Water productivity of RCAR6 and RCAR10 lines under well-watered

Former analyses based on gravimetric methods, carbon isotope discrimination and gas exchange measurements demonstrated the enhanced WUE of RCAR6-3 and RCAR10-4 lines grown under progressive drought, compared with wild type Columbia.

However, enhanced WUE is considered to be associated with a trade-off in yield potential (Blum, 2005). Yield potential here refers to the yield of a crop cultivar when grown in environments to which it is adapted, when nutrients and water are not limiting, and pests and diseases are effectively controlled (Evans and Fischer, 1999).

To investigate whether enhanced WUE is linked to trade-off in yield potential in Arabidopsis, RCAR6-3 and RCAR10-4 lines were grown under well-watered conditions in both low light (maximum photon flux density of 150 μmol m^-2 s^-1) and high light (maximum photon flux density of 900 μmol m^-2 s^-1) to evaluate their growth performance, above-ground biomass, and WUE.

3.2.2.1 Biomass and WUE of plants grown under moderate light

Two experiments were performed under moderate light (a photon flux density of 150 μmol m^-2 s^-1) and well-watered conditions. In the first experiment, seven-day-old

100 seedlings from all lines were transferred to soil and grown with a photoperiod of 16 hours light and 8 hours darkness. Watering was administrated twice a week. The growth performance of eight-week-old RCAR6-3 and RCAR10-4 plants showed little or no visible reduction in comparison to wild type Columbia (Fig. 3-21A higher panel).

After four months of growth, the aerial parts of the plants were harvested. The total biomass was defined as the sum of oven-dried straw weight plus the weight of seeds.

RCAR6-3 did not differ significantly from Col-0 in total biomass while RCAR10-4 lines showed a reduction of biomass by 11% (Fig. 3-21B). In addition, analysis of Δ¹³C of seeds materials displayed a 1.2 ‰ less ¹³C discrimination of RCAR6-3 than Col-0, and therefore RCAR6-3 achieved a 28% higher iWUE than Col-0 according to equation (3) and (4) (Fig. 3-21D and E).

101

Figure 3-21 Biomass production under a well-watered growth regime. A) Representative pictures of wild type Col, RCAR6-3 (R6) and RCAR10-4 (R10) lines at the age of eight weeks (upper row) grown under long-day conditions (16h light / 8h dark photoperiod) and rosettes aged seven weeks (lower panel) grown in short-day conditions (8h light / 16h dark photoperiod). Both scale bars at the top right and bottom right corners represent 3 cm. B) Relative biomass of above-ground biomass (in percentage relative to Col; biomass equals dry straw weight plus weight of seeds; above-ground biomass of Columbia was 1.41 g ± 0.11 g and set to 100%) of Col (n=28), RCAR6-3 (n=28) and RCAR10-4 (n=4) were determined after four-months growth under a well-watered and long-day regime (16h light / 8h dark photoperiod). C) Rosette size of seven-week-old Col-0, RCAR6-3 and RCAR10-4 plants. Δ¹³C D) and Δ¹³C-derived iWUE E) of seeds materials of Col-0 and RCAR6-3 shown in the upper panel of A) and B). A-E) All plants were grown 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. B, D and E) n=28 biological replicates for Col and RCAR6-3; n=4 for RCAR10-4. C) n=3 biological replicates for all. B-E) mean ± SEM, **P<0.001 compared with wild type Col-0.

102 In the second experiment, seven-day-old seedlings of all lines were transferred into the soil and grown with a photoperiod of 8 hours light and 16 hours dark photoperiod. Their pots were covered and water was supplied once a week. Pictures of rosettes after seven weeks indicated a minor reduction in rosette sizes of the RCAR6-3 line (8%) compared to Col-0 while RCAR10-4 line showed a decrease in rosette size (24%) (Fig. 3-21A lower panel and C).

3.2.2.2 Biomass and WUE of plants grown under saturation light

Crops grown in the field always experience full sunlight. High photon flux density promotes the photosynthetic capacity of plants and enhances demand for CO2, therefore imposing a strain on the CO2 gradients between the atmosphere and the site of carboxylation in the plants’ chloroplast. The enhanced CO2 gradient of the RCAR6-3 line found under low light conditions compared with wild type Columbia might be attenuated at saturating light conditions. As a consequence, the increase in the WUE of the overexpression RCAR6-3 line grown under high light might be affected.

To investigate this issue, growth performance, biomass and WUE under high light and well-watered conditions were compared between Col-0 and the RCAR6-3 line. The plants were grown in a sun simulation growth chamber which supplied as much as 900 μmol m^-2 s^-1 light in this experiment (Döhring et al., 1996; Thiel et al., 1996).

After six weeks' growth, Col-0 had obtained 251 cm² ± 17 cm² leaf area and 1.6 g ± 0.1 g dry biomass. The RCAR6-3 line showed only a 1% reduced leaf area at the end of the experiment but had 18% ± 4% reduced above-ground dry biomass compared with Col-0 (Fig. 3-22B-C). Further analysis of Δ¹³C using the dried above-ground biomass revealed 1 ‰ ± 0.1 ‰ less ∆¹³Cbulk and 9% ± 1% higher ¹³C-derived integrated WUE than Col-0 (Fig. 3-22D-E).

103

Figure 3-22 Growth performance, biomass accumulation and Δ¹³C -derived integrated WUE of RCAR6-3 (R6) plants under high photon flux density conditions. A) Pictures of Col and R6 grown under well-watered conditions, an 8h light / 16h dark photoperiod and 22°C, 50%

relative humidity in the daytime and 17°C, 60% relative humidity at night. Illumination began in the morning at 9:00 am with a photon flux density of 140 μmol m^-2 s^-1. It was increased to 330 μmol m^-2 s^-1 at 10:00 am and finally reached 900 μmol m^-2 s^-1 at 11:00 am. Plants grew under the strongest light for four hours till 15:00 pm. Subsequently, light intensity decreased stepwise from 900 to 0 μmol m^-2 s^-1 from 15:00 to 17:00. One hour acclimation was allowed when the light intensity was adjusted to a new level. Text on the left side indicates the age of the plants. The scale bar denotes 10 cm. B) Leaf area values of Col (dotted line) and R6-3 (solid line) as shown in A). C) Biomass obtained by drying the aerial part of plantlets after 42 days of growth. Bulk derived ¹³C discrimination D) and Δ¹³C -derived integrated WUE E) were determined by using dry biomass samples from C). B-E) n=5 for Col and 4 for R6-3 biological replicates for each data point, mean ± SEM, **P<0.001 compared with wild type.