TOND1 confers tolerance to nitrogen deficiency in rice.

Nitrogen (N), the most important mineral nutrient for plants, is critical to agricultural production systems. N deficiency severely affects rice growth and decreases rice yields. However, excessive use of N fertilizer has caused severe pollution to agricultural and ecological environments. The necessity of breeding of crops that require lower input of N fertilizer has been recognized. Here we identified a major quantitative trait locus on chromosome 12, Tolerance Of Nitrogen Deficiency 1 (TOND1), that confers tolerance to N deficiency in the indica cultivar Teqing. Sequence verification of 75 indica and 75 japonica cultivars from 18 countries and regions demonstrated that only 27.3% of cultivars (41 indica cultivars) contain TOND1, whereas 72.7% of cultivars, including the remaining 34 indica cultivars and all 75 japonica cultivars, do not harbor the TOND1 allele. Over-expression of TOND1 increased the tolerance to N deficiency in the TOND1-deficient rice cultivars. The identification of TOND1 provides a molecular basis for breeding rice varieties with improved grain yield despite decreased input of N fertilizers.

Introduction

Nitrogen (N) is the most important macronutrient required for crop growth and development. To enhance crop productivity, the level of N fertilizer used on croplands has globally increased several fold in the last 60 years (Mulvaney et al., 2009). N deficiency affects rice growth and development, producing yellow leaves, dwarf plants and lower grain yields (Borrell et al., 1998, 2001; Lian et al., 2005). As a result of utilization of high-yielding semi-dwarf rice varieties, the quantity of N fertilizer applied for rice production has rapidly increased worldwide. Applications of N fertilizer substantially increase rice yield, which has assisted in averting chronic food shortage. However, agricultural and ecological environments have been gradually polluted because of the excessive use of N fertilizer (Matson et al., 2002; Robertson and Vitousek, 2009; Guo et al., 2010). The necessity of creating crops that require lower input of N fertilizers while producing higher yields has been recognized in the call for a ‘Second Green Revolution’ (Zeigler and Mohanty, 2010; McAllister et al., 2012).

Many quantitative trait loci (QTLs) associated with tolerance of N deficiency in rice have been identified using molecular methods of genetic analysis. Shan et al. (2005) detected 12 QTLs for N concentration/accumulation and N-use efficiency for biomass production in rice plants. Tong et al. (2006) identified 31 QTLs that control plant height, the number of panicles per plant, chlorophyll content, shoot dry weight and grain yield per plant, using a population of chromosome segment substitution lines at the late developmental stage under two N conditions. Cho et al. (2007) mapped 20 QTLs and 58 pairs of epistatic loci for the N concentration in grain, N concentration in straw, N content of shoots, harvest index, grain yield, straw yield and physiological N-use efficiency under both normal and low N conditions. Wang et al. (2009) identified several QTLs related to panicle number and grain yield in field experiments under normal fertilization and low-N treatments. Lian et al. (2005) found that very few QTLs were detected under both low and normal N conditions, and most of the QTLs for the relative measurements were different from QTLs for traits under the two N treatments.

Several genes controlling the absorption, transportation and utilization of N have been identified in rice. OsNRT1 encodes a constitutive component of the low-affinity nitrate uptake transporter (Lin et al., 2000). Knockdown of OsNAR2.1 simultaneously suppressed expression of OsNRT2.1, OsNRT2.2 and OsNRT2.3a (Yan et al., 2011). Knockdown of OsNRT2.3a influenced long-distance nitrate transport from root to shoot under low nitrate supply (Tang et al., 2012). The product of OsNPF2.4 plays a key role in long-distance nitrate transport in rice (Xia et al., 2015). The product of the OsAMT1;1 gene affects ammonium uptake and content (Hoque et al., 2006). OsAMT2;2 is N-inducible, and its expression was detected in both roots and shoots (Li and Shi, 2006). Over-expression of rice NADH-dependent glutamate synthases encoded by NADH-GOGAT genes enhanced grain filling (Yamaya et al., 2002), but knockout of NADH-GOGAT1 in rice decreased overall biomass and panicle production, as well as yield (Tamura et al., 2010). Dof1, the transcription factor proposed to affect N-use efficiency, influenced N assimilation under low N condition (Yanagisawa et al., 2004). Recently, qNGR9, a major rice QTL for N-use efficiency, was cloned and found to be synonymous with the previously identified DEP1 (DENSE AND ERECT PANICLE 1) gene (Sun et al., 2014).

