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Article

Membrane-Bound Transcription Factor ZmNAC074 Positively Regulates Abiotic Stress Tolerance in Transgenic Arabidopsis

by
Yexiong Qian
1,*,
Yan Xi
1,
Lingxue Xia
1,
Ziling Qiu
1,
Li Liu
1 and
Hui Ma
2,*
1
Anhui Provincial Key Laboratory of Conservation and Exploitation of Important Biological Resources, College of Life Sciences, Anhui Normal University, Wuhu 241000, China
2
Key Laboratory of Rice Genetic Breeding of Anhui Province, Rice Research Institute, Anhui Academy of Agricultural Sciences, Hefei 230031, China
*
Authors to whom correspondence should be addressed.
Int. J. Mol. Sci. 2023, 24(22), 16157; https://doi.org/10.3390/ijms242216157
Submission received: 17 October 2023 / Revised: 2 November 2023 / Accepted: 8 November 2023 / Published: 10 November 2023
(This article belongs to the Special Issue Recent Advances in Molecular Breeding for Drought and Salt Stress Tolerance in Crops)
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Abstract

:
Maize (Zea mays L.) is one of the most widely cultivated crops for humans, making a vital contribution to human nutrition and health. However, in recent years, due to the influence of external adverse environments, the yield and quality of maize have been seriously affected. NAC (NAM, ATAF1/2 and CUC2) transcription factors (TFs) are important plant-unique TFs, which are crucial for regulating the abiotic stress response of plants. Therefore, it is of great biological significance to explore the underlying regulatory function of plant NAC TFs under various abiotic stresses. In this study, wild-type and ZmNAC074-overexpressed transgenic Arabidopsis were used as experimental materials to dissect the stress-resistant function of ZmNAC074 in transgenic Arabidopsis at phenotypic, physiological and molecular levels. The analyses of seed germination rate, survival rate, phenotype, the content of chlorophyll, carotenoids, malondialdehyde (MDA), proline and other physiological indexes induced by distinct abiotic stress conditions showed that overexpression of ZmNAC074 could confer the enhanced resistance of salt, drought, and endoplasmic reticulum (ER) stress in transgenic Arabidopsis, indicating that ZmNAC074 plays an important regulatory role in plant response to abiotic stress, which provides an important theoretical foundation for further uncovering the molecular regulation mechanism of ZmNAC074 under abiotic stresses.

1. Introduction

Global agriculture is seriously threatened by various ecological and environmental conditions, resulting in substantial declines in crop yields. These yield losses are expected to increase further with global climate change [1]. Plants in terrestrial environments must face a myriad of unpredictable and extreme environmental challenges, particularly various abiotic stresses such as high temperature, drought, and high salinity, which greatly limit the distribution, growth, and development of plants [2,3]. The process of resisting stress in plants is complex and involves molecular, metabolic, and physiological levels [2,4,5]. When confronted with adverse environments, plants have evolved a series of adaptive strategies that can perceive environmental stress stimuli and thus respond to naturally occurring stress signals through specific regulatory mechanisms to improve plant tolerance [3,6,7].
In general, plants have developed distinct regulatory mechanisms to cope with different abiotic stresses [7]. For example, to adapt to the water deficiency in the soil, plants change their physiological function and then alter the growth and structure of the root system, and also close the leaf stomata in the aboveground part [8,9]. Furthermore, previous research has demonstrated that drought tolerance in plants is greatly influenced by redox regulation and antioxidant systems [10,11]. Similarly, plants have evolved appropriate mechanisms to adapt to salt environments, including regulating ion homeostasis, activating osmotic stress pathways, mediating plant hormone signals, and pathways dependent on reactive oxygen species (ROS) homeostasis [12,13,14]. In addition, when plants are stimulated by the external environment, it can lead to ER stress. Generally, under low ER stress, plants can positively regulate protein folding and degradation through the unfolded protein response (UPR), so as to restore the function of the ER [15,16].
Transcription factors are essential for plants to respond to a variety of environmental stresses [17,18,19]. Increasing studies have shown that specific NAC proteins not only participate in the regulation of plant growth and development, but also act as key regulatory factors to regulate various stress signaling pathways [20,21,22,23]. A substantial number of NAC family members are capable of responding to various abiotic stresses in plants [21,24]. For instance, overexpression of SNAC3 and ONAC066 can enhance the drought resistance of transgenic rice compared to wild-type rice [25,26]. The drought tolerance of wheat plants is weakened by knocking out TaNAC071-A, while overexpression of TaNAC071-A enhances drought resistance in wheat [27]. In addition, rice ONAC022 can play a significant role in salt stress by regulating ABA in various ways [28]. Potato NAC053 overexpression in Arabidopsis may enhance salt tolerance of transgenic Arabidopsis by promoting the expression of stress-associated genes [29]. Moreover, the membrane-binding transcription factor NAC062 of Arabidopsis transmits ER stress signals from the plasma membrane to the nucleus and thereby activates and up-regulates the expression of UPR-associated genes [30]. The NAC103 gene in Arabidopsis transmits transcriptional regulatory signals through the ER stress cis-element UPRE-III to downstream genes of UPR through its encoded protein, positively regulating ER stress [31].
Maize is one of the most widely cultivated crops and the most abundant grain crop in the world, and it also has become an important model monocot species for functional genomics analysis [32]. Studies have shown that drought or salt stress can seriously affect the growth and development of maize, which in turn affects the livelihood and economy of millions of people around the world [33]. Plant tolerance to various environmental stresses can be enhanced by NAC TFs, and thus they have potential use in crops to acquire stress tolerance and further develop stress-resilient varieties [34,35]. In our previous studies, ZmNAC074 has been demonstrated to respond in maize to heat, drought and salt stresses, and it can enhance the heat tolerance of ZmNAC074-overexpressing transgenic Arabidopsis [36]. In this study, ZmNAC074-overexpressing transgenic Arabidopsis were further used as experimental materials to dissect the underlying regulatory function of ZmNAC074 under salt, drought, and ER stress from the phenotype, physiology, and molecular levels. This study will provide an important basis for an in-depth exploration of the stress resistance function and molecular regulation mechanism of ZmNAC074 in maize.

