Silicon-Mediated Growth, Physiological, Biochemical and Root Alterations to Confer Drought and Nickel Stress Tolerance in Maize (Zea mays L.)

Abiotic stresses are the leading environmental factors which adversely affect plant growth and development particularly drought and nickel stress. Maize is susceptible to drought and nickel stress from germination to final kernel development stage. The purpose of the current study was to evaluate the role of silicon to improve drought and nickel stress tolerance in maize. Different treatments of the study were i) two drought stress levels (100% field capacity and 60% field capacity) ii) nickel stress (100 mg/kg) and iii) combined stress (drought and nickel stress) were used along with two levels of silicon applications, i) control and ii) 50 mg/kg). The result showed that silicon had significant effects on plant growth attributes, including plant height (cm), leaf area (g), and leaf dry weight (g), stem fresh weight (g), and stem dry weight (g). Plant height reduced up to 24% under drought stress, and 13% under nickel stress. While silicon application mitigated the adverse effects of these stresses and increased the shoot length up to 35% as averaged of both stresses. Leaf water potential decreased under drought by 25% and nickel stress by 11% and combined stress showed 53% reduction as compared with control, but silicon application significantly improved the leaf water potential up to 12%. Gas Exchange Parameters i.e., photosynthetic rate, transpiration rate, stomatal conductance and respiration rate were significantly reduced under stress conditions. photosynthetic rate showed more reduction in combined stress (drought and nickel stress). as compared with the control (no stress). Silicon application @ 50 mg/kg improved the gas exchange parameters, protein contents, chlorophyll a and b under stress and non-stress conditions. Chlorophyll a and b increased up to 21% and 32% respectively as compared to control (no silicon application). The H2O2 values increased under the drought and nickel stress conditions and decreased in control-no stress. Drought and nickel stress decreased the levels of the catalase (CAT), peroxide dismutase (POD) and superoxide dismutase (SOD). Soil application of silicon 50 mg/kg improved the values of SOD, POD and CAT. In crux, the present investigation suggested that silicon application @ 50 mg/kg mitigated the harmful effects of drought and nickel alone and in combination by improving the morpho-physiological and biochemical attributes in maize. Si-applied plants significantly improved the performance of maize against these stresses, which was linked to maintaining plant water status and photosynthetic pigments, lower oxidative damage, and higher activities of antioxidant enzymes under drought and nickel stress.


Introduction
Global climate change and its adverse factors such as floods, droughts, severe and low temperatures, salts and pollutants are the serious threat to field crops [1].All of these factors negatively impact crop physico-biochemical function, the increase and yield of important crops [2,3].Among various abiotic factors, drought stress and metal stress affect soil fertility, water quality and the productivity of field crops [3,4].
Water stress decreases plant growth around the world.Drought mainly occurs due to constantly low availability of fresh water for crop growth [5].It reduces the fresh and dry weight of the plant, parameters of water relation, and antioxidants [6,7].Moreover, water stress decreases the leaf water potential, photosynthetic rate, relative water contents, stomatal conductance, rate of transpiration [8] and a loss of cell volume, which disturb the normal photosynthetic activities [5].
Heavy metals are found naturally in soil ecosystems as a result of weathering processes of parent materials [9].Moreover, anthropogenic activities may be the main cause to pollute soil by different containments are due to the increasing trends of world population, land use changes, modern agriculture practices, and industrialization processes [10,11].However, various soil variables i.e., pH, Cation Exchange Capacity and organic matters concentration influence metal availability for plants.Nickel is an essential trace element for optimum plant growth, but its high accumulation causes toxic and serious damage in crop production [12].Metal enters vegetal tissues at toxic levels disrupt many morpho-physio and biochemical functions of crop plants and reduce crop yield [12,13].Researchers need to explore the way to ameliorate the osmotic stress caused by nickel and drought stress to support plant growth and development.
Many agronomic, breeding, physicochemical and biological strategies have been developed that are helpful in managing and providing suitable environment for plant growth.Among various strategies application of chemical nutrients such as silicon (Si) play important role in biotic and abiotic stresses [14,15].Silicon is the most common element on the outermost layer of earth [16] and famous for improving the overall performance of plants under stress conditions.Furthermore, exogenous silicon application has been shown to improve the relative water content and enhance seedling growth of sorghum and sunflower [17].Silicon applications increases stomatal conductance, rate of photosynthesis and antioxidant defense in wheat [18].Silicon application under drought and metal stress improves the root growth in plants by upregulating the plant physiological and biochemical attributes [19].
The application of Si has been reported to improve abiotic stress in plants.However, the physicochemical role of Si applications in drought, nickel stress, and combined stress tolerance has never been done for maize.
The hypothesis of the study was that the application of Si may improve the nickel and drought stress tolerance in maize.The objective of current research was to evaluate the impact of soil applications of silicon on morpho-physiological and biochemical traits to impart drought stress, nickel stress, and combined stresses tolerance in maize.

