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Article

Morphological, Histological, and Transcriptome Analysis of Doubled Haploid Plants in Poplars (Populus simonii × Populus nigra)

by
Yiran Wang
1,†,
Jiajie Yu
1,†,
Xiang Zhang
1,
Yaxin He
1,
Song Chen
1,
Erqin Fan
1,2,
Guanzheng Qu
1,
Su Chen
1,* and
Caixia Liu
1,3,*
1
State Key Laboratory of Tree Genetics and Breeding, Northeast Forestry University, Harbin 150040, China
2
State Key Laboratory of Tree Genetics and Breeding, Chinese Academy of Forestry, Beijing 100091, China
3
College of Life Science, Northeast Forestry University, Harbin 150040, China
*
Authors to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Forests 2023, 14(8), 1535; https://doi.org/10.3390/f14081535
Submission received: 30 May 2023 / Revised: 19 July 2023 / Accepted: 21 July 2023 / Published: 27 July 2023
(This article belongs to the Section Genetics and Molecular Biology)
Browse Figures
Versions Notes

Abstract

:
In this study, the poplar doubled haploid (DH) plants were used as the experimental material to explore the huge phenotypic differences between homozygous DH plants and the paternal plants, and the molecular regulation mechanism of the differential phenotypes. In this experiment, through morphological and histological observation and statistics, we found that the double haploid plants had significantly reduced plant height and ground diameter, increased leaf aspect ratio, premature senescence phenotype of top bud, and significant changes in the shape and cell area of the shoot apical meristem. Significantly differentially expressed genes were obtained using RNA-seq transcriptome sequencing. They were subjected to GO enrichment and KEGG analysis. Transcription factors with key functions were screened out for qRT-PCR to verify gene expression changes to predict gene function. The results showed that after the IAA and ABA treatment, the expression levels of some hormone-responsive genes in wild type plants were significantly changed with different treatment time. In the dihaploid plants, the corresponding genes also changed to different degrees, which reflected the changes in the response of the dihaploid plants to hormones. Compared to in WT, the differential expressed genes in the double haploids were involved in multiple physiological process such as response to oxidative stress, response to salicylic acid, plant pathogen interaction, and plant hormone signal transduction. A TF–centered gene regulatory network for phytohormone synthesis and plant senescence was constructed with the expression patterns of differentially expressed transcription factors (TFs). This study increases researchers’ understanding of the regulation of poplar growth and development and provides new research ideas for the creation of new species of poplar.

1. Introduction

Poplars (Populus simonii × Populus nigra) has been widely applied in Northeast China because of its excellent cold resistance, drought resistance, and fast growth. In addition, due to its excellent wood structure, it is also widely used as industrial raw materials, civil construction, and agricultural materials. However, its reproduction mode is mainly asexual reproduction, so seed breeding becomes extremely important. In conclusion, poplars (Populus simonii × Populus nigra) were chosen as pollen donors and control material for double haploid plants in this study.
Due to the long growth cycle, large number of genes, and complex gene regulatory networks in woody plants [1], progress in germplasm resource creation and gene function research is slow. If haploid breeding technology is combined with tree breeding, the problems of forest germplasm resource innovation and gene function research will be effectively alleviated. The use of haploid breeding can quickly obtain the desired trait homozygotes, shorten the breeding period, and speed up the breeding process [2]. It can provide a good material basis for the creation of new varieties and the study of gene function [3,4]. Breaking the inherent way of reverse genetics research, using forward genetics to explore the gene function of forest trees is a more rapid and targeted approach for investigating the regulatory relationship between phenotype and gene.
Haploid or doubled haploid (DH) plants have the advantages of genome homozygosity and high mutation rates, which make them important materials for genetic research, molecular biology research, gene function analysis, and transformation research. The long growth cycle of poplar, the large number of genes, and the complex gene regulatory network limit the creation of germplasm resources and gene function research. Therefore, the application of haploid or doubled haploid in forest trees can solve this problem.
An apical bud is typically located at the top of the main stem or lateral branch of a plant, and its growth status directly affects the overall morphology and structure of the plant. As one of the most active growing points in plants, the apical bud is not only a reproductive organ but also an important environmental perception organ [5]. It can adjust the growth rhythm of plants by sensing changes in the surrounding environment. For example, under environmental stresses such as low temperature and drought, plants can form dormant buds to avoid serious damage caused by environmental changes [6]. Additionally, the apical bud can regulate tissue and organ development by promoting or inhibiting stem cell differentiation through hormone regulation [7]. Therefore, it is of great significance to study the regulatory mechanism of tree terminal bud development for understanding the hormonal regulation of tree growth and development and tree response to abiotic stress. The activity of apical meristem tissue is controlled with a complex regulatory network, which involves transcription factors, miRNAs, peptides, kinases, and epigenetic markers [8]. Many key genes that determine the intrinsic characteristics of apical meristem tissue have been identified through genetic methods in arabidopsis, and the genes related to apical bud growth and development have been well-characterized in rice [9] (Oryza sativa L.), arabidopsis [10] (Arabidopsis thaliana), and maize [11] (Zea mays L.) The study of apical bud growth has not been fully explored in poplar [6].
In this study, we compared the difference in terminal bud growth between doubled haploid and paternal Populus simonii × P. nigra. At the molecular level, we revealed the primary cause of significant differences in growth and development of top buds among plants with different genotypes. This will further our understanding of tree top bud regulation to hormones and response to environmental stress, facilitating accelerated forest breeding processes and further exploring the regulation mechanism of plant top-bud growth and development.

