A method for identification of biomarkers from uv-b tolerant plants and development of a metabolite formulation

Abstract

The present invention is a method to develop a suitable formulation (Fl) from identified potential metabolites that can be termed as biomarkers isolated from specific UV-B tolerant plant varieties and to provide the invention as a suitable UV-resistant technology to varies companies and pharma industries. The formulation can be a precise plant supplement formulation that can act as bio-stimulant and help to relieve high light stress and perform better against UV-B stress. The isolation of potential biomarkers involves screening of metabolites from susceptible and tolerant plant genotypes using metabolomics approach. Comprehensive metabolomics of heat map analysis and GC-MS spectra used to show the effectiveness of the formulation (Fl) in modulating secondary metabolites and relative accumulation of Gamma-amino butyric acid (GABA) under UV-B radiations. The formulations acts as the precursor in central metabolic network and play a crucial role in the glutathione metabolism of plants.

Description

[0001] A METHOD FOR IDENTIFICATION OF BIOMARKERS FROM UV-B TOLERANT PLANTS AND DEVELOPMENT OF A METABOLITE FORMULATION

[0002] FIELD OF THE INVENTION

[0003] The present invention in general relates to the development of a futuristic solution to promote UV-B tolerance in plants. More particularly, the invention relates to formulate a suitable biomarker formulation from high UV-B tolerant genotypes to promote the UV-B tolerance technology for providing it to various companies and pharma industries.

[0004] BACKGROUND OF THE INVENTION

[0005] Plants regulate their metabolism against different kinds of stresses by monitoring central carbon regulation. The carbon regulation mechanism in plants involves identification of new specialized metabolites to resist the effects of stress. In general, the plant stress factors include both abiotic and biotic stresses. Most of the existing literature only presents bio-stimulants but the specific bio formulations targeting high light radiations and crop health using inherit metabolites have not yet been formulated and invented.

[0006] Plants regulate their metabolism to cope with different kinds of stresses that include regulation of the carbon metabolism by synthesizing metabolites or accumulating a certain class of metabolites to resist the stress levels. UV-B radiations in high doses are detrimental to plant health as it inhibits the growth and photosynthetic efficiency of plants. The mode of action of metabolites is based on the increase the carbon influx to each node of central carbon metabolism that provides resistant against UV-B radiations. The present invention discloses a solution to plant health against UV-B radiation, which is projected to be a major cause for the drop in agricultural yields. Being sessile, plants cannot escape the exposure to the solar UV-B light and therefore, there is always a need of proper tolerance technology.

[0007] There are plants with engineered B-Box (BBX) proteins which have the capacity to modulate their metabolism by the genetic manipulation of BBX proteins leading to the accumulation of metabolites that can act as sunscreens against UV-B radiations. The need for such formulations is needed as high wavelength UV-B radiations are projected to be a major cause for the drop in agricultural yields under constantly changing climatic conditions.

[0008] The aspect of the present invention is based to provide a route to prepare novel bioformulations to enhance the fitness in plants under high light stress, drought conditions, and other abiotic stresses. Comprehensive multi-omics approach is adapted to screening of potential biomarker metabolites and synthesis of the potential formulation that can be extracted from high UV-B tolerant varieties. The identified metabolites and the compositions made from the marker metabolites enable enhancing of the carbon flux in each node and can enhance the fitness of plants under UV-B stresses.

[0009] The metabolomic investigation involves analysis in lab experiments either as a single molecule or in combination to identify pyroglutamic acid or pyroglutamate (PGA), valine, leucine and isoleucine, and phenylalanine (PHE) and some of their chemical derivatives that have been observed to be highly modulated and showed significant accumulation under high UV-B radiations by participating as a precursor in the glutathione metabolism of plants thereby playing a greater role in UV protection.

[0010] Various concentrations and metabolic formulations of pyroglutamic acid (PGA), valine, leucine and isoleucine and phenylalanine (PHE) have been tested under normal light and high UV-B radiation. Further, the metabolic formulations with better survival and biochemical parameters were selected to examine different plant growth and development parameters to arrive at a suitable bio formulation (Fl) with specific combinations of pyroglutamic acid, valine, leucine, isoleucine, phenylalanine and its derivatives.

