| The summer maize(Zea mays L.) hybrid Denghai 618 was grown from 2013 to 2014 on a farm and state key laboratory of crop biology at Shandong Agricultural University, Shandong Province in eastern China(N36°18′,E117°12′). The average content of the total nitrogen(N), rapidly available phosphorous(P), and rapidly available potassium(K) were 1.03 g kg-1, 43.05 mg kg-1 and 78.91 mg kg-1, respectively. Four treatments were designed, involving the application of nitrogen at rates of 0, 180, 360 and 540 kg hm-2(N0, N1, N2 and N3). Both treatments had three replicate plots, with each plot area measuring 3×15 m. The presence of disease, pests and weeds were well controlled by managers. We applied a comparative proteomics study with the aim of explaining the molecular mechanisms that affect senescence and nitrogen regulation. The main results are as followes: 1. The effect of nitrogen on yield and physiological characteristicsThe yield of maize was affected significantly by different nitrogen treatments. Plants had significantly greater yield with the increasing N-fertilizer in the range of 0 to 360 kg·hm-2. However, maize yield was decreased significantly when the nitrogen supply arrived 540 kg·hm-2. Maize had significantly greater kernel number and 1000-grain weight with the increasing N-fertilizer in the range of 0 to 360 kg·hm-2. In this study, the highest maize yield was 16369.9 kg·hm-2 in N2 treatment, which was 27.9%, 3.9% and 11.3% higher than N0, N1 and N3 treatment.The content of chlorophyll was affected significantly by different nitrogen treatments. Maize leaves had significantly greater chlorophyll content with the increasing N-fertilizer in the range of 0 to 360 kg·hm-2. However, leaf chlorophyll content was decreased significantly when the nitrogen supply arrived 540 kg·hm-2. In N2 treatment at flowering stage, leaf chlorophyll content was 2.4%, 3.3% and 3.5% higher than N1, N3 and N0 treatment; In N2 treatment at 20 days after flowering stage, leaf chlorophyll content was 0.8%, 2.0% and 7.5% higher than N1, N3 and N0 treatment. In N2 treatment at 40 days after flowering stage, leaf chlorophyll content was 3.9%, 9.1% and 15.2% higher than N1, N3 and N0 treatment.The change trend of photosynthetic ability of leaf was similar to the leaf chlorophyll content. After flowering stage, the Gs was decreased during senescence at all nitrogen treatments, and the Ci was increased during senescence at all nitrogen treatments. The Pn was decreased during senescence in N0, N1 and N2 treatment. However, the Pn was increased at 20 days after flowering srage and it was decreased at 40 days after flowering stage in N3 treatment. Compared to other nitrogen treatments, the Gs and Pn keep higher level and the Ci keep lower level in N2 treatment.The activity of protective enzyme in maize leaves had significantly greater chlorophyll content and lower membrane lipid peroxide level with the increasing N-fertilizer in the range of 0 to 360 kg·hm-2. The activity of protective enzyme was decreased and the membrane lipid peroxide level was increased when the nitrogen supply arrived 540 kg·hm-2. 2. Nitrogen effects on leaf proteome after flowering stageWe identified 71 ―nitrogen-regulated proteins‖ in this study, and most of them were up-regulation in N2 treatment compared to N0 treatment. We classified the 71 nitrogen-regulated proteins into 10 functional groups, including energy(56.3%), disease/defence(11.3%), protein synthesis(5.6%), metabolism(4.2%), transcription(4.2%), protein destination and storage(4.2%), secondary metabolism(2.8%), signal transduction(1.4%), cell growth/division(1.4%) and unclear functions(8.5%). These results showed that nitrogen chiefly affected proteins involved in energy, disease/defence and protein synthesis. 3. Sensecence effects on leaf proteome after flowering stage and nitrogen regulationIn this study, we identified 32 ―senescence-associated proteins‖. We classified this 32 senescence-associated proteins into 9 functional groups, including energy(50%), disease/defence(12.5%), metabolism(9.4%), cell growth/division(3.1%), protein synthesis(3.1%), transcription(3.1%), protein destination and storage(3.1%), secondary metabolism(3.1%), and unclear functions(12.5%). Based on our study of the maize leaf proteome, leaf senescence induces complex responses including the degradation of senescence-associated proteins that are involved in many biological processes, especially energy, metabolism and cell rescue, defense and virulence pathways. Although similar conclusions have been highlighted in other crops, this study filled the knowledge gap in maize leaf senescence. Moreover, we discovered, for the first time, 29 ―nitrogen-regulated senescence proteins‖ had significant(P ≤ 0.05) interaction term for nitrogen × stage. Although further study of nitrogen-related senescence proteins, such as 30 S ribosomal protein, will be required to fully elucidate their complex functions, the surprising results in our study provide a new vision to research the relationship between nitrogen and senescence. |
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