Introduction

Shortages of mineral nutrients and micronutrient imbalances seriously affect crop productivity under various environmental conditions (Hajiboland 2012). Among others, boron (B) is one of the most essential micronutrients for the development and growth of vascular plants (Wimmer et al. 2020). Boron maintains plasma membrane integrity, cell wall composition, pollen tube development, and root elongation (Mühling et al. 1998; Brown et al. 2002; Wimmer et al. 2003, 2009; Landi et al. 2019). However, the demand for B is not similar to all plant species, with monocots having a lower demand compared to dicot plants. In this regard, rapeseed (Brassica napus L.) is an important oilseed crop with a known high B demand for growth and seed production (Wimmer and Eichert 2013). However, worldwide, in more than 80 countries B deficiency [< 0.25 mg B (kg soil−1)] is a major constraint, not least as rapeseed production requires at least 0.5 mg B (kg soil–1) (Pommerrenig et al. 2018). To intervene and correct the deficiency of B diminishing yields, high B fertilization is a necessary practice. However, as the margin between B deficiency and toxicity is extremely narrow within one plant species, high B fertilizer applications can also be detrimental. The predominant chemical form of B is the uncharged boric acid [B(OH)3] that plants easily absorb (Broadley et al. 2012). But B underlies a high pH-dependency, by which the equilibrium between boric acid [B(OH)3] and borate [B(OH)4–] in the growth medium determines the relative proportion of boric acid to borate anions and thus plant availability. For example, at soil pH < 7, 99% of B is primarily present as unaltered boric acid, which is readily available for passive plant uptake by the root system (Dinh et al. 2021b). On the other hand, the predominance of borate anions is higher in alkaline soil than in neutral or acidic soil (Wimmer et al. 2015; Koohkan and Maftoun 2016). Besides the media pH, high B availability triggers passive B uptake as boric acid, whereas lower availability facilitates active B uptake as borate (Wimmer et al. 2015; Dinh et al. 2021b).

The root is the main medium for the uptake of B through xylem loading. Having a high selectivity of membrane protein aquaporin to transport water with boric acid and ammonia (Kishchenko et al. 2023). B uptake channels such as nodulin26-like intrinsic protein (NIP) belong to the aquaporin subfamily localized in the root (Hua et al. 2016; Verwaaijen et al. 2023). In addition, several members of the NIP subfamily are involved in plant B uptake and transport, with NIP5;1 (Takano et al. 2005; Tanaka et al. 2008; Pommerrenig et al. 2015) being one of them that facilitates boric acid diffusion under limited boron supply. However, active uptake and distribution of B in plants by xylem loading is also mediated by the B transport protein BOR1 (Miwa and Fujiwara 2010; Onuh and Miwa 2021), which allows the transport of the borate anion rather than the boric acid-like NIP5;1.

In the course of a sustainable and optimal B supply, B uptake must, therefore, always be seen as a concert between dosage and external media pH. The latter, although is also readily affected by accompanying elements within the growth medium. In this context, nitrogen (N) is one of the most prevalent macronutrients in plants. Nitrate (NO3–) and ammonium (NH4+) are the most common inorganic forms of nitrogen that plants may absorb and use directly (Xu et al. 2012; Zhu et al. 2021), but they also directly affect soil pH. It is well known that the pH of the growth medium can be altered by N-fertilizers through physiological acidification or alkalinization of the rhizosphere (Zeng et al. 2012; Jampeetong et al. 2013; Dreyer et al. 2020; Li et al. 2023a), and N sources responsible for the expression of several aquaporin genes, thus involved in the transport of water, other studies also shows that the mixture of NO3– and NH4+ facilitated higher water transport by enhanced aquaporin (AQP) expression (Wang et al. 2016), which directly affects B uptake.

Since N input in the form of NO3− causes a pH elevation in the rhizosphere (Wang and Tang 2018), active B uptake in form of borate will be improved under conditions of low or limited B availability. On the other hand, N supply in the cationic form as NH4+ causes a decrease in rhizosphere pH (Dreyer et al. 2020), resulting in boosted B uptake under adequate or high B supply when passive and facilitated uptake pathways of boric acid are more relevant. Suggesting that different N forms might influence B uptake by plants depending on the B supply level. N-sources such as NH4+ enhance root B uptake in olive trees by causing an increase in root B accumulation as compared to NO3− (Chatzissavvidis et al. 2007); nevertheless, Dinh et al. (2021b) discovered that NH4+-indued acidity then impacts oilseed rape’s B uptake, like Carr et al. 2020 noted for coffee plants. Conversely, Yang et al. (2022) identified two limiting factors: a lack of B and soil acidity, which significantly impacts citrus yield and quality in South China which was in line with the findings of Cheng et al. (2022) and Du et al. (2018) the decrease in pH limits the absorption of B and inhibits the growth of citrus plants in acidic soil. In addition, research on many plant species has shown that changes in root zone pH may affect root hydraulic conductivity, aquaporin-mediated water transport, or both (Siemens and Zwiazek 2011; Kapilan et al. 2018).

