Studies of macromolecular trafficking across Arabidopsis homografts

Micrografting was used to study the restoration of symplasmic transport at the graft union and to examine the long-distance transport of macromolecules between scion and rootstock. New techniques were established, such as correlative imaging and single-cell analysis in microfluidic devices, to study graft development both in vivo and in vitro. Imaging of Arabidopsis homografts showed that a symplasmic domain develops in the callus stele whose function may be to contain the spread of auxin into the surrounding ground tissue. It was demonstrated, also, that recent reports of organelle transfer at the graft union cannot be explained by the formation of secondary plasmodesmata (PD) at the graft interface. While fused calli did not exchange organelles in vitro, large aggregates of the SIEVE-ELEMENT OCCLUSION RELATED protein fused to YFP (SEOR-YFP; 112 kDa) were unloaded from mature sieve tubes into living cells of the graft partner in vivo, suggesting that vascular remodelling may be a prerequisite for the exchange of organelles at the graft interface. Fusion proteins expressing organelle-targeting signals were found to translocate across the graft junction, unloading into cell files adjacent to the root protophloem. The phloem mobility of a given fusion protein was assessed using bioinformatic and statistical analysis of publicly available data. The size of a protein and its relative abundance in CCs both emerged as defining factors for subsequent phloem transport. The recipient tissue for phloem-unloaded macromolecules was identified as the phloem-pole pericycle (PPP). This cell layer is required to remove macromolecules from the terminus of the protophloem. Induced callose deposition at the PD that connect protophloem SEs to the PPP caused a restriction in unloading and a subsequent arrest in root growth. A non-cell autonomous protein of CC origin, NaKR1-1, is proposed to affect the unloading of macromolecules either by increasing the size exclusion limit (SEL) of PD within the PPP or by enabling a build-up in pressure at the protophloem terminus, due to SUC2 activity, thus allowing phloem unloading