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. 2016 Apr 20;171(2):1156–1168. doi: 10.1104/pp.16.00112

The WD-Repeat Protein CsTTG1 Regulates Fruit Wart Formation through Interaction with the Homeodomain-Leucine Zipper I Protein Mict1

Chunhua Chen 1,2, Shuai Yin 1,2, Xingwang Liu 1, Bin Liu 1, Sen Yang 1, Shudan Xue 1, Yanling Cai 1, Kezia Black 1, Huiling Liu 1, Mingming Dong 1, Yaqi Zhang 1, Binyu Zhao 1, Huazhong Ren 1,*
PMCID: PMC4902597  PMID: 27208299

CsTTG1 regulates the initiation of fruit bloom trichomes and warts and morphogenesis of the fruit spines.

Abstract

The cucumber (Cucumis sativus) fruit is covered with bloom trichomes and warts (composed of spines and tubercules), which have an important impact on the commercial value of the crop. However, little is known about the regulatory mechanism underlying their formation. Here, we reported that the cucumber WD-repeat homolog CsTTG1, which is localized in the nucleus and cytomembrane, plays an important role in the formation of cucumber fruit bloom trichomes and warts. Functional characterization of CsTTG1 revealed that it is mainly expressed in the epidermis of cucumber ovary and that its overexpression in cucumber alters the density of fruit bloom trichomes and spines, thereby promoting the warty fruit trait. Conversely, silencing CsTTG1 expression inhibits the initiation of fruit spines. Molecular and genetic analyses showed that CsTTG1 acts in parallel to Mict/CsGL1, a key trichome formation factor, to regulate the initiation of fruit trichomes, including fruit bloom trichomes and spines, and that the further differentiation of fruit spines and formation of tubercules regulated by CsTTG1 is dependent on Mict. Using yeast two-hybrid assay and bimolecular fluorescence complementation assay, we determined that CsTTG1 directly interacts with Mict. Collectively, our results indicate that CsTTG1 is an important component of the molecular network that regulates fruit bloom trichome and wart formation in cucumber.

Cucumber (Cucumis sativus; 2n = 2x = 14) is one of the most economically important vegetable crops and is cultivated worldwide (Huang et al., 2009). The fruit is usually consumed freshly (Ando et al., 2012), and its visual appearance is an extremely important determinant of its economic value. During early fruit development, deep ridges along the length of the fruit cover the fruit surface and densely spaced fruit trichomes are randomly scattered relative to the ridges (Ando et al., 2012; Chen et al., 2014). Cucumber fruit have two types of trichomes, both of which are multicellular (Chen et al., 2014). Type I are small, glandular trichomes (called bloom trichomes), with a three-to-five-cell base topped with a four-to-eight-cell head. These type I trichomes produce fine, white powdery secretions, giving cucumber a coarse outer appearance (Yamamoto et al., 1989; Samuels et al., 1993; Chen et al., 2014). Type II, which predominate, are much larger, nonglandular trichomes (called spines), which are composed of a base and a stalk (Chen et al., 2014). When spines are combined with tubercules, typically formed at the base of the spine, a mound-like joint connected to the fruit, cucumber fruits have a characteristic warty (Wty) trait. Smooth fruits that lack spines and tubercules, are more convenient to clean, transport, pack and store (Zhang et al., 2010; Yang et al., 2014; Li et al., 2015), and there is therefore great interest in identifying the molecular mechanism that controls the formation of cucumber bloom trichomes and warts (composed of spines and tubercules).

The model experimental plant Arabidopsis (Arabidopsis thaliana) does not exhibit the Wty fruit trait (Yang et al., 2014), and has single-celled, nonglandular trichomes that develop from epidermal cells on the surfaces of aerial organs, including leaves, stems, and sepals (Hülskamp et al., 1994; Marks, 1997; Szymanski et al., 2000). Trichome development in Arabidopsis is dependent on signaling mediated by the hormone GA and has been shown to be regulated by a number of genes, including positive and negative regulators (Perazza et al., 1998; Larkin et al., 2003; Ishida et al., 2008). The three positive regulators TRANSPARENT TESTA GLABRA1 (TTG1; Galway et al., 1994; Walker et al., 1999), GLABRA1 (GL1; Oppenheimer et al., 1991), and GLABRA3/ENHANCER OF GLABRA3 (GL3/EGL3; Payne et al., 2000; Szymanski et al., 2000; Zhang et al., 2003) form a complex to activate trichome formation by enhancing the expression of two target genes, GL2 and EGL2 (Larkin et al., 2003; Serna, 2004).

In contrast to Arabidopsis, very little is known about the regulation of trichome formation and development in cucumber. It has been shown that the phenotype of the csgl cucumber mutant is controlled by a single recessive nuclear gene and that csgl1 is epistatic to the Tuberculate fruit (Tu) gene (Cao et al., 2001; Yang et al., 2014). Tu and Mict/CsGL1 were both identified, and Mict/CsGL1 was shown to be required for further differentiation of cucumber trichomes in all aerial parts of the plant, including leaves, stems, tendrils, floral organs, and fruits, but not for their initiation (Li et al., 2015; Zhao et al., 2015). Recently, a homeodomain-leucine zipper gene, Tril, involved in multicellular trichome initiation in cucumber, was identified (Wang et al., 2016). However, to date, the regulatory mechanisms underlying the development of fruit bloom trichomes and warts are largely unknown.

