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Influence of nitrogen source and concentrations on wheat growth and production inside “Lunar Palace-1”
Accepted Manuscript Influence of nitrogen source and concentrations on wheat growth and production inside “Lunar Palace-1” Chen Dong, Zhengpei Chu, Minjuan Wang, Youcai Qin, Zhihao Yi, Hong Liu, Yuming Fu PII:
S0094-5765(16)30689-0
DOI:
10.1016/j.actaastro.2017.12.043
Reference:
AA 6623
To appear in:
Acta Astronautica
Received Date: 24 July 2016 Revised Date:
17 December 2017
Accepted Date: 29 December 2017
Please cite this article as: C. Dong, Z. Chu, M. Wang, Y. Qin, Z. Yi, H. Liu, Y. Fu, Influence of nitrogen source and concentrations on wheat growth and production inside “Lunar Palace-1”, Acta Astronautica (2018), doi: 10.1016/j.actaastro.2017.12.043. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT 1
Influence of nitrogen source and concentrations on wheat growth and
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production inside “Lunar Palace-1”
3 a,b,d,e,1
Chen Dong
, Minjuan Wang
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Zhihao Yi a,b,c, Hong Liu a,b,c,∗, Yuming Fu a,b,c,∗
6 a
Beijing 100191, China b
International Joint Research Center of Aerospace Biotechnology&Medical
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Engineering, Beihang University, Beijing 100191, China d
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Higher Education Evaluation Center of the Ministry of Education, Beijing
100081, China e
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College of Information and Electrical Engineering, China Agricultural
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University, 100083, Beijing, China
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Email address: [email protected], [email protected],
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[email protected], [email protected], [email protected]
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These authors contributed equally to this work.
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,
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University, Beijing 100191, China c
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a,b,c
Institute of Environmental Biology and Life Support Technology, Beihang
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, Youcai Qin
School of Biological Science and Medical Engineering, Beihang University,
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e,1
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, Zhengpei Chu
a,b,c,1
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* Corresponding Author:
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Hong Liu:
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Tel: 86-10-82339837 Fax: 86-10-82339837
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E-mail: [email protected]
27
Yuming Fu:
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E-mail: [email protected] 1
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Abstract:
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Minimizing nitrogen (N) consumption and maximizing crop productivity are major challenges to growing plants in Bioregenerative Life Support System
32
(BLSS) for future long-term space mission. Plants cultivated in the controlled
33
environments are sensitive to the low recyclable N (such as from the urine).
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The purpose of this study is to investigate the effects of nitrogen fertilizer
35
(NH4+-N and NO3--N) disturbance on growth, photosynthetic efficiency,
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antioxidant defence systems and biomass yield and quality of wheat (Triticum
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aestivum L.) cultivars during ontogenesis. Experiments were divided into 4
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controlled groups,Ⅰ: NO3--N: NH4+-N=7:1 mmol•L-1; Ⅱ: NO3--N:
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NH4+-N=14:0.5 mmol•L-1; Ⅲ: NO3--N: NH4+-N=7:0.5 mmol•L-1 and CK: NO3--N:
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NH4+-N=14:1 mmol•L-1, and other salt concentrations were the same. The
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results showed that heading and flowering stages in spring wheat are sensitive
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to low N concentration, especially NO3--N in group Ⅰ and Ⅲ. NO3- is better
43
to root growth than to shoot growth. The plants were spindling and the output
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was lower 21.3% when spring wheat was in low N concentration solution.
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Meanwhile, photosynthetic rate of low N concentrations is worse than that of
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CK. The soluble sugar content of the edible part of wheat plants is influenced
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with NO3- : NH4+ ratio. In addition, when N concentration was lowest in group
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Ⅲ, the lignin content decreased to 2.58%, which was more beneficial to
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recycle substances in the processes of the environment regeneration.
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Keywords: NO3- ; NH4+; spring wheat; nutrient solution; biomass yield
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1. Introduction Humanity’s plans to further explore space strongly suggest the
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development of bioregenerative life support systems (BLSS) fully incorporated
60
into space stations, transit vehicles and eventually in habitats on the Moon and
61
Mars [1, 2]. These concepts aim to decrease the (re-)supply mass by
62
(re-)generating essential resources for humans through biological processes.
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Within a BLSS, the cultivation of higher plants takes a crucial role as they can
64
contribute to all major functional aspects (e.g. food production, carbon dioxide
65
reduction, oxygen production, water recycling and waste management)[3]. As
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a primary input source, nutrient solution is one of the most important
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environmental factors for plant growth. In particular, wheat (Triticum aestivum
68
L.), which is a core crop in BLSS [4], is often restricted in growth by nitrogen
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fertilizer disturbance from urine treatment module [5]. Thus, enhancing the
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crop yield/quality and nitrogen fertilizer recycled are matters of interest for
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researchers in both space and urban agriculture settings [6].
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Mineral metabolism plays a significant role in photosynthesis, respiration
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and carbohydrate accumulation of wheat plants. Ammonium N (NH4+-N) and
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nitrate N (NO3--N) are the main inorganic N absorbed and utilized by wheat
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roots. Root uptake of NH4+-N and NO3- is an active process and it can be
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blocked by metabolic inhibitor. As assimilation of NH4+ requires less energy
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than that of NO3- many plants prefer NH4+ as their source of N [7]. Some
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studies have shown that particularly plants growing on acid or waterlogged
79
soils, where NH4+ prevails, prefer NH4+ over NO3- and have high uptake rates
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and vigorous growth when supplied with NH4+ [8]. However, when NH4+ is
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supplied as the exclusive N-source at high concentrations, NH4+ is toxic and
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impairs plant growth [9].
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Ion concentration and transpiration rate have an effect on ion exchange
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properties of roots and ionic interactions within the root apoplasm. Plants 3
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the form of ammonium (NH4+) and nitrate (NO3−), which is fundamental for the
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photosynthesis and respiration process. Considerable variation exists in the
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reported effects of NO3- and NH4+ nutrition on photosynthetic activity. Both
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NO3- and NH4+ nutrition have been severally reported to produce higher
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photosynthetic rates than used alone [10] or to
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photosynthetic
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impacts of different inorganic N concentrations on the growth and development
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of wheat plants in the natural ecosystem [12, 13]. However, little is known
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about the effects of nitrogen fertilizer disturbance on the crops in the artificial
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ecosystem. What effects will have different N forms and concentrations,
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especially those of combined NO3- and NH4+ nutrition, on the growth and
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development of spring wheat plants? And which stage will be more important
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not only suitable to the plants cultivation, but also beneficial to yield and quality?
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For these reasons, it is necessary to investigate the nitrogen availability effects
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on the space agricultural production and evaluate different consequences
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caused by the nitrogen disturbance in the artificial condition.
have
no influence
on
[11]. Previous researches mainly focused on the
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Hydroponic method of growing plants is the biotechnological process of
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obtaining the high-quality crops because it allows rational control of the mineral
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composition through the regulation of their mineral nutrition during ontogenesis
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[14]. Therefore, we cultivated wheat plants in hydroponics and investigated the
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influences of different N forms and concentrations on the wheat growth,
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photosynthetic characteristics, antioxidant capacity, biomass yield and quality
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during their life cycle.
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2 Materials and methods
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2.1 Plant material and cultivation conditions
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Spring wheat plants (Triticum aestivum L) were planted in plant cabin of
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“Lunar Palace-1” [4]. The porous tube nutrient delivery system with water 4
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seeds per m2. The growth period of the wheat was 70 days. For all treatments
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lighting was continuous (24/0 h light/dark). Photosynthetic photon flux density
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(PPFD) levels were measured daily at the top of plant canopy with a quantum
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sensor (Li-250A, Li-Cor, USA). PPFD was about 500 µmol·m–2·s–1 for all the
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treatments. The relative humidity was maintained at 55 ± 4.6%, with a
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temperature of 21 ± 1.3 ℃ during daytime and night. The modified Hoagland
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nutrient solution was the basic culture medium. During the whole life cycle of
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wheat plants, different N supplies were designed and listed in Table 1.
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2.2 Morphological and physiological analyses
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2.2.1 Morphology
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The height and root length of wheat plants were measured every two days
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by straight scale and vernier caliper. Ten samples of those wheat plants were
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selected randomly when the measurement was in process[17], Wheat plants
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state was analyzed as precisely as possible[18].
