PFGE analysis of changed mt-nucleoid DNA movement at different stages of cotyledon development
To investigate mt-nucleoid structure alterations at different stages of cotyledon development, we used PFGE analysis of purified mt-nucleoids from dry seeds, 4 °C-12 hr, 27 °C-12 hr, day 1, day 2 and day 3. The mtDNA–protein complex (mitochondria lysed and assayed without protease treatment) separated into two sub-populations: one stayed in the sample loading well was defined as the “well-bound DNA protein complex” (wb-DNA protein complex) and the other that migrated as a smeared zone was defined as “the fast-moving DNA protein complex” (fm-DNA protein complex) (Cheng et al. 2017; Dai et al. 2005). The fm-DNA protein complex was present in mt-nucleoids at all stages, from 27 °C-12 hr to day-3 cotyledon mitochondria, and migrated to the area equivalent to a 50- to 300-kb DNA position on PFGE analysis. However, under equal amounts of mitochondria (in terms of protein content), the wb-DNA protein complex was found in samples with 27 °C 12-hr immersion in water and day 1, 2, and 3 samples but not easily found in dry seeds or 4 °C 2-, 6- and 12-hr water-immersed cotyledons (Fig. 1A).
To make sure completely get the pure mt-nucleoid in different-aged cotyledon mitochondria, preliminary testing was carried out by sucrose gradient analysis. Samples were recovered from the supernatant and the 15% to 30%, 30% to 50%, 50% to 60% boundary of the sucrose gradient for each stage of developing cotyledon mitochondria. Day-3 seedling mt-nucleoids were a control. After agarose gel separation on each fraction obtained after sucrose gradient separation, mtDNA appeared only in the supernatant and 15% to 30% boundary collection (Fig. 1B). Our previous study also demonstrated that the mt-nucleoids collected from 15% to 30% sucrose gradient had a chromatin-like structure and associated with a membrane component. These mt-nucleoids showed DNA replication and RNA transcription ability (Dai et al. 2005). In this study, mt-nucleoids collected from 15% to 30% sucrose gradient underwent LC MS/MS analysis.
Mt-nucleoid proteins related to transcription and translation are present in different-aged cotyledon mitochondria during seed germination
Purified mt-nucleoid samples collected from the 15% to 30% boundary of the sucrose gradient were dissolved in tris-buffer for LC-MS/MS analysis. Almost 50 proteins identified were repeatedly associated with mt-nucleoids (Supplementary Table 1). We classified these proteins according to their potential functions and attempted to understand the possible function of these proteins involved in different-aged mt-nucleoids during seed germination.
60S ribosomal protein appeared in dry seed mt-nucleoids. DEAD-box ATP-dependent RNA helicase and mediator of RNA polymerase II transcription subunits appeared in the stages of dry seeds. DEAD-box ATP-dependent RNA helicase and mediator of RNA polymerase II transcription subunits were also found in 4 °C 12-hr samples. They all disappeared in later cotyledon developing stages. These proteins are associated with transcription factors or editing at early stages before seed germination, these important factors may pre-exist in dry seeds and bind with mtDNA during mt-nucleoid purification (Table 1). With seeds at 27 °C 12 hr and cotyledon mitochondria executing their function, these transcription factors no longer bind to mtDNA. However, we found the DNA-directed RNA polymerase subunit of transcription in mt-nucleoids of 27 °C 12-hr cotyledon mitochondria, so transcription activity is in progress in this stage and new transcription factors can be produced during mitochondria/nucleus functioning.
Other factors associated to mtDNA that could be found in 27 °C 12-hr to day-3 data included elongation factor Tu and Lon protease homolog protein, which suggests that these proteins may bind with mtDNA directly or indirectly in active states of cotyledon mitochondria.
Mt-nucleoid-associating membrane proteins
It is known that mtDNA anchors to the inner mitochondrial membrane by nucleoid proteins. In our results, some membrane-binding proteins, such as ATPaseα, ATPaseβ, and adenine nucleotide transporter 1 (ANT1), appeared in mt-nucleoids at all developing stages (Table 2). ANT and ATPase alpha were not found in day 3 cotyledon mt-nucleoids. MtDNA may directly or indirectly bind with inner membrane proteins. Outer-membrane protein Porin was also found in mt-nucleoids at all stages as previously found (Dai et al. 2005).
Metabolic proteins related to respiration appear in mt-nucleoids from dry seeds to day-3 cotyledons
Numerous metabolic proteins appeared in mt-nucleoids from dry seeds to day-3 cotyledons (Table 3). Most of these metabolic proteins are related to respiration: complex I protein NADH dehydrogenase, complex II protein succinate dehydrogenase, complex III protein cytochrome c1, and complex IV protein cytochrome c oxidase. We also found glycolysis intermediate proteins: dihydrolipoyl dehydrogenase, superoxide dismutase, dihydrolipoyllysine-residue acetyltransferase component 2, serine hydroxymethyltransferase 1; and citric acid cycle intermediate protein: malate dehydrogenase, aconitate hydratase 2, formate dehydrogenase 1, isocitrate dehydrogenase, succinyl-coa ligase (probable), pyruvate dehydrogenase E1 component subunit beta and succinate-semialdehyde dehydrogenase. We found no evidence that those metabolic-associated nucleoid proteins are directly related to mtDNA maintenance.
Other proteins found in cotyledon mt-nucleoids functioning in protein quality control reported in mammal cells and yeast studies (Table 4). Chaperonin 60 (CPN60) belonging to Hsp60 appeared in 4 °C 12 hr to day-3 cotyledon mt-nucleoids. CPN60 is involved in mitochondrial protein homeostasis functioning in protein refolding (Voos 2013). Hsp70 was also found in human and yeast mt-nucleoids in previous studies (Kucej and Butow 2007; Voos 2013). It was found in mt-nucleoids during the whole period of cotyledon mitochondria development we investigated.
Catalase is peroxisomal enzyme and functions in reactive oxygen species (ROS) homeostasis. It is found in peroxisomes and also in mitochondria in yeast, maize, Arabidopsis and animal cells (Mhamdi et al. 2010; Zamocky et al. 2008). We found the homolog in dry seeds, 4 °C-12 hr, day-1 and -3 cotyledon mt-nucleoids (Table 4), which indicates a high level of ROS involved during these stages.