In this study, we used overexpression and RNAi to study the function of a WD-repeat protein, CsTTG1, in the formation of bloom trichomes and warts in cucumber fruit. WD-repeat proteins belong to a large and highly conserved protein family and are defined by the WD motif, which is typically present as several (4-10) tandemly repeated units containing a conserved core of approximately 40 amino acids that usually ends with Trp-Asp (WD) motif (Neer et al., 1994; Yu et al., 2000; van Nocker and Ludwig, 2003). The loss of function of the WD-repeat protein TTG1 in Arabidopsis was shown to result in severe defects in trichome differentiation (Koornneeff, 1981; Larkin et al., 1999; Walker et al., 1999); however, it is not known whether cucumber fruit trichome formation is regulated by WD-repeat proteins. The results presented here provide important insights into the role of CsTTG1 in the regulatory network regulating cucumber fruit bloom trichome and wart formation.

RESULTS

Cloning and Sequence Analysis of the Cucumber WD-Repeat Homolog CsTTG1

CsTTG1 (Csa4M097650), a TTG1-like gene from cucumber, was previously shown to complement the Arabidopsis ttg1 mutant that is defective in trichome development; it was therefore suggested that CsTTG1 may participate in the formation of trichomes and fruit spines (Guan, 2008). A search of the cucumber genome revealed that CsTTG1 has the highest similarity of predicted cucumber proteins to Arabidopsis TTG1. The CsTTG1 cDNA was derived from mRNA extracted from female cucumber flower buds. The full-length CsTTG1 transcript is 1,591 bp and comprises an open reading frame of 1,026 bp, a 144-bp 5′-untranslated region, and a 421-bp 3′-untranslated region. As is the case with AtTTG1, CsTTG1 contains no introns (Walker et al., 1999; Supplemental Fig. S1A). The CsTTG1 open reading frame encodes a putative WD-repeat protein of 303 amino acids with four WD-repeat domains, and the full-length CsTTG1 protein has 78% sequence identity to AtTTG1 (Supplemental Fig. S1B).

The maize (Zea mays) PALE ALEURONE COLOR1 (PAC1) and MP1 genes encode WD40-repeat proteins closely related to AtTTG1, and PAC1 is required for anthocyanin pigment in the aleurone and scutellum of the maize seed (Hernandez et al., 2000; Carey et al., 2004). Carey et al. (2004) used the deduced PAC1 and MP1 protein sequences as queries to build a phylogenetic tree of homologous WD40-repeat proteins, thereby revealing an ancestral gene duplication leading to two plant clades: the PAC1 clade and the MP1 clade. To understand the evolutionary relationship between CsTTG1 and other WD40-repeat proteins, we also constructed a phylogenetic tree using the neighbor-joining (NJ) method (Saitou and Nei, 1987; Fig. 1). CsTTG1 was found to be clustered within the PAC1 clade that includes ZmPAC1 (maize), PhAN11 (petunia [Petunia hybrida]), GhTTG1 (Gossypium hirsutum), GhTTG3, AtTTG1, and others for which anthocyanin and/or trichome mutant phenotypes have been identified (de Vetten et al., 1997; Carey et al., 2004; Humphries et al., 2005). Within the PAC1 clade, CsTTG1 and CmTTG1 (Cucumis melo), which belong to the Cucurbitaceae family, were grouped within the same subclade. CsTTG1 is closely related to AtTTG1, which regulates unicellular trichome development, suggesting that CsTTG1 may be involved in trichome development in cucumber.

Figure 1.

Figure 1.

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Phylogenetic analysis of CsTTG1. Phylogenetic analysis of CsTTG1 homologs in various species. This NJ phylogenetic tree was constructed using MEGA5 and 1000 replicates. The lengths of the branches refer to the amino acid variation rates. The tree shows two well-supported clades: PAC1 clade and MP1 clade (red labels represent the different clades), consisting of 48 WD40-repeat proteins from 39 plant species. TTG1 homolog from cucumber (CsTTG1) is indicated in the red box. The GenBank accession numbers for the WD40-repeat proteins used for this analysis are listed in Supplemental Table S1.

CsTTG1 Expression Pattern

To better understand the function of CsTTG1, we used quantitative real-time PCR (qRT-PCR) to evaluate its expression in various organs of cucumber line 3407: roots, stems, young leaves, female flower buds, male flower buds, fruits, and tendrils. As shown in Figure 2A, CsTTG1 expression was detected in all examined organs, with the highest levels in female flower buds, male flower buds, and young leaves. The transcript levels were also analyzed in different parts of the cucumber ovary at 7 d before anthesis (DBA; the stage of fruit spine initiation and development) and was found to be expressed at higher levels in the epidermis than in the spine or pulp (Fig. 2B). This result was supported by in situ hybridization analysis, which showed that CsTTG1 transcripts were abundantly expressed in the epidermis, spines, bloom trichomes, and pulp adjacent to the epidermis of 7 DBA ovary (Fig. 2, C–E). In addition, the pCsTTG1-GUS-expressing cucumber line 3407 showed GUS activity in the fruit epidermis, spines, and pulp (Fig. 2, F and G), but the signal was weak in the fruit pulp. No obvious signal was detected in the wild-type cucumber control (Fig. 2H). These results suggested that CsTTG1 plays an important role in epidermal cell differentiation and/or development of fruit spines and bloom trichomes.

