國立臺灣大學生物資源暨農學院農藝學系 博士論文
Department of Agronomy
College of Bioresources and Agriculture National Taiwan University
Doctoral Dissertation
光質對水稻幼苗形態與光合生理之影響 The Effect of Light Quality on Morphology and
Photosynthetic Physiology in Rice Seedling
陳昶璋
Chang-Chang Chen
指導教授：黃文達 博士 楊棋明 博士 Advisor: Dr. Wen-Dar Huang
Dr. Chi-Ming Yang
中華民國 103 年 6 月
June 2014
國立臺灣大學博士學位論文
口試委員會審定書
光質對水稻幼苗形態與光合生理之影響 The Effect of Light Quality on Morphology and
Photosynthetic Physiology in Rice Seedling
本論文係陳昶璋君（D99621103）在國立臺灣大學農藝學系完成 之博士學位論文，於民國一百零三年六月三十日承下列考試委員審 查通過及口試及格，特此證明
口試委員：
國立台灣大學農藝系助理教授
黃 文 達 博士（本論文指導教授）
中央研究院生物多樣性研究中心副研究員 楊 棋 明 博士（本論文指導教授）
農業委員會特有生物研究保育中心助理研究員 翁 韶 良 博士
台灣中油股份有限公司煉製研究所研究員 許 明 晃 博士
農業委員會桃園區農業改良場助理研究員
楊 志 維 博士
誌 謝
本論文完成，首要感謝兩位指導教授 黃文達老師以及 楊棋明多年來的指 導、愛護與包容。論文修改過程，感謝翁韶良博士、許明晃博士與楊志維博士提 供寶貴建議。求學期間感謝 葉學文老師多年來耐心指導、訓練與提攜。並感謝 國立台灣大學農藝學系與中央研究院生物多樣性研究中心提供良好研究環境與資 源，使研究得以順利進行。
在台大農藝完成博士學位，乃因緣際會。彭雲明老師與盧虎生老師的推薦函 成為博士班的契子。回顧博士班生涯，與盧老師雖然師生緣分不長，但在那三個 學期，受到朱鈞老師、盧虎生老師與張孟基老師在邏輯思維上的訓練，奠定良好 研究基礎。在提交論文此刻，對於當時老師的諄諄教誨仍深深烙印在心裏，在未 來亦時時自我警惕。感謝作物生理實驗室的夥伴在實驗上提供協助，特別是李佳 諭博士及楊蕓瑋博士。
2005 年還是大學生的我開始踏入 308 實驗室，轉眼就是 2014 年，今日能完成 學業，猶如站在巨人的肩膀上，看得更廣，卻也更顯渺小。張新軒教授、蔡養正 教授、蔡文福教授與曾美倉教授平時的指導與薰陶，讓我明白學海無涯。感謝黃 秀鳳博士，從試驗的方法學乃至生活細節，都照顧得無微不至。感謝黃盟元博士 多年的關懷及照顧。
感謝實驗室歷年助理、研究生與大學部的研究夥伴：彭懷慈、許毓鈞、林柏 齡、彭元慶、陳傑君、郭宇翔、張瀞文、李雅蓁、羅啟元、陳怡夙、林奎庭、藍 秋月、陳相全、周庭聿、陳煥文、徐芷陶、楊敬屏，你們的協助、討論、陪伴與 關懷都是論文完成的推手。台大農藝全職博士生王群山、林亞平、楊琇淳、華德 揚同學，感謝你們，文字敘述在這裡是累贅卻又不足。
I have to thank Dr. Karyne Rogers, Andy Phillips and all SIL people. I am grateful for your patient during the training in National Isotope Centre, GNS science.
And thank Simon Stewart for sample running and calibration of rough isotopic data.
Meanwhile, I am thankful for all of your kinds and let me feel welcome and at home in New Zealand.
最後，感謝我的祖母與父母親，你們的愛就如同無盡的海洋，包容了我的一 切。謝謝我生命中的所有人，成就今天的我。感謝上天。
陳昶璋 謹誌 July, 2014
摘要
本研究主要評估光質對於水稻幼苗之形態與光合生理之影響。先行探討幼苗 生長、發育與碳、氮代謝方面對於不同光質之反應。水耕幼苗栽培於紅(R)、綠(G)、
藍(B)與紅藍(RB)發光二極體(LED)照射之生長箱。紅光誘導地上部伸長。藍光抑 制伸長並促進壯苗指數。相較於紅藍混和，葉片總蛋白在藍光照射下含量較高。
另外光合生理方面，藍光可提高幼苗葉片之光系統 II 有效光量子產量(ΦPSII)與光化
學消散(qP)，同時降低非光化學消散(NPQ)。水稻幼苗對紅光與綠光的反應相當類
似。幼苗葉片花青素含量以 RB 最高，R、B 卻低於 G。葉片中葉綠素 a/b 比例則 受不同光質波段所調控。
另外結果顯示，不同光質對於光合作用與氮素代謝具不同效果，而此類生理 反應與水分利用效率 (WUE)、氮素吸收具相關性。進一步探討水稻在不同光質處
理下，WUE、穩定性碳同位素分辨率(Δ13C)與氮素吸收反應，另以螢光燈(FL)為對
照。發現 R 之幼苗 WUE 最高，而後依序為 G、RB、B。除了 FL 處理之外，WUE
與Δ13C 具顯著正相關(P<0.01)。另利用氮含量與氮同位素值(δ15N)評估不同光質對
於氮肥吸收之結果顯示，幼苗中化學肥貢獻之氮素(Nf)以 B 最高而 R 最低。因此，
推測藍光可促進氣孔導度與蒸散作用，造成 WUE 降低而促進根部氮素吸收。
在此 水稻 幼苗葉 片之葉 綠素 (Chl) 與其生 合成 中間產 物(protoporphyrin IX, PPIX; magnesium protoporphyrin IX, MGPP; protochlorophyllide, Pchlide)、降解代謝 產物 (chlorophyllide, Chlide; pheophytin, Phe; pheophorbide, Pho)以及胡蘿蔔素 (Car) 。葉片 中 Chl 與 Car 在綠光 下較 低。光 質並 未影響 生合 成途徑 中卟啉 (porphyrins) 莫爾百分比。Phe/Chlide 在 G 與 FL 照明下數值較低，顯示綠光較高 之環境會促進葉片中 Chlilde 分解途徑。
為了釐清綠光在 Chl 分解途徑之效應，將幼苗在固定紅、藍光強度 40 μmol m-2
s-1)下生長，分別以 4 個綠光等級 (0, 20, 40 and 60 μmol m-2 s-1) 處理。同時調查部
具較長之葉鞘且葉片角度較為直立，具避蔭效應(shade avoidance symptoms, SAS)。
且增強綠光亦會造成葉綠素、ФPSII降低與 NPQ 提高。另外增強綠光下，也發現較
高之 Chlide 與較低之 Phe/Chlide 比例。以上結果顯示綠光可誘導水稻幼苗之 SAS 產生且調節 Chl 分解途徑。
關鍵字：水稻幼苗、光質、光形態發生、降解代謝、穩定性同位素、水分利用效 率、氮素吸收、葉綠素分解途徑、避蔭效應
Abstract
Our objective in this study was to evaluate the effect of light quality on the morphology and photosynthetic physiology of rice seedlings. We examined the growth, development, and metabolic responses of rice seedlings to varying light quality first ly.
