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. 2024 Nov 6;25:1047. doi: 10.1186/s12864-024-10966-z

Molecular cloning and characterization of a brassinosteriod biosynthesis-related gene PtoCYP90D1 from Populus tomentosa

Juntao Song 1, Jie Tan 1, Tao Long 1, Yuanshuai Shi 1, Xu Luo 1, Yang Liu 1,✉
PMCID: PMC11539751  PMID: 39506673

Abstract

Brassinosteroids (BRs), one of the major classes of phytohormones are essential for various processes of plant growth, development, and adaptations to biotic and abiotic stresses. In Arabidopsis, AtCYP90D1 acts as a bifunctional cytochrome P450 monooxygenase, catalyzing C-23 hydroxylation in the brassinolide biosynthetic pathway. The present study reports the functional characterizations of PtoCYP90D1, one of the AtCYP90D1 homologous genes from Populus tomentosa. The qRT-PCR analysis showed that PtoCYP90D1 was highly expressed in roots and old leaves. Overexpression of PtoCYP90D1 (PtoCYP90D1-OE) in poplar promoted growth and biomass yield, as well as increased xylem area and cell layers. Transgenic plants exhibited a significant increase in plant height and stem diameter as compared to the wild type. In contrast, the CRISPR/Cas9-generated mutation of PtoCYP90D1 (PtoCYP90D1-KO) resulted in significantly decreased biomass production in transgenic plants. Further studies revealed that cell wall components increased significantly in PtoCYP90D1-OE lines but not in PtoCYP90D1-KO lines, as compared to wild-type plants. Overall, the findings indicate a positive role of PtoCYP90D1 in improving growth rate and elevating biomass production in poplar, which will have positive implications for its versatile industrial or agricultural applications.

Keywords: Populus tomentosa, Biomass, Brassinosteroid, CYP90D1, Secondary cell wall

Introduction

Brassinosteroid hormones (BRs) are a new class of polyhydroxylated sterols, which are distributed in roots, stems, leaves, seeds, fruits and other organs of plants, and have high biological activity, which can play crucial roles in many aspects of the growth and development process of plants [1]. These were first discovered from the pollens of Brassica napus [2, 3]. BRs can be involved in the regulation of plant cell elongation and division, vascular bundle differentiation [4], chloroplast development and photosynthesis [5], and the enhancement of plant stress tolerance [6, 7]. Genetic regulation of BR content promotes lignin formation in plants [8]. Exogenous application of BR enhances in-plant biosynthesis and signal transduction and increases crop yield [9].

The BR signalling pathway is currently one of the best understood plant signalling pathways. The biosynthetic pathways of brassinolide (BL) are complex and diverse, consisting of early and late C-22 oxidation pathways, early and late C-6 oxidation pathways [10]. Meanwhile, the specific molecular mechanisms of signal transduction in BRs are gradually being unravelled [11, 12]. The receptor for BRs, BRASSINOSTEROID-INSENSITIVE1 (BRI1), is rich in leucine repeats and encodes a single transmembrane protein kinase, which is phosphorylated by BRs upon binding to BRI1, and the phosphorylated BRASSINOSTEROID .INSENSITIVE1 (BRI1) interact s with the BRI1-ASSOCIATED RECEPTOR KINASE (BAK1) to phosphorylate BAK1, which further transmits the signals, thus initiating and regulating BR signal transduction [13]. Many studies have identified some key proteins in the BR synthesis pathway. Phosphorylated BSK1 can further activate another phosphatase, BRI1-SUPPRESSOR1 (BSU1) [14], and phosphorylated BSU1 can dephosphorylate and inactivate Tyr200 of BRASSINOSTEROID-INSENSITIVE2 (BIN2) [15], and the inactivation of BIN2 reduces BRI1-EMS-SUPPRESSOR1 (BES1) and BRASSINAZOLE-RESISTANT1 (BZR1), both of which play important roles in transcriptional regulation in this pathway. Inactivation of BIN2 reduced the phosphorylation of BRI1-EMS-SUPPRESSOR1 (BES1) and BRASSINAZOLE-RESISTANT1 (BZR1), which are important transcriptional regulators in this pathway [16]. 14-3-3 proteins are highly conserved and ubiquitous proteins present in all eukaryotic organisms, 14-3-3 proteins have shown to be closely linked to the regulation of plant hormone signalingDephosphorylated BZR1 can separate from 14-3-3 proteins and enter the nucleus to regulate BR synthesis-related genes through negative feedback [17].

