Cross-regulatory mechanisms in hormone signaling
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- Kuppusamy, K.T., Walcher, C.L. & Nemhauser, J.L. Plant Mol Biol (2009) 69: 375. doi:10.1007/s11103-008-9389-2
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Recent studies suggest that hormones act through a web of interacting responses rather than through isolated linear pathways. This signal integration architecture may be one mechanism for increasing the specificity of outcomes in different cellular contexts. Several common themes for cross-regulation between pathways can be observed. Here, we propose a classification scheme for different levels of signaling pathway cross-regulation. This scheme is based on which parts of the individual pathways are acting as information conduits between pathways. Examples from the recent plant hormone biology literature are used to illustrate the different modes of interaction.
KeywordsCross-regulationCross-talkPlant hormonesAuxinBrassinosteroidsGibberellinsEthyleneJasmonatesAbscisic acid
One of the classic paradoxes of plant hormone biology is how a small number of simple organic molecules transmit a wide variety of developmental, environmental, and metabolic cues throughout the organism. To complicate matters, subsets of these hormones carry seemingly redundant information; yet, in most cases, they cannot substitute for one another. To take only one example, if any of the three plant hormones auxin, brassinosteroids or gibberellins is supplied exogenously, they are sufficient to trigger elongation of cells in the seedling stem. In other words, each of these hormones carries the ‘grow’ signal to the same organ and can produce largely similar effects. Following this logic, auxin, brassinosteroids and gibberellins can be thought of as nodes in a growth network, rather than as individual pathways acting in isolation. But auxin, brassinosteroids and gibberellins are not interchangeable. Each is necessary for normal growth—blocking perception or biosynthesis of any one of them results in significant growth inhibition. A fundamental question is how hormone signaling pathways robustly trigger specific outcomes, while operating in a communication network with other pathways.
To produce a systems-level understanding of hormone signaling pathways, it is clear that we need a common language to precisely describe the ways in which pathways connect. The term cross-talk is often used to mean one pathway influencing another. In electrical engineering, cross-talk is when the signal on one circuit causes an undesired effect on another circuit. In yeast, mutants have been identified which allow for an analogous type of “undesirable” cross-talk (Elion et al. 2005). The mating pheromone and the signal for invasive growth share many signal transduction components; yet, in wild-type yeast the signals maintain strict specificity (Elion et al. 2005). Single point mutations disrupt the insulation between pathways and allow mating pheromone to induce invasive growth (Bao et al. 2004b; Chou et al. 2004). We suggest the alternative term cross-regulation to describe cases where, under normal conditions, multiple input signals influence a common biological outcome.
Using this nomenclature, the architecture of a successful signaling network facilitates signal integration through cross-regulation while protecting against cross-talk. By combining information from multiple input pathways, the strength and nature of the biological outcome can be fine-tuned, amplified, or attenuated (Komarova et al. 2005; Bardwell et al. 2007). Two key measures of network function are specificity and fidelity (Bardwell et al. 2007). Signal specificity for pathway X can be described as the degree to which Xin promotes Xout as compared with Yout. Fidelity for pathway X can be defined as the degree to which Xin promotes Xout, when compared to Yin. Mathematical models show that different pathway architectures result in different levels of specificity and fidelity, and that the addition of cross-regulation improves both factors (Bardwell et al. 2007).
Examples of primary cross-regulation are still rare in the plant literature, although global transcriptome studies frequently uncover significant regulation of transcription factors involved in disparate pathways. One well-studied example of positive primary cross-regulation comes from studies of plant responses to ethylene and jasmonates. Both hormones are known to work together to activate defense responses (Kunkel and Brooks 2002). Co-regulation of the same transcription factor, ETHYLENE RESPONSE FACTOR1 (ERF1), is responsible for a large share of this coordination (Lorenzo et al. 2003). Expression of ERF1 was previously shown to be a key downstream target of the ethylene-induced defense response (Berrocal-Lobo et al. 2002). Work by Lorenzo and colleagues shows that ERF1 is also induced by jasmonates and that ERF1-regulated genes are generally involved in defense pathways (Lorenzo et al. 2003). Ethylene and jasmonates synergistically induce ERF1 expression, and expression of ERF1 requires both hormone-signaling pathways to be functional.
