Plant and Soil

, Volume 419, Issue 1–2, pp 219–236 | Cite as

Involvement of reactive oxygen species and Ca2+ in the differential responses to low-boron in rapeseed genotypes

Regular Article
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Abstract

Background and aims

Boron (B) deficiency significantly inhibits plant growth and development. Oilseed rape (Brassica napus L.) is highly susceptible to B deficiency. Reactive oxygen species (ROS) and Ca2+ play pivotal roles in plant responses to environmental stresses. We aim to identify the differential Ca2+ fluxes and ROS bursts of a B-efficient genotype ‘QY10’ and a B-inefficient genotype ‘W10’ to B deficiency, and establish a signalling pathway involving Ca2+ and ROS implicated in the low-B-induced cell death.

Methods

Under both plant and suspension cell systems, the ROS production was investigated histochemically, cytochemically and biochemically; K+ and Ca2+ effluxes were assayed using the Non-invasive Micro-test Technology (NMT); the expression of ROS-producing genes and the activity assays of antioxidant enzymes were tested, and the ROS scavengers and Ca2+ channel inhibitors were used to characterize the roles of ROS and Ca2+ in response to low-B, respectively.

Results

The cell death was mainly responsible for rapeseed growth inhibition under B deficiency. Low-B induced O2 accumulation, whose distribution was similar to the cell death regions in the plant roots. The increase in O2 production was much stronger in ‘W10’ than in ‘QY10’. The change trend of H2O2 was similar to that of O2 , whereas less significant. The enhancement of lipid peroxidation, ion leakage and K+ efflux indicated that low-B caused cell death through the induction of oxidative damages, particularly in ‘W10’. Pretreatment with O2 scavenger increased the cell viabilities. Low-B induced Ca2+ influx, which worked upstream of ROS. It was not the antioxidant enzymes but the ROS-generating enzymes that determined the differential oxidative damages in rapeseed genotypes.

Conclusions

Low-B induced Ca2+ influx, which then stimulated the ROS burst and eventually caused cell death. The present study enriches our understanding of the involvement of ROS and Ca2+ in the differential responses to B deficiency in rapeseed genotypes.

Keywords

Boron deficiency Brassica napus Ca2+ Genotypes Reactive oxygen species 

Abbreviations

ASC

Ascorbate

B

Boron

CAT

Catalase

DAB

3, 3′-diaminobenzidine

DCF-DA

2′,7′-dichlorofluorescein diacetate

DHE

Dihydroethidium

FDA

Fluorescein diacetate

H2O2

Hydrogen peroxide

MDA

Malondialdehyde

NBT

Nitroblue tetrazolium

NMT

Non-invasive Micro-test Technology

O2

Superoxide radical

PCD

Programmed cell death

PI

Propidium iodide

PM

Plasma membrane

POD

Peroxidase

qRT-PCR

Quantitative real-time PCR

RBOH

Respiratory Burst Oxidase Homolog

ROS

Reactive oxygen species

RGII

Rhamnogalacturonan II

SOD

Superoxide dismutase

TMP

Tetramethyl piperidinooxy

Introduction

Boron (B) is a micronutrient essential for normal growth and development of higher plants (Warington 1923). Direct evidence shows B is required for the effective cross-linking of rhamnogalacturonan II (RG-II) and pectin assembly in the cell wall (CW), whereby B is indispensable for CW structure and plasma membrane (PM) integrity (O'Neill et al. 2004; Hänsch and Mendel 2009). B is difficult to re-translocate from mature to newly developing organs, under the circumstances, plants have to continually absorb B from soils and distribute it into developing tissues (Brown and Shelp 1997). Due to the immobility in plants, B-deficiency symptoms are usually observed in the young tissues (Dell and Huang 1997). B concentrations in soils vary from 2 to 200 mg kg−1, whereas generally less than 5–10% of which is available to plants (Diana 2006). B deficiency has been recognized as the second most severe micronutrient (following Zn) constraint in crop production on a global scale (Ahmad et al. 2012). Under B deficiency, for the growing parts of plants, growth and development defects are primarily attributed to cell elongation rather than cell division (Dell and Huang 1997).

Reactive oxygen species (ROS) are key signaling factors that are involved in the modulation of plant growth and development in response to environmental cues (Foreman et al. 2003; Apel and Hirt 2004; Ray et al. 2012; Choudhury et al. 2016). However, high concentrations of ROS induce oxidative damages and subsequent cell death, therefore repressing plant growth and development. Together with ROS, the changes of Ca2+ in the cytosol are common signals during biological processes (Dodd et al. 2010), and the link of ROS to Ca2+ changes has long been studied in previous researches (Levine et al. 1996; Evans et al. 2005; Gilroy et al. 2014). Demidchik et al. (2009) established that the production of ROS is involved in the regulation of Ca2+ influx channels. However, abiotic stresses are shown to cause Ca2+ fluxes in cells, which further activates ROS production (Choudhury et al. 2016). Thus, the up- and downstream relationships between ROS and Ca2+ appears to be controversial under different environmental stresses. Recently, B-deficiency-caused ROS and Ca2+ signaling has been brought up separately using ROS histochemical staining in the roots of Arabidopsis (Oiwa et al. 2013; Camacho-Cristóbal et al. 2015) and Ca2+ aequorin-expressing tobacco Bright Yellow-2 (BY-2) cells (Koshiba et al. 2010), respectively. The differential accumulations of O2 were also identified in rapeseed genotypes in response to B deficiency (Hua et al. 2016b). Therefore, a possible interaction may exist among B deficiency, Ca2+ flux and ROS burst. However, the delicate signaling pathway remained unknown, including (i) which ROS is (are) principally responsible for the inhibition of plant growth caused by low-B, (ii) the up- and downstream relationships between ROS and Ca2+ and (iii) whether they are directly related to B-efficiency.