BRs are produced from campesterol via a series of enzymatic processes. Numerous genes encoding these pivotal enzymes in the BR-biosynthetic pathway have been identified in various plant species, including Arabidopsis, rice (Oryza sativa), tomato (Solanum lycopersicum), and poplar (Populus tomentosa) [18]. Studies of BR-deficient mutants in different plant species have revealed that BR deficiency leads to leaf crumpling, plant dwarfism, reduced fertility, delayed senescence, and altered vascular structure and photomorphogenesis. For example, in Arabidopsis, the DE-ETIOLATED(DET2) mutant exhibits a dwarf growth phenotype [19]. DET2 encodes a protein with high similarity to mammalian steroid 5α-reductase [20]. The Arabidopsis CPD mutant, on the other hand, showed a more severe dwarfing phenotype than DET2 and also exhibited male sterility [21]. BRs affects rice pollen maturation by controlling starch accumulation and male fertility [22]. BR plays a crucial role in the regulation of male and female fertility in crops by description. The pea LKB mutant also showed a dwarfing phenotype [23]. Overexpression of CZP72457 in rice increased rice yield by increasing starch accumulation in the seed grain and thereby improving rice yield [24]. Overexpression of DWARF in tomato increased the photosynthetic efficiency of the plant, which in turn enhanced the yield and quality of the gains [25].

In the biosynthetic pathway of BRs, the AtCYP90D1 is involved in C-23 hydroxylation [26]. Two rice genes OsCYP90D2/D3, homologous to AtCYP90D1 were cloned, and their mutants exhibited a typical BR-deficient phenotype [27, 28]. Additionally, OsCYP90D2 can participate in the C-3 dehydrogenation reaction. CYP90D1 encodes a cytochrome P450 protein which catalyzes the C-23 hydroxylations (22-OH-3-one, 3-epi-6-deoxoTY to 6-deoxo3DT, 6-deoxoTY) during BR biosynthesis [29].

The effects of CYP90D1 in Arabidopsis thaliana and rice have been studied, but the role of CYP90D1 in poplar growth and development has not been established yet. In this study, the PtoCYP90D1, homologous to Arabidopsis CYP90D1, was isolated from Populus tomentosa Carr. The effect of overexpression of PtoCYP90D1 in transgenic plants was investigated as compared to the wild type. Similarly, the impact of its absence on the growth rate of poplars was elucidated by knocking out PtoCYP90D1 via CRISPR/Cas9-mediated mutation of PtoCYP90D1. Thus, the role of PtoCYP90D1 in the growth and biomass regulation of poplar was comprehensively encompassed in the present study.

Materials and methods

Plant materials and growth conditions

Transgenic (PtoCYP90D1-OE and PtoCYP90D1-KO) and WT (wild-type) Populus tomentosa Carr. were generously provided by Tobacco College Laboratory of Guizhou University. Plants of Populus tomentosa Carr. were grown in a greenhouse under a 16/8-hour light/dark cycle with supplemental light of 4500 lx at temperatures ranging from 22 to 25 °C and 60% relative humidity. Watering was adjusted based on evapotranspiration requirements at various growth stages, and fertilization was performed using ½-strength Hoagland nutrient solutions.

Sequence alignment and phylogenetic analysis

Total RNA was extracted from leaf samples using plant Trizol Reagent (Tiangen, China). The full-length PtoCYP90D1 cDNA was cloned from P. tomentosa, verified by sequencing, and inserted into the plant binary vector pCXSN under the control of the 35 S promoter. The PCR reaction was carried out for 34 cycles with DNA polymerase (Takara, Dalian, China) in a total volume of 50 µl. The reaction system contained 25 µl of Primer star Max DNA Polymerase (Takara, Dalian, China), 2 µl of each primer (Forward primer and Reverse primer), 2 µl of cDNA as DNA template, and 19 µl of nuclease-free water. The steps constituted of initialization at 98 °C for 2 min, denaturation at 98 °C for 15 s, annealing at 56 °C for 30 s, and extension at 72 °C for 1 min 30 s, followed by final extension of 72 °C for 10 min. The PCR products were purified and ligated into pSH737 plasmid (Takara, Dalian, China).

