Journal of Experimental Botany, Vol. 51, No. 342, pp. 115-122,
January 2000
© 2000 Oxford University Press
Disturbance in the allocation of carbohydrates to regenerative organs in transgenic Nicotiana tabacum L.
1 Department of Biochemistry, The University of Arizona, Biosciences West, 1041 E. Lowell St, Tucson, Arizona, 85721-0088 USA
2 Department of Plant Sciences, The University of Arizona, Biosciences West, 1041 E. Lowell St, Tucson, Arizona, 85721-0088 USA
3 Department of Molecular and Cellular Biology, The University of Arizona, Biosciences West, 1041 E. Lowell St, Tucson, Arizona, 85721-0088 USA
Received 19 January 1999; Accepted 14 April 1999
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Abstract |
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Transgenic tobacco (Nicotiana tabacum L, cv. SR-1) expressing mannitol 1-phosphate dehydrogenase, MTLD, in chloroplasts and myo-inositol O-methyltransferase, IMT1, in the cytosol after crossing of lines which expressed these foreign genes separately has been analysed. Plants expressing both enzymes accumulated mannitol and
-ononitol in amounts comparable to those following single gene transfer and showed phenotypically normal growth during the vegetative stage. Induction of flowering for transgenovar and wild-type occurred at the same time, but during flowering the phenotype of the transformed plants changed. Compared to wild-type, transgenic plants were characterized by curled, smaller upper leaves and elongated stems during flowering; incomplete development of flower buds with shorter sepals and pedicels resulted in increased abortion. Flowers completing development were normal. The vegetative biomass of the transformed plants was slightly higher than that of wild-type. Concentrations of soluble sugars and potassium were lower than in wild-type only in the apical parts of the transgenic plants. Both enzymes, under control of the CaMV 35S promoter, promoted accumulation of mannitol and -ononitol in the youngest leaves close to the vegetative meristem and in flowers, suggesting that their presence could signal lower sink demand leading to a decrease in carbon import to flowers and developing seed capsules. The interpretation here is that increases of inert carbohydrates in developing sinks interfere with metabolism, such as respiration or glycolysis. This interference may be less significant in source tissues during vegetative growth than in sink tissues during seed development. Key words: transgenic tobacco, mannitol, D-ononitol, source–sink disturbance, seed production.
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Introduction |
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Metabolic effects in tobacco after single-gene transfers leading to either mannitol or D-ononitol production have previously been studied, with emphasis on possible protective effects under salinity stress conditions. The accumulation of mannitol and/or D-ononitol has been correlated with increased resistance to salinity stress and/or drought (Tarczynski et al., 1992
; Vernon et al., 1993; Shen et al., 1997a; Sheveleva et al., 1997). Transgenic tobacco plants containing mannitol in the cytosol or in chloroplasts (1–8 µmol g-1 fw) were phenotypically normal and exhibited rates of photosynthesis identical to those of wild-type SR1 (Tarczynski et al., 1992; Shen et al., 1997a, b). Under in vitro conditions mannitol protected membranes and phosphoribulokinase against oxygen radical damage (Smirnoff and Cumbes, 1989; Shen et al., 1997a, b). D-ononitol-producing plants (I5A) also displayed a normal phenotype (Vernon et al., 1993; Sheveleva et al., 1997) and the plants accumulated high amounts of D-ononitol, up to 35 µmol g-1 fw, only when they experienced stress conditions
(Sheveleva et al., 1997). This was enabled by an increase in substrate availability for the methyltransferase reaction leading to D-ononitol. The substrate, myo-inositol, for the methylation reaction increases in tobacco under stress. Preconditioning I5A plants in 50 mM NaCl increased D-ononitol amounts and resulted in increased protection of photosynthesis compared to the wild type, when the plants were stressed subsequently with 150 mM NaCl.
In previous experiments, transgenic tobacco was characterized which expressed an apple cDNA, encoding sorbitol 6-phosphate dehydrogenase, Stl-6PDH, under 35S promoter control resulting in the accumulation of sorbitol (Sheveleva et al., 1998). In several independently transformed lines low expression of the transcript and low amounts of sorbitol resulted in normal plants, comparable to those that contained mannitol. However, many other lines were recovered in which sorbitol amounts, for unknown reasons, were high or extremely high, up to 130 µmol g-1 fw, and these plants were either non-viable
(unable to produce roots) or severely growth-inhibited with necrotic lesions (Sheveleva et al., 1998). Based on the determination of carbohydrates in the necrotic plants, it is suggested that a disturbance of sink unloading was the major cause for this high-sorbitol phenotype. One conclusion from these experiments was that plant transformation resulting in stress-inducible solute accumulation provided better protection against drought and salt-stress conditions than strategies using osmotic adjustment by metabolites that are constitutively present.