Oilseed rape (Brassica napus L.) is the third largest oleaginous crop in the world (after palm and soybean) (FAO 2010), whereas it is highly susceptible to B deficiency (Yang et al. 2013). When exposed to B limitations, B. napus exhibits ‘flowering without seed setting’ during the reproductive development, which seriously hinders seed yield (Wang et al. 2007). In previous studies, a B-efficient (B-deficiency tolerant) rapeseed genotype ‘Qingyou10’ (‘QY10’) keeps excellent growth under low-B conditions, which outperforms a B-inefficient (B-deficiency sensitive) rapeseed genotype ‘Westar10’ (‘W10’) (Hua et al. 2016a, 2016b). Most previous studies into B deficiency have been carried out under plant systems. However, the plant is considerably complicated because diverse types of differentiated cells probably have differential requirements for B in plant tissues. In addition, within a plant tissue, only a small number of the cells are directly in touch with the outer environment, thus most cells may remain insusceptible to the changes in the environmental B concentrations (Koshiba et al. 2010). To address these experimental problems, suspension cultured cells are widely used in the scientific research due to their homogeneity and easy manipulation in the surrounding media (Koshiba et al. 2010; He et al. 2013).

Here, we established a rapeseed suspension cell culture system from ‘QY10’ and ‘W10’, and identified great differences in the low-B-induced cell death between ‘QY10’ and ‘W10’. Subsequently, we investigated the roles of ROS and Ca2+ in low-B-induced cell death under both plant and suspension cell systems. Our results showed that the B-inefficient genotype triggered more serious Ca2+ influx and suffered from severe oxidative damages, thus causing more severe cell death than the B-efficient genotype. The findings revealed a series of ROS- and Ca2+-mediated physiological, biochemical and transcriptional responses of plants and suspension cells to B deficiency, which also enrich our understanding of the involvement of ROS and Ca2+ in the differential responses to B deficiency in rapeseed genotypes.

Materials and Methods

Cell culture and plant growth

Suspension-cultured cell lines of rapeseed (cv.‘QY10’ and ‘W10’) were established according to the following processes: plump seeds were surface-sterilized with 75% ethanol for 30 s, 0.1% mercury chloride for 12 min and rinsed completely with sterilized water. Sterilized seeds were incubated at 24 °C in the dark for 6 d in B5 medium (pH: 5.8) (Gamborg et al. 1968). The hypocotyl segments (0.5–1.0 cm) were sub-cultured twice for the formation of embryo-genic calli in a modified B5 medium (Induction and subculture medium; pH: 5.8). The calli were transferred to 150 mL plastic Erlenmeyer flasks, which contain 40 mL liquid BA medium (pH: 5.8) supplemented with 2, 4-D (3 mg L−1), which were cultured on a rotary shaker (120 rpm) at 25 °C in the dark. During cultivation, the liquid medium was refreshed every three days. All the chemical solutions were prepared using ultrapure water (>18.25 MΩ·cm). All the reagents were purchased from Sigma-Aldrich, St. Louis, USA. The solution compositions of different culture media are listed in the Supplemental Table S1.

For the hydroponic culture experiment, plump seeds of the B-efficient genotype ‘QY10’ and the B-inefficient genotype ‘W10’ (Hua et al. 2016a, 2016b), were surface-sterilized using 0.5% (w/v) NaClO for 10 min, and then they were rinsed completely with sterilized ultrapure water (>18.25 MΩ·cm). The seeds were sown in gauze after being soaked in ultrapure water overnight. After germination for 5 d, the uniform seedlings were transplanted into black plastic containers with Hoagland and Arnon (1950) solution. The nutrient solution was replaced every three days. The rapeseed seedlings were first grown in one quarter-strength solution, afterwards progressing to one-half-strength and eventually full-strength. The plants were grown in an illuminated culture room with a temperature regime of 24/22 °C (day/night) and a photoperiod of 14/10 h (day/night) and a light intensity of 300–320 μmol m−2 s−1.

B treatments

After germination for 5 d, the uniform seedlings were transplanted to different B treatment. For B gradient treatments, the rapeseed plants were subjected to 0, 0.10, 0.25, 1.0, 10, 25 or 50 μM B for 12 d. For cell death tests, the plants were grown with 0.25 or 25 μM B, and the suspension cells were grown with 0.25 or 50 μM B for 3 d. For the assays of B concentrations, the suspension cells were cultured in 0.25 or 50 μM B for 10 d, respectively. For the tests of PM integrity, the plants were grown in 25 or 0.25 μM B for 3 d, respectively. For the determination of ion leakage, the plants were grown in 25 or 0.25 μM B for 12 d, respectively. For MDA determination, quantification of B concentrations, short-term gene expression analyses, the measurements of O2 and H2O2 concentrations, histochemical and cytochemical staining of O2 and H2O2, the assays of antioxidant enzyme activities, first, the plants were cultured in 25 μM B and the suspension cells were cultured in 30 μM B for 10 d, and then the roots and cells were washed with B-free solution to remove residual B, and then transferred to B-free condition for 0 h, 3 h, 12 h and 72 h. For long-term gene expression analysis, the plants were hydroponically cultured in 25 μM B or 0.25 μM B for 12 d, respectively. For the measurement of K+ and Ca2+ fluxes, the plants were cultured in 25 μM B for 7 d, and then the roots were washed with B-free solution to remove residual B, and then transferred to 25 μM B or B-free condition for 1 d; the suspension cells were cultured in 30 μM B for 7 d, and then the cells were washed with B-free solution to remove residual B, and then transferred to 30 μM B or B-free conditions for 1 d.

Cell viability assays

The viabilities of suspension cells were determined by FDA-PI staining (He et al. 2013). The living cells were stained with FDA, which can generate a green fluorescence on the plasma membranes. In terms of the dead cells, they are stained with PI, which can be excited a red fluorescence on the nucleus. The cell death in the root tips was determined by PI staining. The cells stained with FDA-PI were observed with a confocal laser scanning microscope (CLSM, OLYMPUS FV 1200; Tokyo, Japan), and the roots stained with PI were captured with a light microscope (Nikon Eclipse 80i; Tokyo, Japan). Each experiment was conducted with at least eight biological replicates.

Determination of the root system architecture

The rapeseed roots were imaged using a scanner (Epson Perfection V800 photo), and the root length was measured by hand using a ruler. All the measurements were conducted with six replicates.

