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Fusion to GFP blocks intercellular trafficking of the sucrose transporter SUT1 leading to accumulation in companion cells.

Lalonde S, Weise A, Walsh RP, Ward JM, Frommer WB - BMC Plant Biol. (2003)

Bottom Line: The 3'-UTR of SUT1 affected intracellular distribution of GFP but was insufficient for trafficking of SUT1, GFP or their fusions to SEs.A fusion with GFP prevents targeting of the sucrose transporter SUT1 to the SE while leading to accumulation in the CC.The 3'-UTR of SUT1 is insufficient for mobilization of either the fusion or GFP alone.

View Article: PubMed Central - HTML - PubMed

Affiliation: ZMBP Tübingen, Plant Physiology, Auf der Morgenstelle 1, D-72076 Tübingen, Germany. sylvie.lalonde@zmbp.uni-tuebingen.de

ABSTRACT

Background: Plant phloem consists of an interdependent cell pair, the sieve element/companion cell complex. Sucrose transporters are localized to enucleate sieve elements (SE), despite being transcribed in companion cells (CC). Due to the high turnover of SUT1, sucrose transporter mRNA or protein must traffic from CC to SE via the plasmodesmata. Localization of SUT mRNA at plasmodesmatal orifices connecting CC and SE suggests RNA transport, potentially mediated by RNA binding proteins. In many organisms, polar RNA transport is mediated through RNA binding proteins interacting with the 3'-UTR and controlling localized protein synthesis. To study mechanisms for trafficking of SUT1, GFP-fusions with and without 3'-UTR were expressed in transgenic plants.

Results: In contrast to plants expressing GFP from the strong SUC2 promoter, in RolC-controlled expression GFP is retained in companion cells. The 3'-UTR of SUT1 affected intracellular distribution of GFP but was insufficient for trafficking of SUT1, GFP or their fusions to SEs. Fusion of GFP to SUT1 did however lead to accumulation of SUT1-GFP in the CC, indicating that trafficking was blocked while translational inhibition of SUT1 mRNA was released in CCs.

Conclusion: A fusion with GFP prevents targeting of the sucrose transporter SUT1 to the SE while leading to accumulation in the CC. The 3'-UTR of SUT1 is insufficient for mobilization of either the fusion or GFP alone. It is conceivable that SUT1-GFP protein transport through PD to SE was blocked due to the presence of GFP, resulting in retention in CC particles. Alternatively, SUT1 mRNA transport through the PD could have been blocked due to insertion of GFP between the SUT1 coding sequence and 3'-UTR.

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GFP Localization in RolC-GFP transgenic plants. GFP fluorescence was detected on longitudinal hand sections of transgenic tobacco petiole under CLSM and co-stained with DAPI and aniline blue. A) All constructs were prepared in a modified pGPTV-HPT under the control of the CC-specific, RolC, promoter. The asterisk indicates the position of the start codon. T, nos terminator. B) RolC-GFP petiole, C) RolC-GFP-3'-UTR petiole, D) major vein of mature leaf of RolC-GFP transgenic plant, E) sink leaf (1 cm long) of RolC-GFP transgenic plant, F) leaf petiole of a sink leaf of RolC-GFP transgenic plant, G) Stem of RolC-GFP transgenic plant, H) Veins of an unopened flower of RolC-GFP transgenic plant. I) Untransformed wild type tobacco leaf petiole visualized with the average PMT level used for the above images. CC, companion cell; SE, sieve element; sp, sieve plate; n, nucleus. Bar equals to 10 μm.
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Figure 3: GFP Localization in RolC-GFP transgenic plants. GFP fluorescence was detected on longitudinal hand sections of transgenic tobacco petiole under CLSM and co-stained with DAPI and aniline blue. A) All constructs were prepared in a modified pGPTV-HPT under the control of the CC-specific, RolC, promoter. The asterisk indicates the position of the start codon. T, nos terminator. B) RolC-GFP petiole, C) RolC-GFP-3'-UTR petiole, D) major vein of mature leaf of RolC-GFP transgenic plant, E) sink leaf (1 cm long) of RolC-GFP transgenic plant, F) leaf petiole of a sink leaf of RolC-GFP transgenic plant, G) Stem of RolC-GFP transgenic plant, H) Veins of an unopened flower of RolC-GFP transgenic plant. I) Untransformed wild type tobacco leaf petiole visualized with the average PMT level used for the above images. CC, companion cell; SE, sieve element; sp, sieve plate; n, nucleus. Bar equals to 10 μm.

