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. 2024 Dec 17;30(12):1983–1999. doi: 10.1007/s12298-024-01541-7

Genome-wide identification, evolution and expression analysis unveil the role of Dendrocalamus farinosus NRT genes in nitrogen utilization and nitrogen allocation

Boya Wang 1, Siyuan Ren 1, Sen Chen 1, Suwei Hao 1, Gang Xu 1, Shanglian Hu 1,✉, Ying Cao 1,✉
PMCID: PMC11685372  PMID: 39744329

Abstract

The rapid growth of Bamboo made the uptake and allocation of nitrogen much important. Nitrate is the main form that plant utilized nitrogen by nitrate transporters (NRTs) as well as ammonium salt. In this study, we identified 155 DfNRT genes which mapped to 32 chromosomes out of 35 chromosomes in Dendrocalamus farinosus. Collinearity analysis showed most NRT genes in D. farinosus paired with NRT genes in D. farinosus and P. edulis, which another two sequenced woody bamboo species, and the divergence was similar to the woody bamboo whole-genome duplication event. Through the 15N-nitrate trace analysis, we found that the nitrogen absorbed by roots in D. farinosus was preferentially distributed to above-ground parts, especially transported to leaves. DfNPF2.13 and DfNPF6.9 exhibited higher expression in leaf, and upregulated with extra N supply, suggesting they might be participating in N allocation between leaves in D. farinosus. This study provides a foundation for understanding the mechanism of nitrate transport and distribution in bamboo, and provide valuable information for improving bamboo nitrate absorption and promoting efficient nitrogen utilization.

Supplementary Information

The online version contains supplementary material available at 10.1007/s12298-024-01541-7.

Keywords: Dendrocalamus farinosus, Nitrate transporter, 15N trace, Gene family, Evolutionary relationships

Introduction

Nitrogen (N) is an essential macronutrient for sustaining plant growth and development (Hawkesford et al. 2023). N is commonly taken up from the soil in one of two inorganic forms: ammonium and nitrate (Glass et al. 2002; S. Guo et al. 2007). Of the two forms, nitrate is the main nitrogen source for most plants, which is absorbed by plant roots through nitrate transporters (NRTs) by sensing external nitrogen signals and then translocated in plants (Glass 2009; Wang et al. 2012a). Plant roots have developed high-affinity transport system (HATS) and low-affinity transport system (LATS) to cope with low (< 1 mM) or high (> 1 mM) concentrations of nitrate in soil, respectively (Dechorgnat et al. 2011; Wang et al. 2012a). The transporters protein involved in those system were segregated.

With evolution and gene duplication, most plants have a large gene family wihch encode NRTs. Usually, NRT family is composed of NRT1/PTR (nitrate/peptide transporters, also known as nitrate transporter1/ peptide transporter family, NPF), NRT2, and NRT3 members, which play multifunctional roles in nitrate uptake and transportation from soil to plant (Dechorgnat et al. 2011; O’Brien et al. 2016). In Arabidopsis genome, 62 NRT genes were identified, of which 53 members belong to NRT1/PTR subfamily, 7 and 2 members belong to NRT2 and NRT3 family, respectively (Okamoto et al. 2003). NRT1 proteins were thought to normally function as components of the LATS, which was activated in high nitrate concentrations (Tsay et al. 2007). The first identified NRT1 gene in Arabidopsis was AtNRT1.1 (CHL1/AtNPF6.3), which was a dual-affinity nitrate transporter in both low- and high-nitrate concentrations, and was modulated when response to fluctuations of nitrate level (Liu et al. 1999). NPF7.2/NRT1.8 and NPF7.3/NRT1.5 were shown to be involved in nitrate uptake by the roots and root-to-shoot nitrate transport in Arabidopsis (Cui et al. 2019), while another root transporter NPF2.3 was proved contributing to nitrate translocation to shoots under salt stress (Taochy et al. 2015). In addition, some NRT1 members, such as AtNPF6.2/AtNRT1.4, AtNPF2.13/AtNRT1.7, and AtNPF1.2/AtNRT1.11, were demonstrated to function in leaf nitrate homeostasis. For example, AtNRT1.7 was reported to participate in nitrate transport from old leaves to new leaves (Fan et al. 2009). NRT2 and NRT3 members were commonly joining with each other in HATS to implicate in nitrate absorption and remobilization under N starvation stress in many plant species. (Okamoto et al. 2006).

In rice, members of NRT1 gene family also had an important role of nitrogen use in the field. Two NRT1 genes were identified that were homologous to AtNRT1.1 in rice with different functions (Plett et al. 2010). OsNRT1.1A/OsNPF6.3 is plastid localized and induced by ammonium (Wang et al. 2018a).  Protein encoded by OsNRT1.1B gene was localized to cell membrane, and sensed an external nitrate signal, and contributed for grain yield and nitrogen use efficiency (NUE) in rice (Hu et al. 2015). There were two more OsNRT1 subfamily genes involved in nitrate transport and encoded low-affinity nitrate transporter. OsNPF2.2/OsPTR2 that function in nitrate transport from root to stem and vascular development (Li et al. 2015), whiling OsNPF4.5 participated in nitrogen acquisition by a conserved mycorrhizal pathway (Wang et al. 2020). With the rapid accumulation of plant genome data, NRT genes were characterized in many more plants, such as poplar (Bai et al. 2013), cucumber (Migocka et al. 2013), apple (Wang et al. 2018b), sugarcane (Wang et al. 2019) and spinach (Wang et al. 2021).

Bamboo belongs to the subfamily Bambusoideae of Poaceae family, and is widely distributed in tropical and subtropical zones in Asia, Africa and America (Soreng et al. 2015; Yen 2016). Woody bamboos have tree-like lignified culms, and exhibit the property of rapid growth and high output, therefore they are considered as one of the most important lignocellulose feedstocks in biofuel industry (Engler et al. 2012; Nayak & Mishra 2016). The fast growth of bamboo shoot and the potential ecosystem benefits including carbon sequestration, soil and water conservation (Nath et al. 2015) are highly concerned. Bamboo needs high amount of nitrogen from the outside to support its rapid growth, therefore the availability of nitrogen in the soil is an important factor for bamboo morphogenesis and biomass (McIntyre 1987; Long et al. 2020).

In recent years, several bamboo speices were genome sequenced and all of them exhibit obvious variation in ploidy levels, comprising diploid herbaceous (Olyra latifolia and Raddia guianensis, HH), tetraploid woody lineages (G. angustifolia BBCC, Phyllostachys edulis CCDD), and hexaploid woody lineages (Bonia amplexicaulis, Dendrocalamus latiflorus, AABBCC) (Guo et al. 2019; Peng et al. 2013; Zheng et al. 2022). Dendrocalamus farinosus is a sympodial woody bamboo and considered as a preferable wood substitute for chemical pulping because of its high cellulose content and good fiber quality (Du et al. 2021). In our previous research, we generated a chromosome-scale genome of D. farinosus using the PacBio Sequel platform and Hi-C chromosome conformation capture, and found that the D. farinosus genome was hexaploid AABBCC (2n = 6x = 70), corresponded well with the closely related species D. latiflorus (Zheng et al. 2022). As perennial sympodial bamboo, D. farinosus mostly grow on mountains whose nitrogen nutrition are hard to get replenished without applying extra nitrogen fertilizer. Thus, in the present study, a genome-wide identification and evolution analysis of NRT genes in D. farinosus was performed. Moreover, the expression profiles of DfNRTs in response to different nitrate supplies were detected. Our results present a comprehensive characterization of DfNRT genes, and offer the foundation for the complex genetic dissection of nitrate transport system in D. farinosus.

Materials and methods

Identification and phylogenetic analysis of DfNRTs

The sequences of NRTs in Arabidopsis and rice were obtained from UniProt (https://www.uniprot.org/) and National Center for Biotechnology Information (https://www.ncbi.nlm.nih.gov/). Arabidopsis NRTs were used as the initial query set to search against the D. farinosus genome database (http://150.158.144.134/DFA-genome/) with e value < 1E-20. The candidate sequences removing the redundant and too short sequences (CDS length < 300 bp) were further used for the phylogenetic analysis (Maximum likelihood method, bootstrap value n = 1000). The phylogenetic tree was further annotated using Interactive Tree of Life (https://itol.embl.de). ExPASy ProtoParam (https://web.expasy.org/protparam/) was used to predict the number and composition of amino acids, relative molecular weight (MW), theoretical isoelectric point (pI) of NRT1 and NRT2 proteins. DeepTMHMM version 1.0.13 (https://dtu.biolib.com/DeepTMHMM) was used to predict the putative trans-membrane (TM) spanning regions. The core domains were predicted using NCBI conserved domains database (https://www.ncbi.nlm.nih.gov/Structure/bwrpsb/bwrpsb.cgi) and gene structures were constructed using Gene Structure Display Server 2.0 (http://gsds.cbi.pku.edu.cn/). DfNRT protein sequences were annotated using the eggNOG-mapper tool (http://eggnog-mapper.embl.de/) in D. farinosus genome and the annotated results were visualized using the TBtools program (https://github.com/CJ-Chen/TBtools). The DfNRT sequences described above are available in the supplementary Table S1.