Measurement of ion leakage

Membrane leakage was determined by the electrolytes (or ions) leakage from the plant roots according to the method described by Sakuraba et al. (2014). The roots were immersed in 5 mL of 0.4 M mannitol at 25 °C with gentle shaking for 4 h, and the solution conductivity was assayed with a conductivity meter (INESA DDB-303A, Shanghai, China). Total conductivity was determined after sample incubation at 85 °C for 20 min. The ion leakage is reflected by the percentage of the initial conductivity that is divided by total conductivity. All the experiments were performed with four biological replicates.

Quantification of B concentration

All the samples were killed out at 105 °C for 30 min, and then oven-dried to constant weight at 65 °C. The dry samples were then ground to fine powder in a carnelian mortar. B was extracted from the dry powder by shaking it in 8 mL of 1 M HCl for 2 h. The extract was filtered and then diluted 10 times. B concentration was determined by inductively coupled plasma mass spectrometry (ICP-MS, NexION™ 350X; PerkinElmer, Massachusetts, USA). Each experiment was conducted with four biological replicates.

Histochemical staining

The loss of PM integrity in the plant roots was histochemically stained with the 0.25% aqueous Evan’s blue solution according to Schützendübel et al. (2001). The generation of O2 was in situ detected by nitroblue tetrazolium (NBT) staining (Sung and Hong 2010). H2O2 production was detected by 3, 3′-diaminobenzidine (DAB) staining (Thordal-Christensen et al. 1997). The plant roots were observed under a light microscope (Nikon Eclipse 80i; Tokyo, Japan). All the experiments were performed with six replicates.

Determination of endogenous MDA, O2 and H2O2 concentrations

The young leaves and roots (~ 150 mg) were individually harvested and immediately frozen at the indicated time points. Lipid peroxidation was estimated by the determination of malondyaldehyde (MDA) content with the method of the thiobarbituric acid reaction established by Buege and Aust (1978). The quantification of O2 and H2O2 concentrations were performed using the O2 and H2O2 assay kit (Comin, Suzhou, China) according to the manufacturer’s instructions. All the experiments were performed with four replicates.

Detection of O2 and H2O2 in suspension cells

O2 and H2O2 was detected by the staining of 10 μM dihydroethidium (DHE) and 25 μM 2′,7′-dichlorofluorescein diacetate (DCF-DA), respectively (Rodríguez-Serrano et al. 2009). The suspension cells were observed with a CLSM (OLYMPUS FV 1200; Tokyo, Japan). The signal intensity was quantified using the Image J software (ImageJ v.1.47; http://rsbweb.nih.gov/ij/download.html). All the experiments were performed with eight biological replicates.

Total RNA extraction and gene expression analyses

Total RNA of the suspension cells was extracted according to the methods of tobacco BY-2 cells (Sun et al. 2015). Total RNA of the roots was extracted with Trizol Reagent (Invitrogen, CA, USA). The gene sequences were retrieved from the Brassica Database (BRAD) (http://brassicadb.org/brad) and the primers were designed using Primer Premier 5.0. Quantitative real-time PCR (qRT-PCR) assays for the detection of the relative expression of genes were performed using the SYBR Premix Ex Taq™ II (Toyobo, Osaka, Japan) and the CFX96™ Real-Time PCR Detection System (Bio-Rad, Hercules, CA, USA). The sequences of the primers used for mRNA quantifications are shown in the Supplemental Table S2, and the actin gene was used as an internal control. The amplification efficiencies of the gene-specific primers were calculated according to the “Serial dilutions” methods (Rebrikov and Trofimov 2006). The PCR regimes were as follows: 95 °C for 1 min, 40 cycles of 95 °C for 5 s, 60 °C for 30 s, and 72 °C for 30 s; 95 °C for 15 s, melting curve 60 °C - 95 °C, +0.5 °C/cycle, 15 s. All the experiments were performed in triplicate.

Measurement of K+ and Ca2+ fluxes

The net fluxes of K+ and Ca2+ were in situ determined using the Non-invasive Micro-test Technology (NMT) (NMT100 Series; Younger USA Sci. & Tech. Corp., Amherst, MA, USA; Applicable Electronics Inc., Sandwich, MA, USA; and Science Wares Inc., Falmouth, MA, USA) at Xu-Yue Sci. & Tech. Co. Ltd. (Beijing, China; http://www.xuyue.net). The Ca2+-selective microelectrodes were prepared as described by Xu et al. (2006). Before the flux measurement, the microelectrodes were calibrated with the culture media that contain different concentrations of K+, 0.05 mM and 0.5 mM and Ca2+, 0.05 mM and 0.5 mM, respectively. In this study, for the detection of K+ fluxes, we only used the electrodes with a Nernstian slope > 50 mV/decade; for the detection of Ca2+ fluxes, we only used the electrodes with a Nernstian slope > 22 mV/decade. The suspension cells (200 μL) were added to glass coverslips that were pretreated with a poly-L-lysine (Sigma-Aldrich) solution, which then were transferred to the measuring chamber containing 3 mL solution (0.1 mM KCl, 0.05 mM CaCl2, 0.05 mM MgCl2, 0.5 mM NaCl, 0.1 mM Na2SO4, 0.3 mM 2-(N-morpholino) ethanesulfonic acid (MES), and 0.1% sucrose; pH 5.7). The plant roots were fixed gently on a chamber containing 3 mL solution (0.1 mM KCl, 0.1 mM CaCl2, 0.1 mM MgCl2, 0.5 mM NaCl, 0.2 mM Na2SO4, 0.3 mM MES; pH 5.7), and the measurement chamber was mounted on the micromanipulator, and the electrodes were positioned close to the surfaces of plant roots (1 cm from the root tip) and suspension cells, and left to equilibrate for 15 min. Gradients of K+ and Ca2+ adjacent to the cells were measured by moving the selective microelectrodes of K+ and Ca2+ between two positions in a pre-set excursion of 30 μm for the roots and 10 μm for the suspension cells, respectively. The fluxes of K+ and Ca2+ were recorded for a period of 5 min. The flux data were obtained with the ASET software under the scanning ion-selective electrode (SIET) system. They were analyzed with an Excel (Microsoft Office 2010) spread sheet to convert data from the background potential (mV) estimation of concentration and the microvolt difference estimation of the local gradient into specific ion flux (pmol cm−2 s−1) using MAGEFLUX, developed by Xu-Yue Sci. & Tech. Co. Ltd. (http://xuyue.net/mageflux). All the experiments were performed with at least eight biological replicates.

