3. Results
3.1 System establishment
3.1.3 Thermal imaging and gas exchange analysis during progressive drought .71
Figure 3-3 Thermal imaging and gas exchange analysis during progressive drought. A) Leaf temperatures of wild type Columbia plants were recorded at six-day intervals. Single plants were grown for 18 days prior to the discontinuation of watering at SWP = -0.02 bar. n=4 biological replicates per data point, mean ± SEM. B-D) Whole rosette gas exchange measurements using Col. Stomatal conductance (gs) B), net carbon assimilation rate (An) C) and insWUE D) defined as the ratio of An to gs were analyzed in response to soil water content during progressive drought. Gas exchange measurements were conducted at photon flux density of 150 μmol m-2s-1, 420 μmol mol-1 external CO2 and vapor pressure deficit of 13 Pa kPa-1± 2 Pa kPa-1. A-D) Growth condition of plants as described in Fig. 3-1. B-D) Single plant measurements with 10 technical replicates per data point.
Most of the water loss in plants occurs through stomata. This process can be detected by IR thermogram (IRT), which allows non-contact and high throughout
72 visualization of leaf surface temperatures, and therefore reflects the transpiration of plants (Hamlyn G. Jones, 2004). When water is transpired in C3 species, CO2
simultaneously diffuses along its concentration gradient in the atmosphere and the site of carboxylation (Chaves et al., 2008). The gas exchange can be traced by a gas exchange system which is designed to trace the water vapor as well as CO2 at the inlet and outlet of the cuvette where plant samples are clamped. Analysis of the thermal imaging and the gas exchange during progressive drought using single leaves or whole rosettes, facilitates identification of plants with high WUE and elucidates possible mechanisms.
During progressive drought, the leaf surface temperatures of Col plants varied by more than three degrees (Fig. 3-3A). The initial drop of soil water content from 80%
to 60% did not result in elevated leaf surface temperatures, but rather a one-degree decrease. Further soil water depletion led to a consistent elevation in leaf surface temperatures, indicating that water shortage had emerged and stomata had started to react to it (Fig. 3-3A). Consistent with the leaf temperature results, the stomatal conductance (gs) of the whole Col rosette showed a continuous reduction when soil water content was below 60% (Fig. 3-3B). In contrast to stomatal conductance (gs), net carbon assimilation rate (An) responded little to the drop in soil water content from 68% to 30% (Fig. 3-3C). As a result, insWUE (the ratio of net carbon assimilation rate to stomatal conductance) was enhanced by a factor of up to 2.4 under the progressive drought compared with well-watered conditions (Fig. 3-3D).
3.2 Enhanced water use efficiency conferred by ectopic expression RCARs
When adapting to their habitats, plants need to regulate the relationship between growth and water availability. This relationship can be expressed as WUE. High WUE is an attractive agronomic trait in agriculture. The enhancement of WUE by a factor of 1.5 to 2.5 under drought conditions has been reported in wheat and other crops (Gu et al., 2013; Medrano, 2002; Rizza et al., 2012; Torrecillas et al., 1999). Likewise, a two-fold increase in WUE under drought conditions was also found in Arabidopsis thaliana accessions Columbia (Col) and Landsberg (Ler) (Des Marais et al., 2014;
Easlon et al., 2014; Juenger et al., 2005; Masle et al., 2005). These facts indicate that high WUE is achievable and can be modulated by drought stress. However, the
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underlying molecular mechanisms still have not been fully elucidated. Genes involved in regulating stomatal density or size were found to control WUE (Des Marais et al., 2014; Masle et al., 2005; Yoo et al., 2010), whereas genes controlling open and close movements of stomatal pores are rarely reported. In addition, whether increased WUE is associated with reduced growth and trade-offs in yield is still debated (Blum, 2005). Some studies found that plants with enhanced WUE are always linked to reduced growth and yield (Blum, 2005; Hausmann et al., 2005;
Martin et al., 1999; Munoz et al., 1998), while few studies showed both enhanced WUE and yield potential (Rebetzke et al., 2008).
Drought induces ABA synthesis (Iuchi et al., 2001a). Increments in levels of endogenous ABA trigger ABA-dependent signal transduction after being recruited by an ABA-binding regulatory component, RCAR/PYR1/PYL, and an associated protein phosphatase of type 2C (PP2C) (Kang et al., 2010; Kuromori et al., 2010; Ma et al., 2009; Park et al., 2009). OPEN STOMATA1 (OST1/SRK2E/SnRK2.6) protein kinase is subsequently activated, which results in phosphorelay of OST1 protein to SLAC1 ion channel, KAT1 cation channel in guard cells to trigger stomatal closure. Moreover, OST1 and other related SnRK2 protein kinases phosphorylates transcription factors (AREBs/ABFs/ABI5/ABI4) to activate downstream gene expression in the nucleus (Fujii et al., 2007; Fujii et al., 2009; Fujita et al., 2009; Geiger et al., 2009; Lee, S. C. et al., 2009; Ma et al., 2009; Park et al., 2009; Raghavendra et al., 2010; Sato et al., 2009; Yoshida et al., 2010). As a consequence, adaption responses of plants to drought was mediated in both short term and long term manners. Analogously, the adaption responses of plants can also be modulated by constitutively enhanced ABA signaling through ectopic expression of RCAR genes. There are fourteen RCAR proteins in Arabidopsis thaliana, and they are classified into three subclades (Ma et al., 2009; Park et al., 2009; Raghavendra et al., 2010). Transgenic plants in Columbia background with ectopic expression single RCARs induced using the cauliflower mosaic virus 35S promoter were generated, and the expression level of these lines was analyzed by my colleague Stefanie V. Tischer (2016). Water productivity of all ectopic expression RCAR lines is investigated in this chapter. Growth performance, biomass, and WUE were evaluated during progressive drought on three levels:
immediate net carbon assimilation and transpiration, short-term growth and leaf surface temperature, and long-term growth and water use. Certain RACRs were
74 found to combine maintained growth with enhanced WUE compared with wild type Col-0. The underlying mechanism was also studied to elucidate when, where and why plants benefit from overexpressing certain ABA receptors. In addition, yield potential was analyzed in both low light and high light intensity conditions, and trade-offs under variable ambient temperatures and deficit irrigation were investigated.