ARTICLE IN PRESS

Flora 205 (2010) 144–151

Contents lists available at ScienceDirect

Flora

0367-25

doi:10.1

� Corr

E-m

journal homepage: www.elsevier.de/flora

An integrated diagnostic approach to understand drought tolerance inmulberry (Morus indica L.)

Anirban Guha, Debashree Sengupta, Girish Kumar Rasineni, Attipalli Ramachandra Reddy �

Department of Plant Sciences, School of Life Sciences, University of Hyderabad, Hyderabad 500046, India

a r t i c l e i n f o

Article history:

Received 3 December 2008

Accepted 21 January 2009

Keywords:

Drought tolerance

Photosynthetic rate

a-Tocopherol

Lipid peroxidation

Anatomy

Mulberry

30/$ - see front matter & 2009 Elsevier Gmb

016/j.flora.2009.01.004

esponding author. Fax: +9140 23010120.

ail address: arreddy@yahoo.com (A. Ramacha

a b s t r a c t

Four popular mulberry cultivars (Morus indica L. cvs.V-1, MR-2, S-36 and K-2) were assessed for drought

tolerance with an integration of selective approaches. The potted plants were subjected to two watering

treatments for 75 days: control pots were watered up to 100% field capacity (FC) and stressed pots were

maintained at 25–30% FC. Net photosynthetic rate (Pn), stomatal conductance (gs), transpiration rate (E)

and instantaneous water use efficiency (WUEi) were the key parameters to assess photosynthetic gas

exchange performance. Drought caused marked down-regulation in leaf gas exchange in all cultivars

(cvs) except V-1 which maintained better Pn, gs, E and higher WUEi under severe water deficit. All the

four cvs also showed differential antioxidative responses under water stress. Higher concentrations of

carotenoids, ascorbic acid, glutathione, a-tocopherol and proline were observed in the leaf extracts of V-

1, while minimum accumulation of those metabolites was recorded with K-2 and S-36. An endogenous

loss of a-tocopherol and higher lipid peroxidation were encountered in K-2, S-36 and MR-2, whereas V-

1 showed minimum lipid peroxidation under water deficit regimes. Comparative morpho-anatomical

analysis revealed a well-developed root system and a better anatomical architecture in V-1 which could

further contribute tolerance during drought stress.

& 2009 Elsevier GmbH. All rights reserved.

Introduction

Plants acquire stress tolerance by altering internal metabolismand the expression of genes. In some cases, innate developmentaland phenotypic traits also confer plants the ability to functionbetter in critical environmental situations (Pathan et al., 2004).However, development of crop plants tolerant to various environ-mental stresses is a burgeoning field of stress biology andconsidered to be the most challenging. Among various abioticstresses, restricted water availability is one of the over-ridinglimitations for achieving potential crop productivity. Coping withwater limitation occurs either by morphological or by physiolo-gical acclimations to drought that make plants resistant andproductive. Reduction in plant biomass, increased root/shoot ratio,increased stomatal resistance, resulting in reduced transpirationand net photosynthetic rate (and this, in turn, in a reduced growthrate) are the most common morpho-physiological responses todrought stress in higher plants (Sharp et al., 2004; Susiluoto andBerninger, 2007). Involved are also biochemical responses thatenable plants to circumvent damage from toxic reactive oxygenspecies (ROS) under water limited conditions. The accumulationof stress metabolites like poly-sugars, proline, glycinebetaine

H. All rights reserved.

ndra Reddy).

(GB), abscicic acid (ABA) as well as up-regulation in synthesis ofenzymatic and non-enzymatic antioxidants like superoxide dis-mutase (SOD), catalase (CAT), ascorbate peroxidase (APX),glutathione reductase (GR), ascorbic acid (AA), a-tocopherol andglutathione are some of the significant biochemical responses inhigher plants under water limiting situations (Cattivelli et al.,2008; Ramachandra Reddy et al., 2004; Valliyodan and Nguyen,2006). A high degree of tolerance to drought stress is also offeredby constitutive anatomical features which are stable in higherplants and hence act as better-observed indicators (Kulkarni et al.,2008; Rhizopoulou and Psaras, 2003). For a comprehensiveunderstanding of water stress tolerance mechanisms in higherplants, aspects of physiology and cellular biochemistry should beinvestigated in combination with morpho-anatomical traits inorder to find out the subtle links leading to better droughtresistance. Such integrated traits, expressed at a higher level oforganization are suggested to be quintessential in crop improve-ment programs.

Mulberry (Morus spp. L.), a promising cash crop of India, iscultivated under the risk of either intermittent or terminaldrought, as 50% of the country’s mulberry acreage falls underarid and semi-arid conditions. Moisture stress frequently limitsboth the quality and yield of mulberry leaf. A limited number ofstudies primarily investigated the role of osmoprotectants andantioxidants in mulberry under varying degree of drought stress(Kotresha et al., 2007; Ramachandra Reddy et al., 2005). Few

ARTICLE IN PRESS

A. Guha et al. / Flora 205 (2010) 144–151 145

reports were focused upon the identification of physiologicalcharacters and morpho-anatomical features that impart droughttolerance to mulberry (Susheelamma and Datta, 1995). In anattempt to better understand the responses of mulberry plantsunder severe water deprivation, we analyzed selected sets ofphysiological, biochemical and morpho-anatomical drought stressmarkers in four popular mulberry cultivars (cvs) that frequentlyare used in cultivation. The goal of the present research was toobtain new insights into the inter-linked mechanism underlyingdrought tolerance in mulberry as well as to screen for efficientcv(s) that could be used for producing better leaf yield even underadverse environmental conditions.

