Next Article in Journal
Effect of Postharvest Ripening on the Phytochemical Composition and Antioxidant Properties of Fruits from Ten Plum (Prunus domestica L.) Cultivars
Previous Article in Journal
Potential for Enhancing Seed Yield and Quality of Spring Oat and Hull-Less Barley Through Intercropping with Pea Under the Pannonian Climate
 
 
Search for Articles:
Title / Keyword
Author / Affiliation / Email
Journal
Article Type
 
 
Advanced Search
 
Section
Special Issue
Volume
Issue
Number
Page
 
Journals
Agronomy
Volume 15
Issue 6
10.3390/agronomy15061347
agronomy-logo
Submit to this Journal Review for this Journal Propose a Special Issue
► Article Menu

Article Menu

share Share announcement Help format_quote Cite question_answer Discuss in SciProfiles
first_page
settings
Order Article Reprints
Font Type:
Arial Georgia Verdana
Font Size:
Aa Aa Aa
Line Spacing:
Column Width:
Background:
Article

Overexpression of PagLAR3 in Populus alba × P. glandulosa Promotes Resistance to Hyphantria cunea

by
Zhibin Fan
1,
Luxuan Hou
1,
Zheshu Wang
1 and
Lijuan Wang
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
*
Author to whom correspondence should be addressed.
Agronomy 2025, 15(6), 1347; https://doi.org/10.3390/agronomy15061347
Submission received: 24 March 2025 / Revised: 21 May 2025 / Accepted: 29 May 2025 / Published: 30 May 2025
Browse Figures
Versions Notes

Abstract

:
Poplar is a vital ecological and economic tree species. In recent years, poplar plantations in China have been increasingly threatened by the fall webworm (Hyphantria cunea). Developing resistant varieties through genetic engineering is an environmentally friendly and cost-effective approach to controlling this pest. Although some exogenous toxic genes have been used in insect-resistant poplar breeding, endogenous defense genes remain scarce. This study focused on tannins, key defensive metabolites in poplar, and explored the role of PagLAR3, a gene encoding a crucial enzyme in condensed tannin biosynthesis, in poplar’s defense against the fall webworm. The findings revealed that overexpression of PagLAR3 significantly increased levels of catechin, gallocatechin, procyanidin B3, and procyanidin C2 in poplar leaves. Feeding assays with fall webworm larvae demonstrated that, compared with an 84 K (P. alba × P. glandulosa) control, transgenic lines overexpressing PagLAR3 significantly reduced larval and pupal weight, prolonged larval duration, and caused a decrease in adult emergence. Development retardation caused by overexpression of PagLAR3 in fall webworm is expected to effectively control the pest population, thereby mitigating damage to poplar trees. PagLAR3 represents a potential target for enhancing poplar resistance to the fall webworm.

1. Introduction

Poplar is a crucial economic and ecological forest species in China, serving as a primary source of raw materials for wood-based products and playing a significant role in ecological protection and restoration efforts [1,2,3]. However, the expansion of monoculture poplar plantations has exacerbated pest pressures, particularly from lepidopteran insects, posing a major challenge to poplar production [4]. Among these pests, Hyphantria cunea Drury (Lepidoptera: Erebidae), a leaf-feeding insect native to North America and introduced to China in 1979, has become a significant threat to deciduous tree species in northern China. While this pest feeds on a wide range of tree species, it exhibits a strong preference for poplar [5]. In recent years, H. cunea has emerged as a major issue in poplar plantations nationwide [6]. Breeding pest-resistant poplar varieties is widely regarded as the most economical, environmentally friendly, and effective strategy for controlling this pest. Transgenic approaches, characterized by short breeding cycles and high precision, offer a powerful and promising method for enhancing the resistance of poplar clones used in plantations [7,8].
Currently, the genes used in transgenic poplar breeding for lepidopteran resistance are predominantly derived from exogenous sources. For instance, the Cry1Ac, Cry1Ah, and Cry9Aa3 genes from Bacillus thuringiensis exhibit insecticidal activity against lepidopterans and have been extensively utilized in developing insect-resistant transgenic poplar [9,10]. Additionally, genes such as the insect neurotoxin AaIT from Androctonus australis, as well as the arrowhead proteinase inhibitor (API), and cowpea trypsin inhibitor (Cpti) from plants and animals, also show insecticidal activity against lepidopteran insects. Co-transformation of these genes with Bt genes into poplar has significantly enhanced tree resistance to lepidopterans [11,12,13]. In contrast to exogenous genes, the use of endogenous poplar genes for improving insect resistance is considered to offer greater genetic stability and lower potential risks [14]. Focusing on the defense mechanisms inherent in poplar trees, uncovering potential defense genes, and verifying their resistance to insects through transgenic methods will lay a solid foundation for applying genetic engineering to the breeding of insect-resistant forest trees.
Chemical defense is one of the most effective strategies employed by plants to combat insect herbivory [15]. Metabolomic analyses of poplar trees before and after H. cunea feeding have revealed that phenolic compounds, such as flavonoids and tannins, play a critical role in induced insect resistance [16,17]. Comparative studies on the effects of various phenolic compounds on H. cunea food utilization have demonstrated that tannins significantly inhibit the food utilization efficiency of H. cunea larvae [18]. Another study also found that tannins in hosts negatively correlated with the performance of H. cunea, highlighting their vital role in host plant defense [19]. Leucoanthocyanidin reductase (LAR), which catalyzes the synthesis of catechin, a key monomer in biosynthesis of condensed tannins (CTs) or proanthocyanidins (PAs) from 3,4-cis-leucocyanidin, represents the first committed step in CT biosynthesis [20]. In poplar, overexpression of PtrLAR3 has been shown to increase CT levels in leaves, which in turn inhibits infection by Marssonina brunnea f. sp. multigermtubi and reduces the incidence of leaf black spot disease [21]. However, it remains unclear whether LAR influences poplar resistance to chewing leaf-eating pests such as H. cunea.
In this study, the full-length cDNA of PagLAR3 was cloned from the poplar clone 84 K (P. alba × P. glandulosa) and transformed into 84 K via Agrobacterium-mediated transformation. Transgenic plants were confirmed through the expression of PagLAR3 and quantification of CTs. Resistance to H. cunea was evaluated, revealing that transgenic plants effectively reduced larval feeding and hindered larval growth and development.

