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Improved tolerance to post-anthesis drought stress by pre-drought priming at vegetative stages in drought-tolerant and -sensitive wheat cultivars
Plant Physiology and Biochemistry 106 (2016) 218e227
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Research article
Improved tolerance to post-anthesis drought stress by pre-drought priming at vegetative stages in drought-tolerant and -sensitive wheat cultivars Muhammad Abid, Zhongwei Tian, Syed Tahir Ata-Ul-Karim, Yang Liu, Yakun Cui, Rizwan Zahoor, Dong Jiang, Tingbo Dai* Key Laboratory of Crop Physiology, Ecology and Production Management, Nanjing Agricultural University, Nanjing 210095, Jiangsu Province, PR China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 7 April 2016 Received in revised form 4 May 2016 Accepted 4 May 2016 Available online 5 May 2016
Wheat crop endures a considerable penalty of yield reduction to escape the drought events during postanthesis period. Drought priming under a pre-drought stress can enhance the crop potential to tolerate the subsequent drought stress by triggering a faster and stronger defense mechanism. Towards these understandings, a set of controlled moderate drought stress at 55e60% field capacity (FC) was developed to prime the plants of two wheat cultivars namely Luhan-7 (drought tolerant) and Yangmai-16 (drought sensitive) during tillering (Feekes 2 stage) and jointing (Feekes 6 stage), respectively. The comparative response of primed and non-primed plants, cultivars and priming stages was evaluated by applying a subsequent severe drought stress at 7 days after anthesis. The results showed that primed plants of both cultivars showed higher potential to tolerate the post-anthesis drought stress through improved leaf water potential, more chlorophyll, and ribulose-1, 5-bisphosphate carboxylase/oxygenase contents, enhanced photosynthesis, better photoprotection and efficient enzymatic antioxidant system leading to less yield reductions. The primed plants of Luhan-7 showed higher capability to adapt the drought stress events than Yangmai-16. The positive effects of drought priming to sustain higher grain yield were pronounced in plants primed at tillering than those primed at jointing. In consequence, upregulated functioning of photosynthetic apparatus and efficient enzymatic antioxidant activities in primed plants indicated their superior potential to alleviate a subsequently occurring drought stress, which contributed to lower yield reductions than non-primed plants. However, genotypic and priming stages differences in response to drought stress also contributed to affect the capability of primed plants to tolerate the postanthesis drought stress conditions in wheat. © 2016 Published by Elsevier Masson SAS.
Keywords: Pre-drought priming Post-anthesis Drought stress Photosynthesis Antioxidants Winter wheat
1. Introduction Drought stress has become the most devastating constraint to crop productivity due to aridifying and warming climatic trends, and its prevalence is expected to diversify in future at regional and global scales (Backhaus et al., 2014). Wheat is one of the foremost staple food crops and is reported to be highly susceptible to drought stress which often occurs at post-anthesis phase consequencing considerable yield penalties (Wang et al., 2014a). Grain yield is the ultimate product of photosynthesis and closely interrelated physiological processes. Fluctuations around the
* Corresponding author. E-mail address: [email protected] (T. Dai). http://dx.doi.org/10.1016/j.plaphy.2016.05.003 0981-9428/© 2016 Published by Elsevier Masson SAS.
normal values of photosynthesis and its interlinked processes are the key indicators of plant fitness and extent of environmental stress (Zlatev and Lidon, 2012). Mild drought stress declines the rate of photosynthesis due to limited stomatal conductance, meanwhile the photosynthetic apparatus is not significantly affected (Cornic, 2000). In contrast, under severe drought stress, stomatal limitation, the poor efficiency of photosystemII (PSII) as well as declined activities of CO2 assimilating enzymes such as ribulose-1, 5-bisphosphate carboxylase/oxygenase (Rubisco) have been reported as the primary constraints to lower photosynthetic rates (Bota et al., 2004). In addition to direct drought-induced damages to the photosynthetic process, it also leads to the light-induced oxidative stress by the generation of reactive oxygen species (ROS) in the plant cells (Reddy et al., 2004). If the drought stress proceeds and ROS
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accumulation is not quenched by antioxidants, it results in protein oxidation, membrane lipid peroxidation, inhibition of DNA, RNAs and hormonal activities, eventually turning the cell into a state called as “oxidative stress”, which adversities the normal growth or even causes the death of plants (Liu et al., 2015). The limited wheat yield under environmental stresses is mostly attributed to the downregulation of key yield influential photosynthesis and other interdependent physiological processes functioning in the flag leaf during grain filling (Bruce et al., 2007; Zlatev and Lidon, 2012). Therefore, improving the tolerance to drought stress, especially at post-anthesis phase is of great significance in yield production of wheat. Previous research efforts provide promising evidence that priming “the pre-exposure of plants to a stimulus stress enabling them to mobilize their rapid and intense defense mechanism” can induce improved tolerance in plants to later-occurring stress events (Bruce et al., 2007). To date, various priming techniques have been used in different crop species against a range of environmental disasters. So far, mainly attention has been focused to exogenously applied chemical-induced priming such as nitric oxide for drought priming in rice (Farooq et al., 2010), hydrogen sulphide for drought priming in wheat (Shan et al., 2011), hydrogen peroxide for salt stress priming in wheat (Li et al., 2011a,b) and b-aminobutyric acid for pathogens attack priming in Arabidopsis (Zimmerli et al., 2000). Similarly, the priming state has also been achieved by bioticinduced colonization of plants with beneficial micro-organisms (Abdel Latef and Chaoxing, 2011) and by the epigenetic changes in plants for the initiation and regulation of their potential defense metabolism (Beckers et al., 2009). Some recent studies in various crop species have revealed that the plants pre-exposed to environmental stress can also attain the potential to display a faster and stronger activation of their defense system in response to the subsequent challenge stress events (Backhaus et al., 2014; Vu et al., 2015). For example, the plants of Arrhenatherum elatius experiencing an early drought episode showed an improved photo-protection and higher biomass under a second drought event than non pre-exposed plants (Walter et al., 2011). Similarly, pre- exposure of vegetative stage rice plants to sub-lethal heat stress improved the thermo-tolerance to heat stress during grain filling (Shi et al., 2015). Likewise, these beneficial prestress imprints have also been reported in tobacco (Choi and Sano, 2007), radish (Vu et al., 2015) and some grass species (Meisner et al., 2013). A few current studies addressing these legacy effects of prestress priming are also available in wheat. For example, wheat plants exposed to early stage heat stress, displayed an improved antioxidative capacity and higher grain yields against terminal high-temperature stress (Wang et al., 2014b). Another study on wheat investigated that waterlogging pre-treatment during vegetative growth stage enhanced dry matter accumulation and its distribution to grain formation resulting in a noticeably improved grain yield (Li et al., 2011a,b). The studies exploring the response of pre-drought stressinduced drought priming to post-anthesis drought events in wheat are rare (Wang et al., 2014a). Moreover, the response of primed plants to succeeding drought stress may vary due to the interval between priming and the reoccurring stress (Backhaus et al., 2014), due to response difference of growth stages to drought stress (Wang et al., 2015) as well as due to genotypic differences in tolerance to drought stress (Rampino et al., 2006; Khanna-Chopra and Selote, 2007). So, we suggest that drought priming study should be extended to gain insights into the resilience response difference of wheat cultivars and priming growth stages to the subsequent drought stress. This study was aimed to investigate that (1) whether drought priming during vegetative growth stages in
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wheat plants develops a memory to adapt the subsequent drought occurrence, and (2) whether the drought priming effect varies under varying cultivars and vegetative growth stages selected for priming. Hopefully, the projected results of the study would be supportive for research programs seeking to develop anti-drought stress practices for wheat.
2. Materials and methods 2.1. Plant culture and growth conditions A greenhouse experiment was carried out at Pailou Experimental Station of Nanjing Agricultural University, China (32 040 N, 118 760 E), during the growing season of 2014e2015. Two winter wheat cultivars with contrasting attitude towards drought stress namely Luhan-7 (drought resistant) and Yuangmai-16 (drought sensitive) (Wang et al., 2007), were used as experimental material in the present study. Fifteen surface sterilized uniform seeds were planted in the free-draining plastic pots having 22 cm and 25 cm height and diameter, respectively. Each pot was filled with 8 kg airdried, sieved (0.5 mm) and uniformly mixed clay loam soil having 13% soil moisture. At the time of soil filling, 0.8 g N, 0.5 g P2O5 and 1.1 g K2O/pot were applied for each treatment. Further, 0.4 g/pot N was applied at jointing and booting, respectively. Thinning was carried out 10 days after germination at 10 seedlings per pot. Then a week later, second thinning was carried out and seven uniform seedlings per pot were selected for the subsequent studies. Each pot was irrigated to 80% field capacity (FC) with tap water characterized with 7.5 pH, 2.8 dsm1electrical conductivity (EC) and 1200 mg L1 total soluble salts (TSS) until start of the treatments. Soil water status was measured before the application of water to the pots. The amount of water required for irrigation was calculated by using the following equation:
W ¼ Y H A ðFC1 FC0Þ
(1)
where, W is amount of irrigation water, Y is soil bulk density, H is soil depth, A is the area of the pot, FC1 is the desired soil FC, and FC0 is the actual soil FC before irrigation. Each treatment had 20 replicates (pots). The pots with different treatments were rotated on every alternate day to ensure that all the pots received equal radiation and other environmental exposures till maturity.
2.2. Treatments application and management 2.2.1. Drought priming treatments The drought priming treatments were carried out at tillering (40 days after planting, Feekes 2.0, beginning of tillering, 4 leaf stage) and at jointing (125 days after planting, Feekes 6.0, when first node was visible, 6 leaf stage), as described in experimental description under Fig. 1. At each priming stage, the irrigation to pots was withheld until a moderate drought stress stage at 55e60% FC reached. This moderate drought stress level as priming treatment at tillering and jointing respectively was maintained for 10 days by compensating the lost water. Meanwhile, the control pots kept on irrigating at 80% FC. After priming at both stages, the pots were rewatered to the level of control pots until the application of the subsequent drought stress. Thus, four priming treatments for each cultivar were designated as PT and PJ for priming at tillering and jointing, meanwhile non-primed plants were designated as NT and NJ during tillering and jointing, respectively. The overall nonprimed plants were designated as NN.
