Trees (2012) 26:393–404 DOI 10.1007/s00468-011-0600-8
ORIGINAL PAPER
Autumnal N storage determines the spring growth, N uptake and N internal cycling of young peach trees Marie-Odile Jordan • Renate Wendler Peter Millard
•
Received: 18 January 2011 / Revised: 19 July 2011 / Accepted: 25 July 2011 / Published online: 28 August 2011 Ó Springer-Verlag 2011
Abstract Although N storage determines early spring growth in trees, the usefulness of autumn N supply remains unclear as N uptake decreases in autumn, but could be restored earlier in spring to compensate for low N cycling. We intended here to evaluate the effects of autumn N supply on N uptake, storage and cycling, and spring growth. Four levels of N fertilisation were applied to 1-year-old peach trees, between the end of shoot growth and leaf fall. In spring, N supply was 15N labelled. Organ dry weights and concentrations of 14N, 15N, starch and soluble sugars were evaluated after the first growth flush. Bud development had previously been described in the same trees by Jordan et al. (Trees-Struct Func 23:235–245, 2009). Fertilisation promoted autumn N uptake, spring N uptake and growth up to a threshold level, since no differences were evidenced between the three highest N treatments. The variability in tree 14N contents was related to the number of phytomers per tree in autumn, i.e. to tree size. In spring, the depletion of the perennial structures was independent of treatment, indicating a complete mobilisation of the N stores. Spring growth was related to the amounts of cycling N, and spring N uptake was in turn proportioned to shoot and fruit growth. The lower N uptake of the N limited trees was not due to a C shortage since
Communicated by H. Rennenberg. M.-O. Jordan (&) UR1115 Plantes et Syste`mes de Culture Horticoles, INRA, Domaine Saint-Paul Agroparc, 84914 Avignon Cedex 9, France e-mail:
[email protected] R. Wendler P. Millard MLURI, Macaulay Institute Craigiebuckler, Aberdeen AB15 8QH, UK
these trees displayed the highest starch concentrations. We conclude that a moderate autumn fertilisation improved spring growth and fruit production (Jordan et al. in TreesStruct Func 23:235–245, 2009) and that a deficit of N storage could not be compensated for by an increase in spring N uptake. Keywords Nitrogen fertilisation N uptake N cycling Spring growth Non-structural carbohydrate Abbreviations C Carbon DW Dry weight N Nitrogen TNC Total non-structural carbohydrate
Introduction Optimising fertilisation is a challenge of increasing importance to orchard management. Fall N supply may be preferred to spring fertilisation for practical reasons such as time organisation and also because it is assumed to enable better control of vegetative growth. Nevertheless, the efficiency of fall N fertilisation is still debated in the literature. According to Niederholzer et al. (2001), the leaf N resumption of adult peach trees may be sufficient to ensure optimum spring growth because fall N uptake efficiency is low (18%) and leaf N resumption is responsible for 80% of N accumulation in the perennial parts. On the other hand, Dong et al. (2002) and Amponsah et al. (2004) suggested that up to 33% of supplied N could be recovered by the trees in late fall. For Cheng et al. (2002), apple trees may absorb up to 45% of their total N content within the
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2 months that precede leaf fall. It is nonetheless accepted that the mobilisation of stores in the spring is proportional to the N accumulated prior to leaf fall (Millard and Neilsen 1989; Millard and Proe 1993; Millard 1996). Similarly, no clear consensus has been reached regarding the efficiency of early spring fertilisation. In pears (Quartieri et al. 2002) N uptake between burst and May is found to be about seven times higher than between June and September. But more frequently, N uptake is assumed to become significant and dependent on soil N availability only after several weeks (Millard and Neilsen 1989; Munoz et al. 1993), i.e. after the start of rapid shoot growth (Weinbaum et al. 1978). The dynamics of the restoration of N uptake in spring is dependent on tree species, but whether it is affected by store mobilisation in spring has been the subject of little investigation. According to Niederholzer et al. (2001), supplying N in early spring rather than in autumn has no impact on tree growth. The consensus most widely accepted is that shoot growth depends on store mobilisation during the first weeks after burst, but is determined as earlier by spring N uptake as the amounts of cycling N are small (Deng et al. 1989; Millard and Proe 1993; Niederholzer et al. 2001). Depending on the situation, cycling N accounts for between 36 and 76% of the N incorporated into current year shoots by the end of April (Weinbaum et al. 1978; Millard and Proe 1993; Tagliavini et al. 1997). The contribution of N store mobilisation to spring development cannot be precisely quantified due to discrepancies in the data. First, variations in tree response with species prevent any generalisation of results. Second, the effect of cycling N on spring growth is usually evaluated by manipulating tree N status through fertilisation treatment applied before leaf fall. Tree preconditioning can last for between 2 weeks and the entire vegetative period and affects plant growth immediately. Tree biomass at leaf fall thus varies according to treatment from almost nil (Niederholzer et al. 2001) to a factor of four (Millard and Proe 1991). Early spring development therefore varies not only according to the differences in tree N status, but also in tree size induced by treatment. The latter is rarely taken into account, despite its effects on the volume of N reservoirs (i.e. the stems and roots) and on spring growth potential (i.e. the number of vegetative buds that may develop). Third, most studies only compare two fertilisation treatments; in other words, threshold levels of (1) N supply corresponding to uptake saturation, i.e. at which N uptake levels off, and (2) maximum N cycling corresponding to either store fulfilment or a peak of store mobilisation, have never been determined. Fourth, fall N supply affects not only spring development but also TNC (total non-structural carbon) stores. Thus, it delays leaf senescence (Rosecrance et al. 1998; Niederholzer et al.
