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. 2005 Aug;138(4):1877–1895. doi: 10.1104/pp.105.066019

Table II.

Distribution of immunogold labeling with GFP, BiP, and TMV antiserum in transgenic N. tabacum leaves

Transgenea
Antiserab
Fieldsc
Total CW
Mean ± se of Number of Gold Particles in:d
PM ER Golgie Vesiclesf
Experiments using single antisera
    GFP:TGBp2 GFP 41 0.8 + 0.3b 0.3 + 0.1b 3.4 ± 0.5a 0 ± 0b 1.3 ± 0.5b
    GFP:TGBp3 GFP 31 0.8 ± 0.4b 0.7 ± 0.4b 4.3 ± 0.7a 0 ± 0b NO
    mGFP5-ER GFP 37 0.4 ± 0.2b 0.1 ± 0.1b 2.4 ± 0.4a 0.4 ± 0.2b NO
    Nontransgenic GFP 36 0.3 ± 0.1a 0.2 ± 0.1a 0.3 ± 0.1a 0 ± 0a NO
    GFP:TGBp2 BiP 8 0.3 ± 0.3b 1.4 ± 0.5a 1.6 ± 0.5a – –
    GFP:TGBp3 BiP 12 0 ± 0b 0.3 ± 0.2b 2.5 ± 0.3a – NO
    mGFP5-ER BiP 12 0 ± 0b 0.1 ± 0.1b 1.5 ± 0.3a – NO
    Nontransgenic BiP 15 0.1 ± 0.1b 0.4 ± 0.2b 2.3 ± 0.4a – NO
    GFP:TGBp2 NoAb 22 0 ± 0a 0 ± 0a 0 ± 0a – –
    GFP:TGBp3 NoAb 20 0 ± 0a 0 ± 0a 0.1 ± 0.1a – –
    mGFP5-ER NoAb 13 0 ± 0b 0 ± 0b 0.6 ± 0.2a – –
    Nontransgenic NoAb 10 0 ± 0a 0 ± 0a 0 ± 0a – –
Experiments using two antisera
    GFP:TGBp2 GFP 51 0.8 ± 0.2b 1.1 ± 0.2b 3.5 ± 0.4a 0 ± 0b 2.6 ± 0.2b
BiP 51 0 ± 0c 0.2 ± 0.1c 0.6 ± 0.1a 0 ± 0c 0.3 ± 0.1b
    GFP:TGBp3 GFP 28 1.1 ± 0.6b 0.4 ± 0.2b 7.3 ± 0.9a 0.4 ± 0.4b NO
BiP 28 0.2 ± 0.1b 0 ± 0b 1.2 ± 0.2a 0.0 ± 0b NO
    mGFP5-ER GFP 29 2.0 ± 0.6b 1.3 ± 0.4b 9.1 ± 0.7a 0.3 ± 0.2b NO
BiP 29 0 ± 0b 0.1 ± 0.1b 1.5 ± 0.3a 0.0 ± 0.0b NO
    GFP:TGBp2 GFP 16 0.1 ± 0.1b 0 ± 0b 4.6 ± 1.1a – –
TMV 16 0 ± 0a 0 ± 0a 0 ± 0a – –
    GFP:TGBp3 GFP 17 1.2 ± 0.6b 0.5 ± 0.3b 5.8 ± 1.0a – NO
TMV 17 0 ± 0a 0 ± 0a 0 ± 0a – NO
    mGFP5-ER GFP 13 0 ± 0b 0 ± 0b 0.6 ± 0.2a – NO
TMV 13 0 ± 0a 0 ± 0a 0 ± 0a – NO
a

Transgenic tobacco leaf samples were embedded in LR White, sectioned, and analyzed by immunogold labeling and electron microscopy to assess the subcellular accumulation patterns of GFP:TGBp2 and GFP:TGBp3. Control samples included nontransgenic leaves and transgenic leaves expressing GFP targeted to the ER (mGFP5-ER). Since we lack antisera to PVX TGBp2 and TGBp3, transgenic plants expressing GFP-fused genes were used. Immunogold labeling was then conducted using commercially available anti-GFP sera.

b

In the top half of the table, samples of transgenic or nontransgenic tobacco leaves were treated with either full-length mouse monoclonal AV antiserum (BD living colors; CLONTECH Laboratories) to detect GFP, BiP antiserum, or no primary antiserum (NoAb). All samples were treated with 10-nm gold-conjugated anti-mouse or anti-rabbit sera. In the bottom half of the table, samples were dual-labeled using antiserum to detect GFP and BiP. As a control, some samples were treated with antiserum to detect GFP and TMV. These samples were treated with 10 nm and 25 nm of gold-conjugated anti-mouse and anti-rabbit sera.

c

Fields are defined as areas of 1 μm2 (using an ultrastructure size calculator) that contain gold particles. Gold particles in each field were counted manually. The total numbers of fields analyzed for each transgenic plant sample are indicated.

d

The numbers of gold particles detected in the cell wall (CW), plasma membrane (PM), and ER were determined for all fields treated with each antiserum. Means within the same row with the same letter are not significantly different using Fisher's protected lsd procedure at a 0.05 significance level on square-root-transformed data. Zeros indicate samples that had the CW, PM, or ER, but no labeling.

e

There were few Golgi viewed in all of the samples analyzed. The averages shown in this table were derived from 5 to 6 Golgi for each sample. –, Antibody treatments in which their reaction with Golgi was not quantified.

f

The vesicles reported in GFP:TGBp2 samples were abundant. Of the total fields analyzed (41 treated with one antiserum and 51 treated with two antisera), there were a total of 72 vesicles viewed. –, Treatments in which the reaction of the antibodies with vesicles was not quantified. NO, None observed.