1 Introduction

Biochar application has been introduced and utilized due to its potential to enhance soil properties and plant productivity during drought stress (Joseph et al. 2021). In general, biochar serves as a soil conditioner, enhancing soil water retention, nutrient availability, and carbon sequestration (Peng et al. 2011). A 2.5% biochar application rate increases plant-available phosphorus (P) and potassium (K) contents by approximately 10% and 80%, respectively, due to biochar ash input or biochar-activated soil nutrient release (Liu et al. 2018). Besides, biochar has the potential to increase soil cation exchange capacity through its organic groups and specific surface area, facilitating the sorption of salts in alkaline soils (Akhtar et al. 2015; Limwikran et al. 2018). Numerous studies have demonstrated that these effects of biochar amendment eventually improve root system development and crop productivity (Viger et al. 2015; Xiang et al. 2017). It remains of great concern to understand how biochar application enhances plant functioning under drought stress.

The application of biochar may effectively improve plant water status and water-use efficiency during drought stress (Ali et al. 2017). It is suggested that additional K+ supply in biochar-amended soils directly contributes to osmotic protection, preventing water loss in plant tissues (Viger et al. 2015). Akhtar et al. (2015) reported that the increased K+ concentration in the xylem due to biochar application alleviated osmotic stress on potato, as plants likely used biochar’s K for osmotic adjustment and maintenance of turgor pressure. However, the large number of cations from biochar application could inhibit plant growth under stress conditions (Gascó et al. 2016; Joseph et al. 2021). Geilfus (2017) found that an increase in the cation/anion ratio led to an increase in apoplastic pH, which, in turn, caused stomatal closure and inhibited root growth during drought stress. Sun et al. (2017) observed that biochar application up to a 30% dose highly inhibited lateral root formation due to changes in soil chemical properties under biochar addition. Both positive and negative connections have previously been mentioned between the modification of soil properties by biochar application and the plants themselves during drought stress, warranting further investigation into plant physiological status and metabolic processes to explain the underlying mechanisms.

Biochar’s K may serve as a drought stress signal within plants, thereby activating plant responses to drought stress (Viger et al. 2015). Previous studies indicated that external K+ application resulted in the accumulation of compatible solutes (e.g., proline and soluble sugars) during stress (Chakraborty et al. 2016), contributing significantly to osmotic protection. Therefore, biochar application could change the osmoregulators in maize to adapt to drought stress (Cong et al. 2023). However, some studies have reported that biochar application reduced osmotically active solutes or antioxidant enzyme activities, thereby lessening the stress defense of plants (Mehdizadeh et al. 2019; Viger et al. 2015). This could be attributed to the fact that biochar application has the potential to increase plant-available water content, thereby enhancing soil water retention under drought stress, resulting in lower stress levels and reduced stress damage (Baronti et al. 2014). Yet, the exact role of biochar application in plant drought response remains largely elusive.

Additionally, plant genotypes may determine whether growth inhibition is induced by biochar application. For example, biochar application was more effective in improving plant growth for stress-tolerant plants, while it had no or adverse effects on plant growth for stress-sensitive plants (Liu et al. 2022a; Zhang et al. 2019). Plants have evolved various physiological mechanisms to survive under drought stress. For sensitive plants, growth inhibition has the potential to benefit by protecting plants from stress conditions (Kim et al. 2014). Since stomatal closure is effective in preserving water in plants and root growth inhibition may decrease water consumption (Geilfus 2017), inhibiting growth during drought periods could conserve more water in the soil for later use during the growing season (Bechtold and Field 2018). For tolerant plants, the stress response may not come at the cost of reduced growth. For instance, the overexpression of components of the circadian clock could confer drought tolerance without penalizing plant growth (Grundy et al. 2015).

Therefore, in this study, two maize genotypes with contrasting drought tolerance, drought-sensitive KN5585 and -tolerant Mo17, were selected as experimental materials to investigate biochar–soil ion exchange, root ion concentrations, apoplastic pH regulation, metabolic signatures, transcriptomic changes, and physiological traits under drought stress. The objective of this study was to analyze the responses of distinct maize genotypes to biochar application and to elucidate the regulatory mechanisms of biochar application on plant drought response. It was hypothesized that biochar application could enhance drought tolerance in two maize genotypes, attributed to osmotic protection and root growth regulation during drought stress.

