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. 2025 Aug 25;15:31257. doi: 10.1038/s41598-025-16483-7

Physiological response of safflower to water deficit and genome-wide identification of its NAC transcription factor family

Jialing Yang 1,#, Long Han 1,#, Xueyi Zhao 1, Ziteng Zhao 1, Jing Yang 1,2, Fawei Wang 1,✉, Linna Du 1,✉
PMCID: PMC12378207  PMID: 40854944

Abstract

Accumulated evidence demonstrated the major role of NAC transcription factors in plants response to drought stress. However, there are limited studies on the identification of safflower NAC genes and their functions in response to abiotic stress, i.e., water deficit. In the present study, a total of 85 CtNACs were identified and categorized into 17 subfamilies, and the vast majority of the CtNACs were annotated in biological process. Their promoters contained cis-regulatory elements related to light, hormonal and stress responses, and plant development. Moreover, physiological response of safflower to water deficit was assessed. Plant growth of safflower was downregulated during water deficit. The contents of osmotic adjustment substances (soluble sugar, soluble protein, proline), reactive oxygen species, malondialdehyde, hydrogen peroxide, and antioxidant enzymes activities, i.e., superoxide dismutase, peroxidase, catalase in safflower leaves were enhanced under water deficit. Additionally, the photosynthetic pigments in safflower were decreased during water stress. Moreover, quantitative real-time PCR detection revealed that the expression levels of most CtNACs were upregulated under water deficit, indicating that these genes may involved in the response of safflower to water deficit. These data facilitate the in-depth study of the biological functions of CtNACs and the breeding of drought-tolerant safflower varieties.

Keywords: NAC, Transcription factor, Photosynthetic responses, Safflower, Water deficit, Genome-wide identification

Subject terms: Biological techniques, Computational biology and bioinformatics

Introduction

Safflower (Carthamus tinctorius L.) is an annual herb belonging to the Compositae family1. Due to its broad application potential in pharmaceutical, foods and other fields, safflower is considered as a major economic crop globally2,3. A large number of studies have shown that the seeds, leaves and petals of safflower have great medicinal and edible value4. The seeds of safflower are rich in oil, protein, crude fiber, mineral elements, vitamins, etc5. Additionally, safflower is famous for its flowers, which are traditionally used in dyeing and medicine to treat lung injury, cancer and other diseases6. However, the development of safflower and its yield are affected by various environmental factors. A wide range of abiotic stresses can interfere with crop growth, of which drought is generally considered to the most destructive7. The scale of seasonal drought is predicted to enhance progressively in the upcoming time period due to era of climate change8. In higher plants, complex and precise gene expression regulation is necessary for different biological processes9. It is widely known that multiple genes in plants are used to encode transcription factors (TFs), which are key regulators playing a major role in gene transcription regulation10. TFs are capable of activating or suppressing the expression of their target genes by recognizing and combining with the cis-regulatory elements in promoters to further control the flow of genetic information11,12. At present, various TF families are grouped together depending on their DNA-binding domains13. Among them, the NAC gene family has received extensive attention in research on plants14.

Since Souer et al. first cloned NAC TFs from Petunia in 1996, functional characterization of NAC family proteins had been carried out in soybean, tomato and other plants15–17. The naming of this family of proteins comes from the initials of proteins with similar conserved structures, such as NAM (no apical meristem), ATAF1/2 (Arabidopsis transcription activation factor) and CUC (cup-shaped cotyledon)15,18. The highly conserved structural domain at the N-terminus of NAC proteins can be divided into five subdomains (A-E)19. Subdomain A can be associated with the formation of dimerization of NAC proteins, subdomains C and D contain nuclear localization signals for binding to DNA, while the conserved B and E subdomains are usually considered to associated with the diversity of NAC proteins20. Meanwhile, the functional diversity of NAC proteins is due to the existence of transcriptional activation regulatory region at the C-terminus of NAC TFs with high variability21. NAC TFs can interact with the NAC recognition sequence in the promoter of target genes, which contains the CACG core DNA-binding motif, to regulate the transcription of these genes, thereby affecting plant senescence, secondary cell wall biosynthesis, sustainable yield, lateral root development and various stress tolerance processes22–25. For instance, Yuan et al.26 revealed that ONAC066 exerts a positive effect when rice is subjected to drought stress. Similarly, various NAC TFs (GmNAC12, TaNAC071-A) have been identified from different plants that are closely associated to drought tolerance27,28. Therefore, it is particularly important to identify the NAC genes in safflower and further investigate their relationships with safflower’s response to drought stress.

Among various varieties of safflower, we are particularly interested in safflower cultivar ‘Jihong 01’, which is widely grown in the western region of China. To gain a deeper understanding of the safflower, the whole-genomic sequencing of this safflower cultivar ‘Jihong 01’ was conducted in our preliminary research29. This variety differs from the genome used for identifying the safflower NAC TFs reported by Zhan et al30. In their study, 87 NAC genes were identified based on the genome of the safflower cultivar ‘Anhui-1’, safflower variety distributed in eastern region of China. In the present study, to discover the CtNAC TFs, and their relationship with drought stress response, genome-wide identification and systematic analysis of the NAC family in safflower cultivar ‘Jihong 01’ were carried out using the latest genomic data. Furthermore, the changes in morpho-physiological and biochemical properties of safflower under water deficit, as well as the expression of CtNACs under water deficit was also investigated.

