Genome-Wide Identification of NAC Transcription Factors in Chimonanthus praecox and Transgene CpNAC30 Affects Salt and Drought Tolerance in Arabidopsis

86-23-6825-0086 Abstract: NAC (NAM, ATAF1/2, and CUC2) transcription factors regulate plant growth and development and response to various stresses. However, there is still limited insight into the NAC family in Chimonanthus praecox . This study performed a genome-wide characterization of the NAC transcription factor family members in C. praecox . A total of 105 NAC family members were identified from the C. praecox genome. The phylogenetic tree categorized the CpNACs into nine groups and the accuracy of this classification was confirmed by the analysis results of conserved motifs, conserved domain, and gene structure. Cis -acting element analysis revealed that the promoters of CpNACs were abundant in elements responsive to various hormones and stresses, implying the functional diversity and complexity of CpNACs . Furthermore, we investigated the function of the CpNAC30 . The expression level of CpNAC30 could be significantly induced by abiotic stress and the CpNAC30 was the highest expressed in mature leaves of C. praecox . Overexpression of CpNAC30 reduced salt stress tolerance of transgenic Arabidopsis . Nevertheless, the drought stress tolerance of transgenic plants was enhanced. This study lays a foundation for further understanding the function of CpNACs genes and provides insights for abiotic stress tolerance breeding of C. praecox and other woody plants.


Introduction
Transcription factors (TFs) are protein molecules that regulate gene transcription, acting as key regulatory factors that control gene expression in various biological and metabolic processes [1].Many TFs that are responsive to abiotic stresses, including bZIP, bHLH, DREB, MYB, WRKY, NAC [2], etc., have been identified and analyzed in various plants.NAC is a class of plant-specific TFs with multiple biological functions.The NAC TFs were named by combining the first letters of three genes, Petunia NAM, Arabidopsis ATAF1/2, and CUC2 proteins, which were the initial proteins reported to have a conserved amino acid sequence at the N-terminus, known as the NAM domain [3].The NAM domain can be divided into five subdomains (A, B, C, D, and E), in which subdomain A is involved in the formation of functional dimers, including homodimers or heterodimers; subdomains C and D contain nuclear localization signals, which are closely related to promoter-specific element binding; and subdomains B and E are relatively weakly conserved, associated with the functional diversity of NAC transcription factors [4].
NAC proteins are widely involved in the activation of specific target proteins and play multiple important biological functions at different developmental stages of plants and under various environmental factors [4].With the advancement in genome sequencing in recent years, the NAC several TF family members have been identified in model plants, including 205 NAC family genes in soybean [5], 117 in Arabidopsis [6], 151 in rice [7], 154 in tobacco [8], and 163 in poplar [9].It was found that most of the NAC family genes positively regulate abiotic stress tolerance in plants.For instance, overexpressing OsNAC10 in rice significantly enhanced its resistance to drought, low temperature, and high salt stress at the vegetative stage and increased the yield of transgenic plants under drought conditions [10].Similarly, overexpressing Pinus tabuliformis PtNAC3 increased the tolerance of transgenic Arabidopsis to multiple abiotic stresses and facilitated reproduction under abiotic stresses by shortening the lifespan [11].Conversely, RNAi-mediated suppression of ONAC066 diminished drought and oxidative stress tolerance and abscisic acid (ABA) sensitivity in rice, whereas the rice overexpressing ONAC066 exhibited the opposite effects.This indicated that ONAC066 is a positive regulator of drought and oxidative stress tolerance in rice [12].Likewise, CaNAC035-silenced pepper seedlings exhibited enhanced sensitivity to cold, salt, and drought stress treatments, demonstrating that CaNAC035 is a positive regulator of abiotic stress tolerance in pepper [13].
A few NAC family genes have also been reported to negatively regulate abiotic stress tolerance in plants.For example, the silencing of tomato NAC family gene SlSRN1, which is induced by Botrytis cinerea, jasmonic acid (JA), salicylic acid (SA), and drought stress, resulted in improved oxidative and drought resistance, proving that it is a negative regulator of the oxidative and drought response [14].Similarly, Arabidopsis lines overexpressing the AtNAC016 gene, which is involved in the drought stress response, were more sensitive to drought, while the nac016 mutant lines showed high drought tolerance [15].In rice, OsNAC2 was significantly induced by ABA, drought, and high salt stress and overexpressing OsNAC2 reduced plant resistance to salt and drought stress, whereas RNAi transgenic lines showed enhanced tolerance to salt and drought at both the vegetative and flowering stages [16].It has been shown that the functions of the NAC family members, as well as the complex regulatory mechanisms of NAC proteins, exhibit diversity and therefore, even the homologous NAC family genes in different plants, might have diverse functional characteristics [4].
Chimonanthus praecox, a deciduous shrub or a small tree in the genus Chimonanthus of the family Calycanthaceae, is a traditional ornamental flower native to China.As a rare winter-flowering plant, C. praecox has high ornamental and economic value [17].Additionally, C. praecox has high abiotic stress tolerance, which may be regulated by molecular mechanisms different from other plants [18].The genome of the varieties C. praecox 'H29', C. praecox 'Hongyun', and C. praecox var.concolor have been sequenced and assembled, making it possible to analyze the resistance mechanisms of C. praecox at the genome-wide level [19][20][21].The genome-wide characterization of NAC TFs offers substantial potential for understanding their biological functions.However, there is still limited insight into the NAC family in C. praecox, with only a few reports focusing on CpNAC68 and CpNAC1 [17,22].Ectopic overexpression of CpNAC68 enhanced the tolerance of transgenic Arabidopsis plants to cold, heat, salinity, and osmotic stresses with no effects on growth and development [22].On the contrary, CpNAC1 was induced by various abiotic stresses and ABA treatments and negatively regulated the drought stress response in transgenic Arabidopsis [17].In this study, we performed the genome-wide identification of the NAC family members in C. praecox, from which we renamed CpNAC68 to CpNAC11 and CpNAC1 to CpNAC55 based on their genetic locus.Subsequently, the expression pattern of CpNAC30 was investigated and the CpNAC30 overexpression vector was constructed and transformed into Arabidopsis to explore its stress resistance function.This study aimed to enrich the understanding of the NAC family and provide a theoretical basis for further investigating the biological functions of CpNACs in C. praecox.It also promotes the development of genetic engineering strategies for inducing abiotic stress tolerance in C. praecox and other woody plants.

