Journal of Experimental Botany, Vol. 53, No. 369, pp. 699-705,
April 1, 2002
© 2002 Oxford University Press
Original Papers |
Engineering for drought avoidance: expression of maize NADP-malic enzyme in tobacco results in altered stomatal function
Pioneer Hi-Bred International, Inc., A DuPont Company, 7300 N.W. 62nd Avenue, Johnston, Iowa 50131, USA
Received 6 July 2001; Accepted 20 November 2001
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Abstract |
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Water is a principal limitation to agricultural production during drought and in arid regions of the world. Mechanisms that plants use to cope with drought can be grouped into two different strategies: drought tolerance and drought avoidance. Previous efforts toward engineering plants for improved performance during drought have focused on drought tolerance, the ability to adjust to dry conditions. This report addresses the engineering of a drought-avoidance phenotype, which allows for the conservation of water during plant growth. The majority of water lost from plants occurs through stomata. When stomata are open, potassium, chloride and/or malate are present at high concentrations in guard cells. The accumulation of large numbers of ions during stomatal opening increases the turgor pressure of the guard cells, which results in increased pore size. Expression of a single gene from maize, NADP-malic enzyme (ME), which converts malate and NADP to pyruvate, NADPH, and CO2, resulted in altered stomatal behaviour and water relations in tobacco. The ME-transformed plants had decreased stomatal conductance and gained more fresh mass per unit water consumed than did the wild type, but they were similar to the wild type in their growth and rate of development. Providing chloride via the transpiration stream partially reversed the effects of ME expression on stomatal aperture size, which is consistent with the interpretation that expression of ME altered malate metabolism in guard cells. These results suggest a role for malic enzyme in the mechanism of stomatal closure, as well as a potential mechanism for genetically altering plant water use.
Key words: Drought, drought avoidance, guard cells, NADP-malic enzyme, stomata, transgenic plants.
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Introduction |
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Plants respond to variations in soil water availability by altering stomatal aperture size, which, in turn, alters the rate of water loss from leaves. The size of the stomatal aperture is determined by guard-cell volume. When guard-cell volume increases as a result of movement of ions and water into the cell, the size of the stomatal pore increases. The dominant cation accumulated in guard cells during stomatal opening is potassium, which is acquired by the cell via specific potassium channels (Schroeder et al., 1994
; MacRobbie, 1997). To balance the influx of positive charge, organic anions, principally malate, and chloride are also accumulated (Zeiger, 1983; Raschke, 1975). Malate is synthesized in the cytosol from starch stored in the guard-cell chloroplast by a unique, regulated form of phosphoenolpyruvate carboxylase (Du et al., 1997). When stomata are open, malate is stored in the vacuole of guard cells (Gotow et al., 1985; Pei et al., 1996). The fate of malate during stomatal closure is unclear. However, several lines of evidence support the hypothesis that a form of ME located in guard cells and the cells around them may facilitate malate degradation during stomatal closure (Outlaw et al., 1981; Maurino et al., 1997).
To study the effect of altered malate metabolism on stomatal aperture and plant water use, NADP-malic enzyme, which converts malate and NADP+ to pyruvate, NADPH, and CO2, was expressed in tobacco. The form of ME expressed was the primary decarboxylating enzyme in C4 photosynthesis. Because ME is located in the chloroplast of maize (Maurino et al., 1997), the enzyme was targeted for expression in the chloroplasts of tobacco. The extensive mobility of the intracellular malate pool (Martinoia and Rentsch, 1994) should allow for a change in concentration of malate in the chloroplast to affect the concentration of malate in many or all compartments of the cell, including the vacuole, where malate is stored in open stomata. To ensure expression in a variety of tissues, including guard cells, a modified mannopine synthase promoter (Ni et al., 1995) was used. The hypothesis that expression of ME would decrease stomatal aperture and alter plant water use by either (i) decreasing the intracellular malate concentration in guard cells, thereby depriving them of the counter-ion necessary for proper stomatal opening; or (ii) increasing the internal CO2 concentration in whole leaves as a result of ME activity in mesophyll cells, was addressed.
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Materials and methods |
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Gene construction and transformation
A maize NADP-malic enzyme cDNA was identified from an EST collection (Pioneer Hi-Bred International, Inc., Johnston, IA, USA) based on its homology to the published sequence (Rothermel and Nelson, 1989
). To ensure correct targeting of the transgenic protein into a C3 chloroplast, the maize chloroplast transit peptide was replaced with a Petunia rbcS transit peptide (Dean et al., 1987). The maize sequence was spliced between the nucleotides encoding amino acids 65 and 66 of the ME protein. The chimeric gene was fused to a modified mas promoter (Ni et al., 1995) and pinII terminator (An et al., 1989). The ME construct was cloned into a binary vector linked to the neomycin phosphotransferase II gene (Herrera-Estrella et al., 1983). The binary vector was introduced into Agrobacterium tumefaciens LBA 4404 using a freeze–thaw method. The construct was introduced into tobacco by Agrobacterium-mediated transformation. Individuals that were homozygous for the ME construct and null segregates derived from five independent transgenic events were analysed after two generations of selfing. Homozygosity was confirmed by evaluation of segregation ratios after selection on kanamycin (data not shown).
