ACTA BOT. CROAT. 78 (2), 2019 125 Acta Bot. Croat. 78 (2), 125–134, 2019 CODEN: ABCRA 25 DOI: 10.2478/botcro-2019-0021 ISSN 0365-0588 eISSN 1847-8476 Variation in morpho-physiological, biochemical and molecular responses of two Eucalyptus species under short-term water stress Sivanantham Amrutha1, Abdul Bari Muneera Parveen1, Muthusamy Muthupandi1, Veerasamy Sivakumar1, Raman Nautiyal2, Modhumita Ghosh Dasgupta1* 1 Institute of Forest Genetics and Tree Breeding, R. S. Puram, Coimbatore 641002, India 2 Indian Council of Forestry Research and Education, P. O. New Forest, Dehradun 248006, Uttarakhand, India Abstract – The genus Eucalyptus occurs in a wide range of environmental conditions, including rainforests, subal- pine, arid/semi-arid and moist temperate zones. It includes species with the capacity to cope with extremely low water potential. This study aims to screen water stress tolerance in two Eucalyptus species under nursery condi- tions. Inter-specific variation in morphological, physiological, biochemical and molecular parameters in two Euca- lyptus species (E. tereticornis and E. camaldulensis) with contrasting levels of tolerance to progressive short term water-deprived condition was evaluated. Water stress reduced growth measured in terms of root:shoot ratio and specific leaf area (SLA), photosynthetic parameters, leaf water potential and relative water content (RWC) in both genotypes. Biochemical parameters including total sugars, phenol, phytohormones (indole acetic acid and ab- scisic acid) and proline were found to significantly increase during stress in both genotypes. Water responsive transcripts like osmotin and DREB/CBF registered significant expression variation in the two genotypes, sug- gesting their key role in regulating water stress tolerance in Eucalyptus. Keywords: drought stress, leaf temperature, osmotin, sugars, transcription factors * Corresponding author e-mail: ghoshm@icfre.org Introduction The genus Eucalyptus with over 900 species forms a dominant canopy of woodland ecosystems (Boland et al. 2006). The ecological range of Eucalyptus species encompasses high rainfall, temperate, semi arid and extreme arid conditions (Merchant et al. 2006). Several species belonging to the subgenus Symphyomyrtus are adapted to prolonged water deficit conditions, determining the species distribution (Adams 1996). Eucalypts are widely introduced as a plantation crop due to their rapid growth, adaptability and superior wood properties for the paper and pulp industries. Eucalyptus grandis, E. urophylla, E. globulus, E. camaldulensis, and their hybrids account for 80% of plantations globally. Seven species, E. camaldulensis, E. grandis, E. globulus, E. pellita, E. tereticornis, E. urophylla and Corymbia citriodora, were reported to be suitable for Indian agro-climatic conditions and are widely planted in the subcontinent (Kallarackal and Somen 1997, Kallarackal et al. 2002). The high potential productivity of Eucalyptus has resulted in the deployment of genetically improved planting stock in mono-specific plantations to maximize harvesting performance (Forrester et al. 2010). However, this practise renders the plantations vulnerable to abiotic and biotic stresses that affect establishment and growth (White et al. 2009). Further, the potential productivity in eucalypts is governed by stem growth, biomass allocation and water availability (Stape et al. 2008). Response to water-deprived conditions in Eucalyptus species has been extensively studied. Significant changes in osmotic potential, gas-exchange parameters, cell turgor ad- justment (White et al. 1996), stomatal function (Costa e Silva et al. 2004), chlorophyll and carotenoid contents (Michelozzi et al. 1995) in leaf tissues are reported during water-deprived conditions. Other parameters used to assess water stress tol- erance included changes in leaf temperature, photosynthetic rate, leaf water potential, and transpiration rate (Dye 1996, AMRUTHA S., PARVEEN A.B.M., MUTHUPANDI M., SIVAKUMAR V., NAUTIYAL R., GHOSH DASGUPTA M. 126 ACTA BOT. CROAT. 