Results

Identification of NAC family genes

To identify the NAC genes in safflower, BLAST and Hidden Markov Models were used to search the genome database using the Arabidopsis NAC sequences as queries. A total of eighty-five NAC gene family members were found in this crop, which were designated as CtNAC1-CtNAC85. The various basic properties of the 85 CtNAC proteins were further analyzed. As listed in Table A1, it can be clearly seen that the amino acid number of proteins encoded by the CtNAC genes varied greatly, ranging from 83 (CtNAC18) to 784 (CtNAC22) amino acids. This difference was confirmed in the analysis of the molecular weight (MW) of the proteins encoded by these genes, which was in the range of 9.40 KDa (CtNAC18) to 87.00 KDa (CtNAC22). Additionally, the isoelectric points (PI) of most CtNAC proteins were acidic (53, 4.31–6.64), while those of the remaining 32 proteins were neutral and or alkaline (7.05–9.79). The aliphatic index of all CtNAC proteins exceeded 40, these CtNACs were considered unstable. It is widely known that hydrophobicity is one of the important factors determining the tertiary structure of proteins. Hence, the negative grand average of hydropathicity (GRAVY) of these CtNAC proteins were determined, and the results indicated that these identified proteins were hydrophilic. Furthermore, online tool was used to forecast the subcellular localization of the CtNACs in cells. It was found that the majority of the CtNAC family genes (61 out of 85) were localized in the nucleus, while the number of CtNACs located in the cytoplasm, chloroplast, mitochondria, peroxisome and golgi were 15, 4, 2, 2 and 1, respectively. These results suggest that the structure of CtNAC proteins is highly diversified, which also provides the possibility for CtNACs to present a wide range of biological functions.

Phylogenetic analysis and classification of CtNAC proteins

To reveal the evolutionary characteristics of the CtNAC gene family, phylogenetic analysis of the NAC protein sequences obtained from safflower and Arabidopsis was conducted. A total of 17 distinct subfamilies were found in the phylogenetic tree (Fig. 1). Referring to other studies, the subfamilies were named as NAM, NAC1, OsNAC7, NAC011, ATAF, AtNAC3, NAP, ONAC022, NAC2, ONAC003, ANAC063, TREN, ANAC001, OsNAC8, SEU5, TIP and ANAC077. Among them, the vast majority of subfamilies were shared by Arabidopsis and safflower. It could be observed that only one CtNAC protein was grouped in the subfamily OsNAC8, while the subfamily OsNAC7 contained up to 14 CtNAC proteins. It is worth noting that no NAC member from the ANAC001 subfamily was identified in safflower, which was different from the subgroup analysis of NAC proteins from Arabidopsis.

Fig. 1.

Fig. 1

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Phylogenetic analysis of NAC proteins in safflower and Arabidopsis. The neighbor-joining (NJ) phylogenetic tree was constructed using MEGA X with 1000 bootstrap replications. The tree divided the CtNAC proteins into 17 subfamilies represented by different colored clusters within the tree. CtNACs indicates the NAC proteins from safflower. ANACs represents the NAC proteins from A. thaliana.

Gene structure and conserved protein motifs detection of CtNACs

As typical markers of gene family evolution, the exons/introns of the CtNAC genes were further analyzed31. As illustrated in Fig. 2a, the NAC gene family members of safflower were grouped into 16 subgroups, which was consistent with above results. Figure 2b shows that almost all the CtNAC genes contain multiple introns except for CtNAC72. Intron loss is a widespread event in the evolution of eukaryotes, which is often considered to be related to intron turnover or mature mRNA homologous recombination with intron-containing alleles after reverse transcription32. Among them, the one with the highest number of introns is CtNAC53. A putative motif analysis was performed on all 85 members using an online procedure, and 20 conserved motifs were found (Fig. 2c). The specific amino acid sequences and the identification for each motif are presented in Table A2 and Figure A1. Motifs 3, 5, 1, 4, 2 and 6 correspond to the NAM domain located at the N-terminus of NAC proteins in safflower, and most CtNACs have NAM domain. Some of the CtNAC members in the same subfamily have analogous motif arrangements, and it is speculated that they may exhibit similar biological functions. Additionally, markedly differences in the analytical characteristics of CtNAC proteins were also observed, which might be due to differences in their non-conserved regions. In addition, according to the distribution of the conserved motifs in the subfamilies of CtNAC proteins (Figure A2), some specific conserved motif compositions were found in some specific subgroups. For example, motifs 10, 12, 16 and 19 were found only in the OsNAC7 subfamily, motifs 9 and 11 in the NAC2 subgroup, motif 15 in the NAM subgroup, etc. It is hypothesized that the motifs unique to these subgroups may confer specific functions on safflower.

Fig. 2.

Fig. 2

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Gene structure and conserved motif distribution of CtNAC gene family. The phylogenetic tree was constructed by MEGA-X using the NJ method. The phylogenetic subfamilies are marked with different color backgrounds (a). Gene structure analysis (b). The red color indicates the exons, the purple color indicates the untranslated 5′and 3′ regions, and black lines indicate the introns. Conserved motif composition analysis (c). The conserved motifs of safflower were represented by boxes. Each motif is represented by a number in the colored box.

