Progressive drought

Soil preparation and water status evaluation

46 Figure 2-1 Soil preparation and evaluation of water status. A) The soil preparation scheme used in the progressive drought experiment. The original coarse soil was used as a source of cultivation substrate and saturated soil B) was obtained and followed by a drying and sieving processes. C) A pF-meter was used to measure water potential in soil.

The coarse soil was dried in the oven and sieved to obtain the fine soil that was used to prepare the soil saturated with water (Fig. 2-1A and B). The density of the soil used in this study was 0.17 g cm^-3. Therefore, 33.4 g of dried, fine soil were loaded in 200 mL pots or 100.2 g of soil were loaded for 600 mL pots (Figure 2-1B). All the pots with the same amount of soil were then saturated in a large water tank for 2 to 3 days. After saturation, the soil water content (SWC) of each pot was determined to reach the field capacity. To evaluate the soil water availability of the plants, a gravimetric method and a pF-meter (Fig.2-1C) were used to obtain readouts on SWC and soil water potential (SWP).

It is impractical to use a pF-meter to measure SWP when an experiment is ongoing because of the interruption of plant growth, but SWC is easy to be determined by weighing pots and this method does not disturb plants. A calibration curve of SWC against SWP was determined and was utilized to convert SWC to SWP. The calibration curve follows a power function relationship (Figure 2-2A). 66% SWC has about -0.03 bar SWP. Decreasing SWC led to a reduced SWP.

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Figure 2-2 The calibration curve of the soil water content and soil water potential. A) Calibration curve of soil water content (SWC) against soil water potential (SWP). SWP was measured by pF-meter and SWC was determined by the gravimetric method. B) Classification of the severity of drought throughout progressive drought was based on the leaf temperature (filled circles) and leaf area (open circles) results of Arabidopsis wild type Columbia plants. Progressive drought was classified into non-stress (well-watered; SWC ˃ 60%

or SWP ˃ -0.03 bar; blue box), mild stress (mild drought; 15% < SWC < 60% or -0.5 bar < SWP

< -0.03 bar; green box) and severe stress (severe drought; SWC < 15% or SWP < -0.5 bar; red box) phases. A) Single measurement. B) n=4 biological replicates, mean ± SEM.

Progressive drought

Two phases are included in a progressive drought experiment: a preparation phase and a progressive drought phase. The preparation phase included germination of seeds, seedling growth and covering the pots with Parafilms. The progressive drought phase included repeated measurements of pot weight, leaf area, and leaf temperature. Seeds were sown first on 1/2 MS medium and incubated at 4 °C for 2 days to let the seeds germinate well. Afterward, the seeds were incubated at 22°C with continuous illumination (60 μmol m^-2 s^-1) for 7 days. At this moment, two cotyledons emerged, and the seedlings were transferred into the fine soil with saturated water in 200 mL pots (SWC ≈ 77%; SWP ≈ -0.02 bar ). The plantlets were then allowed to grow under short day conditions with an 8-hour light and 16-hour dark photoperiod and at a photon flux density of 150 μmol m^-2 s^-1. The temperature and relative humidity in the plants' growth cabinet stabilized at 22°C and 50% in the daytime, and 17°C and 60% at night. To avoid stressing the seedlings, each pot was covered with a transparent plastic wrap to maintain high humidity. The wraps were removed after 7 days of growth of the plantlets. At this time, the plantlets had four to five fully expanded leaves (two cotyledons and two to three true leaves).

Subsequently, the surface of the soil was covered with two pieces of Parafilm (5 cm x

48 10 cm for each piece) to prevent soil water evaporation and the holes at the bottom of the pots were also sealed with adhesive tape. In order to ensure that each pot had a similar soil water status at the onset of the progressive drought, a certain amount of water was injected into the soil using a syringe. At this point, the progressive drought was started, and no water was administrated to the soil. In this experimental setup, the progressive drought lasted two months, during which the weight of the pots was recorded and, optical photos and thermal pictures were taken in 5-day intervals. After day 58 of progressive drought, the plant shoots were harvested to determine the dry biomass.

In addition, the progressive drought was classified into well-watered phase (SWC ˃ 60% or SWP ˃ -0.03 bar), mild drought phase (15% < SWC < 60% or -0.5 bar < SWP <

-0.03 bar) and severe drought phase (SWC < 15% or SWP < -0.5 bar) (Fig. 2-2A and B).

60% SWC or -0.03 bar was used as a boundary to classify the well-water phase and the mild drought phase because the leaf temperature of plants of Arabidopsis wild type Columbia began to increase (Fig. 2-2A and B). The determination of 15% SWC or -0.5 bar as the boundary between the mild drought phase and the severe drought phase was due to that the leaf area of plants began to wilt (Fig. 2-2A and B).

Progressive drought under variable ambient temperatures

The progressive drought experiments were also performed at different ambient temperatures in growth chambers. The preparation phase was the same as in the progressive drought at 22 °C. In the progressive drought phase, the ambient temperatures were set to 17 °C, 27 °C and 32 °C in the daytime; the temperatures at night were 5°C less than the corresponding daytime temperatures. All plants were grown under short day conditions (8 hours light / 16 hours dark photoperiod) at a photon flux density of 150 μmol m^-2 s^-1 and 50% relative humidity in the daytime and 60% relative humidity at night. 18-day-old plantlets were subjected to progressive drought for 70 days at 17 °C, 50 days at 27 °C, and 40 days at 32 °C. Using these experimental setups, the weight of the pots was recorded and optical photos and thermal pictures were taken in 5-day intervals. At the end of each progressive drought experiments, the shoot parts of the plants were harvested to determine the dry biomass.

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The progressive drought experiment using Col-0, RCAR10-4 and their grafts were performed under short day conditions with an 8 hours light and 16 hours dark photoperiod and at a photon flux density of 150 μmol m^-2 s^-1. The temperature and relative humidity in the plants' growth cabinet stabilized at 22°C and 50% in the daytime, and 17°C and 60% at night. 28-day-old plants were transferred into the soil saturated with water and covered with Parafilms. In this experimental setup, the progressive drought lasted ten weeks. Thermograms were taken two weeks after the onset of the progressive drought. After the 70th day of progressive drought, plant shoots were harvested to determine the dry biomass.

The progressive drought experiment performed to assess the response of Arabidopsis accessions RLD-0, Ler-0, Col-0, and Cvi-0 to progressive drought differed from the progressive drought experiments shown above. In this experiment, pots with a volume of 600 cm³ volume were used, and 100.2 g of dry soil were loaded into each pot. No water was administered after the soil was saturated with water.

Seven-day-old seedlings were transferred into pots and grew under domes for seven days. Subsequently, the domes were removed and all plantlets were allowed to grow for another four days. At this point, the pots were covered, and the progressive drought was started. Around 220 g of water were present in the soil in each pot (37%

SWC) at the onset of this progressive drought experiment. All plants were grown under short day conditions (8 hours light / 16 hours dark photoperiod) at a photon flux density of 150 μmol m^-2 s^-1 and at 22°C and 50% relative humidity in the daytime and 17°C and 60% relative humidity at night. Using this experimental setup, the weight of pots was recorded and optical photos and thermal pictures were taken in 7-day intervals. After the 49th day of progressive drought, plant shoots were harvested to determine the dry biomass.