Experimental Conditions, Treatment, Design, and Crop Husbandry
The factorial arrangements and three replications of the pot experiment were set up using a Completely Randomized Design (CRD).The research project was carried out in the Crop Stress Physiology Lab, Department of Agronomy, University of Agriculture, Faisalabad in an open environment under sheltered net house.The plastic pots (15 × 10 cm) with 1.5 kg of sand were filled with ten seeds.Sand was used as growth medium.Maize seeds were obtained from the Ayub Agriculture Research Institute, Faisalabad.After sieving the sand to eliminate all impurities, the field capacity was determined using the correct methodology.Metal stress was imposed by using nickel sulphate with 94.3% purity as it is most toxic form of nickel which was applied two months before sowing in each pot as per treatment level.The average air temperature was 33 °C during light period and 22 °C during the dark period.Different treatments of the study were i) two drought stress levels (100% field capacity and 60% field capacity) ii) nickel stress (100 mg kg −1 ) and iii) combined stress (drought and nickel stress) were used along with two levels of silicon applications, i) control and ii) 50 mg kg −1 .Field capacity was determined by following the standard procedure described by Pennypacker et al. [20].After seedling establishment (three-four leaf stage) five seedlings were maintained in each pot, all the pots were divided into two main sets, first was kept under well water or full field capacity (control) second under 60% field capacity as per treatment level..EDTA 0.1 mM.) was used for the nutritional needs of maize.At time of sowing, Hoagland solution was applied and finished off after every two weeks.The experiment used 24 pots divided into four groups of six pots each, which were raised under similar circumstances until one pair was subjected to drought.After imposition of drought, seedling growth parameters, water relations and gaseous exchange attributes and data regarding biochemical and root morphological was recorded as mentioned below.Silicon (2023) 15:6579-6589 1 3

Seedling Growth Attributes
Growth attributes were measured by selecting two plants from each replicate randomly.These same plants were gently uprooted in order to measure the height of plants and length of roots by using a metre rod.Using an electric weighing balance, to determine the plant's shoot and root fresh weight (Kern 440-49A, Germany).These plants were oven dried at 70 °C (Memmert-110, Schawabach, Germany) until their weight remained constant, and then the dry weight was measured (Shimadzu AW-320, Kyoto, Japan).

Plant Water Relation Attributes
To determine the water potential of the leaves, from each treatment by following the procedure used by Ahmad et al. [21], we took the upper third of fully expanded leaves.Data was recorded between the early hours of 8:00 and 10:00 using a Scholander-type pressure chamber (ARIMAD-2, EL International, Japan).The solute/osmotic potential (Ψs) of the same leaf was measured after it had been frozen at 20 °C.By thawing and crushing the frozen substance, a syringe was used to extract the cell sap after it had been collected in a glass tube.An osmometer was used to measure the osmotic potential of this cell fluid.(Wescor-5500, USA).Using the formula, the pressure potential was determined.
For measurement of relative water contents, three leaves per treatment were harvested in order to obtain the data.Each sample was first given its fresh weight (FW) by using a digital scale (Shimadzu AW-320, Kyoto, Japan), after which it was placed in a test tube with distilled water for 24 h to soak.Water was wiped off from the samples using tissue paper to get turgid weight (TW).Dry weight (DW) was measured by oven drying at 65 °C for 72 h.The leaf RWC for each treatment was calculated [22].

