Boron stress inhibits beet (Beta vulgaris L.) growth through influencing endogenous hormones and oxidative stress response

ABSTRACT

Objective: This study aimed to evaluate the pleiotropic effects of boron stress on beet (Beta vulgaris L.) growth.

Methods: Beet seedlings were treated with varying concentrations of H₃BO₃ (0.05, 0.5, 2, and 30 mg/L) for 30 d for evaluation of diverse morphological parameters related to shoot and root growth. Transverse sections of beet leaves were observed under a microscope, and the leaf thickness was measured. Photosynthesis in beet was evaluated by measuring the photosynthetic rate, intercellular carbon dioxide concentration, and chlorophyll content; chloroplast morphology was observed by transmission electron microscopy. In addition, the levels of diverse endogenous hormones, as well as oxidative stress parameters, were also analyzed in beet leaves.

Results: We found that boron accumulated in different beet tissues but was observed mainly in mature leaves, followed by young leaves, mature petioles, young petioles, and roots. Boron stress (boron toxicity, 30 mg/L; boron deficiency, 0.05 mg/L) significantly inhibited beet shoot, root growth, and leaf expansion while promoting leaf thickening. Additionally, the levels of indole-3-acetic acid, gibberellin A3, and zeatin in beet leaves decreased significantly under boron stress, whereas the level of abscisic acid increased. Moreover, the levels of superoxide dismutase, peroxidase, catalase, malondialdehyde, and proline in beet leaves increased significantly under boron stress.

Conclusion: Together, our results indicate that boron stress significantly inhibited normal beet growth via the influence of endogenous hormone levels and oxidative stress responses.

KEY WORDS:

• Boron
• beet
• morphology
• endogenous hormone
• oxidative stress

1. Introduction

Boron (B) is an essential micronutrient required for the normal growth and development of higher plants (Zhou et al. 2017; Riaz et al. 2018a). Boron is the only element among the diverse and necessary micronutrients that is taken up by plants as an uncharged molecule, but not as an ion (Ahmad et al. 2012). Since B availability is inadequate in a large tract of land worldwide, B deficiency is a global agricultural concern (Eggert and Von Wirén 2017). In plants, B is involved mainly in carbohydrate metabolism and cell division (Gupta and Solanki 2013), and B deficiency can result in a series of phenotypic and physiological changes in plants (Riaz et al. 2018b). For example, B deficiency reduces plant dry mass, root number, root length, root surface area, and leaf area expansion in citrus rootstocks (Mei et al. 2011). Boron deficiency also decreases the leaf net photosynthetic rate (Pn), plant height, leaf area, fruiting sites, and dry matter accumulation, and increases fruit abscission in cotton (Zhao et al. 2003). Furthermore, B deficiency inhibits root cell elongation in Arabidopsis thaliana seedlings by regulating ethylene, auxin, and the levels of reactive oxygen species (ROS) (Camacho et al. 2015). Consequently, B deficiency contributes to considerable yield reductions in crops as diverse as cotton, rice, maize, wheat, soybean, groundnut, and citrus (Ahmad et al. 2012), making B fertilizer application necessary to improve crop yields, especially in B-deficient soils.

Many agricultural fields contain naturally high B concentrations, especially in arid and semi-arid regions (Nasim et al. 2015); overapplication of B fertilizers, as well as deposition of mining wastes, fly ash, and industrial chemicals also contribute to excessive B in the soil (Kato et al. 2009; Yau and Ryan 2008). Like B deficiency, B toxicity may also influence the quality and yield of plants. It has been reported that, under B toxicity, Citrus grandis exhibits reduced chlorophyll levels, inhibition of photosynthesis, irregular cell wall thickening in leaf cortex cells, and accelerated the death of phloem and root epidermal cells (Huang et al. 2014). Boron toxicity also inhibits the accumulation of shoot dry matter in soybean and increases the levels of superoxide dismutase (SOD), catalase (CAT), and ascorbate peroxidase (APX) (Hamurcu et al. 2013). Additionally, B toxicity was reported to significantly decrease leaf biomass, relative leaf growth rate, organic N and soluble protein levels, and nitrate reductase, and nitrite reductase activities in tomato leaves (Cervilla et al. 2010). Together, these results indicate that an appropriate B concentration in the soil is necessary for normal plant growth and development.

