Construction and Validation of the pOp6/LhG4AtO Expression System in the Embryo and Endosperm of Arabidopsis Seed

doi:10.13560/j.cnki.biotech.bull.1985.2026-0021

Abstract

Abstract:

Objective To construct the pOp6/LhG4^AtO expression system for studying gene function in the embryo and endosperm of Arabidopsis seeds, and to validate its feasibility to provide a reliable tool for tissue-specific gene expression. Method Based on the pOp6/LhG4^AtO binary transcriptional activation system, previously reported embryo-specific (TWS1) and endosperm-specific (FWA, ESH1) promoters were selected to construct the driver vectors. Meanwhile, a fluorescent reporter gene with enhanced nuclear localization (H2B-Clover) was employed to generate the effector vector. Genetic transformation was performed via Agrobacterium-mediated floral dip method to obtain positive transgenic lines. By crossing the driver lines with the effector line, the tissue-specific expression of the reporter gene was observed in F1 seeds at 2–5 d after pollination, and the expression patterns were compared with publicly available microarray transcriptome data (NCBI GEO; accession number GSE12404). Result The pOp6/LhG4^AtO expression system was successfully constructed, and the required independent transgenic lines were obtained, including driver lines (pTWS1::LhG4^AtO, pFWA::LhG4^AtO, pESH1::LhG4^AtO ) and an effector line (pOp6::H2B-Clover). After crossing the driver line with the effector line, green fluorescence signals driven by pTWS1::LhG4^AtO, were specifically and continuously observed at high levels in the nuclei of embryos cells only at 3‒5 d after pollination (DAP). Similarly, green fluorescence signals driven by pFWA::LhG4^AtO and pESH1::LhG4^AtO were observed specifically and continuously at high levels in the nuclei of endosperm cells at 2‒5 DAP after pollination. These results demonstrate that the fluorescence expression patterns driven by the pOp6/LhG4^AtO system are highly consistent with published microarray transcriptome data. Conclusion This study established and validated the pOp6/LhG4^AtOsystem for tissue-specific driver gene expression in the embryo and endosperm of Arabidopsis seeds, providing a reliable tool for functional studies of key genes during seed development.

Key words: pOp6/LhG4^AtO system, tissue-specific expression, embryo, endosperm, seed development

FU Cai-xia, ZHANG Rui-hua, ZHU Yun-jun, LI Jing, YANG Ke, WANG Xuan-peng. Construction and Validation of the pOp6/LhG4^AtO Expression System in the Embryo and Endosperm of Arabidopsis Seed[J]. Biotechnology Bulletin, 2026, 42(9): 120-127.

Figures/Tables 5

Fig. 1 Vector design and hybrid combination of pOp6/LhG4AtOA: Design of pop6/LhG4Ato vector structure. Promoter: promoter. Ω: Omega, translation enhancement element. Ter: Terminator. TEV: Translation enhancer of tobacco etch virus. pAt2S3: Seed storage albumingene SESA3 promoter. B: Crossing combinations between driver lines and effector lines in the pop6/LhG4Ato system

Fig. 2 Cloning of embryo and endosperm promoter fragments and identification of binary vector digestionA: Cloning of tissue-specific promoter (M: 2 000 DNA marker. 1-4: Cloned fragments were, pTWS1, pFWA, pESH1, H2B-Clover). B: Restriction enzyme digestion identification of recombinant plasmid (M: 2 000 DNA marker. 1-4: Plasmids were pTWS1::LhG4AtO, pFWA::LhG4AtO, pESH1::LhG4AtO, pOp6::H2B-Clover, respectively)

Fig. 3 Screening and identification of transgenic materialsA: Screening of transgenic positive seeds. Scale bar=1 mm. B: Detection of positive identification of transgenic lines by agarose gel electrophoresis (M: 2 000 DNA marker. 1-4: The fragments were pTWS1::LhG4AtO, pFWA::LhG4AtO, pESH1::LhG4AtO, pOp6::H2B-Clover, respectively)

