overexpression increases drought and salinity

Plant Science 232 (2015) 23–34

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Erianthus arundinaceus HSP70 (EaHSP70) overexpression increases drought and salinity tolerance in sugarcane (Saccharum spp. hybrid) Sruthy Maria Augustine a , J. Ashwin Narayan a , Divya P. Syamaladevi b , C. Appunu a , M. Chakravarthi a , V. Ravichandran c , N. Subramonian a,∗ a

Sugarcane Breeding Institute (ICAR), Coimbatore, Tamil Nadu, India Indian Grass and Fodder Research Institute Regional Station, Avikanagar, Rajasthan, India c Department of Rice, Tamil Nadu Agricultural University, Coimbatore, Tamil Nadu, India b

a r t i c l e

i n f o

Article history: Received 1 November 2014 Received in revised form 9 December 2014 Accepted 15 December 2014 Available online 23 December 2014 Keywords: Sugarcane Transformation Drought tolerance Salinity tolerance HSP70 Erianthus arundinaceus

a b s t r a c t Heat shock proteins (HSPs) have a major role in stress tolerance mechanisms in plants. Our studies have shown that the expression of HSP70 is enhanced under water stress in Erianthus arundinaceus. In this paper, we evaluate the effects of overexpression of EaHSP70 driven by Port Ubi 2.3 promoter in sugarcane. The transgenic events exhibit significantly higher gene expression, cell membrane thermostability, relative water content, gas exchange parameters, chlorophyll content and photosynthetic efficiency. The overexpression of EaHSP70 transgenic sugarcane led to the upregulation of stress-related genes. The transformed sugarcane plants had better chlorophyll retention and higher germination ability than control plants under salinity stress. Our results suggest that EaHSP70 plays an important role in sugarcane acclimation to drought and salinity stresses and its potential for genetic engineering of sugarcane for drought and salt tolerance. © 2014 Elsevier Ireland Ltd. All rights reserved.

1. Introduction Erianthus arundinaceus, a wild relative of the genus Saccharum is a broad-leaved species and well known for its drought tolerance [1], high fiber and biomass [2]. It also has resistance to pests and diseases with good ratooning ability. Our previous studies have shown that there is a seven-fold increase in the relative expression of heat-shock protein 70 (HSP70) under water stress in E. arundinaceus when compared to the commercial sugarcane variety Co 86032. HSPs belong to a class of proteins that are conserved in prokaryotes and eukaryotes and are abundant in plants. Because of the heat shock induction, HSPs are highly expressed in all organisms including plants [3]. Based on the molecular weight, HSPs are divided into five families: HSP100, HSP90, HSP70, HSP60 and sHSP (small heat shock protein) with a molecular weight ranging from 15 to 42 kDa [4]. The members of the HSP70 family are highly conserved with one HSP70 domain, which have a 44 kDa ATP-binding N-terminal region and a 25–30 kDa substrate-binding C-terminal region. The role of HSP70 family proteins under abiotic stresses has

∗ Corresponding author. E-mail address: monian [email protected] (N. Subramonian). http://dx.doi.org/10.1016/j.plantsci.2014.12.012 0168-9452/© 2014 Elsevier Ireland Ltd. All rights reserved.

been reported earlier [5]. HSP70 plays different roles during stress and normal conditions. HSP70 stabilizes the proteins by binding to its protein substrates and prevents denaturation or aggregation. In addition, it helps in certain proteins subcellular transport, folding of newly synthesized proteins, the unwanted proteins degradation and protein complexes formation and disassociation. Thus, both during normal and stress conditions, HSP has a major role in protein homeostasis by protein quality controlling and turnover [6]. Previous studies have demonstrated that NtHSP70-1 overexpression in Nicotiana tabacum plants conferred tolerance to drought stress [7]. The overexpression of HSP70/DnaK gives enhanced heat tolerance as well as early growth and suppression of programmed cell death in transgenic tobacco and rice [8–11]. The HSP70 overexpression in Arabidopsis enhances the tolerance to heat stress and leads to increased transcript levels of two stress-related genes SOS1 and APX1 associated with salinity tolerance [12]. The overexpression of Chrysanthemum HSP70 in Arabidopsis conferred tolerance to abiotic stresses [13]. We have isolated the gene coding for HSP70 from E. arundinaceus and incorporated it into the sugarcane genome. Our results demonstrate that overexpression of EaHSP70 enhances tolerance to drought and salt stresses in sugarcane (Saccharum spp. hybrid). This is the first report on the overexpression of HSP70 imparting salinity tolerance in food/bioenergy crop plants in general and drought tolerance in sugarcane in particular.

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S.M. Augustine et al. / Plant Science 232 (2015) 23–34

