New Forests 14: 19–31, 1997.c 1997 Kluwer Academic Publishers. Printed in the Netherlands.

Physiological and morphological responses to nitrogenlimitation in jack pine seedlings: potential implicationsfor drought tolerance

WEIXING TAN� and GARY D. HOGANNatural Resources Canada, Canadian Forest Service, Ontario Region, P.O. Box 490, 1219Queen St. E., Sault Ste. Marie, Ontario, Canada, P6A 5M7 (�Author for correspondence)

Received 10 January 1995; accepted 1 June 1996

Key words: apoplasmic fraction, dry weight fraction, dry weight partitioning, seedling quality,tap root, turgor maintenance

Application. Nitrogen concentrations less than 24 mg g�1 dry weight in needles of jackpine seedlings induced physiological and morphological changes advantageous to enhancedtolerance to drought. This suggests that: 1) there are opportunities for modifying nurseryfertilization regimes to produce stock for use on particularly droughty sites; and 2) considerationof the severity of drought at a particular site should be made prior to fertilization.

Abstract. The morphological and physiological responses to nitrogen (N) limitation in jackpine (Pinus banksiana Lamb.) seedlings were studied following the initiation of four differentdynamic N treatments for six and 15 weeks. The N treatments produced needle N concentrationsfrom 11 to 31 mg g�1 dry weight, and seven-fold difference in dry weight at 15 weeks. Low-Njack pine seedlings: 1) had an higher root/shoot ratio; 2) extended their tap root more rapidly;3) were better able to maintain turgor when shoot water potential declined; and 4) had a largerdry weight fraction and apoplasmic fraction than seedlings with higher foliar N concentrations.These responses may contribute collectively to enhance drought tolerance in N-limited plants,thereby affecting seedling quality. Modifying nursery fertilization regimes, other than optimalas usually applied, may thus be needed to produce stock for use on particularly droughty sites.Knowledge of the nature of drought at a particular site could be an important considerationwhen making decisions related to fertilization.

Introduction

Nutritional status is one of the most significant “material attributes” ofseedling quality (Ritchie 1984). Early relative growth rate of many tree specieshas been shown to be linearly correlated with plant nitrogen (N) concentra-tion under controlled conditions (e.g., Ingestad and Kahr 1985). Increasingnutrient reserves in nursery stock through nutrient loading, or through late-season nutrient additions, often resulted in improved outplanting performance(Benzian et al. 1974; Fisher and Mexal 1984).

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A tree’s ability to respond to fertilization, however, is often constrainedby other environmental conditions, particularly soil moisture availability(Fisher and Mexal 1984). For example, nitrogen fertilization has beenshown to decrease yield and/or growth if moisture was severely limited(Schomacher 1969; Boyer 1983). Linder et al. (1987) found that fertilizationwith N in the field increased the mortality of outplanted Pinus radiata D.Don during droughty years, and Smith et al. (1966) reported similar resultsfor Pseudotsuga menziesii (Mirb.) Franco seedlings. In two separate studies,van den Driessche (1980; 1988) reported that Pseudotsuga menziesii (Mirb.)Franco seedlings with a foliar N concentration above 2% showed poorsurvival and growth after outplanting.

Studies with both loblolly pine (Pinus taeda L.) (Pharis and Kramer 1964)and lodgepole pine (Pinus contorta Douglas) (Etter 1969) under controlledenvironments showed that seedlings receiving the lowest N treatment wereleast affected by drought stress. Photosynthesis of high-N treated hybridpoplar (Populus deltoides� nigra) declined dramatically during water stressand was very vulnerable to the subsequent drought cycles when comparedwith the low-N plants (Liu and Dickmann 1993).

In spite of its practical and theoretical importance, the role of N as amodifier of morphological and physiological responses, particularly inrelation to drought tolerance, remains unclear. In the following study, weexamined turgor and apoplasmic adjustments, dry weight partitioning, androot development in response to N limitation in jack pine (Pinus banksianaLamb.) seedlings. These responses have been shown to be involved in planttolerance to drought (ability to survive) by modifying either dehydration post-ponement or dehydration tolerance (Kramer 1980) and may provide a partialexplanation as to why N-limited seedlings tend to be more drought-tolerant.This information is also crucial in guiding foresters in the management oftree nutrition in the nursery and field.

