Biology subjects

Brutnell, T. P.

Publications and source records attributed to Brutnell, T. P..

7 recordsLinked to original sources

bioRxiv2023

Maize COI1 quadruple-knockout mutants exhibit elevated DELLA protein accumulation, stunted growth, and reduced photosynthetic efficiency

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.

plant biology↗

bioRxiv2022

A SHR-IDD-PIN regulatory network mediates minor vein differentiation in rice

C3 and C4 grasses directly and indirectly provide the vast majority of calories to the human diet, yet our understanding of the molecular mechanisms driving photosynthetic productivity in grasses is largely unexplored. Here we define a genetic circuit comprised of SHR, IDD and PIN family members that specify vascular identify and ground cell proliferation in leaves of both C3 and C4 grasses. Ectopic expression and loss-of-function mutant studies of SHORT ROOT (SHR) paralogs in C3 Oryza sativa (rice) and C4 Setaria viridis (green millet) revealed a role in both minor vein formation and ground cell differentiation. Genetic and in vitro studies further suggest that SHR regulates this process through its interaction with Indeterminate Domain (IDD) IDD 12 and 13. We further show a direct interaction of these IDD proteins with a putative regulatory element within the auxin transporter PIN5c gene. Collectively, these studies indicated that a SHR-IDD regulatory circuit mediates auxin flow through the negative regulation of PIN protein expression to modulate minor vein patterning in the grasses.

plant biology↗

bioRxiv2020

The maize Aliphatic Suberin Feruloyl Transferase genes affect leaf water movement but are dispensable for bundle sheath CO2 concentration

C4 grasses often outperform C3 species under hot, arid conditions due to superior water and nitrogen use efficiencies and lower rates of photorespiration. A method of concentrating CO2 around the site of carbon fixation in the bundle sheath (BS) is required to realize these gains. In NADP-malic enzyme (NADP-ME)-type C4 grasses such as maize, suberin deposition in the BS cell wall is hypothesized to act as a diffusion barrier to CO2 escape and O2 entry from surrounding mesophyll cells. Suberin is a heteropolyester comprised of acyl-lipid-derived aliphatic and phenylpropanoid-derived aromatic components. To disrupt BS suberization, we mutated two paralogously duplicated, unlinked maize orthologues of Arabidopsis thaliana ALIPHATIC SUBERIN FERULOYL TRANSFERASE, ZmAsft1 and ZmAsft2, using closely linked Dissociation transposons. Loss-of-function double mutants revealed a 97% reduction in suberin-specific omega-hydroxy fatty acids without a stoichiometric decrease in ferulic acid. However, BS suberin lamellae were deficient in electron opaque material, and cohesion between the suberin lamellae and polysaccharide cell walls was attenuated in double mutants. There were no other morphological phenotypes under ambient conditions. Furthermore, there was no significant effect on net CO2 assimilation at any intercellular CO2 concentration, and no effect on 13C isotope discrimination relative to wild type. Thus, ZmAsft expression is not required to establish a functional CO2 concentrating mechanism in in maize. Double mutant leaves exhibit elevated cell wall elasticity, transpirational, and stomatal conductance relative to WT. Thus, the ZmAsft genes are dispensable for gas exchange barrier function but may be involved in regulation of leaf water movement. One-sentence SummaryDouble mutants of two paralogously duplicated maize Aliphatic Suberin Feruloyl Transferase (ZmAsft) genes exhibit reduced aliphatic suberin content, cell wall cohesion defects, and elevated leaf transpiration, but no changes in CO2 assimilation relative to wild type.

plant biology↗

bioRxiv2017

Characterization and Validation of a Novel Group of Type V, Class 2 Nucleases for in vivo Genome Editing

