PHB3 Is Required for the Assembly and Activity of Mitochondrial ATP Synthase in Arabidopsis

Mitochondrial ATP synthase is a multiprotein complex, which consists of a matrix-localized F1 domain (F1-ATPase) and an inner membrane-embedded Fo domain (Fo-ATPase). The assembly process of mitochondrial ATP synthase is complex and requires the function of many assembly factors. Although extensive studies on mitochondrial ATP synthase assembly have been conducted on yeast, much less study has been performed on plants. Here, we revealed the function of Arabidopsis prohibitin 3 (PHB3) in mitochondrial ATP synthase assembly by characterizing the phb3 mutant. The blue native PAGE (BN-PAGE) and in-gel activity staining assays showed that the activities of ATP synthase and F1-ATPase were significantly decreased in the phb3 mutant. The absence of PHB3 resulted in the accumulation of the Fo-ATPase and F1-ATPase intermediates, whereas the abundance of the Fo-ATPase subunit a was decreased in the ATP synthase monomer. Furthermore, we showed that PHB3 could interact with the F1-ATPase subunits β and δ in the yeast two-hybrid system (Y2H) and luciferase complementation imaging (LCI) assay and with Fo-ATPase subunit c in the LCI assay. These results indicate that PHB3 acts as an assembly factor required for the assembly and activity of mitochondrial ATP synthase.


Introduction
Mitochondria produce the bulk of the energy used by almost all eukaryotic cells through oxidative phosphorylation (OXPHOS), which is composed of five membrane complexes (Complex I-V) [1,2]. Complex I-IV, defined as the electron transport chain [3][4][5], can pump protons from the mitochondrial matrix to the intermembrane space (IMS) and undertake the task of producing a proton concentration gradient [6,7]. After that, Complex V (also named ATP synthase or ATPase) harvests the electrochemical energy from the proton motive force across the mitochondrial inner membrane and catalyzes the synthesis of ATP from ADP and phosphoric acid by proton translocation and subunit rotation [8][9][10][11][12]. Therefore, mitochondrial ATP synthase plays a key role in energy production.
Mitochondrial ATP synthase is a multiprotein complex, which is mainly divided into two domains, a matrix-soluble F1 domain (F1-ATPase) and an inner membrane-embedded Fo domain (Fo-ATPase). In yeast, the F1 domain consists of five kinds of subunits: α, β, γ, δ, and ε. The heterohexamer (αβ) 3, composed of subunits α and β, forms the catalytic head, and the subunits γ, δ, and ε constitute the central stalk [13]. The Fo domain includes subunits a, b, c, d, e, f, g, h, i/j, k, 8, and OSCP. The subunit c oligomerizes to form a ringlike homologous polymer, named c-ring, which attaches to subunit a to form a stator [14]. The rest of the Fo domain subunits form a peripheral stalk to fasten the F1 domain into the Fo domain stator. The peripheral stalk and central stalk are considered to be parts of the Fo domain and F1 domain, respectively [15][16][17]. The structure and composition including the nucleus and mitochondria, and the loss of the function of PHB3 results in a slow-growth phenotype in Arabidopsis [47][48][49]. Previous studies indicate that PHB3 is involved in cell production, cell proliferation, phytohormone signal transduction, and so on [50][51][52][53]. Transmission electron microscopy (TEM) results reveal that the mitochondria are swollen, and the inner mitochondrial membrane cristae disappear in the phb3 mutant [47]. In the absence of PHB3, alternative oxidase genes (AOX1A and AOX1C) and NAD(P)H dehydrogenase genes (NDA1, NDB2, NDB3, and NDB4) of alternative pathways are induced [51]. In plants, the induction of alternative pathway genes is considered as the retrograde signals when mitochondria are impaired, implying that the mitochondria are damaged in the phb3 mutant.
In this paper, we elucidated the mitochondrial function of PHB3 in Arabidopsis. We found that the deletion of PHB3 resulted in the significantly reduced activities of mitochondrial ATP synthase and F1-ATPase. Western blotting showed that the abundance of subunit a was increased in Fo-ATPase, while decreased in the ATP synthase monomer in the phb3 mutation. In addition, the loss of PHB3 leads to the accumulation of F1-ATPase by hybridization with primary antibodies against subunits α and β. Meanwhile, we performed Y2H and LCI assays and discovered that PHB3 could interact with the subunit c of Fo-ATPase, and the subunits β and δ of F1-ATPase. Together, our studies provided insight into the assembly of mitochondrial ATP synthase in Arabidopsis. PHB3 might act as an assembly factor and is required for the assembly and activity of mitochondrial ATP synthase.

