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Zinnia (Z. elegans) is one of the most popular cut flowers and bedded plant of the summer season in Pakistan, partly because of its diverse color range and potential to survive drought periods in arid regions (Riaz et al. 2013). Most of the tall varieties are used as cut flowers and dwarf varieties as potted plants. The long stem (15-100 cm) and reasonable shelf life have resulted in increased demand for tall cultivars popular as cut flowers (Ahmad & Dole 2014).
Plants for flower production are mostly grown in pots where irrigation management is more critical due to limited soil volume (Grant et al. 2012). The drought stress exposure restricts the water supply to plant rhizosphere that results in reduced root growth and prompted flower abortion. Nutrient uptake and net photosynthesis declines (Praba et al. 2009, Anjum et al. 2011). The production of reactive oxygen species (ROS) increases, resulting in increased damage to soluble proteins, lipids, ATP synthesis, and the photo-stabilizing efficiency of chlorophyll (Shehzad et al. 2020). Plants reduce impacts of drought by developing extensive root systems, leaf rolling to reduce stomatal water loss, and lowering tissue water potential through the accumulation of organic osmolytes and phytohormones (Forni et al. 2017). Furthermore, supplemental use of nutrients and growth regulators is considered essential for alleviating the drought effects, particularly, in flowering plants (Prerostova et al. 2018, Waqas et al. 2019).
Salicylic acid (SA) is an important phytohormone that serves as signaling molecule in drought mediated resistance mechanisms of plants (Pieterse et al. 2012). Exogenous SA is an effective non-enzymatic antioxidant that relieves the plants from drastic effects of drought through its involvement in vital physiological processes (Belkadhi et al. 2014). The up-regulation of secondary metabolites and stomatal closure, and reduction in transpiration rate is also simulated by SA (Khan et al. 2015). Ghasemi et al. (2016) and Shahmoradi & Naderi (2018) showed positive effects of supplemental SA in improving growth rate in jasmine (Jasminum nudiflorum L.), amaranth (Amaranthus tricolor L.), starflower (Borago officinalis L.) and chamomile (Matricaria chamomilla L.) under drought conditions. In fact, SA application directly influences the cellular contents and volume of chloroplast to overcome chlorophyll degradation and fragmentation in drought-prone plants (Uzunova & Popova 2000, Cheng et al. 2016). Positive effects of SA in reducing the impact of stress conditions may vary with the application rate and timing, and environmental growth conditions. SA supplementation directly influences the expansion of grana, coagulation of stroma, and increases the chloroplast volume to overcome chlorophyll degradation and fragmentation under drought stress (Uzunova & Popova 2000, Cheng et al. 2016).
The up-regulation of expression of stress-responsive genes involved in signal transduction, photosynthesis and protein metabolism under drought stress also attributed to SA supplementation (Kanget al. 2012). Recent study involving various petunia varieties also showed that SA application helped to maintain photosynthetic pigments by decreasing electrolyte leakage in drought stressed seedlings. Habibi (2012) also proposed that SA contributes to photoprotection and maintains photosynthetic pigments in plants under drought stress. Similarly, Aldesuquyet al. (2012) suggested that enhanced drought tolerance in drought-prone plants treated with SA was correlated with improved photosynthetic activity.
The positive effects of exogenous SA on growth and regulatory mechanisms in plants have been well reported, but flowering plants like Zinnia still are scant. The present study focused on the major physiological and biochemical processes that are considered vital for improved drought tolerance characteristics in flowering plants. A better understanding of such physio-biochemical stress regulatory mechanisms would contribute to reducing drought-induced losses in ornamental Zinnia.
Materials and methods
Experimental setup. Healthy, uniform seeds of Z. elegans var. ‘Dreamland’ was purchased at Chanan Seeds Store Lahore, Pakistan and sown in 24 cm diameter, 30 cm high earthen pots at MNS-University of Agriculture, Multan, Punjab, Pakistan. The earthen pots were filled with 3 kg air-dried and sieved (2 mm mesh) mixture of silt (sweet soil) and slug (70:30 w/w), with pH 8.0, organic matter 0.39 %, electrical conductivity (ECe) 10.91 mS cm−1, saturation 18 %, total available N 0.021 %, available P 5.20 mg kg-1 and available K 110 mg kg-1. The pots were placed under natural conditions from March 20th to May 20th, 2018. The environmental variables (air temperature, and relative humidity) are presented in Figure 1. Seedlings at the 2-4 leaf stage (30 days after sowing) were transplanted to earthen pots of similar size and allowed to establish for 15 days before the initiation of drought stress. Fresh ground water (EC 642 uScm−1) was used for irrigation. All cultural practices like hoeing, weeding, and fertilizing were evenly applied on all treatments throughout the study. The experiment comprised of four treatments, arranged in completely randomized design (CRD), and repeated four times with one plant per replicate.