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Fig. 1. Experimental design for the study: Drought priming was applied at tillering (PT) and jointing (PJ) 11 by exposing the wheat plants to moderate drought stress (55-60% FC) for 10 days and followed by re12 watering. NT, NJ and NN indicated no drought priming at tillering, jointing and at any growth stage, 13 respectively. At 7 days after anthesis, severe drought stress was applied at 35-40% FC for 7 days and 14 followed by re-watering. The treatments during post-anthesis drought stress were designated as PTD=15 priming at tillering þ post-anthesis drought stress, PTC= priming at tillering þ no post-anthesis drought 16 stress, PJD= priming at jointing þ post-anthesis drought stress, PJC= priming at jointing þ no post17 anthesis drought stress, NND= no priming entirely þ post-anthesis drought stress, and NNC= no priming 18 entirely þ no post-anthesis drought stress. Tillering, jointing and post-anthesis were the stages of 19 treatments application, measurements and sampling. After measurements and sampling at last day of 20 priming and post-anthesis drought stress treatments, the plants were re-watered immediately at 75-80% 21 FC for recovery.
2.2.2. Post-anthesis drought stress treatments At anthesis, tillering and jointing stage primed (PT and PJ) and entirely non-primed (NN) plants were divided into two halves. One half plants of PT, PJ and NN were exposed to severe drought stress (D) at 35e40% FC at 7 days after anthesis (160 days after planting, Feekes 11.0), meanwhile other half plants kept on watering (C). The drought stress at this FC was maintained for 7 days by compensating the water lost through evapo-transpiration to the desired FC. After stress completion, the pots were re-watered to 75e80% FC up to maturity. Thus, six post-anthesis severe drought stress treatments for each cultivar were designated as PTD (priming at tillering þ post-anthesis drought stress), PTC (priming at tillering þ no post-anthesis drought stress), PJD (priming at jointing þ post-anthesis drought stress), PJC (priming at jointing þ no post-anthesis drought stress), NND (non-priming entirely þ post-anthesis drought stress), and NNC (non-priming entirely þ no post-anthesis drought stress). 2.3. Plant sampling Plant sampling and measurements were carried out at the last day of drought priming at tillering and jointing as well as at the last day of post-anthesis drought stress. The top fully expanded leaves from three randomly selected pots/replicates were detached for each treatment. One leaf from each replicate was used to measure the leaf water potential (Jw), and other leaves were immediately put into liquid nitrogen and shifted to the university laboratory to store at 40 C for quantifying the total chlorophyll, Rubisco, lipid peroxidation and enzymatic antioxidants. One pot plants were used only once a time measurements and sampling and then were discarded from the experiment. At each sampling point, leaf gas exchange and chlorophyll fluorescence were also measure. Dry matter and grain yield traits were calculated at maturity. 2.4. Physiological measurements and chemical analysis 2.4.1. Leaf water potential Leaf water potential (Jw) was measured according to the method given by (Bruggink and Huang, 1997) by using a pressure chamber (PMS Instrument Co., Corvallis, OR, USA). Leaf was firmly fixed in the sealing sleeve of specimen holder of the instrument and pressure was built up until the appearance of sap from the exposed cut of the leaf. The pressure reading was noted at this point and expressed in eMPa. 2.4.2. Chlorophyll contents Frozen leaf samples (0.2 g) were placed for 24 h in a vial with
4 ml of dimethyl sulphoxide for pigment extraction to determine chlorophyll content according to Huang and Zhao (2001). The absorbance of the supernatant was measured using a Pharmacia Ultra Spec Pro UV/VIS spectrophotometer (Pharmacia, Cambridge, England), at a wavelength of 470 and 648 nm for chlorophyll a and b, respectively. The sum of chlorophyll a and b was used as total chlorophyll contents. 2.4.3. Rubisco contents Rubisco contents were measured with Western-blot analysis using the SDS-PAGE by following the method given by Makino et al. (1985). Leaf samples of 0.5 g were homogenized in a 50 mM TriseHCL buffer (pH 8.0) containing 12.5% (v/v) glycerol and 5 mM b-mercaptoethanol, and centrifuged for 15 min at 15,000 g. SDS, b-mercaptoethanol, and glycerol with a final concentrations of 1% (w/v), 2% (v/v), and 5% (v/v), respectively were added to the supernatant and the mixture was boiled for 5 min. A 10 mL of enzyme extract was used for electrophoresis. The gels were stained with 0.1% (w/v) Coomassie Brilliant Blue R-250 solution. The stained bands from the gels were excised and eluted in 1 mL of formamide at room temperature with agitation for 8 h with an unstained gel as a standard. The blots were finally washed three times in phosphate buffered solution as above and developed with Super Sigmal West Pico Chemiluminescent Substrate (Pierce, USA). Images of the blots were scanned using a CCD imaging system (Fluor SMax, Bio-Rad, USA) and Quantity One software (Bio-Rad, Hercules, CA, USA) was used to calculate the optical density. 2.4.4. Leaf gas exchange Leaf gas exchange measurements were done on leaf blades of evenly oriented and maximum light-exposed top leaves from five plants in each treatment with an open IRGA LI-COR 6400 system (LI- 6400, Li-Cor Inc. USA) at 9:00e11:00 h (local time). Net photosynthetic rate (Pn) and stomatal conductance (gs) were noted under light saturated conditions at photosynthetic photon flux density of 1000 mmol photon m2 s1, at 25 C and 380 mol mol1 of CO2. 2.4.5. Chlorophyll fluorescence On the day of gas exchange measurements, chlorophyll fluorescence was also recorded on the same leaves by using a portable modulated fluorescence measuring system (FMS2; Hansatech, King’s Lynn, Norfolk, UK). The base values of the minimum (F0) and maximum (Fm) chlorophyll fluorescence taken in 30 min fully dark adapted leaves were used to calculate the maximum efficiency of photosystemII (Fv/Fm ¼ Fm F0/Fm) as proposed by Maxwell and
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Johnson (2000).
HIð%Þ ¼ 2.4.6. Enzymatic antioxidants activities The activities of enzymatic antioxidants such as superoxide dismutase (SOD) and ascorbate peroxidase (APX) were determined according to the methods given by Jiang and Zhang (2002). Frozen leaf samples of 0.5 g were sliced and homogenized in a mortar, and pestle with 5 mL ice-cold extraction buffer. The homogenate was centrifuged at 10,000 g for 30 min at 4 C and the supernatant was used as crude extract for the above and MDA assays. Superoxide dismutase (SOD) activity was determined by adding 0.1 mL enzyme extract to a reaction mixture of 1.5 mL 50 mM sodium phosphate (pH 7.8), 0.3 mL 130 mM methionine, 0.3 mL 750 mM nitro-blue tetrazolium (NBT), 0.3 mL 100 mM EDTA-Na2, 0.300 mL 20 mM riboflavin and 100 mL distilled water, and illuminated in light of 4000 flux for 20 min and the sample absorbance was determined at 560 nm UV/visible spectrophotometer (UltrospecTM 2100 pro; Amersham Bio-sciences). One unit of SOD activity was considered as the amount of enzyme used for 50% inhibition of the NBT reduction. For APX activity, 0.2 mL crude enzyme extract was added to a reaction mixture of 50 mmol L1 potassium phosphate buffer (pH 7.0), 0.5 mmol L1 ASC and 0.1 mmol L1 H2O2. And APX activity was determined by observing the decrease at 290 nm for 1 min in 1 mL of the reaction mixture. 2.4.7. Lipid peroxidation estimation Lipid peroxidation was estimated by measuring the malondialdehyde concentration (MDA) following the thiobarbituric acid reaction solution (TBA) analysis (Jiang and Zhang, 2002). A mixture of 1 ml of extract and 4 ml of reaction solution was incubated at 95 C for 30 min, and cooled immediately. The mixture was centrifuged for 10 min at 12,000 g, and supernatant was used for determining the MDA content at 532 nm and 600 nm. 2.5. Morphological traits determination 2.5.1. Dry matter At maturity, three randomly selected pots were manually cut at the ground level using pruning-scissors from each treatment and oven dried at 70 C to a constant weight for measuring the total above-ground dry matter (DM; g/pot). 2.5.2. Grain yield From three randomly selected pots, spikes were cut and manually threshed. The grains were oven dried at 40 C for 24 h and then grain yield/pot was calculated. 2.5.3. Drought index Drought index (DI) was estimated as grain yield differences between drought priming and drought stress treatments as compared to control pots according to the following method;
DI ¼
YD YN
Grain yield 100 Dry weight of above ground biomass
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(3)
2.6. Statistical analysis The analysis of variance (ANOVA) was performed by using General Linear Model procedure to calculate the homogeneity of variance under drought priming and post-anthesis drought stress treatments, respectively. Means were compared by using Duncan’s multiple comparison test (P < 0.05). A two-way ANOVA was performed to evaluate the level of significance of drought priming, drought stress, cultivars and their interactive effect by using SPSS statistical package (SPSS Inc., Chicago, IL, USA). Graphs were plotted by using Sigma Plot 10.0 software (Systat Software Inc., Chicago, IL, USA). 3. Results 3.1. Morphological traits 3.1.1. Dry matter production Dry matter production (DM) calculated at maturity was significantly (P < 0.05) reduced by post-anthesis drought stress treatments (PTD, PJD, and NND) in both cultivars as compared to NNC (control) (Table 1). However, decrease in DM was relatively lower in primed plants than the non-primed plants. The plants of Luhan-7 showed lower decrease in DM under drought stress than those of Yangmai-16. In relation to priming stages, there was no significant difference in DM reduction in PTD and PJD in both cultivars. The DM was decreased by 14, 15 and 19% and by 19, 19 and 25% under PTD, PJD, and NND as compared to NNC in Luhan-7 and Yangmai-16, respectively. 3.1.2. Grain yield production Post-anthesis drought stress resulted in a significant decrease in grain yields. Yangmai-16 exhibited higher yield reduction than that Luhan-7 as compared to NNC, respectively. PTD and PJD significantly decreased the extent of yield reduction in both cultivars, however, the positive effect of drought priming to decrease the grain yield reduction was higher in Luhan-7 than that Yangmai-16. In both cultivars, PTD showed lower yield reduction than PJD. Overall, grain yield was reduced by 37, 25 and 20% in Luhan-7 whereas in Yangmai-16, it was reduced by 49, 28, and 24% under NND, PJD, and PTD as compared to NNC, respectively. 3.1.3. Drought index Under post-anthesis drought stress, higher drought index (DI) values were observed in primed plants than those non-primed plants. PTC gave higher DI in Luhan-7 (0.98) and Yangmai-16 (0.96). However, under NND, Yangmai-16 exhibited lower DI (0.55) than that Luhan-7 (0.65). Overall, DI was reduced by 14, 22 and 33% in Luhan-7 and by 20, 26, and 41% in Yangmai-16 under PTD, PJD and NND as compared to PTC, respectively.