2. Materials and Methods

2.1. Gene Cloning and Agrobacterium-Mediated Transformation

The full-length cDNA sequence of PagLAR3 was amplified from 84 K using gene-specific primers (Table S1: PagLAR3_F/R) designed with Primer 6.0 software, based on the homologous PtrLAR3 sequence from P. trichocarpa. The PagLAR3 coding sequence was first cloned into the Gateway® entry vector pDNOR207 and subsequently transferred into the destination vector pMDC32 using the gateway cloning system (Life Technologies, Paisley, UK). The construct 35S:PagLAR3, which includes the PagLAR3 open reading frame driven by the cauliflower mosaic virus 35S promoter and the hygromycin phosphotransferase gene (Hpt) as a selectable marker for hygromycin resistance in plants, was introduced into A. tumefaciens strain GV3101 (Weidi Biotechnology, Shanghai, China) by electroporation [22].

2.2. Transgenic Plant Verification and Cultivation Conditions

After transformation, transgenic plants were identified and verified by examining transgene expression using gene-specific primers (Table S1: PagLAR3_qF/qR). Tissue culture seedlings of 84 K and transgenic lines were grown on 1/2 MS medium (PhytoTech, KS, USA) supplemented with 5 g/L agar, 30 g/L sucrose, 50 mg/L indole-3-butyric acid, and 20 mg/L naphthaleneacetic acid (pH 5.8–6.0) (Sinopharm Chemical Reagent Co.,Ltd, Beijing, China). After 20 days, seedlings were transplanted into soil and maintained under controlled conditions: 16 h/8 h light/dark cycles, 60–70% relative humidity, and temperatures of 22–25 °C.

2.3. Gene Expression Analysis

The relative expression levels of PagLAR3 in leaf, stem, and root were analyzed on 2-month-old 84 K seedlings using RT-qPCR. The response of PagLAR3 in leaf to herbivory was detected by analyzing expression levels at 0 h, 12 h, 24 h, 48 h, 72 h, and 96 h following feeding of H. cunea larvae. Transgenic plants overexpressing PagLAR3 were identified by RT-qPCR. Total RNA was extracted from each sample using the RNAprep Pure Plant Kit (Tiangen, Beijing, China), followed by reverse transcription of 1 μg RNA with the PrimeScript™ II 1st Strand cDNA Synthesis Kit (Takara, Dalian, China). RT-qPCR was conducted on a LightCycler480 system using KAPA Library Amplification Kits, with PagACTIN serving as the internal reference gene. Relative gene expression levels were calculated using the 2−ΔΔCt method, with four technical replicates per gene. Three independent biological replicates were included for all samples.

2.4. Relative Quantification of CTs in Leaves

For extraction of CTs, leaves were ground in liquid nitrogen and 1 g of each powdered material was extracted with a buffer (70% acetone and 0.5% acetic acid) using a previously described method [23]. The extracts were analyzed using a UPLC-ESI-MS/MS system (UPLC, SHIMADZU Nexera X2; MS, Applied Biosystems 4500 Q TRAP, Shimadzu, Kyoto, Japan) [24]. Qualitative analyses of the metabolites were performed by referencing secondary mass spectrometry data against the self-built database MWDB V2.0 (Metware Biotechnology Co., Ltd., Wuhan, China) which includes common plant flavonoids and tannins. Quantitative analyses were carried out by the multiple reaction monitoring mode (MRM) using triple quadrupole mass spectrometry according to the method described previously [25]. The relative contents of CTs in each sample were represented with chromatographic peak area integrals.

2.5. Evaluation of H. cunea Resistance

Resistance evaluation of 84 K and the two transgenic lines was performed according to the method described by Wang et al. [26]. Eggs of the fall webworm were sterilized using 0.1% sodium hypochlorite dissolved in 1% Tween-80 (Sinopharm Chemical Reagent Co., Ltd., Beijing, China). Insect feeding assays were carried out in plastic bottles (440 mL capacity each) in an insectarium (25 ± 1 °C; 65% relative humidity; 14 h light/10 h dark cycle). Twelve seedlings were used for each genotype. Leaves from 4 seedlings were collected and placed into one rearing bottle containing 30 larvae, with three replicate bottles prepared for each material. Fresh leaves were replaced every 2–3 days until the newly hatched larvae developed to the pupal stage. To control for the influence of leaf age, only mature leaves from near the top of the tree were selected. The weight of larvae at each instar, along with larval duration, pupal weight, pupation success, and emergence rate, were recorded.

