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Article

Transcriptome Analysis of Developing Xylem Provides New Insights into Shade Response in Three Poplar Hybrids

by
Min Zhang
1,
Xinglu Zhou
1,
Xiaodong Xiang
1,
Changjian Du
1,
Xiaolan Ge
1,
Jiujun Du
1,
Lei Zhang
1,2,* and
Jianjun Hu
1,2,*
1
State Key Laboratory of Tree Genetics and Breeding, Key Laboratory of Tree Breeding and Cultivation of National Forestry and Grassland Administration, Research Institute of Forestry, Chinese Academy of Forestry, Beijing 100091, China
2
Co-Innovation Center for Sustainable Forestry in Southern China, Nanjing Forestry University, Nanjing 210037, China
*
Authors to whom correspondence should be addressed.
Forests 2022, 13(8), 1261; https://doi.org/10.3390/f13081261
Submission received: 21 June 2022 / Revised: 3 August 2022 / Accepted: 5 August 2022 / Published: 10 August 2022
(This article belongs to the Section Genetics and Molecular Biology)
Browse Figures
Versions Notes

Abstract

:
Poplars have a strong response to light, and shade is one of the main environmental factors that limits the growth and development of poplars. Exploring the response mechanism of the developing xylem of poplar under shady conditions is of great reference significance for improving wood yields. In this study, three excellent hybrids of poplar (Populus euramericana ‘Zhonglin46’ (Pe), Populus deltoides ‘27-17’ (Pd), Populus × ‘Wq156’ (Pw) were studied under shady conditions. Based on the phenotypic data and developing a xylem transcriptome analysis, the molecular mechanism of poplars’ response to shade was preliminarily revealed, and the core regulatory genes responding to shade were identified by a weighted co-expression network analysis (WGCNA). The results showed that Pw growth was significantly affected by shade, while Pe growth was slightly affected by shade. An enrichment analysis of 13,675 differentially expressed genes (DEGs) found that shade affected the expression of genes related to the glutathione metabolic pathway. The WGCNA analysis identified two modules (“Brown” and “Purple”) related to the shade response and discovered seven hub genes. These hub genes were related to xylem development, vascular cambium division, stomatal development, and phytochrome A signal transduction. These results provide important basic information for gaining insight into the molecular response to shade in different poplar hybrids.

1. Introduction

Light is a basic resource in nature that limits plants’ growth and development [1]. Plants grow in dynamic vegetation and provide mutual shade amongst each other, leading to the reduction of photosynthetically active radiation (PAR) and changes in spectral composition, especially a decrease in the ratio of red to far-red light (R:FR) and other spectral changes [2], which will activate shade avoidance or shade tolerance [3]. For example, the elongation of various organs, early flowering, and reduced branching, collectively known as shade avoidance syndrome (SAS), will strongly affect the development and structure of plants [4,5,6]. The aboveground light environment is the key factor affecting the biomass accumulation in tree plantations [7]. Plant stem growth and development are sensitive to shade. However, the specific mechanisms behind the shade-induced responses in plant stems remains unclear.
There are considerable intraspecific and interspecific differences in the response of plant bodies to shade. They respond to a variety of different and variable light conditions, and natural genetic variations in photoreceptors are involved in their adaptation to the environment [8]. These adaptations are often accompanied by reductions in leaf area, stem biomass, and fruit weight or number [9,10]. There is growing evidence that multiple photoreceptors (including red and far-red receptor photochromes, the UV-A/blue-light receptor cryptochromes, and phototropins) converge on a shared signal network to modulate the response to shade [11].
Transcriptomics has been extensively used to compare and identify specific regulatory networks in specific tissues to mine the underlying genes and identify similar regulatory pathways [12]. Weighted co-expression network analysis (WGCNA) is extensively used to explain the molecular mechanisms of traits such as growth, yield, and resistance [13]. Transcriptomic analysis identified a set of core shade-inducing genes in maize, tomatoes, brassica, cabbage, and conifers (pines and spruces) [14,15,16]. Jin et al. [17] confirmed through WGCNA that UV is the key factor promoting the biosynthesis of flavonol glycosides in tea buds. Simultaneously, the combination of WGCNA methods and RNA-sequence data has been used to elucidate the molecular mechanisms of abiotic stress in Arabidopsis [18], grapes [19], maize [20], rice [21], and soybeans [22].
Poplar is one of the fast-growing tree species with respect to the development of plantations in the world, and it is a renewable resource [23]. Elite poplar (Populus spp.) varieties are created through interspecific hybridization, followed by clonal propagation [24]. Some interspecific poplar hybrids exhibit strong heterosis, outperforming their parental species [25]. Among the many poplar crosses, the effect of the interspecific hybridization was remarkable in Section Leuce and Aigeiros, but the most remarkable result of hybridization is the cross between Section Aigeiros and Tacamahaca [26]. Tomasella et al. [27] found that shade reduced stem NSCs’ (nonstructural carbohydrates) concentration and increased xylem vulnerability, resulting in growth restriction and reduced biomass compared with light-exposed poplar. The functional diversity of phytochromes B and the downstream phytochrome interaction factors in the shade response of poplars have been demonstrated [28,29]. At present, most studies on shade have focused on leaves and roots [14,15,16], but there are few studies have explored plant shade responses using the developing xylem as a material.
To better understand the molecular events leading to growth inhibition by shade, the developing xylem of three poplar hybrids (Section Aigeiros hybrids of Populus euramericana ‘Zhonglin46’ and intraspecific hybrids Populus deltoides ‘27-17’, and interspecific Populus × ‘Wq156’ of Section Aigeiros and Tacamahaca) under full sun and shade conditions were used as the experimental materials to explore the genes associated with shade by a combination of RNA-seq data and WGCNA methods. The mechanism and significance of the poplar shade response were explained from the molecular aspect, which provides a molecular basis for future research on poplars’ shade response.

