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Sensing of tomato plant response to hypoxia in the root environment
Scientia Horticulturae 122 (2009) 17–25
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Sensing of tomato plant response to hypoxia in the root environment Hans-Peter Kla¨ring a,*, Manuela Zude b a b
Leibniz Institute of Vegetable and Ornamental Crops Großbeeren/Erfurt e.V., Theodor-Echtermeyer-Weg 1, 14979 Großbeeren, Germany Leibniz Institute for Agricultural Engineering Potsdam-Bornim e.V., Max-Eyth-Allee 100, 14469 Potsdam-Bornim, Germany
A R T I C L E I N F O
A B S T R A C T
Article history: Received 12 December 2008 Received in revised form 25 March 2009 Accepted 30 March 2009
A severe drawback in hydroponic production systems and irrigated field cultivation arises due to the risk of hypoxia, provoked by water logging in the root environment. The effects of hypoxia become temporarily visible when plants are irreversibly damaged. For this reason, non-invasive methods are required for detecting hypoxia in good time. In five experiments, tomato plants at two stages of development were grown in containers in aerated nutrient solution. Aeration was interrupted to trigger hypoxic conditions in the root environment. Whereas young plants were able to adapt to hypoxia in the root environment and survived, mature plants wilted two days after aeration interruption and died rapidly. A decrease in leaf photosynthesis, leaf transpiration rates and efficiency of the photosystem II was observed in older plants, while leaf diffuse reflectance changed slowly. On the other hand, if young plants were able to adapt to hypoxia in the root environment and survived, no clear reduction of leaf photosynthesis and the efficiency of the photosystem II arose, although the dry matter growth was decreased by 50%. Changes in leaf colour and reflectance spectra occurred. The latter indicated changes in the profile of the carotenoids. The ratio of intensities at 550 and 455 nm in particular provided a sensitive and diagnostic parameter for hypoxia in the root zone of adapted plants which, nevertheless, displayed severe growth limitation. ß 2009 Elsevier B.V. All rights reserved.
Keywords: Anoxia Carotenoids Chlorophyll fluorescence Colour Diffuse reflectance spectra Dissolved oxygen Non-invasive measurement Photosynthesis
1. Introduction Cultivated plant species for greenhouses production were mainly selected for their high yield in optimum growing conditions. In the selection process, we can assume that tolerances to many stress factors were reduced, since such mechanisms often require extra energy and thus potentially decrease the assimilate availability for the harvest organs. This also concerns sensitivity to hypoxia in the root environment (Crawford and Braendle, 1996). Irrigation strategies do not only have to avoid water deficiency – they must also prevent hypoxic conditions from occurring in the root environment. Both water and oxygen concentrations of soil or substrate depend on irrigation control. Increasing the rate or frequency of irrigation may increase the water content of the substrate or soil, thus decreasing oxygen availability in the root environment. On the other hand, it increases the availability of water to the plants and decreases the osmotic potential by leaching the substrate or soil, avoiding accumulations of salt. Unfavourable growing conditions – hypoxia and osmotic stress – visually result in similar plant responses. Particularly under hypoxic conditions, symptoms often become visible when plants are already severely damaged. There appears to be a lack of feasible methods for the
* Corresponding author. Tel.: +49 33701 78351; fax: +49 33701 55391. E-mail address: [email protected] (H.-P. Kla¨ring). 0304-4238/$ – see front matter ß 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.scienta.2009.03.029
timely detection of critical hypoxia levels in the root environment affecting the plants, preferably by non-invasive sensing. Oxygen concentrations in the root environment can be measured by a variety of methods; however, the influence of the apparent oxygen concentration on plants depends on exogenous variables such as temperature as well as endogenous factors. The limiting effect of anoxia in the root environment on photosynthesis was shown for several crops, including tomato (Bradford, 1983) and many investigations focussed on the mode of action (Vartapetian and Jackson, 1997; Gibbs and Greenway, 2003), but a decrease in the photosynthesis rate was also observed in several different stress conditions, rendering it no good for practical decision-making (Zude and Kla¨ring, 2009). The present study was aimed at approaching non-invasive sensing methods that show potential for plant monitoring, particularly to detect oxygen deficiency in the root environment of tomato plants. Readings of the leaf apparent photosynthesis rate, chlorophyll fluorescence kinetic, leaf colour and leaf diffuse reflectance were evaluated. 2. Material and methods 2.1. Plant material and treatments Five experiments (Table 1) were carried out on tomato (Solanum lycopersicum cv. ‘Vanessa’ in experiments 1 and 5; cv. ‘Liberto’ in
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Table 1 Duration of the treatments, number of unfolded leaves at treatment start, number of replications per treatment, non-invasive sensing methods tested and mean environmental conditions. Experiment
Period (d)
Number of leaves
Replications
Sensing methoda
PAR (mol m2 d1)
Temperature (8C)
rH (%)
1 2 3 4 5
14 14 14 2 3
6 4 4 8 28
6 14 14 12 4
PH, CF, CF, CF, PH,
15.1 15.0 17.2 23.4 29.2
22.0 21.8 23.5 23.1 28.6
70 71 64 52 61
a
CF, R R C R R
Non-invasive sensing methods were tested for: PH – apparent leaf photosynthesis rate, CF – chlorophyll fluorescence kinetic, R – leaf diffuse reflectance, and C – leaf colour.
experiments 2–4). In experiments 1–4 seedlings were planted in 2l containers in an aerated nutrient solution. The containers were covered with black-and-white plastic foil (with the white side facing the outside) to prevent algae growth. The plant stems were adjusted in polystyrene discs, floating on the surface of the solution, and later mechanically supported by strings hanging from a wire. In experiment 5, mature plants were grown in 15-l containers in an aerated nutrient solution covered with black-andwhite plastic foil. Plants were trained using strings hanging from a wire. The nutrient solution was based on recommendations for tomato production in hydroponic growing systems (De Kreij et al., 1997), with an air supply to each container. In experiment 1, plants from each treatment were distributed randomly in two growth chambers and were grown at a photosynthetic active photon flux density (PPFD) of 300 mmol m2 s1 for 14 h d1, with an air temperature of 22 0.2 8C and 70 2% relative humidity. In the other experiments, the plants were randomly distributed in a greenhouse – the small containers in experiments 2–4 on a table and the larger containers in experiment 5 on the floor. After between three and ten days, when the plants were established in the environment, aeration was interrupted in stress-treatment to trigger hypoxic conditions in the root zone. Additionally in experiment 1, aeration was interrupted for one week and subsequently re-established. Treatments were scheduled for two weeks, if the plants had not already faded away by then. All of the plants in experiments 1–3 were destructively measured after completion of the experiments. Additionally, samples were removed from experiment 1 before treatments commenced and after re-aeration. The shoot and root mass were measured before and after drying the samples in a ventilated oven at 80 8C for two days. Non-invasive readings were recorded throughout the experimental phase (Table 1). Unless otherwise stated, the measurements in experiments 1–4 were carried out on the mid part of one lateral leaflet with the already expanded leaf blade of the highest leaf, which completely filled the leaf chamber. Leaf diffuse reflectance measurements required an enhanced leaf area and could therefore not be performed on these young plants at the start of experiments 1–3. The leaf number was the same for all treatments. In experiment 5, the second leaflet of the leaves below the second and third truss (counted from the top) was used.
