Localization of CmPP16 mRNA and protein in C. maxima vascular tissue.

(A) Schematic transverse section of a pumpkin stem. Vascular bundles are composed of internal and external phloem (IP and EP, respectively), cambium (CA), and xylem (X). Such bundles are distributed in a ring around the outer region of the stem (or petiole).

(B) Northern blot analyses establish that the CmPP16 RNA is located in a variety of plant tissues. Total RNA (10 µg) from the indicated tissue was electrophoresed, blotted, probed with 32P-labeled CmPP16 open reading frame at 65°C, and washed with 0.1× SSC (0.015 M NaCl and 0.0015 M sodium citrate).

(C) Cellular arrangement within the IP illustrating functional (arrowheads) and immature (asterisks) SE derived from the CA. Semithin section stained with toluidine blue.

(D) Confocal laser scanning microscope (CLSM) image of a pumpkin semiserial section [see (C)] labeled with antiserum to R-CmPP16-1 reveals the presence of CmPP16 within the phloem. CmPP16 signal (green fluorescence) is present at the periphery of mature and immature SE. Tissue structure was observed (red fluorescence) with Safranin O used as a histochemical stain. Controls with preimmune sera yielded images devoid of fluorescent signal.

(E to H) CLSM images of transverse pumpkin petiole sections processed for in situ RT-PCR. To identify cellular structure, we collected images (E and G) before removal of unincorporated CF-labeled deoxyuridine triphosphate. Asterisks identify immature SE.

CmPP16 mRNA is detected (green fluorescent signal) within immature CC-SE complexes and, predominantly, in mature CC (F); note low but detectable amount of green fluorescent signal in SE.

White asterisks in (E) and (F) facilitate identification of the same cell in the two images. In control experiments, tissues were treated in the same manner except that primers were omitted (H).

Red fluorescence in (F) and (H) represents autofluorescence. Scale bars, 50 µm; bar in (D) is common to (C) and bar in (F) is common to (E), (G), and (H).

Reference: Science January 1, 1999, Vol. 283, No. 5398.