Targeted Proteomics Approach Toward Understanding the Role of the Mitochondrial Protease FTSH4 in the Biogenesis of OXPHOS During Arabidopsis Seed Germination

Abstract

Seed germination provides an excellent model to study the process of mitochondrial biogenesis. It is a complex and strictly regulated process which requires a proper biogenesis of fully active organelles from existing promitochondrial structures. We have previously reported that the lack of the inner mitochondrial membrane protease FTSH4 delayed Arabidopsis seed germination. Here, we implemented a targeted mass spectrometry-based approach, Multiple Reaction Monitoring (MRM), with stable-isotope-labeled standard peptides for increased sensitivity, to quantify mitochondrial proteins in dry and germinating wild-type and ftsh4 mutant seeds, lacking the FTSH4 protease. Using total seed protein extracts we measured the abundance of the peptide targets belonging to the OXPHOS complexes, AOX1A, transport, and inner membrane scaffold as well as mitochondrial proteins that are highly specific to dry and germinating seeds. The MRM assay showed that the abundance of these proteins in ftsh4 did not differ substantially from that observed in wild-type at the level of dry seed and after stratification, but we observed a reduction in protein abundance in most of the examined OXPHOS subunits in the later stages of germination. These changes in OXPHOS protein levels in ftsh4 mutants were accompanied by a lower cytochrome pathway activity as well as an increased AOX1A amount at the transcript and protein level and alternative pathway activity. The analyses of the steady-state transcript levels of mitochondrial and nuclear genes encoding OXPHOS subunits did not show significant difference in their amount, indicating that the observed changes in the OXPHOS occurred at the post-transcriptional level. At the time when ftsh4 seeds were fully germinated, the abundance of the OXPHOS proteins in the mutant was either slightly lowered or comparable to these amounts in wild-type seeds at the similar developmental stage. By the implementation of an integrative approach combining targeted proteomics, quantitative transcriptomics, and physiological studies we have shown that the FTSH4 protease has an important role in the biogenesis of OXPHOS and thus biogenesis of mitochondria during germination of Arabidopsis seeds.

Long-read sequencing characterizes mitochondrial and plastid genome variants in Arabidopsismsh1mutants

“…During seed germination, normal mitochondrial biogenesis and mtDNA copy number are tightly controlled (Paszkiewicz et al, 2017). This connection between seed germination and mitochondrial defects was also reported in many other studies (Han et al, 2010; Virdi et al, 2015; Van Aken et al, 2016; Heidorn-Czarna et al, 2018; Golin et al, 2020). We showed that variation in root lengths at 5 DAS was heritable among different msh1 mutant families.…”

Conserved and Opposite Transcriptome Patterns during Germination in Hordeum vulgare and Arabidopsis thaliana

“…Likewise, the abundance of mitochondrial-encoded subunits in maize and rice was already high at the early stages in germination, as was porin [ 21 , 23 ], compared to in barley where it steadily increased over time to peak at 24 or 48 h. Moreover, overall, organelle translation machineries were upregulated in Arabidopsis compared to barley ( Figure 5 B), suggesting that mitochondrial capacity was overall increasing rapidly in Arabidopsis. Given the important role of components involved in mitochondrial biogenesis for the timing of germination, such as Tim17-1 ([ 44 ], and overall the importance of mitochondrial function for germination [ 14 , 60 , 61 , 62 ]), the kinetics of mitochondrial biogenesis during germination may play an important role in determining the speed of germination.…”

Cytochrome c and the transcription factor ABI4 establish a molecular link between mitochondria and ABA‐dependent seed germination

“…Mutations in genes encoding several mitochondrial components affect the time of germination, revealing the importance of mitochondrial function for the process (Meyer et al ., 2009; Zmudjak et al ., 2013; Wang et al ., 2014; Zhu et al ., 2014; Sechet et al ., 2015; Sew et al ., 2016; Lee et al ., 2017, 2019; Heidorn‐Czarna et al ., 2018; Ma et al ., 2019). The use of inhibitors during respiratory measurements in Arabidopsis seeds suggested that most oxygen consumption during seed germination relies on the activity of the cyanide‐sensitive pathway (Sew et al ., 2013; Paszkiewicz et al ., 2017; Nietzel et al ., 2020).…”