2. Materials and Methods

2.1. Plant Materials

Double haploid plants are homozygous diploid plants obtained from the pollen of paternal poplars (Populus simonii × Populus nigra) after cultivation and chromosome doubling [12]. DH (DH1588, DH1207, DH1716, and DH1717) and the paternal plants were grown in soil in a growth chamber (25 ± 1 °C, 16-h-light/8-h-dark cycle). Three-month-old plants were used for part of further experiments such as morphological observation and bioinformatics analysis. Apical bud tissue for RNA-seq were collected directly into liquid nitrogen and stored in liquid nitrogen. Nondormant shoot apical meristem (SAM) of 1.5-month-old plants were used for histological analysis.

2.2. Morphological and Histological Analysis

Plant height and ground diameter were measured by transferring established plants from WPM medium to soil and incubated at 23 °C with 16 h: 8 h, light: dark photoperiod for three months. The height of the plant (the height from the ground to the top bud of the plant) and the ground diameter of the plant (the diameter of the plant stem close to the soil) were measured. The aspect ratio of a blade is measured by measuring the ratio of the blade’s longitudinal maximum point (from the base of the blade to the tip of the blade) to its transverse widest point. The circularity is calculated by importing the shape of the blade and SAM into ImageJ 1.52p (National Institutes of Health, USA) software and calculating the similarity with the circle. The closer the value is to 1, the closer the shape is to the circle. The 1.5-month-old greenhouse-grown DH (DH1588, DH1207, DH1716, and DH1717) and the paternal plants SAM were observed under a stereoscopic microscope. For that purpose, SAM and stem were collected (stem cut into 2 mm fragments with the aid of razor blades). The SAM and stem fragments were fixed with FAA solution, dehydrated in a graded ethanol series, and incubated in xylene sequentially as described previously [13]. The embedded SAM and stem fragments were cut into 16 μm sections via a rotary microtome (Leica, RM2245) and stained with 0.1% toluidine blue. The sections were imaged and analyzed with an M8 scanner (Precipoint, FM34F056) and Viewpoint 4.6.0 software. Cell area was measured with ImageJ software. Data are presented as the means and standard errors of three plants, and the statistical significance of mean differences was assessed using Students’s t-test. Values are the means and the standard deviation, and asterisks indicate statistical significance at different levels (*, p < 0.05; **, p < 0.01).

2.3. Sample Preparation, cDNA Library Construction and Illumina Sequencing

Total RNA was isolated from SAM samples with the traditional CTAB (Cetyltri-methylammonium Bromide) method as described previously. The RNA samples used for cDNA libraries construction were assessed using the Qubit Fluorometer and the Agilent 2100 Bioanalyzer. The qualified RNA samples were then used for cDNA libraries construction according to the user manual of RNA-Seq Library Preparation Kit (Illumina) and subsequent sequencing. The paired-end read sequencing was constructed by BGI-Wuhan (BGI, Wuhan, China) with the aid of the MGISEQ-2000 platform.

2.4. Differential Expression Profile

The RNA-Seq data were analyzed using the RSEM (RNA-Seq by Expectation-Maximization) pipeline [14]. The reference genome of the populus was downloaded from the phytozme website [15]. After removing reads of low quality with fastp [16], each sample’s data were separately aligned to the Populus trichocarpa v4.1 genome [17]. STAR [18] was selected as the aligner to map sequencing reads to reference genome. The RSEM was used to estimate the gene-level abundance between both isoforms and genes. Gene expression levels were normalized using the trimmed mean of the M-values (TMM) method. Based on the expression matrix output by RSEM, we have identified the differentially expressed genes (DEGs) using edgeR 3.36.0 [19] with the threshold of FDR < 0.05. The fold changes of DEGs were calculated with the average TMM of each DH line divided by the average TMM of the wild type. The repeat samples of each DH line were 3, and the minimum value of count per million was selected to be 1 to filter out the samples with a low expression. We then used the ClusterProfiler program to undertake the enrichment analysis of the significant DEGs considered. GO and KEGG terms with corrected p-values less than 0.05 were reasoned obvious enriched. About the enrichment analysis, we used the DEGSeq R package (1.12.0). p-values were adjusted using the Benjamini & Hochberg method. Corrected p-value of 0.05 and log2 (fold change) of 1 were set as the threshold for visible differential expression. Gene Ontology (GO) enrichment analysis of differentially expressed genes was implemented with the Goseq v1.46.0 R package, in which gene length bias was corrected. GO terms with corrected p-value less than 0.05 were considered significantly enriched by DEGs (Table S1).