Chromosome Mapping and Basic Characteristics of CpNACs
A total of 105 NAC family members were identified from the genome of C. praecox and they were sequentially renamed CpNAC1~CpNAC105 based on their genetic locus.The CpNAC genes were unevenly distributed on 11 chromosomes, except for four numbers, which could not be localized to a particular chromosome.Chr1 had the highest number of CpNAC genes (19), named CpNAC1~CpNAC19, while Chr3, Chr9, and Chr11 had the fewest CpNAC genes (6 each) (Figure 1).

Chromosome Mapping and Basic Characteristics of CpNACs
A total of 105 NAC family members were identified from the genome of C. praecox and they were sequentially renamed CpNAC1~CpNAC105 based on their genetic locus.The CpNAC genes were unevenly distributed on 11 chromosomes, except for four numbers, which could not be localized to a particular chromosome.Chr1 had the highest number of CpNAC genes (19), named CpNAC1~CpNAC19, while Chr3, Chr9, and Chr11 had the fewest CpNAC genes (6 each) (Figure 1).Based on the physicochemical properties analysis of CpNAC proteins, the numbers of amino acids ranged from 78 (CpNAC97) to 951 (CpNAC31) and the molecular weights ranged from 8.98 (CpNAC97) to 105.82 kDa (CpNAC31).The molecular weights were positively correlated with the number of amino acids.Additionally, the theoretical pIs varied from 4.33 (CpNAC42) to 9.81 (CpNAC22), with 66 proteins being acidic (pI < 7) and the rest being basic (pI > 7).The instability indices varied from 28.09 (CpNAC8) to 70.14 (CpNAC64) and the GRAVY of CpNAC proteins ranged from −1.061 (CpNAC32) to −0.252 (CpNAC83).Most of the CpNAC proteins were hydrophilic (GRAVY < 0.5), with up to 90 family members.Predictions of subcellular localization showed that 93 CpNAC proteins were localized to the nucleus, 10 to the cytoplasmic cytoplasm, and 1 to mitochondria and chloroplasts (Table 1).Based on the physicochemical properties analysis of CpNAC proteins, the numbers of amino acids ranged from 78 (CpNAC97) to 951 (CpNAC31) and the molecular weights ranged from 8.98 (CpNAC97) to 105.82 kDa (CpNAC31).The molecular weights were positively correlated with the number of amino acids.Additionally, the theoretical pIs varied from 4.33 (CpNAC42) to 9.81 (CpNAC22), with 66 proteins being acidic (pI < 7) and the rest being basic (pI > 7).The instability indices varied from 28.09 (CpNAC8) to 70.14 (CpNAC64) and the GRAVY of CpNAC proteins ranged from −1.061 (CpNAC32) to −0.252 (CpNAC83).Most of the CpNAC proteins were hydrophilic (GRAVY < 0.5), with up to 90 family members.Predictions of subcellular localization showed that 93 CpNAC proteins were localized to the nucleus, 10 to the cytoplasmic cytoplasm, and 1 to mitochondria and chloroplasts (Table 1).

Phylogenetic Tree of CpNACs
To explore the interrelatedness of CpNACs during evolution, we used 105 CpNAC protein sequences to construct a phylogenetic tree based on the maximum likelihood method.The phylogenetic tree categorized the NAC family of C. praecox into 9 groups, with each group exhibiting a large disparity in the number of CpNAC members.Group 5 contained the most members of up to 22, while group 7 contained the least members (only 4).Group 9 had large variations among its members, which were relatively distantly homologous to other CpNAC proteins (Figure 2).
protein sequences to construct a phylogenetic tree based on the maximum likelihood method.The phylogenetic tree categorized the NAC family of C. praecox into 9 groups, with each group exhibiting a large disparity in the number of CpNAC members.Group 5 contained the most members of up to 22, while group 7 contained the least members (only 4).Group 9 had large variations among its members, which were relatively distantly homologous to other CpNAC proteins (Figure 2).

Protein and Gene Structure Analysis of CpNACs
Conserved motif analysis revealed that the 105 CpNAC proteins contained different numbers of motifs, ranging from 1 (CpNAC97) to 9 (CpNAC5).The CpNAC proteins in the same group contained the same types and numbers of conserved motifs.The majority of CpNAC proteins contained motif1~motif6, with the conserved NAM domain having the highest frequency of occurrence.Motif1, motif2, and motif3 represented the conserved

Protein and Gene Structure Analysis of CpNACs
Conserved motif analysis revealed that the 105 CpNAC proteins contained different numbers of motifs, ranging from 1 (CpNAC97) to 9 (CpNAC5).The CpNAC proteins in the same group contained the same types and numbers of conserved motifs.The majority of CpNAC proteins contained motif1~motif6, with the conserved NAM domain having the highest frequency of occurrence.Motif1, motif2, and motif3 represented the conserved subdomains A, B, and C, respectively, while motif4 and motif5 represented subdomain D and motif6 represented subdomain E of the NAC family (Figure 3a,b).Conserved domain analysis showed that each of the CpNAC proteins contained either complete or partially conserved NAM domains, suggesting that they all belong to the NAC TF family (Figure 3c).
Gene structure analysis showed that the 105 CpNAC genes contained different numbers of introns and CDS, with the number of CDS ranging from 2 to 15 and the number of introns ranging from 1 to 14. CpNAC58 contained the highest number of introns and seven CpNAC genes contained only one intron.The majority of the CpNAC genes (64) contained two introns and three CDS, accounting for 60.95% of the total genes.CpNAC genes in the same group exhibited the same numbers and lengths of CDS and introns, suggesting that the gene structures of CpNACs are relatively conserved (Figure 3a,d).
bers of introns and CDS, with the number of CDS ranging from 2 to 15 and the number of introns ranging from 1 to 14. CpNAC58 contained the highest number of introns and seven CpNAC genes contained only one intron.The majority of the CpNAC genes (64) contained two introns and three CDS, accounting for 60.95% of the total genes.CpNAC genes in the same group exhibited the same numbers and lengths of CDS and introns, suggesting that the gene structures of CpNACs are relatively conserved (Figure 3a,d).

Pivotal Cis-Acting Elements in the Promoters of CpNACs
To predict the upstream regulators of CpNACs, we identified cis-acting elements of the promoter sequences that were located 2000 bp upstream of the start codon (ATG) of the CpNAC genes using the PlantCARE database.We found that the promoters of CpNAC genes contained abundant cis-acting elements, of which 135 were ABA-responsive elements, 116 were methyl jasmonate (MeJA)-responsive elements, 40 were GA-responsive elements, 32 were salicylic acid SA-responsive elements, and 29 were auxin-responsive elements.This suggested that CpNACs might be involved in the regulation of various hormonal pathways in plants.Furthermore, there were 31 low-temperature-responsive elements, 28 defense-and stress-responsive elements, 32 drought-induced elements, and 4 trauma-responsive elements, suggesting that CpNACs might be involved in the regulation of diverse biotic and abiotic stress responses in C. praecox (Figure 4).In addition, response elements related to light, circadian rhythm, meristem expression, and mesophyll cell differentiation were identified, suggesting that CpNACs may play important regulatory functions at various stages of plant growth and development.