2 Materials and methods

2.1 Soil and biochar materials

A fluvo-aquic soil (Soil Survey Staff 2015) was used in the experiment, comprising 70.7% sand, 18.1% silt, and 11.3% clay, with a pH of 8.3, an organic C content of 0.9%, and a field capacity of 0.25 g g–1. The soil was collected from the 0–30 cm layer at the Experimental Station (40°15′N, 116°55′E) of the Chinese Academy of Agricultural Sciences located in Shunyi District, Beijing, China. This area experiences an average annual temperature of 12.5 °C and an average annual precipitation of 623.5 mm. The soil samples were air-dried and sieved through a 5-mm mesh. Biochar was produced from maize straw in a sealed oven at 400 °C for 4 h, with the size range of 0.5–5.0 mm, pH of 8.7, total C content of 549.5 g kg–1, total N content of 9.5 g kg–1, total K content of 29.4 g kg–1, total Ca content of 33.1 g kg–1, and total Na content of 6.4 g kg–1.

2.2 Plant material and growth conditions

The drought-sensitive inbred line (KN5585) and the drought-tolerant inbred line (Mo17) were selected, and the drought tolerance of these two maize genotypes was determined as described in Fig. S1. Maize plants were cultivated in soils with and without biochar. Biochar was applied at rates equivalent to field applications of 0 t ha–1 (designated as the Soil treatment) and 100 t ha–1 (designated as the Soil+Biochar treatment), assuming a soil bulk density of 1.20 g cm–3 to a depth of 30 cm, achieved by mixing soil with 0% and 2.8% biochar. Seeds were germinated on moist, and sterilized filter papers for 2 days in the dark at 28 °C. Following uniform germination, seedlings were transplanted into PVC pots (6.0 cm in diameter × 12.0 cm in height), and placed in a growth chamber, with a 16 h photoperiod at 1000 μmol m–2 s–1 light intensity, 60% relative humidity, and 28 °C temperature during the day, and 50% relative humidity, and 22 °C temperature at night. The soil water content was maintained at 0.16 g g–1 (Fig. S2, period 1, approximately 60% field capacity) daily without the application of any fertilizers. Each treatment consisted of 20 plants per genotype. Following a 10-day growth period, drought stress was initiated by withholding water for 4 days until the soil water content decreased to 0.07 g g–1 (Fig. S2, period 2, approximately 30% field capacity). Soil and plant samples were collected after 4-day drought stress.

2.3 Soil exchangeable cation analyses

Soil pH, electrical conductivity (EC), and exchangeable cations were measured in quadruplicate for soils with or without biochar. Soil pH and EC were analyzed in an aqueous solution suspension with a 1:2.5 (soil: water) ratio. Exchangeable cations were extracted using 0.1 mol L–1 NH4Cl in a 70% C2H5OH solution. The exchangeable Ca2+, Mg2+, K+, and Na+ contents were measured using an atomic absorption spectrometer (iCE 3500, Thermo Fisher Scientific, Waltham, MA, USA).

2.4 Osmolality, K+ and Ca2+ quantifications

Following drought stress, leaf sap osmolality, sap K+ and Ca2+ concentrations were measured using six plants as individual biological replicates. Leaf sap was extracted using a centrifugation method from the leaves that were frozen and then thawed. Thawed leaves were placed in 5 ml microcentrifuge tubes with a basal opening, allowing cell sap but not fragments to enter the collectors. Then the collected cell sap was further centrifuged at 10,000 × g for 3 min. The osmolality was determined using a vapor pressure osmometer (model 5520, Wescor Inc., Logan, UT, USA). The K+ and Ca2+ concentrations in cell sap were measured using an atomic absorption spectrometer (iCE 3500, Thermo Fisher Scientific, Waltham, MA, USA).

2.5 Root growth and apoplastic pH measurements

Following drought stress, six biological replicates, consisting of 10 root segments, were carefully extracted by washing the soil to preserve the integrity of the root tips for microscopic imaging (Eclipse Ci, Nikon Instruments Inc., Tokyo, Japan). The lengths of root extension zone, defined as the distance from the root apex to the beginning of the root hair (Han et al. 2015), were analyzed using the Fiji software (https://fiji.sc/).