[0011] The selected bio-formulation (Fl) is tested for studying its effect on plant growth factors; percentage of survival; chlorophyll and anthocyanin levels in plants under UV- B stress conditions and the formulation is found to conserve the levels of both the factors. In accordance to the above tested experiments, the selected bio marker (Fl) is also checked for ROS assay and Cell death assay revealing decreased cell death and lowered the ROS in plants.

[0012] Therefore, to overcome this situation, there is a need to develop a natural formulation to resist the harmful UV-B radiations. In the present study, the supplementation of Fl found to provide better growth parameters, high percentage survival and better postrecovery and opened up a fruitful pathway to develop effective UV-B protectant solutions to provide to bio-fertilizer companies and indoor agricultural industries.

[0013] OBJECTIVES OF THE INVENTION

[0014] The principal objective of the present invention is to formulate a potential biomarker formulation by the extraction of significant metabolites from high UV-B tolerant genotypes and to develop an effective combination of them to achieve excellent plant health technology.

[0015] Another objective of the invention is to utilize the metabolomics approach for screening of the potential metabolites from high UV-B tolerant plants such as susceptible Col-0 and tolerant B-box engineered plants.

[0016] Another objective of the invention is to carry out the metabolomics profiling and screening of 200 metabolites from most susceptible and tolerant genotypes of plants to extract the plant metabolites and utilize their optimum concentrations for preparation of a novel bio-formulation (Fl).

[0017] Another objective of the invention is to provide the formulation as a pioneering and safe bio fertilizer that can protect crops from the harmful effects of UV-B light, high- intensity light, drought stress, and under low water availability.

[0018] Another objective of the invention is to develop such UV-B protectant technology that can be used as a supplement from outside to act as bio-stimulants and to provide the technology to bio-fertilizer companies, indoor agriculture companies, and pharma industries interested in plant-based products. These and other objectives of the present invention will be apparent from the descriptions herein. Every objective of the invention is attained by at least one embodiment of the present invention.

[0019] SUMMARY OF THE INVENTION An aspect of the present invention is to synthesize a suitable futuristic bio-formulation (Fl) from isolated potential metabolic biomarkers from specific UV-B tolerant plant varieties and develop a UV-resistant technology that can be utilized by various biofertilizer companies, indoor agriculture companies, and pharma industries.

[0020] Another aspect of the present invention is to carry out the identification and screening of 200 metabolites from different susceptible and tolerant plant genotypes using mass spectrometry and metabolomics approach including analytical lab experiments to test the isolated metabolites either as single molecule or in combination to prepare the bio formulations.

[0021] Another aspect of the present invention is to use the selected metabolites pyroglutamic acid (PGA), valine, leucine, isoleucine and phenylalanine (PHE) and their specific combinations for the selection of a particular formulation (Fl) having suitable concentration combination of metabolites for various physiochemical and biochemical parameters such as Chlorophyll levels, Anthocyanin levels, ROS and cell assay levels to monitor the survival and fitness of plant types.

[0022] Another aspect of the present invention is to study the effect of all the aforesaid mentioned plant parameters for one of the major metabolites pyro glutamate (PGA) that have been used in making of the formulation for representation of a comparative investigative study of the effect of pyroglutamate (PGA) and the formulation on the growth parameters of the plant varieties.

[0023] Another aspect involves the use of three plant / crop varieties for the comparative investigative studies to be named as Arabidopsis, Spinach, and Tomato crops and the study revealed that the bio-formulation application decreased the cell death probability and lowered the ROS levels in plants much effectively than the sole application of Pyro glutamate (PGA).

[0024] Another aspect is the selection of proper concentration combination for the development of formulation which comprises a wide concentration range of (XmM PGA + YmM PHE + ZmM BCCAs) and their derivatives. In particular, X, Y and Z are a range of concentrations of bio formulation starting from nanomolar to millimolar / litre and optimized as per plant’s nutrition requirements and the UV-B range selected to be of 0.5mW / cm2.