Synteny analysis and calculation of Ka/Ks values of NRT genes

Multiple Collinearity Scan Toolkit (MCScanX) with default parameters was used to analyze the tandem repeats and segmental duplication events of the DfNRT genes family in D. farinosus genome and NRT syntenic pairs between the bamboo and rice genome (https://www.ncbi.nlm.nih.gov/assembly/GCF_001433935.1/). The Intra-species and inter-species collinearity analysis were drawn by TBtools program. The synonymous (Ks) and non-synonymous (Ka) nucleotide substitutions rate ratios between the paralogs NRT pairs of bamboo were calculated according to the TBtools program. The results of synonymous nucleotide substitutions were visualized using the R program (https://www.r-project.org/).

RNA sequencing

In order to obtain conveniently tissues samples from the below ground and aboveground parts of bamboo, one-year-old D. farinosus plantlet potted in the growing chamber were used for RNA sequencing after 30 days of new shoots germinated from rhizome lateral buds on Aug 2021. Root and fully expanded leaf from maternal plant, rhizome neck (a joint of rhizome between maternal plant and new shoot), as well as the basal 4 th internode and node of new shoots were sampled. Each sample had three biological duplications. Total RNA was extracted using the E.Z.N.A. ® Plant RNA Kit (Omega), and the quality and quantity of RNA were detected using a NanoDrop 2000 spectrophotometer. A total of 15 samples were subjected to the Illumina HiSeq 2500 platform (Illumina, Beijing, China). Data of RNA sequencing is available at Table S2. Heatmap and GO enrichment of the DfNPF genes were constructed based on this RNA-seq data.

15N isotope tracing and nitrate treatments

In the current research, one-year-old D. farinosus plantlet potted in growing chamber were used to 15N isotope tracing and nitrate treatments. The cultivation condition was 16 h light /8 h dark, and the temperature was 23 ~ 25 °C. The 15N-sodium nitrate with 99 atom% 15N was purchased from Wuhan Newradar Special Gas Co., Ltd (Wuhan, China). Plantlet per plot were added with 15N-sodium nitrate solution of 20 mg in 500 mL deionized water. Each experiment was repeated three times. After 15 days of 15N treatment each, 4 samples from maternal plants including root, leaf, branch and rhizome were collected for analysis of 15N content. At the same time, samples from new-emerged shoots also were detected, including underground samples (rhizome and rhizome neck) and aboveground parts samples (leaf, internode and node); among them, aboveground parts samples were collected from base and upper part of the new-born shoots, respectively. These plant components were over-dried (60°C for 72 h), ground and analyzed for total 15N enrichment with the Isoprime 100 mass spectrometer instrument (Elementar Analysensysteme GmbH Inc., Germany).

Nitrate treatments were performed according the modified method reported by Long et al. (2020). The following three treatments were set up which were: the control group (CK) represent no extra fertilizer, N1 and N2 applied 0.1 mol N-NO3 and 0.14 mol N–NO3 per pot, respectively. In detail, N1 treatment was performed with supply of 7.737 g Ca(NO3)2, 0.874 g Ca(H2PO4)2 and 0.499 g K2SO4 per pot, while N2 treatment was supplied with 11.605 g Ca(NO3)2, 1.311 g Ca(H2PO4)2 and 0.749 g K2SO4. After 15 days of N treatment, 4 samples from new-emerged shoots including root, leaf, internode 4th (base) and internode 10th (upper) aboveground were collected. Each sample included three independent biological replicates. All the samples were cleaned by distillation, then frozen immediately in liquid N and stored at − 80 °C.

Total RNA was extracted as described before and then the cDNA was synthesized using the PrimeScript™ RT Master Mix (TAKARA). qRT-PCR was performed using SYBR® Premix Ex Taq™ II (TAKARA) and gene-specific primers for qRT-PCR are listed in Table S3. The values were calculated via the 2−ΔΔCt method.

Results

Genome-wide identification of D. farinosus NRT genes

By HMM search against D. farinosus genome and BLASTP analysis, 155 DfNRT genes were identified in the current research (Table 1). According to the cluster analysis and comparison with Arabidopsis and rice in evolution, these DfNRT genes were divided into 3 subfamilies, 147 DfNRT1(DfNPFs), 5 DfNRT2 and 3 DfNRT3 (Fig. 1). Further, gene characteristics, the length of protein sequences, protein molecular weight (MW), isoelectric point (pI), and gene locus in chromosome were analyzed (Table 1). Since NRT1 and PTR (peptide transporter) members have high sequence homology, they are placed in NPF (NRT1 / PTR) family (Tsay et al. 2007). Plant NPF proteins can transport nitrate nitrogen, polypeptides, abscisic acid (ABA) and other substrates, which can be divided into 8 subfamilies (Wittgenstein et al. 2014; Corratgé-Faillie & Lacombe 2017). D.farinosus was found to contain all of NPF subfamilies; especially for NPF8 subfamily, it contained 45 members, higher than other NPF families that only having 4 ~ 27 members (Table 1). Bioinformatics analysis showed that the MW of these DfNRT proteins ranged from 20.81 kDa (DfNRT3.2) to 116.93 kDa (DfNPF4.11), and the pI ranged from 4.92 (DfNPF8.21) to 10.33 (DfNPF2.8). Among 155 DfNRT proteins, DfNPF4.11 was the largest one with 1067 aa and 21 transmembrane domains, whereas NRT3 subfamily with three members were carrying the smallest protein in range of 200 amino acid (aa) with only 1 or 2 tans-membrane domains.

Table 1.

