Characterization of a Potential Ripening Regulator, SlNAC3, from Solanum Lycopersicum.

NAC (for NAM, ATAF1-2, and CUC2) proteins are one of the largest transcription factor families in plants. They have various functions and are closely related to developmental processes of fruits. Tomato (Solanum lycopersicum) is a model plant for studies of fruit growth patterns. In this study, the functional characteristics and action mechanisms of a new NAC-type transcription factor, SlNAC3 (SGN-U568609), were examined to determine its role in tomato development and ripening. The SlNAC3 protein was produced by prokaryotic expression and used to immunize New Zealand white rabbits to obtain a specific polyclonal antibody against SlNAC3. By co-immunoprecipitation and MALDI-TOF-MS assays, we showed that there was an interaction between the SlNAC3 protein and Polygalacturonase-2 (PG-2), which is related to the ripening and softening of fruit. Chromatin immunoprecipitation assays revealed the genome of the highly stress-tolerant Solanum pennellii chromosome 10 (sequence ID, HG975449.1), further demonstrating that SlNAC3 is a negative regulator of drought and salinity stress resistance in tomato, consistent with previous reports. These results indicate that SlNAC3 is not only involved in abiotic stress, but also plays a necessary role in mediating tomato ripening.

Introduction

Fruits represent significant dietary sources of sugars, fiber, vitamins, and minerals, and therefore are important components of balanced diets, contributing to good health. Fruit developmental processes can generally be divided into five stages: organogenesis, expansion, maturation, ripening, and senescence [1]. Among these, fruit ripening is an irreversible process [2]. It is also a complex developmental program coordinating the color, texture, flavor, and aroma of fruit flesh. Studies have provided evidence about the genetic mechanisms that mediate fruit pigment synthesis, sugar metabolism, and cell wall metabolism [3, 4]. Tomato (Solanum lycopersicum) is an experimental model for investigations of fleshy fruit developmental processes owing to its favorable features, such as its well-characterized ripening mutants, efficient transient and stable transformation, short life cycle, and complete genome sequence [5, 6].

Ripening processes in plants that are regulated by ethylene occur at the physiological, biochemical, and molecular levels. Specifically, the regulatory process includes signal transduction and the regulation of numerous target genes, including those involved in ethylene biosynthesis and ethylene perception [7, 8]. Many genes involved in fleshy fruit ripening have been identified. These include genes related to ethylene synthesis and signal transduction, e.g., Never-ripe (NR), ETHYLENE RESPONSE6 (ETR6), and Green-ripe (GR) [9, 10]. In tomato, in addition to hormones, ripening is mediated by numerous transcription factors (TFs), such as Ripening inhibitor (RIN), Colorless Non-Ripening (CNR), and Non-ripening (NOR), which have been confirmed to act upstream of ethylene biosynthesis [11, 12, 13]. In addition, ripening-related TFs are also targets of RIN, such as basic helix–loop–helix (bHLH), NAM, ATAF1/ATAF2, and CUC2 (NAC) [14, 15, 16, 17, 18], and a recently reported gene, APETALA2a, functions as a regulator of fruit ripening activity and carotenoid flux via the negative regulation of ethylene biosynthesis [19, 20]. The discovery of these genes has provided recent insight into the transcriptional control of fleshy fruit development and ripening via ethylenedependent and independent processes.

The NAC gene family is among the largest families of plant-specific transcription factors. Notable members of the NAC gene family play important roles in diverse developmental processes. For example, ZmNAM1 in Zea mays inhibits the growth of specific cells, promotes the boundary formation of organization, and also enhances SAM [21]. NAC members are also reported to function in other processes; over 25% of NAC proteins are upregulated in dark-treated leaves of Arabidopsis, indicating potential roles in leaf senescence [22]. NAM-B1 in wheat can accelerate senescence and increase protein, iron, and zinc contents in wheat grain by altering nutrition remobilization [23]. Additionally, the expression of many NAC genes is induced by abiotic and biotic stresses [24, 25, 26]. ANAC016 has been reported as a positive regulator of the response to drought stress in Arabidopsis [27]. Overexpression of SNAC3 (ONAC003) or OsNAC6 in rice enhances the tolerance to drought and high-salt stress by up-regulating the expression of downstream stress-related genes [28]. Overexpression of SlNAC1 in tomato can improve chilling stress resistance by reducing the accumulation of ROS and up-regulating the expression of CBF1 [29].

