| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
|
First published online February 2, 2007; 10.1104/pp.106.093559 Plant Physiology 143:1905-1917 (2007) © 2007 American Society of Plant Biologists OPEN ACCESS ARTICLE
Overproduction of Abscisic Acid in Tomato Increases Transpiration Efficiency and Root Hydraulic Conductivity and Influences Leaf Expansion1,[OA]Warwick HRI, Wellesbourne, University of Warwick, Warwickshire CV35 9EF, United Kingdom (A.J.T., J.A., B.J.M., J.M.T.M., H.W.H., J.S.H.); Environmental Biology Group, Research School of Biological Sciences, Australian National University, Canberra, Australian Capital Territory 2601, Australia (G.D.F.); and Department of Plant Sciences, School of BioSciences, University of Nottingham, Sutton Bonington Campus, Loughborough, Leicestershire LE12 5RD, United Kingdom (R.C.S., I.R.A.S., C.R.B., I.B.T.)
Overexpression of genes that respond to drought stress is a seemingly attractive approach for improving drought resistance in crops. However, the consequences for both water-use efficiency and productivity must be considered if agronomic utility is sought. Here, we characterize two tomato (Solanum lycopersicum) lines (sp12 and sp5) that overexpress a gene encoding 9-cis-epoxycarotenoid dioxygenase, the enzyme that catalyzes a key rate-limiting step in abscisic acid (ABA) biosynthesis. Both lines contained more ABA than the wild type, with sp5 accumulating more than sp12. Both had higher transpiration efficiency because of their lower stomatal conductance, as demonstrated by increases in  13C and 18O, and also by gravimetric and gas-exchange methods. They also had greater root hydraulic conductivity. Under well-watered glasshouse conditions, mature sp5 plants were found to have a shoot biomass equal to the wild type despite their lower assimilation rate per unit leaf area. These plants also had longer petioles, larger leaf area, increased specific leaf area, and reduced leaf epinasty. When exposed to root-zone water deficits, line sp12 showed an increase in xylem ABA concentration and a reduction in stomatal conductance to the same final levels as the wild type, but from a different basal level. Indeed, the main difference between the high ABA plants and the wild type was their performance under well-watered conditions: the former conserved soil water by limiting maximum stomatal conductance per unit leaf area, but also, at least in the case of sp5, developed a canopy more suited to light interception, maximizing assimilation per plant, possibly due to improved turgor or suppression of epinasty.
Crop yield is often limited by water availability, and global climate change, together with increasing competition for water resources, makes the genetic improvement of water-use efficiency an increasingly important goal (Parry et al., 2005  ). Transpiration efficiency (TE) can be defined as crop yield per unit of water transpired or, at the whole-plant level, as the biomass gained per unit of water transpired (TEp). At the individual leaf level, the ratio between CO2 assimilation rate (A) and stomatal conductance (gs) (A/gs) gives the intrinsic TE (TEi; Condon et al., 2002). Progress has been made in defining the genetic loci that control TE (Teulat et al., 2002; Hall et al., 2005; Juenger et al., 2005), and a quantitative trait locus that influences TE has been identified as the erecta gene (Masle et al., 2005). Overexpression of candidate genes has produced improvements in TE, for example, by overexpression of the ABA-inducible group 3 LEA gene HVA1 in wheat (Triticum aestivum; Sivamani et al., 2000; Bahieldin et al., 2005) or the maize (Zea mays) NADP-malic enzyme in tobacco (Nicotiana tabacum; Laporte et al., 2002). There are many reports of mutants and transgenic lines where overexpression of candidate genes reduces gs or improves survival on withholding water (Zhang et al., 2004), but it has been pointed out that little progress has been made in demonstrating that transgenic manipulations provide useful improvements in crop species (Passioura, 2006). It is clearly important to understand the effects of gene manipulation on whole-plant and crop physiology so that predictions of agronomic utility can be made and tested.