However, most B absorption and translocation research has been at acidity or alkalinity, with little data on the differential effects of ammonia and nitrate at constant N source with buffering media pH. External medium pH regulation (i.e., buffering) becomes a significant regulatory mechanism for water transport. However, information regarding buffering the media with different constant N forms to grow plants and especially how boron will respond to such pH variations is scarcely known. Moreover, the underlying genetic mechanisms at the low and high B supplies with differently buffered pH at the constant N forms (NH4+ and NO3−) by rapeseed plants have not been explored yet or poorly identified.

In this context, we hypothesize that buffered pH at N-source (NH4+ compared to NO3−) increases B uptake and the growth of rapeseed. Thus, this study aims to elucidate the response of B transporter BnaBOR1;2 and the channel BnaNIP5;1 whether the effects on B uptake and plant growth were due to physiological acidification caused by N-forms and/or a single pH effect of the external solution or levels of B supply.

Materials and Methods

Growth Condition

Rapeseed (Brassica napus L. cv. Kaliber; Norddeutsche Pflanzenzucht Lembke, Hohenlieth, Germany) seeds were soaked in a well-aerated 1 mM CaSO4 solution for 24 h and germinated on filter paper moistened with 1 mM CaSO4 at 22 °C for 5 days in the dark before being exposed to light for another 3 days. Eight-day-old seedlings of uniform size were transplanted to plastic pots with 2 L of an aerated a quarter-strength modified Hoagland nutrient solution (Hoagland and Arnon 1950). After 4 and 8 days of cultivation, the concentration of the nutrient solution was increased to half and full strength. The full-strength solution had the following composition: 2.0 mM Ca(NO3)2, 0.5 mM K2SO4, 0.25 mM KH2PO4, 0.5 mM MgSO4, 50 μM NaCl, 1.0 μM H3BO3, 2.0 μM MnSO4, 0.4 μM ZnSO4, 0.4 μM CuSO4, 0.1 μM NH4Mo7O24, 40 μM Fe-EDTA, 2.0 mM CaCl2⋅2H2O (pH ≈ 5.5). The nutrition solution was changed every 2 days to replenish the nutrients consumed by the plants. The plants were cultivated in a growth chamber set to 14 h d–1 photoperiod, 350 μmol photon m–2 s–1 light intensity (measured by a light meter, Li–198, Lincoln, USA), 60% relative humidity, and 22 °C (day)/18 °C (night) temperature.

Experimental Design and Treatments

Based on a pilot study 1 and 100 µM B were used as low and high B treatments, respectively, in this study. After 14 days of growth under homogeneous conditions, the roots of plants were washed completely with double-deionized water and transferred to a nutrient solution containing either 1 µM B or 100 µM B in form of boric acid.

Two identical sets of experiments were conducted for two N-forms, in which either nitrate Ca(NO3)2 or ammonium (NH4)2SO4 was provided as the only N source to the plants at a concentration of 2 mM at each B level. A lack of calcium in the ammonium treatment was compensated by using 2 mM CaSO4. After the application of the N and B treatments, the plants were cultivated for a total of 5 days. Throughout the treatment phase, the nutrient solutions pH adjusted to 5.0, 6.0, and 7.0, respectively, by using 1 M 2-(N-morpholino) ethane sulfonic acid (MES) and 2 M N-(2-hydroxyethyl)-piperazine-N′–2-ethane sulfonic acid (HEPES), which was tested every 12 h (Table 1).

Table 1 The treatments were used in the experiment for 5 days
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The nutrient solution was changed once a day. To prevent the contamination of B, all solutions were prepared in plastic ware using double-deionized water (18.2 MΩ). Both experiments were carried out with a two-factor full factorial layout. In which individual treatments containing two plants were replicated four times. NH4+-fed and NO3−-fed buffered two identical sets of pots were grown simultaneously under the same conditions. For both buffered-fed plants, one set of plants was used to measure growth parameters and nutrient status, and the other was used for B transporters studies.

Harvesting

Prior to treatment application, the initial B content for the computation of B uptake rates was determined by harvesting plants from four pots after 14 days of growth under identical conditions. Roots and shoots were collected 5 days after treatment application, rinsed carefully with deionized water (18.2 MΩ), and dried at 65 °C for 72 h to determine the dry matter. Shoots and roots were also taken from the second set of the experiment, instantly frozen in liquid nitrogen, and then placed in a refrigerator set at − 80 °C for gene expression analysis.

According to Dinh et al. (2021b), the growth rate of the shoots and roots was determined based on using the formula:

$${\text{GR(}}g{\text{ DM }}d^{ - 1} {)}\, = \,(lnW2 - lnW1)/(D),$$
(1)

where W1 and W2 are the dry matter (g DM) before and after the treatment period, and D represents the total days of the treatment application.

According to Bellaloui and Brown (1998), a specific B uptake rate (IM) was calculated:

$$I_{M} (\mu g \, B \, g^{ - 1} root{\text{ DM }}d^{ - 1} )\, = \,[(lnR_{2} - lnR_{1} )/(T)] \times [(M_{2} - M_{1} )/(R_{2} - R_{1} )],$$
(2)

where R1 and R2 are the root dry matter at the beginning and end of the treatment period, respectively, M1 and M2 are the B contents at the beginning and at the end of the treatment period, respectively, and T refers to the total number of treatment days.

