Differential occurrence of epicuticular wax and its role in leaf tissues of three edible aroids hails


 Localization of epicuticular wax (EW) content in leaf tissues and its interaction on leaf protective mechanisms of three edible aroids, Alocasia, Colocasia and Xanthosoma were assessed. Scanning electron microscopy depicted the occurrence of EW in leaf tissues which was higher in Colocasia (10.61 mg dm-2) and Xanthosoma (11.36 mg dm-2) than in Alocasia (1.36 mg dm-2). The result highlighted the interface of EW between the leaves and its internal and external environments. EW acted as a protecting barrier against deleterious solar radiation in term of sun protecting factor (SPF). Occurrence of EW also effectively managed leaf pigmentation, moisture retention, cellular membrane integrity against the invaders. Colocasia exhibited superhydrophobic properties with higher static contact angle (CA) >150o than hydrophobic Xanthosoma and Alocasia with CA ranged between 99.0o to 128.7o. Colocasia EW highly influenced the qualitative and protective mechanisms of leaf. Aroids are the cheapest sources of edible EW among the terrestrial plants could be used in food, agricultural and industrial applications.


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The surface area was measured by digital image analysis using Image J software, and the amount of wax 72 was obtained by extraction dipping the leaves in chloroform during different times 18 , then followed by the 73 evaporation of chloroform 15 . The results were calculated using the following equation. Samples were analysed in 74 triplicate.
Where, Ww is the weight of the wax in mg, and AL is the area of the leaf in cm 2 .

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The wax extracted from the three plants were dissolved in methanol at different concentrations (4 mg ml -1 , 79 2 mg ml -1 , 1 mg ml -1 and 0.5 mg ml -1 ). Samples were analysed using a absorbance scan (Eppendorf, Germany) 80 measuring every 5 nm from 290 to 320 nm in a UV-Vis spectrophotomete 19 . SPF was calculated using the following 81 equation,

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= CF x ∑ 320 Where, Abs is the absorbance of the sample, CF is a correction factor (=10), and EE(λ) x I(λ) is the 84 product of erithermal efficiency spectrum and the solar simulator intensity spectrum, which was tabulated following  Leaves with and without waxes were (n=8 each) fastened to a flat surface with tape in front of a white 88 background. A drop of water (0.01ml) was placed on the surface of the leaves with and without wax. A digital 89 camera with macro lens placed perpendicularly to the sample was used to capture an image. Contact angle value was 90 determined by Image J software 20 . The experiment was repeated thrice with three replications.

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In order to observe the wettability, the extracted wax was dissolved in chloroform at different 92 concentrations (100 mg ml -1 , 75 mg ml -1 , 50 mg ml -1 , 25 mg ml -1 and 0 mg ml -1 ). 0.25ml of each solution was poured 93 in 3x3 cm 2 filter paper. Once the chloroform was completely evaporated, 0.01ml droplet was placed on top of each 94 sample and the time until its completely absorbed was measured.

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were analised using a SPAD-502 portable leaf greenness meter (Minolta Corp, Romsey, NJ). Samples were exposed 98 to 56°C for 30min in a water bath to determine the pigment stability. CSI was calculated following the equation as

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The algorithms for pre-processing of full images, image segmentation and colour quantification were

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Leaves were dipped in distilled water at 22°C during 4 h to obtain the turgid weight (TW) and the samples 116 were dried in a hot air oven (REMI, India] at 70°C for four days. RWC was calculated using the following equation,

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In the present study, we have optimized the wax extraction process for the three aroid species by dipping 147 the leaf pieces in chloroform for 1 min to obtain pure white wax crystals (

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Alocasia registered higher mean SPF (2.02) when compared to Xanthosoma (1.35) and Colocasia (0.24). Sun 154 protection activity depends on the ability to prevent the plants from deleterious UV radiation led mutagenesis 31 .

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Higher SPF was positively correlated with the protective mechanisms and negatively correlated with adverse effect 156 of ultraviolet (UV) radiations 32 .

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Our results revealed that, Alocasia leaves showed 10-fold higher SPF than Colocasia and 2-fold higher 158 SPF than Xanthosoma, which could be explored as a potential natural sun protector.

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Wettability test showed the capacity of epicuticular wax to repel environmental water and protect the leaf 175 surface. In our study, the sample filter paper piece coated with aroid wax persisted the water resistance significantly 176 (https://drive.google.com/file/d/1SlAchDLY1aveMY2A0PSjhvHoXyLSKHIB/view?usp=sharing). Results

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showed that filter paper without wax coating instantly absorbed the water droplet when compared to the filter paper 178 with wax. The resistivity varied significantly (p<0.05) among the three types of aroid wax coating.

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Wettability showed a linear tendency of higher wax concentration correlated with higher water resistance

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Colocasia leaves showed a high dehydration rate in wax and de-waxed conditions, which can be related with the 217 thinner leaf structure. Rapid moisture loss is one of the major factors that affect the leaf quality and EW evidently 218 helped leaf moisture retention in the tested aroids.

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On the other hand, the wax solubility might be another reason of rapid cellular depletion. However, the de-231 waxed leaves showed higher incidence when compared to waxed leaves which could be used to predict the role of 232 EW on Pc prevention. Evidence of natural wax preventing disease incidence was reported by several authors 44,45 .

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Results showed that the presence of EW in leaf tissues sustainably inhibit electrolytic leakage which in turns defends