Systematic prompting the new physiological and morphological, biological responses of modied metabolic induction process of Calendula ocinalis L under heat stress with couple of supplementary nutritional applications as safety of oriculture crops

Purpose the objective of study to modied the metabolism of Calendula ocinalis L. under heat stress couple with N and P application in two years of study. Methods the trail was conducted during peak summer season (months of May, June, and July) couple with different levels of N & P application to monitor the quality and quantity characterization of marigold (Calendula ocinalis L.). The traits of three doses of N and P (0.6 g, 0.9 g, and 0.8 g, 1 − 1 g) given to Calendula ocinalis L.) to display the vegetative, reproductive, physiological parameters such as Malondialdehyde (MDA), Chlorophyll contents, Lipid Peroxidation Assay in leaves, Li and K contents, phytochemicals and nitrogen and phosphorus used ecacy Results showed that maximum values of Malondialdehyde (MDA), Chlorophylls, Lipid Peroxidation Assay in leaves observed followed by the alteration in Li and K measured in the month of May, June with slight differences July. The phytochemicals like total phenolic contents (84.41 mg GAE/g), total antioxidants (36.3% DPPH), total carotenoids and total avonoids contents (16.2 and 0.9 mg/100 g) were measured by 0.9 g of Nitrogen application and followed by 1 g of Phosphorus in both years of study. The respirational changes were observed in the higher rate of P levels. The liner changes of N and P rates showed some uctuation in heat stress months. It was concluded that the higher doses of both N and P fertilizers were effective in controlling the heat stress and mentioned the quality of orets by various induction and biological active process. randomized block design pot experiment conducted with various concentrations of N and P followed by the monitoring of their interaction under stress condition with the plants by analysis of variance (ANOVA). The post-hoc test with student newman Kuehl’s (SNK) multiple-range test (* p < 0.05) were calculated. All samples were normal, and homogeneous variances were tested using the Levene and Cochran tests were implemented on SPSS 21.0 Inc., Chicago, USA software. The mineralization process was measured from the cumulative amounts of NH 4+ -N with non-liner regression procedure.


