Volume 8, Issue 11, Pages 1635-1650 (November 2015) An-2 Encodes a Cytokinin Synthesis Enzyme that Regulates Awn Length and Grain Production in Rice  Benguo Gu, Taoying Zhou, Jianghong Luo, Hui Liu, Yongchun Wang, Yingying Shangguan, Jingjie Zhu, Yan Li, Tao Sang, Zixuan Wang, Bin Han  Molecular Plant  Volume 8, Issue 11, Pages 1635-1650 (November 2015) DOI: 10.1016/j.molp.2015.08.001 Copyright © 2015 The Author Terms and Conditions

Figure 1 Phenotypic Comparison in GLA4, SL4 and CSSLs. (A) A comparison of panicles in GLA4, SL4, and CSSLs. Bar represents 10 mm. (B) A comparison of awn lengths in GLA4, SL4, and CSSLs. Bar represents 10 mm. (C) Chromosome 4 structures of GLA4, SL4, and CSSLs. Red bars indicate segments from cultivated rice GLA4, blue bars indicate segments from wild rice W1943, and orange circles indicate the position of QTLs (Awn-1 and Awn-2) or genes (An-1 and An-2). Bar represents 2 Mb. (D–G) Statistical comparisons of awn ratios per panicle (D), awn lengths (E), tiller number per plant (F), and grain number per panicle (G) in GLA4, SL4, and CSSLs. An asterisk (*) indicates a significant difference. The statistical significance was set at P < 0.01 based on a two-tailed Student t-test. Error bars represent the SD. Wild-type GLA4 was set as the control and all lines were compared with GLA4 (n = 20). Molecular Plant 2015 8, 1635-1650DOI: (10.1016/j.molp.2015.08.001) Copyright © 2015 The Author Terms and Conditions

Figure 2 Map-Based Cloning and Identification of the An-2 Gene. (A) Fine mapping of An-2. The CI of An-2 was between markers M6229 and M1160. An-2 was finally mapped to a 56-kb region between markers FM5 and FM6, according to the Nipponbare genomic sequence. The black arrows indicate gene direction, predicted by the Rice Annotation Project (RAP) (http://rapdb.dna.affrc.go.jp). (B) The gene structure of An-2 and constructs used for the complementation test. pCP_An-2 contains a 6-kb genomic sequence including the whole gene region of W1943. pOX_An-2 represents cDNA of An-2 from SL4, driven by maize Ubiquitin (UBI) promoter, used for ectopic and over-expression. Bar represents 0.5 kb. Molecular Plant 2015 8, 1635-1650DOI: (10.1016/j.molp.2015.08.001) Copyright © 2015 The Author Terms and Conditions

Figure 3 Complementation Test of An-2. (A) A comparison of panicles in GLA4, NIL_An-2, complementation and over-expression lines of the GLA4 genetic background. Bar represents 10 mm. (B) A comparison of awn lengths in GLA4, NIL_An-2, complementation and over-expression lines of the GLA4 genetic background. Bar represents 10 mm. (C) A comparison of the tiller number per plant in GLA4, NIL_An-2, complementation and over-expression lines of the GLA4 genetic background. Bar represents 10 cm. (D–K) Statistical comparisons of awn ratios (D), awn lengths (E), tiller number per plant (F and G), grain number per panicle (H and I), 1000 grain weight (J), and yield per plant (K) in GLA4 genetic background lines (D–F, H, J, K) and Nipponbare background lines (G and I). An asterisk (*) indicates a significant difference. The statistical significance was set at P < 0.01 based on a two-tailed Student t-test. Error bars represent the SD. Wild-type GLA4 and N_p1301were set as controls (n = 20). G_CP_An-2 indicates GLA4 transformed by pCP_An-2 and G_OX_An-2 indicates GLA4 transformed by pOX_An-2. Molecular Plant 2015 8, 1635-1650DOI: (10.1016/j.molp.2015.08.001) Copyright © 2015 The Author Terms and Conditions

Figure 4 Typical Haplotypes of An-2/an-2 in O. rufipogon and O. sativa. (A) The An-2 sub-haplotype in W1943 and most accessions of wild rice. (B) The an-2 sub-haplotype in most cultivated varieties, including indica, aus, temperate japonica, tropical japonica, and aromatic, such as GLA4. (C) The an-2(J) sub-haplotype only in temperate japonica varieties including Nipponbare. (D) The an-2(A) sub-haplotype mainly in aus variety Kasalath. The common variations between cultivated varieties and wild rice accessions are indicated in this figure. Black bars represent 5′ upstream regions and introns. Light-gray bars represent 5′ and 3′ untranslated regions. Dark-gray bars represent coding regions. The short dashes represent single base deletions. The asterisks (*) indicate stop codons. The filled star represents the one-nucleotide deletion in an-2. The open star represents single nucleotide substitution (GGC:Gly→GTC:Val) in an-2(A). The black right-angled triangles indicate a 29-bp insertion. Bar represents 0.5 kb. Molecular Plant 2015 8, 1635-1650DOI: (10.1016/j.molp.2015.08.001) Copyright © 2015 The Author Terms and Conditions

Figure 5 Functional Comparisons of An-2 Alleles. (A) Statistical comparison of cytokinin base content between GLA4 and NIL_An-2 (15-day seedlings). iP, isopentenyladenosine; tZ, trans-zeatin; cZ, cis-zeatin; DHZ, dihydrozeatin; ND, not detected. NIL_An-2 was set as the control (n = 3). (B) Statistical comparison of relative reactivity between protein An-2 and an-2(A). Enzymes were incubated at pH 5.0 with 100 μM nucleotide. The reactivity of An-2 was set as the control. An-2 protein was set as the control (n = 3). An asterisk indicates a significant difference. The statistical significance was set at P < 0.01 based on a two-tailed Student t-test. Error bars represent the SD. Molecular Plant 2015 8, 1635-1650DOI: (10.1016/j.molp.2015.08.001) Copyright © 2015 The Author Terms and Conditions

