
==== Front
Nat Commun
Nat Commun
Nature Communications
2041-1723
Nature Publishing Group UK London

39256370
52339
10.1038/s41467-024-52339-w
Article
HASTY-mediated miRNA dynamics modulate nitrogen starvation-induced leaf senescence in Arabidopsis
http://orcid.org/0000-0002-0432-6767
Sakuraba Yasuhito
http://orcid.org/0009-0005-3536-006X
Yang Mailun
http://orcid.org/0000-0002-3758-5933
Yanagisawa Shuichi asyanagi@mail.ecc.u-tokyo.ac.jp

https://ror.org/057zh3y96 grid.26999.3d 0000 0001 2169 1048 Plant Functional Biotechnology, Agro-Biotechnology Research Center, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Yayoi 1-1-1, Bunkyo-ku, Tokyo 113-8657 Japan
10 9 2024
10 9 2024
2024
15 791311 9 2023
30 8 2024
© The Author(s) 2024
2024
https://creativecommons.org/licenses/by-nc-nd/4.0/ Open Access This article is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License, which permits any non-commercial use, sharing, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if you modified the licensed material. You do not have permission under this licence to share adapted material derived from this article or parts of it. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by-nc-nd/4.0/.
Nitrogen (N) deficiency responses are essential for plant survival and reproduction. Here, via an expression genome-wide association study (eGWAS), we reveal a mechanism that regulates microRNA (miRNA) dynamics necessary for N deficiency responses in Arabidopsis. Differential expression levels of three NAC transcription factor (TF) genes involved in leaf N deficiency responses among Arabidopsis accessions are most significantly associated with polymorphisms in HASTY (HST), which encodes an importin/exportin family protein responsible for the generation of mature miRNAs. HST acts as a negative regulator of N deficiency-induced leaf senescence, and the disruption and overexpression of HST differently modifies miRNA dynamics in response to N deficiency, altering levels of miRNAs targeting transcripts. Interestingly, N deficiency prevents the interaction of HST with HST-interacting proteins, DCL1 and RAN1, and some miRNAs. This suggests that HST-mediated regulation of miRNA dynamics collectively controls regulations mediated by multiple N deficiency response-associated NAC TFs, thereby being central to the N deficiency response network.

Here Sakuraba et al. show that HASTY-mediated regulation of miRNA dynamics controls regulations mediated by multiple N deficiency response-associated NAC transcription factors, thereby being central to the N deficiency response network in Arabidopsis.

Subject terms

Plant signalling
Natural variation in plants
Plant stress responses
https://doi.org/10.13039/501100003382 MEXT | JST | Core Research for Evolutional Science and Technology (CREST) JPMJCR 15O5 Yanagisawa Shuichi https://doi.org/10.13039/501100001691 MEXT | Japan Society for the Promotion of Science (JSPS) 22K05368 Sakuraba Yasuhito issue-copyright-statement© Springer Nature Limited 2024
==== Body
pmcIntroduction

Nitrogen (N) is a vital plant nutrient and a principal constituent of molecules necessary for plant growth, such as amino acids and chlorophyll1. Therefore, N availability greatly affects plant growth and development. In natural ecosystems and agricultural fields, plants often encounter N-deficient environments, and thus exhibit N deficiency responses to efficiently acquire and use available N in the soil2. N deficiency responses include modifications of root architecture3 and induction of genes associated with high-affinity nitrate and ammonium transport systems2,4. Leaf yellowing due to the remobilization of N sources from older leaves to developing organs, such as young leaves and sink organs, is another N deficiency response. In the early, middle, and late stages of growth and development, plants employ leaf yellowing as a tactical strategy for enhancing nutritional fitness via nutrient relocation to sustain growth and productivity in N-deficient environments5. However, N starvation-induced leaf yellowing at the early seedling stage causes severe growth defects6. Therefore, understanding the molecular mechanisms underlying N starvation-induced leaf yellowing is necessary to sustain plant growth in N-deficient environments.

The mechanisms underlying N starvation-induced leaf yellowing have been widely studied, especially in Arabidopsis, revealing that N starvation-induced leaf yellowing is a highly complex process intricately regulated at the transcriptional, post-transcriptional, and post-translational levels7. Leaf yellowing is accompanied by the degradation of N-containing compounds, including chlorophyll. Chloroplastic proteins also undergoes degradation via multiple proteolytic pathways under N deficiency stress, because approximately 80% of N in the mesophyll cells of C3 plants is stored as chloroplastic proteins8. The degradation of N-containing compounds results in the release of amino acids, which are transported from older leaves to developing leaves via the phloem sap7,9. In Arabidopsis, several nitrate transporters, such as NITRATE TRANSPORTER1.7 (NRT1.7), NRT1.11, and NRT1.12, are involved in the N starvation-induced N remobilization10,11. Furthermore, enhanced expression of NRT1.1, encoding a dual-affinity nitrate transporter12, in shoots was shown to delay N starvation-induced leaf senescence6. However, despite the progress made in understanding the N deficiency response process, the mechanisms regulating N deficiency responses remain largely unknown.

Many transcription factors (TFs) involved in natural and dark-induced leaf senescence have been identified and characterized13,14. However, whether these TFs also regulate N starvation-induced leaf senescence has yet to be thoroughly investigated. Therefore, a limited number of TFs have been shown to play roles in regulating N starvation-induced leaf senescence. For example, ORESARA1 (ORE1)/ANAC092, one of the NAM/ATAF1/2/CUC2 (NAC) family TFs15, has been shown to act as an enhancer of N starvation-induced leaf senescence in Arabidopsis. Thus, under N-deficient conditions, leaves of ore1 knockout mutants remained green, while those of ORE1 overexpressors turned yellow much earlier than those of the wild type (WT)16. PHYTOCHROME-INTERACTING FACTOR4 (PIF4) and PIF5, basic helix-loop-helix (bHLH)-type TFs acting as negative regulators of red light signaling17, have also been shown to promote N starvation-induced leaf senescence18. Furthermore, B-BOX DOMAIN PROTEIN14 (BBX14), a B-box (BBX)-type TF, has been identified as a negative regulator of N starvation-induced leaf senescence19.

MicroRNAs (miRNAs) are key signaling molecules that regulate gene expression at the post-transcriptional level20. Some miRNAs play essential roles in regulating N deficiency responses. For instance, in Arabidopsis, three isoforms of miR164 (miR164a, miR164b, and miR164c) act as negative regulators of N starvation-induced leaf senescence by targeting the ORE1 mRNA and repressing its expression at the post-transcriptional level15. Consistently, under N deficiency stress, the expression levels of miR164s and ORE1 significantly decreased and increased, respectively16. Similarly, miR169 level decreased during N deficiency, and transgenic Arabidopsis plants overexpressing miR169 exhibited accelerated leaf yellowing phenotype under N-deficient conditions21. Furthermore, miR826 was strongly upregulated upon N deficiency, and Arabidopsis plants overexpressing miR826 exhibited improved growth with delayed leaf yellowing under N-deficient conditions22. These findings suggest that several miRNAs play a positive or negative role in regulating N deficiency-induced leaf yellowing. Moreover, small RNA sequencing (sRNA-seq) analysis of Arabidopsis plants grown under N-deficient conditions revealed that N deficiency induces dynamic changes in miRNA expression levels; the levels of more than 20 miRNAs were elevated, while those of several miRNAs were decreased upon N deficiency23,24. However, the mechanisms underlying these changes remain to be elucidated.

To investigate the molecular mechanisms regulating N deficiency responses, we identified three NAC genes involved in N starvation-induced leaf senescence, and conducted expression genome-wide association study (eGWAS) using 52 Arabidopsis accessions exhibiting natural diversity in the expression levels of these three NAC genes. Expression levels of the three NAC genes showed a strong association with polymorphisms in HASTY gene (HST), which encodes an importin/exportin family protein that physically interacts with proteins involved in the generation of mature miRNAs. Functional characterization of the HST revealed the essential role of HST-mediated regulation of miRNA dynamics in N deficiency-induced leaf senescence.

Results

Three senNAC TFs are involved in the acceleration of N starvation-induced leaf yellowing

In Arabidopsis, more than 10 NAC TFs have been characterized as regulators of leaf yellowing during natural and dark-induced leaf senescence, and therefore are referred to as senNAC TFs25–27. Since we found in previous transcriptome data28 that the expression levels of three genes encoding senNAC TFs, NAC-LIKE, ACTIVATED BY AP3/PI (NAP)/ANAC029, and ANAC055, were reduced by nitrate supplementation in N-starved seedlings, we hypothesized that some senNAC TFs play vital roles in the regulation of N starvation-induced leaf yellowing. To test this hypothesis, expression levels of 11 genes encoding senNACs were examined in WT (Col-0) Arabidopsis seedlings grown first under N-sufficient (6 mM N) and then under N-deficient (0.3 mM N) conditions for 3 or 5 days. N deficiency treatment significantly induced the expression of all genes examined, especially NAP, ANAC055, and ORE1 (Supplementary Fig. 1a). N deficiency-induced expression patterns of NAP, ANAC055, and ORE1 were very similar to that of NRT2.5, a typical N deficiency-inducible gene4 (Fig. 1a). Furthermore, among the knockout mutants for each of the 11 NAC genes (Supplementary Data 1), the nap, ore1, and anac055 mutants showed higher chlorophyll contents than that in the WT under N-deficient conditions (Supplementary Fig. 1b, d). The Fv/Fm ratio, a measure of the maximum quantum yield of photosystem II (PSII), was also higher in nap, ore1, and anac055 mutants than in the WT under N-deficient conditions (Fig. 1c, d). Consistently, expression levels of two leaf yellowing marker genes, STAY-GREEN1 (SGR1) and SGR229, were significantly lower in these mutants than in the WT (Fig. 1e, f). These results indicated that NAP, ORE1, and ANAC055 are senNAC TFs involved in the acceleration of N starvation-induced leaf yellowing.Fig. 1 Role of NAP, ORE1, and ANAC055 in N starvation-induced leaf senescence.

a Expression levels of NAP, ORE1, ANAC055, and NRT2.5 in Arabidopsis rosette leaves. RT-qPCR analysis was performed on wild-type (WT) Col-0 seedlings grown initially on 1/2 MS agar plates for 5 days and then on N-deficient (0.3 mM N) medium for the indicated number of days. Expression levels were normalized first against ACT2 transcript levels and then against the value obtained at time zero. n = 4 biologically independent samples. The same letters above each bar indicate that means did not differ significantly at the 0.05 level in Tukey’s multiple comparison test. (b–f) Total chlorophyll content (b), Fv/Fm ratio (c), shoot and Fv/Fm ratio images (d), and SGR1 (e) and SGR2 (f) expression levels of WT, nap, ore1, and anac055 seedlings. The seedlings were initially grown on 1/2 MS agar plates for 5 days and then under control (6 mM N) or N-deficient (0.03 mM N) conditions for additional 7 days. n = 5 (b, c) or 4 (e, f) biologically independent samples. b, c, e, f Asterisks above each bar indicate significant differences between the WT and other samples (**p < 0.01; two-tailed Student’s t-test). In (e, f), expression levels were normalized first against ACT2 transcript levels and then against the value obtained in WT rosette leaves. In (d), scale bars = 1 cm. g Expression genome-wide association study (eGWAS) of 52 Arabidopsis accessions using NAP, ORE1, and ANAC055 expression levels as traits of interest. Seedlings were initially grown on 1/2 MS agar plates for 5 days and then on N-deficient (0.03 mM N) medium for 5 days. In Manhattan plots, horizontal green lines indicate the significant genome-wide threshold (p-value = 5 × 10−6). Peaks consisting of single nucleotide polymorphisms (SNPs) located in the vicinity of the HASTY (HST) locus (At3G05040) are marked with a blue rectangle. Source data are provided as a Source data file.

eGWAS based on natural variation in NAP, ORE1, and ANAC055 expression levels

To identify regulatory genes controlling N deficiency responses, eGWAS was conducted using 52 Arabidopsis accessions with NAP, ORE1, and ANAC055 expression levels as traits of interest (Supplementary Fig. 2a). Wide-ranging natural variation was observed in the expression levels of these genes among the 52 Arabidopsis accessions, and this variation was highly correlated with the previously determined natural variation in reduced chlorophyll content due to N deficiency6 (Supplementary Fig. 2). Additionally, several peaks were detected in the Manhattan plot, which appeared to be associated with the expression levels of NAP, ORE1, and ANAC055 (Supplementary Fig. 3). Interestingly, the strongest association were identified in the genomic region harboring the HASTY (HST) gene (At3g05040), which encodes an importin/exportin family protein, in the eGWAS of each of the three genes (Fig. 1g), implying that HST may be involved in activation of NAP, ORE1, and ANAC055 under N-deficient conditions.

HST acts as a negative regulator of N deficiency-induced leaf yellowing

HST has been suggested previously to regulate the accumulation of miRNAs in both the nucleus and cytosol30. Furthermore, HST physically interacts with DICER-LIKE1 (DCL1), which acts as a microprocessor that generates mature miRNA duplexes from pre-miRNAs31,32. However, a relationship between HST and N deficiency responses has yet to be reported. To investigate the role of HST in regulating N deficiency responses, we expressed the HST-MYC fusion under the control of the cauliflower mosaic virus (CaMV) 35S RNA promoter in two distinct hst knockout mutants in the Col-0 background (hst-6 and hst-7). The HST-MYC transcript levels in each of the resultant transgenic lines (35S:HSTCol-0-MYC/hst-6 and 35S:HSTCol-0-MYC/hst-7) were nearly or more than 10-fold higher than the HST transcript level in Col-0 (WT) plants (Supplementary Fig. 4), indicating that these two transgenic lines are HST overexpressors.