2. Results

2.1. Germination of ZmNAC074-Overexpressing Transgenic Arabidopsis Seeds under Salt Stress

To investigate the germination rate of wild-type and transgenic Arabidopsis under salt stress, wild-type and transgenic Arabidopsis were seeded on 1/2 MS plates with 0, 100, and 200 mM NaCl, and the germination rate was counted seven days after vernalization. Under normal conditions, there was no significant difference in the germination rates of all Arabidopsis seeds, and the germination rates were all close to 95% (Figure 1A,D). Under 100 mM NaCl treatment, the germination rate of transgenic Line 1 and 9 was significantly higher than that of the wild-type (Figure 1B,D). In addition, when the NaCl concentration reached 200 mM, although the germination rates of all wild-type and transgenic Arabidopsis lines were lower than 50%, the germination rate of transgenic Arabidopsis lines was still higher than that of the wild-type (Figure 1C,D). In addition, under the two concentrations of salt stress, except for transgenic Line 9, which had a small number of green leaves after germination, the other lines hardly grew any green leaves. In conclusion, under salt stress, ZmNAC074 in Arabidopsis can improve the germination rate of transgenic Arabidopsis seeds.

2.2. The Phenotype of ZmNAC074-Overexpressing Transgenic Arabidopsis Seedlings under Salt Stress and the Determination of Physiological Indexes

To further determine the relationship between ZmNAC074 and salt tolerance of transgenic Arabidopsis, WT and transgenic Arabidopsis were treated with 200 and 400 mM NaCl for 10 days. Before salt stress treatment, there was no significant difference in the phenotype between transgenic Arabidopsis and WT (Figure 2A). However, under 200 mM NaCl stress treatment, the leaves of the WT showed obvious yellowing, but only a few of the leaves of transgenic Arabidopsis yellowed (Figure 2A). Under 400 mM NaCl stress treatment, the leaves of the WT and overexpression lines were yellowed in most plants, but the leaves of the WT also showed withering largely, while the yellowing degree of leaves of transgenic Arabidopsis was significantly lower than that of WT Arabidopsis. The above phenotypic observations showed that the salt tolerance of transgenic Arabidopsis was stronger than that of WT Arabidopsis.
To further explore the factors that may affect the salt tolerance of transgenic Arabidopsis, the physiological indexes of some Arabidopsis leaves under normal conditions and under 200 and 400 mM NaCl stress treatments were measured, respectively. Under normal conditions, there was no significant difference in the contents of chlorophyll, carotenoid, MDA, soluble protein, and proline between the wild-type (WT) and transgenic Arabidopsis (Figure 2B–F). However, after treatment with two different concentrations of salt stress for 10 days, except that there was no significant difference in chlorophyll content between Line 1 and WT under 200 mM NaCl stress treatment, the chlorophyll content of other transgenic lines was significantly higher than that of WT Arabidopsis (Figure 2B,C). In addition, except for the carotenoid content of Line 9 under 200 mM NaCl treatment and Line 2 under 400 mM NaCl treatment being higher than that of WT Arabidopsis, there was no significant difference in carotenoid content between WT and transgenic Arabidopsis under other salt stress treatments. Furthermore, the MDA content of transgenic Arabidopsis under 200 mM NaCl treatment was significantly lower than that of the WT (Figure 2D), while under 400 mM NaCl stress, only the MDA content of Line 9 was significantly lower than that of WT Arabidopsis. Additionally, except for the proline content of Line 2 under 200 mM NaCl treatment and the soluble protein content of Line 2 under 400 mM NaCl treatment not being significantly different from those of WT Arabidopsis, the proline and soluble protein contents of transgenic lines under other salt stress treatments were significantly higher than those of WT Arabidopsis (Figure 2E,F). To conclude, overexpression of ZmNAC074 enhanced the salt tolerance of transgenic Arabidopsis.
The leaves of all Arabidopsis lines under 200 and 400 mM NaCl stress were stained with DAB and NBT, respectively (Figure 2G,H). Under normal conditions, there was no significant difference in leaf color stained with DAB and NBT between the WT and the transgenic Arabidopsis lines. However, the leaves of dyed WT and transgenic Arabidopsis lines became darker after 10 days of salt stress, indicating that the contents of H2O2 and O2− increased under salt stress. In addition, the color of Arabidopsis leaves treated under 400 mM NaCl stress was darker than that under 200 mM NaCl stress. However, under either the treatment of 200 mM NaCl or 400 mM NaCl, the leaf color of the transgenic Arabidopsis lines was lighter than that of WT Arabidopsis, indicating that the contents of H2O2 and O2− in the transgenic lines were lower than those in WT Arabidopsis under salt stress. Correspondingly, by measuring the content of H2O2, it was found that the content of H2O2 of transgenic Arabidopsis was indeed lower than that of WT Arabidopsis after salt stress (Figure 2I). Similarly, after the determination of CAT and SOD enzyme activity, the corresponding results were obtained, that is, under two concentrations of salt stress, the CAT and SOD enzyme activity of transgenic Arabidopsis was higher than that of WT Arabidopsis (Figure 2J,K). However, the activities of CAT and SOD in WT and transgenic Arabidopsis leaves treated with 400 mM NaCl were lower than those treated with 200 mM NaCl. Collectively, compared with WT Arabidopsis, the ROS level of transgenic Arabidopsis was lower, which further indicated that transgenic Arabidopsis had stronger salt tolerance.

