Food Chemistry
Volume 123, Issue 1, 1 November 2010, Pages 32-40

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doi:10.1016/j.foodchem.2010.03.122 | How to Cite or Link Using DOI Copyright © 2010 Elsevier Ltd All rights reserved.	Cited By in Scopus (0)
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Effects of 1-MCP and ethylene on expression of three CAD genes and lignification in stems of harvested Tsai Tai (Brassica chinensis)

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Lubin Zhang^{a, b}, Guang Wang^b, Jinmei Chang^a, Jianshuang Liu^b, Jianghua Cai^b, Xiuwen Rao^b, Lijuan Zhang^b, Juanjuan Zhong^b, Jianghui Xie^a and Shijiang Zhu^{b, ,}

^a Hainan Key Laboratory for Postharvest Physiology and Technology of Tropical Horticultural Products, South Subtropical Crops Research Institute, Chinese Academy of Tropical Agricultural Sciences, Zhanjiang 524091, Guangdong Province, China

^b Guangdong Province Key Laboratory of Postharvest Physiology and Technology of Fruits and Vegetables, College of Horticulture, South China Agricultural University, Guangzhou 510642, Guangdong Province, China

Received 7 December 2009;  revised 8 February 2010;  accepted 30 March 2010.  Available online 2 April 2010.
   

Abstract

Tsai Tai (Brassica chinensis) is produced mainly in China but also consumed overseas. The stems of Tsai Tai are subject to toughening due to lignification. Cinnamyl alcohol dehydrogenase (CAD) is a key enzyme involved in lignin biosynthesis. Three full-length CAD cDNAs were isolated, encoding proteins of 288, 323, and 323 amino acids, respectively. Sequence analysis showed that they share a highly conserved putative NAD/NADP(H)-binding site at the amino terminus. All the three BcCAD genes responded to 1-MCP and ethylene within 2 h. Ethylene up-regulated expression of BcCAD1-1 and BcCAD2, while 1-MCP down-regulated them. Ethylene increased yellow leaves and stem lignin, and 1-MCP decreased them. These results suggest ethylene is involved in lignin growth in Tsai Tai, and induced expression of BcCAD1-1 and BcCAD2 could contribute to lignification. They also suggest removal of ethylene and application of 1-MCP could extend shelf-life and knockout of certain CAD genes could produce lines that store well.

Keywords: Cinnamyl alcohol dehydrogenase; Ethylene; 1-MCP; Senescence; Lignification; Brassica chinensis

Article Outline

1.

Introduction

2.

Materials and methods

2.1. Plant material and treatment

2.2. Extraction of RNA and isolation of full-length cDNA of CAD genes

2.3. Sequence analysis

2.4. CAD gene expression analysis

2.5. Lignin content assay

2.6. Design and analysis

3.

Results

3.1. Isolation and analysis of Tsai Tai BcCAD cDNA sequences

3.2. Effect of 1-MCP and ethylene on expression of BcCAD genes during and after treatment

3.3. Effect of 1-MCP and ethylene treatment on leaf yellowing of Tsai Tai shoots

3.4. Effect of 1-MCP and ethylene treatment on lignin contents of Tsai Tai stems

4.

Discussion

4.1. BcCAD1-1, BcCAD1-2 and BcCAD2 sequence analysis

4.2. Patterns of response of BcCAD to duration of treatments

4.3. Role of regulation of BcCAD genes in senescence-related lignification

4.4. Potential application of inhibition of lignification in Tsai Tai industry

5.

Conclusion

Acknowledgements

References

Fig. 1. Nucleotide and deduced amino acid sequence of BcCAD1-1 (A), BcCAD1-2 (B) and BcCAD2 (C). The LRGYTV motif is shaded in B and C. The untranslated regions are shown in lower case and signal peptide (M1-V32) of BcCAD1-2 and BcCAD2 is shown in a rectangular box. EKWL motif of CAD1-1 (E284–L287), CAD1-2 (E318–L321) and CAD2 (E318–L321) is single underlined. Numbers on the left margins correspondingly represent nucleotide and deduced amino acid sequences. GenBank accession numbers of the BcCAD1-1, BcCAD1-2 and BcCAD2 sequences are FJ865474, FJ865475 and FJ865476, respectively.
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Fig. 2. Multiple alignment of BcCAD amino acid sequences with CAD and DFR from other plants. Similar amino acid regions were aligned by Clustal W multiple alignment software. Identical amino acids are represented with dots. Double-underlined amino acids indicate conserved amino acid residues. The putative NAD/NADP(H) binding domain is underlined which is located in the N-terminal region.
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Fig. 3. Phylogenetic tree illustrating the genetic relationships between of CAD, dihydroflavonol-4-reductase (DFR), cowpea responsive to dehydration (CPRD), cinnamoyl-CoA reductase (CCR), alcohol dehydrogenase-like protein (ADL), cinnamoyl-CoA reductase-like protein (CCRL), phenylacetaldehyde reductase (PAR) from various species. Brassica chinensis CAD are underlined. Gene name and accession numbers are shown. All sequences with full-length prepropeptides were used for both the alignment and the generation of the tree. The abbreviations are as follow: CaCCR, Capsicum annuum CCR; EgsCAD, Elaeis guineensis CAD; EgiCAD, E. gunnii CAD; EuCAD, Eucommia ulmoides CAD; EsCCR, E. saligna CCR; LpCCR, Lolium perenne CCR; MdCAD, Malus × domestica CAD; NtCAD1, N. tabacum CAD1; ObADL, Ocimum basilicum ADL; PtCCRL, P. trichocarpa CCRL; PmCAD, Plantago major CAD; QiCAD, Quercus ilex CAD; SlPAR, Solanum lycopersicum PAR; StCCR, S. tuberosum CCR; RcCCR, Ricinus communis CCR; VcCCR, Vaccinium corymbosum CCR; VuCPRD14, Vigna unguiculata CPRD14; ZmDFR, Z. mays DFR; PmuCAD, P. mume CAD. A. thaliana (At).
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Fig. 4. Semi-quantitative RT-PCR analysis showing gene expression in stems of Tsai Tai (Brassica chinensis) treated with air (control), 1-MCP and ethylene, respectively. Equal amount of total RNA used in the experiment under 27 cycles. Tsai Tai shoots were treated for 2, 6 and 12 h, respectively, and sampled at each time point after treatment. Data for 1 day through 4 days were from materials exposed to treatments for 12 h.
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Fig. 5. Effect of 1-MCP and ethylene on leaf yellowing of Tsai Tai stored at 25 °C. Tsai Tai shoots were exposed for 12 h to 1-MCP (0.9 ppm) and ethylene (1000 ppm). (A) Pictures taken 2 days after treatment, where air is control. (B) Yellow leaf rate assessed 2 days after treatment. Significance of differences is indicated by letters above the bars (P < 0.05). Standard errors are shown (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.).
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Fig. 6. Effect of 1-MCP and ethylene (Eth) treatment on lignin contents of stems of harvested Tsai Tai stored at 25 °C. Tsai Tai shoots were exposed for 12 h to 1-MCP (0.9 ppm) and ethylene (1000 ppm). Significance of differences is indicated by letters above the bars (P < 0.05). Standard errors are shown.
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  Table 1.

Primers used in the 5′- and 3′-RACE of the CAD cDNA.

Note: FA, first amplification and SA, second amplification.

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  Table 2.

Sequences of primers for semi-quantitative RT-PCR.

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Corresponding author. Tel.: +86 20 38902512/13632355208; fax: +86 20 85288280.

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Food Chemistry Volume 123, Issue 1, 1 November 2010, Pages 32-40