Under control N conditions (6 mM N), the color of hst-6, hst-7, 35S:HSTCol-0-MYC/hst-6 (line 1), and 35S:HSTCol-0-MYC/hst-7 (line 3) leaves was similar to that of WT leaves; however, under N-deficient conditions (0.03 mM N), the leaves of both hst mutants turned yellow much faster than those of the WT, while the leaves of 35S:HSTCol-0-MYC/hst-6 and 35S:HSTCol-0-MYC/hst-7 seedlings, unlike WT leaves, remained green (Fig. 2a). Even under mild N deficiency (0.3 mM N), the leaves of hst seedlings turned slightly yellow, while WT leaves remained green (Fig. 2a). Furthermore, under N-deficient conditions, the Fv/Fm ratio (Fig. 2a, b), chlorophyll contents (Fig. 2c), and levels of three chloroplast proteins (Lhcb1, Lhca1, and PsbC) (Fig. 2d) were significantly lower in hst-6 leaves than in WT and 35S:HSTCol-0-MYC/hst-6 leaves. During the late-vegetative growth phase, the hst-6 mutants showed earlier leaf senescence (Fig. 2e), lower chlorophyll contents (Fig. 2f) and Fv/Fm ratio (Fig. 2g, h), and decreased shoot and root fresh weight (Fig. 2i, j) under N-deficient conditions compared with the WT; by contrast, the 35S:HSTCol-0-MYC/hst-6 plants exhibited the opposite phenotypes (Fig. 2e–j). Furthermore, seed yield per plant under N-deficient conditions was significantly lower in hst-6 than in WT plants, probably due to the reduced number of siliques (Supplementary Fig. 5), suggesting that the amount of N source translocated to sink organs may be reduced in the hst-6 mutant. Collectively, these results indicate that HST acts as a negative regulator of N starvation-induced leaf yellowing during N deficiency.Fig. 2 HST-mediated regulation of N starvation-induced leaf senescence.

a–c Shoot and Fv/Fm ratio images (a), Fv/Fm ratio (b), and total chlorophyll content of the shoots (c) of WT, hst-6, hst-7, 35S:HSTCol-0-MYC/hst-6 line 1 (L1), and 35S:HSTCol-0-MYC/hst-7 line 3 (L3) seedlings initially grown on 1/2 MS agar plates for 5 days and then under control (6 mM N) or N-deficient (0.3 mM and 0.03 mM N) conditions for 5 days. In (a), scale bars = 1 cm. d Immunoblot analysis of photosynthesis-associated proteins (Lhcb1, Lhca1, and PsbC) in rosette leaves of WT, hst-6, and 35S:HSTCol-0-MYC/hst-6 L1 seedlings initially grown on 1/2 MS agar plates for 5 days and then under control (6 mM N) or N-deficient (0.03 mM N) conditions for 5 days. α-Tubulin was served as the loading control. e–j Whole-plant images (e), total chlorophyll content (f), Fv/Fm ratio images (g), Fv/Fm ratio (h), and fresh shoot (i) and root (j) weights of 4-week-old WT, hst-6, and 35S:HSTCol-0-MYC/hst-6 L1 plants grown hydroponically under N-sufficient (6 mM N) and then under N-free conditions for 2 weeks. The 3rd or 4th rosette leaves were used for Fv/Fm ratio and chlorophyll content measurements. In (e), scale bars = 5 cm. b, c, f, g, i, j n = 5 biologically independent samples, and asterisks above each bar indicate significant differences between the WT and other samples (*p < 0.05; **p < 0.01; two-tailed Student’s t-test). Source data are provided as a Source data file.

Disruption of HST affects miRNA dynamics under N deficiency

HST is involved in the generation of mature miRNA duplexes. Thus, we performed miRNA sequencing (miRNA-seq) analysis of WT and hst-6 seedlings grown under control (6 mM N) and N-deficient (0.3 mM N) conditions. Consequently, 160 miRNAs were identified, including 107 known miRNAs and 53 novel miRNAs (NovelmiRNAs) from the non-annotated reads of the sRNA library33 (Supplementary Data 2). Statistical analysis using DESeq234 revealed that 118 miRNAs were differentially expressed under N-deficient conditions (Supplementary Data 2). Among them, the accumulation levels of 78 miRNAs were significantly decreased (FC [fold change] <0.5, p-value < 0.05), while those of 8 miRNAs were significantly increased (FC > 2, p-value < 0.05) (Fig. 3a), revealing N deficiency-induced dynamic changes in miRNA accumulation. Furthermore, the statistical analysis revealed that 109 and 102 miRNAs were differentially expressed in the hst-6 seedlings compared with WT seedlings under control and N-deficient conditions, respectively (Supplementary Data 2). Among them, the hst-6 seedlings exhibited a significant decrease (FC < 0.5, p-value < 0.05) in the accumulation levels of 84 and 79 miRNAs under control and N-deficient conditions, respectively. They also exhibited a significant increase (FC > 2, p-value < 0.05) in the accumulation levels of 5 and 5 miRNAs under control and N-deficient conditions, respectively (Fig. 3a, Supplementary Data 3–6). Furthermore, among the 79 miRNAs that significantly decreased in abundance under N-deficient conditions, 54 miRNAs were significantly downregulated in hst-6 mutants under both control and N-deficient conditions. In contrast, only NovelmiRNA-14 was significantly upregulated by N and downregulated by HST disruption (Fig. 3a). Thus, we speculated that HST might be responsible for the generation of the majority of miRNAs whose levels were reduced in response to N deficiency. Hierarchical average linkage cluster analysis and scatter plot analysis further supported this speculation (Fig. 3b, c). The miRNAs whose accumulation was downregulated by both N deficiency treatment and HST disruption included functionally characterized miRNAs, such as miR164a, miR169a, miR399f, and miR82716,21, uncharacterized miRNAs, such as miR773 and miR781, and several NovelmiRNAs.Fig. 3 HST-dependent dynamic changes in miRNA accumulation under N deficiency stress.

a Venn diagrams showing differential changes in N deprivation-induced miRNA accumulation levels in WT and hst-6 leaves. Total RNA was extracted from the shoots of WT and hst-6 seedlings grown initially on 1/2 MS agar plates for 5 days and then on N-sufficient (6 mM N; control) or N-deficient (0.3 mM N; low N [LN]) medium for 3 days. Overlaps between genes whose expression increased (fold change [FC] > 2, p < 0.05) and decreased (FC < 0.5, p < 0.05) in hst-6 shoots and those whose expression increased (FC > 2, p < 0.05) or decreased (FC < 0.5, p < 0.05) during N deficiency in WT shoots are shown by gene number. b Hierarchical clustering analysis of 168 miRNAs based on the N starvation-induced change in accumulation of each miRNA in WT shoots (WTLN/WTcontrol) and the relative value of accumulation in hst-6 shoots to that in WT shoots of each miRNA under control N conditions (hst-6control/WTcontrol) and LN conditions (hst-6LN/WTLN). Green and red rows represent downregulated genes and upregulated genes, respectively. c Scatterplot representing the relationship between the effects of N deficiency and HST disruption on the accumulation of 168 miRNAs. d Time-course analysis of miR164a, miR169a, miR397b, miR399f, miR781, and miR827 accumulation levels in WT, hst-6, and 35S:HSTCol-0-MYC/hst-6 L1 seedlings. The analysis was performed using total RNA prepared from the nuclear fractions of the shoots of WT, hst-6, and 35S:HSTCol-0-MYC/hst-6 L1seedlings grown initially on 1/2 MS agar plates for 5 days and then on control (6 mM N) or N-deficient (0.3 mM N [LN]) medium for 3 or 5 days. n = 4 biologically independent samples, and asterisks above each bar indicate significant differences between the WT and other samples (*p < 0.05; **p < 0.01; two-tailed Student’s t-test). Source data are provided as a Source data file.

To verify the results of miRNA-seq analysis, the N deficiency-induced dynamic changes in the levels of six miRNAs (miR164a, miR169a, miR397b, miR399f, miR781, and miR827) were examined in WT, hst-6, and 35S:HSTCol-0-MYC/hst-6 seedlings (Fig. 3d). The levels of these six miRNAs decreased under N deficiency, and levels of these six miRNAs were significantly lower in hst-6 seedlings than in WT seedlings, although some of these miRNAs (miR397b and miR827) were retained at high levels in 35S:HSTCol-0-MYC/hst-6 seedlings even under N deficiency (Fig. 3d). These results were consistent with the results of miRNA-seq analysis, clarifying that the knockout mutation and overexpression of HST significantly affect the dynamic changes in miRNA accumulation levels under N deficiency. Note that expression levels of NAP, ORE1, and ANAC055 as well as SGR1, a leaf yellowing-associated marker gene, were higher in hst-6 seedlings and lower in 35S:HSTCol-0-MYC/hst-6 seedlings than in WT seedlings (Supplementary Fig. 6).

miRNA781 is a negative regulator of N starvation-induced leaf yellowing

We investigated whether an uncharacterized miRNA found to be biosynthesized under the control of HST is genuinely involved in regulating N deficiency-induced leaf senescence. According to the Plant microRNA database (http://bioinformatics.cau.edu.cn/PMRD/), miR781 was predicted to bind to NAP mRNA, because of the presence of a potential miR781-binding site in the second exon of the NAP gene (Fig. 4a). Thus, RNA Ligase-Mediated Rapid Amplification of Complementary DNA 5’ End (RLM-5’RACE) was performed to investigate the miR781-directed cleavage of NAP mRNA. The main product of RLM-5’RACE was an approximately 500 bp DNA fragment (Fig. 4b). DNA sequencing analysis of 6 independent cDNA clones from the RLM-5’RACE product revealed that the 5’ ends of these cDNA clones mapped to an identical position of the sequence predicted to form base pairs with miR781 (Fig. 4a). These results suggested that miR781 binds to NAP mRNA and regulates its abundance.Fig. 4 NAP mRNA is a target of miR781.

a Positions of the miR781 sequence and the miR781-guided cleavage site in NAP mRNA. The cleavage site determined using RLM-5’RACE products is indicated by a vertical arrow in the alignment of the miR781 sequence with the miR781-like sequence in the NAP gene. b Agarose gel electrophoresis of RLM-5’RACE products. An arrowhead indicated the position of the main product. M, DNA size marker. The experiments were repeated 6 times with similar results. c Time-course analysis of NAP expression levels in the shoots of WT and 35S:miR781 L1 seedlings grown initially under 1/2 MS agar plates for 5 days and then on N-deficient (0.3 mM N) medium for 2, 3, 5, or 7 days. d–f Shoot and Fv/Fm ratio images (d), total chlorophyll content (e), and Fv/Fm ratio (f) of WT, 35S:miR781 (L1 and L2), and nap seedlings grown initially on 1/2 MS agar plates for 5 days and then on control (6 mM N) or N-deficient (0.03 mM N) medium for another 5 days. Scale bars = 1 cm. g Image showing natural leaf senescence in 6-week-old WT, 35S:miR781 (L1 and L2), and nap plants grown hydroponically under continuous white light irradiation. Scale bar = 5 cm. h Images of dark-induced leaf senescence of detached leaves collected from 3-week-old WT, 35S:miR781 (L1 and L2), and nap plants. The detached leaves after 0 and 3 days of dark incubation (DDI) are shown. Scale bar = 2 cm. i, j Changes in the miR781 accumulation level in WT plants during natural (i) and dark-induced leaf senescence (j). k, l NAP expression levels in WT and 35S:miR781 (L1) plants during natural leaf senescence (k) and dark-induced leaf senescence in detached leaves incubated in the dark for the indicated time periods (l). c, i–l Relative miR781 accumulation and NAP expression levels. Data were normalized first against ACT2 transcript levels and then against the value obtained at time 0 (c, j, l) or in 1- or 2-week-old plants (i, k). n = 4 biologically independent samples in (c, i–l) and 5 biological independent samples in (e, f). Significant differences between WT and other plants grown under the same nutrient conditions are indicated with asterisks (*p < 0.05, **p < 0.01; two-tailed Student’s t-test) in (c, e, f, k, l), and with different letters (p < 0.05; Tukey’s multiple comparison test) in (i, j). Source data are provided as a Source data file.

