Plant growth and sample collection
Switchgrass (cv. Alamo) seeds were surface-sterilized and germinated as
described in Ramamoorthy & Kumar (2012). Seedlings of comparable size
were transplanted into growth cones (Stuewe & Sons, Inc. Tangent, OR,
United States; 6.9 cm in diameter X 25.4 cm in depth) filled with 40%
all-purpose sand (The QUIKRETE® Companies, Atlanta,
GA, United States), 40% Q-ROK silica sand (American Plants & Services,
Inc. Oklahoma City, OK, United States) and 20% fine (2-4 mm particle
size) perlite (Ambient Minerals Inc. Benton, AR, United States). Plants
were watered daily to field capacity during a 4-week growth period with
a nutrient solution containing 600 µM (P-replete control), 200 µM (mild
stress), 60 µM (moderate stress) or 20 µM Pi (severe stress), supplied
as KH2PO4. Each nutrient solution also
contained the following macro- and micronutrients: 6 mM
KNO3, 1 mM CaCl2, 0.25 mM
MgSO4·7H2O, 25 μM NaCl, 12.5 μM
H3BO3, 1 μM ZnSO4·7H2O,
1 μM MnSO4·H2O, 0.05 μM
CuSO4·5H2O, 0.25 μM
(NH4)6Mo7O24·4H2O,
and 10 μM Fe(III)-EDTA. 1 mM 2-(N-morpholino) ethanesulfonic acid (MES)
was added as a buffering agent. The pH of the solution was adjusted to
6.5. Cones were randomly arranged on a greenhouse bench and plants grown
in a 14 h light /10 h dark period at temperatures of 29 °C (light) and
24 °C (dark). Plant samples for metabolic and transcriptional analysis
were rinsed in Milli-Q water, blotted dry and immediately frozen in
liquid nitrogen before storage at -80 °C until use. Plant samples for
biomass determination were placed in pre-weighed paper bags and dried at
65 °C until weights were constant.