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Raffinose family oligosaccharides (RFOs) perform several physiological functions in plants. In addition to accumulating during seed formation, raffinose and stachyose are translocated in the phloem and may accumulate in response to low temperatures, drought, or salt stress. Although the synthesis of galactinol, as mediated by galactinol synthase (GAS), is the first committed step in RFO formation, its expression patterns are poorly understood in most species. We have cloned and characterized the expression of two galactinol synthase gene family members in melon (Cucumis melo L. Cantalupensis Group). Both CmGAS1 and CmGAS2 are highly expressed in mature leaves. Galactinol synthase transcription in leaves was not upregulated by either water or low temperature stresses. Transcripts of CmGAS1 were present in developing melon seeds at a time coincident with the formation of raffinose and stachyose. Based on the GAS expression and RFO accumulation patterns, we propose that RFOs in melon function in carbon translocation and seed desiccation tolerance. and ajugose (a hexasaccharide RFO) accumulates in the mesophyll cells of Ajuga reptans after exposure to low temperatures (Bachmann and Keller, 1995; Sprenger and Keller, 2000). These acclimation responses may be critical for the survival of low temperature tolerant species. Although the presence of RFOs has been documented in a variety of systems, there are few studies demonstrating the expression of the GAS genes. Sprenger and Keller (2000) published a study that described the expression patterns of two galactinol synthase genes, GolS-1 and GolS-2, in Ajuga reptans. Ajuga is a frost-hardy evergreen labiate that both accumulates and translocates RFOs. The gene, GolS-1 was primarily expressed in the mesophyll in response to low temperatures while GolS-2 was expressed in intermediary cells and most likely catalyzes the formation of RFOs destined for translocation. The Arabidopsis GAS genes AtGolS1 and AtGolS2 are expressed in response to drought and salt stress while AtGolS3 accumulates in response to low temperature stress (Taji et al., 2002). The expression patterns of other Arabidopsis GAS genes have not been characterized. We have cloned two GAS genes in melon (Cucumis melo L. Cantalupensis Group). One gene, CmGAS1, is highly homologous to the zucchini CpGAS1 gene identified by Kerr et al. (1993). A CmGAS1 promoter-gusA fusion was made and used to transform Arabidopsis and tobacco plants. In these transgenic plants, GUS expression localized to the smallest veins of mature Arabidopsis and tobacco leaves (Haritatos et al., 2000). Although galactinol is not normally synthesized in wild-type tobacco leaves, stain localized to the companion cells and sieve elements in the minor veins (Haritatos et al., 2000), indicating that the CmGAS1 promoter drives vein- specific gene expression. There are no previous reports document- ing the expression pattern of the second melon GAS gene, CmGAS2. In melon, raffinose and stachyose serve as phloem translocation sugars and also accumulate in maturing seeds. Other functions of RFOs in melon have not been documented. We have used RNA blots and RT-PCR techniques to characterize the mRNA expression patterns of the CmGAS1 and CmGAS2 genes in melon.

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 thegusA 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.

Leaf and minor vein structure were studied in Arabidopsis thaliana (L.) Heynh. to gain insight into the mechanism(s) of phloem loading. Vein density (length of veins per unit leaf area) is extremely low. Almost all veins are intimately associated with the mesophyll and are probably involved in loading. In transverse sections of veins there are, on average, two companion cells for each sieve element. Phloem parenchyma cells appear to be specialized for delivery of photoassimilate from the bundle sheath to sieve element-companion cell complexes: they make numerous contacts with the bundle sheath and with companion cells and they have transfer cell wall ingrowths where they are in contact with sieve elements. Plasmodesmatal frequencies are high at interfaces involving phloem parenchyma cells. The plasmodesmata between phloem parenchyma cells and companion cells are structurally distinct in that there are several branches on the phloem parenchyma cell side of the wall and only one branch on the companion cell side. Most of the translocated sugar in A. thaliana is sucrose, but raffinose is also transported. Based on structural evidence, the most likely route of sucrose transport is from bundle sheath to phloem parenchyma cells through plasmodesmata, followed by efflux into the apoplasm across wall ingrowths and carrier-mediated uptake into the sieve element-companion cell complex.

Raffinose, stachyose, and galactinol are synthesized in intermediary cells (specialized companion cells) of the minor-vein phloem of cucurbits. To better understand the role of these carbohydrates and the regulation of their synthesis and transport, we measured the concentrations of each of the components of the raffinose oligosaccharide synthetic pathway in mesophyll and sieve element-intermediary cell complexes (SE-ICCs) in the leaves of melon (Cucumis melo L. cv. Hale's Best Jumbo). These concentrations are consistent with a polymer-trapping mechanism for phloem loading, with sucrose diffusing from mesophyll into intermediary cells and being made into raffinose and stachyose, which are too large to diffuse back to the mesophyll. To determine carbohydrate concentrations, we developed a method involving microdissected tissues. Blind endings of areoles, and mesophyll surrounding these veins, were separately removed from lyophilized leaf tissue. Carbohydrates were quantitated by high-performance liquid chromatography with pulsed amperometric detection. A small amount of mesophyll remained attached to the blind endings; the carbohydrate contribution of these cells to the vein sample was eliminated by subtraction, based on the amount of chlorophyll. Volumes of cells and subcellular compartments were calculated by morphometric analysis and were used to calculate carbohydrate concentrations. Assuming no subcellular compartmentation, the additive concentration of sugars in the SE-ICCs of minor veins is about 600 mM. Stachyose and raffinose concentrations are about 330 mM and 70 mM, respectively, in SE-ICCs; concentrations of these sugars are much lower in mesophyll (0.2 and 0.1 mM). This is consistent with the view that stachyose and raffinose are unable to pass through the plasmodesmata between intermediary cells and bundle-sheath cells. Sucrose levels appear to be higher in the SE-ICC (about 130mM) than in the mesophyll (about 10 mM), but if compartmentation is taken into account the gradient for sucrose is probably downhill from mesophyll to intermediary cells. Flux through plasmodesmata between the bundle sheath and intermediary cells was calculated and was found to be within the range of values of flux through plasmodesmata reported in the literature.