Sugar beet (Beta vulgaris L.) is one of the most important sucrose-producing crops and the second most prevalent sugar crop in China (Geng and Yang 2015). Foliar B application has been shown to increase root yield and sucrose concentration, as well as improve the seed yield and quality of sugar beet (Dordas et al. 2007; Armin and Asgharipour 2011). However, related studies on the specific roles of B stress on beet growth are still limited. In this study, beet seedlings were treated with varying concentrations of H₃BO₃ (0.05, 0.5, 2, and 30 mg/L) for 30 d. Morphology, photosynthesis, levels of endogenous hormones, oxidative stress response, and B accumulation were all evaluated in beet under B stress. Our findings reveal the pleiotropic inhibitory effects of both B deficiency and toxicity on beet growth that may be helpful for agricultural production.

2. Materials and methods

2.1. Plant materials and B treatments

Seeds of sugar beet Beta vulgaris ‘HI0099’ were purchased from Syngenta (Basel, Switzerland). Sterilized beet seeds (0.2% sodium hypochlorite) were planted in vermiculite until germination (fully stretched cotyledons). The germinated seeds were then moved into plastic containers containing a half-strength nutrient solution. When the cotyledons were fully stretched, the seedlings were moved into full-strength nutrient solution (236 g/L Ca(NO₃)₂ · 4H₂O, 102 g/L KNO₃, 98 g/L MgSO₄ · 7H₂O, 27 g/L KH₂PO₄, 7.45 g/L Na₂-EDTA, 5.57 g/L FeSO₄ · 7H₂O, 0.079 g/L CuSO₄ · 5H₂O, 0.088 g/L (NH₄)₆MO₇O₂₄, 1.81 g/L MnCL₂ · 4H₂O, and 0.22 g/L ZnSO₄ · 7H₂O, pH 6.5) containing varying concentrations of H₃BO₃ (T1, 0.05 mg/L; T2, 0.5 mg/L; T3, 2 mg/L; T4, 30 mg/L). The nutrient solution was renewed every 5 d. After 30 d of B treatment, beet plants of different groups were assayed. All the plants were cultured in a greenhouse at the Key Laboratory of Sugar Beet Genetic Breeding of Heilongjiang Province (Harbin, Heilongjiang, China) under a 12-h light/12-h dark cycle, 26°C day/18°C night temperature, and 40–60% relative humidity.

2.2. Detection of morphological parameters

Plant height, petiole length, and root diameter (the widest section) were measured using a Vernier caliper. The petiole number was counted directly under naked eyes. The root volume was measured according to the drainage method (Zhao et al. 2013). Shoots and roots were dried at 105°C for 10 min and then at 70°C until constant weight, after which the dry weight was measured using an electronic analytical balance. The leaf area (the latest fully stretched leaf) was measured using an image analysis system (LA-S, Wanshen, Shenzhen, China). Root vitality was measured according to the α-naphthyl amine method (Tachibana and Nakayama 2009).

2.3. Microscopy

Transverse sections of beet leaves in different groups were observed under a microscope. Briefly, fresh leaves (the latest fully stretched leaves) in the widest section were cut into 1-cm pieces and fixed in FAA solution (5% formalin, 5% acetic acid, 45% ethyl alcohol, and 45% distilled water) for 24 h. The samples were then dehydrated with graded ethanol (50%, 70%, 85%, 95%, and 100%), vitrificated in xylene, embedded in paraffin, and sliced into 400-µm transverse sections. The sample sections were subsequently dewaxed with xylene, rehydrated with graded ethanol (100%, 95%, 85%, 70%, and 50%), and stained with safranin and fast green. Stained samples were observed under a microscope (BH-2, Olympus, Japan), and the leaf thickness was measured using an image analysis system (LA-S).