Materials and methods

Seedling culture

Two newly-germinated jack pine seedlings were planted in each Leach tube(0.16 L; Stuewe & Sons Inc., Corvallis, OR, USA) containing sand, asdescribed by Tan and Hogan (1995). Seedlings were grown in a controlledenvironment room (Conviron, Winnipeg, Manitoba, Canada) under thefollowing day/night conditions: temperature, 24/18 � 1 �C; RH 60/80 �10%; photoperiod, 16/8 h; 450 �mol m�2 s�1 PPFD at the top of Leachtubes. Plants were watered with a semi-automatic, drip irrigation system four

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times each day during the photoperiod (Tan and Hogan 1995). The amount ofsolution (100 mL for the first irrigation, and 50 mL for each of the remainingapplications) delivered to the top of each individual Leach tube was abovethe capacity of the sand. Highly purified water, containing less than 0.03 mgL�1 major nutrients, from an Aqua-Cheer Series S reverse-osmosis system(Culligan of Canada Ltd., Mississauga, Ontario, Canada), was used through-out the experiment. Using Ingestad and Kahr’s (1985) technique, we appliedonly water to the seedlings for the first two weeks following germination.They were then thinned to one seedling per Leach tube with an average totaldry weight of 10.4 mg per seedling and N concentration of 12.9 mg g�1 dryweight.

N treatment

Four different dynamic rates of N supply were initiated after two weeksgrowth without nutrients (week 0). Nitrogen (NH4NO3) concentration (mgL�1) in solution varied as follows; treatment I, increased from 3.0 to 3.4 overthe course of the 15 week experiment; treatment II from 9.2 to 12.6; treatmentIII from 27.8 to 51.2; and treatment IV from 46.4 to 100. Concentration of Nin each treatment was increased every two days according to a predeterminedschedule, to satisfy the increased demand for N as the seedlings grew, andto produce a range of growth rates under conditions of restricted N supply(Ingestad and Kahr 1985; Tan and Hogan 1995). A range of internal Nconcentrations in jack pine seedlings was achieved, from supra-optimal todeficient, over the 15 week growth period (see Table 1).

The nutrient solution used in this study was based on a fixed proportion(mass basis) of nutrients, relative to 100 mg L�1 N (Tan and Hogan 1995),and followed the suggestion of Ingestad and Kahr (1985) for pine species.This solution allowed for the manipulation of nitrogen concentrations whilekeeping an optimal availability of other nutrients, thereby avoiding potentialconfounding effects from limitations of nutrients other than N (Radin andMatthews 1989).

Seedling morphology and nutrient status

Seedlings were harvested six and 15 weeks after the initiation of N fertiliza-tion, oven-dried at 80 �C for 48 h, and separated into roots and shoots (needlesand stems) for dry weight (g) determination. At week six, the length (mm)of tap root was measured. Needle total N was determined by micro-Kjeldahldigestion and other major macro-nutrients by Inductively Coupled PlasmaEmission Spectrometry (Tan and Hogan 1995).

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Osmotic potential at turgor loss point, apoplasmic fraction and dry weightfraction

Seedling water relations were analyzed using pressure-volume (P-V)analysis (Tyree and Jarvis 1982). Either entire shoots or side shoots from4 different seedlings in each treatment and age were used for P-V analysis,following free transpiration procedures as described by Tan and Hogan(1995). Since this experiment concentrated on examining practical impli-cations for seedling quality, we chose to report just a few parameters closelyrelated to drought tolerance: 1) osmotic potential at turgor loss point (MPa),an indication of turgor adjustment; and 2) dry weight fraction (ratio of dryweight to saturated fresh weight in shoot, g g�1) and apoplasmic fraction (%)to estimate a potential change in cell wall thickness and/or cell size (Tyreeand Jarvis 1982; Correia et al. 1989). The relationship between plant N statusand water relations has been discussed in depth in another experiment (Tanand Hogan 1995).

