extfieldsofstudy:
Materials Science
Physics

provenance: AgriculturalAndFoodSciences,2019-2020-Agricultural And Food Sciences-2019.jsonl.gz.gz

text:
The authentication of peaberry and civet ground roasted robusta coffee using UV-visible spectroscopy and PLS-DA method with two different particle sizes

The objective of this research was to investigate the influence of particle size of ground roasted coffee samples in the authentication of peaberry and civet coffee using UV-visible spectroscopy combined with partial least squares-discriminant analysis (PLS-DA) method. For this purpose, we provide a total of 80 samples of peaberry and civet coffee with two different particle size of 1680 micrometer (mesh 12) and 297 micrometer (mesh 50). The all coffee samples were extracted using hot distilled water (90-98°C). UV-visible spectral data of the all coffee samples were acquired using a UV-visible spectrometer in transmittance mode. The result showed that the different absorbance spectra were observed for the different particle sizes of peaberry and civet coffee samples. The absorbance of samples having particle size of 297 micrometer (mesh 50) is higher than that of particle size of 1680 micrometer (mesh 12). The calibration model was developed using PLS-DA method for samples having same particle size and resulted in acceptable result with high coefficient of determination (R2) (R2=0.99 both for mesh 12 and 50, respectively), low root mean square error of calibration (RMSEC), and high residual prediction deviation (RPD). However, the performance of the prediction results was very low for samples having different particle size with high bias and high root mean square error of prediction (RMSEP).

Introduction
Peaberry coffee ('kopi lanang' in Indonesian language) and civet coffee ('kopi luwak' in Indonesian language) are one of the expensive specialty coffees in the world. A peaberry (also called caracol or snail in Spanish) is a natural mutation of the coffee bean inside the cherry. Normally two coffee beans grow in a fruit (dicotyledonous)-flat against each other like halves of a peanut; however, on rare occasions a single bean is produced (monocotyledon) [1]. Civet coffee is any coffee bean (arabica or robusta) which has been eaten and passed through the digestive tract of Asian palm civet (Paradoxurus , which uses its keen senses to select only the best and ripest berries [2][3].The production of both peaberry and civet coffees are very limited. For this reason, peaberry and civet coffees have been a target of food adulteration. Nowadays, the keyword of food authentication is becoming popular. This authentication system may give benefit to both producers and customers. For this reason, the area of research for food authentication is attracting many researchers. The development of new analytical method for food authentication system is one of the research focus in this area. No exception for coffee authentication system. Several analytical methods have been proposed for coffee authentication such as NIR and mid spectroscopy [4][5][6][7][8][9], fluorescence spectroscopy [10][11], image processing/computer vision [12], nuclear magnetic resonance (NMR) spectroscopy [13], and electronic nose [14]. However, most of these methods are expensive and not easy to follow.
Previously, Suhandy and coworkers has developed an authentication and quality evaluation of ground roasted coffee using UV-visible spectroscopy and chemometrics [1][2][15][16][17]. This method is simpler and cheaper. However, all those reported studies used coffee samples with same particle size (same mesh) for developing calibration and validation model. Those researches involved procedure of sieving of ground roasted coffee that is laborious and time consuming. Therefore, for practical application, it is needed to remove the procedure of sieving and establish calibration model of coffee authentication which can be universally used for any particle sizes. However, there is lack of research regarding to relationship between particle sizes of ground roasted coffee and quality of spectral data in UV-visible region. In this research, an investigation on the influence of particle size of ground roasted coffee samples in the authentication of peaberry and civet coffee was conducted using UV-visible spectroscopy combined with partial least squares-discriminant analysis (PLS-DA) method.

Civet and peaberry coffee samples
In this research, 80 samples of peaberry and civet coffee with two different particle size of 1680 micrometer (mesh 12) and 297 micrometer (mesh 50) were prepared (1.0 gram weight for each samples). The samples were collected from coffee farmer in West Lampung, Lampung province of Indonesia. The extraction of coffee samples was performed by using hot distilled water. The samples were divided into two groups: calibration and validation set (30 samples for peaberry and civet, respectively) and prediction set (10 samples for peaberry and civet, respectively).

Acquisition of UV-visible spectral data
The spectral data of aqueous civet and peaberry coffee samples were acquired in the range of 190-1100 nm by using a UV-Vis spectrometer (Genesys™ 10S UV-Vis, Thermo Scientific, USA). This spectrometer was equipped with a quartz cell with optical path of 10 mm. The spectral acquisition was done at spectral resolution of 1 nm at a room temperature (about 27-28ºC). The original spectra were used for chemometric analysis.

Chemometric analysis
To study the influence of particle sizes of ground roasted coffee for authentication of peaberry and civet coffee, two methods were attempted. First is using principal component analysis or PCA (unsupervised chemometric analysis) method. The PCA was performed using all samples (80 samples) using original spectral data in the range of 250-350 nm. The second method is using PLS-DA or partial least squaresdiscriminant analysis. In this PLS-DA method, a PLS regression was employed with a dummy variable as a reference value (variable Y). In this research, the response variable Y is composed of 0´s and 1´s, where the value 1 is assigned to peaberry coffee samples and the value 0 to civet coffee samples. To evaluate the performance of PLS-DA model, the coefficient of determination (R 2 ), root mean square error of calibration (RMSEC) and root mean square error of cross-validation (RMSECV) are used in this research. It is expected to have ideal models with lower RMSEC and RMSECV as well as higher In the prediction, two statistic parameters were used to assess the model: SEP (standard error of prediction) and bias. Those two parameters should be as low as possible. PCA and PLS-DA were performed using the multivariate software of the Unscrambler 9.7 (CAMO software AS, Oslo, Norway).

Results and Discussion
3.1. UV-visible spectral data of civet and peaberry coffee with different particle sizes UV-visible spectral data of peaberry coffee with different particle sizes was depicted in Figure 1. Each data (peaberry mesh 12 and 50) was averaging of 10 spectral data. From Figure 1, we can notice that there is a different of peaberry spectral data between mesh 12 and 50 especially in the wavelength of 250-350 nm. The intensity of absorbance of peaberry with mesh 50 is higher than that of mesh 12. The similar phenomenon was observed for civet coffee as seen in Figure 2. This result showed that the quality of spectral data in UV-visible spectroscopy of coffee samples is highly influenced by particle sizes of ground roasted coffee samples.   Figure 3 showed the result of PCA analysis of all samples (80 samples) using original spectra in the range of 250-350 nm. Using two PCs (PC1 and PC2), a clear separation between civet and peaberry coffee samples was observed. The total of PC1 and PC2 can explain 100% of the total variance of original data. From here, we cannot see clearly the influence of different particle sizes on separation of (ICoSITeR) 2018 IOP Conf. Series: Earth and Environmental Science 258 (2019) 012043 IOP Publishing doi:10.1088/1755-1315/258/1/012043 4 civet and peaberry. More quantitative analysis should be conducted using PLS-DA in order to see the influence of particle sizes on discrimination between civet and peaberry ground roasted coffee.

Prediction samples of mesh 12 and 50 using PLS-DA model mesh 12
The performance of PLS-DA model mesh 12 was evaluated in two ways. First, the developed PLS-DA model mesh 12 was used to predict samples having mesh 12. Second, it was used to predict the samples having mesh 50. The result was showed in Figure 5. In term of SEP and bias, the prediction of samples having mesh 12 resulted in low SEP and bias (SEP = 0.085681 and bias = 0.030608). However, the result of the prediction of samples having mesh 50 became worst with increasing both the value of SEP and bias (SEP = 0.258729 and bias = 0.089416). This result has confirmed that the performance of PLS-DA model for civet and peaberry discrimination is highly influenced by particle sizes of the ground roasted coffee samples. For this, in the future for practical application, it is highly needed to develop PLS-DA model that can compensate the influence of particle sizes of coffee samples.

Conclusion
In this research, we showed the influence of particle sizes of ground roasted coffee samples in the authentication of peaberry and civet coffee. The influence was not so clear based on the result of PCA. However, using PLS-DA method, the influence was seen clearly. It is confirmed that the performance of PLS-DA model for civet and peaberry discrimination is highly influenced by particle sizes of the ground roasted coffee samples. For this, in the future for practical application, it is highly needed to develop PLS-DA model that can compensate the influence of particle sizes of coffee samples. By doing this, a procedure of sieving may be removed. Finally, faster authentication process of coffee samples using UV-visible spectroscopy could be established.


extfieldsofstudy: Medicine Biology

text:
A family of auxin conjugate hydrolases from Solanum lycopersicum and analysis of their roles in flower pedicel abscission

Background Auxin conjugates are hydrolyzed to release free auxin to ensure defined cellular auxin levels or gradients within tissues for proper development or response to environmental signals. The auxin concentration in the abscission zone (AZ) is thought to play an important role in mediating the abscission lag phase. Results In this study, the full cDNA sequences of seven tomato ILR1-like SlILL genes were identified and characterized, All SlILLs were found to have auxin conjugate hydrolysis activity. The effects of different auxin conjugates on abscission identified IAA-Ile as a candidate to determine the auxin conjugate and auxin conjugate hydrolysis functions in abscission. Treatment of pedicel explants with IAA-Ile for different times showed that application before 6 h could effectively delay abscission. IAA-Ile pre-incubation for 2 h was sufficient to inhibit abscission. These results showed that there is not sufficient auxin conjugates in the AZ to inhibit abscission, and the optimal time to inhibit abscission by the application of exogenous auxin conjugates is before 6 h. Treatment with cycloheximide (CHX, a protein biosynthesis inhibitor) indicated that de novo synthesis of auxin conjugate hydrolases is also required to delay abscission. During abscission, SlILL1, 5, and 6 showed abscission-related gene expression patterns, and SlILL1, 3, 5, 6, and 7 showed increasing expression trends, which collectively might contribute to delay abscission. Silencing the expression of SlILL1, 3, 5, 6, and 7 using virus-induced gene silencing showed that SlILL1, 5, and 6 are major mediators of abscission in tomato. Conclusions In the process of abscission, auxin inhibition is concentration dependent, and the concentration of auxin in the AZ was regulated by hydrolyzed auxin conjugates. SlILR1, 5, and 6 play a key role in flower pedicel abscission. Electronic supplementary material The online version of this article (10.1186/s12870-019-1840-9) contains supplementary material, which is available to authorized users.

