УДК 581.1

CLONING AND CHARACTERIZATION OF ETHYLENE INSENSITIVE 2 (EIN2) GENE FROM Cucumis melo

© 2013 F. Gao*, **, 2, J. Hao*, **, 2, Y. Yao*, **, X. Wang*, A. Hasi*, **

* College of Life Sciences, Inner Mongolia University, Hohhot, P.R. China

** Inner Mongolia Key Laboratory of Herbage and Endemic Crop Biotechnology, Hohhot, 

P.R. China

Received July 26, 2012

Melon is an ideal alternative model fruit to examine ethylene perception and sensitivity. Ethylene insensitive 2 (EIN2), an integral membrane protein in the endoplasmic reticulum, is an important regulator of ethylene and other phytohormone signaling. We isolated a cDNA clone that encoded EIN2 homolog for the first time on the basis of melon (Cucumis melo L. cv. Hetao) fruit total RNA by in silico cloning and reverse-transcription PCR (RT-PCR). The cDNA contained an open reading frame of 3876 bp corresponding to a polypeptide of 1291 amino acids with a predicted mol wt of 141 kD. The expression patterns of different developmental stages of fruit, vegetative organs, and reproductive tissues and upon the treatment with IAA and ABA were analyzed. CmEIN2 mediates ethylene signals in many processes and is a component of signal transduction by ethylene, auxin, and abscisic acid.

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1 This text was submitted by the authors in English.

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2 These authors contributed equally to this work.

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Abbreviations: CTR1  constitutive triple response1; DAP  days after pollination; EBF  EIN3-binding F-box; EIL  ethylene-insensitive3-like; EIN  ethylene insensitive; ETP  EIN2-targeting protein. 

Corresponding author: A. Hasi. College of Life Sciences, Inner Mongolia University, Hohhot 010021, P.R. China. E-mail: hasind@sina.com

 Keywords: Cucumis melo  EIN2  ethylene  gene expression

 

INTRODUCTION

The plant hormone ethylene triggers a wide range of physiological and morphological responses in plant, including the inhibition of cell expansion, the promotion of leaf and flower senescence, the induction of fruit ripening and abscission, the resistance to pathogen and insect attack, and the adaptation to stress conditions [1]. Many components in the ethylene signaling pathway have been identified, using genetic mutants of the model plant Arabidopsis thaliana, and a largely linear pathway in its early steps has been established and expanded into an increasingly complex signaling system [2].

Ethylene insensitive 2 (EIN2), an integral membrane protein in the endoplasmic reticulum [3], is a central regulator of the ethylene signaling pathway and its gene is the only one in this pathway whose loss-of-function mutations effect complete insensitivity to ethylene [4]. Genetic data indicate that EIN2 mediates an essential step in ethylene signaling between CTR1 and EIN3/EIL [5]. Recent studies have demonstrated that ethylene-induced stabilization of EIN3 is mediated by proteasomal degradation of EBF1 and EBF2, which requires EIN2 [6], and that the degradation of EIN2 is triggered by the ethylene-controlled F-box proteins ETP1 and ETP2 [7]. However, few EIN2 genes have been isolated from plants, such as Arabidopsis thaliana [4], Petunia × hybrida [8], Oryza sativa [9], Lycopersicon esculentum [10], Medicago truncatula [11], Prunus persica [12], and Dianthus caryophyllus [13], and little is known about the function of EIN2 in species other than Arabidopsis.

One of the most frequently studied examples of ethylene regulation is the ripening of climacteric fruit. Melon is an ideal, alternative model fruit to examine ethylene perception and sensitivity with regard to fruit ripening, because the ethylene-dependent and -independent regulatory pathways coordinate ripening in melon fruit [14, 15]. Based on the whole-genome shotgun sequence of the cucumber, an important cultivated plant of Cucubitaceae, the mean sequence similarity over coding regions between cucumber and melon is 95% [16]. This information provided the additional genomic approach to learn melon. To increase our understanding of the function of EIN2 in ethylene signaling and responses, we isolated a homolog of Arabidopsis EIN2 from melon by in silico cloning and RT-PCR and analyzed its expression patterns.