2.3. Overexpression of ZmNAC074 Regulates the Expression of Stress-Responsive Genes under Salt Stress

To explore the possible role of ZmNAC074 in salt stress, the expression of ZmNAC074, stress response genes and AtAPX2 was determined, respectively (Figure 3). After salt stress, the expression of ZmNAC074 in transgenic Arabidopsis was higher than that in normal conditions (Figure 3A). In addition, under 200 mM NaCl stress, the expression of ZmNAC074 of Line 1 and 9 was slightly lower than that of Line 2, and the expression of ZmNAC074 in Line 9 was the highest under 400 mM NaCl stress. However, compared with 200 mM NaCl treatment, the expression of ZmNAC074 in transgenic Arabidopsis was down-regulated under 400 mM NaCl stress. In addition, under normal conditions, except that the expression of AtDREB2B in Line 1 and 2 was higher than that of WT Arabidopsis, there was no significant difference in the expression of other stress-responsive genes between the two Arabidopsis (Figure 3B–F). After salt stress, the expression of stress response genes and AtAPX2 increased in all Arabidopsis lines, but the expression of stress response genes and AtAPX2 under 200 mM NaCl stress was higher than that under 400 mM NaCl stress. In addition, after salt stress, the expression of AtP5CS2 in Line 9 under 200 mM NaCl stress, AtP5CS1 and AtDREB2B in Line 1 under 400 mM NaCl stress and AtP5CS2 in Line 2 was not significantly different from those in WT Arabidopsis, but the expression of related genes in other transgenic lines was significantly higher than those in WT Arabidopsis. These results suggest that ZmNAC074 may directly or indirectly regulate the expression of stress-responsive genes in transgenic Arabidopsis to enhance its salt tolerance.

2.4. Germination of ZmNAC074-Overexpressing Transgenic Arabidopsis Seeds under Drought Stress

To explore the effects of drought stress on the germination of WT and transgenic Arabidopsis, WT and transgenic Arabidopsis were seeded in 1/2MS plates containing 0 and 150 mM mannitol. Under normal conditions, the germination rates of WT and transgenic Arabidopsis lines were not significantly different after 7 days (Figure 4A,C). Under the treatment of 150 mM mannitol, the germination rate of transgenic Line 1 and Line 9 was significantly higher than that of WT Arabidopsis on the 7th day (Figure 4B,C). Based on the above results, the overexpression of ZmNAC074 in Arabidopsis could improve the germination rate of Arabidopsis seeds under mannitol treatment.

2.5. Phenotypic of ZmNAC074-Overexpressing Transgenic Arabidopsis Seedlings under Drought Stress and Determination of Physiological Indexes

To further determine the relationship between ZmNAC074 and drought tolerance of transgenic Arabidopsis, WT and transgenic Arabidopsis were subjected to a water deficit for 10 days, and then watered again for one week, respectively. It was observed that under water deficiency, the leaves of WT Arabidopsis showed large-scale curl and withering, and nearly half of the leaves showed obvious yellowing, while the overexpressed Arabidopsis lines only showed slight yellowing at the leaf tip, but the yellowing degree was not as deep as that of WT Arabidopsis (Figure 5A). After resuming watering, some leaves of WT Arabidopsis returned to normal, but most of the leaves of WT Arabidopsis were yellowed, while the leaves of transgenic lines were only slightly yellowed, and the degree of yellowing was much lower than that of WT Arabidopsis. In addition, the plant height of transgenic Arabidopsis was significantly higher than that of WT Arabidopsis, especially Line 2. According to the above phenotype and plant height analysis, the drought resistance of transgenic Arabidopsis was stronger than that of WT Arabidopsis. Under normal conditions, there was no significant difference in the contents of chlorophyll, carotenoid, MDA, soluble protein and proline between transgenic Arabidopsis and WT Arabidopsis. However, under water deficiency treatment, only the transgenic Line 1 had no significant difference compared with WT Arabidopsis, but the chlorophyll and carotenoid contents of Line 2 and 9 were higher than those of WT Arabidopsis (Figure 5B,C). In addition, under the condition of water deficiency, the MDA content of Line 2 was not significantly different from that of WT, but the MDA content of Line 2 and 9 was lower than that of WT Arabidopsis (Figure 5D). Moreover, the contents of proline and soluble protein in transgenic Arabidopsis were higher than those in WT Arabidopsis (Figure 5E,F). To sum up, the drought tolerance of transgenic Arabidopsis with the overexpression of ZmNAC074 was stronger than that of WT Arabidopsis.
DAB and NBT chemical staining were carried out on Arabidopsis leaves under normal growth and water stress treatment (Figure 5G,H). Under normal conditions, there was no significant difference in leaf color among all Arabidopsis lines after DAB and NBT staining, but under water deficiency, the color of stained WT and transgenic Arabidopsis leaves became darker, indicating that the production of H2O2 and O2− increased. However, the color of WT Arabidopsis leaves was still darker than that of transgenic Arabidopsis, indicating that the accumulation of H2O2 and O2− of WT Arabidopsis was higher than that of transgenic Arabidopsis after drought stress. Correspondingly, by measuring the H2O2 content after drought stress, it was found that the content of H2O2 of the transgenic lines was lower than that of WT Arabidopsis (Figure 5I). Similarly, the activities of CAT and SOD in transgenic Arabidopsis were higher than those in WT Arabidopsis (Figure 5J,K). Collectively, the antioxidant capacity of transgenic Arabidopsis is stronger than that of WT Arabidopsis.

2.6. Overexpression of ZmNAC074 Regulates the Expression of Stress-Responsive Genes under Drought Stress

To explore the possible role of ZmNAC074 under drought stress, the expression of some stress response genes and AtAPX2 under normal conditions and after drought stress were determined (Figure 6). After drought stress, the expression of ZmNAC074 in transgenic Arabidopsis increased significantly, but the level of Line 1 was slightly lower than that of Line 2 and 9 (Figure 6A). Under normal conditions, only the overexpression of AtDREB2B in Arabidopsis was higher than that of WT Arabidopsis, and there was no significant difference in the expression of other stress response genes compared with WT Arabidopsis (Figure 6B–F). Moreover, although the expression of these stress response genes and AtAPX2 was up-regulated in all Arabidopsis after drought stress, the expression of these genes in transgenic Arabidopsis lines was significantly higher than that in WT Arabidopsis lines. To sum up, ZmNAC074 may directly or indirectly activate and up-regulate the expression of stress-responsive genes in Arabidopsis, thus enhancing the drought tolerance of transgenic Arabidopsis.