Background
Indole-3-acetic acid (IAA) is an essential regulator of many aspects of plant growth and development [1]. In most tissues, auxin responses are concentration dependent, and different tissues respond in a distinct manner to varying amounts of exogenous auxins [2,3]. Low IAA concentrations stimulate growth, while high concentrations can be inhibitory to organ development or are toxic to the plant [4,5]. In plant organs, local auxin concentrations are altered to maintain optimum concentrations using various mechanisms, such as biosynthesis, degradation, transport, and conjugate formation. Auxin conjugates are formed to turn free auxin into bound forms, in which the carboxyl group is conjugated to sugars via ester linkages, or to amino acids or peptides via amide linkages [6,7]. However, little is known about the spectrum of auxin conjugates present in a given tissue because of the difficulty in analyzing individual conjugates. The function of auxin conjugates has been mainly determined using mutant analysis, implying that auxin conjugates are connected with development of the embryo, shoot, and vasculature through their possible roles in storage and transport of auxins and protecting free IAA from degradation [2,8]. The content of free IAA and that stored as conjugates are maintained in a balance. Under normal conditions, only very small amounts, approximately 5%, of the auxin in a plant is free; the majority of the auxin is conjugated into amide-linked and ester-linked conjugates of IAA as storage forms [5,9,10]. However, only a fraction of the IAA conjugates, such as IAA-Ala, IAA-Leu, IAA-Phe, and IAA-Ile are hydrolyzed back to free IAA via auxin amino acid conjugate hydrolyses, and most intriguingly, IAA-Ala can act as an auxin without being hydrolyzed to release free IAA in tomato cell culture, whereas the amino acid conjugates IAA-Asp and IAA-Glu are thought to be precursors for a degradation pathway. Thus the different IAA conjugates vary in their effects on plant development and responses, such as seed germination and root elongation [2,[11][12][13].
The hydrolysis of amino acid-type IAA conjugates has been studied in great detail, revealing gene families from various plant species with distinct, yet overlapping, substrate specificities for various conjugates of both IAA and indole-3-butyric acid (IBA) [4,12,14,15]. In Arabidopsis, ILR1 cleaves IAA-Leu and IAA-Phe preferentially, whereas the ILL1, ILL2, and resistant IAR3 enzymes prefer IAA-Ala as a substrate. ILR1, IAR3, ILL1, and ILL2 encode active IAA-amino acid hydrolases, and there are three additional amidohydrolase-like genes (ILL3, ILL5, and ILL6). Single mutants of ILL1 and IAR3 show a normal plant phenotype. MtIAR31, − 32, − 33, and − 34 from Medicago truncatula have hydrolytic activity against IAA-aspartate and IBA-alanine. Only MtIAR36 is highly active against IAA-glycine, −alanine, and -isoleucine (IPA) [4,14,16,17]. The different enzyme activities and expression patterns make it reasonable to deduce that induction of one specific hydrolytic activity in response to a particular challenge, suggesting that various conjugate hydrolases might supply free IAA in response to a variety of needs [1,2,7].
When the abscission zone (AZ) auxin concentration drops below a certain threshold, abscission is initiated [18][19][20]. The AZ auxin is mostly derived from de novo synthesis in the young organ. Removal of flowers or leaves to deplete the AZ auxin source will induce abscission rapidly compared with the normal abscission caused by the absence of pollination or pathogen attack [5,7,21]. However, even in this process, abscission is not initiated immediately, and the delay stage can last for at least two hours [20,22]. This is a problem if stored auxin conjugates in the AZ are not converted to active free IAA, because free IAA is very unstable and would oxidase or degrade in several minutes. Previous studies have shown that there are peaks of increased IAA levels after flower removal during abscission, which further confirms the view that hydrolysis of auxin conjugates plays an important role in mediating organ abscission [20,22]. However, because the ILL protein family members in tomato are not well characterized, little information is available concerning the function of auxin conjugate hydrolysis in mediating flower pedicel abscission. The auxin conjugate hydrolase family members are present in both monocot and dicot plants [22]. The entire gene families have been well characterized in some species; for example, seven members of this family have been identified from Arabidopsis, six from Brassica rapa [15], and six from Medicago truncatula [14].
In this study, we isolated the full cDNA sequences of seven novel SlILLs from tomato (Solanum lycopersicum L.). Enzyme assays confirmed that these SlILLs have auxin conjugate hydrolysis activity. Treatment with IAA-Ile for various times in the presence of cycloheximide (CHX, a protein biosynthesis inhibitor) showed that there is inadequate auxin conjugate and auxin conjugate hydrolyzing enzyme levels in the AZ to inhibit abscission, and the optimal time to inhibit abscission by applying exogenous auxin conjugate is before 6 h. Virusinduced gene silencing (VIGS) of SlILL1, 3, 5, 6 and 7 expression showed that the SlILL1, 5, and 6 genes play major roles in mediating tomato abscission.

Identification, isolation, and sequence analysis of the SlILL gene family in tomato
To identify the tomato ILL family genes, BLAST searches of the tomato sequences at the SGN database were performed using the ILL domains of the seven Arabidopsis proteins as queries. A total of seven ILL-domain-containing unigenes were obtained using TBLASTN searches. The full-length cDNA sequences of all seven putative SlILLs were isolated using PCR-based approaches. The lengths of the open reading frames (ORFs) of the SlILL genes varied from 1628 bp (SlILL2) to 1902 bp (SlILL7), and they are predicted to encode polypeptides of 430-485 aa. These genes were named Solanum lycopersicum IAA-amino acid hydrolase/IAA-leucine resistant-like (SlILL) genes (Table 1).
We used the PSORT program (http://www.genscript. com/psort.html) to predict protein localization sites in cells; analyses of the predicted protein products suggested that none of the hydrolases are localized to the endoplasmic reticulum (ER). The SlILL4 and SlILL7 proteins contain the N terminal HDEL sequence that has been annotated as an ER retention signal, but according to PSORT, the probability of ER localization is low. The sequences of SlILL4 and SlILL6 appear to possess an uncleavable N-terminal signal sequence, while the other SlILL proteins all have a cleavable N-terminal signal sequence, after which the mature proteins are predicted to start ( Table 1). The percent identity between the different tomato and Arabidopsis ILR1-like proteins ranges between 44 and 87% (Additional file 1: Figure S1, Table 2). Phylogenetic analysis of the predicted ILL protein sequences revealed that the AtILR and SlILL proteins are divided into two well defined clades; SlILL2, 4, 6, and 7 are in cluster I, and SlILL 4 and SlILL7 show a close relationship to AtIAR3 and AtILL5, while SlILL6 clusters with AtILL6. SlILL1, 3, and 5 are in cluster II, with SlILL1 and 3 in a subcluster with AtILR1, and SlILL5 clusters with AtILL3 ( Fig. 1).

Auxin conjugate hydrolase activity of the SlILL proteins
The SlILL proteins were purified using affinity chromatography (Additional file 2: Figure S2) and tested for IAA conjugate hydrolysis activity using different IAA amino acid conjugates. All SlILLs hydrolyzed IAA-Ala efficiently, and SlILL1 had the highest activity. Similar to IAA-Ala, all of the SlILLs were able to hydrolyze IAA-Ile and IAA-Gly, but had lower activities against these substrates. Only SlILL1 and SlILL4 hydrolyzed IAA-Asp, and the relative activities were very low. None of the SlILLs could hydrolyze IAA-Trp. (Table 3).