Pivotal Cis-Acting Elements in the Promoters of CpNACs
To predict the upstream regulators of CpNACs, we identified cis-acting elements of the promoter sequences that were located 2000 bp upstream of the start codon (ATG) of the CpNAC genes using the PlantCARE database.We found that the promoters of CpNAC genes contained abundant cis-acting elements, of which 135 were ABA-responsive elements, 116 were methyl jasmonate (MeJA)-responsive elements, 40 were GA-responsive elements, 32 were salicylic acid SA-responsive elements, and 29 were auxin-responsive elements.This suggested that CpNACs might be involved in the regulation of various hormonal pathways in plants.Furthermore, there were 31 low-temperature-responsive elements, 28 defenseand stress-responsive elements, 32 drought-induced elements, and 4 trauma-responsive elements, suggesting that CpNACs might be involved in the regulation of diverse biotic and abiotic stress responses in C. praecox (Figure 4).In addition, response elements related to light, circadian rhythm, meristem expression, and mesophyll cell differentiation were identified, suggesting that CpNACs may play important regulatory functions at various stages of plant growth and development.

Expression Pattern Analysis of CpNAC30 in C. praecox
Due to the large number of NAC family members in C. praecox, we selected the CpNAC30 for gene expression analysis under different abiotic stresses and hormone treatments.It was found that the expression of CpNAC30 was induced to varying levels in the

Expression Pattern Analysis of CpNAC30 in C. praecox
Due to the large number of NAC family members in C. praecox, we selected the CpNAC30 for gene expression analysis under different abiotic stresses and hormone treatments.It was found that the expression of CpNAC30 was induced to varying levels in the leaves treated with various abiotic stresses and hormones (Figure 5a).In particular, the expression of CpNAC30 was remarkably induced and reached the highest level at 24 h after NaCl treatment, which was about four-fold higher than the pre-treatment level.Moreover, CpNAC30 could also be significantly induced by 4 • C, 42 • C, and PEG treatments.The expression of CpNAC30 was very significantly induced by ABA treatment, reaching the maximum level at 24 h.Moreover, the expression of CpNAC30 significantly increased, reaching the maximum level at 36 h after GA treatment.
leaves treated with various abiotic stresses and hormones (Figure 5a).In particular, the expression of CpNAC30 was remarkably induced and reached the highest level at 24 h after NaCl treatment, which was about four-fold higher than the pre-treatment level.Moreover, CpNAC30 could also be significantly induced by 4 °C, 42 °C, and PEG treatments.The expression of CpNAC30 was very significantly induced by ABA treatment, reaching the maximum level at 24 h.Moreover, the expression of CpNAC30 significantly increased, reaching the maximum level at 36 h after GA treatment.
Furthermore, to investigate the tissue expression pattern of CpNAC30, we analyzed the expression levels of the CpNAC30 gene in different tissues.It was shown that the CpNAC30 gene was expressed in the roots, stems, leaves, and floral organs of C. praecox, with the highest expression in mature leaves, followed by pistils (Figure 5b).

CpNAC30 Exhibits Transcriptional Activation Ability
The positive control plasmid pGBKT7-VP16, the negative control plasmid pGBKT7, and the recombinant plasmids pGBKT7-CpNAC30-ORF and pGBKT7-CpNAC30-VP16 were transformed separately into yeast strain AH109 and cultured in SD/His+X-α-gal medium (divided into four equal portions) by streaking.The yeast strains containing pGBKT7-VP16, pGBKT7-CpNAC30-ORF, and pGBKT7-CpNAC30-VP16 could grow normally and showed blue color, while the yeast strains containing pGBKT7 empty vector Furthermore, to investigate the tissue expression pattern of CpNAC30, we analyzed the expression levels of the CpNAC30 gene in different tissues.It was shown that the CpNAC30 gene was expressed in the roots, stems, leaves, and floral organs of C. praecox, with the highest expression in mature leaves, followed by pistils (Figure 5b).

CpNAC30 Exhibits Transcriptional Activation Ability
The positive control plasmid pGBKT7-VP16, the negative control plasmid pGBKT7, and the recombinant plasmids pGBKT7-CpNAC30-ORF and pGBKT7-CpNAC30-VP16 were transformed separately into yeast strain AH109 and cultured in SD/His+X-α-gal medium (divided into four equal portions) by streaking.The yeast strains containing pGBKT7-VP16, pGBKT7-CpNAC30-ORF, and pGBKT7-CpNAC30-VP16 could grow normally and showed blue color, while the yeast strains containing pGBKT7 empty vector could not grow normally and did not show blue color (Figure 6a,b).This result indicated that VP16, CpNAC30, and CpNAC30-VP16 could activate the LacZ and HIS3 reporter genes.The transcriptional activation ability of the CpNAC30 protein was significantly higher than that of VP16, as shown by the β-galactosidase activity assay, indicating that the CpNAC30 protein is a transcriptional activator (Figure 6c).
could not grow normally and did not show blue color (Figure 6a, b).This result indicated that VP16, CpNAC30, and CpNAC30-VP16 could activate the LacZ and HIS3 reporter genes.The transcriptional activation ability of the CpNAC30 protein was significantly higher than that of VP16, as shown by the β-galactosidase activity assay, indicating that the CpNAC30 protein is a transcriptional activator (Figure 6c).

