How calmodulin binding transcription activators (CAMTAs) mediate auxin responses

Abstract

Phenotypic plasticity is an adaptive feature of all organisms, which, in land plants, entails changes in orientation of growth (tropism), patterns of development, organ architecture, timing of developmental processes and resource allocation. However, little is known about the molecular components that integrate exogenous environmental cues with internal hormonal signaling pathways. This addendum describes a role for calcium-regulated calmodulin-binding transcription 1 (CAMTA1) in auxin signaling and stress responses. We discuss possible mechanisms that may underlie this role of CAMTA1, and speculate on the more general roles of CAMTAs in auxin responses and phenotypic plasticity.

Introduction

The ability of land plants to monitor and respond to their environment is manifested in tremendous plasticity of their growth and development. No doubt, Ca²⁺ signaling plays important roles in conveying changes in environmental cues to cellular machineries that underlie such phenotypic plasticity. Transcription factors regulated by Ca²⁺ and Ca²⁺ sensors are likely important factors in this phenomenon. Calmodulin (CaM) is one of the prominent Ca²⁺ sensors in eukaryotic cells including plants.¹ To date several types of transcription factors in plants were shown to interact with CaM,² among them CAMTAs (for Calmodulin-Binding Transcription Activators^3,4). The six members of the CAMTA family in Arabidopsis thaliana respond rapidly and differentially to various environmental stresses and investigation of camta mutants show their roles in both biotic and abiotic defenses.^5–7 What recently became apparent is that CAMTAs link environmental cues with phytohormone-dependent growth responses.⁸ Here we summarize evidence for the role of CAMTA1 in auxin responses⁸ and further evidence for major CAMTA-regulated gene clusters that link environmental cues to auxin responses.

Several lines of evidence point towards the role of CAMTA1 in auxin responses.⁸ First, the expression pattern conferred by a CAMTA1 promoter fragment is reminiscent of auxin distribution as typically shown by the DR5::GUS reporter system. Second, camta1 mutants show major changes in the expression of genes associated with auxin signaling, including the enhanced expression of AUX/IAA (IAA29) transcriptional repressors and an F-box gene homologous to proteins that bind auxin and function as activators of auxin responses by promoting the degradation of AUX/IAA repressors. Loss-of-function mutants of camta1 also differ from wt in the repression of genes encoding auxin conjugating enzymes (detailed below). Moreover, an auxin-dependent phenotype of camta1 mutants is apparent. Hypocotyl elongation in dark-grown seedlings was inhibited by exogenous auxin in both wt and camta1 mutants. However, the effect of auxin was substantially stronger in the mutants.⁸ In addition, transgenic lines ectopically expressing an engineered CAMTA1 repressor (CAMTA1 fused to the SRDX transcription repressor amino acids) also showed substantially greater auxin-dependent inhibition compared to the effect of auxin in wild-type plants.⁸ Recent investigations show that the phenotype of short hypocotyls in camta1 mutants is also apparent in the absence of exogenous auxin (unpublished results).

Hypocotyl elongation involves mostly cell elongation without significant cortical or epidermal cell division.⁹ In the dark, hypocotyl elongation was shown to be inhibited by the addition of exogenous auxin⁸ and by elevation of endogenous auxin resulting from different types of genetic manipulations.^10,11 In early experiments the polar auxin transport inhibitor NPA was suggested not to affect hypocotyl elongation in the dark,¹² which raised questions about the role of polar auxin transport in hypocotyl elongation in the dark.¹² However, later experiments showed that in the auxin-resistant mutants axr2-1 and axr3-1, which carry gain-of-function mutations in AUXI/IAA transcription repressors,¹³ hypocotyls of dark-grown plants are very short compared to wild-type hypocotyls,^10,13 indicating that AUX/IAA and probably auxin per se, are involved in hypocotyl elongation in the dark. At this point, it is not clear how CAMTA1 mediates auxin responses. Nevertheless, it is plausible that changes in AUX/IAA, either by mutation or by alterations of levels of gene expression, may affect hypocotyl elongation in the dark. CAMTA1 may also be interacting with factors other than auxin such as gibberellin,¹⁴ ethylene,¹⁵ and brassinosteroids.¹⁶

Involvement of CAMTA1 in Auxin-mediated Hypocotyl Elongation through Regulation of Auxin Signaling Intermediates