2.7. Survival Rate of Transgenic Arabidopsis under ER Stress

WT and three transgenic Arabidopsis lines were grown in normal 1/2MS plates for 10 days, and then transplanted into 1/2MS plates containing 0, 1, 2 and 5 mM DTT for 24 h, respectively. The phenotypic changes were observed and the survival rate was counted (Figure 7). Before transplanting, there was no significant difference between WT and transgenic Arabidopsis. All Arabidopsis survived at 0 mM DTT (Figure 7A). After DTT treatment, a part of Arabidopsis leaves turned white, and the survival rate was calculated on the basis of keeping the green Arabidopsis. Under 1 mM DTT treatment, although there was no significant difference in the survival rate between Line 2 and WT, the survival rate of Line 1 and 9 was higher than that of WT Arabidopsis (Figure 7B). Under 2 mM DTT treatment, the survival rate of Line 2 and 9 was much higher than that of WT Arabidopsis (Figure 7C). In addition, although there was no significant difference in the survival rate between transgenic Arabidopsis Line 9 and WT Arabidopsis under 5 mM DTT treatment, the survival rate of transgenic Arabidopsis Line 9 was still higher than that of WT Arabidopsis (Figure 7D). Based on the above results, the overexpression of ZmNAC074 in transgenic Arabidopsis can improve the survival rate of Arabidopsis seedlings under ER stress.

2.8. Phenotypic and Physiological Indexes of Transgenic Arabidopsis under ER Stress

To further investigate whether ZmNAC074 can improve the tolerance to ER stress in transgenic Arabidopsis, it was sprayed with 5 and 10 mM DTT for 24 h, respectively. It was observed that a large number of yellowish-brown spots appeared in the leaves of WT after DTT treatment, while there were only a few yellow brown spots in the leaves of transgenic lines, and the difference was more obvious in 10 mM DTT treatment (Figure 8A). Based on the above phenotypic observations, it is suggested that Arabidopsis with overexpression of ZmNAC074 has stronger tolerance to ER stress than WT Arabidopsis.
To further explore the factors affecting the tolerance of Arabidopsis to ER stress, the contents of chlorophyll, carotenoid, MDA, soluble protein and proline under normal and two kinds of DTT treatment were measured, respectively (Figure 8B–F). Under normal conditions, there was no significant difference in the physiological indexes among all the WT and transgenic Arabidopsis lines. There was no significant difference in the contents of chlorophyll and carrots in the Arabidopsis lines under 5 mM DTT treatment for 24 h, but the chlorophyll and carotenoid contents of transgenic lines were significantly higher than those of the WT after 24 h of 10 mM DTT treatment (Figure 8B,C). In addition, under 5 mM DTT treatment, except that the MDA content of Line 2 was not significantly different from that of WT Arabidopsis, the content of MDA of other transgenic lines was lower than that of WT Arabidopsis (Figure 8D). Similarly, except that there was no significant difference in the soluble protein and proline content between transgenic Arabidopsis Line 1 and 2 under 5 mM DTT treatment and WT Arabidopsis, the soluble protein and proline contents of other transgenic lines under DTT treatment were higher than those of WT Arabidopsis (Figure 8E,F). In summary, the overexpression of ZmNAC074 can better alleviate the ER stress in transgenic Arabidopsis.
All leaves of Arabidopsis under ER stress induced by 5 mM and 10 mM DTT were stained with DAB and NBT, respectively (Figure 8G,H). Under normal conditions, there was no significant difference in the color of the Arabidopsis leaves after staining. However, the color of stained WT and transgenic Arabidopsis leaves darkened significantly, indicating that the content of H2O2 and O2− increased after 24 h of DTT treatment. The color of all Arabidopsis leaves under 10 mM DTT treatment was darker than that of 5 mM DTT treatment, but the color of WT Arabidopsis leaves was still darker than that of transgenic Arabidopsis. Correspondingly, by measuring the content of H2O2, it was found that except that the content of H2O2 of Line 2 under 5 mM DTT treatment was not significantly different from that of WT Arabidopsis, the content of H2O2 of other transgenic lines under DTT treatment was lower than that of WT (Figure 8I). After determining the activities of CAT and SOD, the corresponding results were also obtained, that is, after DTT treatment, the activities of CAT and SOD in transgenic Arabidopsis were significantly higher than those in WT Arabidopsis (except Line 2 under 5 mM DTT treatment) (Figure 8J,K). In summary, the overexpression of ZmNAC074 may alleviate ER stress.

2.9. Expression Analysis of Stress-Responsive Genes in Transgenic Arabidopsis under ER Stress

To clarify the possible role of ZmNAC074 under ER stress, the expression of ZmNAC074 and some stress response genes in WT and transgenic lines were determined (Figure 9). The expression of ZmNAC074 in transgenic lines increased after DTT treatment. In addition, compared with 5 mM DTT treatment, the expression of ZmNAC074 increased more than that of 10 mM DTT treatment, and the expression of ZmNAC074 of Line 9 increased the most (Figure 9A). In addition, under normal growth conditions, except that the expression of AtBIP3 and AtCNX1 in transgenic lines was significantly higher than that in WT, the expression of other UPR-related genes in transgenic lines was not significantly different from those in WT (Figure 9B–F). After DTT treatments, the expression of all ER stress response genes increased, but the expression regularity of ER stress genes in two lines under DTT treatment was not strong. For example, the expression of AtbZIP60 was the highest in Line 2 under 5 mM DTT treatment, but the overexpression Line 1 had the highest expression under 10 mM DTT treatment. The expression of AtBIP3 in Line 9 under 5 mM DTT treatment and in Line 2 under 10 mM DTT treatment was slightly lower than that of WT treated with corresponding DTT concentration. However, the expression of UPR-related genes in transgenic Arabidopsis lines was higher than that in WT after DTT treatments. These results suggest that ZmNAC074 may alleviate ER stress in transgenic Arabidopsis.