Li and colleagues provided one of the first examples of negative primary cross-regulation (Li et al. 2004). Auxin and ethylene act antagonistically in the development of an apical hook during seedling growth in the dark. HOOKLESS1 (HLS1) is an ethylene-responsive gene required for proper hook formation. Loss of AUXIN RESPONSE FACTOR 2 (ARF2), a member of the Auxin Response Factor family of transcription factors, partially suppresses the effects of loss of HLS1, including restoration of auxin-responsive reporter expression in the apical hook. Li and colleagues showed that the antagonism between the auxin and ethylene pathways was at least partially mediated by ethylene-induced degradation of ARF2 protein. Moreover, light exposure also causes degradation of HLS1 protein and increases in ARF2 levels, suggesting an additional level of primary cross-regulation between the light, ethylene and auxin pathways.
Another example of negative primary cross-regulation was recently described between the light and gibberellin signaling pathways (Alabadi et al. 2008; de Lucas et al. 2008; Feng et al. 2008). Gibberellins have long been known to promote many processes of plant development such as seed germination, elongation growth, flowering time and floral development (Schwechheimer 2008). Current models suggest that in the absence of gibberellins, a group of nuclear-localized repressors called DELLA proteins inhibit growth (Schwechheimer 2008). When gibberellins are present, they bind to GIBBERELLIN INSENSITIVE DWARF1 (GID1), encoded by three functionally redundant paralogs in Arabidopsis. The GA-GID1 complex interacts with the DELLAs to relieve their repressor activity by gibberellin-dependent degradation via the E3 ubiquitin ligase SCFGID2/SLY1. Recent work by three groups shows evidence for crosstalk between gibberellins and light to promote photomorphogenesis (Alabadi et al. 2008; de Lucas et al. 2008; Feng et al. 2008). Two groups report that in the absence of gibberellins, nuclear-localized DELLA proteins interact with two PHYTOCHROME-INTERACTING FACTORS, PIF3 and PIF4. PIFs are transcription factors that generally act in opposition to the light response (Castillon et al. 2007). The DELLA-PIF interaction prevents the PIFs from binding to DNA. When gibberellins are present, GID1 interacts with and promotes degradation of the DELLA proteins. This releases the PIFs and promotes hypocotyl elongation (de Lucas et al. 2008; Feng et al. 2008).
In a similar study, Alabadi and colleagues provide evidence for primary cross-regulation between gibberellins, the COP1-mediated pathway and the PIF family (Alabadi et al. 2008). In the absence of light, COP1 actively promotes degradation of transcriptional activators of photomorphogenesis, such as ELONGATED HYPOCOTYL 5 (HY5), and promotes accumulation of transcription factors that promote cell elongation, such as PIF3 (Castillon et al. 2007). In the light, gibberellin levels are decreased, leading to accumulation of DELLA proteins. DELLAs interact with PIF3, inhibiting PIF3 DNA-binding and negatively regulating cell elongation in the hypocotyl. At the same time, COP1 is repressed, allowing accumulation of HY5 and activation of the photomorphogenetic program (Jiao et al. 2007). HY5 has been shown to bind to the promoters of genes encoding some DELLA members (Sibout et al. 2006), adding a re-enforcing secondary cross-regulation layer to this network.
Higher order protein complexes are another potential mechanism of primary cross-regulation, as suggested for the TOPLESS (TPL) co-repressor family and the auxin response pathway (Szemenyei et al. 2008). In Arabidopsis, the dominant negative topless mutant tpl1-1 transforms the embryonic shoot into a second embryonic root (Long et al. 2002). TPL is related to the Groucho/Tup1 family of co-repressors and acts in coordination with chromatin remodeling factors to regulate transcription at specific target genes (Long et al. 2006). Seedlings with milder tpl phenotypes exhibit a loss of basal structures, highly reminiscent of the defects observed in plants with loss of MONOPTEROS/ARF5 (MP/ARF5) or stabilized versions of BODENLOS/IAA12 (BDL/IAA12) (Szemenyei et al. 2008). ARF5 is a member of the Auxin Response Factor family of transcription factors and is known to regulate genes required for establishment of polarity during embryogenesis (Hardtke and Berleth 1998; Hardtke et al. 2004). IAA12 is a transcriptional repressor that directly binds to ARF5 and blocks its ability to activate transcription (Hamann et al. 2002; Hardtke et al. 2004; Weijers et al. 2005). Auxin activates ARFs by facilitating the degradation of Aux/IAA proteins like IAA12 (Guilfoyle and Hagen 2007). Specific pairs of ARF and Aux/IAA proteins have been proposed to direct the development of different plant tissues (Weijers et al. 2005). Elegant work by Szemenyei and colleagues demonstrates a direct interaction between TPL and IAA12 through an ETHYLENE RESPONSE FACTOR-associated Amphiphilic Repression (EAR) motif in domain I of IAA12 (Szemenyei et al. 2008). In planta transcriptional repression assays show that TPL acts as a transcriptional corepressor both necessary and sufficient for repression of ARF5. tpl-1 bdl-1 double mutants form hypocotyl and roots, indicating that tpl-1 can suppress bdl-1 basal patterning defects. Fusion of the ARF-interacting domain of IAA12 to the C-terminal repression domain of TPL reproduces the phenotypes caused by stabilized IAA12 or loss of ARF5. Differential expression of TPL family members, potential protein-protein affinity differences, and interaction of TPL with other transcription factor families, may together play a critical role in the spatio-temporal modulation of auxin sensitivity.