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2.2.2 Determination of relative water content (RWC)
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At vegetative growth stage, heading stage, flowering stage and maturity
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stage, the RWCs of leaves were separately measured [19]. Samples were
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excised from the leaves of ten wheat plants at the second leaf at the terminal
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bud for each treatment. The fresh leaves were weighted about 0.5 g (m1) and
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soaked in double distilled water at room temperature for 4 h. Then the leaves
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were weighted as m2 and put in the drying oven (65 ℃) for 48 h. The dried
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leaves were expressed as m3. RWC was calculated as according to the
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following equation:
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m1 − m3 × 100% m2 − m3
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RWC =
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2.2.3 Determination of membrane stability index (MSI)
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Samples were excised from the leaves of ten wheat plants at the second leaf at the terminal bud for each treatment. To measure the MSI of leaves at
141
different stages [20], the sample was divided into two equivalent parts (about
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0.1g for each) and soaked in 10 mL double distilled water. Then one part was
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heated at 40 ℃ for 30 min. Conductivity C1 was determined by conductivity
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meter (HI8733, Hanna instruments, Italy). The other part was heated at 100 ℃
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for 10 min, and conductivity C2 was determined. MSI was calculated as the
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following:
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2.3 Stomata observation
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Samples were excised from the leaves of ten wheat plants at a similar
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position for each treatment at vegetative growth stage. To observe the stomata,
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samples were taken from fully expanded leaves in each plant. The slides made
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by the leaf epidermal fingerprint of cotton with the transparent nail polish
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method were observed using an optical microscope [21]. Slides were analyzed
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with an Olympus DP71 microscope (Olympus Inc., Japan). The length, width
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and frequency of stomata were measured with Motic Images Plus 2.0. 10
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images per leaf, one leaf per plant and 10 plants per treatment were analyzed.
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2.4 Photosynthetic characteristics analyses
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2.4.1 Chlorophyll contents
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Samples were excised from the leaves of ten wheat plants at the second
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leaf at the terminal bud for each treatment. The content of chlorophyll a and
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chlorophyll b was measured with an ultraviolet spectrophotometer (SP-75,
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Shanghai spectrum instruments co., LTD, China) at vegetative growth stage,
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heading stage, flowering stage and maturity stage, respectively [22]. Leaf
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samples were frozen in liquid nitrogen and stored at -80 °C until measured.
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2.4.2 Photosynthetic efficiency 6
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instrument (Li-6400XT, Li-Cor, USA) was used for the determination of
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photosynthetic characteristics. Leaf gas-exchange parameters included
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photosynthetic rate (A), stomatal conductance (gs) and and intercellular CO2
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concentration (Ci) using the second leaf at the wheat terminal bud. Water use
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efficiencies (A/gs) were calculated by dividing A by gs and the instantaneous
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carboxylation efficiencies (A/Ci) were also calculated [23].
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2.5 Antioxidant capacity analyses
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2.5.1 Peroxidase (POD) activity
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POD activity during vegetative growth stage, heading stage, flowering
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stage and maturity stage was analyzed spectrophotometrically at 470 nm
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using guaiacol as a phenolic substrate with hydrogen peroxide [24]. Samples
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were excised from the leaves of ten wheat plants at the second leaf at the
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terminal bud for each treatment. The reaction mixture contained 0.15 mL of 4%
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(v/v) guaiacol, 0.15 mL of 1% (v/v) H2O2, 2.66 mL of 0.1 M phosphate buffer
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(pH= 7.0) and 40 µL of enzyme extract. Blank sample contained the same
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mixture without enzyme extract.
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2.5.2 Catalase (CAT) activity
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CAT activity was determined according to the method described by Kumar
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and Knowles [25]. Samples were excised from the leaves of ten wheat plants
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at the second leaf at the terminal bud for each treatment. CAT reaction solution
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consisted of 100 mM Na2HPO4-NaH2PO4 buffer solution (pH=7.0) and 0.1 M
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H2O2. The optical density was determined every 1 min at λ=240 nm.
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2.5.3 Malonaldehyde (MDA) content
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Samples were excised from the leaves of ten wheat plants at the second
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leaf at the terminal bud for each treatment. Determination of MDA depended
192
on the method of Stewart and Bewley [26]. Briefly, at vegetative growth stage,
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heading stage, flowering stage and maturity stage, 10 mL 0.1% trichloroacetic 7
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4000 rpm for 10 min. 2 mL supernatant was added to 4 mL 5% thiobarbituric
196
acid (TBA) which was made up by 20% TCA. The mixture was heated at 95 °C
197
for 30 min and then cooled in ice-bath rapidly. The supernatant was obtained
198
by centrifuging at 3000 rpm for 10 min. The absorbency of the supernatant was
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recorded at 532 nm. The value for non-specific absorption at 600 nm was
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subtracted. The MDA content was calculated using its extinction coefficient of
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155 mM-1 cm-1.
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2.6 Biomass yield analyses
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2.6.1 Edible biomass
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The thousand-kernel weight (TKW) of wheat seeds was weighed
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respectively under 4 different treatments. At maturity, above-ground biomass
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(AGB) and grain yield per plant (GY) were also recorded. Harvest index (HI =
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GY/AGB) was then calculated.
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2.6.2 Inedible biomass
209
For determination of inedible biomass components, plant tissues were
210
dried in an oven for 48 h at 70 °C before weighing. The content of Neutral
211
detergent solution (NDS), neutral detergent fiber (NDF), acid detergent fiber
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(ADF), acid detergent lignin (ADL) and acid-insoluble ash (Ash) in wheat straw
213
was determined according to Van Soest et al. method [27] using FIWE six raw
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fiber extractor (VelpScientifica, Italy). The contents of hemicellulose, cellulose,
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lignin, ash and NDS were analyzed.
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2.7 Data statistics
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The experiment was setup in a completely randomized design. All
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experiments were performed in triplicate. The average value of total 6
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measurements ± standard deviation was regarded as the final result. All
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statistical analyses were performed using SPSS 18.0. Comparison among
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means performed at significance level P=0.05. 8
ACCEPTED MANUSCRIPT 3. Results
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3.1 The response of wheat growth to different treatments
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It was found that the morphology of the wheat plants was noteworthily
225
influenced under different N forms and concentrations of different growth
226
periods (Fig 1A). Particularly at the heading stage of seedling, straw height of
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wheat under low concentrations was 5-10 cm greater than CK. However, after
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that, the growth momentum of wheat plants under low concentrations had
229
been restricted, which might probably due to the adaptation to low
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concentration. The most obvious influence of different N forms and
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concentrations on RWC of wheat leaves happened when wheat heading (Fig
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1B). With the condition of Ⅰ, Ⅱ and CK, the RWC was the higher at heading
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stage wherein the transpiration strengthened and the plant growth was
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vigorous. However, the more RWC existed in leaves, the less reflectivity of
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leaves was, which would affect the optical property. At flowering stage, RWC
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of Ⅲ group plants was at the lowest level of 79% and it is 7% lower than the
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value for CK. The RWC occurred lower, which was more beneficial for
238
accumulating energy to perform self-pollination. Therefore, photosynthesis,
239
transpiration and water-use efficiency are strongly linked to the water regime of
240
plants. MSI gradually reduced during the development and growth of wheat
241
plants (Fig 1C). The largest difference happened at vegetative growth stage
242
and flowering stage. The highest CK value reached 73.2% and the lowest Ⅲ
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group value was 70.1% at vegetative growth stage. The gap was larger when
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flowering, the MSI of Ⅲ group was 4.9% lower than that in CK. But significant
245
difference disappeared at the maturity stage at last.
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At vegetative growth stage, the size of wheat leaf stomata was small due
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to NO3--N decreased(Fig 2A), and the length of epidermal cells between
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stomata was getting longer, with the tendency of growth elongation (Fig 2C),
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leading to the reduction of CO2 assimilation efficiency when photosynthesis 9
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was on. The epidermal stomatal density in the Ⅲ group was sharply lower
251
than that in other conditions. These observations demonstrate that different N
252
concentrations and forms impact differently the morphology and the
253
distribution of epidermal cells and stomata of wheat leaves. Transpiration is the main driving force of nutrient transportation. Therefore,
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inorganic N changes the transport of nutrients, which promotes the absorption
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of nutrients by plant roots. In addition, the selectivity of plants to exogenous
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nutrient ions decreases with the increase in their concentrations; thus, higher
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NH4+ and NO3- concentrations result in more significant promoting effects of
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increased transpiration on ion uptake rate. However, plants gradually adapt to
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external environment (such as reducing stomatal aperture and the number and
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distribution of stomata), and the degree of the promoting effect of increased
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transpiration on nutrient uptake may also decrease gradually.