2.5. Hormone Treatment

To detect the response of differentially expressed genes to hormones, WT plants were transferred from WPM solid medium to WPM liquid medium and incubated at 23 °C with 16 h: 8 h, light: dark photoperiod for one month. The one-month-old wild type was used as the experimental plant, which was soaked and sprayed with 20 μmol IAA and 100 μmol ABA, respectively, and the infected apical bud were collected at 0 h, 3 h, 6 h, 12 h, 24 h, 48 h, and 72 h after inoculation. Apical bud samples were immediately ground into powder in liquid nitrogen. For the soaking treatment, IAA was mixed into liquid rooting medium and plant roots were put into the medium for rooting. For the spraying treatment, hormones were evenly sprayed in the culture bottle without changing the original culture conditions. Each treatment has five biological repeats.

2.6. qRT-PCR Analysis

One microgram of total RNA was reverse-transcribed using the PrimeScript RT Reagent Kit with gDNA Eraser (Takara, RR047Q). qRT-PCR was performed with TB Green Premix Ex Taq II (Tli RNaseH Plus) (Takara Biomedical Technology Co. Ltd., Beijing, China) on a 7500 Fast Real-time PCR System machine (Applied Biosystems, Waltham, MA, USA) according to the manufacturer’s instructions. The primers are listed in additional Table S2.

3. Results

3.1. Phenotypic Characteristics of the DH Plants

To investigate the growth characteristics, we compared four lines of DH plants, DH1588, DH1717, DH1716, and DH1207, with the paternal diploid plants. We found that although the DH plants can grow in the soil, they exhibited obvious growth defects (Figure 1a). The plant heights of DH1717, DH1716, and DH1207 were much lower than the diploid plants. DH1588 had a relatively good growth vigor, but it was also significantly lower than the diploid plants (Figure 1b).
We then measured plant heights (Figure 1b) and ground diameters (Figure 1d) to quantify the growth differences between the diploid and DH plants. The results showed that the average heights of the one-year-old diploid plants and DH1588 were 193.96 and 152.3 cm, and the ground diameters were 9.69 and 7.16 mm, respectively. For DH1207, the plant heights were only one third of that of the diploid plants, and the ground diameters were half of the diploid plants. For DH1717 and DH1716, their plant height was only one-sixth that of the diploid plant, and the ground diameters were one-third that of the wild-type plant. Due to the obvious difference in plant heights, we measured the internode lengths of these plants (Figure 1c) Each treatment has more than three biological repeats. The results showed that the average internode lengths of the internode of the fifth to eighth stem segments of DH1717, DH1716, and DH1207 were 12 mm, 19 mm, and 26 mm, respectively. In contrast, the average internode lengths of the internode of the fifth to eighth stem segments of the diploid plants was 40 mm, and that of DH1588 was 33.68 mm, which was similar to the diploid plants. Therefore, the reduced internode lengths of the DH plants may be responsible for their dwarf phenotypes. In addition to the defective growth, leaf shapes of the DH plants were also altered. The leaves of the diploid plants had a rounded shape with the aspect ratio of 1 (Figure 1e), while the leaves of the DH plants were oval in shape with a greater aspect ratio.
Apical buds are the primary growing points of plants whose structures are important for plant growth and development. The apical buds of the DH and diploid plants were dissected and observed anatomically and histologically (Figure 2a,b). We found that the shape and degree of differentiation in the shoot apical meristems of DH plants differed from those of diploid plants. Specifically, the conical structure of the apical meristem in DH plants was sharper than that in wild–type plants (Figure 2c). Since the degree of differentiation of shoot apical meristem’s differentiated cells was positively correlated with cell size, larger cells exhibited more complete differentiation. DH1717, DH1716, and DH1207 showed a higher degree of cell differentiation (Figure 2d).