We subsequently generated transgenic Arabidopsis lines expressing miR781 under the control of the 35S promoter (35S:miR781 lines). The miR781 levels in 35S:miR781 lines were approximately 20-fold higher than that in the WT (Supplementary Fig. 7). In the N deficiency treatment, NAP expression level was significantly lower in 35S:miR781 seedlings than in WT seedlings at all time points examined (Fig. 4c), indicating that miR781 downregulates the NAP gene. Furthermore, like the nap knockout mutant, two independent 35S:miR781 lines exhibited delayed leaf yellowing (Fig. 4d), highly retaining the chlorophyll content (Fig. 4e) and Fv/Fm ratio (Fig. 4f) under N-deficient conditions. Moreover, like the nap mutant35, the two 35S:miRNA781 lines also exhibited delayed natural (Fig. 4g) and dark-induced (Fig. 4h) leaf senescence, retaining the chlorophyll content and Fv/Fm ratio (Supplementary Fig. 8). Consistently, the miR781 expression level dramatically decreased during the natural (Fig. 4i) and dark-induced (Fig. 4j) leaf senescence, while the NAP expression level increased. Although the increase in NAP expression during natural and dark-induced leaf senescence was observed in both WT and 35S:miR781 plants, NAP expression was much more mildly enhanced in 35S:miR781 plants (Fig. 4k, l). Note that the expression level of ETHYLENE INSENSITIVE3 (EIN3) encoding a transcriptional activator of NAP36 was only slightly reduced in 35S:miR781 seedlings after 3 days of N deficiency treatment (Supplementary Fig. 9). These results suggest that miR781 acts as a negative regulator of N starvation-induced, natural, and dark-induced leaf senescence and directly regulates the NAP mRNA level, thus clarifying the HST–miR781–NAP pathway. To obtain genetic evidence in support of this pathway, genetic epistasis between HST and NAP and between HST and miR781 was investigated by the phenotypic characterization of the hst-6 nap double mutant and the 35S:miR781/hst-6 line, a crossbreeding line generated by crossing of the hst-6 mutant with the miR781-OX line. The result indicated that effects of HST mutation in hst-6 on N starvation-induced responses were attenuated in the nap mutant or in the 35S:miR781 background (Supplementary Figs. 10 and 11), supporting the presence of the HST–miR781–NAP pathway.

T1006A amino acid substitution exerts an essential effect on HST activity in planta

To investigate whether polymorphisms in the HST coding sequence are associated with the differences in expression levels of NAP, ORE1, and ANAC055 among Arabidopsis accessions, we compared the nucleotide sequence of HST (from the translation initiation site to the stop codon) among the 52 Arabidopsis accessions. Several accessions exhibiting higher NAP, ORE1, and ANAC055 expression levels than others possessed common polymorphisms causing amino acid substitutions. HST proteins in five accessions showing high expression of NAP, ORE1, and ANAC055, i.e., Yeg-3, Yeg-4, Yeg-6, Yeg-7, and Yeg-8 (Supplementary Fig. 2), carried the K48N, V816L, and T1006A substitutions. Unlike the V816L and T1006A substitutions, the K48N substitution was found in 29 of the 52 accessions, including those displaying low NAP, ORE1, and ANAC055 expression levels (Supplementary Data 7). Therefore, the V816L and/or T1006A substitution, but not the K48N substitution, were likely critical for HST activity.

To reveal the relationship between HST polymorphisms and HST activity, the phenotypes of Yeg-3, Yeg-4, Yeg-6, Yeg-7, Yeg-8, Bur-0, and Bak-7 accessions, as well as a reference accession (Col-0), were compared under N-deficient conditions; Bur-0 and Bak-7 accessions were included in this analysis, because HST of Bur-0 also contains the K48N and V816L substitutions (Supplementary Fig. 2, Supplementary Data 7), while that of Bak-7, which was not included in the previous phenome analysis shown in Supplementary Fig. 2, also contains the K48N, V816L, and T1006A substitutions. Five days after the transfer to N-deficient conditions (0.03 mM N), the leaves of six accessions (Yeg-3, Yeg-4, Yeg-6, Yeg-7, Yeg-8, and Bak-7) turned yellow much faster than those of Col-0 (Fig. 5a). Moreover, the chlorophyll contents and Fv/Fm ratio of these six accessions were significantly lower than those of Col-0 (Fig. 5b, c). On the other hand, the leaf color, chlorophyll contents, and Fv/Fm ratio of Bur-0 and Col-0 seedlings were comparable under N-deficient conditions (Fig. 5a–c). In addition, petioles of 3-week-old Yeg-3, Yeg-4, Yeg-6, Yeg-7, Yeg-8, and Bak-7 seedlings, as well as hst-6 seedlings, were significantly longer than those of Col-0 seedlings (Supplementary Fig. 12). Thus, the T1006A substitution, which was not found in Bur-0, likely contributes to the differences in HST activity among Arabidopsis accessions.Fig. 5 Effects of the T1006A substitution in HST on N starvation-induced leaf senescence.

a–c Shoot and Fv/Fm ratio images (a), total chlorophyll content (b), and Fv/Fm ratio (c) of Col-0, Yeg-3, Yeg-4, Yeg-5, Yeg-6, Yeg-7, Yeg-8, Bak-7, and Bur-0 seedlings grown initially on 1/2 MS agar plates for 5 days and then on control (6 mM N) or N-deficient (0.03 mM N) medium for 5 days. In (a), scale bars = 1 cm. b, c n = 5 biologically independent samples. d HST transcript levels in 10-day-old pHST:HSTV816L-MYC/hst-6, pHST:HSTT1006A-MYC/hst-6, and pHST:HSTCol-0-MYC/hst-6 seedlings grown on 1/2 MS agar plates. Transcript levels of HST were normalized against those of ACT2. e–g Shoot and Fv/Fm ratio images (e), total chlorophyll content (f), and Fv/Fm ratio (g) of pHST:HSTCol-0-MYC/hst-6, pHST:HSTV816L-MYC/hst-6, and pHST:HSTT1006A-MYC/hst-6 seedlings grown initially on 1/2 MS agar plates for 5 days and then under control (6 mM N) or N-deficient (0.03 mM N) conditions for 5 days. To obtain the data shown in (d, f, and g), five or more independent lines of each transgenic genotype were analyzed (Supplementary Fig. 10), with four biological replicates per line. In (e), WT and hst-6 seedlings were used as references, and the scale bar was set at 1 cm. In (d, f, g), the whiskers indicate the range from minimum to maximum values, while the boxes extend from the 25th to 75th percentiles with the middle lines indicating the median values. n = 8 (pHST:HSTT1006A-MYC/hst-6), 5 (pHST:HSTV816L-MYC/hst-6), and 6 (pHST:HSTCol-0-MYC/hst-6) biologically independent samples, respectively. h, i Analysis of the correlation of HST expression level with total chlorophyll content (h) and Fv/Fm ratio (i). Green, blue, and red dots represent the data obtained from five or more independent lines of pHST:HSTCol-0-MYC/hst-6, pHST:HSTV816L-MYC/hst-6, and pHST:HSTT1006A-MYC/hst-6 plants, respectively. A black dot and white circle represent data from WT and hst-6 plants, respectively. j–l Shoot and Fv/Fm ratio images (j), total chlorophyll content (k), and Fv/Fm ratio (l) of Col-0, Yeg-3, pHST:HSTCol-0-MYC/Yeg3, Bak-7, and pHST:HSTCol-0-MYC/Bak-7 seedlings grown initially on 1/2 MS agar plates for 5 days and then on control (6 mM N) or N-deficient (0.03 mM N) medium for 5 days. In (j), scale bars = 1 cm. k, l n = 5 biologically independent samples. In (b–d, f–i, k, and l), asterisks above each bar indicate significant differences between WT and other samples in (b, c, k, and l) or between HSTT1006A and other samples (d, f, g) (*p < 0.05, **p < 0.01; two-tailed Student’s t-test). Source data are provided as a Source data file.

To further examine whether the T1006A substitution affects HST activity in planta, we generated transgenic Arabidopsis plants expressing Col-0-type HST or its mutant variant (harboring the V816L or T1006A substitution) under the control of the native promoter (−3000 to −1 bp, relative to the translational start site) in the hst-6 mutant background. Using the resultant transgenic plants (pHST:HSTCol-0-MYC/hst-6, pHST:HSTV816L-MYC/hst-6, and pHST:HSTT1006A-MYC/hst-6 plants), we detected variations in HST expression level, which were independent of the construct used and were probably caused by positional effects (Fig. 5d, Supplementary Fig. 13). However, the early leaf yellowing phenotype of hst-6 seedlings under N-deficient conditions (Fig. 2a) was complemented in pHST:HSTCol-0-MYC/hst-6 and pHST:HSTV816L-MYC/hst-6 plants, even when the expression levels of the introduced genes were low; this conclusion was supported by the chlorophyll content and Fv/Fm ratio of pHST:HSTCol-0-MYC/hst-6 and pHST:HSTV816L-MYC/hst-6 plants (Fig. 5e–g). Correlation analysis revealed that the chlorophyll content and Fv/Fm ratio are closely related to the expression level of HST in pHST:HSTCol-0-MYC/hst-6 and pHST:HSTV816L-MYC/hst-6 plants (Fig. 5h, i), indicating that the active HST likely has a dosage effect on leaf senescence. However, unlike pHST:HSTCol-0-MYC/hst-6 and pHST:HSTV816L-MYC/hst-6 plants, the pHST:HSTT1006A-MYC/hst-6 plants continued to show the earlier leaf yellowing phenotype of the hst-6 seedlings (Fig. 5d). Therefore, we concluded that the T1006A substitution markedly damaged the functionality of HST.

We also obtained additional evidence supporting the essential role of the HST T1006A substitution in the early leaf yellowing phenotype of some Arabidopsis accessions. We generated transgenic Arabidopsis plants expressing Col-0-type HST under the control of the native HST promoter in the Yeg-3 or Bak-7 background (pHST:HSTCol-0-MYC/Yeg-3 and pHST:HSTCol-0-MYC/Bak-7, respectively, Supplementary Fig. 14). Unlike Yeg-3 and Bak-7 accessions, the pHST:HSTCol-0-MYC/Yeg-3 and pHST:HSTCol-0-MYC/Bak-7 lines showed Col-0-like phenotypes, i.e., early leaf yellowing and reduced chlorophyll content and Fv/Fm ratio, under N deficiency stress (Fig. 5j–l).

T1006A substitution negatively affects HST-mediated miRNA accumulation

The effects of T1006A substitution on HST-mediated accumulation of miRNAs were investigated by analyzing the accumulations of 20 miRNAs that were modified in the hst-6 mutant (Fig. 6a). The result indicated that 18 miRNAs downregulated in the hst-6 mutants were similarly downregulated in hst-6, Yeg-3, Bak-7, and pHST:HSTT1006A-MYC/hst-6 seedlings and similarly accumulated in pHST:HSTV816L-MYC/hst-6 and Col-0 seedlings. In addition, 2 miRNAs upregulated in the hst-6 mutants were similarly upregulated in hst-6, Yeg-3, Bak-7, and pHST:HSTT1006A-MYC/hst-6 seedlings and similarly accumulated in pHST:HSTV816L-MYC/hst-6 and Col-0 seedlings. Time-course analysis of the accumulation levels of three miRNAs (miR164a, miR399f, and miR781) during the N deficiency treatment revealed that levels of these miRNAs were significantly lower in Yeg-3, Bak-7, and pHST:HSTT1006A-MYC/hst-6 seedlings than in Col-0 seedlings independently of the N deficiency treatment. However, levels of these miRNAs were comparable between pHST:HSTV816L-MYC/hst-6 and Col-0 seedlings (Fig. 6b). Consistently, during the N deficiency treatment, the expression levels of NAP, ORE1, and ANAC055 more dramatically increased in Yeg-3, Bak-7, and pHST:HSTT1006A-MYC /hst-6 seedlings than in Col-0 seedlings but similarly increased in pHST:HSTV816L-MYC/hst-6 and Col-0 seedlings (Fig. 6c). These results indicated that the T1006A substitution significantly decreases the accumulation of miRNAs and enhances NAP, ORE1, and ANAC055 expression.Fig. 6 T1006A substitution attenuates HASTY activity.

a Relative miRNA accumulation levels in the shoots of 10-day-old Yeg-3, Bak-7, pHST:HSTT1006A-MYC/hst-6, pHST:HSTV816L-MYC/hst-6, and hst-6 seedlings grown on 1/2 MS agar plates. The data of 20 miRNAs whose accumulation was significantly up- or downregulated in the hst-6 mutant are shown. Levels of each miRNA were normalized first against the transcript levels of ACT2 and then against the value obtained from Col-0 seedlings. b, c Time-course analysis of three miRNAs (miR164a, miR399f, and miR781) (b) and three NAC genes (NAP, ORE1, and ANAC055) (c) in the shoots of Col-0, Yeg-3, Bak-7, pHST:HSTT1006A-MYC/hst-6, pHST:HSTV816L-MYC/hst-6, and hst-6 seedlings. The seedlings were grown initially on 1/2 MS agar plates for 5 days and then on N-deficient (0.3 mM N) medium for 3 or 5 days. n = 4 biologically independent samples with three rosette leaves per sample. Asterisks indicate significant differences between WT and other samples (*p < 0.05; **p < 0.01; two-tailed Student’s t-test). d Yeast two-hybrid assays performed with HST or HSTT1006A as prey and DCL1 as bait. The DNA-binding domain (BD) and transactivation domain (AD) of yeast Gal4 served as controls. Transformants were grown on non-selective (-LW) and selective (-LWHA) media. The experiment was repeated two times with similar results. e Bimolecular fluorescence complementation (BiFC) assays. The HST–DCL1, HST1006A–DCL1, HST–RAN1, and HAT1006A–RAN1 interactions were examined in protoplasts isolated from the rosette leaves of 3-week-old Col-0 plants. The N-terminal of GFP (nGFP) was fused to HST and HST1006A, while the C-terminal region of GFP (cGFP) was fused to DCL1 and RAN1. Scale bar = 10 μm. The experiment was repeated two times with similar results. f RNA immunoprecipitation (RIP)–RT-qPCR assays for the quantitative evaluation of the interactions of HST or HST1006A with six miRNAs (miR164a, miR169a, miR395f, miR397b, miR399f, and miR781). HST-MYC and HST1006A-MYC fusion proteins were immunoprecipitated using anti-MYC antibodies from the cell lysates of 35S:HSTCol-0-MYC/Col-0 (L1) and 35S:HSTT1006A-MYC/Col-0 (L5) seedlings, respectively, grown on 1/2 MS agar plates for 12 days. UBQ10 served as a negative control. n = 4 biologically independent samples. g RNA immunoprecipitation (RIP) –RT-qPCR assays for the quantitative evaluation of the interactions between HST and three miRNAs (miR164a, miR399f, and miR781) in the presence or absence of DCL1. Cell lysates were prepared from 35S:HSTCol-0-MYC/Col-0 (L1) seedlings and from seedlings homozygous for the introduced 35S:HSTCol-0-MYC construct and dcl1 alleles (35S:HSTCol-0-MYC/Col-0 x dcl1) that were grown on 1/2 MS agar plates for 12 days. The HST-MYC fusion protein was immunoprecipitated from the cell lysate. UBQ10 served as a negative control. n = 4 biologically independent samples. Asterisks above each bar indicate significant differences between Col-0 and other samples (b, c) or between 35S:HSTCol-0-MYC/Col-0 (L1) and other samples (f, g) (*p < 0.05, **p < 0.01; two-tailed Student’s t-test). Source data are provided as a Source data file.