Figure 1 The maximum and minimum air temperature and relative humidity during the growth period of experimentation
Drought stress and SA application. Two soil moisture treatments of 100 % (2,590 ml / pot) and 60 % (1,554 ml / pot) field capacity (FC) were maintained using the gravimetric method (Shehzad et al. 2020). After one-week foliar applications of SA (100 mg L-1) (C7H6O3; Purity ≥ 99 %; Sigma-Aldrich Ltd. USA), with Tween-20 (0.1 %), were applied to half the plants in each soil moisture treatment (repeated one week later) and the other half (control) were sprayed with distill water + Tween 20.
Biomass. The number of leaves (NOL) of each plant were counted manually and leaf area (LA) was calculated using a portable leaf area meter (Model CI-202, CID Inc. USA). At the end of experiment, the plants were cut at soil level to measure shoot length (SL), shoot fresh and dry weight (SFW & SDW), and root fresh and dry weights (RFW & RDW). The roots were cleaned by careful rinsing in water and root length (RL) measured by measuring scale. Shoots and roots were placed in the oven at 65 oC for 72 h to determine the dry weight of shoots and roots.
Leaf water status. To measure leaf relative water content (RWC), young fully expanded leaves from each treatment were selected and the fresh weight (FW) immediately recorded. The leaves were then soaked in autoclaved water at 4 oC for 24 h to determine turgid weight (TW). They were then dried at 65 °C for 72 h to estimate dry weight (DW). RWC was calculated using the formula 1 (Mayak et al. 2004).
For excised leaf water retention (ELWR), young leaves from each treatment were weighed (FW) and incubated at room temperature (25 oC) for six hours and the leaf wilted weight (LW) recorded following the formula 2 (Lonbani & Arzani (2011)
Chlorophyll pigments concentration. The stored leaves (-80 oC) of each treatment (0.5 g) were thawed in ice buckets, then chopped and extracted in 5 mL acetone solution (80 %) at 4 oC overnight to determine the chlorophyll (Chl a, and b) and carotenoids (Car) concentrations (Taiz & Zeiger 2002). The extract was centrifuged for 15 min at 4 oC, and absorbance measured at 645, 663 and 680 nm wavelength using a double beam spectrophotometer (Pharmacia, LKB-Novaspec II). The leaf Chl a, b and Car contents were calculated using the formulas 3, 4 and 5 respectively.
Antioxidant enzymes. Catalase (CAT) activity was measured following the procedure of Chance & Maehly (1955). The methods of Nakano & Asada (1981) and Elia et al. (2003) were used to measure the guaiacol peroxidase (GPX) activity, and SOD activity was measured following the method of Ekler et al. (1993).
Statistical analysis. Two-way-factorial (water stress by SA) analysis was employed to statistically analyze the data using STATISTICA Computer Software (Version 8.1) through ANOVA technique. LSD test at 5% probability was employed to compare the treatments’ means. A correlation analysis was also done using STATISTICA to find the strong relationship among different variables under drought stress conditions.
Results
Biomass. Drought stress markedly reduced the NOL and LA of Zinnia by 12.66 and 31.21 cm2, respectively (Table 1). Foliar SA application (100 mg L-1) decreased the negative effects of water stress by increasing the NOL (15.33), LA (46.89 cm2). A significant reduction in SL (21.67 cm) and RL (18.33 cm) was recorded under water stress whereas, SA application (100 mg L-1) improved SL and RL to 25 cm and 26.67 cm respectively. Under water stress conditions, the plants of Zinnia supplemented with 100 mg L-1 SA produced maximum SFW (7.60 g) and SDW (0.81 g), whereas minimum SFW (5.60 g) and SDW (0.59 g) in nontreated plants. Likewise, water stress also reduced the RFW, and RDW by 1.22 g and 0.30 g, respectively. In the water-stressed plants SA treatment increased the RFW and RDW by 1.45 g and 0.41 g respectively (Table 1).