2. Materials and Methods

2.1. Experimental Design and Sample Collection

The experiment consisted of full sun and shade, with the former involving poplar hybrids that were planted in full sun with no surrounding vegetation to provide shade. The latter involved the poplar hybrids planted in a north-south direction relative to the adjacent willows. The adjacent willows had an average height of 11.5 m, an average breast diameter of 10.52 cm, and an average crown of 3.43 m. The shade time was approximately 10:00 a.m.–15:00 p.m. from March to August.
In our study, the poplar hybrids were cut for rooting in the greenhouse of the Chinese Academy of Forestry in March 2021, and the poplar seedlings were transplanted to the field after two months of growth. The field was divided into two plots, six replicates per hybrid in each plot, and with a plant and row spacing of 40 cm × 50 cm. The poplar hybrids (P. euramericana ‘Zhonglin46’ (Pe), P. deltoides ‘27-17’ (Pd), and Populus × ‘Wq156’ (Pw)) (Table 1) were greatly affected by shade and were selected prior to September 2021, where Pe and Pw were triploids [30]. Three hybrids, each in full sun and shade and with three replicates per hybrid, had their bark peeled from the breast height of the stem with a double-edged blade; then, the developing xylem was removed via scaping, was immediately flash frozen in liquid nitrogen, and then stored at −80 °C for RNA sequencing. The plant heights and ground diameters of poplar hybrids were measured one week before sampling, and the dry weight of stems and leaves were measured after sampling. Full sun is represented by F and shade by S.

2.2. Illumina Sequencing and Mapping

Total RNA was extracted from the developing xylem using the RNAprep Pure Plant Plus Kit (TIANGEN, Beijing, China). The Agilent 2100 bioanalyzer was used to detect the quality and integrity of RNA. The sequencing libraries preparation were prepared using three micrograms of high-quality RNA per sample using the NEBNext® Ultra™ RNA Library Prep Kit for Illumina®. Paired-end reads (150 bp) were generated on an Illumina NovaSeq 6000 platform. The raw reads were filtered using fastp (version 0.19.7) to obtain high-quality clean reads, and the clean reads were mapped to the reference genome of P. trichocarpa v4.1 using HISAT2 [31]. Gene expression levels were calculated using featureCounts (1.5.0-p3) to calculate reads per kilobase of transcript sequence per million base pairs sequenced (FPKM) [32].

2.3. Differential Expression Genes (DEGs) and Functional Analysis

Differentially expressed genes (DEGs) in 9 control groups (FPe vs. FPd, FPe vs. FPw, FPd vs. FPw, SPe vs. SPd, SPe vs. SPw, SPd vs. SPw, FPe vs. SPe, FPd vs. SPd, and FPw vs. SPw) were analyzed using the DESeq2 software package [33]. A threshold of false discovery rate (FDR) ≤ 0.01 and |log2(FoldChange)| ≥ 1 and padj ≤ 0.05 were used to retrieve DEGs. ClusterProfile 3.8.1 software was used for GO and KEGG enrichment analysis of differentially expressed genes, and padj < 0.05 was used to evaluate the significance of GO category and KEGG pathway [34].

