Functional Analysis Of The Arabidopsis RUS4 Gene In Anther Dehiscence And Pollen Development

ROOT UV-B SENSITIVE4(RUS4)is a member of the Domain of Unknown Function 647(DUF647)protein family that is conserved in diverse eukaryotic organisms,but has no known function.The Arabidopsis genome encodes six DUF647 family members,namely RUS1(At3g45890),RUS2(At2g31190),RUS3(At1g13770),RUS4(At2g23470),RUS5(At5g01510)and RUS6/EMB1879(At5g49820).Two of these,RUS1/WXR3 and RUS2/WXR1,play vital roles in Arabidopsis early seedling morphogenesis and development;they are thought to interact and are required for UV-B response and polar auxin transport.However,the biological functions of the remaining four members of the DUF647 family have not yet been characterized.In this study,we generated constitutive knockdown RUS4 plants using an artificial micro RNA(ami RNA)approach,and demonstrated that the DUF647-containing gene RUS4 plays an essential role in anther dehiscence and pollen development.The results are as follows:1.Knockdown of RUS4 leads to anther indehiscence in Arabidopsisq PCR showed that the expression of RUS4 in ami R-rus4 flower buds was markedly downregulated,leading to severely reduced fertility.Reciprocal crosses between wild-type and ami R-rus4 plants showed that the semi-sterility was due to the defective stamen development.Light microscopy of anther sections revealed a significantly reduced secondary wall thickening in the anther endothecium.In ami R-rus4 flower buds,the expression of NAC SECONDARY WALL–PROMOTING FACTOR1(NST1)and NST2,which have been shown to regulate secondary wall thickenings in the endothecium,were strongly down-regulated.Furthermore,the expression of IRREGULAR XYLEM1(IRX1),IRX3,IRX5 and IRX8,which are responsible for cellulose synthesis and xylan synthesis,were down-regulated.However,the expression of IRX4,which has been shown to be associated with lignin monomer formation,did not show a major change.The reduced fertility of ami R-rus4 plants was rescued by 35Spro:RUS4.Overexpression of RUS4 increased NST1 and NST2 transcripts,leading to enhanced secondary thickening in anther endothecium and filament.However,no ectopic secondary thickening was seen in RUS4 overexpression lines.2.Knockdown of RUS4 impairs pollen developmentAlexander staining showed that wild-type pollen grains were stained purple,while most ami R-rus4 pollen grains were stained green,implying that pollen viability was compromised in RUS4 knockdown plants.Pollen taken from ami R-rus4 anthers at anthesis showed in vitro germination 4.4% compared with 72.7% germination for pollen from wild-type plants.Pollen tube length of ami R-rus4(227.0 ?m)was significantly shorter than that of wild type(394.4 ?m);the pollen tube growth defect was further validated in vivo germination by aniline blue staining.Transmission electron microscopy observation indicated that wild-type elaioplasts and tapetosomes deposited onto pollen surface and filled the cavities of the exine to form pollen coat.However,in ami R-rus4 pollen,these two organelles were absent within the spaces created by exine and aggregated in the locule.DPBA and Nile red staining revealed that ami R-rus4 pollen lacked pollen coat composed mainly of flavonoids and lipids,which is the main reason for reduced pollen viability.3.RUS4 is expressed throughout the plants,but is highly expressed in anthersSemi-quantitative PCR analysis showed that RUS4 transcript was detected in all plant organs tested,including roots,stems,leaves,seedlings,floral buds and open flowers.Further GUS staining indicated that RUS4 was expressed in the cell wall and vascular bundle of these tissues,but was highly expressed in anthers.In anthers younger than stage 10,RUS4 was mainly expressed in the tapetum;the strongest expression of RUS4 was detected in anthers at stage 11 and 12;RUS4 expression declined in anthers at stage 13 and 14,and was present mainly in pollen.In addition,RUS4 was expressed in pollen exine and pollen coat.Observation of 35Spro:RUS4-GFP plants showed that RUS4 was localized to the chloroplast.Taken together,these data suggest that RUS4 plays an important role in anther dehiscence and pollen development,both of which contribute to the male sterility phenotype of ami R-rus4 plants.The expression pattern is in agreement with the proposed function of RUS4 in anther and pollen development.