Gas Exchange Parameters
The gas exchange attributes were measured by using a photosynthesis system, CI-340 portable Infrared Gas Analyzer, following the method used by Ahmad et al. [23].The measurements were taken from the top third leaf at 9.00 a.m. to 11.00 a.m., when ambient temperature was ranged from 22.4-27.9°C, by retaining the molar flow of air per unit leaf area at 403.3 mmol m −2 s −1 , the atmospheric pressure was 99.9 K Pa, ambient CO 2 concentration was at 352 mol mol −1 , while the water vapor pressure into the chamber was between 6.0-8.9 m bar, and leaf temperature of 28.4-32.4°C [23].

Chlorophyll Contents
Each treatment yielded samples that were tested for chlorophyll concentration.A scissor was used to cut off the leaves of plants that were arbitrarily tagged.Using fresh leaf samples of maize, the chlorophyll concentrations were determined by Arnon [24] and Davies [25].Fresh sample leaves weighed (0.1 g) were cut into pieces of 0.5-cm, ground in 10 mL of acetone (80%) at 0 °C, and then centrifuged at 15,000 rpm for five minutes.The absorption of chlorophyll a and b was measured at 663, 645, and 480 nm, by using spectrophotometer (Hitachi, 220, Japan).

Antioxidants
Superoxide dismutase (SOD) was measured in leaf samples from each treatment using techniques described by Nakano and Asada [26] and Stagner and Popovic [27], respectively.

Hydrogen Peroxide (H 2 O 2 )
A 50 mM phosphate buffer solution with a pH of 6.5 was used in 3 mL for the determination of H 2 O 2 in 50 mg of maize leaf samples.Centrifugation of the combination (6000 g) took place at 4 °C for 30 m.The solution was then centrifuged (6000 g for 20 min at 4 °C) after being added titanium sulfate (0.1%, 1 mL) in 20% (v/v) H 2 SO 4 .At 410 nm, the supernatant's absorbance was determined.The concentration of H 2 O 2 was then determined using a formula (0.28 mol −1 cm −1 ) [28].

Root Attributes
Roots of all treatments were obtained from each pot by washing the pots after 40 days of sowing and then the roots were washed in water and cleaned.Root traits were recorded using root scanner using WINRHIZO'S Companion Software for Data Analysis and Visualization.

Statistical Analysis
In a factorial experiment with three replications and a completely randomized design (CRD), data were analyzed using Statistix 10.0 and the analysis of variance (ANOVA) method.The mean comparison at 5% level of significance was performed by using the least significant difference test [29].

Seedling Growth Attributes
Analysis of variance showed a great effect of silicon under drought and nickel stress for seedling growth parameters i.e., shoot length, shoot fresh weight, shoot dry weight, root fresh weight, root dry weight and stem diameter.Drought and nickel stress significantly decreased all seedling growth parameters in maize.Maximum reduction was observed at non-silicon applications (Table 1).Shoot length, shoot fresh weight, shoot dry weight, root fresh weight, root dry weight and stem diameter decreased up to 24%, 94%, 54%, 29%, 27% and 29%in drought stress, 13%, 24%, 27%, 5.2%, 5% and 17% in nickel stress, and 44%, 117%, 79%, 49%, 64% and 33% in combined drought and nickel stress, respectively, as compared with control.Among the stresses, combined stress of nickel and drought showed the maximum reduction in all seedling growth parameters in maize.However, silicon applications improved the shoot length, shoot fresh weight, shoot dry weight, root fresh weight, root dry weight and stem diameter by 33%, 33%, 33%, 21%, 39% and 18% respectively in maize (Table 1).The interaction of silicon and stressed conditions was non-significant in shoot length, root fresh weight and stem diameter, highly significant in shoot fresh weight, and significant in root and shoot dry weight.

