Nitrogen is an essential macronutrient that is absorbed by roots and stored in leaves, mainly as ribulose-1,5-bisphosphate carboxylase/oxygenase1,2. During nitrogen deficiency (−N), plants activate leaf senescence for source-to-sink nitrogen remobilization for adaptative growth3,4,5,6. However, how −N signals perceived by roots are propagated to shoots remains underexplored. We found that ELF18-INDUCED LONG NONCODING RNA 1 (ELENA1) is −N inducible and attenuates −N-induced leaf senescence in Arabidopsis. Analysis of plants expressing the ELENA1 promoter β-glucuronidase fusion gene showed that ELENA1 is transcribed specifically in roots under −N. Reciprocal grafting of the wild type and elena1 demonstrated that ELENA1 functions systemically. ELENA1 dissociates the MEDIATOR SUBUNIT 19a–ORESARA1 transcriptional complex, thereby calibrating senescence progression. Our observations establish the systemic regulation of leaf senescence by a root-derived long non-coding RNA under −N in Arabidopsis.
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All data generated or analysed in this study are included in this Letter and its Supplementary Information files. The materials and transgenic plants generated in this study are available from the corresponding author on reasonable request. Source data are provided with this paper.
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We thank X. Qi for the gift of pYAO CRISPR–Cas9 vectors and C. Xiangbin for guidance on Arabidopsis grafting. This work was supported by core funding from Temasek Life Sciences Laboratory and Disruptive & Sustainable Technology for Agricultural Precision (DiSTAP), an interdisciplinary research group (IRG) of the Singapore MIT Alliance for Research and Technology (SMART) Centre supported by the National Research Foundation (NRF), Prime Minister’s Office, Singapore, under its Campus for Research Excellence and Technological Enterprise (CREATE) programme.
The authors declare no competing interests.
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Extended Data Fig. 1 ELENA1 transcript levels in ELENA1 and ORE1 genotypes under nitrogen deficiency (-N).
RT-qPCR analysis of ELENA1 transcript levels in WT (Nitrogen sufficient; +N), WT (-N), EL-KD#10, EL-KD#20, EL-OE#16, EL-OE#29, ore1 and ORE1 OE on -N. The value of WT ( + N) was set to 1. Data are means ± SD. n = 3 (biologically independent repeats) and individual data points as overlays. ns, no statistical difference. Asterisks indicate statistically significant difference compared with WT (-N). **P < 0.01; one-way ANOVA, Dunnett’s multiple comparison analysis.
Extended Data Fig. 2 ELENA1 does not influence growth, nitrate content and expression of ORE1 target genes under nitrogen sufficient (+N) conditions.
(a) Average fresh weight of shoots and roots in the indicated genotypes grown under +N conditions. (b) Shoot-to-root ratio of fresh weight in the indicated genotypes. (c) Total nitrate content in shoots and roots of the indicated genotypes grown under +N conditions. (d) Shoot-to-root ratio of total nitrate content in the indicated genotypes. (e) Transcript analysis of ORE1 target genes BFN1, RNS3, SAG29, SINA1 and VNI2 in the indicated genotypes treated on +N medium. ns indicated not statistically significant, P > 0.05; two-way ANOVA, multiple comparison with Dunnett post hoc analysis. Value of each gene in WT ( + N) was set to 1. (a, b, c, d, e) Data are means ± SD. n = 3 and individual data points as overlays. ns, no statistical difference; one-way ANOVA (a,b,c,d), two-way anova (e) Dunnett’s multiple comparison analysis.
(a) -N induced leaf senescence phenotype of empty vector transgenic plant, EL-OE#29, EL5M-OE #1 and EL8M-OE #1. Scale bar represents 1 cm. (b) Total chlorophyll content in different leaf groups of the indicated genotypes treated on -N medium. Leaf number, L. FW, fresh weight. (c) Expression level of ELENA1 in the indicated genotypes treated on -N medium. Value of EV ( + N) was set to 1. (b, c) Data are means ± SD. n = 3 (biologically independent samples). Each sample contained 20 seedlings and individual data points as overlays. ns, no statistical difference; one-way ANOVA, Dunnett’s multiple comparison analysis.
