- Email: [email protected]
Enhanced drought resistance in fructan-producing sugar beet
Plant Physiol. Biochem., 1999, 37 (4), 313−317
Enhanced drought resistance in fructan-producing sugar beet Elizabeth A. H. Pilon-Smitsa*§, Norman Terrya, Tobin Searsa, Kees van Dunb a
Department of Plant and Microbial Biology, University of California, 111 Koshland Hall, Berkeley CA 94720, USA
b
D.J. Van der Have b.v., Van der Haveweg 2, 4410 AA Rilland, the Netherlands
* Author to whom correspondence should be addressed (fax +1 970 491 0649; e-mail [email protected])
(Received June 15, 1998; accepted February 22, 1999) Abstract — Fructans are soluble polymers of fructose that are produced by approximately 15 % of the flowering plant species. Production of bacterial fructans in tobacco has been shown previously to lead to improved biomass production under polyethylene glycol-mediated drought stress. Here, we used the same SacB gene from Bacillus subtilis to produce bacterial fructans in sugar beet (Beta vulgaris L.). The transgenic sugar beets accumulated fructans to low levels (max. 0.5 % of dry weight) in both roots and shoots. Two independent transgenic lines of fructan-producing sugar beets showed significantly better growth under drought stress than untransformed beets. Drought stressed fructan-producing plants attained higher total dry weights (+25–35 %) than wildtype sugar beet, due to higher biomass production of leaves (+30–33 %), storage roots (+16–33 %) and fibrous roots (+37–60 %). Under well-watered conditions, no significant differences were observed between the transgenic and wildtype beets. In conclusion, the introduction of fructan biosynthesis in transgenic plants is a promising approach to improve crop productivity under drought stress. © Elsevier, Paris Beta vulgaris / drought stress / fructan / sugar beet
1. INTRODUCTION Drought is the single most limiting factor for agriculture in general [1], including sugar beet production [2]. One approach to improve drought stress resistance has been the introduction of fructan biosynthesis in transgenic plants [11]. Fructans are polymers of fructose, and function as the main storage carbohydrate in approximately 15 % of the flowering plant species [5, 6, 10]. Besides being a storage carbohydrate, fructans have had attributed to them an additional role as a defense mechanism against drought stress. In addition to the solubility of fructan, this idea was based on the geographical distribution of fructan flora in drier areas, as well as the historical rise of prominent fructan-producing taxa concomitant with a climatological shift towards seasonal drought [5, 6]. In support of a possible role of fructans in drought resistance was the observation that the production of bacterial fructans in transgenic tobacco plants increased plant biomass production under polyethylene glycol-mediated drought stress [11]. The gene construct used for these studies consisted of the Bacillus §
subtilis SacB gene, with the CPY yeast vacuolar targeting sequence under the control of the constitutive 35S CaMV promoter [3]. Here, the same gene construct was used to introduce fructan biosynthesis in sugar beets. The resulting transgenic sugar beet plants were compared with their wildtype relatives with respect to growth performance under drought stress.
2. RESULTS 2.1. Production of transgenic fructan-accumulating sugar beet plants Fourteen independent transgenic lines were obtained when sugar beet cotelydons were transformed with the SacB gene from Bacillus subtilis. The transgenic sugar beet plants showed fructan accumulation in both storage roots and in older leaves. Fructan accumulated with time in leaves of the transgenic plants: the oldest leaves contained fructan levels comparable to those in the storage roots, while there was no detectable fructan in young leaves (figure 1). Simi-
Present address : Biology Department, Anatomy/Zoology Building, Colorado State University, Fort Collins, CO 80523, USA.
Plant Physiol. Biochem., 0981-9428/99/4/© Elsevier, Paris
314
E.A.H. Pilon-Smits et al.
Figure 1. Fructan levels in different parts of sugar beet plants (line A30-1), and in untransformed control plants. For each sample, 1 µL undiluted cell sap was applied onto the TLC plate, separated and stained for fructose residues (scale 1:1). Fru, Fructose; Suc, sucrose; Fructan, fructan. 1, Untransformed plant, oldest leaf; 2–7, A30-1 leaves of increasing age; 8, A30-1 storage root.
larly in untransformed sugar beet plants, no fructan could be detected. The accumulated fructan levels varied about 20-fold among the transgenic lines (figure 2); the maximum foliar fructan levels obtained were ∼5 mg⋅g–1 DW in line A30-1. Unfortunately, not enough seeds could be obtained from this line. Instead, lines A30-3 and T72-1 were selected for further analysis. Roots from A30-3 plants contained ∼0.9 mg fructan⋅g–1 DW, and roots of T72-1 plants ∼0.3 mg fructan⋅g–1 DW. These transgenic lines were shown by Southern blotting to contain single T-DNA inserts and to be independent transgenics (figure 3).