Net flux of H+ was measured using noninvasive micro-test technology (NMT, YoungerUSA LLC, Amherst, MA, USA). Six biological replicates of roots were washed with running Milli-Q water, and then horizontally immobilized in a 5 ml measuring plate filled with a basic salt solution (0.1 mM KCl, 0.1 mM CaCl2, 0.1 mM MgCl2, 0.5 mM NaCl, 0.3 mM MES, 0.2 mM Na2SO4, pH 6.0) for 30 min. Measurements were recorded from root epidermal cells at the root cap, meristematic zone, elongation zone, and mature zone for each genotype. Net H+ flux was calculated by NMT software based on the measured differences in the electrochemical gradient between the two positions using cylindrical diffusion geometry (Chen et al. 2015). Steady-state results were recorded for 5 min to ensure that the steady-state condition was reached. The sign convention considered efflux as positive.

The apoplastic pH was determined using the HPTS (8-hydroxypyrene-1,3,6-trisulfonic acid, AAT Bioquest) staining method. Six biological replicates, consisting of 18 root tips, were transferred to half-strength Murashige and Skoog (1/2 MS) liquid growth medium containing 1 mM HPTS for 20–30 min. Imaging of mature zone epidermal cells in the root tips was performed on a Zeiss LSM 700 confocal microscope (Zeiss LSM 700 META, Jena, Germany). Fluorescent signals for the protonated HPTS form (excitation 405 nm) and the deprotonated HPTS form (excitation 488 nm) were detected under a × 20/0.8 NA air objective with the same emission peak at 514 nm (Li et al. 2021). The images were analyzed using the Fiji software (https://fiji.sc/) and the relative pH was calculated as the background-subtracted intensity of the deprotonated dye divided by that of the protonated dye.

The activity of the root plasma membrane (PM) H+-ATPase was determined using an enzyme-linked immunosorbent assay kit (LabRe Bio Co., Wuhan, China). Entire root samples from four biological replicates were ground using a frozen tissue grinder (MM400, Retsch Technology, Haan, Germany). A 10% tissue homogenate was prepared according to the manufacturer’s recommendations. Each sample of 10 μl was loaded onto a polystyrene microtiter plate that was coated beforehand with purified plant PM H+-ATPase antibody. The yellow color was measured at 450 nm in a microplate reader (Infinite F50, Tecan Group Ltd., Männedorf, Switzerland).

2.6 Metabolite profiling analyses

A total of four biological replicates from the roots subjected to drought stress were harvested and frozen in liquid nitrogen. Entire root samples were ground using a frozen tissue grinder (MM400, Retsch Technology, Haan, Germany). Root samples (50 mg) were mixed with 400 μl of 80% methanol premixed with L-2-chlorophenylalanine (0.02 mg mL–1) and subsequently homogenized. The samples were centrifuged for 15 min at 13,000 g (4 °C) and the resulting supernatant was transferred into fresh tubes. An ExionLCTMAD system (AB Sciex, Framingham, Massachusetts, USA) equipped with an ACQUITY UPLC BEH C18 column (100 mm × 2.1 mm i.d., 1.7 µm; Waters, Milford, USA) was used to perform chromatographic separation of the metabolites.

The original data were imported into the Progenesis QI 2.3 software (Nonlinear Dynamics, Waters, USA) to detect and align peaks after UPLC-TOF/MS analyses. The metabolite identification was based on reliable biochemical databases (Human Metabolome Database (HMDB) (http://www.hmdb.ca/) and the KEGG database (http://www.genome.jp/kegg/) and should meet a mass error within 10 ppm and MS/MS spectra with high scores. We applied median sample normalization and log data transformation and filtered features if their relative standard deviations (RSDs) were greater than 30% in QC samples. The R package from Bioconductor on the Majorbio Cloud Platform (https://cloud.majorbio.com) was used to conduct a multivariate statistical analysis. The metabolites with VIP (variable importance in the projection) value in orthogonal partial least squares discriminate analysis greater than 1 and a P value less than 0.05 were considered as the significant metabolites (Soil+Biochar vs. Soil, for each genotype).