2. Materials and methods 2.1. Vector construction and plant transformation The HSP70 gene was isolated from E. arundinaceus (accession no. IK 76-81) from the germplasm collection being maintained at the Sugarcane Breeding Institute, Coimbatore, India, using gene-specific primers designed from Sorghum bicolor (NCBI accession no. U41653) and Zea mays (NCBI accession no. NM 001196236) through polymerase chain reaction (PCR) (1 cycle at 94 ◦ C for 5 min, followed by 35 cycles of 94 ◦ C for 45 s, 62 ◦ C for 30 s and 72 ◦ C for 2 min). Port Ubi 2.3 promoter was amplified from pPORT-UBI-GFP [14] using two specific primers: forward 5 -GATCGGATCCACTATCACCCTCGAGGTG-3 (BamHI site underlined) and reverse, 5 -ATCCCATGGCTGCAAGAAATAATCACCAA-3 (NcoI site underlined), and cloned into pCAMBIA-3301 between the multiple cloning site SacI and SbfI. The newly constructed plasmid was named pPORT-UBI2.3-3301. After sequencing and confirmation, the full-length sequence of EaHSP70 was amplified using two specific primers: forward 5 GATTCCTGCAGGATGGCCAAGGGCGAG-3 (SbfI site underlined) and reverse 5 -GCTCAAGCTTTTAGTCGACCTCCTCGATCTTGG-3 (HindIII site underlined), then cloned into pPORT-UBI2.3-3301 between the SbfI and HindIII and the resultant construct pSBI-HSP70 (Fig. 1) was transferred to Agrobacterium tumefaciens (LBA4404) by freeze–thaw method [15]. For Agrobacterium-mediated transformation in sugarcane, the method described by Arvinth et al. [16] was followed. Meristematic leaf whorls from the shoot tips of 5- to 8-month-old field-grown plants were co-cultivated with A. tumefaciens (LBA4404) harboring pSBI-HSP70, and the transformed cells were selected with repeated sub-culturing in a callusing medium (supplementary Table 3) containing phosphinothricin (3 mg/l). After six rounds of selection when the calli have attained a size, the selected calli were transferred to the regeneration medium (supplementary Table 3) and the regenerated plants were rooted in the rooting medium (supplementary Table 3) and subsequently hardened and planted in pots containing a mixture of sand, soil and farmyard manure (1:1:1 ratio). 2.2. Molecular analyses and selection of transgenic events PCR analysis was carried out using the genomic DNA isolated from phosphinothricin-tolerant plants. DNA from wild type (WT) plants was used as negative control and plasmid DNA was used as positive control. For PCR, plasmid DNA (10 ng) and genomic DNA (50 ng) were used as templates. The promoter-gene fusion primer sequences 5 -GCATGTGTGAATGGTGCGATTTG-3 and 5 -ATGACACCGGCATCCTTGGT-3 were used for the PCR reaction to distinguish EaHSP70 from host plant HSP70. Plants with the expected size amplicon (1.2 kb) were selected for further experiments. Further, these transgenic events were again confirmed by amplifying the 300-bp amplicon marker gene (bar gene) by using primers 5 -AACCACTACATCGAGACAA-3 and 5 CAACCACGTCTTGAAGCCC-3 (1 cycle at 94 ◦ C for 5 min, followed by 35 cycles of 94 ◦ C for 45 s, 51 ◦ C for 30 s and 72 ◦ C for 30 s). 2.3. Plant material and stress treatments Twenty-five independent transgenic events (V0 ) of the sugarcane variety Co 86032 were further propagated through planting single bud cuttings from the V0 plants, thus taking the transgenic events to V1 stage (V1 – vegetative generation). Morphological differences were not observed between the transgenic events and WT plants (untransformed Co 86032). Five biological replications from each transgenic event and WT plants were planted in 16 pots containing soil, sand and farmyard manure in 1:1:1 ratio for moisture

stress screening. Plants were grown with normal irrigation and recommended fertilizer doses for 120 days. Plants were subjected to water stress at the tillering phase (120 days after planting) by withholding irrigation for 10 days. Plants received regular watering from the 11th day of water stress, and subsequently regular watering was continued as required. Relative water content and cell membrane thermostability tests were carried out in the transgenics and WT plants on 0th and 10th day of drought induction. EaHSP70 transgene expression was analyzed under irrigated condition, and endogenous HSP70 in WT sugarcane was assessed in different soil moisture stresses, i.e. 0th day (25% soil moisture), 10th day after stress (8.1% soil moisture) and 5 days after the release of stress (25% soil moisture). Stress-related gene expression and gas exchange parameters were measured in the transgenics and WT plants on the 10th day after inducing drought stress. Chlorophyll content and photosynthetic efficiency (Fv/Fm) were recorded on the 0th day, 10th day after withholding the irrigation and 15 days after releasing the drought. All the physiological experiments were conducted for the 25 independent transgenic events with five biological and three technical replications. The molecular experiment, i.e. gene expression analysis, was conducted in five independent transgenic events with five biological and three technical replications. Soil moisture percentage was measured through gravimetric method using a moisture analyser (A&D model Mx-50) in samples collected from three different layers (10, 20 and 30 cm). To evaluate the extent of salinity tolerance of the transgenic events, leaf disk and bud germination assays were carried out. Leaf disk assay was carried out in the leaves collected from the transgenics and WT plants grown under normal irrigated conditions. The assay was conducted for 25 independent transgenic events with five biological and three technical replications. The bud germination assay was carried out for 10 independent transgenic events selected from leaf disk assay with five biological and three technical replications. 2.4. Morphological analysis (visual scoring) of the transgenic events The transgenic events along with the WT were visually observed for wilting of leaves on the 5th and 10th day after drought induction. Leaf wilting was scored on a scale of 1–4 modified from [17] as follows: 1 = no wilting; 2 = slightly wilting; 3 = wilting, wherein the plant showed leaf wilting only during hot hours from which the leaves recovered; and 4 = severe wilting, wherein wilted leaves did not recover. The mean score was computed from five plants from each event. 2.5. Cell membrane thermostability analysis The cell membrane thermostability test was carried out for estimating the cell membrane injury percentage, following the method described by Martineau et al. [18]. The cell membrane injury percentage is considered as one of the indications of the ability of the plant to tolerate drought [19]. This parameter was studied in both V0 and V1 transgenic generations and compared with WT plants. The third fully opened leaf was collected from transgenics and WT plants on 0th and 10th day after inducing drought condition, as mentioned above. Leaf discs (0.5 cm diameter) weighing 200 mg were washed with 20 ml distilled water three times, each wash extending to 2 min. Immediately after washing, 20 ml of distilled water was added to each of the control and treatment tubes (2.5 cm × 15 cm), the tube mouths were covered with aluminum foil, the tubes were incubated at 60 ◦ C in a thermostatically controlled water bath for 20 min and thereafter both treatment and control sets were placed at 10 ◦ C for 12 h to allow the diffusion of electrolytes into the water. After recording the initial conductance at 30 ◦ C, the tubes were heated at 100 ◦ C for 20 min, and


