs have also evolved multiple post-transcriptional regulatory processes, including miRNA-mediated cleavage, alternative splicing, and trans-splicing, in regulating the transcripts of NAC functional genes, such as the microRNA164 (miR164)-mediated and organ boundary formation regulatory genes (e.g., CUC1/AtNAC054, GOBLET/GOB, and SlNAM2) (Samad et al., 2017), the petal size regulatory genes (e.g., RhNAC100 in Rosa hybrid) (Pei et al., 2013), and the cell wall thickening genes (e.g., PtrWND1B-s and PtrWND1B-l in Populus trichocarpa) produced by intron-mediated alternative splicing of the wood-associated NAC TF1B (PtrWND1B) (Zhao et al., 2014).

Nuclear localization is a prerequisite for a protein to function as a TF; thus, NACs contain a bipartite NLS within the D subdomain. However, the Arabidopsis NTL4/AtNAC053 protein accumulates early in the ER, and its C-terminal portion may move to the nucleus after its cleavage from the ER (Kim et al., 2007), as confirmed by several similar reports on NACs targeted to the nucleus by homomerization/heteromerization (Seo et al., 2010; Ng et al., 2013; Lee et al., 2014; Mathew et al., 2016). These findings uncover an important regulatory mechanism of the activity of NACs from early PM/ER localization to nucleus translocation.

In addition, NACs participate in post-translational modifications through reversible acetylation and phosphorylation, protein–protein interactions, and ubiquitin-mediated proteolysis. For instance, the phosphorylation of the membrane-associated NTL6/AtNAC062 by SnRK2.8 (SNF1-related protein kinase) promotes the entry of NTL6 into the nucleus during drought conditions (Robertson, 2004; Kim et al., 2012; Guan et al., 2014). NAC proteins can form both homo- and heterodimers through the conserved β-sheet domain, which is influenced by the TRR/TAR (Zhu et al., 2012). The rice OsNAC29 and OsNAC31 independently interact with OsSLR1 (SLENDER RICE1), a repressor for OsMYB61, which promotes cellulose synthesis by increasing the transcripts of a cellulose synthase gene (Huang et al., 2015). The involvement of NAC1 in auxin signaling is ubiquitinated by SlNAT5, an E3 ubiquitin ligase, which leads to the degradation of NAC1 and the downregulation of the auxin signal in plant cells (Xie et al., 2002; Guo et al., 2015). In summary, NAC TFs have shown varied regulatory mechanisms at the transcriptional, post-transcriptional, translational, and post-translational levels.

The first landmark finding for NAC function in ripening was from the non-ripening mutant (nor) of tomato (Solanum lycopersicum) fruit, known as NAC-SlNOR, which plays a central role in its ripening (Giovannoni, 2004). However, recent studies have considered that nor is a gain-of-function, rather than a loss-of-function, mutant of SlNOR, and SlNOR positively regulates fruit ripening by binding to the promoter of SlACS2 as an activator (Wang et al., 2019; Gao et al., 2020). In addition, SlNOR is a direct target gene of SlRIN/SlMADS-RIN (RIPENING INHIBITOR) and SlAREB1 (a TF in ABA signaling). SlRIN and SlAREB1 bind to the SlNOR promoter to activate its expression and to promote the expression of SlACS2/SlACS4 and SlACO1 for ETH burst, suggesting that the SlAREB1-mediated transcriptional regulation of SlNOR is involved in the crosstalk between ETH and ABA in ripening (Fujisawa et al., 2013; Mou et al., 2018).