2.4. Photosynthesis assay

Photosynthesis in beet was evaluated in the different groups by measuring the Pn, intercellular CO₂ concentration (Ci), and chlorophyll content. The latest fully stretched leaves were used for assays. Pn and Ci were detected using the LI-6400 portable photosynthetic system (Li-Cor, Lincoln, NE, USA) under a 1500 µmol∙m⁻² s⁻¹ photon flux density. Following extraction in 80% acetone in the dark overnight, the optical density (OD) was detected at 663 and 645 nm using a spectrophotometer (U4100, Hitachi, Tokyo, Japan). The chlorophyll content was calculated as 8.02(OD₆₆₃) + 20.2(OD₆₄₅) × V/(W × 1000), where V and W represent extract volume and sample weight, respectively (Arnon 1949).

2.5. Transmission electron microscopy (TEM)

Chloroplast morphology was observed in beet leaves of different groups by TEM. Briefly, fresh leaves (the latest fully stretched leaves) in the widest section were cut into pieces (1 mm × 5 mm) and fixed in 2.5% glutaraldehyde for 48 h. The samples were then fixed in 2% osmic acid for 2 h, dehydrated with graded ethanol (50%, 70%, 85%, 95%, and 100%), embedded in epoxy resin (Epon 812), and cut into 50-nm sections using an ultramicrotome (LKB, Uppsala, Sweden). Following staining with uranyl acetate and lead citrate, chloroplast morphology was observed under a transmission electron microscope (H-7650, Hitachi).

2.6. Endogenous hormone assay

Endogenous hormones, including indole-3-acetic acid (IAA), zeatin (ZT), gibberellin A3 (GA₃), and abscisic acid (ABA), were detected in beet leaves (the latest fully stretched leaf) of different groups by high-performance liquid chromatography (HPLC). HPLC was performed on an HPLC system (1200, Agilent, CA, USA) using a C18 column (XDB-C18, Agilent). The detection wavelength for IAA, GA₃, and ABA was 210 nm, and the mobile phase was methanol, acetonitrile, and citrate buffer solution (pH 3.0) (20:20:60). The detection wavelength of ZT was 254 nm, and the mobile phase was methanol, acetonitrile, and phosphate buffer (pH 7.0) (15:15:70).

2.7. Oxidative stress assay

Oxidative stress-related parameters, including SOD, CAT, peroxidase (POD), malondialdehyde (MDA), and proline levels, were evaluated in beet leaves (the latest fully stretched leaf) of different groups. Detection of SOD, POD, CAT, MDA, and proline was performed using the nitroblue tetrazolium photochemical reduction, guaiacol, ultraviolet absorption, thiobarbituric acid, and sulfosalicylic acid methods, respectively (Ding et al. 2014).

2.8. Boron accumulation assay

Boron accumulation was analyzed in different beet tissues, including the mature leaf, young leaf, mature petiole, young petiole, and root. Briefly, 0.5 g dry samples were digested with 15 mL nitric and 5 mL perchloric acids (volume ratio 3:1, pH 6.8–7.0) for 3 h, and diluted to 100 mL with ultrapure water (pH 6.8–7.0). Then, 4 mL solution samples were mixed with 4 mL ammonium acetate and 2 mL azomethine (volume ratio 2:2:1) for 1 h under darkness. The OD₄₂₀ was detected using a spectrophotometer (U4100), and the B concentration was calculated according to a standard curve (established using standard boric acid).

2.9. Statistical analyses

All data are expressed as means ± standard deviation (SD). Statistical analysis was performed with SPSS version 17.0 (SPSS Inc., Chicago, IL, USA). Comparison between different groups was determined by one-way ANOVA. A p-value less than 0.05 was considered to be significantly different.

