nitrate ratios involve auxin distribution in two tobacco cultivars

Journal of Integrative Agriculture 2019, 18(12): 2703–2715 Available online at www.sciencedirect.com

ScienceDirect

RESEARCH ARTICLE

Differential responses of root growth to nutrition with different ammonium/nitrate ratios involve auxin distribution in two tobacco cultivars MENG Lin1*, DONG Jian-xin1*, WANG Shu-sheng1, SONG Ke1, LING Ai-fen2, YANG Jin-guang1, XIAO Zhi-xin3, LI Wei4, SONG Wen-jing1, LIANG Hong-bo1 1

Key Laboratory of Tobacco Biology and Processing, Ministry of Agriculture and Rural Affairs/Tobacco Research Institute, Chinese Academy of Agricultural Sciences, Qingdao 266101, P.R.China 2 Liangshan Branch of Sichuan Tobacco Corporation, Xichang 615000, P.R.China 3 Baoshan Branch, Yunnan Tobacco Company, Baoshan 678000, P.R.China 4 Hongyunhonghe Tobacco (Group) Co., Ltd., Kunming 650231, P.R.China

Abstract Nitrogen (N), the major forms of which are nitrate (NO3–) and ammonium (NH4+), plays an important role in plant growth and mediation of root development. However, the role of auxin in root growth in response to different NH4+/NO3– ratios remains unclear. Two tobacco cultivars (Nicotiana tabacum L.) were adopted in this study, which displayed variant growth features under the situations with sole NO3– nutrition ratio (NH4+/NO3– ratio: 0/100), low NO3– nutrition ratio (NH4+/NO3– ratio: 97/3), and optimal NH4+/NO3– ratio (50/50). We investigated the effects of the different NH4+/NO3– ratios on the formation and elongation of lateral roots (LRs), auxin concentration, DR5::GUS expression, 3H-labeled indole acetic acid ([3H]IAA) transport, and the expression of six PIN genes in tobacco roots. We also examined the effects of exogenous auxin and a transport inhibitor on LRs growth. The results are shown as follows, compared to optimal N nutrition conditions, the biomass and nitrogen (N) accumulation were largely reduced by sole and low NO3– nutrition treatment in NC89, but no difference was observed in Zhongyan 100. In most cases, sole and low NO3– nutrition impaired the elongation and formation of firstorder lateral roots (1° LRs), only in NC89, thus reducing the root growth. IAA concentration and DR5::GUS expression levels decreased in roots when NC89 was subjected to sole and low NO3– nutrition media, suggesting that different NH4+/ NO3– ratios affect the transport of auxin from leaves to roots. Results were similar following exogenous NAA application to low NO3– nutrition treated seedlings. Based on direct [3H]IAA transport measurement, the transport of polar auxin from shoots to roots decreased due to low NO3– nutrition. PIN4 expression levels were markedly decreased in roots of NC89

Received 23 July, 2018 Accepted 10 December, 2018 MENG Lin, E-mail: [email protected]; DONG Jian-xin, E-mail: [email protected]; Correspondence SONG Wen-jing, Tel/ Fax: +86-532-66715918, E-mail: [email protected]; LIANG Hong-bo, E-mail: [email protected] * These authors contributed equally to this study. © 2019 CAAS. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http:// creativecommons.org/licenses/by-nc-nd/4.0/). doi: 10.1016/S2095-3119(19)62595-5

2704

MENG Lin et al. Journal of Integrative Agriculture 2019, 18(12): 2703–2715

by sole and low NO3– nutrition, while they were unaffected in Zhongyan 100 roots. Overall, our findings suggest that LRs formation in tobacco seedlings is regulated by NH4+/NO3– ratios via modifying polar transport of auxin. Keywords: auxin, lateral root, formation and elongation, tobacco, NH4+/NO3− ratio

1. Introduction Among all the mineral nutrients, nitrogen (N) is needed in the greatest quantity by plants; and it most frequently limits plant growth and crop yields (Vidal and Gutierrez 2008). Typically, there is 1.5 to 4.5% N in the whole dry matter of herbaceous plants. With the exception of plants that develop N-fixing root nodules and insectivorous plants, N is normally absorbed through the roots, primarily in an inorganic form, such as nitrate (NO3−) or ammonium (NH4+), and it is the only plant nutrient available in significant quantities in both cationic and anionic forms. With NH4+ and NO3− as its major forms, N is important to plant growth, which plays a role in the mediation of root development. Previous studies have shown that NO3− and NH4+ have differential effects on morphology, physiology, and biochemical processes of plants and hence major impacts on their growth (Wiesler 1997); however, plant responses to different N sources are species-dependent. The majority of plants preferentially absorbs N from NO3− (Raven et al. 1992; Britto and Kronzucker 2002); by contrast, only a few grow well when taking N only from NH4+ (Qian et al. 2004). Moreover, for some plants, mixed NH4+ and NO3− nutrition is reported to be more beneficial than only NH4+-N or NO3−-N sources (Marschner 2012). There are many primary functions in plant root systems including water and nutrient uptake. As we all know, root development is greatly sensitive to changes in N distribution and supply in the soil. Also, plants are able to self-modify the root morphology under different environments (Sattelmacher et al. 1993; Zhang et al. 2007). Root architecture mainly depends on the pattern of root branching as well as the trajectory and rate of growth of individual roots. For example, when nutrients (NO3−, NH4+, or inorganic phosphate) are distributed unevenly, the lateral roots of many plant species proliferate preferentially within nutrient-optimal zones (Robinson 1994; Leyser and Fitter 1998). As reported, the growth rate of lateral roots can be stimulated by the local supply of NO3− (Drew 1975; Zhang et al. 1999); while uniformly applying NO3− larger than 10 mmol L–1 to roots may systemically inhibit the growth of both primary root (Linkohr et al. 2002; Tian et al. 2005) and lateral root (Stitt and Feil 1999; Zhang and Hasenstein 1999; Guo et al. 2005). For tomato plants, their root volume

