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b ratios to different nitrogen supply rates in paddy field
Field Crops Research 114 (2009) 426–432
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Short communication
Responses of rice leaf thickness, SPAD readings and chlorophyll a/b ratios to different nitrogen supply rates in paddy field Li Jinwen a, Yang Jingping b,*, Fei Pinpin b, Song Junlan c, Li Dongsheng c, Ge Changshui d, Chen Wenyue d a
College of Life Sciences, Zhejiang University, Kaixuan Road, Hangzhou 310029, China College of Environmental and Resource Sciences, Zhejiang University, Hangzhou 310029, China c College of Measurement and Test Engineering, China Jiliang University, Hangzhou 310018, China d Hangzhou Academy of Agricultural Sciences, Hangzhou 310003, China b
A R T I C L E I N F O
A B S T R A C T
Article history: Received 24 February 2009 Received in revised form 9 September 2009 Accepted 12 September 2009
The objective of this paper was to investigate the influence of different nitrogen (N) application rates to some morphological and physiological features of leaf blades, including leaf thickness, chlorophyll content at different leaf ages and chlorophyll a/b ratios. A paddy field and a cement tank experiments were conducted simultaneously. Rice leaf thickness was measured through a specially developed displacement sensor. Meanwhile, chlorophyll content was estimated using chlorophyll meter (SPAD) and spectrophotometer after ethanol extraction of leaf samples. With the increase of N application, leaf thickness became thinner and chlorophyll a/b ratios decreased. Moreover, the sensitivity of the SPAD readings of the same leaf at different leaf ages to N rates was assessed through coefficients of variation (CV). CV of SPAD readings increased from 8.8% to 21.6% during leaf lifetime, which indicates that SPAD readings became more and more sensitive to nitrogen rates as leaf aged. Therefore, SPAD readings of the lower leaves, which were physiologically older than the upper ones, were more sensitive to nitrogen rates. Published by Elsevier B.V.
Keywords: Specific leaf mass Nitrogen Leaf thickness
1. Introduction Leaf thickness and photosynthetic pigments including chlorophyll (chl) a and b are vital components to the uptake rate of CO2. Thicker rice leaf, containing more photosynthetic apparatus per unit area, is an important morphological trait for ‘‘ideotype’’ (plant type that has greater yield potential due to its morphological configuration) (Peng et al., 2008). Moreover, the influence of leaf thickness to the estimate of nitrogen (N) content (based on per unit weight) by SPAD is noteworthy (Peng et al., 1993; Chapman and Barreto, 1997), and the devices for estimating leaf thickness could be incorporated into the SPAD meter to provide SPAD readings adjusted for leaf thickness (Yamamoto et al., 2002). Light intensity is one of the environmental factors influencing rice leaf thickness (Murchie et al., 2005). High light intensity induces thicker leaf, higher chl a/b ratios and chl content per unit area, but lower chlorophyll content per unit weight or volume (Boardman, 1977; Khan et al., 2000; Terashima et al., 2006). Besides light intensity, N supply is another fundamental component participating in plant metabolism. Therefore, investigating the effect of N application rates on leaf thickness, chl content
* Corresponding author. Tel.: +86 15924176980. E-mail address: [email protected] (Y. Jingping). 0378-4290/$ – see front matter . Published by Elsevier B.V. doi:10.1016/j.fcr.2009.09.009
and chl a/b ratios is indispensible to understand the physiological activities of rice leaves. An accurate measurement of leaf thickness is difficult and timeconsuming because leaf blade changes under pressure. Furthermore, close protruding veins in rice leaf and wide variation in leaf thickness from the base to the tip of the leaf blade limit precision of measurement (Chen et al., 2007). Leaf chl content can be rapidly estimated in situ by SPAD (SoilPlant Analysis Development) readings. The chl content is affected by available N in leaf as N is one of the staple elements to chl. Therefore, N status in rice plant could be indicated by chl contents or SPAD readings. That means that the SPAD readings can reflect plant N requiring status at different growth stages. In the past two decades the use of SPAD to monitor rice N status was widely applied to judging rice N demand at different growth stages to improve grain yield and N use efficiency (Huang et al., 2008; Khurana et al., 2007; Peng et al., 2006). Measuring SPAD readings of the uppermost fully expanded leaf to reveal plant N status has been accepted as a common practice, although it was found that leaves in lower positions could be more suitable to serve as testing sample for N status diagnosis, as the lower leaves were much better than the upper leaves in separating N level in case the total N was used as an indicator (Zhou and Wang, 2003). Moreover, compared to the upper leaves, the SPAD readings of the lower ones are better
L. Jinwen et al. / Field Crops Research 114 (2009) 426–432
correlated with the total N in whole leaves and plant (Li et al., 2007). Nevertheless, to our knowledge, a satisfied explanation to the aforesaid phenomenon has not been made. When SPAD was used to diagnose N deficiencies during various growth stages of corn (Zea mays L.), the difference of plant physiological age induced by factors such as tillage, soil organic matter could introduce unexpected errors to the diagnosis (Zhang et al., 2008). Inspired by this discovery, we supposed that the responses of the same leaf at different growth stages to status of N supply should deserve attention when SPAD is used to diagnose plant N status. In addition, N rates influence not only chl contents but also the composition of chl, namely the ratio of chl a/b (Kitajima and Hogan, 2003). Greater chl a/b ratio taken as a bioassay to assess the light environment of a plant for leaves receiving intense light are characteristic of higher chl a/b ratios (Dale and Causton, 1992). There was also a report about a distinct subgroup of genotypes existing under different chl a/b ratios (Fritschi and Ray, 2007). The increase in chl a/b ratio reflects the increase in the ratio of [PSII] (amount of core complex of photosystem II, mmol PSII m2) to [LHCII] (amount of light-harvesting complex of photosystem II, mmol LHCII m2), and if the N is limited under strong light, the N allocated to LHCII is maintained at a similar level while N allocated to PSII will increase at the cost of a decline in N that should have been allocated to Rubisco, so the ratio of [PSII] to [LHCII] and chl a/b ratio will increase when N availability decreases (Hikosaka and Terfashima, 1995). However, whether there is an occurrence of the variation in the ratios in rice leaf has not been confirmed yet. In this study, a leaf thickness measuring instrument developed by China Jiliang University was applied to measuring the leaf thickness of rice nondestructively and accurately. The leaf chl content in different genotypes of rice with a wide range of leaf thickness was determined. Furthermore, the effect of different N application rates on leaf thickness was explored. To ascertain the effect of leaf age on rice N status diagnosis, SPAD readings at different leaf ages were investigated through long-term observation of the same leaves under different N rates, and the alteration in ratios of chl a/b under different N supply levels was also examined. 2. Materials and methods 2.1. Field experiments A field experiment was conducted on the experimental farm of Hangzhou Academy of Agriculture, Hangzhou in 2008. Rice (Oryza sativa L.) cv. ‘‘Bing 9363’’ was planted and treated with 6 N rates in
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Fig. 1. Diurnal change of leaf thickness with ambient temperature.