The oxygen concentration in the nutrient solution was measured using an electrochemical oxygen meter (GMH 3630, Greisinger, Regenstauf, Germany). 2.2. Gas exchange analyses Leaf apparent photosynthesis and transpiration rates were measured using a portable infrared gas analyser with leaf chamber (LI-6400XT, Licor Inc., USA). Conditions in the leaf chamber were adjusted to the same levels as in the growth chambers (experiment 1: 300 mmol m2 s1 PPFD, 400 mmol mol1 CO2 concentration, 22 8C, approximately 70% relative humidity) or close to the conditions in the greenhouse (experiment 5: 1000 mmol m2 s1 PPFD, 400 mmol mol1 CO2 concentration, 25 8C, approximately 60% relative humidity). After closing the leaf chamber, data were recorded every 10 s for 2 min. The steady state appeared rapidly and the average of the last four measurements was used for the data analysis. 2.3. Optical readings Chlorophyll fluorescence was measured using a pulse–amplitude modulated system (Mini-Pam, Walz, Effeltrich, Germany) after 10 min of dark adaptation (Krause and Weis, 1984, 1991). Readings of light saturation curves were taken to analyse the yield (y = variable fluorescence/maximum fluorescence) and maximum electron transport rate (ETR ¼ y PPFD 0:84 0:5). A portable, hand-held spectrophotometer device (Pigment Analyzer PA-1101, CP, Falkensee, Germany) equipped with photodiode arrays was applied to record the leaf spectra in the UV and visible wavelength range from 190 to 720 nm (MMS1 UV/ VIS, Carl Zeiss, Jena, Germany) or in the visible and near infrared range from 320 to 1120 nm (MMS1 NIR enh., Carl Zeiss, Germany), providing a spectral resolution of 2.2 nm in experiments 1 and 5 and 3.3 nm in experiments 2 and 3. In the present study, an integrated light cup equipped with light-emitting diodes, capturing the entire wavelength range recorded, served as the light source. Spectralon (20% certified, Labsphere Ltd., North Sutton, USA) was used as the white reference for the calibration. The leaf raw spectra were used to calculate the indices (Table 2). The leaf colour was measured using a portable spectrophotometer (CM-508d, Minolta Camera Co. Ltd., Osaka, Japan)
Table 2 Indices applied for analysing the leaf spectra recorded in diffuse reflectance geometry using the intensity (I) values at specified wavelengths. Index
Equation
Indicator for
Literature
PRI Car-ratio Red-edge NChlI RVSI
Carotenoids Carotenoids Chlorophyll Chlorophyll Chlorophyll
˜ uelas et al. (1998) Gamon et al. (1997); Pen Zude and Kla¨ring (2009) Lichtenthaler et al. (1996) Adapted from Richardson et al. (2002) Merton (1999)
RII
(I531 I570)/(I531 + I570) I550/I455 I00 (l650–710) = 0 (I718 I660)/(I718 + I660) ((I714 + I752)/2)/I733 R 750 Iy 705 I7051 dy
Chlorophyll
Richardson et al. (2002); Gitelson and Merzlyak (1994)
Chlorophyll ratio Lichtenthaler’s index PSRI SIPI
I698/I760 I750/I550 (I678 I500)/I750 (I800 I445)/(I800 I680)
Chlorophyll Chlorophyll Chlorophyll to carotenoids ratio Chlorophyll to carotenoids ratio
Carter (1994); Moran and Moran (1998) Lichtenthaler et al. (1996) Merzlyak et al. (1999) ˜ uelas et al. (1995) Pen
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on the second day after aeration interruption, lapsed into lethargy (L) and died. Highlighting the different responses, experiments 1, 2, . . ., 5 were denoted in the following by A1, A2, A3, L4 and L5, respectively. 3.2. Concentration of dissolved oxygen in the nutrient solution
and analysed in the L*a*b* colour space (CIE, 1978). The colours were compared by taking the Euclidian distances DE* in the L*a*b* space. Data recorded from the treatments were evaluated by ANOVA, followed by Student’s or Dunnett’s multiple range procedure at significance level a = 0.05.
The concentration of dissolved oxygen in the aerated nutrient solution at the start of the experiments ranged from 7 to 8 mg l1. When aeration ceased it dropped to hypoxic conditions. At a certain level, equilibrium was attained between oxygen consumption by root respiration and the dissolution of oxygen from the atmosphere. The duration of dropping and the steady state probably depended in part on environmental conditions, such as temperature, but mainly on the age of the plants (Table 1) and the corresponding root size. In experiment A1, the steady state was reached at 1 mg l1, one day after aeration interruption (Fig. 1), while in experiments A2 and A3 the steady state appeared at 2 mg l1 after six days. In experiments L4 and L5, the oxygen concentration dropped rapidly to 0.6 and 0.2 mg l1, respectively, within a few hours (data not shown). When root mass increased during the experiments, the total root respiration rate increased and the oxygen supply in the nutrient solution was hampered, resulting in a slight decrease in the oxygen concentration in both aerated and non-aerated treatments of experiments A1 (Fig. 1), A2 and A3 (data not shown).
3. Results
3.3. Changes in the morphology of plants adapting to hypoxia
3.1. Adaptation to hypoxia or fall into lethargy
The young tomato plants in experiments A1–A3 developed lateral adventive roots at the stem above the polystyrene disc. The shoot and root dry matters, however, were dramatically reduced by the low oxygen concentration in the root environment (Fig. 2a, c and Table 3). The plant organ’s dry matter content and shoot-to-
Fig. 1. Time courses of the concentration of dissolved oxygen in the treatments aerated control (N – normoxia), hypoxia (H) and re-aeration after one week of hypoxia (R) in experiment A1. The bars indicate the standard error of the mean when greater than the symbol, while full black symbols indicate significant differences of the treatment compared to the control (Dunnett’s multiple range procedure at significance level a = 0.05).
Two responses by the plants were observed in the five experiments. While in experiments 1–3 the plants were able to adapt to hypoxia (A), in experiments 4 and 5 they started wilting
Fig. 2. Shoot dry matter (a), root dry matter (c), shoot dry matter content (b) and shoot-to-root ratio (d) of tomato plants affected by hypoxia (H) and re-aeration after one week of hypoxia (R) in the root environment compared with the aerated control (N – normoxia) in experiment A1. The bars represent the standard error of the mean when greater than the symbol, while full black symbols indicate significant differences of the treatment compared to the control (Dunnett’s multiple range procedure at significance level a = 0.05).
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Table 3 Growth characteristics of tomato plants measured at the end of experiments A1–A3. The upper values are the aerated (normoxia) plants, while the lower values are the non-aerated (hypoxia) plants. Bold numbers indicate a significant effect of hypoxia on the corresponding characteristic (Student’s t-procedure at significance level a = 0.05). Characteristic
Values in experiment A1
A2
14.011 6.303
4.536 3.144
6.018 3.923
2.017 0.740
0.863 0.384
1.073 0.342
1
0.075 0.113
0.081 0.115
0.091 0.119
Root dry matter content (g g1)
0.053 0.058
0.053 0.061
0.058 0.063
Shoot-to-root ratio (g g1)
6.950 8.518
5.219 8.280
5.670 11.566
Shoot dry matter (g)
Root dry matter (g)
Shoot dry matter content (g g
)
A3
root ratio significantly increased when conditions in the root environment became hypoxic (Table 3), but dropped back to normal values one week after re-aeration (Fig. 2b, d).
compared to all of the treatments. The leaf transpiration rate showed similar patterns. Differences were more pronounced compared to apparent photosynthesis analysis. On the other hand, when the plants were unable to adapt to hypoxia, such as under the conditions of experiment L5, both net photosynthesis and transpiration rates decreased dramatically just one day after interrupting aeration, and dropped to virtually zero on the second day (Fig. 3c, d). 3.5. Chlorophyll fluorescence kinetic The efficiency of photosystem II in the leaves was dramatically affected by hypoxia when the plants were unable to adapt to oxygen deficiency in the root environment. Just two days after aeration interruption, the yield in experiment L4 was reduced by 34% and the maximum electron transport rate by 51% (Fig. 4g, h). However, when plants were able to adapt to hypoxia, no clear effect on chlorophyll fluorescence kinetics could be observed. The yield was virtually unaffected, while the electron transport rate indicated an upward trend in experiments A2 and A3 (Fig. 4a–f). The yield and electron transport rate followed the trend of the apparent photosynthesis rate, but showed a certain amount of variability between the experiments. 3.6. Leaf diffuse reflectance
3.4. Photosynthesis and transpiration rates No clear effect of hypoxia in the root environment was observed regarding the gas exchange rates of plants adapting to hypoxia in experiment A1. The leaf net photosynthesis rate decreased slightly after three days of hypoxia in the root environment compared to the control plants, but then very soon convalesced. Three days after re-aeration, it was even significantly higher compared to the control plants (Fig. 3a). At the end of the experiment, the leaf apparent photosynthesis rate of the control plants was lowest
When tomato plants were able to adapt to hypoxia in the root environment, the diffuse reflectance of the leaves was changed at wavelengths from 420 to 760 nm and from 880 to 1030 nm, while the lethargic plants maintained quite stable intensity values in the whole leaf spectrum (Fig. 5). Interesting effects were observed at the carotenoids’ absorption bands. The upper leaves of the plants that adapted to hypoxia in experiments A1–A3 showed a significant decrease of the car-ratio, indicating a change in the carotenoids’ profile (Table 4). This effect was more pronounced in the upper,
Fig. 3. Apparent leaf photosynthesis rate (a) and leaf transpiration rate (b) of young tomato plants in experiment A1 affected by low oxygen concentrations (hypoxia, H) and re-aeration after one week of hypoxia (R) in the root environment compared with the aerated control (N – normoxia) measured in a leaf gas exchange chamber at 300 mmol m2 s1 PPFD and leaf net photosynthesis (c) and transpiration (d) of mature tomato plants in experiment L5 measured at 1000 mmol m2 s1. The bars represent the standard error of the mean when greater than the symbol, while full black symbols indicate significant differences of the treatment compared to the control (Dunnett’s (a, b) or Student’s (c, d) multiple range procedure at significance level a = 0.05).
