CsNPF7.2 Has a Potential to Regulate Cucumber Seedling Growth in Early Nitrogen Deficiency Stress

Abstract

Cucumber is an economically important horticultural crop that is highly dependent on nitrogen fertilizer. Nitrate is the main nitrogen source for cucumber; however, the effects of nitrogen signaling on the early-stage growth of cucumber seedlings and the related regulatory mechanisms are still unclear. To compare seedling growth status at different nitrate levels, we performed a growth experiment using cucumber seedlings that had nearly exhausted their nitrogen reserves under nitrogen deficiency conditions (NO3-N/NH4+-N = 0 in MS medium). Using qPCR and in situ RNA hybridization localization of candidate CsNPF genes, we found that short-term nitrogen deficiency promoted changes in root vascular bundle morphology and xylem growth in cucumber seedlings, thereby enhancing their growth potential. Among the candidate genes, CsNPF7.2, a gene located in the vascular cambium was found to be induced by short-term nitrogen deficiency. Considering the abundance of vasculature development marker genes, we speculated that the function of CsNPF7.2 might relate to the development of vascular bundles in plants suffering from nitrogen stress. The objective of our study was to investigate the growth changes in cucumber seedlings in response to different nitrogen levels, and to examine the mRNA accumulation and expression patterns of nitrate transporter CsNPF genes, so that critical genes can be identified to improve nitrogen use efficiency in cucumber cultivation. The results of this study provides a novel theoretical basis for optimizing cultivation, regulating rational fertilization levels, and improving nitrogen use efficiency in production. In addition, our study also provides new avenues for the further study of the function of CsNPF7.2 on regulating vasculature development in response to nitrogen stress.

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Abbreviations

NPFs:

nitrate transporter/peptide transporter family proteins

CESA:

cellulose synthase

WOX4:

Wuschel-related homeobox 4

PXY:

phloem intercalated with xylem

NEN4:

NAC45/86-dependent exonuclease-domain protein 4

qPCR:

quantative RT-PCR

RNA-Seq:

RNA sequencing

References

  1. Alboresi A, Gestin C, Leydecker MT et al (2005) Nitrate, a signal relieving seed dormancy in Arabidopsis. Plant Cell Environ 28:500–512

    CAS  PubMed  Google Scholar 

  2. Bi Y, Wang R, Zhu T, Rothstein SJ (2007) Global transcription profiling reveals differential responses to chronic nitrogen stress and putative nitrogen regulatory components in Arabidopsis. BMC Genomics 8:821

    Google Scholar 

  3. Calvo P, Zebelo S, McNear D et al (2019) Plant growth-promoting rhizobacteria induce changes in Arabidopsis thaliana gene expression of nitrate and ammonium uptake genes. J Plant Interact 14:224–231

    CAS  Google Scholar 

  4. Chen J, Zhang Y, Tan Y et al (2016) Agronomic nitrogen-use efficiency of rice can be increased by driving OsNRT2.1 expression with the OsNAR2.1 promoter. Plant Biotechnol J 14:1705–1715. https://doi.org/10.1111/pbi.12531

  5. Chi H, Wang D, Fang Q et al (2011) Yield and quality response of cucumber to irrigation and nitrogen fertilization under subsurface drip irrigation in solar greenhouse. Sci Agric Sin 10(6):921–930

    Google Scholar 

  6. Dechorgnat J, Nguyen C, Armengaud P, Jossier M, Diatloff E, Filleur S, Daniel-Vedele F (2010) From the soil to the seeds: the long journey of nitrate in plants[J]. J Exp Bot 62(4):1349–1359

    PubMed  Google Scholar 

  7. Dechorgnat J, Francis K, Dhugga K et al (2018) Root ideotype influences nitrogen transport and assimilation in maize. Front Plant Sci. https://doi.org/10.3389/fpls.2018.00531

  8. Drew M, Saker L (1975) Nutrient supply and the growth of the seminal root system in barley: II. Localized, compensatory increases in lateral root growth and rates op nitrate uptake when nitrate supply is restricted to only part of the root system. J Exp Bot 24(6):1189–1202. https://doi.org/10.1093/jxb/26.1.79

  9. Enya V E, Agba O (2006) Economic optimum of nitrogen fertilizer application in cucumber production: the case of Cross River University of Technology (CRUTECH) farm, Obubra campus. 49(8):080211-080211-3