[0025] Another aspect is to provide a safe bio fertilizer formulation that can protect crops from the harmful effects of UV-B light, high-intensity light, drought stress, and under low water availability.

[0026] BRIEF DESCRIPTION OF THE DRAWINGS

[0027] The present invention will be described in more detail herein with the detailed description that relates well with the preferred embodiments of the invention as explained with reference to the following accompanying schematic drawings:

[0028] FIG. 1 illustrates a method for the identification of metabolites from tolerant plants and the development of a formulation.

[0029] FIG. 2 illustrates the metabolomics-guided selection of potential biomarkers against UV-B radiations.

[0030] FIG. 3 (A) shows the images of the effect of Pyro glutamate (PGA) in presence of white light from Day 1 to Day 8; (B) the results of the effect of Pyro glutamate (PGA) on Chlorophyll levels; (C) the results of the effect of Pyro glutamate (PGA) on Anthocyanin levels in Arabidopsis seedlings.

[0031] FIG. 4 (A) shows the images of plates of Arabidopsis Col-0 seedlings exposed under UV-B light of lmW / cm2 when supplemented with various PGA concentrations over a duration of (10-28) days; and (B) images of the study of foliar applications under UV-B light of 0.5 mW / cm2 with PGA concentrations of (0.01 mM to O. lmM) on both wild type or genetically engineered genotypes.

[0032] FIG 5 (A) shows plates of Arabidopsis Col-0 exposed to UV-B for seven days; (B) plates exposed to UV-B for fourteen days; (C) shows the survival rate up to (80-90%) in plants when treated with UV-B for seven days; (D) shows the survival rate up to (70- 80%) in plants when treated with UV-B for seven days; (E) monitoring of the chlorophyll levels in plants that are subjected to UV-B radiation; and (F) monitoring of the anthocyanin levels in plants that are subjected to UV-B radiation.

[0033] FIG. 6 shows the (A) appearance of PGA under 14-day white light and 24 hours of UV- B light; (B) images of the ROS assay of the Arabidopsis seedlings when exposed to 14- day white light and 24 hours of UV-B light; (C) and (D) Cell death assay of Arabidopsis seedlings when exposed to (14 day white light (WU), 24 hours UV-B) light and (18 day WU, 48 hours of UV-B light).

[0034] FIG. 7 Study of plant fitness on application of Pyroglutamate (PGA) metabolite for UV- B protection as: (A) images of heat map analysis done using metabolomics approach; (B) image of metabolomic analysis of Phenylalanine (PHE); (C) image of metabolomic analysis of Valine; and (D) image of metabolomic analysis of Ueucine on plant types.

[0035] FIG. 8 Study of plant fitness on application of formulation (Fl) for UV-B protection as: (A) images of heat map analysis done using metabolomics approach; (B) image of metabolomic analysis of Phenylalanine (PHE); (C) image of metabolomic analysis of Valine; and (D) image of metabolomic analysis of Leucine on plant types.

[0036] FIG. 9 shows the study of 18-day old plant fitness parameters on application of formulation (Fl) as: (A) image of only the Arabidopsis plants in presence of water and exposed to UV-B light; (B) Percentage survival of the plant with formulation Fl under UV-B for 7 days and post recovery; and (C) ROS levels of the plant with formulation Fl under UV-B.

[0037] FIG. 10 shows images of Mustard seedlings (18 days old) with PGA under UV-B (lmW / cm2) for 7 days and post-recovery; and (B) images of Wheat plants (14 days old) with Fl under UV-B (0.5 mW / cm2) for 7 days and 14 days respectively.

[0038] FIG. 11 shows the effect of applying the Fl formulation on drought tolerance capacity in A) & B) Arabidopsis, C) Spinach, and D) Tomato crop plants. FIG. 12 shows (A) images of Spinach plants with Fl in presence of water; and (B) 1H NMR results revealing the effect of the application of bio formulation (Fl) on Arabidopsis roots.

