RtNAC055 promotes drought tolerance via a stomatal closure pathway linked to methyl jasmonate/hydrogen peroxide signaling in Reaumuria trigyna

Abstract The stomata regulate CO2 uptake and efficient water usage, thereby promoting drought stress tolerance. NAC proteins (NAM, ATAF1/2, and CUC2) participate in plant reactions following drought stress, but the molecular mechanisms underlying NAC-mediated regulation of stomatal movement are unclear. In this study, a novel NAC gene from Reaumuria trigyna, RtNAC055, was found to enhance drought tolerance via a stomatal closure pathway. It was regulated by RtMYC2 and integrated with jasmonic acid signaling and was predominantly expressed in stomata and root. The suppression of RtNAC055 could improve jasmonic acid and H2O2 production and increase the drought tolerance of transgenic R. trigyna callus. Ectopic expression of RtNAC055 in the Arabidopsis atnac055 mutant rescued its drought-sensitive phenotype by decreasing stomatal aperture. Under drought stress, overexpression of RtNAC055 in poplar promoted ROS (H2O2) accumulation in stomata, which accelerated stomatal closure and maintained a high photosynthetic rate. Drought upregulated the expression of PtRbohD/F, PtP5CS2, and PtDREB1.1, as well as antioxidant enzyme activities in heterologous expression poplars. RtNAC055 promoted H2O2 production in guard cells by directly binding to the promoter of RtRbohE, thus regulating stomatal closure. The stress-related genes RtDREB1.1/P5CS1 were directly regulated by RtNAC055. These results indicate that RtNAC055 regulates stomatal closure by maintaining the balance between the antioxidant system and H2O2 level, reducing the transpiration rate and water loss, and improving photosynthetic efficiency and drought resistance.