 

MATERIALS AND METHODS

Plant material and hormonal treatments. Melon (Cucumis melo L. cv. Hetao) plants were grown on a farm. Self-pollination was performed manually, and the pollination time was recorded and controlled between 9:00 and 11:00 a.m. Only one fruit was remained on each plant. Fruits were harvested at 9:0010:00 a.m. every 5 days from 15 to 30 DAP and daily from 31 DAP until rotting, based on the DAP and maturity indices. Other tissues, such as roots, stems, young leaves, petals, and ovaries, were also collected from the greenhouse.

To minimize the effects of endogenous hormones (ethylene, auxin, and ABA) on CmEIN2 transcription, sterile young leaves were transferred to 250-mL flasks that contained 100 mL of 0.5 MS liquid medium with the appropriate treatment. The flasks were incubated on a rotary shaker (100 rpm) for 2 h at 30°C. The treatments with IAA and ABA (at 0.4, 4, and 40 µM) were performed. Leaves that were kept in basal medium without any hormone were used as the control. At the end of the treatment, all leaves were removed and briefly blotted dry.

All plant materials were frozen immediately in liquid nitrogen and stored at 80°C for RNA analysis.

Internal ethylene measurements. The endogenous gas sample was collected with a syringe from the fruit cavity at 15, 20, 25, and 30 DAP and daily from 31 DAP until rotting and measured by gas chromatography (GC-9A, “Shimadzu”, Japan) to determine the internal ethylene production.

Extraction of total RNA. Total RNA from mesocarp tissue was isolated as described [17]. For other tissues, total RNA was extracted using RNAiso for polysaccharide-rich plant tissue (“Takara”, Japan) according to the manufacturer’s instructions. All RNA extracts were analyzed by agarose gel electrophoresis and UV spectrophotometry.

Cloning and in silico analysis of melon CmEIN2 cDNA. Mesocarp RNA was used to clone full-length melon cDNA homologs of Arabidopsis EIN2 by RT-PCR. First-strand cDNA synthesis was performed using the ThermoScriptTM RT-PCR System (“Invitrogen”, United States) according to the manufacturer’s instructions.

Degenerate gene-specific primers were designed as follows. (1) In silico analysis was used to extend the melon CmEIN2 sequence in the Cucurbit Genomics Database (http://www.icugi.org/), based on the sequence of CmEIN2 EST (GenBank ID: AM717510) [18]. (2) The extended CmEIN2 sequence was searched against the NCBI whole-genome shotgun contigs (wgs) database using BLASTN (http://www.ncbi.nlm.nih.gov/blast/Blast.cgi), optimizing for discontiguous megablast, to obtain cucumber contigs that contain the genomic DNA sequence of the putative CsEIN2. (3) The gene prediction program Softberry of FGENESH (http://linux1.softberry.com/berry.phtml) was used to predict multiple (alternative splice) variants of potential CsEIN2 genes in the cucumber genome contig to identify the initiation and termination codons of the putative CsEIN2.

Using first-strand cDNA as a template, the predicted CmEIN2 cDNA was amplified with the degenerate primers 5'-GATGGAATCTACGACATTGCATAC-3' (forward) and 5'-ACTATGAGCTATAAGGAACGGATG-3' (reverse), using PrimeSTARTM HS DNA polymerase (“Takara”). The PCR product was purified, ligated into pMD19-T (“Takara”), transformed into Escherichia coli JM109, and sequenced. Full-length amino acid alignments were performed using DNAMAN (“Lynnon Biosoft”, United States). The phylogenetic analysis was conducted with ClustalX (2.0.12) (ftp://ftp.ebi.ac.uk/pub/software/clustalw2/2.0.12/). The phylogenetic tree was constructed using MEGA v. 5.

