Effect of different forms of nitrogen on the activity of nitrate reductase and expression of the relative genes in Citrus sinensis ×Poncirus trifoliate

Abstract: 【Objective】Citrus is one of the main fruit trees in the world, and China is the biggest country inits production. There are a number of factors affectting growth and fruitting of Citrus. The nutrition deficiency can cause physiological disorder. Nitrogen is the most important element for Citrus. At present,seedling rootstocks are used for citrus production in China. The ability of absorption and transportation ofinorganic nutrients varies with the species of rootstocks. Thus, it is necessary to investigate the ability ofnitrogen absorption of the main rootstock. Citrus sinensis (L.) Osbeck×Poncirus trifoliate (L.) Raf. is a hybrid of trifoliate and sweet orange being widely used due to its anti-aging disease characteristic. NO3- andNH4+ are the two main forms of inorganic nitrogen used. The influence of the forms of nitrogen and the ratio of NO3- and NH4+ on C. sinensis (L.) Osbeck×P. trifoliate (L.) Raf. and the nitrogen metabolism mecha⁃nism remains unknown at present. Nitrate reductase (NR) plays an important role in the metabolism process of nitrogen. Therefore, this research aims to study the nitrate absorption ability and mechanism in C. sinensis (L.) Osbeck × P. trifoliate (L.) Raf. to provide a scientific basis for improving efficiency of nitrogen usage in citrus production.【Methods】C. sinensis (L.) Osbeck × P. trifoliate (L.) Raf., were planted by hy⁃droponic method with Hoagland nutrient solution. Five treatments were made as follows: No.1（NO3-∶NH4+=10∶0）, No.2（NO3-∶NH4+=7∶3）, No.3（NO3-∶NH4+=5∶5）, No.4（NO3-∶NH4+=3∶7）and No.5（NO3-∶NH4+=0∶10）. The seedlings were planted in environmental chamber, and the culture medium for each treatmentwas added 7 μmol·L^-1 C2H4N4 in the nutrient solution to inhibit nitrate. The pH value of media was 6 or so,ventilation time was 15 min every 3 h. The seedlings were cultured for different times (0, 20, 40, 60, 80 d)with the solution adjustments, the effect of different ratio of nitrogen forms on the NR activity of the seedlings and the expression of the relative genes were studied.【Results】The NR activity of the leaves and theroots of the seedlings increased with the increase of cultivation time. The NR activity of the leaves washigher than that of the roots. The NR activity of the leaves under the treatments of No.2 and No.3 werehigher than that under other treatments. While the NR activity of the roots under the treatments No.1 and No.2 were significantly higher than that under the other treatments; The NR activity of the leaves and theroots under the treatments of No.4 and No.5 increased firstly and then decreased. The highest value of theNR activities was found in the seedlings cultured for 60 d. The lowest value was found in the seedlings cul⁃tured for 80 d under No.5 treatment. The related NR genes were cloned in many plants besides orange.The Nia gens analysis showed that CitNia1, CitNia2, CitNia OA were all found in the seedlings under alltreatments. The expression of CitNia1 of the roots in the seedlings under all treatments were higher thanthat of the other two genes, inferring that it was might be the main NR gene in the roots. The expression of CitNia2 of the leaves in the seedlings was higher than the other genes, and might be the major NR genesin the leaves. CitNia1 and CitNia2 were all induced by the different ratios of NO3- and NH4+, and the highest expression of CitNia1 and CitNia2 was found when the ratio was 5∶5 (treatment No.3). It indicated thatthe treatment 3 was suitable for nitrate absorption and metabolism in Citrus.【Conclusion】The main organof nitrate metabolism in the seedlings of C. sinensis (L.) Osbeck× P. trifoliate (L.) Raf. was the leaf. CitNia2 was the major gene in the leaves, its expression was affected by the regulation of the ratio of NO3-and NH4+.