Figure 2. Fructan levels in undiluted leaf extracts from different transgenic sugar beet lines, assayed as in figure 1 (scale 1:1). 1, A28-1; 2, A28-3; 3, A29-1; 4, A29-3; 5, A30-1; 6, A30-2; 7, A30-3; 8, A31-1; 9, A31-2; 10, A38-1; 11, A38-2; 12, T72-1; 13, T81-1.
Plant Physiol. Biochem.
Figure 3. Southern blot of different transgenic fructan-producing sugar beet lines (scale 1:1). Total leaf tissue DNA was digested with EcoRI and hybridized with a GUS probe, resulting in one border fragment per inserted T-DNA. 1, T81-1; 2, T72-1; 3, A41-1; 4, A38-2; 5, A38-1; 6, A31-2; 7, A31-1; 8, A30-3; 9, A30-2; 10, A30-1; 11, A29-3; 12, A29-1; 13, A28-3; 14, A28-1.
2.2. Drought stress experiments The first drought stress experiment was performed with line A30-3. After having been grown under drought stress for a month, the fructan-producing sugar beets had reached a 35 % higher total dry weight than their wildtype relatives (figure 4 A, P < 0.05). Under well-watered conditions no significant differences were observed between transgenic and wildtype plants (results not shown). The difference in total biomass production under drought stress was mainly due to increased production of leaves (+30 %) and fibrous roots (+60 %, figure 4 C, F). The fibrous root biomass of the transgenic plants under drought stress was more than twice that of unstressed transgenics (P < 0.05), whereas wildtype sugar beets produced similar size roots under both conditions. The dry weight attained by the transgenic plants grown under drought stress was 80 % of the dry weight produced by these plants under well-watered conditions, and not significantly different. In contrast, the yield of drought-stressed wildtype sugar beets was only 49 % of the yield of unstressed wildtype plants (P < 0.0001, figure 4 B). The 27 % higher leaf dry weight of the fructan-producing plants under drought stress (P < 0.01) corresponded with a 24 % increase in leaf area (P < 0.001, figure 4 D). A second drought stress experiment was performed using line A72-1, which was shown previously to contain 3-fold lower fructan levels than line A30-3. This time the sugar beet plants were grown for a longer time under drought stress, to be able to better study the effect of fructans on storage root growth under drought stress. The longer growth period resulted in a higher biomass at harvest, especially with respect to the storage roots. Again, the fructan-
Enhanced drought resistance in fructan-producing sugar beet
315
Figure 4. Biomass production and leaf area of wildtype (WT) and fructan-producing (A30-3) sugar beets after 1 month of drought treatment. A, Total DW; B, DW of drought-stressed plants relative to unstressed plants; C, leaf DW; D, leaf area; E, storage root DW; F, fibrous root DW. The values shown are the averages of ten plants and their standard errors (in C and E, the SE is too small to be visible).
Figure 5. Biomass production and leaf area of wildtype (WT) and fructan-producing (A72-1) sugar beet plants after 6 weeks of drought treatment. A, Total DW; B, DW of drought-stressed plants relative to unstressed plants; C, leaf DW; D, leaf area; E, storage root DW; F, fibrous root DW. The values shown are the averages of ten plants and their standard errors (in C and E, the SE is too small to be visible).
producing plants showed better growth under drought stress than wildtype plants, although the effect was less pronounced for the T72-1 plants than for A30-3 plants. After 6 weeks of drought stress, the T72-1 plants had attained 25 % more total dry weight than their wildtype relatives (P < 0.05, figure 5 A). No significant differences in growth were observed under wellwatered conditions (results not shown). The T72-1 plants produced 33 % more leaf dry weight (P < 0.01) and 31 % more storage root biomass (P < 0.05, figure 5 C, E) than their wild type; the fibrous roots were 37 % heavier (NS, figure 5 F).
fructan biosynthesis to improve plant productivity under drought stress has now been shown to be successful for more than one plant species; and (b) the enhanced growth of fructan-producing plants has now been shown to also occur under more realistic drought stress conditions, compared to polyethylene glycolinduced drought stress. Moreover, the effect of fructans on drought resistance was shown to be correlated with the fructan level: of the two transgenic lines tested, A30-3, which had the highest fructan levels, also showed the largest increase in productivity under drought stress (+35 % for A30-3, relative to +25 % for T72-1). The amount of fructan produced by these transgenic sugar beet plants was low compared to the fructan levels obtained in tobacco and, especially, potato transformed with the same construct [3, 15], but comparable to the fructan levels obtained in transgenic Arabidopsis thaliana (unpubl. results). We do not know what causes these different fructan levels in different species. The foreign levansucrase protein may be degraded to different extents in these species. Alternatively, the levels of its substrate, sucrose, may differ among these species at the intracellular location
3. DISCUSSION The main finding of this study is that the introduction of fructan biosynthesis in sugar beet plants resulted in enhanced biomass production under drought stress. The results presented here are in agreement with the earlier report that fructan accumulation in tobacco leads to improved growth under polyethylene glycol-induced drought stress [11]. The new information presented here is: (a) the approach of introducing
vol. 37 (4) 1999
316
E.A.H. Pilon-Smits et al.
