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Boron deficiency and toxicity altered the subcellular structure and cell wall composition architecture in two citrus rootstocks
Scientia Horticulturae 238 (2018) 147–154
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Boron deficiency and toxicity altered the subcellular structure and cell wall composition architecture in two citrus rootstocks Xiuwen Wu, Xiaopei Lu, Muhammad Riaz, Lei Yan, Cuncang Jiang
T
⁎
Microelement Research Center, College of Resources and Environment, Huazhong Agricultural University, Wuhan, Hubei, PR China
A R T I C LE I N FO
A B S T R A C T
Keywords: Boron stresses Leaf Ultrastructure Cell wall components FTIR 13 C-NMR
Trifoliate orange and citrange are two important rootstock resources to citrus, and citrange has a stronger tolerance to boron (B) deficiency and toxicity than trifoliate orange. In this study, we described how B deficiency and toxicity depressed the variations of cell wall B location, subcellular structure, cell wall components and structure of the two citrus rootstocks, to evaluate the mechanisms of different B tolerance of rootstocks on the cellular and structural levels. The results showed that citrange had better growth and lower symptoms of B stresses than trifoliate orange. What is more, citrange had a stronger ability to allocate more B to cell walls than trifoliate orange at deficient-B level. Under B deficiency and excessive conditions, severe damages in subcellular structure with obvious irregular thickening of cell walls and higher accumulation of plastoglobulus were observed in leaf cells of both two rootstocks. Additionally, it also showed obvious variations in the mode of hydrogen bonding, and more accumulation of cellulose, phenols and carbonhydrates in cell walls of trifoliate orange leaf under B starvation, while lighter changes on cell wall components were observed in citrange. As for B toxicity, comparing to trifoliate orange, citrange showed a lighter damage on pectin crosslinking structure in cell walls of leaf tissue. These results gained some novel mechanisms of different citrus rootstocks to B stress tolerance, and provide a theoretical basis for cultivating improved citrus rootstocks.
1. Introduction Boron (B) is an essential microelement for higher plants and B plays an important role in the structure of plant cell walls, and its deprivation causes a wide range of variations in the cell function, the physiology and biochemistry of cell walls, and plant metabolites (Pan et al., 2012; Dordas and Brown, 2005; Liu et al., 2014). It has been demonstrated that the accepted physiological function of B is to cross-link two chains of rhamnogalacturonan II (RG-II) by the formation of borate diol diester in pectin of cell wall, and then affect the biochemical and mechanical properties of the plant wall (O’Neill et al., 2004). Cell walls have an important influence on the structural integrity of the cells and determine the cells shape and size (Hayot et al., 2012). Boron starvation is a widespread problem for many agricultural crops, including citrus (Shorrocks, 1997), is responsible for considerable loss of productivity and poor fruit quality in many citrus orchards (Xiao et al., 2007). Conclusive evidence shows that B deficiency results in curling of the leaves, leaf chlorosis, weakened photosynthesis, abnormal anatomic structure and metabolic disturbance in leaves (Liu et al., 2014; Han et al., 2009; Lu et al., 2017a,b; Dong et al., 2016).
Application of B-enriched fertilizers is an effective solution for resolving B deficiency, but B toxicity is a more difficult problem to manage and the damage of B toxicity to plants is irreversible. At excessive B level, B tends to accumulate in leaf tips and leaf margins, causing margin damage (Reid et al., 2004; Shapira et al., 2013). Under such conditions, many plants show inhibited growth and the symptoms of B toxicity like chlorotic and necrotic patches, which are usually found on the margins and tips of older leaves (Nable et al., 1997; Han et al., 2009). Several studies have demonstrated the destruction of leaf structure, oxidative damage, and interference to synthesis of cell wall components with excessive B supply (Papadakis et al., 2004a; Shah et al., 2017; Mesquita et al., 2016). Citrus is one of the most important economic crops in China, and the rootstocks have been used to optimize plant growth, and improved fruit production and quality (Xiao et al., 2007; Papadakis et al., 2004a). Early studies have indicated that citrus plants are sensitive to B deficiency or toxicity (Papadakis et al., 2003; Liu et al., 2011), and rootstocks vary greatly in their effects on tolerance to B deficiency (Boaretto et al., 2008) and toxicity (Papadakis et al., 2004a,b). Trifoliate orange [Poncirus trifoliata (L.) Raf.] and citrange [Citrus sinensis (L.) Osb. ×
Abbreviations: CK, control; BD, boron deficiency; BT, boron toxicity; FTIR, fourier-transform infrared spectroscopy; 13C-NMR, 13C nuclear magnetic resonance; TEM, transmission electron microscope; PBS, glutaraldehyde in phosphate buffer solution; CWM, cell wall material; Sg, starch grain; Pg, plastoglobulus; Tri, trifoliate orange; Cir, citrange ⁎ Corresponding author at: College of Resources and Environment, Huazhong Agricultural University, No.1 Shizishan Street, Wuhan, 430070, Hubei Province, PR China. E-mail address: [email protected] (C. Jiang). https://doi.org/10.1016/j.scienta.2018.04.057 Received 6 March 2018; Received in revised form 25 April 2018; Accepted 25 April 2018 0304-4238/ © 2018 Elsevier B.V. All rights reserved.
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5.8 ∼ 6.2 every day with 0.5 M H2SO4 or 1 M NaOH.
Poncirus trifoliata (L.) Raf.] are two vital rootstock resources to citrus, and the two rootstocks have different tolerances to B stress (FornerGiner et al., 2003; Liu et al., 2013). It has been reported that citrange has a stronger tolerance to B deficiency than trifoliate orange (Sheng et al., 2009) and evaluated the mechanisms of difference by studying cellular B allocation and pectin composition (Liu et al., 2013), structure and components of leaf (Lu et al., 2017a,b). Huang et al. (2014) investigated the changes on leaf photosynthesis, chlorophyll, plant B absorption and distribution induced by B toxicity, using two citrus species differing in B sensitivity, to elucidate the possible mechanisms of B tolerance in different citrus plants. Under high B conditions, better growth status was observed in “Newhall” plants grafted on citrange than in those grafted on trifoliate orange by Liu et al. (2011), which suggesting that plants grafted on citrange were more tolerant to B toxicity than the trifoliate orange-grafted plants. However, there have been few studies performed on the differences and changes in the cellular structure and the architecture of cell wall components in leaves of trifoliate orange and citrange rootstocks under B deficiency and excess conditions, the relationship between variations on cell walls and Btolerant is not clear. Fourier-transform infrared spectroscopy (FTIR) has been used to gather information about the chemical composition of almost all substances (Liu et al., 2014), and it is also extensively applied in analyzing plant cell walls (Abidi et al., 2014). In recent years, solid-state 13C nuclear magnetic resonance (13C-NMR) spectroscopy has become a powerful tool to identify the structure of organic compounds and provide comprehensive structural information (Mao et al., 2008). Despite its wide application in characterization of the structure of soil organic matter, wood lignin and cell wall polysaccharides, etc. (RondeauMouro et al., 2003; Salati et al., 2008; Balakshin et al., 2016), the13CNMR technique has rarely been used to investigate the cell wall organic carbon structure of citrus under different B stress conditions. Therefore, the trifoliate orange and citrange were selected as research materials, and chemical analysis methods combined with transmission electron microscope (TEM), FTIR and solid-state 13C-NMR were conducted to examine (1) how B deficiency and toxicity altered B allocated in cell walls and cellular structure of the two citrus rootstocks; (2) varied changes on cell wall architecture and components betwwen trifoliate orange and citrange treated with deficient-B and toxic-B, and ultimately to gain the structural changes of cell walls in different citrus rootstocks under different B stresses.
