SEASONAL TRANSLOCATION OF
FOLIAR NITROGEN WITHIN BEECH TREES

Introduction 

From fifty years, drought periods and heat waves have increased in worldwide as a consequence of global climate change and this trend is expected to increase in the coming decades (Coumou et al., 2013, Wagner et al., 2013, IPCC, 2013). These climate hazards can induce strong reductions of primary productivity (Piovesan et al., 2008) and sometimes forest decline and tree mortality. Understanding of the physiological processes involved in tree dysfunctions and forest tree decline is a major concern (Bréda et al., 2006, McDowell et al., 2008, Sala et al., 2010) Two main but non-exclusive physiological mechanisms have been proposed: hydraulic failure and carbon starvation (McDowell et al., 2008, Sala et al., 2012, Hartmann et al., 2013). On one side, hydraulic failure may lead to mortality when partial or total loss of xylem function occurs in response to drought. On the other side, a carbon starvation is also possible when drought lasts long enough to create a carbon imbalance between sink demand and source supplies (photosynthesized or remobilized carbon). Besides these two hypotheses, some authors have recently suggested a reduced nutrient availability in response to drought, especially nitrogen (N) which could also play an important role in tree dysfunction (Gessler et al., 2016). However experimental evidence of how N cycling of adult forest trees could be modified by harsh conditions is still missing today. Available studies focused only on seedlings but these results are difficult to generalize if ontology have an impact on tree N balance, as noticed on C balance (Cavender and Bazzaz, 2000, Gilson et al., 2014). Forest ecosystems have often developed on poor soils which were not convenient for optimal growth due to the low N availability (Raven and Andrews, 2010). To cope with this N deficiency, trees adopted dedicated strategy by optimizing N use efficiency and internal recycling (Vitousek, 1982). The internal N cycling consists in N storage in perennial structures (wood and roots) when N requirements are low and remobilization of these N reserves towards sinks (mainly the foliage) in case of N shortage when current demand exceeds root N absorption capacity due to the seasonality of plant growth (Chapin et al., 1990). In fact, in early spring, N uptake by roots is not sufficient to sustain spring growth and trees rely on stored N as largely demonstrated on fruit trees (O’Kenney, 1975, Millard and Nielsen, 1989, Neilsen et al., 1997, Tagliavini et al., 1998, Cheng et al., 2002, Dong et al., 2002) and more recently on mature forest trees (El Zein et al., a & b 2011, Valenzuela Nunes et al., 2011, Bazot et al., 2013). This net decrease of stored N observed in spring cannot be recovered fastly and so, during the vegetative season, trees rely mainly on N soil uptake by roots (Bazot et al., 2013, Villar-Salvador et al., 2015). In autumn, a mechanism called N resorption occurs and consists in the degradation of foliar proteins into 123 amino acids and the transport via the phloem towards bark and wood parenchyma where they are stored during winter under amino acids and vegetative storage proteins forms (Sauter et al., 1989, Wetzel et al., 1989, Stepien et al., 1994, Millard, 1996). Mobilization and refilling of N stores lead to seasonal fluctuations in these storage pools. As N soil is often a growth-limiting factor in natural forest ecosystems (Rennenberg et al., 1998) and the ability of trees to store and redistribute N resources internally is a fundamental process conditioning their survival. A limited soil N availability caused by a stress like drought will end to an inability of roots to explore the soil and to a decrease of microbial activity (Kreuzwieser and Gessler, 2010, Creeger et al., 2014), increasing the risk that N uptake will not be sufficient enough to refill tree internal reserves. Consequently, Gessler et al., (2016) hypothesized that the internal N cycling, could become critical for tree survival when drought occurs in early spring and summer especially in deciduous tree species. In relation to the Gessler’s hypothesis previously presented (2016), we explore in our study how the tree internal N cycle is modified by a prolonged drought or a yearly manual defoliation in eight-year-old beech trees submitted to two years of constraints. As mentioned above, a soil water deficit is expected to decrease both N soil availability and uptake by trees. In another way, any drastic defoliation may cause a harsh loss of N in trees because thirty-height percent of N are located in leaves in June on beech trees (El Zein, 2011). So by these two severe constraints, we expect to decrease the tree N availability leading to adjustments on internal tree N metabolism to maintain fundamental tree functions (growth, maintenance and storage). To follow these adjustments, we labeled the whole foliage of control, defoliated and water stressed beech trees with 15N-urea before leaf senescence in autumn. Then, we followed the fate of 15N from senescing leaves toward perennial organs in the whole tree during the winter storage, and then its remobilization for spring growth. We chose to work on European beech trees (Fagus sylvatica L.) trees, one of the most widespread and abundant species in Europe, because it is known to be more drought sensitive than other European broad-leaves species (Zang et al., 2014; Zimmermann et al., 2015) but paradoxaly it is also one of the most resistant to mortality. Given its survival capacity, we hypothesized that its resources management is particular efficient to face to constrains. Various authors also emphasize the remarkable potential for recovery after drought stress of Fagus sylvatica (Elling et al., 2007). In the present study, we made the following hypotheses: (1) drought and defoliation applied repeatedly for two years will create a significant decrease of the tree N pool; (2) due to this N reduction, the stressed tree will intensify its leaf N recycling   and export more N in autumn toward perennial organs compared to a control tree in an attempt to counterbalance a marked decrease in total N winter storage level. (3) The expected N storage decrease in the stressed trees will impact the level of remobilized N available for growth increment and canopy establishment in spring. (4) Finally, if the N remobilization is source driven (Millard and Grelet, 2010), a reduced growth without any changes in N mobilization intensity and its partitioning between perennial organs and new formed organs (twigs, leaves) will be expected in stressed trees compared to controls

