Increased photosynthetic capacity in response to

Increased photosynthetic capacity in response to nitrate is correlated with enhanced cytokinin levels in rice cultivar with high responsiveness to nitrogen nutrients Wenjing Song, Jiao Li, Huwei Sun, Shuangjie Huang, Xianpo Gong, Qunyu Ma, Yali Zhang & Guohua Xu Plant and Soil An International Journal on Plant-Soil Relationships ISSN 0032-079X Volume 373 Combined 1-2 Plant Soil (2013) 373:981-993 DOI 10.1007/s11104-013-1867-x

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Author's personal copy Plant Soil (2013) 373:981–993 DOI 10.1007/s11104-013-1867-x

REGULAR ARTICLE

Increased photosynthetic capacity in response to nitrate is correlated with enhanced cytokinin levels in rice cultivar with high responsiveness to nitrogen nutrients Wenjing Song & Jiao Li & Huwei Sun & Shuangjie Huang & Xianpo Gong & Qunyu Ma & Yali Zhang & Guohua Xu

Received: 17 May 2013 / Accepted: 26 July 2013 / Published online: 9 August 2013 # Springer Science+Business Media Dordrecht 2013

Wenjing Song and Jiao Li contributed equally to this paper.

of exogenous cytokinin (6-BA) on leaf growth and photosynthetic activity was examined. Results Cell expansion and CO2 assimilation in the first fully expanded leaf were enhanced by PNN in Nanguang but not in Elio. The concentrations of cytokinins in roots, xylem sap, and leaves of Nanguang increased approximately 25–34 % with PNN compared with sole NH4+, but no difference was observed in Elio. Exogenous 6-BA counteracted the effects of sole NH4+ on leaf growth and photosynthetic activity in both cultivars. OsIPT3 was the key NO3–-responsive cytokinin synthesis gene in cv. Nanguang. Conclusions High NO3– responsiveness is associated with increased cytokinin synthesis and transport from the root to the leaf and is strongly related to a higher photosynthetic capacity in cv. Nanguang.

Electronic supplementary material The online version of this article (doi:10.1007/s11104-013-1867-x) contains supplementary material, which is available to authorized users.

Keywords Cytokinin . Leaf growth . Nitrate . Photosynthetic capacity . Rice

Abstract Background and aims Ammonium (NH4+) is the preferred nitrogen nutrient over nitrate (NO3–) in Oryza sativa L. (rice), but photosynthetic capacity is enhanced by partial NO3– nutrition (PNN). The role of cytokinin in the effects of PNN on photosynthetic capacity is unknown. Methods We investigated effects of PNN on six cytokinin fractions in roots, xylem sap, and leaves and on the expression of eight cytokinin synthesis genes in the roots of Nanguang and Elio rice cultivars. The effect

Responsible Editor: Hans Lambers.

W. Song : J. Li : H. Sun : S. Huang : X. Gong : Q. Ma : Y. Zhang (*) : G. Xu Key Laboratory of Plant Nutrition and Fertilization in Low-Middle Reaches of the Yangtze River, Ministry of Agriculture, Nanjing Agricultural University, Nanjing 210095, China e-mail: [email protected] W. Song Key Laboratory of Tobacco Biology and Processing, Ministry of Agriculture, Tobacco Research Institute of Chinese Academy of Agriculture Sciences, Qingdao 266101, China

Abbreviations A CO2 assimilation rate Ci intercellular CO2 partial pressure 6-BA 6-benzylaminopurine gs stomatal conductance iP N6-(Δ2-isopentenyl) adenine iPA iso-pentenyl adenosine NUE nitrogen use efficiency OsIPT rice adenosine phosphateisopentenyltransferase

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PNN Tr Z ZR

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partial nitrate nutrition transpiration rate zeatin zeatin riboside

Introduction Nitrogen (N) is a major limiting nutrient for plant growth and development. The most abundant source of N for plant roots in aerobic soils is nitrate (NO3–). In contrast, ammonium (NH4+) is the main form of available N in flooded paddy soils due to the anaerobic soil conditions (Sasakawa and Yamamoto 1978). For most plants, mixed NH4+ and NO3– nutrition is documented to be superior to NH4+ and/or NO3– sources (Marschner 1995). The proportions of NH4+ and NO3− that are optimal for plant growth differ among plant species, developmental stages, and concentrations of total supplied N (Chaillou et al. 1991; Claussen 2002; Lu et al. 2009; Stratton et al. 2001; Xu et al. 2002; Zou et al. 2005). Maximum growth and yield are usually obtained with an NH4+ concentration not more than 30 % of total N in aerobically grown crops and a NO3– concentration not more than 35 % of total N in anaerobically grown crops (Claussen 2002; Lu et al. 2009; Sandoval-Villa et al. 2001; Song et al. 2011b; Takács and Técsi 1992; Xu et al. 2002). No difference was observed in plant growth for different N forms under low light intensity, but differences in growth were pronounced under high-light intensity, indicating that the effects of different N forms on plant growth are related to photosynthetic capacity (Seel et al. 1993; Zhu et al. 2000). The type of N source affects photoenergy consumption and due to reduction of NO3– in the leaf, a substantial portion of electron transport chain products can be used for NO3– assimilation (Bloom et al. 1989; Huppe and Turpin 1994; Salsac et al. 1987). The photoenergy cost for NH4+ supply is 145 % less than for NO3– supply (Raven 1985). Furthermore, the effects of different N forms on photosynthesis are also associated with gas exchange parameters such as stomatal conductance (gs) and intercellular CO2 partial pressure (Ci) (Guo et al. 2007; Raven and Farquhar 1990). Gao et al. (2010) observed that the CO2 assimilation rate (A) and carboxylation efficiency in rice were significantly enhanced by combined NH4+ and NO3– nutrition compared to sole NH4+ nutrition.

