Scientia Horticulturae 122 (2009) 17–25

Sensing of tomato plant response to hypoxia in the root environment

Hans-Peter Klaring a,*, Manuela Zude b

a Leibniz Institute of Vegetable and Ornamental Crops Großbeeren/Erfurt e.V., Theodor-Echtermeyer-Weg 1, 14979 Großbeeren, Germanyb 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

Article history:

Received 12 December 2008

Received in revised form 25 March 2009

Accepted 30 March 2009

Keywords:

Anoxia

Carotenoids

Chlorophyll fluorescence

Colour

Diffuse reflectance spectra

Dissolved oxygen

Non-invasive measurement

Photosynthesis

A B S T R A C T

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.

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1. Introduction

Cultivated plant species for greenhouses production weremainly selected for their high yield in optimum growingconditions. In the selection process, we can assume that tolerancesto many stress factors were reduced, since such mechanisms oftenrequire extra energy and thus potentially decrease the assimilateavailability for the harvest organs. This also concerns sensitivity tohypoxia 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 theroot environment. Both water and oxygen concentrations of soil orsubstrate depend on irrigation control. Increasing the rate orfrequency of irrigation may increase the water content of thesubstrate or soil, thus decreasing oxygen availability in the rootenvironment. On the other hand, it increases the availability ofwater to the plants and decreases the osmotic potential by leachingthe substrate or soil, avoiding accumulations of salt. Unfavourablegrowing conditions – hypoxia and osmotic stress – visually resultin similar plant responses. Particularly under hypoxic conditions,symptoms often become visible when plants are already severelydamaged. There appears to be a lack of feasible methods for the

* Corresponding author. Tel.: +49 33701 78351; fax: +49 33701 55391.

E-mail address: klaring@igzev.de (H.-P. Klaring).

0304-4238/$ – see front matter � 2009 Elsevier B.V. All rights reserved.

doi:10.1016/j.scienta.2009.03.029

timely detection of critical hypoxia levels in the root environmentaffecting the plants, preferably by non-invasive sensing. Oxygenconcentrations in the root environment can be measured by avariety of methods; however, the influence of the apparent oxygenconcentration on plants depends on exogenous variables such astemperature as well as endogenous factors. The limiting effect ofanoxia in the root environment on photosynthesis was shown forseveral crops, including tomato (Bradford, 1983) and manyinvestigations focussed on the mode of action (Vartapetian andJackson, 1997; Gibbs and Greenway, 2003), but a decrease in thephotosynthesis rate was also observed in several different stressconditions, rendering it no good for practical decision-making(Zude and Klaring, 2009). The present study was aimed atapproaching non-invasive sensing methods that show potentialfor plant monitoring, particularly to detect oxygen deficiency inthe root environment of tomato plants. Readings of the leafapparent 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

Table 1Duration 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 m�2 d�1) Temperature (8C) rH (%)

1 14 6 6 PH, CF, R 15.1 22.0 70

2 14 4 14 CF, R 15.0 21.8 71

3 14 4 14 CF, C 17.2 23.5 64

4 2 8 12 CF, R 23.4 23.1 52

5 3 28 4 PH, R 29.2 28.6 61

a Non-invasive sensing methods were tested for: PH – apparent leaf photosynthesis rate, CF – chlorophyll fluorescence kinetic, R – leaf diffuse reflectance, and C – leaf colour.

H.-P. Klaring, M. Zude / Scientia Horticulturae 122 (2009) 17–2518

experiments 2–4). In experiments 1–4 seedlings were planted in 2-l containers in an aerated nutrient solution. The containers werecovered with black-and-white plastic foil (with the white sidefacing the outside) to prevent algae growth. The plant stems wereadjusted in polystyrene discs, floating on the surface of thesolution, and later mechanically supported by strings hanging froma wire. In experiment 5, mature plants were grown in 15-lcontainers in an aerated nutrient solution covered with black-and-white plastic foil. Plants were trained using strings hanging from awire.

The nutrient solution was based on recommendations fortomato production in hydroponic growing systems (De Kreij et al.,1997), with an air supply to each container. In experiment 1, plantsfrom each treatment were distributed randomly in two growthchambers and were grown at a photosynthetic active photon fluxdensity (PPFD) of 300 mmol m�2 s�1 for 14 h d�1, with an airtemperature of 22 � 0.2 8C and 70 � 2% relative humidity. In theother experiments, the plants were randomly distributed in agreenhouse – the small containers in experiments 2–4 on a tableand the larger containers in experiment 5 on the floor.