3. Discussion

Plants respond to abiotic stress in unique ways, including physiological and biochemical reactions, gene transcription, and a large number of stress-specific transcripts and metabolites [37]. Plants have evolved complex regulatory networks to adapt to adverse external environments [7]. Under abiotic stress, NAC TFs from various plants, such as ANAC087, NAC019 and AtNTL7 from Arabidopsis [38,39,40], OsNTL3 protein, SNAC3 and ONAC066 from rice [25,26,41] and TaNAC2L from wheat [42], can perform their functions under abiotic stress. In this study, the assays of seed germination rate at the plate stage and phenotypic identification of seedlings at the age of three weeks from the transgenic Arabidopsis overexpressing ZmNAC074 were firstly performed to explore the underlying regulatory function of ZmNAC074 under various abiotic stresses.
Further, some physiological indexes such as chlorophyll [43], MAD [44], proline [45] and ROS [4] are usually utilized to assess plant stress resistance. For example, MDA is the product of lipid peroxidation of biofilm, and the higher the content of MDA indicates the greater the degree of membrane damage in plant cells [44]. In this study, the transgenic Arabidopsis overexpressing ZmNAC074 showed a lower MDA content compared to WT Arabidopsis under various abiotic stresses, indicating that ZmNAC074 can enhance the stress resistance of transgenic Arabidopsis by reducing cellular lipid peroxidation. Moreover, proline can be utilized as a penetration agent or free radical scavenger to reduce cell damage [45]. For example, overexpression of MfbHLH38 can increase the proline content of transgenic Arabidopsis under drought and salt stresses, which indicates that transgenic Arabidopsis exhibits stronger drought and salt tolerance than wild Arabidopsis [46]. In addition, Arabidopsis proline biosynthesis representative genes AtP5CS1 and AtP5CS2 can promote proline biosynthesis [47]. In this study, the transgenic Arabidopsis overexpressing ZmNAC074 showed a large amount of proline accumulation under drought and salt stress compared to WT Arabidopsis, and the expression of AtP5CS1 and AtP5CS2 was also higher than that of WT Arabidopsis, indicating that ZmNAC074 may enhance the stress resistance of transgenic Arabidopsis via promoting proline biosynthesis.
Furthermore, the degradation and synthesis of chlorophyll is impacted by abiotic stress [48]. Under abiotic stress, plants with a higher chlorophyll content can make better use of light energy for photosynthesis [43]. In addition, plant responses to environmental changes and stressors can be regulated by hormones and signaling molecules, including carotenoid-derived metabolites [2]. For example, sweet potato IbARF5 has been revealed to be involved in carotenoid biosynthesis, salt and drought tolerance [49]. Moreover, carotenoids can also reduce the harmful heat-induced ROS [50]. In this study, the contents of chlorophyll and carotenoids in transgenic Arabidopsis were higher than those in WT Arabidopsis under abiotic stresses, indicating that ZmNAC074 might enhance photosynthesis and stress resistance of transgenic Arabidopsis by promoting the synthesis of chlorophyll and carotenoids or reducing their degradation. However, the detailed mechanism for regulating photosynthetic pigments in transgenic Arabidopsis must be further explored by measuring the expression changes in certain chlorophyll and carotenoid synthesis and degradation genes.
Under stress conditions, the content of ROS increases rapidly, which disturbs the cell redox dynamic balance [51]. On the other hand, when the rate of ROS production decreases below the threshold level, it can regulate the redox signal pathway that promotes plant growth, development and adaptation to stress [52]. However, excessive ROS produced by various abiotic stresses is generally toxic and can cause oxidative damage to proteins, nucleic acids, cell membranes, lipids and so on [53]. In addition, SOD and CAT can protect plant cells from oxidative damage [51]. In this study, the transgenic Arabidopsis overexpressing ZmNAC074 has a stronger ability to alleviate ROS damage under a variety of abiotic stresses. It can be speculated that ZmNAC074 may be involved in modulating the level of ROS through ROS-mediated stress-responsive signal pathways to enhance the stress resistance of transgenic Arabidopsis. However, the specific relationship between ZmNAC074 and ROS homeostasis needs to be further clarified, for example, by knocking out maize ZmNAC074 to investigate the change in the ROS level in mutant plants.
In addition, the regulation of ROS levels usually involves antioxidant enzymes such as GPX, SOD and APX, which act as antioxidants and regulate oxidative signal transmission [54]. In this study, under abiotic stress, the expression of representative AtAPX2 in transgenic Arabidopsis was significantly higher than that in WT Arabidopsis. This result is consistent with the cumulative content of ROS, indicating that ZmNAC074 may improve the ROS scavenging ability of transgenic Arabidopsis by up-regulating the expression of some antioxidant enzyme genes, thus enhancing the stress resistance of transgenic Arabidopsis. In addition, other peroxidase activity and the expression of corresponding genes can be also determined to further clarify the mechanism of regulating ROS homeostasis in transgenic Arabidopsis.
The transcription factor AtDREB2A can activate the expression of downstream genes involved in drought and salt stress responses in Arabidopsis [55]. For example, drought-tolerant transgenic Arabidopsis with MlNAC10 may be attributed to the up-regulated expression of AtDREB2A under drought and salt stresses [56]. In this study, under drought and salt treatments, the expression of AtDREB2A and AtDREB2B in transgenic lines was higher than that in WT Arabidopsis, indicating that ZmNAC074 may trigger the expression of downstream genes in drought and salt stresses through the up-regulated expression of AtDREB2A and AtDREB2B, thus enhancing drought and salt tolerance in Arabidopsis.
Additionally, AtbZIP60 encodes a UPR-associated transcription factor and can respond to dithiothreitol (DTT) and tunicamycin (TM), thus positively regulating the ER stress response [57]. Previous studies have revealed the close relationship between plant NAC proteins and ER stress [58]. For example, AtNAC062, AtNAC089 and AtNAC103 can be activated in response to ER stress and modulate different UPR-associated genes in Arabidopsis [30,31,59]. Similarly, it has been demonstrated that OsNTL3 protein in rice, which has a high homology with ZmNAC074 protein in maize, can directly bind to the promoter of the OsbZIP74 gene and thereby regulate its expression in response to heat stress [41]. In this study, we found that the expression of AtbZIP60 in transgenic lines treated with two concentrations of DTT was significantly higher than that of WT Arabidopsis, indicating that the ZmNAC074 protein may interact with the AtbZIP60 protein to alleviate ER stress. However, further experiments including yeast two hybrids are needed to reveal the interaction between them. In addition, UPR-associated genes AtbZIP28, AtBIP3, AtPDI5 and AtCNX1 in transgenic Arabidopsis were up-regulated in this study, and these genes can further modulate the expression of genes downstream of the UPR pathway [57,60,61,62]. Therefore, ZmNAC074 may alleviate ER stress by regulating the expression of UPR-associated genes in transgenic lines, but the specific regulatory mechanism needs to be further elucidated, such as to determine the potential interaction between the ZmNAC074 protein and UPR-associated proteins.
Taken together, our results showed that ZmNAC074 is a stress-responsive NAC transcription factor in maize, as shown in the proposed model in this study (Figure 10). Phenotypic and physiological analyses displayed that overexpression of ZmNAC074 in transgenic Arabidopsis confers enhanced abiotic stress tolerance significantly through modulating the accumulation of a variety of stress metabolites, including reactive oxygen species (ROS), antioxidants, malondialdehyde (MDA), proline, soluble protein, chlorophyll and carotenoid. Further, quantitative real-time PCR analysis revealed that the expression levels of some stress-responsive genes in transgenic Arabidopsis were significantly up-regulated under various abiotic stress treatments, suggesting that ZmNAC074 may function as a positive regulator that triggers the expression of stress-associated genes to enhance plant stress tolerance under various abiotic stress conditions.