Although the process of photosynthetic acclimation by plants to different N forms has been extensively studied, little is known about the signaling mechanisms by which acclimation is regulated. Transpiration rate (Tr) is significantly affected by different N forms in several plant species (tobacco, Lu et al. 2005; cucumber and rice, Zhou et al. 2011). Photosynthetic acclimation to environmental factors may be perceived indirectly through changes in the Tr and the import of compounds via the xylem (Boonman et al. 2007; Pons et al. 2001). Among the compounds carried in the xylem sap, the hormone cytokinin is likely to have a regulatory role in photosynthetic acclimation for three reasons. First, import of rootsynthesized cytokinin into the shoot depends on the shoot Tr (Aloni et al. 2005; Boonman et al. 2007), so cytokinin distribution among leaves with different Trs is likely to be regulated similarly. Second, cytokinin is known to increase photosynthetic capacity through increases in leaf expansion, A, gs, and Rubisco, and to stimulate the development of functional chloroplasts (Cortleven and Valcke 2012; Lazova and Yonova 2010; Monakhova and Chernyad’ev 2007). Third, a link between cytokinin and N was indicated by a close correlation between N supply and cytokinin content in several species (tobacco, WalchLiu et al. 2000; Arabidopsis, Takei et al. 2004; tomato, Lu et al. 2009; rice,Ding et al. 2012). The stimulatory effect of NO3− on shoot growth in tobacco and wheat was shown to correspond to an increase in zeatin (Z) and zeatin riboside (ZR) levels in leaves and xylem sap, and the inhibitory effect of a sole NH4+ supply corresponded to decreases in Z and ZR levels (Garnica et al. 2010; Rahayu et al. 2005; Walch-Liu et al. 2000). In contrast, Lu et al. (2009) showed that the stimulatory effect of a mixture of NO3− and NH4+ on shoot growth in tomato was linked to a decrease in the total concentration of cytokinin, especially N6-(Δ2-isopentenyl) adenine (iP), iso-pentenyl adenosine (iPA), and Z in xylem sap compared with solely the NO3− supply. These results suggested that cytokinins may be key players in root-toshoot communication and involved in nutrition provided by different N forms nutrition . NH4+ is the preferred nutrient form of N over NO3– in rice (Oryza sativa L.) due to its waterlogged growth environment. Although the predominant form of mineral N in the bulk soil of rice paddy fields is likely to be NH4+, rice roots are exposed to partial NO3– nutrition (PNN) by nitrification in the rice rhizosphere (Li et al. 2007). A significantly higher positive response to PNN was observed in rice cultivars with high N-use efficiency (NUE)

Author's personal copy Plant Soil (2013) 373:981–993

than in cultivars with low NUE (Duan et al. 2007a, b; Song et al. 2011a, b). Zhang et al. (2011) showed that the effects of different N forms on plant growth are closely related to photosynthetic activities regulated by PNN. Little work has been undertaken to clarify the mechanism behind changes in photosynthetic capacity in response to PNN or to what extent that response is linked to cytokinin. We propose that changes in photosynthetic capacity in response to NO3– supply correlates with parallel changes in cytokinin synthesis and translocation in the Nanguang and Elio rice cultivars, which show high and low responsiveness to NO3– respectively. The concentrations of six cytokinin fractions in roots, xylem sap, and leaves and the relative expression levels of eight rice adenosine phosphate-isopentenyltransferase (OsIPT) genes were examined in the Nanguang and Elio cultivars under NH4+ nutrition with and without NO3–. Leaf area, cell size, cell numbers in leaves, and photosynthetic activity were observed in response to exogenous application of 6-benzylaminopurine (6-BA) to plants grown solely under NH4+ nutrition.

Materials and methods Plant material Two japonica rice cultivars, Nanguang and Elio, were selected from 177 japonica rice cultivars based on their similar growth duration and differential responses to N application in field trials conducted in 2003 and 2004. Actually, at the first seven rice cultivars with high NUE and three with low NUE were selected from 177 japonica rice cultivars to study their responsiveness to PNN. Further study under hydroponic condition showed that five out of seven rice cultivars with high NUE were sensitive to PNN (Duan et al. 2007b) and other three rice cultivars with low NUE were insensitive to PNN (Song et al. 2011a). Finally, cvs Nanguang and Elio were selected as representative rice cultivars respectively in our experiment. Nanguang was identified as one of high NO3–-response cultivars because it exhibited high N accumulation and grain yield under PNN compared with NH4+ as the sole N source (Duan et al. 2007a, b) and was determined to be one of rice cultivars with high NUE due to high biomass production and grain yield under low N supply and a positive response to increasing N supply (Zhang et al. 2009). Conversely, Elio was identified as one of low NO3–-response cultivars due to low N

