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Analysis of genes required for magnesium homeostasis through characterization of low-magnesium-sensitive Arabidopsis thaliana mutants

馮, 志航 東京大学 DOI:10.15083/0002001610

2021.09.08

概要

Introduction
Magnesium ion (Mg2+) is among the most abundant divalent cations in living cells and involved in a number of physiological and biochemical processes such as energy metabolism, DNA replication, transcription and ribosome aggregation. In plants, in addition to the conserved functions among organisms including enzyme activation, nucleic acid and protein synthesis, the most well-known characteristic of Mg is serving as the central atom of chlorophyll and functions as a reaction center in photosystem.

To maintain Mg at reasonable physiological levels, living organisms utilize multiple transport systems for Mg homeostasis. Especially, numerous transporters have been identified to play essential roles in Mg transportation processes. And these Mg transporters exhibit different expression patterns at transcriptional and translational levels in response to Mg availability. In plants, two types of Mg transporter family, MRS2 (Mitochondrial RNA splicing 2) and MHX (Mg2+/H+ exchanger), have been discovered and their physiological roles have been characterized to a certain extent. In contrast, compared to the Mg2+ transportation system identified in other organisms, the regulatory mechanisms of Mg transporters in plant remain largely unknown.

As a part of efforts to reveal mechanisms of Mg transportation and homeostasis, a battery of EMS-mutagenized A.thaliana mutants exhibited suppressed shoot growth on low-Mg medium were previously isolated our laboratory. By analyzing these mutants, I identified novel genes which play essential roles in regulating Mg transporters at either transcriptional or translational levels.

Chapter 1. SMU1 and SMU2 mediate MRS2-7 pre-mRNA splicing and are required for low-Mg adaptation in A. thaliana
In this study, a low-Mg sensitive A. thaliana mutant was isolated and the causal gene was identified to be SMU1 (suppressor of mec-8 and unc-52 1), an auxiliary splicing factor. Disruption of SMU1 leaded to suppressed growth and reduced Mg accumulation in shoot under low-Mg condition. In addition, disruption of SMU2 (suppressor of mec-8 and unc-52 1), one of the SMU1 interacting proteins, resulted in similar phenotypes under low-Mg condition. In both smu1 and smu2 mutants, one of the Mg transporter gene, MRS2-7, showed altered splicing patterns. Genetic evidence indicated that MRS2-7 functions in the same pathway with SMU1 and SMU2 on plant low-Mg tolerance. in vivo experiments demonstrated that overexpression of either SMU1 or SMU2 alone promoted MRS2-7 pre-mRNA splicing, suggesting a function redundancy of SMU1 and SMU2 in pre-mRNA splicing processing. In addition, SMU1-SMU2 interaction was observed in restricted nucleus compartments presumably speckles, which are considered as storage or assembly sites of spliceosomal components.

Taken together, the present study establishes the requirement of SMU1 and SMU2 in plant Mg homeostasis through splicing regulation of a Mg transporter gene.

Chapter 2. A CRIM domain-containing protein physically associates with ER-localized Mg transporters and plays an essential role in extending Mg stress adaptation range in A. thaliana
In addition to the expression regulation at the transcriptional level, protein modification and degradation control the activity and the amount of transporters. In case of Mg transporters, several genes have been found to play essential roles in regulating synthesis and turnover of Mg transporter proteins in bacteria, yeast and animals. While in plant, to my knowledge, no such kind of regulatory factors have been discovered.

In this chapter, a novel gene AT1G70780 encoding a 140 a.a protein was found to be required for low-Mg tolerance in A. thaliana. AT1G70780 disruption lines exhibited repressed shoot growth under low-Mg condition. Shoot and root Mg accumulations were significantly lower than those of wild-type independent of the supplied Mg concentrations. BiFC result showed that AT1G70780 physically associates with MRS2-1, MRS2-3, MRS2-5 and MRS2-10 on ER membrane, suggesting a potential function of AT1G70780 on protein properity modulation of these Mg transporters. However, in the yeast mrs2 mutant carrying either A. thaliana MRS2-1, MRS2-3, MRS2-5 or MRS2-10, expression of AT1G70780 did not affect the yeast cell growth on either normal or non-fermentable medium. Further analysis demonstrated that the predicted secondary structure of AT1G70780 protein shows similarities to the CRIM (Conserved region In the Middle) domain of yeast SIN1/AVO1 proteins, the latter of which is essential for efficient phosphorylation of TORC2 (Target of Rapamycin Complex 2) substrates. In addition, MRS2-1, MRS2-5 and MRS2-10 were detected to be phosphorylated on serine residues within a relatively conserved region in previous phosphoproteomic studies (Whiteman et al., 2008; Chen et al., 2010; Nakagami et al., 2010). In consistent, expression of phospho-mimicking MRS2-1, MRS2-5 and MRS2-10 conferred yeast mrs2 mutant with improved growth on non-fermentable medium than those expressing wild-type and non-phosphomimic versions. In addition, overexpression of AT1G707080-GFP increased the A. thaliana Mg accumulation and growth tolerance to both low- and high-Mg stress.

