Os02g0313400

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Annotated Information

Function

Please input function information here. APOPTOSIS INHIBITOR5 (API5), which encodes a putative homolog of antiapoptosis protein Api5 in animals,with 537 amino acids, including a nuclear localization signal, a LXXLL motif, and a transcription activation domain(Figure1), results in delayed degeneration of the tapetum due to inhibition of the tapetal PCD process leading to defects in formation of male gametophytes.A functional transcription activation domain might exist in Os API5 and that the LXXLL motif is a likely suppressor of the transcription activationOs API5 interacts with two DEAD-box ATP-dependent RNA helicases, API5-INTERACTING PROTEIN1 (AIP1) and AIP2 which is directly regulates the expression of CP1. Summing up, the API5 is required for normal expression of CP1 and that AIP1/2 directly regulates the expression of CP1, which might regulate tapetum degeneration during pollen development. Zhao002.jpg

Mutantion

Please input mutantion information here. The api5-1 mutant plants were normal during vegetative and floral development stages but produced smaller anthers (Figures 1A to 1D). The pollen grains of api5-1 lacked starch, as shown by staining with iodine (Figures 1E and 1F).Degeneration of the tapetum is delayed in api5-1(Figures 1G to 1N),but that pollen wall development and orbicule formation are not affected(Figures 2A to 2P). The cross sections of anthers from the wild-type and api5-1 mutant plants according to a recent classification consisting of 14 anther developmental stages . The typical anther structure with pollen mother cells surrounded by four layers of somatic cells is differentiated from the anther primordia during stages 1 to 5. Subsequently, the pollen mother cells undergo meiosis and dyads and tetrads of haploid microspores are then formed during stages 7 to 8. At stage 9, the middle layer is degenerated and almost invisible; the tapetum is more condensed, with dark staining, and has started to degenerate. Young microspores are finally released from the tetrad. No obvious differences were observed in api5-1 anthers compared with the wild type from stages 4 to 9 (Figures 1G and 1H). However, the morphologic defects were observed at stage 10. At this stage, vacuolated microspores and more condensed tapetum were visible in wildtype anthers (Figure 1I), whereas in anthers of api5-1, the anther lobes were filled with irregular microspores and debris and the tapetum seemed slightly swollen (Figure 1J). During stage 11, the tapetum and the endothecium were completely degenerated,whereas the microspores completed mitosis in the wild type (Figure 1K). At this stage in api5-1, highly condensed cytoplasm and intact cell walls of tapetal cells were observed, along with fewer microspores, which had irregular shapes, and more debris and unknown particles in anther lobes (Figure 1L). At stage 12,the wild-type anther wall layer was completely degraded except for the epidermis and endothecium layer, and mature pollen grains were formed inside anther lobes (Figure 1M). However, the middle layer and belatedly degenerated tapetal cells were still visible in api5-1 anthers, which were filled with shrunken,empty sterile microspores (Figure 1N). These observations indicate that degeneration of the tapetum is delayed in api5-1.To understand better the api5-1 anther defects, we compared anthers between the wild type and api5-1 at the level of ultrastructureusing transmission electron microscopy (TEM). Consistent with the analysis for semi-thin sections of anthers, no obvious morphological changes were observed between anthers of the wild type and api5-1 up to stages 8 and 9 (Figures 2A to2D). At stage 10, the tapetal cells were degenerated and it was difficult to distinguish organelles such as the nuclei and mitochondria from cellular debris in wild-type anthers (Figures 2E and 2G). However, in api5-1 anthers, intact membrane and mitochondria were visible, while the tapetal cells were vacuolated (Figures 2F and 2H). At stage 11, most of the tapetal cells had disappeared and almost all organelles were degenerated in the remaining tapetal cells of the wild-type anthers (Figures 2I and 2K). At the same stage, the condensed cytoplasm with a surrounding membrane was detached from the tapetal cell wall in api5-1 (Figure 2J), and within the condensed cytoplasm, intact nuclear membranes and mitochondria were also observed (Figure 2L). At stage 12, the middle layer and tapetal cells were completely degraded in the wild-type anthers (Figure 2M), and mature pollen grains were produced (Figure 2O). However, the middle layer and tapetal cells persisted in api5-1 (Figure 2N), and highly condensed cytoplasm surrounding the obvious nuclear membrane was observed in api5-1 anthers (Figure 2P). Despite the presence of nuclear membrane, most of the organelles were degraded at this stage. However, the cells of the epidermis in api5-1 anthers were expanded. Although the middle layer and tapetum developed abnormally in api5-1 anthers, orbicules and the cuticle of the outer epidermis cell wall seemed normally formed (Figures 2M and 2N). Furthermore, pollen wall development was not affected in api5-1 compared with the wild-type anthers during stages 8 to 12 .


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Expression

Please input expression information here. API5 is expressed in all tissues analyzed, including root, leaf blades, leaf sheath, culms, and panicles (Figure 5A). Unexpectedly, it was expressed more highly in roots than in panicles.Its transcripts were detected in the parietal anther wall layers and microsporocyte, with stronger signal in tapetum in all the examined stages (Figures 5B to 5D). In vegetative tissues, transcripts of API5 were detected in epidermal cells in roots (Figures 5F and 5G), vascular tissues in culms (Figure 5I), and mesophyll cells in leaf blades (Figure 5J). As expected, sense probe used as a negative control gave rise to only a faint, almost undetectable, signal (Figures 5E, 5H, and 5K). The preferential expression of API5 in the tapetum is consistent with its inferred function in the differentiation of the tapetum during anther development. The expression of API5 in vegetative tissues suggests that it has other functions in growth and development that may be complemented by other gene product(s) in the api5 mutant.

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Using transgenic lines harboring the full-length coding sequence (CDS) of Os API5 driven by the maize Ubiquitin promoter in the genetic background of japonica cultivar Zhonghua11 to gain more knowledge regarding the function of API5. The relative expression level of API5 between transgene-positive and -negative plants of the T1 generation derived from a single copy transgenic T0 plant was verified by qRT-PCR. The amount of API5 transcripts in positive plants was significantly enhanced (>80-fold) compared with negative plants . The normal fertility of both positive and negative plants suggested that the development of reproductive organs was normal in API5-overexpressing plants .The morphology of the positive plants relative to their negative counterparts at the seedling and reproductive stages, and also the cytologic characteristics of roots, culms, and leaves were no obvious differences between them.

Evolution

Please input evolution information here.

API5 proteins exist in the majority of eukaryotes Make phylogenetic tree using API5 homologs from protists, plants, and animals. 32 API5 protein sequences were classified into three clusters according to the phylogenetic tree (Figure 6). There are three major groups in the phylogenetic analyses may indeed reflect the systematization of animals, plants, and protists.

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Labs working on this gene

Please input related labs here.National Key Laboratory of Crop Genetic Improvement and National Center of Plant Gene Research (Wuhan),Huazhong Agricultural University

References

Please input cited references here.Xingwang Li, Xinqiang Gao, YiWei, Li Deng, Yidan Ouyang, Guoxing Chen, Xianghua Li, Qifa Zhang,and ChangyinWu.Rice APOPTOSIS INHIBITOR5 Coupled with Two DEAD-Box Adenosine 59-Triphosphate-Dependent RNA Helicases Regulates Tapetum Degeneration.The Plant Cell, Vol. 23: 1416–1434, April 2011.

Structured Information