1. Volume 7, Issue 5, Pages 856-873 (May 2014)
Perturbation of Auxin Homeostasis Caused by Mitochondrial FtSH4 Gene-Mediated Peroxidase Accumulation Regulates Arabidopsis Architecture 
Zhang Shengchun , Wu Juelin , Yuan Dongke , Zhang Daowei , Huang Zhigang , Xiao Langtao , Yang Chengwei  
Molecular Plant 
Volume 7, Issue 5, Pages (May 2014)
DOI: /mp/ssu006
Copyright © 2014 The Authors. All rights reserved. Terms and Conditions

2. Figure 1 The Phenotype of mas Mutant Plants.
(A) Two-week-old seedlings. Bar = 1cm.
(B) Four-week-old plants.
(C) Six-week-old plants. Bar = 2cm.
(D) Eleven-week-old plants. Left, wild-type plant; right, mas mutant plants. Bar = 2cm.
(E) Axillary branches (tillers) from the rosette leaves of 11-week-old mas plants. Main stem, the main inflorescence stems with axillary shoots from cauline leaves.
(F) Cauline leaves show more than one axillary bud in mas plants.
(G) The average terminal height of mas mutant plants.
(H) The average axillary branch number of the mas plants from the rosette leaves.
(I) The number of secondary axillary branches present on mas cauline leaves. (G–I), week-old plants were analyzed for each of three biological replicates.
Molecular Plant 2014 7, DOI: ( /mp/ssu006)
Copyright © 2014 The Authors. All rights reserved. Terms and Conditions

3. Figure 2 Identification of mas Mutant Plants.
(A) The localization of the T-DNA inserted in the genomic DNA. The light gray line and black line before the ATG is the promoter region, the black box is the exon, and the dark gray line is the intron. The left primers (LP) and right primers (RP) are the genomic primers used for verification.
(B) Genomic DNA verification by PCR with three primers. Lanes 1 and 4: left border (LB) and genomic LP; lanes 2 and 5: LB and genomic RP; lanes 3 and 6: LP and RP. The positions of LP and RP are shown in (A).
(C)FtSH4 gene completely reverses the ftsh4-4 mutant plant phenotypes. Top, photographs of 7-week-old plants. 1#, 2#, and 3# indicate ftsh4-4 plants transformed with pFtSH4: FtSH4 constructs. Bottom, real-time PCR verification of the FtSH4 gene expression in different genotype plants in the top. For real-time PCR, three technical replicates were performed for each of three biological replicates.
(D) Protein expression verification of the ftsh4-4 mutant and complementation plants by Western blot. C1 indicates the 1# line complementation plants.
(E) AAA-domain is necessary for FtSH4 function in plant growth and development. Photograph of the 4-week-old different ftsh4-4 mutant plants.
(F) Axillary branch number of the different ftsh4-4 mutant plants.
Molecular Plant 2014 7, DOI: ( /mp/ssu006)
Copyright © 2014 The Authors. All rights reserved. Terms and Conditions

4. Figure 3 Expression Patterns of FtSH4 Gene.
(A)FtSH4 expressed stably in different growth stages detected by real-time PCR. The numbers 1–5 indicate 1–5-week-old wild-type plants.
(B)FtSH4 expressed in different tissues detected by real-time PCR. Seedling and root are from 15-day-old plants, and the other tissues were from 35-day-old plants. For real-time PCR in (A) and (B), three technical replicates were performed for each of three biological replicates.
(C)pFtSH4:GUS expressed in the 5-day-old seedling cotyledons, shoot apex, and hypocotyls.
(D)pFtSH4:GUS expressed in the 15-day-old seedling rosette leaves.
(E)pFtSH4:GUS expressed in the branching sites and branches of the 25-day-old seedlings on MS plates.
(F)pFtSH4:GUS expressed in the branching sites and branches from the rosette axillary buds of 35-day-old seedlings. Red arrows in (E) and (F) indicate the branching sites and branches.
Molecular Plant 2014 7, DOI: ( /mp/ssu006)
Copyright © 2014 The Authors. All rights reserved. Terms and Conditions

5. Figure 4 Excessive Branch Production Is Inhibited by Exogenous Auxin.
(A) Six-week-old plants sprayed with 5 μM NAA every 4 d from 3 weeks old for a further 3 weeks.
(B) IAA levels were determined in the 7-day-old and 4-week-old wild-type plants, homozygous ftsh4-4 mutant plants, and pFtSH4: FtSH4-ftsh4-4 plants. Data are means of three biological repetitions ± SE.
(C) Auxin synthesis gene iaaM overexpression completely reverses the ftsh4-4 mutant phenotypes. The iaaM-ftsh4-4–5 indicates the fifth-line plants that overexpressed iaaM in the ftsh4-4 mutant. All plants are 35 d old. Bar = 2cm.
Molecular Plant 2014 7, DOI: ( /mp/ssu006)
Copyright © 2014 The Authors. All rights reserved. Terms and Conditions