Introduction
Horticultural crops play a vital role in international trade and getting incentive in our country for as source of earing (Khan, 2001). The oriculture crops had impacts on the development of business in all over the world through their highly demands values (Khan, 2001). Medicinal and ornamental plant like Calendula o cinalis L, contained signi cant bioactive compounds (Pérez Gutiérrez et al., 2006). belongs to family Asteraceae (Khan, 2001) although its origin is west Asia and Mediterranean of the world (Mozafariyan, 2003) but also cultivated in 17th century as an herbal medicine in Europe followed by its cultivation in growing areas of Germany, Czech Republic, Slovakia, Austria and Switzer Land, Hungary and recently in Egypt and Syria due to its signi cance (Paul et al., 2002). Calendula o cinalis L, is the rich resource of essential oil, triterpene saponins, avonoids, carotenoids, saponins, polyacetylenes, sterols, carbohydrates, vitamin C, mucilages and minerals, e.g., manganese (Grant et al., 1985). The physiological responses of Photosynthetic function is recognized as one of the most sensitive indicators of physiological processes to elevated temperatures changes in marigold ower the critical months are seriously issue in which the stomatal and non-stomatal gaseous exchanges under heat stress months (Paul et al., 2002). The heat stress was alter the stomata closure to reduce water loss, altering stomata and trichome density are the most common anatomical changes observed under high environmental temperatures in oriculture crops, the heat stress is a critical issue the high-temperature stress during summer months severely reduces the marketable quality and landscape survival capacity of marigold, the reduction in the ower diameter, and ower number in calendula has been reported with an increase in temperature (Warner & Erwin, 2005). The results of our previously reported study also showed that morphological characteristics (plant height, leaf area, shoot and root weight, ower diameter, ower number and ower longevity) of calendula cultivars were reduced by high temperature stress, especially in prolonged exposure. However, the extent to which growth and owering parameters were reduced differed among cultivars. High ambient temperatures causes heat stress and is a serious problem of the agricultural world (Hall, 2001). Temperature stress even high or low, are signi cant environmental factors that in uence growth, development and physiological and biochemical structure of oriculture plants (IPCC, 2007). It speeds up the kinetic energy and movement of molecules in the membrane. The lipid bilayer produced more uid either denaturing of protein, results an increased in the contents of fatty acid (Savchenko et al., 2002). Secondary metabolites play a role in tolerance during abiotic stress condition (Wahid, 2007). Stress protein, chemicals, and modi cation of hormonal are more prominent for supplemental application of nutrients (Mozafariyan, 2003;Liu et al., 2006;Feussner et al., 1997). Chemical fertilizers like Phosphate fertilizers put impact on growth of Calendula o cinalis L (Mozafariyan, 2003) as quality and quantity of plants is effected by nutrients (Paul et al., 2002).
In stress condition presence of nitrogen plays a very crucial role to overcome the pressure of environment (Huang et al., 2004) in prevention of the photo oxidative damages (Kato et al. 2003;Waraich et al. 2011;Zhang et al., 2006). The nitrogen is considered as a muscularly mobile nutrient in a plant that moves with living cells, and its de ciency caused a problem in owering and quality of plants (Marschner, 1995;Olson and Swallow, 1984). Phosphorous plays a dynamic part in minimizing heat stress and available to plants as an orthophosphate ions. It is a mobile nutrient in plant and transferred to sites of new growth and promotes the plant growing structures like (Cell division, cell elongation). Reduced crop yield observed under N de ciency, whereas supplements of N fertilizers can considerably upsurge productivity of the plants (Olson and Swallow, 1984;Grant et al., 1985 ;Olson and Swallow, 1984). The application of N fertilizers increased growth as it is primary constituent of proteins and are extremely liable to a loss in the range of 20 to 50% for dry matter production (Olson and Swallow, 1984 ;Grant et al., 1985). The electron ux such as Li and K play a vital role in abiotic stress condition, these uxes have potential to attained a particular ion exchange capacity these ions should be bound to the heat radiation structure and help for heat tolerance mechanism in owers and removed the heat in any part of plants (Grant et al., 1985).
The aims and objective of this article to explore active the role of induced modi ed mechanism of N & P application on quality, quantity and physiological responses under heat stress environment of marigold (Calendula o cinalis L) with advance monitoring and evaluation process. The non-reports are available on marigold nutritional requirements supplementary dozes with active nutritional responses under abiotic stress condition with trigger responses in metabolism changes under heat stress and its control through N and P fertilizers reported for the rst time up to my best knowledge with modern and advances modelling and biocenology methods was performed in this study. The Systematic prompting the new physiological and morphological, biological responses of modi ed metabolic induction process of Calendula o cinalis L under heat stress periods couple with supplementary nutritional applications as safety of oriculture crops with e cient modelling of Potential nitrogen mineralization (PNM) of media were reported in the relevant section of this article and discussed with all necessary physiological responses under altered metabolism changes during stress periods with advanced biotechnology approaches and methods.

Materials And Methods
Experimental site and location: The growing condition of Calendula o cinalis L. were monitored in current investigation under stress at Horticultural Nursery, The University of Haripur, and Khyber Pakhtunkhwa, Pakistan. It is located 34.00 latitude and 72.93 longitudes and it is situated at elevation 537 meters above sea level.
Seed sowing in pots with modern monitoring system The diameter of plastic container 200 mm deep was used, and the seed of Calendula o cinalis L was shifted into the pots with a mixture of different media shown in Table 1. The solarization of media was done for removal of any contamination before shifting into pots.

Media Preparation with active materials
The seeds were planted in a medium possess a cocopeat and sand (40:40 v/v) shown in Table 1. After 47 days of planting the seedling in four leaves stage were transplanted into large ower pots with the mixture of sand mould and cocopeat ratio of (1:1:1).

Treatments and Responses under stress periods
The improvements of orets quality of marigold was planned to monitor under N and P dozes in heat stress conditions where different levels of N and P (N 2 0.6 & 0.8 g and P 2 0.9 &1 g) per pot were applied at four leaves stages of plant to evaluate the responses of these foliar sprays. The physiological responses of these fertilizers under the heat stress condition of Pakistan were implemented according to the growing periods of marigold.