(2)
where, YD is the grain yield under a treatment condition and YN is the grain yield under fully irrigated conditions. 2.5.4. Harvest index Harvest index (HI) was calculated as the ratio between grain weight and the total dry weight of the above ground biomass;
3.1.4. Harvest index Harvest index (HI) was also significantly reduced under postanthesis drought stress. Lowest values of HI (25% and 29%) were obtained under NND in Yangmai-16 and Luhan-7, respectively. Nevertheless primed plants, especially PTD showed higher HI in both cultivars. The primed plant of Luhan-7 gave higher HI than those of Yangmai-16 under drought stress. HI was decreased by 10, 14 and 30% in Luhan-7, whereas in Yangmai-16 it was decreased by
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Table 1 Effects of drought priming during vegetative growth stages on dry matter and grain yield traits under post-anthesis severe drought stress in Luhan-7 and Yangmai-16 wheat cultivars. Cultivars
Treatments
Dry matter (g/pot)
Grain yield (g/pot)
Drought index
Harvest index (%)
Luhan-7
PTD PTC PJD PJC NND NNC PTD PTC PJD PJC NND NNC
107.4d 121.1c 106.1d 117.8c 103.4d 125.5b 107.4d 126.8b 107.4d 123.5c 100.4d 132.3a
39.0d 49.7ab 35.1e 47.8b 30.5f 50.5a 34.5e 49.4ab 33.0e 45.6c 24.3g 51.1a
0.84e 0.98a 0.75f 0.94c 0.65g e 0.76d 0.96ab 0.71e 0.89cd 0.55h e
36.3d 41.3a 33.1e 40.1b 29.4h 40.3b 31.4f 39.4bc 29.0g 37.5d 25.3i 38.8c
FCultivars FDrought stress FC DS
15.0 ns 192.5* 10.5 ns
219.4* 552.1* 32.9*
1.7 ns 459.9* 21.7*
151.5* 406.5* 43.0*
Yangmai-16
Fvalues
Note: Within the columns of ‘Treatments’, PT, PJ and NN indicate drought priming at tillering and jointing, and no priming at either stage, respectively. C and D indicate no post-anthesis drought stress and post-anthesis drought stress, respectively. Data are means ± SE, (n ¼ 3). Different lowercase letters following the data within the same column indicate significant differences at P < 0.05. A two-way analysis of variance was performed to evaluate the effect of cultivars (C), drought stress (DS) and their interaction (C DS). ns ¼ not significant; * ¼ significant at P < 0.05.
17, 25 and 41% under PTD, PJD, and NND as compared to NNC, respectively.
than PJD. As compared to NNC, Jw was reduced by 245, 195 and 181% in Luhan-7, whereas in Yangmai-16, it was reduced by 350, 210, and 190% in NND, PJD, and PTD respectively.
3.2. Physiological traits 3.2.1. Leaf water potential Drought priming treatments during vegetative stages (PT and PJ), significantly decreased the leaf water potential (Jw) as compared to NT and NJ (Fig. 2A). This decrease was higher in case of Yangmai-16 than Luhan-7. During priming, plants at jointing showed more sensitivity to reduce Jw than tillering. Under subsequent drought stress at post-anthesis, NND, PJD and PTD significantly showed lower Jw as compared to NNC (Fig. 2B). However, PTD and PJD maintained significantly higher Jw in both cultivars. Under NND, Yangmai-16 showed higher decrease than that Luhan7. In relation to priming stages, PTD showed higher Jw regulation
3.2.2. Chlorophyll and Rubisco contents During drought priming at vegetative stages, a slight decrease in chlorophyll (Chl) and Rubisco contents in both cultivars was observed under PT and PJ as compared to NT and NJ (Fig. 3; A and C). This decrease was higher in Yangmai-16 as compared to Luhan-7. PT showed lower decrease than PJ in both cultivars. In contrast, under post-anthesis drought stress, Chl and Rubisco contents were significantly reduced in both cultivars as compared to NNC, respectively (Fig. 3; B and D). The primed plant PTD and PJD significantly showed higher Chl and Rubisco contents against NND, which were higher in Luhan-7 than that Yangmai-16. The Chl and Rubisco contents were reduced by 51, 40, and 38%, 60, 47 and 44% in
Fig. 2. Leaf water potential as affected by drought priming during vegetative growth stages (A) and post-anthesis drought stress (B) in Luhan-7 and Yangmai-16 wheat cultivars. In “treatments” PT, PJ, NT and NJ indicate drought priming and no priming at tillering and jointing stages, respectively. C and D indicate no post-anthesis drought stress and postanthesis drought stress, respectively. Different lowercase letters indicate significant differences at P < 0.05. A two-way analysis of variance was performed to evaluate the effect of drought priming (DP), cultivars (C) and their interaction (DP x C) during priming, and the effect of drought stress (DS), cultivars (C) and their interaction (DS x C) during postanthesis drought stress. ns ¼ not significant; * ¼ significant at P < 0.05.
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Fig. 3. Chlorophyll (Chl a þ b) and Rubisco contents (mg/g fresh weight) as affected during drought priming at vegetative stages (A and C), and post-anthesis drought stress (B and D) in Luhan-7 and Yangmai-16 wheat cultivars. In “treatments” PT, NT, PJ and NJ indicate drought priming and no drought priming at tillering and jointing, respectively. C and D indicate no post-anthesis drought stress (control) and post-anthesis drought stress, respectively. Different letters indicate significant differences at P < 0.05. A two-way analysis of variance was performed to evaluate the effect of drought priming (DP), cultivars (C) and their interaction (DP x C), and the effect of drought stress (DS), cultivars (C) and their interaction (DS x C). ns ¼ not significant; * ¼ significant at P < 0.05.
Luhan-7, whereas these were decreased by 60, 45 and 41% and 70, 51, and 48% in Yangmai-16, respectively under NND, PJD, and PTD as compared with NNC.
16 Pn, gs and Fv/Fm were reduced by 70, 53 and 43%, 63, 49, and 45% and by 29, 17 and 15% under NND, PJD, and PTD, respectively as compared to NNC.
3.2.3. Leaf gas exchange and chlorophyll fluorescence During drought priming, PT and PJ showed a significant decline in net photosynthetic rate (Pn), stomatal conductance (gs), and Fv/ Fm was observed as compared to NT and NJ (Fig. 4; A, C and E). Pn and gs during priming were more decreased in Yangmai-16 as compared to Luhan-7. Pn during priming was decreased by 32 and 36% in Yangmai-16 and by 22 and 27% in Luhan-7 under PT and PJ than NT and NJ, respectively. Whereas, gs was decreased by 30 and 34% in Yangmai-16 and by 22 and 27% in Luhan-7 under PT and PJ as compared to NT and NJ, respectively. At post-anthesis, the subsequent drought stress significantly downregulated the Pn, gs and Fv/ Fm values as compared to NNC (Fig. 4; B, D and F). Yangmai-16 showed more decline in these parameters under pos-anthesis drought stress treatments than that Luhan-7. In relation to priming stages, PTD in both cultivars maintained higher Pn, gs and Fv/Fm than PJD. The Pn, gs and Fv/Fm were reduced by 66, 48, and 41%, 59, 43 and 41% and by 21, 15, and 12% in Luhan-7, whereas in Yangmai-
3.2.4. Lipid peroxidation and enzymatic antioxidant activities During drought priming, the lipid peroxidation (MDA), activities of SOD and APX were increased in PT and PJ as compared to NT and NJ in both cultivars (Fig. 5A, C and E). However, Luhan-7 contained comparatively lower MDA contents and higher SOD and APX activities than that Yangmai-16. Under PT and PJ, MDA contents, SOD and APX activities were increased by 28 and 32%, 31 and 29%, and 18 and 20% in Yangmai-16, whereas in Luhan-7 MDA contents, SOD and APX activities were increased by 20 and 27%, 39 and 34%, and 25 and 20%, respectively as compared to NT and NJ. Under postanthesis drought stress, NND showed a significant increase in MDA contents, however, in case of SOD, it showed a slight increase but in APX activities, it showed a non-significant increase as compared with NND (Fig. 5B, D and F). In contrast, priming treatments PTD and PJD showed significantly lower contents of MDA and higher activities of SOD and APX as compared to NND in both cultivars. The higher upregulated SOD and APX activities and lower
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Fig. 4. Net photosynthetic rate (Pn), stomatal conductance (gs) and maximum photosynthetic efficiency of PSII (Fv/Fm) as affected by drought priming during vegetative stages (A, C and E, respectively) and post-anthesis drought stress (B, D and F, respectively) in Luhan-7 and Yangmai-16 wheat cultivars. In “treatments” PT, NT, PJ and NJ indicate drought priming and no drought priming at tillering and jointing, respectively. C and D indicate no post-anthesis drought stress (control) and post-anthesis drought stress, respectively. Different letters indicate significant differences at P < 0.05. A two-way analysis of variance was performed to evaluate the effect of drought priming (DP), cultivars (C) and their interaction (DP x C), and the effect of drought stress (DS), cultivars (C) and their interaction (DS x C). ns ¼ not significant; * ¼ significant at P < 0.05.
MDA contents under PTD and PJD were observed in Luhan-7 against Yangmai-16. Among priming stages, PTD showed higher SOD and APX activities, but lower MDA contents than that PJD in both cultivars. Under NND, PJD and PTD, the MDA contents, SOD and APX activities were increased by 67, 41 and 36%, 12, 56 and 61% and by 10, 51 and 60% in Luhan-7, whereas in Yangmai-16, these were increased by 85, 46, and 40%, 9, 51, and 57% and by 7, 43 and
47%, respectively, as compared with NNC. 4. Discussion In this study, the plants of two wheat cultivars were preexposed to drought stress for priming at tillering and jointing, respectively and then simultaneously subjected to severe drought
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Fig. 5. Lipid peroxidation (MDA contents), activities of superoxide dismutase (SOD) and ascorbate peroxidases (APX) as affected by drought priming during vegetative growth stages (A, C and E, respectively) and post-anthesis drought stress (B, D and F, respectively) in Luhan-7 and Yangmai-16 wheat cultivars, FW ¼ fresh weight. In “treatments” PT, NT, PJ and NJ indicate drought priming and no drought priming at tillering and jointing, respectively. C and D indicate no post-anthesis drought stress (control) and post-anthesis drought stress, respectively. Lowercase letters indicate significant differences at P < 0.05. A two-way analysis of variance was performed to evaluate the effect of drought priming (DP), cultivars (C) and their interaction (DP x C), and the effect of drought stress (DS), cultivars (C) and their interaction (DS x C). ns ¼ not significant; * ¼ significant at P < 0.05.