In our previous study, we isolated a new NAC-type gene, SlNAC3, from a tomato flower cDNA library, and reported its cloning, characterization, expression, and subcellular localization. Proteins that interact with SlNAC3 were identified using a yeast two-hybrid (Y2H) system by screening the tomato cDNA library, using the SlNAC3 protein as the bait protein. The results revealed that 7 proteins interacting with SlNAC3 were filtered out, which include cell wall structural protein, transcription related proteins, and ethylene signal transduction pathway related proteins [30, 31]. In this study, interacting proteins of the SlNAC3 protein were fished using co-immunoprecipitation (Co-IP) and analyzed by MALDITOF-MS assays to further validate the yeast two-hybrid results. Interacting DNA was detected by chromatin immunoprecipitation (ChIP) assays. Ultimately, these studies clarified the role of SlNAC3 in the developmental processes of tomatoes.

Materials and methods

Plant materials and growth conditions

The tomato (S. lycopersicum) cultivar Ailsa Craig (National Clonal Germplasm Repository, USA) was grown in an environment-controlled glasshouse under sodium lights set to 16 h days (24°C) and 8 h nights (18°C) according to previous methods [18].

Expression and purification of the SlNAC3 protein

Based on the PCR-based Accurate Synthesis method, full-length splice primers were initially designed and then protective bases were designed at both ends of the primers to synthesize SlNAC3. By double digestion, SlNAC3 was ligated between the NdeI and XbaI sites of the pCzn1 vector. Next, the resulting recombinant plasmid pCzn1-SlNAC3 was transferred into the TOP10 clonal strain and ArcticExpress expression strain. IPTG was used to induce the expression of the target SlNAC3 protein and the induction conditions were adjusted to 11°C, with gentle shaking overnight. The expressed protein was identified by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Subsequently, the SlNAC3 protein was solved by the denaturation and refolding method and purified by affinity chromatography on a Ni column. Finally, the purified protein was analyzed by 12% SDS-PAGE and confirmed by a Western blot analysis. The gel was analyzed by western blotting with the mouse anti-His mAb and peroxidase-conjugated goat anti-mouse IgG as the primary and secondary antibodies, respectively.

Preparation of the SlNAC3 polyclonal antibody

To prepare polyclonal antibodies against the SlNAC3 protein, SlNAC3 protein was used as an antigen to immunize two New Zealand white rabbits (2–2.5 kg) (Zoonbio Biotechnology Co., Ltd., China) by subcutaneous administration of 400 μg, and immunized 4 times at 3 weeks interval. After the fourth immunization, blood sampling was performed to determine the titer of the antiserum against the SlNAC3 protein by indirect enzyme linked immunosorbent assay (ELISA). When the titer exceeded 1:50000, the antiserum was prepared and purified. Then the specific polyclonal antibodies (pAbs) against the SlNAC3 protein were obtained by antigen affinity purification. The SlNAC3 protein was conjugated to agarose medium to prepare an antigen-affinity chromatography column, and the resulting antiserum was mixed with an equal amount of phosphate-buffered saline (PBS) and slowly loaded into the column. After the antibody was bound to the column and eluted with glycine elution buffer, the purified SlNAC3 pAbs (anti-SlNAC3) was obtained. Ultimately, the purified antibody was identified by 12% SDS-PAGE and Coomassie brilliant blue staining.

