Transcriptome profiling reveals genes involved in spine development during CsTTG1-regulated pathway in cucumber (Cucumis sativus L.)

Journal Pre-proof Transcriptome profiling reveals genes involved in spine development during CsTTG1-regulated pathway in cucumber (Cucumis sativus L.) Pei Guo, Hualin Chang, Qiang Li, Lina Wang, Zhonghai Ren, Huazhong Ren, Chunhua Chen

PII:

S0168-9452(19)31527-4

DOI:

https://doi.org/10.1016/j.plantsci.2019.110354

Reference:

PSL 110354

To appear in:

Plant Science

Received Date:

25 July 2019

Revised Date:

27 October 2019

Accepted Date:

21 November 2019

Please cite this article as: Guo P, Chang H, Li Q, Wang L, Ren Z, Ren H, Chen C, Transcriptome profiling reveals genes involved in spine development during CsTTG1-regulated pathway in cucumber (Cucumis sativus L.), Plant Science (2019), doi: https://doi.org/10.1016/j.plantsci.2019.110354

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Transcriptome profiling reveals genes involved in spine development during CsTTG1-regulated pathway in cucumber (Cucumis sativus L.)

Pei Guo1, Hualin Chang1, Qiang Li1, Lina Wang1, Zhonghai Ren1, Huazhong Ren2, *, Chunhua Chen1, *

State Key Laboratory of Crop Biology; College of Horticulture Science and

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Engineering, Shandong Agricultural University, Tai’an, Shandong 271018, PR China 2

Beijing Key Laboratory of Growth and Developmental Regulation for Protected

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Vegetable Crops, College of Horticulture, China Agricultural University, Beijing,

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*Corresponding author:

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100193, PR China

Highlights

Our study provides a foundation for dissecting the molecular regulatory networks

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E-mail: [email protected] ; [email protected]

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of multicellular trichome (fruit spine) control in cucumber. 

XTH23 and Cyclin family genes were significantly activated by CsTTG1, and may be involved in fruit spine development.

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Transcription factors (TFs) participate in regulation of spine size in 35S:CsTTG1 transgenic plants.

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In 35S:CsTTG1 transgenic plants, GA was implicated in the regulation of fruit spine

development in cucumber.

Abstract The cucumber (Cucumis sativus L.), a type of fleshy fruit, is covered with spines (multicellular trichomes), which have a crucial impact on the economic value of the crop. Previous studied have found that CsTTG1 plays important roles in the initiation

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and further differentiation of cucumber spines, but how spine formation is regulated at the molecular level by CsTTG1 remains poorly understood. In this study, we

characterized a cucumber 35S:CsTTG1 transgenic T2 line, OE-2, which bears

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relatively large and long spines compared with the small and short spines of the wild type (WT). Phenotypic measurements and histological analyses revealed that this

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phenotypic change was attributed to significant increases in cell number and size.

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Comparison of ovary epidermis transcriptomes between OE-2 and WT by DGE (Digital Gene Expression) analysis identified 1241 differentially expressed genes,

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among which, 712 genes were dramatically upregulated and 529 downregulated in the ovary epidermis of OE-2. XTH23 and Cyclin family genes were significantly

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activated in OE-2, and transcription factors (TFs) were found to participate in spine

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size regulation in OE-2. Further analyses confirmed that GA was implicated in the regulation of fruit spine development in cucumber. Thus, our study provides a foundation for dissecting the molecular regulatory networks of fruit spine control in cucumber.

Keywords: gene regulatory networks; transcription factors; fruit spine; Cucumber

1. Introduction Cucumber (Cucumis sativus L.), one of the most economically important vegetable crops, produces fruits that are harvested immature and consumed fresh [1, 2]. The visual appearance of cucumber fruits is an extremely important determinant of their economic value, and the surface of immature cucumber fruit is covered with densely

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spaced fruit trichomes, specialized structures originating from the epidermal cells [3, 4]. In cucumber, fruit trichome morphology varies widely in shape, size, and structure. By examining 200 cucumber varieties, researchers identified eight

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morphologically distinct types (I-VIII) of fruit trichomes, all of which are

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multicellular [5]. Types I and VI-VIII are tiny and invisible to the naked eye. Types IIV, which predominate, are much larger, non-glandular trichomes (called spines)

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composed of a base and a stalk [5, 6]. Cucumber fruits with dense and large spines are

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preferred by consumers in China, whereas fresh cucumber fruit cultivated in European greenhouses (English) or Mediterranean (beit alpha or mini) usually have no visible

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trichomes [5, 7]. Hence, as a key trait of appearance quality, spine size, which is determined by the stalk length and the base transverse and longitudinal diameter or

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transverse/longitudinal diameter ratio, greatly affect the commercial value of the fruit and varietal improvement in cucumber. According to a previous study, CsTTG1 is involved in fruit spine initiation and morphogenesis in cucumber [3]. In plants, cell size and cell number, which are attributed to cell expansion and cell division, act as crucial determinants of organ size

and shape [8, 9]. The development of cotton fibers, which are seed trichomes differentiated from ovule epidermal cells, is a complex process involving numerous genes functioning in concert to regulate cell differentiation and expansion. Key proteins involved in cell wall loosening and various other processes have been shown to be involved in cotton fiber development [10, 11]. Xyloglucan is the major hemicellulose matrix polysaccharide in the primary cell walls, and xyloglucan