stress event at post-anthesis. It is evident from the results of the study that wheat plants with priming and non-priming treatments displayed a range of response processes to drought stress for regulating their survival, growth and final grain yield. Drought priming during vegetative growths proved to be a valuable strategy for facilitating the plants to initialize an efficient tolerance
mechanism, which in turn permitted the plants to tolerate the subsequent severe drought conditions adeptly. The findings of our study are in accordance with those, which reported stress imprint properties or stress memories developed by the stress priming in plants (Walter et al., 2011; Backhaus et al., 2014). In the present study, different physiological investigations were carried out which
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might be the sophisticate indicators and contributors to the drought tolerance in wheat plants. Nevertheless, the response of drought tolerant and sensitive cultivars primed at a particular growth stage was different towards the subsequent drought stress. It was observed that drought primed plants (PTD and PJD) reduced the grain yield reductions caused by the post-anthesis drought stress as compared to the non-primed plants (Table 1). Thus the priming strategy proved to be a great contribution to grain yield sustainability by enhancing the potential of plants to alleviate the drought effects at reproductive phase. The higher grain yield in primed plants against the non-primed plants seemed to be parallaly associated with their capability of retaining higher photosynthetic substrates (chlorophyll) and the regulation of higher leaf water potential (Jw) during stress period. Since, maintenance of favorable Jw under drought stress is vital to develop drought s et al., 2007; Khannatolerance mechanism in crop plants (Galme Chopra and Selote, 2007). The higher Jw observed in primed plants as compared to non-primed plants might be the prime reason for the leaf photosynthetic apparatus keep on functioning to maintain the grain development during drought stress period under the persistence of higher photosynthetic pigment and more CO2 assimilating enzyme (Rubisco) activity. Since, the quantity of coexisting photosynthates in the leaf depends on the performance of photosynthesis (Pn) (Cornic, 2000). The primed plants, showed a superior ability over the non-primed plants to develop the grains with higher weight by sustaining a higher rate of carbon accumulation through Pn in relatively long lasting canopy during grain filling period. Our findings of extended period of flag leaf Pn capacity in primed plants during grain-filling phase correlated with the production of higher grain yield were in consensus with previous reports by Foulkes et al. (2007). In contrast, in the non-primed plants, the plant canopy tended to senescence earlier with reduced grain filling duration. Under these contexts, the non-primed plants forcedly finished their life cycles earlier to produce the offspring with reduced grain yield rather than die and remain barren due to post-anthesis drought stress. The positive effects of drought priming to tolerate post-anthesis drought stress were more pronounced in plants primed at tillering than those primed at jointing. It might be because that during tillering, the wheat plants had lower leaf area, slower plant growth, less water demand, low transpiration rate as well plants were more flexible to get higher recovery after stress (Izanloo et al., 2008). In contrast, during jointing, the plant growth was rapid and the most important part of photosynthates reserves those contribute up to 57% of the grain yield of wheat is assimilated during this period (May, 1976). Moreover, the number of grains, which is a vital determinant of yield in drought conditions, depends on the plant growth during stem development and plant growth rate a few weeks before anthesis (Ercoli et al., 2008). So, the plants primed at jointing (PJD) even though showed higher grain yields than NND, but could not reach the levels of PTD in both cultivars. A rapid decline in photosynthetic rate of non-primed plants under drought stress metabolically might be related to lower Rubisco carboxylation activity, its higher degradation as well as more activation as oxygenase than as carboxylase. Since, a major consequence of abiotic stresses is the induced degradation and lower generation of Rubisco in the crop plants (Crafts-Brandner and Law, 2000). Our these findings are in accordance with studies reporting that decreased RuBP generation by declined supply of NADPH and energy (ATP) are the limiting factors for Pn under drought stress (Flexas et al., 2002; Wang et al., 2014b). Photosynthetic limitation/and regulation under drought stress events are the absolute indicators of plant growth, survival and yield under the s et al., 2007). During stress when Pn is drought stress (Galme
inhibited, meanwhile the photosynthetic reserves are depleted due s et al., 2007). So, an imbalance to continuous respiration (Galme occurs between photosynthates accumulation and their utilization through photorespiration resulting in the reduced translocation of photosynthates towards grain development. These facts may give the explanation that why harvest index was more declined than that dry matter production, particularly in non-primed plants on account of post-anthesis drought stress (Akbar et al., 2010; Ercoli et al., 2008). The higher Fv/Fm values observed in primed plants against the nonprimed plants in both cultivars indicated that drought priming upregulated the maximum efficiency of PSII and the electron flow to the photosystem during subsequent drought stress as well as protected the primary photochemistry from the deleterious effects of drought stress. However these effects were higher in the plants of Luhan-7 than that of Yangmai-16. Additionally, the reduction in Pn was less pronounced than the limitation in gs in primed plants in relation to nonprimed plants. This result reflected a better protection of leaf metabolic apparatus in the primed plants than that in the non-primed plants, which was associated to the better adjustment of stomatal conductance as well as to the regulation of the non-stomatal activities of the leaf (Bota et al., 2004). The better efficiency of photosystem under drought stress also prevents the generation of reactive oxygen species, as well as assists a rapid and complete photosystem recovery after re-watering et al., 2007). The higher MDA contents observed during post(Galle anthesis drought stress in non-primed plants indicate the suppressed capacity of sub-cellular reactive oxygen species (ROS) scavenging antioxidant system and higher accumulation of ROS in non-primed plants. Similar findings have been reported by Khanna-Chopra and Selote (2007). The tight control of ROS generation and accumulation in the plant cell is essential; otherwise, over-produced ROS can cause cells death reducing the plant growth and productivity (Bieker and Zentgraf, 2013). The higher SOD and APX activities with lower MDA contents identified in primed plants indicated their improved redox defense status to scavenge ROS damage by down-regulating peroxidation of cell membrane lipids under drought stress. The development of the efficient ROS detoxifying antioxidant system in the pre-drought stressed wheat plants indicated the pre-existing stress memory, which made them familiar to initiate their potential drought tolerance mechanism to drought stress within their next lifespan. However, drought tolerant cultivar exhibited better advantages of drought priming in response to drought stress for sustaining its productivity. 5. Conclusions It was concluded that wheat plants pre-exposed to moderate drought stress retained a long-lasting drought stress memory that triggered a faster and more efficient stress scavenging mechanism towards post-anthesis severe drought stress. Plants subjected to drought priming showed enhanced photosynthesis through improved leaf water potential, more chlorophyll, and Rubisco contents, improved photoprotection and efficient enzymatic antioxidant system leading to less yield reduction in wheat plants. However, drought tolerant cultivar exhibited better effects of drought priming in response to drought stress indicating that the involvement of genotypic differences towards drought tolerance can also contribute to the capability of the primed wheat plants to tolerate the subsequent severe drought stress conditions. The tillering stage primed plants showed higher photosynthetic rates and effectively alleviated photo-inhibition than those jointing stage primed plants. In addition to physiological studies, molecular and epigenetic elucidations will be helpful to better understand the
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drought priming-induced drought tolerance mechanism in wheat. Since, to cope with the future challenges to limited water resourceagriculture depends not only on breeding new wheat varieties well adapted to water-limited conditions, but also on the application of improved crop management practices, which in turn can enhance the crop potential to withstand the scarce water scenarios for sustainable crop production. Authors’ contributions MA, TD, JD and ZT planned the experiment. MA conducted the study, collected and analyzed the data, and prepared the draft. RZ, LY and CK helped in sampling and measurements of physiological parameters. ST and ZT helped in drafting the manuscript and interpretation the results. Acknowledgement We deeply acknowledge the financial support from the National Natural Science Foundation of China (Grant no. 31471443) for this study as a part of a Project by the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD). References Abdel Latef, A.A.H., Chaoxing, H., 2011. Effect of arbuscular mycorrhizal fungi on growth, mineral nutrition, antioxidant enzymes activity and fruit yield of tomato grown under salinity stress. Sci. Hortic. 127, 228e233. Akbar Moayedi, A., Nasrulhaq Boyce, A., Shahar Barakbah, S., 2010. The performance of durum and bread wheat genotypes associated with yield and yield component under different water deficit conditions. Aust. J. Basic Appl. Sci. 4, 106e113. Backhaus, S., Kreyling, J., Grant, K., Beierkuhnlein, C., Walter, J., Jentsch, A., 2014. Recurrent mild drought events increase resistance toward extreme drought stress. Ecosystems 17, 1068e1081. Beckers, G.J.M., Jaskiewicz, M., Liu, Y., Underwood, W.R., He, S.Y., Zhang, S., Conrath, U., 2009. Mitogen-activated protein kinases 3 and 6 are required for full priming of stress responses in Arabidopsis thaliana. Plant Cell 21, 944e953. Bieker, S., Zentgraf, U., 2013. Plant senescence and nitrogen mobilization and signaling. Senescence Senescence-Relat. Disord. 104, 53e83. Bota, J., Medrano, H., Flexas, J., 2004. Is photosynthesis limited by decreased Rubisco activity and RuBP content under progressive drought stress? New Phytol. 162, 671e681. Bruce, T.J.A., Matthes, M.C., Napier, J.A., Pickett, J.A., 2007. Stressful memories of plants: evidence and possible mechanisms. Plant Sci. 173, 603e608. Bruggink, E., Huang, C., 1997. Vessel contents of leaves after excision d a test of S Cholander’S. Am. J. Bot. 84, 1217e1222. Choi, C.S., Sano, H., 2007. Abiotic-stress induces demethylation and transcriptional activation of a gene encoding a glycerophosphodiesterase-like protein in tobacco plants. Mol. Genet. Genomics 277, 589e600. Cornic, G., 2000. Drought stress inhibits photosynthesis by decreasing stomatal aperture e not by affecting ATP synthesis. Trends Plant Sci. 5, 187e188. Crafts-Brandner, S.J., Law, R.D., 2000. Effect of heat stress on the inhibition and recovery of the ribulose-1, 5-bisphosphate carboxylase/oxygenase activation state. Planta 212, 67e74. Ercoli, L., Lulli, L., Mariotti, M., Masoni, A., Arduini, I., 2008. Post-anthesis dry matter and nitrogen dynamics in durum wheat as affected by nitrogen supply and soil water availability. Eur. J. Agron. 28, 138e147. Farooq, M., Kobayashi, N., Ito, O., Wahid, A., Serraj, R., 2010. Broader leaves result in better performance of indica rice under drought stress. J. Plant Physiol. 167, 1066e1075. Flexas, J., Bota, J., Escalona, J.M., Sampol, B., Medrano, H., 2002. Effects of drought on photosynthesis in grapevines under field conditions: an evaluation of stomatal and mesophyll limitations. Funct. Plant Biol. 29461e29471. Foulkes, M.J., Sylvester-Bradley, R., Weightman, R., Snape, J.W., 2007. Identifying physiological traits associated with improved drought resistance in winter
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Water stress-induced abscisic acid accumulation triggers the increased generation of reactive oxygen species and up-regulates the activities of antioxidant enzymes in maize leaves. J. Exp. Bot. 53, 2401e2410. Khanna-Chopra, R., Selote, D.S., 2007. Acclimation to drought stress generates oxidative stress tolerance in drought-resistant than -susceptible wheat cultivar under field conditions. J. Environ. Exp. Bot. 60, 276e283. Li, C., Jiang, D., Wollenweber, B., Li, Y., Dai, T., Cao, W., 2011a. Waterlogging pretreatment during vegetative growth improves tolerance to waterlogging after anthesis in wheat. Plant Sci. 180, 672e678. Li, J.T., Qiu, Z.B., Zhang, X.W., Wang, L.S., 2011b. Exogenous hydrogen peroxide can enhance tolerance of wheat seedlings to salt stress. Acta Physiol. Plant. 33, 835e842. Liu, H., Sultan, M.A.R.F., Liu, X.L., Zhang, J., Yu, F., Zhao, H.X., 2015. 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Plant Physiology and Biochemistry journal homepage: www.elsevier.com/locate/plaphy
Research article
Improved tolerance to post-anthesis drought stress by pre-drought priming at vegetative stages in drought-tolerant and -sensitive wheat cultivars Muhammad Abid, Zhongwei Tian, Syed Tahir Ata-Ul-Karim, Yang Liu, Yakun Cui, Rizwan Zahoor, Dong Jiang, Tingbo Dai* Key Laboratory of Crop Physiology, Ecology and Production Management, Nanjing Agricultural University, Nanjing 210095, Jiangsu Province, PR China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 7 April 2016 Received in revised form 4 May 2016 Accepted 4 May 2016 Available online 5 May 2016
Wheat crop endures a considerable penalty of yield reduction to escape the drought events during postanthesis period. Drought priming under a pre-drought stress can enhance the crop potential to tolerate the subsequent drought stress by triggering a faster and stronger defense mechanism. Towards these understandings, a set of controlled moderate drought stress at 55e60% field capacity (FC) was developed to prime the plants of two wheat cultivars namely Luhan-7 (drought tolerant) and Yangmai-16 (drought sensitive) during tillering (Feekes 2 stage) and jointing (Feekes 6 stage), respectively. The comparative response of primed and non-primed plants, cultivars and priming stages was evaluated by applying a subsequent severe drought stress at 7 days after anthesis. The results showed that primed plants of both cultivars showed higher potential to tolerate the post-anthesis drought stress through improved leaf water potential, more chlorophyll, and ribulose-1, 5-bisphosphate carboxylase/oxygenase contents, enhanced photosynthesis, better photoprotection and efficient enzymatic antioxidant system leading to less yield reductions. The primed plants of Luhan-7 showed higher capability to adapt the drought stress events than Yangmai-16. The positive effects of drought priming to sustain higher grain yield were pronounced in plants primed at tillering than those primed at jointing. In consequence, upregulated functioning of photosynthetic apparatus and efficient enzymatic antioxidant activities in primed plants indicated their superior potential to alleviate a subsequently occurring drought stress, which contributed to lower yield reductions than non-primed plants. However, genotypic and priming stages differences in response to drought stress also contributed to affect the capability of primed plants to tolerate the postanthesis drought stress conditions in wheat. © 2016 Published by Elsevier Masson SAS.
