Accumulation of hexoses in leaf vacuoles: Studies with transgenic tobacco plants expressing yeast-derived invertase in the cytosol,vacuole or apoplasm

The subcellular distribution of hexoses, sucrose and amino acids among the stromal, cytosolic and vacuolar compartments was analysed by a nonaqueous fractionation technique in leaves of tobacco (Nicotiana tabaccum L.) wild-type and transgenic plants expressing a yeast-derived invertase in the cytosolic, vacuolar or apoplasmic compartment. In the wild-type plants the amino acids were found to be located in the stroma and in the cytosol, sucrose mainly in the cytosol and up to 98% of the hexoses in the vacuole. In the leaves of the various transformants, where the contents of hexoses were greater than in wild-type plants, again 97–98% of these hexoses were found in the vacuoles. It is concluded that leaf vacuoles contain transporters for the active uptake of glucose and fructose against a high concentration gradient. A comparison of estimated metabolite concentrations in the subcellular compartments of wild-type and transformant plants indicated that the decreased photosynthetic capacity of the transformants is not due to an osmotic effect on photosynthesis, as was shown earlier to be the case in transformed potato leaves, but is the result of a long-term dedifferentiation of tobacco leaf cells to heterotrophic cells.Abbreviations apo-inv tobacco plant with yeast invertase in the apoplasm - Chl chlorophyll - cy-inv tobacco plant with yeast invertase in the cytosol - vac-inv tobacco plant with yeast invertase in the vacuole - WT wild-type tobacco plant The authors thank A. Großpietsch for her able technical assistance. This work has been supported by the Bundesminister für Forschung und Technologie.     

A role for 'futile cycles' involving invertase and sucrose synthase in sucrose metabolism of tomato fruit.

Current concepts of the factors determining sink strength and the subsequent regulation of carbohydrate metabolism in tomato fruit are based upon an understanding of the relative roles of sucrose synthase, sucrose phosphate synthase and invertase, derived from studies in mutants and transformed plants. These enzymes participate in at least four futile cycles that involve sugar transport between the cytosol, vacuole and apoplast. Key reactions are (1) the continuous rapid degradation of sucrose in the cytosol by sucrose synthase (SuSy), (2) sucrose re-synthesis via either SuSy or sucrose phosphate synthase (SPS), (3) sucrose hydrolysis in the vacuole or apoplast by acid invertase, (4) subsequent transport of hexoses to the cytosol where they are once more converted into sucrose, and (5) rapid synthesis and breakdown of starch in the amyloplast. In this way futile cycles of sucrose/hexose interchange govern fruit sugar content and composition. The major function of the high and constant invertase activity in red tomato fruit is, therefore, to maintain high cellular hexose concentrations, the hydrolysis of sucrose in the vacuole and in the intercellular space allowing more efficient storage of sugar in these compartments. Vacuolar sugar storage may be important in sustaining fruit cell growth at times when less sucrose is available for the sink organs because of exhaustion of the carbohydrate pools in source leaves.     

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     

Manipulation of sink-source relations in transgenic plants

Since 1980, the use of transgenic plants in modern plant science has become a powerful tool to study whole plant physiology. In this review, we try to summarize the data obtained in the field of photoassimilate partitioning. Attempts to study sink-source interactions concern factors which might limit sink strength and source capacity. Transgenic plants have been used to manipulate the sucrose to starch ratio in order to produce plants with higher sucrose levels in their source leaves. Alterations in partitioning were achieved by manipulating Calvin cycle enzymes, transport proteins and sucrose biosynthetic enzymes. The ability of sink tissues to attract photoassimilates has been altered by either increasing or decreasing sucrose hydrolytic activities. The increase of sucrose hydrolysis was achieved by creating transgenic potato plants with tuber specific yeast-derived invertase. Decreased sucrose utilization was achieved by antisense inhibition of sucrose synthase in potato tubers.     

Correlation between arrested secondary plasmodesmal development and onset of accelerated leaf senescence in yeast acid invertase transgenic tobacco plants

Mature leaves of a transgenic tobacco plant ( Nicotiana tabacum L var. Samsun, line A41-10) that constitutively express a yeast-derived acid invertase gene develop symptoms which are characterized by the presence of greenish-yellow and green sectors in the same leaf, and onset of early, leaf senescence. Previous studies indicated that invertase activity was two- to threefold higher in the greenish-yellow sectors than in the green sectors. Our structural analyses revealed that development of secondary plasmodesmata, via modification of existing primary plasmodesmata, between mesophyll cells was inhibited severely in the greenish-yellow sectors, but only marginally in the green sectors. In contrast, the structure and function of primary plasmodesmata in the same symptomatic sectors remained unaltered as determined by structural and dye coupling studies. It is hypothesized that secondary plasmodesmata differ from primary plasmodesmata in having special abilities to traffic information molecules to coordinate leaf development and physiological function(s). Arrest of secondary plasmodesmal development by high invertase activity in the transgenic tobacco leaf may have prevented this type of trafficking and hence resulted in early leaf senescence. The results also indicate that the yeast acid invertase-expressing tobacco may provide an effective experimental system for the molecular characterization of cellular mechanisms that regulate the development, function, and possible turnover of secondary plasmodesmata.     

