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. 2010 Jul;5(7):907–910. doi: 10.4161/psb.5.7.12099

NAC genes

Time-specific regulators of hormonal signaling in Arabidopsis

Michael Krogh Jensen 1, Trine Kjaersgaard 1, Klaus Petersen 1, Karen Skriver 1,✉
PMCID: PMC3014545  PMID: 20484991

Abstract

Environmental stresses on both animals and plants impose massive transcriptional perturbations. Successful adaptations to such stresses are being orchestrated by both activating and repressing effects of transcription factors on specific target genes. We have recently published a systematic characterization of members of the large NAC gene transcription factor family in the model weed Arabidopsis thaliana. Our analysis revealed interesting sub-groupings of the Arabidopsis NAC genes, relating structure and function. Here we present a meta-analysis revealing distinct temporal expression profiles of NAC genes upon stimuli with seven phytohormones. Our analysis could be a first indication of NAC-centered transcriptional networks, which coordinate timely hormonal signaling in plants.

Key words: Arabidopsis, NAC genes, hormonal signaling, transcription factor, network

Phytohormones play pivotal roles in many developmental and stress-related processes in plants.1,2 Adequate perception and adaptation to hormonal fluctuations are to a large extent governed by regulatory proteins, including transcription factors.3,4 In plants, NAC transcription factors constitute one of the largest transcription factor families with over 100 family members in Arabidopsis.5 Several NAC proteins have been reported to be involved in hormone-related processes during plant development and environmental stresses.6–9

To provide a comprehensive overview of hormone-inducible NAC genes a metaanalysis of NAC gene expression patterns was performed using genome-wide expression datasets at the AtGenExpress repository: (www.arabidopsis.org/info/expression/ATGenExpress.jsp).

This revealed prominent time- and hormone-specific regulation of NAC genes in response to hormone treatments applied at three different time-points by Goda et al.10 (Fig. 1). By unsupervised hierarchical clustering of 21 experiments (three time-points, seven hormones) arrays representing the same time-point clustered together; i.e., strong time-specific regulation of many NAC genes by all hormones tested (Clusters II–IV). Other NAC genes displayed time-independent and narrower hormonal induction (Clusters I and V). Overlapping regulation by several hormones of genes encoding regulatory proteins is in agreement with observations by Goda et al.10 supporting the notion on massive inter-connectivity between different hormonal signaling pathways sharing target genes at the transcriptional level.10–12 However, and most importantly, all hormones tested not only regulate the expression of a large fraction of the NAC genes; hormonal regulation also displays distinct time-specificity. The AtGenExpress data, presented here, correlates with data available from the less extensive hormone expression study using the full-genome Arabidopsis TILING array13 (data not shown).

Figure 1.

Figure 1

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Expression and promoter analysis of Arabidopsis NAC genes. Eighty-nine ATH1 probe-sets matching Arabidopsis NAC genes were hierarchical clustered using Affymetrix GeneChip expression data from the AtGenExpress Consortium (http://www.arabidopsis.org/info/expression/ATGenExpress.jsp) corresponding to 7 different hormone treatments (ABA; abscisic acid, MeJA; methyl-jasmonate, IAA; auxin-analog, ACC; ethylene-analog, BRS; brassinosteroid, GA; gibberelic acid, Zea; cytokinin-analog) sampled at 0.5, 1 and 3 hrs after treatment.10 All raw data sets were normalized and interpreted using the GCRMA function24 of the Bioconductor microarray analysis package (http://www.bioconductor.org/).25 Average of three replicate treatments relative to average of appropriate control mean values were used. Color bars at the top indicates identical sampling times after treatment which also group based upon unsupervised hierarchical clustering26 using a scaled correlation of rows and complete linkage clustering. A color key in the lower right corner displays genes repressed relative to their control in red, those induced white, whereas genes unchanged in their expression are colored orange. Color key displays correlation between color and expression level. AGI names and clusters, defined in this analysis, are given to the right. To the far right a grey-scaled color bar illustrates the number of selected cis-elements in the 1 kb upstream promoter of the 89 NAC genes. Selected elements are NAC binding site consensus (NACBS; [TA][TG]XCGT[GA]), jasmonate-responsive element (JRE; TGACG), ABA-responsive element (ABRE; CCGTGT), ethylene-responsive element (ERE; AGCCGCC), and GA-repression element (GA_down; ACGTGC). Color key below displays grey-scale illustrating number of selected promoter elements.