3. Results

3.1. Boron stress inhibited beet growth

The effects of different concentrations of B on beet growth were analyzed. As shown in Table 1, plant height, petiole length, petiole number, shoot dry weight, root dry weight, root volume, root diameter, and root vitality were all significantly increased in a dose-dependent manner with increasing concentrations of B, attaining the highest levels at 2 mg/L (T3) (p < 0.05). Plant height, petiole length, petiole number, shoot dry weight, and root vitality were significantly higher in beet under B toxicity (T4, 30 mg/L) than under B deficiency (T1, 0.05 mg/L) (p < 0.05); however, the root dry weight, root volume, and root diameter were significantly lower in beet under B toxicity (T4, 30 mg/L) than under B deficiency (T1, 0.05 mg/L) (p < 0.05) (Table 1, Fig. S1).

Table 1. The morphological parameters of beet under the treatment of different concentrations of boron (N = 10, from independent plants)

Parameters	Boron concentrations
	T1 (0.05 mg/L)	T2 (0.5 mg/L)	T3 (2 mg/L)	T4 (30 mg/L)
Plant height (cm)	20.40 ± 1.21a	25.81 ± 1.08b	27.69 ± 1.45c	22.01 ± 1.33d
Petiole length (cm)	11.70 ± 0.98a	13.92 ± 1.02b	15.42 ± 1.16c	12.66 ± 0.87d
Petiole number (N)	12 ± 2a	14 ± 2b	16 ± 2c	12 ± 2a
Shoot dry weight (g)	12.14 ± 0.71a	16.47 ± 1.07b	18.43 ± 1.21c	13.85 ± 1.06d
Root dry weight (g)	5.36 ± 0.42a	6.35 ± 0.61b	7.42 ± 0.49c	4.43 ± 0.35d
Root volume (mL)	54.28 ± 1.54a	66.45 ± 1.76b	72.69 ± 1.65c	49.08 ± 1.32d
Root diameter (cm)	3.07 ± 0.09a	3.25 ± 0.05b	3.48 ± 0.08c	2.71 ± 0.07d
Root vitality [µg NA/(g.FW·h)]	81.06 ± 6.06a	140.74 ± 10.4b	218.61 ± 18.17c	90.99 ± 7.06d

3.2. Boron stress inhibited leaf expansion and promoted leaf thickening

The effects of different B concentrations on leaf growth in beet were analyzed. As shown in Table 2, the leaf area increased significantly in a dose-dependent manner with increasing B concentrations, reaching a peak at 2 mg/L (T3) (p < 0.05). The beet exhibited smaller leaf area under B toxicity (T4, 30 mg/L) than under 0.5 and 2 mg/L boron treatment (T2 and T3) (p < 0.05). In addition, B deficiency (T1, 0.05 mg/L) and B toxicity (T4, 30 mg/L) in beet resulted in significantly increased leaf thickness compared to B treatments at 0.5 and 2 mg/L (T2 and T3) (p < 0.05). The palisade tissue (PT) and spongy tissue (ST) thickness were significantly reduced in a dose-dependent manner with increasing B concentrations, reaching the lowest levels at 2 mg/L (T3) (p < 0.05). The beet exhibited significantly thicker PT and ST under B toxicity (T4, 30 mg/L) than under 2 mg/L boron treatment (T3) (p < 0.05). However, upper epidermis (UE) and lower epidermis (LE) thickness decreased continuously with increasing B concentrations (p < 0.05) (Fig. 1 and Table 2).

