Effect of heat use efficiency on bioproduction of drought resistance eliciting phytochemicals in groundnut (Arachishypogaea L.) genotypeGKVK-5

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K Raagavalli ¹ , K Pradeepa¹ , T Msoumya² , T D karthik³ , B U Sourabh Giri³ , S Ravi Kumar³ , V Krishna³

¹Department of PG Studies and Research in Biotechnology, Sahyadri Science College, Kuvempu University, Shivamogga-577203, Karnataka, India.

²Department of Agronomy, Keladi Shivappa Nayaka University of Agricultural and Horticultural Sciences, Shivamogga-577204, Karnataka, India.

³Department of PG Studies and Research in Biotechnology, Jnana Sahyadri, Kuvempu University, Shankaraghatta-577451, Karnataka, India.

Corresponding Author Email: krishnabiotech2003@gmail.com

DOI : https://doi.org/10.51470/AGRI.2024.3.2.12

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INTRODUCTION

Heat stress caused by increased temperature is a major problem in world agriculture. Plants experience a range of morpho-anatomical, physiological, and biochemical changes in response to brief or persistently high temperatures. These changes can have a significant effect on the growth of plants. and development and potentially result in a sharp decline in economic yield. Genetic factors, crop type, and the crop’s capacity for the biosynthesis of secondary metabolites during dry seasons and drought all affect how well heat is utilized in terms of dry matter accretion. The world’s most significant oilseed and cash crop, groundnut (Arachishypogaea L.), is grown in semi-arid regions. However, fluctuations in the agroclimatic conditions and the chemical composition of biological components directly impact the crop’s growth and output.In Central Punjab, India, the studies of [1] have documented the impact of sowing dates on the groundnut cultivar’s thermal use and heat use efficiency.

The thermal requirements and heat consumption efficiency under various sowing circumstances and row orientations were also reported in pea cultivars [2].. A delay in crop sowing significantly reduced the length of phenological stages and the accumulation of agro-climatic indices (GDD, PTU, and HTU).The researchers noticed that the plants sustained in drought due to biosynthesis of the key secondary metabolite molecules and plant metabolic responses to drought stress have been comprehensively profiled by metabolic profile analysis [3]. Thus, utilizing the environmental parameters of growing degree days (GDD), heliothermal units (HTU), and photothermal units (PTU), an attempt was made to assess the growth and yield of groundnut genotypes GKVK-5 and TMV-2 in the current study.

Many secondary metabolites are essential for plants to adapt and increase their chances of survival during drought and other environmental stresses.Hence analysis of metabolic profiles of the crop plant and their role in drought tolerance has sought attention towards the improvement of crop plants.Hence, in the present study, HR-LCMS analysis was carried out to identify phytochemicals of A.hypogaeathatplay a significant role in drought tolerance and oxidative stress.

MATERIAL AND METHODS

Heat use efficiency of groundnut genotypes:

A field study was carried out under rainfed conditions during the Kharifseasonon sandy loam soils of AHRS, Bavikere, UAHS, Shivamogga (13º 42’ latitude and 75 º 51’ longitude and 695 m above MSL). The experiment was laid out in a whole block design that is randomized with a factorial concept[4].. Testing was done on the groundnut genotypes, GKVK-5 and TMV-2, as well as the four sowing windows, which are the second and first weeks of June, July, and August.For each genotype experiments were conducted with three replications.  Seeds were sown at a depth of 5 cm with 30 x 15 cm spacing. The recommended dose of fertilizers (25: 50: 25 kg N: P₂O₅: K₂O) was applied as a basal dose (50 % N). The remaining 50 % of N was applied during the time of earthing up (30 DAS). The weather parameters were obtained from AHRS, Bavikere. The HUE (Heat use efficiency) and HTU (Helio-thermal units) were calculated from GDD obtained using the weather parameters.

HTU is the product of accumulated heat units and corresponding actual sunshine hours, which was calculated using the formula,

HTU = ∑GDD x Actual sunshine hours (0 to 90 DAS)       

HTUE is the ratio of yield and HTU,

HUE is the ratio of total dry matter to the accumulated GDD,

Based on the result of HUE and HTU experiments the drought-resistant genotype was subjected to qualitative and quantitative phytochemical screening.

Preparation of Extract:

The fresh and healthy leaves of the ground nut genotypes GKVK-5 were collected from the experimental plots of AHRS, Bavikere, UAHS, and Shivamogga. About 30 g of powdered leaves of each genotype were subjected to sequential Soxhlet extraction using the solvents, petroleum ether, chloroform, and methanol. The extracts were sieved through Whatman paper no.1 and then concentrated using a rotary flash evaporator.

Qualitative phytochemical screening:

The dried residue of petroleum ether, ethyl acetate, and methanol extracts were resuspended in Mili-Q water and qualitatively examined for various secondary metabolites viz., alkaloids, flavonoids, terpenoids, glycosides, saponins, phenols using standard tests [5].

Quantitative estimation of phytochemicals:

Estimation of total flavonoid content in sequential extracts of leaves of ground nut was determined [6]. The amounts of total phenolic contents of extracts were determined by the spectrophotometric method[7]. Estimation of total alkaloids was performed by gravimetricmethod [8] and total terpenoids in the extracts was also  determined by spectrophotometric method [9]. 