Functional annotation of the CtNAC gene family

To comprehensively understand the functional differentiation of safflower NAC gene family members, the identified 85 CtNAC genes were annotated and functionally categorized. In terms of GO analysis, 80 CtNAC genes out of the 85 genes analyzed were successfully annotated. As illustrated in Fig. 3a, among the 80 CtNAC genes annotated, 19 genes were classified into all three main categories, including biological process (BP), molecular function (MF) and cellular component (CC). A total of 68.75% (55) of CtNAC genes were grouped into BP and MF categories, while only 6 CtNAC genes (7.5%) were categorized into one of the three main categories. Furthermore, the annotated 80 CtNAC genes were classified into a total of 21 subcategories, with 16 subcategories in BP, 3 subcategories in MF and 2 subcategories in CC (Fig. 3b). The majority of the CtNAC genes were markedly enriched in six subcategories, including cellular process, biological regulation, regulation of biological process, metabolic process, binding and cellular anatomical entity. The genes in the subfamily ANAC077 mapped up to 17 subcategories, which demonstrated the functional differentiation of the members in this subfamily (Fig. 3c). Additionally, CtNAC56 was annotated with the highest number of 15 subcategories, indicating that this gene has multiple functions in biological processes (Fig. 3d). However, CtNAC63 was only annotated to the cellular anatomical entity of CC. We also noted that most of the CtNAC genes were mapped to bioregulatory subclasses, indicating that CtNACs might be particularly important for drought tolerance in safflower.

Fig. 3.

Fig. 3

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GO functional annotation and enrichment analysis of CtNAC genes. Venn diagram of CtNAC genes’ functional classification (a). Functional categorization of the CtNAC genes at Level 2 and enrichments (b). Distribution of each subfamily in 21 subcategories of CtNAC genes (c), and variation of the functional categories of the 85 CtNAC genes (d).

Cis-regulatory element analysis of CtNAC genes

To gain a preliminary understanding of the possible transcriptional regulatory mechanisms of CtNAC genes during the stress response of safflower, the predictive analysis of cis-regulatory elements in the promoters of CtNAC genes was carried out. The detail distribution of the cis-acting elements of all CtNAC genes is illustrated in Fig. 4a. The cis-acting elements contained in all NAC genes of safflower can be divided into the following four classes, such as light response, hormonal response, stress response, and plant growth and development-related elements (meristem, endosperm, circadian and seed). Among the identified CtNAC genes, the highest number of cis-regulatory elements was associated with hormones, 82 were associated with light, 83 associated with stress, and the lowest number associated with plant growth and development, with a total of 48. To clarify the number distribution of each element in the four major categories of cis-regulatory elements, a classification analysis was performed (Fig. 4b). In the light response category, there were eight subcategories, namely G-box, GT1-motif, GATA-motif, MRE, TCCC-motif, I-box, Box-4 and CHS-CMA cis-elements. Additionally, a variety of cis-elements responsive to plant hormones were found. Similarly, six cis-elements (ARE, MBS, LTR, TC-rich repeats, WUN-motif, and W-box cis-elements) were also identified, which are common cis-regulatory elements involved in various stress responses, such as anaerobic stress, drought and low-temperature response, pathogen defense and wound stress response. The cis-elements in the plant growth and development category predicted in this study were metabolism regulation-related cis-elements (O2-site, HD-ZIP and MSA-like), meristem-related cis-elements (CAT-box and CCGTCC-box), endosperm development-related cis-elements (GCN4-motif), and circadian and seed-related cis-elements (RY elements). Thus, these findings reveal that the identified CtNAC genes may contribute prominently to several biological processes in safflower.

Fig. 4.

Fig. 4

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The analyses of cis-acting elements in CtNAC genes. The number of cis-acting elements of CtNAC genes and divided into four categories, i.e., light responsive, hormone responsive, stress responsive and plant growth and development (a). The percentage of each cis-elements in each group (b).

Analysis of interaction networks among members of the CtNACs family

BioLayout Express3D software was used to construct an interaction network based on the correlation between the NAC genes (Fig. 5). The interaction network showed a complex family, consisting of 80 nodes and 838 edges. There was a certain degree of correlation among the genes in the network, and this trend was statistically significant. These findings suggest that although the functions and expressions of the NAC family members in safflower are quite different, there is a relationship among them in terms of expression and functional collaboration.

Fig. 5.

Fig. 5

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Network analysis of the CtNAC genes. The co-expression network of the 85 CtNAC genes constructed at P ≤ 0.05. It consists of 80 nodes and 838 edges.

Changes in the growth and physiological indicators of safflower after exposure to water deficit

Changes in the growth indices of safflower under water deficit condition

The detection of different growth indices of safflower under water deficit treatment was performed, and the results are illustrated in Fig. 6. It is evident that drooping and wilting leaves were observed in the treatment groups exposed to different degrees of water deficit, especially the severe water deficit (SD) group (Fig. 6a). Furthermore, as illustrated in Fig. 6b-d, the relative water content (RWC), fresh weight (FW), dry weight (DW) of safflower treated with moderate (MD) and severe water deficit condition were significantly lower than control group (CK, P < 0.001). Meanwhile, there were statistically significant differences in the above three indicators between MD and SD groups (P < 0.001). Moreover, the roots of safflower were collected and photographed. An obviously less developed root system was found in the water deficit groups compared to control group (Fig. 6e). Similarly, regarding to the fresh and dry weight of the roots, the two water deficit-treated groups were detected to be lower than those of CK group (Fig. 6f, g; P < 0.001). Additionally, the root length of safflower in the MD group and the SD group was 24.07% and 48.1% shorter than that in the CK group, respectively (Fig. 6h). The above data reveal that the growth of safflower affected by the water deficit condition.