Table 2. The thickness of beet leaves under the treatment of different concentrations of boron (N = 10, from independent plants)

Parameters	Boron concentrations
	T1 (0.05 mg/L)	T2 (0.5 mg/L)	T3 (2 mg/L)	T4 (30 mg/L)
Leaf area (cm^2)	202.49 ± 6.41a	256.19 ± 8.48b	274.81 ± 6.85c	223.19 ± 7.30d
Leaf thickness (µm)	290.42 ± 8.48a	209.77 ± 6.79b	147.77 ± 5.23c	222.89 ± 5.22b
UE thickness (µm)	18.44 ± 3.12a	15.87 ± 2.01b	14.28 ± 2.99b	13.01 ± 2.14c
PT thickness (µm)	103.68 ± 12.39a	79.33 ± 9.18b	50.09 ± 5.04c	88.74 ± 6.66b
ST thickness (µm)	148.12 ± 14.94a	99.35 ± 12.87b	70.35 ± 9.92c	108.86 ± 10.06d
LE thickness (µm)	20.18 ± 3.46a	15.22 ± 3.09b	13.05 ± 2.97c	12.28 ± 2.03c

Figure 1. Microscopic observation of the transverse section of leaves (×400, bar = 100 µm). Beet plants were treated with different concentrations of Boron (T1, 0.05 mg/L; T2, 0.5 mg/L; T3, 2 mg/L; T4, 30 mg/L) for 30 days. The representative images are shown (N = 10, from independent plants). UE, upper epidermis; PT, palisade tissue; ST, spongy tissue; LE, lower epidermis

3.3. Boron stress inhibited photosynthesis in beet

Beet photosynthesis was analyzed under different B concentrations. As shown in Table 3, the Pn increased significantly in a dose-dependent manner with increasing B concentrations, peaking at 2 mg/L (T3) (p < 0.05); in contrast, Ci presented a significant decrease. The beet exhibited significantly lower Pn and higher Ci under B toxicity (T4, 30 mg/L) than under B treatments at 0.5 and 2 mg/L (T2 and T3) (p < 0.05). The chlorophyll content in the beet leaves exhibited a trend consistent with the Pn (Table 3). The chloroplasts in the beet leaves were observed by TEM. Chloroplast number and size both increased significantly in a dose-dependent manner with increasing B concentrations, peaking at 2 mg/L boron treatment (T3) (p < 0.05). The beet exhibited significantly lower chloroplast number and size under B toxicity (T4, 30 mg/L) than under treatments at 0.5 and 2 mg/L boron (T2 and T3) (p < 0.05) (Fig. 2 and Table 3).

Table 3. The photosynthesis parameters in beet leaves under the treatment of different concentrations of boron (N = 5, from independent plants)

Parameters	Boron concentrations
	T1 (0.05 mg/L)	T2 (0.5 mg/L)	T3 (2 mg/L)	T4 (30 mg/L)
Pn (µmol CO2/m^2/s)	10.91 ± 0.87a	14.68 ± 0.63b	18.60 ± 0.80c	13.09 ± 0.85d
Ci (µmol/m^2/s)	465.75 ± 15.6a	380.95 ± 10.3b	359.71 ± 9.2c	418.50 ± 11.2d
Chlorophyll content (mg/g.FW)	1.73 ± 0.09a	2.29 ± 0.18b	2.86 ± 0.14c	2.01 ± 0.19d
Chloroplast number (N/10^3µm^2)	7 ± 2a	11 ± 3b	18 ± 4c	6 ± 2a
Chloroplast size (µm^2)	3.72 ± 0.75a	8.48 ± 1.34b	13.88 ± 1.53c	5.19 ± 1.22d

Figure 2. Microscopic observation of chloroplasts in beet leaves by transmission electron microscope (× 4000, bar = 10 µm). Beet plants were treated with different concentrations of Boron (T1, 0.05 mg/L; T2, 0.5 mg/L; T3, 2 mg/L; T4, 30 mg/L) for 30 days. The representative images are shown (N = 5, from independent plants). For analysis of chloroplasts, five images from five independent plants were used (five cells for each image)

