Arabidopsis NAC Transcription Factor JUNGBRUNNEN1 Exerts Conserved Control Over Gibberellin and Brassinosteroid Metabolism and Signaling Genes in Tomato

Abstract

The Arabidopsis thaliana NAC transcription factor JUNGBRUNNEN1 (AtJUB1) regulates growth by directly repressing GA3ox1 and DWF4, two key genes involved in gibberellin (GA) and brassinosteroid (BR) biosynthesis, respectively, leading to GA and BR deficiency phenotypes. AtJUB1 also reduces the expression of PIF4, a bHLH transcription factor that positively controls cell elongation, while it stimulates the expression of DELLA genes, which are important repressors of growth. Here, we extend our previous findings by demonstrating that AtJUB1 induces similar GA and BR deficiency phenotypes and changes in gene expression when overexpressed in tomato (Solanum lycopersicum). Importantly, and in accordance with the growth phenotypes observed, AtJUB1 inhibits the expression of growth-supporting genes, namely the tomato orthologs of GA3ox1, DWF4 and PIF4, but activates the expression of DELLA orthologs, by directly binding to their promoters. Overexpression of AtJUB1 in tomato delays fruit ripening, which is accompanied by reduced expression of several ripening-related genes, and leads to an increase in the levels of various amino acids (mostly proline, β-alanine, and phenylalanine), γ-aminobutyric acid (GABA), and major organic acids including glutamic acid and aspartic acid. The fact that AtJUB1 exerts an inhibitory effect on the GA/BR biosynthesis and PIF4 genes but acts as a direct activator of DELLA genes in both, Arabidopsis and tomato, strongly supports the model that the molecular constituents of the JUNGBRUNNEN1 growth control module are considerably conserved across species.

Keywords: Arabidopsis; DELLA proteins; brassinosteroid; fruit; gibberellic acid; growth; tomato; transcription factor.

FIGURE 1

Analysis of AtJUB1-OX transgenic plants. (A) Expression level of AtJUB1 in leaves of transgenic lines OX1, OX2, and OX3 compared to wild type (WT). Mean ± SD (n = 3). (B) Expression of AtJUB1 in fruits of line OX2 at mature green (MG), breaker (B), and breaker+7d (B+7) stages. Mean ± SD (n = 3). For quantitative real-time polymerase chain reaction (qRT-PCR) analysis, values are expressed as the difference between an arbitrary value of 40 and dCt, so that high 40-dCt value indicates high gene expression level. (C) Nuclear localization of GFP-tagged AtJUB1 protein in leaves of transgenic tomato plants, imaged by confocal microscopy. Top left, fluorescence microscopy; top right, DAPI staining; bottom, overlay. Some AtJUB1-GFP protein appears to reside in the cytoplasm surrounding the central vacuole, likely due to incomplete nuclear import.

FIGURE 2

Phenotype of AtJUB1-OX tomato plants at different developmental stages. (A) Reduced growth in 3-week-old AtJUB1-OX transgenic plants strongly overexpressing AtJUB1, compared to wild type (WT). (B) Leaf parameters: length (L) and width (W) of leaflet blade; petiolule (P) length; hypocotyl length of 3-week-old AtJUB1-OX and WT tomato seedlings. Leaf parameters were determined for the two subterminal leaflets of leaf number 1. (C) Vegetative growth phenotype of 6-week-old plants and their leaves (lower panel). (D) Height of plants. (E) Flowering time of AtJUB1-OX and WT plants measured by counting the number of leaves on the date that the first inflorescence arose. (F) Phenotype of plants at reproductive stage. (G) Flowers and fruits of plants shown in (F). (H) Fruit ripening was scored in AtJUB1-OX plants compared to WT by counting the days after pollination (DAP) to reach the mature green and breaker stages. Data in graphs represent mean ± SD (n = 10) and ^∗p < 0.05, ^∗∗p < 0.01 by Student’s t-test. Asterisks denote significant differences to WT.

FIGURE 3

Direct regulation of GA- and BR-associated genes by AtJUB1. (A) Heat map showing the expression of orthologs of Arabidopsis DWF4, GA3ox1, GAI, RGL1, and PIF4 in tomato line OX2 at mature green (MG), breaker (B), breaker+7d (B+7) stages and leaves of mature plants compared to wild type (WT). The log₂ fold change scale is indicated under the heat map. Complete data are given in Supplementary Table S2. (B) Schematic diagram showing positions of AtJUB1 binding sites (numbered I, II and III if three sites were present) in the promoters of genes orthologous to the AtJUB1 targets in Arabidopsis. ChIP amplicons are indicated as black thick underlines. (C) ChIP assays showing the direct binding of AtJUB1 to the promoters of SlDWF4-1, SlGA3ox1-1, SlGA3ox1-2, SlGAI, SlGAI-like and SlPIF4. Values were normalized to the values for Solyc01g090460 (promoter lacking AtJUB1 binding site), as a negative control. Error bars represent mean ± SD (two independent biological replicates, each with three technical replicates). FC, fold change.

FIGURE 4

Quantitative real-time polymerase chain reaction analysis of AtJUB1-regulated genes in tomato fruits. Heat map showing differentially expressed genes in AtJUB1-OX fruits compared to wild type. Means of three experiments are shown; complete data are given in Supplementary Table S3. The log₂ fold change scale is indicated under the heat map. Transcription factors, hormone metabolism-related, cell wall-related and peroxidase categories are shown.

FIGURE 5

Heat map showing the metabolite changes in fruits of AtJUB1-OX plants in comparison with wild type at three ripening stages. Mature green fruit (MG), breaker stage (B), and red ripe fruit (B+7) were analyzed. Red or blue indicate that the metabolite content is increased or decreased, respectively. Asterisks denote significant difference (p ≤ 0.01, Student’s t-test) in AtJUB1-OX compared to wild type; complete data are given in Supplementary Table S4.