[0039] FIG. 13 illustrates (A) Gas chromatography-mass spectrometry (GC-MS) analysisbased metabolic profding of (UV-B+F1) treated plants; and (B) Principal component analysis (PC A) analysis of (UV-B+F1) treated plants.

[0040] FIG. 14 shows (A); (B); and (C) images for the comprehensive metabolomics showing Fl modulates secondary metabolites and accumulates GABA under UV-B light.

[0041] DETAILED DESCRIPTION OF THE INVENTION

[0042] The preferred embodiments are provided so that the disclosure will be thorough and will fully convey the scope to those who are skilled in the art. Various specific details are set as specific components to provide an overall understanding of the preferred embodiments of the present disclosure. It will be apparent to those skilled in the art that the specific details need not be employed and the embodiments may be embodied in many different forms and the steps followed do not limit the scope of the disclosure. It is also to be understood that the terminology used herein is for the purpose of describing only the particular embodiments of the invention and is not intended to limit the scope of the invention in any manner.

[0043] The present invention discloses the process for the development of a futuristic solution to promote UV-B tolerance in plants by formulating a suitable and precise potential biomarker from high UV-B tolerant genotypes to promote UV-B tolerance technology for various companies and pharma industries.

[0044] The profiling and selection of the potential plant biomarkers have been carried out by a metabolomics profiling approach in which the selected metabolites and their derivatives have been tested to prepare an effective and novel bio-formulation after the screening of 200 metabolites.

[0045] The initial step involves the isolation of the potential metabolites from two engineered Col-0 and tolerant B-box plant varieties. The metabolomics profiling procedure involves the use of water, methanol, and chloroform as solvents and the techniques of Gas chromatography-mass spectrometry (GC-MS) data and GC-MS library or commercial standards to decipher the profiling of all the 200 metabolites.

[0046] Based on profiling studies, the metabolites are listed, and based on the accumulation capacity few metabolites are identified as potential UV-B markers as a single molecule or in combinations to prepare several compositions with concentrations in the range of nanomolar to millimolar per liter of metabolites under UV-B light exposure of 0.5mW / cm2.

[0047] On completion of profiling procedure, one of the significant biomarkers is selected and along with Fl formulation is applied to several plant varieties and tested under white light (WL) and UV-B light (0.5mW / cm2to 1.0mW / cm2) to determine the physicochemical and biochemical parameters, and the compatibility with water.

[0048] One of the selected metabolite pyroglutamic acid / pyroglutamates (PGA) and their derivatives showed a higher shift in plant metabolism than other selected metabolites and in furtherance the metabolite has been investigated along with Fl formulation for the survival percentage and to examine the growth parameters such as chlorophyll; anthocyanin levels; and ROS assay levels.

[0049] EXPERIMENTS

[0050] The present invention will be illustrated with the help of the following experiments, which are not intended to limit the scope of the invention and any such modification therein falls within the scope of the invention. It is to be understood that both the foregoing general description and the following details description are exemplary and explanatory only and are intended to provide further explanation of the subject matter.

[0051] EXPERIMENT -1: Genetic engineering and high throughput metabolomics guided potential biomarkers against UV-B

[0052] Metabolomics profiling of 200 metabolites isolated from susceptible UV-B plant varieties including both wild and genetically engineered plants have been done using several techniques of Gas chromatography-mass spectrometry (GC-MS); High- g performance liquid chromatography (HPLC); and Liquid chromatography-mass spectrometry (LC-MS).

[0053] Several concentrations of each metabolite starting from nanomolar scale to milli molar scale optimized in view of the aforesaid studies to develop an efficient bio-formulation that facilitates better results against stress. The basis of development of formulations is based on glutathione metabolism and carbon flow at the nodal points wherein the aforesaid factors are known to have a greater role in UV protection capacity.

