Winter-hardiness describes the ability of plants to survive winter. In the Triticeae group of cereal crop plants, there is a general trend: rye is more winter-hardy than wheat, and wheat is more winter-hardy than barley. Thus, in areas of colder winters, or in more northly regions where it is too cold for wheat, rye tends to predominate. Similarly, in regions where winter temperatures are too cold for barley, wheat survives. Survivability parallels the differences these plants exhibit in lab-based freezing experiments, rye can be frozen to colder temperatures than wheat, and wheat can be frozen to colder temperatures than barley. Triticale, a synthetic hybrid in which the entire genome of rye is combined with the entire genome of wheat is only as freezing tolerant as the wheat parent used to generate the synthetic hybrid. These properties were established decades ago, yet we still lack a clear mechanistic understanding of the underlying genomic properties that control winter hardiness across these cereal crops. In addition to the variability in cold tolerance exhibited across these taxonomic groups there are also differences within each taxonomic group. It is this diversity, within taxonomic groups which provides the greatest mechanistic insight to winter-hardiness.
Wheat, barley, and rye can each be separated into two broad categories that are descriptive of their growth habits, winter types and spring types. Spring types are planted in the spring and will complete their life cycle that summer. Winter types are planted in the autumn because they won’t flower unless they have been exposed to a prolonged period of cold temperatures, a phenomenon known as vernalization. Hence they must be able to survive the winter temperature and other environmental conditions in the region where they are grown. We know that there are differences across winter varieties in their ability to survive winter, but the challenge we face is that the true test winters that enable us to discriminate these differences do not occur with recurring frequency. The figure below shows winterkill occurring in winter barley varieties following the 2013–14 winter, a true test winter. All spring lines exhibited 100% winterkill that winter, while percent differences were observed across winter lines. Note that the 'Puffin' and 'Signal' increase strips appear to suggest greater survival of 'Puffin', while the stand counts represented in the bar graph more accurately quantify this difference.
|Time course through winter. The left side shows two 225' long, 5' wide strips each of 'Signal and 'Puffin'. The right side shows 3' long individual headrow plots of approximately 1,000 winter barley lines. The latter experiment is described in greater detail in Muñoz-Amatriaín et al., 2021.||Percent survival of plants grown in 5' × 10' plots. Error bars show stand counts from three independent plots planted in randomized complete block design, except MO B2191, MO B2797, and the three NT lines, which were each in a single plot. Red arrows identify AMBA-recommended malting barley lines and year recommended. (This figure is from Stockinger, 2021.)|
Spring types are less winter hardy than their winter counterparts. This has long been known and was first described in writings from plant breeders and researchers credited with rediscovering Mendel’s work on inheritance around the turn of the 20th Century. Today we know that several regions on the homoeologous group 5 chromosomes play a major role in determining the differences in winter hardiness. One of these regions encompasses a cluster of genes known as C-Repeat Binding Factors (CBFs) The CBF proteins are transcription factors that bind to a short sequence motif and activate expression of the adjacent gene. They act as regulatory switches, activating and reconfiguring gene expression, much like a railroad switch that redirects trains from one track to another. Although we currently have substantial insight into the genes and pathways the CBFs alter and reconfigure in Arabidopsis, we have only minimal insight in the cereal crop plants. But it is already noteworthy that the cereals possess a multitude of unique aspects regarding the CBFs.
The CBF gene cluster on chromosome 5 spans about 1 cM in genetic units and approximately 1 MB of physical distance and consists of at least 13 unique CBF genes (Francia et al., 2007; Knox et al., 2008; Skinner et al., 2006; Skinner et al., 2005; Tondelli et al., 2006). The entire region is referred to as the Frost Resistance-H2 (FR-H2) locus. Many of the CBF genes at FR-H2 exhibit copy number variation. Multiple copies of CBF2A and CBF4B in barley result from a tandem iteration of a 22 kb region that encompasses these two genes – some lines may contain eight or more copies of this region (Dhillon et al., 2017; Knox et al., 2010). Higher copy number is associated with higher CBF2 and CBF4 transcript levels and greater winter-hardiness (Dhillon et al., 2017; Jeknić et al., 2014; Stockinger et al., 2007). Currently we know that the non-hardy spring types possess only a single copy of CBF2 and CBF4, and unlike the winter types, the expression of these genes is repressed by VRN-H1 (VERNALIZATION-H1), which like the CBFs, is a key regulatory switch – one that directs the plant to transition to the reproductive phase (Dhillon et al., 2010). The VRN-H1 gene is also known as the Frost Resistance-H1 (FR-H1) gene because its key role in winter-hardiness was identified genetically before the advent of genomic sequencing efforts.
The figure below shows a working model of the winter-hardy state (left) and the non-winter-hardy state (right). A key difference being the CBFs are expressed in the winter-hardy state but not in the nonwinter-hardy state. In the winter hardy state the VRN-H1 gene is not expressed until induced by vernalization.
|The CBF gene cluster comprising a winter allele of the FR-H2 locus. Three iterations of the CBF2A–CBF4B genomic region are shown boxed. Some barley lines harbor higher copy numbers. A consequence of the increased copy numbers is the increased expression of other CBF genes at the locus, identified in green text.||CBF gene expression is repressed by the action of the VRN-H1 protein. Once induced by vernalization, the VRN-H1 protein product acts to repress expression of the CBFs by directly binding to target sites in the CBF genomic region.|
Greater insight and depth on winter hardiness alongside current working model of the CBFs and the VERNALIZATION1 gene can be found in a recent review (Stockinger, 2021).
