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First published online 10 July 2006
doi: 10.1242/dev.02463

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Regulation of Arabidopsis tapetum development and function by DYSFUNCTIONAL TAPETUM1 (DYT1) encoding a putative bHLH transcription factor

Wei Zhang^1,*, Yujin Sun^1,*, Ljudmilla Timofejeva², Changbin Chen¹, Ueli Grossniklaus³ and Hong Ma^1,

¹ Department of Biology and the Huck Institutes of Life Sciences, the Pennsylvania State University, University Park, PA 16802, USA.
² Department of Gene Technology, Tallinn University of Technology, Akadeemiatee 15, Tallinn 19086, Estonia.
³ Institute of Plant Biology, Zollikerstrasse 107, Zurich CH-8008, Switzerland.

View larger version (47K): [in a new window]   Fig. 1. Phenotypes of the wild type (Ler), dyt1 mutant and transgenic plants for complementation. (A) A Ler plant. (B) A dyt1 plant, with very small siliques (arrows). (C) A Ler flower. (D) A dyt1 flower. (E) A flower of the dyt1 plant with the DYT1 transgene. (F) A wild-type anther, with viable pollen grains (stained). (G) A dyt1 anther, no viable pollen. (H) An anther from a dyt1 plant with the DYT1 transgene, with a large number of viable pollen grains and some microspores (arrowheads). Scale bars: 10 mm in A,B; 500 µm in C-E; 20 µm in F-H.

View larger version (40K): [in a new window]   Fig. 2. Meiosis in Ler and dyt1 anthers. (A,B) Pachytene images of the Ler (A) and dyt1 (B) meiocytes with condensed chromosomes. (C,D) Diakinesis images of the Ler (C) and dyt1 (D) meiocytes, each with five bivalents of attached homologous chromosomes. (E,F) Telophase II images of the Ler (E) and dyt1 (F) meiocytes. Both show four decondensed chromosome clusters. Scale bar: 5 µm.

View larger version (99K): [in a new window]   Fig. 3. Anther development from stage 4 to stage 8 in the wild type (Ler) and dyt1 mutant. Locules from anther sections: (A,C,E,G,I,K) wild type; (B,D,F,H,J,L) dyt1 mutants. (A,B) Stage 4 anthers. (C,D) Late stage 4 or very early stage 5. Vacuolization in the dyt1 mutant occurred in more cells and the vacuoles were larger than those in the wild type. (E,F) Stage 5 anthers, with more and larger vacuoles in the tapetum and middle layer of the dyt1 anther (F) than the wild type (E). (G,H) Stage 6 anthers. The vacuolization in cells of the mutant tapetum and middle layer became more extensive. The mutant meiocytes had a much thinner callose layer around them (arrowheads). (I,J) Stage 7 anthers. Wild-type meiocytes undergo cytokinesis to form tetrads. In dyt1 anther, the tapetum and middle layer cells were swollen and had excess vacuolization, and meiocytes generally collapsed before cytokinesis (arrowheads). (K,L) At stage 8, in the wild-type anther locules, microspores were released from the tetrad; in the dyt1 anther, almost all meiocytes degenerated. E, epidermis; En, endothecium; ISP, inner secondary parietal cells; SS, secondary sporogenous cells; ML, middle layer; T, tapetum; Ms, meiocytes; Tds, tetrads; Msp, microspores; D-Ms, degenerated meiocytes. Scale bars: 10 µm.