and surface area reach the maximum when supplied with 25% NH4+ and 75% NO3− (Lu et al. 2009). Watermelon root growth was significantly decreased as the proportion of NH4+ was increased when grown in hydroponic culture treated with different NH 4+/NO 3− ratios (100/0, 75/25, 50/50, 25/75, and 0/100) (Liu et al. 2014). Different plant species exhibit various root growth responses to various NH4+/NO3– ratios (Chaillou et al. 1994; Zou et al. 2005). Although these responses at the physiological level are well documented, the corresponding mechanisms of regulation are still unknown. A variety of environmental and endogenous signals act in combination to alter the polar transport of hormones, so as to mediate the changes in auxin distribution, and in this way, lateral roots (LRs) formation is regulated (Vanneste and Friml 2009; Lavenus et al. 2013). Auxin is mostly synthesized in aboveground tissues, such as young leaves and shoot apices (Ljung et al. 2001). It is redistributed via the interaction between the auxin influx carriers (e.g., the LAX/AUX1 family proteins) and auxin efflux carriers (e.g., the ABCB/PGP family proteins and PIN) (Friml 2003; Blakeslee et al. 2005; Zazimalova et al. 2010; Peret et al. 2012). Among them, PIN exhibits polar localization at the plasma membrane; and its polarity is decisive to the direction of the auxin flux (Wisniewska et al. 2006). According to Zhang et al. (1999), local NO3− supply does not stimulate the lateral root elongation of the mutant axr4 which is not sensitive to auxin. This indicates that, the signaling pathways of auxin and NO3− overlap to a certain extent. Yet, there is no difference in lateral root elongation of axr4 and wild-type plants when responding to local NO3− supply (Linkohr et al. 2002). The initiation restraint of lateral roots by low N supply and high sucrose has possible relation to the inhibition of transporting auxin from shoots to roots (Malamy and Ryan 2001). Results of Tian et al. (2008) showed that the major reason for the inhibition of root growth by high NO3− supply may possibly be the reduction of auxin concentrations in roots. However, the mechanism by which the ratio of NH4+/NO3− supply modulates root auxin levels is still unclear. Tobacco (Nicotiana tabacum L.) is a crop with economic significance worldwide. Lateral roots account for a large portion in the root system of its seedlings; different from most dicotyledonous plants, an obvious tap root is lacking. When the apex is removed from tobacco plants, they develop

MENG Lin et al. Journal of Integrative Agriculture 2019, 18(12): 2703–2715

larger root systems than intact plants, and application of α-naphthylacetic acid (NAA) to the decapitated plants rescues dry root weight, thereby revealing a relationship between auxin and root growth (Jiang et al. 2001). In this study, we measured auxin concentrations in leaves and roots of the seedling by high-pressure liquid chromatography (HPLC). Transgenic plants carrying the auxin reporter construct DR5::GUS were used to identify the pattern of auxin distribution in response to the different NH4+/NO3– ratios. We also examined the effects of the auxin polar transport inhibitor N-1-naphthylphthalamic acid (NPA) and NAA on LRs formation and elongation in tobacco seedlings. In this paper, two tobacco cultivars were examined which displayed variant growth features when treated with different NH4+/NO3– ratios. In addition, the tolerance mechanism of tobacco to NH4+/NO3– ratio changes was explored.

2. Materials and methods 2.1. Plant growth Two tobacco genotypes (N. tabacum L.), NH 4+/NO 3– ratio susceptible NC89 and NH 4+/NO 3– ratio tolerant Zhongyan 100, were taken as subjects in the experiments. These genotypes of tobacco were chosen based on various responses of 35 tobacco genotypes to three types of N treatments in preliminary experiments (unpublished data). Trays with a mixture of peat and vermiculite (1:1, v/v) were prepared for the germination of seeds. The seeds were grown in a greenhouse under natural light at temperatures of 28/22°C for day and night, respectively. Individual 25-day-old seedlings with uniform size and vigor were transferred into plastic boxes through holes in the lids (10 holes per pot; one seedling per hole). Hoagland’s nutrient solution (Hoagland and Arnon 1950) at one-quarter strength was provided for 7 days. Based on preliminary experiments (Appendix A), seedlings provided with either sole NO3– nutrition (0/100: 0 mmol L–1 NH4+ and 3.75 mmol L–1 NO3–), low NO3– nutrition (97/3: 3.64 mmol L–1 NH4+ and 0.11 mmol L–1 NO3–), or an optimum NH4+/NO3– ratio (50/50: 1.875 mmol L–1 NH4+ and 1.875 mmol L–1 NO3–) for 9 days in hydroponic culture. The forms of N in the nutrient medium were Ca(NO3)2, (NH4)2SO4, or NH4NO3. The nutrient solution was replaced daily and aerated for 30 min to maintain the optimal content of oxygen. Each treatment had six-fold replication and replicates were assigned randomly to avoid edge effects. Furthermore, there were three independent biological replicates in all the experiments. The tobacco plants were harvested at different times in the 9-day period following treatment. The plant samples were flash-frozen and kept in a freezer at –40°C before the determination of indole-3-acetic acid (IAA) levels as well as the relative

2705

expression levels of the genes described below. In the treatments, NAA as exogenous auxin was dissolved in 1 mol L–1 NaOH and NPA as the auxin transport inhibitor was dissolved in dimethylsulfoxide (DMSO) (Thomson et al. 1973; Reed et al. 1998). Both were applied through the plant growth hydroponic medium. The 33-day-old tobacco seedlings were grown for 9 days within the hydroponic medium including NAA, and the seedlings were treated for 9 days to determine the effects of NPA on plant growth. Localized application of NPA was performed by dispensing diluted agar containing 20 mmol L–1 NPA directly across the root-shoot junction using a pipette.

2.2. Measurement of root system architecture Tobacco belongs to the dicotyledons, but its taproot is not well differentiated compared to those of typical dicotyledonous species (Song et al. 2015). A significant development of first-order lateral roots (1° LRs) and secondary (2° LRs) was observed, but taproots exhibited no change after the treatments in hydroponic culture. Hence, LR responses to the NH4+/NO3– ratio was selected for measurement. Total root length was measured by an image analysis system based on WinRhizo Scanner (Regent Instruments, Montreal, QC, Canada). A ruler was used to measure the lengths of 1° LRs, the counts of 1° LRs were counted visually, and the counts 2° LRs were counted using a colour CCD camera (Olympus, at ×160 magnification, Olympus Corporation, Japan). The average lengths of 1° LRs and 2° LRs densities were calculated as: The average lengths of 1° LRs (cm)=The lengths of 1° LRs (cm)/The counts of 1° LRs (no.) The 2° LRs density (no. cm–1)=The counts of 2° LRs (no.)/ The lengths of 1° LRs (cm)

2.3. Measurement of N content and accumulation in plants N content was determined in the whole plants of tobacco seedlings following the procedures described by Zou et al. (2005). The total N concentration in the assay solution was determined with an N-analysis meter after the plant materials were digested using H 2SO 4-H 2O 2 methods. Nitrogen accumulation was calculated as: N accumulation (mg/pot)=Dry matter (mg/pot)×Plant N content (%)

2.4. Measurement of IAA IAA content was determined by high performance liquid chromatography (HPLC). Fresh samples were obtained and then immediately frozen in liquid nitrogen. Based on

2706

MENG Lin et al. Journal of Integrative Agriculture 2019, 18(12): 2703–2715

the procedure by Song et al. (2013), free IAA was purified

2.8. Data analysis

and quantified. Finally, a standard IAA sample was acquired from Sigma-Aldrich (St Louis, MO, USA).

