Overexpression of γ-glutamylcysteine synthetase gene from Caragana korshinskii decreases stomatal density and enhances drought tolerance

Background Gamma-glutamylcysteine synthetase (γ-ECS) is a rate-limiting enzyme in glutathione biosynthesis and plays a key role in plant stress responses. In this study, the endogenous expression of the Caragana korshinskii γ-ECS (Ckγ-ECS) gene was induced by PEG 6000-mediated drought stress in the leaves of C. korshinskii. and the Ckγ-ECS overexpressing transgenic Arabidopsis thaliana plants was constructed using the C. korshinskii. isolated γ-ECS. Results Compared with the wildtype, the Ckγ-ECS overexpressing plants enhanced the γ-ECS activity, reduced the stomatal density and aperture sizes; they also had higher relative water content, lower water loss, and lower malondialdehyde content. At the same time, the mRNA expression of stomatal development-related gene EPF1 was increased and FAMA and STOMAGEN were decreased. Besides, the expression of auxin-relative signaling genes AXR3 and ARF5 were upregulated. Conclusions These changes suggest that transgenic Arabidopsis improved drought tolerance, and Ckγ-ECS may act as a negative regulator in stomatal development by regulating the mRNA expression of EPF1 and STOMAGEN through auxin signaling. Supplementary Information The online version contains supplementary material available at 10.1186/s12870-021-03226-9.


Background
Water deficiency is one of the main reasons for poor plant performance and crop yields worldwide [1]. The global losses in crop yields due to drought totaled ~$30 billion in the past decade. Drought causes a wide range of changes including reduced leaf sizes, loss of root hairs, low water-use efficiency, and reduced photosynthesis [2][3][4]. Plants adopt multiple inherent strategies to (i) escape (acceleration of the reproductive phase before stress could impact its survival), (ii) avoid (increase internal water content), and (iii) tolerate drought (sustain growth with low internal water content during drought period) [5]. For example, redox homeostasis, root-associated microbiome, and transcription factors are all involved in plant resistance to drought. Oxidative damages can occur due to excessive reactive oxygen species (ROS) accumulated during water shortage [6]. ROS are the results of the partial reduction of atmospheric O 2 and are metabolic products in all cells. Under physiological conditions, ROS function as intracellular messengers in redox signaling and many cellular processes, and there is a frail balance between ROS production and breakdown. Under stress, slight enhancement in ROS production is sensed by the plant as an alarm and triggers defense responses; while Open Access *Correspondence: baijuan@nwsuaf.edu.cn excessive accumulation of ROS and unrestricted oxidation will cause damages to DNA, proteins, and lipids, and ultimately cause cell death [7,8].
The antioxidant glutathione (GSH) alleviates ROS damages, mainly through the redox signaling pathways [9][10][11]. The biosynthesis of GSH is tightly regulated by the rate-limiting enzyme gamma-glutamylcysteine synthetase (γ-ECS) [12]. In Arabidopsis, overexpression of γ-ECS (from S. cerevisiae strain S288C) led to a higher tolerance to heavy metals and the accumulation of arsenic and cadmium ions [13]. Over-expression of bacterial γ-ECS in poplar (Populus tremula × P. alba) and tobacco affected the photosynthesis and improved adaptability under mild drought and metal exposure [14][15][16]. In transgenic Arabidopsis overexpressing γ-ECS without seed vernalization, the level of flowering repressor FLOWERING LOCUS increased, suggesting that floral transition requires redox changes in GSH [17].
