1. Volume 8, Issue 12, Pages 1795-1808 (December 2015)
A Rare SNP Identified a TCP Transcription Factor Essential for Tendril Development in Cucumber 
Shenhao Wang, Xueyong Yang, Mengnan Xu, Xingzhong Lin, Tao Lin, Jianjian Qi, Guangjin Shao, Nana Tian, Qing Yang, Zhonghua Zhang, Sanwen Huang 
Molecular Plant 
Volume 8, Issue 12, Pages (December 2015)
DOI: /j.molp
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2. Figure 1 Distribution of Rare SNPs in Cucumber Population and Genome.
(A) The distribution of rare SNPs in each accession from four cucumber groups. The number of rare SNPs of each group is under the group name. The order of accession is the same as listed in Supplemental Data Set 1.
(B) Average number of rare SNPs per accession in the four groups.
(C) The distribution of rare SNPs in the cucumber genome. UTR, untranslated region.
Molecular Plant 2015 8, DOI: ( /j.molp )
Copyright © 2015 The Author Terms and Conditions

3. Figure 2 Morphology of Normal and Modified Tendrils.
(A) The cucumber line 404, bearing typical tendrils, has the ability to climb. Helically coiling tendrils tightly attach to the support, providing stability for the climbing cucumber plant.
(B) The tendril-less cucumber line CG9192 loses the climbing ability.
(C) Arrangement of organs at nodes of cucumber line 404 (top) and CG9192 (bottom). Four components from proximal to distal to the leaf base: leaf, flower (staminate or pistillate flower), lateral shoot, and tendril/modified tendril.
(D–K) Scanning electron micrographs of the development process of normal tendril in 404 (D–G) is compared with modified tendril in CG9192 (H–K). Axillary bud complex from 404 (D) and CG9192 (H) showing similar primordium initiation at early developmental stage. At a later stage, their morphology starts to differ. In 404, primordium develops a normal tendril tip (E) and its epidermal cells are stem-like (F); later a curling structure emerges (G). In CG9192, primordium develops a leaf-like tip (I) and its epidermal cells resemble leaf epidermal cells with obvious stomata (arrows) (J); later a new leaf primordium emerges (K).
L, leaf primordium; T, tendril primordium; MT, modified tendril primordium; F, flower primordium; Ax, axillary bud. Scale bars: (D) and (H), 100 μm; (E) and (I), 300 μm; (F) and (J), 20 μm; (G) and (K), 1 mm.
Molecular Plant 2015 8, DOI: ( /j.molp )
Copyright © 2015 The Author Terms and Conditions

4. Figure 3 Identification of the TEN Gene.
(A) Transcriptome heat map of the 75 genes carrying rare non-synonymous SNPs. The heat map is derived from a transcriptome map of 9930 that contains seven organs/tissues: tendril, leaf, stem, root, male flower, female flower, and fruit. Scaled log2 expression values are shown, and expression levels are indicated by shades of red, where white indicates no expression. Note that Csa5G is specifically expressed in tendril.
(B) High-resolution mapping of TEN. The numbers on the top of the markers indicate the physical position. Numbers below the map indicate the numbers of recombinants detected between the corresponding markers.
(C) Variation map of four cucumber lines in the SNP4–rSNP2 interval. The dots and triangles represent the SNPs and InDels that are polymorphic among the four cucumber lines; the red ones represent the CG9192 alleles and the black ones represent non-CG9192 alleles. Line CG3079, which is far from CG9192 in the phylogenetic tree (Supplemental Figure 2), was used as control.
Molecular Plant 2015 8, DOI: ( /j.molp )
Copyright © 2015 The Author Terms and Conditions

5. Figure 4 TEN is a TCP Protein with a Unique Motif.
(A) Phylogenetic tree of cucumber TEN (CsTEN) and other TCP family members in Arabidopsis thaliana (At), Oryza sativa (Os), Antirrhinum majus (Am), Pisum sativum (Ps), and Zea mays (Zm). The phylogenetic tree was constructed using the maximum-likelihood method implemented in the MEGA program (v5.2), and only the TCP domain was used for multiple alignment.
(B) Transcript structure of the TEN gene. Gray boxes represent 5′- and 3′-UTRs, black boxes represent exons, the black line represents an intron, orange boxes represent conserved domains (TCP and R), and the red box represents the mutated region.
(C) A key mutation changed a conserved amino acid of the TEN protein. The seven amino acids (CNNFYFP) in the red box are conserved in Cucurbitaceae species. The 338th amino acid residue of the TEN protein in the tendril-less cucumber changed from an Asn residue (N) to a Tyr residue (Y), while cucumbers with normal tendrils and another six Cucurbitaceae species all carry the Asn residue (N). TCP proteins of other species do not have the CNNFYFP motif.
(D) Localization of TEN-GFP in the nucleus in Arabidopsis protoplasts. Scale bar, 10 μm.
(E) The protein TEN shows stronger transactivation activity than the mutated form (ten) in a transcriptional activity assay in tobacco (N. benthamiana). The C terminus of TEN and ten were fused to the GAL4 binding domain (GAL4BD). Activity of the firefly luciferase gene driven by the GAL4 binding element UPSTREAM ACTIVATION SEQUENCE (UAS) was measured. Renilla LUC was used as reference and VP16 as a positive control. Error bar indicates SE.
** stands for significant difference (P < 0.01).
(F) Transactivation activity assay in yeast. The C terminus of TEN and ten showed a serial dilution series grown on two different selective media.
Molecular Plant 2015 8, DOI: ( /j.molp )
Copyright © 2015 The Author Terms and Conditions

6. Figure 5 In Situ Analysis of TEN Expression.
Cross sections of 4-week-old cucumber buds of the cucumber line 404 (TEN/TEN) were hybridized with digoxigenin-labeled antisense probes to TEN transcripts (A–C), or digoxigenin-labeled sense probes to TEN transcripts (D).
(A) TEN expression was detected in tendril primordia from the early stage of tendril development.
(B) At a slightly later stage, TEN mRNA accumulated in the growing tendril primordium.
(C) At a later stage of tendril development, TEN mRNA were preferentially enriched in dorsa of coiled tendril.
(D) Sense control of developing tendril.
L, leaf primordium; T, tendril primordium; F, flower primordium; SAM, shoot apical meristem. Scale bar, 0.5 mm.
Molecular Plant 2015 8, DOI: ( /j.molp )
Copyright © 2015 The Author Terms and Conditions

7. Figure 6 Genes Regulated by TEN.
(A) Selected enrichment of GO terms of up-regulated genes in-modified tendril compared with normal tendril.
(B) Quantitative RT–PCR analysis showed that CsYAB1 was not expressed in normal tendril while highly expressed in modified tendril.
(C) The expression of CsYAB1 was repressed significantly by TEN compared with the mutated form (ten) in tobacco (N. benthamiana). The empty pCAMBIA-1300 vector was used as a control. Different letters indicate significant difference.
(D) Selected enrichment of GO terms of down-regulated genes in modified tendril compared with normal tendril.
(E) Quantitative RT–PCR analysis showed that the expression quantity of Csa5G in normal tendrils was several-fold that of modified tendrils.
(F) Coiling response of cucumber tendril treated with GABA (1 mM in 1/4 Hogland nutrient solution), 0.1% Ach (acetylcholine, 0.1% in 1/4 Hogland nutrient solution), and 1/4 Hogland nutrient solution as control.
Molecular Plant 2015 8, DOI: ( /j.molp )
Copyright © 2015 The Author Terms and Conditions