Sugar beet cultivar ‘BS02’, a homozygous cultivar with a high sucrose content, was bred by the Sugar Beet Physiological Research Institute, Inner Mongolia Agricultural University, China, and used in this study. The seeds were sown in vermiculite with one seedling per 8×8×8 cm³ float tray, and the seedlings were grown in an artificial climate chamber at 22°C with 60% humidity and a 16/8 h light/dark cycle. Plants with eight leaves were treated with 0.1 mg/L of epibrassinolide (Sigma, USA) or 20 µmol/L of Brz (Sigma, USA) for 4 h. The control group was treated with water. The samples were collected to investigate BvBZR1 gene expression patterns.

Sterile BS02 seedlings were obtained through the following steps: the seeds were artificially sanded to ensure a smooth seed shell. Seeds were washed for 30 min, treated with 0.1% HgCl₂ solution for 10 min, rinsed five times with sterile water, dried on a filter paper for 5–10 min, and planted in germination medium (1 mg/L 6-BA + 8.0 g/L agar; pH 5.8). Seeds were cultured at 22°C with a 16 h light/8 h dark photoperiod and a photon flux density of 45 mmol m⁻² s⁻¹. Ten-day-old sugar beet seedlings were used for genetic transformation experiments.

The BvBZR1 gene (Bv1_fxre) was isolated in our previous research (Wang W. et al., 2019). To analyze the function of BvBZR1, a pCAMBIA1300-BvBZR1 vector was constructed. The open reading frame (ORF) of BvBZR1 was amplified using the BvBZR1-BamHI-F and BvBZR1-SalI-R primers ( Supplementary Table S1 ) and inserted between the BamHI and SalI sites of the pCAMBIA1300 vector under control of the CaMV 35S promoter to generate pCAMBIA1300-BvBZR1. pCAMBIA1300-BvBZR1 was introduced into Agrobacterium tumefaciens (strain LBA4404) using the freeze–thaw method (Jyothishwaran et al., 2007), and sugar beet was transformed using the cotyledon node infiltration method described by Guo et al. (2023). Briefly, 10-day-old sterile seedling cotyledon nodes were cut off and pre-cultured (MS + 30 g/L sucrose + 1 mg/L 6-BA + 0.2 g/L casein + 7 g/L agar) for 24 h, followed by 10 min of Agrobacterium infection, and incubated on co-culture medium (MS + 30 g/L sucrose + 1 mg/L 6-BA + 0.2 g/L casein + 100 µM AS + 7 g/L agar) for 2–3 days in the dark. After washing the cotyledonary nodes, they were cultured on a suppressive medium (MS + 30 g/L sucrose + 1 mg/L 6-BA + 0.25 mg/L NAA + 0.5 mL/L PPM + 8.0 g/L agar + 400 mg/L temetin) for 5–7 days and then transferred to a screening medium (MS + 30 g/L sucrose + 1 mg/L 6-BA + 0.25 mg/L NAA + 0.5 mL/L PPM + 8.0 g/L agar + 6 mg/L hygromycin) for 15–20 days to screen resistant beet shoots. The resistant shoots were then transferred to a rooting medium (MS + 30 g/L sucrose + 2 mg/L NAA + 0.5 mL/L PPM + 8.0 g/L agar) to induce root formation. After 1 month, the seedlings were refined and transplanted into nutrient soil. Transgenic lines were confirmed using genomic PCR. The vector-specific primers Hgy2-F and Hgy2-R (Supplementary Table S1) were used for PCR under the following conditions: denaturation at 94°C for 10 min, 33 cycles of 94°C for 1 min, 55°C for 30 s, and 72°C for 1 min, followed by a final extension period at 72°C for 5 min. Selected hygromycin-resistant plants were used for further validation and subsequent functional identification.

