AP-3-mediated Arabidopsis PAT10 Vacuolar Trafficking And Pollen Tube Growth

Plant vacuoles as special and versatile organelles are critical for plant growth and responses to environment by maintaining ion homeostasis and turgor regulation whereas luminal proteins inside the vacuoles participate in nutrient recycling and signal termination.Vacuolar proteins are transported from the endoplasmic reticulum(ER)via multiple routes in plants.Two classic routes bear great similarity to other phyla with major regulators known,such as COP? and Rab5 GTPases.By contrast,vacuolar trafficking mediated by adaptor protein-3(AP-3)or the ER to vacuole direct route has few recognized cargos and none of the regulators.In this study,we find that the tonoplast targeting of Arabidopsis PROTEIN S-ACYL TRANSFERASE 10(PAT10)is not impaired in Rab5-DN,implying that PAT10 is transported to the vacuolar membrane via unconventional routes.To determine the trafficking route of PAT10 to the tonoplast and in search of novel regulatory components in vacuolar trafficking routes,we performed a fluorescence-based forward genetic screen,genetic and pharmacological assays.The main results and conclusions presented in this thesis are as follows:(1)Targeting of PAT10 to the tonoplast is independent of classic Rab5-mediated trafficking routesBecause the classic post-Golgi trafficking routes both require the activity of Rab5,we thus expressed a dominant negative(DN)Rab5 to inhibit Rab5-dependent post-Golgi trafficking.To verify that the genetic interference worked as it should,we examined the effect of Rab5-DN on INT1(Inositol Transporter 1)whose tonoplast localization relies on Rab5-dependent vacuolar trafficking.As contrast to the tonoplast association of INT1 in wild type,both the PM and the tonoplast were labeled with INT1 signals in cells expressing Rab5-DN.The expression of Rab5-DN also did not alter the tonoplast association of PAT10.These results indicated that PAT10 did not take the classic Rab5-depedent vacuolar trafficking routes.(2)The enzymatic domain and C-terminal non-catalytic domain are both required for its subcellular targetingThe construction of PAT10 was simple,which consist of four transmembrane domain,enzymatic domain,N-terminal and C-terminal domain.In this study,we performed domain dissection by removing individual domains,such as N-ternimal,En,and C-terminal regulatory domain and fused with GFP.Following their subcellular localization,we demonstrated that removing N-terminal of PAT10 did not alter the tonoplast targeting,while deletion of En or C-terminal domain impaired the trafficking of PAT10.These results indicated that the enzymatic domain and C-terminal non-catalytic domain are both required for its subcellular targeting.(3)The tonoplast targeting of PAT10 is mediated by AP-3To determine the trafficking route of PAT10 to vacuoles and identify novel regulatory components,we performed fluorescence-based forward genetic screening.Because PAT10 is expressed in pollen and pollen tubes,an in vitro pollen germination assay at the M1 generation would be able to detect mutations that resulted in the mis-targeting of PAT10.We screened around 3000 M1 plants from an ethyl methane-sulfonate(EMS)-mutagenized PAT10g-GFP;pat10-1 population using fluorescent microscopy for pollen tubes and found two mutants,named reva1-1 and reva1-2.Map-based cloning and sequencing,we identified a base pair change in the coding sequence of At1g48760,which encodes the ? subunit of adaptor protein 3(AP-3?).Allele test and complementation assays demonstrated that the tonoplast association of PAT10 relies on AP-3 and mutations at AP-3 resulted in retention of PAT10 at the Golgi.(4)AP-3-dependent vacuolar trafficking involves COP?COP? mediates vesicle trafficking from ER to the Golgi.Sar1 is a COP?-recruiting guanosine triphosphate hydrolyzing protein(GTPase).In fact,the expression of DN-Sar1 altered targeting of INT1,which confirmed the functionality of DN-Sar1.As AP-3-mediated tonoplast protein cargo,SUC4 and PAT10 were relocalized into cis-Golgi stacks in AP-3 mutants.To identify whether COP? mediated the vesicle trafficking between ER and Golgi,we introduce PAT10-GFP and GFP-SUC4 into DN-Sar1 and find that DN-Sar1 reduced the tonoplast association of PAT10 and SUC4.These results suggested that AP-3-mediated tonoplast proteins were selectively transported at the ER by COP?.(5)HOPS complex is required for AP-3-mediated vacuolar traffickingIn yeast,the homotypic fusion and vacuole protein sorting(HOPS)complex controls membrane fusion at the vacuoles.We thus considered the possibility whether the function of HOPS was required for AP-3-mediated tonoplast targeting.It was recently reported that functional loss of VPS41 resulted in male gametophytic lethality by interfering pollen tube growth.To examine the effect of VPS41 functional loss on the tonoplast targeting of AP-3 cargos,we introduced PAT10g-GFP and CBL2-RFP into vps41-1/+.PAT10 was distributed into cytoplasmic vesicles and CBL2 was detected mostly at the PM and cytoplasmic vesicles in vps41-1.Oregon green staining showed that vacuolar morphology in vps41-1 was affected.These data indicated that VPS41-participated HOPS complex is crucial for tonoplast targeting from both AP-3-mediated.(6)AP-3 mediates pollen tube growth by coordinating vacuolar targeting and organizationPollen tube growth is an essential step leading to successful plant reproduction.Vacuolar trafficking and dynamic organization are important for pollen tube growth although key proteins involved are unclear.We report here that adaptor protein-3(AP-3)and its two tonoplast cargos are critical for pollen tube growth.Arabidopsis AP-3 regulates tonoplast targeting of several cargo proteins,two of which,PROTEIN S-ACYL TRANSFERASE10(PAT10)and the R-SNARE protein VAMP711,participates in calcium signaling and vesicular fusion respectively.Functional loss of either AP-3 subunit caused reduced fertility.We show that reduced male transmission of the ap-3 mutants was due to compromised pollen tube growth both in vitro and in vivo whereas pollen development was normal.Functional loss of PAT10 and VAMP711 caused a similar reduction in pollen tube growth,suggesting that the tonoplast association of PAT10 and VAMP711 mediated by AP-3 is crucial for this process.We further show that AP-3 loss-of-function affected vacuolar organization of pollen tubes such that vacuoles of mutant pollen tubes were less complex tubules attached with bulbous structures.Results reported here revealed the role of AP-3 in plant reproduction and provided insights into the role of vacuoles in polarized cell growth.