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2001) and increases carbon acquisition (Kubiske et al. 1998; Veberic et al. 2002). TNC availability has no impact on spring growth (Cheng et al. 2002), but does affect the restoration of N uptake (Weinbaum et al. 1978; Adamowicz and Le Bot 2008). The regulation of the TNC and of the N stores is therefore interdependent. The aim of the present study was to assess the effects of an autumn N supply on spring development, N uptake and TNC availability. For this purpose, young peach trees received four levels of N supply in late autumn, i.e. between the cessation of shoot growth in terms of elongation, and leaf fall. The following spring, N was supplied in quantities that exceeded growth needs and was labelled with 15N. The impact of fall N supply on (1) N uptake in fall and spring, (2) N store mobilisation in spring, (3) growth and (4) tree TNC contents was quantified at the end of the first growth flush. Only data on gross growth are detailed in the Results section, but the effects of fall N supply on the tree architecture and on the balance between vegetative and reproductive growth were assessed in the same trees in a previous study (Jordan et al. 2009).
Materials and methods Experimental design The study was carried out at the INRA Research Centre in Avignon (southern France). Forty 1-year-old peach rootstocks (Prunus persica Batsch, cv. GF305) with a diameter of between 6 and 8 mm were grafted with pushing buds of peach (cv. RO52) on March 16, 1999 and transplanted into 10 dm3 pots filled with a 50% vermiculite and 50% peat mixture. The trees were left in a greenhouse for 1 month and then moved outside. During the growth period, chemical treatments were applied regularly to deter pests. Two drippers per pot, each with a delivery rate of 2 dm3 h-1, supplied a nutrient solution at a concentration of 1 g dm-3 of a commercial 14/7/27% NPK fertiliser. The trees were irrigated for 6 min, ten times each day. At the end of shoot elongation, i.e. when all leaves were fully expanded, each tree was described by counting the number of phytomers of each axis, i.e. on the trunk and its ramifications. Phytomers are the elementary base units that form the axes and are composed of a node, an internode, a leaf and an axillary bud (White 1979). Their number is therefore representative of tree size or vigour. Twenty-four trees were selected for their homogeneity and divided into four groups of six individuals; the total mean number of phytomers was 196 (SE or standard error: 4.5) distributed between the main axis (41 phytomers, SE: 1.6) and the 8.3 secondary axes. Six further trees were kept under automatic
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irrigation for subsequent evaluation of the natural abundance of 15N. Each group of six trees received a different level of nitrate fertilisation between September 13 and leaf-fall (November 10). Nitrate and other nutrients were supplied three times a week (on Monday, Wednesday and Friday) in 0.3 dm3 nutrient solution which, depending on the treatment, contained 1.5, 3, 4.5 or 6 g NO3 dm-3 as Ca(NO3)2, corresponding to a weekly supply ranging from 1.3 to 5.4 g NO3plant-1. These treatments were designated 1.5N, 3N, 4.5N and 6N, respectively. This solution also contained the following in mol m-3: MgSO4 1; KCl 0.2; K2SO4 1.5; KH2SO4 0.5; Fe EDDHA (Ethylenediamine-di(o-hydroxyphenylacetic Acid)) 0.1; and in lmol m-3: H3BO3 206.58; MnCl2 116.57; CuSO4 4.72; ZnSO4 32.41; MoNH4 28.15. No excess solution drained from the pots. On the four remaining days of each week, the field capacity was restored by automatic irrigation with tap water for ten sequences of 6 min (corresponding to a daily supply of 4 dm3 tree-1). The number of irrigations per day was reduced to five (29 September), then to three (15 October) and finally to 0 (2 November). No irrigation was supplied during the winter. On February 24, 2000, after soaking the roots in tap water for 3 h, the trees were transplanted into 15 dm3 pots containing a substrate composed of 60% sand (Biot B4, ref 16.14.2) and 40% pozzolana. The trees were left outside. The NO3- concentration in the nutrient solution was labelled with 2.6 atom % 15N and adjusted to 1.5 mmol dm-3. The concentrations of the other nutrients were the same as in the solution used the previous autumn. Each tree received 0.3 dm3 day-1 from March 2 to April 2, then 0.5 dm3 day-1 until April 18, 1 dm3 day-1 until May4, 1.5 dm3 day-1 until May 15 and 2 dm3 day-1 until tree sampling. The supply was adjusted so as to maintain some solution available to the plants throughout the day, in saucers placed under the pots. Two destructive samplings of three individuals per treatment were made at the end of the first growth flush, or the presumed end of the N cycling period, i.e. on May 29 and June 13, respectively. The six trees used to evaluate the natural abundance of 15N were sampled on February 25. Tree sampling and chemical analyses The trees were subsampled as follows for biochemical analyses: thin and thick roots (less than and more than 0.5 cm in diameter, respectively), rootstock trunk, main axis, secondary axis, stems of current year shoots, leaves and fruits. Because N is stored preferentially in bark, and non-structural C mainly accumulates in wood, the wood and bark were separated for biochemical analyses. All samples were kept at -20°C before freeze drying and weighing. The samples were ground in a stainless steel
