Multispectral Phloem-Mobile Probes: Properties and Applications

Abstract

Using Arabidopsis (Arabidopsis thaliana) seedlings, we identified a range of small fluorescent probes that entered the translocation stream and were unloaded at the root tip. These probes had absorbance/emission maxima ranging from 367/454 to 546/576 nm and represent a versatile toolbox for studying phloem transport. Of the probes that we tested, naturally occurring fluorescent coumarin glucosides (esculin and fraxin) were phloem loaded and transported in oocytes by the sucrose transporter, AtSUC2. Arabidopsis plants in which AtSUC2 was replaced with barley (Hordeum vulgare) sucrose transporter (HvSUT1), which does not transport esculin in oocytes, failed to load esculin into the phloem. In wild-type plants, the fluorescence of esculin decayed to background levels about 2 h after phloem unloading, making it a suitable tracer for pulse-labeling studies of phloem transport. We identified additional probes, such as carboxytetraethylrhodamine, a red fluorescent probe that, unlike esculin, was stable for several hours after phloem unloading and could be used to study phloem transport in Arabidopsis lines expressing green fluorescent protein.

Phloem unloading in Arabidopsis roots is convective and regulated by the phloem-pole pericycle

“…Published images of phloem unloading in Arabidopsis give the impression that the unloading zone is a relatively broad region (Oparka et al, 1994; Wright and Oparka, 1996; Knoblauch et al, 2015). However, such images represent a ‘snapshot’ of unloading, taken at a defined time point following the application of fluorescent solutes to the leaf.…”

“…Carboxyfluorescein diacetate (CFDA) is the most widely utilized phloem-mobile probe (Oparka et al, 1994; Wright and Oparka, 1996; Knoblauch and van Bel, 1998). This probe is non-fluorescent when applied to source leaves but is subsequently cleaved by endogenous esterases to produce fluorescent, membrane-impermeant, carboxyfluorescein (CF; Knoblauch et al, 2015). The dye travels with the phloem translocation stream to sink tissues, where it can be visualized (Figure 1A).…”

“…When studied in real time, the dye characteristically shows preferential movement outwards into the cortex relative to the stele (Figure 1A; see also Oparka et al, 1994). Unloading of the dye in the root tip indicates a symplastic (plasmodesmata-mediated) pathway (Oparka et al, 1994; Wright and Oparka, 1996; Knoblauch et al, 2015). The cells in the unloading zone sequester the dye rapidly into their vacuoles where it becomes trapped.…”

“…The many unknowns surrounding phloem unloading in plants prompted us to conduct a detailed structure/function study of the terminal PSEs in the phloem unloading zone which, in Arabidopsis, is amenable to non-invasive imaging (Oparka et al, 1994; Knoblauch et al, 2015). We combined a detailed ultrastructural analysis of the cellular interfaces in the phloem unloading zone with the kinetics of phloem unloading of fluorescent solutes and macromolecules obtained by real-time imaging of growing roots.…”

Cellulose Deficiency Is Enhanced on Hyper Accumulation of Sucrose by a H+-Coupled Sucrose Symporter

“…Esculin is a coumarin b-glucoside that is readily transported by type I Suc transporters like SUC1 (Sivitz et al, 2007). Previously, it was used as a fluorescent probe to visualize phloem loading and transport in planta (Knoblauch et al, 2015) and heterologous Suc transporter activity in Saccharomyces cerevisiae (Gora et al, 2012). After 30 min of incubation with 1 mM esculin, we observed blue fluorescent signal accumulating in roots of wild-type plants, indicating intracellular accumulation of esculin (Fig.…”