PpCBF3 from Cold-Tolerant Kentucky Bluegrass Involved in Freezing Tolerance Associated with Up-Regulation of Cold-Related Genes in Transgenic Arabidopsis thaliana

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.

Fig 1. Sequence homology analysis of PpCBF3 with DREB/CBFs from Arabidopsis, rice, wheat, barley, tall fescue and perennial ryegrass.

Conserved AP2 DNA-binding domain is indicated by black line, two signature sequence of CBF are marked by asterisk and filled triangle. ‘LWSY’ domain at the end of C terminal is indicated by black dots. Accession numbers of the proteins are as follows: AtDREB1B/CBF1 (AAC49662), AtDREB1C/CBF2 (AAD15976), AtDREB1A/CBF3 (AAD15977), OsDREB1B/CBF1 (AAN02488), OsDREB1A/CBF3 (AAN02486), HvCBF2 (AAM13419), FaDREB1 (AAQ98965), TaCBF6 (AAX28964), HvCBF3 (AAG59618), LpCBF3 (AAX57275), HvCBF1 (AAL84170), TaCBF1 (AAL37944), PpCBF3 (KP258182). Sequence alignment was done by Cluster W.

Fig 2. Phylogenetic tree of PpCBF3 and other DREB/CBF proteins.

The tree is constructed by MEGA 4.0 software (Tamura et al. 2007) based on alignment of complete protein sequences. Black dot indicates PpCBF3 protein. Accession number of protein sequences used here are the same as in Fig 1.

Fig 3. Subcellular localization of PpCBF3 protein.

(A) Fluorescence image of Arabidopsis mesophyll protoplast expressing the PpCBF3-eGFP fusion protein. (B) Fluorescence image of nucleus in protoplast stained with DAPI. (C) Fluorescence image of mesophyll in Arabidopsis protoplast. (D) Image of Arabidopsis mesophyll protoplast under bright field. (E) Merged fluorescence image of Arabidopsis protoplast expressing the PpCBF3-eGFP fusion protein and stained with DAPI. (A)-(C), dark field. (D), (E), bright field. All bar = 10 μm.

Fig 4. Relative mRNA expression level of PpCBF3 in leaves of P.

pratensis at different time scale under 4 ^o C by RT-PCR and qRT-PCR. (A) qRT-PCR analysis of PpCBF3 expression level. Three independent experiments show the similar results and here shows one of the results. Values are means ± SD of three technical repetitions. (B) RT-PCR analysis of PpCBF3 expression level.

Fig 5. Expression analysis of PpCBF3 and phenotype of WT and transgenic lines under normal and cold treatment conditions.

(A) Shows 4-week-old seedlings of WT and transgenic lines grow under normal condition. (B) Phenotype of WT and transgenic lines after -20°C freezing stress. Red tick represents the stable transformed lines finally selected for further analysis. (C) Percentage of chlorotic leaves is calculated in (B). More than half yellow color of one leaf is designated as chlorolic leaf. Values are means ± SD of fifteen independent plants. Different letters on the top of bars indicate significant differences (P < 0.05) between WT and transgenic lines under the same growth condition. (D) Detection of the relative mRNA expression level of PpCBF3 in WT and transgenic lines grow under normal condition. (E) PpCBF3-GFP fused protein is detected by western blot in transgenic lines but not in WT.

Fig 6. Physiological index including relative electrolyte leakage (EL), total chlorophyll content (Chl) and photochemical efficiency (Fv/Fm) in WT and transgenic plants under normal and freezing treated.

Values are means ± SD of twelve independent plants. The same letters atop bars indicate that there is no significant difference at P < 0.05.

Fig 7. Detection of ROS in WT and transgenic Arabidopsis plants.

(A) NBT staining for superoxide in WT and transgenic Arabidopsis under normal condition and (B) freezing stress. (C) DAB staining for hydrogen peroxide in WT and transgenic Arabidopsis under normal condition and (D) freezing stress.

Fig 8. Relative mRNA expression level of downstream genes of DREB/CBF in WT and transgenic plants under normal and cold treatment (4°C).

(A)-(D) Relative mRNA expression level of cold responsive gene (COR) in WT and transgenic plants under normal and cold treatment (4°C). (E) Relative mRNA expression level of P5CS controlling the rate- limiting step of glutamate- derived proline biosynthesis in WT and transgenic plants under normal and cold treatment (4°C).

Fig 9. Growth characteristic of 6-week-old WT and transgenic Arabidopsis under normal growth condition.

All transgenic plants show late flowering phenotype compared with WT. The growth chamber condition is set at 23 ^oC, 16h/8h light/dark, 70% humidity, 120 μmol m^-2 s^-1 light density.