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Root restriction-induced limitation to photosynthesis in tomato (Lycopersicon esculentum Mill.) leaves
Scientia Horticulturae 117 (2008) 197–202
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Scientia Horticulturae journal homepage: www.elsevier.com/locate/scihorti
Root restriction-induced limitation to photosynthesis in tomato (Lycopersicon esculentum Mill.) leaves Kai Shi a, Xiao-Tao Ding a, De-Kun Dong c, Yan-Hong Zhou a, Jing-Quan Yu a,b,* a
Department of Horticulture, Huajiachi Campus, Zhejiang University, Kaixuan Road 268, Hangzhou 310029, PR China Key Laboratory of Horticultural Plants Growth, Development and Biotechnology, Agricultural Ministry of China, Kaixuan Road 268, Hangzhou 310029, PR China c The Institute of Crop and Nuclear Technology Utilization, Zhejiang Academy of Agricultural Sciences, Hangzhou 310021, PR China b
A R T I C L E I N F O
A B S T R A C T
Article history: Received 12 February 2007 Received in revised form 19 April 2008 Accepted 30 April 2008
Root restriction often depresses photosynthetic capacity and the mechanism for this reduction, however, remains unclear. To identify the mechanism by which root restriction affects the photosynthetic characteristics, tomato (Lycopersicon esculentum Mill.) seedlings were subjected to root restriction stress with or without supplemental aeration to the nutrient solution. With the development of the root restriction stress, CO2 assimilation rate was decreased only in confined plants without supplemental aeration. There were also significant decreases in leaf water potential, stomatal conductance (gs), intercellular CO2 concentration (Ci), and increases in the stomatal limitation (l) and the xylem sap ABA concentration. Meanwhile, the maximum carboxylation rate of Rubisco (Vcmax) and the capacity for ribulose-1,5-bisphosphate regeneration (Jmax) also decreased, followed by substantial reductions in the quantum yield of PSII electron transport (FPSII). Additionally, root restriction resulted in accumulation of carbohydrates in various plant tissues irrespective of aeration conditions. It is likely that root restrictioninduced depression of photosynthesis was mimicked by water stress. ß 2008 Elsevier B.V. All rights reserved.
Keywords: Carbohydrates Chlorophyll fluorescence Leaf water potential Lycopersicon esculentum Photosynthesis Root restriction
1. Introduction Root restriction is a powerful approach to improve the utilizing efficiency of agricultural resources, and to control the size of shoot and partitioning of assimilates between vegetative and reproductive organs (Carmi, 1986). However, growing plant in a restricted rooting media frequently brings about a down-regulation in photosynthetic capacity (Pezeshki and Santos, 1998; Kharkina et al., 1999; Goto et al., 2002). To date, the mechanism by which root restriction reduces CO2 assimilation and photosynthetic productivity remains unclear. It is commonly interpreted as the imbalance in the supply and demand
* Corresponding author at: Department of Horticulture, Huajiachi Campus, Zhejiang University, Kaixuan Road 268, Hangzhou 310029, Zhejiang, PR China. Tel.: +86 57186971120; fax: +86 57186049815. E-mail address: [email protected] (J.-Q. Yu). Abbreviations: ABA, abscisic acid; Asat, light-saturated rate of the CO2 assimilation; Ci, intercellular CO2 concentration; F0 m, Fs, maximal and steady-state fluorescence yield in a light-adapted state; gs, stomatal conductance; Jmax, maximum potential rate of electron transport contributed to RuBP regeneration; l, stomatal limitation; Rubisco, ribulose-1,5-bisphosphate carboxylase/oxygenase; RuBP, ribulose-1,5-bisphosphate; Tr, transpiration rate; Vcmax, maximum carboxylation rate of Rubisco; FCO2 , quantum efficiency for CO2 assimilation; FPSII, relative quantum efficiency of PSII photochemistry. 0304-4238/$ – see front matter ß 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.scienta.2008.04.010
for carbohydrates, leading to feedback inhibition of photosynthesis by carbohydrates (Pezeshki and Santos, 1998; Kharkina et al., 1999). Decrease in the net CO2 assimilation rate in alder and cotton seedlings has also been found to be associated with stomatal closure caused by the internal water stress (Tschaplinski and Blake, 1985) and the decrease in the carboxylation efficiency of Calvin cycle (Thomas and Strain, 1991), respectively. In cucumber, root restriction resulted in a reduction in CO2 assimilation rate, followed by an accumulation of carbohydrates in leaves, and decreases in stomatal conductance and transpiration rate (Kharkina et al., 1999). Will and Teskey (1997), however, found that the decrease in photosynthesis of loblolly pine seedlings in small pots was not associated with increased starch or sugar concentrations. Apparently, there are species differences in term of their photosynthetic response to root restriction (Will and Teskey, 1997). Otherwise, it is worth to note that few studies have systematically measured the various processes involved in the photosynthesis. Accordingly, a simultaneous analysis of the photosynthetic processes is necessary before we could account for this discrepancy. It is well known that photosynthesis rate could be impaired by photosynthetic carbon fixation, thylakoid electron transport, stomatal limitation of CO2 supply, feedback inhibition by carbohydrate metabolism, and others (Sharkey, 1985; Lawlor and Cornic, 2002). In our previous study, we found
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that depressed shoot growth of tomato plants under root restriction condition was associated with O2 deficit in a hydroponic system (Shi et al., 2007). In this study, leaf gas exchange, chlorophyll fluorescence, water relations, xylem sap abscisic acid (ABA) concentration and carbohydrates status were analyzed to elucidate the mechanism by which root restriction affects the leaf photosynthesis of tomato plants. 