Extended Data Fig. 4 Time course analysis of ELENA1 and ORE1 transcripts in individual leaf during nitrogen deficiency (-N) in WT plants.
RT-qPCR analysis of ELENA1 and ORE1 transcripts in various leaves of WT plants Data are means ± SD. n = 3 (biologically independent samples) and individual data points as overlays. The expression level of L1 + N at each indicated day was set at 1.
(a) Schematic of ELENA1 genomic loci in WT and elena1. In elena1, the genomic region between -297 and -7 upstream of transcriptional start site of ELENA1 was excised by CAS9. (b) DNA sequencing result of indicated genomic loci in (A) of elena1 aligned to that of WT. (c) ELENA1 expression level of WT and elena1 treated on nitrogen deficient (-N) medium. Data are means ± SD. n = 3 (biologically independent samples). Each sample contained 20 seedlings and individual data points as overlays. Expression level in day 0 WT was set as 1. Asterisks indicate statistically significant difference compared with WT. **P < 0.01; one-way ANOVA, Dunnett’s multiple comparison analysis.
RT-qPCR analysis of ORE1 target genes BFN1, RNS3, SAG29, SINA1 and VNI2 in the indicated genotypes treated on +N or -N medium. Data are means ± SD. n = 3 (biologically independent samples). Value of each gene transcript in WT ( + N) was set to 1. Each sample contained 20 seedlings and individual data points as overlays. Asterisks indicate statistically significant difference compared with WT/WT ( + N). ns, no statistical difference.**P < 0.01; two-way ANOVA, Dunnett’s multiple comparison analysis.
Extended Data Fig. 7 Transgenic ELENA1 transcripts are root-to-shoot mobile under nitrogen deficient (-N) condition.
(a) Schematic of primer design for specific detection of transgenic ELENA1. (b) ELENA1 transcripts root-to-shoot mobility assay. Indicated graft chimeras were generated and treated on -N. Shoot and root RNA and gDNA of the graft chimeras were analysed by RT-PCR, no RT PCR, gDNA-PCR and ACT2 correspondingly for 35x PCR cycles. Numbers indicate biological repeat sample. (c) DNA sequencing of the PCR amplicon in RT-PCR reaction in (b).
Extended Data Fig. 8 Comprehensive genetic analysis of MED19a/ORE1/ELENA1 under nitrogen deficient (-N) condition.
(a)-N induced leaf senescence phenotype of the indicated genotypes. Scale bar represents 1 cm. (b) Total chlorophyll content in different leaf groups of the indicated genotypes treated on -N medium. Leaf number, L. FW, fresh weight. Data are means ± SD. n = 3 (biologically independent samples). Each sample contained 20 seedlings and individual data points as overlays. Alphabets indicate statistically significant groups with P < 0.05. One-way ANOVA, Tukey’s multiple comparison analysis.
Extended Data Fig. 9 ELENA1 transcripts show systemic root-to-shoot movement under nitrogen deficiency (-N) is independent of MED19a and ORE1.
ELENA1 expression levels in shoots and roots of WT ( + N), WT (-N), med19a-2 (-N), and ore1 (-N). Data are means ± SD. n = 3 (biologically independent samples). Value of WT ( + N) shoot was set to 1. Each sample contained 20 seedlings and individual data points were shown. ns, no statistical difference; one-way ANOVA, Dunnett’s multiple comparison analysis.
Extended Data Fig. 10 Effects of increasing concentrations of ELENA1 and antisense ELENA1 transcripts on MED19a-ORE1 complex in vitro.
Left panel, sense ELENA1 RNA; Right panel, antisense ELENA1 RNA. Experiment was repeated 3 times with similar results.
Source data and P values.
Source data and P values.
Source data and P values.
Source data and P values.
Source data and P values.
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Cheng, S.L.H., Xu, H., Ng, J.H.T. et al. Systemic movement of long non-coding RNA ELENA1 attenuates leaf senescence under nitrogen deficiency. Nat. Plants 9, 1598–1606 (2023). https://doi.org/10.1038/s41477-023-01521-x