2.2. Plant sampling and boron analysis After completing 90-days, the plants were harvested and plant samples were divided into roots, stems, and leaves. All the leaves of each plant were further separated into three parts: one part was used for determination of B concentration, another part was stored for extraction of cell wall materials, and the third part was used for transmission electron microscope analyses. After drying to a constant weight at 75 °C, the dry weight of roots, stems and leaves were measured. Then the samples were ground to a fine powder and ashed at 500 °C for 5 h, followed by dissolving the ashes in 0.1 M HCl. The B contents in roots, stems and leaves were measured at 540 nm spectrophotometrically (Hitachi UV-3100 UV–vis; TECHCOMP, Shanghai, China), according to the curcumin colorimetric method (Dible et al., 1954). 2.3. Transmission electron microscope (TEM) analysis The TEM slices were prepared by the method of Kong et al. (2013) with slight modification. The TEM analysis was performed as follows: briefly, leaves from the same parts under different B treatments were cut into small pieces (1 × 1 mm), then fixed in 2.5% glutaraldehyde in phosphate buffer solution (PBS) for 12 h at 4 °C. Next, the tissue blocks were rinsed four times with 0.1 M PBS (pH 7.4) and post-fixed for 2–3 h with 1% buffered osmium tetroxide, followed by rinsing three times with 0.1 M PBS (pH 7.4), and dehydration three times in an increasing acetone concentration series [50, 70 and 90 (three times)] and then in a mixture of 90% ethanol and 90% acetone for 15 min. After being stained with 2% uranyl acetate and lead citrate, ultrathin sections were examined with a JEM-100CX II transmission electron microscope. 2.4. Preparation of cell wall materials (CWM) The cell wall was separated from the leaves as reported by Hu and Brown (1994) with some modifications. Firstly, the leaf samples were homogenized in liquid nitrogen with a mortar, after homogenizing in 10 volumes of ice-cold ultrapure water and centrifuging at 5000g for 10 min at 4 °C, the precipitate was washed with 10 volumes of ice-cold ultrapure water and centrifuged again. Next, the residue was washed three times with 10 volumes of 80% ethanol and once with 10 volumes of mixture of methanol/chloroform (1/1, v/v). Finally, the precipitate was washed with 10 volumes of acetone. The final insoluble pellet was dried at 50–60 °C, weighed and defined as CWM. The dry CWM was divided into two portions: one was dried to ashes at 500 °C for determining B allocated in cell walls as described above, and the other portion was used for FTIR and 13C-NMR analyses.
2. Materials and methods 2.1. Plant culture and growth conditions The experiment was conducted in a greenhouse located at Huazhong Agricultural University, Wuhan, China, from March to August 2016. The citrange [Citrus sinensis (L.) Osb. × Poncirus trifoliata (L.) Raf.] and trifoliate orange [Poncirus trifoliata (L.) Raf.] seedlings used were uniform in size and obtained from a commercial nursery (Ganzhou, Jiangxi, China). After being washed with distilled water, plants were transplanted to 4-liter black plastic pots (three plants per pot), which had been immersed in 1 M HCl for 24 h and washed with distilled water. In this experiment, all the seedlings were cultured in a nutrient solution modified from Hoagland and Arnon (1950), containing the following macronutrients in mM: KNO3, 2.00; Ca (NO3)2, 1.23; MgSO4, 0.50; Na2HPO4, 0.14; NaH2PO4, 0.32; and the following micronutrients in μM: MnCl2, 4.45; ZnSO4, 0.80; CuSO4, 0.16; Na2MoO4, 0.18; EDTA-Fe, 37.30. Two B contents were supplied at 10 μM (CK, control treatment) and 400 μM (BT, boron toxicity) using boric acid, accompanied with a treatment without B (BD, boron deficiency). The solution was aerated for 20 min at a 4-h interval and replaced once a week. The experiment was conducted in a completely randomized with six treatments, and each treatment was replicated six times with one replication contained one plant. The pH was adjusted to
2.5. Fourier transform infrared spectroscopy (FTIR) analyses of cell wall According to the method described by Liu et al. (2014), disks for FTIR spectroscopy were prepared using a Graseby-Specac Press. A small amount of cell wall powder of leaves was mixed with KBr (1:100 m/m) and pressed into tablets. IR spectra (4000-400 cm−1) were recorded using a VERTEX 70 spectrometer by a resolution of 4 cm−1 and 32 scans per sample. The 6 copies of the spectra of cell walls with different B treatments were normalized and baseline-corrected with OMNIC 32 software. Graphical data were processed with Microsoft Excel 2010 and Origin 8.6. 2.6. Solid-State nuclear magnetic resonance (13C-NMR) spectroscopy analysis of cell wall For the 13C-NMR analysis, the cell wall of leaf samples was ground to fine powder and passed through a 0.2 mm sieve. The 13C-NMR spectra were obtained on a fully automatic nuclear magnetic resonance 148
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Furthermore, the dry mass accumulations in trifoliate orange and citrange plants were both hindered by either B deficiency or excess relative to the control (Table 1). The total dry weights of trifoliate orange and citrange treated with deficient B were decreased by 24.57 and 15.71%, respectively, while decreased by 14.85 and 12.86%, respectively, under B excessive conditions. The B concentration was much higher in leaves than in any other plant parts, regardless of the different types of rootstocks or B treatments (Table 1). Significant differences in different parts of the two rootstock seedlings were found between the control and the other two B treatments. Compared with the control, B deprivation significantly decreased the B concentration in roots, stems and leaves by 47.02, 42.49 and 79.61% in trifoliate orange, but only 40.96, 41.16 and 70.47% in citrange, indicating that the response of B contents to boron deficiency is smaller in citrange than that in trifoliate orange plants. Additionally, B toxicity notably increased the content of B in roots, stems and leaves, especially in leaves, and citrange had significantly lower B concentration of corresponding parts. It should be pointed out that B contents in every parts of trifoliate orange were higher compared with citrange under B-deficient, normal and excessive conditions, which suggested that citrange need less B to satisfy the growth than trifoliate orange.
spectrometer of 400 MB (Bruker Avance III) using a 4 mm magic angle probe at 100.63 MHz. The cross polarization-total suppression of sidebands (CP/TOSS) NMR spectrum was recorded at speed 5 kHz, sampling time 5 ms, a 1 s recycle delay, contact time 1 ms, 1024 sampling points and 4096 scans (Wu et al., 2017). 2.7. Data analysis The data were submitted to analysis of variance (ANOVA) using SAS 9.1.3 (SAS Institute, Cary, NC, USA). Unless otherwise noted, results were presented as mean ± SD. When a significant (P < 0.05) treatment effect was observed, the mean values were compared using the LSD test (P < 0.05), and significant differences (P < 0.05) within each group were indicated by different lower case letters (a, b, c……). Graphs were prepared using Origin pro.8.6 (Origin Lab Corporation, USA). FTIR spectra were normalized and baseline-corrected with OMNIC 32 software (Thermo Fisher Scientific Inc., USA), and then patterns were exported using Origin 8.6. Integrations of 13C-NMR data were conducted with Topspin 3.2 (Bruker, Germany) and then plotted with Adobe Illustrator CS5. 3. Results
3.2. Cell wall material (CWM) and allocation of boron in cell walls of leaf 3.1. Plant dry mass accumulation and boron concentration of the two citrus rootstocks
The results presented in Table 2 showed that CWM per unit of leaf fresh weight in trifoliate orange and citrange were both increased with deficient and excess B supplying, and the content of CWM on a leaf fresh weight basis was generally higher in trifoliate orange than in citrange under the same B treatment. The result suggested a lower B requirement of the cell wall in citrange. Compared with the control, wall B on a dry wall weight basis and wall B on a dry leaf weight basis in trifoliate orange and citrange leaf were both significantly decreased due to B deprivation, while were both increased due to B excess (Table 2). What is more, remarkably increases and decreases of the proportions of wall B in leaf B in two citrus rootstocks respectively caused by B deficiency and toxicity were observed. In control plants, leaves contained only 18.20 and 22.87% of cellular B in cell walls of trifoliate orange and citrange, respectively. These proportions were increased to 50.70% in trifoliate orange and
As shown in Fig. 1, citrus rootstocks of both trifoliate orange and citrange treated under either B deficient or toxicity conditions had shorter roots and shoots compared to the control treatment, and this phenomenon was more obvious in trifoliate orange. The results indicated that B stresses had a lighter suppression on the growth of citrange and also further illustrate that citrange has a stronger tolerance to B deficiency and toxicity than trifoliate orange. Under B deficiency condition, leaf chlorosis was observed at the top in trifoliate orange seedlings, but not in citrange plants. Comparatively, under B excessive condition, symptoms of B-toxicity appeared in old leaves of trifoliate orange as tip-yellowing, followed by marginal, and finally, these chlorotic leaves became necrotic and shed prematurely, while the visible symptoms occurred later in citrange and only on the leaf tip.