Determination of Boron Concentration

B concentration was measured according to the procedure followed by Dinh et al. (2021a). In short, 200 mg of finely ground (Cyclotec 1093, Foss Tecator, Höganäs, Sweden) to powder plant samples were digested in 10 mL 69% HNO3 in a microwave oven (MARS 6 Xpress, CEM Corporation, Matthews, USA) as follows:

2 min at 100 °C, 1 min at 120 °C, 20 min at 180 °C, and 20 min cooling time. The digested samples were then diluted with 100 mL of 18.2 MΩ cm conductivity Milli-Q water, and afterward stored at 4 °C temperature, Due to abstaining from B contamination, only plasticware was allowed to use, and B concentration was analyzed by inductively coupled plasma mass spectrometer (ICP-MS; Agilent 7700, Agilent Technologies Inc., USA). Boron content was estimated:

$$Boron \, content\left( {\mu g \, plant^{{ - { 1}}} } \right)\, = \,B \, concentration\, \times \,corresponding \, dry \, weight,$$
(3)

Determination of Nitrate

Nitrate (NO3−) anions in the shoot were measured by anion exchange chromatography using an ICS–5000 system equipped with a Carbo Pac PA-100 column and an integrated amperometric detector (Dionex, Sunnyvale, CA, USA) followed by slightly modified method of Cataldi et al. (2000). Each sample was prepared from the extract of (≈ 20 mg) of dry material in 1.5 mL of double-deionized water and was cooked in a shaking bath (Medingen SWB20, Dresden, Germany) that was preheated to 100 °C for 5 min. Afterward, vortexed thoroughly for mixing. Subsequently, the sample extract was cooled on ice for 30 min and thereafter centrifuged at 12,000 rpm at 4 °C temperature for 10 min. The supernatant was diluted 10 times using chloroform and mixed one more time with the vortex (5 s), and then again centrifuged at 12,000 rpm for 5 min for cleaning up and running them over C18 columns (Strata®. 8B-S001-DAK, Phenomex, Torrance, CA, USA).

Determination of Macro- and Micronutrient Concentrations

Approximately, 200 mg of fine ground (Cyclotec 1093, Foss Tecator, Höganäs, Sweden) oven-dried (65 °C, 72 h) plant material each replicate was digested with 10 mL of 69% HNO3 (ROTIPU-RAN Supra for ICP, 69%) and dissolved for 45 min in an 1800 W microwave-oven (MARS 6 Xpress; CEM, Matthews, MC, USA) stated by Jezek et al. (2015). Next, cooled digested samples were diluted with 100 mL of deionized water (18.2 MΩ cm conductivity) and before analysis kept at 4 °C. Finally, the shoots’ macronutrients (phosphorus, P; potassium, K; calcium, Ca; magnesium, Mg) and micronutrients (iron, Fe; manganese, Mn; copper, Cu; zinc, Zn), concentration were evaluated by inductively coupled plasma optical emission spectrometry (ICP-OES; Agilent 5800, Agilent Technologies Inc., USA), Following the Dumas combustion method, the total N concentration in shoots was measured by a CNS elemental analyzer (Flash EA 1112 NCS, Thermo Fisher Scientific, Waltham, MA, USA) using the ground, finely powdered samples weighted 20 mg and then placed into aluminum capsules for analysis (Supplementary Tables 1 and 2).

Gene Expression Analysis

Samples of roots and shoots were taken individually from the third set of plants, roots were washed three times in running deionized water, immediately frozen in liquid nitrogen after separation from the shoots, and stored at − 80 °C. The frozen samples were ground into a fine powder using liquid nitrogen, mortar, and pestle. Following Sagervanshi et al. (2020), a reagent TRIZOL (Invitrogen, CA, USA) was used to isolate total RNA from the powdered roots sample. A NanoDrop spectrophotometer (ND1000, Thermo Fisher Scientific, USA) was used to measure the amount of RNA and kept at − 80 °C for further analysis. According to the manufacturer’s instructions, the cDNA was synthesized using the Thermo Fisher Scientific VersoTM cDNA Kit. A total volume of 20 μL was used for the reaction. After 30 min of reverse transcription at 42 °C, the reverse transcriptase was inactivated at 95 °C for two minutes. After being aliquoted, the cDNA was kept for further use at − 20 °C. The cDNA amplification was performed according to the instructions provided by Dream taqTM DNA polymerase from Fermentas. The components of the PCR reaction were mixed on ice and then transferred to reaction tubes. The tubes were then placed into a T Gradient Thermocycler (Bio-metra, Germany). To control the specificity and reliability of the PCR, negative controls (NTC) were employed. The Polymerase Chain Reaction (PCR) was performed as follows: An initial denaturation step was carried out at 95 °C for 5 min, followed by 35 cycles of denaturation at 95 °C for 10 s, primer annealing at 56 °C for 15 s, and extension at 72 °C for 20 s. PCR amplification was used to verify and confirm each primer. This was done to ensure specificity. To ensure the correct length of the amplicon, PCR products from the above reaction were run on agarose gels.