Introduction
Plant growth and yield are severely affected by drought.The effect of drought on crop and woody plant biomass is increasing worldwide, especially in arid and semiarid regions, as a result of climate change problems, such as limited rainfall, excessive vaporization, and global warming [1,2].The adverse effects of drought are mediated by membrane damage, osmotic stress, and reduced photosynthetic and respiratory rates, which cause retardation of plant growth and metabolism [3].In the leaf epidermis, guard cells are arranged in pairs, forming stomatal pores that mediate entry of CO 2 (photosynthetic raw material) together with water transpiration [4].Stomatal transpiration contributes to ∼95% of overall plant-based water losses [5].Stomatal closure reduces water loss under drought conditions.Movement of guard cells is induced by reactive oxygen species (ROS), abscisic acid (ABA), methyl jasmonate (MeJA), CO 2 and water status, and controls both transpiration and photosynthesis [6][7][8].
ROS, involved in stomatal closure regulation, comprise ubiquitous metabolites produced in all organisms [9].Their accumulation in the apoplast and chloroplast serves as the earliest signs of stomatal closure.Excess ROS first accumulate in the guard cell apoplast, and the subsequent sensing and signaling activities contribute to the activation of K + /Ca 2+ channels [10].As reported for ABA, MeJA can induce accumulation of ROS in Arabidopsis thaliana guard cells, and this process is regulated by plasma membrane NAD(P)H oxidase Atrboh/F.Next, ROS act as signaling molecules in the plasma membrane and activate non-selective Ca 2+ -permeable cation channels [11][12][13][14][15]. MYC2, a bHLH transcription factor (TF), the master TF in JA signaling, is essential in a variety of JA-induced processes, such as lateral root development [16], saline stress [17], cold stress response [18], wounding/pathogen invasion [19,20], leaf and fruit senescence [21][22][23][24], plant photomorphogenesis [25], ROS accumulation [26], and secondary metabolite biosynthesis [23,[27][28][29].MYC2s participate in the mediation of responses against drought stress [30,31], though such a regulatory network and direct target gene(s) involved in the response to stomatal opening and closure under drought stress are unclear.
Environmental stress perception and transmission lead to the activation of transcriptional control [32].NAC, MYB/MYC, WRKY, DREB, and nuclear factor TFs are implicated in reactions following abiotic/biotic stress conditions [33][34][35][36][37][38][39][40][41][42][43].The TFs involved in guard cell movements in plants have been reported [44][45][46][47][48].The JAZ2 mutations in Arabidopsis restrain stomatal reopening by coronatine (COR) and promote resistance to bacterial invasion.In addition, MYC2/3/4 as JAZ2 target genes directly control ANAC19/55/72 expression and modulate the stomatal aperture [49].Two homologous NAC TFs in tomato, JA2 and JA2-like (JA2L), are specifically expressed in guard cells.JA2 regulates ABA accumulation and is implicated in ABA-meditated guard cell movement, and JA2L inhibits salicylic acid biosynthesis by regulating SA metabolism-related genes and controls pathogentriggered stomatal closure [50].Loss of function of MYB61, an Arabidopsis R2R3-MYB TF predominantly expressed in guard cells, reduces stomatal aperture, together with reductions in water loss and wilting symptoms during drought stress [51].Repression of DST, a C2H2 zinc finger TF, results in downregulation of H 2 O 2 homeostasis-related genes such as peroxidase 24 precursor, whose promoter contains a DST-binding element.This event triggers H 2 O 2 production and accelerates stomatal closure, consequently promoting drought/salt tolerance within Oryza sativa [52].In many species, NAC TFs are implicated in the regulation of guard cell movement.For instance, the stressresponse NAC1, SNAC1, controls the expression of the SRO protein OsSRO1c specifically produced in stomata under abiotic stress.Overexpression of OsSRO1c lowers transpiration-mediated water loss via triggering H 2 O 2 production in guard cells, which reduces the aperture of open stomata [53].Under drought stress, MusaSNAC1 expression is upregulated in guard cells of banana, where it triggers stomatal closure by elevating the H 2 O 2 concentration in the guard cells [54].However, the network of NAC TFs implicated in stomatal movement in halophytes, particularly recretohalophytes, is unclear.
Inner Mongolia is a harsh environment characterized by high soil salinity (up to 0.7% salts), drought (annual average precipitation 140.9-302.2mm), low temperatures (annual average temperature 6.0-9.2 • C), and scorching summers (maximum surface temperature 68.5 • C) [55][56][57].Many plants with tolerance to extreme environments grow in this area, and an in-depth study of the drought tolerance mechanisms of recretohalophytes would accelerate the generation of drought-resilient plants for use in agriculture [42,58].To adapt to these environmental stresses, Reaumuria trigyna has evolved distinct morphological and physiological features, including succulent, acicular leaves, salt-excreting glands, a highly efficient antioxidant system, and strong osmoregulatory capacity [59][60][61].Reaumuria trigyna is a typical recretohalophyte in this area and can act as a reference species for those that thrive in the severely adverse environment of the Eastern Alxa-Western Or dos desert.In previous studies, we identified 35 NAC TFs from a transcriptome database of R. trigyna.RtNAC100 was implicated in mediating salt tolerance by triggering ROS accumulation and programmed cell death (PCD) induction in plants [42].However, no study has investigated the role of NAC TFs in stomatal movements during drought stress in R. trigyna.In this study, an NAC TF, RtNAC055, was identified from R. trigyna, and was predominantly expressed in guard cells and directly regulated by MYC2.RtNAC055 regulated the expression of RtR-bohE/DREB1.1/P5CS1by directly binding to their promoters, which increased H 2 O 2 accumulation in stomata, maintained the balance between the antioxidant system and ROS level, and promoted stomatal closure.Thus, plant drought tolerance is enhanced by a reduction in the transpiration rate and water loss, maintenance of the balance between the antioxidant system and ROS level, and promotion of photosynthetic efficiency.

Jasmonic acid biosynthesis induced by drought and expression patterns of RtNAC055 in R. trigyna
JA biosynthesis-related genes were upregulated in R. trigyna seedlings after 400 mM NaCl treatment [42].In order to probe JA activity following R. trigyna drought stress, qRT-PCR was performed.PEG treatment led to a significant upregulation in JA biosynthesis-related gene expression (RtMYC2/AOS1.2/AOC4/LOX3/AOS1.1) in leaves (Supplementary Data Fig.S1), implicating MeJA in the drought response of R. trigyna.
The role of AtNAC055 in MeJA-related biological functions has been investigated [27,49].Bioinformatics analysis showed that RtNAC055 had a conserved NAM domain at amino acids 1-150 and had higher homology with AtNAC055 than other NAC proteins in Arabidopsis (Supplementary Data Figs S2 and S3).In this work, RtNAC055 expression was upregulated in increasing order by PEG, NaCl, and exogenous MeJA (Fig. 1A).RtNAC055 expression first increased and decreased thereafter following NaCl and PEG exposure, with peaks at 12 and 6 h.Exogenous MeJA induced RtNAC055 expression, which peaked at 3 h (Fig. 1D).Therefore, RtNAC055 was induced by JA and environmental stresses, and may be an upstream signal in the regulation of RtNAC055 expression following drought/salinity stress.
Analysis for RtNAC055 promoter recognized multiple putatively stress-response cis-elements, including LTRs, ABREs, drought-related MYB-binding sites, TATC boxes, GARE motifs, and TGACG motifs (Supplementary Data Table S1).We determined the tissue localization of gene expression by transferring the RtNAC055 promoter into Columbia-0.A GUS staining assay showed that RtNAC055 was predominantly expressed in roots and stomatal guard cells, implicating this in root development and stomatal movement (Fig. 1E-G).