78 (2), 2019 Roden and Ball 1996, Rolando and Little 2003, Stokes 2004). Concurrently, reduction in plant growth (Chaves et al. 2003, Bedon et al. 2012), photosynthesis (Warren et al. 2007) and hormone production (Granda et al. 2011) have also been re- ported during water stress conditions. The Institute of Forest Genetics and Tree Breeding, Co- imbatore, India initiated its program on the genetic im- provement of Eucalyptus in 1997 in association with CSIRO, Australia. Productive clones with superior pulping quality were tested under multi-environment conditions and sub- sequently released and deployed in plantation programs. In the present study two Eucalyptus species were evaluated for their response to water deficit conditions and variations in growth, physiological, biochemical and transcript expres- sion patterns were documented to identify key parameters governing water stress tolerance in the species studied. Materials and methods Plant material The two species used in the present study are second generation clones derived from the Eucalyptus breeding pro- gram implemented by the Institute of Forest Genetics and Tree Breeding, Coimbatore, India. Eucalyptus tereticornis (Et88) was selected from the seed production area estab- lished and maintained by the Institute at Pudukottai, Tamil Nadu, India. Eucalyptus camaldulensis (Ec226) is of Kather- ine, Northern Territory, provenance and was selected from the seed orchard at Satyavedu, Andhra Pradesh, India, which was established and maintained by the Institute. The selec- tion of the genotypes in the present study was based on a preliminary phenotypic screening of 60 clones for water stress tolerance (data not shown). Eucalyptus camaldulensis (Ec226) was used as the susceptible genotype, while E. tereti- cornis (Et88) served as the tolerant genotype. Experiment design and water stress imposition Coppice cuttings of both species were rooted and main- tained in polybags containing a soil mixture of Pallam soil, River sand and Red earth in the ratio of 2:1:0.5. The cuttings were fortified with Hoagland’s media (Hoagland and Arnon 1950) supplemented to its field capacity (FC) (30 mL) on al- ternate days for a period of 5 days prior to being subjected to water stress conditions. Subsequently, six month old cut- tings were transferred to a controlled environmental cham- ber and maintained at 32 ± 3 °C with relative humidity at 70–80 ± 5%. Fifteen ramets of each genotype were used for the study in a randomized design. Six ramets per genotype were sub- jected to a well-watered (WW) regime wherein the cuttings were watered to 100% FC every day, while nine ramets were subjected to progressive water stress (WS) regime. The stress treatment was initiated by complete water withdrawal for a period of 15 days and the soil moisture content was record- ed everyday using a tensiometer. On day 6 when soil mois- ture content was 0.005 ± 0.0011 MPa, the susceptible geno- type Ec226 showed symptoms of leaf curling and hence all parameters except root:shoot ratio were assessed on days 5 and 6 after stress imposition. The root:shoot ratio was docu- mented on day 15 after the imposition of water stress treat- ment with soil moisture content of 0.0215 ± 0.0017 MPa. All parameters were recorded in three randomly selected ramets and used as biological replicates. Physiological parameters Root:shoot ratio analysis of clones subjected to WW and WS conditions was conducted on oven dried samples after completion of the experiment on day 15 post stress imposi- tion. Specific leaf area (SLA) was determined from scanned images of 4–5 leaves and the area was determined using Image J software (Schindelin et al. 2015). Digital images of leaves (with ruler) were uploaded in the software and the scale was fixed using the ‘set scale’ option in the tool. Sub- sequently, under the option ‘Image’, colour threshold was adjusted to shade the leaf area followed by measuring the area using the Wand tool. SLA was calculated as area divid- ed by dry weight determined by oven drying of leaf mate- rial at 45 °C for 2 days and represented as cm–2 g–1 leaf dry weight (LDW). The leaf surface temperature and chlorophyll content were measured on day 6 after initiation of stress, since com- plete leaf curling was recorded after day 6 of stress impo- sition in the susceptible genotype Ec226. Temperature was manually recorded in mature leaves each day between 11.00 a. m. – 12.00 p. m. using an infrared