Statistical Analysis

The data were statistically analyzed using the two-way analysis of variance (ANOVA) using the R studio (version. 4.2.2) (R Core Team 2022). The means were compared by using Tukey’s HSD test with a confidence level of 95% (p ≤ 0.05) for statistically significant variations between the means. The data are shown as the mean accompanied by the standard error (SE), and the graphs were created using GraphPad Prism 8 (Version 9.5.0, GraphPad Software, San Diego, California, USA). The Pearson correlations between the response variables were evaluated using the “corrplot” package in R (version. 4.2.2). 

Results

Changes in pH of the Nutrient Solution

The changes in pH of the nutrient solution followed a similar trend but in opposite directions in case of both N-forms (Fig. 1). On the first day of the treatment period, the pH values of NO3− and NH4+ containing non-buffered nutrient solution were 5.8 and 4.5 (initial), respectively (Fig. 1). NO3−containing non-buffered nutrient solution pH values were increased gradually and reached 6.4 after 36 h. In contrast, the pH of the NH4+ containing non-buffered nutrient solution declined and reached 3.2 after 36 h. These changes were similar in both levels of supplied B (Fig. 1).

Fig. 1
figure 1

pH changes of the non-buffered nutrient solution containing NO3− and NH4+ during a period of 36 h

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Roots and Shoots Dry Matter

Roots and shoots dry matter content of rapeseed varied significantly at 100 μM B and 1 μM B supply among various pH levels irrespective of the N forms (Fig. 2). Overall, at the 100 μM B level, roots and shoots dry biomass were substantially higher than those recorded at the 1 μM B level. Among the NO3−-fed plants (Fig. 2A), the maximum shoot dry matter was recorded at 100 μM B level at non-buffered pH which was statistically similar to the value at buffered pH (6 and 7). Similarly, NO3−-fed plants with a nonbuffered pH had the highest amount of root dry matter at 100 μM B (Fig. 2B). While NO3−-fed plants with a buffered pH of 5 had the lowest amount of shoot and root dry matter at both 1 μM and 100 μM B (Fig. 2A, B). In contrast, NH4+-fed plants (Fig. 2C, D) with an unbuffered pH had the lowest root and shoot dry matter content at both 1 μM B and 100 μM B levels, while plants with a buffered pH (6) had the highest root and shoot dry matter content. Moreover, root and shoot dry weights were comparatively lower with NH4+-fed plants than with NO3−-fed plants (Fig. 2).

Fig. 2
figure 2

Shoot dry matter and root dry matter of NO3− (A, B) and NH4+-fed (C, D) rapeseed plants with low and high levels of boron at buffered and non-buffered pH. Different letters denote significant differences (Tukey’s HSD test; p ≤ 0.05)

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Growth Rate

The shoot growth rate of rapeseed varied significantly at 1 μM B and 100 μM B supply at various pH levels (Fig. 3). Among the NO3−-fed plants, the maximum shoot growth rate was recorded at 100 μM B level at non-buffered pH and pH 6. On the other hand, NO3−-fed plants with a buffered pH of 5 had the lowest shoot growth rate at both 1 μM B and 100 μM B levels (Fig. 3A). In contrast to NO3−-fed plants, NH4+-fed plants with a non-buffered pH had the lowest shoot growth rate at both 1 μM B and 100 μM B levels, while plants with a buffered pH (6) had the maximum shoot growth rate (Fig. 3C). Among the NO3−-fed plants, maximum root growth was recorded in non-buffered pH at both 1 μM and 100 μM B levels, whereas the minimum was observed with a buffered pH of 5 (Fig. 3B). However, NH4+-fed plants showed contrasting results to NO3−-fed plants where the minimum root growth rate was found at non-buffered pH (Fig. 3D). The maximum root growth was recorded at pH level 6 for both 1 μM and 100 μM B level (Fig. 3).

Fig. 3
figure 3

Shoot growth rate and root growth rate of NO3− (A, B) and NH4+-fed (C, D) rapeseed plants with low and high levels of boron at buffered and non-buffered pH. Different letters denote significant differences (Tukey’s HSD test; p ≤ 0.05)

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Boron Concentration in Shoots and Roots

In comparison to plants treated with 1 μM B, plants treated 100 μM B, showed significantly increased B concentration in their roots and shoots, regardless of the N form supplied (Fig. 4A, D). The highest B concentration in NO3−-fed plants was observed in the shoots at unbuffered pH, whereas the lowest was reported at a pH of 5 (Fig. 4A). Similarly, in the roots the highest concentration of B was measured at non-buffered pH and the least concentration at a pH of 5 (Fig. 4B). However, NH4+-fed plants followed a different pattern compared to NO3−-fed plants, with the lowest B concentration in shoots reported at non-buffered pH and the highest at buffered pH 6 and 7 (Fig. 4C). Similarly, the B concentration in the roots of NH4+-fed plants was lowest at unbuffered pH, whereas it was highest at pH 6 and 7 for both low and high levels of boron treatments (Fig. 4D). Overall, NO3−-fed plants with or without buffered showed a higher value of B concentration in shoots and roots compared to NH4+-fed with or without buffered plants (Fig. 4).