Figure 2.

Figure 2.

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The expression pattern of CsTTG1. A, qRT-PCR analysis of CsTTG1 expression in different tissues. The cucumber gene ubiquitin extension protein (UBI-EP) was used as an internal control for normalization, and three independent samples were used for these experiments. Error bars indicate se. B, qRT-PCR analysis of CsTTG1 expression in different parts of the cucumber ovary at 7 DBA. C to E, mRNA in situ hybridization of CsTTG1 in cucumber line 3407 ovaries at 7 DBA. A strong signal was detected in the spine, epidermis, and pulp adjacent to the epidermis (C) and bloom trichomes (D). Black arrows in C and D indicate expression locations of CsTTG1 in fruit trichomes. E, Negative control using the sense probe at 7 DBA. F, Transverse sections of pCsTTG1-GUS cucumber ovaries. G, GUS expression (blue staining) patterns in cucumber spines from the pCsTTG1-GUS transgenic lines. H, Negative control, GUS staining in wild-type fruit spines. R, Root; S, stem; YL, young leaf; MFB, male flower bud; FFB, female flower bud; O, ovary at 7 DBA; T, tendril; Sp, spines; Ep, epidermis; Pu, pulp; Bt, bloom trichome. Scale bars: C to F = 200 μm, G and H = 1 mm.

Subcellular Localization of CsTTG1

To determine the subcellular localization of CsTTG1, a translational fusion between the full-length CsTTG1 coding region and the coding sequence of the GFP reporter, under the control of the 35S promoter (35S:CsTTG1-GFP), was constructed. Cucumber plants expressing this fusion protein showed a fluorescent signal in both the nucleus and the plasma membrane of the fruit spines (Fig. 3, A–C), in contrast to the control expressing 35S:GFP where a signal was observed throughout the whole cell (Fig. 3, D–F).

Figure 3.

Figure 3.

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Subcellular localization of the CsTTG1 protein. Shown are fluorescence micrographs of the spine cells of the transgenic lines expressing 35S:CsTTG1-GFP (A–C) and 35S:GFP (D–F). Scale bars: 100 μm.

CsTTG1 Regulates the Formation of Bloom Trichomes, Ridges, and Warts in Cucumber Fruits

We next fused the full-length coding region of CsTTG1 to the Cauliflower mosaic virus 35S promoter to obtain the 35S:CsTTG1 construct, which was transformed into cucumber line 3413, which has a sparse fruit warts phenotype, and line 3407, which has a dense fruit wart phenotype. Transgenic plants were screened on hygromycin-containing medium, and the presence of the transgene was confirmed by genomic PCR. A total of eight and seven independent positive T1 transgenic lines were obtained for 3413 and 3407, respectively (Fig. 4D; Supplemental Fig. S2D).

Figure 4.

Figure 4.

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Phenotypic analysis of 35S:CsTTG1 transgenic cucumber line 3413 plants. A to C, External morphology of different 35S:CsTTG1 lines. A, Whole cucumber ovaries at 5 DBA. B, Localized regions at 5 DBA. C, Whole cucumber fruits at 9 DPP. D, Relative expression levels of CsTTG1 measured by qRT-PCR analysis of wild-type (WT; 3413) and 35S:CsTTG1 plants. The cucumber UBI-EP gene was used as an internal control. At least three independent samples were used for gene expression analyses. Error bars represent ± se. E and F, Scanning electron microscopy images of the fruit surface of wild-type plants (3413; E) and a 35S:CsTTG1-overexpressing line (F) at 0 DPP. G and H, Cucumber carpopodium of a wild-type plant (3413; G) and the 35S:CsTTG1-overexpressing line OX-1 (H). I and J, Wty fruit in wild-type plants (3413; I) and the 35S:CsTTG1 transgenic line OX-1 (J). Red arrows indicate fruit warts. K, The density of fruit spines at 0 DPP in three 35S:CsTTG1 lines: OX-1, OX-2, and OX-3. Error bars represent ± se. Significant differences were determined by Student’s t test (**P < 0.01). Scale bars: A to C, I, and J = 0.5 cm, E and F = 400 μm, G and H = 1 mm.

Three representative T1 lines (OX-1, OX-2, and OX-3) were selected for detailed studies from the sparsely Wty line 3413 transformants. These lines exhibited much higher levels of CsTTG1 expression than wild-type plants (4.6-, 2.6-, and 3.2-fold, respectively; Fig. 4D). We observed a substantial increase in all three lines, in the number of spines on the surface of the fruit, and carpopodium throughout fruit development (Fig. 4, A–C, G, and H). Specifically, the number of fruit spines at 0 d post pollination (DPP) was 113%, 44%, and 88% higher in OX-1, -2, and -3, respectively, than in wild-type plants (Fig. 4K; Supplemental Fig. S2A). The numbers of bloom trichomes on the surface of the fruit of the transgenic lines were also substantially greater than on those of the wild-type plants (Fig. 4, E and F). Furthermore, the transgenic plants had more fruit tubercules and prominent ridges at 9 DPP than the wild type (Fig. 4, C, I, and J). From the transformants of the high trichome and spine density Wty cucumber line 3407, three representative T1 lines were also selected for detailed studies: OX-13, OX-14, and OX-15. Surprisingly, the average density of fruit spines at 0 DPP was lower in the transgenic lines (Supplemental Fig. S2, A–C). While in the T1 generations of OX-14 and OX-15, only two plants survived. For other lines selected for analysis, fruit spines were counted at 0 DPP on three or more plants. Therefore, we did not show analysis results of OX-14 and OX-15.