3.2. Identification of Differentially Expressed Genes between the DH Plants and the Diploid Plants

RNA-seq was used to identify differentially expressed genes (DEGs) in the apical buds of the DH plants compared to the diploid plants. Using |Fold Change| ≥ 1 and FDR < 0.05 as thresholds, we identified 11,246, 7534, 3732, and 3212 DEGs in DH1717, DH1716, DH1588, and DH1207, respectively (Figure 3a). DH1717 contained the largest number of DEGs, including 5545 down-regulated genes and 5701 up-regulated genes. DH1716, which contained 2783 down-regulated genes, and 4751 up-regulated genes. In DH1588, there were 2469 up-regulated genes and 1263 down-regulated genes. DH1207 contained the least number of DEGs, including 2065 up-regulated genes and 1147 down-regulated genes (Figure 3b). Further analysis indicated that 1059 genes were differentially expressed in all the four DH lines, including 995 structural genes and 64 transcription factors (TFs). Among the 64 TFs, 18 were down-regulated and 46 were up-regulated. They were distributed in 24 families, including ARF, B3 and bHLH which are related to plant growth and development (Figure 3c).
To test the accuracy of RNA-seq data, we performed qRT-PCR quantification validation for 37 differentially expressed transcription factors. The quantitative data is generally consistent with the results of the RNA-seq data; therefore, we consider the RNA-seq data to be accurate and reliable (Figure 5a).

3.3. GO Enrichment and KEGG Pathway Analysis of the DEGs

The homozygous genomes of the DH plants resulted in significant alterations in plant phenotype and gene expression. To reveal the potential molecular mechanisms underlying the transcriptional changes in these DH lines, GO enrichment was performed on the 1059 common DEGs. A total of 85 biological processes were enriched, including “response to stress”, “secondary metabolite biosynthetic process”, “response to salicylic acid”, “regulation of cell death”, “leaf senescence”, “plant organ development”, “indole-containing compound metabolic process”, “response to abscisic acid”, “response to salt stress”, etc. A total of 18 molecular functions were enriched, including “methyl indole-3-acetate esterase activity”, “oxidoreductase activity”, “polysaccharide binding”, “transmembrane receptor protein kinase activity”, “RNA binding”, etc. A total of eight cellular components were significantly enriched, including “plant-type cell wall”, “plasma membrane”, “intracellular non-membrane-bounded organelle”, “extracellular region”, etc. The numerous enriched GO terms indicated that the biosynthetic process of the DH plants were drastically different from diploid (Figure 4a).
The results of GO enrichment analysis showed that a total of 77 DEGs were involved in plant tissue development, senescence, and secondary metabolic regulation (Table S1). Among them, 20 significantly up-regulated DEGs affected not only plant senescence, but also plant resistance against stress. Three homologous genes, PsnNHL10, PsnMKK9, and PsnGLN1;1 may act as immunity-related genes that affect the accumulation of ROS and H2O2 and negatively regulate salicylic acid signals. The up-regulation of PsnPAD4 and PsnWRKY70 [20] may lead to the accumulation of salicylic acid and the expression level of PsnDMR6, which ultimately leads to the reduction in plant size and early senescence. The up-regulation of PsnNAC087 [21], PsnNAC074 [22], PsnSAG12, and PsnSRG1, regulators of early senescence, directly or indirectly led to programmed cell death, and affected the survival of leaves, root tips, and stigma. Functioning in the senescence of leaf and root tip cells, PsnNAC083 [23] and PsnAIN1 [24] were up-regulated, which affected the formation of xylem vessel and the development of primary roots. The tendency of the changes on gene expression described above can well explain the phenotypic differences of the DH plants compared to WT plants, such as early senescence and decreased size.
KEGG pathway enrichment analysis was also performed on the common DEGs, and the top three enriched pathways were “Biosynthesis of secondary metabolites”, “Plant hormone signal transduction”, and “Plant-pathogen interaction”. A total of 55, 22 and 20 DEGs were enriched in the three pathways, respectively (Figure 4b). The KEGG result is consistent with the GO enrichment result.