Effects of T1006A substitution on HST–DCL1 interaction

HST physically interacts with DCL1, a microprocessor involved in the generation of mature miRNA duplexes31. Thus, by yeast two-hybrid assay, we examined whether the T1006A substitution affects the HST–DCL1 interaction. The results revealed that the HST–DCL1 interaction was significantly weakened by the T1006A substitution in yeast (Fig. 6d), although both HST-GFP and HSTT1006A-GFP were localized in the nucleus (Supplementary Fig. 15). Consistent results were obtained in the bimolecular fluorescence complementation (BiFC) assay performed using Arabidopsis mesophyll protoplasts; co-expression of HST-nGFP with DCL1-cGFP in protoplasts resulted in GFP fluorescence, but no GFP fluorescence was observed when HSTT1006A-nGFP was co-expressed with DCL1-cGFP (Fig. 6e). Similar results were obtained using RAS-RELATED NUCLEAR PROTEIN-1 (RAN1), another HST-interacting protein37 (Fig. 6e).

Since HST physically interacts with miRNAs32, we also examined whether the T1006A substitution of HST affects its interaction with miRNAs by the RNA immunoprecipitation (RIP) assay using Arabidopsis transgenic plants expressing HST-MYC or HSTT1006A-MYC under the control of the 35S promoter in Col-0 background (35S:HSTCol-0-MYC/Col-0 and 35S:HSTT1006A-MYC/Col-0 plants) (Supplementary Fig. 16). HST interacted with miR164a, miR169a, miR395f, miR397b, miR399f, and miR781, but the T1006A substitution greatly decreased these interactions (Fig. 6f). Furthermore, the interactions between HST and miRNAs weakened in the dcl1-9 mutant38 background (Fig. 6g), suggesting the requirement of DCL1 in the interaction of HST with miRNAs. Collectively, these results demonstrate that the T1006A substitution decreases the interaction ability of HST with DCL1 and miRNAs, leading to reduced accumulation of specific miRNAs.

N deficiency decreases the interaction of HST with RAN1 and DCL1

To understand how N deficiency modulates HST activity to control N deficiency responses, we first investigated the changes in HST expression and HST protein levels during N deficiency. However, expression levels of HST and DCL1 only slightly decreased in 7 days of N deficiency treatment (Fig. 7a). Similarly, the protein levels of HST-MYC and HSTT1006A-MYC in pHST:HSTCol-0-MYC/hst-6 and pHST:HSTT1006A-MYC/hst-6 seedlings, respectively, hardly changed during the N deficiency treatment (Fig. 7b). Given these results, we hypothesized that the HST activity is modulated post-translationally by N deficiency stress. Thus, we examined whether N deficiency treatment alters the HST-DCL and HST-RAN1 interactions in vivo. In the BiFC assay, GFP fluorescence derived from the interaction between HST and DCL1 or RAN1 was observed in protoplasts isolated from plants grown under control (6 mM N) conditions but hardly detected in protoplasts isolated from plants grown under N-deficient (0.3 mM) conditions (Fig. 7c), suggesting that N deficiency alters the interaction of HST with DCL1 and RAN1. Furthermore, co-immunoprecipitation (Co-IP) assay using the 35S:HSTCol-MYC/35S:DCL1-FLAG plants, which were generated by crossing 35S:HSTCol-MYC/Col-0 plants with transgenic plants expressing DCL1-FLAG under the control of the 35S promoter (35S:DCL1-FLAG/Col-0 plants line 1; Supplementary Fig. 17), revealed that DCL1-FLAG protein is co-immunoprecipitated with HST-MYC protein in cell lysates from 35S:HSTCol-MYC/35S:DCL1-FLAG plants grown under control conditions but barely co-immunoprecipitated in cell lysates from 35S:HSTCol-MYC/35S:DCL1-FLAG plants exposed to N deficiency (Fig. 7d). Moreover, the RIP assay performed using 35S:HSTCol-MYC/Col-0 seedlings revealed that N deficiency treatment decreased the amounts of miRNAs co-immunoprecipitated with HST, indicating that N deficiency also reduces the interactions between HST and miRNAs involved in N deficiency-induced leaf senescence (Fig. 7e). We also examined whether DCL1 is involved in N starvation-induced leaf senescence by phenotypic analysis of dcl1-9 mutant38 under N deficiency. As a result, the leaves of dcl1-9 seedlings, similar to hst-6 seedlings, were found to turn yellow faster than those of WT seedlings (Supplementary Fig. 18). Collectively, these results suggest that N deficiency significantly reduces the interactions of HST with DCL1 and RAN1, thereby reducing the generation of leaf senescence-related miRNAs.Fig. 7 N deficiency-induced reduction in the interactions of HST with its partner proteins and miRNAs.

a Time-course analysis of the expression levels of HST and DCL1 in the rosette leaves of WT (Col-0) seedlings grown initially on 1/2 MS agar plates for 5 days and then on N-deficient (0.3 mM N) medium for the indicated number of days. Each gene expression level was normalized first against the ACT2 transcript level and then against the value obtained from the samples at time zero. n = 4 biologically independent samples. Significant differences among samples are indicated with different letters (p < 0.05; Tukey’s multiple comparison test). b Immunoblot analysis. HST-MYC and HST1006A-MYC fusion proteins were extracted from the cell lysates of pHST:HSTCol-0-MYC/hst-6 and pHST:HSTT1006A-MYC/hst-6 seedlings grown initially on 1/2 MS agar plates for 5 days and then on N-deficient (0.3 mM N) medium for the indicated number of days, and detected using anti-MYC antibodies. α-Tubulin was used as the loading control. c BiFC assays. The HST-nGFP construct was co-transfected with the DCL1-cGFP or RAN1-cGFP construct into protoplasts isolated from the rosette leaves of 3-week-old Col-0 plants grown hydroponically in control (6 mM N) or N-deficient (0.3 mM N) medium. Scale bar = 10 μm. d Co-immunoprecipitation (co-IP) assay of HST-MYC and DCL1-FLAG proteins using 35S:HSTCol-0-MYC/35S:DCL1-FLAG seedlings grown initially on 1/2 MS agar plates for 5 days and then on N-deficient (0.3 mM N) medium for the indicated number of days. α-Tubulin was used as the loading control. The 35S:HSTCol-0-MYC/35S:DCL1-FLAG plant is the T3 progeny of the 35S:HSTCol-0-MYC/Col-0 (L1) × 35S:DCL1-FLAG/Col-0 (L1) cross, homozygous for the two introduced constructs: 35S:HSTCol-0-MYC/Col-0 and 35S:DCL1-FLAG/Col-0. e RIP–RT-qPCR assays. The HST-MYC protein and six miRNAs (miR164a, miR169a, miR395f, miR397b, miR399f, and miR781) were co-immunoprecipitated using the cell lysates of 35S:HSTCol-0-MYC/Col-0 seedlings that were grown initially on 1/2 MS agar plates for 5 days and then on N-deficient (0.3 mM N) medium for the indicated number of days. Co-immunoprecipitated miRNAs and UBQ10 transcripts (negative control) were quantified by RT-qPCR. n = 4 biologically independent samples. Asterisks above each bar indicate significant differences between samples that were collected before and after 3 or 5 days of low N treatment (*p < 0.05, **p < 0.01; two-tailed Student’s t-test). b–d These experiments were repeated at least two times with similar results. Source data are provided as a Source data file.

Discussion

Previous studies revealed the fundamental pathway of miRNA biogenesis in plants, which proceeds by a step-by-step RNA processing mechanism. In this pathway, miRNA genes are first transcribed by RNA polymerase II to synthesize primary transcripts (pri-miRNAs) with a hairpin structure. Next, the pri-miRNAs undergo processing by DCL1 with the help of the double-stranded RNA (dsRNA)-binding protein HYPONASTIC LEAVES1 (HYL1) and the zinc-finger protein SERRATE (SE) to produce pri-miRNA intermediates and subsequently miRNA/miRNA* duplexes39. Then, miRNA duplexes are 2’-O-methylated and exported from the nucleus to the cytoplasm to interact with ARGONAUTE1 (AGO1) and form the RNA-induced silencing complex (RISC)40, which inspects target mRNAs, triggering gene silencing39. Previous studies also identified a number of factors involved in the regulation of miRNA abundance in plants, revealing that the intracellular miRNA abundance in plants is under multiple levels of tight control, including transcription, processing, RISC assembly, and miRNA–mRNA interaction41. Furthermore, it has been shown that both developmental and environmental factors induce large-scale changes in miRNA profiles in various organisms. For instance, aberrant expression of miRNAs is one of the common features of a variety of human diseases42. However, despite identifying the miRNA biogenesis pathway and observing large-scale changes in miRNA profiles in response to various stimuli, the physiological importance of dynamic changes in miRNA profiles under stress conditions and the molecular mechanisms responsible for the dynamic changes have yet to be clarified.

In the present study, we showed, by the eGWAS of Arabidopsis accessions with differential expression levels of NAP, ORE1, and ANAC055 that encode N deficiency response-associated senNAC TFs, that HST activity modulates the biogenesis of N deficiency-related miRNAs, including miR164 targeting the ORE1 transcripts and miRNA781 targeting the NAP transcripts, in response to N deficiency. Therefore, we propose that HST-mediated regulation of miRNA dynamics is a regulatory mechanism that controls N deficiency responses in plants. This mechanism is likely central to the network regulating N deficiency responses, because it regulates the activity of multiple N deficiency-responsive TFs. On the other hand, the finding that HST activity is closely linked to the biogenesis of selective miRNAs provides a clue to reveal how miRNA profiles dynamically change in response to developmental and environmental stimuli.

Given its high homology with animal exportin proteins, HST was assumed to be involved in the nuclear export of miRNAs30; however, subsequent studies revealed that HST acts as a component of the microprocessor complex in the early step of miRNA biogenesis32,43. In this study, we showed that HST was required for the biogenesis of most of the 78 miRNAs whose accumulation levels were reduced by N deficiency stress and that the biogenesis of 63 out of 93 miRNAs whose accumulation levels were lower in the hst-6 mutant than in the WT was reduced by N deficiency stress (Fig. 3a, b). This result supports the idea that HST is involved in miRNA biogenesis and also suggests that HST activity is more closely associated with the biogenesis of miRNAs negatively acting on N deficiency responses. In this context, the reduced interactions of HST with DCL1, RAN1, and miRNAs under N-deficient conditions (Fig. 7c–e) are an important clue for revealing how HST regulates N deficiency responses, because a reduction in such interactions is probably responsible for the large-scale changes in the miRNA profile in response to N deficiency, including reduced miRNA781 expression leading to an increase in NAP mRNA abundance (Fig. 3, Supplementary Fig. 19). Elucidating how such interactions are altered in response to N deficiency would help us to understand how the biogenesis of specific miRNAs associated with particular biological processes is regulated in response to developmental and environmental factors for achieving optimal growth in each environment. HASTY homologs are widely conserved in plants44, and the rice HASTY homolog, CROWN ROOT DEFECT1 (CRD1), was shown to act as a regulator of crown root development45. However, it is unknown whether CRD1 and HASTY homologs in other plant species are involved in N deficiency responses and other environmental responses. Functional characterization of HASTY homologs in other plant species would clarify whether HASTY-mediated miRNA dynamics is an important process common to many plant species for N deficiency responses.