Assays of antioxidant enzyme activities

The plant roots (~ 150 mg) were individually harvested and immediately frozen at the indicated time points. Total SOD activities were quantified through the determination of its competence to inhibit the photochemical reduction of NBT (Beauchamp and Fridovich 1971). CAT activity was assayed based on the reduction of H2O2 by monitoring the decrease in absorbance at 240 nm (de Azevedo Neto et al. 2006). POD activities were determined through the assays of the oxidation of guaiacol at 470 nm (Han et al. 2008). All the experiments were performed with four biological replicates.

Pre-treatments of plants and suspension cells with ROS scavengers and LaCl3

The plant roots and suspension cells were pre-incubated with 1 mM TMP (a O2 scavenger), 1 mM ASC (a H2O2 scavenger) (Pérez-Chaca et al. 2014) for 1 h and 1 mM lanthanum chloride (LaCl3, a Ca2+ channel blocker) (Monshausen et al. 2009) for 5 min, respectively; and then they were transferred to sufficient B or B-free conditions for 3 d. The assays of cell death were performed as the above-mentioned ‘cell viability assay’ section.

Statistical analysis

The software Statistical Product and Service Solutions 17.0 (SPSS, Chicago, IL, USA) were used for the statistical analyses. Among the B treatments and rapeseed genotypes, the differences were determined using Duncan’s tests at P < 0.05.

Results

Low-B caused cell death in plant roots and suspension cells

To examine the effect of B on rapeseed growth, the rapeseed seedlings were hydroponically grown under 0, 0.10, 0.25, 1.0, 10, 25 or 50 μM B conditions for 12 d, respectively, and the primary root length was measured. With the decrease in B concentrations, the B-deficiency symptoms became more and more obvious, especially in the B-inefficient genotype ‘W10’ (Fig. 1a, b). There was no significant phenotypic difference between ‘QY10’ and ‘W10’ when B concentration in the solution was more than 25 μM, and the greatest differences in the plant performance were shown under the 0.25 μM B condition (Fig. 1a, b). Therefore, for the following hydroponic experiments, 25 and 0.25 μM B were selected as the sufficient and low-B conditions of the plant growth, respectively. Under the low-B condition, the B-efficient genotype ‘QY10’ possessed a larger root system compared with the B-efficient genotype ‘W10’ (Fig. 1c). Through the propidium iodide (PI) staining, large numbers of dead cells were identified in the meristematic zone, transition zone and elongation zone under low-B, especially in ‘W10’, which may lead to the inhibition of root growth (Fig. 1d).
Fig. 1

Growth performance and cell viabilities of plant roots and suspension cells of the Brassica napus genotypes ‘QY10’ (B-efficient) and ‘W10’ (B-inefficient). (a) Growth performance of the seedlings of ‘QY10’ and ‘W10’ under different B conditions. Scale bar = 5 cm. (b) Primary root length of ‘QY10’ and ‘W10’. For (a) and (b), the plants were grown hydroponically under 0, 0.10, 0.25, 1.0, 10, 25 –or 50 μM B for 12 d. (c) Root system architecture of ‘QY10’ and ‘W10’ under sufficient B (25 μM, +B) and low-B (0.25 μM, −B) conditions for 12 d. (d) Representative images of the primary roots stained with propidium iodide (PI). The plants of ‘QY10’ and ‘W10’were grown hydroponically under sufficient - B or low-B for 3 d. Scale bar = 200 μm. (e) Representative images of suspension cells stained with propidium iodide/ fluorescein diacetate (PI/FDA). Green staining with fluorescein diacetate (FDA) denotes live cells, and red staining with propidium iodide (PI) indicates dead cells. The suspension cells were cultivated in 50 or 0.25 μM B for 10 d. The images from the top to the bottom represent QY10–50 μM B, QY10–0.25 μM B, W10–50 μM B and W10–0.25 μM B, respectively. Scale bar = 20 μm. Values denote means (n = 6), and error bars denote standard error (SE). * represents the significant difference at P < 0.05 (Duncan’s test)

Under the suspension cell system, for both ‘QY10’ and ‘W10’, when treated with 50 μM B for 10 d, most of the cells were viable with green fluorescence, which also presented spherical morphology (Fig. 1e). However, under 0.25 μM B, the suspension cells became morphologically abnormal, together with higher death rates that are reflected by red fluorescence in the nucleus, particularly in ‘W10’ (Fig. 1e).

Low-B induced membrane damages in plant primary roots

It was assumed that the low-B signal was transduced to the cell nucleus through the PM (Goldbach and Wimmer 2007). Due to the great sensitivity of PM to lipid peroxidation (Escriba et al. 2015), we measured the oxidative damages in the PM. The plant roots were stained with Evan’s blue reagents, and the higher loss of PM integrity was indicated by an intense dark blue color in the roots of the low-B-treated seedlings (Fig. 2a). Obviously, ‘W10’ suffered from more pronounced oxidative PM damages under low-B. Subsequently, increased ion leakage in the plant roots was detected under B limitations (Fig. 2b). Then, we measured the concentrations of malondialdehyde (MDA), which was used as an indicator of lipid peroxidation (Buege and Aust 1978). The MDA concentration in the roots of ‘W10’ increased significantly at 3 h, while no significant increase was detected in ‘QY10’. Subsequently, a 35.9% and 80.1% increase in the MDA concentrations were detected in the roots of ‘QY10’ and ‘W10’ at 72 h, respectively (Fig. 2c). The ICP-MS assay showed that B concentrations of plant leaves deceased with the time of B deprivation, while the concentrations in the roots showed no significant decrease under 3-d B deprivation (Supplemental Fig. S1a, b). Meanwhile, the quantitative real-time PCR (qRT-PCR) results revealed that B-deprivation significantly increased the expression levels of B channel genes in the plant roots, particularly in ‘QY10’ (Supplemental Fig. S1c, d). These results confirmed that the PM was severely damaged by the oxidative stress under low-B conditions, particularly in the roots of ‘W10’.
Fig. 2