Stomata are vital to the drought tolerance of plants. Stomatal closure and the consequent limitation on CO 2 fixation are ways of ROS accumulation under drought conditions [8]. As a gateway for water transpiration and photosynthesis CO 2 exchange, stomata open and close pores according to the turgidity of a pair of guard cells [18,19]. The development of stomata undergoes several stages. In Arabidopsis thaliana, stomatal lineage initiates from asymmetric divisions of meristemoid mother cells to generate meristemoids, which reiterate several asymmetric divisions to produce neighboring nonstomatal cells, before finally producing the guard mother cells (GMC). GMC undergoes a single symmetric division to produce a set of paired guard cells to form mature stomata [20]. These divisions require the basic helixloop-helix (bHLH) transcription factors SPEECHLESS (SPCH), MUTE, and FAMA [21,22]. The activities of SPCH, MUTE, and FAMA are regulated by an intrinsic signaling pathway including the secreted peptides, EPI-DERMAL PATTERNING FACTORS (EPFs), receptorlike kinases ERECTA (ER) family, receptor-like protein TOO MANY MOUTHS (TMM) and MAPK cascades [20,23,24]. EPF1 and EPF2 expressed in the epidermis are negative regulators of stomatal development in Arabidopsis. Their activities depend on the TMM and ER family receptor kinases. They are genetically upstream of the genes for TMM, ER family, and the MAPKs cascades [25,26]. EPF2 is primarily perceived by ER and the co-receptor TMM to inhibit stomatal development [27]. TMM is a negative regulator of stomatal formation in cotyledons and leaves [28,29]. Transgenic Arabidopsis, tomato, and rice overexpressing ER showed improved heat tolerance and increased biomass in the greenhouse and field tests [30]. STOMAGEN, which is expressed in mesophyll and is a member of the EPFL family of proteins (EPF-LIKE9) [31], migrates to the epidermis where it is proposed to positively regulate stomatal density and stomatal index by competitively inhibiting TMM-mediated signaling [32]. Auxin signaling is a fundamental part of many plant growth processes and stress responses and operates through auxin/indole-3-acetic acid (Aux/IAA) protein degradation and the transmission of the signal via auxin response factors (ARFs) [33][34][35]. ARF5 directly binds to the STOMAGEN promoter to inhibit its expression, suggesting auxin negatively regulates stomatal development through ARF5 repression of the mobile peptide gene STOMAGEN in the mesophyll [36]. It has been shown that guard cells accumulate more GSH than other epidermal cells [37]. However, the precise function of GSH remains uncharacterized.
C. korshinskii is a leguminous shrub with strong tolerance to drought, cold, salt, and other biotic and abiotic stresses, and is widely distributed in arid and semiarid areas in the wilderness of Northwestern China [38]. Here, we isolated γ-ECS from C. korshinskii and characterized its role in mediating drought stress tolerance. The results suggest that Ckγ-ECS may participate in the negative regulation of auxin-mediate signaling to regulate stomatal development.

