Introduction

Increasing pressure on global irrigation water resources has forced prolonged irrigation intervals due to water shortage. This is undoubtedly one of the most challenging issues that agriculture, and particularly the horticulture industry, faces today (Mehran et al. 2017; Ahmadi et al. 2018). Water stress may have morpho-physiological, metabolic, and molecular responses on plants, ending to reducing growth and yield. These changes are associated with mechanisms of resistance to water stress that include tolerance, avoidance, and escape (Nilsen and Orcutt 1996; Li et al. 2018). Drought tolerance may be defined as the ability of the plant to maintain growth despite water stress. This can be accomplished by accumulating solutes (e.g., K), a condition termed osmotic adjustment (Huang et al. 2014). Drought avoidance targets increasing water uptake and/or minimizing water loss by reducing leaf area and/or increasing root growth (Nilsen and Orcutt 1996; Huang et al. 2014; Gonçalves et al. 2017). Drought escape is achieved by shortening the plant life cycle or by undergoing dormancy during periods of stress (Huang et al. 2014). Drought-tolerance and drought-avoidance mechanisms are common in plants and may interact or co-occur with metabolic and molecular regulation (Yu et al. 2012; Li et al. 2018). Plant metabolic performance under drought conditions includes increased carbohydrates (Pallas et al. 2013), synthesis of specific proteins (Chen et al. 2017), increased stress-related nutrient uptake (e.g., K), and accumulation of antioxidants that neutralize reactive oxygen species (ROS) (El-Esawi et al. 2017). Plant responses to water deficit also include proteomic regulation by activating specific antioxidant-related genes such as SOD, CAT, and APX (El-Esawi et al. 2017; Ju et al. 2018) and by activating stress-related genes such as those reported previously (Gupta and Huang 2014; Ali et al. 2017; Li et al. 2018).

Developing novel tools that may enable crops to cope with effects of water stress are growing worldwide, including the use of trinexapac-ethyl (Elansary 2017), seaweed extracts (Elansary et al. 2017), nanoparticles (Saxena et al. 2016), myo-inositol (Yildizli et al. 2018), chitosan (Yang et al. 2009), and robinin.

Chitosan is a biostimulant manufactured by treating chitin with high temperature, and then following by deacetylation process that removes proteins and calcium (Sharp 2013; Pichyangkura and Chadchawan 2015). Chitosan oligosaccharides may be prepared and sold as a liquid solution or as water-soluble powder. They are known as plant elicitors that increase secondary metabolites production (Karuppusamy 2009), particularly polyphenols (Yin et al. 2012). Chitosan also has antimicrobial effects and, in the same time, may enhance beneficial microbes’ growth (Ramírez et al. 2010). Additionally, increased crop yield had been reported following chitosan treatment (Sharp 2013). However, the mechanism whereby chitosan improves the tolerance in agricultural crops is not well understood. Furthermore, no additive effects of chitosan and robinin on plant growth have been previously described.

Robinin (syn. Kaempferol 3-O-robinoside-7-O-rhamnoside) is a flavone glycoside (Fig. 1) that was first isolated from Vinca erecta (Akhmedzhanova 1986) as well as other plants (Elansary et al. 2018) and is an important natural phenolic compound. Sergiev et al. (2004) reported that robinin has growth-stimulatory activities on wheat coleoptiles; this is the only report on robinin application in plants, to our knowledge.

Fig. 1
figure 1

Robinin chemical structure

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Chrysanthemum (Dendranthema grandiflorum), which belongs to the family Asteraceae, is an important horticultural plant worldwide that is used as ornamental plant suitable to arid and semi-arid regions. Chrysanthemum genes related to abiotic stress are well characterized, including DREB1, which is strongly related to freezing and drought stresses (Tong et al. 2009); BBX24, which influences flowering and abiotic stresses (Yang et al. 2014); and ProDH, which encodes a mitochondrial protein representing the key enzyme in proline degradation (Xu et al. 2013). The response of this species to robinin and/or chitosan oligosaccharide elicitor under water stress has not yet been investigated.