Mentions: To use a smaller reporter previously shown to move between CC and SE, GFP was fused to the SUT1 3'-UTR under control of the RolC promoter (Fig. 3A). Western analysis was first used to screen for plants expressing high levels of GFP, and selected plants were then analyzed by confocal laser scanning microscopy (CLSM). In both cases (RolC-GFP and RolC-GFP-3'-UTR), ~60 transgenic plants were analyzed, from which three lines with the highest expression levels were selected for further analysis. All lines showed similar expression patterns (data not shown) and none of the transgenic plants analyzed showed obvious phenotypic alterations under tissue culture or greenhouse conditions. Identification of phloem cell types was performed using DAPI staining for CC nuclei and aniline blue staining for the callose of sieve plates. Using CLSM, 3D images could be scanned from which xz or yz 2D images were obtained. In all RolC-GFP plants analyzed, GFP fluorescence was found in the CCs of petiole, leaf midrib, and stem (Fig. 3B,3D,3G). Sink tissues such as sink leaves or unopened flowers did not accumulate GFP (Fig. 3E,3F,3H). In all tissues, GFP protein was found in the cytoplasm and in the proximity of the nucleus. In plants transformed with the GFP-3'-UTR constructs, GFP was also localized in the CC (Fig. 3C), moreover the addition of the 3'-UTR resulted in the formation of particle-like structures and in the loss of the perinuclear localization (Fig. 3C). Thus the 3'-UTR affects GFP distribution in the cell, but is insufficient for trafficking to SE.


Fusion to GFP blocks intercellular trafficking of the sucrose transporter SUT1 leading to accumulation in companion cells.

Lalonde S, Weise A, Walsh RP, Ward JM, Frommer WB - BMC Plant Biol. (2003)

GFP Localization in RolC-GFP transgenic plants. GFP fluorescence was detected on longitudinal hand sections of transgenic tobacco petiole under CLSM and co-stained with DAPI and aniline blue. A) All constructs were prepared in a modified pGPTV-HPT under the control of the CC-specific, RolC, promoter. The asterisk indicates the position of the start codon. T, nos terminator. B) RolC-GFP petiole, C) RolC-GFP-3'-UTR petiole, D) major vein of mature leaf of RolC-GFP transgenic plant, E) sink leaf (1 cm long) of RolC-GFP transgenic plant, F) leaf petiole of a sink leaf of RolC-GFP transgenic plant, G) Stem of RolC-GFP transgenic plant, H) Veins of an unopened flower of RolC-GFP transgenic plant. I) Untransformed wild type tobacco leaf petiole visualized with the average PMT level used for the above images. CC, companion cell; SE, sieve element; sp, sieve plate; n, nucleus. Bar equals to 10 μm.
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Related In: Results  -  Collection