Abscisic acid (ABA) is a phytohormone that mediates plant responses to abiotic stresses, including drought, salinity, and low temperature, and acts with other phytohormones to regulate plant growth (Sharp, 2002
It is thus clear that overproduction of ABA limits gs, but the impact on TE and other physiological traits that influence productivity has not been reported. Because crop yield is often positively related to the total amount of water transpired, it is possible that increasing ABA production and reducing transpiration will simply suppress biomass production (Condon et al., 2004
Exogenous application of ABA and the study of ABA-deficient mutants have established that ABA can have both positive and negative effects on growth, depending on tissue, applied concentration, and interactions with the environment (Arteca and Tsai, 1987
Exogenously applied ABA is known to increase root hydraulic conductivity (Lpr) both at the organ and the cellular level (Glinka, 1980
The production and analysis of transgenic genotypes with elevated ABA content provides an approach for the investigation of the function of ABA in whole plants that has advantages over either the use of periodic or short-term exogenous application of ABA, or the study of ABA-deficient mutants where ethylene effects (Sharp, 2002
We previously described transgenic lines that overexpress LeNCED1 driven by the Gelvin Superpromoter and have a high ABA content (Thompson et al., 2000 ). In this study, we further investigate two of these high ABA lines, sp5 and sp12, in a homozygous state. Note that sp12 was previously named d9 (see "Materials and Methods"). As sp5 has higher transgene expression than sp12 (Thompson et al., 2007), its responses have been found to be more extreme than sp12 in almost all physiological assays.
Line sp12 was compared to the wild type in a drought experiment in which plants were either irrigated with a fixed volume of water delivered twice daily throughout the experimental period (the well-watered treatment) or not watered to provide a drought treatment. Leaf conductance (gl), leaf water potential (
In the well-watered treatment, gl remained high in the wild type on days 1 to 3 and, as expected, was significantly higher than in sp12 (Thompson et al., 2000 ). However, by days 4 and 5, there was evidence of mild water stress in wild-type plants, even in the well-watered treatment, because both gl and  w declined (P < 0.05 on day 5 for gl and days 4 and 5 for w). Wild-type plants had both a larger leaf area and higher gl on day 1 in the well-watered treatment and would have withdrawn water more rapidly from the vermiculite substrate than the sp12 plants; if we assume that gl did not vary with leaf age, it can be calculated that gl on a per plant basis on day 1 would have been 14.9 and 6.8 mmol plant–1 s–1 for the wild type and sp12, respectively. In addition, mean leaf area for well-watered wild-type plants increased from 272 to 589 cm2 between days 1 and 4. One explanation for the decrease in gl and w on days 4 and 5 in well-watered wild-type plants is that, as leaf area increased, transpiration began to exceed the unvarying daily supply of irrigation water and a water deficit consequently began to develop during each day between irrigations. In the case of sp12, with its 28% lower leaf area and 38% lower gl than the wild type on day 1, irrigation is more likely to have been sufficient to avoid a decline in w as leaf area increased.
In the drought treatment,
In the well-watered treatment, sp12 exhibited higher leaf, root, and xylem sap ABA at all time points, significantly for root and xylem sap ABA on days 1 to 3 (P < 0.05), before the wild-type plants became mildly stressed; there was also a general trend of increasing ABA content over the 5-d observation period in the well-watered plants. However, when water was withheld, despite the delayed decline of
In summary, in nonstressed, well-watered conditions, sp12 had values for ABA content and gl that were comparable to those of mildly stressed wild-type plants, i.e. similar to wild-type plants on days 4 and 5 in the well-watered treatment in which The relationship between xylem sap ABA concentration and gl was similar in the wild type and sp12, indicating that the sensitivity to ABA was not influenced by the higher ABA concentration (Fig. 1B).