View larger version (29K): [in a new window]   Fig. 4. The DYT1 gene structure and annotated conserved domain. (A) The genomic region of the DYT1 gene. The dyt1 insertion is flanked by a direct repeat of 6 bp ACTTCT, which correspond to nucleotides 109-104 upstream of the annotated ATG (nucleotides 1-3) codon. The DYT1 gene has three exons represented as white boxes. (B) The annotated amino acid sequence of the DYT1 protein, with 207 amino acid residues. From Phe29 to the Gln78 is the conserved bHLH domain, which corresponds to the black region in the schematic box image and is underlined in the amino acid sequence. (C) An unrooted neighbor-joining tree of Arabidopsis, rice, and Poplar bHLH genes in the same subfamily as DYT1. Gene ID numbers starting with `At' indicate genes from Arabidopsis thaliana; names of genes with functional information are given after the gene ID numbers. Gene ID numbers starting with `Os' indicates genes from rice (Oryza sativa); UDT1 is shown as OsUDT1. `Pt' indicates genes from poplar (Populus trichocarpa), with temporary names given according to gene ID from the Floral Genome Project. Bootstrap values are shown near the relevant nodes.

View larger version (81K): [in a new window]   Fig. 5. The DYT1 expression pattern. (A) Detection of DYT1 expression using real-time PCR in Ler background. DYT1 expression was not detected in any vegetative tissues or stage-12 flower, was detected at low levels in the young inflorescence and siliques, and was at the highest level in the anther. (B) Detection of DYT1 expression using real-time PCR in Ler, ems1 and spl inflorescences. The DYT1 expression was not detected in spl, but was detected in ems1 at about 17% of the normal level. (C-K) RNA in situ hybridization with a DYT1 probe. (C-F,H) DYT1 expression in the Ler background. (C) The DYT1 signal was detected in the floral meristem. (D) An anther at stage 4 to early stage 5. The DYT1 signal can be detected mainly within the newly formed tapetum and meiocytes. (E,H) At late stage 5, a strong signal is detected in the tapetal cell layer, whereas the signal in the meiocytes is much weaker. (F) At late stage 6, the DYT1 signal is greatly reduced, with residual expression in some meiocytes. (I) A dyt1 mutant anther at late stage 5; the DYT1 signal is low and non-specific in the entire anther. (J) A spl mutant anther at late stage 5. The DYT1 signal is at the background level. (K) An ems1 mutant anther locule at late stage 5. Uniformly weak DYT1 signal can be detected in meiocytes and little signal in cells surrounding the meiocytes. (G) The sense control with a Ler late stage 5 anther. Only background signal is seen. Rt, root; Sm, stem; Lf, leaf; Se, silique; S12, stage 12 flower; Inf, inflorescence; Ar, anther; WT-Inf, wild-type inflorescence; ems1-Inf, ems1 inflorescence; spl-Inf, spl inflorescence; T, tapetum; Ms, meiocytes; I, indeterminate cells; E-Ms, excess meiocytes. Scale bars: 20 µm in C-K.

View larger version (38K): [in a new window]   Fig. 6. Expression of anther and tapetum genes in the wild type and dyt1 mutant. (A) Each of the 11 genes, including the controls UBQ1 and MYB4, show either normal or increased expression in the dyt1 mutant background compared with the wild type. (B,C) Twenty-one genes show significantly decreased expression in the dyt1 mutant background. (D) Real-time PCR of selected anther genes in both wild-type and dyt1 backgrounds. (E) Effects of overexpression of DYT1 on selected anther genes. No significant effects were observed by DYT1 overexpression, even though the DYT1 expression levels were elevated. (F) Expression pattern of selected putative regulatory genes in spl, ems1 and dyt1 background.

View larger version (90K): [in a new window]   Fig. 7. The ems1 dyt1 double mutant anther morphology at late stage 5. (A) Wild-type. (B) dyt1.(C) ems1. (D) ems1 dyt1 double mutant. The double mutant lacks the tapetum. T, tapetum; ML, middle layer; Ms, meiocytes; E-Ms, excess meiocytes; I, indeterminate cells. Scale bar: 10 µm.

View larger version (17K): [in a new window]   Fig. 8. A model for DYT1 function and tapetum specification. The thin arrows indicate a positive genetic regulation. The arrowheads represent gene functions controlling a developmental stage. The open arrows indicate development from one stage to the next.