In all experiments, plants were grown in one of two available climate chambers (Typ Z-1800 / 10-45DU-PI, Weiss Technik, Lindenstruth, Germany; or Ernst Schütt jun. Laborgerätebau, Göttingen, Germany).
The day temperature for all experiments was 22 - 25 °C and the air humidity was in the range of 40 – 70 %. All plants were kept under long day conditions (16 h light, 80 - 100 µmol PAR m-2 s-1) (light bulb: Osram HQI-T 250W/D and Osram L 18W/640 both Osram, Munich, Germany). Aerated hydroponics containing Long-Ashton media (Tab. 2.3) were used for all experiments. For all experiments, each plant was placed in a 1 l pot, if not described otherwise.
In general, P. euphratica and P. x canescens were adapted to high NaCl concentration by increasing the concentration in weekly steps of 0 mM, 25 mM, 50 mM, 100 mM to 150 mM for P. euphratica and of 0 mM, 25 mM, 50 mM and 75 mM NaCl for P. x canescens (Fig.
2.2). Deviations from this scheme have been noted in the results section as appropriate.
Control plants were maintained in aerated hydroponic solution for the same time in absence of added NaCl.
P. euphratica
150 mM
Basic experimental setup
4.Week
25 mM 50 mM 100 mM 0 mM
P. x canescens
25 mM 50 mM 75 mM 0 mM
1.Week 2.Week 3.Week
pre-cultivation
Fig. 2.2: Basic experimental setup for all experiments. The plants were adapted to high NaCl concentration by increasing the weekly external NaCl concentration, starting with 25 mM to final concentrations of 150 mM NaCl for P. euphratica and 75 mM NaCl for P. x canescens, respectively.
2.3.1 Short term 22Na+ uptake experiments
Two independent experiments were carried out, in which the uptake of 22Na+ in one single root tip for short exposure times of 1 min – 30 min and 30 min – 8 hours were measured.
Five – 8-week-old P. euphratica were adapted to 150 mM NaCl as described under 2.3.
Afterwards, plants were exposed to their final NaCl concentration for three weeks.
Single root tips attached to the plant were placed each in one Falcon tube (Sarstedt, Nümbrecht, Germany) containing 150 mM NaCl with additional 57 – 76 KBq 22Na+. The remaining roots were removed from hydroponics container and placed in 1 l pots containing 150 mM NaCl (NaCl adapted) or 0 mM NaCl (control), respectively. After the exposure, the labelled root tip was immediately cut from the main root and washed for 30 s in non-labelled 150 mM NaCl solution to remove surface-bound radioactive label. Digital picture (Minolta Dimage 7, Konica Minolta Holdings Inc., Tokyo, Japan) of the root tip were taken for measurements of the surface area of the root tip (see 2.4.2). Afterwards, the harvested plant tissue was dried at 60 °C for 72 h and measurements of the radioactive tracer (see 2.6.5) were performed.
2.3.2 Split root experiments
For split root experiments, 5 – 8 weeks old P. euphratica and P. x canescens were adapted to high NaCl concentrations for four weeks (P. euphratica) or three weeks (P. x canescens) as described under 2.3.
NaCl adapted plants were placed into the split root system (Fig. 2.3), that was designed for this experiment. The split root system (25 cm x 11 cm area x 8.3 cm height) is divided in three separated chambers (Fig. 2.3 C). The two outer chambers contain nutrient solution, whereas the small, middle chamber (2 x 10 x 8.3 cm) remained empty to avoid transfer of liquid from outer chamber to the other.
Fig. 2.3: Split root system. (A) P. x canescens and (B) P. euphratica in split root boxes containing hydroponics. (C) Empty split root box. LB is the removable labelling box, which was used to minimize the volume of radioactive labelled solution. It was replaced after labelling by GB, a growth box without a transparent front side (A, B). Plants were placed in the middle opening (1). Roots were aerated using an aeration system (2) through separated holes (3). The loss of water was measured using water level indicator (4).
The growth box (10 x 10 x 8.3 cm) (Fig. 2.3 C, GB) contained 600 ml hydroponics and the smaller chamber LB (10 x 10 x 3.4 cm) 200 ml hydroponics. The small chamber was only used for radioactive labelling and replaced by a growth box afterwards (10 x 10 x 8.3 cm).
The split root system was covered by lids to prevent the evaporation of hydroponic solution.