Experimental design and growth conditions

The study was conducted on European beech trees. In 2006, beech seeds were collected in several forests in the Lorraine region of France and sown in 2007 in biodegradable horticultural pots made of wood fiber and filled with a peat and sand mixture. The seedlings were grown for one year in a nursery (INRA Grand-Est Nancy, France). In 2008, about 1000 of the seedlings were transplanted and grown for 7 more years in open ground at the INRA Grand-Est nursery (Champenoux, France, 48°75’N, 6°34’E, 229m asl). In 2014, a rain exclusion system was built above the 8-year-old trees: a semi-rigid structure supporting a transparent roof built with polycarbonate sheets and nets installed around the roof to intercept lateral rain. The trees under the roof were subjected to four different treatments for two years (2014, 2015): (1) control (C) in which the trees were regularly irrigated; (2) defoliation (D) in which the trees were submitted to a yearly defoliation and regularly irrigated: manual defoliation of the trees in treatment D was done each year in June (Figure VII.1.A); 75% of the total foliage was removed and the removal was homogeneously distributed throughout the tree crown; (3) moderate drought (MD) and (4) severe drought (SD), where the trees were submitted to two levels of soil water deficit. The soil in the drought treatments was isolated by a rigid waterproof plastic sheet 1.80 meters depth buried vertically around the area. The two drought stress levels were not designed to realistically simulate a climate change scenario, but rather to create drought conditions that were so unfavorable that they would likely cause beech tree dysfunction and mortality. In fact, lateral rain entering under the roof created some variability in soil water status in the drought treatment at the time of labeling and this allowed us to select trees with contrasting levels of water stress. The hydraulic status of the chosen trees for the experiment in each treatment (8 trees in C and D, 5 in MD and SD in September 2015 and 6 trees in C and D, 3 in MD and SD in June 2016) was checked by measuring pre-dawn water potential in twigs (ψpd) in September 2015 and in June 2016. We sampled the twigs (one per tree) before sunrise and performed the ψpd measurement with a pressure chamber (PMS Instruments, Albany, OR, USA).

