Cloning and expression analysis of cinnamoyl-CoA reductase (CCR) genes in sorghum

Abstract

Cinnamoyl-CoA reductase (CCR) is the first enzyme in the monolignol-specific branch of the lignin biosynthetic pathway. In this research, three sorghum CCR genes including SbCCR1, SbCCR2-1 and SbCCR2-2 were cloned and characterized. Analyses of the structure and phylogeny of the three CCR genes showed evolutionary conservation of the functional domains and divergence of function. Transient expression assays in Nicotiana benthamiana leaves demonstrated that the three CCR proteins were localized in the cytoplasm. The expression analysis showed that the three CCR genes were induced by drought. But in 48 h, the expression levels of SbCCR1 and SbCCR2-2 did not differ between CK and the drought treatment; while the expression level of SbCCR2-1 in the drought treatment was higher than in CK. The expression of the SbCCR1 and SbCCR2-1 genes was not induced by sorghum aphid [Melanaphis sacchari (Zehntner)] attack, but SbCCR2-2 was significantly induced by sorghum aphid attack. It is suggested that SbCCR2-2 is involved in the process of pest defense. Absolute quantitative real-time PCR revealed that the three CCR genes were mainly expressed in lignin deposition organs. The gene copy number of SbCCR1 was significantly higher than those of SbCCR2-1 and SbCCR2-2 in the tested tissues, especially in stem. The results provide new insight into the functions of the three CCR genes in sorghum.

Biochemical and Expression Analyses of the Rice Cinnamoyl-CoA Reductase Gene Family

“…Of OsCCRs with appropriate peptide lengths, OsCCR19 and 20 exhibited the fully conserved catalytic motif, and OsCCR4, 5, 17, 18, and 21 had the signature motif with one amino acid variation (G to A) (Figure 3 ). The G to A variation in the CCR catalytic motifs has been frequently found in other active CCRs, such as PvCCR2a and ZmCCR2 (Figure 3 ) (Pichon et al, 1998 ; Escamilla-Treviño et al, 2010 ; Li et al, 2016 ). Indeed, our biochemical assays confirmed that OsCCR17, 19, 20 and 21 had CCR activity to hydroxycinnamoyl-CoAs (Table 2 and Supplementary Table 4 ).…”

“…A large member of CCR gene family in plants has also been supposed to because of their substrate diversity (Xu et al, 2009 ). All previously characterized CCRs showed similar peptide lengths ranging from 332 to 374 amino acids in A. thaliana , wheat, sorghum, switchgrass, and E. gunnii (Lacombe et al, 1997 ; Lauvergeat et al, 2001 ; Ma, 2007 ; Escamilla-Treviño et al, 2010 ; Li et al, 2016 ). Twenty-four OsCCRs had peptide lengths similar to known CCRs (Table 1 ).…”

“…In particular, OsCCR20 and 19 were closely related to PvCCR1 ( P. virgatum CCR1), SbCCR1, ZmCCR1, LpCCR ( Lolium perenne CCR), HvCCR ( Hordeum vulgare CCR) and TaCCR1 ( T. aestivum CCR1). These CCRs have been suggested as monocot functional CCRs involved in developmental lignification (Figure 2 and Supplementary Figure 2 ) (Pichon et al, 1998 ; Larsen, 2004a , b ; Ma, 2007 ; Escamilla-Treviño et al, 2010 ; Tamasloukht et al, 2011 ; Li et al, 2016 ). OsCCR17, 18, and 21 were grouped with ZmCCR2, SbCCR2, TaCCR2, and PvCCR2, suggesting that they play a role in defense-related processes under biotic and abiotic stresses (Figure 2 and Supplementary Figure 2 ) (Pichon et al, 1998 ; Escamilla-Treviño et al, 2010 ; Li et al, 2016 ).…”

“…Rather, the exon-intron structure of OsCCR21 was more similar to Pattern 2 (Pattern 2-like), with the length of the fourth exon equaling that seen in Pattern 2 (Figure 1 ). SbCCR2-2 has been reported to also exhibit a Pattern 2-like exon-intron structure (Figure 1 ) (Li et al, 2016 ). The biochemically active OsCCR17 was composed of four exons with an exceptionally long fourth exon (Pattern 5) (Figure 1 ).…”

Establishment and Characterization of Callus and Cell Suspension Cultures of Selected Sorghum bicolor (L.) Moench Varieties: A Resource for Gene Discovery in Plant Stress Biology

“…The genes and their gene products are SORBI_3008G134700 (aspartic peptidase), SORBI_3001G514200 (thioredoxin), SORBI_3003G136200 (germin), SORBI_3006G135500 (galactose oxidase), SORBI_3007G149600 (histone H2B), SORBI_3001G313200 (histone H4), SORBI_3001G073900 (malate dehydrogenase), and SORBI_3003g322400 (ribosomal protein S25). Data analysis was carried out using the REST2009 version 2.0.13 software (Qiagen, Manchester, UK) with sorghum genes Sb03g038910 [32] and Sb04g003390 [33] as constitutive reference controls. Primers were designed using the Primer-BLAST tool [34] and the sequences are listed in Table 1.…”