Fig. 4
figure 4

Concentration of boron in shoot and root of NO3− (A, B) and NH4+-fed (C, D) rapeseed plants with low and high levels of boron at buffered and non-buffered pH. Different letters denote significant differences (Tukey’s HSD test; p ≤ 0.05)

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Specific Boron Uptake Rate and Distribution in Root and Shoot

The specific boron uptake rate of NO3− and NH4+-fed rapeseed plants also varied significantly with 1 μM and 100 μM levels of boron at buffered and unbuffered pH (Fig. 5A, B). NO3−-fed plants treated with 100 μM B showed almost doubled B uptake rate compared to plants treated with 1 μM B. The maximum boron uptake rate was recorded in nonbuffered pH which was statistically equal to pH of 6 and 7. However, the lowest uptake was observed in plants with a pH of 5 (Fig. 5A). In contrast to NO3−-fed plants, NH4+-fed plants with 100 μM of boron at buffered pH 6 and 7 showed a maximum uptake rate, whereas the minimum was reported at unbuffered pH (Fig. 5B). NO3−-fed plants with 100 μM B supply accumulated relatively more B in roots compared to root boron accumulation at 1 μM B supply (Fig. 6A). However, the NH4+-fed plants retained higher B in roots at 1 μM B compared to root B accumulation at 100 μM B supply (Fig. 6B). In addition, the results suggest that among NH4+-fed plants, buffering of pH had a major effect on root and shoot B accumulation at 1 μM B supply, as all buffered pH values from 5 to 7 exhibited significantly higher root B distribution (Fig. 6).

Fig. 5
figure 5

Specific boron uptake rate of NO3− (A) and NH4+-fed (B) rapeseed plants with low and high levels of boron at buffered and unbuffered pH. Different letters denote significant differences (Tukey’s HSD test; p ≤ 0.05)

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Fig. 6
figure 6

Distribution of boron in root and shoot of NO3− (A) and NH4+-fed (B) rapeseed plants with low and high levels of boron at buffered and non-buffered pH. Different letters denote significant differences (Tukey’s HSD test; p ≤ 0.05)

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Nitrate Concentration in Shoots

Compared to NH4+-fed plants, NO3−-fed plants accumulated relatively more NO3− in shoots, at both B supply levels (Fig. 7). NO3−-fed plants accumulated almost 5 times higher NO3− compared to NH4+-fed plants. The maximum NO3− accumulation was found on plants with a pH of 6 which was statistically more or less similar to all other pH levels at both 100 μM and 1 μM low B supply (Fig. 7A). This clearly indicates that buffering of pH has minimum/no impact on NO3− accumulation in shoots. However, among the NH4+-fed plants, maximum accumulation was recorded in plants with a pH of 6 and 7, whereas minimum was found on the unbuffered pH (Fig. 7B).

Fig. 7
figure 7

Concentration of nitrate in the shoot of NO3− (A) and NH4+-fed (B) rapeseed plants with low and high levels of boron at buffered and non-buffered pH. Different letters denote significant differences (Tukey’s HSD test; p ≤ 0.05)

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Relative mRNA Expression of BnaNIP5; 1 and BnaBOR1; 2 in Roots

On an individual set of experimental basis, the relative expression of both BnaNIP5;1 and BnaBOR1;2 were significantly influenced by each B level and constant N sources with buffer state in the root of rapeseed plant (Fig. 8). At the limited supply of B in the root of NO3−-fed plants, relative expression of BnaNIP5;1 and BnaBOR1;2 was higher at both non-buffered and buffered pH except buffered pH 5.0 (Fig. 8A, B). However, buffered pH 7 expressed 3.8-fold higher compared to buffered pH 5 (Fig. 8A, B). Furthermore, at a high B level, no significant change was observed in NO3−-fed plants (Fig. 8A, B). In comparing NH4+-fed plants at buffered and non-buffered pH, the relative expression of BnaNIP5;1 and BnaBOR1;2 was expressed significantly in both supplied B levels (Fig. 8C, D). At a low and high B levels, the relative expression of BnaNIP5;1 and BnaBOR1;2 was increased 2.7, 3.4, 2.43, and 3.34-fold, respectively, at buffered pH 7 compared to non-buffered pH (Fig. 8C, D). Overall, both supplied B levels of NH4+-fed plants with buffering showed a positive affinity in roots for higher expression of both genes compared to NO3−-fed plants (Fig. 8C, D).