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Fig. 4. Chlorophyll fluorescence kinetic of tomato plants affected by hypoxia (H) and re-aeration after one week of hypoxia (R) in the root environment compared with the aerated control (N – normoxia) in experiments A1–A3 and L4 (from top to bottom). The bars represent the standard error of the mean when greater than the symbol, while full black symbols indicate significant differences of the treatment compared to the control (Dunnett’s (a, b) or Student’s (c–h) multiple range procedure at significance level a = 0.05).
younger leaves than in the lower leaves (Fig. 6a, b). Interestingly, in contrast to the hypoxia-adapted plants, the car-ratio of the lethargic plants remained stable or tended to increase (Table 4). Some of the reflectance indices attributed to chlorophyll absorption, such as RVSI, RII, the chlorophyll ratio, Lichtenthaler’s index and NChlI, tended to indicate an increase in the chlorophyll content – for each index a significant effect of hypoxia was observed in one of the experiments at one leaf position (Table 4 and
Fig. 6c–f). Sometimes differences were more pronounced in the upper leaves (RVSI), while with other indices (RII, the chlorophyll ratio, Lichtenthaler’s index) significant differences were found in the middle leaves only. Further chlorophyll-related indices, such as red-edge did not change as a response to the treatments (Table 4). Chlorophyll-related indices were not influenced by hypoxia in the root environment when plants lapsed into lethargy in experiments L4 and L5 (Table 4).
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significantly when plants adapted to hypoxia in the root environment in experiment A2. In contrast, no effect on this index was observed with the lethargic plants (Table 4 and Fig. 6h). 3.7. Leaf colour
Fig. 5. The effect of hypoxia in the root environment on leaf diffuse reflectance intensities in the wavelength range from 320 to 1120 nm. The curves depict the ratio of the reflectance intensities of the leaves of the tomato plants in the nonaerated and aerated nutrient solution (Ihypoxia/Inormoxia), measured in experiment L4 and at different times and leaf positions in experiment A2.
Indices attributed to the ratio of chlorophyll to the carotenoids contents, such as PSRI and SIPI, were not affected by hypoxia in the root environment in adapted and lethargic plants (Table 4 and Fig. 6g). A clear response by the hypoxia-adapted plants was observed in the near infrared range (NIR) from 900 to 1000 nm, the absorption band of carbohydrates and water (Fig. 5). The empirical derived ratio of reflectance intensities at 940 and 718 nm increased Table 4 The effect of hypoxia in the root environment on indices for analysing the leaf spectra recorded in diffuse reflectance geometry. The upper data depict the relative differences between the indices of plants in non-aerated and aerated nutrient solution ([Indexhypoxia Indexnormoxia]/Indexnormoxia), while the lower values show the probability level P of Student’s t-procedure for the hypothesis that the difference is zero. Bold numbers indicate a significant effect of hypoxia on the corresponding index (Student’s t-procedure at significance level a = 0.05). All measurements were performed on the uppermost leaves, which filled the measurement chamber. Index
Relative differences and P in experiment A1a
A2
L4
L5a
PRI
0.053 0.965
0.212 0.003
0.246 0.636
0.230 0.031
Car-ratio
0.089 0.007
0.328 0.003
0.145 0.192
0.269 <0.001
Red-edge
0.004 0.752
<0.001 0.956
0.001 0.623
0.001 0.962
0.403 0.038
0.137 0.059
0.082 0.521
0.032 0.803
RVSI
0.079 0.008
0.008 0.365
RII
0.031 0.798
0.048 0.587
0.173 0.225
0.002 0.977
Lichtenthaler’s index
0.343 0.056
0.038 0.752
PSRI
0.145 0.627
0.099 0.494
SIPI
0.026 0.633
0.134 0.435
I940/I718
0.491 0.002
0.057 0.471
NChlI
Chlorophyll ratio
a Diffuse reflectance geometry was measured from 190 to 720 nm with 2.2 nm resolution.
The tomato plants responded to hypoxia in the root environment with significant changes in the leaf colour (Fig. 7). The younger the leaf, the more pronounced the observed effect was. Evaluating in the L*a*b* colour space, the leaf colour changed from green to red (Fig. 7b) and from yellow to blue (Fig. 7c), while the lightness only decreased slightly in the upper leaves (Fig. 7a). Two weeks after aeration interruption, the Euclidian difference DE* between the upper leaves was 5.6, which is considered to be two different colours. Just eight days after aeration interruption, an Euclidian difference DE* of 6.2 was observed for the leaves at the top of the plants (data not shown). 4. Discussion The short-term responses of the tomato plants measured in experiments L4 and L5 correspond with most of the literature on anoxia-sensitive species. Thus, the photosynthesis rate decreased markedly within one or two days, as reported, for example, for tomato (Bradford, 1983) and mango (Zude-Sasse et al., 2001). This reduction in photosynthetic capacity was accompanied by a modification of the chlorophyll fluorescence pattern, indicating limitations in the PSII reaction centre and subsequent electron transport rate (Fig. 4g, h), which is in agreement with observations on citrus and mango cuttings (Larson and Schaffer, 1991; ZudeSasse and Lu¨dders, 2000; Zude-Sasse et al., 2001). Furthermore, significant reductions in the transpiration rate, as in experiment L5 (Fig. 3d), were reported for tomato (Bradford and Hsiao, 1982). In the short-term readings, however, the leaf diffuse reflectance profile remained unaffected in the chlorophylls and the carotenoids’ absorption bands (Table 4, Figs. 5 and 6). Leaf diffuse reflectance only responded on the third day of hypoxia, when the photosynthesis and transpiration rates had dropped close to zero and the leaves had wilted considerably. The significant increase in the car-ratio and the decrease of PRI indicate changes in the leaf carotenoids’ contents. However, surprisingly, none of the indices related to leaf chlorophyll content was affected. On the other hand, the tomato plants in experiments A1–A3 adapted to hypoxia in the root environment and survived. Nonetheless, such behaviour was accompanied by a significant decrease in root and shoot growth. The marked reduction in shoot and root dry matter and the increased dry matter content and shoot-to-root ratio under hypoxic conditions (Table 3 and Fig. 2) are in agreement with many reports on several crops, including tomato (Jackson and Drew, 1984; Rong and Tachibana, 1997; Shi et al., 2007). The re-establishment of normoxia-like dry matter contents and shoot-to-root ratios one week after re-aeration (Fig. 2b, d) is an indication of the plant’s ability to cope with hypoxia, if normoxic conditions are adjusted in good time. Although often described as a sensitive indicator of stress conditions, and in contradiction to the results in experiments L4 and L5, neither the chlorophyll fluorescence kinetic (Fig. 4a–f) nor the leaf apparent photosynthesis rate (Fig. 3a, b) changed markedly due to hypoxic conditions in the root zone in experiments A1–A3. The young tomato plants might have been able to adapt to the decreasing oxygen supply in the short term by increasing anaerobic respiration, such as the fermentation of lactic acid and ethanol (Davies, 1980; Perata and Alpi, 1993) supported in the longer term by developing adventive roots above the water level (Zude et al., 1998). In addition, the slow drop of the O2 concentration may act as a hypoxic pretreatment, which is known to improve adaptation to anoxic
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Fig. 6. Spectral characteristics PRI (a), car-ratio (b), RVSI (c), RII (d), chlorophyll ratio (e), Lichtenthaler’s index (f), SIPI (g) and I940/I718 (h) of tomato leaves at two different positions in experiment A2. Hatched columns of the hypoxia (H) treatment indicate a significant difference compared to the normoxia (N) treatment (Student’s t-procedure at significance level a = 0.05.). The bars represent the standard error of the mean.