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3.2 The response of photosynthetic characteristics to different treatments
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Chlorophyll a concentrations increased as N concentrations increased
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during the whole life cycle (Fig 3A). However, the chlorophyll b concentration
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was opposite, especially at the heading and flowering stages (Fig 3B).
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Chlorophyll b contents of Ⅲ group at the heading and flowering stages were
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0.81 and 1.02 mg/g, respectively. And chlorophyll a/b ratio of leaves in low
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concentration treatments is considerably below the control level (Fig 3C).
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During flowering, the relative content of chlorophyll of all the samples gradually
271
reached the peak. Our results showed that low N concentrations tended to
272
increase the chlorophyll b content in wheat leaves during heading and
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flowering, and then the ratio chlorophyll a/b decreased. Starting from heading,
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the total content of chlorophyll (especially Chl a) changed significantly, with the
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level of Ⅱ and CK treatments was higher than that of Ⅲ group. When the
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NO3- : NH4+ ratios were both 14:1 (Ⅲ and CK), during flowering, chlorophyll b
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production was 1.02 and 0.89 mg·g-1, respectively. This suggests that total N
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wheat plants. The NO3- : NH4+ ratio is secondary for the chlorophyll production.
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Wheat plants sense and respond to low N concentrations through
281
changed photosynthesis (A) and stomatal conductance (gs) (Fig 4A-B). Both A
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and gs maximized at the heading stage and then decreased later in senescent
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leaves. Photosynthetic rate had a significant difference from the heading stage
284
to the flowering stage, which means the two growth stages are more sensitive
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than other stages during the wheat life cycle. At the heading stage,
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photosynthetic rate of Ⅲ group was 12.57% lower than CK, and then the
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distance increased to 21.12% at the flowering stage. In particular, the increase
288
in A at the vegetative growth stage, was smaller than the decrease in gs, so
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A/gs increased (Fig 4C), allowing the wheat plants to use water more
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efficiently. However, the instantaneous carboxylation efficiency (A/Ci) of Ⅲ
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group was at lowest level across the entire life cycle (Fig 4D).
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3.3 The response of antioxidant system to different treatments
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We also studied the activity of the antioxidant enzymes POD and CAT and
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the production of MDA in wheat leaves during ontogenesis. POD and CAT
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activities increased during early leaf development, reaching their maximal
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levels at flowering stage and decreasing later in senescent leaves. Also, these
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antioxidant enzymatic activities were higher in the plants grown under low N
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concentrations condition throughout leaf development (Fig. 5A-B). NH4+-N and
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NO3--N both are plant-needed N source. Either of NH4+ and NO3- can be well
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absorbed by wheat plants. As shown in Fig.5C, MDA production increased
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with leaf aging, especially under low NO3--N condition, suggesting that NO3--N
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may play an important role in regulating leaf senescence in wheat plants by
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increasing reactive oxygen species (ROS) production. Alternatively, lower
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ROS production may decrease the incidence of oxidative stress, translating as
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a reduced stimulus for antioxidant production. Such reduced oxidative
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may translate into greater CO2/O2 ratio in the chloroplast, causing lower
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oxygenation rates by Rubisco and therefore lower rates of photorespiration
309
and associated ROS production in wheat cultivar. However, the mechanism
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needs to be further investigated. Moreover, responses of plant organs to
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environmental factors should be different during different growth stages. Plants
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have adaptability to environmental changes.
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3.4 The responses of biomass yield and quality to different treatments
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The analysis of variance revealed significant differences among
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treatments for wheat yields per square meter (Fig 6A), harvest index (HI) (Fig
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6B) and thousand-kernel weight (TKW) of wheat seeds (Fig 6C). It was found
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that there was no significant difference among Ⅱ (1830.8 g/m2) and CK
318
(1899.6 g/m2) treatments on yields per square meter, but the yield of Ⅲ
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(1493.4 g/m2) treatment was lower (Fig 6A). That is to say, the growth and
320
development of wheat plants had been affected by N concentration in the
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solution, especially NO3--N. Similarly, TKW and HI in Ⅰ , Ⅱ and CK
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treatments were larger than that in the Ⅲ treatment (Fig 6B-C).
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The results of impact of different N forms and concentrations on the dry
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weight of each part of wheat plants showed that NH4+ nutrition was conductive
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to the growth of overground part in wheat plants, including the thickening of
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stem, the increase of overground biomass (Table 2). Adding more NO3- was
327
beneficial for the development and growth of crop root system. In more NO3-
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concentration, wheat seeding was easy to form roots and the root system was
329
strong. The underground part of biomass was increased by 1.14% compared
330
with that in CK treatment.
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Nitrogen deficiency has many impacts on the root system development:
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although total weight decreases, the plant preferentially allocates biomass to
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the root system so that the root/shoot ratio increases. It is well known that NH4+ 12
ACCEPTED MANUSCRIPT nutrition results in the acidification of the root environment, in these
335
experiments the pH of the hydroponic solutions was carefully controlled to
336
exclude this factor as an influence on plant growth. Changes in the proportion
337
of plant dry mass found in the shoot and root components can only be
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attributed to alter partitioning of carbon within the plant. The roots of wheat
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plants are particularly sensitive to the form of nitrogen nutrition resulting in
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large differences in the shoot/root ratios. The lower shoot/root ratios of wheat
341
plants supported in Table 2 that in general NO3- is better to root growth than to
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shoot growth. The soluble sugar content of the edible part of wheat plants
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decreased with NO3- : NH4+ ratio such as in Ⅱ or Ⅲ treatments, but the
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accumulation of carbohydrate has no significant difference (Table 3). Since
345
nitrogen
346
may
347
acquisition by plants is the result of two processes: root system development
348
from which depends soil colonization, and nitrate uptake capacity of the root
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system.
is translocated the
to a large extent
movement
of
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the
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It is noteworthy that inedible biomass also plays one of the most important
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roles in the ecosystem, which means the compositions of biomass may have a
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significant impact on biological cycle degradation. To investigate the influence
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of different conditions on inedible part of wheat plants, we determined the
354
contents of lignin, cellulose and hemicelluloses (Fig 7). The results showed
355
that the higher N concentration promoted the increase of lignin content, with
356
maximal mass fraction of 3.99% (CK). However, the lignin content in group Ⅲ
357
decreased to 2.58% with N concentration lower, which would be helpful for
358
wheat straw degradation. ,
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4. Discussion
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At vegetative growth stage, weak N supply reduced the mean realized
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photosynthetic rate. Besides, more biomass seems to be allocated to the 13
ACCEPTED MANUSCRIPT overground portion for stem growth so that catch up the growth of leaves and
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the full absorption of limited energy to meet the demand for plant
364
photosynthesis (Fig 1A, Table 2). At the same time, leaf temperature was also
365
dropped down because of the lower RWC and MSI to meet the requirement of
366
plant growth. The difference of biomass accumulation and biomass allocation
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in organs of wheat plants under different N forms was integrated with
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performance of efficiency utilization of environmental N resources.
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Pigments are integrally related to the physiological function of leaves.
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Chlorophylls absorb light energy and transfer it into the photosynthetic
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apparatus and carotenoids can also contribute energy to the photosystem [28],
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which finally effect plant growth and yield. It is beneficial for Chlorophyll b to
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trap scattered light with short wavelength to harness its quantum properties
374
effectively [29]. Previous study indicated that photosystems I and II of the
375
plants
376
chlorophyll b than the corresponding photosystems of spinach [30]. The shade
377
plant chloroplasts contained very large grana stacks and the extent of grana
378
formation in higher plant chloroplasts appears to be related to the total
379
chlorophyll content of the chloroplast. The effects of the form and
380
concentration of the nutrient nitrogen on photosynthetic carbon dioxide
381
assimilation may arise from the influence of the nitrogen source on
382
photosynthetic enzymes，photorespiration，stomatal conductance or other
383
aspects of photosynthetic metabolism.
light
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had
lower
proportions
of
chlorophyll a to
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Stimulation of photosynthesis is the driving force for increased growth and
385
yield of wheat in Ⅰ, Ⅱ and CK groups. However, at the heading and
386
flowering stage, the effect of low N concentrations became more significant,
387
which may be because the stages from vegetative growth to reproductive
388
growth are more sensitive. Ion sensing is an intrinsic property of guard cells,
389
which are thought to be sensitive to the intercellular carbon dioxide 14
ACCEPTED MANUSCRIPT concentration (Ci) rather than CO2 concentration at the wheat leaf surface. Ion
391
and organic solute concentrations mediate the turgor pressure in the guard
392
cells that determines stomatal aperture (Fig 2). Stomata closure implies lower
393
CO2 availability, and ultimately lower CO2 fixation by Rubisco, which may
394
finally result in lower biomass production [31]. The uptake of NH4+ and NO3- is
395
an active process, which is energy-related and can be blocked by metabolic
396
inhibitor. Plant up take of ions needs energy provided by respiratory
397
metabolism, and the energy required for uptake of NO3- is higher than that for
398
uptake of NH4+; therefore, many plants tend to utilize NH4+ [7]. More,
399
differences between NO3- and NH4+-fed plants with respect to photosynthetic
400
rates may be due to effects of the form of nitrogen on the activity of the
401
photosynthetic enzymes.