On the other hand, clarifying the physiological role of each miRNA whose accumulation is modulated in response to N deficiency, depending on HST activity, may assist in fully understanding the regulation of N deficiency responses in plants. Although we identified 78 and 8 miRNAs showing reduced and increased accumulation levels, respectively, under N deficiency (Fig. 3a), the roles of most of these miRNAs still need to be elucidated. However, previous studies revealed that miR164 and miR169 regulate N starvation-induced leaf senescence16,21. Furthermore, we found that miR781 reduces the mRNA level of NAP, encoding a NAC TF-type central regulator of leaf senescence35, and acts as a negative regulator of leaf senescence (Fig. 4). Since the expression levels of these three miRNAs were strongly downregulated in the hst-6 mutant (Fig. 3d), at least these three miRNAs play important roles in the HST-mediated regulation of N starvation-induced leaf senescence. By a survey of the conservation of miRNAs detected in 74 plant species, we found that miR164 and miR169, two of the three miRNAs of interest in this study, are widely conserved in plants, including monocots (Supplementary Table 8). Furthermore, miR169 in rice has been shown to be involved in the regulation of N deficiency responses by directly targeting mRNA of NF-YA genes45. Thus, several miRNA-target gene pairs may be widely conserved modules in the plant N deficiency response network. On the other hand, miR781 is conserved only in some Brassica species (Supplementary Table 8), suggesting that the miR781-NAP module is critical for N deficiency only in some Brassica species. Further characterization of miRNAs, whose expression is regulated by N deficiency in an HST-dependent manner, will lead to a full understanding of the significance of miRNA-mediated regulation in N deficiency responses. Given the nature of miRNAs, the identification of the target genes of each miRNA may reveal the miRNA-mediated N deficiency response network in plants.

Deletion of a part of the N-terminal region (107–322 aa) of HST altered its subcellular localization, reducing its ability to interact with DCL1 and miRNAs, while the deletion of part of its C-terminal region (963–1202 aa), including the 1006th threonine residue (T1006), did not affect the nuclear localization of HST32. For this reason, the C-terminal region of HST was not well characterized previously. However, we found that the T1006A substitution of HAT significantly reduced its interaction with DCL1 (Fig. 6d, e) and miRNAs (Fig. 6f), indicating that not only the N-terminal region but also the 1006th threonine residue in the C-terminal region is crucial for HST function. X-ray crystallographic analysis of the EXPORTIN5 protein revealed that the N-terminal and C-terminal regions of EXPORTIN 5 are located close together due to its U-shaped structure for binding to miRNAs46. Accordingly, the T1006A substitution may affect the function of its N-terminal region, leading to the reduced ability to interact with DCL1 and miRNAs. Alternatively, it is still possible that even though the T1006A substitution does not affect the nuclear localization of HST, it is vital for HST activity in the nucleus. By comparing the amino acid sequences of HST proteins encoded by different HST alleles, the significance of the 1006th threonine residue was first identified. Then, the T1006A substitution was found to induce different HST activity levels in distinct Arabidopsis accessions, which were at least partly responsible for the different speeds of N starvation-induced leaf senescence in these accessions. Thus, T1006 is undoubtedly critical for HST activity. Therefore, investigating the role of T1006 in maintaining biochemical and structural properties, and consequently the activity of HST would be precious for understanding the mechanism that regulates HST activity and then HST-mediated miRNA biogenesis in response to N deficiency.

DCL1 forms aggregates, called dicing bodies, with HYL1, SE, and DEAD-box RNA helicases, such as RNA HELICASE 6 (RH6), RH8, and RH12, through the SE-mediated liquid–liquid phase separation in Arabidopsis47,48. The formation of dicing bodies is believed to be required for the storage and assembly of miRNA processing complexes. However, the nuclear localization pattern of the HST-GFP protein appears to be different from those of DCL1-YFP, HYL-YFP, and SE-YFP fusion proteins, which were enriched in dicing bodies with a diameter of 0.2–0.8 μm32,43. Since the nuclear localization pattern of the HST-GFP protein has been analyzed only under N-sufficient conditions32, it is currently unknown whether dicing bodies also contain the HST. Therefore, examining the formation of dicing bodies under various nutrient stress conditions and their relationship with HST-mediated regulation of N deficiency responses may provide a clue to understand the mechanism of HST-mediated N deficiency responses.

GWAS has been conducted in several studies to identify N deficiency response-associated genes in plants. In fact, GWAS using Arabidopsis accessions showing differences in lateral root growth revealed a strong association between lateral root growth under N deficiency and polymorphisms in the auxin biosynthetic gene YUCCA849. Similarly, GWAS of root morphology traits in rice seedlings grown under different N conditions revealed N deficiency response-associated genes50. Furthermore, through the GWAS of Arabidopsis accessions showing differences in N starvation-induced reduction of chlorophyll content, we found a correlation between N deficiency responses and NRT1.1 function in shoots6. However, in the present study, we employed eGWAS, which has been used extensively in animal studies51 but less so in plants52, to identify regulatory genes involved in the gene regulatory network responsible for N deficiency responses. Using this approach, we found a mechanism regulating N deficiency responses, and thus demonstrated the potential of eGWAS in plant studies. The HST-mediated regulation of the biogenesis of selective miRNAs is an unanticipated mechanism distinct from the known regulatory mechanisms of N deficiency responses, which are controlled by single or multiple homologous TFs. However, this mechanism integrates regulations caused by different TFs and is superordinate. Therefore, eGWAS based on expression levels of TF genes may be one of the best experimental approaches to discover mechanisms integrating regulations by individual TFs as well as key regulatory genes involved in specific physiological processes in plants.

Although additional peaks were found in our eGWAS, their relevance to N deficiency responses was not examined in this study. However, these peaks may indicate gene loci involved in the regulation of N deficiency responses. In fact, another peak was coincident with SNPs at the NITRATE INDUCIBLE GARP-TYPE TRANSCRIPTIONAL REPRESSOR1.1 (NIGT1.1) locus (Supplementary Fig. 3), which encodes a GARP-type transcriptional repressor that downregulates nitrate-inducible genes53 and controls N starvation-responsive nitrate uptake28. Therefore, the characterization of additional peaks observed in our eGWAS may reveal other genes and polymorphisms related to N starvation-induced leaf senescence.

Methods

Plant materials and growth conditions

In this study, 52 Arabidopsis accessions, including Col-0, Alst-1, Bay-0, Boot-1, Bu-0, Bur-0, Can-0, Chat-1, Com-1, Da-0, Di-1, Dra-2, Edi-0, Ga-2, Go-0, Gr-5, Gu-1, H55, Is-1, Ka-0, Kn-0, Kro-0, Kz-9, Lag2-2, Lc-0, Ler-0, Lis-0, Lm-2, N7, Per-1, Pog-0, Pu2-7, Rubezhone-1, S96, Sei-0, Sorbo, Sq-8, Ta-0, Tscha-1, Tu-0, Ty-0, Uk-2, Ullapool-4, Ullapool-8, Wc-2, Yeg-3, Yeg-4, Yeg-6, Yeg-7, Yeg-8, and Zu-1, were used for phenotypic analysis. The above Arabidopsis accessions and Arabidopsis mutants, including hst-6 (CS24279), hst-7 (CS24280), dcl1-9 (CS3828), onac002/ataf1 (SALK_090242), anac016 (SALK_001597), anac019 (SALK_096310), anac029/nap (SALK_005010), anac032 (SALK_128894), anac046 (SAIL_857_B11), anac055 (SALK_014331), anac059/ORS1 (SALK_204517), anac072 (SALK_063576), anac081/ataf2 (SALK_015750), and anac092/ore1 (SALK_090154), were obtained from the Arabidopsis Biological Resource Center (ABRC) at Ohio State University, OH, USA. Since all Arabidopsis mutants and all generated transgenic plants, except for the 35S:HSTCol-0-MYC/Yeg3 and 35S:HSTCol-0-MYC/Bak-7 lines, were in the Col-0 background, Col-0 was used as the WT.

Arabidopsis seeds were surface-sterilized with 0.5% NaClO, followed by 99.5% ethanol, and then rinsed with distilled water three times. The surface-sterilized seeds were cold-stratified at 4 °C for 3–4 days, and then sown on 1/2 MS agar plates that contained half-strength Murashige-Skoog (1/2 MS) salts54, 0.9% [w/v] agar, 0.5% [w/v] sucrose, and 3 mM MES-KOH (pH 5.7). Then, seedlings were grown under continuous light conditions (70 μmol m−2s−1) at 23 °C. To examine N deficiency-induced leaf senescence, 5-day-old seedlings grown on 1/2 MS agar plates were grown for 5 or 7 days on agar plates containing 6, 0.3, or 0.03 mM N nutrients, which were prepared by replacing 30 mM N nutrients (10 mM KNO3 and 10 mM NH4NO3) in 1/2 MS medium with 6 mM (2 mM KNO3 and 2 mM NH4NO3), 0.3 mM (0.1 mM KNO3 and 0.1 mM NH4NO3), or 0.03 mM (0.01 mM KNO3 and 0.01 mM NH4NO3) N nutrients, under continuous light conditions (70 μmol m−2s−1) at 23 °C. To examine N-deficiency-induced leaf senescence during the late-vegetative growth phase, seedlings were hydroponically grown in solution containing 1/2 MS salts and 3 mM MES-KOH (pH 5.7) for 2 weeks and then in solution containing 1/2 N-free MS salts, 3 mM MES-KOH (pH 5.7), and 6 mM N (2 mM KNO3 and 2 mM NH4NO3) or that containing 1/2 N-free MS salts and 3 mM MES-KOH (pH 5.7) for another 2 weeks under continuous light conditions (70 μmol m−2s−1) at 23 °C. On the other hand, seedlings hydroponically grown in nutrient solution containing 1/2 MS salts and 3 mM MES-KOH (pH 5.7) for 2 weeks were further grown in nutrient solution containing 1/2 N-free MS salts, 3 mM MES-KOH (pH 5.7), and 6 mM N (2 mM KNO3 and 2 mM NH4NO3) or that containing 1/2 N-free MS salts and 3 mM MES-KOH (pH 5.7) for 5 weeks to investigate seed development under different N conditions. To examine the dark-induced leaf senescence phenotype, plants were grown on nutrient-containing peat moss (Jiffy-7; Sakata Seed Co., Yokohama, Japan) under continuous light conditions (70 μmol m−2s−1) at 23 °C for 3 weeks. Then, rosette leaves were detached from plants and floated on 3 mM MES-KOH buffer (pH5.7) in the dark for 3 days. To examine natural leaf senescence, plants were grown on nutrient-containing peat moss under continuous light conditions (70 μmol m−2s−1) at 23 °C for 6 weeks.

Determination of chlorophyll contents

Rosette leaves were homogenized with zirconia beads in 80% ice-cold acetone55. Then, the homogenates were centrifuged at 12,000 × g for 10 min to collect supernatants. According to the formula of Porra et al.56, the absorbance of each extract was measured at 647 and 664 nm, and chlorophyll contents were calculated.

Reverse transcription and quantitative PCR analysis

Extraction of total RNA from plant materials was performed using the ISOSPIN Plant RNA Kit (cat. no. 310–08171, NIPPON GENE Co., Ltd., Tokyo, Japan), according to the manufacturer’s instructions. Reverse transcription (RT) was performed from 500 ng of total RNA using SuperScript™ II reverse transcriptase (cat. no. 18064014, Thermo Fisher Scientific, Waltham, MA) and oligo(dT)15 primer. Then, quantitative PCR (qPCR) was conducted on the StepOnePlus instrument (Applied Biosystems, Foster City, CA) using the KAPA SYBR FAST qPCR Kit (cat. no. KK4600, KAPA Biosystems Inc., Wilmington, MA) and gene-specific primers (Supplementary Data 9). Transcript levels of each gene were normalized relative to that of ACTIN2 (ACT2). The number of biological replicates in each experiment is indicated in the figure legends.

Measurement of Fv/Fm ratio

Chlorophyll fluorescence and Fv/Fm ratio were measured using a kinetics multispectral fluorescence imaging system (FluorCam 800 MF; Photon System Instruments, Brno, the Czech Republic), according to the manufacturer’s instructions.

Plasmid construction and plant transformation

All plasmids used to generate transgenic plants were constructed with PCR-amplified DNA fragments and the Gateway® cloning system. PCRs were performed using genomic DNA (to clone promoter regions) or cDNA libraries (to clone cDNAs) and gene-specific primers (Supplementary Data S9).

To generate 35S:HSTCol-0-MYC/Col-0, 35S:HSTCol-0-MYC/hst-6, and 35S:HSTCol-0-MYC/hst-7 transgenic Arabidopsis lines, a binary vector was generated by cloning the HST cDNA (encoding Col-0-type HST; HSTCol-0) between the 35S promoter and a sequence encoding a 4-copy MYC epitope tag (4 × MYC) in the pGWB17 Gateway binary vector (Nakagawa et al., 2007). To generate 35S:HSTT1006A-MYC/Col-0 lines, a binary vector was generated by cloning a mutant version of HSTCol-0 cDNA, which carried point mutation for the T1006A substitution, between the 35S promoter and a sequence encoding a 4-copy MYC epitope tag (4 × MYC) in the pGWB17 Gateway binary vector. To generate pHST:HSTCol-0-MYC/hst-6, pHST:HSTV816L-MYC/hst-6, pHST:HSTT1006A-MYC/hst-6, pHST:HSTCol-0-MYC/Yeg-3, and pHST:HSTCol-0-MYC/Bak-7 lines, a DNA fragment of the Col-0-type HST promoter (−3000 to −1 bp, relative to the translational start codon) was cloned upstream of 4 × MYC in the pGWB16 Gateway binary vector57, together with HSTCol-0 cDNA or its mutant versions with point mutations for the V816L or T1006A substitution (HSTV816L or HSTT1006A, respectively). The mutant versions of HST cDNA were generated by introducing the corresponding point mutations into HSTCol-0 cDNA using the megaprimer PCR method58. To construct a binary vector for generating 35S:miR781 lines, a genomic DNA containing the pre-miR781 sequence was PCR-amplified from Col-0 genomic DNA and then cloned downstream of the 35S promoter in the pMDC32 Gateway binary vector59. To construct a binary vector containing the 35S:DCL1-FLAG cassette for the generation of 35S:DCL1-FLAG lines, the Col-0-type DCL1 cDNA was cloned upstream of the sequence encoding a FLAG epitope tag in the pGWB11 gateway vector55. All constructs generated were verified by sequencing. Agrobacterium-mediated transformation of Arabidopsis plants was performed using the Agrobacterium tumefaciens strain GV3101 and the floral-dip method60. Transgenic plants in the T2 generation with T-DNA insertion(s) at a single locus were selected, and homozygous T3 plants were used for all analyses.

eGWAS

eGWAS was performed using the easyGWAS web interface (https://easygwas.ethz.ch/). The genome sequence information of the 52 naturally occurring Arabidopsis accessions used in this study was obtained from the 1001 genome website (https://1001genomes.org/). A Manhattan plot was generated using the accelerated mixed model.

