The Effects Of Nitrogen Nutrition On Glucosinolate Concentration In Chinese Kale (Brassica Alboglabra L.)

Chinese kale(Brassica alboglabra L.) is a kind of Chinese native Brassica vegetables,which was consumed originally in southern China for its rich nutrition and delicious taste and now has been gaining popular in whole China and Japan as well as Europe and America due to its high content of glucosinolates(GSs) with anti-carcinogenic function.GSs are secondary metabolites derived from amino acids. The concentration and composition of GSs in plant tissue is not only determined by genetic,environmental and seasonal factors,but also by the amount and form of macro- and micro-nutrient elements supplied.The growth and high biomass accumulation of Chinese kale required a large amount of nitrogen nutrition. However it is still unclear whether the GS concentration could be increased by improving nitrogen fertilizing strategy.To reveal this issue,after modifying the methods for GS separation and extraction from Chinese kale bolting stems,the effects of nitrogen levels,nitrogen forms in culture solution,and top dressing of methionine and tryptophan with or without surfactant as well as the interactions between CO₂ concentration and N levels on the GS concentration based on hydroponic and artificial medium culture experiments were investigated.The main results were as follows:(1) To improve the procedure of GSs separation by HPLC,the different mobile phases and different lengths of C18 column were compared.The results showed that desulpho-GS compounds could be completely separated by HPLC with C18 column of 250 mm×4.6 mm and the mobile phase of the acetonitrile/water system.The concentration of GSs extracted from Chinese kale under different extract temperatures,methanol concentrations of extract solution,extract times,and ratios of sample to extract solution were compared.The results revealed that the concentration of the total GSs,the total aliphatic GSs as well as the total indolyl GSs extracted were significantly affected by ratio of sample to solution and extract temperature.The optimal extracting temperature and ratio of sample to extract solution were 75℃and 1:45(g:mL),respectively.And 11 GS compounds were detected in 10 genotypes of Chinese kale bolting stems,including 7 aliphatic and 4 indolyl GSs,while no aromatic GS was identified.(2) The results of the hydroponics experiment with five nitrogen(N) levels of 5 mmol L^-1 to 30 mmol L^-1 showed that the biomass of Chinese kale and the concentrations of total GSs,total aliphatic GSs and total indolyl GSs were significantly affected by N levels in culture solution.Although the highest concentration of total GSs was obtained at the lowest N level of 5 mmol L^-1,the growth of Chinese kale was badly suppressed because of nitrogen deficiency.The highest biomass and relative higher total GS concentration were gained at middle N level of 15 mmol L^-1.The biomass did not significantly increase while the total GS concentration was obviously decreased under the higher N levels of 20～30 mmol L^-1 compared to that at 15 mmol N L^-1 treatment.It suggested that the highest GS concentration at lowest N level of 5 mmol L^-1 maybe caused by the "concentration effect" of low biomass,while the lowest GS concentration at higher N levels of 20～30 mmol N L^-1 perhaps resulted from the imbalance of N/S ratio due to over uptake of nitrogen.(3) The hydroponics experiment with 5 ratios of ammonium(NH₄⁺-N) to nitrate (NO₃^--N) from 100/0 to 0/100 was conducted for Chinese kale.It is found that Chinese kale supplied with complete NH₄⁺-N grown very slowly and appeared obvious ammonium toxic symptom with partial plants died.Besides no bolting stem formed the lowest biomass was observed in this treatment.Except for the biomass of Chinese kale,the concentrations of total GSs,total aliphatic GSs and total indolyl GSs were all significantly different among the other four treatments supplied with partial or complete NO₃^--N.Nitrate content sharply decreased with increasing percentages of ammonium.The highest concentration of total GSs and total aliphatic GSs in bolting stems were detected at the treatment supplied with 50%NH₄⁺-N and the highest total indolyl GS concentration was achieved at the treatment with 75% NH₄⁺-N,which indicated that the appropriate ratio of ammonium to nitrate in nutrient solution may be helpful for the GS synthesis and accumulation in Chinese kale bolting stems.The sulfur content and N/S ratio but not nitrogen content in bolting stems were not significantly affected by the ratio of ammonium to nitrate, which indicated that the differences of GS concentrations among the treatments with different ratio of ammonium to nitrate was not caused by the change of N/S ratio. Detailed mechanism of ammonium to nitrate ratio on the GS concentration in Chinese kale needs to be further explored.(4) The artificial medium culture experiment was carried out to investigate the top dressing effects of methionine and tryptophan with or without surfactant on the GS concentrations in Chinese kale bolting stems.The results revealed that top dressing of methionine,tryptophan and tryptophan with surfactant did not affect the concentrations of total GSs,total aliphatic and total indolyl GSs as well as the biomass.Top dressing of tryptophan combined with surfactant significantly increased the total GSs and the total aliphatic GS concentrations,especially the concentration of gluconapin,but not for total indolyl concentration.It implied that methionine absorbed from leaves surface may promote the synthesis and accumulation of GSs and the surfactant could assistant the absorption of methionine through the Chinese kale leaves covered with a heavy layer of waxiness.(5) The results of the hydroponic experiment with two CO₂ concentrations of 350 and 800μL L^-1 and three N levels of 5,10 and 20 mmol L^-1 showed that at both normal and high CO₂ concentration,the growth of Chinese kale was suppressed at the lowest N level and the biomass was increased significantly as N level increased. Furthermore,enrichment of CO₂ concentration considerably enhanced the biomass under higher three nitrogen levels and fresh weight of bolting stems at N levels of 10 and 20 mmol L^-1.The concentrations of total GSs and total aliphatic GSs were decreased significantly as nitrogen level increased,while the total indolyl GS concentration increased.Enrichment of CO₂ concentration from 350 to 800μL L^-1 elevated concentrations of total GSs and total aliphatic GSs under low and middle N levels,but not at high N level.The C content and C/N ratios of Chinese kale bolting stems were increased as CO₂ enriched under all three nitrogen levels,while both the N content and the S contents decreased.The N/S ratios decreased at lower N levels of 5 and 10 mmol L^-1 because the degree of N content reduced was larger than that of S content,which may be one of the reasons why the total aliphatic GS concentrations under lower N levels with lower N/S ratio were higher than that at the higher nitrogen level when CO₂ elevated.