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The F-box protein Coronatine Insensitive (COI) is a receptor for the jasmonic acid signaling pathway in plants. To investigate the functions of the 6 maize (Zea mays) COI proteins (COI1a, COI1b, COI1c, COI1d, COI2a, and COI2b), we generated single, double, and quadruple loss-of-function mutants. The pollen of the coi2a coi2b double mutant was inviable. The coi1 quadruple mutant (coi1-4x) exhibited shorter internodes, decreased photosynthesis, leaf discoloration, microelement deficiencies, and accumulation of DWARF8 and/or DWARF9, 2 DELLA family proteins that repress the gibberellic acid (GA) signaling pathway. Coexpression of COI and DELLA in Nicotiana benthamiana showed that the COI proteins trigger proteasome-dependent DELLA degradation. Many genes that are downregulated in the coi1-4x mutant are GA-inducible. In addition, most of the proteins encoded by the downregulated genes are predicted to be bundle sheath- or mesophyll-enriched, including those encoding C4-specific photosynthetic enzymes. Heterologous expression of maize Coi genes in N. benthamiana showed that COI2a is nucleus-localized and interacts with maize jasmonate zinc-finger inflorescence meristem domain (JAZ) proteins, the canonical COI repressor partners. However, maize COI1a and COI1c showed only partial nuclear localization and reduced binding efficiency to the tested JAZ proteins. Together, these results show the divergent functions of the 6 COI proteins in regulating maize growth and defense pathways.

 

The cell plasma membrane is a two-dimensional, fluid mosaic material composed of lipids and proteins that create a semipermeable barrier defining the cell from its environment. Compared with soluble proteins, the methodologies for the structural and functional characterization of membrane proteins are challenging. An emerging tool for studies of membrane proteins in mammalian systems is a “plasma membrane on a chip,” also known as a supported lipid bilayer. Here, we create the “plant-membrane-on-a-chip,″ a supported bilayer made from the plant plasma membranes of Arabidopsis thaliana, Nicotiana benthamiana, or Zea mays. Membrane vesicles from protoplasts containing transgenic membrane proteins and their native lipids were incorporated into supported membranes in a defined orientation. Membrane vesicles fuse and orient systematically, where the cytoplasmic side of the membrane proteins faces the chip surface and constituents maintain mobility within the membrane plane. We use plant-membrane-on-a-chip to perform fluorescent imaging to examine protein–protein interactions and determine the protein subunit stoichiometry of FLOTILLINs. We report here that like the mammalian FLOTILLINs, FLOTILLINs expressed in Arabidopsis form a tetrameric complex in the plasma membrane. This plant-membrane-on-a-chip approach opens avenues to studies of membrane properties of plants, transport phenomena, biophysical processes, and protein–protein and protein–lipid interactions in a convenient, cell-free platform. 

An enormous diversity of specialized metabolites is produced in the plant kingdom, with each individual plant synthesizing thousands of these compounds. Previous research showed that benzoxazinoids, the most abundant class of specialized metabolites in maize, also function as signaling molecules by regulating the production callose as a defense response.

We searched for additional benzoxazinoid‐regulated specialized metabolites, characterized them, examined whether they too function in herbivore protection, and determined howSpodoptera frugiperda(fall armyworm), a prominent maize pest, copes with these metabolites.

We identified catechol acetylglucose (CAG) as a benzoxazinoid‐regulated metabolite that is produced from salicylic acid via catechol and catechol glucoside. Genome‐wide association studies of CAG abundance identified a gene encoding a predicted acetyltransferase. Knockout of this gene resulted in maize plants that lack CAG and over‐accumulate catechol glucoside. Upon tissue disruption, maize plants accumulate catechol, which inhibitsS. frugiperdagrowth. Analysis of caterpillar frass showed thatS. frugiperdadetoxifies catechol by glycosylation, and the efficiency of catechol glycosylation was correlated withS. frugiperdagrowth on a catechol‐containing diet.

Thus, the success ofS. frugiperdaas an agricultural pest may depend partly on its ability to detoxify catechol, which is produced as a defensive metabolite by maize.

 

Chemical defense systems involving tryptophan-derived secondary metabolites (TDSMs) and salicylic acid (SA) are induced by general nonself signals and pathogen signals, respectively, in Arabidopsis thaliana. Whether and how these chemical defense systems are connected and balanced is largely unknown. In this study, we identified the AVRRPT2-INDUCED GENE2A (AIG2A) and AIG2B genes as gatekeepers that prevent activation of SA defense systems by TDSMs. These genes also were identified as important contributors to natural variation in disease resistance among A. thaliana natural accessions. The loss of AIG2A and AIG2B function leads to upregulation of both SA and TDSM defense systems. Suppressor screens and genetic analysis revealed that a functional TDSM system is required for the upregulation of the SA pathway in the absence of AIG2A and AIG2B, but not vice versa. Furthermore, the AIG2A and AIG2B genes are co-induced with TDSM biosynthesis genes by general pathogen elicitors and nonself signals, thereby functioning as a feedback control of the TDSM defense system, as well as limiting activation of the SA defense system by TDSMs. Thus, this study uncovers an AIG2A- and AIG2B-mediated mechanism that fine-tunes and balances SA and TDSM chemical defense systems in response to nonpathogenic and pathogenic microbes.

 

The chemical arms race between plants and insects is foundational to the generation and maintenance of biological diversity. We asked how the evolution of a novel defensive compound in an already well‐defended plant lineage impacts interactions with diverse herbivores.Erysimum cheiranthoides(Brassicaceae), which produces both ancestral glucosinolates and novel cardiac glycosides, served as a model.

We analyzed gene expression to identify cardiac glycoside biosynthetic enzymes inE. cheiranthoidesand characterized these enzymes via heterologous expression and CRISPR/Cas9 knockout. UsingE. cheiranthoidescardiac glycoside‐deficient lines, we conducted insect experiments in both the laboratory and field.

EcCYP87A126 initiates cardiac glycoside biosynthesis via sterol side‐chain cleavage, andEcCYP716A418 has a role in cardiac glycoside hydroxylation. In EcCYP87A126knockout lines, cardiac glycoside production was eliminated. Laboratory experiments with these lines revealed that cardiac glycosides were highly effective defenses against two species of glucosinolate‐tolerant specialist herbivores, but did not protect against all crucifer‐feeding specialist herbivores in the field. Cardiac glycosides had lesser to no effect on two broad generalist herbivores.

These results begin elucidation of theE. cheiranthoidescardiac glycoside biosynthetic pathway and demonstratein vivothat cardiac glycoside production allowsErysimumto escape from some, but not all, specialist herbivores.