Immunoblot analysis

Seedling samples were ground in liquid N2, and then 10-mg aliquots of the ground samples were homogenized in 100 μL of SDS-PAGE sample loading buffer (10% [w/v] glycerol, 50 mM of Tris-HCl [pH 8.0], 2% [w/v] SDS, 6% [v/v] 2-mercaptoethanol, and 0.003% [v/v] bromophenol blue). The homogenates were centrifuged at 10,000 × g for 3 min, and the obtained supernatants were incubated at 80 °C for 5 min. A 4 μL aliquot of each supernatant was subjected to SDS-PAGE. The separated proteins were electroblotted onto immobilon-P transfer membranes (cat. no. IPVH00010, Millipore), and then detected using antibodies directed against Lhca1 (cat. no. 01005, 1:10000 dilution, Agrisera, Vännäs, Sweden), Lhcb1 (cat. no. AS09522, 1:10000 dilution, Agrisera), PsbC (cat. no. AS111787, 1:5000 dilution, Agrisera) and α-tubulin (cat. no. AS10680, 1:2000 dilution, Agrisera). Peroxidase activity of the secondary antibody (anti-rabbit IgG HRP-linked antibody; cat. no. 7074, 1:10000 dilution, Cell Signaling Technology Japan, Tokyo, Japan) was visualized with Supersignal West Dura Extended Duration Substrate (cat. no. 34075, Thermo Fisher Scientific).

miRNA-seq

To prepare libraries for miRNA-seq, total RNA was extracted from the shoots of Col-0 and hst-6 seedlings initially grown on 1/2 MS agar plates for 5 days and then grown on control (6 mM N) and N-deficient (0.3 mM N) medium for 3 days. Then, library preparation and miRNA-seq using the Illumina HiseqXten/Novaseq/MGI2000 System were performed by Azenta Japan Corp. (Tokyo, Japan). Raw data were produced by calculating the average of three independent biological replicates. Statistical analysis of differential expression was conducted utilizing DESeq234, and a difference was considered significant if the p-value was less than 0.05. The miRNA-seq data were deposited in the Gene Expression Omnibus repository of the National Center for Biotechnology Information database under the accession number GSE266624.

miRNA quantitation

Five-day-old Arabidopsis seedlings grown on 1/2 MS agar plates were further grown under control (6 mM N) or N-deficient (0.3 mM N) conditions for 3–5 days. Leaf tissue samples were frozen in liquid N2 and ground to powder using a motor, and the cell wall-disrupting buffer (10 mM K2PO4 [pH 7.0], 100 mM NaCl, 10 mM 2-mercaptoethanol, 1 M hexylene glycol)30 was added. The mixtures were then filtered through the Miracloth (cat. no. 475855, Millipore) to remove remaining tissues and cell debris and centrifugated at 1500 × g for 10 min to obtain nuclear pellets. RNA extraction from the nuclear pellets was performed using the Plant RNeasy Mini kit (cat. no. 74904, Qiagen K.K., Tokyo, Japan). Subsequently, miRNA quantitation was performed as described previously61. In brief, RT reaction mixtures containing 1 μL of RNA sample (200 ng) and 1 μM of a stem-loop RT primer, which contained a common backbone (a 41-nt sequence) and an 8–10 nt sequence at the 3’ end of an individual miRNA (Supplementary Data 9), at 16 °C for 30 min. Then, RT was performed under the following conditions: 45 cycles of 30 s at 30 °C, 30 s at 42 °C, and 1 s at 50 °C. To terminate RT, the reaction mixtures were incubated at 85 °C for 5 min. Then, qPCR analysis was carried out using miRNA-specific forward primers and a universal reverse primer (Supplementary Data 9).

RLM-5’RACE

Mapping of miR781-directed cleavage in the mRNA sequence of NAP was conducted by the RNA Ligase-Mediated Rapid Amplification of Complementary DNA 5’ End (RLM-5’RACE) method62, using the GeneRacer Advanced RACE Kit (cat. no. L150001, Thermo Fischer Scientific) and poly (A)+-enriched RNA extracts isolated from Col-0 seedlings. A 1 kb DNA ladder (cat. no. N3232S, New England Biolabs, MA, USA) was used as a DNA marker in DNA electrophoresis to detect 5’RACE products. Gene-specific and adapter-specific primers listed in Supplementary Data 9 were used in RLM-5’RACE.

Yeast two-hybrid assay

Y2H assays were performed using yeast (Saccharomyces cerevisiae) strain AH109, as described previously63. Yeast was transformed with plasmids constructed based on pGBT9 and pGADT7 using lithium acetate-polyethyleneglycol solution. After transformation with the plasmids of interest, the transformants were selected at 30 °C on synthetic defined (SD) medium lacking leucine (Leu) and tryptophan (Trp) (SD/-Leu/-Trp), and single colonies were streaked on plates containing SD/-Leu/-Trp or SD medium lacking Leu, Trp, histidine (His), and adenine (Ade) (SD/-Leu/-Trp/-His/-Ade). Plates were photographed after 5 days of incubation at 30 °C.

BiFC assay

To construct plasmids for BiFC assays, the HSTCol-0, HSTT1006A, DCL1, and RAN1 cDNAs were first cloned into pENTR/D-TOPO and then introduced into the Gateway-compatible BiFC vector pB4cGGW or pB4nGGW64. Protoplasts were isolated from the rosette leaves of Col-0 seedlings grown hydroponically under control (6 mM N) or N-deficient (0.3 mM N) conditions, as described previously55. Co-transfection of pB4cGGW derivatives with pB4nGGW derivatives into Arabidopsis protoplasts was performed as described previously65. The transfected protoplasts were incubated at room temperature in the dark for 18 h before observing under a fluorescence microscope (BX51; Olympus Co., Tokyo, Japan).

Co-immunoprecipitation (Co-IP) assay

35S:HSTCol-MYC/35S:DCL1-FLAG plants, which were generated by crossing 35S:HSTCol-MYC/Col-0 with 35S:DCL1-FLAG/Col-0 lines, were initially grown on 1/2 MS agar plates for 5 days and then grown on N-deficient (0.3 mM N) medium for the indicated number of days. Then, total protein extracts were prepared from whole seedlings as previously described66, and incubated with protein G agarose beads (Roche, Basel, Switzerland) conjugated with anti-MYC antibody (cat. no. #05-419, clone 9E10, Millipore, Burlington, MA) for 2 h. Then, the beads were washed with Co-IP buffer66, and proteins were eluted from the beads with SDS-PAGE sample loading buffer at 80 °C for 3 min. The extracted proteins were separated on SDS-polyacrylamide gels, transferred onto an Immobilon-P transfer membrane (Merck Millipore), and detected using anti-MYC (cat. no. ab32072, 1:1000 dilution, abcam, Cambridge, UK), anti-FLAG (cat. no. ab213519, 1:1000 dilution, abcam), and -α-tubulin (cat. no. AS10680, 1:2000 dilution, Agrisera) antibodies, followed by an anti-mouse or -rabbit IgG HRP-linked antibody (cat. no. 7074, 1:2000 dilution, Cell Signaling Technology Japan) and Supersignal West Dura Extended Duration Substrate (Thermo Fisher Scientific).

RIP assay

Transgenic (35S:HSTCol-0-MYC/hst-6 and 35S:HSTT1006A-MYC/hst-6) and WT seedlings grown on 1/2 MS agar plates for 12 days were incubated in 1% (v/v) formaldehyde for 15 min under vacuum. Then, nuclear extracts were prepared, as described previously67, and sonicated using a Biorupter II (COSMO BIO CO., LTD, Tokyo, Japan). Protein G-agarose beads conjugated to anti-MYC polyclonal antibody (cat. no. ab32072, abcam) were used for immunoprecipitation. Proteinase K (cat. no. 3115836001, Merck, Burlington, MA) and RNase-free DNase (cat. no. M6101, Promega K.K., Tokyo, Japan) were used to digest the protein and genomic DNA, respectively. RNA was purified using the RNeasy Mini Kit, and qPCR was performed on the StepOnePlus™ Real Time PCR System using the KAPA SYBR Fast qPCR Kit. Four biological replicate samples were analyzed with consistent results.

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

Supplementary information

Supplementary Information

Peer Review File

Description of Additional Supplementary Files

Supplementary Data 1

Supplementary Data 2

Supplementary Data 3

Supplementary Data 4

Supplementary Data 5

Supplementary Data 6

Supplementary Data 7

Supplementary Data 8

Supplementary Data 9

Reporting Summary

Source data

Source Data

Supplementary information

The online version contains supplementary material available at 10.1038/s41467-024-52339-w.

Acknowledgements

The work was supported, in part, by the Core Research for Evolutional Science and Technology, Japan Science and Technology Agency (grant no. JPMJCR 15O5 to S.Y.), and the Japan Society for the Promotion of Science (KAKENHI grant nos. 22H04977 to S.Y. and 22K05368 to Y.S.).

Author contributions

S.Y. and Y.S. initiated the project and designed experiments. Y.S. and M.Y. performed experiments and analyzed the data. S.Y. and Y.S. wrote the manuscript.

Peer review

Peer review information

Nature Communications thanks Hikmet Budak, Zhongtao Jia and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. A peer review file is available.

Data availability

The datasets generated and/or analyzed in this study are available from the corresponding author upon request. The miRNA-seq data were deposited in the Gene Expression Omnibus repository of the National Center for Biotechnology Information database under the accession number GSE266624. Source data are provided with this paper.

Competing interests

The authors declare no competing interests.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
==== Refs
References

