Transcriptome analysis of germinating maize kernels exposed to smoke-water and the active compound KAR1

Abstract

Background: Smoke released from burning vegetation functions as an important environmental signal promoting the germination of many plant species following a fire. It not only promotes the germination of species from fire-prone habitats, but several species from non-fire-prone areas also respond, including some crops. The germination stimulatory activity can largely be attributed to the presence of a highly active butenolide compound, 3-methyl-2H-furo[2,3-c]pyran-2-one (referred to as karrikin 1 or KAR1), that has previously been isolated from plant-derived smoke. Several hypotheses have arisen regarding the molecular background of smoke and KAR1 action.

Results: In this paper we demonstrate that although smoke-water and KAR1 treatment of maize kernels result in a similar physiological response, the gene expression and the protein ubiquitination patterns are quite different. Treatment with smoke-water enhanced the ubiquitination of proteins and activated protein-degradation-related genes. This effect was completely absent from KAR1-treated kernels, in which a specific aquaporin gene was distinctly upregulated.

Conclusions: Our findings indicate that the array of bioactive compounds present in smoke-water form an environmental signal that may act together in germination stimulation. It is highly possible that the smoke/KAR1 'signal' is perceived by a receptor that is shared with the signal transduction system implied in perceiving environmental cues (especially stresses and light), or some kind of specialized receptor exists in fire-prone plant species which diverged from a more general one present in a common ancestor, and also found in non fire-prone plants allowing for a somewhat weaker but still significant response. Besides their obvious use in agricultural practices, smoke and KAR1 can be used in studies to gain further insight into the transcriptional changes during germination.

Figure 1

Effect of smoke and KAR₁ on the germination time course of maize kernels. Each treatment consisted of four independent experiments with three biological replicates (n = 30). The kernels were treated with water (control), 1:1000 or 1:2000 (v/v) dilution of smoke-water, and 0.1 or 0.01 μM KAR₁. Germinated kernels were scored every day for 10 d. Error bars represent standard error (SE) of the mean germination percentage.

Figure 2

Hierarchical clustering of data from the microarray analysis of gene expression in smoke- and KAR₁-treated germinating maize kernels. The data represents control vs. smoke, control vs. KAR₁, control vs. smoke-treated for 3 h after a 3 h delay, control vs. smoke-treated for 6 h after a 3 h delay, and KAR₁ vs. smoke comparisons. Samples with similar patterns of expression of the genes studied cluster together, as indicated by the dendrogram. The hierarchical clustering of the 21 genes that seemed to be the most relevant in all experiments is illustrated (expression changed in all experiments with fold-change ≥ 2, and at least in one experiments the corrected p-values < 0.1). Yellow indicates up-, and blue indicates downregulation.

Figure 3

Principal component analysis of the genes distinctly differentially expressed between smoke and KAR₁ treatments. Biplot representation of the principal component analysis. The figure shows the 199 genes at 1.5 h, 3 h, 6 h, 9 h, 12 h and 24 h that were distinctly differentially expressed (fold-change ≥ 4 and a corrected p-value < 0.1) in at least one experiment. All of the genes were plotted with respect to the first and second principal components and they are represented with a light gray text. The originally observed variables are plotted as red (smoke- related) and blue (KAR₁-related) responsive arrows. The arrows represent the association of the measured variables (fold-change) with the samples in the visualization: the length and location are proportional to the variable loadings on the two first principal components. The analysis suggests that much of the variability and difference between the two gene sets can be attributed to the two different treatments (smoke and KAR₁). Also see Additional File 10.

Figure 4

Validation of microarray results via quantitative real-time PCR. A, Quantitative real-time PCR analysis was performed for 14 genes under the same conditions and design used for microarray analysis. Real-time PCR data were obtained from three independent experiments with similar results, and four amplification reactions. Microarray data (least-square means) were plotted against data from qRT-PCR and fitted into a linear regression. Both x- and y-axes are shown in log2 scale. B, The biological variation of the expression (assessed by quantitative real-time PCR analysis) of 8 selected master genes are shown. Experimental design is as for Fig. 5A. Error bars represent standard deviation. Asterisks indicate significant difference (p < 0.05) from the control samples (statistical analysis was assessed by a t-test).

Figure 5

The list of GO terms overrepresented in the group of genes up- and downregulated (fold change ≥2 and corrected p-value < 0.2). Data obtained from 1.5 h, 3 h and 24 h smoke-water treated samples are not included on the map. The frequency of each GO term was calculated [42] and multiplied by 100 to make the plotting easier on the heat map. Light colours indicate low representation; blue/deep blue colours show overrepresentation. Red squares: raw p-value < 0.05; orange squares: raw p-value of 0.05 - 0.1; yellow squares: raw p-value 0.1 - 0.2.

Figure 6

Effect of smoke, KAR₁, AgNO₃ and their combinations on the seedling vigour of 5-d-old maize seedlings. A, Typical phenotypes of the treated kernels. B, Frequency distribution of two growth variables (coleoptile and root length in mm) in control, smoke (1:1000 dilution), KAR₁ (0.1 μM), AgNO₃ (10 μM), smoke+AgNO₃, KAR₁+AgNO₃. The data were grouped into bins as presented on the graph. For statistical analysis (see Additional File 9), the Mann-Whitney U-test was applied (R 2.9.0.) and p < 0.05 was regarded as significant (n = 120).

Figure 7

SDS-PAGE and immunoblotting analysis. Maize kernels were germinated in water (c, control) or were treated with smoke (s, 1:1000 dilution) or KAR₁ (KAR₁, 0.1 μM) for 3, 4.5, 6 and 7.5 h and proteins were extracted from the embryos (n = 15). The experiments were repeated three times with similar results. Molecular masses (kDa) of standard proteins are indicated on the left. A, Immunoblotting analysis with anti-ubiquitin antibody. B, Protein pattern obtained by SDS-PAGE and Coomassie Blue staining.