Characterization and expression of genes involved in the ethylene biosynthesis and signal transduction during ripening of mulberry fruit.

Although ethylene is well known as an essential regulator of fruit development, little work has examined the role ethylene plays in the development and maturation of mulberry (Morus L.) fruit. To study the mechanism of ethylene action during fruit development in this species, we measured the ethylene production, fruit firmness, and soluble solids content (SSC) during fruit development and harvest. By comparing the results with those from other climacteric fruit, we concluded that Morus fruit are probably climacteric. Genes associated with the ethylene signal transduction pathway of Morus were characterized from M. notabilis Genome Database, including four ethylene receptor genes, a EIN2-like gene, a CTR1-like gene, four EIN3-like genes, and a RTE1-like gene. The expression patterns of these genes were analyzed in the fruit of M. atropurpurea cv. Jialing No.40. During fruit development, transcript levels of MaETR2, MaERS, MaEIN4, MaRTE, and MaCTR1 were lower at the early stages and higher after 26 days after full bloom (DAF), while MaETR1, MaEIL1, MaEIL2, and MaEIL3 remained constant. In ripening fruit, the transcripts of MaACO1 and MaACS3 increased, while MaACS1 and MaACO2 decreased after harvest. The transcripts of MaACO1, MaACO2, and MaACS3 were inhibited by ethylene, and 1-MCP (1-methylcyclopropene) upregulated MaACS3. The transcripts of the MaETR-like genes, MaRTE, and MaCTR1 were inhibited by ethylene and 1-MCP, suggesting that ethylene may accelerate the decline of MaETRs transcripts. No significant changes in the expression of MaEIN2, MaEIL1, and MaEIL3 were observed during ripening or in response to ethylene, while the expressions of MaEIL2 and MaEIL4 increased rapidly after 24 h after harvest (HAH) and were upregulated by ethylene. The present study provides insights into ethylene biosynthesis and signal transduction in Morus plants and lays a foundation for the further understanding of the mechanisms underlying Morus fruit development and ripening

Gene structure and predicted functional domains of <i>Morus notabilis</i> ethylene signaling genes.

<p>Gene structures were obtained by aligning the cloned cDNA sequences with the <i>M</i>. <i>notabilis</i> genome data and functional domains were predicted in SMART (<a href="http://smart.embl-heidelberg.de/smart/set_mode.cgi?NORMAL=1" target="_blank">http://smart.embl-heidelberg.de/smart/set_mode.cgi?NORMAL=1</a>). Gene structures were displayed by Fancy Gene (<a href="http://bio.ieo.eu/fancygene/" target="_blank">http://bio.ieo.eu/fancygene/</a>)</p

Information of genes involved in ethylene signaling in <i>Morus notabilis</i>.

<p>^a The CDS length was predicted according to the predicted <i>Morus</i> genes.</p><p>^b The number of exon was predicted based on the <i>Morus</i> genomic data.</p><p>Information of genes involved in ethylene signaling in <i>Morus notabilis</i>.</p

The expression profiles of genes involved in ethylene signal transduction in <i>Morus notabilis</i>.

<p>Expression analysis of genes involved in ethylene signal transduction based on the RPKM (reads per kilobase of exon model per million mapped reads) profile of five tissues (root, bark, bud, flower, and leaf). Cluster 3.0 software was used to normalize the expression level of the ethylene signal-related genes from RNA sequencing data. Sample names are shown above the heat maps. Color scale indicates the degree of expression: green, low expression; red, high expression; grey, no expression.</p

Effects of exogenous C₂H₄ and 1-MCP on postharvest fruit of <i>Morus atropurpurea</i> cv. <i>Jialing</i> No.40 at 23°C.

<p>The batch 2 fruits were divided into three groups. Two groups were treated with ethylene and 1-MCP, respectively. The third group of fruits was sealed in a similar container of the same volume as a control. Error bars on each column indicate SDs from eight replicates.</p

Primers used for cloning the genes involved in the ethylene signal transduction in <i>Morus atropurpurea</i> cv. <i>Jialing</i> No.40.

<p>Primers used for cloning the genes involved in the ethylene signal transduction in <i>Morus atropurpurea</i> cv. <i>Jialing</i> No.40.</p

Expression of the genes involved in ethylene biosynthesis and signaling pathway during fruit ripening of <i>Morus atropurpurea</i> cv. <i>Jialing</i> No.40.

<p>The batch 2 fruits were used for qRT-PCR. Error bars on each column indicate SDs from three replicates. Significant differences (<i>P</i><0.05) among treatment are marked with different letters above bars.</p

Expression of different components of the ethylene signaling pathway during fruit development of <i>Morus atropurpurea</i> cv. <i>Jialing</i> No.40.

<p>The batch 1 fruits were used for qRT-PCR. Each column height indicates relative mRNA abundance. Error bars on each column indicate SDs from three replicates. Significant differences (<i>P</i><0.05) are marked with different letters above bars.</p

Phylogenetic analysis of MnETRs (A), MnCTR1 (B), MnRTE (C), and MnEILs(D).

<p>The amino acid sequences were analyzed with MUSCLE 3.6 and the phylogenetic tree constructed with MEGA 5.0 using a bootstrap test of phylogeny with minimum evolution test and default parameters. AtETR1 (AAA70047.1), AtERS1 (NP_181626.1), AtETR2 (NP_188956.1), AtERS2 (AAC62209.1), AtEIN4(AAD02485.1), AtCTR1 (AAA32780.1), AtEDR1 (AAG31143.1), AtRTE1(F4ITL6.1), AtRTE2(Q9SD42.1), AtEIN3 (NP_188713.1), AtEIL1 (NP_180273.1), AtEIL2 (NP_197611.1), AtEIL3 (NP_177514.1) and AtEIL4(NP_196574.1) in <i>Arabidopsis</i>; LeETR1 (AAC02213.1), LeETR2 (AAC02214.1), LeETR3 (AAC49124.1), LeETR4 (AAU34076.1), LeETR5 (AAD31397.1), LeETR6 (AAL86614.1), LeCTR1(AAL87456.1), TCTR2 (CAA06334.1), LeCTR3 (AAR89820.1), LeCTR4 (AAR89822.1), LeEIL1 (AAK58857.1), LeEIL2 (AAK58858.1), LeEIL3 (AAK58859.1) and LeEIL4 (BAC99307.1) in <i>Solanum lycopersicum</i>; PmRTE (XP_008220470.1) in <i>Prunus mume</i>; ObRTE (XP_006654719.1) in <i>Oryza brachyantha</i>; CsRTE (XP_006475936.1) in <i>Citrus sinensis</i>; StRTE (XP_006349393.1) in <i>Solanum tuberosum</i>; SiRTE (XP_004961370.1) in <i>Setaria italic</i>; CaRTE (XP_004498900.1) in <i>Cicer arietinum</i>; FaRTE (XP_004291036.1) in <i>Fragaria vesca subsp</i>. <i>Vesca</i>; CsRTE1 (XP_004157357.1) in <i>Cucumis sativus</i>; CsRTE2 (XP_004141314.1) in <i>Cucumis sativus</i>; DcRTE1 (ADW80941.1) in <i>Dianthus caryophyllus</i>.</p