Structural Changes in Senescing Oilseed Rape Leaves at Tissue and Subcellular Levels Monitored by Nuclear Magnetic Resonance Relaxometry through Water Status

Abstract

Nitrogen use efficiency is relatively low in oilseed rape (Brassica napus) due to weak nitrogen remobilization during leaf senescence. Monitoring the kinetics of water distribution associated with the reorganization of cell structures, therefore, would be valuable to improve the characterization of nutrient recycling in leaf tissues and the associated senescence processes. In this study, nuclear magnetic resonance (NMR) relaxometry was used to describe water distribution and status at the cellular level in different leaf ranks of well-watered plants. It was shown to be able to detect slight variations in the evolution of senescence. The NMR results were linked to physiological characterization of the leaves and to light and electron micrographs. A relationship between cell hydration and leaf senescence was revealed and associated with changes in the NMR signal. The relative intensities and the transverse relaxation times of the NMR signal components associated with vacuole water were positively correlated with senescence, describing water uptake and vacuole and cell enlargement. Moreover, the relative intensity of the NMR signal that we assigned to the chloroplast water decreased during the senescence process, in agreement with the decrease in relative chloroplast volume estimated from micrographs. The results are discussed on the basis of water flux occurring at the cellular level during senescence. One of the main applications of this study would be for plant phenotyping, especially for plants under environmental stress such as nitrogen starvation.The main physiological outcome of leaf senescence is the recycling of organic resources and the provision of nutrients to sink organs such as storage and growing tissues (Buchanan-Wollaston, 1997;Hikosaka, 2005;Krupinska and Humbeck, 2008). In crop plants, senescence progresses from the lower older leaves to the younger top leaves. Macromolecular degradation and the mechanism of reallocation of breakdown products are mediated by the up-regulation of senescence-related genes (Lee et al., 2001) in close relationship with both developmental and environmental conditions (Gombert et al., 2006). This leads to remobilization of carbon and nitrogen (N) compounds mostly from plastidial compartments (Martínez et al., 2008;Guiboileau et al., 2012), involving proteolytic activity in plasts, vacuole, and cytosol (Adam and Clarke, 2002;Otegui et al., 2005), chlorophyll breakdown (Hoertensteiner, 2006), galactolipid recycling (Kaup et al., 2002) in the plastoglobules (Brehelin et al., 2007), and loading of Suc and amino acids into the phloem through appropriate transporters (Wingler et al., 2004;Masclaux-Daubresse et al., 2008). In terms of leaf senescence at the cell level, where chloroplasts are degraded sequentially, relative organelle volume does not seem to be greatly modified, the vacuole remains intact, and in darkness-induced senescence the number of chloroplasts per cell decreases only slightly (Keech et al., 2007). However, major changes in metabolic fluxes and cell water...

Leaf Development Monitoring and Early Detection of Water Deficiency by Low Field Nuclear Magnetic Resonance Relaxation in Nicotiana tabacum Plants

“…The transverse relaxation of each water pool can be distinguished thanks to the slow diffusion process between cell compartments compared to the NMR relaxation process. In oilseed rape leaves, a new attribution was recently demonstrated [30]. The palisade and spongy cells of young oilseed rape leaves are similar in size but develop differently, resulting in cells of very different size between the tissues in old leaves.…”

“…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].…”

“…The palisade and spongy cells of young oilseed rape leaves are similar in size but develop differently, resulting in cells of very different size between the tissues in old leaves. NMR relaxation was shown to be able to finely describe the changes in cell size that occur during leaf development and can be used to monitor the water in vacuoles of palisade or spongy cells in old leaves separately [30]. Applied on a wide leaf panel collected from plant grown in both controlled and field conditions, the low field NMR relaxometry was shown to provide precise and robust markers of development of oilseed rape leaves [30].…”

“…NMR relaxation was shown to be able to finely describe the changes in cell size that occur during leaf development and can be used to monitor the water in vacuoles of palisade or spongy cells in old leaves separately [30]. Applied on a wide leaf panel collected from plant grown in both controlled and field conditions, the low field NMR relaxometry was shown to provide precise and robust markers of development of oilseed rape leaves [30]. NMR relaxation is thus a powerful tool to monitor changes in water relations inside the leaf caused by development or different stress as Nitrogen deficiency [35] or thermal stress [36].…”

“…These effects of drought stress can be effectively monitored using NMR (nuclear magnetic resonance) relaxometry. NMR relaxation is a powerful technique to study changes in the status and distribution of water in plant tissues and has also been used to investigate water status in seeds [26], fruit [27], stem [28] leaves [29][30][31] or to monitor water relations in trees [32]. In plant tissues, the NMR transverse relaxation (T 2 relaxation) is always described by multi-exponential decay.…”