Fig. 4 Relative expression of TWS1 at different developmental stages of seeds and the expression of H2B-Clover reporter gene driven by TWS1A: Expression pattern of TWS1 in seed development stage (NCBI GEO; accession number GSE12404). Stage pG: Pre-globular stage. Stage G: Globular stage. Stage H: Heart stage. Stage IC: Linage cotyledon stage. Stage bC: Bent cotyledon stage. Em: Embryo. Mi En: Micropylar endosperm. Pe En: Peripheral endosperm. Ch En: Chalazal endosperm. Ch SC: Chalazal seed coat. Ge SC: General seed coat. The Y-axis value is a logarithmic scale value based on 10. The same below. B: Fluorescence expression of the TWS1 driven effector line in F1 hybrid seeds at 2‒5 d after pollination. The nuclear-localized green fluorescent signal in the embryo indicates the TWS1 driven target signal, whereas the autofluorescence of plastids in the seed coat and endosperm appears as a red fluorescent signal. DAP is days after pollination. Scale bar, 50 μm. The same below

Fig. 5 Expression pattern analysis of FWA and ESH1 genes at different developmental stages of seeds and the expression of H2B-Clover reporter gene driven by themA: Expression patterns of FWA and ESH1 during seed development (NCBI GEO; accession number GSE12404). B‒C: Fluorescence expression of the FWA and ESH1 effector lines in F1 hybrid seeds at 2‒5 d after pollination, respectively. The nuclear-localized green fluorescent signal in the endosperm represents the target signal driven by FWA and ESH1, whereas the autofluorescence of plastids in the seed coat, embryo, and endosperm appears as a red fluorescent signal. Scale bar, 50 μm