To quantify BvBZR1 expression in sugar beet under BR treatment and transgenic plants, real-time PCR was performed using BvBZR1-specific primer sets (BvBZR1-RT-F/R; Supplementary Table S1 ) with the BvActin gene as an internal control. To examine the expression of genes related to cell wall synthesis and sucrose metabolism in transgenic sugar beet, cDNA was synthesized using RNA extracted from different tissues from WT and transgenic plants. The expression levels of the BvCEL1, BvCESA6, BvFAD3, BvXTH33, BvSPS, and BvINV genes were quantified using real-time RT-PCR. The BvActin gene was used as an internal control. The sequences of all primers used are listed in Supplementary Table S1 . Total RNA was reverse-transcribed into first-strand cDNA using the TransScript One-Step gDNA Removal and cDNA Synthesis Super Mix (TransGen Biotech, Inc.) according to the manufacturer’s protocol. Three separate cDNA sample dilutions were used as templates for real-time PCR analysis. The cDNA was amplified using TransStart Tip Green qPCR SuperMix (TransGen Biotech, Inc.) in a 480 II real-time PCR Light Cycler (Roche) under the following reaction conditions: denaturation at 95°C for 30 s, followed by 48 cycles of 95°C for 10 s, 58°C for 15 s, and 72°C for 20 s. The threshold cycle (CT) values of triplicate samples were averaged, and fold-change values in the transcription level of the target gene relative to the reference gene were calculated using the comparative CT (2^–△△CT) method, as previously described by Livak and Schmittgen (2001). All experiments were repeated three times using cDNA prepared from three batches of plants.

To investigate BvBZR1 expression in transgenic sugar beet plants, RT-PCR was performed using the specific primers BvBZR1-RT-F and BvBZR1-RT-R ( Supplementary Table S1 ). The BvActin gene was used as an internal control. RNA was prepared from leaves of transgenic sugar beet seedling according to the operating instructions of the TransZol Up Plus RNA Kit (TransGen Biotech, Inc.). The cDNA was synthesized from 1 µg of total RNA at 42°C for 30 min with 1 µL of oligo (dT) using an RT-PCR kit (TransGen Biotech, Inc.). The PCR mix included 2.5 µL of 10×TransStart Taq buffer, 2 µL of 2.5 mM dNTPs, 2.5 U of TransStart Taq DNA polymerase, 1 µL of 10 mM primers, and 1 µL of cDNA for a 25 µL volume. The reaction conditions were 35 cycles of 94°C for 30 s, 56°C for 30 s, and 72°C for 1 min, followed by a final extension period of 10 min at 72°C.

Morphological indicators of sugar beet were measured when transplanted transgenic sugar beet seedlings were incubated in an artificial climate chamber for 80 days. Three plants were sampled from each line. The fresh weight of the total plant, leaf, petiole, and root was determined using an electronic balance. The plant height, leaf length, and root length were measured with a tape measure, and the root diameter was measured with a vernier caliper.

Samples were collected from the root 1 cm from the root apex. All specimens were fixed in 70% formaldehyde acetic acid for 24 h and dehydrated in an increasing ethanol series. Transverse 12-µm-thick sections were obtained with a rotating microtome, stained with Safranin and Astra Blue, and mounted on paraffin (Maia et al., 2018). The slides were observed under a microscope (Pannoramic DESK, Hungary) and photomicrographed. The images were analyzed with CaseViewer. The following histological parameters were evaluated: ring spacing, area of parenchyma cells, and number of parenchyma cell layers. The distance between the outermost xylem of two vascular forming layers was defined as the spacing between the rings of forming layers. CaseViewer was used to randomly select 10 adjacent cells to determine the total area, and the average area was calculated. The number of parenchyma cell layers between the rings of two forming layers was counted, and ten groups of data were randomly selected for analysis. All experiments had 10 biological replicates.

To investigate the function of BvBZR1, total RNA was isolated from 60-day-old WT and transgenic line 2 (OE2) using a total RNA Isolation System (Takara, Shanghai, China) according to the manufacturer’s instructions. The concentration and quality of RNA were determined on a 2100 Bioanalyzer (Agilent Technologies), and 3 mg of RNA from each sample was used for library construction. Sequencing of three independent biological replicates per sample type was performed using the NovaSeq 6000 platform (Illumina). Approximately 4.0 Gb of clean data were generated per sample. Clean reads were mapped to a genome reference (RefBeet-1.1) using BWA (Burrows-Wheeler Alignment Tool) and Bowtie (Dohm et al., 2014). The differentially expressed genes (DEGs) were filtered using the following thresholds: |log²(Fold change)| > 1 and adjusted P (q-value) < 0.05. Functional annotation of the DEGs was conducted using the Blast2GO program and the non-redundant protein database (NR; NCBI). A significance level of P ≤ 0.05 was used to confirm Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway, and MapMan analysis results.

