Increased Abscisic Acid Sensitivity And Drought Stress By Overexpression of Abscisic Acid Receptors In Arabidopsis Thaliana

Abscisic acid (ABA) is a key plant hormone that regulates plant growth development and stress response. ABA is recognized and bound by ABA Receptor PYR/PYL/RCAR (referred to as PYLs). However, little is known about the PYLs gene family in Populus euphratica. Here, we identied 12 PYLs in P. euphratica and named PePYL1-12. Phylogenetic analysis divided the 12 PePYLs into three subfamilies. Subcellular localization showed that PePYL2, PePYL4, PePYL5, PePYL6, and PePYL9 were located in the cytoplasm and nucleus, PePYL10 localized in the nucleus. The promoter of 12 PePYLs contains hormones- and abiotic stress-related cis-acting elements. Moreover, ABA and drought signicantly up-regulation the expression of PePYL6 and PePYL9. To study the performance of PePYLs under ABA and drought stress, we generated transgenic Arabidopsis plants overexpressing PePYL6 and PePYL9. Compared with wild type, transgenic Arabidopsis enhanced ABA sensitivity during seed germination and root growth, improved water use eciency and drought resistance. Taken together, our results conrmed that PePYL6 and PePYL9 play a positive role in ABA-mediated stress responses in P. euphratica. Tomato PYR/PYL/RCAR abscisic acid receptors show high expression in root, differential sensitivity to the abscisic acid agonist quinabactin, and the capability to enhance plant drought resistance. 4451-4464.

PYLs, sensing and binding ABA, belongs to the START (star-related lipid transfer) domain/Bet v 1-fold proteins superfamily, the proteins of this family have a ligand-binding pocket formed by the four conserved loops of CL1-CL4 that contribute to ABA signaling (Melcher et al., 2009;Park et al., 2009). In Arabidopsis, there are 14 AtPYLs. According to the oligomeric nature of the apo receptors, 14 AtPYLs can be divided into monomeric: AtPYL4-AtPYL12, dimeric: AtPYR1/AtPYL1/AtPYL2, AtPYL3 has two forms: monomeric or dimeric (Hao et al., 2011). Dimeric receptors are strictly ABA-dependent for their function, and monomeric receptors have different binding characteristics with ABA, and selectively interact with PP2Cs (Ma et al., 2009). 14 AtPYLs can also be divided into three subfamilies based on sequence similarity (Tischer et al. 2017). Since the discovery of the AtPYLs family in Arabidopsis, homologous genes of PYLs in other plants have been identi ed at genome-wide levels, including 6 PYLs in sweet orange (Romero et

Phylogenetic analysis of the PePYLs gene family
The PYLs amino acid sequences of Arabidopsis and P. euphratica were used to construct a phylogenetic tree. Multiple alignments of all protein sequences were analyzed by the neighbor-join (NJ) algorithm using MEGA 7 software. Accession numbers in Table S2.
Analysis of cis-acting elements in the promoter of the PePYLs gene family The cis-elements of 2000 base pairs (bp) upstream of PePYLs start code (ATG) were analyzed using PlantCare Online (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/). The cis-acting elements in Table S3.

Subcellular localization
For subcellular localization of PePYLs in plants, GFP fusion proteins were observed using a laser confocal uorescence microscopy (Leica TCS SP8).

Quantitative Real-Time qRT-PCR Analysis
Gene expression analysis in different tissues. P. euphratica seedlings about 50 cm high. Collect young leaves, adult leaves, old leaves, stems, and roots, and immediately immerse them in liquid nitrogen for tissue expression analysis. The total RNA was extracted by the CTAB method described in this article (Springer, 2010). Use ABI StepOnePlus Real-Time PCR System (ABI, Foster City, CA) according to the manufacturer's speci cations to perform Quantitative Real-Time RT-PCR (qRT-PCR). PeActin and AtActin as internal control, to quantify the relative expression level of genes in samples.

