YGENO-08719; No. of pages: 8; 4C: Genomics xxx (2015) xxx–xxx
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Transcriptome analysis in Cucumis sativus identifies genes involved in multicellular trichome development Jun-Long Zhao, Jun-Song Pan, Yuan Guan, Jing-Tao Nie, Jun-Jun Yang, Mei-Ling Qu, Huan-Le He, Run Cai ⁎ School of Agriculture and Biology, Shanghai Jiao Tong University, 800 Dongchuan Road, Minhang District, Shanghai 200240, China
a r t i c l e
i n f o
Article history: Received 19 November 2014 Accepted 23 January 2015 Available online xxxx Keywords: Cucumis sativus DNA-binding proteins Gene regulatory networks Transcription factors Transcriptome Trichomes
a b s t r a c t The regulatory gene network of unicellular trichome development in Arabidopsis thaliana has been studied intensively, but that of multicellular remains unclear. In the present study, we characterized cucumber trichomes as representative multicellular and unbranched structures, but in a spontaneous mutant, mict (micro-trichome), all trichomes showed a micro-size and stunted morphologies. We revealed the transcriptome profile using Illumina HiSeq 2000 sequencing technology, and determined that a total of 1391 genes exhibited differential expression. We further validated the accuracy of the transcriptome data by RT-qPCR and found that 43 genes encoding critical transcription factors were likely involved in multicellular trichome development. These 43 candidate genes were subdivided into seven groups: homeodomain, MYB-domain, WRKY-domain, bHLH-domain, ethylene-responsive, zinc finger and other transcription factor genes. Our findings also serve as a powerful tool to further study the relevant molecular networks, and provide a new perspective for investigating this complex and species-specific developmental process. © 2015 Elsevier Inc. All rights reserved.
1. Introduction Trichomes are highly specialized structures developed from the protodermal cells of most terrestrial plants. They play important biotic and abiotic roles in many aspects, such as insect, herbivore and microbe deterrence; water regulation through transpiration; light reflectance (including UV) [1,2]; pollen collection and dispersal; absorption of water and nutrients [3]; secretion of ions and pollutant metals [4,5]; reduced mechanical abrasion; and regulation of surface temperature [2]. Trichomes can be categorized according to whether they are unicellular or multicellular, glandular or glandless, and branched or unbranched [3]. They also provide a suitable model system for researching cell differentiation at the single-cell level, including cell fate determination, cell cycle developmental control, and cell morphogenesis [6,7]. In Arabidopsis thaliana, the unicellular trichome differentiation is thought to be regulated by a competitive system comprising promoting and limiting activities. The promoting activity up-regulates both activities, and the limiting activity spreads via cell-to-cell communication and inhibits trichome differentiation [8–10]. Critical positive transcription factors include GL1 (GLABRA1), which acts as an R2R3 MYB protein [11]; GL3 (GLABRA3) and its homolog EGL3 (ENHANCER OF GLABRA3), which act as basic helix-loop-helix proteins [12]; and TTG1 (TRANSPARENT TESTA GLABRA1), which acts as a WD40-repeat protein [13]. In contrast, negative transcription factors include TRY (TRIPTYCHON), ⁎ Corresponding author. Fax: +86 21 34206938. E-mail address: [email protected] (R. Cai).
CPC (CAPRICE), and ETC (ENHANCER OF TRY AND CPC), which belong to the small R3 single-repeat MYB family [14,15]. In this system, GL1, GL3/EGL3 and TTG1 constitute the promoting activity, TRY/CPC/ETC, GL3/EGL3 and TTG1 constitute the limiting activity, and GL2 (GLABRA2), a homeodomain protein, acts as a quantitative factor directly regulated by both activities [6,7,9,16]. Cucumber (Cucumis sativus L., 2n = 2x = 14), an annual sprawling herbaceous plant, is one of the most commercially important vegetable crops worldwide. Cucumber also serves as a model plant for sex determination studies due to its diverse sex types [17]. Trichomes are widely found on leaves, stems, flowers, tendrils and fruits of the wild type cucumber plants. The trichomes on the fruits are commonly called “fruit spines” (Fig. 1A, B). Cucumber fruits are economically valuable and fruit spines directly affect the appearance quality. Here, a spontaneous mutant, which originated from North China inbred line 06-1, presented a glabrous phenotype on leaves, stems, flowers, tendrils and fruits (Fig. 1C, D). Scanning electron microscopy revealed that all trichomes in this mutant exhibited a micro-size and stunted morphologies, and are only visible under at least 20 times magnification, thus, we named this gene “Mict (Micro-trichome)” and this line “mict”. Compared with unicellular trichomes, the regulatory mechanisms of multicellular trichome development in plants are much less understood. In this study, we characterized cucumber trichomes as multicellular and unbranched. We further used Illumina HiSeq 2000 sequencing technology to reveal the transcriptome changes between the mict mutant and wild type, and identified a series of candidate genes encoding critical transcription factors which are likely involved in multicellular trichome development.
http://dx.doi.org/10.1016/j.ygeno.2015.01.010 0888-7543/© 2015 Elsevier Inc. All rights reserved.
Please cite this article as: J.-L. Zhao, et al., Transcriptome analysis in Cucumis sativus identifies genes involved in multicellular trichome development, Genomics (2015), http://dx.doi.org/10.1016/j.ygeno.2015.01.010
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segregating population, 2032 of 7936 exhibited the mutated phenotype (χ2 = 1.548 b χ20.05,1 = 3.84); in addition, a test cross yielded 122 descendants comprising 59 mutated individuals (χ2 = 0.131 b χ20.05,2 = 5.991), which closely fit the segregation ratios of 3:1 and 1:1, respectively, indicating that the mutation is recessive and Mict acts as a single dominant nuclear gene. 2.3. Mapped reads and annotated genes We sequenced the apical leaf (1.5 cm length, 21 days old, trichomes attached with epidermis) transcriptome between the mict mutant and its wild type background line on the Illumina HiSeq 2000 platform. Sequences of the two cDNA libraries generated 102.33 and 105.40 million high quality reads, respectively, and the average read length was 100 bp. A total of 93.89 (91.75%) and 98.16 (93.13%) million reads were mapped to the cucumber genome, including 88.09 (93.82%) and 92.03 (93.76%) million unique reads, and 5.80 (6.18%) and 6.12 (6.24%) million multiple reads, in which 91.23 (82.47%) and 95.67 (83.22%) million were mapped to genes, 88.99 (97.54%) and 93.17 (97.38%) million were mapped to exons, and 19.39 (17.53%) and 19.29 (16.78%) million were mapped to intergenic regions, respectively (Supplementary Table S1). All useful reads were assembled by Cufflinks program and annotated via BLAST against the cucumber database. As a result, a total of 23,454 genes were predicted including 20,040 annotated and 3414 unannotated (Supplementary dataset). 2.4. eggNOG functional category analysis
Fig. 1. Trichome phenotypes between the wild type and mict mutant. (A, B) Line 06-1 (wild type). (C, D) Line 06-2/mict (the mict mutant). The absence of normal trichomes can be seen in the leaf, stem, branch, flower and fruit of the mict mutant. Scale bars represent 5 mm.
