A novel thioredoxin h is secreted in Nicotiana alata and reduces S-RNase in vitro.
Thioredoxins type h are classified into three subgroups. The subgroup II includes thioredoxins containing an N-terminal extension, the role of which is still unclear. Although thioredoxin secretion has been observed in animal cells, there is no evidence suggesting that any thioredoxin h is secreted in plants. In this study, we report that a thioredoxin h, subgroup II, from Nicotiana alata (NaTrxh) is secreted into the extracellular matrix of the stylar transmitting tract tissue. Fractionation studies showed that NaTrxh is extracted along with well characterized secretion proteins such as S-RNases and NaTTS (N. alata transmitting tissue-specific protein). Moreover, an NaTrxh-green fluorescent fusion protein transiently expressed in Nicotiana benthamiana and Arabidopsis thaliana leaves was also secreted, showing that NaTrxh has the required information for its secretion. We performed reduction assays in vitro to identify potential extracellular targets of NaTrxh. We found that S-RNase is one of the several potential substrates of the NaTrxh in the extracellular matrix. In addition, we proved by affinity chromatography that NaTrxh specifically interacts with S-RNase. Our findings showed that NaTrxh is a new thioredoxin h in Nicotiana that is secreted as well as in animal systems. Because NaTrxh is localized in the extracellular matrix of the stylar transmitting tract and its specific interaction with S-RNase to reduce it in vitro, we suggest that this thioredoxin h may be involved either in general pollen-pistil interaction processes or particularly in S-RNase-based self-incompatibility.
Juárez-Díaz
Javier Andrés
JA
Departamento de Bioquímica y Biología Molecular de Plantas, Facultad de Química, Universidad Nacional Autónoma de México (UNAM), DF 04510, México.
McClure
Bruce
B
Vázquez-Santana
Sonia
S
Guevara-García
Arturo
A
León-Mejía
Patricia
P
Márquez-Guzmán
Judith
J
Cruz-García
Felipe
F
D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000201:enzymology; D001483:Base Sequence; D002846:Chromatography, Affinity; D018076:DNA, Complementary / Q000378:metabolism; D004220:Disulfides / Q000737:chemistry; D004305:Dose-Response Relationship, Drug; D015180:Electrophoresis, Gel, Two-Dimensional; D004591:Electrophoresis, Polyacrylamide Gel; D005109:Extracellular Matrix / Q000378:metabolism; D015971:Gene Expression Regulation, Enzymologic; D018506:Gene Expression Regulation, Plant; D017343:Genes, Plant; D005982:Glutathione Transferase / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000378:metabolism; D015151:Immunoblotting; D066298:In Vitro Techniques; D008969:Molecular Sequence Data; D010802:Phylogeny; D010940:Plant Proteins / Q000378:metabolism*; D011485:Protein Binding; D025941:Protein Interaction Mapping; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry; D012260:Ribonucleases / Q000378:metabolism*; D013347:Subcellular Fractions / Q000378:metabolism; D054480:Thioredoxin h; D013879:Thioredoxins / Q000096:biosynthesis / Q000737:chemistry* / Q000378:metabolism*; D014026:Nicotiana / Q000201:enzymology / Q000378:metabolism*
D018076:DNA, Complementary; D004220:Disulfides; D010940:Plant Proteins; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D054480:Thioredoxin h; D049452:Green Fluorescent Proteins; D013879:Thioredoxins; D005982:Glutathione Transferase; D012260:Ribonucleases; C489908:S-like RNase protein, plant