Introduction

Boron (B) is essential for the growth and development of higher plants. There are numerous reports of B deficiency affecting the productivity and quality of many agricultural crops in high precipitation areas in the past decades (Goldbach and Wimmer 2007; Han et al. 2008, 2009; Camacho-Cristóbal et al. 2008a; Ahmad et al. 2012; Bogiani et al. 2014). B deficiency causes many anatomical, physiological, and biochemical changes, such as impaired cell elongation, pollen germination and pollen tube growth. A particular role of B in cell wall formation was found to be attributed to B crosslinking with rhamnogalacturonan II molecules, and regulation of the transcript level of genes involved in cell wall synthesis and modification (Kobayashi et al. 1996; Camacho-Cristóbal et al. 2011). Although this has advanced our understanding, most of the research on physiological processes in response to B deficiency has focused on aboveground structures, and more research is required on the effect of B deficiency on root cell structure and nutrient absorption.

The roots are the main organs that penetrate the soil and are thus directly affected by B deficiency. It is, therefore, expected that the roots are the first to undergo B-deficient signals and develop corresponding symptoms (Goldbach et al. 2001). Such symptoms mainly include inhibition of root tip cell division and elongation (Dell et al. 1997), and suppression of the growth of the taproot and lateral root, which results in stubby root branching, root cracking or hollowing, or even necrosis (Martín-Rejano et al. 2011). Other researchers reported that B deficiency affects Citrus root morphology and physiological processes (Lu et al. 2014; Zhou et al. 2014). For example, it has been shown that B deficiency led to a decline in root and leaf nitrate contents (Camacho-Cristóbal and González-Fontes 2007) or the concentrations of other macronutrients such as magnesium, calcium, potassium or phosphate (Camacho-Cristóbal et al. 2005). However, knowledge on the changes in plant apical subcellular structure and root functions (e.g., mineral nutrient uptake) caused by the structural collapse of root cells is limited.

Boron is generally regarded as the most widespread limitation to citrus spp. productivity and yield stability in rain-fed production systems. Trifoliate orange (Poncirus trifoliata), which is the most widely used rootstock in citrus cultivation, the characteristics are of fundamental importance to soil exploration and below-ground resource acquisition, and hence are strongly related to plant adaptation to sub-optimal conditions such as B deficiency stress in citrus. The objectives of this study were (1) to determine the effects of B deficiency on root tip subcellular structure and mineral nutrient absorption, and (2) to clarify the relationships between vessel element structure and mineral nutrient absorption and allocation in response to B deficiency of trifoliate orange.

Materials and methods

Plant culture and treatments

Poncirus trifoliata seeds were surface sterilized for 20 min in 20 % sodium hypochlorite solution and then rinsed repeatedly in deionised water, placed on a porcelain tray with moistened gauze and transferred to an incubator at 30 °C. The seeds were moistened with deionised water during the entire incubation period to hasten seed germination.

Germinated seeds were transferred into plastic pots filled with vermiculite; 20 days (d) later, 200 uniform seedlings were selected, and the individual seedlings were solution-cultured in a plastic barrel covered with black plastic bags. The seedlings were fixed on a black foam board with holes and transferred to a greenhouse. The seedlings were cultured in whole Hoagland’s solution for 10 d, and then the solution was replaced with 0.25 mM H3BO3 (control) or 0 mM H3BO3 (B deficiency). The modified B-deficient full-strength nutrient solution contained 6 mM KNO3, 4 mM Ca(NO3)2, 1 mM NH4H2PO4, 2 mM MgSO4, 9 μM MnCl2, 0.8 μM ZnSO4, 0.3 μM CuSO4, 0.01 μM H2MoO4, and 50 μM Fe-ethylenediaminetetraacetic acid or EDTA. All of the experimental studies were conducted in a greenhouse under controlled conditions of light, humidity, and temperature. The solution was ventilated for 2 h everyday and replaced once a week. The pH of all of the nutrient solutions was adjusted to 6.0 using 0.1 M KOH. The design of the experiment was completely randomized.

Plant growth parameters and mineral nutrient analysis

Six seedlings from both the control and B-deficient treatments were harvested at 0, 5, and 30 d after treatment. The seedlings were separated into roots, stems, and leaves and then weighed separately. Root morphological parameters were determined using an Epson scanner and WinRHIZO analysis system (Pro 2010a).

