Phloem sap proteins from Cucurbita maxima and Ricinus communis have the capacity to traffic cell to cell through plasmodesmata

Abstract

In angiosperms, the functional enucleate sieve tube system of the phloem appears to be maintained by the surrounding companion cells. In this study, we tested the hypothesis that polypeptides present within the phloem sap traffic cell to cell from the companion cells, where they are synthesized, into the sieve tube via plasmodesmata. Coinjection of f luorescently labeled dextrans along with sizefractionated Cucurbita maxima phloem proteins, ranging in size from 10 to 200 kDa, as well as injection of individual f luorescently labeled phloem proteins, provided unambiguous evidence that these proteins have the capacity to interact with mesophyll plasmodesmata in cucurbit cotyledons to induce an increase in size exclusion limit and traffic cell to cell. Plasmodesmal size exclusion limit increased to greater than 20 kDa, but less than 40 kDa, irrespective of the size of the injected protein, indicating that partial protein unfolding may be a requirement for transport. A threshold concentration in the 20-100 nM range was required for cell-to-cell transport indicating that phloem proteins have a high affinity for the mesophyll plasmodesmal binding site(s). Parallel experiments with glutaredoxin and cystatin, phloem sap proteins from Ricinus communis, established that these proteins can also traffic through cucurbit mesophyll plasmodesmata. These results are discussed in terms of the requirements for regulated protein trafficking between companion cells and the sieve tube system. As the threshold value for plasmodesmal transport of phloem sap proteins falls within the same range as many plant hormones, the possibility is discussed that some of these proteins may act as long-distance signaling molecules.

Binding Properties of the N-Acetylglucosamine and High-Mannose N-Glycan PP2-A1 Phloem Lectin in Arabidopsis

“…They were first identified as components of the socalled P-proteins in cucurbits and shown to act as dimeric chitin-binding lectins in these plants (Beyenbach et al, 1974;Sabnis and Hart, 1978;Allen, 1979;Read and Northcote, 1983a;Smith et al, 1987;Bostwick et al, 1992), leading to the suggestion of a role in defense mechanisms. In C. maxima, CbmPP2 presents several properties associated with NCAPs (Bostwick et al, 1994;Balachandran et al, 1997;Golecki et al, 1998Golecki et al, , 1999, suggesting a role in the trafficking of macromolecules. However, it remains unclear whether PP2s from species other than cucurbits present such properties.…”

“…Therefore, we generated recombinant PP2-A1 by heterologous expression in E. coli. This bacterium is a suitable host because there is no evidence that PP2 phloem lectins require glycosylation for activity in vivo (Allen, 1979;Balachandran et al, 1997). The PP2-A1 cDNA was amplified from Arabidopsis total RNA, and the coding region was fused in frame with a 63 His tag (His 6 ) in the C-terminal position to facilitate its subsequent purification on a Ni 2+ -nitriloacetic affinity column.…”

“…In Cucurbita maxima, the P-proteins are the two most abundant sap proteins: Phloem Protein1 (PP1), which belongs to a gene family found only in cucurbits (Clark et al, 1997), and PP2, which belongs to a large gene family in angiosperms (Bostwick et al, 1992(Bostwick et al, , 1994Dinant et al, 2003). In cucurbits, PP2s display additional properties, including RNA binding Pallás, 2001, 2004;Owens et al, 2001;Gó mez et al, 2005), trafficking from cell to cell, and modification of the size exclusion limit of the plasmodesmata (Balachandran et al, 1997). These observations suggest that PP2 might play a role in the cell-to-cell trafficking of macromolecules.…”

Collection and Analysis of <em>Arabidopsis</em> Phloem Exudates Using the EDTA-facilitated Method

“…There are four major methods used to collect phloem exudates, yet they work only in select species: 1) In cucurbits it is possible to obtain reasonable amounts of phloem exudate through cuts of the petiole. Once the initial drop with contamination of injured cells is removed, it is possible to obtain reasonably large amounts of pure phloem sap 1,15 . However, with increasing collection time, this exudate thickens, making it increasingly unsuitable for liquid chromatography approaches (unpublished).…”

Metabolic networks of Cucurbita maxima phloem

“…This mechanism might inhibit size-increased oligosaccharides to flow back into mesophyll tissues. However, such strict control of size exclusion limits is hard to understand (Schaffer et al, 1996) since it is known that a range of far larger molecules may pass plasmodesmata (Alachandran et al, 1997), and basic physiological considerations (Stitt, 1996) do not back this hypothesis. Phloem concentrations of raffinose family oligosaccharides (RFO) in collecting minor veins were shown to be in the range of hundreds of mM using microdissection (Schmitz, 1970;Haritatos et al, 1996) or aphid exudates (Knop et al, 2001) for phloem collection whereas monosaccharides and sucrose were only present at rather low amounts.…”