Differential palisade and spongy parenchyma structural changes in oilseed rape leaf were demonstrated. These dismantling processes were linked to early senescence events and associated to remobilization processes. During leaf senescence, an ordered cell dismantling process allows efficient nutrient remobilization. However, in Brassica napus plants, an important amount of nitrogen (N) in fallen leaves is associated with low N remobilization efficiency (NRE). The leaf is a complex organ mainly constituted of palisade and spongy parenchyma characterized by different structures and functions concerning water relations and carbon fixation. The aim of the present study was to demonstrate a specific structural evolution of these parenchyma throughout natural senescence in B. napus, probably linked to differential nutrient remobilization processes. The study was performed on 340 leaves from 32 plants during an 8-week development period under controlled growing conditions. Water distribution and status at the cellular level were investigated by low-field proton nuclear magnetic resonance (NMR), while light and electron microscopy were used to observe cell and plast structure. Physiological parameters were determined on all leaves studied and used as indicators of leaf development and remobilization progress. The results revealed a process of hydration and cell enlargement of leaf tissues associated with senescence. Wide variations were observed in the palisade parenchyma while spongy cells changed only very slightly. The major new functional information revealed was the link between the early senescence events and specific tissue dismantling processes.

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...

Improvement of nutrient use efficiency is a major goal for several crop plants, especially Brassica napus. Indeed, the low nitrogen use efficiency (NUE) in this crop results in negative economic and ecological consequences. The low NUE of oilseed rape is mainly due to low remobilization of nitrogen from vegetative parts to growing organs. Remobilization of leaf nitrogen takes place during senescence, a process known to strongly modify cell and tissue structure. This study focused on the impact of moderate N depletion, expected to induce 30 % reduction of seed yield, on these structural modifications. Two genotypes (Aviso and Express) were studied, with different tolerance of nitrogen depletion, evaluated through seed yield and dry mass production. Structural modifications of leaf cells and tissues were investigated through NMR relaxometry and light microscopy. Lower tolerance of N depletion was associated with higher impact on senescence associated structural modification pattern. The link between leaf structure modifications and nutrient remobilization is discussed. It is proposed that leaf structure monitoring during senescence through NMR device could be developed to select genotypes with high NUE.Electronic supplementary materialThe online version of this article (doi:10.1186/s40529-016-0125-y) contains supplementary material, which is available to authorized users.

SUMMARYIn order to obtain insights into the regulatory pathways controlling phloem development, we characterized three genes encoding membrane proteins from the G sub-family of ABC transporters (ABCG9, ABCG11 and ABCG14), whose expression in the phloem has been confirmed. Mutations in the genes encoding these dimerizing 'half transporters' are semi-dominant and result in vascular patterning defects in cotyledons and the floral stem. Co-immunoprecipitation and bimolecular fluorescence complementation experiments demonstrated that these proteins dimerize, either by flexible pairing (ABCG11 and ABCG9) or by forming strict heterodimers (ABCG14). In addition, metabolome analyses and measurement of sterol ester contents in the mutants suggested that ABCG9, ABCG11 and ABCG14 are involved in lipid/sterol homeostasis regulation. Our results show that these three ABCG genes are required for proper vascular development in Arabidopsis thaliana.

Drought is the main abiotic stress worldwide affecting harvest quality and quantity of numerous crops. To enable better water management, low field NMR (nuclear magnetic resonance) relaxometry was assessed as a developmental marker and a new method for early detection of water deficiency. The effect of a foliar biostimulant against water stress was also investigated. Two leaves of different ranks (four and eight) were studied. The leaves of different ranks were characterized by different NMR T 2 spectra which validated the ability of NMR to describe the developmental stage of tobacco. Results also showed that T 2 NMR relaxation spectra allow the detection of mild water stress (80% of the field capacity) through the precise characterization of the leaf water status while other water stress markers (relative water content, photosynthetic related parameters . . . )were not yet impacted. The agricultural impact of the mild water stress was determined through the nitrogen rate in shoots and amino acids assay six weeks after the beginning of the stress and results shows that foliar application of biostimulant limits the negative consequences of drought. Our results demonstrate the sensitivity of NMR to detect slight changes triggered in the leaf by water stress at the tissue level.

BackgroundLow field NMR has been used to investigate water status in various plant tissues. In plants grown in controlled conditions, the method was shown to be able to monitor leaf development as it could detect slight variations in senescence associated with structural modifications in leaf tissues. The aim of the present study was to demonstrate the potential of NMR to provide robust indicators of the leaf development stage in plants grown in the field, where leaves may develop less evenly due to environmental fluctuations. The study was largely motivated by the need to extend phenotyping investigations from laboratory experiments to plants in their natural environment.MethodsThe mobile NMR laboratory was developed, enabling characterization of oilseed rape leaves throughout the canopy without uprooting the plant. The measurements made on the leaves of plants grown and analyzed in the field were compared to the measurements on plants grown in controlled conditions and analyzed in the laboratory.ResultsThe approach demonstrated the potential of the method to assess the physiological status of leaves of plants in their natural environment. Comparing changes in the patterns of NMR signal evolution in plants grown under well-controlled laboratory conditions and in plants grown in the field shows that NMR is an appropriate method to detect structural modifications in leaf tissues during senescence progress despite plant heterogeneity in natural conditions. Moreover, the specific effects of the environmental factors on the structural modifications were revealed.ConclusionThe present study is an important step toward the selection of genotypes with high tolerance to water or nitrogen depletion that will be enabled by further field applications of the method.

Consumption of fresh‐cut vegetables has rapidly increased over the past decades. Among salads, escarole is one of the most popular varieties. Specific packaging limits gas exchange and consequently water loss and bacterial respiration, increasing the shelf life of salads. Although the major cause of quality loss for minimally processed salads is the leaf textural changes, this aspect has rarely been investigated. Therefore, investigating structural changes of leaves during storage is important in order to understand and minimize quality loss of salads. In this study, we focused on the impact of storage duration and temperature on the escarole leaf structure. The complex leaf structure was investigated by relaxation NMR, via transverse relaxation times, which allows the specific description of vacuolar water compartment of the cell. The storage duration (maximum 12 days) and temperatures (4°C, 7°C, 10°C, and 12°C) have been chosen in order to represent the conditions registered in factory. The results showed that the temperature did not have significant impact on the salad structure during the first week. During the second week, changes in the water distribution and changes in the relaxation time T2 have been observed. The changes in transverse relaxation times associated with vacuolar water are related to lost of cell membrane and wall integrity. The NMR results confirmed the effect of storage temperature on the degradation process of the cell before visual detection of the salad leaf degradation. The present study confirmed the sensibility of NMR relaxometry for monitoring water changes in the leaf.

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