2. Results 2.1. Trichome morphologies controlled by Mict Scanning electron microscopy imaging showed that the wild type cucumber trichomes consisted of three distinct cell types: (a) apical cell: the apical cell could be classified into two types, the majority was a single-celled, non-glandular, and pyramid-shaped cell; the minority was a glandular secreting head (Fig. 2A); (b) stalk cell: the stalk was generally composed of two to four elongated cylindrical-shaped cells; and (c) base cell: the base was pie-shaped at the bottom connected with the epidermal cells (Fig. 2C). The trichomes in the mict mutant, however, were much smaller and only visible under a microscope (at least 20 times magnification). They presented two morphologies, the majority (type i) had only a small papillar-shaped head, and the minority (type ii) divided abnormally, resulting in a structure consisted of one to five rounded cells, but without the pyramid-shaped head and the pieshaped base (Fig. 2B, D, F). The fruit spine had a similar structure with the leaf trichome, but the base was much more inflated and divided into multiple spherical cells (Fig. 2E). We also characterized the root hairs as single-celled, unbranched, elongated and soft structure with numerous small tumors attached, but there was no difference between the wild type and mict mutant (Fig. 2G, H), indicating that Mict functions in trichome differentiation of the aerial organs, such as leaves and fruits, rather than the underground organ root. 2.2. Genetic analysis of Mict A cross between the wild type and mict mutant generated F1 descendants that all presented a wild type trichome phenotype, and in their F2
We used eggNOG (evolutionary genealogy of genes: Non-supervised orthologous groups) to classify orthologous genes with functional descriptions. A total of 14,857 (74.14%) genes were categorized into 25 eggNOG (Fig. 3). Among all eggNOG, unfortunately, “Function unknown” and “General function prediction only” still represented the largest clusters in cucumber species, which had 4020 (27.06%) and 2627 (17.68%) genes, respectively. “Signal transduction mechanisms” 1059 (7.13%), “Posttranslational modification, protein turnover, chaperones” 987 (6.64%), and “Transcription” 789 (5.31%) clusters were following. “Extracellular structures” 11 (0.07%) and “Cell motility” 7 (0.05%) clusters had the fewest orthologous genes (Supplementary Table S2). 2.5. Differential expression, function and pathway enrichment analyses Gene expression levels were calculated by baseMean values and differential expression was defined by statistical parameters (P b 0.05 and fold change N2 or b−2). As a result, a total of 1391 genes exhibited differential expression, including 966 up-regulated and 425 downregulated (Fig. 4, Supplementary dataset). To explore the biological functions of these differentially expressed genes, GO (Gene ontology) enrichment analysis was carried out. Among all 53 GO terms, “Sequence-specific DNA binding transcription factor activity” (P = 1.19E − 09, 29 up-regulated, 7 down-regulated) was the most enriched cluster, “Extracellular region” (P = 1.77E−04, 7 up-regulated, 7 down-regulated) and “External encapsulating structure” (P = 1.26E − 03, 8 up-regulated, 4 down-regulated) clusters were also significantly enriched (Fig. 5, Supplementary Table S3). We also performed KEGG (Kyoto encyclopedia of genes and genomes) enrichment analysis to determine whether multicellular trichome-related genes were involved in specific pathways, and a total of 4720 genes were annotated into 24 KEGG categories. The most enriched category was “Biosynthesis of other secondary metabolites” (P = 5.33E−03, 106 genes including 7 differentially expressed), “Environmental adaptation” (P = 5.53E−02, 103 genes including 5 differentially expressed) and “Lipid metabolism” (P = 6.25E − 02, 249 genes including 9 differentially expressed) categories were following (Fig. 6, Supplementary Table S4).
Please cite this article as: J.-L. Zhao, et al., Transcriptome analysis in Cucumis sativus identifies genes involved in multicellular trichome development, Genomics (2015), http://dx.doi.org/10.1016/j.ygeno.2015.01.010
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Fig. 2. Scanning electron microscopy images of leaf trichome, fruit spine and root hair morphologies between the wild type and mict mutant. (A, C) Trichomes on the wild type leaf. (A) Arrowheads indicate the cuticle (left) and glandular secreting trichome (right), respectively; (C) arrowheads indicate the apical cell (upper), stalk cell (middle) and base cell (lower), respectively. (B, D) Micro-trichomes on the mict mutant leaf. (B) Arrowheads indicate the two morphologies: type i (left) and type ii (right), respectively. (E) Trichomes on the wild type fruit. Arrowheads indicate the apical cell (left), stalk cell (middle) and base cell (right), respectively. (F) Micro-trichomes on the mict mutant fruit. Arrowheads indicate the two morphologies: type i (left) and type ii (right), respectively. (G) Root hair morphology of the wild type. (H) Root hair morphology of the mict mutant.
Please cite this article as: J.-L. Zhao, et al., Transcriptome analysis in Cucumis sativus identifies genes involved in multicellular trichome development, Genomics (2015), http://dx.doi.org/10.1016/j.ygeno.2015.01.010
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Fig. 3. eggNOG functional category analysis. A total of 14,857 (74.14%) genes were categorized into 25 eggNOG. The details of eggNOG functional category analysis can be found in Supplementary Table S2.
2.6. Transcriptome validation by RT-qPCR
2.7. Candidate genes involved in multicellular trichome development
To validate the correctness of the differentially expressed genes identified by transcriptome, we conducted RT-qPCR using separately grown apical leaf samples at the same developmental stage as those analyzed in the transcriptome. We randomly selected 15 differentially expressed genes and used three biological replicates to conduct each reaction. As shown in Table 1 and Supplementary dataset, all RT-qPCR results were in good agreement with the transcriptome data, indicating that our transcriptome provided a credible reference for further studies.
We further screened out 43 candidate genes encoding critical transcription factors that are likely involved in multicellular trichome development for future research. Interestingly, of 43 candidates, 3 genes (WRKY transcription factor 65-like, Ethylene-responsive transcription factor TINY-like, and PHD finger protein MALE STERILITY 1-like) were not expressed at all (baseMean = 0) in the mict mutant, but were highly expressed in the wild type background. These 43 genes could be subdivided into seven groups: homeodomain, MYB-domain, WRKY-
Fig. 4. Differential gene expression analysis. (A) Volcano with log2(fold change) plotted versus −log10(P-value), horizontal line represents P = 0.05. (B) MA with log2(fold change) plotted versus baseMean fold change. Genes colored blue represent differentially expressed, and genes colored orange expressed no significant difference. The details of differential expression analysis can be found in Supplementary dataset. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
Please cite this article as: J.-L. Zhao, et al., Transcriptome analysis in Cucumis sativus identifies genes involved in multicellular trichome development, Genomics (2015), http://dx.doi.org/10.1016/j.ygeno.2015.01.010
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Fig. 5. GO enrichment analysis of differentially expressed genes. Red line represents P = 0.05. The details of GO enrichment analysis can be found in Supplementary Table S3. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
domain, bHLH-domain, ethylene-responsive, zinc finger and other transcription factor genes (Table 2). For example, “Homeodomain-leucine zipper protein GLABRA2-like”, GLABRA2 in Arabidopsis regulates the epidermal cell differentiation including trichomes and root hairs [16]; “Homeodomain-leucine zipper protein ATHB-51-like”, ATHB-51 protein acts together with LEAFY as a meristem regulator to induce CAULIFLOWER expression, and has additional roles in bract formation, floral meristem determinacy, and leaf morphogenesis in Arabidopsis [18,19]; “Transcription factor RAX2-like”, RAX2 (REGULATOR OF AXILLARY MERISTEMS2) belongs to the class R2R3 MYB family and regulates the axillary meristem formation in Arabidopsis [20,21]; and “Transcription factor MYB76-like”, its homolog LMI2 (LATE MERISTEM IDENTITY2) plays a role in the meristem transition from vegetative growth to flowering in Arabidopsis, and is also a target of LEAFY [22]. 3. Discussion Cucumber has a narrow hereditary basis and high similarities in genetic background. There has no report about a gene which is characterized to regulate trichome development in cucumber to date. Unlike the common regulatory mechanism of unicellular trichomes in Arabidopsis or cotton, that of multicellular involves plant-specific genes that function distinctively. For example, the cotton GaMYB2 gene, a homolog of the Arabidopsis GL1, controls cotton fiber development and could rescue the phenotype of the Arabidopsis gl1 mutant [23]; another cotton GaHOX1 gene, is a homolog of the Arabidopsis GL2 and could rescue the phenotype of the Arabidopsis gl2 mutant [24]. These results suggest that cotton and Arabidopsis use similar transcription factors to regulate unicellular