Vegetative and Reproductive growth of Marigold owers under hot climatic condition
Measurements of vegetative characters like plant height determined via a ruler at the end of the experiment by cutting above ground parts at the soil surface. Leaves, branches, petals numbers were also counted at the same time. Two dissimilar extract prepared, using 100% pure acetic acid and mixed 50% acetic with 50%water. The 1 mL solution of TBA and 1 mL standard MDA was taken in a 10 mL test and mixed then heat on water bath at 95°C for 60 minutes followed by cooling at room temperature. The absorbance of the mixture recorded at 532 nm UV/Visible spectrophotometer, a blank sample of acetic acid or water was also run. The calibration curve prepared through standard solutions. The concentration of MDA calculated through from the calibration curve.

Chlorophyll contents
Chlorophyll contents were measured in leaves (100 mg) and crushed into motor piston in acetone (80%) followed by centrifugation about 2000 rpm done for 3 mins. until colorless ltrate obtained. Absorbance of the ltrate recorded with blank 80% acetone on (UV-1800A) Shimadzu spectrophotometer. Pigment contents were determined by the method of Ahmed et al., 2018. Determination of K + contents of membrane The advanced technique Inductively coupled plasma (ICP) optical emission spectrometry used to measure the K + content in leaves which were dried in oven at 70 °C for 48 h and then agitated in 5% H 2 SO 4 overnight followed by the determination of K + content using standard curves.
Flux membrane stability of Li + and K + The Net uxes of Li + and K + were measured in leaf samples using the NMT technique by moving microelectrodes and calculated by Fick's law of diffusion (Saadalla et al. 1990).

Analysis of Phytochemical of marigold ower under heat stress periods
Total phenolic contents (TPC) Total phenolic contents (TPC) was measured using a Folin-Ciocalteu assay method where 1 mL of Folin-Ciocalteu reagent added into a diluted to 5 times in which 0.2 mL of sample and 0.8 mL 7.5% Na 2 CO 3 was added. The mixture were kept in dark for 20 mins then absorption was recorded at 765 nm against control sample (Stintzing et al. 2005). The gallic acid used to prepare the standard curve for determination of the concentration in mg equivalent of gallic acid (GAE) per gram.
Determination of DPPH radicals scavenging activity Brand-Williams et al. (1995) method applied for the determination of DPPH radicals scavenging activity using with the 4 mg DPPH dissolved in 100 mL of methanol and kept in dark place. The different concentrations were mixed with 5 mL of 0.004% DPPH solutions. The absorbance was measured at 517 nm after 20 minutes of reaction against the corresponding blank solution. The assay was performed in triplicates reactions.
Percentage inhibition of free radical DPPH was calculated based on control reading by following equation A con -is the absorbance of the control reaction A test -is the absorbance in the presence of the sample of the extracts.

Responses of Nitrogen and phosphorus used e ciency methods in heat stress period
The total nitrogen analyzed by the Kjeldahl method (Ladha et al., 2005) using 1 g leaves sample, digested with a mixture of H 2 SO 4 -H 2 O 2 . The phosphorus content was determined calorimetrically in digested samples with a solution of perchloric acid (72%), ammonium molybdate (5%, aminonaphtholsulphonic acid (0-2 %), 0.5 g of sodium bisulphite and 6 g crystalline sodium sulphite. The total N and P contents determined through method given by (Dobermann, (2007). .