Auxin responses that are involved in cell expansion are mediated by two distinct cellular pathways, one that operates at the plasma membrane and activates ATP-driven proton pumps and inward potassium channels and another operating in the nucleus activating transcription of several genes, among them plasma membrane H⁺-ATPAses, K⁺ channels and cell wall proteins.¹⁷ According to the acid-growth hypothesis,¹⁸ acidification of the cell wall enables its loosening by activating cell wall proteins such as expansins and other hydrolases.¹⁷ Potassium-driven entrance of water builds up turgor pressure, which eventually leads to cell elongation. Auxin-mediated transcription regulation involves a family of repressors (AUX/IAA) that prevent auxin-responsive transcription factors (ARFs) from activating their target genes under low levels of auxin. Auxin binds to an F-box protein which is part of a multisubunit protein complex that sends AUX/IAA to the proteasome to be degraded in the presence of higher levels of auxin thus allowing the transcription of auxin-responsive genes. Plants possess multi-gene families of ARFs, AUX/IAA and F-box proteins, and it is the subtle balance between activators and repressors that determines the outcome of the auxin response.^17,18

The transcription of family members of Aux/IAA genes is rapidly enhanced by auxin.¹⁹ Auxin doesn't have the same effect on genes encoding members of the F-box family.²⁰ Therefore, we speculate that in the presence of high auxin in the medium, the high ratio of AUX/IAA repressors to F-box proteins results in repression of auxin-responsive genes that are necessary for cell expansion. In the camta1 mutants, the enhanced expression of AUX/IAA repressors⁸ may further enhance the inhibition of cell elongation compared to wt seedlings. The F box gene that is upregulated in camta1 mutants was shown to be enhanced by IAA treatments,^8,20 although not to the extent of the enhancement of the expression of AUX/IAA genes.

Possible Effects of CAMTA1 on Auxin Transport and Homeostasis

One of the previously identified targets of AtCAMTA1 is the gene encoding AVP1, an H⁺-pyrophosphatase.²¹ In addition to maintaining vacuolar pH, the protein regulates auxin transport and cell wall acidification by mediating the recycling of PIN1 and H⁺-ATPase to the plasma membrane. Therefore it controls auxin-dependent development.²² Indeed, AVP1 overexpression causes increased auxin transport while avp1-1 null mutants have reduced auxin transport and disrupted organ development.²² Addition of auxin to the medium, or when levels of endogenous auxin rise, could activate the transcription of CAMTA1²³ and consequently may enhance the transcription of AVP1.²¹ This would facilitate the recycling and the presence of PIN1 and H⁺ATPase in the plasma membrane. As a result, auxin transport would be enhanced, thus changing the spatial distribution of free auxin. These changes are expected to be attenuated in camta1 mutants.

Finally, transcriptome analysis of camta1 mutants revealed reduced expression of genes encoding auxin-conjugating enzymes including UDP-glucosyl transferases and IAA amido synthetase⁸ suggesting that in wild type plants CAMTA1 is an activator of these genes. Consequently in the camta1 mutants we expect reduced activity of these enzymes resulting in higher levels of free auxin. In summary, CAMTA1 responds to auxin and stresses and, directly or indirectly, represses the expression of specific genes involved in auxin homeostasis, transport and signaling (Fig. 1).

Figure 1.

CAMTA1 links stress responses to auxin-mediated growth. Evidence for auxin-responsive expression of the CAMTA1 gene and its promoter analysis were described earlier.⁸ CAMTA1 target genes are derived from transcriptome analysis of camta1 mutants.⁸ The involvement of CAMTA 1 in cellular pH control and auxin transport were also reported.²¹,²² SR, Stress-responsive; SRE, SR DNA elementl; AR, Auxin-responsive; ARE, AR DNA element; TF, transcription factor; CaM, calmodulin. CAMTA protein domains were previously reviewed in ref. 4, as follows: CaMBD, CaM-binding domain; ANK, Ankyrin repeat; TIG, transcription factor immunoglobulin-like DNA-binding domain; TAD, Transcription activation domain; DNA-BD, DNA-binding domain.

High-order Regulation of Gene Clusters by CAMTA Members Link Environmental Cues to Auxin Homeostasis and Signaling

Microarray analyses were performed on all camta mutants. In the absence of gene-function redundancy it is expected that CAMTA target genes would be up- or downregulated in camta mutants. According to the microarray expression level data, camta4, camta5 and camta6 have a common transcriptome profile (up and downregulated genes). Similarly, camta1, camta2 and camta3 have a common transcriptome profile (up- and downregulated genes), which is broadly opposite in expression-level changes (relative to the wt transcriptome) to the profile of camta4, camta5 and camta6. To investigate this phenomenon in greater detail, the genes composing the two transcriptome profiles were grouped in two clusters. Cluster I includes 790 genes that are upregulated in camta1, camta2 and camta3 and downregulated in camta4, camta5 and camta6. Cluster II includes 570 genes that are downregulated in camta1, camta2 and camta3 and upregulated in camta4, camta5 and camta6. The opposite profile patterns of the two clusters may point to a regulatory mechanism involving all six AtCAMTAs and might involve regulation of specific CAMTAs by other CAMTAs by protein-protein or protein-DNA interactions, or by mechanisms involving regulatory RNA elements.