trichome development, but plants with multicellular trichomes seem different. For example, the MIXTA gene, which regulates the floral papillae development in Antirrhinum majus and belongs to the MYB family, could not rescue the Arabidopsis gl1 mutant, but regulates the trichome differentiation in tobacco [25]; in tomato, Wo (Woolly), which encodes a class IV homeodomain-leucine zipper protein and is a homolog of the Arabidopsis GL2, has an additional role in embryo development: embryo lethality occurs when Wo becomes homozygous [26]. These results indicate that unicellular and multicellular trichomes are not homologous structures, their differentiation is controlled by distinct regulatory genes, and homologs could have diverse roles that are specific to different plant species. That is why we focused on these 43 critical transcription factor genes. To our knowledge, genes involved in trichome development, which have been characterized to date, all encode transcription factors. The 43 differentially expressed candidate genes are subdivided into seven groups: homeodomain, MYB-domain, WRKY-domain, bHLH-domain, ethylene-responsive, zinc finger and other transcription factor genes. Homeodomain-leucine zipper transcription factors are unique to plants and participate in a wide variety of biologic roles, such as lateral organ initiation [27], vascular system development [28], floral meristem regulation [29], and responses to environmental conditions [30,31]. For example, “Homeodomain-leucine zipper protein ATHB-51-like”, ATHB-51 protein acts together with LEAFY as a meristem regulator, and has additional roles in bract formation, floral meristem determinacy, and leaf morphogenesis in Arabidopsis [19]; and whereas the homolog of ATHB-51, Tl (Tendril-less), also has a plant-specific role which regulates tendril formation in pea leaves [32]. Thus, the real biological function of
Please cite this article as: J.-L. Zhao, et al., Transcriptome analysis in Cucumis sativus identifies genes involved in multicellular trichome development, Genomics (2015), http://dx.doi.org/10.1016/j.ygeno.2015.01.010
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Fig. 6. KEGG enrichment analysis of differentially expressed genes. Red line represents P = 0.05. The details of KEGG enrichment analysis can be found in Supplementary Table S4. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
ATHB-51-like gene in cucumber, is likely involved in trichome development or another plant-specific process, therefore, further studies are required. Phytohormones are also thought to have important roles in trichome regulation. For example, jasmonic acid and salicylic acid increase and decrease the number of trichomes on leaves, respectively, in Arabidopsis [33]; transient exposure to gaseous ethylene can stimulate cell division and alter the fate and polarity of the epidermal cells, resulting in aberrant guard cell and trichome formation in cucumber [34]; ZFP6 (Zinc Finger Protein 6), a newly identified C2H2 transcription factor gene, regulates inflorescence trichome initiation by integrating cytokinin and gibberellin signals in Arabidopsis [35]. In our transcriptome data, a series of ethylene-responsive and zinc finger genes were identified, indicating that these phytohormone-related genes could be helpful to understand the regulatory mechanism of multicellular trichome development for further research.
In conclusion, the present study provides an overall view of the transcriptomic changes of the loss of Mict function and identifies a series of candidate genes that are likely involved in multicellular trichome development. Although the exact biological roles of these candidate genes in cucumber remain unclear, these findings serve as a powerful tool to further investigate the relevant molecular networks and provide a new platform perspective for studying this complex and speciesspecific developmental process. 4. Materials and methods 4.1. Plant materials Cucumber (C. sativus) North China inbred line 06-1 was used as the wild type background, the spontaneous mict mutant line 06-2 (mict) was originated from line 06-1. Cucumber plants were grown with
Table 1 RT-qPCR validation of differentially expressed genes identified by transcriptome. Gene ID
Gene description
P-value (transcriptome)
P-value (RT-qPCR)
101212616 101221983 101207090 101208089 101208551 101218948 101222436 100256609 101221341 101203107 101212451 101213234 101220998 101216829 101211180
Transcription factor MYB76-like Homeobox-leucine zipper protein ATHB-51-like Ethylene-responsive transcription factor WIN1-like Mitogen-activated protein kinase A-like Early nodulin-like protein 1-like Auxin-induced protein AUX28-like Transcription repressor MYB6-like LRR receptor-like serine/threonine-protein kinase ERL2-like Homeobox-leucine zipper protein GLABRA2-like Transcription factor bHLH35-like Auxin-induced protein 5NG4-like MADS-box protein SVP-like MADS-box protein CMB1-like Auxin transporter-like protein 1-like GLABRA2 expression modulator-like
7.92E−23 3.56E−19 1.82E−15 1.49E−09 1.51E−05 0.000278769 0.000651305 0.002933638 0.005979729 0.006585425 0.00942616 0.011535995 0.015524079 0.017598343 0.038194762
7.56E−08 6.96E−06 4.57E−08 0.011319072 3.10E−06 1.57E−06 0.000232127 0.004623258 1.52E−05 0.000244103 0.009918712 0.000510179 6.71E−05 6.77E−05 7.55E−05
Please cite this article as: J.-L. Zhao, et al., Transcriptome analysis in Cucumis sativus identifies genes involved in multicellular trichome development, Genomics (2015), http://dx.doi.org/10.1016/j.ygeno.2015.01.010
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Table 2 Candidate genes associated with multicellular trichome development. Gene ID
Gene description
baseMean (WT)
baseMean (mict)
Fold change
P-value
Homeodomain transcription factor genes 101218828 WUSCHEL-related homeobox 9-like 101216890 Homeobox-leucine zipper protein ATHB-21-like 101218216 Homeobox-leucine zipper protein ANTHOCYANINLESS2-like 101218782 Homeobox-leucine zipper protein HAT22-like 101215049 Homeobox protein BEL1 homolog 101215660 Homeobox-leucine zipper protein HDG11-like 101221341 Homeobox-leucine zipper protein GLABRA2-like 101221983 Homeobox-leucine zipper protein ATHB-51-like
67.78333366 1201.701672 606.1766696 1932.793343 1343.07834 2079.98001 643.9416698 790.1600038
1129.776242 5583.820971 1629.604123 4079.173818 471.9449203 690.8777935 208.605851 2.065404465
16.66746354 4.64659499 2.688331974 2.110506968 0.351390463 0.332155978 0.323951471 0.002613907
8.89E−12 1.57E−08 0.001570765 0.004966457 0.001466776 0.000253505 0.005979729 3.56E−19
MYB-domain transcription factor genes 101209599 Myb-related protein 305-like 101219416 Transcription factor MYB86-like 101213982 Transcription factor MYB3-like 101210516 Myb-related protein A-like 101215587 Transcription factor RAX2-like 101205677 Transcription factor MYB44-like 101212616 Transcription factor MYB76-like
0.968333338 5.810000028 54.22666693 109.4216672 65.84666699 1552.238341 996.4150048
168.3304639 185.8864019 542.1686721 810.6712525 299.4836474 6165.232328 2.065404465
173.8352459 31.99421703 9.998192822 7.408690374 4.548197519 3.971833555 0.002072836
1.55E−05 0.000150695 4.35E−06 1.92E−06 0.008817435 2.23E−07 7.92E-23
WRKY-domain transcription factor genes 101212174 WRKY transcription factor 51-like 101218366 WRKY transcription factor 40-like 101223628 WRKY transcription factor 54-like 101203902 WRKY transcription factor 65-like
48.4166669 356.3466684 205.2866677 173.3316675
2832.702224 6375.903584 1499.483642 0
58.50675821 17.89241811 7.304340115 0
bHLH-domain transcription factor genes 101226097 Transcription factor bHLH128-like 101219379 Transcription factor bHLH67-like 101202980 Transcription factor bHLH130-like 101208845 Transcription factor bHLH13-like 101203107 Transcription factor bHLH35-like
3386.261683 972.2066714 947.9983379 5116.673358 4114.448353
8950.430249 2199.655755 2107.745257 10124.61269 2001.376927
2.643159651 2.262539252 2.223363873 1.978749078 0.486426552
0.00012126 0.004991273 0.00630532 0.005871155 0.006585425
Ethylene-responsive transcription factor genes 101219627 Ethylene-responsive transcription factor ERF109-like 101202953 Ethylene-responsive transcription factor ERF025-like 101206564 Ethylene-responsive transcription factor 1B-like 101213288 Ethylene-responsive transcription factor ERF017-like 101210659 Ethylene-responsive transcription factor 9-like 101215527 Ethylene-responsive transcription factor ERF012-like 101221306 Ethylene-responsive transcription factor 5-like 101207090 Ethylene-responsive transcription factor WIN1-like 101222354 Ethylene-responsive transcription factor TINY-like
13.55666673 0.968333338 18.39833342 154.9333341 523.8683359 19.36666676 1446.690007 603.2716696 144.2816674
48.82926933 126.9103942 47.93519534 11.96450408 11.54788191 10.55809162 6.341022532 0.003423672 0
2.01E−12 0.00033357 7.59E−15 1.40E−12 2.34E−17 0.002058041 4.31E−12 1.82E−15 2.43E−05
Zinc finger transcription factor genes 101221218 Zinc finger protein ZAT10-like 101217058 Zinc finger protein ZAT11-like 101203102 Zinc finger CCCH domain-containing protein 29-like 101219115 Zinc finger A20 and AN1 domain-containing stress-associated protein 3-like 101203945 Zinc finger A20 and AN1 domain-containing stress-associated protein 5-like
477.3883357 10.65166672 4687.701689 765.9516704 2739.415013
26.70078572 26.46794318 6.683912034 3.591765452 2.022869902
1.09E−29 8.32E−06 8.41E−14 1.14E−05 0.006260206
Other transcription factor genes 101210622 Transcription factor MYC4-like 101206328 AP2/ERF and B3 domain-containing transcription factor RAV1-like 101217253 NAC transcription factor 29-like 101218318 NAC transcription factor 25-like 101205960 PHD finger protein MALE STERILITY 1-like
123.9466673 276.9433347 103.6116672 193.6666676 1011.908338
16.9052777 12.85734969 9.139781002 0.117312129 0
9.38E−16 2.36E−16 6.42E−08 0.005127066 3.67E−25
appropriate management in the greenhouse under natural photoperiodic condition.
