Impact of temperature stress on secondary metabolite profile and phytotoxicity of Amaranthus cruentus L . leaf extracts

In this study Amaranthus cruentus plants were grown under controlled optimal conditions (28/21 °C) for three months and then subjected to cold (14/7 °C) and hot (33/40 °C) temperatures. We investigated the influence of these temperature regimes on the metabolite profile of the leaves through analyses of data by TLC, HPLC and GC-MS spectrometry. The phytotoxic potential of a methanol-water (MW) and dichloromethane (DCM) extract from the aerial parts were examined through in vitro screening of germination and growth of lettuce and pepper. The optimal extracts displayed the highest diversity of secondary metabolites, and the highest total phenolics and flavonoids content. Through TLC and HPLC analysis the significantly lower phenolic content in the hot temperature treated samples was confirmed. A wide range of metabolites were detected in the DCM extracts through GC-MS analyses. The phytotoxicity of both the MW and DCM extracts were demonstrated, as germination and growth of pepper and lettuce were significantly inhibited, indicating the presence of more than one allelochemical compound which may affect the allelopathic activity of A. cruentus during changes in environmental temperatures.


INTRODUCTION
Several studies documented on the increase of secondary compounds or changes in chemical profile within specimens of the same plant species growing under different or environmental stress conditions (Gobbo-Neto & Lopes, 2007;Ramakrishna & Ravishankar, 2011;Gouvea et al., 2012).The interaction between plants and their environment influence synthesis and accumulation of secondary metabolites and their roles as a response to the environment.(Rhoads et al., 2006).The exposure to various environmental stresses can strengthen the allelopathic potential of many plants (Einhellig, 1987(Einhellig, , 1996;;Gershenzon, 1984;Tang et al., 1995;Kobayashi, 2004) and can affect allelopathy in at least three ways: 1) the production of allelochemicals by the donor species, 2) their bioavailability and 3) modify the effect of an allelochemical on the target plant (Einhellig, 1996, Trezzi et al., 2016).
Amaranth is one of the few multi-purpose crops which can supply grain as well as tasty leafy vegetables of high nutritional quality (Mensah et al., 2008;Maiyo et al., 2010;Nana et al., 2012;Alemayehu et al., 2015).
This study evaluated the influence of temperature on plant secondary metabolite production of A. cruentus L. and whether the chemical-mediated interaction is involved in A. cruentus allelopathy.Thus, phytotoxcitiy of A. cruentus was evaluated with extracts of the different temperature treatments.

Plant material
Amaranthus cruentus 'Anna' seeds were planted in pots containing a soil-compost (

Crude extracts
Methanol-water (70 : 30 v/v) and dichloromethane (DCM) were used as solvents.Ten grams of the powdered A. cruentus leaf material (oven dried at 40 °C) was extracted twice by shaking overnight in the different solvents (1 : 20 w/v).The pooled extracts were dried and kept at 4 °C until further analyses.

Allelopathy determination
A combination of the 'sandwich method' of Fujii et al. (2003) and Hill et al. (2007) was used to determine the in vitro phytotoxicity of the crude leaf extracts from the different temperature treatments of A. cruentus on the vegetable seeds.For this method 5 and 20 mg of each extract was dissolved in 1 ml of their own solvent, and 1 ml pipetted onto a filter paper.The filter papers were allowed to dry then placed on the bottom layer of agar resulting in 0.5 or 2 mg ml -1 extract per well.Controls contain only the solvents on filter paper.
Lettuce (Lactuca sativa L.) and pepper (Capsicum annuum L.) seeds were surface sterilised as described by Allemann et al. ( 2017) and each of the experiments was done in triplicate and presented as the mean of the replicates.

Total phenolic and flavonoid content
Total phenolic content was evaluated in the methanolic extract, using the Folin-Ciocalteu method as reported by Singleton & Rossi (1965).The absorption was measured at 550 nm and the content in phenolics was expressed as mg galllic acid equivalents (GAE) of dry mass extract.Total flavonoid content was determined as reported by Zhishen et al. (1999).The absorption was measured at 510 nm and the content in flavonoids was expressed as mg quercetin equivalents (QE) of dry mass extract.