Materials and methods

Plant material, experimental design and growth conditions

Four mulberry cvs (Morus indica L., V-1, S-36, MR-2 and K-2)were selected for the study. Healthy stem cuttings of the four cvswere planted in 35 L cement pots filled with red loamy soil of pH7.2. After 3 months of growth, all the saplings were pruned at aheight of 20–25 cm from the ground level and transferred to aglass house. All the saplings were well watered until complete leafsprouting occurred and fully expanded leaves were available. Theplants were then randomly submitted to two watering treat-ments; stressed pots were maintained at 25–30% field capacity(FC), whereas the control plants were maintained at 100% FC(Ennahli and Earl, 2005). The study was undertaken for a timeperiod of 75 days calculated from the day after initiation oftreatments. Photosynthetic photon flux density (PPFD) measuredinside the glass house ranged between 900 and 1200mmol m�2

s�1, air temperature ranged from 1871 1C (early morning) to3474 1C (early afternoon) and relative humidity 6075% to8172%. All the sampling and measurements were made usingfully expanded leaves of the third or fourth positions from theapex of top branches. Samplings were conducted periodically at aninterval of 15–18 days and the results reported are the meanvalues of all periodic data.

Leaf water status measurement

Leaf water status was determined by measuring the relativewater content (RWC %) calculated according to the method ofCastillo (1996). RWC(%) ¼ [(fw�dw)/(sw�dw)]�100, where fw isfresh weight, sw is the mass after rehydration obtained by storingleaf samples for 24 h in distilled water and dw is oven dry weight(105 1C) of leaves.

Measurement of photosynthetic gas exchange parameters

The net photosynthetic rates (Pn), transpiration rates (E) andstomatal conductance (gs) were measured between 10 AM and 11AM using a portable infrared gas analyzer system (LC Pro+, ADC,UK). All photosynthetic measurements were performed in situ onclear sunny days. The instantaneous water use efficiency (WUEi)was calculated as Pn/E.

Measurement of chlorophylls and carotenoids

Chlorophylls and carotenoids were extracted from fresh leaves(0.5 g) with 10 ml of 80% (v/v) acetone and centrifuged at 10,000 gfor 5 min. The absorbance of cleared extract was read at 663.2,646.8 and 470 nm in UV–Visible 160A spectrophotometer (Shi-madzu, Tokyo, Japan) for chlorophyll a, chlorophyll b and total

carotenoids, respectively, and calculated according to Lichtentha-ler (1987). The total chlorophyll and carotenoid content wereexpressed in mg g�1 fw.

Measurement of non-enzymatic antioxidants

Foliar ascorbic acid (AA) content was determined according toOmaye et al. (1979) with minor modifications. Fresh leaf tissue(0.5 g) was homogenized with 5 ml of 10% trichloro acetic acid(TCA), centrifuged at 10,000 g for 20 min at room temperature, re-extracted twice, the supernatant made up to 5 ml and used forassay. To 0.5 ml of extract, 1 ml of 2% DNTPH (2,4- dinitrophenylhydrazine in 0.5 N H2SO4) and a drop of 10% thiourea (in 70%ethanol) were added and incubated at 37 1C for 3 h. Afterincubation, 1.75 ml of ice-cold 65% H2SO4 was added, allowed tostand at 30 1C for 30 min and the resulting color was read at520 nm in UV–Visible spectrophotometer. Calibration was doneusing a standard curve prepared with authentic ascorbic acid(Sigma) and results were expressed in mg g�1 fw.

Total glutathione was measured according to Griffith andMeister (1979). Fresh leaf tissue (0.2 g) was ground in a chilledmortar and pestle with 0.8 ml of 10% sulphosalicylic acid andcentrifuged at 15,000 g for 5 min at 4 1C. The supernatant wasneutralized by adding 0.6 ml of 10% sodium citrate. The reactionmixture (1 ml) contained 100ml sample, 100ml distilled water,700ml of 0.3 mM NADPH in potassium phosphate buffer (20 mm,ph 7.5) and 6 mM 50-dithio-bis(2-nitrobenzoic acid)(DNTB). Therate of DNTB reduction was recorded by adding 10ml glutathionereductase (GR) at 412 nm for 3 min. Glutathione content wasexpressed in mmol g�1 fw.

a-Tocopherol was quantified according to Yen et al. (1996)with some modification. Fresh leaf tissue (1 g) was homogenizedin liquid nitrogen with 10 ml of cold 80% methanol. The extractwas transferred to Eppendorf tubes and centrifuged at 3000 g for15 min at 4 1C. The pellets were re-extracted twice with 80%methanol and supernatants were pooled together. The super-natant was filtered through 25 mm millipore filter and the filtratewas stored in ice in dark condition. The a-tocopherol content ofthe filtrate was determined by high performance liquid chroma-tography (HPLC) with a C18 reverse-phase column(250�10.00 mm 5 micron Phenomenax, UK) at a flow rate of1 ml min�1, using isocratic solvent system -methanol and ethylacetate (1:4 v/v) as an eluant. a-Tocopherol level was quantifiedby UV–visible detector system at 295 nm (LC-10 AT VP Shimadzu,Japan). Peak identification was performed by comparing theretention times with pure a-tocopherol standard (Sigma) andconfirmed with characteristic spectra obtained from the photo-diode array detector, which also permitted the confirmation of thepurity of the peaks. a-Tocopherol content was expressed inmg g�1 fw.