Histochemical detection of the loss of plasma membrane integrity, ion leakage and MDA concentrations in the Brassica napus genotypes ‘QY10’ (B-efficient) and ‘W10’ (B-inefficient). (a) Evan’s blue staining of the plant roots. The plants were grown hydroponically under sufficient B (25 μM, +B) or low-B (0.25 μM, −B) for 3 d. Scale bar = 500 μm. (b) The ion leakage in the plant roots. The plants were grown hydroponically under sufficient - B or low-B for 12 d. (c) The MDA concentrations in the plant roots. The plants were grown hydroponically under sufficient B for 10 d, and then transferred to B-free condition for 0, 3, 12 and 72 h, respectively. Bars denote means (n = 4), and error bars denote standard error (SE). Different letters show significant differences at P < 0.05 (Duncan’s test)

Low-B triggered ROS burst and ROS-related signaling components

The stress-induced electrolyte leakage is usually accompanied by the accumulation of ROS, and it often results in programmed cell death (PCD) (Demidchik et al. 2014). Cell death progression is characteristic of increased oxidative damages (Nitschke et al. 2016). To ascertain whether the cell death caused by low-B was related to the oxidative damages or not, we then targeted some major biochemical markers of the oxidative responses in the plant roots and suspension cells. The biochemical markers include the histochemical, cytochemical and spatio-temporal detection of superoxide radical (O2 ) and hydrogen peroxide (H2O2), and the fluxes of K+ and Ca2+.

O2 B deficiency caused a darker blue color in the meristematic and elongation zone of the roots, particularly in ‘W10’, which reflected an O2 burst (Fig. 3a). B deficiency also caused an increase in O2 staining in the young leaves, particularly in ‘W10’ (Supplemental Fig. S2a). Through the assays of O2 concentrations, we found that the O2 concentrations were induced by low-B both in the roots and leaves, particularly in ‘W10’ (Fig. 3b; Supplemental Fig. S2a). In the presence of ROS, DHE can be oxidized to ethidium that can intercalate with DNA to emit fluorescence (Koshiba et al. 2010). B deficiency also led to a higher ratio of DHE staining in the suspension cells of ‘W10’, which was confirmed by the quantification of the higher DHE fluorescence intensity (Fig. 3d), whereas no significant differences were observed by CLSM (Fig. 3c).
Fig. 3

Histochemical localization, cytochemical staining and measurement of O2 in the Brassica napus genotypes ‘QY10’ (B-efficient) and ‘W10’ (B-inefficient). (a, b) NBT staining (a) and concentrations (n = 4) (b) of O2 in the plant roots. The plants were grown hydroponically under sufficient B (25 μM, +B) for 10 d, and then transferred to +B or B-free condition for 72 h, respectively. Scale bar = 1 mm. (c) O2 by DHE staining in the suspension cells. Scale bar = 20 μm. The suspension cells of ‘QY10’ and ‘W10’ were cultivated under 30 μM B for 10 d, and then transferred to 30 μM B or B-free condition for 72 h, respectively. (d) The relative intensity of DHE fluorescence (n = 8) in the suspension cells. Bars denote means, and error bars denote standard error (SE). Different letters show significant differences at P < 0.05, and * represents the significant difference at P < 0.05 (Duncan’s test)

H2O2 B deficiency caused a darker brown color in the meristematic and elongation zone of the roots, though not so significant as O2 , and the allocation pattern was similar to that of O2 (Fig. 4a). H2O2 was evenly distributed in the young leaves, which were not likely responsive to low-B (Supplemental Fig. S2c). The H2O2 concentrations were increased in the roots of ‘W10’ whereas not in ‘QY10’ (Fig. 4b). The H2O2 concentrations in the young leaves remained no change with the time of B deprivation (Supplemental Fig. S2d). The DCF-DA-dependent fluorescence was also evenly distributed in the cytosol, and exposure to the B-free condition for 12 h resulted in a rise of fluorescence intensity in the suspension cells (Fig. 4c, d). According to the above findings, we suggested that ROS, particularly O2 , were likely to play a key role in the regulation of cell viabilities in the plant roots and suspension cells under low-B conditions.
Fig. 4

Histochemical localization, cytochemical staining and measurement of H2O2 in the Brassica napus genotypes ‘QY10’ (B-efficient) and ‘W10’ (B-inefficient). (a, b) DAB staining (a) and concentrations (n = 4) (b) of H2O2 in the plant roots. The plants were grown hydroponically under sufficient B (25 μM, +B) for 10 d, and then transferred to +B or B-free condition for 72 h, respectively. Scale bar = 1 mm. (c) H2O2 by DCF-DA staining in the suspension cells. Scale bar = 20 μm. The suspension cells of ‘QY10’ and ‘W10’were cultivated under 30 μM B for 10 d, and then transferred to 30 μM B or B-free condition for 72 h, respectively. (d) The relative intensity of DHE fluorescence (n = 8) in the suspension cells. Bars denote means, and error bars denote standard error (SE). Different letters show significant differences at P < 0.05 (Duncan’s test)

Further, we identified that the expression of the Respiratory Burst Oxidase Homolog (RBOH) family genes BnaRBOHDs and BnaRBOHFs, encoding the ROS-producing enzymes (Kwak et al. 2003), was induced by low-B, and they were significantly higher in the suspension cells of ‘W10’ than ‘QY10’ (Fig. 5a, b), while the differences narrowed down after a long-term (12 d) B deficiency in the plant roots (Fig. 5c).
Fig. 5

The quantitative real-time PCR results of BnaRBOHDs and BnaRBOHFs in the Brassica napus genotypes ‘QY10’ (B-efficient) and ‘W10’ (B-inefficient). (a, b) The expression of BnaRBOHDs (a) and BnaRBOHFs (b) in the suspension cells. The suspension cells of ‘QY10’ and ‘W10’ were cultivated under 30 μM B for 10 d, and then transferred to B-free condition for 0 h, 3 h, 12 h and 72 h, respectively. (c) The expression of BnaRBOHDs and BnaRBOHFs in the plant roots. The plants were grown hydroponically under sufficient B (25 μM, +B) and low B (0.25 μM, −B) for 10 d. Bars denote means (n = 3), and error bars denote standard error (SE). Different letters show significant differences at P < 0.05 (Duncan’s test)

