Overexpression of Chorispora bungeana CbPLDγ enhances drought tolerance in Arabidopsis


 Phospholipase D (PLD) is a crucial enzyme participated in membrane phospholipid catabolism. In this study, to explore the function of CbPLDγ in drought stress, a CbPLDγ gene, which is a part of CbPLD gene family and from Chorispora bungeana (C. bungeana) was cloned and encoded a protein of 859 amino acids with a calculated molecular weight of 96.3 kDa and with a PI(Isoionic Point) of 7.88. Real-time quantitative PCR (RT-qPCR) and Beta-glucuronidase (GUS) assay showed that CbPLDγ was accumulated dominantly in roots and hypocotyls. Compared with the control, CbPLDγ was positively responsed to the low temperature, salt, mannitol, and exogenous ABA. Subcellular localization analysis showed that the CbPLDγ was localized in the cell membrane. CbPLDγ-overexpression Arabidopsis under drought stress showed higher relative expression of superoxide dismutase (SOD), catalase (CAT) and peroxidase (POD), as well as highe content of proline, soluble proteion and soluble sugar. However, H2O2, malonaldehyde (MDA) content and electrolyte leakage (EL) were lower than wild-type Arabidopsis. These indicated that CbPLDγ was involved in the drought tolerance, and overexpression of CbPLDγ enhanced the drought tolerance in Arabidopsis. This is the first report about cloning and characterizing the gene of CbPLDγ from C. bungeana. It laid a foundation for further research and improvement of the PLD gene family of C. bungeana.


Introduction
Phospholipids are critical in cell membranes and also play a pivotal role in cell development. The phospholipase D (PLD) can hydrolyze phospholipids to produce phosphatidic acid (PA) and soluble head group. Each of the PLD gene superfamilies involves two copies of the conserved HxKxxxxD sequence, which is an active site is known as the HKD motif. Besides, the N-terminal domain contains the binding motif lipid and the C-terminal domain contains the catalytic motif. Different PLDs seem to have different functions but somewhat overlapping functions in cellular processes (Lein and Saalbach, 2001). PLD gene has been cloned from many species such as rice bran, Maize (Ueki et al., 1995), tobacco (Lein and Saalbach, 2001), cowpea (Ben Ali et al., 2007), tomato (Tiwari and Paliyath, 2011), Arabidopsis thaliana . PLD genes are classi ed into six subfamilies in the Arabidopsis genome: α (3), β (2), γ (3), δ, ε and ζ (2) (Wang, 2005). The majority of PIP 2 and Ca 2+ can in uence the activity of PLDγ1 and PLDγ2 , Qin et al., 2006, and previous studies have shown that PLDγ is more tolerant to all stress in the Arabidopsis seedlings (Zhao et al., 2011).
Abiotic stresses trigger the physiological and molecular mechanisms to protect the plant from cell damage or death, such as increasing antioxidant activity, decreasing membrane permeability, and controlling the reactive oxygen homeostasis (Hong et al., 2008, Hong et al., 2010. Many abiotic stresses, such as salt, drought, salicylic acid (SA), abscisic acid (ABA), can in uence the PLD expression , Guo et al., 2013, Kalachova et al., 2013. Chorispora bungeana (C. bungeana) is a cruciferae plant, which is a typical representative of the subnival alpine plant. It can adapt to the environment of low oxygen pressure, cold, drought, and strong ultraviolet radiation. As nature resistant plant, C. bungeana contains abundant stress-related genes. It is also closely related to Arabidopsis. It has a surface lint and wax layer, and contains a lot of free fatty acid, neutral amino acid, soluble sugar, Mg 2+ -ATPase activity, and unsaturated fatty acids (Song Y, et al. 2015). Previous research also showed that C. bungeana has no special morphological characteristics. It is an ideal plant species to study the stress-related molecular mechanisms of C. bungeana.
PLDs are thought to be involved in plant responses to abiotic stresses and have multiple functions during plant growth and development (Pinhero et al., 2003). Water de ciency can trigger PLD activity in Craterostigma plantagineum (Frank et al., 2000). PLD participated in freeze stress response in C. bungeana callus (Yang et al., 2012). In ABA signaling, PLDα1 and PLDδ have a synergistic effect in Arabidopsis guard cells (Kalachova et al., 2013). SA can active the PLD and induce stomatal closure in Arabidopsis (Guo et al., 2013). And the stomata from PLDα1 de cient Arabidopsis plants fail to close in response to ABA, whereas the external supply of PA, promoted the stomatal closure (Hong et al., 2010).
Overexpression analyses suggested isoform PLDδ3 is activated in pollen tubes and PA promotes the binding of PLDδ3 to the plasma membrane in tabacco (Dreßler et al., 2017). Overexpression TaPLDα (Triticum aestivum L) plants can enhance tolerance to drought and osmotic stress in Arabidopsis. TaPLDα is a potential candidate gene to enhance stress tolerance in plant (Wang et al., 2014). Although there are many PLD reports, so far, there is no detailed study about the characterization and function of the CbPLDγ in C. bungeana.
In this work, the full length of the CbPLDγ cDNA sequence from C. bungeana was cloned and analyzed. The localization, expression pattern, and possible function of CbPLDγ were also investigated. It showed a better insight into the molecular mechanisms of CbPLDγ under drought stress and provided a theoretical basis for further research of the PLD gene family.
Arabidopsis and tobacco (Nicotiana benthamiana) seeds were disinfected and germinated on MS medium. After 5 days, the germinated seeds were transferred to the soil, and all the plants were grown in a greenhouse under 22°C light for 16 h and dark light for 8 h.

