Heterologous expression of ZmNF-YA12 confers tolerance to drought and salt stress in Arabidopsis

Drought and salinity are serious environmental factors limiting the growth and productivity of plants worldwide. Therefore, it is necessary to develop ways to improve drought and salinity stress tolerance in plants. In this study, a drought-responsive nuclear factor Y subunit A gene, ZmNF-YA12, was cloned from maize. qPCR revealed ZmNF-YA12 transcript in all vegetative and reproductive tissues, with higher levels in young roots. Expression analyses of maize revealed that ZmNF-YA12 was induced by abscisic acid (ABA), jasmonic acid (JA), and abiotic stresses, including dehydration, high salinity, cold, and polyethylene glycol (PEG) treatment. The heterologous expression of ZmNF-YA12 in Arabidopsis plants resulted in increased root length and better plant growth than in wild-type (WT) plants under conditions of mannitol, salt, and JA stress on 1/2 MS medium. Transgenic Arabidopsis showed improved tolerance to drought and salt stresses in soil, and higher proline content and lower malondialdehyde (MDA) content than WT controls. The transgenic plants also maintained higher peroxidase (POD) activities than WT plants under conditions of NaCl stress. A yeast two-hybrid experiment demonstrated that ZmNF-YA12 interacted with ZmNF-YC1 and ZmNF-YC15. Moreover, the transcript levels of stress-responsive genes (RD29A, RD29B, RAB18, and RD22) were markedly increased in transgenic lines under conditions of drought and salt stress. These observations suggested that the ZmNF-YA12 gene may confers drought and salt stress tolerance by regulating stress-related genes or interacting with ZmNF-YC1 and ZmNF-YC15, and has potential applications in molecular breeding with maintenance of production under conditions of stress.


Introduction
The crop yield of maize (Zea mays L.), the most widely grown cereal crop in the world, is considerably limited by a range of abiotic stress factors (Pechanova et al. 2013). Drought and increased soil salinity are projected to lead to a 50% loss of arable land by the year 2050, which will severely affect crop yields. Therefore, it is becoming increasingly important to improve water use efficiency and salt tolerance for agricultural production in the ever-decreasing area of arable land (Deinlein et al. 2014;Tiburcio et al. 2012). Transcription factors play important roles in abiotic stress responses in plants, and represent promising targets for the genetic engineering of plants with elevated stress resistance (Nowicka et al. 2018). Many plant genes are regulated in response to abiotic stresses, and the products of these genes have functions related to the stress responses and tolerance (Yamaguchi-Shinozaki and Shinozaki 2006).
Nuclear factor Y (NF-Y), also called CBF and CP1, consists of three different subunits (NF-YA, NF-YB, and NF-YC), and is a unique DNA-binding protein that interacts with the CCAAT motif, a common element present in the promoters of a number of mammalian genes (Maity and de Crombrugghe 1998). The core domains of the NF-YC/ NF-YB proteins interact through histone fold motifs. This histone-like pair is closely related to the H2A/H2B and NC2α/NC2β families, both of which have features common 1 3 to this class of proteins and unique to NF-Y (Romier et al. 2003).
The plant NF-Y transcription factors have been reported to be key players in plant-microbe interactions, root development, and stress tolerance. Some members of the NF-Y gene families have been shown to be involved in responses to water and nutrient scarcity in mono-and dicotyledonous plants (Zanetti et al. 2017). AtNF-YB2 and AtNF-YB3 are both essential for the normal flowering induced by long days in Arabidopsis (Kumimoto et al. 2008). Transcription of OsNF-YA7 was shown to be induced by drought stress, and its overexpression in transgenic rice plants enhanced their drought tolerance (Lee et al. 2015). Overexpression of TaNF-YA10-1 in wheat conferred drought tolerance, with longer root length and better whole-plant growth under conditions of drought . Under 200 mM NaCl and 200 mM mannitol stresses, the expression of SbNF-YA1, 2, and 6 were upregulated (Maheshwari et al. 2019). Overexpression of GmNFYA5 in transgenic Arabidopsis and soybean resulted in increased drought tolerance (Ma et al. 2020). In potato, twelve StNF-Y genes were upregulated and another two genes were downregulated under ABA, drought and salinity treatments (Xuanyuan et al. 2021). Transgenic tobacco plants overexpressing CsNF-YA5 showed superior growth and photosynthetic rates under both normal conditions and drought stress (Pereira et al. 2018). Overexpression ShNF-YB2 in sugarcane enhanced tolerance to drought and salinity stresses (Peter et al. 2020). CsNF-YAs had greater impacts than CsNF-YB and C under drought and ABA treatment (Wang et al. 2019). CmNF-YB8-RNAi transgenic chrysanthemum lines enhanced drought resistance, whereas lines overexpressing CmNF-YB8 were less tolerant to drought . PdNF-YB21 overexpression promoted root growth with highly lignified and enlarged xylem vessels in poplar, resulting in increased drought resistance . However, the biological roles of many members of the NF-Y family in maize are not clear. P rev i o u s ly, we re p o r t e d t h a t Z m N F-YA 1 2 (GRMZM5G857944) could respond to abiotic stress (Zhang et al. 2016). To investigate the molecular biology function of ZmNF-YA12, its tissue-specific expression, gene expression patterns under different exogenous stresses, heterologous expression in Arabidopsis, and yeast two-hybrid experiment were performed. Transgenic Arabidopsis lines expressing ZmNF-YA12 showed improved drought and NaCl tolerance. ZmNF-YA12 interacts with ZmNF-YC1 and ZmNF-YC15. Our data suggest that ZmNF-YA12 may represent an important mechanism underlying the function of NF-Y under NaCl and drought stress.