Here we describe the isolation and detailed characterization of Tolerance Of Nitrogen Deficiency 1 (TOND1), located on chromosome 12, which confers tolerance to N deficiency in the indica cultivar Teqing. We show that, among 75 indica and 75 japonica cultivars from 18 countries and regions, only 41 indica cultivars contain TOND1, whereas the remaining 34 indica cultivars and the 75 japonica cultivars do not harbor the TOND1 allele. Over-expression of TOND1 increased the tolerance to N deficiency in TOND1-deficient rice cultivars.

Results and Discussion

Characterization of the N-deficiency intolerant line YIL105

To clone the gene responsible for N-deficiency tolerance, we used a set of introgression lines based on an accession of Yuanjiang common wild rice (YJCWR, Oryza rufipogon) as a donor and an elite indica cultivar Teqing (Oryza sativa) as the recipient (Tan et al., 2007) to identify an introgression line YIL105 that displayed intolerance to N deficiency (Figure1). YIL105 harbored three YJCWR chromosomal segments on the long arms of chromosomes 2, 9 and 12 (Figure S1). The growth, development and production of YIL105 were significantly inhibited under N-deficient conditions compared with N-sufficient conditions. Compared with Teqing, YIL105 exhibited reductions in plant dry weight (DW), N concentration (NC), total N amount per plant (TN), chlorophyll relative content (SPAD), plant height (PH), root length (RL) (Figures1b and S2a), the number of panicles per plant (PN), grain number per plant (GN), grain yield per plant (GY) and the number of tillers (Figures1c and S2b) under N-deficient conditions relative to N-sufficient conditions.

Figure 1

Phenotype of the rice indica cultivar Teqing (Oryza sativa) and the introgression line YIL105. (a) Images of the recipient parent Teqing and YIL105 at 21 days after emergence of the third leaf under hydroponic conditions. +N, N-sufficient conditions; −N, N-deficient conditions. The arrows indicate the top third fully expanded leaf, and the leaf tip is magnified on the right. Scale bars = 10 cm. (b) Comparison of plant dry weight (DW), N concentration (NC) and total N amount per plant (TN) between Teqing and YIL105 under hydroponic conditions. (c) Comparison of panicle number per plant (PN), grain number per plant (GN) and grain yield per plant (GY) between Teqing and YIL105 under field conditions. Values are means ± SD.

Map-based cloning of TOND1

Genetic linkage analysis using 311 F2 individuals derived from the cross between YIL105 and Teqing showed that the N-deficiency tolerance, as indicated by the relative plant dry weight, was controlled by a major QTL, referred to as TOND1, which was located between simple sequence repeat markers RM235 and RM17 on the long arm of chromosome 12 (Figure2a). Using a larger F2 population of 3180 individuals, we further localized TOND1 to within a 269 kb region between the M4 and RM17 markers (Figure2b), in which 45 putative genes are predicted to occur according to the Rice Annotation Project Database (Yuan et al., 2003) (Figure2c).

Figure 2

Map-based cloning of TOND1 in rice. (a) The target gene was mapped between the simple sequence repeat markers RM235 and RM17 on the long arm of chromosome 12 by linkage analysis using 311 F2 individuals created by crossing Teqing (TQ) and YIL105. R is the number of recombinants.(b) TOND1 was fine-mapped between markers M4 and RM17 using recombinants. The number of lines in each recombinant family are shown on the left. The phenotypic differences for each recombinant family compared with Teqing (TQ) and YIL105 are shown on the right. RDW (relative plant dry weight) is the mean value of plant dry weight for each line under N-deficient conditions relative to N-sufficient conditions. (c) Predicted gene contained in the mapping region according to the Nipponbare genome. (d) Real-time quantitative PCR for the relative expression of Os12g0630100 in roots and shoots of Teqing (TQ) and YIL105 after 2 h of hydroponic growth under N-deficient and N-sufficient conditions. (e) Expression of the Os12g0630100 gene in the control (CK1) and RNAi transgenic plants (Ri1 and Ri2) by real-time quantitative PCR after 21 days of N-deficient hydroponic growth. (f) Photos of the control (CK1) and RNAi transgenic plants (Ri1) taken after 21 days of N-deficient hydroponic growth. Scale bar = 10 cm. (g) Comparison of DW, NC and TN between the control and RNAi transgenic plants under N-deficient hydroponic conditions. (h) Comparison of PN, GN and GY between the control and RNAi transgenic plants under various N-deficiency field conditions. Values are means ± SD. Asterisks indicate statistically significant differences compared with +N conditions or CK1 plants, as appropriate (*P < 0.05; **P < 0.01).