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accumulation and grain yield under PNN relative to NH4+ as a sole N source (Duan et al. 2007a, b). Elio produced a lower biomass and grain yield under both low and high N supply and was thus identified as a low NUE cultivar (Zhang et al. 2009). Plant growth and hormonal treatments Plants were grown in a greenhouse under natural light at 30/18 °C day/night temperatures. Seven-day-old seedlings of uniform size and vigor were transplanted into holes in a lid placed over the top of pots (four holes in a lid and two seedlings per hole). All pots were filled with 5 L of a nutrient solution (see below). Seedlings were grown in either of two NH4+-N/NO3–-N ratios [i.e., 100/0 (NH4+) and 75/25 (PNN)] by adding 1.43 mM N in the form of either (NH4)2SO4 or a mixture of (NH4)2SO4 and NH4NO3. Each treatment had 12 replications arranged in a completely randomized design to avoid edge effects in the greenhouse. The composition of the nutrient solution was as follows: 0.3 mM NaH2PO4, 2.0 mM K2SO4, 1.0 mM CaCl2, 1.5 mM MgSO4·7H2O, 1.7 mM Na2SiO3, 20.0 μM Fe-EDTA, 9.1 μM MnCl2, 0.4 μM (NH4)6Mo7O24, 37.0 μM H3BO3, 0.8 μM ZnSO4, and 0.3 μM CuSO4. A nitrification inhibitor (dicyandiamide, 7.0 μM) was added to each pot to prevent NH4+ oxidation. The nutrient solution was renewed every 2 days. Nitrate was not detected in the solely NH4+ nutrient. The pH of all nutrient solutions was adjusted to 5.5 daily with 0.1 M NaOH or 0.1 M HCl. Rice plants were harvested 14 days after treatments. Roots, xylem sap, and leaf samples were snap-frozen in liquid N2 and stored in a −40°C freezer for subsequent determination of cytokinin content and relative expression levels of genes. The 6-benzylaminopurine (6-BA) was widely used in experiments to mimic the effect of cytokinin on plant growth (Ding et al. 2012; Tian et al. 2005). Furthermore, endogenous cytokinin (such as Z and ZR) accumulated rapidly and abundantly under exogenous 6-BA supply according to the result of Ding et al. (2012). Therefore, 6BA, a synthetic cytokinin, was used here due to the effects of 6-BA on rice growth correlative to cytokinin. In the experiment with exogenous 6-BA supply, the concentration of the 6-BA in this study was determined based on our preliminary experiment. And the application time of 6-BA followed the method of Tian et al. (2005). Two rice genotypes were grown initially in nutrient solutions containing sole NH4+ and PNN. After


7 days, 20 μM 6-BA was added to the nutrient solution with only NH4+ nutrition. Plants were harvested 7 days after the 6-BA treatments. Collection of xylem sap Xylem sap was collected as described by Lu et al. (2009) and Song et al. (2011b) and slightly revised as follows. For collection of xylem sap, plant shoots were cut to 2 cm above the root-shoot interface. After 5 min, the cut stem was cleaned with distilled water to avoid contamination with the contents of wounded cells and phloem sap and then a silicon tube was fixed over the stem. The xylem sap driven by root pressure was collected at short intervals with a Pasteur pipette lasting for 3 h with four times from 6:00 pm to 6:00 am and immediately stored on ice. Leaf area, cell size, and cell number Leaf length and width were determined daily at the same time of day using a ruler and the leaf area was calculated using a formula assuming an ellipsoid leaf shape (Walch-Liu et al. 2000): Leaf area ¼ πab; where a is the leaf length×0.5, and b is the leaf width×0.5. Leaf growth rate was expressed as the increase in leaf area per day. The size of epidermal cells in the first fully expanded leaf was estimated by microscopic analysis of signals from the fluorescent probe propidium iodide (SigmaAldrich, St. Louis, MO, USA). Samples of attached cells were fixed with 70 % ethanol and stained with 50 mg L–1 propidium iodide for 2 h. Fluorescence was excited with light (λ>600 nm) and analyzed for red fluorescence with a microscope (Raquel and Rebeccal 1997). Photographs were taken of distinct areas and the average cell size was calculated based on cell counts per unit leaf area. Cell number per leaf was determined by extrapolation of the cell number per unit leaf area to the total leaf area. Gas exchange measurements Gas exchange in newly expanded leaves was measured 2 days before harvest from 09:00 to 15:00 h with a LiCor 6400 (LI-COR Biosciences, Lincoln, NE, USA)