To explore the binding of BvBZR1 to the promoters of the target genes, a yeast one-hybrid kit was used in accordance with the manufacturer’s protocol (Clontech Laboratories, Mountain View, CA, USA). The promoters of the sugar beet genes BvXTH33 and BvCESA6 were cloned into the pAbAi vector to acquire pAbAi-bait vectors. These recombinant pAbAi-bait and pAbAi-53 vectors were transformed into Y1HGold yeast, and the background aureobasidin A (AbA) expression of the bait strain was tested. The pGADT7 prey vector harboring the BvBZR1 ORF (pGAD-BvBZR1) was transformed into the bait strain. Y1HGold yeast cells were used as a negative control, and yeast cells co-transformed with pAbAi-53 and pGADT7-p53 vectors were used as positive controls. The transformed yeast cells were grown in a leucine- and uracil-deficient SD medium containing AbA. After culturing at 30°C for 4 days, the plates were photographed.

The promoters of the sugar beet BvXTH33 and BvCESA6 were inserted into the pGreenII0800-LUC vector as reporter plasmids, and the pGreenII62sk-BvBZR1 were used as effectors plasmid in dual-LUC assays. The mixed effector and reporter plasmids were electrotransformed into Agrobacterium GV3101, and injected into the lower epidermis of tobacco leaves. After three days of low light culture, the leaves were collected and the cells were lysed. The activity of LUC/REN was assessed by employing a kit for dual-luciferase reporter assay (E1910, Promega, Madison, WI, USA). Each sample was measured in triplicate.

Sixty days after sugar beet transplantation, samples were collected from the roots, stems, leaves, and vascular rings and inter-rings of one to three layers of the taproot. The contents of sugar, total soluble sugar, and reducing sugar were measured according to the manufacturer’s instructions (Jiangsu Addison Biotechnology Co., China). The activities of BvCEL, BvCESA, BvFAD, BvXTH, BvSPS, BvSS-C, BvSS-S, and BINV were measured using corresponding assay kits (all from the Jiancheng Bioengineering Institute, Nanjing, China) according to the manufacturer’s instructions.

Data are expressed as means ± standard deviations. Statistical differences were assessed by one-way analysis of variance (ANOVA) using Duncan’s multiple range test (MRT). The significance of differences between the means was assessed using a probability (P) value of < 0.05. All analyses were performed using SPSS 18.0 software (ver. 19; SPSS, Inc., Chicago, IL, USA).

Our previous research showed that BRs increased the spacing between the cambial rings by increasing the size of parenchyma cells between the rings and ultimately increasing sugar beet taproot diameter, and some cell wall biosynthesis-related genes were significantly induced by BR (Wang et al., 2021). To determine whether the expression level of BvBZR1 was regulated by exogenous BR in sugar beet, the roots were sprayed with BR, Brz, or water for 4 h. As shown in Figure 1 , compared with the control group, the expression level of BvBZR1 was significantly increased after spraying with BR, whereas it slightly decreased after Brz treatment, suggesting that BR could promote BvBZR1 expression.

To analyze the function of BvBZR1, the pCAMBIA1300-BvBZR1 plasmid containing the BvBZR1 cDNA sequence under the control of the CaMV35S promoter was constructed and introduced into sugar beet by Agrobacterium-mediated cotyledon node transformation ( Figure 2A ). After screening for hygromycin resistance, three lines overexpressing BvBZR1 (OE1, OE2, and OE3) with hygromycin resistance were validated by Genomic-PCR and RT-PCR ( Figure 2B ). Real-time RT-PCR analysis showed that the BvBZR1 expression levels of transgenic sugar beets were significantly higher than those of the WT ( Figure 2C ).

Transgenic sugar beet overexpressing BvBZR1 displayed faster growth and development compared with the WT ( Figure 3A ). The total fresh weight and height of transgenic sugar beet overexpressing BvBZR1 were significantly higher than those of the WT ( Figures 3B, C ). In the shoot morphology, the petiole weight, leaf weight, and leaf length of transgenic plants were significantly higher than those of WT plants ( Figures 3D–F ). BvBZR1 overexpression enhanced the root growth of transgenic sugar beet and significantly increased the root weight, root length, and root diameter ( Figures 3G–I ). These results suggest that BvBZR1 overexpression can promote the root growth of sugar beet by increasing the root length and diameter.

