Regulation of anthocyanin and sugar accumulation in grape berry through carbon limitation and exogenous ABA application.

To optimize vineyard management practices to adapt viticulture to climate change, knowledge of the regulation mechanism of metabolite accumulation under carbon source limitation and abscisic acid (ABA) application in grapes should be deepened. Here, carbon source limitations were imposed by reducing leaf area from 12 to 2 leaves per vine (at pea sized stage, - 2L-P; or one week prior to veraison - 2L-V) and phloem girdling between the second and third leaf from bottom to top (one week prior to veraison - 12L-girdling) were compared for their effects on berry composition. All three modalities significantly reduced sugar, anthocyanin and ABA content in comparison with berries under sufficient carbon supply (12 leaves per vine - 12L), with 2L-V being the greatest. Allowing leaf area to partially recover (2L-R) or berry ABA application (400 mg. L-1) one week before veraison increased the ratio of anthocyanin to sugar under source limitation. Combined with the analysis of berry metabolites and transcript abundances, our results indicate that source limitation and exogenous ABA co-regulated anthocyanins content through differential gene expression.

Metabolic, Organoleptic and Transcriptomic Impact of Saccharomyces cerevisiae Genes Involved in the Biosynthesis of Linear and Substituted Esters.

Esters constitute a broad family of volatile compounds impacting the organoleptic properties of many beverages, including wine and beer. They can be classified according to their chemical structure. Higher alcohol acetates differ from fatty acid ethyl esters, whereas a third group, substituted ethyl esters, contributes to the fruitiness of red wines. Derived from yeast metabolism, the biosynthesis of higher alcohol acetates and fatty acid ethyl esters has been widely investigated at the enzymatic and genetic levels. As previously reported, two pairs of esterases, respectively encoded by the paralogue genes ATF1 and ATF2, and EEB1 and EHT1, are mostly involved in the biosynthesis of higher alcohol acetates and fatty acid ethyl esters. These esterases have a moderate effect on the biosynthesis of substituted ethyl esters, which depend on mono-acyl lipases encoded by MGL2 and YJU3. The functional characterization of such genes helps to improve our understanding of substituted ester metabolism in the context of wine alcohol fermentation. In order to evaluate the overall sensorial impact of esters, we attempted to produce young red wines without esters by generating a multiple esterase-free strain (Δatf1, Δatf2, Δeeb1, and Δeht1). Surprisingly, it was not possible to obtain the deletion of MGL2 in the Δatf1/Δatf2/Δeeb1/Δeht1 background, highlighting unsuspected genetic incompatibilities between ATF1 and MGL2. A preliminary RNA-seq analysis depicted the overall effect of the Δatf1/Δatf2/Δeeb1/Δeht1 genotype that triggers the expression shift of 1124 genes involved in nitrogen and lipid metabolism, but also chromatin organization and histone acetylation. These findings reveal unsuspected regulatory roles of ester metabolism in genome expression for the first time.

Behind the curtain of the compartmentalization process: Exploring how xylem vessel diameter impacts vascular pathogen resistance.

A key determinant of plant resistance to vascular infections lies in the ability of the host to successfully compartmentalize invaders at the xylem level. Growing evidence supports that the structural properties of the vascular system impact host vulnerability towards vascular pathogens. The aim of this study was to provide further insight into the impact of xylem vessel diameter on compartmentalization efficiency and thus vascular pathogen movement, using the interaction between Vitis and Phaeomoniella chlamydospora as a model system. We showed experimentally that an increased number of xylem vessels above 100 µm of diameter resulted in a higher mean infection level of host tissue. This benchmark was validated within and across Vitis genotypes. Although the ability of genotypes to restore vascular cambium integrity upon infection was highly variable, this trait did not correlate with their ability to impede pathogen movement at the xylem level. The distribution of infection severity of cuttings across the range of genotype's susceptibility suggests that a risk-based mechanism is involved. We used this experimental data to calibrate a mechanistic stochastic model of the pathogen spread and we provide evidence that the efficiency of the compartmentalization process within a given xylem vessel is a function of its diameter.

Dissecting the control of shoot development in grapevine: genetics and genomics identify potential regulators.