2.6. Statistical Analyses

All experiments were repeated at least three times. Data were represented as averages ± SD. Analyses were performed by SPSS 16.0 (SPSS Inc., Chicago, IL, USA) using Student’s t-test to evaluate significant differences, and p-value thresholds were shown as p < 0.05 or p < 0.001.

3. Results

3.1. Confirmation of Transgenic Poplars Overexpressing PagLAR3

RT-qPCR analysis revealed that expression of PagLAR3 was significantly higher in leaves and roots compared with stems (Figure 1a). In response to H. cunea larval feeding, expression of PagLAR3 in leaves was induced, peaking at 24 h after inoculation and subsequently decreasing (Figure 1b). To generate overexpression lines, the CDS of PagLAR3 was cloned into the pMDC32 vector. Fifteen independent transgenic lines were obtained, and RT-qPCR confirmed the overexpression of PagLAR3 at the mRNA level in transgenic poplars. Two lines, OE-4 and OE-11, which showed approximately 25- and 44-fold higher expression levels, respectively, compared with the non-transgenic control (84 K), were selected for further analysis (Figure 1c). Morphological analysis revealed no significant differences in stem diameter, plant height, or numbers of internodes between the transgenic lines and the 84 K control (Figure S1). However, quantitative analysis of metabolites revealed that levels of catechin, gallocatechin, procyanidin B3, and procyanidin C2 in the leaves of transgenic poplars were significantly increased, reaching 1.6 to 3.4 times those of the 84 K control (Figure 1d).

3.2. Transgenic Poplar Trees Overexpressing PagLAR3 Inhibited Larval Development

To investigate whether overexpressing PagLAR3 affects leaf consumption by H. cunea larvae, third-instar larvae were fed with leaves from 84 K and transgenic poplar trees. The results showed that, when identical numbers of larvae were fed for the same duration (4 h), the leaf area consumed on transgenic poplar was less than that in 84 K (Figure 2a). Continuous feeding experiments on newly hatched larvae revealed that larvae fed with transgenic poplar exhibited delayed development, specifically manifested as reduced larval weight and prolonged developmental duration (Figure 2b–d). For fourth-instar larvae, the average weight of those fed with 84 K was 50.76 mg, while those fed with OE-4 and OE-11 weighed 42.62 mg and 36.11 mg, respectively, indicating reductions of 16.04% and 28.86% (Figure 2c). In terms of larval duration, larvae fed with 84 K took 29.96 days on average, whereas those consuming OE-4 and OE-11 required 30.83 days and 31.22 days, respectively, corresponding to increases of 0.87 days and 1.26 days (Figure 2d).

3.3. Overexpression of PagLAR3 in Poplar Decreased the Success of Adult Emergence

Overexpressing PagLAR3 in poplar also affected the pupation and adult emergence of H. cunea. As shown in Figure 3, pupae formed from larvae fed with transgenic lines were smaller than those fed with 84 K (Figure 3a). Although there was no significant difference in the pupation rate, the pupae formed from larvae fed with transgenic line OE-11 were significantly lighter than those fed with 84 K. The average weight of pupae from larvae fed with 84 K was 88.79 mg, while that of larvae fed with OE-11 was 79.07 mg, suggesting a reduction in pupal weight of 10.95%. To further clarify the impact of overexpressing PagLAR3 on pupal development, the success of adult emergence was also examined. It was found that the emergence rate of H. cunea fed with 84 K was 83.33%, while the emergence rates of H. cunea fed with transgenic lines OE-4 and OE-11 were significantly lower, being 56.67% and 33.33%, respectively. These results indicated that overexpressing PagLAR3 significantly decreased the success of adult emergence.