To determine how BvBZR1 promotes the increase in taproot diameter of transgenic sugar beet, we examined taproot cross-sections from transgenic and WT plants ( Figure 4A ). Three cambial rings were found in all groups. The ring spacing of 1st and 2nd formation layers of the taproot in transgenic sugar beet was significantly higher than that in the WT ( Figure 4B ). Compared with the WT, the layers of parenchymal cells of 1st and 2nd cambial rings were significantly increased in BvBZR1-overexpressing beet taproots ( Figure 4B ). The parenchymal cells between 1st and 2nd cambial rings of transgenic plants were significantly larger than those of WT plants ( Figure 4B ). Similar results were found for 2nd and 3rd cambium rings ( Figure 4C ). These results suggest that BvBZR1 overexpression can enhance the spacing between cambial rings by increasing the size and layers of parenchyma cells, thereby increasing the taproot diameter of sugar beet.

To investigate the function of BvBZR1, we performed RNA-seq on WT and OE2, each with three biological replicates. The DEGs were screened according to the |log2 ratio| ≥ 1 (P < 0.001) and false discovery rate ≤ 0.001. We identified 5172 upregulated and 6208 downregulated genes in OE2 versus WT ( Figure 5A ). KEGG pathway analysis was performed using a P-value of less than 0.05 as the cutoff, and the DEGs were significantly enriched in metabolic pathways such as starch and sucrose metabolism, phenylpropanoid biosynthesis, and amino sugar and nucleotide sugar metabolism ( Figure 5B ). Directed acyclic graph analysis was performed on significantly enriched GO entries in cellular components, molecular functions, and biological processes. The results showed that the two largest subcategories were found in the “cellular component” category, including “cell wall” and “plant-type cell wall.” In the “biological processes” category, “plant-type cell wall organization” and “xyloglucan metabolic process” were the most abundant GO terms ( Figure 5C ). In the “molecular function” category, the largest subcategory was “xyloglucan: xyloglucosyl transferase activity.” These results indicate that the BvBZR1 gene may be involved in regulating plant cell wall tissue metabolism and the xyloglucan metabolic process.

To determine whether BvBZR1 could directly regulate the expression of genes related to cell wall metabolism, we selected significantly upregulated genes BvCESA6 and BvXTH33 from the significantly enriched “plant-type cell wall organization” and “xyloglucan metabolic process” entries for yeast single hybridization validation. BvBZR1 directly bound the promoter region of the BvXTH33 and BvCESA6 genes ( Figure 5D ). The dual-LUC assays further confirmed that the BvBZR1 has the ability to activate the transcription of BvCESA6 and BvXTH33 in tobacco protoplasts ( Figure 5E ).

To determine whether the BvBZR1 gene played a regulatory role in cell wall synthesis, the expression of cell wall synthesis-related genes, including BvCEL1, BvCESA6, BvFAD3, and BvXTH33, was detected in transgenic sugar beet taproots, stems, and leaves ( Figures 6A–D ). The expression of BvCESA6, BvFAD3, and BvXTH33 in the leaves of transgenic plants was significantly higher than that of the WT. In the stems, the expression of the four genes in transgenic plants was significantly higher than that in the WT. In the roots, overexpression of the BvBZR1 gene significantly increased the expression levels of BvCESA6 and BvXTH33 genes while decreasing the expression levels of BvCEL1 ( Figures 6A–D ). Similarly, BvCEL, BvCESA, BvFAD, and BvXTH enzyme activities were detected in the roots, stems, and leaves of transgenic plants ( Figures 7A–D ). In the leaves, the BvCESA activity of transgenic plants was significantly increased compared with the WT. In the stems, the BvCEL and BvXTH activities of transgenic plants were significantly higher than those of the WT. In the roots, BvBZR1 overexpression significantly increased the activity of BvCESA and BvXTH but decreased the activity of BvCEL ( Figures 7A–D ). These results indicate that BvBZR1 can regulate the expression of the BvCEL1, BvCESA6, and BvXTH33 genes, improve their enzyme activity, and promote taproot cell wall biosynthesis in sugar beet.