Seedlings were hydroponically cultured under red (R) light-emitting diodes (LED), green LED (G), blue LEDs (B), and red + blue LED (RB) inside growth chambers. Red light induced shoot elongation. B light inhibited shoot elongation and promoted health index values. B light also resulted in higher total protein content in tested leaves
compared to RB. Blue light enhanced the effective quantum yield of PSII photochemistry (ΦPSII) and photochemical quenching (qP) while reducing
non-photochemical quenching (NPQ) in seedling leaves. The responses of rice seedlings to green and red light were quite similar. The anthocyanin content of seedling leaves was observed to be highest in RB but less so in R and B, the latter two being even lower than in G. Different wavelengths mediated the chlorophyll (Chl) a/b ratio of the leaves.
Light quality influenced photosynthetic potential and nitrogen metabolism, which are related to water- use efficiency (WUE) and nitrogen uptake. We further investigated the response of time- integrated WUE, 13C discrimination (Δ13C), and nitrogen uptake in hydroponic seedlings of rice grown under different light treatments with fluorescent light (FL) as the control. The WUE response was highest for seedlings
grown under R light, then (in decreasing order) seedlings grown under G, RB, and B light. WUE had a significantly positive correlation with Δ13C except under FL light (P<0.01). Nitrogen content (%N) and δ15N values were used to estimate the effects of fertilizer uptake under different lighting conditions. The amount of N in seedlings derived from fertilizer (Nf) was highest under B light and lowest under R light.
Therefore, we conclude that blue light may increase stomatal conductance and transpiration, decrease WUE, and promote root N uptake.
The dynamics of Chl, biosynthetic intermediates (protoporphyrin IX, PPIX;
magnesium protoporphyrin IX, MGPP; protochlorophyllide, Pchlide), degradation intermediates (chlorophyllide, Chlide; pheophytin, Phe; pheophorbide, Pho), and carotenoids (Car) in leaves of rice seedlings were also investigated. Lower levels of Chl and Car in leaves were observed under G lighting. Light quality did not mediate the mole percent of porphyrins in biosynthetic pathways. Lower Phe/Chlide ratios were observed under G and FL lighting conditions, indicating that green-enriched environments may up-regulate the Chlide degradation route in leaves.
In order to clarify the effect of green light on the Chl degradation pathway, seedlings were grown under equal intensity (40 μmol m-2 s-1) of red and blue light with four levels of green light intensity (0, 20, 40, and 60 μmol m-2 s-1). Some morphological
traits and photosynthetic physiology were also investigated at the same time. Sheaths of
rice seedling leaves became elongated and leaves grew more erectly under red and blue
light with increasing green light intensity. These morphological traits are known as shade avoidance symptoms (SAS). Lower Chl, decreasing ФPSII, and increasing NPQ
were also observed under increasing green light intensity. Higher Chlide levels and lower Phe/Chlide ratios were observed under increases in green light intensity. These results indicated that green light induced SAS and mediated Chl degradation routes in rice seedlings.
Key words: Rice seedling, Light quality, Photomorphogensis, Metabolism, Stable
isotope, Water-use efficiency, Nitrogen uptake, Chlorophyll degradation pathway, Shade avoidance symptoms
Organization
TCS10 (green leaf) & IR1552 (purple leaf)
Light treatment R, G, B, R+B, (FL)
Rice seedlings (V2-V3 stage)
Growth &
morphology
Photosynthetic pigments &
anthocyanin
Carbon–
nitrogen metabolism
Chlorophyll fluorescence
Water-use efficiency
Stable C &
N isotope
The second stage The first stage
Chlorophyll- related compounds
The third stage
TCN1
four levels of green light intensity (0, 20, 40, and 60 μmol m-2 s-1)
Rice seedlings (V2-V3 stage)
Growth &
morphology
Chlorophyll-related compounds Chlorophyll
fluorescence
Table of Contents
摘要 ...i
Abstract ... iii
Organization... vi
Table of Contents ... vii
List of Figures ... viii
List of Tables ... ix
I. Effects of Light Quality on the Growth, Development and Metabolism of Rice Seedlings (Oryza sativa L.) ... 1
Introduction... 3
Material and Methods ... 6
Results... 12
Discussion ... 17
II. Water-use efficiency and nitrogen uptake in rice seedlings grown under different light treatments ... 31
Introduction... 33
Materials and Methods ... 36
Results... 40
Discussion ... 42
III. Light Quality Influences the Chlorophyll Degradation Pathway in Rice Seedling Leaves ... 52
Introduction... 54
Materials and Methods ... 56
Results... 58
Discussion ... 60
IV. Effects of Green Light Intensity on Shade Avoidance Symptoms and Chlorophyll Degradation in Rice Seedlings... 69
Introduction... 70
Materials and Methods ... 72
Results... 76
Discussions ... 79
Graphical Summary ... 91
References... 92
List of Figures
Figure I-1. The spectral distributions of different light treatments. Spectral scans were recorded at the top of the plant canopy with a spectroradiometer. ... 26 Figure I-2. Effects of light quality on the relative value of chlorophyll
fluorescence... 27 Figure II-1. The relationship between Δ13C and WUE.. ... 47 Figure II-2. Nutrient dynamics (Nf) of TCS10 and IR1552 cultivars under different
light conditions.. ... 48 Figure IV-1. Effects of different light treatments on the maximal photochemical
efficiency of PSII (Fv/Fm, A), relative quantum efficiency of PSII photochemistry (ΦPSII, B), photochemical quenching (qP, C), and
non-photochemical quenching (NPQ, D).. ... 85
List of Tables
Table I-1. The growth parameters of 14 d seedlings cultivated under different light environments. ... 28 Table I-2. The effect of light quality on pigments in 14 d seedling leaves. ... 29 Table I-3. Effects of light quality on the carbon–nitrogen metabolism of seedling
leaves collected from 14 d seedlings under different light environments. ... 30 Table II-1. δ15N values of seeds of two cultivars and three fertilizers in Kimura
solution, and the estimated value of bulk Kimura solution... 49 Table II-2. The response of biomass (BM), total amount of water transpired (W), and
WUE of TCS10 and IR1552 under different light conditions. ... 50 Table II-3. %C, %N, C/N ratio, δ13C, δ15N, and Δ13C of rice seedlings. ... 51 Table III-1. Effects of light quality on the levels of Chl, Car, LP Car, MP Car,
and their ratios in seedling leaves collected from 14 d seedlings under
different lighting environments. ... 65 Table III-2. Effects of light quality on the levels and mole percentages of porphyrins
in leaves collected from 14 d seedlings under different lighting environments.... 66 Table III-3. Effects of light quality on Chlide, Phe, and their ratios in 14 d seedling
leaves. ... 67 Table III-4. Effects of light quality on phytylated and dephytylated pigments and
their ratios in 14 d seedling leaves. ... 68 Table IV-1. Photon flux densities in each treatment. ... 86 Table IV-2. Growth and morphological traits of rice seedlings grown under different
light treatments. ... 87 Table IV-3. Effects of different light treatments on Chl, Car, and their ratios in
seedling leaves... 88 Table IV-4. Effects of different light treatments on the levels and mole percentages
of porphyrins in seedling leaves... 89 Table IV-5. Effects of different light treatments on Chlide, Phe, phytylated, and
dephytylated pigments, and their ratios in seedling leaves. ... 90
I. Effects of Light Quality on the Growth, Development and Metabolism of Rice Seedlings (Oryza sativa L.)
Abstract
The V3 seedlings of two rice cultivars, IR1552 (purple leaf) and Taichung sen 10 (TCS10, green leaf) were hydroponically cultured under 12 h photoperiod at 30/25°C (day/night), 70% relative humidity and 160 μmol m-2 s−1 photon flux density under red light-emitting diodes (LEDs) (R), green LEDs (G), blue LEDs (B) and red + blue LEDs (RB) inside growth chambers for 14 days (starting 2 days after sowing). The results showed that shoot elongation was induced under the exposure of R and G. The maximum health index [(stem diameter/plant height) × biomass)] occurred under B because blue light inhibited shoot elongation. The root length under RB was the shortest.
