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. Hormonal regulation of maize stalk height is influenced by 4 members of a gene family that has similarity to genes previously associated with insect defense induction in numerous other plant species.

Novel alleles can now be directly written into related genomes by sequence identity, thus removing the resource-intensive process of trait introduction by genetic introgression and eliminate the loss of genetic diversity that typically results from introgression bottlenecks.

Gene functional descriptions offer a crucial line of evidence for candidate genes underlying trait variation. Conversely, plant responses to environmental cues represent important resources to decipher gene function and subsequently provide molecular targets for plant improvement through gene editing. However, biological roles of large proportions of genes across the plant phylogeny are poorly annotated. Here we describe the Joint Genome Institute (JGI) Plant Gene Atlas, an update able data resource consisting of transcript abundance assays spanning 18 diverse species. To integrate across these diverse genotypes, we anal yzed e xpression pr ofiles, b uilt gene c lusters that exhibited tissue / condition specific expression, and tested for transcriptional response to environmental queues. We discovered extensive phylogenetically constrained and condition-specific expression profiles f or genes without an y previously documented functional annotation. Such conserved expression patterns and tightly co-expressed gene clusters let us assign expression derived additional biological information to 64 495 genes with otherwise unknown functions. The ever-expanding Gene Atlas resource is available at JGI Plant Gene Atlas ( https://plantgeneatlas.jgi.doe.gov ) and Phytozome ( https://phytozome .jgi.doe .gov/), providing bulk access to data and user-specified queries of gene sets. Combined, these web interfaces let users access differentiall y e xpressed genes, track orthologs across the Gene Atlas plants, graphically represent co-expressed genes, and visualize gene ontology and pathway enrichments.

The F-box protein Coronatine Insensitive (COI) is a receptor for the jasmonic acid signaling pathway in plants. To investigate the functions of the six maize COI proteins (COI1a, COI1b, COI1c, COI1d, COI2a, and COI2b), we made single, double, and quadruple loss-of-function mutants. Double-mutant coi2a coi2b pollen was inviable, and no homozygous mutant plants were obtained. The coi1 quadruple mutant ( coi1-4x ) exhibited shortened internode lengths, decreased photosynthesis, leaf discoloration, microelement deficiencies, and accumulation of DWARF9, a DELLA-family protein that represses the gibberellic acid signaling pathway. Co-expression of maize COI and DWARF9 genes in Nicotiana benthamiana showed that the COI proteins lead to proteasome-dependent DELLA degradation. Many genes expressed at lower levels in the coi1-4x mutant are normally induced by gibberellic acid. The majority of these genes are predicted to be bundle sheath or mesophyll-enriched including those encoding C4-specific photosynthetic enzymes. Ectopic expression of maize COI genes in N. benthamiana showed that COI2a is fully localized in the nucleus and interacts with maize JAZ proteins, the canonical COI repressor partners. However, maize COI1a and COI1c proteins showed only partial nuclear localization and failed to bind to most of the JAZ proteins tested. These results show divergent functions of the six COI proteins in the regulation of maize growth and defense pathways.### Competing Interest StatementThe authors have declared no competing interest.

ABSTRACTGene functional descriptions, which are typically derived from sequence similarity to experimentally validated genes in a handful of model species, offer a crucial line of evidence when searching for candidate genes that underlie trait variation. Plant responses to environmental cues, including gene expression regulatory variation, represent important resources for understanding gene function and crucial targets for plant improvement through gene editing and other biotechnologies. However, even after years of effort and numerous large-scale functional characterization studies, biological roles of large proportions of protein coding genes across the plant phylogeny are poorly annotated. Here we describe the Joint Genome Institute (JGI) Plant Gene Atlas, a public and updateable data resource consisting of transcript abundance assays from 2,090 samples derived from 604 tissues or conditions across 18 diverse species. We integrated across these diverse conditions and genotypes by analyzing expression profiles, building gene clusters that exhibited tissue/condition specific expression, and testing for transcriptional modulation in response to environmental queues. For example, we discovered extensive phylogenetically constrained and condition-specific expression profiles across many gene families and genes without any functional annotation. Such conserved expression patterns and other tightly co-expressed gene clusters let us assign expression derived functional descriptions to 64,620 genes with otherwise unknown functions. The ever-expanding Gene Atlas resource is available at JGI Plant Gene Atlas (https://plantgeneatlas.jgi.doe.gov) and Phytozome (https://phytozome-next.jgi.doe.gov), providing bulk access to data and user-specified queries of gene sets. Combined, these web interfaces let users access differentially expressed genes, track orthologs across the Gene Atlas plants, graphically represent co-expressed genes, and visualize gene ontology and pathway enrichments.