Fig. 6.

Fig. 6

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Effect of water deficit on plant growth indices of safflower. Morphological analysis of safflower under different levels of water deficit (a), fresh weight of plants (b), dry weight of plants (c), relative water content (d), morphological analysis of safflower roots under different levels of water deficit (e), fresh weight of roots (f), dry weight of roots (g), and root length (h). ***P < 0.001, Bar = 1 cm.

Changes in the photosynthetic pigments of safflower leaves during water deficit condition

The differences in the pigment contents in safflower leaves between the treatment and control groups were assessed (Fig. 7). Chlorophyll a, chlorophyll b and total chlorophyll (a + b) contents in safflower leaves showed downregulating trend with increasing severe water deficit, and there were statistically significant differences between the groups (P < 0.001).

Fig. 7.

Fig. 7

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Changes in the photosynthetic pigments of safflower leaves under water deficit condition (***P < 0.001).

Changes in the osmotic substance concentrations of safflower under water deficit condition

Accumulated evidence suggests that accumulation of osmoregulatory substances is the response strategy of plants to cope with arid environments, which has been confirmed in many plants, such as sugar beet33 and wheat34. In the present study, the levels of osmoregulatory substances in safflower collected from different groups were also investigated. It was found that the levels of soluble sugars, proteins, and proline in both treatment groups were all markedly increased by the water deficit treatment (P < 0.001, Fig. 8a-c). Moreover, as the degree of water deficit stress increased, the levels of the detected osmoregulatory substances increased more significantly.These data indicate that safflower can regulate cellular osmotic pressure by accumulating osmoregulatory substances to maintain normal cellular function, thereby reducing its damage caused by water deficit.

Fig. 8.

Fig. 8

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Changes of osmoregulatory substances in safflower under different levels of water stress. Changes in proline content (a), soluble protein (b), and soluble sugar content (c) of safflower during water stress (***P < 0.001).

Antioxidant capability of safflower treated with water deficit

The excessive accumulation of ROS under limited water conditions can seriously damage protein structures, oxidize macromolecules, and ultimately lead to plant cell death33,35,36. To understand the oxidative damage of safflower under water deficit treatment, the content of MDA and H2O2 in the leaves were detected. As presented in Fig. 9a, the MDA accumulation in safflower seedling leaves increased markedly with the severity of water deficit when compared to normal irrigated plants (P < 0.001), and the highest level of MDA was observed in the SD group. Additionally, there was a statistically significant difference in the MDA levels between the MD and SD groups (P < 0.001). Similar results were also obtained regarding the H2O2 levels of safflower under water deficit (Fig. 9b). Moreover, the results of the DAB staining revealed that more intense DAB staining was found in safflower subjected to water deficit stress, especially the SD group (Fig. 9c). These data indicate that a large amount of H2O2 accumulates in safflower in response to water deficit, and the more severe drought stress, the higher the severity of membrane lipid peroxidation in safflower seedling leaves.

Fig. 9.

Fig. 9

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Effect of water deficit on antioxidant capability of safflower. Changes in MDA content of safflower under water deficit (a), H2O2 content (b), DAB staining of safflower leaves (c), and antioxidant enzyme activities, such as CAT, SOD and POD (d-f). ***P < 0.001.

Furthermore, the results showed that the activities of antioxidant enzymes, such as superoxide dismutase (SOD), peroxidase (POD), catalase (CAT) in the mild and severe water deficit groups were clearly higher than CK group (P < 0.001, Fig. 9d-f), suggesting that safflower responds to oxidative damage by enhancing antioxidant enzyme activities to eliminate ROS when adapting to water deficit stress. Moreover, the activities of antioxidant enzymes showed a tendency to enhance and then decrease with an increase in the severity of drought stress. Overall, these findings indicated that the safflower resists oxidative damage caused by water deficit by improving the activities of antioxidant enzymes.

Expression analysis of CtNAC genes

To know the expression changes of CtNACs under water deficit, the expression levels of the CtNAC genes were detected in the plant leaves collected from three groups of safflowers (Fig. 10). Most of the expression levels of the CtNAC genes are affected by water deficit. Compared with normal irrigation group, the expression changes of most CtNACs in the SD group were greater than MD group, except CtNAC21, CtNAC44, CtNAC15, etc. In MD group, 35 CtNACs were up-regulated more than 2 folds, and CtNAC38, CtNAC45, and CtNAC78 were the three most up-regulated genes. 24 CtNACs were down-regulated after mild water deficit condition. Under sever water deficit, 59 and 25 CtNACs were up-regulated and down-regulated, respectively, compared with CK group. Similar, CtNAC38, CtNAC45, and CtNAC78 were the three most up-regulated genes in SD group. It suggests that they may play an important role in the response of safflower to drought stress, but this speculation needs further exploration.

Fig. 10.

Fig. 10

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Expression analysis of CtNAC genes in response to water deficit condition.