Expression patterns of SlILL genes in different organs of tomato
The SlILL gene expression patterns in several tomato organs were also investigated. SlILL1 showed its highest level of expression in the flower, and the lowest expression level in the abscission zone, with intermediate levels of expression in the leaf, stem, and fruit. SlILL2 was most highly expressed in the flower, fruit, and AZ, while extremely low levels of expression were found in the stem. For SlILL3, a similar expression level was found in all tomato organs except for low expression in the AZ. SlILL4 was expressed at a low level in the stem and AZ, while expression was somewhat higher in the other organs. The highest expression levels of SlILL5 were in the flower and root, with lower levels of expression in the other organs. SlILL6 was expressed at high levels in all organs except the leaf, and SlILL7 had relatively high levels of expression in the flower, fruit, and root ( Fig. 2).  The effects of different auxin conjugates on abscission To determine the effect of auxin conjugates on abscission, 30 μM solutions of IAA-Ala, IAA-Asp, and IAA-Ile were applied to pedicel explants. Both IAA-Ala and IAA-Ile could significantly delay abscission, while IAA-Asp had little effect. IAA-Ala has free auxin activity without hydrolysis; therefore, IAA-Ile was used for the subsequent abscission assays (Fig. 3a).
To explore the optimal time of auxin conjugates application for abscission, two experiments were performed. First, explants were pre-incubated on control agar (without IAA-Ile) then transferred to agar containing IAA-Ile. This experiment showed that explants incubated on control agar and transferred to IAA-Ile agar before 6 h showed a significant level of abscission. However, there was no significant inhibition of abscission after 6 h (Fig. 3b). Second, the explants were initially pre-incubated for different times in IAA-Ile and then transferred to agar without IAA-Ile. The results indicated that only 2 h of preincubation with IAA-Ile was sufficient to inhibit abscission. (Fig. 3b, c).
We performed another experiment to test whether there is adequate IAA-amino acid hydrolase activity in the AZ to inhibit abscission if there is an adequate level auxin conjugate present. The explants were incubated with or without CHX for 0, 2, and 4 h, and then transferred to IAA-Ile agar containing CHX. The results indicated that CHX could accelerate abscission compared with the control and drastically reduced the inhibition of abscission by IAA-Ile. Notably, the 0-h CHX treatment shows a higher rate of abscission than the control (Fig. 3d).  Auxin conjugates can modify the free IAA concentration in the AZ After removal of flowers from the auxin reporter DR5::GUS lines, β-Glucuronidase (GUS) staining in the AZ was stable before 2 h, increased at 4 h, and then decreased to 16 h. Incubation with IAA-Ile could significantly increase the auxin content in the AZ, and the significant decrease observed after 4 h showed that the auxin conjugate could enhance free auxin concentration in the AZ. It is notable that there was no significant difference in GUS activity at 2 h in the AZ between the control and the IAA-Ile treatment (Fig. 4). There was adequate auxin conjugate (as 4 h could release more free IAA) present in the AZ and IAA-Ile in the agar; therefore, the only reason for this phenomenon is insufficient auxin conjugate hydrolysis in the AZ before 2 h. Auxin concentration analysis confirmed that IAA-Ile could significantly enhance the AZ auxin concentration after 4 h compared with that in the control (Fig. 5, Additional files 3 and 4). Moreover, in the IAA-Ile treatment, the free auxin did not show a constantly increasing trend, but rather showed a slight decrease after 4 h.

Expression of SlILL genes in response to 1-MCP and auxin treatment during flower-removal pedicel abscission
Abscission rates (AR) of tomato pedicel explants increased significantly after 8 h, reaching 100% at 32 h, while auxin or 1-MCP (1-methylcylopropene, a competitive inhibitor of ethylene action that binds to the ethylene receptor to regulate tissue responses to ethylene) completely inhibited abscission before 48 h (Fig. 6). SlILL1 showed steadily increasing expression in the control AZ during abscission, while its expression remained relatively stable in the NAZ (the proximal part of the AZ). 1-MCP treatment increased the expression of this gene in both the AZ and NAZ. Unlike SlILL1, SlILL2 expression decreased sharply immediately after flower removal in both the AZ and NAZ. 1-MCP treatment did not alter SlILL2 expression. SlILL3 expression showed a slight increase in the control (CK) up to 4 h, followed by a decrease in the AZ, and a similar expression pattern Overall, the expression levels of SlILL1, 5, and 6 showed a continuous increase in the AZ, which is distinct from the NAZ, and was classified as AZ special expression; the expression of these genes was affected by 1-MCP, and they were selected as abscission-related auxin conjugate hydrolase genes for further study (Fig. 7).
Accumulation of SlILLs protein in response to 1-MCP treatment during flower-removal pedicel abscission SlILLs protein accumulation in the AZ was then assayed. The level of SlILL1 protein increased steadily during abscission, and 1-MCP treatment Using VIGS to downregulate SlILL1, SlILL5, and SlILL6 gene expression significantly accelerated pedicel abscission To understand the function of SlILL genes in abscission, the effect of silencing SlILLs on pedicel abscission was examined using VIGS. In this study, we used ten silencing constructs designed to silence SlILL1, SlILL3, SlILL5, SlILL6, and SlILL7 individually, to double-silence SlILL1 + 5, SlILL1 + 6, SlILL5 + 6, and SlILL3 + 7, and to triple silence SlILL1 + 5 + 6 in tomato. The SlILL gene expression levels in the different SlILL VIGS lines were quantified using qRT-PCR. The results confirmed the In the control, the abscission rate was 43% at 16 h, 85% at 24 h, and 100% at 32 h. Silencing SlILL1, SlILL5, and SlILL6 individually significantly accelerated abscission, with 54% pedicel explants abscised at 16 h and 93% at 24 h. The double-silenced SlILL1 + 5, SlILL1 + 6, SlILL5 + 6 lines all showed increased abscission > 60% at 16 h and 100% at 24 h; however, the SlILL3 + 7 doublesilenced line showed no significant difference compared with the control. The triple-silenced SlILL1 + 5 + 6 line showed the most obviously increased abscission rate, which was higher than any of the double-silenced SlILL lines (Fig. 9). The time required for 50% abscission also indicated that SlILL1, SlILL5, and SlILL6 have important and cooperative roles in mediating abscission in tomato ( Table 4).
Downregulation of SlILL1, 5, and 6 significantly altered auxin concentration and polar auxin transporter (PIN) gene expression in the AZ SlILL5 and 6 showed peak expression at 2 h, and SlILL1 expression increased before 4 h in the AZ. Combined with the slight IAA concentration increase in the AZ at 4 h, we evaluated the effects of downregulating SlILL1 + 5 + 6 expression on the auxin concentration and PIN gene expression. The AZ was sampled from VIGS SlILL1 + 5 + 6 lines, which showed the most obviously increased abscission rate. The results showed that the auxin content was significantly decreased in the VIGS triple-silenced SlILL1 + 5 + 6 lines compared with that in the control (Fig. 10, Additional files 3 and 4). In addition, we checked the expression levels of all PINs in the AZ, and only PIN1, 3, 4, 6, 7, 8, and 9 have expression in AZ, and the expression of PIN3, 4, and 8 was significantly decreased in these lines (Fig. 11).

Discussion
Auxin conjugation and hydrolysis play important roles in mediating the auxin balance for proper plant development and response to environment signals [2,23]. Several auxin conjugate hydrolases have been isolated from the dicots Brassica rapa, Medicago truncatula, and Arabidopsis thaliana and the monocots Triticum aestivum and rice, and are involved in root development, pathogenesis, and grain weight [9,16,[24][25][26]. The biochemical characterization of substrate specificity and the different temporal and spatial gene expression  [25,27]. Although our understanding of IAA-regulated abscission processes, including the molecular mechanisms of action, perception, and transport, comes from studies of gene transcription analyzed in pre-abscission or auxin-depleted induced abscission, there remains a lack of knowledge about the potential role of IAA conjugate hydrolases in mediating abscission [20,22,28].
Identification, sequencing, and catalytic activity analysis of ILL genes and proteins in tomato In Arabidopsis, seven genes of the amidohydrolase family have been well characterized: ILR1, ILL1, ILL2, and IAR3 can cleave IAA amino acid conjugates, while ILL3 and ILL6 cannot, and ILL5 is thought to be a pseudo gene [11,26,29]. Genome scanning and DNA sequencing identified seven full-length tomato cDNAs, SlILL1 through SlILL7, that are highly homologous to the IAA-amidohydrolases of Arabidopsis. The IAR3-like (Arabidopsis and Brassica) AtILL1 and AtILL5 proteins contain a C-terminal KDEL sequence that signals retention of plant proteins in the lumen of the ER. However, none of the SlILL proteins contains a terminal KDEL, and are therefore predicted to have a lower probability of being located on the ER, although SlILL1, 2, and 7 have a terminal HDEL motif (similar to Br-ILL6), which is annotated by PSORT as an ER retention signal [15]. The importance of ER localization is not clear, however, because phylogenetic analysis of 66 orthologs from across the plant kingdom resolved two separate monocot clades, one with members possessing ER retrieval sequences and one lacking them [2,30,31]. The ER retention signal is not required for activity, because two of the five amidohydrolases characterized from M. truncatula lack such a tetrapeptide, but still show activity [14], and TaIAR3 has the unusual C-terminal sequence motif RDEL [16]. Moreover, a cleavable N-terminal signal sequence was found in SlILL1, 2, 3, 4, and 7, while SlILL5 and 6 contain an uncleavable N-terminal sequence.
Previously, the structure of AtILL2 was found to be similar to members of the M20 peptidase family, which suggests that ILL2 likely uses a catalytic mechanism similar to that established for the M20 peptidase enzymes family. All SlILLs have the M20 dimerization domain, which implies that all of them have auxin conjugate hydrolysis activity. The experiment further indicated that all SlILLs are able to hydrolyze IAA-Ala. The different enzymes also showed distinct patterns of hydrolase activity. AtILL2 preferentially catalyzes the hydrolysis of IAA-Ala and the hydrolytic activity is higher than that of SlILLs, while AtILL1, which shares 87% sequence identity with AtILL2, showed a lower activity than the SlILLs toward IAA-Ala. AtILR1 prefers IAA-Leu, IAA-Tyr, and IAA-Phe, while showing less activity toward IAA-Ile, and less activity than SlILLs. ILL3 and ILL6/GR1 have no such catalytic activity towards IAA conjugates [11,30]. In Triticum aestivum, TaIAR3 has far less activity than SlILLs toward all of IAA-Ala, IAA-Ile, IAA-Asp and IAA-Gly substrates [4]. In Medicago truncatula, MtIAR31, − 32, − 33, and − 34 have hydrolytic activity toward IAA-aspartate and IBA-alanine, and shows higher activity than SlILLs toward IAA-Asp [16]. No hydrolase activity was found for IAA-Asp in vitro for all seven SlILLs; IAA-Asp may be an uncleavable auxin conjugate, and has no effect on abscission, hence it is possible that there is no specific auxin conjugate hydrolase for IAA-Asp in tomato.