4.2. Scanning electron microscopy analysis Juvenile leaf (3 cm length old) and fruit (6 cm length old) samples were fixed in FAA (Formaldehyde acetic acid-ethanol which contained 50% (v/v) ethanol, 5% (v/v) acetic acid and 3.7% (v/v) formaldehyde dissolved in water) at 4 °C for 24 h, then dehydrated through gradient ethanol elution (50, 60, 70, 85, 90, 95 and 100% (v/v)), critical-point dried by Leica EM CPD030 desiccator, and coated with gold palladium by Hitachi E-1045 ion sputter and carbon coating unit, and then observed under JSM-6360LV scanning electron microscope.
661.962131 122.8915657 881.9277066 1853.700507 6049.569678 204.475042 9173.493931 2.065404465 0
12746.64366 281.9277095 31332.18573 2751.118747 5541.48018
2095.35283 3560.757298 946.9879472 22.71944912 0
4.75E−28 2.85E−22 1.57E−08 3.15E−06
4.3. Illumina HiSeq 2000 transcriptome sequencing Total RNA was purified with poly-T oligo-attached magnetic beads, and then fragmented into approximate 100 bp of average insert size to create cDNA libraries. The standard operating procedure was followed by TruSeq RNA Sample Preparation Kit (Illumina) instruction. Quality control was conducted using Pico green fluorescence spectrophotometry and Agilent 2100 bioanalyzer.
4.4. Gene annotation, expression, classification and enrichment analyses Spliced reads were mapped by TopHat program (http://ccb.jhu.edu/ software/tophat/), and transcripts were assembled by Cufflinks program (http://cufflinks.cbcb.umd.edu/), and then annotated via BLAST
Please cite this article as: J.-L. Zhao, et al., Transcriptome analysis in Cucumis sativus identifies genes involved in multicellular trichome development, Genomics (2015), http://dx.doi.org/10.1016/j.ygeno.2015.01.010
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(http://blast.ncbi.nlm.nih.gov/Blast.cgi/) against the cucumber database. The reference cucumber genome was derived from ftp://ftp.ncbi. nlm.nih.gov/genomes/Cucumis_sativus/. Expression level of each gene was calculated by baseMean value, which is the sequencing depth for each transcript normalized to library size. Differential gene expression was measured by HTSeq (http://www-huber.embl.de/users/anders/ HTSeq/) and DESeq (http://www-huber.embl.de/users/anders/DESeq/ ) programs. eggNOG functional category (http://eggnog.embl.de/), GO enrichment (http://www.geneontology.org/) and KEGG enrichment (http://www.genome.jp/kegg/) analyses were conducted as described by Powell et al., Ashburner et al., and Kanehisa et al., respectively [36–38]. 4.5. Extraction of nucleic acids and RT-qPCR Total RNA was extracted using RNeasy Plant Mini Kit (Qiagen). The first strand cDNA was prepared according to PrimeScript RT reagent Kit with gDNA Eraser (TaKaRa) protocol. RT-qPCR was conducted using SYBR Premix Ex Taq II Kit (TaKaRa). Primers used in this study are listed in Supplementary Table S5. 4.6. Accession numbers Transcriptome raw data from this manuscript can be found in the NCBI BioProject database under accession numbers: SAMN03276490 (WT) and SAMN03276492 (mict). Acknowledgments We thank Hui-Ming Chen for providing us with the mict mutant. We thank Li-Da Zhang for his zealous help on bioinformatics analysis. This work was supported by China 973 Program (no. 2012CB113900), National Natural Science Foundation of China (no. 31271291, 31471156), Shanghai Municipal Committee of Science and Technology (no. 13JC1403600), and Specialized Research Fund for the Doctoral Program of Higher Education (no. 20120073110051). No conflict of interest is declared. Appendix A. Supplementary data Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.ygeno.2015.01.010. References [1] R.N. Bennett, R.M. Wallsgrove, Tansley review No. 72: secondary metabolites in plant defence mechanisms, New Phytol. 127 (1994) 617–633. [2] G.J. Wagner, E. Wang, R.W. Shepherd, New approaches for studying and exploiting an old protuberance, the plant trichome, Ann. Bot. 93 (2004) 3–11. [3] E. Werker, Trichome diversity and development, Adv. Bot. Res. 31 (2000) 1–35. [4] Y.E. Choi, E. Harada, M. Wada, H. Tsuboi, Y. Morita, T. Kusano, H. Sano, Detoxification of cadmium in tobacco plants: formation and active excretion of crystals containing cadmium and calcium through trichomes, Planta 213 (2001) 45–50. [5] H. Küpper, E. Lombi, F.J. Zhao, S.P. McGrath, Cellular compartmentation of cadmium and zinc in relation to other elements in the hyperaccumulator Arabidopsis halleri, Planta 212 (2000) 75–84. [6] M. Hülskamp, Plant trichomes: a model for cell differentiation, Nat. Rev. Mol. Cell Biol. 5 (2004) 471–480. [7] D.B. Szymanski, A.M. Lloyd, M.D. Marks, Progress in the molecular genetic analysis of trichome initiation and morphogenesis in Arabidopsis, Trends Plant Sci. 5 (2000) 214–219. [8] J.C. Larkin, M.D. Marks, J. Nadeau, F. Sack, Epidermal cell fate and patterning in leaves, Plant Cell 9 (1997) 1109–1120. [9] Y. Ohashi, A. Oka, I. Ruberti, G. Morelli, T. Aoyama, Entopically additive expression of GLABRA2 alters the frequency and spacing of trichome initiation, Plant J. 29 (2002) 359–369. [10] A. Schnittger, U. Folkers, B. Schwab, G. Jürgens, M. Hülskamp, Generation of a spacing pattern: the role of TRIPTYCHON in trichome patterning in Arabidopsis, Plant Cell 11 (1999) 1105–1116.
