Engineering transgenic Populus with enhanced biomass, wood quality and pest resistance through dual gene expression

Abstract

Wood, one of the most abundant renewable natural resources globally, plays a crucial role in the timber, papermaking and bioenergy industries (Chutturi et al., 2023). Wood (i.e. secondary xylem) is derived from vascular cambium, which is pivotal in determining the wood biomass in woody plants (Tang et al., 2022). Reactive oxygen species (ROS) act as signalling molecules that regulate plant development, growth and responses to abiotic and biotic stresses (Wang et al., 2024). Numerous studies underscore the significance of ROS in maintaining the root and shoot stem cell niches (Wang et al., 2024). A recent study has indicated that LATERAL ORGAN BOUNDARIES DOMAIN 11 (LBD11) governs several ROS metabolic genes to manage the specific distribution of ROS within the cambium, thus affecting cambial cell proliferation in Arabidopsis root and shoot (Dang et al., 2023). However, there remains a lack of clarity on the biological functions of ROS accumulation in tree vascular cambium activity. Additionally, the localized accumulation of ROS is required for lignin biosynthesis (Wang et al., 2024). Therefore, ROS homeostasis enables woody plants to fine-tune the activity of cambium, increase wood yield and improve their quality.

In plants, various forms of ROS exist, including singlet oxygen (¹O₂), superoxide anion (O₂^·−), hydrogen peroxide (H₂O₂), hydroxyl radical (HO^·) and others. Among them, O₂^·− and H₂O₂ play a crucial role in regulating stem cell fate in shoot apical meristem (SAM) and root apical meristem (RAM) (Wang et al., 2024). Superoxide dismutases (SODs) are a group of metalloenzymes that scavenge ROS by converting O₂^·− radicals into H₂O₂. In SAM and RAM, the balance between O₂^·− and H₂O₂ plays a critical role in the maintenance and differentiation of stem cells (Zeng et al., 2017). Since the development of vascular cambium originates from the peripheral region of SAM, the balance between O₂^·− and H₂O₂ may also contribute significantly to vascular cambium activity. In this study, 11 SOD genes were identified in Populus genome (Figure S1). As revealed by the cell-type transcriptome analysis of the poplar stem (Dai et al., 2023), among the 11 SOD genes, CSD2 has a higher specific expression level in the cambium other than in the xylem or phloem, indicating a potential role of CSD2 in vascular cambium development (Figure 1a).

Figure 1

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Engineering transgenic poplar with enhanced biomass, wood quality and pest resistance. (a) Transcript abundance of SOD encode gene family in cambium, xylem and phloem cells in the poplar stems, the 8th internode was collected by LCM for RNA-seq analysis. (b) Phenotypes of 3-month-old PdCSD2 overexpressing (PdCSD2OE) transgenic plants. Bar, 10 cm. (c) Cross-sectioning and staining with toluidine blue of the 20th internode stem of 3-month-old WT and PdCSD2OE transgenic plants (middle). Detailed observation of the cambial cells (left) and secondary xylem (right) in WT and PdCSD2OE plants. Bars, 25 μm (right); 100 μm (middle); 25 μm (right). (d) Histochemical staining using phloroglucinol–HCl (left) and lignin content (e) in the cell wall of the 20th internode stem of 3-month-old WT and PdCSD2OE transgenic plants. The cellulose was counter-stained with S4B (middle), and the whole mount immunolabelling of crystallized cellulose was performed with the CBM3a antibody (right) and cellulose content (f) in the cell wall of the 20th internode stem of 3-month-old WT and PdCSD2OE transgenic plants. Bars, 25 μm (right); 50 μm (middle and right). Data represent means ± SD from at least six plants from each line. (g) Saccharification of stem cell wall materials from WT and PdCSD2OE transgenic plants with or without pretreatment. Data represent the means ± SD from at least six plants from each line. (h) Fibre cells were hydrolyzed from basal internodes of 3-month-old plants and stained by TBO. Bars = 50 μm. (i, j) Fibre cell length and width among WT and PdCSD2OE. More than 60 fibre cells of each line were randomly chosen and measured. (k) Phenotypes of 3-month-old PdCSD2 and Cry3A overexpressing (PdCSD2/Cry3AOE) transgenic plants. Bar, 10 cm. (l) Cry3A protein assay from young leaves of PdCSD2/Cry3AOE lines (1# and 7#). Data represent means ± SD from at least three plants from each line. (m) Mortality data obtained for third-instar P. versicolora feeding on WT, PdCSD2OE and PdCSD2/Cry3AOE leaves Data represent means ± SD from three biological replicates of each line. (n) A comparison of leaves from WT, PdCSD2OE and PdCSD2/Cry3AOE after 2 days of P. versicolora feeding. Statistical differences were determined using Duncan's test, different letters indicate significant differences at P < 0.05.

