Antisense Repression of Vacuolar and Cell Wall Invertase in Transgenic Carrot Alters Early Plant Development and Sucrose Partitioning

Abstract

To unravel the functions of cell wall and vacuolar invertases in carrot, we used an antisense technique to generate transgenic carrot plants with reduced enzyme activity. Phenotypic alterations appeared at very early stages of development; indeed, the morphology of cotyledon-stage embryos was markedly changed. At the stage at which control plantlets had two to three leaves and one primary root, shoots of transgenic plantlets did not separate into individual leaves but consisted of stunted, interconnected green structures. When transgenic plantlets were grown on media containing a mixture of sucrose, glucose, and fructose rather than sucrose alone, the malformation was alleviated, and plantlets looked normal. Plantlets from hexose-containing media produced mature plants when transferred to soil. Plants expressing antisense mRNA for cell wall invertase had a bushy appearance due to the development of extra leaves, which accumulated elevated levels of sucrose and starch. Simultaneously, tap root development was markedly reduced, and the resulting smaller organs contained lower levels of carbohydrates. Compared with control plants, the dry weight leaf-to-root ratio of cell wall invertase antisense plants was shifted from 1:3 to 17:1. Plants expressing antisense mRNA for vacuolar invertase also had more leaves than did control plants, but tap roots developed normally, although they were smaller, and the leaf-to-root ratio was 1.5:1. Again, the carbohydrate content of leaves was elevated, and that of roots was reduced. Our data suggest that acid invertases play an important role in early plant development, most likely via control of sugar composition and metabolic fluxes. Later in plant development, both isoenzymes seem to have important functions in sucrose partitioning.

Induction of male sterility in plants by metabolic engineering of the carbohydrate supply

“…Thus, the apoplastic carbohydrate supply pathway for the pollen is controlled both by the surrounding tissues of the anther and by the developing pollen in a sequential manner (21). This study strongly supports the importance of apoplastic cleavage of sucrose by extracellular invertase for supplying carbohydrates to sink tissues and for normal development of plants, which has been suggested by an increasing number of studies in recent years (42)(43)(44)(45). A number of studies have shown that cell wall invertases are sink tissue specifically expressed and up-regulated by stimuli that are associated with an increased demand for carbohydrates such as stress-related stimuli and growth-stimulating phytohormones (25).…”

“…It also has been shown that an arrest in pollen development in wheat because of a water deficit during meiosis correlated with alterations in carbohydrate metabolism and a drastic decrease in invertase activity (23). Antisense repression of an extracellular invertase in carrot under control of the constitutive 35S promoter strongly interferes with regeneration of the transformed plants and resulted in pleiotropic effects (43). This further supports the highly restricted expression pattern of the Nin88 promoter during anther development because we could not observe any effect during plant growth and development, and even anther formation was phenotypically normal.…”

Vacuolar invertase regulates elongation of Arabidopsis thaliana roots as revealed by QTL and mutant analysis

“…Vac-Inv is important for osmotic purposes and to supply energy. In Daucus carota, antisense suppression of CWInv or Vac-Inv resulted in altered source-sink ratios and carbohydrate levels, although the effects were more pronounced in antisense CW-Inv plants (11). In tomato fruits, a QTL for total soluble solids turned out to be a CW-Inv (18).…”

“…The relative usage of Inv or Susy pathways might also be due to energetic reason, because Susy requires less ATP to breakdown sucrose as compared to Inv, in combination with hexokinase (9). When trying to establish a possible role for these enzymes in the regulation of sink activities, several problems may arise, including: (i) all plant species tested so far have multiple genes for both Susy and Inv (10)(11)(12), and it is unlikely that they are all involved in regulation of sink strength in different organs, and (ii) expression of these genes may have profound organ-specific patterns and may vary during plant and͞or organ development (5,10,12,13).…”

In Arabidopsis thaliana, the invertase inhibitors AtC/VIF1 and 2 exhibit distinct target enzyme specificities and expression profiles

“…The first enzyme yields UDP-glucose and fructose in a reversible reaction in the cytosol, whereas invertases in the cytosol, the vacuole or the cell wall release glucose and fructose in an irreversible reaction. Both types of enzymes have been shown to be essential for plants, especially during growth, storage compound accumulation, and stress responses [1,2].…”