To further verify that CsTTG1 regulates the initiation of cucumber fruit spines, an RNAi construct was transformed into Wty line 3407, which has a high spine density. Ten independent T1 transgenic plants were obtained, and the expression of CsTTG1 was shown to be 10% to 75% lower than that in wild-type plants, as determined by qRT-PCR analysis (Fig. 5C). As expected, fruit spine production was reduced at 0 DPP (Fig. 5, A, B, and D), and since there was a correlation between the reduction in spine numbers and CsTTG1 transcript levels, we concluded that this reflected a causal relationship.

Figure 5.

Figure 5.

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Phenotypic analysis of CsTTG1-RNAi cucumber line 3407 plants. A and B, Morphology of the fruit surface of whole fruits (A) and localized regions (B) in different CsTTG1-RNAi lines at 0 DPP. The observations in the framed regions of A are shown in a magnified view in B. C, qRT-PCR analysis of CsTTG1 expression in wild-type plants (WT; 3407) and CsTTG1-RNAi lines. The cucumber gene UBI-EP was used as an internal control for normalization, and the experiments were repeated in three independent samples. Error bars represent ± se. D, The density of fruit spines at 0 DPP in three CsTTG1-RNAi lines: RNAi-3, RNAi-4, and RNAi-10. Error bars represent ± se. Significant differences were determined by Student’s t test (*P < 0.05, **P < 0.01). Scale bars = 0.5 cm.

Overexpression of CsTTG1 Causes Abnormal Fruit Spine Development

The size of the fruit spine in 35S:CsTTG1 plants was much greater than in wild-type plants (Fig. 4B; Supplemental Fig. S2B), and to better assess the role of CsTTG1 in spine development, we characterized their morphology. At 0 DPP, measurements of the transverse diameter (TD) and longitudinal diameter (LD) of the spines from the 35S:TTG1 and wild-type plants showed that the transgenic plants had much larger fruit spine bases (Fig. 6, A–D; Supplemental Fig. S3A). The length (L), cell number (N), and average length of a single cell (L/N) of the stalk were also examined (Fig. 6A). Compared with wild-type plants, the length and L/N of fruit spines from the overexpressors (OX-1, OX-2, OX-3, and OX-13) were 30% to 70% and 13% to 32% greater, respectively (Fig. 6, E and F; Supplemental Fig. S3, B and C). In addition, the stalks had mostly six or seven cells in the wild-type plants of 3413 and 3407 (Table I). All the overexpressing lines had an increased number of stalk cells (Table I), with OX-1 and OX-3 having mostly eight or nine cells and OX-1 up to 11 cells in some cases (Table I).

Figure 6.

Figure 6.

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Morphological characterization of fruit spines at 0 DPP. The length (L), cell number (N), and average length of a single cell (L/N) of the stalk were examined, and the transverse diameter (TD) and longitudinal diameter (LD) of the base were determined. A, A fruit spine shows the measurement sketch. B and C, Paraffin-embedded sections of cucumber spines in wild-type plants (WT; 3413; B) and the 35S:CsTTG1-overexpressing line OX-1 (C). D to F, The size of the base (D), the length of the stalk (E), and the average length of a single cell (L/N) of the stalk (F) were measured in wild-type plants (3413) and 35S:CsTTG1-overexpressing lines (OX-1, OX-2, and OX-3). Scale bars: A = 500 μm; B and C = 200 μm.

Table I. Effect of CsTTG1 on stalk cell numbers of the fruit spines.

WT, Wild type; –, not found.

Genotype Cell No. per Stalk of the Fruit Spinea
 No. of Spinesb
5 6 7 8 9 10 11
WT(3413) 6.9 39.7 36.2 17.2 – – – 116
OX-1 – 2.1 11.7 30.9 41.5 12.7 1.1 94
OX-2 2.9 17.6 29.5 30.9 17.6 1.5 – 68
OX-3 – 2.7 19.8 42.4 29.7 4.5 0.9 111
WT(3407) 4.3 38.6 47.1 10.0 – – – 70
OX-13 – 3.8 33.3 37.2 20.5 5.2 – 78
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a

Percentage of spines.

b

Fruit spines were counted at 0 DPP on three or more plants.

Expression Analysis of CsTTG1 in Different Cucumber Lines

The expression level of CsTTG1 in different cucumber lines, including cucumber lines with the densely or sparsely Wty/nonwarty fruit trait, was analyzed by qRT-PCR using at least three independent RNA samples from 11 DBA female buds (the stage of fruit spine initiation). The expression level of CsTTG1 was found to be higher in some densely Wty lines, such as 3407, 4, 8, and 11, than in other densely Wty lines, such as 5, 6, and 10, and all the sparsely Wty/nonwarty lines (Fig. 7). Thus, a high CsTTG1 expression level correlated with the densely Wty fruit phenotype, but cucumber lines with this phenotype also can have low CsTTG1 expression.

Figure 7.

Figure 7.