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Fig. 1. Schematic diagram of the plant expression vector.

final conductance was recorded after cooling. Membrane injury % = 1 − [(1 − T1 /T2 )/(1 − C1 /C2 )] × 100, where T and C refer to the values for treatment and control samples, and the subscripts 1 and 2 denote the initial and final conductance, respectively.

of photosynthesis (Asat). Measurements were recorded every 30 s over a period of 2 h. Data were recorded for 25 independent events with five biological replications and three technical replications per event.

2.6. Plant water status

2.8. Leaf chlorophyll content measurements

The relative water content (RWC) of the leaf was evaluated to determine the leaf hydration status. The excised third leaves of 120day-old transgenics and WT plants were subjected to measurement for leaf RWC on the 0th and 10th day after the induction of drought stress. Leaf RWC was calculated based on fresh (FW), turgid (TW) and dry weights of 200 mg leaf samples. Fresh weights of the leaf samples were determined on a mass balance immediately after being excised from the plants. TWs were determined after soaking the leaf discs in de-ionized water for 4 h at room temperature in a closed Petri dish and weighed immediately after blot drying. Samples were then dried at 90 ◦ C for 72 h and the dry weight was recorded. Then, the RWC was determined according to the method of Barrs and Weatherley [20] based on the following calculation:

Total chlorophyll content was recorded on the 0th and 10th day after the induction of the drought (soil moisture 25% and 8.1%, respectively) and 15 days after the release of drought (re-irrigation, 25% soil moisture) from the transgenics and WT plants using a chlorophyll meter (SPAD-502; Konica Minolta, Japan).

RWC =

FW − DW × 100 TW − DW

2.7. Gas exchange parameters Gas exchange parameters, photosynthesis rate (A), stomatal conductance (gs ) and transpiration rate (E), were recorded from the middle part of the fully opened third leaf on the 10th day of drought induction (soil moisture 8.1%) from the transgenics and WT plants using a portable photosynthesis system (Li-6400, Li-COR Inc., Lincoln, Nebraska, USA) with a leaf chamber of 2 cm × 3 cm and an integrated light source (LI-6400-02B). Gas exchange parameters were measured at 40 ◦ C (closed cabinet incubated to 40 ◦ C), 130 ␮mol m−2 s−1 [CO2 ] and RH ∼60–70%. The external CO2 concentration in air was maintained at 380-␮mol mol−1 in the reference cuvette. All the gas exchange parameters were measured at a leaf temperature of 32 ± 2.0 ◦ C, and photon flux density was controlled at 1500 ␮mol m−2 s−1 , which gave the light-saturated rates

2.9. Photosynthetic efficiency (Fv/Fm) of the HSP70 transgenic sugarcane The maximum potential photochemical efficiency, defined as the ratio of variable to maximum fluorescence emitted by chlorophyll (Fv/Fm), was recorded using a portable chlorophyll fluorimeter (OS-30P; Optisciences, USA). Photosynthetic efficiency (Fv/Fm) was recorded on the 0th and 10th day after the induction of drought (soil moisture 8.1%) and 15 days after the release of drought (re-irrigation, soil moisture 25%) from the transgenics and WT plants. The plants were dark-adapted for 20 min before measurement. The maximum fluorescence under light exposure (Fm ) was obtained by imposing a 1-s saturating flash to the leaf in order to reduce the entire PS II reaction center after attaining steady-state fluorescence (Ft). The minimum fluorescence immediately after light exposure (Fo ) was determined by imposing dark while simultaneously switching on a far red light to oxidize PS II rapidly by drawing electrons from PS II to PS I. 2.10. Gene expression analysis using quantitative real time PCR (qPCR) Total RNA was isolated using the method described by Chomczynski and Mackey [21]. RNA was isolated by using Trizol reagent (Sigma Chemicals, USA) and DNAse (Fermentas International Inc., Ontario, Canada) treatments were carried out. First-strand cDNAs

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Ct = Ct sample − Ct actin [24]. The Ct values reflect the relative expression of the target gene upon exposure to drought stress.

Table 1 Visual scoring of pSBI-HSP70 transgenic events for soil moisture stress. Event no.

