Os04g0599300

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Annotated Information

Function

ETERNAL TAPETUM 1(EAT1), a basic helix-loop-helix transcription factor conserved in land plants, positively regulates programmed cell death in tapetal cells in rice anthers. eat1 exhibits delayed tapetal cell death and aborted pollen formation, causing complete male sterility[1]. EAT1 directly regulates the expression of OsAP25 and OsAP37, which encode aspartic proteases that induce programmed cell death. In addition, EAT1 can interact with the TAPETUM DEGENERATION RETARDATION (TDR)protein and acts downstream of TDR[1]. TDR is also a key factor in regulates programmed cell death in tapetal cells[2].

Mutation

There are three kinds of EAT1 Mutation.

The mutant exhibits normal vegetative development and female organ formation, but is completely male sterile and has shrunken anthers and aborted pollen grains . All F1 plants of reciprocal crosses between the wild type and the mutant were fertile, and the F2 plants had an approximate 3:1 ratio for phenotypic segregation (fertility: sterility¼160: 47, w2¼0.58, P40.05, w2 test used), suggesting that this male sterile phenotype is caused by a single recessive mutation Similar to the wild type, the eat1-1 mutant anthers appeared to undergo normal meiosis, forming tetrads of haploid microspores at late stage 8 (stage 8b) Consistent with this observation, normal chromosome separation during meiosis and regular tetrads could be seen in eat1-1 microspore mother cells, as revealed by 40,6-diamidino-2-phenylindole staining. After stage 10, the wild-type tapetal cells had condensed cytoplasm, then became thinner and were eventually degenerated before mature pollen grains were formed. In contrast, the eat1-1 mutant had thicker and darkly stained tapetal cells, and abnormal abortion of the anther locule and microspores. These results together show that in the eat1 mutant, anther development and microspore formation are aborted.The TUNEL-positive signal in wild-type tapetal cells commenced at stage 8 during late meiosis, became intense at stage 9 when the microspore is released from the tetrad, and weakened at stage 12 when mature pollen grains are formed. However, no obvious DNA fragmentation signal was observed in eat1 tapetal cells till stage 10, when the vacuolated microspore forms. At stage 11 during mitosis I, weak DNA fragmentation signal was seen in all four layers of the eat1 anthers, suggesting delayed and abnormal PCD.

TEM analysis also revealed a delay in PCD in the mutant. At stage 10, the wild-type tapetal cells showed typical signs of PCD, such as condensed cytoplasm and the presence of poorly defined organelles. In contrast, eat1-1 tapetal cells contained abundant distinctive organelles, including the endoplasmic reticulum (ER), Golgi apparatus, a large number of ribosomes attached to the rough-surfaced ER, and an increased number of enlarged mitochondria, suggesting the retention of highly active metabolism and/or delayed cell death processes in the tapetum. At stage 11, the wild-type tapetal cells became more degenerated with the breakage of the nuclear membrane. By contrast, at this stage, the eat1-1 tapetal cells appeared less disintegrated, with intact nuclear membranes and increased number of mitochondria and vacuoles.At stage 10, the internal surface of the wild-type tapetum contained abundant Ubisch bodies . In eat1-1, however, the Ubisch bodies were abnormally round-shaped and covered by a layer of tubular structures. As a result, eat1-1 displayed irregular pollen exine patterning, such as no obvious inter-layer space between the nexine (foot layer) and tectum .

Expression

The result of qRT–PCR indicated that in the wild type, EAT1 is weakly expressed in roots, shoots and leaves, and highly expressed in the anther from stage 7 to 12, while a dramatic reduction in expression was detected in the anthers in all three eat1 alleles. In GUS stained EAT1pro:GUS transgenic flowers, GUS signals started to appear in anthers at stage 7, became stronger from stage 8 to 9, and were nearly undetectable at stage 12. Further in situ RNA hybridization indicated that EAT1 is highly expressed in the tapetum.

Two aspartic protease-encoding genes, OsAP25 and OsAP37, showed a significant reduction in expression, which was further confirmed by qRT–PCR.Given that bHLH transcription factors are predicted to regulate gene expression by binding to the E-box (CANNTG) in the promoter of target genes7, we used chromatin immunoprecipitation (ChIP)-PCR and electrophoretic mobility shift assay (EMSA) assays to test whether EAT1 could directly regulate OsAP25 and OsAP37.