K+ K + is the main cation in cells, and the efflux of K+ happens in the process of cell apoptosis (Bortner et al. 1997). The kinetics of ROS production are similar to the K+ efflux in response to a certain stress (Demidchik et al. 2014). This points to the existence of a link between ROS production and K+ efflux. To examine K+ flux in response to B-deprivations, we used the non-invasive micro-test technology (NMT) on the plant roots and suspension cells, we found that the B-efficient genotype ‘QY10’ did not induce K+ release as much as the B-inefficient genotype ‘W10’ in both the plant roots and suspension cells (Fig. 6). Ben-Hayyim et al. (1987) found that the cytosolic K+ level is directly linked to the growth of cultured cells. Consistent with this finding, we identified that more K+ efflux was induced in the plant roots and suspension cells of ‘W10’, which also presented a greater severity in root growth inhibition and cell death (Fig. 1).
Fig. 6

The K+ flux in the plant roots and suspension cells of the Brassica napus genotypes ‘QY10’ (B-efficient) and ‘W10’ (B-inefficient). (a, b) The representative measurement graphs of K+ flux in the plant roots (scale bar = 100 μm) (a) and suspension cells (scale bar = 50 μm) (b) using the non-invasive micro-test technology. (c, d) The kinetics of K+ fluxes in the plant roots (c) and suspension cells (d), respectively. (e, f) The mean rates of K+ fluxes in the plant roots (e) and suspension cells (f). The plants were cultured in 25 μM B and the suspension cells were cultured in 30 μM B for 7 d, and then transferred to +B (25 μM B for plants, 30 μM B for suspension cells) or B-free condition for 1 d, respectively. Bars denote means (n = 8–12), and error bars denote standard error (SE). Different letters show significant differences at P < 0.05 (Duncan’s test)

Low-B induced Ca2+ influx that worked upstream of ROS

Ca2+, an essential mineral nutrient and a key second messenger (Dodd et al. 2010), plays a crucial role in the maintenance of cell wall structure and PM integrity (Bose et al. 2011). We characterized the involvement of Ca2+ signaling in low-B-induced cell death using the NMT. Under B-free conditions, remarkable net Ca2+ influxes were induced in both the plant roots and suspension cells of the B-inefficient genotype ‘W10’ (Fig. 7). By contrast, less remarkable changes in the NMT signals were detected in the B-efficient genotype ‘QY10’, though not significant statistically (Fig. 7). The finding indicated a stronger Ca2+ signal was excited in ‘W10’ under low-B compared with ‘QY10’. The production of ROS was partly inhibited by the pre-treatment with LaCl3 (Fig. 8), which was used as a Ca2+ channel blocker, and it suggested that Ca2+ likely worked upstream of ROS.
Fig. 7

The Ca2+ flux of the plant roots and suspension cells of the Brassica napus genotypes ‘QY10’ (B-efficient) and ‘W10’ (B-inefficient). (a, b) The representative measurement graphs of Ca2+ flux in the plant roots (scale bar = 100 μm) (a) and suspension cells (scale bar = 50 μm) (b) using the non-invasive micro-test technology (NMT). (c, d) The kinetics of Ca2+ fluxes in the plant roots (c) and suspension cells (d). (e, f) The mean rate of Ca2+ fluxes in the plant roots (e) and suspension cells (f). The plants were cultured in 25 μM B, and the suspension cells were cultured in 30 μM B for 7 d, and then transferred to +B (25 μM B for plants, 30 μM B for suspension cells) or B-free condition for 1 d, respectively. Bars denote means (n = 8–12), and error bars denote standard error (SE). Different letters show significant differences at P < 0.05 (Duncan’s test)

Fig. 8

Histochemical localizations of O2 and H2O2 in the plant roots pretreated with LaCl3 of the Brassica napus genotypes ‘QY10’ (B-efficient) and ‘W10’ (B-inefficient). (a) O2 by NBT staining. (b) H2O2 by DAB staining. The plants were pre-incubated with 0 or 1 mM LaCl3 (a Ca2+ channel blocker) for 5 min, and then transferred to sufficient B (25 μM, +B) and low B (0.25 μM, −B) for 72 h, respectively. Scale bar = 500 μm

Endo-antioxidases seemed not to be involved in the differential differences in ROS accumulations in rapeseed genotypes

The ROS homeostasis is controlled by a delicate balance between their production and scavenging. The above findings suggested that the B-inefficient genotype ‘W10’ accumulated more ROS than the B-efficient genotype ‘QY10’ under B deficiency (Figs 3, 5). Then, we were wondering whether ‘QY10’ was more capable of detoxifying ROS compared with ‘W10’ under low-B conditions. Surprisingly, in response to B deprivation, the activities of superoxide dismutase (SOD) were increased in the plant roots of ‘W10’, which reached its maximum on the 3rd day of B deprivation, 4.2-fold above its basal level at 0 h (Fig. 9a). However, the SOD activities showed no significant increase with the time of B-deprivation going on (Fig. 9a). The activities of catalase (CAT) seemed to be induced by low-B in the roots, which were also significantly higher in ‘W10’ than in ‘QY10’ (Fig. 9b). The activities of peroxidase (POD) were increased in the roots, whereas no significant difference between the B-efficient and -inefficient genotypes was detected (Fig. 9c). The increase in the activities of the anti-oxidant enzymes was slower than the increase in ROS production (Figs 3c, 4c and 9), which indicated the increase in the activities of antioxidant enzymes lagged behind the ROS production. Generally, the increase in the activities of antioxidant enzymes was even higher in ‘W10’ than ‘QY10’ (Fig. 9a, b). The gene expression of the antioxidant enzymes also showed no significant superiority in ‘QY10’ over ‘W10’ in the plant roots (Supplemental Fig. S3). Thus, it was likely not the different abilities of detoxifying ROS but the differential ROS productions that led to the differential ROS accumulations in the B-efficient and -inefficient rapeseed genotypes.
Fig. 9

