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Summary

Four-year old clones (FG1 and FG11) of teak (Tectonagrandis Linn. f.), differing in rejuvenation capacity weregrown in glazed earthenware pots. Drought treatmentswere imposed by withholding water for 20 days and re-watered to the field capacity daily for 5 days and thepossible role of biochemical alteration and antioxidantmetabolism in conferring photosynthetic capacity wasdetermine by measuring photosynthetic traits, cellulardamage and assaying activities of the superoxide dismu-tase (SOD) and peroxidase (PER) enzymes. Growth, rel-ative water content (RWC), net photosynthetic rate (Pn),stomatal conductance (gs), chlorophyll fluorescence(Fv/Fm) and chlorophyll a, b, total chlorophyll and solu-ble protein content decreased significantly with increas-ing drought treatments from 5 to 20 days. Drought-induced stress significantly increased the carotenoidscontent, relative electrolyte leakage and malondialde-hyde (MDA) content, and, at the same time, accumulat-ed free proline, free amino acid and soluble sugars inboth clones. After re-watered to the field capacity dailyfor 5 days, both clones were shown significant recoveryin the studied parameters. As compared with the FG11,the FG1 clone was more tolerant to drought as indicatedby higher level of antioxidant enzyme activities as wellas lower MDA content and electrolyte leakage. Similar-ly, drought stress caused less pronounced inhibition of

Pn in FG1 than in FG11 clone. After re-hydration, therecovery was relatively quicker in FG1 than in FG11clone. FG1 clone showed significant recovery in maxi-mum quantum yield or photochemical efficiency of PSII(Fv/Fm) after 5 days of re-watering. The FG11 comparedto the FG1, the former clone was less tolerant todrought than the latter. These results demonstratedthat the different physiological strategies includingantioxidative enzymes employed by the FG1 and FG11clones of T. grandis to protect photosynthetic apparatusand alleviate drought stress. Furthermore, this studyalso provides ideas for teak improvement programmesand may be useful in breeding or genetic engineering fortheir tolerance to drought stress.

Key words: Antioxidative enzymes, Chlorophyll fluorescence,Drought stress, Leaf oxidative damage, Osmolyte accumula-tion, Photosynthetic capacity, Teak clones.Abbreviations: Car Carotenoids

Chl ChlorophyllDW Dry weightE Transpiration rate FAA Free amino acidFv/Fm Maximum quantum yield or photo-

chemical efficiency of PSIIFW Fresh weightgs Stomatal conductanceMDA MalondialdehydePER Peroxidase Pn Net photosynthetic rateRWC Relative water contentSOD Superoxide dismutaseTSP Total soluble protein TSS Total soluble sugar

Growth Characteristics, Physiological and Metabolic Responses of Teak (Tectona Grandis Linn. f.) Clones Differing

in Rejuvenation Capacity Subjected to Drought Stress

By AZAMAL HUSEN*)

(Received 8th August 2009)

*) Department of Biology, Faculty of Natural and ComputationalSciences, University of Gondar, P.O. Box 196, Gondar,Ethiopia. Phone: 00251-918-806304. Fax: 00251-58-1141931.E-Mail: adroot92@yahoo.co.in

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Introduction

Among naturally occurring abiotic stresses, drought isone of the most widespread global environmental prob-lems leading to low water availability for plants.Drought causes significant losses in growth, productivi-ty of plants, and finally their yields (LUDLOW andMUCHOW, 1990; JONES and CORLETT, 1992). Global cli-mate change will likely make water scarcity an evengreater limitation to plant productivity across anincreasing amount of land. It is expected that extremeweather conditions, particularly in terms of temperatureand precipitation, will be more frequent in the future.Fluctuations in water availability, including periods ofdrought, may become more common, and due to this theavailable water in the soil is reduced and adverseatmospheric conditions are cause of continuous loss ofwater in plants (SAXE et al., 2001). It is well known thatdrought affects morphological, physio-biochemical andmolecular processes in plants resulting in growth inhibi-tion, stomata closure with consecutive reduction of tran-spiration, decrease in chlorophyll content and inhibitionof photosynthesis, protein changes and differentialresponses of antioxidative enzymes (ZHU, 2002; ASHRAF

et al., 2004; YIN et al., 2005; DUAN et al., 2005; XIAO etal., 2008; CHAVES et al., 2009) to cope with osmoticchanges in their tissues. Teak (Tectona grandis Linn. f.)wood is considered, the noblest among the all timberproducing not simply because of its golden hue and won-derful texture, but even more because of its durability. Itoccurs naturally only in India and its neighboring coun-tries, and is naturalized in Java, Indonesia, where itwas probably introduced some 400 to 600 years ago. Inaddition, it has been established throughout tropicalAsia, as well as in tropical Africa, Latin America and theCaribbean. Teak has also been introduced in someislands in the Pacific region and in northern Australiaat trial levels. At present, it is a diverse and widely dis-tributed genus (TEWARI, 1992; PANDEY and BROWN,2000), and has been extensively clonally propagated(HUSEN and PAL, 2003a, b, c, 2006, 2007a, b, c; HUSEN,2008a). Its growth is often affected by a number of eco-climatic factors in India (TEWARI, 1992).