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Fig. 7. Colour of leaves in the L*a*b* colour space measured at different positions of aerated and non-aerated tomato plants two weeks after aeration interruption in experiment A3. Hatched columns of the hypoxia (H) treatment indicate a significant difference compared to the normoxia (N) treatment (Student’s t-procedure at significance level a = 0.05.). The bars represent the standard error of the mean.
conditions (Germain et al., 1997). It can be assumed that the apparent photosynthesis rate and chlorophyll fluorescence kinetic are not feasible for detecting hypoxia in the case of plant adaptation, although, nonetheless, growth and yield are reduced. Diffuse reflectance indices related to the leaf chlorophyll content were not repeatedly affected by hypoxia in the root environment (Table 4 and Figs. 6c–g). Reproducible differences were obtained from the hypoxia-adapted plants in the carotenoids’ absorption band (Table 4 and Fig. 6a, b). In the present study, the ratio of the reflectance intensities at 550 and 455 nm (car-ratio) appeared to be sensitive. Such a result might have been expected, since the total carotenoids content is less affected than changes in the carotenoids’ profile. Adaptation to oxygen deficiency always includes coping with an energy crisis, due to the lack of the terminal electron acceptor in the respiratory chain, as well as protecting the cell against the reducing pressure (Gibbs and Greenway, 2003; Zude-Sasse et al., 2001). The latter can be obtained by the response of the xanthophyll cycle, resulting in changes in the composition of the carotenoids.
Beside the car-ratio, the empirically derived ratio of the reflectance at 940 and 718 nm responded significantly when plants adapted to hypoxia in the root environment. In previous studies, NIR spectroscopy has been successfully employed to nondestructively predict fruit quality parameters, such as soluble solids and water contents in citrus and apple, as shown by many authors over the past 15 years (Nicolai et al., 2007; Walsh and Kawano, 2008). Thus, the changes in the NIR reflectance profile of the hypoxia-adapted plants are probably associated with the increase in dry matter content of the tomato leaves, as well as with changes in the water content. However, changes of OH bonds absorbing in the NIR may occur due to many stress factors. For diagnostic purposes, the differences in the carotenoids are assumed to be more feasible. Dramatic changes in the leaf colour were observed in experiment A3 (Fig. 7). However, it is difficult to ascribe this response to a specific plant metabolite. Leaf colour in terms of leaf greenness is used in several applications, mainly in precision farming, as an indication of the N-nutrition status, which is assumed to be related to the leaf chlorophyll content (e.g. Blackmer et al., 1994). However, it is recognised that colour values are influenced by the plant growth stage, cultivar, leaf thickness, plant population and any soil or climate factor causing leaf chlorosis (Turner and Jund, 1994). After the sudden decrease of oxygen concentration in experiment L5, the apparent leaf photosynthesis and transpiration rates of the mature plants fell dramatically. The plants were damaged irreversibly. This response seems to be more similar to most other experiments, where strong effects of anoxia on photosynthesis, water relations and stomatal conductance were found (Bradford, 1983). From the perspective of practical application in irrigation control, this situation is of less interest because the crop is often lost before corresponding management strategies take effect. In irrigated crops, a change from aerobic to anaerobic conditions in the root environment usually evolutes within a few days, and absolute anoxia hardly ever occurs. The roots may be protected by a hypoxic pre-treatment that has been found for many crops, including tomato (Germain et al., 1997). During the pre-treatment, several enzyme activities increased, including that of sucrose synthase. In hypoxia-pre-treated roots, ethanol was produced immediately after transfer to anoxia; little lactic acid accumulated in the tissues and sucrose was able to sustain glycolytic flux via the sucrose synthase pathway (Germain et al., 1997). This protection may be a survival mechanism of the plants, but is often unable to fully compensate for growth and yield reductions. Measuring sensitive symptoms in plants adapting to hypoxia is therefore highly important, particularly if no visible symptoms appear while yield reductions may already occur. The measured differences in leaf diffuse reflectance in the carotenoids’ absorption bands may provide a sensitive tool under these conditions. Also, reflectance in the NIR wavelength range and colour readings responded sensitively to hypoxia. However, the latter were tested in one experiment only, and both are difficult to interpret in terms of changes in the plant’s metabolism. The main difficulty with all of the tested non-invasive methods is the absence of the (normoxia) reference in practical applications. Although this reference is present in all experiments, its absolute value depends on the cultivar, growth stage and several environmental conditions, and therefore cannot be used comprehensively. Evaluating profiles may be a solution to this problem. For example, the car-ratio significantly increased towards the top of the tomato plants when the roots were supplied with oxygen, while the values were equal under hypoxia (Fig. 6b); or, the leaf colour parameter b* increased from the lower to the upper leaves under aerobic conditions, while it decreased under hypoxia (Fig. 7c). Nonetheless, this approach requires further investigation.
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The feasibility of gas exchange sensors is rather limited in greenhouse production. A portable infrared gas analyser with leaf chamber is relatively large and heavy, and the leaf needs a certain amount of time to adapt to the conditions in the chamber, especially when aiming at transpiration. Also, the chlorophyll fluorescence technique is rather complicated to use, due to the requirement of the leaf’s adaptation to the dark prior to the measurement. In contrast, the leaf diffuse reflectance and leaf colour can be measured quickly on many leaves using easily-touse, hand-held spectrophotometer devices. Acknowledgements This research was supported by the German Federation and the federal states of Brandenburg and Thuringia. We thank Jutta Lenk for her excellent technical support. References Blackmer, T.M., Schepers, J.S., Varvel, G.E., 1994. Light reflectance compared with other nitrogen stress measurements in corn leaves. Agron. J. 86, 934–938. Bradford, K.J., Hsiao, T.C., 1982. Stomatal behaviour and water relations of waterlogged tomato plants. Plant Physiol. 70, 1508–1513. Bradford, K.J., 1983. Effects of soil flooding on leaf gas exchange of tomato plants. Plant Physiol. 73, 475–479. Carter, G.A., 1994. Ratios of leaf reflectances in narrow wavebands as indicators of plant stress. Int. J. Remote Sensing 15, 697–703. CIE, 1978. Recommendations on uniform color spaces—color difference equations, psychometric color terms. Supplement 2 to CIE publication No. 15 (E-1.3.1) 1971/(TC-1-3). Commission Internationale de l’Eclairage, Paris. Crawford, R.M.M., Braendle, R., 1996. Oxygen deprivation stress in a changing environment. J. Exp. Bot. 47, 145–159. Davies, D.D., 1980. Anaerobic metabolism and the production of organic acids. Biochem. Plants 2, 581–611. De Kreij, C., Voogt, W., Van den Bos, A.L., Baas, R., 1997. Nutrient solutions for the growth of tomato in closed systems (In Dutch). Proefstation voor Bloemistrij en Glasgroente. Naaldwijk, The Netherlands. Brochure VG 2, 21. Gamon, J.A., Serrano, L., Surfus, J.S., 1997. The photochemical reflectance index: an optical indicator of photosynthetic radiation use efficiency across species, functional types, and nutrient levels. Oecologia 112, 492–501. Germain, V., Ricard, B., Raymond, P., Saglio, P.H., 1997. The role of sugars, hexokinase, and sucrose synthesis in the determination of hypoxically induced tolerance to anoxia in tomato roots. Plant Physiol. 114, 167–175. Gibbs, J., Greenway, H., 2003. Mechanisms of anoxia tolerance in plants. I. Growth, survival and anaerobic catabolism. Funct. Plant Biol. 30, 1–47. Gitelson, A.A., Merzlyak, M.N., 1994. Spectral reflectance changes associated with autumn senescence of Aesculus hippocastanum L. and Acer platanoides L. leaves. Spectral features and relation to chlorophyll estimation. J. Plant Physiol. 143, 286–292.
