Mitigating Effects of Beta-Carotene on Hydroponic Growth and Productivity of Amaranthus Hybridus L. under Aluminium Toxicity Induced Stress

Aluminium (Al) toxicity is one of the major sources of environmental stress that limit plant growth and productivity in many acidic soils, especially in the tropics and sub-tropics. Al toxicity subsequently leads to food insecurity in developing countries, especially in sub-Saharan Africa. Though plants can synthesize their antioxidants; the need exists to investigate whether under Al toxicity-induced stress; beta-carotene (β-Carotene) supplementation could ameliorate the stress situation and enhance growth and productivity. A 3× 10 − 2 mM aluminium chloride (AlCl 3 ) at pH 4.6 was used to stress plants. β-Carotene was extracted from carrots and high-performance liquid chromatography (HPLC) was used to determine its peak absorbance at 295 nm. β-Carotene's activity was determined using the thiobarbituric acid method. The effects of 50 and 200 µM concentrations of β-Carotene on the hydroponic growth of Amaranthus hybridus, subjected to Al stress, were evaluated in this study. Pre- and post- β-Carotene treatments were applied to A. hybridus seedlings before and after Al treatment for 72 h. Results showed that post- β-Carotene treatments signicantly ameliorated plants from Al stress when compared to pre- β-Carotene treatments. Higher doses of β-Carotene signicantly increased leaf number, plant height, length and number of inorescence, fresh and dry weights of shoot, root and inorescence but signicantly decreased root length. The present study suggests that plants of A. hybridus were susceptible to Al toxicity-induced stress and post-β-Carotene supplementation could signicantly ameliorate the stress situation and enhance growth and productivity. However, intrinsic antioxidants could be adequate for plants not subjected to stress.


Introduction
Aluminium (Al) toxicity is an important growth-limiting factor for plants in acid soils below pH 5.0 and is responsible for shortages in food production (Blue and Dantzman, 1977 The general population is exposed to Al from its widespread use in water treatment, food additive, various Al-based pharmaceuticals, toothpaste, antiperspirants, pollutants from electrical power stations, industrial activities and automobile exhaust as well as from Al containers/packaging materials and cooking utensils (Harris et al. 1996 toxicity have focused only on Al being the major cause of stress limiting crop productivity but in reality, low pH sets the stage for Al to become toxic. Al toxicity not only depends on its total concentration but also its chemical forms that wholly depend on low pH (Kochian 1995 There is growing evidence from in vitro and in vivo laboratory animal studies that β-Carotene can protect phagocytic cells from auto-oxidative damage, enhance T and B lymphocyte proliferating responses, stimulate effector T-cell functions, and enhance macrophage, cytotoxic T-cell and natural killer cell tumoricidal capacities, as well as increase the production of certain interleukins (Bendich 1989;Mueller and Boehm 2011;Esrefoglu et al. 2016). Many of these effects have also been seen with carotenoids lacking provitamin A activity but having the antioxidant and singlet oxygen quenching capacities of β-Carotene, however, reports have it that β-Carotene could be converted into vitamin A, which is essential for normal growth and development (Lemmens et al. 2010; Giuliano et al. 2017)). Since vitamin A is a relatively poor antioxidant and cannot quench singlet oxygen, β-Carotene may have more importance as a nutrient than simply serving as a precursor of vitamin A (Bendich 1989). Carotenoids, including β-Carotene, are very e cient at quenching singlet oxygen (Di Mascio et al. 1989;Baltschun et al. 1997). Stahl and Sies (2003) have shown that β-Carotene undergo different con gurations (either cis or trans isomers) because of their double bonds. Growing observations have also showed that most carotenoids isomerize to their cis-con guration during thermal procession and extraction (Lemmens et al. 2010), storage conditions due to light acids and oxygen (Rao and Rao 2007), thus leading to loss of colour and reduction in biological activities. However, the all-trans isomer may exist predominantly in nature.
Though evidence for carotenoids roles' in animals and humans are ubiquitous, studies about the supplementation of plants with β-Carotene are scarce. However, lycopene had ameliorating effects on chromosome aberrations in Allium cepa (Aslanturk and Celik 2005); while a higher concentration of lycopene alleviated and bolstered plants of A. hybridus from the devastating effects of Al-induced stress (Udengwu and Egedigwe 2014). It is well known that animals cannot synthesize carotenoids because they lack chromoplasts and thus depend on plants for the nutritious and protective values of carotenoids.
However, Moran and Jarvik (2010) showed that some aphids manufacture toluene de novo.
A. hybridus, being a staple vegetable eaten in the tropics, is sensitive to low concentrations of Al and such low concentrations subjected the same plant to stress and reduced growth and productivity (Udengwu and Egedigwe 2014). In another study, Osaki et al. (1997) classi ed A. hybridus and some other tropical plants under the Al-sensitive group. Despite the intrinsic antioxidants inherent in plants, some plants, including staple crops, may not be capable of synthesizing high levels of antioxidants during stress situations and thus cause devastating damages to plant growth and yield. Excessive reduction in crop yield and productivity have consequently led to food insecurity in sub-Saharan regions. The current study, therefore, explores the role of supplementary β-Carotene in mitigating plants of A. hybridus exposed to Al toxicity via pre-and post-β-Carotene applications.