Water Relation Attributes
Analysis of variance showed a great effect of silicon under drought and nickel stress for plant water relation attributes i.e., leaf water potential, leaf osmotic potential, leaf turgor potential and relative water content.Drought and nickel stress significantly decreased all plant water relation attributes in maize.Maximum reduction was observed at non-silicon applications (Fig. 1).Leaf water potential, leaf osmotic potential, leaf turgor potential and relative water content decreased up to 25%, 25%, 57% and 35% in drought stress, 11%, 14%, 36% and 37% in nickel stress, and 53%, 52%, 20% and 68% in combined drought and nickel stress, respectively, as compared with control.Among the stresses, combined stress of nickel and drought showed the maximum reduction in all seedling growth parameters in maize.However, silicon applications improved leaf water potential, leaf osmotic potential, leaf turgor potential and relative water content 12%, 15%, 65% and 25% respectively in maize (Fig. 1).The interaction of silicon and stressed conditions was significant in leaf water potential, leaf osmotic potential and in relative water content.While in case of turgor potential it was highly significant.

Gas Exchange Attributes
Analysis of variance showed a significant effect of silicon under drought and nickel stress for gaseous exchange attributes i.e., plant photosynthetic rate, transpiration rate, stomatal conductance, and internal carbon dioxide concentration in maize.Drought and nickel stress significantly decreased all gaseous exchange attributes in maize.Maximum reduction was observed at non-silicon applications (Fig. 2).Plant photosynthetic rate, transpiration rate, stomatal conductance and internal carbon dioxide contents decreased up to 41%, 38%, 34% and 12% in drought stress, 31%, 22%, 17% and 12% in nickel stress, and 43%, 57%, 22% and 16% in combined drought and nickel stress, respectively, as compared with control.Among the stresses, combined stress of nickel and drought showed the maximum reduction in all gaseous exchange attributes in maize.However, silicon applications improved the plant photosynthetic rate, stomatal conductance, transpiration rate and internal carbon dioxide contents in maize by 51, 26, 64 and 36% respectively in maize (Fig. 2).The interaction of silicon and stressed conditions

Chlorophyll Contents
Analysis of variance showed a significant effect of silicon under drought and nickel stress for chlorophyll a and b.Drought and nickel stress significantly decreased the chlorophyll a and chlorophyll b content in maize.Maximum reduction was observed at non-silicon applications (Table 2).Chlorophyll a and b decreased up to 14 and 17% in drought stress, 10 and 9% in nickel stress, and 23 and 31% in combined drought and nickel stress, respectively, as compared with control.Among the stresses, combined stress of nickel and drought showed the maximum reduction in chlorophyll a and b in maize.However, silicon applications improved the chlorophyll a and b content by 21% and 32.51%, respectively in maize (Table 2).The interaction of silicon and stressed conditions was non-significant in chlorophyll b and significant in chlorophyll a.

H 2 O 2 , SOD, POD and CAT
Analysis of variance showed a significant effect of silicon under drought and nickel stress for SOD, POD and CAT .  in drought stress, 9%, 16% and 14% in nickel stress, and 35%, 42% and 35% in combined drought and nickel stress, respectively, as compared with control.Among the stresses, nickel stress showed the maximum reduction in SOD, POD and CAT in maize.However, silicon applications improved the SOD, POD and CAT content by 52%, 26% and 15% respectively in maize (Table 2).The interaction of silicon and stressed conditions was significant in H 2 O 2, highly significant in SOD and POD, and non-significant in CAT.

Root Attributes
Analysis of variance showed a significant effect of silicon under drought and nickel stress for total root length, root surface area and average diameter.Drought stress significantly increased the total root length, root surface area up to 40% and 39% in maize.While the average diameter decreased under drought stress conditions.Maximum reduction was observed at non-silicon applications (Fig. 3).Average diameter decreased up to 28% in drought stress, 0.9% in nickel stress, and 45% in combined drought and nickel stress, respectively, as compared with control.Drought showed the maximum increase in total root length and root surface area.However, silicon applications also increased the total root length and root surface area content by 13% and 22%, respectively (Fig. 3).The interaction of silicon and stressed conditions was non-significant in total root length, root surface area and average diameter.

