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

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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

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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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Author notes

  1. Xi Hu, Jiali Zhang and Wenqian Liu contributed equally to this work.

Authors and Affiliations

  1. Beijing Key Laboratory of Growth and Developmental Regulation for Protected Vegetable Crops, China Agricultural University, Beijing, China

    Xi Hu, Jiali Zhang, Wenqian Liu, Qing Wang, Tao Wang, Xiaojun Li, Xiaohong Lu, Lihong Gao & Wenna Zhang

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  1. Xi Hu
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  2. Jiali Zhang
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  3. Wenqian Liu
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  4. Qing Wang
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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

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)

High Resolution Image (TIFF 7980 kb)

Supplemental Fig. 2

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)

High Resolution Image (TIFF 3009 kb)

Supplemental Fig. 3

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)

High Resolution Image (TIFF 3005 kb)

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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