Fig. 8
figure 8

The relative expression of BnaNIP5;1 and BnaBOR1;2 in the roots of NO3− (A, B) and NH4+-fed (C, D) rapeseed plants with low and high levels of boron at buffered and non-buffered pH. Different letters denote significant differences (Tukey’s HSD test; p ≤ 0.05)

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Relative mRNA Expression of BnaNIP5; 1 and BnaBOR1; 2 in Shoots

The relative mRNA expression of both BnaNIP5;1 and BnaBOR1;2 was greatly affected by NH4+-fed buffered plants in roots (Fig. 8C, D). Therefore, it was more interesting to observe the expression levels of both genes in shoots for NH4+-fed buffered rather than NO3−-fed buffered plants at low and high B supply (Fig. 9) and the results for shoots showed significant difference between the treatments in NH4+-fed plants (Fig. 9A, B). At low B levels, pH 6.0 and 7.0 showed higher BnaNIP5;1 expression compared to other treatments, where 3.44 and 2.34-fold, respectively (Fig. 9A). However, a higher B level at pH 7 was a 3.1-fold change for the relative expression of BnaNIP5;1, while others had no significant difference with non-buffered pH (Fig. 9A). In shoots, BnaBOR1;2 expressed highly at higher B levels than low (Fig. 9B). In addition, no significant difference was observed at low B levels, but expression levels were 1.38, 1.39, and 1.53-fold higher than non-buffered pH, respectively. At a higher B level at pH 7.0, BnaBOR1;2 2.46-fold highly expressed in the shoots compared to non-buffered pH (Fig. 9B). Overall, On an individual gene basis, the BnaNIP5;1 was expressed more strongly in the most obvious B level in the shoots of NH4+-fed plants with a different buffered pH compared to BnaBOR1;2 (Fig. 9).

Fig. 9
figure 9

The relative expression of BnaNIP5;1 and BnaBOR1;2 in the shoots of NH4+-fed (A, B) rapeseed plants with low and high levels of boron at buffered and non-buffered pH. Different letters denote significant differences (Tukey’s HSD test; p ≤ 0.05)

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Pearson Correlation

A Pearson correlation analysis was performed (Fig. 10) to find out the general relationships among the response variables in NO3−- and NH4+-fed nonbuffered and buffered plants. Growth characters showed the highest correlation among them, and a similar pattern was observed for B content and uptake in shoot and root for both individual sets of experiments, such as NO3− and NH4+-fed plants (Fig. 10). Furthermore, In NO3−-fed buffered plants, relative expression of BnaNIP5;1 and BnaBOR1;2 was strongly correlated; however, root B concentrations were negatively correlated with relative expression of BnaNIP5;1 (− 0.72) and BnaBOR1;2 (− 0.67) but positively correlated with root growth rate (Fig. 10A). In comparison, NH4+-fed buffered plants showed a strong and positive correlation of relative expression of BnaNIP5;1 and BnaBOR1;2 with root B concentration, except specific B uptake rate negatively but moderately correlated (− 0.50) with shoot relative expression of BnaBOR1;2 and positively with shoot B concentration (Fig. 10B).

Fig. 10
figure 10

Correlation analysis among the growth, B concentration, uptake, distribution, and relative expression of BnaNIP5;1 and BnaBOR1;2 of NO3− (A) and NH4+-fed (B) rapeseed plants with low and high levels of boron at buffered and unbuffered pH. Here, SDW shoot dry weight, RDW root dry weight, SGR shoot growth rate, RGR root growth rate, SBC shoot B concentration, RBC root B concentration, SBUR specific B uptake rate, RNIP1;5 relative expression of BnaNIP5;1 in root, RBOR1;2 relative expression of BnaBOR1;2 in root, SNIP1;5 relative expression of BnaNIP5;1 in shoot, SBOR1;2 relative expression of BnaBOR1;2 in shoot

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Discussion

The novelty of this current study is to differentiate the impact of pH and/or sources of N-form from two distinct N-sources at varying buffered pH levels, with both low and high supplies of B. The key distinction lies in the consistent N-sources, while the pH level is adjusted to determine whether the boron uptake is influenced by pH or the sources of N-form.

N-Forms at Buffered pH Regulating B Uptake and Plant Growth

The acidity or alkalinity of the rooting media is of the utmost significance for the formation of dry matter in plants. This is because a significant number of processes, such as the availability of nutrients and their absorption rate, as well as the availability of hazardous ion species, are intimately connected to this parameter (Marschner 2012; Zhao and Ling 2007). Earlier studies already indicated that the primary factor that inhibits plant growth in acidic conditions is pH itself (Kochian et al. 2015), which supports the results of our most recent investigation. Despite this, regardless of the N forms, the lowest amounts of dry matter were found to accumulate in plants with a pH of 5 (buffered) or less than 5 (non-buffered) (Fig. 2). As Pou et al. (2022) reported, nitrogen concentration influenced root hydraulic parameters and regulated the intercellular flow of water by aquaporins. Kapilan et al. (2018) and Sade et al. (2012) showed that the reduction of aquaporin-mediated by higher protonation thus negatively affects the transpiration stream of water, thus altering plant water relations (Wimmer and Eichert 2013).

Different N-forms define the pH of the nutrient solution, and this might affect B uptake and translocation. Similarly, different nitrogen forms had varying effects on the concentrations of B (Fig. 4) and the accumulation of B, thus well in line with the specific B uptake rate (Fig. 5) in rapeseed plants. Our results also illustrated that buffering pH of NH4+-based medium increased the growth rate (Fig. 3C, D) and upregulated transcriptomic expression of nodulin 26-like intrinsic protein 5; 1 (BnaNIP5;1) and BOR1 transporters BnaBOR1;2 (Fig. 8C, D) compared to NO3−-fed plants. Moreover, medium alkalization mediated by nitrate increases root-to-shoot B transport under B limitation by enhancing root expression of BnaBOR1;2 and BnaNIP5;1 (Dinh et al. 2021b; Wang et al. 2022b; Li et al. 2023b). Additionally, in our present study, the highest accumulation of macro- and micronutrients (N, P, K, Mg, Fe, Mn, and Zn) occurred at a buffered pH of 6 and 7, while the lowest was observed at a non-buffered pH (Supplementary Tables 1 and 2) thus supported the findings of Wang et al. (2022a) changes of pH in an acidic environment improved growth status by improving nutrient status of sugar beet plants.

