Figure 2. Expression and localization of AtMRP4.
(a) Results of RT–PCR analysis suggest that AtMRP4 is expressed in low amounts in all tissues tested. cDNAs from different Arabidopsis tissues were synthesized from total RNA and transcripts specific for AtMRP4 and 40S ribosomal protein S16 were detected by PCR. Five micrograms of total RNA and equal volumes of PCR products were separated on 1% formaldehyde (RNA; lowest panel) and 2.5% native agarose gels (PCR products; upper two panels), respectively.
(b) Histochemical localization of GUS activity (i–vii) and subcellular localization of AtMRP–GFP (viii–xiii). (i-vi) GUS activity in A. thaliana plants stably transformed with approximately 2 kbp of the 5′ UTR of AtMRP4 fused to the uidA gene. (i) Eight-day-old seedling grown in 8 h light/16 h dark showing GUS activity in the cotyledons mainly in punctuate structures, parts of the hypocotyls, and the primary root. The arrow points to the root tip, which is not stained. (ii) Rosette leaves taken from independent 21-day-old seedlings showing different GUS staining patterns mainly with respect to the petiole and vascular tissue, which are not always stained. In all cases, punctuate structures representing stomata are strongly stained. (iii) Surface view of the abaxial side of a rosette leaf viewed with differential interference contrast optics exhibiting a strong GUS activity in the stomata. (iv) Eight-day-old etiolated seedling. Arrow, in comparison to (i), the root tip exhibits strong GUS activity. (v,vi) Flowers and siliques of 5-week-old transgenic A. thaliana plants. Stomata of the carpel wall are strongly stained. (vii) GUS staining in guard cells of a V. faba leaf 2 days after particle gun bombardment of a leaf with the promoter–GUS fusion construct. (viii–xiii) Laser scanning confocal microscope analysis of plant material expressing a GFP-tagged version of AtMRP4. Images represent internal optical sections generated by CLSM from onion epidermis cells expressing transiently (ix, x) and Arabidopsis seedlings expressing ectopically AtMRP4–EGFP (ix–xiii). AtMRP4-specific GFP fluorescence was found mainly in the periphery of cells, suggesting that AtMRP4 protein is located at the plasma membrane. (viii) Onion epidermis cell expressing the vacuolar marker KCO1–GFP labeling the tonoplast surrounding the central vacuole. The position of the nucleus is indicated by an arrow. (ix) Onion epidermis cell expressing AtMRP4–EGFP shows fluorescence at the periphery of cells. In some onion cells bombarded with AtMRP4–EGFP, dense vesicles, filled with GFP fluorescence (indicated by an arrow), were observed. These may have been caused by overloading the cells' secretory pathway with excessive quantities of fusion protein. (x) Same cells in a bright field. (xi) Close up of root epidermis cells showing fluorescence at the periphery of cells. (xii) Close up of an Arabidopsis guard cell pair showing fluorescence at the periphery of cells. Note that circular green fluorescence of chloroplasts (marked by an asterisk) is not because of GFP fluorescence but because of autofluorescence of bleaching chlorophyll. (xiii) Same cell in a bright field. Scale bars are 2 mm (i, ii, iv), 50 µm (iii, vii), 20 µm (viii, ix), and 5 µm (xi, xii).
(c) Arabidopsis microsomal fractions overexpressing MRP4–GFP were separated by linear sucrose gradient centrifugation, and Western blots were imunoprobed with anti-GFP antisera. Origin of immunopositive fractions was ascertained using antisera against the marker proteins vacuolar V-type H+-ATPase, ER-localized BIP and the plasma membrane-bound P-type H+-ATPase (Geisler et al., 2000).