Relative quantitative real-time RT-PCR. CmEIN2 mRNA levels were measured by quantitative PCR. Primers were designed with a calculated Tm of 60~65°C, and the amplification product was 155 bp: forward primer 5'-ATGGTCAAGGATGTGGAGATAGC-3'; reverse primer 5'-TCGTGAGTGGCAACTGGTTTA-3'. For each trial, 6 independent experiments were used as three biological replicates and two records, and all relative fold-differences in expression were normalized to GAPDH (GenBank ID: AB033600) (forward primer 5'-ATCATTCCTAGCAGCACTGG-3' and reverse primer 5'-TTGGCATCAAATATGCTTGACCTG-3').

First-strand cDNA was synthesized using the PrimeScript® RT Reagent Kit (Perfect Real Time) (“Takara”) according the manufacturer’s protocol. For the cDNA synthesis, 0.5 µg of total RNA from melon fruit at various stages of development and ripening and other tissues was used as a template in a 10-µL of the reaction mixture. SYBR® Premix ex TaqTM (Perfect Real Time) (“Takara”) was used for the real-time RT-PCR, including 5 µM of each primer, and the reactions were run on an Opticon 3 Real-Time PCR System (“BioRad”, United States). Melting curves were generated immediately after the last cycle to exclude any influence of primer dimers. Cycle numbers, at which the fluorescence passed the cycle threshold (Ct), were analyzed, and the relative expression was calculated by 2(Ct)-method.

 

RESULTS

Cloning of CmEIN2 and sequence analysis

 

The melon CmEIN2 sequence could be only extended to 1255 bp (matched with melon unigene MU49291, Database: melon_unigene_v. 4) in the Cucurbit Genomics Database. Thus, it was necessary to search for cucumber whole-genome shotgun contigs that harbored CsEIN2. The BLAST results matched with cucumber contig 6472 (GenBank ID: ACHR01006472), which contained a region of 7 exons, encoding 1290 amino acids and the putative CsEIN2. By RT-PCR, we isolated a cDNA that contained a full-length ORF and designated this gene as CmEIN2.

The CmEIN2 cDNA (which has been submitted to GenBank under the accession no. HQ451896) is 3876 bp long and contains an ORF that encodes a polypeptide of 1291 amino acids with a predicted mol wt of 141 kD. The deduced CmEIN2 amino acid sequence of the amplified fragment shared 96.7, 63.9, 55.5, 54.8, and 55.1% identity with those of Cucumis sativus (CsEIN2, predicted from cucumber-shotgun contig sequences), Prunus persica (PpEIN2, GU120631), Petunia × hybrida (PhEIN2, AY353249), Solanum lycopersicum (SlEIN2, AY566238), and Arabidopsis thaliana (AtEIN2, AF141203), respectively (fig. 1).

We noted highly conserved regions in EIN2 in its carboxy-terminus (fig. 1), which is believed to be essential for signal transduction to downstream components [4]. By phylogenetic analysis, CmEIN2 was more closely related to CsEIN2 (Cucumis sativus) and PpEIN2 (Prunus persica) than OsEIN2 (Oryza sativa) and ZmEIN2 (Zea mays) (fig. 2).

 

Expression patterns of CmEIN2 mRNA

To determine the function of CmEIN2 in plant development, its expression was quantified by real-time RT-PCR to examine its accumulation patterns in various plant organs, including root, stem, young leaves, petal, ovary, and fruit at different developmental stages. CmEIN2 had similar expression patterns in young leaves and petal tissues  approximately two times higher than in the root; minimum and maximum expression were observed in the stem and ovary, respectively (fig. 3a). During fruit development, CmEIN2 mRNA increased slightly from 20 DAP, peaked at 35 DAP (the stage of pre-ethylene climacteric), and declined, followed by the burst of ethylene (40 DAP). CmEIN2 was up-regulated during the transition from the immature to mature stage of melon fruit (figs. 3b, 4). Leaves that were treated with IAA and ABA had the lower levels of CmEIN2 transcripts. The expression patterns were similar between various concentration of IAA, whereas its accumulation increased slightly at 4 µM ABA and decreased dramatically at the higher concentration (40 µM) (fig. 3c).

