Volume 6, Issue 2, Pages 503-513 (March 2013) Requirement and Functional Redundancy of Ib Subgroup bHLH Proteins for Iron Deficiency Responses and Uptake in Arabidopsis thaliana  Ning Wang, Yan Cui, Yi Liu, Huajie Fan, Juan Du, Zongan Huang, Youxi Yuan, Huilan Wu, Hong-Qing Ling  Molecular Plant  Volume 6, Issue 2, Pages 503-513 (March 2013) DOI: 10.1093/mp/sss089 Copyright © 2013 The Authors. All rights reserved. Terms and Conditions

Figure 1 Protein–Protein Interaction Assay of AtbHLH100 and AtbHLH101 with FIT by Yeast Two-Hybrid and Bimolecular Fluorescence Complementary Analysis.(A) The protein–protein interaction assay of AtbHLH100 and AtbHLH101 with FIT via yeast two-hybrid. The coding sequences of AtbHLH100, AtbHLH101, and FIT were fused to the GAL4–AD or GAL4–BD vectors to construct the yeast expression plasmids pAD–FIT, pBD–AtbHLH100, and pBD–AtbHLH101, respectively. The plasmids were differently combined, and transformed into yeast YRG-2 cells. The transformants were subjected to auxotrophic screening and filter-shift assay. The yeast cells containing pAD–FIT and with either pBD–AtbHLH100 or pBD–AtbHLH101 grew well on the selective medium (SD/-Leu/-Trp/-His) and the corresponding yeast clones showed β-galactosidase activity in the filter-lift assay. The cells containing pAD–FIT with either pBD–AtbHLH38 or pBD–AtbHLH39 were used as positive controls, and the cells with the plasmid combination of pAD–FIT and pBD–AtbHLH40 was used as the negative control (Yuan et al., 2008).(B) Bimolecular fluorescence complementary assay (BiFC) of AtbHLH100 and AtbHLH101 with FIT in the leaf mesophyll protoplasts of Arabidopsis. The coding sequences of AtbHLH100, AtbHLH101, and FIT were fused to the vectors of nYFP and cCFP to generate transient expression plasmids pAtbHLH100–nYFP, pAtbHLH100–cCFP, pAtbHLH101–nYFP, pAtbHLH101–cCFP, pFIT–nYFP, and pFIT–cCFP, respectively. The Arabidopsis mesophyll protoplasts were transformed with different combinations of the plasmids and investigated under a confocol microscope after incubation of 16–20h. The protoplasts transformed with pFIT–nYFP and pAtbHLH100–cCFP or pAtbHLH101–cCFP together displayed green fluorescence in the nucleus according to the co-localization of the 4’,6-diamido-2-phenylindole (DAPI) staining and GFP fluorescence. Molecular Plant 2013 6, 503-513DOI: (10.1093/mp/sss089) Copyright © 2013 The Authors. All rights reserved. Terms and Conditions

Figure 2 Transcription Activation Assay of GUS Reporter Gene Driven by FRO2 and IRT1 Promoters via Transcription Factor FIT, AtbHLH100, and AtbHLH101 in Yeast Cells. (A) GUS staining of the yeast strains carrying different plasmid combinations shifted from SD/-Leu/-Trp agar plate.(B, C) Quantification of the GUS activity of the yeast strains. The numbers 1–24 indicate the plasmid combinations as follows: (1) pAD/pBD–PFRO2:GUS, (2) pAD–FIT/pBD–PFRO2:GUS, (3) pAD–AtbHLH100/pBD–PFRO2:GUS, (4) pAD–AtbHLH101/pBD–PFRO2:GUS, (5) pAD–AtbHLH40/pBD–PFRO2:GUS, (6) pAD/pBD–FIT-PFRO2:GUS, (7) pAD–FIT/pBD–FIT-PFRO2:GUS, (8) pAD–AtbHLH100/pBD–FIT-PFRO2:GUS, (9) pAD–AtbHLH101/pBD–FIT-PFRO2:GUS, (10) pAD–AtbHLH40/pBD–FIT-PFRO2:GUS, (11) pAD/pBD–PIRT1:GUS, (12) pAD–FIT/pBD–PIRT1:GUS, (13) pAD–AtbHLH100/pBD–PIRT1:GUS, (14) pAD–AtbHLH101/pBD–PIRT1:GUS, (15) pAD–AtbHLH40/pBD–PIRT1:GUS, (16) pAD/pBD–FIT-PIRT1:GUS, (17) pAD–FIT/pBD–FIT-PIRT1:GUS, (18) pAD–AtbHLH100/pBD–FIT-PIRT1:GUS, (19) pAD–AtbHLH101/pBD–FIT-PIRT1:GUS, (20) pAD–AtbHLH40/pBD–FIT-PIRT1:GUS, (21) pAD–AtbHLH38/pBD–FIT-PFRO2:GUS, (22) pAD–AtbHLH39/pBD–FIT-PFRO2:GUS, (23) pAD–AtbHLH38/pBD–FIT-PIRT1:GUS, (24) pAD–AtbHLH39/pBD–FIT-PIRT1:GUS. (1)–(9) are assaying for transcriptional activation of FRO2. (10) is a negative control, (21) and (22) are the positive control (Yuan et al., 2008). (11)–(19) are assaying for transcriptional activation of IRT1. (20) is a negative control, (23) and (24) are positive control (Yuan et al., 2008). * and ** indicate the Student’s t-test, with p<0.05 and p<0.01, respectively. Molecular Plant 2013 6, 503-513DOI: (10.1093/mp/sss089) Copyright © 2013 The Authors. All rights reserved. Terms and Conditions

