Article Figures & Data
Figures
- Fig. 1.
Plant expression cassettes for targeting TDC to the chloroplast (pT-CHL), cytosol (pT-CYT), and ER (pT-ER). ChS, 5′-Untranslated region (UTR) of chalcone synthase; CTS, chloroplast-targeting signal of the potato (Solanum tuberosum) granule-bound starch synthase; Ω, 5′-UTR Ω sequence of tobacco mosaic virus; tags, c-myc/His6 tags; LPL*, plant codon optimized light chain leader peptide of the murine antibody 24 (Voss et al., 1995); Lnk, linker; K, KDEL sequence; 35SS, double enhanced cauliflower mosaic virus (CaMV) 35S promoter; Ter, CaMV termination sequence.
- Fig. 2.
Fluorometric detection of tryptamine in the crude extract of tobacco SR1 leaves transiently expressing recombinant TDC targeted to different subcellular compartments. Bars represent the average value of fluorescence intensity g−1fresh weight (fw) of leaf material calculated from the total integral of the 300- to 400-nm emission scan area of four infiltrated leaves per plant expression cassette. Control leaves were infiltrated with nonrecombinant agrobacteria. ses are shown.
- Fig. 3.
Immunoblot analysis of the crude TSP extract of independent transgenic tobacco plants expressing targeted TDC to the chloroplast (A), cytosol (B), and ER (C). The 9E10 anti-c-myc antibody (A and B) or the MA-GRP78 anti-KDEL antibody (C) was used to detect the recombinant TDC. M, Prestained protein marker. Lanes 1 through 6A and lanes 1 through 8, B and C, Leaf crude extract of transgenic T1 (A and B) and T0 plants (C). W, TSP of wild-type tobacco SR1 plants.
- Fig. 4.
In vivo TDC function in transgenic tobacco plants. Bars represent the average amount of tryptamine in the leaf crude extract of 25 T0 and 15 T1tobacco plants accumulating TDC in different subcellular compartments. Tryptamine amounts are in micrograms per milligram of TSP.ses are shown.
- Fig. 5.
Leaf necrotic areas in transgenic plants expressing TDC in the chloroplast. Necrosis started to appear in the form of small HR-like necrotic lesions (A), and they increased in number and size as soon as the plants approached flowering when diffused necrosis with strong deformation of the leaf surface developed (B and C). The fertility of the plants was not affected, and seeds from T0 plants were used to cultivate T1 plants that showed the same phenotype with no reduction of fertility.
- Fig. 6.
Immunoblot analysis and in vitro assay of TDC and marker enzymes in the H, S, and MP of chloroplast preparations from two T1 plants (17/2 and 25/3) accumulating TDC in the chloroplast and a wild-type tobacco plant (W). Bars represent the average value of the specific enzymatic activity expressed in units (catalase and GAPDH), milliunits (fumarase), or nanokatals (TDC) per milligram of chlorophyll.
Tables
pT-CTS pT-CYT pT-ER T0 T1 T0 T1 T0 Mean1-a 50.584 (258.966) 99.359 (660.784) 20.366 (159.244) 34.019 (227.981) 0.591 (4.016) se 8.097 (39.154) 12.255 (110.704) 2.464 (25.103) 4.949 (23.318) 0.132 (0.766) Minimum 6.061 (61.92) 32.757 (321.367) 10.407 (61.881) 14.953 (76.025) 0.041 (0.759) Maximum 98.949 (570.124) 196.966 1-b (1,277.465) 37.327 (346.991) 62.604 (353.936) 1.380 (10.701) -
↵F1-a Data represent statistical analysis of the fluorimetric quantitation of tryptamine synthesized in vivo in transgenic plants harboring cassettes for targeting TDC to different subcellular compartments. Levels of tryptamine are in microgram per milligram of TSP or microgram per gram fresh wt of leaf material (in parentheses).
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↵F1-b The highest amount of tryptamine detected in fully expanded leaves of a transgenic T1 plant expressing recombinant TDC in the chloroplast is underlined.
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