Environmental and Experimental Botany 60 (2007) 318–323
The effects of rate and timing of N fertilizer on growth, photosynthesis, N accumulation and yield of mustard (Brassica juncea) subjected to defoliation P.M. Lone, N.A. Khan ∗ Department of Botany, Aligarh Muslim University, Aligarh 202002, India Received 1 January 2006; received in revised form 21 July 2006; accepted 28 December 2006
Abstract Mustard (Brassica juncea L.) is characterized by large number of broad oblong shaped leaves in the lower layers. Our earlier studies have shown that removal of these shaded lower leaves on mustard plant axis enhanced growth, photosynthetic capacity and yield of the crop. We now present evidence that soil-applied nitrogen (N) at pre- or post-flowering stage following defoliation of lower leaves influences plant growth, photosynthesis and assimilation balance. Following defoliation at pre-flowering, i.e. 40 d after sowing (DAS) and N applied at the rate of 100 kg ha−1 at the time of sowing and 50 kg ha−1 at post-flowering (60 DAS) enhanced the characteristics maximally. The defoliation treatment together with N combinations and the time of its application, N at 150 kg ha−1 applied as single dose at the time of sowing or N applied in split; 100 kg ha−1 at the time of sowing and 50 kg ha−1 at 40 DAS or 75 kg ha−1 at the time of sowing or 75 kg ha−1 at pre- or post-flowering time proved less effective. The plants which were not defoliated and received 75 kg N ha−1 at the time of sowing and 75 kg ha−1 at 60 DAS showed lowest values. Furthermore, N assimilation was more efficient in plants following defoliation at 40 DAS. The results suggest that split N application (100 kg ha−1 at sowing and 50 kg ha−1 at post-flowering) enhances substantially growth, photosynthesis, N assimilation and yield of mustard following defoliation. This management practice could be adopted in mustard culture for increasing seed yield together with minimizing N loss. © 2007 Elsevier B.V. All rights reserved. Keywords: Brassica juncea; Defoliation; Nitrogen
1. Introduction The productivity of crops depends on soil inputs and leaf area index and efficiency of leaves for conversion of light and carbon dioxide absorbed into biomass. Photosynthetic photon flux density decreases from top to lower leaves in an axis. Therefore, the photosynthetic potential of lower leaves is less compared to upper leaves (Nobel et al., 1993). Moreover, adequate supply of nitrogen (N) is required for compensating N loss by crop removal or leaching (Tabachow et al., 2001). This is normally attained by a judicious supply of input and its timing (Rice et al., 1995). Earlier research has shown that removal of 50% of leaves on lower regions at pre-flowering stage, i.e. 40 d after sowing (DAS) improved photosynthetic efficiency of the remaining leaves and increase in dry mass and yield of mustard (Khan
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and Ahsan, 2000; Khan, 2002, 2003; Khan and Lone, 2005). However, defoliation at post-flowering stage (60 DAS) was less effective (Khan and Lone, 2005). Assuming that N is used more efficiently by the crop after defoliation, it was decided to test the effect of the rate and timing of N application to the crop following defoliation on growth, photosynthesis, N assimilation and yield of mustard. 2. Material and methods 2.1. Plant material and treatments Seeds of mustard (Brassica juncea L. Czern and Coss. cv. Alankar) were sown in 10 m2 (5 m × 2 m) experimental plots of Aligarh Muslim University, Aligarh, India (27◦ 52 N, 78◦ 51 E and 187.4 m asl) in the winter season of 2002–2003. Plant population of 12 m−2 was maintained by keeping distance of 30 cm between rows and 15 cm between plants. The soil was sandy loam (Alfisols with Ustochrept type). Available soil N measured