of the enzyme. The location of the enzyme has up until now not been determined with certainty. The fructan levels in the transgenic sugar beet plants increased with leaf age, from below the detection limit in young leaves to ∼5 mg⋅g–1 DW in the oldest leaves. A similar increase of fructan level with leaf age was reported for fructan-producing tobacco and potato plants [3, 15]. Apparently, these plants lack specific fructan hydrolases to degrade these fructans, nor can they transport them. Thus, the level of accumulation is determined mainly by the age of the organ, and by the level of sucrose, as sucrose is the substrate for fructan synthesis. The fructan levels in cell sap were relatively high in the storage roots of the beet plants, compared to other plant parts. This may have been caused by the higher sucrose levels in storage roots, and perhaps by the lower water content in root tissues. The A30-3 and T72-1 sugar beets had 60 and 37 % larger fibrous roots, respectively, than their wild types. A similar increase in root production was found for fructan-producing tobacco plants [11]. Perhaps fructans stimulate root growth, thereby promoting water uptake under drought stress. How fructans would promote root production will need more investigation. The amount of fructan present in these plants was probably not enough to have any significant effect on the osmotic potential. Improved growth performance under osmotic stress was also observed when other foreign sugars were introduced in plants, and accumulated to levels that were too low to be osmotically significant [8, 9, 12, 14]. Perhaps foreign sugars can function as regulators or signal molecules and thus affect plant metabolism, or maybe they can function as free radical scavengers, as was reported for mannitol [13]. The introduction of fructan biosynthesis in crop plants appears to be a promising approach to improve crop productivity under drought stress. This is of significance because water is the most limiting factor for sugar beet production, and for agriculture in general. Under our growth conditions, the beet storage roots were 15 % heavier in A30-3 plants and 31 % heavier in T72-1 plants than in their wildtype controls. The T72-1 plants were harvested at a later developmental stage, indicating that the positive effect of fructans on storage root biomass may be even more pronounced when the storage root reaches its maximum size. The fructans are not expected to influence the sucrose concentrations in the sugar beet: in other transgenic fructan-accumulating species, the levels of soluble sugars (including sucrose) were the same or somewhat higher than in wildtype plants [3, 11, 15]. Plant Physiol. Biochem.
4. METHODS 4.1. Gene construct The binary vector pMOG23 (BIN19 derivative), containing the nptII gene driven by the nos promoter as well as a multiple cloning site, was kindly provided by MOGEN International N.V. (Leiden, the Netherlands). A chimeric gene encoding for β-glucuronidase containing an intron (35S-GUSint) was inserted into the HindIII site of pMOG23, resulting in pVDH65. The chimeric SacB gene [3] was inserted in pVDH65 after digestion with EcoRI and XhoI. The resulting binary transformation vector is called pVDH124. pVDH124 was transferred to Agrobacterium tumefaciens LBA4404 by triparental mating, and the functionality of the construct was tested by transformation to tobacco (Nicotiana tabacum). The transgenic tobacco was shown to accumulate high molecular weight fructans, identical to the results described by Ebskamp et al. [3].
4.2. Plant transformation Cotelydons of Beta vulgaris L. (diploid O-type population) were transformed according to Fry et al. [4]. Plants from the first generation of transgenics were crossed back with untransformed plants of the same variety. The seeds resulting from two of these crossings were used for these physiological studies: these lines are referred to as A30-3 and T72-1.
4.3. Fructan analysis Samples for fructan analysis were homogenized in a 1.5-mL microcentrifuge tube using a micropestle, and 1 µL undiluted cell sap was applied onto a silica thin layer chromotography plate (Schleicher and Schuell). The thin layer chromatography plates were run twice in 90 % acetone and stained specifically for fructose residues using the method of Wise et al. [16]. The fructan spot at the application site was quantified using a standard of commercially available levan from Erwinia herbicola (Sigma).