HR-LCMS analysis of leaves methanol extract:

A High-resolution liquid chromatograph mass spectrometer (HR-LCMS) G6550A (Agilent technologies) instrument was used to examine the bioactive components of groundnut genotypes of leaves methanol extract. The method used for chromatography was 30 mins ± ESI 10032014_MSMS.m. The temperature of the gas utilized for analysis was 250 °C. Identification was done using the protonated compound’s theoretical mass. The compounds expressed with more than 10 Hits were considered for comparative evaluation. The lead compounds expressed repeatedly were used for spectral studies and characterization. HR-LC-MS analysis was performed at the Sophisticated Analytical Instrument Facility (SAIF), Indian Institute of Technology, Mumbai, India. The compounds were identified through a comparison of their mass and retention time (RT) with the stored Metlin Library maintained at IIT Bombay. The ESI’s positive and negative ionization modes were used for the MS analysis. Gas temperature of 250 °C, drying gas flow of 13 L/minute, nebulizer pressure of 35 psi, and capillary voltage of 3,500 V were the parameters of the MS source that were employed. Acquisition of Q-TOF data and mass spectrometric analysis were done using Agilent Mass Hunter software.

RESULTS AND DISCUSSION

Influence of different sowing windows on GDD of groundnut crop:

Weather data obtained from the Agricultural and Horticultural Research Station, Bavikere, Karnataka, India (Fig. S1) showed minimal variation in actual maximum and minimum temperatures and relative humidity from normal values, while rainfall and sunshine hours varied significantly. The computed weather parameter, GDD, was derived from these temperatures and varied accordingly (Table 1). Groundnut sown in the second fortnight of June recorded higher GDD (1721.95 ºC day-1) at harvest, decreasing with delayed sowing.

Groundnut dry matter depends on environmental factors like solar radiation, temperature, relative humidity, and moisture availability. The early sown crop (second fortnight of June) received more sunshine, rainfall, and longer day-length conditions, allowing it to intercept more radiation, utilize more moisture, and produce more photosynthates. Similar results, was also noticed by the previous investigator [10] where  delayed sowing extended the time required for 50% of the population to initiate flowering.

DAS: Days After Sowing

An increase in the total dry matter accumulation significantly increased the heat use efficiency (HUE) between the genotypes at 60 DAS and 90 DAS in GKVK-5 (Table 2). Similarly, an increase in the efficiency adds up to the increased photosynthates accumulation and further increases the pod yield. Thus, the genotype GKVK-5 produced a higher pod yield (16.73q ha-1). The decrease in the HUE in TMV-2 might be due to the decrease in total dry matter content which further decreased the pod yield. The increase in the total dry matter content increased the haulm yield in GKVK-5 (26.99 q ha-1), while it decreased in TMV-2 (15.07 q ha-1). This might be due to the stay-green nature of the varieties GKVK-5[11 and 12].. The increase in yield and total dry matter was noticed in the genotype GKVK-5 due to increased Helio-thermal use efficiency (HTUE) indicating the drought-resistant characteristics GKVK-5 to genotypes TMV-2, when sown during the second fortnight of June compared to delay sowing.

The crop sown during the second fortnight of June had taken more days for initiation of flower in the 50 percent of the population,where there was initially a better accumulation of the dry matter and could thus produce a higher pod yield of 15.2 q ha-1 (Table 2). An increase in the HUE was observed during 60 DAS and 90 DAS due to a drastic increase in the total dry matter content and GDD during the period. An increase in total dry matter provides a pre-requisite to increase the yield in any crop, as it indicates the maximum translocation of photosynthates to the sink. Based on the results of the HTUE experiment, the genotype GKVK-5 has been subjected to phytochemical investigation for the identification of metabolites that act as elicitors for acquiring drought-resistant characteristics.

Qualitative and quantitative phytochemical analysis:

Preliminary qualitative phytochemical screening of the crude extracts (LPE-Leaves Petether Extract, LEE-Leaves Ethyl  AcetateExtract, LME-Leaves Methanol Extract,) showed that alkaloids, terpenoids, and saponins were present in LPE. The LEE showed a positive test for alkaloids, Flavonoids, phenolics, terpenoids, and glycosides, whereas, tannins and saponins are absent. In LME except terpenoids all the phytochemical groups were present. In the quantitative determination of compounds,concentration of total alkaloids in LPE, LEE, and LME was estimated as 12.32, 36.24, and 32.44 μg/mg respectively. In LPE flavonoids and phenolics were absent. The concentration of total flavonoids in LEE and LME was38.61 and 28.56 μg/mg respectively similarly the total phenolics were estimated as 24.02 μg/mg in LEE and 47.32 μg/mg in LME. In the quantitative estimation of compounds, the LME showed higher concentrations of total alkaloids, and phenolic compounds therefore, the LME was subjected to HRLCMS analysis for the identification of compounds.