Loss of Function of PHB3 Impairs the Abundance of Multiple Mitochondrial Proteins
To further explore the biological function of Arabidopsis PHB3 (AT5G40770) in mitochondria, we obtained the T-DNA mutant, phb3 (SALK_020707), harboring an insertion in the first exon of this gene [51,52]. We first analyzed the abundance of several mitochondrial proteins in the phb3 mutants. These proteins are involved in various metabolic pathways in mitochondria, such as the complex assembly, electron transport, TCA cycle, and antioxidant system. Total mitochondrial proteins of the wild-type and phb3 mutants were extracted, separated in SDS-PAGE, and analyzed with the Western blotting assay. As shown in Figure 1, the levels of most proteins were decreased in the phb3 mutant compared with the wild-type ( Figure 1). Among these, the subunits of mitochondrial complex I (V1, A5, and CA2) [54,55], complex IV (COX3), as well as complex I assembly factor GLDH (l-galactone-1,4-lactone dehydrogenase) [54,55] in abundance were significantly reduced ( Figure 1). The levels of SHMT (serine hydroxymethyltransferase) [56], heat shock protein HSP90 [57], and mitochondrial ribosomal protein L16 were also significantly decreased, together with the potent antioxidant MnSOD and FeSOD [58] (Figure 1). In contrast, several proteins were marginally decreased in the phb3 mutant (Figure 1), including the complex I subunit Nad9 [55], complex III subunit Cyt c1 [59], GDC-H (mitochondrial glycine decarboxylase complex) [60], IDH (isocitrate dehydrogenase) [61], and GR (glutathione reductase) [62]. This result indicates that mitochondrial function is impaired in the phb3 mutant.

The Loss of PHB3 Results in the Decrease in Mitochondrial ATP Synthase Activity
The main function of mitochondria is to generate energy via the mitochondrial respiratory chain [2,3]. Therefore, we investigated whether the loss of Arabidopsis PHB3 affects the function of the mitochondrial respiratory complex. Mitochondria were isolated from the wild-type and phb3 mutant seedlings grown in the dark for 12 days, as previously described [63]. The mitochondrial membrane complexes were solubilized with n-Dodecyl β-D-maltoside (β-DM) and then separated by blue native polyacrylamide gel electrophoresis (BN-PAGE), and the in-gel complex activity was analyzed [64]. Coomassie brilliant blue (CBB) staining showed an equal protein loading ( Figure 2E). The results showed that the activity of ATP synthase was decreased in the phb3 mutant compared with the wild-type ( Figure 2D). The F1-ATPase activity was also reduced ( Figure 2D), while the activities of complex I and complex IV were slightly decreased (Figure 2A,C). In addition, no significant differences in the activity of complex II between the phb3 mutant and wild-type were observed ( Figure 2B). These results indicate that the loss of function of PHB3 affects mitochondrial ATP synthase. Figure 1. The abundance of mitochondrial proteins in the phb3 mutant. Crude mitochondrial total proteins of the wild-type and the phb3 mutant were separated by SDS-PAGE and transferred to the polyvinylidene difluoride (PVDF) membrane. Western blotting analysis of total mitochondrial proteins with antibodies against various mitochondrial proteins. CI, complex I; CI-AF, complex I assembly factor; GLDH, l-galactone-1,4-lactone dehydrogenase; CIII, complex III; CIV, complex IV; ET, electron transport; Cyt c, cytochrome c; GDC-H, glycine decarboxylase-H protein; SHMT, serine hydroxymethyltransferase; TCA, tricarboxylic acid cycle; IDH, isocitrate dehydrogenase; HSP, heat shock protein; TP, transport pathway; VDAC1, voltage-dependent anion-selective channel protein 1; M-ribo, mitochondria ribosome protein; MnSOD, Mn superoxide dismutase; FeSOD, Fe superoxide dismutase; GR, glutathione reductase. The intensity value of the immune signals in the wildtype and the phb3 mutant are measured by ImageJ software (version 1.46r). The percentage of relative intensity value behind each group indicates the relative abundance of the phb3 mutant to the wild-type (phb3/WT). Thick arrows represent a more than 2-fold decrease in the abundance in the phb3 mutant, and thin arrows represent a 1.5-2 fold decrease. CBB (coomassie brilliant blue) staining gels were used as the sample loading control.