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Dangoumeau grinder (Prolabo France) cooled with liquid N2. Total N concentrations and 15N excess were determined using a Tracer-MAT continuous flow mass-spectrometer (Finnigan MAT, Hemel Hempstead, UK). The 15 N enrichment was used to calculate the amount of labelled N taken up from the fertilizer solution in 2000, as described by Millard and Neilsen (1989). The determinations of non-structural C concentrations were limited to the 1.5N and 3N treatments, since no differences between the three highest N treatments, i.e. the 3N, 4.5N and 6N ones, were evidenced with respect to spring development (Jordan et al. 2009), N uptake and allocation (see Results). The extractions and determinations of soluble sugar concentrations were performed as described by Gomez et al. (2002): extraction in a methanol–chloroform–water medium and determination by HPLC (Sugar PaK 1 column at 80°C and refractometer, Waters, Milford, MA, USA). The starch concentration was determined on pellets, as described by Jordan and Habib (1996): solubilisation by autoclaving, depolymerisation and determination of the resulting glucose using the reference enzymatic method. The total non-structural carbohydrate (TNC) was assumed to be the sum of soluble sugars and starch. The content and concentration of the each compound thus determined were then calculated from subsample DW (dry weights) and concentrations for (1) the roots, (2) the old axes composed of the bark and wood of the rootstock trunk, the main and secondary axes, (3) current year shoots: stems and leaves and (4) the whole tree. Fruits were not fractionated before chemical determinations. Data analyses Since the number of repetitions per treatment was small, i.e. limited to three trees per harvest, randomisation (or permutation) tests (Manly 1991) were used to evaluate the effects of treatment and harvest date on the concentrations and contents of starch, soluble sugars, TNC, 14N, 15N and total N. Its use was justified on the null hypothesis that the treatments had no effect since, before treatment, the trees were (1) raised under the same conditions, (2) equivalent in terms of size and number of buds and (3) randomly allocated to groups. Each test, performed at 5% level, consisted of the generation of 2500 random orders (rearrangements of the dataset). Two treatments were assumed to be significantly different when the differences between their means were greater for the observed value than for the estimates in 95% of the cases (for more than 2375 random orders). The homogeneity of variances was tested along with the mean comparisons. Computations (Rplus software) were performed first at tree level and then for each tree fraction: roots, old axes, shoots and fruits, which were reanalysed one by one.
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Linear regressions (Draper and Smith 1998) were also calculated in order to assess how far individual variations in 14N, 15N and total N contents could be explained (1) by the initial vigour of a tree approximated by the number of phytomers at experiment start, (2) or by growth approximated by tree or organ biomass. The normal distribution, homogeneity of variance and functional dependence between residuals and predicted values of the residuals have been verified (Scherrer 1984) using the qqnorm procedure, i.e. comparing the quantile– quantile plots of the residuals to a normal quantile– quantile plot.
Results N uptake Tree 14N content corresponded to the N absorbed during the treatment period and the previous growing period. It increased first with fall N supply and then levelled off, indicating that autumnal N uptake increased with fertilisation up to a threshold level of supply corresponding to the 3N treatment (Fig. 1). In 3N trees, it varied moreover with sampling date, increasing from 2675 to 3378 mg 14N tree-1 between May 29 and June 13. This was probably an artefact resulting from tree vigour, since the sampled 3N trees had a mean number of 184 and 211 phytomers on May 29 and June 13, respectively. Thus (Fig. 2), the tree 14 N contents (Ct 14N in mg) among trees under the same treatment were related to the number of phytomers on their constitutive axes (Phyto). The following linear models were significant at 1% (**) and 5% (*) levels, respectively. Nonetheless, in 1.5N trees only the explanatory variable was significant, not the intercept (noted ns or not significant).
