Sucrose-mediated transcriptional regulation of sucrose symporter activity in the phloem

Abstract

A proton-sucrose symporter mediates the key step in carbon export from leaves of most plants. Sucrose transport activity and steady-state mRNA levels of BvSUT1, a sugar beet leaf sucrose symporter, are negatively regulated specifically by sucrose. Results reported here show that BvSUT1 mRNA was localized to companion cells of the leaf's vascular system, which supports its role in the systemic distribution of photoassimilate. Immunoblot analysis showed that decreased transport activity was caused by a reduction in the abundance of symporter protein. RNA gel blot analysis of the leaf symporter revealed that message levels also declined, and nuclear run-on experiments demonstrated that this was the result of decreased transcription. Further analysis showed that symporter protein and message are both degraded rapidly. Taken together, these data show that phloem loading is regulated by means of sucrose-mediated changes in transcription of a phloem-specific sucrose symporter gene in a regulatory system that may play a pivotal role in balancing photosynthetic activity with resource utilization.

Fig 1.

Companion cell-specific localization of BvSUT1 mRNA. In sections of mature sugar beet leaf hybridized with antisense BvSUT1 cRNA probe, staining was localized to cytoplasmically dense companion cells proximal to the sieve elements, whereas no cells were stained in sections hybridized with the sense probe. The veins shown are directly embedded in the mesophyll and lack supporting parenchyma, indicating they are minor veins involved in phloem loading.

Fig 2.

BvSUT1 protein abundance and sucrose transport activity as modulated by sucrose feeding. (A) In immunoblots of PMV isolated from leaves fed increasing concentrations of sucrose for 24 h. (B) Proton motive force-dependent sucrose (♦) and alanine transport (□) in the same PMV.

Fig 3.

Sucrose symporter BvSUT1 protein stability in leaves treated with cycloheximide (cyclohex). Immunoblots of PMV isolated from leaves fed water or 100 μm cycloheximide for 4 and 8 h. BvSUT1 protein abundance decreased to 42% and 34% of water-fed control levels, respectively.

Fig 4.

BvSUT1 transcription in response to sucrose feeding. (A) Individual excised leaves were fed 100 mM sucrose for 0, 2, 4, 6, or 24 h, then flash-frozen in liquid nitrogen. Nuclei were isolated from each leaf and transcription of BvSUT1, ACT2, and EF1A was assayed by using nuclear run-on analysis. BvSUT1 and ACT2 transcription activity was normalized within each assay using EF1A transcription activity as an internal standard. Normalized BvSUT1 transcription activity for each time point was then expressed as a percentage of normalized BvSUT1 transcription activity in the 0-h leaf. Normalized BvSUT1 transcription decreased to around 50% of 0-h levels after just 2 h of sucrose feeding and remained depressed for up to 24 h. Error bars represent ± SE, and each time point is the mean of 3–5 independent experiments. (B) In this representative autoradiograph from a nuclear run-on assay performed on leaves fed 100 mM sucrose for 0 or 24 h, EF1A-normalized BvSUT1 transcription activity in the 24-h sucrose-fed leaf was 31% ± 9% of 0-h BvSUT1 transcription activity. Normalized ACT2 transcription activity was not affected by sucrose feeding.

Fig 5.

Effect of sucrose feeding on BvSUT1 mRNA stability. BvSUT1 mRNA stability was examined in leaves fed water or sucrose for 3 h, then treated with 150 μg/ml cordycepin. BvSUT1 mRNA levels were measured before and after cordycepin treatment. BvSUT1 message decayed at about the same rate in sucrose-fed (♦) as in water-fed (□) leaves. (Inset) RNA gel blot analysis of BvSUT1 mRNA abundance in leaves fed water (W) or sucrose (S) for 3 h followed by cordycepin for 3 h (WC and SC).