COMPLETE: "The following specification particulady describes
the invention and the manner in which it is to be performed"
Anthropogenic activities are the major factors contributing in increased amount of chromium (Cr) concentrations in the soil and water. Among various forms of heavy metal contamination, Cr contamination is of significant scientific interest as it affects plant productivity and human health as well. In the environment, various forms of Cr are known of which trivalent [Cr(III)] and hexavalent Cr [Cr(VI)] are more common. Hexavalent chromium is more toxic than Cr(III) and has capability of penetrating plasma membranes. Hexavalent chromium is a non-essential element so plants do not
have any specific transport for it. However, it is reported that Cr(VI) is transported through sulfate transporters in plants. After its entry in the plant system, Cr(VI) creates wide ranges of physiological, biochemical and molecular alterations starting from seed germination to the plant death. Thus, continuous investigation on toxicological impact of Cr(VI) in plants is needed in order to develop strategies for its toxicity mitigation.
Since under certain circumstances, Cr(VI) uptake occurs through the sulfate transporters so it is possible to reduce its entry in the plant system by using appropriate amount of additional S sources. In the literature, this strategy for mitigating metal toxicity by using essential elements has been considered as nutrient management approach. Though in the recent years, there is an increasing amount of literatures which have advocated efficiency of essential elements in mitigating metal toxicity with greater importance on Cd; however, mechanism(s) associated with metal toxicity alleviation are still poorly known. Glutathione (GSH), a tripeptide (y-glu-cys-gly), severs as a major pool of reduced sulfur species and also discharges various functions in plants by maintaining cellular redox homeostasis. Study has shown that embryonic defects in GSH deficient mutant of Arabidopsis can be rescued by the addition of GSH which indicates its crucial role in plant development. Besides this, GSH pool can also manage negative consequence of abiotic stresses such as heavy metal. In this connection, in the recent years several studies have reported metal toxicity alleviatory role of GSH in plants. However, role of exogenous applied GSH in mitigating Cr(VI) toxicity remains elusive. Besides this, in recent years hydrogen sulfide (H2S), a colorless gaseous signaling molecule, has gained much attention because of its implication in regulating wide ranges of plant physiological processes. Under abiotic stress, metal toxicity alleviatory role of H2S has increasingly been reported. However, implication of H2S signaling in regulating essential elements like S-mediated mitigation ^o^Qrj^Vl^t^xJciiy'jn p|ai!|s,-tCL Qjir-kSdwlIdge, il still' nbf%nown. Tomato (Solanum lycopersicum L.) and

: brinjal (Solarium melongena L.) are widely used as vegetables all over the world as they are rich sources of - - minerals, vitamins, fibers and antioxidants. Besides this, immature pea (Pisum sativum L.) seeds are also
popularly used as a vegetable and are rich sources of protein, fats, vitamins, fibers and antioxidants.
Therefore, in the present patent, potential of formulation of additional sulfur (prepared formulation) in
mitigating/managing Cr(VI) toxicity in tomato, pea and brinjal seedlings is presented with a focus on
involvement of GSH and H2S signaling.