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at the depth of 30 cm was 100 kg N ha−1 . The plots were treated with 150 kg N ha−1 as a single dose or in its split application. The treatment of 150 kg N ha−1 was based on earlier experience (Lone, 2004) in which plants grown with 250 kg N ha−1 (100 kg N ha−1 as available soil N and 150 kg N ha−1 as additional N treatment) increased growth and yield maximally. Split application of 150 kg N ha−1 was given as 100 kg N ha−1 at the time of sowing (BN100) and 50 kg N ha−1 as top dressing at 40 DAS or 60 DAS, and 75 kg N ha−1 at the time of sowing and 75 kg N ha−1 as top dressing at 40 DAS or 60 DAS. A sufficient amount of P and K fertilizers were applied as single super phosphate and muriate of potash, respectively as these nutrients were non-limiting. Defoliation of 50% leaf number on lower layers on plant axis was done at 40 DAS (pre-flowering). The details of the defoliation procedure have been described elsewhere (Khan et al., 2002a; Khan, 2005). This stage of defoliation has been found more effective than the other stages (Khan and Lone, 2005). Intact plants also received these N treatments and served as the control. The treatments were arranged in a randomized block design with five replications. Data on growth, photosynthesis, N assimilation were recorded at 80 DAS (pod-fill), and yield characteristics at 120 DAS (maturity). The environmental conditions during the crop growth monitored at 40, 60 and 80 DAS were: PAR, 1035 ± 10, 1021 ± 7 and 1016 ± 6 mol m−2 s−1 ; humidity, 61 ± 3, 59 ± 2 and 60 ± 3%; temperature, 23 ± 2, 22 ± 1 and 20 ± 2 ◦ C, respectively. 2.2. Determination of growth characteristics Number of functional leaves on plant axis was counted and recorded as leaf number per plant, and area of these leaves was determined with a leaf area meter (LA21, Systronics, Ahmedabad, India). The dry mass of the leaves and plants was determined after drying them in an oven at 80 ◦ C till constant weight. 2.3. Photosynthetic characteristics Carbonic anhydrase (CA) activity was determined in leaves used for photosynthesis measurement. Leaves were homogenized in 5 mM Tris–HCl (pH 8.5), containing 1 mM MgCl2 , 1 mM EDTA, and 1% polyvinylpyrrolidone. Homogenate was passed through Whatman 42 filter paper and centrifuged first at 1000 × g for 10 min and then at 5000 × g for 30 min. The CA activity was determined by an electrometric method (Rickli et al., 1964) in the supernatant. Net photosynthetic rate (PN ) and stomatal conductance (gS ) in fully expanded uppermost leaves (four plants in each treatment) were measured at light saturating intensity between 11:00 and 12:00 h using a portable photosynthesis system (LiCOR-6200, NE, USA). The atmospheric conditions during measurement were PAR, 1016 ± 6 mol m−2 s−1 ; relative humidity 60 ± 3%, atmospheric temperature 20 ± 2 ◦ C and atmospheric CO2 360 ± 4 mol mol−1 . The ratio of atmospheric CO2 to intercellular CO2 concentration was constant. The data on PN and gS were used for calculat-
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ing water use efficiency (WUE) as described by Dudley (1996). 2.4. N assimilation For N assimilation, the activities of nitrate reductase (NR), nitrite reductase (Ni R) and glutamine synthetase (GS) were assayed. Leaves used for photosynthetic measurements were selected for the assay of enzyme activities. Plant N content was determined as a product of plant dry mass and its N concentration, determined in acid-peroxide digested leaf sample according to Lindner (1944). Activity of NR in leaves was estimated by the method of Jaworski (1971). Fresh 200 mg leaf tissue was incubated for 2 h at 30 ◦ C in reaction mixture containing 2.5 ml phosphate buffer (pH 7.5), 0.5 ml of 0.2 M potassium nitrate solution and 2.5 ml of 5% isopropanol. To 0.4 ml reaction mixture, 0.3 ml each of 1% sulphanilamide and 0.02% NED-HCl were added and absorbance was read on spectrophotometer at 540 nm (SL-164 UV–vis, Elico, Hyderabad, India). The activity of Ni R was determined by the Methyl Viologen method (Lillo, 1984). Enzyme extract was prepared by homogenizing 5 g leaf tissue in 