ClNAC100 Is a NAC Transcription Factor of Chinese Fir in Response to Phosphate Starvation

Phosphate (Pi) deficiency is one of the most limiting factors for Chinese fir growth and production. Moreover, continuous cultivation of Chinese fir for multiple generations led to the reduction of soil nutrients, which hindered the yield of Chinese fir in southern China. Although NAC (NAM, ATAF, and CUC) transcription factors (TFs) play critical roles in plant development and abiotic stress resistance, it is still unclear how they regulate the response of Chinese fir to phosphate (Pi) starvation. Based on Pi-deficient transcriptome data of Chinses fir root, we identified a NAC transcription factor with increased expression under Pi deficiency, which was obtained by PCR and named ClNAC100. RT-qPCR confirmed that the expression of ClNAC100 in the root of Chinese fir was induced by phosphate deficiency and showed a dynamic change with time. It was positively regulated by ABA and negatively regulated by JA, and ClNAC100 was highly expressed in the roots and leaves of Chinese fir. Transcriptional activation assay confirmed that ClNAC100 was a transcriptional activator. The promoter of ClNAC100 was obtained by genome walking, which was predicted to contain a large number of stress, hormone, and growth-related cis-elements. Tobacco infection was used to verify the activity of the promoter, and the core promoter was located between −1519 bp and −589 bp. We identified 18 proteins bound to the ClNAC100 promoter and 5 ClNAC100 interacting proteins by yeast one-hybrid and yeast two-hybrid, respectively. We speculated that AHL and TIFY family transcription factors, calmodulin, and E3 ubiquitin ligase in these proteins might be important phosphorus-related proteins. These results provide a basis for the further study of the regulatory mechanism and pathways of ClNAC100 under Pi starvation.


Introduction
Chinese fir (Cunninghamia lanceolata (Lamb.) Hook), as an important fast-growing and high-yield tree species, is widely cultivated in southern China. However, the concentration of available phosphate (Pi) in the soil of the Chinese fir cultivation area is low, and the continuous plantation of Chinese fir leads to the decrease of soil nutrients and environmental pollution caused by excessive application of phosphate fertilizer [1,2]. These factors affect the growth and yield of Chinese fir directly. Plants have evolved complex physiological and molecular mechanisms to cope with Pi starvation, optimizing the high-affinity acquisition, translocation, utilization, and control of Pi homeostasis [3][4][5]. It has been reported that Chinese fir adapted to Pi starvation by adjusting root morphological traits to improve Pi acquisition and effective utilization [6]. The study found that the Pi mobilization capacity of Chinese fir decreased with stand age, but the Pi recycling capacity within Chinese fir increased [7]. Although the response of Chinese fir to phosphorus starvation has been 2. Results

Cloning and Bioinformatics Analysis of ClNAC100 Gene from Chinese Fir
Based on the transcriptome data of Chinese fir with phosphate deficiency, specific primers were designed, and the PCR products were sequenced to obtain the NAC gene sequence (Supplementary File S1). By contrast, it was found that the NAC gene had the closest relationship with AtNAC100 in Arabidopsis which is from the NAM family, so it was named ClNAC100. It has the typical NAC-family-conserved domain NAM, and this gene was identified as an NAC family gene (Figure 1a). The protein encoded by the ClNAC100 gene had a total of 415 amino acids, and its molecular weight was 46,861.65. The protein's isoelectric point was 6.72, the instability coefficient was 51.13, and the average hydrophilicity (GRAVY) was −0.723, indicating that the protein was classified as unstable. ClNAC100 protein has no signal peptide and transmembrane structure (Figure 1b,c).

