ABSTRACT
Well-defined motifs often make it easy to investigate protein function and localization. In plants, peroxisomal proteins are guided to peroxisomes mainly by a conserved type 1 (PTS1) or type 2 (PTS2) targeting signal, and the PTS1 motif is commonly used for peroxisome targeting protein prediction. Currently computational prediction of peroxisome targeted PTS1-type proteins are mostly based on the 3 amino acids PTS1 motif and the adjacent sequence which is less than 14 amino acid residue in length. The potential contribution of the adjacent sequences beyond this short region has never been well investigated in plants. In this work, we develop a bi-profile Bayesian SVM method to extract and learn position-based amino acid features for both PTS1 motifs and their extended adjacent sequences in plants. Our proposed model outperformed other implementations with similar applications and achieved the highest accuracy of 93.6% and 92.6% for Arabidosis and other plant species respectively. A large scale analysis for Arabidopsis, Rice, Maize, Potato, Wheat, and Soybean proteome was conducted using the proposed model and a batch of candidate PTS1 proteins were predicted. The DNA segments corresponding to the C-terminal sequences of 9 selected candidates were cloned and transformed into Arabidopsis for experimental validation, and 5 of them demonstrated peroxisome targeting.
Subject(s)
Arabidopsis/genetics , Computer Simulation , Peroxisomes/metabolism , Plant Proteins/genetics , Receptors, Cytoplasmic and Nuclear/metabolism , Algorithms , Amino Acid Motifs , Amino Acids/metabolism , Arabidopsis Proteins/genetics , Bayes Theorem , Computational Biology/methods , Genome, Plant , Microscopy, Confocal , Oryza/genetics , Peroxisome-Targeting Signal 1 Receptor , Probability , Protein Sorting Signals/genetics , Proteome , Solanum tuberosum/genetics , Glycine max/genetics , Triticum/genetics , Zea mays/geneticsABSTRACT
Programmed cell death (PCD)-triggered degradation of plant tapetum is essential for microspore development and pollen coat formation; however, little is known about the cellular mechanism regulating tapetal PCD Here, we demonstrate that Rab7-mediated vacuolar transport of tapetum degradation-related cysteine proteases is crucial for tapetal PCD and pollen development in Arabidopsis (Arabidopsis thaliana), with the following evidence: (1) The monensin sensitivity1 (mon1) mutants, which are defective in Rab7 activation, showed impaired male fertility due to a combined defect in both tapetum and male gametophyte development. (2) In anthers, MON1 showed preferential high level expression in tapetal cell layers and pollen. (3) The mon1 mutants exhibited delayed tapetum degeneration and tapetal PCD, resulting in abnormal pollen coat formation and decreased male fertility. (4) MON1/CALCIUM CAFFEINE ZINC SENSITIVITY1 (CCZ1)-mediated Rab7 activation was indispensable for vacuolar trafficking of tapetum degradation-related cysteine proteases, supporting that PCD-triggered tapetum degeneration requires Rab7-mediated vacuolar trafficking of these cysteine proteases. (5) MON1 mutations also resulted in defective pollen germination and tube growth. Taken together, tapetal PCD and pollen development require successful MON1/CCZ1-mediated vacuolar transport in Arabidopsis.
Subject(s)
Apoptosis , Arabidopsis Proteins/metabolism , Arabidopsis/cytology , Arabidopsis/metabolism , Guanine Nucleotide Exchange Factors/metabolism , Pollen/cytology , Pollen/metabolism , Vesicular Transport Proteins/metabolism , rab GTP-Binding Proteins/metabolism , Cysteine Proteases/metabolism , Enzyme Activation , Genes, Dominant , Germination , Models, Biological , Mutation/genetics , Plant Infertility/genetics , Plant Leaves/metabolism , Pollen/ultrastructure , Pollen Tube/growth & development , Pollen Tube/metabolism , Protoplasts/metabolism , Vacuoles/metabolism , rab7 GTP-Binding ProteinsABSTRACT
Spatially and temporally regulated membrane trafficking events incorporate membrane and cell wall materials into the pollen tube apex and are believed to underlie the rapid pollen tube growth. In plants, the molecular mechanisms and physiological functions of intra-Golgi transport and Golgi integrity maintenance remain largely unclear. The conserved oligomeric Golgi (COG) complex has been implicated in tethering of retrograde intra-Golgi vesicles in yeast and mammalian cells. Using genetic and cytologic approaches, we demonstrate that T-DNA insertions in Arabidopsis COG complex subunits, COG3 and COG8, cause an absolute, male-specific transmission defect that can be complemented by expression of COG3 and COG8 from the LAT52 pollen promoter, respectively. No obvious abnormalities in the microgametogenesis of the two mutants are observed, but in vitro and in vivo pollen tube growth are defective. COG3 or COG8 proteins fused to green fluorescent protein (GFP) label the Golgi apparatus. In pollen of both mutants, Golgi bodies exhibit altered morphology. Moreover, γ-COP and EMP12 proteins lose their tight association with the Golgi. These defects lead to the incorrect deposition of cell wall components and proteins during pollen tube growth. COG3 and COG8 interact directly with each other, and a structural model of the Arabidopsis COG complex is proposed. We believe that the COG complex helps to modulate Golgi morphology and vesicle trafficking homeostasis during pollen tube tip growth.