a randomized block design with 3 replications. Seedlings with fully expanded 5 leaves were transplanted on 27 June. Hill spacing was 0.23 m 0.13 m with 2 seedlings per hill in the 3 m 6 m plot. Superphosphate (225 kg ha1) and potassium (75 kg ha1) chloride were incorporated into each plot on the transplanting day, and another 75 kg ha1 potassium chloride was top-dressed on 40 DAT (days after transplanting). Plants received 0, 75, 150, 225, 300, or 375 kg pure N ha1 respectively as urea, and each rate of N was applied in 4 doses according to rice growing stages: 3 July (plant reviving, 20%), 10 June (tillering, 30%), 6 August (panicle initiation, 30%) and 30 August (grain filling, 20%). The paddy field has loam soil with organic matter content at 35.50 g kg1 and total N at 2.05 g kg1. 2.2. Cement tank experiment A cement tank trial was conducted in an open-air field synchronizing with the field experiment. All tanks were filled with 1-m depth of homogeneous soil. A total of 9 genotypes of rice with various degrees of greenness were selected, consisting of 2 japonica varieties (Guihuahuang and Nanjing 42), 1 indica variety (Zhongzu 53), and 6 hybrid varieties (Yongyou 6, Yongyou 9, Qianyou 1, Qianyou 63, Qianyou 0508, and Liangyoupeijiu). Varieties Nanjing 42, Liangyoupeijiu, Qianyou 63 and Yongyou 9
Fig. 2. Relationship of leaf thickness with chl content based on per unit weight (A) and per unit area (B). Data were pooled from 9 varieties.
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received 75 and 225 kg pure N ha1 as urea to create different crop N status, and the remaining varieties received 75 kg N pure ha1. All other farming management practices were the same as the field experiment. Soil in tanks was silt loam, with the content of organic matter and total N of 22.50 and 1.66 g kg1 respectively. 2.3. Measurements In the field experiment, a chlorophyll meter (Minolta SPAD502) was adopted to take SPAD values (SPAD units) from the 4 upper fully expanded functional leaves on each plant at an interval of approximately 10 days (according to weather). Total of 10 plants were measured in every plot, and 3 SPAD readings per leaf, including one reading around the midpoint of leaf blade and 2 readings of points 3 cm apart from the midpoint were averaged as the mean SPAD reading of the leaf (Peng et al., 1993). To observe temporal change of the SPAD readings for the same leaf at different N rates, when the tenth leaves (numbered acropetally) were just fully expanded, 10 of them in each plot were marked with numbered labels. For the purpose of more accurate estimation of chl content, a leaf SPAD reading was calculated by taking the mean of 10 SPAD readings from leaf base to apex. From August 19 to 22 and from October 6 to 10, 10 leaves were removed with sheaths from every plot, and were submerged into water in a bottle immediately to prevent leaf from curling. Based
Fig. 3. Relationship between leaf thickness and FSLM (fresh specific leaf mass) measured in field during August 19–22 and October 6–10 respectively. The uppermost leaves in the two growth stages were selected.
Fig. 4. Relationship between FSLM (fresh specific leaf mass), leaf thickness measured during August 19–22 (A and C), October 6–10 (B and D) and N supply level. Each datum is mean of 30 values measured in 3 plots under the same N rate.
L. Jinwen et al. / Field Crops Research 114 (2009) 426–432
on single-chip measuring system, a leaf thickness measuring instrument using displacement sensor was applied to determining leaf thickness in 15 min after removing (Li et al., 2006). The pressure exerted by the sensor to leaf blade surface was only 0.1 N to reduce the variability in leaf thickness. It was discovered that diurnal changes in leaf thickness are mainly caused by ambient temperature changes (Schroeder, 1980). The diurnal change in leaf thickness is shown in Fig. 1. When the ambient temperature fluctuated, leaf thickness varied inversely. The leaf thickness was measured at a stable temperature approximately 30 8C to reduce the measurement error induced by variation of ambient temperature. Both SPAD readings and leaf thickness were measured from leaf base to apex for at least 10 times (according to leaf length), and means of all readings were taken as the SPAD reading and thickness of a leaf. Leaf area was measured using leaf area meter (AM100, ADC, UK). Five leaves from each plot were extracted by 96% (v/v) ethanol and chl a and b were determined by measuring absorbance at 649 and 665 nm wavelengths on a spectrophotometer (Fritschi and Ray, 2007). In cement tank trial, the 5 uppermost fully expanded leaves were removed from every tank during August 11–15, and 15 leaves were removed during September 26–30 from the tanks with Nanjing 42, Liangyoupeijiu, Qianyou 63 and Yongyou 9. All the leaves were disposed and measured the same way as field experiment. 3. Results 3.1. The interspecific relationship between chl content and leaf thickness Fig. 2 shows the relationship between chl content and leaf thickness in 9 varieties planted in tanks. When the content was expressed based on the basis of fresh weight, it was not significantly correlated with the leaf thickness (P = 0.189; Fig. 2A), but if the content was expressed on the basis of leaf area, the relationship between them was significantly positive (R = 0.37406, P = 0.00214; Fig. 2B).