The Loss of PHB3 Results in the Decrease in Mitochondrial ATP Synthase Activity
The main function of mitochondria is to generate energy via the mitochondrial respiratory chain [2,3]. Therefore, we investigated whether the loss of Arabidopsis PHB3 affects the function of the mitochondrial respiratory complex. Mitochondria were isolated from the wild-type and phb3 mutant seedlings grown in the dark for 12 days, as previously described [63]. The mitochondrial membrane complexes were solubilized with n-Dodecyl β-D-maltoside (β-DM) and then separated by blue native polyacrylamide gel electrophoresis (BN-PAGE), and the in-gel complex activity was analyzed [64]. Coomassie brilliant blue (CBB) staining showed an equal protein loading ( Figure 2E). The results showed that the activity of ATP synthase was decreased in the phb3 mutant compared with the wildtype ( Figure 2D). The F1-ATPase activity was also reduced ( Figure 2D), while the activities of complex I and complex IV were slightly decreased (Figure 2A,C). In addition, no significant differences in the activity of complex II between the phb3 mutant and wild-type were observed ( Figure 2B). These results indicate that the loss of function of PHB3 affects mitochondrial ATP synthase. Figure 1. The abundance of mitochondrial proteins in the phb3 mutant. Crude mitochondrial total proteins of the wild-type and the phb3 mutant were separated by SDS-PAGE and transferred to the polyvinylidene difluoride (PVDF) membrane. Western blotting analysis of total mitochondrial proteins with antibodies against various mitochondrial proteins. CI, complex I; CI-AF, complex I assembly factor; GLDH, l-galactone-1,4-lactone dehydrogenase; CIII, complex III; CIV, complex IV; ET, electron transport; Cyt c, cytochrome c; GDC-H, glycine decarboxylase-H protein; SHMT, serine hydroxymethyltransferase; TCA, tricarboxylic acid cycle; IDH, isocitrate dehydrogenase; HSP, heat shock protein; TP, transport pathway; VDAC1, voltage-dependent anion-selective channel protein 1; M-ribo, mitochondria ribosome protein; MnSOD, Mn superoxide dismutase; FeSOD, Fe superoxide dismutase; GR, glutathione reductase. The intensity value of the immune signals in the wild-type and the phb3 mutant are measured by ImageJ software (version 1.46r). The percentage of relative intensity value behind each group indicates the relative abundance of the phb3 mutant to the wildtype (phb3/WT). Thick arrows represent a more than 2-fold decrease in the abundance in the phb3 mutant, and thin arrows represent a 1.5-2 fold decrease. CBB (coomassie brilliant blue) staining gels were used as the sample loading control.

PHB3 Is Essential for the Assembly of Mitochondrial ATP Synthase
The reduced activity of the mitochondrial ATP synthase in phb3 mutants might be caused by defects in its assembly. To test this possibility, we analyzed the abundance of the ATP synthase subunits in the blue native gels using Western blotting (WB). CBB staining gels were used as the sample loading control ( Figure 3A). Antibodies against the Fo-ATPase subunit a (Fo-ATPa), F1-ATPase subunit α (F1-ATPα), and subunit β (F1-ATPβ) were used. The result showed that the abundance of Fo-ATPase subunit a in ATP synthase monomer was decreased in the phb3 mutant compared with the wild-type, whereas the level of Fo-ATPase was increased ( Figure 3B). This result suggests that subunit a of Fo-ATPase cannot be efficiently assembled into the intact ATP synthase in the absence of PHB3. In contrast, the abundance of F1-ATPα and F1-ATPβ in ATP synthase did not change in the phb3 mutant, while both were significantly increased in F1-ATPase ( Figure 3C,D). These results indicate that the assembly process of ATP synthase is impaired in the phb3 mutant, probably due to the blocked assembly of the Fo-ATPase into ATP synthase. On the other hand, the PHB3 deficiency resulted in the decreased abundance of Fo-ATPa in ATP synthase ( Figure 3B) but not the abundance of F1-ATPα and F1-ATPβ ( Figure 3C,D), implying that the ATP synthase lacks subunit a in the phb3 mutant. Meanwhile, we detected the presence of complex I subunit CA2 and complex III 2 subunit Cyt c1, and their distribution remained unchanged between the phb3 mutant and wild-type ( Figure 3E,F). Together, these results suggest that PHB3 is involved in the assembly of ATP synthase.