It is well documented fact that one of the primaryphysiological consequences of drought is photosynthesisinhibition (CHAVES, 1991; LAWLOR, 1995). There is nowconsiderable consensus that reduced CO2 diffusion fromthe atmosphere to the site of carboxylation in the leaf,as a result of both stomatal closure and reduced meso-phyll conductance, is the main cause of decreased photo-synthesis under most water stress conditions (GRASSI

and MAGNANI, 2005; FLEXAS et al., 2006, 2007). Stomatalclosure leads to lowering of the influx of CO2 resultingin inactivation of electron transport reactions in leaves(LAWLOR, 2002). This inequity between electron trans-port and CO2 fixation rates may result in over-reductionof the components of the electron transport chain andhence, facilitates the transfer of electrons to reactiveoxygen species (ROS), including superoxide radical,hydroxyl radical and hydrogen peroxide (BAISAK et al.,1994; POLLE and RENNENBERG, 1994; ITURBE-ORMAETXE

et al., 1998) which ultimately leads to oxidative stress(ASADA, 1994; BAISAK et al., 1994). High concentration of

ROS causes oxidative damage to photosynthetic appara-tuses, bio-molecules such as lipids, proteins and nucleicacids, leakage of electrolytes via lipid peroxidation,which results in the disruption of the cellular metabo-lism (ASADA, 1994; REDDY et al., 2004; ANJUM et al.,2008). Plants protect cellular and sub-cellular systemincluding photosynthetic apparatus from the cytotoxiceffects of active oxygen radicals with antioxidativeenzymes such as superoxide dismutase, catalase, ascor-bate peroxidase and peroxidase, and low molecularweight antioxidants (ascorbate, glutathione, proline,carotenoids, α-tocopherols and phenolics etc.) (ALSCHER

et al., 2002; REDDY et al., 2004). These components helpplants to cope with the menace of ROS, thus minimizingthe oxidative damages, during exposure to stress factorsincluding drought (FOYER et al., 1994; XIAO et al., 2008,2009). Furthermore, reports are also available ondrought-induced accumulation of osmolyte compoundssuch as inorganic ions (especially K+), sugars and aminoacids (e.g. proline) within the cell is often associatedwith a possible mechanism to tolerate the harmful effectof water stress (TURNER and JONES, 1980; ELSHEERY andCAO, 2008; XIAO et al., 2009). These accumulations ofosmolytes in plant cells might contribute, via loweringthe cell osmotic potential, to maintain several physiolog-ical processes under drought stress (BAJJI et al., 2001) orimportant in adaptive processes, i.e. osmotic adjust-ments (YIN et al., 2005; XIAO et al., 2008, 2009).

The frequency and intensity of droughts is increasingin tropical regions due to high irradiance and high tem-perature, which may leads to alterations in growth, pho-tosynthesis and antioxidative defence system in variousplant species. Like many other plant species that growin the tropical environments, teak plantations are alsoexposed to long periods of drought stress. Therefore, ithypothesized that there would be a large set of parallelchanges in the morphological, physiological and bio-chemical responses when plants were exposed todrought stress, these changes may enhance the capabili-ty of plants to survive and grow during drought periods.The objective of this study was to compare the effect ofwater-withholding and subsequent re-hydration ongrowth, photosynthetic gas exchange, chlorophyll fluo-rescence, osmotic compound accumulation, membraneperoxidation and antioxidant enzyme systems in twoteak clones to determine which clone is more tolerant todrought, and to aid in breeding and genetic engineeringprogrammes for drought resistance.

Materials and Methods

Experimental site and plant material

The study was conducted at the Plant Physiologynursery of New Forest campus (30°20’40“N, 77°52’12“Eat 640 m above mean sea level), Forest Research Insti-tute (FRI), Dehra Dun, India. Plantlets (clones FG 1 andFG11) of Tectona grandis Linn. f. (Verbenaceae) wasobtained from the rooted cuttings (HUSEN and PAL,2003a). The rooted cuttings were transplanted in poly-bags (9’’ x 6’’) in July 1999, filled with the mixture ofloam soil, sand and farmyard manure in 2:1:1 (1.5 kg).These polybags with rooted cuttings were kept inside a

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green house; the upper portion of the green house wascovered with green plastic shade, while the other partsremained open. In January 2003, both clonal materials(healthy and approximately equal height) were trans-ferred to the glazed earthenware pots (9’’ x 9’’) @ 4.00 kgmedium per pot. Standard method was used to analyzethe soil, e.g. pH in water, N by micro-Kjeldhal method(JACKSON, 1962), P by KURTZ and BRAY method (1945),C by WALKLEY and BLACK method (1934), and Ca, Mg,Na, and K by atomic absorption. Soil texture was deter-mined by the methods of BOYOCOUS (1962). Data are pre-sented in Table 1. Complete protection was providedagainst diseases and insects by foliar spray with insecti-cides and fungicides, as and when required. Furtherthese plants were maintained carefully by regularwatering and weeding.