4. Discussion

The fall webworm currently poses a significant threat to poplar cultivation. There is an urgent need for resistant varieties to control the damage caused by this pest. Genetic engineering based on endogenous defense genes holds promise for developing insect-resistant poplar varieties with low ecological risk in the short term. In this study, the role of PagLAR3 in poplar defenses against H. cunea was investigated. Through genetic transformation of poplar trees, transgenic poplars overexpressing PagLAR3 were obtained and resistance to H. cunea was evaluated. The results revealed that overexpression of PagLAR3 reduced the feeding behavior of H. cunea, inhibited larval development, and decreased the success of adult emergence. This developmental retardation of H. cunea caused by PagLAR3 suggested significant levels of resistance against this insect in transgenic poplars.
CTs, which are also referred to as proanthocyanidins (PAs), are complex flavonoid polymers, typically found in woody plants. The biological effects of CTs on insects mainly include antinutritive and toxic effects. Antinutritive effects relate to interactions with proteins, which reduce nitrogen availability for herbivores [27,28], whereas toxic effects could be linked to oxidation reactions which are either spontaneous or mediated by plant polyphenol oxidases (PPOs) [29]. Comparative studies on the performance of H. cunea feeding on different host plants revealed a negative correlation between host tannin content and larval growth and development [15]. Another study, which involved adding various phenolics to artificial diets, found that tannins significantly reduced food utilization, and inhibited growth and development in H. cunea [18]. These findings suggested the possibility that enhancing accumulation of tannins in poplar trees might improve resistance to H. cunea. LAR is a key enzyme in the flavonoid and tannin biosynthesis pathways. Although overexpression of LAR in some species does not increase content levels of CTs [30], implying the existence of alternative biosynthetic pathways, studies on poplar have demonstrated that LAR does play a crucial role in CT synthesis [21]. In the present study, overexpression of PagLAR3 increased the contents of several poplar CTs, including catechin, gallocatechin, procyanidin B3 and procyanidin C2, further confirming the role of LAR in poplar CT biosynthesis. These findings establish LAR and CTs as important targets for breeding insect-resistant poplar varieties.
In most northern-hemisphere plants, CTs are composed of the four flavan-3-ols catechin, epicatechin, gallocatechin, and epigallocatechin [31]. A study on Artocarpus lacucha found that isolated catechin exhibited significant contact toxicity against Spodoptera litura larvae (24-h LD50 value of ~8.37 μg/larva). This toxicity mainly involved significantly inhibiting the activity of detoxification enzymes, including acetylcholinesterase, carboxylesterase, and glutathione S-transferase [32]. In addition to the monomeric form, CTs typically exist as oligomers (2–10 monomeric units) or polymers (>10 monomeric units), such as dimers (e.g., procyanidin B3), trimers (e.g., procyanidin C2). The structural complexity of CTs underpins their functional versatility in plant defense. A previous study found that tannins with a higher degree of polymerization exhibit stronger protein-precipitating capacity [33]. That means that dimers or trimers may exert more potent anti-nutritional effects on insect digestive enzymes than monomers like catechin through stronger protein binding. In future, we will use artificial diet incorporation experiments to compare the effects of catechin, gallocatechin, procyanidin B3, and procyanidin C2 on the development of H. cunea, in order to determine the CT with the strongest toxic effect on this insect.
Over the past few decades, many studies have explored the potential defensive roles of CTs against herbivores. However, this defensive relationship is often context-specific [31,34]. Some studies have suggested that CTs might act as deterrents to tree-feeding specialist insects but exhibit limited effects on certain generalist species [35,36,37]. Nevertheless, this is not universally applicable. Several studies have demonstrated that CTs can significantly defend against generalist insects in Lepidoptera. For instance, CTs in Quercus robur were found to negatively correlate with the growth and survival of Operophtera brumata [38]. In another study, genetic modifications of the flavonoid pathway in Betula pendula showed that CTs (or their monomers) were physiologically harmful to Epirrita autumnata larvae [39]. In addition, studies on two tree-adapted generalists, Japanese beetles and H. cunea, indicated that CTs have little or no adverse effect on the former but might negatively impact the performance of the H. cunea [40]. Structural differences among CTs, as well as specific adaptations in certain herbivores, may explain the variable activity of tannins across different systems [37,41].
In addition to their insect defense functions, CTs also exhibit broad-spectrum antimicrobial activities. They can disrupt cell membrane formation and inhibit the growth of both Gram-positive and Gram-negative bacteria [42]. Previous studies have demonstrated that elevated CT concentrations in poplar root systems or leaf litter may suppress pathogenic or competitive microorganisms, thereby enhancing disease resistance [43]. However, some investigations have also indicated that such inhibitory effects may occasionally impair beneficial symbionts [43]. One study examining Compsilura concinnata, a kind of parasitoids of Lymantria dispar, revealed that high-CT diets significantly prolonged host larval development and reduced pupal weight, yet exerted no direct toxicity on the parasitoids’ survival or developmental cycles [44]. This indicates that CTs indirectly affect parasitoids. For predators, a limited body of evidence suggests that CTs rarely cause lethal effects but may indirectly reduce predator populations by diminishing healthy prey availability [45]. These studies suggested that CTs may exert complex effects in ecosystems. Consequently, comprehensive safety assessments targeting non-target insects, natural enemies, and soil microorganisms are imperative prior to the field application of PagLAR3-overexpressing transgenic plants.
The current study was limited to evaluating resistance to H. cunea and the growth performance of PagLAR3-overexpressing transgenic poplar in seedlings. Although no significant growth differences were observed between two-month-old transgenic lines and non-transgenic control, these findings cannot be extrapolated to transgenic trees. Furthermore, uncertainties remain regarding potential developmental influences on PagLAR3 expression levels and accumulation of CTs in trees, both of which may subsequently affect resistance against H. cunea. Additionally, the dynamic field environment may present additional challenges to the expression of insect resistance traits. Therefore, comprehensive field evaluations of both insect resistance and growth characteristics in transgenic trees are essential for a thorough assessment of PagLAR3-overexpressing poplars.

5. Conclusions

This study demonstrated that overexpression of PagLAR3 in poplar significantly increased the accumulation of tannins, including catechin, gallocatechin, procyanidin B3, and procyanidin C2, in leaves, without significantly affecting growth during the seedling stage. Transgenic poplar seedlings overexpressing PagLAR3 suppressed weight gain in larvae and also extended larval duration. This retardation in larval development led to a reduction in pupal weight and negatively impacted the success of adult eclosion. The present study demonstrates that PagLAR3-overexpressing transgenic poplars exhibit significant resistance against H. cunea. These transgenic poplars will become part of potential strategies to control this insect. However, field evaluations of insect resistance and growth performance need to be conducted before practical application.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/agronomy15061347/s1, Figure S1: Comparison of growth parameters between two-month-old 84 K and transgenic lines. (a) Plant height. (b) Basal stem. (c) Numbers of internodes. p-values from Student’s t-test are denoted with asterisks. “ns” denotes no significant difference. Table S1: The primers used in this study.

