title: An iNTT system for the large-scale screening of differentially expressed, nuclear-targeted proteins: cold-treatment-induced nucleoproteins in Rye (Secale cereale L.).
abstract: BACKGROUND
  Nuclear proteins play critical roles in regulating mRNA transcription and processing, DNA replication, and epigenetic genome modification. Therefore, the ability to monitor changes in nuclear proteins is helpful not only to identify important regulatory proteins but also to study the mechanisms of actions of nuclear proteins. However, no effective methods have been developed yet. Rye is strongly resistant to various biotic and abiotic stresses; however, few genes have been functionally characterized to date due to the complexity of its genome and a lack of genomic sequence information.
  
  
  RESULTS
  We developed an integrative Nuclear Transportation Trap (iNTT) system that includes an improved nuclear transportation trap and utilizes the "after suppression subtraction" method. Oligonucleotides encoding a nuclear localization signal (NLS) or a transcription factor, GmAREB, were inserted into pLexAD or pLexAD-NES, respectively, and then transformed into yeast cells (EGY48). We showed that the pLexAD vector expressing a cDNA library in the iNTT system was more efficient for screening than the vector pLexAD-NES, which has previously been used in an NTT system. We used the iNTT system to screen a cDNA library of cold-treated rye. A total of 241 unique genes were identified, including 169 differentially expressed proteins; of these, 106 were of known and 63 were of unknown function. Moreover, 82 genes (49 %) among the 169 differentially expressed genes were predicted to contain an NLS domain. Thirty-three (31 %) of the 106 functionally known proteins have DNA-binding activity. To test the specificity of the nuclear proteins identified using the iNTT screen, four of the proteins differentially expressed in response to temperature stress, ScT1 (a heat shock protein), ScT36 (a MYB-like transcription factor), ScT133 (an ERF-like transcription factor) and ScT196 (a protein of unknown function), were studied in more depth. These proteins were shown to exclusively localize to the nucleus, and their expression levels were increased in response to low-temperature stress. To identify the function of these screened nuclear proteins, ScT1- and ScT36-transgenic Arabidopsis plants were constructed, and ScT1 or ScT36 overexpression was found to enhance tolerance to high-temperature or freezing stresses, respectively.
  
  
  CONCLUSIONS
  The newly developed iNTT system provides an effective method for identifying nuclear-targeted proteins and monitoring induced expression levels. ScT1 and ScT36 might be good candidate genes for improving the stress tolerance of plants by genetic transformation.
authors.affiliation: National Key Facility for Crop Genetic Resources and Genetic Improvement, Key Laboratory of Crop Genetics and Breeding, Ministry of Agriculture/Institute of Crop Sciences, Chinese Academy of Agricultural Sciences, Beijing, 100081, P.R. China. cxytvs@163.com.|Crop Research Institute, Shandong Academy of Agricultural Sciences/National Engineering Laboratory for Wheat and Maize/Key Laboratory of Wheat Biology and Genetic Improvement in North Yellow and Huai River Valley, Ministry of Agriculture, 250100, Jinan, China. cxytvs@163.com.
authors.forename: Xinyou
authors.initials: X
authors.lastname: Cao
authors.affiliation: Crop Research Institute, Shandong Academy of Agricultural Sciences/National Engineering Laboratory for Wheat and Maize/Key Laboratory of Wheat Biology and Genetic Improvement in North Yellow and Huai River Valley, Ministry of Agriculture, 250100, Jinan, China. chxytvs@163.com.|College of Agronomy, Northwest A&F University, Yangling, 712100, P.R. China. chxytvs@163.com.
authors.forename: Xueyan
authors.initials: X
authors.lastname: Chen
authors.affiliation: National Key Facility for Crop Genetic Resources and Genetic Improvement, Key Laboratory of Crop Genetics and Breeding, Ministry of Agriculture/Institute of Crop Sciences, Chinese Academy of Agricultural Sciences, Beijing, 100081, P.R. China. 337108138@qq.com.
authors.forename: Yangna
authors.initials: Y
authors.lastname: Liu
authors.affiliation: National Key Facility for Crop Genetic Resources and Genetic Improvement, Key Laboratory of Crop Genetics and Breeding, Ministry of Agriculture/Institute of Crop Sciences, Chinese Academy of Agricultural Sciences, Beijing, 100081, P.R. China. xuzhaoshi@caas.cn.
authors.forename: Zhaoshi
authors.initials: Z
authors.lastname: Xu
authors.affiliation: National Key Facility for Crop Genetic Resources and Genetic Improvement, Key Laboratory of Crop Genetics and Breeding, Ministry of Agriculture/Institute of Crop Sciences, Chinese Academy of Agricultural Sciences, Beijing, 100081, P.R. China. liliancheng@caas.cn.
authors.forename: Liancheng
authors.initials: L
authors.lastname: Li
authors.affiliation: National Key Facility for Crop Genetic Resources and Genetic Improvement, Key Laboratory of Crop Genetics and Breeding, Ministry of Agriculture/Institute of Crop Sciences, Chinese Academy of Agricultural Sciences, Beijing, 100081, P.R. China. zhouyongbin@caas.cn.
authors.forename: Yongbin
authors.initials: Y
authors.lastname: Zhou
authors.affiliation: Crop Research Institute, Shandong Academy of Agricultural Sciences/National Engineering Laboratory for Wheat and Maize/Key Laboratory of Wheat Biology and Genetic Improvement in North Yellow and Huai River Valley, Ministry of Agriculture, 250100, Jinan, China. ljjsaas@163.com.
authors.forename: Jianjun
authors.initials: J
authors.lastname: Liu
authors.affiliation: Crop Research Institute, Shandong Academy of Agricultural Sciences/National Engineering Laboratory for Wheat and Maize/Key Laboratory of Wheat Biology and Genetic Improvement in North Yellow and Huai River Valley, Ministry of Agriculture, 250100, Jinan, China. zhaozhendong925@163.com.
authors.forename: Zhendong
authors.initials: Z
authors.lastname: Zhao
authors.affiliation: National Key Facility for Crop Genetic Resources and Genetic Improvement, Key Laboratory of Crop Genetics and Breeding, Ministry of Agriculture/Institute of Crop Sciences, Chinese Academy of Agricultural Sciences, Beijing, 100081, P.R. China. chenming02@caas.cn.
authors.forename: Ming
authors.initials: M
authors.lastname: Chen
authors.affiliation: National Key Facility for Crop Genetic Resources and Genetic Improvement, Key Laboratory of Crop Genetics and Breeding, Ministry of Agriculture/Institute of Crop Sciences, Chinese Academy of Agricultural Sciences, Beijing, 100081, P.R. China. mayouzhi@caas.cn.
authors.forename: Youzhi
authors.initials: Y
authors.lastname: Ma
mesh_terms: D021581:Active Transport, Cell Nucleus; D017360:Arabidopsis / Q000235:genetics; D002467:Cell Nucleus / Q000378:metabolism; D003080:Cold Temperature; D005615:Freezing; D018506:Gene Expression Regulation, Plant; D015723:Gene Library; D009698:Nucleoproteins / Q000235:genetics / Q000378:metabolism*; D010940:Plant Proteins / Q000235:genetics / Q000378:metabolism*; D030821:Plants, Genetically Modified; D012434:Secale / Q000235:genetics* / Q000378:metabolism; D013312:Stress, Physiological; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism
chemical_list: D009698:Nucleoproteins; D010940:Plant Proteins; D014157:Transcription Factors