Plant Physiol. (1998) 117: 831-839

Sorbitol-6-Phosphate Dehydrogenase Expression in Transgenic Tobacco¹
High Amounts of Sorbitol Lead to Necrotic Lesions

Elena V. Sheveleva^*, Sheila Marquez, Wendy Chmara, Abreeza Zegeer, Richard G. Jensen, and Hans J. Bohnert

Departments of Biochemistry (E.S., S.M., W.C., A.Z., R.G.J., H.J.B.), Plant Sciences (R.G.J., H.J.B.), and Molecular and Cellular Biology (H.J.B.), Biosciences West, The University of Arizona, Tucson, Arizona 85721-0088

	ABSTRACT

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We analyzed transgenic tobacco (Nicotiana tabacum L.) expressing Stpd1, a cDNA encoding sorbitol-6-phosphate dehydrogenase from apple, under the control of a cauliflower mosaic virus 35S promoter. In 125 independent transformants variable amounts of sorbitol ranging from 0.2 to 130 µmol g¹ fresh weight were found. Plants that accumulated up to 2 to 3 µmol g¹ fresh weight sorbitol were phenotypically normal, with successively slower growth as sorbitol amounts increased. Plants accumulating sorbitol at 3 to 5 µmol g¹ fresh weight occasionally showed regions in which chlorophyll was partially lost, but at higher sorbitol amounts young leaves of all plants lost chlorophyll in irregular spots that developed into necrotic lesions. When sorbitol exceeded 15 to 20 µmol g¹ fresh weight, plants were infertile, and at even higher sorbitol concentrations the primary regenerants were incapable of forming roots in culture or soil. In mature plants sorbitol amounts varied with age, leaf position, and growth conditions. The appearance of lesions was correlated with high sorbitol, glucose, fructose, and starch, and low myo-inositol. Supplementing myo-inositol in seedlings and young plants prevented lesion formation. Hyperaccumulation of sorbitol, which interferes with inositol biosynthesis, seems to lead to osmotic imbalance, possibly acting as a signal affecting carbohydrate allocation and transport.

	INTRODUCTION

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Under water-stress conditions plants in many families accumulate metabolites that are thought to provide osmotic adjustment, i.e. their presence leads to water retention under water-limiting conditions (LeRudulier and Bouillard, 1983). Another view assumes that the accumulating metabolites might have specific protective functions, e.g. in the protection of membranes or protein complexes, in enzyme stabilization, or in radical scavenging (Smirnoff and Cumbes, 1989; Galinski, 1993; Smirnoff, 1993; Asada, 1994; Papageorgiou and Murata, 1995). These concepts, originally based on correlative evidence and in vitro studies, can now be tested in transgenic plants. The engineered expression of mannitol, ononitol, fructans, Pro, Gly betaine, and trehalose has been reported mostly in transgenic tobacco (Nicotiana tabacum) (Tarczynski et al., 1993; Kishor et al., 1995; Nomura et al., 1995; Pilon-Smits et al., 1995; Holmström et al., 1996; Hayashi et al., 1997). In all cases accumulation of the metabolite had some effect, often monitored as marginal protection of plant performance under water- or salt-stress conditions, but it is unknown by which mechanism(s) the accumulating metabolites function. Considering that many engineered metabolites accumulate to low, osmotically irrelevant amounts, we wanted to explore the effect of an extremely high accumulation of an osmolyte.

We studied the consequences of mannitol and ononitol production on plant performance under salt-stress conditions (Tarczynski et al., 1993; Vernon et al., 1993; Shen et al., 1997a; Sheveleva et al., 1997). Transgenic tobacco plants containing mannitol in chloroplasts (1-8 µmol g¹ fresh weight) are phenotypically normal and exhibit rates of photosynthesis identical to those of the wild type (Shen et al., 1997a), but growth seems to decrease as mannitol increases (Karakas et al., 1997). Ononitol-accumulating (up to 10 µmol g¹ fresh weight) plants also display a normal phenotype (Vernon et al., 1993; Sheveleva et al., 1997). When these plants are exposed to drought stress and high salinity, they accumulate even higher amounts of ononitol (up to 35 µmol g¹ fresh weight) as a result of a stress-induced increase of myo-inositol, which then serves as the substrate for additional ononitol production (Sheveleva et al., 1997). Growth of the plants is normal during this stress-inducible accumulation.