Cloning and Bioinformatics Analysis of ClNAC100 Gene from Chinese Fir
Based on the transcriptome data of Chinese fir with phosphate deficiency, specific primers were designed, and the PCR products were sequenced to obtain the NAC gene sequence (Supplementary File S1). By contrast, it was found that the NAC gene had the closest relationship with AtNAC100 in Arabidopsis which is from the NAM family, so it was named ClNAC100. It has the typical NAC-family-conserved domain NAM, and this gene was identified as an NAC family gene (Figure 1a). The protein encoded by the ClNAC100 gene had a total of 415 amino acids, and its molecular weight was 46,861.65. The protein's isoelectric point was 6.72, the instability coefficient was 51.13, and the average hydrophilicity (GRAVY) was −0.723, indicating that the protein was classified as unstable. ClNAC100 protein has no signal peptide and transmembrane structure ( Figure  1b ClNAC100 was compared with amino acid sequences of homologous genes of eight species of plants, including Arabidopsis thaliana, Tamarix hispida, Liquidambar formosana, Zea mays, Gossypium arboreum, Larix kaempferi, Taxus chinensis, and Camellia lanceoleosa, to explore the similarity of ClNAC100 sequence with other species. The results are shown in Figure 1d. It was found that these sequences had a highly conserved NAC domain in the ClNAC100 was compared with amino acid sequences of homologous genes of eight species of plants, including Arabidopsis thaliana, Tamarix hispida, Liquidambar formosana, Zea mays, Gossypium arboreum, Larix kaempferi, Taxus chinensis, and Camellia lanceoleosa, to explore the similarity of ClNAC100 sequence with other species. The results are shown in Figure 1d. It was found that these sequences had a highly conserved NAC domain in the N-terminal region, and a diverse array of different amino acid sequences in the C-terminal region, which was consistent with the structure of NAC protein. In addition, the C terminus also contained some sequences with high homology, indicating that its function also had a certain similarity.

ClNAC100 Transactivation Activity Analysis
To verify whether the ClNAC100 protein had transcriptional activation, the full-length sequence of ClNAC100, the NAC domain (N-terminal sequence) from 1 to 169 amino acids (aa), and the TR region (C-terminal sequence) from 170 to 415 were inserted into the pGBKT7 vector ( Figure 2a). When the yeast containing four different plasmids was transferred to SD/-Trp/-His/-Ade triple-deficient medium containing X-α-gal, the control empty plasmid pGBKT7 and the recombinant plasmid inserted with N-terminal fragment could not grow normally and showed no chromogenic reaction. The recombinant plasmid containing the full-length and C-terminal fragments of the inserted gene grew normally and showed a blue color (Figure 2b). The results showed that the protein encoded by the ClNAC100 gene had transcription activation activity, and the transcription activation domain was located at the C-terminus, which belonged to the transcription activator with transcription activation activity.
ol. Sci. 2023, 24, x FOR PEER REVIEW Figure 2. Analysis of ClNAC100 transcriptional activity: (a) Cloning of ClNAC100 NAC do TR-region. (b) Validation of transcriptional autoactivation of ClNAC100: different trun ClNAC100 fused to the GAL4 DNA binding domain in pGBKT7 vector. Transactivation a say of ClNAC100 in yeast strain Y2H Gold on the SD/-Trp/-Leu/-Ade/X-α-gal medium.

ClNAC100 Expression Pattern Analysis
To verify the accuracy of transcriptome data, we performed RT-qPCR to v expression of ClNAC100, and the results showed that its expression was increase consistent with the expression trend in the transcriptome, and the expression l 3.17 times that of CK (Figure 3a).

