RICE PHLOEM THIOREDOXIN-H HAS THE CAPACITY TO MEDIATE ITS OWN CELL-TO-CELL TRANSPORT THROUGH PLASMODESMATA

Rice (Oryza sativa L.) phloem sieve tubes contain RPP13-1, a thioredox in h protein that moves around the plant via the translocation stream. Such phloem-mobile proteins are thought to be synthesized in the comp anion cells prior to being transferred, through plasmodesmata, to the enucleate sieve-tube members. In this study, in-situ hybridization exp eriments confirmed that expression of RPP13-I is restricted to compani on cells within the mature phloem. To test the hypothesis that RPP13-1 enters the sieve tube, via plasmodesmata, recombinant RPP13-1 was exp ressed in Escherichia coli, extracted, purified and fluorescently labe led with fluorescein isothiocyanate (FITC) for use in microinjection e xperiments into tobacco (Nicotiana tabacum L.) mesophyll cells. The FI TC-RPP13-1 moved from the injected cell into surrounding cells, wherea s the E, coli thioredoxin, an evolutionary homolog of RPPI3-1, when si milarly labeled and injected, failed to move in this same experimental system. In addition, co-injection of RPP13-1 and FITC-dextrans establ ished that RPP13-1 can induce an increase in plasmodesmal size exclusi on limit to a value greater than 9.4 but less than 20 kDa. Nine mutant forms of RPP13-I were constructed and tested for their capacity to mo ve from cell to cell, two such mutants were found to be incapable of m ovement. Crystal-structure prediction studies were performed on wild-t ype and mutant RPP13-1 to identify the location of structural motifs r equired for protein trafficking through plasmodesmata. These studies a re discussed with respect to plasmodesmal-mediated transport of macrom olecules within the companion cell-sieve tube complex.