Stress treatment
The C. bungeana seedlings were grown according to 2.1. A week later, the seedlings in vitro were subjected to 0.3 M mannitol, 150 mM NaCl, 4°C, and 100 Mm ABA, for 72 h respectively. All the samples were immediately frozen in liquid nitrogen for further experiments. The CbPLDγ overexpressing Arabidopsis seedlings were grown according to 2.1. Two weeks later, the seedlings were growing without watering for 1 week and rehydrated for ve days.

Cloning of CbPLDγ gene
RNAiso Plus kit (Takara, Dalian) was used to extract total RNA from the plantlets of C. bungeana according to the manufacturer's instructions. The rst strand of cDNA was synthesized using the PrimeScript™ II 1st Strand cDNA Synthesis Kit (Takara, Dalian) according to the manufacturer's instructions.
Based on other PLDγ homologous species, two primers (CbPLDγ1-F and CbPLDγ1-R) (Supplement Table  1) corresponding to sequences of the conserved regions of PLD genes were used to amplify part of the CbPLDγ sequence. The ampli ed product was linked to the pMD19-T vectors (Takara, Dalian) for further sequencing. The primers (CbPLDγ2-F and CbPLDγ2-R) (Supplement Table 1) were designed from the 5' end of the rst ampli ed fragment to amplify the middle fragment of CbPLDγ. The product was linked to the pMD19-T vectors (Takara, Dalian) for further sequencing. The three sequences were combined to obtain the middle part of CbPLDγ.
The 5′ and 3′ rapid ampli cation of cDNA ends (RACE) were performed using the SMARTer TM RACE cDNA Ampli cation (Invitrogen, USA). Based on the instruction manual, total RNA was isolated and produced templates for 5′ and 3′ race from the C. bungeana. The primers of 5′GSP, 5′NGSP, 3′GSP and 3′NGSP (Supplement Table 1) were designed to amplify the RACE products. And the nest PCR products were cloned into the pMD19-T vector and sequenced.
By comparing and aligning the sequences, the full length of the CbPLDγ gene sequence was obtained. The full length of the CbPLDγ gene was veri ed by PCR using Primer Star HS DNA polymerase (Takara, Dalian) and sequencing.

Cloning of the CbPLDγ promoter
Genomic DNA was isolated from the C. bungeana using the TaKaRa MiniBEST Plant Genomic DNA Extraction Kit (Takara, Dalian) to clone the CbPLDγ promoter sequence. The CbPLDγ promoter was cloned by using Genome Walking Kit (Takara, Dalian). Adaptors were provided by the kit and SP primers (Supplement Table 1) were designed according to the sequence of CbPLDγ. The PCR products were puri ed using TIANGEN Midi Puri cation Kit (TIANGEN, Beijing), cloned into a pMD19-T vector, and sequenced.