Co-immunoprecipitation assay

Fresh tomato fruit (10 g) in the breaker stage was ground with liquid nitrogen into a powder and supplemented with 50 mL of extraction buffer [10 mM HEPES (pH 7.5), 100 mM NaCl, 1 mM PMSF, 1 mM EDTA, 2 mM DTT, 10% glycerol, 0.2% TritonX-100, 0.1% SDS] [32], and placed on ice for 2 h for the complete lysis of proteins. The supernatant was obtained after centrifugation at 12000 × g for 15 min and the protein concentration of the supernatant was measured using the BCA assay. Anti-SlNAC3 (1 mL of 1 mg/mL solution was added to 1 μg of anti-SlNAC3) was added to the supernatant, and samples were incubated overnight at 4°C. The complex was recovered by incubating the samples with 60 μL of protein A/G Sepharose beads for 4 to 8 h at 4°C with gentle shaking. After centrifugation at 1000 × g for 3 minutes and removal of the supernatant, beads were collected, then washed five times with 1 mL of washing buffer (0.01 M PBS); the subsequent steps were as described above. After the last washing and centrifugation, the precipitate was added to 40 μL of 1× loading buffer for SDS-PAGE and silver stained. After obtaining an unknown protein that interacts with the SlNAC3 protein, it was analyzed by MALDI-TOF-MS to predict functionality.

Chromatin immunoprecipitation assay

Healthy young leaves were used for a chromatin immunoprecipitation assay by Ch-IP Assay Kit (Beyotime, China); the procedure can mainly be divided into the following steps. First, cross-linking was performed. Leaves (3 g) were harvested, submerged in cold extraction buffer 1 and 1% formaldehyde, vacuum infiltrated for 20 min, supplemented with glycine, and reverse crosslinked for 10 min. Second, nuclear extraction was performed. Leaves were ground in liquid nitrogen and added to extraction buffer 1. Samples were filtered through a nylon mesh and spun for 20 min at 2880 × g at 4°C. The supernatant was removed, and the pellet was resuspended in 10 mL of extraction buffer 2 and spun for 10 min at 12,000 × g at 4°C. After the supernatant was removed, the pellet was resuspended in 4 mL of nuclei resuspension buffer, spun for 10 min at 12,000 × g at 4°C, and the supernatant was still discarded. Third, sonication was performed. The pellet was resuspended in 1 mL of nuclei lysis buffer, and then lysed for 20 min on ice. Sonication was performed for 5 × 10 s at 15% power, followed by centrifugation for 5 min at 21,000 × g at 4°C. Fourth, immunoprecipitation was performed. Samples were treated with protein A/G agarose beads for at least 1 h at 4°C, with gentle shaking. The input control was removed and SlNAC3 pAbs was added to all samples, followed by incubation overnight at 4°C. The beads were pelleted and washed for 10 min sequentially with 1 mL of low-salt buffer, high-salt buffer, LiCl wash buffer, and TE buffer (two washes). Finally, DNA was eluted and purified. Complexes were eluted for 15 min at 65°C, reverse crosslinked for 6 h at 65°C, and treated with proteinase K for 1 h at 65°C. DNA was extracted with an equal volume of phenol/chloroform/isoamyl alcohol.

After the DNA fragments that interact with the SlNAC3 protein were obtained by Ch-IP assays, they were cloned, sequenced, and searched against the tomato NCBI database to determine the regulatory role of the DNA fragment.

Ethical approval: The research related to animals use has been complied with all the relevant national regulations and institutional policies for the care and use of animals.