In the present study, the objective was to study the different mechanisms involved in the effects of robinin and/or chitosan on Chrysanthemum grown under two different irrigation intervals using morphological, physiological, metabolic, and molecular markers. We hypothesized that stress tolerance, as well as stress avoidance mechanisms, co-occurs with the antioxidant pathway to control plant growth. In addition, enhanced molecular regulation of antioxidant enzymes and stress-related genes in plants subjected to robinin and/or chitosan do exist. We measured several physiological markers that reflect stress and stress alleviation in plants such as proline, carbohydrates, antioxidants, and leaf water potential. The information obtained from this study will contribute to our understanding of robinin and/or chitosan effects in plant subjected to water stress.

Material and methods

Plant material and treatments

Chrysanthemum morifolium Ramat plants that height is 10 cm (short-day flowering) were purchased from private commercial nurseries in January (for 2 successive years of 2017 and 2018). A polyethylene-covered greenhouse was used for growing the plants in Alexandria (Alexandria-Cairo Desert Road), Egypt. Identified plants by Hosam Elansary were registered at the Faculty of Agriculture-Elshatby, Alexandria. The plants were transplanted to 2.1 L pots that contained growing media (brown peat and perlite, 3:1 w/w). A compound fertilizer was used (Crystalon®; 20% N: 20% P: 20% K, 2 g/L media). The growing temperature ranged from 15.2 °C (night) to 27.6 °C (day); relative humidity between 58 and 68%; photosynthetically active radiation (PAR) around 1000 m−2 μmol m−2 s−1 at 12.00 p.m.; and manual daily watering of 38–50 mL/plant. Two groups of plants were used, one of which was watered at 2-day intervals (2 DWI) with 38–40 mL/plant to reach 100% of evapotranspiration (ET), while the other group was watered with 40–50 mL/plant at 6-day intervals (6 DWI) for 6 weeks to reach 100% ET. Day length control was applied to maintain vegetative growth by extending day length to 14 h using 1000 m−2 μmol m−2 s−1 incandescent light.

To investigate the optimal concentrations of robinin (Sigma-Aldrich, Germany) that boost plant growth (pilot study), 48 Chrysanthemum seedlings were watered separately for 3 weeks with a solution containing different concentrations of robinin. Then, in the final experiment, the plants were watered weekly with a solution containing robinin (Fig. 1) at 25, 100, and 200 ppm during extended irrigation interval conditions (every 6 days, 6DWI). Untreated plants with robinin/chitosan were considered as controls. Chitosan oligosaccharide (deacetylation > 95%, powder, Aldebeiky Group Co., Cairo, Egypt) water solution was applied using a sprayer at concentrations of 50 and 200 ppm until drop-off during the experiment and started 2 weeks prior to extending the watering interval from 2 days (2DWI) to 6DWI. The experiment was designed as split plot. Irrigation treatments were considered as the main plot and robinin and/or chitosan treatments as the subplot. Three blocks/repetitions (n = 3) were formed from plants containing five replicates for each treatment to reach a total of 360 plants in Randomized Complete Block Design (RCBD) experiment.

Morphology and physiology

After applying treatments for 6 weeks, the plant height and the leaf number were determined. Leaf area was determined by a scanner and AutoCAD program. Soil-free plants were dried in the oven at 40 °C, and then, the total constant dry weight was determined. Freeze-dried ground samples were also obtained and kept at 20°. The carbohydrates composition was quantified following Dubios et al. (1956) as percentage. The K+, Ca2+, and proline concentrations were measure in leaves following Elansary et al. (2017). Leaf midday water potential and relative water content were determined at 12 pm (solar time) following Elansary et al. (2016a).

Antioxidants, chlorophyll, phenols, and enzyme activities

The leaves were air dried and then ground into powder, and then, methanolic extracts were prepared and the antioxidant effects were measured by the 2,2′-diphenylpicrylhydrazyl (DPPH) and β-carotene-bleaching tests, which determine OH− radical effects (Elansary et al. 2017, 2019). Total phenolic and total chlorophyll contents were determined following Elansary et al. (2016b). Peroxidase (POD) activity was quantified in leaves following He et al. (2011). Superoxide dismutase (SOD), ascorbate peroxidase (APX), and H2O2 accumulation were quantified in leaf tissues following Elansary et al. (2017). One unit represented the enzyme amount inhibiting 50% of nitroblue tetrazolium (NBT).

