Expression of a yeast-derived invertase in the cell wall of tobacco and Arabidopsis plants leads to accumulation of carbohydrate and inhibition of photosynthesis and strongly influences growth and phenotype of transgenic tobacco plants.

Chimeric genes consisting of the coding sequence of the yeast invertase gene suc 2 and different N-terminal portions of the potato-derived vacuolar protein proteinase inhibitor II fused to the 35S CaMV promoter and the poly-A site of the octopine synthase gene were transferred into tobacco and Arabidopsis thaliana plants using Agrobacterium based systems. Regenerated transgenic plants display a 50- to 500-fold higher invertase activity compared to non-transformed control plants. This invertase is N-glycosylated and efficiently secreted from the plant cell leading to its apoplastic location. Whereas expression of the invertase does not lead to drastic changes in transgenic Arabidopsis thaliana plants, transgenic tobacco plants show dramatic changes with respect to development and phenotype. Expression of the invertase leads to stunted growth due to reduction of internodal distances, to development of bleached and/or necrotic regions in older leaves and to suppressed root formation. In mature leaves, high levels of soluble sugars and starch accumulate. These carbohydrates do not show a diurnal turnover. The accumulation of carbohydrate is accompanied by an inhibition of photosynthesis, and in tobacco, by an increase in the rate of respiration. Measurements in bleached versus green areas of the same leaf show that the bleached section contains high levels of carbohydrates and has lower photosynthesis and higher respiration than green sections. It is concluded that expression of invertase in the cell wall interrupts export and leads to an accumulation of carbohydrates and inhibition of photosynthesis.     

Cell wall-bound invertase limits sucrose export and is involved in symptom development and inhibition of photosynthesis during compatible interaction between tomato and Xanthomonas campestris pv vesicatoria

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.     

Transgenic tobacco plants expressing yeast-derived invertase in either the cytosol, vacuole or apoplast: a powerful tool for studying sucrose metabolism and sink/source interactions

In higher plants sucrose plays a central roles with respect to both short-term storage and distribution of photoassimilates formed in the leaf. Sucrose is synthesized in the cytosol, transiently stored in the vacuole and exported via the apoplast. In order to elucidate the role of the different compartments with respect to sucrose metabolism, a yeast-derived invertase was directed into the cytosol and vacuole of transgenic tobacco plants. This was in addition to the targeting of yeast-derived invertase into the apoplast described previously. Vacuolar targeting was achieved by fusing an N-terminal portion (146 amino acids long) of the vacuolar protein patatin to the coding region of the mature invertase protein. Transgenic tobacco plants expressing the yeast-derived invertase in different subcellular compartments displayed dramatic phenotypic differences when compared to wild-type plants. All transgenic plants showed stunted growth accompanied by reduced root formation. Starch and soluble sugars accumulated in leaves indicating that the distribution of sucrose was impaired in all cases. Expression of cytosolic yeast invertase resulted in the accumulation of starch and soluble sugars in both very young (sink) and older (source) leaves. The leaves were curved, indicating a more rapid cell expansion or cell division at the upper side of the leaf. Light-green sectors with reduced photosynthetic activity were evenly distributed over the leaf surface. With the apoplastic and vacuolar invertase, the phenotypical changes induced only appear in older (source) leaves. The development of bleached and/or necrotic sectors was linked to the source state of a leaf. Bleaching followed the sink to source transition, starting at the rim of the leaf and moving to the base. The bleaching was paralleled by the inhibition of photosynthesis.     

