Fig 2: Impacted mitochondrial complexes in zmnmat1 kernels at 10 DAP. (A) BN-PAGE analysis of mitochondrial complexes. The positions of complex I+III2, complex I, complex III and complex V were indicated. About 100 μg of mitochondrial protein was loaded in each lane. (B) In-gel NADH dehydrogenase activity test of complex (I) The positions of complex I+III2 and complex I was indicated. About 100 μg of mitochondrial protein was loaded in each lane. (C) Western blot analysis with antibodies against NAD9 (subunit of complex I), Cytc (Cytochrome c), COX2 (subunit of complex IV), ATPase-B (subunit of complex V) and AOX2 (stands for alternative oxidase) in mitochondrial protein from WT and zmnmat1 immature seeds at 10 DAP. Tubulin was used as a loading control. About 25 μg of mitochondrial protein was loaded in WT and zmnmat1 lanes, and about 12.5 μg of mitochondrial protein was loaded in ½WT lane.

Fig 3: Cleavage analysis of atp6c transcripts mediated by ZmRF5. (a) Strategy for ligation‐mediated amplification of the 5′ ends of cleaved atp6c mRNA for analysis of the cleavage site. Solid line, mitochondrial RNA; dotted line, cDNA; orange box, 5′‐race adapter; green box, reverse transcription primer; and yellow boxes, PCR primers. (b) Agarose gel electrophoresis analysis of the 5′ RACE PCR products from C6233R and CMo17A. Adapters can be ligated to matured and cleaved mitochondrial RNA at 5′ terminus, except for nascent mitochondrial RNA. Red arrows, cleaved fragments of C6233R (251 bp) and CMo17A (113 bp). (c) The physical location of primers used in the qRT‐PCR experiment. (d, e) qRT‐PCR analysis of the expression of atp6c in anther during meiosis of C6233R and CMo17A using primer atp6c‐1F/R (514–908) and atp6c‐2F/R (26–236). Each experiment was replicated three times. Values are the means with SEs (*P < 0.05, **P < 0.01, student's t‐test). (f, g) The accumulation of ATP6C and ATP6 in the different lines, respectively. COX2 was used as an internal control. NMo17B, the maintainer line, N(zmrf4zmrf4zmrf5zmrf5); CMo17A, the sterile line, C(zmrf4zmrf4zmrf5zmrf5); C6233R, the restorer line, C(zmrf4zmrf4ZmRf5ZmRf5); CF1, crossing CMo17A with C6233R, C(zmrf4zmrf4ZmRf5zmrf5); YU87‐1A, the sterile line, C(zmrf4zmrf4zmrf5zmrf5); YU87‐1B, the maintainer line, N(zmrf4zmrf4zmrf5zmrf5).

Fig 4: Epidermal Mitochondrial Proteome of High Light‐Treated Plants. (A) Representative confocal images of mitochondria in a transgenic line expressing GFP‐TOM5 under the epidermis‐specific promoter pATML1 show GFP fluorescence exclusively in epidermal tissues. In contrast, MitoTracker Red staining reveals a similar mitochondrial density in both epidermal and mesophyll cells, further confirming the tissue specificity of pATML1:GFP‐TOM5 expression. (B) Western blot analysis of mitochondrial proteins isolated from epidermal peels and mesophyll tissues of pATML1:GFP‐TOM5 plants using GFP antibodies. Total lysate and enriched mitochondrial fractions were probed with antibodies against RbcL, IDH, and COX2 to validate mitochondrial enrichment. (C) Volcano plots depicting differential protein abundance in mitochondrial‐enriched samples between HL and control conditions, as well as between FUM‐treated and control plants. The curved lines represent threshold boundaries for statistical significance and fold change, visually identifying differentially expressed proteins (n = 3, using Student's t‐tests, with an S0 value of 0.5 applied to stabilize the test statistic, FDR threshold = 0.05) each biological replicate was derived from an individual mitochondria enrichment from separate pools of biological material. (D) Venn diagram comparing proteins with increased abundance identified in treatments stated in (C). (E) Cellular component gene ontology (GO) term enrichment of 248 proteins with significantly increased abundance after HL exposure. (F) Table of proteins significantly associated with the mitochondrial‐ER interface in Arabidopsis or with homologs in other model organisms linked to mitochondrial‐ER interactions or mitochondrial morphology regulation [114, 115].