2.5. β-Glucuronidase (GUS) histochemical assay To identify distribution patterns of IAA in tobacco plants, a DR5::GUS construct (provided by the Prof. Wu Ping group at Zhejiang University, Hangzhou, China) was transformed into tobacco by Agrobacterium tumefaciens Conn. (strain EHA105) (Smith & Townsend). The natural GH3 promoter and DR5 exhibited similar and strong responses to auxin in tobacco (Tao et al. 2002). The tobacco plant tissues were treated with ethanol before the observation to remove the chlorophyll pigmentation. The stained tissues were photographed under an Olympus SZX2-ILLK stereomicroscope (Olympus, Tokyo, Japan) which was installed with a color CCD camera.

2.6. [3H]IAA transport assay As reported by Song et al. (2013), 3H-labeled indole acetic acid ([3H]IAA) polar transport was assayed after N treatment. The [3H]IAA solution included 0.25% agar, 25 mmol L–1 2-ethanesulfonic acid (MES) (pH 5.2) and 0.5 µmol L–1 [3H]IAA (20 Curie (Ci) mmol–1) in 2% dimethyl sulphoxide (DMSO). The shoots of the tobacco plant at 2 cm above the root-shoot junction were removed and 20 µL [3H]IAA solution was added to the cut surface. Ten replicate roots were incubated for 18 h in darkness, and then sampled and weighed. The root samples were subjected to an 18-h incubation in 4 mL of scintillation solution. A Beckman Coulter LS6500 Multipurpose Scintillation Counter was used to detect [3H]IAA radioactivity.

2.7. qRT-PCR analysis of gene expression The comparison of transcript levels was conducted between the tobacco auxin carrier genes (NtPIN1 (KC347302.1), NtPIN3 (KC425459.1), NtPIN1b (KC460399.1), NtPIN3b (KC438370.1), NtPIN9 (KC433528.1), and NtPIN4 (KC433529.1)) and NtL25 (L18908.1), which was a reference gene with stable expression (Schmidt and Delaney 2010). Total RNA was extracted from the roots of tobacco seedlings after treatment with various NH4+/NO3– ratios for 9 days. RNA extraction, qRT-PCR and reverse

transcription were carried out based on the methods used by Chen et al. (2012). The works of Meng et al. (2015) were referenced for the primer sets of PIN genes. We used thrice replication.

Data needed to calculate standard errors (SE) and means were collected. Then one-way analysis of variance was carried out and LSD multiple comparison tests were conducted for the detection of significant pairwise differences (P≤0.05) between means. SAS 9.2 Software (from SAS Institute; Cary, NC, USA) was employed for all statistical analyses.

3. Results 3.1. Responses to NH4+/NO3– ratio changes differ between the two tobacco cultivars The three N treatments produced distinct differences in plant growth responses. In comparison with the optimum NO3–/NH4+ ratio (50/50), the growth of root and shoot was largely reduced by sole and low NO3– nutrition treatments, but only in NC89 (Fig. 1). The sole NO3– nutrition markedly reduced dry weights of the root and shoot in NC89 by 48 and 44%, respectively. The low NO3– nutrition significantly decreased the dry weights of shoot and root in NC89 by 60 and 58%, respectively. Interestingly, no significant differences were detected in plant growth in Zhongyan 100 between the three treatments, except that root dry weight was reduced slightly by the low NO3– nutrition compared with the optimum NH4+/NO3– ratio. We determined the N concentrations in roots and shoots of each tobacco cultivar (Fig. 2). The low NO3– nutrition treatment significantly increased N concentration in the roots and shoots of tobacco, while no distinct differences in N concentration were detected between the other two treatments. However, this effect was not observed for N accumulation in roots and shoots. Sole and low NO3– nutrition significantly decreased N accumulation in NC89 seedlings by 23 and 33%, respectively, compared with plants grown in the optimum NH4+/NO3– ratio. No significant differences were observed in Zhongyan 100 for the three treatments.

3.2. NH4+/NO3– ratios affected 1° LRs elongation and formation only in NC89 Sole and low NO3– nutrition remarkably reduced root growth in NC89 relative to the optimum NH4+/NO3– ratio treatment at 9 days, while this difference was not detected in root growth in Zhongyan 100 (Fig. 3). The sole NO3– nutrition markedly reduced total root length, average length, and number of 1° LRs in NC89 by 36, 20, and 15%, respectively. The low NO3– nutrition significantly decreased total root length, 1° LRs number, and average length of 1° LRs in NC89 by 59, 23, and 26%, respectively, compared with the optimum

2707

MENG Lin et al. Journal of Integrative Agriculture 2019, 18(12): 2703–2715

0/100

50/50

a

0.75

a

a a

b

0.50

c

0.25

Root dry weight (g)

Shoot dry weight (g)

1.00

0

97/3 0.25 a

0.20 a a a

0.15

b c

0.10 0.05

Zhongyan 100

0

NC89

Zhongyan 100

NC89

Fig. 1 Biomass of two tobacco cultivars responding to three NH4+/NO3– ratios. Seedlings were subjected to sole NO3– nutrition (0/100: 0 mmol L–1 NH4+ and 3.75 mmol L–1 NO3–), low NO3– nutrition (97/3: 3.64 mmol L–1 NH4+ and 0.11 mmol L–1 NO3–) or provided with the optimum NH4+/NO3– ratio (50/50: 1.875 mmol L–1 NH4+ and 1.875 mmol L–1 NO3–) for 9 days. Values are means of six replications±SE and bars with different letters indicate significant differences at P<0.05 among the three treatments for each cultivar, as determined by ANOVA followed by the LSD test.

0/100

50/50 24

b

b

a

b b

a

2

0

Zhongyan 100

NC89

N accumulation (mg/pot)

N concentration (%)

6

4

97/3

18

a

a

a a

b c

12

6

0

Zhongyan 100

NC89

Fig. 2 N concentration and accumulation in two tobacco cultivars in response to three NH4+/NO3– ratios. Seedlings were subjected to sole NO3– nutrition (0/100: 0 mmol L–1 NH4+ and 3.75 mmol L–1 NO3–), low NO3– nutrition (97/3: 3.64 mmol L–1 NH4+ and 0.11 mmol L–1 NO3–) or provided with the optimum NH4+/NO3– ratio (50/50: 1.875 mmol L–1 NH4+ and 1.875 mmol L–1 NO3–) for 9 days. Values are means of six replications±SE and bars with different letters indicate significant differences at P<0.05 among the three treatments but same cultivar, as determined by ANOVA followed by the LSD test.