GUS staining and activity assay
Histochemical GUS staining. Transgenic Arabidopsis were incubated in GUS staining solution at 37°C, and then 75% ethanol was used to remove chlorophyll. The activity of GUS was detected by the uorescence of 4-methylumbelliferone (4-186 MU) produced from the β-glucuronidase substrate 4methylumbelliferyl β-D187 glucuronide (Jefferson et al., 1987), and the protein concentration was quanti ed according to the previous protocol (Bradford, 1976).

Cloning of PePYLs gene and transformation of Arabidopsis
The cDNA of PePYL6 and PePYL9 were ampli ed by PCR using primers PePYL6 F/R and PePYL9 F/R. The primer sequences in Table S4. Germination and root length analysis 100 seeds of different lines were sown on 1/2 MS medium containing 0, 0.5, and 1.0 µM ABA each time, and the germination rate was counted once every 12 h to con rm the germination rate. The Col-0, transgenic, and mutant plants germinated were transplanted into 1/2 MS medium containing 0, 5, and 10 µM ABA was grown vertically for 10 d, and then the primary root length was measured.

Drought experiments
Drought stress experiments were conducted by controlling plant water in the greenhouse. The seedlings of the Col-0, transgenic and mutant plants were transplanted into the soil and watered for 15 d. One seedling was planted in each pot. After 4 weeks of growth, the Arabidopsis plants with the same growth were selected to carry out the water-withheld experiment for 8 d.

Physiological analysis
The net photosynthetic rate (P n ), transpiration rate (T r ) of the Col-0, transgenic, and mutant plants under the same conditions were measured by Li-Cor portable photosynthesis meter (LI-COR 6400) at an ambient CO 2 concentration of 500 µmol mol − 1 , the photosynthetic photon ux density of 800 µmol m − 2 s − 1 , and a chamber temperature of 22°C. Instantaneous water use e ciency (iWUE) (iWUE = P n /T r ).
Water loss analysis. The Col-0, transgenic, and mutant plants , detached rosette leaves were weighed immediately and incubated at room temperature. Losses of fresh weight in rosette leaves were weighted every half an hour until the weight remains constant. The percentage of initial fresh weight represents the water loss rate.
After 20 min of dark adaptation, the photosynthetic activity of leaves before and after the drought was

Statistical analysis
All data were analyzed with SPSS software, and Student's t-test (*P ≤ 0.05; **P ≤ 0.01) was used to test whether there was a signi cant difference between the measured dataset and the control dataset.

Results
Genome-wide identi cation and characterization of PePYLs in poplar We identi ed 12 putative PYLs from the NCBI P. euphratica genome based on 14 AtPYLs amino acid sequences and named PePYL1-PePYL12 according to sequence similarity. The characteristics of PePYLs, including gene name, gene ID, coding DNA sequence (CDS) length, theoretical isoelectric point (pI), molecular weight (MW), and protein length are presented in (Table S1).
To investigate the phylogenetic relationship between PePYLs and AtPYLs, the Neighbor-Joining method was used and the tree was constructed using MEGA 7 (Fig. 1a). The Gene Structure Display Server (GSDS v2.0) further analyzed the exon-intron structure of the PePYLs and AtPYLs gene (Fig. 1b). The conserved domain of the PePYLs was analyzed by DNAMAN software. The results showed that 12 PePYLs proteins can be divided into three subfamilies, among them, PePYL7 to PePYL11 belong to subfamily I; PePYL3 to PePYL6 belong to subfamily II; PePYL1, PePYL2, and PePYL12 belong to subfamily III. The amino acid sequences of 12 PePYLs all contained four highly conserved surface loops CL1-CL4 (Fig. 1c).

The promoter cis-acting elements analysis of PePYLs in poplar
To study the potential functions of PePYLs, the cis-acting elements on the PePYLs promoter sequence were analyzed by PlantCARE online (Table S3). The results showed that the 12 PePYLs promoter sequences mainly include ve hormone response-related elements ( Fig. 2): abscisic acid-responsive element, MeJA-responsive element, salicylic acid-responsive element, auxin-responsive elements and, gibberellin-responsive elements; three abiotic stress-related elements: defense and stress-responsive elements, low-temperature-responsive element and MYB binding site involved in drought-inducibility.
Some PePYLs promoters contain elements related to growth and development, such as, circadian elements, which are involved in circadian control; MBSI elements, which are MYB binding sites involved in the regulation of avonoid biosynthetic genes; HD-Zip 1 elements, which regulate the differentiation of the palisade mesophyll cells. In summary, PePYLs with different cis-acting elements may be involved in a variety of physiological processes.