thermometer (Gaby Instruments, India) at 3 zonal levels of the plant (apex, mid- level and basal zone). The ambient temperature of the cham- ber was also recorded. Three fully expanded leaves of each ramet were selected for estimating the chlorophyll content. The chlorophyll con- tent index (CCI) was recorded using the chlorophyll concen- tration meter MC-100 (Apogee Instruments Inc., UT, USA) for both the WW and WS regime. Photosynthetic rate (A), stomatal conductance (gs), in- tercellular CO2 concentration (Ci) and transpiration rate (E) were measured using an LCi-SD photosynthesis system (ADC BioScientific Ltd. UK). Measurements were taken be- tween 10 a. m. to 12 p. m. in mature leaves on day 6 after im- position of stress in both WW and WS plants. Relative water content of the leaf (% RWC) was deter- mined from detached leaf segments of 1 – 2 cm length and their fresh weight (Fw) was recorded on day 6 after impo- sition of water stress. The leaf segments were placed in de- ionised water for a period of 4 h and turgid weight (Tw) was recorded. Subsequently, leaves were dried at 60 °C overnight and dry weight was determined (Dw). RWC was estimated according to Morgan (1984) using the formula 100 × (Fw- Dw)/(Tw-Dw) . Mid-day leaf water potential (Ψw) was assessed from a single mature leaf from three different ramets using the PSY1 stem psychrometer (ICT International, Australia) on day 5 RESPONSE OF EUCALYPTUS SPECIES UNDER WATER STRESS CONDITION ACTA BOT. CROAT. 78 (2), 2019 127 after imposition of stress. The psychrometer chamber was vacuum fixed over the non-cuticularized leaf surface and water potential was measured in MPa. Biochemical parameters Leaf tissues from both clones subjected to WW and WS conditions were assayed for total phenols, flavonoids, total sugars, proline content, free ABA and IAA content. Leaves were collected from three ramets each for both conditions on day 6 after imposition of water stress and processed in- dependently for biochemical estimation. Fifty mg of leaf tis- sues were ground in liquid nitrogen and subsequently used for biochemical assay. Total phenols were estimated using the protocol described by Malik and Singh (1980), while to- tal sugars were quantified by anthrone method (Hedge and Hofreiter 1962). Total flavonoids were estimated by alumin- ium chloride method (Kiranmaiet et al. 2011). The phyto- hormones, ABA and IAA were estimated using the proto- col described by Unyayar et al. (1996), while leaf proline was determined using the procedure described by Ábrahám et al. (2010). Expression profiling of water stress responsive transcripts Total RNA was isolated from the leaves of WW and WS plants of both genotypes on day 6 of stress imposition. Sin- gle leaves from three independent ramets were harvested for RNA isolation and maintained as three biological replicates. RNA was isolated using RNAqueous®-Micro Total RNA Iso- lation Kit (Thermo Scientific, USA). The concentration and purity of RNA were evaluated using NanoDrop ND-1000 UV–Vis Spectrophotometer (Thermo Scientific, USA). Ap- proximately 1 µg of total RNA was treated with DNAse I and used to synthesize the first strand cDNA with RevertAid M- MuLV Reverse Transcriptase (Thermo Scientific, USA) fol- lowing the manufacturer’s protocol. In the present study, 12 drought responsive transcripts were selected according to their role in water stress response. Transcripts from hormone signaling pathways (AR, GER3, LEA6); osmoprotectant pathways (GOLS2, SIP, OSM34); sucrose pathway (TPPB); antioxidant (GPX6); membrane intrinsic protein (PIP) and drought related transcription factors (DREB, CBF1c, CBF2) were selected. Transcript se- lection was based on an earlier study in E. grandis (Ghosh Dasgupta and Dharanishanthi 2017). The expression of PEG induced water stress responsive transcripts were analysed by quantitative real time (qRT-PCR ; On-line Suppl. Tab. 1). qRT- PCR was performed using ABI PRISM 7500 Step one plus Sequence Detection System (Applied Biosystems, USA) using the following program: one cycle of 95 °C for 10 min; 40 cycles of 95 °C for 15 s and 60 °C for 1 min. The qRT-PCR reactions (10 µL) included 200 ng of cDNA, 5.0 mL SYBR Green JumpStartTaq ReadyMix (Sigma, St. Louis, Tab. 1. Effect of