We are interested in understanding the role the individual CBF gene family members play in winter-hardiness, and how copy number variation plays into it.
Dhillon, T., Pearce, S. P., Stockinger, E. J., Distelfeld, A., Li, C., Knox, A. K., Vashegyi, I., Vágújfalvi, A., Galiba, G., and Dubcovsky, J. (2010) Freezing tolerance and flowering regulation in cereals: the VRN-1 connection. Plant Physiol. 153: 1846-1858. http://www.ncbi.nlm.nih.gov/pubmed/20571115
Dhillon, T., and Stockinger, E.J. (2013). Cbf14 copy number variation in the A, B, and D genomes of diploid and polyploid wheat. Theor. Appl. Genet. 126, 2777-2789. http://www.ncbi.nlm.nih.gov/pubmed/23918064
Dhillon, T., Morohashi, K., and Stockinger, E.J. (2017) CBF2A-CBF4B genomic region copy numbers alongside the circadian clock play key regulatory mechanisms driving expression of FR-H2 CBFs. Plant Mol. Biol. 94: 333-347. https://www.ncbi.nlm.nih.gov/pubmed/28434151
Francia, E., Barabaschi, D., Tondelli, A., Laidò, G., Rizza, F., Stanca, A.M. Busconi, M., Fogher, C., Stockinger, E.J., and Pecchioni, N. (2007) Fine mapping of a HvCBF gene cluster at the frost resistance locus Fr-H2 in barley. Theor. Appl. Genet. 115:1083-1091. http://www.ncbi.nlm.nih.gov/pubmed/17763839
Jeknić, Z., Pillman, K.A., Dhillon, T., Skinner, J.S., Veisz, O., Cuesta-Marcos, A., Hayes, P.M., Jacobs, A.K., Chen, T.H.H., and Stockinger, E.J. (2014). Hv-CBF2A overexpression in barley accelerates COR gene transcript accumulation and acquisition of freezing tolerance during cold acclimation. Plant Mol. Biol. 84: 67-82. http://www.ncbi.nlm.nih.gov/pubmed/23949371
Knox, A.K., Li, C., Vagujfalvi, A., Galiba, G., Stockinger, E.J., and Dubcovsky, J. (2008) Identification of candidate CBF genes for the frost tolerance locus Fr-Am2 in Triticum monococcum. Plant Mol Biol. 67, 257-270. http://www.ncbi.nlm.nih.gov/pubmed/18317935
Knox A.K., Dhillon, T., Cheng, H., Tondelli, A., Pecchioni, N., and Stockinger, E.J. (2010) CBF gene copy number variation at Frost Resistance-2 is associated with levels of freezing tolerance in temperate-climate cereals. Theor. Appl. Genet. 121: 21-35. http://www.ncbi.nlm.nih.gov/pubmed/20213518
Munoz-Amatriain, M., Hernandez, J., Herb, D., Baenziger, P. S., Bochard, A.M., Capettini, F., Casas, A., Cuesta-Marcos, A., Einfeldt, C., Fisk, S., Genty, A., Helgerson, L., Herz, M., Hu, G., Igartua, E., Karsai, I., Nakamura, T., Sato, K., Smith, K., Stockinger, E., Thomas, W., Hayes, P. (2020). Perspectives on low temperature tolerance and vernalization sensitivity in barley: prospects for facultative growth habit. Front. Plant Sci. 11 (1625). https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7814503/
Stockinger, E.J., Skinner, J.S., Gardner, K.G., Francia, E., and Pecchioni, N. (2007) Expression levels of barley Cbf genes at Frost resistance-H2 are dependent upon alleles at Fr-H1 and Fr-H2. Plant J. 51:308-321. http://www.ncbi.nlm.nih.gov/pubmed/17559507
Skinner, J.S., Szűcs, P., von Zitzewitz, J., Marquez-Cedillo, L., Filichkin, T., Stockinger, E.J., Chen, T.H.H., and Hayes P.M. (2006) Mapping of barley homologs to genes that regulate low temperature tolerance in Arabidopsis. Theor. Appl. Genet. 112:832-842. http://www.ncbi.nlm.nih.gov/pubmed/16365758
Skinner, J.S., von Zitzewitz, J., Szűcs, P., Marquez-Cedillo, L., Filichkin, T., Amundsen, K., Stockinger, E.J., Thomashow, M.F., Chen, T.H.H., and Hayes P.M. (2005) Structural, functional and phylogenetic characterization of a large CBF gene family in barley. Plant Mol. Biol. 59:533-551. http://www.ncbi.nlm.nih.gov/pubmed/16244905
Tondelli, A., Francia, E., Barabaschi, D., Aprile, A., Skinner, J.S., Stockinger, E.J., Stanca, A.M., and Pecchioni, N. (2006) Mapping regulatory genes as candidates for cold and drought stress tolerance in barley. Theor. Appl. Genet. 112:445-454. http://www.ncbi.nlm.nih.gov/pubmed/16315028