Fig. 1 Mean tree content (mg tree-1) as a function of treatment of 14 N (stars, full line) and 15N (dotted lines). For 15N, trees sampled on May 29 (full dots) and June 13 (full triangles) are separated. Each symbol is the mean of 6 (14N) or 3 (15N) trees plotted with standard errors. Means coded with different letters are significantly different at 5% level. Comparisons were made using a randomization test involving the generation of 2500 random orders. Each N fraction was computed separately
In 1:5N trees : Ct14 N ¼ 9:01 Phyto þ 553ðnsÞ r2 ¼ 0:92
ð1Þ
In 3N; 4:5N and 6N trees : Ct14 N ¼ 9:66 Phyto þ 1172
r2 ¼ 0:47 :
ð2Þ
Spring N uptake was evaluated using 15N labelling. The differences between treatments became significant on June 13 (Fig. 1), since the N uptake rates of 1.5N trees did not increase after May 29, as was the case in high N trees (i.e. 3N, 4.5N and 6N trees). Thus, during the 14 days between the two samplings, 1.5N trees absorbed 15% of their final 15 N content, although this proportion reached 31% in high N trees. The remainder had been absorbed during the previous 94 days, i.e. between February 24 and May 29. In
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Fig. 2 Relationship between the number of phytomers per tree and 14N contents (mg organ-1, mg tree-1) of either whole trees (stars and closed symbols) or trees perennial structures: i.e. roots and woody axes (crosses and open symbols). Each symbol represents a single tree under the following treatments: 1.5N (crosses, stars), 3N (squares), 4.5N (dots), 6N (triangles). The lines plot the regression thus calculated
contrast with what had been observed for 14N, no relationship could be evidenced between tree 15N content and number of phytomers.
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Tree biomass Spring growth was important, insofar as shoot and fruit DW accounted for at least 50% of tree DW at harvest. Shoot and fruit DW varied with treatment and sampling date (Fig. 3), but were not affected by the number of phytomers per tree (results not shown). On May 29, 1.5N trees had a lower shoot DW than the 3N and 4.5N trees (Table 1), but no differences between treatments could be evidenced for fruit DW. Between the two sampling dates, shoot DW increased significantly in 1.5 trees, and fruit DW in high N trees. On June 13, 1.5N trees had a shoot DW almost as high as that of high N trees, but a significantly lower fruit DW. The DW of the perennial structure, i.e. the roots and old axes, was not affected by treatment (Table 1), with one exception: on June 13, the 3N trees had a higher root DW than the 4.5N. Between the two sampling dates root growth was almost nil, and old axes increased their dry biomass by 18%, but the difference was only significant in 4.5N trees. The shoot to root ratio also increased with fall N supply, as treatment only affected shoot and fruit growth. On June 13, it was found to be 3.4 for 1.5N trees and 5.15 for high N trees. Respective contributions of N cycling and current year uptake to shoot and fruit growth N mobilisation was completed on May 29. Thus no differences in the 14N content of perennial structures could be
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evidenced between sampling dates or treatments, with one exception: 1.5N trees sampled on May 29 had significantly less 14N in their perennial structures than 3N trees sampled on June 13 (Table 2). Whatever the treatment, only 850 mg 14 N remained in the roots and axes after spring mobilisation (Fig. 2). This amount (St 14N) varied nonetheless (significance level of 5%) with the number of phytomers per tree. In all trees : St14 N ¼ 2:78 Phyto þ 318 r2 ¼ 0:43 ð3Þ The remaining 14N, which accounted for between 63 and 71% of the tree 14N content, was incorporated in the current year shoots and fruits. Depending on the treatments, it represented between 1447 and 2247 mg 14 N tree-1. Shoots received between 55% (high N trees) and 68% (1.5 N trees) of that amount, while fruits received the remainder. Shoots and fruits also contained an average of 89% of the tree 15N content, which corresponded to an amount comprised between 1.03 and 1.76 g 15N. Shoot and fruit dry DW (NewOrg in g), or current year organ growth, was related to the tree contents in 14N (Ct 14N in mg) and 15N (Ct 15N in mg), i.e. to both current year N uptake and amount of cycling N (tree 14N content minus ±850 mg). The linear models (Fig. 4) were not affected by sampling date, but by treatment when computed for 14N. Thus growth enabled by 14N cycling was limited in the 1.5N trees because of the high proportion of 14N incorporated in the shoots, i.e. in the organs with the highest N concentration (Fig. 3). In all trees : NewOrg ¼ 5:25 Ct15 N þ 144:7ðnsÞ r2 ¼ 0:93
ð4Þ
In 1. 5N trees : NewOrg ¼ 2:93 Ct14 N þ 1633 r2 ¼ 0:77
ð5Þ
In 3N; 4:5N and 6N trees :
Fig. 3 Dry biomass of organs (DW g organ-1) in 1.5N trees (stars and crosses), 3N trees (squares) 4.5N trees (dots) and 6N trees (triangles); trees were sampled on May 29 (crosses, opens symbols) and June 13 (stars, closed symbols). Each symbol is the mean of three trees plotted with standard errors. The results of the mean comparisons are shown in Table 1
NewOrg ¼ 5:12 Ct14 N þ 1560 r2 ¼ 0:69 ð6Þ The best fit was obtained for 15N which continued to be assimilated between May 29 and June 13, when 14N incorporation into shoots and fruits had ceased. During that period, shoot and fruit growth remained small enough not to affect the linear models computed for 14N. Thus the dilution of 14N over time was limited, because the proportion of 14N in the N pool decreased by less than 4% in the growing organs, i.e. in the shoots of 1.5N trees and the fruits of high N trees. Shoot and fruit DW were also proportional to their 14N and 15N contents. The coefficients of determination of the linear models were, respectively, 0.91*** (shoot and fruit 15 N content), 0.96*** (shoot and fruit 14N content of 1.5N trees) and 0.67** (shoot and fruit 14N content of high N trees).
