Genome-Wide Identification, Expression Analysis, and Subcellular Localization of DET2 Gene Family in Populus yunnanensis

(1) Background: Brassinosteroids (BRs) are important hormones involved in almost all stages of plant growth and development, and sterol dehydrogenase is a key enzyme involved in BRs biosynthesis. However, the sterol dehydrogenase gene family of Populus yunnanensis Dode (P. yunnanensis) has not been studied. (2) Methods: The PyDET2 (DEETIOLATED2) gene family was identified and analyzed. Three genes were screened based on RNA-seq of the stem tips, and the PyDET2e was further investigated via qRT-PCR (quantitative real-time polymerase chain reaction) and subcellular localization. (3) Results: The 14 DET2 family genes in P. yunnanensis were categorized into four groups, and 10 conserved protein motifs were identified. The gene structure, chromosome distribution, collinearity, and codon preference of all PyDET2 genes in the P. yunnanensis genome were analyzed. The codon preference of this family is towards the A/U ending, which is strongly influenced by natural selection. The PyDET2e gene was expressed at a higher level in September than in July, and it was significantly expressed in stems, stem tips, and leaves. The PyDET2e protein was localized in chloroplasts. (4) Conclusions: The PyDET2e plays an important role in the rapid growth period of P. yunnanensis. This systematic analysis provides a basis for the genome-wide identification of genes related to the brassinolide biosynthesis process in P. yunnanensis, and lays a foundation for the study of the rapid growth mechanism of P. yunnanensis.

The DET2 gene of A. thaliana, known as the DWF6 gene, is found in det2 (deetiolated2) mutants and is highly consistent with steroid 5α-reductase, which catalyzes the synthesis of dihydrotestosterone in animals and catalyzes the development of the male genitalia and prostate during animal embryonic development [18].The DET2 gene in plants is the rate-limiting gene in three pathways of BRs biosynthesis, and all the intermediates in the BR biosynthetic pathway after the DET2 reaction can be used to rescue the det2 mutant phenotype [19].Many studies have speculated that the DET2 gene affects cell division and growth during plant development by regulating BR content and then regulating cell wall synthesis and extension.The expression of CycD3 was significantly increased after treatment with 24-epibrassinolide (24-EBL) in A. thaliana, which indicated that brassinolide might have some effect on cell division, and the in vitro application of brassinolide to the DET2 mutant of A. thaliana could increase the number and size of its leaf cells [20].The study of BR-related mutants (det2-1 and bril-301) in A. thaliana showed the expression of cellulose synthase gene and the content of cellulose decreased significantly [21].When the amino acid Glu240 in the DET2 protein was substituted, the activity of 5α-dehydrogenase was completely lost, resulting in a decrease in BR biosynthesis, the mutant cells reduced, and the plant appeared yellowing and dwarfing [22].The over expression of the DET2 gene showed the elongation of fibroblasts [23], the improvement of endogenous castasterone (CS) levels, and the enhancement of cell activity in cambium meristem and xylem (XY) differentiation to promote stem growth [24,25].
The fast growth of forest trees is determined by the comprehensive action of many factors of external and endogenous signals [26,27].As a representative species of Populus in China, Populus yunnanensis is adaptable, fast-growing, and easily survives through cuttings.It is primarily distributed in mountainous regions ranging from 1600 to 3200 m in Southwest China.So it plays an important role in forestry production, ecological protection, industrial raw materials, and building materials [28].Because of its stronger photosynthetic ability and tissue-life ability than other poplar species, it grows more rapidly.The peak of plant height growth occurs from June to October.Even at altitudes of 3000 to 4000 m, the tree can achieve a height growth of 1.0 to 2.4 m, demonstrating robust and rapid development, along with a significant value for utilization [29].
In the organs, tissues, or cells of plants, there are some morphological and biochemical gradients in different axes, which is called polarity.Polarity is a basic phenomenon in plant differentiation.Once it is established, the growth and development of plants have a specific direction, and it is difficult to reverse.Previous studies have shown that upright and inverted cuttings of P. yunnanensis could survive, and both grew faster in September, so their height and stem diameter were significantly higher than those in July.In this study, the steroidal dehydrogenase family of P. yunnanensis was identified and other bioinformatics were analyzed.The growth of the stem is caused by the continuous or periodic cell division of the meristem at the end of the stem, which makes the stem grow.The differentially expressed genes of this family were screened based on the transcriptome of the stem tip, and the expression and subcellular localization were analyzed to elucidate the molecular mechanism of PyDET2e involved in the rapid growth of P. yunnanensis.

