Molecular framework integrating nitrate sensing in root and auxin-guided shoot adaptive responses

Abstract

Mineral nutrition is one of the key environmental factors determining plant development and growth. Nitrate is the major form of macronutrient nitrogen that plants take up from the soil. Fluctuating availability or deficiency of this element severely limits plant growth and negatively affects crop production in the agricultural system. To cope with the heterogeneity of nitrate distribution in soil, plants evolved a complex regulatory mechanism that allows rapid adjustment of physiological and developmental processes to the status of this nutrient. The root, as a major exploitation organ that controls the uptake of nitrate to the plant body, acts as a regulatory hub that, according to nitrate availability, coordinates the growth and development of other plant organs. Here, we identified a regulatory framework, where cytokinin response factors (CRFs) play a central role as a molecular readout of the nitrate status in roots to guide shoot adaptive developmental response. We show that nitrate-driven activation of NLP7, a master regulator of nitrate response in plants, fine tunes biosynthesis of cytokinin in roots and its translocation to shoots where it enhances expression of CRFs. CRFs, through direct transcriptional regulation of PIN auxin transporters, promote the flow of auxin and thereby stimulate the development of shoot organs.

Keywords: macronutrient; nitrate; plant development.

Fig. 1.

Modulation of PIN expression is part of nitrate-mediated transcriptional reprogramming. (A and B) Imaging (A) and surface area quantification (B) of cotyledons from 7-d-old wild-type (Col 0), pin4-3, and pin7-2 seedlings grown on AS (0.5 mM) (D0), and 24 HAT to either ammonium (AS)- or nitrate (5 mM KNO₃)-containing media. Relative size was quantified as mean surface area of cotyledons on AS or KNO₃ 24 HAT compared to D0 (n = 5 to 8 seedlings per treatment). The experiment was repeated three times; the result from one representative experiment is presented. Significant differences were determined by one-way ANOVA followed by a significant Tukey’s multiple comparison test; *P < 0.05, ***P < 0.001. (C) qRT-PCR expression analysis of PIN genes normalized to UBQ10 (AT4G05320) in Col 0 shoots, 1 and 6 HAT to AS- or KNO3-containing media. All qRT-PCR reactions were carried out with biological and technical triplicates. Statistical difference was determined by Student’s t test; *P < 0.05, **P < 0.01. (D) Expression analyses of PIN::GUS reporters in shoots of 7-d-old seedlings, 6 HAT to AS and KNO₃. (E–G) Monitoring (E) and quantification of PIN1::PIN1-GFP (F) and PIN7::PIN7-GFP (G) membrane signal in vasculature (yellow arrows) and adaxial epidermal cells of cotyledons, respectively, 3 HAT on AS- or KNO₃-supplemented media. PIN-GFP signal measured in n = 2 epidermal cells, and at least two cells of vasculature in six to seven seedlings per treatment. Significant differences were determined by Student’s t test; ****P < 0.0001 (F and G). (Scale bars in A, 400 μm; D, 200 μm; and E, 24 μm.)

Fig. 2.

Cytokinin response factors mediate shoot developmental adaptations to nitrate. (A–D) Expression analyses of CRFs using qRT-PCR (A, B, and D) and CRF::GUS reporters (C) in wild-type (Col 0) (A), (Col 8) (B), and nlp7 (D) shoots of 7-d-old seedlings 1 HAT (A–D) and 6 HAT (A and B) to AS and KNO₃. Expression of CRF genes normalized to UBQ10 (AT4G05320) 1 HAT and 6 HAT on AS- or KNO₃-containing media. All qRT-PCR reactions were carried out with biological and technical triplicates. Statistical difference was determined by Student’s t test; *P < 0.05, **P < 0.01. (E and F) Imaging (E) and surface area quantification (F) of cotyledons from 7-d-old wild type (Col 0), mutants, and CRF overexpressors grown on AS (D0), and 24 HAT to AS-or KNO₃-containing media. Relative size was quantified as mean surface area of cotyledons on AS or KNO₃ 24 HAT compared to D0 (n = 5 to 8 seedlings per treatment). The experiment was repeated twice; the result from one representative experiment is presented. Significant differences were determined by one-way ANOVA followed by Tukey’s multiple comparison test; lowercase letters indicate significant differences of at least *P < 0.05. (Scale bars in C , 200 μm and E, 100 μm.)