Author Contributions

Conceptualization, Z.F. and L.W.; methodology, Z.F. and L.W.; formal analysis, Z.F. and L.H.; investigation, Z.F., L.H. and Z.W.; writing—original draft preparation, L.W. and Z.F.; writing—review and editing, Z.F., L.H., Z.W. and L.W.; funding acquisition, L.W. All authors have read and agreed to the published version of the manuscript.

Funding

This work was supported by Biological Breeding-National Science and Technology Major Project (2022ZD04015).

Data Availability Statement

The complete dataset underlying this research is fully contained within the main text and Supplementary Information files.

Conflicts of Interest

The authors declare no conflicts of interest.

References

  1. Zhang, J.M.; Jia, G.D.; Liu, Z.Q.; Wang, D.D.; Yu, X.X. Populus simonii Carr. Reduces Wind Erosion and Improves Soil Properties in Northern China. Forests 2019, 10, 315. [Google Scholar] [CrossRef]
  2. Wang, Z.; Yan, W.D.; Peng, Y.Y.; Wan, M.; Farooq, T.H.; Fan, W.; Lei, J.J.; Yuan, C.L.; Wang, W.C.; Qi, Y.Q.; et al. Biomass Production and Carbon Stocks in Poplar-Crop Agroforestry Chronosequence in Subtropical Central China. Plants 2023, 12, 2451. [Google Scholar] [CrossRef]
  3. He, M.J.; Zhang, J.; Li, Z.; Li, M.L. Production and Mechanical Performance of Scrimber Composite Manufactured from Poplar Wood for Structural Applications. J. Wood Sci. 2016, 62, 429–440. [Google Scholar] [CrossRef]
  4. Liu, B.; Yan, J.Y.; Wang, D.; Wang, Y.; Zhou, Y.T.; Chen, Y.F. Occurrence of Major Forest Pests in China in 2023 and Prediction for Trend in 2024. For. Pest. Dis. 2024, 43, 41–45. (In Chinese) [Google Scholar] [CrossRef]
  5. Ning, J.; Lu, P.F.; Fan, J.T.; Ren, L.L.; Zhao, L.L. American Fall Webworm in China: A New Case of Global Biological Invasions. Innovation 2022, 3, 100201. [Google Scholar] [CrossRef] [PubMed]
  6. Xue, M.Y.; Hao, D.J.; Zhao, X.D.; Geng, Y.S.; Hu, T.Y.; Xie, C.X. Effects of Extreme Climate on the Distribution and Potential Habitat of Hyphantria cunea in China. J. Nanjing For. Univ. (Nat. Sci. Ed.) 2024, 48, 197–203. (In Chinese) [Google Scholar] [CrossRef]
  7. Sharan, S.; Chakraborty, A.; Roy, A.; Singh, I.K.; Singh, A. Transgenic Poplar for Resistance against Pest and Pathogen Attack in Forests: An Overview. Front. For. Glob. Change 2024, 7, 1490562. [Google Scholar] [CrossRef]
  8. Ding, L.P.; Chen, Y.J.; Wei, X.L.; Ni, M.; Zhang, J.W.; Wang, H.Z.; Zhu, Z.; Wei, J.H. Laboratory Evaluation of Transgenic Populus davidiana × Populus bolleana Expressing Cry1Ac + SCK, Cry1Ah3, and Cry9Aa3 Genes against Gypsy Moth and Fall Webworm. PLoS ONE 2017, 12, e0178754. [Google Scholar] [CrossRef]
  9. Tian, Y.C.; Li, T.Y.; Mang, K.Q.; Han, Y.F.; Li, L.; Wang, X.P.; Lu, M.Z.; Dai, L.Y.; Han, Y.N.; Yan, J.J.; et al. Insect Tolerance of Transgenic Populus nigra Plants Transformed with Bacillus thuringiensis Toxin Gene. Chin. J. Biotechnol. 1993, 9, 291–297. (In Chinese) [Google Scholar] [CrossRef]
  10. Xu, C.; Wei, H.; Wang, L.K.; Yin, T.M.; Zhuge, Q. Optimization of the cry1Ah1 Sequence Enhances the Hyper-Resistance of Transgenic Poplars to Hyphantria cunea. Front. Plant Sci. 2019, 10, 335. [Google Scholar] [CrossRef]
  11. Zhu, G.Q.; Fang, D.; Li, X.F.; Sun, W.B.; Huang, M.R.; Wang, M.X. Transformation of Populus × Euramericana Cv. ‘Nanlin895’ Sing Bt and CpTI Insect-Resistant Genes. Mol. Plant Breed. 2006, 4, 819–824. (In Chinese) [Google Scholar]
  12. Li, K.Y.; Fan, J.F.; Zhao, Z.; Li, L.; Zhu, H.L. Transformation of CryIAc and API Two Insect-Resistant Genes to Poplar (Populus alba × P. glandulosa). For. Res. 2007, 20, 699–704. (In Chinese) [Google Scholar]
  13. Zuo, L.L.; Wang, Z.Y.; Liang, C.; Xie, S.P. Transformation of Populus davidiana × P. bollena with Bt+ Spider Toxin Gene. J. Northeast. For. Univ. 2009, 37, 112–114. (In Chinese) [Google Scholar] [CrossRef]
  14. Domingo, J.L.; Bordonaba, J.G. A Literature Review on the Safety Assessment of Genetically Modified Plants. Environ. Int. 2011, 37, 734–742. [Google Scholar] [CrossRef] [PubMed]
  15. Negin, B.; Jander, G. Convergent and Divergent Evolution of Plant Chemical Defenses. Curr. Opin. Plant Biol. 2023, 73, 102368. [Google Scholar] [CrossRef] [PubMed]
  16. Wang, Z.S.; Qu, L.J.; Fan, Z.B.; Hou, L.X.; Hu, J.J.; Wang, L.J. Dynamic Metabolic Responses of Resistant and Susceptible Poplar Clones Induced by Hyphantria cunea Feeding. Biology 2024, 13, 723. [Google Scholar] [CrossRef]
  17. Ding, Y.T.; Shen, J.J.; Li, H.X.; Sun, Y.; Jiang, T.B.; Kong, X.B.; Han, R.; Zhao, C.L.; Zhang, X.X.; Zhao, X.Y. Physiological and Molecular Mechanism of Populus pseudocathayana × Populus deltoides Response to Hyphantria cunea. Pestic. Biochem. Phys. 2024, 202, 105969. [Google Scholar] [CrossRef]
  18. Liu, X.X.; Jiang, D.; Meng, Z.J.; Yan, S.C. Effects of Secondary Substances on Food Utilization by Hyphantria cunea Larvae. J. Northeast. For. Univ. 2020, 48, 99–103. (In Chinese) [Google Scholar] [CrossRef]