Quantitative real-time PCR
Total RNA was isolated from C. bungeana were subjected to salt, mannitol, 4°C, and abscisic acid (ABA) induction. The cDNAs were synthesized using the FastKing RT Kit (With gDNase) (TIANGEN, Beijing). Gene-speci c RT primer pairs designed based on the CbPLDγ sequence (Supplement Table 1) were subjected to RT-qPCR using TBGreen® Premix Ex Taq™ II (Tli RNaseH Plus) (Dalian Takala). ACTIN gene (GenBank Accession No.AY_825362) was used as internal controls for normalizing gene expression levels. The results were displayed using the 2 −ΔΔct method.

Histochemical location of CbPLDγ
The promoter of CbPLDγ was cloned (Supplementary Table 1) by the Genomic DNA of C. bungeana, and the correct sequences were inserted into vectors. The nal construct of pBIB-CbPLDγ-GUS was introduced into Arabidopsis. The homozygous seedlings were screened by herbicide Basta and were used to detect the histochemical location of GUS activity. Plant tissues were incubated for 16 h at 37°C in X-Gluc solution, then were decolorized in destaining solution for approximately 10 h. The seedlings were washed and then observed under Olympus CX31 Microscope (Olympus, Japan).

Subcellular localization of CbPLDγ
The coding region of CbPLDγ was ampli ed from C. bungeana by RT-PCR using primer CDS-F and CDS-R (Supplementary Table 1), and the PCR products were sequencing. The cloned gene fragment was recombined into the pMDC83 vector with recombinase. Competent DH5α cells were transformed and plasmids were veri ed by sequencing. The correct plasmids containing the pMDC83-CbPLDγ-GFP vector were introduced into Agrobacterium tumefaciens GV3101. The recombinant plasmids were transformed into tobacco for transient expression (Sparkes I A, et al. 2006), and the transformed tobaccos were cultured in darkness at 22°C for 48 h. The GFP uorescence was captured by the confocal microscope (Leica, German).

Transformation of Arabidopsis
The CbPLDγ promoter and coding regions were ampli ed by nested PCR using primer Promoter-F and Promoter-R (Supplementary Table 1). The cloned fragment was recombined into the pBIB vector with recombinase. Competent DH5α cells were transformed and plasmids were veri ed by sequencing. The correct plasmids containing the pro-pBIB-CbPLDγ-GUS vector were introduced into Agrobacterium tumefaciens GV3101. Then, the recombinant plasmids were transformed into the Arabidopsis Col-0 according to the oral dip method and the transformed Arabidopsis was cultured in darkness at 22°C for 24 h. The transformants were selected by MS medium containing Basta. The fourth generation of transgenic Arabidopsis plant was used for further analyses (Hong et al., 2010). The seedlings of transgenic Arabidopsis which have been screened to the fourth generation were selected for RT-PCR identi cation and RT-qPCR analysis.

Statistical analyses
All experiments were repeated at least three biological times. Data were analyzed by one-way ANOVA using SPSS 19.0 for windows (SPSS Inc. Chicago, IL, USA). Data were shown with the mean ± SD of three biological replicates.

Cloning of CbPLDγ gene
Based on the PLDγ gene sequence in other homologous species, the middle fragments of the CbPLDγ gene were cloned from C. bungeana. And the fragments about 480 bp, 712 bp, 524 bp were obtained in sequence. 5′GSP and 5′NGSP were designed beside the middle region sequence for 5′-RACE. A 160 bp 5′ untranslated region (UTR) of the ATG codon upstream was obtained. Then the 3′GSP and 3′NGSP for 3′-RACE were designed, the 1373 bp was cloned which contained 197 bp 3′UTR in the TAA codon downstream (including a poly-A tail of 12 bp). By comparing and aligning, the 2937 bp full-length cDNA was deduced (Supplementary Fig. 2A).
Analysis by DNAstar, the full-length cDNA contained a 2580 bp ORF (GenBank accession No. MF951104), which encoded a protein of 859 amino acids with a calculated molecular weight of 96.3 kDa and with a pI of 7.88. Besides, CbPLDγ had the conserved motif "FIYIENQFF", which may be related to the hydrophobic interaction between the methyl groups in the choline group. The motif "FIYIENQFF" was as important as the HKD domain ( Supplementary Fig. 2B). Phylogenetic analysis showed that the CbPLDγ gene had high homology with other plant PLDγ genes. Also, among the PLDγ subtypes, C. bungeana was closely related to Arabidopsis PLDγ gene ( Supplementary Fig. 3).