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Currently, it is extensively accepted that reactive oxygen species are
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responsible for kinds of damage (stress-induced) of macro-molecules, finally of
404
the cellular structure [32]. Physiological and genetic evidence clearly indicates
405
that the reactive oxygen intermediate scavenging systems of plants are an
406
important component of the stress protective mechanism [20]. As for the
407
response of antioxidant enzyme to different N concentrations conditions, the
408
level of POD and CAT in wheat plants was an important index of ageing and
409
death. At the flowering stage, the response of CAT activity to different
410
treatments was quite sensitive (Fig.5B). The reason may be that the
411
expression of wheat enzyme genes is promoted more during the flowering
412
stage, ensuring a good growth of plants. Meanwhile, it also indicates that there
413
might be complementary and / or additive effect in different treatments. The
414
level of these enzymes reflects the situation of physiological activity of plants.
415
POD has been proven to have the function of oxidase IAA [33]. Low level of
416
POD promoted the growth of overground part of plants, especially for the
417
elongation growth. POD also could prevent the toxic levels of internal
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of reactive oxygen. Moreover, the activities of POD and CAT were related to
420
plant senescence. With the senescence of plants, the activity of them dropped
421
very fast. As a product of membrane lipid peroxidation, MDA is used to assess
422
the extent of oxidative stress in plants, and its level is increased under stress
423
conditions. ROS such as O2•− and endogenous H2O2 can be overproduced in
424
plants under stress conditions and will thereby increase MDA content [34].
425
These highly reactive ROS could alter normal cellular metabolism by oxidative
426
damage to nucleic acids, proteins and lipids [35]. Plants should maintain
427
efficient antioxidant defense system for opposing the oxidative damage.
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The relationship between total biomass and harvestable yield is very close.
429
From the ecological view, the differences are reflected in the range of
430
individual size under the 4 conditions. HI is the primary result of biomass
431
production, but not generally determined in a direct way. The nutrient uptake in
432
wheat aboveground and belowground biomass varied widely among the
433
different N forms and concentration treatments. Biomass/yield increased
434
linearly with increasing N availability then after a certain level of N availability,
435
there is no further increase when more N is available [36]. Wheat N uptake has
436
been found strongly correlated to shoot biomass (Table 2). The faster
437
above-ground growth is often associated with a faster accumulation of root
438
biomass, root length and surface area; traits which could be expected to
439
enhance the capacity of wheat to capture NO3 before it is leached, whether
440
through a capacity to explore deeper soil layers or greater root length density.
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These findings are useful for designing and operating growth environments for
442
enhanced crop production.
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5. Conclusion
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Our results clearly demonstrate that, N forms and concentrations are very
445
significant factors that affect spring wheat growth. At heading and flowering 16
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447
photosynthetic rate and transpiration rate, and enhanced water used efficiency.
448
The plants were spindling and the output was very low when wheat was in low
449
N concentration solution. Meanwhile, some photosynthetic characteristics of
450
plants at low N concentrations are worse than that of control. The soluble
451
sugar content of the edible part of wheat plants is influenced with NO3- : NH4+
452
ratio. In addition, when N concentration was lower, the lignin content
453
decreased, which would be helpful for wheat straw degradation. It also offers
454
new thoughts about straw degradation.
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Acknowledgements
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This work was financially supported by Defense Industrial Technology
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Development Program (JCKY2016601C010).
458 459
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ACCEPTED MANUSCRIPT Table 1：Major nutrient solution parameters of different treatments -
+
NO3 -- KNO3
NH4 --NH4H2PO4
NO3 :
（mmol•L ）
（mmol•L ）
NH4
Ⅰ
7
1
7:1
8
Ⅱ
14
0.5
28:1
14.5
Ⅲ
7
0.5
14:1
7.5
CK
14
1
14:1
15
-1
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Total N （mmol•L ）
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ACCEPTED MANUSCRIPT Table 2 The proportion of different parts of wheat plants (DW, %) Treatment
Root 1 2
Ⅰ Ⅱ Ⅲ CK
7.80±0.56 b 9.13±0.96a 7.23±0.69b 7.99±0.19b
Leaf
Stem
Spike
8.27±0.68a 8.29±0.78a 8.28±0.63a 8.30±0.41a
27.19±2.75b 25.64±1.98c 30.29±2.35a 26.66±1.56bc
56.74±4.78a 56.94±4.56a 54.44±4.74b 57.05±4.65a
1
Mean±SE. Mean values with the same letter were not significantly different, based on ANOVA followed by Tukey’s test at P≤0.05.
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ACCEPTED MANUSCRIPT Table 3 The contents of nutrients of wheat seed in different treatments （g/100g） Treatment
Soluble sugar Carbohydrate Rough protein Rough fat Ash NDS Hemicellulose Cellulose Lignin
Ⅰ 1 2
8.15±0.19 a 72.09±3.95a 21.78±1.79a 2.09±0.21a 0.61±0.04a 34.35±3.45a 3.12±0.25b 4.02±0.13b 0.79±0.05b
Ⅱ
Ⅲ
CK
7.35±0.09b 71.27±3.17a 22.03±1.89a 2.05±0.39a 0.33±0.09b 33.46±3.21a 3.52±0.33ab 3.19±0.24c 0.49±0.06c
7.23±0.17b 69.99±5.16a 22.04±1.98a 2.11±0.49a 0.63±0.15a 34.71±3.78a 3.01±0.29b 4.04±0.23b 1.18±0.06a
8.17±0.13a 73.87±4.64a 21.97±1.12a 2.01±0.44a 0.31±0.11b 32.78±2.56a 3.71±0.21a 4.97±0.09a 0.52±0.07c
1
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Mean±SE. Mean values with the same letter were not significantly different, based on ANOVA followed by Tukey’s test at P≤0.05.
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Fig 1：Response of straw height of wheat plants (A), relative water content
(B), membrane stability index (C) of leaves in wheat plants at different stages of ontogenesis to different treatments. Vertical bars are means ± SD. Different lowercase letters indicates a significant difference at p=0.05 between the means.
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Fig 2: Effects of different treatments on wheat leaf stomata. A (Ⅰ), B (Ⅱ),
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Fig 3: Response of chlorophyll a (A) and chlorophyll b (B) and chlorophyll
ratio (C) of wheat leaves at different stages of ontogenesis to different treatments. Vertical bars are means ± SD. Different lowercase letters indicates a significant difference at p=0.05 between the means.
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Fig 4: Response of photosynthetic rate (A), stomatal conductance (B), A/gs (C) and instantaneous carboxylation efficiency (D) of wheat leaves at
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Fig 5: Response of POD activity (A), CAT activity (B) and MDA content (C) of wheat leaves at different stages of ontogenesis to different treatments. Vertical bars are means ± SD. Different lowercase letters indicates a significant difference at p=0.05 between the means.
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Fig 6: Response of unit yield (A), harvest index (B) and thousand-kernel weight (C) of wheat plants to to different treatments. Vertical bars are means ± SD. Different lowercase letters indicates a significant difference at p=0.05 between the means.
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Fig 7: Components of inedible biomass in different treatments (DW, %).
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ACCEPTED MANUSCRIPT ► NO3- and NH4+ concentration and ratio have significant effects on wheat root and shoot growth.
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► Soluble sugar content of wheat edible part is affected by NO3- : NH4+ ratio.
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► When heading and flowering, wheat is sensitive to N concentration.