Apoplastic pH and growth in expanding leaves of Vicia faba under salinity

Salinity affects water availability in the soil and subsequently the plant uptake capacity. Upon exposure to salt stress, leaf growth in monocot plants has been shown to be reduced instantaneously, followed by a gradual acclimation. The growth reactions are caused by an initial water deficit and an accompanied osmotic effect, followed by an IAA-induced sequestration of protons into the apoplast that increases leaf growth again as explained by the acid growth theory. In this study, we investigated the dynamics of growth reactions and apoplastic pH in leaves of the dicot Vicia faba in the presence of NaCl during the initiation of salt stress. Concurrent changes in apoplastic pH were detected by ratiometric fluorescence microscopy using the fluorescent dye fluorescein tetramethylrhodamine dextran. To elucidate the possible relation between the dynamics of leaf growth and apoplastic pH, results of the ratio imaging technique were combined with an in vivo growth analysis imaging approach. Leaf growth rate of V. faba was highest in the dusk and the early night phase; at this time a concomitant decrease of the apoplastic pH was observed. Under salinity, the apoplastic pH in leaves of V. faba increased with a simultaneous decrease of leaf growth towards increasing developmental stages, but with complex aberrations in the 24-h-leaf-growth pattern compared to control leaves. In conclusion, these results show that salt stress leads to an increase in apoplastic pH and to a declined leaf growth activity with complex 24-h-interactions of growth and pH in V. faba.     

The involvement of apoplastic invertase in the formation of resistance of cold-tolerant plants to hypothermia

Involvement of apoplastic invertase in the formation of resistance of cold-tolerant plants to hypothermia was established for Solanum tuberosum L. cv. Désirée and a transformant of the same cultivar expressing the yeast suc2 gene encoding apoplastic invertase. The dependence of the formation of increased constitutive and stress-induced cold-tolerance on the activity of apoplastic invertase and, consequently, on the level of intracellular sugars in the roots and leaves was demonstrated.     

The impact of reduced vacuolar invertase activity on the photosynthetic and carbohydrate metabolism of tomato

The impact of reduced vacuolar invertase activity on photosynthetic and carbohydrate metabolism was examined in tomato (Solanum lycopersicon L.). The introduction of a co-suppression construct (derived from tomato vacuolar invertase cDNA) produced plants containing a range of vacuolar invertase activities. In the leaves of most transgenic plants from line INV-B, vacuolar invertase activity was below the level of detection, whereas leaves from line INV-A and untransformed wild-type plants showed considerable variation. Apoplasmic invertase activity was not affected by the co-suppression construct. It has been suggested that, in leaves, vacuolar invertase activity regulates sucrose content and its availability for export, such that in plants with high vacuolar invertase activity a futile cycle of sucrose synthesis and degradation takes place. In INV-B plants with no detectable leaf vacuolar invertase activity, sucrose accumulated to much higher levels than in wild-type plants, and hexoses were barely detectable. There was a clear threshold relationship between invertase activity and sucrose content, and a linear relationship with hexose content. From these data the following conclusions can be drawn. (i) In INV-B plants sucrose enters the vacuole where it accumulates as hydrolysis cannot take place. (ii) There was not an excess of vacuolar invertase activity in the vacuole; the rate of sucrose hydrolysis depended upon the concentration of the enzyme. (iii) The rate of import of sucrose into the vacuole is also important in determining the rate of sucrose hydrolysis. The starch content of leaves was not significantly different in any of the plants examined. In tomato plants grown at high irradiance there was no impact of vacuolar invertase activity on the rate of photosynthesis or growth. The impact of the cosuppression construct on root vacuolar invertase activity and carbohydrate metabolism was less marked.Abbreviations CaMV Cauliflower Mosaic Virus - WT wild type     

Solute accumulation and decreased photosynthesis in leaves of potato plants expressing yeast-derived invertase either in the apoplast, vacuole or cytosol