Data analysis

All the analyses were completed using General Linear Model or Regressionprocedures in a SAS-PC software (SAS 1988). Treatment differences in totaldry weight, needle N concentration, and tap root length were determined byan analysis of variance and the least significant difference between means bypairwise t-tests (P < 0.05). Means of osmotic potential at turgor loss point,apoplasmic fraction, and dry weight fraction from each treatment and weekwere correlated with needle N concentration. Regression models were chosenbased on simplicity, fit, and distribution of errors.

Comparisons of N treatments in dry weight partitioning consider the allo-metric correlation between plant organ and total dry weight (Bongarten andTeskey 1987; Tan et al. 1995). Briefly, ln-transformed organ dry weight orroot/shoot ratio were analyzed using ln-transformed total dry weight as acovariate (Tan et al. 1995). Significant N treatment effects indicate differ-ences in treatment means. Differences in regression slopes between N treat-ments are shown by a significant treatment � ln(total dry weight) interaction(Bongarten and Teskey 1987). To demonstrate the extent of the difference,the N treatments were also compared in adjusted (least squares) means at themean value of ln(total dry weight) (Tan et al. 1995). The preliminary resultsindicated that: 1) there is no difference between treatments III and IV; and 2)treatments I and II did not differ significantly (P = 0.05) on many occasionsbecause of variation among individuals. To improve the level of precision,we combined the observations in treatments III and IV as a high N group, andI and II as a low N group in analyzing dry weight partitioning.

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Results

Total dry weight and nutrient status

Total dry weight growth and needle N concentration in jack pine seedlingsvaried significantly and corresponded to the respective N treatments at bothweek six and 15 (Table 1). Seedlings in treatment I, which received the lowestlevel of N supply, consistently had the lowest total dry weight and needle Nconcentrations. Total dry weight and needle N concentration in treatment IIwere invariably intermediate between treatments III and I (Table 1). Althoughneedle N concentration in the seedlings of treatment IV was significantlyhigher than that in the treatment III, their total dry weight did not differ(Table 1). This suggested that luxury consumption of N was occurring atthe highest rate of N supply. The average total dry weight in treatment IVwas three and seven times larger than that found in treatment I at weeks sixand 15, respectively (Table 1). There was a significant decline in needle Nconcentration across all treatments from week six to 15. The concentrationsof total P, K, Ca, Mg, and S in needles at week 15 exceeded 2.7, 11.0, 1.5,1.7, and 1.8 mg g�1 dry weight, respectively, in all samples.

Table 1. Total dry weight (g) and needle N concentration (mg g�1

dry weight) six and 15 weeks after the initiation of four different low(I) to high (IV) nitrogen treatments in jack pine seedlings. Meansfollowed by the same letter within a row are not significantly differentas determined by pairwise t-tests (P < 0.05, n = 6). Part of this tablewas adapted from Tan and Hogan (1995) to demonstrate the growthand nutrient differences among treatments for readers’ convenience.

Treatment

Week I II III IV

Total dry weight

6 0.218 c 0.332 b 0.598 a 0.641 a15 1.635 c 4.658 b 13.82 a 11.81 a

Needle N concentration

6 14.80 d 19.87 c 26.12 b 31.08 a15 11.34 d 12.88 c 22.51 b 25.57 a

Dry weight partitioning and tap root length

Analysis of variance generally showed a significant N treatment effect onallometric growth of shoot, root, needle (except at week 15), and stem dry

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Table 2. Analysis of variance for the effects of N supply treatments on allometric growth ofshoots, roots, needles and stems dry weight (DW, g) and root/shoot ratio using ln(total dryweight [TDW]) as a covariate. The two low (I and II) or high (III and IV) N treatments weregrouped to improve the level of precision. F-ratio significant levels: �, P < 0.05; ��, P <0.01; ���, P < 0.001.