 

DISCUSSION

EIN2 is an important component in ethylene signal transduction in Arabidopsis [4]. Gene silencing of LeEIN2 delays tomato fruit development and ripening, causing down-regulation of ethylene-related and ripening-related genes, such as EILs, PG, E4, and LoxB [19, 20], and transgenic plants with the lower PhEIN2 expression experience significant delays in flower senescence and fruit ripening [8]. However, the ortholog of Arabidopsis EIN2 has not been cloned in melon, an ideal model system for studying the function of ethylene during fruit ripening. Recently, the completion of shotgun sequences of the cucumber genome [16] has allowed us to compare and clone melon genes in silico, saving time and cost. In this study, we isolated the melon homolog of Arabidopsis EIN2 and analyzed its expression patterns over time.

Our comparison of gene structures suggests that CmEIN2 is an ortholog of AtEIN2, and its peptide sequence shares a high identity with CsEIN2 and PpEIN2. During melon fruit ripening, CmEIN2 mRNA levels increased slightly before ethylene climacteric and decreased after. During ripening process, minimum and maximum CmEIN2 levels did not differ by more than fourfold, similar to data by Trainotti et al. for peach fruit [21]. CmEIN2 transcripts were found in all vegetative and reproductive tissues  roots, stems, young leaves, and petals  peaking in ovaries, indicating that CmEIN2 regulates the development of various tissues. EIN2 is believed to mediate signal transduction by other hormones. The decrease of CmEIN2 mRNA in melon treated with IAA and ABA indicates that, CmEIN2 is important for the cross-talk between IAA, ABA, and ethylene signaling and that these interactions must be complex, meriting further investigation.

In conclusion, we report cloning and expression analysis of CmEIN2, encoding a key regulator in signaling by ethylene and other hormones. The results should prompt further research on melon plant hormone signaling and genetic engineering to improve fruit shelf-life, resistance, and adaptation to stress.

This research was supported by the National Natural Science Foundation of China (no. 30960159) and the National Science Foundation for Fostering Talents in Basic Research of the National Natural Science Foundation of China (no. J0730648).

 REFERENCES

1. Bleecker A.B., Kende H. Ethylene: a gaseous signal molecule in plants // Annu. Rev. Cell Dev. Biol. 2000. V. 16. P. 118.

2. Stepanova A.N., Alonso J.M. Ethylene signaling and response: where different regulatory modules meet // Curr. Opin. Plant Biol. 2009. V. 12. P. 548555.

3. Bisson M.M., Bleckmann A., Allekotte S., Groth G. EIN2, the central regulator of ethylene signalling, is localized at the ER membrane where it interacts with the ethylene receptor ETR1 // Biochem. J. 2009. V. 424. P. 16.

4. Alonso J.M., Hirayama T., Roman G., Nourizadeh, S., Ecker J.R. EIN2, a bifunctional transducer of ethylene and stress responses in Arabidopsis // Science. 1999. V. 284. P. 21482152.

5. Roman G., Lubarsky B., Kieber J.J., Rothenberg M., Ecker J.R. Genetic analysis of ethylene signal transduction in Arabidopsis thaliana: five novel mutant loci integrated into a stress response pathway // Genetics. 1995. V. 139. P. 13931409.

