SummaryDuring the storage phase, cotyledons of developing pea seeds are nourished by nutrients released to the seed apoplasm by their maternal seed coats. Sucrose is transported into pea cotyledons by sucrose/H + symport mediated by PsSUT1 and possibly other sucrose symporters. PsSUT1 is principally localised to plasma membranes of cotyledon epidermal and subepidermal transfer cells abutting the seed coat. We tested the hypothesis that endogenous sucrose/H + symporter(s) regulate sucrose import into developing pea cotyledons. This was done by supplementing their transport activity with a potato sucrose symporter (StSUT1), selectively expressed in cotyledon storage parenchyma cells under control of a vicilin promoter. In segregating transgenic lines, enhanced [ 14 C]sucrose in¯ux into cotyledons above wild-type levels was found to be dependent on StSUT1 expression. The transgene signi®cantly increased (approximately 2-fold) transport activity of cotyledon storage parenchyma tissues where it was selectively expressed. In contrast, sucrose in¯ux into whole cotyledons through the endogenous epidermal transfer cell pathway was increased by only 23% in cotyledons expressing the transgene. A similar response was found for rates of biomass gain by intact cotyledons and by excised cotyledons cultured on a sucrose medium. These observations demonstrate that transport activities of sucrose symporters in¯uence cotyledon growth rates. The attenuated effect of StSUT1 overexpression on sucrose and dry matter¯uxes by whole cotyledons is consistent with a large proportion of sucrose being taken up at the cotyledonary surface. This indicates that the cellular location of sucrose transporter activity plays a key role in determining rates of sucrose import into cotyledons.

Pyruvate orthophosphate dikinase (PPDK) is a key enzyme of C4 photosynthesis providing the acceptor molecule for the primary CO2 fixation in the mesophyll cells. Here we present the isolation and characterisation of the corresponding gene (termed pdk) from the C4 plant Flaveria trinervia (Asteraceae). Southern analysis indicates that in contrast to maize pdk sequences in F. trinervia are present as single copy. Sequence analysis of the entire gene reveals that its coding sequence is identical to the previous isolated PPDK-cDNA from this species. The gene spans about 13 kb and consists of 21 exons, it thus contains two additional exons compared to the maize gene. As in maize, a long intervening sequence of 6.1 kb is positioned at the boundary of the transit peptide segment and the mature protein region. Pdk transcripts accumulate abundantly in leaves, but are also detectable in stems and roots. While the leaf and stem transcripts are 3.4 kb in size and encode the chloroplastic PPDK isoform, a 3.0 kb transcript lacking the region encoding the plastidic transit peptide accumulates in roots. Thus two different transcripts can be produced from a single pdk gene most likely by use of alternative promoters and not by alternative splicing. The accumulation of the 3.4 kb transcript is under light control. Darkening leads to a drastic depletion of this transcript in both leaves and stems. Instead, the 3.0 kb transit peptide-lacking pdk transcript accumulates, but only in stems and roots, not in leaves.

We have isolated full-size cDNA sequences encoding the photosynthetic isoform of pyruvate orthophosphate dikinase (PPDK) of the C3 plant Flaveria pringlei. The encoded protein shares 96% identical amino acid residues with the C4 isoform of PPDK in the C4 species F. trinervia. The differing amino acid residues are evenly distributed along the polypeptide chain. Genomic Southern analysis of photosynthetic PPDK sequences in F. pringlei (C3), F. chloraefolia (C3-C4), F. linearis (C3-C4), F. floridana (C3-C4), F. brownii (C4-like) and F. trinervia (C4) reveals a simple hybridization pattern which is suggestive of a single gene. Northern hybridization experiments show that the abundance of PPDK transcripts in leaves correlates with the degree of C4 characteristics expressed in the various photosynthetic types analysed. This finding demonstrates that the increase in expression levels must have played a crucial role in evolving the C4-PPDK gene in the genus Flaveria.

To study the molecular evolution of NADP-dependent malic enzyme (NADP-ME) in the genus Flaveria a leaf-specific cDNA library of the C3 plant F. pringlei was screened for the presence of sequences homologous to the C4 isoform gene (named modA) of the C4 plant F. trinervia. The cDNAs isolated contained varying numbers of identical restriction fragments suggesting that they were derived from a single gene. This was supported by Southern hybridisation experiments with genomic DNA from F. trinervia and F. pringlei. Nucleotide sequence analysis of a full-size clone identified the presence of a typical plastidic transit peptide and revealed that the mature modA proteins of F. trinervia (C4) and F. pringlei (C3) are 90% similar. These findings indicate that C3 plants, like C4 species, possess a plastidic isoform of NADP-ME and that the modA genes of the two species represent orthologous genes. Northern analyses showed that modA transcripts accumulate to similar levels in leaves, stems and roots of F. pringlei. The expression of this gene in F. pringlei thus appears to be rather constitutive. In contrast, the modA gene of F. trinervia is abundantly expressed in leaves, but maintains its expression in stems and roots. It has to be concluded from these data that the leaf-specific increase in the expression level was a key step which was taken during the evolution of the C4 isoform modA gene starting from a C3 ancestral gene.

Correlative physiological evidence suggests that membrane transport into storage parenchyma cells is a key step in determining hexose levels accumulated in tomato (Lycopersicon esculentum Mill.) fruit (Ruan et al. 1997). Expression of three previously identified hexose transporter genes (LeHT1, 2 and 3) demonstrated that LeHT3, and to a lesser extent LeHT1, are the predominant transporters expressed in young fruit (10 d after anthesis; DAA). Expression of both transporters dropped sharply until 24 DAA, after which only LeHT3 expression remained at detectable levels through to fruit ripening. LeHT2 was not expressed substantially until the onset of fruit ripening. For fruit at both 10 and 30 DAA, LeHT3 transcripts were detected in storage parenchyma cells of the outer pericarp tissue, but not in vascular bundles or the first layer of parenchyma cells surrounding these bundles. In contrast to LeHT gene expression, hexose transporter protein levels were maximal between 20 and 30 DAA, which corresponded to the period of highest hexose accumulation. The delayed appearance of transporter protein is consistent with some form of post-transcriptional regulation. Based on these analyses, LeHT3 appears to be responsible for the rapid hexose accumulation in developing tomato fruit.