S0094-5765(16)30689-0
DOI:
10.1016/j.actaastro.2017.12.043
Reference:
AA 6623
To appear in:
Acta Astronautica
Received Date: 24 July 2016 Revised Date:
17 December 2017
Accepted Date: 29 December 2017
Please cite this article as: C. Dong, Z. Chu, M. Wang, Y. Qin, Z. Yi, H. Liu, Y. Fu, Influence of nitrogen source and concentrations on wheat growth and production inside “Lunar Palace-1”, Acta Astronautica (2018), doi: 10.1016/j.actaastro.2017.12.043. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT 1
Influence of nitrogen source and concentrations on wheat growth and
2
production inside “Lunar Palace-1”
3 a,b,d,e,1
Chen Dong
, Minjuan Wang
5
Zhihao Yi a,b,c, Hong Liu a,b,c,∗, Yuming Fu a,b,c,∗
6 a
Beijing 100191, China b
International Joint Research Center of Aerospace Biotechnology&Medical
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Engineering, Beihang University, Beijing 100191, China d
13 14
Higher Education Evaluation Center of the Ministry of Education, Beijing
100081, China e
16
College of Information and Electrical Engineering, China Agricultural
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15
University, 100083, Beijing, China
17
Email address: [email protected], [email protected],
19
[email protected], [email protected], [email protected]
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18
20
22
These authors contributed equally to this work.
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1
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,
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University, Beijing 100191, China c
11
a,b,c
Institute of Environmental Biology and Life Support Technology, Beihang
9 10
, Youcai Qin
School of Biological Science and Medical Engineering, Beihang University,
7 8
e,1
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, Zhengpei Chu
a,b,c,1
4
23
* Corresponding Author:
24
Hong Liu:
25
Tel: 86-10-82339837 Fax: 86-10-82339837
26
E-mail: [email protected]
27
Yuming Fu:
28
E-mail: [email protected] 1
ACCEPTED MANUSCRIPT 29
Abstract:
30
Minimizing nitrogen (N) consumption and maximizing crop productivity are major challenges to growing plants in Bioregenerative Life Support System
32
(BLSS) for future long-term space mission. Plants cultivated in the controlled
33
environments are sensitive to the low recyclable N (such as from the urine).
34
The purpose of this study is to investigate the effects of nitrogen fertilizer
35
(NH4+-N and NO3--N) disturbance on growth, photosynthetic efficiency,
36
antioxidant defence systems and biomass yield and quality of wheat (Triticum
37
aestivum L.) cultivars during ontogenesis. Experiments were divided into 4
38
controlled groups,Ⅰ: NO3--N: NH4+-N=7:1 mmol•L-1; Ⅱ: NO3--N:
39
NH4+-N=14:0.5 mmol•L-1; Ⅲ: NO3--N: NH4+-N=7:0.5 mmol•L-1 and CK: NO3--N:
40
NH4+-N=14:1 mmol•L-1, and other salt concentrations were the same. The
41
results showed that heading and flowering stages in spring wheat are sensitive
42
to low N concentration, especially NO3--N in group Ⅰ and Ⅲ. NO3- is better
43
to root growth than to shoot growth. The plants were spindling and the output
44
was lower 21.3% when spring wheat was in low N concentration solution.
45
Meanwhile, photosynthetic rate of low N concentrations is worse than that of
46
CK. The soluble sugar content of the edible part of wheat plants is influenced
47
with NO3- : NH4+ ratio. In addition, when N concentration was lowest in group
48
Ⅲ, the lignin content decreased to 2.58%, which was more beneficial to
49
recycle substances in the processes of the environment regeneration.
51
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Keywords: NO3- ; NH4+; spring wheat; nutrient solution; biomass yield
52 53 54 55 56 2
ACCEPTED MANUSCRIPT 57
1. Introduction Humanity’s plans to further explore space strongly suggest the
59
development of bioregenerative life support systems (BLSS) fully incorporated
60
into space stations, transit vehicles and eventually in habitats on the Moon and
61
Mars [1, 2]. These concepts aim to decrease the (re-)supply mass by
62
(re-)generating essential resources for humans through biological processes.
63
Within a BLSS, the cultivation of higher plants takes a crucial role as they can
64
contribute to all major functional aspects (e.g. food production, carbon dioxide
65
reduction, oxygen production, water recycling and waste management)[3]. As
66
a primary input source, nutrient solution is one of the most important
67
environmental factors for plant growth. In particular, wheat (Triticum aestivum
68
L.), which is a core crop in BLSS [4], is often restricted in growth by nitrogen
69
fertilizer disturbance from urine treatment module [5]. Thus, enhancing the
70
crop yield/quality and nitrogen fertilizer recycled are matters of interest for
71
researchers in both space and urban agriculture settings [6].
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Mineral metabolism plays a significant role in photosynthesis, respiration
73
and carbohydrate accumulation of wheat plants. Ammonium N (NH4+-N) and
74
nitrate N (NO3--N) are the main inorganic N absorbed and utilized by wheat
75
roots. Root uptake of NH4+-N and NO3- is an active process and it can be
76
blocked by metabolic inhibitor. As assimilation of NH4+ requires less energy
77
than that of NO3- many plants prefer NH4+ as their source of N [7]. Some
78
studies have shown that particularly plants growing on acid or waterlogged
79
soils, where NH4+ prevails, prefer NH4+ over NO3- and have high uptake rates
80
and vigorous growth when supplied with NH4+ [8]. However, when NH4+ is
81
supplied as the exclusive N-source at high concentrations, NH4+ is toxic and
82
impairs plant growth [9].
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Ion concentration and transpiration rate have an effect on ion exchange
84
properties of roots and ionic interactions within the root apoplasm. Plants 3
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86
the form of ammonium (NH4+) and nitrate (NO3−), which is fundamental for the
87
photosynthesis and respiration process. Considerable variation exists in the
88
reported effects of NO3- and NH4+ nutrition on photosynthetic activity. Both
89
NO3- and NH4+ nutrition have been severally reported to produce higher
90
photosynthetic rates than used alone [10] or to
91
photosynthetic
92
impacts of different inorganic N concentrations on the growth and development
93
of wheat plants in the natural ecosystem [12, 13]. However, little is known
94
about the effects of nitrogen fertilizer disturbance on the crops in the artificial
95
ecosystem. What effects will have different N forms and concentrations,
96
especially those of combined NO3- and NH4+ nutrition, on the growth and
97
development of spring wheat plants? And which stage will be more important
98
not only suitable to the plants cultivation, but also beneficial to yield and quality?
99
For these reasons, it is necessary to investigate the nitrogen availability effects
100
on the space agricultural production and evaluate different consequences
101
caused by the nitrogen disturbance in the artificial condition.
have
no influence
on
[11]. Previous researches mainly focused on the
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Hydroponic method of growing plants is the biotechnological process of
103
obtaining the high-quality crops because it allows rational control of the mineral
104
composition through the regulation of their mineral nutrition during ontogenesis
105
[14]. Therefore, we cultivated wheat plants in hydroponics and investigated the
106
influences of different N forms and concentrations on the wheat growth,
107
photosynthetic characteristics, antioxidant capacity, biomass yield and quality
108
during their life cycle.
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2 Materials and methods
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2.1 Plant material and cultivation conditions
111
Spring wheat plants (Triticum aestivum L) were planted in plant cabin of
112
“Lunar Palace-1” [4]. The porous tube nutrient delivery system with water 4
ACCEPTED MANUSCRIPT supply on demand was used [15, 16]. The wheat planting density was 800
114
seeds per m2. The growth period of the wheat was 70 days. For all treatments
115
lighting was continuous (24/0 h light/dark). Photosynthetic photon flux density
116
(PPFD) levels were measured daily at the top of plant canopy with a quantum
117
sensor (Li-250A, Li-Cor, USA). PPFD was about 500 µmol·m–2·s–1 for all the
118
treatments. The relative humidity was maintained at 55 ± 4.6%, with a
119
temperature of 21 ± 1.3 ℃ during daytime and night. The modified Hoagland
120
nutrient solution was the basic culture medium. During the whole life cycle of
121
wheat plants, different N supplies were designed and listed in Table 1.
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2.2 Morphological and physiological analyses
123
2.2.1 Morphology
124
The height and root length of wheat plants were measured every two days
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by straight scale and vernier caliper. Ten samples of those wheat plants were
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selected randomly when the measurement was in process[17], Wheat plants
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state was analyzed as precisely as possible[18].