RtNAC055 improves drought tolerance of transgenic R. trigyna callus and Arabidopsis
To investigate the function of RtNAC055, we obtained transgenic R. trigyna callus of RtNAC055.We carried out real-time f luorescence quantification and a GUS staining assay to certify that RtNAC055overexpressing (OE) and RNAi vector were transferred into callus of R. trigyna successfully (Fig. 2B).Then we analyzed the tolerance of different lines of R. trigyna callus to mannitol treatments.Wildtype (WT) and RtNAC055 OE/RNAi callus of the same size and fresh weight were grown on MS medium without or containing 400 mM mannitol for 15 days, and then the callus phenotypes and growth rates were analyzed.The growth rates of the WT and RtNAC055 OE/RNAi grape callus on MS medium were similar (Fig. 2A).Compared with the WT, the RtNAC055 RNAi transgenic callus was more sensitive to mannitol, and the growth rate was slower.However, the RtNAC055 OE callus had higher tolerance of mannitol and a faster growth rate (Fig. 2C).JA and H 2 O 2 contents in OE RtNAC055 callus were higher under mannitol treatment than in the WT and RtNAC055 RNAi groups (Fig. 2D and E).Therefore, our results suggested that RtNAC055 accelerated JA and H 2 O 2 synthesis and enhanced tolerance of mannitol in R. trigyna.AtNAC055 is implicated in drought tolerance [70,71].To investigate RtNAC055 function, a vector containing RtNAC055 was introduced into the atnac055 mutant to generate complementary lines.After drought treatment, Col-0 and the complementary lines showed longer roots and enhanced stress tolerance compared with atnac055 plants, and the drought-sensitive phenotype of mutant lines was rescued in the complementary lines (Fig. 3A and B).
Because water loss by transpiration decreases the temperature of the leaf surface, we measured temperature in Col-0, atnac055 mutant, and atnac055-OE RtNAC055 plants.The leaf temperature and survival rate of the atnac055 mutant were lower than those of Col-0 and atnac055-OE RtNAC055 plants under drought treatment.Overexpression of RtNAC055 rescued the phenotype of leaf surface temperature reduction in the mutant (Fig. 3C-F).
Stomatal closure was insensitive to drought in the atnac055 mutant.To determine whether RtNAC055 regulates stomatal closure in the transgenic lines under drought treatment, we examined the stomatal aperture in atnac055-OE RtNAC055 plants.The insensitive phenotype of stomatal closure of atnac055 in response to drought was partially rescued (Fig. 3G and Supplementary Data Fig.S6).
The above results indicate that RtNAC055 restored the temperature of the leaf surface of complementary lines by reducing the stomatal aperture and decreasing transpiration and water loss.This increased the survival rate of atnac055-OE RtNAC055 plants under drought treatment (Fig. 3F).

RtNAC055 overexpression improves water use efficiency and photosynthetic rate by reducing stomatal conductance
Stomatal movement is closely related to water use efficiency (WUE) and photosynthesis.To clarify the function of RtNAC055 in pant drought tolerance, a recombinant plasmid harboring the RtNAC055 coding sequence drove through the 35S promoter was introduced into poplar.GUS stain-step, genomic PCR, and qRT-PCR were performed to identify RtNAC055 transgenic poplar (Supplementary Data Fig.S8).To determine whether RtNAC055 regulates photosynthesis, we generated photosynthesis-light curves for WT and OE RtNAC055 poplar under normal watering conditions.The net CO 2 assimilation was similar for OE lines and WT (Fig. 4A).Leaf transpiration rate and stomatal conductance were markedly lower for OE lines in comparison with WT (Fig. 4B and E).Furthermore, OE lines demonstrated higher instantaneous WUE than the WT (Fig. 4D).No major variations were found within vapor pressure deficit across WT and OE lines (Fig. 4C), indicating that transpiration rate was unaffected by vapor pressure deficit.We speculate that RtNAC055 regulates stomatal movements to reduce the transpiration rate and increase the WUE and photosynthetic rate.