PHB3 Is Essential for the Assembly of Mitochondrial ATP Synthase
The reduced activity of the mitochondrial ATP synthase in phb3 mutants might be caused by defects in its assembly. To test this possibility, we analyzed the abundance of the ATP synthase subunits in the blue native gels using Western blotting (WB). CBB staining gels were used as the sample loading control ( Figure 3A). Antibodies against the Fo-ATPase subunit a (Fo-ATPa), F1-ATPase subunit α (F1-ATPα), and subunit β (F1-ATPβ) were used. The result showed that the abundance of Fo-ATPase subunit a in ATP synthase monomer was decreased in the phb3 mutant compared with the wild-type, whereas the level of Fo-ATPase was increased ( Figure 3B). This result suggests that subunit a of Fo-ATPase cannot be efficiently assembled into the intact ATP synthase in the absence of PHB3. In contrast, the abundance of F1-ATPα and F1-ATPβ in ATP synthase did not change in the phb3 mutant, while both were significantly increased in F1-ATPase ( Figure  3C,D). These results indicate that the assembly process of ATP synthase is impaired in the phb3 mutant, probably due to the blocked assembly of the Fo-ATPase into ATP synthase. On the other hand, the PHB3 deficiency resulted in the decreased abundance of Fo-ATPa in ATP synthase ( Figure 3B) but not the abundance of F1-ATPα and F1-ATPβ ( Figure  3C,D), implying that the ATP synthase lacks subunit a in the phb3 mutant. Meanwhile, we detected the presence of complex I subunit CA2 and complex III2 subunit Cyt c1, and their distribution remained unchanged between the phb3 mutant and wild-type ( Figure 3E,F). Together, these results suggest that PHB3 is involved in the assembly of ATP synthase.

PHB3 Interacts with ATP Synthase Subunits
In yeast, mitochondrial ATP synthase assembly factors ATP11 and ATP12 can bind to the subunits β and α, respectively [29,30], promoting the formation of (αβ)3 hexamer in F1-ATPase [32]. In Arabidopsis, the homologs of ATP11 and ATP12 also interact with the corresponding subunits [41]. Our results show that the loss of the PHB3 function impairs the assembly and activity of the mitochondrial ATP synthase (Figures 2D and 3B). Therefore, it is reasonable to speculate that PHB3 might interact with some mitochondrial ATP synthase subunits. To test this notion, we first examined the interaction between PHB3