The performance of a cross between two transgenic tobacco lines, which led to the expression of two genes, mannitol-1-phosphate dehydrogenase and myo-inositol O-methyltransferase (termed OB line) is reported here. The expression of both genes is controlled by the CaMV 35S promoter which leads to a gradient of expression throughout the plant with highest expression of the transgenes and highest accumulation of the products in meristems and young leaves. In the OB line subtle phenotypic effects of the overexpression of the two osmolytes were observed during flowering and seed production, but vegetative growth was normal or even slightly enhanced.
Introducing novel genes and altering the endogenous enzyme network can disturb metabolism in subtle ways that can be used to gain a better understanding of source–sink relationships. The results of the experiments suggest, most importantly, that strategies for constructing plants with altered metabolism need to pay closer attention to promoter strength and tissue-, cell- and developmental specificity of expression.
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Materials and methods |
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Plant transformation and growth
The transgenic tobacco line (termed OB) which was used for this investigation was obtained by cross-pollination of the lines I5A (D-ononitol in the cytosol; Sheveleva et al., 1997
) and B1-31 (mannitol in chloroplasts; Shen et al., 1997a), with I5A as the pollen donor. I5A had been obtained by transforming tobacco (Nicotiana tabacum L., cv. SR1) with the Imt1 cDNA from Mesembryanthemum crystallinum L. expressed under the control of a CaMV 35S promoter/enhancer element to produce the enzyme, D-myo-inositol methyltransferase (Vernon et al., 1993). B1-31 had been obtained by fusing the mannitol-1-phosphate dehydrogenase gene from Escherichia coli, mtlD (Novotny et al., 1984) with the transit peptide sequence of the pea RbcS3A cDNA (Shen et al., 1997a). The B1-31 line also expressed the foreign gene under the control of a CaMV 35S promoter. The line OB was used after three cycles of selfing and selection on kanamycin. Each plant was checked for the presence of both novel products, mannitol and 
ononitol. Seeds of the T4 generation of the line called I5A after repeated selfing were used for crossing. Plants of wild-type tobacco and the transformed lines, OB, I5A, and B1-31 were germinated and grown in vermiculite for approximately 3 weeks before they were transferred to hydroponic culture.
Two hydroponic systems, one with salt added and one without, were set up for plant cultivation as described (Sheveleva et al., 1997, 1998). The plants were irrigated in 0.25x Hoagland's solution in a greenhouse, with midday light intensity of 1 600 µmol quanta m-2 s-1, 60–80% humidity and 28±3 °C. Inorganic ion content of the hydroponic solutions was determined every second day by ion exchange chromatography and depleted compounds were added. Six-week-old plants were subjected to NaCl stress in nine tubs with a total volume, including the reservoir, of 640 l and ten plants per tub. The second system was stocked similarly, but without addition of NaCl. Mature leaves were considered leaves five and older, counted from the meristem, while immature leaves were leaves 1–3, counted from the meristem, which were less than 4–5 cm in length.
Analytical methods
Tissue was extracted in ethanol : chloroform : water
(12 : 5 : 3, by vol.) and analysed by HPLC separation with pulsed amperometric detection of carbohydrates (Adams et al., 1992). Cations were analysed similarly using a 50 µl injection loop. The cation profile was separated on an Alltech Universal Cation Column (Alltech Associates, Deerfield, MI) without guard column with 3 mM methane sulphonic acid at a flow rate of 1.0 ml min-1.
Xylem sap was extracted by using a pressure bomb. The sap was collected from the upper part of the stem, the top 10 cm below meristem or flowers. Phloem sap was obtained from stem segments of mature plants. Phloem sap was collected in 10 mM TRIS-Cl, pH 7.3, 10 mM EDTA. For 2 h after cutting, the solution was replaced several times and sap was collected for analysis for the next 1.5 h, dried, resuspended and analysed for the presence of carbohydrates and ions.
Gas-exchange measurements and 14CO2 labelling
Net CO2 assimilation rates in air were measured with attached leaves in the greenhouse under saturating light conditions using an infrared gas analyser (Li-6400; Li-Cor, Inc.). Leaf temperature was maintained at 28 °C with CO2 at 360 ppm. 14C labelling experiments were conducted with 5-week-old plants. Plants were placed in a sealed chamber and 14CO2 was released by the reaction of Ba14CO3 with lactic acid. Plants were labelled for 4 h. After the pulse the plants or individual leaves were frozen in liquid nitrogen, ground in an ethanol/chloroform/water mixture and aliquots were used for HPLC detection. Fractions containing sugars and sugar alcohols were collected and the specific radioactivity was determined (Beckman LS7000; Beckman Instruments Inc., Irvine, CA). Some plants were held in normal air for 24 h after labelling and processed as indicated.