RtNAC055 contributes positively to drought tolerance of transgenic poplars
OE and WT plantlets were cultured in liquid Hoagland media supplemented with 300 mM mannitol.After 10 days some leaves from the WT group had died and fresh weight had decreased.By contrast, OE3 and OE8 plants exhibited better growth with little discoloration (Fig. 5A and B).In addition, the proline and H 2 O 2 contents and POD/SOD activities were elevated for OE lines in comparison with WT after drought treatment; by contrast, the MDA content was lower (Fig. 5C-H).Therefore, RtNAC055 overexpression enhanced the drought tolerance of poplar.
To further analyze RtNAC055 function under drought stress, a short-term drought stress in soil assay was performed.On day 9, WT plant leaves were seriously wilted and had a decreased chlorophyll content, whereas OE poplar leaves remained turgid.After rewatering for 2 days, OE plant leaves seemed refreshed and remained upright, whereas WT plant leaves were severely wilted, with abscission and senescence (Fig. 6A).Moreover, the leaf relative water content (RWC) for OE plants was elevated in comparison with WT plants after drought stress (Fig. 6E).
After 9 days of drought stress, F v /F m , leaf RWC, and chlorophyll content were significantly higher while water losses were reduced for OE lines in comparison with WT (Fig. 6).By contrast, there was little difference in these parameters under normal conditions.Delayed f luorescence (DF) is predominantly emitted from PSII, which is a sensitive phenomenon highly associated with changes in different photosynthetic processes induced by environmental factors.After drought treatment, DF signals for OE poplar leaves were stronger than those for the WT (Fig. 6B), indicating that PS II had a higher chlorophyll content and photosynthetic efficiency.Therefore, RtNAC055 enhanced the drought tolerance of transgenic poplar.7B).Stomatal aperture was smaller in transgenic poplar in comparison with WT during drought stress (Fig. 7A).Therefore, RtNAC055 accelerated H 2 O 2 production in stomata, triggering stomatal closure.

RtNAC055 promotes H 2 O 2 -induced stomatal closure
We investigated time-course changes in the signaling events under ABA or JA treatment.Within 30 min after stimulation with ABA, the ROS production of stomata in transgenic and WT poplar was significantly induced, but there were no remarkable differences between different lines (Fig. 7E and F).However, MeJA could induce more ROS production of stomata in transgenic lines than WT poplar.Thus, MeJA content increased within 30 min in transgenic poplar after drought treatment to induce ROS production and stomatal closure (Fig. 7G and H).ABA content in OE RtNAC055 poplar was not significantly different between the PEG treatment group and the WT group (Fig. 8B).Meanwhile, we investigated the gene expression level in the ABA signaling pathway (PtNCED3/ABF3/ABI2/PP2C.D2) under drought treatment.The expression of ABA biosynthesis-related genes in leaves was not significantly induced in transgenic poplar after drought treatment (Fig. 8A).Therefore, we speculated that ABA may not be involved in the signaling to regulate RtNAC055 and enhance drought tolerance.The DREB, P5CS, and RBOH genes are drought-responsive and promote drought tolerance.The expression levels of PtP5CS2, PtDREB2.2/2.6, and PtRbohD/F in OE poplars were upregulated significantly by drought stress.expression was significantly induced by drought (Supplementary Data Fig.S7).Y1H assays showed that after co-transformation of RtNAC055 and the promoter of RtRbohE, DREB1.1, and P5CS1, Y1H yeast grew normally in selective medium but not control medium.Therefore, RtNAC055 binds to the NACRS promoter in target genes.Dualluciferase assays showed that coexpression of 35S::RtNAC055 and RtRbohE, DREB1.1, and P5CS1pro::LUC significantly increased luminescence intensity, which was decreased by pGreen-62sk empty vector and RtRbohE, DREB1.1, and P5CS1pro::LUC as controls (Fig. 9).Therefore, RtNAC055 positively regulates RtRbohE, DREB1.1, and P5CS1 expression by directly binding to their promoters.

