Transcriptional regulation of the CRK/DUF26 group of receptor-like protein kinases by ozone and plant hormones in Arabidopsis

Abstract

Background: Plant Receptor-like/Pelle kinases (RLK) are a group of conserved signalling components that regulate developmental programs and responses to biotic and abiotic stresses. One of the largest RLK groups is formed by the Domain of Unknown Function 26 (DUF26) RLKs, also called Cysteine-rich Receptor-like Kinases (CRKs), which have been suggested to play important roles in the regulation of pathogen defence and programmed cell death. Despite the vast number of RLKs present in plants, however, only a few of them have been functionally characterized.

Results: We examined the transcriptional regulation of all Arabidopsis CRKs by ozone (O3), high light and pathogen/elicitor treatment - conditions known to induce the production of reactive oxygen species (ROS) in various subcellular compartments. Several CRKs were transcriptionally induced by exposure to O3 but not by light stress. O3 induces an extracellular oxidative burst, whilst light stress leads to ROS production in chloroplasts. Analysis of publicly available microarray data revealed that the transcriptional responses of the CRKs to O3 were very similar to responses to microbes or pathogen-associated molecular patterns (PAMPs). Several mutants altered in hormone biosynthesis or signalling showed changes in basal and O3-induced transcriptional responses.

Conclusions: Combining expression analysis from multiple treatments with mutants altered in hormone biosynthesis or signalling suggest a model in which O3 and salicylic acid (SA) activate separate signaling pathways that exhibit negative crosstalk. Although O3 is classified as an abiotic stress to plants, transcriptional profiling of CRKs showed strong similarities between the O3 and biotic stress responses.

Figure 1

Transcriptional regulation of the CRKs in response to O₃. Expression of all members of the CRK group of plant RLKs was analyzed by quantitative real-time RT-PCR (qPCR) in Col-0 plants exposed to 250 ppb O₃ for 6 h. Samples were harvested at 1 or 8 h (6 h followed by 2 h recovery under clean air conditions) after the onset of the O₃ treatment. Transcript levels were calculated by comparison of O₃-exposed plants with corresponding control plants grown under clean air conditions harvested in parallel with the O₃-treated plants. An expression level of one indicates no change in expression, increased expression is indicated by values larger than one while decreased expression is shown by values smaller than one. Increase in expression by 2-fold or higher is high-lighted in red and decrease in expression by 2-fold or more in green. NR - no reproducible data could be obtained for this gene. The experiment was repeated four times; fold change was calculated from the average normalized cycle difference of all biological repeats. Statistical significance (Benjamini-Hochberg FDR corrected p-value ≤ 0.1) is indicated with asterisks (see additional file 1).

Figure 2

Phylogenetic tree of the CRK kinase domains indicates that O₃ regulation is distributed throughout group. The kinase domains of all CRKs were aligned using ClustalW2 and a Neighbour-joining tree was constructed using MEGA4 [84]. DUF26 44 (At4g11500) and CRK9 (At4g23170) were not included in the analysis. Genes with increased expression by O₃ treatment are indicated in red and genes with decreased expression in green (statistically significant changes are indicated by an asterisk).

Figure 3

Transcriptional downregulation of CRKs in response to light stress. Expression of APX2 (a marker for light stress) and CRKs was analyzed by qPCR in plants after 1 h and 2 h exposure to light stress conditions and after 4 h light stress followed by 4 hours recovery at normal growth light conditions. Transcript levels were calculated by comparison of light stress-treated plants with corresponding control plants grown under normal light conditions. An expression level of one indicates no change of expression, increased expression is indicated by values larger than one while decreased expression is shown by values smaller than one. Increase of expression by 2-fold or higher is high-lighted in red and decrease in expression by 2-fold or more in green. NR - no reproducible data could be obtained for this gene. The experiment was repeated twice; fold change was calculated from the average normalized cycle difference of all biological repeats. Statistical significance (Benjamini-Hochberg FDR corrected p-value ≤ 0.1) is indicated with asterisks (see additional file 1).

Figure 4

Bayesian hierarchical clustering of the CRKs in abiotic and biotic stress experiments. Biotic and abiotic stress data sets were down loaded from public databases and included O₃, norflurazon, paraquat, BTH (benzothiadiazole S-methylester), various elicitors and pathogens (see materials and methods for complete details). Red and green indicate increased or decreased expression compared to untreated plants, respectively. The intensity of the colours is proportional to the absolute value of the fold difference.

Figure 5

Expression of CRKs is changed in hormone mutants. The expression of all CRKs was analyzed by qPCR in the SA mutants sid2 and npr1, the ET mutant ein2, the JA mutant fad3/7/8 and the cell death mutant dnd1 by qPCR and compared to Col-0 under two different growth conditions. (A) Weiss chamber conditions. (B) Phytotron. Transcript levels were calculated by comparison between mutants and Col-0 grown under control conditions. An expression level of one indicates no change of expression, increased expression is indicated by values larger than one while decreased expression is shown by values smaller than one. Increase of expression by 2-fold or higher is high-lighted in red and decrease in expression by 2-fold or more in green. NR - no reproducible data could be obtained for this gene. ND - The dnd1 mutant did not grow in the phytotron. Fold-change is shown for the geometric mean of all biological repeats (n = 4). Statistically significance (Benjamini-Hochberg FDR corrected p-value ≤ 0.1) is indicated with asterisks (see additional file 1).