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Expression of CsTTG1 in different cucumber lines. A, qRT-PCR analysis of CsTTG1 expression in different cucumber lines. The expression of the cucumber UBI-EP gene was used as an internal control for normalization, and three plants were sampled for gene expression analyses. Error bars indicate ± se. B and C, Morphology of the fruit surface of different cucumber lines. B, At 0 DPP. C, At approximately 10 DPP. Scale bars = 2 cm.

CsTTG1 Interacts with Trichome Formation Regulator Mict/CsGL1 to Regulate Wart Formation

Overexpression of CsTTG1 not only increased the number of fruit spines, but also increased the number of fruit tubercules. Tu, which is required for the Wty fruit phenotype (Yang et al., 2014), was expressed at 12.1- and 4.2-fold higher levels in OX-1 and OX-3, respectively, than in the wild type (Supplemental Fig. S4A).

To explore the regulatory mechanism of CsTTG1 in fruit bloom trichome and wart formation, the genetic relationship between CsTTG1 and Mict/CsGL1, which is required for further differentiation of cucumber trichomes (Li et al., 2015), was examined. The relative expression level of Mict in the 35S:CsTTG1 plants was lower than in wild-type plants (Supplemental Fig. S4A). To further study the genetic relationship between CsTTG1 and Mict in the fruit trichome and tubercule formation pathway, the CsTTG1 gene was overexpressed in the mict mutant background. This caused a higher density of fruit trichomes than in the mict mutant; however, the morphology of the trichomes and the initiation of tubercules was not rescued (Fig. 8). These results showed that the function of CsTTG1 in the regulation of fruit trichome initiation seems independent of Mict/CsGL1. For further differentiation of spine and tubercule formation, CsTTG1 is dependent on Mict/CsGL1, which shows epistatic recessiveness to the Tu gene. It therefore meant that CsTTG1 acts upstream of Mict to regulate the spine further differentiation and upstream of Tu to regulate the tubercule formation.

Figure 8.

Figure 8.

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Phenotypic analysis of a CsTTG1-overexpressing mict/csgl1 mutant. Morphological features of mict (A) and 35S:CsTTG1/mict (B). Left: Whole fruit and carpopodium, and the morphology of the spines on the carpopodium in the framed regions are magnified in C and D, respectively. Scale bars = 0.6 cm. Middle: Scanning electron microscopy images of the spines on the carpopodium. Scale bars = 500 μm. Right: Scanning electron microscopy images of the fruit surface. Scale bars = 200 μm.

Finally, we examined the potential physical interaction between CsTTG1 and Mict. Mict is known to localize to the cell nucleus in onion (Allium cepa) epidermal cells when expressed heterologously (Li et al., 2015), and we also observed here that CsTTG1-GFP was localized mainly to the nucleus and plasma membrane of onion epidermal cells (Fig. 9A). Yeast two-hybrid analysis indicated that CsTTG1 undergoes weak autoactivation because yeast (Saccharomyces cerevisiae AH109) cells continued to grow well on synthetic dextrose (SD)/-Leu,-Trp,-His medium when harboring DNA binding domain (BD)-CsTTG1 and activation domain (AD) empty plasmid. However when grown on SD/-Leu,-Trp,-His medium with 2 mm 3-amino-1,2,4-triazole or SD/-Leu,-Trp,-His,-Ade medium, autoactivation was inhibited. The clear positive results for the combination of CsTTG1-BD and Mict-AD indicated that CsTTG1 interacts physically with Mict (Fig. 9B; Supplemental Fig. S5). To further verify the interactions between CsTTG1 and Mict, bimolecular fluorescence complementation (BiFC) assays were performed in the tobacco (Nicotiana tabacum) leaves. As shown in Figure 9C, fluorescence was observed for both the CsTTG1-YFPN and Mict-YFPC combination as well as the CsTTG1-YFPC and Mict-YFPN combination, but not for the negative control (Supplemental Fig. S6), suggesting that CsTTG1 indeed interacts with Mict. These results indicated that spine further differentiation and tubercule formation may be regulated by the CsTTG1-Mict complex.

Figure 9.

Figure 9.

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Physical interaction between CsTTG1 and Mict. A, Subcellular localization of CsTTG1 protein in onion epidermal cells. B, Yeast two-hybrid assay for the physical interaction between CsTTG1 and Mict. The protein interaction was examined using various combinations of prey and bait vectors. Mating with empty vector pGBKT7 or/and pGADT7 was used as a negative control. The combination of HAN-BD and HAN-AD was used as positive control. Yeast cells were grown on SD/-Leu,-Trp medium. Interactions were confirmed by an X-α-GAL assay on medium. Dilutions (1 and 10−1) of saturated cultures were spotted onto the plates. C, BiFC analysis of the physical interaction between CsTTG1 and Mict. CsTTG1 and Mict were fused with either the C or N terminus of yellow fluorescent protein (YFP; designated as YFPC or YFPN, respectively). INDEHISCENT (IND)-YFPC and SPATULA (SPT)-YFPN were used as positive controls (Girin et al., 2011). Different combinations of the fused constructs were used to cotransform into tobacco cells, and the cells were then visualized using confocal microscopy.