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endotransglucosylase/hydrolases (XTHs) are thought to participate in the expansion

and/or assembly of plant cell walls [12, 13]. The expression of XTH was found only in cotton fibers, indicating that XTH plays an important role in cotton fiber development

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[11]. In addition, expansins (EXPs), plant cell wall proteins, regulate cell division and

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plant growth by mediating cell wall extension. EXPs of plants can be divided into four families, a-expansins (EXPAs), b-expansins (EXPBs), expansin-like A (EXLA)

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and expansin-like B (EXLB) [14, 15]. Previous studies have shown that EXPs

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maintain the looseness of the fiber cell wall during the elongation stage [16, 17]. Cyclins and cyclin-dependent protein kinases (CDKs) play critical roles in the

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progression of the cell cycle, which regulates cell division and expansion in plants [18-20]. Plant cyclins are involved in regulating mitotic activity and have been

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grouped into 10 types, including A- to D-, H-, L-, T-, U-, SDS-, and J18-types, among which D-type cyclins control the transition progression from G1 to S phase in response to growth factors and nutrients, whereas very little is known about the roles of U-type cyclins in the cell cycle [21-23]. In rice (Oryza sativa), the U-type cyclin CYC U4;1 participates in the cell cycle at the early G1/S stage [24]. Notably, the

catalytic activity of CDKs is dependent on the binding and activation of cyclin, and can be further controlled by several additional mechanisms [18, 22]. Plant trichome development is also influenced by phytohormones, such as gibberellins (GA), salicylic acid (SA), jasmonic acid (JA), and cytokinin (CTK). In Arabidopsis and tomato, GA, CTK, and JA were reported to play positive roles in trichome production, whereas SA had negative influences on Arabidopsis trichome

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production [25-31]. In cucumber, GA and CTK were found to be capable of

increasing the trichome numbers of fruit, with distinct effects under different concentrations [5].

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In this study, we further studied the function and regulatory network of a

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cucumber gene, CsTTG1, encoding a putative WD-repeat protein that is highly conserved in plants. Functional study of CsTTG1 by constitutive overexpression using

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the 35S promoter revealed positive effects on fruit trichome development, including

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density and morphological alterations of the fruit trichomes. Both the division and elongation of spine cells were promoted by overexpressed CsTTG1. In addition,

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35S:CsTTG1 ovaries exhibited altered levels of GA and CTK. Both physiological tests and genome-wide expression profiling suggested that CsTTG1 may act as

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regulatory proteins involved in mediating the control of fruit spine development by key genes involved in cell wall loosening and phytohormone GA.

2. Materials and Methods 2.1 Plant Materials and Growth Conditions For 35S:CsTTG1 transgenic plants, seeds were first sown on nutritive media, and then, transgenic plants were confirmed by genomic PCR and were transferred to the greenhouse when the seedlings had grown two leaves. All plants were grown in a greenhouse under natural sunlight in the experimental field of the China Agricultural

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University in Beijing. 2.2 Paraffin sectioning

The ovaries or fruits at 0 days post pollination (DPP) were collected and fixed with

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4% paraformaldehyde and left overnight. The fixed samples were dehydrated using

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graded ethanol : 50% ethanol for 2 h, 70% ethanol for 2 h, 80% ethanol for 2 h, 90% ethanol for 2 h, 95% ethanol for 1 h, ethanol for 30 min, and ethanol for 30 min.

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Then, the samples were treated with dimethylbenzene (a transparent agent) and

microscope.

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embedded in paraffin. Paraffin sections were made for morphological analysis by

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2.3 Plant materials for digital gene expression analysis T2 35S:CsTTG1 transgenic and control plants were grown in the same conditions. For

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the three biological replicates, the control plants were from three independent vector lines, and 35S:CsTTG1 plants were from line OE-2. Pericarps approximately 0.2 cm thick were peeled from 1.0–1.3 cm cucumber ovaries at the same time on the same day. 2.4 DGE library construction and sequencing

Total RNA was extracted using a Huayueyang RNA extraction kit (Huayueyang, China). Then, 3 μg of total RNA from six samples (WT-1, WT-2, WT-3, OE-2-1, OE2-2, OE-2-3) was used to prepare the cDNA library according to the manufacturer’s instructions. First, mRNA was enriched from total RNA using oligo-dT magnetic beads, and then the fragmentation buffer was added to break them into short fragments. Fragments of mRNA were used as a template to synthesize the first-strand

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of cDNA using random hexamer primers, and the second strand of cDNA was

synthesized using dNTPs, RNase H and DNA polymerase I. Double- stranded cDNA

fragments were purified using a QIAQuick PCR kit and washed with EB buffer. After

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end repair and the addition of A overhangs and sequencing adapters, the target

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fragments were selectively enriched through agarose gel electrophoresis and PCR. Finally, RNA sequencing was performed on an Illumina HiSeq 2500 platform to

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generate 50-100 bp single-end reads.