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Fig. 5. FSLM (fresh specific leaf mass) and leaf thickness under 2N rates in 4 varieties. NJ42, LYPJ, QY63 and YY9 were abbreviations of Nanjing 42, Liangyoupeijiu, Qianyou 63 and Yongyou 9. N1 and N2 were 75 and 225 kg N ha1. Vertical bars represent leaf thickness (mm). Each bar was an average of 20 data.
different (P < 0.001). Averaged leaf thickness of all uppermost fully expanded leaves measured on August 19–22 and October 6–10 were 144.71 mm and 179.35 mm respectively. Averaged FSLM measured during the same two periods were 147.23 g m2 and152.61 g m2 respectively. As N rate increased, FSLM became smaller (Fig. 4A and B), whereas leaf thickness trended to be thinner (Fig. 4C and D). On contrary, the higher N rate, the greater of the leaf in length and width, is associated with less variation in the two dimensions (data were not shown). In the other 4 varieties, smaller leaf FSLM and thickness were also associated with higher N rates (Fig. 5).
3.2. Leaf thickness affected by N supply rates Fresh specific leaf mass (fresh leaf weight/leaf area, FSLM) is an effective surrogate for leaf thickness (Vile et al., 2005). In the field trial, FSLM and leaf thickness were positively correlated in two growth stages (Fig. 3). The slopes of linear regression equations established at the two growth stages respectively did not differ significantly (P = 0.627) whereas the intercepts were significantly
3.3. Temporal variation of SPAD readings of labeled leaves under different N rates and the top functional leaves at different growth stage Fig. 6 shows temporal changes in SPAD readings of the 10th leaves (numbered acropetally) under 6 N rates (ranged from 0 to 375 kg ha1 N) between DAT35 (the 10th leaves were just expanded) to DAT65 (leaves under low N rates started senescence).
Fig. 6. SPAD readings temporal variation of labeled leaves under different N rate in field; each bar was an average of 30 measured values in 3 plots under the same N rate; N1– N6 were N rates from 0 to 375 kg N ha1. Vertical bars represent SPAD readings.
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Table 1 Temporal SPAD readings (standard deviation) and coefficients of variation (CV) from top 3 or 4 leaves (functional leaves) on different days after transplanting (DAT). Each value was an average of 180 (30 6) SPAD readings from the same leaf position under a range of 6N rates. On DAT105 most 4th leaves were dead; therefore, the data were not presented. Within a column, data followed by different letters are significantly different at P < 0.05 by Duncan’s multiple range test. Leaf relative positiona
1st CV 2nd CV 3rd CV 4th CV a b
Days after transplantation (DAT) DAT30
DAT40
DAT53
DAT65b
DAT78
DAT89
DAT105
39.07 2.93a 7.50% 41.61 3.45b 8.29% 41.93 3.55b 8.47% 43.11 4.28 c 9.92%
40.45 3.37a 8.34% 41.79 3.57b 8.54% 42.11 3.91bc 9.28% 40.28 4.97a 12.33%
41.16 2.58ab 6.27% 41.46 3.44ab 8.30% 41.87 4.27a 10.19% 40.69 5.34b 13.12%
46.07 3.02a 6.56% 44.13 3.10b 7.02% 43.24 3.74b 8.65% 41.94 4.98c 11.88%
46.01 3.10a 6.73% 43.52 3.22b 7.40% 41.82 3.94c 9.42% 39.54 5.35d 13.54%
44.65 5.74a 12.86% 41.41 6.11b 14.77% 39.73 6.46c 16.27% 35.87 7.14d 19.90%
34.22 8.30a 24.24% 29.12 9.31b 31.98% 28.07 9.21c 32.69% –
Numbered basipetally and corresponding to different plant growth stage. Day that all leaves had already shot and no other new leaves would appear.
Table 2 Chl content (per unit weight and area), SPAD readings, and chl a/b ratios measured during August 19–22 and October 6–10 under different N rates. Mean (standard deviation) of chl content and chl a/b ratios were indicated for 15 leaves with SPAD readings for 30 leaves. Within a column, data followed by different letters are significantly different at P < 0.05 by Duncan’s multiple range test. N rate
Chl content
SPAD reading 1
Per unit weight (mg g
N1 N2 N3 N4 N5 N6
)
Per unit area (mg dm
2
)
August 19–22
October 6–10
August 19–22
October 6–10
1.87 0.19a 2.23 0.21b 2.52 0.22c 2.50 0.13c 2.54 0.12c 2.52 0.12c
1.09 0.72a 1.15 0.56a 1.42 0.38ab 1.60 0.35ab 1.76 0.45b 1.65 0.37b
3.00 0.32a 3.36 0.34b 3.63 0.28c 3.60 0.28c 3.61 0.23c 3.64 0.23c
1.70 1.12a 1.82 0.84a 2.14 0.60ab 2.37 0.53b 2.54 0.60b 2.48 0.51b
Coefficients of variation (CV) in SPAD readings measured at each time increased as leaf aged, which meant that SPAD readings became more and more sensitive to nitrogen rates following leaf aging. On the other hand, N application also prolonged lifespan of leaves. On DAT65, the 10th leaves without receiving any N were tended to die (SPAD readings = 24), but those receiving 375 kg N ha1 were still vigorous (SPAD readings = 42.3). There were totally about 15 leaves on effective stem tillers of Bing 9363. At each plant growth stage, the top 4 leaves acted as functional leaves and undertook main dry matter production. Prior to the panicle initiation, the leaf development from emergence to full size took 3–5 days, whereas it took 6–8 days post the panicle initiation. Correspondingly, a lower leaf was 3–5 or 6–8 days physiologically older than the neighboring leaf above it. Distinction in SPAD readings also existed in different leaf positions, as shown in Table 1, with standard deviation (SD) and CV of readings increasing from 1st to 4th (numbered basipetally) leaf. Starting from August 28 (DAT62) the 15th leaf (the last one) was just fully expanded and no other leaf would develop, so during DAT65 to 105, the leaf position of top 3 or 4 leaves did not vary. During DAT65 to 105, SPAD readings decreased from the upper to lower leaves while SD and CV of SPAD readings in the same leaf position raised (Table 1). 3.4. Chl a/b ratios varied with chl content As revealed in Table 2, chl content per unit weight or area were elevated by increase of N rates, whereas chl a/b ratios were significantly higher when fertilizer N was not or lightly topdressed. The chl a/b ratios were negatively related to chl content (expressed either as per unit weight or area) (Fig. 6A–D) and to SPAD readings (Fig. 6E and F). Nevertheless, linear regression equations generated by data measured during October 6–10 (Fig. 7B, D and F) were poorly fit as compared with the data measured on August 19–22 (Fig. 7A, C and E). Chl a/b ratios
Chl a/b ratio
August 19–22
October 6–10
August 19–22
October 6–10
36.58 2.92a 38.54 2.59b 40.41 1.52c 41.61 1.99d 42.83 2.00e 43.02 1.83e
30.37 9.52a 30.42 9.69a 33.75 4.81b 35.51 4.20bc 37.07 4.48c 36.72 4.48bc
2.69 0.30a 2.34 0.27b 2.09 0.28b 1.71 0.46c 1.49 0.27cd 1.34 0.20d
2.48 0.30a 2.58 0.20a 2.37 0.23ab 2.34 0.28ab 2.11 0.36bc 2.02 0.31c