2. Materials and methods 2.1. Plant material and cultural methods Tomato (Lycopersicon esculentum Mill. cv. Hezuo 903) seeds were sown in a growth medium containing a mixture of vermiculite and perlite (1:1, v:v) in trays in a greenhouse. Twenty days later, groups of six seedlings were transplanted into a 15-l tank (40 cm 25 cm 15 cm) filled with half-strength Enshi nutrient solution (Yu and Matsui, 1997). The solution was continuously aerated with an air pump. The greenhouse environmental conditions were as follows: a 12-h photoperiod, mean daily air temperature of 29 8C/17 8C (day/night), 70% relative humidity, and average photosynthetic photoflux density (PPFD) of 800 mmol m2 s1. After 1 week of preculture, seedlings were subjected to following three treatments: control, plants grown directly in the 15-l tank; RR treatment, roots of each plant were moved into a small cylinder which was suspended in the tank; RR + O2 treatment, in which cylinders for root-restricted plants were supplemented with additional aeration by an air pump. The small cylinders were made from nylon bags (mesh < 0.05 mm in diameter) with a capacity of 100 ml (3 cm in diameter, 14 cm in depth) and was sustained with a stainless steel skeleton, thus allowing solution exchanges but not root penetration. The rhizosphere oxygen concentration in the nutrient solution for control, RR and RR + O2 plants was ca. 248, 180, and 230 mM after 20 d of treatment (Shi et al., 2007). 2.2. Growth measurement On days 5, 10, 15, 20 and 25 after treatment, five plants from each treatment were sampled randomly for determination of biomass and leaf area. Total leaf area per plant was determined by measuring the length and width of each leaf and calculating leaf area using the equation of Schwarz and Kla¨ring (2001). Leaves, stems and roots were separated and dried to constant mass in an oven at 80 8C before the determination. 2.3. Leaf gas exchange and chlorophyll fluorescence analysis Leaf gas exchange measurements were coupled with measurements of chlorophyll fluorescence using an open gas exchange system (LI-6400; LI-COR, Inc., Lincoln, NE, USA) with an integrated fluorescence chamber head (LI-6400-40 leaf chamber fluorometer; LI-COR, Inc.) on the second fully developed leaves. For all cases, unless otherwise stated, gas exchange and chlorophyll fluorescence parameters were measured at 25 8C, 80% relative humidity, 1–1.3 kPa leaf-to-air vapor pressure deficit, 390 mmol mol1 CO2 and 1000 mmol m2 s1 incident PPFD, respectively. The quantum efficiency of CO2 fixation, FCO2 , was determined under non-photorespiratory conditions (2% O2) by dividing the rate of net CO2 assimilation rate (A) by the rate at which quanta were absorbed (Fryer et al., 1998). Leaf absorption was measured as described by Miyake and Yokota (2000). Responses of A versus intercellular CO2 concentration (Ci) (A/Ci) were made on at least four plants. The leaves were initially allowed to equilibrate at 390 mmol mol1 reference CO2, then the
CO2 concentration was reduced stepwise to 50 mmol mol1 and subsequently increased again from 390 in eight steps to 1800 mmol mol1 allowing 2–3 min for equilibration at each CO2 concentration. Estimation of maximum carboxylation rate of Rubisco (Vcmax) and maximum potential rate of electron transport contributed to RuBP regeneration (Jmax) were made according to Ethier and Livingston (2004). Stomatal limitation (l), which is the proportionate decrease in light-saturated net CO2 assimilation attributable to stomata, was calculated by the method of Farquhar and Sharkey (1982). Fluorescence parameters were calculated on the basis of the light-adapted fluorescence measurements. The quantum efficiency of PSII (FPSII) were calculated as ðF 0m F s Þ=F 0m (Genty et al., 1989). 2.4. Low oxygen measurements To estimate chloroplastic inorganic phosphate limitation, light saturated CO2 assimilation rate (Asat) was determined under both photorespiratory (21% O2) and non-photorespiratory (2% O2) conditions. Inorganic phosphate limitation is characterized by reduced sensitivity to O2 (Sage and Sharkey, 1987). 2.5. Xylem sap ABA analysis To collect xylem sap from the plants, the main stem was cut 2 cm above the root–shoot interface below the cotyledons. Then the procedure of Mencuccini et al. (2000) was used to maximize the chances that only true xylem sap was sampled. Sap from 3 or 4 plants was bulked for each sample and stored at 80 8C. Prior to further analysis of ABA concentration, the sap samples were thawed, purified, dried, and methylsilylanized with fresh N,Obis(trimethylsilyl)trifluoroacetamide (BSTFA) and pyridine before being analyzed by gas chromatography (Shimazu GC14B, Kyoto, Japan). A DB-1 capillary column (30 m 0.25 mm, J and W Scientific) was used for the analysis with N2 as carrier gas. 2.6. Leaf water potential determination Leaf water potential measurements were taken on the same leaf immediately after measuring gas exchange. Leaf discs (5.0 mm in diameter) were sampled and placed into a C-52 chamber (Wescor Inc., Logan, Utah, USA) connected to a dew-point microvoltmeter (HR-33T, from the same company). Water potential was determined after half an hour of thermal and water vapor equilibrium (Bahrun et al., 2002). 2.7. Carbohydrate concentration Carbohydrate concentrations in various tissues on 25 d were extracted from 200 mg of freeze-dried samples in 50 ml 80% (v/v) ethanol by five extraction steps and determined using anthrone (Yemm and Willis, 1954). The supernatant was analyzed for sucrose and soluble sugars, and the residue was boiled for 3 h in 10 ml 2% HCl (v/v) in order to hydrolyse starch as described in our early study (Zhou et al., 2004). 2.8. Statistical methods Measurements were performed on randomly samples from four replicates. SAS 8.0 (SAS Institute, Cary, NC) for windows was used for statistical analysis. The data were analyzed with a one-way analysis of variance and differences between treatment means were separated by Tukey’s test at the P < 0.05 level.
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the 253.1 23.0 and 250.8 7.17 mmol m3 in control and RR + O2 plants, respectively. 3.3. Effects of root restriction on leaf gas exchange RR treatment greatly reduced Asat from 15 d to the end of the experiment (Fig. 3A). The reduction in Asat was accompanied with reductions in stomatal conductance (gs) (Fig. 3B), intercellular CO2 concentration (Ci) (Fig. 3C), and transpiration rate (Tr) (Fig. 3D). However, these four parameters for RR + O2 plants were not significantly different from those of controls (Fig. 3). The response of net CO2 assimilation rate to changes in Ci was examined and analyses of A/Ci curves allowed the determination of Vcmax, Jmax, and l (Fig. 4). RR plants showed significant reductions in Vcmax from 20 to 25 d and in Jmax on 25 d (Fig. 4A and B). The l of RR treatment was significantly increased from 15 d to the end of the experiment (Fig. 4C).
Fig. 1. Changes in root dry mass (A), shoot dry mass (B), leaf area (C), and specific leaf area (D) for control (open circles), root restriction (RR, open triangles) and root restriction + O2 (RR + O2, filled triangles) plants. Data are the means of four replicates with standard deviations shown by vertical bars.