After between three and ten days, when the plants wereestablished in the environment, aeration was interrupted instress-treatment to trigger hypoxic conditions in the root zone.Additionally in experiment 1, aeration was interrupted for one weekand subsequently re-established. Treatments were scheduled fortwo weeks, if the plants had not already faded away by then.

All of the plants in experiments 1–3 were destructively measuredafter completion of the experiments. Additionally, samples wereremoved from experiment 1 before treatments commenced and afterre-aeration. The shoot and root mass were measured before and afterdrying the samples in a ventilated oven at 80 8C for two days.

Non-invasive readings were recorded throughout the experi-mental phase (Table 1). Unless otherwise stated, the measure-ments in experiments 1–4 were carried out on the mid part of onelateral leaflet with the already expanded leaf blade of the highestleaf, which completely filled the leaf chamber. Leaf diffusereflectance measurements required an enhanced leaf area andcould therefore not be performed on these young plants at the startof experiments 1–3. The leaf number was the same for alltreatments. In experiment 5, the second leaflet of the leaves belowthe second and third truss (counted from the top) was used.

Table 2Indices applied for analysing the leaf spectra recorded in diffuse reflectance geometry

Index Equation Indicator for

PRI (I531 � I570)/(I531 + I570) Carotenoids

Car-ratio I550/I455 Carotenoids

Red-edge I00(l650–710) = 0 Chlorophyll

NChlI (I718 � I660)/(I718 + I660) Chlorophyll

RVSI ((I714 + I752)/2)/I733 Chlorophyll

RIIR 750

705Iy

I705�1

� �dy Chlorophyll

Chlorophyll ratio I698/I760 Chlorophyll

Lichtenthaler’s index I750/I550 Chlorophyll

PSRI (I678 � I500)/I750 Chlorophyll to ca

SIPI (I800 � I445)/(I800 � I680) Chlorophyll to ca

The oxygen concentration in the nutrient solution wasmeasured using an electrochemical oxygen meter (GMH 3630,Greisinger, Regenstauf, Germany).

2.2. Gas exchange analyses

Leaf apparent photosynthesis and transpiration rates weremeasured using a portable infrared gas analyser with leaf chamber(LI-6400XT, Licor Inc., USA). Conditions in the leaf chamber wereadjusted to the same levels as in the growth chambers (experiment 1:300 mmol m�2 s�1 PPFD, 400 mmol mol�1 CO2 concentration, 22 8C,approximately 70% relative humidity) or close to the conditionsin the greenhouse (experiment 5: 1000 mmol m�2 s�1 PPFD,400 mmol mol�1 CO2 concentration, 25 8C, approximately 60%relative humidity). After closing the leaf chamber, data were recordedevery 10 s for 2 min. The steady state appeared rapidly and theaverage of the last four measurements was used for the data analysis.

2.3. Optical readings

Chlorophyll fluorescence was measured using a pulse–ampli-tude 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 maximumelectron transport rate (ETR ¼ y� PPFD� 0:84� 0:5).

A portable, hand-held spectrophotometer device (PigmentAnalyzer PA-1101, CP, Falkensee, Germany) equipped withphotodiode arrays was applied to record the leaf spectra in theUV and visible wavelength range from 190 to 720 nm (MMS1 UV/VIS, Carl Zeiss, Jena, Germany) or in the visible and near infraredrange from 320 to 1120 nm (MMS1 NIR enh., Carl Zeiss, Germany),providing a spectral resolution of 2.2 nm in experiments 1 and 5and 3.3 nm in experiments 2 and 3. In the present study, anintegrated light cup equipped with light-emitting diodes, captur-ing the entire wavelength range recorded, served as the lightsource. Spectralon (20% certified, Labsphere Ltd., North Sutton,USA) was used as the white reference for the calibration. The leafraw spectra were used to calculate the indices (Table 2).

The leaf colour was measured using a portable spectro-photometer (CM-508d, Minolta Camera Co. Ltd., Osaka, Japan)

using the intensity (I) values at specified wavelengths.