4. Materials and Methods

4.1. Plant Material and Growth Conditions

The ZmNAC074-overexpressing transgenic Arabidopsis lines used in this study were generated from our previously obtained seeds kept in our laboratory via transgenic methods. The Arabidopsis ecotype Columbia (Col) was used as original material for genetic transformation to obtain these transgenic plants and perform further functional analysis [36]. The seeds of the Arabidopsis Columbia ecotype (WT) and transgenic Arabidopsis were sterilized with 75% ethanol for 10 min and washed with deionized water three times. The sterilized seeds were placed on a medium containing 1/2 strength Murashige and Skoog (MS) containing 0.7% (w/v) agar and 2% (w/v) sucrose at a pH of 5.8–6.0. After two days of vernalization at 4 °C, they were placed in a light incubator for about 10 days. The seedlings were transplanted into pots filled with nutrient soil in the growing room and treated with drought and stress for three weeks under conditions of 22 °C, long photoperiods (16 h day/8 h night) and relative humidity of approximately 60%.

4.2. RNA Extraction and Quantitative Real-Time PCR

Total RNA was isolated using Trizol reagent (TIANGEN, Beijing, China) and the first cDNA strand was synthesized from 2 mg of total RNA using the FastKing RT Kit (Mona, China) [36]. The Light-CykerR96 Real-Time PCR system (BioRad, Hercules, CA, USA) was used for real-time quantitative PCR (qRT-PCR). All experimental data were obtained through three technical replicates, and the relative expression was calculated using the 2−ΔΔCT method. The primer sequences used are listed in Table S1.

4.3. Germination Assay

To perform the characteristic analysis of seed germination under various abiotic stresses, the seeds of wild-type and transgenic Arabidopsis were firstly sterilized and uniformly germinated on a petri dish and grown in a controlled chamber (22 °C, 16 h light (120 μmol m−2s−1)/8 h darkness, and 60% relative humidity). For salt stress treatment, the petri dish was equipped with 1/2 MS solid medium containing 0 mM NaCl (serving as the control), 100 mM NaCl and 200 mM NaCl for seven days under salt stress, respectively. For drought stress treatment, the petri dish was equipped with 1/2 MS solid medium containing 0 mM mannitol (serving as the control), and 150 mM mannitol for seven days under drought stress, respectively. For ER stress treatment, the petri dish was equipped with 1/2 MS solid medium containing 0 mM DTT (serving as the control), 1 mM DTT, 2 mM DTT, and 5 mM DTT for 24 h under ER stress, respectively. Three biological replicates were conducted for the germination of Arabidopsis seeds in each treatment. After treatment, the germination potential and rate of seeds were counted in each treatment, respectively.

4.4. Salt, Drought and ER Stress Treatments

In order to treat salt stress, 3-week-old wild-type (WT) and transgenic Arabidopsis seedlings were placed in 200 mM and 400 mM NaCl, respectively, for 10 days. Then, the physiological characteristics and related gene expression of all the Arabidopsis lines were determined. For drought stress, seedlings of 3-week-old WT and transgenic Arabidopsis were subjected for 10 days to water deficiency, followed by one week of watering, and the plant phenotypes were recorded. For ER stress treatment, Arabidopsis seedlings with normal growth for 10 days were transplanted into 1/2MS plates containing 0 mM, 2 mM, and 5 mM DTT 24 h later. The phenotype and survival rate of Arabidopsis seedlings were observed on different concentrations of DTT plates. Moreover, four-week-old transgenic Arabidopsis and WT seedlings were evenly sprayed with 5 mM DTT and 10 mM DTT, respectively, and the phenotypes of the plants were recorded after 24 h of treatment.

