Plant Mol Biol Rep(2011)29:525-532 531 ■BrICE1口BrCBF BrCOR14 120 elements that had been found to be involved in ABA- 100 ABA regulated gene expression in a number of genes.The other 80 intriguing hypothesis thus raised is that BrCOR/4 was not o involved in the ABA-response pathway. 90 Further studies in ABA,salt and drought stresses also carried out (Yamaguchi-Shinozaki and Shinozaki 1994). 30 Results showed that with salt and drought treatments.the 120 three genes all showed ascending expression,while the NaCl highest expression of the three genes all appeared at 4 h under drought treatment,not coming from ICE,CBF,and % COR in that order.which might likely indicate that the pathway of salt and drought stresses did not completely 40 resembled that of the cold pathway.Only when treated with 20 ABA within 0.5 h did Br/CE/show higher expression than the basal while the other two genes showed no significant 120 difference at different time points.These results resembled 100 Mannitol those of COR genes involved in the expression of cold-, 80 salt-,and drought-regulated genes through an ABA- independent pathway in Arabidopsis at the same time % verifying the hypothesis that BrCOR/4 is not involved in % the ABA-response pathway.While BrICEl was slightly up- 0 Oh 0.5h 2h 4h 8h 24h regulated by drought stress which is different from 4d Time Arabidopsis that ICE was not induced by drought stress Fig.6 Real-time fluorescence quantitative PCR analysis of Br/CE/, (Yang et al.2005).The reason need to be determined BrCBF,and BrCOR/4 genes in non-heading Chinese cabbage in further. response to ABA,salt (NaCl)and drought(Mannitol)stresses Through the above-mentioned bioinformatics analysis and cold acclimation assay,it was found that BrICEl,BrCBF,and genes or proteins,for instance Br/CEl,as enhancers and BrCOR/4 have many common characteristics with Arabi- some downstream genes or their products,specifically dopsis ICEl,CBF3,and CORI5B genes.There is also a BrCOR14,as suppressors.BrICEl increased quickly and BrLOS2 gene being cloned from non-heading Chinese then decreased immediately,while BrCBF and BrCOR/4 cabbage.Therefore,it is highly presumed that non-heading can maintain the higher expression for a relatively long time, Chinese cabbage may have similar cold acclimation process which also verified the hypothesis that BrICEl protein is strongly resembling that of Arabidopsis (Jiang et al.2007c; unstable whereas BrCBF and BrCOR/4 are stable. Yang et al.2005).Results also suggest that BrCBF and In addition,BrCBF contains a dehydration-responsive BrCOR14 are involved in the expression of cold-,salt-and element (Liu et al.1998)which suggests that BrCBF might drought-regulated genes through an ABA-independent path- be responsible not only to cold stress but also to way.So these genes might be the potential breeding dehydration.While a potential ABA-responsive element resources through transformation to improve the cold,salt, (ABRE).CACGTG (Guiltinan et al.1990:Williams et al. as well as drought tolerance.It is important to bear in mind, 1992)is not found in BrCOR/4.ABREs are cis-acting however,that constitutive high-level overexpression of the CBF genes can result in undesirable agronomic traits.In Cold Arabidopsis,high-level CBF overexpression can cause a BrICE1 bHLH poly(A) "stunted"growth phenotype,a decrease in seed yield,and a delay in flowering (Liu et al.1998;Gilmour et al.2000), Binding whether strategies such as using stress-inducible promoters BrCBF- CACCTG AP2 poly(A) to drive BrCBF expression can be developed to attain the Potential MAC domain Binding potential positive effects of CBF regulon engineering without BrCOR14 CGCCGTC poly(A) incurring undesirable negative traits remains to be deter- mined(Kasuga et al.1999).Recently,studies on cold stress Potential GCC box signaling and tolerance also revealed that post-transcriptional Increased Freezing Tolerance regulation at pre-mRNA processing and export from nucleus Fig.7 The anticipated non-heading Chinese cabbage CBF cold- plays a role in cold acclimation.Cold stress-regulated response pathway miRNAs have been identified in Arabidopsis and rice 鱼Springergenes or proteins, for instance BrICE1, as enhancers and some downstream genes or their products, specifically BrCOR14, as suppressors. BrICE1 increased quickly and then decreased immediately, while BrCBF and BrCOR14 can maintain the higher expression for a relatively long time, which also verified the hypothesis that BrICE1 protein is unstable whereas BrCBF and BrCOR14 are stable. In addition, BrCBF contains a dehydration-responsive element (Liu et al. 1998) which suggests that BrCBF might be responsible not only to cold stress but also to dehydration. While a potential ABA-responsive element (ABRE), CACGTG (Guiltinan et al. 1990; Williams et al. 1992) is not found in BrCOR14. ABREs are cis-acting elements that had been found to be involved in ABA￾regulated gene expression in a number of genes. The other intriguing hypothesis thus raised is that BrCOR14 was not involved in the ABA-response pathway. Further studies in ABA, salt and drought stresses also carried out (Yamaguchi-Shinozaki and Shinozaki 1994). Results showed that with salt and drought treatments, the three genes all showed ascending expression, while the highest expression of the three genes all appeared at 4 h under drought treatment, not coming from ICE, CBF, and COR in that order, which might likely indicate that the pathway of salt and drought stresses did not completely resembled that of the cold pathway. Only when treated with ABA within 0.5 h did BrICE1 show higher expression than the basal while the other two genes showed no significant difference at different time points. These results resembled those of COR genes involved in the expression of cold-, salt-, and drought-regulated genes through an ABA￾independent pathway in Arabidopsis at the same time verifying the hypothesis that BrCOR14 is not involved in the ABA-response pathway. While BrICE1 was slightly up￾regulated by drought stress which is different from Arabidopsis that ICE was not induced by drought stress (Yang et al. 2005). The reason need to be determined further. Through the above-mentioned bioinformatics analysis and cold acclimation assay, it was found that BrICE1, BrCBF, and BrCOR14 have many common characteristics with Arabi￾dopsis ICE1, CBF3, and COR15B genes. There is also a BrLOS2 gene being cloned from non-heading Chinese cabbage. Therefore, it is highly presumed that non-heading Chinese cabbage may have similar cold acclimation process strongly resembling that of Arabidopsis (Jiang et al. 2007c; Yang et al. 2005). Results also suggest that BrCBF and BrCOR14 are involved in the expression of cold-, salt- and drought-regulated genes through an ABA-independent path￾way. So these genes might be the potential breeding resources through transformation to improve the cold, salt, as well as drought tolerance. It is important to bear in mind, however, that constitutive high-level overexpression of the CBF genes can result in undesirable agronomic traits. In Arabidopsis, high-level CBF overexpression can cause a “stunted” growth phenotype, a decrease in seed yield, and a delay in flowering (Liu et al. 1998; Gilmour et al. 2000), whether strategies such as using stress-inducible promoters to drive BrCBF expression can be developed to attain the potential positive effects of CBF regulon engineering without incurring undesirable negative traits remains to be deter￾mined (Kasuga et al. 1999). Recently, studies on cold stress signaling and tolerance also revealed that post-transcriptional regulation at pre-mRNA processing and export from nucleus plays a role in cold acclimation. Cold stress-regulated miRNAs have been identified in Arabidopsis and rice Fig. 7 The anticipated non-heading Chinese cabbage CBF cold￾response pathway Fig. 6 Real-time fluorescence quantitative PCR analysis of BrICE1, BrCBF, and BrCOR14 genes in non-heading Chinese cabbage in response to ABA, salt (NaCl) and drought (Mannitol) stresses Plant Mol Biol Rep (2011) 29:525–532 531