Cassava Nitrate Transporter NPF5.4 promotes both yield potential and salt tolerance in rice

doi:10.21203/rs.3.rs-2206489/v1

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Cassava Nitrate Transporter NPF5.4 promotes both yield potential and salt tolerance in rice

https://doi.org/10.21203/rs.3.rs-2206489/v1

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Nitrogen is a major driving force for the improvement of crop yield worldwide, but brings detrimental effects on ecosystems, thus future agricultural sustainability demands enhanced nitrogen use efficiency (NUE). The nitrate transporter (NRT/NPF) family associated with nitrogen uptake and utilization is indispensable to the improvement of NUE in crops. Because cassava (Manihot esculenta) has high-affinity to absorb nitrate, the NUE of the NPF genes in cassava might be higher than other crops. Here we identified and systematically analyzed the NPF gene family in cassava, including phylogenetic relationship, chromosome location, gene duplication, and gene expression in response to different nitrogen supply. Gene expression analysis revealed that MeNPF5.4 and MeNPF6.2 were specifically expressed in stem, and have diverse expression in different nitrogen conditions. To well study the roles of these two genes, we constructed their overexpression (OE) lines in rice. A NO₃⁻ flux assay showed that MeNPF5.4 OE lines exhibited a significant NO₃⁻ influx, which suggests that they might have contributed to NUE improvement of rice. Notably, overexpressing MeNPF5.4 not only results in increased grain size and weight but also enhanced tolerance to salt. Compared with MeNPF5.4, MeNPF6.2 OE lines showed higher salt stress tolerance but had smaller grain size. Taken together, our results demonstrated that MeNPF5.4 can potentially improve the NUE and salt stress tolerance of rice, which reveals valuable breeding targets to improve crop yield and stress tolerance.

Nitrate transporter

Rice (Oryza sativa L.)

Nitrogen use efficiency

Stress tolerance

Nitrogen (N) is the essential macronutrient for plant growth, development and reproduction. Increasing nitrogen fertilizer application has contributed to greatly increased crop yield, but causes some serious problems such as soil acidification, water eutrophication, greenhouse gas emission, waste of resources, allele dropout of high nitrogen use efficiency (NUE) (Liu et al. 2021). It is estimated that crop NUE only accounts for approximately 30–50% (Wang et al. 2018b). Therefore, improving crop NUE is crucial for food security and sustainable agriculture.

NUE is a complex agronomic trait which involves N uptake, transport, assimilation, and remobilization in crop plants (Xu et al. 2012). Four nitrate transporter (NRT) families are responsible for nitrate uptake from the soil and nitrate distribution within the whole plant, including NPF (nitrate transporter 1/peptide family), NRT2 (nitrate transporter 2), CLC (chloride channel), and SLAC/SLAH (slow anion channel-associated 1 homologues) (Dechorgnat et al. 2010; Krapp et al. 2014; Zhang et al. 2020). The NPF family is one of the most important NRTs in plants, and play governing role for N uptake and distribution (Vidal et al. 2020). To date, there are 53 and 103 NPF genes in the model plants Arabidopsis and rice, respectively (Fan et al. 2017). NPF genes have diverse functions in plant N uptake and utilization. Most of the NPF gene family members have low affinity for nitrate, while other NPF family members, such as NPF6.3 (NRT1.1/CHL1) in Arabidopsis and NRT1.3 in Medicago truncatula exhibit dual affinity in either low or high nitrate concentrations (Liu et al. 1999; Morère-Le Paven et al. 2011). Notably, OsNRT1.1B has one amino acid mutation between indica and japonica rice varieties and contributes to nitrate use divergence (Hu et al. 2015; Zhang et al. 2019). In Arabidopsis, AtNPF6.3, AtNPF2.7 (NAXT1) and AtNPF4.6 are mainly involved in root nitrate uptake (O'Brien et al. 2016). Among them, AtNPF2.7 is responsible for nitrate efflux, whereas AtNPF4.6 mainly participates in nitrate influx (Segonzac et al. 2007; Huang et al. 1999). Some members of the NRT1 family play important roles in root-to-shoot nitrate transport, including AtNPF2.3, AtNPF2.9, AtNPF7.2 and AtNPF7.3 (Dechorgnat et al. 2010; Wang et al. 2012; Krouk et al. 2010a; Krapp et al. 2014). After transportation to shoots, nitrate can be assimilated in cytosol or stored in vacuoles. Thereinto, AtNRT1.4 mediates leaf nitrate homeostasis and leaf development (Chiu et al. 2004). AtNPF2.13 has a role in remobilizing nitrate from older leaves into younger leaves (Fan et al. 2009). In addition, AtNPF2.12 and AtNPF5.5 play a role in nitrogen storage and seed development (Tegeder and Masclaux-Daubresse 2018).