Subcellular localization of PePYLs
To clarify the localization of PePYLs in cells, 35S:PePYLs-GFP (green uorescent protein) fusion protein was transiently transfected into tobacco leaves. The results showed that: PePYL2, PePYL4, PePYL5, PePYL6, and PePYL9 are located in the cytoplasm and nucleus, the uorescence signals of PePYL10 and PePYL12 were weak, among which PePYL10 is more obvious in the nucleus, while PePYL1, PePYL3, PePYL7, PePYL8, and PePYL11 did not detect the uorescence signal, and its localization in cells cannot be determined (Fig. 3).

The expression patterns of PePYL6 and PePYL9
The expression of PePYL6 and PePYL9 was detected in different tissues (Fig. 4a, d). Previous studies showed that the transcription levels of PePYL6 and PePYL9 were up-regulated under ABA and mannitol treatment. To further determine the expression patterns of PePYL6 and PePYL9, we generated Arabidopsis plants with GUS driven by PePYL6 and PePYL9 promoter. The results showed that ABA and mannitol treatment enhanced β-glucuronidase (GUS) staining (Fig. 4b, e), GUS activity further con rmed GUS staining (Fig. 4c, f), PePYL6 and PePYL9 were induced by ABA and mannitol.
To further investigate the performance of the PYLs gene in response to drought stress, PePYL6 and PePYL9 were stably transformed into Arabidopsis. Col-0, pyl6 and pyl9 were used in this study. Transgenic Arabidopsis was veri ed by PCR and qRT-PCR, the high expression levels plants were selected for experiments ( Fig. 4g-i) (Czechowski et al., 2004).
Overexpression of PePYLs increases ABA sensitivity of seed germination and root growth As a potential ortholog of AtPYLs, the function of PePYLs in the process of seed germination and root growth regulated by ABA has been studied. The inhibition of seed germination by exogenous ABA changed with the concentration of ABA, and the higher ABA concentration, the lower seed germination rate (Fig. 5a). On the 1/2 MS medium, the Col-0, pyl6, and pyl9 plants germinated normally, while OEPePYLs plants showed delayed germination (Fig. 5b). On the medium supplemented with 0.5 µM ABA, the germination rates of Col-0, pyl6, and pyl9 plants were comparable, OEPePYLs plants were lower than those of Col-0 and mutant plants (Fig. 5c). After 24 h of growth on the medium supplemented with 1.0 µM ABA, the germination of OEPePYLs plants was about 15.35%, while the germination of Col-0 plants was 55.95%, and the difference of germination rate between Col-0 and OEPePYLs plants was signi cant with time (Fig. 5d). Therefore, overexpression of PePYL6 and PePYL9 increased the ABA sensitivity during seed germination, even without ABA treatment.
Overexpression of PePYLs improves the drought resistance of transgenic Arabidopsis It has been reported that increasing ABA sensitivity enhances the drought resistance of plants (He et al., 2018). To investigate whether overexpression of PePYL6 and PePYL9 increases ABA sensitivity affects drought resistance of transgenic Arabidopsis. Col-0, OEPePYLs, and mutant plants were transplanted into the soil for 8 d without water. Before the drought stress, the phenotypes of Col-0, OEPePYLs, and mutant plants were not signi cantly different. After the drought stress, Col-0 and mutant plants showed more severe wilt, however, leaves of OEPePYLs plants did not wither, remained green (Fig. 7a). The physiological analysis showed that OEPePYLs plants had a higher net photosynthetic rate (P n ) than the Col-0 and mutant plants, mutant plants had the lowest net photosynthetic rate (Fig. 7b), the transpiration rate (T r ) of OEPePYLs transgenic plants were lower than that of Col-0 and mutant plants (Fig. 7c), which resulted in higher instantaneous water use e ciency (iWUE) (iWUE = P n /T r ) of OEPePYLs transgenic plants under the same conditions (Fig. 7d).
The survive rate, water loss, leaf RWC, and Maximal PSII quantum yield (F v /F m ) of all lines before and after the drought were measured. The survive rate of all lines was 95%-97% under the control condition, however, the survive rate of OEPePYLs plants was 70%-85%, higher than the Col-0 and mutant plants (Fig.   7e). The data of water loss of OEPePYLs plants showed that transgenic plants were lower than that of Col-0 and mutant plants (Fig. 7f), and the RWC and F v /F m values were also higher than other plants ( proline content than that of Col-0 and mutant plants (Fig. 7i). Therefore, overexpression of PePYL6 and PePYL9 enhances drought resistance of transgenic plants.

