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Perception of root-derived peptides by shoot LRR-RKs mediates systemic N-demand signaling

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Science  17 Oct 2014:
Vol. 346, Issue 6207, pp. 343-346
DOI: 10.1126/science.1257800

Abstract

Nitrogen (N) is a critical nutrient for plants but is often distributed unevenly in the soil. Plants therefore have evolved a systemic mechanism by which N starvation on one side of the root system leads to a compensatory and increased nitrate uptake on the other side. Here, we study the molecular systems that support perception of N and the long-distance signaling needed to alter root development. Rootlets starved of N secrete small peptides that are translocated to the shoot and received by two leucine-rich repeat receptor kinases (LRR-RKs). Arabidopsis plants deficient in this pathway show growth retardation accompanied with N-deficiency symptoms. Thus, signaling from the root to the shoot helps the plant adapt to fluctuations in local N availability.

Getting to the root of a root problem

Although a plant's root system reaches through the soil in search of nutrients, its search is not indiscriminate. If some section of the root is unable to deliver the amount of nitrogen that the rest of the plant demands, other sections of the root compensate and ramp up their delivery of nitrogen. Tabata et al. have now found a small peptide that delivers a signal involved in this process (see the Perspective by Bisseling and Scheres). Only with perception of the signal by the matching receptor in the shoot can the root system compensate for unproductive members.

Science, this issue p. 343; see also p. 300

Nitrogen is an essential macronutrient in plants. Plants take up inorganic N directly from the soil in the form of nitrate or ammonium and assimilate these ions into amino acids. Most of the inorganic N in natural soils is, however, present as nitrate as a result of microbial nitrification. Additionally, there is spatial variation in soil nitrate distribution at scales relevant to individual plants due to uptake by plants or leaching by rain. Plants, therefore, have evolved sophisticated strategies allowing them to modulate the efficiency of root N acquisition in response to fluctuating external N availability and their own nutritional status.

Nitrate uptake systems are under control by both cell-autonomous local signaling triggered by nitrate itself and systemic long-distance signaling that transduces external and internal N status across spatially distant root compartments (1). N starvation on one side of the root system leads to an up-regulation of nitrate uptake on the other side of the root system (24). This compensatory N acquisition response is accompanied by the transcriptional up-regulation of nitrate transporter genes such as NRT2.1 and is postulated to be mediated by a systemic signal, the N-demand signal (24).

Small molecules such as secreted peptides can mediate long-distance signaling, as exemplified by Rhizobium-induced CLAVATA3/ESR-related (CLE) peptides involved in autoregulation of nodulation (5). Similarly, peptide signaling may coordinate root development with needs for N. The genes that encode small peptide signals are often parts of large families of genes with overlapping and redundant functions (6). To overcome the experimental limitations associated with gene redundancy on signaling peptides, we explored the function of these peptides from the receptor side by a binding assay against an expression library of Arabidopsis leucine-rich repeat receptor kinases (LRR-RKs). Here, we clarify the functions of an Arabidopsis-secreted peptide family consisting of multiple functionally redundant members and identify peptide ligands and receptors mediating long-distance N-demand signaling.

The Arabidopsis C-terminally encoded peptide (CEP) family was identified by in silico screening for a family of secreted peptides that share short, conserved domains near the C terminus, a feature that is common to several posttranslationally modified small peptide signals in plants (7). CEP1 is secreted as a 15–amino acid peptide originating from a C-terminal conserved domain (the CEP domain) through posttranslational proline hydroxylation and proteolytic processing. A total of 15 CEP family genes (CEP1 through CEP15) have been found in the Arabidopsis genome (79). Expression of CEP1 through CEP5 is mainly found in the basal region of the lateral roots, but minor expression has also been detected in the aboveground tissues. Overexpression of several CEP family genes leads to repression of root growth accompanied with morphological alterations of shoots and, conversely, loss-of-function mutation of CEP3 promotes root development (79). However, genetic redundancy obscures the functions of CEPs.

To gain insight into the function of CEP family peptides, we prepared a CEP1 analog derivatized with 125I-labeled photoreactive 4-azidosalicylic acid ([125I]ASA-CEP1) (Fig. 1A). ASA-CEP1 exhibited biological activity comparable to that of CEP1 in a root growth assay (fig. S1A). We also generated an expression library of Arabidopsis LRR-RKs subfamily X and XI by overexpressing individual proteins in tobacco BY-2 cells. Photoaffinity labeling of Arabidopsis LRR-RKs by [125I]ASA-CEP1 revealed that two related LRR-RKs, At5g49660 and At1g72180, in subfamily XI directly interact with CEP1 (Fig. 1B and fig. S1B). This binding was competitively inhibited by excess unlabeled CEP1, supporting the specificity of this interaction. We confirmed that the double loss-of-function mutant of At5g49660 and At1g72180 was insensitive to CEP1 in a root growth assay (Fig. 1C and fig. S2A). We concluded that these two LRR-RKs function as CEP receptors and named them CEPR1 (At5g49660) and CEPR2 (At1g72180). At5g49660 has been characterized as XIP1, and a loss-of-function mutant displays anthocyanin accumulation in the leaves and ectopic lignification in phloem of inflorescence stems (10), although we did not observe the latter phenotype under our culture conditions.