Potato (Solanum tuberosum cv. Désirée) plants expressing yeast invertase directed either to the apoplast, vacuole or cytosol were biochemically and physiologically characterised. All lines of transgenic plants showed similarities to plants growing under water stress. Transformants were retarded in growth, and accumulated hexoses and amino acids, especially proline, to levels up to 40-fold higher than those of the wild types. In all transformants rates of CO₂ assimilation and leaf conductance were reduced. From the unchanged intercellular partial pressure of CO₂ and apoplastic cis-abscisic acid (ABA) content of transformed leaves it was concluded that the reduced rate of CO₂ assimilation was not caused by a limitation in the availability of CO₂ for␣the ribulose-1,5-bisphosphate carboxylase-oxygenase (Rubisco). In the transformants the amount of Rubisco protein was not reduced, but both activation state and carboxylation efficiency of photosynthesis were lowered. In vacuolar and cytosolic transformants this inhibition of Rubisco might be caused by a changed ratio of organic bound and inorganic phosphate, as indicated by a doubling of phosphorylated intermediates. But in apoplastic transformants the pattern of phosphorylated intermediates resembled that of leaves of water-stressed potato plants, although the cause of inhibition of photosynthesis was not identical. Whereas in water-stressed plants increased contents of the phytohormone ABA are supposed to mediate the adaptation to water stress, no contribution of ABA to reduction of photosynthesis could be detected in invertase transformants. Received: 29 May 1996 / Accepted: 30 December 1996     

The fate of apoplastic sucrose in sink and source leaves of Urtica dioica

Experiments were performed with developing and mature leaves of Urtica dioica L. to trace differences which could be interpreted in terms of cell wall-bound acid invertase (EC 3.2.1.26) participating in phloem unloading in a sink leaf. The pH of apoplastic fluid that was collected by gentle centrifugation of entire leaves was identical (7.1) in the two types of leaves; also, fluorometric determination with esculetin showed a neutral apoplastic pH between 7.0 in the source and 7.2 in the sink leaf. To detect whether differences in apoplastic pH occur within limited leaf areas, such as of the tissue surrounding the veins, the metabolic fate of [¹⁴C]–(fructosyl)-sucrose that was administered via the xylem was investigated. In source leaves, there was a large transitory decrease in [¹⁴C]-sucrose followed by a substantial resynthesis of this compound. In sink leaves, resynthesis was less significant and carbon was incorporated mainly in starch, charged soluble compounds and cell walls. However, after correction for resynthesis, the two types of leaves showed an identical capacity for sucrose cleavage. Finally, activation of the apoplastic invertase by administering labelled sucrose in buffered solution of pH 5.0 did not result in an enhanced degradation. By contrast, apoplastic fluid collected from leaves which had been infiltrated with buffer solutions of pH 5.5 and 8.0, respectively, showed a rapid adjustment of the pH close to the natural neutral value by the mesophyll tissue. The results are incompatible with the idea of an active invertase in the sink (and the source) leaves apoplast, and hence do not lend support to the theory of apoplastic cleavage of sucrose being required for phloem unloading in this kind of a utilization sink.     

Effect of sugars on the development of oxidative stress induced by hypothermia in potato plants expressing yeast invertase gene

The influence of sugars on the development of oxidative stress induced by hypothermia was investigated in the leaves of two genotypes of potato (Solanum tuberosum L.) grown in vitro on the Murashige and Skoog medium supplemented with 2% sucrose. We used wild-type plants of potato, cv. Désirée, and potato plants expressing a yeast invertase gene under the control of the B33 class I patatin promoter and carrying a sequence of proteinase inhibitor II leader peptide for the apoplastic enzyme localization. At temperature of 22°C optimal for growth, expression of the yeast invertase gene in the leaves of transformed plants brought about a modification in the carbohydrate metabolism manifested in the activation of acid forms of invertase and accumulation of intracellular sugars (predominantly of sucrose because of its resynthesis). The exposure of plants to light under prolonged hypothermia (5°C, 6 days) activated all the forms of invertase (predominantly of acid invertase) and induced accumulation of sugars. In the leaves of potato expressing the yeast invertase gene, these processes were more intense. Under chilling, superoxide dismutase activity and the rate of lipid peroxidation in the leaves of investigated potato genotypes depended on the level of accumulated intracellular sugars. It was concluded that sugars play an important role as stabilizers of cellular membranes and scavengers of reactive oxygen species decelerating the processes of free radical oxidation of biomolecules upon the development of oxidative stress induced by hypothermia.     

