Volume 7, Issue 1, Pages 170-186 (January 2014) The Plastid-Localized NAD-Dependent Malate Dehydrogenase Is Crucial for Energy Homeostasis in Developing Arabidopsis thaliana Seeds  Jennifer Selinski, Nicolas König, Benedikt Wellmeyer, Guy T. Hanke, Vera Linke, H. Ekkehard Neuhaus, Renate Scheibe  Molecular Plant  Volume 7, Issue 1, Pages 170-186 (January 2014) DOI: 10.1093/mp/sst151 Copyright © 2013 The Authors. All rights reserved. Terms and Conditions

Figure 1 Subcellular Localization of plNAD–MDH. Protoplasts were isolated from the mature leaves of Arabidopsis plants and transformed using a plNAD–MDH–GFP fusion construct to identify subcellular localization of plNAD–MDH. Chlorophyll autofluorescence marks the areas filled with thylakoids in the mesophyll plastids. Scale bars were calibrated to 10 μm. Molecular Plant 2014 7, 170-186DOI: (10.1093/mp/sst151) Copyright © 2013 The Authors. All rights reserved. Terms and Conditions

Figure 2 Histochemical Localization of GUS Expression under the Control of the plNAD–MDH Promoter in Arabidopsis Seedlings and Mature Plants. (A–G) GUS expression in plNAD–MDH-promoter::GUS plants. GUS staining of seedlings at 1 d (A), 2 d (B), and 7 d (C), as well as after 2 weeks (D), 4 weeks (E) after germination, and a flowering plant (F) are shown. Scale bars were calibrated to 1mm (A–C) and 2mm (D–F). Molecular Plant 2014 7, 170-186DOI: (10.1093/mp/sst151) Copyright © 2013 The Authors. All rights reserved. Terms and Conditions

Figure 3 Molecular Characterization of Heterozygous plnad–mdh/plNAD–MDH Knockout Mutants. (A) Analysis of the plnad–mdh-T-DNA-insertion line ET8629 (designated line 159). The insertion in line 159 is localized at the 3′ end of the gene. The positions of the primers used for PCR analysis are marked by arrows. (B) PCR analysis on genomic DNA of wild-type (WT), heterozygous plnad–mdh/plNAD–MDH mutants (159), and complemented homozygous plnad–mdh knockout plants (Compl.). The detection of the WT gene by primer combination NAD–MDHdsF and NAD–MDHdsR (P1) or plNAD–MDH Intron for and NAD–MDHdsR (P3; 1412bp), for the T-DNA insertion by primer combination simorowski Ds5-1 and NAD–MDHdsR (P2) and for the construct of complementation by primer combination NADMDHrtR and pGPTV S (P4). (C) Transcript level of plNAD–MDH in line 159 normalized to RAN3 analyzed by qRT–PCR. Asterisks indicate that the differences (** P < 0.01) between WT and line 159 are statistically significant as determined by t-test. Molecular Plant 2014 7, 170-186DOI: (10.1093/mp/sst151) Copyright © 2013 The Authors. All rights reserved. Terms and Conditions

Figure 4 Phenotype of Line 159. Comparison of different developmental stages of wild-type (WT) and plnad–mdh/plNAD–MDH plants (159) grown on soil (A). Lipid (B), starch (C), and protein (D) contents of mature seeds were determined. Scale bars were calibrated to 2cm. Molecular Plant 2014 7, 170-186DOI: (10.1093/mp/sst151) Copyright © 2013 The Authors. All rights reserved. Terms and Conditions

Figure 5 Autotrophic Growth of A. thaliana Wild-Type (WT) and Heterozygous plnad–mdh/plNAD–MDH Knockout Plants (159) after 6 Weeks and after 3 Months on 2.5mM ammonium sulfate or 5mM potassium nitrate (A). Transcript levels of plNAD–MDH, NTT1, NTT2, GAPCP1, GAPCP2, and GLT1 in line 159 were analyzed by qRT–PCR in leaves of 2-week-old plants grown on half-strength MS medium (B). The dashed line represents WT transcript levels. Asterisks (* P < 0.05; ** P < 0.01) and crosses († P < 0.1) indicate that the differences between WT and line 159 are statistically significant as determined by t-test. Scale bars were calibrated to 1cm. Molecular Plant 2014 7, 170-186DOI: (10.1093/mp/sst151) Copyright © 2013 The Authors. All rights reserved. Terms and Conditions