Two measures of TEi were made for well-watered plants of the wild type, sp12, and sp5: first, instantaneous leaf gas exchange using an infrared gas analyzer (IRGA) to record both A and gl, and, second, carbon isotope composition (
Similarly, in Figure 2B, instantaneous gs was measured by IRGA and also by measurement of oxygen stable isotope composition ( 18O) of leaf dry matter, an integrative, surrogate measure that is inversely related to gs, assuming constant relative humidity (RH; Barbour and Farquhar, 2000). Again, these two approaches are in agreement, showing that the two transgenic lines had significantly lower gs (P < 0.001) and higher 18O (significant for the comparison of the wild type to sp5, P < 0.05).
A plot of A against gs for a single genotype where variation in gs is driven by environmental factors typically has a hyperbolic relationship where A is limited by declining gs (Wong et al., 1979 In addition to measurements of TEi, whole-plant TE (TEp) was measured gravimetrically in a separate experiment where plants were grown in pots in a glasshouse (Table I ). Compared to the wild type, TEp was 27% and 79% higher in sp12 and sp5, respectively (significant at P < 0.05 in both cases).
Response to RH Wild-type and sp12 plants were established under the same low RH conditions (65%) as used in Figures 1 and 2, and then transferred to controlled environment cabinets, either at the same low RH or at higher RH (88%–92%), and kept well-watered. During the light period, the low and high RH treatments were equivalent to 0.92 and 0.48 kPa vapor pressure deficit (VPD). Gas-exchange and leaf chlorophyll measurements were made (Fig. 3 ). Considering both genotypes together, low humidity reduced gl (P < 0.001), but sp12 showed a greater response than the wild type as low humidity reduced gl by 19% in the wild type compared to 50% in sp12. Thus, as previously observed under low RH (Figs. 1A and 2B), sp12 had lower gl than the wild type (in this case 42% lower). However, there was no genotype effect on gl at high humidity (P > 0.05), and so a significant genotype x humidity interaction was detected (P = 0.023). Assimilation was reduced at lower humidity (P = 0.007), but there was no significant genotype effect (P > 0.05). As a result of lower gl, A/gl was 2-fold higher in sp12 than in the wild type at low humidity (P < 0.05), but there was no significant difference at high humidity, giving a genotype x humidity interaction (P = 0.004). SPAD readings were not significantly different between genotypes (Fig. 3, P > 0.9).
We previously reported increased guttation and interveinal chlorosis when sp12 and sp5 plants were grown at high humidity (Thompson et al., 2000 ); from closer observation of both lines, it is now apparent that the interveinal chlorosis mapped to the same leaf regions where intercellular air spaces had previously been flooded with sap (leaf flooding). Here, we report observations of guttation and leaf flooding at both RH levels for the wild type and sp12. No plants growing at 65% RH exhibited guttation or leaf flooding, but almost all plants of both sp12 and the wild type guttated during the predawn period under high RH; sp12 plants produced a much larger volume of guttation fluid than the wild type, with a 6.6-fold greater volume recorded on day 5 (Fig. 4B
). The guttation fluid had a 7.8-fold higher ABA concentration in sp12 (Fig. 4A). Leaf flooding occurred only in sp12 plants at high RH, and was recognizable as sectors that were dark when observed under reflective light (Fig. 4C) but pale and translucent when observed by transmitted light (not shown). Flooded air spaces were predominantly bordered by veins (Fig. 4C) and were more prevalent in the lower leaves (Fig. 4D). Two weeks after transfer to the high humidity environment, most sp12 plants ceased to show guttation and leaf flooding, presumably due to adaptation, or perhaps the increasing leaf area allowed greater dissipation of root pressure. Figure 4E illustrates that tomato leaves are heterobaric (Lawson and Morison, 2006), i.e. the substomatal air spaces are subdivided by veins or bundle sheath extensions connecting the lower epidermis to the palisade layer, thus providing a barrier to sap flow and explaining the interveinal nature of the flooding.