NH 4+/NO 3– ratio treatment. However, no remarkable differences were detected in the average length and density of 2° LRs between the three treatments.

3.3. NH4+/NO3– ratio affected endogenous IAA concentration in the shoot/root junction and roots in NC89, but not in Zhongyan 100 For the purpose of determining whether auxin is involved in the elongation and formation of 1° LRs subjected to sole or low NO3– nutrition, the endogenous IAA concentrations in the two topmost fully-expanded leaves and roots of seedlings

from the three treatments were measured (Fig. 4). Values from NC89 leaves were markedly reduced by sole NO3– nutrition (16%) and low NO3– nutrition (30%) compared with the optimum NH4+/NO3– ratio treatment. A similar trend was seen in roots, with IAA concentrations markedly reduced by sole NO3– nutrition (15%) and low NO3– nutrition (40%) compared with the optimum NH4+/NO3– ratio. However, no significant differences were recorded in either roots or shoots of Zhongyan 100 under the three treatments. A specific reporter was identified by Ulmasov et al. (1997), which included seven repeats of an auxin response element synthesized with high activity. They indicated that

2708

MENG Lin et al. Journal of Integrative Agriculture 2019, 18(12): 2703–2715

0/100

Total root length (cm)

1 200

600

Average length of 1° LRs (cm)

0

1

3

16

7

8 4

1

3

5

7

9

1 500 1 000

Number of 1° LRs

9

3 5 7 Treatment time (d)

0/100

7

9

NC89

15 10 5 0

1

3

5

7

9

3 5 7 Treatment time (d)

9

NC89

27 18 9

50/50

a

a

a

a

a

Zhongyan 100

NC89

a

Average length of 2° LRs (cm)

Density of 2° LRs (no. cm–1)

5

1

97/3 3

0.6

0

3

20

0

9

1.8

1.2

1

36

18

1

500 0

Zhongyan 100

27

0

NC89

2 000

9

12

36 Number of 1° LRs

5

Zhongyan 100

0

97/3

2 500

Zhongyan 100

Average length of 1° LRs (cm)

Total root length (cm)

1 800

50/50

a

a a

2

a

a

a

1

0

Zhongyan 100

NC89

Fig. 3 Root morphology of two tobacco cultivars (Zhongyan 100 and NC89) in response to three NH4+/NO3– ratios. Seedlings were subjected to sole NO3– nutrition (0/100: 0 mmol L–1 NH4+ and 3.75 mmol L–1 NO3–), low NO3– nutrition (97/3: 3.64 mmol L–1 NH4+ and 0.11 mmol L–1 NO3–) or provided with the optimum NH4+/NO3– ratio (50/50: 1.875 mmol L–1 NH4+ and 1.875 mmol L–1 NO3–) for 9 days. 1° and 2° LRs, first-order and secondary lateral roots, respectively. Values are means of six replications±SE and bars with different letters indicate significant differences at P<0.05 among the three treatments but same cultivar, as determined by ANOVA followed by the LSD test.

2709

MENG Lin et al. Journal of Integrative Agriculture 2019, 18(12): 2703–2715

IAA concentration (ng g–1)

20

15

a a a

a b c

10

5

0

Zhongyan 100

NC89

50/50

97/3

B

16

IAA concentration (ng g–1)

0/100 A

12

a

a

a

a b

8

c

4

0

Zhongyan 100

NC89

Fig. 4 Indole-3-acetic acid (IAA) concentration in the roots (A) or shoots (B) in two tobacco cultivars (Zhongyan 100 and NC89) in response to three NH4+/NO3– ratios. Seedlings were subjected to sole NO3– nutrition (0/100: 0 mmol L–1 NH4+ and 3.75 mmol L–1 NO3–), low NO3– nutrition (97/3: 3.64 mmol L–1 NH4+ and 0.11 mmol L–1 NO3–) or provided with the optimum NH4+/NO3– ratio (50/50: 1.875 mmol L–1 NH4+ and 1.875 mmol L–1 NO3–) for 9 days. Values are means of six replications±SE and bars with different letters indicate significant differences at P<0.05 among the three treatments but same cultivar, as determined by ANOVA followed by the LSD test.

3.4. Low and high NH4+/NO3– ratios inhibit the expression of PIN genes and [3H]IAA polar transport It was observed that LRs elongation and formation in tobacco seedlings treated with sole and low NO3– nutrition are related to the reductions in root auxin concentrations. Direct auxin transport was measured by radio-labeled IAA, with the aim of examining the impacts of NH4+/NO3– ratio on auxin polar transport from leaves to roots (as shown in Fig. 5). There was a remarkable decrease in the values of roots, by 39 and 61% under sole and low NO3– nutrition, respectively, compared with the optimum ratio. These results demonstrated that the polar auxin transport from shoots to roots increased under the optimum NH4+/NO3– ratio in NC89 (Fig. 4). Polar transport of auxin from shoots to roots is controlled mainly through PIN family proteins. The transcript levels of PIN genes were quantified in the roots of low and high

90 [3H]IAA activity in roots (dpm mg–1 FW)

the expression of a DR5::GUS gene construct can be used to monitor the changes in in vivo auxin levels, as this expression shows a relation to root auxin concentrations (Casimiro et al. 2001). In this case, the expression of DR5::GUS in tobacco seedlings was examined under the three treatments, and results were congruent with those in Fig. 4. Sole and low NO3– nutrition depressed DR5::GUS expression in the root/ shoot junction as well as in root tips, respectively (compared with the optimum NH4+/NO3– ratio), suggesting that sole and low NO3– nutrition probably attenuate root/shoot auxin levels compared with the optimum ratio.

a

72

54

b

c

36

18

0

0/100

50/50 NH4+/NO3– ratio

97/3

Fig. 5 3H-labeled indole acetic acid transport from the junction to lateral roots (LRs) in tobacco seedlings. Seedlings of cv. NC89 were subjected to sole NO3– nutrition (0/100: 0 mmol L–1 NH4+ and 3.75 mmol L–1 NO3–), low NO3– nutrition (97/3: 3.64 mmol L–1 NH4+ and 0.11 mmol L–1 NO3–) or provided with the optimum NH4+/NO3– ratio (50/50: 1.875 mmol L–1 NH4+ and 1.875 mmol L–1 NO3–) for 9 days. Radiolabeled IAA was given after 9 days of different ammonium nitrate ratio treatments. Plants were kept in the dark for 18 h before being assayed for radioactivity in the roots. Values are mean±SE (n=5) and bars with different letters indicate significant differences at P<0.05 among the three treatments but same cultivar, as determined by ANOVA followed by the LSD test..