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 WT

Score after stress induction 5th Day

10th Day

2 1 1 1 1 1 1 1 1 1 1 2 1 1 1 1 1 1 1 1 1 1 1 1 1 3

2 2 1 1 3 1 1 1 1 1 2 2 2 2 3 1 1 2 3 3 2 1 1 1 2 4

were synthesized from total RNA using the Fermentas firststrand cDNA synthesis kit using oligo (dT) primers (Fermentas International Inc., Ontario, Canada) following the manufacture’s instruction. The relative expression of the transgene EaHSP70 was assessed in transgenics and WT under normal irrigated and drought stress conditions. In addition, the expression of the endogenous HSP70 was also analyzed in WT sugarcane at different soil moisture conditions, i.e. 25% (irrigated condition), 8.1% (drought stressed) and 25% (5 days after the release of stress). The relative expressions of six important abiotic stress (salt, cold and heat) responsive genes, i.e. RD29 (response to dehydration), LEA (late embryogenesis abundant protein), COR15 (cold regulated protein), ERD (early responsive to dehydration)] [22], ERF (ethylene responsive factor) [23] and BRICK/HSPC300 (unpublished data), were assessed in transgenics and WT plants under drought stress condition. The ␤-actin transcript was used as an internal control to quantify the relative transcript levels. The expression of ␤-actin was determined in both irrigated and drought-stressed samples to make sure that this gene did not respond to drought stress, to use it as an internal control. cDNA fragments and the actin gene were amplified with gene-specific primers. The primers were designed using Primer Express, version 3 software (Applied Biosystems, Burlington, ON, Canada) (supplementary data Table 1). The stress-related genes used for primer synthesis and their accession numbers are given in Table 1 (supplementary data Table 1). For each primer set, the primer efficiency was analyzed by constructing standard curves with correlation coefficients of 0.99 or higher, and PCR efficiencies (calculated by efficiency = [10(−1/slope) ] − 1) between 97% and 101% were accepted. Prior to the qPCR, primer concentrations were optimized through melt-curve analysis. All PCRs were performed for 40 cycles. qPCR was performed using MESAGREEN Master Mix (12.5 ␮l), Taq DNA polymerase (1 U), gene-specific primers (10 pmol each) and the StepOne real-time PCR system (Applied Biosystems, Burlington, ON, Canada). The CT values for both the target and internal control genes were used for quantification of transcripts by comparative CT method normalization. All reactions were performed in triplicates, and the expression of target gene was calculated using the formula: 2−Ct , where

2.11. Leaf disk assay for assessing salinity tolerance Leaf disk assay was carried out to evaluate the sensitivity of the transformed and WT plants to sodium chloride (NaCl) stress as described by Fan et al. [25]. Two grams of fresh leaf sample (each 3.0 cm × 2.0 cm size) was excised from healthy and fully expanded third leaves of the 25 independent transgenic events and WT plants (120 days old). The discs were floated in a 10 ml solution of 250 mM NaCl and experimental control (0 mM NaCl), kept in water for 72 h, and then used for measuring chlorophyll content spectrophotometrically after extraction in methanol [26]. The treatment was carried out in continuous white light at 25 ± 2 ◦ C. 2.12. Bud germination assay Ten independent transgenic events were selected for bud germination assay based on the leaf disk senescence assay. Five biological replications of transgenic events and WT plants single bud setts were planted in cavity trays and grown under identical controlled conditions in a green house. To evaluate the germination under salt stress conditions, single bud setts were irrigated daily with 0, 100, 200 and 300 mM NaCl solution for a period of 26 days. The number of buds germinated and their shoot heights were recorded on the 26th day of planting. 2.13. Statistical analysis For statistical analysis of the data, five biological and three technical replicates from each of the transgenic events were used. Mean value, standard deviation and Student’s t-test were evaluated using the XLSTAT 2013.5 program to analyze all the data to compare the transgenic events and WT plants under normal and stress conditions. A P-value of (P ≤ 0.05) was considered statistically significant. 3. Results 3.1. Isolation of HSP70 gene from E. arundinaceus and development of transgenic sugarcane overexpressing EaHSP70 gene The HSP70 gene (1.9 kb) was isolated from E. arundinaceus and the sequence was submitted to Genbank (accession no. KJ670162). The primers were designed from Z. mays and S. bicolor, so the amplified HSP70 may be possibly a chimera. The gene was named EaHSP70 and did not have any intron sequence. The isolated gene sequence had high sequence homology (98–100%) with the other reported HSP70 gene sequences of Z. mays and S. bicolor. The nucleotide sequences translated to amino acid sequence showed high sequence homology (95–98%) with other reported HSP70 protein sequences. To establish the functional significance of the EaHSP70 gene, the complete gene sequence was overexpressed in sugarcane by using Agrobacterium-mediated transformation. The preliminary screening of phosphinothricin-resistant sugarcane transformants was confirmed through PCR using promoter-gene fusion primers (supplementary Fig. 1A) to distinguish the EaHSP70 from host plant HSP70 and marker gene (supplementary Fig. 1B) at V0 and V1 stages. Thirty-five independent events were obtained, and 25 events were selected based on the cell membrane thermostability for subsequent analysis. The morphological and growth characteristics of the transgenic plants were similar to those of WT (Fig. 2A).


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Fig. 2. Screening of EaHSP70 transgenic events along with the WT for soil moisture stress and bud germination assay for salinity stress. Screening for soil moisture stress (A) at 25% soil moisture and (B) at 8.1% soil moisture. Bud germination assay for salinity tolerance (C) WT and (D) EaHSP70 transgenic events.

3.2. Cell membrane thermostability is higher in EaHSP70 transgenic sugarcane events at V0 stage A standard test of cellular membrane thermostability was performed at the V0 stage to study drought tolerance in transgenic sugarcane containing EaHSP70 (Fig. 3A). Though there was variation between the transgenic events, high membrane stability was

observed in HSP70 transgenics under normal irrigated condition. A high membrane injury indicates low cell membrane stability. Sixty percent of the events had 60–70% membrane injury, whereas in WT plants the membrane injury was 95.5%. The highest stability was observed in the second event, with 50.8% injury. The lowest stability was in the seventh event, with 95.11% membrane injury.