RNA isolation and quantitative real-time PCR

The RNA was obtained using the RNeasy Plant Mini Kit (Qiagen, Germany) from fresh leaves as well as the roots 12 h after the last application of robinin and/or chitosan, and the cDNA was obtained using a Reverse Transcription Kit (Qiagen, Germany). Real-Time PCR (qRT-PCR) were performed in duplicates (SYBR Green, Qiagen, Germany) (Al-Ghamdi and Elansary 2018) to calculate the expression levels of genes. The expression of zinc-finger transcription factor gene (Cm-BBX24) (Yang et al. 2014), Chrysanthemum roots dehydration responsive element binding factor 1 (DREB1A-1) (Tong et al. 2009), Chrysanthemum heat shock protein CgHSP70 (Song et al. 2014), pyrroline-5-carboxylate synthetases (P5CS), pyrroline-5-carboxylate reductase (P5CR), and proline dehydrogenase (ProDH) (Xu et al. 2013) were examined. PCR conditions followed Al-Ghamdi and Elansary (2018). Amplification specificity was tested using melting curve analysis. A housekeeping gene (Actin) was used as a reference gene following Gu et al. (2011).

Statistical analyses

The data of 2017 and 2018 seasons showed no significant differences, and then, they were pooled and expressed as means, and least significant difference (LSD) was determined using SPSS (PASW Ver. 21) at P ≤ 0.05.

Results

Morphological performance

Under the 2 DWI, robinin and/or chitosan had no effect on chrysanthemum leaf numbers except at high doses of 100/200 ppm robinin + 200 ppm chitosan (Figs. 2 and 3). However, leaf area, dry weight, and height showed higher variation compared to leaf numbers in response to robinin and/or chitosan doses. The leaf area increased in response to the additive effects of robinin + chitosan. For example, the leaf area increased from 308.1 (control) to 360.4 cm2 plant−1, plant dry weight from 10.6 (control) to 13.9 g plant−1, and plant height from 27.2 (control) to 33.9 cm in response to 200 ppm robinin + 200 ppm chitosan application. In general, under 2 DWI, the application of high doses (robinin + chitosan) had effects on dry weight, leaf area and number, and height.

Fig. 2
figure 2

Effect of prolonged irrigation, robinin, and chitosan treatments on leaf number and leaf area in Chrysanthemum. Values are expressed as means of replicates (± sd)

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Fig. 3
figure 3

Effect of prolonged irrigation, robinin, and chitosan treatments on plant dry weight and plant height in Chrysanthemum. Values are expressed as means of replicates (± sd)

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Under 6 DWI, there were increases in the morphological performance of plants subjected to robinin + chitosan at 200 ppm of each. For example, application at 200 ppm robinin + 200 ppm chitosan increased leaf numbers from 12.5 (control) to 19.5 leaf plant−1. In the same manner, the leaf area increased from 121.6 (control) to 150.9 to cm2 plant−1, the plant dry weight increased from 5.5 (control) to 6.4 g plant−1), and plant height increased from 17.2 (control) to 21.9 cm.

Physiological performance

Carbohydrates, K+, Ca2+, proline, and leaf water potential

Under 2 DWI, carbohydrates, K+, Ca2+, and proline compositions were the highest in chrysanthemum plants subjected to 200 ppm robinin + 200 ppm chitosan (Figs. 4 and 5). For example, total carbohydrates increased from 12.43% (control) to 14.35%, K+ increased from 2.4 (control) to 2.6 mg g−1 DW, Ca2+ increased from 1.81 (control) to 2.41 mg g−1 DW, and proline increased from 14.9 (control) to 19.7 mg g−1 DW.