Expression profiling using an Affymetrix whole-genome array and real-time quantitative PCR assays showed that, among the 45 predicted genes, only a single gene (Os12g0630100) in the TOND1 mapping region exhibited a significant expression change. Os12g0630100 expression in YIL105 was obviously lower than that in Teqing, and the relative expression of Os12g0630100 under N-deficiency conditions was significantly decreased in YIL105 (Figure2d).

To genetically examine whether Os12g0630100 is responsible for N-deficiency tolerance, we generated an RNA interference (RNAi) construct that was designed to target the Os12g0630100 gene of Teqing (Figure2e,f). The results, shown in Figure2, demonstrate that down-regulation of the Os12g0630100 gene in Teqing leads to a decreased tolerance to N deficiency. The RNAi transgenic plants (Ri1 and Ri2) exhibited an obvious reduction in DW, NC, TN, SPAD, PH and RL, in comparison with that of the control plants, under N-deficient hydroponic conditions (Figures2g and S3a). Furthermore, PN, GN and GY of the RNAi transgenic plants were lower than that of control plants under a variety of N-deficiency levels (Figure2h). These results suggest that the Os12g0630100 gene in Teqing corresponds to TOND1.

Subcellular localization and expression pattern of TOND1

The TOND1 gene comprises 549 bp with a single exon (Figure S4), and encodes a thaumatin protein containing an N-terminal signal peptide and the thaumatin motif (Figure S5a). Using the full-length protein sequence of TOND1 as a query for blastp searches against the non-redundant protein database (http://www.ncbi.nlm.nih.gov/BLAST/), we identified 27 putative homologs in other crop species (Figure S5b). The sequences for thaumatin motif were highly conserved between TOND1 and its homologs in other crop plants (Figure S5c). A TOND1–GFP fusion protein localized to the plasma membrane (Figure3a). Histochemical analysis of japonica cultivar Zhonghua 17, which is transgenic for the GUS reporter gene driven by the TOND1 2 kb regulatory region, indicated that TOND1 is expressed in leaves at the seedling and heading stages, in spikelet hulls and anthers (Figure3b–d), and particularly in the vascular bundles of leaf blades and pollen grains (Figure3e,f). Real-time quantitative PCR also detected higher expression levels of TOND1 in the leaf blades, leaf sheathes and spikelet hulls (Figure3g).

Figure 3

Subcellular localization and expression pattern of the TOND1 gene in rice. (a) Subcellular localization of TOND1. The TOND1–GFP fusion gene was expressed transiently in onion epidermal cells. DIC, a differential interference contrast image; GFP, the same cells showing TOND1-GFP green fluorescence in the plasma membrane; Merged, the merged image. Scale bars = 50 μm. (b–f) Tissue localization of TOND1. Expression of TOND1 was detected in a young leaf at the seedling stage (b), a leaf at the heading stage (c) and anthers (d) by GUS staining. (e) Magnification of cross section from the yellow box in (c). x, xylem; p, phloem. (f) Magnification of cross section from the yellow box in (d). Scale bars = 50 μm. (g) Real-time quantitative PCR indicating the relative expression of TOND1.

Sequence comparison and functional site analysis of TOND1

Sequence comparison between Teqing and YIL105 revealed seven single nucleotide polymorphisms (SNP1–7) in the 2 kb regulatory region, and three SNPs (SNP8–10) in the coding region of TOND1 (Figure4a). Further sequence verification of these ten SNPs in 75 indica and 75 japonica cultivars from 18 countries and regions showed that only 41 indica cultivars, 27.3% of the tested cultivars, harbor a TOND1 allele identical to Teqing, whereas 72.7% cultivars, including the remaining 34 indica and the 75 japonica cultivars, have sequences similar to the tond1 allele of YIL105 (Table S1).

