Os12g0472500

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Annotated Information

Function

• OsTDL1A, the closest rice homolog of the Arabidopsis (TAPETUM DETERMINANT1)TPD1 gene. In Arabidopsis TPD1, which encodes a small protein hypothesized to be an extracellular ligand of EXS/EMS1.

• OsTDL1A binds to the LRR domain of rice receptor kinase (MULTIPLE SPOROCYTES1)MSP1, and is required to limit sporocyte numbers. MSP1 encodes a leucine-rich-repeat receptor kinase, which is orthologous to EXS/EMS1 in Arabidopsis. Like mac1 and msp1, exs/ems1 mutants produce extra sporocytes in the anther instead of a tapetum, causing male sterility. OsTDL1A binds MSP1 in order to limit sporocyte numbers. OsTDL1A-RNAi lines may be suitable starting points for achieving synthetic apospory in rice.

• OsTDL1A has another name is MIL2. MIL2 is responsible for the differentiation of primary parietal cells into secondary parietal cells in rice anthers.

• MIL2 works upstream of UDT1(is an early regulator of rice tapetum development) in controlling anther wall cell development.

MIL2 sequence analysis

The predicted MIL2 protein has 226 amino acids, including a 34- amino-acid signal peptide at the N terminus.A BLAST search of the database indicated that MIL2 is well conserved in land plants and generally has two homologs in many angiosperm species, including maize (Zea mays), rice, soybean Glycine max) and grape (Vitis vinifera). The closest homolog of MIL2 is the maize MULTIPLE ARCHESPORIAL CELLS 1 (MAC1) gene. However, no clear MIL2 homologs could be detected in the Chlorophyta. The predicted MIL2 homolog proteins all have a highly conserved region (corresponding to positions 112–220 in AK713941), which we termed the MIL2 homology domain. These proteins are typically c. 160–230 amino acids in length and carry the MIL2 homology region at the C terminus. In addition, based on Signal IP predictions, most of the homologs have N-terminal signal peptides similar to MIL2.

Mutation and Phenotype

Rice contains two close homologues of TPD1 (OsTDL1A and OsTDL1B), . The sites of expression of these genes relative to MSP1, examine the affinity of OsTDL1A and OsTDL1B proteins for the LRR domain of MSP1, and study the effect of RNA interference of OsTDL1A on anther and ovule. MSP1 and its close paralog MSP1-like1 (MSL1) are structurally the most similar rice proteins to EXS/EMS1.

MIL2 has three kinds of mutations mil2-1, mil2-2 and mil2-3. The mil2-1 genome contains a one-nucleotide-deletion in the marked site in exon 4. In mil2-2, a point mutation occurs in exon 3, causing a Q83P reversion. The mil2-3 genome contains a 280-kb deletion of a region on chromosome 12 in which MIL2 gene is located.

Plants of the mil2 mutant line showed typical sterility phenotypes with very low seed set. The mil2 anthers were white and never dehisced compared with wild-type anthers. A closer examination revealed that these anthers were apparently devoid of pollen grains. When mil2 mutant plants were pollinated with pollen from wild-type plants, only a few seeds resulted, suggesting that the mil2 mutation affected both male and female fertility.

In mil2-1 anthers, PPCs and sporogenous cells were generated from archesporial initials, with PPCs actually forming a sequential ring around a central core of sporogenous cells. Most of the mil2-1 PPCs did not engage in further periclinal division, although evidence of a periclinal division could be observed occasionally in some PPCs . Therefore, in most of the mil2-1 anthers, only one visible layer of somatic cells could be clearly observed beneath the epidermis. Enveloped by two somatic cell layers, the mil2-1 sporogenous cells divided to form an increased number of microsporocytes occupying the center of the locules. Followed, both the DAPI result and the result of the expression of marker gene OsREC8 (it is a meiotic-specific gene, which is often used as a marker for the identification of meiotic cells)、 MIL1 (controls meiotic entry of the microsporocytes) indicate the mil2-1 microsporocytes entered meiosis, but did not complete meiotic division.