Discussion

With the advancement of sequencing technologies and genome research, the identification of NAC gene family members in different plants has been widely carried out. In the present study, a total of eighty-five CtNACs were identified from safflower widely grown in the western region of China. Considering the differences in genome size among species, the number of NAC gene family members identified in different plants varied. For example, the number of NAC genes identified from the genome databases of Arabidopsis, sunflower, Nelumbo nucifera, Melilotus albus, and tomato was 117, 150, 97, 101 and 93, respectively37–41. Compared with these plants, the limited NAC genes in safflower may interpreted as a reduction in this gene family during genome duplication or evolution. This evolutionary divergence likely reflects species-specific gene duplication events occurring after speciation from their common ancestor. Moreover, many initially predicted plant transcription factors have been subsequently excluded due to either extensive duplications or annotation errors in earlier genomic analyses42. Notably, in comparison to the 87 CtNAC genes reported by Zhan et al. in safflower, our study identified fewer in the safflower cultivar ‘Jihong 01’30. It is speculated that the difference in the number of genes may due to variations in genome annotation results or evolution. Similar phenomena have also been found in other plants43. For instance, compared with the CrRLK1L subfamily members in previous research, Ma and his coworkers identified more CrRLK1L genes in tomato44. Similarly, discrepancies exist in the reported numbers of NAC members in Brassica rapa (L.), with Li et al. and Chen et al. reporting different counts45.

As well known, phylogenetic analysis represents a critical framework in modern biology, providing essential insights into evolutionary relationships among species45. More importantly, phylogenetic analysis provides a robust framework for predicting drought-tolerant candidate genes within TF families46. It is reported that NAC genes with analogical functions are more possible to be grouped into the identical subfamily47. For instance, A. thaliana NAC transcription factors belonging to the ATAF, NAP and AtNAC3 subfamilies have been demonstrated to mediate critical stress response pathways37. Our classification identified 17 phylogenetically distinct subfamilies, with co-orthologous CtNAC and AtNAC proteins present in most subfamilies, suggesting the NAC proteins within the same subfamily may exhibit similar functions. For instance, it is speculated that the CtNAC39 belonging to the NAP subfamily may have stress-responsive functions similar to the ANAC05637. Moreover, the analysis cis-regulatory elements indicated that many identified safflower NAC genes might be involved in response to stress of this crop, as evidenced by the presence of numerous stress-associated cis-elements. Taken MBS element as example, Kaur et al. revealed the funcional importance of genes harboring MBS cis-element in drought stress adaptation48. These findings suggest that multiple CtNAC genes may be invovled in safflower’s stress response. Additionally, the above results also indicate that the CtNAC genes exhibit diverse functional roles. The phylogenetic analysis revealed that no safflower NAC member was identified in the ANAC001 subfamily, the results of which was similar to the phylogenetic analysis results of tartary buckwheat and tea plant NAC proteins49,50. It is speculated that the proteins in this subfamily were lost during the evolution of safflower, tartary buckwheat and tea plants.

Considering the major role of NAC TFs in various plants’ response to drought, this study further investigated the physiological and morphological variation of safflower under water deficit, as well as the expression changes of CtNACs. The plants undergo varying degrees of changes in morphological, physiological, biochemical, and metabolic characteristics during stress condition51. Due to the damages of cellular components, drought leads to changes in plant morphology. Leaf drooping, wilting, rolling and even abnormal abscission are common morphological changes in plants subjected to drought, and wilting leaves were observed in water deficit treated safflowers52. The morphological response of leaves to drought stress is one of the important strategies for plants to adapt to arid environments, reduce water loss, and enhance water use efficiency (WUE). When plants perceive insufficient water, their leaves will noticeably droop, reducing water loss through passive movement. Evidently, the phenotype of plants treated with drought vary among species, which is closely related to various factors (the tolerance level, developmental stage, etc.)53. In this work, the safflowers grown under water deficit exhibited more wilted leaves, lower RWC, FW, DW, and root biomass, all of which may closely related to decreased intracellular turgor pressure under drought stress54. These changes are in accordance with multiple previous investigations55.

Osmotic adjustment is considered another strategy for plants to cope with osmotic stress caused by drought treatment. Under limited water irrigation, plants mitigate turgor loss by accumulating osmotic adjustment substances (proline, soluble proteins and soluble sugar, etc.) to reduce the cell osmotic potential and further maintain cell turgor pressure56. Accumulation of proline, soluble sugars, and soluble proteins in the leaves has been widely documented in stressed plants, such as Cocos nucifera57, Tartary buckwheat58, pepper59. In the present study, the levels of proline, soluble proteins and soluble sugar were enhanced in MD and SD groups, indicating that in response to adverse effects caused by water deficit, osmotic adjustment substances were accumulated in safflower. However, previous studies have mentioned that there are differences in the frequency and degrees of solutes accumulation under drought conditions among different crop species60. For instance, it was reported that the proline content in drought tolerant plant varieties is higher than stress sensitive varieties61. Undoubtedly, the accumulation of proline may be attributed to the decreased protein synthesis and enhanced protein degradation, as well as an increase in proline synthesis and decrease in its degradation62. Previous studies have revealed that the increase in proline synthesis caused by drought may be related to the deficiency of feedback inhibition of P5CS (△1 -pyrroline-5-carboxylate synthetase, a key enzyme for proline synthesis) by proline, and the upregulation of P5CS expression63. In addition to balancing osmotic pressure, it is worth noting that osmotic adjustment substances can also exert the functions of scavenging ROS, stabilizing cell membranes and proteins in adverse environmental conditions64. Moreover, proline can also serve as a regulator of cytosolic acidity, a source of carbon and nitrogen, and signaling molecule that activates the defense systems of plants63. Similarly, in arid environments, soluble sugars can not only serve as substrates for cellular respiration, but also regulate osmotic pressure, protect organic molecules and membranes, and improve LWC65. The accumulation of soluble sugars reduces the water potential of cells, which is beneficial for plants to absorb limited water in the rhizospheric soil66. Likewise, increased soluble protein in plants treated with drought was widely found by others. However, the findings that stress leads to reduced in soluble proteins in plants have also been observed67.