Developmental changes of antioxidative systems in tobacco leaves as affected by limited sucrose export in transgenic plants expressing yeast-invertase in the apoplastic space

It is generally believed that a restricted export of carbohydrates from source leaves causes oxidative stress because of an enhanced utilisation of O₂ instead of NADP⁺ as electron acceptor in photosynthesis. To test this hypothesis, developmental changes of antioxidative systems were investigated in wild-type and transgenic tobacco (Nicotiana tabacum L.) suffering from disturbed sink-source relations by expression of yeast invertase in the apoplastic space. Young expanding leaves of the wild type contained higher activities of Superoxide dismutase (EC 1.15.1.1), ascorbate peroxidase (EC 1.11.1.11), catalase (EC 1.11.1.6), dehydroascorbate reductase (EC 1.8.5.1), glutathione reductase (EC 1.6.4.2) and a higher glutathione content than mature source leaves. The activity of monodehydroascorbate-radical reductase (EC 1.1.5.4) and the ascorbate content remained unaffected by the developmental stage in the wild type. In young expanding leaves of the transgenic plants the capacity of the antioxidative systems was similar to or higher than in corresponding leaves from the wild type. Source leaves of transgenic tobacco with an increased carbohydrate content showed a small chlorophyll loss, an increased malondialdehyde content, a selective loss of the activities of Cu/Zn-superoxide dismutase isoenzymes and a fourfold decrease in ascorbate compared with the wild type. There was no evidence that the protection from H₂O₂ was insufficient since source leaves of transgenic tobacco contained increased activities of catalase, ascorbate peroxidase, and monodehydroascorbate-radical reductase and an increased ascorbate-to-dehydroascorbate ratio compared with source leaves of the wild type. In severely chlorotic leaf sections of the transgenic plants, most components of the antioxidative system were lower than in green leaf sections, but the ascorbate-to-dehydroascorbate ratio was increased. These results suggest that carbohydrate-accumulating cells have an increased availability of reductant, which can increase the degree of reduction of the ascorbate system via glutathione-related systems or via the activity of monodehydroascorbate-radical reductase. At the same time, transgenic tobacco leaves seem to suffer from an increased oxidative stress, presumably as a result of a decreased consumption of O ₂ ^.- by Cu/Zn-superoxide dismutases in the chloroplasts. There was no evidence that carbohydrate-accumulating leaves acclimated to enhanced O ₂ ^.- production rates in the chloroplasts.     

Morphological features of the transgenic tobacco plant shoot expressing the 3-hydroxy-3-methylglutagyl-CoA reductase (<Emphasis Type="Italic">HMG1</Emphasis>) gene in the direct and reverse orientations towards the promoter

3-Hydroxy-3-methylglutaryl-CoA reductase (HMG1) catalyzes the formation of mevalonic acid, the key intermediate of the cytosolic isoprenoid synthesis pathway. The parameters of stem and leaf growth were studied in the transgenic tobacco plants that express the HMG1 gene in both sense and antisense orientations towards the constitutive promoter. The transgenic plant height did not significantly differ from that of the control plants, though the plants carrying the sense copy of the HMG1 gene were considerably taller than plants that carried the antisense gene copy. Plants carrying an extra copy of the HMG1 gene were also characterized by increased leaf area. The number of mesophyll cells calculated per square unit of transgenic plants leaves was smaller than in the control plant leaves, though their volume was not considerably changed in any of the variants, suggesting changes in the cell packing density in leaves.     