F-ratio

Week Source ln(shoot DW) ln(root DW) ln(needle DW) ln(stem DW) ln(root/shoot)

Treatment 10.05�� 6.87� 6.56� 1.61 7.68��

6 ln(TDW) 305.87��� 77.96��� 186.47��� 83.07��� 4.30�

Treatment � 0.72 0.21 0 2.78 0.32ln(TDW)

Treatment 8.54�� 7.87�� 0.80 10.31�� 8.25��

15 ln(TDW) 1313.14��� 68.46��� 641.15��� 115.52��� 2.93Treatment � 9.28�� 5.87� 1.42 7.97�� 6.75�

ln(TDW)

weight (except at week six) and root/shoot ratio when the large effect of totaldry weight was constrained by using the ln(total dry weight) as the covariate(Table 2). Interaction of treatment and ln(total dry weight) was not significantfor any of the parameters at week six, but was significant at week 15 (Table2).

At week six, seedlings in the low N treatment group clearly partitioned asignificantly smaller amount of dry weight towards shoots and needles andmore towards roots compared with the high N group, as shown from theadjusted means at the same mean ln(total dry weight) (Table 3). As a result,root/shoot ratio was twice as large in the low N treatments (0.525) as in thehigh (0.261) (Table 3). Interpretation of the results at week 15 in Table 3 needsto be exercised with caution, as the slopes differed significantly between theN treatments (Table 2). However, when compared at the mean ln(total dryweight), low N-treated seedlings still allocated more dry weight to their rootsand less to shoots (P< 0.1), and thus had a significantly larger root/shoot ratiocompared to the high N-treated seedlings, results similar to those at week six(Table 3).

As N supply and needle N concentration declined, tap root length graduallyincreased (Figure 1 and Table 1). Tap root length in treatment I was almost25 mm longer than that in the treatment IV (P < 0.05).

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Table 3. Comparison of N supply treatments in adjusted means of ln(shoot dry weight[DW, g]), ln(root DW), ln(needle DW), ln(stem DW), and ln(root/shoot) using ln(total dryweight) as a covariate. The two low (I and II) or high (III and IV) N supply treatmentswere grouped to improve the level of precision. Back-transformed values are shown in thebrackets below each mean to demonstrate actual differences. Pair means for each weekfollowed by the same letter do not differ significantly (P< 0.05) as determined by a t-test.

Week 6 Week 15

Low High Low High

ln(shoot DW) –1.3381 b –1.1493 a 1.5239 a 1.5961 a(0.262) (0.317) (4.590) (4.934)

ln(root DW) –1.9830 a –2.4927 b 0.1603 a –0.2611 b(0.138) (0.083) (1.174) (0.770)

ln(needle DW) –1.5228 b –1.2731 a 1.3519 a 1.3452 a(0.218) (0.280) (3.865) (3.839)

ln(stem DW) –3.1195 a –3.2491 a –0.3767 b 0.0916 a(0.044) (0.039) (0.681) (1.096)

ln(root/shoot) –0.6449 a –1.3434 b –1.3636 a –1.8573 b(0.525) (0.261) (0.256) (0.156)

Figure 1. Tap root length (mm) of jack pine seedlings six weeks after the initiation of fourdifferent low (I) to high (IV) nitrogen treatments. Means with the same letter do not differsignificantly (P < 0.05). Vertical bars are SEM (n = 6).

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Figure 2. Relationship between needle N concentration (mg g�1 dry weight) and osmoticpotential at turgor loss point (MPa) in jack pine seedlings six (�) and 15 (#) weeks afterthe initiation of four different low (I) to high (IV) nitrogen treatments. Vertical bars are SEM(n = 4).

Osmotic potential at turgor loss point, apoplasmic fraction and dry weightfraction

Osmotic potential at turgor loss point was unaffected when needle N con-centration declined from 31 to about 24 mg g�1 dry weight but droppedsignificantly with the further reductions in needle N concentration (Figure2). At the lowest needle N concentration the osmotic potential at turgor losspoint (close to –1.9 MPa) was almost 0.5 MPa lower than that at the highestN concentration (–1.4 MPa; Figure 2).