CpNAC30 Enhances Plant Sensitivity to Salt Stress
To further investigate the function of CpNAC30, we ectopically transformed the gene into Arabidopsis and three T3 generation transgenic lines with high, medium, and low expression, named OE#7, OE#4, and OE#3, were selected for subsequent experiments.No significant differences were observed between the transgenic and wild-type lines in terms of growth parameters, such as plant height, time to first flower, time to first pod, and number of cauline leaves (Supplementary Figure S1).Therefore, the ectopic transformation of the CpNAC30 gene did not affect the growth of transgenic Arabidopsis and could be used for abiotic stress analysis.
CpNAC30-overexpressing and wild-type Arabidopsis plants were seeded on the MS medium containing different concentrations of NaCl so as to observe the germination and survival of the seeds under salt stress.There was no significant difference in the germination rates of transgenic and wild-type seeds on MS medium with 0 mmol•L -1 NaCl, whereas the transgenic Arabidopsis showed enhanced NaCl sensitivity with increasing NaCl concentration (Figure 7a).On the MS medium containing 100 mmol•L -1 NaCl, the germination rate of the wild-type plants was 2%~7% higher than that of the transgenic plants and when the NaCl concentration was 150 mmol•L -1 , the germination rate of the wild-type plants increased by 10%~23% compared with that of the transgenic plants.Similarly, when the NaCl concentration was 175 mmol•L -1 , the germination rate of the wildtype plants increased by 37% to 62% compared with that of the transgenic lines.Overall, the germination rate of transgenic plants was significantly lower than that of wild-type plants under salt-stress conditions (Figure 7b).
Meanwhile, wild-type and transgenic Arabidopsis grown normally for 2 weeks were treated with 300 mmol•L -1 NaCl solution.After 25 d of salt stress, most CpNAC30 transgenic plants failed to shoot normally compared with wild-type Arabidopsis and most of the plants that had already started shooting had atrophied and whitened inflorescences, resulting in a higher mortality rate (Figure 7c).The survival rates after 25 d of salt stress were 22.2%, 33.3%, and 40.7% for the transgenic lines OE#7, OE#4, and OE#3, respectively, and 66.7% for the wild-type Arabidopsis (Figure 7d).In addition, the plant height of transgenic plants was significantly lower than that of the wild-type plants after salt stress treatment (Figure 7e).These results indicated that overexpression of the CpNAC30 gene in Arabidopsis weakened the plant tolerance to salt stress.

CpNAC30 Enhances Plant Sensitivity to Salt Stress
To further investigate the function of CpNAC30, we ectopically transformed the gene into Arabidopsis and three T3 generation transgenic lines with high, medium, and low expression, named OE#7, OE#4, and OE#3, were selected for subsequent experiments.No significant differences were observed between the transgenic and wild-type lines in terms of growth parameters, such as plant height, time to first flower, time to first pod, and number of cauline leaves (Supplementary Figure S1).Therefore, the ectopic transformation of the CpNAC30 gene did not affect the growth of transgenic Arabidopsis and could be used for abiotic stress analysis.
CpNAC30-overexpressing and wild-type Arabidopsis plants were seeded on the MS medium containing different concentrations of NaCl so as to observe the germination and survival of the seeds under salt stress.There was no significant difference in the germination rates of transgenic and wild-type seeds on MS medium with 0 mmol•L −1 NaCl, whereas the transgenic Arabidopsis showed enhanced NaCl sensitivity with increasing NaCl concentration (Figure 7a).On the MS medium containing 100 mmol•L −1 NaCl, the germination rate of the wild-type plants was 2%~7% higher than that of the transgenic plants and when the NaCl concentration was 150 mmol•L −1 , the germination rate of the wild-type plants increased by 10%~23% compared with that of the transgenic plants.Similarly, when the NaCl concentration was 175 mmol•L −1 , the germination rate of the wild-type plants increased by 37% to 62% compared with that of the transgenic lines.Overall, the germination rate of transgenic plants was significantly lower than that of wild-type plants under salt-stress conditions (Figure 7b).
Meanwhile, wild-type and transgenic Arabidopsis grown normally for 2 weeks were treated with 300 mmol•L −1 NaCl solution.After 25 d of salt stress, most CpNAC30 transgenic plants failed to shoot normally compared with wild-type Arabidopsis and most of the plants that had already started shooting had atrophied and whitened inflorescences, resulting in a higher mortality rate (Figure 7c).The survival rates after 25 d of salt stress were 22.2%, 33.3%, and 40.7% for the transgenic lines OE#7, OE#4, and OE#3, respectively, and 66.7% for the wild-type Arabidopsis (Figure 7d).In addition, the plant height of transgenic plants was significantly lower than that of the wild-type plants after salt stress treatment (Figure 7e).These results indicated that overexpression of the CpNAC30 gene in Arabidopsis weakened the plant tolerance to salt stress.

CpNAC30 Enhances Plant Tolerance to Drought Stress
After 12 d of drought stress on wild-type and CpNAC30-overexpressing Arabidopsis, wild-type Arabidopsis showed more yellowing and wilting compared with transgenic lines.After 4 d of rewatering, most of the wild-type Arabidopsis died, whereas most of the CpNAC30-transgenic plants were able to survive, and the leaves turned green and plants grew well (Figure 8a).It was found that the survival rate of wild-type plants was only 14.8%, while the survival rates of three transgenic lines OE#7, OE#4, and OE#3 were 74%, 63%, and 55.6%, respectively (Figure 8b).Meanwhile, the relative electrolyte leakage and chlorophyll content of wild-type and transgenic Arabidopsis leaves were detected separately after drought stress treatment.Compared with the wild-type Arabidopsis, the relative electrolyte leakage of transgenic plants was found to be significantly lower by 23%~30% (Figure 8c), while the chlorophyll content of transgenic plants was 2.3~2.7 times higher than that of wild-type plants (Figure 8d).These results indicated that overexpression of the CpNAC30 gene in Arabidopsis significantly improved drought tolerance.

CpNAC30 Enhances Plant Tolerance to Drought Stress
After 12 d of drought stress on wild-type and CpNAC30-overexpressing Arabidopsis, wild-type Arabidopsis showed more yellowing and wilting compared with transgenic lines.After 4 d of rewatering, most of the wild-type Arabidopsis died, whereas most of the CpNAC30-transgenic plants were able to survive, and the leaves turned green and plants grew well (Figure 8a).It was found that the survival rate of wild-type plants was only 14.8%, while the survival rates of three transgenic lines OE#7, OE#4, and OE#3 were 74%, 63%, and 55.6%, respectively (Figure 8b).Meanwhile, the relative electrolyte leakage and chlorophyll content of wild-type and transgenic Arabidopsis leaves were detected separately after drought stress treatment.Compared with the wild-type Arabidopsis, the relative electrolyte leakage of transgenic plants was found to be significantly lower by 23%~30% (Figure 8c), while the chlorophyll content of transgenic plants was 2.3~2.7 times higher than that of wild-type plants (Figure 8d).These results indicated that overexpression of the CpNAC30 gene in Arabidopsis significantly improved drought tolerance.

