Molecular cloning, characterization and expression of two rapeseed (Brassica napus L.) cDNAs orthologous to Arabidopsis thaliana phenylalanine ammonia-lyase 1

Abstract

Phenylalanine ammonia-lyase (PAL) plays important roles in determining flavonoid-and lignin-related traits. Two PAL full-length cDNAs, BnPAL1-1 and BnPAL1-2, were isolated from Brassica napus. BnPAL1-1 is 2460 bp encoding a 722-aa protein, while BnPAL1-2 is 2396 bp encoding a 719-aa protein. BnPAL1-1 shares 86.8% nucleotide identities and 94.9%/97.0% identities/positives at protein level to BnPAL1-2. NCBI BLAST, pairwise alignment and preferential amino acid analysis indicated that they are orthologs of Arabidopsis thaliana PAL1 (AtPAL1), suggesting that the divergence of AtPAL1 and AtPAL2 preceded the divergence of genera Arabidopsis and Brassica. Southern hybridization and EST analysis showed that B. napus has 2-6 orthologs of AtPAL1. High ratios of synonymous to non-synonymous substitutions suggest that in Brassicaceae PAL1 orthologs evolve under high pressure of purification selection, while among PAL1 paralogs this pressure is a little lower. Escherichia coli-expressed 6 · His-tagged BnPAL1-1 and BnPAL1-2 were both bioactive, but BnPAL1-2 was much higher in tested catalytic activity. BnPAL1-1 and BnPAL1-2 are transcribed in all the 11 organs tested, but most abundant in flower. BnPAL1-1 shows declined expression in late-stage seed, and BnPAL1-2 shows stronger organ specificity. Transcription of these two genes in middle and late stages of seed development of yellow-seeded line L2 is obviously lower than near-isogenic black-seeded line L1, indicating the correlation of yellow-seed trait to down regulation of PAL transcription, but this is probably caused by suppressed seed-specific upstream regulatory signal or by seed-specific feedback inhibition from downstream pathways.

TRANSPARENT TESTA 12 genes from Brassica napus and parental species: cloning, evolution, and differential involvement in yellow seed trait

“…In previous studies, we isolated and characterized the key enzyme loci for F3'H and PAL1 and the regulatory loci TTG1 and TT2 from B. napus. Interestingly, we found that these genes showed no or limited downregulation in the yellow seed line L2 bred by our group Lu et al 2007;Wei et al 2007;Ni et al 2008). …”

Genome-Wide Survey of Flavonoid Biosynthesis Genes and Gene Expression Analysis between Black- and Yellow-Seeded Brassica napus

“…Moreover, several key loci isolated from B . napus by our group, such as F3'H, PAL1, TTG1 , and TT2 , showed no or limited down-regulation in the yellow-seeded lines (Wei et al, 2007; Xu et al, 2007; Ni et al, 2008; Lu et al, 2009). Based on a marker closely linked with a major QTL for seed fiber and color in B .…”

“…Hence, identifying candidate genes by cloning Brassica TT genes involved in the flavonoid biosynthetic pathway and conducting comparative studies of these genes is a reasonable approach, and many homologs of these genes have also been identified in B. napus (Wei et al, 2007; Xu et al, 2007; Ni et al, 2008; Chai et al, 2009; Lu et al, 2009; Chen et al, 2013). However, little is known about the mechanism underlying seed color formation in B. napus .…”

Molecular Mapping and QTL for Expression Profiles of Flavonoid Genes in Brassica napus

“…napus (Theander et al, 1977; Marles and Gruber, 2004; Akhov et al, 2009; Qu et al, 2013), and dozens of homologous genes in the B . napus flavonoid biosynthesis pathway have been cloned and characterized (Wei et al, 2007; Xu et al, 2007; Ni et al, 2008; Akhov et al, 2009; Auger et al, 2009; Chai et al, 2009; Lu et al, 2009; Chen et al, 2013). Prior to this study, no comprehensive analysis of the flavonoid biosynthesis pathway had been conducted in B. napus .…”

“…Moreover, some homologs of genes involved in flavonoid biosynthesis have been cloned and characterized in B . napus (Wei et al, 2007; Xu et al, 2007; Ni et al, 2008; Akhov et al, 2009; Auger et al, 2009; Chai et al, 2009; Lu et al, 2009; Chen et al, 2013). These results provide a foundation for further studies of the molecular and regulatory mechanisms underlying seed coat color formation in B. napus .…”