Thin Layer Chromatography
Thin layer chromatography (TLC) was carried out using silica gel 60 F 450 -aluminium backed pre-coated plates.Extracts (50 mg ml -1 ) were dissolved in their appropriate extraction solvents and 10 μl applied to the TLC.The mobile phase for development of the MW extracts was chloroform-methanol-water-acetic acid (65:35:5:1), while for the DCM extracts, plates were developed in toluene-ethyl acetate (93:7).Compounds resolved on the plate were visualized using ultraviolet light (UV) at 365 nm and 254 nm, ninhydrin (Pifrung, 2006), p-anisaldehyde-sulphuric/acetic acid, 5 % ferric chloride and dragendorf reagents, prepared according to the standard methods described by Wagner & Bladt (1996).

High Pressure Liquid Chromatography
The MW extracts (20 mg ml -1 ) were separated and identified through high pressure liquid chromatography (HPLC) by comparing the retention times to standard phenolic compounds.Standards were prepared in methanol (3 mg ml -1 ) and absorption measured between 200 and 400 nm.Ten micro litre of extracts and 2 µl of standards were injected while the flow rate was kept at 1 ml min -1 .A Shimadzu instrument with a Photo Diode Array Detector (PDA) and an elution procedure as described by Vidović et al. (2015) with a C18 column (Phenomenex C18, 250mm × 4.6mm, 5µm diameter), was used to achieve acceptable separation of all compounds.The mobile phase consisted of: A, acetonitrile and B, a mixture of acetic acid-acetonitrilephosphoric acid-water (10:5:0.1:84:9,by vol.).

GC-MS analysis
The DCM extracts (10 mg) were dissolved in 1 ml hexane.Analyses was done through GC-MS using a Shimadzu GC-MS QP-2010 gas chromatography equipped with a DB-5 MS column (30 m length × 0.32 mm diameter × 0.25 µm film thickness) and injecting 1 µl of sample.The GC operating conditions were the following: 5 min at 60 °C, then gradually increased to 280 °C at a rate of 2 °C min -1 , and held for 10 min.Helium was used as the carrier gas (1.5 ml min -1 flow rate).Spectra analysis was conducted using the library "National Institute of Standard and Technology (NIST) version 5.0.

Statistical analysis
The experiments were carried out adopting a completely randomized design with three replications.The results were expressed as means with least significant difference (LSD).Analysis of variance (ANOVA) was performed using SAS 9.3 (Institute Inc., Cary, NC, USA, 2008) statistical programme for data and Tukey-Kramer's LSD procedure for comparison of means.Significance of differences compared to the control groups was determined using the t-test (Steel & Torrie, 1980).