To determine the number of lateral root primordia (LRP), root tips without lateral roots were fixed using a formalin–acetic acid–ethanol solution (FAA) at 60 °C in 10 % HCl for 10 min, then transparented and dehydrated with a glycerol gradient series of 10, 30, 50, and 80 %. Transparent roots were placed on glass slides and photographed, and the numbers of LRP were counted using an Olympus differential interference contrast microscope.

The fresh tissues were dried in a forced oven at 70 °C for 48 h, and accurately weighed to determine the dry weight. A dried sample (0.50 g) was ashed in a muffle furnace at 500 °C for 6 h, followed by dissolution in 0.1 N HCl. B and other mineral nutrient contents of the plant samples were determined using ICP-AES (Thermo Inc, IRIS Advan, United States, Miwa et al. 2006).

Apical slice preparation and anatomical structure observation

Five days after the B-deficient treatment was imposed, small pieces of root tips (1 mm length, zone of maturation) and mature leaves were cut lengthways and initially fixed for 3 h at room temperature with 5 % glutaraldehyde in 0.1 M phosphate buffer (pH 7.2). Ultrathin sections for electron microscopy were cut using a Reichert–Jung Ultracut E ultramicrotome, stained with uranyl acetate and lead citrate and examined under a JEM 2000 FX II transmission electron microscope (TEM). Entire roots were observed with a Zeiss M63A stereoscope.

After washing in the buffer, some specimens were post-fixed for 2 h with 2 % osmium tetroxide similarly buffered. The samples were dehydrated in an ascending ethanol series followed by critical-point drying with carbon dioxide. Then, the dried samples were fixed on aluminum slides using double-sided adhesive tape. Finally, the samples were coated with gold and examined using a scanning electron microscope (SEM) (XL30 S-FEG, FEI).

Vessel element separation, photographs and parameter measurements

Different root orders above the root tips (zone of cell maturation) were sampled 30 days after treatment, treated with Jeffrey’s Fluid (10 % chromic acid:10 % nitric acid = 1:1) at 37 °C for 72 h, then rinsed with deionized water, and stored in 70 % alcohol. Separated vessel elements were stained with safranin for 15–20 min and rinsed with deionized water, placed on microscope slides and examined under an Olympus BH-2 light microscope. A total of 100 vessel elements were randomly selected from each sample and photographed. Image-Pro Plus software was used to measure the length, diameter, wall thickness, and caliber of 100 vessel elements that were randomly selected.

Statistical analysis

The effects of B deficiency on the mineral nutrient content in different tissues were tested by factorial analysis of variance using the Systat v. 8.0 software package (SPSS Inc., Evanston, IL, USA). Significant differences between treatments were compared using the least significant difference test at the P < 0.05 probability level. The data are presented as mean values ± standard error (SE, n = 6).

Results

Plant growth and root morphology

The dry weights of the leaf, stem, and root were decreased in the B-deficient seedlings compared with the control (Table 1). The root number and total root length per plant were decreased and the lateral root diameter increased significantly compared with the control (P < 0.05) (Table 1; Fig. 1). The root tip swelling and growth point necrosis are shown in Fig. 1. Transparent root tissue showed that number of LRP was not affected although the lateral root number was decreased, lateral root elongation was inhibited, and the density of the LRP was increased in B-deficient seedlings.

Table 1 Plant dry weight (g plant−1), root morphological parameters, number of lateral root primordia (LRP) (number per lateral root) and root density (No./cm) (n = 3, p < 0.05) 30 days after treatment
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Fig. 1
figure 1

Root morphology (a) and anatomy section (b, c) in control (CK) and boron deficiency (−B) treatments of trifoliate orange (Poncirus trifoliata). CK control, −B boron deficiency; the same convention is used in the figures below. Triangle indicates the necrosis in growth point of boron deficiency treatment

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Anatomical characteristics of root and leaf cells

SEM micrographs of the root tips showed the thickened cell wall and more attachments on the cell wall in the B-deficient treatment compared with the control (Fig. 2a, b). TEM micrographs of root cells showed that the cell wall thickened and separated, inside which materials accumulated in the B-deficient treatment. In addition, the cell nucleus was deformed, organelles were degraded or had disappeared, and there was a high density of electrical particles that were dispersed in the vacuole or combined with the cell wall (Fig. 2d). By contrast, the control had a thin cell wall and intact organelles that were structured and full of cytolymph (Fig. 2c). The leaf cells in the B-deficient treatment also had a thickened and separated cell wall, degraded chloroplasts, and more high-density electronic particles (Fig. 2e, f).