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Jackson, M.B., Drew, M.C., 1984. Effect of flooding on growth and metabolism of herbaceous plants. In: Kozlowski, T.T. (Ed.), Flooding and Plant Growth. Academic Press, Orlando, pp. 47–128. Krause, G.H., Weis, E., 1984. Chlorophyll fluorescence as a tool in plant physiology. 2. Interpretation of fluorescence signals. Photosynth. Res. 5, 139–157. Krause, G.H., Weis, E., 1991. Chlorophyll fluorescence and photosynthesis—the basics. Ann. Rev. Plant Physiol. Plant Mol. Biol. 42, 313–349. Larson, K.D., Schaffer, B., 1991. Flooding, leaf gas exchange, and growth of mango in containers. J. Am. Soc. Hortic. Sci. 116, 156–160. Lichtenthaler, H.K., Gitelson, A., Lang, M., 1996. Non-destructive determination of chlorophyll content of leaves of a green and an aurea mutant of tobacco by reflectance measurements. J. Plant Physiol. 148, 483–493. Merton, R.N., 1999. Multi-temporal analysis of community scale vegetation stress with imaging spectroscopy. PhD Theses. University of Auckland, New Zealand. http://hdl.handle.net/2292/539. Merzlyak, M.N., Gitelson, A.A., Chivkunova, O.B., Rakitin, V.Y., 1999. Non-destructive optical detection of pigment changes during leaf senescence and fruit ripening. Physiol. Plant 106, 135–141. Moran, J.A., Moran, A.J., 1998. Foliar reflectance and vector analysis reveal nutrient stress in prey-deprived pitcher plants (Nepenthes rafflesiana). Int. J. Plant Sci. 159, 996–1001. Nicolai, B., Beullens, K., Bobelyn, E., Peirs, A., Saeys, W., Theron, K.I., Lammertyn, J., 2007. Nondestructive measurement of fruit and vegetable quality by means of NIR spectroscopy, a review. Postharvest Biol. Technol. 46, 99–118. ˜ uelas, J., Baret, F., Filella, I., 1995. Semi-empirical indices to assess carotenoids/ Pen chlorophyll a ratio from leaf spectral reflectance. Photosynthetica 31, 221–230. ˜ uelas, J., Filella, I., Llusia`, J., Siscart, D., Pin ˜ ol, J., 1998. Comparative field study of Pen spring and summer leaf gas exchange and photobiology of the Mediterranean tree Quercus ilex and Phillyrea latifolia. J. Exp. Bot. 49, 229–238. Perata, P., Alpi, A., 1993. Plant responses to anaerobiosis. Plant Sci. 93, 1–17. Richardson, A.D., Duigan, S.P., Berlyn, G.P., 2002. An evaluation of non-invasive methods to estimate foliar chlorophyll content. New Phytol. 153, 185–194. Rong, G.S., Tachibana, S., 1997. Effect of dissolved O-2 levels in a nutrient solution on the growth and mineral nutrition of tomato and cucumber seedlings. J. Jpn. Soc. Hortic. Sci. 66, 331–337 (in Japanese). Shi, K., Hu, W.H., Dong, D.K., Zhou, Y.H., Yu, J.Q., 2007. Low O2 supply is involved in the poor growth in root-restricted plants of tomato (Lycopersicon esculentum Mill.). Environ. Exp. Bot. 61, 181–189. Turner, F.T., Jund, M.F., 1994. Assessing the nitrogen requirements of rice crops with a chlorophyll meter. Aust. J. Exp. Agric. 34, 1001–1005. Vartapetian, B.B., Jackson, M.B., 1997. Plant adaptation to anaerobic stress. Ann. Bot. 79, 3–20. Walsh, K.B., Kawano, S., 2008. Near-infrared spectroscopy. In: Zude, M. (Ed.), Optical Monitoring of Fresh and Processed Agricultural Crops. CRC Press, Boca Raton, Florida, USA, pp. 192–233. Zude, M., Ebert, G., Lu¨dders, P., 1998. Influence of flooding on growth and gas exchange of mango rootstocks (Mangifera indica L.) and proposed selection criteria for flood tolerance. Appl. Bot. 72, 148–151. Zude-Sasse, M., Lu¨dders, P., 2000. Citrus roots’ responses to anoxia. Acta Hortic. 531, 121–128. Zude-Sasse, M., Hartmond, U., Lu¨dders, P., 2001. Pyridine nucleotide charge reduces photosynthesis under short-term oxygen deficiency. J. Am. Soc. Hortic. Sci. 125, 703–709. Zude, M., Kla¨ring, H.P., 2009. Two case studies of precision horticulture approaches in the citrus production. Tree For. Sci. Biotechnol. 3, 139–151.
Contents lists available at ScienceDirect
Scientia Horticulturae journal homepage: www.elsevier.com/locate/scihorti
Sensing of tomato plant response to hypoxia in the root environment Hans-Peter Kla¨ring a,*, Manuela Zude b a b
Leibniz Institute of Vegetable and Ornamental Crops Großbeeren/Erfurt e.V., Theodor-Echtermeyer-Weg 1, 14979 Großbeeren, Germany Leibniz Institute for Agricultural Engineering Potsdam-Bornim e.V., Max-Eyth-Allee 100, 14469 Potsdam-Bornim, Germany
A R T I C L E I N F O
A B S T R A C T
Article history: Received 12 December 2008 Received in revised form 25 March 2009 Accepted 30 March 2009
A severe drawback in hydroponic production systems and irrigated field cultivation arises due to the risk of hypoxia, provoked by water logging in the root environment. The effects of hypoxia become temporarily visible when plants are irreversibly damaged. For this reason, non-invasive methods are required for detecting hypoxia in good time. In five experiments, tomato plants at two stages of development were grown in containers in aerated nutrient solution. Aeration was interrupted to trigger hypoxic conditions in the root environment. Whereas young plants were able to adapt to hypoxia in the root environment and survived, mature plants wilted two days after aeration interruption and died rapidly. A decrease in leaf photosynthesis, leaf transpiration rates and efficiency of the photosystem II was observed in older plants, while leaf diffuse reflectance changed slowly. On the other hand, if young plants were able to adapt to hypoxia in the root environment and survived, no clear reduction of leaf photosynthesis and the efficiency of the photosystem II arose, although the dry matter growth was decreased by 50%. Changes in leaf colour and reflectance spectra occurred. The latter indicated changes in the profile of the carotenoids. The ratio of intensities at 550 and 455 nm in particular provided a sensitive and diagnostic parameter for hypoxia in the root zone of adapted plants which, nevertheless, displayed severe growth limitation. ß 2009 Elsevier B.V. All rights reserved.
Keywords: Anoxia Carotenoids Chlorophyll fluorescence Colour Diffuse reflectance spectra Dissolved oxygen Non-invasive measurement Photosynthesis
1. Introduction Cultivated plant species for greenhouses production were mainly selected for their high yield in optimum growing conditions. In the selection process, we can assume that tolerances to many stress factors were reduced, since such mechanisms often require extra energy and thus potentially decrease the assimilate availability for the harvest organs. This also concerns sensitivity to hypoxia in the root environment (Crawford and Braendle, 1996). Irrigation strategies do not only have to avoid water deficiency – they must also prevent hypoxic conditions from occurring in the root environment. Both water and oxygen concentrations of soil or substrate depend on irrigation control. Increasing the rate or frequency of irrigation may increase the water content of the substrate or soil, thus decreasing oxygen availability in the root environment. On the other hand, it increases the availability of water to the plants and decreases the osmotic potential by leaching the substrate or soil, avoiding accumulations of salt. Unfavourable growing conditions – hypoxia and osmotic stress – visually result in similar plant responses. Particularly under hypoxic conditions, symptoms often become visible when plants are already severely damaged. There appears to be a lack of feasible methods for the
* Corresponding author. Tel.: +49 33701 78351; fax: +49 33701 55391. E-mail address: [email protected] (H.-P. Kla¨ring). 0304-4238/$ – see front matter ß 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.scienta.2009.03.029
timely detection of critical hypoxia levels in the root environment affecting the plants, preferably by non-invasive sensing. Oxygen concentrations in the root environment can be measured by a variety of methods; however, the influence of the apparent oxygen concentration on plants depends on exogenous variables such as temperature as well as endogenous factors. The limiting effect of anoxia in the root environment on photosynthesis was shown for several crops, including tomato (Bradford, 1983) and many investigations focussed on the mode of action (Vartapetian and Jackson, 1997; Gibbs and Greenway, 2003), but a decrease in the photosynthesis rate was also observed in several different stress conditions, rendering it no good for practical decision-making (Zude and Kla¨ring, 2009). The present study was aimed at approaching non-invasive sensing methods that show potential for plant monitoring, particularly to detect oxygen deficiency in the root environment of tomato plants. Readings of the leaf apparent photosynthesis rate, chlorophyll fluorescence kinetic, leaf colour and leaf diffuse reflectance were evaluated. 2. Material and methods 2.1. Plant material and treatments Five experiments (Table 1) were carried out on tomato (Solanum lycopersicum cv. ‘Vanessa’ in experiments 1 and 5; cv. ‘Liberto’ in
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Table 1 Duration of the treatments, number of unfolded leaves at treatment start, number of replications per treatment, non-invasive sensing methods tested and mean environmental conditions. Experiment
Period (d)
Number of leaves
Replications
Sensing methoda
PAR (mol m2 d1)
Temperature (8C)
rH (%)
1 2 3 4 5
14 14 14 2 3
6 4 4 8 28
6 14 14 12 4
PH, CF, CF, CF, PH,
15.1 15.0 17.2 23.4 29.2
22.0 21.8 23.5 23.1 28.6
70 71 64 52 61
a
CF, R R C R R
Non-invasive sensing methods were tested for: PH – apparent leaf photosynthesis rate, CF – chlorophyll fluorescence kinetic, R – leaf diffuse reflectance, and C – leaf colour.