Fig. 3. Cell membrane thermostability in EaHSP70 transgenics along with WT (A) V0 stage at 25% soil moisture and (B) V1 stage with and without soil moisture stress. Data shows a significant difference in the CMS between transgenic events and untransformed control, ns: not significant; at P ≤ 0.05, by Student’s t-test. Data are presented as mean ± SD (n = 5) and error bars represent SD.

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Fig. 4. Relative water content in EaHSP70 transgenics and WT with and without soil moisture stress. Data followed by an (*) showed a significant difference with respect to control Co 86032 (P ≤ 0.05; Student’s t-test). Data are presented as mean ± SD (n = 5) and error bars represent SD.

3.3. Overexpression of EaHSP70 in sugarcane results in enhanced cell membrane thermostability and drought tolerance at V1 stage The wilting symptoms in each of the transgenics on 5th and 10th day (Fig. 2B) were recorded, and are given in Table 1. In transgenics, 36% of plants were almost near normal showing very little wilting which was rated as 1, whereas in WT plants all the plants were scored as 4 (representing severe wilting). The membrane injury was found to decrease with the increasing moisture stress in all the transgenic events at V1 stage (Fig. 3B). The membrane injury varied from 50.8% to 92.41% at 25% soil moisture. At 8.1% soil moisture, the injury percentage ranged between 27.17% and 58.45%. Sixty-eight percent of the events showed 50–70% injury at 25% soil moisture, and 56% of the transgenic events showed 30–40% of injury at 8.1% soil moisture. The lowest injury percentage (i.e. suggesting outstanding drought tolerance) at 8.1% was in the event 1 (27.17%). The 7th (92.41%) and 25th (90.53%) events had the same injury as that of the WT plants at 25% soil moisture. In WT plants, the membrane injury increased from 93.2% to 98.71% when the soil moisture decreased from 25% to 8.1%. 3.4. Higher leaf water content in EaHSP70 overexpressed sugarcane transgenics at V1 stage Transgenic events showed a significantly higher RWC under water stress when compared to WT plants at V1 stage (Fig. 4). On the day before withholding the irrigation, the RWC of each plant was monitored to ensure that the water status of transgenic and WT plants was similar at this condition. At 8.1% soil moisture, out of 25 independent transgenic events, in four events (3, 10, 11 and 18) the reduction in RWC from the normal irrigated condition did not exceed 3%. Fifty-two percent of the transgenic events had a maximum of 10% reduction in RWC. In addition, the 13th event showed reduced RWC, almost as much as that of the WT plants, i.e. 30% reductions from the normal irrigated condition. In WT plants, the RWC decreased by 30% when the soil moisture was reduced from 25% to 8.1%. 3.5. Higher rate of gas exchange parameters, chlorophyll content and photosynthetic efficiency at V1 stage The gas exchange parameters, stomatal conductance, transpiration rate and photosynthesis rate were significantly higher in transgenics when compared to the WT plants at V1 stage (Fig. 5A–C). Three events 14, 15 and 22 showed 2 times higher stomatal conductance and 1.5 times higher transpiration rate than the WT plants.

Fig. 5. Gas exchange parameters in EaHSP70 transgenics and WT at 8.1% soil moisture: (A) stomatal conductance, (B) transpiration rate and (C) photosynthesis rate. All the transgenic events differ significantly with respect to control Co 86032 (P ≤ 0.05; Student’s t-test). Data are presented as mean ± SD (n = 3) and error bars represent SD.


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Fig. 6. (A) Chlorophyll content in EaHSP70 transgenic events and WT with and without stress. All the transgenic events differ significantly with respect to control Co 86032 (P ≤ 0.05; Student’s t-test). (B) Photosynthetic efficiency of EaHSP70 transgenic events and WT with and without stress. Data shows a significant difference in the photosynthetic efficiency between transgenics and untransformed control, ns: not significant at P ≤ 0.05, by Student’s t-test. Data are presented as mean ± SD (n = 5) and error bars represent SD.

Sixty-eight percent of the events had 1–2 times higher stomatal conductance and transpiration rate. The 14th event had 5 times higher photosynthesis rate than WT plants. All the events maintained significantly higher photosynthesis rate than WT plants. The chlorophyll content and photosynthetic efficiency were significantly higher in transgenics than WT plants at V1 stage (Fig. 6A and B). On the day before withholding the irrigation, the chlorophyll content (SPAD values) in transgenics varied between 35 and 45 SPAD units, whereas in WT sugarcane it was 35 SPAD units, indicating that there was not much variation in the chlorophyll content at this stage. After the induction of drought stress, out of the 25 transgenic events screened, 36% of the transformants exhibited only a maximum reduction of 3 SPAD units. Fifty-six percent of the transgenic plants showed a maximum reduction of 5 SPAD units in chlorophyll content and it varied between 30 and 40 SPAD units (Fig. 6A). However, an increased level of chlorophyll was observed after re-irrigating for 15 days (36–42 SPAD units). On the 10th day of stress, there was a reduction of 10 SPAD units in WT plants (25 SPAD unit), and after re-irrigation for 15 days the value was around 27 SPAD units in these plants (Fig. 6A). The Fv/Fm values were measured at V1 stage, which varied between 0.55 and 0.75 at 8.1% soil moisture (Fig. 6B). The fourth event showed a higher value (0.75) at 8.1% soil moisture. Sixty percent of the events were in the range 0.6–0.7 at 8.1% soil moisture. The 13th event showed a reduced value similar to that of the WT plants at 8.1% soil moisture (0.55). After the release of stress (reirrigation for 15 days), the values varied between 0.71 and 0.73 at