3. Results 3.1. Effects of root restriction on plant growth Root restriction treatment resulted in reduced plant biomass, leaf area, and the specific leaf area (Fig. 1). This effect was manifest 10 d after root confinement. However, suppression of plant growth by root restriction was alleviated to some extent by aeration (RR + O2). Root and shoot dry mass were 93.4% and 43.8% higher than those of RR by the final harvest. 3.2. Effects of root restriction on leaf water potential and xylem sap ABA concentration
3.4. Effects of root restriction on inorganic phosphate limitation and carbohydrate concentrations The possibility that root restriction-induced decrease in Asat were associated with changes in photorespiration or limitations in inorganic phosphate supply to the chloroplasts was also examined by changing the atmospheric O2 concentration from 21% to 2% (i.e. non-photorespiratory condition) (Table 1). Increases (between 11% and 51%) were observed in Asat measured at low O2 partial pressure in all measurements regardless of the treatment. RR treatment had no significant effect on sucrose or starch concentration in the roots (Fig. 5A and C), but it significantly increased sucrose, total soluble sugars and starch concentrations in stems and leaves at the final harvest (Fig. 5). Sucrose and total soluble sugars concentrations for RR + O2 plants were similar to those of RR plants for all the three plant organs expressed either on plant dry weight basis or on leaf area basis. RR + O2 plants, however, showed significantly higher starch concentration than RR plants in roots, stems, and leaves when expressed on a dry weight basis.
The change in leaf water potential is shown in Fig. 2. Initially, all plants showed similar leaf water potential. However, from 15 d onward, a sharp and significant decline of leaf water potential was observed in RR plants, while for control and RR + O2 treatments, it remained constant throughout the experiment. Interestingly, xylem sap ABA concentration was also significantly higher for RR plants on day 15, it was 696.1 81.4 mmol m3 compared with
Fig. 2. Change in the leaf water potential for control (open circles), root restriction (RR, open triangles) and root restriction + O2 (RR + O2, filled triangles) plants. Data are the means of four replicates with standard deviations shown by vertical bars.
Fig. 3. Changes in light-saturated rate of CO2 assimilation (Asat, A), stomatal conductance (gs, B), intercellular CO2 concentration (Ci, C), and transpiration rate (Tr, D) for control (open circles), root restriction (RR, open triangles) and root restriction + O2 (RR + O2, filled triangles) plants. Leaf temperature was maintained at 25 8C with 390 mmol mol1 CO2 and 1000 mmol m2 s1 incident PPFD for the gas exchange measurement. Data are shown as the means of four replicates and the standard deviations are shown when larger than the symbols.
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Fig. 4. Changes in maximum carboxylation rate of Rubisco (Vcmax, A), maximum potential rate of electron transport contributed to RuBP regeneration (Jmax, B), stomatal limitation (l, C) for control (open circles), root restriction (RR, open triangles) and root restriction + O2 (RR + O2, filled triangles) plants. Leaf temperature was maintained at 25 8C with 390 mmol mol1 CO2 and 1000 mmol m2 s1 incident PPFD for the gas exchange measurement. Data are the means of four replicates with standard deviations shown by vertical bars. Fig. 5. Concentrations of sucrose (A), total soluble sugar (B), starch (C) and leaf carbohydrate content on leaf area basis (D) in control, root restriction (RR), root restriction + O2 (RR + O2) plants 25 d after treatment. Data are the means of four replicates with standard deviations shown by vertical bars. Different letters are significantly different between the treatments at 5% level according to Tukey’s test.
3.5. Effects of root restriction on leaf chlorophyll fluorescence parameters Root restriction had little effects on the maximal photochemical efficiency of PSII (Fv/Fm, data not shown). Meanwhile, quantum yield of PSII (FPSII) for all plant groups did not differ from each other during the initial phase of root restriction stress (Fig. 6A).
However, on 25 d, FPSII for RR plants declined by 31.4% as compared to that of control leaves. Furthermore, RR treatment significantly increased the value of FPSII =FCO2 from 15 to 25 d
Table 1 Effects of root restriction on light-saturated rate of the CO2 assimilation (Asat) in tomato leaves at an O2 partial pressure of either 21% or 2% with 1000 mmol m2 s1 PPFD Time (d)
Control
Root restriction
21% O2 5 10 15 20 25
27.1 1.0 23.7 2.4 25.2 0.5 21.1 1.7 21.9 0.9
2% O2 b a b a b
35.9 1.1 26.3 2.5 29.6 0.8 24.6 2.2 27.7 1.0
21% O2 a a a a a
25.0 2.2 25.5 2.3 14.6 2.4 16.2 0.9 10.8 1.7
Root restriction + O2 2% O2
a a b b b
32.3 5.4 29.6 3.4 19.5 2.5 20.1 0.5 16.2 3.0
21% O2 a a a a a
27.0 1.7 25.5 0.5 24.6 2.9 20.2 1.4 20.1 1.9
2% O2 b b a b b
Values are the means S.D. (n = 4). Values followed by same letters in a treatment are not significantly different according to Tukey’s tests at 5% level.
33.9 3.3 28.4 0.5 29.5 4.1 24.1 0.7 24.8 2.0
a a a a a
K. Shi et al. / Scientia Horticulturae 117 (2008) 197–202
Fig. 6. Changes in the quantum efficiency of PSII (FPSII, A) and the ratio of quantum efficiency of PSII to the maximum apparent quantum efficiency for CO2 assimilation (FPSII =FCO2 , B) for control (open circles), root restriction (RR, open triangles) and root restriction + O2 (RR + O2, filled triangles) plants. Leaf temperature was maintained at 25 8C with 390 mmol mol1 CO2 and 1000 mmol m2 s1 incident PPFD for the gas exchange and chlorophyll fluorescence measurement. Data are the means of four replicates with standard deviations shown by vertical bars.
