The Function Of Arabidopsis RUS4 Gene In Pollen Maturation

The DUF647(Domain of unknown function 647)domain protein family is found in diverse eukaryotic organisms,including plants,animals,and some fungi.There are six members of the DUF647-containing proteins in Arabidopsis.Two members of the DUF647 domain protein,RUS1(ROOT UVB SENSITIVE 1)and RUS2(ROOT UVB SENSITIVE 2),have previously been associated with root UV-B-sensing pathway that play a major role in Arabidopsis early seedling morphogenesis and development.The additional RUS genes were named based on their genome locations: RUS3(At1g13770),RUS4(At2g23470),RUS5(At5g01510)and RUS6 / EMB1879(At5g49820).However,the biological functions of the remaining four members of the DUF647 family have not yet been characterized.We have previously generated constitutive knockdown Arabidopsis DUF647 family gene RUS4 plants,called ami R-RUS4,using an artificial micro RNA(ami RNA)approach.The ami R-RUS4 plants showed severely reduced male fertility with defect in anther dehiscence.However,ami R-RUS4 plants have additional phenotypes that we investigate in this study.Here,we show that pollen grains from ami R-RUS4 plants was strongly reduced.Promoter-reporter analysis shows that RUS4 is highly expressed in tapetal layer,developing microspores,mature and germinating pollen,strongly suggesting its role in the process of pollen maturation.To determine the nature of the defects in the pollen coat in the ami R-RUS4 pollen grains,we examined the flavonoids and lipids that are major components of the pollen coat by confocal laser scanning microscopy(CLSM)after diphenylboric acid 2-aminoethyl ester(DPBA)and Nile Red double staining.We observed a clear reduction in both flavonoids and lipids levels in ami R-RUS4 pollen compared with the wild-type.These observations indicate that pollen coat defects of the ami R-RUS4 plants was due,at least in part,to deficient lipids and flavonoids content in the pollen coat.To investigated whether knockdown of RUS4 may cause altered auxin responses,we crossed the DR5:GUS plants to ami R-RUS4 plants,and then analyzed the auxin-responsive DR5 promoter activity in the ami R-RUS4 background by GUS staining.Compared with the DR5:GUS plants in wild-type background,the DR5:GUS activity in the ami R-RUS4 background was more weak in both roots and anthers.Our results suggest that RUS4 may be involved in auxin-mediated pollen maturation in Arabidopsis.