Functional Study of SmHD-ZIP25 and SmHD-ZIP29 Genes in Flower Development of Sapindus mukorossi

doi:10.13560/j.cnki.biotech.bull.1985.2026-0174

Abstract

Abstract:

Objective To investigate the regulatory roles of SmHD-ZIP25 and SmHD-ZIP29 in Sapindus mukorossi flower development and sexual differentiation, and to provide valuable reference genes for its molecular design breeding. Method Using S. mukorossi as a model plant, two members of the HD-ZIP I subfamily, SmHD-ZIP25 and SmHD-ZIP29, were cloned. These genes were subjected to sequence alignment, phylogenetic analysis, subcellular localization, and expression pattern analysis. Their functions were further investigated through heterologous transformation in Arabidopsis. Result Sequence alignment and phylogenetic tree analysis revealed that SmHD-ZIP25 and SmHD-ZIP29 possessed complete Homeodomain and Leucine Zipper domains and exhibited high homology with the sex differentiation-related persimmon MeGI gene. Subcellular localization studies indicated that both proteins were localized in the nucleus. Expression pattern analysis revealed distinct expression patterns of SmHD-ZIP25 and SmHD-ZIP29 during S. mukorossi male and female flower development, while they showed similar expression levels in root, stem, and leaf tissues. Compared with wild-type Arabidopsis, overexpression of either SmHD-ZIP25 or SmHD-ZIP29 exhibited phenotypes including premature flowering, rosette leaf curling, and pod developmental abnormalities, significantly impairing floral organ development. Specifically, pistils exhibited relative elongation while stamen development was suppressed (shortened anthers, pollen abortion, and severely reduced pollen viability). Cytological observations further confirmed abnormal anther sac development and collapsed pollen grain morphology in transgenic plants. RT-qPCR analysis indicated that these two genes synergistically regulated female-promoting and male-suppressing developmental processes by upregulating the expression of the flowering integrator AtFT and of the carpel developmental genes AtSHP2 and AtCRC, while simultaneously suppressing the expression of the key jasmonic acid signaling pathway gene AtCOL1 and its downstream male organ developmental core factors AtAMS and AtMYB21. Conclusion SmHD-ZIP25 and SmHD-ZIP29 are key factors involved in regulating floral organ developmental balance, providing new theoretical support for elucidating the molecular mechanisms underlying S. mukorossi flower development and sexual differentiation.

Key words: Sapindus mukorossi, HD-ZIP transcription factor, flower development, sex differentiation, pistil development, stamen development, pollen fertility, molecular regulatory mechanism

CHEN Ning-yi, WU Wen-tong, ZHAO Guo-chun, JIA Li-ming, CHEN Zhong. Functional Study of SmHD-ZIP25 and SmHD-ZIP29 Genes in Flower Development of Sapindus mukorossi[J]. Biotechnology Bulletin, 2026, 42(9): 210-220.

Figures/Tables 10

Fig. 1 Sequence alignment (A) and phylogenetic analysis (B) of SmHD-ZIP25, SmHD-ZIP29 and their homologs from Diospyros speciesIn Fig. A, the red and blue lines indicate the conserved Homeodomain (HD) and Leucine Zipper (LZ) domains, respectively

Fig. 2 Subcellular localization of SmHD-ZIP25 and SmHD-ZIP29

Fig. 3 Expression of SmHD-ZIP25 and SmHD-ZIP29 in different organs of S. mukorossiFF1-FF7 represent seven developmental stages of female flowers, and MF1-MF7 represent seven developmental stages of male flowers. * P < 0.05, ** P < 0.01, *** P < 0.001, the same below

Fig. 4 Molecular characterization and phenotypic analysis of Arabidopsis plants overexpressing SmHD-ZIP25A: PCR identification of transgenic plants. B: Relative expression of SmHD-ZIP25 in wild-type and transgenic plants. C: Overall phenotype comparison between wild-type and overexpressing plants at the flowering stage. D: Vegetative growth morphology of 20-day-old seedlings. E: Morphology of rosette leaves. F: Statistical analysis of rosette leaf number and flowering time. G: Silique morphology (intact and dissected). H: Curling phenotype of cauline leaves. Scale bar: 1 cm. The same for Fig. 5

Fig. 5 Molecular characterization and phenotypic analysis of Arabidopsis plants overexpressing SmHD-ZIP29

Fig. 6 Overexpression of SmHD-ZIP25 leads to stamen development inhibition and floral organ morphological changesA: Comparative phenotypes of the whole flower, pistil, and stamens. B: Pollen load on the stigma. C: Statistical analysis of pistil length and stamen number. Scale bars: 250 µm (A), 50 µm (B). The same for Fig. 7

Fig. 7 Overexpression of SmHD-ZIP29 leads to stamen development inhibition and floral organ morphological changes

Fig. 8 Paraffin sections of anther cross-sections from SmHD-ZIP25 and SmHD-ZIP29 overexpressing plantsStage 9: Uninucleate microspore vacuolation stage. Stage 10: Late uninucleate microspore stage. Stage 11: Tapetum degradation initiation stage. Stage 12: Pollen maturation stage. Stage 13: Anther dehiscence stage. E: Epidermis. En: Endothecium. ML: Middle layer. T: Tapetum. Msp: Microspore. Scale bars: 50 μm

Fig. 9 Microstructural and fertility analysis of anthers and pollen in SmHD-ZIP25 and SmHD-ZIP29 overexpressing plantsA: Scanning electron micrograph of anthers. B: Scanning electron micrograph of pollen grain surface morphology. C: Alexander staining of anthers. D: Pollen germination in vitro. Scale bars: 300 μm (A), 10 μm (B), 50 μm (C), 100 μm (D)

Fig. 10 Changes in endogenous gene expression during flower development in SmHD-ZIP25 (A) and SmHD-ZIP29 (B) transgenic lines

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