2.7 Transcriptomic profiling analyses

For transcriptome sequencing, four biological replicates from the entire roots subjected to drought stress were ground as described above. RNA was extracted from root samples (100 mg) using Plant RNA Purification Reagent (Invitrogen) according to the manufacturer’s instructions (Thermo Fisher, Waltham, MA, USA). The NanoDrop2000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA) and the Agilent 2100 Nano bioanalyzer (Agilent Technologies, Santa Clara, CA, USA) were used to analyze the integrity and quality of total RNA. cDNA synthesis was prepared with the Truseq™ RNA Sample Prep Kit (Illumina, San Diego, CA, USA). The samples were sequenced at Shanghai Majorbio Bio-Pharm Technology Co., Ltd (Shanghai, China) using the Illumina Novaseq 6000 platform.

The clean reads obtained for root samples of KN5585 and Mo17 were respectively mapped to two maize reference genomes. The results of reads are shown in Table S2, and the accuracy and quality of the data mapping efficiency were sufficient for further analysis. The data were analyzed on the free online platform of the Majorbio Cloud Platform (https://cloud.majorbio.com/). Differentially expressed genes were filtered for adjusted P value ≤ 0.05 (adjusted by Benjamini–Hochberg correction) and log2FC >|1|. Goatools (https://github.com/tanghaibao/Goatools) was used to carry out gene ontology (GO) functional enrichment analysis. Statistical values of the enrichment were obtained using a Fisher’s test (adjusted P value ≤ 0.05, Benjamini–Hochberg correction).

2.8 Physiological parameters

Fresh leaves and roots were harvested after drought stress and weighed with eight biological replicates. The leaf and root water potentials were measured using a dewpoint potentiometer (WP4C, Decagon Devices Inc., Pullman, WA, USA). Assay kits (http://geruisi-bio.com) were used to determine the O2– content, H2O2 content, malondialdehyde content (MDA), total antioxidant capacity (T-AOC), superoxide dismutase activity (SOD), catalase activity (CAT), and peroxidase activity (POD) of the root samples.

2.9 Omics data integration and statistical methods

The Kyoto Encyclopedia of Genes and Genomes (KEGG) database was used to link transcriptomic changes in the metabolic signature to metabolic pathways. Significant enrichment in the pathway was considered when the adjusted P value was ≤ 0.05 (adjusted by Benjamini–Hochberg correction). All the differences between biochar application treatments were evaluated with a t-test (Origin 2022).

3 Results

3.1 Biochar effect on ion concentrations and osmolality under drought stress

In the alkaline soil (initial pH of 8.3), soil pH significantly decreased (Table 1, P < 0.01) in the Soil+Biochar treatment compared to the Soil treatment. Furthermore, the decrease in soil pH corresponded to a significant reduction in soil exchangeable Na+ and Ca2+ contents in the Soil+Biochar treatment compared to the Soil treatment (P < 0.05). However, the Soil+Biochar treatment resulted in a significantly higher soil exchangeable K+ content (2.7-fold) and soil electrical conductivity (4.0-fold) compared to the Soil treatment (P < 0.01).

Table 1 Effect of biochar application on soil pH, electrical conductivity and exchangeable cations
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Sap K+ concentration in the leaves under drought stress significantly increased with the Soil+Biochar treatment by 1.7 and 3.1 folds for KN5585 and Mo17, respectively, compared to the Soil treatment (Fig. 1b, P < 0.01). Nonetheless, sap Ca2+ concentration in the leaves under drought stress significantly decreased with the Soil+Biochar treatment for both KN5585 and Mo17 compared to the Soil treatment (Fig. 1c, P < 0.05). Leaf osmolality considerably increased in the Soil+Biochar treatment under drought stress for Mo17 compared to the Soil treatment (Fig. 1a, P < 0.05), whereas it remained similar between the Soil and Soil+Biochar treatments for KN5585.

Fig. 1
figure 1

Effect of biochar application on sap osmolality (a), K+ (b), and Ca2+ (c) concentrations in the leaves of two maize genotypes under drought stress. Standard error was presented for each treatment (n = 6). Asterisks indicate significant differences between the biochar and no biochar treatments (*, P < 0.05; **, P < 0.01)

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3.2 Biochar effect on root growth and apoplastic pH under drought stress

As shown in the images of root tips of the two maize genotypes (Fig. 2), the Soil+Biochar treatment inhibited root elongation for KN5585 under drought stress compared to Mo17. The length of the root extensible region was significantly reduced by 30.7% in the Soil+Biochar treatment for KN5585 under drought stress compared to the Soil treatment (Fig. 3c, P < 0.05).