NH4+/NO3– ratio-treated seedlings (Fig. 6). According to qRT-PCR, the expression levels of PIN1, PIN1b, and PIN3b

2710

MENG Lin et al. Journal of Integrative Agriculture 2019, 18(12): 2703–2715

as well as PIN4 were markedly decreased in roots of NC89 by sole and low NO3– nutrition compared with the optimum ratio, and were the lowest in the low NO3– nutrition treatment. Expression levels of PIN3, PIN4, and PIN9 in roots of Zhongyan 100 experienced no impact from the treatments. Since only the PIN4 expression patterns of the two tobacco cultivars in different treatments are consistent with the root growth, we conclude that PIN4 may play a pivotal role in down-regulating auxin shoot-root transport under sole and low NO3– nutrition treatments.

transport inhibitor NPA were examined to explore whether auxin levels (regulated by the three N treatments) resulted in underground changes in tobacco seedling morphology (Figs. 7 and 8). 1° LRs number and length in optimum NH4+/ NO3– ratio-treated seedlings decreased significantly with increasing concentrations of applied NPA (range tested: 0–500 nmol L–1) (Appendix B). The number and lengths of 1° LRs in seedlings treated with optimum N or 100 nmol L–1 NPA were reduced to a level similar to those of seedlings in low NO3– nutrition treatments (Fig. 7). 1° LRs formation and growth were promoted in seedlings treated with low NO3– nutrition and increasing concentrations of applied NAA (range tested: 0–100 nmol L–1) (Appendix C). 10 nmol L–1 NAA was applied to seedlings in the low NO3– nutrition treatment, increasing the number and the length of 1° LRs to levels similar to those of seedlings in the optimum N treatment (Fig. 7).

3.5. Exogenous applications of NPA and NAA reduce and increase 1° LRs formation and elongation, respectively The responses of 1° LRs formation and elongation to the application of exogenous NAA and the auxin polar 0/100 A

2.4

50/50

97/3

Zhongyan 100

Relative expression

a 1.8 a 1.2

b

a

a a

a

0.6

0 B

b

a

b

b

PIN1

PIN1b

a a

a

a

a a

c

PIN3

PIN3b

2.5

NC89 a

PIN4

PIN9

a

Relative expression

2.0 a

1.5 1.0

b

b

b

a a

c

PIN1b

PIN3

a

c

c

PIN1

a a

b c

b

0.5 0

a

PIN3b

PIN4

PIN9

Fig. 6 qRT-PCR analysis of PIN family gene expression levels in the roots of two tobacco cultivars (Zhongyan 100 and NC89) in response to three NH4+/NO3– ratios. Seedlings were subjected to sole NO3– nutrition (0/100: 0 mmol L–1 NH4+ and 3.75 mmol L–1 NO3–), low NO3– nutrition (97/3: 3.64 mmol L–1 NH4+ and 0.11 mmol L–1 NO3–) or provided with the optimum NH4+/NO3– ratio (50/50: 1.875 mmol L–1 NH4+ and 1.875 mmol L–1 NO3–) for 9 days in hydroponic culture. Values mean±SE (n=6) and bars with different letters indicate significant differences among the three treatments at the same time point or for the same gene at P<0.05, as determined by ANOVA followed by the LSD test.

2711

MENG Lin et al. Journal of Integrative Agriculture 2019, 18(12): 2703–2715

30

a b

180 a b

20 10 0

–NAA +NAA 97/3

–NPA +NPA 50/50

Average 1° LRs length (mm)

1° LRs number (no.)

40

a

a

b

b

120

60

0

–NAA +NAA 97/3

–NPA +NPA 50/50

Fig. 7 Effects of exogenous 1-naphthlcetic acid (NAA) and 1-N-naphthylphthalamic acid (NPA) on first-order lateral root (1° LRs) number and length in cv. NC89 seedlings. Seedlings were subjected to low NO3– nutrition (97/3: 3.64 mmol L–1 NH4+ and 0.11 mmol L–1 NO3–) or provided with the optimum NH4+/NO3– ratio (50/50: 1.875 mmol L–1 NH4+ and 1.875 mmol L–1 NO3–) for 9 days with or without either 10 nmol L–1 NAA or 100 nmol L–1 NPA. Values are mean±SE (n=5) and bars with different letters indicate significant differences among the three treatments at the same time point or for the same gene at P<0.05, as determined by ANOVA followed by the LSD test.

4. Discussion N is the mineral element largest demand by plants. Its availability has close relationships to plant productivity in managed and natural ecosystems. NO3– and NH4+ are the forms of N mainly taken up by the roots of plants at higher altitude (Guo et al. 2007). According to the responses to NH4+, there are two kinds of higher plants: tolerant and sensitive species (Britto and Kronzucker 2002).

4.1. Biomass accumulation In this paper, variable growth features of two tobacco genotypes responding to NH 4 + /NO 3 – ratio changes were displayed. Compared to the optimum NH 4 + / NO3– ratio treatment, an NH4+/NO3– ratio tolerant cultivar (Zhongyan 100) maintained shoot and root growth under sole and low NO3– nutrition while biomass accumulation in shoots and roots was inhibited by 44–60% in the NH4+/NO3– ratio-susceptible cultivar (NC89). Furthermore, lower dry mass of plants was observed in low NO3– nutrition compared with high NO3– treatment. This may related to the decrease of leaf number and area as well as shoot height, which conforms with previous observations: the plants subjected to NO3– grow larger leaf areas and leaf numbers than those subjected to NH4+ (Guo et al. 2007; Uysal and Kuru 2013). It was also found that leaf growth of tobacco subjected to NH4+ was limited due to cell elongation and the decrease of cell number (Walch-Liu et al. 2000). Dry matter accumulation is greatly dependent on photosynthesis. Plants can regulate their photosynthetic metabolism to adapt to various forms of N nutrition (Claussen and Lenz 1995). Moreover, it has been suggested that in photosynthesizing tissues, NH4+ is able

Junction

RT

Junction

50/50

50/50+NPA

97/3

97/3+NAA

RT

Fig. 8 Effects of synthetic 1-naphthlcetic acid (NAA) and 1-N-naphthylphthalamic acid (NPA) on DR5::GUS expression in the shoot/root junction and RT (root tip) of NC89 tobacco cultivar. Seedlings were subjected to low NO3– nutrition (97/3: 3.64 mmol L–1 NH4+ and 0.11 mmol L–1 NO3–) or provided with the optimum NH4+/NO3– ratio (50/50: 1.875 mmol L–1 NH4+ and 1.875 mmol L–1 NO3–) for 9 days with or without 10 nmol L–1 NAA or 100 nmol L–1 NPA. Bar=1 mm.

to uncouple electron transport from photophosphorylation (Peltier and Thibault 1983). This process may explain why the dry mass of tobacco plants taking NH4+ as the major N source was decreased.