The expression of CbPLDγ under abiotic stress
The expression of transcription level of CbPLDγ was analyzed to explore the CbPLDγ responding to low temperature, salt, mannitol, and exogenous ABA. The relative expression levels of the CbPLDγ gene uctuated under different abiotic stresses. It peaked at 12 h under 0.3 M mannitol and 150 mM NaCl, and was 2.97 fold and 3.34 fold compared to the control, respectively (Fig. 1A&B). The relative expression levels of the CbPLDγ gene peaked at 24 h under 4°C and 100 mM ABA and was 4.3 fold and 6.5 fold compared to the control (Fig. 1A &B). This result showed that the CbPLDγ was up-regulated under low temperature, salt, mannitol, and ABA, and the CbPLDγ actively responded to different abiotic stress.

The expression patterns of CbPLDγ in different tissues
To explore the expression patterns of CbPLDγ in different tissues, the histochemical location was carried out. The GUS activity results con rmed that CbPLDγ was expressed in all the tissues, but with signi cant enrichment in root tips and anther (Fig. 2).

Subcellular localization of CbPLDγ
The recombinant plasmids were transformed into tobacco according to the transient expression method for epidermal cells. The results showed that the CbPLDγ was located in the cell membrane (Fig. 3).

Identi cation of transgenic Arabidopsis
Four transgenic plants (T 1 -T 4 ) were selected from Basta screening to the fourth generation, and genomic DNA was extracted for RT-PCR identi cation (Fig. 4A). The successful heterologous expression of the CbPLDγ gene in Arabidopsis was con rmed. Take 18-day-old Arabidopsis seedlings (T 1 -T 4 ) for RT-qPCR analysis (Fig. 4B).In subsequent experiments, select the two highest expression lines (T 1 , T 3 ) for analysis.

Overexpression of CbPLDγ enhances the drought stress tolerance of Arabidopsis
To study the effect of CbPLDγ on the cell membrane and plant osmoregulatory substances, we measured the EL, MDA, proline, soluble protein, and soluble sugar in CbPLDγ transgenic Arabidopsis plants under drought stress. In this experiment, the EL and MDA of WT were signi cantly higher than those of transgenic plants under drought stress, indicating that overexpress of CbPLDγ decreased the membrane damage under stress (Fig. 5A & B). Compared with the WT, the transgenic plants had higher contents of proline, soluble sugar and soluble protein, suggesting that overexpression of CbPLDγ enhanced the plant osmoregulatory substances to protect the plant from drought stress (Fig. 5C, D & E ).
Under normal conditions, there was no signi cant difference in SOD, POD, and CAT between WT and CbPLDγ transgenic lines. However, the drought signi cantly increased the relative expression levels of the antioxidant enzyme. The relative expression levels of SOD, POD, and CAT in transgenic lines were higher than that in WT signi cantly (Fig. 6A, B & C ). The H 2 O 2 contents in the transgenic plants were also lower than that in WT signi cantly (Fig. 6D ).

