Evaluation of Genetic Diversity of Cis-acting Elements of Abscisic Acid Responsive Element Binding Protein ( ABRE-BP ) in Selected Sri Lankan Rice varieties

Salinity is a major abiotic stress that affects rice cultivation. Osmotic stress caused by salinity activates tolerance mechanisms in rice. The Abscisic Acid Responsive Element Binding Protein (ABRE-BP), transcribes for a bZIP family transcription factor that binds to the cis-regulatory element Abscisic Acid Responsive Element (ABRE) at the promoter of downstream salinity responsive genes and regulates their expression. Hence, the study was focused on analyzing the nucleotide diversity of a region in ABRE-BP promoter. Analysis of the ABRE-BP sequences of 47 Sri Lankan rice varieties and two reference varieties retrieved from a public database revealed many insertions and deletions (INDELs) and single nucleotide polymorphisms (SNPs) in the promoter. Eleven, abiotic stress related cis-elements were identified, but none of the INDELs spanned over them. However, SNPs either deleted existing cis-elements or created new cis-elements. The salinity stress responsive elements, MYBCORE and GT1GMSCAM4 were detected in few varieties, and were not associated with salinity tolerance based on current available salinity ratings. The presence of SNPs in cis-elements clustered them to nine groups at 68% similarity. The DNA polymorphisms on stress responsive cis-acting elements did not show a strong association with the known salinity ratings of many varieties. Nevertheless, the presence of INDELs affects the relative distance between elements, and thus may alter the expression of the ABRE-BP that regulates downstream stress responsive genes.


INTRODUCTION
Rice (Oryza sativa L.) is cultivated in regions with diverse environmental conditions.In some parts of the world, rice is grown under various abiotic stress conditions such as: drought, submergence, frost prone conditions, saline, acidic and toxic soils (Sankar et al., 2011).These conditions directly affect the plant growth and its productivity.Out of the persisting abiotic stress factors, drought and salinity are the major contributors to crop failure in rice cultivations (Bartels and Sunkar, 2005).Due to the climatic changes, rising of sea levels, geo-chemical weathering of rocks and improper drainage in cultivated lands, rapid expansion of salinity affected land is expected (El-Swaify, 2000).It is hard to treat and amend a salinity affected soil and thus, only the crops that are tolerant to such adverse conditions can be cultivated (Pitman and Lauchli, 2002).Due to the fact that 20% of irrigated lands under cultivation are affected by soil salinity, development of rice cultivars that are tolerant to salinity is a priority in rice breeding programs all over the world (Pitman and Lauchli, 2002).
When a plant is under stress, the stress signal gets transmitted to responsive sites via many pathways including regulation of ion homeostasis via salt overly sensitive (SOS) regulatory pathway for ions and hormones induce signal transduction pathways such as abscisic acid (ABA)-dependent pathway (Sairam and Tyagi, 2004).Increasing soil salinity changes the osmotic potential of the plant cell and creates a water deficit condition, a potential stressful condition triggering the activation of salinity tolerance response gene cascades (Shinozaki and Yamaguchi-Shinozaki, 1997).The activation of the synthesis of the plant stress hormone ABA, in turn activates a cascade of stress responsive genes to deliver plant tolerance/resistance responses (Nakashima et al., 2009).The ABA inducible stress responsive genes such as, salt, GST and rab 16-A delivering salinity tolerance have been reported in rice (Mundy and Chua, 1988;Garcia et al., 1998;Taji et al., 2002;Rabbani et al., 2003;Walia et al., 2005;Jain et al., 2010).
The abscisic acid responsive element (ABRE) is a commonly found cis-element in stress responsive genes (Uno et al., 2000).A bZIP-family transcription factor ABRE-BP binds to the ABRE cis-element(s) and induces the expression of ABA-inducible salinity responsive genes in rice (Nakashima et al., 2009;Todaka et al., 2015).Thus, the expression of the salinity responsive genes in ABRE-BP regulon is dependent upon the level of expression of the ABRE-BP.The cis-elements found in the promoter of ABRE-BP and the distance between those elements are crucial in mediating its expression.To our knowledge, the ABRE-BP promoter region of different rice varieties has not been characterized.Identifying and comparing the cis-elements, their relative distance and DNA polymorphisms may shed insight on the contribution of these elements for the salinity responses in these varieties.The salinity responses of most Sri Lankan varieties are not well characterized and of those that have been characterized the actual salinity ratings reported in previous work have been at times contradictory (de Costa et al., 2012;Pradheeban et al., 2014).However, varieties such as Pokkali and Kuruluthudu are well known for their salinity tolerance.
In the current in silico analysis, sequences covering the upstream region of ABRE-BP of 47 Sri Lankan rice varieties and two reference varieties (cultivar 93-11(indica) and Nipponbare (japonica)) were used to detect DNA polymorphisms of the ABRE-BP promoter region, specially at cis-elements related to abiotic stresses and their possible contribution to salinity responses.