RtNAC055 interacts directly with
MYC2 is a bHLH TF, the master TF in JA signaling, typically bound to the G-box (CACGTG) in their target genes [75].In Arabidopsis, MYC2 directly regulates ANAC019/055/072 expression by binding specifically to the promoter of the MYC recognition sequence (G-box) [27,49].Bioinformatic analysis identified three potential MYC recognition elements (G-box) in the promoter region of RtNAC055.Y1H and dual-luciferase assays demonstrated RtMYC2 directly regulates RtNAC055 expression (Fig. 9).Therefore, RtMYC2 may bind to G-box elements to regulate RtNAC055 expression.

Discussion
TFs function in senescence [42,76,77], root formation [78,79], fruit ripening [22,[80][81][82], secondary cell wall development [74,[83][84][85], and responses to biotic/abiotic stress [37,38,[86][87][88].Some stressrelated TFs enhance plant abiotic stress tolerance by directly regulating stomatal movements and modulating photosynthesis.For instance, PdGNC directly regulates PdHXK1 expression in poplar to mediate stomatal closure through NO and H 2 O 2 production in guard cells, thereby enhancing WUE and improving drought tolerance [68].PeABF3 enhances drought tolerance and maintains high photosynthetic activity by directly regulating PeADF5 expression and triggering ABA-induced stomatal closure [66].A salicylic acid biosynthesis-related TF, PtrWRKY75, stimulates ROS accumulation in leaves and increases the expression of the salicylic acid biosynthesis gene PtrPAL1.This phenomenon results in stomatal closure and reduction in transpiration, eventually enhancing WUE and drought tolerance [89].NAC TFs are implicated in the regulation of stomatal movements and photosynthesis, particularly NAC055 in Arabidopsis and Brassica napus.In Arabidopsis, AtNAC055 promotes the drought response by enhancing proline accumulation to regulate stomatal closure [71].BnaNAC55 triggers ROS biosynthesis and defense-related genes, inducing ROS accumulation and hypersensitive response-like cell death against biotic stress [90].In this study, RtNAC055, which has higher homology to AtNAC055 than other NAC proteins in Arabidopsis, was isolated from R. trigyna, a typical recretohalophyte thriving in the Eastern Alxa-Western Ordos Desert.The expression of RtNAC055 was induced by drought, salt, and MeJA, and its promoter harbored stress-related cis-elements.The transgenic R. trigyna callus assay suggested that RtNAC055 accelerated JA and H 2 O 2 synthesis and enhanced tolerance of mannitol in R. trigyna.RtNAC055 was transfected into atnac055 mutant Arabidopsis and Populus davidiana × P. bolleana.The complementary lines had a narrower stomatal aperture and higher leaf surface temperature and survival rate than the atnac055 mutant under drought, which suggests that RtNAC055 rescued the drought-sensitive phenotype of the atnac055 mutant via regulation of stomatal movements.Compared with the WT, transgenic poplar overexpressing RtNAC055 showed higher drought tolerance and had higher proline content and SOD/POD activities and a lower MDA level.In addition, it had higher chlorophyll and RWC contents and lower water loss, increasing photosynthetic efficiency and F v /F m , and DF with PSII under drought stress.Drought markedly upregulated a proline synthesis-related gene (PtP5CS2), ROS production-related genes (PtRbohD/F), and dehydration-responsive genes (PtDREB2.2/2.6) in transgenic poplar.In addition, H 2 O 2 accumulation in stomatal guard cells was enhanced in the OE lines under drought, accelerating stomatal closure.Therefore, RtNAC055 reduced the transpiration rate and increased the WUE and photosynthetic rate of transgenic plants by modulating ROS levels and promoting stomatal closure.Stomatal movements control plant transpiration and photosynthesis and optimize photosynthetic CO 2 uptake, thus modulating vegetative growth and biomass accumulation [91].H 2 O 2 functions as a secondary messenger in stomatal closure induced by ABA.In addition, MeJA stimulates ROS production via plasma membrane NAD(P)H oxidases [14].Heterotrimeric G-protein is induced by MeJA, increasing the Ca 2+ content and H 2 O 2 accumulation and causing stomatal closure [92].MeJA induces ROS production and increases the Ca 2+ level in guard cellsin a manner mediated by COI1, and MPK9 and MPK12 accelerate MeJAinduced stomatal closure by activating S-type anion channels [93].However, the mechanism by which MeJA affects TFs and regulates ROS production is unclear.MYC2, the master TF in JA signaling, is essential in JA-induced physiological processes.In this study, the expression of JA biosynthesis-related genes (RtMYC2/AOS1.2/AOC4/LOX3/AOS1.1) in leaves was significantly induced in R. trigyna under drought and RtNAC055 responded to MeJA but not ABA.ABA contents and ABA signaling pathway genes (PtNCED3/ABF3/ABI2/PP2C.D2) activity