Microbial HemG-type protoporphyrinogen IX oxidase enzymes for biotechnology applications in plant herbicide tolerance traits.

BACKGROUND: Protoporphyrinogen IX oxidase (PPO)-inhibiting herbicides act by inhibiting a key enzyme in the heme and chlorophyll biosynthetic pathways in plants. This enzyme, the PPO enzyme, is conserved across plant species. However, some microbes are known to utilize a unique family of PPO enzymes, the HemG family. This enzyme family carries out the same enzymatic step as the plant PPO enzymes, but does not share sequence homology with the plant PPO enzymes. RESULTS: Bioinformatic analysis was used to identify putative HemG PPO enzyme variants from microbial sources. A subset of these variants was cloned and characterized. HemG PPO variants were characterized for functionality and tolerance to PPO-inhibiting herbicides. HemG PPO variants that exhibited insensitivity to PPO-inhibiting herbicides were identified for further characterization. Expression of selected variants in maize, soybean, cotton and canola resulted in plants that displayed tolerance to applications of PPO-inhibiting herbicides. CONCLUSION: Selected microbial-sourced HemG PPO enzyme variants present an opportunity for building new herbicide tolerance biotechnology traits. These traits provide tolerance to PPO-inhibiting herbicides and, therefore, could provide additional tools for farmers to employ in their weed management systems. © 2019 Society of Chemical Industry.

Directions for research and training in plant omics: Big Questions and Big Data.

A key remit of the NSF-funded "Arabidopsis Research and Training for the 21st Century" (ART-21) Research Coordination Network has been to convene a series of workshops with community members to explore issues concerning research and training in plant biology, including the role that research using Arabidopsis thaliana can play in addressing those issues. A first workshop focused on training needs for bioinformatic and computational approaches in plant biology was held in 2016, and recommendations from that workshop have been published (Friesner et al., Plant Physiology, 175, 2017, 1499). In this white paper, we provide a summary of the discussions and insights arising from the second ART-21 workshop. The second workshop focused on experimental aspects of omics data acquisition and analysis and involved a broad spectrum of participants from academics and industry, ranging from graduate students through post-doctorates, early career and established investigators. Our hope is that this article will inspire beginning and established scientists, corporations, and funding agencies to pursue directions in research and training identified by this workshop, capitalizing on the reference species Arabidopsis thaliana and other valuable plant systems.

Development of enzymes for robust aryloxyphenoxypropionate and synthetic auxin herbicide tolerance traits in maize and soybean crops.

BACKGROUND: Effective management of weedy species in agricultural fields is essential for maintaining favorable growing conditions and crop yields. The introduction of genetically modified crops containing herbicide tolerance traits has been a successful additional tool available to farmers to better control weeds. However, weed resistance challenges present a need for additional herbicide tolerance trait options. RESULTS: To help meet this challenge, a new trait that provides tolerance to an aryloxyphenoxypropionate (FOP) herbicide and members of the synthetic auxin herbicide family, such as 2,4-dichlorophenoxyacetic acid (2,4-D), was developed. Development of this herbicide tolerance trait employed an enzyme engineered with robust and specific enzymatic activity for these two herbicide families. This engineering effort utilized a microbial-sourced dioxygenase scaffold to generate variants with improved enzymatic parameters. Additional optimization to enhance in-plant stability of the enzyme enabled an efficacious trait that can withstand the higher temperature conditions often found in field environments. CONCLUSION: Optimized herbicide tolerance enzyme variants with enhanced enzymatic and temperature stability parameters enabled robust herbicide tolerance for two herbicide families in transgenic maize and soybeans. This herbicide tolerance trait for FOP and synthetic auxin herbicides such as 2,4-D could be useful in weed management systems, providing additional tools for farmers to control weeds. © 2019 Society of Chemical Industry.

Endogenous tassel-specific small RNAs-mediated RNA interference enables a novel glyphosate-inducible male sterility system for commercial production of hybrid seed in Zea mays L.

Hybrid crops produce higher yields than their inbred parents due to heterosis. For high purity of hybrid seeds, it is critical to eliminate self-pollination. Manual or mechanical removal of male parts (such as detasseling in maize) is labor-intensive, fuel and time-consuming, and can cause physical damage to female plants, resulting in significant seed yield reductions. Many male-sterility systems either require a maintainer for male-sterile line propagation or are often affected by environmental factors. Roundup® Hybridization System (RHS) utilizes glyphosate to induce male sterility, which effectively eliminates the need for maintainer lines and removal of male parts for commercial hybrid seed production. The first-generation RHS (RHS1) is based on low expression of a glyphosate-insensitive 5-enolpyruvylshikimate-3-phosphate synthase (CP4 EPSPS) in pollen. This report presents the second-generation RHS (RHS2) technology built on RNA interference (RNAi) combined with CP4 EPSPS. It utilizes maize endogenous male tissue-specific small interfering RNAs (mts-siRNAs) to trigger cleavage of the CP4 EPSPS mRNA specifically in tassels, resulting in glyphosate-sensitive male cells due to lack of the CP4 EPSPS protein. Male sterility is then induced by glyphosate application at the stages critical for pollen development, and the male-sterile plants are used as the female parent to produce hybrid seed. The endogenous mts-siRNAs are conserved across maize germplasms, and the inducible male sterility was replicated in representative germplasms through introgression of a CP4 EPSPS transgene containing the mts-siRNA target sequence. This technology combines the relative simplicity and convenience of a systemic herbicide spray methodology with targeted protein expression to create an inducible male sterility system for industrial production of row crop hybrid seeds in an environmentally-independent manner.