Metabolites
Comparison of the compounds in the MW and DCM leaf extracts of the different temperature treatments of A. cruentus plants, are illustrated by TLC in Figure 1.It is clear that temperature played an obvious role in the production of secondary compounds, as clear differences in compounds between the treatments were visible in both the polar and non-polar extracts (Fig. 1A  & B).Different compounds with varying R f values were visible when spraying the TLC's with p-anisaldehydesulphuric/acetic acid reagent.Colours of compounds range from green, yellow, pink, blue and purple with different R f values for the polar and non-polar extracts.
The diverse coloured compounds with varying R f values visible in both polar and non-polar extracts on TLC (Fig. 1) may indicate many different compounds, including terpenes, saponins, sugars and flavonoids amongst others (Wagner & Bladt 1996).
In the optimal treatment of the MW extract, 11 compounds were noted, compared to 8 and 5 in the cold and hot treated samples respectively.Prominent spots, including a dark purple (R f = 0.055), a blue-purple (R f = 0.49) and a light blue spot (R f = 0.6) were only present in the optimal extract (Fig. 1A).From these results one can deduct that the stress temperatures, particularly the hot, inhibited the biosynthesis of some of the more polar compounds.
Differences were also visible in the non-polar samples (Fig. 2B), with a noticeable blue coloured compound visible at R f = 0.83, solely in the hot treatment DCM extract (Fig 1B).Less green pigment, probably chlorophyll was also observed in the hot treatment extract, indicating the effect the hot treatment had on photosynthesis.
Several studies were conducted on the impact of increased temperatures on secondary metabolite production of plants (Morrison & Lawlor, 1999).Phenolic compounds are important and common plant allelochemicals in the ecosystem and the main phenolic compounds are water soluble (Li et al., 2010).Kraujalis et al. (2013) reported on the antioxidant properties and phytochemical composition of amaranth extracts isolated by acetone and methanol-water from plant leaves, flowers, stems and seeds.They found that the methanol-water extract of the leaves possessed the highest antioxidant activities and various phenolic compounds and flavonoids e.g.rutin, nicotiflorin, isoquercitrin, 4-hydroxybenzoic and p-coumaric acids were identified as major constituents.In the review article by Mroczek (2015) it is reported that saponins were isolated from a diversity of Amaranthaceae genera and species.In this study, the total phenolic and flavonoid content significantly declined in A. cruentus plants exposed to hot temperatures compared to plants grown at the optimal temperature (Table 1).The decrease in total phenolic and flavonoid content are in contrast with findings of many authors who reported an increase in production of phytotoxic phenolic compounds in plant tissues exposed to high temperatures and solar radiation (Koeppe et al., 1969;Wender, 1970;Einhelig & Eckrich, 1984).Rudikovskaya et al. (2008), however reported that low growth temperature decreased the content of some phenolic compounds in pea seedling roots and according to Król et al. ( 2014), long-term drought stress caused a decrease in particular components of secondary metabolism in the leaves and roots of grapevine.It seems therefore that one cannot expect generalized patterns of phenolic compounds in stress situations.Flavonoids are not usually seen as allelopathic compounds but they have other roles in plants such as attractants to pollinators, protection against ultraviolet light (Li et al., 1993) and as an anti-inflammatory, antiallergic and anti-viral activities (Miller, 1996).Some flavonoids do however have allelopathic properties such as quercetin (Inderjit & Dakshini, 1995), catechin (Bais & Kaushik, 2010;Chobot et al., 2009) and rutin (Basile et al., 2000), which has been found in both A. hybridus and A. cruentus.From our HPLC results, catechin and rutin were identified in the optimal and cold treated amaranth The influence of temperature on the expressed compounds in the different DCM extracts were clearly visible after analyses through gas chromatography and mass spectrometry (GC-MS).Major compounds made up a total composition of 75.69 % (9 compounds), 90.44 % (7 compounds) and 91.89 % (9 compounds), of the optimal, cold and heat treated samples respectively (Table 2).Neophytadiene and hexadecanoic acid were the only compounds present in all three extracts, although the concentrations of these compounds varied substantially between the treatments (Table 2).The highest concentration of neophytadiene (27.53 %) was found in the cold treated sample, while hexadecanoic acid (13.52 %) was maximum in the heat treatment extract.Squalene, trans-phytol and the phytosterol, stigmasta-7,22-dien-3-ol were present in only cold and heat treated samples.Gamel et al. (2007) found high squalene concentrations in oil fractions of A. caudatus L. and A. cruentus, while Shah (2005) reported on the presence of stigmasta-7,22dien-3-ol (α-spinasterol) in A. spinosus L.. According to Szakiel et al. (2010), lower soil temperatures triggered an increase in levels of steroidal furostanol and spirostanol saponins.Area (%) of compound = height of peak x width of peak at ½height ≤ x Total area -1 * Identification by Library: NIST 05.LIB

Phytotoxcitiy
Germination Phytotoxic activity of A. cruentus extracts may be ascribed to a wide range of biologically active phytochemicals such as phenolic acids, flavonoids and fatty acids which are known for their phytotoxic and allelochemical activities.When these compounds are released into the soil by leaf litter decomposition there may be a change in both the physical and chemical properties and therefore affecting the organization and growth of plant communities.At different concentrations both the MW and DCM extracts of the different temperature treatments, significantly inhibited germination of both lettuce and pepper (LSD (T≤0.05)= 1.88) (Table 3).The polar MW extract was more effective in lowering germination percentages in both pepper and lettuce than the non-polar DCM extract (Table 3).