4. DESCRIPTION {Description shall start from next page.)
In the present work, we have presented the potential of formulation of additional S in mitigating/managing Cr(VI) stress in some vegetable crops i.e. tomato (Solarium lycopersicum L.), pea (Pisum sativum L.) and brinjal (Solarium melongena L.) by analyzing growth attributes, Cr accumulation, endogenous H2S accumulation, cell death, oxidative stress indices, glutathione biosynthesis and ascorbate-glutathione cycle. Further in this study, we have also kept our emphasis on involvement of GSH and H2S signalling in additional S-governed mitigation of Cr(VI) stress in vegetable crops. In the present work, we have chosen tomato, brinjal, and pea as they are commonly consumed in India, and are rich sources of proteins, minerals, vitamins, fibres, flavonoids, anthocyanins, antioxidants, and other nutrients.
2.1. Plant materials and growth conditions
Seeds of Solarium lycopersicum L. var. Damini-131 (tomato), Pisum sativum L. var. VS-10 (pea) and Solarium melongena L. var. Indam Supriya (brinjal) were purchased from certified supplier of Baikunthpur, Chhattisgarh, India. Healthy seeds were surface sterilized with 2% (v/v) sodium hypochlorite solution for 15 min followed by repeated washing with distilled water. Thereafter, seeds were soaked in distilled water for 1 h. Thereafter, seeds were wrapped in sterilized cotton cloth and kept overnight for germination in the dark at 26 ± 1 °C and then sprouting seeds were sown in sand in plastic trays. Trays were placed in a plant growth chamber (Impact model IIC 129D, New Delhi) under photosynthetically active radiation (PAR) of 200 umol photons m"2 s"1 with 12:12 h day-night regime and 65-70% relative humidity at 26 ± 1 °C. Seedlings were allowed to grow for 30 days in order to develop secondary leaves. Seedlings were given half strength Hoagland nutrient medium whenever required. After 30 days of growth, seedlings were harvested for experimental set up. Seedlings having secondary leaves (30 days old) were gently up rooted, and roots were washed with tap water. Thereafter, uniform sized seedlings were acclimatized in half strength Hoagland nutrient medium for 24 h. After this, prepared formulation [additional sulfur as potassium sulfate 1.5 mM (48 mg/L)+ half strength Hoagland nutrient solution] was applied to mitigate/manage (Cr(VI) (25 uM, as a potassium dichromate) toxicity in the selected Vegetables. Used concentration of Cr(VI) is environmentally relevant and significantly declined growth (fresh weight) of tomato, pea and brinjal and thus selected for this study. In the case of S, as per dose response curve, 1.5 mM of S maximally alleviated Cr(VI) toxicity and thus selected for this study. Treatments of Cr(VI) and S were given in plastic pots having 40 ml of half strength Hoagland nutrient medium in each. Each plastic pot contained five uniform sized seedlings.
Furthermore, to test whether glutathione^ (GSH) and hydrogen sulfide (H2S) are involved in S-
_ — -. — ■ ?_s T ~ f- - I 2 - 2 G- 2. I. I 7 - i s rleai'ated MMgaticjn-of (Sr(^I) toxicity iff tomato, pea and brinjal seedlings, we have used various inhibitors,

;. donor and scavenger. For instance, we used 50 uM of hydroxylamine (HA, an inhibitor of cysteine - - desulfhydrase activity) and 500 uM of L-buthionine sulfoximine (BSO, GSH biosynthesis inhibitor). Besides
this, we have also used 100 uM of GSH (reduced glutathione), 50 uM of sodium hydrosulfide (NaHS, a , donor of H2S) and 100 uM of hypotaurine (HT, a scavenger of H2S). Thus, following combinations were
made: control (only half strength Hoagland nutrient medium), Cr(VI), Cr(VI)+S, Cr(VI)+S+BSO, 1. Cr(VI)+S+BSO+GSH, Cr(VI)+S+HA, Cr(VI)+S+HA+NaHS and Cr(VI)+S+HA+NaHS+HT. All reagents
like Cr(VI), S, BSO, GSH, HA, NaHS and HT were prepared in half strength Hoagland nutrient medium.
The Cr(VI), S, BSO, GSH, HA, NaHS and HT treated seedlings were placed in a growth chamber for further
growth for 7 days. After this, seedlings were harvested and various morphological, physiological and
biochemical attributes were determined.
2.2. Determination of growth and chromium content
Morphological attribute like growth was determined in terms of fresh weight and length of root and shoots. For this, treated and untreated seedlings were harvested and their fresh weight and length were determined using digital balance and centimeter scale, respectively. Chromium content was determined in acid digested solution of roots by using atomic absorption spectrometer.
2.3. Detection of Cr(VI) and cell death in root tips
Fluorescent histochemical detection of Cr(VI) in roots was carried out using 1,5-diphenylcarbazide as per the procedure described in Singh and Prasad (2019). Detection of cell death in treated and untreated roots was carried out by the method of Cruz-Ramirez et al. (2004).
2.4. Determination of endogenous H2S and cysteine contents, and DES activity
For determination of L-cysteine desulfhydrase activity (DES) activity, and contents H2S and cysteine in leaf and root of tomato, pea and brinjal seedlings, the protocols of Bloem et al. (2004), Nashef et al. (1977) and Gaitonde (1967), respectively were adopted. For detection of endogenous H2S in root tips, a H2S specific fluorescent probe 7-azido-4-methylcoumarin (AzMC) was used following the method as described byLiuetal.(2017).
2.5. Determination of total chlorophyll and photosynthetic quantum yield
Total chlorophyll (chlorophyll a and b) was estimated according to the method of Lichtenthaler (1987). Briefly, fresh leaves (20 mg) from treated and untreated seedlings were extracted in 80% acetone, and centrifuged at 5,000 g for 5 min under cool condition. The absorbance of supernatant was read at 663.2 and 646.5 nm.