3.4. Expression Analysis of Hormone-Responsive Genes in WT

According to the GO enrichment and KEGG results, hormone response pathways played an important role in it. Among them, ABA and IAA play an extremely important role in the growth and development of plants. In Arabidopsis, AtWRKY41 [25], AtMYB73/77 [26], AtIAA12 [27], and AtARF18 [28] regulate vegetative growth of plants by directing gene expression in response to IAA and ABA plant hormones, respectively. To further explore this molecular mechanism, we examined the expression patterns of poplar PsnWRKY41, PsnMYB73/77, PsnIAA2, and PsnARF18 genes in wild types.
The qRT-PCR results showed that the gene expression of PsnWRKY41 and PsnMYB73/77 were significantly up-regulated at the third hour after 100 μmol ABA hormone treatment. At the sixth hour, the expression of PsnWRKY41 was 65 times that of the control group, and the expression of PsnMYB73/77 was 3 times that of the control group. Their gene expression levels dropped significantly at the twelfth hour, but under short-time hormone immersion treatment, the gene expression exceeded the peak at the sixth hour at 72 h of treatment, and the gene expression was 75 times and 6.5 times higher than that of the control group, respectively (Figure 5b,c).
After soaking 20 μmol IAA hormone treatment (Figure 5f), the expression level of PsnIAA12 was 2.3 times that of the control group at the sixth hour, and the gene expression level of PsnARF18 was 2.5 times that of the control group. There was a gradual decline at 12 h and 24 h, and the expression level was even lower than that of the control group when it was the lowest. However, under short-time hormone immersion treatment, the gene expression levels showed a second peak at 72 h and 48 h, and the gene expression levels at this time were 2 times and 1.4 times that of the control group, respectively (Figure 5d,e).
Forests 14 01535 g005 550
Figure 5. qRT−PCR results of differentially expressed transcription factors. (a) Heat maps of 37 differentially expressed TF in WT and DH. In the legend, red represents higher gene expression and blue represents lower gene expression. Right legend: different colors represent different gene families. The figure is the result of a standardized calculation of gene expression. (b) PsnWRKY41 gene expression at 1 h, 3 h, 6 h, 12 h, 24 h, 48 h, and 72 h after hormone treatment. (c) PsnMYB73/77 gene expression at 1 h, 3 h, 6 h, 12 h, 24 h, 48 h, and 72 h after hormone treatment. (d) PsnIAA12 gene expression at 1 h, 3 h, 6 h, 12 h, 24 h, 48 h, and 72 h after hormone treatment. (e) PsnARF18 gene expression at 1 h, 3 h, 6 h, 12 h, 24 h, 48 h, and 72 h after hormone treatment. (f) Schematic diagram of the treatment of WT with ABA and IAA hormones, respectively. * indicate p value < 0.05 (t test), ** indicate p value < 0.01 (t test).
Figure 5. qRT−PCR results of differentially expressed transcription factors. (a) Heat maps of 37 differentially expressed TF in WT and DH. In the legend, red represents higher gene expression and blue represents lower gene expression. Right legend: different colors represent different gene families. The figure is the result of a standardized calculation of gene expression. (b) PsnWRKY41 gene expression at 1 h, 3 h, 6 h, 12 h, 24 h, 48 h, and 72 h after hormone treatment. (c) PsnMYB73/77 gene expression at 1 h, 3 h, 6 h, 12 h, 24 h, 48 h, and 72 h after hormone treatment. (d) PsnIAA12 gene expression at 1 h, 3 h, 6 h, 12 h, 24 h, 48 h, and 72 h after hormone treatment. (e) PsnARF18 gene expression at 1 h, 3 h, 6 h, 12 h, 24 h, 48 h, and 72 h after hormone treatment. (f) Schematic diagram of the treatment of WT with ABA and IAA hormones, respectively. * indicate p value < 0.05 (t test), ** indicate p value < 0.01 (t test).
Forests 14 01535 g005