The stomatal density of C. korshinskii leaves decreased with drought
The lowest stomata density was measured in leaves from Dongsheng, the driest site of the sampling sites. Under natural conditions, the stomatal density of C. korshinskii leaves decreased with intensified drought stress (P < 0.05, Fig. 1).

Expression of stomatal development-related genes was changed after PEG 6000 treatment in C. korshinskii
The relative expressions of stomatal developmentrelated genes (CkFAMA, CkSTOMAGEN, CkARF5, and CkAXR3) were evaluated by qRT-PCR (Fig. 2). Under polyethylene glycol-6000 (PEG 6000) induced drought to C. korshinskii seeds, the expressions of CkFAMA and CkSTOMAGEN were decreased (Fig. 2a, d); the expression of CkARF5 was increased slightly without a statistical difference (Fig. 2c), and the expression of CkAXR3 was increased (Fig. 2b). The results suggested that auxin signaling may be involved in the stomatal development of C. korshinskii. Under drought. The expression of Ckγ-ECS and the γ-ECS activities also increased significantly with drought stresses (2.17-fold for 5% PEG and 4.6-fold for 10% PEG, Fig. 2e, f ). These findings suggest that Ckγ-ECS is related to stomata development by auxin signaling to adapt to drought in C. korshinskii.

Isolated Ckγ-ECS genes share a high identity with other Leguminosae plant γ-ECS
The Ckγ-ECS gene was isolated from C. korshinskii using reverse-transcription PCR (RT-PCR), and the template was taken from the transcriptome data of C. korshinskii (Figs. S1 and S2). The Ckγ-ECS has an open reading frame of 1527 nucleotides, encoding 508 amino acids with an estimated molecular weight of 57.59 kDa and a calculated pI of 6.85. Sequence alignments indicated that the putative protein shared higher sequence identity with its homologous sequences, including (  (Fig. 3a). The putative protein contained the HELICc, ELK, FN2, and UBA domains, which were highly conserved amongγ-ECS proteins (Fig. 3a). A phylogenetic analysis was performed to evaluate the evolutionary relationshipwith other species (Fig. 3b), and the results showed that Ckγ-ECS had a high degree of identity with the γ-ECS from other Leguminosae plants such as Medicago truncatula, Phaseolus vulgaris, Vigna angularis, Vigna radiata (Fig. 3b).

Generation and identification of Ckγ-ECS transgenic Arabidopsis lines
In order to verify the function of Ckγ-ECS, we generated transgenic Arabidopsis overexpressing the Ckγ-ECS Fig. 1 The stomatal density of Caragana korshinskii with different precipitation areas from Loess Plateau in China. Scanning electron micrographs of C. korshinskii leaves in Ansai, Shenmu, Dongsheng (a-c). The stomatal density of the C. korshinskii leaf with three sites (d). The data were shown above as means±SD (n ≥ 3). * and ** indicate that compared with Ansai, P < 0.05 and P < 0.01 cDNA without the termination under the control of the CaMV35S promoter and GFP fusion protein (Fig. S3a). Plants survived on the kanamycin selection medium and homozygous transgenic lines were obtained (Fig. S3b). After subsequent screening in T1 and T2 plant lines, homozygous T3 lines without resistance segregation on the selective medium were obtained and identified by the reverse transcription PCR amplification of the Ckγ-ECS gene using specific primers (Fig. S3c). The western blotting analysis showed that the Ckγ-ECS was expressed in the transgenic lines with an anti-GFP (Fig. S3d). T3 transgenic lines were used for further physiological studies.

Stomatal density and aperture sizes decreased in Ckγ-ECS overexpressing Arabidopsis
The Ckγ-ECS overexpressing Arabidopsis leaves were longer and narrower (Fig. S4), and their stomatal density and leaf aperture sizes were significantly reduced compared with those of WT plants (26.04 and 26.66% respectively, P < 0.05, Fig. 4). Overexpression of Ckγ-ECS led to a reduction in Arabidopsis stomatal density. The phenotypes of transgenic lines and WT plants under drought stress are shown in Fig. 5. The RWC of WT plants was significantly reduced (Fig. 5c). However, plants overexpressing Ckγ-ECS exhibited slower water loss in detached rosette leaves (Fig. 5d). Compared with WT, the MDA content of transgenic plants was significantly reduced (Fig. 5e). Compared with WT plants, the Ckγ-ECS transgenic Arabidopsis had a longer main root length withholding water for 7 days, and a shorter plant height at 60 days (Fig. 6). Together, the data indicate that overexpression of Ckγ-ECS in transgenic Arabidopsis can significantly improve drought tolerance.

Relative expression of stomatal development-related genes was changed in Ckγ-ECS overexpressing Arabidopsis
The levels of AtFAMA and AtSTOMAGEN mRNA in the transgenic plants were significantly lower than those in the WT (Fig. 7). The expressions of AtAXR3, AtARF5 and AtEPF1 in the transgenic plants increased significantly. There was no significant difference in the expression of Atγ-ECS between WT and transgenic plants (Fig. 7b), suggesting that overexpression Ckγ-ECS did not affect the expression of endogenous genes. However, γ-ECS activity in transgenic Arabidopsis was significantly increased (Fig. 7b).