Sequence analysis
The retrieved ABRE-BP promoter sequences of 47 Sri Lankan varieties and two reference varieties, were aligned using ClustalW (cost matrix: IUB, gap open cost: 15 and gap extend cost: 6.66) in Geneious v7.1.3(Biomatters Ltd., New Zealand) with manual editing.The nucleotide polymorphisms of the promoter region were analyzed using DnaSP v5.10.1 (University of Bacelona, Spain).The cis-elements present in the promoter of the ABRE-BP were identified and was annotated using the PLACE database (https://sogo.dna.affrc.go.jp;Higo et al., 1999).A cluster analysis (complete linkage method and euclidean distance) was carried out considering the single nucleotide polymorphisms (SNPs) present within the identified cis-elements, using Minitab v15 (Minitab Inc., USA).

RESULTS
The  The in silico analysis revealed presence of 57 SNP sites among the 49 varieties (data not shown).The frequency of occurrence of all SNP sites in the considered region was 7.4%.Out of that, 6.6% (51 SNPs) were parsimony informative sites and 0.8% (six SNPs) were singleton variables.Out of the 51 parsimony informative sites, 42 sites lead to transition mutations and nine to transversion mutations.From the six singletons, five lead to transition mutations and one to a transversion mutation.
In the 800 bp region of the ABRE-BP promoter considered in the current study, the detected SNPs created and deleted cis-elements.Considering all 49 varieties, a total of 14 abiotic stresses related cis-elements were identified (Table 2; Fig. 2).Three of the cis-elements ACGTATERD1 (involved in expression of early response to dehydration), DPBFCOREDCDC3 (bZIP transcription factor binding sequence present in ABA inducible genes) and MYCCONSENSUSAT (present in dehydration responsive genes) were conserved across all 49 varieties.Based on the remaining 11 cis-acting elements related to abiotic stress responses that showed SNP variations, the 49 varieties were clustered in to nine groups at a similarity percentage cut -off of 68 % (Fig. 3).Rice Varieties

Similarity %
The tolerant variety Pokkali carried nine abiotic stress response related cis-elements and did not carry the stress responsive elements GT1GMSCAM4 (two copies found in other varieties) and the element LTRE1HVBLT49D found in other varieties.Further, five of the identified abiotic-stress related elements in Pokkali (ACGTATERD1, DRE2COREZMRAB17, DRECRTCOREAT, LTRECOREATCOR15 and MYBCORE) were deleted in some other varieties.
Based on the current salinity ratings surveyed through available literature, the varieties Pokkali, Kuruluthuda, Kahata wee, and Kotteyaran has been reported as tolerant (de Costa et al., 2012;Pradheeban et al., 2014) and Nipponbare and Hondarawalu as susceptible (de Costa et al., 2012).The rating of Pachchaperumal remained ambiguous as de Costa et al., (2012) reported it as susceptible and Pradheeban et al., (2014) as tolerent at seedling stage.To our knowledge the remaining varieties were not evaluated for salinity responses.

