Multiple Interactions between Glucose and Brassinosteroid Signal Transduction Pathways in Arabidopsis Are Uncovered by Whole-Genome Transcriptional Profiling

Abstract

Brassinosteroid (BR) and glucose (Glc) regulate many common responses in plants. Here, we demonstrate that under etiolated growth conditions, extensive interdependence/overlap occurs between BR- and Glc-regulated gene expression as well as physiological responses. Glc could regulate the transcript level of 72% of BR-regulated genes at the whole-genome level, of which 58% of genes were affected synergistically and 42% of genes were regulated antagonistically. Presence of Glc along with BR in medium could affect BR induction/repression of 85% of BR-regulated genes. Glc could also regulate several genes involved in BR metabolism and signaling. Both BR and Glc coregulate a large number of genes involved in abiotic/biotic stress responses and growth and development. Physiologically, Glc and BR interact to regulate hypocotyl elongation growth of etiolated Arabidopsis (Arabidopsis thaliana) seedlings in a dose-dependent manner. Glc may interact with BR via a hexokinase1 (HXK1)-mediated pathway to regulate etiolated hypocotyl elongation. Brassinosteroid insensitive1 (BRI1) is epistatic to HXK1, as the Glc insensitive2bri1-6 double mutant displayed severe defects in hypocotyl elongation growth similar to its bri1-6 parent. Analysis of Glc and BR sensitivity in mutants defective in auxin response/signaling further suggested that Glc and BR signals may converge at S-phase kinase-associated protein1-Cullin-F-box-transport inhibitor response1/auxin-related f-box-auxin/indole-3-acetic acid-mediated auxin-signaling machinery to regulate etiolated hypocotyl elongation growth in Arabidopsis.

Figure 1.

Effect of Glc on BR-regulated genes in dark-grown seedlings. A, In etiolated seedlings, BR can altogether up- or down-regulate a total of 303 genes in Glc-free medium (cutoff, 2-fold). Glc alone can independently affect 217 (72%) genes out of a total of 303 BR-regulated genes. B, Out of these 217 genes, 125 (58%) genes were synergistically, and the remaining 92 (42%) genes were antagonistically regulated by BR and Glc treatment alone. C, Presence of Glc can also change the extent of BR induction or repression by more than 2-fold for almost 85% of BR-affected genes (i.e. 256 genes out of a total of 303 BR-regulated genes). Out of these 256 genes, the extent of 110 (43%) genes was affected synergistically, and the extent of 146 (57%) genes was affected antagonistically in the presence of Glc. Glc can also affect BR regulation of those genes that are themselves not regulated by Glc alone.

Figure 2.

Validation of microarray results using real-time PCR. A, The relative transcript levels of a few selected genes from microarray data. Real-time PCR results of selected BR (B), auxin (C), and cell wall-related genes (D and E). Values represent the average from two biological replicates, and error bars represent se (Student’s t test, P < 0.05; *, control versus treatment). ARF6, AUXIN RESPONSE FACTOR6; GH3.1, GRETCHEN HAGEN3; LAX3, LIKE AUXIN RESISTANT3; ATEXPA17, EXPANSIN A17; ATEXLA2, EXPANSIN-LIKE A2; XTH6, XYLOGLUCAN ENDOTRANSGLUCOSYLASE/HYDROLASE6.

Figure 3.

Glc interacts antagonistically with BR to regulate TCH4 expression. A, The relative abundance of TCH4 transcript upon BR and Glc treatments in 7-d-old etiolated wild-type (Col-0) seedlings. B, GUS expression analysis in hypocotyls and cotyledons of 7-d-old dark-grown pTCH4:TCH4::GUS seedlings in the absence or presence of Glc (0% and 3% [w/v]) and/or BR (0 m and 100 nm). BR regulation of TCH4 expression was lost in presence of Glc, suggesting, mainly, an antagonistic interaction between BR and Glc response. Values represent the average from two biological replicates, and error bars represent se (Student’s t test, P < 0.05; *, control versus treatment).

Figure 4.

Glc and BR regulation of hypocotyl elongation growth in etiolated seedlings. Quantification of hypocotyl elongation in 7-d-old etiolated wild-type (Col-0) seedlings treated without or with increasing concentrations of BR (A), increasing concentrations of Glc (B), and Glc-free medium or increased Glc concentration containing one-half-strength MS medium supplemented without or with increasing concentrations of BR (C). Glc and BR work antagonistically at lower Glc concentrations but act synergistically at higher Glc concentrations for regulation of hypocotyl elongation growth. Values represent the average from two biological replicates, each having 30 seedlings, and error bars represent se (Student’s t test, P < 0.001; *, control versus treatment).

Figure 5.

Involvement of AtHXK1-dependent Glc signaling during BR regulation of hypocotyl elongation growth. Quantification of hypocotyl elongation in 7-d-old etiolated seedlings of the wild type (Ler) and AtHXK1-dependent Glc sensor mutant gin2-1 growing on Glc-free (0%) medium or increasing concentrations of Glc (1%, 3%, and 5% [w/v]) containing one-half-strength MS medium supplemented without or with increasing BR concentrations (10 nm, 100 nm, and 1 µm). The gin2-1 mutant showed less response to BR in terms of hypocotyl elongation growth compared with the wild type. Values represent the average from two biological replicates, each having 30 seedlings, and error bar represents se (Student’s t test, P < 0.001; *, control versus treatment; and **, wild type versus mutant).