Different wavelengths mediated the chlorophyll (Chl) a/b ratio of the leaves.
The content of anthocyanin (Ant) in seedling leaves was observed to be highest in RB but less in R and B, the latter pair being even lower than in G. B light LEDs enhanced effective quantum yield of PSII photochemistry (ΦPSII) and photochemical quenching (qP), but reduced non-photochemical quenching (NPQ) of seedling leaves. B LEDs also showed higher total protein content in the tested leaves compared to B plus R.
In summary, precise management of irradiance and wavelength may hold promise in maximizing the economic efficiency of plant growth, development and metabolic
potential of rice seedlings grown in controlled environments.
Keywords: Light-emitting diode, Light quality, Rice, Photomorphogensis, Metabolism
Introduction
Light is the main energy source for plant photosynthesis and is an environmental signal used to trigger growth and structural differentiation in plants. Light quality, quantity and photoperiod control the morphogenesis, growth and differentiation of plant
cells, tissue and organ cultures (Abidi et al., 2013). Plant development is strongly influenced by light quality which refers to the colors or wavelengths reaching a plant’s
surface (Johkan et al., 2010). Red (R) and blue (B) lights have the greatest impact on plant growth because they are the major energy sources for photosynthetic CO2
assimilation in plants. It is well known that spectra have action maxima in the B and R ranges (Kasajima et al., 2008). The integration, quality, duration and intensity of red light/far red light, blue light, mixed red and blue lights (RB), UV-A (320–500 nm) or UV-B (280–320 nm) and hormone signaling pathways have a profound influence on plants by triggering or halting physiological reactions and controlling the growth and development of plants (Clouse, 2001; Shin et al., 2008). Recent studies reported that green (G) light also affects the morphology, metabolism and photosynthesis of plants (Johkan et al., 2012; Zhang et al., 2011).
Light sources such as fluorescent, metal- halide, high-pressure sodium and incandescent lamps are generally used for plant cultivation. These sources are applied to increase photosynthetic photon flux levels but contain unnecessary wavelengths that are
located outside the photosynthetically active radiation spectrum and are of low quality for promoting growth (Kim et al., 2004b). Compared to those conventional light sources, gallium-aluminum- arsenide light-emitting diode (LED) lighting systems have several unique advantages, including the ability to control spectral composition, small size, durability, long operating lifetime, wavelength specificity, relatively cool emitting surfaces and photon output that is linear with electrical input current. These solid-state light sources are therefore ideal for use in plant lighting designs and allow wavelengths to be matched to plant photoreceptors for providing more optimal production and influencing plant morphology and metabolism (Bourget, 2008; Massa et al., 2008;
Morrow, 2008).
The LED light spectra in many reported experiments were inconsistent with light intensity being non-uniform because the investigators were unable to precisely modulate and quantify spectral energy parameters (Liu et al., 2011). Furthermore, experimental results may have been influenced in part by differences in light intensity and this often presents a problem when comparing results from experiments conducted under inconsistent lighting parameters. While it is widely understood that light intensity can positively affect photochemical accumulation (Fu et al., 2012; Li and Kubota, 2009), the effects of light quality are more complex and mixed results have often been reported.
Spectral light changes evoke different morphogenetic and photosynthetic responses that can vary among different plant species. Such photo responses are of practical importance in recent plant cultivation technologies since the feasibility of tailoring illumination spectra enables one to control plant growth, development and nutritional quality. The effects of LED light sources on several plants such as maize (Felker et al., 1995), cotton (Li et al., 2010) and peas (Wu et al., 2007) have been reported and indicate that LED lights are more suitable for plant growth than fluorescent lights.
Rice (Oryza sativa L.) is a staple food in Asia. During the vegetative growth stage, rice plants grow better under RB lights than under R alone (Matsuda et al., 2004;
Ohashi-Kaneko et al., 2006b). The quality of V3 seedlings during growth is therefore a n important factor in rice production, especially when mechanically transplanting seedlings to the field. The seedlings incubated under RB LEDs were more robust than when incubated under other LED spectra in terms of root number, stem diameter, health index and soluble sugars (Guo et al., 2011b). Therefore, in order to apply the findings to rice seedling quality and production, we considered it important to investigate the effects of light quality when provided by R, B, G and RB LED systems to meet different purposes. Hence, in this study, the growth, development and quality of rice hydroponically grown under various LEDs at the same light intensity were investigated
to determine the efficacy of this promising radiation source.
In order to clarify the different response of green and purple leaf rice, rice seedlings of two indica rice varieties, IR1552 (purple leaf) and Taichung sen 10 (TCS10, green leaf), were cultivated under different light environments at the same light density.
Fourteen day old seedlings were collected to investigate the effects of light quality on growth and metabolism of rice seedlings. Controlled climates and LEDs may be practical issue for rice seedling stages before transplanting to field conditions. An optimal strategy of light quality regulation will help in designing growth chambers or greenhouse light environments to obtain maximum economic benefit for rice growers.
Material and Methods
Plant materials and growth conditions
Seeds of indica rice (Oryza sativa L.) cultivar, IR1552, were donated by Dr.
Su-Jein Chang, Miaoli District Agricultural Research and Extension Station, Taiwan.
IR1552 is famous for its purple leaf. In addition, Taichung shen 10 (TCS10, green leaf), one of the most widely grown rice cultivars in Taiwan, was also used in this study.
Seeds were sterilized with 2% sodium hypochlorite for 20 min, washed extensively with distilled water and then germinated in Petri dishes with wetted filter paper at 37°C in the dark. After 48 h of incubation, uniformly germinated seeds were selected and cultivated
in a 250 ml beaker containing a half-strength Kimura B nutrient solution with the following macro and microelements: 182.3 μM (NH4)2SO4, 91.6 μM KNO3, 273.9 μM
MgSO4·7H2O, 91.1 μM KH2PO4, 182.5 μM Ca(NO3)2, 30.6 μM Fe-citrate, 0.25 μM
H3BO3, 0.2 μM MnSO4·H2O, 0.2 μM ZnSO4·7H2O, 0.05 μM CuSO4·5H2O and 0.07 μM H2MoO4.