Plant Materials and Vector
The cuttings of 3 clones of P. yunnanensis were cultivated in the greenhouse for one year (Southwest Forestry University, Kunming, China.102.76 E, 25.06 N).In February 2022, we obtained about 15 cm cuttings and preserved these in the soil.The cuttings with different tissues were used in experiments performed in July 2023.We obtained roots, stem, lateral buds, branches, young leaves (1st to 3rd leaves counted from the stem tip), and the stem tip.All plant samples collected were brought back in liquid nitrogen and then stored at −80 • C until the next use.The plant expression vector pSuper1300-GFP was kept in our laboratory.

Date Sources
The complete genome sequence and annotation files of P. yunnanensis wereretrieved from our laboratory (Southwest Forestry University, Kunming 6500224, China) (BioProject:PRJNA886471).The RNA-seq data used in this study were obtained from previous studies in our laboratory.The DET2 protein sequences of A. thaliana and P. trichocarpa were obtained from the phytozome database (https://phytozome-next.jgi.doe.gov/,accessed on 15 June 2023).

Genome-Wide Identification of DET2 Gene Members in P. yunnanensis
The HMM data of the steroid dehydrogenase structural domain (PF02544) were obtained from the online server of the Inter Pro database (https://www.ebi.ac.uk/interpro/, accessed on 15 June 2023).Then, the protein sequences of the DET2 gene family members of A. thaliana and P. trichocarpa were downloaded from the Phytozome database (https://phytozome-next.jgi.doe.gov/,accessed on 15 June 2023) using PF02544.Proteome files were extracted using TBtools (v2.008) based on the genome and GFF files of P. yunnanensis [30].First, we queried the homologous sequences in the P. yunnanensis genome based on the known AtDET2 protein sequences by performing a BLASTp search (E-value < 10 −14 ) with TBtools (v2.008).Then, we further screened the conserved structural domains using the NCBI Conserved Structural Domain Search tool (CD-Search) (https://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi, accessed on 15 June 2023).After removing incomplete and redundant sequences, the existence of the DET2 gene family of P. yunnanensis was determined.

Chromosomal Location and Collinearity Analysis
The location of PyDET2s genes on chromosomes and collinearity analysis were visualized using TBtools (v2.008).

Codon Bias and Influence Factors Analysis
CodonW and Emboss (http://emboss.toulouse.inra.fr/cgi-bin/emboss/chips,accessed on 17 June 2023) were used to calculate the codon bias parameters of the coding proteins of the DET2 gene family of P. yunnanensis, and then the analysis ENc-Plot, PR2-plot, and Neutral mapping was conducted using R Studio (V3.6.0)[32].

Expression Pattern Analysis of PyDET2e
Expression patterns were analyzed using TBtools software by constructing PyDET2s expression heatmaps based on RNA-seq data.The primers for qRT-PCR were designed from CDS sequences using the NCBI primer design tool (https://www.ncbi.nlm.nih.gov/tools/primer-blast/, accessed on 19 June 2023) (Table S1).
Firstly, the total RNA extraction was performed using the E.Z.N.A@ Plant RNA kit (Omega Bio-tek Inc., Beijing, China), and the quality and purity of RNA were detected using K5800C (KAIAO, Beijing, China).The 1-st cDNA synthesis was performed using Hifair ® III Reverse Transcriptase (YEASEN, Shanghai, China) on 500ng of RNA from each sample.The cDNA was then diluted 7-fold for qRT-PCR.The Applied Biosystems 6800 real-time PCR machine and Hieff SYBR Green Master Mix (Yeasen Biotechnology Co. Ltd., Shanghai, China) were used to perform qRT-PCR following the specified system and procedures.The reaction system (20.0 µL) included the following: 1.0 µL cDNA template, 0.4 µM primer (F/R), 10 µL mix, and 8.2 µL RNase-free water.The qRT-PCR thermal cycle conditions were as follows: denaturation at 95 • C (2 min), 45 cycles at 95 • C (10 s), and 56 • C (30 s).Fluorescence intensities were measured for qRT-PCR at the end of each cycle.The relative transcript abundance values were calculated using the 2 −∆∆Ct method.The data statistical analysis and visualization were performed by SPSS21 (p-values equal to 0.05 were kept statistically significant) and GraphPad (v8.0.2).

Subcellular Localization of PyDET2e
The localization of PyDET2e was determined by injecting tobacco epidermal cells using the pSuper1300-GFP vector with the CaMV35S promoter.For this purpose, we designed appropriate primers with restriction sites (Xbal 1 and Hind III) according to the CDS sequence of PyDET2e and the multiple cloning site sequence in the vector and cloned this fragment into the pMD-19T vector.Then, positive bacteria were sent for sequencing (Shanghai Biotechnology Co., Ltd., Shanghai, China) after transformation.After sequencing, the gene fragments were digested with the restriction enzymes (Xbal 1 and Hind III) and ligated into the pSuper1300-GFP vector digested by Xbal 1 and Hind III using T4 ligase (Takara Bio, Shanghai, China).The over expression vector PyDET2e-pSuper1300-GFP was successfully transformed into tobacco epidermal cells, while pSuper1300-GFP alone was transformed to use as a control.Three days after injection into the abaxial epidermis of tobacco leaves, protein expression was observed using fluorescence confocal microscopy.