Plant materials, growth conditions, and treatments
For tissue-specific analysis, leaves, stems, and roots were harvested from three-leaf seedlings grown in a greenhouse (28 °C, 16/8 h day/night cycle). Mature leaves, roots, silks, tassels, and embryos were harvested at the grain-filling stage from plants grown in the field. All harvested materials were frozen immediately in liquid nitrogen and stored at − 80 °C.
To determine the ZmNF-YA12 expression patterns under various stress conditions, maize seedlings were grown in 12-cm hydroponic barrels containing nutrient solution. Three-leaf maize seedlings were subjected to dehydration, NaCl, polyethylene glycol (PEG), cold, abscisic acid (ABA), and jasmonic acid (JA) treatments. For dehydration treatment, the whole seedling was removed, washed, and placed on an experimental table for natural dehydration at room temperature (25 °C). For salt and PEG treatments, the roots of the seedlings were immersed in solutions containing 200 mM NaCl and 20% PEG, respectively. For cold treatment, seedlings were kept at 4 °C. For each of the above four treatments, the shoots and roots were collected at 0, 1, 2, 5, 10, and 24 h after treatment. For the ABA and JA treatments, the leaves of the seedlings were sprayed with solutions containing 100 μM ABA or 100 μM JA and covered with plastic film. The leaves were then collected at 0, 1, 2, 5, and 10 h after treatment. The samples were immediately frozen with liquid nitrogen for isolation of RNA.
Arabidopsis thaliana ecotype Columbia (Col-0) was used for transformation in this study. After vernalization treatment, seeds were surface-sterilized in a solution of 0.5% NaClO for 10 min, and washed five times with sterile distilled water. Following this treatment, the seeds were germinated and grown on half-strength Murashige and Skoog (1/2 MS) medium (pH 5.8-6.0). The plates were transferred to a growth chamber at 22 °C for germination.