Results

Expression and Purification of the SlNAC3 protein

After the SlNAC3 gene was inserted into the prokaryotic expression vector pCzn1, the recombinant SlNAC3 protein was successfully expressed at a high level in Escherichia coli. It was predicted using Lasergene software to encode a recombinant protein with a molecular weight of approximately 17.514 kDa. To induce protein expression, 0.5 mM IPTG was added, followed by gentle shaking overnight at 11°C to detect expression by SDS–PAGE. These results showed that the target protein was mainly detected in the form of inclusion bodies, and the supernatant exhibited minor expression (Fig. 1A) After refolding and purification, the target SlNAC3 protein was enriched and no other proteins existed in the collection liquid (Fig. 1B) The Western blotting assay further confirmed that the recombinant protein was successfully expressed and the molecular weight of the target protein was approximately the same as that predicted by Lasergene software (Fig. 1C)

Fig. 1

Expression and Purification of the SlNAC3 protein Expressions of the recombinant protein with IPTG induction were analyzed by 12% SDS–PAGE. Lane M, protein marker (Thermo); Lane 1, un-induced; Lane 2, induced; Lane 3, supernatant of 11° induction with 0.5 mM IPTG; Lane 4, precipitate of 11° induction with 0.5 mM IPTG. (B) Purification of the inclusion body protein by affinity chromatography on a Ni column. Lane 1, un-purified; Lane 2, flow through; Lane 3, elution. (C) Identification of the purified protein by western blotting. Lane 1, the purified protein.

Preparation of the SlNAC3 Polyclonal Antibody

After the fourth immunization, ELISA was used to test the reactivity of serum from New Zealand white rabbits immunized with the recombinant SlNAC3 protein, and the antiserum was tested at different dilutions from 1:500 to 1:512,000. The antibody titer was defined as the highest dilution of serum at which the A450 ratio (A450 of post-immunization serum/A450 of pre-immunization serum) was greater than 2.1, which is of biological significance. In accordance with the above criterion, the titer was greater than 512,000 (Table 1). Therefore, the New Zealand white rabbits could be used to produce the SlNAC3 pAb, and the antisera showed a high specificity against the SlNAC3 protein.

Table 1

The specific antibody titers in antiserum of the immunized rabbits

Dilution	OD450
	0.6 mg/mL antiserum	0.3 mg/mL antiserum
500	3.723	3.052
1000	3.598	2.875
2000	3.422	2.839
4000	3.355	2.822
8000	3.246	2.790
16000	3.024	2.775
32000	2.954	2.542
64000	2.706	2.380
128000	2.556	2.175
256000	2.428	2.036
512000	1.914	1.526
Negative	0.094	0.103

*The antibody titer was defined as the highest dilution of serum at which the A450 ratio (A450 of post-immunization serum/A450 of pre-immunization serum) is greater than 2.1, and the pre-immunization serum as negative control.

The SlNAC3 pAb was purified by affinity chromatography and confirmed by SDS-PAGE. For two different concentrations of the SlNAC3 pAb (Lane 1, Anti-SlNAC3, 0.3 mg/mL; Lane 2, Anti-SlNAC3, 0.6 mg/mL), a 55 kDa (heavy chain) band and a 28 kDa (light chain) band were observed on the membrane (Fig. 2) after SDS-PAGE electrophoresis. These results demonstrate that a pAb specific to the SlNAC3 protein was successfully obtained.

Fig. 2

Purification of the SlNAC3 polyclonal antibody. The purified SlNAC3 pAb was confirmed by 12% SDS-PAGE. Two different concentrations of anti-SlNAC3 in the gel: Lane 1, Anti-SlNAC3, 0.3 mg/mL; Lane 2, Anti-SlNAC3, 0.6 mg/mL; M, protein marker (Therno).