[0054] Experiment 2: Effect of Pyroglutamate (PGA) on growth parameters and survival under UV-B light and white light (WL)

[0055] The selected genotype of Arabidopsis Col-0 seedlings has been subjected to different concentrations in the range of (0.01 to 0.5mM) of Pyro glutamate (PGA) for 7 days to monitor the effect on the growth and fitness of the plant varieties irrespective of whether it is a wild variety or a plant genotype and to identify the optimal concentration range of metabolite that promotes chlorophyll and anthocyanin levels in Arabidopsis seedlings and on the percentage survival of the plants under UV-B light.

[0056] EXPERIMENT -3: Effect of Pyroglutamate (PGA) on ROS and cell assay levels

[0057] The isolated plant varieties were subjected to white light for four days, followed by UV- B treatment with UV radiations of 0.5 mW / cm2for 72 hours in order to measure the reactive oxygen species (ROS) and cell death levels with 3, 3 '-diaminobenzidine (DAB) and Trypan Blue Staining, respectively.

[0058] EXPERIMENT-4: Trials for various formulations and designing of bio formulation (Fl) for UV-B protection

[0059] Multiple tests supplementing either pyro glutamate (PGA), phenylalanine (PHE) or invented formulation (Fl) including a derivative of pyro glutamic acid, and a combination of branch chain amino acids in certain ratios (e.g., valine, leucine, and isoleucine) and at least one aromatic amino acid. One of the bio formulations (Fl) has been zeroed in with the combination of (XmM PGA+ YmM PHE + ZmM BCCA) wherein X, Y, and Z are in the concentration range of metabolites as: Pyro glutamic acid from 0.1 mM to 0.5 mM for UV-B survival; Phenylalanine in the range of (0.01 mM to 0.25 mM); and branch chain amino acids required in a range of (0.01 mM to 0. 1 mM) for best results.

[0060] EXPERIMENT-5: Investigative study on enhancement of plant fitness by the application of formulation (Fl) on plant type

[0061] For the investigation of the comparative effectiveness of the formulation (Fl) and the metabolites on plant biochemical parameters. All the metabolite components and the formulation have been separately applied on an 10 day old and 14 day old ‘Arabidopsis seedlings’ in presence of UV-B light (0.5 mW / cm2) that have been irradiated for 7 days. The biochemical parameters that have been studied after the application of the metabolites and the Fl are Chlorophyll levels, Anthocyanin levels, percentage survival and the post recovery status of the plants.

[0062] EXPERIMENT-6: Specific study of effectiveness of formulation (Fl) and the metabolite (PGA) on plant fitness parameters

[0063] For the study of the effectiveness of the formulation (Fl) in comparison to the most accumulative metabolite (PGA) have been achieved by selecting two plant types namely Mustard (18 days old) and Wheat (14 days old) under UV-B light for an irradiation duration of 7 days under UV-B light of (1.0 Mw / cm2) for mustard plant and (0.5 Mw / cm2) for wheat plant.

[0064] EXPERIMENT-7: Study of effectiveness of formulation (Fl) on plant fitness parameters and ROS assay levels under drought conditions

[0065] For the study of the effectiveness of the formulation (Fl) have been achieved by selecting three plant types namely Arabidopsis, Spinach, and Tomato crop plants under UV-B light for irradiation duration of 18 days.

[0066] EXPERIMENT-8: Metabolic Profiling

[0067] Gas chromatography-mass spectrometry (GC-MS) and 'H-NMR techniques have been utilised for the collection of metabolic profiling of (UV-B+F1) treated plant varieties. Specifically, Spinach plants have been grown in water in presence of Fl formulation to determine how well the bio formulation was taken in by the plants.

[0068] Results:

[0069] In the present invention, the metabolic profiling results shown in Fig 1 and Fig 2 demonstrate the Pyroglutamate (PGA) and its derivatives to show effective accumulation under UV-B light. The potential metabolites take part as precursor of glutathione metabolism intermediates, and such metabolites of GS-GOGAT play a more significant role in UV protection. Furthermore, the heat map analysis showed pyro glutamic acid, valine, leucine, and isoleucine, as well as phenylalanine and their derivatives among the potential metabolites. Under high UV-B radiation exposure, exceptionally Pyroglutamate found to have the highest relative abundance and accumulation in plants in comparison to other selected metabolites.