Root growth assay
For root growth assay, transgenic and wild-type (WT) seeds were placed on 1/2 MS agar plates for germination. Seven days later, five germinated seedlings of the same size from each line were carefully transferred to 1/2 MS agar plates supplemented with 150 mM NaCl, 150 mM mannitol, 50 μM JA, or 10 μM ABA. Seedling root lengths were measured using ImageJ software (NIH, Bethesda, MD, USA) after 8 days of upright growth in treatment medium.

Drought and NaCl treatment of transgenic Arabidopsis
Drought and NaCl tolerance assays were performed on seedlings grown in pots in a greenhouse. Transgenic and WT seeds were germinated on 1/2 MS medium. Oneweek-old seedlings were planted in 7-cm pots containing mixed soil (vermiculite: humus = 1:1) of equal quality and well-watered for 3 weeks. For drought stress treatment, the seedlings were subsequently cultured without watering for 3 weeks and then re-watered for 2 days. For NaCl stress treatment, the plants were irrigated with a solution containing 450 mM NaCl for 1 week. Drought and NaCl tolerance experiments were performed in triplicate. Samples of Arabidopsis leaves were collected after the seedlings exhibited distinct phenotypes under drought and salt treatments. The peroxidase (POD) activity, proline, malondialdehyde (MDA), and chlorophyll contents were measured using a commercial assay kit (Solarbio, Beijing, China) according to the manufacturer's instructions. The sixth from last rosette leave was used to measure chlorophyll contents and all leaves were measured to calculate the rate of green leaves.

Statistical analysis
The experiments were repeated three times and the data are presented as the mean ± SEM. The significance of the differences in the data was determined using SPSS statistical software (v. 25.0; SPSS Inc., Chicago, IL, USA). In all analyses, p < 0.05 was taken to indicate statistical significance.

Isolation and characterization of ZmNF-YA12
The full-length ZmNF-YA12 cDNA is 816 bp in length and encodes 271 amino acid residues with a predicted molecular mass of 29.3 kDa and isoelectric point (pI) of 10.96. Protein structure alignment showed that the ZmNF-YA12 sequence included an NF-Y transcription factor conserved domain (Fig. 1a). The results indicated that ZmNF-YA12 is a member of the NF-YA transcription factor family. The deduced amino acid sequence of ZmNF-YA12 was further compared to other NF-YA proteins from various organisms by phylogenetic analysis (Fig. 1b). The results indicated that ZmNF-YA12 is closely related to OsHAP2E. Analysis of the ZmNF-YA12 promoter using the PlantCARE database revealed a series of light-related and hormone stress response elements, including CAT-box, G-box, and CGTCA motif (Supplementary Fig. 2). The results suggest that ZmNF-YA12 may play important roles in responses to environmental stresses and regulation of plant growth and development.

Expression pattern of maize ZmNF-YA12
The expression levels of ZmNF-YA12 in different tissues under various stresses were determined by qPCR. The results showed that ZmNF-YA12 was expressed at higher levels in young roots than in other tissues (Fig. 2), and the expression levels of ZmNF-YA12 in shoots and roots were upregulated by dehydration treatments (Fig. 3a). For PEG treatment, the expression of ZmNF-YA12 was significantly upregulated in roots and increased rapidly at 1 h and then declined in shoots (Fig. 3b). Under conditions of cold and NaCl treatment, the expression of ZmNF-YA12 was markedly induced in roots but not in shoots (Fig. 3c, d). As shown in Fig. 3e, the ZmNF-YA12 transcript level was downregulated at 1, 3, and 5 h, and upregulated at 10 h, with ABA treatment. With JA treatment, the expression of ZmNF-YA12 first decreased and then increased, peaking at 3 h (Fig. 3f).

Tolerance of transgenic Arabidopsis plants to salt, mannitol, JA, and ABA stress
To assess the effects of ZmNF-YA12 in responses to abiotic stresses, ZmNF-YA12 transgenic Arabidopsis plants (L-1, L-2, L-3) and WT seedlings were grown on 1/2 MS medium with different treatments. WT and transgenic plants showed similar root lengths under normal and 10 μM ABA conditions. However, the roots of transgenic lines were much longer than those of WT plants in the presence of 150 mM NaCl, 150 mM mannitol, or 50 μM JA ( Fig. 4; Supplementary Figs. 3-6).