2.4. Co-Expression Network Construction

The co-expression network was analyzed using the weighted gene co-expression network analysis (WGCNA) R package. Firstly, we performed hierarchical clustering based on FPKM of DEGs and the outlier samples were removed. Then, we used the Soft Threshold function to set the soft thresholding to 10. The modules were divided by Dynamic Shear method, and the minimum number of genes in the module was 30 [35]. The modules with similar expression patterns were combined according to the epigengene similarity (0.75), and then the association analysis between each module and phenotypic data was performed. The graphic network was created by Cytoscape 3.8.1 software [36].

2.5. Quantitative Real-Time PCR (qRT-PCR)

Eight light-related genes were selected from the DEGs among the nine comparisons, and PtrActin (Potri.001G309500) transcripts were used as an internal reference for qRT-PCR to verify the accuracy of the RNA-seq results [37]. Primer pairs were designed using Primer 3 (Table S1). A qRT-PCR was performed using a TB Green Premix Ex TaqTM Ⅱ qPCR (TaKaRa, Dalian, China) according to the manufacturer’s instructions. The relative gene expression was calculated by the 2−ΔΔCt method [38]. Three biological replicates and three technical replicates were used in all experiments.

3. Results

3.1. Growth Response of Three Poplar Hybrids to the Shade

To clarify the differences among the different hybrids, P. euramericana ‘Zhonglin46’ (Pe), P. deltoides ‘27-17’ (Pd), and Populus × ‘Wq156’ (Pw) were selected for analysis. Firstly, four growth traits of three poplar hybrids were compared (Table S2). Under full sun conditions, the plant height of Pd was better than that of Pe and Pw. Pw was superior to Pe and Pd in ground diameter and aboveground biomass. Under shade conditions, Pe and Pd were significantly higher than Pw in plant height, ground diameter, and stem dry weight (Figure 1).
Compared with full sun, Pe, Pd, and Pw under shade conditions had significant reductions in plant height and ground diameter, which decreased by 17.95%, 30.05%, and 50.53% (Figure 1A), and 14.31%, 20.17%, and 44.94% (Figure 1B), respectively. The stem dry weight of Pd and Pw were significantly decreased by 53.67% and 81.91%, respectively (Figure 1C). Pw showed a significant reduction in leaf dry weight (decreased by 38.27%), and the others had no significant difference (Figure 1D). In summary, the responses of the growth traits of the different poplar hybrids to shade was different, Pw was more sensitive to shade than the others, and their growth change was the most significant.

3.2. RNA-Seq Profiling and Differentially Expressed Genes (DEGs) Analysis

A total of 18 samples from the full sun and shade groups produced raw reads of 397,394,618 and 410,743,436, respectively. After the quality control of the raw sequencing reads, a total of 75,148,898,996 clean reads (including 371,871,356 (Full Sun) and 379,618,540 (Shade)) were generated, of which 83.43% were successfully mapped to the P. trichocarpa reference genome. Approximately 6.17 billion of the reads (82.13%) could be uniquely mapped to the genome, and 9.77 million of the reads (1.30%) were multiple mapped. The Q30 was 93.37% and the GC content was 42.60% (Table S3).
To understand the changes of different poplar hybrids under shade at the transcriptional level, we analyzed all the DEGs. The principal component analysis (PCA) (Figure 2A) and the Pearson correlation coefficient (Figure S1) were used to detect the correlation between the biological repetitions of each sample. The results showed that the three biological replicates had high repeatability, with PC1 and PC2 explaining 59.58% of the total variance, and the Pearson correlation coefficient between the biological repeats was 0.826–0.982, indicating that the RNA-seq data had high reliability.
To investigate the intraspecific and interspecific poplar hybrids’ differences under full sun and shade conditions, we examined the DEGs between nine comparison groups. A total of 13,675 DEGs were identified (Figure 2B). Under full sun, FPd vs. FPw had the largest DEG set (4892 DEGs in total, including 2053 up- and 2839 down-regulated genes). Under Shade conditions, SPd vs. SPw had the largest DEG set (6344 DEGs in total, including 2304 upregulated genes and 4040 downregulated genes), indicating a large difference between Pd and Pw. The DEGs of SPe vs. FPe, SPd vs. FPd, and SPw vs. FPw were 3579, 3705, and 1842, respectively (Figure 2C).
The intersection of all the up- and down-regulated genes in group Ⅰ within hybrids (SPe vs. FPe, SPd vs. FPd, and SPw vs. FPw), group Ⅱ under full sun (FPe vs. FPd, FPe vs. FPw, and FPd vs. FPw), and group Ⅲ under shade (SPe vs. SPd, SPe vs. SPw, and SPd vs. SPw) were analyzed to identify the common set of genes associated with shade in all the comparison groups. The results showed that there were 447 and 1271 common up- and down-regulated genes, respectively (Figure 2D,E). The former mainly involved plant–pathogen interactions, brassinosteroid biosynthesis, nitrogen metabolism, and secondary metabolism (Figure S2A). The latter involved only nitrogen metabolism and flavonoid metabolism pathways (Figure S2B).