Besides being a nutrient element, nitrate is also an important signaling molecule that triggers nitrogen responses in regulating plant growth and metabolic processes (Ho and Tsay 2010; Fan et al. 2017; Hachiya and Sakakibara 2017). NRT1.1 is proposed as a nitrate sensor to perceive nitrate and regulate primary nitrate response (PNR) in Arabidopsis (Armijo and Gutierrez 2017; Bouguyon et al. 2012). As a dual affinity transporter, NRT1.1 displays nitrate transport activity in either low or high nitrate concentrations, and its dual affinity for nitrate is regulated by phosphorylation and dephosphorylation at threonine residue 101 (Thr101) (Liu and Tsay 2003). When Thr101 is phosphorylated by calcineurin-interacting protein kinase 23 (CIPK23), NRT1.1 shows high affinity activity in response to low external nitrate. When environmental nitrate concentration is high, it forms a dimer and acts as a low affinity nitrate transporter through dephosphorylation (Parker and Newstead 2014; Wang et al. 2020). Moreover, some transcription factors, such as NIN-like protein 7 (NLP7), nitrate regulated 1 (ANR1), lateral boundary domain 37/38/39 (LBD37/38/39), BTB and TAZ domain protein (BT), SQUAMOSA promoter binding protein-like 9 (SPL9) and elongated hypocotyl 5 (HY5) are also be involved in PNR and directly interact with nitrate-related target genes (Gan et al. 2012; Rubin et al. 2009; Araus et al. 2016; Krouk et al. 2010b; Chen et al. 2016).

Rice is one of the three major staple crops for more than half of the world’s population and its yield is often limited by high N fertilizer input and low-NUE problems. Cassava grows well in nutrient-poor soils because of its high nitrate-absorption activity. Therefore, excavating the high-NUE-related genes in cassava is crucial for the improvement of rice high-NUE genetic resources. In this study, we identified NPF genes in cassava and systematically analyzed their phylogenetic relationship, chromosomal location, gene duplication, and gene expression in response to different nitrogen supply. Two genes, MeNPF5.4 and MeNPF6.2, are localized in the plasma membrane and mainly expressed in stem, and were proposed as candidate NUE-related genes. We further identified the function of MeNPF5.4 and MeNPF6.2, and found that MeNPF5.4 might have contributed to NUE improvement of rice. Intriguingly, overexpression of these two genes enhanced the plants tolerance to salt stress. Our results provided new insight in rice breeding to improve NUE and stress tolerance in crops.

Plant materials and growth conditions

Cassava cultivar ‘Huanan5’ was used in this study. Cassava seedings were grown on nitrogen-free Murashige and Skoog (MS) media respectively supplemented with 0 and 10 mM NO₃⁻ for 30 days under 16 h light (30°C)/8 h (22°C) dark cycle and 70% relative humidity conditions. The roots, stems and leaves were collected and immediately frozen in liquid nitrogen, and stored at -80℃.

Japonica rice cultivar Nipponbare was used for all rice experiments. For overexpression construct, the coding sequence (CDS) region of MeNPF5.4 or MeNPF6.2 were cloned into the binary vector pCambia1300 driven by the cauliflower mosaic virus 35S promoter, respectively. For hydroponic culture, plants were grown in International Rice Research Institute (IRRI) nutrient solution supplied with different N concentration (0.5 mM NH₄NO₃, NN; 0.01 mM NH₄NO₃, LN), pH 5.5. All plants were grown in 16 h light (28°C)/8 h dark (26°C) photoperiod and ~ 60% relative humidity. The nutrient solution was changed every three days.