short-term progressive water stress on different parameters in Eucalyptus tereticornis (Et88) and Eucalyptus camaldulensis (Ec226). Data presented are mean of triplicate values ± SD (standard deviation). WW – Well watered condition; WS ‒ water stress condi- tion. SLA ‒ specific leaf area; LDW ‒ leaf dry weight; CCI ‒ chlorophyll content index; RWC ‒ relative water content; ABA ‒ abscisic acid; IAA ‒ indole acetic acid; FW ‒ fresh weight. Clone ID Et 88 Ec 226 Treatments WW WS WW WS SLA (cm–2 g–1 LDW) 46.56±9.28 45.09±7.99 47.04±1.50 43.47±4.58 Root : shoot ratio 0.42±0.06 0.45±0.08 0.76±0.4 0.98±0.2 Leaf temperature (°C) 30.70±0.70 31.78±0.39 30.52±0.69 31.60±0.31 CCI 11.12±1.94 13.36±1.69 9.63±0.98 15.61±1.50 RWC (%) 82.78±2.68 78.79±3.50 76.09±3.35 40.07±5.64 Ψw (MPa) –2.34±0.57 –4.25±0.05 –1.68±0.26 –2.94±0.08 Transpiration rate (mmol m–2 s–1) 0.85±0.09 0.80±0.12 0.82±0.14 0.42±0.07 Stomatal conductance (cm s–1) 0.11±0.01 0.10±0.02 0.13±0.03 0.06±0.02 Photosynthetic rate (µmol m–2 s–1) 22.39±4.84 17.18±1.71 21.21±11.8 8.39±3.46 Total carbohydrate (mg g–1 FW) 2.04±0.23 5.28±1.65 4.23±3.05 9.04±1.59 Flavonoids (mg g–1 FW) 7.83±1.58 11.31±2.69 11.28±0.43 10.01±1.19 Phenols (mg g–1 FW) 0.40±0.14 0.97±0.43 0.67±0.01 1.13±0.18 Proline (μmol g–1 FW) 4.86±1.83 9.03±3.89 3.56±0.10 8.44±5.76 ABA (mg g–1 FW) 0.04±0.01 0.09±0.06 0.03±0.01 0.06±0.00 IAA (mg g–1 FW) 0.29±0.18 0.60±0.08 0.24±0.15 0.50±0.03 AMRUTHA S., PARVEEN A.B.M., MUTHUPANDI M., SIVAKUMAR V., NAUTIYAL R., GHOSH DASGUPTA M. 128 ACTA BOT. CROAT. 78 (2), 2019 reduction in RWC in WS condition when compared to Et88 (Tab. 1). Mid-day leaf water potential (Ψw) was also found to reduce in WS condition in both Et88 and Ec226 when com- pared to WW condition (Tab. 1). The photosynthetic parameters including transpiration rate, stomatal conductance and photosynthesis were record- ed and a decrease in WS conditions in both genotypes was registered (Tab. 1). However, the decrease documented in Ec226 for transpiration rate, stomatal conductance and pho- tosynthetic rate was high as compared to Et88 (Tab. 1). Biochemical parameters Six biochemical parameters including total sugars, fla- vonoids, phenols, proline, ABA and IAA content were re- corded after 6 days of water stress imposition. All param- eters were found to increase under WS condition in both genotypes except flavonoid content. Increase in total sugars was documented in clone Ec226 in WS condition as com- pared to the WW condition. A similar trend was observed in Et88 (Tab. 1). A decrease in the flavonoid content was re- corded in Ec226 under WS condition, while it increased in Et88 (Tab. 1). Significant differences across species and treatments were found in nested ANOVA for each measured param- eter, at least in one of the factors (treatment or genotype or both) (Tab. 2). Leaf temperature and total phenol con- tent were significantly different only for the treatment fac- MO, USA), 200 nM each of forward and reverse primer and 0.4 µL of 20 mg mL–1 BSA. All reactions were conducted in three biological replicates. The melting curve was generated to ensure product specificity and to differentiate between the product and primer dimer. Actin related protein 3 (ARP3) was used as reference gene for normalization of data (Kar- paga Raja Sundari and Ghosh Dasgupta 2012). The relative quantification value (RQ) was calculated using the formula 2–ΔΔCT (Livak and Schmittgen 2001) and the fold decrease was calculated as the reciprocal of the fold change (Peirson et al. 2003). Statistical analyses All parameters were determined for three independent ramets and the data was represented as mean ± SD. Statisti- cal analysis for morphological, physiological and biochemi- cal parameters was performed using Statistica version 7.0. The effect of species, treatment and their interaction on all parameters was tested using Nested ANOVA. Ramets were nested within treatment and treated as random effect in analyses, while the water stress treatment and species were considered fixed factors. The post hoc test with Bonferroni correction was conducted when an effect was found to be significant. In transcript expression studies, delta CT for WW and WS conditions across species were statistically analyzed us- ing t-test implemented