3.3. GO Enrichment and KEGG Pathway Analysis

To further characterize the biological function of the DEGs, a GO enrichment analysis was performed. The significant GO terms of the DEGs included 38 terms of biological process (BP), 58 terms of molecular function (MF), and 9 terms of cellular components (CC). Some of these GO terms were significantly enriched in two or more comparisons, while others were unique in each comparison group. In the BP category, multiple carbohydrate metabolic processes, peptide biosynthesis and metabolic processes, translation-related processes, and several stress responses (e.g., responses to chemical, stress) were mainly in group Ⅰ among the hybrids, while others were not enriched. The steroid biosynthetic process was significantly enriched only within FPe vs. FPw. In the MF category, the GO terms of ADP binding, iron ion binding, and oxidoreductase activity (acting on paired donors, with the incorporation or reduction of molecular oxygen) were significantly enriched in most of the comparison groups, and the terms of enzyme activity (e.g., carbon-oxygen lyase and terpene synthase activity), DNA binding transcription factor activity, sequence-specific DNA binding, tetrapyrrole binding, and heme binding were significantly enriched only within group Ⅰ. The dioxygenase activity, hydrolase activity (acting on glycosyl bonds), and phosphoric ester hydrolase activity were mainly enriched in group Ⅱ that was under full sun. The GO terms of protein-disulfide reductase activity and serine hydrolase activity were mainly enriched in group Ⅲ that was under shade. In the CC category, the cell walls, ribosomes, external encapsulating structure, and intracellular non-membrane-bounded organelles were mainly enriched in group Ⅰ. The extracellular region part was significantly enriched only in FPe vs. FPd. In conclusion, the differentially expressed genes involved in GO terms may play an essential role in the shade response (Figure 3).
To further understand the molecular response of different poplar hybrids to shade at the level of metabolic pathways, we performed an enrichment analysis of the KEGG pathway. In the process, a total of 21 pathways were significantly enriched (Figure S3). Many pathways were affected by shade. The pathways of flavonoid biosynthesis, nitrogen metabolism, and plant–pathogen interactions were significantly enriched in most of the comparison groups. In addition, some unique metabolic pathways were significantly enriched in each comparison group. The glutathione metabolism pathway was only significantly enriched in group Ⅱ, and the expression levels of most genes in this metabolic pathway among the hybrids under full sun were higher than those under shade (Figure S4), indicating that this metabolic pathway was affected by shade. The KEGG enrichment analysis showed that different poplar hybrids had different adaptation mechanisms in response to shade, and the DEGs enriched in these pathways may play an essential role in poplars’ response to shade.