Auxin conjugates are involved in flower pedicel abscission
Cellular IAA is mainly present as amide derivatives and, to a lesser extent, as ester-linked conjugates, in Arabidopsis thaliana [32]. The most common auxin conjugates in Arabidopsis are IAA-Ala, IAA-Leu, IAA-Asp, IAA-Glu, IAA-glucose, and protein/peptide conjugates [23,[32][33][34][35]. Auxin conjugation is reversible through the actions of specific hydrolases, with the subsequent release of free IAA. Therefore, IAA conjugates have auxin activity when applied exogenously and show physiological activity in regulating different developmental processes, such as seed germination and root elongation.
The level of endogenous auxin must fall below a certain threshold in the abscission zone to initiate abscission [36,37]. Endogenous auxin would be synthesized de novo and is mainly derived from young organs such as flowers, young fruits, and seeds; therefore, the local auxin concentration in the AZ is mainly mediated by auxin flux from flowers to stems. Flower removal abolishes auxin influx and then activates conjugate hydrolyase enzymes to release active IAA; however, this is insufficient to keep the AZ insensitive to ethylene and prevent abscission, which usually only lags the initial abscission by several hours. For complete inhibition of abscission, exogenous auxin is required. In this study, abundant IAA-Ala and IAA-Ile could completely inhibit abscission, while IAA-Asp had little effect. As previously reported, IAA-Ala possesses auxin activity without hydrolysis, so we chose to use IAA-Ile to study the effects of an auxin conjugate on abscission. IAA-Asp is thought to be involved in a degradation pathway and, because it does not release free auxin, it has a minimal role in abscission. In auxin-depleted induced tomato pedicel abscission, IAA in the AZ declined during the early stages (before 8 h) when abscission was initialized, then increased at 16 h, which is an ethylene-accelerated abscission stage. About 60% percent of the flowers abscised during this stage. The low auxin concentration in the AZ is linked to increased ethylene sensitivity, and might also result in secondary cell wall deposition at the site of the reduced auxin response, which is instrumental during the separation process [38,39]. The different times of treatment showed that before 6 h, IAA-Ile could effectively delay abscission, while there was little effect after 8 h. Moreover, incubation with IAA-Ile for 2 h was sufficient to inhibit abscission. These results showed that there is not enough auxin conjugate in the AZ to inhibit abscission, and that the optimal time to inhibit  To determine whether de novo synthesized auxin conjugates are required for abscission, the tomato pedicel explants were incubated in MS agar for different times then transferred to IAA-Ile medium containing 10 mM CHX (cycloheximide). Inhibition of de novo synthesis of auxin conjugate hydrolase enzymes would decrease the auxin concentration and accelerate abscission. Our results showed that induced auxin conjugate hydrolases are required for the release of more free auxin to delay abscission. The results also indicated that there is not enough auxin conjugate hydrolase activity before 4 h and insufficient levels of auxin conjugate after 8 h. In our Fig. 11 The effects of downregulated expression of SlILL1, SlILL5, and SlILL6 on the expression of seven PIN genes in AZ during abscission. The results are means of three biological replicates and three technical replicates ± SD previously study, the early abscission stage was found before 8 h, and the dynamics of free auxin in the AZ seems to be a major regulator for this process [22].

Expression of tomato SlILL genes during abscission
Assaying SlILL gene expression in the different tomato organs showed that SlILL1, 3, 4, and 7 are expressed at a relatively low levels, while the expression of SlILL2 and 5 is somewhat higher in the AZ compared with that in other organs. Notably, only SlILL6 had a high level of expression in the AZ. Moreover, SlILL1, 5, and 6 showed unique expression patterns in the AZ, and the expression of all three genes was altered by 1-MCP and auxin treatments, showing that gene expression is highly associated with abscission. A previous report showed that wounding could induce the expression of auxin conjugate hydrolases, such as IAR3 in Arabidopsis, and a nonspecific increase in expression of both SlILL3 and 7 in the AZ and NAZ might be a manifestation of the wound response. However, in another view, SlILL1, 3, 5, 6, and 7 all showed a trend of increasing expression during abscission in the AZ, which may not exclude their potential role in releasing auxin to delay abscission. This also draws attention to the fact that expression of all SlILL genes is inhibited under auxin treatment. The expression of SlILL genes is mediated by free auxin, which might be required for precise auxin signaling in mediating AZ abscission. In addition, the auxin conjugate hydrolase enzymes are regulated by the free auxin content at the transcriptional level, which might be important to determine the precise auxin concentration required for cell development and for responses to environmental stimuli.
Transcription and translation of tomato SlILLs protein during abscission SlILL1, 5, and 6 showed special expression profiles during abscission; therefore, they were selected as abscission-related auxin conjugate hydrolase genes for western blotting analysis. The pattern of SlILL1, SlILL5 and SlILL6 protein levels in response to 1-MCP and IAA indicated that they were possibly related to abscission.
SlILL1, 5, and 6 are major regulators of delayed abscission The plants in which single SlILL genes were silenced by VIGS showed a normal phenotype, but had reduced sensitivity to specific auxin conjugates. The more hydrolase genes that were downregulated, the less sensitive the plants became to exogenous auxin conjugates [11,40]. Double and triple mutants of Arabidopsis auxin conjugate hydrolases showed lower auxin levels in seedlings and defective responses to exogenous auxin conjugates [11,26]. In this study, all tomato hydrolases showed distinct expression patterns and cleavage activities, implying that they might have distinct, yet overlapping, functions during abscission. Silencing SlILL1, SlILL5, and SlILL6 individually resulted in significantly accelerated abscission in pedicel explants, while double silencing showed that SlILL3 and 7 had little effect on abscission. The double-silenced SlILL1 + 5, SlILL1 + 6, and SlILL5 + 6 explants showed a significant increase in abscission compared with that of the single genesilenced explants, implying that SlILL1, 5, and 6 play important roles in the abscission process. The triplesilenced lines further supported this hypothesis, since they showed a further accelerated abscission rate compared to the double-silenced VIGS lines. Taken together, these results indicated that the auxin conjugate hydrolase enzymes encoded by SlILL1, SlILL5, and SlILL6 coordinate to mediate pedicel abscission in tomato. We also used VIGS1 + 5 + 6 lines to investigate restoring the phenotype, The result showed that IAA addition could restore the phenotype of the VIGS lines (Additional file 5: Figure S3). The concept that the AZ local auxin concentration plays a negative role in mediating abscission procession has been understood for 50 years. Artificial activation of the bacterial auxin biosynthetic genes iaaL and iaaM in the AZ further supports this view [41]. Moreover, auxin is transported in a polar manner to other organs mediated by PIN efflux carriers that determine the direction of flux [42]. It is also important to prevent abscission by maintaining this constant auxin flux [43]. The effect of auxin on PIN expression has been well-studied. Below the optimal concentration, the auxin-induced PIN expression effect was correlates positively with the amount of auxin, while PIN expression was reduced when the auxin concentration was far in excess of the optimal concentration. In AZ, the auxin concentration is relatively low, and depressed SlILR1, 5, and 6 expression in the triple silenced line showed further downregulation of the AZ auxin concentration, leading to a low expression of PIN3, 4, and 8, which might decrease auxin polar transport [44]. The mechanism by which SlILL1, SlILL5, and SlILL6 mediate abscission, not only via altered auxin content, also via auxin flux by affecting PIN gene expression. It is reasonable to propose that the local auxin content in the AZ and auxin transport are tightly connected to regulate abscission.

Conclusions
In this study, we identified ILR genes in the tomato genome. Their protein structures and activities were also characterized, their expression level and protein accumulation in different abscission stage were also investigated using qRT-PCR and western blotting, respectively. Further, three candidates ILR genes were identified to function in tomato abscission. Our identification and characterization of ILRs genes in tomato revealed that certain ILRs might fine-tune the levels of auxin in tomato AZ, which is critical for abscission. These findings provide a new way to inhibit abscission by applying auxin conjugations to flowers.

Plant material
The Solanum lycopersicum cultivar 'Zhongshu NO 6' and transgenic line 'DR5' were grown in soil in a greenhouse (25 ± 3°C day, 15 ± 3°C night) under natural light. Newly opened flowers with fresh yellow petals at an opening angle of 90°were screened to obtain pedicel explants. The explants were trimmed to 2 cm in length and contained the proximal side, the abscission zone, and the distal side of the flower after the petals and base were removed.
Auxin, 1-MCP, and auxin-conjugate treatments, and abscission rate assessment Fifty explants were inserted by their proximal ends into 1% w/v agar medium in a 40 × 25 × 20 cm glass container at 25°C. The agar medium contained deionized water (control) or 50 μg·g − 1 IAA in IAA treatment (dissolved in ethanol and the control also contain a equal amount of ethanol), and for phenotype restoration experiment in the VIGS lines, the IAA were added at 10 μg·g − 1 and 50 μg·g − 1 respectively. For the 1-MCP treatment, 1-MCP (20 μl l − 1 ) was applied for 24 h to treat the whole plant before the flowers were removed and incubated. For auxin conjugate treatments, the agar medium was supplemented individually with 10 to 100 μM IAA-Ala, IAA-Asp, and IAA-Ile (dissolved in ethanol and the control also contain equal amounts of ethanol). The explants were incubated in medium containing IAA-Ile for 1, 2, 4, 6, 8, and 12 h then transferred to the 1% w/v agar medium without IAA conjugate to investigate abscission. The explants were also incubated in control medium (without IAA conjugate) for 1, 2, 4, 6, 8, and 12 h then transferred to medium containing the IAA conjugate. To determine whether the IAA conjugate hydrolytic enzymes depended on de novo synthesis during abscission, the explants were first incubated in medium agar for 1, 2 and 4 h and then transferred to IAA conjugate medium containing 5 μm cycloheximide (CHX). Pedicel abscission rates were recorded every 8 h for 40 h, as described by Wang et al. (2005).

