Pollen grains undergo dramatic changes in cellular water potential as they deliver the male germ line to female gametes, and it has been proposed that mechanosensitive ion channels may sense the resulting mechanical stress. Here, we identify and characterize MscS-like 8 (MSL8), a pollen-specific, membrane tension–gated ion channel required for pollen to survive the hypoosmotic shock of rehydration and for full male fertility. MSL8 negatively regulates pollen germination but is required for cellular integrity during germination and tube growth. MSL8 thus senses and responds to changes in membrane tension associated with pollen hydration and germination. These data further suggest that homologs of bacterial MscS have been repurposed in eukaryotes to function as mechanosensors in multiple developmental and environmental contexts.

InArabidopsis thaliana, the acyl acid amido synthetase Gretchen Hagen 3.5 (AtGH3.5) conjugates both indole-3-acetic acid (IAA) and salicylic acid (SA) to modulate auxin and pathogen response pathways. To understand the molecular basis for the activity of AtGH3.5, we determined the X-ray crystal structure of the enzyme in complex with IAA and AMP. Biochemical analysis demonstrates that the substrate preference of AtGH3.5 is wider than originally described and includes the natural auxin phenylacetic acid (PAA) and the potential SA precursor benzoic acid (BA). Residues that determine IAA versus BA substrate preference were identified. The dual functionality of AtGH3.5 is unique to this enzyme although multiple IAA-conjugating GH3 proteins share nearly identical acyl acid binding sites.In plantaanalysis of IAA, PAA, SA, and BA and their respective aspartyl conjugates were determined in wild-type and overexpressing lines ofA.thaliana. This study suggests that AtGH3.5 conjugates auxins (i.e., IAA and PAA) and benzoates (i.e., SA and BA) to mediate crosstalk between different metabolic pathways, broadening the potential roles for GH3 acyl acid amido synthetases in plants.

From domestication and breeding to the genetic engineering of crops, plants provide food, fuel, fibers, and feedstocks for our civilization. New research and discoveries aim to reduce the inputs needed to grow crops and to develop plants for environmental and sustainability applications. Faced with population growth and changing climate, the next wave of innovation in plant biology integrates technologies and approaches that span from molecular to ecosystem scales. Recent efforts to engineer plants for better nitrogen and phosphorus use, enhanced carbon fixation, and environmental remediation and to understand plant-microbiome interactions showcase exciting future directions for translational plant biology. These advances promise new strategies for the reduction of inputs to limit environmental impacts and improve agricultural sustainability.

Various phytohormones control plant growth and development and mediate biotic and abiotic stress responses. Gretchen Hagen 3 (GH3) acyl acid amido synthetases are plant enzymes that typically conjugate amino acids to indole-3-acetic acid (IAA) or jasmonic acid (JA) to inactivate or activate these phytohormones, respectively; however, the physiological and biological roles of many of these enzymes remain unclear. Using a biochemical approach, we found that the GH3.15 (AtGH3.15) preferentially uses indole-3-butyric acid (IBA) and glutamine as substrates. The X-ray crystal structure of the AtGH3.15·AMP complex, modeling of IBA in the active site, and biochemical analysis of site-directed mutants provide insight on active site features that lead to AtGH3.15's preference for IBA. Assay-based analysis of AtGH3.15-overexpressing lines indicated that their root elongation and lateral root density were resistant to IBA treatment but not to treatment with either IAA or JA. These findings suggest that AtGH3.15 may play a role in auxin homeostasis by modulating the levels of IBA for peroxisomal conversion to IAA. Analysis of promoter-driven yellow fluorescent protein reporter lines revealed that is expressed at significant levels in seedlings, roots, and parts of the siliques. We conclude that AtGH3.15 is unique in the GH3 protein family for its role in modifying IBA in auxin homeostasis and that it is the first GH3 protein shown to primarily modify a plant growth regulator other than IAA and JA.

Herbicide-resistance traits are the most widely used agriculture biotechnology products. Yet, to maintain their effectiveness and to mitigate selection of herbicide-resistant weeds, the discovery of new resistance traits that use different chemical modes of action is essential. In plants, the Gretchen Hagen 3 (GH3) acyl acid amido synthetases catalyze the conjugation of amino acids to jasmonate and auxin phytohormones. This reaction chemistry has not been explored as a possible approach for herbicide modification and inactivation. Here, we examined a set of Arabidopsis GH3 proteins that use the auxins indole-3-acetic acid (IAA) and indole-3-butyric acid (IBA) as substrates along with the corresponding auxinic phenoxyalkanoic acid herbicides 2,4-dichlorophenoxylacetic acid (2,4-D) and 4-(2,4-dichlorophenoxy)butyric acid (2,4-DB). The IBA-specific AtGH3.15 protein displayed high catalytic activity with 2,4-DB, which was comparable to its activity with IBA. Screening of phenoxyalkanoic and phenylalkyl acids indicated that side-chain length of alkanoic and alkyl acids is a key feature of AtGH3.15's substrate preference. The X-ray crystal structure of the AtGH3.15·2,4-DB complex revealed how the herbicide binds in the active site. In root elongation assays, Arabidopsis AtGH3.15-knockout and -overexpression lines grown in the presence of 2,4-DB exhibited hypersensitivity and tolerance, respectively, indicating that the AtGH3.15-catalyzed modification inactivates 2,4-DB. These findings suggest a potential use for AtGH3.15, and perhaps other GH3 proteins, as herbicide-modifying enzymes that employ a mode of action different from those of currently available herbicide-resistance traits.

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