Discussion
We examined the effects of a GSH rate-limiting enzyme γ-ECS on the plant drought tolerance both in C. korshinskii. and in Ckγ-ECS transgenic Arabidopsis lines, and revealed that Ckγ-ECS improved plant drought tolerance and the possible mechanism was stomatal development inhibition through regulating the auxin signaling (changed EPF1 and STOMAGEN expressions). The γ-ECS gene isolated from C. korshinskii and the putative proteins shared a high identity with those from other Leguminosae species. Stomata play crucial roles in plant responses to various abiotic stresses. Under the natural environment of the three sampling sites in the Loess Plateau, the stomatal density of C. korshinskii decreased with increased drought stress. Under drought treatment, the expressions of stomatal development positive regulators CkFAMA and CkSTOMAGEN in C. korshinskii were inhibited, and the expresson of auxin-response gene CkAXR3 was enhanced (CkARF5 was not enhanced). Furthermore, the expression of Ckγ-ECS and activity of γ-ECS were significantly increased under drought treatment. Previous study show that drought stress positively affect auxin-response genes (such as Aux/IAA, ARFs, Gretchen Hagen3 (GH3), small auxin-up RNAs, and lateral organ boundaries (LBD)), to regulate plant growth and development [35,[39][40][41], which was consistent with our In the Ckγ-ECS overexpressing Arabidopsis thaliana, the stomatal density and leaf aperture size was significantly reduced. In Arabidopsis thaliana, STOMAGEN positively and EPF1 and EPF2 negatively regulate the leaf stomatal density, and they are perceived by a receptor complex composed of the receptor-like proteins TMM, ER, and ERL1/2 [42]. STOMAGEN positively regulates stomatal formation and SPCH protein levels, [42,43] while EPF1 and EPF2 act antagonistically to STOMA-GEN, they activate MAP kinase that phosphorylates and destabilizes the SPCH [42]. The effect of FAMA on epidermal stomatal density and frequency was consistent with that of STOMAGEN [44]. In this study, the expression of AtSTOMAGEN and AtFAMA significantly decreased while the expression of AtEPF1 was enhanced in transgenic lines (Fig. 7a), which explained the reduction in stomatal density.
Many stress tolerance mechanisms are related to hormone signaling, such as BR [45], ABA [19], and auxin [36]. The phytohormone auxin signaling is a fundamental part of plant growth processes and stress responses [46]. Auxin accumulation activates signaling pathways and inducts auxin-responsive genes, which are mediated by the Aux/IAA and ARFs protein families [33][34][35]. Another report showed that auxin inhibited stomatal development through auxin response factors. ARF5 directly binds to the STOMAGEN promoter to inhibit its expression and thereby suppresses the development of stomat a [36]. As shown in Fig. 7a, ARF5 and STOMAGEN mRNA levels were opposite in overexpressing Ckγ-ECS plants. This result was consistent with a previous study in which the transgenic line with overexpression of the OsIAA6 gene improved tolerance to drought stress via the regulation of auxin biosynthesis genes [40]. In the present study, the expression of AtAXR3 (an auxin-inducible gene, same as IAA17 protein) is promoted, in a large part, which owes to the genetical position of AXR3 in upstream of the YDA MAP kinase cascade to regulate stomata formation in response to light and auxin signals [47]. There are some Aux/IAA members induced in different plants under drought. SbIAA8, SbIAA11, SbIAA 22 in leaves and SbIAA23 in roots were significantly up-regulated exposed to drought conditions in Sorghum bicolor [39]. 15 (OsIAA1,2,4,6,7,9,13,16,18,19,20,21,22,27,and 30) genes were induced by drought treatment in rice  [48]. Furthermore, γ-ECS activity in transgenic plants of overexpressing Ckγ-ECS was significantly increased (Fig. 7b), in line with the previous report [16]. Previous studies indicated that overexpression of the γ-ECS gene enhanced tolerance to drought in poplars [16]. These results suggested that γ-ECS could regulate stomatal development by mediating the auxin signaling pathway.
Under drought stress, Ckγ-ECS overexpressing plants grew better than wild-type plants (Fig. 5b), they had higher RWC, lower water loss, and lower MDA content (Fig. 5c-e). RWC and water loss are typical phenotypic and physiological parameters used to assess the water status of plants under drought stress [49,50]. MDA content in tissues can be used as a biomarker to estimate the degree of lipid peroxidation and tolerance to oxidative damage caused by dehydration stress [51]. These results indicated that overexpression of Ckγ-ECS can enhance plant resistance and tolerance to drought stress.