Construction of the PtoCYP90D1-OE vector and generation of transgenic poplar plants

The full-length coding sequence (CDS) of PtoCYP90D1 was digested with BamHl restriction enzyme (Takara, Dalian, China), and ligated into the plant binary vector pCXSN [30] via the same restriction site, under the control of Cauliflower mosaic virus (CaMV) 35 S promoter. The resultant construct PtoCYP90D1-OE was introduced into Agrobacterium tumefaciens GV3101 by the freeze-thaw method [31]. P. tomentosa transformation was carried out using the Agrobacterium-mediated method [32]. Briefly, after being infected with Agrobacterium, poplar leaf discs were co-cultured in Murashige and Skoog Medium (MS), containing 2.0 mg/L zeatin (ZT), 1.0 mg/L naphthalene acetic acid (NAA) and 100 µM-Acetosyringone (AS), in dark for 48 h. Two days later, the leaf discs were transferred to Woody Plant Medium (WPM) with 2.0 mg/L ZT, 1.0 mg/L NAA, and 90 mg/L Kanamycin (Kana) for callus induction. After one month of subculturing in the dark, these leaf discs with calli were transferred to a selective medium containing 2.0 mg/L ZT, 0.1 mg/L NAA, and 90 mg/L Kana. Subsequently, regenerated shoots were transferred to rooting medium with 0.1 mg/L NAA, and 50 mg/L Hygromycin (Hyg). After the regeneration of roots, the seedlings were planted in the greenhouse. Genomic DNA isolated from leaves of transgenic plants was used for PCR analysis, using Kana gene-specific primers. The expression levels in transgenic plants were evaluated by quantitative real-time PCR (qRT-PCR) using total RNA extracted from leaves.

CRISPR/Cas9-mediated mutation of PtoCYP90D1 in poplar

For mutation of PtoCYP90D1 in poplar, the pYLCRIPSR/Cas9 vectors for multiplex genome targeting vector system of CRISPR/Cas9 were used [33]. Based on their position and GC percentage, two CRISPR/Cas9 target sites of PtoCYP90D1 were chosen for the sgRNA sequences. These sgRNA cassettes were driven by Arabidopsis AtU3b, AtU6-1, and AtU6-29 promoters, respectively. The entire construction process was followed by Golden Gate Cloning, resulting in PtoCYP90D1-KO [34].

To identify the CRISPR/Cas9-mediated mutation of PtoCYP90D1 in transgenic poplar plants, genomic DNA was extracted from stems via the CTAB method. The PtoCYP90D1 genomic fragment was amplified using the gene-specific primers of PtoCYP90D1. The amplified products were cloned into the pSH737 simple vector (Takara, Dalian, China), followed by sequencing. The PtoCYP90D1-Cas9 transgenic lines (KO) were chosen for propagation. For further validation, PCR genotyping and DNA sequencing were confirmed in six regeneration plants of KO transgenic lines.

Quantification of xylem relative area

Xylem relative area was measured by ImageJ software [34]. Three biological replicates were measured for each line.

Quantitative RT-PCR

Total RNA was extracted from various tissues of 3-month-old P. tomentosa plants, including roots, the sixth stem internode, and seven leaves, by using the Trizol Reagent (Tiangen, China). The sixth internode of stems was selected for separating the xylem and phloem. DNase treatment was utilized to remove genomic DNA from total RNA. DNA was synthesized by PrimeScript™ RT reagent Kit(Takara, Dalian, China) with a genomic DNA Eraser (Takara, Dalian, China). The qRT-PCR was conducted on a TP700 Real-Time PCR machine (Takara, Japan), employing the SYBR Green PCR master mix (Takara, Dalian, China). The poplar 18 S rRNA gene served as the reference gene for the internal standard.

Quantitative estimation of BRs

The content of BRs in vivo was measured using stems from 3-month-old PtoCYP90D1-OE, PtoCYP90D1-KO, and wild-type plants. Following cutting, stem samples were immersed in PBS(phosphate-buffered saline, pH 7.4) and subsequently frozen using liquid nitrogen. BR levels were determined using the Plant Brassinolide ELISA Kit (Beijing Chenglin Biotechnology Company, China) [35].