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.     

Involvement of cell-wall invertase in low-temperature hardening of tobacco plants

Specific features of low-temperature hardening (6 days at 8°C) of cold-sensitive tobacco plants (Nicotiana tabacum, cv. Samsun) related to changes in the cell-wall invertase activity were studied. During cold hardening, oppositely directed changes in this enzyme activity occurred in tobacco leaves and roots. In the leaves, cell-wall invertase was activated (approximately by 30%), the content of sugars increased (approximately by 25%), and the content of sucrose, the main transport form of sugars, in the apoplast reduced by three times; all these changes indicate that assimilate outflow from leaves to roots was inhibited. In contrast, in the root system, enzyme activity was decreased almost twice and the content of sugars in them was essentially unchanged. It is suggested that a strategy of low-temperature adaptation of cold-sensitive tobacco plants aimed at creating the high cold tolerance of aboveground parts, even at the expense of the root system, which, under conditions of native vegetation, is not practically exposed to damaging low temperatures.     

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.     

Expression of a luteoviral movement protein in transgenic plants leads to carbohydrate accumulation and reduced photosynthetic capacity in source leaves

Elucidating the role of viral genes in transgenic plants revealed that the movement protein (MP) from tobacco mosaic virus is responsible for altered carbohydrate allocation in tobacco and potato plants. To study whether this is a general feature of viral MPs, the movement protein MP17 of potato leafroll virus (PLRV), a phloem-restricted luteovirus, was constitutively expressed in tobacco plants. Transgenic lines were strongly reduced in height and developed bleached and sometimes even necrotic areas on their source leaves. Levels of soluble sugars and starch were significantly increased in source leaves. Yet, in leaf laminae the hexose—phosphate content was unaltered and ATP reduced to only a small extent, indicating that these leaves were able to maintain homeostatic conditions by compartmentalization of soluble sugars, probably in the vacuole. On the contrary, midribs contained lower levels of soluble sugars, ATP, hexose—phosphates and UDP-glucose supporting the concept of limited uptake and catabolism of sucrose in the phloem. The accumulation of carbohydrates led to a decreased photosynthetic capacity and carboxylation efficiency of ribulose-1,5-bisphosphate carboxylase/oxygenase (rubisco) probably owing to decreased expression of photosynthetic proteins. In parallel, levels of pathogenesis-related proteins were elevated which may be the reason for the obtained limited resistance against the unrelated potato virus Y (PVY)^N in the transgenic tobacco plants. Ultrathin sections of affected leaves harvested from 2-week-old plants revealed plasmodesmal alterations in the phloem tissue while plasmodesmata between mesophyll cells were indistinguishable from wild-type. These data favour the phloem tissue to be the primary site of PLRV MP17 action in altering carbohydrate metabolism.     

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.     

A carboxy-terminal plant vacuolar targeting signal is not recognized by yeast

Three different classes of signals for plant vacuolar targeting have been defined. Previous work has demonstrated that the carboxyl-terminal propeptide (CTPP) of barley lectin (BL) is a vacuolar targeting signal in tobacco plants. When a mutant BL protein lacking the CTPP is expressed in tobacco, the protein is secreted. In an effort to determine the universality of this signal, the CTPP was tested for its ability to target proteins to the vacuole of Saccharomyces cerevisiae. Genes encoding fusion proteins between the yeast secreted protein invertase and BL domains were synthesized and transformed into an invertase deletion mutant of yeast. Invertase assays on intact and detergent-solubilized cells demonstrated that invertase+CTPP was secreted, while nearly 90% of the invertase::BL+CTPP (fusion protein between invertase and BL containing the CTPP) and invertase::BL-CTPP proteins (fusion between invertase and BL lacking the CTPP) were retained intracellularly. These fusions were secreted in a mutant of yeast that normally secretes proteins targeted to the vacuole. With this and previous work, proteins representing all three classes of plant vacuolar targeting signals have now been tested in yeast, and in all cases, the experiments indicate that the plant proteins are directed to the yeast vacuole using signals other than those recognized by plants.     

Slow-growth phenotype of transgenic tomato expressing apoplastic invertase

The growth of transgenic tomato (Lycopersicon esculentum) plants that express in their apoplast yeast invertase under the control of the cauliflower mosaic virus 35S promoter is severely inhibited. The higher the level of invertase, the greater the inhibition of growth. A second phenotypic characteristic of these transgenic plants is the development of yellow and necrotic spots on the leaves, and leaf curling. Again the severity of the symptoms is correlated with the level of invertase. These symptoms do not develop in shaded leaves indicating the need for photosynthesis. Keeping the plants in the dark for a prolonged period (24 hours) results in the disappearance of leaf starch from the control plants, but not from the plants with apoplastic invertase. These results are consistent with the interpretation that apoplastic invertase prevents photosynthate export from source leaves and that phloem loading includes an apoplastic step.     