Setaria viridis is an emerging model for cereal and bioenergy grasses because of its short stature, rapid life cycle and expanding genetic and genomic toolkits. Its close phylogenetic relationship with economically important crops such as maize and sorghum positions Setaria as an ideal model system for accelerating discovery and characterization of crop genes that control agronomically important traits. The Second International Setaria Genetics Conference was held on March 6-8, 2017 at the Donald Danforth Plant Science Center, St. Louis, MO, United States to discuss recent technological breakthroughs and research directions in Setaria (presentation abstracts can be downloaded at https://www.brutnelllab.org/setaria). Here, we highlight topics presented in the conference including inflorescence architecture, C-4 photosynthesis and abiotic stress. Genetic and genomic toolsets including germplasm, mutant populations, transformation and gene editing technologies are also discussed. Since the last meeting in 2014, the Setaria community has matured greatly in the quality of research being conducted. Outreach and increased communication with maize and other plant communities will allow broader adoption of Setaria as a model system to translate fundamental discovery research to crop improvement.

Transposon tagging populations are important resources for characterizing gene function in model plant systems. In Setaria viridis, a transposon tagging population utilizing the miniature Ping (mPing) element was generated by introducing a construct containing mPing and the transposase Ping into accession A10.1. Transformants were screened for the presence of the transgene and mPing insertions were identified using nested inverse-PCR coupled with Illumina sequencing. Here we describe step-by-step protocols for generating the Setaria viridis mPing population, introduce methods for genotyping mPing insertion alleles, and discuss future implementations of the system.

Millet is a common name for a group of polyphyletic, small-seeded cereal crops that include pearl, finger and foxtail millet. Millet species are an important source of calories for many societies, often in developing countries. Compared to major cereal crops such as rice and maize, millets are generally better adapted to dry and hot environments. Despite their food security value, the genetic architecture of agronomically important traits in millets, including both morphological traits and climate resilience remains poorly studied. These complex traits have been challenging to dissect in large part because of the lack of sufficient genetic tools and resources. In this article, we review the phylogenetic relationship among various millet species and discuss the value of a genetic model system for millet research. We propose that a broader adoption of green foxtail (Setaria viridis) as a model system for millets could greatly accelerate the pace of gene discovery in the millets, and summarize available and emerging resources in S. viridis and its domesticated relative S. italica. These resources have value in forward genetics, reverse genetics and high throughput phenotyping. We describe methods and strategies to best utilize these resources to facilitate the genetic dissection of complex traits. We envision that coupling cutting-edge technologies and the use of S. viridis for gene discovery will accelerate genetic research in millets in general. This will enable strategies and provide opportunities to increase productivity, especially in the semi-arid tropics of Asia and Africa where millets are staple food crops.

JASMONATE ZIM-DOMAIN (JAZ) proteins are primary transcriptional repressors in the jasmonate (JA) signaling pathway that regulate a broad range of JA-dependent responses. A mechanistic mode of action is well established, but what are the biological roles of individual JAZs, and how do they contribute to the specification of JA signaling outputs? Two recent articles in Journal of Experimental Botany reveal roles of JAZ7 in regulating dark-induced senescence and susceptibility to fungal pathogens. Jasmonates (JAs) regulate a broad range of processes, from growth and development to biotic and abiotic stress responses. JASMONATE ZIM-DOMAIN (JAZ) proteins are key regulators in controlling JA signaling outputs. Since the discovery of JAZs in 2007, over 100 research articles have been published on the characterization of JAZ functions, genomic or transcriptomic analyses of JAZs, or the identification of down-stream transcription factors in the JA signaling pathway through yeast two-hybrid screens using JAZs as bait (reviewed in Pauwels and Goossens, 2011; Shyu and Brutnell, 2015). Biochemical and molecular functions of JAZ proteins have been extensively studied, but surprisingly little is known about the biological role of JAZs and how they contribute to specific JA signaling outputs.