CRISPR-based genome editing is an enabling technology with potential to dramatically transform multiple industries. Identification of additional editing tools will be imperative for broad adoption and application of this technology. A novel Type V, Class 2 CRISPR nuclease system was identified from Microgenomates and Smithella bacterial species (CRISPR from Microgenomates and Smithella, Cms1). This system was shown to efficiently generate indel mutations in the major crop plant rice (Oryza sativa). Cms1 are distinct from other Type V nucleases, are smaller than most other CRISPR nucleases, do not require a tracrRNA, and have an AT-rich protospacer-adjacent motif site requirement. A total of four novel Cms1 nucleases across multiple bacterial species were shown to be functional in a eukaryotic system. This is a major expansion of the Type V CRISPR effector protein toolbox and increases the diversity of options available to researchers.

molecular biology↗

bioRxiv2017

Precise insertion and guided editing of higher plant genomes using Cpf1 CRISPR nucleases

Introduction Paragraph Introduction Paragraph Main Body Online Methods References Precise genome editing of plants has the potential to reshape global agriculture through the targeted engineering of endogenous pathways or the introduction of new traits. To develop a CRISPR nuclease-based platform that would enable higher efficiencies of precise gene insertion or replacement, we screened the Cpf1 nucleases from Francisella novicida and Lachnospiraceae bacterium ND2006 for their capacity to induce targeted gene insertions via homology directed repair. Both nucleases, in the presence of guide RNA and repairing DNA template, were demonstrated to generate precise gene insertions as well as indel mutations at the target site in the rice genome. The frequency of targeted insertions for these Cpf1 nucleases, up to 8%, is higher than most other genome editi ...

plant biology↗

bioRxiv2016

Identification of the maize Mediator CDK8 module, and Dissociation insertional mutagenesis of ZmMed12a

Mediator is a conserved transcriptional co-activator that links transcription factors bound at enhancer elements to RNA Polymerase II. Mediator-RNA Polymerase II interactions can be sterically hindered by the Cyclin Dependent Kinase 8 (CDK8) module, a submodule of Mediator that acts to repress transcription in response to discrete cellular and environmental cues. The CDK8 module is conserved in all eukaryotes and consists of 4 proteins: CDK8, CYCLIN C (CYCC), MED12, and MED13. In this study, we have characterized the CDK8 module of Mediator in maize. The maize genome contains single copy genes for Cdk8, CycC, and Med13, and two genes for Med12. Analysis of expression data for the CDK8 module demonstrated that all five genes are broadly expressed in maize tissues, with ZmMed12a, ZmMed12b, and ZmMed13 exhibiting similar expression patterns. We performed a Dissociation (Ds) insertional mutagenesis, recovering two independent insertions in the ZmMed12a gene. One of these Ds insertions results in a truncation of the ZmMed12a transcript. Our molecular characterization of the maize CDK8 module, as well as transposon tagging of ZmMed12a, establish the basis for molecular and functional studies of these important transcriptional regulators in Zea mays.

plant biology↗

bioRxiv2016

Time dependent genetic analysis links field and controlled environment phenotypes in the model C4 grass Setaria

Vertical growth of plants is a dynamic process that is influenced by genetic and environmental factors and has a pronounced effect on overall plant architecture and biomass composition. We have performed twelve controlled growth trials of an interspecific Setaria italica x Setaria viridis recombinant inbred line population to assess how the genetic architecture of plant height is influenced by developmental queues, water availability and planting density. The nondestructive nature of plant height measurements has enabled us to monitor vertical growth throughout the plant life cycle in both field and controlled environments. We find that plant height is reduced under water limitation and high density planting and affected by growth environment (field vs. growth chamber). The results support a model where plant height is a heritable, polygenic trait and that the major genetic loci that influence plant height function independent of growth environment. The identity and contribution of loci that influence height changes dynamically throughout development and the reduction of growth observed in water limited environments is a consequence of delayed progression through the genetic program which establishes plant height in Setaria. In this population, alleles inherited from the weedy S. viridis parent act to increase plant height early, whereas a larger number of small effect alleles inherited from the domesticated S. italica parent collectively act to increase plant height later in development.

plant biology↗