Abstract
Nitrogen metabolism in citrus has received increased attention due to its effects on plant growth and productivity. However, little is known about the effects of nitrogen fertilization on nitrogen metabolism in young trees of citrus cultivar ‘Huangguogan’ (Citrus reticulata × Citrus sinensis). Here, genes encoding nitrate reductase (NR), nitrite reductase (NiR), glutamine synthetase (GS), glutamate dehydrogenase (GDH), and asparagine synthetase (AS), represented as HgNR, HgNiR, HgGS, HgGDH, and HgAS, respectively, were cloned from Huangguogan. Deduced protein sequences were analyzed and proteins were confirmed to be localized in their respective cellular organelles. Moreover, pot-cultured ‘Huangguogan’ seedlings were fertilized with 0 (N1), 1.36 (N2), 1.81 (N3), 2.26 (N4), or 2.72 (N5) kg N/year, for 12 months. Enzyme activity and enzyme-gene expression were studied in roots, leaves, and fruits at different stages. Finally, the effects of N application rate on root activity, leaf N, soluble protein, yield, and fruit quality at the ripening stage were measured. The results showed that: 1) HgNR, HgNiR, HgGDH, and HgAS gene products were found mainly in the cytoplasm and plasma membrane, while HgGS gene product was found mainly in cytoplasm and mitochondria. 2) Gene expression and enzyme activity differed among plant organs. As the root is in permanent direct contact with the soil we suggest that root gene expression and enzyme activity can be used as reference to determine N application rate. 3) Yield, fruit quality, enzyme activity, and enzyme-related gene expression were considerably lower at low than at high-N supply. However, they were all inhibited by excess nitrogen (i.e., 2.72 kg/year). Therefore, we recommend 1.81 kg N/year as the optimal N application rate for young ‘Huangguogan’ trees.
Highlights
Our results showed that the expression of key genes and enzymes involved in N metabolism were significantly responsive to the amount of N applied, and we propose an optimal N application rate for best seedling growth of the citrus cultivar ‘Huangguogan’
Cloning and subcellular localization of gene products involved in N metabolism Full-length CDSs of various genes amplified using cDNA prepared from total RNA isolated from seedlings of the citrus cultivar ‘Huangguogan’ were cloned and their sequences confirmed through nucleotide sequencing
Phylogenetic tree analysis was applied to determine the phylogenetic positions of HgNR, HgNiR, HgGS, HgGDH, and HgAS from the cultivar ‘Huangguogan’ in relation to those of 10 different species
Summary
Effects of nitrogen supply on nitrogen metabolism in citrus trees metabolism, N is a major limiting factor for plant production [2] and an essential structural constituent of proteins, RubisCO, nucleic acids, and chlorophyll, in addition to some hormones [3]. N fertilizer plays a vital role in citrus tree growth, fruit yield and quality, as shown by several reports on the effects of N application rate on citrus growth [4,5]. N deficiency leads to poor plant growth [9], small fruit size [10], reduced photosynthetic capacity [11], and production [12]. A reduction in the use of nitrogen fertilizers is required that does not impact crop productivity [19]
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