Conclusions
We cloned and characterized the Ckγ-ECS gene from C. korshinskii. The overexpression of Ckγ-ECS resulted in increased tolerance to drought stress in transgenic Arabidopsis lines. The function of Ckγ-ECS may be to negatively regulate stomatal development in response to drought by affecting the level of auxin-related genes. However, a detailed molecular mechanism by which Ckγ-ECS regulates stomatal density to adapt to drought remains unclear and requires further study.

Plant materials and growth conditions
The three sampling sites selected for natural C. korshinskii seeds were Ansai, Shenmu, and Dongsheng (with the annual precipitation and relative humidity of 506.5 mm, 60.8%; 440.8 mm, 54%; and 325.8 mm, 48.5%; respectively; Data available online (http:// data. cma. cn/ data/ cdcde tail/ dataC ode/A. 0029. 0004. html) or in the provincial Statistical Yearbook published by the Statistics Bureau), along a precipitation gradient from south to north across the Loess Plateau in north-west China. The sampling procedures were carried out in accordance with the institute guidelines by professor Fang Xiangwen from Lanzhou University. Sampled C. korshinskii seeds were grown in sterile distilled water under 16 h of light at 25 °C and 8 h of dark at 22 °C. The drought treatments included mock, 5% (w/v) PEG 6000, and 10% PEG 6000. After 20 days of growth, plants were sampled and stored with different treatments at − 80 °C for future tests. Seeds of Arabidopsis thaliana (Col-0) were produced in our lab in the Northwest A&F University and maintained by Dr. Yang Yazhou. The wild-type Arabidopsis thaliana (WT, ecotype Col-0) and overexpressing (OE) plants were germinated under long-day conditions (16 h/8 h, day/night cycles) at a controlled temperature (20 °C, day or night) and relative air humidity of 65%. These plants were grown in plastic square pots (6.8 cm × 6.8 cm × 7.8 cm) filled with soil (Pindstrup Sphagnum Moss Peat, Ryomgaard, Denmark) in a growth chamber. After 4 weeks, shoots were collected and stored in liquid N2. Some Arabidopsis plants were grown in Petri dishes on half-strength Murashige and Skoog medium (1/2 MS medium, pH 5.8) with 1% sucrose and 0.8% plant agar on 90 mm circular plates and stored for 3 days in cold and dark to synchronize germination. Then, the plates were placed in a plant growth chamber with 16 h day photoperiod and 20 °C. After 10 days the shoots were collected and stored in liquid N 2 . After growing the plants in the soil for 4 weeks under normal conditions, stress treatment was started. Then, drought was induced by withholding water to both the overexpression plants and wild-type plants (all 12 replicates of each group were analyzed).

Stomatal density
Leaf samples (width: 5 -7 mm) were fixed in 4% glutaraldehyde (Sigma, USA) at 4 °C overnight, and then washed four times with 0.1 M phosphate buffer (PBS, pH 6.8) for 10 min. The samples were dehydrated with a series of ethanol mixtures (30%, 50%, 70%, 80%, and 90% ethanol) for 15 min, and then washed twice with 100% ethanol and isoamyl acetate for 30 min, respectively. The stomatal density was observed by scanning electron microscopy (SEM6360LV). Image analysis was performed using ImageJ (NIH, Bethesda, MD, USA). All experiments were carried out with three biological replicates and three technical experiments.  Table 1.

Isolation and sequence analysis of Ckγ-ECS
The primers for Ckγ-ECS were designed based on the sequences of transcriptome data (Fig. S1). The PCR product was cloned into the pGEM-T Easy Vector System (Promega, USA) using PCR primers ( Table 1). The products were inserted into the SacI/PstI-digested pCAMBIA2301 binary vector (modified). After that, the plasmids were introduced into Agrobacterium strain GV3101. The putative amino acid sequences were deduced using BioEdit and ExPASy (https:// web. expasy. org/ protp aram/). The multiple protein alignment was performed using Clustal X software (version 2.0). DNA-MAN (version 5.2.2) was used for sequence assembling and SMART (http:// smart. embl-heide lberg. de/ smart/ set_ mode. cgi? NORMAL=1) was used for conserved domain analysis. The phylogenetic tree was constructed with the neighbor-joining (NJ) method for molecular evolutionary analysis using TreeView in MEGA (version 5.10).