Figure 3 Phenotype, Iron Content, and Gene Expression Profiles of the Plants Overexpressing AtbHLH100, AtbHLH101, FIT/AtbHLH100, and FIT/AtbHLH101.(A) Phenotype of the overexpression lines on one-half-strength MS agar plate without iron supply for 4d.(B, C) Iron contents in shoots of the overexpression lines grown on agar plates without (B) and with (C) iron supply for 7d. Values shown in the figure are means of three biological repeats.(D, E) Expression profiles of IRT1 (D) and FRO2 (E) in the roots of the overexpression lines treated on one-half-strength MS agar plate with (+Fe) and without (–Fe) iron supply for 7d by multiplex RT–PCR analysis. The expression data were normalized by comparison to the expression of actin8.(F) Determination of ferric–chelate reductase activity in roots of the overexpression lines under iron-deficiency conditions.(G) Detection of IRT1 protein accumulation in roots of the overexpression lines grown under the conditions with (+R) and without (–R) iron supply for 7d. * and ** indicate the Student’s t-test, with p<0.05 and p<0.01, respectively, compared to wild-type (WT). Molecular Plant 2013 6, 503-513DOI: (10.1093/mp/sss089) Copyright © 2013 The Authors. All rights reserved. Terms and Conditions

Figure 4 Phenotype, Iron Contents, Ferric–Chelate Reductase Activity, and Gene Expression Profiles in the Double Knockout Mutants of AtbHLH38, AtbHLH39, AtbHLH100, and AtbHLH101.(A) Phenotype of the seedlings grown in vermiculites (upper part) and on one-half-strength MS agar plate without iron supply (lower part). (B) Fe content in the shoots of seedlings grown on one-half-strength MS agar plate without (–Fe) and with (+Fe) iron supply for 14d. (C) Expression of IRT1 and FRO2 in the roots. Different letters indicate significant difference at the level of p<0.05.(D) Ferric–chelate reductase activity in the roots of seedlings grown on one-half-strength MS agar plate without (–Fe) and with (+Fe) iron supply.(E) Western blot analysis of IRT1 protein accumulation in the roots of seedlings grown under iron-sufficient (+R) and -deficient (–R) conditions. WT, wild-type. * and ** indicate the Student’s t-test, with p<0.05 and p<0.01, respectively, compared to WT. Molecular Plant 2013 6, 503-513DOI: (10.1093/mp/sss089) Copyright © 2013 The Authors. All rights reserved. Terms and Conditions

Figure 5 Phenotype, Iron Contents, Ferric–Chelate Reductase Acti vity, and Gene Expression Profiles in the Triple Knockout Mutants of AtbHLH38, AtbHLH39, AtbHLH100, and AtbHLH101. (A) Phenotype of the seedlings grown in vermiculites (upper part) and on one-half-strength MS agar plate without iron supply (lower part).(B) Fe content in the shoots of seedlings grown on one-half-strength MS agar plate without (–Fe) and with (+Fe) iron supply for 14d.(C) Expression profiles of IRT1 and FRO2 in the roots.(D) Ferric–chelate reductase activity in the roots of seedlings grown on one-half-strength MS agar plate without (–Fe) and with (+Fe) iron supply.(E) Western blot analysis of the IRT1 protein accumulation in the roots of the seedlings treated by iron sufficiency (+R) and deficiency (–R) for 7d. fit1-2 is the null mutant of FIT. Different letters indicate significant difference at the level of p<0.05. Molecular Plant 2013 6, 503-513DOI: (10.1093/mp/sss089) Copyright © 2013 The Authors. All rights reserved. Terms and Conditions