Volume 5, Issue 1, Pages 131-142 (January 2012) Glycerol-3-Phosphate Acyltransferase 6 (GPAT6) Is Important for Tapetum Development in Arabidopsis and Plays Multiple Roles in Plant Fertility  Li Xiao-Chuan , Zhu Jun , Yang Jun , Zhang Guo-Rui , Xing Wei-Feng , Zhang Sen , Yang Zhong-Nan   Molecular Plant  Volume 5, Issue 1, Pages 131-142 (January 2012) DOI: 10.1093/mp/ssr057 Copyright © 2012 The Authors. All rights reserved. Terms and Conditions

Figure 1 Expression Analysis of GPAT6 and GPAT1. (A–D) Promoter–GUS staining of (A) inflorescence, (B) stamen, (C) pollen tube, (D) pistil, where the GUS staining signal is found specifically in the stigma, but not in the carpels. Scale bar = 200 μm. (E–I) In-situ hybridization of GPAT6 transcript with antisense probe within the (E) stage 5 anther, (F) stage 7 anther, (G) stage 8 anther, (H) stage 11 anther. (I) In-situ hybridization of GPAT6 transcript with sense probe. The signal is detected initially in stage 5, peaking in stages 7 and 8. (J–N) In-situ hybridization of GPAT1 transcript with antisense probe within the (J) stage 5 anther, (K) stage 7 anther, (L) stage 8 anther, (M) stage 11 anther. (N) In-situ hybridization of GPAT1 transcript with sense probe. The signal of GPAT1 transcript is similar with GPAT6 transcript. Bar = 20 μm. Molecular Plant 2012 5, 131-142DOI: (10.1093/mp/ssr057) Copyright © 2012 The Authors. All rights reserved. Terms and Conditions

Figure 2 Characterization of the Wild-Type (Col) and gpat6-3 Mutant. (A) Phenotypes of the 35-day-old wild-type Arabidopsis ecotype Columbia (WT) with normal fertility; 35-day-old gpat6-3 mutant (gpat6) with very small siliques (white arrow head) containing no seeds; gpat6-3 reproductive organs at the 55-day-old development stage (L-gpat6) with some siliques rescued and bearing seeds; and a gpat6-3 plant containing the GPAT6 transgene (C-gpat6) with normal fertility. (B) Gene structure of GPAT6 and T-DNA insertion. The left (LB) and right (RB) borders of the T-DNA sequences are shown. The T-DNA contains a kanamycin resistance (KanR) gene. Black box, exon; gray line, intron; light gray line, untranslated regions. L-TEST F and L-TEST R primers used for linkage analysis. Molecular Plant 2012 5, 131-142DOI: (10.1093/mp/ssr057) Copyright © 2012 The Authors. All rights reserved. Terms and Conditions

Figure 3 Examination of Stamens in Wild-Type, gpat1, gpat6-3, and gpat1 gpat6 Double Mutant. (A–E) The length of stamen filaments in (A) wild-type, (B) gpat6-3, (C) gpat1, and (D, E) gpat1 gpat6 double mutant. (A) The stamen filaments in the wild-type are slightly longer than the pistils. (B, C) In gpat1 and gpat6-3, the stamen filaments are shorter than the pistils. (D) In the gpat1 gpat6 double mutant, distinctively short stamen filaments are generated with shrunken anthers (arrow head). (E) This phenotype was partly rescued in later-generated flowers. (F–J) Examination of pollen production in (F) wild-type, (G) gpat6-3, (H) gpat1, and (I, J) gpat1 gpat6 double mutant anthers by Alexander’s staining. The production of pollen grains in gpat1 and gpat6-3 are diminished compared to wild-type pollen production, and no pollen grains can be detected in the double mutant in both early (I) and late (J) generated flowers. S, stigma; SF, stamen filament; PGs, pollen grains. Bar = 50 μm. Molecular Plant 2012 5, 131-142DOI: (10.1093/mp/ssr057) Copyright © 2012 The Authors. All rights reserved. Terms and Conditions