DISCUSSION
Osmotic stress responses in plants controlled by different regulons involves a cascade of genes that get up-regulated and down-regulated based on environmental cues (Nakashima et al., 2009).The stress responses can get delivered via an ABA dependent or independent signal transduction pathway.These pathways involve specific cis-elements such as: dehydration responsive element (DRE), C-repeat (CRT), and ABRE that are responsible for regulating the expression of osmotic stress responsive genes.The DRE and CRT are known to be involved in both cold and drought stresses, while ABRE is mainly engaged with salinity and drought stress (Shinozaki and Yamaguchi-Shinozaki, 2000).Phytohormone ABA produced under osmotic stress is recognized by ABA receptors in the cell and the signaling components induce the ABA-dependent gene ABRE-BP (Sairam and Tyagi, 2004).The ABRE-BP codes for a bZIP family transcription factor which binds to the ABRE elements in the promoter of stress responsive genes up-regulating their expression (Chaves et al., 2003;Tuteja, 2007;Nakashima et al., 2014).The ABRE-BP acts as an early responsive gene in ABA dependent stress responsive regulon (Sairam and Tyagi, 2004) and thus, the ABRE-BP acts as a master regulator in ABA dependent stress response.The ciselements present, their relative distance and DNA polymorphisms involved in the promoter region could alter the expression of a gene (Dubouzet et al., 2003).Thus, such variations in the ABRE-BP could alter its expression and affect the expression of the down-stream genes that are under its regulation.Hence, in the current study the diversity of cis-elements of ABRE-BP were evaluated.
The analysis of the ABRE-BP promoter sequences of 49 rice varieties retrieved from public databases showed several INDELs and SNPs.The sequence over the major INDEL (INDEL1-25 bp) was found to be absent only in two varieties, PuttuNellu and cultivar 93-11 (indica reference).However, the salinity ratings of PuttuNellu and cultivar 93-11 are not known.Among the varieties that have a sequence over the major INDEL, varieties such as Pokkali, Kuruluthudu, Kahata Wee and Kotteyaran were already reported as salinity tolerant and Hondarawalu as a susceptible variety (de Costa et al., 2012).Thus, the INDEL1 did not show a direct association to the currently reported salinity tolerance ratings.Similarly, none of the other INDELs and SNPs identified in the current study showed strong associations with available salinity ratings.Salinity is not under the control of a single gene; rather, it is controlled by several quantitative trait loci (QTL; Saltol (Mohammadi-Nejad et al., 2008;Thomson et al., 2010) and qDW1.1 in chromosome 1, qDW2.1 and qDW2.2 in chromosome 2, qDW6.1 in chromosome 6 (Bimpong et al., 2014), qSNC3 in chromosome 3, qSNC9 in chromosome 9 and qSNC11 in chromosome 11 (Wang et al., 2012) and rab-16A (Mundy et al., 1990)).Thus, the overall salinity response is a cumulative effect of the responses coming from many QTL/genes.However, only the genes carrying the ABRE cis-elements are under the regulation of ABRE-BP.Only the promoter region of the gene rab16A has been characterized to carry ABRE elements (Mundy et al., 1990).It is not known if the other QTL/genes responsible for salinity tolerance are carrying ABRE cis-elements or not.
Among various regulatory elements found in a promoter region, cis-elements play a key role in regulating gene transcription.The cis-elements get regulated by the status of various cellular responses, including signal molecules related to abiotic stresses (Chaves et al., 2003).The six INDEL sites detected in the current study did not fall over any cis-elements that were previously known to be associated with abiotic stress tolerance.However, an INDEL can cause a change in the relative positions of the adjacent cis-acting elements and thus could have an effect on the overall expression rate of the gene.Hence, it is recommended that an expression analysis be carried out for ABRE-BP of different varieties to see if these INDELs have an effect on the relative expression of the gene.Out of the identified 11 abiotic stress responsive cis-elements, the elements MYBCORE (CNGGTTA) and GT1GMSCAM4 (GAAAAA) are known to be related to salinity stress responses.These cis-elements are present in the C3HC4-type RING finger gene family, which plays a key role in growth and development, and abiotic stress responses (Ma et al., 2009).The ciselement MYBCORE is also known to be involved in stress response regulation in rice plants under dehydration, salinity, drought and low temperature stress (Ma et al., 2009).Further, the cis-element GT1GMSCAM4 is also known to be related to salinity stress and biotic stress tolerance (Park et al., 2004;Ma et al., 2009;Trivedi et al., 2013).In stress responsive genes a GT-1 like transcription factor binds to the cis-element GT1GMSCAM4 and regulates responses under salinity stress (Park et al., 2004).Previous studies indicates the presence of GT1GMSCAM4 in most of the highly expressing genes in rice sperm cells (Sharma et al., 2011).The GT1GMSCAM4 element is found in known susceptible varieties such as Nipponbare and Honderawala and several other varieties with unknown salinity ratings, but not in known salinity tolerant varieties such as Pokkali (IRGC 8948-1), Kuruluthudu (IRGC 36304-1), Kahata Wee (IRGC 12004-1), Kotteyaran (IRGC 47383-1) and in several varieties with unknown salinity responses.Similarly a direct association could not be established with respect to the cis-element MYBCORE, due to lack of information related to salinity stress responses.
Based on the abiotic stress related cis-elements carrying SNP sites (11 sites; Fig. 2), the studied rice varieties were clustered into nine clusters (Fig. 3).The salinity tolerant variety Pokkali was clustered into haplotype 1 with tolerant varieties Kuruluthudu, Kahata Wee, Kotteyaran and moderately salinity tolerant variety Samba (de Costa et al., 2012;Pradheeban et al., 2014).However, within the same haplotype were the salinity susceptible variety Honderawala (de Costa et al., 2012) , Pachchaperumal with an ambiguous salinity rank (de Costa et al., 2012;Pradheeban et al., 2014) and 20 other varieties of which the tolerance level is unknown.Thus, the SNPs in these elements are not strongly associated with salinity responses of these varieties.
Even though this in silico study revealed the nucleotide diversity of the cis-elements present in ABRE-BP promoter region, the association of the detected polymorphisms to salinity tolerance ratings was not possible as the salinity responses of many of the tested varieties were not available.Therefore, it is recommended that a systematic phenotypic study be carried out to clearly identify the salinity rank of the used accessions at different life stages.The current study is an initial step to identify the nucleotide diversity of the cis-elements present in ABRE-BP among a panel of Sri Lankan rice varieties, and based on the already available salinity ratings preliminary inferences were made on the associations of the DNA polymorphisms detected in the promoter region of ABRE-BP with emphasis on salinity stress response related cis-elements.