For improved understanding of the observed expression patterns, we analyzed the 1 kb upstream promoter sequences of NAC gene clusters I–V using Promomer site: (bar.utoronto.ca/ntools/-cgi-bin/BAR_Promomer.cgi).14 The analysis highlighted promoters of Cluster I genes to be significantly enriched in both ABA-responsive elements (ABRE), and cis-elements of GA repressed genes (GA_down)(p-values > 0.001 and 0.0001, respectively), as noted by Christianson et al.15 and in accordance with the antagonistic effects of ABA and GA.16 Interestingly, with the exception of At1g69490, this gene cluster corresponds to the ATAF subgroup of NAC genes defined by structure-based phylogenetic analysis,17 and could reflect evolution of the NAC gene family by gene duplication creating paralogous genes with a high degree of sequence similarity and functional redundancy. A consequence of redundancy among paralogous genes is a high degree of co-expression during development and stress perception in which they share functionality.18 It should be noted that apart from the significant hormonal-regulation of the 6 ATAF genes shown in Figure 1, McGrath et al.19 verified MeJA-induction of At3g15500 by qRT-PCR, and, moreover, showed that expression of At5g08790 (not present on the ATH1 GeneChip) was also induced by MeJA. Hence, both ABA and MeJA induce the entire ATAF subgroup. Another prominent co-expression cluster (Cluster II) contains NAC genes, which show early induction of expression by all hormone treatments. Surprisingly, early hormone-responsive NAC genes are not over-represented in any known hormone responsive elements. Neither are the promoters of NAC genes with expression induced late by most hormones tested (Cluster IV). Hormone-responsive NAC genes induced at 1 hr after hormone application (Cluster III), however, are significantly over-represented in both jasmonate-responsive elements (JRE) (p value > 0.005) and ethylene-responsive elements (ERE) (p > 0.005). The small cluster of ABA-only responsive genes (Cluster V) has an over-representation of JRE (p values > 0.001), whereas ABRE is not significantly over-represented. Finally, most NAC genes contain one or more NAC-binding sites (NACBS) in their 1 kb upstream promoter. In conclusion, three of five NAC clusters have significant over-representation of specific hormone-response elements in their promoters reflecting pronounced hormonal regulation of NAC gene expression.

To further investigate whether clustering of NAC gene expression in response to hormone-treatments is representative of overall NAC gene expression-based clustering, we used PRIMe software (prime.psc.riken.jp/)20 to analyze expression datasets involving the entire 1388 Arabidopsis ATH1 GeneChip arrays in the AtGenExpress project.21,22 This analysis identified 36 NAC genes, which correlate in their expression profile with one, two or three other NAC genes (Fig. 2). Again, Cluster I ATAF-like genes form a significant pattern with several interconnectivities representing co-expression. In addition to pronounced intra-cluster connectivities, members of this cluster connect with At5g39610 (NAC2/ORE1), also known to respond to hormonal exposures.7 Also, the CUC genes (At1g76420, At5g53950 and At3g15170) are co-expressed in accordance with their structural relationships.17 However, this is not reflected in the overall hormonal regulation due to significant differences in the time-dependence of induction by specific hormones. Altogether this illustrates that for the ATAF-like NAC genes, clustering based on hormone-induced expression profiles correlates with their overall expression perturbations during development and stress. However, the PRIMe analysis also reveals co-expression patterns not reflected in those resulting from hormone-treatments.

Figure 2.

Figure 2

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A NAC gene co-expression network built using PRIMe (http://prime.psc.riken.jp/).20 A stringent cut-off (0.6) was used to identify NAC co-expression profiles from 1388 Arabidopsis ATH1 array samples from the AtGenExpress Consortium (www.arabidopsis.org/info/expression/ATGenExpress.jsp). AGIs from the CUC subgroup are in bold, and members of the ATAF subgroup in bold and labeled with an asteriks.

In summary, the time-specific expression patterns of many NAC genes in response to all hormones tested reflects an interesting ‘NACome’-mediated fine-tuning of hormonal perception, in which a set of early NAC hormonal signaling regulators (Cluster II) potentially regulates a secondary intermediate class of co-expressed NAC genes (Cluster III), that eventually targets a third class of late hormone-inducible NAC genes. The late hormone-inducible NAC genes seem to have a more narrow range of inducers (Cluster IV), than the early and intermediate hormone-inducible NAC genes. Previous studies have already shown that NAC proteins can regulate expression of the corresponding or other NAC genes.9,23 However, further studies of time-specific DNA-binding are needed to determine if NAC genes are direct targets of other NAC family members in hormonal-signaling.

Acknowledgements

This study was supported by the Danish Agency for Science (274-07-0173 and 09-064140/FTP).

Abbreviations

ABA

abscisic acid

MeJA

methyl-jasmonate

GA

gibberelic acid

NAC

NAM-ATAF1,2-CUC2

Addendum to: Jensen MK, Kjaersgaard T, Nielsen MM, Galberg P, Petersen K, O'Shea C, Skriver K. The Arabidopsis thaliana NAC transcription factor family: structure-function relationships and determinants of ANAC019 stress signaling. Biochemical J. 2010;426:183–196. doi: 10.1042/BJ20091234.

Footnotes

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