Discussion
Plant-specific NAC proteins are a major TF family that play critical roles in plant growth, development, and responses to abiotic and biotic stresses [4].Mining the information and elucidating the functions of the NAC TF family at the genomic level could provide essential data for the functional genomics study.This could also offer the theoretical basis for analyzing the molecular mechanism of plant stress tolerance, thereby presenting candidate genes conferring salt tolerance.However, there have been no comprehensive studies on the NAC TF family in C. praecox, in which only two NAC family members, CpNAC11 (previously named CpNAC68) [22] and CpNAC55 (previously named CpNAC1) [17], have been reported.The sequencing of the complete C. praecox genome provides the possibility of identifying, comparing, and characterizing the CpNAC family at the genome-wide level [19].Here, we identified a total of 105 NAC family members containing conserved NAM domains from the genome of C. praecox.As reported in previous studies, the number of NAC family members varies widely among different plant species.We found that the number of CpNAC members in C. praecox was more than that of Apocynum venetum (74) [23], Ginkgo biloba (50) [24], Salvia miltiorrhiza (84) [25], and Liriodendron chinense (85) [26].However, the number was less than that of A. thaliana (117) [6], Oryza sativa (151) [7], Populus trichocarpa (163) [9], and Malus domestica (180) [27] but similar to that of Theobroma cacao (102) [28] and Solanum lycopersicum (105) [29].The differences in the number of NAC family members between species may be attributed to evolutionary mechanisms like gene duplication and variations in genome size [30].

Discussion
Plant-specific NAC proteins are a major TF family that play critical roles in plant growth, development, and responses to abiotic and biotic stresses [4].Mining the information and elucidating the functions of the NAC TF family at the genomic level could provide essential data for the functional genomics study.This could also offer the theoretical basis for analyzing the molecular mechanism of plant stress tolerance, thereby presenting candidate genes conferring salt tolerance.However, there have been no comprehensive studies on the NAC TF family in C. praecox, in which only two NAC family members, CpNAC11 (previously named CpNAC68) [22] and CpNAC55 (previously named CpNAC1) [17], have been reported.The sequencing of the complete C. praecox genome provides the possibility of identifying, comparing, and characterizing the CpNAC family at the genome-wide level [19].Here, we identified a total of 105 NAC family members containing conserved NAM domains from the genome of C. praecox.As reported in previous studies, the number of NAC family members varies widely among different plant species.We found that the number of CpNAC members in C. praecox was more than that of Apocynum venetum (74) [23], Ginkgo biloba (50) [24], Salvia miltiorrhiza (84) [25], and Liriodendron chinense (85) [26].However, the number was less than that of A. thaliana (117) [6], Oryza sativa (151) [7], Populus trichocarpa (163) [9], and Malus domestica (180) [27] but similar to that of Theobroma cacao (102) [28] and Solanum lycopersicum (105) [29].The differences in the number of NAC family members between species may be attributed to evolutionary mechanisms like gene duplication and variations in genome size [30].
The nucleus is the main localization site of TFs [4].Subcellular localization analysis indicated that CpNAC TFs exhibited a moderate distribution in the cell, with approximately 89% of all CpNAC proteins localized in the nucleus and small amounts in the cytoplasm, mitochondria, and chloroplasts.In particular, both CpNAC11 and CpNAC55 were localized in the nucleus, consistent with the published experimental results [17,22].The different subcellular locations imply that the proteins may behave differently, suggesting that CpNACs may play additional roles in other locations, like the cytoplasm, besides their transcriptional regulatory functions in the nucleus.This is consistent with findings on the Dendrobium nobile and Passiflora edulis NAC families [31,32].
The classification of NAC family members varies across species; for example, 183 PbNAC genes were classified into 33 groups in Pyrus bretschneideri [33], 154 NtNAC genes were classified into 15 groups in Nicotiana tabacum [34], and 249 TdNAC genes were divided into 7 clades in Triticum turgidum ssp.Dicoccoides [35].In this study, the phylogenetic tree classified the 105 CpNAC family members into 9 groups, which were supported by the results of conserved motifs, conserved domains, and gene structure analyses.The N-terminal of the NAC transcription factors is highly conserved during evolution and the majority of the CpNACs contained the conserved five sub-structural domains (A~E), suggesting that they share a common evolutionary origin and may have similar physiological functions, similar to previous studies.Variations in conserved motifs assist in grouping proteins and reflect the specific functions performed by members of each group.Some CpNAC proteins have unique conserved motifs, such as CpNACs in group 2 that contained the motif10 not present in other groups, suggesting that the CpNACs in this group performed specific functions during the evolution of C. praecox.Furthermore, CpNACs showed overall large variations in gene structure and relatively small within-group variations, suggesting that intron gains and deletions may have contributed to the differences between the CpNAC groups.
TFs regulate the initiation and efficiency of gene transcription by specifically binding to cis-acting elements in downstream gene promoters [36].Therefore, the type of cis-acting elements in gene promoters could reflect the gene function.As reported, the promoters of LcNAC genes in Liriodendron chinense mainly contain stress response elements (low temperature, high temperature, and drought response elements) and growth and development-related elements (light response elements and auxin response elements) [26].Meanwhile, the DnNAC genes in D. nobile have abundant cis-acting elements related to photosensitivity, hormonal responses, and biotic and abiotic stress responses [31].Likewise, the CpNAC genes in C. praecox contain numerous cis-acting elements that are responsive to various hormones (including ABA, MaJA, and GA), stress responses (such as low temperature and drought), and elements related to growth and development (like light and circadian rhythms).Accordingly, it is hypothesized that CpNACs play multiple roles in hormone signaling, biotic and abiotic stress response, and the growth and development of C. praecox.However, the definite functions of each CpNAC in C. praecox need to be further investigated.
To analyze the expression changes in CpNAC30 during different stages of stress and hormone treatments, we examined its expression levels at multiple time points after treatments and the results were largely consistent with previous studies [37,38].Extensive studies have demonstrated that the NAC TF family genes are variably expressed in response to abiotic stresses.There were some NAC TFs that mainly responded to one abiotic stress, such as the two NAC TFs in Hevea brasiliensis that were only induced by drought stress [39].On the contrary, many NAC TFs were found to simultaneously respond to several stresses and be involved in the regulation of multiple stress tolerances [40].For example, the expression of the SlNAC8 gene was significantly induced by drought and salt stress in Suaeda liaotungensis [37].AtSOG1, a homolog of CpNAC30 in A. thaliana, has been reported to induce adaptive responses to salt stress by down-regulating the expression of key cell cycle regulators while upregulating the expression of internal replication regulators [41].In this study, the CpNAC30 gene was significantly upregulated by various stresses in C. praecox, especially under NaCl treatment.ABA-dependent and ABA-independent pathways are the two major pathways associated with the stress-related NAC gene family [42,43].In Scutellaria baicalensis, ABA and GA treatments significantly upregulated the expression of eight SbNAC genes (SbNAC9/32/33/40/42/43/48/50), which regulate the expression of biosynthesis genes through diverse mechanisms [38].Since the CpNAC30 gene was significantly induced by both ABA and GA treatments to varying degrees, it was hypothesized that the CpNAC30 gene might be involved in the response of C. praecox to multiple abiotic stresses through the ABA and GA signaling pathways.Moreover, it has been reported that NAC TF genes are diversely expressed in different plant tissues.The CpNAC30 was expressed in all tissues and organs of C. praecox, while the highest expression level was found in mature leaves.Similarly, the GhNAC8 gene of Gossypium hirsutum showed the highest expression in senescence leaves, which could respond to various abiotic stresses [44].The TaNAC22 gene in wheat and the SlNAC7 gene in Suaeda liaotungensisis were also expressed at high levels in leaves; such NAC genes are mostly associated with abiotic stress response [45,46].
It was reported that the majority of heterologously introduced NAC genes did not affect the growth and development of transgenic plants.For instance, transgenic plants did not show growth impacts from the ectopic overexpression of rice ONAC063 and eucalyptus EgNAC141 in Arabidopsis [47,48].In the present study, the ectopic transformation of the CpNAC30 gene did not affect the growth and development of transgenic Arabidopsis.Furthermore, the germination rate of Arabidopsis seeds overexpressing CpNAC30 and the survival rate of transgenic plants were significantly lower than that of wild-type plants under salt stress, suggesting that CpNAC30 overexpression enhances the sensitivity of Arabidopsis to salt stress, thereby negatively regulating the salt stress tolerance of the plants.Opposite results were reported by previous studies, where most NAC genes positively regulated salt stress tolerance in plants.For instance, overexpressing the StNAC053 gene enhanced salt stress tolerance in transgenic Arabidopsis by activating and upregulating a series of stress-related genes [49].Picea wilsonii PwNAC1 interacted with PwRBP1 and synergistically enhanced plant resistance to salt stress through the ABA-dependent CBF pathway [50].In sweet potato plants, IbNAC087 directly activated the expression of JA synthesis-related genes, IbLOX and IbAOS, and increased JA content, leading to more intense stomatal closure and ROS scavenging mediated by the JA signaling pathway, thereby enhancing salt and drought tolerance in transgenic plants [51].Nevertheless, a few investigations found that NAC genes negatively regulate salt stress tolerance in plants.For example, Solanum habrochaites ShNAC1 was upregulated by drought, cold, and salt stresses, and overexpressing ShNAC1 in tomato plants resulted in decreased cold, drought, and salt tolerance, with further studies revealing that ShNAC1 might negatively regulate abiotic stress tolerance in tomato plants by regulating ethylene biosynthesis and signal transduction pathways [52].Similarly, rice OsNAC2 was intensely induced by ABA and osmotic stresses like drought and high salt, and OsNAC2-overexpressing rice lines exhibited decreased resistance to high salt and drought conditions, whereas RNAi plants showed enhanced tolerance to high salt and drought stresses at both the nutrient growth and flowering stages [16].
Previous studies have shown that NAC genes play important roles in response to drought stress in plants.In this study, we found that overexpression of the CpNAC30 gene in Arabidopsis significantly increased the survival rate of plants after drought stress and the relative electrolyte leakage and chlorophyll content of leaves indicated that the CpNAC30 gene positively regulated the drought tolerance of Arabidopsis, which is consistent with many recent studies [53][54][55].For example, cotton GhNAC2-A06 was significantly induced by PEG, drought stress, and ABA treatments and played a positive regulatory role in cotton under drought stress by affecting the expression of drought stress-related genes [53].Rose RcNAC091, a drought-and ABA-induced NAC transcription factor, positively regulated ABA signaling and the drought response in rose plants by activating the transcription of RcWRKY71 [54].Similarly, the sorghum NAC gene, SbNAC9, was highly induced by PEG treatment and directly activated a putative peroxidase gene, SbC5YQ75, and a putative ABA biosynthesis gene, SbNCED3, thereby enhancing drought tolerance in transgenic sorghum [55].In this study, the CpNAC30 could be extremely significantly induced by ABA, which is closely related to seed germination under salt stress, and might lead to difficulties in seed germination [56], so that the germination rate of CpNAC30-transgenic seeds was lower than that of the wild type, exhibiting more sensitivity to salt stress.We speculated that the involvement of the CpNAC30 gene in the stress response might be dependent on the ABA signaling pathway and might be a synergistic effect of multiple signals; therefore, transgenic Arabidopsis is more sensitive to salt stress but more resistant to drought.The molecular mechanisms by which NAC genes perform their functions are complex and diverse [57] and the molecular regulatory mechanism of CpNAC30 involved in abiotic stress resistance remains to be further investigated.