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Table 1 Results of randomization tests on mean dry biomass per organ (g organ-1) as a function of treatment and sampling date May 29 1.5N trees
June 3N trees
4.5N trees
6N trees
1.5N trees
3N trees
4.5N trees
6N trees 248.8 bc
Fruits
136.5 a
167.8 ab
171.9 ab
182.3 abc
129.2 a
265.7 c
270.8 c
Shoots
57.8 a
72.2 bc
72.1 bc
66.3 ab
79.3 bc
80.1 bc
74.8 bc
80.8 c
Old axes
92.4 ab
103.8 abcd
90.2 a
101.5 abc
118.3 bcd
127.6 d
122.8 cd
106.6 abcd
Roots
66.4 a
79.9 ab
73.6 ab
77.5 ab
80.6 ab
97.6 b
69.6 a
87.2 ab
Mean values and standard errors are plotted in Fig. 3. Each test involved the generation of 2,500 random orders and the results were assumed to be significant at 5% level (i.e. if fewer than 125 estimates were higher than the real value). The tests were performed organ by organ, comparing the treatments along with harvest dates. Each line thus represents a single test and means with the same letter are not significantly different. The numbers are mean of three replicates Table 2 Amounts of 14N (mg organ-1) remaining in the perennial structure (roots and axes) after spring mobilisation for the different treatments and sampling dates 1.5N trees
3N trees
4.5N trees
6N trees
May 29
709 (a) ± 33
814 (ab) ± 29
809 (ab) ± 39
874 (ab) ± 89
June 13
856 (ab) ± 162
919 (b) ± 98
867 (ab) ± 80
893 (ab) ± 68
The numbers are mean ± standard errors of three replicates. Letters differing between treatments indicate that mean values were significantly different at 5% level. Mean were compared using a randomization test. The test involved the generation of 2,500 random orders and the results were assumed to be significant at 5% level (i.e. if fewer than 125 estimates were higher than the real value)
Fig. 4 Tree 14N (stars and closed symbols) and 15N (crosses and open symbols) contents (mg tree-1) according to the dry biomass of current year organs: i.e. of shoots and fruits (DW g organ-1). Each symbol represents a single tree under the following treatments: 1.5N (crosses, stars), 3N (squares), 4.5N (dots), 6 N (triangles). The lines plot the regression thus calculated
treatments emerged between the two sampling dates; on June 13, the 1.5N trees were N and C limited (Fig. 1, Table 4) when compared with high N trees. In terms of N, the differences in concentrations between treatments were the greatest in fruits and shoots, i.e. in current year organs. Total N and 14N were the most markedly affected. For 15N, supplied in excess compared with spring growth needs, no difference was observed in shoots, but fruits on 1.5N trees were less concentrated than those on 3N and 6N trees. For C, the situations differed with organs and C compounds. Despite their low TNC contents, 1.5N trees accumulated more than twice as much starch as 3N trees. In 1.5N trees, concentrations were lower for all C compounds in fruits, but higher for starch and TNC in vegetative organs than in 3N trees. The old axes of 1.5N trees also contained lower concentrations of soluble sugars than 3N trees. This general description of the tree response to autumn N supply suffered from several exceptions, as detailed in Table 3. For instance, no difference between treatments could be observed in the root TNC concentrations on June 13, and higher concentrations of total N and 14N were observed in the roots of 4.5N trees on June 13.