Characterization of NRT genes family in Dendrocalamus farinosus

Gene Name Gene identifier pI MW (kDa) Amino Acid Length (aa) Coding Sequence Length (bp) Gene Locus No. of transmembrane domains Subcellular location
DfNPF1.1 DfaA01G009870.1 8.81 62.29 569 1707 ChrA01 8 Plasma membrane
DfNPF1.2 DfaB01G009620.1 9.16 63.98 585 1755 ChrB01 9 Plasma membrane
DfNPF1.3 DfaB01G009630.1 8.48 69.50 644 1932 ChrB01 9 Plasma membrane
DfNPF1.4 DfaC10G002050.1 9 64.94 600 1800 ChrC10 10 Plasma membrane
DfNPF2.1 DfaA02G000120.1 9.44 58.84 541 1623 ChrA02 9 Plasma membrane
DfNPF2.2 DfaA05G004600.1 8.4 66.05 616 1848 ChrA05 11 Plasma membrane
DfNPF2.3 DfaA08G020510.1 9.08 64.31 591 1773 ChrA08 8 Plasma membrane
DfNPF2.4 DfaA10G000320.1 8.91 64.35 584 1752 ChrA10 10 Plasma membrane
DfNPF2.5 DfaB02G000090.1 8.73 62.37 572 1716 ChrB02 8 Plasma membrane
DfNPF2.6 DfaB02G010620.1 8.99 60.98 571 1713 ChrB02 11 Plasma membrane
DfNPF2.7 DfaB08G020550.1 9.15 64.03 587 1761 ChrB08 8 Plasma membrane
DfNPF2.8 DfaB10G000200.1 10.33 64.31 592 1776 ChrB10 6 Plasma membrane
DfNPF2.9 DfaC01G004560.1 9.09 63.57 579 1737 ChrC01 9 Vacuole
DfNPF2.10 DfaC01G006800.1 9.23 62.05 580 1740 ChrC01 10 Plasma membrane
DfNPF2.11 DfaC04G016300.1 8.95 65.95 607 1821 ChrC04 11 Plasma membrane
DfNPF2.12 DfaC04G017510.1 9.52 66.11 604 1812 ChrC04 12 Plasma membrane
DfNPF2.13 DfaC08G021460.1 9.07 67.44 617 1851 ChrC08 12 Plasma membrane
DfNPF2.14 DfaC11G013980.1 8.72 64.21 588 1764 ChrC11 8 Plasma membrane
DfNPF3.1 DfaA02G007910.1 9.44 65.05 596 1788 ChrA02 6 Plasma membrane
DfNPF3.2 DfaA02G007930.1 8.43 62.42 577 1731 ChrA02 10 Plasma membrane
DfNPF3.3 DfaA02G007940.1 9.39 62.06 569 1707 ChrA02 8 Plasma membrane
DfNPF3.4 DfaA02G007950.1 7.66 64.21 593 1779 ChrA02 10 Plasma membrane
DfNPF3.5 DfaA02G007960.1 8.33 65.20 602 1806 ChrA02 9 Plasma membrane
DfNPF3.6 DfaA02G011040.1 8.56 65.10 601 1803 ChrA02 9 Plasma membrane
DfNPF3.7 DfaA11G011890.1 8.63 65.03 596 1788 ChrA11 10 Plasma membrane
DfNPF3.8 DfaB02G008380.1 6.48 64.44 588 1764 ChrB02 6 Plasma membrane
DfNPF3.9 DfaB02G008390.1 9.04 64.93 598 1794 ChrB02 7 Plasma membrane
DfNPF3.10 DfaB02G011380.1 8.87 65.59 608 1824 ChrB02 9 Plasma membrane
DfNPF3.11 DfaC02G007340.1 9.4 65.09 595 1785 ChrC02 8 Plasma membrane
DfNPF3.12 DfaC02G007360.1 8.89 64.95 603 1809 ChrC02 9 Plasma membrane
DfNPF3.13 DfaC11G003610.1 8.75 65.18 597 1791 ChrC11 10 Plasma membrane
DfNPF4.1 DfaA01G010620.1 9.21 60.17 545 1635 ChrA01 12 Plasma membrane
DfNPF4.2 DfaA03G012390.1 8.48 62.51 593 1779 ChrA03 12 Plasma membrane
DfNPF4.3 DfaA04G008350.1 9.48 53.53 508 1524 ChrA04 8 Plasma membrane
DfNPF4.4 DfaA04G010870.1 8.82 58.36 539 1617 ChrA04 11 Plasma membrane
DfNPF4.5 DfaA11G006110.1 8.84 63.78 586 1758 ChrA11 12 Plasma membrane
DfNPF4.6 DfaA12G005260.1 8.54 67.15 628 1884 ChrA12 12 Plasma membrane
DfNPF4.7 DfaA12G006910.1 8.38 63.33 579 1737 ChrA12 11 Plasma membrane
DfNPF4.8 DfaA12G006980.1 8.83 63.67 583 1749 ChrA12 11 Plasma membrane
DfNPF4.9 DfaB03G012070.1 8.08 62.90 596 1788 ChrB03 12 Plasma membrane
DfNPF4.10 DfaB04G011090.1 8.74 62.40 579 1737 ChrB04 12 Plasma membrane
DfNPF4.11 DfaB04G013540.1 8.53 116.93 1067 3201 ChrB04 21 Plasma membrane
DfNPF4.12 DfaB10G006890.1 8.04 62.79 572 1716 ChrB10 12 Plasma membrane
DfNPF4.13 DfaB11G006410.1 8.94 63.71 589 1767 ChrB11 12 Plasma membrane
DfNPF4.14 DfaB12G004400.1 8.84 65.74 617 1851 ChrB12 12 Plasma membrane
DfNPF4.15 DfaB12G005330.1 8.72 64.54 587 1761 ChrB12 12 Plasma membrane
DfNPF4.16 DfaC04G010250.1 8.35 66.79 607 1821 ChrC04 11 Plasma membrane
DfNPF4.17 DfaC04G010260.1 8.46 61.65 561 1683 ChrC04 12 Plasma membrane
DfNPF4.18 DfaC08G004860.1 9.22 63.17 581 1743 ChrC08 12 Plasma membrane
DfNPF4.19 DfaC10G007020.1 9.05 67.11 624 1872 ChrC10 12 Plasma membrane
DfNPF4.20 DfaC10G007750.1 8.56 62.47 568 1704 ChrC10 11 Plasma membrane
DfNPF4.21 DfaC10G007760.1 8.96 64.52 586 1758 ChrC10 11 Plasma membrane
DfNPF4.22 DfaC10G010560.1 6.91 62.67 573 1719 ChrC10 12 Plasma membrane
DfNPF4.23 Dfa0G006350.1 9.05 66.36 609 1827 Contig00179 11 Plasma membrane
DfNPF5.1 DfaA01G005150.1 9.37 57.61 534 1602 ChrA01 12 Nucleus
DfNPF5.2 DfaA02G007180.1 8.99 61.19 571 1713 ChrA02 8 Plasma membrane
DfNPF5.3 DfaA04G019440.1 6.45 58.11 545 1635 ChrA04 10 Plasma membrane
DfNPF5.4 DfaA05G006320.1 9.2 63.21 578 1734 ChrA05 9 Plasma membrane
DfNPF5.5 DfaA08G002200.1 8.96 65.88 598 1794 ChrA08 10 Plasma membrane
DfNPF5.6 DfaA08G026640.1 9.31 47.11 431 1293 ChrA08 5 Plasma membrane
DfNPF5.7 DfaA09G004530.1 9.13 66.51 605 1815 ChrA09 10 Plasma membrane
DfNPF5.8 DfaA09G004540.1 8.68 65.21 599 1797 ChrA09 12 Plasma membrane
DfNPF5.9 DfaA11G012490.1 8.34 56.93 515 1545 ChrA11 10 Plasma membrane
DfNPF5.10 DfaB01G008400.1 8.9 59.21 545 1635 ChrB01 11 Plasma membrane
DfNPF5.11 DfaB01G008420.1 7.53 55.44 507 1521 ChrB01 10 Plasma membrane
DfNPF5.12 DfaB02G010680.1 8.86 68.19 614 1842 ChrB02 10 Plasma membrane
DfNPF5.13 DfaB08G026660.1 9.01 63.54 583 1749 ChrB08 12 Plasma membrane
DfNPF5.14 DfaB09G004350.1 9.23 66.54 605 1815 ChrB09 10 Plasma membrane
DfNPF5.15 DfaB09G004370.1 8.93 62.77 580 1740 ChrB09 10 Plasma membrane
DfNPF5.16 DfaC01G009880.1 8.55 54.13 504 1512 ChrC01 10 Plasma membrane
DfNPF5.17 DfaC01G009890.1 9.03 54.12 504 1512 ChrC01 9 Plasma membrane
DfNPF5.18 DfaC01G009900.1 8.75 57.07 535 1605 ChrC01 8 Plasma membrane
DfNPF5.19 DfaC01G009910.1 9.09 56.09 516 1548 ChrC01 8 Plasma membrane
DfNPF5.20 DfaC02G006680.1 8.34 57.42 536 1608 ChrC02 11 Plasma membrane
DfNPF5.21 DfaC02G009030.1 9.28 67.31 610 1830 ChrC02 10 Plasma membrane
DfNPF5.22 DfaC08G005940.1 9.05 48.21 436 1308 ChrC08 7 Plasma membrane