Plant Soil (2013) 373:981–993

portable photosynthesis open system. The measurements were conducted on different plants in one pot. Prior to the initial measurements, the ambient CO2 concentration in the cuvette was adjusted to atmospheric CO2 concentration (400 μmol CO2 mol–1 air) with a photosynthetic photon flux density (PPFD) of 1500 μmol m–2 s–1. Leaf temperature was controlled at about 27 °C, and relative humidity in the leaf chamber was roughly 45 % throughout the measurements. Data were recorded after steadystate equilibration was reached. One leaf was enclosed in a chamber to measure the light response curve at an ambient CO2 level of 400 μmol CO2 mol–1 air. Determination of six cytokinin fractions The sample preparation and measurement of six cytokinin fractions in root, xylem sap, and the first fully expanded leaf by high-performance liquid chromatography (HPLC) were carried out according to the method described by Song et al. (2011b) and Lu et al. (2009) with some modifications. The plant tissues were ground with quartz sand and butyleret hydroxytoluen (BHT) in liquid N2 and lixiviated in 80 % methanol (23–30 mL) for 12–16 h. The extracted fluid was collected and concentrated in a rotary evaporator from 40 mL to 10 mL, and the concentrated fluid was extracted with an equal volume of petroleum ether. Concentrated fluid from plant tissues and xylem sap was adjusted to pH 8.5, polyvinylpyrrolidone (0.2 g) was added, and the mixture was vibrated for 30 min, filtered through a 0.45-μm filter, and passed over an OASIS HLB cartridge (Waters, Milford, MA, USA). The cartridge was initially washed with 0.1 M acetic acid and eluted with 4 mL of a mixture of 25 % (v/v) methanol and 0.1 M acetic acid followed by 70 % (v/v) methanol only. After vacuum evaporation, the purified samples were brought to a volume of 1 mL with mobile phase and loaded onto a reverse-phase HPLC column. Standard cytokinin fraction samples [zeatin (Z), zeatin riboside and dihydrozeatin riboside (ZR+(diH)ZR), kinetin (KT), N6-(Δ2-isopentenyl) adenine (iP), and iso-pentenyl adenosine (iPA)] were obtained from Sigma-Aldrich. The chromatographic conditions used were as described by Lu et al. (2009): Waters 600 Series HPLC system with a 2487 detector; Hibar column RT 250×4.6 mm; Purospher STAR RP-18 (5 μm); a column temperature of 45 °C; fluid phase: methanol:1 % acetic acid (v/v, 40/60), isocratic elution; fluid rate: 0.6 mL min−1; UV detector, l=269 nm; and an injection volume of 20 μL. A


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TCCGTGCG-3′ (reverse) for OsIPT3 (accession number AB239799); 5′-TGGATGTGGTGACGAACAAG GTGAC-3′ (forward) and 5′-GATCTACGTCGACCC AGAGGAAGCA-3′ (reverse) for OsIPT4 (accession number AB239800); 5′-AGGTGATCAACGCCGA CAAGCTGCA-3′ (forward) and 5′-TCGACGAGCTC CTCGATGTAGGAGT-3′ (reverse) for OsIPT5 (accession number AB239801); 5′-GATCGATGCGGCAT ATCTCATCACC-3′ (forward) and 5′-CCTCCAATT GCCCAAAAGGATCCAC-3′ (reverse) for OsIPT6 (accession number AB239803); 5′-TGGACGACATGGT GGACGCTGGCAT-3′ (forward) and 5′-GCTTTGATG TCGTCGATCGCCTCGG-3′ (reverse) for OsIPT7 (accession number AB239804); 5′-GTCGACGACGAT GTTCTCGACGAAT-3′ (forward), 5′-TGTTGGCCTT GATCTCGTCTATCGC-3′ (reverse) for OsIPT8 (accession number AB239805); and 5′-GGAACTGGTAT GGTCAAGGC-3′ (forward) and 5′-AGTCTCATG GATAACCGCAG-3′ (reverse) for OsACT (accession number AB047313). Two independent plant cultivations were conducted and all samples were analyzed three times by this technique.

0.22-μm filter was used for filtration of both the buffer and the samples before HPLC analysis. Semiquantitative real-time PCR analysis Total RNA from plant tissues was isolated using the guanidine thiocyanate extraction method with TRIzol reagent (Invitrogen, Shanghai, China). First-strand cDNA synthesis using M-MLV reverse transcriptase (Promega Madison, WI, USA) was performed with approximately 1 μg total RNA and oligo-DT primer (Invitrogen) according to the manual. The mRNA levels were analyzed by semiquantitative real-time PCR. The primers used for amplification were as follows: 5′ACCAAGCCCAAGGTTATCTTCGTGC-3′ (forward) and 5′-TCGTCGGTGACCTTGTTGGTGATGA-3′ (reverse) for OsIPT1 (accession number AB239797); 5′-AGTCACCCAAGCCCAAGGTCGTCTT-3′ (forward) and 5′-CTCCTCGGTGACCTTGTTCG TGATG-3′ (reverse) for OsIPT2 (accession number AB239798); 5′-GAGCTGTGCTTCCTGTGGGTGG ACT-3′ (forward) and 5′-GCGACCTTGTACTTGTC a

c a

NH4+

b

PNN

3.6

c 2.7 f f

f f

ef ef

ee

d

1.8 0.9 0

b

0.2

a

a

a

0.15 b 0.1

0.05

0 Nanguang

6

Elio

aa

5

Leaf area (cm 2 )

Leaf growth rate (cm 2 /d)

Leaf area (cm 2 )

4.5

bb

4 dd

cd cd

cc

cc

3 2 1 0 1

3 5 7 10 T reatment time (d)

14

Fig. 1 Leaf area in cvs Nanguang (a) and Elio (b) and leaf growth rate (c). Seedlings were grown under NH4+-only and partial NO3– nutrition (PNN) for 14 days. Data are means of 12

replications±SE. Means with the same letter are not significantly different at P≤0.05 according to one-way ANOVA followed by the LSD test


