The phenomena of nighttime stomatal opening and stomatal

The nighttime stomatal opening and stomatal oscillation were observed (Fig. 3-43 A and B) through the day-night-day, whole-rosette gas exchange measurements using well-watered, 20 days ± 8 days old Arabidopsis plants. For each gas exchange measurement, the air with 380 μmol mol^-1 of CO2 and 12 mmol mol^-1 of water vapor were continuously supplied. No external light source was mounted over either the ring chamber or the cuvette, and temperature control was switched off. Therefore, the photon flux density and temperature in the ring chamber and the cuvette were dependent on the light source and temperature in the growth chamber. The light regime, light intensity, and leaf temperature of the gas exchange measurements are displayed in Fig. 3-43C and D. According to the illumination regime, three phases of the gas exchange measurement were defined: the dark phase, the phase of transition between dark and daytime, and the daytime phase.

During the dark phase (from 18:00 to 9:00), no illumination occurred, and temperatures in the ring chamber (Tring) were around 22 °C (Fig. 3-43C and D). CO2

concentration in the ring chamber (Ca) was maintained at 380 μmol mol^-1 (Fig. 3-43E) and the concentration of water vapor (Wa) was stabilized at 14 mmol mol^-1 (Fig.

3-43E), somehow higher than the supplied value, indicating that the stomata were not fully closed. The Wa and the nighttime Tring resulted in leaf-to-air VPDs reaching approximately 15 Pa kPa^-1 at night (Fig. 3-43F). Under these conditions, the nighttime stomatal opening of both the Col-0 and Cvi-0 was reflected by the nighttime transpiration rate (Enight) and nighttime stomatal conductance (gs-night). Col-0 had a Enight ranging from 0.2 mmol m^-2 s^-1 to 0.6 mmol m^-2 s^-1, a gs-night ranging from 13 mmol m^-2 s^-1 to 35 mmol m^-2 s^-1, while Enight of Cvi-0 varied from 0.4 mmol m^-2 s^-1 to 0.8 mmol m^-2 s^-1, and gs-night varied from 25 mmol m^-2 s^-1 to 60 mmol m^-2 s^-1. (Fig.

3-43A and B). In addition, Cvi-0 had a predawn (from 2:00 to 9:00) increase in Enight

from 0.5 mmol m^-2 s^-1 to 0.8 mmol m^-2 s^-1 and gs-night from 25 mmol m^-2 s^-1 to 60 mmol m^-2 s^-1.

138 Figure 3-43 Nighttime stomatal opening and stomatal oscillation of Arabidopsis accessions Col-0 and Cvi-0. Nighttime stomatal opening and stomatal oscillation were indicated by A) the transpiration rate (E) and B) stomatal conductance (gs) of 20 ± 8 days old Col-0 (black lines) and Cvi-0 (red lines).

Determination of E and gs were achieved simultaneously in the gas exchange measurements. Individual plants are labeled with numbers #1, #2, and #3. The light regime and photon flux density C), ring chamber temperature (Tring) D), CO2 (Ca) and H2O concentration (Wa) E), and leaf-to-air VPD F) over time of the day-and-night gas exchange measurements are depicted. Bars on the top of each figure represent the illumination phase (white), the phase of transition from illumination to darkness or darkness to illumination (gray), and the dark phase (black). Black arrows indicate the predawn stomatal opening in Cvi-0 accession. All plants were grown under short day (8h light / 16h dark photoperiod) at a photon flux density of 150 μmol m^-2 s^-1, at a temperature of 22°C and 50% relative humidity in the daytime and a temperature of 17°C and 60% relative humidity at night, and under well-watered conditions (SWP ≥ -0.02 bar).

A-F) Each curve is a single measurement of an individual plant. n=3 biological replicates for Col-0 and Cvi-0.

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During the phase of transition from dark to light (9:00 to 9:30), the light intensity increased stepwise from 0 μmol m^-2 s^-1 to 150 μmol m^-2 s^-1 (Fig. 3-43C), and Tring

increased from 22 °C to 27 °C (Fig. 3-43D). The ambient CO2 in the ring chamber decreased slightly (Fig. 3-43E), while Wa increased steadily from 12.5 mmol mol^-1 to up to 17 mmol mol^-1 (Fig. 3-43E). The increase of Tring and Wa caused an increase of leaf-to-air VPDs from 15 Pa kPa^-1 to up to 27 Pa kPa^-1 (Fig. 3-43F). The unsteady state of the environmental conditions at the transition phase triggered the oscillation of the E (Fig. 3-43A) and gs (Fig. 3-43B) of Col-0 and Cvi-0.

Figure 3-44 Stomatal oscillations of Arabidopsis accessions Col-0 and Cvi-0. The enlargement

142 of stomatal conductance between 9:00 and 12:00 in Fig. 3-43B presents the details of the stomatal conductance oscillations of Col-0 (black lines) and Cvi-0 (red lines). Individual lines are labeled in the top left corner of each figure. The vertical dotted lines indicate the times of the crests of each oscillation event and the number between the dotted lines indicates the period (length in minutes) of the corresponding oscillation cycle. The gas exchange measurement conditions and growth conditions of plants were as described as in Fig. 3-42.

Curves shown in A-F) were single measurements.

During the daytime phase (9:30 to 17:30), light intensity stabilized at 150 μmol m^-2 s^-1 (Fig. 3-43C) and Tring remained at 27 °C (Fig. 3-43D). Other conditions such as Ca, Wa, and VPD also became stable (Fig. 3-43E and F). The oscillations in E and gs of Col-0 as well as Cvi-0 were damped and finally disappeared. (Fig. 3-43A and B).

The enlargement of stomatal conductance between 9:00 and 12:00 in Fig. 3-43B provides details about the cycles, amplitude, and periods of oscillation. The cycles of oscillation varied considerably (Fig. 3-44A-F). Two of three individual Col-0 plants had one cycle (Fig. 3-44A and B), while the other one had two cycles (Fig. 3-44C).

Moreover, one of three independent Cvi plants experienced only one cycle (Fig.

3-44D), while the others had more than three cycles (Fig. 3-44E-F). The half of the difference between the stomatal conductance at the first crest and stomatal conductance at the first trough was used to describe the amplitude of each oscillation. Using this method of calculation, the three Col-0 plants had amplitudes of 0.2 mmol m^-2 s^-1, 0.4 mmol m^-2 s^-1, and 4 mmol m^-2 s^-1 (Fig. 3-44A-C), while the three independent Cvi plants had amplitudes of 1.5 mmol m^-2 s^-1, 15 mmol m^-2 s^-1, and 10 mmol m^-2 s^-1 (Fig. 3-44D-F). These results do not suggest that differences in the cycles and amplitude of the oscillations were caused by genetic differences between Col-0 and Cvi-0. However, the periods of the oscillations were quite stable in the Col and Cvi accessions (Fig. 3-44A-F). Col #1 and Col #2 had periods of 20 and 21 minutes respectively (Fig. 3-44A and B). The oscillations of Col #3 occurred over a period of 22 minutes in the first oscillation cycle and 16 minutes in the second cycle (Fig. 3-44C).

Cvi had longer oscillation periods than Col-0 (Fig. 3-44D-F). Cvi #1 had only one cycle with an oscillation period of 26 minutes (Fig. 3-44D); Cvi #2 and Cvi #3 had several oscillation cycles and displayed period lengths of 30, 28, 25, 20 minutes and 30, 28, 25 minutes respectively (Fig. 3-44E-F).

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