Changes of apoplasmic fraction and dry weight fraction in response toreduced N supply were similar (Figure 3). There was little change in eithervariable when needle N concentration declined from 31 to about 24 mgg�1 dry weight, but both increased significantly with the further reduction inneedle N levels (Figure 3). Apoplasmic fraction (30%) and dry weight fraction(0.28) found at the lowest needle N concentration were nearly double thosefound at the highest N concentration (15% and 0.17 respectively; Figure 3).

Discussion

The range of needle N concentrations (11 to 31 mg g�1; Table 1) achievedin this study covered the range of foliar N concentrations (low, critical and

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Figure 3. Relationship between needle N concentration (mg g�1 dry weight) and dry weightfraction (g g�1) and apoplasmic fraction (%) in jack pine seedlings six (�) and 15 (#) weeksafter the initiation of four different low (I) to high (IV) nitrogen treatments. Vertical bars areSEM (n = 4).

adequate) previously found in jack pine seedlings (Swan 1970). Internal Nstatus of treatment IV seedlings had reached the supra-optimal level, sincedry weight growth did not differ between treatments III and IV (Table 1).The growth and/or foliar N concentration differences observed among the 4treatments persisted over the 15 week experiment (Table 1). There was noindication of nutrient deficiency, other than that attributed to N, and levelsof the other macro-nutrients in all samples exceeded the levels required fornormal growth of jack pine according to Swan (1970).

Jack pine seedlings with lower N levels grew far slower than N-sufficientones (Table 1) – a phenomenon well-known since the time of Liebig (cf.,Epstein 1971). Slow growth rates are a common, central feature of plantsadapted to a variety of other low-resource environments, including thosecharacterized by drought (Chapin 1991a). A recent analysis of the ecolog-ical consequences of variation in plant growth rate has suggested a closelinkage between a slow growth rate and drought (stress) tolerance (Lambersand Poorter 1992). Thus, the slow growth rate in the seedlings with low Nconcentrations (Table 1) could probably confer advantages to their tolerance(survival) to drought, a suggestion supported by the physiological and mor-phological results found in this experiment and by a number of previousstudies with other species (e.g., Pharis and Kramer 1964; Etter 1969; Liu andDickmann 1993). However, it should be emphasized that the mechanismswhich promote survival may not necessarily be advantageous to growth oryield under drought (Kramer 1980; Tan et al. 1992).

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Using the allometric principle (Bongarten and Teskey 1987; Tan et al.1995), this study was able to demonstrate a clear shift of dry weight parti-tioning from shoots to roots in response to insufficient N, thereby resultingin a significant increase in root/shoot ratio when potential ontogenic effectswere taken into consideration (Table 2 and 3). This result confirms previ-ous findings for a number of tree species (e.g., Linder et al. 1987; van denDriessche 1988; Dewald et al. 1992). The shift in allocation towards the rootsystem is a common response to drought as well (Bongarten and Teskey 1987;Tan et al. 1995), that serves to increase a plant’s accessibility to water andnutrients alike (Mooney and Winner 1991; Tan et al. 1995). This commonresponse, therefore, promotes tolerance to both drought (through dehydrationpostponement) and nutrient limitations, a situation that often occurs at thesame site. In addition, N-limited jack pine seedlings were also able to extendtheir tap root faster than N-sufficient seedlings (Figure 1), a result rarelyreported previously in other trees. A deeper tap root would further enhancethe capacity of N-limited seedlings to access water and nutrients in the soil.

Low-N jack pine seedlings were better able to maintain turgor as waterpotential declined (Figure 2), an outcome which agrees with findings forRosa rugosa L. (Auge et al. 1990). The increased capacity to maintain tur-gor could be largely attributed to an increase in cell wall elasticity (Tan andHogan 1995). The magnitude of turgor maintenance in N-limited jack pineseedlings (up to 0.5 MPa) was comparable to the observations in N-stressed(Auge et al. 1990) and water stressed plants (Turner 1986; Tan et al. 1992).An increased ability to maintain turgor during water stress helps plants totolerate dehydration (Kramer 1980; Turner 1986), since turgor is crucial forthe functioning of most, if not all, biochemical, physiological and develop-mental processes (Jones et al. 1981; Tan et al 1992). This could help explainfindings that N-limited plants had better survival (Etter 1969), and superiorrelative growth (Pharis and Kramer 1964), than N-sufficient seedlings whensubjected to drought stress.