Keywords: Pre-drought priming Post-anthesis Drought stress Photosynthesis Antioxidants Winter wheat
1. Introduction Drought stress has become the most devastating constraint to crop productivity due to aridifying and warming climatic trends, and its prevalence is expected to diversify in future at regional and global scales (Backhaus et al., 2014). Wheat is one of the foremost staple food crops and is reported to be highly susceptible to drought stress which often occurs at post-anthesis phase consequencing considerable yield penalties (Wang et al., 2014a). Grain yield is the ultimate product of photosynthesis and closely interrelated physiological processes. Fluctuations around the
* Corresponding author. E-mail address: [email protected] (T. Dai). http://dx.doi.org/10.1016/j.plaphy.2016.05.003 0981-9428/© 2016 Published by Elsevier Masson SAS.
normal values of photosynthesis and its interlinked processes are the key indicators of plant fitness and extent of environmental stress (Zlatev and Lidon, 2012). Mild drought stress declines the rate of photosynthesis due to limited stomatal conductance, meanwhile the photosynthetic apparatus is not significantly affected (Cornic, 2000). In contrast, under severe drought stress, stomatal limitation, the poor efficiency of photosystemII (PSII) as well as declined activities of CO2 assimilating enzymes such as ribulose-1, 5-bisphosphate carboxylase/oxygenase (Rubisco) have been reported as the primary constraints to lower photosynthetic rates (Bota et al., 2004). In addition to direct drought-induced damages to the photosynthetic process, it also leads to the light-induced oxidative stress by the generation of reactive oxygen species (ROS) in the plant cells (Reddy et al., 2004). If the drought stress proceeds and ROS
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accumulation is not quenched by antioxidants, it results in protein oxidation, membrane lipid peroxidation, inhibition of DNA, RNAs and hormonal activities, eventually turning the cell into a state called as “oxidative stress”, which adversities the normal growth or even causes the death of plants (Liu et al., 2015). The limited wheat yield under environmental stresses is mostly attributed to the downregulation of key yield influential photosynthesis and other interdependent physiological processes functioning in the flag leaf during grain filling (Bruce et al., 2007; Zlatev and Lidon, 2012). Therefore, improving the tolerance to drought stress, especially at post-anthesis phase is of great significance in yield production of wheat. Previous research efforts provide promising evidence that priming “the pre-exposure of plants to a stimulus stress enabling them to mobilize their rapid and intense defense mechanism” can induce improved tolerance in plants to later-occurring stress events (Bruce et al., 2007). To date, various priming techniques have been used in different crop species against a range of environmental disasters. So far, mainly attention has been focused to exogenously applied chemical-induced priming such as nitric oxide for drought priming in rice (Farooq et al., 2010), hydrogen sulphide for drought priming in wheat (Shan et al., 2011), hydrogen peroxide for salt stress priming in wheat (Li et al., 2011a,b) and b-aminobutyric acid for pathogens attack priming in Arabidopsis (Zimmerli et al., 2000). Similarly, the priming state has also been achieved by bioticinduced colonization of plants with beneficial micro-organisms (Abdel Latef and Chaoxing, 2011) and by the epigenetic changes in plants for the initiation and regulation of their potential defense metabolism (Beckers et al., 2009). Some recent studies in various crop species have revealed that the plants pre-exposed to environmental stress can also attain the potential to display a faster and stronger activation of their defense system in response to the subsequent challenge stress events (Backhaus et al., 2014; Vu et al., 2015). For example, the plants of Arrhenatherum elatius experiencing an early drought episode showed an improved photo-protection and higher biomass under a second drought event than non pre-exposed plants (Walter et al., 2011). Similarly, pre- exposure of vegetative stage rice plants to sub-lethal heat stress improved the thermo-tolerance to heat stress during grain filling (Shi et al., 2015). Likewise, these beneficial prestress imprints have also been reported in tobacco (Choi and Sano, 2007), radish (Vu et al., 2015) and some grass species (Meisner et al., 2013). A few current studies addressing these legacy effects of prestress priming are also available in wheat. For example, wheat plants exposed to early stage heat stress, displayed an improved antioxidative capacity and higher grain yields against terminal high-temperature stress (Wang et al., 2014b). Another study on wheat investigated that waterlogging pre-treatment during vegetative growth stage enhanced dry matter accumulation and its distribution to grain formation resulting in a noticeably improved grain yield (Li et al., 2011a,b). The studies exploring the response of pre-drought stressinduced drought priming to post-anthesis drought events in wheat are rare (Wang et al., 2014a). Moreover, the response of primed plants to succeeding drought stress may vary due to the interval between priming and the reoccurring stress (Backhaus et al., 2014), due to response difference of growth stages to drought stress (Wang et al., 2015) as well as due to genotypic differences in tolerance to drought stress (Rampino et al., 2006; Khanna-Chopra and Selote, 2007). So, we suggest that drought priming study should be extended to gain insights into the resilience response difference of wheat cultivars and priming growth stages to the subsequent drought stress. This study was aimed to investigate that (1) whether drought priming during vegetative growth stages in
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wheat plants develops a memory to adapt the subsequent drought occurrence, and (2) whether the drought priming effect varies under varying cultivars and vegetative growth stages selected for priming. Hopefully, the projected results of the study would be supportive for research programs seeking to develop anti-drought stress practices for wheat.
2. Materials and methods 2.1. Plant culture and growth conditions A greenhouse experiment was carried out at Pailou Experimental Station of Nanjing Agricultural University, China (32 040 N, 118 760 E), during the growing season of 2014e2015. Two winter wheat cultivars with contrasting attitude towards drought stress namely Luhan-7 (drought resistant) and Yuangmai-16 (drought sensitive) (Wang et al., 2007), were used as experimental material in the present study. Fifteen surface sterilized uniform seeds were planted in the free-draining plastic pots having 22 cm and 25 cm height and diameter, respectively. Each pot was filled with 8 kg airdried, sieved (0.5 mm) and uniformly mixed clay loam soil having 13% soil moisture. At the time of soil filling, 0.8 g N, 0.5 g P2O5 and 1.1 g K2O/pot were applied for each treatment. Further, 0.4 g/pot N was applied at jointing and booting, respectively. Thinning was carried out 10 days after germination at 10 seedlings per pot. Then a week later, second thinning was carried out and seven uniform seedlings per pot were selected for the subsequent studies. Each pot was irrigated to 80% field capacity (FC) with tap water characterized with 7.5 pH, 2.8 dsm1electrical conductivity (EC) and 1200 mg L1 total soluble salts (TSS) until start of the treatments. Soil water status was measured before the application of water to the pots. The amount of water required for irrigation was calculated by using the following equation:
W ¼ Y H A ðFC1 FC0Þ
(1)
where, W is amount of irrigation water, Y is soil bulk density, H is soil depth, A is the area of the pot, FC1 is the desired soil FC, and FC0 is the actual soil FC before irrigation. Each treatment had 20 replicates (pots). The pots with different treatments were rotated on every alternate day to ensure that all the pots received equal radiation and other environmental exposures till maturity.
2.2. Treatments application and management 2.2.1. Drought priming treatments The drought priming treatments were carried out at tillering (40 days after planting, Feekes 2.0, beginning of tillering, 4 leaf stage) and at jointing (125 days after planting, Feekes 6.0, when first node was visible, 6 leaf stage), as described in experimental description under Fig. 1. At each priming stage, the irrigation to pots was withheld until a moderate drought stress stage at 55e60% FC reached. This moderate drought stress level as priming treatment at tillering and jointing respectively was maintained for 10 days by compensating the lost water. Meanwhile, the control pots kept on irrigating at 80% FC. After priming at both stages, the pots were rewatered to the level of control pots until the application of the subsequent drought stress. Thus, four priming treatments for each cultivar were designated as PT and PJ for priming at tillering and jointing, meanwhile non-primed plants were designated as NT and NJ during tillering and jointing, respectively. The overall nonprimed plants were designated as NN.