Plant Soil (2013) 373:981–993

a

NH4+

NH4++6-BA

PNN

b

NH4+

Cell size (×102 µm 2 )

21

Nanguang

NH4++6-BA

PNN

b

a

ab bc bc

c 14

7

0 a

Cell number (×104 )

c

Elio

b

b

a

a

b

22

11

0 Nanguang

Fig. 2 Epidermis cells (a), cell number (b), and cell size (c) in first fully expanded leaf of cvs Nanguang and Elio. Seedlings were grown under NH4+-only and partial NO3– nutrition (PNN) for 14 days. In the experiment with exogenous 6-benzylaminopurine (6-BA) supply, seedlings were grown initially in nutrient solution

Elio

containing NH4+-only for 7 days and 20 μM 6-BA was added to the NH4+-only treatment for 7 more d. Bar=50 μm. Data are means of 12 replications±SE. Means with the same letter are not significantly different at P≤0.05 according to one-way ANOVA followed by the LSD test

Leaf area (cm 2 )

Data analysis 6.5

NH4+

5.2

PNN NH4++6-BA

c 3.9

a b

b

c

d

2.6 1.3

Data from experiments were pooled for calculations of means and standard errors (SEs) and analyzed by oneway ANOVA followed by the LSD test at P≤0.05 to determine the statistical significance of the differences between individual treatments. All statistical evaluations were conducted using SPSS (version 11.0) statistical software (SPSS Inc., Chicago, IL, USA).

0 Nanguang

Elio

Fig. 3 Leaf area of the first fully expanded leaf in cvs Nanguang and Elio. Seedlings were grown under NH4+-only and partial NO3– nutrition (PNN) for 14 days. In the experiment with exogenous 6-benzylaminopurine (6-BA) supply, seedlings were grown initially in nutrient solution containing NH4+ only for 7 day and 20 μM 6-BA was added to the NH4+-only treatment for 7 more d. Data are means of 12 replications±SE. Means with the same letter are not significantly different at P≤0.05 according to one-way ANOVA followed by the LSD test

Results Leaf growth Leaf area of the first fully expanded leaf was more responsive to PNN than to NH 4 + alone in cv. Nanguang. The increase in leaf area was 16 % greater at 10 days and 26 % greater at 14 days for PNN than for NH4+ alone in cv. Nanguang (Fig. 1). However, no


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Table 1 CO2 assimilation rate (A), stomatal conductance (gs), intercellular CO2 concentration (Ci), transpiration rate (Tr), and apparent quantum yield (α) in response to NH4+ only, partial

nitrate nutrition (PNN), and NH4+ only with 20 μM 6-BA added in cvs Nanguang and Elio

Nanguang

Elio

NH4+

PNN

NH4+ + 6-BAa

NH4+

PNN

A (μmol CO2 m–2 s–1)

18.91±0.61b

24.19±0.83a

24.12±1.22a

16.61±0.24c

16.91±0.55c

gs (mol H2O m–2 s–1)

0.32±0.01b

0.39±0.02a

0.39±0.01a

0.28±0.01c

0.27±0.02c

284.23±10.13a

260.18±10.36a

264.60±10.23a

Ci (μmol CO2 mol–1 air)

276.45±11.45a

Tr (mmol H2O m–2 s–1) α

280.23±9.37a

NH4++6-BA 22.86±0.72a 0.38±0.01a 277.90±10.36

6.04±0.34b

7.22±0.12a

7.20±0.14a

5.14±0.15c

5.07±0.22c

7.14±0.12a

0.041±0.01a

0.041±0.01a

0.042±0.01a

0.041±0.01a

0.042±0.01a

0.041±0.01a

Data are means of 12 replications±SE; means with the same letter are not significantly different at P≤0.05 according to one-way ANOVA followed by the LSD test a

Treatments described in Fig. 2

Th e ex u d atio n accu m u latio n o f x y lem sap during 12 h (ml)

significant differences were detected between the two N treatments for cv. Elio. The leaf growth rate was lower under sole NH4+ nutrition for cv. Nanguang than for cv. Elio, but similar leaf growth rates were observed for both cultivars under PNN. Leaf area was higher for cv. Elio than for cv. Nanguang after 14 days with both N treatments. To test whether the effect of PNN on leaf growth was due to changes in cell division, cell expansion, or both, we determined the numbers of cells and cell sizes in leaves of both cultivars. Partial NO3- nutrition enhanced cell size in cv. Nanguang but not in cv. Elio compared to treatment with NH4+ alone (Fig. 2a). The increase in cell size was 18 % greater for PNN than for NH4+ alone in cv. Nanguang (Fig. 2b). However, the number of cells