Identification of proteins that interact with SlNAC3

Protein–protein interactions in plants are studied using Co-IP and MALDI-TOF-MS assays. In our experiments, under non-denatured conditions, the SlNAC3 polyclonal antibody was immobilized with protein A/G Sepharose beads for binding to the SlNAC3 protein, thereby indirectly yielding an unknown protein interacting with the SlNAC3 protein. Next, the immunoprecipitate was subjected to SDS–PAGE and silver staining. Compared to the other lanes, the bands appearing in the lane of the immunoprecipitates (Lane 4) were consistent with the SlNAC3 pAbs, the SlNAC3 protein, and an unknown protein that interacted with SlNAC3 (Fig. 3). This unknown protein was analyzed by MALDI-TOF-MS to predict its functionality and the result indicated that the unknown protein was Polygalacturonase-2 (PG-2) (Fig. S1). According to previous reports, PG is a major cell wall-degrading enzyme in tomato fruits and it is developmentally regulated and is synthesized de novo in ripening fruits [33]. Based on the above series of experiments, we successfully obtained a protein that interacts with the SlNAC3 protein and preliminarily found that it functions in the process of tomato ripening.

Fig. 3

Identification of proteins that interact with SlNAC3. The immunoprecipitates were subjected to SDS–PAGE and silver staining. M, protein marker (Thermo); Lane 1, input; Lane 2, SlNAC3 protein; Lane 3, Anti-SlNAC3; Lane 4, the protein A/G Sepharose beads; Lane 5, total input incubated with protein A/G Sepharose beads; Lane 6, anti-SlNAC3 incubated with protein A/G Sepharose beads. Lane 7, the immunoprecipitates (including the SlNAC3 pAbs, the SlNAC3 protein, and polygalacturonase-2, which interacted with the SlNAC3 protein).

Genomic interactions with the SlNAC3 protein

The ChIP assay can be used to identify the DNA-binding sites in genes or genomic regions of the model species, tomato (S. lycopersicum). This method is based on the fixation of protein–DNA interactions in vivo, random fragmentation of chromatin, immunoprecipitation of protein–DNA complexes with specific antibodies, and quantification of the DNA associated with the protein of interest by PCR techniques. Here, we immunoprecipitated the SlNAC3 protein–DNA complexes with the specific SlNAC3 pAbs to obtain DNA fragments interacting with the SlNAC3 protein (Fig. S2). In order to identify the regulatory sites, the DNA fragment was cloned and sequenced (Table S1), and the sequence was searched against the tomato NCBI database. By BLAST analysis and sequence alignment, a sequence related to tomato was found on Solanum pennellii chromosome 10 (Sequence ID, HG975449.1), which was related to differential stress resistance in the wild tomato species Solanum pennellii [34]. Thus, SlNAC3 may be a negative regulator of tomato stress tolerance.

Discussion

In our study, an unknown protein interacting with the SlNAC3 protein was identified as polygalacturonase-2 (PG-2) by chromatin immunoprecipitation and MALDI-TOF-MS analysis. PG-2 is a structural component of cell walls, which is consistent with the result of the yeast two-hybrid assay. During the ripening of fruits, several polysaccharide-degrading enzymes are known to increase their activity, including cellulose [35], pectinesterase [36], and polygalacturonase (PG) [37, 38]. These last two enzymes are associated with cell walls and are involved in the substantial softening of the fruit during ripening. The identification and functional analysis of PG genes have shown that in the late stages of maturity of many climacteric fruits, PG genes mediate fruit softening by degrading pectin [39, 40, 41]. According to the results of the present study, PG is not detectable in green tomatoes and its activity appears at the onset of ripening and in ripe fruit and its protein levels tend to peak and then gradually decline as the fruit softens [42]. In our experiment, we focused on the tomato fruits at the color breaking stage based on the above studies. However, the activity of two isoenzymes (PG-1 and PG-2) only appears sequentially during ripening and they contain similar polypeptides, so it is probable that PG-1 is a dimer of PG-2 [33, 43]. In addition, the expression of cell wall metabolism-related genes such as PG is positively correlated with the degree of softening of the fruit and is regulated by ethylene. In the ethylene pathway, PG-2 is a receptor protein that mediates fruit ripening, such as NR, ETR, etc. As a transcription factor, SlNAC3 is upstream of the ethylene pathway and regulates PG-2. But the mechanism of transcriptional regulation has not been reported, which is an attention-worthy point for further study.