DfNPF5.23 DfaC09G006950.1 8.9 67.51 620 1860 ChrC09 10 Plasma membrane
DfNPF5.24 DfaC11G004280.1 6.33 56.88 516 1548 ChrC11 10 Plasma membrane
DfNPF5.25 DfaC11G019560.1 9.45 63.98 588 1764 ChrC11 10 Plasma membrane
DfNPF5.26 Dfa0G017230.1 8.44 57.85 538 1614 Contig00517 11 Plasma membrane
DfNPF5.27 Dfa0G076280.1 8.34 56.93 515 1545 Contig02216 10 Plasma membrane
DfNPF6.1 DfaA03G013450.1 8.56 64.42 601 1803 ChrA03 10 Plasma membrane
DfNPF6.2 DfaA04G009580.1 8.82 62.35 590 1770 ChrA04 11 Plasma membrane
DfNPF6.3 DfaA07G003050.1 9 65.24 604 1812 ChrA07 11 Plasma membrane
DfNPF6.4 DfaA08G000280.1 9.51 60.85 568 1704 ChrA08 11 Plasma membrane
DfNPF6.5 DfaA09G001220.1 8.21 63.99 595 1785 ChrA09 11 Plasma membrane
DfNPF6.6 DfaB01G018430.1 9.07 63.46 587 1761 ChrB01 9 Plasma membrane
DfNPF6.7 DfaB03G013370.1 9.31 64.84 602 1806 ChrB03 11 Plasma membrane
DfNPF6.8 DfaB06G011960.1 9.18 65.13 602 1806 ChrB06 11 Plasma membrane
DfNPF6.9 DfaB07G002170.1 8.79 65.08 604 1812 ChrB07 11 Plasma membrane
DfNPF6.10 DfaB09G001090.1 8.57 63.22 589 1767 ChrB09 12 Plasma membrane
DfNPF6.11 DfaC01G000350.1 9.06 69.37 628 1884 ChrC01 10 Plasma membrane
DfNPF6.12 DfaC01G021920.1 8.71 62.82 581 1743 ChrC01 10 Plasma membrane
DfNPF6.13 DfaC09G000230.1 9.35 62.16 580 1740 ChrC09 11 Plasma membrane
DfNPF6.14 DfaC09G003700.1 8.57 63.94 596 1788 ChrC09 12 Plasma membrane
DfNPF6.15 Dfa0G070670.1 9.07 63.46 587 1761 Contig02063 9 Plasma membrane
DfNPF7.1 DfaA03G018750.1 6.12 66.17 607 1821 ChrA03 10 Plasma membrane
DfNPF7.2 DfaA03G019950.1 6.23 63.93 584 1752 ChrA03 11 Plasma membrane
DfNPF7.3 DfaC03G016260.1 8.73 69.16 639 1917 ChrC03 12 Plasma membrane
DfNPF7.4 DfaC03G017400.1 6.6 65.42 596 1788 ChrC03 11 Plasma membrane
DfNPF7.5 DfaC09G000100.1 7.21 66.41 607 1821 ChrC09 11 Plasma membrane
DfNPF7.6 DfaC11G002430.1 8.26 64.66 584 1752 ChrC11 11 Plasma membrane
DfNPF8.1 DfaA01G019140.1 5.42 62.35 582 1746 ChrA01 8 Plasma membrane
DfNPF8.2 DfaA01G029690.1 6.8 63.90 581 1743 ChrA01 10 Plasma membrane
DfNPF8.3 DfaA03G019140.1 7.1 63.77 581 1743 ChrA03 10 Plasma membrane
DfNPF8.4 DfaA03G019150.1 8.44 64.24 581 1743 ChrA03 8 Plasma membrane
DfNPF8.5 DfaA04G015260.1 6.33 62.46 580 1740 ChrA04 10 Plasma membrane
DfNPF8.6 DfaA04G015270.1 5.04 61.14 564 1692 ChrA04 9 Plasma membrane
DfNPF8.7 DfaA05G001200.1 6.7 61.13 563 1689 ChrA05 9 Plasma membrane
DfNPF8.8 DfaA05G001210.1 7.96 64.88 599 1797 ChrA05 10 Plasma membrane
DfNPF8.9 DfaA08G006590.1 8.01 50.59 458 1374 ChrA08 7 Plasma membrane
DfNPF8.10 DfaA08G006600.1 5.7 64.65 587 1761 ChrA08 8 Plasma membrane
DfNPF8.11 DfaA08G014650.1 5.59 67.53 621 1863 ChrA08 8 Plasma membrane
DfNPF8.12 DfaA08G021770.1 5.04 65.13 592 1776 ChrA08 10 Plasma membrane
DfNPF8.13 DfaA09G000170.1 8.66 62.87 581 1743 ChrA09 10 Plasma membrane
DfNPF8.14 DfaA09G000180.1 7.53 61.20 563 1689 ChrA09 9 Plasma membrane
DfNPF8.15 DfaA09G009090.1 8.81 65.25 598 1794 ChrA09 11 Plasma membrane
DfNPF8.16 DfaA09G009130.1 6.23 63.63 583 1749 ChrA09 12 Plasma membrane
DfNPF8.17 DfaA09G009140.1 6.33 62.58 578 1734 ChrA09 12 Plasma membrane
DfNPF8.18 DfaB01G003580.1 7.59 60.04 554 1662 ChrB01 10 Plasma membrane
DfNPF8.19 DfaB01G030130.1 7.11 63.34 578 1734 ChrB01 10 Plasma membrane
DfNPF8.20 DfaB03G018460.1 8.3 53.84 483 1449 ChrB03 8 Plasma membrane
DfNPF8.21 DfaB03G018470.1 4.92 61.90 576 1728 ChrB03 10 Plasma membrane
DfNPF8.22 DfaB04G018310.1 5.4 60.26 554 1662 ChrB04 12 Plasma membrane
DfNPF8.23 DfaB08G007520.1 6.67 67.08 606 1818 ChrB08 9 Plasma membrane
DfNPF8.24 DfaB08G007530.1 6.99 63.65 572 1716 ChrB08 10 Plasma membrane
DfNPF8.25 DfaB09G010150.1 6.46 63.14 575 1725 ChrB09 12 Plasma membrane
DfNPF8.26 DfaB09G010570.1 7.55 62.72 574 1722 ChrB09 11 Plasma membrane
DfNPF8.27 DfaB11G001140.1 6.41 63.43 584 1752 ChrB11 12 Plasma membrane
DfNPF8.28 DfaC01G001310.1 8.24 63.75 581 1743 ChrC01 10 Plasma membrane
DfNPF8.29 DfaC01G004060.1 5.54 65.66 598 1794 ChrC01 10 Plasma membrane
DfNPF8.30 DfaC01G004070.1 8.05 60.40 553 1659 ChrC01 12 Plasma membrane
DfNPF8.31 DfaC01G022870.1 5.43 62.17 582 1746 ChrC01 10 Plasma membrane
DfNPF8.32 DfaC03G016570.1 5.88 63.39 586 1758 ChrC03 10 Plasma membrane
DfNPF8.33 DfaC04G014790.1 5.95 62.36 574 1722 ChrC04 11 Plasma membrane
DfNPF8.34 DfaC05G003820.1 7.49 62.87 574 1722 ChrC05 12 Plasma membrane
DfNPF8.35 DfaC05G014130.1 7.05 64.20 592 1776 ChrC05 10 Plasma membrane
DfNPF8.36 DfaC05G014260.1 7.05 64.24 592 1776 ChrC05 10 Plasma membrane
DfNPF8.37 DfaC07G000960.1 8.05 63.82 588 1764 ChrC07 8 Plasma membrane
DfNPF8.38 DfaC07G000980.1 5.95 61.99 568 1704 ChrC07 12 Plasma membrane
DfNPF8.39 DfaC07G001000.1 6.6 63.32 578 1734 ChrC07 12 Plasma membrane
DfNPF8.40 DfaC08G010460.1 5.46 67.94 613 1839 ChrC08 10 Plasma membrane
DfNPF8.41 DfaC08G017910.1 5.38 67.12 623 1869 ChrC08 10 Nucleus
DfNPF8.42 DfaC11G000260.1 10.23 54.27 503 1509 ChrC11 8 Plasma membrane
DfNPF8.43 DfaC11G015130.1 5.15 58.70 533 1599 ChrC11 8 Plasma membrane
DfNPF8.44 Dfa0G053380.1 6.31 45.69 415 1245 Contig01553 6 Plasma membrane
DfNPF8.45 Dfa0G069870.1 7.6 60.31 553 1659 Contig02037 10 Plasma membrane
DfNRT2.1 DfaA01G018440.1 7.98 50.04 483 1449 ChrA01 10 Plasma membrane
DfNRT2.2 DfaA03G000530.1 8.20 53.64 495 1485 ChrA03 10 Plasma membrane
DfNRT2.3 DfaB01G012120.1 8.62 55.70 518 1554 ChrB01 10 Plasma membrane
DfNRT2.4 DfaB03G000430.1 7.94 53.28 493 1479 ChrB03 10 Plasma membrane
DfNRT2.5 DfaC03G010230.1 10.02 46.23 417 1251 ChrC03 8 Plasma membrane
DfNRT3.1 DfaA03G013960.1 9.24 20.98 202 606 ChrA03 2 Vacuole
DfNRT3.2 DfaA04G010290.1 9.19 20.81 199 597 ChrA04 1 Golgiosome
DfNRT3.3 DfaB03G013890.1 9.36 21.27 204 612 ChrB03 1 Vacuole
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Fig. 1.