Fig. 1. Transmission electron micrographs of leaves in trifoliate orange and citrange rootstock seedlings under different B treatments [CK (control): 10 μM B; BD (boron deficiency): 0 μM B; BT (boron toxicity): 400 μM B]. Explanation of plate (Ⅰ: B-deficient trifoliate orange; Ⅱ: control trifoliate orange; Ⅲ: B-toxic trifoliate orange; Ⅳ: B-deficient citrange;Ⅴ: control citrange; Ⅵ: B-toxic citrange ;Sg: Starch grain; Pg: Plastoglobulus; Cw: Cell wall). 149
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Table 1 Effect of different B treatments on dry matter (g/plant) and B content(mg/kg)in different parts of trifoliate orange and citrange rootstock seedlings. Rootstock
Treatment
Trifoliate orange
BD CK BT BD CK BT
Citrange
Dry mass (g/plant)
Boron concentration (mg/kg)
Root
Stem
Leaf
Whole plant
Root
Stem
Leaf
0.41d 0.56b 0.50c 0.53b 0.67a 0.62a
0.55c 0.71ab 0.52c 0.69ab 0.74a 0.64b
0.40c 0.55b 0.51b 0.55b 0.75a 0.60b
1.32c 1.75b 1.49c 1.77b 2.10a 1.83b
11.11e 20.97c 42.72a 8.51f 14.43d 30.51b
9.99e 17.37c 31.17a 8.42e 14.31d 22.79b
29.36e 144.02c 467.55a 17.80f 60.27d 416.33b
Note: CK (control): 10 μM B; BD (boron deficiency): 0 μM B; BT (boron toxicity): 400 μM B. Columns with different letters (a, b……) indicate significant differences among different treatments (n = 6, P < 0.05).
proteins and carbohydrates (cellulose, hemicellulose) in trifoliate orange leaves, but not in citrange or under excessive B condition. Meanwhile, significant decreases of intensity of peak at 1736 cm−1, which is characteristic of the C]O stretching vibration of alkyl esters in pectin (Abidi et al., 2008), were showed in B-toxicity leaf, especially in trifoliate orange leaf. The variations on peaks at 1,640, 1541 and 1250 cm−1 corresponding to amideⅠ, amide II, and amide III of proteins, respectively (Kong and Yu, 2007; Abidi et al., 2008) indicated that B stress destroyed protein structure of cell walls from leaf. Spectra from B-deficient cell walls had a small but distinct peak at 1518 cm−1 in both rootstocks, which is characteristic of phenolic ring structures. Vibrations located at 1436 and 1380 cm−1 were attributed to CeH deformation and represented cellulose in the cell walls. B deficiency significantly increased the intensity of the two peaks in trifoliate orange leaves but exhibited a lower intensity in citrange. Spectra of cell walls from B-toxic leaves had a lower relative absorbance and an obvious shift from 1436 to 1425 cm−1 in trifoliate orange, suggesting that B toxicity influenced the structure of cellulose and reduced the cellulose content in leaf cell walls of trifoliate orange. B deficiency significantly increased the relative concentration of carbohydrates in trifoliate orange leaves due to the enhanced characteristic peaks of polysaccharides (1,157, 1,105, 1065 and 1028 cm−1), while have no notably changes on citrange (Fig. 2). These results suggested that the effects of B deficiency and toxicity on cell wall structure and components were both more significant in trifoliate orange than citrange.
69.74% in citrange under B deficient conditions. The increased percentage of wall B/leaf B in citrange was 204.94%, notably higher than that of trifoliate orange (178.57%) treated with deficient-B, while there was no obvious difference in the percentage of wall B/leaf B between the citrus rootstock seedlings under the B excessive condition. These results suggested that B in leaves is assigned preferentially to cell walls under limited-B conditions, and cell walls of citrange has more RG-Ⅱto maintain a stronger ability to combine with B under B deficiency. 3.3. Subcellular structure changes and TEM analysis of leaf As shown in Fig. 1 I, IV TEM micrographs of the leaf showed thickened cell walls, accumulation of starch grains and plastoglobulus under B-deficiency treatment compared with the control (Fig. 1 II, V), regardless of the rootstock type. With excessive B supply, except thickened cell walls and increase of plastoglobulus, significant injury to chloroplasts in leaf with disruption of thylakoids was also observed (Fig. 1 III, VI). The increased number and elongation of plastoglobulus occupied the entire chloroplast, making the chloroplasts nearly circular in shape. 3.4. Changes in composition and structure of cell wall materials in leaf FTIR was performed between 4000 and 400 cm−1 to demonstrate the changes in the material composition and structure of leaf cell walls in response to different B stresses (Fig. 2). The differences in cell wall structure and composition were mainly revealed in the region of 1800800 cm−1 and were more evident in trifoliate orange than in citrange (Fig. 2). As shown in Fig. 4 and Table 1, the peaks located around 3420 cm−1 corresponded to OeH and NeH stretching vibrations mainly derived from carbohydrates (Yang and Yen, 2002). The vibration located at 3424 cm−1was present in the spectra of control plant cell walls in trifoliate orange leaves but shifted to 3400 cm−1 under B deficiency. In contrast, no visible changes occurred in the cell wall of citrange leaves or the two excessive-B-treated rootstocks (Fig. 2), suggesting that B-deficiency influences the hydrogen bonding between
3.5. Changes in organic carbon in leaf cell walls The 13C-NMR CP/TOSS spectra of cell walls in Fig. 3 can be divided into 4 resonance regions (Table 3): the alkyls C (0–45 ppm), the O-alkyl C (45–112 ppm), the aromatic C (112–160 cm) and the carbonyls C (160–190 ppm). Specifically, the O-alkyl C consisted of the methoxyl C (45–62 ppm), the carbohydrates C (62–92 ppm) and the Di-O-alkyl C (92–112 ppm), while the aromatic C consisted of the aryl-C (112–141 ppm) (containing the resonance of CeH and CeC) and the phenolic C (141–160 ppm). The integration results based on the different resonance regions are
Table 2 Cell wall material and B allocation in cell walls of leaves in trifoliate orange and citrange rootstock seedlings. Rootstock
Trifoliate orange
Citrange
Treatment
BD CK BT BD CK BT
Cell wall material (mg/g FW)
19.51a 16.06 cd 18.07ab 17.00bc 14.78d 15.55 cd
Wall B per unit dry wall (ug/g)
31.69d 41.2c 47.64a 28.03e 39.03c 44.02b
Wall B per unit dry leaf (ug/g)
14.75c 17.00b 21.34a 12.07d 15.28bc 17.20b
Wall/Leaf %
Increase (%)
Decrease (%)
50.70b 18.20c 4.87d 69.74a 22.87c 4.21d
178.57b – – 204.94a – –
– – 73.24b – – 81.59a
Note: CK (control): 10 μM B; BD (boron deficiency): 0 μM B; BT (boron toxicity): 400 μM B. Rows with different letters (a, b……) indicate significant differences among different treatments (n = 6, P < 0.05). 150
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Fig. 2. FTIR spectra of the cell walls from leaves of trifoliate orange and citrange rootstock seedlings (1800-800 cm−1) under different B treatments [CK (control): 10 μM B; BD (boron deficiency): 0 μM B; BT (boron toxicity): 400 μM B].
in trifoliate orange, compared with increases of only 8.62, 6.96 and 2.91% in citrange, which suggested more obvious increases of cellulose, lignin, carbohydrates and phenols in cell walls of trifoliate orange. However, B toxicity had no remarkably effects on organic carbon constitution in leaf of the two rootstock seedlings. These results suggested that B deficiency induced variations in the organic carbon structure of leaf cell walls and were more significant in trifoliate orange than citrange, while B toxicity had no obvious adverse effects on organic carbon.