Growth condition and experimental design

Experiment was executed inside the green house asdescribed above. Four-year old healthy clones of uniformheight were obtained. Drought and re-hydration treat-ment was conducted from 2 to 26 July 2003, twenty-fivepotted clones were used for each treatment and in total125 plants of each clone (FG1 and FG11) were taken.FG1 and FG11 clones were differing in their reju-venation capacity (HUSEN and PAL, 2003a). The com-pletely randomized factorial design was used for thisexperimentation. Five replications, each of five plants(5 x 5 = 25) were used for each treatment. All the plantswere irrigated to the field capacity on the day prior tothe start of the drought treatment. Drought wasimposed by withholding water for 20 days. Immediatelyafter these 20 days, all the plants were re-watered tothe field capacity daily for 5 days (stress relief phase).Pot soil surfaces were covered with rice mulch to mini-mize evaporation, thus allowing a slower establishmentfor the water stress. The soil of the pots was mulchedwith plastic to avoid roots spreading into the ground.During drought and re-hydration treatment inside thegreen house, the relative humidity was 78 ± 2% whilemaximum and minimum day/night temperature was33 ± 1 and 23 ± 1°C respectively.

Determination of growth and physiological parameters

The growth and physiological measurements weredone on the day before treatment (control), on 5, 10, 15,

20 day since the initiation of the drought treatment andsubsequent 5 day of re-hydration. The height (cm) wasmeasured from the ground line of earthenware pots upto the tip of FG1 and FG11 clone. Similarly, the groundline basal diameter (mm) was measured.

Net photosynthetic rate (Pn), stomatal conductance(gs) and transpiration rate (E) were measured using aLi 6400 photosynthetic system (Li-Cor, Inc., Nebraska,USA). From each replication fully mature and expandedleaves of each clone were measured during the periodof 1000 to 1200 h in the morning, under photo-synthetic flux density of 1,000 µmol m–2 s–1 that wasprovided by a LED light source. The air humidity in theleaf chamber was about 60%, with CO2 concentration of370–380 µmol mol–1, with ambient air temperature of26–28°C, and flow rate of 500 µmol s–1.

Chlorophyll fluorescence measurements were per-formed during 1000 to 1100 h in the morning with thehelp of a portable Hansatech Plant Efficiency Analyser(Hansatech, King’s Lynn, England). The leaves werepre-darkened with leaf clips for 20 min before measure-ment. Fluorescence was excited by red (actinic) radia-tion with 650 nm peak wavelength obtained from lightemitting diodes. The irradiance used was 2000 µmolm–2S–1. Maxmium quantum yield of PSII was estimatedby the ratio Fv/Fm = (Fm – Fo)/ Fm according to GENTY

et al. (1989).

Determination of leaf water status

After growth, gas exchange and chlorophyll fluores-cence measurements, leaf discs from the same clone(FG1 and FG11) leaves were sampled. Five replicatesper clone were obtained. Leaf relative water content(RWC) was calculated as (WEATHERLEY, 1950):

RWC = [(fresh weight – dry weight) ÷ (turgid weight –dry weight)]

Turgid weight was determined by placing samples indistilled water and maintaining them at 5°C in dark-ness until they reached a constant weight. Full turgorwas typically reached after 12 h. Dry weight wasobtained after placing the samples in an oven at 70°Cfor 48 h.

Determination of electrolyte leakage

Electrolyte leakage was assayed by estimating theions leaching from leaf into distilled water (SAIRAM etal., 1998). Five replicates per clone were obtained. Leafmaterial of 300 mg was taken in 10 ml of deionizedwater in two sets. One set was subjected to room tem-perature (approximately 25°C) for 4 h and its electricalconductivity (C1) was determined. The other set waskept in a boiling water bath (100°C) for 10 min and itselectrical conductivity was also determined (C2). Per-cent electrolyte leakage was calculated as below:

Electrolyte leakage = [1 - (C1/C2)] x 100

Determination of photosynthetic pigments, TSP, TSS,FAA, proline, MDA content, SOD and PER enzyme activities

Fully mature and expanded leaves were randomlyselected from each replication for the estimation of bio-

Table 1. – Chemical and physical properties of soil (pottingmedium) used in the experiment.

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chemical contents. For estimating biochemical contentsin leaves five replication, each of five leaves (5 x 5 = 25)were randomly chosen from FG1 and FG11 clones.

The content of chlorophyll (Chl) ‘a’, ‘b’ and total Chl inleaves was analysed by keeping 0.1 g samples in 7 ml ofdimethyl sulfoxide (DMSO) in the oven at 65°C for 2 h.Then 3 ml of DMSO was added in 1 ml of aliquot. Opti-cal density was measured using Perkin Elmer UV/VISspectrophotometer Lamda 25 (HISCOX and ISRAELSTAM,1979). The contents of Chl ‘a’, ‘b’ and total Chl were esti-mated by the formula given by DUXBURY and YENTSCH

(1956) and MACLACHLAN and ZALICK (1963) respectively.

The content of total soluble proteins (TSP) in leaveswas measured in phosphate buffer extracts at 595 nmafter reaction with trichloroacetic acid (TCA) and Brad-ford reagent (BRADFORD, 1976). Bovine serum albuminwas used as a standard. The amount of TSP wasexpressed as mg g–1 FW.