DISCUSSION

CsTTG1 Is Involved in the Regulation of Fruit Wart Formation

In cucumber fruits, the CsTTG1 transcript was found to be more abundantly expressed in the epidermis than in the spine and pulp, and both GUS staining of pCsTTG1-GUS-expressing plants and in situ hybridization clearly showed that CsTTG1 is also expressed in fruit spines and bloom trichomes (Fig. 2, B–K). These results suggested that CsTTG1 may regulate epidermal cell differentiation and fruit spine and bloom trichome development.

The impact of CsTTG1 silencing or overexpression did not affect the gross morphology of the plant. The primary effects were on the production of warts and bloom trichomes in the fruit. In the 3413 line with sparse fruit spines, the expression level of CsTTG1 was low (Fig. 7), and when overexpressing CsTTG1 in this background, more fruit warts and bloom trichomes were formed (Fig. 4) and a gradual increase in spine number in individual lines was correlated with increased CsTTG1 expression levels. Furthermore, RNAi suppression of CsTTG1 in the 3407 line, which has a high density of fruit spines, resulted in a significant decrease in their number (Fig. 5), again transcript expression correlating with spine numbers in individual lines. These results suggested that CsTTG1 regulates fruit wart formation, and that this regulation is dosage-dependent, with higher CsTTG1 expression levels causing more fruit warts. However, we noted that overexpressing CsTTG1 in the densely Wty line 3407 that only exhibited approximately 2.0-fold higher levels of CsTTG1 expression than sparsely Wty line 3413 (Fig. 7) led to significantly decreased numbers of fruit spines (Supplemental Fig. S2, A–C). These results may be partially explained by the following reasons. (1) Based on the activator-inhibitor model, the activator activates factors that inhibit trichome development in neighboring cells (Hülskamp and Schnittger, 1998; Hülskamp, 2004; Larkin et al., 1994). Therefore, CsTTG1 may be involved in promoting the formation of cucumber fruit spine as well as the activation of some inhibiting factor(s). In addition, the promotion of spine formation is more sensitive to increased expression levels of CsTTG1 than the activation of inhibiting factor(s). (2) The polymorphisms of CsTTG1 sequences may exist among different cucumber cultivars, and it will be an interesting area for further investigation.

It seems that, within limits, high CsTTG1 expression is correlated with the densely Wty fruit phenotype but that lines with this phenotype can also have low CsTTG1 expression (Fig. 7). These results indicated that there is no very strict correlation between CsTTG1 expression levels and spine/wart formation, suggesting that other factors are also involved in the regulation of fruit spine/wart formation.

The CsTTG1 Gene Is Involved in a Regulatory Network Controlling the Formation of Fruit Warts

Trichome formation is known to be influenced by GAs in maize (Evans and Poethig, 1995) and Arabidopsis (Chien and Sussex, 1996; Perazza et al., 1998). In cucumber, CsGA20ox1, a gene putatively encoding GA 20-oxidase, has been found to be a negative regulator of fruit spine growth, and transgenic lines with significantly increased expression of CsGA20ox1 were reported to have shorter fruit spines than the wild type (Li et al., 2015). In this study, we found that 35S:CsTTG1 transgenic lines had longer fruit spines (Fig. 6E; Supplemental Fig. S3B), and that the expression of CsGA20ox1 in the transgenic OX-1 and OX-3 lines was down-regulated (Supplemental Fig. S4), indicating that the phenotype was likely regulated by CsGA20ox1, which needs to be confirmed by further studies. The relationship between CsTTG1 and CsGA20ox1 has not been characterized; however, since CsTTG1 interacts with Mict and the regulation of the abnormal spine phenotype is dependent on Mict, CsTTG1 may form a complex with Mict to regulate the expression of CsGA20ox1.

Mict/CsGL1 is required for the further differentiation of cucumber trichomes. Notably, despite BiFC and yeast two-hybrid assay, results support interaction between CsTTG1 and Mict proteins, but the transcript level of Mict was reduced in the 35S:CsTTG1 transgenic plants. Thus, the causal reason is needed for further study. Previous studies have found that Mict displays epistatic recessiveness to the Tu gene (Cao et al., 2001; Li et al., 2015). Tu was up-regulated in the 35S:CsTTG1 transgenic plants, which showed an enhanced Wty fruit trait. CsTTG1 was found to be dependent on Mict to regulate the formation of tubercules and could interact with Mict. Therefore, the formation of tubercules may also be regulated by the CsTTG1-Mict complex.

Proteins containing WD repeats (WDRs) are known to serve as platforms for the assembly of protein complexes or to be mediators of transient interplays between other proteins (van Nocker and Ludwig, 2003). In Arabidopsis, the WDR protein TTG1 interacts with the bHLH transcription factor GL3 while recruiting the R2R3 MYB transcription factor GL1, thereby forming a WD40-bHLH-MYB complex that regulates trichome development (Payne et al., 2000; Szymanski et al., 2000; Morohashi et al., 2007; Zhao et al., 2008). In this study, we observed that cucumber CsTTG1 interacted with the HD-ZIP I protein Mict, suggesting that cucumber trichome formation is regulated by a mechanism that is distinct from that in Arabidopsis. It would be of interest to investigate whether one or more additional proteins are recruited to form this complex and regulate fruit wart formation. In addition, Tril required for the initiation of multicellular trichome in cucumber is recessive and epistatic to the Mict gene (Wang et al., 2016). And in this study, the expression of Tril in the transgenic OX-1 and OX-3 lines had no obvious changes. Based on the results of these studies, we hypothesized a formation mechanism of the fruit spines and tubercules in cucumber (Fig. 10).

