Nitrate reductase (NR) is a key enzyme for nitrogen assimilation in plants, and its activity is regulated by posttranslational phosphorylation. To investigate the effects of dephosphorylation of the NIA1 protein on the growth and the physiological and biochemical characteristics of rice under different forms of nitrogen supplies, the phenotypes, nitrogen metabolism and reactive oxygen metabolism were measured in NIA1 phosphorylation site-directed mutant lines (S532D and S532A), an OsNia1 over-expression line (OE) and Kitaake (wild type, WT). Compared with WT and OE, S532D and S532A have stronger nitrogen assimilation capacities. When ammonium nitrate served as the nitrogen source, the plant heights, dry weights of shoots and chlorophyll (Chl) contents of S532D and S532A were lower than those of the WT and OE, whereas hydrogen peroxide (H₂O₂), malondialdehyde (MDA) and nitrite contents were higher. When potassium nitrate served as the nitrogen source, the plant heights, dry weights of shoots and Chl contents of S532D and S532A were higher than those of the WT and OE, there were no significant differences in the contents of H₂O₂ and MDA in the leaves of the test materials, and the difference in nitrite contents among different lines decreased. When ammonium sulfate served as the nitrogen source, there were no significant differences in the physiological indexes of the test materials, except NR activity. Compared with ammonium nitrate and ammonium sulfate, the content of NH₄⁺-N in the leaves of each plant was lower when potassium nitrate was used as the nitrogen source. The qPCR results showed that OsGS and OsNGS1 were negatively regulated by downstream metabolites, and OsNrt2.2 was induced by nitrate. In summary, when ammonium nitrate served as the nitrogen source, the weak growth of NIA1 phosphorylation site-directed mutant lines was due to the toxicity caused by the excessive accumulation of nitrite. When potassium nitrate served as the nitrogen source, the assimilation rates of nitrate, nitrite and ammonium salt were accelerated in NIA1 phosphorylation site-directed mutant lines, which could provide more nitrogen nutrition and improve the tolerance of rice to ammonium nitrogen deficiency. These results could provide a possible method to improve the efficiency of nitrogen utilization in rice under low-nitrogen conditions.

Fe is a trace element considered to be essential for rice, and it drives several metabolic processes. Fe toxicity occurs due to excessive Fe ions (Fe2+) and which, disturb cellular homeostasis and dramatically reduces the rice yield. A set of 118 BRILs made from a cross of japonica cv.’02428’ and indica ‘Changhui 891’ was used with high density bin map constructed by using high quality SNP to identify the QTL for Fe toxicity tolerance. As a whole total of 23 QTL were identified for various seedling traits, 3 under control with phenotypic difference ranging from 14.21% to 62.46%, 11 QTL under stress with phenotypic difference ranging from 7.89% to 47.39% and 9 under stressed/control ratio with phenotypic variance ranging from 9.17% to 183.50%. LOD values of QTL ranging from 4.05 to 17.04 in control, 3.41 to 8.09 in stress and 2.84 to131.63 in stress/control ratio. Shoot length (SL), root length (RL), shoot fresh weight (SFW), root fresh weight (RFW), shoot dry weight (SDW), and root dry weight (RDW), were used to estimate the degree of Fe tolerance. Many stable QTL, qSSDW-4, qSSDW-6, qRSDW-4 and qRSDW-6 affecting SDW were detected and beside this some new QTL, qRSFW-1, qRRFW-10 and qRRDW-1 were successfully identified significantly contributing to Fe toxicity tolerance in rice. The results of current study indicated that these novel regions could be transferred via markers assisted section and QTL pyramiding to develop Fe resistant lines in rice.

Phosphorus (P) is one of the essential macronutrients for rice. In this study, we used 120 rice backcross recombinant inbred lines (BRILs) derived from a cross indica cv. Changhui 891 and japonica cv. 02428. To elucidate the genetic control of P deficiency tolerance in rice, we have used high quality SNPs bin markers to identify some important loci underlying phosphorus deficiency. The bin map was generated which includes 3057 bins covering distance of 1266.5 cM with an average of 0.41 cM between markers. Based on this map, 50 loci, including four novel loci, qSL-3, qRL-11, qSDW-1, qRDW-1 with phenotypic variance 23.26%, 12.06%, 9.89% associated with P deficiency-related seedling traits were identified. No significant QTLs was found for root length under P+, shoot fresh weight P− and root length, shoot fresh weight for P+, P− and their ratio respectively. Root fresh weight, and root dry weight were strongly correlated to each other, and QTLs for these variables were located on the same chromosome 1 at the same region. Notably, 3 pleiotropic regions is the pioneer of our study, and these regions would facilitate map-based cloning to expedite the MAS selection for developing low phosphorous tolerant varieties. This study not only improves our knowledge about molecular processes associated with P deficiency, but also provides useful information to understand the genetic architecture of low phosphorous tolerance.

The function of NR in modulating NO production and cold tolerance was elucidated through the analysis of physiological changes in NR mutants. The results indicated that the cold tolerance of osnia1 and osnia1nia2 was diminished, compared with the wild type (WT) and osnia2, and these mutants also showed lower NR activity. The hydrogen peroxide (H₂O₂) and malondialdehyde (MDA) levels were higher in the osnia1 and osnia1nia2 rice seedlings compared with WT and osnia2. The proline, ascorbic acid (AsA), and glutathione (GSH) contents, as well as antioxidant and AsA-GSH cycle-related enzyme activities, were reduced. The application of nitric oxide synthase (NOS) inhibitor (L-NAME) and NO scavenger (cPTIO) promoted the accumulation of H₂O₂ and MDA while decreasing the antioxidant content and enzyme activities. Conversely, sodium nitroprusside (SNP) administration induced opposite effects. The NO content exhibited significant or highly significant positive correlations with the proline, AsA, GSH contents, and the activities of ascorbate peroxidase (APX) and glutathione reductase (GR). In contrast, it exhibited negative correlations with the levels of hydrogen peroxide and MDA, and the activities of related reactive oxygen species enzymes. In summary, the NR gene OsNia1 is a crucial regulator of NR activity and NO production. Low temperature stress can directly or indirectly induce NR activity through reactive oxygen species (ROS), subsequently mediating NO production. The production of NO alleviates oxidative stress under low temperature conditions and enhances the antioxidant defense system, thereby improving the tolerance of rice seedlings to low temperature stress.