12

NH4+

a

a

PNN

b

9 c 6

3

0 Nanguang

Elio

Fig. 4 Effect of partial nitrate nutrition (PNN) on xylem sap during 12 h in cvs Nanguang and Elio. Seedlings were grown under NH4+ only and PNN for 14 days; Data are means of 12 replications±SE. Means with the same letter are not significantly different at P≤0.05 according to one-way ANOVA followed by the LSD test

per leaf was the same with both N treatments in cv. Nanguang (Fig. 2c). These results indicated that the increased leaf growth in cv. Nanguang in response to PNN was due primarily to enhanced cell expansion. Similar cell numbers and cell sizes were observed in cv. Elio with both N treatments. Effect of exogenous 6-BA on leaf growth and photosynthetic activity in plants grown solely with NH4+ nutrition To confirm whether the increased rate of leaf growth in response to PNN was related to changes in endogenous cytokinin levels, we examined the effects of exogenous cytokinin, 6-BA, on leaf growth in both cultivars. Exogenous 6-BA increased leaf growth markedly with NH4+ nutrition-only in both rice cultivars (Fig. 3). Similar leaf areas were observed in cv. Nanguang under both PNN and in NH4+ plus exogenous 6-BA. The increase in cell size in response to exogenous 6-BA was 25 % greater in both cv. Nanguang and Elio relative to NH4+ nutrition only (Fig. 2a, b). Cell numbers in the first expanded leaf were similar in both cultivars with NH4+ nutrition exclusively, with or without exogenous 6-BA. The values of A, gs, and Tr in cv. Nanguang were markedly higher than those in cv. Elio under both of the N treatments (Table 1). PNN treatment significantly increased A, gs, and Tr in cv. Nanguang compared to NH4+ only. No differences were detected in A or gs between the two N treatments for cv. Elio. When 6-BA was exogenously applied to roots grown under NH4+ nutrition the effects on A, gs, and Tr were significantly reversed in both cultivars (Table 1).


Plant Soil (2013) 373:981–993

Table 2 Effect of partial nitrate nutrition (PNN) on the concentrations (μg g–1) of zeatin (Z), zeatin riboside and dihydrozeatin riboside (ZR+(diH)ZR), kinetin (KT), N6-(Δ2-isopentenyl) Z

ZR+(diH)ZR

adenine (iP), iso-pentenyl adenosine (iPA), and total cytokinin concentration in roots, xylem sap, and leaves of cvs Nanguang and Elio KT

iP

iPA

Total

Root Nanguang Elio

NH4+

0.14±0.02b

0.55±0.02c

0.30±0.03b

0.1±0.02c

0.05±0.01b

1.25±0.08c

PNN

0.19±0.03a

0.74±0.02b

0.34±0.01b

0.3±0.02b

0.06±0.01b

1.68±0.03b

NH4+

0.21±0.02a

0.95±0.02a

0.43±0.04a

0.4±0.01a

0.08±0.01a

2.09±0.07a

PNN

0.24±0.01a

0.97±0.03a

0.44±0.02a

0.4±0.02a

0.08±0.01a

2.18±0.04a

NH4+

0.07±0.01c

0.39±0.07c

0.06±0.01b

0.09±0.01c

0.04±0.01b

0.65±0.07c

PNN

0.10±0.01b

0.50±0.06b

0.07±0.01b

0.12±0.02b

0.04±0.01b

0.83±0.05b

NH4+

0.13±0.02a

0.65±0.04a

0.07±0.03a

0.13±0.03a

0.06±0.01a

1.04±0.06a

PNN

0.15±0.02a

0.65±0.02a

0.08±0.02a

0.14±0.01a

0.06±0.01a

1.08±0.02a

NH4+

0.16±0.02c

0.37±0.04c

0.51±0.01b

0.7±0.02b

0.17±0.02a

2.00±0.04b

PNN

0.23±0.03b

0.49±0.07b

0.53±0.03b

1.1±0.01a

0.12±0.04a

2.50±0.02a

NH4+

0.28±0.03a

0.60±0.07a

0.67±0.03a

0.9±0.07a

0.19±0.04a

2.72±0.53a

PNN

0.30±0.03a

0.63±0.05a

0.68±0.03a

1.0±0.15a

0.15±0.01a

2.82±0.06a

Xylem sap Nanguang Elio Leaf Nanguang Elio

Data are means of 12 replications±SE; means with the same letter are not significantly different in the same organ in the same cytokinin fraction at P≤0.05 according to one-way ANOVA followed by the LSD test

Xylem exudation rate PNN treatment caused a 22 % increase in xylem exudate accumulation after 12 h compared to the NH4+-only treatment in cv. Nanguang plants (Fig. 4). However, xylem exudate accumulation after 12 h was the same with both N treatments in cv. Elio plants. Higher xylem exudate accumulation was observed after 12 h in cv. Elio than in cv. Nanguang with both treatments. Cytokinin concentration in roots, xylem sap, and leaves Significant increases in the total concentration of six cytokinin fractions in roots, xylem sap, and leaves were observed in cv. Nanguang in response to the PNN treatment compared to the NH 4 + -only treatment (Table 2). The NO3–-induced increases in total cytokinin concentration in roots, xylem sap, and leaves were about 34 %, 28 %, and 25 %, respectively, for cv. Nanguang compared to NH4+-only nutrition. However, the total concentrations of cytokinin fractions were the same for both N treatments in cv. Elio. The rank of total concentrations of cytokinins in tissues from both cultivars was leaves>roots>xylem sap. Higher total cytokinin