Extracts from leaves were prepared as per the methoddescribed by SAWHNEY et al. (1968), and total solublesugars (TSS) content was estimated by the phenol-sulfu-ric-acid method (DUBOIS et al., 1956). The amount ofTSS was expressed as mg g–1 DW.

Free amino acids in leaves were determined using themethod suggested by LEE and TAKAHASHI (1966). Theamount of free amino acids was expressed as mg g–1 FW.

Extraction and estimation of free proline were con-ducted according to the procedures described by BATES

et al. (1973). The corresponding concentration of prolinewas determined against the standard curve processed inthe same manner by using L-proline. The amount of pro-line in leaf samples was expressed as µmol g–1 FW.

Leaf oxidative damage to lipids was expressed asequivalents to malondialdehyde (MDA). Its content wasdetermined as described by HODGES et al. (1999) withslight modification, for taking into account the possibleinfluence of interfering compounds in the assay for thio-barbituric acid (TBA)-reactive substances. Leaf tissueswere repeatedly extracted with 4 ml 5% (w/v) TCA. Thehomogenate was centrifuged at 15,000 x g for 15 minand an aliquot of appropriately diluted sample wasadded to a test tube with an equal volume of either: (1) –TBA solution containing 20% (w/v) TCA and 0.01%butylated hydroxytoluene (BHT), or (2) +TBA solutioncontaining the above solution plus 0.65% (w/v) TBA.Samples were heated at 95°C for 25 min, and thencooled immediately. The absorbance was read at 440,532 and 600 nm using Perkin Elmer UV/VIS spec-trophotometer Lamda 25. MDA equivalents were calcu-lated as 106 x [(A -B)/157,000], where A = [(OD532 + TBA)– (OD600 + TBA) – (OD532 – TBA – OD600 – TBA)], and B = [(OD440 + TBA – OD600 + TBA) x 0.0571].

An enzyme extract for superoxide dismutase (SOD) ata cold temperature was prepared by first freezing aweighed amount of leaf sample (1 g) in liquid nitrogen toprevent proteolytic activity, followed by grinding with10 ml of extraction buffer (0.1M phosphate buffer,pH7.5, containing 0.5mM EDTA and 1mM ascorbicacid). Brie was passed through four layers of cheeseclothand filtrate was centrifuged for 20 min at 15,000 x g,4°C and the supernatant was used as enzyme (PROC-

HAZKOVA et al., 2001). SOD activity was estimated byrecording the decrease in optical density of nitrobluetetrazolium dye by the enzyme (DHINDSA et al., 1981).Three millilitres of the reaction mixture contained13mM of methionine, 25mM of nitroblue tetrazoliumchloride, 0.1mM of EDTA, 50mM of phosphate buffer(pH7.8), 50mM of sodiumcarbonate and 0.1 ml ofenzyme. The reaction was started by adding 2 mM ofriboflavine and placing the tubes under two 15W fluo-rescent lamps for 15 min. A complete reaction mixturewithout enzyme, which gave the maximal colour, servedas a control. The reaction was stopped by switching offthe light and placing the tubes in the dark. A non-irradi-ated complete reaction mixture served as a blank. Theabsorbance was recorded at 560 nm using Perkin ElmerUV/VIS spectrophotometer Lamda 25, and one unit ofenzyme activity was taken as that amount of enzymewhich reduced the absorbance reading to 50% in com-parison with tubes lacking enzyme.

Peroxidase (PER) enzyme activity was measuredusing guaiacol as substrate following the methods ofHUSEN (2008b). The assay mixture contained 0.1 Mphosphate buffer (pH 6.1), 4.0 mM guaiacol as donor,3.0 mM H2O2 as substrate, and 0.4 ml crude enzymeextract. The total reaction volume was 1.2 ml. The opti-cal density was measured at 420 nm using a PerkinElmer UV/VIS spectrophotometer Lamda 2S. Theenzyme activity was expressed as the rate of optical den-sity change min–1 mg–1 protein (BARNETT, 1974).

Statistical analysis

Statistical analysis was performed with the StatisticalPackage for Social Sciences (SPSS) windows® softwarepackage. The data recorded on the growth, physiologicaland biochemical parameters were subjected to two-wayANOVA. Means were compared by using Tukey’s test atsignificance level P < 0.05 (the same letters indicate thatmeans within a row or column are not significantly dif-ferent at P > 0.05 level).

Results

Growth parameters, relative water content and photosynthetic traits

Plant height and basal diameter of two clones showeda significant relationship with increasing number ofdays since drought treatment (Table 2 and 3). After 20days withholding water, plant growth in terms of heightof FG1 and FG11 were 1.22% and 0.61% respectivelycompared to control. In contrast, basal diameter incre-ment of FG1 and FG11 were 1.52% and 2.07%, respec-tively compared to control. The interaction effectbetween clone and drought treatment was significant forheight increment (Table 3).

Relative water content (RWC) of two clones progres-sively decreased with increasing number of days sincedrought treatment (Table 2). After 20 days withholdingwater, compared to control, the decrease in leaves RWCwas more in FG11 (51.56%) than FG1 (49.59%). Afterre-watering for 5 days, a full recovery of leaf water sta-tus was achieved in FG1 while at the same time FG11did not show complete recovery. The interaction effect

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between clone and drought treatment was also signifi-cant for RWC (Table 3).