Potential nitrogen mineralization (PNM) of media monitored and evaluation process
The sample media was incubated under waterlogged at 30˚C in a controlled temperature cabinet for 11 days (a 2: 1 water: media ratio was maintained during incubation). Three models, i.e. the single rst-order kinetics model, the double rst order kinetics model and the mixed rst-order and zero-order kinetics model were used to simulate the cumulative mineralized N (NH4 + -N and TSN) in the laboratory and waterlogged incubation. The complete model of PNM shown in Table 5.
Analysis of Growth media an nutritional pro ling in growth and developmental stages The analysis of growth media comprises of total organic matter and total nitrogen of the soil. For total nitrogen of soil sample Kjeldahl method was used where H 2 SO 4 used for titration. Organic matter estimated by dissolving 1g air-dried soil sample in 10 ml 1N potassium dichromate solution and 20 ml concentrated sulfuric acid in a 500 ml beaker, using a dispenser to mix the suspension for 30 minutes, then 200 mL of distilled water and `10 ml concentrated orthophosphoric acid added followed to cool it. and mixture was allowed to cool. Titration of the mixture carried out by ammonium sulfate solution using diphenylamine

Responses of Respirational changes under heat stress condition
The respirational rate was determined using a portable infrared gas analyzer 6250, LI-COR, Inc., Lincoln, Nebr.), connected with automatic software to record the reading during critical months.

Statistical Analysis
A complete randomized block design pot experiment conducted with various concentrations of N and P followed by the monitoring of their interaction under stress condition with the plants by analysis of variance (ANOVA). The posthoc test with student newman Kuehl's (SNK) multiple-range test (* p < 0.05) were calculated. All samples were normal, and homogeneous variances were tested using the Levene and Cochran tests were implemented on SPSS 21.0 Inc., Chicago, USA software. The mineralization process was measured from the cumulative amounts of NH 4 + -N with nonliner regression procedure.

Results And Dissuasion
Morphological and metabolism responses under growth and developmental changes by N &P doses It was observed that the growth of marigold plant showed modi cation under N and P application where substantial variations reported in height and number of leaves of plant in comparison with control plant (Table 2)  Chlorophyll is a green pigment and obligates part in photosynthesis activity of plants and considered as a food supply component of owering plants (Jain et al., 2001;Gupta et al., 2017). Heat stress period is a critical part for photosynthetic pigments. The structures may damage and reverse food in stored parts of organs are lost. The N and P nutrients play a vital role to minimise these oxidative damages Falk et al. (1999). The application of these additional nutrients through fertilizers improved the carbon exchange activities in owering plants and enhanced the pigments of chlorophyll in all parts of plants (Sofo et al., 2004).

Application of N& P on Phytochemicals changes of Pot marigold responses with advance biological changes
The higher contents of total phenolic compound (84.41 mg GAE/g), total carotenoids (16.2 mg/100g), and Total avonoids content (0.90 mg/100g) and total antioxidants (36.3 %DPPH) were noted in N 2 0.9 g application over control (P>0.05) shown in table 4. Phytochemical play a vital role in oxidative stress (Jain et al. 2001). The higher content of Phenyl ammonia lyase and decreased peroxidase activity by the application of nutrient in oxidative stress periods was similar to report of Rivero et al.,(2001). The phytochemicals are produced in roots and translocate into the other parts of owering plants. The current study showed that the enhance contents of these phytochemicals produce in root after application of these nutrients ions, and transferred into leaves, nodes, internodes, buds and nal involves in covering the injured organ couple with removal of heat stress injuries in plants (Jain et al. 2001). Application of N and P play a role in oxidative stress and a signi cant role in heat stress months in the owering plants which develop the ability to tolerate heat stress condition (Falk et al. 1999).

Net changes of Dry weight of ower and leaves responses of marigold
The dry weight of owers and leaves were presented in Fig 3. The higher number of Flowers noted under 1 g of P application over control. The higher number of dry leaves was observed in 1 g of P application. The fact is evident that the higher rate of phosphate fertilizer showed maximum contents of orets parts and leaf materials (Jain et al., 2001;Gupta et al., 2017). Sofo et al., (2004) also report that the dry weights were increased due to extra supply of nutrients.
Adavnce modeling of net rate of N and P E ciency up take induction process of marigold: The net e ciency rate of N and P of marigold shown in Fig 4. The net rate of N and P e ciency was increased due to a higher supply of nutrients of both doses of N and P. The higher content of both rates were measured by the application of (N 2 0.9 g and P 2 1 g). While in Fig 5 showed that N rate of e ciency was lower before application of N supply as compared to after application to marigold. The higher contents of Net N rate e ciency were recorded in 0.9 g of N 2 followed by 1g of P over control, and minor increased is reported after application. The similar trend in Phosphate fertilizer reported in earlier reported work (Dobermann, 2007). The nutrient supply increased the NUE and PUE after application of N and P (Cassman et al., 2002;Dobermann, 2007). The amount of N increased after estimation 5 to 20 % in different crops (Howarth et al., 2002) and 52% (Janzen et al., 2003). Application of N improves the NUE in cereals crops Dobermann, 2007).