shown in Table 3. The organic carbon of leaf cell walls under three different B treatments was dominated by carbohydrates C. Additionally, compared with B-toxic treatment, more substantial variations were observed in the cell walls under B-deficient condition. The different C functional groups consist of CH3, OeCH3, CαeOR, OCHO, CeH and CeC, COO represent different compounds in Tables 2. B starvation significantly decreased the signals of CH3 (17.28%), OeCH3 (11.68%), CeH and CeC (8.18%) and COO groups (17.36%) in trifoliate orange versus 14.56, 7.25, 5.29 and 15.16% in citrange, respectively, indicating B deficiency decreased the contents of wax, amino acids, polyphenol, carboxylic acid and organic acid in cell walls, especially of trifoliate orange. Meanwhile, B starvation significantly increased the signals of CαeOR (11.39%), OCHO (8.28%) and CeO (16.50%) groups
Fig. 3. 13C-NMR spectra of cell walls from leaves of trifoliate orange and citrange rootstock seedlings under different B treatments [CK (control): 10 μM B; BD (boron deficiency): 0 μM B; BT (boron toxicity): 400 μM B]. 151
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Table 3 The relative content (%) of different types of organic carbon in leaf cell walls from trifoliate orange and citrange rootstocks with different B treatments. Rootstock
Treatment
0–45 CH3
45–62 O-CH3
62–92 Cα-OR
92–112 OCHO
112–121 C-H
121–141 C-C
141–160 C-O
160–190 COO
Trifoliate orange
BD CK BT BD CK BT
17.42bc 21.06a 19.17ab 16.78c 19.64a 19.24ab
10.21b 11.56a 10.89ab 10.10b 10.89ab 10.60b
49.67a 44.59c 46.46bc 50.15a 46.17bc 47.15b
11.90a 10.99b 10.50b 11.84a 11.07b 10.91b
1.32b 1.43a 1.37ab 1.31b 1.43a 1.40ab
2.15b 2.35a 2.35a 2.27a 2.35a 2.27a
1.20a 1.03b 1.19a 1.06ab 1.03b 0.99b
5.84b 7.09a 7.16a 5.93b 6.99a 6.89a
Citrange
Note: CK (control): 10 μM B; BD (boron deficiency): 0 μM B; BT (boron toxicity): 400 μM B. Columns with different letters (a, b……) indicate significant differences among different treatments (n = 6, P < 0.05).
4. Discussions
contents of methoxyl C, aryl C and carboxyl C, and increased the carbohydrate C, Di-O-alkyl C and phenolic C, which can represent a reduction of amino acid, polyphenol, carboxylic acid, organic acid and a raisin of carbohydrates, cellulose and lignin, respectively (Wang et al., 2013). The cell walls contained a variety of wall-associated proteins, and the structural proteins supports in the mechanical strength of the wall and facilitate the proper asse mbly of other wall components (Jamet et al., 2006). The decreased amino acids revealed by the B-deficient 13C-NMR spectra indicated B deficiency affected the synthesis of wall-associated proteins. Additionally, B could participate in lignin metabolism (Bellaloui, 2012) and it has been reported that lignin biosynthesis was obviously increased in trifoliate orange roots under Bdeprived conditions (Dong et al., 2016). Our 13C-NMR spectral results showed a higher level of phenolic C which presented accumulated lignin in cell walls under B deprivation, and subsequently resulted in the lignification of cell wall. Under B deficiency, the vibrations located at 1,200–800 cm−1 (fingerprint region for polysaccharides) showed that the relative concentration of carbohydrate in trifoliate orange leaves was much higher than that of citrange due to the enhanced characteristic peaks of polysaccharides, which was in agreement with the obvious increase in the content of carbohydrate C in the cell walls of trifoliate orange leaves. The peaks at 1380 and 1030 cm−1 are attributed to cellulose (Zhang et al., 2008). The intensities of the two peaks were higher in trifoliate orange with no B supply, suggesting the synthesis of cellulose in cell walls was promoted or degradation was inhibited, allowing the cells to slow down and stop division, and finally leading to the inhibition of the cell physiological metabolism in trifoliate orange under the B-deficient condition. In addition, the cell wall extraction rate of trifoliate orange leaves under B deficiency was much higher than that of citrange (Table 2), which was another reason for the inhibition of physiological metabolism by the increase of the cell wall material content (mainly carbohydrate). Therefore, alteration on the architecture of cell walls may destroy the integrity of cell walls in leaf. Phenolic compounds which are generated in pentose phosphate pathway (PPP) are important secondary metabolites in plants. B deficiency obviously promoted the accumulation of phenols in the cell walls of trifoliate orange leaves, while had no remarkably promotion in citrange (Fig. 2 and Table 3). Increased phenols caused a rapid rise of polyphenol oxidase activity and a large number of oxyradical and active quinone derivatives (Shkol’nik et al., 1981). The increase of phenols due to B starvation may be a secondary reaction to the death of cells, suggesting more cells died in trifoliate orange than in citrange. These results suggest that the changes induced by B starvation and excess in the celluar structure and the architecture of cell walls determine, to some extent, chlorosis, necrotic and shed prematurely of leaf. A schematic summary, describing the key responses on B allocated in cell walls, cell wall structure and components in B-deficient and Bexcessive of rootstocks, is proposed in Fig. 4.
4.1. Changes on allocation of boron in cell walls It has been reported that B is required for the structural integrity of cell walls (O’Neill et al., 2004). In this study, B deficiency significantly decreased the B concentration in the leaf cell walls of the two citrus rootstock seedlings (Table 1), which was in agreement with the report about other plant species (Goldbach et al., 2000; Li et al., 2007). Additionally, the increased percentage of wall B/leaf B was much higher in citrange, indicating that much more B was allocated to the cell walls in citrange than in trifoliate orange under B deficiency condition, enabling citrange seedlings to maintain its growth under the B stress. Under excessive B conditions, citrange showed a much lower increase in B concentration of their leaf cell walls on a dry weight basis than trifoliate orange (Table 1). These results suggested that citrange leaves can allocate more B to cell walls under a relatively low B supply level to maintain the relatively normal cell wall structure. This may be partially responsible for the less serious symptoms of B deficiency in citrange compared to trifoliate orange. 4.2. Changes in subcellular structure and the architecture of cell wall components In the present study, the cell wall extraction rate increased in trifoliate orange and citrange under B deficiency and excess conditions, which implies that both B deficiency and toxicity increased the content of cell wall material in the two rootstock seedlings (Table 2). This result was supported by the TEM micrographs of the thickened cell wall in cells from the leaves treated under B deficiency and excess treatments (Fig. 1). Similar results were also observed in rape and navel orange under B-limited conditions (Pan et al., 2012; Liu et al., 2015) and irregular cell wall thickening were observed in leaf cortex cells and phloem tissue of B-toxic C. grandis and C. sinensis leaves (Huang et al., 2014). It has been reported that the disintegration of thylakoid system lead to replacement of few non chlorophyllous single thylakoids, with accumulation of large osmiophilic plastoglobules (Zhu and Fang, 1995). At deficient-B and excessive-B levels, increased of plastoglobule numbers and volumes in leaf cells indicated that disintegration of the thylakoid in leaf and the photosynthetic characteristics affected by B stresses. FTIR and 13C-NMR spectra showed that the material composition and organic carbon of cell walls varied between the two citrus rootstocks under B stresses. The 13C-NMR spectra revealed that the organic carbon of leaf cell walls under different B treatments was dominated by O-alkyl C (70–80%), which consists of the methoxyl C, the carbohydrates C (44–50%) and the Di-O-alkyl C (Tables 2 and 3), indicating that carbohydrate is the most important organic compound in the organic carbon of leaf cell walls in the two rootstock seedlings (Wang et al., 2013). Under B-toxicity conditions, there was no obvious increase of Cα-OR in the two rootstocks as represented by carbohydrate in the 13 C-NMR spectra (Table 3), While B deficiency decreased the relative
5. Conclusions Under B deficiency, the cellular B in cell walls of the two citrus 152
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Fig. 4. Schematic diagram in the changes of cell walls in trifoliate orange and citrange due to B deficiency and toxicity. Explanation of plate (Tri: trifoliate orange; Cir: citrange).
Appendix A. Supplementary data
rootstocks were 2.79 and 3.05 folds of their controls, and citrange allocated a larger proportion (69.74%) of B in cell walls than trifoliate orange (50.70%). Additionally, B deficiency and toxicity affected the subcellular structure and the architectural compositions of cell walls in the two citrus rootstocks, and more severe in trifoliate orange. No matter under control or B stresses, the largest amount of organic carbon in leaf cell walls is carbohydrate C (Cα-OR), about 44–50%. B-deficiency-induced decreases of methoxyl C, aryl C, carboxyl C and increases of carbohydrate C, Di-O-alkyl C, phenolic C represented less amino acid, polyphenol, carboxylic acid, organic acid and more carbohydrates, cellulose, lignin, phenols in cell walls of the two citrus rootstocks, and changes were also more remarkably in trifoliate orange, while the adverse effects of B toxicity on cellulose structure and the linkage pattern among pectin in cell walls were lighter in citrange. Therefore, variations of changes on the cellular B allocation, cell ultrastructure, cell wall compositions and structure may determine the different tolerance to B deficiency and toxicity in the two rootstocks.
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Conflicts of interest The authors declare that they have no conflict of interest.
