Expression of GFP‐fusions in Arabidopsis companion cells reveals non‐specific protein trafficking into sieve elements and identifies a novel post‐phloem domain in roots

Abstract

SummaryTransgenic Arabidopsis plants were constructed to express a range of GFP-fusion proteins (36-67 kDa) under the companion cell (CC)-specific AtSUC2 promoter. These plants were used to monitor the trafficking of these GFP-fusion proteins from the CCs into the sieve elements (SEs) and their subsequent translocation within and out of the phloem. The results revealed a large size exclusion limit (SEL) (>67 kDa) for the plasmodesmata connecting SEs and CCs in the loading phloem. Membrane-anchored GFP-fusions and a GFP variant targeted to the endoplasmic reticulum (ER) remained inside the CCs and were used as 'zero trafficking' controls. In contrast, free GFP and all soluble GFP-fusions, moved from the CCs into the SEs and were subsequently translocated through the phloem. Phloem unloading and post-phloem transport of these mobile GFP-fusions were studied in root tips, where post-phloem transport occurred only for the free form of GFP. All of the other soluble GFP-fusion variants were unloaded and restricted to a narrow zone of cells immediately adjacent to the mature protophloem. It appears that this domain of cells, which has a peripheral SEL of about 27-36 kDa, allows protein exchange between protophloem SEs and surrounding cells, but restricts general access of large proteins into the root tip. The presented data provide additional information on phloem development in Arabidopsis in relation to the formation of symplasmic domains.

Arabidopsis NPCC6/NaKR1 Is a Phloem Mobile Metal Binding Protein Necessary for Phloem Function and Root Meristem Maintenance

“…Compared with free GFP (27 kD), NaKR1-GFP (61 kD) was more restricted to the phloem in mature roots. This is similar to results for multiple soluble GFP fusions of 36 to 67 kD analyzed by Stadler et al (2005). Compared with free GFP, NaKR1-GFP mobility in the root tip was more restricted.…”

“…SUC2 is specifically expressed in companion cells, and GFP fluorescence was observed in the phloem and within two cell layers surrounding the phloem in the mature region of roots ( Figure 6G) as previously reported (Stadler et al, 2005). Free GFP diffused through the symplast into the whole root meristem ( Figure 6H) (Stadler et al, 2005;Benitez-Alfonso et al, 2009). Compared with free GFP (27 kD), NaKR1-GFP (61 kD) was more restricted to the phloem in mature roots.…”

“…Interestingly, NaKR1-GFP driven by the native promoter produced a different localization, consistent with transport through PD. In mature parts of the root, NaKR1-GFP fluorescence was observed in files of cells consistent with sieve elements and appeared similar to other soluble GFP fusions that move from companion cells into sieve elements (Stadler et al, 2005). In mature regions of the root, NaKR1-GFP was more restricted to sieve elements compared with free GFP, which is able to move one to two cell layers laterally ( Figure 6G; Stadler et al, 2005).…”

“…Observing cell-to-cell movement of green fluorescent protein (GFP) fusion proteins has been instrumental in identifying symplasmic domains (groups of cells connected by PD). GFP fusions of 36 to 67 kD expressed under the control of the companion cell-specific SUC2 promoter were translocated in the phloem and entered the protophloem but did not move throughout the root tip, although free GFP (27 kD) was able to move throughout the root tip (Stadler et al, 2005). This sets a size exclusion limit of 27 to 36 kD for PD connecting cells of the root tip with protophloem cells.…”

“…Companion cells and sieve elements are connected by specialized PD with a size exclusion limit of at least 67 kD (Stadler et al, 2005). Observing cell-to-cell movement of green fluorescent protein (GFP) fusion proteins has been instrumental in identifying symplasmic domains (groups of cells connected by PD).…”