Later, many NAC proteins of SlNOR homologs were identified in ripening, such as SlNOR-like1 (SlNAC3/SlNAC4/SlNAC48) that favors ripening by binding to the promoter of multiple target genes involved in both ETH and ABA biosynthesis and signaling, including SlACS2, SlACS4, SlACS8, SlACO1, SlACO6, SlCYP707A1, SAPK3, and SlPYL9. In particular, SlNAC9 interacts with SlPYL9 and SlAREB1 (Kou et al., 2016; Gao et al., 2018; Yang et al., 2021). In contrast, SlNAC1 (SlNAC033) can directly bind to the promoter regions of SlACS2 and SlACO1 and has multiple effects on tomato fruit ripening by inhibiting the biosynthesis of ETH while enhancing the expression of SlNCED1 and SlNCED2 for the accumulation of ABA (Ma et al., 2014; Meng et al., 2016). It has been reported that SlNAC4 may be upstream of ETH biosynthesis by regulating the activities of SlRIN and SlNOR (Zhu M. et al., 2014). On the other hand, the expression of SlNAC7 was induced by ETH and ABA during color breaking, and, together with SlNAC6, it induced the expression of SlACS2, SlACS4, SlACO1, SlNCED1, SlNCED2, SlABA2, and aldehyde oxidase (SlAAO1/2) (Zhu M. K. et al., 2014; Jian et al., 2021). Similarly, a recent report has shown that the new NAC, SlNAM1, is a positive regulator of ripening initiation by promoting the expression of SlACS2 and SlACS4 (Gao et al., 2021). In addition, an Arabidopsis NAC, JUNGBRUNNEN1 (AtJUB1), was overexpressed in tomatoes and resulted in dwarf plants and delayed ripening fruits by directly binding to a line of gene promoters of GA3ox1 (GA 3-oxidase 1) and DWF4 (DWARF4), which are responsible for the biosynthesis of both gibberellin (GA) and brassinosteroid (BR), and indirectly inhibited the expression of ACS, ACO, SlERF.H15, and SlRIN (Shahnejat-Bushehri et al., 2017).

Notably, a recent study has found that the NAC-NOR target gene encoding lipoxygenase (SlLOXC) is involved in fatty acid-derived volatile synthesis by epigenetics. NAC-NOR activated the expression of SlDML2 (DNA demethylase 2) by directly binding to its promoter both in vitro and in vivo (Gao et al., 2022). On the other hand, the reduced NAC-NOR expression in the sldml2 mutant was accompanied by the hypermethylation of its promoter, demonstrating a relationship between SlDML2-mediated DNA demethylation and NAC-NOR during tomato fruit ripening (Gao et al., 2022).

In summary, a series of NAC TFs constitute a complex regulation network in tomato fruit ripening, mainly through ETH and ABA homeostasis and signaling levels, confirming that the NAC family plays a central role in fruit ripening.

In addition to tomatoes, the NAC family also plays vital roles in many climacteric fruits. For instance, at least 13 NAC genes have been found during apple (Malus domestica) fruit ripening (Wang et al., 2011; Zhang et al., 2018; Migicovsky et al., 2021). In banana fruits (Musa acuminata), a total of six NACs (MaNAC1–MaNAC6) were associated with fruit ripening (Shan et al., 2012; Li et al., 2020; Shan et al., 2020; Wei et al., 2021). In pear fruits (Pyrus pyrifolia), 185 NACs were found in the genome, among which 1) PpNAC56 may be related to fruit ripening (Ahmad et al., 2018); 2) PpNAP1, PpNAP4, and PpNAP6 may regulate ripening through ETH and ABA; 3) PpNOR controls fruit ripening in a conservative manner, similar to SlNOR; and 4) PpNAC.A59 directly promotes PpERF.A16 by binding to its promoter and facilitating the expression of PpACS1 and PpACO1, confirming that it plays an important role in fruit ripening through ETH biosynthesis and signaling (Li et al., 2016; Ahmad et al., 2018; Guo et al., 2021; Tan et al., 2021).

Moreover, during ripening in papaya fruits (Carica papaya L.), CpEIN3a could interact with CpNAC2 to activate the biosynthesis of carotenoids, while CpNAC3 could interact with CpMADS4 to activate the expression of pERF9 and CpEIL5 (Fu et al., 2017; Fu C. C. et al., 2021). In apricot (Prunus sibirica) fruits, 102 NACs were identified in the genome, among which PsNAC6/13/46/51/41/67/37/59 were highly expressed during the ripening stage (Xu et al., 2021). In kiwifruit (Actinidia deliciosa/Actinidia chinensis), AcNAC2–AcNAC4 are positive regulators of ripening through ETH production, while AdNAC2/AdNAC3 are promoted by methyl jasmonate (MeJA), suggesting a crosstalk between MeJA and ETH via AdNAC2 and AdNAC3 (Wang et al., 2020). In addition, the positive regulators of AdNAC6 and AdNAC7 in ripening are degraded by miR164, while this degradation is inhibited by ETH. Interestingly, the miR164–NAC pathway is conserved in both climacteric and non-climacteric fruits, including apple, pear, banana, peach, strawberry, citrus, and grape (Wang et al., 2020; Wu et al., 2020; Nieuwenhuizen et al., 2021).