Identification of the DET2 Genes in P. yunnanensis
Fourteen DET2 genes were identified in P. yunnanensis and named PyDET2a-PyDET2n based on their positions on the chromosome (Table S2).They differed in sequence length, isoelectric point, and molecular weight.The amino acid length variedfrom 253 aa (PyDET2b) to 351 aa (PyDET2f ).The molecular weight ranged from 20.71 kDa (PyDET2k) to 40.35 kDa (PyDET2f ) with an average of 31.44 kDa, and the pI ranged from 8.79 (PyDET2a) to 9.66 (PyDET2e) with an average of 9.32.The analysis of the total average index of hydrophobicity (GRAVY) showed that all proteins were hydrophobic.Most of the proteins were unstable with an instability index ranging from 22.63 (PyDET2l) to 46.84 (PyDET2a).The secondary structure of all proteins was dominated by α-helices (Table S2 and Figure S1).Subcellular localization predictions indicated that most were mainly located in chloroplasts and cell membranes.

Phylogenetic Analysis and Motif Elicitation of PyDET2s
The phylogenetic tree to understand the evolutionary relationships of three species using the protein sequences of the members was built, which consisted of 7 AtDET2s, 14 PyDET2s, and 10 PtDET2s, which were unevenly divided into five subgroups (Figure 1).We also found that the genes in the same group had similar structures (Figure 2). of 29 amino acids, which appears as a dark green block (motif 1), which is presumed to be conservative in this family, and this motif was present at the N-terminus of all members.The composition of the motif is similar in the same subgroup.The first subgroup contained motif 1, 2, 3, 4, 9, and 10.The second subgroup contained motif 9 and 10.The third subgroup contained motif 1, 2, 5, 6, 7, 9, and 10.The fourth subgroup only contained motif 1.The conserved protein motifs of the DET2 proteins are shown in Figure 2. It was found that approximately all sequences contained a conserved protein motif with a width of 29 amino acids, which appears as a dark green block (motif 1), which is presumed to be conservative in this family, and this motif was present at the N-terminus of all members.The composition of the motif is similar in the same subgroup.The first subgroup contained motif 1, 2, 3, 4, 9, and 10.The second subgroup contained motif 9 and 10.The third subgroup contained motif 1, 2, 5, 6, 7, 9, and 10.The fourth subgroup only contained motif 1.
We analyzed the collinear blocks and gene duplication types.Five duplication PyDET2 gene pairs were observed (PyDET2a-PyDET2f, PyDET2b-PyDET2c, PyDET2b-PyDET2g, PyDET2b-PyDET2j, and PyDET2c-PyDET2g), and located on different chromosomes (chromosome 4, 5, 8, 9, 10, and 13) in P. yunnanensis (Figure 4).During the evolution of PyDET2s, the replication events including whole genome duplication and segmental duplication were identified.A total of 6 pairs of collinearity genes were found between P. yunnanensis and A. Thaliana, and 14 pairs of collinearity genes were found between P. yunnanensis and P. trichocarpa, but no collinearity was found between PyDET2d, h, i and k (Figure 5).We analyzed the collinear blocks and gene duplication types.Five duplication PyDET2 gene pairs were observed (PyDET2a-PyDET2f, PyDET2b-PyDET2c, PyDET2b-PyDET2g, PyDET2b-PyDET2j, and PyDET2c-PyDET2g), and located on different chromosomes (chromosome 4, 5, 8, 9, 10, and 13) in P. yunnanensis (Figure 4).During the evolution of PyDET2s, the replication events including whole genome duplication and segmental duplication were identified.A total of 6 pairs of collinearity genes were found between P. yunnanensis and A. Thaliana, and 14 pairs of collinearity genes were found between P. yunnanensis and P. trichocarpa, but no collinearity was found between PyDET2d, h, i and k (Figure 5).
We analyzed the collinear blocks and gene duplication types.Five duplication PyDET2 gene pairs were observed (PyDET2a-PyDET2f, PyDET2b-PyDET2c, PyDET2b-PyDET2g, PyDET2b-PyDET2j, and PyDET2c-PyDET2g), and located on different chromosomes (chromosome 4, 5, 8, 9, 10, and 13) in P. yunnanensis (Figure 4).During the evolution of PyDET2s, the replication events including whole genome duplication and segmental duplication were identified.A total of 6 pairs of collinearity genes were found between P. yunnanensis and A. Thaliana, and 14 pairs of collinearity genes were found between P. yunnanensis and P. trichocarpa, but no collinearity was found between PyDET2d, h, i and k (Figure 5).

