Nitrogen Demand Pattern For Tea Plant And CsATG2,CsATG18f-1,CsATG18f-2 Associated With High Efficiency Use Of Nitrogen

Tea plant is a perennial plant for leaf-use,repeatedly picking a year takes away a amount of nutrient.Nitrogen（N）is the highest content of mineral elements in tea plant,and it is also an element of important organic components in tea.Nitrogen fertilizer is highly required for tea plant plantation.However,the excessive application of N fertilizer causes low tea quality and soil deterioration.Therefore,improvement for nitrogen utilization efficiency（NUtE）and nitrogen remobilization efficiency（NRE）is the key to solve this problem.Autophagy includes degradation of macromolecular substances such as proteins and organelles,nutrient redistribution and then to improve nutrition remobilization efficiency under nutrition deficient or other abiotic stresses.Firstly,we determined the natural variation of N concentration of one bud with two leaves among 20 tea plant cultivars in the growing season,from April to October,and N utilization efficiency for one bud with two leaves and N remobilization efficiency for leaves attached to green-red stem and grey stem were investigated respectively.Three autophagy-related genes（ATG genes）,CsATG2,CsATG18f-1,CsATG18f-2,were identified based on the PacBio transcriptome of tea plant.Overexpressed CsATGx transgenic Arabidopsis thaliana plants were developed to dissect their NUtE and NRE in response to various N levels.The main results were as follows:（1）Natural Variation of N concentration in one bud with two leaves,leaves attached to green-red stem and grey stem in 20 tea plant cultivars in growing season（April-October）.The dynamic changes of different leaf positions in the growing season within a tea cultivar were compared.The results showed that the trend of N concentration,one bud with two leaves>leaves attached to green-red stem>leaves attached to grey stem.The sequence of N concentration in one bud with two leaves was as follows,in spring（April,May,April>May）>in summer（July,August,July<August）>in autumn（October）.The average value presented the highest in April（44.29 g/kg）and the lowest in October（31.06 g/kg）.The overall trend of N concentration in leaves attached to green-red stem was the highest in autumn（October）,followed by that in spring（April>May）,in summer（August>July）,the highest in October（29.42 g/kg）and the lowest in July（22.67 g/kg）.The sequence of N concentration in the leaves attached to grey stem was as follows,May（24.5 g/kg）>August（21.93 g/kg）>October（21.7 g/kg）>July.（21.1 g/kg）>April（20.79g/kg）,and no significant difference was observed between the months of April,July,August and October.（2）Natural variations of NUtE among the tea plant cultivars were investigated.The NUtE among the tea plant germplasm showed abundant genetic variation across the growing season.The NUtE was the highest in autumn（October）,followed by summer（July>August）and spring（May>April）.The tea plant cultivars of Jiaming 1,Echa 10 and Jinmudan in spring,Fuandabai,Mingxuan131 and Qianmei502 in summer and Meizhan,Yingshuang,Fuandabai,Jinmudan and Duokangxiang in autumn presented higher NUtE.The average NUtE were 32.99 g/g in October and 22.76 g/g in April.（3）Natural variations among the tea plant cultivars NRE were calculated..Seventeen,fifteen and five cultivars in the leaves attached to green-red stem and five,fourteen,eight and twelve cultivars in the leaves attached to grey stem showed NRE<0in April-May,May-July,July-August（no cultivar in August-October）,respectively.It was interesting that the NRE revealed a significant regularity of NRE>0 as‘sink’organ during April-May and July-August,NRE<0 during May-July and August-October as‘source’organ in the leaves attached to grey stem,indicating alternative during growing seasons.Additionally,the NREs were higher in April-May and May-July in the leaves attached to green-red stem,and higher in May-July and August-October in the leaves attached to grey stem respectively.（4）Autophagy-related genes,CsATG2,CsATG18f-1 and CsATG18f-2,were isolated and characterized in response to various N levels.The ORF of CsATG2,CsATG18f-1 and CsATG18f-2 are 1935 bp,1740 bp and 1572 bp,and encode 644,579 and 523 amino acids,respectively.Subcellular localization showed that CsATG2 was located in plasma membrane,and CsATG18f-1 and CsATG18f-2 were located in mitochondria.The NUEs were significantly improved in the overexpressed Arabidopsis thaliana lines（OX）in response to N deficiency（N₁）.The growth performance of OX lines was better than that of wildtype at seedling and silique stage,exhibiting larger rosette leaves and significant biomass compared with wild type（P<0.05）.OX3 and OX4 increased significantly at N₁and N₃ levels,and OX7 and OX10 increased significantly at N₁ and N₂ level,suggesting that OX3 and OX4 lines were characterized by low-N-tolerant and high-N-potential lines,and OX7 and OX10 lines were low N tolerant.It was found that the amino nitrogen concentrations in the young rosette leaves and old rosette leaves in the OX lines were significantly higher than that of wildtype under N₁ level（P<0.05）,but no significant difference was observed in roots.No significant difference was observed between OX lines and wildtype at potential N deficiency（N₂）and normal N（N₃）levels.The density autophagosomes in the overexpressed lines were obviously higher than those in the wildtype at the levels of N₁,N₂ and N₃（P<0.05）.The above results showed that under N₁ condition,OX lines improved the production of autophagic bodies and enhanced autophagic activity in Arabidopsis thaliana,therefore improved the NRE of transgenic Arabidopsis and increased plant biomass accumulation.This study indicated that CsATG2,CsATG18f-1 and CsATG18f-2 play important roles via autopgagy to improve N remobilization and to further improve NUE in response to low N conditions.