Free proline measurement

Free proline content was measured according to Bates et al.(1973). Fresh leaf tissue (0.5 g) was homogenized in 10 ml of 3%sulphosalicyclic acid. The homogenate was centrifuged at 9000 gfor 15 min at room temperature. The reaction mixture containing2 ml acid ninhydrin, 2 ml glacial acetic acid and 1 ml filtrate wasincubated for 1 h in boiling water bath. After incubation, 4 ml oftoluene was added to the reaction mixture and mixed vigorouslyfor 15–20 s. The upper reddish pink colored phase was separatedand absorbance was read at 520 nm in UV–Visible spectro-photometer. A standard curve was prepared using authenticproline. Proline content was expressed in mg g�1 fw.

ARTICLE IN PRESS

Table 1Relative water content (RWC %) in the leaves of four mulberry cultivars.

Cultivars RWC (%)

Control Drought

V-1 86.872.7 74.471.9

MR-2 83.473.7 70.771.3

S-36 85.372.8 68.671.3

K-2 82.173.8 68.570.9

Means71 SE. (n ¼ 4), po0.05.

A. Guha et al. / Flora 205 (2010) 144–151146

Lipid peroxidation

Lipid peroxidation was determined by measuring the mal-ondialdehyde (MDA) equivalents as described by Fu and Huang(2001). Fresh leaves (0.5 g) were homogenized in 5 ml of 0.1%trichloroacetic acid (TCA). The homogenate was centrifuged at5000 g for 10 min at 4 1C. The reaction mixture contained 500ml ofthe supernatant and 4 ml of 20% TCA with 0.5% thiobarbituric acid(TBA). The mixture was heated at 95 1C for 30 min, quickly cooledon ice, and centrifuged at 5000 g for 15 min. The absorbance of thesupernatant was read at 532 and 600 nm. After subtracting thenon-specific absorbance at 600 nm, MDA concentration wascalculated using an extinction coefficient of 155 mM cm�1. Lipidperoxidation was expressed in nmol MDA g�1 fw.

Specimen preparation for scanning electron microscopy (SEM)

Freshly harvested leaves were cut into 3�3 mm squares andfixed in FAA (10% formalin:0.5% acetic acid:50% ethyl alcohol:35%distilled water) for 4 h at 4 1C. The samples were post fixed for 2 hin 2% osmium tetroxide and dehydrated in a graded ethyl alcohol-acetone series for 4 h. Specimens were critical point dried withcarbon dioxide as a transition fluid and were mounted on copperstubs using double stick cellophane tape. To observe cross-sectional area, leaf pieces were vertically mounted exposing theircut surfaces. The mounted samples were gold-coated in a Polaronsputter coater (Quorum technologies, UK). Observation andphotography were made with a transmission electron microscope(JEOL, Tokyo, Japan) fitted with a scanning attachment (Hitachi S-570, Japan) at 20 kV. The thickness of leaf and mesophyll tissueswas recorded from SEM images.

Histochemical studies of stem and root

Samples were collected from stems and roots of similar age,size and position and fixed in FAA (10% formalin:0.5% aceticacid:50% ethyl alcohol:35% distilled water) for 24 h and kept in70% alcohol until cutting. Freehand sections were made using arazor blade and the samples were stained by immersion intoluidine blue just for 20–30 s, followed by three washes indistilled water. After washing, the sections were mounted in 50%aqueous glycerol and the cover slips sealed with DPX to makesemi-permanent slides. The slides were then observed under aLeitz Diaplan light microscope and microphotographed.

Growth and yield measurements

At the end of the experiment (75th day) the plants wereharvested to obtain growth and yield measurements. Leaves ofeach plant were harvested separately and the weight recorded asfw of leaves in grams. Likewise, the stems of each plant werepruned separately and the weight recorded as fw in grams. Leafweight and stem weight were added to get shoot weight (aboveground biological yield). Root weight of each plant was takenseparately after washing and the weight recorded as fw of roots ingrams. Root to shoot ratio was calculated using the data of rootweight and shoot weight. Harvest index (HI) was calculated as theratio of leaf yield to above ground biological yield (Donald, 1962).

Statistics

The experiment was arranged in a completely randomizedblock design (CRBD) with four replications. Four independentdeterminants from individual plants were used for statisticalanalysis. Student’s t-test and analysis of variance (ANOVA) were

used for analyzing significant differences between control andtreated plants (po0.05). Correlations between the investigatedparameters were established by calculating simple pair-wisecorrelation coefficient (r) and coefficient of determination (r2).Relationships between different parameters were plotted usingSigmaplot 2000.

Results

Leaf water status

Under low water regime the RWC was reduced in all the cvscompared to control (Table 1). However, V-1 showed tolerance todrought with highest RWC of 74%, whereas, S-36 and K-2 werefound to be drought sensitive with RWCs of 68.49% and 68.63%,respectively under drought stress.