N and TNC concentrations in tree organs
Discussion
On May 29, organ concentrations of N and non-structural C (Fig. 5; Table 3) were similar in all trees, with one exception: the axes of 4.5N trees had a higher total N concentration than in other trees. The difference between
The effects of an autum N supply on N store management, N uptake, accumulation of non-structural C and spring growth were evaluated in trees of similar size. The response, summarised Fig. 6, could therefore be attributed
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solely to the impact of N treatment on tree N status (or organ N concentrations) at bud burst, not on differences in spring growth potential, defined by the number of phytomers per tree, i.e. the number of axillary buds that could potentially develop. Overall growth could also be related to individual bud development, described in relation to their position in a previous paper (Jordan et al. 2009). Since treatments were applied after the cessation of shoot elongation (i.e. of leaf emergence), autumnal growth was restricted to axes thickening and root development, which are despite their importance
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(Niederholzer et al. 2001; Kaelke and Dawson 2005) only little (Kubiske et al. 1998; Dong et al. 2005) or not (Niederholzer et al. 2001; Jordan et al. 2011) affected by soil N availability. We found no difference between treatments in terms of root and old axes DW (Fig. 3), indicating that growth was independent of treatment for these organs not only in late autumn but also in early spring. The existence of growth correlations probably limited the thickening of old axes and root growth once shoot elongation had ceased in autumn (Sone et al. 2005; Osone et al. 2008). Indeed, root and axis biomass could
Fig. 5 Organ concentrations (% in DW) of 1.5N trees (stars and crosses), 3N trees (squares), 4.5N trees (dots) 6N trees (triangles); trees were sampled on May 29 (crosses, opens symbols) and June 13 (stars, closed symbols). a 15N concentration, b 14N concentration, c total N concentration, d soluble sugars concentration, e starch concentration and f TNC concentration. Determinations were restricted to the 1.5 N and 3 N treatments for the C fractions: soluble sugars, starch and TNC. Each symbol is the mean of three trees plotted with standard errors. The results of mean comparisons are shown in Table 3
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Table 3 Results of randomization tests for the mean concentration (% DW) of (a) 15N, (b) (f) total non-structural carbohydrates (TNC) as a function of treatment and sampling date May 29
14
N, (c) total N, (d) soluble sugars, (e) starch and
June 13
1.5N trees
3N trees
4.5N trees
6N trees
1.5N trees
3N trees
4.5N trees
6N trees
Fruits
0.32 ab
0.30 ab
0.29 ab
0.29 ab
0.21 a
0.35 b
0.30 ab
0.35 b
Shoots Old axes
0.99 a 0.07 a
1.01 a 0.07 a
1.05 a 0.07 a
1.03 a 0.06 a
0.96 a 0.08 ab
0.93 a 0.14 b
1.15 a 0.08 ab
1.15 a 0.10 ab
Roots
0.11 abc
0.09 ab
0.09 ab
0.07 a
0.13 bc
0.08 ab
0.15 c
0.11 ab
Fruits
0.39 ab
0.43 b
0.48 b
0.48 b
0.25 a
0.43 b
0.42 b
0.49 b
Shoots
1.46 ab
1.58 b
1.64 b
1.71 b
1.28 a
1.77 b
1.57 ab
1.54 ab
Old axes
0.30 ab
0.31 b
0.35 b
0.33 b
0.31 b
0.24 a
0.30 ab
0.30 b
Roots
0.65 ab
0.63 a
0.67 ab
0.69 ab
0.62 a
0.62 a
0.74 b
0.66 ab
Fruits
0.71 b
0.73 b
0.77 b
0.77 b
0.46 a
0.78 b
0.73 b
0.84 b
Shoots
2.45 b
2.59 b
2.70 b
2.74 b
2.24 a
2.69 b
2.72 b
2.69 b
Old axes
0.37 a
0.37 a
0.42 b
0.39 ab
0.39 ab
0.38 a
0.38 a
0.40 ab
Roots
0.76 a
0.72 a
0.76 a
0.76 a
0.76 a
0.71 a
0.89 b
0.77 a
Fruits
24.23 ab
24.63 ab
–
–
18.07 a
34.73 b
–
–
Shoots Old axes
8.69 ab 2.71 ab
9.17 b 2.48 ab
– –
– –
8.14 ab 2.41 a
7.69 a 3.19 b
– –
– –
Roots
4.71 a
4.61 a
–
–
4.57 a
5.29 a
–
–
a:
b:
15
N
14
N
c: Total N
d: Soluble sugars
e: Starch Fruits
0.66 b
0.62 b
–
–
0.12 a
0.39 ab
–
–
Shoots
5.42 ab
4.95 ab
–
–
6.47 b
3.34 a
–
–
Old axes
4.38 b
3.33 ab
–
–
4.17 b
1.84 a
–
–
Roots
5.13 ab
3.86 ab
–
–
6.39 b
1.60 a
–
–
Fruits
24.89 ab
25.25 ab
–
–
18.19 a
35.12 b
–
–
Shoots
14.12 ab
14.13 ab
–
–
14.61 b
11.04 a
–
–
Old axes
7.09 b
5.81 ab
–
–
6.58 b
5.03 a
–
–
Roots
9.84 ab
8.47 ab
–
–
10.96 b
6.89 a
–
–
f: TNC
Mean and standard errors are plotted in Fig. 5. Determinations were restricted to the 1.5N and 3N treatments for the C fractions: soluble sugars, starch and TNC. Each test involved the generation of 2,500 random orders and the results were assumed to be significant at 5% level (i.e. if fewer than 125 estimates were higher than the real value). The tests were performed organ by organ, comparing treatments along with harvest dates. Each line thus represents a single test and means with the same letter are not significantly different. The numbers are mean of three replicates
Table 4 Tree contents of soluble sugars (g tree-1), starch (g tree-1) and TNC or total non structural carbon (g tree-1) May 29 1.5N trees Soluble sugars Starch TNC
43.8 (a) ± 11.9 11.5 (ab) ± 1.6 55.3 (a) ± 10.3
June 13 3N trees
1.5N trees
3N trees
54.3 (a) ± 3.9
49.0 (a) ± 19.2
107.9 (b) ± 8.7
11.2 (ab) ± 1.2
17.6 (b) ± 5.0
65.5 (a) ± 3.7
66.6 (a) ± 14.2
7.5 (a) ± 0.5 115.4 (b) ± 8.4
The results are mean ± standard errors of three replicates. Letters differing between treatments indicate that mean values were significantly different at 5% level. Mean were compared using a randomization test. The test involved the generation of 2,500 random orders and the results were assumed to be significant at 5% level (i.e. if fewer than 125 estimates were higher than the real value). Each C compound was computed separately to compare the treatments along with harvest dates. Each line represents a single test and means with the same letter are not significantly different