3.4. Boron stress influenced endogenous hormone levels in beet leaves

Since B stress significantly inhibited leaf expansion, the levels of endogenous hormones were evaluated in beet leaves of different groups. As shown in Table 4, the levels of IAA, GA₃, and ZT increased significantly in a dose-dependent manner with increasing B concentrations, peaking at 2 mg/L (T3) (p < 0.05). The beet exhibited significantly lower levels of IAA, GA₃, and ZT under B toxicity (T4, 30 mg/L) than under treatments at 0.5 and 2 mg/L (T2 and T3) (p < 0.05). Meanwhile, the lowest ABA level was recorded in beet treated with 2 mg/L boron (T3) (p < 0.05). Both B deficiency (T1, 0.05 mg/L) and B toxicity (T4, 30 mg/L) significantly increased the ABA level compared to 0.5 and 2 mg/L boron treatments (T2 and T3) (p < 0.05) (Table 4).

Table 4. The endogenous hormone parameters in beet leaves under the treatment of different concentrations of boron (N = 5, from independent plants)

Parameters	Boron concentrations
	T1 (0.05 mg/L)	T2 (0.5 mg/L)	T3 (2 mg/L)	T4 (30 mg/L)
IAA (µg/g.FW)	5.46 ± 0.54a	8.28 ± 0.58b	9.94 ± 0.96c	6.43 ± 0.45d
GA3 (µg/g.FW)	2115.56 ± 107.45a	3238.22 ± 145.56b	3845.17 ± 128.98c	2489.38 ± 123.56d
ZT (µg/g.FW)	58.12 ± 7.89a	73.72 ± 8.82b	94.50 ± 10.45c	62.67 ± 6.38a
ABA (µg/g.FW)	1.08 ± 0.34a	0.56 ± 0.12b	0.38 ± 0.11c	1.04 ± 0.33a

3.5. Boron stress activated the oxidative stress response in beet leaves

The response of beet to B stress was analyzed through the measurement of several oxidative stress parameters. As shown in Table 5, B deficiency (T1, 0.05 mg/L) and B toxicity (T4, 30 mg/L) both significantly increased SOD, POD, CAT, and MDA levels in beet leaves compared to B treatment at 0.5 and 2 mg/L (T2 and T3) (p < 0.05). No significant differences were found in SOD, POD, CAT, and MDA levels between the 0.5 and 2 mg/L boron treatments (T2 and T3). In addition, MDA and proline levels decreased significantly in a dose-dependent manner with increasing concentrations of B, reaching their lowest levels at 2 mg/L boron treatment (T3) (p < 0.05). Beet MDA and proline content were highest under B toxicity (T4, 30 mg/L) (p < 0.05) (Table 5).

Table 5. The oxidative stress parameters in beet leaves under the treatment of different concentrations of boron (N = 5, from independent plants)

Parameters	Boron concentrations
	T1 (0.05 mg/L)	T2 (0.5 mg/L)	T3 (2 mg/L)	T4 (30 mg/L)
SOD (u/g.FW/min)	376.60 ± 13.34a	356.73 ± 12.81b	343.25 ± 15.89b	395.08 ± 7.35c
POD (u/g.FW/min)	444.34 ± 22.86a	325.90 ± 16.80b	350.88 ± 24.16b	425.85 ± 22.89a
CAT (u/g.FW/min)	15.39 ± 1.24a	12.93 ± 2.83b	11.57 ± 1.16b	17.89 ± 3.63a
MDA (nmol/g.FW)	3.62 ± 0.14a	3.36 ± 0.19b	2.76 ± 0.14c	4.21 ± 0.21d
Proline (µg/g.FW)	22.84 ± 1.45a	14.89 ± 1.58b	12.30 ± 0.81c	25.84 ± 1.45d

3.6. Boron concentration in different beet tissues

Boron concentration in different beet tissues was further analyzed. As shown in Table 6, B accumulated mainly in mature leaves, followed by young leaves, mature petioles, young petioles, and roots. Boron concentration in these tissues increased significantly in a dose-dependent manner with increasing B concentrations (p < 0.05). With B treatment at 30 mg/L (T4), B concentration in the roots, young leaves, mature leaves, young petioles, and mature petioles was 87.70, 31.15, 1335.21, 46.03, and 62.62 µg/g, respectively (Table 6).