in GraphPad Prism® version 7 and the difference between the conditions were considered sta- tistically significant when P < 0.05. Results The effect of water stress on two species of Eucalyptus was evaluated and the variations in growth, physiological, biochemical and transcript expression were recorded in WW and WS conditions. Physiological parameters Percent leaf curling was recorded in both genotypes un- der WW and WS conditions. In tolerant genotype Et88, 5.71% ± 0.48 leaf curling was documented, while suscepti- ble genotype Ec226 recorded 96% ± 2.71 leaf curling under WS conditions (Fig. 1). The root:shoot ratio was variable between the genotypes and Ec226 showed a decrease in shoot growth upon stress imposition, while no difference was registered in Et88 (Tab. 1). SLA was recorded in both genotypes under WW and WS conditions and a marginal decrease was documented in Et88, while a decrease in SLA was recorded in Ec226 under WS conditions when compared to WW condition (Tab. 1). Leaf temperature showed a marginal increase under WS condition in both genotypes tested, while CCI was found to increase in Ec226 under WS condition when compared to WW condition. An increase in CCI in clone Et88 was also recorded (Tab. 1). Percent RWC was found to vary in both genotypes subjected to WS condition, where Ec226 showed a Fig. 1. Effect of progressive water stress in six month old rooted cuttings of (A) Eucalyptus tereticornis (Et88) and (B) Eucalyptus camaldulensis (Ec226); WW ‒ Well watered condition; WS ‒ Water deprived condition. RESPONSE OF EUCALYPTUS SPECIES UNDER WATER STRESS CONDITION ACTA BOT. CROAT. 78 (2), 2019 129 tor, whereas significant differences for the species were re- corded for root:shoot ratio. Significant differences in RWC, leaf water potential and total sugars content were found for both treatment and species. Expression profiling of water stress responsive transcripts Relative quantification (RQ) of twelve transcripts related to water deficit condition was determined using qRT-PCR in both genotypes. Osmotin (OSM34), dehydration responsive element binding proteins (DREB), C-repeat/DRE-binding factor (CBF1c and CBF2), glutathione peroxidase (GPX6), and raffinose synthase family protein (SIP) showed signifi- cant up-regulation in Et88 when compared to Ec226 (Fig. 2). Maximum expression was recorded for OSM34 with Et88 registering a 54-fold increase compared to the 5-fold increase in Ec226. Further, Ec226 showed down regulation of all the transcripts profiled except OSM34 (6.31) and SIP (2.10). Down regulation of CBF2 (-22-fold), CBF1c (-11-fold) and DREB (-10-fold) was documented in Ec226 when com- pared to their expression level in Et88 which was up-regulat- ed 20-fold, 28-fold and 15-fold, respectively (Fig. 2). Discussion A growing concern in commercial plantation programs has been the depletion in water resources and area of culti- vation. Managing tree plantations to maximize wood pro- duction from available water resources is the critical param- eter in tree breeding programs. In Eucalyptus, the concept of plantation water productivity (PWPWOOD) is gaining impor- tance in the provision of social license for wood production from plantations (White et al. 2014). Hence, several species of Eucalypts have been screened for their water stress toler- ance and variations in response to low water potential are reported among species (Myers and Neales 1986) among provenances (Li 1998) and between clones (Pita and Pardos 2001). The inter- and intraspecific variation in response to changes in osmotic potential has been used as a selection cri- terion in genetic improvement programs in eucalypts (van der Moezel et al. 1991, Lemcoff et al. 1994). The imposition of a water-deprived condition elicits multi-dimensional responses at morphological, physiologi- cal, biochemical and molecular levels (Granda et al. 2014). In Eucalyptus species and their hybrids, adjustments dur- ing water stress conditions are mediated by hormonal bal- ance (Granda et al. 2011), variation in pigment content (Sh- valeva et al. 2006, 2008), osmotic changes (Merchant et al. Tab. 2. Statistical analysis of some morphological, physiological and biochemical parameters, in Eucalyptus tereticornis (Et88) and Eucalyptus camaldulensis (Ec226) exposed to water stress, across treatment and species. Df ‒ degrees of freedom; SS ‒ sum of squares; MS ‒ mean of squares; F ‒ F value. Level of significance: *P < 0.01, **P < 0.05. Parameter Species Treatment Leaf temperature Df 1 2 SS 0.11 3.40 MS 0.11 1.70 F 0.61 9.18* RWC Df 1 2 SS 1428.41 2043.93 MS 1428.41 1021.96 F 816.91* 583.95* Leaf water potential Df 1 2 SS 2.90 7.80 MS 2.90 3.90 F 28.47* 3.905* Total carbohydrates Df 1 2 SS 2658.55 5036.65 MS 2658.55 2518.32 F 7.24** 6.85** Phenols Df 1 2 SS 7.79 124.199 MS 7.79 62.099 F 0.58 4.61** Root : Shoot Df 1 2 SS 0.562 0.076 MS 0.562 0.038 F 8.32** 0.56 Fig. 2. Expression profiling of water stress responsive transcripts in leaves of Eucalyptus tereticornis (Et88) and Eucalyptus camaldulensis (Ec226). Values are mean of data from three biological replicates and the error bars represent SD. Gene IDs are given in On-line Suppl. Tab. 1. Asterisk indicates statistical significance between well watered and water stressed plants when P < 0.05. AMRUTHA S., PARVEEN A.B.M., MUTHUPANDI M., SIVAKUMAR V., NAUTIYAL R., GHOSH DASGUPTA M. 130 ACTA BOT. CROAT. 78 (2), 2019 2006, Warren et al. 2007, Callister et al. 2008), photosynthe- sis adjustment (Warren et al. 2007) and growth modifica- tions (Drew et al. 2009). In general, water stress reduces growth rate in all spe- cies by modifying the biomass allocation in above and below ground tissues, which determines the morphological plastic- ity of the genotype (Maseda and Fernández 2016). In euca- lypt species, reduction in growth and biomass under water stress conditions is reported in E. globulus (Costa e Silva et al. 2004, Correia et al. 2014, Coopman et al. 2008), E. camal- dulensis (Maseda and Fernández 2016) and E. microtheca (Li 1998). In the present study, the genotype (Et88) showed marginal reduction in SLA and root:shoot ratio during stress imposition, while Ec226 documented significant reduction in both parameters, revealing that the tissue biomass was significantly reduced in Ec226 during stress conditions. The photosynthetically active leaf is the plant part that is critically affected by water limitation, which causes reduction in leaf emergence/expansion and shrinkage/curling (Burling et al. 2013, Scoffoni et al. 2014). The maintenance of balance be- tween the areas involved in transpiration and absorption is a key determinant of survival and productivity as reported in E. globulus (Marron et al. 2003, Costa e Silva et al. 2004). Hence, reduction in leaf area during water stress condition is a response in all eucalypt species as reported in E. globu- lus (Costa e Silva et al. 2004, Maseda and Fernández 2016), E. camaldulensis (Lemcoff et al. 2002, Maseda and Fernán- dez 2016) and E. microtheca (Susiluoto and Berninger 2007). Specific leaf area (SLA), which essentially describes the dis- tribution of leaf biomass relative to area, is a major trait that correlates with whole plant growth (Cheng et al. 2016). It links carbon allocation and water status and thus is a criti- cal parameter to assess soil moisture content (Pierce et al. 1994). In a recent study by Wellstein et al. (2017) in grass- es and forbs from temperate and sub-Mediterranean eco- systems, it was hypothesized that phenotypic adjustments like SLA show variable response during short-term water stress and reduced SLA was associated with enhanced water use efficiency, while increased SLA indicated better growth performance. Hence, it was concluded that the differential response of SLA during drought conditions had to be in- terpreted according to the evolutionary history and envi- ronmental niche of the species. In agreement with this hy- pothesis, SLA was reported to decrease in E. globulus (Costa e Silva et al. 2004, Coopman et al. 2008) and increase in E. microtheca (Susiluoto and Berninger 2007) subsequent to stress imposition. In another study, no significant difference in SLA was reported in either E. camaldulensis or E. globulus seedlings during water stress (Maseda and Fernández 2016). In the present study, decrease in SLA was documented in both E. camaldulensis and E. tereticornis. Leaf relative water content (RWC) is a key indicator of plant water status since it reflects the balance between water availability and transpiration