50 ml Tris–HCl buffer (pH 7.5) in blender. The homogenate was passed through multilayered cheese cloth. To 0.3 ml enzyme extract, a reaction mixture of 6.25 ml of Tris–HCl buffer, 2 ml of sodium nitrite, 2 ml of methyl viologen solution and 14.75 ml of distilled water were added. Freshly prepared 0.2 ml of 0.29 M dithionite sodium bicarbonate solution was added to start the reaction and incubated at 30 ◦ C for 15 min. To stop the reaction, the mixture was vigorously shaken on vortex mixer till the blue colour disappeared. To 20 l aliquot, 1.0 ml each of sulphanilamide and NED-HCl was added, and the absorbance was noted spectrophotometrically at 540 nm (SL-164 UV–vis, Elico). The enzyme extract for GS assayed by the method of Farnden and Robertson (1980) was prepared by homogenizing 1.0 g leaf material in 5 ml of 50 mM inidazole–acetate buffer (pH 7.8) containing 0.5 mM EDTA, 1 mM dithiothreitol, 2 mM MnCl2 and 20% glycerol. The enzyme extract was centrifuged at 10,000 rpm at 4 ◦ C (CPR 24 Remi, New Delhi, India) for 3 min. For purification the enzyme was precipitated with (NH4 )2 SO4 at 60% saturation and the precipitate was resuspended in extraction buffer and then desalted over sephadex G25. To 0.2 ml enzyme extract, 2.0 ml of 0.2 M l-glutamine, 0.5 ml of 20 mM sodium arsenate and 0.3 ml of 2 mM MnCl2 were added followed by the addition of 0.5 ml of 1 mM ADP and 50 mM hydroxylamine and then incubated at 37 ◦ C for 30 min. The reaction was stopped by adding 1.0 ml ferric chloride to the reaction mixture, and the absorbance was read on spectrophotometer at 540 nm (SL-164 UV–vis, Elico). 2.5. Yield characteristics At maturity, plants in 1 m2 land area were harvested, sundried and pod number per plant was counted. Seeds were
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Table 1 Leaf number, leaf area, leaf dry mass and plant dry mass of mustard (Brassica juncea) at pod-fill, i.e. 80 d after sowing (DAS) following 50% defoliation of lower leaves at 40 DAS and treated with single or split N at 40 (pre-flowering) or 60 (post-flowering) DAS Treatment
Leaf number
Leaf area (cm2 plant−1 )
Leaf dry mass (g plant−1 )
Plant dry mass (g plant−1 )
No defoliation N levels (kg ha−1 ) BN150 BN100 + N50 (40 d) BN75 + N75 (40 d) BN100 + N50 (60 d) BN75 + N75 (60 d)
57fg 63cd 59ef 63cd 52h
1142f 1285d 1189e 1292d 1063g
9.1g 9.6e 9.4ef 10.0d 7.9h
28.7g 30.7ef 30.0f 32.3d 27.7h
Defoliation N levels (kg ha−1 ) BN150 BN100 + N50 (40 d) BN75 + N75 (40 d) BN100 + N50 (60 d) BN75 + N75 (60 d) P
62de 66b 64bc 70a 55g <0.001
1317c 1348b 1339bc 1425a 1181e <0.001
10.4c 11.1b 10.9b 12.8a 9.3fg <0.001
33.7c 35.7b 35.2b 38.7a 31.7de <0.001
The statistical evaluation using analysis of variance (ANOVA). Data followed by the same letter within a column are significantly not different at P < 0.05 as determined by LSD.
collected after thrashing pods to record 1000 seed weight and seed yield. 2.6. Data analysis Data presented are mean of five replicates per treatment. As four plants per treatment were analyzed for photosynthetic characteristics and N assimilation, the total number of samples for these purposes was twenty. Statistical analysis was carried out by analysis of variance (ANOVA) using SPSS (10.0 for Windows). The least significant difference (LSD) at p < 0.05 was calculated for the significant data to identify significant difference in the mean of the treatment. The treatment mean was separated using Duncan’s multiple range test. Different letters indicate significant difference at p < 0.05.
3. Results and discussion Application of 100 kg N ha−1 at the time of sowing and 50 kg N ha−1 at 60 DAS [BN100 + N50 (60 d)] to plants subjected to defoliation proved more effective in enhancing growth, photosynthesis, N assimilation and yield than other N doses applied to plants irrespective of defoliation at 40 (pre-flowering) or 60 (post-flowering) DAS (Tables 1–4). Other splits of N given at pre-flowering or post-flowering proved less effective.