Heterologous expression of ZmNF-YA12 confers enhanced drought and salt tolerance in Arabidopsis
Under control conditions, both WT and ZmNF-YA12 transgenic plants exhibited a similarly normal growth phenotype. Drought and salt stress significantly inhibited the growth of WT plants, which exhibited more wilted and smaller leaves ( Fig. 5a and Fig. 6a). However, ZmNF-YA12 transgenic Arabidopsis plants showed less wilted leaves and more green and larger leaves ( Fig. 5b and Fig. 6b). For drought treatment, after re-watering for 2 days, the ZmNF-YA12 transgenic lines recovered more quickly, grew more green leaves and appeared to be healthier than WT plants (Fig. 5c).
To further characterize the function of ZmNF-YA12, we examined the MDA, proline, chlorophyll contents and POD activity under drought and salt stress treatment in transgenic lines and WT plants. As shown in Fig. 5d, MDA content was lower in the transgenic plants than WT controls under drought stress. As expected, the proline content was much higher in the transgenic lines than in WT plants (Fig. 5e). Under NaCl treatment, the activities of POD in the transgenic lines were much higher than in WT plants (Fig. 6c). However, there were no significant differences in chlorophyll contents and POD activity between WT and transgenic plants with drought treatment ( Fig. 5f and g) and in chlorophyll, MDA and proline contents with salt treatment (Fig. 6d, e and f). These results suggested that the heterologous expression of ZmNF-YA12 improves drought and salt tolerance in Arabidopsis.
To determine the mechanism underlying the involvement of ZmNF-YA12 in the stress response, the expression levels of stress-related genes were analyzed by qPCR in ZmNF-YA12 transgenic and WT plants grown under normal, NaCl, and drought conditions. As shown in Fig. 7, the expression levels of the stress-related genes RD29A, RD29B, RAB18, and RD22 in the transgenic lines were much higher than in WT plants under NaCl and drought conditions.

Discussion
The growth, development, and productivity of maize are seriously affected by abiotic stresses, such as drought, salinity, high and low temperatures, and by biotic stresses, such as fungi, viruses, and pests (Gong et al. 2014). The NF-Y transcription factors are important regulators of plant development and responses to environmental stress (Petroni et al. 2012). The maize genome includes 50 ZmNF-Y genes (14 ZmNF-YA, 18 ZmNF-YB, and 18 ZmNF-YC) (Zhang et al. 2016). In this study, we identified and characterized a gene, ZmNF-YA12, related to stress tolerance. ZmNF-YA12 transcript levels were significantly induced by dehydration, PEG, cold, NaCl, ABA, and JA treatments (Fig. 3). The expression of ZmNF-YA12 in shoots was induced at 1 h and then declined, similar result was also observed in other previously reported gene, such as GmNF-YA3 (Ni et al. 2013). However, the expression of ZmNF-YA12 was significantly induced in roots but not in shoots with cold and NaCl treatments, which may be related to its tissue-specific expression (Fig. 2).
Several studies have indicated that NF-Y genes are involved in stress responses. OsHAP2E, a homolog of ZmNF-YA12, confers biotic and abiotic resistance, and increased photosynthesis and tiller numbers in rice (Alam et al. 2015). Overexpression of SiNF-YA1 in transgenic tobacco lines enhanced drought and salt tolerance (Feng et al. 2015). Transgenic Arabidopsis plants overexpressing