Lpr Wild type, sp12, and sp5 plants were grown in a hydroponic system, and exudate was collected from root stumps between 1 to 3 and 3 to 6 h after detopping the plants. Osmolarities of exudates and the hydroponic media were determined and Lpr was calculated (Fig. 5 ). Exudation rate expressed on a root dry weight basis (JVr) and Lpr were both higher in sp12 and sp5 than in the wild type during the 1 to 3 h time interval, although only sp5 differed significantly from the wild type (P < 0.05). However, during the 3 to 6 h period, both transgenic lines had significantly higher Lpr and JVr values than the wild type (P < 0.05; Fig. 5, A and C). Taking both time intervals together, Lpr values were 2.4- and 3.6-fold higher than the wild type in sp12 and sp5, respectively (Fig. 5C). Both lines had lower exudate osmolarities than the wild type at both time intervals (P < 0.05 for all comparisons to the wild type, and P < 0.001 for the overall genotype effect; Fig. 5B). Exudates from sp12 and sp5 contained significantly more ABA than those from wild-type plants (2.7- and 4.3-fold, respectively; P < 0.001 for the overall genotype effect; Fig. 5D).
When transpiration is low, root pressure may contribute significantly to sap flow to the shoot, although this will be influenced by both the mass of the root system and the JVr. Based on the mean values from the two hydroponic experiments, the fraction of dry matter partitioned to the roots, i.e. the root mass fraction (Poorter and Nagel, 2000 ), for the wild type, sp12, and sp5 was 0.058, 0.056, and 0.068, respectively. The 17% greater root mass fraction in sp5 was significant (P < 0.05, n = 12 plants per genotype) and would have further increased delivery of exudate to the shoot, although the 49% mean increase in JVr would have had a greater impact.
It was clear from numerous observations under glasshouse conditions that sp12 and sp5 take longer to establish than wild-type plants, by about 4 to 10 d between radicle emergence and the four-leaf stage; however, growth rate is subsequently similar in all genotypes under well-watered conditions and transgenic plants often appeared to produce longer leaves. To investigate leaf growth and biomass partitioning within the shoot, germination of wild-type and sp5 seeds was synchronized and plants were harvested after 13 weeks of growth under standard glasshouse conditions. Final shoot dry weight and plant height were not significantly different between genotypes (21.6 and 21.4 g for wild-type and sp5 shoot dry weight, respectively; P = 0.871). However, the sp5 plants partitioned significantly more biomass to the lamina (P = 0.021) and petiole (P < 0.001) and significantly less to the trusses (P = 0.012), although the developing fruit had only progressed to the immature green fruit stage in the lower trusses at harvest (Fig. 6A ). Leaf area was higher in sp5 (2,114 and 2,683 cm2 for the wild type and sp5, respectively; P < 0.001), and there was a suggestion of higher specific leaf area (258.3 and 285.3 cm2 g–1 dry weight for the wild type and sp5, respectively; P = 0.065). There was a noticeable increase in petiole length in sp5 (P < 0.001), which was significant for leaf numbers 8 to 15 (Fig. 6, B and C) in this experiment; similar leaf length data were obtained from plants grown in the hydroponic system (data not shown). There was also a reduction in leaf epinasty in sp5 (Fig. 6D), as was also apparent in many other glasshouse experiments. The angle between the petiole and lower stem was measured for leaves 3, 4, and 5; the mean angle for sp5 (124°) was higher (P = 0.007) than that for the wild type (110°).
In another experiment where irrigation was set at a suboptimal level for growth of the wild type, mean leaf lengths at final harvest were 33.2, 40.2, and 42.5 cm for the wild type, sp12, and sp5, respectively (P = 0.006 for overall genotype effect, LSD [5%] = 3.7), indicating that the effect on petiole extension is common to the two transgenic lines (R. Smeeton and I. Taylor, unpublished data).