BACKGROUND: Grapevine is a crop of major economic importance, yet little is known about the regulation of shoot development in grapevine or other perennial fruits crops. Here we combine genetic and genomic tools to identify candidate genes regulating shoot development in Vitis spp. RESULTS: An F2 population from an interspecific cross between V. vinifera and V. riparia was phenotyped for shoot development traits, and three Quantitative Trait Loci (QTLs) were identified on linkage groups (LGs) 7, 14 and 18. Around 17% of the individuals exhibited a dwarfed phenotype. A transcriptomic study identified four candidate genes that were not expressed in dwarfed individuals and located within the confidence interval of the QTL on LG7. A deletion of 84,482 bp was identified in the genome of dwarfed plants, which included these four not expressed genes. One of these genes was VviCURLY LEAF (VviCLF), an orthologue of CLF, a regulator of shoot development in Arabidopsis thaliana. CONCLUSIONS: The phenotype of the dwarfed grapevine plants was similar to that of clf mutants of A. thaliana and orthologues of the known targets of CLF in A. thaliana were differentially expressed in the dwarfed plants. This suggests that CLF, a major developmental regulator in A. thaliana, also controls shoot development in grapevine.

Vine nitrogen status and volatile thiols and their precursors from plot to transcriptome level.

BACKGROUND: Volatile thiols largely contribute to the organoleptic characteristics and typicity of Sauvignon blanc wines. Among this family of odorous compounds, 3-sulfanylhexan-1-ol (3SH) and 4-methyl-4-sulfanylpentan-2-one (4MSP) have a major impact on wine flavor. These thiols are formed during alcoholic fermentation by the yeast from odorless, non-volatile precursors found in the berries and the must. The present study investigates the effects of vine nitrogen (N) status on 3SH and 4MSP content in Sauvignon blanc wine and on the glutathionylated and cysteinylated precursors of 3SH (Glut-3SH and Cys-3SH) in the berries and the must. This is paralleled by a RNA-seq analysis of gene expression in the berries. The impact of N supply on the expression of the glutathione-S-transferase 3 and 4 (VviGST3 and VviGST4) and the γ-glutamyltranspeptidase (VviGGT), considered as key genes in their biosynthesis, was also evaluated. RESULTS: N supply (N100 treatment) increased the 3SH content in wine while no effect was noticed on 4MSP level. Furthermore, N supply increased Glut-3SH levels in grape berries at late berry ripening stages, and this effect was highly significant in must at harvest. No significant effect of N addition was noticed on Cys-3SH concentration. The transcript abundance of the glutathione-S-transferases VviGST3 and VviGST4 and the γ-glutamyltranspeptidase (VviGGT), were similar between the control and the N100 treatment. New candidate genes which might be implicated in the biosynthetic pathway of 3SH precursors were identified by whole transcriptome shotgun sequencing (RNA-seq). CONCLUSIONS: High vine N status has a positive effect on 3SH content in wine through an increase of Glut-3SH levels in grape berries and must. Candidate GSTs and glutathione-S-conjugates type transporters involved in this stimulation were identified by RNA-seq analysis.

Vine Nitrogen Status Does Not Have a Direct Impact on 2-Methoxy-3-isobutylpyrazine in Grape Berries and Wines.

Methoxypyrazines (MP) constitute a large family of compounds that contribute to the vegetative varietal aroma of many grapevine varieties and wines. The berry content in 2-methoxy-3-isobutylpyrazine (IBMP), a major MP reminiscent of green-pepper aroma, can be influenced by environmental factors or cultural practices such as water status or mineral nutrition. To date, no study has investigated a possible direct effect of nitrogen (N) on IBMP synthesis without possible interference from water status and vigor variations. In this study, only vine nitrogen status was significantly different among treatments. Water status was controlled during the season, and vine vigor was similar among treatments. IBMP level was maximal at bunch closure and decreased during the season. There was no significant effect of nitrogen nutrition on this metabolite. Moreover, the expression profiles of VvOMT3 and VvOMT4, key genes in the IBMP biosynthetic pathway, were similar between treatments. This result indicates that when an effect of N on IBMP was found in previous studies, it was likely mediated through the modification of bunch-zone microclimate, induced by the higher vigor of high N-status vines.

Genetic analysis of the biosynthesis of 2-methoxy-3-isobutylpyrazine, a major grape-derived aroma compound impacting wine quality.