Acknowledgements This research project was financially supported by the National Natural Science Foundation of China (41271320) and the Fundamental Research Funds for the Central Universities (2017PY055). 153
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Boron deficiency and toxicity altered the subcellular structure and cell wall composition architecture in two citrus rootstocks Xiuwen Wu, Xiaopei Lu, Muhammad Riaz, Lei Yan, Cuncang Jiang
T
⁎
Microelement Research Center, College of Resources and Environment, Huazhong Agricultural University, Wuhan, Hubei, PR China
A R T I C LE I N FO
A B S T R A C T
Keywords: Boron stresses Leaf Ultrastructure Cell wall components FTIR 13 C-NMR
Trifoliate orange and citrange are two important rootstock resources to citrus, and citrange has a stronger tolerance to boron (B) deficiency and toxicity than trifoliate orange. In this study, we described how B deficiency and toxicity depressed the variations of cell wall B location, subcellular structure, cell wall components and structure of the two citrus rootstocks, to evaluate the mechanisms of different B tolerance of rootstocks on the cellular and structural levels. The results showed that citrange had better growth and lower symptoms of B stresses than trifoliate orange. What is more, citrange had a stronger ability to allocate more B to cell walls than trifoliate orange at deficient-B level. Under B deficiency and excessive conditions, severe damages in subcellular structure with obvious irregular thickening of cell walls and higher accumulation of plastoglobulus were observed in leaf cells of both two rootstocks. Additionally, it also showed obvious variations in the mode of hydrogen bonding, and more accumulation of cellulose, phenols and carbonhydrates in cell walls of trifoliate orange leaf under B starvation, while lighter changes on cell wall components were observed in citrange. As for B toxicity, comparing to trifoliate orange, citrange showed a lighter damage on pectin crosslinking structure in cell walls of leaf tissue. These results gained some novel mechanisms of different citrus rootstocks to B stress tolerance, and provide a theoretical basis for cultivating improved citrus rootstocks.
1. Introduction Boron (B) is an essential microelement for higher plants and B plays an important role in the structure of plant cell walls, and its deprivation causes a wide range of variations in the cell function, the physiology and biochemistry of cell walls, and plant metabolites (Pan et al., 2012; Dordas and Brown, 2005; Liu et al., 2014). It has been demonstrated that the accepted physiological function of B is to cross-link two chains of rhamnogalacturonan II (RG-II) by the formation of borate diol diester in pectin of cell wall, and then affect the biochemical and mechanical properties of the plant wall (O’Neill et al., 2004). Cell walls have an important influence on the structural integrity of the cells and determine the cells shape and size (Hayot et al., 2012). Boron starvation is a widespread problem for many agricultural crops, including citrus (Shorrocks, 1997), is responsible for considerable loss of productivity and poor fruit quality in many citrus orchards (Xiao et al., 2007). Conclusive evidence shows that B deficiency results in curling of the leaves, leaf chlorosis, weakened photosynthesis, abnormal anatomic structure and metabolic disturbance in leaves (Liu et al., 2014; Han et al., 2009; Lu et al., 2017a,b; Dong et al., 2016).
Application of B-enriched fertilizers is an effective solution for resolving B deficiency, but B toxicity is a more difficult problem to manage and the damage of B toxicity to plants is irreversible. At excessive B level, B tends to accumulate in leaf tips and leaf margins, causing margin damage (Reid et al., 2004; Shapira et al., 2013). Under such conditions, many plants show inhibited growth and the symptoms of B toxicity like chlorotic and necrotic patches, which are usually found on the margins and tips of older leaves (Nable et al., 1997; Han et al., 2009). Several studies have demonstrated the destruction of leaf structure, oxidative damage, and interference to synthesis of cell wall components with excessive B supply (Papadakis et al., 2004a; Shah et al., 2017; Mesquita et al., 2016). Citrus is one of the most important economic crops in China, and the rootstocks have been used to optimize plant growth, and improved fruit production and quality (Xiao et al., 2007; Papadakis et al., 2004a). Early studies have indicated that citrus plants are sensitive to B deficiency or toxicity (Papadakis et al., 2003; Liu et al., 2011), and rootstocks vary greatly in their effects on tolerance to B deficiency (Boaretto et al., 2008) and toxicity (Papadakis et al., 2004a,b). Trifoliate orange [Poncirus trifoliata (L.) Raf.] and citrange [Citrus sinensis (L.) Osb. ×
Abbreviations: CK, control; BD, boron deficiency; BT, boron toxicity; FTIR, fourier-transform infrared spectroscopy; 13C-NMR, 13C nuclear magnetic resonance; TEM, transmission electron microscope; PBS, glutaraldehyde in phosphate buffer solution; CWM, cell wall material; Sg, starch grain; Pg, plastoglobulus; Tri, trifoliate orange; Cir, citrange ⁎ Corresponding author at: College of Resources and Environment, Huazhong Agricultural University, No.1 Shizishan Street, Wuhan, 430070, Hubei Province, PR China. E-mail address: [email protected] (C. Jiang). https://doi.org/10.1016/j.scienta.2018.04.057 Received 6 March 2018; Received in revised form 25 April 2018; Accepted 25 April 2018 0304-4238/ © 2018 Elsevier B.V. All rights reserved.
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5.8 ∼ 6.2 every day with 0.5 M H2SO4 or 1 M NaOH.
Poncirus trifoliata (L.) Raf.] are two vital rootstock resources to citrus, and the two rootstocks have different tolerances to B stress (FornerGiner et al., 2003; Liu et al., 2013). It has been reported that citrange has a stronger tolerance to B deficiency than trifoliate orange (Sheng et al., 2009) and evaluated the mechanisms of difference by studying cellular B allocation and pectin composition (Liu et al., 2013), structure and components of leaf (Lu et al., 2017a,b). Huang et al. (2014) investigated the changes on leaf photosynthesis, chlorophyll, plant B absorption and distribution induced by B toxicity, using two citrus species differing in B sensitivity, to elucidate the possible mechanisms of B tolerance in different citrus plants. Under high B conditions, better growth status was observed in “Newhall” plants grafted on citrange than in those grafted on trifoliate orange by Liu et al. (2011), which suggesting that plants grafted on citrange were more tolerant to B toxicity than the trifoliate orange-grafted plants. However, there have been few studies performed on the differences and changes in the cellular structure and the architecture of cell wall components in leaves of trifoliate orange and citrange rootstocks under B deficiency and excess conditions, the relationship between variations on cell walls and Btolerant is not clear. Fourier-transform infrared spectroscopy (FTIR) has been used to gather information about the chemical composition of almost all substances (Liu et al., 2014), and it is also extensively applied in analyzing plant cell walls (Abidi et al., 2014). In recent years, solid-state 13C nuclear magnetic resonance (13C-NMR) spectroscopy has become a powerful tool to identify the structure of organic compounds and provide comprehensive structural information (Mao et al., 2008). Despite its wide application in characterization of the structure of soil organic matter, wood lignin and cell wall polysaccharides, etc. (RondeauMouro et al., 2003; Salati et al., 2008; Balakshin et al., 2016), the13CNMR technique has rarely been used to investigate the cell wall organic carbon structure of citrus under different B stress conditions. Therefore, the trifoliate orange and citrange were selected as research materials, and chemical analysis methods combined with transmission electron microscope (TEM), FTIR and solid-state 13C-NMR were conducted to examine (1) how B deficiency and toxicity altered B allocated in cell walls and cellular structure of the two citrus rootstocks; (2) varied changes on cell wall architecture and components betwwen trifoliate orange and citrange treated with deficient-B and toxic-B, and ultimately to gain the structural changes of cell walls in different citrus rootstocks under different B stresses.
