In silico promoter and expression analyses of rice Auxin Binding Protein 57 (ABP57)

Auxin Binding Protein 57 (ABP57) is one of the molecular components involved in rice response to abiotic stress. The ABP57 gene encodes an auxin receptor which functions in activating the plasma membrane H+-ATPase. Biochemical properties of ABP57 have been characterized; however, the function of ABP57, particularly on stress and hormone responses is still limited. This study was conducted to understand the regulation of ABP57 expression under abiotic stress. Thus, in silico identification of cis-acting regulatory elements (CAREs) in the promoter region of ABP57 was performed. Several motifs and transcription factor binding site (TFBS) that are involved in abiotic stress such as ABRE, DRE, AP2/EREBP, WRKY and NAC were identified. Next, expression analysis of ABP57 under drought, salt, auxin (IAA) and abscisic acid (ABA) was conducted by reverse transcription-PCR (RT-PCR) to verify the effect of these treatments on ABP57 transcript level. ABP57 was expressed at different levels in the shoot and root under drought conditions, and its expression was increased under IAA and ABA treatments. Moreover, our results showed that ABP57 expression in the root was more responsive to drought, auxin and ABA treatments compared to its transcript in the shoot. This finding suggests that ABP57 is a drought-responsive gene and possibly regulated by IAA and ABA.


Introduction
As sessile organisms, plants adapt to the environment by maximizing the growth potential to escape stressful conditions or undergo growth reduction for survival (1). Drought promotes abscisic acid (ABA) synthesis, which subsequently initiates signal transduction and affects cellular responses to stress (2). ABA is involved in plant adaptation to abiotic stress, especially in regulating physiological responses such as root elongation and stomatal aperture (3). Additionally, ABA modulates root architecture under water scarcity by regulating root meristem function and root cell length (4). Plant physiological changes during stress conditions are regulated by ABA interactions with other hormones such as auxin, ethylene and jasmonate (3).
Gene expression is diverse among plant stages of development, tissues and response to external stimulus. A stress-responsive gene expression is regulated by specific sequences present at the promoter known as cisacting regulatory elements (CAREs). These regulatory elements served as motifs or binding sites for proteins called transcription factors (TFs), activating or repressing the expression of stress-responsive genes (5)(6)(7). For example, abscisic acid responsive element (ABRE) is involved in transcriptional responses induced by phytohormone ABA (5,8) and this element is recognized by ABA-RESPONSIVE ELEMENT BINDING PROTEIN (AREB) and ABRE BINDING FACTOR (ABF) (9)(10)(11). These TFs play a role in regulating plant response to osmotic stress such as drought and high salinity (9,10).
Auxin is a key phytohormone that regulates various processes in plant growth, development and stress response (12,13). Together with ABA and ethylene, auxins act as communication signals between roots and shoots in rice (14,15). Auxin signaling is facilitated by AUXIN RESPONSE FACTORS (ARFs), which bind to the Aux/IAA motif and activate the transcription of auxininducible genes (16,17). However, not all auxin responses employ this pathway, especially some rapid responses at the plasma membrane (18). This response involved another candidate of the auxin receptor, which is AUXIN BINDING PROTEIN 1 (ABP1). ABP1 localizes mainly at the endoplasmic reticulum (ER), and it is involved in regulating the membrane potential at the plasma membrane (18,19).
Other than ABP1, another putative auxin receptor known as AUXIN BINDING PROTEIN 57 (ABP57) has been isolated from the shoot of rice (20). Apparently, this protein exists only in monocots such as Oryza sativa, Zea mays, Sorghum bicolor and Setaria viridis, but not in the dicot plant (21,22). The binding of ABP57 to indole-3-acetic acid (IAA) leads to the activation of the plasma membrane H + -ATPase by direct interaction (21). Previously, a study showed that the ABP57 overexpression had increased the resistance of transgenic rice to drought stress (23). Moreover, the 35S::ABP57 transgenic Arabidopsis possessed root elongation changes and also restoration of root cells under drought and submergence conditions (24)(25)(26).
These previous studies suggest that ABP57 is involved in abiotic stress and root development. However, more studies are needed to uncover the role of ABP57 in plant development and stress response. To date, no information regarding ABP57 transcriptional regulation is available, particularly on the characterization of its promoter. Nevertheless, the effect of stress and hormone treatments on ABP57 expression has yet to be determined. Hence, the objectives of this study were to characterize the CAREs present at the promoter region of ABP57 via in silico analysis and to determine the effect of osmotic stresses (drought and salt) and hormones (IAA and ABA) on the transcript level of ABP57 in the rice at various time points.
By performing in silico promoter analysis, CAREs including transcription factor binding sites (TFBS) involved in stress response were identified. In addition, our expression analysis showed that ABP57 responds to drought, high salinity, auxin and ABA. It confirms that the presence of several motifs is required to activate ABP57 expression under these treatments. Taken together, our study provided insight into the transcriptional regulation of ABP57 under stress conditions.