Figure 4

Sequence comparison between Teqing and YIL105, and functional sites analysis of TOND1. (a) Sequence comparison of the 2 kb upstream regulatory region and coding region between Teqing and YIL105. (b) Association testing of 10 variants in the regulatory region and coding region of TOND1. The black dots represent five variations; the red dots represent five proposed functional variant sites. (c) A triangle matrix of pairwise linkage disequilibrium calculated for 45 samples. (d) Comparison of mean values of relative plant dry weight (RDW) between varieties containing the TOND1 allele and varieties containing the tond1 allele after 21 days of hydroponic growth under N-deficient conditions relative to N-sufficient conditions. (e) Comparison of mean values of relative expression of TOND1 between varieties containing the TOND1 allele and varieties containing the tond1 allele. (f) Relative expression of TOND1 in 93-11, ZH17 and NIP. (g) Photographs of 93-11, ZH17 and NIP taken after 21 days of hydroponic growth under N-deficient and N-sufficient conditions. Scale bar = 10 cm. (h) Comparison of relative plant dry weight, relative plant height, relative root length and relative SPAD between 93-11, ZH17 and NIP under N-deficient conditions relative to N-sufficient conditions. ZH17, Zhonghua 17; NIP, Nipponbare. Values are means ± SD. Asterisks indicate statistically significant differences compared with TOND1 or 93-11, as appropriate (**P < 0.01).

To determine whether rice cultivars harboring the TOND1 allele exhibit stronger tolerance to N deficiency, 45 rice cultivars clustered in four haplotypes (H1–H4) were used to evaluate N-deficiency tolerance (Figure4b and Table S2). We found that, similar to Teqing, H1 haplotypes (including 93-11), exhibited tolerance to N deficiency and showed higher TOND1 expression, while other haplotypes (including Zhonghua 17 and Nipponbare) that are similar to YIL105 were intolerant to N deficiency and exhibited lower tond1 expression (Figure4d–h). A further test of association between N-deficiency tolerance and the ten SNPs identified between Teqing and YIL105 revealed that the strongest signal was present at SNP1, SNP4, SNP7, SNP8 and SNP9 (P = 4.43 × 10−19; Figure4b,c). These results suggest that rice cultivars harboring the favorable TOND1 allele exhibit tolerance to N deficiency, and that five SNPs (SNP1, SNP4, SNP7, SNP8 and SNP9) lead to the increase in N-deficiency tolerance in cultivars harboring TOND1.

Over-expression of TOND1 enhances tolerance to N deficiency

As 72.7% of cultivars did not harbor the favorable TOND1 gene, we investigated whether TOND1 is able to enhance tolerance to N deficiency in TOND1-deficient rice. As shown in Figure5, over-expression of the TOND1 gene enhanced tolerance of N deficiency in YIL105. TOND1 over-expressing transgenic plants (Ox1 and Ox2) in the YIL105 background exhibited an obvious increase in DW, NC, TN, SPAD, PH and RL in comparison with control plants under N-deficient hydroponic conditions (Figures5a,b and S3b). Furthermore, compared with control plants, PN, GN and GY of the over-expressing transgenic plants were significantly increased by approximately 17.3, 19.8 and 20.2%, respectively, under certain N-deficiency conditions (1/3N) (Figure5c).

Figure 5

The phenotype of over-expressing transgenic rice plants. (a) Expression of TOND1 in over-expressing transgenic plants (Ox1 and Ox2) in the YIL105 background was assessed by real-time quantitative PCR. (b) Comparison of DW, NC and TN between control (CK2) and over-expressing transgenic plants (Ox1 and Ox2) after 21 days of hydroponic growth under N-deficient conditions. (c) Comparison of PN, GN and GY between control (CK2) and over-expressing transgenic plants under various N-deficiency field conditions. (d) Expression of TOND1 in over-expressing transgenic plants (Oe) in the japonica cultivar Zhonghua 17 background was assessed by real-time quantitative PCR. (e) Comparison of DW, NC and TN between control (CK3) and over-expressing transgenic plants (Oe1 and Oe2) after 21 days of hydroponic growth under N-deficient conditions. Values are means ± SD. Asterisks indicate statistically significant differences compared with +N conditions or control plants, as appropriate (*P < 0.05; **P < 0.01).