4.4. Drought treatment Two drought stress experiments were done, the first one with transgenic line A30-3 and the second with line T72-1. Each experiment was done with ten replicates per treatment. For each experiment, one hundred seeds obtained from a crossing between a transgenic primary transformant and an untransformed sugar beet plant were sown in soil (Davis mix), and watered daily
Enhanced drought resistance in fructan-producing sugar beet
with half-strength Hoagland’s solution [7]. After 3 weeks, twenty-five GUS (β-glucuronidase) positive seedlings and twenty-five negative seedlings (‘wildtype’ controls) were selected. These plants were transplanted to equal amounts of soil (Davis mix) in 15-cm diameter pots, and grown in a growth chamber at 20 °C, at a photoperiod of 14 h and a light intensity of ∼100 µE⋅m–2. When the plants were 7-week-old, the drought treatment was started with ten plants from each line while the same number of plants from each line were kept well-watered as controls. Drought stress was imposed by reducing the supply of nutrient solution. During drought treatment, the wildtype and transgenic plants received the same amount of water, which corresponded with 10–20 % of the field capacity of the soil. This resulted in reversible wilting. After 4 1/2 weeks of drought stress, the A30-3 plants and their controls were harvested; the T72-1 plants and their controls were harvested after 6 weeks of drought treatment. At harvest, the total leaf area was determined using a Delta-T Devices leaf area meter (Santa Clara, CA, USA). In addition, the fresh weight and dry weight of leaf blades, stem and petioles, storage root, and fibrous roots were measured.
Acknowledgments E.A.H. Pilon-Smits was supported by a TALENT stipend from the Dutch Organization for Scientific Research (N.W.O.).
REFERENCES [1] Boyer J.S., Plant productivity and environment, Science 218 (1982) 443–448. [2] Clarke N., Hetschkun H., Jones C., Boswell E., Marfaing H., Identification of stress tolerance traits in sugar beet, in: Jackson M.B., Black C.R. (Eds.), Interacting Stresses on Plants in a Changing Climate, vol I16, NATO ASI Series, Springer Verlag, Berlin Heidelberg, 1993, pp. 511–524. [3] Ebskamp M.J.M., Van der Meer I.M., Spronk B.A., Weisbeek P.J., Smeekens S.J.M., Accumulation of fructose polymers in transgenic tobacco, Biotechnology 12 (1994) 272–275.
317
[4] Fry J.E., Barnason A.R., Hinchee M., Genotypeindependent transformation of sugar beet using Agrobacterium tumefaciens, Poster Abstract of the 3rd Congress of ISPMB, Tucson, AZ, 1991, p. 384. [5] Hendry G.A.F., The ecological significance of fructan in a contemporary flora, New Phytol. 106 (1987) 201–216. [6] Hendry G.A.F., Evolutionary origins and natural functions of fructans – a climatological, biogeographic and mechanistic appraisal, New Phytol. 123 (1993) 3–14. [7] Hoagland D., Arnon D.I., The water culture method for growing plants without soil, Calif. Agric. Exp. Stn. Circ. 347 (1938) 1–39. [8] Holmstrom K.O., Mantyla E., Welin B., Mandal A., Tapio Palva E., Tunnela O.E., Drought tolerance in tobacco, Nature 379 (1996) 683–684. [9] Kishor P.B.K., Hong Z., Miao G., Hu C.A., Verma P.S., Overexpression of pyrroline-5-carboxylate synthetase increases proline production and confers osmotolerance in transgenic plants, Plant Physiol. 108 (1995) 1387–1394. [10] Nelson C.J., Smith D., Fructans: their nature and occurrence, Curr. Top. Plant Biochem. Physiol. 5 (1986) 1–16. [11] Pilon-Smits E.A.H., Ebskamp M.J.M., Paul M.J., Jeuken M.J.W., Weisbeek P.J., Smeekens S.C.M., Improved performance of transgenic fructanaccumulating tobacco under drought stress, Plant Physiol. 107 (1995) 125–130. [12] Pilon-Smits E.A.H., Terry N., Sears T., Kim H., Zayed A.M., Hwang S.B., van Dun K., Voogd E., Verwoerd T.C., Krutwagen W.H.H., Goddijn O.J.M., Trehaloseproducing transgenic tobacco plants show improved growth performance under drought stress, J. Plant Physiol. 152 (1998) 525–532. [13] Shen B., Jensen R.G., Bohnert H.J., Increased resistance to oxidative stress in transgenic plants by targeting mannitol biosynthesis to chloroplasts, Plant Physiol. 113 (1997) 1177–1183. [14] Tarczynski M.C., Jensen R.G., Bohnert H.J., Stress protection of transgenic tobacco by production of the osmolyte mannitol, Science 259 (1993) 508–510. [15] Van der Meer I.M., Ebskamp M.J.M., Visser R.G.F., Weisbeek P.J., Smeekens S.C.M., Fructan as a new carbohydrate sink in transgenic potato plants, Plant Cell 6 (1994) 561–570. [16] Wise C.S., Dimler R.J., Davis H.A., Rist C.E., Determination of easily hydrolyzable fructose units in dextran preparations, Anal. Biochem. 27 (1955) 33–36.