(Fig. 6B). For RR + O2 plants, the values of chlorophyll fluorescence parameters were close to those of control levels (Fig. 6). 4. Discussion Reduced photosynthesis as a result of root restriction is a wellrecognized phenomenon (Goto et al., 2002). In agreement with previous reports, this study showed that Asat significantly decreased as root restriction progressed. The decline in photosynthesis in root-restricted condition was often interpreted as feedback inhibition by carbohydrate accumulation (Pezeshki and Santos, 1998). In this study, carbohydrate levels in various tissues for the RR + O2 plants was several-fold larger than that in control plants while the Asat was similar to the controls. Accordingly, it seems illogical to attribute the reduction in Asat for RR treatment to carbohydrate feedback inhibition since the carbohydrates accumulated in these plants were similar or even significantly lower (e.g. the starch concentration expressed on dry weight basis) than the RR + O2 plants (Figs. 3 and 5). Changes in CO2 assimilation may be attributable to either stomatal or non-stomatal factors or both. In this study, the reduction in CO2 assimilation rates in RR plants from 15 d onward was largely dependent on stomatal factors. RR treatment reduced gs and Tr which were accompanied by a significant decrease of Ci and increase of l (Figs. 3 and 4C). The l was highly correlated with decreased leaf water potential (Fig. 2). All these results suggest that the decrease in Asat of RR plants was mimicked by water stress. It has been demonstrated that root restriction increased the resistance to water movement as a consequence of increased root hydraulic resistance (Hameed et al., 1987). The increased xylem sap ABA concentration in RR plants, might participate in regulating the stomatal conductance to prevent large losses of water (Fig. 3B and D) (Bahrun et al., 2002). In accordance with this, Will and Teskey (1997) also found that the reduction in photosynthesis of loblolly pine seedlings in small pots was closely correlated with decreased needle water potentials. The percentage decrease of Ci and Asat in RR plants was proportional on 15 and 20 d but not for 25 d. On 25 d, RR treatment decreased Ci by 15.8% while Asat was decreased by 50.8%. This suggests the occurrence of some transient non-stomatal (namely mesophyll) limitation of photosynthesis in the late stage of root restriction stress. In the present work, root restriction did not reduce the sensitivity of Asat to O2 concentration, indicating that Pilimitation could not play a role in the decrease in photosynthesis observed in RR leaves (Table 1). Meanwhile, there was no evidence to support the notion that photorespiration caused the photosynthesis reduction as the loss of Rubisco carboxylation (Fig. 4) is
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mirrored by a decline in oxygenase activity and therefore a reduction in photorespiration (Allen and Ort, 2001). At the later stages of the experiment, reductions in Asat of RR plants were accompanied by large decreases both in Vcmax and Jmax (Fig. 4). This is in accordance with the observation made by Thomas and Strain (1991) on cotton seedlings in which they found that there was a significant decrease in carboxylation efficiency and RuBP regeneration capacity in plants grown in a small pot. Inactivation or loss of Rubisco would reduce the carboxylation efficiency and accounts for decrease in Vcmax (Nogue´s and Baker, 2000). The decrease of Jmax on 25 d could be associated with a diminution of other Calvin-cycle enzymes, e.g. sedoheptulose-1,7bisphosphate, a key regulatory enzyme of the Calvin cycle (Nogue´s and Baker, 2000). Following the decrease of Vcmax, RR treatment also significantly decreased FPSII (Fig. 6A) Accordingly, the reduced FPSII in response to root restriction stress is attributed to downregulation of electron transport to match the lower demand for ATP and NADPH which was indicated by the decreased Vcmax and Jmax. RR treatment decreased Asat from 15 d onward (Fig. 3), but did not significantly affect FPSII (Fig. 6) during the first 20 d of the experiment. This suggests that the rate of non-cyclic electron transport is higher than that required to maintain CO2 assimilation. The large increase in FPSII =FCO2 observed in leaves of RR treatment (Fig. 6B) suggested that root restriction increased the rate of photosynthetic electron transport to alternative electron sinks such as nitrate reaction and Mehler reactions to protect the photosynthetic apparatus from photoinhibition (Brestic et al., 1995). Even though the photosynthetic capacity of RR + O2 plants was not affected, the biomass production of these plants was depressed (Figs. 1 and 3) due to the limited space for root growth. The reduction in the leaf area could be associated with a decreased ability of plants to capture photosynthetically active radiation and, consequently, be one of the main factors in determining the decreased photosynthetic productivity of these plants. Since reductions in the shoot biomass production and leaf area occurred earlier than that in Asat (Fig. 1C), factors other than photosynthesis were also involved in the growth reduction for the RR plants. Additionally, there was a slight drop of plant growth on day 15. This might be due to the poor light condition at that time and so was also true for Asat, Ci and gs, especially in RR plants (Figs. 1 and 3). In conclusion, this study has shown that the limitation to leaf photosynthesis by root restriction is mimicked by water stress. The decrease in CO2 assimilation is primarily due to the stomatal limitation. As the root restriction stress progressed, the capacity of CO2 assimilation in the Calvin cycle was also affected. Acknowledgements We thank Dr. Demetriades-Shah of Li-Cor for his critical reading of the manuscript. This work was supported by the National Natural Science Foundation of China (40571083), National Key Project of Scientific and Technical Supporting Programs Funded by Ministry of Science & Technology of China (2006BAD07B02, 2006BAD07B03; 2008BADA6B02. References Allen, D.J., Ort, D.R., 2001. Impact of chilling temperatures on photosynthesis in warm climate plants. Trends Plant Sci. 6, 36–42. Bahrun, A., Jensen, C.R., Asch, F., Mogensen, V.O., 2002. Drought-induced changes in xylem pH, ionic composition, and ABA concentration act as early signals in fieldgrown maize (Zea mays L.). J. Exp. Bot. 53, 251–263. Brestic, M., Cornic, G., Fryer, M.J., Baker, N.R., 1995. Does photorespiration protect the photosynthetic apparatus in French bean leaves from photoinhibition during drought stress? Planta 196, 450–457.