Nutrient solutions (pH 4.7) were replaced every 3 d. Hydroponically cultivated rice seedlings were raised in growth chambers with the LED lighting system set at 30°C and 25°C for day and night respectively and 70% relative humidity under a 12 h photoperiod.
Light treatments
LED lighting systems designed by GRE Technology Co. (Taipei, Taiwan) were used to control light quality. The spectral distribution of the rela tive energy of the blue (peak at 460 nm), red (peak at 630 nm) and green (peak at 530 nm) regions were measured using a spectroradiometer (LI-COR1800, Lincoln, NE, USA) in the 300-800 nm range. These peak emissions of LEDs closely coincide with the absorpt ion peaks of chlorophylls a and b and the reported wavelengths are at their respective maximum photosynthetic efficiencies (McCree, 1972). Light treatments for rice seedlings, proliferation and differentiation included red LEDs (R), blue LEDs (B), green LEDs (G)
and red + blue LEDs (R:B = 4:1 by photon flux density; RB) (Figure I-1), with photon flux density (PPFD) being set at 160 μmol m-2s-1. The experiment was independently
performed three times for a randomized design of growth conditions and measurements representing the means of 15 plants (three reps consisting of five plants each) were taken.
Plant growth parameters
Rice seedlings were sampled after 14 d of growth after reaching the V3 stage according to Counce et al. (2000). Three seedlings for each beaker and 3 beakers for each light treatment were randomly selected for growth analysis. Plant height and root length were measured from the base of the seedling to the top of the third leaf and from the root base to the seed root tip respectively. Column diameter was measured in the seedling base with a Vernier caliper. Fresh we ights (FW) and dry weights (DW) of seedlings were measured with an electronic balance. To determine DW, seedlings were dried at 80°C until constant weights were achieved. Moisture content (%) was calculated as [1- (DW/FW)] × 100%. The health index was calc ulated as (stem diameter / plant height) × biomass according to Guo et al. (2011b).
Chlorophyll fluorescence measurements
Seedlings were kept in the dark for approximately 20 min before measurement.
Chlorophyll fluorescence was measured at the middle portion of the second leaf of the seedlings taken at ambient temperatures with a Portable Chlorophyll Fluorometer
PAM-2100 (Walz, Effeltrich, Germany). Actinic light and saturating light intensities were set at 280 μmol m-2s-1 and 2500 μmol m-2s-1 photosynthetically active radiation
(PAR) respectively. The maximal photochemical efficiency of PSII (Fv/Fm), relative quantum efficiency of PSII photochemistry (ΦPSII), photochemical quenching (qP) and
non-photochemical quenching (NPQ) were measured and calculated according to the method described previously (Kooten and Snel, 1990).
Chlorophyll (Chl), carotenoid (Car) and anthocyanin (Ant) contents
Chl and Car contents were eluted from the second leaf DW samples (0.01 g) with 5 ml of 80% acetone at 4°C overnight and determined using the methods by Porra et al. (1989) and Holm (1954) respectively. Samples were then centrifuged at 13,000 g for 5 min. Supernatants were tested to determine the absorbances of Chl a, Chl b and Car in acetone as measured with a spectrophotometer (U-2000, Hitachi, Tokyo, Japan) at wavelengths of 663.6, 646.6 and 440.5 nm respectively. Concentrations (μg g-1 DW)
of Chl a, Chl b and Car were determined using the following equations:
Chl a = (12.25 × OD663.6 – 2.55 × A646.6) × volume of supernatant (ml) / sample weight (g)
Chl b = (20.31 × A646.6 – 4.91 × A663.6) × volume of supernatant (ml) / sample weight (g) Car = [(4.69 × A440.5 × volume of supernatant (ml) / sample weight (g)) – 0.267 × (Chl a + Chl b).
Ant content was measured according to the protocol of Mancinelli et al. (1975).
A mixture of 80% methanol containing 1% HCl of solvent was used to extract the powder samples. The mixture was then centrifuged at 4°C and 3,000 rpm for 5 min and the supernatant was used to measure the absorbance at 530 nm and 657 nm. Ant content (μg g-1 DW) was calculated as (A530 - 0.33 × A657 / 31.6) × volume of supernatant (ml) /
sample weight (g).
Free amino acid, soluble sugar and starch contents
DW samples of the second leaf (0.05 g) were placed into 15 ml tubes and then 5 ml of distilled water was added and mixed in. The supernatant was collected after 30 min in a water bath at 85°C. This step was repeated once and then distilled water was added to obtain 10 ml of the extract for use in determining soluble sugar and free amino
acid contents (mg g-1 DW). The soluble sugar content was determined using the sulfuric acid anthrone method at a wavelength of 630 nm (Morris, 1948). Free amino acid content was determined using the ninhydrin method at a wavelength of 570 nm (Moore and Stein, 1948). Starch was extracted according to the procedures from Takahashi et al.
(1995).
The residue obtained after distilled water extraction was dried and then 1 ml of distilled water was added. The mixture was placed in a water bath for 30 min at 100°C.
The gelatinized starch was digested after cooling with 1 ml 9.2 N perchloric acid for 10 min. Two ml of distilled water was added and the mixture centrifuged at 8,000 g for 6 min. After the extract was transferred to a 15 ml tube, 1 ml of 4.6 N perchloric acid was added and stirred for 10 min. Three ml of distilled water were added to the final volume after centrifugation. Starch contents (mg g-1 DW) were determined using the same method for soluble sugar.
Total protein content
Total proteins were measured using the method of Bradford (1976). Samples (0.05 g FW) were ground in a mortar with liquid nitrogen to which 3 ml of a phosphate buffered solution (pH 7.0) was added. The extract was centrifuged at 13,000 g for 15 min at 4°C and 0.1 ml of the supernatant was combined with 5 ml of Coomassie
brilliant blue G-250 solution (0.1 g l-1). The soluble protein content (mg g-1 FW) was determined after 2 min at a wavelength of 595 nm.
Statistical analysis
All measurements were evaluated for significance using analysis of variance (ANOVA) followed by the least significant difference (LSD) test at the P < 0.05 level.
All statistical analyses were conducted using SAS 9.2 (SAS Institute; Cary, NC, USA).
Results
Plant growth and morphology
The effects of light quality treatments (T) on the two rice varieties (V) were monitored by measuring changes in plant height, root length, stem diameter, shoot and root biomasses, moisture content and health index at 14 d seedling. In this experiment, a factorial experiment design with a completely randomized arrangement was used. Table 1 presents that all the measured components of growth parameters were significant at the 5% level for the main effects, except for plant height and shoot moisture content in V and shoot biomass and root moisture content in both V and T which showed negligible differences. Moreover, when the V × T interaction was examined for significance, all parameters significantly differed except the plant height, shoot biomass,
moisture content of shoot and root and health index.
Plant heights of both varieties were significantly shorter (12.9 and 13.1 cm) and stem diameters were larger (0.19 and 0.16 cm) under B than other lighting treatments (Table I-1). Root lengths of both varieties were significantly shorter (12.2 and 9.1 cm) under RB than under other lighting conditions. However, shoot biomass and root moisture contents were not significantly different among all lighting environments.