measured in other 4 rice varieties under higher N rate were smaller too (Fig. 8). 4. Discussion The maximum rate of CO2 assimilation (per unit area), an important component to crop productivity, is almost proportional or somewhat curvilinear to amount of N per unit leaf area (specific leaf N, SLN), so leaf thickness is an important trait related to photosynthetic activity because thicker leaf contain much more chl per unit area (Fig. 2B). Moreover, since N expressed based on per unit area in different varieties could be more precisely estimated by SPAD than based on per unit weight (Peng et al., 1993), inherent difference in chl or N content (based on per unit area) accompanied by diverse leaf thickness (Fig. 2) among different varieties needs to be taken into account fully when SPAD is applied to diagnose plant N status. However, the poor linear regression describing relationships between chl content per unit area and leaf thickness might be due to the differences in chl content per unit weight among different varieties. FSLM was proved to be a proper estimate of leaf thickness as shown in Fig. 3, but regression equations describing relation between leaf thickness and FSLM in two growth stages differed in their intercepts, which might be the consequence of different leaf density (mass per volume) formed at different plant growth stages (Witkowski and Lamont, 1991). In other words, FSLM in different growth stage are incomparable if they are used as surrogates of leaf thickness. Our data demonstrated that higher N supply caused thinner leaves, which would be another inducement to leaf droop. It has been reported that nutrient supply does not affect epidermal thickness and differences in leaf thickness reflects differences in mesophyll thickness (David, 1980). The change in anatomical structure of rice leaf caused by N supply deserves further investigation.
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Fig. 7. Relationship of chl a/b ratios with chl content based on per unit weight (A and B), per unit area (C and D) and SPAD readings (E and F). A, C and E were for data measured during August 19–22 and B, D and F for October 6–10.
Temporal SPAD readings of the same leaves under different N rates indicated that leaf age is an important factor deserving to be checked when SPAD measurements are used to plant N status diagnosis. We infer that the effect of N supply on SPAD readings
would be manifested more and more distinctly as leaf ages, so that CV of SPAD readings measured on the same leaves increased as leaf ages. Therefore, it seems more reasonable to use the lower leaves (physiologically older) than the upper leaves as indicators of plant
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also be given to Mr. Wang Zhumei, retired research fellow at Hangzhou Academy of Agricultural Sciences for his kind guidance to field management. References
Fig. 8. Chl a/b ratios under 2N rates in 4 varieties. NJ42, LYPJ, QY63 and YY9 were abbreviations of Nanjing 42, Liangyoupeijiu, Qianyou 63 and Yongyou 9, and N1 and N2 were 75 and 225 kg N ha1. Each bar was an average of 15 data. Vertical bars represent chl a/ratios.
N status. This conclusion is consistent with previous research (Zhou and Wang, 2003). The inference is further confirmed by temporal SPAD readings of the top 4 leaves (as shown in Table 1): constantly greater CV of SPAD readings measured on the lower leaves, as well as the aged leaves (data after DAT65). One important reason that created the differences in SPAD readings at various leaf position (Table 1) might be associated with the heterogeneous distribution of radiation flux in rice canopy. The distribution of radiation flux could be described as: IL ¼ I0 ekL where I0 and IL are the radiation flux above the canopy and at a point above which there are L layers of leaf, and k is the extinction coefficient (Fitter and Hay, 1981; Murchie et al., 2002). Upper leaves receive greater radiation flux than lower leaves, which result in higher chl a/b ratios, thicker leaves and chl content. Other factors, such as different leaf age and humidity probably also contribute to the differences. The increase in the chl a/b ratios reflects the relative increase in the ratios of [PSII] to [LHCII], and the increase of chl a/b ratios and hence [PSII]/[LHCII] ratios with decrease in [N] under high irradiance were predicted by Hikosaka and Terfashima (1995). Data presented in our paper is consistent with the speculation. A much poorer linear regression equation describing relationship of chl a/b ratios with chl content measured in October 6–10 (Fig. 7A, C and E) might be due to leaf aging and lower light intensity in leaf lifespan (from late August to early November). A decrease in chl a/b ratios has been reported during leaf senescence in rice (Jun et al., 1991), which blurs the relationship between chl content and chl a/ b ratios, together with the effect of lower light intensity. Chl a/b ratios of youthful leaf under high intensity light could be regarded as an indicator revealing rice N status if the leaf receives homogeneous light intensity, because leaf lack of N is characteristic of high chl a/b ratios. However, this report is only preliminary report and more in-depth physiological research should be conducted in the future. Acknowledgments The research was financially supported by National Natural Science Foundation of China (No. 60574046) and Zhejiang Natural Science Foundation (No. Y507005). Our heartfelt thanks should be given to Professor Zhang Jun of Wright University, U.S.A., Dr. Niu Genhua of Texas AgriLife Research and Extension Center at El Paso, U.S.A., for their critical reading of the manuscript, and Professor Wang Shaohua of Nanjing Agricultural University and Mr. Liu Xin of Seed Extension Centre of Zhejiang Province for their kind donation of rice seed. Many thanks should
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Field Crops Research journal homepage: www.elsevier.com/locate/fcr
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Responses of rice leaf thickness, SPAD readings and chlorophyll a/b ratios to different nitrogen supply rates in paddy field Li Jinwen a, Yang Jingping b,*, Fei Pinpin b, Song Junlan c, Li Dongsheng c, Ge Changshui d, Chen Wenyue d a
College of Life Sciences, Zhejiang University, Kaixuan Road, Hangzhou 310029, China College of Environmental and Resource Sciences, Zhejiang University, Hangzhou 310029, China c College of Measurement and Test Engineering, China Jiliang University, Hangzhou 310018, China d Hangzhou Academy of Agricultural Sciences, Hangzhou 310003, China b
A R T I C L E I N F O
A B S T R A C T
Article history: Received 24 February 2009 Received in revised form 9 September 2009 Accepted 12 September 2009