Carbon Partitioning and Growth of a Starchless Mutant of Nicotiana sylvestris

We have further characterized the photosynthetic carbohydrate metabolism and growth of a starchless mutant (NS 458) of Nicotiana sylvestris that is deficient in plastid phosphoglucomutase (Hanson KR, McHale NA [1988] Plant Physiol 88: 838-844). In general, the mutant had only slightly lower rates of photosynthesis under ambient conditions than the wild type. However, accumulation of soluble sugars (primarily hexose sugars) in source leaves of the mutant compensated for only about half of the carbon stored as starch in the wild type. Therefore, the export rate was slightly higher in the mutant relative to the wild type. Starch in the wild type and soluble sugars in the mutant were used to support plant growth at night. Growth of the mutant was progressively restricted, relative to wild type, when plants were grown under shortened photoperiods. When grown under short days, leaf expansion of the mutant was greater during the day, but was restricted at night relative to wild-type leaves, which expanded primarily at night. We postulate that restricted growth of the mutant on short days is the result of several factors, including slightly lower net photosynthesis and inability to synthesize starch in both source and sink tissues for use at night. In short-term experiments, increased “sink demand” on a source leaf (by shading all other source leaves) had no immediate effect on starch accumulation during the photoperiod in the wild type or on soluble sugar accumulation in the mutant. These results would be consistent with a transport limitation in N. sylvestris such that not all of the additional carbon flux into sucrose in the mutant can be exported from the leaf. Consequently, the mutant accumulates hexose sugars during the photoperiod, apparently as the result of sucrose hydrolysis within the vacuole by acid invertase.     

Apoplastic expression of yeast-derived invertase in potato : effects on photosynthesis, leaf solute composition, water relations, and tuber composition

In potato plants (Solanum tuberosum), a chimeric yeast-derived invertase gene fused to a 35S cauliflower mosaic virus promoter has been expressed. The protein was targeted to the cell wall by using the signal peptide of proteinase inhibitor II fused to the amino terminus of the yeast invertase. The transformed plants had crinkled leaves, showed a reduced growth rate, and produced fewer tubers. Although in the apoplast of the leaves of the transformed plants the content of glucose and fructose rose by a factor of 20, and that of sucrose declined 20-fold, 98% of the carbohydrate in the phloem sap consisted of sucrose, demonstrating the strong specificity of phloem loading. In the leaf cells of the transformed plants, glucose, fructose, and amino acids, especially proline, were accumulated. Consequently, the osmolality of the cell sap rose from 250 to 350 mosmol/kg. Our results show that the observed 75% decrease of photosynthesis is not caused by a feedback regulation of sucrose synthesis and is accompanied by an increase in the osmotic pressure in the leaf cells. In the transformed plants, not only the amino acid to sucrose ratio in the phloem sap, but also the amino acid and protein contents in the tubers were found to be elevated. In the tubers of the transformed plants, the protein to starch ratio increased.     

High-Level Transient Expression of ER-Targeted Human Interleukin 6 in Nicotiana benthamiana

Tobacco plants can be used to express recombinant proteins that cannot be produced in a soluble and active form using traditional platforms such as Escherichia coli. We therefore expressed the human glycoprotein interleukin 6 (IL6) in two commercial tobacco cultivars (Nicotiana tabacum cv. Virginia and cv. Geudertheimer) as well as the model host N. benthamiana to compare different transformation strategies (stable vs. transient expression) and subcellular targeting (apoplast, endoplasmic reticulum (ER) and vacuole). In T₀ transgenic plants, the highest expression levels were achieved by ER targeting but the overall yields of IL6 were still low in the leaves (0.005% TSP in the ER, 0.0008% in the vacuole and 0.0005% in the apoplast). The apoplast variant accumulated to similar levels in leaves and seeds, whereas the ER-targeted variant was 1.2-fold more abundant in seeds and the vacuolar variant was 6-fold more abundant in seeds. The yields improved in subsequent generations, with the best-performing T₂ plants producing the ER-targeted IL6 at 0.14% TSP in both leaves and seeds. Transient expression of ER-targeted IL6 in leaves using the MagnICON system resulted in yields of up to 7% TSP in N. benthamiana, but only 1% in N. tabacum cv. Virginia and 0.5% in cv. Geudertheimer. Although the commercial tobacco cultivars produced up to threefold more biomass than N. benthamiana, this was not enough to compensate for the lower overall yields. The recombinant IL6 produced by transient and stable expression in plants was biologically active and presented as two alternative bands matching the corresponding native protein.