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2.5 Bioinformatics analysis of DGE data

Raw reads were filtered to generate clean reads as previously described [32]. Clean

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reads were mapped to the cucumber reference genome (http://cucurbitgenomics.org/organism/2) using TopHat [1, 33]. The transcript level of

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each gene was measured by FPKM (fragments per kilobase of transcript sequence per millions base pairs sequenced). The genes were considered to be significantly differentially expressed when the false discovery rate (FDR) < 0.05 and foldchange≥2. 2.6 Gene Ontology (GO) enrichment analysis

Because GO terms are not well annotated for cucumber genes, Blast2GO was used to assign GO terms to differentially expressed genes. GO terms with corrected P-values of less than 0.01 were considered to be significantly enriched. 2.7 Quantitative real-time PCR To verify the results of the DGE analyses, we independently collected pericarps of cucumber ovaries from WT and transgenic plants, which were the same batch of

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plants at the same time as in the DGE analyses. We used the Huayueyang RNA

extraction kit (Huayueyang, China) for the isolation of total RNA and the PrimeScript RT reagent Kit (TaKaRa, China) for the reverse transcribed cDNA. Quantitative RT-

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PCR (qRT-PCR) analyses were performed with SYBR Premix Ex Taq (Mei5bio,

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China) on an ABI 7500 Real-Time PCR System (Applied Biosystems, USA). For normalization of the expression data, the cucumber ACTIN gene (Csa6G484600) was

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used as a reference control. Each qRT-PCR assay was repeated with three

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independent biological samples. The primers used for qRT-PCR are listed in Table S1.

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2.8 In situ hybridization

Cucumber ovaries from WT were fixed and hybridized as described previously [6].

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Sense and anti-sense in situ probes were synthesized through PCR amplification using gene-specific primers with T7 and SP6 RNA polymerase-binding sites. The primers are listed in Table S1. 2.9 Quantification of phytohormones To examine the gibberellin and cytokinin productions in cucumbers, 0.5-1.0 g ovaries

were excised at 0.5 cm and 3.0 cm of WT and 35S:CsTTG1 plants, respectively. The samples were homogenized in extracting solution (80% methanol containing 1 mmol/L butylated hydroxytoluene). After extraction for 4 h at 4°C, the supernatant was filtered off, and the residue was washed again with extracting solution. The combined filtrates were run through a C-18 SPE (solid phase extraction) column, methanol was removed by vacuum drying or blow drying with nitrogen, and the

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volume was determined using sample diluent. Then, the samples were determined

through coating, washing the plate, competition, washing the plate, adding the second

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antibody, washing the plate, adding the substrate color, and colorimetry.

3. Results 3.1 Phenotypic analysis of fruit spines in 35S:CsTTG1 transgenic plants CsTTG1 (Csa4G097650), which is mainly expressed in the epidermis of cucumber ovary, was previously shown to actively regulate the density of fruit bloom trichomes and spines, thereby promoting the warty fruit trait [3]. T2 plants of overexpression transgenic line OE-2 (from the sparsely Wty line 3413 transformants) were selected

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for detailed studies. The cucumber ovary/fruit of the OE-2 transgenic plant was

characterized by large and dense spines, whereas that of the WT 3413 plant was

covered with smaller and sparser spines. Notably, these morphological differences

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were apparent throughout ovary/fruit development (Fig. 1). As shown in Fig. 1F, the

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spine number of one ovary in OE-2 at 0 days post pollination (DPP) was higher than that in WT. We compared some traits of the spine morphology between OE-2

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transgenic plants and WT. Measurements of the length (L) of the stalk from the OE-2

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and WT plants showed that the transgenic lines had much longer fruit spines (Fig. 2AB; Table 1). Compared with WT plants, the average stalk cell number (N) from the

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overexpressor (OE-2) was approximately one more cell (Table 1). In addition, the average length of a single cell (L/N) of the stalk was also examined, and found that

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L/N obviously increased in the OE-2 (Fig. 2A-B; Table 1). As shown in Table 1, the transverse diameter (TD) and longitudinal diameter (LD) of the spine bases significantly increased in the OE-2 compared with that of WT, leading to the much larger fruit spine bases. The number of base cell layers and the size of the base cell were both significantly higher than those in WT fruits (Fig. 2C-D; Table 1). These

results were identical to the measuring results of the T1 lines (OX-1, OE-2, and OX3) that we published previously [3], indicating that the increased cell number and size of the base and stalk contributed to the spine phenotype of the ovary/fruit in the OE-2 transgenic plant. 3.2 Differentially expressed genes (DEGs) in the epidermis and spines of ovaries To further elucidate the molecular mechanism of CsTTG1 that is involved in the

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regulation of the development of fruit spines, we performed genome-wide expression analyses using the DGE (Digital Gene Expression) approach. Given that the rapid

expansion of the spine base occurred when the fruit length was between 0.5 and 1.5

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cm [6], we chose ovaries at 5 DBA (days before anthesis; approximately 1.0-1.3 cm,

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the key stage for fruit spine expansion) and peeled pericarps of approximately 0.2 cm thick for RNA-Seq analyses. Three biological replicates were performed for OE-2

