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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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Structural analysis of the genomic sequence of LeSUT1. LeSUT1 genomic sequence contains three introns and has three polyadenylation signals predicted based on sequence comparison with different cDNA clones. The numbers under the introns/exon boxes indicate the size in bp.
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Figure 1: Structural analysis of the genomic sequence of LeSUT1. LeSUT1 genomic sequence contains three introns and has three polyadenylation signals predicted based on sequence comparison with different cDNA clones. The numbers under the introns/exon boxes indicate the size in bp.

Mentions: In yeast and animals, the 3'-UTR is often necessary and sufficient as a cis-element for RNA transport. These cis-elements are also implicated in the blocking of translation during transport in cells where the RNA originates. To study a potential role of the SUT1 3'-UTR in SUT1 trafficking, a genomic clone containing a 1.2 kb of downstream region of SUT1 was isolated. The DNA sequence has been deposited in Genbank with the accession number AY380824. To determine the position of transcriptional termination, cDNAs were isolated from a leaf library of Lycopersicum esculentum (tomato). Of 15 cDNAs isolated and representing LeSUT1, twelve had a 3'-UTR of 484 bp, 2 of 294 bp and one of 269 bp; in all cases, the sequences are identical (Fig. 1). The canonical polyadenylation sequence AAUAAA found in animals is much less conserved in plants [17]. Furthermore, upstream sequences are necessary for optimum polyadenylation [17,18]. Sequence analysis of the LeSUT1 clones reveals the presence of polyA signals in the vicinity of the cleavage site in all cDNAs. StSUT1, which is similarly localized to the potato SE (protein) and CC (mRNA) [5], shows a similar number and positions of polyA signals in the mRNA transcript.


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)

Structural analysis of the genomic sequence of LeSUT1. LeSUT1 genomic sequence contains three introns and has three polyadenylation signals predicted based on sequence comparison with different cDNA clones. The numbers under the introns/exon boxes indicate the size in bp.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC319702&req=5

Figure 1: Structural analysis of the genomic sequence of LeSUT1. LeSUT1 genomic sequence contains three introns and has three polyadenylation signals predicted based on sequence comparison with different cDNA clones. The numbers under the introns/exon boxes indicate the size in bp.
Mentions: In yeast and animals, the 3'-UTR is often necessary and sufficient as a cis-element for RNA transport. These cis-elements are also implicated in the blocking of translation during transport in cells where the RNA originates. To study a potential role of the SUT1 3'-UTR in SUT1 trafficking, a genomic clone containing a 1.2 kb of downstream region of SUT1 was isolated. The DNA sequence has been deposited in Genbank with the accession number AY380824. To determine the position of transcriptional termination, cDNAs were isolated from a leaf library of Lycopersicum esculentum (tomato). Of 15 cDNAs isolated and representing LeSUT1, twelve had a 3'-UTR of 484 bp, 2 of 294 bp and one of 269 bp; in all cases, the sequences are identical (Fig. 1). The canonical polyadenylation sequence AAUAAA found in animals is much less conserved in plants [17]. Furthermore, upstream sequences are necessary for optimum polyadenylation [17,18]. Sequence analysis of the LeSUT1 clones reveals the presence of polyA signals in the vicinity of the cleavage site in all cDNAs. StSUT1, which is similarly localized to the potato SE (protein) and CC (mRNA) [5], shows a similar number and positions of polyA signals in the mRNA transcript.

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