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Synergistic effects of nitrogen and potassium on quantitative limitations to photosynthesis in rice (Oryza sativa L.) Wenfeng Hou, Jinyao Yan, Bálint Jákli, Jianwei Lu, Tao Ren, Rihuan Cong, and Xiaokun Li J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b01135 • Publication Date (Web): 01 May 2018 Downloaded from http://pubs.acs.org on May 4, 2018
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Journal of Agricultural and Food Chemistry
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Synergistic effects of nitrogen and potassium on quantitative
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limitations to photosynthesis in rice (Oryza sativa L.)
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Wenfeng Hou,1 Jinyao Yan,1 Bálint Jákli,2 Jianwei Lu,1 Tao Ren,1 Rihuan Cong,1
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Xiaokun Li*1
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1
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Yangtze River), Ministry of Agriculture/Microelement Research Center/College of
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Resources and Environment, Huazhong Agricultural University, Wuhan 430070,
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China
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2
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Key Laboratory of Arable Land Conservation (Middle and Lower Reaches of
Institute of Applied Plant Nutrition, University of Göttingen, Carl-Sprengel-Weg 1,
37075 Göttingen, Germany
1
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ABSTRACT: The inhibition of the net CO2 assimilation (A) during photosynthesis is
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one of the major physiological effects of both nitrogen (N) and potassium (K)
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deficiencies on rice growth. Whether the reduction in A arises from a limitation in
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either the diffusion and biochemical fixation of CO2 or photochemical energy
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conversion is still debated in relation to N and K deficiencies. In this study, the gas
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exchange parameters of rice under different N and K levels were evaluated, and
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limitations within the photosynthetic carbon capture process were quantified. A was
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increased by 17.3% and 12.1% for the supply of N and K. The suitable N/K ratio
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should be maintained from 1.42 to 1.50. The limitation results indicated that A is
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primarily limited by biochemical process. The stomatal conductance (LS), mesophyll
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conductance (LM) and biochemical (LB) limitations were regulated by 26.6-79.9%,
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24.4-54.1% and 44.1-75.2%, respectively, with the N and K supply.
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KEYWORDS: nitrogen, potassium, rice, photosynthesis, limitation
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1. INTRODUCTION
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The global demand for food is expected to increase by 85% by 2050 relative to 2013,
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implying a need to improve the efficiency of agricultural production.1 Rice (Oryza
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sativa L.) is the staple food for more than 50% of the global population.2 To ensure
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future food security in Asia, Africa and Latin America, the production of rice is of
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central importance.1,3 Improving photosynthetic efficiency is regarded as one of the
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major targets for enhancing yield potentials, because over 90% of the crop biomass is
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derived from the products of photosynthesis.4-6 Photosynthesis is sensitive to the
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availability of essential mineral nutrients.7 Among them, nitrogen (N) and potassium
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(K) are present in the highest quantities within photosynthetic tissues.7-8 A deficiency
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of either N or K eventually results in a substantial decline in photosynthetic carbon
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assimilation.9-11
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More than half of the leaf N is allocated to the photosynthetic apparatus, and
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deficient N concentrations in the photosynthetic tissues limit the net CO2 assimilation
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rate (A). Several mechanisms contribute to the overall decline in A under N deficiency.
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N is a substantial part of the chlorophyll molecule and is therefore involved in the
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photochemical absorbance of light energy that is utilized during photosynthesis.12-13 N
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is crucial for the production of proteins and the regulation of enzyme activity. 14-15 As
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a consequence, N deficiency inhibits the activity of the CO2-fixing enzyme ribulose
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bisphosphate
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accelerates leaf senescence, reducing the effective carbon assimilation period of each
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leaf and therefore the accumulation of photosynthetic products.15, 18-19 Additionally, N
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deficiency is associated with reduced stomatal conductance and an inhibition of the
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leaf area expansion.20
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carboxylase/oxygenase
(Rubisco).16-17
Moreover,
N
deficiency
In contrast to N, K is not covalently bound in organic molecules, but it is the most 3
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abundant cellular cation and therefore mediates plant homeostasis by regulating
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stomatal movement, osmotic adjustment, the electric charge balance and enzyme
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activation.21-22 Furthermore, K is critically involved in the maintenance of
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photosynthesis. It has frequently been shown that the leaf stomatal conductance (gs) is
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reduced when the K concentrations in the leaf tissue are low.23-24, 26 However, recent
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studies indicate that A is not primarily limited by gs, but rather by the reduced
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conductance of the leaf mesophyll to CO2 diffusion (i.e., mesophyll conductance, gm),
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limiting the quantity of CO2 that is available for carboxylation.23-26 In addition to the
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constraints on CO2 diffusion, K deficiency limits the biochemical capacity for
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photosynthesis by reducing the activity of Rubsico.26-28 Additionally, increased
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production rates of reactive oxygen species accelerate chloroplast degradation and
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therefore reduce the overall capacity for photochemical energy conversion, the rate of
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electron transport and eventually carboxylation.23, 26, 29
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Apart from photosynthesis, numerous studies report significant interaction effects
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for N and K on the crop growth and yield.30-31 Although their individual functions in
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photosynthesis are intensively studied, the synergistic effects of N and K on A are still
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not well understood. As described above, A can be limited by CO2 diffusion (gs, gm),
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by the biochemical capacity or by a combination of these factors. Previous studies
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have systematically addressed the individual effects of N and K on photosynthesis,
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and they have quantified the stomatal, mesophyll and biochemical limitations to
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A.23-24, 26 However, the fractional contribution of each individual factor to the overall
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limitation of photosynthesis has not been quantified to date with respect to the
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interactions of N and K nutrition.