[0070] The optimal growth study of the isolated plant genotypes on application of pyro glutamate showed that the concentration range of (0. 1 to 0.5mM) as demonstrated in Fig 3 and found to promote the chlorophyll and anthocyanin levels in Arabidopsis seedlings.

[0071] In furtherance, Fig 4 shows that on application of optimum concentration of Pyro glutamate (PGA) on Arabidopsis Col-0 seedlings under high UV-B exposures resulted in bright appearances on the leaf of the plants. Specifically, a concentration of (0.01 to 0. ImM) found to be optimal for foliar application and improved plant growth.

[0072] The invention of identification of plant effective metabolites and formulation of more effective solution is based on the fact that all these metabolites take part in the Carbon flow mechanism in plants. All the metabolomics profiling clearly confirmed that Pyroglutamate (PGA) and derivative along with valine, leucine, and isoleucine, as well as phenylalanine (PHE) showed highest relative abundance in plants when compared to other selected metabolites.

[0073] The investigative study on the effect of the selected metabolite PGA on the survival percentage and on the cell death and ROS assay levels clearly shown in Fig. 5 and Fig. 6 revealed an optimal concentration of (0.1 to 0.5) mM promotes B) chlorophyll and C) anthocyanin levels in Arabidopsis seedlings. The figures shows the plates of Arabidopsis Col-0 seedlings when exposed to UV-B for seven days, and for fourteen days, the survival rate was observed to be up to (80-90%) and (70-80%) in plants respectively and the metabolite Pyroglutamate (PGA) known to maintain and enhance the Chlorophyll and Anthocyanin levels in plants under high UV-B light.

[0074] The reactive oxygen species (ROS) and cell death study using 3, 3 '-diaminobenzidine (DAB) and Trypan Blue staining dyes in Fig 6, confirmed Pyroglutamate (PGA) and its derivatives to reduce the amount of ROS in plant leaves and prevent tissue damage in Arabidopsis seedlings.

[0075] Heat maps analysis using a metabolomics approach for the screening of 200 metabolites showed a high degree of regulation (as shown in Fig 7). All the potential metabolites identified by the processes subjected for the fine tuning of the concentration range from nano molar to mill molar concentrations so that the plants experienced an improved growth when tested against the effects of stress.

[0076] Effect of various concentrations and metabolic formulations of pyroglutamic acid (PGA), valine, leucine and isoleucine, and phenylalanine (PHE) were tested in presence of normal light and high UV-B radiation have been shown in Fig 8 which supported the study in fine tuning of the concentrations of each metabolite in the range of nanomolar to mill molar concentrations for the plants to experience improved growth when tested against the effects of stresses. In addition, metabolic formulations that had better survival and biochemical parameters effects selected in order to investigate various growth and development parameters. Out of all the metabolites, supplementation of PGA concentration, either on its own or in a variety of different combinations found to result in a higher percentage of plants surviving. The formulation (Fl) developed by combining a particular combination of pyro glutamic acid, valine, leucine, isoleucine, phenylalanine, and its derivatives in particular range helped plants to maintain and up regulate higher anthocyanin levels when exposed to UV-B. The positive effects of the formulation (Fl) include the plants displayed improved growth parameters; up regulate higher anthocyanin levels when exposed to UV-B; a high percentage of survival; and post-recovery towards lesser levels of ROS levels. In Fig 9, beter plant fitness is achieved through the use of Fl supplementation. When the formulation applied to the Arabidopsis plants exposed to UV-B light, the plants displayed improved growth parameters, high percentage of survival, and postrecovery with less levels of ROS.

[0077] In Fig 9, beter plant fitness is achieved through the use of Fl supplementation. When the formulation applied to the Arabidopsis plants exposed to UV-B light, the plants displayed improved growth parameters, high percentage of survival, and postrecovery with less levels of ROS.