in OE RtNAC055  [53].A banana NAC transcription factor (MusaSNAC1) strongly expressed in guard cells under drought accelerated stomatal closure by promoting H 2 O 2 production in guard cells [54].In this study, the RtNAC055 promoter was expressed specifically in root and stomatal guard cells.Y1H and dual-luciferase assays revealed the direct binding of RtNAC055 to an ROS production-related gene (RtRbohE).We speculate that RtNAC055 controls stomatal closure by modulating ROS production in guard cells.Indeed, H 2 O 2 accumulation in stomata was increased in the RtNAC055-OE lines, and the ROS productionrelated gene (PtRbohD/F) was enhanced and stomatal closure was accelerated under drought stress.In addition, the transgenic poplar showed enhanced drought tolerance with reduced water loss and ROS accumulation, and increased F v /F m , DF, and biomass.Therefore, RtNAC055 controls stomatal closure by modulating the ROS level in stomatal guard cells under drought stress, reducing the transpiration rate and increasing WUE and photosynthesis.NAC TFs also promote ROS accumulation in plant tissues to response to abiotic stress.GmSIN1, a salt-induced NAC TF in Glycine max, directly activates expression of GmNCED3s and GmRbohBs to increase the ABA and H 2 O 2 levels in leaves and roots, resulting in increased salt tolerance and root length [94].Herein, the H 2 O 2 content was higher in transgenic poplar leaf and R. trigyna OE callus than in the WT.Therefore the higher H 2 O 2 level in OE lines may act as a secondary messenger regulating stomatal closure and maintaining antioxidant system balance to defend against drought stress.
NAC TFs regulate DREB expression to alter drought tolerance in plants [34,95,96].In Arabidopsis, DREB1A/DREB2 regulate drought-related gene expression to enhance drought tolerance [97].DNA demethylation in the DREB2A promoter may enhance drought tolerance and promote root system development in Malus prunifolia [98].MsDREB6.2 enhances the expression of two AQP genes and accelerates stomatal closure and density, which reduces water loss under drought stress [99].In Arabidopsis and Solanum lycopersicum, JUNGBRUNNEN1 (JUB1), an NAC TF, improves drought tolerance by directly binding to the promoters of DREB1, DREB2, and DELLA to induce the expression of droughtresponse genes [100].In G. max, DREB1A is regulated by GmNAC20 and promotes lateral root formation to improve drought tolerance [101].CsATAF1, an NAC TF induced by ABA, improves drought tolerance by activating CsDREB2C expression to reduce ROS production [102].qRT-PCR results showed an upregulation in RtDREB1.1 expression in response to drought treatment and had a similar pattern to RtNAC055.Y1H and dual-luciferase assays showed the direct binding between RtNAC055 and the promoter of RtDREB1.1.In addition, the expression of the dehydrationresponsive gene PtDREB2.2/2.6 was higher in RtNAC055 OE poplar lines.Therefore, RtNAC055 directly regulates members of the DREB family to enhance drought tolerance.
Proline protects against abiotic stresses and oxidative damage and accumulates in plants experiencing environmental stress.Together with its osmolyte function, proline has multiple antioxidant activities [103,104].NAC TFs regulate proline accumulation to inf luence ROS production and modulate antioxidant enzyme activities.GmNAC8 improves SOD activity by altering the proline content to increase drought tolerance.BpNAC012 activates the core cis-element CGT[G/A] to promote expression of the prolinesynthesis gene P5CS1/2, increasing antioxidant enzyme activities in BpNAC012 OE transgenic birch [74].AtNAC055 enhances drought tolerance by increasing P5CS1 expression, triggering proline production [71].Here, RtP5CS1 expression was significantly increased by drought in R. trigyna seedlings.RtNAC055 directly bound to the RtP5CS1 promoter.Moreover, the OE poplar lines accumulated more proline because of upregulation of a proline biosynthesis gene (PtP5CS2), compared with WT poplar under drought stress.Therefore, RtNAC055 regulates P5CS expression to increase POD and SOD activity to enhance plant drought tolerance.
In conclusion, notably, the R. trigyna responed to drought required RtMYC2 and was regulated by MeJA.RtNAC055 directly binds to an ROS production-related gene (RtRbohE), leading to greater H 2 O 2 accumulation in stomata and accelerated stomatal closure.The relationships among RtNAC055, RtDREB1.1, and RtP5CS1 constitute a feed-forward loop responsible for rapid amplification of drought stress signals and maintain higherlevel antioxidant enzyme activities.RtNAC055 regulated stomatal closure, reduced the transpiration rate and water loss, and enhanced photosynthetic efficiency and drought resistance by maintaining the oxidant-antioxidant balance in a manner mediated by MeJA (Fig. 10).