Table 1 Effect of foliar applied salicylic acid on number of leaves (NOL), leaf area (LA), shoot length (SL), root length (RL), shoot fresh weight (SFW), shoot dry weight (SDW), root fresh weight (RFW), root dry weight (RDW) in Zinnia elegans under drought stress.
| Drought stress/ Salicylic acid |
NOL | LA (cm2) |
SL (cm) |
RL (cm) |
SFW (g) |
SDW (g) |
RFW (g) |
RDW (g) |
|---|---|---|---|---|---|---|---|---|
| Normal conditions | ||||||||
| N | 20.33±0.90a | 58.78±1.74a | 30.33±1.89a | 18.67±0.90b | 9.81±0.33b | 1.27±0.04a | 1.72±0.03a | 0.47±0.02a |
| SA | 19.67±1.22a | 63.24±1.39a | 34.33±1.22a | 19.66±1.22b | 11.47±0.47a | 1.34±0.03a | 1.71±0.02a | 0.52±0.01a |
| Drought stress | ||||||||
| N | 12.66±1.23b | 31.21±2.27c | 21.67±1.21b | 18.33±1.80b | 5.60±0.73b | 0.59±0.02c | 1.22±0.07c | 0.30±0.03c |
| SA | 15.33±0.68b | 46.89±2.18b | 25.00±1.56b | 26.67±1.24a | 7.60±0.43c | 0.81±0.04b | 1.45±0.03b | 0.41±0.02b |
| P-value | ||||||||
| D | 0.0004 | < 0·0001 | 0·0003 | 0·0509 | < 0·0001 | < 0·0001 | < 0·0001 | < 0·0001 |
| SA | 0.3528 | 0·0007 | 0.0375 | 0·0124 | 0.0066 | 0.0032 | 0.0069 | 0.0026 |
| D × SA | 0.1388 | 0·0181 | 0.8265 | 0·0356 | 0.7536 | 0.0659 | 0.0050 | 0.0791 |
| CVa | 10.33 | 6.55 | 9.16 | 12.08 | 10.10 | 5.95 | 3.41 | 7.05 |
Values are mean ± SE and letters represent significant differences at P < 0.05 according to LSD test. N, normal water application; SA, salicylic acid under normal water application; D, drought stress without salicylic acid; D+SA, salicylic acid under drought stress. a CV, Coefficient of variation.
Leaf water content. SA treated Zinnia plants retained maximum 88.46 % and 75.98 % leaf RWC both under normal (100 % FC) and water stress (60 % FC) conditions, respectively (Figure 2A). Whereas, minimum 66.41 % leaf RWC was recorded in water stress Zinnia plants. ELWR was also maximum 23.82 % and 31.15 % in SA treated Zinnia plants grown under normal (100 % FC) and water stress (60 % FC) conditions respectively. Water stress reduced ELWR of Zinnia plants by 22.67 % (Figure 2B).
Figure 2 Effect of foliar applied salicylic acid on leaf relative water contents (RWC) (A) and excised leaf water retention (ELWR) (B) in Zinnia elegans under drought stress. Values are mean ± SE and letters above the bars represent significant differences at P < 0.05 according to LSD test. N, normal water application; SA, salicylic acid under normal water application; D, drought stress without salicylic acid; D+SA, salicylic acid under drought stress
Pigments content. Water stress (60 % FC) reduced Chla, Chlb and Car concentrations by 0.52, 0.25 and 0.96 mg g-1 respectively (Figure 3A-B-C). SA treatment of the water stressed treatments resulted in higher leaf Chla (0.68 mg g-1), Chlb (0.38 mg g-1) and Car (1.10 mg g-1) concentrations. Well water (100 % FC) Zinnia plants treated with SA improved Chla, Chlb and Car to 0.83, 0.50 and 1.27 mg g-1 respectively.
Figure 3 Effect of foliar applied salicylic acid on chlorophyll (a, b, A.B), and carotenoids (Car, C) contents in Zinnia elegans under drought stress. Values are mean ± SE and letters above the bars represent significant differences at P < 0.05 according to LSD test. N, normal water application; SA, salicylic acid under normal water application; D, drought stress without salicylic acid; D+SA, salicylic acid under drought stress
Antioxidant activity. Water stress (60 % FC) significantly (P < 0.05) increased the activities of the antioxidant-related enzymes of CAT, GPX and SOD by 91.33 mg g-1, 46.33 U min-1 mg-1 protein and 74.33 U min-1 mg-1 protein respectively (Figure 4A-C). Foliar applied SA (100 mg L-1) of the water stressed treatments (60 % FC) resulted in considerably improved activity of 116.67 mg g-1, 72 U min-1 mg-1 protein and 93 U min-1 mg-1 protein for CAT, GPX and SOD respectively. Foliar SA application also produced maximum CAT (78.07 mg g-1), GPX (35.33 U min-1 mg-1 protein) and SOD (58.33 U min-1 mg-1 protein) in well water (100 % FC) Zinnia plants.