In addition to turgor loss, osmotic stress caused by stress can also triggers to increase the oxidative stress53. In water deficient environments, the accumulation of ROS is major responsive change in plants, which further affect the membrane structure, photosynthetic pigment levels, macromolecular amount68. Insufficient water leads to decrease in the absorption of CO2 by plants, followed by the reduction in carbon fixation, saturation of the electron transport system and ultimately accumulation of ROS69. In this study, optimum ROS, MDA and H2O2 levels were found in the safflowers treated with drought, and this phenomenon became more pronounced with increasing severity of stress. It suggests that the membrane lipid peroxidation of safflowers became more severe with increasing drought intensity. Similar results were also found in other plants70. These changes might be attributed to the oxidative stress and lipid peroxidation for the organelles of plant, including mitochondria, chloroplast, and cell membrane71.

However, the antioxidant enzyme system is the main strategy for plants to resist oxidative stress. In present study, obvious increases in the activities of SOD, POD, and CAT were detected in the stress treated safflower plants, suggesting that the redox defense ability of safflower was enhanced to cope with oxidative stress caused by drought. However, as the intensity of stress increased, the activities of these enzymes markedly downregulated, in comparison with MD group. These results were similar to the results of other studies72,73. SOD activity in maize hybrids did not enhance with the severity of stress72. Continuously increasing MDA level and initial increase and then reduction in POD and CAT activities were observed in soybeans during stress intensity increased74. It is speculated that sustained high-intensity stress disrupted the ROS scavenging system and chloroplast structure of safflower, leading to significant accumulation of ROS and intensified membrane lipid peroxidation36,75. Moreover, it is reported that the changes of antioxidant enzyme after drought treatment are related to various factors, such as the species and metabolic state of plants, stress intensity and treatment frequency76. For example, with the extension of drought treatment, the activities of SOD and CAT enzymes in Castanopsis fissa showed the change of initial increasing and then reducing73. The degree of changes in antioxidant enzyme activity varied among different genotypes of soybeans after drought treatment77. Additionally, the severity of drought increases, the POD activity that first decreased and then obviously increased was also observed78.

In the present study, the levels of photosynthetic pigments (chl. a, chl. b and chlorophyll a + b) were decreased in the leaves of safflowers treated with water deficit. It is widely known that chlorophyll is the main pigment that affects plant photochemical efficiency, and maintaining high chlorophyll content is another way to improve the stress tolerance efficiency. However, the phenomenon of chlorophyll degradation in plants under drought conditions has been demonstrated in different studies. For example, the chlorophyll content in Oudeneya africana grown under water deficit was significant decreased79. Lei et al. found that with the aggravation of drought stress, the chlorophyll level in Cunninghamia lanceolata leaves showed markedly downward trends80. These changes are mainly attributed to the damage of ROS to chloroplasts, subsequently causing the photooxidation of pigment or the degradation of chlorophyll81. Taken together, the above findings further demonstrate that there are differences in physiological responses of different plant varieties to drought stress. Meanwhile, understanding the physiological responses of plants to drought is particularly important for plant cultivation.

Furthermore, the changes in expression of CtNACs under water deficit was detected by qRT-PCR. The data revealed that the expression of most CtNACs were upregulated under water deficit stress. According to the results provided by others, it is speculated that many members of the NAC subfamilies in safflower may be involved in safflower’s response to drought. Among these up-regulated CtNACs, CtNAC38 and CtNAC45 belonging to the OsNAC7 subfamily, as well as the CtNAC78 belonging to the AtNAC3 subfamily were the most up-regulated genes. Previous studies revealed that the genes from some NAC subfamilies have been found to be associated with the drought stress response. It is particularly prominent that the member from subfamilies ATAF, AtNAC3 and OsNAC7 are commonly considered stress-responsive NAC protein82. For instance, in Chrysanthemum nanking, Wang and coworkers83 found that the OsNAC7 subfamily members were involved the regulation of drought stress. Other research studies have provided evidence for the important role of Arabidopsis AtNAC3 subfamily in physiological processes, such as leaf senescence and stress tolerance84,85. It was reported that the overexpression of the ANAC019, ANAC055 and ANAC072 genes in this subfamily enhanced drought tolerance and conferred abiotic stress tolerance in Arabidopsis86. Additionally, there is a positive correlation between the expression of CaNAC46 gene belonging to ATAF subfamily and the drought resistance of A. thaliana87. In this work, it was found that the expression levels of CtNACs (CtNAC77 and CtNAC80) belonging to the ATAF subfamily exhibited up-regulation under water deficiency. These findings further suggest that many CtNAC genes may be involved in the safflower’s drought stress. In recent years, more and more research has focused on the key role of NAC TFs in plant stress tolerance and their regulatory mechanisms. Previous works have revealed the function of several NAC TFs (PgNAC10388, PwNAC189, MdNAC190, etc.) in regulating plant ROS levels under drought conditions. Shim et al. OsNAC14 plays a major role in improving drought tolerance in transgenic rice by regulating the genes related to DNA repair, defense, and strigulates biosynthesis91. Additionally, NAC TFs has been reported to improve plant stress tolerance by affecting the development of plant roots92, stomata93, the accumulation of osmotic substances94, etc. In addition to the ABA independent pathway, the NAC TFs can also participate in the responses to drought stress through ABA dependent pathways95. These data reveal that NAC TFs are associated in the regulation of several complex signaling pathways under conditions of insufficient moisture96. However, the regulatory mechanism of CtNACs in response to drought and other abiotic stresses in safflower still need to be further explored in our subsequent work.

