A 14-3-3 Protein-Encoding Gene, BdGF14g, Confers Better Drought Tolerance by Regulating ABA Biosynthesis and Signaling

Abscisic acid (ABA), a phytohormone, enacts a cardinal function in coping with abiotic stress. 14-3-3 proteins can interact with ABA-responsive-element-binding transcription factors (ABFs), a chief constituent of ABA signaling, and play critical roles in the dehydration response involving ABA signaling. Meanwhile, whether and how 14-3-3 proteins regulate ABA signaling to respond to aridity stress is yet to be fully investigated. Herein, BdGF14g, a 14-3-3 gene induced by ABA, H2O2, and PEG treatments, was identified in Brachypodium distachyon (B. distachyon). Overexpression of BdGF14g improved drought stress tolerance in tobacco plants, with a higher survival rate, longer root length, enhanced cell membrane stability, and increased antioxidase activity compared with non-transgenic controls in coping with dehydration. Both drought and exogenous ABA treatments resulted in smaller stomatal apertures in BdGF14g-transgenic lines. Additionally, when an ABA biosynthesis inhibitor was added, the better growth statuses, less H2O2 accumulation, and higher activities of catalase and superoxide dismutase under mannitol stress disappeared. Moreover, BdGF14g interacted with NtABF2, upregulated the endogenous ABA content, and enhanced the transcription of ABA-related genes, including NtNCED1, a crucial ABA biosynthesis gene, under drought conditions. In conclusion, BdGF14g acts as a positive factor in the water deficiency response by affecting ABA biosynthesis and signaling in tobacco plants.


Introduction
Harsh environments, including drought, restrict plant growth and development, especially for cultivating staple crops, and cause agricultural yield loss. Plants use a series of signal transduction networks to cope with various stress conditions.Recent research has reported that a group of adaptor proteins, 14-3-3s, play crucial roles in responses to stresses.According to the numbers of introns and exons, the 14-3-3 family genes are divided into non-ε and ε types [1].The 14-3-3 family proteins usually form homologous or heterologous dimers to exhibit their functions, and some plant crystal structures that are similar to those in animals have been obtained [2][3][4].Additionally, 14-3-3 proteins are highly conserved in different plant species and within the same species of plants, and their interactions with respective phosphorylated forms lead to the modulation of their activities and stabilities, Plants 2023, 12, 3975 2 of 15 the localization of targets, the substrate specificity of themselves or their derivatives, and the complex formation or disassembly of macromolecules [5,6].
Several studies have shown the vital role of 14-3-3 proteins in coping with abiotic stresses including salt and drought stress.In Arabidopsis, 14-3-3λ/κ functions under salt conditions via interacting with SOS2 [21].The heterologous expression of At14-3-3λ in cotton imparts more drought stress tolerance to transgenic cotton than that of wild-type (WT) cotton [22].The increased tolerance of GRF9, ZmGF14, and OsGF14f transgenic plants to drought stress indicates that 14-3-3 proteins play a core role under water-deficient conditions [23][24][25].Hv14-3-3A responds to drought conditions via rapid stomatal regulation in barley [3].OsGF14b plays a key function in responding to salt stress by interacting with OsPLC1 and promoting phospholipase C1 stability [26].OsGF14b confers resistance to rice under drought stress, and the mechanism is partially dependent on abscisic acid (ABA) [27,28].In transgenic tobacco, TaGF14b strengthens the resistance to drought and salt stress by increasing the ABA content and promoting ABA signaling [29].
Although progress has been made in elucidating the roles of plant 14-3-3 proteins, few 14-3-3 proteins have been functionally studied in B. distachyon, an emerging monocotyledonous plant model [37].In this research, BdGF14g, a 14-3-3 gene, was identified in B. distachyon.BdGF14g-expressing tobacco exhibited enhanced tolerance to drought because of an improved reactive oxygen species (ROS)-scavenging ability, rapid stomatal closure, and regulated ABA accumulation and signaling.Moreover, we verified the connection between the B. distachyon 14-3-3 gene and ABA signaling in coping with water deficiency.

BdGF14g Is Located in the Cytoplasm and Nucleus
The mixture of complementary DNA (cDNA) templates from two-week-old B. distachyon seedlings after treatments was used to clone the sequence of a 14-3-3 gene named BdGF14g, belonging to the non-ε subgroup, with an ORF of 789 bp and encoding a 262 aa protein (BRADI5G12510.2;GenBank: KU933263.1).The ORF of BdGF14g was cloned into pBI121-GFP (VC) driven by the promoter of CaMV 35s, with gene-specific primers listed in Table S1.To find the subcellular location of BdGF14g in plant cells, VC and pBI121-BdGF14g-GFP plasmids were transferred into tobacco (Nicotiana tabacum L.) from the abaxial surface of leaves via Agrobacterium transformation.Consistent with the control (pBI121-GFP), a green fluorescence signal of pBI121-BdGF14g-GFP was detected in tobacco cells (Figure 1).These results indicate that BdGF14g was located throughout the tobacco plant cell.
pBI121-GFP (VC) driven by the promoter of CaMV 35s, with gene-specific in Table S1.
To find the subcellular location of BdGF14g in plant cells, VC and pB GFP plasmids were transferred into tobacco (Nicotiana tabacum L.) from the a of leaves via Agrobacterium transformation.Consistent with the control (p green fluorescence signal of pBI121-BdGF14g-GFP was detected in tobacc 1).These results indicate that BdGF14g was located throughout the tobacco

