Assessing the utility of photoswitchable fluorescent proteins for tracking intercellular protein movement in the Arabidopsis root.

One way in which cells communicate is through the direct transfer of proteins. In plants, many of these proteins are transcription factors, which are made by one cell type and traffic into another. In order to understand how this movement occurs and its role in development, we would like to track this movement in live, intact plants in real-time. Here we examine the utility of the photoconvertible proteins, Dendra2 and (to a lesser extent) EosFP as tags for studying intracellular and intercellular protein movement in the Arabidopsis root. To this end, we made fusions between Dendra2 and six mobile transcription factors. Our results show that Dendra2 is an effective tool for studying protein movement between plant cells. Interestingly, we found that Dendra2 could not simply be swapped into existing constructs that had originally contained GFP. Most of the fusions made in this way failed to produce a fluorescent fusion. In addition we found that the optimal settings for photoconversion of Dendra2 in stably transformed roots were different from what has been published for photoconversion in transient assays in plants or in animal cells. By modifying the confocal setting, we were able to photoconvert Dendra2 in all cell layers in the root. However the efficiency of photoconversion was affected by the position of the cell layer within the root, with more internal tissues requiring more energy. By examining the Dendra2 fusions, we confirmed the mobility of the SHORT-ROOT (SHR) and CAPRICE (CPC) transcription factors between cells and we further discovered that SHR movement in stele and CPC movement in the epidermis are non-directional.

An essential protein that interacts with endosomes and promotes movement of the SHORT-ROOT transcription factor.

Plant cells can communicate through the direct transport of transcription factors [1-7]. One of the best-studied examples of this phenomenon is SHORT-ROOT (SHR), which moves from the stele cells into the endodermis and root tip of Arabidopsis, where it specifies endodermal cell identity and stem cell function, respectively [8-10]. In the endodermis, SHR upregulates the transcription factors SCARECROW (SCR) [2] and JACKDAW (JKD), which in turn inhibit movement of SHR from the endodermis [11]. Although much is known about the regulatory pathways that mediate expression and activity of SHR [1, 8-14], little is known about the factors that promote its movement or the movement of other transcription factors. We have identified a novel protein, SHORT-ROOT INTERACTING EMBRYONIC LETHAL (SIEL), that interacts with SHR, CAPRICE (CPC), TARGET OF MONOPTEROUS 7 (TMO7), and AGAMOUS-LIKE 21 (AGL21). Null alleles of SIEL are embryonic lethal. Hypomorphic alleles produce defects in root patterning and reduce SHR movement. Surprisingly, both SHR and SCR regulate expression of SIEL, so that siel/scr and siel/shr double mutants have extremely disorganized roots. SIEL localizes to the nucleus and cytoplasm of root cells where it is associated with endosomes. We propose that SIEL is an endosome-associated protein that promotes intercellular movement.