Materials and methods

Identification of NAC genes in safflower

NAC family sequences of Arabidopsis (ANAC) were retrieved from the Arabidopsis Information Resource (TAIR) database (https://www.arabidopsis.org/). The whole genome sequence data of safflower supporting the results of this study have been deposited in the NCBI database (https://www.ncbi.nlm.nih.gov), and the biological project accession number (PRJNA399628). To identify potential NAC proteins in safflower, BLASTp analyses with all ANACs as queries were carried out31. Hidden Markov Models (HMMs) of the conserved structural domains of NAM (PF02365) and NAC (PF01489) were obtained from the Pfam database (https://pfam.xfam.org/) to screen for target genes, and these models were used to further screen for putative NAC sequences using HMMER3.0 (http://hmmer.janelia.org/)97. Subsequently, to eliminate the interference of sequences without NAC domains, all candidate CtNAC proteins were verified using the Conserved Domains Database (CDD) (http://www.ncbi.nlm.nih.gov/cdd/) and SMART tool (http://smart.embl-heidelberg.de/)98. Sequences with NAC or NAM domains were considered NAC proteins in safflower.

Protein feature analysis of NAC gene family members in safflower plants

With the help of the ExPASy online tool (https://www.expasy.org/), the physicochemical properties of the identified NAC proteins in safflower were analyzed, including the length of amino acids, molecular weight, isoelectric point, aliphatic index and grand average of hydropathicity. Additionally, Wolfpsort (https://wolfpsort.hgc.jp/) was employed to assess the subcellular localization of all selected CtNACs.

Phylogenetic analysis of NAC proteins

To investigate the evolutionary relationship of NAC proteins in safflower, phylogenetic tree of the identified CtNAC proteins was built. Briefly, the obtained full-length NAC protein sequences of Arabidopsis were aligned with the identified NAC protein sequences of safflower using Clustal X (version 1.83). Afterward, MEGA X program was employed to build a neighbor-joining (NJ) phylogenetic tree with 1000 bootstrap replications and pairwise detection99. Furthermore, to visualize the phylogenetic tree, the iTOL software (https://itol.embl.de/login.cgi) was also used41.

Gene structure and conserved motif analyses of safflower NAC gene family members

The Gene Structure Display Server (GSDS) 2.0 program (http://gsds.gao-lab.org/) was used to elucidate the intron/exon structures of safflower NAC genes as previous studied86. Furthermore, the conserved motifs of CtNAC proteins were generated using MEME (Multiple Expectation Maximization for Motif Elicitation) tool (https://meme-suite.org/meme/tools/meme)100.

Functional annotation of safflower NAC gene family

To preliminarily analyze the role of CtNACs, GO analysis was further conducted. All the NAC proteins in safflower were functionally categorized and annotated with gene ontology (GO) terms using the Omicsbox platform (https://www.biobam.com/omicsbox-update-1-4/).

Identification of cis-regulatory elements of NAC genes in safflower

To predict the cis-regulatory elements of the obtained NAC genes in safflower, all promoter sequences of CtNACs (the 2000 bp sequences upstream of the transcription start) were obtained from the safflower genome. Then, the plantCARE software (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/) was used to analyze the cis-regulatory elements of these target genes, which were visually displayed in the promoters using the Tbtools software101,102.

Network analysis of the CtNAC genes

The interaction network between the CtNAC gene family, the Pearson correlation coefficient was calculated using the R programming language and software (http://www.rproject.org/). Gene co-expression networks were constructed using BioLayout Express software (Version 3.2).

Treatment design and morphological traits responses

The safflower seeds (cultivar ‘Jihong 01’) used in the present work were bought from the Honghuayuan Co. Ltd. (Xinjiang, China) and preserved in our laboratory. This variety is a widely planted safflower in Xinjiang, with plant height of 85–115 cm, branch height of about 40 cm, and fruit diameter of 1.9–3.2 cm. Safflower cultivar ‘Jihing 01’ usually used for oil and flower purposes. The seeds are white and conical, with husk rate of 58%, and oil content of 26.3%. The whole growth period was 115 days.