vol. 37 (4) 1999
Enhanced drought resistance in fructan-producing sugar beet Elizabeth A. H. Pilon-Smitsa*§, Norman Terrya, Tobin Searsa, Kees van Dunb a
Department of Plant and Microbial Biology, University of California, 111 Koshland Hall, Berkeley CA 94720, USA
b
D.J. Van der Have b.v., Van der Haveweg 2, 4410 AA Rilland, the Netherlands
* Author to whom correspondence should be addressed (fax +1 970 491 0649; e-mail [email protected])
(Received June 15, 1998; accepted February 22, 1999) Abstract — Fructans are soluble polymers of fructose that are produced by approximately 15 % of the flowering plant species. Production of bacterial fructans in tobacco has been shown previously to lead to improved biomass production under polyethylene glycol-mediated drought stress. Here, we used the same SacB gene from Bacillus subtilis to produce bacterial fructans in sugar beet (Beta vulgaris L.). The transgenic sugar beets accumulated fructans to low levels (max. 0.5 % of dry weight) in both roots and shoots. Two independent transgenic lines of fructan-producing sugar beets showed significantly better growth under drought stress than untransformed beets. Drought stressed fructan-producing plants attained higher total dry weights (+25–35 %) than wildtype sugar beet, due to higher biomass production of leaves (+30–33 %), storage roots (+16–33 %) and fibrous roots (+37–60 %). Under well-watered conditions, no significant differences were observed between the transgenic and wildtype beets. In conclusion, the introduction of fructan biosynthesis in transgenic plants is a promising approach to improve crop productivity under drought stress. © Elsevier, Paris Beta vulgaris / drought stress / fructan / sugar beet
1. INTRODUCTION Drought is the single most limiting factor for agriculture in general [1], including sugar beet production [2]. One approach to improve drought stress resistance has been the introduction of fructan biosynthesis in transgenic plants [11]. Fructans are polymers of fructose, and function as the main storage carbohydrate in approximately 15 % of the flowering plant species [5, 6, 10]. Besides being a storage carbohydrate, fructans have had attributed to them an additional role as a defense mechanism against drought stress. In addition to the solubility of fructan, this idea was based on the geographical distribution of fructan flora in drier areas, as well as the historical rise of prominent fructan-producing taxa concomitant with a climatological shift towards seasonal drought [5, 6]. In support of a possible role of fructans in drought resistance was the observation that the production of bacterial fructans in transgenic tobacco plants increased plant biomass production under polyethylene glycol-mediated drought stress [11]. The gene construct used for these studies consisted of the Bacillus §
subtilis SacB gene, with the CPY yeast vacuolar targeting sequence under the control of the constitutive 35S CaMV promoter [3]. Here, the same gene construct was used to introduce fructan biosynthesis in sugar beets. The resulting transgenic sugar beet plants were compared with their wildtype relatives with respect to growth performance under drought stress.
2. RESULTS 2.1. Production of transgenic fructan-accumulating sugar beet plants Fourteen independent transgenic lines were obtained when sugar beet cotelydons were transformed with the SacB gene from Bacillus subtilis. The transgenic sugar beet plants showed fructan accumulation in both storage roots and in older leaves. Fructan accumulated with time in leaves of the transgenic plants: the oldest leaves contained fructan levels comparable to those in the storage roots, while there was no detectable fructan in young leaves (figure 1). Simi-
Present address : Biology Department, Anatomy/Zoology Building, Colorado State University, Fort Collins, CO 80523, USA.
Plant Physiol. Biochem., 0981-9428/99/4/© Elsevier, Paris
314
E.A.H. Pilon-Smits et al.
Figure 1. Fructan levels in different parts of sugar beet plants (line A30-1), and in untransformed control plants. For each sample, 1 µL undiluted cell sap was applied onto the TLC plate, separated and stained for fructose residues (scale 1:1). Fru, Fructose; Suc, sucrose; Fructan, fructan. 1, Untransformed plant, oldest leaf; 2–7, A30-1 leaves of increasing age; 8, A30-1 storage root.
larly in untransformed sugar beet plants, no fructan could be detected. The accumulated fructan levels varied about 20-fold among the transgenic lines (figure 2); the maximum foliar fructan levels obtained were ∼5 mg⋅g–1 DW in line A30-1. Unfortunately, not enough seeds could be obtained from this line. Instead, lines A30-3 and T72-1 were selected for further analysis. Roots from A30-3 plants contained ∼0.9 mg fructan⋅g–1 DW, and roots of T72-1 plants ∼0.3 mg fructan⋅g–1 DW. These transgenic lines were shown by Southern blotting to contain single T-DNA inserts and to be independent transgenics (figure 3).
