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

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.

Gene expression profiling reveals the effects of light on adventitious root formation in lotus seedlings (Nelumbo nucifera Gaertn.).

BACKGROUND: Lotus is an aquatic horticultural crop that is widely cultivated in most regions of China and is used as an important off-season vegetable. The principal root of lotus is degenerated, and adventitious roots (ARs) are irreplaceable for plant growth. We found that no ARs formed under darkness and that exposure to high-intensity light significantly promoted the development of root primordia. Four differential expression libraries based on three light intensities were constructed to monitor metabolic changes, especially in indole-3-acetic acid (IAA) and sugar metabolism. RESULTS: AR formation was significantly affected by light, and high light intensity accelerated AR development. Metabolic changes during AR formation under different light intensities were evaluated using gene expression profiling by high-throughput tag-sequencing. More than 2.2 × 104 genes were obtained in each library; the expression level of most genes was between 0.01 and 100 (FPKF value). Libraries constructed from plants grown under darkness (D/CK), under 5000 lx (E/CK), and under 20,000 lx (F/CK) contained 1739, 1683, and 1462 upregulated genes and 1533, 995, and 834 downregulated genes, respectively, when compared to those in the initial state (CK). Additionally, we found that 1454 and 478 genes had altered expression in a comparison of libraries D/CK and F/CK. Gene transcription between libraries D/F ranged from a 5-fold decrease to a 5-fold increase. Twenty differentially expressed genes (DEGs) were involved in the signal transduction pathway, 28 DEGs were related to the IAA response, and 35 DEGs were involved in sugar metabolism. We observed that the IAA content was enhanced after seed germination, even in darkness; this was responsible for AR formation. We also observed that sucrose could eliminate the negative effect of 150 µMol IAA during AR development. CONCLUSIONS: AR formation was regulated by IAA, even in the dark, where induction and developmental processes could also be completed. In addition, 36 genes displayed altered expression in carbohydrate metabolism and ucrose metabolism was involved in AR development (expressed stage) according to gene expression and content change characteristics.

Transcriptome analysis of miRNAs expression reveals novel insights into adventitious root formation in lotus (Nelumbo nucifera Gaertn.).

MicroRNA (miRNA)-regulated gene expression plays an important role in various plant metabolic processes. Although adventitious roots are critical to plant growth in lotus, the role of miRNA in AR formation remains unclear. Expression profiling of miRNAs was carried out during three different developmental stages of ARs in lotus: no induction of AR stage, initial stage of ARs, and maximum number of ARs. These data are referenced with the whole lotus genome as already identified through high-throughput tag-sequencing. 1.3 × 107 tags were achieved, of which 11,035,798, 11,436,062, and 12,542,392 clean tags were obtained from each stage, respectively. miRNA analysis revealed that miRNAs were less than 10% among all small RNAs. In total, 310 miRNAs (240 up-regulated and 70 down-regulated miRNAs) exhibited expression changes from the no induction stage to the initial stage. Moreover, expression of 140 miRNAs was increased and that of 123 miRNAs was decreased between the initial and maximum AR stages, mostly by ~ - 4-4-fold. miRNAs involved in metabolic pathways differed between the initial stage/no induction stage and the maximum number stage/initial stage. Several miRNAs in the initial stage/no induction stage were related to plant hormone metabolism and pyruvate and MAPK pathways, while major miRNAs in the maximum number stage/initial stage were involved in carbohydrate metabolism. All differentially expressed miRNAs associated with AR formation from the initial stage to maximum stage were also analyzed. The expression of 16 miRNAs was determined using qRT-PCR. This work provides a general insight into miRNA regulation during AR formation in lotus.