Different light quality treatments affected the growth of rice seedlings and blue light likely inhibited the elongation of rice seedlings. The shoot moisture content of both varieties was lower (83.3 ~84.5%) under blue light than without (85.0~ 86.2%) indicating that blue light could increase water transport. Root biomass of TCS10 was significantly higher (0.019 g) under B compared to other lighting environments.
Lighting environments not only affected shoot growth but also mediated root elongation and root biomass accumulation.
The shoot/root dry weight ratios of TCS10 under B (1.91) and RB (2.16) were significantly lower than without blue light, but there was no significant difference in the S/R DW ratios of IR1552 among all treatments. A normal appearance and compact morphology with vigorous roots in TCS10 seedlings treated with B LED light were observed. However, seedlings grown under B light looked small or even severely dwarfed (photos not shown). The health index was used to describe the morphological
quality of rice seedlings and a higher index number contributed to shorter shoot height and larger stem diameter. Under B LED light, values were significantly higher (0.525 and 0.431) than under other lighting colors.
Chlorophyll (Chl), carotenoid (Car) and anthocyanin (Ant) contents
ANOVA was used to uncover the main effects of variety (V) and light quality treatment (T) and their interaction effects (V × T) for different pigments as summarized in Table I-2. All pigments displayed significant differences (P< 0.05) for the main effects, with the exception of Car levels. Only total Chl and Ant contents constituted significant differences for the interaction effect.
Pigment content in leaves was influenced by different lighting e nvironments.
Total Chl content in leaves of TCS10 was not significantly different among all treatments but in IR1552 it was highest (18.25 mg g-1 DW) under RB and lowest (13.24 mg g-1 DW) under R condition (Table I-2). The Chl a/b ratio of both varieties was higher (2.81 and 2.47 mg g-1 DW) under B than other lighting treatments.
The Car content in leaves of both varieties was not significantly different among all lighting environments. Changes in the Car/Chl ratio were therefore attributed to the level of total Chl content in the leaves. The level of Ant in the purple leaves of IR1552 was sensitive to lighting. Ant content in IR1552 was significantly higher (150 μg g-1
DW) under RB as compared to other conditions, indicating that light quality affected the synthesis of pigments (Chl and Ant) in rice seedling leaves.
Chlorophyll (Chl) fluorescence
Chl fluorescence components were used to indirectly measure the different functional levels of photosynthesis. Figure I-2 shows the effects of light quality on Chl fluorescence in 14 d rice leaves. The Fv/Fm ratios of both varieties were not significantly different among all lighting conditions. In healthy leaves, the Fv/Fm ratio is close to 0.8, a value typical for uninhibited plants. A lower value indicates that a portion
of the PSII reaction center is damaged (Jung et al., 1998; Somersalo and Krause, 1989).
The ΦPSII and qP of the two varieties under B were highest (0.85~0.87) among all
lighting treatments and those under R and G were at similar levels. The exception was that the qP of IR1552 under R (0.82) was significantly higher than under G (0.71).
Therefore, blue light might promote the photosynthetic potential of rice seedlings. The seedlings of TS10 under R (1.4) and G (1.3) exhibited higher NPQ than those grown in the blue light environment (1.0). This indicated thermal energy dissipation in the antennae. In IR1552, there was no significant difference in the NPQ among the R (1.0), G (0.8) and B (0.8) lighting but NPQ under RB (1.2) was slightly higher than under other lighting qualities. In general, cultivars responded differently to
light quality due to a different photosynthetic apparatus and the Chl fluorescence of two varieties varied in response to RB LED conditions.
Free amino acid, soluble sugar, starch and total protein contents: ANOVAs for variety (V), light quality treatment (T) and their interaction (V × T) for carbon–nitrogen metabolism in 14 d seedlings are tabulated in Table I-3. There were significant differences in soluble sugar, free amino acid and total protein content between the two varieties. Moreover, total protein levels were significantly affected by T and soluble sugar appeared to significantly differ in V × T.
The soluble sugar content of IR1552 was significantly greater in seedling leaves under R and G (47~48.6 mg g-1 DW) than under B and RB (37~38.7 mg g-1 DW) (Table I-3). A similar trend was observed in starch levels where R and G (23~25.4 mg g-1 DW) were greater than B and RB (14.0~15.4 mg g-1 DW), suggesting that R and G lights might stimulate carbohydrate accumulation. The soluble sugar content in TS10 seedling leaves was not significantly different among all treatments but the starch content was greatest (54.3 mg g-1 DW) when exposed to G. The only significant difference in free amino acid content was the value from TS10 seedling leaves under RB which showed the lowest level (15.3 mg g-1 DW) among all lighting qualities. The total protein of leaves was greatest (43.7 mg g-1 DW) in IR1552 under B and lowest (33.1 mg g-1 DW) in TS10 under R. Furthermore, total proteins of IR1552 under R (35.6 mg g-1 DW) were
significantly lower in comparison to other LED conditions (41.1~43.7 mg g-1 DW) indicating that blue light might promote protein synthesis in seedling leaves.
Discussion
Growth and morphological quality
Rice is widely grown in Taiwan and its production is very impor tant economically and commercially. The spectral quality of lighting is defined as the relative intensity and quantity of different wavelengths emitted by a light source and perceived by photoreceptors within a plant. Plant yields and quality are the result of interactions of various environmental factors under which plants are grown. The present study examined the effects of different spectral lighting conditions on growth parameters, pigments, chlorophyll fluorescence and carbon–nitrogen metabolism of two genotypes of rice seedling plants grown under identical environmental conditions.
Plants showed distinct growth responses to different light-quality treatments. Results from table 1 demonstrated that light quality influenced the growth and morphology of rice seedlings and blue light inhibited shoot elongation. Seedling height was shortest and stem diameter greatest under B LED conditions.
Similar results were observed in rice seedlings (Guo et al., 2011b), strawberry plantlets (Nhut et al., 2003a), sprouting broccoli (Kopsell and Sams, 2013), grapes
(Poudel et al., 2008), roses (Abidi et al., 2013) and Cymbidium plantlets (Tanaka et al., 1998b). In addition, several studies (Johkan et al., 2010; Kim et al., 2004b; Lee et al., 2007; Li et al., 2010; Nhut et al., 2003b; Ohashi-Kaneko et al., 2006b; Tanaka et al., 1998b) showed that blue and red mixed LEDs increased biomass accumulation. In our study, however, shoot biomass was unaffected by light quality. It is unlikely that red and blue mixed LEDs could promote shoot biomass. Root length was the shortest and root biomass the lowest in seedlings of the two varieties under RB LED conditions. This agrees with the reports by Guo et al. (2011b) and Nhut et al. (2003b) but differs from previous studies (Johkan et al., 2010; Lee et al., 2007; Tanaka et al., 1998b) in which red and blue mixed LEDs were shown to induce root elongation.
Liu et al. (2011) reported that a different red to blue ratio affected root morphology and an increase in blue radiation caused a longer root length. The B:R (3:1) LED light was suitable for rapeseed plantlet growth and can be used as a priority light source in the rapeseed culture system (Li et al., 2013). In our study, the energy distribution of RB LEDs was 80% red and 20% blue in PPFD (data not shown). B LED light is important for leaf expansion and enhances biomass production (Hogewoning et al., 2010; Johkan et al., 2012; Li et al., 2010). Yorio et al. (2001) also reported that there was higher dry weight accumulation in lettuce grown under R light supplemented with B light than in lettuce grown under R light alone. These results indicate that plant
responses to light quality are species- or cultivar- dependent.
