Virus-induced gene silencing of 14-3-3 genes abrogates dark repression of nitrate reductase activity in Nicotiana benthamiana.
In order to study the effect of repression of 14-3-3 genes on actual activity of the nitrate reductase (NR) in Nicotiana benthamiana leaves, Nb14-3-3a gene was silenced by virus-induced gene silencing (VIGS) method using potato virus X (PVX). Expression of Nb14-3-3a as well as Nb14-3-3b genes was altogether repressed in the leaves of PVX-14-3a-infected plants. Furthermore, two-dimensional gel electrophoresis and immunoblot analysis with anti-14-3-3 antiserum suggested that the expressions of Nb14-3-3a and Nb14-3-3b proteins are accordingly repressed in PVX-14-3a-infected plants. It is well known that binding of 14-3-3 proteins to phosphorylated NR leads to substantial decrease in NR activity of leaves under darkness. Therefore, we studied the changes in NR activity in response to light/dark transitions in the leaves of PVX-14-3a-infected plants. NR activation state was kept at a high level under darkness in PVX-14-3a-infected plants, but not in PVX-green fluorescent protein (GFP)-infected and control plants. This result suggests that Nb14-3-3a and/or Nb14-3-3b proteins are indeed involved in the inactivation of NR activity under darkness in N. benthamiana.
Hirano
Tatsuya
T
Faculty of Agriculture, Meijo University, 1-501 Shiogamaguchi, Tenpaku, Nagoya, Japan. thirano@ccmfs.meijo-u.ac.jp
Ito
Akiko
A
Berberich
Thomas
T
Terauchi
Ryohei
R
Saitoh
Hiromasa
H
D048948:14-3-3 Proteins / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism; D001483:Base Sequence; D003624:Darkness; D015180:Electrophoresis, Gel, Two-Dimensional; D004794:Enzyme Repression; D018506:Gene Expression Regulation, Plant / Q000502:physiology*; D020868:Gene Silencing; D008027:Light; D008969:Molecular Sequence Data; D050901:Nitrate Reductase / Q000096:biosynthesis*; D009569:Nitric Oxide / Q000378:metabolism; D010802:Phylogeny; D018515:Plant Leaves / Q000201:enzymology*; D030821:Plants, Genetically Modified; D017863:Potexvirus / Q000378:metabolism*; D018749:RNA, Plant; D012689:Sequence Homology, Nucleic Acid; D014026:Nicotiana / Q000201:enzymology* / Q000821:virology
D048948:14-3-3 Proteins; D018749:RNA, Plant; D009569:Nitric Oxide; D050901:Nitrate Reductase