For our hydroponic system under salt stress, 4-day-old rice seedlings under normal conditions were transferred into 150 mM NaCl solution for 10 days. The survival rate of rice seedlings was determined after another 7-day recovery period. Plant height, root length, shoot weight and root weight of rice seedlings were measured after 10 days of salt treatment. For PEG treatment, the rice seedings were planted with 20% (w/v) PEG (polyethylene glycol 6000) for 10 days.

Identification and phylogenetic analysis of NPF genes in cassava

The protein sequences of cassava were downloaded from EnsemblePlants (http://plants.ensembl.org/index.html). The Hidden Markov Model (HMM) profile (PTR2, PF00854) was downloaded from Pfam database (http://pfam.xfam.org/) and was used as query to search NPF proteins in cassava. The putative MeNPF protein sequences were submitted to NCBI Conserved Domain Search (https://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi) and Pfam database to verify whether they contain PTR2 domain. The candidate sequences were considered to be the final protein sequences of MeNPF gene family. The nomenclature of MeNPF genes was performed based on the relationships with orthologues of Arabidopsis.

The NPF protein sequences of Arabidopsis was downloaded from TAIR (https://www.arabidopsis.org/). The rice NPF protein sequences were obtained from Leran et al. (2014). The candidate sequences of cassava and rice were aligned with AtNPF protein sequences through Clustal Omega. The phylogenetic tree was constructed by IQ-TREE using the Maximum Likelihood (ML) method with 1000 replicates of bootstrap alignments.

Chromosomal localization and gene duplication of MeNPF gene family

The chromosomal positions of NPF genes were obtained according to the genome sequences of cassava and the sequences of MeNPF genes. MG2C (http://mg2c.iask.in/mg2c_v2.1/) was used to draw the chromosomal distribution of NPF genes. Collinearity analysis of the NPF gene family was performed using MCScanX. The location of NPF genes on the chromosome and gene duplication information were visualized by circus in TBtools software.

Expression analysis of MeNPF gene family from RNA-seq data

The total RNA of roots, stems and leaves in cassava was extracted and sequenced using an Illumina HiSeq platform. For RNA-seq data, all reads were mapped to cassava reference genome using HISAT2 with default parameters. To count the uniquely mapped reads, featureCounts was used to calculate the read count per gene. The differentially expressed genes (DEGs) were identified by DESeq2 package with standard parameters (log₂|fold change| > 1, force discovery rate (FDR) < 0.05). The phylogenetic tree and heatmap were constructed by iTOL website (https://itol.embl.de/).

Subcelluar localization of MeNPF5.4 and MeNPF6.2

The open reading frames (ORFs) of MeNPF5.4 and MeNPF6.2 were amplified and cloned into 35S-eYFP vector between the XhoI and HindIII sites. The resulting clone places the MeNPF5.4 and MeNPF6.2 ORFs under control of 35S promoter. A plasma membrane marker (ZmCDPK7-mcherry) was used in this study (Zhao et al. 2021). The MeNPF5.4-eYFP/MeNPF6.2-eYFP and ZmCDPK7-mcherry fusion construct were transiently expressed in rice protoplasts using polyethylene glycol mediated method. Empty 35S-eYFP vector was served as a control. Transfected protoplasts were observed by confocal laser scanning microscope after 16 h incubation. The primers used to generate these constructs were listed in Supplemental Table S4.

RT-qPCR analysis

For quantitative real-time PCR (RT-qPCR), total RNA was extracted from leaves using TRNzol reagent (Tiangen, DP424). First strand cDNA was synthesized from 1 µg total RNA using PrimeScript RT reagent Kit with gDNA Eraser (Takara, RR047A). RT-qPCR was performed on LightCycler 480 Green I Master Mix (Roche). The target gene expression level was normalized with OsActin2 for rice and was calculated by the ΔΔCT method (Livak and Schmittgen 2001). The gene-specific primers were given in Supplementary Table S4.