1. Marschner, H. M. Mineral nutrition of higher plants, 2nd edn (Academic press, London, UK, 1995).
2. Kiba T Krapp A Plant nitrogen acquisition under low availability: regulation of uptake and root architecture Plant Cell Physiol. 2016 57 707 714 10.1093/pcp/pcw052 27025887
Kiba, T. & Krapp, A. Plant nitrogen acquisition under low availability: regulation of uptake and root architecture. Plant Cell Physiol. 57, 707–714 (2016).27025887 10.1093/pcp/pcw052
3. Gruber BD Giehl RFH Friedel S von Wirén N Plasticity of the Arabidopsis root system under nutrient deficiencies Plant Physiol. 2013 163 161 179 10.1104/pp.113.218453 23852440
Gruber, B. D., Giehl, R. F. H., Friedel, S. & von Wirén, N. Plasticity of the Arabidopsis root system under nutrient deficiencies. Plant Physiol. 163, 161–179 (2013).23852440 10.1104/pp.113.218453
4. Lezhneva L The Arabidopsis nitrate transporter NRT2.5 plays a role in nitrate acquisition and remobilization in nitrogen-starved plants Plant J. 2014 80 230 241 10.1111/tpj.12626 25065551
Lezhneva, L. et al. The Arabidopsis nitrate transporter NRT2.5 plays a role in nitrate acquisition and remobilization in nitrogen-starved plants. Plant J. 80, 230–241 (2014).25065551 10.1111/tpj.12626
5. Masclaux-Daubresse C Nitrogen uptake, assimilation and remobilization in plants: challenges for sustainable and productive agriculture Ann. Bot. 2010 105 1141 1157 10.1093/aob/mcq028 20299346
Masclaux-Daubresse, C. et al. Nitrogen uptake, assimilation and remobilization in plants: challenges for sustainable and productive agriculture. Ann. Bot. 105, 1141–1157 (2010).20299346 10.1093/aob/mcq028
6. Sakuraba Y Chaganzhana Mabuchi A Iba K Yanagisawa S Enhanced NRT1.1/NPF6.3 expression in shoots improves growth under nitrogen deficiency stress in Arabidopsis Commun. Biol. 2021 4 1 14 10.1038/s42003-021-01775-1 33398033
Sakuraba, Y., Chaganzhana, Mabuchi, A., Iba, K. & Yanagisawa, S. Enhanced NRT1.1/NPF6.3 expression in shoots improves growth under nitrogen deficiency stress in Arabidopsis. Commun. Biol. 4, 1–14 (2021).33398033 10.1038/s42003-021-01775-1
7. Sakuraba Y Molecular basis of nitrogen starvation-induced leaf senescence Front. Plant Sci. 2022 13 3729 10.3389/fpls.2022.1013304
Sakuraba, Y. Molecular basis of nitrogen starvation-induced leaf senescence. Front. Plant Sci. 13, 3729 (2022).10.3389/fpls.2022.1013304
8. Makino A Sakuma I Sudo E Mae T Differences between maize and rice in N-use efficiency for photosynthesis and protein allocation Plant Cell Physiol. 2003 44 952 956 10.1093/pcp/pcg113 14519777
Makino, A., Sakuma, I., Sudo, E. & Mae, T. Differences between maize and rice in N-use efficiency for photosynthesis and protein allocation. Plant Cell Physiol. 44, 952–956 (2003).14519777 10.1093/pcp/pcg113
9. Hayashi H Chino M Chemical composition of phloem sap from the uppermost internode of the rice plant Plant Cell Physiol. 1990 31 247 251
Hayashi, H. & Chino, M. Chemical composition of phloem sap from the uppermost internode of the rice plant. Plant Cell Physiol. 31, 247–251 (1990).
10. Fan SC Lin CS Hsu PK Lin SH Tsay YF The Arabidopsis nitrate transporter NRT1.7, expressed in phloem, is responsible for source-to-sink remobilization of nitrate Plant Cell 2009 21 2750 2761 10.1105/tpc.109.067603 19734434
Fan, S. C., Lin, C. S., Hsu, P. K., Lin, S. H. & Tsay, Y. F. The Arabidopsis nitrate transporter NRT1.7, expressed in phloem, is responsible for source-to-sink remobilization of nitrate. Plant Cell 21, 2750–2761 (2009).19734434 10.1105/tpc.109.067603
11. Tsay YF Chiu CC Tsai CB Ho CH Hsu PK Nitrate transporters and peptide transporters FEBS Lett. 2007 581 2290 2300 10.1016/j.febslet.2007.04.047 17481610
Tsay, Y. F., Chiu, C. C., Tsai, C. B., Ho, C. H. & Hsu, P. K. Nitrate transporters and peptide transporters. FEBS Lett. 581, 2290–2300 (2007).17481610 10.1016/j.febslet.2007.04.047
12. Liu KH Huang CY Tsay YF CHL1 is a dual-affinity nitrate transporter of Arabidopsis involved in multiple phases of nitrate uptake Plant Cell 1999 11 865 874 10.1105/tpc.11.5.865 10330471
Liu, K. H., Huang, C. Y. & Tsay, Y. F. CHL1 is a dual-affinity nitrate transporter of Arabidopsis involved in multiple phases of nitrate uptake. Plant Cell 11, 865–874 (1999).10330471 10.1105/tpc.11.5.865
13. Woo HR Kim HJ Lim PO Nam HG Leaf senescence: systems and dynamics aspects Annu. Rev. Plant Biol. 2019 70 347 376 10.1146/annurev-arplant-050718-095859 30811218
Woo, H. R., Kim, H. J., Lim, P. O. & Nam, H. G. Leaf senescence: systems and dynamics aspects. Annu. Rev. Plant Biol. 70, 347–376 (2019).30811218 10.1146/annurev-arplant-050718-095859
14. Guo Y Leaf senescence: progression, regulation, and application Mol. Hortic. 2021 1 1 25 10.1186/s43897-021-00006-9 37789402
Guo, Y. et al. Leaf senescence: progression, regulation, and application. Mol. Hortic. 1, 1–25 (2021).37789402 10.1186/s43897-021-00006-9
15. Kim J Trifurcate feed-forward regulation of age-dependent cell death involving miR164 in Arabidopsis Science 2009 323 1053 1057 10.1126/science.1166386 19229035
Kim, J. et al. Trifurcate feed-forward regulation of age-dependent cell death involving miR164 in Arabidopsis. Science 323, 1053–1057 (2009).19229035 10.1126/science.1166386
16. Park BS Arabidopsis NITROGEN LIMITATION ADAPTATION regulates ORE1 homeostasis during senescence induced by nitrogen deficiency Nat. Plants 2018 4 898 903 10.1038/s41477-018-0269-8 30374089
Park, B. S. et al. Arabidopsis NITROGEN LIMITATION ADAPTATION regulates ORE1 homeostasis during senescence induced by nitrogen deficiency. Nat. Plants 4, 898–903 (2018).30374089 10.1038/s41477-018-0269-8
17. Leivar P Quail PH PIFs: pivotal components in a cellular signaling hub Trends Plant Sci. 2011 16 19 28 10.1016/j.tplants.2010.08.003 20833098
Leivar, P. & Quail, P. H. PIFs: pivotal components in a cellular signaling hub. Trends Plant Sci. 16, 19–28 (2011).20833098 10.1016/j.tplants.2010.08.003
18. Fan H A molecular framework underlying low-nitrogen-induced early leaf senescence in Arabidopsis thaliana Mol. Plant 2023 16 756 774 10.1016/j.molp.2023.03.006 36906802
Fan, H. et al. A molecular framework underlying low-nitrogen-induced early leaf senescence in Arabidopsis thaliana. Mol. Plant 16, 756–774 (2023).36906802 10.1016/j.molp.2023.03.006
19. Buelbuel S Arabidopsis BBX14 negatively regulates nitrogen starvation- and dark-induced leaf senescence Plant J. 2023 116 251 268 10.1111/tpj.16374 37382898
Buelbuel, S. et al. Arabidopsis BBX14 negatively regulates nitrogen starvation- and dark-induced leaf senescence. Plant J. 116, 251–268 (2023).37382898 10.1111/tpj.16374
20. Jones-Rhoades MW Bartel DP Bartel B MicroRNAs and their regulatory roles in plants Annu. Rev. Plant Biol. 2006 57 19 53 10.1146/annurev.arplant.57.032905.105218 16669754
Jones-Rhoades, M. W., Bartel, D. P. & Bartel, B. MicroRNAs and their regulatory roles in plants. Annu. Rev. Plant Biol. 57, 19–53 (2006).16669754 10.1146/annurev.arplant.57.032905.105218
21. Zhao M Ding H Zhu JK Zhang F Li WX Involvement of miR169 in the nitrogen-starvation responses in Arabidopsis New Phytol. 2011 190 906 915 10.1111/j.1469-8137.2011.03647.x 21348874
Zhao, M., Ding, H., Zhu, J. K., Zhang, F. & Li, W. X. Involvement of miR169 in the nitrogen-starvation responses in Arabidopsis. New Phytol. 190, 906–915 (2011).21348874 10.1111/j.1469-8137.2011.03647.x
22. He H Liang G Li Y Wang F Yu D Two young microRNAs originating from target duplication mediate nitrogen starvation adaptation via regulation of glucosinolate synthesis in Arabidopsis thaliana Plant Physiol. 2014 164 853 865 10.1104/pp.113.228635 24367020
He, H., Liang, G., Li, Y., Wang, F. & Yu, D. Two young microRNAs originating from target duplication mediate nitrogen starvation adaptation via regulation of glucosinolate synthesis in Arabidopsis thaliana. Plant Physiol. 164, 853–865 (2014).24367020 10.1104/pp.113.228635
23. Liang G He H Yu D Identification of nitrogen starvation-responsive microRNAs in Arabidopsis thaliana PLoS One 2012 7 e48951 10.1371/journal.pone.0048951 23155433
Liang, G., He, H. & Yu, D. Identification of nitrogen starvation-responsive microRNAs in Arabidopsis thaliana. PLoS One 7, e48951 (2012).23155433 10.1371/journal.pone.0048951
24. Nguyen GN Rothstein SJ Spangenberg G Kant S Role of microRNAs involved in plant response to nitrogen and phosphorus limiting conditions Front Plant Sci. 2015 6 629 10.3389/fpls.2015.00629 26322069
Nguyen, G. N., Rothstein, S. J., Spangenberg, G. & Kant, S. Role of microRNAs involved in plant response to nitrogen and phosphorus limiting conditions. Front Plant Sci. 6, 629 (2015).26322069 10.3389/fpls.2015.00629
25. Kim YS Sakuraba Y Han SH Yoo SC Paek NC Mutation of the Arabidopsis NAC016 transcription factor delays leaf senescence Plant Cell Physiol. 2013 54 1660 1672 10.1093/pcp/pct113 23926065
Kim, Y. S., Sakuraba, Y., Han, S. H., Yoo, S. C. & Paek, N. C. Mutation of the Arabidopsis NAC016 transcription factor delays leaf senescence. Plant Cell Physiol. 54, 1660–1672 (2013).23926065 10.1093/pcp/pct113
26. Kim HJ Nam HG Lim PO Regulatory network of NAC transcription factors in leaf senescence Curr. Opin. Plant Biol. 2016 33 48 56 10.1016/j.pbi.2016.06.002 27314623
Kim, H. J., Nam, H. G. & Lim, P. O. Regulatory network of NAC transcription factors in leaf senescence. Curr. Opin. Plant Biol. 33, 48–56 (2016).27314623 10.1016/j.pbi.2016.06.002
27. Kim HJ Time-evolving genetic networks reveal a NAC troika that negatively regulates leaf senescence in Arabidopsis Proc. Natl. Acad. Sci. USA. 2018 115 E4930 E4939 29735710
Kim, H. J. et al. Time-evolving genetic networks reveal a NAC troika that negatively regulates leaf senescence in Arabidopsis. Proc. Natl. Acad. Sci. USA. 115, E4930–E4939 (2018).29735710
28. Kiba T Repression of nitrogen starvation responses by members of the Arabidopsis GARP-type transcription factor NIGT1/HRS1 subfamily Plant Cell 2018 30 925 945 10.1105/tpc.17.00810 29622567
Kiba, T. et al. Repression of nitrogen starvation responses by members of the Arabidopsis GARP-type transcription factor NIGT1/HRS1 subfamily. Plant Cell 30, 925–945 (2018).29622567 10.1105/tpc.17.00810
29. Sakuraba Y Arabidopsis STAY-GREEN2 is a negative regulator of chlorophyll degradation during leaf senescence Mol. Plant 2014 7 1288 1302 10.1093/mp/ssu045 24719469
Sakuraba, Y. et al. Arabidopsis STAY-GREEN2 is a negative regulator of chlorophyll degradation during leaf senescence. Mol. Plant 7, 1288–1302 (2014).24719469 10.1093/mp/ssu045
30. Park M Gang W Alfredo G-S Hervé V Scott, P R Nuclear processing and export of microRNAs in Arabidopsis Proc. Natl. Acad. Sci. USA. 2005 102 3691 3696 10.1073/pnas.0405570102 15738428
Park, M., Gang, W., Alfredo, G.-S., Hervé, V. & Scott, P, R. Nuclear processing and export of microRNAs in Arabidopsis. Proc. Natl. Acad. Sci. USA. 102, 3691–3696 (2005).15738428 10.1073/pnas.0405570102
31. Achkar NP Cambiagno DA Manavella PA miRNA biogenesis: a dynamic pathway Trends Plant Sci. 2016 21 1034 1044 10.1016/j.tplants.2016.09.003 27793495
Achkar, N. P., Cambiagno, D. A. & Manavella, P. A. miRNA biogenesis: a dynamic pathway. Trends Plant Sci. 21, 1034–1044 (2016).27793495 10.1016/j.tplants.2016.09.003
32. Cambiagno DA HASTY modulates miRNA biogenesis by linking pri-miRNA transcription and processing Mol. Plant 2021 14 426 439 10.1016/j.molp.2020.12.019 33385584
Cambiagno, D. A. et al. HASTY modulates miRNA biogenesis by linking pri-miRNA transcription and processing. Mol. Plant 14, 426–439 (2021).33385584 10.1016/j.molp.2020.12.019
33. Friedländer MR Discovering microRNAs from deep sequencing data using miRDeep Nat. Biotechnol. 2008 26 407 415 10.1038/nbt1394 18392026
Friedländer, M. R. et al. Discovering microRNAs from deep sequencing data using miRDeep. Nat. Biotechnol. 26, 407–415 (2008).18392026 10.1038/nbt1394
34. Andersm S Huber W Differential expression analysis for sequence count data Genome Biol. 2010 11 R106 10.1186/gb-2010-11-10-r106 20979621
Andersm, S. & Huber, W. Differential expression analysis for sequence count data. Genome Biol. 11, R106 (2010).20979621 10.1186/gb-2010-11-10-r106
