Optimization of TaDREB3 gene expression in transgenic barley using cold‐inducible promoters

Abstract

SummaryConstitutive over-expression of the TaDREB3 gene in barley improved frost tolerance of transgenic plants at the vegetative stage of plant development, but leads to stunted phenotypes and 3-to 6-week delays in flowering compared to control plants. In this work, two cold-inducible promoters with contrasting properties, the WRKY71 gene promoter from rice and the Cor39 gene promoter from durum wheat, were applied to optimize expression of TaDREB3. The aim of the work was to increase plant frost tolerance and to decrease or prevent negative developmental phenotypes observed during constitutive expression of TaDREB3. The OsWRKY71 and TdCor39 promoters had low-to-moderate basal activity and were activated by cold treatment in leaves, stems and developing spikes of transgenic barley and rice. Expression of the TaDREB3 gene, driven by either of the tested promoters, led to a significant improvement in frost tolerance. The presence of the functional TaDREB3 protein in transgenic plants was confirmed by the detection of strong up-regulation of cold-responsive target genes. The OsWRKY71 promoter -driven TaDREB3 provides stronger activation of the same target genes than the TdCor39 promoter. Analysis of the development of transgenic plants in the absence of stress revealed small or no differences in plant characteristics and grain yield compared with wild-type plants. The WRKY71-TaDREB3 promoter-transgene combination appears to be a promising tool for the enhancement of cold and frost tolerance in crop plants but field evaluation will be needed to confirm that negative development phenotypes have been controlled.

Modulating AtDREB1C Expression Improves Drought Tolerance in Salvia miltiorrhiza

“…However, constitutively expressing DREB genes often has various pleiotropic effects on the growth and development of plants, such as growth retardation, dwarfism, and delayed flowering (Sharabi-Schwager et al, 2010; Shavrukov et al, 2015; Yamasaki and Randall, 2016; Wu et al, 2017). Several attempts have been made to overcome the problem of severe growth retardation by reducing the duration of DREB overexpression through the use of stress-inducible promoters (Morran et al, 2011; Datta et al, 2012; Kovalchuk et al, 2013; Ravikumar et al, 2014). …”

“…DREB TFs, some of the earliest TFs found to be involved in abiotic stress, comprise 56 members divided into six groups (A-1 to A-6) (Hichri et al, 2015). All DREB TFs, which contain a single AP2 domain, bind to the consensus sequence A/GCCGAC in the C repeat or dehydration response element (DRE) in the promoters of genes that are induced under low temperatures and/or water stress (Kovalchuk et al, 2013; Licausi et al, 2013; Dou et al, 2015; Park et al, 2015; Jia et al, 2016). Overexpressing DREB TFs in transgenic plants markedly improves plant survival under both drought and low-temperature conditions (Shan et al, 2007; Byun et al, 2015; Kidokoro et al, 2015; Shavrukov et al, 2015).…”

“…Misexpression of the targeted transgene(s) in the absence of stress may negatively affect crop performance (Kovalchuk et al, 2013; Shavrukov et al, 2015). For instance, ectopic expression of CBF/DREB genes via fusion to the constitutive 35S CaMV promoter in Arabidopsis (Yamasaki and Randall, 2016), tobacco (Zhou et al, 2014), potato (Storani et al, 2015), and tomato (Li et al, 2012) improved plant performance under abiotic stress but resulted in developmental abnormalities that, under non-stress environments, penalized plant performance.…”

“…Therefore, precise regulation of the timing and expression level of the transgene is an important consideration when designing constructs to be introduced into a plant genome. For example, when stress-inducible promoters were used in transgenic cassettes carrying transgenes thought to improve abiotic stress tolerance in rice (Ravikumar et al, 2014), barley (Kovalchuk et al, 2013), wheat (Morran et al, 2011), and tomato (Rai et al, 2013), phenotypic development was normal in the transgenic lines under non-stress conditions, yet plant performance was still improved under stress conditions.…”

P gas , a Low-pH-Induced Promoter, as a Tool for Dynamic Control of Gene Expression for Metabolic Engineering of Aspergillus niger

“…Transcription levels at pH 6.0, 5.0, and 4.0 increased slowly, but the expression level at pH 3.0 increased 15-fold above that at pH 7.0. In addition, the expression level at pH 2.0 increased sharply by 122-fold, indicating that Pgas was a low-pH-induced promoter and that a new transduction pathway may exist (25). The relationship between the relative expression level and pH was calculated by linear regression analysis using the following equation: log(E) ϭ [(6.042/pH) Ϫ 0.9059], where E is the relative expression level compared with expression at pH 7.0; the correlation coefficient factor (R 2 ) is 0.9962.…”