Chemical analysis of secondary cell wall (SCW) components

The sixth internode of 3-month-old poplars was used for chemical analysis of SCW components. The absorbance was measured by a Bio UV-visible spectrophotometer (Shimadzu UV-2401 PC UV-VIS). The concentration of acetyl bromide (AcBr)-soluble lignin was determined by an extinction coefficient of 20.01 g− 1 ·cm− 1. The content of cellulose and lignin was determined by the Van Soest method [35]. All experiments were conducted in triplicates.

Statistical analysis

Statistical analysis of qRT-PCR data among the different tissue types, in response to exogenous hormones, and decapitation was performed in Excel 2010. The Statistical Program for Social Science 18 (SPSS, Chicago, IL, USA) was used for ANOVA and Student’s t-tests (two tail distribution and two samples with unequal variances). The statistical significance was considered at *0.01 < P < 0.05 and **P < 0.01.

Results

Isolation and characterization of PtoCYP90D1 from P. tomentosa

The sequencing analysis revealed that PtoCYP90D1, which contained a full-length CDS of 1,497 bp, encoded a putative protein of 493 amino acid residues with a predicted molecular weight of 56.7 kDa and isoelectric point (pI) of 8.97. The predicted amino acid sequence of PtoCYP90D1 contained three conserved domains, including the Proline-rich domain (pxgxxgwpxxget), oxygen-binding domain (mipxxk, exxr, and pxrx), steroid-binding domain (ftqxvitetlrxgn), and heme-binding domain [36]. Multiple sequence alignment revealed that PtoCYP90D1 shared a high identity with PtCYP90D1 (76.4%) in Populus trichocarpa, and AtCYP90D1 (74%) in Arabidopsis (Fig. 1B).

Fig. 1.

Fig. 1

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Bioinformatics analysis. (A) Phylogenetic tree of BR’s gene of P. tomentosa with closest components in Populus trichocarpa, Arabidopsis thaliana, and Oryza sativa. M: methionine start codon, pxgxxgwpxxget: proline-rich domain. Mipxxk, exxr, and pxrx: dioxygen binding domain. Ftqxvitetlrxgn: steroid binding domain. Fgggrlcpgldlarl: hemo binding domain. (B) Multiple sequence alignment of CYP90D1 amino acid sequence. PtCYP90D1: Potri.003G038200.1. AtCYP90D1: AT3g13730.1. OsCYP90D2: LOC_Os01g10040.1. OsCYP90D3: LOC_Os05g11130.1

Previous studies have reported that CYP90D1 belongs to the cytochrome P450 superfamily D [29]. phylogenetic (Fig. 1A) analysis revealed that PtoCYP90D1 was grouped into Class I together with AtCYP90D1, PtCYP90D1, and OsCYP90D2/3, which have been identified to participate in BR biosynthesis and promote the growth and development of plants [27, 28].

Expression patterns of PtoCYP90D1 in poplar

To determine the expression profiles of PtoCYP90D1, total RNA was extracted from different tissues of 3-month-old P. tomentosa. The findings of qRT-PCR showed that the expression level of the PtoCYP90D1 gene in the root and old leaf was significantly higher than in shoot, bud, juvenile leaf, and internode (Fig. 2), suggesting that PtoCYP90D1 is involved in the development and maturation of leaves as well as in the growth and development of roots.

Fig. 2.

Fig. 2

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Expression analysis of PtoCYP90D1 in different tissues (root, old leaf, bud, stem, internode, and juvenile leaf) of P. tomentosa. Significant difference analysis using Duncan’s test, n = 3. Error bars represent the SDs, and the different lowercase letters indicate statistically significant differences at P < 0.01

Construction and identification of transgenic poplars

To explore PtoCYP90D1’s role in poplar growth and development regulation, the coding sequence of PtoCYP90D1 was isolated and fused with the CaMV 35 S promoter to generate a plant binary expression vector pSH737-35 S-PtoCYP90D1 (Fig. 3A). After transformation, putative transgenic plants were selected for confirming the integration of the transgene by PCR analysis using the primers of the Kana gene. Two transgenic lines PtoCYP90D1-OE and PtoCYP90D1-KO were propagated and used for further studies (Fig. 3B).