Drought Response Under nonstressed, well-watered conditions, the LeNCED1 overexpression line sp12 exhibited increased xylem sap ABA concentrations and reduced gl values equivalent to mildly stressed wild-type plants (Fig. 1). When a root-zone water deficit was imposed by withholding irrigation, ABA and gl responded, reaching the same final levels in the wild type and sp12, albeit from different basal levels. The response of both the wild type and sp12 to drought was greater than the difference between these two genotypes under well-watered conditions. In agreement with this pattern, the constitutive transgenic expression of LeNCED1 in leaves of well-watered sp12 and sp5 plants was lower than that of the endogenous gene in wilted leaves (S.A. Tung, unpublished data). The important characteristic of these two lines in this context, then, is that they behave differently than the wild type under well-watered conditions, whereas, at least in the study of sp12, the wild-type and transgenic genotypes exhibited similar final values for ABA content and gl, such that differences were no longer apparent once a particular severity of stress was reached and the endogenous mechanisms for ABA accumulation were triggered.
Under well-watered conditions, lines overexpressing LeNCED1 had greatly improved TEi, driven by the reduction in gs, as demonstrated by both instantaneous and integrative methods. There was a positive relationship between
The decline in gs and transpiration is generally proportionally greater than the associated reduction in A (Jones, 1976
A simulation analysis for sorghum (Sorghum bicolor; Sinclair et al., 2005
Setting a maximum limit for E was particularly effective in the simulations described by Sinclair et al. (2005)
It is well known that stomatal closure is induced by increases in VPD (Grantz, 1990
We previously reported observations of increased guttation in the two NCED-overexpressing lines (Thompson et al., 2000
The most likely mechanism for the increase in Lpr is an ABA-induced increased activity of aquaporins (Hose et al., 2000
A key question is whether the limit on assimilation imposed by low gs has a negative effect on biomass accumulation under optimum conditions. At least under the environmental conditions used here, there was no reduction in aboveground biomass in mature plants when comparing the wild type and sp5; we therefore propose that, although assimilation rate per unit leaf area was reduced, there were counteracting positive effects of ABA on growth. We have also observed in several experiments where roots were washed from soil (A.J. Thompson and J. Andrews, unpublished data) or where plants were grown hydroponically (data given in "Results" section "Lpr") that the fraction of biomass partitioned to the roots in sp5 or sp12 was either not significantly different to the wild type or was slightly higher, allowing the conclusion that shoot biomass was not maintained in sp5 at the expense of root biomass.
The observed increases in shoot biomass partitioning to the lamina and petiole, reduced epinastic leaf curling, and higher specific leaf area (thinner leaves) in sp5 might be predicted to increase light interception and assimilation per plant (Poorter and Nagel, 2000
The reduction in epinasty in these high ABA lines concurs with observations of exaggerated epinasty in ABA-deficient tomato mutants (Tal and Imber, 1970
Partitioning of biomass to reproductive tissues was reduced in sp5; our observations suggest that this is likely to be attributable either to a small delay in the initiation of the first truss or to greater flower abortion in the lower trusses. In a collection of Arabidopsis ecotypes, McKay et al. (2003)
Plant water status often declines around midday, and stomata may close due to the inability of the root system to supply sufficient water to the shoot during periods of high evaporative demand. In the lines with elevated ABA (sp5 and sp12), it is likely that water status would have been increased by reductions in transpiration and because their greater Lpr would have improved water supplies to the shoot when atmospheric demand for water was high and Lpr might otherwise have become limiting. In the experiment shown in Figure 6, where plants were grown in 18-cm-diameter pots in a free-draining soil with a single irrigation in the morning, it is likely they experienced diurnal cycles of soil water deficit that increased in severity as the plants grew. The more conservative use of water by sp5 may have had the effect of maintaining a higher soil water status during the afternoon, with positive effects on shoot water status. For these reasons, positive effects of ABA on growth are likely to arise from improvements in water status and enhanced turgor-driven growth.