Table 6. Boron concentration in different tissues of beet under the treatment of different concentrations of boron (N = 5, from independent plants)

Parameters	Boron concentrations
	T1 (0.05 mg/L)	T2 (0.5 mg/L)	T3 (2 mg/L)	T4 (30 mg/L)
Root (µg/g.DW)	1.65 ± 0.51a	8.57 ± 0.52b	23.74 ± 0.53c	87.70 ± 11.19d
Young leaf (µg/g.DW)	16.34 ± 1.09a	26.56 ± 2.88b	62.31 ± 5.21c	231.15 ± 10.44d
Mature leaf (µg/g.DW)	15.07 ± 1.09a	30.07 ± 5.11b	117.84 ± 17.22c	1335.21 ± 40.55d
Young petiole (µg/g.DW)	2.30 ± 0.29a	10.92 ± 1.27b	36.77 ± 3.77c	46.03 ± 1.62d
Mature petiole (µg/g.DW)	5.49 ± 0.18a	11.24 ± 0.71b	43.15 ± 1.99c	62.62 ± 3.34d

4. Discussion

Boron is an important non-metal micronutrient required for normal plant growth and development (Gupta and Solanki 2013). Globally, there are large tracts of arable land that are deficient in B while others are restricted by B toxicity (Reid 2014). Boron deficiency and toxicity can both induce diverse morphological and physiological changes in plants (Shah et al. 2017). In this study, we evaluated the effects of B stress on beet growth. We found that B deficiency and toxicity both significantly affected beet growth parameters, including morphology, rate of photosynthesis, endogenous hormone levels, and oxidative stress responses.

Morphology is an important indicator of plant growth status. Reduced shoot and root dry weight under B stress have been observed in diverse species such as cotton (Zhao et al. 2003), citrus (Huang et al. 2014), and soybean (Hamurcu et al. 2013). In this study, we found that B deficiency and toxicity both elicited significantly reduced beet plant height, petiole length, petiole number, shoot and root dry weight, root volume, root diameter, and root vitality. Our findings are consistent with previous studies, and further illustrate that B stress is detrimental to beet growth. In addition, we also found that the inhibitory effects of B deficiency on shoot growth were stronger than those of B toxicity, while the inhibitory effects of B deficiency on root growth were weaker than those of B toxicity. This result may be explained by the higher demand for B in the shoot than in the root, suggesting that the root may be more sensitive to high concentrations of B than the shoot.

We also analyzed beet leaf morphology under B stress in this study and found that the leaf area was significantly decreased with B deficiency and toxicity, whereas leaf thickness was increased. Microscopic observation showed that B deficiency and toxicity significantly increased PT and ST thickness, consistent with previous studies in citrus. It has been reported that, under B deficiency, citrus exhibits thickened leaves, big cell gaps, and increased ST number and volume (Liu et al. 2015). Moreover, B toxicity has been shown to increase citrus leaf thickness by increasing the numbers of PT and ST cells (Huang et al. 2014). We suspect that the thickened PT and ST may help to enhance beet inner tissue resistance to B stress.