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Carmi, A., 1986. Effects of root zone volume and plant density on the vegetative and reproductive development of cotton. Field Crops Res. 13, 25–32. Ethier, G.J., Livingston, N.J., 2004. On the need to incorporate sensitivity to CO2 transfer conductance into the Farquhar-von Caemmerer-Berry leaf photosynthesis model. Plant Cell Environ. 27, 137–153. Farquhar, G.D., Sharkey, T.D., 1982. Stomatal conductance and photosynthesis. Annu. Rev. Plant Physiol. 33, 317–345. Fryer, M.J., Andrews, J.R., Oxborough, K., Blowers, D.A., Baker, N.R., 1998. Relationship between CO2 assimilation, photosynthetic electron transport, and active O2 metabolism in leaves of maize in the field during period of low temperature. Plant Physiol. 116, 571–580. Genty, B., Briantais, J.M., Baker, N.R., 1989. The relationship between the quantum yield of photosynthetic electron transport and quenching of chlorophyll fluorescence. Biochim. Biophys. Acta 990, 87–92. Goto, T., Matsuno, T., Yoshida, Y., Kageyama, Y., 2002. Photosynthetic, evapotranspiratory and leaf morphological properties of chrysanthemum grown under root restriction as affected by fertigation frequency. J. Jpn. Soc. Hortic. Sci. 71, 277–283. Hameed, M.A., Reid, J.B., Rowe, R.N., 1987. Root confinement and its effects on the water relations, growth and assimilate partitioning of tomato (Lycopersicon esculentum Mill.). Ann. Bot. 59, 685–692. Kharkina, T.G., Ottosen, C.O., Rosenqvist, E., 1999. Effects of root restriction on the growth and physiology of cucumber plants. Physiol. Plant. 105, 434–441. Lawlor, D.W., Cornic, G., 2002. Photosynthetic carbon assimilation and associated metabolism in relation to water deficits in higher plants. Plant Cell Environ. 25, 275–294. Mencuccini, M., Mambelli, S., Comstock, J., 2000. Stomatal responsiveness to leaf water status in common bean (Phaseolus vulgaris L.) is a function of time of day. Plant Cell Environ. 23, 1109–1118. Miyake, C., Yokota, A., 2000. Determination of the rate of photoreduction of O2 in the water–water cycle in watermelon leaves and enhancement of the rate by limitation of photosynthesis. Plant Cell Physiol. 41, 335–343.
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Contents lists available at ScienceDirect
Scientia Horticulturae journal homepage: www.elsevier.com/locate/scihorti
Root restriction-induced limitation to photosynthesis in tomato (Lycopersicon esculentum Mill.) leaves Kai Shi a, Xiao-Tao Ding a, De-Kun Dong c, Yan-Hong Zhou a, Jing-Quan Yu a,b,* a
Department of Horticulture, Huajiachi Campus, Zhejiang University, Kaixuan Road 268, Hangzhou 310029, PR China Key Laboratory of Horticultural Plants Growth, Development and Biotechnology, Agricultural Ministry of China, Kaixuan Road 268, Hangzhou 310029, PR China c The Institute of Crop and Nuclear Technology Utilization, Zhejiang Academy of Agricultural Sciences, Hangzhou 310021, PR China b
A R T I C L E I N F O
A B S T R A C T
Article history: Received 12 February 2007 Received in revised form 19 April 2008 Accepted 30 April 2008
Root restriction often depresses photosynthetic capacity and the mechanism for this reduction, however, remains unclear. To identify the mechanism by which root restriction affects the photosynthetic characteristics, tomato (Lycopersicon esculentum Mill.) seedlings were subjected to root restriction stress with or without supplemental aeration to the nutrient solution. With the development of the root restriction stress, CO2 assimilation rate was decreased only in confined plants without supplemental aeration. There were also significant decreases in leaf water potential, stomatal conductance (gs), intercellular CO2 concentration (Ci), and increases in the stomatal limitation (l) and the xylem sap ABA concentration. Meanwhile, the maximum carboxylation rate of Rubisco (Vcmax) and the capacity for ribulose-1,5-bisphosphate regeneration (Jmax) also decreased, followed by substantial reductions in the quantum yield of PSII electron transport (FPSII). Additionally, root restriction resulted in accumulation of carbohydrates in various plant tissues irrespective of aeration conditions. It is likely that root restrictioninduced depression of photosynthesis was mimicked by water stress. ß 2008 Elsevier B.V. All rights reserved.
Keywords: Carbohydrates Chlorophyll fluorescence Leaf water potential Lycopersicon esculentum Photosynthesis Root restriction
1. Introduction Root restriction is a powerful approach to improve the utilizing efficiency of agricultural resources, and to control the size of shoot and partitioning of assimilates between vegetative and reproductive organs (Carmi, 1986). However, growing plant in a restricted rooting media frequently brings about a down-regulation in photosynthetic capacity (Pezeshki and Santos, 1998; Kharkina et al., 1999; Goto et al., 2002). To date, the mechanism by which root restriction reduces CO2 assimilation and photosynthetic productivity remains unclear. It is commonly interpreted as the imbalance in the supply and demand
* Corresponding author at: Department of Horticulture, Huajiachi Campus, Zhejiang University, Kaixuan Road 268, Hangzhou 310029, Zhejiang, PR China. Tel.: +86 57186971120; fax: +86 57186049815. E-mail address: [email protected] (J.-Q. Yu). Abbreviations: ABA, abscisic acid; Asat, light-saturated rate of the CO2 assimilation; Ci, intercellular CO2 concentration; F0 m, Fs, maximal and steady-state fluorescence yield in a light-adapted state; gs, stomatal conductance; Jmax, maximum potential rate of electron transport contributed to RuBP regeneration; l, stomatal limitation; Rubisco, ribulose-1,5-bisphosphate carboxylase/oxygenase; RuBP, ribulose-1,5-bisphosphate; Tr, transpiration rate; Vcmax, maximum carboxylation rate of Rubisco; FCO2 , quantum efficiency for CO2 assimilation; FPSII, relative quantum efficiency of PSII photochemistry. 0304-4238/$ – see front matter ß 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.scienta.2008.04.010
for carbohydrates, leading to feedback inhibition of photosynthesis by carbohydrates (Pezeshki and Santos, 1998; Kharkina et al., 1999). Decrease in the net CO2 assimilation rate in alder and cotton seedlings has also been found to be associated with stomatal closure caused by the internal water stress (Tschaplinski and Blake, 1985) and the decrease in the carboxylation efficiency of Calvin cycle (Thomas and Strain, 1991), respectively. In cucumber, root restriction resulted in a reduction in CO2 assimilation rate, followed by an accumulation of carbohydrates in leaves, and decreases in stomatal conductance and transpiration rate (Kharkina et al., 1999). Will and Teskey (1997), however, found that the decrease in photosynthesis of loblolly pine seedlings in small pots was not associated with increased starch or sugar concentrations. Apparently, there are species differences in term of their photosynthetic response to root restriction (Will and Teskey, 1997). Otherwise, it is worth to note that few studies have systematically measured the various processes involved in the photosynthesis. Accordingly, a simultaneous analysis of the photosynthetic processes is necessary before we could account for this discrepancy. It is well known that photosynthesis rate could be impaired by photosynthetic carbon fixation, thylakoid electron transport, stomatal limitation of CO2 supply, feedback inhibition by carbohydrate metabolism, and others (Sharkey, 1985; Lawlor and Cornic, 2002). In our previous study, we found