4.2. N accumulation In this study, we found that Zhongyan 100 maintained higher N accumulation under both sole and low NO3– nutrition compared to NC89. These results agree with previous studies in citrus (Serna et al. 1992) and watermelons (Liu et al. 2014) under different NH4+/NO3– ratios. This suggests that element accumulation may have a significant positive correlation to dry matter. Furthermore, NO3− and NH4+ have

2712

MENG Lin et al. Journal of Integrative Agriculture 2019, 18(12): 2703–2715

different transporters and assimilation patterns. There is one high-affinity transporter system (HATS) for NH4+, and one HATS for NO3− for the regulation of their transport in higher plant roots (Glass et al. 2002). The assimilation of NH4+ occurs in the roots, while the assimilation of NO3− occurs in both roots and shoots. The intra-plant variation in δ15N was not observed in tomato plants treated with NH4+; by contrast, the δ15N of roots was relatively low compared to that of roots in plants supplied with NO3− (Evans et al. 1996). Bennett and Adams (1970) suggest that the critical concentration for incipient NH3 toxicity is 0.15–0.20 mmol L–1, based on experiments with sudan grass and cotton. However, a study using wheat showed that NH4+ toxicity occurred when its concentration was far below the critical concentration reported by Bennett and Adams (1970); also, with the increase of NH4+ levels, the uptake of N rises at certain concentrations (Cox and Reisenauer 1973). These different results may be attributed to variations in the environmental conditions and plant species tested. The disturbance in mineral nutrition has certain correlations to the inhibition of NH4+ nutrition and the two transport systems. It is interesting to observe higher N concentrations in plants absorbing N mainly from NH4+; while N accumulation in this treatment was the lowest, mainly as a result of the diluting effects caused by enhancement of plant biomass in optimum NH4+/NO3– ratio treatments.

4.3. Root morphological There are many primary functions in plant root systems, like water and nutrient uptake (Lu et al. 2009). Roots are able to perceive their environments in an efficient manner and then adjust morphology accordingly (Sattelmacher et al. 1993; Zhang et al. 2007). Zhang et al. (2007) summarized four morphological adaptations in the root system to N supply changes in Arabidopsis. They are: (i) external NO3− nutrition localized stimulatory effect on lateral root elongation, (ii) a systemic inhibition effect of high tissue NO3− concentrations on the activation of lateral root meristems, (iii) a suppression of lateral root initiation by high C (carbon):N ratios, and (iv) inhibition of primary root growth and stimulation of root branching by external L-glutamate. There are differences in root architecture between tobacco and most other dicotyledonous plants (Hochholdinger et al. 2004). LRs account for a large portion in the root system of tobacco seedlings because of the absence of a taproot. Commonly, the growth of roots in higher plants is more sensitive to excessive NH4+ (Li et al. 2010). Liu et al. (2014) put forward that NH4+ leads to smaller root systems than NO3–. In this study, inhibition of root growth in an NH4+/NO3– ratiosusceptible cultivar by sole and low NO3– nutrition mainly

resulted from the reduction of formation and elongation of 1° LRs rather than 2° LRs. These results indicated that the strategy of tobacco root morphological adaptation to sole or low NO3– nutrition was to arrest 1° LRs growth but maintain 2° LRs growth.

4.4. Polar auxin transport and root growth Based on previous studies, ethylene signaling (You and Barker 2004) and auxin transport (Cao et al. 1993) have relations to NH 4+-mediated inhibition of primary root development. Li et al. (2010) found that instead of cell division, cell elongation was the principal target of NH4+ inhibition of primary root growth. The stimulation was reported to occur when the root was exposed to a localized source of NO3– directly because the root derived the most benefits from the increase of N supply (Hackett 1972). Alternatively, a growth-stimulating influx of auxin and carbohydrates is caused by the increase of metabolic activity in those same roots (Wiersum 1958; Drew et al. 1973; Granato and Raper 1989; Sattelmacher et al. 1993). Zhang et al. (1999) demonstrated that the response pathways of auxin and NO3– overlapped. Various environmental and endogenous signals are integrated to adapt to changes in auxin distribution via the alteration of hormone polar transport. In this way, LRs formation is regulated (Vanneste and Friml 2009; Lavenus et al. 2013). However, the mechanism of root growth response to different NH4+/NO3– ratios remains unclear. In this study, IAA concentrations in NC89 leaves and roots were markedly reduced through sole and low NO3– nutrition, compared with the optimum NH4+/ NO3– ratio. A similar trend between IAA concentrations and 1° LRs phenotypes in different NH4+/NO3– ratio treatments suggests that IAA may be important in the response of root growth to N nutrition changes; and various DR5::GUS expression levels in the root/shoot junction and root tips provided corroborative evidence (Fig. 5). The synthesis of auxin mostly occurs in aboveground tissues (such as young leaves and shoot apices) (Ljung et al. 2001); and the auxin is then redistributed via the interaction between auxin influx carriers (LAX/AUX1 family proteins), and auxin efflux carriers (including ABCB/PGP and PIN family proteins) (Friml 2003; Blakeslee et al. 2005; Zazimalova et al. 2010; Peret et al. 2012). Among them, PIN at the plasma membrane displayed a polar localization and the direction of auxin flux is determined by its polarity in certain cases (Wisniewska et al. 2006). In our study, application of NPA decreased the number and length of 1° LRs in NC89 seedlings treated with optimal NO3–/NH4+ ratio to levels similar to those in seedlings grown in low NO3– nutrition medium; and it is noteworthy that these

MENG Lin et al. Journal of Integrative Agriculture 2019, 18(12): 2703–2715

two treatments had similar DR5::GUS expression levels as well. When applying NAA to NC89 seedlings treated with low NO3– nutrition, the results were similar. Direct measurement of auxin transport using radio-labeled IAA confirmed the increased polar auxin shoot-root transport under an optimum NH4+/NO3– ratio in NC89 (Fig. 4). Our qRT-PCR analyses showed that only the PIN4 expression level was markedly decreased in roots of NC89 by sole and low NO3– nutrition compared to the optimum ratio, and was the lowest in the high ratio treatment, while roots of Zhongyan 100 showed no effects. Thus, we can conclude that PIN4 may be vital to downregulating auxin shoot-root transport under sole and low NO3– nutrition conditions.

5. Conclusion The optimum NH4+/NO3– ratio treatment enhanced auxin polar transport from shoot to root, induced greater auxin flux into the LRs zone and enhanced LRs proliferation in susceptible cultivar NC89. These results indicated that stronger NO3– responses are associated with greater auxin accumulation in the LRs zone and a higher rate of LRs initiation in NC89.