2.2. Plant sampling and boron analysis After completing 90-days, the plants were harvested and plant samples were divided into roots, stems, and leaves. All the leaves of each plant were further separated into three parts: one part was used for determination of B concentration, another part was stored for extraction of cell wall materials, and the third part was used for transmission electron microscope analyses. After drying to a constant weight at 75 °C, the dry weight of roots, stems and leaves were measured. Then the samples were ground to a fine powder and ashed at 500 °C for 5 h, followed by dissolving the ashes in 0.1 M HCl. The B contents in roots, stems and leaves were measured at 540 nm spectrophotometrically (Hitachi UV-3100 UV–vis; TECHCOMP, Shanghai, China), according to the curcumin colorimetric method (Dible et al., 1954). 2.3. Transmission electron microscope (TEM) analysis The TEM slices were prepared by the method of Kong et al. (2013) with slight modification. The TEM analysis was performed as follows: briefly, leaves from the same parts under different B treatments were cut into small pieces (1 × 1 mm), then fixed in 2.5% glutaraldehyde in phosphate buffer solution (PBS) for 12 h at 4 °C. Next, the tissue blocks were rinsed four times with 0.1 M PBS (pH 7.4) and post-fixed for 2–3 h with 1% buffered osmium tetroxide, followed by rinsing three times with 0.1 M PBS (pH 7.4), and dehydration three times in an increasing acetone concentration series [50, 70 and 90 (three times)] and then in a mixture of 90% ethanol and 90% acetone for 15 min. After being stained with 2% uranyl acetate and lead citrate, ultrathin sections were examined with a JEM-100CX II transmission electron microscope. 2.4. Preparation of cell wall materials (CWM) The cell wall was separated from the leaves as reported by Hu and Brown (1994) with some modifications. Firstly, the leaf samples were homogenized in liquid nitrogen with a mortar, after homogenizing in 10 volumes of ice-cold ultrapure water and centrifuging at 5000g for 10 min at 4 °C, the precipitate was washed with 10 volumes of ice-cold ultrapure water and centrifuged again. Next, the residue was washed three times with 10 volumes of 80% ethanol and once with 10 volumes of mixture of methanol/chloroform (1/1, v/v). Finally, the precipitate was washed with 10 volumes of acetone. The final insoluble pellet was dried at 50–60 °C, weighed and defined as CWM. The dry CWM was divided into two portions: one was dried to ashes at 500 °C for determining B allocated in cell walls as described above, and the other portion was used for FTIR and 13C-NMR analyses.
2. Materials and methods 2.1. Plant culture and growth conditions The experiment was conducted in a greenhouse located at Huazhong Agricultural University, Wuhan, China, from March to August 2016. The citrange [Citrus sinensis (L.) Osb. × Poncirus trifoliata (L.) Raf.] and trifoliate orange [Poncirus trifoliata (L.) Raf.] seedlings used were uniform in size and obtained from a commercial nursery (Ganzhou, Jiangxi, China). After being washed with distilled water, plants were transplanted to 4-liter black plastic pots (three plants per pot), which had been immersed in 1 M HCl for 24 h and washed with distilled water. In this experiment, all the seedlings were cultured in a nutrient solution modified from Hoagland and Arnon (1950), containing the following macronutrients in mM: KNO3, 2.00; Ca (NO3)2, 1.23; MgSO4, 0.50; Na2HPO4, 0.14; NaH2PO4, 0.32; and the following micronutrients in μM: MnCl2, 4.45; ZnSO4, 0.80; CuSO4, 0.16; Na2MoO4, 0.18; EDTA-Fe, 37.30. Two B contents were supplied at 10 μM (CK, control treatment) and 400 μM (BT, boron toxicity) using boric acid, accompanied with a treatment without B (BD, boron deficiency). The solution was aerated for 20 min at a 4-h interval and replaced once a week. The experiment was conducted in a completely randomized with six treatments, and each treatment was replicated six times with one replication contained one plant. The pH was adjusted to
2.5. Fourier transform infrared spectroscopy (FTIR) analyses of cell wall According to the method described by Liu et al. (2014), disks for FTIR spectroscopy were prepared using a Graseby-Specac Press. A small amount of cell wall powder of leaves was mixed with KBr (1:100 m/m) and pressed into tablets. IR spectra (4000-400 cm−1) were recorded using a VERTEX 70 spectrometer by a resolution of 4 cm−1 and 32 scans per sample. The 6 copies of the spectra of cell walls with different B treatments were normalized and baseline-corrected with OMNIC 32 software. Graphical data were processed with Microsoft Excel 2010 and Origin 8.6. 2.6. Solid-State nuclear magnetic resonance (13C-NMR) spectroscopy analysis of cell wall For the 13C-NMR analysis, the cell wall of leaf samples was ground to fine powder and passed through a 0.2 mm sieve. The 13C-NMR spectra were obtained on a fully automatic nuclear magnetic resonance 148
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Furthermore, the dry mass accumulations in trifoliate orange and citrange plants were both hindered by either B deficiency or excess relative to the control (Table 1). The total dry weights of trifoliate orange and citrange treated with deficient B were decreased by 24.57 and 15.71%, respectively, while decreased by 14.85 and 12.86%, respectively, under B excessive conditions. The B concentration was much higher in leaves than in any other plant parts, regardless of the different types of rootstocks or B treatments (Table 1). Significant differences in different parts of the two rootstock seedlings were found between the control and the other two B treatments. Compared with the control, B deprivation significantly decreased the B concentration in roots, stems and leaves by 47.02, 42.49 and 79.61% in trifoliate orange, but only 40.96, 41.16 and 70.47% in citrange, indicating that the response of B contents to boron deficiency is smaller in citrange than that in trifoliate orange plants. Additionally, B toxicity notably increased the content of B in roots, stems and leaves, especially in leaves, and citrange had significantly lower B concentration of corresponding parts. It should be pointed out that B contents in every parts of trifoliate orange were higher compared with citrange under B-deficient, normal and excessive conditions, which suggested that citrange need less B to satisfy the growth than trifoliate orange.
spectrometer of 400 MB (Bruker Avance III) using a 4 mm magic angle probe at 100.63 MHz. The cross polarization-total suppression of sidebands (CP/TOSS) NMR spectrum was recorded at speed 5 kHz, sampling time 5 ms, a 1 s recycle delay, contact time 1 ms, 1024 sampling points and 4096 scans (Wu et al., 2017). 2.7. Data analysis The data were submitted to analysis of variance (ANOVA) using SAS 9.1.3 (SAS Institute, Cary, NC, USA). Unless otherwise noted, results were presented as mean ± SD. When a significant (P < 0.05) treatment effect was observed, the mean values were compared using the LSD test (P < 0.05), and significant differences (P < 0.05) within each group were indicated by different lower case letters (a, b, c……). Graphs were prepared using Origin pro.8.6 (Origin Lab Corporation, USA). FTIR spectra were normalized and baseline-corrected with OMNIC 32 software (Thermo Fisher Scientific Inc., USA), and then patterns were exported using Origin 8.6. Integrations of 13C-NMR data were conducted with Topspin 3.2 (Bruker, Germany) and then plotted with Adobe Illustrator CS5. 3. Results
3.2. Cell wall material (CWM) and allocation of boron in cell walls of leaf 3.1. Plant dry mass accumulation and boron concentration of the two citrus rootstocks
The results presented in Table 2 showed that CWM per unit of leaf fresh weight in trifoliate orange and citrange were both increased with deficient and excess B supplying, and the content of CWM on a leaf fresh weight basis was generally higher in trifoliate orange than in citrange under the same B treatment. The result suggested a lower B requirement of the cell wall in citrange. Compared with the control, wall B on a dry wall weight basis and wall B on a dry leaf weight basis in trifoliate orange and citrange leaf were both significantly decreased due to B deprivation, while were both increased due to B excess (Table 2). What is more, remarkably increases and decreases of the proportions of wall B in leaf B in two citrus rootstocks respectively caused by B deficiency and toxicity were observed. In control plants, leaves contained only 18.20 and 22.87% of cellular B in cell walls of trifoliate orange and citrange, respectively. These proportions were increased to 50.70% in trifoliate orange and
As shown in Fig. 1, citrus rootstocks of both trifoliate orange and citrange treated under either B deficient or toxicity conditions had shorter roots and shoots compared to the control treatment, and this phenomenon was more obvious in trifoliate orange. The results indicated that B stresses had a lighter suppression on the growth of citrange and also further illustrate that citrange has a stronger tolerance to B deficiency and toxicity than trifoliate orange. Under B deficiency condition, leaf chlorosis was observed at the top in trifoliate orange seedlings, but not in citrange plants. Comparatively, under B excessive condition, symptoms of B-toxicity appeared in old leaves of trifoliate orange as tip-yellowing, followed by marginal, and finally, these chlorotic leaves became necrotic and shed prematurely, while the visible symptoms occurred later in citrange and only on the leaf tip.
