MRNA degradation‐based biosensors for boron

Abstract

Boron is an essential micronutrient for plants, mainly because it is required for cross-linking of pectin at rhamnogalacturonan II regions in cell walls (Funakawa and Miwa, 2015). Soils in humid areas of the world tend to be deficient in boron, while soils in arid regions tend to accumulate too much. Boric acid can diffuse into cells, but plants also use boric acid importers and exporters to modulate boron levels, as there is a narrow window between deficiency and toxicity (Yoshinari and Takano, 2017). In high boron soils, the BOR4 transporter acts to exclude excess boron from roots. In low boron soils, both an importer, NIP5;1, a member of the aquaporin group of channels, and another exporter, BOR1, facilitate transfer of boron to the xylem. Both NIP5;1 and BOR1 are down-regulated post-transcriptionally when boron levels are high; BOR1 levels are reduced via ubiquitination and protein degradation, while NIP5;1 mRNA is degraded (Tanaka et al., 2016), as illustrated in the top panel of Figure 1. The highlighted article from Junpei Takano's group (Fukuda et al., 2018) took advantage of this post-transcriptional control of NIP5;1 to develop two biosensors for boron, as illustrated in the bottom panels of Figure 1. Junpei has worked on various aspects of plant nutrition since his Ph.D. studies at the University of Tokyo. He started to develop these boron biosensors when he was an assistant professor in Hokkaido University, and since 2016 has been a professor at Osaka Prefecture University (OPU). Co-first author Shinji Wakuta, a postdoc when the lab was at Hokkaido, generated the biosensors and carried out the initial experiments to analyze boron distribution and uptake. Toru Fujiwara, a professor at the University of Tokyo, heard about their results and asked to use these biosensors to validate their mathematical model for boron flow (Sotta et al., 2017). Therefore Makiha Fukuda, a postdoc in Fujiwara's lab, introduced the biosensors into some mutants Fujiwara's lab were studying. Since moving from Hokkaido was time consuming, Junpei asked Makiha if she would characterize the biosensors in BY2 cells and Arabidopsis. Jio Kamiyo, a graduate student in Junpei's lab at OPU, confirmed Shinji's results (always a good idea when moving labs) and together with Junpei established a method to measure relative boron levels. The first biosensor (Figure 1, bottom left) is comprised of the ubiquitin promoter, the NIP5;1 5′ UTR sequence, and a fluorescent reporter with a nuclear localization signal (proUBQ10:NIP5;1 5′UTR:NLS-3×mVenus). It was introduced into BY2 cells and the fluorescence intensity of the mVENUS reporter assessed at varying concentrations of boron. The biosensor had a physiologically relevant dynamic range (30–500 μm). In stably transformed Arabidopsis plants, the fluorescence intensity was strong when the boron concentration was low and gradually declined as the boron concentration increased (Figure 1, bottom left). They then examined roots using confocal microscopy. At low boron concentrations the fluorescence was stronger in epidermal, cortical, and endodermal cells than in the stele and, as expected, when the boron concentration increased, the fluorescence intensity declined in the epidermis, cortical, and endodermal cells, but not in the stele. From this they concluded that probably BOR1 and the Casparian strip help maintain high boron levels in the stele. The time resolution of the first biosensor was not ideal (taking several hours to start a decline in fluorescence intensity upon a change in boron concentration from low to high), so they designed the second biosensor (Figure 1, bottom right), replacing the Venus reporter with a Luciferase with a protein destabilization sequence added, so that the reporter protein would have a shorter half-life. The dynamic range of this reporter in transgenic Arabidopsis roots was 10–100 μm, and differences in the reporter intensity were detectable within 1 h after changing the boron concentration (from low to high, or high to low). Lastly, they introduced this Luciferase construct into Arabidopsis plants that are more tolerant to excess boron (Wakuta et al., 2016) because they express a weakly polar and stabilized variant of the BOR1 transporter (BOR1 K590A-GFP-HPT) that is not targeted for ubiquitination and degradation. Indeed, they showed that the weakly polar and stabilized variant exported boron and thereby reduced the intercellular boron concentration. What's next? The uNIP5;1-Venus sensor is useful for comparing the signal in a cell with the signal in neighboring cells, while the uNIP5;1-Luc sensor is useful for time courses. However, neither is suitable for reporting precise concentrations in a given cell, so they are working to make ratiometric sensors. They plan to study boron homeostasis and transport in other tissues, for example in pollen, where they are interested in measuring the boron concentration dynamically during pollen tube growth and in boron transport mutants. Other researchers will be able to use these biosensors to determine if phenotypes they are studying are influenced by boron concentration differences.