concentration was observed in cv. Elio than in cv. Nanguang in response to both N treatments. PNN treatment increased Z, ZR+(diH)ZR, and iP concentrations in roots, xylem sap, and leaves of cv. Nanguang compared to NH4+-only nutrition. However, similar concentrations of KT and iPA were measured in cv. Nanguang and the concentrations of the six cytokinin fractions were similar with both N treatments in cv. Elio. ZR+(diH)ZR was the dominant cytokinin form in roots and xylem sap and iP was the dominant form in leaves of both cultivars (Table 2), indicating that ZR+ (diH)ZR is an active or mobile form and iP is a storage form in the leaf. ZR+(diH)ZR in xylem sap accounted for up to 60–80 % of the total cytokinin concentration in both cultivars with both N treatments, which is in agreement with the results of Takei et al. (2001). N6-(Δ2isopentenyl) adenine (iP) accounted for up to 33–44 % of the total cytokinin concentration in the leaves of both cultivars with both N treatments. Expression of OsIPT genes The expression patterns were analyzed for eight OsIPT genes, OsIPT1–OsIPT8. Real-time PCR analysis showed that the expression levels of the OsIPT1, OsIPT2,


989

Fig. 5 Real-time PCR analysis of OsIPT gene expression in cv. Nanguang. a Expression patterns of OsIPT genes in two root sections (0–4 and 4–8 cm) of cv. Nanguang plants under NH4+-only and partial NO3– nutrition (PNN). The numbers of PCR cycles are indicated on the right. Seedlings were cultured under NH4+only nutrition for 7 days after germination and treated with NH4+ or PNN for 24 h. b Relative mRNA levels for individual genes relative to OsACT (control). Data are shown as means±SE. Data with the same letter are not significantly different for the same gene at P≤0.05 according to one-way ANOVA followed by the LSD test

a

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NH4+ PNN

0-4cm

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OsIPT1

30 C

OsIPT2

32 C

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30 C

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32 C

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30 C

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a b

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OsIPT4, and OsIPT5 genes were unaffected by either of the N treatments in cv. Nanguang (Fig. 5). PNN enhanced OsIPT7 expression in the 0–4-cm root section, OsIPT8 expression in the 4–8-cm root section, and OsIPT3 expression in both root sections relative to NH4+-only nutrition in cv. Nanguang. Real-time PCR analysis showed that the expression level of OsIPT1-8 was unaffected in cv. Elio by either N treatment (Fig. 6).

Discussion Cytokinin is considered to be the plant hormone group most involved in N nutrition (Ding et al. 2012; Patterson et al. 2010; Sakakibara 2003; Sakakibara et al. 2006; Takei et al. 2004). Cytokinins are synthesized mainly in the root and are translocated via the xylem (Marschner 1995). The concentration and form of N both have

OsIPT2

OsIPT3

OsIPT4

OsIPT5

OsIPT7

OsIPT8

important effects on endogenous cytokinin synthesis (Garnica et al. 2010; Singh et al. 1992). Expression levels of the cytokinin synthesis genes, IPT3 and IPT5, are differentially regulated by N availability in Arabidopsis, and IPT3 plays a key role in cytokinin biosynthesis in response to rapid changes in NO3– availability (Takei et al. 2004). We showed that of the eight OsIPT genes, only OsIPT3 expression was stronger in response to PNN than to NH4+-only in root sections of cv. Nanguang. This result indicates that OsIPT3 is the key NO3–-responsive cytokinin synthesis gene in rice cultivars that have high NUE, consistent with the results in Arabidopsis (Takei et al. 2004). The level of ZR in roots of N-limited barley is largely nonresponsive to NO3– over the long term, but shows a transient positive response to increased NO3– supply (Samuelson and Larsson 1993). Similarly, Z and ZR levels increased in tobacco xylem sap after 24 h in response to PNN

Author's personal copy 990 NH4+ PNN

a

NH4+ PNN

0-4cm

4-8cm

OsIPT1

30 C

OsIPT2

32 C

OsIPT3

30 C

OsIPT4

32 C

OsIPT5

30 C

OsIPT6

30 C

OsIPT7

32 C

OsIPT8

30 C

ACT

25 C

b

NH4+;0-4cm

NH4+;4-8cm

PNN; 0-4cm

PNN; 4-8cm

1.8 a a

Relative expression

Fig. 6 Real-time PCR analysis of OsIPT gene expression in cv. Elio. a Expression patterns of OsIPT genes in two root sections (0–4 and 4–8 cm) of cv. Elio plants under NH4+-only and partial NO3– nutrition (PNN). The numbers of PCR cycles are indicated on the right. Seedlings were cultured under NH4+-only nutrition for 7 days after germination and supplied with NH4+ or PNN for 24 h. b Relative mRNA levels for individual genes relative to OsACT (control). Data are shown as means ±SE. Data with the same letter are not significantly different for the same gene at P≤0.05 according to oneway ANOVA followed by the LSD test

Plant Soil (2013) 373:981–993

1.2

a b

b

a

a a

a

a a a

a

a

a

aa

a

a

a

a aa a

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0 OsIPT1

compared to plants fed solely with NH4+; however Z and ZR levels declined to almost zero within the next 4 days after NH4+ treatment. In this work, the levels of Z, ZR+(diH)ZR, and iP were significantly enhanced in the roots, xylem sap, and leaves of cv. Nanguang after 14 days of PNN treatment compared to the NH4+-only treatment. Furthermore, the stimulatory effect of NO3− on shoot growth in cv. Nanguang was associated with a corresponding increase in the levels of Z and ZR in leaves and xylem sap, which was consistent with previous results in tobacco and wheat (Garnica et al. 2010; Rahayu et al. 2005; Samuelson and Larsson 1993; Walch-Liu et al. 2000). Previous studies showed that cytokinin levels were increased by mixed N forms (Smiciklas and Below 1992; Wang and Below 1996). However, cytokinin levels were reduced in the xylem sap of tomato plants by a mixture of N forms compared to NO3– alone (Lu et al. 2009). A higher Tr in response to mixed N forms

