Signal peptide-dependent targeting of a rice alpha-amylase and cargo proteins to plastids and extracellular compartments of plant cells

Abstract

alpha-Amylases are important enzymes for starch degradation in plants. However, it has been a long-running debate as to whether alpha-amylases are localized in plastids where starch is stored. To study the subcellular localization of alpha-amylases in plant cells, a rice (Oryza sativa) alpha-amylase, alphaAmy3, with or without its own signal peptide (SP) was expressed in transgenic tobacco (Nicotiana tabacum) and analyzed. Loss-of-function analyses revealed that SP was required for targeting of alphaAmy3 to chloroplasts and/or amyloplasts and cell walls and/or extracellular compartments of leaves and suspension cells. SP was also required for in vitro transcribed and/or translated alphaAmy3 to be cotranslationally imported and processed in canine microsomes. alphaAmy3, present in chloroplasts of transgenic tobacco leaves, was processed to a product with Mr similar to alphaAmy3 minus its SP. Amino acid sequence analysis revealed that the SP of chloroplast localized alphaAmy3 was cleaved at a site only one amino acid preceding the predicted cleavage site. Function of the alphaAmy3 SP was further studied by gain-of-function analyses. beta-Glucuronidase (GUS) and green fluorescence protein fused with or without the alphaAmy3 SP was expressed in transgenic tobacco or rice. The alphaAmy3 SP directed translocation of GUS and green fluorescence protein to chloroplasts and/or amyloplasts and cell walls in tobacco leaves and rice suspension cells. The SP of another rice alpha-amylase, alphaAmy8, similarly directed the dual localizations of GUS in transgenic tobacco leaves. This study is the first evidence of SP-dependent dual translocations of proteins to plastids and extracellular compartments, which provides new insights into the role of SP in protein targeting and the pathways of SP-dependent protein translocation in plants.

Figure 1.

Expression of αAmy3 in transgenic tobacco leaves. A, Diagram shows the amino acid sequence of αAmy3 SP and constructs containing the CaMV35S promoter and coding regions of αAmy3 without SP. Arrowhead indicates the putative SP cleavage sites. Amino acids with hydrophobic (○) or positively charged (+) chain are indicated. B, Two independent transgenic tobacco plants expressing αAmy3 (A2 and A4) or αAmy3ΔSP (B1 and B2) were analyzed. Total proteins were isolated from tobacco leaves and subjected to western-blot analysis using anti-rice α-amylase antibodies (Chen et al., 1994). Fifty micrograms of total proteins were loaded in each lane. NT, Nontransformed control.

Figure 2.

SP-dependent localization of αAmy3 in the chloroplast and cell wall of transgenic tobacco leaves. Immunocytochemical localization of αAmy3 in leaf samples using anti-rice α-amylase antibodies. A and B, Labeling of αAmy3 in leaf mesophyll cells of transgenic tobacco line A2 expressing αAmy3. C, Labeling of αAmy3 in leaf mesophyll cells of transgenic tobacco line A4 expressing αAmy3. αAmy3 detected in the stroma and over starch granules within a chloroplast and in cell walls. Arrowheads indicate positions of αAmy3. Scale bar represents 1 μm. Abbreviations: Cpt, chloroplast; Cyt, cytoplasm; CW, cell wall; M, mitochondria; S, starch granule; V, vacuole.

Figure 3.

SP-dependent localization of αAmy3 in the chloroplast and cell wall of transgenic tobacco leaves. Immunocytochemical localization of αAmy3 in leaf samples using anti-rice α-amylase antibodies. A to D, Labeling of αAmy3 in leaf mesophyll cells of transgenic tobacco line B2 expressing αAmy3ΔSP. αAmy3 mainly detected in the cytoplasm. E, The αAmy3 antibodies do not label starch grains of nontransformant. Arrowheads indicate positions of αAmy3. Scale bar represents 1 μm. Abbreviations: Cp, chloroplast; Cyt, cytoplasm; CW, cell wall; M, mitochondria; S, starch granule; V, vacuole.

Figure 4.

SP-dependent localization of αAmy3 in the amyloplasts and cell walls of transformed tobacco suspension cells. Immunocytochemical localization of αAmy3 in suspension cell samples using anti-rice α-amylase antibodies. A and B, Labeling of αAmy3 in suspension cells of transgenic tobacco line A2 expressing αAmy3. αAmy3 mainly detected over starch granules and in the cell wall. C and D, Labeling of αAmy3 in suspension cells of transgenic tobacco line B2 expressing αAmy3ΔSP. αAmy3 mainly detected in the cytoplasm. E, Preimmune serum does not label αAmy3 in starch grains and cell wall in transgenic tobacco line A2. F, The αAmy3 antibodies do not label starch grains of nontransformant. Arrowheads indicate positions of αAmy3. Scale bar represents 1 μm. Abbreviations: Amy, amyloplast; CW, cell wall; Cyt, cytoplasm; M, mitochondria; ML, middle lamella; S, starch granule; V, vacuole.