Overexpression PePYL6 and PePYL9 altered the expression of downstream genes
To study whether overexpression of PePYL6 and PePYL9 enhanced the drought tolerance of transgenic Arabidopsis by regulating the expression of downstream stress-related genes, we compared the expression of ABF2, RAB18, P5CS1, RD29A, and RD29B genes (Cutler et al., 2010) previously reported to be induced by drought and ABA in Col-0 and OEPePYLs plants under water and withholding water conditions. The data showed that the expression of all these genes in the transgenic plants was signi cantly higher than those of the Col-0 plants (Fig. 8), which suggested that PePYLs overexpressed altered the expression patterns of downstream stress-related genes and thus contributed to the enhancement of drought tolerance of transgenic plants.

Discussion
ABA regulates plant growth development and stress response. PYLs, as the ABA signaling pathway core regulatory component, play a major role in ABA perception and signal transduction (Cutler et al., 2010). In this study, a total of 12 PePYLs were identi ed in poplar. Amino acid sequence analysis showed that 12 PePYLs contained the conserved loops of the PYLs family, among them, CL2 and CL3 represent conserved gate and latch domains (Fig. 1c), which are important for ABA signal transduction (Melcher et al., 2009). The structure of exon-intron in PePYLs gene was similar to AtPYLs (Fig. 1b), and phylogenetic analysis further con rmed that the PePYLs could be grouped into three subfamilies (Fig. 1a) (Tischer et al., 2017). Thus, the function of PYLs gene in the same subfamily in Arabidopsis and P. euphratica may be conserved. Given that many AtPYLs' functions have been reported in Arabidopsis, the potential function of PePYLs in poplars can be better investigated.
The cis-acting elements of PePYLs promoters have been analyzed. ABA, MeJA, SA, GA, and auxin ve hormone-responsive elements were found in the 12 PePYLs promoter regions, and most PePYLs promoter regions existed one or more hormone-related elements (Fig. 2), suggesting that PePYLs may be involved in the hormone crosstalk pathway. Previous studies reported that NtPYL4 (Nicotiana tabacum) is involved in jasmonate signaling transduction and regulates metabolic reprogramming in Arabidopsis and tobacco to balance growth and defense processes (Lackman et al., 2011), AtPYL8 directly interact with MYB77 to enhance the MYB77-dependent transcription of auxin-responsive genes (Zhao et al., 2014). Therefore, understanding the existence of cis-acting elements of the PYLs gene promoter will be helpful to further study the functional characteristics of the PYLs gene in poplar or other species.
The expression of PePYL6 and PePYL9 were different in leaves, stems, and roots contribute to understanding the characteristics of these genes in depth. PePYL6 was expressed abundantly in mature leaves, PePYL9 was mainly expressed in leaves and roots (Fig. 4a, d), the tissue expression of the gene may be closely related to its function. It has been reported that the pRD29A::PYL9 transgenic Arabidopsis and rice promote the senescence of old leaves by inducing the expression of senescence-related genes through ABA-responsive element-binding factors (ABFs) and Related to ABA-Insensitive 3/VP1 (RAV1) transcription factors , the pyl8-pyl9 double mutant signi cantly reduced ABA sensitivity to primary root growth and lateral root formation, while the overexpression of PYL8 and PYL9 restore lateral roots by directly interacting with MYB77 and MYB44 under ABA treatment . Therefore, the tissue expression of genes helps to explore genes' function accurately.