OsIPT2

OsIPT3

OsIPT4

OsIPT5

OsIPT7

OsIPT8

may counteract the reduction in the cytokinin concentration in the xylem sap. The amount of cytokinin transported is dependent on cytokinin concentration in the xylem sap and on Tr. Gao et al. (2010) showed that a mixture of N forms increased water uptake and Tr in rice plants relative to a NH4+-only treatment. Previous studies showed that Tr controls the import cytokinin from the root at the whole-shoot level (Aloni et al. 2005; Boonman et al. 2007; Dieleman et al. 2005). Tr is controlled by stomatal aperture in direct response to environmental stimuli such as nutrition or drought. Zhang et al. (2011) showed that the Tr increase in cv. Nanguang plants grown with PNN corresponded to an increase in gs compared with NH4+ nutrition only. Xylem sap flow was increased by 22 % in response to PNN compared solely to NH 4 + nutrition in cv. Nanguang (Fig. 4). At the same time, the total concentration of five cytokinin fractions in the xylem sap was significantly enhanced by PNN in cv. Nanguang relative


to NH4+. This demonstrated that cytokinin delivery and concentration in the xylem sap and xylem sap flow were increased by PNN only in cv. Nanguang, the high NUE rice cultivar. The active cytokinin forms, Z and ZR, were the dominant cytokinin fractions in the xylem sap, which was in agreement with the findings of Lu et al. (2009) and Garnica et al. (2010), who proposed that the presence of NO3− was associated with significant increases in the active forms of cytokinin. Root-synthesized cytokinin is recognized as a longdistance signal for leaf growth in response to NO3− (Rahayu et al. 2005; Ruffel et al. 2011; Sakakibara et al. 2006; Takei et al. 2001; Walch-Liu et al. 2000), although Dodd and coworkers (Dodd and Beveridge 2006; Dodd et al. 2004) proposed that leaf growth in response to N deprivation was independent of xylemborne cytokinin. Leaf growth is a complex process of cell division and cell expansion (Gonzalez et al. 2012). Walch-Liu et al. (2000) showed that cytokinin fractions in the xylem sap of tobacco are involved in the regulation of both cell division and cell expansion in response to NO3– supply. In this study, leaf growth in PPN-treated cv. Nanguang plants was attributable to increased cell expansion rather than cell division (Fig. 2). Exogenous application of 6-BA to both cultivars solely under NH4+ nutrition mimicked the change in cell size in the leaves of cv. Nanguang in response to PNN, further indicating that the increased cytokinin concentration in leaves led to the enhancement of leaf growth in cv. Nanguang. Our previous results showed that the stimulation of shoot growth in response to NO3– application could be attributable to a significant increase in photosynthetic activity in cv. Nanguang in addition to NO3–-increased leaf area (Zhang et al. 2011). In this study, we showed that A increased in both rice cultivars in response to exogenous application of 6-BA compared with NH4+-only nutrition. No difference was observed in cv. Nanguang between PNN and NH4+-only plus 6-BA. This further indicated that the increased cytokinin concentration in leaves led to the enhancement of photosynthetic capacity in cv. Nanguang. However, our results showed that the effect of PNN on leaf cell size cannot be fully explained by increased cytokinin import. The effect of PNN on leaf cell size was counteracted, but not completely rescued, in cv. Nanguang by the application of 6-BA (Fig. 2a). This may result from that cytokinin fractions increased by 6-BA application were not totally similar to those increased by PNN treatment according to the result of Ding et al. (2012). These observations

991

also suggest that other signal transduction pathways may be involved. Other compounds carried in the xylem sap, such as NO3-, might be delivered to leaves in proportion to transpiration rates and act as signals. Song et al. (2011b) showed that the concentration of NO3– was higher in the xylem sap of cv. Nanguang in response to PNN than solely to NH4+ nutrition. This suggested that NO3− and cytokinin may be key players in root-to-shoot communication (Sakakibara 2003). Synergistic and antagonistic interactions of cytokinins with auxins, which are involved in the regulation of cell expansion, have been demonstrated (Coenen and Lomax 1997). Song et al. (2011b) showed that auxin concentration was higher in the leaves of cv. Nanguang in response to PNN than to NH4+-only nutrition. In conclusion, PNN increased the concentration of Z, ZR+(diH)ZR, and iP in roots, xylem sap, and leaves and increased cell expansion and photosynthetic capacity in the leaves of cv. Nanguang. Higher NO3– responsiveness is associated with elevated cytokinin synthesis and transport from the root to the leaf and higher photosynthetic capacity in cv. Nanguang. Acknowledgments This work was funded by the National Nature Science Foundation of China (No. 31071846 and 31172022), by the Ministry of Science and Technology of China (No.2011CB100302),by State Key Laboratory of Soil and Sustainable Agriculture, Institute of Soil Science (No.Y052010013), by A Project Funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD).

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