| Adaptor proteins(Aps)are heterotetrameric complexes.Eukaryotic cells contain five types of AP complexes(AP-1 to AP-5),which play critical roles in membrane trafficking.Recent studies have shown that Arabidopsis AP-1 and AP-2 mediate cargo selection and transportation in clathrin-dependent trafficking at the trans-Golgi network(TGN)and the plasma membrane(PM),respectively.AP-1 is required for trafficking to the PM and vacuoles,and AP-2 mediates endocytosis.AP-1 complex consists of AP1γ,AP1/2β,AP1μ,and AP1σ;whereas AP-2 is composed of AP2α,AP1/2β,AP2μ and AP2σ.In general,subunits(adaptins)are unique for each AP complex,with the exception of β subunits that are shared by AP-1 and AP-2 in some invertebrates.The roles of some AP-1 and AP-2 adaptins in vegetative and reproductive development have been characterized in plants.However,the function of the large β subunits and whether they are shared by the two AP-1 and AP-2 complexes in plants are currently unknown.Pollen development is regulated by both the pollen itself and the tapetum.The PM-localized ABCG(ATP binding cassette G)family transporters ABCG9 and ABCG16 of tapetal cells transport nutrients for the pollen wall formation during pollen development,but it is not clear that how these transporters are transported to the PM of tapetal cells.In this study,by using bioinformatics,biochemistry,genetics,cell biology,and pharmacology,we demonstrated that the AP1/2β subunit is shared by AP-1 and AP-2 complexes in Arabidopsis;elaborated the important role of AP1/2β adaptor protein in plant reproduction and development and its special function in specific cell types;and deeply analyzed the new regulation mechanisms of tapetum-controlled pollen development mediated by vesicle trafficking.The main results are as follows:1.Bioinformatic analysis showed that the AP1/2β subunit was encoded by β1and β2 in Arabidopsis genome.Similar to mammalian AP1β and AP2β,Arabidopsisβ1 and β2 also contain the Alpha＿adaptin C2 domain and the clathrin-binding domain B2-adapt-app＿C.Alignment analysis of amino acid sequence showed that the Arabidopsis β1 and β2 share ～93% amino acid identity with each other.2.Biochemical and cell biology analyses showed that both β1 and β2 could coassemble with the other three subunits of AP-1 and AP-2 complexes,and participated in endocytosis and exocytosis in root tip cells.3.RT-q PCR and GUS staining analyses showed that β1 was mainly expressed in the tapetum,pollens and pollen tubes,while β2 was mainly expressed in embryos.Colocalization analysis showed that β1 and β2 were localized to TGN and PM in root and pollen cells,but only to TGN in tapetal cells.4.Disruption of β1 and β2 affects the plant vegetative and reproductive growth,such as delayed growth of rosette leaves and plants,decreased seed setting rate,reduced pollen germination rate,and slowed pollen tube elongation.Self and reciprocal cross analyses showed that the simultaneous deletion of β1 and β2 resulted in the lethality of male gametophytes and abnormal transmission of female gametophytes.Further study found that loss of AP1/2β function led to abnormal pollen wall formation.5.Genetic rescue experiments showed that β1 played a critical role in the tapetum,and exogenous expression of β1 in the tapetum could restore the pollen defect of the β1-1 β2-1/+ mutant,but not the reduced pollen germination rate.6.The loss of β1 and β2 function led to the blocked transportation of the ABCG9 and ABCG16 transporters to the PM of the tapetal cells,thereby ABCG9 and ABCG16 were retained on the intracellular TGN.Further biochemical analyses showed that β1 and β2 can directly interact with ABCG9 and ABCG16 and mediated the exocytosis of these two transporters.These results showed that AP1/2β play a key role in the pollen wall formation by regulating vesicle trafficking activities in tapetal cells. |