25% soil moisture, which was same as the 0th day value (0.73–0.77). The Fv/Fm for the WT plants at 8.1% soil moisture was 0.5 and after re-irrigation, it reached 0.71, which was not much different from the 0th day value (0.73). The pooled data of all the physiological parameters along with the visual scoring for transformants with EaHSP70 are given in Table 2 (supplementary data). Based on the physiological parameters, five independent events showing a higher level of tolerance to drought were selected for further gene expression (qPCR) analysis. Table 2 Bud germination (%) of EaHSP70 transgenic plants in pots supplied with 0 mM (water), 100 mM NaCl, 200 mM NaCl and 300 mM NaCl solution for 26 days. The data for WT plants could not be obtained in 100 mM NaCl, 200 mM NaCl and 300 mM NaCl solution because these plants failed to sustain growth in the presence of NaCl. Event no.

NaCl concentration 0 mM

5 6 8 10 13 19 20 22 23 24 WT

100% 80% 80% 100% 100% 80% 100% 100% 100% 100% 100%

100 mM 100% 60% 100% 100% 60% 80% 60% 80% 80% 100% –

200 mM 60% 40% 60% 60% 40% 20% 40% 60% 80% 100% –

300 mM 60% 40% 20% 60% 20% 20% 20% 20% 60% 100% –

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Fig. 7. (A) Relative expression of EaHSP70 gene in transgenic events under normal irrigated condition. (B) Relative expression of endogenous ScHSP70 in WT plants. (C) Relative expression of EaHSP70 gene in transgenic events under water stress condition. (D–H) Relative expression of the abiotic stress responsive genes in individual transgenic sugarcane plants. ␤-actin was used as a reference gene to measure the relative quantification. Data is presented as mean ± SD (n = 5) and error bars represent SD.

3.6. Expression analysis of the stress-responsive genes in EaHSP70 overexpressed sugarcane transgenics by qPCR at V1 stage Relative expression of the EaHSP70 gene under normal irrigated condition was evaluated in twenty independent transgenic events, and assessed through comparative CT method at the V1 stage. Each event had a 40-fold or more expression of EaHSP70 compared to the WT (Fig. 7A). At 8.1% soil moisture all the events have 500-fold and above EaHSP70 expression (Fig. 7C). The ScHSP70 gene expression was assessed in WT under different soil moisture conditions, and it showed only 13-fold upregulation under drought stress (Fig. 7B). The relative transcript abundance of eight

abiotic stress-responsive genes (DREB2, DNA helicase, ERD, ERF, RD29, LEA, Cor15 and BRICK) known to be induced by drought were evaluated in five independent transgenic events which were selected through relative expression data, physiological studies and assessed through comparative CT method at V1 stage (Fig. 7D–H). qPCR analysis of the EaHSP70 gene was performed in leaf samples collected on the 10th day of drought stress. In each of the selected five HSP70 transgenic events, at 8.1% soil moisture the increase in the EaHSP70 gene expression varied from 2000- to 2680-fold compared to WT plants. The relative expression of DREB2, DNA helicase, ERD, ERF, RD29, LEA, Cor15 and BRICK were 800–1880, 300–470, 100–190, 700–1010, 1000–1860, 100–660, 400–1000,


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Fig. 8. (A) Representative picture to show phenotypic differences in the leaf of EaHSP70 transgenics (13th event) and WT after incubation in 0 and 250 mM NaCl solution for 72 h. (B) Chlorophyll content from leaf disks of EaHSP70 transgenics and WT after incubation in 0 and 250 mM NaCl solution for 72 h. Data shows a significant difference in the chlorophyll content between transgenics and untransformed control at P ≤ 0.05, by Student’s t-test. Data is presented as mean ± SD (n = 5) and error bars represent SD.

and 1500–2100 fold upregulated in HSP70 transgenic events compared to WT. 3.7. Overexpression of EaHSP70 in sugarcane results in tolerance to excess salinity at V1 stage To understand the level of tolerance of the transgenic events to salinity, leaf disk assay was carried out at the V1 stage. Leaf discs from V1 transgenics and WT plants were subjected to 0 and 250 mM NaCl for 72 h. The damage caused by salinity stress was reflected in the degree of bleaching observed in the leaf tissue after 72 h (Fig. 8A). Measurement of the chlorophyll content provided further support for a positive relationship between the overexpression of HSP70 and tolerance to excess salinity (Fig. 8B). In the WT plants, there was complete bleaching of the excised leaf under salinity stress, and the chlorophyll reduction was as high as 9 ␮g. The chlorosis rate was lower because the salt stress was much lower in EaHSP70-overexpressing events compared with WT plants, and it varied between events. The lowest chlorosis rate was observed in the 20th event with 0.21 ␮g reduction of chlorophyll (i.e. higher tolerance to salinity), and the highest rate of chlorosis was in the 16th event with 11.54 ␮g reduction of chlorophyll. 3.8. EaHSP70-overexpressed sugarcane has better bud germination ability under excess salinity at V1 stage V1 transgenic events and WT plants were morphologically similar when grown without NaCl (Fig. 9A and B). Increasing the

Fig. 9. Screening of EaHSP70 transgenic events along with the WT for bud germination assay for salinity stress. Bud germination assay for salinity tolerance: (A) WT and (B) EaHSP70 transgenic events.

salt concentrations significantly affected the germination percentage and shoot length of the transgenic events to varying degrees. Ten independent events were selected from the leaf disk assay and screened along with the WT plants. The WT plants failed to

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Table 3 Shoot height (cm) of EaHSP70 transgenic plants in pots supplied with 0 mM (water), 100 mM NaCl, 200 mM NaCl and 300 mM NaCl solution for 26 days. Event no.