4.5. Measurements of Physiological Indexes

The acetone extraction method was used to determine the contents of chlorophyll and carotenoid in leaves. The contents of soluble protein, proline, and MDA were also determined according to the kit instructions. In addition, the accumulations of H2O2 and O2− in the leaves of the control and transgenic plants before and after heat stress were detected using 3,3′-diaminobenzidine (DAB) and Nitro blue tetrazolium (NBT), respectively. The H2O2 contents, activities of superoxide dismutase (SOD) and catalase (CAT) in stressed and control plants were determined using kits purchased from the Nanjing Jiancheng Bioengineering Institute (Nanjing, China).

4.6. Statistical Analysis

SPSS 20 software was used for data analysis; one-way ANOVA was used to analyze and compare the differences of the data of each group, and the new Duncan’s multiple range test difference method was used to compare the data of each group. Compared to the control group, p < 0.05 was considered to be statistically significant and p < 0.01 was considered extremely significant.

5. Conclusions

In conclusion, our results in this study demonstrate that ZmNAC074 may be involved in the regulation of the abiotic stress response of Arabidopsis and some important physiological processes, thus enhancing the stress resistance of transgenic Arabidopsis. This study laid a foundation for further study of the function of ZmNAC074 in maize, and provided an important basis for further functional analysis of the exact mechanism of crop stress resistance regulated by ZmNAC074. However, the special stress-resistant mechanism of ZmNAC074 remains to be further elucidated.

Supplementary Materials

The supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/ijms242216157/s1.

Author Contributions

Conceptualization, Y.Q. and H.M.; methodology, Y.X. and Y.Q.; formal analysis, Y.X., L.L. and Y.Q.; investigation, Y.X., Y.Q., L.X., Z.Q. and L.L.; resources, Y.X. and H.M.; data curation, Y.X. and Y.Q.; writing—original draft preparation, Y.Q. and Y.X.; writing—review and editing, Y.Q. and H.M.; supervision, Y.Q. and H.M. All authors have read and agreed to the published version of the manuscript.

Funding

This study was supported by grants from the National Natural Science Foundation of China (NSFC) (Grant NO. 31571673) and Anhui Provincial Natural Science Foundation (Grant NO. 2308085MC86) and Anhui Provincial Key Laboratory of the Conservation and Exploitation of Biological Resources (Grant NO. Swzy202003) and Anhui Provincial Academic Funding Project for Top Talents in Disciplines (Majors) (Grant NO. gxbjZD 2021044) and Anhui Academy of Agricultural Sciences Young Talent Programme (Grants No. QNYC-201902). The funders had no role in the study design, collection, analysis and interpretation of data, or in the writing of the report or decision to submit the article for publication.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Data are contained within the article and Supplementary Materials.

Acknowledgments

The authors are grateful to the editor and reviewers for critically evaluating the manuscript and providing constructive comments for its improvement.

Conflicts of Interest

The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.