experiments 2–4). In experiments 1–4 seedlings were planted in 2l containers in an aerated nutrient solution. The containers were covered with black-and-white plastic foil (with the white side facing the outside) to prevent algae growth. The plant stems were adjusted in polystyrene discs, floating on the surface of the solution, and later mechanically supported by strings hanging from a wire. In experiment 5, mature plants were grown in 15-l containers in an aerated nutrient solution covered with black-andwhite plastic foil. Plants were trained using strings hanging from a wire. The nutrient solution was based on recommendations for tomato production in hydroponic growing systems (De Kreij et al., 1997), with an air supply to each container. In experiment 1, plants from each treatment were distributed randomly in two growth chambers and were grown at a photosynthetic active photon flux density (PPFD) of 300 mmol m2 s1 for 14 h d1, with an air temperature of 22 0.2 8C and 70 2% relative humidity. In the other experiments, the plants were randomly distributed in a greenhouse – the small containers in experiments 2–4 on a table and the larger containers in experiment 5 on the floor. After between three and ten days, when the plants were established in the environment, aeration was interrupted in stress-treatment to trigger hypoxic conditions in the root zone. Additionally in experiment 1, aeration was interrupted for one week and subsequently re-established. Treatments were scheduled for two weeks, if the plants had not already faded away by then. All of the plants in experiments 1–3 were destructively measured after completion of the experiments. Additionally, samples were removed from experiment 1 before treatments commenced and after re-aeration. The shoot and root mass were measured before and after drying the samples in a ventilated oven at 80 8C for two days. Non-invasive readings were recorded throughout the experimental phase (Table 1). Unless otherwise stated, the measurements in experiments 1–4 were carried out on the mid part of one lateral leaflet with the already expanded leaf blade of the highest leaf, which completely filled the leaf chamber. Leaf diffuse reflectance measurements required an enhanced leaf area and could therefore not be performed on these young plants at the start of experiments 1–3. The leaf number was the same for all treatments. In experiment 5, the second leaflet of the leaves below the second and third truss (counted from the top) was used.
The oxygen concentration in the nutrient solution was measured using an electrochemical oxygen meter (GMH 3630, Greisinger, Regenstauf, Germany). 2.2. Gas exchange analyses Leaf apparent photosynthesis and transpiration rates were measured using a portable infrared gas analyser with leaf chamber (LI-6400XT, Licor Inc., USA). Conditions in the leaf chamber were adjusted to the same levels as in the growth chambers (experiment 1: 300 mmol m2 s1 PPFD, 400 mmol mol1 CO2 concentration, 22 8C, approximately 70% relative humidity) or close to the conditions in the greenhouse (experiment 5: 1000 mmol m2 s1 PPFD, 400 mmol mol1 CO2 concentration, 25 8C, approximately 60% relative humidity). After closing the leaf chamber, data were recorded every 10 s for 2 min. The steady state appeared rapidly and the average of the last four measurements was used for the data analysis. 2.3. Optical readings Chlorophyll fluorescence was measured using a pulse–amplitude modulated system (Mini-Pam, Walz, Effeltrich, Germany) after 10 min of dark adaptation (Krause and Weis, 1984, 1991). Readings of light saturation curves were taken to analyse the yield (y = variable fluorescence/maximum fluorescence) and maximum electron transport rate (ETR ¼ y PPFD 0:84 0:5). A portable, hand-held spectrophotometer device (Pigment Analyzer PA-1101, CP, Falkensee, Germany) equipped with photodiode arrays was applied to record the leaf spectra in the UV and visible wavelength range from 190 to 720 nm (MMS1 UV/ VIS, Carl Zeiss, Jena, Germany) or in the visible and near infrared range from 320 to 1120 nm (MMS1 NIR enh., Carl Zeiss, Germany), providing a spectral resolution of 2.2 nm in experiments 1 and 5 and 3.3 nm in experiments 2 and 3. In the present study, an integrated light cup equipped with light-emitting diodes, capturing the entire wavelength range recorded, served as the light source. Spectralon (20% certified, Labsphere Ltd., North Sutton, USA) was used as the white reference for the calibration. The leaf raw spectra were used to calculate the indices (Table 2). The leaf colour was measured using a portable spectrophotometer (CM-508d, Minolta Camera Co. Ltd., Osaka, Japan)
Table 2 Indices applied for analysing the leaf spectra recorded in diffuse reflectance geometry using the intensity (I) values at specified wavelengths. Index
Equation
Indicator for
Literature
PRI Car-ratio Red-edge NChlI RVSI
Carotenoids Carotenoids Chlorophyll Chlorophyll Chlorophyll
˜ uelas et al. (1998) Gamon et al. (1997); Pen Zude and Kla¨ring (2009) Lichtenthaler et al. (1996) Adapted from Richardson et al. (2002) Merton (1999)
RII
(I531 I570)/(I531 + I570) I550/I455 I00 (l650–710) = 0 (I718 I660)/(I718 + I660) ((I714 + I752)/2)/I733 R 750 Iy 705 I7051 dy
Chlorophyll
Richardson et al. (2002); Gitelson and Merzlyak (1994)
Chlorophyll ratio Lichtenthaler’s index PSRI SIPI
I698/I760 I750/I550 (I678 I500)/I750 (I800 I445)/(I800 I680)
Chlorophyll Chlorophyll Chlorophyll to carotenoids ratio Chlorophyll to carotenoids ratio
Carter (1994); Moran and Moran (1998) Lichtenthaler et al. (1996) Merzlyak et al. (1999) ˜ uelas et al. (1995) Pen
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on the second day after aeration interruption, lapsed into lethargy (L) and died. Highlighting the different responses, experiments 1, 2, . . ., 5 were denoted in the following by A1, A2, A3, L4 and L5, respectively. 3.2. Concentration of dissolved oxygen in the nutrient solution
and analysed in the L*a*b* colour space (CIE, 1978). The colours were compared by taking the Euclidian distances DE* in the L*a*b* space. Data recorded from the treatments were evaluated by ANOVA, followed by Student’s or Dunnett’s multiple range procedure at significance level a = 0.05.
The concentration of dissolved oxygen in the aerated nutrient solution at the start of the experiments ranged from 7 to 8 mg l1. When aeration ceased it dropped to hypoxic conditions. At a certain level, equilibrium was attained between oxygen consumption by root respiration and the dissolution of oxygen from the atmosphere. The duration of dropping and the steady state probably depended in part on environmental conditions, such as temperature, but mainly on the age of the plants (Table 1) and the corresponding root size. In experiment A1, the steady state was reached at 1 mg l1, one day after aeration interruption (Fig. 1), while in experiments A2 and A3 the steady state appeared at 2 mg l1 after six days. In experiments L4 and L5, the oxygen concentration dropped rapidly to 0.6 and 0.2 mg l1, respectively, within a few hours (data not shown). When root mass increased during the experiments, the total root respiration rate increased and the oxygen supply in the nutrient solution was hampered, resulting in a slight decrease in the oxygen concentration in both aerated and non-aerated treatments of experiments A1 (Fig. 1), A2 and A3 (data not shown).
3. Results
3.3. Changes in the morphology of plants adapting to hypoxia
3.1. Adaptation to hypoxia or fall into lethargy
The young tomato plants in experiments A1–A3 developed lateral adventive roots at the stem above the polystyrene disc. The shoot and root dry matters, however, were dramatically reduced by the low oxygen concentration in the root environment (Fig. 2a, c and Table 3). The plant organ’s dry matter content and shoot-to-
Fig. 1. Time courses of the concentration of dissolved oxygen in the treatments aerated control (N – normoxia), hypoxia (H) and re-aeration after one week of hypoxia (R) in experiment A1. The bars indicate the standard error of the mean when greater than the symbol, while full black symbols indicate significant differences of the treatment compared to the control (Dunnett’s multiple range procedure at significance level a = 0.05).