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Please cite this article as: J.-L. Zhao, et al., Transcriptome analysis in Cucumis sativus identifies genes involved in multicellular trichome development, Genomics (2015), http://dx.doi.org/10.1016/j.ygeno.2015.01.010
Contents lists available at ScienceDirect
Genomics journal homepage: www.elsevier.com/locate/ygeno
Transcriptome analysis in Cucumis sativus identifies genes involved in multicellular trichome development Jun-Long Zhao, Jun-Song Pan, Yuan Guan, Jing-Tao Nie, Jun-Jun Yang, Mei-Ling Qu, Huan-Le He, Run Cai ⁎ School of Agriculture and Biology, Shanghai Jiao Tong University, 800 Dongchuan Road, Minhang District, Shanghai 200240, China
a r t i c l e
i n f o
Article history: Received 19 November 2014 Accepted 23 January 2015 Available online xxxx Keywords: Cucumis sativus DNA-binding proteins Gene regulatory networks Transcription factors Transcriptome Trichomes
a b s t r a c t The regulatory gene network of unicellular trichome development in Arabidopsis thaliana has been studied intensively, but that of multicellular remains unclear. In the present study, we characterized cucumber trichomes as representative multicellular and unbranched structures, but in a spontaneous mutant, mict (micro-trichome), all trichomes showed a micro-size and stunted morphologies. We revealed the transcriptome profile using Illumina HiSeq 2000 sequencing technology, and determined that a total of 1391 genes exhibited differential expression. We further validated the accuracy of the transcriptome data by RT-qPCR and found that 43 genes encoding critical transcription factors were likely involved in multicellular trichome development. These 43 candidate genes were subdivided into seven groups: homeodomain, MYB-domain, WRKY-domain, bHLH-domain, ethylene-responsive, zinc finger and other transcription factor genes. Our findings also serve as a powerful tool to further study the relevant molecular networks, and provide a new perspective for investigating this complex and species-specific developmental process. © 2015 Elsevier Inc. All rights reserved.
1. Introduction Trichomes are highly specialized structures developed from the protodermal cells of most terrestrial plants. They play important biotic and abiotic roles in many aspects, such as insect, herbivore and microbe deterrence; water regulation through transpiration; light reflectance (including UV) [1,2]; pollen collection and dispersal; absorption of water and nutrients [3]; secretion of ions and pollutant metals [4,5]; reduced mechanical abrasion; and regulation of surface temperature [2]. Trichomes can be categorized according to whether they are unicellular or multicellular, glandular or glandless, and branched or unbranched [3]. They also provide a suitable model system for researching cell differentiation at the single-cell level, including cell fate determination, cell cycle developmental control, and cell morphogenesis [6,7]. In Arabidopsis thaliana, the unicellular trichome differentiation is thought to be regulated by a competitive system comprising promoting and limiting activities. The promoting activity up-regulates both activities, and the limiting activity spreads via cell-to-cell communication and inhibits trichome differentiation [8–10]. Critical positive transcription factors include GL1 (GLABRA1), which acts as an R2R3 MYB protein [11]; GL3 (GLABRA3) and its homolog EGL3 (ENHANCER OF GLABRA3), which act as basic helix-loop-helix proteins [12]; and TTG1 (TRANSPARENT TESTA GLABRA1), which acts as a WD40-repeat protein [13]. In contrast, negative transcription factors include TRY (TRIPTYCHON), ⁎ Corresponding author. Fax: +86 21 34206938. E-mail address: [email protected] (R. Cai).
CPC (CAPRICE), and ETC (ENHANCER OF TRY AND CPC), which belong to the small R3 single-repeat MYB family [14,15]. In this system, GL1, GL3/EGL3 and TTG1 constitute the promoting activity, TRY/CPC/ETC, GL3/EGL3 and TTG1 constitute the limiting activity, and GL2 (GLABRA2), a homeodomain protein, acts as a quantitative factor directly regulated by both activities [6,7,9,16]. Cucumber (Cucumis sativus L., 2n = 2x = 14), an annual sprawling herbaceous plant, is one of the most commercially important vegetable crops worldwide. Cucumber also serves as a model plant for sex determination studies due to its diverse sex types [17]. Trichomes are widely found on leaves, stems, flowers, tendrils and fruits of the wild type cucumber plants. The trichomes on the fruits are commonly called “fruit spines” (Fig. 1A, B). Cucumber fruits are economically valuable and fruit spines directly affect the appearance quality. Here, a spontaneous mutant, which originated from North China inbred line 06-1, presented a glabrous phenotype on leaves, stems, flowers, tendrils and fruits (Fig. 1C, D). Scanning electron microscopy revealed that all trichomes in this mutant exhibited a micro-size and stunted morphologies, and are only visible under at least 20 times magnification, thus, we named this gene “Mict (Micro-trichome)” and this line “mict”. Compared with unicellular trichomes, the regulatory mechanisms of multicellular trichome development in plants are much less understood. In this study, we characterized cucumber trichomes as multicellular and unbranched. We further used Illumina HiSeq 2000 sequencing technology to reveal the transcriptome changes between the mict mutant and wild type, and identified a series of candidate genes encoding critical transcription factors which are likely involved in multicellular trichome development.
http://dx.doi.org/10.1016/j.ygeno.2015.01.010 0888-7543/© 2015 Elsevier Inc. All rights reserved.
Please cite this article as: J.-L. Zhao, et al., Transcriptome analysis in Cucumis sativus identifies genes involved in multicellular trichome development, Genomics (2015), http://dx.doi.org/10.1016/j.ygeno.2015.01.010
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segregating population, 2032 of 7936 exhibited the mutated phenotype (χ2 = 1.548 b χ20.05,1 = 3.84); in addition, a test cross yielded 122 descendants comprising 59 mutated individuals (χ2 = 0.131 b χ20.05,2 = 5.991), which closely fit the segregation ratios of 3:1 and 1:1, respectively, indicating that the mutation is recessive and Mict acts as a single dominant nuclear gene. 2.3. Mapped reads and annotated genes We sequenced the apical leaf (1.5 cm length, 21 days old, trichomes attached with epidermis) transcriptome between the mict mutant and its wild type background line on the Illumina HiSeq 2000 platform. Sequences of the two cDNA libraries generated 102.33 and 105.40 million high quality reads, respectively, and the average read length was 100 bp. A total of 93.89 (91.75%) and 98.16 (93.13%) million reads were mapped to the cucumber genome, including 88.09 (93.82%) and 92.03 (93.76%) million unique reads, and 5.80 (6.18%) and 6.12 (6.24%) million multiple reads, in which 91.23 (82.47%) and 95.67 (83.22%) million were mapped to genes, 88.99 (97.54%) and 93.17 (97.38%) million were mapped to exons, and 19.39 (17.53%) and 19.29 (16.78%) million were mapped to intergenic regions, respectively (Supplementary Table S1). All useful reads were assembled by Cufflinks program and annotated via BLAST against the cucumber database. As a result, a total of 23,454 genes were predicted including 20,040 annotated and 3414 unannotated (Supplementary dataset). 2.4. eggNOG functional category analysis
Fig. 1. Trichome phenotypes between the wild type and mict mutant. (A, B) Line 06-1 (wild type). (C, D) Line 06-2/mict (the mict mutant). The absence of normal trichomes can be seen in the leaf, stem, branch, flower and fruit of the mict mutant. Scale bars represent 5 mm.
