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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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LeSUT1 immunolocalization. SUT1 was immunolocalized in different transgenic tobacco lines using antibody against StSUT1 [5] and detected using a secondary antibody conjugated to FITC. SUT1 localized to sieve element in wild type tobacco and tomato; but appears present in companion cells of transgenic plants. A) Wild type tobacco, B) Wild type tomato, C) RolC-LeSUT1-GFP, D) RolC-LeSUT1-GFP-3'-UTR. CC, companion cell; SE, sieve element; sp, sieve plate, nucleus. Bar indicates 10 μm.
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Figure 6: LeSUT1 immunolocalization. SUT1 was immunolocalized in different transgenic tobacco lines using antibody against StSUT1 [5] and detected using a secondary antibody conjugated to FITC. SUT1 localized to sieve element in wild type tobacco and tomato; but appears present in companion cells of transgenic plants. A) Wild type tobacco, B) Wild type tomato, C) RolC-LeSUT1-GFP, D) RolC-LeSUT1-GFP-3'-UTR. CC, companion cell; SE, sieve element; sp, sieve plate, nucleus. Bar indicates 10 μm.

Mentions: The above results suggested that the 3'-UTR is insufficient by itself to mobilize reporter activity to the SE, indicating that other targeting signals may be required in the LeSUT1 mRNA or protein. To test this hypothesis, GFP was fused translationally to the C-terminus of LeSUT1 (Fig. 4A). Intact folding and targeting of the sucrose transporter-GFP fusion to the plasma membrane was shown by complementation of a sucrose-uptake deficient yeast strain (Fig. 5). Transgenic plants were generated and selected as described above. In lines expressing either LeSUT1-GFP or LeSUT1-GFP-3'-UTR, GFP fluorescence was detected in CC, whereas only very low fluorescence levels were found in the SE (Fig. 4B,4C). As compared to the RolC-GFP plants, the SUT1-GFP fusion constructs showed much lower fluorescence. As a consequence, the sensitivity of the CLSM photomultipliers had to be increased. Thus, the fluorescence seen in the SE is interpreted as an increase in background noise based on similar observations in untransformed plants (compare Fig. 3I with Fig. 4D). The pattern of GFP particles at the cell periphery in LeSUT1-GFP-3'-UTR lines was similar to that described before in the GFP-3'-UTR lines (Fig. 4C, and 3C). Thus in contrast to the native SUT1 protein, which was found exclusively in SE, the SUT1-GFP fusion protein is found almost exclusively in CC. To confirm the presence of SUT1 in the CC, LeSUT1 was also immunolocalized using a SUT1-specific antibody affinity-purified against solanaceous SUT1 proteins [5]. As shown previously, SUT1 protein localizes to the sieve elements in wild type tobacco and tomato plants, (Fig. 6A,6B and [5]). In contrast, SUT1 protein was detected in CC of both the RolC-LeSUT1-GFP and RolC-LeSUT1-GFP-3'-UTR transgenic lines (Fig. 6C,6D). The cross-reactivity detected in the SE can be attributed to the presence of NtSUT1 (Fig. 6A and 6B). Consistent with the GFP localization, a LeSUT1-GUS-3'-UTR construct (including a 2.3 kb LeSUT1 promoter; Fig. 7A) displayed GUS activity localized to the CC (Fig. 7B), suggesting that the fusion protein is produced in the CC.


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)

LeSUT1 immunolocalization. SUT1 was immunolocalized in different transgenic tobacco lines using antibody against StSUT1 [5] and detected using a secondary antibody conjugated to FITC. SUT1 localized to sieve element in wild type tobacco and tomato; but appears present in companion cells of transgenic plants. A) Wild type tobacco, B) Wild type tomato, C) RolC-LeSUT1-GFP, D) RolC-LeSUT1-GFP-3'-UTR. CC, companion cell; SE, sieve element; sp, sieve plate, nucleus. Bar indicates 10 μm.
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Figure 6: LeSUT1 immunolocalization. SUT1 was immunolocalized in different transgenic tobacco lines using antibody against StSUT1 [5] and detected using a secondary antibody conjugated to FITC. SUT1 localized to sieve element in wild type tobacco and tomato; but appears present in companion cells of transgenic plants. A) Wild type tobacco, B) Wild type tomato, C) RolC-LeSUT1-GFP, D) RolC-LeSUT1-GFP-3'-UTR. CC, companion cell; SE, sieve element; sp, sieve plate, nucleus. Bar indicates 10 μm.
Mentions: The above results suggested that the 3'-UTR is insufficient by itself to mobilize reporter activity to the SE, indicating that other targeting signals may be required in the LeSUT1 mRNA or protein. To test this hypothesis, GFP was fused translationally to the C-terminus of LeSUT1 (Fig. 4A). Intact folding and targeting of the sucrose transporter-GFP fusion to the plasma membrane was shown by complementation of a sucrose-uptake deficient yeast strain (Fig. 5). Transgenic plants were generated and selected as described above. In lines expressing either LeSUT1-GFP or LeSUT1-GFP-3'-UTR, GFP fluorescence was detected in CC, whereas only very low fluorescence levels were found in the SE (Fig. 4B,4C). As compared to the RolC-GFP plants, the SUT1-GFP fusion constructs showed much lower fluorescence. As a consequence, the sensitivity of the CLSM photomultipliers had to be increased. Thus, the fluorescence seen in the SE is interpreted as an increase in background noise based on similar observations in untransformed plants (compare Fig. 3I with Fig. 4D). The pattern of GFP particles at the cell periphery in LeSUT1-GFP-3'-UTR lines was similar to that described before in the GFP-3'-UTR lines (Fig. 4C, and 3C). Thus in contrast to the native SUT1 protein, which was found exclusively in SE, the SUT1-GFP fusion protein is found almost exclusively in CC. To confirm the presence of SUT1 in the CC, LeSUT1 was also immunolocalized using a SUT1-specific antibody affinity-purified against solanaceous SUT1 proteins [5]. As shown previously, SUT1 protein localizes to the sieve elements in wild type tobacco and tomato plants, (Fig. 6A,6B and [5]). In contrast, SUT1 protein was detected in CC of both the RolC-LeSUT1-GFP and RolC-LeSUT1-GFP-3'-UTR transgenic lines (Fig. 6C,6D). The cross-reactivity detected in the SE can be attributed to the presence of NtSUT1 (Fig. 6A and 6B). Consistent with the GFP localization, a LeSUT1-GUS-3'-UTR construct (including a 2.3 kb LeSUT1 promoter; Fig. 7A) displayed GUS activity localized to the CC (Fig. 7B), suggesting that the fusion protein is produced in the CC.

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