Identification of auxin conjugate hydrolase genes
To find previously identified ILL family genes in tomato, "auxin conjugate hydrolase" was used as a query to search the SGN database (http://solgenomics.net). We also used the amino acid sequences of the conserved ILL domains from known ILL proteins (including AtILLs (Arabidopsis), MeILLs (Medicago truncatula) and BrILLs (Brassica rapa)) as queries to identify all members of the tomato ILL family. DNASTAR and FGENESH (http:// www.softberry.com/) were then used to analyze and predict the unknown SlILLs. Putative open reading frames and functional domains were identified using ORF finder and BLASTP searches (blast.ncbi.nlm.nih.gov), respectively.
Isolation of full-length ILL cDNA sequences using RT-PCR Total RNA was extracted from tomato seedlings using the TRIZOL reagent (Invitrogen, Carlsbad, CA, USA). First-strand cDNA was synthesized using a reverse transcription system. The seven novel full-length SlILL cDNA sequences were amplified by PCR using specific primers (Additional file 6: Table S1). The PCR conditions consisted of denaturation at 94°C for 4 min; followed by 30 cycles of 30 s at 94°C, 30 s at 55°C, and 120 s at 72°C; with a final 10-min elongation at 72°C.

Multiple sequence alignments and phylogenetic analysis
DNAStar software and the web service ExPASy Proteomics Server (http://ca. expasy.org) were used to analyze the gene sequences. Multiple sequence alignments were performed using Clustal X v1.81 [45]. Phylogenetic analysis was performed using the neighbor-joining (NJ) method as implemented in the MEGA 4.1 program [46]. Conserved motifs were identified using Clustal W.

RNA extraction and expression analysis
Total RNA was extracted from AZ and NAZ (the proximal part) using the TRIZOL reagent (Invitrogen). Quantitative real-time reverse transcription PCR (qRT-PCR) assays were performed as described by Jain [47]. Firststrand cDNA was synthesized using the High Capacity cDNA Archive Kit (Applied Biosystems, Foster City, CA, USA) in reactions containing 2 μg of total RNA. For qRT-PCR, each 20 μl reaction system contained 1 μl of cDNA, 200 nM of each primer, and SYBR Green PCR Master Mix (Qiagen) (Additional file 4: Table S1). Amplifications were performed on an ABI 7500 fast system (Applied Biosystems). Two independent RNA samples and three technical replicates for qRT-PCR were performed for each sample.

Protein extraction and Western blotting analysis
For western blotting, synthetic peptides: FFFLCIFLLVLTS, SSYFDQEFVKQIL, and DCSIWTKECSNEI were used to raise polyclonal antibodies targeting SlILL1, SlILL5, and SlILL6, respectively, by Abmart (Berkeley Heights, NJ, USA). The ß-actin conjugated antibody was purchased from Abbkine Company (Wuhan, China) and the antirabbit conjugated antibody was purchased from Sigma (St. Louis, MI, USA. Total proteins were extracted from the AZ (control and 1-MCP treatment) at 0, 2, 4, and 16 h using the Minute™ total protein extraction kit (Invent Biotechnologies, Plymouth, MN, USA). The protein concentration was assessed using the BCA assay (Takara company). Each sample was boiled for 5 min with loading buffer, separated in a 10% SDS-PAGE, and transferred to polyvinylidene difluoride (PVDF) membranes (Bio-Rad, Hercules, CA, USA). Then, the PVDF membranes were blocked with 5% non-fat dried milk for 2 h at room temperature, probed with the primary antibodies at 4°C overnight, and then with anti-rabbit antibody for 1 h at room temperature. Chemiluminescent detection was performed on an Azure c600 system (Azure Biosystems, Dublin, CA, USA) (Additional file 7). Quantitative analysis of the western blotting data was performed using Image J software of selected gel band intensities.

Synthesis and purification of his-tagged fusion proteins
The amplified cDNAs were cloned into the pET 30a (+) expression vector, which contains both N-terminal and C-terminal His tags for heterologous expression in Rosetta Escherichia coli. Single colonies of transformed and untransformed E. coli TOP10 (as control), were inoculated into 150 ml of Luria-Bertani broth containing 50 μg·ml − 1 ampicillin and grown at 28°C overnight.
Isopropyl β-D-1-galactopyranoside was used at a concentration of 1 mM to induce the E. coli cultures, which were incubated at 28°C for 8 h. The cells were recovered by centrifugation and resuspended in 1.5 ml lysis buffer (50 mM NaH 2 PO 4 , 300 mM NaCl, 10 mM imidazole, 50 μg·ml − 1 phenylmethanesulfonyl fluoride, 1 mg·ml − 1 lysozyme, 0.1% Triton; pH 8.0) For enrichment of the tagged proteins, samples were incubated on ice for 30 min, then frozen and thawed three times in liquid nitrogen. E. coli DNA was removed by adding 500 U DNAse I (Takara) and incubating at room temperature for 20 min. After centrifugation, the cell lysate was transferred to a new tube, and 20 μl Ni-NTA Agarose (Qiagen, Valencia, CA, USA) was added. The lysate was then incubated with rotation for 1 h at 4°C and the agarose was subsequently washed three times with wash buffer (300 mM NaCl, 50 mM NaH 2 PO 4 and 20 mM imidazole; pH 8.0). Proteins were eluted three times with 30 μl of elution buffer (300 mM NaCl, 50 mM NaH 2 PO 4 , 250 mM imidazole; pH 8.0). The proteins were analyzed using denaturing polyacrylamide gel electrophoresis (SDS-PAGE) on 12% gels and quantified using the BCA assay Protein Quantification Kit (Beyotime, Jiangsu, China).

IAA conjugate hydrolase enzyme assays
For the enzyme assays, 2 μg samples of the enriched protein fraction were mixed with assay buffer (100 mM Tris, 200 μM MnCl 2 , 1 mM DTT; pH 8.0), and the IAA conjugates, IAA-Ala, IAA-Leu, IAA-Asp, IAA-Gly and IAA-Trp, were added individually at a concentration of 1 mM, as substrates. The reactions were incubated at 37°C for 1 h and terminated by the addition of 20 μl 1 N HCl. The aqueous phases were then extracted with 250 μl of ethyl acetate, evaporated, and the reaction products were dissolved in 100 μl of methanol for ultra performance liquid chromatography (UPLC) analysis. Aliquots (0.2 μl) of the methanol extracts were then injected into a UPLC instrument (Waters H-class, Waters, Millford, MA, USA) coupled to an autosampler (Jasco AS-1550), and equipped with a 2.1 × 100-mm Cortecs C18 2.7 μm, reversed phase column. 1% aqueous acetic acid and 100% methanol were used as the solvents. The solvent program was 15% methanol for 3 min, followed by a gradient from 15 to 90% methanol for 4 min, then a decreasing gradient from 90 to 15% for 3 min with 1% acetic acid throughout. Compounds were detected using ACQUITY FLR fluorescence (excitation 278 nm, emission 350 nm) and quantitated by the peak area.

Auxin concentration measurements
The IAA concentration was measured using gas chromatography-mass spectrometry (GC-MS) according to the method described by Barkawi. In short, 100 μl of extraction buffer (65% isopropanol, 35% 0.2 M imidazole, pH 7.0) that contained 5 ng of a [ 13 C 6 ] (Isoreag, Shanghai, China) IAA standard from 100 mg of homogenized frozen tissue. Tissue were centrifuged at 14,000×g for 5 min after incubation on ice for 1 h, using solid phase extraction and ethereal diazomethane for the methylated forms. GC-MS (Thermo, USA) was run in select ion monitoring mode and IAA was quantified using isotope dilution analysis [48].


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N Isotope Fractionation in Tree Tissues During N Reabsorption and Remobilization in Fagus crenata Blume

Background and Motivation: Nitrogen content in tissues of Fagus crenata Blume is key for flowering and seed production. However, there is a lack of information on seasonal intra-plant nitrogen partitioning in this representative tree species typical of heavy snowfall regions in Japan. Therefore, the objective of this study was to elucidate Fagus crenata intra-plant nitrogen movement by means of nitrogen content, nitrogen isotope analysis, and amino acids temporal variability. Materials and Methods: Nitrogen content, isotope ratio, and free amino acids content were measured in coarse roots, sapwood, leaves, and litter in four phenological stages in nine adult Fagus crenata trees and upscaled to the whole-tree level.Results: Nitrogen was reabsorbed to and stored in coarse roots during the pre-abscission stage, as was revealed by the depletion of the δ15N ratio of coarse roots, which coincided with an enrichment of 15N found in leaves. During the post-abscission stage, N was stored in the sapwood, where an enrichment in 15N was found coinciding with the depletion of the δ15N ratio in leaves. It seemed that 15N-enriched nitrogen was initially reabsorbed from leaves to coarse roots during the pre-abscission period, followed by the reabsorption of 15N-enriched nitrogen from leaves to sapwood shortly before leaf abscission. Free amino acids content and their dynamics could mostly explain seasonal δ15N fractionation in leaves, coarse roots, and partially in sapwood. At the whole-tree level, N content stored in coarse roots and sapwood was similar. Furthermore, reabsorbed leaf N accounted for 32% of all nitrogen stored during leaf senescence.Conclusion: We found three phases of nitrogen storage revealed by δ15N fractionation during leaf senescence: (1) reabsorption of leaf 15N-depleted nitrogen to coarse roots, followed by (2) reabsorption of leaf 15N-enriched nitrogen to sapwood and (3) soil 15N-depleted nitrogen uptake to coarse roots. Further, changes in free amino acids, which are the result of enzyme activities involved in amino acids synthesis, partially explained δ15N fractionation in plant tissues.