Figure 6 Comparisons of Awn Length under a Genetic Background Harboring An-1. (A) A comparison of panicles of NILs. Bar represents 10 mm. (B) A comparison of awn lengths of NILs. Bar represents 10 mm. (C) and (D) Statistical comparisons of awn ratios (C) and awn lengths (D) in NILs. NIL_An-1 was set as the control. An asterisk (*) indicates a significant difference. The statistical significance was set at P < 0.01 based on a two-tailed Student t-test. Error bars represent the SD (n = 20). Molecular Plant 2015 8, 1635-1650DOI: (10.1016/j.molp.2015.08.001) Copyright © 2015 The Author Terms and Conditions

Figure 7 Awn Development and Expression Analysis of Awn Associated Genes and Histone Hs. (A) Scanning electron microscopy (SEM) images of spikelets at different developmental stages in SL4. Bar represents 50 μm. (B) Expression patterns of An-1 during spikelet development in SL4. Arrows point to An-1 expression in awn primordia. Bar represents 50 μm. (C) Expression patterns of An-2 during spikelet development in SL4. Arrows point to An-2 expression in awn primordia. Bar represents 50 μm. (D) Expression patterns of Histone H4 during spikelet development in SL4. Bar represents 50 μm. (E) The developmental model of rice long awn. The upper panel indicates the developmental stage of rice spikelet. The second panel indicates rice long awn developmental stage. The third and fourth panels indicate the expression pattern of An-1 (third) and An-2 (fourth) in awn primordia. Dark indicates a high expression level and light indicates a low expression level. (F) Statistical comparisons of Histone H1 relative expression levels between NIL_An-1 and NIL_An-1+An-2 in young panicles (from 0 to 4 cm) (n = 3). (G) Statistical comparisons of ETTIN2 relative expression levels between NIL_An-1 and NIL_An-1+An-2 in young panicles (from 0 to 1 cm) (n = 3). (A1) (B1) (C1) Sp4-Sp5, formation of lemma and the palea primordia stage. (A2) (B2) (C2) Sp6, formation of the stamen primordial stage. (A3) (B3) (C3) (D1) Sp7, formation of the carpel primordial stage. (A4) (B4) (C4) (D2) Sp8e (Sp8 early), differentiation of the ovule and pollen stages. (A5) (B5) (C5) (D3) Sp8l (Sp8 late),differentiation of the ovule and pollen stages. (A6) (B6) (C6) (D4) aSp8 (after Sp8), awn elongation. An asterisk indicates a significant difference. The statistical significance was set at P < 0.01 based on a two-tailed Student t-test. NIL_An-1 was set as the control. Error bars represent the SD. In situ sense controls are in Supplemental Figure 13. Molecular Plant 2015 8, 1635-1650DOI: (10.1016/j.molp.2015.08.001) Copyright © 2015 The Author Terms and Conditions

Figure 8 Comparisons of Gene Expression Levels in Branching Crowns and Young Panicles. (A) The expression pattern of An-2 at the branching (tiller formation) stage. Development of a branching niche. Black arrows indicate branching niches (A1) to (A4). (A5) shows a newly formed branch (tiller). (A6) shows a shoot apical meristem (SAM) of a new branch (tiller). Bar represents 100 μm. The sense control is in Supplemental Figure 13C. (B) Expression pattern of An-2 in a young panicle. (B1) Rachis meristem stage; (B2) primary branch formation stage; (B3) secondary branch formation stage; (B4) spikelet primordia formation stage; (B5) spikelet development stage. Bar represents 100 μm. The sense control is in Supplemental Figure 13D and 13E. (C–H) Statistical comparisons of the relative expression levels of An-2 (C and E), OsRRs and OsCKX3 (D and F), and branching genes (G and H) in 15-day seedlings. Branching crowns (C, D, and G) and young panicles (1–3 cm) (E, F, and H) of Nipponbare genetic background lines. All lines were compared with the vector control N-p1301 (n = 3). CK, cytokinin. An asterisk (*) indicates a significant difference. The statistical significance was set at P < 0.01 based on a two-tailed Student t-test. Error bars represent the SD. Molecular Plant 2015 8, 1635-1650DOI: (10.1016/j.molp.2015.08.001) Copyright © 2015 The Author Terms and Conditions

Figure 9 Phylogenetic Tree of An-2 in Rice. The gray shaded area indicates a cluster of wild rice accessions. Bootstrap values were estimated (with 1000 replicates) to assess the relative support for each branch. Bootstrap values of 50% and above are indicated on the tree. W3104 (O. glaberrima) and W3106 (O. barthii) are used as outgroups. Molecular Plant 2015 8, 1635-1650DOI: (10.1016/j.molp.2015.08.001) Copyright © 2015 The Author Terms and Conditions

Figure 9 Phylogenetic Tree of An-2 in Rice. The gray shaded area indicates a cluster of wild rice accessions. Bootstrap values were estimated (with 1000 replicates) to assess the relative support for each branch. Bootstrap values of 50% and above are indicated on the tree. W3104 (O. glaberrima) and W3106 (O. barthii) are used as outgroups. Molecular Plant 2015 8, 1635-1650DOI: (10.1016/j.molp.2015.08.001) Copyright © 2015 The Author Terms and Conditions