3.4. Gene Co-Expression Network Construction

To investigate the gene regulatory networks of the developing xylem in three poplar hybrids under shade, we performed WGCNA on 18 samples based on RNA-seq results (DEGs). The genes with the same function tended to have the same expression tendency, and highly interconnected gene clusters were divided into the same module. The results showed that the genes were clustered into 13 co-expressed modules (labeled with different colors), ranging in size from 45 (‘grey’) to 2963 (‘Turquoise’) (Figure 4A). A correlation analysis was conducted between the modules and phenotypic traits. We found that among the 13 modules, the “Brown” module contained 1971 DEGs that were significantly negatively correlated with above-ground biomass and significantly positively correlated with light (r2 = 0.7, p = 0.002), while the “Blue” module contained 2554 DEGs that were significantly positively correlated with above-ground biomass. The “Purple” module contained 517 DEGs that were significantly negatively correlated with plant height and close to being significantly negatively correlated with ground diameter (p < 0.05) (Figure 4B). These differences suggested that the specific gene sets that responded to shade exist among hybrids. According to the above results, we speculated that the DEGs in the “Brown” module might have played a key role in the plants’ response to shade, and the DEGs in the “Purple” module might have been affected by the differences of the poplar hybrids themselves or the shade environment. However, due to the excessive number of genes in the “Blue” module, some important information may be lost, so the subsequent analysis will only focus on the “Brown” and “Purple” modules.
To reveal the specific functions of the genes in the “Brown” and “Purple” modules, we conducted a functional enrichment analysis on the DEGs in the modules. The GO enrichment analysis showed that the DEGs of the “Brown” module were significantly enriched in peptide biosynthesis and metabolism, microtubule-based processes, non-membrane-bounded organelles, protein–DNA complexes, chromatin, ribosomes, and so on (Figure S5A). The DEGs of the “Purple” module were significantly enriched in some glucose metabolism processes (e.g., glucose, hexose, and monosaccharide metabolic process), phosphatase complexes, isoprenoid biosynthesis and metabolism, and so on (Figure S5B). In addition, the KEGG pathway enrichment analysis showed that the DEGs of the “Brown” module were significantly enriched towards the pathway of the proteasome (Figure S5C). The DEGs of the “Purple” module were significantly enriched in the pentose phosphate pathway (Figure S5D).
Transcription factors (TFs) are important components of transcriptional regulation in plant growth and stress responses. To further explore the important genes in the “Brown” and “Purple” modules, the transcription factors in both modules were analyzed. A total of 127 transcription factors from 32 gene families were identified, including 13 bHLHs, 16 MYBs, 18 bZIPs, 9 bZIPs, 8 GRASs, 7 ERFs, and so on (Figure S6A). These TFs in the hybrids under shade were expressed oppositely to those under full sun (Figure S6B). Some TFs in the bHLH [39], WRKY [15], MYB [40], bZIP [41], GRAS [42], and FAR1 [43] gene families have been confirmed to play a crucial role in plants’ response to shade.
According to the co-expression relationship, we screened three and four hub genes with the highest connectivity from the “Brown” and “Purple” modules, respectively, to construct the network (Figure 5, Table S4). The three hub genes in the “Brown” module included the following transcription factors: WOX4 (Potri.014G025300), ICE1 (Potri.012G106000), and DEL1 (Potri.015G070300) (Figure 5A). Four hub genes in the “Purple” module contained G2-like family protein (Potri.014G000700), GL19 (Potri.001G112400), GRAS53 (Potri.001G361700), and NAC128 (Potri.001G206900) (Figure 5C). A heatmap of the expression showed that the hub gene of the “Brown” and “Purple” modules was transcriptionally upregulated by shade (Figure 5B,D), and combined with the phenotypic data, it was further demonstrated that these genes had a potential negative regulatory effect on the growth and development of the three poplar hybrids.

3.5. Quantitative Real-Time PCR

The expression levels of the light-related genes in the different hybrids in the shade were detected using quantitative Real-Time PCR (qRT-PCR), and the accuracy of the transcriptome data was verified by comparing the results of qRT-PCR and RNA sequencing. The eight genes selected from the DEGs included the Chromatin modification-related protein EAF1B (EAF1B, Potri.002G243350), 60S ribosomal protein L39 (RPL39, Potri.018G112301), Cryptochrome 1 (CRY1, Potri.002G096900), Cryptochrome 2 (CRY2, Potri.010G071200), Phytochrome A (PhyA, Potri.013G000300), Phytochrome interacting factor 4 (PIF4, Potri.002G055400), Phytochrome B (PhyB, Potri.008G105200), and Photosystem II subunit R (psbR, Potri.011G142300). The expression levels of RPL39, CRY2, and psbR in the hybrids under shade were higher than those under full sun, which were consistent with the RNA-seq. The expression levels of other genes were also consistent (Figure 6).