Acknowledgements This work was funded by the Agricultural Science and Technology Innovation Program, Chinese Academy of Agricultural Sciences (ASTIP-TRIC03), the Science Foundation for Young Scholars of Tobacco Research Institute of Chinese Academy of Agricultural Sciences (2018B01), the National Nature Science Foundation of China (3601818), the Liangshan Branch of Sichuan Tobacco Corporation, China (LSYC201805), and the Hongyunhonghe Tobacco (Group) Co., Ltd., China (HYHH2016YL02). Appendices associated with this paper can be available on http://www.ChinaAgriSci.com/V2/En/appendix.htm

References Bennett A C, Adams F. 1970. Concentration of NH3 (aq) required for incipient NH3 toxicity to seedlings. Soil Science Society of America Journal, 34, 259–263. Blakeslee J J, Peer W A, Murphy A S. 2005. Auxin transport. Current Opinion in Plant Biology, 8, 494–500. Britto D T, Kronzucker H J. 2002. NH4+ toxicity in higher plants: A critical review. Journal of Plant Physiology, 159, 567–584. Casimiro I, Marchant A, Bhalerao R, Beeckman T, Dhooge S, Swarup R, Graham N, Inzé D, Sandberg G, Casero P J, Bennett M. 2001. Auxin transport promotes Arabidopsis lateral root initiation. The Plant Cell, 13, 843–852. Cao Y W, Glass A D M, Crawford N M. 1993. Ammonium

2713

inhibition of Arabidopsis root-growth can be reversed by potassium and by auxin resistance mutations aux1, axr1, and axr2. Plant Physiology, 102, 983–989. Chaillou S, Rideout J W, Raper C D, Morotgaudry J F. 1994. Responses of soybean to ammonium and nitrate supplied in combination to the whole root-system or separately in a split-root system. Physiologia Plantarum, 90, 259–268. Chen Y N, Fan X R, Song W J, Zhang Y L, Xu G H. 2012. Overexpression of OsPIN2 leads to increased tiller numbers, angle and shorter plant height through suppression of OsLAZY1. Plant Biotechnology Journal, 10, 139–149. Claussen W, Lenz F. 1995. Effect of ammonium and nitrate on net photosynthesis, flower formation, growth and yield of eggplants (Solanum melongena L.). Plant and Soil, 171, 267–274. Cox W J, Reisenauer H M. 1973. Growth and ion uptake by wheat supplied nitrogen as nitrate, or ammonium, or both. Plant and Soil, 38, 363–380. Drew M C. 1975. Comparison of the effects of a localized supply of phosphate, nitrate, ammonium and potassium on the growth of the seminal root system, and the shoot in barley. New Phytologist, 75, 479–490. Drew M C, Saker L R, Ashley T W. 1973. Nutrient supply and the growth of the seminal root system in barley. Journal of Experimental Botany, 24, 1189–1202. Evans R D, Bloom A J, Sukrapanna S S, Ehleringer J R. 1996. Nitrogen isotope composition of tomato (Lycopersicon esculentum Mill. cv. T-5) grown under ammonium or nitrate nutrition. Plant Cell and Environment, 19, 1317–1323. Friml J. 2003. Auxin transport-shaping the plant. Current Opinion in Plant Biology, 6, 7–12. Glass A D, Britto D T, Kaiser B N, Kinghorn J R, Kronzucker H J, Kumar A, Okamoto M, Rawat S, Siddiqi M Y, Unkles S E, Vidmar J J. 2002. The regulation of nitrate and ammonium transport systems in plants. Journal of Experimental Botany, 53, 855–864. Granato T C, Raper J R. 1989. Proliferation of maize (Zea mays L.) roots in response to localized supply of nitrate. Journal of Experimental Botany, 40, 263–275. Guo S, Zhou Y, Shen Q, Zhang F. 2007. Effect of ammonium and nitrate nutrition on some physiological processes in higher plants - Growth, photosynthesis, photorespiration, and water relations. Plant Biology, 9, 21–29. Guo Y, Chen F, Zhang F, Mi G. 2005. Auxin transport from shoot to root is involved in the response of lateral root growth to localized supply of nitrate in maize. Plant Science, 169, 894–900. Hackett C. 1972. A method of applying nutrients locally to roots under controlled conditions, and some morphological effects of locally applied nitrate on the branching of wheat roots. Australian Journal of Biological Sciences, 25, 1169–1180. Hoagland D R, Arnon D I. 1950. The water-culture method for growing plants without soil. Circular California Agricultural Experiment Station, 347, 357–359. Hochholdinger F, Park W J, Sauer M, Woll K. 2004. From weeds

2714

MENG Lin et al. Journal of Integrative Agriculture 2019, 18(12): 2703–2715

to crops: Genetic analysis of root development in cereals. Trends in Plant Science, 9, 42–48. Jiang F, Li C J, Jeschke W D, Zhang F S. 2001. Effect of top excision and replacement by 1-naphthylacetic acid on partition and flow of potassium in tobacco plants. Journal of Experimental Botany, 52, 2143–2150. Lavenus J, Goh T, Roberts I, Guyomarc’h S, Lucas M, De Smet I, Fukaki H, Beeckman T, Bennett M, Laplaze L. 2013. Lateral root development in Arabidopsis: Fifty shades of auxin. Trends in Plant Science, 18, 450–458. Leyser O, Fitter A. 1998. Roots are branching out in patches. Trends in Plant Science, 3, 203–204. Li Q, Li B H, Kronzucker H J, Shi W M. 2010. Root growth inhibition by NH4+ in Arabidopsis is mediated by the root tip and is linked to NH4+ efflux and GMPase activity. Plant Cell and Environment, 33, 1529–1542. Linkohr B I, Williamson L C, Fitter A H, Leyser H M C. 2002. Nitrate and phosphate availability and distribution have different effects on root system of architecture of Arabidopsis. The Plant Journal, 29, 751–760. Liu N, Zhang L, Meng X X, Neelam A, Yang J H, Zhang M F. 2014. Effect of nitrate/ammonium ratios on growth, root morphology and nutrient elements uptake of watermelon (Citrullus lanatus) seedlings. Journal of Plant Nutrition, 37, 1859–1872. Ljung K, Bhalerao R P, Sandberg G. 2001. Sites and homeostatic control of auxin biosynthesis in Arabidopsis during vegetative growth. The Plant Journal, 28, 465–474. Lu Y L, Xu Y C, Shen Q R, Dong C X. 2009. Effects of different nitrogen forms on the growth and cytokinin content in xylem sap of tomato (Lycopersicon esculentum Mill.) seedlings. Plant and Soil, 315, 67–77. Malamy J E, Ryan K S. 2001. Environmental regulation of lateral root initiation in Arabidopsis. Plant Physiology, 127, 899–909. Marschner H. 2012. Mineral nutrition of higher plants. Journal of Ecology, 76, 1250–1251. Meng L, Song W J, Liu S J, Dong J X, Zhang Y L, Wang C D, Xu Y M, Wang S S. 2015. Light quality regulates lateral root development in tobacco seedlings by shifting auxin distributions. Journal of Plant Growth Regulation, 34, 574–583. Peltier G, Thibault P. 1983. Ammonia exchange and photorespiration in chlamydomonas. Plant Physiology, 71, 888–892. Peret B, Swarup K, Ferguson A, Seth M, Yang Y, Dhondt S, James N, Casimiro I, Perry P, Syed A. 2012. AUX/LAX genes encode a family of auxin influx transporters that perform distinct functions during Arabidopsis development. The Plant Cell, 24, 2874–2885. Qian X Q, Shen Q R, Xu G H, Wang J, Zhou M Y. 2004. Nitrogen form effects on yield and nitrogen uptake of rice crop grown in aerobic soil. Journal of Plant Nutrition, 27, 1061–1076. Raven J A, Wollenweber B, Handley L L. 1992. A comparison of ammonium and nitrate as nitrogen-sources for photolithotrophs. New Phytologist, 121, 19–32.

