NnARF17 and NnARF18 from lotus promote root formation and modulate stress tolerance in transgenic Arabidopsis thaliana

Abstract

Auxin response factors (ARFs) play a crucial role in regulating gene expression within the auxin signal transduction pathway, particularly during adventitious root (AR) formation. In this investigation, we identified full-length sequences for ARF17 and ARF18, encompassing 1,800 and 2,055 bp, encoding 599 and 684 amino acid residues, respectively. Despite exhibiting low sequence homology, the ARF17- and ARF18-encoded proteins displayed significant structural similarity and shared identical motifs. Phylogenetic analysis revealed close relationships between NnARF17 and VvARF17, as well as NnARF18 and BvARF18. Both ARF17 and ARF18 demonstrated responsiveness to exogenous indole-3-acetic acid (IAA), ethephon, and sucrose, exhibiting organ-specific expression patterns. Beyond their role in promoting root development, these ARFs enhanced stem growth and conferred drought tolerance while mitigating waterlogging stress in transgenic Arabidopsis plants. RNA sequencing data indicated upregulation of 51 and 75 genes in ARF17 and ARF18 transgenic plants, respectively, including five and three genes associated with hormone metabolism and responses. Further analysis of transgenic plants revealed a significant decrease in IAA content, accompanied by a marked increase in abscisic acid content under normal growth conditions. Additionally, lotus seedlings treated with IAA exhibited elevated levels of polyphenol oxidase, IAA oxidase, and peroxidase. The consistent modulation of IAA content in both lotus and transgenic plants highlights the pivotal role of IAA in AR formation in lotus seedlings.

Keywords: Arabidopsis; NnARF17; NnARF18; Adventitious root; lotus.

Fig. 1

The role of exogenous application of IAA on ARs formation in lotus seedlings. a. Observation of root growth from morphology of seedlings treated with 10 µM IAA for 0 d, 2 d, 4 d, 6 d, and 8 d. Analysis of microstructure on ARs formation at the hypocotyls of lotus seedlings treated with 10 µM IAA for 0 d, 2 d, 4 d, 6 d, and 8 d. Ellipses represents ARs or ARs primordium

Fig. 2

Comparison and phylogenetic tree analysis of NnARF17 and NnARF18. (a) Comparison of NnARF17 and NnARF18 with amino acid sequences. (b) Domain analysis of NnARF17 and NnARF18 encoded proteins, and the box different color represents conserved region. (c) Motifs analysis of NnARF17 and NnARF18 encoded proteins. Boxes of different colors represent the ten putative motifs, and the boxes with the same color represent the same motif in structure of these three genes. (d) Phylogenetic tree analysis of NnARF17 and NnARF18 encoded proteins with ARF17 and ARF18 encoded proteinof other species, and total eleven groups with different color were detected. The red triangle represents the positions of the NnARF17 and NnARF18 encoded proteins in the phylogenetic tree

Fig. 3

Expression patterns of ARF17 and ARF18 with different treatments and in different organs, as determined by qRT-PCR. (a) Expression analysis of NnARF17 and NnARF18 after IAA treatment. (b) Identification of NnARF17 and NnARF18 expression in lotus seedlings treated with ethephon. (c) Determination of NnARF17 and NnARF18 expression in lotus seedlings treated with sucrose. (d) Organ-specific expression analysis in roots, stems, leaves, flowers and fruit of lotus plants. The data were recorded as means ± SEs of three biological replicates with about five seedlings in each experiment. Significant differences were carried out and determined by presented as * p < 0.05

Fig. 4

Subcellular localization of NnARF17 and NnARF18 in tobacco plants

Fig. 5

Functional analysis of NnARF17 and NnARF18 in transgenic Arabidopsis plants. (a) Assessment of root development in transgenic plants with constitutive NnARF17, NnARF18 and wild-type plants. (b) Effect of NnARF17 and NnARF18 on stem growth in transgenic Arabidopsis plants. (c) Statistic analysis of NnARF17 and NnARF18 role on root and stem development. The mean values were calculated from three replicated experiments, and error bars showed standard deviation. Significant differences were determined by Student’s t-test. Statistically significant difference between two samples was presented as * p < 0.05

Fig. 6

Statistical analysis of DEGs and pathway enrichment in transgenic plants. (a) Number of DEGs following overexpression of NnARF17 and NnARF18 in Arabidopsis plants. (b) Distribution of DEGs between NnARF17 and NnARF18 transgenic plants. (c) Genes involved in plant hormone transduction pathway are counted in transgenic plants expressing NnARF17 and NnARF18.

Fig. 7

Determination of IAA, GA3, ABA, and POD contents in transgenic plants with NnARF17 and NnARF18 and in wild-type plants. For statistical analysis, the data were recorded as means ± SEs of three biological replicates with about twenty seedlings in each experiment. Significant differences were determined by Student’s t-test. Statistically significant difference between two samples was presented as * p < 0.05

Fig. 8

Survival rates of transgenic NnARF17 and NnARF18 plants and wild-type plants in response to waterlogging, drought and salt stresses. (a) Survival rates of transgenic NnARF17, NnARF18 and wild type Arabidipsis plants after waterlogging treatment. (b) Survival rates of transgenic NnARF17, NnARF18 plants and wld-type plants after drought treatment. (c) Survival rates of NnARF17, NnARF18 transgenic plants and wild type plants after salt treatment. The data were recorded as means ± SEs of three biological replicates with about fifty seedlings in each experiment. Significant differences were determined by Student’s t-test. Statistically significant difference between two samples was presented as * p < 0.05

Fig. 9

Role of IAA on the development of lotus seedlings. a. Effect of exogenous IAA on the content of IAA, ABA, GA3, POD, PPO and IAAO in lotus seedlings at 0, 2, 4, 6 and 8 d after 10 µM IAA treatment. Each experiment was carried out with three replicates, and the data represents means ± SEs for about 20 seedlings. Significant differences were determined by Student’s t-test. Significant difference between two samples was presented as * p < 0.05