Perlite

Arabidopsis thaliana plants were grown in a mixture of vermiculite, perlite, and peat moss at a 1:1:1 ratio in an environmentally-controlled chamber with a long photoperiod (16 hr light and 8 hr dark) at 22°C. The transgenic lines and plasmids were obtained from the Arabidopsis Biological Resource Center (ABRC, Columbus, OH), and had been deposited by Dr. B.K. Nelson [8 (link)].

Seeds of Amaranthus hypochondriacus cultivar Revancha and of accession 38040 (origin: India) were kindly provided by E. Espitia (INIFAP, México) and D. Brenner (USDA, Iowa State University, Ames, IA), respectively. Seeds were germinated in 60-well germinating trays filled with a sterile soil preparation composed of a general soil mixture (three parts Sunshine Mix 3TM [SunGro Horticulture, Bellevue, WA], one part loam, two parts mulch, one part vermiculite [SunGro Hort] and one part perlite [Termolita S.A., Nuevo León, México] and coconut paste [Hummert de México, Morelos, México] in a 1:1 v/v relation). The trays were maintained in a growth chamber kept at 26°C, ≈75% R.H. and with a 16: 8 h light (at approximately 300 μmol m^-2 s^-1) dark photoperiod. Amaranth plantlets were subsequently transplanted to 1.3-L plastic pots, containing sterile general soil mixture, 21 days after germination. They were fertilized once, one week after transplant, with a 20:10:20 (N: P: K) nutrient soil drench solution according to the manufacturer's instructions (Peters Professional; Scotts-Sierra Horticultural Products, Marysville, OH, USA). Plants having six expanded leaves were employed for experimentation. Total RNA was obtained from leaves (A. hypochondriacus cv. Revancha) or pigmented stems (A. hypochondriacus India 38040) using the Trizol reagent (Invitrogen Corp., Carlsbad, CA, USA) as instructed, treated with RNAase-free DNAase and re-purified with the RNeasy kit (Qiagen, Valencia, CA, USA) following the manufacturer's protocol. Different sources of RNA were used to generate the six cDNA libraries employed for pyrosequencing runs: i) leaves of intact plants grown under natural greenhouse conditions in the summer of 2009 (Source 1, S1) ; ii) pooled damaged leaf tissue from plants subjected to herbivory for 1, 4 and 12 h (≈20% maximum leaf-tissue loss) by larvae of the salt marsh caterpillar Estigmene acrea (S2); iii ) leaves of noticeably wilted plants resulting from the drought-stress imposed after withholding watering for 3 days (S3) (drought-stress was most probably caused by the confinement of the treated plants in pots, which impeded taproot elongation, a known morphological response to drought in amaranth [see above]), and iv) leaves of plants, showing increased thickness and coarser leaf texture as a result of the acute salt-stress produced by watering the plants for three straight days with 100 ml of a 400 mM NaCl solution, (S4). Leaf material was also obtained from leaves of plants infected with Pseudomonas argentinensis, a bacterial amaranth pathogen, as described previously [51 ] (S5) and from pigmented (red) stem tissue of un-stressed 38040 plants (S6). RNA source S1 to S5 were obtained exclusively from plants of the Revancha cultivar.

Arabidopsis thaliana (L.) Heynh. var. Columbia (Col-0) and var. Landsberg (erecta mutant) (Ler) were used as wild-type strains. The sub-1 mutant was described previously [25] (link). Plants were grown in a greenhouse under Philips SON-T Plus 400 Watt fluorescent bulbs on a long day cycle (16 hrs light). Dry seeds were sown on soil (Patzer Einheitserde, extra-gesiebt, Typ T, Patzer GmbH & Co. KG, Sinntal-Jossa, Germany) overlying perlite, stratified for 4 days at 4°C and then placed in the greenhouse. Plant trays were covered for 7–8 days to increase humidity and support equal germination. Ler seeds mutagenized with ethylmethane sulfonate (EMS) were obtained from Lehle Seeds (Round Rock, TX, USA). 60'000 M2 plants, corresponding to about 7'500 M1 plants, were screened for plants exhibiting a sub-like phenotype. All sub-like mutants described in this paper were outcrossed three times to Ler prior to further analysis. Two qky T-DNA insertion mutants (line SALK_140123 and SALK_043901) [112] (link) were obtained from the ABRC (http://www.arabidopsis.org). The GL2::GUS line [33] (link) in Ler was crossed into slm mutants for analysis of root hair specification.