In this analysis of polyol production we report on the performance of tobacco after the expression of Stpd1, a cDNA encoding sorbitol-6-phosphate dehydrogenase from apple (Kanayama et al., 1992). After analyzing a large number of primary transformants we detected sorbitol accumulation ranging from 0.2 to approximately 130 µmol g¹ fresh weight. Even under the assumption that sorbitol might be at least in part partitioned to the vacuole, the high amounts would be osmotically significant. Within this range, we monitored plant performance under optimal growth conditions and during salt-stress treatment. The results indicated that high amounts of sorbitol reduced growth and led to symptoms similar to those that have been reported for plants expressing extracellular invertase (von Schaewen et al., 1990). However, the symptoms produced by invertase expression, patchiness of photosynthesis, loss of chlorophyll, and necroses, predominantly affected mature source leaves, whereas the symptoms observed with the high sorbitol producers already appeared in immature leaves. Although different foreign polyols might have different effects in transgenic plants, one interpretation is that generating high amounts of osmolytes is not necessarily the best strategy for osmotic stress protection in an organism that is not adapted for the metabolite that accumulates. In another hypothesis, and possibly specific to sorbitol accumulation, the adverse effect of sorbitol could be caused by a disturbance of the Glc-6-P pool by sorbitol-6-phosphate dehydrogenase, which might then affect either UDP-Glc or Glc amounts, leading to altered sugar sensing in the plants.

	MATERIALS AND METHODS

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Plant Transformation

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View larger version (14K): [in this window] [in a new window]   Figure 1. Schematic presentation of the gene construction leading to the expression of sorbitol-6-phosphate dehydrogenase (S6PDH) in transgenic tobacco. The gene cassette was subcloned into pBIN19 and introduced into plants by A. tumefaciens-based transformation.

Plant Growth

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Other experiments were conducted in a growth room in soil composed of potting mix:vermiculite:sand (3:2:1). Photon flux density (400-700 nm) was maintained at 400 µmol m² s¹ with a day/night cycle of 12/12 h, RH < 20%, and temperatures of 27°C during the light period and 23°C during the dark period.

Sugar, Polyol, Pro, and Starch Analysis

Gas-Exchange Measurements

Growth on Artificial Medium

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	RESULTS

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Correlation between Amount of Sorbitol and Lesion Formation

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View this table: [in this window] [in a new window]   Table I. Sorbitol amounts in a selection of independent transformants of tobacco SR1 (T0 generation) Plantlets (T0) were transferred from agar to soil and kept in a growth room (450 µmol quanta m2 s1). After 10 d the first leaf, which had completely unfolded from the meristem, was sampled. No sorbitol could be detected in SR1 plants at any developmental stage. Transformants with sorbitol levels higher than approximately 30 µmol g1 fresh weight never developed into mature plants. The letters a and b denote individual transformed plants that originated from the same callus.

When transferred to soil, plantlets producing the highest amount of sorbitol failed to develop roots and died. Those with lower concentrations of sorbitol (5-50 µmol g¹ fresh weight) developed necrotic lesions in their leaves and the leaves remained small (Fig. 2). Plants with sorbitol concentrations less than approximately 2 µmol g¹ fresh weight had normal growth patterns, whereas plants with more than approximately 3 µmol g¹ fresh weight sorbitol showed stunted growth. Lesion formation in immature leaves was also correlated with the sorbitol concentration. Plants containing less than 3 µmol sorbitol g¹ fresh weight showed no lesions, and occasional lesions occurred in plants accumulating sorbitol at 3 to 7 µmol g¹ fresh weight. The number of lesions and the size of the affected areas increased as sorbitol increased (Table I).

View larger version (66K): [in this window] [in a new window]   Figure 2. Phenotype of S5C plant and examples of leaves with necrotic lesions. The top panel shows the habitus of a 10-week-old plant typical of plants with a sorbitol concentration of more than approximately 15 µmol g1 fresh weight. The bottom panel shows mature leaves typical of plants that accumulated sorbitol to 5 to 10 µmol g1 fresh weight in their young, expanding leaves.

Plants from the segregating T₂ generation of S5C were used in further analyses after selection for kanamycin resistance and screening for sorbitol amounts. The higher the sorbitol concentration, the more numerous the lesions and the larger the areas of necrotic tissue (Fig. 2; Table II). The appearance of lesions depended on sorbitol amount when the leaves were young. Initially, affected tissue patches lost chlorophyll, yellowed, and later became necrotic. After the leaves became fully expanded, no new lesions formed, necrotic areas expanded slightly, and, because of inequal expansion in the presence of necrotic spots, the leaves became distorted (Fig. 2). In older leaves sorbitol amounts always declined, which we interpret as a consequence of altered CaMV 35S promoter activity. The examples provided in Table II represent typical behavior. Plants with high amounts of sorbitol contained the highest polyol concentrations in leaf number 2 and sorbitol amounts declined from leaf number 3 on. Leaves of one of these plants, at 10% to 20% of the area of a mature leaf, might show lesions or yellowing in patches in a prelesion stage. In plants accumulating intermediate or low sorbitol amounts (e.g. plant no. 4, Table II), the two youngest leaves showed the highest amount of sorbitol, although lesions never formed at a comparable frequency compared with plants with higher sorbitol concentrations, and sorbitol declined gradually in the older leaves.