35. Guo Y Gan S AtNAP, a NAC family transcription factor, has an important role in leaf senescence Plant J. 2006 46 601 612 10.1111/j.1365-313X.2006.02723.x 16640597
Guo, Y. & Gan, S. AtNAP, a NAC family transcription factor, has an important role in leaf senescence. Plant J. 46, 601–612 (2006).16640597 10.1111/j.1365-313X.2006.02723.x
36. Kim HJ Gene regulatory cascade of senescence-associated NAC transcription factors activated by ETHYLENE-INSENSITIVE2-mediated leaf senescence signalling in Arabidopsis J. Exp. Bot. 2014 65 4023 4036 10.1093/jxb/eru112 24659488
Kim, H. J. et al. Gene regulatory cascade of senescence-associated NAC transcription factors activated by ETHYLENE-INSENSITIVE2-mediated leaf senescence signalling in Arabidopsis. J. Exp. Bot. 65, 4023–4036 (2014).24659488 10.1093/jxb/eru112
37. Bollman KM HASTY, the Arabidopsis ortholog of exportin 5/MSN5, regulates phase change and morphogenesis Development 2003 130 1493 1504 10.1242/dev.00362 12620976
Bollman, K. M. et al. HASTY, the Arabidopsis ortholog of exportin 5/MSN5, regulates phase change and morphogenesis. Development 130, 1493–1504 (2003).12620976 10.1242/dev.00362
38. Kurihara Y Watanabe Y Arabidopsis micro-RNA biogenesis through Dicer-like 1 protein functions Proc. Natl. Acad. Sci. USA. 2004 101 12753 12758 10.1073/pnas.0403115101 15314213
Kurihara, Y. & Watanabe, Y. Arabidopsis micro-RNA biogenesis through Dicer-like 1 protein functions. Proc. Natl. Acad. Sci. USA. 101, 12753–12758 (2004).15314213 10.1073/pnas.0403115101
39. Li M Yu B Recent advances in the regulation of plant miRNA biogenesis RNA Biol. 2021 18 2087 2096 10.1080/15476286.2021.1899491 33666136
Li, M. & Yu, B. Recent advances in the regulation of plant miRNA biogenesis. RNA Biol. 18, 2087–2096 (2021).33666136 10.1080/15476286.2021.1899491
40. Bolonga NG Nucleo-cytosolic shuttling of ARGONAUTE1 prompts a revised model of the plant microRNA pathway Mol. Cell 2018 69 709 719 10.1016/j.molcel.2018.01.007 29398448
Bolonga, N. G. et al. Nucleo-cytosolic shuttling of ARGONAUTE1 prompts a revised model of the plant microRNA pathway. Mol. Cell 69, 709–719 (2018).29398448 10.1016/j.molcel.2018.01.007
41. Mencia R Gonzalo L Tossolini I Manavella PA Keeping up with the miRNAs: current paradigms of the biogenesis pathway J. Exp. Bot. 2023 74 2213 2227 10.1093/jxb/erac322 35959860
Mencia, R., Gonzalo, L., Tossolini, I. & Manavella, P. A. Keeping up with the miRNAs: current paradigms of the biogenesis pathway. J. Exp. Bot. 74, 2213–2227 (2023).35959860 10.1093/jxb/erac322
42. Erson AE Petty EM MicroRNAs in development and disease Clin. Genet. 2008 74 296 306 10.1111/j.1399-0004.2008.01076.x 18713256
Erson, A. E. & Petty, E. M. MicroRNAs in development and disease. Clin. Genet. 74, 296–306 (2008).18713256 10.1111/j.1399-0004.2008.01076.x
43. Fang Y Spector DL Identification of nuclear dicing bodies containing proteins for microRNA biogenesis in living Arabidopsis Plants Curr. Biol. 2007 17 818 823 10.1016/j.cub.2007.04.005 17442570
Fang, Y. & Spector, D. L. Identification of nuclear dicing bodies containing proteins for microRNA biogenesis in living Arabidopsis Plants. Curr. Biol. 17, 818–823 (2007).17442570 10.1016/j.cub.2007.04.005
44. Zhu J CRD1, an Xpo1 domain protein, regulates miRNA accumulation and crown root development in rice Plant J. 2019 100 328 342 10.1111/tpj.14445 31257621
Zhu, J. et al. CRD1, an Xpo1 domain protein, regulates miRNA accumulation and crown root development in rice. Plant J. 100, 328–342 (2019).31257621 10.1111/tpj.14445
45. Seo J The rice NUCLEAR FACTOR0YA5 and MICRORNA169a module promotes nitrogen utilization during nitrogen deficiency Plant Physiol. 2019 194 491 510 10.1093/plphys/kiad504
Seo, J. et al. The rice NUCLEAR FACTOR0YA5 and MICRORNA169a module promotes nitrogen utilization during nitrogen deficiency. Plant Physiol. 194, 491–510 (2019).10.1093/plphys/kiad504
46. Wu K He J Pu W Peng Y The role of Exportin-5 in microRNA biogenesis and cancer Genomics Proteomics Bioinformatics 2018 16 120 126 10.1016/j.gpb.2017.09.004 29723684
Wu, K., He, J., Pu, W. & Peng, Y. The role of Exportin-5 in microRNA biogenesis and cancer. Genomics Proteomics Bioinformatics 16, 120–126 (2018).29723684 10.1016/j.gpb.2017.09.004
47. Xie D Phase separation of SERRATE drives dicing body assembly and promotes miRNA processing in Arabidopsis Nat. Cell Biol. 2021 23 32 39 10.1038/s41556-020-00606-5 33288888
Xie, D. et al. Phase separation of SERRATE drives dicing body assembly and promotes miRNA processing in Arabidopsis. Nat. Cell Biol. 23, 32–39 (2021).33288888 10.1038/s41556-020-00606-5
48. Li Q DEAD-box helicases modulate dicing body formation in Arabidopsis Sci. Adv. 2021 7 eabc6266 10.1126/sciadv.abc6266 33910901
Li, Q. et al. DEAD-box helicases modulate dicing body formation in Arabidopsis. Sci. Adv. 7, eabc6266 (2021).33910901 10.1126/sciadv.abc6266
49. Jia Z Giehl RFH von Wirén N Local auxin biosynthesis acts downstream of brassinosteroids to trigger root foraging for nitrogen Nat. Commun. 2021 12 1 12 10.1038/s41467-021-25250-x 33397941
Jia, Z., Giehl, R. F. H. & von Wirén, N. Local auxin biosynthesis acts downstream of brassinosteroids to trigger root foraging for nitrogen. Nat. Commun. 12, 1–12 (2021).33397941 10.1038/s41467-021-25250-x
50. Li J Mapping of candidate genes in response to low nitrogen in rice seedlings Rice 2022 15 1 16 10.1186/s12284-022-00597-x 34982277
Li, J. et al. Mapping of candidate genes in response to low nitrogen in rice seedlings. Rice 15, 1–16 (2022).34982277 10.1186/s12284-022-00597-x
51. Puig-Oliveras A Expression-based GWAS identifies variants, gene interactions and key regulators affecting intramuscular fatty acid content and composition in porcine meat Sci. Rep. 2016 6 1 12 10.1038/srep31803 28442746
Puig-Oliveras, A. et al. Expression-based GWAS identifies variants, gene interactions and key regulators affecting intramuscular fatty acid content and composition in porcine meat. Sci. Rep. 6, 1–12 (2016).28442746 10.1038/srep31803
52. Agrahari RK Expression GWAS of PGIP1 identifies STOP1-dependent and STOP1-independent regulation of PGIP1 in aluminum stress signaling in Arabidopsis Front. Plant Sci. 2021 12 2934 10.3389/fpls.2021.774687
Agrahari, R. K. et al. Expression GWAS of PGIP1 identifies STOP1-dependent and STOP1-independent regulation of PGIP1 in aluminum stress signaling in Arabidopsis. Front. Plant Sci. 12, 2934 (2021).10.3389/fpls.2021.774687
53. Maeda Y A NIGT1-centred transcriptional cascade regulates nitrate signalling and incorporates phosphorus starvation signals in Arabidopsis Nat. Commun. 2018 9 1 14 10.1038/s41467-018-03832-6 29317637
Maeda, Y. et al. A NIGT1-centred transcriptional cascade regulates nitrate signalling and incorporates phosphorus starvation signals in Arabidopsis. Nat. Commun. 9, 1–14 (2018).29317637 10.1038/s41467-018-03832-6
54. Murashige T Skoog F A revised medium for rapid growth and bioassays with tobacco tissue cultures Physiol. Plant. 1962 15 473 497 10.1111/j.1399-3054.1962.tb08052.x
Murashige, T. & Skoog, F. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plant. 15, 473–497 (1962).10.1111/j.1399-3054.1962.tb08052.x
55. Zhuo M Sakuraba Y Yanagisawa S A iasmonate-activated MYC2–Dof2.1–MYC2 transcriptional loop promotes leaf senescence in arabidopsis Plant Cell 2020 32 242 262 10.1105/tpc.19.00297 31641025
Zhuo, M., Sakuraba, Y. & Yanagisawa, S. A iasmonate-activated MYC2–Dof2.1–MYC2 transcriptional loop promotes leaf senescence in arabidopsis. Plant Cell 32, 242–262 (2020).31641025 10.1105/tpc.19.00297
56. Porra RJ Thompson WA Kriedemann PE Determination of accurate extinction coefficients and simultaneous equations for assaying chlorophylls a and b extracted with four different solvents: verification of the concentration of chlorophyll standards by atomic absorption spectroscopy Biochim. Biophys. Acta. Bioenerg. 1989 975 384 394 10.1016/S0005-2728(89)80347-0
Porra, R. J., Thompson, W. A. & Kriedemann, P. E. Determination of accurate extinction coefficients and simultaneous equations for assaying chlorophylls a and b extracted with four different solvents: verification of the concentration of chlorophyll standards by atomic absorption spectroscopy. Biochim. Biophys. Acta. Bioenerg. 975, 384–394 (1989).10.1016/S0005-2728(89)80347-0
57. Nakagawa T Ishiguro S Kimura T Gateway vectors for plant transformation Plant Biotechnol. 2009 26 275 284 10.5511/plantbiotechnology.26.275
Nakagawa, T., Ishiguro, S. & Kimura, T. Gateway vectors for plant transformation. Plant Biotechnol. 26, 275–284 (2009).10.5511/plantbiotechnology.26.275
58. Ke SH Madison EL Rapid and efficient site-directed mutagenesis by single-tube “megaprimer” PCR method Nucleic Acids Res. 1997 25 3371 3372 10.1093/nar/25.16.3371 9241254
Ke, S. H. & Madison, E. L. Rapid and efficient site-directed mutagenesis by single-tube “megaprimer” PCR method. Nucleic Acids Res. 25, 3371–3372 (1997).9241254 10.1093/nar/25.16.3371
59. Curtis MD Grossniklaus U A gateway cloning vector set for high-throughput functional analysis of genes in planta Plant Physiol. 2003 133 462 469 10.1104/pp.103.027979 14555774
Curtis, M. D. & Grossniklaus, U. A gateway cloning vector set for high-throughput functional analysis of genes in planta. Plant Physiol. 133, 462–469 (2003).14555774 10.1104/pp.103.027979
60. Zhang X Henriques R Lin SS Niu QW Chua NH Agrobacterium-mediated transformation of Arabidopsis thaliana using the floral dip method Nat. Protoc. 2006 1 641 646 10.1038/nprot.2006.97 17406292
Zhang, X., Henriques, R., Lin, S. S., Niu, Q. W. & Chua, N. H. Agrobacterium-mediated transformation of Arabidopsis thaliana using the floral dip method. Nat. Protoc. 1, 641–646 (2006).17406292 10.1038/nprot.2006.97
61. Varkonyi-Gasic E Wu R Wood M Walton EF Hellens RP Protocol: A highly sensitive RT-PCR method for detection and quantification of microRNAs Plant Methods 2007 3 1 12 10.1186/1746-4811-3-12 17207290
Varkonyi-Gasic, E., Wu, R., Wood, M., Walton, E. F. & Hellens, R. P. Protocol: A highly sensitive RT-PCR method for detection and quantification of microRNAs. Plant Methods 3, 1–12 (2007).17207290 10.1186/1746-4811-3-12
62. Jeong DH Abiotic stress-associated miRNAs: detection and functional analysis Methods Mol. Biol. 2010 592 203 230 10.1007/978-1-60327-005-2_14 19802598
Jeong, D. H. et al. Abiotic stress-associated miRNAs: detection and functional analysis. Methods Mol. Biol. 592, 203–230 (2010).19802598 10.1007/978-1-60327-005-2_14
63. Ueda Y NIGT1 family proteins exhibit dual mode DNA recognition to regulate nutrient response-associated genes in Arabidopsis PLoS Genet. 2020 16 e1009197 10.1371/journal.pgen.1009197 33137118
Ueda, Y. et al. NIGT1 family proteins exhibit dual mode DNA recognition to regulate nutrient response-associated genes in Arabidopsis. PLoS Genet. 16, e1009197 (2020).33137118 10.1371/journal.pgen.1009197
64. Tanaka, Y. et al. Gateway vectors for plant genetic engineering: overview of plant vectors, application for bimolecular fluorescence complementation (BiFC) and multigene construction. In: Barrera-Saldaña HA, editor. Genetic Engineering-Basics, New Applications and Responsibilities. InTechOpen; (2012).
65. Sakuraba Y STAY-GREEN and chlorophyll catabolic enzymes interact at light-harvesting complex II for chlorophyll detoxification during leaf senescence in Arabidopsis Plant Cell 2012 24 507 518 10.1105/tpc.111.089474 22366162
Sakuraba, Y. et al. STAY-GREEN and chlorophyll catabolic enzymes interact at light-harvesting complex II for chlorophyll detoxification during leaf senescence in Arabidopsis. Plant Cell 24, 507–518 (2012).22366162 10.1105/tpc.111.089474
66. Lee BD The F-box protein FKF1 inhibits dimerization of COP1 in the control of photoperiodic flowering Nat. Commun. 2017 8 2259 10.1038/s41467-017-02476-2 29273730
Lee, B. D. et al. The F-box protein FKF1 inhibits dimerization of COP1 in the control of photoperiodic flowering. Nat. Commun. 8, 2259 (2017).29273730 10.1038/s41467-017-02476-2
67. Terzi LC Simpson GG Arabidopsis RNA immunoprecipitation Plant J. 2009 59 163 168 10.1111/j.1365-313X.2009.03859.x 19419533
Terzi, L. C. & Simpson, G. G. Arabidopsis RNA immunoprecipitation. Plant J. 59, 163–168 (2009).19419533 10.1111/j.1365-313X.2009.03859.x
