Biology subjects

Holland, C. K.

Publications and source records attributed to Holland, C. K..

3 recordsLinked to original sources

bioRxiv2025

2-Oxoglutarate-dependent dioxygenases contribute to cardenolide biosynthesis in Erysimum cheiranthoides (wormseed wallflower)

O_LICardenolide biosynthesis evolved convergently in several plant lineages, including wallflowers (Erysimum, Brassicaceae). Although the first steps of the biosynthetic pathway, which involve conversion of sterols to pregnane derivatives, have been characterized in Erysimum cheiranthoides and other species, key enzymes that catalyze the 14{beta}- and 21-hydroxylation of the steroid core remained elusive. C_LIO_LIIn this study, we used comparative transcriptomic analysis of different Erysimum species to identify 2-oxoglutarate dependent dioxygenases (2OGDs) that catalyze these reactions in E. cheiranthoides: Erche03g034150 (CARDENOLIDE METABOLISM 5; CARD5), which is related to ALKENYL HYDROXYALKYL PRODUCING 1 (AOP1), and Erche01g020322 (CARDENOLIDE METABOLISM 6; CARD6), which arose from a duplication of DIOXYGENASE FOR AUXIN OXIDATION 1 (DAO1). C_LIO_LIKnockout mutants of both genes are deficient in cardenolide biosynthesis and instead accumulate pathway intermediates. Based on transient expression and activity in Nicotiana benthamiana, and we identified CARD5 as the likely 14{beta}-hydroxylase, and CARD6 as the 21-oxygenase. Finally, enzyme modeling and substrate docking identified key residues that may allow shifts to substrate recognition during neofunctionalization. C_LIO_LIThe results of this research provide new insight into the evolution of cardenolide biosynthesis and have potential practical applications in the engineering of steroid-derived compounds for medical uses. C_LI

plant biology↗

bioRxiv2024

Molecular basis of one-step methyl anthranilate biosynthesis in grapes, sweet orange, and maize

Plants synthesize an array of volatile compounds, many of which serve ecological roles in attracting pollinators, deterring herbivores, and communicating with their surroundings. Methyl anthranilate is an anti-herbivory defensive volatile responsible for grape aroma that is emitted by several agriculturally relevant plants, including citrus, grapes, and maize. Unlike maize, which uses a one-step anthranilate methyltransferase, grapes have been thought to use a two-step pathway for methyl anthranilate biosynthesis. By mining available transcriptomics data, we identified two anthranilate methyltransferases in Vitis vinifera (wine grape), as well as one ortholog in Concord grape. Many angiosperms methylate the plant hormone salicylic acid to produce methyl salicylate, which acts as a plant-to-plant communication molecule. Because the Citrus sinensis (sweet orange) salicylic acid methyltransferase can methylate both anthranilate and salicylic acid, we used this enzyme to examine the molecular basis of anthranilate activity by introducing rational mutations, which identified several active site residues that increase activity with anthranilate. Reversing this approach, we introduced mutations that imparted activity with salicylic acid in the maize anthranilate methyltransferase, which uncovered different active site residues from those in the citrus enzyme. Sequence and phylogenetic analysis revealed that one of the Vitis anthranilate methyltransferases shares an ancestor with jasmonic acid methyltransferases, similar to the anthranilate methyltransferase from strawberry (Frageria sp.). Collectively, these data demonstrate the molecular mechanisms underpinning anthranilate activity across methyltransferases and identified one-step enzymes by which grapes synthesize methyl anthranilate. Significance StatementWhile the two-step pathway responsible for the biosynthesis of the grape aroma molecule, methyl anthranilate, has remained incomplete in Vitis spp., we identified two one-step anthranilate methyltransferases in wine and one in Concord grapes that can methylate the tryptophan pathway intermediate anthranilate. Tracing the molecular basis of anthranilate activity in the maize and sweet orange methyltransferases uncovered distinct active site amino acids that impart substrate specificity.

plant biology↗

bioRxiv2019

Rapid and independent evolution of ancestral and novel defenses in a genus of toxic plants (Erysimum, Brassicaceae)

Phytochemical diversity is thought to result from coevolutionary cycles as specialization in herbivores imposes diversifying selection on plant chemical defenses. Plants in the speciose genus Erysimum (Brassicaceae) produce both ancestral glucosinolates and evolutionarily novel cardenolides as defenses. Here we test macroevolutionary hypotheses on co-expression, co-regulation, and diversification of these potentially redundant defenses across this genus. We sequenced and assembled the genome of E. cheiranthoides and foliar transcriptomes of 47 additional Erysimum species to construct a highly resolved phylogeny, revealing that cardenolide diversity increased rapidly rather than gradually over evolutionary time. Concentrations, inducibility, and diversity of the two defenses varied independently among species, with no evidence for trade-offs. Closely related species shared similar cardenolide traits, but not glucosinolate traits, likely as a result of specific selective pressures acting on distinct molecular diversification mechanisms. Ancestral and novel chemical defenses in Erysimum thus appear to provide complementary rather than redundant functions.

plant biology↗