Gas exchange

Effect of drought stress on Pn, gs and E depicted contrastingresponses among the cvs. Significant genotypic variation wasnoticed for Pn among the four cvs in both water regimes (Fig. 1A).In control conditions, V-1 had the highest Pn followed by S-36,MR-2 and K-2. Compared to control, Pn of V-1 and MR-2 wasreduced by 14% and 17%, respectively under water deficit,whereas, maximum reduction in Pn was recorded in K-2 (40%)(Fig. 1B). The gs ranged from 106 to 190 mmol m�2 s�1 and from 52to 139 mmol m�2 s�1 in control and drought treatments,respectively (Fig. 1C). Drought stress reduced gs in all the cvs(Fig. 1D). However, V-1 showed minimum reduction in gs (26%),whereas maximum reduction was recorded in K-2 (50%) undersevere moisture stress. Drought caused drastic reduction in E of allthe cvs (Fig. 1E) with an average decline of 41% (Fig. 1F). K-2showed significantly lower E in both irrigation regimes comparedto other cvs. V-1 performed best for E under low water regime.Relative to unstressed plants, drought led to an increase in WUEi

in all the mulberry cvs (Fig. 1G). V-1 exhibited highest WUEi of3.98 and 5.41 mmol CO2 mol�1 H2O under control and droughttreatments, respectively. K-2 performed poor exhibiting minimumincrease in WUEi (12%) compared to V-1 (27%) and MR-2 (25%)under low water regime (Fig. 1H).

Photosynthetic pigments

Changes in total chlorophyll and carotenoid accumulation wererecorded under two water regimes (Table 2). Total chlorophyllcontent was affected under drought stress and decreased up to25% and 20% in S-36 and K-2, respectively. Chlorophyll loss wasminimum in V-1 (8%) followed by MR-2 (14%). An increasing trendin total carotenoids accumulation was recorded in all the cvsunder drought stress. V-1 showed maximum level of total

ARTICLE IN PRESS

Fig. 1. Effect of drought stress on (A) net photosynthetic rates, (B) percentage decrease in net photosynthetic rates, (C) stomatal conductance, (D) percentage decrease in

stomatal conductance, (E) transpiration rates, (F) percentage decrease in transpiration rates, (G) instantaneous water use efficiency and (H) percentage increase in

instantaneous water use efficiency in four mulberry cultivars (V-1, S-36, MR-2 and K-2). Vertical bars represent 71 SE. (n ¼ 4), po0.05.

Table 2Total chlorophyll and carotenoid contents in the leaves of four mulberry cultivars

subjected to drought stress.

Cultivars Total chlorophyll (mg g�1 fw) Total carotenoids (mg g�1 fw)

Control Drought Control Drought

V-1 3.6770.05 3.3670.06 0.59670.06 0.69570.01

MR-2 3.3470.23 2.8770.51 0.53470.02 0.61770.02

S-36 3.5670.01 2.6470.26 0.52270.02 0.60370.03

K-2 3.1770.97 2.5170.08 0.51570.01 0.56670.01

Means71 SE. (n ¼ 4), po0.05.

A. Guha et al. / Flora 205 (2010) 144–151 147

carotenoids 0.596 mg g�1 fw and 0.695 mg g�1 fw under controland drought conditions, respectively.

Free proline and non-enzymatic antioxidants

Differential changes in endogenous proline content of mulber-ry leaves were monitored in all the four mulberry cvs in bothstressed and well-watered plants (Fig. 2A). Proline content rangedfrom 0.6 to 1.4 mg g�1 fw and from 1.3 to 3.8 mg g�1 fw in controland drought treatments, respectively. In general, free prolinecontent was significantly elevated in all the cvs under low waterregime. V-1 showed inherently high proline content compared toother cvs during unstressed condition and also accumulatedproline significantly (3-fold increase) under low water regimescompared to other cvs.

Under well-watered conditions, the concentration of glu-tathione did not show much significant differences among thecvs. However, under water deficit conditions, V-1 exhibited more

glutathione content (7mmol g�1 fw), whereas K-2 showed lowestcontent of glutathione (4mmol g�1 fw) (Fig. 2B). The foliar AAconcentration of well-irrigated plants was not significantlydifferent among the tested cvs (AA~1.05 mg g�1 fw). However, anincreasing trend in AA level was witnessed in the cvs subjected towater stress (Fig. 2C). AA concentration in the leaves of stressedV-1 plants showed maximum elevation resulting in 36% increasein AA content compared to K-2 (8%). a-Tocopherol content showedmixed variation among the four mulberry cvs (Fig. 2D). V-1exhibited an elevated level of a-tocopherol under drought stresswith respect to control, whereas the endogenous a-tocopherollevels of MR-2, K-2 and S-36 were affected severely and a neta-tocopherol loss was visible in those three mulberry genotypes.Water deficiency augmented the MDA content of all cvs rangingfrom 19.7 to 38.2 nmol MDA g�1 fw (Fig. 3). However, V-1exhibited minimum MDA accumulation followed by MR-2,whereas MDA levels were high in S-36 and K-2 under severewater stress conditions. Interestingly, a-tocopherol levels shared apositive relationship with foliar AA contents under severemoisture stress (Fig. 4A). V-1 with a high AA contentmaintained an elevated state of foliar a-tocopherolconcentration under water deprivation. In the other threemulberry genotypes the a-tocopherol level showed apparentreduction in accordance to low level of AA concentration duringsevere water stress. A strong negative relationship was recordedbetween endogenous a-tocopherol levels and quantity of MDAaccumulated in mulberry leaves under water deficit conditions(Fig. 4B). V-1, with maximal a-tocopherol content showed thelowest lipid peroxidation in terms of MDA accumulation. In theother three mulberry cvs MDA levels were augmented whilehaving low levels of a-tocopherol.

ARTICLE IN PRESS

Fig. 2. Influence of drought stress on (A) proline, (B) glutathione, (C) ascorbic acid and (D) a-tocopherol contents in four mulberry cultivars. Vertical bars represent 71 SE.

(n ¼ 4), po0.05.

Fig. 3. Effect of drought stress on lipid peroxidation in four mulberry cultivars.

Vertical bars represent 71 SE. (n ¼ 4), po0.05.