ClNAC100 Expression Pattern Analysis
To verify the accuracy of transcriptome data, we performed RT-qPCR to verify the expression of ClNAC100, and the results showed that its expression was increased; it was consistent with the expression trend in the transcriptome, and the expression level was 3.17 times that of CK (Figure 3a).  In the RT-qPCR experiment, the expression of ClNAC100 in the roots of Chinese fir seedlings was significantly increased at 2 h after Pi deficiency treatment, which was 10.3 times that of the normal Pi supply (CK). The expression of ClNAC100 decreased to 2.7 times that of CK after 4 h of treatment. The expression of ClNAC100 increased during the 12 h-3 d interval of P deficiency treatment, and the expression reached the highest level at 3 d. The expression of ClNAC100 decreased with the time of phosphorus deprivation treatment, but it was still higher than CK. Pi deficiency induced the up-regulation of ClNAC100 expression in the root of fir and showed dynamic changes with time ( Figure 3b).
The transcriptional expression of ClNAC100 may be induced by some hormones. Exogenous hormones ABA and JA were applied to the roots of Chinese fir. The expression of ClNAC100 was analyzed by RT-qPCR. After treatment for 3 h, it reached 1.62 times that of CK. After 6 h of treatment, the expression of ClNAC100 was significantly higher than that of CK, and its expression was 2.02 times that of CK. After 12 h of treatment, the expression of ClNAC100 was down. When the roots were treated with JA for 1 h, the expression of ClNAC100 was significantly down, only 0.49 times that of CK. The expression of ClNAC100 decreased continuously at 3 h and 6 h, and reached the lowest value at 6 h, and the expression was only 0.41 times that of CK. These results indicated that the application of ABA could induce ClNAC100 expression (Figure 3c), while the application of JA could inhibit ClNAC100 expression ( Figure 3d) in the roots of Chinese fir treated with exogenous hormones.
The expression level of ClNAC100 was different in different parts. Because the ClNAC100 was found in Chinese fir roots, its relative expression level in roots was used as a control. The part-specific expression of ClNAC100 was analyzed in the root, stem, and leaf of Chinese fir seedlings ( Figure 3e). The results showed that the expression level of ClNAC100 in the root and leaf was significantly higher than that in the stem. In conclusion, ClNAC100 was specifically expressed in different parts of Chinese fir.

Promoter Cloning and Activity Analysis of ClNAC100
By using the PCR-based genome walking procedure, we obtained about 2200 bp promoter fragment upstream of the start codon of the ClNAC100 gene (Supplementary File S1). We predicted two TSSS (Table 1), located 1882 bp and 800 bp upstream of the translation start codon, respectively. In addition, by prediction, it was found that there were a large number of hormone response elements (Table S2) such as ABA response elements: AAGAA-motif and ABRE on the ClNAC100 promoter. Meja response elements: CGTCAmotif and TGACG-motif as well as a salicylic acid response element as-1, ethylene response element ERE, and auxin-related element GC-motif. In addition, there were some lightresponsive elements (I-box, GT1-motif), stress-and damage-related elements (STRE, LTR, MYB, WUN-motif), and stress-related transcription factor binding sites (W-box, MYC, and MBS) on the promoter. Furthermore, we performed a visual analysis of these cis-elements ( Figure 4a). The four promoter fragments were cloned and replaced the 35S promoter of the PBI121 vector. The PBI121 empty vector was used as a positive control, and tobacco leaves without treatment were used as a negative control (Figure 4b). The results of GUS staining showed that five different deletion promoter fragments could drive the expression of the GUS gene (Figure 4c), and the leaves of P1-PBI121 infection turned light blue. The leaves of infected P2-PBI121 and P3-PBI121 were mostly dark blue, and P3-PBI121 was darker. Only a small number of extremely light blue or blue dots could be observed on tobacco leaves infected with P4-PBI121. Combined with the above TSS and cis-element prediction results, the ClNAC100 promoter has the strongest activity in the −1053 bp to −589 bp part and the ClNAC100 TSS is located in this part, which may contain key elements to enhance the promoter activity and drive the high expression of GUS gene in the −1519 bp to −589 bp region. Additionally, the promoter of other fragments was significantly different. However, the promoter from −589 bp to −1 bp showed only weak activity, indicating that there was no element to drive gene expression.