Fig. 1. Transmission electron micrographs of leaves in trifoliate orange and citrange rootstock seedlings under different B treatments [CK (control): 10 μM B; BD (boron deficiency): 0 μM B; BT (boron toxicity): 400 μM B]. Explanation of plate (Ⅰ: B-deficient trifoliate orange; Ⅱ: control trifoliate orange; Ⅲ: B-toxic trifoliate orange; Ⅳ: B-deficient citrange;Ⅴ: control citrange; Ⅵ: B-toxic citrange ;Sg: Starch grain; Pg: Plastoglobulus; Cw: Cell wall). 149
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Table 1 Effect of different B treatments on dry matter (g/plant) and B content(mg/kg)in different parts of trifoliate orange and citrange rootstock seedlings. Rootstock
Treatment
Trifoliate orange
BD CK BT BD CK BT
Citrange
Dry mass (g/plant)
Boron concentration (mg/kg)
Root
Stem
Leaf
Whole plant
Root
Stem
Leaf
0.41d 0.56b 0.50c 0.53b 0.67a 0.62a
0.55c 0.71ab 0.52c 0.69ab 0.74a 0.64b
0.40c 0.55b 0.51b 0.55b 0.75a 0.60b
1.32c 1.75b 1.49c 1.77b 2.10a 1.83b
11.11e 20.97c 42.72a 8.51f 14.43d 30.51b
9.99e 17.37c 31.17a 8.42e 14.31d 22.79b
29.36e 144.02c 467.55a 17.80f 60.27d 416.33b
Note: CK (control): 10 μM B; BD (boron deficiency): 0 μM B; BT (boron toxicity): 400 μM B. Columns with different letters (a, b……) indicate significant differences among different treatments (n = 6, P < 0.05).
proteins and carbohydrates (cellulose, hemicellulose) in trifoliate orange leaves, but not in citrange or under excessive B condition. Meanwhile, significant decreases of intensity of peak at 1736 cm−1, which is characteristic of the C]O stretching vibration of alkyl esters in pectin (Abidi et al., 2008), were showed in B-toxicity leaf, especially in trifoliate orange leaf. The variations on peaks at 1,640, 1541 and 1250 cm−1 corresponding to amideⅠ, amide II, and amide III of proteins, respectively (Kong and Yu, 2007; Abidi et al., 2008) indicated that B stress destroyed protein structure of cell walls from leaf. Spectra from B-deficient cell walls had a small but distinct peak at 1518 cm−1 in both rootstocks, which is characteristic of phenolic ring structures. Vibrations located at 1436 and 1380 cm−1 were attributed to CeH deformation and represented cellulose in the cell walls. B deficiency significantly increased the intensity of the two peaks in trifoliate orange leaves but exhibited a lower intensity in citrange. Spectra of cell walls from B-toxic leaves had a lower relative absorbance and an obvious shift from 1436 to 1425 cm−1 in trifoliate orange, suggesting that B toxicity influenced the structure of cellulose and reduced the cellulose content in leaf cell walls of trifoliate orange. B deficiency significantly increased the relative concentration of carbohydrates in trifoliate orange leaves due to the enhanced characteristic peaks of polysaccharides (1,157, 1,105, 1065 and 1028 cm−1), while have no notably changes on citrange (Fig. 2). These results suggested that the effects of B deficiency and toxicity on cell wall structure and components were both more significant in trifoliate orange than citrange.
69.74% in citrange under B deficient conditions. The increased percentage of wall B/leaf B in citrange was 204.94%, notably higher than that of trifoliate orange (178.57%) treated with deficient-B, while there was no obvious difference in the percentage of wall B/leaf B between the citrus rootstock seedlings under the B excessive condition. These results suggested that B in leaves is assigned preferentially to cell walls under limited-B conditions, and cell walls of citrange has more RG-Ⅱto maintain a stronger ability to combine with B under B deficiency. 3.3. Subcellular structure changes and TEM analysis of leaf As shown in Fig. 1 I, IV TEM micrographs of the leaf showed thickened cell walls, accumulation of starch grains and plastoglobulus under B-deficiency treatment compared with the control (Fig. 1 II, V), regardless of the rootstock type. With excessive B supply, except thickened cell walls and increase of plastoglobulus, significant injury to chloroplasts in leaf with disruption of thylakoids was also observed (Fig. 1 III, VI). The increased number and elongation of plastoglobulus occupied the entire chloroplast, making the chloroplasts nearly circular in shape. 3.4. Changes in composition and structure of cell wall materials in leaf FTIR was performed between 4000 and 400 cm−1 to demonstrate the changes in the material composition and structure of leaf cell walls in response to different B stresses (Fig. 2). The differences in cell wall structure and composition were mainly revealed in the region of 1800800 cm−1 and were more evident in trifoliate orange than in citrange (Fig. 2). As shown in Fig. 4 and Table 1, the peaks located around 3420 cm−1 corresponded to OeH and NeH stretching vibrations mainly derived from carbohydrates (Yang and Yen, 2002). The vibration located at 3424 cm−1was present in the spectra of control plant cell walls in trifoliate orange leaves but shifted to 3400 cm−1 under B deficiency. In contrast, no visible changes occurred in the cell wall of citrange leaves or the two excessive-B-treated rootstocks (Fig. 2), suggesting that B-deficiency influences the hydrogen bonding between
3.5. Changes in organic carbon in leaf cell walls The 13C-NMR CP/TOSS spectra of cell walls in Fig. 3 can be divided into 4 resonance regions (Table 3): the alkyls C (0–45 ppm), the O-alkyl C (45–112 ppm), the aromatic C (112–160 cm) and the carbonyls C (160–190 ppm). Specifically, the O-alkyl C consisted of the methoxyl C (45–62 ppm), the carbohydrates C (62–92 ppm) and the Di-O-alkyl C (92–112 ppm), while the aromatic C consisted of the aryl-C (112–141 ppm) (containing the resonance of CeH and CeC) and the phenolic C (141–160 ppm). The integration results based on the different resonance regions are
Table 2 Cell wall material and B allocation in cell walls of leaves in trifoliate orange and citrange rootstock seedlings. Rootstock
Trifoliate orange
Citrange
Treatment
BD CK BT BD CK BT
Cell wall material (mg/g FW)
19.51a 16.06 cd 18.07ab 17.00bc 14.78d 15.55 cd
Wall B per unit dry wall (ug/g)
31.69d 41.2c 47.64a 28.03e 39.03c 44.02b
Wall B per unit dry leaf (ug/g)
14.75c 17.00b 21.34a 12.07d 15.28bc 17.20b
Wall/Leaf %
Increase (%)
Decrease (%)
50.70b 18.20c 4.87d 69.74a 22.87c 4.21d
178.57b – – 204.94a – –
– – 73.24b – – 81.59a
Note: CK (control): 10 μM B; BD (boron deficiency): 0 μM B; BT (boron toxicity): 400 μM B. Rows with different letters (a, b……) indicate significant differences among different treatments (n = 6, P < 0.05). 150
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Fig. 2. FTIR spectra of the cell walls from leaves of trifoliate orange and citrange rootstock seedlings (1800-800 cm−1) under different B treatments [CK (control): 10 μM B; BD (boron deficiency): 0 μM B; BT (boron toxicity): 400 μM B].
in trifoliate orange, compared with increases of only 8.62, 6.96 and 2.91% in citrange, which suggested more obvious increases of cellulose, lignin, carbohydrates and phenols in cell walls of trifoliate orange. However, B toxicity had no remarkably effects on organic carbon constitution in leaf of the two rootstock seedlings. These results suggested that B deficiency induced variations in the organic carbon structure of leaf cell walls and were more significant in trifoliate orange than citrange, while B toxicity had no obvious adverse effects on organic carbon.
shown in Table 3. The organic carbon of leaf cell walls under three different B treatments was dominated by carbohydrates C. Additionally, compared with B-toxic treatment, more substantial variations were observed in the cell walls under B-deficient condition. The different C functional groups consist of CH3, OeCH3, CαeOR, OCHO, CeH and CeC, COO represent different compounds in Tables 2. B starvation significantly decreased the signals of CH3 (17.28%), OeCH3 (11.68%), CeH and CeC (8.18%) and COO groups (17.36%) in trifoliate orange versus 14.56, 7.25, 5.29 and 15.16% in citrange, respectively, indicating B deficiency decreased the contents of wax, amino acids, polyphenol, carboxylic acid and organic acid in cell walls, especially of trifoliate orange. Meanwhile, B starvation significantly increased the signals of CαeOR (11.39%), OCHO (8.28%) and CeO (16.50%) groups
Fig. 3. 13C-NMR spectra of cell walls from leaves of trifoliate orange and citrange rootstock seedlings under different B treatments [CK (control): 10 μM B; BD (boron deficiency): 0 μM B; BT (boron toxicity): 400 μM B]. 151
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Table 3 The relative content (%) of different types of organic carbon in leaf cell walls from trifoliate orange and citrange rootstocks with different B treatments. Rootstock
Treatment
0–45 CH3
45–62 O-CH3
62–92 Cα-OR
92–112 OCHO
112–121 C-H
121–141 C-C
141–160 C-O
160–190 COO
Trifoliate orange
BD CK BT BD CK BT
17.42bc 21.06a 19.17ab 16.78c 19.64a 19.24ab
10.21b 11.56a 10.89ab 10.10b 10.89ab 10.60b
49.67a 44.59c 46.46bc 50.15a 46.17bc 47.15b
11.90a 10.99b 10.50b 11.84a 11.07b 10.91b
1.32b 1.43a 1.37ab 1.31b 1.43a 1.40ab
2.15b 2.35a 2.35a 2.27a 2.35a 2.27a
1.20a 1.03b 1.19a 1.06ab 1.03b 0.99b
5.84b 7.09a 7.16a 5.93b 6.99a 6.89a
Citrange
Note: CK (control): 10 μM B; BD (boron deficiency): 0 μM B; BT (boron toxicity): 400 μM B. Columns with different letters (a, b……) indicate significant differences among different treatments (n = 6, P < 0.05).