Figure 2. Fructan levels in undiluted leaf extracts from different transgenic sugar beet lines, assayed as in figure 1 (scale 1:1). 1, A28-1; 2, A28-3; 3, A29-1; 4, A29-3; 5, A30-1; 6, A30-2; 7, A30-3; 8, A31-1; 9, A31-2; 10, A38-1; 11, A38-2; 12, T72-1; 13, T81-1.
Plant Physiol. Biochem.
Figure 3. Southern blot of different transgenic fructan-producing sugar beet lines (scale 1:1). Total leaf tissue DNA was digested with EcoRI and hybridized with a GUS probe, resulting in one border fragment per inserted T-DNA. 1, T81-1; 2, T72-1; 3, A41-1; 4, A38-2; 5, A38-1; 6, A31-2; 7, A31-1; 8, A30-3; 9, A30-2; 10, A30-1; 11, A29-3; 12, A29-1; 13, A28-3; 14, A28-1.
2.2. Drought stress experiments The first drought stress experiment was performed with line A30-3. After having been grown under drought stress for a month, the fructan-producing sugar beets had reached a 35 % higher total dry weight than their wildtype relatives (figure 4 A, P < 0.05). Under well-watered conditions no significant differences were observed between transgenic and wildtype plants (results not shown). The difference in total biomass production under drought stress was mainly due to increased production of leaves (+30 %) and fibrous roots (+60 %, figure 4 C, F). The fibrous root biomass of the transgenic plants under drought stress was more than twice that of unstressed transgenics (P < 0.05), whereas wildtype sugar beets produced similar size roots under both conditions. The dry weight attained by the transgenic plants grown under drought stress was 80 % of the dry weight produced by these plants under well-watered conditions, and not significantly different. In contrast, the yield of drought-stressed wildtype sugar beets was only 49 % of the yield of unstressed wildtype plants (P < 0.0001, figure 4 B). The 27 % higher leaf dry weight of the fructan-producing plants under drought stress (P < 0.01) corresponded with a 24 % increase in leaf area (P < 0.001, figure 4 D). A second drought stress experiment was performed using line A72-1, which was shown previously to contain 3-fold lower fructan levels than line A30-3. This time the sugar beet plants were grown for a longer time under drought stress, to be able to better study the effect of fructans on storage root growth under drought stress. The longer growth period resulted in a higher biomass at harvest, especially with respect to the storage roots. Again, the fructan-
Enhanced drought resistance in fructan-producing sugar beet
315
Figure 4. Biomass production and leaf area of wildtype (WT) and fructan-producing (A30-3) sugar beets after 1 month of drought treatment. A, Total DW; B, DW of drought-stressed plants relative to unstressed plants; C, leaf DW; D, leaf area; E, storage root DW; F, fibrous root DW. The values shown are the averages of ten plants and their standard errors (in C and E, the SE is too small to be visible).
Figure 5. Biomass production and leaf area of wildtype (WT) and fructan-producing (A72-1) sugar beet plants after 6 weeks of drought treatment. A, Total DW; B, DW of drought-stressed plants relative to unstressed plants; C, leaf DW; D, leaf area; E, storage root DW; F, fibrous root DW. The values shown are the averages of ten plants and their standard errors (in C and E, the SE is too small to be visible).
producing plants showed better growth under drought stress than wildtype plants, although the effect was less pronounced for the T72-1 plants than for A30-3 plants. After 6 weeks of drought stress, the T72-1 plants had attained 25 % more total dry weight than their wildtype relatives (P < 0.05, figure 5 A). No significant differences in growth were observed under wellwatered conditions (results not shown). The T72-1 plants produced 33 % more leaf dry weight (P < 0.01) and 31 % more storage root biomass (P < 0.05, figure 5 C, E) than their wild type; the fibrous roots were 37 % heavier (NS, figure 5 F).
fructan biosynthesis to improve plant productivity under drought stress has now been shown to be successful for more than one plant species; and (b) the enhanced growth of fructan-producing plants has now been shown to also occur under more realistic drought stress conditions, compared to polyethylene glycolinduced drought stress. Moreover, the effect of fructans on drought resistance was shown to be correlated with the fructan level: of the two transgenic lines tested, A30-3, which had the highest fructan levels, also showed the largest increase in productivity under drought stress (+35 % for A30-3, relative to +25 % for T72-1). The amount of fructan produced by these transgenic sugar beet plants was low compared to the fructan levels obtained in tobacco and, especially, potato transformed with the same construct [3, 15], but comparable to the fructan levels obtained in transgenic Arabidopsis thaliana (unpubl. results). We do not know what causes these different fructan levels in different species. The foreign levansucrase protein may be degraded to different extents in these species. Alternatively, the levels of its substrate, sucrose, may differ among these species at the intracellular location
3. DISCUSSION The main finding of this study is that the introduction of fructan biosynthesis in sugar beet plants resulted in enhanced biomass production under drought stress. The results presented here are in agreement with the earlier report that fructan accumulation in tobacco leads to improved growth under polyethylene glycol-induced drought stress [11]. The new information presented here is: (a) the approach of introducing
vol. 37 (4) 1999
316
E.A.H. Pilon-Smits et al.