Reed R C, Brady S R, Muday G K. 1998. Inhibition of auxin movement from the shoot into the root inhibits lateral root development in Arabidopsis. Plant Physiology, 118, 1369–1378. Robinson D. 1994. The responses of plants to non-uniform supplies of nutrients. New Phytologist, 127, 635–674. Sattelmacher B, Gerendas J, Thoms K, Brück H, Bagdady N H. 1993. Interaction between root growth and mineral nutrition. Environmental and Experimental Botany, 33, 63–73. Schmidt G W, Delaney S K. 2010. Stable internal reference genes for normalization of real-time RT-PCR in tobacco (Nicotiana tabacum) during development and abiotic stress. Molecular Genetics and Genomics, 283, 233–241. Serna M D, Borras R, Legaz F, Primomillo E. 1992. The influence of nitrogen concentration and ammonium-nitrate ratio on N-uptake, mineral-composition and yield of citrus. Plant and Soil, 147, 13–23. Song W J, Liu S J, Meng L, Xue R, Wang C D, Liu G L, Dong C X, Wang S S, Dong J X, Zhang Y L. 2015. Potassium deficiency inhibits lateral root development in tobacco seedlings by changing auxin distribution. Plant and Soil, 396, 163–173. Song W J, Sun H, Li J, Gong X P, Huang S J, Zhu X, Zhang Y L, Xu G H. 2013. Auxin distribution is differentially affected by nitrate in roots of two rice cultivars differing in responsiveness to nitrogen. Annals of Botany, 112, 1383–1393. Stitt M, Feil R. 1999. Lateral root frequency decreases when nitrate accumulates in tobacco transformants with low nitrate reductase activity: Consequences for the regulation of biomass partitioning between shoots and root. Plant and Soil, 215, 143–153. Tao L Z, Cheung A Y, Wu H M. 2002. Plant Rac-like GTPases are activated by auxin and mediate auxin-responsive gene expression. The Plant Cell, 14, 2745–2760. Thomson K S, Hertel R, Müller S, Tavares J E. 1973. 1-N-naphthylphthalamic acid and 2,3,5-triiodobenzoic acid. Planta, 109, 337–352. Tian Q, Chen F, Zhang F, Mi G. 2005. Possible involvement of cytokinin in nitrate-mediated root growth in maize. Plant and Soil, 77, 185–196. Tian Q Y, Chen F J, Liu J X, Zhang F S, Mi G H. 2008. Inhibition of maize root growth by high nitrate supply is correlated with reduced IAA levels in roots. Journal of Plant Physiology, 165, 942–951. Ulmasov T, Murfett J, Hagen G, Guilfoyle T J. 1997. Aux/IAA proteins repress expression of reporter genes containing natural and highly active synthetic auxin response elements. The Plant Cell, 9, 1963–1971. Uysal A, Kuru B. 2013. Magnesium ammonium phosphate production from wastewater through box-behnken design and its effect on nutrient element uptake in plants. CleanSoil Air Water, 41, 447–454. Vanneste S, Friml J. 2009. Auxin: A trigger for change in plant development. Cell, 136, 1005–1016.

MENG Lin et al. Journal of Integrative Agriculture 2019, 18(12): 2703–2715

Vidal E A, Gutierrez R A. 2008. A systems view of nitrogen nutrient and metabolite responses in Arabidopsis. Current Opinion in Plant Biology, 11, 521–529. Walch-Liu P, Neumann G, Bangerth F, Engels C. 2000. Rapid effects of nitrogen form on leaf morphogenesis in tobacco. Journal of Experimental Botany, 51, 227–237. Wisniewska J, Xu J, Seifertova D, Brewer P B, Ruzicka K, Blilou I, Rouquie D, Benkova E, Scheres B, Friml J. 2006. Polar PIN localization directs auxin flow in plants. Science, 312, 883–883. Wiesler F. 1997. Agronomical and physiological aspects of ammonium and nitrate nutrition of plants. Journal of Plant Nutrition and Soil Science, 160, 227–238. Wiersum L K. 1958. Density of root branching as affected by substrate and separate ions. Acta Botanica Neerlandica, 7, 174–190. You W Q, Barker A V. 2004. Ethylene evolution and ammonium accumulation by tomato plants after root-applied glufosinateammonium treatment in the presence of ethylene inhibitors. Communications in Soil Science and Plant Analysis, 35,

2715

1957–1965. Zazimalova E, Murphy A S, Yang H, Hoyerova K, Hosek P. 2010. Auxin transporters - Why so many? Cold Spring Harbor Perspectives in Biology, 2, a001552. Zhang H, Jennings A, Barlow P W, Forde B G. 1999. Dual pathways for regulation of root branching by nitrate. Proceedings of the National Academy of Sciences of the United States of America, 96, 6529–6534. Zhang H M, Rong H L, Pilbeam D. 2007. Signalling mechanisms underlying the morphological responses of the root system to nitrogen in Arabidopsis thaliana. Journal of Experimental Botany, 58, 2329–2338. Zhang N G, Hasenstein K H. 1999. Initiation and elongation of lateral roots in Lactuca sativa. International Journal of Plant Sciences, 160, 511–519. Zou C Q, Wang X F, Wang Z Y, Zhang F S. 2005. Potassium and nitrogen distribution pattern and growth of flue-cured tobacco seedlings influenced by nitrogen form and calcium carbonate in hydroponic culture. Journal of Plant Nutrition, 28, 2145–2157.

Executive Editor-in-Chief LI Shao-kun Managing editor WANG Ning