Measurement of net NO₃⁻ flux

Net NO₃⁻ flux was measured using Non-invasive Micro-test Technology (NMT) according to the description of Sun et al. (2021) with minor modification. The microelectrode was calibrated in the solution containing 0.05 mM and 0.5 mM NO₃⁻. Roots sampled from rice seedlings of wild type and transgenic lines were equilibrated in measuring solution for 20 min and transferred into the fresh solution containing 0.05 mM NO₃⁻. The net influxes of NO₃⁻ in the root meristem zone about 600 µm from root tip was examined for 16 min. All measurement results were imported and converted into net NO₃⁻ flux using JCal V3.3.

Physiological measurements of transgenic rice lines

Rice seedlings of wild type and transgenic lines were grown under NN and LN conditions for 2 weeks, then were harvested and immediately calculated plant height, root length, shoot weight, and root weight. The chlorophyll was extracted from rice leaf samples by soaking them in 95% ethanol for 24 hours. The absorbance of supernatant was recorded at 665 and 649 nm in spectrophotometer against the ethanol blank. The content of chlorophyll a, chlorophyll b and total chlorophyll were calculated using the following equation: Chla = 13.95(A₆₆₅)-6.88(A₆₄₉), Chlb = 24.96(A₆₄₉)-7.32(A₆₆₅), Total Chlorophyll = Chla + Chlb. The activity of POD was measured using 4-methylcatechol as substrate. SOD activity was determined by nitro blue tetrazolium (NBT) method. The CAT activity assay was carried out by monitoring the decrease at 240 nm.

Total nitrogen was determined using the Kjeldahl method. Rice samples were ground through a suitable laboratory mill. The samples (around 0.2 g) were digested in the digestion flasks filled with 10 mL of concentrated H₂SO₄ and 0.5 to 1.0 g catalyst granules at 420°C for 2 hours. After cooling 15 to 20 min to room temperature, total nitrogen was determined using Kjeltec 8400. Every sample had four replicates.

Statistical analysis

All of the bar charts were presented using GraphPad Prism 8. The statistical analysis by One-way analysis of variance (ANOVA) was performed using IBM SPSS 26. Different letters or asterisks represent statistical significances determined by ANOVA (P < 0.05).

Identification and gene duplication of NPF genes in cassava

To identify the entire NPF genes in cassava, we used a HMM profile (PTR2, PF00854) to search NPF proteins. Finally, we identified a total of 72 NPF genes in cassava genome. In order to clarify the phylogenetic relationship of NPF genes among Arabidopsis, cassava and rice, we performed multiple sequence alignment of NPF sequences using Clustal Omega, and a phylogenetic tree was constructed by IQ-TREE (Fig. 1a). According to the similarity of NPF sequences and the classification of members in Arabidopsis, the cassava NPF family was obviously divided into eight subfamilies. Among the cassava NPF gene family, NPF5 subfamily contained the most abundant NPF genes (20 members), followed by NPF2 and NPF4 (12 members of each subfamily), NPF6 (7 members), NPF3 (6 members), NPF1, NPF7 and NPF8 (5 members of each subfamily) (Fig. 1a; Supplementary Table S1-3).

Chromosomal localization analysis revealed that 72 MeNPF genes were widely distributed on 18 chromosomes, but not evenly distributed in the cassava genome (Fig. S1). Chromosome 1 (Chr1) had the highest density of NPF genes with 8 members, while Chr2, Chr3 and Chr12 only contained one gene, respectively. Due to the incomplete assembly and annotation of the current version of cassava genome, MeNPF5.14 was temporarily located at Scaffold01657. In addition, it was also found that the density of MeNPF genes at both ends of the chromosome was higher than that in the middle region.

Gene families are expanded mainly through whole genome duplication (WGD), tandem duplication (TD) and segmental duplication (SD)(Panchy et al. 2016). Collinearity analysis revealed that there were collinear relationships between some NPF genes. In this study, we found 21 pairs of segmental duplicated genes which were distributed on different chromosomes (Fig. 1b). There were four pairs of tandem duplicated genes. The above results indicate that segmental duplication events participate in the expansion of NPF gene family in cassava.