Figure 10.

Figure 10.

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A proposed genetic network mechanism for the formation of the fruit spines and tubercules in cucumber. CsTTG1 is parallel to Mict/CsGL1 in the regulation of fruit trichome initiation. The spine further differentiation and tubercule formation are regulated by the CsTTG1-Mict complex (indicated by a yellow star). The question mark indicates one or more additional proteins may be recruited to form this complex. Tril, required for the initiation of multicellular trichome, is recessive and epistatic to the Mict/CsGL1 gene. Mict/CsGL1, required for the further differentiation of cucumber trichomes, shows epistatic recessiveness to the Tu gene. The corresponding regulatory network is depicted with green arrows, indicating epistatic recessiveness. CsTTG1 is highlighted in red type and red circle.

CsTTG1 Has Both Conserved and Divergent Functions Compared with Homologs from Other Species

WDR proteins have been found to play key roles in a variety of processes (Li and Roberts, 2001; van Nocker and Ludwig, 2003). The loss of function of TTG1 in Arabidopsis results in severe defects in trichome differentiation, anthocyanin pigmentation, seed coat pigmentation, and seed coat mucilage (Koornneeff, 1981; Larkin et al., 1999; Walker et al., 1999). The petunia An11, pomegranate (Punica granatum) WD40, Medicago truncatula WD40-1, and maize PAC1 genes, encoding WD-repeat proteins, all control anthocyanin pigmentation and do not regulate trichome development, but they can complement Arabidopsis ttg1 mutant defect in trichomes (de Vetten et al., 1997; Carey et al., 2004; Pang et al., 2009; Ben-Simhon et al., 2011). In addition, the tobacco NtTTG1 protein, a trichome protein with a high degree of sequence identity to AtTTG1, plays an essential role in hypersensitive cell death signal transduction from leaf trichomes to mesophyll cells (Glover et al., 1998; Wang et al., 2009). However, NtTTG2, a paralog of NtTTG1, not only plays a role in suppressing SA/NPR1-regulated defense against pathogens but also regulates development by affecting the expression of ARF genes (Li et al., 2012; Zhu et al., 2013). These studies suggest that WDR proteins, which are structurally strongly conserved, have diverse functions in different species.

In our study, we found that CsTTG1 was expressed in all examined tissues, with the highest expression in female flower buds, male flower buds, and young leaves. This corresponds well with the observed expression pattern of the Arabidopsis AtTTG1 gene, which is highly expressed in floral buds (Walker et al., 1999). CsTTG1 was found to regulate the initiation of cucumber fruit trichomes, including fruit bloom trichomes and spines, supporting the hypothesis that CsTTG1 has a conserved role in trichome formation. However, CsTTG1 also plays an important role in fruit tubercule and ridge formation, and can interact with the HD-ZIP protein Mict, indicating that it has both conserved and divergent functions compared to its homologs from other species.

MATERIALS AND METHODS

Plant Materials and Growth Conditions

The following cucumber (Cucumis sativus) inbred lines were used: the sparsely Wty line 3413, the densely Wty line 3407, and the glabrous and wart-free mutant line mict. All plants were grown in a greenhouse under natural sunlight in the experimental field of the China Agricultural University in Beijing.

Sequence Alignments and Phylogenetic Analysis

A 1,026-bp PCR fragment containing the complete CsTTG1 coding sequence was amplified from female flower bud cDNA using primers CsTTG1-F and CsTTG1-R (Supplemental Table S1). Sequence alignment and phylogenetic analysis was performed based on WD-repeat protein sequences from other species, retrieved using BLAST analysis in the National Center for Biotechnology Information nucleotide database (http://www.ncbi.nlm.nih.gov/nucleotide/), with the deduced amino acid sequence of CsTTG1. The GenBank accession numbers for the WD40-repeat proteins used for this analysis are listed in Supplemental Table S2. The multiple sequence alignment of CsTTG1 and related WD-repeat proteins was performed as reported previously (Zhang et al., 2014). The phylogenetic tree was constructed using the NJ method with the Poisson model and 1,000 bootstrap replicate tests, using the MEGA5 software (Saitou and Nei, 1987).

Spatial and Temporal Expression Analysis of CsTTG1 by qRT-PCR

Total RNA was extracted from specified tissues using Total RNA Isolation System (Huayueyang). One microgram of total RNA was used to synthesize cDNA with the PrimeScript First Strand cDNA Synthesis Kit (Takara). qRT-PCR was performed using the SYBR green detection protocol (Takara) with an Applied Biosystems 7500 real-time PCR system. Expression data were normalized to those of the UBIQUITIN EXTENSION PROTEIN (UBI-EP) gene (Wan et al., 2010). For expression dynamics analysis, three biological replicates were used. All primers used in this study are listed in Supplemental Table S1.

GUS Expression and Staining of Transgenic Cucumber Plants

The putative promoter region of CsTTG1, a 2,000-bp fragment upstream of the start codon (ATG), was cloned and fused upstream of the GUS gene between the PstI and BamHI sites in the pCAMBIA1391 vector (Hajdukiewicz et al., 1994), to generate pCsTTG1-GUS. The construct was introduced into Agrobacterium tumefaciens strain C58 by electroporation and was then transformed into cucumber line 3407 using the cotyledon transformation method as described previously (Wang et al., 2014). Histochemical staining for GUS activity was performed as described by Jefferson et al. (1987). Some samples were made into paraffin section for morphological analysis by microscope.

