RESUMEN
The normal development of pollen grains and the completion of double fertilization in embryos are crucial for both the sexual reproduction of angiosperms and grain production. Actin depolymerizing factor (ADF) regulates growth, development, and responses to biotic and abiotic stress by binding to actin in plants. In this study, the function of the ZmADF1 gene was validated through bioinformatic analysis, subcellular localization, overexpression in maize and Arabidopsis, and knockout via CRISPR/Cas9. The amino acid sequence of ZmADF1 exhibited high conservation and a similar tertiary structure to that of ADF homologs. Subcellular localization analysis revealed that ZmADF1 is localized mainly to the nucleus and cytoplasm. The ZmADF1 gene was specifically expressed in maize pollen, and overexpression of the ZmADF1 gene decreased the number of pollen grains in the anthers of transgenic Arabidopsis plants. The germination rate of pollen and the empty seed shell rate in the fruit pods of the overexpressing plants were significantly greater than those in the wild-type (WT) plants. In maize, the pollen viability of the knockout lines was significantly greater than that of both the WT and the overexpressing lines. Our results confirmed that the ZmADF1 gene was specifically expressed in pollen and negatively regulated pollen quantity, vigor, germination rate, and seed setting rate. This study provides insights into ADF gene function and possible pathways for improving high-yield maize breeding.
Asunto(s)
Arabidopsis , Destrina , Polen , Zea mays , Secuencia de Aminoácidos , Arabidopsis/metabolismo , Destrina/genética , Destrina/metabolismo , Gelsolina/metabolismo , Regulación de la Expresión Génica de las Plantas , Polen/genética , Polen/crecimiento & desarrollo , Zea mays/metabolismoRESUMEN
Actin-depolymerizing factor (ADF) is a small class of actin-binding proteins that regulates the dynamics of actin in cells. Moreover, it is well known that the plant ADF family plays key roles in growth, development and defense-related functions. Results: Thirteen maize (Zea mays L., ZmADFs) ADF genes were identified using Hidden Markov Model. Phylogenetic analysis indicated that the 36 identified ADF genes in Physcomitrella patens, Arabidopsis thaliana, Oryza sativa japonica, and Zea mays were clustered into five groups. Four pairs of segmental genes were found in the maize ADF gene family. The tissue-specific expression of ZmADFs and OsADFs was analyzed using microarray data obtained from the Maize and Rice eFP Browsers. Five ZmADFs (ZmADF1/2/7/12/13) from group V exhibited specifically high expression in tassel, pollen, and anther. The expression patterns of 13 ZmADFs in seedlings under five abiotic stresses were analyzed using qRT-PCR, and we found that the ADFs mainly responded to heat, salt, drought, and ABA. Conclusions: In our study, we identified ADF genes in maize and analyzed the gene structure and phylogenetic relationships. The results of expression analysis demonstrated that the expression level of ADF genes was diverse in various tissues and different stimuli, including abiotic and phytohormone stresses, indicating their different roles in plant growth, development, and response to external stimulus. This report extends our knowledge to understand the function of ADF genes in maize.
Asunto(s)
Destrina/genética , Regulación de la Expresión Génica de las Plantas , Estrés Fisiológico , Zea mays/genética , Actinas/metabolismo , Arabidopsis/genética , Bryopsida/genética , Cromosomas de las Plantas/ultraestructura , Destrina/metabolismo , Sequías , Perfilación de la Expresión Génica , Estudios de Asociación Genética , Genoma de Planta , Análisis de Secuencia por Matrices de Oligonucleótidos , Oryza/genética , Filogenia , Reguladores del Crecimiento de las Plantas/metabolismo , Polen/químicaRESUMEN
The Actin Depolymerizing Factor (ADF) gene family of Arabidopsis thaliana encodes 11 functional protein isovariants in four ancient subclasses. We report the characterization of the tissue-specific and developmental expression of all Arabidopsis ADF genes and the subcellular localization of several protein isovariants. The four subclasses exhibited distinct expression patterns as examined by qRT-PCR and histochemical assays of a GUS reporter gene under the control of individual ADF regulatory sequences. Subclass I ADFs were expressed strongly and constitutively in all vegetative and reproductive tissues except pollen. Subclass II ADFs were expressed specifically in mature pollen and pollen tubes or root epidermal trichoblast cells and root hairs, and these patterns evolved from an ancient dual expression pattern comprised of both polar tip growth cell types, still observed in the monocot Oryza sativa. Subclass III ADFs were expressed weakly in vegetative tissues, but were strongest in fast growing and/or differentiating cells including callus, emerging leaves, and meristem regions. The single subclass IV ADF was constitutively expressed at moderate levels in all tissues, including pollen. Immunocytochemical analysis with subclass-specific monoclonal antibodies demonstrated that subclass I isovariants localize to both the cytoplasm and the nucleus of leaf cells, while subclass II isovariants predominantly localize to the cytoplasm at the tip region of elongating root hairs and pollen tubes. The distinct expression patterns of the ADF subclasses support a model of ADF s co-evolving with the ancient and divergent actin isovariants.