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that depressed shoot growth of tomato plants under root restriction condition was associated with O2 deficit in a hydroponic system (Shi et al., 2007). In this study, leaf gas exchange, chlorophyll fluorescence, water relations, xylem sap abscisic acid (ABA) concentration and carbohydrates status were analyzed to elucidate the mechanism by which root restriction affects the leaf photosynthesis of tomato plants. 2. Materials and methods 2.1. Plant material and cultural methods Tomato (Lycopersicon esculentum Mill. cv. Hezuo 903) seeds were sown in a growth medium containing a mixture of vermiculite and perlite (1:1, v:v) in trays in a greenhouse. Twenty days later, groups of six seedlings were transplanted into a 15-l tank (40 cm 25 cm 15 cm) filled with half-strength Enshi nutrient solution (Yu and Matsui, 1997). The solution was continuously aerated with an air pump. The greenhouse environmental conditions were as follows: a 12-h photoperiod, mean daily air temperature of 29 8C/17 8C (day/night), 70% relative humidity, and average photosynthetic photoflux density (PPFD) of 800 mmol m2 s1. After 1 week of preculture, seedlings were subjected to following three treatments: control, plants grown directly in the 15-l tank; RR treatment, roots of each plant were moved into a small cylinder which was suspended in the tank; RR + O2 treatment, in which cylinders for root-restricted plants were supplemented with additional aeration by an air pump. The small cylinders were made from nylon bags (mesh < 0.05 mm in diameter) with a capacity of 100 ml (3 cm in diameter, 14 cm in depth) and was sustained with a stainless steel skeleton, thus allowing solution exchanges but not root penetration. The rhizosphere oxygen concentration in the nutrient solution for control, RR and RR + O2 plants was ca. 248, 180, and 230 mM after 20 d of treatment (Shi et al., 2007). 2.2. Growth measurement On days 5, 10, 15, 20 and 25 after treatment, five plants from each treatment were sampled randomly for determination of biomass and leaf area. Total leaf area per plant was determined by measuring the length and width of each leaf and calculating leaf area using the equation of Schwarz and Kla¨ring (2001). Leaves, stems and roots were separated and dried to constant mass in an oven at 80 8C before the determination. 2.3. Leaf gas exchange and chlorophyll fluorescence analysis Leaf gas exchange measurements were coupled with measurements of chlorophyll fluorescence using an open gas exchange system (LI-6400; LI-COR, Inc., Lincoln, NE, USA) with an integrated fluorescence chamber head (LI-6400-40 leaf chamber fluorometer; LI-COR, Inc.) on the second fully developed leaves. For all cases, unless otherwise stated, gas exchange and chlorophyll fluorescence parameters were measured at 25 8C, 80% relative humidity, 1–1.3 kPa leaf-to-air vapor pressure deficit, 390 mmol mol1 CO2 and 1000 mmol m2 s1 incident PPFD, respectively. The quantum efficiency of CO2 fixation, FCO2 , was determined under non-photorespiratory conditions (2% O2) by dividing the rate of net CO2 assimilation rate (A) by the rate at which quanta were absorbed (Fryer et al., 1998). Leaf absorption was measured as described by Miyake and Yokota (2000). Responses of A versus intercellular CO2 concentration (Ci) (A/Ci) were made on at least four plants. The leaves were initially allowed to equilibrate at 390 mmol mol1 reference CO2, then the
CO2 concentration was reduced stepwise to 50 mmol mol1 and subsequently increased again from 390 in eight steps to 1800 mmol mol1 allowing 2–3 min for equilibration at each CO2 concentration. Estimation of maximum carboxylation rate of Rubisco (Vcmax) and maximum potential rate of electron transport contributed to RuBP regeneration (Jmax) were made according to Ethier and Livingston (2004). Stomatal limitation (l), which is the proportionate decrease in light-saturated net CO2 assimilation attributable to stomata, was calculated by the method of Farquhar and Sharkey (1982). Fluorescence parameters were calculated on the basis of the light-adapted fluorescence measurements. The quantum efficiency of PSII (FPSII) were calculated as ðF 0m F s Þ=F 0m (Genty et al., 1989). 2.4. Low oxygen measurements To estimate chloroplastic inorganic phosphate limitation, light saturated CO2 assimilation rate (Asat) was determined under both photorespiratory (21% O2) and non-photorespiratory (2% O2) conditions. Inorganic phosphate limitation is characterized by reduced sensitivity to O2 (Sage and Sharkey, 1987). 2.5. Xylem sap ABA analysis To collect xylem sap from the plants, the main stem was cut 2 cm above the root–shoot interface below the cotyledons. Then the procedure of Mencuccini et al. (2000) was used to maximize the chances that only true xylem sap was sampled. Sap from 3 or 4 plants was bulked for each sample and stored at 80 8C. Prior to further analysis of ABA concentration, the sap samples were thawed, purified, dried, and methylsilylanized with fresh N,Obis(trimethylsilyl)trifluoroacetamide (BSTFA) and pyridine before being analyzed by gas chromatography (Shimazu GC14B, Kyoto, Japan). A DB-1 capillary column (30 m 0.25 mm, J and W Scientific) was used for the analysis with N2 as carrier gas. 2.6. Leaf water potential determination Leaf water potential measurements were taken on the same leaf immediately after measuring gas exchange. Leaf discs (5.0 mm in diameter) were sampled and placed into a C-52 chamber (Wescor Inc., Logan, Utah, USA) connected to a dew-point microvoltmeter (HR-33T, from the same company). Water potential was determined after half an hour of thermal and water vapor equilibrium (Bahrun et al., 2002). 2.7. Carbohydrate concentration Carbohydrate concentrations in various tissues on 25 d were extracted from 200 mg of freeze-dried samples in 50 ml 80% (v/v) ethanol by five extraction steps and determined using anthrone (Yemm and Willis, 1954). The supernatant was analyzed for sucrose and soluble sugars, and the residue was boiled for 3 h in 10 ml 2% HCl (v/v) in order to hydrolyse starch as described in our early study (Zhou et al., 2004). 2.8. Statistical methods Measurements were performed on randomly samples from four replicates. SAS 8.0 (SAS Institute, Cary, NC) for windows was used for statistical analysis. The data were analyzed with a one-way analysis of variance and differences between treatment means were separated by Tukey’s test at the P < 0.05 level.
