The transcription factor OsNAC25 regulates potassium homeostasis in rice

Abstract

Over-application of potassium (K) fertilizer in fields has a negative impact on the environment. Developing rice varieties with high KUE will reduce fertilizer for sustainable agriculture. However, the genetic basis of KUE in a more diverse and inclusive population remains largely unexplored. Here, we show that the transcription factor OsNAC25 enhances K⁺ uptake and confers high KUE under low K⁺ supply. Disruption of OsNAC25 by CRISPR/Cas9-mediated mutagenesis led to a considerable loss of K⁺ uptake capacity in rice roots, coupled with reduced K⁺ accumulation in rice and severe plant growth defects under low- K⁺ conditions. However, the overexpression of OsNAC25 enhanced K⁺ accumulation by regulating proper K⁺ uptake capacity in rice roots. Further analysis displayed that OsNAC25 can bind to the promoter of OsSLAH3 to repress its transcription in response to low- K⁺ stress. Nucleotide diversity analyses suggested that OsNAC25 may be selected during japonica populations' adaptation of low K⁺ tolerance. Natural variation of OsNAC25 might cause differential expression in different haplotype varieties, thus conferring low K⁺ tolerance in the Hap 1 and Hap 4 -carrying varieties, and the japonica allele OsNAC25 could enhance low K⁺ tolerance in indica variety, conferring great potential to improve indica low K⁺ tolerance and grain development. Taken together, we have identified a new NAC regulator involved in rice low K⁺ tolerance and grain development, and provide a potential target gene for improving low K⁺ tolerance and grain development in rice.

Keywords: K+ uptake; OsNAC25; Potassium; low K+ stress; natural variation; rice.

Figure 1

Growth retardation of Osnac25 mutants under K⁺‐deficient conditions. (a) Growth retardation of the Osnac25 mutants under K⁺‐deficient conditions. The seeds of the wild‐type plants (WT) and Osnac25 mutants (ko‐1, ko‐2) germinated in water for 5 days, then grown in hydroponic solutions containing 0.01, 1, or 10 mM K⁺ and photographed after 14 days. Osnac25 mutants (ko‐1, ko‐2) displayed growth retardation under the K⁺‐deficient conditions. Bars = 4 cm. (b) Root length of wild‐type and Osnac25 mutants under various K⁺ concentrations. (c) Shoot lengths of wild‐type and Osnac25 mutants. (d) Fresh weight of wild‐type and Osnac25 mutants. (e) ChlorophyII content of wild‐type and Osnac25 mutants. Growth conditions are described in a. Significant differences were found between the wild‐type and Osnac25 knockout plants (*P < 0.01 by Student's t‐test). Data are means of three replicates of one experiment. The experiment was repeated three times with similar results. Error bars represent ± SD. Asterisks represent significant differences.

Figure 2

Expression pattern of OsNAC25. (a) qRT‐PCR analyses showing OsNAC25 mRNA accumulation in roots, stems, leaves, panicle, and glume. Rice seedlings (Nipponbare) were grown in soil for 10 weeks. (b) The transcriptional expression of OsNAC25 in rice under different K⁺ concentrations treatment. 3‐days‐old Nipponbare rice seedlings were cultivated in hydroponic solutions with 10 mM K⁺ for 7 days and then transferred to the culture containing 0.01 mM K⁺ for 2 days. Total RNAs were isolated from the rice seedlings, and the mRNA levels of OsNAC25 were examined by real‐time qRT‐PCR. OsActin was used as an internal reference. A significant difference was found between 0.01 or 10 mM K⁺ samples and is indicated in rice seedlings (*P < 0.01 by Student's t‐test). (c) Time course of OsNAC25 mRNA accumulation in roots, as shown by qRT‐PCR analysis. 3‐days‐old Nipponbare rice seedlings were cultivated in hydroponic solutions with 10 mM K⁺ for 7 days, and then transferred to the culture containing 0 mM (−K) or 10 mM K⁺ (+K) for the indicated times. (d) Histochemical analysis of OsNAC25 promoter‐driven GUS reporter expression in transgenic rice plants. (a) GUS staining of seedlings. 3‐days‐old Nipponbare rice seedlings were cultivated in hydroponic solutions with 10 mM K⁺ for 2 days and then transferred to the solution containing 0.01 mM K⁺ for 2 days, and GUS activities (indicated in blue) were examined using histochemical staining. (b) GUS staining of the root of 10 weeks transgenic plants. (c) GUS staining of the stem. (d) GUS staining of the leaf. (e) GUS staining of the panicle. (f) GUS staining of brown grain. Bar in (a) 1 cm, (b) 1 mm, bar in (c) 5 mm, bar in (d) 5 mm, bar in (e) 1 mm, bar in (f) 5 mm. (e) GFP and OsNAC25‐GFP in rice mesophyll protoplasts. For each localization experiment, ≥40 individual cells were analysed using a Zeiss LSM880 confocal laser scanning microscope (Carl Zeiss). Bar = 5 μm. The experiment was repeated three times with similar results. Data are means of three replicates of one experiment. Error bars represent ± SD.