Secondary regulation of signaling pathway involves components of one pathway affecting the levels of, or sensitivity to, input into a second pathway (Fig. 1b). This is perhaps the most widespread mechanism of cross-regulation for plant hormone signaling. Many hormones have been shown to regulate the expression of metabolic genes of other hormones (e.g. Nordstrom et al. 2004). In a recent global transcriptome survey, evidence for this type of cross-regulation was found for all growth regulators examined (Nemhauser et al. 2006).
Recent identification of the putative transcription factor BREVIS RADIX (BRX) provides molecular details for one example of secondary cross-regulation of the brassinosteroid pathway by auxin (Mouchel et al. 2006). A loss of function allele of BRX exhibits a short root phenotype with reduced levels of endogenous brassinosteroids and severely impaired auxin response. This decrease in brassinosteroid levels is correlated with reduced expression of CONSTITUTIVE PHOTOMORPHOGENIC DWARF (CPD), a rate-limiting enzyme in the brassinosteroid biosynthetic pathway. Both the severe root defects and impaired auxin response of brx mutants can be rescued by exogenously supplying brassinosteroids or constitutively expressing CPD. In wild-type seedlings, BRX is strongly induced by application of auxin but slightly repressed with brassinosteroid treatment. This suggests a negative feedback loop to maintain homeostasis between the two hormone pathways. The mechanism of cross-regulation by which brassinosteroids contribute to auxin sensitivity is still an open question.
Secondary cross-regulation can also occur when the output from one pathway influences the sensitivity of a cell to a second input. Previous studies have connected the transcriptional effects of the light-regulated transcription factor HY5 with altered seedling sensitivity to auxin (Sibout et al. 2006). Recent work has shown that HY5 mediates abscisic acid response in seed germination, seedling growth and root development (Chen et al. 2008). The abscisic acid-activated transcription factor, ABA INSENSITIVE 5 (ABI5), regulates the expression of AtEM genes encoding the LATE EMBRYOGENESIS-ABUNDANT (LEA) proteins required for normal seed maturation. In this study, HY5 was shown to bind with high affinity to the ABI5 promoter and was required for expression of both ABI5 and its downstream targets. In hy5 mutants, mRNA levels of ABI5 itself and several direct ABI5 targets are greatly reduced. In addition, chromatin immunoprecipitation assays show that abscisic acid treatment significantly enhances HY5 binding to the ABI5 promoter. Overexpression of ABI5 restores abscisic acid sensitivity to hy5 mutants during seed germination and seedling growth. Overexpression of ABI5 in wild-type also enhances seedling responses to blue, red and far-red light, supporting a model of mutual cross-regulation of light and abscisic acid signaling.
Polar auxin transport is a crucial determinant of the initiation, direction, and extent of growth throughout plant development (Vieten et al. 2007). Three recent studies have demonstrated that the hormone ethylene mediates root growth by modulating auxin transport (Ruzicka et al. 2007; Stepanova et al. 2007; Swarup et al., 2007). Ethylene affects root growth primarily by inhibiting cell expansion in the elongation zone (Le et al. 2001). Mutants defective in basipetal transport of auxin, such as auxin resistant 1 (aux1) and pin-formed 2 (pin2), show ethylene insensitive root growth (Ruzicka et al. 2007; Swarup et al. 2007). Under conditions where the ethylene response pathway was activated, the region of expression of auxin-responsive genes expanded into the root meristem. Enhanced expression of these genes is abolished in ethylene insensitive mutant like ethylene response 1 (etr1) and ethylene insensitive 2 (ein2). Stepanova and colleagues used the auxin-responsive reporter DR5 to show more specifically that auxin-responsive gene expression is normally limited to the columella cells and the quiescent zone of the root tip, but spreads into the elongation zone in the presence of ethylene (Stepanova et al. 2007). This study also found that the auxin-dependent ethylene effects observed in roots do not occur in hypocotyls. Previous studies have indicated that ethylene and auxin effects on root growth act through modulation of the stability of DELLA proteins (Achard et al. 2003; Fu and Harberd 2003), adding cross-regulation of the gibberellin pathway to this multi-level network.