Expresson

qRT-PCR shows MIL2 was expressed at a very low level, it was observed in most of the tissues analyzed, including leaf, internode, leaf sheath and panicle at early developmental stages. MIL2 expression in panicles declined gradually as the panicles elongated, and hardly any transcripts could be detected in panicles that were 10 cm in length.(R, root; I,internode; L, leaf; Sh, sheath; S, seedling; P1, 1-cm panicles; P2, 3-cm panicles; P3, 6-cm panicles; P4, 10-cm panicles; P5, panicles near maturity.)

RNA in situ hybridization shows MIL2 was expressed conspicuously in archesporial cells located in the four corners of young anthers. As archesporial cells developed into sporogenous cells and parietal cells, MIL2 mRNA preferentially accumulated in parietal cells, first in PPCs, and then in inner SPCs; hybridization signals in sporogenous cells were considerably weaker. After differentiation of the four somatic cell layers, MIL2 was expressed mainly in the somatic cell layers embracing the reproductive cells, including the middle and tapetal layers. A low level of expression was observed in microsporocytes in the center of each anther lobe, whereas, in the outer two layers, hardly any hybridization signal above background was detectable. Expression of MIL2 in the anther lobes decreased substantially as the microsporocytes underwent meiosis.

Evolution

OsTDL1A and OsTDL1B are rice homologs of TPD1 of Arabidopsis

The full-length protein sequence of Arabidopsis TPD1 [AAR25553, 176 amino acids (aa)] was used as the query in a tblastn search of the rice genome. We detected two TPD1-like genes and named them OsTDL1A (blaste-value: 5 × 10−27) and OsTDL1B (blaste-value: 1 × 10−18). They are located on chromosomes 12 and 10, respectively, and the corresponding full-length cDNAs are AK108523 and AK121594. When their predicted protein sequences (NP_001066753, 226 aa; NP_001064316, 169 aa) were used as tblastn queries of the Arabidopsis genome, the best hits were to TPD1 and another Arabidopsis protein (ABF59206, 179 aa), which we have named AtTDL1. TPD1 is more similar to AtTDL1 (e-value: 2 × 10−35) than OsTDL1A is to OsTDL1B (e-value: 2 × 10−14), a conclusion supported by CLUSTALW analysis (Figure 1A). The four proteins cluster separately from the next most similar proteins encoded by the rice and Arabidopsis genomes (the boxed proteins are from rice). Single amplicons of the expected size for OsTDL1A and OsTDL1B (546 and 485 bp, respectively) were generated from RNA of roots and spikelets (1 mm, 3 mm and 5 DAF). Three amplicon sizes were seen for MSP1, as was also reported by Nonomura et al. (2003). The sizes of these amplicons are consistent with the sizes expected for the fully spliced transcript (507 bp), a transcript in which only the first intron has been removed by splicing (752 bp) and an unspliced transcript (1298 bp), based on the full-length cDNA sequence (AK120933). The smallest amplicon was amplified most strongly from 1- and 3-mm spikelets. The largest amplicon was not a PCR product derived from possible DNA contamination, because the RNA preparations lacked DNA contamination as judged by the failure of the GAPDH primers to produce the genomic amplicon (Figure 2, open arrow). We conclude that MSP1, OsTDL1A and OsTDL1B are all expressed in spikelets before and during meiosis.

Lab

• State Key Laboratory of Plant Genomics and Center for Plant Gene Research, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing, 100101, China;

• Biotechnology Research Institute/National Key Facility for Gene Resources and Gene Improvement, Chinese Academy of Agricultural Sciences, Beijing, 100081, China

• College of Agriculture and Biotechnology, Zhejiang University, Hangzhou, China

• Plant Breeding, Genetics and Biotechnology Division, International Rice Research Institute, Manila, Philippines

• Division of Plant Industry, Commonwealth Scientific and Industrial Research Organization, Canberra, Australia

References

• Xinai Zhao, Justina de Palma, Rowena Oane, et al. OsTDL1A binds to the LRR domain of rice receptor kinase MSP1, and is required to limit sporocyte numbers. The Plant Journal (2008) 54, 375–387
• Lilan Hong, Ding Tang, Yi Shen, et al. MIL2 (MICROSPORELESS2) regulates early cell differentiation in the rice anther. New Phytologist (2012) 196: 402–413