Grace Zi Hao Tan

Hyperspectral imaging is an imaging technique that allows for acquisition of high-resolution spectral information beyond that of the visible spectrum. When applied to plants, it effectively enables non-invasive characterization of physiological status and has been widely used in agricultural settings. Marchantia is a model bryophyte species whose flat morphology and visually distinct stress-response phenotypes makes it an ideal candidate for imaging studies. Here, we provide a comprehensive protocol for hyperspectral imaging for Marchantia plants, which encompasses hardware configuration, data acquisition, and computations processing. This protocol features a streamlined data processing pipeline hosted on a web-based development platform that automates 1) the segmentation of plant area into spatially distinct regions for localized analysis of intra-specimen physiological gradients, and 2) classification of plant pixels based on their spectral signatures. All results are exported as structured CSV files for ease of further analysis as desired by the user.

The broad-spectrum herbicide L-phosphinothricin (PPT) irreversibly inhibits glutamine synthetase (GS) activity. Here we present cationic amino acid transporters (CAT) as mediators of PPT accumulation and susceptibility. Comparative analysis of Arabidopsis thaliana, Marchantia polymorpha, rice, and Klebsormidium nitens reveals conserved responses to GS inhibition, with absence of efficient PPT uptake underlying resistance in M. polymorpha. Transcriptomic analysis identifies four candidate transporters in A. thaliana which, when overexpressed in M. polymorpha, confer varying levels of sensitivity corresponding with PPT accumulation. AtCAT1 and AtCAT5 confer the greatest sensitivities and are required for glutamic acid uptake and endogenous nitrogen metabolism in A. thaliana, and PPT susceptibility. Molecular dynamics simulation of AtCAT5 identifies key residues involved in PPT binding, which induce the rotational flexibility of helices H1 and H6 which form an intracellular access tunnel. These findings highlight the natural diversity underlying PPT accumulation and susceptibility, which can guide herbicide resistance management strategies.

Gamma-aminobutyric acid (GABA) is a metabolite involved in plant growth and stress responses, with its synthesis regulated by glutamate decarboxylase (GAD). Plant GAD enzymes have an autoinhibitory α-helix at the C-terminus, which calmodulin (CaM) binding typically relieves. Eliminating this C-terminal motif usually increases GABA levels in crops. In this case study, we generated a CRISPR/Cas9-edited lettuce line with a 14-amino acid deletion in the C-terminal helix of LsGAD2, the isozyme primarily expressed in most tissues. This targeted truncation removes CaM-binding residues while retaining the key Lys cluster (Lys489, Lys490, Lys491) responsible for autoinhibition, resulting in a significant reduction in GABA content without affecting growth. The LsGAD1/2-ΔC line showed a transcriptomic profile resembling stress responses in the wildtype under unstressed conditions. Reduced GABA levels appeared to upregulate genes involved in stress perception, signalling, and defense-related metabolic and hormonal changes, potentially mediated by WRKY-family transcription factors. Likely due to lower GABA levels and altered defense responses, LsGAD1/2-ΔC plants showed increased Agrobacterium-mediated transient expression of β-glucuronidase. Overall, our study suggests that targeted genetic manipulation of the C-terminal helix of GAD enzymes can reduce GABA levels while enhancing transformation efficiency in lettuce, thus presenting a means for engineering for such purposes.

Fluorescent nanosensors operating in planta have shown recent success toward informing basic plant biology and agricultural applications. We developed near-infrared (NIR) fluorescent nanosensors using the Corona Phase Molecular Recognition (CoPhMoRe) technique that distinguish Fe(II) and Fe(III) species with limit of detection as low as 10 nM. An anionic poly(p-phenyleneethynylene) (PPE) polyelectrolyte wrapped single-walled carbon nanotube (SWNT) shows up to 200% turn-on and 85% turn-off responses to Fe(II) and Fe(III), respectively, allowing spatial and temporal analysis of iron uptake in both foliar and root-to-shoot pathways. Our findings reveal species-dependent iron uptake efficiency, mobility, and utilization rates, which we show is primarily affected by the chelation status of iron source and by plant physiological conditions such as iron deficiency and treatment with the stress hormone, abscisic acid (ABA). The broad applicability of this sensor across important plant species highlights the potential of nanotechnology-enabled sensors to enable precise and sustainable nutrient management.

Plant responses to nitrate, phosphate and sucrose form a complex molecular network crucial for terrestrial adaptation. However, the origins, functional diversity and evolvability of this network during plant terrestrialization remain scarcely understood. Here we compare the transcriptomic response to these nutrients in the bryophyte Marchantia polymorpha and the streptophyte alga Klebsormidium nitens. We show that the largely species-specific nutrient response pattern is driven by gene regulatory network (GRN) alterations. Intriguingly, while pathways governing the GRNs exhibit modest conservation, M. polymorpha GRNs exhibit more regulatory connections through the redeployment of ancient transcription factor CSD. In M. polymorpha, functional analyses reveal the involvement of pre-existing cytokinin machineries in downstream targets, orchestrating plastic morpho-physiological responses to nutrient status. Our findings implicate the genetic co-option events facilitating successful land plant establishment.