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Figure 1. Germination of WT and transgenic Arabidopsis under salt stress. (A–D) The phenotype and germination percentage of Arabidopsis under 0 (control), 100 and 200 mM NaCl treatments. The error line (T) represents the standard deviation (±SD). Asterisks indicate significant differences, that is, * for p < 0.05 and ** for p < 0.01.
Figure 1. Germination of WT and transgenic Arabidopsis under salt stress. (A–D) The phenotype and germination percentage of Arabidopsis under 0 (control), 100 and 200 mM NaCl treatments. The error line (T) represents the standard deviation (±SD). Asterisks indicate significant differences, that is, * for p < 0.05 and ** for p < 0.01.
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Figure 2. Analysis of salt tolerance of transgenic Arabidopsis. (A) Phenotypes analysis of WT and transgenic Arabidopsis under 0 (control), 200 and 400 mM NaCl treatments. (B–F) The content of chlorophyll, carotenoid, MDA, soluble protein and proline of Arabidopsis under salt stress. (G,H) DAB and NBT staining. (I–K) H2O2 content and SOD, CAT activity before and after salt stress treatment. The error line (T) represents the standard deviation (±SD). Asterisks indicate significant differences, that is, * for p < 0.05 and ** for p < 0.01.
Figure 2. Analysis of salt tolerance of transgenic Arabidopsis. (A) Phenotypes analysis of WT and transgenic Arabidopsis under 0 (control), 200 and 400 mM NaCl treatments. (B–F) The content of chlorophyll, carotenoid, MDA, soluble protein and proline of Arabidopsis under salt stress. (G,H) DAB and NBT staining. (I–K) H2O2 content and SOD, CAT activity before and after salt stress treatment. The error line (T) represents the standard deviation (±SD). Asterisks indicate significant differences, that is, * for p < 0.05 and ** for p < 0.01.
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Figure 3. The expression of stress-responsive genes under salt stress in WT and transgenic Arabidopsis. (A–F) The expression of ZmNAC074, AtP5CS1, AtP5CS2, AtDREB2A, AtDREB2B and AtAPX2. The error line (T) represents the standard deviation (±SD). Asterisks indicate significant differences, that is, * for p < 0.05 and ** for p < 0.01.
Figure 3. The expression of stress-responsive genes under salt stress in WT and transgenic Arabidopsis. (A–F) The expression of ZmNAC074, AtP5CS1, AtP5CS2, AtDREB2A, AtDREB2B and AtAPX2. The error line (T) represents the standard deviation (±SD). Asterisks indicate significant differences, that is, * for p < 0.05 and ** for p < 0.01.
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Figure 4. Germination of WT and transgenic Arabidopsis under normal (control) and mannitol treatment. (A,B) The germination phenotype of WT and transgenic Arabidopsis treated in drought simulation. (C) Statistics of germination percentage of Arabidopsis under drought stress treatment. The error line (T) represents the standard deviation (±SD). Asterisks indicate significant differences, that is, ** for p < 0.01.
Figure 4. Germination of WT and transgenic Arabidopsis under normal (control) and mannitol treatment. (A,B) The germination phenotype of WT and transgenic Arabidopsis treated in drought simulation. (C) Statistics of germination percentage of Arabidopsis under drought stress treatment. The error line (T) represents the standard deviation (±SD). Asterisks indicate significant differences, that is, ** for p < 0.01.
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Figure 5. Drought tolerance analysis of transgenic Arabidopsis. (A) Phenotypic changes in WT and transgenic Arabidopsis under normal (control) and drought treatment. (B–F) The contents of chlorophyll, carotenoid, MDA, soluble protein and proline of Arabidopsis under drought stress. (G,H) DAB and NBT staining. (I–K) H2O2 content and SOD, CAT activity under water deficiency treatment. The error line (T) represents the standard deviation (±SD). Asterisks indicate significant differences, that is, * for p < 0.05 and ** for p < 0.01.
Figure 5. Drought tolerance analysis of transgenic Arabidopsis. (A) Phenotypic changes in WT and transgenic Arabidopsis under normal (control) and drought treatment. (B–F) The contents of chlorophyll, carotenoid, MDA, soluble protein and proline of Arabidopsis under drought stress. (G,H) DAB and NBT staining. (I–K) H2O2 content and SOD, CAT activity under water deficiency treatment. The error line (T) represents the standard deviation (±SD). Asterisks indicate significant differences, that is, * for p < 0.05 and ** for p < 0.01.
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Figure 6. Analysis of the expression of stress-responsive genes under normal (control) and drought stress treatment. (A–F) The expression of ZmNAC074, AtP5CS1, AtP5CS2, AtDREB2A, AtDREB2B and AtAPX2. The error line (T) represents the standard deviation (±SD). Asterisks indicate significant differences, that is, * for p < 0.05 and ** for p < 0.01.
Figure 6. Analysis of the expression of stress-responsive genes under normal (control) and drought stress treatment. (A–F) The expression of ZmNAC074, AtP5CS1, AtP5CS2, AtDREB2A, AtDREB2B and AtAPX2. The error line (T) represents the standard deviation (±SD). Asterisks indicate significant differences, that is, * for p < 0.05 and ** for p < 0.01.
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Figure 7. The phenotypic and survival rate of Arabidopsis under normal (control) and DTT treatment. (A–D) Phenotypic and survival rate of WT and transgenic Arabidopsis under 0, 1, 2 and 5 mM DTT treatments. The error line (T) represents the standard deviation (±SD). Asterisks indicate significant differences, that is, * for p < 0.05 and ** for p < 0.01.
Figure 7. The phenotypic and survival rate of Arabidopsis under normal (control) and DTT treatment. (A–D) Phenotypic and survival rate of WT and transgenic Arabidopsis under 0, 1, 2 and 5 mM DTT treatments. The error line (T) represents the standard deviation (±SD). Asterisks indicate significant differences, that is, * for p < 0.05 and ** for p < 0.01.
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Figure 8. Analysis of the tolerance to RE stress in transgenic Arabidopsis. (A) Phenotype of WT and transgenic Arabidopsis under 0 (control), 5 and 10 mM DTT treatments, respectively. (B–F) The contents of chlorophyll, carotenoid, MDA, soluble protein, and proline of WT and transgenic Arabidopsis under DTT treatments. (G,H) DAB and NBT staining. (I–K) H2O2 content and the activity of SOD and CAT under DTT treatments. The error line (T) represents the standard deviation (±SD). Asterisks indicate significant differences, that is, * for p < 0.05 and ** for p < 0.01.
Figure 8. Analysis of the tolerance to RE stress in transgenic Arabidopsis. (A) Phenotype of WT and transgenic Arabidopsis under 0 (control), 5 and 10 mM DTT treatments, respectively. (B–F) The contents of chlorophyll, carotenoid, MDA, soluble protein, and proline of WT and transgenic Arabidopsis under DTT treatments. (G,H) DAB and NBT staining. (I–K) H2O2 content and the activity of SOD and CAT under DTT treatments. The error line (T) represents the standard deviation (±SD). Asterisks indicate significant differences, that is, * for p < 0.05 and ** for p < 0.01.
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Figure 9. Changes in the expression of stress-responsive genes under normal (control) and DTT treatment. (A–F) The expression of ZmNAC074, AtbZIP60, AtbZIP28, AtBIP3, AtPDI5 and AtCNX1. The error line (T) represents the standard deviation (±SD). Asterisks indicate significant differences, that is, * for p < 0.05 and ** for p < 0.01.
Figure 9. Changes in the expression of stress-responsive genes under normal (control) and DTT treatment. (A–F) The expression of ZmNAC074, AtbZIP60, AtbZIP28, AtBIP3, AtPDI5 and AtCNX1. The error line (T) represents the standard deviation (±SD). Asterisks indicate significant differences, that is, * for p < 0.05 and ** for p < 0.01.
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Figure 10. A proposed model for the potential roles of ZmNAC074 in response to abiotic stress in transgenic Arabidopsis.
Figure 10. A proposed model for the potential roles of ZmNAC074 in response to abiotic stress in transgenic Arabidopsis.
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Qian, Y.; Xi, Y.; Xia, L.; Qiu, Z.; Liu, L.; Ma, H. Membrane-Bound Transcription Factor ZmNAC074 Positively Regulates Abiotic Stress Tolerance in Transgenic Arabidopsis. Int. J. Mol. Sci. 2023, 24, 16157. https://doi.org/10.3390/ijms242216157

AMA Style

Qian Y, Xi Y, Xia L, Qiu Z, Liu L, Ma H. Membrane-Bound Transcription Factor ZmNAC074 Positively Regulates Abiotic Stress Tolerance in Transgenic Arabidopsis. International Journal of Molecular Sciences. 2023; 24(22):16157. https://doi.org/10.3390/ijms242216157

Chicago/Turabian Style

Qian, Yexiong, Yan Xi, Lingxue Xia, Ziling Qiu, Li Liu, and Hui Ma. 2023. "Membrane-Bound Transcription Factor ZmNAC074 Positively Regulates Abiotic Stress Tolerance in Transgenic Arabidopsis" International Journal of Molecular Sciences 24, no. 22: 16157. https://doi.org/10.3390/ijms242216157

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