  10. Guan P (2017) Dancing with hormones: a current perspective of nitrate signaling and regulation in Arabidopsis. Front Plant Sci 8:1697

    PubMed  PubMed Central  Google Scholar 

  11. Heffer P (2009a) Assessment of fertilizer use by crop at the global level. International fertilizer industry association, Paris, www fertilizer org/ifa/home-page/LIBRARY/publication-database html/assessment-of-fertilizer-use-by-crop-at-the-global-level-2006-07-2007-08 html2

  12. Heffer P (2009b) Assessment of fertilizer use by crop at the global level

  13. Huang N, Chiang C, Crawford N, Tsay Y (1996) CHL1 encodesacomponentofthelow-affinity nitrate uptake system in Arabidopsis and shows cell type specific expression in roots. Plant Cell 8:2183–2191

    CAS  PubMed  PubMed Central  Google Scholar 

  14. 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 47:834–838. https://doi.org/10.1038/ng.3337

    CAS  Article  PubMed  Google Scholar 

  15. Hu R, Qiu D, Chen Y et al (2016) Knock-down of a tonoplast localized low-affinity nitrate transporter OsNPF7.2 affects rice growth under high nitrate supply. Front Plant Sci 7:1529

    PubMed  PubMed Central  Google Scholar 

  16. Jackson R, Caldwell M (1993) The scale of nutrient heterogeneity around individual plants and its quantification with geostatistics. Ecology 74:612–614

    Google Scholar 

  17. Krouk G, Crawford NM, Coruzzi GM, Tsay YF (2010a) Nitrate signaling: adaptation to fluctuating environments. Curr Opin Plant Biol 13:265–272

    Google Scholar 

  18. Krouk G, Lacombe B, Bielach A, Perrine-Walker F, Malinska K, Mounier E, Hoyerova K, Tillard P, Leon S, Ljung K, Zazimalova E, Benkova E, Nacry P, Gojon A (2010b) Nitrate-regulated auxin transport by NRT1.1 defines a mechanism for nutrient sensing in plants. Dev Cell 18:927–937

    CAS  PubMed  Google Scholar 

  19. Kumar S, Stecher G, Tamura K (2016) MEGA7: molecular evolutionary genetics analysis version 7.0 for bigger datasets. Mol Biol Evol 33:1870–1874

    CAS  Google Scholar 

  20. Léran S, Varala K, Boyer JC, Chiurazzi M, Crawford N, Daniel-Vedele F, David L, Dickstein R, Fernandez E, Forde B, Gassmann W, Geiger D, Gojon A, Gong JM, Halkier BA, Harris JM, Hedrich R, Limami AM, Rentsch D, Seo M, Tsay YF, Zhang M, Coruzzi G, Lacombe B (2014) A unified nomenclature of nitrate transporter 1/peptide transporter family members in plants. Trends Plant Sci 19:5–9

    PubMed  Google Scholar 

  21. Li H, Du X, Li H et al (2017) NRT1.5/NPF7.3 functions as a poton-coupled H+/K+ antiporter for K+ loading into the xylem in Arabidopsis. Plant Cell 29:2016–2026

    CAS  PubMed  PubMed Central  Google Scholar 

  22. Li J, Chen C, Gassmann W et al (2010) The Arabidopsis nitrate transporter NRT1.8 functions in nitrate removal from the xylem sap and mediates cadmium tolerance. Plant Cell 22:1633–1646

    CAS  PubMed  PubMed Central  Google Scholar 

  23. Lian X, Wang S, Zhang J, Feng Q, Zhang L, Fan D, Li X, Yuan D, Han B, Zhang Q (2006) Expression profiles of 10,422 genes at early stage of low nitrogen stress in rice assayed using a cDNA microarray. Plant Mol Biol 60:617–631

    CAS  PubMed  Google Scholar 

  24. Lin S, Kuo H, Canivenc G, Lin CS, Lepetit M, Hsu PK, Tillard P, Lin HL, 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 20:2514–2528

    CAS  PubMed  PubMed Central  Google Scholar 

  25. Little D, Rao H, Oliva S et al (2005) The putative high-affinity nitrate transporter NRT2.1 represses lateral root initiation. Proc Natl Acad Sci 102:13693–13698

    CAS  PubMed  Google Scholar 

  26. Liu K, Niu Y, Konishi M et al (2017) Discovery of nitrate-CPK-NLP signalling in central nutrient-growth networks. Nature 545:311