Methoxypyrazines (MPs) are strongly odorant volatile molecules with vegetable-like fragrances that are widespread in plants. Some grapevine (Vitis vinifera) varieties accumulate significant amounts of MPs, including 2-methoxy-3-isobutylpyrazine (IBMP), which is the major MP in grape berries. MPs are of particular importance in white Sauvignon Blanc wines. The typicality of these wines relies on a fine balance between the pea pod, capsicum character of MPs and the passion fruit/grapefruit character due to volatile thiols. Although MPs play a crucial role in Sauvignon varietal aromas, excessive concentrations of these powerful odorants alter wine quality and reduce consumer acceptance, particularly in red wines. The last step of IBMP biosynthesis has been proposed to involve the methoxylation of the nonvolatile precursor 2-hydroxy-3-isobutylpyrazine to give rise to the highly volatile IBMP. In this work, we have used a quantitative trait loci approach to investigate the genetic bases of IBMP biosynthesis. This has led to the identification of two previously uncharacterized S-adenosyl-methionine-dependent O-methyltransferase genes, termed VvOMT3 and VvOMT4. Functional characterization of these two O-methyltransferases showed that the VvOMT3 protein was highly specific and efficient for 2-hydroxy-3-isobutylpyrazine methylation. Based on its differential expression in high- and low-MP-producing grapevine varieties, we propose that VvOMT3 is a key gene for IBMP biosynthesis in grapevine.

Transcriptional analysis of late ripening stages of grapevine berry.

BACKGROUND: The composition of grapevine berry at harvest is a major determinant of wine quality. Optimal oenological maturity of berries is characterized by a high sugar/acidity ratio, high anthocyanin content in the skin, and low astringency. However, harvest time is still mostly determined empirically, based on crude biochemical composition and berry tasting. In this context, it is interesting to identify genes that are expressed/repressed specifically at the late stages of ripening and which may be used as indicators of maturity. RESULTS: Whole bunches and berries sorted by density were collected in vineyard on Chardonnay (white cultivar) grapevines for two consecutive years at three stages of ripening (7-days before harvest (TH-7), harvest (TH), and 10-days after harvest (TH+10)). Microvinification and sensory analysis indicate that the quality of the wines made from the whole bunches collected at TH-7, TH and TH+10 differed, TH providing the highest quality wines.In parallel, gene expression was studied with Qiagen/Operon microarrays using two types of samples, i.e. whole bunches and berries sorted by density. Only 12 genes were consistently up- or down-regulated in whole bunches and density sorted berries for the two years studied in Chardonnay. 52 genes were differentially expressed between the TH-7 and TH samples. In order to determine whether these genes followed a similar pattern of expression during the late stages of berry ripening in a red cultivar, nine genes were selected for RT-PCR analysis with Cabernet Sauvignon grown under two different temperature regimes affecting the precocity of ripening. The expression profiles and their relationship to ripening were confirmed in Cabernet Sauvignon for seven genes, encoding a carotenoid cleavage dioxygenase, a galactinol synthase, a late embryogenesis abundant protein, a dirigent-like protein, a histidine kinase receptor, a valencene synthase and a putative S-adenosyl-L-methionine:salicylic acid carboxyl methyltransferase. CONCLUSIONS: This set of up- and down-regulated genes characterize the late stages of berry ripening in the two cultivars studied, and are indirectly linked to wine quality. They might be used directly or indirectly to design immunological, biochemical or molecular tools aimed at the determination of optimal ripening in these cultivars.

Isolation and expression analysis of salt induced genes from contrasting grapevine (Vitis vinifera L.) cultivars.

Salt stress adversely affects the growth of grapevine plants. In order to understand the molecular basis of salt stress response in grapevine plants, suppression subtractive hybridization (SSH) and microarray based screening approaches were combined. Two leaf-specific subtractive cDNA libraries were constructed from grapevine plants subjected to a moderate, incremental salt stress treatment. SSH were performed 6h and 24h after NaCl peaked at 100mM using cDNAs prepared from leaves of a salt tolerant cultivar (Razegui) as testers and cDNAs from unstressed leaves as drivers. Then, a pre-screened subset of cDNA clones from these SSH libraries were used to construct a Vitis vinifera cDNA array, in order to verify the expression changes of the genes upon salt treatment. Expression profiles were compared between the salt tolerant and a susceptible cultivar (Syrah) under both control conditions and after salt stress treatment. Seven cDNA clones were identified which were up-regulated by salt stress in two independent growth experiments and confirmed by RNA blot analysis. The transcript expression patterns of the selected genes differed between the contrasting grapevine cultivars tested with respect to stress-regulation. The possible relationship of individual cDNAs with salinity tolerance mechanisms is discussed.