BdGF14g Improves Drought Tolerance at the Young Seedling Stage
BdGF14g driven by CaMV 35s was transformed into model tobacco p mine the role of BdGF14g in coping with drought stress.Three stable and i transgenic lines, detected using relative quantitative PCR (RT-qPCR), desig OE2, and OE3 (Figure S1), were selected for stress-related studies, and the assay was performed.BdGF14g transgenic plants and control lines had s statuses, and the root lengths were not significantly different on 1/2 MS, wh of the three BdGF14g-expressing tobacco plants were much longer than th trols when grown on media containing 250 mM and 350 mM of mannitol These results show that BdGF14g-expressing tobacco plants grew better u treatment at the seedling stage.

BdGF14g Improves Drought Tolerance at the Young Seedling Stage
BdGF14g driven by CaMV 35s was transformed into model tobacco plants to determine the role of BdGF14g in coping with drought stress.Three stable and independent T 3 transgenic lines, detected using relative quantitative PCR (RT-qPCR), designated as OE1, OE2, and OE3 (Figure S1), were selected for stress-related studies, and then a root length assay was performed.BdGF14g transgenic plants and control lines had similar growth statuses, and the root lengths were not significantly different on 1/2 MS, whereas the roots of the three BdGF14g-expressing tobacco plants were much longer than those of the controls when grown on media containing 250 mM and 350 mM of mannitol (Figure 2A,B).These results show that BdGF14g-expressing tobacco plants grew better under mannitol treatment at the seedling stage.
pBI121-GFP (VC) driven by the promoter of CaMV 35s, with gene-specific prime in Table S1.
To find the subcellular location of BdGF14g in plant cells, VC and pBI121-B GFP plasmids were transferred into tobacco (Nicotiana tabacum L.) from the abaxia of leaves via Agrobacterium transformation.Consistent with the control (pBI121 green fluorescence signal of pBI121-BdGF14g-GFP was detected in tobacco cell 1).These results indicate that BdGF14g was located throughout the tobacco plan

BdGF14g Improves Drought Tolerance at the Young Seedling Stage
BdGF14g driven by CaMV 35s was transformed into model tobacco plants mine the role of BdGF14g in coping with drought stress.Three stable and indepe transgenic lines, detected using relative quantitative PCR (RT-qPCR), designated OE2, and OE3 (Figure S1), were selected for stress-related studies, and then a ro assay was performed.BdGF14g transgenic plants and control lines had similar statuses, and the root lengths were not significantly different on 1/2 MS, whereas of the three BdGF14g-expressing tobacco plants were much longer than those of trols when grown on media containing 250 mM and 350 mM of mannitol (Figu These results show that BdGF14g-expressing tobacco plants grew better under m treatment at the seedling stage.

BdGF14g Improves Drought Tolerance at the Mature Seedling Stage
To further confirm the responses of BdGF14g transgenic tobacco plants at the mature seedling stage to drought conditions, three-week-old controls and three BdGF14g transgenic tobacco plants growing in the soil were subjected to water-deficient conditions.After the 25 d treatment, the BdGF14g-expressing tobaccos exhibited better growth than the control lines, whereas the leaves of most wild-type (WT) and vacant vector control (VC) plants were wilting and yellowish (Figure 2C).After re-watering for 7 d, some transgenic plants recovered to a normal growth status, and the statistical analyses of the survival rates showed that those of the BdGF14g-expressing plants were remarkably higher (66-91%) than those of the controls (12-17%) (Figure 2D).These results show that BdGF14g transgenic tobacco exhibited increased resistance to water deficiency at the adult stage, which is in line with the results at the young seedling stage.

BdGF14g Increases ROS-Scavenging Ability under Water Deficiency
The stability of the cell membrane was reduced and the ion balance was destroyed under abiotic stress.Ion leakage (IL) and malondialdehyde (MDA), two membrane damage indices, arising from ROS, as well as H 2 O 2 content and relative water content (RWC) were measured.No pronounced differences were observed between the three BdGF14gexpressing plants and controls under normal conditions (Figure 3).However, compared with the controls, lower IL, MDA, and H 2 O 2 contents, as well as higher RWCs, were observed in BdGF14g transgenic tobacco plants deprived of water (Figure 3A-D).These results indicate that BdGF14g transgenic tobacco plants suffered less cell damage caused by ROS than WT and VC plants when subjected to drought.Therefore, the enzyme activities of the antioxidant system, including catalase (CAT), peroxidase (POD), superoxide dismutase (SOD), and total antioxidant capacity (T-AOC), were measured.Under normal conditions, there was no statistical difference between any of the detected groups.When subjected to a water deficit, BdGF14g-expressing tobacco plants exhibited higher CAT, POD, SOD, and T-AOC activities than the controls (Figure 3E-H).The above results show that BdGF14g could mitigate oxidant-induced cell injury by increasing the activities of enzymes related to ROS scavenging, including CAT, SOD, POD, and T-AOC, under drought stress.