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transgenic plants and WT, and thus six DGE libraries were sequenced. As indicated in

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Table 2, 12.18-12.94 million raw reads from each library were generated. When adapter sequences and low-quality reads were removed, we obtained 11.23-11.86

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million clean reads. Of these clean reads, Q30 (with a base quality of more than 30) was 96.89%-97.18%. Afterwards, we clustered these clean reads into unique reads

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and mapped to the cucumber genome with TopHat [33]. In the six libraries, approximately 10.89-11.17 million clean reads (96.44%-97.15% of total clean reads) were mapped uniquely to the cucumber genome (Table 2). After deep sequencing, we obtained 23,248 genes from these six libraries. Using the FDR < 0.05 and fold change > 2 as the significance cut-off, 1,240 DEGs were

identified. Among these DEGs, we found that the number of significantly upregulated and downregulated genes was 712 and 529 in the pericarps of the OE-2 transgenic plant compared with those of the WT, respectively (Table S2). To validate the DEGs identified by DGE, qRT-PCR assays were performed using the independent cucumber epidermis and fruit spine samples of both genotypes during the same developmental stages of spine as those used for the DGE. Moreover,

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12 DEGs were randomly selected for qRT-PCR analysis, of which 7 were upregulated genes and 5 were downregulated genes. Despite differences in the exact fold change

values, the expression patterns of these selected genes in the qRT-PCR and DGE data

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indicated that the DGE data were reliable.

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were consistent (Fig. 3). The pearson correlation coefficient (0.81, P = 8E-04)

3.3 Cell elongation/expansion- and cell division-related genes implicated in the

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development of the cucumber fruit spine

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To analyse the functions of DEGs identified by DGE profiling, a total of 1,241 DEGs were grouped into three major categories, namely, biological process, cellular

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component and molecular function, through gene ontology (GO) term enrichment analysis (Fig. S1). The top six enriched GO terms were “cellular process” (490

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genes), “metabolic process” (476 genes), “cell part” (610 genes), “organelle” (417 genes), “binding” (513 genes), and “catalytic” (428 genes) in these three major categories (Fig. 4; Fig. S1 and S2). And the number of genes that were downregulated in these significantly enriched groups was significantly lower than that were upregulated (Fig. 4; Fig. S2; Table S2). These results indicated that CsTTG1

significantly affected the expression of genes related to cell part, consistent with the phenotypes of the fruit spines in 35S:CsTTG1 transgenic plant and the WT. Given that the increased cell size in the base and stalk contributed to the larger fruit spine phenotype in the 35S:CsTTG1 transgenic plant (Fig. 1 and 2; Table 1), we screened for cell elongation/expansion-related genes involved in the cellular component in the DEGs. EXPs encode EXPANSINs for wall loosening and are

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generally known as key regulators of wall extension during growth [34, 35]. We

found that the transcript levels of EXPs (EXPA5, EXPA8, EXPB3, EXP12) were all reduced by 2.02-, 3.05-, 6.25-, 2.21-, and 4.74-fold, respectively, in the OE-2

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transgenic plant compared with those in the WT (Table 3). Xyloglucan

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endotransglycosylases/hydrolases (XTHs) split and reconnect xyloglucan chains or suitable xyloglucanderived oligosaccharides (XGOs) in the primary wall, leading to

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cell wall expansion [12, 36]. Five XTH genes were identified among the DEGs, and

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the transcript levels of these XTHs, except for XTH23, were all downregulated (Table 3). According to the normalized results of these XTH genes in DGE profiling, we

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found that the expression level of upregulated gene XTH23 was much higher than those of the other XTH genes (Fig. S3A). We used qRT-PCR to evaluate the

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expression of these XTHs in different tissues and organs: tendrils, roots, stems, leaves, male flower buds, female flower buds, 5 and 0 DBA ovaries. As shown in Fig. 5A, a heatmap of 5 XTH genes, represented by values of the relative expression, was established by TBtools. In FFB and ovary, XTH23 showed the highest transcript abundances. The transcript level of XTH23 was also analyzed in different parts of the

cucumber ovary at 5 DBA and 2 DBA (approximately 3.0 cm; the stage of fruit spine during slow expansion), and was found to be expressed at higher levels in the epidermis and spine than in the pulp at 5 DBA. And its expression level was reduced along with the fruit/spine development (Fig. 5B). In addition, in situ hybridization analysis showed that XTH23 transcripts were abundantly expressed in the epidermis and spines of 5 DBA ovary (Fig. 5C-E). This was consistent with high level in XTH23

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expression in the epidermis and spines revealed by RT-PCR data. Accordingly, we speculated that XTH23 might positively influence the formation of the fruit spine

phenotype, particularly through increased cell size. In 35S:CsTTG1 transgenic plants,

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CsTTG1 may upregulated the transcript level of XTH23 to affect the size of the spine.

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Moreover, the increased cell number in the base and stalk also contributed to the spine phenotype of the ovary/fruit in the OE-2 transgenic plant. In the OE-2, we

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further identified eight CDK (Cyclin-dependent Kinase) and Cyclin genes, which are

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the key regulators in the cell cycle machinery [37], and six of these genes were upregulated by 2.02- to 5.04-fold, respectively, compared with those in WT (Table 3).