The loss of nutrient solution was controlled using the water level indicator (Fig. 2.3 A, B).
Roots were aerated using compressed-air through aeration bars (Fig. 2.3 B). The front side of the boxes were transparent to control the growth of the roots (Fig. 2.3 C).
A B
C
12
3 3
4
GB GB
GB LB
The plants were placed in the middle opening of the lid and the roots were separated from each other, placing one single root into the small, labelling chamber and the main root part into the growth chamber (see Fig. 2.3 C). Radioactive labelling was conducted for 7 days during the week before the final NaCl concentration for each poplar species was reached (P.
euphratica: 100 mM NaCl; P. x canescens: 50 mM NaCl). P. euphratica were exposed to 185.3 KBq of 22Na+ and P. x canescens were exposed to 108.4 KBq of 22Na+ in the hydroponics. After radioactive labelling, the hydroponic solution was removed and radioactive labelled root tips were washed with non-labelled hydroponic solution, containing the same NaCl concentration, to remove surface-bound radioactive label. The nutrient solution was renewed and the NaCl concentration increased to final concentrations of 150 mM NaCl or 75 mM NaCl for P. euphratica or P. x canescens, respectively. After a chase period of 21 (P. euphratica) or 14 days (P. x canescens), respectively, plants were harvested.
The plant shoots were divided into three part (top, middle, bottom) and the roots were divided into two parts (labelled root and non-labelled root). Plant tissue was weighed, immediately dried at 60 °C for 72 h and measurements of the radioactive tracer (see 2.6.5) were performed.
2.3.3 Leaf feeding experiments
For leaf feeding, one single leaf in the middle of the stem of each plant was fixed with modelling clay (“Nakiplast”, Pelikan, Hannover, Germany) onto a Petri dish. Afterwards, the surface of the leaf was rubbed with silicium carbid (“Carborund”, ESK-SIC GmbH, Frechen, Germany) for at least 10 s. Thereby, the surface of the leaf was injured and leaf uptake of
22Na+ enabled.
Fig. 2.4: Leaf feeding of P. euphratica. Leaves were placed in a Petri dish and fixed with modelling clay (B,C and D). Leaves were exposed to 35 ml 22Na+ labelled nutrient solution containing 150 mM NaCl or 75 mM NaCl for P. euphratica or P. x canescens, respectively, for 24 h.
A B
C
D E
35 ml of the nutrient solution with 150 mM NaCl, labelled with radioactive 22Na+ (7.16 - 28.28 KBq per plant) was added into the Petri dish and the set-up was incubated for 24 h.
After the incubation, the Na-fed leaf was harvested and washed with ddH2O for 5 sec to remove radioactive solution from the surface. The whole plant was harvested after further case period of 48 h. The plants shoots were divided into three parts (top, middle, bottom).
To determine 22Na+ phloem content, the middle shoot part was taken and treated as described under 2.6.3 for the collection of phloem sap. Plant tissue was weighed and dried at 60 °C for 72 h. Afterwards, radioactive tracer were performed as described under 2.6.5.
2.3.4 Experiments with 45Ca2+
P. euphratica and P. x canescens were adapted to final concentrations of 150 mM NaCl with weekly increasing NaCl concentrations starting with 25 mM NaCl (25 mM, 50 mM, 100 mM and 150 mM NaCl). In the first experiment, both poplar species were labelled with 526.2 KBq
45Ca2+ in the hydroponics during the last three days before the start of NaCl treatment.
In the second experiment, P. euphratica plants were adapted to 100 mM NaCl as described above and plants were labelled during the last three days of the exposure to 100 mM NaCl with 520 KBq 45Ca2+. Afterwards the plants were exposed to 150 mM NaCl for a chase period of 1 week. For both experiments, plant roots were washed after radioactive labelling with non-radioactive nutrient solution containing the respective NaCl concentration for 30 s and placed in new 1 l pots, to avoid radioactive contamination.
2.3.5 Ca2+ deficiency experiments
P. euphratica and P. x canescens were exposed to two different Ca2+ concentrations (0 mM and 1 mM) in combination with three different NaCl treatments (0 mM, 25 mM and 150 mM NaCl).
Modified Long-Ashton media with 1.8 mM KNO3 replacing Ca(NO3) in the hydroponic solution were used for treatments without Ca2+. For all other treatment, supplementary 1 mM CaCl2 was added.