Fig. 3.

Fig. 3

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Generation of transgenic poplars. (A) Schematic representation of the PtoCYP90D1-OE vector. (B) The expression levels of PtoCYP90D1 in the transgenic and wild-type plants. Error bars represent the SD (n = 3). Statistical analyses were performed using Student’s t-test as ** P < 0.01 and * 0.01 < P < 0.05. (C) Determination of the mutations in the coding region of PtoCYP90D1 generated by the CRISPR/Cas9 system. The text on the right summarizes mutation details in the independent CRISPR/Cas9-generated lines

Furthermore, the PtoCYP90D1 mutant lines were generated using the CRISPR/Cas9-based genome editing system. Two 20 bp sequences followed by a trinucleotide (5’-NGG-3’) protospacer adjacent motif (PAM) located in the first three exons of PtoCYP90D1 were assigned (Fig. 3D). The Agrobactium-mediated leaf disc method was used to introduce PtoCYP90D1-knock-out construct into P. tomentosa. More than 2 putative transgenic plants were generated and PCR analysis showed that the PtoCYP90D1 fragments containing two sgRNA-targeted sites were amplified from all transgenic lines (Figure S2B available as supplementary data at Tree Physiology Online). A total of 20 PCR clones for each mutant were randomly chosen for DNA sequencing. The results revealed that deletions (-), insertions (+), and substitutions of one or a few nucleotides were detected at the three sgRNA-targeted sites in one transgenic line (PtoCYP90D1-KO). In PtoCYP90D1-KO, small deletions (1 bp) were detected at the two target sites, suggesting the efficiency of mutagenesis. These deletions detected in the PtoCYP90D1-KO lines resulted in translational frame-shift or premature termination of PtoCYP90D1 (Fig. 3C), indicating the successful generation of PtoCYP90D1 loss-of-function mutants using CRISPR/Cas9-based genome editing.

Overexpression of PtoCYP90D1 promotes the growth and development of poplar

All transgenic and wild-type poplar plants were grown under the same environmental conditions. The 3-month-old transgenic PtoCYP90D1-OE plants exhibited rapid growth and development, as compared to the wild type. Inversely, transgenic PtoCYP90D1-KO lines exhibited delayed morphological changes, including retarded growth, and reduced plant weight (Fig. 4).

Fig. 4.

Fig. 4

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Growth comparisons of wild-type and transgenic plants. (A) Plants after growing for 3 months. (B) Plant height. (C) Stem diameter. (D) Fresh weight. (E) The leaf length and width at the seventh node of wild-type and transgenic plants. (F-G) Internode number and length. Statistical analysis using Student’s t-test, n ≥ 3: *, significant difference, 0.01 < P < 0.05, **, extremely significant difference, P < 0.01

The quantitative measures showed that the plant height and stem diameter were significantly increased in PtoCYP90D1-OE plants, but not dramatically reduced in PtoCYP90D1-KO lines in comparison to the wild-type control (Fig. 4B and C). Fresh weight increased by 46.94% in transgenic PtoCYP90D1-OE lines and reduced by 42.54% in PtoCYP90D1-KO lines, as compared to the wild type (Fig. 4D). These phenotypical alterations are in line with previous studies, which reported that BRs-related genes could enhance cell elongation and promote plant growth [14, 37]. The findings of the present study also indicate the crucial role PtoCYP90D1 plays in regulating poplar growth and development.

PtoCYP90D1 is involved in the positive regulation of wood formation in poplar

To determine the potential role of PtoCYP90D1 in wood formation, SCW development was analyzed in the same internode of 3-month-old wild-type and transgenic plants. The paraffin section analysis showed that transgenic PtoCYP90D1-OE exhibited a larger stem diameter compared to the wild-type control (Fig. 5).

Fig. 5.

Fig. 5

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Cross sections of the stem in the 8th internode showing xylem and phloem area, Longitudinal cutting of the pith. (A, D, G) wild-type, (B, E, H) PtoCYP90D1-OE and (C, F, L) PtoCYP90D1-KO. P: phloem, X: xylem

To further verify the effect of PtoCYP90D1 on xylem development, the content of the chemical components, including lignin and cellulose, was examined in transgenic lines. The findings revealed that lignin contents in PtoCYP90D1-OE lines were significantly increased by 18.98% and decreased by 11.19% in PtoCYP90D1-KO lines, compared to wild-type plants (Table 1). These results indicated that PtoCYP90D1 is a positive regulator for improving xylem development in poplar.

