Chapter three The phenylpropanoid pathway in arabidopsis: Lessons learned from mutants in sinapate ester biosynthesis

Summary and Future Directions The analysis of mutants of the phenylpropanoid pathway in Arabidopsis , asoutlined in this review, has led to numerous revisions of the pathway over the past decade. The presently accepted pathway clarifies some of the contradictory data of the past, but also poses new questions for which we do not yet have answers. For example, a growing body of evidence suggests that neither ferulic acid nor sinapic acid are intermediates in phenylpropanoid biosynthesis. This is problematic in that many plant cell walls contain esterified ferulic acid, 10,11 and sinapic acid esters are major soluble secondary metabolites in Arabidopsis leaves and seeds. 31 If the most current model of the pathway is correct, how are these molecules synthesized? Another challenge will be to assign function to individual members ofenzymes that belong to gene families, which include CAD, CCR, and 4CL. 122 Different isoforms may exhibit specific spatial or temporal expression during development. Alternatively, individual members of a gene family may possess different substrate specificities towards intermediates of the pathway, which in turn may control the flux of the pathway towards different phenylpropanoid end products. The analysis of mutants with null alleles of these isoforms, either from publicly available T-DNA insertion lines or developed utilizing RNAi, will be necessary to elucidate their roles. Evidene that supports the assembly of multi-enzyme complexes responsible for the metabolic channeling of intermediates during flavonoid biosynthesis has been described in Arabidopsis . 123, 124 Multi-enzyme assemblies, or “metabolons”, would concentrate substrate pools for each reaction, leading to an overall more efficient production of final products. Such a, complex has recently been proposed to operate in the production of monolignols, 125 in which P450s would provide an anchor to which the soluble enzymes of the pathway would be tethered via protein/protein interactions. 126 It has been further suggested that these metabolons may be differentially assembled for the production of either H, G, or S monolignols. If this proves to be the case, it will provide significant new opportunities for the study of phenylpropanoid biosynthetic regulation. To date, most of the phenylpropanoid pathway genes isolated from Arabidopsis using genetic approaches encode enzymes. In contrast, little is known regarding the transcriptional regulatory elements of monolignol and sinapate ester biosynthesis. 127 This is in stark contrast to our understanding of the regulation of flavonoid and anthocyanin biosynthesis, which has been elucidated in detail through the analysis of maize and petunia mutants. 128 Recently, a number of Arabidopsis flavonoid regulatory mutants and their corresponding genes have been described. 129–134 In contrast, the sole regulatory element shown to be required for sinapate ester and monolignol biosynthesis is AtMyb4, an ortholog of the Antirrhinum majus gene Am MYB308, 135 which represses C4H trnascription in response to low UV levels. 136,137 Only a few MYB regulatory proteins are found in yeast and animals, whereas the Arabidopsis genome contains at least 123 MYBs. 138 It seems clear that this class of proteins has evolved to regulate an array of functions in plants, including secondary metabolism. 139 The assignment of function to this class of proteins may, thus, shed further light onto the regulation of secondary metabolism in plants. Finally, further research into the structural and regulatory aspects ofphenylpropanoid biosynthesis in Arabidopsis may lead to interesting insights into the evolution of land plants. It is generally accepted that lignin biosynthesis was crucial for the colonization of land by plants. 140, 141 The knowledge gained by studies in Arabidopsis will permit the isolation and functional characterization of enzymes and regulatory factors from a wide array of genera, including pteridophytes and lycophytes, that arose before seed plants. These studies will reveal the similarities and differences in phenylpropanoid biosynthesis and its regulation that have arisen over the past 400 million years. In doing so, we may gain further appreciation for ancient evolutionary events that allowed for the spectacular diversity in plant life that we see today.