Nutrient dose-responsive transcriptome changes driven by Michaelis-Menten kinetics underlie plant growth rates

Abstract

An increase in nutrient dose leads to proportional increases in crop biomass and agricultural yield. However, the molecular underpinnings of this nutrient dose-response are largely unknown. To investigate, we assayed changes in the Arabidopsis root transcriptome to different doses of nitrogen (N)-a key plant nutrient-as a function of time. By these means, we found that rate changes of genome-wide transcript levels in response to N-dose could be explained by a simple kinetic principle: the Michaelis-Menten (MM) model. Fitting the MM model allowed us to estimate the maximum rate of transcript change (V_max), as well as the N-dose at which one-half of V_max was achieved (K_m) for 1,153 N-dose-responsive genes. Since transcription factors (TFs) can act in part as the catalytic agents that determine the rates of transcript change, we investigated their role in regulating N-dose-responsive MM-modeled genes. We found that altering the abundance of TGA1, an early N-responsive TF, perturbed the maximum rates of N-dose transcriptomic responses (V_max), K_m, as well as the rate of N-dose-responsive plant growth. We experimentally validated that MM-modeled N-dose-responsive genes included both direct and indirect TGA1 targets, using a root cell TF assay to detect TF binding and/or TF regulation genome-wide. Taken together, our results support a molecular mechanism of transcriptional control that allows an increase in N-dose to lead to a proportional change in the rate of genome-wide expression and plant growth.

Keywords: Michaelis–Menten kinetics; nitrogen dose; transcriptome regulation.

Fig. 1.

Michaelis–Menten (MM) kinetics underlie transcriptomic responses to N-dose in Arabidopsis roots. (A) A factorial treatment matrix systematically varied both the exposure time to N and the N-dose provided. Colors indicate how many genes were differentially expressed (fold change cutoff ± 1.5) in response to N-dose in each condition. (B) Differentially expressed genes were detected using a multivariate linear model. Seventy-seven percent of genes found by linear modeling (3,818 genes) were fit by a model holding an N$\times$T term. (C) Heatmap displaying rates of transcript change for each of the 3,818 genes under each N-dose. Genes whose rate change in N-dose–responsive expression significantly fit the MM model (1,153 genes) are indicated in red. (D) GDH1 is an example of a gene fit by model holding a “N$\times$T” term. (E) GDH1 is also an example of a gene whose rate of N-dose–responsive expression is significantly fit by the MM model.

Fig. 2.

N-dose–response rates of transcripts mediated by changes in TGA1 levels in planta. (A) A ranking in expression fold change for mRNAs of all plant TFs after 15 min of exposure to N treatment. (B) cis-regulatory analysis identifies overrepresented motifs among the 1,153 N-dose–responsive genes captured by the MM model. (C) Changes in N-dose–dependent transcription rate of PDX1.1 is fit by the MM model in wild type, and V_max is altered in the 35S::TGA1 and tga1/tga4 lines. (D–F) A histogram of log₂ fold changes in V_max shown for 192 MM-modeled genes up-regulated by N-dose across wild-type and TGA1 genotypes. D compares these changes in V_max between 35S::TGA1 and wild type; E, between tga1/tga4 and wild type; and F, between 35S::TGA1 and tga1/tga4. A binomial test was used to assess bias toward an increase or decrease in V_max.

Fig. 3.

TGA1 is a transcriptional activator that regulates both TFs and N-uptake/assimilation genes in root cells. (A) Workflow of TARGET TF-perturbation assay that can detect the gene targets that TGA1 binds to and regulates in isolated root cells. (B) The TARGET TF-perturbation assay reveals TGA1 directly up-regulates (yellow, 77%) or down-regulates (blue, 23%) the expression of 584 genes in root cells (SI Appendix, Table S4). (C) NLP8 and CIB3 TFs are examples of direct targets transcriptionally activated by TGA1. The expression of NLP8 and CIB3 is affected by TGA1 nuclear import, as assayed by both RNA-seq (steady-state mRNA) and by 4tU affinity capture (de novo mRNA) (green bars). TGA1 binding at these loci was captured by ChIP-seq (red bars). (D) TGA1 transcriptional subnetwork in root cells, where nodes represent genes and edges represent regulatory interactions detected by our assay. TGA1 directly or indirectly—through intermediate TFs (triangles)—regulate the expression genes involved in N uptake/metabolism (circles).

Fig. 4.

Changes in TGA1 levels impacts N-dose–responsive growth rates in planta. (A–C) Growth rates of (A) wild-type, (B) tga1/tga4, and (C) 35S::TGA1 seedling growth over time (days) under different N-doses. The impact of N-dose on growth rates differs significantly between wild-type and the 35S::TGA1 line (three-way ANCOVA, P = 4.1 $\times$ 10⁻⁵), and between wild-type and the tga1/tga4 line (P = 1.5 $\times$ 10⁻⁷). (D–F) Rates of plant growth of (D) wild-type, (E) tga1/tga4, and (F) 35S::TGA1 plants, fit with the MM model. (G) GLU1 gene expression assayed under these N-dose conditions in wild-type and 35S::TGA1. (H) Genes differentially expressed in response to N-dose in 35S::TGA1 plants significantly intersect with direct and indirect targets of TGA1, as well as MM-modeled genes (Monte Carlo test). (I) Phenotype of 15-d-old wild-type plants, a tga1/tga4 mutant, and two independent TGA1-overexpressing lines grown on plates containing 10 mM N.

Fig. 5.

The molecular basis for Michaelis–Menten (MM) kinetics that underlies N-dose–dependent regulation of plant growth. The MM model (upper gray panel) can explain the effect of N-dose on rates of N uptake (A), N signaling (B), and N growth (C). (A) The rate of N uptake by plant nitrate (NRTs) and ammonium (AMTs) transporters follows MM kinetics (11). The N-regulated expression of the NRT1.1 nitrate transceptor has also been shown to follow MM kinetics (53). (B) Results presented herein show that transcriptome responses to N-dose follows MM kinetics (1,153 genes) (Fig. 1). Moreover, a portion of this MM-mediated transcriptome response (192 genes) to N-dose is affected by TGA1, as shown via changes in V_max and K_m in tga1/4 and 35S::TGA1 lines (Figs. 2 and 4 and SI Appendix, Fig. S7). TGA1’s role in signaling N-dose at the molecular level impacts genes involved in translation, glucose metabolism, and energy metabolism. The N response of TGA1 mRNA levels is altered in the nrt1.1 transceptor mutant (SI Appendix, Fig. S12) (18). (C) Overexpression of TGA1 (35S::TGA1) leads to higher N-dose–dependent growth rates, while it is reduced in the tga1/4 mutant (Fig. 4 D–F). Thus, the role of TGA1 in mediating rate of transcript change in response to N-dose provides a molecular basis for how N-dose regulates rates of plant growth, a phenotype that can also be explained by MM kinetics (Fig. 4 D–F) (10).