Plant materials and growth conditions
The indica rice cv. IR64 and MR219 were obtained from the Malaysian Agricultural Research and Development Institute (MARDI). Drought treatment was conducted in a greenhouse with relative humidity was 85%, and the maximum and minimum temperatures were 30 ºC and 25 ºC respectively. Salt, auxin and ABA treatments were conducted at 25 ºC under 16

Drought treatments
Drought treatment was conducted as per standard procedure (31). The seeds (cv. IR64) were soaked in distilled water for two days to break seed dormancy before they were sowed in a petri dish with wet cotton to allow germination. The 10-daysold rice seedlings were transferred into pots (D 12 cm x H 13 cm) that contain mixed soils (topsoil: organic matter: sand with ratio 3: 2: 1). Five pots were prepared for each control and treatment, and each pot contained three seedlings. All seedlings were watered daily for three weeks before drought treatment begins. Control plants were watered continuously, whereas water withholding was imposed on treatment plants for four weeks. The mature leaf (L5) and root samples from three seedlings were collected weekly (Week 1, 2, 3 and 4). All samples were flash-frozen before total RNA extraction.

Salt, auxin and abscisic acid treatments
Seeds of rice (cv. MR219) were surface-sterilized with sodium hypochlorite for 15 min before they were soaked in distilled water for two days. Seeds were planted on moist cotton and they were grown for 10 days. These 10-days-old rice seedlings were used for salt (NaCl), IAA and ABA treatments. The seedlings were transferred separately into 50 ml centrifuge tubes containing 35 ml of 250 mM NaCl, 100 μM IAA and 100 μM ABA solutions respectively. In each tube, three seedlings were transferred and whole seedlings were soaked for 0, 6 and 24 hrs. Three replicates were prepared in each treatment. The root and leaf (L3) samples were cut and flashfrozen in liquid nitrogen prior to total RNA isolation.

Semi-quantitative expression analysis of ABP57
To determine the expression pattern of ABP57 during osmotic stress and hormone treatments, RT-PCR was carried out. Total RNAs from the leaf and root were extracted using Trizol ® Reagents (Thermo Scientific, USA) following the manufacturer's instruction. The quality and quantity of the total RNA were determined by a spectrophotometer (NanoDrop 1000, USA). DNase treatment was performed on 1 mg of total RNA with RQ1 RNase-Free DNase (Promega Corp, USA) to ensure that RNA samples were free from genomic DNA contaminations. The cDNA was synthesized by reverse transcription on the total RNA using the SuperScript cDNA Synthesis kit (Invitrogen, Life Technologies, USA) following the manufacturer's instruction. The synthesized cDNA was then used as a template for PCR.

Statistical analysis
The mean data was recorded by taking average value and subjected to analysis of variance (ANOVA) by using the SAS programme version 9.4. The ANOVA was used to determine the variation for all parameters such as type of organ (leaf and root), genotypes and treatments. Mean comparisons were performed using Duncan's New Multiple Range Test (DNMRT). Differences among the treatments mean were considered statistically significant when the p-value is less than 0.01 (33). All data represent an average of three replicates and the standard error.