Plant responses of Net rate of NUE and PUE under Heat Stress Periods its supply and changes
The net rate of NUE and PUE under heat stress conditions represented in Fig 5, 6 ,7. It was found that the high doses of both N and P were effective on metabolism of the plant in the Month of May which was (1.4%) under 0.9 g of N 2 followed by the (14.5%) in the month of June. While PUE was 32% under 1 g of P application. The month of July, the most critical period of heat stress where the assembles rate of N e cacy rate was (1.7%), over Phosphate application which showed a maximum rate increased (34%) over control for both N and P net rate e ciency.
The summer season is critical for plants growth due to high temperature. The additional application of these nutrients plays a role in minimizing heat stress in these months as reported by Ladha et al. (2005)  The results showed that the maximum respirational rates were recorded in control while lower values were obtained in N 2 0.6 g which was slow at next dose than the increase in turn at P application. These results are according to the ndings of Hussein et al. (2011) who reported signi cant role of respiration on growth and yield of marigold under N and P application. The improvement in the photosynthetic pigments compounds which enhanced the production of dry matter, seems to be a valuable tool to interpret the relationship between photosynthesis, respiration, and dry matter production, and, therefore, quality and its yield improves (Hussein et al. (2011. Growth e ciency may be associated with the morphology of plants as well as with the chemical nature of respiration which is governed by the environmental conditions and genetic modi cation and heat tolerance system (Jain et al., 2001).

New Potential nitrogen mineralization (PNM) model of media and Organic residues of media responses under heat stress changes in marigold
The results indicated that the zero-order kinetics model were used to simulate the cumulative mineralized formed of N (NH4 + -N and TSN) in the laboratory and waterlogged incubation was measured in marigold. The complete model of PNM shown in Table 5. The higher value of NH 4 + N (mg kg -1 ) 190 was found in 1 g of P application, NH 3+ N (mg Kg -1 ) 2.22 was found in same doze of P application. The net uptake of N kg.g -1 4.20 was reordered in application of 0.9 N similar in organic matters and C:N were found here in application of 1g p and higher doses of N. The higher value of N org (media) and N org (media) + min 4.1 and 5.5 was measured in application of N 0.9 g per pot shown in table 6. The lower values of N in org and media response were lower due to addition nitrogen was less in media and residue based changes were found as reported by Araus in his experiment. The mineral response in media was changed to less update of N while this critical periods were the N its update is major response of growth in owers Lima et al. 2015, Rehman, 2016.

Conclusion
Exposure the plant parts for prolonged heat stress periods has negative effects on phytochemical, physiological process of calendula under critical months of summer in Asia regions. The study was prove the application of Nitrogen and Phosphorus fertilizers play an effective role in minimising the damages during summer seasons periods as supplementary dozes apply on their active growth and developmental stages. The new and advanced biotechnology systematic changes were held in this study as new science of era for safely of orets. The application of Nitrogen 1 g showed a potential improvement of Systematic prompting the new physiological and morphological, biological responses with a positive aspect on phytochemical and physiological improvement by Phosphorus apply at 1 g. The foliar sprays of N and P has potential effects on physiological changes and tagger the response of growth and developmental process at active periods by the timely application of nutritional dozes at stress periods and proved the NUE and PUE was best method for plant health response's The e cacy of Nitrogen and Phosphorus is clear indicator of plants observed nutrients at critical response.

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Competing interests
The other authors declare that they have no competing interests.      Calculation of N and P e ciency rates before and after sowing of marigold Figure 6