Chlorophyll (Chl) a, b and total Chl were reducedseverely with increasing number of days since droughttreatment (Table 2) in both clones. After 20 days with-holding water, content of Chl a in FG1 and FG11 were47.83% and 48.89% respectively, compared to control.After 5 days of re-watering a full recovery in content ofChl a was achieved in FG1 while at the same time FG11did not show complete recovery. After 20 days withhold-ing water, in FG1, the decrease in Chl b was 48.65%,and in FG11 was 50.00%. However, after 5 days of re-watering, content of Chl b was fully recovered in bothclones. Total Chl content, after 20 days withholding, thedecrease in FG1 and FG11 were 43.82% and 44.83%respectively, compared to control. After 5 days of re-watering, total Chl was fully recovered in FG1 while,FG11 show partial recovery. Drought treatment signifi-cantly increased the carotenoids (Car) content in bothclones. The increase in FG1 and FG11 were 78.13% and75.00%, respectively. However, after re-watering for 5days, both clones did not show complete recovery in con-tent of Car.

Exposing 20 days of drought treatment over teakclones produced significant changes in the net photosyn-thetic rate (Pn), stomatal conductance (gs) and transpi-ration rate (E) chlorophyll fluorescence (Fv/Fm) (Table 2and 3). The decrease of Pn, gs and E in FG1 were69.22%, 82.56%, and 72.17%, respectively as comparedto control. However, in FG11, the decrease of Pn, gs andE were 74.25%, 86.21% and 73.33%, respectively. FG11was more affected by drought treatment than FG1

clones. Both clones did not show a complete recoveryafter 5 days of re-watering as indicated by the restitu-tion of water status and gas exchange rates to the con-trol values. Inhibition of photosynthetic rate was accom-panied by parallel decrease in chlorophyll fluorescence(Fv/Fm). After 20 days withholding water, Fv/Fm valuesdecreased by 22.67% in FG1 and by 23.33% in FG11.After 5 days of re-watering a full recovery in Fv/Fm wasachieved in FG1 while at the same time FG11 did notshow complete recovery (Table 2). The interaction effectbetween clone and drought treatment was significant forPn, gs, E and Fv/Fm (Table 3).

The content total soluble protein (TSP) was sensitiveto drought treatment. As shown in Table 3, treatmentwith drought stress at 20 days withholding water, grad-ually decrease the content of TSP. Content of TSPdecreased by 39.27% and 38.81% in FG1 and FG11clones, respectively. This decrease of TSP was more inFG11. TSP content in both clones did not show a com-plete recovery after 5 days of re-watering as comparedto the control values. However, TSP recovery was nearto the 5 days withholding water, i.e. drought treatment.

Osmotic adjustment

Exposing the teak clones to the drought treatmentcaused a significant change in proline, free amino acids(FAA) and total soluble sugar (TSS) content (Figure 1and Table 3). The content of proline, FAA and TSS wereincreased progressively during the withholding water inboth the clones. On 20 days, proline content increasedby 87.09% in FG1 and by 82.09% in FG11. At the sametime, FAA content increase by 60.18% in FG1 and by

Table 2. – Growth, relative water content (RWC), net photosynthetic rate (Pn), stomatal conductance (gs), transpirationrate (E), chlorophyll fluorescence (Fv/Fm), photosynthetic pigments (Chl a, b, total Chl and Car) and protein (TSP) intwo clone of Tectona grandis on the day before treatment (control), 5, 10, 15, 20th day since the start of the droughttreatment and on 5th day of re-hydration (RHD). Values followed by the same letter indicate no significant differences atP < 0.05 level according to Tukey’s test. Each value represents the mean ± SE of five replicates.

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59.34% in FG11. After 5 days of re-watering, both clonesshow a complete recovery in content of proline. While,FG11 shows incomplete recovery in FAA content as com-pared with the control. In terms of recovery, the value ofFAA content in FG11 was near to the 5 days withholdingwater, i.e. drought treatment. Drought treatment also,leads to an increase in TSS content by 27.36% in FG1and by 26.81% in FG11. After 5 days of re-watering,both FG1 and FG11 teak clones did not show a completerecovery. However, TSS content recovery situation wasnear to the 5 days withholding water.

Electrolyte leakage and leaf oxidative damage

To evaluate the drought stress-induced oxidative dam-age to membranes, the electrolyte leakage, which is anindicator of lipid peroxidation, and malondialdehyde(MDA) which is a product of lipid peroxidation, weredetermined. Due to exposure of drought treatmentcaused a significant increase in electrolyte leakage aswell as MDA (Figure 2 and Table 3). The increase inelectrolyte leakage in FG1 was found 72.29%, while inFG11 accumulated 89.05% as compared with the con-trol. However, after re-watering for 5 days, FG11 clonesdid not show complete recovery. Further there was a sig-nificant interaction effect between clone and droughttreatment in electrolyte leakage from the leaves of teak(Table 3). The increase MDA content was noticed by172.73% in FG1 and by 182.78% in FG11, after 20 dayswithholding water. Both FG1 and FG11 clones shows acomplete recovery after 5 days of re-watering as com-pared to the control values of MDA content.