Fig. 2
figure 2

Ultrathin section micrographs of root and leaf cells 5 days after treatment. Explanation of plate: a, b SEM micrographs of root cell (zone of maturation); c, d TEM micrographs of root cell (zone of maturation); e, f TEM micrographs of leaf cell (mature mesophyll cells). CK a, c, e; −B b, d, f. CW cell wall, N nucleus, S starch, V vacuole, Chl chloroplast; triangle indicates the isolation between cell wall; star indicates the accumulated materials

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Root vessel element morphology and anatomical characteristics

Vessel elements that originated from the secondary xylem of the root were alternate pitted vessels with simple perforation (Fig. 3a–c). There were three different types of vessel elements: double tailed (Fig. 31, 2, 7, 8, 13, 14), single tailed (Fig. 33, 4, 9, 10, 15, 16) and those without a tail (Fig. 35, 6, 11, 12, 17, 18). In the whole root system, the proportion of double-tailed vessel elements was the largest, followed by those with a single tail and those without a tail. The proportions of vessel elements without a tail and those with a single tail increased while the proportion of those with double tails decreased from the taproot to the second-order lateral roots. Vessel length did not vary significantly between the different order roots, but the average diameter, inner diameter and vessel wall thickness decreased significantly from the taproot to the second-order lateral roots (Table 2).

Fig. 3
figure 3

Vessel elements dispersed from different order roots (zone of cell maturation) 30 days after treatment. a Taproot (CK 1, 3, 5; −B: 2, 4, 6); b first-order root (CK 7, 9, 11; −B 8, 10, 12); c second-order root (CK 13, 15, 17; −B 14, 16, 18)

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Table 2 Morphological traits of the vessel elements in the control and B-deficient treatments (n = 100, p < 0.05) 30 days after treatment
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The vessel length decreased in the first- and second-order roots in the B-deficient treatment. The vessel wall in all order roots thickened markedly and the inner vessel diameter of the first- and second-order roots decreased in the B-deficient treatment (Figs. 3, 4; Table 2, P < 0.05). TEM micrographs showed the material accumulated in the inner vessel walls, which led to the thickening of the vessel wall and a thin inner diameter in the B-deficient treatment compared with the control (Fig. 4). The proportion of double-tailed vessel elements decreased whereas those without a tail or a single tail increased in the B-deficient treatment (Table 2).

Fig. 4
figure 4

TEM photographs of transaction vessel elements (VE) in root tips (zone of maturation) 5 days after treatment. a CK, b −B. VE vessel elements. Triangle indicates the accumulated materials

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Mineral nutrient allocation

The foliar B, N, Ca, and Mg contents were significantly higher than those in the stem and root, while the Fe and Mn contents in the roots were higher than those in the stem and leaf of Poncirus trifoliata (P < 0.05). Under the condition of B deficiency, the B and Ca contents in the leaf, stem, and root were significantly decreased, while the N, Fe, and Mg contents showed no significant difference between treatments. The P content also decreased significantly in the leaf but not in the stem and root in the B-deficient treatment of Poncirus trifoliata. The Mg content exhibited a downward trend in all of the organs, but this was not significant in the leaf and root (Table 3, P > 0.05). Fe and Mn were mainly allocated to the root, while B, N, P, Ca, Mg were mainly allocated to the leaves. Under the condition of B deficiency, higher ratios of B and P were allocated to the roots (Table 3).

Table 3 Effect of B deficiency on mineral nutrient contents in different organs (n = 3, p < 0.05) 30 days after treatment
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Discussion

B deficiency effect on root development and cell anatomy structure

The results of this research indicated that B deficiency inhibited the lateral root elongation of Poncirus trifoliata, which is consistent with the research results on other citrus root stocks (Mei et al. 2011; Zhou et al. 2014). In addition, our results showed that the root tips swelled and coarsened, and the number of LRP did not decrease in the B-deficient treatment (Table 1). The research results of Martín-Rejano et al. (2011) also indicate that a low B supply caused an inhibition of primary root (PR) growth without altering the number of lateral roots (LRs). These results showed that the effect of B deficiency on root elongation was greater than that on the number of lateral roots.