2.0

1.8

1.6

1.4

1.2

1.0

0.8

40

35

30

25

20

150 5 10 15 20 25 30 35

V-1MR-2S-36K-2

α- Tocopherol (μg g-1 fw)

MD

A (n

mol

g-1

fw)

AA

(mg

g-1fw

)V-1MR-2S-36K-2

Fig. 4. (A) Relationship between a-tocopherol content and ascorbic acid

(r2¼ 0.97) and (B) relationship between endogenous a-tocopherol and MDA

accumulation (r2¼ 0.96) in four mulberry cultivars under drought stress. Each

point represents mean of four independent measurements from individual plant.

The correlations were significant at po0.001.

A. Guha et al. / Flora 205 (2010) 144–151148

Morpho-anatomical characters

In order to understand the contribution of anatomical traits ofmulberry that could probably improve drought stress resistance,we selected only two mulberry genotypes (V-1 and K-2) differingwidely in their response to water stress for the purpose ofmorpho-anatomical studies. Samples were taken from controlplants and analyzed for their innate anatomical differences. Thefindings are summarized in Table 3. Leaf thickness wassignificantly greater in V-1 compared to K-2. The height andthickness of palisade cells also showed marked variation betweenthe two genotypes (Fig. 5A). While the V-1 leaves had a palisadethickness of 55mm, K-2 possessed leaves with reduced palisadethickness of 32mm. Moreover, the percentage ratio of the palisadepart of mesophyll was higher in V-1 (48%) compared to K-2 (35%).The phloem width in the stem of V-1 was apparently larger(360mm), with a prominent layer of secondary phloem, whereasin K-2, the phloem width was only 275mm (Fig. 5B). Thetransverse section of roots also exhibited contrasting differencesin the number of xylem vessels between the two cvs (Fig. 5C).Maximum xylem poles were recorded in V-1. The genotypes alsovaried significantly in respect to root length and root weight.Longer root length was recorded in V-1 with maximum rootweight compared to K-2. The V-1 root system had more density ofsecondary roots with innumerable entangled mass of tertiaryroots (feeder roots), compared to K-2 (Fig. 5D).

A parallel attempt was made to understand the influence ofleaf thickness on WUEi. We measured the leaf thickness of all the

mulberry cvs grown under severe drought stress. A positiverelationship was encountered between the leaf thickness andWUEi of mulberry genotypes during progressive moisture stress(Fig. 6). Cvs maintaining thicker leaves under severe water stressalso exhibited higher values of WUEi. V-1 with higher leafthickness performed best in WUEi, whereas, the reverse wastrue in case of K-2.

ARTICLE IN PRESS

A. Guha et al. / Flora 205 (2010) 144–151 149

Growth and yield

Discrete differences in root:shoot ratios were recorded amongthe mulberry cvs between the two treatments. Compared tocontrol, the root:shoot ratio increased in all the cvs under droughtstress (Fig. 7A). The maximum value of root:shoot was recorded inV-1, whereas in K-2, the ratio was less. Significant reduction in HIwas recorded among all the cvs under drought stress with valuesranging from 47% to 25% (Fig. 7B). V-1 recorded the best HI (47%)under water deficit conditions, whereas, K-2 proved to be droughtsensitive with a minimum HI of 25%.

Discussion

The present study clearly demonstrated different physiological,biochemical, and morpho-anatomical responses among mulberrycultivars subjected to drought stress. The cvs selected for the

Table 3Comparative analysis of selected morpho-anatomical parameters in two mulberry

cultivars.

Morpho-anatomical traits V-1 K-2

Leaf thickness (mm) 11672.6 9374.0

Palisade thickness (mm) 5576.0 3274.7

% palisade tissue/leaf cross-section 4874.5 3575.1

Phloem width of stem (mm) 36074.0 27576.5

Xylem poles/root cross-section 3575.8 22 7 4.7

Root length (cm) 4575.6 2875.0

Root weight (g) 18476.8 142716.6

Means71 SE. (n ¼ 3), po0.05.

Fig. 5. Variabilities in morpho-anatomical features in two mulberry cultivars: V-1 and K

arrangement of palisade mesophyll (PM) and spongy mesophyll (SM) cells. (Bar ¼ 20

distribution of palisade mesophyll cells between the two cultivars. (B) Cross-sections th

(P), phloem (Ph), secondary phloem (S Ph), cambium (C) and xylem vessels (X). Note th

(C) Variation in root xylem density within the cultivars. (Bar ¼ 100mm). (D) Differences

tertiary roots in V-1 compared to K-2.

experiment were reported to be high yielding under well-irrigatedconditions and accredited with high HI (Susheelamma andDandin, 2006). However, in our present study, except V-1, theothers exhibited a sharp decline in HI under drought conditions.The reduction in HI can be explained as a consequence of declinedPn resulting from down-regulation in gs and concomitant decreasein also E (similar to findings by, e.g., Dias et al., 2007; Nunes et al.,2008). Stomatal resistance and marked reduction in ribulose-1,5-bisphosphate carboxylase severely can limit Pn during severewater stress (Chaitanya et al., 2003; Flexas and Medrano, 2002).The cellular strategies by which plants cope with soil drying canbe categorized as ‘drought avoidance’ and ‘drought tolerance’.Plants preferring ‘drought avoidance’ minimize water loss bysubstantial reduction of gas exchange parameters, mainly con-trolled by gs. By contrast, a drought tolerant genotype canmaintain stomatal conductance and photosynthesis rather high,despite development of a large water deficit (Mundree et al.,2002; Tardieu, 2005). The drought combating strategy found inV-1 can be termed ‘drought tolerance’ rather than ‘droughtavoidance’, since this cv showed the least inhibition in Pn andmaintained higher leaf gas exchange under severe water stress, ifcompared to other genotypes. Similar findings about suchdifferences between cultivars were recorded in drought tolerantcotton and ramie plants (Liu et al., 2005; Ullah et al., 2008).