Fig. 2
figure 2

Representative images of root tips of two maize genotypes subjected to biochar application under drought stress. Red line segments indicate the length of root extensible region that is defined from the root apex to the beginning of the root hair

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Fig. 3
figure 3

Effect of biochar application on apoplastic pH regulation in the roots of two maize genotypes under drought stress. a Relative apoplastic pH of epidermal cells in the root mature zone (n = 18). b PM H+-ATPase activity of entire root samples (n = 4). c Length of root extensible region (n = 10). d and e Net H.+ steady-state fluxes of KN5585 and Mo17 roots, respectively (n = 6). f Model for putative pathway of apoplastic pH regulation for KN5585. Boxplot depicts the range from maximum to minimum and points experimental observations. Asterisks indicate significant differences between the biochar and no biochar treatments (*, P < 0.05; **, P < 0.01)

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A significant increase in the root relative apoplastic pH was observed in the Soil+Biochar treatment for KN5585 under drought stress compared to the Soil treatment (Fig. 3a, P < 0.05). In contrast, there was no difference in apoplastic pH between the Soil and Soil+Biochar treatments for Mo17 under drought stress. The net H+ flux dynamics of maize roots under drought stress were strongly correlated with changes in apoplastic pH (Fig. 3d, e). The results showed that, for KN5585 under drought stress, the Soil+Biochar treatment significantly increased H+ efflux in the root elongation zone but decreased H+ efflux in the root meristematic zone compared to the Soil treatment (P < 0.05); however, no differences in H+ fluxes were found between the Soil and Soil+Biochar treatments for Mo17 under drought stress. Similarly, PM H+-ATPase activity in KN5585 roots under drought stress was significantly activated in the Soil+Biochar treatment compared to the Soil treatment (Fig. 3b, P < 0.05). However, the Soil+Biochar treatment had no significant effect on PM H+-ATPase activity in Mo17 roots under drought stress compared to the Soil treatment.

3.3 Biochar effects on root metabolic signature, transcriptomic and physiological parameters under drought stress

A total of 45 and 52 significant metabolites (Table 2) were identified in KN5585 and Mo17 roots, respectively, based on variable importance in projection (VIP > 1, P < 0.05). For KN5585 roots under drought stress, Fig. 4a illustrates two clusters of metabolites. The first cluster showed metabolites significantly increased in the Soil+Biochar treatment compared to the Soil treatment, including norepinephrine (class: phenols), cGMP (3′,5′-cyclic guanosine monophosphate, class: purine nucleotides), galactaric acid (class: organic oxygen compounds), and inulobiose (class: organic oxygen compounds). The second cluster in Fig. 4a depicts the metabolites significantly decreased in the Soil+Biochar treatment compared to the Soil treatment, including sucrose (class: organic oxygen compounds) and glucosamine 6-sulfate (class: organic oxygen compounds). In contrast, half of all metabolites whose concentration increased in the Soil+Biochar treatment for Mo17 under drought stress (Fig. 4b, the second cluster) were identical to those in KN5585, including galactaric acid and inulobiose. Unique metabolites such as isocitric acid (class: carboxylic acids and derivatives), gamma-glutamylcysteine (class: carboxylic acids and derivatives), and trehalose (class: organic oxygen compounds) were observed in Mo17 compared to the Soil treatment. Thirteen of thirty-two metabolites whose concentration decreased in the Soil+Biochar treatment for Mo17 under drought stress (Fig. 4b, the first cluster) were identical to those in KN5585, including sucrose and glucosamine 6-sulfate.