4. Discussion

Light is one of the essential factors for plant survival. It plays a crucial role in the regulation of plant morphogenesis, photosynthesis, substance metabolism, and gene expression [44]. When the light in the environment in which plants live changes, plants adapt to the new light in the environment through a series of complex response mechanisms. This adaptability is usually accompanied by a reduction in growth, leaf area, stem biomass, and fruit weight or quantity [10]. In this study, the height and ground diameter of the three poplar hybrids under shade conditions were significantly lower than those under full sun. Populus × ‘Wq156’ had the most significant change in the above biomass, followed by P. deltoides ‘27-17’. The data showed that shade had a negative effect on growth performance, and the change of the plants’ external morphology evidenced the adaptive performance of the plants to shade environment. Previous studies of conifer species found that Norwegian spruce seedlings were also affected by shade, with an overall decrease in biomass [16]. The cause of these phenomena may be related to differences in carbon redistribution in plants. In our comparison, the response of P. euramericana ‘Zhonglin46’, P. deltoides ‘27-17’, and Populus × ‘Wq156’ growth traits to shade further reflected the shade tolerance levels of different hybrids. Based on the above results, we speculated that P. euramericana ‘Zhonglin46’ was a shade tolerant cultivar and that P. deltoides ‘27-17’ and Populus × ‘Wq156’ were two light-demanding poplar hybrids. At the same time, we found that P. euramericana ‘Zhonglin46’ and Populus × ‘Wq156’ were triploid, but their shade tolerance levels were opposite, indicating that the shade tolerance level of the poplar hybrids was not significantly related to the level of cell ploidy, which might be related to the differences between the hybrids.
In addition to the morphological changes and adaptation, extensive transcriptomic reprogramming occurs in plants to survive adverse environmental conditions [45]. The growth and development of plant stems are sensitive to shade. In our study, a total of 13,675 DEGs were detected by RNA-sequencing in the developing xylem of three poplar hybrids. The sequence analysis showed that the glutathione metabolic pathway was affected by shade and was significantly enriched only in the comparison group under full sun, and the expression levels of most of the genes in this metabolic pathway for the hybrids under full sun were higher than those under shade (Figure S4). Glutathione (GSH) is necessary for plants to function with respect to a variety of processes, including growth processes, stress tolerance, and programmed cell death [46]. GSH can act as an electron donor to scavenge reactive oxygen, such as in photosynthesis and respiration [47]. The ratio of R:FR is a major regulator of GSH [48]. Liu et al. [49] showed through combined transcriptional and metabolic analysis that blue and red-light affect glutathione metabolism in maize seedling leaves through three transcription-signaling pathways, and further affect photosynthesis. Toldi et al. [50] found that different light intensities and spectral compositions also affected glutathione and amino acid metabolism in wheat, further affecting the yield. Combined with the results of our study, we speculated that shade may inhibit the glutathione-dependent redox-reduction state and the photosynthesis of poplars to a certain extent, decrease the carbohydrate content, and inhibit the metabolism of young stems, thus affecting the growth and development of the plant.
Shade initiates a complex transcriptional regulatory network [51]. In recent years, WGCNA has received a lot of attention for its ability to mine candidate genes of the target traits. Based on a WGCNA analysis, we identified two modules related to the shade response. We found three transcription factors co-expressed with most of the genes in the “Brown” module, including the WOX family protein (Potri.014G025300, WOX4), bHLH family protein (Potri.012G106000, ICE1), and E2F/DP family protein (Potri.015G070300, DEL1). Members of the WUSCHEL (WUS)-related homeobox (WOX) protein family play essential roles in the maintenance and proliferation of the stem cell niches in the shoot apical meristem, root apical meristem, and cambium [52]. WOX4 played an important role in maintaining vascular cambium organization during secondary growth [53]. Kucukoglu et al. [54] found that WOX4 genes (WOX4a/b) in poplars control the cell division activity of the vascular cambium, and thus control the stem’s girth. Zheng et al. [55] studied the dormant and active cambium cells in the stems of the poplar clone Nanlin95 and found that WOX4, CLE12/13, and other genes were expressed in the active cambium, which promoted the differentiation of the stem cells in poplars’ vascular cambium.
In the “Brown” module, the Inducer of CBF Expression (ICE) transcription factors were core components of the gene regulatory networks that specify stomatal differentiation through interactions with the bHLH transcription factors of SPEECHLESS, MUTE, and FAMA [56]. In Arabidopsis, light triggers the accumulation of ICE proteins and integrates light and developmental signals into stomatal development. COP1can direct the degradation of various target proteins during plant photomorphogenesis [57]. Studies have proven that light can inhibit the COP1-Mediated degradation of ICE1. The light-mediated stabilization of ICE1 was crucial for stomatal development [58]. In addition, Arabidopsis contains six transcription factors, subdivided into typical (E2Fa, E2Fb, and E2Fc) and atypical E2Fs (DP-E2F-LIKE1 [DEL1]/E2Fe, DEL2/E2Fd, and DEL3/E2Ff). DEL1 has been identified as a key negative regulator of the endocycle’s onset, which plays a role in cell division, development, UV-resistance, and metabolism [59]. Studies have found that light controls DEL1 transcription levels, which acts as a mediator of the light-dependent endoreduplication in hypocotyls.
We found four transcription factors co-expressed with most of the genes in the “Purple” module. Among them, the GRAS53 homologous gene (SCL5) co-interacted with PAT1, SCL1, SCL13, SCL21, and other genes in Arabidopsis thaliana to participate in phyA signal transduction in a predominantly negative fashion [60]. NAC128 is a probable transcription factor that affects tracheary elements and xylem development by negatively regulating secondary cell wall fiber synthesis and programmed cell death. In poplars, NAC128 is expressed higher in the secondary xylem than in phloem-formation [61]. Wang et al. [62] found that GRF12a inhibited xylem development by up-regulating XND1a (NAC128) expression in GRF12a transgenic poplar plants. Therefore, we speculated that these hub genes may play an essential role in the regulatory network involved in developing the xylem shade response.