Discussion
Plants growth and development were affected by the abiotic stress including water stress.It decreases the plant height, root shoot fresh, and dry weight and stem diameter.The combined stress of drought and nickel showed negative impacts on growth of the plants and inhibits plant cell expansion.The results demonstrated that combined stress significantly (p ≤ 0.05) hampered the shoot length, root length, and root and shoot fresh and dry weight as compared to the control-no stress.Due to the disruption of stomatal closure and reduced CO 2 assimilation, which inhibits cell division, drought and nickel stress have the negative effect of slowing growth in agricultural plants [30].Using silicon can increase plants' tolerance to abiotic conditions [31].Drought stress reduced shoot length by 24%, nickel stress reduced shoot length by 13%, and combination of drought and nickel stress reduced shoot length by 44%, respectively.The plant growth was enhanced by adding silicon to the soil, water relations, photosynthetic efficiency, roots attributes, and antioxidant activities.Moreover, Similar results recorded by Amin et al. [32] who reported that soil application of Si (100 mg kg −1 ) significantly improved growth under stressed condition by upregulating carbon assimilation and stomatal conductance.
In our study, drought stress, nickel stress, and combined effect of both stresses showed a significant reduction is plant water relations and gas exchange attributes.Leaf water potential, osmotic and turgor potential reduced under drought and nickel stress, as water shortage disrupt the plant water status which adversely affects the development of plants.Low photosynthetic rates in agricultural plants have low CO 2 conductance through the stomata and mesophyll, hampered chloroplast development, and constrained metabolite transport under drought stress.Leaf water potential reduced 25% in drought stress, 11% in nickel stress and 53% in combined stress and seedlings without silicon applications showed greater reduction.Nickel toxicity affected plants water status and efficiency [33].Additionally, plants with silicon applications showed more growth in water relations and gaseous exchange attributes.A possible reason for the reduction in photosynthetic rate is due to decrease the extension of leaf and stomatal conductance that could be an important factor in carbon fixation under stress conditions.The reduction in plant water potential results to decrease the leaf turgor potential, which may have a negative impact on stomatal conductance and consequently reducing the photosynthetic rate that all may led to impair plant growth and development.However, silicon applications may regulate the leaf osmotic potential and assist the plant in maintaining its water balance that may help the plant to upregulate gaseous exchange under stressed conditions [34].Applications of silicon have shown an impact on plant water relations and stomatal conductance without having any physiological consequences because silicon may increase water absorption and transportation to the stem and leaves by upregulating the hydraulic conductance [35].
The results of research demonstrated that drought and nickel stresses significantly reduced the chlorophyll a and chlorophyll b content.Chlorophyll decreased up to 14 in drought stress, 10% in nickel stress, and 23% in combined (drought and nickel) stress, respectively, as compared with control.Silicon applications increased chlorophyll a by 21% as compared with no silicon.Pandey et al. [36] concluded that applications of silicon increased photosynthetic efficacy by preventing oxidative damage to the pigments involved in the photosynthetic processes, enhancing assimilate translocation, and controlling the source-sink relationships within the plant.The application of silicon upregulates leaf chlorophyll contents which has an inverse relationship with the increased photosynthetic efficiency, which increased biomass results.
In the current research, under drought and nickel stress the H 2 O 2 concentration increased which led to a notable decline in the cell membrane stability, it boosted the cells leaking of metabolites into intercellular spaces [37,38].However, maximum increase in H 2 O 2 was observed in combined stress as compared with control.Results have revealed that the increase in ROS damaged the chlorophyll pigments which led to reduced chlorophyll content and photosynthetic rate [39,40].Soil application of silicon has a critical function in scavenging reactive oxygen species (ROS) and lowering oxidative stress in drought and nickel stressed maize plants.Silicon applications improved the stress tolerance by decreasing the concentration of the H 2 O 2 that led to improved abiotic stress tolerance.In addition, results revealed that H 2 O 2 decreased under silicon applications up to 23% in drought, 20% in nickel and 30% in combined (drought and nickel) stress, respectively.To control oxidative stress brought on drought and nickel stress and to scavenge reactive oxygens, plants boost the antioxidant contents through various physio and biochemical mechanisms.
Despite the fact that under many stresses there is no consistency in the production and detoxification of oxygen radicals, as drought and nickel stress increase the contents of ROS, the findings have shown that maize has shown some resilience to drought stress.The rise in H 2 O 2 under multiple stresses may be the cause of such an inequity which causes the plant to sustain damage from oxidative stress.Under combined stress, antioxidant activities (catalase, protease, and ascorbate peroxidase) increased, lowering the amount of H 2 O 2 in stressed plants.Furthermore, silicon increased the defense system of maize plants by improving the activity of catalase, protease and peroxidase under drought and nickel stress.The possible reason for this improvement may be that applications of silicon increased the plant cell redox state, regulated antioxidant activity, and reduced lipid peroxidation products [41,42].Plant metabolism has improved as a result of use of silicon was thought to be essential for reducing the effects of nickel stress and drought by controlling the number of photosynthetic pigments and effectiveness [43].The antioxidants increased under drought and nickel stress as compared with control.Superoxide dismutase increased up to 26% in drought, 9% in nickel stress and 35% in combined stress.However, silicon applications improved the SOD, POD and CAT under stress conditions.
In our results, root diameter reduced under drought stress and nickel stress while the root length and root surface area increase in combined stress (nickel + drought).Average diameter decreased up to 28% in drought stress, 0.9% in nickel stress, and 45% in combined drought and nickel stress, respectively, as compared with control.Maximum reduction was observed at non-silicon applications.Drought stress significantly increased the total root length and root surface area up to 40% and 39%.The possible reason for this increase may be that under drought stress, the ratio of plant roots to shoots usually increases, and as a result, the plant biomass significantly decreases.Same results were also reported by Wang et al. [44].They reported that under water stress, plant roots go in depth in search of water and nutrients, they increase the area of proliferation and also the roots length.However, the application of silicon increased the root mineral nutrient uptake which helps plants to increase its length.
Numerous studies revealed that the exposure to heavy metals adversely impacted the root development of different plant species [45].The Ni accumulation in the root apex significantly inhibits the mitotic cell division which results in decreased growth.However, soil application of silicon increased the total root length and root surface area content by 13% and 22%, respectively.The results showed that root length increased under drought stress conditions with a reduction in root weight as root biomass reduced under stress due to more thin roots [46].Ekinci et al. [47] also revealed that the root development was significantly reduced under drought stress due to inhibited growth in less water conditions that may led to reduce plant growth and development as a deep and prolific root system is a key to perform better under nickel and water stresses [48].However, root length, root fresh and dry weight were increased under silicon applications as silicon has the potential to increase the number of lateral roots in plants.In several crops, plants treated with silicon have shown a rise in root characteristics, like length and diameter.Higher concentration of silicon in aerial parts played a significant role under combined stress through maintaining the roots attributes.