  19. Wang, M.; Jiang, D.; Meng, Z.J.; Yan, S.C. Adaptability of Larval Growth and Development in Hyphantria cunea to Different Host Plant Secondary Metabolites. J. Northeast. For. Univ. 2020, 48, 100–104. (In Chinese) [Google Scholar] [CrossRef]
  20. Tanner, G.J.; Francki, K.T.; Abrahams, S.; Watson, J.M.; Larkin, P.J.; Ashton, A.R. Proanthocyanidin Biosynthesis in Plants: Purification of Legume Leucoanthocyanidin Reductase and Molecular Cloning of Its cDNA. J. Biol. Chem. 2003, 278, 31647–31656. [Google Scholar] [CrossRef]
  21. Yuan, L.; Wang, L.J.; Han, Z.J.; Jiang, Y.Z.; Zhao, L.L.; Liu, H.; Yang, L.; Luo, K.M. Molecular Cloning and Characterization of PtrLAR3, a Gene Encoding Leucoanthocyanidin Reductase from Populus trichocarpa, and Its Constitutive Expression Enhances Fungal Resistance in Transgenic Plants. J. Exp. Bot. 2012, 63, 2513–2524. [Google Scholar] [CrossRef] [PubMed]
  22. Wen, S.S.; Ge, X.L.; Wang, R.; Yang, H.-F.; Bai, Y.E.; Guo, Y.H.; Zhang, J.; Lu, M.Z.; Zhao, S.T.; Wang, L.Q. An Efficient Agrobacterium-Mediated Transformation Method for Hybrid Poplar 84K (Populus alba × P. glandulosa) Using Calli as Explants. Int. J. Mol. Sci. 2022, 23, 2216. [Google Scholar] [CrossRef]
  23. Xin, Y.C.; Meng, S.; Ma, B.; He, W.M.; He, N.J. Mulberry Genes MnANR and MnLAR Confer Transgenic Plants with Resistance to Botrytis cinerea. Plant Sci. 2020, 296, 110473. [Google Scholar] [CrossRef]
  24. Wen, O.Y.; Ning, J.M.; Zhu, X.Z.; Jiang, Y.W.; Wang, J.J.; Yuan, H.B.; Hua, J.J. UPLC-ESI-MS/MS Analysis Revealed the Dynamic Changes and Conversion Mechanism of Non-Volatile Metabolites during Green Tea Fixation. LWT Food Sci. Technol. 2024, 198, 116010. [Google Scholar] [CrossRef]
  25. Li, W.; Wen, L.C.; Chen, Z.T.; Zhang, Z.L.; Pang, X.L.; Deng, Z.C.; Liu, T.; Guo, Y.F. Study on Metabolic Variation in Whole Grains of Four Proso Millet Varieties Reveals Metabolites Important for Antioxidant Properties and Quality Traits. Food Chem. 2021, 357, 129791. [Google Scholar] [CrossRef] [PubMed]
  26. Wang, L.J.; Qu, L.J.; Hu, J.J.; Zhang, L.W.; Tang, F.; Lu, M.Z. Metabolomics Reveals Constitutive Metabolites That Contribute Resistance to Fall Webworm (Hyphantria cunea) in Populus deltoides. Environ. Exp. Bot. 2017, 136, 31–40. [Google Scholar] [CrossRef]
  27. Lattanzio, V.; Lattanzio, V.M.; Cardinali, A. Role of Phenolics in the Resistance Mechanisms of Plants against Fungal Pathogens and Insects. Phytochem. Adv. Res. 2006, 661, 23–67. [Google Scholar]
  28. War, A.R.; Paulraj, M.G.; Ahmad, T.; Buhroo, A.A.; Hussain, B.; Ignacimuthu, S.; Sharma, H.C. Mechanisms of Plant Defense against Insect Herbivores. Plant Signal Behav. 2012, 7, 1306–1320. [Google Scholar] [CrossRef]
  29. Salminen, J.P.; Karonen, M. Chemical Ecology of Tannins and Other Phenolics: We Need a Change in Approach. Funct. Ecol. 2011, 25, 325–338. [Google Scholar] [CrossRef]
  30. Pang, Y.Z.; Peel, G.J.; Wright, E.; Wang, Z.Y.; Dixon, R.A. Early Steps in Proanthocyanidin Biosynthesis in the Model Legume Medicago truncatula. Plant Physiol. 2007, 145, 601–615. [Google Scholar] [CrossRef]
  31. Schweitzer, J.A.; Madritch, M.D.; Bailey, J.K.; LeRoy, C.J.; Fischer, D.G.; Rehill, B.J.; Lindroth, R.L.; Hagerman, A.E.; Wooley, S.C.; Hart, S.C.; et al. From Genes to Ecosystems: The Genetic Basis of Condensed Tannins and Their Role in Nutrient Regulation in a Populus Model System. Ecosystems 2008, 11, 1005–1020. [Google Scholar] [CrossRef]
  32. Ruttanaphan, T.; Songoen, W.; Pluempanupat, W.; Bullangpoti, V. Potential Insecticidal Extracts from Artocarpus lacucha against Spodoptera litura (Lepidoptera: Noctuidae) Larvae. J. Econ. Entomol. 2023, 116, 1205–1210. [Google Scholar] [CrossRef] [PubMed]
  33. Leppä, M.M.; Laitila, J.E.; Salminen, J.-P. Distribution of Protein Precipitation Capacity within Variable Proanthocyanidin Fingerprints. Molecules 2020, 25, 5002. [Google Scholar] [CrossRef] [PubMed]
  34. Barbehenn, R.V.; Constabel, C.P. Tannins in Plant–Herbivore Interactions. Phytochemistry 2011, 72, 1551–1565. [Google Scholar] [CrossRef] [PubMed]
  35. Hwang, S.Y.; Lindroth, R.L. Clonal Variation in Foliar Chemistry of Aspen: Effects on Gypsy Moths and Forest Tent Caterpillars. Oecologia 1997, 111, 99–108. [Google Scholar] [CrossRef]
  36. Osier, T.L.; Hwang, S.Y.; Lindroth, R.L. Effects of Phytochemical Variation in Quaking Aspen Populus Tremuloides Clones on Gypsy Moth Lymantria Dispar Performance in the Field and Laboratory. Ecol. Entomol. 2000, 25, 197–207. [Google Scholar] [CrossRef]
  37. Forkner, R.E.; Marquis, R.J.; Lill, J.T. Feeny Revisited: Condensed Tannins as Anti-herbivore Defences in Leaf-Chewing Herbivore Communities of Quercus. Ecol. Entomol. 2004, 29, 174–187. [Google Scholar] [CrossRef]