Figure 6 Expression of plNAD–MDH and related genes in Leaves of A. thaliana Mutant Lines. (A) Pathways for energy provision in plastids during dark metabolism. (B) Transcript abundance of plNAD–MDH, NTT1, NTT2, GAPCP1, GAPCP2, and GLT1 in various knockout lines, measured by qRT–PCR. Leaves of wild-type (WT), ntt1/2 double mutants, gapcp1/2 double mutants, nadh-gogat knockout plants (glt1), and starchless mutants (pgm) were harvested after 8 weeks (gapcp1/2) or 2 weeks for all other lines. mRNA was extracted and converted to cDNA by reversed transcription. Gene-specific primers were chosen to amplify transcripts of different genes responsible for energy supply in dark plastids. The dashed line represents WT transcript levels. Asterisks (* P < 0.05; ** P < 0.01; *** P < 0.001) and crosses († P < 0.1) indicate that the differences between WT and different knockout mutants are statistically significant as determined by t-test. Molecular Plant 2014 7, 170-186DOI: (10.1093/mp/sst151) Copyright © 2013 The Authors. All rights reserved. Terms and Conditions

Figure 7 Expression of plNAD–MDH in Inflorescences of A. thaliana Wild-Type (WT) Plants by Use of In Situ Hybridization and GUS-Histochemical Staining. (A, B) Transcripts of the plNAD–MDH are mainly detectable in the tapetum (ta), ovules (ov), the gynoecium (gy), and pollen (po). The β-glucuronidase activity is very high in anthers of inflorescences (C) and mature pollen (D). Scale bars were calibrated to 200 μm. Molecular Plant 2014 7, 170-186DOI: (10.1093/mp/sst151) Copyright © 2013 The Authors. All rights reserved. Terms and Conditions

Figure 8 Histochemical Localization of GUS Expression under the Control of the plNAD–MDH Promoter in Arabidopsis Pollen (A–E) and Embryos (F–M) at Different Developmental Stages. Scale bars were calibrated to 5 μm (A–E) and 100 μm (F–M). Molecular Plant 2014 7, 170-186DOI: (10.1093/mp/sst151) Copyright © 2013 The Authors. All rights reserved. Terms and Conditions

Figure 9 Analysis of the Male Gametophyte. (A)In vitro pollen-tube growth of wild-type (WT) and heterozygous plnad–mdh/plNAD–MDH knockout plants (159). Scale bars were calibrated to 10 μm. (B) Recovery of pollen-tube growth in vitro by supplying NADH–GOGAT with its substrates (2-OG and Gln). Molecular Plant 2014 7, 170-186DOI: (10.1093/mp/sst151) Copyright © 2013 The Authors. All rights reserved. Terms and Conditions

Figure 10 Analysis of Embryo Development. (A) Analysis of seeds in siliques and mature seeds from A. thaliana wild-type (WT), heterozygous plnad–mdh/plNAD–MDH knockout (159), and complemented homozygous plnad–mdh knockout plants (Compl.). Plants were grown on soil, and siliques were harvested after approximately 7–8 weeks. (B) Seeds of line 159 at different developmental stages after clearing with Hoyer's medium. Scale bars were calibrated to 100 μm (silique 15, green) and 50 μm (all others). Molecular Plant 2014 7, 170-186DOI: (10.1093/mp/sst151) Copyright © 2013 The Authors. All rights reserved. Terms and Conditions

Figure 11 Crossing Experiments and Evaluation of Progenies. (A) Backcrossing of wild-types with pollen of line 159. (B) Self-fertilization of line 159. P1 and P2 are the primer combinations used to detect the wild-type gene and the T-DNA insertion, respectively (see Figure 3). Molecular Plant 2014 7, 170-186DOI: (10.1093/mp/sst151) Copyright © 2013 The Authors. All rights reserved. Terms and Conditions