Long-term elevation of ABA in plants produced a range of physiological effects that may impact positively or negatively on crop production depending on water availability (Fig. 7 ). In well-watered plants, the negative effects of ABA overaccumulation were reduced assimilation rate, leaf flooding and chlorosis (particularly under high humidity environments in young plants), and delayed germination and establishment. However, under standard glasshouse conditions, these effects were insufficient to reduce biomass production, presumably because of counteracting positive effects on leaf expansion through improvements in water status, turgor, and antagonism of epinastic growth. We also hypothesize that under conditions of limited water availability, additional positive factors become relevant; thus, improvements in TEi should allow continued biomass production by conserving soil water and delaying the onset of shoot water deficits, while increased Lpr will improve the supply of water to the shoot, helping to maintain water status when atmospheric demand is high. However, once high ABA plants begin to experience water deficits, they behave as wild-type plants because loss of turgor activates the native ABA biosynthetic pathways and stimulates a greatly increased level of ABA accumulation, swamping the transgene effect displayed by the two transgenic lines under well-watered conditions.
Optimization of NCED transgene activity for level of expression, developmental timing, and tissue specificity may allow the production of genotypes in which the potential negative physiological effects of high ABA, such as delayed establishment or flowering, can be avoided, while the positive effects on water economy and leaf growth are maintained.
Plant Materials, Germination, and Establishment
Transgenic primary transformants of tomato (Solanum lycopersicum), sp5 and sp12, and the wild type control (cv Ailsa Craig) were used as described previously (Thompson et al., 2000 For germination, tomato seed were surface sterilized by treatment with 10% (v/v) Domestos household bleach (Lever Faberge) for 30 min to inactivate plant viruses, then washed thoroughly in distilled water. Wild-type and sp12 seed were sown directly onto filter paper soaked in distilled water. The more recalcitrant homozygous sp5 seed, in which the high ABA content severely delayed germination, were first washed for 5 d in tap water using an ebb-and-flow device adapted from a pipette washer, and then transferred to filter paper soaked in distilled water plus 0.5 mg L–1 norflurazon (Syngenta; herbicide grade, 80% purity). For all tomato genotypes, seed were germinated in the dark at 25°C and on the day of radicle emergence seed were sown into 30-mL modules filled with Levington's F2 compost (Levington Horticulture). Typically, radicle emergence took 3 to 4, 10 to 13, or 4 to 8 d for the wild type, sp12, and sp5, respectively (note that emergence often took over 20 d for sp5 in the absence of norflurazon). In the case of sp5, seed were washed for 1 h in tap water to remove residual norflurazon before transfer to compost. After a further 2 to 3 weeks, unless otherwise stated, seedlings of all genotypes were transplanted into vermiculite soaked in nutrient solution (Vitafeed 214; Vitax) in 270-mL or 1,000-mL pots. Genotypes sp5 and sp12 typically required 4 to 10 d longer than the wild type to establish after radicle emergence, but, by careful scheduling and selection, all three genotypes were grown such that they reached the three- to four-leaf stage at approximately the same time. Note that a slightly modified protocol for germination and establishment was employed for the glasshouse experiment presented in Table I (see "Gravimetric Measurement of TEp").
Unless otherwise stated, growth cabinet conditions were: 65% RH, 22°C/18°C day/night temperature, 14-h photoperiod, 300 µmol m–2 s–1 photosynthetically active radiation, and 360 µmol mol–1 CO2. Plants were irrigated daily by hand with Vitafeed 214 (Vitax). Side shoots and flower trusses were removed as soon as they were apparent, except for the experiment shown in Figure 6.
Plants were grown to the four- or five-leaf stage in a walk-in controlled environment cabinet, arranged in a split-plot Trojan Square design (Edmondson, 1998
Wild-type and sp12 plants were established in a growth cabinet under the standard controlled environment conditions (65% RH). Plants were selected at the four- to five-leaf stage to provide uniform size between and within genotype. At this time (day 0), plants were split into two growth cabinets, one at low RH (65% day and night) and one at high RH (82%–90% day-night), using standard lighting and temperature regimes. The incidence of guttation and interveinal flooding was scored daily at dawn. For interveinal flooding, the percentage of leaf area that was flooded was estimated in 10% increments for each leaf by visual observation. The mass of guttation fluid was measured following collection by absorption on to preweighed filter paper. Guttation fluid for ABA analysis was harvested by pipette. Chlorophyll measurements were made on day 13 with a chlorophyll meter (SPAD-502; Minolta). On day 26, gas-exchange measurements were taken. Five randomized blocks per cabinet were used throughout.