Soil characteristics and soil water content measurements

 The studied site was characterized by 60cm-deep homogenous soil with an average texture (Silt: 61 ± 1.28%; Clay: 27 ± 0.98%; Sand: 12 ± 0.66%), a pH comprised between 7.5 and 8, an organic matter content between 12.1 and 14.9 g.kg-1 (E Silva, 2010) and a total N comprised 126 between 0.54 to 0.87 g.kg-1 . Below 60cm, the grey marl of the Jurassic inferior (Lotharingian) era was characterized by a swelling heavy clay soil with a relatively high bulk density. We used neutron probes (TROXLER TX 4301, Research Triangle Park, NC, USA) to measure the volumetric water content of the soil. Three neutron probe access tubes (aluminum, closed at their base) were installed in each of the four treatment areas in order to quantify water content at different depths: two ranged from 0-1m in depth and one ranged from 0-1.6m. During the growing season, measurements were carried out every two weeks. Counts were logged every 10 cm for the upper 100 cm, and every 20cm below that. For each depth i (thickness ti), Total Available Water soil Content (TAWC in mm) was calculated by estimating the characteristic points from pedotransfer classes for gravimetric soil moisture at field capacity (θfc) and gravimetric soil moisture at wilting point (θwp). The characteristic points were checked and adjusted with probe measurements, during winter for volumetric soil moisture at field capacity and during summer for volumetric soil moisture at wilting point. Soil bulk density was assessed with the cylinder method. Relative Extractable soil Water (REW in %) was calculated according to Bréda et al., (1995) as follows: REW=100* TAWC-R TAWC where R is the actual volumetric soil water content in mm, and total soil extractable water content down to 1.60m is estimated to 310 mm. The soil in the C and D treatments was irrigated regularly throughout the experiment with an automatic drip watering system which delivered between two and four liters per tree two to three times a week. We adjusted the amount of the water according to the REW measurements in order to avoid any water shortage (REW >0.4), with 40% of the REW corresponding to the critical threshold where trees start to avoid water loss by closing their stomata (Granier et al.,. 1999). 

Foliar 15N labeling procedure 

The labeling experiment was performed at end of September 2015 (DOY: 271), before leaf fall. The timing of labeling is summarized in Figure VII.1. Forty-four trees were randomly chosen for labeling. On each tree, a crown bag made of polyethylene was placed over the total foliage of the tree to isolate it from its local environment. In the late afternoon, an aqueous solution of 15N urea was sprayed inside the bag onto the leaves with a hand sprayer (Zeller et al., 1998). (19) 127 The urea solution (10.4 atom%, 5.0 g.L -1 ) was sprayed in a fine mist, which limited the formation of drops and ensured a homogeneous labeling of the leaves. After the labeling, the plastic bag was kept on all night, then very carefully removed the next morning to avoid any contamination among trees. A net was put all around the tree to collect all the litter from the labeling through the winter fall. 

Sampling protocol Green leaves were sampled in July 2015 

2 months prior the labeling to measure the leaf N concentration in the control, defoliated and water stressed trees (n=12 in each treatment). In October 2015, one month after the labeling, we harvested 8 trees (2 trees per treatment) in view to assess the incorporation of 15N in the internal N cycle and its presence in perennial storage organs. Then, trees were harvested at two key phenological dates (El Zein, 2011) after the labeling: 1) in February 2016, 5 months after labeling at the theoretical highest storage level of N in perennial organs; and 2) in June 2016, 9 months after labeling at the theoretical end of N remobilization, once leaf expansion was done. We harvested 18 trees in February and June (6 C; 6 D; 3 SD; 3 MD), i.e. a total of 44 trees were labeled and harvested during this experiment. Ten unlabeled trees (3 C; 3 D; 2 MD; 2 SD) were also harvested in October 2015 to assess the natural abundance of 15N in each tree compartment. Each tree was separated in its compartments (leaves, branches, trunk and roots). Roots were separated according to their diameter: fine roots (d<1mm), lateral roots (13mm). We collected the litter in February 2016 with use of litter net. Each compartment was weighted to get the fresh mass, immediately frozen in liquid nitrogen, and then stored at -80°C. Then compartments were freeze-dried (Dura-Top (r), Dura-Dry (r), FTS Systems (r), Stone Ridge, NY, USA), weighed to determine the dry matter (DM) and ground into a fine powder with a ball mill (CEPI SODEMI CB2200, Cergy, France). The timing of labeling and harvest is displayed in Figure VII.1.