PHB3 Interacts with ATP Synthase Subunits
In yeast, mitochondrial ATP synthase assembly factors ATP11 and ATP12 can bind to the subunits β and α, respectively [29,30], promoting the formation of (αβ) 3 hexamer in F1-ATPase [32]. In Arabidopsis, the homologs of ATP11 and ATP12 also interact with the corresponding subunits [41]. Our results show that the loss of the PHB3 function impairs the assembly and activity of the mitochondrial ATP synthase (Figures 2D and 3B). Therefore, it is reasonable to speculate that PHB3 might interact with some mitochondrial ATP synthase subunits. To test this notion, we first examined the interaction between PHB3 and ATP synthase subunits using the yeast two-hybrid (Y2H) system. The open reading frame (ORF) sequences of Fo-ATPase subunits (ATPa, ATPc, ATPd, OSCP) and PHB3 were fused to the expression vector pGADT7 (AD). The ORF sequences of F1-ATPase subunits (ATPα, ATPβ, ATPγ, ATPδ, and ATPε) and PHB3 were cloned into the expression vector pGBKT7 (BD). The results showed that PHB3 interacted with subunits ATPβ and ATPδ of F1-ATPase in the Y2H system ( Figure 4A). We also performed the luciferase complementation imaging (LCI) assay in the Nicotiana benthamiana leaf epidermal cells. The result showed that the co-expression of nLUC-PHB3/cLUC-ATPβ and nLUC-PHB3/cLUC-ATPδ in tobacco leaves reconstituted strong luciferase activities, compared with the negative controls ( Figure 4B). These results indicate that PHB3 could interact with the F1-ATPase subunits β and δ in Arabidopsis.  In the Y2H system, PHB3 did not interact with subunit a ( Figure 4A). However, the absence of PHB3 seriously affected the assembly of Fo-ATPase subunit a, resulting in its accumulation in Fo-ATPase and reduction in ATP synthase ( Figure 3B). Therefore, it is possible that PHB3 affects the assembly of subunit a through its neighboring subunits, i.e., other Fo-ATPase subunits. Subunit a and the c-ring are attached in the mitochondrial inner membrane and form a proton translocating channel at their binding interface [65]. Therefore, we further tested the interaction between PHB3 and subunit c. The results showed that PHB3 could interact with subunit c in the LCI assay ( Figure 4B). These results imply that PHB3 directly binds to subunit c, which is required for the assembly between subunit a and the c-ring. When PHB3 is absent, subunit a either cannot bind to the c-ring or the binding is unstable. Consequently, it leads to the decreased abundance of subunit a in the In the Y2H system, PHB3 did not interact with subunit a ( Figure 4A). However, the absence of PHB3 seriously affected the assembly of Fo-ATPase subunit a, resulting in its accumulation in Fo-ATPase and reduction in ATP synthase ( Figure 3B). Therefore, it is possible that PHB3 affects the assembly of subunit a through its neighboring subunits, i.e., other Fo-ATPase subunits. Subunit a and the c-ring are attached in the mitochondrial inner membrane and form a proton translocating channel at their binding interface [65]. Therefore, we further tested the interaction between PHB3 and subunit c. The results showed that PHB3 could interact with subunit c in the LCI assay ( Figure 4B). These results imply that PHB3 directly binds to subunit c, which is required for the assembly between subunit a and the c-ring. When PHB3 is absent, subunit a either cannot bind to the c-ring or the binding is unstable. Consequently, it leads to the decreased abundance of subunit a in the ATP synthase monomer ( Figure 3B). In conclusion, PHB3 affects the assembly process of mitochondrial ATP synthase, possibly through direct binding to its subunits.

PHB3 Acts as an Assembly Factor in the Assembly of Mitochondrial ATP Synthase
PHB3 has been localized in the mitochondrion and nucleus [47,48]. Nuclear-localized PHB3 acts as a negative or positive co-regulator of transcription, affecting cell cycle and cell proliferation to regulate plant development [50][51][52][53]. However, its mitochondrial function remains unclear. In this study, we found that PHB3 may function as an assembly factor of the mitochondrial ATP synthase and is required for the assembly of subunit a from the Fo domain to ATP synthase ( Figure 5). Two pieces of evidence support this conclusion. First, our results showed that the loss of PHB3 blocked the assembly of the Fo domain into ATP synthase via subunit a ( Figure 3B) by interacting with its neighboring subunit c ( Figure 4B). In yeast, OXA1 is an assembly factor of mitochondrial ATP synthase, which connects with subunit c. In the oxa1 mutant, the c-ring combines with the F1 domain to form the F1-c-ring subcomplex, while a further assembly of the F1-c-ring with subunit a is limited [40]. This is consistent with the molecular phenotype of PHB3 deficiency in this study ( Figure 5). In addition, the function of the assembly factor of mitochondrial ATP synthase in yeast, INA22 (inner membrane assembly protein 22) [20], lends support to this conclusion as well. In yeast, INA22 is required for the combination of the c-ring and subunits a/8 by directly binding to these subunits. Mutation of INA22 leads to an accumulation of the assembly intermediate containing subunits a/8. Meanwhile, the subunit a is decreased in the monomer of ATP synthase [20]. Similarly, these changes were also found in the phb3 mutant using the immunoblotting assay with anti-ATPa ( Figure 3B). In addition, ATP23 and ATP10 mediate the assembly of subunit a into the Fo domain in the assembly of ATP synthase. The loss of ATP23 and ATP10 promotes the accumulation of the F1 domain and Fo-containing assembly intermediates but decreases the abundance of ATP synthase [36][37][38], which is analogous to the results observed in the phb3 mutant ( Figure 3B-D). Second, PHB3 is not present in the assembled ATP synthase, i.e., not a subunit of the mature holoenzyme. In Arabidopsis, PHB3 mainly forms a~1 MDa complex with other PHB proteins (PHB1, PHB2, PHB4, and PHB6), as shown in the mitochondrial complexome data [66]. PHB3 and PHB4 are also detected in the~1 MDa PHB complex by Western blotting using antibody PHB3/PHB4 [67], showing that the size of the PHB complex is larger than~620 kDa ATP synthase in Arabidopsis [66]. In that case, a small amount of PHB3 could comigrate with the partial ATP synthase subunits at~500 and~100 kDa positions corresponding to the bands in the blue native gels [66]. However, whether PHB3 is present in the assembly intermediates of ATP synthase requires further investigation.