Enzymatic antioxidant activities

The significant drought treatment responses on activi-ties of superoxide dismutase (SOD) and peroxidase

(PER) enzymes were noted in both FG1 and FG11 clones(Figure 3 and Table 3). After 20 days withholding waterresulted in elevated expression of SOD activity by67.50% and 64.12% for FG1 and FG11 clone, respective-ly. After 5 days of re-watering, FG1 had higher SODantioxidant activity than FG11 clones and did not showa complete recovery as compared with the control. Simi-lar trend of variation was noticed for PER activity, itincreased by 27.53% and 25.44% in FG1 and FG11clone, respectively. After 5 days of re-watering, FG11clone show a complete recovery, while FG1 was not fullyrecovered. However, in FG1 clone, the PER activityrecovery was near to the 5 days withholding water con-dition. Both SOD and PER antioxidant activity wasfound higher in FG1 clones during the drought treat-ment and also after 5 days of re-hydration.

Discussion

The availability of water is one of the major limitingfactors for normal plant growth. Plants growing in tropi-cal climate often face certain degree of drought stress.As a result, it influences plant growth by affecting celldivision, enlargement and differentiation and also theplant genetic make-up (PATEL and GOLAKIA, 1988). Inthe present study, drought stress significantly slowsdown and reduced the plant height and basal diameterin both the clone of teak. This may be due to the avoid-ance mechanism through adjustment of plant growthrate or decreased rate of photosynthesis. Reduction interms of growth was obtained for many other plantspecies (ZHANG et al., 2005; YIN et al., 2005; XIAO et al.,2009). In this study, RWC in leaves decreased signifi-cantly. However, RWC was more reduced in FG11 thanFG1 clone. After 5 days of re-hydration FG11 did notshow complete recovery, this suggests that FG1 needs

Table 3. – ANOVA result on the effect of clone, drought treatment and their combination on variousgrowth, physiological and biochemical parameters (MSS mean square value; * and ** significance levelat P < 0.05 and P < 0.01 respectively; ns non significant).

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lower RWC maintenance. Drought stress-induceddecrease in RWC has been reported in many previousstudies (DUAN et al., 2005; ELSHEERY and CAO, 2008;XIAO et al., 2009).

The contents of photosynthetic pigments in plantssubjected to drought stress are known to decline (DUAN

et al., 2005; ELSHEERY and CAO, 2008). Drought condi-

tion decreased the chlorophyll content considerably inboth the teak clones. This may be due to reduction inthe lamellar content of the light harvesting Chl a/b pro-tein that accounts for the elevated Chl a/b ratio (2.8:4.5)and/ or due to water stress-induced decrease in theavailability of mineral nutrients, and/or to the damagesto chloroplasts by ROS, and or either slow synthesis or

Figure 1. – The content of free amino acids (FAA), proline and total soluble sug-ars (TSS) in two clones of Tectona grandis (FG1 white bars and FG11 gray bars)on the day before treatment (Control), 5, 10, 15, 20th day since drought treatmentand 5th day since re-hydration (RHD). Values followed by the same letter indicateno significant differences at P < 0.05 level according to Tukey’s test. Each valuerepresents the mean ± SE of five replicates.

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fast breakdown of chlorophyll pigments (KHANNA-CHOPRA et al., 1980; ASHRAF, 2003). However in contrast,carotenoid contents significantly increased in both FG1and FG11 clones. This confirm to earlier report on Popu-lus cathayana, where under moderate drought stresscarotenoids contents was increased (XIAO et al., 2008).The carotenoids content has essential functions in pho-tosynthesis and photoprotection. In addition to structur-al roles, they are well known for their antioxidant activi-ty by quenching 3Chl and 1O2, inhibiting lipid peroxida-tion, and stabilizing membranes (DEMMIG-ADAMS andADAMS, 1992; FRANK and COGDELL, 1996; NIYOGI, 1999).Carotenoids also play a critical role in the assembly ofthe light-harvesting complex and in the radiationlessdissipation of excess energy (STREB et al., 1998; MUNNÉ-BOSCH and ALEGRE, 2000).

In the present study, Pn, gs, E decreased significantlyunder drought stress. Reductions in the photosyntheticactivity induced by drought were first triggered by stom-atal closure, resulting in limitation of ambient CO2 dif-fusion to the mesophyll and thus reduction of photosyn-thesis. Further, results showed that gs was more sensi-

tive in response to drought treatment before the changein the leaf water content is detectable, as has been alsoshown in other species (GOLLAN et al., 1985; SOCÍAS etal., 1997). This reveals that these teak clones employthe physiologically drought-avoidance strategy (GULIAS

et al.. 2002). At later stage of drought treatment, the Pnreduction may be caused by both stomatal and non-stomatal mechanisms (EPRON et al., 1992; MAXWELL andJOHNSON, 2000). After 5 days of re-watering, gasexchange parameters did not show a complete recoverymay be due to limitation of stomatal and mesophyll con-ductance and biochemical limitations (GALMÉS et al.,2007). In many species, PSII is quite resistant todrought (GENTY et al., 1987; CORNIC et al., 1992). Maxi-mum quantum yield of PSII (Fv/Fm) did not change sig-nificantly under moderate drought stress in both clones,indicating no photodamage to PSII reaction centers(FOYER et al., 1994). After 5 days of re-watering, bothclones showed rapid recovery in Fv/Fm in parallel withthat of Pn. However, FG1 teak clones recovered com-pletely, but FG11 was recovered only up to the moderatedrought stress treatment, i.e. 5 days of drought treat-