6. Figure 5
Auxin Reporter ProDR5:GUS Gene Expression Changed in the ftsh4-4 Inflorescence Stem.
(A) Localization of the ProDR5:GUS activity in the 7-day-old wild-type Col-0 and in ftsh4-4 cotyledon, hypocotyl, root, root apex, and the 6-week-old inflorescence stems and whole plants.
(B, C) The ftsh4-4 mutant plants are less susceptible to the de novo shoot induction system. Explants or callus from the wild-type or ftsh4-4 mutant plants in the callus-inducing medium (B) or the shoot-inducing medium (C).
(D) GUS activity of ProDR5:GUS plants inflorescence stems measured by a MUG assay. MUG, 4-methylumbelliferyl β-D-glucuronide; three technical replicates were performed for each of three biological replicates.
Molecular Plant 2014 7, DOI: ( /mp/ssu006)
Copyright © 2014 The Authors. All rights reserved. Terms and Conditions

7. Figure 6
Auxin Synthesis and Signaling Gene Transcript Levels in ftsh4-4 Mutants Compared to Wild-Type Plants.
(A) IAA biosynthesis gene transcript levels did not significantly change in ftsh4-4 compared to wild-type plants.
(B) Aux/IAA gene transcript levels were down-regulated in ftsh4-4 mutant plants.
(C) GH3 gene transcript levels did not change in ftsh4-4 mutants. Three technical replicates were performed for each of three biological replicates.
Molecular Plant 2014 7, DOI: ( /mp/ssu006)
Copyright © 2014 The Authors. All rights reserved. Terms and Conditions

8. Figure 7
Exogenous Antioxidants Restore the Branching Phenotypes of ftsh4-4 Mutant Plants.
(A) AsA can rescue the ftsh4-4 mutant plant branching phenotype. Eight-week-old plants were photographed, and these plants were sprayed by 1mM AsA solution every 3 d from 3 weeks old for a further 2 weeks. There were 32 plants analyzed for each of three biological replicates.
(B) The abundance of the FtSH4 gene transcripts was not affected by H2O2 or antioxidants. The 15-day-old seedlings treated by water with or without 1 mM H2O2, 1 mM AsA, or 1mM GSH respectively for 3h. AsA, ascorbic acid; GSH, reduced glutathione.
(C) H2O2 concentration increased significantly in mas mutant plants from the third week onwards compared to wild-type plants.
(D) H2O2 concentration increased significantly in the ftsh4-4 mutant plants, and restored to normal levels by FtSH4 expression. The 21-day-old plants were stained by DAB solution. Bar = 2cm. Three technical replicates were performed for each of three biological replicates.
Molecular Plant 2014 7, DOI: ( /mp/ssu006)
Copyright © 2014 The Authors. All rights reserved. Terms and Conditions

9. Figure 8
FtSH4 Regulates the Plant Development through Peroxidase Metabolism.
(A)Real-time PCR for determining expression of the four peroxidase genes in wild-type and ftsh4-4 mutant plants. Four-week-old plants were analyzed, and three technical replicates were performed for each of three biological replicates.
(B) Peroxidase isozyme content increased significantly in the ftsh4-4 mutant plants, but was reduced by PRX34 and PRX33 knockdown. Top: phenotypes of plants used for peroxidase assays; middle: PAGE assay and benzidine staining for peroxidase content of each plant shown in the top panel; bottom: Coomassie blue-stained gel for the peroxidase isozyme PAGE assay of the wild-type and mutant plants is shown.
(C)PRX34 and PRX33 knockdown reduced the ftsh4-4 dwarfism.
(D)PRX34 and PRX33 knockdown reduced the number of ftsh4-4 axillary branches. The 11-week-old plants were analyzed, and 32 plants measured for each of three biological replicates in (C) and (D).
(E) IAA content increased significantly in the ftsh4-4 mutant plants by PRX34 and PRX33 knockdown expression. Four-week-old plants were analyzed, and three technical replicates were performed for each of three biological replicates.
Molecular Plant 2014 7, DOI: ( /mp/ssu006)
Copyright © 2014 The Authors. All rights reserved. Terms and Conditions

10. Figure 9
Exogenous IAA Reverses the H2O2 Effects on the ftsh4-4 Mutant Plants.
(A) A low concentration of H2O2 (0.1mM) shortened the ftsh4-4 mutant plant hypocotyls (middle), and the low concentrations of IAA (0.01 μM) can reverse phenotype changes (bottom). The asterisk indicates significant differences compared with control plants (P < 0.005).
(B) Hypocotyls length statistics of the seedlings in (A).
(C) Exogenous IAA did not decrease the peroxidase level in the ftsh4-4 mutant. Five-week-old plants, sprayed with 1mM H2O2 or 5 μM IAA every 3 d from 3 weeks old for a further 2 weeks, were analyzed.
Molecular Plant 2014 7, DOI: ( /mp/ssu006)
Copyright © 2014 The Authors. All rights reserved. Terms and Conditions

11. Figure 10 Simplified Model of FtSH4 Function.
Loss of FtSH4 function resulted in a reduction in protein stability in the mitochondria, and led to a H2O2 rise. High levels of H2O2 induced high levels of peroxidase production, which may function as an IAA oxidase. Increased peroxidase resulted in free IAA degradation and auxin transport and signaling perturbation, which promote the production of excessive axillary branches and dwarfism phenotypes. IM, inner membrane; OM, outer membrane; Prxs, peroxidases.
Molecular Plant 2014 7, DOI: ( /mp/ssu006)
Copyright © 2014 The Authors. All rights reserved. Terms and Conditions