of the enzyme. The location of the enzyme has up until now not been determined with certainty. The fructan levels in the transgenic sugar beet plants increased with leaf age, from below the detection limit in young leaves to ∼5 mg⋅g–1 DW in the oldest leaves. A similar increase of fructan level with leaf age was reported for fructan-producing tobacco and potato plants [3, 15]. Apparently, these plants lack specific fructan hydrolases to degrade these fructans, nor can they transport them. Thus, the level of accumulation is determined mainly by the age of the organ, and by the level of sucrose, as sucrose is the substrate for fructan synthesis. The fructan levels in cell sap were relatively high in the storage roots of the beet plants, compared to other plant parts. This may have been caused by the higher sucrose levels in storage roots, and perhaps by the lower water content in root tissues. The A30-3 and T72-1 sugar beets had 60 and 37 % larger fibrous roots, respectively, than their wild types. A similar increase in root production was found for fructan-producing tobacco plants [11]. Perhaps fructans stimulate root growth, thereby promoting water uptake under drought stress. How fructans would promote root production will need more investigation. The amount of fructan present in these plants was probably not enough to have any significant effect on the osmotic potential. Improved growth performance under osmotic stress was also observed when other foreign sugars were introduced in plants, and accumulated to levels that were too low to be osmotically significant [8, 9, 12, 14]. Perhaps foreign sugars can function as regulators or signal molecules and thus affect plant metabolism, or maybe they can function as free radical scavengers, as was reported for mannitol [13]. The introduction of fructan biosynthesis in crop plants appears to be a promising approach to improve crop productivity under drought stress. This is of significance because water is the most limiting factor for sugar beet production, and for agriculture in general. Under our growth conditions, the beet storage roots were 15 % heavier in A30-3 plants and 31 % heavier in T72-1 plants than in their wildtype controls. The T72-1 plants were harvested at a later developmental stage, indicating that the positive effect of fructans on storage root biomass may be even more pronounced when the storage root reaches its maximum size. The fructans are not expected to influence the sucrose concentrations in the sugar beet: in other transgenic fructan-accumulating species, the levels of soluble sugars (including sucrose) were the same or somewhat higher than in wildtype plants [3, 11, 15]. Plant Physiol. Biochem.
4. METHODS 4.1. Gene construct The binary vector pMOG23 (BIN19 derivative), containing the nptII gene driven by the nos promoter as well as a multiple cloning site, was kindly provided by MOGEN International N.V. (Leiden, the Netherlands). A chimeric gene encoding for β-glucuronidase containing an intron (35S-GUSint) was inserted into the HindIII site of pMOG23, resulting in pVDH65. The chimeric SacB gene [3] was inserted in pVDH65 after digestion with EcoRI and XhoI. The resulting binary transformation vector is called pVDH124. pVDH124 was transferred to Agrobacterium tumefaciens LBA4404 by triparental mating, and the functionality of the construct was tested by transformation to tobacco (Nicotiana tabacum). The transgenic tobacco was shown to accumulate high molecular weight fructans, identical to the results described by Ebskamp et al. [3].
4.2. Plant transformation Cotelydons of Beta vulgaris L. (diploid O-type population) were transformed according to Fry et al. [4]. Plants from the first generation of transgenics were crossed back with untransformed plants of the same variety. The seeds resulting from two of these crossings were used for these physiological studies: these lines are referred to as A30-3 and T72-1.
4.3. Fructan analysis Samples for fructan analysis were homogenized in a 1.5-mL microcentrifuge tube using a micropestle, and 1 µL undiluted cell sap was applied onto a silica thin layer chromotography plate (Schleicher and Schuell). The thin layer chromatography plates were run twice in 90 % acetone and stained specifically for fructose residues using the method of Wise et al. [16]. The fructan spot at the application site was quantified using a standard of commercially available levan from Erwinia herbicola (Sigma).
