## 4 Discussion
### Impact of Mild Drought Stress  
The impact of water stress depends to a great extent on the development stage of the plant and on the intensity of the stress [5]. The stress applied in this study was low (humidity in the pot was 80% field water capacity) but remained constant over a period of several weeks. As a result of the low intensity of the water stress, most of the parameters measured on leaves were not affected (WC, RWC, FW, etc.). At the first stage (42 DAS), plants were stressed for only two weeks and the water stress was mild, and thus no differences in photosynthetic related parameters were detected. However, the stress did have a negative impact on these parameters at 57 DAS (Figure 2) as expected [48; 49]. At harvest, the nitrogen concentration in the shoots was lower in water stressed plants (Figure 5). Drought affects all water movements and nutrient uptake in the roots and often causes N deprivation [41]. The water stress impact on free amino acids concentration at harvest was very small. At harvest, five weeks after the beginning of the water stress, the primary metabolism was not strongly affected, at the exception of proline, an amino acid known to accumulate in case of water stress [50; 51; 52]. Decrease in serine and glutamine concentration can be linked to the decrease in Nitrogen. In the leaves of both leaf ranks studied, the T\({}_{2}\) value of the first water peak attributed to water in the plastids and small vacuoles remained stable, indicating that this compartment was not affected by the water stress. The increase in LWW\({}_{1}\) between stages only observed in LR 8 was certainly due to the parallel increase in fresh weight. Several mechanisms triggered by water stress could explain variations in the NMR parameters of peaks corresponding to the vacuoles. Indeed, T\({}_{2}\) changes can be explained first by a reduction in the vacuolar water content that will lead to a decrease in T\({}_{2}\), second, an increase in the concentration of soluble compounds in the vacuole that will lead to a decrease in T\({}_{2}\), and finally the permeability of the membrane can also change the T\({}_{2}\). One way to distinguish between all these mechanisms is to also consider the change in LWW. Therefore, this parameter is only sensitive to water content. Thus, a simultaneous increase (or decrease) in T\({}_{2}\) and LWW for a specific water compartment is strong evidence for a change in water content in the cell compartment without taking other mechanisms such as solute concentration or membrane permeability, into account. All of these mechanisms can be affected by water stress, a reduction in the water in the leaf and hence in the vacuoles is a known effect of water stress [25] as is an increase in the concentration of soluble compounds [53]. Moreover, the permeability of the tonoplast increases [54; 55] or decreases [56; 57] depending on the stress and the species. The T\({}_{2}\) and LWW of the palisade and spongy cells were significantly affected by the water stress. In LR 8, at 42 DAS, the T\({}_{2\text{-}2}\) and T\({}_{2\text{-}3}\) decreased, LWW\({}_{2}\) remained constant and LWW\({}_{3}\) increased after water stress. The decrease in T\({}_{2\text{-}3}\) can be explained by osmotic adjustment as the increase in LWW\({}_{3}\) indicated more water in this compartment. Indeed, it is known that water stress induces the accumulation of soluble osmo-protectants in leaf vacuoles, so we can assume that this accumulation process is one of the mechanisms involved in the decrease in T\({}_{2\text{-}3}\) in LR 8 at 42 DAS. At the second stage (57 DAS), water stress induced a decrease in both T\({}_{2}\) and LWW for the two vacuolar peaks. At this stage, the T\({}_{2}\) was mainly explained by the water content of the vacuole, and no effect of water stress on the osmotic potential was detected.