Photosynthesis is vital for dry matter accumulation in plants. Numerous studies have demonstrated that B stress can inhibit photosynthesis in species such as cotton (Zhao et al. 2003), citrus (Huang et al. 2014), mung bean (Jiao et al. 2013), Arabidopsis thaliana (Chen et al. 2014), and wheat (Liu et al. 2017). Consistent with previous reports, we found that B deficiency and toxicity significantly decreased the Pn and increased the Ci in beet leaves. Since the chloroplast is an organelle specialized for photosynthesis, chlorophyll content and chloroplast micromorphology were also analyzed in this study. We found that B deficiency and toxicity significantly decreased chlorophyll content in beet leaves, as well as chloroplast number and size. The inhibitory effect of B deficiency on chloroplast development may have led directly to the decrease in chlorophyll content. Meanwhile, a previous study showed that high B concentrations result in reduced Fe content in apple leaves (Mouhtaridou et al. 2004). Since Fe is an essential element for chlorophyll synthesis, B toxicity may also contribute to the decrease in chlorophyll content by affecting the Fe content.

Endogenous hormones are important regulators of plant growth and development. The levels of both IAA and CK are reportedly reduced in pea plants under B deficiency, as is IAA export from the shoot apex (Wang et al. 2006). Significantly reduced levels of IAA, GA₃, and indole-3-propionic acid (iPA) are observed in wheat under B deficiency, whereas the levels of ABA concentrations are increased (Yan et al. 2003). Consistent with previous studies, we found that, in beet, B deficiency significantly reduced IAA, GA₃, and ZT levels, but increased the levels of ABA (Yan et al. 2003). Meanwhile, B toxicity elicited an inhibitory effect on endogenous hormone levels similar to that of B deficiency, and the changes in endogenous hormone content are consistent with the morphology of beet under B stress.

The oxidative stress response is an antioxidant defense system used by plants to actively adapt to stressful environments. The specific roles of B stress on the oxidative stress response have been identified in various studies. For example, the activities of antioxidant enzymes like CAT, POX, and SOD increase in response to B stress in Artemisia annua (Aftab et al. 2010), and the levels of SOD, CAT, and APX in soybean are increased under B toxicity (Hamurcu et al. 2013). Furthermore, the levels of antioxidant enzymes, including SOD, POD, CAT, and APX, decrease under B deficiency, and to some extent also under B toxicity, in citrange orange plants (Shah et al. 2017). In agreement with the above findings, we found that B deficiency and toxicity significantly increased SOD, POD, CAT, MDA, and proline content in beet, illustrating the occurrence of oxidative damage in beet under B stress. The observed increases in oxidative stress parameters may contribute to protecting cellular membranes and alleviating oxidative stress, thereby enhancing resistance to B stress.

Boron concentration varied according to plant tissue. In this study, we found that B accumulated mainly in mature leaves, followed by young leaves, mature petioles, young petioles, and roots. Our finding is consistent with a previous study showing that the total B concentration in citrus roots was substantially lower than in citrus leaves under B toxicity (Huang et al. 2014). Since B is transported from the root to the petiole, and then to the leaf, B accumulates in the leaves, especially mature leaves. In addition, we also found that B concentration in different beet tissues increased significantly in a dose-dependent manner with increasing B concentrations. Notably, B concentration in mature beet leaves with 30 mg/L boron treatment can reach up to 1335.21 µg/g. Since the safe B intake level for adults is 1–13 mg/d (Nielsen 1998), excessive B concentration in beet may be toxic to human health.

In conclusion, B deficiency and toxicity both significantly inhibited dry matter accumulation in beet shoots and roots while promoting leaf thickening. The inhibitory effects of B deficiency and toxicity on beet growth were closely associated with increasing levels of IAA, GA₃, and ZT and decreasing levels of ABA. Meanwhile, SOD, POD, CAT, MDA, and proline levels were increased in beet under B stress, which may relieve B stress-induced oxidative damage.

Conflict-of-interest statement

The authors declared that they have no conflicts of interest in this work.

Supplementary material

Supplemental data for this article can be accessed here.

Additional information

Funding

This work was supported by a modern agricultural industry system (CARS-170204), and an opening foundation of Heilongjiang provincial key laboratory of modern agricultural cultivation and crop germplasm improvement in China.