It is well known that B is a necessary material for the construction of the cell wall, which combined with pectic polysaccharides, contributes to the structure and function of the cell wall (Liu et al. 2013). B deficiency symptoms in the leaves and root tip cells include nuclear pycnosis, the disappearance of organelles, and pectin in the areas where the cell wall collapses (Fig. 2b, d, f). It is well known that considerable B is needed to construct the cell wall for the rapid cell division of the growing root tip. If B is not supplied promptly, the root tip cells will be severely limited by B, resulting in a reduction in the formation of pectic polysaccharide compounds at the growing point, which in turn affects cell wall construction (Läuchli 2002). Consequently, necrotic areas first appear at the growing point. It has recently been shown that B deficiency causes a rapid decrease in the expression of several AGP genes, which have been suggested to be putative B-binding structures in Arabidopsis roots (Camacho-Cristóbal et al. 2008b, 2011). Therefore, it is important to point out that B might exert its action in membranes not only by stabilizing membrane molecules (Bolãnos et al. 2004), but also by regulating the expression of genes involved in membrane structure and function.

B deficiency effect on root vessel element structure

Vessel elements are the main type of cells involved in the transport of water and nutrients, and the structure of the vessel directly affects the efficiency of water and nutrient transport. Our results showed that the vessel cell wall had sediment, and the cell wall thickness was greater and the inner vessel diameter was smaller in the B-deficient treatment than in the control. These structural changes will reduce the functioning of the vessels in transporting nutrients and water. Covalently cross-linked pectin networks cannot form without B; therefore, the cell wall structure and function will be affected, such as cell wall thickening. Research conducted by Ishii et al. (2001) indicated that considerable callose and carbohydrate accumulation occurred in the sieve tubes and vessels, and the sieve pores were blocked under B deficiency. The accumulation of substances or an embolism in the vessel elements will hinder the upward transport of mineral nutrients, i.e., mineral nutrients absorbed by roots cannot be transported to the aboveground plant parts even if the culture medium is nutrient rich.

A single perforation in the pitted vessel of the Poncirus trifoliata root is a type of evolved trait. The pits of the vessel elements are the main channel for the horizontal transport of water and mineral nutrients, and the perforation plates on the side wall are also involved in the horizontal transport of water and mineral nutrients (Muhammad and Sattle 1982). Vessel elements without a tail or those with a single tail are characteristics of the advanced evolutionary process of the vessel element system (Bailey 1944), which contribute to the water- and mineral nutrient-conducting ability of vessel elements. In this study, the proportion of double-tailed vessel elements was the largest. The longer and greater the diameter of the vessel elements is, the higher the conducting efficiency (Frost 1930). Under B-deficient conditions, the length and diameter of the vessel elements were reduced in the taproot and second-order lateral root, which reduced water and mineral nutrient absorption and transport, limiting the nutrient supply and reducing plant growth.

B deficiency effect on mineral nutrient absorption and allocation

The B content initially decreased significantly (P < 0.05) in the leaves (10 d), and then decreased significantly in the stems (20 d) and roots (30 d) after Poncirus trifoliata was subjected to B-deficient conditions (data not shown). Though there is increasing evidence to support B retranslocation in several plant species (Brown and Shelp 1997; Lehto et al. 2000; Hajiboland et al. 2013), the fact that the B content decreased in the leaves initially showed that B retranslocation to the aboveground part was still restricted to a certain degree when B was limiting.

Mutual promotion or antagonism in plant nutrient absorption will affect the absorption of other mineral nutrients. Research has shown that B deficiency inhibited the absorption of phosphorus and hindered the transfer of phosphorus in the plant, and conversely, phosphorus deficiency inhibited the absorption of B (Kaya et al. 2009). The current study offers further evidence that phosphorus uptake was significantly inhibited in the B-deficient treatment. Calcium is a main chemical component of the cell wall, and more than 90 % of B in plants is present in the cell wall. There are reports that indicate that Ca and B promote each other (Ramon et al. 2000), whereas others believe there is mutual antagonism between them. Several Ca2+ channel/transporter genes were upregulated in response to short-term B deficiency in Arabidopsis roots (Quiles-Pando et al. 2013).

B as well as other mineral nutrients, such as Ca and P, was decreased in the aboveground components in the B-deficient treatment. In addition, B deficiency decreases the photosynthetic rate (Xiao 2001; Pandey and Archana 2013). Stomata appeared closed, collapsed, and sunken underneath the epidermis of soybean (Will et al. 2011) and stomatal conductivity decreased; therefore, the transpiration rate is suppressed in B-deficient plants. The reason for the mineral nutrient content decrease in the aboveground components is, therefore, not only related to the decline in the absorption and transportation functions of the root system, but may also be associated with a slower transpiration stream.

Author contribution statement

Li Mei carried out most of the experiments and drafted the manuscript. Shu-ang Peng and Ou Sheng participated in the design of the study. Li Mei, Qiaohong Li and Huan Wang performed parts of the experiment. All authors have read and approved the final manuscript.