Volume 8, Issue 2, Pages 251-260 (February 2015) Arabidopsis AT-hook Protein TEK Positively Regulates the Expression of Arabinogalactan Proteins for Nexine Formation  Qi-Shi Jia, Jun Zhu, Xiao-Feng Xu, Yue Lou, Zhan-Lin Zhang, Zhi-Ping Zhang, Zhong-Nan Yang  Molecular Plant  Volume 8, Issue 2, Pages 251-260 (February 2015) DOI: 10.1016/j.molp.2014.10.001 Copyright © 2015 The Author Terms and Conditions

Figure 1 TEK Binds the PC-MAR1 In Vitro. (A) Recombinant TEK-His and mTEK-His proteins purified from Escherichia coli. (B) TEK-His was assayed for binding the PC-MAR1 probe. The PC-MAR1 probe is 68 bp long. Nonlabeled competitors reduced the visible shift significantly (lanes 3 and 4). (C) The shift is significantly reduced when using mTEK-His proteins (lane 2). Molecular Plant 2015 8, 251-260DOI: (10.1016/j.molp.2014.10.001) Copyright © 2015 The Author Terms and Conditions

Figure 2 Characterization of Transgenic Lines Expressing TEK-SRDX. (A) Structural representation of the ProTEK:TEK-SRDX construct. The TEK-SRDX fusion is driven by the TEK promoter, and the SRDX domain with its amino acid sequence is shown below. (B–E) Thirty-five-day-old plant of the wild-type (B), tek mutant (C), and transgenic lines expressing TEK-SRDX in the background of tek/+ (D) and wild-type (E). The arrows and arrowheads in (D) and (E) indicate sterile and fertile siliques. (F–W) Semi-thin cross-sectional analysis of anther development of tek (F–K), and transgenic lines expressing TEK-SRDX in the background of tek/+ (L–Q) and wild-type (R–W), with the anther development stages 7–12 indicated above. DPG, degraded pollen grain; Msp, microspore; PG, pollen grain; Tds, tetrads. Scale bars represent 20 μm. Molecular Plant 2015 8, 251-260DOI: (10.1016/j.molp.2014.10.001) Copyright © 2015 The Author Terms and Conditions

Figure 3 MAPMAN Classification of the Differentially Expressed Genes in tek Mutant Anthers. (A) The downregulated genes were displayed on diagrams of the metabolic pathways. The most highly represented pathways included signaling (11.0%), protein metabolism (7.6%), stress response (7.0%), cell wall (6.5%), and transport (6.4%). (B) Functional classification of the upregulated genes. The most highly represented pathways included protein metabolism (11.5%), miscellaneous enzyme families (10.6%), secondary metabolism (9.0%), and RNA regulation of transcription (8.5%). (C) The proportion of genes involved in the cell wall and signaling pathways that are over-represented in the downregulated genes was compared with the proportion in the microarray. Molecular Plant 2015 8, 251-260DOI: (10.1016/j.molp.2014.10.001) Copyright © 2015 The Author Terms and Conditions

Figure 4 Expression Patterns of AGPs in the Wild-Type and tek Mutant. (A) Expression of the four AGPs in the flower buds of the wild-type and tek mutant based on quantitative RT–PCR analysis and microarray data. (B–E) Wild-type anthers from stages 6–9 hybridized with an antisense probe of AGP6. Ms, microsporocyte; Msp, microspore; T, tapetum; Tds, tetrads. Scale bars represent 20 μm. (F) Stage 7 anther hybridized with a sense probe. T, tapetum; Tds, tetrads. Scale bar represents 20 μm. Molecular Plant 2015 8, 251-260DOI: (10.1016/j.molp.2014.10.001) Copyright © 2015 The Author Terms and Conditions

Figure 5 TEK Directly Binds the Promoters of AGP6, AGP11, AGP23 and AGP40. (A) The AT-rich sequences in the promoters of AGP6, AGP11, AGP23, and AGP40. The sequences that are boxed, capitalized, and underlined separately represent the A-box motifs (boxed), WADAWAYAWW motif, and AATATT motif (underlined) present in MARs. (B) ChIP–qPCR analysis of the promoters of four AGPs bound by TEK. Fold enrichment is calculated from the qPCR assays of three independent ChIP experiments. β-Tubulin was used as a control. (C) EMSA assay of the binding of TEK with the promoter region of AGP6. A 69 bp-long (-113 to -45) probe fragment from the AGP6 promoter was labeled with biotin, and TEK was found to be able to bind this probe (lane 2). Five-fold and 25-fold unlabeled probe amounts were used as competitors (lanes 3 and 4). (D) TEK was able to bind the WADAWAYAWW motif present in the AGP11 promoter. The probe fragment from the AGP611 promoter is 168 bp long (-266 to -99) and was labeled with biotin. Nonlabeled competitors are able to reduce the visible shift. Molecular Plant 2015 8, 251-260DOI: (10.1016/j.molp.2014.10.001) Copyright © 2015 The Author Terms and Conditions

Figure 6 Nexine Restoration in ProTEK:AGP6-Terminator Lines. (A) Construct used for genetic transformation. WT, wild-type. (B–D) Plants of the wild-type (B), tek mutant (C), and ProTEK:AGP6-Terminator transgenic lines in the tek background (D). The fertile siliques are indicated in the transgenic lines. The white arrows indicate the siliques with recovered fertility. (E) Statistical analysis of the fertile siliques per plant and seed yield per silique in the wild-type and ProTEK:AGP6-Terminator lines. (F–H) TEM observation of the pollen wall of the wild-type (F), tek mutant (G), and ProTEK:AGP6-Terminator transgenic line (H). The nexine layer is indicated in the wild-type and the nexine-like structure in the ProTEK:AGP6-Terminator transgenic lines. Msp, microspore; Ne, nexine; Se, sexine. Scale bars represent 2 μm. Molecular Plant 2015 8, 251-260DOI: (10.1016/j.molp.2014.10.001) Copyright © 2015 The Author Terms and Conditions

Figure 7 A Working Model for the Functions of TEK in Nexine Formation. In this model, the tapetal specific transcript factor AMS directly regulates the expression of MS188 and TEK. MS188 controls the sexine formation. TEK regulates the expression of AGPs for nexine formation. Solid lines indicate direct gene regulation and dashed lines represent unidentified regulation. Molecular Plant 2015 8, 251-260DOI: (10.1016/j.molp.2014.10.001) Copyright © 2015 The Author Terms and Conditions

Supplemental Figure 1 Molecular Plant 2015 8, 251-260DOI: (10.1016/j.molp.2014.10.001) Copyright © 2015 The Author Terms and Conditions