Introduction
Japanese beech (Fagus crenata Blume) is the most common and widely distributed deciduous broad-leaved tree species in Japan, growing in the cool-temperate zone as a late-successional and climax species [1,2]. It usually grows in pure stands or mixed with Quercus mongolica Fisch. Ex Ledeb. and has an important ecological function, protecting soils from erosion and maintaining biodiversity. Additionally, Fagus crenata is used for afforestation and ceremonial plantations [2]. The importance of level. The driest month is June, with approximately 70 mm of rain and the wettest is December, with 500 mm. Nitrogen deposition with precipitation is relatively low, with 10−15 kg total N ha −1 year −1 (e.g., Kanto region 30−38.5 kg total N ha −1 year −1 ) [2]. The mean annual temperature is 9.7 • C.
The sampling sites were distributed within a distance of 2 km on three slopes and selected by stand purity and accessibility in every season at the Research Forest of Yamagata University. To ensure comparability of the sites, soils and basic parameters were identified prior to tree sampling.

Sample Collection and Treatments
Soil samples were collected on August the 3rd in 2017 from three soil pits. Soil horizons were identified in the field following the FAO guidelines [25]. Genetic horizons were sampled for soil characterization, while samples for nitrogen and δ 15 N analysis were taken at fixed depths from 0−5 cm, 5 −15 cm, and 15−30 cm from three pits as replicates surrounding each individual tree. All the samples were transported in plastic bags after collection in the field, air-dried, sieved (< 2 mm), powdered, and stored until analysis. The samples used for the determination of inorganic N were frozen after transport and stored in the dark. Before analysis, they were thawed overnight in a refrigerator and sieved (< 2 mm).
Nine Fagus crenata trees of a similar age (70−80 years old) in the selected sites were chosen for sampling (Table 1). Coarse roots (> 2 mm), stem, and leaf samples were taken on May 20 (shoot growth stage), August 2 (green leaf stage), October 19 (pre-abscission stage), and November 29 (post-abscission stage), with fresh litter collected from litter traps (size: 1 m 2 ). Sapwood was sampled by using an increment borer (10 mm) and not heartwood, assuming that there was no variation in N content in heartwood throughout the growing season [26]. Leaf samples were taken from the lower canopy from different leaf clusters. The position in the canopy has no significant impact on the reabsorption efficiency [12] and by sampling different leaf clusters, we covered possible variations in N content as leaf clusters' N content is regulated independently for this species [27]. All samples were transported in plastic bags and oven-dried in the laboratory. Coarse roots, leaves, and litter were dried at 70 • C, and sapwood samples at 40 • C, for at least 48 hrs until they were dried. Subsequently, the samples were ground and stored until analysis.

Soil Analysis
Soil texture was measured with a laser diffraction particle size analyzer (Coulter LS200 with an attached Fluid Module, Beckman Coulter GmbH, Germany), after treating the samples with H 2 O 2 and Na 4 P 2 O 7 . The analyses were triplicated. The pH was determined potentiometrically in a 1:2.5 soil:water suspension. The carbon (C) and N contents were analysed by dry combustion using the SUMIGRAPH NC-220F automatic high sensitive NC analyzer SCAS (Japan). The results were expressed on a dry-weight (105 • C) basis. Total soluble N (TNb) was extracted from the soil samples by shaking them with 50 ml of 1M KCl. The supernatant was centrifuged, filtered (0.45 µm), stored in a refrigerator and analysed with a TOC/TNb analyser (vario TOC cube, elementar, Germany) for TNb determination. Ammonium content was determined using the method of Crooke et al. [28], whereas nitrate content was determined by using the method of Mulvaney [29] modified by Miranda et al. [30]. The colorimetric determination of ammonium and nitrate content was conducted with a U-2000 Spectrophotometer (Hitachi, Japan).

Total Nitrogen and δ 15 N Isotope Analysis
For the determination of total N content and δ 15 N in plant tissues, a Thermo Quest EA1110 Elemental Analyzer (Italy) which was connected to an IsoPrime (GV Instruments, UK) was used. The isotopic compositions of samples were expressed relative to atmospheric N 2 (δ 15 N = 0% ) on scales normalized to the known δ 15 N values of laboratory working standards for glycine (δ 15 N = −0.3), which was normalized to L-glutamic acid distributed as USGS-40 (δ 15 N = −0.2% ) by SI Science Inc., Japan. Additionally a tertiary reference material was used, namely the in-house laboratory standard acetanilide (−0.89% ). The three working standards were analysed after every eight to ten samples during CF-IRMS runs to assess the replicability of the isotope measurements and normalization. One pulse of pure N 2 reference gas from a tank reservoir (δ 15 N = −2.5% ) was discharged into the IRMS at the beginning of each chromatogram for both standards and samples. The accuracy obtained for standards and samples during the overall analytical procedure was better than ±0.2% .

Amino Acid Analysis
Amino acids were extracted from the dried powders of plant materials with methanol and separated by liquid/liquid extraction with ultracentrifugation. Nineteen amino acids (Ala, Arg, Asn, Asp, Glu, Gln, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, Val) were quantified by capillary electrophoresis mass spectrometry (CE-MS). The preparation, conditions during sample extraction, and CE-MS analysis followed Oikawa et al. [31].

Whole-Tree N Calculations
Whole-tree biomass was calculated as follows: For root, stem, and branch weight, the allometric equation by Ono et al. [32] for Fagus crenata was used.
where, Y is the biomass [kg], a is a normalization coefficient (roots = 0.0147, stem = 0.0946, branch = 0.0049), DBH is the tree diameter at breast height [cm], and b is a scaling exponent (roots = 2.62, stem = 2.44, branch = 2.78). Ono et al. [32] reported that for smaller trees (DBH < 30 cm), such as those found in our study sites, leaf biomass was underestimated. Thus, for leaf biomass, the equation reported by Tateishi et al. [33] for Fagus sylvatica was used. ln(ML) = p + q ln(DBH) where, (ML) is the leaf mass [g], p is a normalization coefficient, q is a scaling exponent (2.9 and 1.7, respectively), and DBH is expressed as [cm] for Fagus crenata. Fresh litter collected with litter traps verified the estimation of leaf biomass with Equation (2). Lastly, for the determination of the sapwood weight, an equation by Gebauer et al. [34] developed for Fagus sylvatica was used and verified by tree core sampling.
where, A s is the sapwood area (cm 2 ), a and b are species-specific coefficients determined by regression techniques (0.778 and 1.917, respectively), and DBH is in [cm]. Furthermore, we assumed that in the post-abscission stage, all N was in the respective storage tissues (coarse roots and sapwood), while storage was considered empty during the green leaf stage as N is needed in the growing tissues. Thus, the whole-tree N storage was calculated as follows: (4) where, N storage is the amount of N stored in a certain tissue, N post is the N content during the post-abscission stage, and N green is the N content during green leaf stage. This value was calculated for all plant tissues as relative amounts (g N kg −1 ) and as whole-tree absolute N storage (g N).
Reabsorption efficiency (RE) was calculated as Nitrogen was expressed as nitrogen mass per leaf dry mass (mg g −1 ). Litter represents the senesced leaves in November, while the green leaf is the matured August leaf. We used this value to calculate whole-tree total reabsorbed leaf N by considering the total tree leaf weight.

Statistical Analysis
One-way ANOVA was applied to determine the statistical significance of differences in nitrogen content, δ 15 N, and amino acids content. If significant differences were found, and a post-hoc multiple comparison was subsequently conducted, using the Tukey-Kramer test at the significance levels of 0.05 and 0.01. Furthermore, multiple linear regression analysis was conducted with RStudio (Version 1.1.463 -© 2009-2018 RStudio, Inc.) in order to evaluate the effect of the dependent variable (δ 15 N) on the independent variable (free amino acids content).

Soil Characteristics
In all three sites, the soils were identified as brown forest soils (Cambisols), with an average depth of 80 cm and a silty-loamy texture. In the first 30 cm, we found a pH of 3.7 and a carbon to nitrogen ratio of 18 (Table 2). Table 2. Soil characteristics with ± denoting SD. DON stands for dissolved organic nitrogen.

Total Nitrogen Content in Soil and Tissues
Soil N content significantly decreased (min. p < 0.05) with depth from the top to deeper soil layers by 87% (Table 2, Figure 1a).
well as in the pre-abscission stage. However, a significant increase (p < 0.05) in N content of 62% was observed during the post-abscission stage. However, it was 39% lower (p < 0.01) than the value found during the shoot growth stage (Figure 2b).
Leaves' N content significantly decreased (p < 0.01) from the shoot growth to the pre-abscission stage by 35%. Finally, the N content in litter was significantly (p < 0.05) lower in the post-abscission stage (Figure 2c), with an RE of 66%.  Inorganic plant available N decreased similarly (p < 0.01) from top to bottom by 60% and was exclusively formed of ammonium. Nitrate content accounted for less than 1% of the soil total inorganic N. Total soluble N decreased by 50% (p < 0.01) from top to bottom, while dissolved organic nitrogen (DON) accounted for about 25% of that with no clear distribution pattern (Table 2).
N content of coarse roots significantly decreased (p < 0.01) from shoot growth to the green leaf stage by 32%, followed by an increase (p < 0.01) of 28% during the pre-abscission stage. Finally, during the post-abscission stage, no change in N content was observed ( Figure 2a). Sapwood N content decreased by 50% (p < 0.01) from shoot growth to the green leaf stage, as well as in the pre-abscission stage. However, a significant increase (p < 0.05) in N content of 62% was observed during the post-abscission stage. However, it was 39% lower (p < 0.01) than the value found during the shoot growth stage (Figure 2b).
Leaves' N content significantly decreased (p < 0.01) from the shoot growth to the pre-abscission stage by 35%. Finally, the N content in litter was significantly (p < 0.05) lower in the post-abscission stage (Figure 2c), with an RE of 66%.
Leaf δ 15 N enriched significantly during the pre-abscission stage, followed by a significant depletion in litter during the post-abscission stage (Figure 2c).
Soil δ 15 N was the most enriched of all measured plant tissues during any given phenological stage. During the growing season, coarse roots in terms of δ 15 N were the most depleted plant tissue, while leaves and sapwood showed similar δ 15 N values from the shoot growth to the pre-abscission stage. Only during the post-abscission stage was leaf litter significantly more depleted in 15 N than sapwood.