The changes in invertase activity and the content of sugars in the course of adaptation of potato plants to hypothermia

The pattern of changes in the activity of various forms of invertase (acid soluble, alkaline, and acid insoluble) and the content of sugars (glucose, fructose, and sucrose) in the course of plant adaptation to prolonged (6 days) hypothermia (5°C) was investigated in the leaves of potato plants (Solanum tuberosum L., cv. Desiree) produced in vitro. We used the wild-type plants as a control and transformed plants with carbohydrate metabolism modified by inserting the yeast gene for invertase (apoplastic enzyme). In the course of adaptation to hypothermia, the activity of acid invertase was shown to rise and the content of sucrose and glucose to increase in the leaves of both genotypes. The greatest activity of acid invertases by the third day of cold acclimation corresponded to the peak level of sugars; in transformed plants, these characteristics exceeded those in the control plants. The transformed plants were more cold resistant than the control plants as suggested by the lack of disturbance of ion permeability of their membranes. It was concluded that owing to accumulation of low-molecular carbohydrates in the course of cold acclimation caused by activation of acid invertase cold resistant plants better adapt to temperature drop.     

Expression of an abscisic acid-binding single-chain antibody influences the subcellular distribution of abscisic acid and leads to developmental changes in transgenic potato plants

Potato (Solanum tuberosum L. cv. Désirée) plants were transformed to express a single-chain variable-fragment antibody against abscisic acid (ABA), and present in the endoplasmic reticulum at to up to 0.24% of the soluble leaf protein. The resulting transgenic plants were only able to grow normally at 95% humidity and moderate light. Four-week-old plants accumulated ABA to high extent, were retarded in growth and their leaves were smaller than those of control plants. Leaf stomatal conductivity was increased due to larger stomates. The subcellular concentrations of ABA in the chloroplast, cytoplasm and vacuole, and the apoplastic space of leaves were determined. In the 4-week-old transgenic plants the concentration of ABA not bound to the antibody was identical to that of control plants and the stomates were able to close in response to lower humidity of the atmosphere. A detailed analysis of age-dependent changes in plant metabolism showed that leaves of young transformed plants developed in ABA deficiency and leaves of older plants in ABA excess. Phenotypic changes developed in ABA deficiency partly disappeared in older plants.     

Antisense suppression of an acid invertase gene (MAI1) in muskmelon alters plant growth and fruit development

To unravel the roles of soluble acid invertase in muskmelon (Cucumis melo L.), its activity in transgenic muskmelon plants was reduced by an antisense approach. For this purpose, a 1038 bp cDNA fragment of muskmelon soluble acid invertase was expressed in antisense orientation behind the 35S promoter of the cauliflower mosaic virus. The phenotype of the antisense plants clearly differed from that of control plants. The transgenic plant leaves were markedly smaller, and the stems were obviously thinner. Transmission electron microscopy revealed that degradation of the chloroplast membrane occurred in transgenic leaves and the number of grana in the chloroplast was significantly reduced, suggesting that the slow growth and weaker phenotype of the transgenic plants may be due to damage to the chloroplast ultrastructure, which in turn resulted in a decrease in net photosynthetic rate. The sucrose concentration increased and levels of acid invertase decreased in transgenic fruit, and the fruit size was 60% smaller than that of the control. In addition, transgenic fruit reached full-slip at 25 d after pollination (DAP), approximately 5 d before the control fruit (full-slip at 30 DAP), and this accelerated maturity correlated with a dramatic elevation of ethylene production at the later stages of fruit development. Together, these results suggest that soluble acid invertase not only plays an important role during muskmelon plant and fruit development but also controls the sucrose content in muskmelon fruit.     

Opposite Effects on Spodoptera littoralis Larvae of High Expression Level of a Trypsin Proteinase Inhibitor in Transgenic Plants