We further over-expressed TOND1 in the commercial japonica cultivar Zhonghua 17, and found that the transgenic plants (Oe1 and Oe2), with higher expression levels of TOND1, exhibited tolerance of N deficiency, as indicated by increases in DW, NC, TN, SPAD, PH and RL under N-deficient hydroponic conditions (Figures5d,e and S3c,d). These results suggest that over-expression of TOND1 enhances tolerance of N deficiency in TOND1-deficient rice cultivars.

In this study, we identified TOND1, a major QTL in rice controlling tolerance of N deficiency. Increased expression of the TOND1 gene led to a remarkable increase in the N-deficiency tolerance and grain yield of rice plants grown under N-deficient conditions. Previous studies showed that N-deficiency tolerance is associated with the GS/GOGAT cycle (Cai et al., 2009; Tamura et al., 2010). We found that expression levels of the OsGS2, OsNADH-GOGAT1 and OsNADH-GOGAT2 genes, which encode the essential enzymes in the GS/GOGAT cycle for N assimilation (Tabuchi et al., 2005, 2007; Cai et al., 2009, 2010; Tamura et al., 2010, 2011), were significantly decreased in TOND1 RNAi transgenic plants and increased in TOND1 over-expressing transgenic plants, compared with control plants under N-deficiency conditions (Figure S6a–f). It would be interesting to verify whether the TOND1 gene controls N-deficiency tolerance via modulation of the GS/GOGAT cycle.

China is the world's largest consumer of N fertilizers. The annual N fertilizer consumption in China has accounted for 30% of the annual global consumption of N fertilizer since the 1980s (Peng et al., 2006). In recent years, the rate of N application and annual consumption of N fertilizers for rice production in China reached 180 kg ha−1 and 5.29 million tonnes, respectively (Peng et al., 2006). However, only 30–45% of N fertilizer is efficiently used by rice plants (Ladha et al., 1998; Zhu, 2000; Peng et al., 2006), with the majority being released into the environment as non-point source pollution. As 72.7% of the cultivars analyzed do not harbor the TOND1 allele (Table S1), introgression of TOND1 into these TOND1-deficient rice cultivars may significantly decrease the use of N fertilizers, reduce the cost of rice production, alleviate pollution, and protect the environment. It is speculated that the TOND1 gene has broad utilization prospects in rice breeding programs, and will play a vital role in the ‘Second Green Revolution’.

Experimental Procedures

Plant materials

The 150 varieties of cultivated rice, including 75 indica and 75 japonica varieties, used for the sequence comparison analysis are listed in Table S1, and the plant materials used for the association mapping are listed in Table S2.

Primers

The primers used in this study are listed in Table S3.

Phenotypic evaluation

All materials for phenotypic evaluation under hydroponic conditions were grown in nutrient solution under either N-deficient conditions (−N, 0.24 mm NH4NO3) or N-sufficient conditions (+N, 1.44 mm NH4NO3) (Yoshida et al., 1976). Phenotypic evaluation was performed after emergence of the third leaf. The solution was matched with deionized water and refreshed every 2 days. After hydroponic growth for 21 days, the plants were sampled for phenotypic evaluation.

The second generation of transgenic plants (T2) and control plants were planted in the field at the Experimental Station of China Agricultural University, Shangzhuang Road, Haidian District, Beijing, China. All materials were sown on 5 May 2013, and transplanted on 13 June 2013. The field trial followed a randomized block design with four N levels (+N, with 150 kg ha−1 N fertilizer; 2/3N, with 100 kg ha−1 N fertilizer; 1/2N, with 75 kg ha−1 N fertilizer; 1/3N, with 50 kg ha−1 N fertilizer). Under the four N conditions, P and K fertilizers were both applied at 100 kg ha−1. Total P and K and 60% of N fertilizers were applied by top-dressing before transplantation, and the remaining 40% of N fertilizers was applied at the tillering stage. Twenty-one plants were planted for each transgenic line and control line, and the middle five plants were used for phenotypic evaluation. All materials were randomly arranged with three replications. Seedlings were transplanted into the field with a spacing of 13.3 cm between plants and 26.6 cm between rows. Field management followed normal agronomic procedures during the whole growth period (Shen et al., 2007).