Fig. 1 Identification and functional characterization of Arabidopsis CEP receptors.

(A) Structure of [125I]ASA-CEP1 used for photoaffinity labeling. (B) Direct binding of 10 nM [125I]ASA-CEP1 with LRR-RKs At5g49660 and At1g72180. This binding was competitively inhibited by 100-fold excess unlabeled CEP1. (C) Diagram of CEPR1 (At5g49660) and CEPR2 (At1g72180), showing the locations of the transferred DNA (T-DNA) insertions. The deduced primary structure of CEPR1 and CEPR2 includes an N-terminal signal peptide (SP), leucine-rich repeats (LRR), a transmembrane domain (TM), and an intracellular kinase domain (KD). (D) Shoot phenotype of 14-day-old wild-type (left) and cepr1-1 cepr2-1 mutant (right) on the N-rich medium. Scale bar, 5 mm. (E) Root phenotypes of wild type (left) and cepr1-1 cepr2-1 (right) vertically grown on the N-rich medium for 10 days. Scale bar, 1 cm. (F) Histochemical staining of Arabidopsis seedlings transformed with the CEPR1pro:GUS (left) and CEPR2pro:GUS (right) genes. Scale bar, 5 mm.

Seedlings of cepr1-1 cepr2-1 double mutants displayed a pleiotropic phenotype characterized by pale-green leaves and enhanced lateral root elongation (Fig. 1, D and E, and fig. S2B). At the adult stage, compared with wild type, the mutant developed smaller rosette leaves and shorter floral stems, accompanied by anthocyanin accumulation (fig. S2C). An enhanced lateral root elongation phenotype was also observed, albeit to a lesser degree, in the cepr1-1 single mutant, but was absent in the cepr2-1 single mutant, suggesting a major role of CEPR1 and a minor but redundant role of CEPR2 in the relevant signaling pathways (fig. S2D). Confirming this, a genomic fragment containing the 2.0-kb promoter and the entire CEPR1-coding region completely rescued the above phenotype when introduced in the cepr1-1 cepr2-1 double mutant (fig. S2, C and D).

β-glucuronidase (GUS) reporter–aided histochemical analysis revealed CEPR1 promoter activity in the vascular veins of cotyledons and mature leaves, primary roots, and lateral roots, except for the root tip region (Fig. 1F and fig. S3, A and B). CEPR2 promoter activity was detected in mature leaves, primary roots, and the root tips of both primary roots and lateral roots.

Because enhanced lateral root elongation, pale-green leaves, dwarfism, and anthocyanin accumulation are typical responses to N starvation (11), we next analyzed whether cepr1-1 and cepr2-1 mutations affect expression of genes involved in N uptake and assimilation pathways. Using microarray profiling followed by quantitative reverse transcription polymerase chain reaction (qRT-PCR), we found that genes considerably down-regulated in the cepr1-1 cepr2-1 mutant include NRT2.1 (in the top 0.1% of all down-regulated genes), NRT3.1 (in the top 1%), and NRT1.1 (in the top 2%), which encode major components of the root nitrate transport system (table S1 and fig. S4A). These three genes are known to be targets for systemic N-demand signaling (24). Along with the decrease in expression of nitrate transporters, nitrate uptake activity of cepr1-1 cepr2-1 mutant roots was considerably reduced compared with wild type, as indicated by analysis of 15NO3 influx (Fig. 2A).

Fig. 2 CEP signaling underlies N-starvation response.

(A) Nitrate uptake activity of wild-type and cepr1-1 cepr2-1 roots, as determined by 15NO3 influx at 0.2 mM and 10 mM (mean ± SD of triplicates, *P < 0.05 by Student’s t test. (B) qRT-PCR of NRT transcripts in the roots of wild-type Arabidopsis seedlings treated with 1 μM CEP1 for various periods of time. Significant differences between treatments are indicated with different letters (P < 0.05, one-way analysis of variance. (C) qRT-PCR of NRT2.1 transcripts in roots of wild-type, cepr1-1, cepr2-1, and cepr1-1 cepr2-1 seedlings treated with 1 μM CEP1 for 24 hours. (D) qRT-PCR of CEP transcripts in the roots of wild-type seedlings subjected to N starvation for various periods of time. CEP2 transcripts were not detected. (E) Histochemical staining of roots of 10-day-old seedlings transformed with the CEP6pro:GUS gene before (top) and after (bottom) N starvation for 24 hours. Scale bar, 0.2 mm. (F) Cross section of N-starved lateral roots pictured in (E). Scale bar = 2 μm. (G) qRT-PCR of CEP transcripts in the wild-type roots subjected to various degrees of N deprivation for 24 hours.