V-1 possesses a very well-developed root system with manyfine roots and has a high root:shoot ratio. These structural traitsenable a better water status, a well-functioning hydraulicconductivity and, as a consequence, a good gas exchangeperformance. By contrast, K-2 as the most sensitive cultivar hascomparatively shorter tap roots and less root density. Under watershortage these plants experienced a considerable reduction in gs,causing a drastic inhibition in Pn, ultimately resulting in low HI.The hypersensitive physiological changes exhibited by K-2 and

-2. (A) Scanning electron photomicrographs of leaf cross-sections showing a typical

mm). Note the contrasting anatomical differences in respect to length, size and

rough stems of V-1 and K-2 showing different layers of epidermis (E), parenchyma

e conspicuous difference in phloem width within the two cultivars. (Bar ¼ 60mm).

in root morphology showing larger root length and higher density of secondary and

ARTICLE IN PRESS

Fig. 6. Relationship between leaf thickness and WUEi in four mulberry cultivars

under drought stress. (r2¼ 0.91). Each point represents mean of four independent

measurements from individual plants. The correlations were significant at

po0.001.

Fig. 7. Effect of drought stress on (A) root:shoot ratio and (B) harvest index in four

mulberry cultivars under severe drought stress. Vertical bars represent 71 SE.

(n ¼ 4), po0.05.

A. Guha et al. / Flora 205 (2010) 144–151150

S-36 probably will be not sufficient in order to survive severedrought. By contrast, V-1 sustained the photosynthetic gasexchange characteristics even under water shortage making itrather resistant to water stress. Thus, direct screening ofgenotypes for relatively higher gs, Pn, and WUEi under moisturestress should enable to shift the respective populations towardshigher productivity and may be advantageous in selectinggermplasm that brings about deep-rooting plants (Sharp et al.,2004; White et al., 2000).

The biochemical mechanisms that allow higher plants totolerate prolonged periods of water deprivation can involvenumerous attributes. One interesting means of increasing droughttolerance is accumulation of osmoprotectants such as proline andglycine betaine. In the present study, we have reported a positivecorrelation between proline accumulation and drought tolerancein mulberry cultivars. Similar results were also reported inpolyploid wheat, rice leaves, canola leaf discs, and sunflower

leaves (Aziz et al., 2000; Chandrasekar et al., 2000; Paramasivamet al., 2008). Proline is an important osmoticum contributing tostabilizing sub-cellular structures, scavenging free radicals andbuffering cellular redox-potential under stress conditions (Ashrafand Foolad, 2007; Hayashi et al., 2000). The increased accumula-tion of proline observed in stressed V-1 leaves could provide abetter osmotic equilibrium and cell membrane stability duringwater stress period particularly in this cultivar.

Antioxidative defense systems are known to protect the cellstructures against oxygen radicals generated under stress condi-tions (Franc-a et al., 2007; Queval and Noctor, 2007; Shao et al.,2008). Our results indicate a paramount increase in the concen-trations of carotenoids, glutathione, ascorbic acid and a-tocopher-ol in V-1 compared to the other three mulberry genotypes.Tocopherols are low molecular weight, lipophilic antioxidants,which in cooperation with other antioxidants, play an array ofbeneficial roles like scavenging and quenching various ROS andlipid peroxidation products, stabilizing photosynthetic mem-branes and modulating signal transduction (Gonz�alez-Rodrıguezet al., 2001; Ledford and Niyogi, 2005; Munn�e-Bosch, 2005;Munn�e-Bosch and Jon, 2004). In our study, we observed a positivecorrelation between a-tocopherol and AA content. V-1 hadefficient antioxidative protection owing to high level of both thea-tocopherol and the AA pool, whereas, a net a-tocopherol losswas observed in others. An insight into the a-tocopherol recyclingmechanism in relation to the ascorbate–glutathione cycle canprobably justify the incidence of a-tocopherol loss in the sensitivemulberry cvs. The chemical scavenging of 1O2 by a-tocopherolgenerates tocopheroxyl radicals, which are recycled back to a-tocopherol by ascorbate. The AA availability in the leaf extracts ofstressed MR-2, S-36 and K-2 was considerably less in comparisonto V-1, which possibly is responsible for the endogenous a-tocopherol loss in the sensitive cvs. We believe that V-1, by virtueof the high foliar AA pool, was able to recycle back a-tocopherolsuccessfully. Moreover, a 3-fold increase in a-tocopherol level wasalso recorded in stressed V-1, facilitating low MDA accumulation,thus avoiding much oxidative damage in comparison to othermulberry cvs., where increased levels of MDA accumulated as anaftermath of highly jeopardized a-tocopherol recycling, makingthese genotypes more prone to oxidative damage.

The correlation of anatomical structures with physiologicaltraits also supported the superior performance of V-1 whencompared to K-2 under drought stress. V-1 possessed innateanatomical traits, like thicker leaves resulting from a well-developed palisade tissue that would contribute to remaininggreen under stress and that retains higher amounts of photo-synthetic pigments. This again rendered reasonably high photo-synthetic rates and a beneficial WUEi (Kulkarni et al., 2007; Yiotiset al., 2006). The larger phloem area observed in V-1 stems incomparison with K-2 could be an extra capacity to conductphotosynthates in higher amounts during moisture stress situa-tions (Kulkarni and Deshpande, 2006). The higher number ofxylem vessels recorded in V-1 roots would improve the capabil-ities for water transport and be more directly associated withgreater potential for improved lateral root growth.

