The aim of the present study was to gain insight into the contribution of extracellular invertases for sink metabolism in tomato (Lycopersicon esculentum L.). The present study shows that extracellular invertase isoenzymes are encoded by a gene family comprising four members: Lin5, Lin6, Lin7, and Lin8. The regulation of mRNA levels by internal and external signals and the distribution in sink and source tissues has been determined and compared with mRNA levels of the intracellular sucrose (Suc)-cleaving enzymes Suc synthase and vacuolar invertase. The specific regulation of Lin5, Lin6, and Lin7 suggests an important function of apoplastic cleavage of Suc by cell wall-bound invertase in establishing and maintaining sink metabolism. Lin6 is expressed under conditions that require a high carbohydrate supply. The corresponding mRNA shows a sink tissue-specific distribution and the concentration is elevated by stress-related stimuli, by the growth-promoting phytohormone zeatin, and in response to the induction of heterotrophic metabolism. The expression of Lin5 and Lin7 in gynoecia and stamens, respectively, suggests an important function in supplying carbohydrates to these flower organs, whereas the Lin7 mRNA was found to be present exclusively in this specific sink organ.

A cDNA, Wiv-1, for an isozyme of acid invertase (EC 3.2.1.26) was cloned from wounded leaves of tomato (Lycopersicon esculentum). The encoded protein had a basic isoelectric point and strong similarity to the amino acid sequences of plant cell wall-bound invertases. The conserved sequence WECPD that is found in all plant cell wall-bound invertases was also found in the deduced protein. These results suggested that Wiv-1 encoded a cell wall-bound acid invertase of tomato. Wounding increased the levels of mRNAs for soluble and cell wall-bound invertases and the activities of these invertases in leaves of L. esculentum and of a related species, L. peruvianum. The induction of Aiv-1 mRNA for the soluble enzyme in wounded leaves was not very strong, while that of Wiv-1 mRNA for the wall-bound enzyme was prominent. The level of Aiv-1 mRNA reached a maximum 48 h after wounding while that of Wiv-1 mRNA continued to rise for up to 96 h. These findings suggested that the genes for the two isozymes responded independently to wounding. The levels in various organs of Aiv-1 and Wiv-1 mRNAs were higher in L. esculentum than in L. peruvianum. Possible roles of cell wall-bound acid invertase in wound response and in developing plant are discussed.

Comparative analysis of complex developmental pathways depends on our ability to resolve the function of members of gene families across taxonomic groups. LIN5, which belongs to a small gene family of apoplastic invertases in tomato (Lycopersicon esculentum), is a quantitative trait locus that modifies fruit sugar composition. We have compared the genomic organization and expression of this gene family in the two distantly related species: tomato and Arabidopsis. Invertase family members reside on segmental duplications in the near-colinear genomes of tomato and potato (Solanum tuberosum). These chromosomal segments are syntenically duplicated in the model plant Arabidopsis. On the basis of phylogenetic analysis of genes in the microsyntenic region, we conclude that these segmental duplications arose independently after the separation of the tomato/potato clade from Arabidopsis. Rapid regulatory divergence is characteristic of the invertase family. Interestingly, although the processes of gene duplication and specialization of expression occurred separately in the two species, synteny-based orthologs from both clades acquired similar organ-specific expression. This similar expression pattern of the genes is evidence of comparable evolutionary constraints (parallel evolution) rather than of functional orthology. The observation that functional orthology cannot be identified through analysis of expression similarity highlights the caution that needs to be exercised in extrapolating developmental networks from a model organism.

Cell wall-bound invertase (cw-Inv) plays an important role in carbohydrate partitioning and regulation of sink-source interaction. There is increasing evidence that pathogens interfere with sink-source interaction, and induction of cw-Inv activity has frequently been shown in response to pathogen infection. To investigate the role of cw-Inv, transgenic tomato (Solanum lycopersicum) plants silenced for the major leaf cw-Inv isoforms were generated and analyzed during normal growth and during the compatible interaction with Xanthomonas campestris pv vesicatoria. Under normal growth conditions, activities of sucrolytic enzymes as well as photosynthesis and respiration were unaltered in the transgenic plants compared with wild-type plants. However, starch levels of source leaves were strongly reduced, which was most likely caused by an enhanced sucrose exudation rate. Following X. campestris pv vesicatoria infection, cw-Inv-silenced plants showed an increased sucrose to hexose ratio in the apoplast of leaves. Symptom development, inhibition of photosynthesis, and expression of photosynthetic genes were clearly delayed in transgenic plants compared with wild-type plants. In addition, induction of senescence-associated and pathogenesis-related genes observed in infected wild-type plants was abolished in cw-Inv-silenced tomato lines. These changes were not associated with decreased bacterial growth. In conclusion, cw-Inv restricts carbon export from source leaves and regulates the sucrose to hexose ratio in the apoplast. Furthermore, an increased apoplastic hexose to sucrose ratio can be linked to inhibition of photosynthesis and induction of pathogenesis-related gene expression but does not significantly influence bacterial growth. Indirectly, bacteria may benefit from low invertase activity, since the longevity of host cells is raised and basal defense might be dampened.

To elucidate the regulation pattern of extracellular invertase LIN6 of tomato, the corresponding promoter has been cloned and the sink-tissue specific expression and its regulation by sugars, stress stimuli, growth regulators, and the diurnal rhythm is shown. The in situ analysis of transgenic tobacco plants expressing a LIN6 promoter::beta-glucuronidase reporter gene fusion demonstrates LIN6 expression in sink tissues, such as pollen grains and vascular tissues of leaves and stems. LIN6 is up-regulated in close proximity to wounded tissue, and by methyl jasmonate and abscisic acid, global signals known to modulate defence/stress response. Salicylic acid on the other hand, as well as acetyl salicylic acid, suppresses LIN6 expression, supporting the fact that LIN6 is an inducible compound of the defence/stress response pathway that is antagonistically regulated by jasmonates and salicylates. Induction of the LIN6 promoter in stable transformed BY2 suspension cultures by sucrose and the growth-promoting phytohormones cytokinin and auxin along histochemical expression data, showing LIN6 expression in germinating seeds and seedlings, indicates a role of LIN6 invertase during growth processes. In addition, LIN6 is regulated by a diurnal rhythm that drives LIN6 expression in subjective dawn. Transactivation assays with circadian oscillator elements of Arabidopsis Circadian Clock Associated 1 and Late Elongated Hypocotyl demonstrate functional interaction with the LIN6 promoter.