Expression patterns of MeNPF genes under different nitrogen treatments

To determine the expression of MeNPF genes under different nitrogen conditions, we explored the expression patterns of MeNPF gene family based on RNA-seq data. As shown in Fig. 2, some MeNPF genes, such as MeNPF1.1, MeNPF2.5, MeNPF2.10, MeNPF5.18 and MeNPF8.5, were highly expressed in all three tissues, regardless of exogenous NO₃⁻ addition. While MeNPF1.2, MeNPF1.3, MeNPF1.4, MeNPF1.5, MeNPF2.2, MeNPF2.3, MeNPF2.4, MeNPF2.8, MeNPF2.12, MeNPF3.6, MeNPF4.1, MeNPF4.2, MeNPF4.8, MeNPF4.10, MeNPF4.11, MeNPF5.13, MeNPF6.4 MeNPF7.4 and MeNPF8.3 was not detected in all tissues under different nitrogen conditions. Some MeNPF genes showed preferred expression in a specific tissue at different expression levels, including MeNPF2.1, MeNPF4.4, MeNPF5.11, MeNPF6.2, MeNPF7.2, MeNPF7.3, MeNPF7.5. Compared to stems and leaves, MeNPF2.1, MeNPF4.4, MeNPF4.7, MeNPF7.3 and MeNPF7.5 genes were preferentially expressed in roots. Despite this, we still found two genes, MeNPF5.4 and MeNPF6.2, were preferentially and highly expressed in stems. MeNPF5.4 was not induced by nitrogen treatment, whereas MeNPF6.2 was up-regulated in high nitrogen treatment, suggesting these two genes might have diverse roles in nitrogen transportation and remobilization.

MeNPF5.4 and MeNPF6.2 are localized in the plasma membrane

To further characterize the function of MeNPF5.4 and MeNPF6.2, we investigated the subcellular localization of MeNPF5.4 and MeNPF6.2 in rice mesophyll protoplast using the eYFP fusion constructs driven by the 35S promoter. The signal of eYFP alone as a control was distributed throughout the protoplast, whereas both of the MeNPF5.4-eYFP and MeNPF6.2-eYFP fusion protein signals were observed in the plasma membrane, a pattern similar to the membrane-localized protein maker ZmCDPK7-mCherry driven by 35S promoter (Fig. 3). These results indicated that MeNPF5.4 and MeNPF6.2 proteins functioned in the plasma membrane.

MeNPF6.2 mediated NO₃⁻ uptake in transgenic rice

To evaluate the potential value of MeNPF5.4 and MeNPF6.2 in crop genetic improvement, we respectively introduced MeNPF5.4 and MeNPF6.2 into rice (Nipponbare) to produce the MeNPF5.4 and MeNPF6.2 overexpression (OE) lines. From the rice transgenic lines, we selected three pure homozygous lines with variable expressions for MeNPF5.4 and MeNPF6.2, respectively (Fig. 4a-b). As shown in Fig. 4a-b, both of MeNPF5.4-OE and MeNPF6.2-OE showed significantly higher expression compared with the wild type (WT, Nipponbare). To test the effect of MeNPF5.4 and MeNPF6.2 on nitrogen utilization, we investigated the NO₃⁻ flux in wild-type and transgenic rice roots using NMT. As shown in Fig. 4c, we observed that the NO₃⁻ flux differed significantly between WT and transgenic lines. MeNPF6.2-overexpressing line exhibited more significant NO₃⁻ influxes, whereas MeNPF5.4-overexpressing line exhibited a marked decrease in NO₃⁻ effluxes compared with that of the control (Fig. 4c). These findings indicated that both of MeNPF5.4 and MeNPF6.2 can enhance nitrogen utilization by decreasing nitrogen effluxes and increasing nitrogen influxes in rice, respectively.