Enzyme activity and metabolite assays
The total extractable activity of ME was measured in crude leaf extracts from 3-week-old plants as described previously (Kanai and Edwards, 1973; n=10). Whole leaf malate content was measured in extracts of expanded leaves from 6-week-old plants as described previously (Ku et al., 1981; n=3–5). Chlorophyll content was measured according to Wintermans (Wintermans, 1969). The chlorophyll data from the ME-transformed plants were pooled regardless of the line of origin (wild type, n=6; ME-transformed, n=13). All leaf tissue samples were harvested in the greenhouse from illuminated leaves, immediately frozen in liquid nitrogen, and stored at -80 °C until extraction.
Gas exchange measurements
Measurements of stomatal conductance were made with a LI-6400 (Li-Cor, Inc., Lincoln, NE, USA) equipped with a 6400–02B red and blue LED light source. The standard conditions of 1200 µmol PAR m-2 s-1, 25 °C, 60–70% relative humidity, and 400 µmol mol-1 CO2 were used unless otherwise noted. Fully expanded leaves from plants that were 5–7-weeks-old were selected for use in gas exchange experiments. The leaves remained attached to the plant unless otherwise noted. Removing the leaves from the plant and recutting them under water had no effect on the rate of stomatal opening or on the final aperture attained (data not shown).
The correlation between ME activity and stomatal conductance was determined by measuring ME activity as described above, and by measuring stomatal conductance in the greenhouse in the morning using leaves that were already fully illuminated. Under these conditions, stomatal conductance rapidly adjusted to the standard conditions in the leaf chamber and the measurement was recorded within 5–10 min.
Stomatal induction in response to illumination was measured using leaves that were incubated in a darkened leaf chamber for 120 min. These experiments were undertaken in either the morning or the afternoon. The measurements were made under three sets of environmental conditions: (1) the standard conditions (detached leaves; n=3); (2) higher temperature, lower humidity conditions (30 °C, 30–40% relative humidity; n=3, except wild type n=5); and (3) the standard conditions in the presence of 10 mM KCl (detached leaves; n=3).
The response of stomatal conductance to chloride was also measured using leaves that had reached steady-state stomatal conductance in the light prior to the addition of 10 mM KCl to the water in which the petiole was submerged (detached leaves; n=5). Steady-state stomatal conductance at the new level was reached within 5–10 min and maintained for 10–30 min, at which time stomatal conductance began to decline rapidly. The eventual closure of the stomata under these conditions may have been the result of an increase in the concentration of KCl near the guard cells with time as water was transpired from the leaves.
Stomatal closure was measured using attached leaves that had maintained steady-state conductance under the standard conditions for 30 min prior to darkening (n=3).
Hydroponic growth experiments
For measurement of fresh weight gain:water use ratio, three independent experiments were conducted at different times in the same growth chamber (n=6, except experiment 1, event 51, n=3 and null, n=4; experiment 3, event 10, n=7 and null, n=5). The plants were grown in a Conviron growth chamber (model PGR15) equipped with incandescent and fluorescent lamps that provided 400 µmol PAR m-2 s-1 at plant level for a 16 h photoperiod. The chamber was maintained at 25/20 °C day/night temperature and at least 60% relative humidity. The plants were seeded in Strong-Lite High Porosity Plus soil mix (Strong-Lite Horticultural Products, Pine Bluff, AR, USA), and transplanted to foil-covered 1.5 l containers containing 0.5 strength Hoagland's nutrient solution at 4 weeks after seeding. Selected plants were visually similar, and weighed approximately 2 g each at transplant. The plants were allowed to recover from transplant for 3–4 d, and the experiment was conducted over a period of 7 d. During the course of the experiment, the volume of the solution was measured and the plants were gently patted dry and weighed daily. In experiments two and three, the plants were destructively harvested and dried to constant mass at 80 °C in a forced-air drying oven.
Dry-down experiment
For measurement of soil water conservation and drought avoidance, the plants were seeded and maintained as described above. At 4 weeks after seeding, the plants were transplanted to 2.0 l containers, each of which contained the same amount of soil mix by weight. The plants were then allowed to recover for 3–4 d. At the start of the experiment, the soil in each container was saturated with the same amount of water and completely covered with plastic wrap and aluminium foil to prevent evaporation of water from the soil and to prevent excessive heat build-up. Percentage soil moisture was calculated as described previously (Pei et al., 1998; n=6).
