Water productivity of Arabidopsis under deficit irrigation

Ectopic RCAR6 and RCAR10 lines have been proven to have enhanced WUE with little or no reduction in the growth and biomass accumulation under progressive drought

104 conditions. However, an increase in WUE is considered to have benefits under terminal severe drought stress, like progressive drought, but not in a mild drought scenarios (Tardieu, 2011). To investigate whether RCAR6 and RCAR10 lines could still be water-efficient in different drought scenarios, deficit irrigation (60%, 40%, and 20%

soil water content, but not 80% due to unexplained growth see Fig. 3-2) was performed. Seven-day-old seedlings of wild type Columbia, RCAR6-3 and RCAR10-4 were transferred into pots (200 mL) and grown under well-watered conditions (SWC

≥ 77%, SWP ≥ -0.02 bar) for seven days. Subsequently, watering was stopped for another seven days period to allow soil water content to drop to 30%. For pots of plantlets designated to grow with 60% and 40% soil water content were adjusted and pots were covered, and water was applied until their respective levels were reached, while those of 20% soil water content were covered four days later when their soil water content had reached 20%. The time point of covering the 40% and 60% water regime pots were defined as day 0 for the deficit irrigation regime assuming that negligible water was consumed by plantlets grown at the 20% SWC in four extra days.

Subsequently, water was applied according to maintain the designated regimes in three-day intervals. Leaf surface temperatures, leaf growth, above-ground dry biomass and the WUE of those lines were analyzed.

3.2.3.1 Leaf surface temperatures

Figure 3-23 Leaf surface temperatures of wild type Columbia and RCAR lines in response to soil water content. Thermogram A) and leaf temperature values B) of 43-day-old Col, RCAR6-3 (R6) and RCAR10-4 (R10) grown at 60%, 40% and 20% soil water content (See Fig.

3-2). Four plants were grown per line in separate pots at randomized positions and thermal pictures were arranged in groups after imaging. Scale bar indicates 2 cm. All plants were grown under short day conditions (8h light /16h dark photoperiod) at photon flux density of 150 μmol m^-2 s^-1 and 22°C and 50% relative humidity in the daytime and 17°C and 60%

relative humidity at night. n=4 biological replicates for each data point, mean ± SEM.

**P<0.001 compared with wild type Col-0 in each water regime.

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Plants grown for 17 days under water deficit conditions were chosen as examples to show the leaf surface temperatures resulting from different water regimes. In every water regime, both RCAR6-3 and RCAR10-4 lines showed higher leaf temperatures than wild type Columbia. Under the 60% water regime, the leaf temperature of Col averaged 22.2 °C ± 0.1 °C, which was 1 °C lower than RCAR6-3 (23.3 °C ± 0.1 °C) and RCAR10-4 (23.2 °C ± 0.04 °C) (Fig. 3-23A and B). Reducing soil water content to 40%

resulted in an elevation in leaf temperatures for all lines, but the difference was maintained at 1 °C between Col-0 and both other lines (Fig. 3-23A and B). A further decrease of soil water content to 20% diminished the difference, but the leaf temperatures of RCAR6-3 and RCAR10-4 were still higher than Col-0 by at least 0.4 °C (Fig. 3-23A and B).

3.2.3.2 Biomass formation and water use efficiency

In the deficit irrigation experiment, leaf areas of all lines showed a slight increase when soil water content was reduced from 60% to 40% followed by a significant decrease when soil water content was further reduced to 20%. Under the 60% water regime, Col-0 plants consistently increased the leaf area from 1.8 cm² ± 0.2 cm² at the onset of deficit irrigation, to 105 cm² ± 4 cm² at the end, and achieved a maximum leaf expansion rate of 9.4 % d^-1 ± 0.2 % d^-1 (Fig. 3-24A and D). Compared with Col-0, the RCAR6-3 and RCAR10-4 lines showed a somewhat reduction in leaf area and maximum leaf expansion rate (Fig. 3-24A and D). Reducing soil water content to 40% did not result in a reduced in leaf area and maximum leaf expansion rate in Col-0 and RCAR6-3 lines, but the difference between Col-0 and RCAR10-4 lines was attenuated (Fig. 3-24B and D). Further reducing soil water content to 20% led to a significant decrease in both leaf area and maximum leaf expansion rate in all lines, but RCAR6-3 still did not differ from Col-0, while RCAR10-4 showed some reduction (Fig. 3-24C and D).

106 Figure 3-24 Biomass formation and water productivity conferred by ectopic expression of RCAR6 and RCAR10 under deficit irrigation. Leaf area over time of 33 days at A) 60%, B) 40%

and C) 20% soil water content and the maximum leaf expansion rate D) in wild type Columbia (Col, filled circles), RCAR6-3 lines (R6, open squares) and RCAR10-4 (R10, open triangles). E) Above-ground biomass and F) WUE (dry biomass in grams per liter of water consumed) at the end of deficit irrigation. Plants were grown in conditions as described in Fig. 3-23. A-F) n=4 biological replicates, mean ± SEM, **P<0.001 compared with wild type Col in each water regime.

At the end of deficit irrigation, the biomass accumulation of all lines showed the same tendency as leaf area measurements. A slightly higher under the 40% water regime as compared with the 60%, and a significantly lower under the 20% SWC (Fig.

3-24E and F). The WUE of all lines increased consistently with less water, from 60% to

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20% SWC while consumed water showed the opposite trend. Under the 60% water regime, Col-0 generated 0.33 g ± 0.01 g dry biomass and consumed 150 g ± 8 g water, resulting in a WUE of 2.2 g/L ± 0.03 g/L. The overexpression lines RCAR6-3 and RCAR10-4 combined 13% ± 5% and 9% ± 6% reduced biomasses respectively, with 50%

± 2% and 54% ± 4% less water consumption, hence achieving 1.7-fold and 2-fold enhanced WUE (Fig. 3-24E and F). When soil water content was reduced to 40%, both RCAR lines were still water productive. RCAR6-3 and RCAR10-4 combined 1.7-fold and 2-fold enhanced WUEs with 5% ± 5% and 6% ± 5% reduced biomasses respectively, compared with Col-0 (a WUE of 2.5 g/L ± 0.1 g/L and a dry biomass of 0.34 g ± 0.01 g) (Fig. 3-24E and F). Further reducing soil water content to 20%, RCAR6-3 combined a 1.4-fold increase in WUE with 7% less dry biomass compared with Col-0, while RCAR10-4 showed the same increase in WUE with a 25% decline in dry biomass (Fig. 3-24E and F). Taken together, RCAR6-3 line achieved enhanced WUE at no or a minor expense to biomass accumulation under different drought scenarios.