Under a changing climate, including elevated carbon dioxide concentration (e[CO2]), rising vapour pressure deficit (VPD) and intensified soil water deficits, plants have evolved various adaptive strategies to cope with these multiple environmental conditions. In order to improve crop productivity and optimize water use under future drier and CO2-enriched climate, a better understanding of plant water relation characteristics is of critical importance. Although it is believed that e[CO2] enhances plant water use efficiency and improves plant performance when exposure to drought stress, these positive effects could differ among various species or under different stress conditions. The plant hormone abscisic acid (ABA) plays an important role in plant drought response, which is also closely associated with e[CO2]-modulated plant water relations. However, under the combined multiple environmental conditions, the role of ABA in regulating plant hydraulic integrity remains obscure.
Tomato (Solanum lycopersicum) is one of major vegetable crop grown throughout the world, which is sensitive to abiotic stress. The ABA-deficient mutant flacca has been widely used to investigate the function of ABA due to its significantly lower ABA content than its wild type counterpart - Ailsa Craig (AC), and recently the transgenic line sp5 which overproduces ABA was also used in some studies. Using these tomato genotypes with contrasting endogenous ABA levels, the main purpose of the present study was to investigate the plant physiological response under various environmental stimulus and how ABA mediates these regulations. To achieve this research objective, three pot experiments were conducted from 2018 to 2021 in a climate-controlled greenhouse located at the experimental farm of University of Copenhagen, Taastrup, Denmark.
Experiment 1 (Paper II & III): Two tomato genotypes AC and flacca grown under two [CO2] levels (ambient [CO2], a[CO2], 400 ppm; e[CO2], 800 ppm) were exposed to progressive soil drying. The fraction of transpirable soil water (FTSW) was used to determine the soil water status in the pots. During the whole period of soil drying, the changes of leaf and root hydraulic conductance (Kleaf, Kroot) and the transcriptional levels of the plasma membrane intrinsic proteins (PIPs, an important subfamily of aquaporins (AQPs)) at the end of soil drying were determined. The results showed that plant hydraulic regulations by both e[CO2] and drought stress involved PIPs, where some specific PIPs were downregulated by both factors and root PIP2;1 was upregulated by drought stress. In addition, severe drought stress could override the effects of e[CO2] in the absence of ABA. Furthermore, AC and flacca were treated with exogenous ABA for three days before the commencement of the progressive soil drying. The results showed that in flacca the retarded plant growth and abnormal stomatal functionality could be rescued by exogenous ABA application, but through ABA-triggered other indirect metabolic process rather than an ABA direct effect, which might be related to ethylene production. In AC, e[CO2] exerted a positive effect on plant growth and water relations, which counteracted with the ABA spraying effects. Most interestingly, in the two genotypes, e[CO2] had a drag effect on the reduction of leaf ABA concentration after exogenous ABA application, which could explain the reduced stomatal sensitivity of e[CO2]-grown tomato plants to ABA during progressive soil drying in our previous works.
Experiment 2 (Paper IV): As hydroponic system is a good approach to explore the root traits, the overall stomatal, leaf and root morphological traits of hydroponic-grown AC, sp5 and flacca were investigated. In addition, this experiment provided the possibility to determine the Kroot via the root traits. The results showed that ABA accumulation stimulated Kroot through the improvement in specific leaf area and alteration in root distribution pattern, and e[CO2] decreased Kroot as well as stomatal conductance to confer plants a fine-tuned control of water dynamics. As short-term stress response could be different from long-term soil drought, a polyethylene glycol (PEG) 6000-induced 24-h osmotic stress along with long-term e[CO2] exposure was imposed to the three genotypes. The results showed that both ABA and e[CO2] enhanced plant osmotic stress tolerance through a better coordination of stomatal and hydraulic adjustment, an early osmotic adjustment and improved plant water relation characteristics. Moreover, the stomatal closure under fast and severe stress was induced by hydraulic signals (i.e. leaf turgor) rather than ABA.
Experiment 3 (Paper V): High vapour pressure deficit (VPD) causes atmospheric aridity for plants, which was similar to soil drought but also resulted in some different physiological responses. In this experiment, AC and flacca plants grown under two levels of VPD (VPD1, 0.71 kPa; VPD2, 1.25 kPa) were exposed to progressive soil drying. The plant responses at the onset and end of soil drying, including stomatal/ leaf traits, water relations, ABA content and stable isotope compositions as well as the short-term leaf desiccation response were investigated. The results revealed that flacca lost the ability of controlling hydraulic integrity due to impairment of stomatal functionality, and relatively higher VPD caused higher transpiration rate compared to those low VPD-grown plants. Furthermore, in the two genotypes, the change of water use efficiency at leaf and whole-plants levels coincided with the change of leaf carbon and oxygen isotope compositions (δ13C and δ18O), where δ13C was a more robust indicator of stomatal behaviour than δ18O when plants were exposed to combined atmospheric and soil drought.
Based on the above results, it was concluded that:
 The ABA-deficient mutant flacca exhibited impairment stomatal functionality when coping with adverse environmental conditions.
 ABA accumulation in sp5 increased Kroot through regulation of specific leaf area and root distribution pattern, while e[CO2] decreased Kleaf and Kroot to be well-coordinated with the declined stomatal conductance, which required ABA. Although both e[CO2] and drought stress decreased Kleaf and Kroot and involved PIPs, severe stress overrode the effects of e[CO2].
 In short-term osmotic stress, e[CO2] and high endogenous ABA level improved plant drought tolerance through stomatal and hydraulic adjustment, which might be associated with reduced stomatal or hydraulic sensitivity to leaf/ xylem ABA. The ABA insensitivity under e[CO2] could be ascribed to the low ABA degradation rate.
 Hydraulic signal predominantly induced stomatal closure under short-term osmotic stress, and ABA might played a role in maintaining the stomatal closure afterward. Although ABA-treated flacca exhibited recovered stomatal drought responses, exogenous ABA application stimulated these responses via indirect hydraulic way or triggered metabolic process.