Overexpressions of MeNPF6.2 and MeNPF5.4 impact rice physiological characteristics and grain yield

In order to investigate the phenotypic and physiological impacts of transgenic rice, we grow plants hydroponically for two weeks under NN and LN conditions. We measured the plant height, root and shoot weight, chlorophyll contents and the activities of the antioxidant enzymes. As shown in Fig. 5, plant height of MeNPF6.2-OE was lower compared with WT under NN (Fig. 5a). The shoot to root fresh weight ratio was higher in MeNPF6.2-OE lines under LN (Fig. 5b). Both shoot and root biomass showed no significant difference between wild type and MeNPF5.4-OE lines (Fig. S3). Total root length and the number of root tips were decreased in transgenic plants (Fig. S3). The POD activity of MeNPF5.4-OE and MeNPF6.2-OE plants was higher than wild type under both N conditions (Fig. 5c). Moreover, MeNPF6.2-OE plants displayed higher CAT activity (Fig. 5d). There was no significant difference in plant nitrogen content between WT and OE lines under NN conditions, whereas MeNPF6.2-OE line exhibited higher nitrogen accumulation (Fig. 5e). The chlorophyll-b content of OE lines was lower under NN condition, while the OE lines showed higher chlorophyll-b content compared with WT under LN condition (Fig. S3). The OE lines showed higher total chlorophyll content than WT under LN condition (Fig. S3).

To investigate the role of MeNPF5.4 and MeNPF6.2 in rice NUE, we examined agronomic traits related to yield for WT and transgenic lines. Compared to the wild type plants, MeNPF5.4-OE plants had significant increase (P < 0.01) in grain length and thousand-grain weight, whereas no significant differences were observed between wild type and the MeNPF6.2 overexpressing lines (Fig. 6a-d). As shown in Fig. 6g and Fig. 6h, the grain number and grain weight per panicle of MeNPF5.4-OE plants were significantly increased relative to that of the wild type. In addition, the protein and nitrogen accumulation of grain were increased in MeNPF6.2-OE plants (Fig. 6i-j). Overall, our results indicated that elevated MeNPF5.4 expression level significantly enhanced the total grain number and weight per panicle.

Overexpressions of MeNPF5.4 and MeNPF6.2 improve salt stress tolerance

Previous studies have revealed that some nitrate transporters play critical roles in the response of plants to abiotic stress (Zhang et al. 2018). As described above, our RNA-seq identified DEGs that were related to oxidoreductase activity (Fig. S4). To examine whether overexpression of MeNPF5.4 and MeNPF6.2 can improve stress tolerance of transgenic rice, we treated 1-week-old seedlings with 150 mM NaCl and 20% PEG respectively. As shown in Fig. 7, when treated with NaCl, the OE lines mostly still remained green and were more vigorous than wild type under both N conditions. Strikingly, MeNPF6.2-OE showed better performance specifically (Fig. 7a-b). We counted the wilting degree of the latest leaf of wild type and transgenic plants, and found that the leaf wilting degree of transgenic plants was significantly lower than that of the wild type, especially MeNPF6.2-OE plants (Fig. 7d). Compared with NN, we also found that transgenic lines were more tolerant to salt stress under LN. There were no significant differences between WT and OE lines under PEG treatment (data not shown). These findings suggested that MeNPF5.4 and MeNPF6.2 can enhance rice tolerance to salt stress. In particular, MeNPF5.4 coordinates both grain yield and salt stress.

Composition of the NPF gene family in cassava

As an important protein for plants to absorb and transport nitrate from environment, NPF gene family has been identified in more than 30 species. In cassava, NPF genes were previously identified but was not systematically analyzed (Leran et al. 2014). In this study, 72 MeNPF genes were identified based on the latest reference genome of cassava and could be divided into eight clades in the phylogenetic tree, similarly to the NPF genes in other plant species (Fig. 1a). In general, the expansion of gene family is mainly formed by several gene duplication forms including WGD, TD and SD (Panchy et al. 2016). Gene duplication can generate new biological functions, which is an important driving force for the evolution of species genomes. In order to further understand the gene duplication events of cassava MeNPF gene family, MCScanX was used to analyze the segmental duplication gene pairs and tandem duplication gene pairs of MeNPF genes. There were 21 pairs of segmental duplicated genes and 4 pairs of tandem duplicated genes (Fig. 1b), which suggests that segmental duplication may be an important driving force of evolution in the expansion of MeNPF gene family.