Soil Analysis
Topsoil for raising nursery plants was collected from the Botanic Garden, University of Nigeria Nsukka. β-Carotene extraction and puri cation Extraction and puri cation of β-Carotene were carried out following the methods of Udengwu and Egedigwe (2014). Fresh and matured carrots weighing 15kg were purchased from Nsukka local market. β-Carotene crystals were got following the methods of Yaping et al. (2002). Extracted β-Carotene was protected from light and stored at 2 o C to avoid transformation to inactive isomers.

Determination of antioxidant activity
Adapting the methods of Udengwu and Egedigwe (2014), thiobarbituric acid (TBA) value was used to determine the antioxidant activity of β-Carotene. All readings were taken thrice and antioxidant activities were calculated as follows using the method of Hanachi and Sh (2009). % Antioxidant Activity = Where AB sample = Absorbance of sample and AB control = Absorbance of control High-performance liquid chromatography analysis (HPLC) The percentage (%) purity of the extracted β-Carotene was determined using the methods of Udengwu and Egedigwe (2014). HPLC was done in the Department of Pure and Industrial Chemistry, University of Nigeria Nsukka, while UV spectrometry was determined using a UV-visible spectrophotometer. Readings of β-Carotene standard in ethanol were taken to con rm the peak absorbance of extracted β-Carotene.

Stock preparations
Fresh 1 M stock solutions of β-Carotene, AlCl 3, and full Hoagland's nutrient were prepared daily using the methods of Udengwu and Egedigwe (2014). They were stored at 4 o C in the refrigerator before use.
β-Carotene stock solution: One gram of β-Carotene was mixed in 10 mL of ethanol before the addition of 990 mL of distilled water. A 1% alcohol dilution of β-Carotene was used in this study following the protocol of Fiskesjo (1981); who showed that 1% of alcohol dilutions of lipophilic solutes were not toxic to Allium roots.

AlCl 3 stock solution (1 Molar)
This was prepared by dissolving 133.5g of AlCl 3 in little distilled water and the nal volume made up to 1000 mL. The pH of the solution was buffered to 4.6.

Al treatment concentration
Al treatment concentration of 3 × 10 − 2 mM was achieved through serial dilution and pH 4.6 was through adjustments with H 2 SO 4 .

Hoagland's nutrient solution
This was prepared using the formulation of Hoagland and Arnon (1950 revised).

Determination of actual Al concentration in solution
Actual Al in solution was determined using Shull (1960)

Soil analysis
The result of soil analysis was the same as the results of Udengwu and Egedigwe (2014). Results showed no presence of Al (Table 2).

Antioxidant activity
The antioxidant activity of β-Carotene, 72%, differed signi cantly from ethanol blank, which exhibited a lower percentage activity. Rhee (1978) explained that the TBA test was a colorimetric technique used in measuring the absorbance of a red chromogen formed between TBA and malondialdehyde (MDA). Peak absorbance of extracted β-Carotene at 295nm was 0.181.