The grapevine transcription factor WRKY2 influences the lignin pathway and xylem development in tobacco.

Previous work has shown that transgenic tobacco plants constitutively over-expressing the Vitis vinifera L. transcription factor VvWRKY2 exhibit reduced susceptibility to necrotrophic fungal pathogens, suggesting that this transcription factor plays a role in grapevine response to phytopathogens. The work presented here characterizes the modifications in cell wall structure observed in the stems and petioles of these transgenic plants. Histochemical stainings of stem and petiole cross-sections using phloroglucinol or Maüle reagents revealed a delay in xylem formation, particularly in the petioles, and differences in lignin composition. Evaluation of lignin quantity and quality showed a decrease in the syringyl/guaiacyl ratio in both stem and petioles. Expression analysis using RT-PCR and potato microarrays showed that tobacco plants over-expressing VvWRKY2 exhibited altered expression of genes involved in lignin biosynthesis pathway and cell wall formation. The ability of VvWRKY2 to activate the promoter of the VvC4H gene, which is involved in the lignin biosynthetic pathway, was confirmed by transient transcriptional activation assays in tobacco protoplasts. Moreover, in situ hybridization revealed that VvWRKY2 is specifically expressed in cells undergoing lignification in young grapevine stems. Together, these results confirm that VvWRKY2 plays a role in regulating lignification in grapevine, possibly in response to biotic or abiotic stresses.

Expression of cell wall related genes in basal and ear internodes of silking brown-midrib-3, caffeic acid O-methyltransferase (COMT) down-regulated, and normal maize plants.

BACKGROUND: Silage maize is a major forage and energy resource for cattle feeding, and several studies have shown that lignin content and structure are the determining factors in forage maize feeding value. In maize, four natural brown-midrib mutants have modified lignin content, lignin structure and cell wall digestibility. The greatest lignin reduction and the highest cell wall digestibility were observed in the brown-midrib-3 (bm3) mutant, which is disrupted in the caffeic acid O-methyltransferase (COMT) gene. RESULTS: Expression of cell wall related genes was investigated in basal and ear internodes of normal, COMT antisens (AS225), and bm3 maize plants of the INRA F2 line. A cell wall macro-array was developed with 651 gene specific tags of genes specifically involved in cell wall biogenesis. When comparing basal (older lignifying) and ear (younger lignifying) internodes of the normal line, all genes known to be involved in constitutive monolignol biosynthesis had a higher expression in younger ear internodes. The expression of the COMT gene was heavily reduced, especially in the younger lignifying tissues of the ear internode. Despite the fact that AS225 transgene expression was driven only in sclerenchyma tissues, COMT expression was also heavily reduced in AS225 ear and basal internodes. COMT disruption or down-regulation led to differential expressions of a few lignin pathway genes, which were all over-expressed, except for a phenylalanine ammonia-lyase gene. More unexpectedly, several transcription factor genes, cell signaling genes, transport and detoxification genes, genes involved in cell wall carbohydrate metabolism and genes encoding cell wall proteins, were differentially expressed, and mostly over-expressed, in COMT-deficient plants. CONCLUSION: Differential gene expressions in COMT-deficient plants highlighted a probable disturbance in cell wall assembly. In addition, the gene expressions suggested modified chronology of the different events leading to cell expansion and lignification with consequences far beyond the phenylpropanoid metabolism. The reduced availability of monolignols and S units in bm3 or AS225 plants led to plants also differing in cell wall carbohydrate, and probably protein, composition. Thus, the deficiency in a key-enzyme of the lignin pathway had correlative effects on the whole cell wall metabolism. Furthermore, the observed differential expression between bm3 and normal plants indicated the possible involvement in the maize lignin pathway of genes which up until now have not been considered to play this role.

Differential expression of phenylpropanoid and related genes in brown-midrib bm1, bm2, bm3, and bm4 young near-isogenic maize plants.