The Loss of PHB3 Decreases the Activity of ATP Synthase and the F1 Domain
Several studies have shown that the loss of F1 domain assembly factors results in the decreased abundance of F1 domain in yeast, such as FMC1 [68,69]. On the contrary, we found that the loss of PHB3 results in the accumulation of the F1 domain and does not affect the assembly of the F1 domain into ATP synthase in Arabidopsis ( Figure 3C,D). Furthermore, we also found that the activity of the F1 domain is significantly reduced in the phb3 mutant ( Figure 2D). These results imply that PHB3 is mainly responsible for the activity of the F1 domain rather than its assembly. The phenomenon is probably due to the inactive conformation of the F1 domain subunits β and/or δ without the combination with PHB3 in the phb3 mutant. Correct folding and modification of F1 domain subunits α and β are essential for the activity of αβ heterodimer in Acetobacterium woodii [70].
Meanwhile, the activity of ATP synthase was almost undetectable in the phb3 mutant ( Figure 2D), indicating that the decreased activity of ATP synthase may be caused by the decreased activity of the F1 domain and equally by the missing subunit a in the ATP synthase monomer (Figures 3B and 5). In maize, unedited C residue (C635) on the atp6 gene

The Loss of PHB3 Decreases the Activity of ATP Synthase and the F1 Domain
Several studies have shown that the loss of F1 domain assembly factors results in the decreased abundance of F1 domain in yeast, such as FMC1 [68,69]. On the contrary, we found that the loss of PHB3 results in the accumulation of the F1 domain and does not affect the assembly of the F1 domain into ATP synthase in Arabidopsis ( Figure 3C,D). Furthermore, we also found that the activity of the F1 domain is significantly reduced in the phb3 mutant ( Figure 2D). These results imply that PHB3 is mainly responsible for the activity of the F1 domain rather than its assembly. The phenomenon is probably due to the inactive conformation of the F1 domain subunits β and/or δ without the combination with PHB3 in the phb3 mutant. Correct folding and modification of F1 domain subunits α and β are essential for the activity of αβ heterodimer in Acetobacterium woodii [70].
Meanwhile, the activity of ATP synthase was almost undetectable in the phb3 mutant ( Figure 2D), indicating that the decreased activity of ATP synthase may be caused by the decreased activity of the F1 domain and equally by the missing subunit a in the ATP synthase monomer (Figures 3B and 5). In maize, unedited C residue (C635) on the atp6 gene (encoding subunit a) transcript leads to an amino acid substitution and affects the assembly and activity of ATP synthase [71]. Similarly, the maize chimeric gene atp6c, encoding an abnormal ATPa protein with the disordered N-terminal arrangement, results in the decreased activity and abnormal assembly of ATP synthase [72]. These pieces of evidence suggest that subunit a is important for the ATP synthase activity. The severe reduction of ATP synthase activity in the phb3 mutant resulted from the deficiency of subunit a ( Figure 5).
In conclusion, we prove that PHB3 is not only involved in the assembly of the Fo domain to ATP synthase, but is also required for the activities of ATP synthase and the F1 domain in Arabidopsis.

Total RNA Extraction and cDNA Synthesis
Total RNA was extracted from wild-type seedlings according to the manufacturer's protocol of the RNeasy Plant Mini Kit (Vazyme Biotech, Nanjing, China). RNA was further digested by RNase-free DNase I (New England Biolabs, Rowley, MA, USA) to remove residual DNA contamination. The reverse transcription was used by the Transcript First-Strand cDNA Synthesis SuperMix (TransGen Biotech, Beijing, China).

Isolation of Mitochondria
Crude mitochondria of 12 day-old Arabidopsis seedlings that were grown on halfstrength MS medium in the dark were extracted as described previously [63]. Fresh seedling samples were gently ground on ice in an extraction buffer (0.3 M sucrose, 5 mM tetrasodium pyrophosphate, 10 mM KH 2 PO 4 , pH 7.5, 2 mM EDTA, 1% (w/v) polyvinylpyrrolidone 40, 1% (w/v) BSA, 5 mM cysteine, and 20 mM ascorbic acid). The homogenate was centrifuged at 3000× g for 5 min at 4 • C, and the supernatant was centrifuged once at 18,000× g for 30 min at 4 • C. The pellets were resuspended in wash buffer (0.3 M sucrose, 1 mM EGTA, 10 mM MOPS-KOH, pH 7.2). The protein concentration was determined using the Bradford method [73].

