Article Figures & Data
Figures
- Figure 1.
AtPEX10-YFP fusion localizes to peroxisomes in tobacco (Nicotiana tabacum) leaf epidermal cells. A, Tobacco leaf epidermal cells transiently expressing a AtPEX10-YFP fusion. Scale bar = 50 μm. B, Same cells as in A visualized with transmitted light to show cell boundaries. C, Merged image of A and B. D, Higher magnification of structures highlighted with AtPEX10-YFP. Note the absence of fluorescence in the center of these structures (scale bar = 2 μm). E through G, Time-lapse sequence of epidermal cells expressing an AtPEX10-YFP fusion. An arrowhead indicates one of these structures covering a greater distance in comparison with other AtPEX10-YFP bodies present in the same field of view. Time is expressed in seconds at the top right of each frame. Scale bar = 10 μm. H through J, Leaf epidermal cells transiently expressing a GFP-SKL fusion imaged with GFP settings of the confocal microscope (H; scale bar = 50 μm). Inset in H represents a higher magnification comparable with that in D. I, Same cells as in H imaged with transmitted light. The two images are merged in J. K through N, Colocalization of GFP and YFP fusions in epidermal cells of a leaf in which a mixed population of Agrobacterium tumefaciens bearing AtPEX10-YFP and GFP-SKL constructs was inoculated. K, AtPEX10-YFP expression. Scale bar = 20 μm. L, Same cells as in K showing GFP-SKL expression. M, Transmitted light image of cells pictured in K and L. N, In a merged image of K through M, it is possible to identify cells that are coexpressing AtPEX10-YFP and GFP-SKL. In these cells, AtPEX10-YFP colocalizes with peroxisomes highlighted with GFP-SKL (arrowheads).
- Figure 2.
Arabidopsis PEX10 is expressed in a range of tissues. RT-PCR of AtPEX10 from Arabidopsis tissues (indicated above lanes) shows that AtPEX10 is widely expressed. The primers used were P4 and P11, and the expected size of AtPEX10 amplified product is 1.2 kbp.
- Figure 3.
Location and segregation of the dissociation element (Ds) in the Atpex10 line. A, Schematic of genomic sequence of AtPEX10, boxes refer to exons. The Ds element is located in exon 4, and primers used to determine location and orientation of the Ds element in AtPEX10 are displayed. B, Arabidopsis PEX10 (AJ276134) is predicted to have two transmembrane domains (underlined) and a conserved zinc RING finger at the carboxy terminus (boxed residues). The section in bold italics represents the part of the protein that could theoretically be translated from the mutant allele. Five PCR products derived from independent PCR reactions were sequenced. All five contained the substitution M184K relative to the previously published sequences AC002505 and AF119572. In addition, the substitution P199L was found in four out of the five independent PCR products, but the E357K mutation was found in only one PCR product. Therefore, it seems likely that M184K and P199L are genuine polymorphisms, but E357K is a PCR-induced mutation. These three substitutions are shown in bold type. C, Genomic extracts from seven progeny from selfed Atpex10 heterozygotes were genotyped. Two PCR reactions per plant were performed with primer combinations P11 and P18 (black arrowhead) and P11 and DS3 (white arrowhead). P11 and P18 amplifies a 910-bp product from the wild-type allele, and P18 and DS3 amplifies a product of 453 bp from the disrupted allele. Two segregating wild-type (WT) and five heterozygotes (Hh) are shown. Water controls and control PCR reactions from genomic DNA from wild-type plants are also shown.
- Figure 4.
Homozygous Atpex10 mutants are embryo lethal. A and B, Developing seed from siliques from selfed heterozygotes show a higher level of abnormal seeds per silique than seen in wild type. The abnormal seed are white (black arrowhead) or brown and shriveled (white arrowhead). Scale bar = 0.2 mm. C, Embryos were dissected from the developing seed and were genotyped by PCR. Two PCR reactions per embryo were performed using primers GUS1 and GUS2 (white arrowhead) or EM1 and EM2 (black arrowhead). GUS1 and GUS2 amplifies a 1.5-kbp product from the disrupted Atpex10 allele; EM1 and EM2 amplifies a 755-bp product from the wild-type allele.
- Figure 5.
Mutants show developmental delay. A, RT-PCR with AtPEX10-specific primers was performed on whole seed from wild-type plants 2 d postflowering. Normarski image of developing embryo shows that at 2 d postflowering, embryos are at early globular stage of development. The amplified products were blotted and probed with an AtPEX10-specific probe. B through H, Developing seed from selfed heterozygous Atpex10 plants were removed from the silique and viewed using Normarski optics. Seeds were separated based on normal (B and D) or abnormal (C, and E through H) external appearance, where B and C are from the same silique and D and E are from the same silique. F through H are from siliques containing morphologically normal embryos at the same developmental stage as D. Embryos in normal seeds are at a later developmental stage to embryos in abnormal seeds from the same silique, and some of the abnormal embryos appear to be larger (compare E with G, and F with H). Scale bar: A through E = 10 μm; F through H = 50 μm.
