Volume 26, Issue 6, Pages 775-781 (March 2016) Mitogen-Activated Protein Kinase Kinase 3 Regulates Seed Dormancy in Barley  Shingo Nakamura, Mohammad Pourkheirandish, Hiromi Morishige, Yuta Kubo, Masako Nakamura, Kazuya Ichimura, Shigemi Seo, Hiroyuki Kanamori, Jianzhong Wu, Tsuyu Ando, Goetz Hensel, Mohammad Sameri, Nils Stein, Kazuhiro Sato, Takashi Matsumoto, Masahiro Yano, Takao Komatsuda  Current Biology  Volume 26, Issue 6, Pages 775-781 (March 2016) DOI: 10.1016/j.cub.2016.01.024 Copyright © 2016 Elsevier Ltd Terms and Conditions

Figure 1 Map-Based Cloning of MKK3 as the Causal Gene for the Seed Dormancy QTL Qsd2-AK Detected Using RILs from Az and KNG (A) Pictures of Az and KNG spikes soaked with distilled water containing 0.0125% (weight/volume) iminoctadine-triacetate as fungicide and incubated in the dark at 15°C for 5 days under saturated humidity conditions. The spikes with mature seeds were harvested and dried at 30°C for 7 days. (B) Germination percentages of Az, KNG, and NIL #34. Spikes were harvested at the physiological maturity stage, then dried at 30°C for 7 days. Data are shown as mean ± SD; n = 3 spikes. (C) Fine mapping delimited the region of Qsd2-AK to 7 kb on chromosome 5H between SNP434 and SNP7661, which are located in the introns 2 and 10 of MKK3. LOD, logarithm of odds. Genetic map for mapping of Qsd2-AK made with 2,985 segregating F2 plants. Numbers indicate recombination events between DNA markers indicated in the figure. Barley full-length cDNAs, FLbaf165m10: accession #AK252643; NIASHv3004F23: accession #DK691884; NIASHv2059D09: accession #AK367590. In the F2 recombinant figure, yellow rectangles indicate homozygous regions from Az, blue rectangles indicate homozygous regions from KNG, light green rectangles indicate heterozygous regions. See also Figures S1 and S2 and Tables S1 and S2. Current Biology 2016 26, 775-781DOI: (10.1016/j.cub.2016.01.024) Copyright © 2016 Elsevier Ltd Terms and Conditions

Figure 2 In Vitro Kinase Assays of MKK3 (A) The comparison of evolutionary conservation of the substituted amino acids between Az and KNG. The alignment of protein sequences is shown in Figure S3A. (B) An in vitro kinase assay of MKK3. Affinity-purified GST fusion proteins, GST-MPK1K61R and GST-MKK3s, were expressed in E. coli. The K61R amino acid substitution was introduced to prevent auto-phosphorylation of MPK1. GST-Az: GST-fused Az MKK3; GST-KNG: GST-fused KNG MKK3; GST-mutKNG: GST-fused KNG MKK3 (N260T). GST-MKK3s were incubated with (+) or without (−) a substrate GST-MPK1K61R in the kinase reaction mixture, and aliquots of the samples were separated by SDS-PAGE and subjected to autoradiography. Coomassie blue (CBB) staining of the GST-MKK3s and GST-MPK1K61R is shown in the bottom panel. Data are shown as mean ± SD; n = 4 independent experiments. See also Figure S3. Current Biology 2016 26, 775-781DOI: (10.1016/j.cub.2016.01.024) Copyright © 2016 Elsevier Ltd Terms and Conditions

Figure 3 Analysis of the MKK3 Splicing Junction Site Mutation (A) Nucleotide sequence around the two acceptor sites at the 3′ end of the intron 6 and at the 5′ end of the exon 7. Boxed sequence: exon 7. The first acceptor site AG marked with red underline for normal MKK3, the second acceptor site AG marked with green underline for truncated MKK3. Blue arrow indicates nucleotide changes in the splice junction mutant (#17). TGA with red letters: the premature stop codon produced by the alternative splicing. (B) The two types of MKK3 transcripts generated by alternative splicing. KD, kinase domain; NTF2, Nuclear Transport Factor 2 domain; aa, amino acids; ATG, initiation codon; TAG, stop codon. (C) Germination percentages of the mutants. # indicates the TILLING mutant line number. Red bar indicates that the line has a mutation at the splicing junction site; orange bar indicates the line has a non-synonymous mutation (mutant #1: L130F; #3: A173T; #4: V186I; #11:T239I); green bar indicates the line has a synonymous mutation; blue bar indicates the line has a mutation in the non-coding sequence. Germination data for M3 plants are shown as mean ± SD; n = 9 (3 spikes × 3 plants), except for #8 and #11 n = 6 (3 spikes × 2 plants), and #17 n = 6 (3 of the combined seeds from 2 spikes × 2 plants). The spike length of #17 is approximately half the spike length of Barke; thus, number of seeds per spikes was also about half that of Barke. To obtain enough seeds for germination tests, we combined seeds from two spikes for the germination test. The detailed description of these mutants is available in Table S3. See also Figure S4. Current Biology 2016 26, 775-781DOI: (10.1016/j.cub.2016.01.024) Copyright © 2016 Elsevier Ltd Terms and Conditions

Figure 4 Evolution and Worldwide Distribution of the Az Allele of MKK3 in the SV Collection (A) The ancestral and descendant allele of Az. The dash (-) indicates the same nucleotides of the ancestral allele of Az. Only nucleotides with red letters differ from the ancestral allele. The A3041C nucleotide substitution causes the N260T amino acid substitution. (B) The distribution of Az alleles in the SV collection around the world. The number of cultivars is plotted on the country where the cultivar originated. The height of bars in the legend corresponds to five cultivars. Green bar indicates the number of cultivars that have the ancestral allele of Az (Hap_009). Red bar indicates the number of cultivars that have the Az allele (Hap_007). Orange bar indicates the number of cultivars that have a descendant allele of Az (Hap_011). Yellow bar indicates the number of cultivars that have a descendant allele of Az (Hap_018). Light green bar indicates the number of cultivars that have a descendant allele of Az (Hap_019). Blue bar indicates the number of cultivars that have other types of alleles, but all have N260. The world map was obtained from CraftMAP (http://www.craftmap.box-i.net/). See also Table S4. Current Biology 2016 26, 775-781DOI: (10.1016/j.cub.2016.01.024) Copyright © 2016 Elsevier Ltd Terms and Conditions