BN-PAGE
The mitochondrial proteins were solubilized with β-DM and then separated by blue native polyacrylamide gel electrophoresis (BN-PAGE) [76]. BN-PAGE using cathode buffer blue (with 0.02% coomassie Blue G-250 added) was performed at 4 • C in a vertical apparatus. Separation gels consisted of linear gradients of 3% to 12% or 4% to 16% polyacrylamide (Invitrogen, Carlsbad, CA, USA).

Western Blotting
Protein abundance was detected by Western blotting assay. BN-PAGE gels were first treated with denaturation buffer (1% SDS, 50 mM Tris-HCl, 0.05% β-mercaptoethanol) for 30 min. For Western blotting analysis, mitochondrial proteins were transferred onto PVDF membranes (0.45 mm; Millipore, Burlington, MA, USA). The PVDF membranes were incubated with various primary antibodies against wheat Nad9, maize ATP α, Arabidopsis COX2, yeast cyt c1, and pigeon cyt c as described previously [77,78]. Antibodies against maize A5, V1, COX3, ATPβ and ATPa were prepared in our laboratory. The rest of the primary antibodies were purchased from Agrisera company (Agrisera AB, Vännäs, Sweden). Most of these antibodies are reactive to Arabidopsis, except chlamydomonas CA2 and maize GLDH. Signal detection was carried out by ECL reagents (Thermo Fisher Scientific, Waltham, MA, USA) after incubation with the horseradish peroxidase (HRP)-conjugated secondary antibody.

Yeast Two-Hybrid Assay
The open reading frame (ORF) sequences of PHB3, ATPa, ATPc, ATPd, and OSCP were added to the expression vector pGADT7 (AD, prey). The sequences of PHB3, ATPα, ATPβ, ATPγ, ATPδ, and ATPε were cloned into the expression vector pGBKT7 (BD, bait). The resulting bait plasmids were cotransformed with prey plasmids into the yeast strains Y2H Gold (containing HIS3, ADE2, and lacZ as reporters), following the lithium acetate (LiAc)mediated method. Transformants were grown on synthetically defined DDO medium minus Leu and Trp. The strains were screened by QDO media (lacking Leu, Trp, His, and Ade) and QDO/X-α-Gal plates with X-α-Gal. Cells transformed with the clones of p53 (pGBKT7-p53)/T-antigen (pGADT7-T) and parental empty pGBKT7/pGADT7 were used as positive and negative controls, respectively. The primers used are listed in Supplementary  Table S1.

LCI Assay
The coding sequences of PHB3 and genes encoding ATP synthase subunits (ATPc, ATPβ, and ATPδ) were cloned into vectors JW771 (nLUC) and JW772 (cLUC), respectively. The nLUC-and cLUC-related constructs were transformed into the Agrobacterium tumefaciens strain EHA105. Then, we mixed the agrobacterium suspensions containing the nLUC fusion and cLUC fusion in a 1:1 ratio. Both the nLUC-and cLUC-fused proteins were co-infiltrated into N. benthamiana leaves. After infiltration for 48 h, the leaves were soaked with 1 mM Luciferin for 10 min before imaging. The primers used are listed in Supplementary Table S1.

Conclusions
In this study, we uncovered the new roles of PHB3 in Arabidopsis mitochondria. PHB3 could interact with subunits β and δ of F1-ATPase and subunit c of Fo-ATPase. In-gel activity staining assay showed that the loss of function of PHB3 reduced the activities of ATP synthase and F1-ATPase. In the phb3 mutant, the abundance of Fo-ATPase subunit a in ATP synthase monomer was decreased, while Fo-ATPase was accumulated. Meanwhile, when PHB3 was absent, the abundance of F1-ATPase subunits α and β was not decreased in ATP synthase, and both of them were significantly increased in F1-ATPase. These results implied that the loss of PHB3 causes the subunit a of Fo-ATPase cannot be further assembled into the intact ATP synthase. Overall, the above results of this study demonstrated that PHB3 was required for the assembly and activity of mitochondrial ATP synthase in Arabidopsis.