Fig. 3.

NLP7 fine tunes expression of PINs and shoot developmental adaptation to nitrate through CRFs. (A–F) Imaging (A, B, and E) and quantification (C, D, and F) of PIN7-GFP membrane signal in adaxial epidermal cells of cotyledons 3 HAT to medium supplemented with AS or KNO₃ in nlp7-1 (A and C), crf6-1 (B and D), and PIN7::PIN7-GFP, Col 0 and ΔPIN7::PIN7-GFP,RPS5a::CRF2 (E) lines. Different lowercase letters indicate significant difference at one-way ANOVA followed by Tukey’s multiple comparison test (P < 0.05). PIN7-GFP signal measured in n = 2 epidermal cells, in at least five seedlings per treatment. Experiments were repeated at least twice; the result from one experiment is presented (C, D, and F). (Scale bars in A, B, and E, 24 μm.)

Fig. 4.

NLP7-mediated signaling in roots controls cytokinin levels in shoots. (A and B) Imaging (A) and surface area quantification (B) of cotyledons from 7-d-old wild type (Col 0), nlp7-1, ipt3,5,7, abcg14, and ahk2ahk3 seedlings grown on AS (D0), and 24 HAT to either AS, AS supplemented with cytokinin (CK, 100 nM 6-benzylaminopurine), and KNO₃-containing media. Relative size was quantified as mean cotyledon surface area on AS, AS plus CK, or KNO₃ 24 HAT compared to D0 (n = 6 to 8 seedlings per treatment). The xperiment was repeated at least twice; results from one representative experiment are presented. Significant differences were determined by one-way ANOVA followed by Tukey’s multiple comparison test; different lowercase letters indicate P < 0.05 (B). (C) Representative images of ARR5::GUS expression in shoots of 7-d-old seedlings grafted on Col 0 or nlp7-1 roots. Grafted seedlings after 72 h of healing on AS medium were then transferred to KNO₃ for 24 h. (D) qRT-PCR analysis of IPT genes and ABCG14 expression normalized to UBQ10 (AT4G05320) in Col 8 roots 1 HAT to AS- or KNO₃-containing media. All qRT-PCR reactions were carried out with biological and technical triplicates. Statistical difference was determined with a t test; *P < 0.05,**P < 0.01 . (E) Quantification of tZ, tZR, and DHZR in roots and shoots of Col 8 and nlp7-1 seedlings 6 HAT to AS- or KNO₃-containing medium. Different lowercase letters indicate significant one-way ANOVA followed by Tukey’s multiple comparison test (P < 0.05) (n = 5 biological replica/genotype/treatment). (Scale bars in A, 100 μm and C, 200 μm.)

Fig. 5.

Nitrate-dependent fine tuning of PIN expression in shoots is driven by cytokinin translocated from roots. (A) Representative images of PIN7::GUS expression in shoots grafted on roots of PIN7::GUS, nlp7, abcg14, or ipt3,5,7 roots. Grafted seedlings after 48 h of healing on AS, then transferred to KNO₃-containing medium for 24 h. (B–E) Imaging (B and D) and quantification (C and E) of PIN7-GFP membrane signal in adaxial epidermal cells of cotyledons 6 HAT on AS, AS plus cytokinin (CK, 100 nM 6-benzylaminopurine), or KNO₃-supplemented media in nlp7-1 (B and C) and abcg14 (D and E). PIN7-GFP signal was measured in n = 2 epidermal cells, four different seedlings per treatment. Experiments were repeated at least twice; results from one experiment are presented. Different lowercase letters indicate significant one-way ANOVA followed by Tukey’s multiple comparison test (P < 0.05) (C and E). (Scale bars in A, 100 μm and B and D, 24 μm.)