Figure 3

Effects of Osnac25 knockout on K uptake and translocation under different K⁺ supply conditions. The seeds of the wild‐type plants (WT) and Osnac25 mutants (ko‐1, ko‐2) germinated in water for 5 days, then grown in hydroponic solutions containing 0.01, 1 or 10 mM K⁺ for 14 days. The whole plants, roots, and shoots were collected for K⁺ content measurements using inductively coupled plasma analysis. (a) K⁺ contents of wild‐type and Osnac25 mutants. (b) K⁺ contents in root of wild‐type and Osnac25 mutants. (c) K⁺ contents in the shoot of wild‐type and Osnac25 mutants. (d) K⁺ net uptake rate of wild‐type and Osnac25 mutants. (e) K⁺ export rate in xylem. Significant differences were found between the wild‐type and Osnac25 mutants (*P < 0.01 by Student's t‐test). Data are means of three replicates of one experiment. The experiment was repeated three times with similar results. Error bars represent ± SD. Asterisks represent significant differences. DW, dry weight.

Figure 4

Growth phenotype of the overexpression lines (OE‐1, OE‐2, OE‐3) of OsNAC25 under K⁺‐deficient conditions. (a) Growth retardation of the overexpression lines under K⁺‐deficient conditions. The seeds of the wild‐type plants (WT) and the overexpression lines germinated in water for 5 days, then grown in hydroponic solutions containing 0.01, 1 or 10 mM K⁺ and photographed after 14 days. Bars = 4 cm. (b) Plant height of the wild‐type and overexpression lines under various K⁺ concentrations. (c) Root lengths of the wild‐type and overexpression lines. (d) Shoot lengths of the wild‐type and overexpression lines. (e) Fresh weight of the wild‐type and overexpression lines. (f) Chlorophyll of the wild‐type and overexpression lines. Growth conditions are described in Figure 1a. Significant differences were found between the wild‐type and overexpression lines (*P < 0.01 by Student's t‐test). Data are means of three replicates of one experiment. The experiment was repeated three times with similar results. Error bars represent ± SD. Asterisks represent significant differences.

Figure 5

Effects of the overexpression of OsNAC25 on K⁺ uptake and translocation under different K⁺ supply conditions. The seeds of the wild‐type plants (WT) and the overexpression lines (OE‐1, OE‐2, OE‐3) germinated in water for 5 days, then grown in hydroponic solutions containing 0.01, 1 or 10 mM K⁺ for 14 days. The whole plants, roots, and shoots were collected for K⁺ content measurements using inductively coupled plasma analysis. (a) K⁺ contents of wild‐type and the overexpression lines. (b) K⁺ contents in root of wild‐type and the overexpression lines. (c) K⁺ contents in the shoot of wild‐type and the overexpression lines. (d) K⁺ net uptake rate of wild‐type and the overexpression lines. (e) K⁺ export rate in the xylem. Significant differences were found between the wild‐type and the overexpression lines (*P < 0.01 by Student's t‐test). Data are means of three replicates of one experiment. The experiment was repeated three times with similar results. Error bars represent ± SD. Asterisks represent significant differences. DW, dry weight.