Literature

Gamon et al. (1997); Penuelas et al. (1998)

Zude and Klaring (2009)

Lichtenthaler et al. (1996)

Adapted from Richardson et al. (2002)

Merton (1999)

Richardson et al. (2002); Gitelson and Merzlyak (1994)

Carter (1994); Moran and Moran (1998)

Lichtenthaler et al. (1996)

rotenoids ratio Merzlyak et al. (1999)

rotenoids ratio Penuelas et al. (1995)

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).

H.-P. Klaring, M. Zude / Scientia Horticulturae 122 (2009) 17–25 19

and analysed in the L*a*b* colour space (CIE, 1978). The colourswere 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 atsignificance level a = 0.05.

3. Results

3.1. Adaptation to hypoxia or fall into lethargy

Two responses by the plants were observed in the fiveexperiments. While in experiments 1–3 the plants were able toadapt 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-

week of hypoxia (R) in the root environment compared with the aerated control (N – no

greater than the symbol, while full black symbols indicate significant differences of the tre

level a = 0.05).

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

The concentration of dissolved oxygen in the aerated nutrientsolution at the start of the experiments ranged from 7 to 8 mg l�1.When aeration ceased it dropped to hypoxic conditions. At acertain level, equilibrium was attained between oxygen consump-tion by root respiration and the dissolution of oxygen from theatmosphere. The duration of dropping and the steady stateprobably depended in part on environmental conditions, such astemperature, but mainly on the age of the plants (Table 1) and thecorresponding root size. In experiment A1, the steady state wasreached at 1 mg l�1, one day after aeration interruption (Fig. 1),while in experiments A2 and A3 the steady state appeared at2 mg l�1 after six days. In experiments L4 and L5, the oxygenconcentration dropped rapidly to 0.6 and 0.2 mg l�1, respectively,within a few hours (data not shown). When root mass increasedduring the experiments, the total root respiration rate increasedand the oxygen supply in the nutrient solution was hampered,resulting in a slight decrease in the oxygen concentration in bothaerated and non-aerated treatments of experiments A1 (Fig. 1), A2and A3 (data not shown).

3.3. Changes in the morphology of plants adapting to hypoxia

The young tomato plants in experiments A1–A3 developedlateral adventive roots at the stem above the polystyrene disc. Theshoot and root dry matters, however, were dramatically reducedby the low oxygen concentration in the root environment (Fig. 2a, cand Table 3). The plant organ’s dry matter content and shoot-to-

to-root ratio (d) of tomato plants affected by hypoxia (H) and re-aeration after one

rmoxia) in experiment A1. The bars represent the standard error of the mean when

atment compared to the control (Dunnett’s multiple range procedure at significance

Table 3Growth 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 A3

Shoot dry matter (g) 14.011 4.536 6.018

6.303 3.144 3.923

Root dry matter (g) 2.017 0.863 1.073

0.740 0.384 0.342

Shoot dry matter content (g g�1) 0.075 0.081 0.091

0.113 0.115 0.119

Root dry matter content (g g�1) 0.053 0.053 0.058

0.058 0.061 0.063

Shoot-to-root ratio (g g�1) 6.950 5.219 5.670

8.518 8.280 11.566

H.-P. Klaring, M. Zude / Scientia Horticulturae 122 (2009) 17–2520

root ratio significantly increased when conditions in the rootenvironment became hypoxic (Table 3), but dropped back tonormal values one week after re-aeration (Fig. 2b, d).

3.4. Photosynthesis and transpiration rates

No clear effect of hypoxia in the root environment was observedregarding the gas exchange rates of plants adapting to hypoxia inexperiment A1. The leaf net photosynthesis rate decreased slightlyafter three days of hypoxia in the root environment compared tothe control plants, but then very soon convalesced. Three days afterre-aeration, it was even significantly higher compared to thecontrol plants (Fig. 3a). At the end of the experiment, the leafapparent photosynthesis rate of the control plants was lowest

Fig. 3. Apparent leaf photosynthesis rate (a) and leaf transpiration rate (b) of young toma

re-aeration after one week of hypoxia (R) in the root environment compared with t

300 mmol m�2 s�1 PPFD and leaf net photosynthesis (c) and transpiration (d) of mature t

the standard error of the mean when greater than the symbol, while full black symbols in

b) or Student’s (c, d) multiple range procedure at significance level a = 0.05).

compared to all of the treatments. The leaf transpiration rateshowed similar patterns. Differences were more pronouncedcompared to apparent photosynthesis analysis.