  38. Tikkanen, O.P.; Julkunen-Tiitto, R. Phenological Variation as Protection against Defoliating Insects: The Case of Quercus robur and Operophtera brumata. Oecologia 2003, 136, 244–251. [Google Scholar] [CrossRef] [PubMed]
  39. Thitz, P.; Mehtätalo, L.; Välimäki, P.; Randriamanana, T.; Lännenpää, M.; Hagerman, A.E.; Andersson, T.; Julkunen-Tiitto, R.; Nyman, T. Phytochemical Shift from Condensed Tannins to Flavonoids in Transgenic Betula pendula Decreases Consumption and Growth but Improves Growth Efficiency of Epirrita autumnata Larvae. J. Chem. Ecol. 2020, 46, 217–231. [Google Scholar] [CrossRef]
  40. Keathley, C.P.; Potter, D.A. Quantitative Resistance Traits and Suitability of Woody Plant Species for a Polyphagous Scarab, Popillia japonica Newman. Environ. Entomol. 2008, 37, 1548–1557. [Google Scholar] [CrossRef]
  41. Ayres, M.P.; Clausen, T.P.; Stephen, F.M.J.; Redman, A.M.; Reichardt, P.B. Diversity of Structure and Antiherbivore Activity in Condensed Tannins. Ecology 1997, 78, 1696–1712. [Google Scholar] [CrossRef]
  42. Villanueva, X.; Zhen, L.L.; Ares, J.N.; Vackier, T.; Lange, H.; Crestini, C.; Steenackers, H.P. Effect of Chemical Modifications of Tannins on Their Antimicrobial and Antibiofilm Effect against Gram-Negative and Gram-Positive Bacteria. Front. Microbiol. 2023, 13, 987164. [Google Scholar] [CrossRef] [PubMed]
  43. Winder, R.S.; Lamarche, J.; Constabel, C.P.; Hamelin, R. The Effects of High-Tannin Leaf Litter from Transgenic Poplars on Microbial Communities in Microcosm Soils. Front. Microbiol. 2013, 4, 290. [Google Scholar] [CrossRef] [PubMed]
  44. Mallampalli, N.; Barbosa, P.; Weinges, K. Effects of Condensed Tannins and Catalpol on Growth and Development of Compsilura concinnata (Diptera: Tachinidae) Reared in Gypsy Moth (Lepidoptera: Lymantriidae). J. Entomol. Sci. 1996, 31, 289–300. [Google Scholar] [CrossRef]
  45. Zhang, Y.; Bartlow, A.W.; Wang, Z.; Yi, X. Effects of Tannins on Population Dynamics of Sympatric Seed-Eating Rodents: The Potential Role of Gut Tannin-Degrading Bacteria. Oecologia 2018, 187, 667–678. [Google Scholar] [CrossRef]
Agronomy 15 01347 g001
Figure 1. Expression patterns of PagLAR3 and validation of transgenic lines. (a) Relative expression levels of PagLAR3 in leaf, stem, and root, with stem expression set to 1. (b) PagLAR3 expression changes in leaves after feeding by H. cunea larvae, normalized to 0 h. (c) Relative expression levels of PagLAR3 in 84 K, OE-4, and OE-11, with 84 K expression set to 1. (d) Relative contents of CTs in 84 K, OE-4, and OE-11. For each metabolite, content in 84 K was set to 1. p-values from Student’s t-test are denoted with asterisks: * p < 0.05, ** p < 0.01.
Figure 1. Expression patterns of PagLAR3 and validation of transgenic lines. (a) Relative expression levels of PagLAR3 in leaf, stem, and root, with stem expression set to 1. (b) PagLAR3 expression changes in leaves after feeding by H. cunea larvae, normalized to 0 h. (c) Relative expression levels of PagLAR3 in 84 K, OE-4, and OE-11, with 84 K expression set to 1. (d) Relative contents of CTs in 84 K, OE-4, and OE-11. For each metabolite, content in 84 K was set to 1. p-values from Student’s t-test are denoted with asterisks: * p < 0.05, ** p < 0.01.
Agronomy 15 01347 g001
Agronomy 15 01347 g002
Figure 2. Performance of larvae fed with 84 K and transgenic lines. (a) Leaf consumption (circled with red lines) by larvae in 84 K, OE-4, and OE-11 after feeding for 4 h. Bars = 1 cm. (b) Larvae fed with leaves of 84 K, OE-4, and OE-11 for 13 days. Bars = 5 mm. (c) Weight of larvae fed with 84 K, OE-4, and OE-11 for 19 days. (d) Larval duration of larvae fed with 84 K, OE-4, and OE-11. p-values from Student’s t-test are denoted with asterisks: * p < 0.05, ** p < 0.01.
Figure 2. Performance of larvae fed with 84 K and transgenic lines. (a) Leaf consumption (circled with red lines) by larvae in 84 K, OE-4, and OE-11 after feeding for 4 h. Bars = 1 cm. (b) Larvae fed with leaves of 84 K, OE-4, and OE-11 for 13 days. Bars = 5 mm. (c) Weight of larvae fed with 84 K, OE-4, and OE-11 for 19 days. (d) Larval duration of larvae fed with 84 K, OE-4, and OE-11. p-values from Student’s t-test are denoted with asterisks: * p < 0.05, ** p < 0.01.
Agronomy 15 01347 g002
Agronomy 15 01347 g003
Figure 3. Pupation and adult emergence of larvae supplied with 84 K and transgenic lines. (a) Pupae. Bars = 3 mm. (b) Pupal weight. (c) Pupation rate. (d) Rate of adult emergence. p-values from Student’s t-test are denoted with asterisks: * p < 0.05, ** p < 0.01. “ns” denotes no significant difference.
Figure 3. Pupation and adult emergence of larvae supplied with 84 K and transgenic lines. (a) Pupae. Bars = 3 mm. (b) Pupal weight. (c) Pupation rate. (d) Rate of adult emergence. p-values from Student’s t-test are denoted with asterisks: * p < 0.05, ** p < 0.01. “ns” denotes no significant difference.
Agronomy 15 01347 g003
Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