Table 1.

Cell wall composition analysis of the stems in wild-type and transgenic plants

Samples Lignin Cellulose
WT 4.11 ± 0.20 32.66 ± 5.04
PtoCYP90D1-OE 4.89 ± 0.30* 38.78 ± 0.42*
PtoCYP90D1-KO 3.65 ± 0.46 30.30 ± 0.98
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Note: Error bars represent ± SD from three biological repeats. Asterisks indicate significant differences in comparison to WT (Student’s t-test: *P < 0.05;). The unit is mg·100 mg-1

Further magnification of histochemical staining revealed that overexpression of PtoCYP90D1 improved secondary xylem development (Fig. 5E), leading to an increased number of cell layers in xylem (9 layers), phloem (7 layers), cortex (4 layers), and cortical areas (4 layers) (Fig. 6), compared to wild-type plants (Fig. 5D). In contrast, the knock-out of PtoCYP90D1 resulted in reduced vessel development (Fig. 6K). Similar results were also observed in the fifth and seventh internodes between transgenic lines and wild-type plants (Fig. 7).

Fig. 6.

Fig. 6

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Comparison of stem microstructures between transgenic and wild-type poplar plants. The cell layers of (G) cortical (H) cortex (R) xylem and (J) phloem in wild-type and transgenic plants. (K) The vessel counts. (L) The number of piths. * indicates differences in comparison with WT at 0.01 < P < 0.05, **indicates significant differences in comparison with WT at P < 0.01 (Student’s t-test)

Fig. 7.

Fig. 7

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Comparison of leaf vein microstructures between transgenic and wild-type poplar plants. * indicates differences in comparison with WT at 0.01 < P < 0.05, **indicates significant differences in comparison with WT at P < 0.01 (Student’s t-test)

Overexpression of PtoCYP90D1 improves BRs biosynthesis in transgenic poplar

We examined BR levels in transgenic and wild-type plants. Overexpression of PtoCYP90D1 resulted in a significant increase in BR concentrations in transgenic plants. By contrast, in PtoCYP90D1-KO lines, BR levels were decreased by 93.03% of the levels in the wild type, respectively (Table 2).

Table 2.

Total BRs content (ng/g)of transgenic lines and WT

Samples Total BRs content (ng/g)
WT 107.81 ± 5.87
PtoCYP90D1-OE 168.38 ± 12.62*
PtoCYP90D1-KO 100.3 ± 4.46
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Note: Error bars represent ± SD from three biological repeats. Asterisks indicate significant differences in comparison to WT (Student’s t-test: *P < 0.05;)

Discussion

Previous studies have demonstrated that AtCYP90D1 encodes C-23 hydroxylation [29], which catalyzes 22-OH-3-one and 3-epi-6-deoxoTY directly into 6-deoxo3DT and 6-deoxoTY in the late C-6 pathway, reducing the two-step synthesis of BR. CYP90D1 is considered one of the main enzymes in BR biosynthesis. To affirm whether Populus CYP90D1 gene family members have similar functions to AtCYP90D1, homologs were identified in the Populus genome. In this study, PtoCYP90D1 was cloned from P. tomentosa. Amino acid sequence analysis showed that PtoCYP90D1 contains one heme-binding domain, the steroid-binding domain, and oxygen-binding domain [36, 38] (Fig. 1B), suggesting that it was a conserved cytochrome P450 monooxygenase protein. Phylogenetic analysis showed that PtoCYP90D1 was grouped with AtCYP90D1 and OsCYP90D2/3, and had the closest relationship with PtCYP90D1.

As Populus has been taken as an ideal model tree plant [39], a comparative study on the genetic functions of the CYP90D1 gene family between Arabidopsis and rice will provide direct evidence in understanding the mechanism of BR-regulated plant growth and development in herbaceous as well as woody plants. The study found that PtoCYP90D1 was predominantly expressed in roots and old leaves (Fig. 2). Therefore, the high expression of PtoCYP90D1 in old leaves and low expression in young leaves indicated the important role of PtoCYP90D1 in leaf maturation and development. In a previous study, overexpression of AtCYP90D1 in transgenic Arabidopsis plants increased plant height and internode length [40], while internode cell shortening was reported forOsCYP90D1 RNA interference mutant [27]. The present study showed that overexpression of PtoCYP90D1 resulted in an increase in both plant height and stem diameter in poplar, whereas knock-out of PtoCYP90D1 led to reduced plant height and stem diameter compared to the wild type (Fig. 4).