References 30

[1]	Pankaj R, Lima RB, Figueiredo DD. Hormonal regulation and crosstalk during early endosperm and seed coat development [J]. Plant Reprod, 2025, 38: 5.
[2]	Wang W, Xiong HX, Sun KT, et al. New insights into cell-cell communications during seed development in flowering plants [J]. J Integr Plant Biol, 2022, 64(2): 215-229.
[3]	Ingram GC. Family life at close quarters: communication and constraint in angiosperm seed development [J]. Protoplasma, 2010, 247(3/4): 195-214.
[4]	Doll NM, Ingram GC. Embryo-endosperm interactions [J]. Annu Rev Plant Biol, 2022, 73: 293-321.
[5]	Paolo D, Rotasperti L, Schnittger A, et al. The Arabidopsis MADS-domain transcription factor SEEDSTICK controls seed size via direct activation of E2Fa [J]. Plants, 2021, 10(2): 192.
[6]	Ali MF, Shin JM, Fatema U, et al. Cellular dynamics of coenocytic endosperm development in Arabidopsis thaliana [J]. Nat Plants, 2023, 9(2): 330-342.
[7]	Samakovli D, Tichá T, Vavrdová T, et al. HEAT SHOCK PROTEIN 90 proteins and YODA regulate main body axis formation during early embryogenesis [J]. Plant Physiol, 2021, 186(3): 1526-1544.
[8]	Tao Z, Hu HM, Luo X, et al. Embryonic resetting of the parental vernalized state by two B3 domain transcription factors in Arabidopsis [J]. Nat Plants, 2019, 5(4): 424-435.
[9]	Lloyd JPB, Khan A, Lister R. The switch-liker’s guide to plant synthetic gene circuits [J]. Plant J, 2025, 121(5): e70090.
[10]	Benfey PN, Chua NH. The cauliflower mosaic virus 35S promoter: combinatorial regulation of transcription in plants [J]. Science, 1990, 250(4983): 959-966.
[11]	Oh Y, Nagalakshmi U, Dahlbeck D, et al. Heritable virus-induced germline editing in tomato [J]. Plant J, 2025, 122: e70115.
[12]	Nishihara M, Hirabuchi A, Teshima T, et al. Flower color modification in Torenia fournieri by genetic engineering of betacyanin pigments [J]. BMC Plant Biol, 2024, 24: 614.
[13]	Ali ME, Waliullah S. A Core35S promoter of cauliflower mosaic virus drives more efficient replication of turnip crinkle virus [J]. Plants, 2021, 10(8): 1700.
[14]	Samalova M, Brzobohaty B, Moore I. pOp6/LhGR: a stringently regulated and highly responsive dexamethasone-inducible gene expression system for tobacco [J]. Plant J, 2005, 41(6): 919-935.
[15]	Jia HG, Van Loock B, Liao MJ, et al. Combination of the ALCR/AlcA ethanol switch and GAL4/VP16-UAS enhancer trap system enables spatial and temporal control of transgene expression in Arabidopsis [J]. Plant Biotechnol J, 2007, 5(4): 477-482.
[16]	Fu C, Robbins N, Cowen LE. Adaptation of the tetracycline-repressible system for modulating the expression of essential genes in Cryptococcus neoformans [J]. mSphere, 2025, 10(5): e01018-e01024.
[17]	Moore I, Gälweiler L, Grosskopf D, et al. A transcription activation system for regulated gene expression in transgenic plants [J]. Proc Natl Acad Sci U S A, 1998, 95(1): 376-381.
[18]	Craft J, Samalova M, Baroux C, et al. New pOp/LhG4 vectors for stringent glucocorticoid-dependent transgene expression in Arabidopsis [J]. Plant J, 2005, 41(6): 899-918.
[19]	Schürholz AK, López-Salmerón V, Li ZN, et al. A comprehensive toolkit for inducible, cell type-specific gene expression in Arabidopsis [J]. Plant Physiol, 2018, 178(1): 40-53.
[20]	Rutherford S, Brandizzi F, Townley H, et al. Improved transcriptional activators and their use in mis-expression traps in Arabidopsis [J]. Plant J, 2005, 43(5): 769-788.
[21]	López-Salmerón V, Schürholz AK, Li ZN, et al. Inducible, cell type-specific expression in Arabidopsis thaliana through LhGR-mediated Trans-activation [J]. JoVE, 2019(146): e59394.
[22]	Zerin T, Greb T, Wolf S. Inducible, tissue-specific gene expression in Arabidopsis using GR-LhG4-mediated trans-activation [M]//Kaufmann K, Vandepoele K. Plant Gene Regulatory Networks: Methods and Protocols. New York, Springer US, 2023: 13-25.
[23]	Fiume E, Guyon V, Remoué C, et al. TWS1, a novel small protein, regulates various aspects of seed and plant development [J]. Plant Physiol, 2016, 172(3): 1732-1745.
[24]	Doll NM, Royek S, Fujita S, et al. A two-way molecular dialogue between embryo and endosperm is required for seed development [J]. Science, 2020, 367(6476): 431-435.
[25]	Kinoshita T, Miura A, Choi Y, et al. One-way control of FWA imprinting in Arabidopsis endosperm by DNA methylation [J]. Science, 2004, 303(5657): 521-523.
[26]	Yang K, Tang YL, Li Y, et al. Two imprinted genes primed by DEMETER in the central cell and activated by WRKY10 in the endosperm [J]. J Genet Genom, 2024, 51(8): 855-865.
[27]	Li N, Li YH. Signaling pathways of seed size control in plants [J]. Curr Opin Plant Biol, 2016, 33: 23-32.
[28]	Phillips AR, Evans MMS. Maternal regulation of seed growth and patterning in flowering plants [M]//Maternal Effect Genes in Development. Amsterdam: Elsevier, 2020: 257-282.
[29]	Belmonte MF, Kirkbride RC, Stone SL, et al. Comprehensive developmental profiles of gene activity in regions and subregions of the Arabidopsis seed [J]. Proc Natl Acad Sci U S A, 2013, 110(5): E435-E444.
[30]	Mahas A, Aman R, Mahfouz M. CRISPR-Cas13d mediates robust RNA virus interference in plants [J]. Genome Biol, 2019, 20: 263.