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2.2.2 Determination of relative water content (RWC)
129
At vegetative growth stage, heading stage, flowering stage and maturity
130
stage, the RWCs of leaves were separately measured [19]. Samples were
131
excised from the leaves of ten wheat plants at the second leaf at the terminal
132
bud for each treatment. The fresh leaves were weighted about 0.5 g (m1) and
133
soaked in double distilled water at room temperature for 4 h. Then the leaves
134
were weighted as m2 and put in the drying oven (65 ℃) for 48 h. The dried
135
leaves were expressed as m3. RWC was calculated as according to the
136
following equation:
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m1 − m3 × 100% m2 − m3
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RWC =
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2.2.3 Determination of membrane stability index (MSI)
5
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Samples were excised from the leaves of ten wheat plants at the second leaf at the terminal bud for each treatment. To measure the MSI of leaves at
141
different stages [20], the sample was divided into two equivalent parts (about
142
0.1g for each) and soaked in 10 mL double distilled water. Then one part was
143
heated at 40 ℃ for 30 min. Conductivity C1 was determined by conductivity
144
meter (HI8733, Hanna instruments, Italy). The other part was heated at 100 ℃
145
for 10 min, and conductivity C2 was determined. MSI was calculated as the
146
following:
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2.3 Stomata observation
149
Samples were excised from the leaves of ten wheat plants at a similar
150
position for each treatment at vegetative growth stage. To observe the stomata,
151
samples were taken from fully expanded leaves in each plant. The slides made
152
by the leaf epidermal fingerprint of cotton with the transparent nail polish
153
method were observed using an optical microscope [21]. Slides were analyzed
154
with an Olympus DP71 microscope (Olympus Inc., Japan). The length, width
155
and frequency of stomata were measured with Motic Images Plus 2.0. 10
156
images per leaf, one leaf per plant and 10 plants per treatment were analyzed.
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2.4 Photosynthetic characteristics analyses
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2.4.1 Chlorophyll contents
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Samples were excised from the leaves of ten wheat plants at the second
160
leaf at the terminal bud for each treatment. The content of chlorophyll a and
161
chlorophyll b was measured with an ultraviolet spectrophotometer (SP-75,
162
Shanghai spectrum instruments co., LTD, China) at vegetative growth stage,
163
heading stage, flowering stage and maturity stage, respectively [22]. Leaf
164
samples were frozen in liquid nitrogen and stored at -80 °C until measured.
165
2.4.2 Photosynthetic efficiency 6
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167
instrument (Li-6400XT, Li-Cor, USA) was used for the determination of
168
photosynthetic characteristics. Leaf gas-exchange parameters included
169
photosynthetic rate (A), stomatal conductance (gs) and and intercellular CO2
170
concentration (Ci) using the second leaf at the wheat terminal bud. Water use
171
efficiencies (A/gs) were calculated by dividing A by gs and the instantaneous
172
carboxylation efficiencies (A/Ci) were also calculated [23].
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2.5 Antioxidant capacity analyses
174
2.5.1 Peroxidase (POD) activity
175
POD activity during vegetative growth stage, heading stage, flowering
176
stage and maturity stage was analyzed spectrophotometrically at 470 nm
177
using guaiacol as a phenolic substrate with hydrogen peroxide [24]. Samples
178
were excised from the leaves of ten wheat plants at the second leaf at the
179
terminal bud for each treatment. The reaction mixture contained 0.15 mL of 4%
180
(v/v) guaiacol, 0.15 mL of 1% (v/v) H2O2, 2.66 mL of 0.1 M phosphate buffer
181
(pH= 7.0) and 40 µL of enzyme extract. Blank sample contained the same
182
mixture without enzyme extract.
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2.5.2 Catalase (CAT) activity
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CAT activity was determined according to the method described by Kumar
185
and Knowles [25]. Samples were excised from the leaves of ten wheat plants
186
at the second leaf at the terminal bud for each treatment. CAT reaction solution
187
consisted of 100 mM Na2HPO4-NaH2PO4 buffer solution (pH=7.0) and 0.1 M
188
H2O2. The optical density was determined every 1 min at λ=240 nm.
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2.5.3 Malonaldehyde (MDA) content
190
Samples were excised from the leaves of ten wheat plants at the second
191
leaf at the terminal bud for each treatment. Determination of MDA depended
192
on the method of Stewart and Bewley [26]. Briefly, at vegetative growth stage,
193
heading stage, flowering stage and maturity stage, 10 mL 0.1% trichloroacetic 7
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195
4000 rpm for 10 min. 2 mL supernatant was added to 4 mL 5% thiobarbituric
196
acid (TBA) which was made up by 20% TCA. The mixture was heated at 95 °C
197
for 30 min and then cooled in ice-bath rapidly. The supernatant was obtained
198
by centrifuging at 3000 rpm for 10 min. The absorbency of the supernatant was
199
recorded at 532 nm. The value for non-specific absorption at 600 nm was
200
subtracted. The MDA content was calculated using its extinction coefficient of
201
155 mM-1 cm-1.
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2.6 Biomass yield analyses
203
2.6.1 Edible biomass
204
The thousand-kernel weight (TKW) of wheat seeds was weighed
205
respectively under 4 different treatments. At maturity, above-ground biomass
206
(AGB) and grain yield per plant (GY) were also recorded. Harvest index (HI =
207
GY/AGB) was then calculated.
208
2.6.2 Inedible biomass
209
For determination of inedible biomass components, plant tissues were
210
dried in an oven for 48 h at 70 °C before weighing. The content of Neutral
211
detergent solution (NDS), neutral detergent fiber (NDF), acid detergent fiber
212
(ADF), acid detergent lignin (ADL) and acid-insoluble ash (Ash) in wheat straw
213
was determined according to Van Soest et al. method [27] using FIWE six raw
214
fiber extractor (VelpScientifica, Italy). The contents of hemicellulose, cellulose,
215
lignin, ash and NDS were analyzed.
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2.7 Data statistics
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The experiment was setup in a completely randomized design. All
218
experiments were performed in triplicate. The average value of total 6
219
measurements ± standard deviation was regarded as the final result. All
220
statistical analyses were performed using SPSS 18.0. Comparison among
221
means performed at significance level P=0.05. 8
ACCEPTED MANUSCRIPT 3. Results
223
3.1 The response of wheat growth to different treatments
224
It was found that the morphology of the wheat plants was noteworthily
225
influenced under different N forms and concentrations of different growth
226
periods (Fig 1A). Particularly at the heading stage of seedling, straw height of
227
wheat under low concentrations was 5-10 cm greater than CK. However, after
228
that, the growth momentum of wheat plants under low concentrations had
229
been restricted, which might probably due to the adaptation to low
230
concentration. The most obvious influence of different N forms and
231
concentrations on RWC of wheat leaves happened when wheat heading (Fig
232
1B). With the condition of Ⅰ, Ⅱ and CK, the RWC was the higher at heading
233
stage wherein the transpiration strengthened and the plant growth was
234
vigorous. However, the more RWC existed in leaves, the less reflectivity of
235
leaves was, which would affect the optical property. At flowering stage, RWC
236
of Ⅲ group plants was at the lowest level of 79% and it is 7% lower than the
237
value for CK. The RWC occurred lower, which was more beneficial for
238
accumulating energy to perform self-pollination. Therefore, photosynthesis,
239
transpiration and water-use efficiency are strongly linked to the water regime of
240
plants. MSI gradually reduced during the development and growth of wheat
241
plants (Fig 1C). The largest difference happened at vegetative growth stage
242
and flowering stage. The highest CK value reached 73.2% and the lowest Ⅲ
243
group value was 70.1% at vegetative growth stage. The gap was larger when
244
flowering, the MSI of Ⅲ group was 4.9% lower than that in CK. But significant
245
difference disappeared at the maturity stage at last.
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At vegetative growth stage, the size of wheat leaf stomata was small due
247
to NO3--N decreased(Fig 2A), and the length of epidermal cells between
248
stomata was getting longer, with the tendency of growth elongation (Fig 2C),
249
leading to the reduction of CO2 assimilation efficiency when photosynthesis 9
ACCEPTED MANUSCRIPT 250
was on. The epidermal stomatal density in the Ⅲ group was sharply lower
251
than that in other conditions. These observations demonstrate that different N
252
concentrations and forms impact differently the morphology and the
253
distribution of epidermal cells and stomata of wheat leaves. Transpiration is the main driving force of nutrient transportation. Therefore,
255
inorganic N changes the transport of nutrients, which promotes the absorption
256
of nutrients by plant roots. In addition, the selectivity of plants to exogenous
257
nutrient ions decreases with the increase in their concentrations; thus, higher
258
NH4+ and NO3- concentrations result in more significant promoting effects of
259
increased transpiration on ion uptake rate. However, plants gradually adapt to
260
external environment (such as reducing stomatal aperture and the number and
261
distribution of stomata), and the degree of the promoting effect of increased
262
transpiration on nutrient uptake may also decrease gradually.