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Results |
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Plant phenotype
Growth of the OB line appeared slower than wild-type during early development. However, after 11 weeks of growth, the biomass of the OB plants was slightly higher than in the SR1 line, both in unstressed plants and plants stressed with 150 mM NaCl for 4.5 weeks. The length of the stems was significantly different, by 16±5% (Table 1
). Photosynthetic rates of source leaves (data not shown) were similar for both variants, the increase of vegetative growth seemed to be due to the suppression of maturation of regenerative organs (Table 2). Differences which developed in older plants were not due to different environments, because OB and SR1 were grown side by side in the same hydroponic tanks, which were constantly monitored and the amount of nutrient (in 0.25x Hoagland's solution) was adjusted every second day. The concentration of potassium was maintained at 1 mM to reduce the amount of K+ which might be taken up to serve as an external osmoprotectant and then reduce the osmotic effect of polyols in the OB line (as observed when K+ was available at higher amounts; data not shown). No differences in root growth were observed between the two lines.
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After 11 weeks of growth increasingly more differences appeared. At the flowering stage, the top two or three leaves beneath the inflorescence of the OB plants were curled and had less area compared with wild-type leaves of identical developmental age (Fig. 1
). OB flower buds were characterized by short pedicels and sepals, but the number of open flowers was approximately equal in OB and SR1. Flower buds were produced at the same time in both lines and mature flowers developed indistinguishably in both, but most buds, flowers and seedpods were aborted in the OB lines (Fig. 2). By 11 weeks of age, the number of seedpods in OB plants was approximately one-quarter of those in SR1 plants, although new flower primordia constantly appeared in OB (Table 2). The shoot, especially in the upper part, was elongated in OB plants with longer internodes but the same leaf number. Except for these top leaves, which developed at the same time as the inflorescence, all other leaves were indistinguishable from leaves of SR1. After 4.5 weeks of growth in 150 mM NaCl OB plants also showed slight increases in growth over SR1 plants, with the same difference between the lines as under control conditions.
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Polyol concentrations
Abscission of flowers typically indicates disturbance of nutrient influx into the developing sinks of regenerative organs (Dybing et al., 1986; Daie, 1996). The distribution of carbohydrates and major cations in different organs was analysed to establish assimilate availability and to estimate nutrient flux in different organs. Figure 3 shows amounts of sugars and sugar alcohols for OB and SR1 plants. In both lines, different organs were characterized by approximately the same amount of sugars, but myo-inositol amounts were typically lower in the OB plants. It has been shown before that myo-inositol amounts declined significantly and correlated with the production of sorbitol for transgenic tobacco with engineered high sorbitol accumulation (Sheveleva et al., 1998). In the case of the OB plants, myo-inositol is the precursor for D-ononitol, thus the myo-inositol pool should be partly depleted as observed before (Sheveleva et al., 1997). Highest amounts of both polyols were observed in meristematic leaves. Mannitol concentrations in plants grown without salt stress were 5–10 times higher in meristems than in the older source leaves, and D-ononitol concentrations were approximately three times higher. Concentrations for both polyols in developing and mature flowers were higher than the amounts found in source leaves (Fig. 3). As expected, myo-inositol and D-ononitol amounts increased during stress in source leaves, similar to the increases reported before
(Sheveleva et al., 1997), but in meristems and leaves closest to the meristem D-ononitol concentrations did not increase as much as in source leaves under stress conditions.
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Ion relations
Figure 4 shows the distribution of cations for the SR1 and OB lines. No differences in K+ between the two lines were found statistically for all tissues. In the upper stem and the youngest leaves close to the meristem, however, slightly lower concentrations of K+ were measured in OB compared to SR1. With respect to Ca2+, no differences existed in source leaves, sink leaves and flower buds, but the upper stems of the OB line plants accumulated more Ca2+. Magnesium concentrations, shown only for meristematic leaves, were lower in the OB line than in wild-type leaves of comparable age. The most interesting difference between SR1 and OB was observed for the accumulation of Na+ under salt stress conditions. In all tissues analysed, the OB line accumulated significantly less Na+. All cations taken together, the meristems and flowers in OB plants had a significantly lower concentration of cations under normal and stress conditions compared to SR1.