    CAS  PubMed  PubMed Central  Google Scholar 

  27. Marchler-Bauer A, Bo Y, Han L et al (2016) CDD/SPARCLE: functional classification of proteins via subfamily domain architectures. Nucleic Acids Res 45:D200–D203

    PubMed  PubMed Central  Google Scholar 

  28. Melnyk CW, Gabel A, Hardcastle TJ et al (2018) Transcriptome dynamics at Arabidopsis graft junctions reveal an intertissue recognition mechanism that activates vascular regeneration. Proc Natl Acad Sci 115(10):201718263

    Google Scholar 

  29. McClure P, Kochian L, Spanswick R, Shaff J (1990) Evidence for cotransport of nitrate and protons in maize roots: I. effects of nitrate on the membrane potential. Plant Physiol 93:281–289

    CAS  PubMed  PubMed Central  Google Scholar 

  30. McClure P, Kochian L, Spanswick R, Shaff J (2008) Evidence for cotransport of nitrate and protons in maize roots: I. Effects of nitrate on the membrane potential. Plant Physiol 93:281–289

    Google Scholar 

  31. Migocka M, Warzybok A, Klobus G (2013) The genomic organization and transcriptional pattern of genes encoding nitrate transporters 1 (NRT1) in cucumber. Plant Soil 364:245–260

    CAS  Google Scholar 

  32. Nour-Eldin H, Andersen T, Burow M et al (2012) NRT/PTR transporters are essential for translocation of glucosinolate defence compounds to seeds. Nature 488:531

    CAS  PubMed  Google Scholar 

  33. Owen A, Jones D (2001) Competition for amino acids between wheat roots and rhizosphere microorganisms and the role of amino acids in plant N acquisition. Soil Biol Biochem 33:651–657. https://doi.org/10.1016/S0038-0717(00)00209-1

  34. Plett D, Toubia J, Garnett T et al (2010) Dichotomy in the NRT gene families of dicots and grass species. PLoS One 5(12):e15289. https://doi.org/10.1371/journal.pone.0015289

  35. Peng M, Bi Y, Zhu T, Rothstein SJ (2007) Genome-wide analysis of Arabidopsis responsive transcriptome to nitrogen limitation and its regulation by the ubiquitin ligase gene NLA. Plant Mol Biol 65:775–797

    CAS  PubMed  Google Scholar 

  36. Reichman GA, Grunes DL, Viets FG (1966) Effect of soil mositure on ammonification and nitrification in two northern plains soils. Soil Sci Soc Am J 30:363–366

    CAS  Google Scholar 

  37. Saito H, Oikawa T, Hamamoto S et al (2015) The jasmonate-responsive GTR1 transporter is required for gibberellin-mediated stamen development in Arabidopsis. Nat Commun 6:6095

    PubMed  PubMed Central  Google Scholar 

  38. Sakai WS (1973) Simple method for differential staining of paraffin embedded plant material using toluidine blue. Stain Technol 48:247–249

    CAS  PubMed  Google Scholar 

  39. Scott RK, Ogunbemi EA, Ivins JD, Mendham NJ (1973) The effect of sowing date and season on growth and yield of oilseed rape (brassica napus). J Agric Sci 81:277–285

    Google Scholar 

  40. Scheible WR, Morcuende R, Czechowski T, Fritz C, Osuna D, Palacios-Rojas N, Schindelasch D, Thimm O, Udvardi MK, Stitt M (2004) Genome-wide reprogramming of primary and secondary metabolism, protein synthesis, cellular growth processes, and the regulatory infrastructure of Arabidopsis in response to nitrogen. Plant Physiol 136:2483–2499

    CAS  PubMed  PubMed Central  Google Scholar 

  41. Singh GS, Singh BN, Sarma BK et al (2018) Trichoderma asperellum T42 reprograms tobacco for enhanced nitrogen utilization efficiency and plant growth when fed with N nutrients. Front Plant Sci 9:163

    PubMed  PubMed Central  Google Scholar 

  42. Tal I, Zhang Y, Jørgensen ME et al (2016) The Arabidopsis NPF3 protein is a GA transporter. Nat Commun 7:11496

    Google Scholar 

  43. 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 83:466–479

    CAS  PubMed  Google Scholar 

  44. Thompson J, Higgins D, Gibson T (1994) Clustal W-improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res 22:4673–4680