4. Discussion

DH has been widely used in rice. Some pure lines were identified as bridging parents based on the anther culture response of all tested lines and crosses. Field performance trials of 1389 DH lines produced were also conducted. The aims for the application of superior lines with excellent grain quality, blast resistance, and straighthead tolerance were selected and will be advanced as promising breeding lines. In this experiment, the combination of dihaploid and forest trees was innovatively used to explore the growth and development changes of homozygous diploid plants, and many differentially expressed genes were screened. The gene functions of these differentially expressed genes include plant tissue development, plant hormone synthesis, plant tissue aging, and immune regulation.
An apical bud is the most active growing point of a plant. It can percept and transduce the environmental signals via phytohormone, and thereby change the state of plant growth and development and affect the living strategy facing disadvantageous stress. Obvious differences can be observed between DH and WT, including plant height, ground diameter, internode length, leaf shape, and, last but not the least, phenotype of apical buds. The conical structure of the apical meristem in DH plants was sharper than that in wild-type plants, and cells in DH’s apical buds showed a higher degree of cell differentiation. We think it reasonable to explore the potential mechanism by investigating the gene expression patterns and related molecular pathways with apical buds as materials (Figure 6).
After RNA-seq, a co-expression network was constructed with the differentially expressed genes. It showed that 10 DEGs related to the growth and development of meristem (apical bud and root), which has been proven by cumulative previous research. Under the influence of ABA, the expression of PsnMYB73/77 and PsnE2F [29] were suppressed, while that of PsnHFR1/DELLA and PsnRGA/DELLA increased, which inhibited the synthesis of auxin, further inhibiting the growth of roots and buds. The up-regulation of PsnSCL23 [30] affected the development of germinal layers of roots and buds. PsnNAC082/17/90 [31] in response to salicylic acid and melatonin increased the expression levels of PsnAIN1 [24] and PsnWRKY42 and increased the root length. The increased expression level of PsnRAP2.6L [32] also promoted the growth of lateral roots. There were fourteen other DEGs related to the tissue senescence, including five members of WRKY gene family. It has been studied that, upon ABA signal, PsnWRKY41 [25] and PsnWRKY40/18 [33] jointly promote the seed germination. PsnWRKY75 [34], PsnWRKY57 [35], PsnWRKY28 [34], PsnARF18 [28], and PsnIAA12 [27] promoted the leaf senescence under the influence of auxin and jasmonate. The down-regulation of PsnSPL3/4/5/15 increased the expression level of PsnWRKY28/75 [35]. The up-regulation of PsnNTL9 [36], PsnNAC072 [37], and PsnEIN3 [38] also accelerated the leaf senescence. Increased expression level of PsnAGL22/TPL [39] and PsnSVP-AP1 [40] contributed to maintaining the gene expression and function of stem cells, as well as reducing the cell differentiation to delay the development of plant tissues and organs. The up-regulation of PsnRGA/DELLA accelerated cambium senescence and delayed the development of meristem, finally led to senescence of the whole plant. It is widely known that senescence is closely related to immunity. There were six DEGs involved in plant immunity, PsnNAC083, PsnCRF4 [41], PsnWRKY70 [20], PsnWRKY50/51 [42], PsnERF1 [43], and PsnWRKY33, the expression of which were significantly up-regulated, which led to accelerated senescence and distinctive phenotypes.
Phytohormones, as endogenous signals, control plant growth and development and influence the gene transcription and expression, cell division, and cell growth. They play important roles in various aspects, such as embryogenesis, adjustment of organ size, defense against pathogens, and stress tolerance. The GO enrichment analysis showed that 132 DEGs were involved in the responses to phytohormones, 53 of which were responsive to salicylic acid and 74 of which were responsive to ABA. Some other genes were responsive to auxin. Different phytohormone pathways interconnected and overlapped with each other. The results of KEGG analysis only showed 22 phytohormone-related genes (PsnPerx34; PsnPIF3; PsnNPR1; PsnBRI1; PsnGH3.11; PsnGID1; PsnTGA9 [44]; PsnRK2; PsnCYCD3; PsnTGA1; PsnCAPE3.1; PsnCAPE3.2; PsnSAUR20; PsnEIN3; PsnSAUR32.1; PsnSAUR59; PsnGH3.12; PsnARF18 [28]; PsnSAUR32.2; PsnHP2; PsnRGA1 [45]; PsnIAA12). These 22 genes also existed in the results of GO enrichment analysis, seventeen of which were up-regulated and five of which were down-regulated. They were involved in multiple phytohormone signals, including gibberellin (2), salicylic acid (3), jasmonate (3), ethylene (2), ABA (2), auxin (4), brassinolide (3), and cytokinin (3).
According to gene homology analysis, among the up-regulated genes, PsnGID1 and PsnRGA1 were significantly related to gibberellin signal. It has been found that they delayed the vegetative growth and floral morphogenesis induced with gibberellin via interaction with DELLA and PIFs, respectively. PsnNPR1 could response to salicylic acid, ABA, and brassinolide. It contributed to the early senescence by increasing the expression level of downstream gene PsnTGA1 [44]. PsnEIN3 regulated the expression of PsnPerx34, which is a member of ERF/AP2 gene family, to participate in ethylene signal transduction, and finally led to early senescence. This gene jointly with PsnBRI1 could also decelerate the development of root tips under the regulation of auxin and brassinolide. PsnGH3.12 retarded plant growth, delayed flowering, and affected plant resistance. The functions of down-regulated genes were related to the balance between auxin and cytokinin. This dynamic balance is the decisive factor for the direction of cell proliferation or differentiation.
The other 49 DEGs (including the overlapping part with senescence-related genes) participated in leaf or meristem development, forty-three of which were significantly up-regulated, six of which were significantly down-regulated. PsnABCG11, PsnABCG14, and PsnABCG36, as ABC transporter G family members, could induce the formation of cytokinin at plant roots, and directly or indirectly participate in the cytokinin transportation from roots to twigs or buds in xylem and phloem. Cytokinin induced the up-regulation of PsnAVP3 and PsnAVP1, which promoted cell mitosis. The down-regulation of PsnGRF5 and PsnGRF4 inhibited the cell proliferation in leaf development, and the down-regulation of PsnGIF1 retarded cell swelling. The co-regulation of multiple genes caused the decrease in leaf number and chloroplast content, increased senescence rate, and decreased tolerance against N ion. PsnCRF4 responded at the early stage of N stress and was rapidly up-regulated. This gene promoted the utilization efficiency of sucrose and starch and maintained C-N metabolic balance. Furthermore, PsnNAC036.1, PsnNAC036.2, PsnNAC036.3 and PsnNAC090 [16], as NAC gene family members, regulated leaf and floral development. The up-regulation of some DEGs could lead to semi-dwarf phenotypes, which were decreased leaf length, petiole length and plant height, consistent with the phenotypes caused by the up-regulation of PsnSCL23. The tendency of gene expression changing in leaf development described above can well explain the phenotypic differences on leaves of the DH plants compared to WT plants.
However, the gene-regulation networks and related pathways are extremely complicated and remain to be further explored. The molecular reasons behind the phenotypic differences between the DH and WT plants, such as decreased height, internode length, and altered leaf shape are still unclear. It came to us that plants may have evolved exquisite and efficient strategies to balance growth, immunity, and even continuation of species. This study is hoped to shed some light on the research of plant growth and development, phytohormone signal transduction, and plant senescence and immunity with double haploid woody plant as material.