The uptake of NH4+ can lead to an increase in net H+ release, which causes significant intercellular acidification of the roots of rapeseed plants. An excessive amount of hydrogen (H+) ions will be directly harmful to the cell wall structure (Li et al. 2023a; Graças et al. 2021; Zhu et al. 2021). Consequently, reducing root cell elongation led to a reduction of aquaporin function in many plants (Kapilan et al. 2018), thus affecting the plant’s ability to absorb water and vital nutrients such as B constrained, ultimately impairing plant growth (Li et al. 2015; Long et al. 2017). Therefore, the reason that the growth rates of roots (Fig. 3C, D) as impeded in our study when the pH was low could be because the cell death produced by an excessive amount of H+ (Graças et al. 2021; Li et al. 2023a), thus led to lowering hydraulic conductivity in root causes downstream expression of aquaporin (Wang et al. 2016). Our results, in line with the results (Tournaire-Roux et al. 2003; Tornroth-Horsefield et al. 2006), found that under abiotic stress conditions, low cytosolic pH in spinach and Arabidopsis plants caused by higher protonation, thus mediating the reduction of aquaporin function to transport water from root to shoot. Furthermore, Savić et al. (2013) found that probable indications of B shortage in plants include the deformation and brittleness of the leaves, along with growth retardation at the apical meristems of the roots and shoots. The pH of the nutrient solution that contained NH4+ was measured after every 12 h of plant growth, and it was significantly lower than the necessary soil pH values that were reported for canola growth in two different ultisols (Baquy et al. 2017). This result is similar to another study in Arabidopsis by Xiao et al. (2022). It has been demonstrated that the manipulation of external medium pH improves Arabidopsis tolerance to ammonium-induced stress (Britto and Kronzucker 2002; Hachiya et al. 2012; Zeng et al. 2012; Hachiya and Sakakibara 2017). However, Tsadilas et al. (2005) found that NH4+ fertilization did not affect the pH of artificially limed acidic soils. Consequently, the decrease in hot water extractable B and the reduced B accumulation in tobacco plants caused by liming could not be reversed. This highlights the inherent disparities between soil-based and hydroponic investigations. Obviously, when added to soil, lime can buffer the acidic effect of NH4+ uptake by plants before it can affect B uptake.

The most interesting and novel finding of this study is that NO3−-fed plants performed better at non-buffered pH and similar at pH 6 with 7, while NH4+-fed plants prefer buffered pH (6 and 7) for promoting higher root and shoot growth (Fig. 3) which was in line with the B concentration (Fig. 4D). These findings were further supported by the upregulation of BnaNIP5;1 and BnaBOR1;2 in the root (Fig. 8C, D) and higher expression of BnaNIP5;1 in shoots with buffered pH in NH4+-fed plants (Fig. 9A) (with the exception of BnaBOR1;2 in shoots, Fig. 9B). As expected, in our investigation, we also showed that the growth of NH4+-fed plants was better when cultivated at buffered pH of 6 and 7 compared to non-buffered pH (Fig. 11). Improving the pH of media also increased the internal circulation of B in plants under different B treatments. More importantly, rising pH and B addition balances the quantity of lignin and cellulose in the cell wall to preserve the normal growth of the vascular bundle (Cheng et al. 2022). Taken together, we firstly proved that the rise of pH through buffering can stimulate plant growth through accelerating B re-distribution to roots and shoots.

Fig. 11
figure 11

Schematic representation of the genetic mechanism of rapeseed with clear pH dependence in the growth of NH4+-fed rapeseed plants at buffered and unbuffered pH

Full size image

Levels of Boron Mediating B Uptake and Plant Growth

Different levels of B supply confirm the conditions of low or adequate, or high B availability for the plants, possibly also affecting the uptake and translocation of B in the plants, particularly in oilseed rape. There was lowest B level (1 µM) “just-sufficient” growth support for plants in the 5 days following treatment application. Treatment with high levels of B, or “high B (100 μM B),” means that the amount of B available was above the optimum level but below the toxic one. Our results showed that at the 100 μM B level, root and shoot dry biomass were substantially higher than those recorded at the 1 μM B level, indicating that a concentration of 1 μM B may cause B deficiency inhibiting optimum growth in rapeseed (Fig. 2). At the 100 μM B level, the accumulation of B by the rapeseed roots and shoots was substantially higher than those recorded at the 1 μM B level (Fig. 4) confirming previous findings of positive correlation between the external supply of B and its concentration in plant tissues (Eggert and von Wirén 2016; Koohkan and Maftoun 2016; Dinh et al. 2022).