The objective of this paper was to investigate the influence of different nitrogen (N) application rates to some morphological and physiological features of leaf blades, including leaf thickness, chlorophyll content at different leaf ages and chlorophyll a/b ratios. A paddy field and a cement tank experiments were conducted simultaneously. Rice leaf thickness was measured through a specially developed displacement sensor. Meanwhile, chlorophyll content was estimated using chlorophyll meter (SPAD) and spectrophotometer after ethanol extraction of leaf samples. With the increase of N application, leaf thickness became thinner and chlorophyll a/b ratios decreased. Moreover, the sensitivity of the SPAD readings of the same leaf at different leaf ages to N rates was assessed through coefficients of variation (CV). CV of SPAD readings increased from 8.8% to 21.6% during leaf lifetime, which indicates that SPAD readings became more and more sensitive to nitrogen rates as leaf aged. Therefore, SPAD readings of the lower leaves, which were physiologically older than the upper ones, were more sensitive to nitrogen rates. Published by Elsevier B.V.
Keywords: Specific leaf mass Nitrogen Leaf thickness
1. Introduction Leaf thickness and photosynthetic pigments including chlorophyll (chl) a and b are vital components to the uptake rate of CO2. Thicker rice leaf, containing more photosynthetic apparatus per unit area, is an important morphological trait for ‘‘ideotype’’ (plant type that has greater yield potential due to its morphological configuration) (Peng et al., 2008). Moreover, the influence of leaf thickness to the estimate of nitrogen (N) content (based on per unit weight) by SPAD is noteworthy (Peng et al., 1993; Chapman and Barreto, 1997), and the devices for estimating leaf thickness could be incorporated into the SPAD meter to provide SPAD readings adjusted for leaf thickness (Yamamoto et al., 2002). Light intensity is one of the environmental factors influencing rice leaf thickness (Murchie et al., 2005). High light intensity induces thicker leaf, higher chl a/b ratios and chl content per unit area, but lower chlorophyll content per unit weight or volume (Boardman, 1977; Khan et al., 2000; Terashima et al., 2006). Besides light intensity, N supply is another fundamental component participating in plant metabolism. Therefore, investigating the effect of N application rates on leaf thickness, chl content
* Corresponding author. Tel.: +86 15924176980. E-mail address: [email protected] (Y. Jingping). 0378-4290/$ – see front matter . Published by Elsevier B.V. doi:10.1016/j.fcr.2009.09.009
and chl a/b ratios is indispensible to understand the physiological activities of rice leaves. An accurate measurement of leaf thickness is difficult and timeconsuming because leaf blade changes under pressure. Furthermore, close protruding veins in rice leaf and wide variation in leaf thickness from the base to the tip of the leaf blade limit precision of measurement (Chen et al., 2007). Leaf chl content can be rapidly estimated in situ by SPAD (SoilPlant Analysis Development) readings. The chl content is affected by available N in leaf as N is one of the staple elements to chl. Therefore, N status in rice plant could be indicated by chl contents or SPAD readings. That means that the SPAD readings can reflect plant N requiring status at different growth stages. In the past two decades the use of SPAD to monitor rice N status was widely applied to judging rice N demand at different growth stages to improve grain yield and N use efficiency (Huang et al., 2008; Khurana et al., 2007; Peng et al., 2006). Measuring SPAD readings of the uppermost fully expanded leaf to reveal plant N status has been accepted as a common practice, although it was found that leaves in lower positions could be more suitable to serve as testing sample for N status diagnosis, as the lower leaves were much better than the upper leaves in separating N level in case the total N was used as an indicator (Zhou and Wang, 2003). Moreover, compared to the upper leaves, the SPAD readings of the lower ones are better
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correlated with the total N in whole leaves and plant (Li et al., 2007). Nevertheless, to our knowledge, a satisfied explanation to the aforesaid phenomenon has not been made. When SPAD was used to diagnose N deficiencies during various growth stages of corn (Zea mays L.), the difference of plant physiological age induced by factors such as tillage, soil organic matter could introduce unexpected errors to the diagnosis (Zhang et al., 2008). Inspired by this discovery, we supposed that the responses of the same leaf at different growth stages to status of N supply should deserve attention when SPAD is used to diagnose plant N status. In addition, N rates influence not only chl contents but also the composition of chl, namely the ratio of chl a/b (Kitajima and Hogan, 2003). Greater chl a/b ratio taken as a bioassay to assess the light environment of a plant for leaves receiving intense light are characteristic of higher chl a/b ratios (Dale and Causton, 1992). There was also a report about a distinct subgroup of genotypes existing under different chl a/b ratios (Fritschi and Ray, 2007). The increase in chl a/b ratio reflects the increase in the ratio of [PSII] (amount of core complex of photosystem II, mmol PSII m2) to [LHCII] (amount of light-harvesting complex of photosystem II, mmol LHCII m2), and if the N is limited under strong light, the N allocated to LHCII is maintained at a similar level while N allocated to PSII will increase at the cost of a decline in N that should have been allocated to Rubisco, so the ratio of [PSII] to [LHCII] and chl a/b ratio will increase when N availability decreases (Hikosaka and Terfashima, 1995). However, whether there is an occurrence of the variation in the ratios in rice leaf has not been confirmed yet. In this study, a leaf thickness measuring instrument developed by China Jiliang University was applied to measuring the leaf thickness of rice nondestructively and accurately. The leaf chl content in different genotypes of rice with a wide range of leaf thickness was determined. Furthermore, the effect of different N application rates on leaf thickness was explored. To ascertain the effect of leaf age on rice N status diagnosis, SPAD readings at different leaf ages were investigated through long-term observation of the same leaves under different N rates, and the alteration in ratios of chl a/b under different N supply levels was also examined. 2. Materials and methods 2.1. Field experiments A field experiment was conducted on the experimental farm of Hangzhou Academy of Agriculture, Hangzhou in 2008. Rice (Oryza sativa L.) cv. ‘‘Bing 9363’’ was planted and treated with 6 N rates in
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Fig. 1. Diurnal change of leaf thickness with ambient temperature.