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Therefore, to improve the current knowledge of synergistic N and K effects on rice
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photosynthesis, a field experiment was conducted with different combinations of N 4
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and K fertilization to evaluate the combined effects of the N and K tissue status on the
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specific limitations in photosynthesis in a quantitative manner. This knowledge will
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help investigators to develop a better understanding of the mechanisms by which the
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combined use of N and K could have different effects compared with the supply of
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only N or K. In addition, this knowledge will enable better guidance in relation to rice
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photosynthesis and productivity to the supply of N and K. The primary objectives of
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the current study were to (1) evaluate the synergistic effects of N and K on the A of
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rice leaves; (2) quantify the photosynthetic limitation by using the gs, gm and the
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biochemical capacity; and (3) identify the optimal ratio of N to K concentrations in
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the photosynthetically active tissue that will minimize the limitation in A.
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2. MATERIALS AND METHODS
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2.1. Study Site. In 2016, rice (Oryza sativa L. var. Shenliangyou 5814) was
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grown as part of a field experiment that was conducted in Wuxue county, Hubei
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province, central China (30°06'46''N, 115°36'9''E), which is the major production area
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for rice. The average temperature and total precipitation during the rice growing
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season were 27.7 °C and 1118.3 mm, which are typical for a subtropical monsoon
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climate. The plants were grown in a sandy loam soil with the following characteristics:
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clay 16.4%, silt 36.8%, sand 46.8%, pH 5.8 (soil: water = 1: 2.5), organic matter
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content 32.1 g kg-1, total N 1.8 g kg-1, Olsen-P 13.4 mg kg-1, readily available K 44.5
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mg kg-1, slowly available K 302.5 mg kg-1, and cation exchange capacity 4.0 cmol
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kg-1.
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2.2. Experimental Design. The field experiment was designed as a spilt-plot
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experiment with different N and K fertilizer rates. The N treatments were 0 kg N ha-1
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(NL), 180 kg N ha-1 (NM) and 270 kg N ha-1 (NH). The K treatments were 0 kg K2O
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ha-1 (KL), 120 kg K2O ha-1 (KM) and 180 kg K2O ha-1 (KH). The N fertilizer was 5
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applied in three doses, with 50% as basal fertilizer, 25% at the tillering stage and 25%
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at the filling stage. The P fertilizer for all the treatments was applied as basal fertilizer
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in a single dose of 90 kg P2O5 ha-1. The K fertilizer was applied in two doses, with 75%
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as basal fertilizer and 25% at the filling stage. Urea (46% N), superphosphate (12%
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P2O5) and potassium chloride (60% K2O) were used as N, P and K fertilizers. All the
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plots were plowed and leveled after the application of basal fertilizer. The plot area
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was 20 m2 (4 × 5 m). All the possible combinations of the N and K fertilization levels
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were present in the experiment, resulting in a total number of 9 individual treatments,
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with each of them repeated three times.
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2.3. Crop Cultivation. The seeds were sown on May 22 and transplanted on June
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29 with a hill space of (24×15) cm. The plots were separated by soil banks, which
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were covered with plastic film to prevent the exchange of water and fertilizer between
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neighboring plots. The seeds and plants were treated with fungicides, insecticides and
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herbicides to avoid yield losses. No obvious diseases, pests or weeds occurred during
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the rice growing season. The plants were harvested on October 1.