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Fig. 1. Experimental design for the study: Drought priming was applied at tillering (PT) and jointing (PJ) 11 by exposing the wheat plants to moderate drought stress (55-60% FC) for 10 days and followed by re12 watering. NT, NJ and NN indicated no drought priming at tillering, jointing and at any growth stage, 13 respectively. At 7 days after anthesis, severe drought stress was applied at 35-40% FC for 7 days and 14 followed by re-watering. The treatments during post-anthesis drought stress were designated as PTD=15 priming at tillering þ post-anthesis drought stress, PTC= priming at tillering þ no post-anthesis drought 16 stress, PJD= priming at jointing þ post-anthesis drought stress, PJC= priming at jointing þ no post17 anthesis drought stress, NND= no priming entirely þ post-anthesis drought stress, and NNC= no priming 18 entirely þ no post-anthesis drought stress. Tillering, jointing and post-anthesis were the stages of 19 treatments application, measurements and sampling. After measurements and sampling at last day of 20 priming and post-anthesis drought stress treatments, the plants were re-watered immediately at 75-80% 21 FC for recovery.
2.2.2. Post-anthesis drought stress treatments At anthesis, tillering and jointing stage primed (PT and PJ) and entirely non-primed (NN) plants were divided into two halves. One half plants of PT, PJ and NN were exposed to severe drought stress (D) at 35e40% FC at 7 days after anthesis (160 days after planting, Feekes 11.0), meanwhile other half plants kept on watering (C). The drought stress at this FC was maintained for 7 days by compensating the water lost through evapo-transpiration to the desired FC. After stress completion, the pots were re-watered to 75e80% FC up to maturity. Thus, six post-anthesis severe drought stress treatments for each cultivar were designated as PTD (priming at tillering þ post-anthesis drought stress), PTC (priming at tillering þ no post-anthesis drought stress), PJD (priming at jointing þ post-anthesis drought stress), PJC (priming at jointing þ no post-anthesis drought stress), NND (non-priming entirely þ post-anthesis drought stress), and NNC (non-priming entirely þ no post-anthesis drought stress). 2.3. Plant sampling Plant sampling and measurements were carried out at the last day of drought priming at tillering and jointing as well as at the last day of post-anthesis drought stress. The top fully expanded leaves from three randomly selected pots/replicates were detached for each treatment. One leaf from each replicate was used to measure the leaf water potential (Jw), and other leaves were immediately put into liquid nitrogen and shifted to the university laboratory to store at 40 C for quantifying the total chlorophyll, Rubisco, lipid peroxidation and enzymatic antioxidants. One pot plants were used only once a time measurements and sampling and then were discarded from the experiment. At each sampling point, leaf gas exchange and chlorophyll fluorescence were also measure. Dry matter and grain yield traits were calculated at maturity. 2.4. Physiological measurements and chemical analysis 2.4.1. Leaf water potential Leaf water potential (Jw) was measured according to the method given by (Bruggink and Huang, 1997) by using a pressure chamber (PMS Instrument Co., Corvallis, OR, USA). Leaf was firmly fixed in the sealing sleeve of specimen holder of the instrument and pressure was built up until the appearance of sap from the exposed cut of the leaf. The pressure reading was noted at this point and expressed in eMPa. 2.4.2. Chlorophyll contents Frozen leaf samples (0.2 g) were placed for 24 h in a vial with
4 ml of dimethyl sulphoxide for pigment extraction to determine chlorophyll content according to Huang and Zhao (2001). The absorbance of the supernatant was measured using a Pharmacia Ultra Spec Pro UV/VIS spectrophotometer (Pharmacia, Cambridge, England), at a wavelength of 470 and 648 nm for chlorophyll a and b, respectively. The sum of chlorophyll a and b was used as total chlorophyll contents. 2.4.3. Rubisco contents Rubisco contents were measured with Western-blot analysis using the SDS-PAGE by following the method given by Makino et al. (1985). Leaf samples of 0.5 g were homogenized in a 50 mM TriseHCL buffer (pH 8.0) containing 12.5% (v/v) glycerol and 5 mM b-mercaptoethanol, and centrifuged for 15 min at 15,000 g. SDS, b-mercaptoethanol, and glycerol with a final concentrations of 1% (w/v), 2% (v/v), and 5% (v/v), respectively were added to the supernatant and the mixture was boiled for 5 min. A 10 mL of enzyme extract was used for electrophoresis. The gels were stained with 0.1% (w/v) Coomassie Brilliant Blue R-250 solution. The stained bands from the gels were excised and eluted in 1 mL of formamide at room temperature with agitation for 8 h with an unstained gel as a standard. The blots were finally washed three times in phosphate buffered solution as above and developed with Super Sigmal West Pico Chemiluminescent Substrate (Pierce, USA). Images of the blots were scanned using a CCD imaging system (Fluor SMax, Bio-Rad, USA) and Quantity One software (Bio-Rad, Hercules, CA, USA) was used to calculate the optical density. 2.4.4. Leaf gas exchange Leaf gas exchange measurements were done on leaf blades of evenly oriented and maximum light-exposed top leaves from five plants in each treatment with an open IRGA LI-COR 6400 system (LI- 6400, Li-Cor Inc. USA) at 9:00e11:00 h (local time). Net photosynthetic rate (Pn) and stomatal conductance (gs) were noted under light saturated conditions at photosynthetic photon flux density of 1000 mmol photon m2 s1, at 25 C and 380 mol mol1 of CO2. 2.4.5. Chlorophyll fluorescence On the day of gas exchange measurements, chlorophyll fluorescence was also recorded on the same leaves by using a portable modulated fluorescence measuring system (FMS2; Hansatech, King’s Lynn, Norfolk, UK). The base values of the minimum (F0) and maximum (Fm) chlorophyll fluorescence taken in 30 min fully dark adapted leaves were used to calculate the maximum efficiency of photosystemII (Fv/Fm ¼ Fm F0/Fm) as proposed by Maxwell and
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Johnson (2000).
HIð%Þ ¼ 2.4.6. Enzymatic antioxidants activities The activities of enzymatic antioxidants such as superoxide dismutase (SOD) and ascorbate peroxidase (APX) were determined according to the methods given by Jiang and Zhang (2002). Frozen leaf samples of 0.5 g were sliced and homogenized in a mortar, and pestle with 5 mL ice-cold extraction buffer. The homogenate was centrifuged at 10,000 g for 30 min at 4 C and the supernatant was used as crude extract for the above and MDA assays. Superoxide dismutase (SOD) activity was determined by adding 0.1 mL enzyme extract to a reaction mixture of 1.5 mL 50 mM sodium phosphate (pH 7.8), 0.3 mL 130 mM methionine, 0.3 mL 750 mM nitro-blue tetrazolium (NBT), 0.3 mL 100 mM EDTA-Na2, 0.300 mL 20 mM riboflavin and 100 mL distilled water, and illuminated in light of 4000 flux for 20 min and the sample absorbance was determined at 560 nm UV/visible spectrophotometer (UltrospecTM 2100 pro; Amersham Bio-sciences). One unit of SOD activity was considered as the amount of enzyme used for 50% inhibition of the NBT reduction. For APX activity, 0.2 mL crude enzyme extract was added to a reaction mixture of 50 mmol L1 potassium phosphate buffer (pH 7.0), 0.5 mmol L1 ASC and 0.1 mmol L1 H2O2. And APX activity was determined by observing the decrease at 290 nm for 1 min in 1 mL of the reaction mixture. 2.4.7. Lipid peroxidation estimation Lipid peroxidation was estimated by measuring the malondialdehyde concentration (MDA) following the thiobarbituric acid reaction solution (TBA) analysis (Jiang and Zhang, 2002). A mixture of 1 ml of extract and 4 ml of reaction solution was incubated at 95 C for 30 min, and cooled immediately. The mixture was centrifuged for 10 min at 12,000 g, and supernatant was used for determining the MDA content at 532 nm and 600 nm. 2.5. Morphological traits determination 2.5.1. Dry matter At maturity, three randomly selected pots were manually cut at the ground level using pruning-scissors from each treatment and oven dried at 70 C to a constant weight for measuring the total above-ground dry matter (DM; g/pot). 2.5.2. Grain yield From three randomly selected pots, spikes were cut and manually threshed. The grains were oven dried at 40 C for 24 h and then grain yield/pot was calculated. 2.5.3. Drought index Drought index (DI) was estimated as grain yield differences between drought priming and drought stress treatments as compared to control pots according to the following method;
DI ¼
YD YN
Grain yield 100 Dry weight of above ground biomass
221
(3)
2.6. Statistical analysis The analysis of variance (ANOVA) was performed by using General Linear Model procedure to calculate the homogeneity of variance under drought priming and post-anthesis drought stress treatments, respectively. Means were compared by using Duncan’s multiple comparison test (P < 0.05). A two-way ANOVA was performed to evaluate the level of significance of drought priming, drought stress, cultivars and their interactive effect by using SPSS statistical package (SPSS Inc., Chicago, IL, USA). Graphs were plotted by using Sigma Plot 10.0 software (Systat Software Inc., Chicago, IL, USA). 3. Results 3.1. Morphological traits 3.1.1. Dry matter production Dry matter production (DM) calculated at maturity was significantly (P < 0.05) reduced by post-anthesis drought stress treatments (PTD, PJD, and NND) in both cultivars as compared to NNC (control) (Table 1). However, decrease in DM was relatively lower in primed plants than the non-primed plants. The plants of Luhan-7 showed lower decrease in DM under drought stress than those of Yangmai-16. In relation to priming stages, there was no significant difference in DM reduction in PTD and PJD in both cultivars. The DM was decreased by 14, 15 and 19% and by 19, 19 and 25% under PTD, PJD, and NND as compared to NNC in Luhan-7 and Yangmai-16, respectively. 3.1.2. Grain yield production Post-anthesis drought stress resulted in a significant decrease in grain yields. Yangmai-16 exhibited higher yield reduction than that Luhan-7 as compared to NNC, respectively. PTD and PJD significantly decreased the extent of yield reduction in both cultivars, however, the positive effect of drought priming to decrease the grain yield reduction was higher in Luhan-7 than that Yangmai-16. In both cultivars, PTD showed lower yield reduction than PJD. Overall, grain yield was reduced by 37, 25 and 20% in Luhan-7 whereas in Yangmai-16, it was reduced by 49, 28, and 24% under NND, PJD, and PTD as compared to NNC, respectively. 3.1.3. Drought index Under post-anthesis drought stress, higher drought index (DI) values were observed in primed plants than those non-primed plants. PTC gave higher DI in Luhan-7 (0.98) and Yangmai-16 (0.96). However, under NND, Yangmai-16 exhibited lower DI (0.55) than that Luhan-7 (0.65). Overall, DI was reduced by 14, 22 and 33% in Luhan-7 and by 20, 26, and 41% in Yangmai-16 under PTD, PJD and NND as compared to PTC, respectively.
