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

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Abstract

Main conclusion

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.

Abstract

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.

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Abbreviations

EST:

Expressed sequence tag

GPD:

Glyceraldehyde-3-phosphate dehydrogenase

MT:

Metallothionein

ROS:

Reactive oxygen species

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Acknowledgements

This research was supported by a DPST research grant [grant no. 022/2557] to MM from the Institute for the Promotion of Teaching Science and Technology (IPST) and by a research assistantship grant to OP from Faculty of Science, Mahidol University. We acknowledge Dr. Thomas N. Stewart for proofreading of the manuscript.

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  1. Department of Biology, Faculty of Science, Mahidol University, Bangkok, 10400, Thailand

    Orathai Pakdee, Prayad Pokethitiyook & Metha Meetam

  2. Center of Excellence on Environmental Health and Toxicology, CHE, Ministry of Education, Bangkok, 10400, Thailand

    Orathai Pakdee, Prayad Pokethitiyook & Metha Meetam

  3. Department of Plant Science, Faculty of Science, Mahidol University, Bangkok, 10400, Thailand

    Wisuwat Songnuan & Nathinee Panvisavas

  4. Department of Biochemistry, Faculty of Science, Mahidol University, Bangkok, 10400, Thailand

    Kittisak Yokthongwattana

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  1. Orathai Pakdee
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Suppl. Fig. S1

Relative PpACT2 transcript abundance in different tissues or ages of moss or in response to different stress treatments. a Total RNA was extracted from 4-week-old (4 W) or 8-week-old (8 W) protonema (Pro) or gametophore (Gam) tissues cultured on BCDAT medium. b Total RNA was extracted from 4-week-old protonema cultured in normal liquid medium (control) or supplemented with 50 µM CdCl2 or 2 mM H2O2 for 24 h. Relative ACT2 expression was normalized to the geometric mean of three reference genes including histone PpHis3.2, elongation factor 1 alpha PpeF1a, and beta-tubulin 1 PpTub1. Error bars represent SE (n = 3). ns, not significantly different (one-tailed t test, P < 0.05)

Suppl. Fig. S2

Dose response of colony growth inhibition of WT P. patens to ZnSO4, CuSO4 and CdCl2 supplemented to BCDAT agar medium. Equal amounts of protonema were inoculated on medium-containing various concentrations of ZnSO4, CuSO4 and CdCl2. After 4 weeks, moss colonies were photographed and colony areas were quantified by ImageJ software (NIH, USA). Relative colony area was calculated in comparison to the control condition. The fitted curve and the effective concentration (EC) were determined using the built-in “log(inhibitor) - normalized response” equation with a variable slope in GraphPad Prism (GraphPad Software Inc., USA)

Suppl. Fig. S3

Semi-quantitative RT-PCR analysis of PpMT transcript abundance in 4-week-old gametophore tissues of P. patens cultured in normal BCDAT agar medium for 4 weeks using the PpMT gene-specific primers. ACT2 was included as a loading control

Suppl. Fig. S4

Electrolyte leakage of WT P. patens gametophore tissues following treatment with ZnSO4, CuSO4, CdCl2 or H2O2 for 24 h in BCDAT broth medium. Asterisk indicates values that are significantly different from the control (one-tailed t test; *, P < 0.1 or **, P < 0.05). Error bars represent SE (n = 3)

Suppl. Fig. S5

Semi-quantitative RT-PCR analysis of transcript abundance corresponding to the endogenous CUP1 or heterologously expressed MT genes in yeast strains transformed with p424-GDP empty vector (EV) or the vector harboring different transgenes. The PCR amplicons were amplified using CUP1-specific or PpMT gene-specific primers. Yeast ACT1 was used as a loading control

Suppl. Fig. S6

Cadmium sensitivity of S. cerevisiaea ∆ycf1 or b ∆cup1 strains expressing PpMT genes in comparison to controls: empty vector (EV), S. cerevisiae CUP1 (CUP1), or A. thaliana MT2a (AtMT2a). Cultures were grown on normal SC (-trp) agar medium or supplemented with CdCl2 at the indicated concentration

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Pakdee, O., Songnuan, W., Panvisavas, N. et al. Functional characterization of metallothionein-like genes from Physcomitrella patens: expression profiling, yeast heterologous expression, and disruption of PpMT1.2a gene. Planta 250, 427–443 (2019). https://doi.org/10.1007/s00425-019-03173-8

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