An ACC Oxidase Gene Essential for Cucumber Carpel Development Huiming Chen, Jinjing Sun, Shuai Li, Qingzhi Cui, Huimin Zhang, Fengjiao Xin, Huaisong Wang, Tao Lin, Dongli Gao, Shenhao Wang, Xia Li, Donghui Wang, Zhonghua Zhang, Zhihong Xu, Sanwen Huang  Molecular Plant  Volume 9, Issue 9, Pages 1315-1327 (September 2016) DOI: 10.1016/j.molp.2016.06.018 Copyright © 2016 The Author Terms and Conditions

Figure 1 Phenotypic Comparison of the Androecious Mutant 406a with the Wild-Type 406. (A and B) Plants of the line 406 (A) and 406a (B). Plants of 406 bore male and female flowers, whereas 406a bore only male flowers. There was no morphological difference between male flowers from 406 and 406a. The presented male flowers are marked with red rectangles and the female flowers with a white rectangle. Scale bars indicate 10 cm. (C–E) The magnified male flowers from 406a. The orange rectangle indicates the stamen in (D), which is magnified in (E). St, stamen. Scale bars indicate 5 mm in (C) and (D), and 1 mm in (E). (F–H) The magnified female flowers from 406. The orange rectangle indicates the stigma in (G), which is magnified in (H). Sg, stigma. Scale bars indicate 5 mm in (F) and (G), and 1 mm in (H). (I) Sex phenotypes of 406 and 406a. The x axis represents the individual plant and the y axis represents the total number of nodes with female flowers (green bars), male flowers (red bars), or no flowers (blue). The first 40 nodes from 20 plants of 406 and 21 plants of 406a are represented. Molecular Plant 2016 9, 1315-1327DOI: (10.1016/j.molp.2016.06.018) Copyright © 2016 The Author Terms and Conditions

Figure 2 Identification of the Gene Conferring the Androecy Phenotype. (A) SNP-index distribution of the 406a pool. The x axis represents the position of seven chromosomes and the y axis represents the SNP index. The arrow and pink rectangle indicate the region of chromosome 6 with an SNP index of approximately 1. (B) Linkage analysis of the F2 population using dCAPS markers confirmed the causative SNP. Arrows indicate the position of the SNPs that were used for designing the dCAPS markers. The red arrow indicates the only SNP co-segregating with the androecy phenotype. 406a, F2-3, 50, 66, 74, and 97 were all androecious (marked with red) and 406 was monoecious (marked with green). (C) Gene structure of Csa6G511860 (CsACO2). Solid rectangles represent exons; open rectangles represent the untranslated region (UTR); solid lines represent introns. Arrows indicate the mutation sites. The red vertical line represents the C-to-T mutation in the third exon. (D) Alignment of the Fe2+ and 2OG-dependent dioxygenase domain (2OG-FeII_oxy domain) with CsACO2 homologs from diverse species. Amino acid residues displaying >50% identity or similarity between the homologs are shaded black or gray, respectively. Full alignment results are shown in Supplemental Figure 2. The numbers above the alignment indicate the position of the corresponding amino acid site along the CsACO2 protein sequence. Molecular Plant 2016 9, 1315-1327DOI: (10.1016/j.molp.2016.06.018) Copyright © 2016 The Author Terms and Conditions

Figure 3 Enzymatic Activities of the Wild-Type and Mutated CsACO2 Alleles. (A) Initial reaction rate of wild-type CsACO2 protein measured at various concentrations of ACC. Experimental data (circles) were fit to a line representing Michaelis–Menten kinetics. (B) Enzymatic activity of CsACO2 (bright blue bars), P254S (red bars), and the H177Q (light blue bars) protein isoforms at various concentrations of ACC. Error bars represent SD; n = 3. (C) The ACO activity of protein extracts from 406 and 406a shoot tips at the six-leaves stage. Specific activity was measured with 10 μg of total protein in the presence of 1 mM ACC. Error bars represent SD; n = 3. (D) Ethylene production of the 406 and 406a shoot tips at the six-leaves stage. Error bars represent SD; n = 3. (E) Real-time qPCR analysis showed the expression levels of five ACO genes in cucumber flower buds before stage 6. CsTUBULIN was used as the reference. Error bars represent SD; n = 3. Molecular Plant 2016 9, 1315-1327DOI: (10.1016/j.molp.2016.06.018) Copyright © 2016 The Author Terms and Conditions

