mRNA degradation is an important cellular mechanism involved in the control of gene expression. Several genome-wide profiling methods have been developed for detecting mRNA degradation in plants and animals. However, because many of these techniques use poly (A) mRNA for library preparation, degradation intermediates are often only detected near the 3′-ends of transcripts. Previously, we developed the Truncated RNA End Sequencing (TREseq) method using Arabidopsis thaliana, and demonstrated that this method ameliorates 3′-end bias. In analyses using TREseq, we observed G-rich sequences near the 5′-ends of degradation intermediates. However, this finding remained to be confirmed in other plant species. Hence, in this study, we conducted TREseq analyses in Lactuca sativa (lettuce), Oryza sativa (rice) and Rosa hybrida (rose). These species including A. thaliana were selected to encompass a diverse range in the angiosperm phylogeny. The results revealed similar sequence features near the 5′-ends of degradation intermediates, and involvement of translation process in all four species. In addition, homologous genes have similar efficiencies of mRNA degradation in different plants, suggesting that similar mechanisms of mRNA degradation are conserved across plant species. These strong sequence features were not observed in previous degradome analyses among different species in plants.
The brilliant blue color of the Nemophila menziesii flower is derived from metalloanthocyanin, which consists of anthocyanin {petunidin 3-O-[6-O-(trans-p-coumaroyl)-β-glucoside]-5-O-[6-O-(malonyl)-β-glucoside]}, flavone [apigenin 7-O-β-glucoside-4'-O-(6-O-malonyl)-O-β-glucoside] and metal ions (Mg2+, Fe3+). Although the two glucosyl moieties at the apigenin 7-O and 4'-O positions are essential for metalloanthocyanin formation, the mechanism of glucosylation has not yet been clarified. In this study, we used crude protein extract prepared from N. menziesii petals to determine that apigenin is sequentially glucosylated by the catalysis of UDP-glucose:flavone 4'-O-glucosyltrasferase (F4'GT) and UDP-glucose:flavone 4'-O-glucoside 7-O-glucosyltransferase (F4'G7GT). We identified 150 contigs exhibiting homology with a UDP-glucose-dependent GT in the N. menziesii petal transcriptome and isolated 24 putative full-length GT cDNAs which were then subjected to functional analysis. Two GT cDNAs, NmF4'GT and NmF4'G7GT, which are highly expressed during the early stages of petal development and rarely in leaves, were shown to encode F4'GT and F4'G7GT activities, respectively. Biochemical characterization of the recombinant enzymes revealed that NmF4'GT specifically catalyzed 4'-glucosylation of flavonoids and that NmF4'G7GT specifically catalyzed 7-glucosylation of flavone 4'-O-glucosides and flavones. Apigenin 7,4'-O-diglucoside was efficiently synthesized from apigenin in the presence of recombinant NmF4'GT and NmF4'G7GT. Transgenic tobacco BY-2 cells expressing NmF4'GT and NmF4'G7GT converted apigenin into apigenin 7,4'-O-diglucoside, confirming their activities in vivo. Based on these results, we conclude that these two GTs act co-ordinately to catalyze apigenin 7,4'-O-diglucoside biosynthesis in N. menziesii.
Blue flower color of Nemophila menziesii Hook. and Arn. is derived from a metalloanthocyanin, nemophilin, which comprises petunidin-3-O-[6-O-(trans-p-coumaroyl)-β-glucoside]-5-O-[6-O-(malonyl)-β-glucoside], apigenin-7-Oβ-glucoside-4′-O-(6-O-malonyl)-β-glucoside, and Mg 2+ and Fe 3+ ions. The flavonoid biosynthetic pathway of nemophilin has not yet been characterized. RNA-Seq analysis of the petals yielded 61,491 contigs. These were searched using BLAST against petunia or torenia flavonoid biosynthetic proteins, which identified 11 putative full-length protein sequences belonging to the flavonoid biosynthetic pathway. RT-PCR using primers designed on the basis of these sequences yielded 14 sequences. Spatio-temporal transcriptome analysis indicated that genes involved in the early part of the pathway are strongly expressed during early-petal development and that those in the late part at late-flower opening stages, but they are rarely expressed in leaves. Flavanone 3-hydroxylase and flavonoid 3′,5′-hydroxylase cDNAs were successfully expressed in yeast to confirm their activities. Recombinant anthocyanin O-methyltransferase cDNA (NmAMT6) produced using Escherichia coli was subjected to biochemical characterization. Km of NmAMT6 toward delphinidin 3-O-glucoside was 22 µM, which is comparable with Km values of anthocyanin O-methyltransferases from other plants. With delphinidin 3-O-glucoside as substrate, NmAMT6 almost exclusively yielded petunidin 3-O-glucoside rather than malvidin 3-O-glucoside. This specificity is consistent with the anthocyanin composition of Nemophila petals.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.