ABSTRACT
The factors and mechanisms involved in vacuolar transport in plants, and in particular those directing vesicles to their target endomembrane compartment, remain largely unknown. To identify components of the vacuolar trafficking machinery, we searched for Arabidopsis modified transport to the vacuole (mtv) mutants that abnormally secrete the synthetic vacuolar cargo VAC2. We report here on the identification of 17 mtv mutations, corresponding to mutant alleles of MTV2/VSR4, MTV3/PTEN2A MTV7/EREL1, MTV8/ARFC1, MTV9/PUF2, MTV10/VPS3, MTV11/VPS15, MTV12/GRV2, MTV14/GFS10, MTV15/BET11, MTV16/VPS51, MTV17/VPS54, and MTV18/VSR1 Eight of the MTV proteins localize at the interface between the trans-Golgi network (TGN) and the multivesicular bodies (MVBs), supporting that the trafficking step between these compartments is essential for segregating vacuolar proteins from those destined for secretion. Importantly, the GARP tethering complex subunits MTV16/VPS51 and MTV17/VPS54 were found at endoplasmic reticulum (ER)- and microtubule-associated compartments (EMACs). Moreover, MTV16/VPS51 interacts with the motor domain of kinesins, suggesting that, in addition to tethering vesicles, the GARP complex may regulate the motors that transport them. Our findings unveil a previously uncharacterized compartment of the plant vacuolar trafficking pathway and support a role for microtubules and kinesins in GARP-dependent transport of soluble vacuolar cargo in plants.
Subject(s)
Arabidopsis Proteins/metabolism , Protein Transport/genetics , Vacuoles/metabolism , Vesicular Transport Proteins/genetics , Alleles , Arabidopsis/genetics , Arabidopsis/metabolism , Arabidopsis Proteins/genetics , Cytoplasmic Vesicles/genetics , Cytoplasmic Vesicles/metabolism , Endoplasmic Reticulum/genetics , Endoplasmic Reticulum/metabolism , Golgi Apparatus/genetics , Golgi Apparatus/metabolism , Kinesins/genetics , Kinesins/metabolism , Microtubules/genetics , Microtubules/metabolism , Multivesicular Bodies/genetics , Multivesicular Bodies/metabolism , Mutation , Vacuoles/genetics , Vesicular Transport Proteins/metabolismABSTRACT
Lipid droplets (LDs) are ubiquitous organelles in plant cells, but their physiological roles are largely unknown. To gain insight into the function of LDs in plants, we have characterized the Arabidopsis homologs of SEIPIN proteins, which are crucial factors for LD biogenesis in yeast and animals. SEIPIN1 is expressed almost exclusively in embryos, while SEIPIN2 and SEIPIN3 have broader expression profiles with maximal levels in embryos and pollen, where LDs accumulate most abundantly. Genetic analysis demonstrates that all three SEIPINs contribute to proper LD biogenesis in embryos, whereas in pollen, only SEIPIN2 and SEIPIN3 play a significant role. The double seipin2 seipin3 and triple seipin mutants accumulate extremely enlarged LDs in seeds and pollen, which hinders their subsequent mobilization during germination. Interestingly, electron microscopy analysis reveals the presence of nuclear LDs attached to type I nucleoplasmic reticulum in triple seipin mutant embryos, supporting that SEIPINs are essential for maintaining the correct polarity of LD budding at the nuclear envelope, restricting it to the outer membrane. In pollen, the perturbations in LD biogenesis and turnover are coupled to reduced germination in vitro and with lower fertilization efficiency in vivo. In seeds, germination per se is not affected in seipin2 seipin3 and triple seipin mutants, but there is a striking increase in seed dormancy levels. Our findings reveal the relevance of SEIPIN-dependent LD biogenesis in pollen transmission and in adjusting the timing of seed germination, two key adaptive traits of great importance in agriculture.
Subject(s)
Arabidopsis Proteins/metabolism , Arabidopsis/genetics , Lipid Droplets/metabolism , Arabidopsis/physiology , Arabidopsis Proteins/genetics , Chloroplasts/metabolism , GTP-Binding Protein gamma Subunits/genetics , GTP-Binding Protein gamma Subunits/metabolism , Germination , Pollen/genetics , Pollen/physiology , Seeds/genetics , Seeds/physiologyABSTRACT
The potyviruses Plum pox virus (PPV) and Tobacco vein mottling virus (TVMV) have distinct host ranges and induce different symptoms in their common herbaceous hosts. To test the relevance of the P1 protein in host compatibility and pathogenicity, hybrid viruses were constructed in which the P1 coding sequence of PPV was completely or partially replaced by the corresponding sequences from TVMV. Infections induced by these chimeric viruses revealed that the TVMV P1 and a PPV/TVMV hybrid P1 proteins are functionally equivalent in herbaceous plants to the P1 protein of a PPV isolate adapted to these hosts, in spite of having high sequence divergence. Moreover, the presence of TVMV P1 sequences enhanced the competence of a low-infectivity PPV-D-derived chimera in Nicotiana clevelandii. Conversely, all PPV/TVMV hybrids were unable to infect Prunus persicae, a specific host for PPV, suggesting that TVMV P1 is not functionally competent in this plant. Together, these data highlight the importance of the P1 protein in defining the virus host range.