Plants were raised in 9-cm-diameter pots containing F2 compost and then transferred to a recirculating, aerated, nutrient-film hydroponic system (Winsor et al., 1979
ABA was determined by radioimmunoassay directly upon xylem sap or root exudate, or upon aqueous extracts of root or leaf tissues, using appropriate dilutions, as described by Mulholland et al. (2003)
For Figures 1 and 2, plants were analyzed using a CIRAS-1 IRGA (PP-Systems). The leaf chamber was illuminated under ambient photosynthetically active radiation levels (300 µmol m–2 s–1). CO2 concentration was set to the same concentration as the cabinet (360 ppm). For Figure 3, measurements were made with an LCA-4 IRGA (Analytical Development Company) using ambient light, CO2, and humidity. All measurements were taken on the youngest fully expanded leaf.
IRGAs provide measurements of gl, the sum of gs and gc, with gc often considered to be negligible. However, in Figure 2 we plotted A versus gl and fitted a three-parameter rectangular hyperbolae, providing an estimate of gc = 18.9 mmol m–2 s–1 when A = 0, and then calculated gs = gl – gc. In Figure 2C we fitted Amax and k as A = Amax gs/(gs + k), where k/1.6 is equivalent to a carboxylation conductance; this model assumes that gc was the same for each genotype. We have measured the stomatal and cuticular components of water loss in the wild type and sp5 during biphasic "Hygen" drying curves of detached leaflets (Weyers and Meidner, 1990
Wild-type, sp12, and sp5 plants were established so that plant size was synchronized at the beginning of the assessment of TEp (see "Plant Materials, Germination, and Establishment"). In this case, seeds of sp5 were sown 19 d before sp12 and 24 d before the wild type, directly into 7-cm-diameter pots of Levington's M3 compost (Levington Horticulture), with 9-cm petri dish lids placed over the top of the pots to create a humid environment until the hypocotyl emerged. Seeds of sp5 were imbibed in 0.1 mg L–1 norflurazon for 1 d and then thoroughly washed before sowing. During early growth, plants were irrigated each day to field capacity. On the first day of TEp assessment, three plants per genotype were destructively harvested to determine initial aboveground dry weight. Three further plants were potted into 17-cm-diameter, 2-L pots, also using Levington's M3 compost, and Uvi ground cover discs (Growing Technologies) were placed on top of the compost to limit loss of water by evaporation from the soil. The pots were watered until saturated, allowed to drain to reach field capacity, and weighed. On each of the following 25 d, pots were weighed to record the mass of water lost and then returned to field capacity before reweighing. After this period, all plants were then destructively harvested and aboveground dry weights recorded. Biomass production during the 25 d was calculated as the difference between final and initial aboveground dry weight. TEp was calculated for each plant.
Wild-type and sp5 plants that had germinated on the same day were selected and potted into 7-cm and then 18-cm pots using a free-draining compost (John Innes Potting Compost No. 2 made with grit rather than sand). Nine plants per genotype were arranged in three randomized blocks. After 13 weeks growth in the partially environmentally controlled glasshouse, with daily irrigation by hand, leaf angles were measured using a protractor for leaves 3, 4, and 5; the plants were then photographed and their shoots harvested. Whole leaves were detached and their length was measured with a ruler. Shoots were divided into lamina, petiole, stem, and truss; laminar area was determined using a
A whole fully expanded leaf, either the fourth of fifth from the top of the plant (the first leaf greater than 2 cm long was counted as leaf 1), was taken and dried at 80°C for 24 h. The entire sample was ground using an Apex rotary mill (Apex Construction); a subsample was further ground with a ball mill (Glen Creston) and sieved to produce a sample of particle size <0.1 mm. Samples were analyzed for 13C/12C and 18O/16O and expressed relative to Pee Dee Belemnite and Vienna Standard Mean Ocean Water to give
In the drought experiment (Fig. 1), a split-plot Trojan Square design was employed as described above. For other experiments (Figs. 2–6 P values quoted in the text are for overall genotype, or genotype x treatment effects. Additional comparisons between individual means are made at the 5% level using LSD values. For analysis of data for hydraulic conductivity experiments (Fig. 5), two experiments were analyzed together using experiment as a factor; the P values quoted were obtained after taking mean values over the two time intervals (1–3 and 3–6 h) for each experiment, except for ABA where data were only collected for the first time interval.