Amino Acid Analysis in Plant Tissues
Total free amino acids content (FAAC) in coarse roots decreased dramatically (300%, p < 0.01) from the shoot growth to the green leaf stage and decreased another 60% (p < 0.05) in the pre-abscission stage. However, it increased again during the post-abscission stage by 300% (p < 0.05) ( Figure 3).
Leaf δ 15 N enriched significantly during the pre-abscission stage, followed by a significant depletion in litter during the post-abscission stage (Figure 2c).
Soil δ 15 N was the most enriched of all measured plant tissues during any given phenological stage. During the growing season, coarse roots in terms of δ 15 N were the most depleted plant tissue, while leaves and sapwood showed similar δ 15 N values from the shoot growth to the pre-abscission stage. Only during the post-abscission stage was leaf litter significantly more depleted in 15 N than sapwood.

Amino Acid Analysis in Plant Tissues
Total free amino acids content (FAAC) in coarse roots decreased dramatically (300%, p < 0.01) from the shoot growth to the green leaf stage and decreased another 60% (p < 0.05) in the preabscission stage. However, it increased again during the post-abscission stage by 300% (p < 0.05) ( Figure 3).
Sapwood FAAC only showed a significant increase (p < 0.05) of 50% from the green leaf stage to the post-abscission stage ( Figure 3).
Leaf total FAAC decreased (p < 0.05) by 90% from the shoot growth to the green leaf stage. We observed a 15-fold increase (p < 0.01) during the pre-abscission stage and an equally sharp decrease (p < 0.01) in fresh litter during the post-abscission stage ( Figure 3). The most abundant free amino acid in coarse roots was asparagine, which accounted for 60 to 80% of total FAAC along the growing season, with the highest content (p < 0.05) in the shoot growth and post-abscission stage (Table 3). Other free amino acids of minor concentration were alanine and arginine, showing a similar distribution along the growing season as asparagin. Other amino acids were found in negligible concentrations. The results of the multiple linear regression analysis showed that roots' asparagine content alone could not explain roots' δ 15 N. However, the total pool of all amino acids revealed that asparagine content has an increasing effect (p = 0.01) on the δ 15 N value, while all the others have a decreasing effect (p < 0.01) ( Table 4). Sapwood FAAC only showed a significant increase (p < 0.05) of 50% from the green leaf stage to the post-abscission stage ( Figure 3).
Leaf total FAAC decreased (p < 0.05) by 90% from the shoot growth to the green leaf stage. We observed a 15-fold increase (p < 0.01) during the pre-abscission stage and an equally sharp decrease (p < 0.01) in fresh litter during the post-abscission stage (Figure 3).
The most abundant free amino acid in coarse roots was asparagine, which accounted for 60 to 80% of total FAAC along the growing season, with the highest content (p < 0.05) in the shoot growth and post-abscission stage (Table 3). Other free amino acids of minor concentration were alanine and arginine, showing a similar distribution along the growing season as asparagin. Other amino acids were found in negligible concentrations. The results of the multiple linear regression analysis showed that roots' asparagine content alone could not explain roots' δ 15 N. However, the total pool of all amino acids revealed that asparagine content has an increasing effect (p = 0.01) on the δ 15 N value, while all the others have a decreasing effect (p < 0.01) ( Table 4). Asparagine was also predominant in sapwood samples, with an increasing concentration (p < 0.01) from shoot growth to the post-abscission stage. Along with asparagine, alanine was found in high concentrations with similar amounts and increased (p < 0.05) throughout the growing season. Together, they accounted for at least 50% of all free amino acids found in sapwood during any given time. Further amino acids detected in minor concentrations were glutamic acid, glutamine, aspartic acid, proline, and serine. All other amino acids were found in negligible concentrations. The results of the multiple linear regression analysis showed that sapwood asparagine content had an increasing effect (p < 0.05) on sapwood δ 15 N (Table 4).
In leaves, alanine was the most abundant free amino acid, with peak concentrations (p < 0.05) in the shoot growth and pre-abscission stage. All other amino acids were found in negligible concentrations, except during the pre-abscission stage, where asparagine, glutamic acid, glutamine, isoleucine, leucine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine were detected. In fresh litter, during the post-abscission stage, mainly tyrosine, valine, proline, threonine, isoleucine, asparagine, leucine, phenylalanine, and tryptophan remained, while other free amino acids were depleted. The results of the multiple linear regression analysis showed that leaf alanine has the strongest control (p < 0.05) on leaf δ 15 N than all the other free amino acids (Table 4).

Whole-Tree Level
The total biomass of all plant tissues was calculated for four trees of similar DBH in the green leaf stage (Table 5). We assumed that root, sapwood, and branch biomass did not change significantly during one growing season. Furthermore, leaf biomass was assumed not to change significantly during the pre-abscission stage and to equal the litter biomass in the post-abscission stage. We could not make any assumption on the leaf biomass during the shoot growth stage, thus it was not calculated for this stage (Table 5). In the green leaf stage, the whole-tree roots' N content was 688 ± 67 g N, while sapwood, branches', and leaves' total N content was 290 ± 35 g N, 54 ± 8 g N, and 193 ± 20 g N, respectively (Table 5). In the pre-abscission stage, N content in roots increased by 25% (p < 0.05), while sapwood and branches showed no variation. Furthermore, we observed a 25% decrease (p < 0.05) of N in leaves. Finally, in the post-abscission stage, roots' N content did not change, while we found a 60% increase (p < 0.01) in sapwood and branches and another 55% decrease (p < 0.01) in litter.
Roots' N storage was 23% higher (ns) than sapwood, while N stored in branches was the lowest (Figure 4). At the whole-tree level, N stored in all measured tissues was 402 ± 93 g, of which 32% were reabsorbed from senescing leaves during leaf abscission.

Reabsorption, Storage, and Remobilization of N in Plant Tissues
The N cycle in Fagus crenata followed a well-established pattern: in the green leaf stage, most N was bound in leaves' photosynthetic apparatus [35]. In the pre-abscission stage, leaf N was reabsorbed until leaf abscission. Subsequently, fresh litter in the post-abscission stage was depleted in N content (RE 66%). Enta et al. [36] and Yasumura et al. [12] reported a similar value (RE 68-71%) for this species. Subsequently, N was stored in a sequential temporal pattern: first, it was stored in coarse roots during the pre-abscission stage, followed by N storage in the sapwood in the postabscission stage. In previous studies [14,16,35,37], this temporal pattern of N storage in coarse roots and sapwood of deciduous trees has not been shown. In the post-abscission stage, when most N was stored, roots tended to store more N (ns) than sapwood. N reabsorption from leaves contributed 32% to overall N storage in the post-abscission stage.
During the shoot growth stage, N contents peaked in roots (ns) and sapwood (p < 0.01). Together with soil N taken up in late April, stored N supported shoot growth in the shoot growth stage [38]

Reabsorption, Storage, and Remobilization of N in Plant Tissues
The N cycle in Fagus crenata followed a well-established pattern: in the green leaf stage, most N was bound in leaves' photosynthetic apparatus [35]. In the pre-abscission stage, leaf N was reabsorbed until leaf abscission. Subsequently, fresh litter in the post-abscission stage was depleted in N content (RE 66%). Enta et al. [36] and Yasumura et al. [12] reported a similar value (RE 68-71%) for this species. Subsequently, N was stored in a sequential temporal pattern: first, it was stored in coarse roots during the pre-abscission stage, followed by N storage in the sapwood in the post-abscission stage. In previous studies [14,16,35,37], this temporal pattern of N storage in coarse roots and sapwood of deciduous trees has not been shown. In the post-abscission stage, when most N was stored, roots tended to store more N (ns) than sapwood. N reabsorption from leaves contributed 32% to overall N storage in the post-abscission stage.
During the shoot growth stage, N contents peaked in roots (ns) and sapwood (p < 0.01). Together with soil N taken up in late April, stored N supported shoot growth in the shoot growth stage [38] despite heavy snow cover, as is typical for this region. The continuous uptake of soil N during the dormant phase of the tree in the post-abscission stage could also contribute to the high N content found in the shoot growth stage. However, N uptake during dormancy has been reported to be minimal and only few studies have found it for boreal species during warm periods of the winter season [35,39]. Tree internal N storage strongly determines the beech masting period [8,15] and thus, the quantification of N uptake during the dormant period of this species needs to be explored in more detail. In the green leaf stage, after stored N was remobilized to support new shoot and leaf growth, coarse roots and sapwood were left depleted [37,40]. We did not quantify the contribution of stored N to new growth, but Dyckman and Flessa [15] reported that previous-year N contributes to leaf N with about 15% in Fagus sylvatica.