To further determine how BvBZR1 promotes cell wall biosynthesis in the sugar beet taproot, the expression of BvCEL1, BvCESA6, BvFAD3, and BvXTH33 was detected in each cambial ring and in parenchyma cells between each cambium ring of transgenic plant taproots. The expression level of the BvCEL1 gene in parenchyma cells between the first and second/second and third cambium rings and in the second cambium ring of transgenic sugar beet taproot was significantly lower than that of the WT ( Figure 6E ). The expression of the BvCESA6 gene in the first and second cambium rings and in parenchyma cells between the first and second cambium rings of transgenic sugar beet taproot was significantly higher than that of WT ( Figure 6F ). The expression of the BvXTH33 gene in the parenchyma cells between the first and second cambium rings and in the first and third rings of transgenic taproots was significantly higher than that of the WT, but there was no consistent significant difference in the expression of the BvFAD3 gene in each cambial ring of transgenic plant taproots compared with the WT ( Figures 6G, H ). The BvCEL activity in each cambial ring and in parenchyma cells between the second and third rings of transgenic sugar beet taproots was significantly lower than that of the WT ( Figure 7E ). The BvCESA activity in parenchyma cells between each cambial ring of transgenic sugar beet taproots was significantly higher than that of the WT ( Figure 7F ). Compared with the WT, transgenic plant taproots exhibited higher BvFAD activity in the first cambium rings and in parenchyma cells between the first and second cambial rings ( Figure 7G ). The BvXTH activity in parenchyma cells between each cambial ring and in the first cambial ring of transgenic sugar beet taproots was significantly higher than that of the WT ( Figure 7H ). These results suggest that BvBZR1 can be involved in regulating the expression of genes related to cell wall synthesis in each cambium ring and parenchyma cell of sugar beet taproots, improving its enzyme activity, thereby promoting the growth of parenchyma cells and the development of each cambium ring, leading to lateral thickening of the taproot.

Previous transcriptional KEGG enrichment analysis revealed that the DEGs were significantly enriched in starch and sucrose pathways; therefore, the contents of sucrose, soluble sugars, and reducing sugars were detected in the roots, stems, and leaves of transgenic plants ( Figures 8A–C ). In taproots, the accumulation of sucrose and total soluble sugars in transgenic plants was significantly higher than that in WT plants, while there was no significant difference in the accumulation of reducing sugars. In the stems, transgenic plants exhibited higher contents of reducing sugars and total soluble sugars compared with the WT. In the leaves, there was no consistent significant difference in the contents of sucrose, reducing sugar, and soluble total sugar in the leaves ( Figures 8A–C ). These results indicate that BvBZR1 can regulate sucrose accumulation in the taproot.

To better understand how the BvBZR1 gene regulates sucrose accumulation in sugar beets, gene expression and enzyme activity related to sucrose metabolism were detected in transgenic plants. BvSPS expression in transgenic sugar beet taproots, stems, and leaves was significantly higher than that of the WT, while BvINV expression in transgenic plant taproots was significantly lower than that of the WT ( Figures 9A, B ). The activity of enzymes related to sucrose metabolism showed that in the taproot, BvSPS activity was significantly higher in transgenic plants than in the WT. The activities of BvSS-C and BvINV were significantly lower than those in the WT, while BvSS-S activity showed no significant changes. However, in stems and leaves, there was no consistent significant difference between transgenic and WT plants ( Figures 10A–D ). These results indicate that BvBZR1 can regulate the expression levels of the BvSPS and BvINV genes, increase sucrose phosphate synthase activity, and reduce sucrose synthase (decomposition direction) and sucrose invertase activity, thereby increasing sucrose accumulation in taproots.

In addition, the expression levels of genes and the activities of enzymes related to sucrose metabolism were detected in each cambial ring and in inter-ring parenchyma cells of transgenic plant taproots. BvSPS expression in the parenchyma cells between the second and third rings and in the third cambium rings of transgenic taproots was significantly higher than that of the WT. BvINV expression in each inter-ring parenchyma cell and in the third cambium ring of transgenic taproots was significantly lower than that of the WT ( Figures 9C, D ). BvSPS activity was significantly higher in the parenchyma cells between the first and second rings and in each cambium ring of transgenic taproots than in the WT. BvSS-C activity was significantly lower in each inter-ring parenchyma cell and in the third cambium ring of transgenic taproots than in the WT. BvSS-S activity was significantly higher in the parenchyma cells between the second and third rings and in the first cambium rings of the transgenic taproot than in the WT ( Figures 10E–H ). These results indicate that BvBZR1 can increase the activity of sucrose phosphate synthase in the cambium ring and sucrose synthase (synthesis direction) in the inter-ring parenchyma cells and reduce the activity of sucrose synthase (decomposition direction) in the cambium ring and inter-ring parenchyma cells, thus increasing the sucrose content in the taproot.