Promoter Cloning and Activity Analysis of ClNAC100
By using the PCR-based genome walking procedure, we obtained about 2200 bp promoter fragment upstream of the start codon of the ClNAC100 gene (Supplementary File S1). We predicted two TSSS (Table 1), located 1882 bp and 800 bp upstream of the translation start codon, respectively. In addition, by prediction, it was found that there were a large number of hormone response elements (Table S2) such as ABA response elements: AAGAA-motif and ABRE on the ClNAC100 promoter. Meja response elements: CGTCAmotif and TGACG-motif as well as a salicylic acid response element as-1, ethylene response element ERE, and auxin-related element GC-motif. In addition, there were some light-responsive elements (I-box, GT1-motif), stress-and damage-related elements (STRE, LTR, MYB, WUN-motif), and stress-related transcription factor binding sites (W-box, MYC, and MBS) on the promoter. Furthermore, we performed a visual analysis of these cis-elements (Figure 4a).   The four promoter fragments were cloned and replaced the 35S promoter of the PBI121 vector. The PBI121 empty vector was used as a positive control, and tobacco leaves without treatment were used as a negative control (Figure 4b). The results of GUS staining showed that five different deletion promoter fragments could drive the expression of the GUS gene (Figure 4c), and the leaves of P1-PBI121 infection turned light blue. The leaves of infected P2-PBI121 and P3-PBI121 were mostly dark blue, and P3-PBI121 was darker. Only a small number of extremely light blue or blue dots could be observed on tobacco leaves infected with P4-PBI121. Combined with the above TSS and cis-element prediction results, the ClNAC100 promoter has the strongest activity in the −1053 bp to −589 bp part

Combined with ClNAC100 Gene Promoter Protein Screening
Based on the predicted distribution of cis-elements on the ClNAC100 gene promoter, the promoter was divided into three small fragments Pro1, Pro2, and Pro3 (Figure 5a), which were inserted upstream of the AbAir reporter gene in the pAbAi vector. The recombinant vector was linearized and integrated into the YIHGold yeast genome (Figure 5b). The minimum concentrations (Figure 6a) of AbA inhibition for Pro1, Pro2, and Pro3 bait strains were determined to be 600 ng/mL, 800 ng/mL, and 200 ng/mL, respectively. To reduce the occurrence of false positives, the following experiments were performed with AbA at concentrations of 700 ng/mL, 900 ng/mL, and 300 ng/mL to screen the library of Pro1, Pro2, and Pro3 bait strains. with AbA at concentrations of 700 ng/mL, 900 ng/mL, and 300 ng/mL to screen the library of Pro1, Pro2, and Pro3 bait strains.    with AbA at concentrations of 700 ng/mL, 900 ng/mL, and 300 ng/mL to screen the library of Pro1, Pro2, and Pro3 bait strains.   The monoclonal colonies' PCR products were detected by 1% gel electrophoresis (Figure 6b). Finally, 18 proteins were identified (Table 2), including 2 TFs. ClNAC100 was derived from the AHL family related to plant protein phosphorylation and stress (Protein-No.14) and the TIFY family related to the jasmonate-induced signaling pathway

Screening of ClNAC100 Interacting Proteins
By the Mating method, the Y2H yeast strain transfected with the bait plasmid was used to interact with a qualified Chinese fir yeast cDNA library, which was preliminarily screened by SD/-Trp/-Leu plate and rescreened by SD/-Trp/-Leu/-His/-Ade/X-α-gal plate. The blue monoclonal colonies with good growth were selected for PCR, and the PCR products were detected by 1% gel electrophoresis. After screening and comparison, we finally obtained five proteins (Table 3) annotated as heavy metal-related isoprenylated plant protein, protein translation factor, E3 ubiquitin-protein ligase, and potassium transporter.