Hydrogen ion concentration
Using the formula of Stephenson (2010), the H + concentration of extracted β-Carotene at pH 5.8 was determined by taking 10 to the power of the negative pH i.e., 10 − 5.8 which is equivalent to 1.5 × 10 − 6 mol L − 1 while the H + concentration of 3 × 10 − 2 mM of Al at pH 4.6 was calculated to be 2.5 × 10 − 5 mol L − 1 .

Determination of Actual Al concentration in solution
Following the Aluminon protocol, the actual Al concentration in solution used for the study was 1.85 mg/L.
Vegetative and reproductive data.
Before treatment applications, Amaranthus plants stabilized and were acclimatized in full strength Hoagland's nutrient solution (Fig. 1). The rst noticeable symptoms observed three days after Al treatment applications, which was the same as the results of Udengwu and Egedigwe (2014), were prominent inhibition of root growth (Fig. 2), yellowing and wilting of leaves and overall stunting plant growth (Fig. 3).
Number of leaves (NOL) At the end of 21 days, Al-bc 2 (T6) signi cantly yielded a higher number of leaves in comparison with other treatments except control (Ctrl). Only T6, a post-β-Carotene treatment, signi cantly yielded more leaves than that observed in pre-β-Carotene treatments, bc 1 -Al (T3) and bc 2 -Al (T4). Al treatment (Al) (T7) signi cantly reduced the number of leaves by 44.1% in comparison with the control (Fig. 4).
Length of in orescence (LOI) Fig. 7 shows that the least LOI was recorded with T3. Lengths of in orescences from all treatments differed signi cantly from T6. Al (T7) signi cantly reduced the length of in orescence by 37%.
Number of in orescences (NOI) As shown in Fig. 8, T6 yielded the highest number of in orescence and differed signi cantly from other treatments. T7 signi cantly reduced the number of in orescences.
Fresh weight of in orescence (FWI) Responses of plants to all treatments in Fig. 9 signi cantly reduced FWI in comparison with control. There were no signi cant differences in FWI between pre-β-Carotene and post-β-Carotene treatments, except T6. Having a similar trend with the results got in LOI and NOI, T2 and T3 signi cantly reduced FWI by 86.5% and 97% respectively.
Dry weight of in orescence (DWI) Responses of plants to treatments in Fig. 10 followed the exact trend with plants responses to treatments in FWI. The reduction in DWI was not signi cant in comparison with that of control plants. Responses of plants to T6 and T7 did not differ signi cantly from each other but differed signi cantly from other treatments, including control.
Dry weight of shoot (DWS) Fig. 12 13) and control (Fig. 14). The least ROL was recorded with T6. Decreasing β-Carotene concentrations across treatments were directly proportional to longer ROL. Though there were no signi cant differences, pre-β-Carotene treatments prolonged root lengths more than post-β-Carotene treatments.
Fresh weight of root (FWR) Increasing concentrations of pre-β-Carotene treatments did not signi cantly increase FWR except in post-β-Carotene gain in concentration (Fig. 15). There was no signi cant reduction by T7 in comparison with control. Responses of plants to T1 and T6 increased FWR by 20% and 68.2% respectively.