The expression of phenylpropanoid and related genes was investigated in bm1, bm2, bm3, and bm4 near-isogenic maize plants at the 4-5 leaf stage using a gene-specific cell wall macro-array. The bm3 mutant, which is mutated in the caffeic acid O-methyltransferase (COMT) gene, exhibited the lowest number of differentially expressed genes. Although no other phenylpropanoid gene had an altered expression, two distinct OMT and two cytochrome P450 genes were overexpressed suggesting the activation of alternative hydroxylation/methylation pathways. The bm1 mutant had the highest number of differentially expressed genes, all of which were under-expressed. Bm1 mutant plants were affected not only in cinnamyl alcohol dehydrogenase (bm1 related CAD) gene expression as expected, but also in the expression of other CAD/SAD gene family members and several regulatory genes including MYB, ARGONAUTE and HDZip. As originally believed, the bm1 mutation could be localized at the CAD locus, but more probably in a gene that regulates the expression of the CAD gene family. The profile of under-expressed genes in the bm2 mutant is nearly similar to that of bm1. These genes fell under several functional categories including phenylpropanoid metabolism, transport and trafficking, transcription factors and regulatory genes. As the bm2 mutant exhibited a lower guaiacyl (G) unit lignin content, the bm2 mutation could affect a regulatory gene involved, perhaps indirectly, in the regulation, conjugation or transport of coniferaldehyde, or the establishment of G-rich maize tissues. The pattern of gene expression in bm4 plants, characterized by the over-expression of phenylpropanoid and methylation genes, suggests that the bm4 mutation likely also affects a gene involved in the regulation of lignification.

MAIZEWALL. Database and developmental gene expression profiling of cell wall biosynthesis and assembly in maize.

An extensive search for maize (Zea mays) genes involved in cell wall biosynthesis and assembly has been performed and 735 sequences have been centralized in a database, MAIZEWALL (http://www.polebio.scsv.ups-tlse.fr/MAIZEWALL). MAIZEWALL contains a bioinformatic analysis for each entry and gene expression data that are accessible via a user-friendly interface. A maize cell wall macroarray composed of a gene-specific tag for each entry was also constructed to monitor global cell wall-related gene expression in different organs and during internode development. By using this macroarray, we identified sets of genes that exhibit organ and internode-stage preferential expression profiles. These data provide a comprehensive fingerprint of cell wall-related gene expression throughout the maize plant. Moreover, an in-depth examination of genes involved in lignin biosynthesis coupled to biochemical and cytological data from different organs and stages of internode development has also been undertaken. These results allow us to trace spatially and developmentally regulated, putative preferential routes of monolignol biosynthesis involving specific gene family members and suggest that, although all of the gene families of the currently accepted monolignol biosynthetic pathway are conserved in maize, there are subtle differences in family size and a high degree of complexity in spatial expression patterns. These differences are in keeping with the diversity of lignified cell types throughout the maize plant.

Nitrogen availability, local light regime and leaf rank effects on the amount and sources of N allocated within the foliage of young walnut (Juglans nigra x regia) trees.

Early season leaf growth depends largely on nitrogen (N) provided by remobilization from storage, and many studies have tested the effect of N availability to roots on the amount of N provided for new leaf development by remobilization. Although it is well known that the light regime experienced by a leaf influences the amount of N per unit leaf area (LA), the effect of the local light regime on the amount of N derived either directly from root uptake or from remobilization for early season leaf growth has never been tested at an intra- canopy scale. The objective of this study was to quantify the relative importance of (1) N availability to roots, (2) local light regime experienced by the foliage (at the shoot scale) and (3) leaf rank along the shoot, on the total amount of N allocated to leaves and on the proportions of N provided by remobilization and root uptake. To quantify the importance of N uptake and remobilization as sources of leaf N, potted hybrid walnut trees (Juglans nigra L. x regia L.) were grown outdoors in sand and fed with a labeled ((15)N) nutrient solution. By removing the apical bud, the trees were manipulated to produce only two shoots. The experimental design had two factors: (1) high (HN; 8 mol N m(-3)) and low (LN; 2 mol N m(-3)) N availability; and (2) high (HL; 90% of incident photosynthetically active photon flux (PPF)) and low (LL; 10% of incident PPF) light. Total leaf N per tree was unaffected by either N availability or irradiance. The HN treatment increased the amount of leaf N derived from root uptake at the whole-tree scale (typically around 8 and 2% in the HN and LN treatments, respectively). Nitrogen allocation within foliage of individual trees was controlled by the local light regime, which strongly affected individual leaf characteristics as leaf mass per unit LA and area- based amount of leaf (N(a)). Decreasing the light availability to a branch decreased the amount of N allocated to it, benefiting the less shaded branches. In contrast, shading of the lower branch did not affect the fraction of total leaf N remobilized for either the lower, shaded branch or the upper, unshaded branch. The relevance of these findings for tree growth modeling is discussed.

