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For the inhibition of the AQP-mediated water transport pathways, the standard conditions are maintained (plant stage and inhibitor concentration). Plants are grown in hydroponic nutrient solution. The testing is done after the complete development of 6th leaf in case of cereals (sorghum, pearl millet and maize) and between 24 – 27 days after sowing in case of legumes (chickpea, pigeon pea and peanut). On the day of the experiment, the acclimatized plants are given a measured amount of fresh nutrient solution. Then they are positioned on separate balances with 0.01 g accuracy (Kern, Germany) and their weight loss (due to transpiration) recorded every 20 minutes (weighting can be done manually using a single balance for all pots). Inhibitors are applied after 2hrs of initiating the experiment and subsequently transpiration response to treatment is followed up for a minimum of 3 hours. Different inhibitors can be used and the tentative concentrations used are indicated (H2O2 (1.5mM), HgCl2 (200µM), AgNO3 (400µM – cereals and 100µM - legumes). It should be mentioned that these concentrations need to be re-tested for any specific crop or for conditions prior to initiating the routine experiments. In addition, these inhibitors are not specific aquaporins alone and take care while interpreting the transpiration response data. For apoplastic pathway inhibition, nanoparticles (precipitates of insoluble inorganic salts) are used to block the space between root cells in the endodermis (Ranathunge et al., 2005). For this purpose, we enable the reaction between 1mM K4[Fe(CN)6] and CuSO4 (cereals - 0.5mM; legumes 0.25mM) inside the root tissues which produces rusty brown crystals (precipitates) of Cu2[Fe(CN)6]6 or Cu[CuFe(CN)6] and these restrict the apoplastic water flow. As CuSO4 permeates faster than K4[Fe(CN)6], 1mM K4[Fe(CN)6] is applied to the media initially for 3 hours and consequently the solution is exchanged with ½ molar strength of CuSO4 (it is important that all CuSO4 reacts with K4[Fe(CN)6] to limit its poisonous effect). All-over, the experiment then consists of assessment of transpiration for 2 hours prior to CuSO4 application, and then following-up transpiration for at least 3 hours after CuSO4 application.

Data Analysis

In each experiment, transpiration values (water loss per unit of time) are divided by leaf area i.e. transpiration rate (Tr; g of water loss per unit of time per unit of leaf area, i.e. g water loss cm-2 min-1). In inhibition experiments, Tr data are double normalized to a non-treated control, first by dividing the individual Tr data by the mean Tr of the untreated control (Tr ratio, TrR), and then by dividing the TrR values by the mean of TrR value before inhibitor treatment (normalized TrR, NTrR).