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These data indicated that cyclins might be associated with the regulation of fruit spine size and shape in cucumber and play positive roles in the formation of the OE-2 spine

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phenotype.

3.4 GA and CTK are associated with the regulation of fruit spine development It has been reported that 6-benzylaminopurine (BAP, a synthetic cytokinin) and GA can affect spine (trichome) numbers per cucumber fruit, but there are no effects on the size and shape of fruit spines [5]. To test whether there is a correlation between

CsTTG1-regulated and GA- or CTK-modulated spine formation in cucumbers, we first screened for GA- and CTK-related genes in the DEGs. As shown in Table 3, several GA- and CTK-related genes were differentially expressed in the ovary pericarps of the 35S:CsTTG1 transgenic plant compared with those of WT. For GArelated genes, CsTTG1 upregulated the transcripts of Csa7G391240 (GID1B) and Csa1G064730 (G2OX8), but downregulated Csa7G434970 (G3OX) expression in the

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OE-2 ovary. The expression level of upregulated gene Csa1G064730 (G2OX8) was

much higher than that of the downregulated gene Csa7G434970 (G3OX) (Fig. S3B). For CTK-related genes, half of these genes showed significantly downregulated

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expression in 35S:CsTTG1 plants. Then, we measured GA and CTK productions in

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the ovaries of 35S:CsTTG1 plants at 7 DBA (approximately 0.5 cm; the stage of fruit spine initiation and development) and 2 DBA, respectively (Fig. 6). We found that

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GA production had a highly significant change in the 7 DBA ovaries rather than 2

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DBA ovaries compared with the wild type. In contrast, CTK production significantly increased in the 2 DBA ovaries compared with wild type, but it remained almost

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unchanged at 7 DBA (Fig. 6). These results suggested that GA may be involved in the pathway of CsTTG1-regulated cucumber fruit spine formation.

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3.5 Transcription factors associated with the formation of cucumber fruit spines For the differentially expressed genes that were identified by genome-wide expression analyses, the top three enriched GO terms were "cell part", "binding" and "cellular process" (Fig. S1). A total of 196 genes that function in transcriptional regulation were identified, and 141 of these transcription factors (TFs) were found to increase

the level of expression in the ovaries of the 35S:CsTTG1 transformant (Fig. S4; Table S3). These 141 genes could be subdivided into different categories according to the protein structure. As shown in Fig. 7, there are 21, 18, 14, 9, 8 and 8 genes classified into the ERF, Zinc finger, MYB, WRKY, bHLH and MADS families, respectively, that displayed increased transcript levels in the OE-2 transgenic line with denser and larger fruit spines, suggesting that these TFs may function as positive factors involved

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in fruit spine development control. C2H2 zinc finger proteins have been found to play important roles in the control of epidermal cell specification. For example, GLABROUS INFLORESCENCE STEMS (GIS), ZINC FINGER PROTEIN 8

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(ZFP8) and GLABROUS INFLORESCENCE STEMS 2 (GIS2), ZINC FINGER

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PROTEIN 5 (ZFP5), and ZINC FINGER PROTEIN 6 (ZFP6) were discovered as crucial regulators of trichome initiation through the GA signaling pathway

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in Arabidopsis [38]. Among the Zinc finger TFs that we identified, 11 genes were

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C2H2 type. The MYB family of proteins is large and functionally diverse, and several R2R3-MYB proteins are involved in the regulation of cell fate and identity in

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Arabidopsis, including AtMYB0/GL1, AtMYB5, AtMYB23 and AtMYB66 [39]. AtMYB5 regulates trichome branching and extension in combination with AtMYB23

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[39, 40]. We found that the predicted cucumber MYB5 (Csa5G148680) showed a 2.68-fold increase as detected by expression analysis (Table S3). In addition, members of other families of TFs, such as Csa3G119700 (a WRKY transcription factor 30) and Csa3G782720 (a MADS box protein), showed 7.43- and 18.47-fold upregulation in the ovary of OE-2, respectively (Table S3). These data suggest that

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TFs may play important roles in the regulation of spine formation in cucumber.

4. Discussion The development of Arabidopsis trichome, the unicellular trichome, is dependent on signaling mediated by the hormone GA and has been shown to be regulated by the number of genes [26, 41]. Compared to Arabidopsis trichomes, spines are defined as multicellular trichomes, that originate from epidermal cells [6], and the molecular mechanism that regulates the formation of cucumber spines is more complex. Based

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on previous studies, we have shown that CsTTG1, a WD-repeat homologue,

participates in the regulation of the initiation and morphogenesis of spines in

cucumber fruits. In this study, OE-2 plants overexpressing CsTTG1 in the background

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of sparsely Wty line 3413, were selected for detailed studies. Compared with WT

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3413, the cucumber fruit spines of the 35S:CsTTG1 transgenic plants are denser (Fig. 1, Table 1). In terms of their morphological characteristics, the spines of OE-2 plants

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are larger and longer than those of WT (Fig. 1 and 2, Table 1). This phenotype is

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possibly due to the increased cell number and size (Fig. 1 and 2, Table 1), indicating that cell expansion and division may be involved in the transformant phenotype.

