title: Ro-ctrl_2
organism: Triticum turgidum subsp. durum
description: Protocols: Root and leaf tissues were harvested from 10 M Cr stressed and unstressed control plants 50 days after sprouting, frozen in liquid nitrogen, and then stored at -80°C ultrafreezer. Three biological replicates were considered for each of the three conditions for both tissues. For leaf analysis, each biological replicate was composed of a pool of 3-5 fully expanded and green leaves per plant (1 biological replicate = 1 plant). For root analysis, each biological replicate was composed of a pool of 0.5 g of roots for each plant (1 biological replicate = 1 plant).  Seeds were surface sterilized with a 1.2% NaClO solution for 20 minutes, then thoroughly washed and immersed in a 1 mM CaCl2 solution that had been aerated for a full day. Following this, the seeds began the germination process on plastic Petri dishes with vermiculite, maintained in complete darkness at a 10°C temperature. After a period spanning 3 to 4 days, three of these germinated seeds were transferred to plastic containers measuring 10 cm (diameter) x 60 cm (height), which were filled with perlite. They were moistened using deionized water and then integrated into the specified hydroponic setup as described by Sabella et al. (2021). In every pot, three young plants were placed, and for each specific treatment, three separate tubes were set up, acting as three distinct biological replicates. Regular hydration was maintained for the plants using a hydroponic mixture at fixed timeframes (every 4 hours) lasting 5 minutes, ensuring the perlite remained damp while preventing waterlogging. Plants were grown under 40-60% relative humidity, 8 to 22°C temperature day/night of seedling growth to flowering, and photoperiod day/night ranging from 10/14 (seedling growth) to 16/8 (flowering) hours in a Fitotron® Growth Chamber (Weiss Technik, UK), as detailed by Vergine et al. (2017). The durum wheat plants were arranged following a randomized block design, each block consisted of an equal number of plants, systematically allocated to different treatment groups to ensure uniformity across varying environmental conditions within the growth chamber. This arrangement was aimed at minimizing the potential confounding effects of spatial variability on the experimental outcomes. The nutrient solution was prepared using reverse osmosis water (<30 μS/cm-1) and contained 1.1 mM KNO3, 3 mM [Ca(NO3)2 2H2O], 0.2 mM NH4NO3, 1.2 mM K2HPO4, 0.04 g/L FeEDDHA, 2 mM MgSO4, 70 μM H3BO3, 1.2 μM Na2MoO4, 1 μM ZnSO4, 1 μM CuSO4, and 10 μM MnSO4, while the pH was maintained continuously between 5.5 and 6.0 adjusting it every two days.  The treated plant set was cultivated by adding 2.5 and 10 μM Cr to the hydroponic solution. RNA extraction was performed from 50-100 mg of leaves or roots ground with a pestle and mortar in liquid nitrogen using the RNeasy Plant Mini kit (Qiagen, Hilden, Germany). RNA quality was determined using the RNA 6000 nano kit on the Agilent 2100 Bioanalyzer System (Bio-Rad, Hercules, California, USA).  Nine sequencing libraries were obtained following the procedure of Truseq Stranded mRNA Library Prep (Illumina, San Diego, California, USA) and using Truseq RNA Single Indexes (three libraries for each of the experimental conditions or tissues, total 12 libraries). The RNA concentration of each library was determined using the dsDNA High Sensitivity Kit on Qubit™ 4 Fluorometer (Invitrogen, Waltham, Massachusetts, USA).