Share and Cite

MDPI and ACS Style

Fan, Z.; Hou, L.; Wang, Z.; Wang, L. Overexpression of PagLAR3 in Populus alba × P. glandulosa Promotes Resistance to Hyphantria cunea. Agronomy 2025, 15, 1347. https://doi.org/10.3390/agronomy15061347

AMA Style

Fan Z, Hou L, Wang Z, Wang L. Overexpression of PagLAR3 in Populus alba × P. glandulosa Promotes Resistance to Hyphantria cunea. Agronomy. 2025; 15(6):1347. https://doi.org/10.3390/agronomy15061347

Chicago/Turabian Style

Fan, Zhibin, Luxuan Hou, Zheshu Wang, and Lijuan Wang. 2025. "Overexpression of PagLAR3 in Populus alba × P. glandulosa Promotes Resistance to Hyphantria cunea" Agronomy 15, no. 6: 1347. https://doi.org/10.3390/agronomy15061347

APA Style

Fan, Z., Hou, L., Wang, Z., & Wang, L. (2025). Overexpression of PagLAR3 in Populus alba × P. glandulosa Promotes Resistance to Hyphantria cunea. Agronomy, 15(6), 1347. https://doi.org/10.3390/agronomy15061347

Note that from the first issue of 2016, this journal uses article numbers instead of page numbers. See further details here.

Article Metrics

Back to TopTop