We conclude that V-1 performed best under drought amongthe four mulberry cultivars, as it possessed a well-developed rootsystem, improved leaf and stem anatomical characteristics, up-regulation in antioxidative defense mechanisms, osmotic balancemaintenance and better gas exchange capabilities that allowed tocontinue high photosynthetic activity under stressed environ-ments compared to drought sensitive cvs. Our results elucidatethat different mechanisms for drought tolerance are highlyinterconnected with a series of compensatory reactions thatmitigate the effects of drought in a tolerant mulberry plant suchas V-1.

ARTICLE IN PRESS

A. Guha et al. / Flora 205 (2010) 144–151 151

Acknowledgements

We acknowledge Department of Science and Technology,Government of India, New Delhi for financial assistance. (GrantSR/SO/PS-27/05).

References

Ashraf, M., Foolad, M.R., 2007. Roles of glycine betaine and proline in improvingplant abiotic stress resistance. Environ. Exp. Bot. 59, 206–216.

Aziz, P.T., Niogret, M.F., Larher, F., 2000. Proline level is partly under the control ofabscisic acid in canola leaf discs during recovery from hyper-osmotic stress.Physiol. Plant. 110, 376–383.

Bates, L.S., Walderen, R.P., Teare, I.D., 1973. Rapid determination of free proline forwater stress studies. Plant Soil 39, 205–207.

Castillo, F.J., 1996. Antioxidative protection in the inducible CAM plant Sedumalbum L. following the imposition of severe water stress and recovery.Oecologia 107, 469–477.

Cattivelli, L., Rizza, F., Badeck, F., Mazzucotelli, E., Mastrangelo, A.M., Francia, E.,Mar�e, C., Tondelli, A., Stanca, A.M., 2008. Drought tolerance improvement incrop plants: An integrated view from breeding to genomics. Field Crop Res. 105,1–14.

Chaitanya, K.V., Jutur, P.P., Sundar, D., Ramachandra Reddy, A., 2003. Water stresseffects on photosynthesis in different mulberry cultivars. Plant Growth Regul.40, 75–80.

Chandrasekar, V., Sairam, R.K., Srivastava, G.C., 2000. Physiological and biochemicalresponses of hexaploid and tetraploid wheat to drought stress. J. Agron. CropSci. 185, 219–227.

Dias, P.C., Araujo, W.L., Moraes, G.A.B.K., Barros, R.S., DaMatta, F.M., 2007.Morphological and physiological responses of two coffee progenies to soilwater availability. J. Plant Physiol. 164, 1639–1647.

Ennahli, S., Earl, H.J., 2005. Physiological limitations to photosynthetic carbonassimilation in cotton under water stress. Crop Sci. 45, 2374–2382.

Donald, C.M., 1962. In search of yield. J. Aust. Inst. Agric. Sci. 28, 171–178.Flexas, J., Medrano, H., 2002. Drought inhibition of photosynthesis in C3 plants:

stomatal and non stomatal limitations revisited. Ann. Bot. 89, 183–189.Franc-a, M.B., Panek, A.D., Eleutherio, E.C.A., 2007. Oxidative stress and its effects

during dehydration. Comp. Biochem. Physiol. Part A 146, 621–631.Fu, J., Huang, B., 2001. Involvement of antioxidants and lipid peroxidation in the

adaptation of two cool-season grasses to localized drought stress. Environ. Exp.Bot. 45, 105–114.

Griffith, O.W., Meister, A., 1979. Potent and specific inhibition of glutathionesynthesis by buthionine sulfoximine (s-n-butylhomocysteine sulfoximine). J.Biol. Chem. 254, 7558–7560.

Gonz�alez-Rodrıguez, A.M., Tausz, M., Wonisch, A., Jim�enez, M.S., Grill, D., Morales,D., 2001. The significance of xanthophylls and tocopherols in photo-oxidativestress and photoprotection of three Canarian laurel forest tree species on ahigh radiation day. J. Plant Physiol. 158, 1547–1554.

Hayashi, F., Ichino, T., Osanai, M., Wada, K., 2000. Oscillation and regulation ofproline content of P5CS and ProDH gene expressions in the light/dark cycles inArabidopsis thaliana L. Plant Cell Physiol. 41, 1096–1101.

Kotresha, D., Prabhakar, Rao A., Srinivas, N., Vidyasagar, G.M., 2007. Antioxidativeresponse to drought and high temperature stress in selected mulberrygenotypes. Physiol. Mol. Biol. Plants 13, 57–63.

Kulkarni, M., Deshpande, U., 2006. Comparitive studies in stem anatomy andmorphology in relation to drought resistance in tomato (Lycopersiconesculentum). Am. J. Plant Pathol. 1 (1), 82–88.

Kulkarni, M., Borse, T., Chaphalkar, S., 2007. Anatomical variability in grape (Vitisvenifera) genotypes in relation to water use efficiency (WUE). Am. J. PlantPhysiol. 2, 36–43.

Kulkarni, M., Borse, T., Chaphalkar, S., 2008. Mining anatomical traits: A novelmodelling approach for increased water use efficiency under droughtconditions in plants. Czech. J. Genet. Plant Breed. 44, 11–21.