To confirm whether these NRT genes are indeed regulated by CEP, we treated roots of wild-type seedlings with 1 μM CEP1 for 6, 24, and 48 hours. qRT-PCR of transcripts in roots revealed that NRT2.1 expression was up-regulated within 6 hours and transcripts accumulated over 5-fold after 24 hours, even under N-rich conditions (Fig. 2B). This stimulatory activity was dependent largely on CEPR1 (Fig. 2C) and detectable at CEP1 concentrations as low as 100 nM when applied to wild-type plants (fig. S4B). CEP1 also induced other nitrate transporters such as NRT3.1 and NRT1.1 (Fig. 2B), but had lesser effects on expression of transporters for ammonium and other macronutrients (fig. S4C). These phenotypic and transcriptional analyses suggest that CEP signaling is likely to underlie N starvation responses and, accordingly, its overactivation or blockage leads to pleiotropic developmental effects in both roots and shoots.

The Arabidopsis genome includes 11 genes (CEP1 through CEP11) encoding secreted polypeptides containing well-conserved 15–amino acid CEP domains and an additional 4 genes that may encode CEP-like peptides (CEP12 through CEP15) (79). We selected CEP1 through CEP11 for deep analysis. qRT-PCR indicated that 10 of the 11 CEP genes are capable of inducing NRT2.1 expression in roots when overexpressed in Arabidopsis seedlings (fig. S5). We confirmed that several of these CEPs—such as CEP3, CEP5, CEP6, and CEP9—are secreted as 15–amino acid peptides originating from conserved CEP domains (fig. S6, A to M) and that at least the CEP3 and CEP5 peptides interact with CEPR1 and induce NRT2.1 expression at 1 μM (fig. S7, A and B).

In addition to the previously characterized CEP1 through CEP5 (7, 8), we tested expression patterns of CEP6 through CEP11 by generating promoter-GUS reporter lines. We detected promoter activity of CEP6, CEP8, CEP9, and CEP11 both in lateral root primordia and in lateral roots excluding the meristem region (fig. S8A). Promoter activity of CEP6, CEP8, and CEP9 was also detected in the aerial tissues, such as leaf petioles and the shoot apex region (fig. S8B).

We then analyzed whether expression of CEP family peptides are under the control of external N status. When Arabidopsis seedlings cultured under N-rich conditions were transplanted to a medium devoid of N, we observed immediate induction of seven CEP genes (CEP1, CEP3, CEP5, CEP6, CEP7, CEP8, and CEP9) after 6 hours and further up-regulation for up to 24 hours (Fig. 2, D to F). Induction levels correlated well with external N status, being stronger at lower concentrations of nitrate, ammonium, and glutamine in the medium (Fig. 2G and fig. S9A). Deprivation of other macronutrients such as phosphate and potassium showed no effect on CEP expression (fig. S9B). These results indicate that the 7 CEP genes are up-regulated specifically in response to N starvation.

In a heterogeneous N environment, N starvation on one side of the roots can up-regulate nitrate transporter genes in a distant part of the roots exposed to a N-rich medium to compensate for N deficiency (24). This long-distance systemic response is postulated to be controlled by a N-demand signal emitted from the N-starved roots and is prominent when only nitrate is available as a nitrogen source (24). If the CEP family peptides are involved in systemic N-demand signaling, we would expect the compensatory N acquisition response to be abolished in the cepr1-1 cepr2-1 mutant. To test this possibility, we used a split-root culture system, in which the root system of a plant is separated into two parts exposed to different nutrient conditions (Fig. 3A). We transferred one side of the split-root system to a medium lacking N, whereas the other side was kept in a medium containing 10 mM NO3. In wild-type plants, we observed up-regulation of NRT2.1, NRT3.1, and NRT1.1 in the roots exposed to the N-rich medium after 24 hours (Fig. 3B and fig. S10, A and B), accompanied by induction of CEP genes in the portion of the root system directly experiencing N starvation (fig. S10C). In contrast, no such systemic up-regulation of nitrate transporter genes was detected in the roots of the cepr1-1 cepr2-1 mutant, despite local induction of CEP genes in the N-starved roots (Fig. 3B and fig. S10, A, B, and D). These results indicate that CEP family peptides are components for systemic N-demand signaling in roots.