Prediction of Cis-Regulatory Elements in the Promoters of PyDET2s
We studied the potential regulation of cis-acting elements in PyDET2s (Figure 6).Twenty-five cis-regulatory elements were identified, and light responsiveness was the most important element.We also found many hormone responses, and stress elements were abundant in the promoter regions of family members, such as abscisic acid responsiveness (ABRE), anaerobic induction element (ARE), MeJA responsiveness (CGTCA motif), auxin responsiveness (AuxRR-core, TGA-element), gibberellin responsiveness (Pbox), salicylic acid responsiveness (TCA-element), zein metabolism regulation element (O2-site), defense and stress responsiveness (TC-rich repeats), low-temperature responsiveness (LTR), and other elements containing MYB binding sites which regulate light responses and drought stress.A number of elements regulating plant growth and development were also found, including meristem expression element (CAT-box) and endosperm expression element (GCN4-motif).Thus, the members of this family play important roles in the growth and development of P. yunnanensis.

Analysis of Codon Preference and Its Influencing Factors
The codon usage bias in the DET2 family of P. yunnanensis was analyzed (Table S3).The codon adaptation index (CAI) of DET2 family genes ranged from 0.168 to 0.211, with an average of 0.186.The codon preference index (CBI) of DET2 family genes ranged from −0.142 to 0.045, with an average of −0.05, and the optimal codon usage frequency FOP values ranged from 0.323 to 0.420, with an average of 0.372, and less than 0.5.The content

Prediction of Cis-Regulatory Elements in the Promoters of PyDET2s
We studied the potential regulation of cis-acting elements in PyDET2s (Figure 6).Twenty-five cis-regulatory elements were identified, and light responsiveness was the most important element.We also found many hormone responses, and stress elements were abundant in the promoter regions of family members, such as abscisic acid responsiveness (ABRE), anaerobic induction element (ARE), MeJA responsiveness (CGTCA motif), auxin responsiveness (AuxRR-core, TGA-element), gibberellin responsiveness (Pbox), salicylic acid responsiveness (TCA-element), zein metabolism regulation element (O 2 -site), defense and stress responsiveness (TC-rich repeats), low-temperature responsiveness (LTR), and other elements containing MYB binding sites which regulate light responses and drought stress.A number of elements regulating plant growth and development were also found, including meristem expression element (CAT-box) and endosperm expression element (GCN4-motif).Thus, the members of this family play important roles in the growth and development of P. yunnanensis.

Prediction of Cis-Regulatory Elements in the Promoters of PyDET2s
We studied the potential regulation of cis-acting elements in PyDET2s (Figur Twenty-five cis-regulatory elements were identified, and light responsiveness wa most important element.We also found many hormone responses, and stress elem were abundant in the promoter regions of family members, such as abscisic acid res siveness (ABRE), anaerobic induction element (ARE), MeJA responsiveness (CG motif), auxin responsiveness (AuxRR-core, TGA-element), gibberellin responsive (Pbox), salicylic acid responsiveness (TCA-element), zein metabolism regulation ele (O2-site), defense and stress responsiveness (TC-rich repeats), low-temperature res siveness (LTR), and other elements containing MYB binding sites which regulate responses and drought stress.A number of elements regulating plant growth and velopment were also found, including meristem expression element (CAT-box) and dosperm expression element (GCN4-motif).Thus, the members of this family play portant roles in the growth and development of P. yunnanensis.

Analysis of Codon Preference and Its Influencing Factors
The codon usage bias in the DET2 family of P. yunnanensis was analyzed (Table The codon adaptation index (CAI) of DET2 family genes ranged from 0.168 to 0.211, an average of 0.186.The codon preference index (CBI) of DET2 family genes ranged −0.142 to 0.045, with an average of −0.05, and the optimal codon usage frequency values ranged from 0.323 to 0.420, with an average of 0.372, and less than 0.5.The co