Effect of Soil N Uptake on Plant δ 15 N
Soil total N content, as well as inorganic N content, decreased with depth as δ 15 N became heavier due to the input of fresh N on the soil surface: precipitation and microbial turnover of litter, as well as illuviation of 15 N-rich materials to deeper soil layers, are the main mechanisms [41][42][43][44]. Tateno et al. [19] found that foliar δ 15 N of the green leaf stage depends on ammonium and nitrate availability. In this latter study, Fagus crenata trees on upper slopes took up ammonium due to a lack of nitrate, leading to higher foliar δ 15 N values (−1.0% ) than at the bottom of the slope, where nitrate was more abundant, leading to 15 N depletion in leaves (−2.0 % ). In our study, we found a high availability of ammonium and a negligible amount of nitrate, which explained the δ 15 N value of −1.2 ± 0.6% in agreement with Tateno et al.'s [19] findings for trees in upper slope positions. Additionally, we measured low potential mineralization rates (1mg kg −1 soil in 30 days, data not published) in a nearby Cryptomeria japonica stand being similar to Tateno et al.'s [19] results. Thus, we concluded that our trees rather relied on ammonium rather than nitrate, although under different conditions, they would preferably acquire nitrate [45,46], but climatic conditions and the acidic pH strongly limited the nitrification process.
The inorganic soil N taken up by tree roots and mycorrhiza undergoes isotopic fractionation as mycorrhizal fungi selectively accumulate 15 N and thus, a rather depleted 15 N fraction passes to the host plants [19,20,47,48]. Leberecht et al. [18] demonstrated that the ectomycorrhizal fungi (EMF) associated with Fagus sylvatica preferably uptake ammonium from the soil, resulting in a more enriched 15 N accumulation in the EMF in comparison to EMF acquiring nitrate. In both cases, δ 15 N values of the host plants will be lower, but the depletion in 15 N will be less pronounced for N deriving from ammonium. This suggests a stronger control of N supply to the host tree by the EMF acquiring ammonium than would be the case with EMF taking up nitrate. Soil δ 15 N was 5% greater than root δ 15 N, revealing the effect of microbial turn over and uptake of inorganic N via mycorrhiza [20].

Effect of Intra-Plant N Movement on δ 15 N
In general, leaves' δ 15 N ratio falls in line with the findings of Tateno et al. [19], who found similar values for Fagus crenata in lower slope positions under similar soil conditions. In the pre-abscission stage, leaves were significantly enriched in 15 N by 0.5% . At the same time, coarse roots' δ 15 N tended to decrease (ns) by 0.5% . Depleted reabsorbed leaf N seemed to be transported and stored in coarse roots during the pre-abscission stage, possibly together with taken up soil N, forming the bulk of coarse roots' N storage. During the post-abscission stage, N storage only increased marginally (ns); however, a significant decrease in coarse roots' δ 15 N suggests that only 15 N-depleted soil N was stored, since the reabsorption of leaf N was already concluded.
After leaf abscission, N content significantly decreased in litter, depleting the δ 15 N value by 1.5% . Thus, shortly before leaf senescence, 15 N-enriched N was reabsorbed and subsequently stored in sapwood, increasing it by 1.5% . In contrast to the results of Kolb and Evans [10], we found N isotope fractionation during reabsorption in leaves.
Thus, three phases of N storage could be observed: (1) 15 N-depleted N was reabsorbed from leaves to coarse roots in the pre-abscission stage, which was followed by (2) 15 N-enriched leaf N reabsorption, significantly enriching sapwood in the heavier isotope during the post-abscission stage. (3) At the same time, coarse roots' δ 15 N decreased, suggesting a strong N isotope fractionation in the EMF during the uptake and storage of soil N, as it occurs under ammonium-rich soil conditions [18].
In the transition from the post-abscission to the shoot growth stage, coarse roots were enriched in 15 N, while sapwood was depleted. This suggests a movement of depleted N from coarse roots to sapwood, which eventually would be used for new shoot growth and leaf production. Only during the green leaf stage did all measured tissues show similar δ 15 N values. Further analysis is necessary to confirm whether foliar δ 15 N during the green leaf stage could be used as a proxy for whole-tree δ 15 N in other tree species, as has been suggested in previous studies [10].

Plant Tissues Free Amino Acids
During the green leaf stage, free amino acid content (FAAC) was generally low in all tissues. However, coarse roots' FAAC was the highest among all tissues, possibly because of soil N uptake [49]. DON represents a major soluble N pool in the soil and is mostly composed of amino acids, which can be directly taken up by roots [50]. In our study, DON made up 21% of all soluble N of the top 30 cm. Roberts and Jones [51] reported that DON made up about 40% of all plant available N in a eutric cambisol, with free amino acids accounting for 10%. The low amino acid concentration in the soil solution contributed to the extremely fast rate at which these are removed from the soil, thus explaining the high values found in coarse roots of our study.
During the pre-abscission stage, FAAC increased sharply in leaves as a result of protein hydrolysis [37]. During the post-abscission stage, as leaf N reabsorption ended, a sharp decrease in total N and FAAC content in the fresh litter was observed, corresponding with significant increases in sapwood and coarse roots. This was likely linked to the continuous uptake of soil N [48] and further storage during the dormant phase of trees in the post-abscission stage. FAAC was high in all plant tissues during the shoot growth stage, suggesting that in coarse roots, N was internally remobilized within the tree and also absorbed from the soil. N is transported via sapwood to the emerging leaves to synthesise proteins for building cell walls and the photosynthetic apparatus [35].
In coarse roots, the most abundant free amino acid throughout the phenological stages was asparagine. Asparagine is known to act as a transport and storage form of N in plants [22] and its peak in the shoot growth stage revealed its high importance for N remobilization in spring, as has also been found in the case of apple trees [52]. Other major compounds were alanine and arginine. Alanine is closely connected with N metabolism [53] and is commonly found under stress conditions (e.g., temperature stress), yet its precise function is unclear. As alanine is high in all tissues in the shoot growth, as well as in the pre-and post-abscission stage, it might be linked not only to temperature stress caused by the long snow cover in our plots, but also to the transport of N. Arginine seemed to be linked to stress and transport as well, as it contributes to building and degrading proteins [22], which coincides with its occurrence during the shoot growth and pre-abscission stage.
Similar patterns were found in the sapwood for asparagine and alanine being linked to N transport during remobilization and storage. Although only low contents of glutamic acid and glutamine were found, these two amino acids are also associated with the transport and storage of N [51]. The occurrence of proline in the shoot growth stage was likely linked to snow-related temperature stress [54]. Finally, leaves predominantly contained alanine, which largely occurred during the shoot growth and pre-abscission stage, supporting our findings of its transport function linked to N storage and remobilization.

Correlation between Free Amino Acid Content and Plant Tissues δ 15 N
We observed four synchronized events between FAAC variation and δ 15 N fractionation in leaves. (1) In the pre-abscission stage, free amino acids are released after protein hydrolysis [37] and fractionation of 15 N occurred. (2) In the post-abscission stage, as all the released amino acids were reabsorbed from leaves, δ 15 N significantly decreased. (3) During the shoot growth stage, 15 N increased in leaves and leaf FAAC increased in comparison to litter. (4) During the green leaf stage, FAAC was low, as free amino acids were synthesised into proteins [35], and leaf δ 15 N showed no variation.
Therefore, the seasonal increase in leaf FAAC appears to be the reason for the seasonal enrichment of leaf 15 N. However, a decrease in FAAC, as observed during the green leaf and post-abscission stage, does not automatically mean a depletion in leaf 15 N. A decrease in leaf δ 15 N in the green leaf stage was not observed because the free amino acids were bound in proteins and are present in the leaf [55]. In contrast, in the post-abscission stage, the small leaf FAAC reflects their absence because they have been reabsorbed to storage tissues. The single most abundant free amino acid in leaves was alanine, which suggests that the seasonal variability of this amino acid is mainly responsible for leaf δ 15 N fractionation. Singling out seasonal measurements of alanine δ 15 N during the pre-abscission stage could clarify its contribution to leaf 15 N fractionation [23].
We found a less clear connection between FAAC and δ 15 N in sapwood and coarse roots, since the source of stored N originates from leaf N reabsorption and soil N uptake, as reflected by the difference in free amino acids' composition in these tissues [56], resulting in a lower adjusted R 2 in the linear regression analysis. However, we could link root and sapwood asparagine content to cause an enrichment in δ 15 N. In contrast to leaves and roots where FAAC were found in abundance, sapwood seemed to be dominated by other N-containing compounds, e.g., linked to the incomplete breakdown of leaf N storage to free amino acids [10,56]. Future studies should include measurements of protein in order to increase the R 2 , especially in the case of sapwood.

Conclusions
The results obtained in this study showed that, on average, adult Fagus crenata trees had an N reabsorption efficiency of 66%. The reabsorbed foliar N was sequentially stored in coarse roots during the pre-abscission stage and in the sapwood in the post-abscission stage, from where it was remobilized to growing tissues during the shoot growth stage. Leaf N made up 32% of the total storage in roots and sapwood, making leaf N reabsorption vital for the species' survival. Nitrogen was evenly stored between coarse roots and sapwood. N isotope fractionation occurred when N reabsorption took place before leaf abscission. First, 15 N-depleted N from leaves was traced to 15 N-depleted N stored in coarse roots, followed by the reabsorption of 15 N-enriched N that was traced to 15 N-enriched N stored in sapwood. Free amino acids' dynamics could partially explain changes in δ 15 N in all measured tissues.