Genetic and molecular basis of grass cell wall biosynthesis and degradability. II. Lessons from brown-midrib mutants.

The brown-midrib mutants of maize have a reddish-brown pigmentation of the leaf midrib and stalk pith, associated with lignified tissues. These mutants progressively became models for lignification genetics and biochemical studies in maize and grasses. Comparisons at silage maturity of bm1, bm2, bm3, bm4 plants highlighted their reduced lignin, but also illustrated the biochemical specificities of each mutant in p-coumarate, ferulate ester and etherified ferulate content, or syringyl/guaiacyl monomer ratio after thioacidolysis. Based on the current knowledge of the lignin pathway, and based on presently developed data and discussions, C3H and CCoAOMT activities are probably major hubs in controlling cell-wall lignification (and digestibility). It is also likely that ferulates arise via the CCoAOMT pathway.

Colocation between a gene encoding the bZip factor SPA and an eQTL for a high-molecular-weight glutenin subunit in wheat (Triticum aestivum).

The quality of wheat grain is largely determined by the quantity and composition of storage proteins (prolamins) and depends on mechanisms underlying the regulation of expression of prolamin genes. The endosperm-specific wheat basic region leucine zipper (bZIP) factor storage protein activator (SPA) is a positive regulator that binds to the promoter of a prolamin gene. The aim of this study was to map SPA (the gene encoding bZIP factor SPA) and genomic regions associated with quantitative variations of storage protein fractions using F7 recombinant inbred lines (RILs) derived from a cross between Triticum aestivum "Renan" and T. aestivum "Récital". SPA was mapped through RFLP using a cDNA probe and a specific single nucleotide polymorphism (SNP) marker. Storage protein fractions in the parents and RILs were quantified using capillary electrophoresis. Quantitative trait loci (QTLs) for protein were detected and mapped on six chromosome regions. One QTL, located on the long arm of chromosome 1B, explained 70% of the variation in quantity of the x subunit of Glu-B1. Genetic mapping suggested that SPA is located on chromosome arm 1L and is also present in the confidence interval of the corresponding QTL for Glu-B1x on 1BL, suggesting that SPA might be a candidate gene for this QTL.

Genetic and molecular basis of grass cell-wall biosynthesis and degradability. III. Towards a forage grass ideotype.

Lignification of cell walls is the major factor controlling the digestibility of forage grasses. Thus far, from QTL analysis, about 15 locations involved in cell-wall lignification or digestibility have been identified in the maize genome, many of which colocalise with QTLs involved in corn borer susceptibility. Genetic diversity for enhancing cell-wall digestibility in maize must be identified in novel germplasm, but genetic engineering is also a relevant way both to design specific cell-wall characteristics for improved digestibility and to identify genes involved in these traits for further discovery of alleles of interest in grass germplasm.

Coupling sap flow velocity and amino acid concentrations as an alternative method to (15)N labeling for quantifying nitrogen remobilization by walnut trees.

The temporal dynamics of N remobilization was studied in walnut (Juglans nigra x regia) trees growing in sand culture. Trees were fed with labeled N ((15)N) during 1999 and unlabeled N in 2000. Total N and (15)N contents in different tree compartments were measured during 80 d after bud burst and were used to estimate N remobilization for spring growth. The seasonal (and occasionally diurnal) dynamics of the concentration and (15)N enrichment of the major amino acids in xylem sap were determined concurrently. Sap flow velocity was also measured for sample trees. A new approach coupling amino acid concentrations to sap flow velocity for quantifying N remobilization was tested. A decrease of the labeled N contents of medium roots, tap roots, and trunk was observed concurrently to the increase in the labeled N content of new shoots. Remobilized N represented from previous year storage 54% of N recovered in new shoots. Arginine, citruline, gamma-amino butyric acid, glutamic acid, and aspartic acid always represented around 80% of total amino acid and amide N in xylem sap and exhibited specific seasonal trends and significant diurnal trends. N translocation was mainly insured by arginine during the first 15 d after bud burst, and then by glutamic acid and citruline. The pattern of N remobilization estimated by the new approach was consistent with that measured by the classical labeling technique. Implications for quantifying N remobilization for large, field-growing trees are discussed.