Figure 4 Treatment of Androecious Plants 406a and Erez with Ethephon and ACC. (A–D) 406a androecious plants (A and C) treated with 100 ppm ethephon (B) or 500 μM ACC (D). Ethephon treatment can induce female flowers but ACC cannot. Scale bar indicates 5 mm. (E–H) Erez androecious plants (E and G) treated with 100 ppm ethephon (F) or 500 μM ACC (H). Both ethephon and ACC treatment can induce female flowers. Scale bar indicates 5 mm. (I) Summary of the flowers sex types in 406a and EreZ when treating with CK (water), ACC or ethephon. The x-axis represents the individual plant and the y-axis represents the number of nodes with female flowers (green bars) and male flowers (orange bars). For each treatment, results from 5 representative 406a and EreZ plants were summarized. Molecular Plant 2016 9, 1315-1327DOI: (10.1016/j.molp.2016.06.018) Copyright © 2016 The Author Terms and Conditions

Figure 5 Expression Patterns of CsACO2, CsWIP1, and CmACO3. (A–E) Expression pattern of CsACO2. (A) A shoot tip containing early initiated flower buds. (B and C) A flower bud at stage 2 (B) and stage 5 (C). (D) A female flower bud between stage 6 and stage 7. (E) A male flower buds at stage 8. (F–J) Expression pattern of CsWIP1. (F) A shoot tip containing early initiated flower buds. (G and H) A flower bud at stage 2 (G) and stage 5 (H). (I) A female flower bud at late stage 8. (J) A male flower bud at stage 8. (K–O) Expression pattern of CmACO3 in line 502 (GG background, andromonoecious). (K) A shoot tip containing early initiated flower buds. (L and M) A flower bud at stage 2 (L) and stage 5 (M). (N) A flower bud at late stage 7 with developed carpel. (O) A male flower bud at stage 8. (P–T) Expression pattern of CmACO3 in line 304 (gg background, bisexual). (P) A shoot tip containing early initiated flower buds. (Q–T) A flower bud at stage 2 (Q) stage 5 (R), stage 7 (S), and stage 8 (T). S, sepal; P, petal; St, stamen; C, carpel. Scale bars indicate 50 μM. Molecular Plant 2016 9, 1315-1327DOI: (10.1016/j.molp.2016.06.018) Copyright © 2016 The Author Terms and Conditions

Figure 6 CsWIP1/CmWIP1 Directly Regulate CsACO2/CmACO3. (A) Schematic diagram of the constructs using dual luciferase (LUC) analysis in a tobacco transient expression system. Effector constructs contained the CaMV 35S promoter fused to the transcription factors CsWIP1 or CmWIP1. Reporter constructs contained 2 kb of the promoters of either CsACO2 and CmACO3, upstream of the translation initiation sites, fused to the LUC reporter gene. Renilla luciferase (REN) was used as the internal control. (B) Effects of CsWIP1 and CsWIP1G242R on the activity of the CsACO2 promoter. Error bars indicate SD (n = 8). *p < 0.05, Student's t-test. (C) Effects of CmWIP1 on the activity of the CmACO3 promoter. Effects on the corresponding promoter fragments are presented as a ratio of LUC to REN. Error bars indicate SD (n = 8). *p < 0.05, Student's t-test. (D) Yeast one-hybrid assay. Yeast cells were co-transformed with the coding sequence of CsWIP1 and CsWIP1G242R and 2 kb of the CsACO2 promoter sequence, and selected on Simple Dropout (SD) medium lacking Trp and Leu. Also, coding sequence of CmWIP1 and 2 kb of the CmACO3 promoter sequence were co-transformed and selected in the same way. 3-AT (40 mM) was added to the SD/−His/−Trp/−Leu medium for the CsACO2 promoter and 20 mM 3-AT for CmACO3 promoter. (E) Schematic diagram of the regions used in ChIP–qPCR (P1 to P3) and probes used in an EMSA assay (Probe 1 and Probe 2). Specific primer and probe sequences are listed in Supplemental Table 4. (F) ChIP–qPCR assays with pro35S:CsWIP1-myc transformed cucumber protoplasts. Untransformed cucumber protoplasts were used as a negative control. CsTUBULIN was used as internal reference for normalization. Error bars represent the SD from three biological replicates. (G) EMSA assays with the purified CsWIP1 protein and the biotin-labeled probes, designed according to the enriched P1 and P2 fragments. The red triangle indicates the increasing amounts of CsWIP1 protein. The blue triangle indicates the increasing amounts of unlabeled competitor probes. Molecular Plant 2016 9, 1315-1327DOI: (10.1016/j.molp.2016.06.018) Copyright © 2016 The Author Terms and Conditions