Rearrangement of nitrogen metabolism in rice (Oryza sativa L.) under salt stress

ABSTRACT

Salt stress is an important environmental condition limiting the agricultural production. The reprogram of protein expression is one of the strategies of plants to cope with salt stress. We have previously analyzed the photosynthesis, antioxidant and oxidative phosphorylation involved in the carbon metabolism and the redox metabolism in rice seedlings under salt stress. Here, we focused on the proteins involved in nitrogen metabolic response. As it was reported that the nitrite uptake was enhanced in Arabidopsis PII knock-out mutants, the down-regulation of P-II nitrogen sensing protein in rice probably contributes to the accumulation of amino acids under stress. In addition, the protein synthesis is limited by the decrease of related proteins, and more amino acids could be used as the compatible solute. Hence, our study indicates that the rearrangement of nitrogen metabolism under salt stress could accumulate more amino acids as the compatible solute rather than the components of proteins. This study provides information for an improved understanding of nitrogen metabolic response to salt stress in rice.

Abiotic stress, such as drought, salinity and low temperatures can adversely affect the growth and development of plant. In agriculture, those stresses reduce crop yield, and, in extreme cases, cause total crops failure. Metabolic rearrangement in plant is a strategy to adjust the adverse growth conditions.¹ For example, plants alter amino acid metabolism (e.g. proline) to stabilize the structure of proteins,² and regulate redox metabolism to remove excess ROS and balance the cellular redox level.³ But the metabolic response to abiotic stress is complex and diverse among plant species.¹ To reveal the proteomic response of rice to salt stress, we treated salt-sensitive rice cultivar Zhonghua11 (ZH11) plants at 4-leaf stage with 150 mM NaCl for 2 four hours and applied the isobaric Tags for Relative and Absolute Quantitation (iTRAQ) approach to reveal the proteomic response of rice to salt stress. We previously focused on the response of photosynthesis, antioxidant and oxidative phosphorylation pathways, which were involved in the carbon metabolism and the redox metabolism.⁴ But salt stress response of rice was not fully revealed for the complexity of it. Hence, other information was further dug, and the nitrogen metabolic response was showed in the present study.

A total of 56 significantly differentially expressed proteins were detected in the quantitative proteomic analysis.⁴ It was found in Gene Ontology (GO) biological process analysis, 27 of those proteins were enriched in metabolic process of Gene Ontology (GO) biological process, and the proportion was 48.21% (Fig. 1). Among them, P-II nitrogen sensing protein (gi|50878396) is involved in nitrogen metabolism, while acidic ribosomal protein P1a (gi|50725625), putative ribosomal protein S18 (gi|25553579), ribosomal protein L10a (gi|50252685), ribosomal protein S2 (gi|11974), ribosomal protein L34 (gi|37805854), 26S proteasome regulatory particle non-ATPase subunit 9 (Rpn9, gi|14495192), and 26S proteasome regulatory particle triple-A ATPase subunit4 (Rpt4, gi|11094192) participate in protein synthesis and degradation respectively (Table 1).

Figure 1.

Distribution of protein's biological process function. The proportion of the proteins in each GO biological process to the 56 significantly differentially expressed proteins was shown in the diagram.

Table 1.

The differentially expressed proteins involved in nitrogen metabolism, protein synthesis and protein degradation.

Accession	Os ID	Description	Fold ratio (NaCl/CK)
gi|50878396	Os05g0133100	putative P-II nitrogen sensing protein	0.632
gi|50725625	Os08g0116500	putative acidic ribosomal protein P1a	0.372
gi|25553579	Os03g0794700	putative ribosomal protein S18	0.57
gi|50252685	Os02g0321900	putative ribosomal protein L10a	0.604
gi|11974	None	ribosomal protein S2	0.614
gi|37805854	Os08g0156800	putative ribosomal protein L34	1.563
gi|14495192	Os01g0511300	putative 26S proteasome subunit RPN9b	0.519
gi|11094192	Os02g0199900	26S proteasome regulatory particle triple-A ATPase subunit4	0.622