Table 2 The number of significant metabolites and differentially expressed genes for the comparisons between Soil+Biochar and Soil treatment
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Fig. 4
figure 4

Effect of biochar application on metabolic signature in the roots of two maize genotypes under drought stress. a and b Heatmap illustrating levels of significant metabolites for KN5585 and Mo17, respectively (the Soil+Biochar treatment vs. the Soil treatment). Significant metabolites were selected with variable importance in the projection (VIP) value greater than 1 and P value less than 0.05 (n = 4)

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The transcriptomic changes (Table 2) revealed 1475 differentially expressed genes in the Soil+Biochar treatment in KN5585 roots under drought stress (259 up-regulated and 1216 down-regulated; adjusted P value ≤ 0.05 and log2FC >|1|; supplementary data 1), and 36 differentially expressed genes in the Soil+Biochar treatment in Mo17 roots under drought stress (26 up-regulated and 10 down-regulated; supplementary data 2) compared to the Soil treatment. GO-enrichment analysis showed no significant enrichment in the type of biological processes for up-regulated genes in KN5585 roots under drought stress, and similarly, no significant enrichment was found in the type of biological processes for all differentially expressed genes in Mo17 roots under drought stress. Significant enrichment in several GO terms (Fig. 5; term type: biological processes) was observed only among genes down-regulated in the Soil+Biochar treatment for KN5585 roots, including regulations of responses to stress, metabolic process, biological process, water deprivation, and water and inorganic substances.

Fig. 5
figure 5

Most representative GO terms within the type of biological processes enriched in KN5585 roots from differentially expressed genes subjected to biochar application under drought stress. The enrichments within the type of biological processes were filtered with adjusted P value ≤ 0.05 (adjusted by Benjamini–Hochberg correction, n = 4)

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The input of all differentially expressed genes and significant metabolites into the KEGG pathway identified four and two significantly enriched pathways for KN5585 and Mo17 roots, respectively (Fig. 6a, b, adjusted P value ≤ 0.05). In KN5585 roots under drought stress, the MAPK (mitogen-activated protein kinase) signaling pathway was significantly identified by several down-regulated genes in the Soil+Biochar treatment, with MAPKKK17 and WRKYs being the most notable (Fig. 6c and supplementary data 4); the significant enrichment in plant-pathogen interaction was primarily attributed to the down-regulated CBP, CMLs (calmodulin-like genes), CPKs (calcium dependent protein kinase genes), KCSs (3-ketoacyl-CoA synthase genes), and WRKYs in the Soil+Biochar treatment (Fig. 6e and supplementary data 5); plant hormone signal transduction was significantly enriched by the down-regulated genes in the Soil+Biochar treatment, including TIFYs, SAURs (small auxin-up RNA genes), XTHs (xyloglucan endotransglucosylase/hydrolase genes), and PP2Cs (type 2 C protein phosphatase genes) (Fig. 6g and supplementary data 6); and  phenylpropanoid biosynthesis was enriched by the down-regulated genes in the Soil+Biochar treatment (mainly PERs, POD-encoding genes) and up-regulated genes in the Soil+Biochar treatment (including PRX74) (Fig. 6h and supplementary data 7). In Mo17 roots under drought stress, starch and sucrose metabolism was significantly identified with a decrease in sucrose concentration but an increase in trehalose concentration in the Soil+Biochar treatment (Fig. 6d); and circadian rhythm was significantly enriched by up-regulated LHY (late elongated hypocotyl) and PRR73 (pseudo-response regulator 37) in the Soil+Biochar treatment (Fig. 6f and supplementary data 8).

Fig. 6
figure 6

Integrative enrichment analyses of metabolic signature and transcriptomic changes in the roots of two maize genotypes subjected to biochar application under drought stress. a and b KEGG pathways enriched in KN5585 and Mo17 roots from significant metabolites and differentially expressed genes, which were obtained with adjusted P value ≤ 0.05 (adjusted by Benjamini–Hochberg correction, n = 4). c–h The metabolites and genes that were involved in the pathways regulated in KN5585 and Mo17 roots. Gray color represents down-regulation (the Soil+Biochar treatment vs. the Soil treatment); yellow color denotes up-regulation (the Soil+Biochar treatment vs. the Soil treatment)

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The Soil+Biochar treatment exerted a greater influence on the physiological activities of KN5585 than Mo17 under drought stress compared to the Soil treatment (Table 3 and Fig. S3). Root O2– and H2O2 contents, total antioxidant capacity, as well as SOD and POD activities under drought stress were all decreased in the Soil+Biochar treatment compared to the Soil treatment for KN5585 (P < 0.05). Nevertheless, leaf fresh weight and root water potential under drought stress were increased in the Soil+Biochar treatment compared to the Soil treatment for KN5585 (P < 0.05). Leaf and root fresh weight under drought stress significantly increased by the Soil+Biochar treatment for Mo17 compared to the Soil treatment (P < 0.05).

