Functional characterization of metallothionein-like genes from Physcomitrella patens: expression profiling, yeast heterologous expression, and disruption of PpMT1.2a gene.

Abstract

Physcomitrella patens contains four metallothionein-like genes. Three were shown to confer metal tolerance in yeast. Transcript profiling suggests their roles in senescence and reproductive development or cadmium and oxidative stress. Metallothioneins (MTs) have been suggested to play various roles including metal detoxification, nutrient remobilization, ROS scavenging, stress tolerance, and plant development. However, little is known about the forms and functions of MTs in bryophytes. The moss Physcomitrella patens genome was found to contain four MT-like genes. Amino acid sequence composition showed that the P. patens MTs (PpMTs) were clustered with Type 1 plant MTs, and could be further classified into two sub-types, herein referred to as sub-type 1: PpMT1.1a and PpMT1.1b and sub-type 2: PpMT1.2a and PpMT1.2b. Transcript abundance of PpMT1.1b and PpMT1.2b was upregulated in the gametophore compared to protonema, and all, except PpMT1.2a, were highly induced in senescing gametophytes. PpMT1.1a and PpMT1.1b transcripts were upregulated in protonema treated with cadmium and hydrogen peroxide. Unlike many higher plant MTs, the PpMT transcript abundance was not strongly induced in response to copper and zinc. These results suggest that PpMTs may play a role in protecting P. patens from cadmium and oxidative stress and may be involved in tissues senescence and reproductive development. The PpMTs, except PpMT1.2b, were also able to confer metal tolerance and accumulation when heterologously expressed in the ∆cup1 yeast. A P. patens mutant lacking PpMT1.2a through targeted gene disruption was generated. However, it did not show any alteration in growth phenotypes under senescence-induced conditions or hypersensitivity to cadmium, copper, zinc, H2O2, and NaCl stresses. Further characterization of additional P. patens mutants lacking single or multiple PpMTs may provide insight into the physiological roles of bryophytic MTs.