BRs play an important role in regulating plant growth and development [41]. It has been verified that genetic modification of BR synthesis promotes plant growth [18]. Plants with BRs metabolic mutants and overexpression exhibited rapid growth, including larger leaves, more height, and increased biomass [18, 34]. These phenotypic alterations are consistent with previous reports on transgenic Arabidopsis overexpressing AtCYP90D1, which displayed increased inflorescence height and seed and branch counts [40]. Similarly, overexpression of Populus trichocarpa CYP85A3, a key enzyme gene for BRs synthesis, in tomatoes increased the height of transgenic plants by 10 ~ 20 cm, which was 2.17 times that of wild type. Overexpression of PtCYP85A3 in poplar also increased plant height and stem diameter. Overexpression of PtoDET2 transgenic poplars resulted in faster growth rates and agronomic traits such as the number of internodes, leaves, plant height, and stem diameter increased [34]. The present findings revealed that PtoCYP90D1-OE transgenic plants increased plant height and stem diameter relative to wild-type, while knockout ones did not change significantly. At the same time, it was found that in PtoCYP90D1-OE transgenic plants, the length and width of leaves, as well as the fresh weight, increased significantly, whereas it decreased significantly in PtoCYP90D1-KO plants. These results imply that PtoCYP90D1 overexpression promotes the growth and development of poplar.

Previous studies have shown that brassinolide plays an important role in xylem formation and vascular development [18]. In BR biosynthesis mutants, the cell wall synthesis genes were down-regulated, while exogenous BRs promoted the differentiation of vessel molecules and xylem formation [42]. Overexpression of BRI1 in transgenic poplar increased the expression of CESA, a gene related to SCW biosynthesis, and promoted wood formation [43]. In this study, the paraffin sections of stem and vein tissues of PtoCYP90D1 plants showed that the xylem and phloem area of the overexpression plants was greater than that of the wild type, while the knockout plants did not change significantly. Likewise, the number of xylems and phloem cell layers in the stem, as well as the number of pith cells in the overexpressed PtoCYP90D1 transgenic plants, increased significantly, while no substantial alterations occurred in the knockouts. Overexpression of PtoCYP90D1 resulted in a significant increase in BR concentrations in transgenic plants. By contrast, in PtoCYP90D1-KO lines, BR levels were decreased to 93.03% of the levels in the wild type, respectively. These findings suggest that PtoCYP90D1 overexpression promotes the growth and development of plant stems by increasing the number of tissue cells.

Author contributions

Y.L. supervised the overall study and designed the experiment.All authors participated in the ex-perimental procedure and analyzed all data. J.S., T.L. and J.T. wrote the main manuscript text. J.S. and T.L. prepared Figs. 1, 2, 3, 4, 5, 6 and 7. J.T., Y.S. and X.L. prepared Table 1, and 2. J.S. wrote the paper. Y.L. wrote sections of the manuscript.All authors contributed to manuscript revision and read and approved the submitted version.

Funding

This work was supported by the National Natural Science Foundation of China (3226180451, 3226180488); Guizhou Province Outstanding Young Scientific and Technological Talent Cultivation Project (Qiankehe PlatformTalent [2019]5651); Guizhou Province Science and Technology Planning Project (Qiankehe Support [2021] General 111).

Data availability

The dataset generated in the current study is available in the NCBI repository under the accession number LOC18096744 (XM_024597059.2).

Declarations

Competing interests

The authors declare no competing interests.

Ethics approval and consent to participate

We ensured that the collection of plant material and experimental research and field studies on plants complied with relevant institutional, national, and international guidelines and legislation.

Consent for publication

Not Applicable.

Conflict of interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Footnotes

Publisher’s note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

References

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Data Availability Statement

The dataset generated in the current study is available in the NCBI repository under the accession number LOC18096744 (XM_024597059.2).

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