JAsmonate ZIM-domain (JAZ) proteins repress the activity of transcription factors that execute responses to the plant hormone jasmonoyl-L-isoleucine (JA-Ile). The ZIM protein domain recruits the co-repressors NINJA and TOPLESS to JAZ-bound transcription factors, and contains a highly conserved TIF[F/Y]XG motif that defines the larger family of TIFY proteins to which JAZs belong. Here, we report that diverse plant species contain genes encoding putative non-TIFY JAZ proteins, including a previously unrecognized JAZ repressor in Arabidopsis (JAZ13, encoded by At3g22275). JAZ13 is most closely related to JAZ8 and includes divergent EAR, TIFY/ZIM, and Jas motifs. Unlike JAZ8, however, JAZ13 contains a Ser-rich C-terminal tail that is a site for phosphorylation. Overexpression of JAZ13 resulted in reduced sensitivity to JA, attenuation of wound-induced expression of JA-response genes, and decreased resistance to insect herbivory. JAZ13 interacts with the bHLH transcription factor MYC2 and the co-repressor TOPLESS but, consistent with the absence of a TIFY motif, neither NINJA nor other JAZs. Analysis of single and higher-order T-DNA insertion jaz null mutants provided further evidence that JAZ13 is a repressor JA signaling. Our results demonstrate that proteins outside the TIFY family are functional JAZ repressors and further suggest that this expansion of the JAZ family allows fine-tuning of JA-mediated transcriptional responses.

Phenotyping has become the rate-limiting step in using large-scale genomic data to understand and improve agricultural crops. Here, the Bellwether Phenotyping Platform for controlled-environment plant growth and automated multimodal phenotyping is described. The system has capacity for 1140 plants, which pass daily through stations to record fluorescence, near-infrared, and visible images. Plant Computer Vision (PlantCV) was developed as open-source, hardware platform-independent software for quantitative image analysis. In a 4-week experiment, wild Setaria viridis and domesticated Setaria italica had fundamentally different temporal responses to water availability. While both lines produced similar levels of biomass under limited water conditions, Setaria viridis maintained the same water-use efficiency under water replete conditions, while Setaria italica shifted to less efficient growth. Overall, the Bellwether Phenotyping Platform and PlantCV software detected significant effects of genotype and environment on height, biomass, water-use efficiency, color, plant architecture, and tissue water status traits. All ∼79 000 images acquired during the course of the experiment are publicly available.

Growth-defence balance is the selective partitioning of resources between biomass accumulation and defence responses. Although it is generally postulated that reallocation of limited carbon pools drives the antagonism between growth and defence, little is known about the mechanisms underlying this regulation. Jasmonates (JAs) are a group of oxylipins that are required for a broad range of responses from defence against insects to reproductive growth. Application of JAs to seedlings also leads to inhibited growth and repression of photosynthesis, suggesting a role for JAs in regulating growth-defence balance. The majority of JA research uses dicot models such as Arabidopsis and tomato, while understanding of JA biology in monocot grasses, which comprise most bioenergy feedstocks, food for human consumption, and animal feed, is limited. Interestingly, JA mutants of grasses exhibit unique phenotypes compared with well-studied dicot models. Gene expression analyses in bioenergy grasses also suggest roles for JA in rhizome development, which has not been demonstrated in Arabidopsis. In this review we summarize current knowledge of JA biology in panicoid grasses-the group that consists of the world's emerging bioenergy grasses such as switchgrass, sugarcane, Miscanthus, and sorghum. We discuss outstanding questions regarding the role of JAs in panicoid grasses, and highlight the importance of utilizing emerging grass models for molecular studies to provide a basis for engineering bioenergy grasses that can maximize biomass accumulation while efficiently defending against stress.