Figure 2. – Electrolyte leakage and content of malondialdehyde (MDA) in twoclones of Tectona grandis (FG1 white bars and FG11 gray bars) on the day beforetreatment (Control), 5, 10, 15, 20th day since drought treatment and 5th day sincere-hydration (RHD). Values followed by the same letter indicate no significantdifferences at P < 0.05 level according to Tukey’s test. Each value represents themean ± SE of five replicates.

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ment. Therefore, this indicated no irreversible damagesto photosynthetic reaction centers occurred. After 20days withholding water, the FG1 clone quickly recoveredin a few days (5 days) and FG11 clones are very near torecovery. Moreover, the recovery of photochemistry anddown regulation of heat dissipation occurred in theshort period. The role of stomatal closure and biochemi-cal processes in decreasing net photosynthetic rates dur-ing drought stress is well established. The same holdalso in teak clones when drought treatment varies from0 to 20 days of treatment and after re-hydration. Thedrought stress-dependent decrease in the content of sol-uble proteins in the FG1 and FG11 clones studied couldbe due to enhanced degradation of proteins as a result ofprotease activity (PALMA et al., 2002) and polysomeactivity and/or due the generation of ROS (BAISAK et al.,1994; KRAMER and BOYER, 1995). Excess light energy canenhance production of free oxygen radicals, which leadto peroxidation of fatty acids in cell membranes (PARKIN

et al., 1989; FOYER et al., 1994). In the present study,drought stress-induced considerable damage on the cel-lular membranes in both clones is assessed by lipid per-oxidation and electrolyte leakage. Increased electrolyte

leakage percentage and higher content of MDA, which isthe product of membrane lipid peroxidation, were foundin the leaves of the teak clones after drought treatment.However, increase in electrolyte leakage percentage wasmore pronounced in FG11 clones than FG1. Thisincrease in cellular damage appeared to reflect impair-ments in the equilibrium between the ROS productionand antioxidant defence systems, which also indicatedthat photoprotection and antioxidant substances werenot sufficient enough to protect against cell membranedamage caused by ROS. Drought stress-induced damageon the cellular membranes has been reported also inmany previous studies (YIN at al., 2005; DUAN et al.,2005; ELSHEERY and CAO, 2008; XIAO et al., 2009).

The physiological mechanisms that allow a species totolerate prolonged periods of drought stress can involvenumerous attributes including the accumulation ofosmotically active solutes (free amino acid, proline andtotal soluble sugar), so that turgor and turgor-depend-ent processes may be maintained during the episodes ofdry-down. In this study, FAA content increased underdrought treatment, as compared with control. This con-forms to earlier reports of YADAV et al. (2005) and MANI-

Figure 3. – Activities of superoxide dismutase (SOD) and peroxidase (PER)enzymes in two clones of Tectona grandis (FG1 white bars and FG11 gray bars)on the day before treatment (Control), 5, 10, 15, 20th day since drought treatmentand 5th day since re-hydration (RHD). Values followed by the same letter indicateno significant differences at P < 0.05 level according to Tukey’s test. Each valuerepresents the mean ± SE of five replicates.

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VANNAN et al. (2007). Accumulation of amino acids maybe due to the hydrolysis of protein and may occur inresponse to the change in osmotic adjustment of cellularcontents (GREENWAY and MUNNS, 1980), an importantmechanism that alleviates some of the detrimentaleffects of water-deficit stress (MORGAN, 1984). Accumula-tion of free amino acids under water deficit stress indi-cates the possibility of their involvement in osmoticadjustment, as reported earlier (YADAV et al., 2005;MANIVANNAN et al., 2007; ANJUM et al., 2008). The con-tent of proline, a protective solute in teak leavesincreased due to drought treatment in both the clonesstudied. Proline is an amino acid and known to act as anosmolyte, protecting the plasma membrane integrity(MANSOUR, 1998), as a sink of energy or reducing power(VERBRUGGEN et al., 1996), as a scavenger of ROS andtheir derivatives (HONG et al., 2000; BASHIR et al., 2007)and as a source for carbon and nitrogen (PENG et al.,1996), and thereby helping the plants to tolerate stresseffects (MANIVANNAN et al., 2007). In the present teakclonal materials proline accumulation thus appears tobe related to mechanisms associated with tolerance todesiccation (MANIVANNAN et al., 2007). Drought treat-ment also enhanced the accumulation of other compati-ble solute such as total soluble sugars in both clones.This was in agreement with the results observed inother studies (ELSHEERY and CAO, 2008; XIAO et al.,2009). Correlation between sugar accumulation andosmotic stress tolerance has been widely reported,including transgenic experiments (CHAVES et al., 2003;BARTELS and SUNKAR, 2005). Sugars have different func-tions in plants from energy storage to signaling, andplants utilize several sugar-based strategies to adapt toenvironmental stresses (ANDERSON and KOHORN, 2001;CHAVES et al., 2003). The current hypothesis is that sug-ars act as osmotica and/or protect specific macromole-cules and contribute to the stabilization of membranestructures (BARTELS and SUNKAR, 2005). Generally, solu-ble sugar content tends to be maintained in the leaves ofdrought-stressed plants, although rates of carbon assim-ilation were partially reduced. The maintenance of solu-ble sugar content may be achieved at the expense ofstarch which drastically declines (CHAVES, 1991).Increase in soluble sugar contents through inversion ofsome carbohydrates may contribute to enhanced desic-cation tolerance and allows metabolic activity to bemaintained. Overall, in general the higher accumulationof compatible solutes was found in FG1 teak clones.