CONCLUSION
The nucleotide diversity analysis of the ABRE-BP promoter region revealed DNA polymorphisms among the studied rice varieties, and some of these polymorphisms were found within the cis-elements.A total of 11cis-elements related to abiotic stress responses were detected and the identified SNPs deleted existed cis-elements or generated new ciselements.No INDELs fell on the cis-elements.The groups created based on a cluster analysis of these SNPs indicated that the SNPs in cis-elements of the ABRE-BP may not be a strong regulator of salinity tolerance.However, the INDELs changed the relative distance between cis-elements and their position from the TSS, imposing possible effects on the expression of the ABRE-BP that regulate stress responsive genes enriched with ABRE element(s).

Fig. 1 .
Fig. 1.Illustration of the size and the positions of six insertions/deletions (INDEL) starting from the transcription start site (TSS) in the promoter region of abscisic acid responsive element binding protein .

Fig. 2 .Fig. 3 .
Fig. 2. Illustration of haplotypes based on abiotic stress related cis-elements and theirsingle nucleotide polymorphisms in the promoter region of the abscisic acid responsive element binding protein among the selected Sri Lankan rice varieties

Table 2 . Sequences of abiotic stress responsive cis-acting elements present in an 800 bp upstream region of the abscisic acid responsive element binding protein (Source: PLACE database)
Among the analyzed sequences there were two entries from the variety Hondarawalu (accessions IRGC 67631 and IRGC 47372).Accession level differences involving INDELs and SNPs were detected in the promoter region of Hondarawalu including, four INDELs and 14 SNPs.The variations detected among the Hondarawalu accessions indicated heterogeneity of the germplasm.