The morphological quality of rice seedlings can be described by the S/R DW ratio and the health index. TCS10 seedlings under B exhibited the lowest S/R ratio (1.91) which contributed to an increase in root biomass. A higher seedling root biomass supports shoot growth by fully supplying the plant with water and mineral nutrition a nd may increase successful transplantation into the field. Poor roots cannot supply sufficient water for large shoots so plants with high S/R ratios are unsuitable for active growth (Johkan et al., 2010). In our study, the S/R DW in TS10 was not optimal under G (2.80) compared to other light colors. This observation is indicative of the poor growth of roots under G light and also indicates that root induction is probably also dependent on the spectral quality of lighting.
In addition, a growth-retarding effect might have been caused by an insufficient quality of light. The seedling health index was greatest under B which is in agreement with Guo et al. (2011b). The higher health index under the blue light environment contributed to the shorter shoot height a nd larger stem diameter which can provide a higher lodging resistance potential. Consequently, B LED light was an effective light source for plant growth and development and light spectra, intensities and durations can easily be controlled by growers in artificial growing environments.
Photosynthetic pigments and chlorophyll fluorescence
Plant pigments have specific wavelength absorption patterns known as absorption spectra. Biosynthetic wavelengths for the production of plant pigments are referred to as action spectra (Wang et al., 2009). Chl and Car have high light absorptions at 400–500 and at 630–680 nm respectively and low light absorption at 530–610 nm.
Previous studies (Guo et al., 2011b; Johkan et al., 2010; Lee et al., 2007; Lin et al., 2011;
Liu et al., 2011; Nhut et al., 2003a; Tanaka et al., 1998b) demonstrated that blue light induces the synthesis of Chl and Car. In our study, light quality also affected photosynthetic pigments in rice seedling leaves (Table I-2). Total Chl in IR1552 seedling leaves under RB was higher than other light conditions but Car in seedling leaves was not responsive to different light qualities. Although different quality lighting for all treatments were applied at the same PPFD level, plants showed similar absorption spectra of photosynthetic pigments, total Chl and Car (Table I-2).
Perhaps the applied PPFD level (160 μmol m−2 s−1) had reached a certain
minimum that is necessary for sufficient synthesis and activity of photosynthetic pigments and electron carriers. The Chl a/b ratio was mediated by lighting treatments in seedling leaves of two varieties and was higher under B compared to other lighting environments. This result is consistent with those of previous studies (Johkan et al., 2010; Lee et al., 2007; Lin et al., 2011; Wang et al., 2009). An increase in Chl a/b is
usually observed in higher irradiation environments (Evans and Poorter, 2001) suggesting it as an indicator for estima ting relative photosystem stoichiometry (Pfannschmidt et al., 1999). Plants grown under all treatments appeared to synthesize more Chl a because it has a wider spectrum compared to that of Chl b and Chl a is the molecule that makes photosynthesis possible (Calatayud and Barreno, 2004).
Furthermore, a change in the Chl a/b ratio is usually correlated with variation in PSII light-harvesting antenna size and PSII:PSI content (Leong and Anderson, 1984).
This inference is strengthened by our findings on Chl fluorescence (Figure I-2). The qP
and ΦPSII of the tested samples under B were higher than those of under R and G which
may indicate non-radiative (thermal) energy dissipation. The thermal dissipation process is called non-photochemical quenching (NPQ), referring to the fact that the thermal dissipation of Chl's excited states competes with fluorescence emission as well as with photochemistry (i.e. photosynthesis). The decreases in NPQ are associated with decreases in non-photochemical quenching. PSII activity may regulate the response of photosynthesis to light quality changes. Blue light promoted the ΦPSII and qP of seedling leaves and is in agreement with the findings by Wang et al. (2009) who indicated that the decrease in ΦPSII was due to the lower qP. This might be caused by rate- limiting
processes including the PSI and cytochrome b6/f complex processes (Wang et al., 2009).
Yu and Ong (2003) found a reduction of ΦPSII and qP in leaves under red or yellow light
compared with blue light.
In addition, blue light is essential for high light acclimation and photoprotection in the diatom Phaeodactylum tricornutum (Costa et al., 2013). These results imply that the Chl fluorescence parameters were genotype- and light quality-specific and were not expressed solely in response to an increasing excess of photon energy. Chloroplast development in TCS10 may be particularly sensitive to blue lights. Electron transport would be inhibited under conditions without blue light and NPQ would increase in TS10 seedling leaves. Both genotypes behaved similarly when their leaves were developed at 30/25°C and 160 μmol m−2s−1 PPFD inside growth chambers for 14 d and hence the genotypic differences might be related to adaptation mechanisms induced by light quality.
Carbon–nitrogen metabolism
The selected LED lights differentially affected the metabolic system of the investigated rice varieties. Seedlings under B were observed to have higher total protein content in leaves than under other monochromatic lights (Table I-3) which is in agreement with the findings by Lin et al. (2011), Guo et al. (2011b), Wang et al. (2009) and (Eskins et al., 1991). Blue light influences nitrate reductase activity for mediating the rate of nitrogen assimilation in radish plants (Maevskaya and Bukhov, 2005).
Ohashi-Kaneko et al. (2006b) and (Matsuda et al., 2004) reported that a red light environment with a supplemental blue light caused an increase in the total N content of rice leaves. This included Rubisco, cytochrome f and light- harvesting complex II and was positively correlated with photosynthetic rate and stomatal conductance. These results were consistent with our findings on Chl a/b (Table I-2) and Chl fluorescence (Figure I-2). Previous studies found that the density, length and width of stomata were enhanced in blue light-enriched environments (Kim et al., 2004b; Li et al., 2010; Poudel et al., 2008; Wang et al., 2009).
In contrast, leaves in the blue light-enriched environments of our study exhibited stronger water transport, contributing to lower moisture content. The accumulation of carbohydrates in IR1552 leaves was promoted significantly under R and G LED conditions (Table I-3). This outcome was similar to those published for rice seedlings (Guo et al., 2011b), Oncidium (Liu et al., 2011) and upland cotton plantlets (Li et al., 2010) but was opposite to the findings of (Wang et al., 2009) which indicated blue light- induced carbohydrates were accumulated in leaves. Red light induces the accumulation of carbohydrates which is attributed to inhibiting the translocation of photosynthetic products from leaves (Sæ bø et al., 1995).
IR1552 had the higher soluble sugar and starch contents under R and G LEDs, so these light sources might be beneficial for the accumulation of soluble sugars and
starches in plants. However, the amount of free amino acids in all plant leaves showed no significant differences among all treatments except for RB in TS10 leaves. This suggests that the light spectrum might not be advantageous for free amino acids synthesis.
Function of green light
Anthocyanins are one group of polyphenols that are thought to protect plants against unsuitable environments (Winkel-Shirley, 2001). A study of red leaf lettuce discovered that blue light induced the synthesis of Ant in seedling leaves (Johkan et al., 2010). Our results showed that RB lighting also induced Ant synthesis in purple leaf IR1552; however, the efficiency of green light was higher than other monochromatic lights (Table I-2). Johkan et al. (2012) tested the effects of green light wavelengths on red leaf lettuce and found that green LEDs (peak wavelength 510 nm) induced Ant synthesis in baby lettuce leaves. In our study, G LEDs had a peak wavelength of 525 nm and induced more Ant synthesis than red or blue light (Figure I-1).