In Situ Hybridization

Female flower buds from greenhouse-grown cucumber line 3407 were fixed, embedded, sectioned, and hybridized with digoxigenin-labeled probes as described previously (Zhang et al., 2013). Digoxigenin-labeled sense and antisense RNA probes were PCR amplified with T7 and SP6 RNA polymerases (Roche) using gene-specific primers (Supplemental Table S1).

Fluorescence Microscopy of Spines in Transgenic Cucumber Lines Expressing 35S:TTG1-GFP

The binary vector pCAMBIA1300 (CAMBIA) was modified for this study. CsTTG1, with PCR-amplified XbaI and SpeI sites at the ends, was cloned and fused upstream of the GFP sequence in a 35S:GFP construct to form the final vector 35S:CsTTG1-GFP. The transformation of cucumber line 3407 was performed as described above. For the in vivo subcellular localization analysis of CsTTG1, fruit spines of 35S:TTG1-GFP and 35S:GFP transgenic plants were examined using an Olympus BX 51 fluorescence microscope.

Subcellular Localization of CsTTG1 in Onion Epidermal Cells

To investigate the subcellular localization of CsTTG1 in onion epidermal cells, the coding region of CsTTG1 without the stop codon was cloned and fused upstream of the EGFP sequence between the KpnI and BamHI sites of the pEZS-NL vector to generate 35S:CsTTG1-GFP. The empty pEZS-NL vector was used as a negative control. The final vectors, 35S:CsTTG1-GFP and 35S:GFP, were used for transient expression in onion epidermal cells as described previously (Varagona et al., 1992). Fluorescent signals were detected using an Olympus BX 51 fluorescence microscope.

Scanning Electron Microscopy

Samples were fixed, washed, postfixed, dehydrated, and coated as described previously (Chen et al., 2014), and observed using a Hitachi S-4700 scanning electron microscope with a 2-kV accelerating voltage.

Construction of the TTG1 Overexpression Vector and Cucumber Transformation

The full-length coding region of CsTTG1 was amplified and inserted into the BglII and SpeI sites of the pCAMBIA1305.1 vector under the control of the 35S promoter. The construct was transformed into cucumber lines 3407 and 3413 and mutant mict, respectively (Wang et al., 2014).

Construction of the CsTTG1 RNAi Vector and Cucumber Transformation

To generate the CsTTG1-RNAi construct, two fragments of CsTTG1 were generated through PCR amplification using specific primers containing AscI (5′ end) and SwaI (3′ end) sites, and SpeI (5′ end) and BamHI (3′ end) sites. These two fragments were inserted in a reverse orientation into the pFGC1008 vector to form the final construct 35S-CsTTG1-RNAi (Wang et al., 2014). Agrobacterium-mediated transformation of the densely Wty cucumber line 3407 was performed as above.

Yeast Two-Hybrid Assay

Full-length cDNA sequences of CsTTG1 and Mict/CsGL1 were subcloned into pGBKT7 and pGADT7 vectors, respectively. The combination of HAN-BD and HAN-AD was used as positive control (Zhang et al., 2013). All constructs were confirmed by sequencing and then transformed into yeast strain AH109. Yeast cells were grown on a minimal medium/-Leu,-Trp according to the manufacturer’s instructions (Clontech). Protein interactions were assayed on selective medium lacking Leu, Trp, and His with 2 mm 3-amino-1,2,4-triazole or selective medium lacking Leu, Trp, His, and Ade.

BiFC Assays

To generate the BiFC constructs, the full-length cDNA sequences of CsTTG1 and Mict/CsGL1 were cloned and inserted into the pSPYNE and pSPYCE vectors between the XbaI and BamHI sites and the BamHI and SmaI sites (Walter et al., 2004). Coexpression studies were conducted in tobacco (Nicotiana tabacum) leaves as described previously (Schütze et al., 2009). The fluorescence of the expressed fusion proteins was detected 2 to 4 d after infiltration, and fluorescence images were acquired using an Olympus BX 51 fluorescence microscope. YFP imaging used 488 nm excitation wavelength.

Supplemental Material

The following supplemental materials are available.

Supplementary Material

Supplemental Data
supp_171_2_1156__index.html (1.3KB, html)

Acknowledgments

The authors thank Dr. Zhongfu Ni (College of Agronomy, China Agricultural University) for constructive comments on the article, Dr. Zhenxian Zhang (College of Horticulture, China Agricultural University) for the gift of the vector pCAMBIA1391, Dr. Xiaolan Zhang (College of Horticulture, China Agricultural University) for the gift of the yeast strain AH109 and the vectors pGBKT7, pGADT7, pSPYNE, and pSPYCE. In addition, we thank PlantScribe (www.plantscribe.com) for editing this article.

Glossary

Wty

warty

BiFC

bimolecular fluorescence complementation

NJ

neighbor-joining

qRT-PCR

quantitative real-time reverse transcription PCR

DBA

days before anthesis

DPP

days post pollination

SD

synthetic dextrose

WDR

WD repeat

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Associated Data

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Supplementary Materials

Supplemental Data
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