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Xylem measurements
The concentrations of sugars, polyols and three cations were determined in xylem sap taken from the upper part of the stem where leaf curling was observed (Fig. 5). Only a trace of sucrose was detected. OB plants contained less glucose, fructose, myo-inositol, K+, and Ca2+ compared with SR1 plants. Glycerol was detected at low concentrations equally in both lines. Salt stress of the plants did not result in significant changes in these ions and metabolites. Low concentrations of sodium were found in stressed plants which were marginally higher in OB plants than in SR1. D-ononitol was present at low concentrations in the xylem, but mannitol was present in significant amount. In phloem sap collected into EDTA from plants maintained at 100 µmol quanta m-2 s-1 mannitol was up to 15 times more abundant compared to D-ononitol in all plants. Phloem measurements are not shown as these data were obtained by the EDTA-technique which may have distorted the amounts of transported carbohydrates (King and Zeevaart, 1974; Chino et al., 1991). However, the ratio of mannitol to D-ononitol increased steadily towards the plant apex which was interpreted as meaning that mannitol is more readily transported than D-ononitol and that it accumulates in the youngest tissues and in flowers.
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Discussion |
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Accumulation of novel products in the OB line generated a phenotype of normal vegetative growth followed by altered internode length, reduced area of young leaves, leaf curling, and dehiscence of flower buds. This phenotype is also correlated with the highest amount of mannitol and D-ononitol in the affected tissues of the plants and is probably due to the expression characteristics of the 35S promoter used in both gene constructs. Each polyol in the OB line accumulated to approximately the same amount as in single polyol-producing lines (Shen et al., 1997a
, b; Sheveleva et al., 1997). In leaves closest to the meristems and flowers, however, mannitol accumulated to the same amount observed in the line that produced only mannitol (Shen et al., 1997a, b), but in the OB line the combined increase of mannitol and D-ononitol produced the observed growth effects. The progenitor lines did not show this phenotype, but were indistinguishable from SR1. Developmental progression was not altered, because when both lines were grown in the same hydroponic tanks they entered flowering at the same time. Also, both lines were characterized by the same initial number of flower primordia, only a few of which developed into seedpods in all transgenic plant lines. Eventually, OB plants produced 1/4 as many seedpods as wild-type.
The disturbance of development in the upper leaves and regenerative organs of the transgenic plants seems to be related with the availability of nutrients to the upper part of the plants. It is suggested that there is a linkage between phenotype and disturbances in sink–source relationships because organs with weak initial sink capacity (flower buds) were most severely affected. Fertilized flowers and developing seeds became stronger sinks and the surviving seedpods resumed normal development, although mannitol and D-ononitol continued to be present in high amounts under these conditions. The increase in sink strength as seed organs matured has been reported (Ho et al, 1989). Sink strength as a measurable term has been controversial, because activity and size of sinks are dependent on photosynthesis, competition between sinks and the transport pathways (Geiger and Shieh, 1993; Stitt, 1993) and demand by the rest of the plant. It may be that these transgenic polyol-accumulating plants could be used to measure sink strength parameters. Note that a change in growth regulator levels could have generated the observed imbalances in carbohydrate transport, but these parameters were not measured.
The osmotic imbalance, most likely caused by polyols, could have affected mainly the transport relationships and not the development of organs. If lower sugar concentrations denote decreased nutrient flux into the developing seeds, it may be that sugar sensing and signalling per se might not be affected by the presence of the two polyols. It is suggested that the OB line suffered from a partial deficiency of carbohydrates as energy sources for metabolism during the early stages of flowering and seed filling. According to measurements of phloem sap in stems, mannitol constituted 10–15% of the flux of sucrose. While it cannot be certain that the phloem sap collected using the EDTA method
(King and Zeevaart, 1974) reported carbohydrate content correctly, mannitol in the phloem increased more than D-ononitol and sucrose in the upper part of the stem and in the flowers. The high amount of mannitol was not channelled into catabolism, although tobacco and celery contain genes which encode a catabolic mannitol dehydrogenase enzyme (Williamson et al. 1995; Everard et al., 1997). In celery the enzyme functions in reducing phosphorylated mannitol which is converted to fru 6-P to be channelled into the Krebs cycle (Everard et al., 1997). Mannitol is a translocated polyol in celery (Keller, 1991). This enzyme seems absent in floral tissues, or only expressed in very low abundance. A connection between the expression of this gene and pathogen attack has been reported (Williamson
et al., 1995).