Figure 6

RNA‐seq analyses of wild‐type and Osnac25 mutants. (a) OsNAC25 is a transcriptional repressor. REN LUC was used as the internal control. The ratio of the luminescent signals from firefly LUC and REN LUC was calculated. Data are expressed as the mean (±SD) of 3 biological replicates. (b) Venn diagram for the numbers of DEGs. (c) Distribution of top 10 biological processes GO terms for differentially expressed genes (DEGs) in the Osnac25 mutant. (d) Heatmap analysis of ion transport‐related genes revealed by RNA‐seq. (e,f) RT‐qPCR analysis of OsHAK21 and OsSLAH3 revealed by RNA‐seq. (G) Yeast one‐hybrid assay. (h) Dual‐luciferase reporter assay. The experiment was repeated three times with similar results. Data are means of three replicates of one experiment. Significant differences were found between the wild‐type and Osnac25 mutants (*P < 0.01 by Student's t‐test). Asterisks represent significant differences. Error bars represent ± SD.

Figure 7

Natural variation analysis of OsNAC25 in rice. (a) Haplotype analysis in 3475 rice germplasm accessions. Orange indicates the reference (Nipponbare) allele sequence, dark blue indicates the alternative allele sequence. (b) Frequencies of different OsNAC25 haplotypes among 5 subgroups of 3475 rice germplasm accessions and 451 wild accessions. (c) A phylogenetic tree of different OsNAC25 haplotypes. (d, e) Nucleotide diversity of different subgroups in the 3475 rice germplasm accessions and 451 WILD accessions of OsNAC25. Nucleotide diversity across the 300 kb upstream and 300 kb downstream regions of OsNAC25 (d). Nucleotide diversity across the 50 kb upstream and 50 kb downstream regions of OsNAC25 (e). Nucleotide diversity of OsNAC25 in the five cultivated rice subgroups of O. sativa and O. rufipogon accessions. AUS, IND, TRI, TEJ, and BAS represent the five ecotypes of cultivated rice, such as aus, indica, tropical japonica, and temperate japonica rice, bas, and wild rice (O. rufipogon). (f) Comparison of low K⁺ tolerance of different haplotypes under K⁺‐sufficient and K⁺‐deficient conditions. The low K⁺ response was further calculated with the value of (sufficient potassium – low potassium)/low potassium. Plant height is used as an index. (g) Expression levels of OsNAC25 in different haplotype rice materials under K⁺‐sufficient to the K⁺‐deficient conditions. The experiment was repeated four times with similar results. Data are means of five replicates of one experiment. Significant differences were found among different haplotype rice materials (*P < 0.01 by Student's t‐test). Asterisks represent significant differences. Error bars represent ± SD. [Correction added on 29 January 2025, after first online publication: Figure 7 has been updated in this version.]

Figure 8

Potential breeding utilization of the japonica allele OsNAC25 in low K⁺ improvement of indica varieties. (a) Growth phenotype of the transgenic plants (COM‐1, COM‐2, and COM‐3) under K⁺‐deficient conditions. The seeds of 9311 and transgenic plants germinated in water for 5 days, then grown in hydroponic solutions containing 0.01, 1, or 10 mM K⁺ and photographed after 14 days. Transgenic plants displayed growth retardation under the K⁺‐deficient conditions. Bars = 4 cm. (b) Root length of 9311 and transgenic plants under various K⁺ concentrations. (c) Shoot lengths of 9311 and transgenic plants. (d) Plant height of 9311 and transgenic plants. (e) Fresh weight of 9311 and transgenic plants. (f) Chlorophyll content of 9311 and transgenic plants. Growth conditions were described in Figure 8a. (g) K⁺ contents in root of 9311 and transgenic plants. The seeds of the 9311 and transgenic plants germinated in water for 5 days, then grown in hydroponic solutions containing 0.01, 1, or 10 mM K⁺ for 14 days. (h) K⁺ contents in the shoot of 9311 and transgenic plants. (i) K⁺ net uptake rate of 9311 and transgenic plants. (j) The transcriptional expression of OsNAC25 in 9311 and transgenic plants under 0.01 mM or 10 mM K⁺ treatment. Significant differences were found between 9311 and transgenic plants (*P < 0.01 by Student's t‐test). Data are means of three replicates of one experiment. The experiment was repeated three times with similar results. Error bars represent ± SD. Asterisks represent significant differences.