In higher plants, the PII protein regulates arginine biosynthesis⁵ and plays a role in nitrogen regulation.⁶ We analyzed the proteins enriched in GO biological process; it was found that the P-II nitrogen sensing protein possessed multifarious functions, such as biological regulation, regulation of biological quality, response to disaccharide stimulus, cellular homeostasis, aromatic compound biosynthetic process, and phenylpropanoid metabolic process (Fig. 2). The subcellular localization analysis showed that the P-II nitrogen sensing protein was localized in the cytoplasm and nucleus of the rice protoplast (Fig. 3). Under salt stress, the P-II nitrogen sensing protein was down-regulated. It has been reported that chloroplast nitrite uptake was enhanced in Arabidopsis PII knockout mutants.⁵ The downregulation of P-II nitrogen sensing protein under salt stress probably increase nitrite uptake and nitrogen metabolism. Considering the accumulation of amino acids under stress,¹ the increased P-II nitrogen sensing protein might contribute to synthesis of amino acids, such as proline and non-protein amino acid γ-aminobutyric acid (GABA) which play important roles in scavenging ROS and stabilizing the structure of proteins.^1,7

Figure 2.

The proteins in enriched GO Biology processes. GO Biology processes were colored. A: cellular macromolecular complex subunit organization, B: nucleosome organization, C: macromolecular complex subunit organization, D: cellular homeostasis, E: regulation of biological quality, F: homeostatic process, G: positive regulation of molecular function, H: biological regulation, I: chromatin organization, J: chromosome organization, K: phenylpropanoid biosynthetic process, L: response to disaccharide stimulus, M: response to abiotic stimulus, N: multicellular organismal development, O: aromatic compound biosynthetic process, P: phenylpropanoid metabolic process, Q: growth, I: post-embryonic development.

Figure 3.

The subcellular localization of P-II nitrogen sensing protein. The subcellular location of SRP1 was analyzed in rice cells. The green fluorescent protein P-II nitrogen sensing protein-GFP fusion product was transiently expressed in rice protoplast. Scale = 50μm.

As the accumulation of amino acids may be increased upon salt stress, the decrease of protein synthesis may be observed. Interestingly, the decreases of some ribosome subunits, such as L10a, P1a and S2, were observed in rice under salt stress. The limited protein synthesis indicates that more amino acids could be released for the compatible solutes. Therefore, the accumulation of amino acids mediated by nitrogen metabolism as well as the limitation of protein synthesis mediated by the down-regulation of ribosome proteins may coordinately contribute to salt tolerance in rice. On the other hand, the salt stress would limit the degradation of protein by decreasing 19S regulatory particle subunits Rpt4 and Rpn9. It may be a mechanism to maintain the necessary protein levels during most of the amino acids are used as the compatible solute. Hence, our results indicate that nitrogen metabolism under salt stress was enhanced and rearranged to synthetize more amino acids as the compatible solute to cope with adverse conditions (Fig. 4).

Figure 4.

The proposed model of nitrogen metabolic rearrangement under salt stress. The downregulation of P-II nitrogen sensing protein under salt stress would increase nitrite uptake and nitrogen metabolism. The decreasing of ribosome subunits L10a, P1a, S2 and S18 under salt stress would limit the protein synthesis, and most of the accumulated amino acids would be used as compatible solute rather than component of protein. On the other hand, the salt stress would limit the degradation of protein by decreasing 19S regulatory particle subunits Rpt4 and Rpn9, and necessary protein level could be maintained during most of the amino acids were used as the compatible solute. In the diagram, green indicates the proteins that were downregulated, real line indicates the enhanced pathway, and broken lines indicate the weaken pathway.

Under the salt stress, the metabolisms in rice are rearranged. It has been reported that the carbon metabolism and the redox metabolism including photosynthesis, antioxidant and oxidative phosphorylation were cooperated and inter-connected to respond to the adverse conditions.⁴ However, the carbon and redox metabolic responses are just parts of the rearrangement while nitrogen metabolic response is also important. In present study, our data indicate that the nitrogen metabolism was rearranged to synthetize more amino acids as the compatible solute. It would be interesting to further elucidate the roles of those proteins involved in nitrogen metabolism under salt stress.

Disclosure of potential conflicts of interest

No potential conflicts of interest were disclosed.

Funding

This research was funded by the National Science Foundation of China (31571627), the National Key Technology R&D Program (2015BAD01B01-2) and Jiangsu Collaborative Innovation Center for Modern Crop Production.