HR-LCMS analysis of leaves methanol extract of groundnut genotype GKVK-5:

Based on the results of the HTUE experiment, the genotype GKVK-5 has been subjected to phytochemical investigation for the identification of metabolites that act as elicitors for acquiring drought-resistant characteristics. Secondary metabolites play an important role in the drought stress tolerance of crop plants.According to the previous investigator [12], under field conditions, plants are subjected to a variety of abiotic stressors. Drought stress, in particular, has an adverse effect on crop output and poses a threat to world food security. Drought stress activates downstream processes, including signaling pathways and phytohormone homeostasis.This in turn triggers the synthesis of many protective secondary metabolites. Multiple stress tolerance, including resistance to biotic and abiotic stressors, is provided by these secondary metabolites.

The HRLCMS of leaves methanol extract of groundnut genotype GKVK-5 showed expression of drought resistance eliciting compounds in both cation and anion mode spectra as shown in (Table 3).The greatest DB hits, longer retention times, and m/z values are expressed for these compounds. (Fig 1).Displays the mass, structure, and MS spectra of the cation compounds.

6-Deoxyfagomine is a polyhydroxylatedpiperidine alkaloid that occurs naturally and is an iminosugar, or pseudo-sugar with an N atom, with intriguing biological characteristics. The compound isolated from Morusbompycis, exhibits strong antihyperglycemic impact on streptozotocin-induced diabetic mice and also increased the  glucose-induced insulin secretion [13].  Maltoxazine, sometimes referred to as daechualkaloid A, is a member of the pyrrolidine class of chemical compounds. Among the HRLCMS-based identified compounds, L-2-Amino-3-methylenehexanoic acid (AMHA) was expressed with repeated folds (Table 3) indicating its synthesis in the groundnut genotype, during drought stress conditions.  AMHA, an endogenous (2S, 3S)-α-amino acid found in nature (Fig. 1B), displays strong anti-extreme temperature stress activity in a variety of plant species. It also induces plant resistance, making it extremely effective against bacterial, viral, and fungal infections.

AMHA, an endogenous (2S, 3S)-α-amino acid found in nature (Fig. 1B), displays strong anti-extreme temperature stress activity in a variety of plant species. Additionally, it induces plant resistance, making it extremely effective against bacterial, viral, and fungal infections. According to [14], AMHA pretreatment provided excellent protection against powdery mildew in wheat, pseudomonas syringae DC3000 in Arabidopsis, and tomato spotted wilt virus in tobacco. With its exceptional potential, AMHA could develop into a special natural elicitor that shields plants from both biotic and abiotic stressors. 2-Amino-3-Methylhexanoic acid is a naturally occurring plant inducer that shields physiological function from the damaging effects of high temperatures on Camellia sinensis [15]. As a result, there is a lot of potential for AMHA to be developed as a naturally occurring commercial plant drought-resistance inducer. Plant cells biosynthesize a significant amount of secondary metabolites from primary metabolites.Plants produce secondary metabolites as a means of adaptation to deal with a variety of biotic and abiotic stressors [16].

In the present study also anion mode of HRLCMS resulted the presence offlavonoid glycosides- Astragalin 7-rhamnoside (Fig.2B), and phenolic compounds,rutin (Fig 2C) and chicoric acid (Fig 2D). These compounds were expressed with the highest DB hits, more retention time, and m/z value (Table 3). A marked accumulation of phenolic compounds chicoric acid chlorogenic acid, and flavonoid compounds. Quercetin-3-O-glucoside and luteolin-7-O-glucoside were also noticdin Lettuce during environmental stresses [17].In the previous study we also noticed that groundnut genotype GKVK-5 showed increased thermal utilization efficiency when it was cultivated in the delayed monsoon season. Drought stress is caused by high transpiration rates combined with low water availability in plants, which lowers their turgor pressure and water potential [18].

Consequently, alteration occurs in the bioproduction of secondary metabolites.The cation mode accumulation of flavonoid compounds, such as astragalin 7-rhamnoside and phenolic compounds, and non-protein amino acid, 2-Amino-3-methylhexanoic acid. In the groundnut genotype GKVK-5, chicoric acid and saponin compounds—Azukisaponin IV, Soyasaponin A3, and Pisumsaponin II—in an anion mode contributed to the development of drought resistance. Strong antioxidant Naringenin-7-O-glucoside down-regulated 3T3-L1 cell diameter and up-regulated intracellular lipid accumulation and adiponectin release.  A flavonoid-7-o-glycoside called astragalin 7-rhamnoside may function as a signaling or defense molecule in a variety of crop species.In many crop species, it plays a major role in acquiring resistance against abiotic and biotic stress [11].

The study concludes that the groundnut genotype GKVK-5 showed increased heat use efficiency and drought stress adaptable characteristics during the second fortnight of June due toits enhanced secondary metabolites production.

Conflict of interest:

The authors declare that there are no conflicts of interest associated with this publication.

Acknowledgment:

The authors are grateful to the Registrar, of Kuvempu University, India for providing the financial support. We are thankful to the Principal, Sahyadri Science College, Shivamogga; and the Dean,KeladiShivappanayaka University of Agricultural and Horticultural Sciences, Shivamogga for providing the facility to carry out this research work.

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