Depending on the amount of supplied B different nitrogen forms had varying effects on the concentrations of B and the accumulation of B in rapeseed plants. As compared to NH4+-fed plants, we found NO3−-fed plants accumulated considerably more B in their roots and shoots at both levels of B supply (Fig. 4); however, in this study, we found that buffering pH greatly influenced the distribution of B from roots to shoots (Fig. 6 B) and more distribution of root at low supply could improve root growth rates in NH4+-fed buffered plants. Similar findings were observed by Carr et al. (2020) for coffee and Dinh et al. (2021b) for oilseed rape; on the other hand, Cheng et al. (2022) documented that changed in pH from an acidic environment changed the distribution of B, thus promote cell wall synthesis in citrus plants. NO3−-fed plants treated with higher B levels showed almost doubled B uptake rates compared to plants treated with lower B levels (Fig. 5A). In contrast to NO3−-fed plants, NH4+-fed rapeseed plants showed maximum uptake rate at buffered pH 6 and 7 with high levels of B, whereas the minimum was reported at pH 5 and non-buffered pH with low levels of B (Fig. 5B). Nevertheless, our presented gene expression results related to B uptake and translocation under limited B supply showed upregulation trends of roots in both constant N-sources with buffered pH (Fig. 8A, D) and shoots in NH4+-fed plants with buffered pH (Fig. 9A, B).

It is known that the channel protein NIP and transporters belong to the BOR families that facilitate and activate B transport under low B conditions (Brown et al. 2002; Miwa and Fujiwara 2010). However, under low B supply, both genes regulate B uptake differently. The BOR1 transport mechanism involves B loading into the xylem, and regulation depends on external B availability, whereas NIP5-1 transports boric acid into the cell via a concentration gradient (Takano et al. 2005). The BnaNIP5;1 and BnaBOR1;2 genes are widely expressed and play a critical role in the uptake and translocation of boron in rapeseed regardless of boron supply (Dinh et al. 2021b; Feng et al. 2019), under limited B supply BnaNIP5;1 express strongly (Dinh et al. 2021a, b) which in line with our present investigation the relative expression of channel protein BnaNIP5;1 in shoots was highly upregulated in NH4+-fed plants with buffered pH (Fig. 9A, B). However, at high B level NO3− restricted upregulation of B transporter (Dinh et al. 2021b), a possible previous study showed that condition B was strongly bound with the cell wall (with positive change), making a stabilized B-complex by divalent cations (O'Neill et al. 2004; Ishii et al. 2002), such results shows that why both genes expression was not upregulated under high supply of our study in NO3− -fed buffered and unbuffered plant (Figs. 7, 8A, B).

The results suggest that buffering of pH had a major effect on root and shoot B concentration of the NH4+-fed plants at lower B supply (Fig. 4). Our results confirm the hypothesis of Wimmer and Eichert (2013) that the transpiration stream strongly induces B dispersion in oilseed rape, which is denoted by the accumulation of B. These observations are consistent with the findings of Dinh et al. (2021b), Koohkan and Maftoun (2016) and as well as Masood et al. (2023). All three groups confirmed that B concentrations in plant parts are directly proportional to the external B concentration. However, the situation was different in the case of B distribution, where NH4+-fed plants retained higher B in roots only at lower B supply (Fig. 6B).

The decrease in the amount of root B under high B supply can be partially explained by the lower dry matter, which most likely occurred as a result of the damaging effects of acidity (Fig. 11). At high B supply, where B is mostly taken up by passive diffusion (Wimmer and Eichert 2013), reduced B accumulation may be considered to be connected with lower transpiration rates. However, in conditions of low pH, B accumulation was found to be significantly reduced. This clearly demonstrated that it was not only due to the decrease in transpiration but also related to the decrease in pH, which restricted the plants’ ability to uptake B. Besides, the level of B supply might have a substantial impact on such anion uptake.

Conclusion

This is the first study that thoroughly documents the benefits of stabilizing pH at constant N-sources, for the uptake of B and growth under hydroponic cultivation of B loving rapeseed. The novelty of the present work is buffering positively heightens B uptake and growth in NH4+-fed plants. The plants fed with NO3− thrived at non-buffered pH, while plants fed with NH4+ thrived at buffered pH for boosting B absorption and distribution, dry matter buildup in roots and shoots, and increased growth. Meanwhile, independent of the B amount administered, NH4+-fed plants benefited from pH buffering by upregulation of the B transport channel protein BnaNIP5;1 in both roots and shoots and BnaBOR1;2 in the root in rapeseed. Here, we could show for the first time that plant growth and uptake of boron correlate positively with NH4+-based nutrition when the pH is stabilized, whereas the opposite or unaffected was found in NO3−-fed plants. NO3−-fed plants showed a higher value of B accumulation in roots and shoots compared to NH4+-fed plants. Overall, the current study’s findings show that adjusting pH with buffers in hydroponic nutrient solutions is critical in ensuring that ammonium fertilization can increase boron uptake. We believe this study would significantly expand knowledge on this topic to understand N-forms (without pH dependence) on B nutrition during vegetative growth stages in plants when N and B are dominantly required.