Analysis of Codon Preference and Its Influencing Factors
The codon usage bias in the DET2 family of P. yunnanensis was analyzed (Table S3).The codon adaptation index (CAI) of DET2 family genes ranged from 0.168 to 0.211, with an average of 0.186.The codon preference index (CBI) of DET2 family genes ranged from −0.142 to 0.045, with an average of −0.05, and the optimal codon usage frequency FOP values ranged from 0.323 to 0.420, with an average of 0.372, and less than 0.5.The content of T3s ranged from 0.3364 to 0.4514, with an average of 0.3967.The content of A3s ranged from 0.1761 to 0.3574, with an average of 0.2851.The content of C3s ranged from 0.1701 to 0.3458, with an average of 0.2674.The content of G3s ranged from 0.2584 to 0.3638, Genes 2024, 15, 148 9 of 14 with an average of 0.2803.The frequency of GC1 (except PyDET2m), GC2, and GC3(except PyDET2n) and the average GC (except PyDET2n) content were below 50%.These results indicated a preference for A/U-ending codons.
A total of 3933 codons (including stop codons) were found in the 14 DET2 genes of P. Yunnanensis.The RSCU value analysis of the codons showed that there were 2331 codons with RSCU > 1, and the RSCU of AGA was 2.32 with the highest frequency among the 59 codons (excluding stop codon and start codon).There were 29 high-frequency codons that showed obvious A/U preference ending (Figure 7).
A total of 3933 codons (including stop codons) were found in the 14 DET2 genes of P. Yunnanensis.The RSCU value analysis of the codons showed that there were 2331 codons with RSCU > 1, and the RSCU of AGA was 2.32 with the highest frequency among the 59 codons (excluding stop codon and start codon).There were 29 high-frequency codons that showed obvious A/U preference ending (Figure 7).
The ENc-plot, Neutral-plot, and PR2-plot analyses are shown (Figure 8).The DET2 family members have GC3s distributions ranging from 0.368 to 0.597, with the exception of PyDET2n with a GC3s content of 0.565 (greater than 0.5), and the distribution of CDSs was not evenly around the center point (A = U/T, G = C).So the formation of codon usage patterns is affected by mutation pressure and natural selection.The correlation coefficient and regression coefficient were 0.13 and 0.394, respectively, which indicated that the correlation between the base composition at the first and second position of the codon and that at the third position of the codon was weak, and the coding gene of the DET2 gene family was highly conserved; codon preference is strongly influenced by selection pressure.The ENc-plot, Neutral-plot, and PR2-plot analyses are shown (Figure 8).The DET2 family members have GC3s distributions ranging from 0.368 to 0.597, with the exception of PyDET2n with a GC3s content of 0.565 (greater than 0.5), and the distribution of CDSs was not evenly around the center point (A = U/T, G = C).So the formation of codon usage patterns is affected by mutation pressure and natural selection.The correlation coefficient and regression coefficient were 0.13 and 0.394, respectively, which indicated that the correlation between the base composition at the first and second position of the codon and that at the third position of the codon was weak, and the coding gene of the DET2 gene family was highly conserved; codon preference is strongly influenced by selection pressure.
Genes 2024, 15, x FOR PEER REVIEW 9 of of T3s ranged from 0.3364 to 0.4514, with an average of 0.3967.The content of A3s range from 0.1761 to 0.3574, with an average of 0.2851.The content of C3s ranged from 0.1701 0.3458, with an average of 0.2674.The content of G3s ranged from 0.2584 to 0.3638, wit an average of 0.2803.The frequency of GC1 (except PyDET2m), GC2, and GC3(excep PyDET2n) and the average GC (except PyDET2n)content were below 50%.These resul indicated a preference for A/U-ending codons.A total of 3933 codons (including stop codons) were found in the 14 DET2 genes of Yunnanensis.The RSCU value analysis of the codons showed that there were 2331 codon with RSCU > 1, and the RSCU of AGA was 2.32 with the highest frequency among the 5 codons (excluding stop codon and start codon).There were 29 high-frequency codon that showed obvious A/U preference ending (Figure 7).
The ENc-plot, Neutral-plot, and PR2-plot analyses are shown (Figure 8).The DET family members have GC3s distributions ranging from 0.368 to 0.597, with the exceptio of PyDET2n with a GC3s content of 0.565 (greater than 0.5), and the distribution of CDS was not evenly around the center point (A = U/T, G = C).So the formation of codon usag patterns is affected by mutation pressure and natural selection.The correlation coeff cient and regression coefficient were 0.13 and 0.394, respectively, which indicated that th correlation between the base composition at the first and second position of the codo and that at the third position of the codon was weak, and the coding gene of the DET gene family was highly conserved; codon preference is strongly influenced by selectio pressure.

Gene Expression Analysis
Based on the RNA-seq transcriptome data, the expression heatmaps of DET2 family genes in the stem tips of P. yunnanensis at two growth stages (initial and rapid growth stages) and two cuttings (upright cuttings and inverted cuttings) were visualized.The result showed that three genes (PyDET2h, PyDET2i, and PyDET2e) showed higher expression in September than in July, both in inserted and upright cuttings (Figure 9).Therefore, three genes play an important role in the rapid growth of P. yunnanensis.We selected PyDET2e for qRT-PCR.The results showed that the transcriptome data were reliable.Subsequently, we analyzed its tissue specificity and found that its expression level in the stem, stem tip, and leaf was significantly higher (Figure 9).