Figure 6

O₃-regulation of CRKs is different in hormone mutants. The expression of 24 O₃-regulated CRKs was analyzed by qPCR in Col-0 and sid2, npr1, dnd1, ein2 and fad3/7/8 exposed to 250 ppb O₃ for 6 h. Samples were harvested at 1 or 8 h (6 h plus 2 h recovery under clean air conditions) after the onset of the O₃ treatment. Transcript levels for Col-0 or each mutant line were calculated by comparison of O₃-exposed plants with corresponding control plants of the same line grown under clean air conditions. An expression level of one indicates no change of expression, increased expression is indicated by values larger than one while decreased expression is shown by values smaller than one. Increase of expression by 2-fold or higher is high-lighted in red and decrease in expression by 2-fold or more in green. NR - no reproducible data could be obtained for this gene. The experiment was repeated four times; fold change was calculated from the average normalized cycle difference of all biological repeats. Statistically significance (Benjamini-Hochberg FDR corrected p-value ≤ 0.1) is indicated with asterisks (see additional file 1).

Figure 7

Clustering and qPCR analysis of the marker genes. (A) The expression of eight O₃-inducible genes and the qPCR normalization gene Actin-2 were analyzed in public array data from biotic and abiotic stress and hormone treatments. Red and green indicate increased or decreased expression compared to untreated plants, respectively. The intensity of the colours is proportional to the absolute value of the fold difference. (B) Markers genes for O₃ responses were analyzed by qPCR in Col-0 and sid2, npr1, dnd1, ein2 and fad3/7/8 exposed to 250 ppb O₃ for 6 h. Samples were harvested at 1 or 8 h (6 h plus 2 h recovery under clean air conditions) after the onset of the O₃ treatment. Transcript levels were calculated by comparison of O₃-exposed plants with corresponding control plants grown under clean air conditions. An expression level of one indicates no change of expression, increased expression is indicated by values larger than one while decreased expression is shown by values smaller than one. Increase of expression by 2-fold or higher is high-lighted in red and decrease in expression by 2-fold or more in green. NR - no reproducible data could be obtained for this gene. The experiment was repeated four times; fold change was calculated from the average normalized cycle difference of all biological repeats. Statistically significance (Benjamini-Hochberg FDR corrected p-value ≤ 0.1) is indicated with asterisks (see additional file 1).

Figure 8

Light stress response in hormone mutants. The expression of 24 O₃-inducible CRKs was analyzed by qPCR in Col-0 and sid2, npr1, ein2 and fad3/7/8 after 1 h and 2 h exposure to light stress conditions, and after 4 h light stress followed by 4 h recovery at normal growth light conditions. Transcript levels were calculated by comparison of light stress-treated plants with the corresponding control plants grown under normal light conditions. An expression level of one indicates no change of expression, increased expression is indicated by values larger than one while decreased expression is shown by values smaller than one. Increase in expression by 2-fold or higher is high-lighted in red and decrease in expression by 2-fold or more in green. NR - no reproducible data could be obtained for this gene. The experiment was repeated twice; fold change was calculated from the average normalized cycle difference of all biological repeats. Statistically significance (Benjamini-Hochberg FDR corrected p-value ≤ 0.1) is indicated with asterisks (see additional file 1).

Figure 9

ROS, elicitor and hormone regulation of O₃-induced CRKs. O₃ enters the leaves through the stomata and immediately reacts with components of the cell wall to generate ROS. O₃ and the ROS induce an active production of ROS in the apoplast which is at least partly depending on membrane bound NADPH oxidases (RBOH), which produce . Similar ROS production in the apoplast takes place after infection of a plant with a pathogen or treatments with pathogen derived elicitors (PAMPs). ROS is hypothetically perceived via a "ROS receptor" which could sense ROS directly via protein modification, or via sensing of modified apoplastic proteins or other molecules that react with ROS. The perception of ROS initiates down-stream signalling events. H₂O₂ is also able to cross the plasma membrane and enter the cells. Inside the cell, the signalling pathway is split into two pathways. In the ROS pathway DND1/CNGC2 mediates a required step of the signalling pathway and JA and ET act as positive regulators, and SA and NPR1 are negative regulators. In the SA pathway ROS or pathogens activate SA biosynthesis via ICS1; and NPR1 is a required component. Since NPR1 is a positive regulator of the SA pathway and a negative regulator of the ROS pathway this implies that the separate signalling pathway use different transcription factors and promoter elements to regulate CRK expression, although it might be possible that two different transcription factors could converge on the same promoter element. In addition the pleiotropic nature of the dnd1 mutant, including high SA-levels, could change the place of DND1/CNGC2 in the model - constitutive SA signalling in dnd1 may limit the possibility for O₃ to activate the ROS pathway. Through the transcription factors EIN3 and EIL1 ET can repress SID2/ICS1 expression and SA levels. Increased ROS production in the chloroplast activates separate signalling pathway(s) leading to repression of CRK expression. One of these pathways could involve ABA and negative cross talk with the SA pathway.