Fig. 4
figure 4

Effect of irrigation intervals, robinin, and chitosan on total carbohydrate and K+ content in the leaves of Chrysanthemum in two successive seasons. Values are expressed as means of replicates (± sd)

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Fig. 5
figure 5

Effect of irrigation intervals, robinin, and chitosan on Ca2+ and proline content in the leaves of Chrysanthemum in two successive seasons. Values are expressed as means of replicates (± sd)

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Under 6 DWI, the carbohydrates increased in chrysanthemum subjected to 200 ppm robinin + 200 ppm chitosan as compared to other doses. In addition, lower doses of robinin such as 100 ppm robinin + 200 ppm chitosan showed comparable results to 200 ppm robinin. Lower doses of robinin and/or chitosan showed much lower carbohydrate compositions.

K+ and Ca2+ compositions showed increases in the robinin + chitosan treatments comparable to robinin/chitosan alone as well as control treatments. The proline content enhanced in robinin + chitosan-treated plants at 200 ppm of each compared to the control (Figs. 4 and 5).

Leaf water potential and relative content of water showed significant increases in plants treated with robinin + chitosan compared to other treatments as well as control (Fig. 6).

Fig. 6
figure 6

Leaf water potential and relative water content of Chrysanthemum. Values are expressed as means of replicates (± sd)

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Antioxidants, phenolics, and chlorophylls

Under 2DWI, the DPPH (IC50) of chrysanthemum decreased from 11.5 (control) to 9.7 µg mL−1 following robinin (200 ppm) + chitosan (200 ppm) treatment, indicating an increase in scavenging activity (Fig. 7). An increase in the scavenging activity in 2 DWI was noted in the β-carotene–linoleic acid test following robinin + chitosan treatments.

Fig. 7
figure 7

Antioxidant activity in leaf methanolic extracts, total phenolic, and total chlorophyll content of Chrysanthemum. Values are expressed as means of replicates (± sd)

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Chrysanthemum leaves in plants growing under 6DWI showed enhanced scavenging activity following application of robinin (200 ppm + chitosan (200 ppm). For example, the application of 200 ppm robinin + 200 ppm chitosan increased the DPPH (IC50) from 9.7 (control) to 7.9 µg mL−1 and the β-carotene–linoleic acid activity from 10.8 (control) to 8.5 µg mL−1.

The total phenolic of chrysanthemum plants peaked following robinin + chitosan treatments under 2 and 6 DWI (Fig. 8). Robinin + chitosan treatments at 200 ppm each boosted the phenolic content from 12.1 (control) to 14.0 mg GAE g−1 and from 14.3 to 16.9 mg GAE g−1 in plants subjected to 2 DWI and 6 DWI, respectively.

Fig. 8
figure 8

Total phenolic and total chlorophyll content of Chrysanthemum. Values are expressed as means of replicates (± sd)

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Total chlorophyll content in chrysanthemum plants was reduced in control plants when the irrigation intervals were prolonged and this chlorophyll content increased from 2.2 (control 2 DWI) to 1.6 (control 6 DWI) mg g−1 DW (Fig. 7). Under 2 DWI, robinin + chitosan (at 200 ppm each) increased the chlorophyll compared to other treatments. Also, under 6 DWI, the robinin and chitosan increased also the chlorophyll of treated plants at different doses.

Antioxidant enzyme activities and gene expression

Major antioxidant POD, SOD, and APX enzymes increased in chrysanthemum subjected to robinin + chitosan (200 ppm each) compared to other treatments under 2 and 6DWI (Fig. 9). The H2O2 in chrysanthemum subjected to robinin + chitosan (200 ppm each) compared to other treatments in both seasons (Fig. 10).

Fig. 9
figure 9

POD, SOD, and APX activities in chrysanthemum plants

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Fig. 10
figure 10

H2O2 content in chrysanthemum plants subjected to prolonged irrigation intervals, robinin, and different chitosan concentrations

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Gene expression levels of Cm-BBX24, DREB1A-1, and CgHSP70 increased in chrysanthemum plants following application of robinin + chitosan at 200 ppm each as compared to other treatments (Fig. 11). There was an additive effect of robinin + chitosan on Cm-BBX24, DREB1A-1, and CgHSP70 under under 2 and 6DWI. Increased expression of P5CS and P5CR was detected under robinin + chitosan treatments (200 ppm each) compared to the other treatments (Fig. 12). ProDH expression was reduced following robinin + chitosan treatment at 100–200 ppm each (Fig. 12).