Figure 4 Anther Development from Stage 5 to Stage 12 in the Wild-Type (Col), the gpat6-3 Mutant, and the gpat1 gpat6 Double Mutant. Anther sections: (A–E, Q, R), wild-type; (F–J, S, T), gpat6-3 mutant; (L–P, U, V), gpat1 gpat6 double mutant. (A, F, L; anthers during stage 6) No differences are detected between these three lines. (B, G; anthers during stage 7) No differences are detected between the wild-type and gpat6-3 mutant lines. (M; anthers during stage 7) The tapetal cells of the double mutant become swollen, highly vacuolated, and the tetrads begin to collapse. (N; after phase m) The locule of the double mutant begins to shrink and the tapetum and tetrads are abolished. (C, H; anthers during stage 8) The gpat6-3 tapetum is more obviously expanded than wild-type. (O; anthers during stage 8) The locule of the gpat1 gpat6 double mutant is shrunken and no microspores can be detected. (D, I; anthers during stage 10) Defective pollen grains have emerged in gpat6-3. (E, J; anthers during stage 12) Both malformed and healthy pollen grains are observed in the gpat6-3 anther locule. (P; anthers during stage 12) The locule of the gpat1 gpat6 double mutant resembles the locule of the double mutant in stage 8. (Q–V) Aniline blue staining of the anthers. Aniline blue fluorescence (arrow head) remains detectable in the (V) double mutant anthers after (U) tetrad stages. Aniline blue fluorescence, detectable in the tetrad stage (Q, S), cannot be detected in either (R) wild-type or (T) gpat6-3 anthers. E, epidermis; En, endothecium; ML, middle layer; Ms, meiocytes; Msp, microspores; SL, shrunken locule; T, tapetum; Tds, tetrads. Bar = 20 μm. Molecular Plant 2012 5, 131-142DOI: (10.1093/mp/ssr057) Copyright © 2012 The Authors. All rights reserved. Terms and Conditions

Figure 5 SEM Examination of Dehiscent Anthers and Pollen Grains. Pollen grains of wild-type (A, B, E) and gpat6-3 (B, D, F, G) plants. (A, B) Emasculated pollen grains. Several gpat6-3 pollen grains are collapsed and aborted. Bar = 5 μm. (C, D) Higher magnification of pollen grains. (D) Defective pollen grains of gpat6-3. Bar = 5 μm. (E) Wild-type pollen grains with a regular reticulate exine pattern. (F, G) The gpat6-3 pollen grains with impaired or flawed exine pattern (white arrow head). Bar = 2 μm. Molecular Plant 2012 5, 131-142DOI: (10.1093/mp/ssr057) Copyright © 2012 The Authors. All rights reserved. Terms and Conditions

Figure 6 TEM Examination of Anthers and Pollen Grains. (A, C, D, G) Tapetum of wild-type and (B, E, F, H) gpat6-3 mutant plants. During stage 7, the ultrastructure of (B) gpat6-3 tapetal cells are similar to (A) wild-type. (C) By late stage 8, little osmophilic material is accumulated in the normal wild-type tapetum, which contains (D) large stacks of endoplasmic reticulum (ER). (E) During late stage 8 in gpat6-3 anthers, large amounts of osmophilic material has accumulated in the tapetal cells and (F) little ER is detectable. The tapetosomes and elaioplasts in both (G) wild-type and (H) gpat6-3 tapetum exhibit no visible difference during stage 10. (I, K, M, O) Pollen wall of wild-type and (J, L, N, P) gpat6-3 mutant. At stage 7, the exine of (J) gpat6-3 tapetal cells are similar to (I) wild-type. Aberrant exine formation is observed in (K) gpat6-3 microspores compared to (L) the wild-type. (N and P) The exine of gpat6-3 pollen grain is irregularly loaded compared to (M, O) the wild-type. (N, P) The gpat6-3 pollen coat loading is inadequate. (N, M) are part of pollen present in (P, O), respectively. In (R), gpat6-3 pollen grains, some mis-shaped vacuoles are detected and hardly any ER can be observed. In (Q), wild-type pollen grains, numerous small storage bodies, and extensive intracellular membrane (arrow head) can be observed. Ba, bacula; E, exine; El, elaioplast; ER, endoplasmic reticulum; In, intine; P, plastid; PC, pollen coat; PR, prime bacula; T, tapetosomes; Te, tectum. V, vacuole. Bar = 2 μm. Molecular Plant 2012 5, 131-142DOI: (10.1093/mp/ssr057) Copyright © 2012 The Authors. All rights reserved. Terms and Conditions

Figure 7 In Vitro and In Vivo Germination of Both Wild-Type and gpat6-3 Pollen Grains. (A) Statistics of the in vitro germination rate of pollen grains from wild-type and gpat6-3 (means of at least 1000 pollen grains from three independent assays). (B) Statistics of length distribution for wild-type and gpat6-3 pollen tubes after in vitro germination for 8 h (means of at least 600 pollen grains from three independent assays). (C, D) In vivo germination of pollen grains of (C) wild-type and (D) gpat6-3 mutant in wild-type pistil for 3 h stained by aniline blue, which shows that the pollen grains of gpat6-3 slow pollen tube elongation. PTs, pollen tubes. Bar = 50 μm. Molecular Plant 2012 5, 131-142DOI: (10.1093/mp/ssr057) Copyright © 2012 The Authors. All rights reserved. Terms and Conditions