Table 3 Effect of biochar application on plant physiological parameters of two maize genotypes under drought stress
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4 Discussion

4.1 Soil property variations as affected by biochar application

Previous studies have indicated that biochar contains a significant amount of water-soluble K readily released from biochar, while the release of Ca from biochar is highly pH-dependent, which is not observed in alkaline soil (Silber et al. (2010). The substantial release of K+ to the soil from biochar displaced sufficient soil exchangeable Na+ and Ca2+ contents, which could subsequently be sorbed by biochar (Limwikran et al. 2018). In line with previous studies, we observed that biochar application increased soil exchangeable K+ content while decreasing soil exchangeable Na+ and Ca2+ contents in the alkaline soil (Table 1). The decrease in soil exchangeable Na+ content, along with an increase in cation exchange capacity due to biochar application, contributed to lowering soil pH and enhancing its buffering capability (Saifullah et al. 2018). Nonetheless, it is noteworthy that a significant increase in soil EC was observed after biochar application, indicating the accumulation of more soluble salts, mainly K+ (Table 1), in the alkaline soil. Due to the accumulation of excessive soluble salts, biochar application may inhibit germination and seedling growth (Joseph et al. 2021).

4.2 Root apoplast alkalinization as induced by biochar application under drought stress

The rise in soil K+ content led to an increase in leaf sap K+ concentration due to biochar application under drought stress for two maize genotypes (Fig. 1). Furthermore, Felle et al. (2005) found that apoplastic pH increased with higher K+ concentration. Geilfus (2017) explained that high cation/anion ratios within xylem sap induced an increase in its pH. Consequently, the provision of K+ by biochar increased root apoplastic pH for KN5585 under drought stress (Fig. 3).

We observed that biochar application increased the accumulation of cGMP in KN5585 roots under drought stress (Fig. 4), which was generally reported to stimulate H+-ATPase activity (Li et al. 2014). Additionally, a decrease in sucrose concentration or an enhanced sucrose utilization could serve as an energy source for the activation of H+-ATPase and H+ efflux for KN5585 roots (Viger et al. 2015). Consequently, H+-ATPase activity and H+ fluxes were altered with biochar application for KN5585 under drought stress (Fig. 3f). The activation of root H+-ATPase, along with the increase in root H+ efflux in the elongation zone, played a role in counteracting apoplast alkalinization (Li et al. 2021). This indicated that the K+ from biochar intensified the regulation of apoplastic pH in drought-sensitive KN5585 during drought stress.

In contrast, apoplastic pH under drought stress for Mo17 was not significantly affected by biochar application. Unlike KN5585, the H+-ATPase activity for Mo17 was relatively higher in the absence of biochar application (Fig. 3). This higher H+-ATPase activity might be relatively sufficient for maintaining apoplastic pH balance in response to K+ supply from the biochar (Wakeel et al. 2011), indicating that biochar application had no effect on the regulation of apoplastic pH for drought-tolerant Mo17.

4.3 Root growth inhibition as induced by biochar application under drought stress

According to classical acid growth theory, apoplast alkalinization is proposed to inhibit cell wall expansion or cell division (Geilfus 2017). For example, increased apoplastic pH significantly decreased primary root elongation and total root length (Li et al. 2021), and this is consistent with the reduced root elongation observed under biochar application for KN5585 (Fig. 3). Meanwhile, genes associated with cell-wall extension and root development, such as XTHs and SAURs, were down-regulated by biochar application for KN5585 (Liu et al. 2022b; Yang et al. 2013). The results demonstrated that biochar application significantly inhibited root growth for sensitive KN5585 under drought stress due to an increase in root apoplastic pH.

Under drought stress, plants may not be capable of acquiring water through their roots. Sensitive plants would limit root growth to conserve water in the leaves (Rowe et al. 2016). With the application of biochar, the inhibited root growth contributed to reducing water consumption and stress damage for KN5585 during drought periods. In consequence, biochar application increased root water potential and leaf fresh weight, resulting in decreased root O2– and H2O2 contents, total antioxidant capacity, as well as SOD, and POD activities for KN5585 (Table 3).