Figure 4 Effect of foliar applied salicylic acid on catalase (CAT, A), guaiacol peroxidase (GPX, B), and superoxide dismutase (SOD, C) in Zinnia elegans under drought stress. Values are mean ± SE and letters above the bars represent significant differences at P < 0.05 according to LSD test. N, normal water application; SA, salicylic acid under normal water application; D, drought stress without salicylic acid; D+SA, salicylic acid under drought stress
The leaf RWC and ELWR was positively correlated with CAT and GPX activities in SA treated plants, but there was no significant correlation with SOD activity under water stress conditions (Table 2). A strong and positive correlation of Chla and Chlb concentrations with LA and SDW was noted in the water stress treatments, but not with Car. Similarly, SA treatment of the water stressed plants showed strong positive correlations with CAT activity, and GPX with LA and SDW while non-significant for SOD.
Table 2 Correlation (r) among different Zinnia elegans traits following salicylic acid (SA) under drought stress conditions
| X-variable | Y-variable | Drought stress |
|---|---|---|
| Water relations | ||
| ELWR | CAT | 0.81* |
| GPX | 0.80* | |
| SOD | 0.67ns | |
| RWC | CAT | 0.88* |
| GPX | 0.95** | |
| SOD | 0.77ns | |
| Pigments | ||
| Chl a | LA | 0.90* |
| SDW | 0.82* | |
| Chl b | LA | 0.92** |
| SDW | 0.78ns | |
| Car | LA | 0.78ns |
| SDW | 0.65ns | |
| Antioxidants | ||
| CAT | LA | 0.89* |
| SDW | 0.85* | |
| GPX | LA | 0.98*** |
| SDW | 0.96** | |
| SOD | LA | 0.90* |
| SDW | 0.76ns |
LA, leaf area; SDW, shoot dry weight; CAT, catalase; GPX, guaiacol peroxidase; SOD, superoxide dismutase; ELWR, excised leaf water retention; RWC, relative water contents; chl, chlorophyll a, b; Car, carotenoids. *P < 0.05, **P < 0.01, ***P < 0.001; ns, non-significant
Discussion
Drought induced growth inhibition of annual flowering (Kaur et al. 2015), however, there is little information on the effects of stress ameliorants on physiological processes of summer annuals plants. The present study identified an effective approach to improve plant growth of Zinnia under water stress.
The present study showed that SA-mediated alterations in physiological processes markedly increased the growth attributes of Zinnia under water stress conditions. A considerable decline in growth of Zinnia under drought stress might be due to a decrease in water relations resulting in significant loss of turgor, thereby preventing the transport of nutrients and photosynthates to and from the leaves (Shehzad et al. 2020). The leaves are one of the primary plant organs that are most vulnerable to severe effects of drought stress. The immediate plant responses to drought stress involve physiological and metabolic changes in leaves (Conti et al. 2019). It was also evident by the reduction in NOL and LA in current study that drought stress significantly reduced photosynthetic capacity causing less CO2 fixation and biomass production. Similar effects have been reported for calendula (Calendula officinalis), brazilian cherry (Eugenia uniflora), purple passionflower (Passiflora incarnate), and red tip photinia (Photinia × fraseri) (Toscano et al. 2016, Garcia-Castro et al. 2017, Akhtar et al. 2019).
Exogenous SA application induces diverse physiological adaptations in plants in relation to water stress (Zarghami et al. 2014). These are associated with biochemical changes responsible for growth inhibition under water stress (Li et al. 2014). In current study, foliar SA application mitigated the effects of water stress by enhancing LA, SFW and SDW. This was likely via increasing photosynthetic capacity since SA plays a critical role in maintaining stomatal regulation and biosynthesis of photo-assimilates (Fakheri et al. 2019).