6. An F., Zhao Q., Ji Y., Li W., Jiang Z., Yu X., Zhang C., Han Y., He W., Liu Y., Zhang S., Ecker J.R., Guo H. Ethylene-induced stabilization of ETHYLENE INSENSITIVE3 and EIN3-Like1 is mediated by proteasomal degradation of EIN3 binding F-box 1 and 2 that requires EIN2 in Arabidopsis // Plant Cell. 2010. V. 22. P. 23842401.

7. Qiao H., Chang K.N., Yazaki J., Ecker J.R. Interplay between ethylene, ETP1/ETP2 F-box proteins, and degradation of EIN2 triggers ethylene responses in Arabidopsis // Genes Dev. 2009. V. 23. P. 512521.

8. Shibuya K., Barry K.G., Ciardi J.A., Loucas H.M., Underwood B.A., Nourizadeh S., Ecker J.R., Klee H.J., Clark D.G. The central role of PhEIN2 in ethylene responses throughout plant development in Petunia // Plant Physiol. 2004.V. 136. P. 29002912.

9. Jun S.H., Han M.J., Lee S., Seo Y.S., Kim W.T., An G.H. OsEIN2 is a positive component in ethylene signaling in rice // Plant Cell Physiol. 2004. V. 45. P. 281289.

10. Wang J., Chen G., Hu Z., Chen X. Cloning and characterization of the EIN2-homology gene LeEIN2 from tomato // DNA Seq. 2007. V. 18. P. 3338.

11. Penmetsa R.V., Uribe P., Anderson J., Lichtenzveig J., Gish J.-C., Nam Y.W., Engstrom E., Xu K., Sckisel G., Pereira M., Baek J.M., Lopez-Meyer M., Long S.R., Harrison M.J., Singh K.B., Kiss G.B., Cook D.R. The Medicago truncatula ortholog of Arabidopsis EIN2, sickle, is a negative regulator of symbiotic and pathogenic microbial associations // Plant J. 2008. V. 55. P. 580595.

12. Begheldo M., Manganaris G.A., Bonghi C., Tonutti P. Different postharvest conditions modulate ripening and ethylene biosynthetic and signal transduction pathways in Stony Hard peaches // Postharv. Biol. Technol. 2008. V. 48. P. 8491.

13. Fu Z., Wang H., Liu J., Liu J., Wang J., Zhang Z., Yu Y. Cloning and characterization of a DCEIN2 gene responsive to ethylene and sucrose in cut flower carnation // Plant Cell, Tissue Organ Cult. 2011. V. 105. P. 447455.

14. Pech J., Bouzayen M., Latche A. Climacteric fruit ripening: ethylene-dependent and independent regulation of ripening pathways in melon fruit // Plant Sci. 2008. V. 175. P. 114120.

15. Ezura H., Owino W.O. Melon, an alternative model plant for elucidating fruit ripening // Plant Sci. 2008. V. 175. P. 121129.

16. Huang S., Li R., Zhang Z., Li L., Gu X., Fan W., Lucas W.J., Wang X., Xie B., Ni P., Ren Y., Zhu H., Li J., Lin K., Jin W., Fei Z., Li G., Staub J., Kilian A., van der Vossen E.A., Wu Y., Guo J., He J., Jia Z., Ren Y., Tian G., Lu Y., Ruan J., Qian W., Wang M., Huang Q., Li B., Xuan Z., Cao J., Asan, Wu Z., Zhang J., Cai Q., Bai Y., Zhao B., Han Y., Li Y., Li X., Wang S., Shi Q., Liu S., Cho W.K., Kim J.Y., Xu Y., Heller-Uszynska K., Miao H., Cheng Z., Zhang S., Wu J., Yang Y., Kang H., Li M., Liang H., Ren X., Shi Z., Wen M., Jian M., Yang H., Zhang G., Yang Z., Chen R., Liu S., Li J., Ma L., Liu H., Zhou Y., Zhao J., Fang X., Li G., Fang L., Li Y., Liu D., Zheng H., Zhang Y., Qin N., Li Z., Yang G., Yang S., Bolund L., Kristiansen K., Zheng H., Li S., Zhang X., Yang H., Wang J., Sun R., Zhang B., Jiang S., Wang J., Du Y., Li S. The genome of the cucumber, Cucumis sativus L. // Nat. Genet. 2009. V. 41. P. 12751281.