Fig. 1

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Phylogenetic relationship of NRT genes among Dendrocalamus farinosus (Df), Arabidopsis (At) and rice (Os). Multiple sequences alignment and phylogenetic tree were performed by MEGA7.0. The value at the nodes represents bootstrap values from 1000 replicates. Different individual subfamilies were shown by different color

Most of members of NPF (NRT1) group contained MFS superfamily-related domain: MFS superfamily, MFS_NPF, MFS_NPF1_2, MFS_NPF4 and MFS_NPF5 (Fig. S1). Interestingly, only two members of NPF viz. DfNPF3.2 and DfNPF3.4 exhibited Ubiquitin_like_fold superfamily domain, while NRT2 and NRT3 members exhibited the PLN0028 single domain, and NAR2 domain, respectively (Fig. S1). These results suggested that NRT1 subfamily had less evolutionarily conserved structure as compared to NRT2 and NRT3. As shown in Fig. S1, NRT1/NPF subfamily possess 1 to 10 exons. The number and location of introns and exons were variable among various members of NPFs, indicating their difference on physiological functions. In contrast, fewer introns were observed in subgroups NRT2 and NRT3. WOLF PSORT (https://wolfpsort.hgc.jp/) analysis showed that most of the DfNRTs were localized in cell membrane, suggesting that they might be responsible for the trans-membrane transport of certain substates (Table 1).

Additionally, GO enrich analysis in biological processes also implied multiple functions of DfNRTs, including nitrogen compound transport (GO: 0071705), phloem nitrate loading (GO:0090408), response to nitrogen compound (GO:1,901,698), as well as transport of glucosinolate (GO:1,901,349) and ions (GO: 0006811). Besides, these DfNRTs were found to be involved in response to biotic (GO: 0009607) and abiotic stimulus (GO: 0009628) (Fig. 2) (Dechorgnat et al. 2012).

Fig. 2.

Fig. 2

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Top 20 enriched GO terms in biological processes from GO enrich analysis of DfNRTs. GO enrichment analyses ranked by q-value

Synteny and evolutionary relationships among NRT genes

Chromosomal location analysis showed that 149 DfNRT genes were unevenly distributed across 32 chromosomes out of 35 chromosomes, where 57, 40 and 52 genes located in A, B, and C sub-genome, respectively (Fig. S2). ChrC1 possess the largest number of DfNRT genes (12), while no NRT gene in the ChrA6, ChrC6 and ChrB5. Most genes (79.9%, 122 out of 155) in DfNRT gene family underwent segmental or tandem duplication events using BLASTP and MCScanX methods. In 130 pairs of DfNRT paralogous genes, 112 pairs from 111 DfNRT genes appeared by segmental duplication events, while only 18 pairs were attributed to tandem duplication (Houb 2001) (Fig. 3a, Table S4). There were more segmental duplication events on chromosomes A08, A03 and C11, including 8, 7 and 7 genes, respectively. Apparently, compared with tandem replication, segmental duplication might be the main driving force for the expansion of DfNRT family.