Figure 5.

SP-dependent import of αAmy3 into canine microsomes. RNAs encoding αAmy3 and αAmy3ΔSP were synthesized in vitro and then translated in rabbit reticulocyte lysate in the presence or absence of canine microsomes plus ³⁵S-Met. The samples were treated with (+) or without (−) proteinase K and/or Triton X-100 and analyzed with SDS-PAGE and fluorography. Dots indicate positions of αAmy3. The molecular masses (kD) of the proteins are indicated to the left of the figure.

Figure 6.

αAmy3 localized in chloroplasts of transgenic tobacco leaves is processed. Leaves were collected from 1-month-old transgenic tobacco line A2 expressing αAmy3 and line B2 expressing αAmy3ΔSP. Chloroplasts were isolated from leaf extracts as described in “Materials and Methods.” Top section, RNA encoding αAmy3 or αAmy3ΔSP was in vitro transcribed from plasmid DNA and translated in the presence of ³⁵S-Met as described in “Methods and Materials.” The ³⁵S-labeled αAmy3 and αAmy3ΔSP were also resolved in the same SDS-PAGE along with leaf and chloroplast extracts, immunoblotted with the rice α-amylase antibodies, and viewed using fluorography. The x-ray film was overlaid onto the gel blot and photographed. Fifty micrograms of leaf extracts and 15 μg of chloroplast extracts were loaded in each lane. Lane 1, ³⁵S-labeled αAmy3 (S); lanes 2 and 3, leaf (L) and chloroplast (C) extracts of line A2, respectively; lane 4, ³⁵S-labeled αAmy3ΔSP; lanes 5 and 6, leaf and chloroplast extracts of line B2, respectively. Bottom section, Protein gel immunoblot analysis of the leaf and chloroplast extracts of lines A2 and B2 using CAB antibodies. Ten micrograms of leaf extract and 3 μg of chloroplast extracts were loaded in each lane.

Figure 7.

The SP of αAmy3 is sufficient for directing GUS to plastids and cell walls. Immunocytochemical localization of GUS in transgenic tobacco leaf and transformed rice suspension cells using anti-GUS antibodies. A, Construct containing the CaMV35S promoter, αAmy3 SP, and GUS coding region was used for tobacco transformation. GUS was detected over starch grains within amyloplast and in the cell wall of leaf mesophyll cells of transgenic tobacco. B, Construct containing the CaMV35S promoter and GUS coding region was used for tobacco transformation. GUS was detected exclusively in the cytoplasm of leaf mesophyll cells of transgenic tobacco. C, Construct containing the αAmy3 promoter and SP and coding region of GUS was used for rice transformation. GUS was detected over starch grains within the amyloplast and in the cell wall. D, GUS was not labeled in any cellular compartment in nontransformed rice suspension cells. Arrowheads indicate positions of GUS. Scale bar represents 1 μm. Abbreviations: Amy, amyloplast; Cp, chloroplast; CW, cell wall; Cyt, cytoplasm; E, endoplasmic reticulum; G, Golgi apparatus; S, starch granule; V, vacuole.

Figure 8.

The SP of αAmy8 is sufficient for directing GUS to plastids and cell walls. Immunocytochemical localization of GUS in transgenic tobacco leaf using anti-GUS antibodies. A, Diagram shows the amino acid sequence of αAmy8 SP and the construct containing the αAmy8 promoter and SP and GUS coding region used for tobacco transformation. Arrowhead (↓) indicates the putative SP cleavage sites. Amino acids with hydrophobic (○) or positively charged (+) chain are indicated. GUS was detected over starch grains within the amyloplast and in the cell wall of leaf mesophyll cells in transgenic tobacco. B, Preimmune serum does not label GUS in leaf mesophyll cells of transgenic tobacco expressing GUS with SP. Arrowheads indicate positions of GUS. Scale bar represents 1 μm. Abbreviations: Cp, chloroplast; CW, cell wall; Cyt, cytoplasm; M, mitochondria; S, starch granule.

Figure 9.

The SP of αAmy3 is sufficient for directing GFP to plastids. Rice calli were transformed with indicated constructs and selected with hygromycin. Protoplasts were isolated from the transformed calli expressing GFP. A to F, Construct containing the αAmy3 promoter and GFP coding region. GFP detected in cytoplasm and nucleus. G to L, Construct containing the αAmy3 promoter and SP and GFP coding region. GFP detected in amyloplasts. A, D, G, and J, Images of bright fields. B, E, H, and K, Images of fluorescent fields. C, F, I, and L, Composite images of fluorescence (green pseudo color) and transmission. Abbreviations: V, vacuole; N, nucleus; Nu, nucleolus. Arrowheads indicate positions of amyloplasts.