Growth
Both the hypocotyl and seminal root were significantly inhibited when exposed to extracts of all the temperature treatments, however the cold stress treatment was the most detrimental (Table 4 & 5).The results also confirmed that root elongation was more sensitive to allelochemicals than stem (hypocotyl) elongation in both species.Furthermore, both polar and non-polar extracts, at 0.5 and 2 mg ml -1 , significantly reduced the growth of lettuce (Table 4) and pepper (Table 5).Allelopathy influences plant succession through root exudation, leaching and volatilization when the plant dies and starts to decompose (Rice 1984;Weston 2005;Minorsky 2002;Bertin et al. 2003).The most frequently reported morphological effects from allelochemicals on sensitive plants is the inhibition or retarded seed germination and retarded development of shoots and roots (Ghafarbi et al. 2012).It has been cited in literature that allelopathy was involved in many natural and manipulated ecosystems and that they play a role in the evolution of different plant communities (Ding et al., 2007).Abiotic stresses can lead to morphological, physiological, biochemical and molecular changes within the plants, and therefore has an impact on plant growth (Wang et al., 2003).Climate change and temperature play a role in the synthesis of allelochemicals, which can affect the growth processes of neighbouring plants (Li et al., 2010).Results by Amini (2009Amini ( , 2012)), proved that root exudates of A. retroflexus had inhibitory effects on shoot length of both crop (wheat) and vegetable (common bean) plants.Aqueous extracts from the leaves, roots and stems of A. retroflexus L. had inhibitory effects on the hypocotyl growth of maize (Konstantinović et al., 2014).Dhole et al. (2013) noticed that seed germination and seedling growth of maize were inhibited when using aqueous extracts from the root, stems and leaves of A. tricolor L.. Phenolic compounds take part in the regulation of seed germination and work together in regulating the growth of plants.The role of several phenolic compounds e.g.lignin, salicylic acid, flavonoids and phytoalexins play important roles in plant resistance, taking part in defence responses during biotic and abiotic stress (Kulbat, 2016).It is therefore possible that the reduction in phenolic compounds caused by temperature stress, could play a role in the germination and growth of lettuce and pepper seeds.Furthermore, it is clear from both literature and our results, that concentration plays a major role in the severity of allelopathic effects on different plants (Qasem, 1995;Obaid & Qasem, 2005).3.87 Different letters along the column indicate significant differences at T ≤ 0.05 (Tukey test).Significant differences within each vegetable; n = 13.
Many studies have been done on allelopathy of the polar extracts of amaranth, but no information is available on the non-polar compounds.This is the first report on the in vitro phytotoxicity of a DCM extract of grain amaranth.The characteristics of allelochemicals are important and play a significant role in their fate in the environment.For example, the mobility of compounds within the soil are influenced by their water solubility; the vapour pressure can impact their volatilization and their chemical structure can affect their affinity with the soil surface (Souza Filho & Alves 2002).The outcome of all these complex interactions results in compounds with allelopathic properties which can be of potential agronomic use.

CONCLUSION
Temperature influenced the chemical composition of A. cruentus and in vitro bioassays proved the negative impact of the extracts on germination and growth of vegetables.This demonstrated that the environment for the cultivation of A. cruentus is important and that more than one compound were responsible for allelopathy, thus both polar and non-polar compounds were involved.Furthermore, with increased concentrations of extracts a decrease in germination and seedling development occurred.Consequently, if more plant residues are left behind in the soil, the growth of the next crop will often be affected with a subsequent decline in yield.It was also clear that vegetables displayed diversity in reaction towards the temperature treatments and type of extract.This information proves that a holistic understanding of the influence of abiotic environmental factors on the production of metabolites in various plant parts are of importance.The release of potentially phytotoxic compounds from A. cruentus leaf litter into soil deserves further investigation as well as the purification of extracts to determine unidentified natural compounds with herbicidal activity.

Figure 1 :
Figure 1: Qualitative TLC profiles of the optimal, cold and hot treated A. cruentus MW (A) and DCM (B) leaf extracts.Detection by p-anisaldehyde reagent

Table 1 :
Total phenolic and flavonoid compounds in temperature stressed amaranth leaf Analyses by HPLC confirmed the decrease in phenolic compounds in the temperature stressed plants (Fig 2).

Figure 2 :
Figure 2: Comparison of HPLC-PDA chromatograms of optimal (A), cold (B) and hot (C) temperature treated A. cruentus methanol-water leaf extracts.1 = Catechin; 2 = Rutin; 3 = Quercetin MW leaf litter extracts (Fig 2A & B), while a small amount of quercetin was detected in only the cold treated sample (Fig 2B).The heat treated sample contained a reduced amount of unidentified compounds (Fig 2C), indicating the role temperature play on the biosynthesis of flavonoids and the possible consequence on allelopathy.

Table 2 :
GC-MS results of compounds present in optimal, cold and hot temperature treated DCM leaf extracts of A. cruentus

Table 3 :
Germination percentage of pepper and lettuce seeds exposed to increasing concentrations of MeOH-H 2 O

Table 4 :
Hypocotyl and seminal root lengths of lettuce seeds exposed to increasing concentrations of MeOH-H 2 O and DCM leaf extracts of A. cruentus grown at optimal, cold and hot temperatures