The seeds were sown in the pots (bottom, 4 × 6 cm, and height 8 cm) with planting soil (mixture prepared by mixing nutrient soil and vermiculite in a mass ratio of 7:3) and grown in a greenhouse (25 ± 1 °C, 16 h/8 h photoperiod (light/dark) and 60% ambient air humidity). After allowing the plants to grow for 45 days, the plants were thoroughly irrigated upto field capacity and then the plants were drought-treated with reference to other studies103. The potted plants were randomly divided into three groups, such as CK group (these plants were grown with regular irrigation, and the water moisture level was maintained at 85–90% of soil moisture capacity), the MD group (the soil water content was decreased to 45–50%, which was maintained to continue the stress treatment), and the SD group (the soil water content was decreased to 20–30%, which was maintained to continue the stress treatment). Specifically, the moisture content of the soil in the potted plants was determined by using the SK-100 soil moisture tester (Shanghai Topsy Electronic Technology Co., Ltd.), by gradually reducing the irrigation volume until the relative moisture content of the soil reaches the expected value, and at the same time, the weight of the potted plants was recorded at this time, and the moisture control was carried out by using the weighing method, and the weight of each potted plant was recorded once every 2 days. Each treatment was replicated three times. After 3-weeks, the morphology of all plants was photographed using a camera (Sony). Meanwhile, the morphological traits, including the leaf relative water content, fresh weight, dry weight, and plant length, fresh weight and dry weight of roots, root length were determined under different degrees of water deficit, with reference to the methods described in the literature104,105. Then, the roots and intact leaves of all groups were harvested. After washing, the samples were placed in liquid nitrogen and stored in a deep freezer at −80℃ for biochemical analysis.

Determination photosynthetic and biochemical activities

After 3 weeks of water deficit treatment, the plant leaves were collected from all groups and used to determine photosynthetic and biochemical parameters by Solarbio analysis kit (Beijing Solarbio Technology Co., Ltd.), including osmotic substance levels (proline, soluble protein, soluble sugar), antioxidant activity (CAT, POD and SOD), lipid peroxidative damage levels and ROS accumulation (MDA and H2O2)106, photosynthetic indicators (chlorophyll a, chlorophyll b and total chlorophyll107). Additionally, the leaves were also stained for hydrogen peroxide using 3,3’-diaminobenzidine (DAB) staining kit (Wuhan Servicebio Technology Co., Ltd.) to detect hydrogen peroxide.

Quantitative real-time PCR analysis

The total RNA was extracted from the safflower leaves under different stress levels using commercially available RNAiso Plus reagent (TaKaRa, Dalian, China), and then reverse transcribed into cDNA by PrimeScript RT kit (TaKaRa, Dalian, China). qRT-PCR was carried out according to the manufacturer’s instructions using SYBR Premix Ex Taq™ (TaKaRa, Dalian, China) according to the manufacturer’s instructions. Ct60srRNA was selected as an internal control. The relative expression of genes was calculated using the 2-ΔΔCt method108. The specific primers for each gene are shown in Table A3.

Statistical analysis

Mean ± standard deviation (S.D.) was employed to represent the data, and one-way ANOVA followed by post hoc multiple comparisons (Dunn’s test) was carried out using GraphPad Prism 8.0 software (GraphPad Software, San Diego, California). Statistically significant differences were found according to P-value.

Conclusion and future recommendations

In the present study, eighty-five CtNAC genes were identified and analyzed in safflower, including their physicochemical properties, phylogenetic relationships, gene structure and conserved protein motifs, cis-elements, etc. Furthermore, the physiological response of safflower to water deficit were further investigated. The results showed that water deficit markedly inhibited safflower growth while increasing the levels of osmotic adjustment substances (soluble sugar, soluble protein, proline), reactive oxygen species (ROS), malondialdehyde, hydrogen peroxide (H2O2), and antioxidant enzymes activities (superoxide dismutase, peroxidase, catalase). Additionally, pigments content in safflower were decreased after water deficit treatment. In addition, most CtNACs were markedly upregulated under water deficit stress, especially the CtNACs belonging to the AtNAC3 and OsNAC7 subfamily, indicating these three genes may be related to the response of safflower to water deficit. These findings lay a foundation for us to understand the role of NAC genes in safflower, which can help improve its water deficit tolerance efficiency through molecular breeding. However, further research is needed to fully elucidate the molecular mechanisms by which CtNACs regulate drought stress responses in safflower. Meanwhile, gene knockout/knockdown studies (using CRISPR-Cas9 or RNAi) should be validate the role of key NAC TFs in drought tolerance in near future.

Supplementary Information

Supplementary Information. (1.2MB, docx)

Author contributions

Conceptualization by J.L.Y., L.H., and L.N.D.; Methodology was performed by J.L.Y., X.Y.Z., and L.H.; The Software is carried out by X.Y.Z and Z.T.Z.; Verification is carried out by J.L.Y. and L.H.; The survey was conducted by J.L.Y. and L.H.; Data management is carried out by J.L.Y., Z.T.Z. and L.H.; Writing – Preparation of the original draft is carried out by J.L.Y., L.H., and L.N.D.; Writing – review and editing by L.N.D and J.Y.; Supervision is carried out by L.N.D and F.W.W.; Project management is carried out by L.N.D; The acquisition of funds was carried out by L.N.D and J.Y. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by National Youth Science Fund Project of National Natural Science Foundation of China (grant number 32201275); Xinjiang safflower industry development fund.

Data availability

All data in the present study are available in the public database as described in the Materials and Methods section.

Declarations

Competing interests

The authors declare no competing interests.

Footnotes

Publisher’s note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Jialing Yang, Long Han are contributed equally to this work.

Contributor Information

Fawei Wang, Email: fw-1980@163.com.

Linna Du, Email: dulinna0918@163.com.

Supplementary Information

The online version contains supplementary material available at 10.1038/s41598-025-16483-7.

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Data Availability Statement

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