Characteristic and Expression Analysis of a Metallothionein Gene, OsMT2b, Down-Regulated by Cytokinin Suggests Functions in Root Development and Seed Embryo Germination of Rice

Abstract

Metallothioneins (MTs) are low molecular mass and cysteine-rich metal-binding proteins known to be mainly involved in maintaining metal homeostasis and stress responses. But, their functions in higher plant development are scarcely studied. Here, we characterized rice (Oryza sativa) METALLOTHIONEIN2b (OsMT2b) molecularly and found that its expression was down-regulated by cytokinins. OsMT2b was preferentially expressed in rice immature panicles, scutellum of germinating embryos, and primordium of lateral roots. In contrast with wild-type plants, OsMT2b-RNA interference (RNAi) transgenic plants had serious handicap in plant growth and root formation, whereas OsMT2b-overexpressing transformants were dwarfed and presented more adventitious roots and big lateral roots. The increased cytokinin levels in RNAi plants and decreased cytokinin levels in overexpressing plants were confirmed by high-performance liquid chromatography quantitative analysis in the roots of wild-type and transgenic plants. In RNAi plants, localization of isopentenyladenosine, a kind of endogenous cytokinin, in roots and germinating embryos expanded to the whole tissues, whereas in overexpressing plants, the isopentenyladenosine signals were very faint in the vascular tissues of roots and scutellum cells of germinating embryos. In vitro culture of embryos could largely resume the reduced germination frequency in RNAi plants but had no obvious change in overexpressing plants. Taken together, these results indicate a possible feedback regulation mechanism of OsMT2b to the level of endogenous cytokinins that is involved in root development and seed embryo germination of rice.

Epidermal Cell Death in Rice Is Confined to Cells with a Distinct Molecular Identity and Is Mediated by Ethylene and H2O2 through an Autoamplified Signal Pathway

“…MT2b belongs to the metallothionein (MT) family of low molecular weight, Cys-rich metal binding proteins (Kä gi, 1991;Kojima, 1991;Mir et al, 2004), which consists of 15 members in rice (Yuan et al, 2008). Plant MT proteins play a role in cellular metal homeostasis and tolerance (Cobbett and Goldsbrough, 2002) and are active as ROS scavengers (Akashi et al, 2004;Wong et al, 2004).…”

“…Rice seeds in cv Nipponbare background carrying the retrotransposon Tos17 insertion in the MT2b gene (NE7013) were obtained from Akio Miyao (Genome Research Center, Ibaraki, Japan). Rice cv Kinmaze wild-type and MT2b-RNAi lines (Yuan et al, 2008) were provided by Hann Ling Wong and Ko Shimamoto (Plant Molecular Genetics, Nara Institute of Science and Technology, Ikoma, Nara, Japan).…”

“…For RNA gel blot analysis, RNA was extracted from young leaves harvested from cv Kinmaze wild-type and MT2b-RNAi plants (Wong et al, 2004;Yuan et al, 2008) or from cv PG56, cv Kinmaze, and cv Nipponbare plants with Tri-reagent (Sigma-Aldrich) following the instructions provided by the manufacturer. RNA was separated and transferred to a Hybond N + membrane (GE-Healthcare) as described (Sauter, 1997).…”

Molecular identification and interaction assay of the gene (OsUbc13) encoding a ubiquitin-conjugating enzyme in rice

“…MTs play important roles in maintaining homeostasis and detoxification of metals in both plants and animals; they are known as a kind of low molecular mass and cysteine-rich metal-binding protein (Yuan J. et al, 2008). In plants, studies on the responses of MTs to stress conditions, such as drought and reactive oxygen species (ROS), have also been conducted widely (Akashi et al, 2004;Wong et al, 2004).…”

“…The protein-protein interactions were verified by co-transformation in yeast AH109 with four genes (out of 20) as representatives. The four genes are OsVDAC (Godbole et al, 2003), OsCROC-1 (Hofmann and Pickart, 1999), OsB22EL8 (Yuan J. et al, 2008), and OsMADS1 (Prasad et al, 2005) of which the function had been defined (Fig. 5b).…”