On the other hand, when the plants were unable to adapt tohypoxia, such as under the conditions of experiment L5, both netphotosynthesis and transpiration rates decreased dramatically justone day after interrupting aeration, and dropped to virtually zeroon the second day (Fig. 3c, d).

3.5. Chlorophyll fluorescence kinetic

The efficiency of photosystem II in the leaves was dramaticallyaffected by hypoxia when the plants were unable to adapt tooxygen deficiency in the root environment. Just two days afteraeration interruption, the yield in experiment L4 was reduced by34% and the maximum electron transport rate by 51% (Fig. 4g, h).However, when plants were able to adapt to hypoxia, no cleareffect on chlorophyll fluorescence kinetics could be observed. Theyield was virtually unaffected, while the electron transport rateindicated an upward trend in experiments A2 and A3 (Fig. 4a–f).The yield and electron transport rate followed the trend of theapparent photosynthesis rate, but showed a certain amount ofvariability between the experiments.

3.6. Leaf diffuse reflectance

When tomato plants were able to adapt to hypoxia in the rootenvironment, the diffuse reflectance of the leaves was changed atwavelengths from 420 to 760 nm and from 880 to 1030 nm, whilethe lethargic plants maintained quite stable intensity values in thewhole leaf spectrum (Fig. 5). Interesting effects were observed at thecarotenoids’ absorption bands. The upper leaves of the plants thatadapted to hypoxia in experiments A1–A3 showed a significantdecrease of the car-ratio, indicating a change in the carotenoids’profile (Table 4). This effect was more pronounced in the upper,

to plants in experiment A1 affected by low oxygen concentrations (hypoxia, H) and

he aerated control (N – normoxia) measured in a leaf gas exchange chamber at

omato plants in experiment L5 measured at 1000 mmol m�2 s�1. The bars represent

dicate significant differences of the treatment compared to the control (Dunnett’s (a,

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).

H.-P. Klaring, M. Zude / Scientia Horticulturae 122 (2009) 17–25 21

younger leaves than in the lower leaves (Fig. 6a, b). Interestingly, incontrast to the hypoxia-adapted plants, the car-ratio of the lethargicplants remained stable or tended to increase (Table 4).

Some of the reflectance indices attributed to chlorophyllabsorption, such as RVSI, RII, the chlorophyll ratio, Lichtenthaler’sindex and NChlI, tended to indicate an increase in the chlorophyllcontent – for each index a significant effect of hypoxia wasobserved in one of the experiments at one leaf position (Table 4 and

Fig. 6c–f). Sometimes differences were more pronounced in theupper leaves (RVSI), while with other indices (RII, the chlorophyllratio, Lichtenthaler’s index) significant differences were found inthe middle leaves only. Further chlorophyll-related indices, such asred-edge did not change as a response to the treatments (Table 4).Chlorophyll-related indices were not influenced by hypoxia in theroot environment when plants lapsed into lethargy in experimentsL4 and L5 (Table 4).

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 non-

aerated and aerated nutrient solution (Ihypoxia/Inormoxia), measured in experiment L4

and at different times and leaf positions in experiment A2.

H.-P. Klaring, M. Zude / Scientia Horticulturae 122 (2009) 17–2522

Indices attributed to the ratio of chlorophyll to the carotenoidscontents, such as PSRI and SIPI, were not affected by hypoxia in theroot environment in adapted and lethargic plants (Table 4 andFig. 6g).

A clear response by the hypoxia-adapted plants was observed inthe near infrared range (NIR) from 900 to 1000 nm, the absorptionband of carbohydrates and water (Fig. 5). The empirical derivedratio of reflectance intensities at 940 and 718 nm increased

Table 4The 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 0.212 �0.246 �0.230

0.965 0.003 0.636 0.031

Car-ratio �0.089 �0.328 0.145 0.269

0.007 0.003 0.192 <0.001

Red-edge 0.004 <0.001 0.001 0.001

0.752 0.956 0.623 0.962

NChlI �0.403 �0.137 0.082 �0.032

0.038 0.059 0.521 0.803

RVSI 0.079 �0.008

0.008 0.365

RII 0.031 0.048

0.798 0.587

Chlorophyll ratio �0.173 0.002

0.225 0.977

Lichtenthaler’s index 0.343 0.038

0.056 0.752

PSRI 0.145 0.099

0.627 0.494

SIPI 0.026 0.134

0.633 0.435

I940/I718 0.491 0.057

0.002 0.471

a Diffuse reflectance geometry was measured from 190 to 720 nm with 2.2 nm

resolution.

significantly when plants adapted to hypoxia in the rootenvironment in experiment A2. In contrast, no effect on thisindex was observed with the lethargic plants (Table 4 and Fig. 6h).

