Two Divergent Members of a Tobacco 4-Coumarate:Coenzyme A Ligase (4CL) Gene Family (cDNA Structure, Gene Inheritance and Expression, and Properties of Recombinant Proteins)

Abstract

Severa1 cDNA clones encoding 4-coumarate:coenzyme A ligase (4CL) were isolated from a tobacco (Nicotiana fabacum) cDNA library and grouped into two classes. Sequencing of one cDNA from each class showed that the clones were similar to other 4CL genes and about 80% identical with each other. Cenomic Southern blots using DNA from Nicofiana sylvesfris, Nicofiana fomentosiformis, and N. tabacum demonstrated the presence of both classes of 4CL sequences (4CL7 and 4CL2) in the progenitor species and in tobacco. Northern blots indicated that 4CL mRNA transcripts are highest in old stems and higher in the unpigmented corolla tubes than i n the pigmented limbs of tobacco flowers. The 4CL genes are developmentally regulated and are wound and methyl jasmonate inducible. The relative abilities of recombinant 4CL1 and 4CL2 proteins to utilize 4-coumarate, ferulate, and caffeate as substrates were similar and comparable with that of 4CL in tobacco stem extracts. Surprisingly, both recombinant 4CL proteins utilized cinnamate as a substrate, an activity not observed in stem extracts. This activity was inhibited by a heat-labile, high-molecular-weight factor found in tobacco stem extracts, suggesting that the substrate specificity of 4CL is, in part, determined by the activity of proteinaceous cellular components.The flow of carbon from primary metabolism into the biosynthesis of an array of phenylpropanoid secondary products involves a minimum of three enzymatic steps, catalyzed by the actions of PAL, cinnamate 4-hydroxylase, and 4CL, which collectively form the general phenylpropanoid pathway. The phenylpropanoid products formed by the action of specific downstream branch pathways include coumarins, flavonoids, lignin, suberin, tannins, and other phenolic compounds, which serve diverse functions as phytoalexins, UV protectants, floral and fruit pigments, structural components of cell walls, and signaling molecules (Hahlbrock and Scheel, 1989).As the last enzyme in the general phenylpropanoid pathway, 4CL converts 4-coumaric acid and other hydroxy-or methoxy-derivatives of cinnamic acid, such as caffeic acid,

The 4-coumarate:CoA ligase gene family in Arabidopsis thaliana comprises one rare, sinapate-activating and three commonly occurring isoenzymes

“…Considering the complexity and the extent of overlapping mRNA expression patterns that have been observed in various species for nearly all tested 4CL isoenzyme combinations (4,7,32,33), including At4CL1-3 (18), the pivotal position of 4CL in plant phenylpropanoid metabolism is probably reflected by a highly diversified metabolic grid (34)(35)(36). Moreover, the combination of distinct substrate preferences with differences in gene-promoter and protein fine structure may facilitate not only a large diversity of genetically programmed metabolic functions of the individual isoenzymes, including pathway-specific associations with biosynthetically related enzymes in metabolite channeling (37-39), but also regular or potential functional overlaps or mutual replacements upon malfunction or down-regulation, as has been investigated so far only for the class I isoforms (4,40,41).…”

Differential Substrate Inhibition Couples Kinetically Distinct 4-Coumarate:Coenzyme A Ligases with Spatially Distinct Metabolic Roles in Quaking Aspen

“…Previous results from single-substrate kinetics (Lee and Douglas, 1996;Allina et al, 1998;Hu et al, 1998;Ehlting et al, 1999) offered no models for how a single or multiple 4CLs would control hydroxycinnamate distribution in a mixed-substrate environment. The present data suggest that exposure of 4CL(s) to different substrate mixtures could constitute a simple mechanism for CoA activation and distribution of hydroxycinnamates.…”

“…Protein modification or competing activities can interfere with measurement of enzyme activity in tissue extracts, potentially causing isoform distribution patterns based on similarity with recombinant enzyme activity to be misleading. In the case of transcriptionally regulated genes like 4CL (Douglas, 1996;Ehlting et al, 1999), transcript distribution would be expected to correlate with isoform activity pattern. A high resolution in situ analysis of these genes in developing tissues was performed to confirm the general distribution of 4CL isoforms derived from enzyme activity, as well as to reveal any discrepancies between enzyme activity and gene expression patterns.…”

Isolation and characterization of the 4-coumarate:coenzyme A ligase (4CL1) promoter from Eucalyptus camaldulensis

“…Gene sequence analysis demonstrated that 4CL is encoded by either single gene in pine (Voo et al 1995), duplicated genes in parsley and potato (Lozoya et al 1988;Becker-André et al 1991), or multiple genes in soybean, poplar (Lindermayr et al 2002;Chen et al 2013;Zhang et al 2015), sorghum (Saballos et al 2012) and Arabidopsis . Although, the expression of the duplicated 4CL genes (4CL1 and 4CL2) was similar in various organs of tobacco, they were divergent in coding sequences (Lee and Douglas 1996). Similarly, Pinta4CL1 and Pinta4CL2 genes of pine (Pinus taeda) were identical in the coding sequences (Zhang and Chiang 1997).…”

“…A number of 4CL and 4CL-like genes have been identified from different species such as parsley (Lozoya et al 1988), potato (Becker-André et al 1991), pine (Voo et al 1995), tobacco (Lee and Douglas 1996), Eucalyptus camaldulensis (Chen et al 2006), Pinus massoniana (Huan et al 2012), and mulberry tree, Morus notabilis (Wang et al 2016).…”