An increase of dry weight fraction by more than 60% in N-limited jack pineseedlings agrees well with results found for other plant species (Radin andParker 1979; Correia et al. 1989). This increase was associated with a similarrise in the apoplasmic fraction (Figure 3). Although not directly measured inthis study, these results suggested that N-limitation may cause an increase incell wall thickness and/or a decline in cell size (Tyree and Jarvis 1982; Correiaet al. 1989), as was found for low-N cotton plants (Gossypium hirsutum L.)(Radin and Parker 1979). Small cell size is a typical characteristic of drought-resistant plants and a common response to drought in many plant species,and could serve to promote plant’s tolerance to dehydration (e.g. Levitt 1980;Turner 1986).

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Many of the physiological and morphological characteristics measured inthis study in jack pine seedlings were unaffected until needle N concentrationdeclined below 24 mg g�1 dry weight (Figures 2 and 3). Dry weight growthand partitioning did not differ between treatment III and IV in spite of theirsignificant difference in needle N concentration (Table 1, 2 and 3). A needle Nconcentration of 24 mg g�1 dry weight coincided closely with the suggestedadequate N level in jack pine seedlings (Swan 1970) and is close to the optimalvalue (20 mg g�1 dry weight) for field survival and growth in outplantedDouglas-fir (van den Driessche 1980 and 1988). The similarity of these valuesraises the possibility that there may be a foliar concentration of N which allowsfor an optimal balance between growth and drought (or perhaps other stressesas well) tolerance, thereby maximizing their capacity to survive and growin droughty environments. The implications and limitations of this strategyneed to be investigated further.

Conclusion

The results of this study support a previous suggestion by Chapin (1991b)that nutrient stress may enhance the tolerance of plants to drought andpossibly some other stresses as well. Our results demonstrated that a varietyof morphological and physiological characteristics, including high root/shootratio (Table 3), rapid tap root expansion (Figure 1), turgor maintenance(Figure 2), and large dry weight fraction and apoplasmic fraction (Figure 3),may collectively contribute to such an increase. Other studies have suggestedthat nutrient absorption capacity (Chapin 1991a), root hydraulic conductivity(Radin and Matthews 1989), growth regulators (Chapin 1991a), and photo-synthesis (Chapin 1991a; Liu and Dickmann 1993) may also play a role.These findings may help to explain why under drought conditions, fertiliza-tion often depressed plant growth (Pharis and Kramer 1964; Fisher and Mexal1984) and caused high mortality (Smith et al. 1966; Etter 1969; Linder et al.1987).

Since drought tolerance is an integral part of seedling quality, this informa-tion highlights the importance of proper nutrition management during stockproduction, in the nursery, and fertilization practice, in the field, especiallyat sites prone to drought. To improve seedling growth rate and shorten theproduction cycle, a high and optimal nutrient regime is usually applied duringstock production. This is probably acceptable only if the stock will be out-planted on the sites where drought is not severe. For outplanting in droughtyenvironments, however, a modified nutrient regime may be necessary to pro-duce stock capable of reasonable initial growth rates, while maintaining anenhanced level of drought tolerance. Some consideration should be given

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to matching the level of fertilization with the nature of drought on the site.Over-fertilization should be avoided on the sites where rather severe droughtmay prevail.

Acknowledgments

Technical assistance from B. Borland, M. Laporte, J. Ramakers, and S. Tayloris gratefully acknowledged. We also would like to thank Dr. K. Brown forhis helpful discussion in nutrient aspects of the present experiment and Dr. R.Sutton and two anonymous reviewers for their useful comments on previousversions of this paper.

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