NnWOX1-1, NnWOX4-3, and NnWOX5-1 of lotus (Nelumbo nucifera Gaertn)promote root formation and enhance stress tolerance in transgenic Arabidopsis thaliana

Abstract

Background: Adventitious roots (ARs) represent an important organ system for water and nutrient uptake in lotus plants because of degeneration of the principal root. The WUSCHEL-related homeobox (WOX) gene regulates plant development and growth by affecting the expression of several other genes. In this study, three WOX genes, NnWOX1-1, NnWOX4-3, and NnWOX5-1, were isolated and their functions were assessed in Arabidopsis plants.

Results: The full lengths of NnWOX1-1, NnWOX4-3, and NnWOX5-1 were 1038, 645, and 558 bp, encoding 362, 214, and 185 amino acid residues, respectively. Phylogenetic analysis classified NnWOX1-1 and NnWOX4-3 encoding proteins into one group, and NnWOX5-1 and MnWOX5 encoding proteins exhibited strong genetic relationships. The three genes were induced by sucrose and indoleacetic acid (IAA) and exhibited organ-specific expression characteristics. In addition to improving root growth and salt tolerance, NnWOX1-1 and NnWOX4-3 promoted stem development in transgenic Arabidopsis plants. A total of 751, 594, and 541 genes, including 19, 19, and 13 respective genes related to ethylene and IAA metabolism and responses, were enhanced in NnWOX1-1, NnWOX4-3, and NnWOX5-1 transgenic plants, respectively. Further analysis showed that ethylene production rates in transgenic plants increased, whereas IAA, peroxidase, and lignin content did not significantly change. Exogenous application of ethephon on lotus seedlings promoted AR formation and dramatically increased the fresh and dry weights of the plants.

Conclusions: NnWOX1-1, NnWOX4-3, and NnWOX5-1 influence root formation, stem development, and stress adaptation in transgenic Arabidopsis plants by affecting the transcription of multiple genes. Among these, changes in gene expression involving ethylene metabolism and responses likely critically affect the development of Arabidopsis plants. In addition, ethylene may represent an important factor affecting AR formation in lotus seedlings.

Keywords: Adventitious root; Arabidopsis; Lotus; NnWOX1-1; NnWOX4-3; NnWOX5-1.

Fig. 1

Comparison and phylogenetic tree analysis of Nnwox1-1, Nnwox4-3, and Nnwox5-1. a Comparison of NnWOX1-1, NnWOX4-3, and NnWOX5-1 with amino acid sequences. The red box represents the homologous region of the three genes. b Domain analysis of NnWOX1-1-, NnWOX4-3-, and NnWOX5-1-encoded proteins, and boxes of different colors represent conserved regions. c Phylogenetic tree analysis of NnWOX1-1-, NnWOX4-3-, and NnWOX5-1-encoded proteins with WOX-encoded proteins of other species. Fourteen groups with different colors were detected. The red triangle represents the positions of the three proteins in the phylogenetic tree

Fig. 2

Conserved motif and chromosomal localization analysis of NnWOX1-1-, NnWOX4-3-, and NnWOX5-1-encoded proteins in the whole lotus genome. a Distribution of conserved motifs in NnWOX1-1-, NnWOX4-3-, and NnWOX5-1-encoded proteins. Boxes of different colors represent the ten putative motifs, and the boxes with the same color represent the same motif in the structure of these three genes. b Chromosomal localization analysis of NnWOX1-1, NnWOX4-3, and NnWOX5-1

Fig. 3

Expression patterns of NnWOX1-1, NnWOX4-3, and NnWOX5-1 with different treatments and in different organs, as determined by RT-qPCR. a Expression analysis after sucrose treatment. b Identification of gene expression in lotus seedlings treated with IAA. c Organ-specific expression analysis in lotus plant roots, stems, leaves, and flowers. The mean expression values were calculated from three independent biological replicates, and “*” indicates values that are significantly different compared with control plants (P < 0.05)

Fig. 4

Functional analysis of NnWOX1-1, NnWOX4-3, and NnWOX5-1 in transgenic Arabidopsis plants. a Assessment of stem and root development in transgenic plants with constitutive NnWOX1-1 expression and wild-type plants. b Effect of NnWOX4-3 on stem growth and root development in transgenic Arabidopsis plants. c Effect of NnWOX5-1 on stem growth and root development in transgenic plants. The mean values were calculated from three replicate experiments, and error bars show standard deviation. Statistical analysis was performed using a Student's t-test, * P < 0.05

Fig. 5

Statistical analysis of upregulated genes and pathway enrichment in transgenic plants with constitutive expression of NnWOX1-1, NnWOX4-3, and NnWOX5-1. a Number of upregulated genes following overexpression of NnWOX1-1, NnWOX4-3, and NnWOX5-1 in Arabidopsis plants. b Genes involved in plant hormone transduction pathway are counted in transgenic plants expressing NnWOX1-1, NnWOX4-3, and NnWOX5-1

Fig. 6

Determination of ethylene, IAA, lignin, and ABA contents in transgenic plants expressing NnWOX1-1, NnWOX4-3, and NnWOX5-1 and in wild-type plants. a Ethylene content in six-leaf-stage transgenic and wild-type plants. b Analysis of IAA content in transgenic and wild type plants at the six-leaf stage. c Lignin contents of the three transgenic and wild-type plants were analyzed to monitor the effect of NnWOX1-1, NnWOX4-3, and NnWOX5-1 expression. d ABA content in NnWOX1-1, NnWOX4-3, and NnWOX5-1 transgenic and wild-type plants. For statistical analysis, the data were recorded as means ± SEs of three experiments with approximately 10 seedlings in each experiment. “*” indicates that values are significantly different between samples (P < 0.05)

Fig. 7

Role of ethylene in the development of lotus seedlings. a Effect of ethylene on the rates of adventitious root formation in lotus seedlings at 0, 2, 4, and 6 d after ethephon treatment. b Stem growth at 0, 2, 4, and 6 d after ethephon treatment in lotus seedlings. c Determination of fresh weight in response to ethephon treatment at 0, 5, 15, and 25 d in lotus seedlings. d Change in dry weight of lotus seedlings after ethephon treatment for 0, 5, 15, and 25 d. Each experiment was carried out with three replicates, and the data represent means ± SEs for approximately 20 seedlings. “*” indicates values that are significantly different between samples (P < 0.05)

Fig. 8

Survival rates of transgenic NnWOX1-1, NnWOX4-3, and NnWOX5-1 plants and wild-type plants in response to drought and salt stress. a Survival rates of transgenic NnWOX1-1 Arabidopsis plants. b Survival rates of transgenic NnWOX4-3 plants and wild-type plants. c Survival rates of NnWOX5-1 transgenic plants. Data are presented as the means ± SE of three replicates, and p < 0.05 was accepted as the level of significance in statistical comparisons