r - The photosynthetic quantum yield (qP=Fv/Fm) was recorded in the dark adapted leaves of tomato, pea ' 'and brinjal grown under various combinations, using hand held FluorPen FP 100, Photon Systems Instruments, Czech Republic.
2.6. Determination of indices of oxidative stress
Qualitative detection of superoxide radical (O2') and hydrogen peroxide (H2O2) in roots was carried out by using their fluorescent probes as per the method of Sandalio et al. (2008) with some modifications, i The O2'"" was quantified by estimating production of nitrite from hydroxylamine in the presence of (V-I (Elstner and Heupel, 1976). The production of H2O2 was quantified according to the method of Velikova et al. (2000). The lipid peroxidation as malondialdehyde (MDA) content was estimated according to the method of Hodges et al. (1999). Protein oxidation (oxidative damage to proteins) was measured by quantifying amount of reactive carbonyl groups (RCG) (Levine et al., 1994). Membrane stability index was measured according to the method of Sairam et al. (2002).
2.7. Determination of antioxidant activity
Superoxide dismutase (SOD; EC 1.15.1.1) activity was measured as inhibition in nitroblue tetrazolium (NBT) reduction method described by Giannopolitis and Ries (1977). Catalase (CAT; EC 1.11.1.6) activity was determined in terms of decrease in absorbance due to decomposition of H2O2 which ) was recorded at 240 nm using an extinction coefficient of 39.4 mM"1 cm-1 (Aebi 1984). Glutathione-5'-transferase (GST; EC 2.5.1.18) activity was assayed according to the method of Habig et al. (1974) using 1-chloro - 2, 4 - dinitrobenzene (CDNB) as a substrate. Protein iri each sample was quantified as per the method of Bradford (1976).
2.8. Determination of components of ascorbate-glutathione cycle
Activities of ascorbate peroxidase (APX, EC 1.11.1.11) and dehydroascorbate reductase (DHAR, EC 1.8.5.1) were measured by monitoring decrease in absorbance at 290 nm and increase in absorbance at 265 nm, respectively (Nakano and Asada, 1981). Glutathione reductase (GR, EC 1.6.4.2) was determined by monitoring decrease in absorbance at 340 nm due to the oxidation of NADPH (Schaedle and Bassham, 1977). Activity of monodehydroascorbate reductase (MDHAR, EC 1.6.5.4) was measured by monitoring decrease in absorbance at 340 nm (Hossain et al., 1984). Amounts of reduced ascorbate and dehydroascorbate, and reduced glutathione and oxidized glutathione in seedlings were quantified by the methods of Gossett etal. (1994) and Brehe and Burch (1976), respectively.
2.9. Determination of glutathione biosynthetic enzymes
. Enzymes of glutathione biosynthetic pathway i.e. cysteine synthase (CS; EC 2.5.1.47), y-glutamylcysteine synthetase (y-ECS^ JEC 6.3.2.2)? and glutathione synthetase (GS; EC 6.3.2.3) were

'determined in seedlings according to the methods of Saito et al. (1994), Seelig and Meister (1984) and • Huang et al. (1995), respectively.

5. CLAIMS (not applicable for provisional specification. Claims should start with the preamble — 'Ywe claim" on separate page)
We claim"
1. A method for reducing hexavalent chromium toxicity [Cr(VI)] in vegetable crops (tomato, pea and brinjal) by using formulation of additional sulfur.
2. A method in which prepared formulation [additional sulfur as potassium sulfate 1.5 mM (48 mg/L)+ half strength Hoagland nutrient solution] was applied to mitigate/manage (Cr(VI) (25 uM, as a potassium dichromate) toxicity in the selected vegetables.
3. A method that showed that in prepared formulation-mediated mitigation/management of Cr(VI) toxicity in studied vegetable, endogenous hydrogen sulfide and reduced glutathione have crucial roles. Further, in accomplishing mitigation of Cr(VI) toxicity by prepared formulation in studied vegetables hydrogen sulfide signaling preceded glutathione biosynthesis.
4. A method for significantly reducing accumulation of chromium in studied vegetables by using formulation of additional sulfur.
5. A method for increasing growth and photosynthesis in vegetable crops under Cr(VI) stress upon application of formulation of additional sulfur.
6. A method for significantly reducing cell death in root tips in studied vegetables under Cr(VI) stress by using formulation of additional sulfur.
7. A method for significantly reducing accumulation of various oxidative stress markers in studied vegetables under Cr(VI) stress by using formulation of additional sulfur.
8. A method for enhancing activity of antioxidant enzyme like glutathione-5-transferase in studied vegetables under Cr(VI) stress by using formulation of additional sulfur.
9. A method for enhancing the activities of enzymes and metabolites (ascorbate and glutathione) of ascorbate-glutathione cycle in studied vegetables under Cr(VI) stress by using formulation of additional sulfur.
10. A method that suggests use of prepared formulation at large scale for the mitigation of Cr(VI) toxicity in vegetable crops at chromium contaminated sites.