2. Results 2.1. Trichome morphologies controlled by Mict Scanning electron microscopy imaging showed that the wild type cucumber trichomes consisted of three distinct cell types: (a) apical cell: the apical cell could be classified into two types, the majority was a single-celled, non-glandular, and pyramid-shaped cell; the minority was a glandular secreting head (Fig. 2A); (b) stalk cell: the stalk was generally composed of two to four elongated cylindrical-shaped cells; and (c) base cell: the base was pie-shaped at the bottom connected with the epidermal cells (Fig. 2C). The trichomes in the mict mutant, however, were much smaller and only visible under a microscope (at least 20 times magnification). They presented two morphologies, the majority (type i) had only a small papillar-shaped head, and the minority (type ii) divided abnormally, resulting in a structure consisted of one to five rounded cells, but without the pyramid-shaped head and the pieshaped base (Fig. 2B, D, F). The fruit spine had a similar structure with the leaf trichome, but the base was much more inflated and divided into multiple spherical cells (Fig. 2E). We also characterized the root hairs as single-celled, unbranched, elongated and soft structure with numerous small tumors attached, but there was no difference between the wild type and mict mutant (Fig. 2G, H), indicating that Mict functions in trichome differentiation of the aerial organs, such as leaves and fruits, rather than the underground organ root. 2.2. Genetic analysis of Mict A cross between the wild type and mict mutant generated F1 descendants that all presented a wild type trichome phenotype, and in their F2
We used eggNOG (evolutionary genealogy of genes: Non-supervised orthologous groups) to classify orthologous genes with functional descriptions. A total of 14,857 (74.14%) genes were categorized into 25 eggNOG (Fig. 3). Among all eggNOG, unfortunately, “Function unknown” and “General function prediction only” still represented the largest clusters in cucumber species, which had 4020 (27.06%) and 2627 (17.68%) genes, respectively. “Signal transduction mechanisms” 1059 (7.13%), “Posttranslational modification, protein turnover, chaperones” 987 (6.64%), and “Transcription” 789 (5.31%) clusters were following. “Extracellular structures” 11 (0.07%) and “Cell motility” 7 (0.05%) clusters had the fewest orthologous genes (Supplementary Table S2). 2.5. Differential expression, function and pathway enrichment analyses Gene expression levels were calculated by baseMean values and differential expression was defined by statistical parameters (P b 0.05 and fold change N2 or b−2). As a result, a total of 1391 genes exhibited differential expression, including 966 up-regulated and 425 downregulated (Fig. 4, Supplementary dataset). To explore the biological functions of these differentially expressed genes, GO (Gene ontology) enrichment analysis was carried out. Among all 53 GO terms, “Sequence-specific DNA binding transcription factor activity” (P = 1.19E − 09, 29 up-regulated, 7 down-regulated) was the most enriched cluster, “Extracellular region” (P = 1.77E−04, 7 up-regulated, 7 down-regulated) and “External encapsulating structure” (P = 1.26E − 03, 8 up-regulated, 4 down-regulated) clusters were also significantly enriched (Fig. 5, Supplementary Table S3). We also performed KEGG (Kyoto encyclopedia of genes and genomes) enrichment analysis to determine whether multicellular trichome-related genes were involved in specific pathways, and a total of 4720 genes were annotated into 24 KEGG categories. The most enriched category was “Biosynthesis of other secondary metabolites” (P = 5.33E−03, 106 genes including 7 differentially expressed), “Environmental adaptation” (P = 5.53E−02, 103 genes including 5 differentially expressed) and “Lipid metabolism” (P = 6.25E − 02, 249 genes including 9 differentially expressed) categories were following (Fig. 6, Supplementary Table S4).
Please cite this article as: J.-L. Zhao, et al., Transcriptome analysis in Cucumis sativus identifies genes involved in multicellular trichome development, Genomics (2015), http://dx.doi.org/10.1016/j.ygeno.2015.01.010
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Fig. 2. Scanning electron microscopy images of leaf trichome, fruit spine and root hair morphologies between the wild type and mict mutant. (A, C) Trichomes on the wild type leaf. (A) Arrowheads indicate the cuticle (left) and glandular secreting trichome (right), respectively; (C) arrowheads indicate the apical cell (upper), stalk cell (middle) and base cell (lower), respectively. (B, D) Micro-trichomes on the mict mutant leaf. (B) Arrowheads indicate the two morphologies: type i (left) and type ii (right), respectively. (E) Trichomes on the wild type fruit. Arrowheads indicate the apical cell (left), stalk cell (middle) and base cell (right), respectively. (F) Micro-trichomes on the mict mutant fruit. Arrowheads indicate the two morphologies: type i (left) and type ii (right), respectively. (G) Root hair morphology of the wild type. (H) Root hair morphology of the mict mutant.
Please cite this article as: J.-L. Zhao, et al., Transcriptome analysis in Cucumis sativus identifies genes involved in multicellular trichome development, Genomics (2015), http://dx.doi.org/10.1016/j.ygeno.2015.01.010
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Fig. 3. eggNOG functional category analysis. A total of 14,857 (74.14%) genes were categorized into 25 eggNOG. The details of eggNOG functional category analysis can be found in Supplementary Table S2.
2.6. Transcriptome validation by RT-qPCR
2.7. Candidate genes involved in multicellular trichome development
To validate the correctness of the differentially expressed genes identified by transcriptome, we conducted RT-qPCR using separately grown apical leaf samples at the same developmental stage as those analyzed in the transcriptome. We randomly selected 15 differentially expressed genes and used three biological replicates to conduct each reaction. As shown in Table 1 and Supplementary dataset, all RT-qPCR results were in good agreement with the transcriptome data, indicating that our transcriptome provided a credible reference for further studies.
We further screened out 43 candidate genes encoding critical transcription factors that are likely involved in multicellular trichome development for future research. Interestingly, of 43 candidates, 3 genes (WRKY transcription factor 65-like, Ethylene-responsive transcription factor TINY-like, and PHD finger protein MALE STERILITY 1-like) were not expressed at all (baseMean = 0) in the mict mutant, but were highly expressed in the wild type background. These 43 genes could be subdivided into seven groups: homeodomain, MYB-domain, WRKY-
Fig. 4. Differential gene expression analysis. (A) Volcano with log2(fold change) plotted versus −log10(P-value), horizontal line represents P = 0.05. (B) MA with log2(fold change) plotted versus baseMean fold change. Genes colored blue represent differentially expressed, and genes colored orange expressed no significant difference. The details of differential expression analysis can be found in Supplementary dataset. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
Please cite this article as: J.-L. Zhao, et al., Transcriptome analysis in Cucumis sativus identifies genes involved in multicellular trichome development, Genomics (2015), http://dx.doi.org/10.1016/j.ygeno.2015.01.010
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Fig. 5. GO enrichment analysis of differentially expressed genes. Red line represents P = 0.05. The details of GO enrichment analysis can be found in Supplementary Table S3. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
domain, bHLH-domain, ethylene-responsive, zinc finger and other transcription factor genes (Table 2). For example, “Homeodomain-leucine zipper protein GLABRA2-like”, GLABRA2 in Arabidopsis regulates the epidermal cell differentiation including trichomes and root hairs [16]; “Homeodomain-leucine zipper protein ATHB-51-like”, ATHB-51 protein acts together with LEAFY as a meristem regulator to induce CAULIFLOWER expression, and has additional roles in bract formation, floral meristem determinacy, and leaf morphogenesis in Arabidopsis [18,19]; “Transcription factor RAX2-like”, RAX2 (REGULATOR OF AXILLARY MERISTEMS2) belongs to the class R2R3 MYB family and regulates the axillary meristem formation in Arabidopsis [20,21]; and “Transcription factor MYB76-like”, its homolog LMI2 (LATE MERISTEM IDENTITY2) plays a role in the meristem transition from vegetative growth to flowering in Arabidopsis, and is also a target of LEAFY [22]. 3. Discussion Cucumber has a narrow hereditary basis and high similarities in genetic background. There has no report about a gene which is characterized to regulate trichome development in cucumber to date. Unlike the common regulatory mechanism of unicellular trichomes in Arabidopsis or cotton, that of multicellular involves plant-specific genes that function distinctively. For example, the cotton GaMYB2 gene, a homolog of the Arabidopsis GL1, controls cotton fiber development and could rescue the phenotype of the Arabidopsis gl1 mutant [23]; another cotton GaHOX1 gene, is a homolog of the Arabidopsis GL2 and could rescue the phenotype of the Arabidopsis gl2 mutant [24]. These results suggest that cotton and Arabidopsis use similar transcription factors to regulate unicellular