The shoot epidermis of land plants serves as a crucial interface between plants and the atmosphere: pavement cells protect plants from desiccation and other environmental stresses, while stomata facilitate gas exchange and transpiration. Advances have been made in our understanding of stomatal patterning and differentiation, and a set of ‘master regulatory’ transcription factors of stomatal development have been identified. However, they are limited to specifying stomatal differentiation within the epidermis. Here, we report the identification of an Arabidopsis homeodomain-leucine zipper IV (HD-ZIP IV) protein, HOMEODOMAIN GLABROUS2 (HDG2), as a key epidermal component promoting stomatal differentiation. HDG2 is highly enriched in meristemoids, which are transient-amplifying populations of stomatal-cell lineages. Ectopic expression of HDG2 confers differentiation of stomata in internal mesophyll tissues and occasional multiple epidermal layers. Conversely, a loss-of-function hdg2 mutation delays stomatal differentiation and, rarely but consistently, results in aberrant stomata. A closely related HD-ZIP IV gene, Arabidopsis thaliana MERISTEM LAYER1 (AtML1), shares overlapping function with HDG2: AtML1 overexpression also triggers ectopic stomatal differentiation in the mesophyll layer and atml1 mutation enhances the stomatal differentiation defects of hdg2. Consistently, HDG2 and AtML1 bind the same DNA elements, and activate transcription in yeast. Furthermore, HDG2 transactivates expression of genes that regulate stomatal development in planta. Our study highlights the similarities and uniqueness of these two HD-ZIP IV genes in the specification of protodermal identity and stomatal differentiation beyond predetermined tissue layers.

The plant hormone jasmonate (JA) activates gene expression by promoting ubiquitin-dependent degradation of jasmonate ZIM domain (JAZ) transcriptional repressor proteins. A key feature of all JAZ proteins is the highly conserved Jas motif, which mediates both JAZ degradation and JAZ binding to the transcription factor MYC2. Rapid expression of JAZ genes in response to JA is thought to attenuate JA responses, but little is known about the mechanisms by which newly synthesized JAZ proteins exert repression in the presence of the hormone. Here, we show in Arabidopsis (Arabidopsis thaliana) that desensitization to JA is mediated by an alternative splice variant (JAZ10.4) of JAZ10 that lacks the Jas motif. Unbiased protein-protein interaction screens identified three related basic helix-loop-helix transcription factors (MYC2, MYC3, and MYC4) and the corepressor NINJA as JAZ10.4-binding partners. We show that the amino-terminal region of JAZ10.4 contains a cryptic MYC2-binding site that resembles the Jas motif and that the ZIM motif of JAZ10.4 functions as a transferable repressor domain whose activity is associated with the recruitment of NINJA. Functional studies showed that the expression of JAZ10.4 from the native JAZ10 promoter complemented the JA-hypersensitive phenotype of a jaz10 mutant. Moreover, treatment of these complemented lines with JA resulted in the rapid accumulation of JAZ10.4 protein. Our results provide an explanation for how the unique domain architecture of JAZ10.4 links transcription factors to a corepressor complex and suggest how JA-induced transcription and alternative splicing of JAZ10 premessenger RNA creates a regulatory circuit to attenuate JA responses.

The lipid-derived hormone jasmonoyl-L-Ile (JA-Ile) initiates large-scale changes in gene expression by stabilizing the interaction of JASMONATE ZIM domain (JAZ) repressors with the F-box protein CORONATINE INSENSITIVE1 (COI1), which results in JAZ degradation by the ubiquitin-proteasome pathway. Recent structural studies show that the JAZ1 degradation signal (degron) includes a short conserved LPIAR motif that seals JA-Ile in its binding pocket at the COI1-JAZ interface. Here, we show that Arabidopsis thaliana JAZ8 lacks this motif and thus is unable to associate strongly with COI1 in the presence of JA-Ile. As a consequence, JAZ8 is stabilized against jasmonate (JA)-mediated degradation and, when ectopically expressed in Arabidopsis, represses JA-regulated growth and defense responses. These findings indicate that sequence variation in a hypervariable region of the degron affects JAZ stability and JA-regulated physiological responses. We also show that JAZ8-mediated repression depends on an LxLxL-type EAR (for ERF-associated amphiphilic repression) motif at the JAZ8 N terminus that binds the corepressor TOPLESS and represses transcriptional activation. JAZ8-mediated repression does not require the ZIM domain, which, in other JAZ proteins, recruits TOPLESS through the EAR motif-containing adaptor protein NINJA. These findings show that EAR repression domains in a subgroup of JAZ proteins repress gene expression through direct recruitment of corepressors to cognate transcription factors.