5. Conclusions

In this study, shade had a negative effect on the growth performance of poplar hybrids, and the responses of different growth traits to shade were significantly different. Populus × ‘Wq156’ was more sensitive to shade. Through the analysis of differentially expressed genes in the developing xylem under shade conditions, the specific biological regulation processes and response modes of the different poplar hybrids to shade were revealed. The glutathione metabolic pathway was affected by shade and was significantly enriched only in the hybrids under full sun. In addition, we identified seven key shade genes through a weighted co-expression network analysis, which may serve as regulatory centers for developing xylem under shady conditions. These findings provided important basic information for an in-depth understanding of the molecular responses of different poplar hybrids to shade and provided an important gene resource for future poplar genetic improvement and breeding programs.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/f13081261/s1, Figure S1. Pearson correlation coefficient of three biological replicates. Figure S2. Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis of common up-/down-regulated differentially expressed genes (DEGs). (A) Top 20 pathways of common up-regulated DEGs. (B) Top 20 pathway of common down-regulated DEGs. Figure S3. KEGG pathway enrichment analysis of DEGs in different comparisons. Group Ⅰ within hybrids, group Ⅱ under full sun, and group Ⅲ under shade. Each node represents the enrichment result of the corresponding pathway for different comparisons. The color and size of the nodes indicate the p-value and number of DEGs showing enriched pathway. Figure S4. Heatmap of Glutathione metabolic pathway. Figure S5. Gene enrichment analysis in the “Brown” and “Purple” module. (A) and (B) Top 20 Gene Ontology (GO) terms of genes in the “Brown” and “Purple” modules, respectively. (C) and (D) KEGG pathway of genes in the “Brown” and “Purple” modules, respectively. Figure S6. Heat map of transcription factors expression in “Brown” and “Purple” module. (A) Summary of differentially-expressed transcription factors (TFs) in the “Brown” and ‘Purple’ module. (B) Heat map of TFs in the “Brown” and “Purple” module. Table S1. The primers used in this study. Table S2. Growth traits of three poplar hybrids under full light and shade conditions. Table S3. Results of quality analysis of the RNA sequencing data. Table S4. Annotation description of the top 7 genes for connectivity (hub genes) in the “Brown” and “Purple” modules.

Author Contributions

M.Z. and J.H. conceived and designed this study. M.Z., X.Z., X.X., C.D., X.G., and J.D. conducted the experiments. M.Z., X.Z., and X.X. performed the data collation and statistical analysis. M.Z. wrote the manuscript. L.Z. and J.H. contributed with suggestions. All authors have read and agreed to the published version of the manuscript.

Funding

This study was supported by the National Natural Science Foundation (32071797), the National Key Research and Development Program of China (2021YFD2200201).