Plant materials and treatments
Reaumuria trigyna seeds were acquired from Eastern Alxa-Western Ordos, a salinized desert in Inner Mongolia, China.Three normal seedlings (similar dimensions) were cultivated in in vitro tubes containing half-strength Hoagland's medium augmented with 20% PEG6000 (w/v) or 400 mM NaCl (stress induction), and 10 μM ABA or 10 μM MeJA treatment, respectively, for different times (0, 3, 6, 12, and 24 h).Stems/leaves were snap-frozen in liquid nitrogen directly following treatment and kept in storage (−80 • C) until analyses.

RNA extraction and qRT-PCR analysis
Total RNA was collected using an Eastep ® Super Total RNA Extraction Kit (Promega) according to kit protocols.cDNA was synthesized using a TRANS RT-PCR assay (TRANS).RT-qPCR was conducted on the Rotor-Gene Q ® platform (Qiagen) using TransStart ® Green qPCR SuperMix (Transgen Biotech).RtActin1 and PtActin1 served as internal controls.Relative gene expression was identified using the 2 -Ct method.Supplementary Data Table 2 lists all involved primers.

Isolation and analysis of RtNAC055
RtNAC055 cDNA corresponding to an ORF was developed by RT-PCR using a TRANS RT-PCR Kit (TRANS).Sequence analysis of RtNAC055 homologs was carried out on NCBI Protein Blast (https://blast.ncbi.nlm.nih.gov/Blast.cgi).Claustal X was used for multiple alignment, and MEGA 5.0 software was used to develop a phylogenetic tree with the neighbor-joining technique.The candidate gene promoter region was amplified using a Universal Genome Walker Kit ® 2.0 (Clontech) in line with kit protocols.

Vector construction and genetic transformation
A plant expression vector was constructed by ligating the coding region of RtNAC055 into pCAMBIA2301-GUS.A 360-bp fragment of RtNAC055 was introduced into the pK7GWIWG2D vector to generate the RNAi plasmid.The recombinant plasmids were introduced into Agrobacterium (EHA105).To probe the RtNAC055 expression profile, cloned fragments of the RtNAC055 promoter were inserted in the pORE-R1 vector using a one-step cloning technique.
Transgenic Populus davidiana × P. bolleana poplars were generated using the Agrobacterium-mediated method [62].35S:RtNAC055 and RtNAC055pro:GUS constructs were introduced into atnac055 (SALK_011069C) mutant and Columbia Arabidopsis, respectively, using the f loral dip technique [65].To generate transgenic callus, wild-type R. trigyna calli were used as the background for Agrobacterium-mediated transformation.The transformed callus was cultured on solid MS medium supplemented with NAA and 6-BA in the dark at 25 • C for 3 days and then cultured on solid MS medium with timentin and kanamycin to obtain positive lines; the transformed callus was subcultured every 14 days.

Subcellular identification/transactivation activity
The full-length (lacking the termination code) RtNAC055 sequence was cloned into pCAMBIA1300-GFP vector to obtain recombinant plasmids.Vectors (recombinant 35S:GFP:RtNAC055 + control 35S:GFP vectors) were transformed into Agrobacterium tumefaciens (GV3101) using electroshock transformation and separately infiltrated into N. benthamiana leaves through a needle-free syringe [66].Images were captured after 72 h with a confocal laser scanning microscope (Zeiss 710 Meta ® , Germany).
The RtNAC055 coding sequence was inserted into pGBKT7 (Clontech) for generating in-frame fusion onto the GAL4 activation domain, while recombinant plasmids were introduced into yeast AH109 (Supplementary Data Table S1).Transactivation activity was identified using previous protocols [42].