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2.4. Leaf Gas Exchange Measurements. To study the effects of the N and K
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supply on the diffusional and biochemical limitations to CO2 assimilation, the leaf gas
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exchange of flag leaves was measured in the field with a Li-6400XT portable
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photosynthesis system (Li-6400XT, Li-Cor, Inc, Lincoln, USA) at the jointing stage,
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at 65 days after sowing. The measurements were performed during the morning
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(8:00-11:00) of a sunny day. The cuvette conditions were set to 1200 µmol m-2 s-1
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photosynthetic photon flux density (PPFD), 400 µmol m-2 s-1 CO2 in the leaf chamber
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(Ca), 60% relative humidity, and 500 µmol s-1 flow rate. The temperature was not
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controlled, and it was 30.0 °C.
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The CO2 response curves (A-Ci curves, where A is the net photosynthesis rate and 6
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Ci is the CO2 concentration in the leaf internal air space) were measured after the
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leaves had been adapted to the cuvette conditions for twenty minutes. Then, the Ca in
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the gas exchange cuvette was reduced stepwise from 400 to 300, 200, 100 and 50
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µmol m-2 s-1, and then increased again from 50 to 400, 600, 800, 1000, 1200 and 1500.
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The A, Ci and stomatal conductance (gs) were recorded for each step of the CO2
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response curves once a steady value for A was reached. The mesophyll conductance
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(gm), CO2 concentration in the chloroplast stroma (Cc) and the transformation of the
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A-Ci curves into A-Cc curves were computed as described by Sharkey.35 The
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maximum rate of Rubisco-catalyzed carboxylation (Vcmax) and the maximum rate of
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electron transport (Jmax) were calculated as defined by Farquhar, von Caemmerer and
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Berry.36
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Based on the limitation analyses presented by Jones36, the limitations to A were
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separated into the stomatal diffusion (LS), mesophyll diffusion (LM) and biochemical
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capacity (LB).37-38 The limitation of A can be approximated as follows:
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ls =
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lm =
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lb =
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where gtot is the total conductance to CO2 diffusion from ambient air into the
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chloroplast, as defined as follows:
. LS, LM and LB are stomatal,
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mesophyll and biochemical limitations to A, respectively, and ls, lm, and lb are the
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corresponding relative limitations (with values from 0 to 1). All the limitations are
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expressed on the basis of the deviation of the observed A from non-limited A (i.e., the
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average A of the optimal fertilized NHKH treatment, where the total limitation is 0). 7
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2.5.
Determination
of
Rubisco
Enzyme
Activity.
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The
extraction
of
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ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco, EC 4.1.1.39) was
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prepared according to Lu et al.30 Fresh leaves (0.15 g) were ground into powder in a
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chilled mortar with some liquid N2 and quartz sand. The powders were homogenized
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with 4 ml of cool extraction buffer containing 1 mM EDTA, 1% PVP (w/v), 10 mM
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β-mercaptoethanol (v/v), 10 mM MgCl2, 12.5% glycerol (v/v) and 50 mM Tris-HCl
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(pH 7.5) at 4 °C. The homogenates were transferred from the mortar into centrifuge
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tubes and centrifuged for 15 min at 4 °C and 15000 g. The supernatant was
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immediately tested for Rubisco activity using an enzyme-linked immunosorbent assay
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method with a Rubisco assay kit (Beijing Solarbio Science and Technology Co., Ltd,
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China) according to the manufacturer’s instructions.
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2.6. Nitrogen and Potassium Concentration Measurements. Three representative
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hills were sampled from each plot directly after the measurement of the gas exchange.
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The leaves of each plant were cut off and the leaf area was measured using ImageJ
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software.39 The plant samples were desiccated at 105 °C for half an hour and then
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dried at 65 °C for 48 h. The dried samples were weighed and milled. Then, 0.10 g of
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each sample was digested with H2SO4-H2O2.40 The N concentration in the digestion
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solution was determined with a continuous flow analyzer (AA3, Seal Analytical Inc.,
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Southampton, UK), and the K concentration in the digestion solution was determined
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with a flame photometer (M-410, Cole-Parmer, Chicago, USA).
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2.7. Statistical Analysis. An analysis of variance (ANOVA) was calculated using
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SPSS 18.0 (SPSS, Chicago, IL, USA). The differences between the mean values were
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compared using Duncan's multiple range test at a P