3.1. Growth characteristics The growth of plants was maximally increased when defoliated and given 100 kg N ha−1 at sowing followed by 50 kg N ha−1 at post-flowering [BN100 + N50 (60 d)]. The treat-
Table 2 Carbonic anhydrase (CA) activity, net photosynthetic rate (PN ), stomatal conductance (gS ) and water use efficiency (WUE) of mustard (Brassica juncea) at pod-fill, i.e. 80 d after sowing (DAS) following 50% defoliation of lower leaves at 40 DAS and treated with single or split N at 40 (pre-flowering) or 60 (post-flowering) DAS Treatment
CA activity (m mol m−2 s−1 )
PN ( mol m−2 s−1 )
gS (m mol m−2 s−1 )
WUE ( mol mol−1 )
No defoliation N levels (kg ha−1 ) BN150 BN100 + N50 (40 d) BN75 + N75 (40 d) BN100 + N50 (60 d) BN75 + N75 (60 d)
18.6f 19.6de 19.4e 20.0c 18.2g
24.9d 25.6c 25.4c 26.1b 24.2e
454ef 458bc 456cd 460b 453f
55.0g 56.0ef 55.8f 56.9cd 55.1g
Defoliation N levels (kg ha−1 ) BN150 BN100 + N50 (40 d) BN75 + N75 (40 d) BN100 + N50 (60 d) BN75 + N75 (60 d) P
19.8cd 20.5b 20.4b 22.5a 19.5e <0.001
25.8c 26.5b 26.1b 29.2a 25.2cd <0.001
457c 459b 458bc 464a 455d <0.01
56.4de 57.8b 57.1bc 63.0a 56.2e <0.001
The statistical evaluation using analysis of variance (ANOVA). Data followed by the same letter within a column are significantly not different at P < 0.05 as determined by LSD.
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Table 3 Activities of nitrate reductase (NR), nitrite reductase (Ni R), glutamine synthetase (GS) and plant N content of mustard (Brassica juncea) at pod-fill, i.e. 80 d after sowing (DAS) following 50% defoliation of lower leaves at 40 DAS and treated with single or split N at 40 (pre-flowering) or 60 (post-flowering) DAS Treatment
NR activity ( mol NO2 g−1 (f.m.) h−1 )
Ni R activity ( mol NH4 g−1 (f.m.) h−1 )
GS activity ( mol ␥-glutamyl hydroxamate g−1 (f.m.) h−1 )
Plant N content (mg per plant)
No defoliation N levels (kg ha−1 ) BN150 BN100 + N50 (40 d) BN75 + N75 (40 d) BN100 + N50 (60 d) BN75 + N75 (60 d)
8.0f 8.5cd 8.4d 8.6bc 7.9f
21.7d 23.6c 21.9d 24.3c 19.3f
38.4g 40.0e 39.8ef 42.2d 36.2h
809.3f 919.7e 874.7e 974.3cd 773.9f
Defoliation N levels (kg ha−1 ) BN150 BN100 + N50 (40 d) BN75 + N75 (40 d) BN100 + N50 (60 d) BN75 + N75 (60 d) P
8.4d 8.7b 8.6bc 8.9a 8.2e <0.001
24.0c 25.6b 23.7c 27.6a 20.6e <0.001
42.5d 45.1b 43.8c 48.7a 39.1fg <0.001
1005.7c 1104.6b 1065.1b 1245.8a 924.8de <0.001
The statistical evaluation using analysis of variance (ANOVA). Data followed by the same letter within a column are significantly not different at P < 0.05 as determined by LSD.
ment increased leaf number by 22.8 and 11.1%, leaf area by 24.7 and 10.3%, leaf dry mass by 40.6 and 28.0% and plant dry mass by 34.8 and 19.8% compared to application of total N (BN150) plus no-defoliation and BN100 + N50 (60 d) plus no-defoliation, respectively (Table 1). The extent of leaf formation and expansion influence the light absorption by the individual leaves within a plant. The plants after defoliation require more assimilates for regrowth which is balanced by the increased leaf assimilatory capacity and efficient N use (Lone, 2004). This leads to the enhancement in the photoassimilate synthesis in leaf increasing leaf and plant dry Table 4 Pod number per plant, 1000 seed weight and seed yield of mustard (Brassica juncea) at harvest, i.e. 120 d after sowing (DAS) following 50% defoliation of lower leaves at 40 DAS and treated with single or split N at 40 (pre-flowering) or 60 (post-flowering) DAS Treatment
Pod number
No defoliation N levels (kg ha−1 ) BN150 BN100 + N50 (40 d) BN75 + N75 (40 d) BN100 + N50 (60 d) BN75 + N75 (60 d)
191de 196d 180gh 182fg 174h
Defoliation N levels (kg ha−1 ) BN150 BN100 + N50 (40 d) BN75 + N75 (40 d) BN100 + N50 (60 d) BN75 + N75 (60 d) P
198cd 212b 205bc 229a 189ef <0.001
1000 seed weight (g)
Seed yield (g m−2 )
4.2f 4.4e 4.1h 4.4e 4.1g
147.2fg 148.6ef 144.1g 156.0c 132.5h
4.7c 4.8b 4.9b 5.6a 4.5d <0.05
151.2de 169.3b 166.6b 180.5a 153.3cd <0.001
The statistical evaluation using analysis of variance (ANOVA). Data followed by the same letter within a column are significantly not different at P < 0.05 as determined by LSD.
