| Mini Chinese cabbage（Brassica pekinensis） is one of the main vegetable crops cultured in Northwestern plateau during the summer season. The growth and development of the crop is usually adversely affected by low light intensity in the process of production. An experiment was conducted to find the moderate NH4+:NO3- ratio for mini Chinese cabbage under different light intensities. The study also explored the physiological and molecular mechanisms of alleviation role of moderate NH4+:NO3- ratios in mini Chinese cabbage seedlings under low light intensity stress. The study provides theoretical bases and technical guidance for the response of mini Chinese cabbage to fertilizer under low light intensity. The hydroponic experiment involved NH4+:NO3- ratios（0:10, 10:90, 15:85 and 25:75;total nitrogen concentration 5 m M） on growth and physio-biochemical characteristics of mini Chinese cabbage seedling under normal(200 μmol·m-2 s-1) and low(100 μmol·m-2 s-1) light intensity. Moreover, the m RNA relative expression levels of related genes and differentially proteomic analyses were conducted to deeply explore the regulation mechanism of NH4+:NO3- ratio. The main results were as following: 1) After 14 days treatment,plants fertilized with NH4+:NO3-（15:85） under normal light intensity were the best in growth potential. The leaf area, growth range,fresh and dry weight of plants improved 33.8%, 30.5%, 77.9% and 72.9% compared with NH4+:NO3-（0:100）, respectively. The root of plants fertilized with NH4+:NO3-（15:85） was well-developed. The leaf area, growth range, fresh and dry weight and root of the plants in low light intensity were lower than those plants under normal light intensity, indicating that low light intensity suppressed the growth of mini Chinese cabbage seedlings. However,the leaf area,growth range,fresh and dry weight of plants fertilized with NH4+:NO3-（10:90） improved by 20.4%,14.9%,55.9% and 36.5%compared with NH4+:NO3-（0:100）,respectively. The root parameters of plants supplied with NH4+:NO3-（10:90） were significantly higher than other treatments. Evaluating the relationship between biomass production of mini Chinese cabbage and the ratios of NH4+:NO3-, we found that the ratio of 0.206–0.226 fertilizer improved biomass production under normal light intensity, while the NH4+:NO3- at 0.14–0.181 improved biomass under low light intensity. 2) Under normal light intensity, plants fertilized with NH4+:NO3-（15:85） had intact chloroplast ultrastructure and a higher degree of granal stacking, larger ratio of aperture stomataand stomatal aperture, higher chlorophyll contents and chlorophyll fluorescence parameters（Fv/Fm, ΦPSII, q P, Pm, α and Ik）, higher activities of Calvin cycle enzymes（Rubisco, GAPDH, FBPase, FBA and TK） and expression levels of genes coding for Calvin cycle enzymes（rbc L, rbc S, FBPase, FBA and TK）, the highest net photosynthetic rate（Pn） and photosynthates（soluble sugar and starch）. Under low light intensity, the cells of plants fertilized with sole NO3-or higher NH4+ concentration appeared out of shape and had different degrees of plasmolysis. The number of osmiophilic globules also appeared to increase in chloroplasts while the degree of granal stacking decreased. However, NH4+:NO3-（10:90） maintained the normal structure of cell and led to higher degree of granal stacking, and effectively reduced the damage of chloroplast under low light intensity. The stomatal density, ratio of aperture stomata and stomatal aperture decreased under low light intensity. However, the addition of NH4+:NO3-（10:90） in the nutrient significantly increased the stomatal density, ratio of aperture stomata and stomatal aperture. The chlorophyll contents of plants fertilized with NH4+:NO3-（10:90 and 15:85） under low light intensity increased, but chlorophyll a/b decreased, making it more beneficial for plants to absorb low light, compared with those under normal light. Moreover, NH4+:NO3-（10:90 and 15:85） suppressed the negative effect of low light on Fv/Fm, ΦPSII, q P, Pm, α and Ik. Pn of plant under low light intensity decreased, but the application of NH4+:NO3-（10:90） suppressed the negative effect of low light on Pn by improving the activities of Calvin cycle enzymes and expression levels of genes coding for Calvin cycle enzymes, ultimately increased the accumulation of photosynthates（soluble sugar and starch） in the leaves. 