Description

Nitrogen is the most essential nutrient to plant growth and grain yield. There are two forms of inorganic nitrogen available to plants: one is NO₃^--form and the other is NH₄⁺-form. NH₄⁺-form is the major form in paddy fields, thus rice plants grown in the fields uptake mainly NH₄⁺-form nitrogen as nitrogen source in roots. Therefore, promoting NH₄⁺-form uptake is one of the major targets to increase grain yield in rice. It is known that NH₄⁺ influx into roots by a high-affinity transport system (HAT) is down-modulated in response to elevated NH₄⁺ concentration around soil surface. We hypothesized that canceling the down-modulation of NH₄⁺ influx by HAT would be beneficial on the uptake of NH₄⁺-form nitrogen in rice. However, no gene that modulates NH₄⁺ influx by HAT has been identified in rice. In this study, we aimed to identify the gene that modulates HAT of rice roots with gene expression analyses. Furthermore, we tried to isolate a gain-of-function gene to promote uptake of NH₄⁺-form nitrogen with rice Tos17 insertion mutants.

To isolate candidate genes concerned in down-modulation of HAT in roots of rice, we performed four-biological-repeat transcriptome analyses. A total of 28,381 out of 36,444 filtered probes were selected as differentially expressed genes based on a false discovery rate of ≤ 0.05. A strong candidate gene for down-modulation of HAT, the coding protein kinase gene OsACTPK1, showed 1,071 times higher expression in roots under NH₄⁺-rich condition as compared with NH₄⁺-deficient condition (Table 1). We then analyzed the detail of Tos17-inserted mutant of OsACTPK1 (actpk1 mutant) to elucidate OsACTPK1 as down-modulator of HAT. Kinetic analyses of NH₄⁺ influx by HAT revealed that Vmax value of actpk1 mutant was 2 times higher than that of wild type, whereas there was no significant difference of Km value between these two lines (Fig. 1). These results indicated that down-modulation of HAT was canceled in actpk1 mutant due to the loss-of-function OsACTPK1. Total nitrogen content of the actpk1 mutant was significantly higher (+32%) than that of wild type in 1,000 mM NH₄Cl condition (NH₄⁺-rich condition), while the significant difference was not observed in 5 mM NH₄Cl condition (NH₄⁺-deficient condition) (Fig. 2A). Furthermore, the longest-root length of the actpk1 mutant was significantly lower (-22%) than that of wild type in 1,000 mM NH₄Cl condition, while the significant difference was observed in 5 mM NH₄Cl condition (Fig.2B). 

We concluded that OsACTPK1 was a down-modulator of HAT and that loss-of-function ACTPK1 (actpk1 gene) could maintain activity of NH₄⁺ influx even in elevated NH₄⁺ concentration. The actpk1 gene could be effective in improving nitrogen use efficiency in a rice molecular breeding program. Also, reduction of root elongation in actpk1 mutant would be used as phenotypic marker in the program. Further analyses to characterize nitrogen use and grain yield of actpk1 are required for the program since OsACTPK1 would function to avoid NH₄⁺ toxicity.

Figure, table

1. Table 1. Details for the OsACTPK1 gene, a strong candidate for down-modulation of HAT

   Item	Description
   Increase of gene expression in response to incease of external nitrogen concentration	1,071-fold
   RAP ID	Os02g0120100
   Protein function	protein kinase

   Rice Oligo DNA Microarray (4X44K RAP-DB) was used in this research. Total RNA was extracted from roots of rice plants grown for 10 days in 5 and 1,000 M NH
2. 

   Fig. 1. Kinetic properties of HAT in actpk1 mutant.
   Vmax value (A) and Km (B) to NH₄⁺ were expressed in blue column for reference, Nipponbare and red column for actpk1. Plants were hydroponically grown for 10 days in 1,000 mM NH₄Cl. Mean value with standard error (n=3-6) was plotted. Asterisks indicate probability of less than 0.01 between reference and actpk1 (ANOVA).
3. 

   Fig. 2. Total nitrogen content (A) and longest root length (B)
   Plants were hydroponically grown for 10 days in two NH₄⁺ conditions, 5 mM as deficient and 1,000 mM as rich condition of NH₄⁺. Mean value with standard deviation (n=6 for total nitrogen content, n=14 for longest root length) was expressed in blue column for Nipponbare as reference and red column for actpk1, respectively. Asterisks indicate probability of less than 0.01 between reference and actpk1 (ANOVA).