mass. It has been reported that defoliation at 40 DAS markedly increased the growth of new leaves that were photosynthetically more active (Khan and Lone, 2005). New leaves emerging after defoliation have been found to have greater efficiency for CO2 assimilation (Alderfer and Eagles, 1976; Caemmerer and Farquhar, 1984; Khan et al., 2002a,b; Khan and Lone, 2005). Goulas et al. (2002) suggested that regrowth of new leaves after defoliation depends upon the supply of carbon and N reserves. Similarly, carbon compounds are also remobilized after defoliation to support leaf growth (Ourry et al., 1988; Thornton et al., 1993, 1994; Thornton and Millard, 1996). Nitrogen is required in sufficient amount to sustain growth after defoliation. Therefore, any change in source–sink relationships is considered as dependent on N reserve accumulation (Lone, 2004). Hilbert et al. (1981) and McPherson and Williams (1998) have reported that remobilization of stored carbohydrate influenced regrowth. The enhanced leaf area has also been reported to be the major mechanism leading to compensatory growth (McNaughton et al., 1983). The increase in leaf area is brought about by large N supply by causing the expansion of individual leaves and branching or tillering in grasses (Gastal and Lemaire, 2002; Trapani and Hall, 1996; Taylor et al., 1993; Vos and Biemond, 1992; Vos et al., 1996) presumably through its effect on cell division and cell expansion (Lemaire, 2001). Increase in biomass accumulation is attributed to the increased CO2 assimilation due to higher rates of photosynthesis by younger leaves (Khan and Lone, 2005). Thus, the increased photosynthetic CO2 assimilation in defoliated plants leads to enhanced leaf and plant dry mass. Gold and Caldwell (1990) and Anten and Ackerly (2001) suggested an increase in light interception by the crop canopy enhanced nutrient availability due to defoliation, which leads to increased photosynthetic rate and unit leaf rate. The increase in unit leaf rate contributed more to compensate for the losses in growth (Anten et al., 2003). Thus, the physiological changes that are related to enhancement of unit leaf rate, including increase in
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leaf photosynthesis, are important for enhancing plant growth and dry mass. 3.2. Photosynthetic characteristics Photosynthetic characteristics significantly increased after defoliation and split N application of 100 kg ha−1 at sowing and 50 kg ha−1 at post-flowering [BN100 + N50 (60 d)] increased CA activity, photosynthesis, stomatal conductance and WUE. The treatment increased CA activity, photosynthesis and WUE by 20.9, 17.2 and 14.5% over no-defoliation plants and 150 kg N ha−1 at sowing (BN150), and 12.5, 11.8 and 10.7% over no-defoliation plants treated with BN100 + N50 (60), respectively (Table 2). Defoliation at early stage of growth reduces the competition between the leaves for efficient utilization of light, water and nutrients. The increase in the photosynthetic characteristics of plants defoliated at 40 DAS was due to the fact that leaf growth following defoliation at this stage of growth had higher requirement of food reserves for growth and development. The other possibility is of accumulation of nutrients like potassium, which helped in maintaining the rate of photosynthesis by improving the relative water content of leaf through osmotic adjustment. Khan et al. (2000) reported that potassium accumulation increased with N supply, causing an increase in photosynthetic rate and dry mass. It is emphasized that an allocation of leaf N due to N application in suitable package [BN100 + N50 (60)] increased the photosynthetic characteristics. The increased N supply has been found to enhance the activities of CA and RuBP carboxylase (Terashima and Evans, 1988; Burnell et al., 1990; Khan et al., 1996). Thus, the plants after defoliation required larger amount of N in compensatory mechanisms, which influenced enzymes of photosynthesis. An increase in CA and ribulose 1–5 bisphosphate (RuBP) carboxylase following defoliation has been observed (Khan, 2002). Considering the effect of defoliation and split N application on stomatal and mesophyll processes, it emerges from the data that the latter were mainly responsible for the substantial increase in photosynthesis. 