BdGF14g Overexpression Enhances Stomatal Closure under Water Deficiency and ABA Treatments
As the stomata are a major gateway for water loss via transpiration in plants, the stomatal aperture is considered another important index of tolerance to drought stress.Therefore, stomatal apertures were measured using WT and OE1 lines as examples.Before treatment, the stomata of controls and BdGF14g-expressing tobacco plants were open, and the stomatal apertures were similar in these lines.However, after dehydration for 40 min, the stomata of the BdGF14g transgenic line OE1 were almost closed, whereas those of the WT were still open, and the transgenic line showed a lower stomatal aperture (Figure 4A,B).These results indicate that BdGF14g transgenic tobacco plants increased the speed of stomatal closure to decrease trans-epidermal water loss and improve the tolerance to water deprivation.
ABA, a phytohormone, performs a vital function in plants coping with adverse environmental stresses [38] and functions in stomatal closure and growth regulation.To identify whether the guard cells of BdGF14g transgenic lines respond to ABA treatment, leaves were exposed to 50 µM of ABA for 2 h.The stomata of BdGF14g-expressing tobacco plants were almost closed, whereas those of most of the control plants were still open.Statistical analysis also exhibited that the controls' stomatal apertures were larger than those of the transgenic plants when exogenous ABA was added (Figure 4A,B).

BdGF14g Overexpression Enhances Stomatal Closure under Water Deficiency and ABA Treatments
As the stomata are a major gateway for water loss via transpiration in plants, the stomatal aperture is considered another important index of tolerance to drought stress.Therefore, stomatal apertures were measured using WT and OE1 lines as examples.Before treatment, the stomata of controls and BdGF14g-expressing tobacco plants were open, and the stomatal apertures were similar in these lines.However, after dehydration for 40 min, the stomata of the BdGF14g transgenic line OE1 were almost closed, whereas those of the WT were still open, and the transgenic line showed a lower stomatal aperture (Figure 4A,B).These results indicate that BdGF14g transgenic tobacco plants increased the speed of stomatal closure to decrease trans-epidermal water loss and improve the tolerance to water deprivation.

Drought Tolerance Conferred by BdGF14g Involving ABA in Transgenic Tobacco Plants
To confirm the role of ABA in the response of BdGF14g transgenic tobacco plants to drought stress, ABA content was measured.Under normal conditions, no statistically sig nificant difference between the controls and three transgenic plants was detected.Endog enous ABA production increased in the tested transgenic tobaccos exposed to dehydration stress compared with that in the controls (Figure 4C).In addition, sodium tungstate (Tu) an ABA biosynthesis inhibitor, was chosen to further study the phenotypes (OE1 and OE2 and physiological indices (OE1, randomly selected) of two-week-old BdGF14g transgenic tobacco plants in response to drought treatment.Under mannitol stress, the BdGF14g transgenic tobacco plants showed a better growth status, with lower H2O2 contents and higher activities of CAT and SOD than the WT plants; however, when Tu was added, the notable differences between transgenic tobacco OE1 and WT plants in growth status, en zyme activities, and H2O2 content under mannitol stress disappeared (Figure 5).The above results indicate that BdGF14g participated in ABA-regulated stomatal closure, and when inhibiting endogenous ABA synthesis in BdGF14g-overexpressing tobacco plants, the tol erance and higher antioxidase activities in transgenic tobacco plants decreased under drought treatment, implying that the higher resistance to drought stress conferred by BdGF14g was dependent on ABA.ABA, a phytohormone, performs a vital function in plants coping with adverse environmental stresses [38] and functions in stomatal closure and growth regulation.To identify whether the guard cells of BdGF14g transgenic lines respond to ABA treatment, leaves were exposed to 50 µM of ABA for 2 h.The stomata of BdGF14g-expressing tobacco plants were almost closed, whereas those of most of the control plants were still open.Statistical analysis also exhibited that the controls' stomatal apertures were larger than those of the transgenic plants when exogenous ABA was added (Figure 4A,B).

