SummaryTransgenic Arabidopsis plants were constructed to express a range of GFP-fusion proteins (36-67 kDa) under the companion cell (CC)-specific AtSUC2 promoter. These plants were used to monitor the trafficking of these GFP-fusion proteins from the CCs into the sieve elements (SEs) and their subsequent translocation within and out of the phloem. The results revealed a large size exclusion limit (SEL) (>67 kDa) for the plasmodesmata connecting SEs and CCs in the loading phloem. Membrane-anchored GFP-fusions and a GFP variant targeted to the endoplasmic reticulum (ER) remained inside the CCs and were used as 'zero trafficking' controls. In contrast, free GFP and all soluble GFP-fusions, moved from the CCs into the SEs and were subsequently translocated through the phloem. Phloem unloading and post-phloem transport of these mobile GFP-fusions were studied in root tips, where post-phloem transport occurred only for the free form of GFP. All of the other soluble GFP-fusion variants were unloaded and restricted to a narrow zone of cells immediately adjacent to the mature protophloem. It appears that this domain of cells, which has a peripheral SEL of about 27-36 kDa, allows protein exchange between protophloem SEs and surrounding cells, but restricts general access of large proteins into the root tip. The presented data provide additional information on phloem development in Arabidopsis in relation to the formation of symplasmic domains.

Abscisic acid (ABA) and sugars regulate many aspects of plant growth and development, and we are only just beginning to understand the complex interactions between ABA and sugar signaling networks. Here, we show that ABA-dependent transcription factors bind to the promoter of the Arabidopsis thaliana AtSUC1 (At1g71880) sucrose transporter gene in vitro. We present the characterization of a cis-regulatory element by truncation of the AtSUC1 promoter and by electrophoretic mobility shift assays that is identical to a previously characterized ABA-responsive element (ABRE). In yeast 1-hybrid analyses we identified ABI5 (AtbZIP39; At2g36270) and AREB3 (AtbZIP66; At3g56850) as potential interactors. Analyses of plants expressing the beta-glucuronidase reporter gene under the control of ABI5 or AREB3 promoter sequences demonstrated that both transcription factor genes are co-expressed with AtSUC1 in pollen and seedlings, the primary sites of AtSUC1 action. Mutational analyses of the identified cis-regulatory element verified its importance for AtSUC1 expression in young seedlings. In abi5-4 seedlings, we observed an increase of sucrose-dependent anthocyanin accumulation and AtSUC1 mRNA levels. This suggests that ABI5 prevents an overshoot of sucrose-induced AtSUC1 expression and confirmed a novel cross-link between sugar and ABA signaling.

Based on analyses in Arabidopsis thaliana ecotype C24, the AtSUC1 protein was previously characterised as a male gametophyte-specific H(+)/sucrose symporter. Later, expression analyses in ecotype Columbia-0 (Col-0) identified AtSUC1 expression also in trichomes (not detected in trichome-less C24 plants) and roots, suggesting ecotype-specific differences in AtSUC1 expression. Here, we present data on additional ecotype-specific differences in AtSUC1 expression in other tissues. Using different AtSUC1 promoter-reporter gene lines, we performed comparative analyses of AtSUC1 expression in floral tissues of C24 and Col-0 plants, and using an AtSUC1-specific antiserum, we performed immunohistochemical analyses on tissue sections from C24, Col-0, Landsberg erecta (Ler) and Wassilewskaija (Ws) ecotypes. We show that AtSUC1 expression occurs in the funicular epidermis of C24, Ler and Ws, but not in Col-0. In contrast, we observed high levels of AtSUC1 protein in pollen grains of Col-0, lower levels in pollen of C24 and Ler, and no AtSUC1 protein in Ws pollen. Moreover, our reporter gene analyses identified a previously undetected expression of AtSUC1 in the female gametophyte, and revealed that AtSUC1 expression in the funicular epidermis is absent from unpollinated siliques and is induced upon successful pollination. The impact of these findings on the potential physiological role of AtSUC1 is discussed.