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The Arabidopsis TTG1, a WD-repeat protein, forms a complex with the bHLH protein GLABRA3/ENHANCER OF GLABRA3 (GL3/EGL3) [42] and the R2R3

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MYB-type transcription factor GLABRA1 (GL1) [43] to control trichome differentiation [44]. TRY, an R3 MYB transcription factor, can suppress the initiation of trichome formation by competing with GL1 to interact with GL3/EGL3 [45, 46]. In cucumber, CsTTG1 was found to play important roles in regulating the differentiation and morphogenesis of fruit trichomes (spines). CsGL1/Mict, encoding an HD-ZIP I

protein, was shown to be involved in regulating further differentiation of cucumber trichomes in all aerial parts of the plant, but not in their initiation [47, 48]. The function of CsTTG1 in the regulation of fruit trichome initiation seems independent of Mict/CsGL1, and dependent on Mict/CsGL1 to regulate further differentiation of spine formation [3]. In cucumber, overexpression of CsMYB6，a MIXTA R2R3MYB homologue, and CsTRY, a R3 MYB transcription factor, revealed that they act as

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negative regulators of trichome initiation [49]. However, the Arabidopsis MIXTA

R2R3MYB homologue MYB106 affects the formation of trichome branches rather than trichome initiation [50]. Thus, these studies suggest that the developmental

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controlled by different regulatory mechanisms.

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processes of cucumber multicellular and Arabidopsis unicellular trichomes are

To further explore the regulatory mechanism of spine developmental processes

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in cucumber, we provided a comprehensive analysis of genes that are involved in CsTTG1-regulated spine formation. A total of 1241 DEGs were found, of which 712

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genes were significantly upregulated and 529 genes were significantly downregulated in the OE-2 with dense and large spines (Fig. 1 and 2; Table 1). The results of qRT-

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PCR confirmed the high reliability of our gene expression profile data (Fig. 3). The functional categories of the DEGs identified by GO term enrichment analysis showed

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that cell cycle-related genes were strongly induced in OE-2 (Fig. 4 and Fig. S1 and S2).

XTHs are enzymes that have two distinct activities on xyloglucan:

endotransglycosylation (XET) and hydrolysis (XEH) [13]. Therefore, they play key roles in cell expansion by catalyzing the cleavage of xyloglucan molecules and the reconnection of their reducing ends to non-reducing ends of other polymeric or

oligomeric xyloglucan molecules [11, 36, 51]. Previous data also showed XTH expression only in fibers of cotton [11]. The gene that encode the cell wall-modifying proteins XTH23 was found to be upregulated in the OE-2 line (Table 3; Fig. S3A). However, the transcript levels of the other four XTH genes that we identified were all downregulated (Table 3; Fig. S3A). We also found that the expression level of XTH23 was much higher than those of the downregulated XTH genes (Fig. S3A). Furthermore, XTH23 showed higher transcript abundances in FFB and ovary than

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other XTH genes (Fig. 5A). For XTH23 the transcript level was expressed at higher levels in the epidermis and spine than in the pulp at 5 DBA (Fig. 5B-E), and was

expressed at lower levels in the all parts of the cucumber ovary at 2 DBA than those

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at 5 DBA (Fig. 5B). It is likely that XTH23 may act as a master regulator for the expansion of spine cells, and its expression levels was reduced along with the

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development of spines slowing down. Moreover, 6 cyclin family genes were activated

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in line OE-2 with long and large spines (Table 3). Previous evidence has shown that the transcription of cyclins and CDKs positively correlates with cell division during early fruit development in tomato and cucumber [4, 21, 52]. EXPs, a primary

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regulator of plant cell enlargement, can induce the extension of cell walls in a pHdependent manner [53]. In our analysis, EXPs genes all exhibited downregulation in

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line OE-2 (Table 3). Given that the samples we used for expression analysis were

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pericarps collected from ovaries at 1.0-1.3 cm, a rapid spine expansion stage [6], together with the fact that the cell number and size of the spines increased in line OE2 compared to the WT (Fig. 1 and 2), it is plausible to infer that the long and large fruit spines in line OE-2 may result from both more rapid cell expansion and division, which correlated with increased expression of XTH23 and cyclin family genes. Furthermore, we concentrated on a new set of 195 TF genes identified in our

expression analysis (Fig. S3). According to their structural domains, upregulated TFs could be subdivided into different categories, including zinc finger, ERF, MYB, WRKY, MADS, NAS, HSF and so on (Fig. 7). In Arabidopsis, several zinc finger and MYB proteins and one WRKY protein, TTG2, are involved in the regulation of trichome development [38, 39, 54]. MADS TFs play important roles in flower and fruit development [55]. NAC proteins have been shown to function in relation to plant development and abiotic and/or biotic stress responses [56]. In this study, genes

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encoding MADS, NAC, and other TF proteins also exhibited significantly differential expression between the OE-2 and WT lines. Thus, these genes were identified as

candidates encoding crucial TFs associated with multicellular spine development and

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regulated by CsTTG1.