Construction of Ckγ-ECS transgenic Arabidopsis plants
The 35S: Ckγ-ECS-GFP vector was introduced into Arabidopsis for overexpression studies. Arabidopsis transformation used the floral dip method [52]. After harvest, the T1 seeds were dried on allochroic silica gel at room temperature and germinated on 1/2 MS medium containing 50 μg ml − 1 carbenicillin to select transgenic seedlings. Seeds obtained from the primary transgenic lines were germinated on an antibiotic medium, and PCR analysis was performed on resistant plants. The surviving positive T1 plantlets were transferred to soil to harvest T2 seeds. T2 and T3 seeds were germinated as previously described. T3 seeds were harvested and used as the materials for all experiments in Arabidopsis.

Detection of transgenic lines
The transgenic lines were detected by PCR and Western blotting. A pair of Ckγ-ECS primers were used to amplify specific sequences. The total protein of the transgenic line was extracted and incubated with anti-GFP antibodies (1:5000; Beijing) and HRP-conjugated IgG secondary antibodies (1:5000; Transgen, Beijing) to detect the GFP fusion protein.

γ-ECS activity analysis
The excised leaves (0.5 g fresh weight) were ground in a mortar to a fine powder with extraction buffer (50 mM Hepes, pH 7.5, 5 mM MgCl 2 , 100 mM NaCl and 10% glycerol) [53]. The enzymatic activities of γ-ECS were determined spectrophotometrically at 25 °C by measuring the rate of ADP formation using a coupling assay of pyruvate kinase and lactate dehydrogenase [54]. The extraction buffer contained 50 mM Tris, pH 8.0, 500 mM NaCl, 25 mM imidazole, 5 mM MgCl 2 , 10% (v/v) glycerol and 1% (v/v) Tween 20. A common reaction mixture (0.5 ml) was used for both proteins and contained 100 mM Hepes (pH 7.5), 150 mM NaCl, 10 mM MgCl 2 , 2 mM sodium phosphoenolpyruvate (PEP), 0.2 mM NADH, 5 units of type III rabbit muscle pyruvate kinase and 10 units of type II rabbit muscle lactate dehydrogenase [54]. The decrease rate in A 340 nm was tracked using a spectrophotometer (V-1100D, Mapada).

Measurement of relative water content, leaf water loss, and MDA content
The WT and overexpressing lines under drought for 7 days were sampled to detect relative water content (RWC), water loss, and MDA content. The RWC was determined as described previously [50]. The water loss determination was carried out according to [55]. MDA was measured by thiobarbituric acid reactive substances (TBARS) assay. Samples of 0.5 g leaves were ground in liquid N 2 and then were added to1.5 ml 20% trichloroacetic acid (TCA). The homogenate was centrifuged at 3000 r/min for 10 min. Then the supernatant was then added to 1.5 ml of 0.5% thiobarbituric acid (TBA). The supernatant was heated to 100 °C for 10 min. The supernatant of 2 ml was sampled for measurement, and 0.5% TBA was used as a control. The absorbance of the supernatant was measured at 450, 532, and 600 nm. The difference was used to calculate the amount of MDA using an extinction coefficient of 6.22 mm − 1 cm − 1 .

Statistical analysis
Data analysis was performed using SPSS 17 (SPSS Inc., Chicago, IL, USA). Data are presented as the mean ± standard deviation. Student's t test and one-way analysis of variance (ANOVA) (LSD model) were used to test differences where it is appropriate. P < 0.05 was considered a statistically significant difference for two-tailed tests. All figures were plotted in Origin 9.0 (Northampton, MA, USA). Table 1 Gene-specific primers used for cloning and qRT-PCR