Overexpression of AtTTP affects ARF17 expression and leads to male sterility in Arabidopsis

Abstract

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.

Fig 1. Phylogenetic analysis of AtTTP and orthologous proteins.

(A) Unrooted phylogenetic tree of NPU and its orthologous proteins. The protein sequences of AtTTP 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 AtTTP and its orthologs. Black triangles, the critical CCCH zinc finger residues: Cysteine and histidine.

Fig 2. Expression analysis of AtTTP.

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

Fig 3. Characterization of AtTTP-OE line (A) Structure of the AtTTP over-expressing gene.

The primers of head, mid and tail are used for real-time PCR amplification. (B) Real-time PCR analysis of AtTTP in wild-type and OE1-3 mutant floral buds. The primer positions were shown in Fig. 3A. Each expression level was normalized to that of TUBULIN. The data and errors bars are representative of 3 replicates. (C) Comparison of reproductive development in 40-d-old wild-type and OE1-3 mutant plants. (D) The percentage of short siliques in the wild-type and OE1-3 mutant plants. (E) Alexander staining of the wild-type anther. (F) Alexander staining of the OE1 mutant anther. (G) Alexander staining of the OE2 mutant anther. (H) Alexander staining of the OE3 mutant anther. (I) Adhered pollen from the OE3 lines under light microscopy. Bars = 10 μm.

Fig 4. The anther development, callose wall, and expression analyses of CalS5, A6 and A6-like in the wild-type and AtTTP-OE line.

(A and F) Anthers at stage 6. The AtTTP-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 AtTTP-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 AtTTP-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 AtTTP-OE line (F). (E and J) Anthers at stage 10. The microspores are disintegrated in the AtTTP-OE line (J). (K and P) Anthers at stage 12. Remnants of microspores and less abnormal microspores were observed in the AtTTP-OE anther locule (P). (L, Q, N, S) The callose wall in the AtTTP-OE line was not obviously different compared with that of the wild-type. (M, R, O, T) The callose wall in the AtTTP-OE line was thinner around the tetrads compared with that of the wild-type. (U) Real-time PCR analysis of CalS5. Each expression level was normalized to that of TUBULIN. The data and errors bars are representative of 3 replicates. (V) Real-time PCR analysis of A6 and A6-like. Each expression level was normalized to that of TUBULIN. The data and errors bars are representative of 3 replicates. Bars = 10 μm.

Fig 5. Expression analysis of NPU, CDKG1, ARFs and miRNAs in the AtTTP-OE line.

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

Fig 6. A putative regulation pathway in anther.

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