Figure 6.

Dependence of Glc regulation of hypocotyl elongation growth upon BR-signaling components. A, Quantification of hypocotyl elongation growth in 7-d-old etiolated seedlings of the wild type (Col-0) growing on Glc-free medium or increasing concentrations of Glc containing one-half-strength MS medium supplemented without or with 1 µm BRZ. B to D, Quantification of hypocotyl elongation growth in the wild type and BR-signaling mutants. BR biosynthesis mutants cpd and det2 and BR receptor mutant bri1-6 were found to be resistant toward Glc-mediated hypocotyl elongation, while BR overproducer mutant ces-D and mutants with endogenously high BR signaling (bzr1-1D and bes1-D) showed more hypocotyl elongation at all Glc concentrations tested compared with their respective wild types. Values represent the average from two biological replicates, each having at least 20 seedlings, and error bar represents se (Student’s t test, P < 0.001; *, control versus treatment; and **, wild type versus mutant).

Figure 7.

BRI1 is epistatic to HXK1. A, Pictures showing phenotypic differences between 7-d-old etiolated seedlings of ecotypes (Ler and En2), wild-type (WT) segregant, Glc sensor mutant gin2-1, BR perception mutant bri1-6, and heterozygous double mutant gin2-1bri1-6 growing on one-half-strength MS medium. B, Difference between hypocotyl elongation growth of the 7-d-old etiolated wild type (Ler and En2), wild-type segregant, Glc sensor mutant gin2-1, BR perception mutant bri1-6, and homozygous double mutant gin2-1bri1-6 seedlings growing on one-half-strength MS medium. C, Analysis of Glc and BR sensitivity of the wild type (Ler and En2), wild-type segregant, Glc sensor mutant gin2-1, BR perception mutant bri1-6, and homozygous double mutant gin2-1bri1-6 seedlings in terms of hypocotyl elongation growth in the dark. The gin2-1bri1-6 double mutant displayed compromised etiolated hypocotyl growth as well as Glc and BR sensitivity similar to its bri1-6 parent rather than gin2-1 parent, suggesting that BRI1 is epistatic to HXK1 during BR-Glc regulation of etiolated seedling hypocotyl elongation growth. Data represent the average of values from 20 seedlings, and error bar represents se (Student’s t test, P < 0.001; *, control versus treatment; and **, wild type versus mutant).

Figure 8.

BR-Glc regulation of DR5::GUS expression in etiolated seedlings. GUS expression analysis in hypocotyls and cotyledons of 7-d-old dark-grown DR5::GUS seedlings in absence or presence of different concentrations of Glc (0%, 1%, 3%, and 5% [w/v]) and/or BR (0 m, 10 nm, 100 nm, and 1 µm). BR induction of DR5::GUS expression was lost in presence of increased Glc concentration (3% and 5% [w/v]), suggesting, mainly, an antagonistic interaction between BR and Glc.

Figure 9.

Involvement of TIR1/AFB mediated auxin perception during Glc-BR for regulation of hypocotyl elongation in etiolated seedlings. Quantification of hypocotyl elongation growth in the 7-d-old etiolated wild type (Col-0 and Wassilewskija), the auxin-signaling mutant tir1 (A), and tir1afb1-3afb2-3afb3-4 quadruple mutant seedlings (B) growing on one-half-strength MS medium supplemented with independent as well as combined treatments of Glc and BR. The tir1 mutants displayed wild-type-like sensitivity for Glc and higher concentrations of BR treatments. However, the tir1afb1-3afb2-3afb3-4 quadruple mutant showed resistance for Glc and BR compared with the wild type in terms of etiolated hypocotyl elongation growth. These results indicate the involvement of TIR1/AFB-dependent auxin perception during BR-Glc cross talk. Values represent the average from two independent biological replicates, each having 20 seedlings, and error bars represent se. (Student’s t test, P < 0.001; *, control versus treatment; and **, wild type versus mutant).

Figure 10.

Involvement of auxin-signaling components in Glc-BR regulation of hypocotyl elongation in etiolated seedlings. A, The BR induction of DR5::GUS expression was inhibited in axr1-3 mutation background. Also, in axr1-3 mutant background, even lower concentrations of Glc were able to inhibit the DR5::GUS expression, suggesting the involvement of AXR1-mediated mechanisms for BR-Glc cross talk. B and C, Quantification of hypocotyl elongation growth in the 7-d-old etiolated wild type (Col-0) and auxin-signaling mutants axr1-12, axr2-1, and axr3-1 seedlings growing on one-half-strength MS medium supplemented with independent as well as combined treatments of Glc and BR in the dark. The auxin-signaling mutant axr1-12 seedlings were found to have significantly reduced sensitivity, whereas axr2-1 and axr3-1 mutants were found highly resistant for different concentrations of Glc and BR for regulation of etiolated hypocotyl elongation growth compared with the wild type. These results indicate the dependence of both BR and Glc action upon these auxin-signaling components for regulation of etiolated hypocotyl growth. Values represent the average from two independent biological replicates, each having at least 15 seedlings (except for axr1-12; data from two biological replicates, 10 seedlings each), and error bars represent se (Student’s t test, P < 0.001; *, control versus treatment; and **, wild type versus mutant).