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, 14 (12), e0225899

Diversity of a Cytokinin Dehydrogenase Gene in Wild and Cultivated Barley


Diversity of a Cytokinin Dehydrogenase Gene in Wild and Cultivated Barley

Beata I Czajkowska et al. PLoS One.


The cytokinin dehydrogenase gene HvCKX2.1 is the regulatory target for the most abundant heterochromatic small RNAs in drought-stressed barley caryopses. We investigated the diversity of HvCKX2.1 in 228 barley landraces and 216 wild accessions and identified 14 haplotypes, five of these with ten or more members, coding for four different protein variants. The third largest haplotype was abundant in wild accessions (51 members), but absent from the landrace collection. Protein structure predictions indicated that the amino acid substitution specific to haplotype 3 could result in a change in the functional properties of the HvCKX2.1 protein. Haplotypes 1-3 have overlapping geographical distributions in the wild population, but the average rainfall amounts at the collection sites for haplotype 3 plants are significantly higher during November to February compared to the equivalent data for plants of haplotypes 1 and 2. We argue that the likelihood that haplotype 3 plants were excluded from landraces by sampling bias that occurred when the first wild barley plants were taken into cultivation is low, and that it is reasonable to suggest that plants with haplotype 3 are absent from the crop because these plants were less suited to the artificial conditions associated with cultivation. Although the cytokinin signalling pathway influences many aspects of plant development, the identified role of HvCKX2.1 in the drought response raises the possibility that the particular aspect of cultivation that mitigated against haplotype 3 relates in some way to water utilization. Our results therefore highlight the possibility that water utilization properties should be looked on as a possible component of the suite of physiological adaptations accompanying the domestication and subsequent evolution of cultivated barley.

Conflict of interest statement

The authors have declared that no competing interests exist.


Fig 1
Fig 1. The barley cytokinin dehydrogenase gene HvCKX2.1.
The top panel shows the structure of the gene and the location of the sequenced region. The lower panel shows the sequence of the gene with lower case letters used for the leader sequence and intron, and upper case letters used for the coding region. Primer annealing positions are indicated: amp1 primers, red lettering; amp2 primers, blue lettering; amp1+2 primers, yellow highlight. The possible ATG initiation codons are shown with green highlight. High confidence SNPs and their amino acid substitutions are highlighted in turquoise and medium confidence SNPs and substitutions in grey.
Fig 2
Fig 2. Network displaying the relationships between the fourteen DNA haplotypes of HvCKX2.1.
Node sizes are proportional to numbers of accessions and empty nodes are shown as black dots. The proportion of wild and domesticated accessions for each haplotype are shown in red and green, respectively. The locations within the network of the four protein variants are shown with the amino acid sequences given in the IUPAC single-letter code.
Fig 3
Fig 3. Secondary structure predictions for the barley HvCKX2.1 protein and various other grass cytokinin dehydrogenases.
The parts of the protein alignments containing the four substitutions (positions 46, 51, 149 and 194) in HvCKX2.1 are shown. Structural codes: pink barrel, α-helix; yellow arrow, β-strand; blue hooked arrow, turn; grey wavy line, coil. The barley 1 and barley 2 sequences are HvCKX2.1 incorporating the alternative versions of each of the four substitutions. The other sequences are taken from Genbank: sorghum, Sorghum bicolor cytokinin dehydrogenase 2, XP_002455003.1; rice, Oryza sativa japonica group cytokinin dehydrogenase 2, XP_015629416; Brachypodium, Brachypodium distachyon cytokinin dehydrogenase 2-like protein, XP_003564990.3; Aegilops, Aegilops tauschii subsp. tauschii cytokinin dehydrogenase 2-like protein, XP_020183514.1; wheat, Triticum aestivum cytokinin oxidase 2, ADG57787.1.
Fig 4
Fig 4. Two views of the X-ray crystallographic structure of a maize cytokinin dehydrogenase protein.
The β-strand–turn–β-strand motif that forms a finger on the surface of the protein is highlighted. The structure is PDB ID IW10 and is viewed using the iCnS3D web-base structure viewer at
Fig 5
Fig 5. Secondary structure of the FAD binding domains of the barley HvCKX2.1 protein and a maize cytokinin dehydrogenase protein.
The Barley 1 and Barley 2 sequences are HvCKX2.1 incorporating the alternative versions of the substitutions at positions 149 and 194. Maize 1 is the secondary structure of a maize cytokinin oxidase/dehydrogenase as predicted from its amino acid sequence, and Maize 2 is the actual secondary structure of this maize cytokinin oxidase/dehydrogenase according to the DSSP analysis of the X-ray crystallographic data of the protein complexed with N6-(3-methoxy-phenyl)adenine (PDB 3DQ0 available at Structural codes: pink barrel, α-helix; yellow arrow, β-strand; blue hooked arrow, turn; grey wavy line, coil. Note that the alignment between and the barley and maize amino acid sequences around barley position 149 is slightly different to that shown in S2 Fig.
Fig 6
Fig 6. Distributions of wild accessions belonging to different haplotypes.
Maps were plotted using ArcMap 10.2.1 of ArcGIS (ESRI. ArcGIS Desktop: Release 10. Redlands, CA: Environmental Systems Research Institute 2011).
Fig 7
Fig 7. Dimensionality reduction analyses using as input data the combined monthly precipitation amounts (WorldClim version 2) for the collection sites of each wild accession.
(A) PCA. Component 1 accounted for 67.8% of the total variability and component 2 accounted for 25.3%. The ellipses indicate the regions within which 95% of the data points for each haplotype are expected to fall. (B) tSNE, run with perplexity = 30, iterations = 2000 and theta = 0.5. (C) UMAP. Black dots and ellipses, haplotype 1; cyan, haplotype 2; orange, haplotype 3.
Fig 8
Fig 8. Average monthly precipitation amounts (WorldClim version 2) at the collections sites for wild accessions of haplotypes 1, 2 and 3.
(A) All accessions; (B) winter barleys; (C) spring barleys. Blue, haplotype 1; red, haplotype 2; green, haplotype 3. Bars indicate standard error.

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    1. Abbo S, Gopher A, Rubin B, Lev-Yadun S. On the origin of Near Eastern founder crops and the ‘dump-heap hypothesis’. Genet Resour Crop Evol. 2005; 52: 491–495.
    1. Zeder MA. Central questions in the domestication of plants and animals. Evol Anthropol. 2006; 15: 105–117.
    1. Zeder MA. Domestication and early agriculture in the Mediterranean Basin: origins, diffusion, and impact. Proc Natl Acad Sci USA. 2008; 105: 11597–11604. 10.1073/pnas.0801317105 - DOI - PMC - PubMed
    1. Brown TA, Jones MK, Powell W, Allaby RG. The complex origins of domesticated crops in the Fertile Crescent. Trends Ecol Evol. 2009; 24: 103–109. 10.1016/j.tree.2008.09.008 - DOI - PubMed
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Grant support

GJ received 269830 from the European Research Council, TAB received 339941 from the European Research Council, The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.