rate (Lugojan and Ciulca 2011). The general trend of decrease in RWC during water-deprived conditions is documented in several eucalypt species includ- ing Eucalyptus camaldulensis (Maseda and Fernández 2016), E. globulus (Pita and Pardos 2001), E. oblique, E. camaldu- lensis, E. rubida, E. polyanthemos, E. tricarpa, E. cladocalyx (Merchant et al. 2007) and Eucalyptus grandis × E. urophyl- la (Valadares et al. 2014). In the present study, a decrease in RWC was recorded in both genotypes but the level of reduc- tion in Ec226 was significantly higher than in the clone Et88. Accumulation of solutes like inorganic cations, organic acids, carbohydrates and free amino acids in response to wa- ter stress is generally reported in most plant species. They contribute to osmotic adjustments, thus maintaining a bal- ance between sucrose synthesis and translocation (Morgan 1984, Akinci and Lösel 2012). Accumulation of soluble sug- ars in water deprived condition was reported in Eucalyptus species (Quick et al. 1992, Warren et al. 2011). It was also re- ported that E. camaldulensis and E. globulus from a ‘mesic’ environment underwent osmotic adjustments through in- creased sugar accumulation, while species from dry (‘xeric’) environments adjusted through increased concentration of quercitol (Merchant et al. 2006). In agreement with the ear- lier report, significant increase in the accumulation of total sugars was documented in both species, which occupy the ‘mesic’ environment. Leaf/canopy temperature is an indicator of stomatal clo- sure in response to soil water stress (Jones et al. 2009, 2002). During water stress condition, closure of stomata to main- tain inner moisture content is an immediate response, re- sulting in increased leaf temperature, decreased transpira- tion and reduced photosynthesis, which adversely affect productivity (Luquet et al. 2003, Jones 2004, Feller 2016). Leaf temperature, water status and stomatal opening are closely connected (Valladares and Pearcy 1997, Feller 2006, Reynolds-Henne et al. 2010, Feller and Vaseva 2014, Teskey et al. 2015) and hence determination of leaf/canopy tem- perature as a measure of stomatal conductance using infra- red thermometry was suggested as early as the 1980s (Jar- vis 1981) to facilitate irrigation scheduling. It was reported that maintenance of lower leaf surface temperature in tol- erant genotypes during stress conditions could lead to fa- vourable water status, sustained transpiration and increased photosynthetic efficiency, resulting in improved yields (Ku- mar 2005). Using leaf temperature as drought index has been reported in potato (Gerhards et al. 2016), maize (Car- roll et al. 2017), sorghum (Blume et al. 1978), wheat (Fisch- er et al. 1998, Talebi 2011), sugarcane (Silva et al. 2007), rice (Hirayama et al. 2006) and Firmiana platanifolia (Yu et al. 2015). However, in woody perennials leaf surface tempera- ture as water stress index is of limited use. In a recent study on Pinus taeda and Populus deltoides × nigra, it was reported that leaf temperature had a direct effect on stomatal opening and an increase in leaf temperature was documented under water deprived condition in both genus irrespective of leaf morphology, xylem structure and physiology (Urban et al. 2017). The effect of leaf temperature on stomatal limitation to photosynthesis, intercellular CO2 concentration and net photosynthesis was demonstrated in the study. Similarly, an increase in leaf temperature was documented in both spe- cies in the present study. RESPONSE OF EUCALYPTUS SPECIES UNDER WATER STRESS CONDITION ACTA BOT. CROAT. 78 (2), 2019 131 Transcriptional regulation of different pathways to main- tain cellular homeostasis during water stress is well docu- mented in plant systems (reviewed by Kaur and Asthir 2017). Several drought responsive genes including dehydrins, mo- lecular chaperones, water channel proteins, transporters and biosynthetic enzymes for compatible solutes, growth regula- tors, enzymes that detoxify reactive oxygen species (Bartels and Sunkar 2005, Wang et al. 2006), transcription factors, protein phosphatases, phospholipid metabolic proteins and protein kinases (Shinozaki and Yamaguchi-Shinozaki 2007, Morran et al. 2011) are reported to play a