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In addition to TFs, spine development is also regulated directly by phytohormones. In Arabidopsis, SA (salicylic acid) and JA (jasmonic acid) promote and suppress the

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formation of trichomes on leaves, respectively [27]; cytokinin (CTK) and gibberellin acid (GA) signals regulate the initiation of trichomes on inflorescences [57], and zinc

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finger proteins GIS, ZFP8 and GIS2, ZFP5, and ZFP6 are important regulators of the CTK and GA signaling pathways. In cucumber, ethylene gas has been discovered as a

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regulator that stimulates epidermal cell division, resulting in aberrant guard cell and

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trichome formation [58]. Xue et al. revealed that BAP (a synthetic cytokinin) and GA are able to stimulate the formation of cucumber spines, but there are no effects on the size and shape of fruit spines. A series of GA- and CTK-related, ethylene-responsive and zinc finger genes were also identified in our expression analysis (Tables 3 and S2), suggesting that the expression of these genes correlated with phytohormone-related

regulation of multicellular trichomes. Compared to WT, GA and CTK production had highly significant changes in 7 DBA and 2 DBA ovaries, respectively (Fig. 6). Therefore, we speculate that GA may be mainly involved in the pathway of CsTTG1regulated cucumber fruit spine initiation. Fruit tubercules were observed from 2 DBA to 13 DPA, during which time tubercule cells were likely to divide rapidly [59]. Thus, CTK may be involved in the pathway of CsTTG1-regulated cucumber fruit tubercules

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formation. As such, gene-expression profiling provides a valuable resource for further

functional characterization and lays a foundation for studying the regulatory networks

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that control multicellular trichome development in plants.

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Author contributions

C. Chen conceived the project. C. Chen and H. Ren designed the

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experiments. P. Guo performed the experiments and the data analysis. C.

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checked it.

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Chen wrote the manuscript, P. Guo, H. Chang, Q. Li, L. Wang and Z. Ren

Conflict of interest

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The authors declared that they have no conflicts of interest to this work. We declare that we do not have any commercial or associative interest that represents a conflict of interest in connection with the work submitted。

Acknowledgments This work was supported by fundings from the National Natural Science Foundation of China (31830080 and 31701923), Natural Science Foundation of Shandong Province

(ZR2017BC085) and the Project of Beijing Agricultural Innovation Consortium

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(BAIC01-2019), and Key R & D plan in Shandong (2018GNC110014).

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Tuberculate fruit gene Tu encodes a C2H2 zinc finger protein that is required for the warty fruit

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phenotype in cucumber (Cucumis sativus L.), Plant J, 78 (2014) 1034-1046

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Fig. 1. Phenotypic analysis of CsTTG1 overexpression line OE-2 plants. (A-E) The external morphological of 35S:CsTTG1 transgenic cucumber T2 line. (A-B) The whole cucumber ovaries at about 4 DBA. (C-D) Cross section of ovaries at 0 DPP. (E) The whole cucumber fruits at 10 DPP. (F) The number of spine on one ovary at 0 DPP. Fruit spines were counted on three or more plants and at

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least three ovaries were chosen from each plant. Error bars represent ± SE. Significant differences were determined by Student’s t-test (**P < 0.01). DBA, days before anthesis. DPP, days post pollination.

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Scale bars: (A-B) = 1.0 cm, (C-D) = 2.0 mm, (E) = 2.0 cm.

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Fig. 2. Morphological characterization of fruit spines. (A-B) The length of the stalk. (C-D) Paraffin-

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embedded sections of cucumber spines. Scale bars = 500 um.

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Fig. 3. Verification of differentially expressed genes by qRT-PCR. Seven DEGs with higher expression and five DEGs with lower expression in transgenic line OE-2 were chosen for qRT-PCR validation.

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The blue and orange bars represent qRT-PCR and RNA-Seq data, respectively.

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Fig. 4. Gene Ontology (GO) terms (P < 0.05) that were significantly enriched in the upregulated genes between the pericarp of line OE-2 and WT. GO terms belong to molecular function (MF), cellular

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components (CC), and biological processes (BP) were shown in green, red, and blue, respectively.

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Fig. 5. The expression pattern of XTH genes. (A) Heat map representation of expression profiles of XTH genes in different tissues and organs. Values of the relative expression were used to established

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this heat map by Tbtools. (B) The expression of XTH23 was analyzed by qRT-PCR in different parts of the cucumber ovary at 5 DBA and 2 DBA, respectively. (C-E) mRNA in situ hybridization of XTH23 in cucumber ovaries at 5 DBA. A strong signal was detected in the epidermis (C)and spine (D). Red

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arrows in (C) and (D) indicate expression locations of XTH23. (E) Negative control using the sense probe at 5 DBA. MFB, male flower bud. FFB, female flower bud. DBA, days before anthesis. Scale bars = 200 um.

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Fig. 6. GA and CTK may be involved in the formation of cucumber spine. (A) Quantification of GA in the ovaries at 0.5cm and 3.0cm, respectively. (B) Quantification of CTK in the ovaries at 7 DBA and 2

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DBA, respectively. DBA, days before anthesis.