Gene Expression Analysis
Based on the RNA-seq transcriptome data, the expression heatmaps of DET2 family genes in the stem tips of P. yunnanensis at two growth stages (initial and rapid growth stages) and two cuttings (upright cuttings and inverted cuttings) were visualized.The result showed that three genes (PyDET2h, PyDET2i, and PyDET2e) showed higher expression in September than in July, both in inserted and upright cuttings (Figure 9).Therefore, three genes play an important role in the rapid growth of P. yunnanensis.We selected PyDET2e for qRT-PCR.The results showed that the transcriptome data were reliable.Subsequently, we analyzed its tissue specificity and found that its expression level in the stem, stem tip, and leaf was significantly higher (Figure 9).

Vector Construction and Subcellular Localization of PyDET2e
Online website predictions show that PyDET2e is localized to chloroplasts.To determine the location of PyDET2e protein in the cells, PyDET2e-pSuper1300-GFP was used as an experimental group and pSuper1300-GFP was used as a control.Three days after the injection of tobacco leaves, the position of the fusion protein was observed by fluorescence confocal microscopy.The results showed that PyDET2e protein was distributed in chloroplasts.However, pSuper1300-GFP alone was distributed in the cytoplasm and the nucleus (Figure 10).

Vector Construction and Subcellular Localization of PyDET2e
Online website predictions show that PyDET2e is localized to chloroplasts.To determine the location of PyDET2e protein in the cells, PyDET2e-pSuper1300-GFP was used as an experimental group and pSuper1300-GFP was used as a control.Three days after the injection of tobacco leaves, the position of the fusion protein was observed by fluorescence confocal microscopy.The results showed that PyDET2e protein was distributed in chloroplasts.However, pSuper1300-GFP alone was distributed in the cytoplasm and the nucleus (Figure 10).

Discussion
BRs have a wide range of physiological functions, such as plant height, plant type, flowering time, plant root growth, stem elongation, leaf extension, microtubule system development, plant morphology in dark conditions, pollen tube elongation, and seed

