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 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.
Ishiwatari
Y
Y
Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, University of Tokyo, Japan.
Fujiwara
T
T
McFarland
K C
KC
Nemoto
K
K
Hayashi
H
H
Chino
M
M
Lucas
W J
WJ
D000595:Amino Acid Sequence; D001692:Biological Transport; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D012275:Oryza / Q000378:metabolism*; D010947:Plants, Toxic; D011487:Protein Conformation; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D054480:Thioredoxin h; D013879:Thioredoxins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014026:Nicotiana / Q000235:genetics
D012333:RNA, Messenger; D054480:Thioredoxin h; D013879:Thioredoxins