Discussion
The results of soil analysis used in this study are the same as the results of Udengwu and Egedigwe There is also an added advantage of the ease in controlling the pH. Using transparent bottles allowed for full monitoring of roots during growth and unwanted cutting of root tips if grown in soil. Despite these advantages, it is important to mention that the growth of Amaranthus plants in nutrient solution will not be as robust as when grown in soil because A. hybridus is not an aquatic plant. It is quite interesting to point out the similar trend plants responded to the respective treatments via the growth parameters except for the situations in ROL and DWR. This shows that responses of experimental plants to independent β-Carotene treatments (T1 and T2) and pre-β-Carotene treatments (T3 and T4) were, perhaps, redundant until Al (T7) was applied (T6). The response of T7 in comparison with T6 signi cantly reduced growth for NOL, PLH, LOI, NOI, FWS, FWR, and DWS but was non-signi cant for DWR and DWI. Udengwu (T5 and  T6), (in almost all parameters except ROL and DWR), was more than that observed in pre-β-Carotene treatments (T3 and T4). The major reason for this has been attributed to stressed plants not needing β-Carotene supplementation when not stressed (T1 and T2) until the introduction of Al stress (T3 and T4) (Udengwu and Egedigwe 2014). They explained that A. hybridus that are not under stress may not need supplemented antioxidants because they are adequately protected by their intrinsic antioxidants and that these antioxidants mitigate stress conditions and not serving as growth stimulants. From the results obtained, it is suggestive that an increased dose of β-Carotene concentration (T2) given to plants for 72 h after Al stress yielded a signi cant ameliorative effect for all cases except ROL and DWR. This suggests that A. hybridus plants signi cantly absorbed β-Carotene when under Al stress and not before Al stress. Perhaps, β-Carotene may have yielded a better ameliorative effect when the dose was further increased (Udengwu and Egedigwe 2014). The difference in plant growth of T1 in comparison with T2 was signi cantly higher for only ROL, but was insigni cant for other growth parameters. Having in mind that T1 and T2 each had a pH of 5.8 and H + concentration of 1.5 × 10 − 6 mol L − 1 , the reason for the lower insigni cant growth decrease of T2 over T1 could be that T1 was optimum in ameliorating stress (caused by H + ) better than T2 where β-Carotene activity could have been redundant. This could buttress the assumption that plants need not be supplemented with β-Carotene except under stress conditions. The The responses of A. hybridus plants to T6 regarding FWR showed a signi cant increase in plant growth while for NOI, an insigni cant growth in plants was recorded. More so, β-Carotene signi cantly ameliorated Al stress for these parameters (NOI and FWR). This shows that the intrinsic antioxidants within plants of A. hybridus could not protect them against stress generated from Al toxicity; rather, plants needed a higher dose of β-Carotene to combat the stress condition. This suggests that under Al stress, the ARE in these plants was increased with higher β-Carotene concentration. Zhang et al. (2010) found that enzyme activities of amylases and esterases, which decreased the levels of MDA and hydrogen peroxide (H 2 O 2 ), were increased in wheat plants under stress when pre-treated with H 2 S. In the situation for DWR, only plants that responded to T3 showed a signi cant increase in plant growth when compared to control and Al treatments. The reason for this is not known, however, there is a possibility that β-Carotene could play important roles in decreasing the uptake of Al ions because β-Carotene readily binds to the free hydroxyl groups of Al, forming β-Carotene -Al complexes (radical adducts and cations), thus reducing free Al that would have been available for root absorption. It is, therefore, possible that β-Carotene could have played vital roles in boosting roots mechanisms of either excluding or chelating Al ions. Nevertheless, Al may exhibit an unpredictable interaction with mineral nutrients and β-Carotene, thus undergoing varying actions in connection with an increase or decrease in concentration when in solution. Al could also affect the secretion or suppression of different hormones that are responsible for different functions in plant growth. The controversy will be whether β-Carotene interacts with Al in a fashion that up-regulates or down-regulates the production of speci c hormones at speci c time frames towards the growth of plants.
Visible symptoms such as leaf stunting, chlorosis and death were characteristic in plants after 9 days of Al treatment. Al could have possibly interfered with essential mineral nutrient uptake in the plants. Some studies have found that Al interference leads to de ciencies of mineral nutrients that consequently manifest symptoms of impaired growth (Delhaize and Ryan 1995;Chang et al. 1999). For all the vegetative and reproductive parameters studied, Al treatment signi cantly impaired plant growth apart from the FWR, DWR and DWI. It may suggest that plants of A. hybridus could have absorbed Al within their leaves, shoots, as well as seeds and there could be a possible transfer of Al, along with the food chain, to its consumers since A. hybridus is a staple vegetable source in Sub-Saharan Africa. Though  Results show that Al stress signi cantly reduced ROL and that β-Carotene could not ameliorate the stress condition. This suggests that the apical roots gave rise to an extensive production of lateral roots as a response signal to Al stress. Llugany et al. (2003) showed that plants of Silene armeria responded rapidly by producing many lateral roots following Cu stress to the root tip meristem. They suggested such responses may prevent the extension of the major root into soil patches with high ion toxicity and favour the extension of lateral roots into the less toxic topsoil. Thus, the production of extensive lateral roots may be responsible for the insigni cant increase in plant growth for DWR. Only the response of plants to T3 (pre-β-Carotene treatment) showed an insigni cant ameliorative effect in combating Al stress. A probable reason could be that β-Carotene boosted roots' exclusion mechanism or exudation of chelating agents in preventing the accumulation of phytotoxic Al in the apoplast and symplast (Gill and Tuteja 2010).
β-Carotene and other carotenoids predominantly occur in their all-trans con guration, however, convert to their cis-isomers during its processing or perhaps when in-vivo. It is not known in this study on which isomer was bioavailable to A. hybridus plants, however, studies have shown that the cis-isomers, though susceptible to oxidation and may present lower bioactivity, are more bio-available to humans and animals after processing (Tyssandier et  and Oreopoulou 2016). The situation may be different in plants but it is hypothesized in their study that the cis-isomer of lycopene (either in-vitro or in-vivo) ameliorated plants of A. hybridus from Al stress (Udengwu and Egedigwe 2014). This now raises the question whether the cis-isomer is transformed back to its all-trans con guration when in-vivo.
This study has shown that plants of A. hybridus responded positively to supplemented β-Carotene. Previous studies have concentrated on the roles of β-Carotene in non-photosynthetic organisms (animal models and humans) because neither animals nor humans can synthesize their antioxidants. Studies concerning the supplementation of in vivo β-Carotene in plants are meagre; however, studies on β-Carotene's role in activating and boosting the antioxidant activities of several plants have been reported.
Plants' intrinsic antioxidants could be boosted by the application of certain compounds ex vivo or through the induction of stressful conditions. Tian et al. (2012) showed that spermicide alleviated the oxidative damage in cucumber seedlings subjected to high temperatures by enhancing the activities of antioxidant enzymes.