Discussion
ClNAC100 was found to have the highest similarity with AtNAC100 of the NAM family in Arabidopsis. The functional study of AtNAC100 in Arabidopsis showed that overexpression of AtNAC100 could improve the tolerance of Arabidopsis to low iron, which increased the primary root length, lateral root number, and iron reductase activity under low iron treatment [27]. There may be some functional similarities between ClNAC100 and AtNAC100.
ClNAC100 was up-regulated in the roots of Chinese fir under Pi deficiency, and other NAC transcription factors found to be related to Pi deficiency stress, such as OsNAC016 and ANAC044, were induced to be up-regulated in roots under this stress, although they were involved in different regulatory mechanisms [25,26]. The ClNAC100 changed dynamically with time in the roots of Chinese fir treated with Pi deficiency. Plant physiology, biochemistry, metabolic activities, and morphogenesis vary at different times under Pi deficiency [28][29][30]; we speculate that the differential expression of the ClNAC100 gene at different time points may be involved in different regulatory functions.
In plants, the promoter of NAC family genes contains a large number of hormoneresponsive elements and their expression can be induced by hormones. RT-qPCR results showed that ABA increased the expression of ClNAC100 in the roots of Chinese fir, while JA decreased its expression. In other plants, for example, the expression of NAC transcription factors in Arabidopsis is induced by different hormones, and these hormones regulate the expression of NAC transcription factors in a time-specific manner [31]. ABA treatment specifically induced StNAC053 expression in potato seedlings [32]. Exogenous MeJA induced a decrease in the expression of most NAM family members in rice [33]. A large number of studies have shown that hormones are involved in the regulation network of Pi starvation of plants, especially in the uptake and utilization of Pi by root growth and development [34,35]. It was possible that ClNAC100 played an important role in the response of Chinese fir to Pi starvation and the hormone-Pi regulation pathway.
Cis-element prediction results show that the ClNAC100 promoter contains many hormone response elements, stress-related response elements, and transcription factor binding sites, which is consistent with previous studies on stress-related NAC gene promoters [31,36,37]. The basic condition of the eukaryotic promoter driving gene expression is that there is a core promoter element, which contains sequence modules such as TATA-box, initiator (Inr), TFIIB recognition element (BRE), and core promoter downstream element (DPE). GUS staining results showed that the promoter activity between −1519 bp and −589 bp was the strongest. Combined with the TSS prediction results, we suggest that this region contains ClNAC100 core promoter elements and a large number of other important elements.
Despite the presence of a large number of recognition sequences for transcription factors in the NAC promoter, it is not clear which transcription factors can drive the transcription of NAC TF. Two transcription factors that recognize the ClNAC100 promoter were identified by yeast one-hybrid analysis, one of which belongs to the AHL family, a class of transcription factors containing the AT-hook motif. Previous studies have shown that the AHL gene is involved in plant growth, development, and response to stress [38]. It was found that most AHL genes were expressed in soybean roots and mainly mediated stress responses in roots [39]. Wong also found that HAI1 is a protein phosphatase involved in abiotic stress and ABA signaling, which can directly dephosphorylate AHL10 protein to participate in the regulation of stress or ABA-induced pathways [40]. Chinese fir may regulate ClNAC100 transcription by AHL for growth or response to stress, but the specific function remains to be verified.
The other belongs to the TIFY family. Besides regulating the development of plants, this family of transcription factors is also widely involved in plant stress responses, such as drought, salt stress, osmotic stress, and signal transduction network regulation of various hormones, especially JA [41][42][43]. The TIFY family members are the transcription factors regulating the MeJA-induced flavonoid biosynthesis, meanwhile, PcMYB10 and PcMYC2 can directly interact with each other and with JAZ proteins, which participate in the transcriptional regulation of jasmonate-mediated flavonoid biosynthesis [44]. In addition, the accumulation of flavonoid compounds is closely related to Pi deficiency in plants [45]. The root system is an important organ for plants to sense Pi starvation and plant hormone signal transduction allows plants to better adapt to stress. ClNAC100 in the roots of Chinses fir was negatively regulated by JA; we speculated that this transcription factor might regulate ClNAC100 in the joint regulation pathway of JA-Pi starvation.
In addition, the ClNAC100 promoter binds to calmodulin, and some calmodulins in Arabidopsis can bind to DNA [46]. The PRAD gene of Arabidopsis thaliana can participate in the rhizosphere acidification induced by low phosphate, and CAM5 can interact with PRAD to enhance its transcriptional activation. The mutation of CAM5 leads to the weakening of the rhizosphere acidification induced by low phosphate, indicating that CAM5 plays an important role in the regulation of this pathway [47]. Zhu found that, in the presence of the calmodulin-binding transcription factor CAMTA, calmodulin CML24 could induce the expression of ALMT1 and promote malate secretion in roots to respond to stress [48]. We hypothesized that this protein was a DNA-binding calmodulin or could co-regulate the expression of ClNAC100 with other proteins.
Protein ubiquitination and degradation pathways are ubiquitous and play an important role in plant life activities. Protein ubiquitination is generally completed by ubiquitinactivating enzyme E1, ubiquitin-conjugating enzyme E2, and ubiquitin-ligase enzyme E3. Some studies have shown that protein ubiquitination is widely involved in molecular regulation in response to Pi starvation. For example, ATL8, a RING E3 ligase, modulates phosphate homeostasis in Arabidopsis; the relative expression levels of the genes involved in the maintenance of Pi homeostasis are differentially modulated in mutant and overexpressed plants compared with the WT under different Pi regimes [49]. Arabidopsis NLA protein belongs to E3 ubiquitinase and interacts with BUC24 to maintain plant Pi homeostasis [50]. E3 ligase PRU1 can interact with WRKY6, and PUR1 can ubiquitinate and degrade WRKY6 under phosphate starvation conditions, relieve the inhibition of WRKY6 on the expression of PHO1, and improve the ability of Arabidopsis to transport Pi under low phosphate stress [51]. NAC protein is also often regulated by ubiquitination. The E3 ligase BRUTUS facilitates degradation of VOZ1/2 transcription factors, especially under drought and cold stress conditions [52]. An E3 ubiquitin ligase was identified in the yeast two-hybrid assay, indicating that ClNAC100 is subject to ubiquitination and degradation, inhibiting its regulation of downstream target genes. Whether ClNAC100 is ubiquitylated in regulatory pathways in response to Pi starvation requires further verification.