Retrieval of CpNAC Family Data
The genome, annotation information, coding sequence (CDS), and protein sequences of C. praecox var.concolor were obtained from Nanjing Agricultural University, China "http://eplantftp.njau.edu.cn(accessed on 18 December 2023)" [19].Using HMMER V3.0 software, all the protein sequences were retrieved by indexing the hidden Markov model (HMM) of the NAM domain (PF02365), which was downloaded from the Pfam database "http://pfam.xfam.org/(accessed on 18 December 2023)" to obtain the sequences with the NAM conserved domain.The retrieved potential NAC family protein sequences were submitted to the NCBI-CDD "https://www.ncbi.nlm.nih.gov/Structure/bwrpsb/bwrpsb.cgi (accessed on 4 January 2024)" online database to verify the presence of NAM domains.All sequences with an NAM domain, including those with an incomplete or atypical NAM domain, were retained to ensure that all possible CpNACs were identified.Based on the identified gene IDs, the relevant annotation information was extracted using the GXF Selector tool of TBtools [58] to obtain the Scaffold number and location information of CpNACs.CpNACs were sequentially named as CpNACn, according to the positional distribution of the members at the start and end of the Scaffold.The CpNAC protein sequences were analyzed for amino acid counts, molecular weight (MW), theoretical pI, Instability Index, Aliphatic Index, and Grand Average of Hydropathicity (GRAVY) using the Protein Parameter Calc tool of TBtools [58].The online tool CELLO "http: //cello.life.nctu.edu.tw/(accessed on 13 January 2024)" was employed to predict the subcellular localization of CpNAC proteins.