5. Conclusions

To date, double haploid plants have been widely used as materials in research of crops, but rarely seen in forest research. In this study, we examined the phenotypic differences between double haploid poplars and wild-type poplars. Bioinformatics methods and experimental verification methods were used to explore the potential molecular mechanism. These results provide us with a deeper understanding of the difference between the growth and development mechanisms of double haploid poplar and that of wild-type poplar. Future studies will further discover and confirm the preliminary conclusions drawn from RNA-seq and qRT-PCR. This study provides a good basis for further study on growth and development and immunity and stress responses of double haploid tree species.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/f14081535/s1, Table S1: GO enrichment detailed data; Table S2: Primers for qRT-PCR of differentially expressed transcription factors.

Author Contributions

Conceptualization, S.C. (Su Chen), G.Q. and C.L.; methodology, S.C. (Su Chen), G.Q. and Y.W.; formal analysis, C.L., Y.W. and Y.W.; investigation, Y.W., J.Y. and Y.H.; resources, S.C. (Su Chen), G.Q. and C.L.; data curation, S.C. (Song Chen), E.F. and S.C. (Su Chen); writing—original draft preparation, Y.W., J.Y. and X.Z.; writing—review and editing, Y.W., J.Y. and X.Z.; visualization, Y.W.; supervision, S.C. (Su Chen) and G.Q.; project administration, S.C. (Su Chen) and C.L. All authors have read and agreed to the published version of the manuscript.

Funding

This work was supported by the National Key R&D Program of China (2021YFD2200203), Heilongjiang Province Key R&D Program of China (GA21B010) and Heilongjiang Postdoctoral Financial Assistance (LBH-Z21097).

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Not applicable.

Conflicts of Interest

The authors declare no conflict of interest.