Consistently, the response to stress, response to water deprivation, and response to water were all down-regulated by biochar application for KN5585 (Fig. 5). Specifically, biochar application down-regulated the genes of MAPKKK17, WRKYs, CMLs, CPKs, KCSs, TIFYs, and PERs for KN5585 (Fig. 6), which were involved in essential signaling and regulatory components for drought stress resistance (Heidari et al. 2021; Li et al. 2017; Ma et al. 2021; Tong et al. 2021; Zhang et al. 2020). These down-regulated genes suggested a reduced level of stress response in KN5585 with the inhibited root growth due to biochar application.

4.4 Plant osmotic protection as induced by biochar application under drought stress

K+ plays indispensable roles in membrane potentials and osmoregulation during drought stress (Zeng et al. 2014). The increases in sap K+ concentration in both two maize genotypes with biochar application likely enhanced plant drought tolerance by increasing osmolality. However, leaf sap osmolality was only enhanced by biochar application in Mo17, not in KN5585, indicating that K+ concentration may not directly contribute to osmoregulation during drought stress.

Mo17 roots accumulated trehalose with biochar application under drought stress (Fig. 4), alongside a decrease in sucrose concentration. Trehalose acts as a protector for proteins and membranes in drought-tolerant plants, serving as a compatible solute for osmoregulation and supporting plant survival under drought stress (Nuccio et al. 2015). Despite the lack of knowledge regarding the relationship between biochar application and trehalose accumulation, it could be assumed that the high sap K+ concentration with biochar application stimulated the biosynthesis of trehalose, given the significant response of trehalose to salt content in plants (Fernandez et al. 2010). Besides, the phosphorylated intermediate of trehalose, trehalose 6-phosphate, is essential for sucrose utilization (Lunn et al. 2014). Upregulation of trehalose 6-phosphate biosynthesis stimulates sucrose utilization, thereby promoting optimum plant growth (Schluepmann et al. 2003). Consequently, biochar application increased trehalose accumulation for osmotic protection and enhanced sucrose utilization for energy supply, enabling Mo17 plants to maintain growth and increase fresh weight under drought stress (Table 3).

The results revealed that the regulation of starch and sucrose metabolism pathway was responsible for plant osmotic protection with biochar application (Fig. 6). Sugar and starch levels are higher during the day because plants produce sugars during photoperiods (Feugier and Satake 2012; Henry et al. 2014). Therefore, it is not surprising that the expression of genes involved in circadian rhythm was altered with biochar application under drought stress for Mo17 roots (Paul et al. 2010). LHY (morning gene) and PRR7 (a repressor of the morning clock gene, Fig. 6), which belong to the major components of the circadian clock (Wu et al. 2016), were up-regulated. Under drought stress, dramatic sucrose utilization and trehalose accumulation occurred during photoperiods with biochar application, providing osmotic protection and possibly strengthening circadian rhythms for Mo17 roots.

5 Conclusions

The results revealed that biochar application decreased soil exchangeable Na+ and Ca2+ contents and soil pH while increasing soil exchangeable K+ content and EC in the alkaline soil. Consequently, biochar application increased sap K+ concentration for both drought-sensitive KN5585 and drought-tolerant Mo17. It is evident that the additional K supply with biochar application induced an increase in apoplastic pH for sensitive KN5585 but not for tolerant Mo17, thereby resulting in the activation of H+-ATPase and greater H+ efflux in the elongation zone for KN5585 roots under drought stress. The root growth of KN5585 was further inhibited by biochar application, likely due to apoplast alkalinization. This contributed to the increased water potential, decreased levels of O2–, H2O2, T-AOC, SOD, and POD, as well as down-regulated genes involved in drought resistance of KN5585 roots. The osmotic protection of Mo17 was enhanced by biochar application, as evidenced by increased sap osmolality under drought stress. The increased osmolality was attributed to trehalose accumulation, which was correlated with sucrose utilization and circadian rhythm with biochar application for Mo17 roots. Overall, these findings suggest that biochar application can be advantageous for sensitive plants under drought condition, as it regulates root growth and water consumption to avoid severe stress damage and response. In comparison, biochar application effectively strengthens osmotic protection for tolerant plants under drought condition without penalizing plant growth. Biochar application emerges as a potential strategy to enhance plant drought tolerance, and further research should consider its practical implications for plants under stress conditions.