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Fig. 7. Family assignment of the 141 up-regulated transcription factors in transgenic line OE-2.

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Number of genes divided to each category is shown behind the family name.

Table 1. Phenotypic characterization of fruit spines at 0 DPP.

The base

WT

OE-2

The length (L)

1420.24 ± 402.11A

1839.06 ± 485.23B

Cell number (N)

6.57 ± 0.88 A

7.44 ± 1.28 B

Average length of a single cell (L/N)

214.55 ± 50.42 A

241.23 ± 44.90 B

The transverse diameter (TD)

715.03 ± 160.83 A

825.57 ± 129.75 B

The longitudinal diameter (LD)

583.14 ± 126.24 A

694.98 ± 147.94 B

Number of cell layers

4.22 ± 0.67 A

6.00 ± 0.82 B

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The stalk

Phenotypic characters

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The values shown are the means ± SD of fruit spines from three or more independent plants, and fruit spines were counted at 0 DPP on three or more fruit for every plant. Different letters (A and B) in column indicate significant differences (P < 0.01) between WT and OE-2 transgenic line determined by Student’s t test.

Table 2. Summary of the transcriptome assembly Samples

OE-rep1

OE-rep2

OE-rep3

WT-rep1

WT-rep2

WT-rep3

Raw reads

12,936,237

12,717,591

12,479,343

12,181,439

12,671,174

12,321,429

11,863,469

11,771,911

11,579,516

11,233,395

11,785,467

11,489,468

(91.71)

(92.56)

(92.79)

(92.22)

(93.01)

(93.25)

96.89

97.18

96.90

97.13

97.16

97.10

11,605,489

11,519,826

11,329,983

10,976,202

11,523,046

11,217,198

(97.83)

(97.86)

(97.85)

(97.71)

(97.73)

(97.63)

11,274,940

11,165,677

10,980,964

10,643,286

11,112,761

10,894,975

(97.15)

(96.93)

(96.92)

(96.97)

(96.44)

(97.13)

Clean reads (％) Clean Q30 bases rate

Mapped clean reads (％) Unique mapped clean

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reads (％)

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(％)

Table 3. List of cell size- and division-related genes that were differentially expressed in the OE-2 and WT. Putative annotation

P value

Fold Change (OE-2 / WT)

Csa7G391240

GID1B (Gibberellin receptor GID1)

8.25E-15

2.76

Csa1G064730

G2OX8

5.96E-24

2.53

Csa7G434970

G3OX (Gibberellin 3-beta hydroxylase)

2.56E-17

-2.73

Gene ID

CKX-related LOG1 (Cytokinin riboside 5'-monophosphate phosphoribohydrolase)

Csa3G150100

IPT5 (cytokinin synthase)

Csa6G030440

IPT (cytokinin synthase)

Csa7G392940

IPT3 (cytokinin synthase)

Csa4G343590 Csa5G175820 Csa6G067360

-2.30

5.59E-05

-5.87 3.49

4.41E-23

-12.66

CKX1 (Cytokinin oxidase/dehydrogenase 1)

8.12E-04

2.81

CKX6 (Cytokinin oxidase/dehydrogenase 1)

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8.60E-05

-4.02

AHP1 (redundant positive regulators of cytokinin signaling)

7.09E-27

2.34

WOX9 (a downstream effector of cytokinin signaling)

0

19.61

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6.69E-04

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Csa6G518270

2.41E-05

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Csa2G367210

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GA-related

Cell elongation/expansion-related XTH2 (Xyloglucan endotransglucosylase/hydrolase protein 2)

5.84E-03

-2.52

Csa1G188670

XTH6 (Xyloglucan endotransglucosylase/hydrolase)

4.41E-15

-21.62

Csa1G188680

XTH7 (Xyloglucan endotransglucosylase/hydrolase)

1.54E-66

-131.39

Csa3G741330

XTH14 (Xyloglucan endotransglucosylase/hydrolase protein 14)

2.00E-22

-2.46

Csa1G422990

XTH23 (Xyloglucan endotransglucosylase/hydrolase)

0

5.53

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Csa6G147660

Csa5G636630

EXPA5 (Expansin protein)

9.43E-53

-2.02

Csa7G047420

EXPB1 (Expansin B1)

1.12E-22

-3.05

Csa5G221940

EXPA8 (Expansin S1)

3.81E-84

-6.25

Csa6G014540

EXPA8 (Expansin)

2.40E-48

-2.21

Csa3G104900

EXP12 (Expansin)

7.97E-04

-4.74

CDKG2 (Cyclin dependent kinase)

Csa1G617380

cyclin-like

Csa6G108560

Cyclin-U2-1

Csa4G025110

Cyclin-U4-1

Csa6G499110

Cyclin d

Csa6G516960

cyclin-like

Csa3G878200

cyclin-like

Jo

re lP

na Cyclin-U1-1

ur

Csa2G422050

2.11E-11

2.02

2.77E-24

2.57

4.08E-05

-2.04

-p

Csa2G350480

ro of

Cell division -related

2.33E-03

-2.89

6.89E-13

2.22

0

5.04

1.55E-11

2.26

8.41E-04

4.76