[0078] In support of the embodiment of the present invention, further tests have been done on Mustard and Wheat seedlings using Pyroglutamate (PGA) and the (Fl) formulation and results shown in Fig 10. The results validated the optimal concentration along with the number of bio-combinations, and formulation against the stress of UV-B radiation and in particular showed that 18 days old Mustard seedlings when exposed to high levels of radiation for 7 days are beter protected with foliar application of PGA showing improved growth even when exposed to UV- B and the 14 days old Wheat plants showed less tip bum when the formulation (Fl) used as their growing medium.

[0079] The formulation (Fl) also subjected for investigation of its effects under drought conditions (in Fig 11) and the effect of Fl was studied on three types of plants namely, Arabidopsis, Spinach, and Tomato and the study duration extended over a period of 18 days. The results showed that plants that received Fl supplements exhibited greener, lower levels of reactive oxygen species (ROS), more anthocyanin, and less cell death.

[0080] Fig 12 shows1H-NMR test results further validated that the Arabidopsis roots uptake the bio formulation (Fl) when Spinach plants were grown in water and Fl.

[0081] GC-MS based metabolite profiling and principal component analysis (PCA) analysis under UV-B light shown in Fig. 13 suggested that (UV-B+F1) possesses unique metabolic processes when implemented to crop / plant varieties and to be specific in regular controlled environmental conditions, the application of the formulation developed in the present invention provides beter plant growth, unique metabolic processes under tolerant plants and higher photosynthetic activity by increasing chlorophyll and anthocyanin biosynthesis.

[0082] Fig. 14 shows the comprehensive metabolomics including heat map analysis and GC-MS spectra on application of Fl modulates secondary metabolites and results in the relative accumulation of Gamma-amino butyric acid (GABA) under UV-B light.

[0083] Although the invention has been described with reference to specific embodiments, this description is not meant to be construed in a limiting sense. Various modifications of the disclosed embodiments, as well as alternate embodiments of the invention will become apparent to persons skilled in the art upon reference to the description of the invention. It is therefore contemplated that such modifications can be made without departing from the scope of the present invention as defined.

Claims

We Claim:

1. A method for identification of metabolites from UV-B tolerant plants and development of a formulation, comprising the steps of:(a) Investigating the unique growth conditions for the plant varieties under UV-B light;(b) Extracting the suitable plant metabolites from a solvent;(c) Screening of the metabolite effects under stress;(d) Identification of the suitable metabolites as potential UV-B markers for the study of the physiochemical and biochemical parameters;(e) Formulating various concentrations of identified markers and recording the compatibility in water and the accumulative growth capacities; and(f) Developing one specific combination as formulation (Fl) from a range of concentrations (from nano molar to milli molar)2. The method as claimed in claim 1, wherein the solvent used for extraction are water; methanol and chloroform.

3. The method as claimed in claim 1, wherein the screening techniques used for identification are Gas chromatography-mass spectrometry (GC-MS); High- performance liquid chromatography (HPEC); Eiquid chromatography-mass spectrometry (EC -MS); 'H-NMR spectroscopy; and Principal component analysis (PCA).

4. The method as claimed in claim 1, wherein the suitable metabolites identified as biomarkers are Pyroglutamate (PGA) and different derivatives; branched chain amino acids like Valine, Leucine, and Isoleucine and at least one aromatic amino acid like Phenylalanine (PHE) or Tyrosine or Tryptophan.

5. The method as claimed in claim 1, wherein the physiochemical and biochemical parameters comprises the study of Chlorophyll level; Anthocyanin level; percentage survival; ROS levels; and Cell assay levels.

6. The method as claimed in claim 1, wherein the concentrations of metabolites used for making Fl involves Pyroglutamate (0.1 mM to 0.5 mM); Phenylalanine (0.01 mM to 0.25 mM) and branched chain amino acids (0.01 mM to 0. 1 mM).

7. The method as claimed in claim 1, wherein the formulation (Fl) follows a central metabolic network under UV-B light and plays a key role in glutathione metabolism and UV-B protection.

Images