The enzyme activities of SOD, CAT and POD in the Brassica napus genotypes ‘QY10’ (B-efficient) and ‘W10’ (B-inefficient). (a-c) The enzyme activities of SOD (a), CAT (b) and POD (c) in the plant roots. The plants of ‘QY10’ and ‘W10’were grown hydroponically under sufficient B (25 μM, +B) for 10 d, and then transferred to B-free conditions for 0, 3, 12 and 72 h, respectively. Bars denote means (n = 4), and error bars denote standard error (SE). Different letters show significant differences at P < 0.05 (Duncan’s test)

Scavenging ROS alleviated cell death in plant root tips and suspension cells

The ROS levels could be decreased by the use of ROS scavengers, such as tetramethyl piperidinooxy (TMP), an O2 scavenger, and ascorbate (ASC), a H2O2 scavenger (Pérez-Chaca et al. 2014). The cell death was significantly alleviated in the elongation zone of the plant roots pretreated with 1 mM TMP, whereas no significant change was observed in the roots pretreated with 1 mM ASC (Fig. 10a). The similar results were observed under the suspension cell system (Fig. 10b). The result indicated that the ROS accumulation, which was induced by low-B, caused cell death, at least in part, and O2 was the predominant ROS that were involved in the cell death induced by low-B.
Fig. 10

Cell death of the plant roots and suspension cells pretreated with ROS scavengers of the Brassica napus genotypes ‘QY10’ (B-efficient) and ‘W10’ (B-inefficient). (a) Representative images of the primary roots stained with propidium iodide (PI). The arrows point to the alleviated cell death in the plant roots pretreated with TMP. Scale bar = 200 μm. (b) Representative images of the suspension cells stained with propidium iodide/ fluorescein diacetate (PI/FDA). Scale bar = 20 μm. The plants and suspension cells of ‘QY10’ and ‘W10’ were pre-incubated with 0 or 1 mM TMP (a O2 scavenger), or 0 or 1 mM ASC (a H2O2 scavenger) for 1 h, and then the plants were transplanted to + B (25 μM for the plants, 50 μM B for the suspension cells) and -B (0.25 μM B) conditions for 72 h, respectively

Discussion

ROS are important signal molecules that are involved in the modulation of plant growth and development; however, high levels of ROS-inducing oxidative damages result in cell death (Foreman et al. 2003; Apel and Hirt 2004; Ray et al. 2012; Choudhury et al. 2016). ROS bursts include the first and secondary bursts. The first burst is instantaneously produced, whereas the intense secondary burst sustainably occurs for hours, which is important for the modulation of cell death (Mur et al. 2008). Previous studies have also suggested that B deficiency immediately leads to ROS accumulation in the plant root elongation zone of Arabidopsis (Oiwa et al. 2013; Camacho-Cristóbal et al. 2015).

Here, we made a detailed research into dynamic spatial and temporal changes of the O2 and H2O2 (two main ROS) in the rapeseed genotypes with differential sensitivities to B deficiency. We found that (i): O2 was mainly distributed in the root cap and meristematic zone. After B-deprivation, the O2 accumulations in the young leaves and roots were increased. The O2 staining even spread to the root elongation zone under B deficiency. The O2 concentrations increased almost two-fold at 72 h and 12 h in the leaves of the B-efficient genotype ‘QY10’ and the B-efficient genotype ‘W10’, respectively; whereas it took 12 h and 3 h in the roots of ‘QY10’ and ‘W10’, respectively. The DHE florescence intensities in the suspension cells of ‘W10’ were significantly higher than those of ‘QY10’ under B limitations (Fig. 3; Supplemental Fig. S2a, b). These results suggested that ‘W10’ produced O2 more drastically than ‘QY10’ in response to low-B; (ii) H2O2 was evenly distributed in the young leaves and the cytoplast of suspension cells, and its distribution pattern in the plant roots was similar to that of O2 . After B-deprivation, a clear darker brown color could be observed in the root elongation zone of ‘W10’. The H2O2 concentrations were also increased in the roots of ‘W10’ with the time of B deficiency going on, while the change was not as significant as O2 (Fig. 4; Supplemental Fig. S2c, d). Consistently, for both the plant roots and suspension cells pretreated with the O2 scavenger TMP, the cell death caused by low-B was significantly alleviated, whereas pretreatment with H2O2 scavenger ASC showed no such significant change (Fig. 10). The results indicated that O2 was the main ROS involved in the low-B-induced cell death, and that ‘W10’ suffered more oxidative damages under limited B conditions. The obvious curved and thickened leaves (young leaves) caused by low-B also showed significant increase in histochemical staining and concentrations of O2 , particularly in ‘W10’, while no change was detected in H2O2 in the young leaves (Supplemental Fig. S2).

In this study, between the B-efficient and -inefficient rapeseed genotypes, the differential low-B-induced losses of PM integrities were highly consistent with the production patterns of ROS (Figs 2, 3, 4). Electrolyte leakage can be widely used for the analysis of the stress-induced injuries in plant tissues and organs, which also can be applied to assess the plant tolerance to environemntal stresses (Levitt 1972; Bajji et al. 2002; Lee and Zhu 2010). Electrolyte leakage is principally attributed to the efflux of K+, which is considered as a physiological marker for cell death (Shabala 2009). Under abiotic and biotic stresses, the stress-tolerant plant species are competent to maintain higher levels of K+ (Shabala and Cuin 2008). Under B deficiency, compared with ‘QY10’, the PM integrity was greatly damaged, and the ion leakages, as well as K+ effluxes were more severe in ‘W10’ (Figs 3, 6). The finding indicated that the differential responses are indeed directly correlated with B-efficiency (or sensitivities to B deficiency), and they were ahead of the obvious emergence of B-deficiency symptoms.

Respiratory burst oxidase homologs (RBOHs), NADPH oxidases that are localized on the PM (Kobayashi et al. 2006), play important roles in the ROS production under various biotic and abiotic stresses (Suzuki et al. 2011). For instance, functioning as key signaling molecules, ROS are involved in the local and systemic resistances against plant pathogens in a RBOH-dependent manner (Miller et al. 2009; Mersmann et al. 2010). The integration of various signaling factors through the RBOH enzymes, such as Ca2+ and protein phosphorylation with ROS production, suggest that RBOHs are crucial for diverse biological reactions in cells, and they function in the core network involving the ROS signaling (Suzuki and Mittler 2012; Kadota et al. 2014). Among the RBOH family members, the NADPH oxidases RBOHD and RBOHF are major ROS-generating enzymes in plants during the defense responses, including the cell death caused by ozone (O3) (Torres et al. 2002; Torres and Dangl 2005; Gilroy et al. 2014; Mignolet-Spruyt et al. 2016).