Drought Tolerance Conferred by BdGF14g Involving ABA in Transgenic Tobacco Plants
To confirm the role of ABA in the response of BdGF14g transgenic tobacco plants to drought stress, ABA content was measured.Under normal conditions, no statistically significant difference between the controls and three transgenic plants was detected.Endogenous ABA production increased in the tested transgenic tobaccos exposed to dehydration stress compared with that in the controls (Figure 4C).In addition, sodium tungstate (Tu), an ABA biosynthesis inhibitor, was chosen to further study the phenotypes (OE1 and OE2) and physiological indices (OE1, randomly selected) of two-week-old BdGF14g transgenic tobacco plants in response to drought treatment.Under mannitol stress, the BdGF14g transgenic tobacco plants showed a better growth status, with lower H 2 O 2 contents and higher activities of CAT and SOD than the WT plants; however, when Tu was added, the notable differences between transgenic tobacco OE1 and WT plants in growth status, enzyme activities, and H 2 O 2 content under mannitol stress disappeared (Figure 5).The above results indicate that BdGF14g participated in ABA-regulated stomatal closure, and when inhibiting endogenous ABA synthesis in BdGF14g-overexpressing tobacco plants, the tolerance and higher antioxidase activities in transgenic tobacco plants decreased under drought treatment, implying that the higher resistance to drought stress conferred by BdGF14g was dependent on ABA.
results indicate that BdGF14g participated in ABA-regulated stomatal closure, and wh inhibiting endogenous ABA synthesis in BdGF14g-overexpressing tobacco plants, the t erance and higher antioxidase activities in transgenic tobacco plants decreased und drought treatment, implying that the higher resistance to drought stress conferred BdGF14g was dependent on ABA.

BdGF14g Interacts with NtABF2 and Increases the Expression of ABA-Related Gene in Transgenic Tobacco Plants
ABF2, an important transcriptional regulator of ABRE-dependent ABA signaling, is involved in the response to water deficit.In our previous studies, BdGF14g was shown to interact with three BdABRE/ABF TFs.Therefore, a yeast two-hybrid (Y2H) assay was performed to detect the interaction between BdGF14g and NtABF2.The results exhibit that NtABF2 interacted with BdGF14g in SD/-Trp-Leu-Ade and SD/-Trp-Leu-His-Ade solid media (Figure 6A).
To further elucidate the molecular mechanism of BdGF14g in response to drought conditions, the expression analyses of NtABF2, NtNCED1, a crucial ABA biosynthesis gene, as well as NtERD10C, an ABA-related stress defense gene, were performed.All detected genes (in addition to NtNCED1 under control conditions) were markedly higher in BdGF14gexpressing tobacco plants compared with WT plants under normal and drought conditions, and their expressions were elevated with dehydration (Figure 6B).These results show that BdGF14g improved drought tolerance by interacting with NtABF2 and upregulating the expression of genes related to ABA signaling.
gene, as well as NtERD10C, an ABA-related stress defense gene, were performed.A tected genes (in addition to NtNCED1 under control conditions) were markedly hig BdGF14g-expressing tobacco plants compared with WT plants under normal and dr conditions, and their expressions were elevated with dehydration (Figure 6B).The sults show that BdGF14g improved drought tolerance by interacting with NtABF upregulating the expression of genes related to ABA signaling.

Discussion
To cope with many abiotic stresses, including drought, plants generate comple nal transduction networks to respond to these abiotic stresses [39,40].The 14-3-3 f