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Trees (2012) 26:393–404
401
Fig. 6 Tree response to autumn N supply with respect to (1) N uptake in fall and spring, (2) N store mobilisation in spring, (3) first growth flush in spring and (iv) tree TNC content. Items surrounded with solid lines refer to measurements. Dotted lines represent the
hypothesis likely to explain modifications to tree TNC contents %,! and & mean, respectively, that N supply has a positive, a negative and no effect
increase significantly if shoot elongation continued late in autumn (Jordan et al. 2011). N treatments affected N uptake and N accumulation in autumn and consequently N cycling in spring (Fig. 2). Thus tree 14N contents varied with treatments by 25%, indicating that N uptake in late autumn could be important, not only in poor forest soils (Amponsah et al. 2004) but also in well-fed orchards (Niederholzer et al. 2001; Cheng et al. 2002; Dong et al. 2005). Nonetheless, uptake saturation occurred rapidly (under 3N treatment), considering that the 6N treatment corresponded to the maximum uptake rate observed in summer on trees of the same origin and age but grown in hydroponic conditions (Me´die`ne et al. 2002). Uptake saturation had previously been described in trees having a high N status, i.e. a leaf N concentration above 2.5% DW (Cheng et al. 2002). This was probably the case in our study, because we had previously found values
of between 2.6 and 3.3% DW in trees grown in similar conditions (Gomez and Jordan, unpublished). Uptake saturation may also be induced by the suppression of an important trophic sink: that relative to shoot elongation, which may represent up to 44% of the increase in autumnal biomass (Jordan et al. 2011). However, these high values were obtained under exceptionally mild temperatures in young tree regrowing after severe late summer pruning. Our results were also able to explain part of the variability in tree 14N content by relating it to the number of phytomers or crown size at experiment start (Fig. 2). The treatments had no effect on the regression slope, i.e. on the ratio between the tree number of phytomers and 14N content, but modified the intercept (553 vs. 1,172) which increased with N supply up to a threshold level corresponding to N uptake saturation. The intercept was not significant for the 1.5N trees indicating that in autumn, N
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402
was distributed evenly between root and axes, i.e. that the amount of N allocated to the roots was a stable proportion to that allocated to the axes and therefore also related to the number of phytomers. For high N trees this relation was no longer respected and N storage in the roots increased independently of tree size. This modification of the allocation scheme possibly indicated a saturation of the axes reservoirs, limited to bark parenchyma and phloem (Gomez and Faurobert 2002). Tree N content in autumn had previously been related to tree size approximated by leaf DW, but without considering the effects of soil N availability (Weinbaum et al. 1978). The following spring, the 14N depletion of perennial structures (Table 2), i.e. of the roots and old axes, was similar in all trees, indicating that N stores were entirely mobilized for shoot and fruit growth. Indeed, the amount of 14 N remaining in the perennial structure at the end of the first growth flush was independent of the autumn N supply but was still related to the tree number of phytomers (Fig. 2). The depletion of old axes was important, since only 27% of the 14N remaining in perennial structures was incorporated into old axes while 73% in the roots. The utilisation of all stored N in order to sustain spring growth, evidenced here in trees receiving a broad range of autumn N supplies, was specific to N, since TNC may be sequestrated for several years (Millard and Grelet 2010). Indeed, starch accumulates more as trees are stressed, even if this penalises growth (Silpi et al. 2007). N store mobilisation in spring impacted plant functioning at three levels, at least (1) new organ emission and growth, (2) N uptake, and (3) tree TNC contents and concentrations (Fig. 6). Shoot growth in spring is known to increase with fall N supply (Millard 1996; Niederholzer et al. 2001; Dyckmans and Flessa 2001). N stores were used to sustain the first growth flush, since 14N mobilisation was achieved on May 29. At that date, spring 15N uptake was similar in all trees, whatever the treatment (Fig. 1) and differences in shoot and fruit production could thus be attributed solely to 14N cycling. Shoot and fruit growth had previously been linked to individual bud development monitored on a weekly basis (Jordan et al. 2009), each bud being located precisely within the structure. Shoot growth adjusted to N availability, thus modulating the number and position of new elongating axes. As a first stage, axis emergence was promoted by autumn N supply at crown top but inhibited in crown centre. As a result, the final number of axes per tree increased with autumn N supply, as did the axis elongation rates, but axis diameter decreased with fall N supply. Indeed, an alteration of axis distribution within the crown affected individual axis growth, since position remained the main determinant of phytomer emergence, i.e. of axis elongation. As a second stage, sylleptic ramification (i.e.