Furthermore, the morphology, photosynthetic pigments, Chl fluorescence and metabolites under G performed similarly to those under R (Table I-1, Table I-2 and Table I-3; Figure I-2), which is in agreement with the trend that was observed in cucumbers (Wang et al., 2009). Green light acts as a signal source affecting the
development of wheat (Kasajima et al., 2008) and the rosette architecture of Arabidopsis (Zhang et al., 2011). However, the function of green light is not clear
(Wang and Folta, 2013); hence the effect of green light on plants is worthy of further evaluation. In addition, it will be interesting to test more rice varieties and lines for seedling growth when illuminated by various monochromatic light spectra and combinations under a wide range of light intensities.
Wavelength (nm)
300 400 500 600 700 800
Relative energy (Watt m-2 s-1 )
0 10000 20000 30000 40000
R G B RB
Figure I-1. The spectral distributions of different light treatments. Spectral scans were recorded at the top of the plant canopy with a spectroradiometer.
R G B RB
qP
0.0 0.2 0.4 0.6 0.8 1.0 1.2
f de
ab cd
bc
ef
a a
Fv/Fm
0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4
TCS10 IR1552
ab ab abab a ab ab
b
PSII
0.0 0.2 0.4 0.6 0.8 1.0 1.2
d cd
a
b b bc
a a
NPQ
0.0 0.5 1.0
1.5 a
ab
cd
e cd
de de
bc
Figure I-2. Effects of light quality on the relative value of chlorophyll fluoresce nce.
Leaves were analyzed from 14 d seedlings under different light environments. Values are the mean of ten plants from two replicates consisting of five plants each. The values followed by the different letter show statistically significant differences at P < 0.05.
Table I-1. The growth parameters of 14 d seedlings cultivated under different light environments.
Variety Treatment
Plant height
(cm)
Root length (cm)
Stem diameter
(cm)
Shoot biomass
(g)
Root biomass
(g)
Shoot/Root Ratio (w/w)
Shoot moisture content (%)
Root moisture content (%)
Health index
TCS10
R 19.5a 13.7 ab 0.16 b 0.038a 0.015b 2.49 b 85.0 ab 92.3a 0.310c
G 20.1a 14.4 a 0.17 ab 0.036a 0.013c 2.80 a 86.1 a 93.2a 0.310c
B 12.9c 14.1 ab 0.19 a 0.035a 0.019a 1.91 c 83.3 d 93.1a 0.525a
RB 19.1a 12.2 c 0.16 b 0.036a 0.017b 2.16 c 84.2 cd 93.0a 0.307c
IR1552
R 19.6a 13.0 bc 0.13 c 0.034a 0.013c 2.66 ab 85.9 ab 92.4a 0.218d
G 19.5a 12.1 c 0.12 c 0.034a 0.013c 2.57 ab 86.2 a 93.3a 0.221d
B 13.1c 11.9 c 0.16 b 0.036a 0.013c 2.79 a 84.5 bcd 92.9a 0.431b
RB 16.8b 9.1 d 0.15 b 0.036a 0.013c 2.69 ab 83.8 cd 92.8a 0.340c
ANOVA F tests
Variety (V) ns <0.0001 <0.0001 ns <0.0001 <0.0001 ns ns 0.0045
Treatment (T) <0.0001 <0.0001 0.0003 ns 0.0016 0.0041 0.0007 ns <0.0001
V × T ns 0.0489 0.0160 ns 0.0007 <0.0001 ns ns ns
Biomass is the total weight of 3 seedlings. Values for ANOVA F tests are type I observed significance levels. Within columns, means followed by the same letter are not significantly different according to LSD (0.05). ns, non-significant at P < 0.05.
Table I-2. The effect of light quality on pigments in 14 d seedling leaves.
Variety Treatment Total Chl
(mg g-1 DW) Chl a/b Car
(mg g-1 DW) Car/Chl Ant (μg g-1 DW)
TCS10
R 14.49 bc 2.55 bc 3.52 ab 0.24 ab nd
G 14.02 bc 2.77 ab 3.70 ab 0.26 a nd
B 13.45 c 2.81 a 3.52 ab 0.26 a nd
RB 13.94 bc 2.53 bc 3.20 ab 0.23 ab nd
IR1552
R 13.24 c 2.19 d 2.84 b 0.22 bc 15 c
G 15.48 b 2.20 d 3.35 ab 0.22 b 33 b
B 15.19 b 2.47 c 3.52 ab 0.23 ab 17 c
RB 18.25 a 2.06 d 3.24 ab 0.18 c 150 a ANOVA F tests
Variety (V) 0.0015 <0.0001 ns 0.0006 <.0001 Treatment (T) 0.0083 0.0060 ns 0.0239 <.0001
V × T 0.0020 ns ns ns <.0001
nd, non-detectable. Values for ANOVA F tests are type I observed significance levels. Within columns, means followed by the same letter are not significantly different according to LSD (0.05); ns, non-significant at P < 0.05.
Table I-3. Effects of light quality on the carbon–nitrogen metabolism of seedling leaves collected from 14 d seedlings under different light environments.
Variety Treatment Soluble sugar (mg g-1 DW)
Starch (mg g-1 DW)
Free amino acid (mg g-1 DW)
Total protein (mg g-1 FW)
TCS10
R 50.3 ab 23.1b 21.4 a 33.1 d
G 53.5 ab 54.3a 19.2 ab 34.7 cd
B 52.3 ab 21.5b 19.1 ab 41.0 ab
RB 57.6 a 21.5b 15.3 b 38.0 bc
IR1552
R 48.6 b 25.4b 19.6 a 35.6 cd
G 47.0 b 23.0b 21.6 a 41.1 ab
B 37.0 c 14.0c 21.4 a 43.7 a
RB 38.7 c 15.4c 21.2 a 41.7 ab
ANOVA F tests
Variety (V) <0.0001 ns 0.0362 0.0034
Treatment (T) ns ns ns 0.0010
V × T 0.0162 ns ns ns
Values for ANOVA F tests are type I observed significance levels. Within columns, means followed by the same letter are not significantly different according to LSD (0.05); ns, non-significant at P < 0.05.
II. Water-use efficiency and nitrogen uptake in rice seedlings grown under different light treatments
Abstract
In this study, our objective was to investigate the response of time- integrated water- use efficiency (WUE), 13C discrimination (Δ13C) and nitrogen uptake in hydroponic seedlings (V2-V3) of rice (Oryza sativa L.) cultivars, Taichung shen 10 (TCS10) and IR1552, grown under different light treatments. Light emitting diode (LED) lighting systems were used to control light quality. Light treatments for rice
seedlings included red (R), blue (B), green (G) and red + blue (RB), with fluorescent light (FL) as control, photon flux density (PPFD) set at 105 μmol m-2 s-1. The WUE
response was highest for seedlings grown under R light, then (in decreasing order)
seedlings grown under G, RB, and B light. WUE had a high positive correlation with Δ13C, except under FL light.