4. Discussions
contents of methoxyl C, aryl C and carboxyl C, and increased the carbohydrate C, Di-O-alkyl C and phenolic C, which can represent a reduction of amino acid, polyphenol, carboxylic acid, organic acid and a raisin of carbohydrates, cellulose and lignin, respectively (Wang et al., 2013). The cell walls contained a variety of wall-associated proteins, and the structural proteins supports in the mechanical strength of the wall and facilitate the proper asse mbly of other wall components (Jamet et al., 2006). The decreased amino acids revealed by the B-deficient 13C-NMR spectra indicated B deficiency affected the synthesis of wall-associated proteins. Additionally, B could participate in lignin metabolism (Bellaloui, 2012) and it has been reported that lignin biosynthesis was obviously increased in trifoliate orange roots under Bdeprived conditions (Dong et al., 2016). Our 13C-NMR spectral results showed a higher level of phenolic C which presented accumulated lignin in cell walls under B deprivation, and subsequently resulted in the lignification of cell wall. Under B deficiency, the vibrations located at 1,200–800 cm−1 (fingerprint region for polysaccharides) showed that the relative concentration of carbohydrate in trifoliate orange leaves was much higher than that of citrange due to the enhanced characteristic peaks of polysaccharides, which was in agreement with the obvious increase in the content of carbohydrate C in the cell walls of trifoliate orange leaves. The peaks at 1380 and 1030 cm−1 are attributed to cellulose (Zhang et al., 2008). The intensities of the two peaks were higher in trifoliate orange with no B supply, suggesting the synthesis of cellulose in cell walls was promoted or degradation was inhibited, allowing the cells to slow down and stop division, and finally leading to the inhibition of the cell physiological metabolism in trifoliate orange under the B-deficient condition. In addition, the cell wall extraction rate of trifoliate orange leaves under B deficiency was much higher than that of citrange (Table 2), which was another reason for the inhibition of physiological metabolism by the increase of the cell wall material content (mainly carbohydrate). Therefore, alteration on the architecture of cell walls may destroy the integrity of cell walls in leaf. Phenolic compounds which are generated in pentose phosphate pathway (PPP) are important secondary metabolites in plants. B deficiency obviously promoted the accumulation of phenols in the cell walls of trifoliate orange leaves, while had no remarkably promotion in citrange (Fig. 2 and Table 3). Increased phenols caused a rapid rise of polyphenol oxidase activity and a large number of oxyradical and active quinone derivatives (Shkol’nik et al., 1981). The increase of phenols due to B starvation may be a secondary reaction to the death of cells, suggesting more cells died in trifoliate orange than in citrange. These results suggest that the changes induced by B starvation and excess in the celluar structure and the architecture of cell walls determine, to some extent, chlorosis, necrotic and shed prematurely of leaf. A schematic summary, describing the key responses on B allocated in cell walls, cell wall structure and components in B-deficient and Bexcessive of rootstocks, is proposed in Fig. 4.
4.1. Changes on allocation of boron in cell walls It has been reported that B is required for the structural integrity of cell walls (O’Neill et al., 2004). In this study, B deficiency significantly decreased the B concentration in the leaf cell walls of the two citrus rootstock seedlings (Table 1), which was in agreement with the report about other plant species (Goldbach et al., 2000; Li et al., 2007). Additionally, the increased percentage of wall B/leaf B was much higher in citrange, indicating that much more B was allocated to the cell walls in citrange than in trifoliate orange under B deficiency condition, enabling citrange seedlings to maintain its growth under the B stress. Under excessive B conditions, citrange showed a much lower increase in B concentration of their leaf cell walls on a dry weight basis than trifoliate orange (Table 1). These results suggested that citrange leaves can allocate more B to cell walls under a relatively low B supply level to maintain the relatively normal cell wall structure. This may be partially responsible for the less serious symptoms of B deficiency in citrange compared to trifoliate orange. 4.2. Changes in subcellular structure and the architecture of cell wall components In the present study, the cell wall extraction rate increased in trifoliate orange and citrange under B deficiency and excess conditions, which implies that both B deficiency and toxicity increased the content of cell wall material in the two rootstock seedlings (Table 2). This result was supported by the TEM micrographs of the thickened cell wall in cells from the leaves treated under B deficiency and excess treatments (Fig. 1). Similar results were also observed in rape and navel orange under B-limited conditions (Pan et al., 2012; Liu et al., 2015) and irregular cell wall thickening were observed in leaf cortex cells and phloem tissue of B-toxic C. grandis and C. sinensis leaves (Huang et al., 2014). It has been reported that the disintegration of thylakoid system lead to replacement of few non chlorophyllous single thylakoids, with accumulation of large osmiophilic plastoglobules (Zhu and Fang, 1995). At deficient-B and excessive-B levels, increased of plastoglobule numbers and volumes in leaf cells indicated that disintegration of the thylakoid in leaf and the photosynthetic characteristics affected by B stresses. FTIR and 13C-NMR spectra showed that the material composition and organic carbon of cell walls varied between the two citrus rootstocks under B stresses. The 13C-NMR spectra revealed that the organic carbon of leaf cell walls under different B treatments was dominated by O-alkyl C (70–80%), which consists of the methoxyl C, the carbohydrates C (44–50%) and the Di-O-alkyl C (Tables 2 and 3), indicating that carbohydrate is the most important organic compound in the organic carbon of leaf cell walls in the two rootstock seedlings (Wang et al., 2013). Under B-toxicity conditions, there was no obvious increase of Cα-OR in the two rootstocks as represented by carbohydrate in the 13 C-NMR spectra (Table 3), While B deficiency decreased the relative
5. Conclusions Under B deficiency, the cellular B in cell walls of the two citrus 152
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Fig. 4. Schematic diagram in the changes of cell walls in trifoliate orange and citrange due to B deficiency and toxicity. Explanation of plate (Tri: trifoliate orange; Cir: citrange).
Appendix A. Supplementary data
rootstocks were 2.79 and 3.05 folds of their controls, and citrange allocated a larger proportion (69.74%) of B in cell walls than trifoliate orange (50.70%). Additionally, B deficiency and toxicity affected the subcellular structure and the architectural compositions of cell walls in the two citrus rootstocks, and more severe in trifoliate orange. No matter under control or B stresses, the largest amount of organic carbon in leaf cell walls is carbohydrate C (Cα-OR), about 44–50%. B-deficiency-induced decreases of methoxyl C, aryl C, carboxyl C and increases of carbohydrate C, Di-O-alkyl C, phenolic C represented less amino acid, polyphenol, carboxylic acid, organic acid and more carbohydrates, cellulose, lignin, phenols in cell walls of the two citrus rootstocks, and changes were also more remarkably in trifoliate orange, while the adverse effects of B toxicity on cellulose structure and the linkage pattern among pectin in cell walls were lighter in citrange. Therefore, variations of changes on the cellular B allocation, cell ultrastructure, cell wall compositions and structure may determine the different tolerance to B deficiency and toxicity in the two rootstocks.
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Conflicts of interest The authors declare that they have no conflict of interest.
Acknowledgements This research project was financially supported by the National Natural Science Foundation of China (41271320) and the Fundamental Research Funds for the Central Universities (2017PY055). 153
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