Phylogenetic Tree Construction of CpNACs
The MUSCLE alignment of CpNAC protein sequences was performed using MEGA 11 software and the alignment results were trimmed using the trimAL Wrapper tool of TBtools [58].According to the best model evaluated by MEGA 11, the phylogenetic tree was constructed using the maximum likelihood (ML) method [Jones-Taylor-Thornton (JTT) mode; Gamma Distributed (G)] with the bootstrap value set to 1000.The evolutionary trees were visualized using the online tool iTOL "https://itol.embl.de/(accessed on 10 January 2024)".

Protein Structure and Gene Structure Analysis of CpNACs
Conserved motif analysis of CpNACs was performed using the MEME Suite V5.5.5 online tool "https://meme-suite.org/meme/tools/meme (accessed on 7 January 2024)", with the number of motifs set to 20.Conserved domain analysis of CpNACs was performed using the CDD online tool.Moreover, the CDS sequences and genomic DNA (gDNA) sequences of CpNACs were extracted according to the ID and position information using the Fasta Extract tool of TBtools [58] and the gene structure map was drawn by the GSDS V4.0 online tool "http://gsds.gao-lab.org/(accessed on 10 January 2024)".Afterward, the Gene Structure View (Advanced) tool of TBtools was used to visualize the protein and gene structures [58].

Promoter Analysis of CpNACs
Based on the positional information of the genomic sequences of CpNACs, the 2 kb upstream sequences of each gene were extracted using the Fasta Extract tool of TBtools [58].The promoter sequences were then submitted to the PlantCARE database "https://bioinformatics.psb.ugent.be/webtools/plantcare/html/(accessed on 13 January 2024)" to predict the cis-acting elements and were visualized via the Basic Biosequence View tool of TBtools.For gene expression profiling, the roots, stems, cotyledons, young leaves, mature leaves, and different floral organs (outer petals, middle petals, inner petals, stamens, and pistils) at the full bloom stage were collected from three plants and flash-frozen in liquid nitrogen to analyze the expression pattern of the CpNAC30 gene.For abiotic stresses and hormone treatments, the seedlings grown for 4 months at the 6-8 leaves stage were transferred to an incubator culture at high temperature (42 • C) and low temperature (4 • C), while 150 mmol•L −1 NaCl (high salt) and 20% PEG6000 (polyethylene glycol to simulate drought stress), ABA (50 µmol•L −1 ), and gibberellic acid (GA) (10 µmol•L −1 ) are used for foliar spray.After 0 h, 6 h, 12 h, 24 h, and 36 h of treatments, the leaves of seedlings were collected for each treatment and immediately stored at −80 • C until RNA extraction, with 3 biological repeats for each sample.

Expression Pattern Analysis of the CpNAC30 Gene
Total RNA was extracted from the collected samples using the RNAprep Pure Total Plant RNA Extraction Kit (Tiangen Biochemical Technology (Beijing) Co., Beijing, China).The cDNA obtained via RNA reverse transcription was used as the template for the RT-qPCR and the Actin and Tublin genes of C. praecox were used as the internal reference genes [59].The RT-qPCR was conducted on a CFX96 (Bio-Rad Laboratories (Shanghai) Co., Ltd., Shanghai, China) using the EvaGreen ® Supermix Kit (Bio-Rad Laboratories Inc., Hercules, CA, USA).All primer sequences are shown in Supplementary Table S1.For each tested tissue, three biological replicates and three technical replicates were set up for the analysis.The data were organized using Bio-Rad Manager TM Software (Version 1.1) and the relative expression of the CpNAC30 gene in different samples was calculated by the delta-delta Ct (2 −∆∆CT ) method [60].

Transcriptional Activation Activity Analysis of CpNAC30
The yeast strain AH109, the VP16 plasmid, the pGBKT7-VP16 recombinant plasmid, and the pGBKT7 vector (Takara Biomedical Technology (Beijing) Co., Ltd., Beijing, China) were provided by the College of Food Science, Southwest University, China.Based on the known sequences of the CpNAC30 gene, a pair of primers was designed to amplify the cDNA sequences of CpNAC30 by PCR (Supplementary Table S1).Specific primers with restriction enzyme cutting sites were designed to amplify the open reading fragments (ORF) sequences of CpNAC30 and CpNAC30 ORF was ligated to the VP16 fragment via two rounds of PCR.The primer sequences are listed in Supplementary Table S1.The two fragments were then inserted into the pGBKT7 vector to construct yeast expression vectors for CpNAC30, named pGBKT7-CpNAC30-ORF and pGBKT7-CpNAC30-VP16.Determination of the transcriptional activation activity was performed according to the instruction of the Yeast β-Galactosidase Assay Kit (Thermo Fisher Scientific Inc., Shanghai, China).

Function Analysis of CpNAC30 in Arabidopsis Thaliana
The ORF sequences of the CpNAC30 were inserted into the pCAMBIA1300 vector to construct the pCAMBIA1300-CpNAC30 plant overexpression vector.The primers used are shown in Supplementary Table S1.The recombinant plasmid was transformed into A. thaliana (ecotype Columbia) using the Agrobacterium-mediated floral dip method [61].To investigate the salt stress tolerance of CpNAC30-transgenic Arabidopsis, we cultured the seeds of wild-type and three transgenic lines with different expression levels on MS medium containing 0, 100, 150, and 175 mmol•L −1 NaCl solution.After 3 d of vernalization, Arabidopsis was cultivated in an artificial incubator and the seed germination rate was determined after 6 d.This experiment was repeated three times.Meanwhile, wild-type and transgenic Arabidopsis grown under normal conditions in soil for 2 weeks were watered with 300 mmol•L −1 NaCl solution and the treatment was repeated every 2 d to ensure sufficient watering volume.The phenotypes of the plants were observed and photographed and the survival rates and plant heights were measured after 2 weeks.To investigate the drought stress tolerance of CpNAC30-transgenic Arabidopsis, the watering of the wild-type and transgenic seedlings at 2 weeks old was stopped.After cessation of watering for 12 d, the phenotypes were observed and the leaves were collected to determine the relative conductivity and chlorophyll content [17].Subsequently, after rewatering for recovery, the survival rate of the plants was counted after 4 d.

Statistical Analysis
The experimental data were analyzed for significant differences using SPSS 20.0 software.A t-test was performed between two independent samples under control and stress treatments, with p < 0.05 considered to be significant and p < 0.01 considered to be highly significant.One-way analysis of variance (ANOVA) was performed using Dunnett's test, and p < 0.05 was considered significant.

