Auxin Induces and Regulates the Expressions of PagHAM4a/PagHAM4b and Affects the Secondary Xylem Development in Poplar

doi:10.13560/j.cnki.biotech.bull.1985.2025-1092

Abstract

Abstract:

Objective Auxin plays a critical role in plant growth and development. This study aims to explore the regulatory role of exogenous auxin (IAA) on the expressions of PagHAM4a and PagHAM4b in 84K poplar (Populus alba × Populus glandulosa), as well as its effect on the secondary xylem development in poplar, providing a basis for exploring the interaction between transcription factors and IAA in the development of secondary xylem in trees. Method Using 84K poplar as experimental material, exogenous IAA was applied. RT-qPCR technology was employed to detect the expressions of PagHAM4a and PagHAM4b. By constructing expression vectors for two genes: one vector containing a GFP reporter driven by the auxin-responsive promoter DR5 and a GUS reporter driven by the PagHAM4a-specific promoter; the other vector containing the DR5-driven GFP reporter and the PagHAM4b-specific promoter-driven GUS reporter, to investigate the relationship between the expression regions of the PagHAM4a/PagHAM4b and auxin distribution. Simultaneously, through measurement and statistical analysis, combined with paraffin sectioning techniques, the external morphology and the microscopic structure of stem secondary xylem in IAA-treated wild-type 84K poplar, PagHAM4a- and PagHAM4b- overexpressing plants (PagHAM4a-OE and PagHAM4b-OE) were observed. Result RT-qPCR result indicated that exogenous IAA application significantly up-regulated the expressions of both PagHAM4a and PagHAM4b. Dual reporter gene assays revealed that expression regions of the PagHAM4a and PagHAM4b in the secondary stem of 84K poplar coincided with the distribution of auxin. Phenotypic analysis showed that, compared with untreated plants, the IAA application significantly increased plant height, leaf area, stem diameter, and internode number in 84K poplar, PagHAM4a-OE and PagHAM4b-OE plants, and also resulted in a significant increase in the width and cell layer number of both the secondary xylem and the cambium in the stems, consistent with the upregulation trend of PagHAM4a and PagHAM4b expression. Conclusion The application of exogenous IAA promotes the expressions of PagHAM4a and PagHAM4b genes and the development of secondary xylem in the stems of 84K poplar. PagHAM4a and PagHAM4b participate in regulating the development of secondary xylem through the auxin signaling pathway.

Key words: 84K poplar, IAA, PagHAM4a/PagHAM4b, dual-reporter gene, secondary xylem

LIU Ran, ZHAO Pan, ZHOU Xin-yi, SU Yu-ting, ZHENG Shu-ya, GAO Hong-bo, GUO Hui-hong. Auxin Induces and Regulates the Expressions of PagHAM4a/PagHAM4b and Affects the Secondary Xylem Development in Poplar[J]. Biotechnology Bulletin, 2026, 42(9): 178-185.

Figures/Tables 8

Fig. 1 Schematic diagram of the dual-reporter gene plant expression vector structureA: A dual-reporter gene plant expression vector in which DR5 promoter drives the GFP reporter gene and the PagHAM4a-specific promoter drives the GUS reporter gene. B: A dual-reporter gene plant expression vector in which DR5 promoter drives the GFP reporter gene and the PagHAM4b-specific promoter drives the GUS reporter gene; DR5pro: Auxin-response promoter. GFP: Green fluorescent protein gene. Tons: Terminator of the nopaline synthase gene. PagHAM4apro/PagHAM4bpro: Specific promoter of the PagHAM4a and PagHAM4b gene. GUS: β-glucuronidase gene

Fig. 2 Expressions of PagHAM4a and PagHAM4b in the stems and leaves of 84K poplar after exogenous IAA treatmentA: PagHAM4a; B: PagHAM4b. Stems and leaves from 3rd‒5th internodes: The internodes during the early stage of secondary growth and the leaves corresponding to those internodes. Stems and leaves from 8th‒10th internodes: The internodes that have showed significant secondary growth and the leaves corresponding to those internodes. *P<0.05; **P<0.01; ***P<0.001. The same below

Fig. 3 PCR identification of transgenic plants containing a dual reporter gene plant expression vectorA: PCR identification of PagHAM4a transgenic plants. B: PCR identification of PagHAM4b transgenic plants. M: DNA marker. P: Plasmids containing the DR5pro::GFP-PagHAM4apro::GUS and DR5pro::GFP-PagHAM4bpro::GUS vector. WT: 84K poplar. #1, #2, #3: Transgenic plants

Fig. 4 GUS assay of transgenic plants

Fig. 5 GFP fluorescence and GUS staining in internodes 7th‒9th of secondary stems from transgenic plants

Fig. 6 Phenotype of wild-type 84K poplar and 84K poplar overexpressing PagHAM4a/PagHAM4b after exogenous IAA treatmentA: External morphological characteristics of plants after exogenous IAA treatment (Bar=10 cm). B: Leaves of plants after exogenous IAA treatment (Bar=10 cm). C: Plant height. D: Leaf area. E: Stem diameter. F: Internode number. WT: 84K plants; PagHAM4a-OE/PagHAM4b-OE: Plants overexpressing PagHAM4a/PagHAM4b in 84K poplar. Different letters indicate significant differences (P<0.05). The same below

Fig. 7 Internal tissue struction phenotype of wild-type 84K poplar and 84K poplar overexpressing PagHAM4a/PagHAM4b after exogenous IAA treatmentA: Cross-sections of the 20th internode of stems from 84K poplar and 84K poplar overexpressing PagHAM4a/PagHAM4b (Bar=100 μm). B: Cambium region of the 20th internode in stems of 84K poplar and 84K poplar overexpressing PagHAM4a/PagHAM4b (Bar=100 μm). C: Xylem width. D: Xylem cell layer number. E: Cambium width. F: Cambium cell layer number. G: Vessel cell area. H: Fiber cell area. Ph: Phloem. Xy: Xylem. Ca: Cambium

Fig. 8 Expressions of PagHAM4a and PagHAM4b in wild-type and transgenic 84K poplar plants after exogenous IAA treatmentA: 3rd‒5th internodes; B: 8th‒10th internodes

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