Identification of phloem involved in assimilate loading in leaves by the activity of the galactinol synthase promoter

Abstract

The definition of "minor" veins in leaves is arbitrary and of uncertain biological significance. Generally, the term refers to the smallest vein classes in the leaf, believed to function in phloem loading. We found that a galactinol synthase promoter, cloned from melon (Cucumis melo), directs expression of the gusA gene to the smallest veins of mature Arabidopsis and cultivated tobacco (Nicotiana tabacum) leaves. This expression pattern is consistent with the role of galactinol synthase in sugar synthesis and phloem loading in cucurbits. The expression pattern in tobacco is especially noteworthy since galactinol is not synthesized in the leaves of this plant. Also, we unexpectedly found that expression in tobacco is limited to two of three companion cells in class-V veins, which are the most extensive in the leaf. Thus, the "minor" vein system is defined and regulated at the genetic level, and there is heterogeneity of response to this system by different companion cells of the same vein.

Figure 1

Staining patterns of Arabidopsis (A–E) and tobacco (F–K) transformed with the GAS promoter CmGAS1-GUS construct pSG3K101. A, Fourteen-day-old seedling. Staining is dense in the veins of the cotyledons and in most of the veins of the first-formed leaves. A progressive, basipetal (tip-to-base) staining pattern in minor veins is evident in the three developing leaves, the most mature of which is stained to approximately the midway point. Basipetal maturation is a characteristic of features associated with the sink-to-source transition in leaves. Hydathodes also stain (arrow). Bar = 1 mm. B, Mature leaf tissue stained for 3 h. Staining is most apparent in the blind endings (arrows) and other small veins. Bar = 200 μm. C, After 24 h, all minor veins are stained. A secondary vein (arrow) is unstained. Bar = 200 μm. D, In this flower, stain is visible in the filaments, in veins of the sepals, and less intensely in those of the petals. Bar = 0.5 mm. E, Faint blue staining near the tip of a lateral root. Bar = 0.5 mm. F, Leaf tissue near the tip of a growing leaf that has completed the sink-source transition. The vein in the lower right hand corner is the midrib, the one in the upper right is a secondary. Bar = 1.8 mm. G, Veins of a mature leaf. The vein classes are numbered. Note the partial staining of a class-IV vein at the junction of a class-V vein (arrow). There is no diffusion of stain from the vein marked with an asterisk. Bar = 400 μm. H, Staining pattern of immature veins in the proximal region of a leaf undergoing the sink-source transition. Bar = 300 μm. I, Tissue from the same leaf as H, but more distal and mature. Bar = 300 μm. J, Localization of stain in a transverse section. Two companion cells, and the small sieve element between them, are stained. The third companion cell (arrow) and its sieve element are unstained. Phloem parenchyma cells are indicated by asterisks. Bar = 10 μm. K, Transverse hand section through the midrib. Stained minor veins can be seen in the flanking lamina, but the vascular tissue of the midrib is unstained. Bar = 0.6 mm.

Figure 2

Phloem loading in leaf discs exposed to [¹⁴C]Suc in plants transformed with the GAS promoter (GAS)-GUS construct pSG3K101. Due to less extensive uptake of label, discs in C and D were exposed to x-ray film for twice as long as the disc in B. A, Developing tobacco leaf the same size as the leaf used for the ¹⁴C-labeling experiments. Leaves of this age have just ceased importing photoassimilate from mature leaves. In developing leaves there is a basipetal gradient in maturity, the distal end being more mature than the base. Bar = 1.5 cm. B, Autoradiograph of a disc from a fully expanded, mature leaf. The disc was exposed to a [¹⁴C]Suc solution prior to freeze-drying. [¹⁴C]Suc has accumulated in the veins. C, Autoradiograph of a disc excised near the tip of a developing leaf, as indicated by the arrow. [¹⁴C]Suc has accumulated in the veins, although not to the same degree as in mature leaf tissue. GUS staining, as illustrated in Figure 1H, was visible from tissue adjacent to this disc but not in more proximal tissues. D, Autoradiograph of a disc excised near the base of a developing leaf, as indicated by the arrow. This is the most proximal tissue in which [¹⁴C]Suc accumulated in the veins. No GUS staining was evident in adjacent tissue. Bar (in B–D) = 1 mm.