Plant Materials and Treatments
The plant materials were 1-year-old Chinese fir seedlings of "061 clones" from Yangkou Forest farm in Fujian Province. The roots of light substrate seedlings were washed and planted in the washed river sand and treated with Hoagland nutrient solution with 1/3 concentration and 1 mM KH 2 PO 4 phosphate for 10 days.
Wild-type tobacco (Nicotiana benthamiana) was cultured in an incubator until 3-4 weeks old with a temperature of 24 • C, the humidity of 60-70%, light intensity of 5000 lx, 16 h light/8 h dark light.

Cloning of the ClNAC100
The roots of Chinese fir with good growth after seedling survival were selected for liquid nitrogen quick-freezing, and total RNA was extracted according to the method provided by TIANGEN RNAprep Pure Plant Plus Kit (Beijing, China). The first Strand cDNA was synthesized using NovoScript ® Plus All-in-one 1st Strand cDNA Synthesis SuperMix (Suzhou, China). According to the transcriptome data of phosphate deficiency in Chinese fir root (Accession number: PRJNA964476), the specific primers ClNAC100-F/ClNAC100-R (Table S1) were designed, and the cDNA sequence with complete ORF was obtained with PCR. After gel electrophoresis verification and gel purification, the pMD-18 vector was connected to transform DH5α, and positive clones were selected for propagation and sent to the sequencing company for sequencing.