a randomized block design with 3 replications. Seedlings with fully expanded 5 leaves were transplanted on 27 June. Hill spacing was 0.23 m 0.13 m with 2 seedlings per hill in the 3 m 6 m plot. Superphosphate (225 kg ha1) and potassium (75 kg ha1) chloride were incorporated into each plot on the transplanting day, and another 75 kg ha1 potassium chloride was top-dressed on 40 DAT (days after transplanting). Plants received 0, 75, 150, 225, 300, or 375 kg pure N ha1 respectively as urea, and each rate of N was applied in 4 doses according to rice growing stages: 3 July (plant reviving, 20%), 10 June (tillering, 30%), 6 August (panicle initiation, 30%) and 30 August (grain filling, 20%). The paddy field has loam soil with organic matter content at 35.50 g kg1 and total N at 2.05 g kg1. 2.2. Cement tank experiment A cement tank trial was conducted in an open-air field synchronizing with the field experiment. All tanks were filled with 1-m depth of homogeneous soil. A total of 9 genotypes of rice with various degrees of greenness were selected, consisting of 2 japonica varieties (Guihuahuang and Nanjing 42), 1 indica variety (Zhongzu 53), and 6 hybrid varieties (Yongyou 6, Yongyou 9, Qianyou 1, Qianyou 63, Qianyou 0508, and Liangyoupeijiu). Varieties Nanjing 42, Liangyoupeijiu, Qianyou 63 and Yongyou 9
Fig. 2. Relationship of leaf thickness with chl content based on per unit weight (A) and per unit area (B). Data were pooled from 9 varieties.
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received 75 and 225 kg pure N ha1 as urea to create different crop N status, and the remaining varieties received 75 kg N pure ha1. All other farming management practices were the same as the field experiment. Soil in tanks was silt loam, with the content of organic matter and total N of 22.50 and 1.66 g kg1 respectively. 2.3. Measurements In the field experiment, a chlorophyll meter (Minolta SPAD502) was adopted to take SPAD values (SPAD units) from the 4 upper fully expanded functional leaves on each plant at an interval of approximately 10 days (according to weather). Total of 10 plants were measured in every plot, and 3 SPAD readings per leaf, including one reading around the midpoint of leaf blade and 2 readings of points 3 cm apart from the midpoint were averaged as the mean SPAD reading of the leaf (Peng et al., 1993). To observe temporal change of the SPAD readings for the same leaf at different N rates, when the tenth leaves (numbered acropetally) were just fully expanded, 10 of them in each plot were marked with numbered labels. For the purpose of more accurate estimation of chl content, a leaf SPAD reading was calculated by taking the mean of 10 SPAD readings from leaf base to apex. From August 19 to 22 and from October 6 to 10, 10 leaves were removed with sheaths from every plot, and were submerged into water in a bottle immediately to prevent leaf from curling. Based
Fig. 3. Relationship between leaf thickness and FSLM (fresh specific leaf mass) measured in field during August 19–22 and October 6–10 respectively. The uppermost leaves in the two growth stages were selected.
Fig. 4. Relationship between FSLM (fresh specific leaf mass), leaf thickness measured during August 19–22 (A and C), October 6–10 (B and D) and N supply level. Each datum is mean of 30 values measured in 3 plots under the same N rate.
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on single-chip measuring system, a leaf thickness measuring instrument using displacement sensor was applied to determining leaf thickness in 15 min after removing (Li et al., 2006). The pressure exerted by the sensor to leaf blade surface was only 0.1 N to reduce the variability in leaf thickness. It was discovered that diurnal changes in leaf thickness are mainly caused by ambient temperature changes (Schroeder, 1980). The diurnal change in leaf thickness is shown in Fig. 1. When the ambient temperature fluctuated, leaf thickness varied inversely. The leaf thickness was measured at a stable temperature approximately 30 8C to reduce the measurement error induced by variation of ambient temperature. Both SPAD readings and leaf thickness were measured from leaf base to apex for at least 10 times (according to leaf length), and means of all readings were taken as the SPAD reading and thickness of a leaf. Leaf area was measured using leaf area meter (AM100, ADC, UK). Five leaves from each plot were extracted by 96% (v/v) ethanol and chl a and b were determined by measuring absorbance at 649 and 665 nm wavelengths on a spectrophotometer (Fritschi and Ray, 2007). In cement tank trial, the 5 uppermost fully expanded leaves were removed from every tank during August 11–15, and 15 leaves were removed during September 26–30 from the tanks with Nanjing 42, Liangyoupeijiu, Qianyou 63 and Yongyou 9. All the leaves were disposed and measured the same way as field experiment. 3. Results 3.1. The interspecific relationship between chl content and leaf thickness Fig. 2 shows the relationship between chl content and leaf thickness in 9 varieties planted in tanks. When the content was expressed based on the basis of fresh weight, it was not significantly correlated with the leaf thickness (P = 0.189; Fig. 2A), but if the content was expressed on the basis of leaf area, the relationship between them was significantly positive (R = 0.37406, P = 0.00214; Fig. 2B).