Conclusions
In crux, the results of current study showed that both drought and nickel stresses negatively affected plant growth, water relation, gas exchange attributes and concertation of antioxidants in maize.Soil application of silicon @ 50 mg/kg mitigated the drought and nickel stress by enhancement of photosynthetic pigments, modulation of gas exchange attributes and improvements in the antioxidant defense which led to improve plant growth and development.Silicon applications improved the plant photosynthetic rate and RWC by 51, and 36%, respectively in maize as compared to control-no Si.The results of this research indicated that after applying silicon to the soil at a rate of 50 mg/kg might be more efficient in improving maize's ability to perform under conditions of nickel stress and drought.

Fig. 1 Fig. 2
Fig. 1 Effect of soil applied silicon on leaf water potential, osmotic potential, turgor potential and relative water content under drought and nickel stress in Maize.Error bars above means indicate the stand-

Table 1
Impact of soil applied Si on seedling growth parameters of maize under drought and nickel stress Means sharing same case letter or without lettering for a parameter do not differ significantly (p ≤ 0.05) by the LSD test.Values represent the average of three replicates per treatment ± SE (Standard error) was significant in stomatal conductance.While, non-significant in case of plant photosynthetic rate, transpiration rate and internal carbon dioxide concentration.

Table 2
Impact 20% in nickel and 30% in combined (drought and nickel) stress.Silicon applications decrease the H 2 O 2 content by 22% respectively in maize.SOD, POD and CAT increases up to 26%, 25% and 20% of soil applied Si on biochemical parameters of maize under drought and nickel stress Means sharing same case letter or without lettering for a parameter do not differ significantly (p ≤ 0.05) by the LSD test.Values represent the average of three replicates per treatment ± SE (Standard error)