Yeast one-hybrid/dual-luciferase assessments
To verify binding of RtNAC055 to the NAC recognized sequence (CGT[A/G]) and the RtRbohE/P5CS1/DREB1.1 promoter, full-length RtNAC055 was cloned into pGADT7 to obtain an effector construct.The target sequence (CGT[A/G]) with tandem repeats was placed in pAbAi as reporter vector.The recombinant effector/reporter vectors were co-introduced into Y1H yeast.Transformants were diluted in a 10-fold series, and consequently grown on dropout medium for 3-5 days at 30 • C.

Physiological measurements and H 2 O 2 histochemical staining
A SPAD-502 chlorophyll meter (Konica Minolta) was employed to determine plant chlorophyll content.Leaf temperature was measured using a thermal imaging camera.POD, SOD, MDA, PRO, and H 2 O 2 activities were determined using corresponding assay kits (Keming Bioengineering Institute).The H 2 O 2 production in guard cells was analyzed by using 50 μM H 2 DCFDA (Coolaber) as described previously [67,68].Leaves were incubated (20 min in darkness) in a staining buffer (loading buffer containing 50 μM H 2 DCFDA and 10 mM MES-Tris, pH 6.15), and washed five times in phosphate-buffered saline to remove excess H 2 DCFDA.A f luorescence microscope was used to detect green f luorescence (Eclipse Ci-S; Nikon).
After 30 min of adaptation to darkness, peak PSII quantum yield (F v /F m ) in leaves from WT and OE lines was automatically monitored and recorded by the Li-Cor 6400XT.A Night-SHADE In Vivo Imaging System (Berthold Technologies) equipped with a CCD camera was used to measure delayed f luorescence (DF) [69].

Statistical analysis
Datasets ref lected means ± standard deviations.Variation significance was probed using one-way analysis of variance (ANOVA)/Duncan's multiple range test.Significance for variations across means was evaluated using * P < 0.05 and * * P<0.01.All such analyses were conducted using SPSS ® 18.0 (SPSS, Inc.).

5 EFigure 2 .
Figure 2. RtNAC055 promoted JA and H 2 O 2 synthesis and drought tolerance of transgenic R. trigyna callus.A Morphological characteristics of WT, RtNAC055 OE, and RNAi, transgenic callus under mannitol treatment.B Relative expression level of RtNAC055 in WT and transgenic callus.C Relative growth rate of R. trigyna callus under mannitol treatment.D, E JA and H 2 O 2 contents of transgenic R. trigyna callus under mannitol treatment.* P < 0.05, * * P < 0.01; Student's t-test.

Figure 3 .
Figure 3.A Root length of RtNAC055 transgenic Arabidopsis seedlings under mannitol treatment and phenotype under drought.B Primary root length under drought.C, D Phenotype and survival rate of RtNAC055 transgenic plants under drought or rewatering conditions.E, F Leaf surface temperature before and after drought.G Stomatal aperture.* P < 0.05, * * P < 0.01; Student's t-test.

Figure 7 .Figure 8 .Figure 9 .Figure 10 .
Figure 7. MeJA and drought induced stomatal closure by triggering production of ROS in RtNAC055 transgenic poplar.A, C Stomatal closure of WT and transgenic poplar under drought.B, D ROS accumulation and f luorescence quantification in WT and transgenic poplar under PEG treatment.E, F Representative images and f luorescence quantification of ROS in guard cells.G Stomatal aperture in WT and transgenic poplar after 30 min of ABA and MeJA treatment.H JA content in WT and transgenic poplar after 30 min of PEG treatment.* P<0.05, * * P < 0.01; ns, no significant difference; Student's t-test.Scale bar, 5 μm.
[9]aluable secondary messenger implicated in stomatal closure under abiotic stresses[9].To evaluate the role of RtNAC055 in H 2 O 2 production to induce stomatal closure, we used 2,7dichlorodihydrof luorescein diacetate (H 2 DCFDA) to measure the endogenous H 2 O 2 level in stomatal guard cells of WT and OE poplars under PEG treatment.Under normal conditions, H 2 O 2 content was similar for both plant types.Following PEG introduction the levels of H 2 O 2 increased in WT and OE lines.OE lines had a considerably higher H 2 O 2 level than WT plants in stomatal guard cells and leaves (Fig.