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the 253.1 23.0 and 250.8 7.17 mmol m3 in control and RR + O2 plants, respectively. 3.3. Effects of root restriction on leaf gas exchange RR treatment greatly reduced Asat from 15 d to the end of the experiment (Fig. 3A). The reduction in Asat was accompanied with reductions in stomatal conductance (gs) (Fig. 3B), intercellular CO2 concentration (Ci) (Fig. 3C), and transpiration rate (Tr) (Fig. 3D). However, these four parameters for RR + O2 plants were not significantly different from those of controls (Fig. 3). The response of net CO2 assimilation rate to changes in Ci was examined and analyses of A/Ci curves allowed the determination of Vcmax, Jmax, and l (Fig. 4). RR plants showed significant reductions in Vcmax from 20 to 25 d and in Jmax on 25 d (Fig. 4A and B). The l of RR treatment was significantly increased from 15 d to the end of the experiment (Fig. 4C).
Fig. 1. Changes in root dry mass (A), shoot dry mass (B), leaf area (C), and specific leaf area (D) for control (open circles), root restriction (RR, open triangles) and root restriction + O2 (RR + O2, filled triangles) plants. Data are the means of four replicates with standard deviations shown by vertical bars.
3. Results 3.1. Effects of root restriction on plant growth Root restriction treatment resulted in reduced plant biomass, leaf area, and the specific leaf area (Fig. 1). This effect was manifest 10 d after root confinement. However, suppression of plant growth by root restriction was alleviated to some extent by aeration (RR + O2). Root and shoot dry mass were 93.4% and 43.8% higher than those of RR by the final harvest. 3.2. Effects of root restriction on leaf water potential and xylem sap ABA concentration
3.4. Effects of root restriction on inorganic phosphate limitation and carbohydrate concentrations The possibility that root restriction-induced decrease in Asat were associated with changes in photorespiration or limitations in inorganic phosphate supply to the chloroplasts was also examined by changing the atmospheric O2 concentration from 21% to 2% (i.e. non-photorespiratory condition) (Table 1). Increases (between 11% and 51%) were observed in Asat measured at low O2 partial pressure in all measurements regardless of the treatment. RR treatment had no significant effect on sucrose or starch concentration in the roots (Fig. 5A and C), but it significantly increased sucrose, total soluble sugars and starch concentrations in stems and leaves at the final harvest (Fig. 5). Sucrose and total soluble sugars concentrations for RR + O2 plants were similar to those of RR plants for all the three plant organs expressed either on plant dry weight basis or on leaf area basis. RR + O2 plants, however, showed significantly higher starch concentration than RR plants in roots, stems, and leaves when expressed on a dry weight basis.
The change in leaf water potential is shown in Fig. 2. Initially, all plants showed similar leaf water potential. However, from 15 d onward, a sharp and significant decline of leaf water potential was observed in RR plants, while for control and RR + O2 treatments, it remained constant throughout the experiment. Interestingly, xylem sap ABA concentration was also significantly higher for RR plants on day 15, it was 696.1 81.4 mmol m3 compared with
Fig. 2. Change in the leaf water potential for control (open circles), root restriction (RR, open triangles) and root restriction + O2 (RR + O2, filled triangles) plants. Data are the means of four replicates with standard deviations shown by vertical bars.
Fig. 3. Changes in light-saturated rate of CO2 assimilation (Asat, A), stomatal conductance (gs, B), intercellular CO2 concentration (Ci, C), and transpiration rate (Tr, D) for control (open circles), root restriction (RR, open triangles) and root restriction + O2 (RR + O2, filled triangles) plants. Leaf temperature was maintained at 25 8C with 390 mmol mol1 CO2 and 1000 mmol m2 s1 incident PPFD for the gas exchange measurement. Data are shown as the means of four replicates and the standard deviations are shown when larger than the symbols.
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Fig. 4. Changes in maximum carboxylation rate of Rubisco (Vcmax, A), maximum potential rate of electron transport contributed to RuBP regeneration (Jmax, B), stomatal limitation (l, C) for control (open circles), root restriction (RR, open triangles) and root restriction + O2 (RR + O2, filled triangles) plants. Leaf temperature was maintained at 25 8C with 390 mmol mol1 CO2 and 1000 mmol m2 s1 incident PPFD for the gas exchange measurement. Data are the means of four replicates with standard deviations shown by vertical bars. Fig. 5. Concentrations of sucrose (A), total soluble sugar (B), starch (C) and leaf carbohydrate content on leaf area basis (D) in control, root restriction (RR), root restriction + O2 (RR + O2) plants 25 d after treatment. Data are the means of four replicates with standard deviations shown by vertical bars. Different letters are significantly different between the treatments at 5% level according to Tukey’s test.
3.5. Effects of root restriction on leaf chlorophyll fluorescence parameters Root restriction had little effects on the maximal photochemical efficiency of PSII (Fv/Fm, data not shown). Meanwhile, quantum yield of PSII (FPSII) for all plant groups did not differ from each other during the initial phase of root restriction stress (Fig. 6A).
However, on 25 d, FPSII for RR plants declined by 31.4% as compared to that of control leaves. Furthermore, RR treatment significantly increased the value of FPSII =FCO2 from 15 to 25 d
Table 1 Effects of root restriction on light-saturated rate of the CO2 assimilation (Asat) in tomato leaves at an O2 partial pressure of either 21% or 2% with 1000 mmol m2 s1 PPFD Time (d)
Control
Root restriction
21% O2 5 10 15 20 25
27.1 1.0 23.7 2.4 25.2 0.5 21.1 1.7 21.9 0.9
2% O2 b a b a b
35.9 1.1 26.3 2.5 29.6 0.8 24.6 2.2 27.7 1.0
21% O2 a a a a a
25.0 2.2 25.5 2.3 14.6 2.4 16.2 0.9 10.8 1.7
Root restriction + O2 2% O2
a a b b b
32.3 5.4 29.6 3.4 19.5 2.5 20.1 0.5 16.2 3.0
21% O2 a a a a a
27.0 1.7 25.5 0.5 24.6 2.9 20.2 1.4 20.1 1.9
2% O2 b b a b b
Values are the means S.D. (n = 4). Values followed by same letters in a treatment are not significantly different according to Tukey’s tests at 5% level.