Discussion
To cope with many abiotic stresses, including drought, plants generate complex signal transduction networks to respond to these abiotic stresses [39,40].The 14-3-3 family proteins participate in various physiological processes, including plant hormone and light signaling, basic metabolism, biotic stress, and so on [6,11,13,20,41,42].In addition, an increasing number of 14-3-3 proteins have been found to play cardinal roles in plants that cope with adverse abiotic stresses.In the present study, a 14-3-3 protein, BdGF14g, was extracted from B. distachyon located in the whole tobacco cell (Figure 1), and it was similar not only to the reported 14-3-3 proteins in B. distachyon, BdGF14d and BdGF14b [24,43], but also to 14-3-3 proteins in other plants such as At14-3-3λ and OsGF14f [14,25,44].The cytoplasm nucleus localization of 14-3-3λ-GFP had no obvious change under different light conditions [14], but significantly strong GFP signals were observed in the nuclei of root cells after 4 • C treatment [44].
BdGF14g can be upregulated by PEG and ABA and downregulated by H 2 O 2 , suggesting that BdGF14g possesses potential functions in response to these stresses [30,45,46].Overexpressing BdGF14a and MdGRF13 exhibited enhanced resistance to drought stress in Arabidopsis [46,47].Likewise, in transgenic tobacco, the function of BdGF14g in abiotic stress was first confirmed by the improved drought-tolerant phenotype in the young and adult seedling stages, with a better growth status, higher survival rate, higher RWC, and longer roots (Figure 2).
Drought treatment can lead to the overproduction of ROS, an important signal molecule in adaptive stress responses to dehydration, the overaccumulation of which may also damage plant cells [48].Therefore, ROS-related physiological indices were determined herein.The results suggest that BdGF14g-expressing tobacco plants exhibited stronger antioxidase activities, including POD, CAT, SOD, and T-AOC, in response to drought stress (Figure 3).Previous studies have shown that GPX, APX, SOD, and CAT are involved in ROS detoxification [29].Therefore, under drought stress, the higher antioxi-Plants 2023, 12, 3975 9 of 15 dase activities in BdGF14g-overexpressing tobacco plants showed that BdGF14g conferred drought tolerance by mediating the ROS-scavenging system.Consequently, lower accumulations of IL (an index of membrane injury), MDA (an index of ROS-mediated lipid peroxidation injury in the membrane), and H 2 O 2 were detected, which indicated that cell membrane stability was strengthened in BdGF14g-overexpressing tobacco plants under drought stress than in WT and VC plants (Figure 3A-H).The above results, in line with those of MdGRF11 and MdGRF13, two 14-3-3 proteins detected in apples under abiotic stress in transgenic plants [47,49], indicate that BdGF14g enhanced antioxidant capacity to improve the ROS-scavenging system under drought conditions.
Avoiding the lowering of the water potential caused by constant exposure to drought is mainly based on the absorption maximization of water from the roots and water loss minimization from evapotranspiration from leaves [39,50].An enhanced root length benefits the uptake of water under adverse conditions, improving plant adaptation to drought stress [50].Furthermore, stomatal regulation is a vital step to decrease the loss of water under water shortage conditions [3,38].In this study, in accordance with the higher RWC, longer roots and lower stomatal apertures of BdGF14g transgenic plants under drought stress were observed (Figures 2A, 3A and 4A,B).These results are partially in line with the previously reported results that At14-3-3λ, ZmGF14-6, GRF9, GsGF14o, TaGF14b, and Hv14-3-3A conferred resistance to plants to extreme stress partly via stomatal regulation and root elongation [3,[22][23][24]29,51].Hence, the above findings show that BdGF14g improved the water retention capacity (higher RWC) and enhanced transgenic tobacco plants' tolerance to dehydration by accelerating stomatal closure and increasing root length.
ABA exhibits a cardinal function in plants that cope with abiotic stresses by participating in stomatal closure, growth regulation, and seed germination inhibition [36,38,52].The 14-3-3 protein interacts with ABF TFs and participates in responding to adverse environmental conditions via the ABA signaling pathway [30][31][32]34,51].In our previous study, BdGF14g was upregulated by exogenous ABA.In this study, endogenous ABA production in BdGF14g transgenic seedlings was higher than in controls subjected to water deficiency, and the stomata of BdGF14g-overexpressing tobacco plants closed faster under drought and exogenous ABA treatments (Figure 4A,B).These findings imply that BdGF14g may participate in ABA signaling under dehydration treatment.We previously reported that BdGF14g interacted with three BdABRE/ABF TFs, including BdbZIP62, an ABA-insensitive 5 (ABI5)like protein [30].In Arabidopsis, ABI5 functions in ABA signaling and ROS homeostasis during seed germination [53].Herein, when Tu, an ABA biosynthesis inhibitor, was added, the better growth status and higher enzyme activity of transgenic tobacco plants than those of WT under mannitol treatment disappeared, and no notable differences were observed (Figure 5).These results are also in accordance with previous results showing that ABA enhances the activities of ROS-scavenging enzymes to alleviate oxidative damage [29,54].The above results indicate that BdGF14g functions in ABA-regulated stomatal closure and suggest that BdGF14g might improve antioxidant enzyme activity in transgenic tobacco via the ABA signaling pathway to achieve tolerance to drought stress.
MdGRF11, a 14-3-3 gene in apples, interacts with MdAREB/ABF TFs, and MdGRF11 improves the drought stress resistance of the MdGRF11-overexpressing plant by altering the transcription of ABA-signaling marker genes [49].OsGF14f positively mediates ABA responses induced by drought stress by interacting with OsbZIP23, a notable AREB/ABF TF, to enhance the transcriptional regulatory activity of downstream target genes [25,55].In line with previous results, in this study, the expression of the ABA-related stress defense genes, NtABF2, a critical ABRE-dependent transcriptional regulator of ABA signaling, NtNCED1, a crucial ABA biosynthesis gene, and NtERD10C, an ABA-related stress defense gene [56,57], were obviously induced in BdGF14g-overexpressing plants under drought treatment (Figure 6B), indicating that BdGF14g positively regulated ABA signal transduction at the transcriptional level.Moreover, similar to the interactions of MdGRF11 and OsGF14f with ABRE/ABFs in response to abiotic stress [25,49], BdGF14g also interacted with NtABF2 (Figure 6A).ABA-induced ABF2 and its homologs play master roles in the presence of drought stress by modulating stomatal aperture, root length, antioxidant activity, ABA content, downstream stress-responsive gene expression, osmotic regulator accumulation, transpiration rate, photosynthetic capacity, and germination [50,[58][59][60].This is similar to the response of BdGF14g to drought in the present study.Moreover, the AREB/ABF TFs regulate the transcription of downstream ABA-responsive genes by interacting with the ABRE elements of the promoters [25,55,58,59].For example, PtrABF2 directly binds to the promoter of pADC containing ABRE core sequences to respond positively to drought stress [59].OsbZIP23 directly promotes the expression of OsNCED4, a vital ABA biosynthesis gene, by binding to its promoter with ABRE elements to regulate the ABA response, and the interaction of OsbZIP23 with OsGF14f can enhance this transcriptional activity [25,55].NtNCED1, whose promoter contains an ABRE element, is a putative NtABF2 direct target gene and might be a critical gene in the feedback regulation of ABA accumulation to enhance the ABA-dependent drought tolerance conferred by BdGF14g.The observed increase in ABA content in BdGF14g-overexpressing lines under drought stress (Figure 4C) partly proved the validity of this hypothesis, although further experiments, including yeast one-hybrid (Y1H), dual luciferase, and electrophoretic mobility shift assays, need to be performed to further confirm this hypothesis.