In silico analysis of the ABP57 promoter
Cis-acting regulatory elements (CAREs) of the promoter regions play a significant role in the transcriptional regulation of stress-inducible genes. To obtain an insight into the regulation of ABP57 expression, in silico promoter analysis was conducted. Many CAREs were identified at the promoter of ABP57 (Table 1). Based on PLACE and PlantCARE databases, several motifs that are related to stress responses were found in the ABP57 promoter, which include ABRE (ABA-responsive element), DRE (dehydration responsive element) and LTR (low temperature-responsive element). Besides that, motifs involved in hormone response such as EREBP (ethylene-responsive binding protein) and GARE (gibberellin-responsive element) were also present.
Several transcription factor binding sites (TFBS) available in the promoter region of ABP57 were listed in Table 1. This TFBS belong to TFs such as WRKY, MYB, MYC, bZIP (Basic Leucine Zipper), HD (Homeodomain) and NAC (NAM, ATAF, CUC). Based on PlantPAN database search, binding sites for other TF families were also discovered, such as B3, bHLH (Basic Helix-Loop-Helix), C2H2 Zinc Finger, GATA Zinc Finger, NF-YB (Nuclear Factor-Y Subunit B) and TCP (TEOSINTE BRANCHED 1, CYCLOIDEA, PCF1) ( Table 2). The listed TFs in Table 2 are involved in stress response and this result suggests that they could be the regulator for ABP57 during adverse conditions.

ABP57 expression pattern under drought and salt treatments
Water withheld of the 4-weeks-old seedlings were carried out for four weeks, and expression patterns of ABP57 under prolonged drought stress were determined (Fig. 1). The ABP57 transcript was slightly expressed in the leaf and root under normal conditions (Fig. 1A). During drought treatment, ABP57 was lowly expressed in the leaf during the first and second week, but its expression was increased dramatically at Week 3 and slightly decreased at Week 4 (Fig. 1B). ABP57 transcript was also observed in the untreated root; however, its expression level was significantly increased (p≤0.01) at Week 2 of drought treatment and noticeably decreased at Week 3 and Week 4 (Fig. 1B).
These results indicate that ABP57 is expressed under normal conditions, but prolonged drought treatment has much induced ABP57 expression in both leaf and root at different time points. ABP57 was highly expressed in the leaf at Week 3, whereas the highest expression in the root can be seen at Week 2 of drought treatment.
On the other hand, ABP57 expression in the seedlings treated with high salinity (250 mM NaCl) was examined for 6 and 24 hrs. Our results showed that ABP57 was expressed in the control but significantly reduced (p≤0.01) in the shoot and root after 6 hrs of NaCl treatment (Fig. 2). After 24 hrs, the expression of ABP57 in both of leaf and root was significantly lowered than its expression at 6 hrs. This result suggests that the expression of ABP57 in the seedlings is suppressed by high salt concentration.

Auxin and abscisic acid treatments induced ABP57 expression
ABP57 acts as an auxin receptor and binds to IAA to activate the plasma membrane H + -ATPase by regulating proton (H + ) translocation activity (21). To examine the effect of IAA treatment on ABP57 expression, we analyzed the ABP57 transcript in the shoot and root of rice treated with this hormone. Our results showed that ABP57 was induced after 6 hrs of IAA treatment in both shoot and root (Fig. 3A). The highest expression of ABP57 in the leaf and root can be seen at 6 hrs and 24 hrs of IAA treatment respectively. Although ABP57 expression in the root was gradually increased from 6 to 24 hrs, its transcript in the shoot was reduced at 24 hrs of IAA treatment (Fig. 3A). These results showed that although ABP57 is responsive to exogenous auxin, it is preferably expressed in the root.