Fig. 11
figure 11

Gene expression levels of Cm-BBX24, DREB1A-1, and CgHSP70 in Chrysanthemum subjected to different robinin and/or chitosan and normal (2 DWI)/prolonged irrigation intervals (6 DWI)

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Fig. 12
figure 12

Gene expression level of P5CS, P5CR, and ProDH in Chrysanthemum subjected to different robinin and/or chitosan and normal (2 DWI)/prolonged irrigation intervals (6 DWI)

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Discussion

Increasing pressure on global irrigation water resources has forced prolonged irrigation intervals due to water shortage. This is undoubtedly one of the most challenging issues that agriculture, and particularly the horticulture industry, faces today (Ali et al. 2017; Ahmad et al. 2018).

We recorded reductions in all the morphological parameters due to extension of the irrigation interval such as shortness of plants and reduction of leaf area and number as drought-avoidance mechanism (Elansary and Salem 2015; Ali et al. 2017; Gonçalves et al. 2017). The reduction in the morphological side was associated with stress-related physiological alterations including leaf water potential, carbohydrate, proline, chlorophylls, K, Ca, and antioxidant (Elansary 2017; Elansary et al. 2017, 2018).

Robinin + chitosan application improved chrysanthemum performance during different irrigation intervals. Previous study on chitosan indicated an increase in essential oil yield and dry matter in Thymus daenensis Celak (Bistgani et al. 2017). However, the mechanisms were not properly understood. Studies reporting robinin application to plants for vegetative stimulation purposes are limited. One study (Sergiev et al. 2004) reported that robinin might have stimulatory activities on the growth of wheat coleoptiles. However, this is the first comprehensive study investigating the physiological and molecular effects of robinin on an ornamental horticultural crop. Furthermore, the additive effects of robinin + chitosan on the vegetative growth of plants are novel and have not been reported previously.

Accumulation of proline, sugars, and ions is a mechanism of osmotic adjustment related to drought tolerance (Ali et al. 2017). The accumulation of proline assists in stabilizing cellular proteins under stress and control free radicals (Seki et al. 2007). Carbohydrate increase indicates stress conditions and helps in osmotic adjustment and free radical control (Yin et al. 2010; Gupta and Huang 2014). Additionally, the accumulation of proline in plant vegetative parts might be an important sign of stress tolerance, because such accumulation balances vacuolar ion osmotic pressure (Elansary and Salem 2015; Ali et al. 2017) and maintains water influx (Hoque et al. 2007). The decrease in leaf water potential and content are strong indicator of stress conditions that was alleviated by robinin and chitosan treatments. In addition, proline accumulation entailed a degree of water stress experienced by the plant tissues; furthermore, proline accumulation was induced in plants treated with robinin (100 and 200 ppm) + chitosan (200 ppm).

In this study, the osmotic adjustment mechanism was strongly present by means of accumulation of K+ and Ca+2 ions during water stress. Ion accumulation is related to carbohydrate, which may increase the growth and enhance cell turgor pressure (Ali et al. 2017; Elansary et al. 2017). Ion accumulation in chrysanthemum increased chlorophyll content (drought resistance mechanism). Low rate applications of robinin + chitosan increased the ion composition in chrysanthemum, and assisted and aided in the attainment of osmotic adjustment.

Excess ROS and related ions are usually present in plants subjected to stress due to electrons production and use imbalance which may end with damage and death of plant cells (Cruz 2008). The antioxidant defense mechanism is composed of enzymatic (POD, SOD, and APX) and non-enzymatic (e.g., phenols) pathways that crosstalk to minimize the intracellular redox (AbdElgawad et al. 2016; Elansary et al. 2017).

The phenolic composition was higher in robinin + chitosan-treated plants. These increases are associated with enhanced antioxidant performance of treated plants. Additionally, chitosan doses of 200 ppm increased the phenolic content of leaves compared to the control plants; these findings concord those reported by Yin et al. (2012). The novelty of this study could be in the additive effects of both substances used in enhancing the antioxidant bioactivities of treated plants. Phenols remove ROS in stressed plants by controlling OH− free radical and H2O2 accumulation.