Plant Materials and Stress Treatments
Nicotiana tabacum L. (Samsun) was used for genetic transformation and stress tolerance analyses.The T 3 seeds of BdGF14g transgenic tobacco, VC, and WT were sterilized according to previously reported instructions [29].The sterilized seeds were germinated on 1/2 MS without vitamins in Petri dishes.The dishes were then kept in a growth chamber (16 h light/8 h dark, 120 µmol photons m −2 s −1 , and 22 • C).The two-week-old tobacco plants were then potted in vermiculite in a phytotron in 16 h light/8 h dark and 150 µmol photons m −2 s −1 at 25 • C.

Cloning and Subcellular Localization Analyses of BdGF14g
BdGF14g was cloned with the cDNA template reverse transcribed from a mixture of RNA extracted from B. distachyon seedlings after ABA, H 2 O 2 , NaCl, and PEG6000 treatments.The ORF of BdGF14g was cloned into pBI121-GFP (VC) driven by the promoter of CaMV 35s.The pBI121-BdGF14g-GFP and VC plasmids were separately transformed into EHA105 (an Agrobacterium tumefaciens strain) and injected into tobacco plants from the lower epidermal cells according to a previously described method [61].Images were obtained after incubation in a growth chamber for 48 h.The gene-specific primers used are listed in Supplementary Table S1.

Tobacco Transformation and Stress Tolerance Analyses
The pBI121-BdGF14g-GFP and VC plasmids were separately transformed into EHA105 and then transformed into tobacco plants via the reported Agrobacterium-mediated transformation [62].After using a medium containing 100 mg/L of kanamycin (Kan) for screening, the expression levels in BdGF14g transgenic lines were assayed with RT-qPCR, with Nt-GAPDH as the control.The T 3 seeds of three stable independent BdGF14g transgenic tobacco plants (OE1, OE2, and OE3) were harvested and sprouted on the 1/2 MS conventional medium (containing 100 mg/L of Kan) for abiotic-stress-tolerance-related assays.The one-week-old seedlings of BdGF14g-expressing lines and controls were transferred to 1/2 MS and 1/2 MS with 250 mM or 350 mM of mannitol, respectively, and the root lengths of at least nine seedlings per line were measured after vertical cultivation for two weeks.For the drought resistance assay during early seedling development, the seedlings were transferred to 1/2 MS, 1/2 MS + 350 mM mannitol, and 1/2 MS + 350 mM mannitol + 1 mM Tu for two weeks, and 0.2 g of plantlets (besides the roots) was harvested for further physiological analysis.For the drought treatment, two-week-old geminated tobacco plants grown in pots (five seedlings/pot) filled with vermiculite for 3 weeks were exposed to dehydration for 25 d and then re-watered for 7 d to take photos, and 0.2 g of leaves from at least three seedlings per line without water supplied for 15 d was sampled for further physiological measurements.

Measurements of Aridity-Responsive Physiological Parameters
To investigate the differences in physiological indices of BdGF14g-expressing tobacco plants in adaptation to dehydration, 0.2 g of leaves or seedlings was collected and ground in 1.8 mL of phosphate-buffered saline on ice.The supernatant was obtained via centrifugation (12,000 rpm for 20 min).The contents of H 2 O 2 and MDA and antioxidase activities of T-AOC, POD, CAT, and SOD, were measured with the respective assay kits (Comin, Suzhou, China).The ABA content was assessed using the ELISA kit (Jiancheng, Nanjing, China).The IL and RWC were also calculated in terms of a priorly reported method [54].The formula IL (%) = C1/C2 × 100 was used to calculate the IL.

Stomatal Closure Assay
A stomatal closure assay was performed according to a prior report [63].The leaves of four-week-old tobacco plants (WT, VC, and OE) were immersed in a solution containing 50 µM of CaCl 2 , 10 mM of MES-KOH, and 30 mM of KCl (PH 6.15) and put under highintensity light (200 µmol m −2 s −1 ) for 6 h to make the stomata open, and then the leaves were removed to dehydrate at 25 • C for 40 min or put into a solution containing 50 µM of ABA for 2 h.After treatments, the stomatal statuses were observed under a fluorescence microscope (IX71, Olympus, Tokyo, Japan) [29].The width/length rates of at least four leaves per line were calculated in each biological replicate to indicate the stomatal aperture.

