RESUMEN
The OsNRT2.3a and OsNRT2.3b isoforms play important roles in the uptake and transport of nitrate during rice growth. However, it is unclear which cis-acting element controls the transcription of OsNRT2.3 into these specific isoforms. In this study, we used a yeast one-hybrid assay to obtain the TATA-box binding protein OsTBP2.1, which binds to the TATA-box of OsNRT2.3, and verified its important role through transient expression and RNA-seq. We found that the TATA-box of OsNRT2.3 mutants and binding protein OsTBP2.1 together increased the transcription ratio of OsNRT2.3b to OsNRT2.3a. The overexpression of OsTBP2.1 promoted nitrogen uptake and increased rice yield compared with the wild-type; however, the OsTBP2.1 T-DNA mutant lines exhibited the opposite trend. Detailed analyses demonstrated that the TATA-box was the key cis-regulatory element for OsNRT2.3 to be transcribed into OsNRT2.3a and OsNRT2.3b. Additionally, this key cis-regulatory element, together with the binding protein OsTBP2.1, promoted the development of rice and increased grain yield.
Asunto(s)
Oryza , Proteínas de Transporte de Anión/metabolismo , Grano Comestible/genética , Regulación de la Expresión Génica de las Plantas , Transportadores de Nitrato , Nitratos/metabolismo , Nitrógeno/metabolismo , Oryza/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Raíces de Plantas/metabolismo , TATA Box , Proteína de Unión a TATA-Box/genética , Proteína de Unión a TATA-Box/metabolismoRESUMEN
The uptake and accumulation of polycyclic aromatic hydrocarbons (PAHs) in crops have gained much attention due to their toxicity to humans. Nitrogen (N) is an essential element for plant growth and has also been implicated in the acquisition and acropetal translocation of PAHs. OsNRT2.3b encodes a nitrate (NO3-) transporter that is involved in the acquisition and mobilization of N in rice. Here, we investigated whether overexpression of OsNRT2.3b would exert any mitigating influence on the uptake and translocation of phenanthrene (Phe, a model PAH) in transgenic rice (Oryza sativa). The wild-type seedlings exhibited a reduction in plant height, primary root length, and shoot biomass when grown hydroponically in a medium supplemented with Phe. Acquisition of Phe by the roots and its subsequent translocation to shoots increased concomitantly with an increase in Phe concentration in the medium and duration of the treatment. OsNRT2.3b-overexpressing lines (Ox-6 and Ox-8) were generated independently. Compared with the wild-type, the concentration of Phe in Ox-6 and Ox-8 were significantly lower in the roots (47%-54%) and shoots (22%-31%) grown hydroponically with Phe (1 mg/L). Further, the wild-type and Ox lines were grown to maturity in a pot soil under Phe conditions and the concentrations of Phe and total N were assayed in the culms and flag leaves. Compared with the wild-type, in Ox lines the concentration of total N significantly increased in the culms (288%-366%) and flag leaves (12%-25%), while that of Phe significantly reduced in the culms (25%-28%) and flag leaves (18%-21%). The results revealed an antagonistic correlation between the concentration of total N and Phe. The concentration of Phe was also significantly lower (29%-38%) in the seeds of Ox lines than the wild-type. The study highlighted the efficacy of overexpressing OsNRT2.3b in mitigating the Phe toxicity by attenuating its acquisition, mobilization, and allocation to the seeds.
Asunto(s)
Oryza , Fenantrenos , Proteínas de Transporte de Anión/genética , Proteínas de Transporte de Anión/metabolismo , Regulación de la Expresión Génica de las Plantas , Humanos , Oryza/genética , Oryza/metabolismo , Proteínas de Plantas/metabolismo , Raíces de Plantas/metabolismo , Semillas/metabolismoRESUMEN
Improving nitrogen use efficiency (NUE) is very important for crops throughout the world. Rice mainly utilizes ammonium as an N source, but it also has four NRT2 genes involved in nitrate transport. The OsNRT2.3b transporter is important for maintaining cellular pH under mixed N supplies. Overexpression of this transporter driven by a ubiquitin promoter in rice greatly improved yield and NUE. This strategy for improving the NUE of crops may also be important for other cereals such as wheat and barley, which also face the challenges of nutrient uptake balance. To test this idea, we constructed transgenic barley lines overexpressing OsNRT2.3b. These transgenic barley lines overexpressing the rice transporter exhibited improved growth, yield, and NUE. We demonstrated that NRT2 family members and the partner protein HvNAR2.3 were also up-regulated by nitrate treatment (0.2 mM) in the transgenic lines. This suggests that the expression of OsNRT2.3b and other HvNRT2 family members were all up-regulated in the transgenic barley to increase the efficiency of N uptake and usage. We also compared the ubiquitin (Ubi) and a phloem-specific (RSs1) promoter-driven expression of OsNRT2.3b. The Ubi promoter failed to improve nutrient uptake balance, whereas the RSs1 promoter succeed in increasing the N, P, and Fe uptake balance. The nutrient uptake enhancement did not include Mn and Mg. Surprisingly, we found that the choice of promoter influenced the barley phenotype, not only increasing NUE and grain yield, but also improving nutrient uptake balance.
Asunto(s)
Proteínas de Transporte de Anión/genética , Transporte Biológico/genética , Hordeum/genética , Oryza/genética , Regulación de la Expresión Génica de las Plantas , Hordeum/crecimiento & desarrollo , Hordeum/metabolismo , Transportadores de Nitrato , Óxidos de Nitrógeno/metabolismo , Nutrientes/genética , Nutrientes/metabolismo , Raíces de Plantas/genética , Raíces de Plantas/crecimiento & desarrollo , Plantas Modificadas Genéticamente/genética , Plantas Modificadas Genéticamente/crecimiento & desarrollo , Regiones Promotoras Genéticas/genéticaRESUMEN
KEY MESSAGE: Overexpression of OsNRT2.3b in rice can increase Pi uptake and accumulation through advanced root system, enhanced OsPT and OsPHR genes expression, and the phloem pH homeostasis. Nitrogen (N) and phosphorus (P) are two essential macronutrients for plants. Overexpression of the rice nitrate transporter, OsNRT2.3b, can improve rice grain yield and nitrogen use efficiency (NUE). Here, OsNRT2.3b overexpression resulted in increased grain yield, straw yield, and grain:straw ratio, accompanied by increased P concentrations in the leaf blade, leaf sheath, culm, and unfilled rice hulls. Overexpression of OsNRT2.3b significantly increased 33Pi uptake compared with WT under 300-µM Pi but not 10-µM Pi condition in 24 h. Moreover, the OsNRT2.3b-overexpressing rice lines showed increased root and shoot biomass, root:shoot ratio, total root length root surface area and N, P accumulation under 300- and 10-µM Pi supply in hydroponic solution. The levels of OsPT2, OsPT8, and OsPHR2 expression in roots and of OsPT1 and OsPHR2 in shoots were upregulated in OsNRT2.3b-overexpressing rice. These results indicated that OsNRT2.3b overexpression can improve rice P uptake and accumulation, partially through the advanced root system, enhanced gene expression, and the phloem pH regulation function.