trichome development, but plants with multicellular trichomes seem different. For example, the MIXTA gene, which regulates the floral papillae development in Antirrhinum majus and belongs to the MYB family, could not rescue the Arabidopsis gl1 mutant, but regulates the trichome differentiation in tobacco [25]; in tomato, Wo (Woolly), which encodes a class IV homeodomain-leucine zipper protein and is a homolog of the Arabidopsis GL2, has an additional role in embryo development: embryo lethality occurs when Wo becomes homozygous [26]. These results indicate that unicellular and multicellular trichomes are not homologous structures, their differentiation is controlled by distinct regulatory genes, and homologs could have diverse roles that are specific to different plant species. That is why we focused on these 43 critical transcription factor genes. To our knowledge, genes involved in trichome development, which have been characterized to date, all encode transcription factors. The 43 differentially expressed candidate genes are subdivided into seven groups: homeodomain, MYB-domain, WRKY-domain, bHLH-domain, ethylene-responsive, zinc finger and other transcription factor genes. Homeodomain-leucine zipper transcription factors are unique to plants and participate in a wide variety of biologic roles, such as lateral organ initiation [27], vascular system development [28], floral meristem regulation [29], and responses to environmental conditions [30,31]. For example, “Homeodomain-leucine zipper protein ATHB-51-like”, ATHB-51 protein acts together with LEAFY as a meristem regulator, and has additional roles in bract formation, floral meristem determinacy, and leaf morphogenesis in Arabidopsis [19]; and whereas the homolog of ATHB-51, Tl (Tendril-less), also has a plant-specific role which regulates tendril formation in pea leaves [32]. Thus, the real biological function of
Please cite this article as: J.-L. Zhao, et al., Transcriptome analysis in Cucumis sativus identifies genes involved in multicellular trichome development, Genomics (2015), http://dx.doi.org/10.1016/j.ygeno.2015.01.010
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Fig. 6. KEGG enrichment analysis of differentially expressed genes. Red line represents P = 0.05. The details of KEGG enrichment analysis can be found in Supplementary Table S4. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
ATHB-51-like gene in cucumber, is likely involved in trichome development or another plant-specific process, therefore, further studies are required. Phytohormones are also thought to have important roles in trichome regulation. For example, jasmonic acid and salicylic acid increase and decrease the number of trichomes on leaves, respectively, in Arabidopsis [33]; transient exposure to gaseous ethylene can stimulate cell division and alter the fate and polarity of the epidermal cells, resulting in aberrant guard cell and trichome formation in cucumber [34]; ZFP6 (Zinc Finger Protein 6), a newly identified C2H2 transcription factor gene, regulates inflorescence trichome initiation by integrating cytokinin and gibberellin signals in Arabidopsis [35]. In our transcriptome data, a series of ethylene-responsive and zinc finger genes were identified, indicating that these phytohormone-related genes could be helpful to understand the regulatory mechanism of multicellular trichome development for further research.
In conclusion, the present study provides an overall view of the transcriptomic changes of the loss of Mict function and identifies a series of candidate genes that are likely involved in multicellular trichome development. Although the exact biological roles of these candidate genes in cucumber remain unclear, these findings serve as a powerful tool to further investigate the relevant molecular networks and provide a new platform perspective for studying this complex and speciesspecific developmental process. 4. Materials and methods 4.1. Plant materials Cucumber (C. sativus) North China inbred line 06-1 was used as the wild type background, the spontaneous mict mutant line 06-2 (mict) was originated from line 06-1. Cucumber plants were grown with
Table 1 RT-qPCR validation of differentially expressed genes identified by transcriptome. Gene ID
Gene description
P-value (transcriptome)
P-value (RT-qPCR)
101212616 101221983 101207090 101208089 101208551 101218948 101222436 100256609 101221341 101203107 101212451 101213234 101220998 101216829 101211180
Transcription factor MYB76-like Homeobox-leucine zipper protein ATHB-51-like Ethylene-responsive transcription factor WIN1-like Mitogen-activated protein kinase A-like Early nodulin-like protein 1-like Auxin-induced protein AUX28-like Transcription repressor MYB6-like LRR receptor-like serine/threonine-protein kinase ERL2-like Homeobox-leucine zipper protein GLABRA2-like Transcription factor bHLH35-like Auxin-induced protein 5NG4-like MADS-box protein SVP-like MADS-box protein CMB1-like Auxin transporter-like protein 1-like GLABRA2 expression modulator-like
7.92E−23 3.56E−19 1.82E−15 1.49E−09 1.51E−05 0.000278769 0.000651305 0.002933638 0.005979729 0.006585425 0.00942616 0.011535995 0.015524079 0.017598343 0.038194762
7.56E−08 6.96E−06 4.57E−08 0.011319072 3.10E−06 1.57E−06 0.000232127 0.004623258 1.52E−05 0.000244103 0.009918712 0.000510179 6.71E−05 6.77E−05 7.55E−05
Please cite this article as: J.-L. Zhao, et al., Transcriptome analysis in Cucumis sativus identifies genes involved in multicellular trichome development, Genomics (2015), http://dx.doi.org/10.1016/j.ygeno.2015.01.010
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Table 2 Candidate genes associated with multicellular trichome development. Gene ID
Gene description
baseMean (WT)
baseMean (mict)
Fold change
P-value
Homeodomain transcription factor genes 101218828 WUSCHEL-related homeobox 9-like 101216890 Homeobox-leucine zipper protein ATHB-21-like 101218216 Homeobox-leucine zipper protein ANTHOCYANINLESS2-like 101218782 Homeobox-leucine zipper protein HAT22-like 101215049 Homeobox protein BEL1 homolog 101215660 Homeobox-leucine zipper protein HDG11-like 101221341 Homeobox-leucine zipper protein GLABRA2-like 101221983 Homeobox-leucine zipper protein ATHB-51-like
67.78333366 1201.701672 606.1766696 1932.793343 1343.07834 2079.98001 643.9416698 790.1600038
1129.776242 5583.820971 1629.604123 4079.173818 471.9449203 690.8777935 208.605851 2.065404465
16.66746354 4.64659499 2.688331974 2.110506968 0.351390463 0.332155978 0.323951471 0.002613907
8.89E−12 1.57E−08 0.001570765 0.004966457 0.001466776 0.000253505 0.005979729 3.56E−19
MYB-domain transcription factor genes 101209599 Myb-related protein 305-like 101219416 Transcription factor MYB86-like 101213982 Transcription factor MYB3-like 101210516 Myb-related protein A-like 101215587 Transcription factor RAX2-like 101205677 Transcription factor MYB44-like 101212616 Transcription factor MYB76-like
0.968333338 5.810000028 54.22666693 109.4216672 65.84666699 1552.238341 996.4150048
168.3304639 185.8864019 542.1686721 810.6712525 299.4836474 6165.232328 2.065404465
173.8352459 31.99421703 9.998192822 7.408690374 4.548197519 3.971833555 0.002072836
1.55E−05 0.000150695 4.35E−06 1.92E−06 0.008817435 2.23E−07 7.92E-23
WRKY-domain transcription factor genes 101212174 WRKY transcription factor 51-like 101218366 WRKY transcription factor 40-like 101223628 WRKY transcription factor 54-like 101203902 WRKY transcription factor 65-like
48.4166669 356.3466684 205.2866677 173.3316675
2832.702224 6375.903584 1499.483642 0
58.50675821 17.89241811 7.304340115 0
bHLH-domain transcription factor genes 101226097 Transcription factor bHLH128-like 101219379 Transcription factor bHLH67-like 101202980 Transcription factor bHLH130-like 101208845 Transcription factor bHLH13-like 101203107 Transcription factor bHLH35-like
3386.261683 972.2066714 947.9983379 5116.673358 4114.448353
8950.430249 2199.655755 2107.745257 10124.61269 2001.376927
2.643159651 2.262539252 2.223363873 1.978749078 0.486426552
0.00012126 0.004991273 0.00630532 0.005871155 0.006585425
Ethylene-responsive transcription factor genes 101219627 Ethylene-responsive transcription factor ERF109-like 101202953 Ethylene-responsive transcription factor ERF025-like 101206564 Ethylene-responsive transcription factor 1B-like 101213288 Ethylene-responsive transcription factor ERF017-like 101210659 Ethylene-responsive transcription factor 9-like 101215527 Ethylene-responsive transcription factor ERF012-like 101221306 Ethylene-responsive transcription factor 5-like 101207090 Ethylene-responsive transcription factor WIN1-like 101222354 Ethylene-responsive transcription factor TINY-like
13.55666673 0.968333338 18.39833342 154.9333341 523.8683359 19.36666676 1446.690007 603.2716696 144.2816674
48.82926933 126.9103942 47.93519534 11.96450408 11.54788191 10.55809162 6.341022532 0.003423672 0
2.01E−12 0.00033357 7.59E−15 1.40E−12 2.34E−17 0.002058041 4.31E−12 1.82E−15 2.43E−05
Zinc finger transcription factor genes 101221218 Zinc finger protein ZAT10-like 101217058 Zinc finger protein ZAT11-like 101203102 Zinc finger CCCH domain-containing protein 29-like 101219115 Zinc finger A20 and AN1 domain-containing stress-associated protein 3-like 101203945 Zinc finger A20 and AN1 domain-containing stress-associated protein 5-like
477.3883357 10.65166672 4687.701689 765.9516704 2739.415013
26.70078572 26.46794318 6.683912034 3.591765452 2.022869902
1.09E−29 8.32E−06 8.41E−14 1.14E−05 0.006260206
Other transcription factor genes 101210622 Transcription factor MYC4-like 101206328 AP2/ERF and B3 domain-containing transcription factor RAV1-like 101217253 NAC transcription factor 29-like 101218318 NAC transcription factor 25-like 101205960 PHD finger protein MALE STERILITY 1-like
123.9466673 276.9433347 103.6116672 193.6666676 1011.908338
16.9052777 12.85734969 9.139781002 0.117312129 0
9.38E−16 2.36E−16 6.42E−08 0.005127066 3.67E−25
appropriate management in the greenhouse under natural photoperiodic condition.