We surveyed plant height (PH), root length (RL), chlorophyll relative content (SPAD), dry weight (DW), N concentration (NC) and total N amount per plant (TN) under hydroponic conditions, and the number of tillers, the number of panicles number (PN), grain number per plant (GN) and grain yield per plant (GY) under field conditions.

Relative plant dry weight, relative plant height, relative root length and relative SPAD, between N-deficient and N-sufficient hydroponic conditions were calculated using the equation:

where R is the relative value, and D and S are the values under N-deficient and N-sufficient conditions, respectively.

To evaluate the tolerance of each F2 individual, 15 plants of the corresponding F3 family were used for phenotyping under N-deficient and N-sufficient conditions.

QTL detection

QTL analysis for low N tolerance was performed using genotypic data and phenotypic data obtained by the single-marker regression method using Map Manager QTXb17 (Manly et al., 2001). A probability of 0.01 was used as the threshold for single-point analysis. The proportion of the observed phenotype variance explained by a particular QTL was estimated by the coefficient of determination (R2) from the corresponding linear model analysis.

Microarray analysis

Total RNA was extracted using Trizol reagent (Invitrogen, http://www.invitrogen.com) and purified using an RNeasy kit (Qiagen, http://www.qiagen.com). The methods for cDNA synthesis, cRNA synthesis, cRNA fragmentation, microarray hybridization, washing and staining are described in the GeneChip® Expression Analysis Technical Manual (http://www.affymetrix.com/). The arrays were scanned using a GeneChip 3000 scanner located in the Bioinformatics Center at China Agricultural University.

Vector construction and transformation

The TOND1 RNAi construct was generated by insertion of a hairpin sequence containing two 487 bp cDNA inverted repeat fragments targeting the TOND1 sequence of Teqing into pJL1460 (Yu et al., 2007), under the control of the ubiquitin promoter. We got 10 independent RNAi transgenic lines, and phenotypically evaluated two of the RNAi lines, Ri1 and Ri2. The construct p35S::TOND1-GFP containing TOND1 fused to GFP under the control of the CaMV 35S promoter was transformed into onion epidermal cells by particle bombardment. The pTOND1::GUS construct was generated by cloning a 2 kb regulatory region harboring the Teqing TOND1 promoter into the vector pCAMBIA1381 (Yu et al., 2007). The over-expression construct harboring only the coding sequence of TOND1 cDNA, a 549 bp fragment including the whole coding region, was inserted into the vector pCAMBIA1301 under the control of the ubiquitin promoter (Yu et al., 2007). This was subsequently transformed into YIL105 and Zhonghua 17, and nine and 16 independent transgenic lines were harvested, respectively. Ox1, Ox2, Oe1 and Oe2 were used for phenotypic evaluation.

Real-time quantitative PCR

Real-time quantitative PCR was performed using the ABI Prism 7900 sequence detection system (Applied Biosystems, http://www.appliedbiosystems.com). cDNA corresponding to 5 ng of total RNA was used as the template, and was amplified using primers TOND1-qRTF and TOND1-qRTR and SYBR Green Master Mix (Applied Biosystems). We normalized the levels of TOND1 transcripts using endogenous ubiquitin transcripts amplified with primers UBIQRT-2F and UBIQRT-2R. Each set of experiments was repeated three times, and the relative quantification method (Zhang et al., 2006) was used to evaluate quantitative variation.

Subcellular and tissue localization

p35S::TOND1-GFP was bombarded into onion epidermal cells using a helium biolistic device (Bio-Rad PDS-1000) (Bio-Rad, http://www.bio-rad.com). We examined the bombarded tissues using a Nikon C1 Si confocal laser-scanning microscope (Nikon, http://www.nikon.com). GUS staining of tissues from plants harboring the pTOND1::GUS construct was performed as previously described (Scarpella et al., 2003).

Association mapping

Association testing was performed by Fisher's exact test using a simple linear model with the r program (Lin et al., 2012).