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Fig. 5. FSLM (fresh specific leaf mass) and leaf thickness under 2N rates in 4 varieties. NJ42, LYPJ, QY63 and YY9 were abbreviations of Nanjing 42, Liangyoupeijiu, Qianyou 63 and Yongyou 9. N1 and N2 were 75 and 225 kg N ha1. Vertical bars represent leaf thickness (mm). Each bar was an average of 20 data.
different (P < 0.001). Averaged leaf thickness of all uppermost fully expanded leaves measured on August 19–22 and October 6–10 were 144.71 mm and 179.35 mm respectively. Averaged FSLM measured during the same two periods were 147.23 g m2 and152.61 g m2 respectively. As N rate increased, FSLM became smaller (Fig. 4A and B), whereas leaf thickness trended to be thinner (Fig. 4C and D). On contrary, the higher N rate, the greater of the leaf in length and width, is associated with less variation in the two dimensions (data were not shown). In the other 4 varieties, smaller leaf FSLM and thickness were also associated with higher N rates (Fig. 5).
3.2. Leaf thickness affected by N supply rates Fresh specific leaf mass (fresh leaf weight/leaf area, FSLM) is an effective surrogate for leaf thickness (Vile et al., 2005). In the field trial, FSLM and leaf thickness were positively correlated in two growth stages (Fig. 3). The slopes of linear regression equations established at the two growth stages respectively did not differ significantly (P = 0.627) whereas the intercepts were significantly
3.3. Temporal variation of SPAD readings of labeled leaves under different N rates and the top functional leaves at different growth stage Fig. 6 shows temporal changes in SPAD readings of the 10th leaves (numbered acropetally) under 6 N rates (ranged from 0 to 375 kg ha1 N) between DAT35 (the 10th leaves were just expanded) to DAT65 (leaves under low N rates started senescence).
Fig. 6. SPAD readings temporal variation of labeled leaves under different N rate in field; each bar was an average of 30 measured values in 3 plots under the same N rate; N1– N6 were N rates from 0 to 375 kg N ha1. Vertical bars represent SPAD readings.
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Table 1 Temporal SPAD readings (standard deviation) and coefficients of variation (CV) from top 3 or 4 leaves (functional leaves) on different days after transplanting (DAT). Each value was an average of 180 (30 6) SPAD readings from the same leaf position under a range of 6N rates. On DAT105 most 4th leaves were dead; therefore, the data were not presented. Within a column, data followed by different letters are significantly different at P < 0.05 by Duncan’s multiple range test. Leaf relative positiona
1st CV 2nd CV 3rd CV 4th CV a b
Days after transplantation (DAT) DAT30
DAT40
DAT53
DAT65b
DAT78
DAT89
DAT105
39.07 2.93a 7.50% 41.61 3.45b 8.29% 41.93 3.55b 8.47% 43.11 4.28 c 9.92%
40.45 3.37a 8.34% 41.79 3.57b 8.54% 42.11 3.91bc 9.28% 40.28 4.97a 12.33%
41.16 2.58ab 6.27% 41.46 3.44ab 8.30% 41.87 4.27a 10.19% 40.69 5.34b 13.12%
46.07 3.02a 6.56% 44.13 3.10b 7.02% 43.24 3.74b 8.65% 41.94 4.98c 11.88%
46.01 3.10a 6.73% 43.52 3.22b 7.40% 41.82 3.94c 9.42% 39.54 5.35d 13.54%
44.65 5.74a 12.86% 41.41 6.11b 14.77% 39.73 6.46c 16.27% 35.87 7.14d 19.90%
34.22 8.30a 24.24% 29.12 9.31b 31.98% 28.07 9.21c 32.69% –
Numbered basipetally and corresponding to different plant growth stage. Day that all leaves had already shot and no other new leaves would appear.
Table 2 Chl content (per unit weight and area), SPAD readings, and chl a/b ratios measured during August 19–22 and October 6–10 under different N rates. Mean (standard deviation) of chl content and chl a/b ratios were indicated for 15 leaves with SPAD readings for 30 leaves. Within a column, data followed by different letters are significantly different at P < 0.05 by Duncan’s multiple range test. N rate
Chl content
SPAD reading 1
Per unit weight (mg g
N1 N2 N3 N4 N5 N6
)
Per unit area (mg dm
2
)
August 19–22
October 6–10
August 19–22
October 6–10
1.87 0.19a 2.23 0.21b 2.52 0.22c 2.50 0.13c 2.54 0.12c 2.52 0.12c
1.09 0.72a 1.15 0.56a 1.42 0.38ab 1.60 0.35ab 1.76 0.45b 1.65 0.37b
3.00 0.32a 3.36 0.34b 3.63 0.28c 3.60 0.28c 3.61 0.23c 3.64 0.23c
1.70 1.12a 1.82 0.84a 2.14 0.60ab 2.37 0.53b 2.54 0.60b 2.48 0.51b
Coefficients of variation (CV) in SPAD readings measured at each time increased as leaf aged, which meant that SPAD readings became more and more sensitive to nitrogen rates following leaf aging. On the other hand, N application also prolonged lifespan of leaves. On DAT65, the 10th leaves without receiving any N were tended to die (SPAD readings = 24), but those receiving 375 kg N ha1 were still vigorous (SPAD readings = 42.3). There were totally about 15 leaves on effective stem tillers of Bing 9363. At each plant growth stage, the top 4 leaves acted as functional leaves and undertook main dry matter production. Prior to the panicle initiation, the leaf development from emergence to full size took 3–5 days, whereas it took 6–8 days post the panicle initiation. Correspondingly, a lower leaf was 3–5 or 6–8 days physiologically older than the neighboring leaf above it. Distinction in SPAD readings also existed in different leaf positions, as shown in Table 1, with standard deviation (SD) and CV of readings increasing from 1st to 4th (numbered basipetally) leaf. Starting from August 28 (DAT62) the 15th leaf (the last one) was just fully expanded and no other leaf would develop, so during DAT65 to 105, the leaf position of top 3 or 4 leaves did not vary. During DAT65 to 105, SPAD readings decreased from the upper to lower leaves while SD and CV of SPAD readings in the same leaf position raised (Table 1). 3.4. Chl a/b ratios varied with chl content As revealed in Table 2, chl content per unit weight or area were elevated by increase of N rates, whereas chl a/b ratios were significantly higher when fertilizer N was not or lightly topdressed. The chl a/b ratios were negatively related to chl content (expressed either as per unit weight or area) (Fig. 6A–D) and to SPAD readings (Fig. 6E and F). Nevertheless, linear regression equations generated by data measured during October 6–10 (Fig. 7B, D and F) were poorly fit as compared with the data measured on August 19–22 (Fig. 7A, C and E). Chl a/b ratios
Chl a/b ratio