Bioinformatics Analysis
The proteins of ClNAC100 were aligned by TAIR, and the protein domain was analyzed by SMART. ProtParam was used for the ClNAC100 gene encoding protein molecular weight, isoelectric point, unstable factor, and physical and chemical properties such as hydrophilicity. Online analysis software SignalP4.1 was used for the analysis and prediction of signal peptides. TMHMM 2.0 was used to measure the transmembrane domain of the protein. The amino acid sequence of the ClNAC100 homologous gene of Chinese fir was downloaded from NCBI public database. The amino acid sequences of Chinese fir ClNAC100 and the above plants were arranged in the format, and Clustal X2.1 software was used for multi-sequence comparison.

Transactivation Activity Analysis
Two fragments of N-NAC (NAC domain), C-NAC (TR-region), and intact ClNAC100 protein divided according to the ClNAC100 protein domain were fused with the GAL4 DNA binding domain. According to the instructions for ClonExpress ® II One Step Cloning Kit (Nanjing, China), a specific primer (Table S1) was designed to insert the full-length gene or fragment between the 5 '-EcoRI and BamHI-3 cleavage sites of vector pGBKT7. The recombinant vector and pGBKT7 transformed into Y2HGold yeast receptive state were coated on SD/-Trp plate and cultured inversely at 30 • C for 3 days. The 2-3 mm well-grown monoclonal yeasts were applied to the SD/-Trp/-His/-Ade/X-α-gal plate. Yeast growth and color were observed.

ClNAC100 Expression Pattern Analysis
The Chinese fir plants with good growth after seedling survival were selected for phosphate deficiency treatment for 7 days. The solution was configured as 1/3 concentration of Hoagland nutrient solution without phosphate, and 1 L was taken and added into 1 mM KH 2 PO 4 solution for normal phosphate supply treatment as the control group. In addition, 1 L Hoagland nutrient solution was added with 1 mM KCl to balance K + for phosphate deficiency treatment.
The Chinese fir plants with good growth after seedling survival were selected for phosphate deficiency and hormone treatment. Phosphate starvation treatment was divided into the normal group and the no phosphate group. These Chinese fir plants were treated for 0, 2, 4, 8, 12, and 24 h and 3, 5, and 7 days, respectively. The concentration of exogenous hormones was set as 0.1 mM ABA (abscisic acid) and 0.1 mM JA (jasmonic acid) [33], and short-term hormone treatment was performed on the roots of Chinese fir, and the treatment time was 0, 1, 3, 6, and 12 h. Three parts (root, stem, and leaf) of normal cultivated Chinese fir seedlings were used as materials, with the root as the control group. Five biological replicates were set for the above-mentioned group of experiments. After treatment, the root was collected and put in liquid nitrogen, then stored in the refrigerator at −80 • C.
Total RNA was extracted from the above samples and reverse-transcribed into cDNA. The expression pattern of ClNAC100 was analyzed by RT-qPCR according to the PerfectStart Green qPCR SuperMix(+Dye II) kit (Nanjing, China) instructions, with the ClActin gene as an internal reference. Primers are listed in Table S1, and the expression of the derived data was calculated using the 2 − CT method. Expression data from different treatments or parts were compared using Duncan's method for multiple comparisons.

Promoter Cloning and Activity Analysis of ClNAC100
In total, 100 mg of well-grown Chinese fir root was selected as material, and genomic DNA was extracted according to the product description by TIANGEN's Plant Genomic DNA Kit (Beijing, China). The purity of isolated genomic DNA was analyzed by running the samples in 0.5% agarose gel. ClNAC100 promoters were cloned from the genomic DNA of Chinese fir using TaKaRa's Genome Walking Kit (Beijing, China). After gel electrophoresis verification and gel purification, the pMD-18 vector was connected to transform DH5α, and positive clones were selected for propagation and sent to the sequencing company for sequencing.
The software TSSP 5.1 and lab tools were used to predict the promoter transcription start site (TSS) of the ClNAC100, and the range of promoter core region was narrowed according to the highest score. Then, PlantCARE was used to analyze the cis-acting elements in the promoter sequence.
The isolated ClNAC100 promoter and its truncations were cloned into the plant expression vector pBI121 using the primer-specific restriction sites 5 -HindIII and BamHI-3 , which replace the 35 s promoter. The vector pBI121 and recombinant vectors were then transformed into Agrobacterium tumefaciens strain GV3101 and confirmed using colony PCR. The positive agrobacterium was propagated, 3-4 week-old tobacco seedlings with good growth were selected, and the bacterial solution was injected into the lower epidermis of tobacco leaves using a 1 mL syringe. At the end of infection, tobacco leaves were stained with GUS, decolorized with 75% alcohol, observed, and photographed.