33.9 3.3 28.4 0.5 29.5 4.1 24.1 0.7 24.8 2.0
a a a a a
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Fig. 6. Changes in the quantum efficiency of PSII (FPSII, A) and the ratio of quantum efficiency of PSII to the maximum apparent quantum efficiency for CO2 assimilation (FPSII =FCO2 , B) for control (open circles), root restriction (RR, open triangles) and root restriction + O2 (RR + O2, filled triangles) plants. Leaf temperature was maintained at 25 8C with 390 mmol mol1 CO2 and 1000 mmol m2 s1 incident PPFD for the gas exchange and chlorophyll fluorescence measurement. Data are the means of four replicates with standard deviations shown by vertical bars.
(Fig. 6B). For RR + O2 plants, the values of chlorophyll fluorescence parameters were close to those of control levels (Fig. 6). 4. Discussion Reduced photosynthesis as a result of root restriction is a wellrecognized phenomenon (Goto et al., 2002). In agreement with previous reports, this study showed that Asat significantly decreased as root restriction progressed. The decline in photosynthesis in root-restricted condition was often interpreted as feedback inhibition by carbohydrate accumulation (Pezeshki and Santos, 1998). In this study, carbohydrate levels in various tissues for the RR + O2 plants was several-fold larger than that in control plants while the Asat was similar to the controls. Accordingly, it seems illogical to attribute the reduction in Asat for RR treatment to carbohydrate feedback inhibition since the carbohydrates accumulated in these plants were similar or even significantly lower (e.g. the starch concentration expressed on dry weight basis) than the RR + O2 plants (Figs. 3 and 5). Changes in CO2 assimilation may be attributable to either stomatal or non-stomatal factors or both. In this study, the reduction in CO2 assimilation rates in RR plants from 15 d onward was largely dependent on stomatal factors. RR treatment reduced gs and Tr which were accompanied by a significant decrease of Ci and increase of l (Figs. 3 and 4C). The l was highly correlated with decreased leaf water potential (Fig. 2). All these results suggest that the decrease in Asat of RR plants was mimicked by water stress. It has been demonstrated that root restriction increased the resistance to water movement as a consequence of increased root hydraulic resistance (Hameed et al., 1987). The increased xylem sap ABA concentration in RR plants, might participate in regulating the stomatal conductance to prevent large losses of water (Fig. 3B and D) (Bahrun et al., 2002). In accordance with this, Will and Teskey (1997) also found that the reduction in photosynthesis of loblolly pine seedlings in small pots was closely correlated with decreased needle water potentials. The percentage decrease of Ci and Asat in RR plants was proportional on 15 and 20 d but not for 25 d. On 25 d, RR treatment decreased Ci by 15.8% while Asat was decreased by 50.8%. This suggests the occurrence of some transient non-stomatal (namely mesophyll) limitation of photosynthesis in the late stage of root restriction stress. In the present work, root restriction did not reduce the sensitivity of Asat to O2 concentration, indicating that Pilimitation could not play a role in the decrease in photosynthesis observed in RR leaves (Table 1). Meanwhile, there was no evidence to support the notion that photorespiration caused the photosynthesis reduction as the loss of Rubisco carboxylation (Fig. 4) is
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mirrored by a decline in oxygenase activity and therefore a reduction in photorespiration (Allen and Ort, 2001). At the later stages of the experiment, reductions in Asat of RR plants were accompanied by large decreases both in Vcmax and Jmax (Fig. 4). This is in accordance with the observation made by Thomas and Strain (1991) on cotton seedlings in which they found that there was a significant decrease in carboxylation efficiency and RuBP regeneration capacity in plants grown in a small pot. Inactivation or loss of Rubisco would reduce the carboxylation efficiency and accounts for decrease in Vcmax (Nogue´s and Baker, 2000). The decrease of Jmax on 25 d could be associated with a diminution of other Calvin-cycle enzymes, e.g. sedoheptulose-1,7bisphosphate, a key regulatory enzyme of the Calvin cycle (Nogue´s and Baker, 2000). Following the decrease of Vcmax, RR treatment also significantly decreased FPSII (Fig. 6A) Accordingly, the reduced FPSII in response to root restriction stress is attributed to downregulation of electron transport to match the lower demand for ATP and NADPH which was indicated by the decreased Vcmax and Jmax. RR treatment decreased Asat from 15 d onward (Fig. 3), but did not significantly affect FPSII (Fig. 6) during the first 20 d of the experiment. This suggests that the rate of non-cyclic electron transport is higher than that required to maintain CO2 assimilation. The large increase in FPSII =FCO2 observed in leaves of RR treatment (Fig. 6B) suggested that root restriction increased the rate of photosynthetic electron transport to alternative electron sinks such as nitrate reaction and Mehler reactions to protect the photosynthetic apparatus from photoinhibition (Brestic et al., 1995). Even though the photosynthetic capacity of RR + O2 plants was not affected, the biomass production of these plants was depressed (Figs. 1 and 3) due to the limited space for root growth. The reduction in the leaf area could be associated with a decreased ability of plants to capture photosynthetically active radiation and, consequently, be one of the main factors in determining the decreased photosynthetic productivity of these plants. Since reductions in the shoot biomass production and leaf area occurred earlier than that in Asat (Fig. 1C), factors other than photosynthesis were also involved in the growth reduction for the RR plants. Additionally, there was a slight drop of plant growth on day 15. This might be due to the poor light condition at that time and so was also true for Asat, Ci and gs, especially in RR plants (Figs. 1 and 3). In conclusion, this study has shown that the limitation to leaf photosynthesis by root restriction is mimicked by water stress. The decrease in CO2 assimilation is primarily due to the stomatal limitation. As the root restriction stress progressed, the capacity of CO2 assimilation in the Calvin cycle was also affected. Acknowledgements We thank Dr. Demetriades-Shah of Li-Cor for his critical reading of the manuscript. This work was supported by the National Natural Science Foundation of China (40571083), National Key Project of Scientific and Technical Supporting Programs Funded by Ministry of Science & Technology of China (2006BAD07B02, 2006BAD07B03; 2008BADA6B02. References Allen, D.J., Ort, D.R., 2001. Impact of chilling temperatures on photosynthesis in warm climate plants. Trends Plant Sci. 6, 36–42. Bahrun, A., Jensen, C.R., Asch, F., Mogensen, V.O., 2002. Drought-induced changes in xylem pH, ionic composition, and ABA concentration act as early signals in fieldgrown maize (Zea mays L.). J. Exp. Bot. 53, 251–263. Brestic, M., Cornic, G., Fryer, M.J., Baker, N.R., 1995. Does photorespiration protect the photosynthetic apparatus in French bean leaves from photoinhibition during drought stress? Planta 196, 450–457.
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