MeNPF5.4 influences nitrate influx, thus positively regulates rice grain size and weight

Crop yield is closely related to N utilization, and it mainly depends on nitrogen absorption by plants before flowering and nitrogen remobilization during seed maturation (Masclaux Daubresse et al. 2008; Kichey et al. 2007). With the gradual deepening of the study, more and more NPF genes related to NUE have been cloned in different species. Using NPF transporters to facilitate nitrate uptake and utilization is an important goal to increase crop yield and NUE. The indica type OsNRT1.1B was introduced into japonica varieties and improved yield and NUE of japonica rice (Zhang et al. 2019). Overexpressing OsNRT2.3b not only enhanced nitrate and iron uptake but also improved grain yield and NUE under varied N supplies in the field (Fan et al. 2016). The natural allele OsNPF6.1^HapB enhanced nitrogen uptake and NUE, thus improved rice yield under low nitrogen condition (Tang et al. 2019). The xylem in the stem participates in root-to-shoot flow of nitrate, hence the stem plays a vital role in nitrogen transportation. In rice, OsNPF2.2 contributes to root-to-shoot nitrate transport and vascular development. The osnpf2.2 mutants present low shoot:root nitrate ratios and exhibited retarded plant growth and abnormal vascular development (Li et al. 2015). In this study, we characterized MeNPF5.4 and MeNPF6.2 genes which were mainly expressed in stem but showed diverse nitrate induction patterns. It is noteworthy that the nitrate influx of OE-lines was higher than wild type (Fig. 4). Interestingly, overexpression of MeNPF5.4 significantly enhanced grain size and thousand-grain weight, whereas MeNPF6.2 overexpression showed no increase in grain size (Fig. 6). These findings indicate that MeNPF5.4 may have an important role in the process of nitrogen transportation, thereby further improving nitrogen utilization efficiency. One previous study demonstrated that elevated expression of OsNPF7.2 enhanced the rate of nitrate influx into root and participated in nitrate allocation from root to shoot, resulting in the increase of rice tiller number and grain yield (Wang et al. 2018a). For MeNPF6.2, although it also showed advantage in nitrogen uptake, there was no difference in the grain compared to WT. This could be due to some unknown reasons that might make it defective in nitrogen transport. AtNPF6.2 is preferentially expressed in leaves and participates in the accumulation of nitrate in the petiole(Chiu et al. 2004). AtNPF6.2 is not regulated by nitrate. As a homologous gene of AtNPF6.2, MeNPF6.2 was induced by nitrate. This difference may be due to their gene structure and species specificity.

MeNPF5.4 and MeNPF6.2 positively enhance rice salinity stress tolerance

It was proposed that some nitrate transporters were in response of plants to abiotic stresses. For example, the Arabidopsis mutant of AtNPF6.3 gene (chl mutant) had reduced stomatal opening and reduced transpiration rates, thus resulted in drought tolerance (Guo et al. 2003). AtNPF2.3 contributes to NO₃⁻ translocation from roots to shoots under salt stress (Taochy et al. 2015). In the present study, we found that transgenic plants enhanced rice salt stress tolerance. Intriguingly, we observed that overexpression of MeNPF5.4 not only increased grain size and weight but also enhanced rice salt tolerance (Fig. 7). These findings indicated that MeNPF5.4 plays a positive role in coordinating grain growth and rice salinity stress. Antioxidant enzymes play a vital part in stress tolerance. Determination of antioxidant enzyme activity showed that MeNPF6.2-OE had higher POD and CAT activity (Fig. 6i-j), thus improved salt stress tolerance. Since overexpression of MeNPF6.2 improved salt stress tolerance, its growth and development were slightly inhibited, and the grain size did not differ significantly between WT and OE lines (Fig. 6, 7). This also revealed the dynamic balance between plant growth and defense in order to maximize survival efficiency.

In conclusion, this study demonstrated that MeNPF5.4 acts as a pivotal transporter by coordinating both rice grain growth and salinity stress, can significantly improve the grain size and salt tolerance, and is a promising candidate gene for improving production and stress tolerance of the crops.

