## 4 Discussion
### Leaves at Two Developmental Stages Monitored through NMR  
In plants, peaks of the T\({}_{2}\) spectra can be associated with the water molecules in different subcellular compartments [44; 45]. The NMR spectra of oilseed rape leaves have been extensively studied and an attribution of the different water peaks has been proposed [30]. In young oilseed rape leaves, the water NMR signal consists of three peaks: the first one characterized by a T\({}_{2}\) around 2 ms was associated with the water interacting with polysaccharides (cell wall, starch), the second one characterized by a T\({}_{2}\) around 15 ms was associated with the water in plastids and other small vacuoles, and the last one, the highest in terms of intensity and with the longest T\({}_{2}\) around 150 ms, was associated with the water inside the cell vacuoles [35]. It has been shown in Brassica napus that vacuoles of palisade cells evolve differently than the spongy ones with leaf age. Consequently, this physiological evolution leads to an increase in the T\({}_{2}\) value of the water in the vacuoles of palisade cells, whereas the T\({}_{2}\) associated with the water in the spongy cells vacuoles remains stable. The consequence is the appearance of a new peak (T\({}_{2}\) of more than 300 ms) that describes only the water in the vacuoles of the palisade cells, whereas the water in the vacuoles of spongy cells is described by a separate peak (T\({}_{2}\) of about 150 ms). The NMR signal of tobacco leaves was similar to the NMR signal of the oilseed rape leaves. The main difference was that no peak around 2 ms was detected in tobacco leaves, whereas the other peaks present in the oilseed rape leaf signal were also present in the NMR signal of tobacco leaf. The absence of T\({}_{2}\) around 2 ms can be explained by lower starch content in young tobacco leaves, which were younger than the oilseed leaves studied. Indeed, [PERSON] et al. [46] reported considerable variation in the amount of starch in leaves from young and mature tobacco plants. Using scanning electron microscopy, they observed that the volume of starch relative to the cell volume was less than 2% in leaves from the early mature plants (12 th leaf rank at 70 DAS) and represented 15% in mature plants (at 100 DAS). Therefore, according to the attribution of the T\({}_{2}\) peaks to water cellular fractionsproposed for oilseed rape leaf and due to the agreement in the relaxation time of the different water fractions, the following attribution can be assumed for tobacco: the first peak with a T\({}_{2\text{-}1}\) of 15 ms could be associated with plastids and small vacuoles, the second one with a T\({}_{2\text{-}2}\) of 120 ms could be associated with the water inside the vacuole of spongy cells, and the last peak characterized by T\({}_{2}\) longer than 300 ms, with the water inside the vacuoles of palisade cells. Unlike in oilseed rape leaves, the vacuolar volume of the two main parenchyma of the young tobacco leaves is already very different [47], resulting in two separate peaks. Based on this similar attribution between oilseed rape and tobacco plants, two different developmental processes can be distinguished in the tobacco leaves from the two leaf ranks. In mature leaves (LR 8), we observed an increase in the T\({}_{2}\) corresponding to the palisade cells (T\({}_{2\text{-}3}\)) between the two stages. Moreover, LWW\({}_{1}\), LWW\({}_{2}\) and LWW\({}_{3}\) increased between stages. This is explained by the enlargement of cells concomitantly with the increase in fresh weight during leaf expansion. Moreover, in these leaves (LR 8), the proportion of total leaf water associated with the palisade cells was higher than the proportion associated with the spongy cells. This change in the water balance detected by NMR showed that the change in leaf fresh weight was mainly associated with the palisade cells. In older leaves (LR 4), the T\({}_{2}\) and LWW of the water from the vacuole of palisade cells were higher than in mature leaves (LR 8).