Table III compares carbohydrates in different leaves of SR1 and S5C plants. The amount of sorbitol in S5C exceeded the amounts of Glc, Fru, and Suc. In all S5C plants sorbitol was highest in the apical leaf. myo-Inositol was lower in the youngest leaves of S5C than in comparable leaves of SR1. In leaves with the highest sorbitol amounts myo-inositol was barely detectable. Concomitant with the decrease in sorbitol as S5C leaves matured, myo-inositol increased again, but did not reach the amount found in leaf number 5 and older leaves of SR1. This behavior is different from that of SR1, in which the youngest leaves contained the highest amount of myo-inositol, which then declined as the leaves became source leaves. In leaves with high sorbitol and low myo-inositol (leaf numbers 1-4) the amount of starch was 2 to 3 times higher in sorbitol-containing plants compared with wild type. The amounts of Glc and Fru for the whole leaf showed no correlation with leaf number, and no significant differences existed between SR1 and S5C plants. Suc amounts, however, were lower in sorbitol-containing plants yet still maintained diurnal fluctuation (data not shown). The ratio of starch to Suc in S5C was higher than that in SR1 by a factor of 7 to 5 in leaf numbers 1 to 4, which contained the highest amounts of sorbitol.

During the entire lifetime of the S5C plants the amounts of the reducing sugars Suc and myo-inositol approached those found in SR1, as the amount of sorbitol gradually decreased in mature leaves (data not shown). Therefore, the plants were able to outgrow the seemingly detrimental effect of sorbitol accumulation in immature leaves. At the flowering stage, S5C presented the habitus of plants that had survived, recognizable by the necrotic lesions, viral or fungal infections, or severe environmental stress as immature plants.

Sugar and Sorbitol Accumulation in Different Parts of One Leaf

View larger version (19K): [in this window] [in a new window]   Figure 3. Relationship between myo-inositol, Glc, Fru, and sorbitol amounts in S5C plants. Plants were grown for 10 weeks in a growth room (see ``Materials and Methods''). The plant material was collected 2 h after the beginning of illumination. The same relationship was found when starch content and sorbitol amounts were compared. gfw, Grams fresh weight.

myo-Inositol and Lesion Formation

View larger version (118K): [in this window] [in a new window]   Figure 4. Seedling growth supplemented with myo-inositol. Seedlings of SR1 (A and B) and S5C (C and D) plants were grown in Murashige and Skoog medium with (B and D) and without myo-inositol (A and C).

View this table: [in this window] [in a new window]   Table V. Amounts of sugars, myo-inositol, and sorbitol in SR1 and S5C plants grown in sterile culture on different media SR1 and S5C seeds were germinated and grown in agar in sterile culture in Murashige and Skoog medium and Gamborg's B5 vitamins including thiamine hydrochloride, pyridoxine, and nicotinic acid, but without myo-inositol. Suc was added at 8.8 mM, Glc at 38.8 mM, and myo-inositol at 1 mM. The plants were grown for 4 weeks under low-light conditions (100 µmol m2 s1). The largest leaf was taken for analysis from six to nine plantlets for each determination and the experiment was repeated twice. In each experiment the same trend was observed, although the absolute values between experiments varied. SR1 with sugars and myo-inositol added grew best; S5C without any additions grew worst and developed lesions in all leaves. S5C with only sugars added showed lesions on the cotyledons.

	DISCUSSION

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Tao et al. (1995) reported the expression of an apple sorbitol-6-phosphate dehydrogenase in tobacco. The work focused on transformants with low amounts of sorbitol (up to 0.45 µmol g¹ fresh weight) and the phenotype of these plants was identical to wild type. In repeated experiments we obtained a large number of independent transformants distinguished by the accumulation of sorbitol over a wide range, up to 130 µmol g¹ fresh weight. Changes in phenotype were correlated with sorbitol accumulation. Plants with low amounts of sorbitol (less than 2-3 µmol g¹ fresh weight) developed normally, but necrotic lesions formed with increasing frequency when sorbitol accumulated to higher amounts. Increased lesion formation was accompanied by increasing severity of compromised growth, reduced or abolished root development, and low fertility or infertility.