The common pathways of the two clusters are associated with auxin, abiotic stresses (salt, heat, cold and osmotic stress) and responses to different spectra of light stimuli (Fig. 2). Genes associated with response to auxin are enriched in cluster I (p < 0.01), whereas indole-3-acetic acid (IAA) amido synthetase activity is enriched in cluster II (p < 0.01). IAA-amido synthetases (e.g., GH3.4) catalyze IAA conjugation and reduce free IAA concentrations.^24,25 Moreover, among camta1 downregulated genes (fold change >[ABS] 1.4, p < 0.01) there is a significant pathway of UDP-glycosyl transferases (UGTs, e.g., AtUGT84B1). UGTs also catalyze IAA conjugation and reduce free IAA concentrations.^25,26 These results suggest that in wild-type plants, CAMTA1, CAMTA2 and CAMTA3 negatively regulate auxin responses. In contrast, CAMTA4, CAMTA5 and CAMTA6 likely have the reverse role of positively regulating auxin signaling and homeostasis.

Figure 2.

Gene cluster regulation by CAMTAs. CAMTA1 to CAMTA6 are divided in two groups according the transcriptome profiles of their respective mutants. On the left, CAMTA1, CAMTA2 and CAMTA3 are shown as one group, on the right CAMTA4, CAMTA5 and CAMTA6 as the second group. Possible direct regulation between the two CAMTA groups is illustrated with a question mark. Blue arrows indicate pathway activation (induction). Red lines indicate negative regulation (suppression). In each pathway the number of genes identified in the specific gene set relative to the number of genes known in the pathway in the whole Arabidopsis genome is indicated in parenthesis. Cluster I of 790 genes (upregulated in camta1, camta2 and camta3 mutants and downregulated in camta4, camta5 and camta6 mutants) are in purple boxes. Cluster II of 570 genes (upregulated genes in camta4, camta5 and camta6 mutants and downregulated in camta1, camta2 and camta3 mutants) are in green boxes. An orange box indicates a pathway specifically downregulated in the camta1 mutant. The black box on top depicts the abiotic stress responses that are common to both clusters I and II. It should be noted that in this schematic illustration genes that were found to be downregulated in camta mutants were considered to be upregulated by the respective CAMTA s in wild type plant and vise versa for genes found to be upregulated in camta mutants.

Furthermore, genes associated with responses to red light and high light are enriched in cluster I, whereas genes associated with responses to blue light and darkness are enriched in cluster II (Fig. 2). Activation of genes associated with darkness by CAMTA1 (e.g., HB52; GDH1) may be involved in the short hypocotyl phenotype of etiolated camta1 mutants.

In addition, based on transcriptome analyses each of the AtCAMTAs is involved in unique pathways that are not included in clusters I and II. For example, CAMTA1 negatively regulates genes involved in shade avoidance and seed germination and positively regulates genes of flavone metabolism. CAMTA2 downregulates genes mediating respiratory burst, indole phytoalexin and camalexin bosynthesis and upregulates genes involved in cell wall components. CAMTA3 downregulates cell death processes and biotic defense responses⁸ and positively regulates response to gibberellin and DNA replication. CAMTA4 upregulates specific toxin metabolic processes, CAMTA5 downregulates genes involved in post-embryonic development and heat acclimation and CAMTA6 may be involved in leaf senescence (Finkler et al., unpublished results).

Summary

The six CAMTA genes in Arabidopsis collectively regulate more than a thousand genes that link environmental cues, including various light signals, biotic and abiotic stresses to growth responses mainly, but not exclusively, through regulation of different aspects of auxin signaling, transport and homeostasis. However, each CAMTA gene has also unique roles, which enabled the identification of phenotypes resulting from mutations in single CAMTAs.^5–8 The finding that the six Arabidopsis CAMTAs may be regarded as two groups of three genes that function in opposite ways to up and downregulate more than a thousand downstream genes linking environmental signals to growth responses is both unique and interesting. Deciphering the mechanisms underlying this complex regulatory property of CAMTA genes should improve our understanding of phenotypic plasticity.

Addendum to: Galon Y, Aloni R, Nachmias D, Snir O, Feldmesser E, Scrase-Field S, et al. Calmodulin-binding transcription activator 1 mediates auxin signaling and responds to stresses in Arabidopsis. Planta. 2010;232:165–178. doi: 10.1007/s00425-010-1153-6.