We generated PePYL6 and PePYL9 transgenic Arabidopsis to gain insight into the performance of the PYLs gene. Our study showed that overexpression of PePYL6 and PePYL9 increased ABA sensitivity during seed germination and root length (Fig. 5, 6). Previous reports have shown that the PYLs gene plays an important role in the perception of ABA during seed germination and root growth. The sextuple mutant pyr1/pyl1/pyl2/pyl4/pyl5/pyl8 exhibited ABA-insensitive phenotype and reduced seed yield (Gonzalez-Guzman et al., 2012), 35S:PYL4 A194T transgenic plants were more sensitive to ABA during seed germination and seedling growth than 35S:PYL4 plants (Pizzio et al., 2013). Our results were consistent with those reported, further suggested that the PYLs are a positive regulator of ABA signaling.
Since ABA perception and signaling pathways also regulate the response to abiotic stress, so many PYLs gene overexpressed has been reported to enhance plant tolerance to abiotic stress. Such as overexpression of rice OsPYL5 (Kim et al., 2014) and Artemisia annua L AaPYL9 enhanced drought resistance of transgenic plants (Zhang et al., 2013), ectopic expression of OsPYL3 enhanced cold tolerance in Arabidopsis (Lenka et al., 2018). Transgenic Arabidopsis with PePYL6 and PePYL9 promoterdriven GUS expression showed deeper GUS staining after ABA and mannitol treatment, supported that PePYL6 and PePYL9 may be involved in drought stress (Fig. 4b, e). Our data further con rmed that overexpression of PePYL6 and PePYL9 enhanced drought resistance in transgenic Arabidopsis by improving WUE. Under the same conditions, the phenotypes of OEPePYLs, Col-0, and mutant plants were not signi cantly different (Fig. 7a), the CO 2 assimilation rates of OEPePYLs plants were higher than Col-0 and mutant plants (Fig. 7b), the transpiration of OEPePYLs plants were lower than other plants (Fi7 8c), which led to higher WUE of transgenic Arabidopsis (Fig. 7d). It is well known that the WUE of C 3 photosynthetic plants varies with water availability, and WUE increases when water is de cient (Medrano et al., 2002). Therefore, after the drought stress, OEPePYLs plants remained green and turgid, Col-0 and mutant lines wilting, transgenic plants were more drought-resistant (Fig. 7). The same conclusions of improving WUE confer drought stress have been reported in the overexpression of TaPYL4 in wheat (Usman et al., 2020).
Generally, the drought resistance of plants was negatively correlated with the water loss of detached leaves (Gupta et al., 2020), the lower water loss rate in OEPePYLs plants, and the more drought resistance in transgenic plants con rmed this view (Fig. 7f) (Fig. 7h). The RWC and proline content of the OEPePYLs plants were also higher than those of Col-0 plants (Fig. 7g), and the high RWC ensured that the photosystem II could still transport electrons properly and maintain normal CO 2 absorption in the transgenic Arabidopsis under severe water stress, the high proline content has a positive effect on regulating protein and antioxidant enzyme stability, ROS scavenging, and the balance of intracellular redox homeostasis (Fig. 7i) (Szabados and Savouré, 2010). More importantly, the up-regulation expression of downstream stress-related genes by PePYL6 and PePYL9 also ensured that the transgenic plants suffered less damage under drought stress. (Fig. 8), Therefore, OEPePYLs plants were better adaptable to drought stress.
In conclusion, a total of 12 PePYLs homologous genes were identi ed in poplar, and overexpression of PePYL6 and PePYL9 increased ABA sensitivity, improved WUE, and enhanced drought resistance of transgenic Arabidopsis, which laid a foundation for further study on biological functions of other PePYLs genes in poplar.

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