This work illustrates potential adverse effects linked with the expression of proteinase inhibitor (PI) in plants used as a strategy to enhance pest resistance. Tobacco (Nicotiana tabacum L. cv Xanthi) and Arabidopsis [Heynh.] ecotype Wassilewskija) transgenic plants expressing the mustard trypsin PI 2 (MTI-2) at different levels were obtained. First-instar larvae of the Egyptian cotton worm (Spodoptera littoralis Boisd.) were fed on detached leaves of these plants. The high level of MTI-2 expression in leaves had deleterious effects on larvae, causing mortality and decreasing mean larval weight, and was correlated with a decrease in the leaf surface eaten. However, larvae fed leaves from plants expressing MTI-2 at the low expression level did not show increased mortality, but a net gain in weight and a faster development compared with control larvae. The low MTI-2 expression level also resulted in increased leaf damage. These observations are correlated with the differential expression of digestive proteinases in the larval gut; overexpression of existing proteinases on low-MTI-2-expression level plants and induction of new proteinases on high-MTI-2-expression level plants. These results emphasize the critical need for the development of a PI-based defense strategy for plants obtaining the appropriate PI-expression level relative to the pest's sensitivity threshold to that PI.     

Winter rye antifreeze activity increases in response to cold and drought, but not abscisic acid

Antifreeze activity increases in winter rye ( Secale cereale L.) during cold acclimation as the plants accumulate antifreeze proteins (AFPs) that are similar to glucanases, chitinases and thaumatin-like proteins (TLPs) in the leaf apoplast. In the present work, experiments were conducted to assess the role of drought and abscisic acid (ABA) in the regulation of antifreeze activity and accumulation of AFPs. Antifreeze activity was detected as early as 24 h of drought treatment at 20°C and increased as the level of apoplastic proteins increased. Apoplastic proteins accumulated rapidly under water stress and reached a level within 8 days that was equivalent to the level of apoplastic proteins accumulated when plants were acclimated to cold temperature for 7 weeks. These drought-induced apoplastic proteins had molecular masses ranging from 11 to 35 kDa and were identified as two glucanases, two chitinases, and two TLPs, by using antisera raised against cold-induced rye glucanase, chitinase, and TLP, respectively. Apoplastic extracts obtained from plants treated with ABA lacked the ability to modify the growth of ice crystals, even though ABA induced the accumulation of apoplastic proteins within 4 days to a level similar to that obtained when plants were either drought-stressed for 8 days or cold-acclimated for 7 weeks. These ABA-induced apoplastic proteins were identified immunologically as two glucanases and two TLPs. Moreover, the ABA biosynthesis inhibitor fluridone did not prevent the accumulation of AFPs in the leaves of cold-acclimated rye plants. Our results show that cold acclimation and drought both induce antifreeze activity in winter rye plants and that the pathway regulating AFP production is independent of ABA.     

Changes in the water binding characteristics of the cell walls from transgenic Nicotiana tabacum leaves with enhanced levels of peroxidase activity

Pore size in the cell wall matrix may affect cell wall–water relations, particularly under osmotic stress. Cross linkage of plant cell wall matrix polymers is an important step in the formation of this structure and peroxidases have been proposed to catalyse the cross-linking of phenolic constituents. Transgenic tobacco ( Nicotiana tabacum ) plants expressing a basic tomato peroxidase gene (TPX2) showed increased apoplastic ferulic acid peroxidase activity in mature leaves. This enhanced activity was not associated with a decreased leaf growth. Differential scanning calorimetry (DSC) of control dried cell walls showed a putative glass transition, after Ca²⁺ removal, that was absent in the transgenic line. This would indicate that transgenic walls were more rigid. DSC analysis of water-hydrated cell wall preparations distinguished two pools of water, freezable and non-freezable water. The amount of non-freezable water, which corresponds to strongly bound water, was higher in the transgenic line (64 versus 55%). DSC thermograms of the transgenic cell wall were displaced to lower temperatures, and this may be interpreted as the result of a stronger interaction between this freezable water and this wall. Water sorption and desorption isotherms, obtained at relative humidity ranging from 5 to 93%, demonstrated the presence of very strongly bound water in the transgenic cell walls that was absent in controls. Water sorption–desorption hysteresis of the isotherms was evident in the control wall but not in the transgenic line. These changes in cell wall–water interaction seem to be relevant at the organ level because leaf discs of transgenic plants maintained higher relative water content than control discs, at water potentials between −1.05 and−2.31 MPa.