Fig. 3.

Fig. 3

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Collinearity and whole-genome duplication of DfNRT genes. a Tandem duplication and segmental duplication of DfNRT genes. Chromosomes of D. farinosus are shown in yellow rectangle. Tandem duplication and segmental duplication are connected by orange lines and red lines respectively; b Synteny analysis of NRT genes between D. latiflorus subgenome (DfA, DfB and DfC) and rice. Chromosomes of bamboo and rice are shown in blue rectangle and green rectangle. Red curves indicate the syntenic relationships of NRT genes between bamboo and rice; c whole-gen me duplication of NRT genes in woody bamboos D.farinosus, D.latiflorus and P.edulis. The y axis indicates the density of calculated Ks value of each duplicated gene pair. The peak of Ks distribution plot represents a recent whole-genome duplication event

In order to characterize selection pressure on DfNRTs during the evolutionary process, the values of synonymous (Ks) and nonsynonymous (Ka) nucleotide substitution rates, and Ka/Ks ratios in paralogous NRT gene pairs were determined (Table S4). Our results showed that all the Ka/Ks ratios of the paralogous NRT family genes in D.farinosus were less than 1 (0.11 ~ 0.87), suggesting that they might have undergone a Darwinian purifying selection (Table S4) (Nekrutenko et al. 2002). Moreover, the phylogenetic mechanism of DfNRT family was investigated, which generated 108 DfNRT genes with 53 OsNRT genes as orthologs by the genomic synteny analysis between bamboo and rice (Fig. 3b, Table S5). There were 42 pairs of syntenic relationships in the bamboo A sub-genome with rice, while there were 31 and 35 pairs in the B and C sub-genome, respectively (Fig. 3b, Table S5). Most syntenic blocks of NRT genes between bamboo and rice were found to be located on rice chromosome 2, 3, 4 and 10. For example, three rice NRT genes, Os01t0871500, Os12t0638200 and Os12t0638200 were associated with six syntenic gene pairs of DfNRT genes; Os01t0871500 paired with DfNPF5.1/DfNPF5.2/DfNPF5.3/DfNPF5.10/DfNPF5.16/DfNPF5.20, Os03t0235700 with DfNPF8.9/DfNPF8.37/DfNPF8.15/DfNPF8.23/DfNPF8.25/DfNPF8.40, Os12t0638200 with DfNPF2.3/DfNPF2.4/DfNPF2.7/DfNPF2.8/DfNPF2.13/DfNPF2.14 (Table S5). In D. farinosus, we also identified one gene DfNPF8.32 that was paired with three rice genes Os10t0579600/Os02t0699000/Os04t0597600 (Table S5). These results implied the occurrence of a recent whole genome duplication (WGD) event or polyploidy event, which generated a large number of DfNRT genes.

In addition, we also investigated NRT genes in other two sequenced woody bamboo species, D. latiflorus and P. edulis by the reciprocal BLASTP analysis, and identified 174 and 166 NRT genes at genome-wide level, respectively (Table S6), which were slightly more than that of D. farinosus (155). Similar to D. farinosus, many duplication events occurred in D. latiflorus (118 events) and P. edulis (104 events). Then, the divergence times of these paralogs pairs of NRTs were investigated. As shown in Fig. 3c, all three bamboo exhibited a significantly large peak of Ks (about 0.15 ~ 0.37), which suggested that the expansion of NRT genes might be related to a recent WGD or polyploidization event in woody bamboos (Table S6).

Nitrogen distribution between bamboo components by 15N tracing

To explore the N-distribution strategies between various components of bamboo, 15N-enrichment after labeling with 15N-NO3− was detected. As shown in Fig. 4, N uptake by roots was incorporated more into newly-emerged culms (bamboo shoots) compared with mature (maternal) plants, and was preferentially distributed to aboveground parts biomass components. Especially for leaves at the base of culms and internode at the upper parts of culms (elongating parts), the 15N accumulation levels of them were significantly higher than those parts, up to 2.32% and 2.44%, respectively.

Fig. 4.

Fig. 4

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15N abundance in different tissues from maternal plants and new-born shoots after.15N-nitrate tracing. Different letters on the bars indicate significant differences in each nitrate treatment (One-way ANOVA, P < 0.01)

Differential expression of DfNRT genes in various tissues

In order to explore the expression patterns of DfNRTs, RNAseq using various tissues of underground and aboveground parts was performed. Based on their expression patterns, the 155 DfNRT genes were classified into three clusters (Fig. 5a). DfNRT genes in cluster I and cluster II were expressed in most of tissues tested, even some of them having tissue-specific expression. For instance, DfNPF2.7 and DfNPF2.14 were highly expressed in internode and node of bamboo culms, while DfNPF2.13 and DfNPF8.3 were higher in leaf, indicating that they might function differently in nitrate transport and distribution. By contrast, DfNRTs in cluster III exhibited low expression in all tissues tested, except for DfNRT3.3, DfNPF2.4, DfNPF4.1 and DfNPF4.5, all of them having a root-specific expression pattern.

Fig. 5.

Fig. 5

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The tissue expression profiles of DfNRTs and different expression analysis. a The heatmap representing the hierarchical clustering of transcript levels of DfNRT genes using log2 (FPKM + 1) in different tissues including root, rhizome neck, leaf, internode and node. b A venn diagram presenting differential gene expression among different tissues; c–e Top 20 enriched GO terms from GO enrich analysis in biology processing of leaf_vs_internode, leaf_vs_root, and root_vs_internode. GO enrichment ranked by q-value

Moreover, differential gene expression analysis revealed 73 and 86 DfNRT genes exhibited different expressions in leaf when compared with root and internode, respectively, while 79 differential expressed genes (DEGs) were detected between internode and root (Fig. 5b). Venn diagram was also created to find 23 DEGs in all pairwise comparisons, with only 2 ~ 4 genes exclusive to leaf_vs_internode, leaf_vs_root, and root_vs_internode (Fig. 5b). GO enrichment analysis in biological process revealed that in all pairwise comparisons, GO terms related to the nitrate and anion transport (GO: 0071705 and GO: 0006820), as well as phloem nitrate and glucosinolate loading (GO: 0090408 and GO: 0090449), were significantly enriched (Fig. 5c–e). GO terms involved in phloem and vascular transport (GO: 0010232 and GO: 0010233) was present in leaf_vs_internode (Fig. 5c), where two putative homologs of AtNPF2.9/AtNRT1.9, DfNPF2.14 and DfNPF2.7 were dominant (Table S8).

The expresson changes under N fertilize treatments

High expression 8 genes were selected and performed quantitative real-time PCR (qRT-PCR) analysis to validate RNAseq data. The results were shown in Fig. 6a that the expression of selected genes in various tissues were similar to RNAseq results. Furthermore, the response of some DfNRTs under N fertilize treatments was investigated. DfNPF2.13, DfNPF2.14 and DfNPF2.7 were homologous to OsNPF2.2 and AtNPF2.9 having function in N-distribution in leaf. Along with DfNPF7.1 and DfNPF7.2, these five genes exhibited higher expression in aboveground parts tissues compared to underground tissue. In addition, DfNPF7.1 and DfNPF7.2 were found to be two closely related transporters according to amino acid composition and phylogeny (Fig. 1). In comparison, DfNPF4.1, DfNPF4.2 and DfNPF6.9 were expressed more in underground tissues according to RNAseq data. We found that their expression pattern also changed under different nitrate treatment. As shown in Fig. 6b–c and g, when supplied by extra nitrate, the expressions of DfNPF2.7, DfNPF2.13 and DfNPF6.9 were significantly induced in leaves, which indicated their role in N allocation between leaves. DfNPF4.1 and DfNPF4.5, which were two root-specific expression genes, along with an internode-specific DfNPF2.14 were repressed in almost all tissues tested under higher N fertilizer treatment (N2) (Fig. 6e and f), suggested they might be as low-affinity nitrate transporters. The moderate N fertilizer treatment (N1) could only induce the expression of DfNPF4.1 to over 20-folds of CK (without extra N supply). Considering that both DfNPF4.1 and DfNPF4.5 were homologous to AtNPF4.6/AtNRT1.2 that participated in the uptake of high nitrate concentrations by roots (Liu et al. 1999). It seemed that only DfNPF4.1 retained original function from AtNPF4.6/AtNRT1.2. Moreover, the extra nitrate treatment characteristically up-regulated the expression of DfNPF7.1 in upper internode (the elongating tissue) and the expression of DfNPF7.2 in root (Fig. 6h and i). These results also mirrored the difference of physiological functions between NRT paralogs.

Fig. 6.