Conclusions
In this study, the NAC TF family members in C. praecox were identified and characterized at the genome-wide level and CpNAC30 was selected for further functional analysis in A. thaliana.A total of 105 NAC family members were identified from the C. praecox genome and the members were unevenly distributed on 11 chromosomes.The phylogenetic tree categorized the CpNACs into 9 groups and the accuracy of this classification was confirmed by the analysis results of the conserved motifs, conserved domain, and gene structure.Cis-acting element analysis revealed that the promoters of CpNACs were abundant in elements responsive to various hormones, stresses, and those related to growth, implying the functional diversity and complexity of CpNACs.Furthermore, the expression of CpNAC30 was significantly induced by salt stress and ABA treatment and overexpressing CpNAC30 in Arabidopsis enhanced the sensitivity of transgenic plants to salt stress, whereas it enhanced the tolerance of the transgenic plants to drought stress, suggesting that CpNAC30 negatively regulates salt stress tolerance but positively regulated the drought stress tolerance in transgenic Arabidopsis.Overall, these findings provide a theoretical basis for further investigating the biological functions of CpNACs and lay a foundation for molecular breeding of stress tolerance in C. praecox and other plants.

Figure 2 .
Figure 2. Phylogenetic tree of CpNACs.The maximum likelihood tree was constructed with MEGA 11 after sequence alignment of CpNAC proteins using MUSCLE.Different colors represent different groups.

Figure 2 .
Figure 2. Phylogenetic tree of CpNACs.The maximum likelihood tree was constructed with MEGA 11 after sequence alignment of CpNAC proteins using MUSCLE.Different colors represent different groups.

Figure 4 .
Figure 4.The cis-acting elements in the promoters of CpNAC genes.

Figure 4 .
Figure 4.The cis-acting elements in the promoters of CpNAC genes.

Figure 5 .
Figure 5. Expression analysis of CpNAC30 in Chimonanthus praecox.(a) Relative expression of CpNAC30 under different abiotic stresses and hormonal treatments.Data represent the mean ± SEM (standard error of the mean) of three biological replicates.Asterisks denote statistically significant differences compared to controls; * p < 0.05 and ** p < 0.01.(b) Relative expression of CpNAC30 in different tissues.The significant difference analysis was conducted using the ANOVA test.Different letters indicate significant differences (p < 0.05).PEG, polyethylene glycol; NaCl, sodium chloride; ABA, abscisic acid; GA, gibberellic acid.

Figure 5 .
Figure 5. Expression analysis of CpNAC30 in Chimonanthus praecox.(a) Relative expression of CpNAC30 under different abiotic stresses and hormonal treatments.Data represent the mean ± SEM (standard error of the mean) of three biological replicates.Asterisks denote statistically significant differences compared to controls; * p < 0.05 and ** p < 0.01.(b) Relative expression of CpNAC30 in different tissues.The significant difference analysis was conducted using the ANOVA test.Different letters indicate significant differences (p < 0.05).PEG, polyethylene glycol; NaCl, sodium chloride; ABA, abscisic acid; GA, gibberellic acid.

Figure 6 .
Figure 6.Transcriptional activation assays of CpNAC30 in yeast cells.(a) Inoculation areas of yeast with different plasmids.(b) Growth and bluing of yeast.(c) β-galactosidase activity of yeast with different plasmids.

Figure 6 .
Figure 6.Transcriptional activation assays of CpNAC30 in yeast cells.(a) Inoculation areas of yeast with different plasmids.(b) Growth and bluing of yeast.(c) β-galactosidase activity of yeast with different plasmids.

Figure 7 .
Figure 7. Salt stress tolerance analysis of CpNAC30-transgenic Arabidopsis.(a) Phenotypes of transgenic Arabidopsis under different sodium chloride (NaCl) concentrations.(b) Germination rate of transgenic Arabidopsis seeds under different NaCl concentrations.Data represent the mean ± SEM (standard error of the mean) of three biological replicates.The significance analysis was conducted using the analysis of variance (ANOVA) test and different letters indicate significant differences (p < 0.05).(c) Phenotypes of transgenic Arabidopsis plants before and after salt stress.(d) Survival rates of transgenic Arabidopsis plants after salt stress.(e) Plant height of transgenic Arabidopsis plants after salt stress.Asterisks denote statistically significant differences compared to controls, * p < 0.05, ** p < 0.01.

Figure 7 .
Figure 7. Salt stress tolerance analysis of CpNAC30-transgenic Arabidopsis.(a) Phenotypes of transgenic Arabidopsis under different sodium chloride (NaCl) concentrations.(b) Germination rate of transgenic Arabidopsis seeds under different NaCl concentrations.Data represent the mean ± SEM (standard error of the mean) of three biological replicates.The significance analysis was conducted using the analysis of variance (ANOVA) test and different letters indicate significant differences (p < 0.05).(c) Phenotypes of transgenic Arabidopsis plants before and after salt stress.(d) Survival rates of transgenic Arabidopsis plants after salt stress.(e) Plant height of transgenic Arabidopsis plants after salt stress.Asterisks denote statistically significant differences compared to controls, * p < 0.05, ** p < 0.01.

Figure 8 .
Figure 8. Drought stress tolerance analysis of CpNAC30-transgenic Arabidopsis.(a) Phenotypes of transgenic Arabidopsis under drought stress.(b) Survival rates of transgenic Arabidopsis plants after drought stress.(c) Relative electrolyte leakage of transgenic Arabidopsis plants after drought stress.(d) Chlorophyll content of transgenic Arabidopsis plants after drought stress.Data represent the mean ± SEM (standard error of the mean) of three biological replicates.Asterisks denote statistically significant differences compared to controls, ** p < 0.01.

Figure 8 .
Figure 8. Drought stress tolerance analysis of CpNAC30-transgenic Arabidopsis.(a) Phenotypes of transgenic Arabidopsis under drought stress.(b) Survival rates of transgenic Arabidopsis plants after drought stress.(c) Relative electrolyte leakage of transgenic Arabidopsis plants after drought stress.(d) Chlorophyll content of transgenic Arabidopsis plants after drought stress.Data represent the mean ± SEM (standard error of the mean) of three biological replicates.Asterisks denote statistically significant differences compared to controls, ** p < 0.01.

4. 5 .
Plant Materials and Growth Condition C. praecox seeds were obtained from Southwest University, Chongqing, China.Seedlings of C. praecox were grown in a greenhouse under the following conditions: 25 • C day/20 • C night, 85% humidity, and 16 h of light and 8 h of darkness, with a light intensity of 25 µmol•m −2 •s −1 .

Table 1 .
Basic characteristics of the NAC family members in Chimonanthus praecox.