T-DNA Insertion in the <i>CLS</i> Gene for Cardiolipin Synthase Affects Development of <i>Arabidopsis thaliana</i>

Abstract

Summary Cardiolipin (CL) is widely distributed in various prokaryotes and eukaryotes as a membrane phospholipid. In eukaryotes, it is localized in the inner membrane and at the contact sites between the inner and outer membranes of mitochondria. CL is suggested to be involved in many mitochondrial functions. We previously identified the CLS gene for CL synthase in Arabidopsis thaliana and showed it to be a useful tool for understanding the physiological roles of CL in higher plants. In this study, we isolated homozygous cls mutants (cls-1 and cls-2) of A. thaliana, in which the CLS gene was disrupted by T-DNA insertion, by observing the development of embryos obtained after self-fertilization of heterozygous cls mutants. Both embryogenesis and growth of the homozygous cls mutants were substantially retarded relative to wild type, and additional phenotypes were observed, namely slow root growth, an abnormal veining pattern in cotyledons, and a low yield of seed production. The delayed embryogenesis and growth of homozygous cls mutants were recovered by introduction of CLS into the mutants. These findings demonstrate that CLS plays important roles in development of A. thaliana, presumably due to the biosynthesis of CL in the mitochondria. Biological membranes required for compartmentalization of cells and organelles are mainly composed of lipids and proteins. Lipids provide a hydrophobic environment in the membranes and form a bilayer that is a fundamental structure of the membranes. The lipid compositions of various biological membranes, such as the mitochondrial and plasma membranes, are different, and there are many lipid classes in a cell. Cardiolipin (CL) is widely distributed in various prokaryotes and mitochondria of eukaryotes as a membrane phospholipid. In eukaryotes, it comprises approximately 10% of the mitochondrial membrane lipids and mainly exists in the inner membrane and the contact sites between the outer and inner membranes of the mitochondria (Frentzen and Griebau 1994). Key wordsAs shown in Fig. 1, CL is synthesized from phosphatidic acid (PA), which is synthesized from glycerol 3-phosphate by a 2-step acylation. PA is converted into CDP-diacylglycerol (CDP-DAG) by CDP-DAG synthase, which transfers the CMP moiety from CTP to PA. The synthesized CDP-DAG reacts with glycerol 3-phosphate to produce phosphatidylglycerophosphate (PGP) and CMP in a reaction catalyzed by PGP synthase. Then, the resulting PGP is converted into phosphatidylglycerol (PG) by dephosphorylation catalyzed by PGP phosphatase. In the final step in the biosynthesis of CL, CL synthase (CLS) transfers a phosphatidyl group from CDP-DAG to PG to produce

Cardiolipin-Mediated Mitochondrial Dynamics and Stress Response inArabidopsis

“…Sequencing of the RT-PCR products showed that transcripts 1 and 2 each retained a partial sequence of exon 5 and were still in frame, and thus may encode truncated proteins lacking part of the catalytic domain, whereas transcript 3 was missing the entire exon 5 and was out of frame ( Figure 3A). Consistent with the previously reported growth phenotypes of cls T-DNA insertion mutants (Katayama and Wada, 2012;Pineau et al, 2013), cls-1 was severely dwarfed ( Figure 3C; Supplemental Figure 4A) and produced very small siliques that contained nonviable seeds (Supplemental Figure 4B). The homozygous cls-1 seeds produced by heterozygous plants were significantly delayed in germination ( Figure 3D).…”

“…CL's role in plants is just beginning to be revealed. CL is essential for normal plant growth and development, as shown by the two previous studies (Katayama and Wada, 2012;Pineau et al, 2013) and this study. Thus, the functions of CL in mitochondrial electron transport, ATP production, and protein import are very likely to be conserved among eukaryotes.…”

“…To test this prediction, we characterized the cls-1 (SALK_049840) mutant, which has a T-DNA insertion in exon 5 ( Figure 3A). A previous report of the same cls mutant allele described extreme retardation in embryo development and plant growth and low fertility of the mutant (Katayama and Wada, 2012), yet the molecular basis of the mutation was not fully characterized. RT-PCR analysis revealed that cls-1 contained three truncated CLS transcripts ( Figure 3B).…”

“…Recent studies of the Arabidopsis CARDIOLIPIN SYNTHASE (CLS) T-DNA insertion mutants demonstrated defects of the mutants in embryo development, growth and reproduction, mitochondrial ultrastructure and respiratory function, and protoplast response to UV light and heat shock (Katayama and Wada, 2012;Pineau et al, 2013). In this study, we investigated the role of CL in organelle dynamics and explored CL's function in protecting plants from stresses at the whole-organism level.…”

Cardiolipin Supports Respiratory Enzymes in Plants in Different Ways

“…Since the bulk of CL research has been conducted on yeast mitochondria and bacteria a picture of the dependence of plant mitochondrial functions on CL is only beginning to emerge. In Arabidopsis CL deficiency has recently been linked with (i) disturbances of plant mitochondrial fusion: fission balance, (ii) altered mitochondrial metabolism and (iii) stress tolerance in Arabidopsis ( Katayama and Wada, 2012 ; Pineau et al, 2013 ; Pan et al, 2014 ). In order to extend current knowledge on the involvement of CL in plant respiration a homozygous knock-out mutant of the final enzyme of CL biosynthesis, CLS, was used for an assessment of respiratory chain components in respect to both, abundance and enzymatic capacity.…”