Notably, a recent study has found that the NAC TF NOR is a master regulator of climacteric fruit ripening. Melon (Cucumis melo) (Rios et al., 2017) has climacteric and non-climacteric fruit ripening varieties, such as the climacteric and non-climacteric haplotypes CmNAC-NOR^S,N and CmNAC-NOR^A,S, respectively. A natural mutation in the transcriptional activation domain of CmNAC-NOR^S,N contributed to climacteric melon fruit ripening by directly activating its carotenoid, ETH, and ABA biosynthesis genes. In addition, CmNAC-NOR knockout in the climacteric-type melon cultivar “BYJH” completely inhibited fruit ripening, while ripening was delayed by 5–8 days in heterozygous cmnac-nor mutant fruits. Finally, CmNAC-NOR mediated the transcription of the “CmNAC-NOR–CmNAC73–CmCWINV2” module to enhance flesh sweetness. Altogether, the transcriptional activation activity of the climacteric haplotype, CmNAC-NOR^S,N, on these target genes was significantly higher than that of the non-climacteric haplotype, CmNAC-NOR^A,S, providing insights into the molecular mechanism of climacteric and non-climacteric fruit ripening in melon (Zhang et al., 2022). In addition, single-cytosine methylome analyses of a DNA demethylase ROS1 (MELO3C024516)-knockout mutant revealed changes in DNA methylation in the promoter regions of the key ripening genes, such as ACS1, ETR1, and ACO1, suggesting the importance of DNA demethylation in melon fruit ripening (Giordano et al., 2022). Thus, the NAC family participates in climacteric fruit ripening at multiple regulation levels.

Strawberry (Fragaria ananassa) is a non-climacteric model plant, and 112 NAC genes have been found in the genome, including six NACs, FaNAC006/021/022/035/042/092, related to fruit development and ripening. FaNAC035/FaRIF regulates ripening via ABA biosynthesis and signaling, demonstrating the presence of not only a FaNAC035-mediated crosstalk among various plant hormones but also a feedback mechanism between the ABA level and its signaling (Moyano et al., 2018; Martín-Pizarro et al., 2021). In addition, FcNAC1 could participate in ripening regulation by responding to upstream hormone signals, such as those of ABA and auxin (Carrasco-Orellana et al., 2018).

In citrus (Citrus reticulata) fruits, an ABA-deficient mutant resulted from the abnormally high expression of CrNAC036, which bound to the CrNCED5 promoter to inhibit its expression. On the other hand, CrMYB68 interacted with CrNAC036 to further inhibit CrNCED5 expression, finally synergistically retarding fruit ripening (Zhu et al., 2020). Although CitNAC62 is located in the cytoplasm, the nucleus-localized CitWRKY1 may interact with CitNAC62 to promote its movement (Li et al., 2017). In jujube (Ziziphus jujuba Mill.) fruits, the NAC LOC107435239 positively regulated the pericarp lignin accumulation during winter jujube fruit coloration by promoting the transcription of F5H (ferulate 5-hydroxylase), and ZjNAC13/14/38/41 showed high expression levels during ripening (Li M. et al., 2021; Zhang Q. et al., 2021). In grape (Vitis vinifera) fruits, VvNAC26 interacted with VvMADS9 to induce the gene expression related to ETH and ABA biosynthesis, resulting in early fruit ripening, suggesting that VvNAC26 promotes ripening by activating the levels of ETH and ABA (Zhang S. et al., 2021). In litchi (Litchi chinensis) fruits, LcNAC13 and LcR1MYB1 may antagonistically regulate the accumulation of anthocyanin during ripening (Jiang et al., 2019). In watermelon (Citrullus lanatus) fruits, ClNAC68 positively regulated sucrose accumulation during ripening by directly binding to the promoters of both the invertase (ClINV) and IAA-amino synthetase (ClGH3.6) to inhibit their expression (Lv et al., 2016; Wang et al., 2021).

In summary, NAC TFs not only directly target genes encoding enzymes related to ripening parameters, softening, sugar, coloring, and aroma but also indirectly affect fruit ripening by regulating the homeostasis of ABA and ETH through conserved and specific mechanisms, which are closely related to fruit quality. ETH-controlled climacteric fruit ripening (such as in tomato) via NAC TFs is directly targeted to the ETH biosynthesis-related genes including ACO and ACS, while also involved in the interaction between ABA and ETH, mostly through ABA to activate NOR transcription and ETH synthesis. With regard to non-climacteric fruit ripening, a homolog of the tomato NOR in strawberry, the NAC TF FaNAC035/FaRIF, can promote ABA biosynthesis via FaNCED3. Thus, NAC TFs play a core role in the two ripening types through the interplay of ETH and ABA.