It was found that the positive effects of SA were also evident on root biomass and root length, possibly through a reduction in reactive oxygen species (ROS) production in roots (Tamaset al. 2015) and increased synthesis of osmolytes such as proline and free fatty acids that promote root growth (Azooz & Youssef 2010, Chen et al. 2014). Our findings are similar to those of Hosseini et al. (2015), which showed SA reduces electrolyte leakage and increases biomass, pigments and antioxidative enzymes activity in lolium (Lolium perenne) under water stress conditions.
Leaf RWC and ELWR can decrease significantly in various chive, turfgrass, and shrubs species subjected to water stress (Toscano et al. 2016). The decreased leaf RWC and ELWR in drought stressed Zinnia might be associated to protoplasm dehydration (Shabbir et al. 2016), damages to cell wall through lipid peroxidation (Cechin et al. 2015), reduction in photosynthetic capacity (Hussain et al. 2016), or increased water retention and enhanced stomatal closure (Miura & Tada 2014). Our study showed a marked effect of SA in maintenance of leaf RWC. Previous studies showed that SA positively interacts with abscisic acid (ABA) signaling pathways to induce stomatal closure, thereby improving drought tolerance in plants. In addition, SA application promotes root growth (also observed in the present study) which will improve water absorption and availability to the shoots
Estimating photosynthetic pigment concentrations including leaf Chla, Chlb and Car provides a good indication of drought-induced damage to photosynthetic activity (Pellegrini et al. 2011, Toscano et al. 2016). Drought reduced photosynthetic pigments in callistemon (Callistemon citrinus) (Álvarez et al. 2011), marigold (Tagetes erecta) (Riaz et al. 2013) phoebe (Phoebe bournei) (Ge et al. 2014), and sunflower (Helianthus annuus) (Shehzad et al. 2020). Similarly, Aldesuquyet al. (2012) suggested that enhanced drought tolerance in plants treated with SA was correlated with improved photosynthetic activity. Similar results were observed in the current study for Chla, Chlb and Car. Foliar SA application substantially improved the pigments in Amaranthus tricolor and Borago officinalis, and Hordeum vulgare (Habibi 2012, Khandaker et al. 2011, Shemi et al. 2021). The protective role of SA during water stress involves regulation of metabolic reactions responsible for maintenance of membrane integrity and photosynthetic pigments (El-Tayeb 2005). Moreover, SA directly influences the expansion of grana, coagulation of stroma and increases the volume of the chloroplast to reduce chlorophyll degradation and fragmentation under water stress (Uzunova & Popova 2000, Cheng et al. 2016), and stimulates the up-regulation of stress-responsive genes involved in signal transduction, photosynthesis and protein metabolism under water stress (Kanget al. 2012).
Exposure to water stress promotes intracellular ROS accumulation that induces oxidative stress and significantly damages membrane integrity (Miura & Tada 2014, Nawaz et al. 2015). Excessive ROS also triggers ABA production that influences K+ transport in guard cells to cause stomatal closure (Kwak et al. 2003), thereby reducing CO2 assimilation and decreasing biomass production in Zinnia (Ge et al. 2014). However, excessive SA application may also promote ROS production by decreasing the ability of antioxidative enzymes to overcome ROS (Miura & Ohta 2010). Hence, it is crucial to apply the appropriate SA dose to improve stress tolerance in plants. In the present study, foliar SA (100 mg L-1) was found effective to reduce the damaging effects of water stress in Zinnia, as evidenced by the increased activities of CAT, GPX and SOD. The accumulation of CAT, SOD, GPX as well as heat shock proteins (HSP) provide an ecological adaptation against ROS-induced oxidative stress (Ramel et al. 2012, Nawaz et al. 2015). Increased antioxidant-related enzymes activity would likely have contributed to lower ROS accumulation and the increased biomass production observed in this study. Similar responses have been observed on tomato (Hayat et al. 2008), sugar beet (Salami & Saadat 2013), zoysia grass Chen et al. (2014), and jasmine (Shahmoradi & Naderi 2018).
Taken together, the current study is the first report on SA mediated physiological and biochemical processes to improve drought tolerance in Zinnia. A marked reduction in growth was observed in Zinnia upon exposure to water stress; however, foliar SA applications significantly mitigated the adversities of water stress through improved leaf water status, photosynthetic pigments, and antioxidant-related enzymes. Our results provide evidence that SA application is an effective, efficient, and feasible approach to reduce water stress in annual flowers like Zinnia. The results would be of relevance to the floriculture industry as well as researchers and breeders interested in developing drought tolerant annual flowers.