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Figure 1. Phenotype of doubled haploid plants. (a) Obtainment of doubled haploid plants in Poplars (Populus simonii × Populus nigra). WT, wild type paternal plants. DH1588, DH1717, DH1716, and DH1207 were doubled haploid plant lines. (b) Plant height of wild–type and doubled haploid plants. (c) Internode lengths of different stem internodes in wild-type and DH plants. Internode length of the fifth to eighth stem nodes. (d) Ground diameters of wild–type and doubled haploid plants. (e) Leaf aspect ratios of wild–type and doubled haploid plants. The leaves are the fifth to eighth leaves starting from the terminal bud. Leaf aspect ratio: the ratio of the longest length of the leaf to the widest part of the leaf. * indicate p value < 0.05 (t test), ** indicate p value < 0.01 (t test). Error bars represent the SD of three independent experiments.
Figure 1. Phenotype of doubled haploid plants. (a) Obtainment of doubled haploid plants in Poplars (Populus simonii × Populus nigra). WT, wild type paternal plants. DH1588, DH1717, DH1716, and DH1207 were doubled haploid plant lines. (b) Plant height of wild–type and doubled haploid plants. (c) Internode lengths of different stem internodes in wild-type and DH plants. Internode length of the fifth to eighth stem nodes. (d) Ground diameters of wild–type and doubled haploid plants. (e) Leaf aspect ratios of wild–type and doubled haploid plants. The leaves are the fifth to eighth leaves starting from the terminal bud. Leaf aspect ratio: the ratio of the longest length of the leaf to the widest part of the leaf. * indicate p value < 0.05 (t test), ** indicate p value < 0.01 (t test). Error bars represent the SD of three independent experiments.
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Figure 2. Phenotype and histological observations on terminal buds of doubled haploid plants. (a) The phenotype of the terminal buds: local close-up of terminal bud tissue of WT, DH1588, DH1717, DH1716, and DH1207, respectively. In the lower left box is the macro form of the terminal bud. Scale bar = 1mm. (b) Histological analyses of SAM stained with 0.1% toluidine blue. From top to bottom, scale bars are 500 μm and 200 μm, respectively. (c) Circularity of the apical meristem in wild-type and doubled haploid plants. Circularity was measured with ImageJ software. (d) Cell area of differentiated cells of wild-type and doubled haploid plants. ** indicated p value < 0.01 (t test). Error bars represent the SD of three independent experiments.
Figure 2. Phenotype and histological observations on terminal buds of doubled haploid plants. (a) The phenotype of the terminal buds: local close-up of terminal bud tissue of WT, DH1588, DH1717, DH1716, and DH1207, respectively. In the lower left box is the macro form of the terminal bud. Scale bar = 1mm. (b) Histological analyses of SAM stained with 0.1% toluidine blue. From top to bottom, scale bars are 500 μm and 200 μm, respectively. (c) Circularity of the apical meristem in wild-type and doubled haploid plants. Circularity was measured with ImageJ software. (d) Cell area of differentiated cells of wild-type and doubled haploid plants. ** indicated p value < 0.01 (t test). Error bars represent the SD of three independent experiments.
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Figure 3. Differentially expressed genes between wild type and DHs. (a) Number of differentially expressed genes between wild type and DHs. The yellow bars represent the number of differentially expressed genes between each line and wild-type plants. The black histogram represents the number of genes identified in the common difference table in the lines marked with black dots. (b) Distribution of up-regulated and down-regulated differentially expressed genes in each line. (c) Expression of all transcription factors and gene families to which they belong. In the left legend, red represents higher gene expression and blue represents lower gene expression. In the right legend, different colors represent different gene families. The figure is the result of a standardized calculation of gene expression.
Figure 3. Differentially expressed genes between wild type and DHs. (a) Number of differentially expressed genes between wild type and DHs. The yellow bars represent the number of differentially expressed genes between each line and wild-type plants. The black histogram represents the number of genes identified in the common difference table in the lines marked with black dots. (b) Distribution of up-regulated and down-regulated differentially expressed genes in each line. (c) Expression of all transcription factors and gene families to which they belong. In the left legend, red represents higher gene expression and blue represents lower gene expression. In the right legend, different colors represent different gene families. The figure is the result of a standardized calculation of gene expression.
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Figure 4. Differentially expressed genes (DEGs) analysis based on Gene ontology and KEGG pathway. (a) GO enrichment results of 1059 differentially expressed genes co-existing in DH plants of the four lines. (b) KEGG-based pathway enrichment of DEGs in wild type and DHs.
Figure 4. Differentially expressed genes (DEGs) analysis based on Gene ontology and KEGG pathway. (a) GO enrichment results of 1059 differentially expressed genes co-existing in DH plants of the four lines. (b) KEGG-based pathway enrichment of DEGs in wild type and DHs.
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Figure 6. The interaction between genes and the functional connection of genes with transcription factors as the main body. Oval: transcription factors, where the bold ones are the differentially expressed transcription factors involved in the article. Rhombus: hormones. Rectangles: functional genes. Hexagons: gene function. Pointed arrow: indicates that there is a direct or indirect mutual promotion relationship between genes, and can also indicate which hormones promote a certain gene function or respond. Flat-ended arrows: indicates that there is a direct or indirect inhibitory effect between genes, inhibiting a certain gene function. The different colors are used to increase the identification factor. Bold fonts represent transcription factors validated with qRT-PCR.
Figure 6. The interaction between genes and the functional connection of genes with transcription factors as the main body. Oval: transcription factors, where the bold ones are the differentially expressed transcription factors involved in the article. Rhombus: hormones. Rectangles: functional genes. Hexagons: gene function. Pointed arrow: indicates that there is a direct or indirect mutual promotion relationship between genes, and can also indicate which hormones promote a certain gene function or respond. Flat-ended arrows: indicates that there is a direct or indirect inhibitory effect between genes, inhibiting a certain gene function. The different colors are used to increase the identification factor. Bold fonts represent transcription factors validated with qRT-PCR.
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MDPI and ACS Style

Wang, Y.; Yu, J.; Zhang, X.; He, Y.; Chen, S.; Fan, E.; Qu, G.; Chen, S.; Liu, C. Morphological, Histological, and Transcriptome Analysis of Doubled Haploid Plants in Poplars (Populus simonii × Populus nigra). Forests 2023, 14, 1535. https://doi.org/10.3390/f14081535

AMA Style

Wang Y, Yu J, Zhang X, He Y, Chen S, Fan E, Qu G, Chen S, Liu C. Morphological, Histological, and Transcriptome Analysis of Doubled Haploid Plants in Poplars (Populus simonii × Populus nigra). Forests. 2023; 14(8):1535. https://doi.org/10.3390/f14081535

Chicago/Turabian Style

Wang, Yiran, Jiajie Yu, Xiang Zhang, Yaxin He, Song Chen, Erqin Fan, Guanzheng Qu, Su Chen, and Caixia Liu. 2023. "Morphological, Histological, and Transcriptome Analysis of Doubled Haploid Plants in Poplars (Populus simonii × Populus nigra)" Forests 14, no. 8: 1535. https://doi.org/10.3390/f14081535

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