Rice phloem thioredoxin h has the capacity to mediate its own cell-to-cell transport through plasmodesmata

Abstract.

Rice (Oryza sativa L.) phloem sieve tubes contain RPP13-1, a thioredoxin h protein that moves around the plant via the translocation stream. Such phloem-mobile proteins are thought to be synthesized in the companion cells prior to being transferred, through plasmodesmata, to the enucleate sieve-tube members. In this study, in-situ hybridization experiments confirmed that expression of RPP13-1 is restricted to companion cells within the mature phloem. To test the hypothesis that RPP13-1 enters the sieve tube, via plasmodesmata, recombinant RPP13-1 was expressed in Escherichia coli, extracted, purified and fluorescently labeled with fluorescein isothiocyanate (FITC) for use in microinjection experiments into tobacco (Nicotiana tabacum L.) mesophyll cells. The FITC-RPP13-1 moved from the injected cell into surrounding cells, whereas the E. coli thioredoxin, an evolutionary homolog of RPP13-1, when similarly labeled and injected, failed to move in this same experimental system. In addition, co-injection of RPP13-1 and FITC-dextrans established that RPP13-1 can induce an increase in plasmodesmal size exclusion limit to a value greater than 9.4 but less than 20 kDa. Nine mutant forms of RPP13-1 were constructed and tested for their capacity to move from cell to cell; two such mutants were found to be incapable of movement. Crystal-structure prediction studies were performed on wild-type and mutant RPP13-1 to identify the location of structural motifs required for protein trafficking through plasmodesmata. These studies are discussed with respect to plasmodesmal-mediated transport of macromolecules within the companion cell-sieve tube complex.