pivotal role in cellular protection and stress alleviation. In Eucalyptus, ex- tensive studies on morpho-physiological and biochemical response to water stress condition is reported, while molec- ular response is limited to transcriptome-wide response in E. globulus and E. cladocalyx (Spokevicius et al. 2017), E. ca- maldulensis (Thumma et al. 2012), E.alba hybrid and E. uro- phylla × E. grandis (Villar et al. 2011), and E. grandis (Ghosh Dasgupta and Dharanishanthi 2017). Osmotin and osmotin- like proteins (OLPs) are known to act as osmotic modulators by compartmentalizing solutes or regulating structural and metabolic alterations (Chowdhury et al. 2017). Biosynthesis of osmotin/OLPs was reported to depend on water status of cells, revealing their key role in osmotic adaptations (Weber et al. 2014, Patade et al. 2013). In transgenic systems, the role of osmotin as an osmoprotectant gene has been extensively reported in crops (reviewed by Khan et al. 2015, Das et al. 2011). Expression of osmotin during water deficit condi- tion is reported in E. cladocalyx (Spokevicius et al. 2017) and E. grandis (Ghosh Dasgupta and Dharanishanthi 2017). In the present study, a significantly high expression of osmotin (OSM34) was registered in the clone Et88 (54-fold) as com- pared to Ec226 (fivefold), indicating that level of osmotin can act as an indicator in screening water-responsive geno- types. The role of transcription factors during dehydration stress has been extensively reviewed in crop plants (Nakashi- ma et al. 2014, Singh and Laxmi 2015). In Eucalyptus, the major transcription factors which regulate water stress in- clude zinc finger transcription factor family protein (Villar et al. 2011), MYB, NAC, ERF, HB12 (Thumma et al. 2012) and HSFs, DREB2A, DEAR3 (Ghosh Dasgupta and Dharanis- hanthi 2017). The ABA independent dehydration responsive element binding/C-repeat binding factors (DREB/CBF) be- longing to the ethylene responsive factor/APETALA2 (ERF/ AP2) family (Shinozaki and Yamaguchi-Shinozaki 2007) are major regulators governing drought response (Agarwal and Jha 2010). Genome-wide analysis in E. grandis revealed the presence of 17 DREB1/CBF genes and 6 DREB2. Further, it was reported that DREB1/CBF was cold-inducible, while DREB2 was induced during water stress (Cao et al. 2015). In another study, ectopic expression of EguCBF1a⁄b from E. gunnii in a Eucalyptus hybrid conferred drought and freezing tolerance (Navarro et al. 2011). An earlier study in E. globu- lus also revealed that drought tolerant genotypes exhibited better cold acclimation due to the accumulation of soluble sugars (Costa e Silva et al. 2009). In the present study, down- regulation of CBF2, CBF1c and DREB in the clone Ec226, in contrast to their significant up-regulation in Et88, revealed the crucial role of DREB/CBF in conferring drought toler- ance in eucalypts. In a similar study in Hevea, a strong cor- relation between fold expressions of DREB with drought tol- erance was reported by Luke et al. (2015). In the present study, several morpho-physiological, bio- chemical and molecular parameters were assessed to docu- ment their response during progressive water stress in two species of eucalypts with contrasting levels of tolerance. Leaf temperature, total phenol, root:shoot ratio, RWC, leaf wa- ter potential and total sugars showed significant variations across the two genotypes. Water responsive transcripts like osmotin and DREB/CBF registered significant expression variation in the two genotypes, indicating their key role in regulating water stress tolerance. Hence, the reported phys- iological (leaf temperature, RWC, leaf water potential and root:shoot ratio), biochemical (total sugars) and molecu- lar (osmotin and DREB/CBF) parameters can be used as an explanatory variables in determining the selection of euca- lypts under progressive short term water stress in nursery condition. Acknowledgements The authors acknowledge Department of Biotechnology, Government of India for funding the research work (proj- ect no. BT/PR10539/PBD/16/1064/2013). Funding support in the form of research fellowships was provided to SA, AMP and MM by the Department of Biotechnology, Government of India. 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