Discussion
The biochemical properties of ABP57 have been characterized before (20,21), but the molecular characterization of this gene is still limited. The overexpression of ABP57 in Arabidopsis and rice had increased their tolerance to high salt and drought, respectively (23,25). These studies suggested that ABP57 is required during the osmotic stress response. Therefore, to obtain more insight into the regulation of ABP57 expression during abiotic stress, we analyzed the ABP57 promoter in silico and its transcript changes under drought, salt, IAA and ABA treatments.
Promoter manipulation is one of the tools used in crop improvement, including stress response (34). It is known that different TFs attach to the promoter of stress-inducible genes in response to abiotic or biotic stresses. Indeed, several types of CAREs are essential in regulating the expression of droughtresponsive genes (35). Thus, identifying CAREs at the promoter of a stress-related gene will help in understanding its expression regulation. In this study, several CAREs involved in stress response were identified at the ABP57 promoter region, such as ABRE, DRE, LTR and others. ABRE contains an ACGT core, which is crucial for DREB2A expression in Arabidopsis (8,35,36) and a recognition site for plant bZIP transcription factors during drought conditions (37). The DRE motif consists of a core sequence A/GCCGAC, which is a conserved sequence in the promoter region of DREBs' direct target genes (38). Meanwhile, the LTR element (CCGAC) is necessary for the activation of low temperature-responsive genes during cold stress (39,40). These results suggest that ABP57 might respond to various environmental stimulus such as water deficit and low temperature.
Besides that, many TF binding sites related to abiotic stress response present at the ABP57 promoter. Osmotic stress activates ABA-independent and ABA-dependent signaling pathways, including TFs like DREB, WRKY, bZIP, MYC, MYB and NAC (3,8,41). These TFs attach to the cis-acting elements in the promoter and activate the transcription of secondary responsive genes, thus, increasing plant tolerance to stress (41,42). The binding site of AP2/EREBP and WRKY was mainly found in the upstream region of ABP57. AP2/EREBPs regulate numerous abiotic stress and hormone responses, and one of its family members, DREB, is a major regulator in plant response to cold, drought, heat and salt (42). Meanwhile, WRKY proteins recognize and bind to Wbox, which is also essential in activating stressresponsive genes (43). With these findings, we suggest that ABP57 might be regulated by ABA, and could be the direct or indirect target gene for TFs such as DREB, WRKY and bZIP during stress response. Therefore, further studies need to be carried out to determine the interaction between ABP57 and its putative transcriptional regulator.
Both ABRE and DRE were found at the ABP57 promoter, and these elements regulate the expression of ABA-, osmotic-and drought-responsive genes (36,38). It suggests that ABP57 could be involved in ABA and osmotic stress response. Therefore, to confirm this hypothesis, ABP57 transcript levels under drought, high salinity and ABA treatments were analyzed. Our results showed that ABP57 expression is induced by drought and ABA preferably in the root, but suppressed by salt treatment, indicating that ABA could regulate ABP57 involvement in drought response. Based on Rice eFP Browser, ABP57 expression is not highly induced by drought, salt, nor cold conditions (http://bar.utoronto.ca/eplant_rice/). Although our result contradicted the expression profile in the database, it might happen because different types of samples were used. In addition, the difference maybe because of the time point of the study, as results showed differential expression at different time point. The expression of ABP57 in this study might be affected by the stage of rice development, types of tissues, duration of drought treatment and concentrations of NaCl and ABA.
Although the AuxRE motif was absent at the promoter region of ABP57, we speculated that auxin still could affect ABP57 expression. It is because other than ARF-mediated transcription, auxin response can also be modulated by an additional signaling pathway (44). Under IAA treatment, the ABP57 transcript level was higher in the root compared to the shoot. This result suggests that ABP57 is induced by auxin and preferentially expressed in the root. The high concentration of exogenous IAA may accelerate the binding of ABP57 to auxin and consequently, may elicit an auxin response of rice plasma membrane (21). Furthermore, the ABP57 response to auxin might be distinct from ARFmediated transcription and might involve different regulation of gene transcriptional activation and post-transcription (17,45,46). Besides, plant response to auxin also depending on auxin concentrations and the types of cells and tissues (46).

Conclusion
In this study, we reported the cis-acting regulatory elements available at the promoter region of ABP57, as well as the effect of drought, salt, auxin and ABA treatments on ABP57 expression patterns at various time points. In silico promoter analysis showed several CAREs essential in stress response were identified, revealing the possible transcriptional regulation of ABP57 under abiotic stress and hormone treatments. Additionally, our expression analysis confirmed that ABP57 is a droughtresponsive gene and may be involved in the root response to drought stress, possibly under auxin and ABA regulation. Drought response in plants involved regulation of gene expression and crosstalk among hormone signaling pathways. Therefore, the ABP57 loss-of-function mutant can be studied to further characterize the role of ABP57 in abiotic stress and hormone response signaling pathways. (Grant No. FRGS/1/2016/STG03/UKM/02/3). We would like to thank MARDI Seberang Prai for providing the seeds, and Universiti Kebangsaan Malaysia (UKM) for providing the facilities to carry out this study.

Authors' contributions
FAB performed expression and statistical analyses, and wrote the manuscript. KZX performed expression analysis. ZZ designed the experiment and wrote the manuscript. NLS designed the experiment, performed in silico promoter analysis and wrote the manuscript. All authors read and approved the final manuscript.