Leaves close to the meristem, the flowers and xylem sap from the upper part of the stem were characterized by lower amounts of reducing sugars, sucrose and ions. This result strongly supports the author's interpretation about disturbances in sink–source relationships responsible for the phenotype. This behaviour was not observed with plants expressing only myo-inositol O-methyltransferase leading to D-ononitol production (Sheveleva et al., 1997), although the amount of D-ononitol in these plants was higher than in plants of the OB line.
The high concentrations of mannitol and D-ononitol could have caused disturbance in the stem and flowers of OB plants, indicating that there may be an upper limit to the concentration of additional osmotically active metabolites that can be achieved in transgenic plants. What this upper limit might be is unclear, but a few data points are available. In mannitol-producing plants (at approximately 4 µmol g-1 fw; Tarczynski et al., 1992) the carbon diverted to mannitol was less than 1% of total carbon fixed. In D-ononitol-producers, line I5A (Sheveleva et al., 1997), after salt stress, approximately 2% of all carbon was found in D-ononitol. Short-time 14C labelling experiments of OB plants (data not included) let us estimate that less than 2–3% of the 14C-carbon was deposited to polyols in source leaves of OB plants. It is expected that this percentage will be higher in meristematic leaves due to the characteristics of the CaMV35S promoter. After pulse labelling of whole plants for 4 h with 14CO2, the specific radioactivity of the reducing sugars and sucrose was approximately 15 times higher than that of mannitol and D-ononitol. After a 24 h chase, however, the expected decline in specific activity of sucrose, glucose and fructose (by 15–20 times) was contrasted by a marginal decline of the specific activities in D-ononitol
(20%) and mannitol (25%), highlighting the slow turnover or gradual conversion of reducing sugars and sucrose into polyols.
The concentration of mannitol and D-ononitol can be compared with sorbitol accumulation in another line expressing apple sorbitol 6-phosphate dehydrogenase (Sheveleva et al., 1998). In more than 100 independently generated lines, sorbitol accumulated to high amounts, up to 130 µmol g-1 fw. At a sorbitol concentration exceeding approximately 15 µmol g-1 fw, these plants developed necrotic lesions and showed stunted growth. The amount of carbon that was laid down in sorbitol in these plants could exceed 20% of the carbon fixed, but even at lower concentrations growth was severely affected. In all probability, the high amount of a foreign polyol affected sugar sensing in this case (Sheveleva et al., 1998). Additional support comes from another experiment (data not shown). When OB plants were grown with full-strength Hoagland's solution instead of 0.25 the concentration of nutrients, the aberrant development of regenerative organs and the upper leaves was largely eliminated. This result again strongly points towards a deficiency of nutrient influx as the main reason for the observed phenotype.
Genetic manipulation of source–sink interactions has been studied in a number of transgenic lines. Disturbance of either symplastic (disturbance of plasmodesmata) or apoplastic (invertase over-expression) loading steps has been achieved (Lucas and Madore, 1988; von Schaewen et al., 1990; Dickinson et al., 1991; Heineke et al., 1992; Bush, 1993; van Bel et al., 1994; Sonnewald et al., 1994). In these experiments, plants typically showed stunted growth, accumulation of reducing sugars, sucrose, and starch in source leaves, and the development of necroses. The phenotype of the tobacco OB line described here is different. An effect of the accumulating polyols is only observed when many weak sinks (e.g. flower buds) develop, while the vegetative growth of these plants was slightly enhanced. The phenotype may indicate the limits of a stress-protecting polyol over-expression strategy in which the authors are interested. As stated before (Sheveleva et al., 1997), it seems that a stress-inducible accumulation strategy is preferable and that organ-specificity of accumulation must equally be considered. From the present study and from earlier work with transgenic tobacco accumulating high sorbitol (Sheveleva et al., 1998), it may be concluded that a limit exists for the amount of polyols that can be diverted into metabolically inert, yet osmotically active, substances. There exist, however, alternative possibilities by which protective polyols, which are synthesized in source tissues and transported in the phloem, could be mobilized again in sink tissues.
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Acknowledgments |
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We wish to thank Mrs Wendy Chmara for help with the HPLC analyses, Abreeza Zegeer for help with greenhouse experiments, and Pat Adams for comments on the manuscript. The work has been supported by a grant from the Department of Energy, Division of Energy Biosciences (DE-FG03–98ER20179.001) and, in part, the Arizona Agricultural Experiment Station and by NEDO, Japan.
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Notes |
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4 To whom correspondence should be addressed. Fax: +1 520 621 1697. E-mail: bohnerth@biosci.arizona.edu
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Abbreviations |
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g fw, gram fresh weight; IMT1, myo-inositol O-methyltransferase..
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