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Results |
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ME expression, malate content, and chlorophyll content
The five transgenic lines studied in detail had 5–18 times more total extractable ME activity on a chlorophyll basis than did wild-type tobacco when measured at 3 weeks after seeding (Fig. 1A
). Whole-leaf chlorophyll content did not differ between wild-type and ME-transformed plants. The chlorophyll content of leaves from wild-type and ME-transformed plants was 503.7±26.0 and 509.6±20.5 µmol m-2, respectively. To control the effects of transformation and regeneration processes, individuals derived from these lines that no longer contained the transgenic insert, null segregates, were also included in the analyses.
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The whole-leaf malate content of ME-transformed plants was 39–77% that of wild type or null segregates when measured at 6 weeks after seeding (Fig. 1B
). This result demonstrates that ME was active and mediated malate decarboxylation in planta.
Stomatal conductance
Stomatal conductance to water vapour, which is a measure of stomatal aperture size based on the rate of water loss from a leaf, was lower in the ME-transformed plants than in wild-type plants under all conditions in which it was measured. When measured in the greenhouse in the morning using previously illuminated and actively photosynthesizing leaves, stomatal conductance was reduced in the ME lines in a manner proportional to the total extractable ME activity (Fig. 2).
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In addition, the time that it took for stomata to open when transferred from darkness to light was increased under diverse environmental conditions (Fig. 3A
, B). To mimic the harsh conditions that are prevalent in agricultural fields and to magnify the differences among the genotypes, stomatal induction under high temperature and low humidity (30 °C, 30–40% relative humidity) was measured. Under these conditions, maximal stomatal conductance attained by the highest expressing ME line (event 10) was only 40% that of wild type (Fig. 3A). Stomatal conductance attained after induction under these conditions was 0.57±0.06 mol H2O m-2 s-1 for wild type, 0.41±0.04 for event 59, moderate ME expression, and 0.25±0.03 for event 10, high ME expression (n=3, except wild type, n=5; P<0.05). Under the milder standard conditions (25 °C, 60–70% relative humidity), maximal conductance in event 10 was 60% that of wild type (Fig. 3B). Stomatal conductance attained under these conditions was 0.69±0.06 mol H2O m-2 s-1 for wild type, 0.57±0.04 for event 59, and 0.42±0.07 for event 10 (n=3; P<0.05). These results are consistent with the recent observation that transgenic modulation of phosphoenolpyruvate carboxylase altered malate metabolism and the rate of stomatal opening (Gehlen et al., 1996).
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) and 10 (
). The arrows indicate the times at which the lamp was turned on (A, B) or off (C). (A) Representative kinetics for stomatal opening at 30 °C and 30–40% relative humidity. (B) Representative kinetics for stomatal opening at 25 °C and 60–70% relative humidity. (C) Representative kinetics for stomatal closure at 25 °C and 60–70% relative humidity.The mechanism of stomatal closure was less affected by expression of ME. The rate of closure in response to darkness was similar among the ME-transformed plants and the wild type, and the extent of closure was unaffected (Fig. 3C
; n=3; P=0.84).
Water conservation during growth and drought avoidance
To assess the effect of ME on whole-plant water relations, plants were grown both under hydroponic conditions in which water was not limiting, and in a dry-down experiment to simulate drought. In three separate hydroponic experiments using different transgenic lines, the fresh weight gain:water use ratio over the course of the experiment was 15–20% greater in the ME-transformed plants than in the wild type or null segregates (Fig. 4A). ME-transformed plants for event 10 (Exp. 3), which showed the highest level of ME activity, were significantly different from both the wild type and null segregates. In these hydroponic experiments, the fresh weights of the plants increased approximately 10-fold, and the final fresh weights were similar among the genotypes (Fig. 4B). There were no differences among the genotypes in fresh:dry weight ratio (data not shown). Neither root:shoot ratio nor time to flowering measurements of plant development differed among the genotypes (data not shown).
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), null segregates (
), and ME-transformed plants derived from event 51 (In the dry-down experiment, the decrease in percentage soil moisture of plants grown in containers over a 3.5 week interval during which the plants were not watered was measured. During this drought simulation, the ME-transformed plants depleted soil moisture more slowly than did the wild type or the null segregates (Fig. 4C
). During the second week of these experiments, wilting was observed in most plants during the light period. However, in all cases, the wild type and null-segregate plants began wilting 2–4 d before the ME-transformed plants, demonstrating that the ME-transformed plants delay or avoid the onset of drought by conserving soil water content. By the third and fourth weeks of the experiment, all plants were visibly water-stressed and began to exhibit signs of irreversible damage. Once stressed, older leaves of plants expressing high levels of ME showed greater development of necrotic spots and yellowing relative to the wild type.
