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

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.

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.

Comparative transcriptome analysis revealed the cooperative regulation of sucrose and IAA on adventitious root formation in lotus (Nelumbo nucifera Gaertn).

BACKGROUND: In China, lotus is an important cultivated crop with multiple applications in ornaments, food, and environmental purification. Adventitious roots (ARs), a secondary root is necessary for the uptake of nutrition and water as the lotus principle root is underdeveloped. Therefore, AR formation in seedlings is very important for lotus breeding due to its effect on plant early growth. As lotus ARs formation was significantly affected by sucrose treatment, we analyzed the expression of genes and miRNAs upon treatment with differential concentrations of sucrose, and a crosstalk between sucrose and IAA was also identified. RESULTS: Notably, 20 mg/L sucrose promoted the ARs development, whereas 60 mg/L sucrose inhibited the formation of ARs. To investigate the regulatory pathway during ARs formation, the expression of genes and miRNAs was evaluated by high-throughput tag-sequencing. We observed that the expression of 5438, 5184, and 5345 genes was enhanced in the GL20/CK0, GL60/CK0, and CK1/CK0 libraries, respectively. Further, the expression of 73, 78, and 71 miRNAs was upregulated in the ZT20/MCK0, ZT60/MCK0, and MCK1/MCK0 libraries, respectively. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis revealed that most of the differentially expressed genes and miRNAs in the GL20/GL60 and ZT20/ZT60 libraries were involved in signal transduction. A large number of these genes (29) and miRNAs (53) were associated with plant hormone metabolism. We observed an association between five miRNAs (miR160, miR156a-5p, miR397-5p_1, miR396a and miR167d) and nine genes (auxin response factor, protein brassinosteroid insensitive 1, laccase, and peroxidase 27) in the ZT20/ ZT60 libraries during ARs formation. Quantitative polymerase chain reaction (qRT-PCR) was used to validate the high-throughput tag-sequencing data. CONCLUSIONS: We found that the expression of many critical genes involved in IAA synthesis and IAA transport was changed after treatment with various concentration of sucrose. Based on the change of these genes expression, IAA and sucrose content, we concluded that sucrose and IAA cooperatively regulated ARs formation. Sucrose affected ARs formation by improving IAA content at induction stage, and increased sucrose content might be also required for ARs development according to the changes tendency after application of exogenous IAA.

Molecular cloning and functional analysis of lotus salt-induced NnDREB2C, NnPIP1-2 and NnPIP2-1 in Arabidopsis thaliana.

Dehydration-responsive element bindings transcription factor (DREBs) and plasma membrane intrinsic proteins (PIPs) have been characterized multi-functions in plant growth and metabolism, as well as in the adaptation to various stresses. In this study, we cloned the full-length cDNA of NnDREB2C from a salt-tolerated lotus species with RT-PCR methods. Analysis of qRT-PCR demonstrated that NnDREB2C mRNA in the leaf dramatically increased after the treatments of NaCl, abscisic acid, low temperature and mannitol. Next, NnDREB2C was cloned into constitutive expression vector pSN1301, which in turn transformed into Arabidopsis thaliana to investigate its function in plants. NnDREB2C overexpression significantly elevated Arabidopsis tolerance against salt and drought stresses, showing higher survival rates, lower conductivity and more chlorophyll content than those of wild-type plants. Moreover, higher germination rates were observed in the NnDREB2C overproducing plants when subjected into the stresses of NaCl and mannitol. Furthermore, we investigate the potential down-stream genes regulated by NnDREB2C and observed a significant increase in expressions of several genes belonging to PIPs family, including PIP1-1, PIP1-2, PIP1-3, PIP1-4 and PIP1-5. Consistently, overexpressed NnPIP1-2 and NnPIP2-1 conferred Arabidopsis the tolerance to stresses. Taken together, we concluded that overexpression of NnDREB2C enhanced the tolerance of salt and drought stresses in plants, which might probably be derived from the increased expression of the genes belonging to PIPs family.

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.

A comparative proteomic analysis for adventitious root formation in lotus root (Nelumbo nucifera Gaertn).

Adventitious roots (ARs) directly affect lotus seedling growth and product quality because principal root is not well developed. However, the details of AR formation at the molecular level have not been determined in lotus. Therefore, three stages were chosen to identify the change of proteins abundant during rhizome formation, using isobaric tags for relative and absolute quantization coupled with liquid chromatography-tandem mass spectrometry to gain insight into the molecular mechanisms involved in AR formation. We totally obtained 323,375 spectra during AR formation. After filtering to eliminate low-scoring spectra, 66,943 spectra, including 53,106 unique spectra, were identified. These unique spectra matched 28,905 peptides, including 24,992 unique peptides, which were assembled into 6686 proteins. In the C0/C1 and C1/C2 stages, 66 and 32 proteins showed enhanced abundance, and 173 and 73 proteins showed decreased abundance, respectively. Seventeen important AR formation-related proteins from the three stages were identified, and the expressions of nine genes from the above-identified proteins were assessed by qRT-PCR. This article provides a comprehensive understanding of the changes in metabolism during AR formation, and is helpful to accelerate the progress of breeding in fulture in lotus root.