Drought and high salt are the main stress factors affecting plant growth and development. When plants are under drought and high salt stress, plant cells sense the external stress and transmit signals to the transcription factor involved in the stress response via a series of complex signaling pathways, such as abscisic acid, salicylic acid, ethylene, and jasmonic acid signaling pathways. Various transcription factors are initiated in response to stress, resulting in changes in gene expression, thereby activating plant stress resistance and reducing or eliminating injury caused by drought and high salt stress. NAC transcription factors (Table 2) are directly involved in the plant response or regulate the expression of drought and high salt response genes, and play an important role in plant abiotic stress responses, such as drought resistance and high salt [7, 44]. Tomato, a vegetable crop

Table 2

NAC transcription factors known to be involved in plant abiotic stress responses.

Gene	Plants	Functions	Reference
SNAC1	Rice	Positive regulation of drought or salt stress response	[48]
SNAC3 / ONAC003	Rice		[28]
OsNAC5	Rice		[49]
ONAC045	Rice		[49]
ONAC131	Rice	Positive regulation of rice blast resistance	[50]
OsNAC6 / SNAC2	Rice		[51]
OsNAC10 / ONAC122	Rice		[50]
ANAC019	Arabidopsis thaliana	Exhibit an enhanced expression of stress-responsive genes and an improved	[52]
ANAC055	Arabidopsis thaliana	tolerance to drought and salinity stress	[52]
ANAC016	Arabidopsis thaliana		[52]
ANAC072 / RD26	Arabidopsis thaliana	Improved tolerance to drought and salinity stress	[52]
ATAF1	Arabidopsis thaliana	Improve tolerance to drought, high salt, and chilling injury	[53]
SlNAC1	Tomato	Positive regulation of drought and temperature stress	[29]
SlNAC4	Tomato		[54]
TaNAC	Wheat	Resistance to drought, high salinity, low temperature	[55]
HvSNAC1	Barley	Improved drought tolerance	[56]
SlNAC35	Tobacco	Positive regulation of drought stress	[57]
SlNAC3	Tomato	Resistance to drought, high salinity	Our study

with important nutritional and economic value, has become an important model plant in botany research since the completion of tomato genome sequencing. The functional identification of NAC family genes has been mainly performed in Arabidopsis thaliana and rice, and the functions of tomato NAC family genes are less well-characterized. SINAC1, SlNAC3 and SINAM1 [18, 45] have been found to be involved in the tomato response to stress. In our study, the function of SlNAC3 in tomato was predicted. ChIP assay showed that SlNAC3 was involved in the highly stress-tolerant phenotype to improve tolerance to drought and salinity stress in the overexpress tomato cultivars, suggesting roles in alterations in cuticle composition and nonrandom associations of specific gene sets with transposable elements. In addition, recent studies have shown the potential of NAC transcription factors in transgenic breeding, e.g., transgenic rice plants that overexpress SNAC1 and SNAC2 also exhibit enhanced drought and salt tolerance [46, 47]. Therefore, it is possible to obtain new tomato cultivars with high resistance to drought and salinity stress by changing the expression level of SlNAC3 using transgenic technology.

Conclusions

In conclusion, we investigated the functional characteristics and action mechanisms of SlNAC3 in the growth of tomatoes. The mechanisms underlying the effects of SlNAC3 were analyzed at the molecular level by Co-IP and ChIP assays. The discovery of PG provided evidence that SlNAC3 is involved in the control of tomato fruit ripening, and the finding of a BLAST hit on Solanum pennellii chromosome 10 further proves that SlNAC3 performs a specific function in the stress monitoring network. Thus, our work indicates that the new NAC-type transcription factor SlNAC3 plays an important role in the developmental process of tomato and paves a theoretical basis for further tomato improvement.