Two responses by the plants were observed in the five experiments. While in experiments 1–3 the plants were able to adapt to hypoxia (A), in experiments 4 and 5 they started wilting
Fig. 2. Shoot dry matter (a), root dry matter (c), shoot dry matter content (b) and shoot-to-root ratio (d) of tomato plants affected by hypoxia (H) and re-aeration after one week of hypoxia (R) in the root environment compared with the aerated control (N – normoxia) in experiment A1. The bars represent the standard error of the mean when greater than the symbol, while full black symbols indicate significant differences of the treatment compared to the control (Dunnett’s multiple range procedure at significance level a = 0.05).
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Table 3 Growth characteristics of tomato plants measured at the end of experiments A1–A3. The upper values are the aerated (normoxia) plants, while the lower values are the non-aerated (hypoxia) plants. Bold numbers indicate a significant effect of hypoxia on the corresponding characteristic (Student’s t-procedure at significance level a = 0.05). Characteristic
Values in experiment A1
A2
14.011 6.303
4.536 3.144
6.018 3.923
2.017 0.740
0.863 0.384
1.073 0.342
1
0.075 0.113
0.081 0.115
0.091 0.119
Root dry matter content (g g1)
0.053 0.058
0.053 0.061
0.058 0.063
Shoot-to-root ratio (g g1)
6.950 8.518
5.219 8.280
5.670 11.566
Shoot dry matter (g)
Root dry matter (g)
Shoot dry matter content (g g
)
A3
root ratio significantly increased when conditions in the root environment became hypoxic (Table 3), but dropped back to normal values one week after re-aeration (Fig. 2b, d).
compared to all of the treatments. The leaf transpiration rate showed similar patterns. Differences were more pronounced compared to apparent photosynthesis analysis. On the other hand, when the plants were unable to adapt to hypoxia, such as under the conditions of experiment L5, both net photosynthesis and transpiration rates decreased dramatically just one day after interrupting aeration, and dropped to virtually zero on the second day (Fig. 3c, d). 3.5. Chlorophyll fluorescence kinetic The efficiency of photosystem II in the leaves was dramatically affected by hypoxia when the plants were unable to adapt to oxygen deficiency in the root environment. Just two days after aeration interruption, the yield in experiment L4 was reduced by 34% and the maximum electron transport rate by 51% (Fig. 4g, h). However, when plants were able to adapt to hypoxia, no clear effect on chlorophyll fluorescence kinetics could be observed. The yield was virtually unaffected, while the electron transport rate indicated an upward trend in experiments A2 and A3 (Fig. 4a–f). The yield and electron transport rate followed the trend of the apparent photosynthesis rate, but showed a certain amount of variability between the experiments. 3.6. Leaf diffuse reflectance
3.4. Photosynthesis and transpiration rates No clear effect of hypoxia in the root environment was observed regarding the gas exchange rates of plants adapting to hypoxia in experiment A1. The leaf net photosynthesis rate decreased slightly after three days of hypoxia in the root environment compared to the control plants, but then very soon convalesced. Three days after re-aeration, it was even significantly higher compared to the control plants (Fig. 3a). At the end of the experiment, the leaf apparent photosynthesis rate of the control plants was lowest
When tomato plants were able to adapt to hypoxia in the root environment, the diffuse reflectance of the leaves was changed at wavelengths from 420 to 760 nm and from 880 to 1030 nm, while the lethargic plants maintained quite stable intensity values in the whole leaf spectrum (Fig. 5). Interesting effects were observed at the carotenoids’ absorption bands. The upper leaves of the plants that adapted to hypoxia in experiments A1–A3 showed a significant decrease of the car-ratio, indicating a change in the carotenoids’ profile (Table 4). This effect was more pronounced in the upper,
Fig. 3. Apparent leaf photosynthesis rate (a) and leaf transpiration rate (b) of young tomato plants in experiment A1 affected by low oxygen concentrations (hypoxia, H) and re-aeration after one week of hypoxia (R) in the root environment compared with the aerated control (N – normoxia) measured in a leaf gas exchange chamber at 300 mmol m2 s1 PPFD and leaf net photosynthesis (c) and transpiration (d) of mature tomato plants in experiment L5 measured at 1000 mmol m2 s1. The bars represent the standard error of the mean when greater than the symbol, while full black symbols indicate significant differences of the treatment compared to the control (Dunnett’s (a, b) or Student’s (c, d) multiple range procedure at significance level a = 0.05).
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Fig. 4. Chlorophyll fluorescence kinetic of tomato plants affected by hypoxia (H) and re-aeration after one week of hypoxia (R) in the root environment compared with the aerated control (N – normoxia) in experiments A1–A3 and L4 (from top to bottom). The bars represent the standard error of the mean when greater than the symbol, while full black symbols indicate significant differences of the treatment compared to the control (Dunnett’s (a, b) or Student’s (c–h) multiple range procedure at significance level a = 0.05).
younger leaves than in the lower leaves (Fig. 6a, b). Interestingly, in contrast to the hypoxia-adapted plants, the car-ratio of the lethargic plants remained stable or tended to increase (Table 4). Some of the reflectance indices attributed to chlorophyll absorption, such as RVSI, RII, the chlorophyll ratio, Lichtenthaler’s index and NChlI, tended to indicate an increase in the chlorophyll content – for each index a significant effect of hypoxia was observed in one of the experiments at one leaf position (Table 4 and
Fig. 6c–f). Sometimes differences were more pronounced in the upper leaves (RVSI), while with other indices (RII, the chlorophyll ratio, Lichtenthaler’s index) significant differences were found in the middle leaves only. Further chlorophyll-related indices, such as red-edge did not change as a response to the treatments (Table 4). Chlorophyll-related indices were not influenced by hypoxia in the root environment when plants lapsed into lethargy in experiments L4 and L5 (Table 4).
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significantly when plants adapted to hypoxia in the root environment in experiment A2. In contrast, no effect on this index was observed with the lethargic plants (Table 4 and Fig. 6h). 3.7. Leaf colour
Fig. 5. The effect of hypoxia in the root environment on leaf diffuse reflectance intensities in the wavelength range from 320 to 1120 nm. The curves depict the ratio of the reflectance intensities of the leaves of the tomato plants in the nonaerated and aerated nutrient solution (Ihypoxia/Inormoxia), measured in experiment L4 and at different times and leaf positions in experiment A2.
Indices attributed to the ratio of chlorophyll to the carotenoids contents, such as PSRI and SIPI, were not affected by hypoxia in the root environment in adapted and lethargic plants (Table 4 and Fig. 6g). A clear response by the hypoxia-adapted plants was observed in the near infrared range (NIR) from 900 to 1000 nm, the absorption band of carbohydrates and water (Fig. 5). The empirical derived ratio of reflectance intensities at 940 and 718 nm increased Table 4 The effect of hypoxia in the root environment on indices for analysing the leaf spectra recorded in diffuse reflectance geometry. The upper data depict the relative differences between the indices of plants in non-aerated and aerated nutrient solution ([Indexhypoxia Indexnormoxia]/Indexnormoxia), while the lower values show the probability level P of Student’s t-procedure for the hypothesis that the difference is zero. Bold numbers indicate a significant effect of hypoxia on the corresponding index (Student’s t-procedure at significance level a = 0.05). All measurements were performed on the uppermost leaves, which filled the measurement chamber. Index
Relative differences and P in experiment A1a
A2
L4
L5a
PRI
0.053 0.965
0.212 0.003
0.246 0.636
0.230 0.031
Car-ratio
0.089 0.007
0.328 0.003
0.145 0.192
0.269 <0.001
Red-edge
0.004 0.752
<0.001 0.956
0.001 0.623
0.001 0.962
0.403 0.038
0.137 0.059
0.082 0.521
0.032 0.803
RVSI
0.079 0.008
0.008 0.365
RII
0.031 0.798
0.048 0.587
0.173 0.225
0.002 0.977
Lichtenthaler’s index
0.343 0.056
0.038 0.752
PSRI
0.145 0.627
0.099 0.494
SIPI
0.026 0.633
0.134 0.435
I940/I718
0.491 0.002
0.057 0.471
NChlI
Chlorophyll ratio
a Diffuse reflectance geometry was measured from 190 to 720 nm with 2.2 nm resolution.
