Volume 1, Issue 4, Pages 675-685 (July 2008) A Mitochondrial Magnesium Transporter Functions in Arabidopsis Pollen Development  Li Le-Gong , Sokolov Lubomir N. , Yang Yong-Hua , Li Dong-Ping , Ting Julie , Pandy Girdhar K. , Luan Sheng   Molecular Plant  Volume 1, Issue 4, Pages 675-685 (July 2008) DOI: 10.1093/mp/ssn031 Copyright © 2008 The Authors. All rights reserved. Terms and Conditions

Figure 1 Complementation of MM281 Mutant by AtMGT5 and AtMGT10. (A) Complementation on solid medium. Growth of different bacterial cells on the N-minimal medium (without Tris-base) containing 10 uM, 100 uM, 1 mM, 1.5 mM, 3 mM, 10 mM MgSO4. The cells include MM281 transformed with AtMGT10 in pTrc99A vector as a positive control (1), MM281 transformed with AtMGT5 cDNA in pTrc99A vector (2), and MM281 transformed with pTrc99A vector only as a negative control (3). (B) Growth in liquid medium. The bacterial cells shown in Figure 1A were cultured in the N-minimal medium containing 100 uM MgSO4. Aliquots of the cultures were taken at different time points to measure the cell density that was used to plot a growth curve. Results are averages (± standard errors) for three independent cultures. pTrc99A, AtMGT5 and AtMGT10 are indicated by square, circle and triangle, respectively. Molecular Plant 2008 1, 675-685DOI: (10.1093/mp/ssn031) Copyright © 2008 The Authors. All rights reserved. Terms and Conditions

Figure 2 Mg2+ Transport Measured by Inhibition of 63Ni2+ Uptake. (A) Mg2+ inhibition of 63Ni2+ uptake. (B) Ni2+ inhibition of 63Ni2+ uptake. Uptake assay was performed using three Salmonella strains described in Figure 1. Tracer is presented as the per cent of the maximal uptake by each strain. Uptake was measured for 30 min at each cation concentration. Data are average values of three independent experiments, each made in triplicate, and presented as mean ± SE. Molecular Plant 2008 1, 675-685DOI: (10.1093/mp/ssn031) Copyright © 2008 The Authors. All rights reserved. Terms and Conditions

Figure 3 AtMGT5 Mediates Mg Efflux in a Concentration-Dependent Manner. (A) 63Ni2+ efflux induced by different concentrations of Mg2+. (B) Time course of 63Ni2+ efflux. Efflux assay was performed using the same Salmonella strains described in Figure 2. After maximal uptake was done, the cells were transferred into a buffer containing different concentrations of Mg2+ (0.1, 1, and 10 mM). Efflux was measured after 30 min incubation at each Mg2+ concentration. Time course was measured at the same concentration of 10 mM Mg2+. Data are average values of three independent experiments and presented as mean ± SE. Molecular Plant 2008 1, 675-685DOI: (10.1093/mp/ssn031) Copyright © 2008 The Authors. All rights reserved. Terms and Conditions

Figure 4 The AtMGT5 Protein Is Localized in the Mitochondria. (A) and (B) show confocal images of root cells carrying AtMGT5::GFP (green) scanned with 1-min intervals. (C–E) AtMGT5 fused to GFP reporter protein (green) targets mitochondria. The GFP (in (C)) and mitochondria-specific dye (Mitotracker, in (D)) are completely overlapping as shown in (E). Scale bar: 10 μm. Molecular Plant 2008 1, 675-685DOI: (10.1093/mp/ssn031) Copyright © 2008 The Authors. All rights reserved. Terms and Conditions

Figure 5 AtMGT5 Is an Anther-Specific Gene and Expressed at the Early Stage of Flower Development. (A) RT–PCR analysis of AtMGT5 transcripts in different floral organs and in flower buds of different stages. Using specific primers for AtMGT5 gene, RT–PCR were performed with total RNA from flower buds of different stages (a) and from different floral organs (b). Lane M, MW markers. Lanes 1–8 in (a), RNA isolated from central flower bud cluster I (1), green bud I (2), green bud II (3), Open flower I (4), Open flower II (5), Stigmata (6), Side flower bud cluster (7), and central flower bud cluster II (8); Lanes 1–6 in (b): Sepal (1), Petal (2), Anther (3), Ovary (4), flower base (5), and silique (6). Primers for the AtACTIN3 gene were used to monitor the cDNA yield and integrity in the samples. (B) AtMGT5 promoter activity in transgenic Arabidopsis plants. GUS histochemical staining in a flower bud cluster from a plant transformed with AtMGT5 promoter-GUS. (a) and (b), GUS activity (indicated by blue color) was observed in young flower buds but was weak or undetectable in open flowers. (c) and (d), GUS staining was only seen in the anther and was absent in the other flower organs. (e) Flower cluster with strong GUS staining at flower stages 9–13. (f–k) Cross and longitudinal sectioning of flowers at various stages revealed specific AtMGT5 promoter/GUS activity in the anther, with distinctive spatial and temporal pattern during pollen developmental stages 5–13 as per Sanders et al. (1999). Stage 5 is the earliest time AtMGT5 promoter activity was detectable. Indicated in the figure with arrows: V, vascular tissue; C, connective; T, tapetum. Bright-field microcopy is used for the images in (B), with the exception of (d) and (k), where Nomarski optics and differential interference contrast (DIC) were applied. Molecular Plant 2008 1, 675-685DOI: (10.1093/mp/ssn031) Copyright © 2008 The Authors. All rights reserved. Terms and Conditions

Figure 6 Phenotypic Analysis of Pollen Grains from Control and Two Independent Alleles of AtMGT5 Mutant Plants. (A) Scheme of AtMGT5 gene structure and positions of the T-DNA insertions. The ATG start codon and TGA stop codon are indicated. The T-DNA insertions are shown as triangles above the gene diagram (a). The size of PCR fragments confirmed the insertion positions (b). (B) Bright-field microscopy images of wild-type (a), and mature pollen grains from T-DNA insertion line in the AtMGT5 gene (b). Approximately 50% of the mature pollens from the AtMGT5/Atmgt5 heterozygous plants were aborted. Two independent T-DNA insertion lines reveal similar phenotype. DAPI staining of 3-nuclear pollen is shown in (c). The cells lacking the typical dotted pattern for the presence of DAPI-stained nuclei have undergone cell death and DNA degradation. (C) Environmental scanning electron microscopy. Whole anther structure at the dehiscence/senescence transition stage Col-0 (a) and AtMGT5/Atmgt5 heterozygous mutant (b, c). The bars is 50 mm in (a) and the scale is the same in (a–c). Higher magnification images reveal healthy WT (d) and about 50% aborted pollen grains in the AtMGT5/Atmgt5 heterozygous mutant (e, f). Scale bars are 10 mm in (d) and (f). Molecular Plant 2008 1, 675-685DOI: (10.1093/mp/ssn031) Copyright © 2008 The Authors. All rights reserved. Terms and Conditions