Five novel transcription factors as potential regulators of OsNHX1 gene expression in a salt tolerant rice genotype
Five novel transcription factors as potential regulators of OsNHX1 gene expression in a salt tolerant rice genotype
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DOI：
10.1007/s11103-016-0547-7
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发表时间：
2017-01-01
影响因子：
5.1
通讯作者：
Saibo, Nelson J. M.
中科院分区：
文献类型：
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作者：
Almeida, Diego M.;Gregorio, Glenn B.;Saibo, Nelson J. M.
本文报道了在耐盐水稻基因型（Hasawi）中与OsNHX 1启动子结合的5个转录因子的鉴定和特征。虽然NHX 1编码基因在转录水平上受到盐胁迫和干旱胁迫的高度调控，但迄今为止只有一种转录因子（TF）与其启动子结合。为了揭示盐胁迫下NHX 1基因表达的调控因子，我们利用Y1 H系统筛选了盐胁迫下水稻Hasawi的cDNA表达文库。这种方法使我们能够鉴定出属于三个不同TF家族的五个TF：一个TCP（OsPCF 2）、一个CPP（OsCPP 5）和三个与OsNHX 1基因启动子结合的NIN样（OsNIN-like 2、OsNIN-like 3和OsNIN-like 4）。我们还发现，这些转录因子作为转录激活因子（OsPCF 2，OsNIN-like 4）或抑制因子（OsCPP 5，OsNIN-like 2）和其编码基因的差异调节盐和PEG诱导的干旱胁迫在两个水稻基因型，日本晴（盐敏感）和Hasawi（耐盐）。OsNIN-like 3的反式激活活性无法确定。OsNHX 1是植物液泡膜中含量最丰富的K+-Na+/H+逆向转运蛋白，其基因表达受盐、干旱和阿坝的诱导。为了研究水稻耐盐基因型（Hasawi）OsNHX 1在盐胁迫下的转录调控，构建了盐胁迫诱导的cDNA表达文库，并以OsNHX 1启动子为诱饵，利用酵母单杂交系统进行筛选。OsNHX 1启动子与5个转录因子（TF）结合，它们分别属于TCP（OsPCF 2）、CPP（OsCPP 5）和NIN样（OsNIN-like 2、OsNIN-like 3和OsNIN-like 4）3个不同的TF家族。在拟南芥和水稻原生质体中进行的反式激活活性分析表明，OsPCF 2和OsNIN-like 4是OsNHX 1基因表达的激活子，而OsCPP 5和OsNIN-like 2则是OsNHX 1基因表达的抑制子。OsNIN-like 3的反式激活活性有待进一步研究。基因表达研究表明，OsNHX 1的转录水平在盐和PEG诱导的干旱胁迫下在日本晴和Hasawi水稻基因型的地上部和根部都受到高度诱导。然而，OsNHX 1似乎在芽响应干旱中发挥特殊作用。OsNHX 1启动子上的转录因子在胁迫条件下表现出一定的转录调控作用，而OsPCF 2在胁迫条件下的转录诱导时间早于OsNHX 1，表明OsPCF 2可能激活OsNHX 1基因的表达。此外，尽管OsNHX 1在两个水稻基因型中对盐和PEG诱导的干旱胁迫的响应非常相似，但OsPCF 2在盐胁迫下的根中的表达和OsNIN-like 4在PEG诱导的根中的表达在Hasawi中主要上调，表明这些TF可能与该基因型观察到的盐和干旱胁迫耐受性相关。
This manuscript reports the identification and characterization of five transcription factors binding to the promoter of OsNHX1 in a salt stress tolerant rice genotype (Hasawi). Although NHX1 encoding genes are known to be highly regulated at the transcription level by different abiotic stresses, namely salt and drought stress, until now only one transcription factor (TF) binding to its promoter has been reported. In order to unveil the TFs regulating NHX1 gene expression, which is known to be highly induced under salt stress, we have used a Y1H system to screen a salt induced rice cDNA expression library from Hasawi. This approach allowed us to identify five TFs belonging to three distinct TF families: one TCP (OsPCF2), one CPP (OsCPP5) and three NIN-like (OsNIN-like2, OsNIN-like3 and OsNIN-like4) binding to the OsNHX1 gene promoter. We have also shown that these TFs act either as transcriptional activators (OsPCF2, OsNIN-like4) or repressors (OsCPP5, OsNIN-like2) and their encoding genes are differentially regulated by salt and PEG-induced drought stress in two rice genotypes, Nipponbare (salt-sensitive) and Hasawi (salt-tolerant). The transactivation activity of OsNIN-like3 was not possible to determine. Increased soil salinity has a direct impact on the reduction of plant growth and crop yield and it is therefore fundamental to understand the molecular mechanisms underlying gene expression regulation under adverse environmental conditions.OsNHX1 is the most abundant K+-Na+/H+ antiporter localized in the tonoplast and its gene expression is induced by salt, drought and ABA. To investigate how OsNHX1 is transcriptionally regulated in response to salt stress in a salt-tolerant rice genotype (Hasawi), a salt-stress-induced cDNA expression library was constructed and subsequently screened using the yeast one-hybrid system and the OsNHX1 promoter as bait. Five transcription factors (TFs) belonging to three distinct TF families: one TCP (OsPCF2), one CPP (OsCPP5) and three NIN-like (OsNIN-like2, OsNIN-like3 and OsNIN-like4) were identified as binding to OsNHX1 promoter. Transactivation activity assays performed in Arabidopsis and rice protoplasts showed that OsPCF2 and OsNIN-like4 are activators of the OsNHX1 gene expression, while OsCPP5 and OsNIN-like2 act as repressors. The transactivation activity of OsNIN-like3 needs to be further investigated. Gene expression studies showed that OsNHX1 transcript level is highly induced by salt and PEG-induced drought stress in both shoots and roots in both Nipponbare and Hasawi rice genotypes. Nevertheless, OsNHX1 seems to play a particular role in shoots in response to drought. Most of the TFs binding to OsNHX1 promoter showed a modest transcriptional regulation under stress conditions, however, in response to most of the conditions studied, the OsPCF2 was induced earlier than OsNHX1, indicating that OsPCF2 may activate OsNHX1 gene expression. In addition, although the OsNHX1 response to salt and PEG-induced drought stress in either shoots or roots was quite similar in both rice genotypes, the expression of OsPCF2 in roots under salt stress and the OsNIN-like4 in roots subjected to PEG was mainly up-regulated in Hasawi, indicating that these TFs may be associated with the salt and drought stress tolerance observed for this genotype.