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Figure 3: GFP Localization in RolC-GFP transgenic plants. GFP fluorescence was detected on longitudinal hand sections of transgenic tobacco petiole under CLSM and co-stained with DAPI and aniline blue. A) All constructs were prepared in a modified pGPTV-HPT under the control of the CC-specific, RolC, promoter. The asterisk indicates the position of the start codon. T, nos terminator. B) RolC-GFP petiole, C) RolC-GFP-3'-UTR petiole, D) major vein of mature leaf of RolC-GFP transgenic plant, E) sink leaf (1 cm long) of RolC-GFP transgenic plant, F) leaf petiole of a sink leaf of RolC-GFP transgenic plant, G) Stem of RolC-GFP transgenic plant, H) Veins of an unopened flower of RolC-GFP transgenic plant. I) Untransformed wild type tobacco leaf petiole visualized with the average PMT level used for the above images. CC, companion cell; SE, sieve element; sp, sieve plate; n, nucleus. Bar equals to 10 μm.
Mentions: To use a smaller reporter previously shown to move between CC and SE, GFP was fused to the SUT1 3'-UTR under control of the RolC promoter (Fig. 3A). Western analysis was first used to screen for plants expressing high levels of GFP, and selected plants were then analyzed by confocal laser scanning microscopy (CLSM). In both cases (RolC-GFP and RolC-GFP-3'-UTR), ~60 transgenic plants were analyzed, from which three lines with the highest expression levels were selected for further analysis. All lines showed similar expression patterns (data not shown) and none of the transgenic plants analyzed showed obvious phenotypic alterations under tissue culture or greenhouse conditions. Identification of phloem cell types was performed using DAPI staining for CC nuclei and aniline blue staining for the callose of sieve plates. Using CLSM, 3D images could be scanned from which xz or yz 2D images were obtained. In all RolC-GFP plants analyzed, GFP fluorescence was found in the CCs of petiole, leaf midrib, and stem (Fig. 3B,3D,3G). Sink tissues such as sink leaves or unopened flowers did not accumulate GFP (Fig. 3E,3F,3H). In all tissues, GFP protein was found in the cytoplasm and in the proximity of the nucleus. In plants transformed with the GFP-3'-UTR constructs, GFP was also localized in the CC (Fig. 3C), moreover the addition of the 3'-UTR resulted in the formation of particle-like structures and in the loss of the perinuclear localization (Fig. 3C). Thus the 3'-UTR affects GFP distribution in the cell, but is insufficient for trafficking to SE.

Bottom Line: The 3'-UTR of SUT1 affected intracellular distribution of GFP but was insufficient for trafficking of SUT1, GFP or their fusions to SEs.A fusion with GFP prevents targeting of the sucrose transporter SUT1 to the SE while leading to accumulation in the CC.The 3'-UTR of SUT1 is insufficient for mobilization of either the fusion or GFP alone.

View Article: PubMed Central - HTML - PubMed

Affiliation: ZMBP Tübingen, Plant Physiology, Auf der Morgenstelle 1, D-72076 Tübingen, Germany. sylvie.lalonde@zmbp.uni-tuebingen.de

ABSTRACT

Background: Plant phloem consists of an interdependent cell pair, the sieve element/companion cell complex. Sucrose transporters are localized to enucleate sieve elements (SE), despite being transcribed in companion cells (CC). Due to the high turnover of SUT1, sucrose transporter mRNA or protein must traffic from CC to SE via the plasmodesmata. Localization of SUT mRNA at plasmodesmatal orifices connecting CC and SE suggests RNA transport, potentially mediated by RNA binding proteins. In many organisms, polar RNA transport is mediated through RNA binding proteins interacting with the 3'-UTR and controlling localized protein synthesis. To study mechanisms for trafficking of SUT1, GFP-fusions with and without 3'-UTR were expressed in transgenic plants.

Results: In contrast to plants expressing GFP from the strong SUC2 promoter, in RolC-controlled expression GFP is retained in companion cells. The 3'-UTR of SUT1 affected intracellular distribution of GFP but was insufficient for trafficking of SUT1, GFP or their fusions to SEs. Fusion of GFP to SUT1 did however lead to accumulation of SUT1-GFP in the CC, indicating that trafficking was blocked while translational inhibition of SUT1 mRNA was released in CCs.

Conclusion: A fusion with GFP prevents targeting of the sucrose transporter SUT1 to the SE while leading to accumulation in the CC. The 3'-UTR of SUT1 is insufficient for mobilization of either the fusion or GFP alone. It is conceivable that SUT1-GFP protein transport through PD to SE was blocked due to the presence of GFP, resulting in retention in CC particles. Alternatively, SUT1 mRNA transport through the PD could have been blocked due to insertion of GFP between the SUT1 coding sequence and 3'-UTR.

Show MeSH
Related in: MedlinePlus