3) The MDA,O2- and H2O2 contents of mini Chinese cabbage under low light intensity increased,resulting in higher membrane lipid peroxidation in plants. However, compared with sole NO3- or higher NH4+ concentration, NH4+:NO3-（10:90） application significantly increased the level of soluble sugar, soluble protein and SOD,POD,CAT and APX activities in mini Chinese cabbage. It decreased the reactive oxide species contents and effectively suppressed membrane lipid peroxidation, which is more beneficial for mini Chinese cabbage growth. 4) Under normal light intensity, the total nitrogen, phosphorus and potassium contents of plants fertilized with NH4+:NO3-（15:85） were significantly greater than those treated with other NH4+:NO3- ratios. The activities of NR and GS were also improved. The relative expression levels of NR reached maximum in plants treated with NH4+:NO3-（25:75） while the relativeexpression levels of GS reached maximum in plants treated with NH4+:NO3-（15:85）. Carbon/ nitrogen ratio was not affected by different NH4+:NO3- ratios. Low light intensity decreased total carbon, carbon/nitrogen, activities of NR and GS, expression levels of NR and GS to different extents. However, the total nitrogen, phosphorus, potassium and carbon contents of plants supplied with NH4+:NO3-（10:90） were the greatest. Furthermore, NH4+:NO3-（10:90） application suppressed the negative effect of low light intensity on carbon/ nitrogen, improved activities of NR and GS. The expression levels of NR was up regulated in plants treated with NH4+:NO3-（10:90）, while the expression levels of GS was up regulated in plants treated with NH4+:NO3-（15:85 and 25:75）. In addition, the nitric oxide（NO） content in leaves and the NO position of root were analyzed. We observed that low light intensity decreased NO content in leaves and NO fluorescence intensity of root tips, while appropriate NH4+:NO3- increased the NO content in leaves and NO fluorescence intensity of root tips. 5) The proteomic analyses of light intensity and NH4+:NO3- ratios responses were conducted in the leaves of mini Chinese cabbage seedling. 36 proteins were identified and they were grouped into ten categories according to their biochemical functions, including photosynthesis（19.44%）, carbon metabolism and energy metabolism（13.89%）, translation and transcription（19.44%）, amino acid metabolism（2.78%）, protein folding, modification and degradation（16.67%）, stress and defense response（11.11%）, signal transduction（5.56%）, cell growth/division（2.78%）, metabolism（2.78%） and unknown（5.56%）. The identified proteins were mainly located in chloroplast（63.9%）, cytoplasm（13.9%）, vacuole（8.3%） and nucleus（8.3%）. Among the proteins related to photosynthesis, only one protein exhibited decreased relative abundance in sole NO3--treated plants under low light intensity. All the other six proteins exhibited increased relative abundance. The up-regulation range in plants fertilized with ammonium was larger than those fed with sole NO3-, suggesting that moderate NH4+:NO3- under low light condition increased plant photosynthesis by enhancing the abundance of proteins related to photosynthesis. The 5-identified proteins related to carbon metabolism and energy metabolism all exhibited increased relative abundance. These proteins were mainly involved in starch synthesis, ATP hydrolysis and glycolysis processes, showing that moderate NH4+:NO3-under low light condition increased carbon metabolism and energy metabolism, thus alleviating the stress-induced energy shortage by ATP hydrolysis and glycolysis processes. Among the 4proteins related to stress and defense response, 3 proteins exhibited increased relative abundance, 1 protein exhibited decreased relative abundance, indicating that moderate NH4+:NO3- under low light condition increased plant oxidation resistance. 1 protein related to amino acid metabolism exhibited increased relative abundance, showing that moderate NH4+:NO3- under low light condition increased plant amino acid metabolism. Among the 13 proteins related to translation and transcription and protein folding, modification and degradation, 6 proteins exhibited decreased relative abundance in plants treated with sole NO3- under low light intensity, 7 proteins exhibited increased relative abundance and the up-regulation range in plants fertilized with ammonium was greater than those fed with sole NO3-. This suggests that the process of protein folding, modification and degradation was retarded, but moderate NH4+:NO3- alleviated the negative effect by enhancing the abundance of related proteins. |
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