17. Asif M., Dhawan P., Nath P. A simple procedure for the isolation of high quality RNA from ripening banana fruit // Plant Mol. Biol. Rep. 2000. V. 18. P. 109115.

18. Gonzalez-Ibeas D., Blanca J., Roig C., González-To M., Picó B., Truniger V., Gómez P., Deleu W., Caño-Delgado A., Arús P., Nuez F., Garcia-Mas J., Puigdomènech P., Aranda M.A. MELOGEN: an EST database for melon functional genomics // BMC Genom. 2007. V. 8. P. 117.

19. Ху Ч.Л., Дэн Л., Чен С.Ц., Ван П.Ц., Чэнь Г.П. Косупрессия гена LeEIN2, гомологичного EIN2, ингибирует созревание плодов и уменьшает чувствительность к этилену у томатов // Физиология растений. 2010. Т. 57. С. 595600.

20. Zhu H.L., Zhu B.Z., Shao Y., Wang X.G., Lin X.J., Xie Y.H., Li Y.C., Gao H.Y., Luo Y.B. Tomato fruit development and ripening are altered by the silencing of LeEIN2 gene // J. Integr. Plant Biol. 2006. V. 48. P. 14781485.

21. Trainotti L., Bonghi C., Ziliotto F., Zanin D., Rasori A., Casadoro G., Ramina A., Tonutti P. The use of microarray µPEACH 1.0 to investigate transcriptome changes during transition from pre-climacteric to climacteric phase in peach fruit // Plant Sci. 2006. V. 170. P. 606613.

 

FIGURE CAPTIONS

Fig. 1. Comparison of the amino acid sequences of EIN2 homologous gene of several plant species.

Alignment was performed using the DNAMAN software (“Lynnon Biosoft”). The predicted EIN2 protein of Cucumis melo (Cm) was compared with those of Cucumis sativus (Cs), Prunus persica (Pp), Petunia × hybrida (Ph), Solanum lycopersicum (Sl), and Arabidopsis thaliana (At).

 

Fig. 2. Phylogenetic tree of EIN2 protein.

Full-length amino acid sequences of the proteins were aligned with the Clustal W, and the phylogenetic tree was constructed using the MEGA v. 5. Bootstrap values from 1000 bootstrap re-samples are given for each branch. The EIN2 sequences were found in GenBank as follows: Cucumis melo (HQ451896), Cucumis sativus (predicted from Cs-contig 6472, ACHR01006472), Prunus persica (GU120631), Petunia × hybrida (AY353249), Solanum lycopersicum (AY566238), Arabidopsis thaliana (AF141203), Populus trichocarpa (XM_002326149, XM_002322846), Zea mays (AY359584), Dianthus caryophyllus (HQ441183), Oryza sativa (AY396568).

 

Fig. 3. Expression analysis of CmEIN2 gene in different organs of melon by real-time PCR.

Each column represents the mean of three biological replicates and two records. Error bar on each column represent the standard error (SE). All the reactions were normalized using the Ct value corresponding to the melon GAPDH gene. a  the expression level, R  root; S  stem; L  young leaves; P  petal; O  ovary; the expression level was expressed as a ratio of the gene in particular organ to that in the root, which was set at 1; b  the expression in melon fruit at different developmental stages; the expression level was expressed as a ratio of the gene in particular organ to that in the root, which was set at 1; c  young leaves treated with differential concentrations of IAA (1) and ABA (2); leaves without any hormone treatment were set at 1.

 

Fig. 4. Changes in the rate of ethylene in melon fruit during development and ripening. 

Data are mean of six fruits ± standard error (SE).