3.3. N assimilation The activity of N assimilation enzymes and plant N content were significantly increased following defoliation and N treatments. Maximal activities of N assimilating enzymes were recorded in defoliated plants treated with 100 kg N ha−1 at sowing and 50 kg N ha−1 at 60 DAS [BN100 + N50 (60 d)]. The activities of enzymes NR, Ni R and GS and N content were found significantly higher in defoliated plants treated with BN100 + N50 (60 d) compared to no-defoliation plants treated with BN150 or BN100 + N50 (60 d). The increase in NR activity due to the treatment BN100 + N50(60 d) was 11.2 and 3.4%, and plant N was 53.8 and 27.9% in comparison to no-defoliation plants treated with BN150 or BN100 + N50(60 d), respectively (Table 3). Nitrogen is important for overall growth, physiology and development of plants. At initial stages of growth, the soilapplied N is liable to losses by volatilization and leaching, etc.
Secondly, further depletion occurs due to a greater demand by the crop at that time. The plants after defoliation required N in large amount to compensate growth. Therefore, application of N partly at 60 DAS stage might have been timely. Higher rates of N incorporation have been reported to increase the activities of N enzymes like NR (Vyas et al., 1995; Khan et al., 1996; Wang et al., 2000). 3.4. Yield characteristics Defoliation and N application significantly increased yield characteristics. Number of pods per plant, 1000 seed weight and seed yield were found greatest in defoliated plants treated with 100 kg N ha−1 at sowing and 50 kg N ha−1 at 60 DAS [BN100 + N50 (60 d)]. An increase of 19.9% in pod number, 33.3% in 1000 seed weight and 22.6% in seed yield over nodefoliation plants treated with 150 kg N ha−1 at sowing (BN150) was recorded. Similarly, these characteristics also showed an increase of 25.8, 27.2 and 15.7%, respectively over the intact plants treated with soil-applied N as [BN100 + N50 (60 d)] (Table 4). Increased seed yield was due to the higher percent increase in pod number and seed weight in defoliated plants. The size of N pools in vegetative parts determines seed set, seed growth and finally seed yield (Marschner, 1995). It can be argued that timely application of N at post-flowering (60 DAS) met the N requirement for seed set, which increased seed yield. The concurrent enhanced regrowth following utilization of soil N more efficiently resulted in higher dry mass and finally seed yield. The increase in seed yield characteristics after defoliation finds support from similar findings of Bruening and Egli (2000), Khan and Ahsan (2000); Khan et al. (2000); Khan (2003) and Khan and Lone (2005). However, the effect of defoliation together with split N application on mustard has not been reported earlier. 4. Conclusion Loss of soil-applied N in terms of leaching, volatilization and surface run off minimizes N use efficiency and causes economic loss together with environmental degradation. It can be concluded from the study that the amount and timing of N application together with defoliation has an important influence in augmenting growth, photosynthesis and yield of mustard. The quantum of increase in the characteristics with defoliation and split N application of 100 kg N ha−1 at sowing and 50 kg N ha−1 as top dressing at 60 DAS (post-flowering) was much higher than no-defoliation and one time N application as 150 kg N ha−1 or no-defoliation followed by split N application at pre- or postflowering. References Alderfer, R.G., Eagles, C.F., 1976. The effect of partial defoliation on the growth and photosynthetic efficiency of bean leaves. Bot. Gaz. 137, 351–355. Anten, N.P.R., Ackerly, D.D., 2001. Canopy-level photosynthetic compensation after defoliation in a tropical understorey palm. Funct. Ecol. 15, 252–262. Anten, N.P.R., Martinez-Ramos, M., Ackerly, D.D., 2003. Defoliation and growth in an understory palm: quantifying the contributions of compensatory responses. Ecology 84, 2905–2918.
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