Isolation of cinnamoyl CoA reductase and cinnamyl alcohol dehydrogenase gene promoters from Leucaena leucocephala, a leguminous tree species, and characterization of tissue-specific activity in transgenic tobacco

Abstract

Promoter sequences of a 795 bp cinnamoyl CoA reductase (LlCCR) and 1,882 bp cinnamyl alcohol dehydrogenase (LlCAD) genes were isolated from Leucaena leucocephala, a leguminous tree species by genome walking, and analysed using bioinformatics tools. This revealed presence of cis-elements such as AC-boxes, XYLAT, WRKY, and MYB binding sites in addition to CAAT and TATA boxes. For functional characterization, each of LlCCR and LlCAD promoter sequences were fused to b-glucuronidase (GUS) reporter gene, immobilized into pBI101 plasmid, and introduced into tobacco via Agrobacterium tumefaciens strain LBA4404. Histochemical observations of transgenic lines indicated tissue-specific expression of GUS in the vascular tissues of leaves, stems, and roots. These results demonstrate that GUS expression driven by either LlCCR or LlCAD promoters were involved in lignifying tissues, and more specifically in differentiating xylem cells. This observed tissue-specific expression driven by either LlCCR or LlCAD promoters is sufficient for reducing the lignin content only in vascular tissues, thus overcoming the risks and challenges associated with down-regulation of lignin content in whole plants.

Plant Promoters: An Approach of Structure and Function

“…GSE2 acts as a regulator only in the sheaths and stem tissues and has a weaker effect on gene expression. Thilmony and Cook [61] characterized the promoter OsGEX2, which is expressed in the sperm cells of mature rice pollen • Leucaena (Leucaena leucocephala): Prashant et al [62] isolated 2 specific promoters, LlCCR and LlCAD, that act in the vascular tissues of leaves, roots, and stamens • Soybean (Glycine max): In the literature, several studies have shown specific promoters isolated from soybean; some of them have been used in transgenic research. Freitas [63] isolated the promoter SPB2 (sucrosebinding protein), which guides the expression of reporter genes in the vascular tissue of transgenic tobacco.…”

“…Promoter sequences can be analyzed using several computational tools currently available, allowing the identification of putative TFs, which are analyzed by alignment using databases available online and further characterization in silico [60,62]. Some database sites are shown in Table 2.…”

A pseudogene of caffeic acid-o-methyltransferase (COMT) in Acacia mangium: Comparative analysis with other COMT plant promoters

“…P-box, a motif implicated in gibberellin response was found at +2. Overall, it is noted that similar sets of cis-acting elements were also reported from promoters of other lignin genes such as cinnamoyl-CoA reductase (CCR) and cinnamyl alcohol dehydrogenase (CAD) (Prashant et al 2012;Lacombe et al 2000).…”

The Alcohol Dehydrogenase Gene Family in Melon (Cucumis melo L.): Bioinformatic Analysis and Expression Patterns

“…Nevertheless, the most abundant motifs detected were the cis-elements involved in the response to biotic and abiotic stresses, including TC-rich repeats, LTR, W-BOX, ARE, HSE and MBS ( Jin et al, 2014 ). At the same time, we still found that there were putative cis -acting regulatory elements involved in the MeJA-responsiveness, namely CGTA-motif, TGACG-motif and CCAAT-motif ( Matton et al, 1990 ; Prashant et al, 2012 ; Cheng H. et al, 2013 ). TCA-element clustered in the promoters of CmFDH1, CmADH1, CmADH3, CmADH5, CmADH7-9 and CmADH12 , and S ARE were also present in CmADH3 promoter, both of which were cis -acting regulatory element involved in the SA-responsiveness ( Prashant et al, 2012 ; Cheng H. et al, 2013 ).…”

“…At the same time, we still found that there were putative cis -acting regulatory elements involved in the MeJA-responsiveness, namely CGTA-motif, TGACG-motif and CCAAT-motif ( Matton et al, 1990 ; Prashant et al, 2012 ; Cheng H. et al, 2013 ). TCA-element clustered in the promoters of CmFDH1, CmADH1, CmADH3, CmADH5, CmADH7-9 and CmADH12 , and S ARE were also present in CmADH3 promoter, both of which were cis -acting regulatory element involved in the SA-responsiveness ( Prashant et al, 2012 ; Cheng H. et al, 2013 ). ERE, ethylene-responsive element, was found in the promoter of CmFDH1, CmADH1, CmADH2, CmADH3, CmADH5, CmADH6, CmADH8 and CmADH12.…”