Fig. 6

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Responses of DfNRTs in transcriptional abundance under different nitrate treatment. a DfNRTs expressions in different tissues determined by qRT-PCR assay; b–i Expression responses of the 8 core DfNRT genes under nitrate treatment. * and ** indicates significant difference at P < 0.05 and P < 0.01 by student’s t test respectively

Discussion

Expansion and evolution of NRT genes in woody bamboo

Polyploidization, or WGD event is the major source of gene duplication, and is widespread in bamboo species (Peer et al. 2017). In the woody bamboos, allopolyploidization events occurred independently and preceded their differentiation. This extensive speciation pattern that led by allopolyploidization is extremely special among plants (Triplett et al. 2014). In current research, 155 NRT genes in D. farinosus were identified at the genome-wide level (Table 1), which was significantly higher than the number of NRT genes in Arabidopsis (60) and rice (127) (Tsay et al. 2007; Lezhneva et al. 2014). In addition, NRT genes in other two sequenced woody bamboo, P. edulis and D. latiflorus were also identified, generated in 174 and 166 NRTs respectively (Table S6). The investigation for the divergence times of these paralogous paired revealed that all three bamboos exhibited a significantly large Ks peak each (with the ks around 0.15 ~ 0.36) (Fig. 3c), indicating the duplication of bamboo NRTs may have occurred around 12 ~ 28 million years ago (Mya) estimated by universal substitution rate of 6.5 × 10−9 mutations per site per year (Zhao et al. 2018). Previous studies had demonstrated that allopolyploidization of P. edulis and D. latiflorus underwent a recent WGD event at about 10 ~ 20 Mya in whole genome level, while the divergence of bambusoideae and rice was estimated at about 45 Mya (Peng et al. 2013; Z. H. Guo et al. 2019; Zheng et al. 2022). Our result is similar to these reports and supported that the expansion of bamboo NRT genes occurred along with the genome duplication event, after the divergence of bamboo and rice.

Functional divergence among NRT paralogs

Gene duplication has been suggested to be an important process in the generation of evolutionary novelty. However, a large proportion of duplicated genes are differentiated through gene expression divergence (Wang et al. 2012b). AtNPF2.9/AtNRT1.9 has been demonstrated that play an important role in phloem nitrate transport (Wang and Tsaya 2011). In our research, two putative homologous genes of AtNPF2.9/AtNRT1.9, DfNPF2.14 and DfNPF2.7 were found to exist in DEGs cluster in leaf_vs_internode (Table S7). The expression of DfNPF2.7 was significantly upregulated in internode at the base under extra N supplies, indicating it might be involved in N transport from base to upper of culm. On the contrary, the expression of DfNPF2.14 were down-regulated in internode when treated with N fertilizer (Fig. 6b and d). Similarly, AtNPF7.3/AtNRT1.5 that had been demonstrated to be a xylem nitrate-loading transporter, was proved to be deeply involved in the regulation of nitrate reallocation conferring plants stress tolerance such as salt, drought, and cadmium stress (Lin et al. 2008). Its orthologous genes in bamboo that DfNPF7.1 and DfNPF7.2 were found to be two closely related transporters according to amino acid composition and phylogeny (Fig. 1), but their functions were greatly different. The expression of DfNPF7.1 was activated just at the upper internode when treated with extra nitrate, while the expression of DfNPF7.2 increased only in root (Fig. 6h and i). These functional divergence among NRT paralogs in rice and pineapple had been reported (Plett et al. 2010; Li et al. 2018), which may play an important role in the preservation of duplicated genes (Wang et al. 2012b).

Nitrogen distribution among bamboo components

As a group of typical clonal plants, bamboos are form clumps or spread via rhizomes. The vegetative reproduction of rhizomatous bamboos depends upon both nutrient elements from soil, the translocation of nutrients and carbohydrates stored in the clonal system (Wu et al. 2013). Nutrient availability in soil often limits bamboo growth and production (Kleinhenz and Midmore 2001). Gao et al. (2016) and Yoo et al. (2017) reported that nitrogen (N) or compound fertilizers (NPK) significantly increased bamboo shoot and culm production. In moso bamboo, N fertilization increased the N concentration and stock of aboveground parts biomass components, while the N stocks of belowground roots were significantly lower (Kim et al. 2018). Through the 15N-nitrate trace analysis, we found that N uptake by roots in D. farinosus was also preferentially distributed to aboveground parts components especially for leaf blades (Fig. 4). Because increasing nitrogen content in leaf can enhance photosynthesis by increasing rubisco content, increasing stomatal aperture, and improving mesophyll conductance (Liu et al. 2021), apparently, N accumulation in leaves is highly related to rapid growth rate and to high biomass accumulation of bamboos. Nevertheless, the distribution of N between mature leaves and from older to younger leaves had been demonstrated to be important factor for leaf and canopy photosynthesis (Gastal and Lemaire 2002). As a perennial sympodial bamboo species, the changes of nitrogen distribution center aboveground parts during fast growing stages helped to reveal the functional NRT in nitrogen utilization. In Arabidopsis, AtNPF6.2/AtNRT1.4, AtNPF2.13/AtNRT1.7, AtNPF1.2/AtNRT1.11, AtNPF1.1/AtNRT1.12, and AtNRT2.4 were found to be involved in leaf nitrate homeostasis (Fan et al. 2009). In D. farinosus, DfNPF2.13 (a putative orthologous gene of AtNPF2.13/AtNRT1.7) exhibited higher expression in leaf, and was found to be induced in leaf when treated by higher N supply (Fig. 6c). Under conditions of both moderate and higher N supplies, the expression level of DfNPF6.9 (an orthologous gene of AtNPF6.3/AtNRT1.1) increased only in the leaf, but decreased in other tissues (Fig. 6g). These results indicated DfNPF2.13 and DfNPF6.9 might participate in N allocation between leaves in D. farinosus. Which were keeping with the nitrogen distribution center changed into the leaf. In addition, we also found that the new-born bamboo shoots (especially for the elongating inter-node at the upper) had more N accumulation compared with maternal plants (Fig. 4). The exchange of N and carbohydrates through rhizome between ramet is pivotal for the survival and expansion of bamboo species, ensuring their ecological successes (Saitoh et al. 2002). Thus, the accumulation of DfNPF2.13 and DfNPF6.9 on the transcription level would lead to a higher efficient use of nitrogen under N-deficiency condition. Along with the evolution and distribution of NRT genes in grass, we have 2 more potential genes for crop directive breeding to get more biomass accumulation with less fertilizer and more environmentally friendly.

Conclusions

We identified a total of 155 NRT genes in a hexaploidy woody bamboo Dendrocalamus farinosus (D. farinosus) according to genome-wide investigation. DfNRT genes displayed various expression profiles in relation to different tissue and nitrate supply, indicating their functional diversity. The analysis of collinearity and evolution revealed the recent whole genome duplication (WGD) event or polyploidization event that might be happened around 12 ~ 28 Mya contributed to the expansion of NRT families in woody bamboos. Through the 15N-nitrate trace, we found that N uptake by roots in D. farinosus was incorporated more into new-born culms (bamboo shoots) compared with maternal plants, and was preferentially distributed to aboveground parts components especially for leaf blades. The exchange of N between interconnected ramets by rhizomes is pivotal for the survival and expansion of bamboo species. The results of this study provide a reference for further analysis of the function of NRT gene and exploration of nitrate transport mechanism in bamboo.

Supplementary Information

Below is the link to the electronic supplementary material.

Supplementary file1 (PDF 1224 KB) (1.2MB, pdf)
Supplementary file2 (PDF 455 KB) (455.2KB, pdf)
Supplementary file3 (XLS 49 KB) (48.5KB, xls)
Supplementary file4 (XLS 204 KB) (204KB, xls)
Supplementary file5 (XLS 72 KB) (72KB, xls)
Supplementary file6 (XLS 21 KB) (21KB, xls)
Supplementary file7 (XLS 39 KB) (38.5KB, xls)
Supplementary file8 (XLS 36 KB) (36KB, xls)
Supplementary file9 (XLS 39 KB) (38.5KB, xls)
Supplementary file10 (XLS 45 KB) (45KB, xls)

Acknowledgements

This work was supported by the National Key Research and Development Program of China, China (grant number 2021YFD2200505-2) and National Science Foundation of Sichuan Province (grant number 2022NSFSC1766). We would also like to thank the editor and anonymous reviewers for their contributions to the peer review of our work.

Abbreviations

N

Nitrogen

NRT

Nitrate transporter

HATS

High-affinity transport system

LATS

Low- affinity transport system

RNA-Seq

RNA sequencing

qRT-PCR

Quantitative real-time PCR

Dendrocalamus farinosus

D. farinosus

Phyllostachys edulis

P. edulis

Dendrocalamus latiflorus

D. latiflorus

Author contributions

Siyuan Ren, Ying Cao and Gang Xu conceived and designed the experiments. Siyuan Ren and Boya Wang performed the experiments and prepared the draft manuscript, Suwei Hao and Sen Chen assisted in the experiments and data visualization. Ying Cao, Shanglian Hu and Boya Wang thoroughly revised the manuscript and approved the final manuscript for publication. All the authors read and approved the manuscript.

Declarations

Conflict of interest

The authors have no relevant financial or non-financial interests to disclose.

Footnotes

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Contributor Information

Shanglian Hu, Email: hushanglian@swust.edu.cn.

Ying Cao, Email: caoying@swust.edu.cn.