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the development of axillary buds on current year axes), which started about 2 months after bud burst, was promoted in N limited trees. Fruit production was in turn adjusted to vegetative growth, maintaining the leaf to fruit ratio independent of treatment. The relationship between N availability and number of growing axes had previously been evidenced (Lobit et al. 2001; Cooke et al. 2005), but we positioned these effects within the structure and related them to fruit production. The root to shoot ratio decreased with the autumn N supply as a consequence of the effect of treatment on shoot growth. This had previously been observed (Millard and Neilsen 1989; Quartieri et al. 2002) and was in accordance with the theory of functional equilibrium. Spring 15N uptake represented about 35% of the tree N content on May 29, which was consistent with the data obtained by Quartieri et al. (2002) and Policarpo et al. (2002). The 15N uptake rates (Fig. 1) increased significantly in high N trees between May 29 and June 13, but not in 1.5N trees, indicating that a deficit of N storage could not be compensated for by an increase in N uptake the following spring. By contrast, the lower 15N uptake rates in 1.5N trees enhanced the effect of the autumn N treatment on plant growth. Indeed, spring N uptake was adjusted to shoot and fruit growth, i.e. to shoot and fruit DW. This relationship was not altered by the difference between treatments observed on June 13 in fruit 15N concentrations, which corresponded to 200 mg 15N, i.e. to a deficit of 15% in the 15N uptake of 1.5N trees. This observation was in contradiction with the findings of Deng et al. (1989), Millard and Proe (1993) and Niederholzer et al. (2001), who found that the restoration of spring N uptake started earlier when the amounts of cycling N were small. It should also be noted that until June 13, spring N uptake was defined by tree N status at burst and growth rather than by soil N availability, which differed from what has been observed in summer (Lobit et al. 2001; Me´die`ne et al. 2002). This probably indicated that N uptake rates reach their maximum only later on in the season (Policarpo et al. 2002). However, the lower spring N uptake of 1.5N trees could not be linked to a shortage of TNC (Fig. 5; Table 4), since these trees had higher starch concentrations than high N trees in all organs except for the fruits. Therefore, the lower concentrations of soluble sugars in these 1.5N trees could not be linked to C shortage, but probably suggested a slowdown in C acquisition and transport due to low sink strength, i.e. to less growth and limited root activity. These trees may also have stored higher amounts of TNC prior to leaf fall in autumn. Thus reducing N storage could increase TNC accumulation since N acquisition and storage is C costly (Bi et al. 2004; Cheng et al. 2002). This seems especially true that leaf fall dynamics in autumn were not
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affected by N treatments (Jordan et al. 2009), as has sometimes been observed after a reduction in N supply (Kubiske et al. 1998; Veberic et al. 2002). In conclusion, early spring growth was defined by the amounts of cycling N, and spring N uptake was in turn proportioned to shoot and fruit growth, at least during the first growth flush (Fig. 6). An N storage deficit could not be compensated for by increasing N uptake during the first growth flush, the main consequence of this being to link fruit production, in terms of fruit number and DW, to overwintering N storage (Jordan et al. 2009). This absence of ‘‘compensatory early spring N uptake’’ was not linked to a C deficit induced by less shoot growth, i.e. by a lower photosynthetic capacity of N limited trees. The amounts of stored N modified not only tree size but also tree shape, modulating the number and position of growing axes, which had consequences in terms of winter pruning costs. For instance, most of the sylleptic axes had to be removed in winter. Nonetheless, an autumn N supply was efficient in improving N storage, but only if it was moderate, insofar as tree N uptake rate decreased after shoot elongation had ceased, but remained proportional to tree size or the number of phytomers. Acknowledgments We would like to thank Josiane Hostalery and Vale´rie Serra for their valuable participation in tree surveys and plant harvests, and Emilie Rubio who carried out biochemical analyses.
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