Discussion
C. bungeana is a typical subnival alpine plant, its living environment is extremely harsh (Wu et al., 2008). In recent years, the research on C. bungeana has been reported from various levels, especially the antiadversity response mechanism. Phospholipase D (PLD) is involved in different plant growth processes, including plant development, the responses to various stresses et al (Distéfano et al., 2015). In this study, a PLDγ gene, named CbPLDγ (GenBank accession No. MF951104), was cloned from C. bungeana for the rst time. The gene is 2937 bp in length and contains an open reading frame (ORF) of 2580 bp, 5' and 3' untranslated regions are 160 bp and 197 bp. The study showed that 4°C low temperature, salt, and mannitol stress can induce the expression of the CbPLDγ gene (Fig. 1), indicating that CbPLDγ is widely involved in the abiotic stress responses of C. bungeana. Plant growth regulators, participate in plant stress response and play an important role (Shaterian et al., 2005). In this study, exogenous ABA can also induce the expression of the CbPLDγ gene (Fig. 1).
The study of the expression pattern of PLD and the subcellular location may be of great signi cance for further understanding the function and regulatory mechanism of PLD. PLDε was found in microsomes, but not in the soluble fraction (Yao et al., 2016). The GmPLDγ-GFP fusion protein is expressed in transgenic Arabidopsis roots and tobacco leaf mitochondria (Bai et al., 2020). The average expression level of PLDα3 in buds, owers, siliques, stems, old leaves, and roots was 1000 times lower than that of PLDα1 in Arabidopsis (Hong et al., 2008). The subcellular localization indicated that the protein was signi cantly clustered on the cell membrane, which implied that the CbPLDγ protein mainly performed its functions on the cell membrane (Fig. 3).
The permeability and stability of plant cell membranes play an important role in plant growth and development, PLD is involved in the degradation of cell membrane lipids. The EL and MDA of WT were always higher than that of CbPLDγ transgenic lines under drought treatment (Fig. 5). This indicates that overexpression CbPLDγ enhanced the drought stress tolerance by alleviating the membrane damage.
As normal plant osmoregulatory substances, proline, soluble proteins, and soluble sugar are used for regulating the osmotic pressure of cell membranes and cell osmotic potential under various stresses (Chun et al., 2018). In this research, compared with the WT, the CbPLDγ transgenic plants had higher contents of proline, soluble sugar, and soluble protein, re ecting that overexpression CbPLDγ could increase the osmoregulatory substances to responding to the drought stress (Fig. 6).
Under environmental stress, the content of reactive oxygen species in plants erupts (Miller et al., 2010, Fichman andMittler, 2020). ROS is a substance produced by plants under stress conditions and is an important medium for plants to respond to stress. H 2 O 2 is the most stable marker of ROS. Plants have an array of antioxidant enzymes, such as SOD, CAT, and POD, which protect cells from oxidative damage. In this study, drought treatment signi cantly increased the H 2 O 2 content and the relative expression of SOD, POD, and CAT in WT and CbPLDγ transgenic plants (Fig. 6A, B & C). The CbPLDγ overexpressed lines had higher antioxidant enzyme relative expression than WT. And the H 2 O 2 contents in the transgenic plants were also lower than that in WT signi cantly (Fig. 6D ). This indicates that CbPLDγ was involved in the regulation of H 2 O 2 and antioxidant enzymes, overexpression CbPLDγ enhanced the drought stress tolerance by regulating the antioxidative system.

Conclusion
In this study, a novel CbPLDγ gene is ampli cation from C. bungeana and is identi ed. Compared with the control, the expression pattern of the CbPLDγ mRNA is up-regulated in response to low temperature, salt, mannitol, and exogenous ABA. CbPLDγ is expressed in all examined tissues and is located in the cell membrane. The overexpression of CbPLDγ enhances the drought stress tolerance by alleviating the membrane damage, increasing the osmoregulatory substances, and regulating the antioxidative system. These support that CbPLDγ is involved in the response to drought stress, and has the potential to improve the drought tolerance of plants. This is the rst report about cloning and characterizing the CbPLDγ gene from C. bungeana. It lays a foundation for further research and improvement of the PLD gene family of C. bungeana and represents a potential candidate gene to enhance stress tolerance in plants.

Declarations
Competing interests   Subcellular localization of CbPLDγ. Constructed pMDC83-CbPLDγ-GFP vector was transformed into tobacco epidermal cells. All images were observed with the confocal microscope.   The effect of drought stress on the antioxidant enzyme and H2O2 in WT and CbPLDγ transgenic plants.
Data are shown with the mean ± SD of three biological replicates. Different letters the signi cant differences at P<0.05.