NaCl concentration 0 mM

5 6 8 10 13 19 20 22 23 24 WT

4.50 5.50 6.87 5.90 4.75 5.50 3.50 4.90 7.50 6.60 6.26

± ± ± ± ± ± ± ± ± ± ±

1.17 1.17 0.62 2.30 1.06 0.70 0.00 0.54 1.73 2.17 0.47

100 mM

200 mM

300 mM

4.25 ± 3.60 ± 5.50 ± 5.30 ± 6.00 ± 5.00 ± 4.00 ± 4.40 ± 7.20 ± 5.90 ± –

5.0 ± 4.3 ± 5.6 ± 4.8 ± 5.5 ± 4.0 ± 2.0 ± 4.3 ± 7.0 ± 5.5 ± –

4.50 ± 4.00 ± 5.50 ± 4.80 ± 5.50 ± 4.30 ± 3.75 ± 3.20 ± 6.50 ± 5.50 ± –

0.64 1.15 1.73 0.50 0.00 0.00 0.00 0.57 0.64 0.00

0.00 1.15 0.47 0.20 1.40 2.12 0.00 0.76 1.40 1.40

0.00 0.00 0.00 1.15 2.60 1.15 0.35 0.26 2.20 0.86

germinate in all the three salt concentrations, indicating susceptibility of this variety to excess salinity. In HSP70 transgenics, four events (5, 8, 10 and 24) had 100%, three events (19, 22 and 23) had 80% and three events (6, 13 and 20) had 60% germination at 100 mM NaCl. At 200 mM NaCl, one event (24) had 100%, one event (23) had 80%, four events (5, 8, 10 and 22) had 60%, three events (6, 13 and 20) had 40% and one event (19) had 20% germination. At 300 mM NaCl, one event (24) had 100%, three events (5, 10 and 23) had 60%, one event (6) had 40% and five events (8, 13, 19, 20 and 22) had 20% germination, proving their tolerance to excess salinity (Table 2). The shoot height is one of the critical growth parameters and was measured as an indicator of salinity stress tolerance in V1 transgenic events along with the WT plants (Table 3). The height of the different transgenics varied between events under different salt concentrations. It should be noted here that similar data for WT plants grown under salinity could not be obtained because these plants failed to sustain growth in the presence of NaCl. 4. Discussion In the present study, HSP70 was isolated from a highly droughttolerant E. arundinaceus accession with a view to engineering the cultivated sugarcane with this gene. Transcriptome engineering has emerged as a promising avenue for the development of abiotic stress-tolerant crops. The overexpression of EaHSP70 enhanced drought and salinity tolerance in sugarcane. In transgenic sugarcane, EaHSP70 overexpressed 2000-fold and above compared to the WT plants. The physiological and molecular traits associated with drought and salinity tolerance showed a significantly enhanced expression in transgenics compared to WT plants. In the present study, a constitutive ubiquitin promoter (Port Ubi 2.3) from Porteresia coarctata was used for driving the transgene. Maize ubi1 promoter has been reported to overexpress under stress in transgenic rice [27,28] and potato Ubi-7 was overexpressed under stress in transgenic potato [29]. It was also reported that Port Ubi 2.3 has many stress responsive cis-elements [14] and our studies have shown the induction of the GUS reporter gene under moisture stress (personal observation). This may be the reason for the drastic upregulation of the EaHSP70 gene under moisture stress. The transgenics overexpressing EaHSP70 were found to have increased membrane stability than WT plants under normal irrigated conditions and even higher stability under moisture stress. Transgenic potato plants expressing DcHSP17.7 were shown to exhibit enhanced cellular membrane stability and tuberization in vitro at elevated temperatures [30]. The overexpression of HSP70 conferred tolerance to heat stress along with improved physiological parameters such as water and nutrient use efficiency, photosynthesis, assimilate partitioning and membrane stability [30–32]. Such improvements make plant growth and development