(2)
where, YD is the grain yield under a treatment condition and YN is the grain yield under fully irrigated conditions. 2.5.4. Harvest index Harvest index (HI) was calculated as the ratio between grain weight and the total dry weight of the above ground biomass;
3.1.4. Harvest index Harvest index (HI) was also significantly reduced under postanthesis drought stress. Lowest values of HI (25% and 29%) were obtained under NND in Yangmai-16 and Luhan-7, respectively. Nevertheless primed plants, especially PTD showed higher HI in both cultivars. The primed plant of Luhan-7 gave higher HI than those of Yangmai-16 under drought stress. HI was decreased by 10, 14 and 30% in Luhan-7, whereas in Yangmai-16 it was decreased by
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Table 1 Effects of drought priming during vegetative growth stages on dry matter and grain yield traits under post-anthesis severe drought stress in Luhan-7 and Yangmai-16 wheat cultivars. Cultivars
Treatments
Dry matter (g/pot)
Grain yield (g/pot)
Drought index
Harvest index (%)
Luhan-7
PTD PTC PJD PJC NND NNC PTD PTC PJD PJC NND NNC
107.4d 121.1c 106.1d 117.8c 103.4d 125.5b 107.4d 126.8b 107.4d 123.5c 100.4d 132.3a
39.0d 49.7ab 35.1e 47.8b 30.5f 50.5a 34.5e 49.4ab 33.0e 45.6c 24.3g 51.1a
0.84e 0.98a 0.75f 0.94c 0.65g e 0.76d 0.96ab 0.71e 0.89cd 0.55h e
36.3d 41.3a 33.1e 40.1b 29.4h 40.3b 31.4f 39.4bc 29.0g 37.5d 25.3i 38.8c
FCultivars FDrought stress FC DS
15.0 ns 192.5* 10.5 ns
219.4* 552.1* 32.9*
1.7 ns 459.9* 21.7*
151.5* 406.5* 43.0*
Yangmai-16
Fvalues
Note: Within the columns of ‘Treatments’, PT, PJ and NN indicate drought priming at tillering and jointing, and no priming at either stage, respectively. C and D indicate no post-anthesis drought stress and post-anthesis drought stress, respectively. Data are means ± SE, (n ¼ 3). Different lowercase letters following the data within the same column indicate significant differences at P < 0.05. A two-way analysis of variance was performed to evaluate the effect of cultivars (C), drought stress (DS) and their interaction (C DS). ns ¼ not significant; * ¼ significant at P < 0.05.
17, 25 and 41% under PTD, PJD, and NND as compared to NNC, respectively.
than PJD. As compared to NNC, Jw was reduced by 245, 195 and 181% in Luhan-7, whereas in Yangmai-16, it was reduced by 350, 210, and 190% in NND, PJD, and PTD respectively.
3.2. Physiological traits 3.2.1. Leaf water potential Drought priming treatments during vegetative stages (PT and PJ), significantly decreased the leaf water potential (Jw) as compared to NT and NJ (Fig. 2A). This decrease was higher in case of Yangmai-16 than Luhan-7. During priming, plants at jointing showed more sensitivity to reduce Jw than tillering. Under subsequent drought stress at post-anthesis, NND, PJD and PTD significantly showed lower Jw as compared to NNC (Fig. 2B). However, PTD and PJD maintained significantly higher Jw in both cultivars. Under NND, Yangmai-16 showed higher decrease than that Luhan7. In relation to priming stages, PTD showed higher Jw regulation
3.2.2. Chlorophyll and Rubisco contents During drought priming at vegetative stages, a slight decrease in chlorophyll (Chl) and Rubisco contents in both cultivars was observed under PT and PJ as compared to NT and NJ (Fig. 3; A and C). This decrease was higher in Yangmai-16 as compared to Luhan-7. PT showed lower decrease than PJ in both cultivars. In contrast, under post-anthesis drought stress, Chl and Rubisco contents were significantly reduced in both cultivars as compared to NNC, respectively (Fig. 3; B and D). The primed plant PTD and PJD significantly showed higher Chl and Rubisco contents against NND, which were higher in Luhan-7 than that Yangmai-16. The Chl and Rubisco contents were reduced by 51, 40, and 38%, 60, 47 and 44% in
Fig. 2. Leaf water potential as affected by drought priming during vegetative growth stages (A) and post-anthesis drought stress (B) in Luhan-7 and Yangmai-16 wheat cultivars. In “treatments” PT, PJ, NT and NJ indicate drought priming and no priming at tillering and jointing stages, respectively. C and D indicate no post-anthesis drought stress and postanthesis drought stress, respectively. Different lowercase letters indicate significant differences at P < 0.05. A two-way analysis of variance was performed to evaluate the effect of drought priming (DP), cultivars (C) and their interaction (DP x C) during priming, and the effect of drought stress (DS), cultivars (C) and their interaction (DS x C) during postanthesis drought stress. ns ¼ not significant; * ¼ significant at P < 0.05.
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Fig. 3. Chlorophyll (Chl a þ b) and Rubisco contents (mg/g fresh weight) as affected during drought priming at vegetative stages (A and C), and post-anthesis drought stress (B and D) in Luhan-7 and Yangmai-16 wheat cultivars. In “treatments” PT, NT, PJ and NJ indicate drought priming and no drought priming at tillering and jointing, respectively. C and D indicate no post-anthesis drought stress (control) and post-anthesis drought stress, respectively. Different letters indicate significant differences at P < 0.05. A two-way analysis of variance was performed to evaluate the effect of drought priming (DP), cultivars (C) and their interaction (DP x C), and the effect of drought stress (DS), cultivars (C) and their interaction (DS x C). ns ¼ not significant; * ¼ significant at P < 0.05.
Luhan-7, whereas these were decreased by 60, 45 and 41% and 70, 51, and 48% in Yangmai-16, respectively under NND, PJD, and PTD as compared with NNC.
16 Pn, gs and Fv/Fm were reduced by 70, 53 and 43%, 63, 49, and 45% and by 29, 17 and 15% under NND, PJD, and PTD, respectively as compared to NNC.
3.2.3. Leaf gas exchange and chlorophyll fluorescence During drought priming, PT and PJ showed a significant decline in net photosynthetic rate (Pn), stomatal conductance (gs), and Fv/ Fm was observed as compared to NT and NJ (Fig. 4; A, C and E). Pn and gs during priming were more decreased in Yangmai-16 as compared to Luhan-7. Pn during priming was decreased by 32 and 36% in Yangmai-16 and by 22 and 27% in Luhan-7 under PT and PJ than NT and NJ, respectively. Whereas, gs was decreased by 30 and 34% in Yangmai-16 and by 22 and 27% in Luhan-7 under PT and PJ as compared to NT and NJ, respectively. At post-anthesis, the subsequent drought stress significantly downregulated the Pn, gs and Fv/ Fm values as compared to NNC (Fig. 4; B, D and F). Yangmai-16 showed more decline in these parameters under pos-anthesis drought stress treatments than that Luhan-7. In relation to priming stages, PTD in both cultivars maintained higher Pn, gs and Fv/Fm than PJD. The Pn, gs and Fv/Fm were reduced by 66, 48, and 41%, 59, 43 and 41% and by 21, 15, and 12% in Luhan-7, whereas in Yangmai-
3.2.4. Lipid peroxidation and enzymatic antioxidant activities During drought priming, the lipid peroxidation (MDA), activities of SOD and APX were increased in PT and PJ as compared to NT and NJ in both cultivars (Fig. 5A, C and E). However, Luhan-7 contained comparatively lower MDA contents and higher SOD and APX activities than that Yangmai-16. Under PT and PJ, MDA contents, SOD and APX activities were increased by 28 and 32%, 31 and 29%, and 18 and 20% in Yangmai-16, whereas in Luhan-7 MDA contents, SOD and APX activities were increased by 20 and 27%, 39 and 34%, and 25 and 20%, respectively as compared to NT and NJ. Under postanthesis drought stress, NND showed a significant increase in MDA contents, however, in case of SOD, it showed a slight increase but in APX activities, it showed a non-significant increase as compared with NND (Fig. 5B, D and F). In contrast, priming treatments PTD and PJD showed significantly lower contents of MDA and higher activities of SOD and APX as compared to NND in both cultivars. The higher upregulated SOD and APX activities and lower
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Fig. 4. Net photosynthetic rate (Pn), stomatal conductance (gs) and maximum photosynthetic efficiency of PSII (Fv/Fm) as affected by drought priming during vegetative stages (A, C and E, respectively) and post-anthesis drought stress (B, D and F, respectively) in Luhan-7 and Yangmai-16 wheat cultivars. In “treatments” PT, NT, PJ and NJ indicate drought priming and no drought priming at tillering and jointing, respectively. C and D indicate no post-anthesis drought stress (control) and post-anthesis drought stress, respectively. Different letters indicate significant differences at P < 0.05. A two-way analysis of variance was performed to evaluate the effect of drought priming (DP), cultivars (C) and their interaction (DP x C), and the effect of drought stress (DS), cultivars (C) and their interaction (DS x C). ns ¼ not significant; * ¼ significant at P < 0.05.
MDA contents under PTD and PJD were observed in Luhan-7 against Yangmai-16. Among priming stages, PTD showed higher SOD and APX activities, but lower MDA contents than that PJD in both cultivars. Under NND, PJD and PTD, the MDA contents, SOD and APX activities were increased by 67, 41 and 36%, 12, 56 and 61% and by 10, 51 and 60% in Luhan-7, whereas in Yangmai-16, these were increased by 85, 46, and 40%, 9, 51, and 57% and by 7, 43 and
47%, respectively, as compared with NNC. 4. Discussion In this study, the plants of two wheat cultivars were preexposed to drought stress for priming at tillering and jointing, respectively and then simultaneously subjected to severe drought
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Fig. 5. Lipid peroxidation (MDA contents), activities of superoxide dismutase (SOD) and ascorbate peroxidases (APX) as affected by drought priming during vegetative growth stages (A, C and E, respectively) and post-anthesis drought stress (B, D and F, respectively) in Luhan-7 and Yangmai-16 wheat cultivars, FW ¼ fresh weight. In “treatments” PT, NT, PJ and NJ indicate drought priming and no drought priming at tillering and jointing, respectively. C and D indicate no post-anthesis drought stress (control) and post-anthesis drought stress, respectively. Lowercase letters indicate significant differences at P < 0.05. A two-way analysis of variance was performed to evaluate the effect of drought priming (DP), cultivars (C) and their interaction (DP x C), and the effect of drought stress (DS), cultivars (C) and their interaction (DS x C). ns ¼ not significant; * ¼ significant at P < 0.05.