Yeast One-Hybrid
The promoter fragment of ClNAC100 was divided into 400~700 bp fragments according to the distribution of cis-elements and inserted into the upstream of the AbAir reporter gene of pbaite-AbAi vector to construct pro1-pAbAi, pro2-pAbAi, and pro3-pAbAi recombinant vectors. BbsB I-linearized recombinant bait plasmids were integrated into the genome of Y1HGold. The transformed competent cells were then transferred onto solid agar SD/-Ura and incubated for 3 days. Single clones were then identified via colony PCR using Matchmaker Insert CheckPCR Mix 1 (Clontech Shanghai, China).
Healthy yeast colonies grown in SD medium-Ura were selected and resuspended in 0.9% NaCl. After adjusting to obtain OD 600nm values of approximately 0.002, the bacterial solutions were plated on SD/-Ura plates containing different concentrations of AbA. The colonies were allowed to grow for 2-3 days at 30 • C. The minimum AbA concentration that completely inhibited colony growth was determined and used for further library screening.
The fresh competent cells prepared from the positive strains transformed with 5 µg of Chinese fir cDNA yeast library plasmid (previously stored in our laboratory) were painted with SD/-Leu/AbA plate at the corresponding concentration, and each plate was painted with 100 µL bacterial solution, then incubated at 30 • C for 3-5 days. Positive clones were relined on SD/-Leu/AbA medium at the corresponding concentration to generate new monoclonals. Clones that could grow normally were selected, and the inserted cDNA fragments in positive clones were amplified according to the instructions of the Matchmaker Insert Check PCR Mix 2 kit (TaKaRa). A total of 5 µL of the PCR product was subjected to electrophoretic analysis on a 1% agarose gel and the products with a band less than 750 bp were screened out, and the other products were sent to sequencing companies for sequencing. The duplicated genes were removed from the sequencing results, and the proteins were annotated by the blast function of NCBI and TAIR, then the hypothetical and uncharacterized proteins were removed.

Yeast Two-Hybrid
Yeast two-hybrid screening library using the Mating method with the N-terminal NAC domain which did not have transcriptional activation. The colonies that grew well on SD/-Trp/-Leu plates were diluted with 0.9%NaCl solution and plated on SD/-Trp/-Leu/-His/-Ade/X-α-gal plates. The negative (pGBKT7-Lam+ pGADT7-T) and positive controls (pGBKT7-53+ pGADT7-T) were used as references. The blue clones were selected for PCR amplification and verification. The products with a band size of less than 750 bp were wiped off, and the other products were sent to sequencing companies for sequencing. The sequencing results were compared with the blast function of NCBI and TAIR.

Conclusions
In conclusion, ClNAC100 is a transcription factor that can be induced by Pi deficiency. It is positively regulated by ABA and negatively regulated by JA and is highly expressed in the roots and leaves of Chinese fir. The ClNAC100 promoter contains a large number of stress-, hormone-, and growth-related cis-elements, and its core promoter is located between −1519 bp and −589 bp. ClNAC100 is regulated by AHL and TIFY family transcription factors as well as calmodulin, and its protein can interact with E3 ubiquitin ligase. These results will help us to further investigate the regulatory mechanism of ClNAC100 under Pi starvation in the future. In particular, we screened some important factors that may be involved in the upstream regulatory pathways of ClNAC100 in response to Pi deficiency