August 19–22
October 6–10
August 19–22
October 6–10
36.58 2.92a 38.54 2.59b 40.41 1.52c 41.61 1.99d 42.83 2.00e 43.02 1.83e
30.37 9.52a 30.42 9.69a 33.75 4.81b 35.51 4.20bc 37.07 4.48c 36.72 4.48bc
2.69 0.30a 2.34 0.27b 2.09 0.28b 1.71 0.46c 1.49 0.27cd 1.34 0.20d
2.48 0.30a 2.58 0.20a 2.37 0.23ab 2.34 0.28ab 2.11 0.36bc 2.02 0.31c
measured in other 4 rice varieties under higher N rate were smaller too (Fig. 8). 4. Discussion The maximum rate of CO2 assimilation (per unit area), an important component to crop productivity, is almost proportional or somewhat curvilinear to amount of N per unit leaf area (specific leaf N, SLN), so leaf thickness is an important trait related to photosynthetic activity because thicker leaf contain much more chl per unit area (Fig. 2B). Moreover, since N expressed based on per unit area in different varieties could be more precisely estimated by SPAD than based on per unit weight (Peng et al., 1993), inherent difference in chl or N content (based on per unit area) accompanied by diverse leaf thickness (Fig. 2) among different varieties needs to be taken into account fully when SPAD is applied to diagnose plant N status. However, the poor linear regression describing relationships between chl content per unit area and leaf thickness might be due to the differences in chl content per unit weight among different varieties. FSLM was proved to be a proper estimate of leaf thickness as shown in Fig. 3, but regression equations describing relation between leaf thickness and FSLM in two growth stages differed in their intercepts, which might be the consequence of different leaf density (mass per volume) formed at different plant growth stages (Witkowski and Lamont, 1991). In other words, FSLM in different growth stage are incomparable if they are used as surrogates of leaf thickness. Our data demonstrated that higher N supply caused thinner leaves, which would be another inducement to leaf droop. It has been reported that nutrient supply does not affect epidermal thickness and differences in leaf thickness reflects differences in mesophyll thickness (David, 1980). The change in anatomical structure of rice leaf caused by N supply deserves further investigation.
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Fig. 7. Relationship of chl a/b ratios with chl content based on per unit weight (A and B), per unit area (C and D) and SPAD readings (E and F). A, C and E were for data measured during August 19–22 and B, D and F for October 6–10.
Temporal SPAD readings of the same leaves under different N rates indicated that leaf age is an important factor deserving to be checked when SPAD measurements are used to plant N status diagnosis. We infer that the effect of N supply on SPAD readings
would be manifested more and more distinctly as leaf ages, so that CV of SPAD readings measured on the same leaves increased as leaf ages. Therefore, it seems more reasonable to use the lower leaves (physiologically older) than the upper leaves as indicators of plant
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also be given to Mr. Wang Zhumei, retired research fellow at Hangzhou Academy of Agricultural Sciences for his kind guidance to field management. References
Fig. 8. Chl a/b ratios under 2N rates in 4 varieties. NJ42, LYPJ, QY63 and YY9 were abbreviations of Nanjing 42, Liangyoupeijiu, Qianyou 63 and Yongyou 9, and N1 and N2 were 75 and 225 kg N ha1. Each bar was an average of 15 data. Vertical bars represent chl a/ratios.
N status. This conclusion is consistent with previous research (Zhou and Wang, 2003). The inference is further confirmed by temporal SPAD readings of the top 4 leaves (as shown in Table 1): constantly greater CV of SPAD readings measured on the lower leaves, as well as the aged leaves (data after DAT65). One important reason that created the differences in SPAD readings at various leaf position (Table 1) might be associated with the heterogeneous distribution of radiation flux in rice canopy. The distribution of radiation flux could be described as: IL ¼ I0 ekL where I0 and IL are the radiation flux above the canopy and at a point above which there are L layers of leaf, and k is the extinction coefficient (Fitter and Hay, 1981; Murchie et al., 2002). Upper leaves receive greater radiation flux than lower leaves, which result in higher chl a/b ratios, thicker leaves and chl content. Other factors, such as different leaf age and humidity probably also contribute to the differences. The increase in the chl a/b ratios reflects the relative increase in the ratios of [PSII] to [LHCII], and the increase of chl a/b ratios and hence [PSII]/[LHCII] ratios with decrease in [N] under high irradiance were predicted by Hikosaka and Terfashima (1995). Data presented in our paper is consistent with the speculation. A much poorer linear regression equation describing relationship of chl a/b ratios with chl content measured in October 6–10 (Fig. 7A, C and E) might be due to leaf aging and lower light intensity in leaf lifespan (from late August to early November). A decrease in chl a/b ratios has been reported during leaf senescence in rice (Jun et al., 1991), which blurs the relationship between chl content and chl a/ b ratios, together with the effect of lower light intensity. Chl a/b ratios of youthful leaf under high intensity light could be regarded as an indicator revealing rice N status if the leaf receives homogeneous light intensity, because leaf lack of N is characteristic of high chl a/b ratios. However, this report is only preliminary report and more in-depth physiological research should be conducted in the future. Acknowledgments The research was financially supported by National Natural Science Foundation of China (No. 60574046) and Zhejiang Natural Science Foundation (No. Y507005). Our heartfelt thanks should be given to Professor Zhang Jun of Wright University, U.S.A., Dr. Niu Genhua of Texas AgriLife Research and Extension Center at El Paso, U.S.A., for their critical reading of the manuscript, and Professor Wang Shaohua of Nanjing Agricultural University and Mr. Liu Xin of Seed Extension Centre of Zhejiang Province for their kind donation of rice seed. Many thanks should
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