Nitrogen content (%N) and δ15N values were used to estimate the effect of fertilizer uptake under different lighting conditions. The results showed that the amount of N in seedlings derived from fertilizer (Nf) was highest under B light, and was lowest under R light. Therefore, we concluded that blue light may increase stomatal conductance and transpiration, decrease WUE, and promote root N uptake. In this study, we also demonstrated the application of stable C and N isotope techniques for crop
physiology.
Key words: Light quality, Rice seedling, Water-use efficiency, 13C discrimination, Nitrogen uptake, 15N isotope, 13C isotope
Introduction
Light is the main energy source for plant photosynthesis and is used as an environmental signal to trigger growth and structural differentiation in plants. Light quality, quantity, and photoperiod controls the morphogenesis, growth, and differentiation of plant cells, tissues, and organ cultures (Abidi et al., 2013). Plant development is strongly influenced by light quality, which refers to the color or wavelengths reaching a plant’s surfaces (Johkan et al., 2010). Red (R) and blue (B) lights have the greatest impact on plant growth because they are the major energy sources for photosynthetic CO2 assimilation in plants. It is well known that spectra have action maxima in the B and R ranges (Kasajima et al., 2008). The integration, quality, duration, and intensity of red light/far red light, blue light, mixed red and blue lights (RB), UV-A (320–500 nm) or UV-B (280–320 nm), and hormone signaling pathways have a profound influence on plants by triggering or halting physiological reactions and controlling the growth and development of plants (Clouse, 2001; Shin et al., 2008).
Recent research and review articles report that green light also affects the morphology, metabolism, and photosynthesis of plants (Johkan et al., 2012; Wang and Folta, 2013;
Zhang et al., 2011).
Light sources such as fluorescent, metal- halide, high-pressure sodium and incandescent lamps are generally used for crop cultivation. These sources are applied to
increase photosynthetic photon flux levels but contain unnecessary wavelengths that are located outside the photosynthetically active radiation spectrum and are of low quality for promoting growth (Kim et al., 2004b). Compared to artificial light sources, light-emitting diode (LED) lighting systems are ideal for use in plant light studies and allow wavelengths to be matched to plant photoreceptors for providing more optimal production and influencing plant morphology and metabolism (Bourget, 2008; Massa et al., 2008). Spectral light changes induce different morphogenetic and photosynthetic responses that can vary among different plant species. Such photo responses are of practical importance in recent plant cultivation technologies since the feasibility of tailoring illumination spectra enables one to control plant growth, development and nutritional quality.
Water‐use efficiency (WUE), which is defined as dry matter produced per unit of water transpired, is perceived as an important attribute for growth and influenced by genetic (Chen et al., 2012; Monclus et al., 2012; Takai et al., 2009) and environmental factors, including water (Grant et al., 2012; Liu et al., 2012; Wang et al., 2013b; Yasir et al., 2013), fertilizer (Guo et al., 2011a; Wang et al., 2013b), and tillage level (Dalal et al., 2013). At the agronomic scale (crop level), WUE is considered as the accumulated dry matter divided by water consumed by the crop during the whole growth cycle (Condon et al., 2004; Tambussi et al., 2007). Carbon isotope discrimination (Δ13C) is an
alternative screening technique for water use efficiency which is highly correlated with transpiration efficiency, and has been demonstrated to be a simple but reliable measure of WUE (Farquhar and Richards, 1984; Farquhar et al., 1982). The advantages of Δ13C expressed on a dry matter basis are that it gives integrated data over a whole period of growth and is suitable for high-throughput screening, because samples can be stored for further measurement (Condon et al., 1987). Several studies (Cabrera‐Bosquet et al., 2007; Chen et al., 2012; Glenn, 2014; Moghaddam et al., 2013; Monclus et al., 2012;
This et al., 2010) have utilized Δ13C as a tool for screening WUE of different C3 plant species. Similarly, intrinsic differences in the nitrogen isotope value (δ15N) of different N sources (such as soil and fertilizer) can be used to estimate their relative contribution to the crop N uptake (Shearer and Kohl 1993). If differences in natural abundance of δ15N values of different N sources occur, then this approach can be successfully used.
Dalal et al. (2013) successful evaluated the N uptake and NUE of wheat using δ15N values.
Our previous studies (Chen et al., 2014a) found that blue light LEDs enhanced relative quantum efficiency of PSII photochemistr y and photochemical quenching, but reduced non-photochemical quenching of seedling leaves, and also showed higher total protein content in the tested leaves compared to B plus RB. Light quality might mediate the photosynthetic potential and nitrogen uptake/metabolism in rice seedling.
Furthermore, WUE is also mediated under different light spectral compositions.
However, there are fewer studies describing the effects of LED lighting on WUE and nitrogen uptake. Our objective is to investigate the effect of light quality on WUE and N uptake of rice seedling using a stable isotope approach.
Materials and Methods
Plant species and growth conditions
In this study, we used rice (Oryza sativa L.) cultivar IR1552, which is famous
for its purple leaves, and rice cultivar Taichung shen 10 (TCS10, green leaf), one of the
most widely grown rice cultivars in Taiwan. Seeds were sterilized with 2% sodium hypochlorite for 20 min, washed extensively with distilled water, and germinated in
Petri dishes on wet filter paper at 37 °C in the dark. After 48 h of incubation, uniformly
germinated seeds were selected and cultivated in a 150 ml beaker containing a
half-strength Kimura B nutrient solution with the following macro and microelements:
182.3 μM (NH4)2SO4, 91.6 μM KNO3, 273.9 μM MgSO4·7H2O, 91.1 μM KH2PO4, 182.5 μM Ca(NO3)2, 30.6 μM Fe-citrate, 0.25 μM H3BO3, 0.2 μM MnSO4·H2O, 0.2 μM ZnSO4·7H2O, 0.05 μM CuSO4·5H2O, and 0.07 μM H2MoO4. Nutrient solutions (pH 4.7)
were replaced every 3 d. Hydroponically cultivated rice seedlings were raised in growth
chambers under LED lights at 30 °C and 25 °C, held day and night, respectively, over a
12 h photoperiod. All hydroponic seedlings were collected on day 14 after reaching stage V2 or V3 according to Counce et al. (2000). The shoots and roots of the seedlings were frozen and freeze-dried before analysis.
Light treatments
LED lighting systems designed by GRE Technology (Taipei, Taiwan) were used to control light quality. Spectral distributions of blue (peak at 460 nm), red (peak at 630 nm), and green (peak at 530 nm) were measured using a spectro-radiometer (LI-COR1800, Lincoln, NE, USA) in the 300-800 nm range. These LED emission peaks closely coincide with the absorption peaks of chlorophyll a and b, and the reported wavelengths are at their respective maximum photosynthetic efficiencies (McCree, 1972). Light treatments for rice seedlings, proliferation, and differentiation consisted of red LEDs (R), blue LEDs (B), green LEDs (G), a mixture of red plus blue LEDs (R:B = 4:1 by photon flux density; RB), and fluorescent lighting (FL).
Photosynthetic photon flux density (PPFD) was uniformly set at 105 μmol m-2 s-1. The
experiment was independently performed three times under randomized growth conditions