Lesion formation in immature leaves was an unexpected outcome of experiments that sought to explore the metabolic tolerance of tobacco to high accumulation of a polyol. Our investigations of polyol accumulation in transgenic plants have mainly pertained to the protective function(s) that carbohydrate-based osmolytes might have (Tarczynski et al., 1993; Shen et al., 1997a, 1997b; Sheveleva et al., 1997). Like mannitol- or ononitol-accumulating plants (Tarzynski et al., 1993; Sheveleva et al., 1997), the sorbitol-accumulating plants tolerated both salt stress and drought better than the wild type, but this effect was difficult to monitor because of the growth retardation and influence on development that characterized high accumulation of sorbitol. Depending on sorbitol amounts in the plants, absolute photosynthesis rates of line S5C in the absence of stress could be less than 50% of those of control plants. However, rates of photosynthesis declined less in S5C than in SR1 controls when the plants were stressed by the addition of 150 mM NaCl (data not shown).

Heineke et al. (1992) have suggested that an increase of osmotic pressure in the leaf sap could cause lesion formation after the accumulation of a foreign carbohydrate in the cytosol. Measurements of osmolality of the leaf sap were, however, identical in S5C and SR1 at approximately 400 mosmol kg¹. Overall osmolality, though, may not reflect the conditions in different compartments. Sorbitol probably accumulated mostly in the cytosol and may be at least partially excluded from chloroplasts, mitochondria, and the vacuole. Osmotic pressure differences between compartments could cause metabolic imbalances. The accumulation of starch in the youngest leaves of high-sorbitol-producing plants (Table III) could be the indicator of such an effect. Sorbitol in the cytosol might interfere with the export of carbohydrates from the chloroplasts, and this could be responsible for starch increases in these leaves. The correlation between sorbitol amount and the frequency of necrotic tissue could point to a causal relationship between osmotic pressure and necrosis symptoms. Compromised membrane integrity or altered carbohydrate export from plastids could lead to a decrease of photosynthesis and altered metabolism in the cytosol, setting in motion signaling events that finally lead to lesions.

The formation of lesions has frequently been reported as a defense reaction to pathogen attack (Walbot et al., 1983; Hahlbrock and Scheel, 1987). Biochemical and metabolic changes after pathogen attack include the accumulation of salicylic acid, callose, and lignin, the synthesis of cell wall-bound phenolics, the biosynthesis of pathogenesis-related proteins, phytoalexin accumulation, and lipid peroxidation (Hahlbrock and Scheel, 1987; Bowles, 1990; Yalpani et al., 1993; Greenberg et al., 1994; Baillieul et al., 1995). A basis for lesion formation has been seen in the perturbation of the ubiquitin system, altered proton pumping, altered hexose concentrations, or the expression of gene VI of CaMV leading to cell death (Takahashi et al., 1989; Becker et al., 1993; Mittler et al., 1995; Herbers et al., 1996). Using probes for a tobacco catalase, Cat1 (Schultes et al., 1994), a sequenced PCR product (E. Sheveleva, unpublished data), and superoxide dismutase (Sod3, cytosolic Cu/Zn superoxide dismutase; Tsang et al., 1991) we observed increases in the transcripts of these genes in S5C (not shown) compared with wild-type tobacco, which seems to support a relationship between lesion formation and the expression of defense-related proteins in the sorbitol-producing plants. Lesions in S5C plants under our growth conditions, however, were not caused by abiotic environmental stresses or senescence or by pathogen infection in the greenhouse because wild-type plants grown interspersed with sorbitol-producing plants never developed comparable symptoms.

Leaf necrosis and lesion formation in the absence of pathogens have also been observed as a result of disturbances in C allocation. Lesions formed on transgenic tobacco leaves expressing the cDNA for yeast invertase targeted to the plant cell wall (von Schaewen et al., 1990; Sonnewald et al., 1991). As a result of invertase activity in the cell wall, Glc and Fru accumulated, leading to a disturbance of source-to-sink relationships, which then inhibited photosynthesis depending on how much the capacity for Suc export was affected. The inhibition of photosynthesis in source leaves inhibited growth, and stunted leaves developed necrotic lesions. Herbers et al. (1996), reporting on the induction of systemic acquired resistance symptoms in tobacco with ectopic expression of differently targeted invertases, point to hexose sensing as a cause of lesion formation. The authors considered a threshold concentration of either Fru or Glc to be responsible for activation of genes of pathogen-related proteins in the absence of pathogens. Accumulating sugars could have directly repressed genes encoding photosynthetic functions and activated defense-related genes. Alternatively, altered levels of hexoses could have affected hexose kinases and acted as signals for Glc-mediated gene regulation (Jang and Sheen, 1994; Jang et al., 1997), but this seems unlikely because sorbitol (and other polyols) are not known to affect hexose kinase signaling.