We thank Steve Quarrie (John Innes Institute) for supplying anti-ABA antibody MAC252; Alison Jackson, Linda Brown, Angela Hambidge, and Colin Clay (Warwick HRI) for technical assistance; Steven Clayton and Sue Woods (Australian National University) for stable isotope analyses; and Rodney Edmondson (Warwick HRI) for statistical advice. Received November 22, 2006; accepted January 24, 2007; published February 2, 2007.
1 This work was supported by the Biotechnology and Biological Sciences Research Council (Competitive Strategic Grant to A.J.T.) and by the Department for Environment, Food and Rural Affairs (project no. HH1332SPC; B.J.M. and A.J.T.). G.F. acknowledges Discovery support from the Australian Research Council. 
2 Present address: Duchy College, Rosewarne, Camborne, Cornwall TR14 OAB, UK. The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Andrew J. Thompson (a.j.thompson{at}warwick.ac.uk).
[OA] Open Access articles can be viewed online without a subscription. www.plantphysiol.org/cgi/doi/10.1104/pp.106.093559 * Corresponding author; e-mail a.j.thompson{at}warwick.ac.uk; fax 44–(0)24–7657–4500.
Arteca RN, Tsai D-S (1987) Effects of abscisic acid on the photosynthesis, transpiration and growth of tomato plants. Crop Res 27: 91–96 Assmann SM, Snyder JA, Lee YRJ (2000) ABA-deficient (aba1) and ABA-insensitive (abi1-1, abi2-1) mutants of Arabidopsis have a wild-type stomatal response to humidity. Plant Cell Environ 23: 387–395[Medline] Aswath CR, Kim SH, Mo SY, Kim DH (2005) Transgenic plants of creeping bent grass harboring the stress inducible gene, 9-cis-epoxycarotenoid dioxygenase, are highly tolerant to drought and NaCl stress. Plant Growth Regul 47: 129–139[CrossRef][ISI] Bahieldin A, Mahfouz HT, Eissa HF, Saleh OM, Ramadan AM, Ahmed IA, Dyer WE, El-Itriby HA, Madkour MA (2005) Field evaluation of transgenic wheat plants stably expressing the HVA1 gene for drought tolerance. Physiol Plant 123: 421–427[CrossRef] Barbour MM, Farquhar GD (2000) Relative humidity- and ABA-induced variation in carbon and oxygen isotope ratios of cotton leaves. Plant Cell Environ 23: 473–485[CrossRef] Blum A (2005) Drought resistance, water-use efficiency, and yield potential: Are they compatible, dissonant, or mutually exclusive? Aust J Agric Res 56: 1159–1168[CrossRef][ISI] Bradford KJ, Sharkey TD, Farquhar GD (1983) Gas exchange, stomatal behavior, and Bray EA (2002) Abscisic acid regulation of gene expression during water-deficit stress in the era of the Arabidopsis genome. Plant Cell Environ 25: 153–161[Medline] Chaves MM, Oliveira MM (2004) Mechanisms underlying plant resilience to water deficits: prospects for water-saving agriculture. J Exp Bot 55: 2365–2384 Condon AG, Richards RA, Rebetzke GJ, Farquhar GD (2002) Improving intrinsic water-use efficiency and crop yield. Crop Sci 42: 122–131 | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
TOP
ABSTRACT
RESULTS
m) and exudates (
T area meter MKII (Delta T Devices) and dry weights were recorded. Weight data are presented as a fraction of total shoot biomass (Poorter and Nagel, 2000