The tomato plants responded to hypoxia in the root environment with significant changes in the leaf colour (Fig. 7). The younger the leaf, the more pronounced the observed effect was. Evaluating in the L*a*b* colour space, the leaf colour changed from green to red (Fig. 7b) and from yellow to blue (Fig. 7c), while the lightness only decreased slightly in the upper leaves (Fig. 7a). Two weeks after aeration interruption, the Euclidian difference DE* between the upper leaves was 5.6, which is considered to be two different colours. Just eight days after aeration interruption, an Euclidian difference DE* of 6.2 was observed for the leaves at the top of the plants (data not shown). 4. Discussion The short-term responses of the tomato plants measured in experiments L4 and L5 correspond with most of the literature on anoxia-sensitive species. Thus, the photosynthesis rate decreased markedly within one or two days, as reported, for example, for tomato (Bradford, 1983) and mango (Zude-Sasse et al., 2001). This reduction in photosynthetic capacity was accompanied by a modification of the chlorophyll fluorescence pattern, indicating limitations in the PSII reaction centre and subsequent electron transport rate (Fig. 4g, h), which is in agreement with observations on citrus and mango cuttings (Larson and Schaffer, 1991; ZudeSasse and Lu¨dders, 2000; Zude-Sasse et al., 2001). Furthermore, significant reductions in the transpiration rate, as in experiment L5 (Fig. 3d), were reported for tomato (Bradford and Hsiao, 1982). In the short-term readings, however, the leaf diffuse reflectance profile remained unaffected in the chlorophylls and the carotenoids’ absorption bands (Table 4, Figs. 5 and 6). Leaf diffuse reflectance only responded on the third day of hypoxia, when the photosynthesis and transpiration rates had dropped close to zero and the leaves had wilted considerably. The significant increase in the car-ratio and the decrease of PRI indicate changes in the leaf carotenoids’ contents. However, surprisingly, none of the indices related to leaf chlorophyll content was affected. On the other hand, the tomato plants in experiments A1–A3 adapted to hypoxia in the root environment and survived. Nonetheless, such behaviour was accompanied by a significant decrease in root and shoot growth. The marked reduction in shoot and root dry matter and the increased dry matter content and shoot-to-root ratio under hypoxic conditions (Table 3 and Fig. 2) are in agreement with many reports on several crops, including tomato (Jackson and Drew, 1984; Rong and Tachibana, 1997; Shi et al., 2007). The re-establishment of normoxia-like dry matter contents and shoot-to-root ratios one week after re-aeration (Fig. 2b, d) is an indication of the plant’s ability to cope with hypoxia, if normoxic conditions are adjusted in good time. Although often described as a sensitive indicator of stress conditions, and in contradiction to the results in experiments L4 and L5, neither the chlorophyll fluorescence kinetic (Fig. 4a–f) nor the leaf apparent photosynthesis rate (Fig. 3a, b) changed markedly due to hypoxic conditions in the root zone in experiments A1–A3. The young tomato plants might have been able to adapt to the decreasing oxygen supply in the short term by increasing anaerobic respiration, such as the fermentation of lactic acid and ethanol (Davies, 1980; Perata and Alpi, 1993) supported in the longer term by developing adventive roots above the water level (Zude et al., 1998). In addition, the slow drop of the O2 concentration may act as a hypoxic pretreatment, which is known to improve adaptation to anoxic
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Fig. 6. Spectral characteristics PRI (a), car-ratio (b), RVSI (c), RII (d), chlorophyll ratio (e), Lichtenthaler’s index (f), SIPI (g) and I940/I718 (h) of tomato leaves at two different positions in experiment A2. Hatched columns of the hypoxia (H) treatment indicate a significant difference compared to the normoxia (N) treatment (Student’s t-procedure at significance level a = 0.05.). The bars represent the standard error of the mean.
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H.-P. Kla¨ring, M. Zude / Scientia Horticulturae 122 (2009) 17–25
Fig. 7. Colour of leaves in the L*a*b* colour space measured at different positions of aerated and non-aerated tomato plants two weeks after aeration interruption in experiment A3. Hatched columns of the hypoxia (H) treatment indicate a significant difference compared to the normoxia (N) treatment (Student’s t-procedure at significance level a = 0.05.). The bars represent the standard error of the mean.
conditions (Germain et al., 1997). It can be assumed that the apparent photosynthesis rate and chlorophyll fluorescence kinetic are not feasible for detecting hypoxia in the case of plant adaptation, although, nonetheless, growth and yield are reduced. Diffuse reflectance indices related to the leaf chlorophyll content were not repeatedly affected by hypoxia in the root environment (Table 4 and Figs. 6c–g). Reproducible differences were obtained from the hypoxia-adapted plants in the carotenoids’ absorption band (Table 4 and Fig. 6a, b). In the present study, the ratio of the reflectance intensities at 550 and 455 nm (car-ratio) appeared to be sensitive. Such a result might have been expected, since the total carotenoids content is less affected than changes in the carotenoids’ profile. Adaptation to oxygen deficiency always includes coping with an energy crisis, due to the lack of the terminal electron acceptor in the respiratory chain, as well as protecting the cell against the reducing pressure (Gibbs and Greenway, 2003; Zude-Sasse et al., 2001). The latter can be obtained by the response of the xanthophyll cycle, resulting in changes in the composition of the carotenoids.
Beside the car-ratio, the empirically derived ratio of the reflectance at 940 and 718 nm responded significantly when plants adapted to hypoxia in the root environment. In previous studies, NIR spectroscopy has been successfully employed to nondestructively predict fruit quality parameters, such as soluble solids and water contents in citrus and apple, as shown by many authors over the past 15 years (Nicolai et al., 2007; Walsh and Kawano, 2008). Thus, the changes in the NIR reflectance profile of the hypoxia-adapted plants are probably associated with the increase in dry matter content of the tomato leaves, as well as with changes in the water content. However, changes of OH bonds absorbing in the NIR may occur due to many stress factors. For diagnostic purposes, the differences in the carotenoids are assumed to be more feasible. Dramatic changes in the leaf colour were observed in experiment A3 (Fig. 7). However, it is difficult to ascribe this response to a specific plant metabolite. Leaf colour in terms of leaf greenness is used in several applications, mainly in precision farming, as an indication of the N-nutrition status, which is assumed to be related to the leaf chlorophyll content (e.g. Blackmer et al., 1994). However, it is recognised that colour values are influenced by the plant growth stage, cultivar, leaf thickness, plant population and any soil or climate factor causing leaf chlorosis (Turner and Jund, 1994). After the sudden decrease of oxygen concentration in experiment L5, the apparent leaf photosynthesis and transpiration rates of the mature plants fell dramatically. The plants were damaged irreversibly. This response seems to be more similar to most other experiments, where strong effects of anoxia on photosynthesis, water relations and stomatal conductance were found (Bradford, 1983). From the perspective of practical application in irrigation control, this situation is of less interest because the crop is often lost before corresponding management strategies take effect. In irrigated crops, a change from aerobic to anaerobic conditions in the root environment usually evolutes within a few days, and absolute anoxia hardly ever occurs. The roots may be protected by a hypoxic pre-treatment that has been found for many crops, including tomato (Germain et al., 1997). During the pre-treatment, several enzyme activities increased, including that of sucrose synthase. In hypoxia-pre-treated roots, ethanol was produced immediately after transfer to anoxia; little lactic acid accumulated in the tissues and sucrose was able to sustain glycolytic flux via the sucrose synthase pathway (Germain et al., 1997). This protection may be a survival mechanism of the plants, but is often unable to fully compensate for growth and yield reductions. Measuring sensitive symptoms in plants adapting to hypoxia is therefore highly important, particularly if no visible symptoms appear while yield reductions may already occur. The measured differences in leaf diffuse reflectance in the carotenoids’ absorption bands may provide a sensitive tool under these conditions. Also, reflectance in the NIR wavelength range and colour readings responded sensitively to hypoxia. However, the latter were tested in one experiment only, and both are difficult to interpret in terms of changes in the plant’s metabolism. The main difficulty with all of the tested non-invasive methods is the absence of the (normoxia) reference in practical applications. Although this reference is present in all experiments, its absolute value depends on the cultivar, growth stage and several environmental conditions, and therefore cannot be used comprehensively. Evaluating profiles may be a solution to this problem. For example, the car-ratio significantly increased towards the top of the tomato plants when the roots were supplied with oxygen, while the values were equal under hypoxia (Fig. 6b); or, the leaf colour parameter b* increased from the lower to the upper leaves under aerobic conditions, while it decreased under hypoxia (Fig. 7c). Nonetheless, this approach requires further investigation.
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