We show that another perturbation in carbohydrate status, high sorbitol accumulation, leads to effects similar to those observed after ectopic invertase expression. The symptoms, including low photosynthesis activity, lesion formation, stunted growth, and altered ratios of Glc and Fru, however, developed earlier than in invertase-expressing tobacco. The correlation between lesions and growth and sorbitol amounts suggests that carbohydrate levels are at the basis of the phenotype, but perturbations of carbohydrates could affect several different pathways. First, sorbitol-6-phosphate dehydrogenase could affect the rate of Suc biosynthesis by competing for Glc-6-P needed for the activation of Suc-P synthase. Second, hexose phosphates will not only serve as substrates for Suc biosynthesis but will be directed toward glycolysis and respiration. Depletion of the hexose phosphate pool would affect both pathways and could alter sink development. In fact, the transformants in which we measured the highest sorbitol amounts were the most severely inhibited in the development of the root and shoot meristems. Third, UDP-Glc as well as myo-inositol are essential precursors for cell wall biosynthesis. Changes in metabolic balance through altered Glc-6-P levels would affect growing tissues. Also, lowering of the Glc-6-P pool might affect the synthesis of myo-inositol through L-myo-inositol-1-phosphate synthase. Finally, draining the Glc-6-P pool might alter the balance in signaling pathways involving Fru kinases and Glc kinases, or sorbitol-6-phosphate/sorbitol might interact with these hexose kinases. Alternatively, when the flux from Glc-6-P to myo-inositol is drastically altered, signaling pathways involving inositol phosphates could be affected. The data do not permit a distinction between the various alternatives, but these sorbitol-producing plants represent an experimental material with which to test these hypotheses. The amounts of Glc and Fru increased in the regions of the leaf that were beginning to show lesion development, likely affecting the expression of photosynthesis-related genes (and activation of pathogenesis-related proteins), but we consider this increase a late consequence of the primary damage.

To our knowledge, the inverse correlation between sorbitol amount and lesion formation versus the amount of myo-inositol has not been reported previously. In S5C and all other lines tested, high sorbitol was invariably correlated with low myo-inositol. Sorbitol-producing plants were free of lesions after the addition of myo-inositol (Fig. 4). A causal relationship is also suggested by the fact that under salt stress and during drought, myo-inositol increases in wild-type tobacco (Sheveleva et al., 1997). This is also observed in sorbitol-producing plants, which showed less lesion formation when stressed by the addition of 150 mM NaCl (data not shown).

In summary, by the introduction of a new enzyme generating a high flux of C into a new pathway originating from Glc-6-P we observed a disturbance in development. The reasons for the metabolic and developmental effects of high sorbitol production seem to be 2-fold: reduced C flux from Glc-6-P to myo-inositol and osmotic imbalance affecting C allocation or transport.

The results permit yet another conclusion, namely, that attempts for the metabolic engineering of osmolyte synthesis may reach an upper limit in the concentration of an accumulating metabolite that is tolerated. Considering that metabolite accumulation has been studied in terms of osmotic or ionic stress protection, we wished to explore how high accumulation affected plant metabolism. Low to moderate accumulation seems to have protective effects and, in addition, cellular location seems to be important (Tarczynski et al., 1993; Shen et al., 1997a, 1997b). Inducible, stress-dependent high accumulation of the methylated inositol, ononitol, did not generate comparable lesions (Sheveleva et al., 1997). High constitutive accumulation, achieved here with sorbitol, may be detrimental. Even in the case of moderate mannitol accumulation, it has been reported that the growth of the transgenic tobacco plants was slower than that of wild type (Karakas et al., 1997). High amounts of metabolites, at least in the case of sorbitol, could imbalance metabolism and development even under normal growth conditions. Flux through basic metabolic pathways may be affected by the high accumulation of products for which the transformed species has not been adapted. Although low constitutive accumulation may be tolerated, the best strategy seems to be inducible accumulation of osmolytes (Sheveleva et al., 1997).

	FOOTNOTES

   Received December 22, 1997; accepted April 15, 1998.

	ABBREVIATIONS

Abbreviation: CaMV, cauliflower mosaic virus.

	ACKNOWLEDGMENTS

We thank Dr. Y. Kanayama (Nagoya University, Japan) for the apple Stpd1 cDNA. We thank Dr. D. Inzé (University of Gent, Belgium) for a probe of tobacco superoxide dismutase, Christine Michalowski for assembling the gene construction, and Pat Adams and Jane Dugas Huff for dedicated assistance.

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