The Molecular Regulation Mechanisms Of Ammonium Of Flowering Chinese Cabbage

Nitrate(NO3-) and ammonium(NH4+) are the two main forms of nitrogen taken up by plants, they differently influence some physiological and biochemical processes differently of plants, including cellular energetics and redox status, nitrogen metabolism, photosynthesis and water uptake. For many cultivated plant species,mixtures of NH4+ and NO3- with the appropriate proportions have shown to increase the growth and yield of crops over that of NO3- or NH4+ application alone,which had been also proved in studys of flowering Chinese cabbage and Chinese kale in our privious studys. In addition, we found that mixtures of NH4+ and NO3- application can promote nitrogen uptake by roots and improve nitrogen use efficiency, while also reduce the loss of nitrogen. However, molecular mechanism of the mixed nitrogen nutrition is unclear.Flowering Chinese cabbage(Brassica campestris L. ssp. chinensis var. Utilis Tsen et Lee), is a subspecies variant of pak choi(Brassica campestris L. ssp.chinensis Makino var. communis Tsen et Lee) and one of the most widely cultivated vegetables in China, especially in southern China.In this previous study, flowering Chinese cabbage was used as materials to study the molecular mechanism of the mixed nitrogen nutrition, and the main results are as follows:1. The regulation of NH4+ on flowering Chinese cabbage growth and nitrogen absorptionTwo experiments: Different ratios of NH4+:NO3-(0:100, 5:95, 25:75, and 50:50)under equal nitrogen level and adding differnet concentrations(0 m M, 0.5 m M, 1 m M,and 2 m M) into 2 m M NO3-, showed that treatments of 5:95, 25:75 of NH4+:NO3-, and0.5 m M, 1 m M NH4+ adding increased shoots biomass of flowering Chinese cabbage significantly, however, treatments of 50:50 of NH4+:NO3- and 2 m M NH4+ inhibitedflowering Chinese cabbage growth. Even 0.1 m M NH4+ application with 4 m M NO3-showed a higher shoots biomass and nitrogen uptake than the treatment of solo 4 m M NO3-.2. Cloning and characterization of 4 Bc AMT1 gene from flowering Chinese cabbageThe full-length open reading frames of Bc AMT1;1、Bc AMT1;3、Bc AMT1;4 and Bc AMT1;5 of flowering Chinese cabbage were cloned. Sequence and protein domain prediction analyzes speculated that these four Bc AMT1 s genes were all transmembrane proteins. The experiment of subcellular localization of Bc AMT1 s in the onion epidermal cells showed that 4 Bc AMT1 s all located in the cell membrane,and the yeast functional complementation experiment showed that 4 Bc AMT1 s proteins could complemented the growth of 31019 b yeast on NH4+ as the single nitrogen source, which suggested the function of NH4+ transporttion. Bc AMT1;1 、Bc AMT1;3 and Bc AMT1;5 all showed the highest expression level in root, and Bc AMT1;3 could not be detected in root. Bc AMT1;4 mainly expressed in flowers and leaves, and no expression was detected in roots.3. Effects of various Nitrogen sources on Bc AMT1 s expressionNitrogen deficiency upregulated the expression of Bc AMT1;1、Bc AMT1;3 and Bc AMT1;5 and promoted the nitrogen absorption of roots. Bc AMT1;1 and Bc AMT1;5were downregulated by NO3- and upregulated by NH4+, but high NH4+ concentration treatment for a long time(48 h) would inhibit the expression of Bc AMT1;1 and Bc AMT1;5. Bc AMT1;3 expression was not affected by NO3- application, but very sensitive to NH4+, and significantly downregulated by NH4+. Both application of NO3-and NH4+ inhibited the nitrogen absorption of roots.4. Agrobacterium-mediated transient expression via root absorption in flowering Chinese cabbageBoth semi-quantitative and fluorescent quantitative RT-PCR confirmed that the target gene Bc AMT1;3 was more highly expressed in plants that were infected with the transformed Agrobacterium strain EHA105-p35S-Bc AMT1;3 than in control plants that were infected with the control Agrobacterium strain EHA105-p35 S. In addition, we found that infection with an Agrobacterium cell density of OD600 = 0.3for 3-6 h, without hyperosmotic pretreatment, yielded the highest transformationefficiency, and under these conditions, the transformation efficiency reached a peak at2 and 4 d after infection.5. Effects of Overexpressing or inhibiting Bc AMT1;3 on Bc AMT and Bc NRT gene expressionTransient overexpression of Bc AMT1;3 upregulated Bc AMT1;1, Bc AMT1;5,Bc NRT1;1 and Bc NRT2;1. Inhibiting expression of Bc AMT1;3 resulting downregulation of Bc AMT1;1, Bc NRT1;1 and Bc NRT2;1, but no significant differences in the expression of Bc AMT1;5.6. De novo assembly and characterization of the flowering Chinese cabbage root transcriptome under various NH4+:NO3- ratiosA large-scale transcriptome sequencing of flowering Chinese cabbage was uptaken using Illumina sequencing technology and then analyzed gene expression in roots under four different ammonium:nitrate ratios(0:100, 5:95, 25:75, and 50:50). In total, 53746 unigenes, with an average length of 803.16 bp, were assembled, among which 78.61% of unigenes were matched to at least one public database.Among the four treatments, 2242 unigenes were differentially expressed, and KEGG pathway analysis showed that the differentially expressed genes covering a total of 97 pathways, of which 13 are significantly enriched pathways, including genes that encoded for components of plant hormones signaling, nitrogen metabolism,and photosynthesis pathways. Additionally, 2913 transcription factors were identified,among which 189 were expressed differentially, including the ERF, MYB, RAV, and LBD transcription factor families, and so on.In summary, NH4+ regulated the growth and nitrogen absorption of flowering Chinese cabbage likely as a signal molecule, and Bc AMT1;3 might participate the NH4+ regulation as a positive regulator of Bc AMT1;1, Bc AMT1;5, Bc NRT1;1, and Bc NRT2;1 expression in root. Hormone signaling pathway related genes and transcription factors are also extensively involved in the metabolic regulation of nitrogen uptake.