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3.2 The response of photosynthetic characteristics to different treatments
264
Chlorophyll a concentrations increased as N concentrations increased
265
during the whole life cycle (Fig 3A). However, the chlorophyll b concentration
266
was opposite, especially at the heading and flowering stages (Fig 3B).
267
Chlorophyll b contents of Ⅲ group at the heading and flowering stages were
268
0.81 and 1.02 mg/g, respectively. And chlorophyll a/b ratio of leaves in low
269
concentration treatments is considerably below the control level (Fig 3C).
270
During flowering, the relative content of chlorophyll of all the samples gradually
271
reached the peak. Our results showed that low N concentrations tended to
272
increase the chlorophyll b content in wheat leaves during heading and
273
flowering, and then the ratio chlorophyll a/b decreased. Starting from heading,
274
the total content of chlorophyll (especially Chl a) changed significantly, with the
275
level of Ⅱ and CK treatments was higher than that of Ⅲ group. When the
276
NO3- : NH4+ ratios were both 14:1 (Ⅲ and CK), during flowering, chlorophyll b
277
production was 1.02 and 0.89 mg·g-1, respectively. This suggests that total N
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10
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279
wheat plants. The NO3- : NH4+ ratio is secondary for the chlorophyll production.
280
Wheat plants sense and respond to low N concentrations through
281
changed photosynthesis (A) and stomatal conductance (gs) (Fig 4A-B). Both A
282
and gs maximized at the heading stage and then decreased later in senescent
283
leaves. Photosynthetic rate had a significant difference from the heading stage
284
to the flowering stage, which means the two growth stages are more sensitive
285
than other stages during the wheat life cycle. At the heading stage,
286
photosynthetic rate of Ⅲ group was 12.57% lower than CK, and then the
287
distance increased to 21.12% at the flowering stage. In particular, the increase
288
in A at the vegetative growth stage, was smaller than the decrease in gs, so
289
A/gs increased (Fig 4C), allowing the wheat plants to use water more
290
efficiently. However, the instantaneous carboxylation efficiency (A/Ci) of Ⅲ
291
group was at lowest level across the entire life cycle (Fig 4D).
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3.3 The response of antioxidant system to different treatments
293
We also studied the activity of the antioxidant enzymes POD and CAT and
294
the production of MDA in wheat leaves during ontogenesis. POD and CAT
295
activities increased during early leaf development, reaching their maximal
296
levels at flowering stage and decreasing later in senescent leaves. Also, these
297
antioxidant enzymatic activities were higher in the plants grown under low N
298
concentrations condition throughout leaf development (Fig. 5A-B). NH4+-N and
299
NO3--N both are plant-needed N source. Either of NH4+ and NO3- can be well
300
absorbed by wheat plants. As shown in Fig.5C, MDA production increased
301
with leaf aging, especially under low NO3--N condition, suggesting that NO3--N
302
may play an important role in regulating leaf senescence in wheat plants by
303
increasing reactive oxygen species (ROS) production. Alternatively, lower
304
ROS production may decrease the incidence of oxidative stress, translating as
305
a reduced stimulus for antioxidant production. Such reduced oxidative
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11
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307
may translate into greater CO2/O2 ratio in the chloroplast, causing lower
308
oxygenation rates by Rubisco and therefore lower rates of photorespiration
309
and associated ROS production in wheat cultivar. However, the mechanism
310
needs to be further investigated. Moreover, responses of plant organs to
311
environmental factors should be different during different growth stages. Plants
312
have adaptability to environmental changes.
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3.4 The responses of biomass yield and quality to different treatments
314
The analysis of variance revealed significant differences among
315
treatments for wheat yields per square meter (Fig 6A), harvest index (HI) (Fig
316
6B) and thousand-kernel weight (TKW) of wheat seeds (Fig 6C). It was found
317
that there was no significant difference among Ⅱ (1830.8 g/m2) and CK
318
(1899.6 g/m2) treatments on yields per square meter, but the yield of Ⅲ
319
(1493.4 g/m2) treatment was lower (Fig 6A). That is to say, the growth and
320
development of wheat plants had been affected by N concentration in the
321
solution, especially NO3--N. Similarly, TKW and HI in Ⅰ , Ⅱ and CK
322
treatments were larger than that in the Ⅲ treatment (Fig 6B-C).
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The results of impact of different N forms and concentrations on the dry
324
weight of each part of wheat plants showed that NH4+ nutrition was conductive
325
to the growth of overground part in wheat plants, including the thickening of
326
stem, the increase of overground biomass (Table 2). Adding more NO3- was
327
beneficial for the development and growth of crop root system. In more NO3-
328
concentration, wheat seeding was easy to form roots and the root system was
329
strong. The underground part of biomass was increased by 1.14% compared
330
with that in CK treatment.
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Nitrogen deficiency has many impacts on the root system development:
332
although total weight decreases, the plant preferentially allocates biomass to
333
the root system so that the root/shoot ratio increases. It is well known that NH4+ 12
ACCEPTED MANUSCRIPT nutrition results in the acidification of the root environment, in these
335
experiments the pH of the hydroponic solutions was carefully controlled to
336
exclude this factor as an influence on plant growth. Changes in the proportion
337
of plant dry mass found in the shoot and root components can only be
338
attributed to alter partitioning of carbon within the plant. The roots of wheat
339
plants are particularly sensitive to the form of nitrogen nutrition resulting in
340
large differences in the shoot/root ratios. The lower shoot/root ratios of wheat
341
plants supported in Table 2 that in general NO3- is better to root growth than to
342
shoot growth. The soluble sugar content of the edible part of wheat plants
343
decreased with NO3- : NH4+ ratio such as in Ⅱ or Ⅲ treatments, but the
344
accumulation of carbohydrate has no significant difference (Table 3). Since
345
nitrogen
346
may
347
acquisition by plants is the result of two processes: root system development
348
from which depends soil colonization, and nitrate uptake capacity of the root
349
system.
is translocated the
to a large extent
movement
of
as amino compounds
carbon within
the
this
plant. Nitrogen
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It is noteworthy that inedible biomass also plays one of the most important
351
roles in the ecosystem, which means the compositions of biomass may have a
352
significant impact on biological cycle degradation. To investigate the influence
353
of different conditions on inedible part of wheat plants, we determined the
354
contents of lignin, cellulose and hemicelluloses (Fig 7). The results showed
355
that the higher N concentration promoted the increase of lignin content, with
356
maximal mass fraction of 3.99% (CK). However, the lignin content in group Ⅲ
357
decreased to 2.58% with N concentration lower, which would be helpful for
358
wheat straw degradation. ,
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4. Discussion
360
At vegetative growth stage, weak N supply reduced the mean realized
361
photosynthetic rate. Besides, more biomass seems to be allocated to the 13
ACCEPTED MANUSCRIPT overground portion for stem growth so that catch up the growth of leaves and
363
the full absorption of limited energy to meet the demand for plant
364
photosynthesis (Fig 1A, Table 2). At the same time, leaf temperature was also
365
dropped down because of the lower RWC and MSI to meet the requirement of
366
plant growth. The difference of biomass accumulation and biomass allocation
367
in organs of wheat plants under different N forms was integrated with
368
performance of efficiency utilization of environmental N resources.
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Pigments are integrally related to the physiological function of leaves.
370
Chlorophylls absorb light energy and transfer it into the photosynthetic
371
apparatus and carotenoids can also contribute energy to the photosystem [28],
372
which finally effect plant growth and yield. It is beneficial for Chlorophyll b to
373
trap scattered light with short wavelength to harness its quantum properties
374
effectively [29]. Previous study indicated that photosystems I and II of the
375
plants
376
chlorophyll b than the corresponding photosystems of spinach [30]. The shade
377
plant chloroplasts contained very large grana stacks and the extent of grana
378
formation in higher plant chloroplasts appears to be related to the total
379
chlorophyll content of the chloroplast. The effects of the form and
380
concentration of the nutrient nitrogen on photosynthetic carbon dioxide
381
assimilation may arise from the influence of the nitrogen source on
382
photosynthetic enzymes，photorespiration，stomatal conductance or other
383
aspects of photosynthetic metabolism.
light
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Stimulation of photosynthesis is the driving force for increased growth and
385
yield of wheat in Ⅰ, Ⅱ and CK groups. However, at the heading and
386
flowering stage, the effect of low N concentrations became more significant,
387
which may be because the stages from vegetative growth to reproductive
388
growth are more sensitive. Ion sensing is an intrinsic property of guard cells,
389
which are thought to be sensitive to the intercellular carbon dioxide 14
ACCEPTED MANUSCRIPT concentration (Ci) rather than CO2 concentration at the wheat leaf surface. Ion
391
and organic solute concentrations mediate the turgor pressure in the guard
392
cells that determines stomatal aperture (Fig 2). Stomata closure implies lower
393
CO2 availability, and ultimately lower CO2 fixation by Rubisco, which may
394
finally result in lower biomass production [31]. The uptake of NH4+ and NO3- is
395
an active process, which is energy-related and can be blocked by metabolic
396
inhibitor. Plant up take of ions needs energy provided by respiratory
397
metabolism, and the energy required for uptake of NO3- is higher than that for
398
uptake of NH4+; therefore, many plants tend to utilize NH4+ [7]. More,
399
differences between NO3- and NH4+-fed plants with respect to photosynthetic
400
rates may be due to effects of the form of nitrogen on the activity of the
401
photosynthetic enzymes.