Discussion
BRs have a wide range of physiological functions, such as plant height, plant type, flowering time, plant root growth, stem elongation, leaf extension, microtubule system development, plant morphology in dark conditions, pollen tube elongation, and seed development [33][34][35].The main signal transduction pathways of BRs have been established, and the key gene functions of these pathways have been verified in A. thaliana; the DET2 gene is a key rate-limiting gene in brassinolide biosynthesis pathway, and its effect is self-evident [36][37][38].At present, the DET2 gene has been identified in many angiosperms and gymnosperms [23,[39][40][41][42], and few studies on woody plants have been reported.Some studies on other plants have been limited to the cloning of related homologous genes of A. thaliana and the analysis of corresponding mutants.Different plants may have different synthetic pathways, so it is necessary to verify the universality of these pathways or reveal new synthetic pathways through the study of many types of plants in the future.As to whether the BR biosynthesis and signal transduction mechanism of woody plants are identical or specific to A. thaliana, and whether there are similarities and differences in the roles played by DET2, nothing is certain.The study on the relationship between the steroid dehydrogenase family and its members in woody plants can provide a molecular basis for the rapid growth of P. yunnanensis and quicken the process of forest genetic breeding.The whole genome sequencing of P. yunnanensis has been completed, which provides important data support for the molecular biology research of P. yunnanensis.
Fourteen DET2 genes were identified in this study.The PyDET2 proteins have different sequence lengths.Most PyDET2 proteins have isoelectric points greater than 7, suggesting that PyDET2 genes may encode an alkaline protein that exerts biological functions in alkaline subcellular environments [43].The 14 genes of the DET2 family were classified into four groups (group 1, group 2, group 3, and group 4).The analysis of the 14 promoter regions of PyDET2 genes suggests that the PyDET2 proteins are involved in many growth and development processes (Figure 6).Codon analysis showed that genes in this family have a clear A/U preference and that natural selection is the main factor influencing their formation (Figure 8).Gene duplication is one of the major forces that act on gene expansion and ultimately drive biological evolution.In total, 14 DET2 genes have evolved repeatedly from WGD and fragments.The two types of gene duplication also contributed to the amplification of DET2s.The collinearity analysis showed that some genes had no collinearity between the DET2 family and A. thaliana and P. trichocarpa; these may be DET2 members with new functions in P. yunnanensis (Figure 5).
The stem tip secretes and accumulates various hormones, and through growth, division, and differentiation, the stem elongates unceasingly and forms a stem-related structure.In our previous research, we found that the height growth and stem diameter of P. yunnanensis cuttings in September were significantly higher than those in July, whether it was upright or inverted cuttings.Based on the RNA-seq of stem-tip and qRT-PCR in the DET2 family, we found that the expression of the PyDET2h, PyDET2i, and PyDET2e was significantly higher.Three genes have similar motifs and gene structures, which suggests that they have similar effects on the growth of P. yunnanensis.These genes are located in group1, and their codon composition is quite similar, so it is inferred that they play similar biological functions (Figure 9).The PyDET2e was selected to clone and construct an over expression vector containing GFP for subcellular localization.The CDS length of PyDET2e is 795 bp, encoding 264 amino acids.It is an unstable hydrophobic protein with no signal peptide and three transmembrane regions composed of α-helices, and it was presumed that the protein is a non-secretory membrane protein with a hydrophobic index of 0.34, which accords with the characteristics of membrane localization protein.Protein phosphorylation is at the end of the signaling chain and can be achieved via the phosphorylation of transcription factors for the purpose of regulating genes, and the site predictions indicate that there are 17 sites at which serine may be phosphorylated.It is speculated that the changes in the conformation of PyDET2e protein may be regulated by the SER phosphorylation site; whether the predicted phosphorylation sites really exist and what roles they play can be further analyzed via protein phosphorylation modification omics and Western blot methods (Figure S2).The subcellular localization results showed that PyDET2e was expressed in the chloroplast and a little in the cytoplasm.To determine which part of the chloroplast it is expressed in, further analysis is needed (Figure 10).
Chloroplasts are the main sites of photosynthesis in plants and are involved in many aspects, such as sulfur and nitrogen assimilation, fatty acid, amino acid, and hormone synthesis [43][44][45].Plant hormones such as brassinolide, cytokinin, auxin, and gibberellin are regulated by light and control chloroplast growth and development [46,47].In plant green tissues, a complex network of transcription factors, light, and hormone signals is formed, and regarding PyDET2e, its expressed within a specific part of the chloroplast and the role of this gene in chloroplast development and BRs synthesis remains to be further studied and demonstrated.In other studies, GmDET2a and GmDET2b genes were widely expressed in the root, leaf, and hypocotyl of soybean [42], and the expression of the LeDET2 gene was highest in the leaves of tomato [41].The tissue specificity of our study indicated that PyDET2e was highly expressed not only in stem tips and stems, but also in leaves, and this may be due to species variability leading to differences in gene function and thus differences in expression tissues.BR does not require polarity transport; it is evident that BR synthesis is active in three tissues, which suggests that it plays an important role in the leaf, stem, and tip of stem development, and the mechanism of DET2 at different tissues needs to be further investigated.

Conclusions
We identified 14 DET2 family genes in P. yunnanensis and analyzed the genetic structure, phylogenetic relationships, cis-regulatory elements, collinearity, subcellular localization, and expression.The result suggested that DET2 genes might be involved in plant growth, development, and hormone signal transduction.The tissue-specific analysis of PyDET2e revealed that PyDET2e in September was significantly higher than that in July (both in upright and inverted cutting), and the expression of it in the stem, stem tip, and leaf was significantly higher than that in other parts; subcellular localization showed that PyDET2e protein was located in the chloroplast.

Figure 2 .
Figure 2. The conserved motifs of proteins and gene structure depending on the phylogenetic relationships of DET2 genes in P. yunnanensis.(a) The phylogenetic tree of 14 DET2 proteins was built.Four subgroups (group 1, group2, group 3, and group 4) are shown in different colors.(b) Analysis of conserved motifs of DET2 genes in P. yunnanensis.Gray lines represent sequences of different lengths, and blocks of different colors represent different conserved motifs.(c) Exon and intron structure analysis of DET2 genes in P. yunnanensis.Black lines represent introns, yellow boxes represent exons, and green boxes represent untranslated regions (UTR).

Figure 2 .
Figure 2. The conserved motifs of proteins and gene structure depending on the phylogenetic relationships of DET2 genes in P. yunnanensis.(a) The phylogenetic tree of 14 DET2 proteins was built.Four subgroups (group 1, group2, group 3, and group 4) are shown in different colors.(b) Analysis of conserved motifs of DET2 genes in P. yunnanensis.Gray lines represent sequences of different lengths, and blocks of different colors represent different conserved motifs.(c) Exon and intron structure analysis of DET2 genes in P. yunnanensis.Black lines represent introns, yellow boxes represent exons, and green boxes represent untranslated regions (UTR).

Figure 3 .
Figure 3.The distribution of PyDET2s in chromosomes.The vertical bar represents the chromosomes of P. yunnanensis.The scale on the left indicates chromosome length.From left to right are 9 chromosomes with DET2 gene distribution.The PyDET2s are shown in red.