    CAS  PubMed  PubMed Central  Google Scholar 

  45. Undurraga SF, Ibarra-Henríquez C, Fredes I, Álvarez JM, Gutiérrez RA (2017) Nitrate signaling and early responses in Arabidopsis roots. J Exp Bot 68:2541–2551

    CAS  PubMed  PubMed Central  Google Scholar 

  46. Walch-Liu P, Neumann G, Bangerth F, Engels C (2000) Rapid effects of nitrogen form on leaf morphogenesis in tobacco. J Exp Bot 51:227–237

    CAS  PubMed  Google Scholar 

  47. Wang J, Lu K, Nie H, Zeng Q, Wu B et al (2018b) Rice nitrate transporter OsNPF7.2 positively regulates tiller number and grain yield. Rice 11:12

    PubMed  PubMed Central  Google Scholar 

  48. Wang J, Wang H, Chen J, Zhang S, Xu J et al (2019) Xylem development, cadmium bioconcentration, and antioxidant defense in Populus × euramericana stems under combined conditions of nitrogen and cadmium. Environ Exp Bot 164:1–9

    Google Scholar 

  49. Wang R (2004) Genomic analysis of the nitrate response using a nitrate reductase-null mutant of Arabidopsis. Plant Physiol 1365:2512–2522. https://doi.org/10.1104/pp.104.044610

    Article  Google Scholar 

  50. 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, Jing H, Li J, Chu C (2018a) Expression of the nitrate transporter gene OsNRT1. 1A/OsNPF6. 3 confers high yield and early maturation in rice. Plant Cell 30:638–651

    CAS  PubMed  PubMed Central  Google Scholar 

  51. Wang Y, Tsay Y (2011) Arabidopsis nitrate transporter NRT1.9 is important in phloem nitrate transport. Plant Cell 23:1945–1957. https://doi.org/10.1105/tpc.111.083618

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  52. Wang Y, Hsu P, Tsay Y (2012) Uptake, allocation and signaling of nitrate. Trends Plant Sci 17:458–467. https://doi.org/10.1016/j.tplants.2012.04.006

    CAS  Article  PubMed  Google Scholar 

  53. Wen Z, Tyerman S, Dechorgnat J, Ovchinnikova E, Dhugga KS, Kaiser BN (2017) Maize NPF6 proteins are homologs of Arabidopsis CHL1 that are selective for both nitrate and chloride. Plant Cell 29:2581–2596. https://doi.org/10.1105/tpc.16.00724

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  54. Withrow A (1945) The interrelationship of nitrogen supply and photoperiod on the flowering, growth and stem anatomy of certain long and short day plants. Butler University Botanical Studies 7(1–13):40–64

    Google Scholar 

  55. Wu T, Qin Z, Fan L et al (2014) Involvement of CsNRT1.7 in nitrate recycling during senescence in cucumber. J Plant Nutr Soil Sci 177:714–721. https://doi.org/10.1002/jpln.201300665

    CAS  Article  Google Scholar 

  56. Xin M, Wang L, Liu Y, Feng Z, Zhou X, Qin Z (2017) Transcriptome profiling of cucumber genome expression in response to long-term low nitrogen stress. Acta Physiol Plant 39:1–11. https://doi.org/10.1007/s11738-017-2429-2

    CAS  Article  Google Scholar 

  57. Yuan S, Zhang Z, Zheng C et al (2016) Arabidopsis cryptochrome 1 functions in nitrogen regulation of flowering. Proc Natl Acad Sci. https://doi.org/10.1073/pnas.1602004113

  58. Zhang J, Liu Y, Zhang N et al (2019) NRT1.1B is associated with root microbiota composition and nitrogen use in field-grown rice. Nature biotechnology. https://doi.org/10.1038/s41587-019-0104-4

  59. Zhang X, Zhou Y, Ding L, Wu Z, Liu R, Meyerowitz EM (2013) Transcription repressor HANABA TARANU controls flower development by integrating the actions of multiple hormones, floral organ specification genes, and GATA3 family genes in Arabidopsis. Plant Cell 25:83–101. https://doi.org/10.1105/tpc.112.107854

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  60. Zhao W, Yang X, Yu H, Jiang W, Sun N, Liu X, Liu X, Zhang X, Wang Y, Gu X (2015) RNA-Seq-based transcriptome profiling of early nitrogen deficiency response in cucumber seedlings provides new insight into the putative nitrogen regulatory network. Plant Cell Physiol 56:455–467. https://doi.org/10.1093/pcp/pcu172

    CAS  Article  PubMed  Google Scholar 

  61. Zheng Y, Drechsler N, Rausch C, Kunze R (2016) The Arabidopsis nitrate transporter NPF7.3/NRT1.5 is involved in lateral root development under potassium deprivation. Plant Signal Behav 11:2832–2847. https://doi.org/10.1080/15592324.2016.1176819

    CAS  Article  Google Scholar 

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Accession Numbers

Sequence data from this article can be found in Cucurbit genomics database (CuGenDB, cucurbitgenomics.org). All accession numbers of related genes in the study are given in Supplemental Table 1.