OsAP25 and OsAP37 can promote cell death.

Evolution

Using the full-length EAT1 protein sequence to search available public databases and retrieved a total of 26 homologues from 10 diverse plant species from moss, pteridophytes, to angiosperms. Phylogenetic analysis showed that EAT1 and the 26 homologues each have a single HLH domain and a conserved but previously uncharacterized motif at the C-terminus, designated as DUF for Domain of Unknown Function. Moreover, EAT1 and three homologues from Sorghum bicolour (Sb04g030850),Zea mays (ZmLOC100282922) and Brachypodium distachyon (BradXP_003580474), respectively, were grouped in a subclade, suggesting that EAT1 and its homologues underwent diversification during grass evolution. EAT1 has one homologue from rice (OsbHLH142), which shares 40.8% identity with EAT1 in the HLH and DUF domains, and three homologues from Arabidopsis (AtbHLH091, AtbHLH089, AtbHLH010), which share an average of B40% identity with EAT1 in these two conserved domains. In addition, seven of the EAT1 homologues identified from Medicago truncatula, Populus trichocarpa, Glycine max, Arabidopsis and rice are also expressed in reproductive organs, such as inflorescences and male organs. These findings suggest a specific and possibly conserved role for EAT1 in male reproductive development in plants

Knowledge Extension

Tapetal cells is the innermost anther wall layer. After the meiosis of microspore mother cells it enter into programmed cell death (PCD). This degradation process is essential for microspore development and provide materials for pollen wall maturation; as a result, premature or delayed tapetal degradation causes male sterility. In rice Several transcriptional regulators have been reported to be associated with tapetal degeneration. GAMYB[3], UNDEVELOPED TAPETUM1[4], TAPETUM DEGENERATION RETARDATION(TDR)[2], PERSISTENT TAPETAL CELL 1 (PTC1)[5], and APOPTOSIS INHIBITOR5 (API5)[6], ETERNAL TAPETUM 1(EAT1)[1]. TDR, the rice ortholog of the Arabidopsis AMS protein[2]. PTC，the rice ortholog of the Arabidopsis MS1 protein[5]. EAT1 shares sequence similarity with AtbHLH089 and AtbHLH091[1,7]. GA is a kind of important plant hormone, plays an important role in the process of plant growth and development, in some experiment show that GA signal is necessary for entry of the tapetum into PCD[3,8].

Labs working on this gene

• State Key Laboratory of Hybrid Rice, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai, China
• Division of Plant Sciences,School of Biosciences, University of Nottingham, Loughborough, Leics, UK
• Department of Energy Plant Research Laboratory, Michigan State University, East Lansing, Michigan, USA.

References

[1] Ningning Niu. et al. EAT1 promotes tapetal cell death by regulating aspartic proteases during male reproductive development in rice. NATURE COMMUNICATIONS. 4:1445. (2013). DOI: 10.1038/ncomms2396

[2] Hui Li. et al. PERSISTENT TAPETAL CELL1 encodes a PHD-finger protein that is required for tapetal cell death and pollen development in rice. Plant Physiol. 156, 615–630 (2011).

[3] Aya, K. et al. Gibberellin modulates anther development in rice via the transcriptional regulation of GAMYB. Plant cell 21 1453–1472 (2009).

[4] Jung, K. H. et al. Rice Undeveloped Tapetum1 is a major regulator of early tapetum development. Plant Cell 10, 2705–2222 (2005).

[5] Li, H. et al. PERSISTENT TAPETAL CELL1 encodes a PHD-finger protein that is required for tapetal cell death and pollen development in rice. Plant Physiol. 156, 615–630 (2011).

[6] Li, X. W. et al. Rice APOPTOSIS INHIBITOR5 coupled with two DEAD-box adenosine 5’-triphosphate-dependent RNA helicases regulates tapetum degeneration. Plant Cell 23, 1416–1434 (2011).

[7] Li, X. X. et al. Genome-wide analysis of basic/helix-loop-helix transcription factor family in rice and Arabidopsis. Plant Physiol. 141, 1167–1184 (2006).

[8] Andrew R.G. Plackett. et al. Gibberellin control of stamen development: a fertile field. cell. 16: 568-578 (2011).