Figure 3 .
Figure 3.The distribution of PyDET2s in chromosomes.The vertical bar represents the chromosomes of P. yunnanensis.The scale on the left indicates chromosome length.From left to right are 9 chromosomes with DET2 gene distribution.The PyDET2s are shown in red.

Figure 3 .
Figure 3.The distribution of PyDET2s in chromosomes.The vertical bar represents the chromosomes of P. yunnanensis.The scale on the left indicates chromosome length.From left to right are 9 chromosomes with DET2 gene distribution.The PyDET2s are shown in red.

Figure 4 .
Figure 4. Synteny analysis of DET2 family members in P. yunnanensis.The genes linked by the gray line represent the homologous DET2s between PyDET2s.

Figure 4 .
Figure 4. Synteny analysis of DET2 family members in P. yunnanensis.The genes linked by the gray line represent the homologous DET2s between PyDET2s.

Figure 5 .
Figure 5. Collinearity analysis of DET2 samong three species.All collinear genes were labeled in gray, while the collinear DET2 gene pairs were labeled in blue.

Figure 6 .
Figure 6.Cis-acting elements in the promoter regions of 14DET2 genes.The black lines represent the promoter length.The different colored boxes represent different elements.

Figure 5 .
Figure 5. Collinearity analysis of DET2s among three species.All collinear genes were labeled in gray, while the collinear DET2 gene pairs were labeled in blue.

Figure 4 .
Figure 4. Synteny analysis of DET2 family members in P. yunnanensis.The genes linked by the line represent the homologous DET2s between PyDET2s.

Figure 5 .
Figure 5. Collinearity analysis of DET2 samong three species.All collinear genes were label gray, while the collinear DET2 gene pairs were labeled in blue.

Figure 6 .
Figure 6.Cis-acting elements in the promoter regions of 14DET2 genes.The black lines repr the promoter length.The different colored boxes represent different elements.

Figure 6 .
Figure 6.Cis-acting elements in the promoter regions of 14 DET2 genes.The black lines represent the promoter length.The different colored boxes represent different elements.

Figure 7 .
Figure 7. Relative codon usage of the DET2 gene family in P. yunnanensis.

Figure 8 .
Figure 8. Analysis of influencing factors of preference for the usage of codons in the DET2 gene family in P. yunnanensis.From left to right: (a) ENc-plot; (b) neutral plot; (c): PR2-plot analysis.

Figure 7 .
Figure 7. Relative codon usage of the DET2 gene family in P. yunnanensis.

Figure 7 .
Figure 7. Relative codon usage of the DET2 gene family in P. yunnanensis.

Figure 8 .
Figure 8. Analysis of influencing factors of preference for the usage of codons in the DET2 gen family in P. yunnanensis.From left to right: (a) ENc-plot; (b) neutral plot; (c): PR2-plot analysis.

Figure 8 .
Figure 8. Analysis of influencing factors of preference for the usage of codons in the DET2 gene family in P. yunnanensis.From left to right: (a) ENc-plot; (b) neutral plot; (c): PR2-plot analysis.

Figure 9 .
Figure 9. (a): The expression patterns ofPyDET2genes.7/9UC: upright cuttings of P. yunnanensis in July/September.7/9IC: invert cuttings of P. yunnanensis in July/September.Blue or red color indicates lower or higher expression levels of each sample, respectively.(b): The relative expression levels ofPyDET2e in the stem tip in two growth phases and cutting methods.(c):The relative expression levels of PyDET2e in different tissues.Statistical significance (p < 0.05) is indicated by lowercase letters.

Figure 9 .
Figure 9. (a): The expression patterns of PyDET2 genes.7/9UC: upright cuttings of P. yunnanensis in July/September.7/9IC: invert cuttings of P. yunnanensis in July/September.Blue or red color indicates lower or higher expression levels of each sample, respectively.(b): The relative expression levels of PyDET2e in the stem tip in two growth phases and cutting methods.(c): The relative expression levels of PyDET2e in different tissues.Statistical significance (p < 0.05) is indicated by lowercase letters.

Genes 2024 , 15 Figure 10 .
Figure 10.Subcellular localization analysis of PyDET2e in the leaves of tobacco; images show an abaxial side view of leaf epidermis with localization of chloroplast marked with chloroplast self-luminescence (Green is GFP fluorescence and red is chloroplast self-luminescence).

Figure 10 .
Figure 10.Subcellular localization analysis of PyDET2e in the leaves of tobacco; images show an abaxial side view of leaf epidermis with localization of chloroplast marked with chloroplast selfluminescence (Green is GFP fluorescence and red is chloroplast self-luminescence).