A variety of abiotic stresses, including drought causedmolecular damage to plant cells either directly or indi-rectly through the formation of ROS. Drought stress-induced generation of ROS triggers activation of compo-nents of antioxidative defence system of plants, whichcomprises both enzymatic and non-enzymatic low molec-ular weight antioxidants. In the present study, duringdrought treatment SOD activity continually increasedand PER activity also activated in both FG1 and FG11clones. However the observed higher antioxidant activi-ties of SOD and PER were fond more in the FG1 clones.This results show that the FG1 clones has stronger abil-ity of scavenging ROS to defence stress/or stronger toler-ance capacity (SOFO et al., 2004). This fact is re-inforced

by the lower levels of lipid peroxidation and electrolyteleakage in the FG1 than those in the FG11 clones.Therefore, the ability to increase the antioxidant SODand PER activity in order to limit cellular damagesmight be an important attribute linked to the FG1clones.

In conclusion, the responses of the two teak clones todrought stress were investigated through some impor-tant morphological, physiological and biochemicalprocesses. The results indicate that drought stress caus-es a pronounced slow down of growth, decreased leafwater status, chlorophyll pigments, soluble protein andincreased carotenoids contents and cellular damage inboth clones. The rate of photosynthesis, stomatal con-ductance, transpiration rate and chlorophyll fluores-cence in FG1 and FG11 clones were also effectivelydecreased due to drought stress. Drought stress-inducedincreased accumulation of osmotically active solutessuch as proline, free amino acids and soluble sugarswere observed. In addition, antioxidant systems includ-ing SOD and PER were also activated by drought treat-ment. On the other hand, there were significant differ-ent responses to drought treatment between the twoclones of T. grandis. In comparison, FG1 teak clones(high rejuvenation capacity) showed higher resistance todrought than FG11 (low rejuvenation capacity) clonesbecause of stronger photoprotection, osmotic adjust-ment, less ion leakage during cellular damage and high-er activity of antioxidative enzymes in response todrought treatments. Further, both teak clones showedthe stable maximum photochemistry during 20 days ofwithholding and rapid recovery in photochemistry in afew days (5 days) after rehydartion, indicating bothclones are quite drought-tolerant on the other hand.Moreover, these differences in drought responses alsoprovide some useful clues for teak improvement pro-gramme and may be useful in breeding or genetic engi-neering for their tolerance to drought stress.

Acknowledgments

This work was partially supported by World BankForest Research Education and Extension Programme(FREEP) and Indian Council of Forestry Research andEducation (ICFRE), Dehra Dun, India.

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Der große Zander. Enzyklopädie der Pflanzen-namen (2 Bände). Von W. ERHARDT, E. GÖTZ,N. BÖDECKER und S. SEYBOLD. 2008. Verlag Ulmer,Stuttgart. 2112 S., 3000 Zeichnungen, geb., ISBN 978-3-8001-5406-7. 99,00 € [D].

The encyclopaedia has more than twice the number ofpages than the dictionary of plant names which is editedby the same publisher. This widened version of the wellknown Zander (plant name dictionary) is divided intotwo volumes.

In volume1 plant families and genera are presented.More than 3000 black and white drawings illustratedthe description of about 3600 genera, which show char-acteristic traits of one plant for each genus. Their namesare explained and main characteristics are listed. Theinternational code of botanical nomenclature (ICBN)and the international code of cultivated plants (ICNCP)are detailed cited.

In volume 2 more than 25000 species and the mostcommon 7500 varieties are listed. The scientific name,the common German, French and English names arewritten. Additionally 10000 synonyms are given. Onabout 450 sides a short biography of all authors is given.

Some mistakes are in this huge book. For example theGerman Abies names are not following the same rule. Inthe plant name biographies the author J. RICHMONDBOOTH (1799–1847) is confounded with JOHN GODFREYBOOTH (*1830). A good reference index and a comprehen-sive figure index close this edition.

By the number of mistakes it seems that the book wasproduced quickly, however, it is worth to purchase itbecause of the huge number of species and cultivars.

MIRKO LIESEBACH, Großhansdorf

Book-Review