4.4. Drought treatment Two drought stress experiments were done, the first one with transgenic line A30-3 and the second with line T72-1. Each experiment was done with ten replicates per treatment. For each experiment, one hundred seeds obtained from a crossing between a transgenic primary transformant and an untransformed sugar beet plant were sown in soil (Davis mix), and watered daily
Enhanced drought resistance in fructan-producing sugar beet
with half-strength Hoagland’s solution [7]. After 3 weeks, twenty-five GUS (β-glucuronidase) positive seedlings and twenty-five negative seedlings (‘wildtype’ controls) were selected. These plants were transplanted to equal amounts of soil (Davis mix) in 15-cm diameter pots, and grown in a growth chamber at 20 °C, at a photoperiod of 14 h and a light intensity of ∼100 µE⋅m–2. When the plants were 7-week-old, the drought treatment was started with ten plants from each line while the same number of plants from each line were kept well-watered as controls. Drought stress was imposed by reducing the supply of nutrient solution. During drought treatment, the wildtype and transgenic plants received the same amount of water, which corresponded with 10–20 % of the field capacity of the soil. This resulted in reversible wilting. After 4 1/2 weeks of drought stress, the A30-3 plants and their controls were harvested; the T72-1 plants and their controls were harvested after 6 weeks of drought treatment. At harvest, the total leaf area was determined using a Delta-T Devices leaf area meter (Santa Clara, CA, USA). In addition, the fresh weight and dry weight of leaf blades, stem and petioles, storage root, and fibrous roots were measured.
Acknowledgments E.A.H. Pilon-Smits was supported by a TALENT stipend from the Dutch Organization for Scientific Research (N.W.O.).
REFERENCES [1] Boyer J.S., Plant productivity and environment, Science 218 (1982) 443–448. [2] Clarke N., Hetschkun H., Jones C., Boswell E., Marfaing H., Identification of stress tolerance traits in sugar beet, in: Jackson M.B., Black C.R. (Eds.), Interacting Stresses on Plants in a Changing Climate, vol I16, NATO ASI Series, Springer Verlag, Berlin Heidelberg, 1993, pp. 511–524. [3] Ebskamp M.J.M., Van der Meer I.M., Spronk B.A., Weisbeek P.J., Smeekens S.J.M., Accumulation of fructose polymers in transgenic tobacco, Biotechnology 12 (1994) 272–275.
317
[4] Fry J.E., Barnason A.R., Hinchee M., Genotypeindependent transformation of sugar beet using Agrobacterium tumefaciens, Poster Abstract of the 3rd Congress of ISPMB, Tucson, AZ, 1991, p. 384. [5] Hendry G.A.F., The ecological significance of fructan in a contemporary flora, New Phytol. 106 (1987) 201–216. [6] Hendry G.A.F., Evolutionary origins and natural functions of fructans – a climatological, biogeographic and mechanistic appraisal, New Phytol. 123 (1993) 3–14. [7] Hoagland D., Arnon D.I., The water culture method for growing plants without soil, Calif. Agric. Exp. Stn. Circ. 347 (1938) 1–39. [8] Holmstrom K.O., Mantyla E., Welin B., Mandal A., Tapio Palva E., Tunnela O.E., Drought tolerance in tobacco, Nature 379 (1996) 683–684. [9] Kishor P.B.K., Hong Z., Miao G., Hu C.A., Verma P.S., Overexpression of pyrroline-5-carboxylate synthetase increases proline production and confers osmotolerance in transgenic plants, Plant Physiol. 108 (1995) 1387–1394. [10] Nelson C.J., Smith D., Fructans: their nature and occurrence, Curr. Top. Plant Biochem. Physiol. 5 (1986) 1–16. [11] Pilon-Smits E.A.H., Ebskamp M.J.M., Paul M.J., Jeuken M.J.W., Weisbeek P.J., Smeekens S.C.M., Improved performance of transgenic fructanaccumulating tobacco under drought stress, Plant Physiol. 107 (1995) 125–130. [12] Pilon-Smits E.A.H., Terry N., Sears T., Kim H., Zayed A.M., Hwang S.B., van Dun K., Voogd E., Verwoerd T.C., Krutwagen W.H.H., Goddijn O.J.M., Trehaloseproducing transgenic tobacco plants show improved growth performance under drought stress, J. Plant Physiol. 152 (1998) 525–532. [13] Shen B., Jensen R.G., Bohnert H.J., Increased resistance to oxidative stress in transgenic plants by targeting mannitol biosynthesis to chloroplasts, Plant Physiol. 113 (1997) 1177–1183. [14] Tarczynski M.C., Jensen R.G., Bohnert H.J., Stress protection of transgenic tobacco by production of the osmolyte mannitol, Science 259 (1993) 508–510. [15] Van der Meer I.M., Ebskamp M.J.M., Visser R.G.F., Weisbeek P.J., Smeekens S.C.M., Fructan as a new carbohydrate sink in transgenic potato plants, Plant Cell 6 (1994) 561–570. [16] Wise C.S., Dimler R.J., Davis H.A., Rist C.E., Determination of easily hydrolyzable fructose units in dextran preparations, Anal. Biochem. 27 (1955) 33–36.
vol. 37 (4) 1999