4.2. Scanning electron microscopy analysis Juvenile leaf (3 cm length old) and fruit (6 cm length old) samples were fixed in FAA (Formaldehyde acetic acid-ethanol which contained 50% (v/v) ethanol, 5% (v/v) acetic acid and 3.7% (v/v) formaldehyde dissolved in water) at 4 °C for 24 h, then dehydrated through gradient ethanol elution (50, 60, 70, 85, 90, 95 and 100% (v/v)), critical-point dried by Leica EM CPD030 desiccator, and coated with gold palladium by Hitachi E-1045 ion sputter and carbon coating unit, and then observed under JSM-6360LV scanning electron microscope.
661.962131 122.8915657 881.9277066 1853.700507 6049.569678 204.475042 9173.493931 2.065404465 0
12746.64366 281.9277095 31332.18573 2751.118747 5541.48018
2095.35283 3560.757298 946.9879472 22.71944912 0
4.75E−28 2.85E−22 1.57E−08 3.15E−06
4.3. Illumina HiSeq 2000 transcriptome sequencing Total RNA was purified with poly-T oligo-attached magnetic beads, and then fragmented into approximate 100 bp of average insert size to create cDNA libraries. The standard operating procedure was followed by TruSeq RNA Sample Preparation Kit (Illumina) instruction. Quality control was conducted using Pico green fluorescence spectrophotometry and Agilent 2100 bioanalyzer.
4.4. Gene annotation, expression, classification and enrichment analyses Spliced reads were mapped by TopHat program (http://ccb.jhu.edu/ software/tophat/), and transcripts were assembled by Cufflinks program (http://cufflinks.cbcb.umd.edu/), and then annotated via BLAST
Please cite this article as: J.-L. Zhao, et al., Transcriptome analysis in Cucumis sativus identifies genes involved in multicellular trichome development, Genomics (2015), http://dx.doi.org/10.1016/j.ygeno.2015.01.010
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(http://blast.ncbi.nlm.nih.gov/Blast.cgi/) against the cucumber database. The reference cucumber genome was derived from ftp://ftp.ncbi. nlm.nih.gov/genomes/Cucumis_sativus/. Expression level of each gene was calculated by baseMean value, which is the sequencing depth for each transcript normalized to library size. Differential gene expression was measured by HTSeq (http://www-huber.embl.de/users/anders/ HTSeq/) and DESeq (http://www-huber.embl.de/users/anders/DESeq/ ) programs. eggNOG functional category (http://eggnog.embl.de/), GO enrichment (http://www.geneontology.org/) and KEGG enrichment (http://www.genome.jp/kegg/) analyses were conducted as described by Powell et al., Ashburner et al., and Kanehisa et al., respectively [36–38]. 4.5. Extraction of nucleic acids and RT-qPCR Total RNA was extracted using RNeasy Plant Mini Kit (Qiagen). The first strand cDNA was prepared according to PrimeScript RT reagent Kit with gDNA Eraser (TaKaRa) protocol. RT-qPCR was conducted using SYBR Premix Ex Taq II Kit (TaKaRa). Primers used in this study are listed in Supplementary Table S5. 4.6. Accession numbers Transcriptome raw data from this manuscript can be found in the NCBI BioProject database under accession numbers: SAMN03276490 (WT) and SAMN03276492 (mict). Acknowledgments We thank Hui-Ming Chen for providing us with the mict mutant. We thank Li-Da Zhang for his zealous help on bioinformatics analysis. This work was supported by China 973 Program (no. 2012CB113900), National Natural Science Foundation of China (no. 31271291, 31471156), Shanghai Municipal Committee of Science and Technology (no. 13JC1403600), and Specialized Research Fund for the Doctoral Program of Higher Education (no. 20120073110051). No conflict of interest is declared. Appendix A. Supplementary data Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.ygeno.2015.01.010. References [1] R.N. Bennett, R.M. Wallsgrove, Tansley review No. 72: secondary metabolites in plant defence mechanisms, New Phytol. 127 (1994) 617–633. [2] G.J. Wagner, E. Wang, R.W. Shepherd, New approaches for studying and exploiting an old protuberance, the plant trichome, Ann. Bot. 93 (2004) 3–11. [3] E. Werker, Trichome diversity and development, Adv. Bot. Res. 31 (2000) 1–35. [4] Y.E. Choi, E. Harada, M. Wada, H. Tsuboi, Y. Morita, T. Kusano, H. Sano, Detoxification of cadmium in tobacco plants: formation and active excretion of crystals containing cadmium and calcium through trichomes, Planta 213 (2001) 45–50. [5] H. Küpper, E. Lombi, F.J. Zhao, S.P. McGrath, Cellular compartmentation of cadmium and zinc in relation to other elements in the hyperaccumulator Arabidopsis halleri, Planta 212 (2000) 75–84. [6] M. Hülskamp, Plant trichomes: a model for cell differentiation, Nat. Rev. Mol. Cell Biol. 5 (2004) 471–480. [7] D.B. Szymanski, A.M. Lloyd, M.D. Marks, Progress in the molecular genetic analysis of trichome initiation and morphogenesis in Arabidopsis, Trends Plant Sci. 5 (2000) 214–219. [8] J.C. Larkin, M.D. Marks, J. Nadeau, F. Sack, Epidermal cell fate and patterning in leaves, Plant Cell 9 (1997) 1109–1120. [9] Y. Ohashi, A. Oka, I. Ruberti, G. Morelli, T. Aoyama, Entopically additive expression of GLABRA2 alters the frequency and spacing of trichome initiation, Plant J. 29 (2002) 359–369. [10] A. Schnittger, U. Folkers, B. Schwab, G. Jürgens, M. Hülskamp, Generation of a spacing pattern: the role of TRIPTYCHON in trichome patterning in Arabidopsis, Plant Cell 11 (1999) 1105–1116.
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