Ledford, H.K., Niyogi, K.K., 2005. Singlet oxygen and photo-oxidative stressmanagement in plants and algae. Plant Cell Environ. 28, 1037–1045.

Lichtenthaler, H.K., 1987. Chlorophylls and carotenoids: pigments of photosyn-thetic biomembranes. Methods Enzymol. 148, 350–382.

Liu, F., Liu, Q., Liang, X., Huang, H., Zhang, S., 2005. Morphological, anatomical andphysiological assessment of ramie [Boehmeria nivea (L.) Gaud.], tolerance tosoil drought. Genet. Resour. Crop Ev. 52, 497–506.

Munn�e-Bosch, S., 2005. The role of a-tocopherol in plant stress tolerance. J. PlantPhysiol. 162, 743–748.

Munn�e-Bosch, S., Jon, F., 2004. New insights into the function of tocopherols inplants. Planta 218, 323–326.

Mundree, S.G., Baker, B., Mowla, S., Peters, Sh., Marais, S., Vander Willigen, C.,Govender, K., Maredza, A., Muyanga, S., Farrant, J.M., Thomson, J.A., 2002.Physiological and molecular insights into drought tolerance. Afr. J. Biotech. 1,28–38.

Nunes, C., Araujo, S.S., da Silva, J.M., Fevereiro, M.P.S., da Silva, A.B., 2008.Physiological responses of the legume model Medicago truncatula cv. Jemalongto water deficit. Environ. Exp. Bot. 63, 289–296.

Omaye, S.T., Turnbull, J.D., Sauberilich, H.E., 1979. Selected methods for thedetermination of ascorbic acid in animal cells, tissues and fluids. MethodEnzymol 62, 3–11.

Paramasivam, M., Cheruth, A.J., Ramamurthy, S., Rajaram, P., 2008. Osmoregulationand antioxidant metabolism in drought-stressed Helianthus annus undertriadimefon drenching. C. R. Biol. 331, 418–425.

Pathan, M.S., Subudhi, P.K., Courtois, B., Nguyen, H.T., 2004. Molecular dissection ofabiotic stress tolerance in sorghum and rice. In: Nguyen, H.T., et al. (Eds.),Physiology and Biotechnology Integration for Plant Breeding. pp. 525–569.

Queval, G., Noctor, G., 2007. A plate reader method for the measurement of NAD,NADP, glutathione, and ascorbate in tissue extracts: Application to redoxprofiling during Arabidopsis rosette development. Anal. Biochem. 363, 58–69.

Ramachandra Reddy, A., Chaitanya, K.V., Vivekanandan, M., 2004. Drought-inducedresponses of photosynthesis and antioxidant metabolism in higher plants. J.Plant Physiol. 161, 1189–1202.

Ramachandra Reddy, A., Chaitanya, K.V., Jutur, P.P., Gnanam, A., 2005. Photosynth-esis and oxidative stress responses to water deficit in five different mulberry(Morus alba L.) cultivars. Physiol. Mol. Biol. Plants 11, 291–298.

Rhizopoulou, S., Psaras, G.K., 2003. Development and structure of drought-tolerantleaves of the mediterranean shrub Capparis spinosa L. Ann. Bot. 92, 377–383.

Shao, H., Chu, L., Shao, M., Jaleel, C.A., Mi, H., 2008. Higher plant antioxidants andredox signalling under environmental stresses. C. R. Biologies 331, 433–441.

Sharp, R.E., Poroyko, V., Hejlek, L.G., Spollen, W.G., Springer, G.K., Bohnert, H.J.,Nguyen, T., 2004. Root growth maintenance during water deficits: physiologyto functional genomics. J. Exp. Bot. 55, 2343–2351.

Susheelamma, B.N., Dandin, S.B., 2006. Improvement for qualitative traits and leafproductivity in mulberry (Morus spp.) and its effect on bivoltine cocoonproduction. Ad. Plant Sci. 19 (I), 23–28.

Susheelamma, B.N., Datta, R.K., 1995. Breeding for drought tolerance in mulberry.In: Sharma, B. (Ed.), Genetic research and education, current trends and nextfifty years. Indian Society of Genetics and Plant Breeding, New Delhi, pp. 641–650.

Susiluoto, S., Berninger, F., 2007. Interactions between morphological andphysiological drought responses in Eucalyptus microtheca. Silva Fenn. 41,221–233.

Tardieu, F., 2005. Plant tolerance to water deficit: physical limits and possibilitiesfor progress. C.R. Geoscience 337, 57–67.

Ullah, I., Rahman, M., Ashraf, M., Zafar, Y., 2008. Genotypic variation for droughttolerance in cotton (Gossypium hirsutum L.): Leaf gas exchange and productiv-ity. Flora 203, 105–115.

Valliyodan, B., Nguyen, H.T., 2006. Understanding regulatory networks andengineering for enhanced drought tolerance in plants. Curr. Opin. Plant Biol.9, 189–195.

White, D.A., Turner, N.C., Galbraith, J.H., 2000. Leaf water relations and stomatalbehaviour of four allelopathic Eucalyptus species planted in mediterraneansouthwestern Australia. Tree Physiol. 20, 1157–1166.

Yen, G.C., Wu, S.C., Duh, P.D., 1996. Extraction and identification of antioxidantcomponents form the leaves of mulberry (Morus alba L.). J. Agric. Food Chem.44, 1687–1690.

Yiotis, C., Manetas, Y., Psaras, G.K., 2006. Leaf and green stem anatomy of thedrought deciduous Mediterranean shrub Calicotome villosa (Poiret) Link.(Leguminosae). Flora 201, 102–107.