Arabidopsis EFD Gene Is Required For Pollen Exine Development

Exine, a chemically and physically resistant structure, is the outermost layer of pollen wall. Exine protects pollen grains from harsh conditions and prevents water loss from pollen grains to keep them viable. Exine also provides physicl support to pollen vegetative cell containing sperm. Besides, proteins existed in exine are proved to be important for recognization and adherence between pollen and stigma. Thus, the exine plays very important roles in pollen development and fertilization.In previous study in our lab, we have found out several genes that specifically expressed in egg and zygoty in tobacco using suppression subtractive hybridization, and identified one of those genes, NtZC8, that specifically expressed in anther and zygote. Bioinformatics analysis indicates that NtZC8is likely to encode a methyltransferase that is invoved in pollen and zygote development.To seek the role of methyltransferase in pollen and zygote development we found six genes in Arabidopsis, which are high homology to NtZC8. Accordinding to their expression pattern revealed by RT-PCR, we then chose one of these genes, named EFD, for further study. The main results are as follows:EFD encodes a323amino acid protein with a methyltransferase domain, structural analysis showed that EFD is conserved in a typical methyltransferase structure and a signature motif of DNA methytransferase is also found in EFD. Evolutionary analysis also indicates that EFD has the closest relationship with proteins of Dnmt3family, which usually function in de novo methylation.Expression vector with EFD fused with sGFP driven by EFD native promoter is constructed and bombarded into onion epidermal cells to detect subcellular location of EFD protein, the result shows that EFD fusion protein is only detected in the nucleus, which is consistent to the nuclear location of DNA methytransferases.Spatial and temporal patterns of EFD expression during anther development are determined by GUS reporter gene. The results show that EFD is strongly expressed in early-staged buds of inflorescence, in both stamens and pistils; sections of stained buds revealed that EFD is expressed in tapetum and microsporocyte, tetrad and early-staged microspore. Later, it is expressed only in tapetum when mitosis begins. The GUS report gene reveals that EFD may be involved in early development in microspores and pistils.To study the functions of EFD, we get a knock-out mutant of EFD and observe whether there are abnormalities in this mutant. We first find that fertility of efd is severely affected, complementation of EFD can rescue this sterility and reciprocal corsses show that male reproductive system is impaired. These results indicate that loss of EFD fuction leads to male steritlity.Ananlysis of efd siliques reveals that63.76%efd siliques on main stalk and75.89%on lateral stalk are totally sterility and only15.17%efd siliques on main stalk and7.92%on lateral stalk look like normal wild type siliques. We also find that aborted siliques are produced in the early stage of florescence and siliques with viable seeds are only produced at the late stage of florescence.We staied nucleus of microspores and pollen grains and find that efd microspores can go through meiosis and mitosis to develop into trinucleate pollen, which indicates that nucleus division is normal in efd microsporogenesis and pollen development.We made anther cross sections to observe anther development in efd mutant. At stage7, the callose wall of efd tetrad looked thinner than that in wide type. At stage8when callose wall degenerated and microspores were released, microspores of efd looked condensed and the callose degeneration was not coinstantaneous in different loculus in the same anther; from stage9to11, some efd microspores looked empty or irregular in shape. At stage12, there were some tapetum residents in anther locule while some pollen grains looked like normal wild type pollen grains in efd mutant.We anatomized anthers of both wild type and efd and compared them with each other. At the late tetrad stage, tetrads from efd were surrounded by the callose layer which was not as thick as that of wild type, and cells in tetrad were not well isolated by the callose wall. When uni-nucleus microspores released from tetrads, efd microspore surface looked coarse. When the mitosis began, some efd microspores could go through such process and developed into mature pollen grains while some stoped at uni-nuclei microspore stage and some collapse. These results indicate that microsporogenesis of efd is aberrant since tetrad pollen stage and callose wall formation is affected in efd, which may lead to exine defects.FDA stating showed that75%mature efd pollen was viable. But both in vivo and in vitro pollen germination were severaly impaired in efd, no pollen tube could be observed in siliques2days after fertilization, and only22.86%efd pollen grains could germinate in vitro. These results indicate efd pollen development is obviously affected so that mature pollen can hardly germinate to carry out double fertilization although most efd pollens are alive.Environmental scanning electron microscopy showed that efd pollen grains were aborted and irregular in shape, the surface of mutant pollen grains were smooth, some membrane-like fragments, which looked like tapetum remnants, were adherent to pollen grains. This resulte indicates that the exine formation is affected in efd mutant. Transmission electron microscopy confirmed that exine was almost totally missing in efd mutant. In efd, callsoe deposited to form a thin callose wall, but no obviouse primexine structure could be observed between callose layer and plasma membrane, no exine-like strcutre can be observed when microspores were released from tetrads; debris of tapetal cells could be found in efd anther, some of which attached to the surface of efd pollen grains and the others just remained in the anther locule. Thus, the failure of primxine formation is supposed to be an important factor resulting in exine development defects.We also investigated the transcriptome of edf and found that most chaning genes are involved in anther and pollen development. Some genes known to be involved in Arabidopsis callose wall formation, primexine formation, sporopollenin syntheisis or transfer are found down-regulated in efd mutant, which indicates that three key factors for exine formation, callose, primexine and sporopollenin are all affected in efd mutant and lead to failure of exine formation in efd.