3.7. Leaf colour

The tomato plants responded to hypoxia in the root environ-ment with significant changes in the leaf colour (Fig. 7). Theyounger the leaf, the more pronounced the observed effect was.Evaluating in the L*a*b* colour space, the leaf colour changed fromgreen to red (Fig. 7b) and from yellow to blue (Fig. 7c), while thelightness only decreased slightly in the upper leaves (Fig. 7a). Twoweeks after aeration interruption, the Euclidian difference DE*between the upper leaves was 5.6, which is considered to be twodifferent colours. Just eight days after aeration interruption, anEuclidian difference DE* of 6.2 was observed for the leaves at thetop of the plants (data not shown).

4. Discussion

The short-term responses of the tomato plants measured inexperiments L4 and L5 correspond with most of the literature onanoxia-sensitive species. Thus, the photosynthesis rate decreasedmarkedly within one or two days, as reported, for example, fortomato (Bradford, 1983) and mango (Zude-Sasse et al., 2001). Thisreduction in photosynthetic capacity was accompanied by amodification of the chlorophyll fluorescence pattern, indicatinglimitations in the PSII reaction centre and subsequent electrontransport rate (Fig. 4g, h), which is in agreement with observationson citrus and mango cuttings (Larson and Schaffer, 1991; Zude-Sasse and Ludders, 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 reflectanceprofile remained unaffected in the chlorophylls and the carote-noids’ absorption bands (Table 4, Figs. 5 and 6). Leaf diffusereflectance only responded on the third day of hypoxia, when thephotosynthesis and transpiration rates had dropped close to zeroand the leaves had wilted considerably. The significant increase inthe car-ratio and the decrease of PRI indicate changes in the leafcarotenoids’ contents. However, surprisingly, none of the indicesrelated to leaf chlorophyll content was affected.

On the other hand, the tomato plants in experiments A1–A3adapted to hypoxia in the root environment and survived.Nonetheless, such behaviour was accompanied by a significantdecrease in root and shoot growth. The marked reduction in shootand root dry matter and the increased dry matter content andshoot-to-root ratio under hypoxic conditions (Table 3 and Fig. 2)are in agreement with many reports on several crops, includingtomato (Jackson and Drew, 1984; Rong and Tachibana, 1997; Shiet al., 2007). The re-establishment of normoxia-like dry mattercontents and shoot-to-root ratios one week after re-aeration(Fig. 2b, d) is an indication of the plant’s ability to cope withhypoxia, if normoxic conditions are adjusted in good time.

Although often described as a sensitive indicator of stressconditions, and in contradiction to the results in experiments L4 andL5, neither the chlorophyll fluorescence kinetic (Fig. 4a–f) nor theleaf apparent photosynthesis rate (Fig. 3a, b) changed markedly dueto hypoxic conditions in the root zone in experiments A1–A3. Theyoung tomato plants might have been able to adapt to the decreasingoxygen 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 developingadventive roots above the water level (Zude et al., 1998). In addition,the slow drop of the O2 concentration may act as a hypoxic pre-treatment, which is known to improve adaptation to anoxic

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.

H.-P. Klaring, M. Zude / Scientia Horticulturae 122 (2009) 17–25 23

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.

H.-P. Klaring, M. Zude / Scientia Horticulturae 122 (2009) 17–2524

conditions (Germain et al., 1997). It can be assumed that theapparent photosynthesis rate and chlorophyll fluorescence kineticare 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 chlorophyllcontent were not repeatedly affected by hypoxia in the rootenvironment (Table 4 and Figs. 6c–g). Reproducible differenceswere obtained from the hypoxia-adapted plants in the carotenoids’absorption band (Table 4 and Fig. 6a, b). In the present study, theratio 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 inthe carotenoids’ profile. Adaptation to oxygen deficiency alwaysincludes coping with an energy crisis, due to the lack of theterminal electron acceptor in the respiratory chain, as well asprotecting the cell against the reducing pressure (Gibbs andGreenway, 2003; Zude-Sasse et al., 2001). The latter can beobtained by the response of the xanthophyll cycle, resulting inchanges in the composition of the carotenoids.