possible under drought/heat stress. The transgenic plants maintained significantly high RWC than WT plants during stress. Generally, osmoregulation is one of the main mechanisms preserving turgor pressure in most plant species against water loss [33]. In [34], it was reported that better osmotic regulation or lower elasticity of the cell walls conferred higher RWC. To reveal the response to stress intensity leaf RWC is one of the best growth/biochemical parameter [35]. The plants that have high RWC under drought stress having higher yield too. The potential of a plant to survive acute water deficits depends on its ability to restrict water loss through the leaf epidermis after the stomata have attained the minimum aperture [36]. Previous studies reported that there is a correlation between photosynthetic rate and leaf water potential, stomatal conductance and transpiration rate [37]. Photosynthetic capacity of the plant is indicated in its chlorophyll content [38,39]. In the present study, the transgenic events with EaHSP70 showed significantly higher chlorophyll content under soil moisture stress. All the transgenic events maintained high, i.e. 30 SPAD units and above, chlorophyll content at 8.1% soil moisture stress which varied between events. During stress, the chlorophyll content stability in the plant tissues allows the cells to maintain functional chloroplast and that helps the plant to revive the photosynthesis after the stress. Hence, the total chlorophyll content estimation can help to understand the tolerance of plants to abiotic stresses. In tomato, genotypes with increased chlorophyll content were observed to be tolerant to high temperatures, indicating that increased chlorophyll content is related to thermotolerance in tomato [31]. The Fv/Fm ratio was significantly higher in EaHSP70 transgenics than in WT plants under moisture stress. Previous reports stated that at least in the early maturing varieties there is a genotypedependent decrease of fluorescence quantum yield (Fv/Fm) in potato under drought, and small decreases were associated with drought tolerance [40]. The initial fluorescence (Fo) decline reflects a regulatory process damage external to P680 (reaction center of PSII), such as photoprotective processes impairment that facilitates the dissipation of excess energy within the leaf [41]. The Fv/Fm reduction indicates either a reversible photo-protective down regulation, or irreversible inactivation of PSII [42,43]. The Fv/Fm ratio was not much reduced in EaHSP70 transgenic plants under moisture stress, suggesting that the function of reaction centers was little affected in transgenic plants relative to the control. It indicated a substantial protection of the photosystem, especially PSII, during drought stress in EaHSP70 transgenic plants. In EaHSP70-overexpressed sugarcane, there was a drastic increase (2000-fold or more) in the upregulation of HSP70 gene compared to the WT plants. In addition, the upregulation of the abiotic stress-responsive genes, i.e. DREB2, DNA helicase 45, LEA, RD29, ERD, ERF, Cor15 and BRICK, was more than 100-fold in each transgenic event when compared to WT plants. Possibly, this might be one of the reasons for its enhanced drought and salinity tolerance. So far, there has been no report on the upregulation of DREB2 while overexpressing HSP70. DREB2 is one of the main droughtresponsive transcription factors determining the expression levels of a number of downstream genes like RD29, HSP70, etc. [44–46]. In the present study, DREB2 was upregulated by 800-fold and more in HSP70-overexpressed transgenics. HSP70 is known to be activated by DREB2, but the mechanism of upregulation of DREB2 in HSP70 transgenics needs to be studied. HSP70 acts as a molecular chaperone by maintaining homeostasis of protein folding and thus helps in maintaining the metabolic and structural integrity of the cells [40,47]. The individual HSP70 proteins specific roles can be determined by their location in different subcellular compartments [48,49], by the differential expression of HSP70s at different stages of development [50] or by their interaction with specific sets of HSP70-associated proteins. For example, the cytosolic HSP70 helps


to prevent protein aggregation, assists folding of de novo proteins and organellar precursor proteins maintenance. Mitochondrial and chloroplastic HSP70 proteins are involved in precursor protein import and translocation. HSP70 also have a role in regulation of other stress-associated gene expression [51]. However, the cellular mechanisms of HSP70 function under stress conditions are not fully understood. HSP70 also has a role in the regulation of signal transducers such as protein kinase A, protein kinase C and protein phosphatase [52]. However, HSP70 chaperones are presumed to play a broad role in regulating many downstream genes expression in signal transduction pathways, under stress and normal growth conditions. The exact role of HSP70 in the modulation of signal transduction in plants has not been proved. The upregulation of DREB2 also helps in the upregulation of many DREB2-responsive genes including LEA, RD29, ERD, ERF and COR15, which was demonstrated in the present study. LEA proteins are quite hydrophilic and are believed to protect plant cells from these stresses. These proteins represent another category of high molecular weight proteins that are abundant during late embryogenesis and accumulate during seed desiccation, water stress [53] and cold stress [54]. RD29 also encodes proteins similar to LEA [55]. ERD is upregulated by dehydration stress and by natural and dark-induced senescence [56,57]. ERF (ethyleneresponsive element-binding factor) is a subfamily of the AP2/EREBP (APETALA2/ethylene responsive element-binding protein) plantspecific transcription factor [58]. Enhanced frost tolerance and induction of cor genes were detected in non-acclimated transgenic Arabidopsis [59]. In addition, DRE elements have been found in the promoter of COR15. The BRICK gene helps in the polymerization of actin [60], and an upregulation of the BRICK gene was identified in the drought-tolerant E. arundinaceus under moisture stress (unpublished data). Overexpression of EaHSP70 in sugarcane also increased the plant’s tolerance against salinity, as reflected by the higher chlorophyll retention in leaf disk assay and improved bud germination. Chlorophyll content measurements revealed that in WT plants the chlorophyll content decreased significantly when compared to the transgenic events under salinity, suggesting the high salinity tolerance potential of the transgenic events. In plants, the reduction of oxidative damage could provide enhanced resistance to salt stress [61]. Overexpression of HSP70 is known to reduce the level of reactive oxygen species (ROS), thus conferring tolerance to salinity [62]. In general, EaHSP70 transgenics showed higher germination rate under high salt conditions (300 mM NaCl) compared to WT plants (Fig. 9A and B). This might be due to the constitutive expression of several salt/osmotic stress-related genes in the transgenic events (Fig. 7C–G). In conclusion, our results showed that transformation of sugarcane with the EaHSP70 gene under the control of the Port Ubi 2.3 promoter enhanced the tolerance of the sugarcane variety Co 86032 to drought and salinity stress to a high extent. As the success of any biotechnological strategy would ultimately be determined by the final yield at field conditions, future studies will involve the analysis of the performance of the sugarcane transgenic events used in this study at realistic field conditions and under different watering regimes. Conflicts of interest The authors have no conflicts of interest to declare. Acknowledgement This work was supported by the Department of Biotechnology (DBT) (grant no. BT/PR6277/AGII/106/882/2012), Government of India.

33

Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.plantsci. 2014.12.012.

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