Acknowledgements

The authors extend their appreciation to the support from Chinese Academy of Tropical Agricultural Sciences.

Author contribution

Li Ji: Writing - original draft, Data curation. Linhu Song: Investigation, Formal analysis. Liangping Zou, Shi Li: Data curation, Investigation, Resources. Runcong Zhang, Jingyu Yang, Changyu Wang, Yan Zhang, Xingmei Wang, Liu Yun, Xiao Qu, Xiang Ji: Data curation, Vadidation. Lanjie Zheng, Mengbin Ruan, Xu Zheng: Project administration, Writing - review & editing, Funding acquisition.

Funding

This work was supported by Hainan Provincial Natural Science Foundation of China (2019RC303), the Major Science and Technology plan of Hainan Province (ZDKJ2021012), the Startup Grant of Henan Agricultural University (No. 30601733), the State Key Laboratory for Managing Biotic and Chemical Treats to the Quality and Safety of Agro-products (2021DG700024-KF202218), the Henan Province Science and Technology Attack Project (222102110465) and the State Key Laboratory for Managing Biotic and Chemical Treats to the Quality and Safety of Agro-products (No. KF20200107).

Data availability

The data generated or used in this study are included in this article and its supplementary materials.

Conflict of interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Ethics approval All authors approved the submission.

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You are reading this latest preprint version

Cassava Nitrate Transporter NPF5.4 promotes both yield potential and salt tolerance in rice

Status:

Version 1

Abstract

Figures

Introduction

Materials And Methods

Plant materials and growth conditions

Identification and phylogenetic analysis of NPF genes in cassava

Chromosomal localization and gene duplication of MeNPF gene family

Expression analysis of MeNPF gene family from RNA-seq data

Subcelluar localization of MeNPF5.4 and MeNPF6.2

RT-qPCR analysis

Measurement of net NO₃⁻ flux

Physiological measurements of transgenic rice lines

Statistical analysis

Results

Identification and gene duplication of NPF genes in cassava

Expression patterns of MeNPF genes under different nitrogen treatments

MeNPF5.4 and MeNPF6.2 are localized in the plasma membrane

MeNPF6.2 mediated NO₃⁻ uptake in transgenic rice

Overexpressions of MeNPF6.2 and MeNPF5.4 impact rice physiological characteristics and grain yield

Overexpressions of MeNPF5.4 and MeNPF6.2 improve salt stress tolerance

Discussion

Composition of the NPF gene family in cassava

MeNPF5.4 influences nitrate influx, thus positively regulates rice grain size and weight

MeNPF5.4 and MeNPF6.2 positively enhance rice salinity stress tolerance

Declarations

References

Supplementary Files

Status:

Version 1

Cassava Nitrate Transporter NPF5.4 promotes both yield potential and salt tolerance in rice

Status:

Version 1

Abstract

Figures

Introduction

Materials And Methods

Plant materials and growth conditions

Identification and phylogenetic analysis of NPF genes in cassava

Chromosomal localization and gene duplication of MeNPF gene family

Expression analysis of MeNPF gene family from RNA-seq data

Subcelluar localization of MeNPF5.4 and MeNPF6.2

RT-qPCR analysis

Measurement of net NO3− flux

Physiological measurements of transgenic rice lines

Statistical analysis

Results

Identification and gene duplication of NPF genes in cassava

Expression patterns of MeNPF genes under different nitrogen treatments

MeNPF5.4 and MeNPF6.2 are localized in the plasma membrane

MeNPF6.2 mediated NO3− uptake in transgenic rice

Overexpressions of MeNPF6.2 and MeNPF5.4 impact rice physiological characteristics and grain yield

Overexpressions of MeNPF5.4 and MeNPF6.2 improve salt stress tolerance

Discussion

Composition of the NPF gene family in cassava

MeNPF5.4 influences nitrate influx, thus positively regulates rice grain size and weight

MeNPF5.4 and MeNPF6.2 positively enhance rice salinity stress tolerance

Declarations

References

Supplementary Files

Status:

Version 1

Measurement of net NO₃⁻ flux

MeNPF6.2 mediated NO₃⁻ uptake in transgenic rice