Purebred tea seeds (Camellia sinensis cv. Fuding dabai) were germinated in perlite and then cultured in nutrient solution (0.75mM (NH₄)₂SO₄, 0.25 mM Ca(NO3)₂•4(H₂O)₃, 0.05 mM KH₂PO₄, 0.35 mM K₂SO₄, 0.395 mM CaCl₂, 0.21 mM MgSO₄, 35.0 μM NaFeEDTA, 46.1 μM H₃BO₃, 2.0 μM MnSO₄, 0.3 μM CuSO₄, 2.0 μM ZnSO₄ and 0.5 μM Na₂MoSO₄). Nitrogen was divided into three concentrations: 0.25 mM (labeled as low nitrogen, LN), 1 mM (labelled as control, CK), and 2.5 mM (labelled as high nitrogen, HN). The control experiment (1 mM) was supplemented with 0.75 mM ammonium and 0.25 mM nitrate using (NH4)₂SO₄ and Ca (NO3)₂•4(H₂O), respectively, which was the best combination of N concentration for seedlings growth. The growth conditions of seedlings in the light culture box were as follows: 28/25°C (day/night), 75% relative humidity, 16/8 h (light/darkness) photoperiod, and 300 μmol⁻² s⁻¹ light intensity. The liquid culture medium was changed every 5 days. The lateral roots of the seedlings were sampled after 10 weeks of seedling growth. The control and nitrogen treatments were repeated three times (CK-1, CK-2, CK-3; LN-1, LN-2, LN-3, HN-1, HN-2, HN-3). The samples were immediately soaked in liquid nitrogen and stored in a refrigerator of -80°C for RNA-seq analysis and qRT-PCR verification.

Both commercial cultivar and mutant line seeds were planted in pots filled with a soil mixture of peat moss and perlite in a 1:2 ratio (v/v). The pots were placed in a growth chamber with a 16-h light/8-h dark photoperiod, an irradiance of 500 μmol m⁻² s⁻¹, and a temperature of 26°C for 28 days. After 2 weeks of germination, irrigation was done on 14-day-old seedlings with a solution containing 1/10 Hoagland and 125 mM NaCl for 2 weeks, with a 3-day interval between waterings (Figure 2). Leaf samples were collected from the seventh-generation mutants and their parent plants (control) for biochemical and molecular analysis. For qPCR, harvesting of the plants was done at 0 and 2 h after treatment, and leaf samples were collected 28 days after treatment. These samples were stored at −80°C after freezing in liquid nitrogen for further procedure of RNA extraction.

The seeds of L. barbarum L. (Cultivar Ningqi 10) were immersed into 1% gibberellin for 30 min, followed by 10 min of disinfection with 0.1% potassium permanganate, then were grown in soil (nutrient soil:perlite:vermiculite = 3:1:1) in an artificial climate chamber (16 h light at 24°C/6 h night at 20°C). After the seedlings had grown two cotyledons, they were transferred to Hoagland’s nutrient solution for hydroponic growth; solutions were refreshed every 3 days. When seedlings reached the 10-leaf stage, salt stress was started by adding different concentrations of NaCl (0, 100, and 200 mmol·L⁻¹) to the nutrient solution for additional 7 days. Leaves for transcriptome analysis and RNA isolation were frozen in liquid nitrogen and stored at −80°C.