Under the suspension cell system, we found the abundances of BnaRBOHDs and BnaRBOHFs were significantly higher in ‘W10’ than in ‘QY10’ after short-term B deprivation (Fig. 5a, b). After 12-d low-B treatment, the expression of BnaRBOHDs and BnaRBOHFs showed no significant change in the roots between the B-efficient and -inefficient genotypes (Fig. 5c), and it indicated that the genes only responded to low-B within a short time. However, a number of studies have shown that the ROS production derived from RBOHD is not directly involved in plant cell death, but it participated in the signaling pathways in the defense responses of plants (Torres and Dangl 2005; Lherminier et al. 2009). Indeed, mutation of the RBOHD gene reduced the ROS production induced by pathogens in Arabidopsis, whereas it did not completely eliminate the stress response (Torres and Dangl 2005). Our study would further probe into the genes of RBOHD and RBOHF, which likely function predominantly in the signaling network involved in the low-B-induced cell death.

Previous studies show that oxidative damages may be minimized in the roots of a Ni hyper-accumulator Alyssum bertolonii by high endogenous anti-oxidant activities compared with the nonhyperaccumulator Nicotiana tabacum (Boominathan and Doran 2002). The Fe-efficient genotypes of apple could keep the redox balance through the activation of ROS scavenging during the long-term Fe deficiency compared with the Fe-inefficient cultivars (Sun et al. 2016). The previous studies indicated that higher anti-oxidant activities confer higher tolerance or efficiency to stresses. However, this mechanism is not suitable for our present study because we found ‘QY10’ did not possess higher activities of antioxidant enzymes, even slower increase and lower activities of SOD and CAT were detected in ‘QY10’ (Fig. 9a, b). The increase in the activities of antioxidant enzymes lagged behind the increase in the ROS production (Figs 3, 4, 9). Together with the expression of ROS-producing genes (Fig. 5a, b), the above findings suggested that it was not the antioxidant enzymes but the ROS-generating enzymes that determined the diffferential oxidative damages between the B-efficient and -inefficient genotypes. During the responses of plants to stresses, the down-regulation of the scavenging systems can lead to an oxidative burst, which further causes cell death (Torres and Dangl 2005; Kasten et al. 2016). In such a scenario, the decrease in SOD and CAT levels (Fig. 9a, b) could be responsible for the amplification of ROS signaling at some time points.

Besides NADPH oxidases-generated-ROS, the activated Ca2+ channels also represent a signaling molecule that is common to many plant responses (Torres and Dangl 2005). In various signaling pathways in plants, Ca2+ functions as the second messenger, and activates the downstream biological processes on the perception of outer environment stimuli (Evans et al. 2005). Ca2+ is also involved in the regulation of the long-distance signaling between the root and shoot, which may be important for the transduction of ROS signals (Choi et al. 2014). The binding of Ca2+ to its EF-hands is activated by RBOHF, which shows a direct interaction between Ca2+ and ROS molecules (Drerup et al. 2013). In the defense response, the oxidative burst has been implicated in the activation of Ca2+ influx (Levine et al. 1996; Demidchik et al. 2009), while another study indicated that Ca2+ fluxes in a cell could activate ROS production (Choudhury et al. 2016). It seems that under different environmental stresses, the up- and down-stream relationship was variant between Ca2+ and ROS signaling. Here, we found B-deprivations induced Ca2+ influx in both the plant roots and suspension cells, and that the trend was more obvious in the B-inefficient genotype ‘W10’ (Fig. 7). Applying La3+ (a Ca2+ channel blocker) could decrease the ROS accumulation in the roots (Fig. 8), which indicated that Ca2+ elevation is a signaling event upstream of ROS. However, the ROS accumulation was still higher under low-B supply than sufficient-B conditions (Fig. 8), suggesting that other signal molecules were likely implicated in the regulation of ROS production under B deficiency.

The possible pathways implicated in low-B-induced cell death of both the plant roots and suspension cells were drawn in Fig. 11. In this study, low-B induced the expression of NADPH-oxidase genes and the influx of Ca2+ in the PM. The expression of NADPH-oxidase genes activated the ROS burst (mainly O2 ), and the influx of Ca2+ promoted this process, which led to serious peroxide damages to the PM. The impaired integrity of the PM caused the loss of ions including K+, and eventually a series of physio-biochemical injuries led to the cell death. Compared with the B-efficient genotype, the B-inefficient genotype sensed more severe B-deficiency and suffered from more serious Ca2+ influx and oxidative damages, eventually resulting in more severe growth disorders under B-deficiency conditions.
Fig. 11

A scheme delineating how ROS and Ca2+ mediate the cell death in response to B deficiency. Low-B induced the expression of NADPH-oxidase genes and the influx of Ca2+ in the plasma membrane (PM). The expression of NADPH-oxidase genes activated the ROS burst (mainly O2 ), and the influx of Ca2+ promoted this process, which led to serious peroxide damages to the PM. The impaired integrity of the PM caused the loss of ions including K+, and eventually a series of physio-biochemical injuries led to the cell death. Compared with the B-efficient genotype, the B-inefficient genotype sensed more severe B-deficiency and suffered from more serious Ca2+ influx and oxidative damages, eventually resulting in more severe growth disorders under B-deficiency conditions

Notes

Acknowledgements

This work was financially supported by the National Natural Science Foundation of China (Grant NO. 31372129, 31572185) and the National Key Research and Development Program of China (Grant NO. 2016YFD0100700).

Supplementary material

11104_2017_3337_MOESM1_ESM.docx (586 kb)
ESM 1 (DOCX 586 kb)

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Copyright information

© Springer International Publishing AG 2017

Authors and Affiliations

  1. 1.National Key Laboratory of Crop Genetic Improvement and Microelement Research CentreHuazhong Agricultural UniversityWuhanPeople’s Republic of China

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