Beside the car-ratio, the empirically derived ratio of thereflectance at 940 and 718 nm responded significantly whenplants adapted to hypoxia in the root environment. In previousstudies, NIR spectroscopy has been successfully employed to non-destructively predict fruit quality parameters, such as solublesolids and water contents in citrus and apple, as shown by manyauthors over the past 15 years (Nicolai et al., 2007; Walsh andKawano, 2008). Thus, the changes in the NIR reflectance profile ofthe hypoxia-adapted plants are probably associated with theincrease in dry matter content of the tomato leaves, as well as withchanges in the water content. However, changes of OH bondsabsorbing in the NIR may occur due to many stress factors. Fordiagnostic purposes, the differences in the carotenoids areassumed to be more feasible.

Dramatic changes in the leaf colour were observed inexperiment A3 (Fig. 7). However, it is difficult to ascribe thisresponse to a specific plant metabolite. Leaf colour in terms of leafgreenness is used in several applications, mainly in precisionfarming, as an indication of the N-nutrition status, which isassumed to be related to the leaf chlorophyll content (e.g. Blackmeret al., 1994). However, it is recognised that colour values areinfluenced by the plant growth stage, cultivar, leaf thickness, plantpopulation and any soil or climate factor causing leaf chlorosis(Turner and Jund, 1994).

After the sudden decrease of oxygen concentration in experi-ment L5, the apparent leaf photosynthesis and transpiration ratesof the mature plants fell dramatically. The plants were damagedirreversibly. This response seems to be more similar to most otherexperiments, where strong effects of anoxia on photosynthesis,water relations and stomatal conductance were found (Bradford,1983). From the perspective of practical application in irrigationcontrol, this situation is of less interest because the crop is oftenlost before corresponding management strategies take effect.

In irrigated crops, a change from aerobic to anaerobicconditions in the root environment usually evolutes within afew days, and absolute anoxia hardly ever occurs. The roots may beprotected by a hypoxic pre-treatment that has been found formany crops, including tomato (Germain et al., 1997). During thepre-treatment, several enzyme activities increased, including thatof sucrose synthase. In hypoxia-pre-treated roots, ethanol wasproduced immediately after transfer to anoxia; little lactic acidaccumulated in the tissues and sucrose was able to sustainglycolytic 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 yieldreductions. Measuring sensitive symptoms in plants adapting tohypoxia is therefore highly important, particularly if no visiblesymptoms appear while yield reductions may already occur.

The measured differences in leaf diffuse reflectance in thecarotenoids’ absorption bands may provide a sensitive tool underthese conditions. Also, reflectance in the NIR wavelength range andcolour readings responded sensitively to hypoxia. However, thelatter were tested in one experiment only, and both are difficult tointerpret in terms of changes in the plant’s metabolism.

The main difficulty with all of the tested non-invasive methodsis the absence of the (normoxia) reference in practical applications.Although this reference is present in all experiments, its absolutevalue depends on the cultivar, growth stage and several environ-mental 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 tomatoplants when the roots were supplied with oxygen, while the valueswere equal under hypoxia (Fig. 6b); or, the leaf colour parameter b*increased from the lower to the upper leaves under aerobicconditions, while it decreased under hypoxia (Fig. 7c). Nonetheless,this approach requires further investigation.

H.-P. Klaring, M. Zude / Scientia Horticulturae 122 (2009) 17–25 25

The feasibility of gas exchange sensors is rather limited ingreenhouse production. A portable infrared gas analyser with leafchamber is relatively large and heavy, and the leaf needs a certainamount of time to adapt to the conditions in the chamber,especially when aiming at transpiration. Also, the chlorophyllfluorescence technique is rather complicated to use, due to therequirement of the leaf’s adaptation to the dark prior to themeasurement. In contrast, the leaf diffuse reflectance and leafcolour can be measured quickly on many leaves using easily-to-use, hand-held spectrophotometer devices.

Acknowledgements

This research was supported by the German Federation and thefederal states of Brandenburg and Thuringia. We thank Jutta Lenkfor her excellent technical support.

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