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AhNF-YC showed increased seedling sensitivity to ABA, and influenced the expression of several genes associated with secondary metabolism, development, and ABA-related responses (Palmeros-Suárez et al. 2015). Heterologous expression of MsNF-YB21 in Arabidopsis led to a longer root length and conferred improved osmotic and salt tolerance (Feng et al. 2021). Our data showed that transgenic Arabidopsis seedlings expressing ZmNF-YA12 had longer roots than WT plants when grown on 1/2 MS medium under mannitol, NaCl, and JA treatments (Fig. 4). Furthermore, seedlings of ZmNF-YA12 transgenic plants grown in soil under drought and high NaCl conditions showed enhanced tolerance in comparison to WT plants ( Fig. 5a and Fig. 6a). Several physiological and biochemical factors, such as Data represent the average root lengths. *p < 0.05; **p < 0.01 compared to the corresponding controls. Bar = 1.5 cm MDA, proline, chlorophyll contents, and POD activity, play essential roles in plant tolerance to abiotic stresses. Proline plays a role as a compatible solute under conditions of environmental stress and contributes to the redox balance of the cell (Lehmann et al. 2010). MDA is the most frequently measured biomarker of oxidative stress, i.e., lipid    (Tsikas 2017). With drought treatment, transgenic ZmNF-YA12 plants showed lower MDA content and higher proline content than WT controls (Fig. 5d, e). Therefore, we concluded that ZmNF-YA12 transgenic plants have enhanced drought tolerance. Peroxiredoxins are thiol PODs with a variety of functions in the oxidation resistance and redox signaling networks of the cell (Liebthal et al. 2018). Leaf chlorophyll content represents the photosynthetic capacity, and high oxidative stress inhibits its synthesis and accumulation (Agathokleous et al. 2020). The levels of POD activity in 35S:ZmNF-YA12 Arabidopsis were much higher than in WT plants under high NaCl conditions (Fig. 6c). These results indicated that ZmNF-YA12 has a positive effect on salinity, osmotic, and drought stress responses in plants.
To investigate the mechanisms of action of ZmNF-YA12 in stress responses, we examined the expression levels of stress-responsive genes. Previous studies showed that RD29A and RD29B can be induced by drought and salt stress, and responded to dehydration and ABA treatments (Msanne et al. 2011;Nakashima et al. 2006). In addition, RD22 and RAB18 are marker genes for ABA-induced gene expression and key nodes in ABA-responsive signaling networks (Rushton et al. 2012;Yao et al. 2020). In the present study, these four stress-related genes showed significantly elevated expression levels in the transgenic ZmNF-YA12 compared to WT plants under drought and high NaCl treatments (Fig. 7a, b, c, d). These results indicated that ZmNF-YA12 improves salt and drought tolerance by inducing the expression of stress-related genes in Arabidopsis.
Proteins interact with other proteins in complex network systems to perform their diverse and targeted functions (Bhardwaj et al. 2016). A previous study showed that the maize NF-Y family gene NF-YA3 could interact with the JA activator MYC4 to improve drought and heat tolerance (Su et al. 2018). PwNF-YB3 can interact with PwHAP5, and heterologous expression PwNF-YB3 in Arabidopsis can induce the expression of drought response genes (Zhang et al. 2015). There have been no previous reports regarding the interactions of ZmNF-YA12 proteins in maize. In the present study, yeast two-hybrid experiments showed that ZmNF-YA12 interacted with ZmNF-YC1 and ZmNF-YC15. Our previous study showed that ZmNF-YC15 was induced by drought stress (Zhang et al. 2016). Therefore, ZmNF-YA12 may respond to stress by interacting with ZmNF-YC15.
In conclusion, we cloned and characterized the NF-Y gene ZmNF-YA12 from Zea mays. ZmNF-YA12 was expressed at high levels in young roots and induced by abiotic stresses. Its heterologous expression conferred enhanced tolerance to drought and salt stress by regulating the expression of stress-related genes. The gene may also perform its diverse and targeted functions by interacting with ZmNF-YC1 and ZmNF-YC15. These results will be helpful to understand the roles of NF-Y in abiotic stress responses.