Chloride supplementation experiments
Chloride, when supplied to isolated leaf epidermal peels, decreases the synthesis and accumulation of intracellular malate in guard cells by supplying an alternative counter-ion pool (Raschke and Schnabl, 1978; Van Kirk and Raschke, 1978). These researchers have shown this response to be relatively specific to chloride. The impermeable zwitterion, iminodiacetate is unable to elicit the response. To test whether the effect of ME on stomatal conductance was the result of altered intracellular malate metabolism in guard cells, chloride was supplied to detached leaves via the transpiration stream. Chloride stimulated further opening of stomata that had already reached steady-state conductance in the light in both wild-type and ME-transformed plants (Fig. 5A). However, the stimulation of conductance was 44% greater for ME-transformed plants than for wild-type plants. The rate of stomatal opening was also partially restored by chloride (Fig. 5B). The increased stimulation of stomatal conductance by exogenous chloride in the ME-transformed plants relative to the wild type supports the hypothesis that the effect of ME on stomatal conductance is mediated by altered intracellular malate metabolism in guard cells.
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Discussion |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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The expression of ME is correlated with a decrease in stomatal aperture and an increase in the amount of fresh mass gained per unit water used. These experiments support a role for malic enzyme in the mechanism of stomatal closure, as first proposed by Outlaw et al. based on measurements of NADP-malic enzyme activity in guard cells and epidermal cells of Vicia faba (Outlaw et al., 1981
). These results suggest that manipulation of organic anion metabolism in guard cells using NADP-malic enzyme may be developed as a practical mechanism for drought avoidance and water conservation during irrigation.
A recent report suggests that NADP-malic enzyme could be detrimental in the development of normal chloroplasts when expressed at high levels (20–70-fold increases) in a C3 plant (Takeuchi et al., 2000). For the plants analysed in this study, which showed 5–18-fold increased ME activity, there was no change in chlorophyll content in the ME-transformed plants under normal culture conditions. However, it is possible that higher levels of ME expression in tobacco may lead to alterations in chloroplast development. Indeed, following exposure to drought, the development of necrosis was more rapid in leaves from plants with the highest ME expression than in leaves from plants with moderate ME expression.
Although the results of the chloride supplementation experiments are consistent with the hypothesis that altered intracellular malate metabolism in guard cells is responsible for the decreased stomatal conductance in the transgenic plants, the use of a constitutive promoter does not preclude other possibilities. The hypothesis that increased internal leaf CO2 concentration also contributes to the reduction in stomatal aperture as a result of ME activity in mesophyll cells cannot be ruled out. Additional experiments using a guard cell-specific promoter to express ME are being undertaken to explore the relative importance of alterations in the guard-cell intracellular malate pool and CO2 production by mesophyll cells. These experiments may also allow for separation of the necrosis observed for mature leaves from the drought avoidance phenotype under conditions of water stress.
The extent to which stomatal conductance can be reduced without affecting plant growth or yield must be determined in the field for crop species. However, both theoretical predictions and experimental results indicate that plant water-use efficiency can be improved with minimal sacrifice in productivity (Farquhar and Sharkey, 1982; Jensen and Cavalieri, 1983; Rademacher et al., 1987). The stomatal data reported here indicate that altering guard-cell malate metabolism may be useful for the maintenance of crop growth during drought in mesic regions of the world that are subject to periodic episodes of drought and for conserving water during irrigation. Delaying the onset of drought through conservative water use during critical periods of plant development, including the days surrounding anthesis, has the potential to raise crop yield toward the genetic potential, which is several fold higher than the actual yield attained during drought (Boyer, 1992). In addition, it should now be possible to engineer plants for a phenotype displaying both drought-avoidance and drought-tolerance mechanisms. Such plants may not only conserve water during growth, but also may be able to respond to drought more effectively once it is detected.
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Acknowledgments |
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The authors thank L Beach, P Anderson, D Byron, and A Cavalieri for project support; D Brockshus, A Davis, and M McConnelee for technical assistance; T Sharkey and C Zinselmeier for technical advice; J Boyer, A Hanson, R Jung, W Outlaw, and T Sharkey for critical reviews of the manuscript.
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Notes |
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1 Present address: Department of Biology, Eastern Michigan University, Ypsilanti, MI 48197, USA.
2 To whom correspondence should be addressed. Fax: +15152542619. E-mail: mitchell.tarczynski{at}pioneer.com
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