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Currently, it is extensively accepted that reactive oxygen species are
403
responsible for kinds of damage (stress-induced) of macro-molecules, finally of
404
the cellular structure [32]. Physiological and genetic evidence clearly indicates
405
that the reactive oxygen intermediate scavenging systems of plants are an
406
important component of the stress protective mechanism [20]. As for the
407
response of antioxidant enzyme to different N concentrations conditions, the
408
level of POD and CAT in wheat plants was an important index of ageing and
409
death. At the flowering stage, the response of CAT activity to different
410
treatments was quite sensitive (Fig.5B). The reason may be that the
411
expression of wheat enzyme genes is promoted more during the flowering
412
stage, ensuring a good growth of plants. Meanwhile, it also indicates that there
413
might be complementary and / or additive effect in different treatments. The
414
level of these enzymes reflects the situation of physiological activity of plants.
415
POD has been proven to have the function of oxidase IAA [33]. Low level of
416
POD promoted the growth of overground part of plants, especially for the
417
elongation growth. POD also could prevent the toxic levels of internal
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15
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419
of reactive oxygen. Moreover, the activities of POD and CAT were related to
420
plant senescence. With the senescence of plants, the activity of them dropped
421
very fast. As a product of membrane lipid peroxidation, MDA is used to assess
422
the extent of oxidative stress in plants, and its level is increased under stress
423
conditions. ROS such as O2•− and endogenous H2O2 can be overproduced in
424
plants under stress conditions and will thereby increase MDA content [34].
425
These highly reactive ROS could alter normal cellular metabolism by oxidative
426
damage to nucleic acids, proteins and lipids [35]. Plants should maintain
427
efficient antioxidant defense system for opposing the oxidative damage.
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The relationship between total biomass and harvestable yield is very close.
429
From the ecological view, the differences are reflected in the range of
430
individual size under the 4 conditions. HI is the primary result of biomass
431
production, but not generally determined in a direct way. The nutrient uptake in
432
wheat aboveground and belowground biomass varied widely among the
433
different N forms and concentration treatments. Biomass/yield increased
434
linearly with increasing N availability then after a certain level of N availability,
435
there is no further increase when more N is available [36]. Wheat N uptake has
436
been found strongly correlated to shoot biomass (Table 2). The faster
437
above-ground growth is often associated with a faster accumulation of root
438
biomass, root length and surface area; traits which could be expected to
439
enhance the capacity of wheat to capture NO3 before it is leached, whether
440
through a capacity to explore deeper soil layers or greater root length density.
441
These findings are useful for designing and operating growth environments for
442
enhanced crop production.
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5. Conclusion
444
Our results clearly demonstrate that, N forms and concentrations are very
445
significant factors that affect spring wheat growth. At heading and flowering 16
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447
photosynthetic rate and transpiration rate, and enhanced water used efficiency.
448
The plants were spindling and the output was very low when wheat was in low
449
N concentration solution. Meanwhile, some photosynthetic characteristics of
450
plants at low N concentrations are worse than that of control. The soluble
451
sugar content of the edible part of wheat plants is influenced with NO3- : NH4+
452
ratio. In addition, when N concentration was lower, the lignin content
453
decreased, which would be helpful for wheat straw degradation. It also offers
454
new thoughts about straw degradation.
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Acknowledgements
456
This work was financially supported by Defense Industrial Technology
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Development Program (JCKY2016601C010).
458 459
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ACCEPTED MANUSCRIPT Table 1：Major nutrient solution parameters of different treatments -
+
NO3 -- KNO3
NH4 --NH4H2PO4
NO3 :
（mmol•L ）
（mmol•L ）
NH4
Ⅰ
7
1
7:1
8
Ⅱ
14
0.5
28:1
14.5
Ⅲ
7
0.5
14:1
7.5
CK
14
1
14:1
15
-1
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+
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-1
Total N （mmol•L ）
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ACCEPTED MANUSCRIPT Table 2 The proportion of different parts of wheat plants (DW, %) Treatment
Root 1 2
Ⅰ Ⅱ Ⅲ CK
7.80±0.56 b 9.13±0.96a 7.23±0.69b 7.99±0.19b
Leaf
Stem
Spike
8.27±0.68a 8.29±0.78a 8.28±0.63a 8.30±0.41a
27.19±2.75b 25.64±1.98c 30.29±2.35a 26.66±1.56bc
56.74±4.78a 56.94±4.56a 54.44±4.74b 57.05±4.65a
1
Mean±SE. Mean values with the same letter were not significantly different, based on ANOVA followed by Tukey’s test at P≤0.05.
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ACCEPTED MANUSCRIPT Table 3 The contents of nutrients of wheat seed in different treatments （g/100g） Treatment
Soluble sugar Carbohydrate Rough protein Rough fat Ash NDS Hemicellulose Cellulose Lignin
Ⅰ 1 2
8.15±0.19 a 72.09±3.95a 21.78±1.79a 2.09±0.21a 0.61±0.04a 34.35±3.45a 3.12±0.25b 4.02±0.13b 0.79±0.05b
Ⅱ
Ⅲ
CK
7.35±0.09b 71.27±3.17a 22.03±1.89a 2.05±0.39a 0.33±0.09b 33.46±3.21a 3.52±0.33ab 3.19±0.24c 0.49±0.06c
7.23±0.17b 69.99±5.16a 22.04±1.98a 2.11±0.49a 0.63±0.15a 34.71±3.78a 3.01±0.29b 4.04±0.23b 1.18±0.06a
8.17±0.13a 73.87±4.64a 21.97±1.12a 2.01±0.44a 0.31±0.11b 32.78±2.56a 3.71±0.21a 4.97±0.09a 0.52±0.07c
1
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Fig 1：Response of straw height of wheat plants (A), relative water content
(B), membrane stability index (C) of leaves in wheat plants at different stages of ontogenesis to different treatments. Vertical bars are means ± SD. Different lowercase letters indicates a significant difference at p=0.05 between the means.
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Fig 2: Effects of different treatments on wheat leaf stomata. A (Ⅰ), B (Ⅱ),
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Fig 3: Response of chlorophyll a (A) and chlorophyll b (B) and chlorophyll
ratio (C) of wheat leaves at different stages of ontogenesis to different treatments. Vertical bars are means ± SD. Different lowercase letters indicates a significant difference at p=0.05 between the means.
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Fig 4: Response of photosynthetic rate (A), stomatal conductance (B), A/gs (C) and instantaneous carboxylation efficiency (D) of wheat leaves at
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different stages of ontogenesis to different treatments. Vertical bars are means ± SD. Different lowercase letters indicates a significant difference at p=0.05
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Fig 5: Response of POD activity (A), CAT activity (B) and MDA content (C) of wheat leaves at different stages of ontogenesis to different treatments. Vertical bars are means ± SD. Different lowercase letters indicates a significant difference at p=0.05 between the means.
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Fig 6: Response of unit yield (A), harvest index (B) and thousand-kernel weight (C) of wheat plants to to different treatments. Vertical bars are means ± SD. Different lowercase letters indicates a significant difference at p=0.05 between the means.
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Fig 7: Components of inedible biomass in different treatments (DW, %).
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ACCEPTED MANUSCRIPT ► NO3- and NH4+ concentration and ratio have significant effects on wheat root and shoot growth.
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► Soluble sugar content of wheat edible part is affected by NO3- : NH4+ ratio.
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► When heading and flowering, wheat is sensitive to N concentration.