Chrysanthemum plants subjected to robinin and chitosan showed increased expression of antioxidant-related genes. A strong indicator of the antioxidant mechanism of stress control is the increased expression of the chrysanthemum-specific Cm-BBX24 (Yang et al. 2014), as found in the current study. The increased expression of Chrysanthemum DREB1A-1 reported here is indicator of the molecular control of robinin + chitosan in treated plants.

These results are novel, and indicate a strong association among morphological, physiological, and genetic chrysanthemum-specific markers. There was robinin + chitosan additive effect on Cm-BBX24, DREB1A-1, and CgHSP70 expression in treated plants and caused increased expression under water stress. This effect was not reported before. Furthermore, P5CS and P5CR increased their expression as molecular control under stress in plants treated with robinin and/or chitosan. Plants treated with 200 ppm robinin + chitosan showed the highest expression of these five genes. Lower doses of robinin + chitosan had lower effects on these genes. In agreement with these results, several investigations have revealed higher expression of these genes under stress conditions in barley (Pérez-López et al. 2009), maize (AbdElgawad et al. 2016), and rosemary (El-Esawi et al. 2017). However, the application of robinin + chitosan is novel and has not been previously reported. Furthermore, drought and salinity stress-related genes such as DREB1 were confirmed to have enhanced expression under robinin + chitosan treatments. In addition, DREB gene overexpression has confirmed stress tolerance in several crops such rosemary (El-Esawi et al. 2017) and ERF3 in soybean (Zhang et al. 2009). The increase in P5CS expression is interesting, because it encodes pyrroline-5-carboxylate synthetase that controls the biosynthesis of proline (Xu et al. 2013). Also, the expression of ProDH reductions is important, because it encodes the main enzyme in the proline degradation pathway. The application of robinin + chitosan has main effect on proline in chrysanthemum plants which is related to water stress tolerance.

The longer irrigation interval of 6 days caused water stress conditions in chrysanthemum plants as found in reduced growth and relative water content of leaves. These conditions were alleviated at the morphological, physiological, and molecular levels by application of robinin + chitosan sprays. Several stress tolerance as well as avoidance mechanisms crosstalk with antioxidant responses at the morphological level as well as the physiological level. The findings of this investigation after water stress and the application of both chemicals were confirmed in several experiments in which we monitored different morphological measurements, all of which, taken together, are considered to be a drought-avoidance mechanism. These morphological changes co-occurred with physiological osmotic adjustment as revealed by carbohydrate, chlorophyll, proline, K+, and Ca+2 accumulation, which constitute the organic basis of a complex drought-tolerance mechanism. The antioxidant mechanism for stress tolerance was investigated by studying overall antioxidant activity, phenolic content, H2O2 content, and POD, SOD, and APX enzyme activities. Overall, the data obtained from these assays were confirmed at the molecular level by screening antioxidant enzyme-related genes as well as chrysanthemum-specific stress tolerance-associated genes. The application of robinin + chitosan during an extended irrigation interval might ameliorate drought stress effects on chrysanthemum. The current knowledge of stress resistance-related mechanisms might help in applying future novel tools that enable us to attenuate the unpleasant effects of earth temperature increase and lack of irrigation water around the world.

Conclusion

A novel finding emerged from this study exploring the mechanisms controlling morpho-physiological, and molecular performance in water-stressed Chrysanthemum treated with robinin + chitosan. The study revealed that several mechanisms interacted to enhance plant overall growth during water stress including drought tolerance, drought avoidance, and antioxidant pathway. Robinin + chitosan application successfully ameliorated the negative effects of water stress on plant growth, suggesting that it may be an excellent way in improving water stress tolerance in agricultural crops and in the future prospects of novel tools and strategies to further attenuate the unpleasant effects of global warming and lack of water resources.

Author contribution statement

HOE and AMEA-H designed and performed experiments. All the authors participated analyzing, writing, revising, and approving the final version of the manuscript.