A study of C - repeat binding factors (CBF) associated with low temperature tolerance locus in winter wheat.

Winter wheat has several advantages over spring varieties, higher (25 % more) yield, efficient use of spring moisture, reduction of soil erosion by providing ground cover during the fall and early spring, rapid initial spring growth to out - compete weeds and circumvent the peak of Fusarium head blight infections by flowering early. Winter wheat is planted in early autumn when it germinates and developing seedlings acclimate to cold. The crown survives under snow cover and in spring rapidly grows into a vigorously growing plant for grain to be harvested in summer. However, the harsh Canadian prairie winters require that winter wheat has increased cold hardiness and improved winter survival to reduce losses from sudden cold snaps during winter and spring. Low temperature (LT) tolerance is one of the major components of cold hardiness. Genetic mapping studies have revealed a major quantitative trait locus (Fr-A2) at wheat chromosome 5A which can explain at least 50 % of LT tolerance in wheat. Physical mapping of 5A LT QTL in a hardy winter wheat cv Norstar revealed a cluster of at least 23 C - repeat binding factors (CBF) coinciding with peak of Fr-A2 QTL. The objective of this study is biochemical, and molecular characterization of CBF co - located at Fr-A2 to identify key CBF participating in conferring LT tolerance in winter wheat. A comparative analysis of CBF gene cluster at the Fr-A2 collinear region among Poaceae members showed an expansion in the number of CBF genes with increased LT tolerance. Rice, a cold sensitive member, had only three CBF genes, whereas cold hardy winter wheat cv Norstar has 23 CBF genes. Amino acid sequence - based cluster analysis of complete CBF genes, or their major functional components such as the AP2 - DNA binding domain and C - terminal trans - activation domain, divide Norstar CBF into Pooideae specific clades. However, analyses of Norstar CBF amino acid sequences of different functional groups revealed a shift in clade members. These results suggest divergence of CBF functions which could lead to possible differences / similarity in the regulon activated by a CBF in a specific group. The 15 CBF genes from winter wheat cv Norstar were expressed in E. coli to produce recombinant TrxHisS - CBF fusion proteins in adequate quantities for structural and functional assays. All CBF fusion proteins could be recovered in the E. coli soluble phase of cell extract, except that the CBF17.0 fusion protein could only be recovered with 6 M urea extraction. Eleven of the 15 CBF fusion proteins were very stable in heat (98 oC), 10 % SDS and 6 M urea treatment. The five other CBF members were very labile under native conditions, but were stable in E. coli cell extracts or when extracted under denaturing conditions. Most of the CBF recombinant proteins in denaturing gel electrophoresis migrated slower than expected from their predicted molecular mass, based on amino acid sequence. The slow migration could be associated to their elongated protein structure as determined by dynamic light scattering (DLS). CBF 12.2 and CBF 17.0 were highly resistant to denaturation and retained their secondary structure in these conditions as determined by circular dichroism (CD) spectra. The high stability of these two CBF proteins may be important for cold acclimation or maintenance of cold hardiness in wheat. CBF proteins are transcription factors that bind to the dehydration-responsive element / C-repeat element (DRE / CRT) motif (CCGAC). Ten of the 15 Norstar recombinant CBFs whether purified under native or denaturing conditions showed in vitro binding to the CRT motif. Within hours of cold exposure (4 oC) the native CBF increased their affinity to CRT interaction which could be due to changes in the CBF secondary structures. Some of the CBF for binding preferred the core GGCCGAC motif while others preferred TGCCGAC. Similarly binding assays with truncated CBF revealed that for some CBF proteins, the second signature motif (DSAWR) and remaining C - terminal were not needed, while for others a considerable portion of the C -terminal region was needed for binding. Norstar CBF 12.1 has a memory of cold experience, and upon exposure to cold, has a high and immediate affinity to CRT elements. A homolog CBF12.2 in less cold - hardy winter wheat cv Cappelle - Desprez had a non - functional protein due to a R → Q substitution in a highly conserved residue within the AP2 domain. Several of the cv Norstar CBFs showed increased activity under LT and denaturing conditions, which may be the reason for the greater cold hardiness in Norstar. In conclusion, detailed and extensive analyses of CBF in this study characterized their structure and function relationships, which are important for understanding and improving LT tolerance in plants. The identification of specific CRT binding motifs and two CBFs which were very stable under adverse conditions may be prime candidates for further study to improve LT tolerance in plants.

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