Y2H Assays
The Y2H assay was performed to detect the interaction between BdGF14g and NtABF2 according to previously reported methods [64].Full-length coding sequences of BdGF14g were cloned into the pGBKT7 (BD) vector, and NtABF2 was transferred to the pGADT7 (AD) vector.Next, these vectors were used to co-transform AH109 (a yeast strain), plated on the SD/-Trp-Leu selection medium for 3 d, spotted on the nutritional selective solid media SD/-Trp-Leu, SD/-Trp-Leu-Ade, and SD/-Trp-Leu-His-Ade, and grown at 30 • C for 3-5 d.The pGBKT7-p53 or pGADT7-T, and pGBKT7-LaminC transformants were taken as the negative and positive control, respectively.The interaction was estimated by the growth ability of yeast cells on selective plates of SD/-Trp-Leu-Ade and SD/-Trp-Leu-His-Ade. Detailed information on the related gene-specific primers is presented in Table S1.

qRT-PCR Assays
To examine the expressions of ABA-related stress-responsive genes in BdGF14gexpressing lines and WT, three-week-old seedlings were transferred to a medium of 1/2MS + 300 mM mannitol for 1 week.These stress-related genes included the ABA-related genes NtABF2, NtNCED1, and NtERD10C.RNA was extracted using an RNA extraction kit (Zoman Biotechnology, Beijing, China), and cDNA was synthesized using a FastQuant RT kit (Tiangen, Beijing, China) [29].Detailed information on the gene-specific primer pairs is provided in Table S2 [29].For data analyses, the 2 −∆∆CT method was used to compute the relative expressions [65].

Statistical Analyses
GraphPad Prism (GraphPad Software 6.01, Inc., La Jolla, CA, USA) was used for statistical analyses following the reported methods [29].

Conclusions
In conclusion, BdGF14g is an adaptor protein that enhances drought resistance in transgenic tobacco via increasing membrane stability, modulating the antioxidase system

Figure 1 .
Figure 1.Subcellular localization of BdGF14g::GFP fusion protein and GFP in tob cells.The plasmids of pBI121-BdGF14g-GFP and PBI121-GFP were introduced into robacterium strain, and then injected into tobacco cells via Agrobacterium transforma bating for 48 h, photos were taken under an inverted fluorescence microscope.

Figure 1 .
Figure 1.Subcellular localization of BdGF14g::GFP fusion protein and GFP in tobacco epidermal cells.The plasmids of pBI121-BdGF14g-GFP and PBI121-GFP were introduced into EHA105, an Agrobacterium strain, and then injected into tobacco cells via Agrobacterium transformation.After incubating for 48 h, photos were taken under an inverted fluorescence microscope.

Figure 1 .
Figure 1.Subcellular localization of BdGF14g::GFP fusion protein and GFP in tobacco e cells.The plasmids of pBI121-BdGF14g-GFP and PBI121-GFP were introduced into EHA1 robacterium strain, and then injected into tobacco cells via Agrobacterium transformation.A bating for 48 h, photos were taken under an inverted fluorescence microscope.

Figure 2 .
Figure 2. Response of BdGF14g transgenic tobacco plants to drought stress.(A) The see wild type (WT), vacant vector (VC) controls, and three BdGF14g-overexpressing lines a

Figure 2 .
Figure 2. Response of BdGF14g transgenic tobacco plants to drought stress.(A) The se wild type (WT), vacant vector (VC) controls, and three BdGF14g-overexpressing lines a days of germination were planted on 1/2 MS with 250 mM or 350 mM of mannitol.Aft

Figure 2 .
Figure 2. Response of BdGF14g transgenic tobacco plants to drought stress.(A) The seedlings of wild type (WT), vacant vector (VC) controls, and three BdGF14g-overexpressing lines after seven days of germination were planted on 1/2 MS with 250 mM or 350 mM of mannitol.After vertical cultivation for two weeks, the root lengths of at least nine seedlings per line were detected and statistically analyzed (B).(C) The two-week-old seedlings growing on MS were transplanted into pots filled with soil for three weeks under normal conditions.After that, watering was stopped for 25 days, and then they were rewatered for a further 7 days.(D) The respective survival rates of BdGF14gexpressing tobacco plants and controls suffering from water withholding were measured based on at least 35 seedlings per line in each replicate.Error bars were calculated from three independent experiments.Asterisks indicate marked differences in statistics (** p < 0.01).