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. Growth traits of three poplar hybrids under full sun and shade conditions: (A) Plant height; (B) Ground Diameter; (C) Stem dry weight; (D) Leaf dry weight. Experiments were performed in three biological replicates. The data were analyzed with one-way ANOVA. * and ** represent the significance of p < 0.05 and p < 0.01 in shade compared with full sun conditions, respectively. Pe—P. euramericana ‘Zhonglin46’, Pd—P. deltoides ‘27-17’, Pw—Populus × ‘Wq156’.
Figure 1. Growth traits of three poplar hybrids under full sun and shade conditions: (A) Plant height; (B) Ground Diameter; (C) Stem dry weight; (D) Leaf dry weight. Experiments were performed in three biological replicates. The data were analyzed with one-way ANOVA. * and ** represent the significance of p < 0.05 and p < 0.01 in shade compared with full sun conditions, respectively. Pe—P. euramericana ‘Zhonglin46’, Pd—P. deltoides ‘27-17’, Pw—Populus × ‘Wq156’.
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Figure 2. Differentially expressed genes (DEGs) in xylem of three poplar hybrids under full sun and shade conditions: (A) Principal component analysis (PCA) of the expressed genes showed the uniformity between biological replicates; (B) Hierarchical cluster analysis of total DEGs—rows and columns in the heatmaps represent genes and samples, respectively; (C) The number of up- and down-regulated genes in pairwise comparisons between three hybrids under different conditions and the same conditions; (D) and (E) Venn diagrams of up- and down-regulated genes within and between poplar hybrids.
Figure 2. Differentially expressed genes (DEGs) in xylem of three poplar hybrids under full sun and shade conditions: (A) Principal component analysis (PCA) of the expressed genes showed the uniformity between biological replicates; (B) Hierarchical cluster analysis of total DEGs—rows and columns in the heatmaps represent genes and samples, respectively; (C) The number of up- and down-regulated genes in pairwise comparisons between three hybrids under different conditions and the same conditions; (D) and (E) Venn diagrams of up- and down-regulated genes within and between poplar hybrids.
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Figure 3. Gene Ontology (GO) enrichment analysis of DEGs in different comparisons. Group Ⅰ within hybrids, group Ⅱ under full sun, and group Ⅲ under shade. Each node represents the enrichment result of the corresponding GO term in different comparisons. Node color and size indicate the P-value and number of DEGs showing enriched terms.
Figure 3. Gene Ontology (GO) enrichment analysis of DEGs in different comparisons. Group Ⅰ within hybrids, group Ⅱ under full sun, and group Ⅲ under shade. Each node represents the enrichment result of the corresponding GO term in different comparisons. Node color and size indicate the P-value and number of DEGs showing enriched terms.
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Figure 4. Co-expression network. (A) Gene cluster dendrogram and 13 gene module divisions, one of the major tree branches represents a module; different colors represent different modules. (B) Module–phenotype associations based on Pearson correlation analysis.
Figure 4. Co-expression network. (A) Gene cluster dendrogram and 13 gene module divisions, one of the major tree branches represents a module; different colors represent different modules. (B) Module–phenotype associations based on Pearson correlation analysis.
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Figure 5. Identification of hub genes in the co-expression network and heatmap of hub genes expressions in the “Brown” and “Purple” modules under shade. (A) Three hub genes in the “Brown” module and their connectivity with co-expressed genes in the network. (B) Heatmap of hub genes in the “Brown” module. (C) Four hub genes in the “Purple” module and their connectivity with co-expressed genes in the network. (D) Heatmap of hub genes in the “Purple” module. (The darker and the thicker the line, the stronger the correlation (weight value) between genes in the module. The larger the node, the darker the color, which indicates that the correlation (weight value) between genes is becoming stronger in the module. TF represents transcription factor).
Figure 5. Identification of hub genes in the co-expression network and heatmap of hub genes expressions in the “Brown” and “Purple” modules under shade. (A) Three hub genes in the “Brown” module and their connectivity with co-expressed genes in the network. (B) Heatmap of hub genes in the “Brown” module. (C) Four hub genes in the “Purple” module and their connectivity with co-expressed genes in the network. (D) Heatmap of hub genes in the “Purple” module. (The darker and the thicker the line, the stronger the correlation (weight value) between genes in the module. The larger the node, the darker the color, which indicates that the correlation (weight value) between genes is becoming stronger in the module. TF represents transcription factor).
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Figure 6. The expression of eight key genes was confirmed by quantitative Real-Time PCR (qRT-PCR). The FPKM values of eight genes obtained by RNA-Sequencing are shown as grey columns, and relative expression levels of target genes by qRT-PCR are shown as red lines with standard error. Three biological replicates and three technical replicates were conducted for each sample.
Figure 6. The expression of eight key genes was confirmed by quantitative Real-Time PCR (qRT-PCR). The FPKM values of eight genes obtained by RNA-Sequencing are shown as grey columns, and relative expression levels of target genes by qRT-PCR are shown as red lines with standard error. Three biological replicates and three technical replicates were conducted for each sample.
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Table
Table 1. Background of three poplar hybrids.
Table 1. Background of three poplar hybrids.
Full Sun/ShadedPoplar Hybrids Crossing ParentsCrossing Year
FPe/SPePopulus × euramericana ‘Zhonglin46’Populus deltoides ‘Lux’ × Populus nigra 1979
FPd/SPdPopulus deltoides ‘27-17’Populus deltoides ‘Danhong’ × Populus deltoides ‘Beiyang’2012
FPw/SPwPopulus × ‘Wq156’Populus deltoides ‘55/65’ × Populus cathayana 1999
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Zhang, M.; Zhou, X.; Xiang, X.; Du, C.; Ge, X.; Du, J.; Zhang, L.; Hu, J. Transcriptome Analysis of Developing Xylem Provides New Insights into Shade Response in Three Poplar Hybrids. Forests 2022, 13, 1261. https://doi.org/10.3390/f13081261

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Zhang M, Zhou X, Xiang X, Du C, Ge X, Du J, Zhang L, Hu J. Transcriptome Analysis of Developing Xylem Provides New Insights into Shade Response in Three Poplar Hybrids. Forests. 2022; 13(8):1261. https://doi.org/10.3390/f13081261

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Zhang, Min, Xinglu Zhou, Xiaodong Xiang, Changjian Du, Xiaolan Ge, Jiujun Du, Lei Zhang, and Jianjun Hu. 2022. "Transcriptome Analysis of Developing Xylem Provides New Insights into Shade Response in Three Poplar Hybrids" Forests 13, no. 8: 1261. https://doi.org/10.3390/f13081261

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