References

  1. Bai H, Euring D, Volmer K, Janz D, Polle A (2013) The nitrate transporter (NRT) gene family in poplar. PLoS ONE 8(8):e72126. 10.1371/journal.pone.0072126 [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Corratgé-Faillie C, Lacombe B (2017) Substrate (un)specificity of arabidopsis NRT1/PTR FAMILY (NPF) proteins. J Exp Bot 68(12):3107–3113. 10.1093/jxb/erw499 [DOI] [PubMed] [Google Scholar]
  3. Cui YN, Li XT, Yuan JZ, Wang FZ, Wang SM, Ma Q (2019) Nitrate transporter NPF7.3/NRT1.5 plays an essential role in regulating phosphate deficiency responses in arabidopsis. Biochem Biophys Res Commun 508(1):314–319. 10.1016/J.BBRC.2018.11.118 [DOI] [PubMed] [Google Scholar]
  4. Dechorgnat J, Nguyen CT, Armengaud P, Jossier M, Diatloff E, Filleur S, Daniel-Vedele F (2011) From the soil to the seeds: the long journey of nitrate in plants. J Experiment Botany 62(4):1349–1359. 10.1093/JXB/ERQ409 [DOI] [PubMed] [Google Scholar]
  5. Dechorgnat J, Patrit O, Krapp A, Fagard M, Daniel-Vedele F (2012) Characterization of the Nrt2.6 gene in Arabidopsis thaliana: a link with plant response to biotic and abiotic stress. PLoS ONE 7(8):e42491. 10.1371/JOURNAL.PONE.0042491 [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Du TT, Li HP, Wang BY, Ou Q, Huang Y, Cao Y, Hu SL (2021) Cloning and promotor analysis of DfMYB3 from Dendrocalamus farinosus. Bull Bot Res 41(5):729. 10.7525/J.ISSN.1673-5102.2021.05.011 [Google Scholar]
  7. Engler B, Schoenherr S, Zhong Z, Becker G (2012) Suitability of bamboo as an energy resource: analysis of bamboo combustion values dependent on the culm’s age. Int J for Eng 23(2):114–121. 10.1080/14942119.2012.10739967 [Google Scholar]
  8. Fan SC, Lin CS, Hsu PK, Lin SH, Tsay YF (2009) The Arabidopsis nitrate transporter NRT1.7, expressed in phloem, is responsible for source-to-sink remobilization of nitrate. Plant Cell 21(9):2750–2761. 10.1105/TPC.109.067603 [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Gao PJ, Zuo ZJ, Zhang RM, Qiu YH, He RH, Gao RF, Gui RY (2016) Optimum nitrogen Fertilization for Phyllostachys edulis productivity and photosynthetic response. Agron J 108(1):448–458. 10.2134/AGRONJ2015.0324 [Google Scholar]
  10. Gastal F, Lemaire G (2002) N uptake and distribution in crops: an agronomical and ecophysiological perspective. J Exp Bot 53(370):789–799. 10.1093/JEXBOT/53.370.789 [DOI] [PubMed] [Google Scholar]
  11. Glass ADM (2009) Nitrate uptake by plant roots. Botany 87(7):659–667. 10.1139/B09-014 [Google Scholar]
  12. Glass ADM, Britto DT, Kaiser BN, Kinghorn JR, Kronzucker HJ, Kumar A, Okamoto M, Rawat S, Siddiqi MY, Unkles SE, Vidmar JJ (2002) The regulation of nitrate and ammonium transport systems in plants. J Exp Bot 53(370):855–864. 10.1093/JEXBOT/53.370.855 [DOI] [PubMed] [Google Scholar]
  13. Guo S, Zhou Y, Shen Q, Zhang F (2007) Effect of ammonium and nitrate nutrition on some physiological processes in higher plants - growth, photosynthesis, photorespiration, and water relations. Plant Bio (Stuttgart, Germany) 9(1):21–29. 10.1055/S-2006-924541 [DOI] [PubMed] [Google Scholar]
  14. Guo ZH, Ma PF, Yang GQ, Hu JY, Liu YL, Xia EH, Zhong MC, Zhao L, Sun GL, Xu YX, Zhao YJ, Zhang YC, Zhang YX, Zhang XM, Zhou MY, Guo Y, Guo C, Liu JX, Ye XY, Chen YM, Yang Y, Han B, Lin CS, Lu Y, Li DZ (2019) Genome sequences provide insights into the reticulate origin and unique traits of woody bamboos. Mol Plant 12(10):1353–1365. 10.1016/J.MOLP.2019.05.009 [DOI] [PubMed] [Google Scholar]
  15. Hawkesford M, Horst W, Kichey T, Lambers H, Schjoerring J, Møller IS, White PJ (2023) Functions of macronutrients. In: marschner’s mineral nutrition of higher plants, 4th edn. ScienceDirec. pp 201–281.
  16. Houb EB (2001) The arms race is ancient history in Arabidopsis, the wildflower. Nature Rev Genetics 2(7):516–527. 10.1038/35080508 [DOI] [PubMed] [Google Scholar]
  17. Hu B, Wang W, Ou S, Tang J, Li H, Che R, Zhang Z, Chai X, Wang H, Wang Y, Liang C, Liu L, Piao Z, Deng Q, Deng K, Xu C, Liang Y, Zhang L, Li L, Chu C (2015) Variation in NRT1.1B contributes to nitrate-use divergence between rice subspecies. Nat Genet 47(7):834–838. 10.1038/NG.3337 [DOI] [PubMed] [Google Scholar]
  18. Kim C, Baek G, Yoo BO, Jung SY, Lee KS (2018) Regular fertilization effects on the nutrient distribution of bamboo components in a Moso Bamboo (Phyllostachys pubescens (Mazel) Ohwi) Stand in South Korea. Forests 9(11):671. 10.3390/F9110671 [Google Scholar]
  19. Kleinhenz V, Midmore DJ (2001) Aspects of bamboo agronomy. Adv Agron 74:99–153. 10.1016/S0065-2113(01)74032-1 [Google Scholar]
  20. Lezhneva L, Kiba T, Feria-Bourrellier AB, Lafouge F, Boutet-Mercey S, Zoufan P, Sakakibara H, Daniel-Vedele F, Krapp A (2014) The Arabidopsis nitrate transporter NRT2.5 plays a role in nitrate acquisition and remobilization in nitrogen-starved plants. Plant J Cell Molecular Biol 80(2):230–241. 10.1111/TPJ.12626 [DOI] [PubMed] [Google Scholar]
  21. Li Y, Ouyang J, Wang YY, Hu R, Xia K, Duan J, Wang Y, Tsay YF, Zhang M (2015) Disruption of the rice nitrate transporter OsNPF2.2 hinders root-to-shoot nitrate transport and vascular development. Sci Rep 5:9635. 10.1038/SREP09635 [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Li W, Yan M, Hu B, Priyadarshani SVGN, Hou Z, Ojolo SP, Xiong J, Zhao H, Qin Y (2018) Characterization and the expression analysis of nitrate transporter (NRT) gene family in pineapple. Tropical Plant Biol 11(3):177–191. 10.1007/S12042-018-9209-Z [Google Scholar]
  23. Lin SH, Kuo HF, Canivenc G, Lin CS, Lepetit M, Hsu PK, Tillard P, Lin HG, Wang YY, Tsai CB, Gojon A, Tsay YF (2008) Mutation of the Arabidopsis NRT1.5 nitrate transporter causes defective root-to-shoot nitrate transport. Plant Cell 20(9):2514–2528. 10.1105/TPC.108.060244 [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Liu KH, Huang CY, Tsay YF (1999) CHL1 Is a dual-affinity nitrate transporter of Arabidopsis involved in multiple phases of nitrate uptake. Plant Cell 11(5):865–874. 10.1105/TPC.11.5.865 [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Liu J, Zhang J, Estavillo GM, Luo T, Hu L (2021) Leaf N content regulates the speed of photosynthetic induction under fluctuating light among canola genotypes (Brassica napus L.). Physiol Plant 172(4):1844–1852. 10.1111/PPL.13390 [DOI] [PubMed] [Google Scholar]
  26. Long Z, Imran M, Hu S, Luo X, Cao Y, Samo N, Gong D (2020) Physiological quantification and molecular detection (BeNRT1.1) of neosinocalamus (bambusa emeiensis) treated with different types of nitrogen fertilizers. Indust Crops Products 150:112323. 10.1016/J.INDCROP.2020.112323 [Google Scholar]
  27. McIntyre GI (1987) Studies on the growth and development of Agropyron repens: interacting effects of humidity, calcium, and nitrogen on growth of the rhizome apex and lateral buds. Canadian J of Botany 65(7):1427–1432. 10.1139/B87-197 [Google Scholar]
  28. Migocka M, Warzybok A, Kłobus G (2013) The genomic organization and transcriptional pattern of genes encoding nitrate transporters 1 (NRT1) in cucumber. Plant Soil 364(1–2):245–260. 10.1007/S11104-012-1345-X/FIGURES/5 [Google Scholar]
  29. Nath AJ, Lal R, Das AK (2015) Managing woody bamboos for carbon farming and carbon trading. Global Ecol Conservation 3:654–663. 10.1016/J.GECCO.2015.03.002 [Google Scholar]
  30. Nayak L, Mishra SP (2016) Prospect of bamboo as a renewable textile fiber, historical overview, labeling, controversies and regulation. Fashion Textiles 3(1):1–23. 10.1186/S40691-015-0054-5/TABLES/3 [Google Scholar]
  31. Nekrutenko A, Makova KD, Li WH (2002) The KA/KS ratio test for assessing the protein-coding potential of genomic regions: an empirical and simulation study. Genome Res 12(1):198. 10.1101/GR.200901 [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. O’Brien JAA, Vega A, Bouguyon E, Krouk G, Gojon A, Coruzzi G, Gutiérrez RAA (2016) Nitrate transport, sensing, and responses in plants. Mol Plant 9(6):837–856. 10.1016/J.MOLP.2016.05.004 [DOI] [PubMed] [Google Scholar]
  33. Okamoto M, Vidmar JJ, Glass ADM (2003) Regulation of NRT1 and NRT2 gene families of Arabidopsis thaliana: Responses to nitrate provision. Plant Cell Physiol 44(3):304–317. 10.1093/PCP/PCG036 [DOI] [PubMed] [Google Scholar]
  34. Okamoto M, Kumar A, Li W, Wang Y, Siddiqi MY, Crawford NM, Glass ADM (2006) High-affinity nitrate transport in roots of Arabidopsis depends on expression of the NAR2-like gene AtNRT3.1. Plant Physiol 140(3):1036–1046. 10.1104/PP.105.074385 [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Peer Y, Mizrachi E, Marchal K (2017) The evolutionary significance of polyploidy. Nat Rev Genetics 18(7):411–424. 10.1038/NRG.2017.26 [DOI] [PubMed] [Google Scholar]
  36. Peng Z, Ying L, Li L, Zhao Q, Feng Q, Gao Z, Hengyun L, Tao H, Yao N, Liu K, Li Y, Fan D, Guo Y, Li W, Yiqi L, Weng Q, Zhou C, Zhang L, Huang T, Zhao Y, Zhu C, Liu X, Yang X, Wang T, Miao K, Zhuang C, Cao X, Tang W, Liu G, Liu Y, Chen J, Liu Z, Yuan L, Liu Z, Huang X, Tingting L, Fei B, Ning Z, Han B, Jiang Z (2013) The draft genome of the fast-growing non-timber forest species moso bamboo (Phyllostachys heterocycla). Nat Genetics 45(4):456–461. 10.1038/ng.2569 [DOI] [PubMed] [Google Scholar]
  37. Plett D, Toubia J, Garnett T, Tester M, Kaiser BN, Baumann U (2010) Dichotomy in the NRT gene families of dicots and grass species. PLoS ONE 5(12):e15289. 10.1371/JOURNAL.PONE.0015289 [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Saitoh T, Seiwa K, Nishiwaki A (2002) Importance of physiological integration of dwarf bamboo to persistence in forest understorey: a field experiment. J Ecol 90(1):78–85. 10.1046/J.0022-0477.2001.00631.X [Google Scholar]
  39. Soreng RJ, Peterson PM, Romaschenko K, Davidse G, Zuloaga FO, Judziewicz EJ, Filgueiras TS, Davis JI, Morrone O (2015) A worldwide phylogenetic classification of the Poaceae (Gramineae). J Syst Evol 53(2):117–137. 10.1111/JSE.12150 [Google Scholar]
  40. Taochy C, Gaillard I, Ipotesi E, Oomen R, Leonhardt N, Zimmermann S, Peltier JB, Szponarski W, Simonneau T, Sentenac H, Gibrat R, Boyer JC (2015) The Arabidopsis root stele transporter NPF2.3 contributes to nitrate translocation to shoots under salt stress. Plant J Cell Molecular Biol 83(3):466–479. 10.1111/TPJ.12901 [DOI] [PubMed] [Google Scholar]
  41. Triplett JK, Clark LG, Fisher AE, Wen J (2014) Independent allopolyploidization events preceded speciation in the temperate and tropical woody bamboos. New Phytol 204(1):66–73. 10.1111/NPH.12988 [DOI] [PubMed] [Google Scholar]
  42. Tsay YF, Chiu CC, Tsai CB, Ho CH, Hsu PK (2007) Nitrate transporters and peptide transporters. FEBS Lett 581(12):2290–2300. 10.1016/J.FEBSLET.2007.04.047 [DOI] [PubMed] [Google Scholar]
  43. von Wittgenstein NJ, Le CH, Hawkins BJ, Ehlting J (2014) Evolutionary classification of ammonium, nitrate, and peptide transporters in land plants. BMC Evol Biol 14(1):11. 10.1186/1471-2148-14-11 [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Wang YY, Tsay YF (2011) Arabidopsis nitrate transporter NRT1.9 is important in phloem nitrate transport. Plant Cell 23(5):1945–1957. 10.1105/TPC.111.083618 [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Wang Y, Wang X, Paterson AH (2012a) Genome and gene duplications and gene expression divergence: a view from plants. Ann N Y Acad Sci 1256(1):1–14. 10.1111/J.1749-6632.2011.06384.X [DOI] [PubMed] [Google Scholar]
  46. Wang YY, Hsu PK, Tsay YF (2012b) Uptake, allocation and signaling of nitrate. Trends Plant Sci 17(8):458–467. 10.1016/J.TPLANTS.2012.04.006 [DOI] [PubMed] [Google Scholar]
  47. Wang Q, Liu C, Dong Q, Huang D, Li C, Li P, Ma F (2018a) Genome-wide identification and analysis of apple NITRATE TRANSPORTER 1/PEPTIDE TRANSPORTER Family (NPF) genes reveals mdnpf65 confers high capacity for nitrogen uptake under low-nitrogen conditions. Int J Molecular Sci 19(9):2761. 10.3390/IJMS19092761 [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Wang W, Hu B, Yuan D, Liu Y, Che R, Hu Y, Ou S, Liu Y, Zhang Z, Wang H, Li H, Jiang Z, Zhang Z, Gao X, Qiu Y, Meng X, Liu Y, Bai Y, Liang Y, Wang Y, Zhang L, Li L, Sodmergen JH, Li J, Chu C (2018b) Expression of the nitrate transporter gene OsNRT1.1A/OsNPF6.3 confers high yield and early maturation in rice. Plant Cell 30(3):638–651. 10.1105/TPC.17.00809 [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Wang J, Li Y, Zhu F, Ming R, Chen LQ (2019) Genome-wide analysis of nitrate transporter (NRT/NPF) family in sugarcane saccharum spontaneum L. Tropical Plant Biology 12(3):133–149. 10.1007/S12042-019-09220-8 [Google Scholar]
  50. Wang S, Chen A, Xie K, Yang X, Luo Z, Chen J, Zeng D, Ren Y, Yang C, Wang L, Feng H, López-Arredondo DL, Herrera-Estrella LR, Xu G (2020) Functional analysis of the OsNPF45 nitrate transporter reveals a conserved mycorrhizal pathway of nitrogen acquisition in plants. Proceed Nat Acad Sci United States of America 117(28):16649–16659. 10.1073/PNAS.2000926117 [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Wang X, Cai X, Xu C, Wang Q (2021) Identification and characterization of the NPF, NRT2 and NRT3 in spinach. Plant Physiol Biochem 158:297–307. 10.1016/J.PLAPHY.2020.11.017 [DOI] [PubMed] [Google Scholar]
  52. Yen TM (2016) Culm height development, biomass accumulation and carbon storage in an initial growth stage for a fast-growing moso bamboo (Phyllostachy pubescens). Bot Stud 57(1):10. 10.1186/S40529-016-0126-X [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Yoo BO, Park JH, Park YB, Jung SY, Lee KS, Kim S (2017) Assessment of expansion characteristics and classification of distribution types for bamboo forests using GIS. J Korean Association Geograph Inform Stud 20(4):55–64. 10.11108/KAGIS.2017.20.4.055 [Google Scholar]
  54. Zhao H, Gao Z, Wang L, Wang J, Wang S, Fei B, Chen C, Shi C, Liu X, Zhang H, Lou Y, Chen L, Sun H, Zhou X, Wang S, Zhang C, Xu H, Li L, Yang Y, Wei Y, Yang W, Gao Q, Yang H, Zhao S, Jiang Z (2018) Chromosome-level reference genome and alternative splicing atlas of moso bamboo (Phyllostachys edulis). Gigascience 7(10):giy115. 10.1093/GIGASCIENCE/GIY115 [DOI] [PMC free article] [PubMed] [Google Scholar]
  55. Zheng Y, Yang D, Rong J, Chen L, Zhu Q, He T, Chen L, Ye J, Fan L, Gao Y, Zhang H, Gu L (2022) Allele-aware chromosome-scale assembly of the allopolyploid genome of hexaploid Ma bamboo (Dendrocalamus latiflorus Munro). J Integr Plant Biol 64(3):649–670. 10.1111/JIPB.13217 [DOI] [PubMed] [Google Scholar]
  56. Zhizhuang W, Xuhua D, Deli X, Shudong W, Yuegou Z, Yan Z (2013) A comparative study on photosynthetic characteristics of different types of bamboos. J Ecol Environ 22(9):1523–1527. 10.16258/j.cnki.1674-5906.2013.09.011 [Google Scholar]

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