Figure 3 .
Figure 3.The physiological index analyses of controls and BdGF14g transgenic plants in response to water withholding.The two-week-old seedlings growing on MS solid medium were grown in potting soil for three weeks under normal conditions, and no water was supplied to treat the seedlings for a further fifteen days.Leaves, 0.2 g in weight, from at least three tobacco plants of test and control groups were sampled to measure the (A) relative water content (RWC), (B) malondialdehyde (MDA) content, (C) hydrogen peroxide (H2O2) content, (D) ion leakage (IL), the enzyme activities of (E) catalase (CAT), (F) peroxidase (POD), (G) superoxide dismutase (SOD), and (H) total antioxidant capacity (T-AOC).Error bars were calculated from three independent experiments.Asterisks indicate marked differences in statistics (* p < 0.05; ** p < 0.01).

Figure 3 .
Figure 3.The physiological index analyses of controls and BdGF14g transgenic plants in response to water withholding.The two-week-old seedlings growing on MS solid medium were grown in potting soil for three weeks under normal conditions, and no water was supplied to treat the seedlings for a further fifteen days.Leaves, 0.2 g in weight, from at least three tobacco plants of test and control groups were sampled to measure the (A) relative water content (RWC), (B) malondialdehyde (MDA) content, (C) hydrogen peroxide (H 2 O 2 ) content, (D) ion leakage (IL), the enzyme activities of (E) catalase (CAT), (F) peroxidase (POD), (G) superoxide dismutase (SOD), and (H) total antioxidant capacity (T-AOC).Error bars were calculated from three independent experiments.Asterisks indicate marked differences in statistics (* p < 0.05; ** p < 0.01).

Figure 4 .
Figure 4.The stomatal movement of WT and BdGF14g-expressing line (OE1) under dehydration and ABA treatments, and abscisic acid (ABA) content under drought stress.(A) The stomatal move ments of potted WT and OE1 lines for six weeks were detected in normal, 40 min dehydration, and 50 µM 2 h ABA conditions.Images were captured under a fluorescence microscope in a bright field (B) The stomatal aperture of at least four leaves per line was examined under dehydration, ABA treatments, and control conditions.(C) The endogenous ABA content under normal and water-with holding conditions.The error bars were calculated based on three independent replicates.Asterisks indicate marked differences in statistics (* p < 0.05).

Figure 4 .
Figure 4.The stomatal movement of WT and BdGF14g-expressing line (OE1) under dehydration and ABA treatments, and abscisic acid (ABA) content under drought stress.(A) The stomatal movements of potted WT and OE1 lines for six weeks were detected in normal, 40 min dehydration, and 50 µM 2 h ABA conditions.Images were captured under a fluorescence microscope in a bright field.(B) The stomatal aperture of at least four leaves per line was examined under dehydration, ABA treatments, and control conditions.(C) The endogenous ABA content under normal and water-withholding conditions.The error bars were calculated based on three independent replicates.Asterisks indicate marked differences in statistics (* p < 0.05).

Figure 5 .
Figure 5.The BdGF14g-overexpressing lines lost the resistance to drought after adding an endogenous ABA inhibitor, sodium tungstate (Tu).Ten OE1, OE2, and WT tobacco plants of two-week-old seedlings were grown in 1/2 MS containing 350 mM of mannitol or 350 mM of mannitol + 1 mM Tu for two weeks, and then photographs were taken (A).Young seedlings (0.2 g, besides roots) of the transgenic line (OE1) and WT without or with Tu treatment were harvested for measurements of (B) H 2 O 2 , (C) CAT, and (D) SOD.Error bars were calculated from three independent experiments.Asterisks indicate marked differences in statistics (* p < 0.05).

Figure 6 .
Figure 6.BdGF14g interacted with NtABF2 in the yeast two-hybrid assay and transcript expr analyses of the ABA signaling pathway.(A) BdGF14g was transferred into the pGBKT7 vecto NtABF2 was cloned into the pGADT7 vector, followed by their co-transfection into the A strain, and spotted onto the nutritional selective solid media SD/-Trp-Leu, SD/-Trp-Leu-Ad SD/-Trp-Leu-His-Ade.The transformants pGADT7-T, pGBKT7-p53, and pGBKT7-LaminC rep the controls.(B) The transcript expression levels of ABA-related genes NtABF2, NtNCED NtERD10C.The error bars were calculated based on three independent replicates.Asterisks in marked differences in statistics (* p < 0.05; ** p < 0.01).

Figure 6 .
Figure 6.BdGF14g interacted with NtABF2 in the yeast two-hybrid assay and transcript expression analyses of the ABA signaling pathway.(A) BdGF14g was transferred into the pGBKT7 vector, and NtABF2 was cloned into the pGADT7 vector, followed by their co-transfection into the AH109 strain, and spotted onto the nutritional selective solid media SD/-Trp-Leu, SD/-Trp-Leu-Ade, and SD/-Trp-Leu-His-Ade.The transformants pGADT7-T, pGBKT7-p53, and pGBKT7-LaminC represent the controls.(B) The transcript expression levels of ABA-related genes NtABF2, NtNCED1, and NtERD10C.The error bars were calculated based on three independent replicates.Asterisks indicate marked differences in statistics (* p < 0.05; ** p < 0.01).