Expression analysis of <i>AtTTP</i>.

<p>(A) qRT-PCR analysis of RNA isolated from various tissues including roots, stems, leaves, and buds. Each expression level was normalized to that of <i>TUBULIN</i>. The data and errors bars are representative of 3 replicates. (B) <i>In situ</i> hybridization of the <i>AtTTP</i> transcript in a stage 4 anther with an antisense probe. (C) <i>In situ</i> hybridization of the <i>AtTTP</i> transcript in a stage 5 anther with an antisense probe. (D) <i>In situ</i> hybridization of the <i>AtTTP</i> transcript in an earlier stage 6 anther with an antisense probe. (E) <i>In situ</i> hybridization of the <i>AtTTP</i> transcript in a later stage 6 anther with an antisense probe. (F) <i>In situ</i> hybridization of the <i>AtTTP</i> transcript in a stage 7 anther with an antisense probe. (G) <i>In situ</i> hybridization of the <i>AtTTP</i> transcript in a stage 8 anther with an antisense probe. (H) <i>In situ</i> hybridization of the <i>AtTTP</i> transcript in a stage 9 anther with an antisense probe. (I) <i>In situ</i> hybridization of the <i>AtTTP</i> transcript in a stage 10 anther with an antisense probe. (J) <i>In situ</i> hybridization of the <i>AtTTP</i> transcript in an earlier stage 6 anther with an sense probe. Bars = 10 μm.</p

Overexpression of <i>AtTTP</i> Affects <i>ARF17</i> Expression and Leads to Male Sterility in Arabidopsis

<div><p>Callose synthesis is critical for the formation of the pollen wall pattern. CalS5 is thought to be the major synthethase for the callose wall. In the Arabidopsis anther, ARF17 regulates the expression of CalS5 and is the target of miR160. Plants expressing miR160-resistant ARF17 (35S:5mARF17 lines) with increased ARF17 mRNA levels display male sterility. Here we report a zinc finger family gene, AtTTP, which is involved in miR160 maturation and callose synthesis in Arabidopsis. AtTTP is expressed in microsporocytes, tetrads and tapetal cells in the anther. Over-expression lines of AtTTP (AtTTP-OE line) exhibited reduced male fertility. CalS5 expression was tremendously reduced and the tetrad callose wall became much thinner in the AtTTP-OE line. Northern blotting hybridization and quantitative RT-PCR analysis revealed that miR160 was decreased, while the expression of ARF17 was increased in the AtTTP-OE line. Based on these results, we propose that AtTTP associates with miR160 in order to regulate the ARF17 expression needed for callose synthesis and pollen wall formation.</p></div

Characterization of <i>AtTTP</i>-OE line (A) Structure of the <i>AtTTP</i> over-expressing gene.

<p>The primers of head, mid and tail are used for real-time PCR amplification. (B) Real-time PCR analysis of <i>AtTTP</i> in wild-type and <i>OE1-3</i> mutant floral buds. The primer positions were shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0117317#pone.0117317.g003" target="_blank">Fig. 3A</a>. Each expression level was normalized to that of <i>TUBULIN</i>. The data and errors bars are representative of 3 replicates. (C) Comparison of reproductive development in 40-d-old wild-type and <i>OE1-3</i> mutant plants. (D) The percentage of short siliques in the wild-type and <i>OE1-3</i> mutant plants. (E) Alexander staining of the wild-type anther. (F) Alexander staining of the <i>OE1</i> mutant anther. (G) Alexander staining of the <i>OE2</i> mutant anther. (H) Alexander staining of the <i>OE3</i> mutant anther. (I) Adhered pollen from the <i>OE3</i> lines under light microscopy. Bars = 10 μm.</p

The anther development, callose wall, and expression analyses of <i>CalS5</i>, <i>A6</i> and <i>A6-like</i> in the wild-type and <i>AtTTP</i>-OE line.

<p>(A and F) Anthers at stage 6. The <i>AtTTP</i>-OE microsporocytes (F) are apparently closely compacted compared with those of the wild-type (A). (B and G) Anthers at stage 7. Wild-type tetrads are surrounded with callose (B), whereas the tetrads lack callose in the <i>AtTTP</i>-OE line (G). (C and H) Anthers at stage 8. Wild-type microspores are released from the tetrads (C), whereas microspores were still adherent in the <i>AtTTP</i>-OE line (H). (D and I) Anthers at stage 9. The microspores have begun to degenerate and still have not been released from the tetrads in the <i>AtTTP</i>-OE line (F). (E and J) Anthers at stage 10. The microspores are disintegrated in the <i>AtTTP</i>-OE line (J). (K and P) Anthers at stage 12. Remnants of microspores and less abnormal microspores were observed in the <i>AtTTP</i>-OE anther locule (P). (L, Q, N, S) The callose wall in the <i>AtTTP</i>-OE line was not obviously different compared with that of the wild-type. (M, R, O, T) The callose wall in the <i>AtTTP</i>-OE line was thinner around the tetrads compared with that of the wild-type. (U) Real-time PCR analysis of <i>CalS5</i>. Each expression level was normalized to that of <i>TUBULIN</i>. The data and errors bars are representative of 3 replicates. (V) Real-time PCR analysis of <i>A6</i> and <i>A6-like</i>. Each expression level was normalized to that of <i>TUBULIN</i>. The data and errors bars are representative of 3 replicates. Bars = 10 μm.</p

Expression analysis of <i>NPU</i>, <i>CDKG1</i>, <i>ARFs</i> and miRNAs in the AtTTP-OE line.

<p>(A) Real-time PCR analysis of <i>NPU</i> and <i>CDKG1</i> in the <i>AtTTP</i>-OE line. Each expression level was normalized to that of <i>TUBULIN</i>. The data and errors bars are representative of 3 replicates. (B) Real-time PCR analysis of <i>ARF10, ARF16</i> and <i>ARF17</i> in the <i>AtTTP</i>-OE line. Each expression level was normalized to that of <i>TUBULIN</i>. The data and errors bars are representative of 3 replicates. (C) Northern blotting hybridization of mature miR160 in the <i>AtTTP</i>-OE line.</p

A putative regulation pathway in anther.

<p>Black arrow: direct regulation has been demonstrated; Dotted arrow: the regulatory mechanism remains unclear.</p

Phylogenetic analysis of <i>AtTTP</i> and orthologous proteins.

<p>(A) Unrooted phylogenetic tree of NPU and its orthologous proteins. The protein sequences of <i>AtTTP</i> and its orthologs were analyzed with the neighbor-joining method by MEGA5.05 software. The numbers at the nodes represent the percentage bootstrap values based on 1,000 replications. The CCCH domains were predicted by the Pfam 26.0 tool online. The protein sequence files are as follows: Drosophila: NP_511141.2; Xenopus: NP_001080610.1; Homo: NP_004917.2; Mus: NP_031590.1; Rattus: NP_058868.1; Vitis: XP_002281139.1; Arabidopsis: NP_176987.1; Ricinus: XP_002526299.1; Populus: POPTR_0010s12860.1; Zea: NP_001148404.1; Sorghum: XP_002440301.1; Oryza: NP_001056400.1; Brachypodium: XP_003569444.1; Ostreococcus: XP_003078184.1; Physcomitrella: XP_001783282.1; Saccharomyces: NP_013237.1; Scheffersomyces: XP_001385679.1. (B) Multiple alignments of <i>AtTTP</i> and its orthologs. Black triangles, the critical CCCH zinc finger residues: Cysteine and histidine.</p