Fig. 3 Perception of root-derived CEP by CEPR in shoots mediates systemic N-demand signaling.

(A) Arabidopsis split-root culture system in which the root system of a plant is separated into left (L) and right (R) parts that are exposed to different nutrient conditions. Scale bar, 1 cm. (B) qRT-PCR of NRT2.1 transcripts on each side of the split-root system of wild-type and cepr1-1 cepr2-1 plants, in which one side of the root system was starved for N for 24 hours (mean ± SD of triplicates). (C) qRT-PCR of NRT2.1 transcripts on each side of a split-root system of wild-type plants, in which one side was treated with 1 μM CEP1 for 24 hours. (D and E) qRT-PCR of NRT2.1 transcripts on each side of a split-root system of reciprocally grafted plants, in which one side was treated with 1 μM CEP1 for 48 hours (D) or starved for N for 48 hours (E).

We then analyzed whether systemic induction of nitrate transporter genes is observed in a split-root system in response to CEP1 treatment instead of N starvation. When both sides of a split-root system were cultured in the presence of 10 mM NO3 and one side was treated with 1 μM CEP1, we observed up-regulation of NRT2.1 in the untreated distant roots as well as the treated roots (Fig. 3C). This systemic response was detectable as early as 6 hours after CEP1 treatment (fig. S10E). NRT3.1 and NRT1.1 also showed similar systemic responses (fig. S10, F and G). These results indicate that CEP1 mediates systemic up-regulation of nitrate transporter genes over long distances in roots. Our observation that, in contrast to N starvation, CEP1 treatment on one side in the presence of 10 mM NO3 causes up-regulation of NRT2.1 on both sides of a split-root system suggests that CEP-mediated up-regulation of nitrate transporters may also reflect the balance of local external NO3 availability and nutritional status of the whole plant.

Finally, to analyze whether root-derived CEP1 is perceived directly in roots or perceived in shoots and then induces a descending secondary signal that up-regulates nitrate transporters, we performed split-root experiments combined with reciprocal grafting between cepr1-1 cepr2-1 and wild-type plants. When cepr1-1 cepr2-1 mutant scions were grafted onto wild-type rootstocks by hypocotyl-to-hypocotyl grafting followed by treating one side of the root system with CEP1, we observed no up-regulation of NRT2.1 on either side of the root system (Fig. 3D). In contrast, when wild-type scions were grafted onto cepr1-1 cepr2-1 mutant rootstocks, root-applied CEP1 caused substantial systemic up-regulation of NRT2.1 on both sides of the root system, even though the rootstocks themselves expressed no CEP receptors (Fig. 3D). In split N-starvation experiments, we confirmed that systemic up-regulation of NRT2.1 in roots was induced only when wild-type scions were grafted onto cepr1-1 cepr2-1 mutant rootstocks (Fig. 3E). These results indicate that CEPRs expressed in the shoots are responsible for the CEP-mediated systemic up-regulation of nitrate transporters in roots and that root-derived CEP is graft-transmissible from roots to shoots. Further supporting the mobile nature of CEP family peptides, we detected endogenous CEPs in xylem sap collected from Arabidopsis plants (fig. S11, A to G). The levels in xylem sap were high under N-starved conditions but lower under N-rich conditions.

Altogether, the available evidence from molecular and physiological analyses of CEP–CEPR ligand receptor pairs suggests that CEP acts as a root-derived ascending N-demand signal to the shoot, where its perception by CEPR leads to the production of a putative shoot-derived descending signal that up-regulates nitrate transporter genes in the roots. This mechanism supports N acquisition, especially when NO3 is unevenly distributed within the soil. CEP family peptides induced on one side of the roots by local N starvation mediate up-regulation of nitrate transporter genes in the distant part of the roots exposed to N-rich conditions to compensate for N deficiency.

The systemic mode of action of CEP family peptides in N-demand signaling is reminiscent of that of Rhizobium-induced, xylem-mobile CLE peptides that suppress excess nodulation in legume plants, although CEP plays a role opposite to that of CLE in terms of lateral organ formation (5, 12, 13). Plants, as sessile organisms, continuously face a complex array of environmental fluctuations and have evolved sophisticated responses to cope with them. Given that CEP family peptides are conserved throughout vascular plants except for ferns (8, 9), peptide-mediated root-to-shoot-to-root long-distance signaling is likely to be a general strategy employed by all higher plants for environmental adaptation.

Supplementary Materials

www.sciencemag.org/content/346/6207/343/suppl/DC1

Materials and Methods

Figs. S1 to S11

Table S1

References (1418)

References and Notes

  1. Acknowledgments: This research was supported by a Grant-in-Aid for Scientific Research (S) from the Ministry of Education, Culture, Sports, Science, and Technology (no. 25221105). The supplementary materials contain additional data.
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