Conclusion
In conclusion, the present study has shown that under Al stress, plants of A. hybridus, 'Inine oma' suffer deteriorating effects if not protected or mitigated with increased concentration of β-Carotene. An interesting aspect of this work was that plants' ght against Al stress was bolstered by supplemented β-Carotene. Higher doses of post-β-Carotene treatment ameliorated Al stress more than other treatments. This is to say that β-Carotene is not only a precursor of vitamin A but may possess strong anti-oxidative properties against Al toxicity in A. hybridus. Independent β-Carotene treatments without Al stress that did not signi cantly affect growth suggested that plants may not require extra antioxidant supplementation unless subjected to Al stress as obtained in pre-and-post-β-Carotene treatments. From the results, it was evident that plants of A. hybridus were not tolerant to Al stress thus, an Al-sensitive plant. This, in turn, could decrease biomass productivity. This study deduces that Al may have interfered with nutrient uptake as well as accumulated in the leaves. Since A. hybridus is a staple vegetable consumed in the Sub-Saharan region, primary concerns should be to check the transfer of high Al ions to consumers via the food chain. Identi cation and characterization of speci c post-β-Carotene Al-induced stress genes and their gene products as well as comparing these candidate genes/gene products with other Al-stress related genes/gene products deposited in global databases, will be a promising approach to future research in combating the menace of Al toxicity in Sub-Saharan Africa.    Effects of treatments on the length of in orescence (LOI). *Bars bearing different letters differ signi cantly (LSD ≤ 0.05) Figure 8 Effects of treatments on the number of in orescences (NOI). *Bars bearing different letters differ signi cantly (LSD ≤ 0.05) Figure 9 Effects of treatments on fresh weight of in orescence (FWI). *Bars bearing different letters differ signi cantly (LSD ≤ 0.05) Figure 10 Effects of treatments on the dry weight of in orescence (DWI). *Bars bearing different letters differ signi cantly (LSD ≤ 0.05) Figure 11 Effects of treatments on fresh weight of shoot (FWI). *Bars bearing different letters differ signi cantly (LSD ≤ 0.05) Figure 12 Effects of treatments on the dry weight of shoot (DWS). *Bars bearing different letters differ signi cantly (LSD ≤ 0.05) Figure 13 Effects of treatments on root length (ROL). *Bars bearing different letters differ signi cantly (LSD ≤ 0.05) Effects of treatments on fresh weight of root (FWR). *Bars bearing different letters differ signi cantly (LSD ≤ 0.05) Figure 16 Effects of treatments on the dry weight of root (DWR). *Bars bearing different letters differ signi cantly (LSD ≤ 0.05)