In conclusion, the present results suggest that AT1G70780 is involved in Mg stress tolerance presumably through regulating the activities of ER-localized Mg transporters.

Chapter 3. A putative methyltransferase physically associates with an ER-localized Mg transporter and is essential for low-Mg tolerance in A. thaliana
In this chapter, through characterizing the A. thaliana low-Mg sensitive mutant kudo10, I identified the gene AT3G13440 encoding a putative S-adenosyl-L-methionine-dependent methyltransferase superfamily protein required for the tolerance to low-Mg condition.

AT3G13440 mutation strongly repressed the growth and Mg accumulation in shoot when grown on low-Mg agar medium. GFP-tagged AT3G13440 fusions were mainly localized to nucleus and cytosol in Arabidopsis mesophyll protoplast and in root cells of transgenic seedlings. AT3G13440 is annotated to have several protein-protein interaction partners including the Mg transporter protein MRS2-2. Our BiFC results showed that, among the nine A.thaliana MRS2 members, AT3G13440 specifically associated with MRS2-2 on ER membrane. Further analysis demonstrated that AT3G13440 and MRS2-2 had similar expression pattern in root and they were at least colocalized on ER-like endomembrane structures when expressed in mesophyll protoplast. In yeast mrs2 mutant, expression of AT3G13440 inhibited the cell growth on both normal and non-fermentable agar media. In contrast, the growth defect was rescued when AT3G13440 co-expressed with A. thaliana MRS2-2. Unlike kudo12, kudo10 showed sensitivity to low-Mg but not to high-Mg and AT3G113440-GFP overexpression only conferred A. thaliana with increased tolerance to low-Mg stress.

Taken together, the results demonstrate the identified putative methyltransferase is essential for A. thaliana low-Mg tolerance and suggest a potential role of this gene in protein modification of Mg transporter MRS2-2.

Conclusion
In this thesis, through characterizing EMS-mutagenized A. thaliana mutants which exhibit sensitivities to low-Mg/high-Ca conditions, three novel genes were identified to be required for plant Mg homeostasis and resistance to low- and/or high-Mg stress. The auxiliary splicing factors SMU1 and SMU2 described in chapter 1 was demonstrated to mediate pre-mRNA splicing of the Mg transporter gene MRS2-7. Genetic evidence indicates that these genes function in the same pathway in shoot Mg accumulation and low-Mg tolerance. In chapter 2, I identified an unknown protein AT1G70780, which physically interacts with ER-localized Mg transporters (MRS2-1, MRS2-3, MRS2-5 and MRS2-10) and is required for plant Mg accumulation and resistance to both low- and high-Mg stress. The putative CRIM domain found in AT1G70780 combined with the detection of phosphorylation in AT1G70780 interacting MRS2 proteins (MRS2-1, MRS2-5 and MRS2-10) implying that, AT1G70780 may regulate the Mg transport activities of these MRS2 proteins through mediating their phosphorylation status. In chapter 3, I identified an uncharacterized gene, AT3G13440, which is essential for plant growth and shoot Mg accumulation under low-Mg condition. The presence of a conserved methyltransferase domain in AT3G13440 suggesting a potential role of this gene in modifying the protein property of its interacting partner, the ER-localized Mg transporter MRS2-2.

Prior to my study, the only known regulatory mechanism of plant Mg transporter is the 5’UTR-mediated expression of A. thaliana MHX gene. Throughout my achievements, the knowledge on plant Mg transporter regulatory mechanisms at transcriptional and translational levels have been extended benefit from the identification of novel MRS2-associated genes in A. thaliana.

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