quantitative-ratiometric-analysis-fret-based-biosensors-arabidopsis

Quantitative Ratiometric Analysis of FRET-Based Biosensors in <em>Arabidopsis thaliana</em> Enables Live Measurement of Analytes in Subcellular Compartments

Fluorescent biosensors provide a non-invasive and versatile approach to monitor dynamic changes in metabolite or ion concentrations within live cells. ...

Texas A&M University

The availability of inorganic phosphate (Pi) limits plant growth and crop productivity on much of the world's arable land. To better understand how plants cope with deficient and variable supplies of this essential nutrient, we used Pi imaging to spatially resolve and quantify cytosolic Pi concentrations and the respective contributions of Pi uptake, metabolic recycling, and vacuolar sequestration to cytosolic Pi homeostasis in Arabidopsis () roots. Microinjection coupled with confocal microscopy was used to calibrate a FRET-based Pi sensor to determine absolute, rather than relative, Pi concentrations in live plants. High-resolution mapping of cytosolic Pi concentrations in different cells, tissues, and developmental zones of the root revealed that cytosolic concentrations varied between developmental zones, with highest levels in the transition zone, whereas concentrations were equivalent in epidermis, cortex, and endodermis within each zone. Pi concentrations in all zones were reduced, at different rates, by Pi starvation, but the developmental pattern of Pi concentration persisted. Pi uptake, metabolic recycling, and vacuolar sequestration were distinguished in each zone by using cyanide to block Pi assimilation in wild-type plants and a vacuolar Pi transport mutant, and then measuring the subsequent change in cytosolic Pi concentration over time. Each of these processes exhibited distinct spatial profiles in the root, but only vacuolar Pi sequestration corresponded with steady-state cytosolic Pi concentrations. These results highlight the complexity of Pi dynamics in live plants and revealed developmental control of root Pi homeostasis, which has potential implications for plant sensing and signaling of Pi.

Spatial Profiles of Phosphate in Roots Indicate Developmental Control of Uptake, Recycling, and Sequestration.

The concentration of inorganic phosphate (Pi) in the chloroplast stroma must be maintained within narrow limits to sustain photosynthesis and to direct the partitioning of fixed carbon. However, it is unknown if these limits or the underlying contributions of different chloroplastic Pi transporters vary throughout the photoperiod or between chloroplasts in different leaf tissues. To address these questions, we applied live Pi imaging to Arabidopsis (Arabidopsis thaliana) wild-type plants and 2 loss-of-function transporter mutants: triose phosphate/phosphate translocator (tpt), phosphate transporter 2;1 (pht2;1), and tpt pht2;1. Our analyses revealed that stromal Pi varies spatially and temporally, and that TPT and PHT2;1 contribute to Pi import with overlapping tissue specificities. Further, the series of progressively diminished steady-state stromal Pi levels in these mutants provided the means to examine the effects of Pi on photosynthetic efficiency without imposing nutritional deprivation. ΦPSII and nonphotochemical quenching (NPQ) correlated with stromal Pi levels. However, the proton efflux activity of the ATP synthase (gH+) and the thylakoid proton motive force (pmf) were unaltered under growth conditions, but were suppressed transiently after a dark to light transition with return to wild-type levels within 2 min. These results argue against a simple substrate-level limitation of ATP synthase by depletion of stromal Pi, favoring more integrated regulatory models, which include rapid acclimation of thylakoid ATP synthase activity to reduced Pi levels.

Genetically manipulated chloroplast stromal phosphate levels alter photosynthetic efficiency.

Engineering plants with reduced lignin content can result in pleiotropic growth defects. In stems of Arabidopsis plants with reduced expression of hydroxycinnamoyl CoA: shikimate hydroxycinnamoyl transferase (HCT), the plastidial glucose 6-phosphate: phosphate co-transporter GPT2 is highly overexpressed, and this coincides with reduced lignin levels and extensive transcriptional and metabolic reprogramming. To explore the potential relationship between GPT2 expression and lignin accumulation, GPT2 transcript levels were evaluated in a suite of Arabidopsis thaliana and Medicago truncatula lignin-defective lines. We also examined lignin levels and composition, and transcriptomic and metabolic profiles in GPT2 loss-of-function, GPT2 overexpression, and wild-type Arabidopsis plants. Loss of GPT2 had no effect on lignin, but its overexpression caused a decrease in stem lignin levels due to reduced accumulation of both guaiacyl and syringyl lignins and their associated monolignol pools. HCT transcript levels were diminished in 35S-GPT2 lines, indicating a potential transcriptional regulatory connection between lignin biosynthesis and GPT2. Based on our transcriptomic and metabolomic analyses, we suggest that GPT2 operates to balance the flux between the biosynthesis of lignin and light-protective phenylpropanoid derivatives.

Article	Year
Spatial Profiles of Phosphate in Roots Indicate Developmental Control of Uptake, Recycling, and Sequestration. Plant physiology\| PubMed ID: 32999006	2020
Genetically manipulated chloroplast stromal phosphate levels alter photosynthetic efficiency. Plant physiology\| PubMed ID: 38701198	2024
A Role for the Plastidial GPT2 Translocator in the Modulation of Lignin Biosynthesis. Plant, cell & environment\| PubMed ID: 40377235	2025

Aditi Subramani Raju

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