To assess the effect of PdCSD2 on wood formation, the PdCSD2 overexpression (OE) lines with substantially elevated PdCSD2 transcript levels were developed in this study (Figure S2). Compared to the wild type (WT), the PdCSD2-OE lines exhibited a significant enhancement of growth with about a 10% increase in height and about a 20% increase in stem diameter (Figure 1b; Figure S3). Analysis of stem cross-sections revealed a 30% rise in the number of cambium cell layers at the 20th internode of PdCSD2OE plants in comparison to WT (Figure 1c; Figure S4). Accordingly, the xylem width at the 20th internode in PdCSD2OE plants was increased by approximately 20% relative to WT. Therefore, PdCSD2OE significantly enhances the wood biomass (Figure 1c; Figure S4). Numerous studies have demonstrated the essential role of ROS in regulating secondary cell wall formation, including lignin and cellulose biosynthesis and deposition (Dang et al., 2023; Wang et al., 2024). The overexpression of the SOD enzyme encoding gene PdCSD2 is suspected to affect ROS accumulation and thus lignin content in xylem. To investigate the effect of PdCSD2 on lignin biosynthesis and xylem cell wall lignification, the xylem of the 20th internode of poplar was stained with phloroglucinol–HCl solution. A diminished staining intensity was observed in PdCSD2-OE plants compared to WT, as discovered for the growth of 1-year-old transgenic plants in fields (Figure 1d; Figure S11). Accordingly, the lignin content in the 20th internodes was reduced by 15% compared to that of WT (Figure 1e). These results provide evidence supporting the association between ROS and lignin biosynthesis. To investigate the effect of ROS on cellulose biosynthesis, pontamine fast scarlet 4B (S4B) staining was conducted on xylem sections of PdCSD2OE and WT plants. An increased staining intensity in PdCSD2OE xylem was observed, indicating elevated cellulose levels (Figure 1d). Furthermore, quantification of cellulose content revealed at least 15% higher cellulose content in PdCSD2OE plants compared to WT (Figure 1f). To evaluate the potential differences in the crystalline cellulose content of PdCSD2OE xylem, whole-mount immunolabelling assays were performed on the xylem section using a family 3 carbohydrate-binding module (CBM3a) antibody that specifically targets crystalline cellulose. The fluorescence intensity was found to be significantly enhanced in the sections of PdCSD2OE xylem, indicating an increase in crystalline cellulose content in PdCSD2OE xylem (Figure 1d).

Lignin is a primary factor that affects cell wall digestibility and saccharification (Halpin, 2019). Therefore, this study also aims to establish whether the reduced lignin content in PdCSD2-OE can improve the efficiency of cell wall saccharification. The level of glucose enzymatically released from both untreated and hot 1.5% H₂SO₄-pretreated PdCSD2-OE stems was found to be higher compared to WT (Figure 1g). As a parameter of papermaking, fibre length directly affects paper strength and performance, as a greater fibre length enhances paper strength and wear resistance. To assess fibre length changes, the basal stem was disintegrated and fibre cell lengths were measured under a microscope. The fibre cells in the basal stem of PdCSD2-OE were found to be approximately 15% longer than those in WT, with no significant difference observed in fibre cell diameter between PdCSD2-OE and WT (Figure 1h,i). These results suggest the possibility to engineer poplars by overexpressing PdCSD2 in the bioethanol and papermaking industries.

The integration of multiple genes for plant transformation provides a novel solution to plant genetic engineering, which allows multiple traits to be modified simultaneously (Naqvi et al., 2010). Pests represent a critical factor that constrains forestry production and development. Originating from the bacterium Bacillus thuringiensis (Bt), the poplar expressing insect-specific toxins exert an inhibitory effect on lepidopteran and coleopteran pests. In this study, Cry3A gene is introduced into the PdCSD2OE plants. Two transgenic poplar lines with high Cry3A expression levels and protein toxin content, 3# and 7#, exhibited normal growth but no discernible phenotypic variances compared to PdCSD2OE plants (Figure 1k; Figures S5–S8). Third-instar Plesioclytus versicolora larvae were fed with the leaves derived from WT, PdCSD2OE and PdCSD2/Cry3AOE poplar plants. After 2 days of larval feeding, PdCSD2/Cry3AOE poplar leaves demonstrated stronger resistance than WT and PdCSD2OE plants (Figure 1n). The mortality rate of third-instar P. versicolora larvae feeding on PdCSD2/Cry3AOE poplar leaves was found to be significantly higher than those feeding on WT and PdCSD2OE leaves for both 4 and 8 days (Figure 1m). These results indicate that PdCSD2/Cry3A-OE poplar plants resisted higher toxicity towards P. versicolora larvae while maintaining a similar trend of growth to PdCSD2OE plants. The phenotype of 1-year-old transgenic plants grown in the field was further verified. As shown in Figures S10 and S11, the difference in lignin content and fibre length is consistent with that exhibited by those transgenic plants grown in greenhouses. Compared to the wild type, higher bending strength is exhibited by the stem of PdCSD2OE and PdCSD2/Cry3AOE poplars (Figure S12).

In summary, PdCSD2 overexpression enhances cambium activity, which increases wood production, reduces lignin content, elevates cellulose content and increases fibre cell length. Furthermore, the use of wood derived from PdCSD2OE poplar trees for bioethanol production and papermaking could lower cost, mitigate pollution and improve paper quality. In this study, PdCSD2Cry3AOE poplar trees are developed through multigene transformation. They exhibit not only an improvement in biomass and wood quality relative to PdCSD2OE poplar trees, but also a better performance in pest resistance. This strategy is expected to be applicable in practice.