Abstract
Dehydration-Responsive Element Binding proteins (DREB)/C-repeat (CRT) Binding Factors (CBF) have been identified as transcriptional activators during plant responses to cold stress. The objective of this study was to determine the physiological roles of a CBF gene isolated from a cold-tolerant perennial grass species, Kentucky bluegrass (Poa pratensis L.), which designated as PpCBF3, in regulating plant tolerance to freezing stress. Transient transformation of Arabidopsis thaliana mesophyll protoplast with PpCBF3-eGFP fused protein showed that PpCBF3 was localized to the nucleus. RT-PCR analysis showed that PpCBF3 was specifically induced by cold stress (4°C) but not by drought stress [induced by 20% polyethylene glycol 6000 solution (PEG-6000)] or salt stress (150 mM NaCl). Transgenic Arabidopsis overexpressing PpCBF3 showed significant improvement in freezing (-20°C) tolerance demonstrated by a lower percentage of chlorotic leaves, lower cellular electrolyte leakage (EL) and H2O2 and O2 .- content, and higher chlorophyll content and photochemical efficiency compared to the wild type. Relative mRNA expression level analysis by qRT-PCR indicated that the improved freezing tolerance of transgenic Arabidopsis plants overexpressing PpCBF3 was conferred by sustained activation of downstream cold responsive (COR) genes. Other interesting phenotypic changes in the PpCBF3-transgenic Arabidopsis plants included late flowering and slow growth or ‘dwarfism’, both of which are desirable phenotypic traits for perennial turfgrasses. Therefore, PpCBF3 has potential to be used in genetic engineering for improvement of turfgrass freezing tolerance and other desirable traits.
Highlights
Low-temperature stresses such as chilling (0–20°C) and freezing stress (< 0°C) are the most common environmental factors limiting plant growth and productivity in cool climatic regions [1,2]
Sequence analysis at http:// blast.ncbi.nlm.nih.gov/Blast.cgi by Blastx programme showed that the gene had high similarity to DREB1/C-repeat binding factors (CBFs) in other plant species (Fig 1); about 81% similarity to LpCBF3 in perennial ryegrass and 64% to OsDREB1A/CBF3 in rice
PpCBF3 belongs to Dehydration-Responsive Element Binding proteins (DREB)/CBF family and responsive to cold stress
Summary
Low-temperature stresses such as chilling (0–20°C) and freezing stress (< 0°C) are the most common environmental factors limiting plant growth and productivity in cool climatic regions [1,2]. During chilling stress and the thawing phase following freezing stress, cells loss water due to the rupture of cellular membranes, leading to dehydration and protecting cellular dehydration is critically important for plant survival of chilling or freezing stress [2]. Enhanced cold tolerance was achieved with DREB1/CBF genes constitutively expressed at high levels in agriculturally important species (i.e. rice and wheat) and model species [i.e. Arabidopsis and tobacco (Nicotiana benthamiana)] [13,14,15,16,17,18]. Heterologenic expression of a CBF3 gene isolated from sweet pepper (Capsicum annuum) conferred enhanced tolerance to chilling stress (4°C) in transgenic tobacco plants through higher accumulations of osmolytes (proline and soluble sugars), higher levels of unsaturated fatty acids, and lower accumulations of H2O2 and O2.-, as well as lower electrolyte leakage and photochemical efficiency [18]. Overexpression of CBF1 enhanced Arabidopsis tolerance to freezing stress (-8°C) as by lower electrolyte leakage and improved whole plant survival [19]. The knowledge of DREB1/CBFs transcriptional control of plant tolerance to severe freezing temperatures is limited
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