stress event at post-anthesis. It is evident from the results of the study that wheat plants with priming and non-priming treatments displayed a range of response processes to drought stress for regulating their survival, growth and final grain yield. Drought priming during vegetative growths proved to be a valuable strategy for facilitating the plants to initialize an efficient tolerance
mechanism, which in turn permitted the plants to tolerate the subsequent severe drought conditions adeptly. The findings of our study are in accordance with those, which reported stress imprint properties or stress memories developed by the stress priming in plants (Walter et al., 2011; Backhaus et al., 2014). In the present study, different physiological investigations were carried out which
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might be the sophisticate indicators and contributors to the drought tolerance in wheat plants. Nevertheless, the response of drought tolerant and sensitive cultivars primed at a particular growth stage was different towards the subsequent drought stress. It was observed that drought primed plants (PTD and PJD) reduced the grain yield reductions caused by the post-anthesis drought stress as compared to the non-primed plants (Table 1). Thus the priming strategy proved to be a great contribution to grain yield sustainability by enhancing the potential of plants to alleviate the drought effects at reproductive phase. The higher grain yield in primed plants against the non-primed plants seemed to be parallaly associated with their capability of retaining higher photosynthetic substrates (chlorophyll) and the regulation of higher leaf water potential (Jw) during stress period. Since, maintenance of favorable Jw under drought stress is vital to develop drought s et al., 2007; Khannatolerance mechanism in crop plants (Galme Chopra and Selote, 2007). The higher Jw observed in primed plants as compared to non-primed plants might be the prime reason for the leaf photosynthetic apparatus keep on functioning to maintain the grain development during drought stress period under the persistence of higher photosynthetic pigment and more CO2 assimilating enzyme (Rubisco) activity. Since, the quantity of coexisting photosynthates in the leaf depends on the performance of photosynthesis (Pn) (Cornic, 2000). The primed plants, showed a superior ability over the non-primed plants to develop the grains with higher weight by sustaining a higher rate of carbon accumulation through Pn in relatively long lasting canopy during grain filling period. Our findings of extended period of flag leaf Pn capacity in primed plants during grain-filling phase correlated with the production of higher grain yield were in consensus with previous reports by Foulkes et al. (2007). In contrast, in the non-primed plants, the plant canopy tended to senescence earlier with reduced grain filling duration. Under these contexts, the non-primed plants forcedly finished their life cycles earlier to produce the offspring with reduced grain yield rather than die and remain barren due to post-anthesis drought stress. The positive effects of drought priming to tolerate post-anthesis drought stress were more pronounced in plants primed at tillering than those primed at jointing. It might be because that during tillering, the wheat plants had lower leaf area, slower plant growth, less water demand, low transpiration rate as well plants were more flexible to get higher recovery after stress (Izanloo et al., 2008). In contrast, during jointing, the plant growth was rapid and the most important part of photosynthates reserves those contribute up to 57% of the grain yield of wheat is assimilated during this period (May, 1976). Moreover, the number of grains, which is a vital determinant of yield in drought conditions, depends on the plant growth during stem development and plant growth rate a few weeks before anthesis (Ercoli et al., 2008). So, the plants primed at jointing (PJD) even though showed higher grain yields than NND, but could not reach the levels of PTD in both cultivars. A rapid decline in photosynthetic rate of non-primed plants under drought stress metabolically might be related to lower Rubisco carboxylation activity, its higher degradation as well as more activation as oxygenase than as carboxylase. Since, a major consequence of abiotic stresses is the induced degradation and lower generation of Rubisco in the crop plants (Crafts-Brandner and Law, 2000). Our these findings are in accordance with studies reporting that decreased RuBP generation by declined supply of NADPH and energy (ATP) are the limiting factors for Pn under drought stress (Flexas et al., 2002; Wang et al., 2014b). Photosynthetic limitation/and regulation under drought stress events are the absolute indicators of plant growth, survival and yield under the s et al., 2007). During stress when Pn is drought stress (Galme
inhibited, meanwhile the photosynthetic reserves are depleted due s et al., 2007). So, an imbalance to continuous respiration (Galme occurs between photosynthates accumulation and their utilization through photorespiration resulting in the reduced translocation of photosynthates towards grain development. These facts may give the explanation that why harvest index was more declined than that dry matter production, particularly in non-primed plants on account of post-anthesis drought stress (Akbar et al., 2010; Ercoli et al., 2008). The higher Fv/Fm values observed in primed plants against the nonprimed plants in both cultivars indicated that drought priming upregulated the maximum efficiency of PSII and the electron flow to the photosystem during subsequent drought stress as well as protected the primary photochemistry from the deleterious effects of drought stress. However these effects were higher in the plants of Luhan-7 than that of Yangmai-16. Additionally, the reduction in Pn was less pronounced than the limitation in gs in primed plants in relation to nonprimed plants. This result reflected a better protection of leaf metabolic apparatus in the primed plants than that in the non-primed plants, which was associated to the better adjustment of stomatal conductance as well as to the regulation of the non-stomatal activities of the leaf (Bota et al., 2004). The better efficiency of photosystem under drought stress also prevents the generation of reactive oxygen species, as well as assists a rapid and complete photosystem recovery after re-watering et al., 2007). The higher MDA contents observed during post(Galle anthesis drought stress in non-primed plants indicate the suppressed capacity of sub-cellular reactive oxygen species (ROS) scavenging antioxidant system and higher accumulation of ROS in non-primed plants. Similar findings have been reported by Khanna-Chopra and Selote (2007). The tight control of ROS generation and accumulation in the plant cell is essential; otherwise, over-produced ROS can cause cells death reducing the plant growth and productivity (Bieker and Zentgraf, 2013). The higher SOD and APX activities with lower MDA contents identified in primed plants indicated their improved redox defense status to scavenge ROS damage by down-regulating peroxidation of cell membrane lipids under drought stress. The development of the efficient ROS detoxifying antioxidant system in the pre-drought stressed wheat plants indicated the pre-existing stress memory, which made them familiar to initiate their potential drought tolerance mechanism to drought stress within their next lifespan. However, drought tolerant cultivar exhibited better advantages of drought priming in response to drought stress for sustaining its productivity. 5. Conclusions It was concluded that wheat plants pre-exposed to moderate drought stress retained a long-lasting drought stress memory that triggered a faster and more efficient stress scavenging mechanism towards post-anthesis severe drought stress. Plants subjected to drought priming showed enhanced photosynthesis through improved leaf water potential, more chlorophyll, and Rubisco contents, improved photoprotection and efficient enzymatic antioxidant system leading to less yield reduction in wheat plants. However, drought tolerant cultivar exhibited better effects of drought priming in response to drought stress indicating that the involvement of genotypic differences towards drought tolerance can also contribute to the capability of the primed wheat plants to tolerate the subsequent severe drought stress conditions. The tillering stage primed plants showed higher photosynthetic rates and effectively alleviated photo-inhibition than those jointing stage primed plants. In addition to physiological studies, molecular and epigenetic elucidations will be helpful to better understand the
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drought priming-induced drought tolerance mechanism in wheat. Since, to cope with the future challenges to limited water resourceagriculture depends not only on breeding new wheat varieties well adapted to water-limited conditions, but also on the application of improved crop management practices, which in turn can enhance the crop potential to withstand the scarce water scenarios for sustainable crop production. Authors’ contributions MA, TD, JD and ZT planned the experiment. MA conducted the study, collected and analyzed the data, and prepared the draft. RZ, LY and CK helped in sampling and measurements of physiological parameters. ST and ZT helped in drafting the manuscript and interpretation the results. Acknowledgement We deeply acknowledge the financial support from the National Natural Science Foundation of China (Grant no. 31471443) for this study as a part of a Project by the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD). References Abdel Latef, A.A.H., Chaoxing, H., 2011. Effect of arbuscular mycorrhizal fungi on growth, mineral nutrition, antioxidant enzymes activity and fruit yield of tomato grown under salinity stress. Sci. Hortic. 127, 228e233. Akbar Moayedi, A., Nasrulhaq Boyce, A., Shahar Barakbah, S., 2010. The performance of durum and bread wheat genotypes associated with yield and yield component under different water deficit conditions. Aust. J. Basic Appl. Sci. 4, 106e113. Backhaus, S., Kreyling, J., Grant, K., Beierkuhnlein, C., Walter, J., Jentsch, A., 2014. Recurrent mild drought events increase resistance toward extreme drought stress. Ecosystems 17, 1068e1081. Beckers, G.J.M., Jaskiewicz, M., Liu, Y., Underwood, W.R., He, S.Y., Zhang, S., Conrath, U., 2009. Mitogen-activated protein kinases 3 and 6 are required for full priming of stress responses in Arabidopsis thaliana. Plant Cell 21, 944e953. Bieker, S., Zentgraf, U., 2013. Plant senescence and nitrogen mobilization and signaling. Senescence Senescence-Relat. Disord. 104, 53e83. Bota, J., Medrano, H., Flexas, J., 2004. Is photosynthesis limited by decreased Rubisco activity and RuBP content under progressive drought stress? New Phytol. 162, 671e681. Bruce, T.J.A., Matthes, M.C., Napier, J.A., Pickett, J.A., 2007. Stressful memories of plants: evidence and possible mechanisms. Plant Sci. 173, 603e608. Bruggink, E., Huang, C., 1997. Vessel contents of leaves after excision d a test of S Cholander’S. Am. J. Bot. 84, 1217e1222. Choi, C.S., Sano, H., 2007. Abiotic-stress induces demethylation and transcriptional activation of a gene encoding a glycerophosphodiesterase-like protein in tobacco plants. Mol. Genet. Genomics 277, 589e600. Cornic, G., 2000. Drought stress inhibits photosynthesis by decreasing stomatal aperture e not by affecting ATP synthesis. Trends Plant Sci. 5, 187e188. Crafts-Brandner, S.J., Law, R.D., 2000. Effect of heat stress on the inhibition and recovery of the ribulose-1, 5-bisphosphate carboxylase/oxygenase activation state. Planta 212, 67e74. Ercoli, L., Lulli, L., Mariotti, M., Masoni, A., Arduini, I., 2008. Post-anthesis dry matter and nitrogen dynamics in durum wheat as affected by nitrogen supply and soil water availability. Eur. J. Agron. 28, 138e147. Farooq, M., Kobayashi, N., Ito, O., Wahid, A., Serraj, R., 2010. Broader leaves result in better performance of indica rice under drought stress. J. Plant Physiol. 167, 1066e1075. Flexas, J., Bota, J., Escalona, J.M., Sampol, B., Medrano, H., 2002. Effects of drought on photosynthesis in grapevines under field conditions: an evaluation of stomatal and mesophyll limitations. Funct. Plant Biol. 29461e29471. Foulkes, M.J., Sylvester-Bradley, R., Weightman, R., Snape, J.W., 2007. Identifying physiological traits associated with improved drought resistance in winter
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