Funding

This work was supported by the National Key Research and Development Program of China (2018YFD1000800 and 2019YFD1000304), National Natural Science Foundation of China (31772358 and 31872158), and Earmarked Fund for China Agriculture Research System (CAS-23).

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Contributions

Conceived and designed the experiments: LG,WZ, and XH. Performed the experiments: XH, WL, and JZ. Analyzed the data: QW and XH. Wrote the paper: WZ and XH.

Corresponding authors

Correspondence to Lihong Gao or Wenna Zhang.

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Key Message

An experiment was conducted with cucumber seedlings grown under nitrogen-deficient conditions to compare seedling growth status at different nitrate levels.

Histological examination of root section, qPCR, and in situ RNA hybridization localization of candidate gene CsNPFs experiments indicated that short-term nitrogen deficiency can promote changes in root vascular bundle morphology and xylem growth, thereby enhancing their growth potential of cucumber seedlings.

CsNPF7.2, a nitrate transporter gene expressed in the vascular cambium, was found to be induced by short-term nitrogen deficiency.

Electronic supplementary material

Supplemental Fig.1
figure10

Homology analysis of CsNPF2.3, CsNPF2.9, CsNPF7.2, CsNPF7.3A and CsNPF7.3B amino acid sequences. Red regions fragments represented the conserved regions of the five proteins, blue regions represent the regions with the most variation, and gray background indicates (PNG 1137 kb)

Supplemental Fig. 2
figure11

Spatiotemporal analysis of CsNPF gene expression in response to nitrogen stress. The relative abundance of CsNPF2.3, CsNPF2.9, CsNPF7.3A, and CsNPF7.3B in roots, stems, and cotyledons of cucumber seedlings in nitrogen supply control (black color labels, MS + N, maintaining normal nitrogen supply), nitrogen deficiency (gray color labels, MS.N, transfer to nitrogen-deficient medium after normal nitrogen supply), nitrogen recovery (red color labels, MS/N, transfer to nitrogen supplied medium after nitrogen-deficient) and nitrogen starvation control (orange color labels, MS-N, no external nitrogen supply); Each experiment included at least 5–6 biological replications and 3–4 technical repeats. Error bars indicate SD. (PNG 175 kb)

Supplemental Fig. 3
figure12

The abundance of CsNPF7.2 transcript in first true leaf of cucumber seedlings in different nitrogen conditions at 11th,15th day. Nitrogen supply control (black color labels, MS + N, maintaining normal nitrogen supply), nitrogen deficiency (gray color labels, MS.N, transfer to nitrogen-deficient medium after normal nitrogen supply), nitrogen recovery (red color labels, MS/N, transfer to nitrogen supplied medium after nitrogen deficiency). MS-N treatment was not included because first true leaves of cucumber seedlings were died. Each experiment included at least 5–6 biological replications and 3–4 technical repeats. Error bars indicate SD. (PNG 96 kb)

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Supplemental Table 1

Genebank accession numbers of NPFs genes from major crop species used in phylogenetic analysis (DOCX 70 kb)

Supplemental Table 2

The formula for MS medium used in this study (without sucrose) (DOCX 53 kb)

Supplemental Table 3

Oligonucleotide primers used in this study (DOCX 15 kb)

Supplemental Table 4

Statistic analysis result in this study (DOCX 49 kb)

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Hu, X., Zhang, J., Liu, W. et al. CsNPF7.2 Has a Potential to Regulate Cucumber Seedling Growth in Early Nitrogen Deficiency Stress. Plant Mol Biol Rep 38, 461–477 (2020). https://doi.org/10.1007/s11105-020-01206-1

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Keywords

  • Cucumber
  • NPFs
  • Nitrate supply
  • Nitrate deficiency
  • Seedling growth