Another example of secondary cross-regulation of polar auxin transport comes from work studying the effect of brassinosteroids on lateral root production (Bao et al. 2004a). Bao and colleagues found that application of brassinosteroids induces lateral root formation, and that a combination of brassinosteroids and auxin produce twice as many lateral roots as compared to treatments with either hormone alone. The authors further demonstrated that brassinosteroids increase auxin transport towards the root tip, and that induction of lateral roots by brassinosteroids is suppressed in the presence of an auxin transport inhibitor. Together, these data provide a likely model for the observed synergism where brassinosteroids act as secondary cross-regulators of polar auxin transport.
Tertiary regulation of pathway activity
Tertiary cross-regulation occurs when the outputs from independent pathways influence one another (Fig 1c). Biophysical outputs are often constrained by multiple parameters and likely targets for tertiary cross-regulation. For example, cell elongation rates depend upon three variables: the extensibility of the cell wall, the amount of turgor pressure, and the yield threshold (Cosgrove 1993). Auxin has long been known to affect the extensibility of the cell wall (Rayle and Cleland 1970), whereas brassinosteroids may regulate the yield threshold (Wang et al. 1993). If true, the ultimate output, which is the extent of growth, could be influenced by the relative strength of the auxin and brassinosteroid signals, regulating the proximal outputs of wall extensibility and yield threshold, respectively.
Another example of tertiary cross-regulation is the control of bud outgrowth by polar auxin transport rates. In a simplified view, the outputs in the main stem and in the bud are the same: the strength of auxin flow (Bennett et al. 2006). When the main stem is at full capacity, the bud’s auxin flow is insufficient to drive a connection, so the bud is arrested. When the capacity for auxin flow in the main stem is increased, through higher levels of auxin efflux carriers or increased distance from the apex, the buds can successfully export auxin and outgrowth is initiated. Control of auxin transport capacity in the main stem is under additional tertiary regulation by the MAX pathway (Bennett et al. 2006). In addition, the auxin in the bud is likely acting to further restrict bud growth by secondary cross-regulation of cytokinin biosynthesis (Bennett et al. 2006).
Some cases of suspected hormonal cross-regulation are still not sufficiently well understood to distinguish between primary, secondary, or tertiary regulation. One example with potential agricultural significance is the relationship among different hormones during nodulation in legumes. The absolute amount of auxin or the rate of polar auxin transport likely limits the number and location of nodules. The super numeric nodules (sunn) mutant in Medicago truncatula forms ten times more nodules than wild-type plants and does not restrict growth to the primary nodulation zone (Penmetsa et al. 2003). Auxin transport measurements reveal that sunn mutants have an increased flux of auxin from the shoot to the root (van Noorden et al. 2006). SUNN is a CLAVATA1-like leucine-rich repeat receptor-like kinase (Schnabel et al. 2005). How it regulates polar auxin transport is still unknown. Studies of the ethylene-insensitive, hypernodulating mutant sickle (skl) found an ethylene-dependent reduction of shoot-to-root polar auxin transport following exposure to rhizobia (Prayitno et al. 2006). SKL also alters local auxin flux by regulating expression of polar auxin transporters (Prayitno et al. 2006).
Past studies have shown that high cytokinin and low auxin levels promoted nodule growth (Hirsch et al. 1989; Wu et al. 1996; Fang and Hirsch 1998), but several recent findings suggest a more complicated relationship between these two hormones. Mathesius and colleagues used a reporter line to visualize a peak of auxin response just before the initiation of cell division in the nodule primordium of white clover (Mathesius et al. 1998). Lohar and colleagues working in Lotus japonicus show a peak of cytokinin response in dividing cells once the nodule primordia is established (Lohar et al. 2004). A gain of function mutation in the cytokinin receptor LOTUS HISTIDINE KINASE 1 (LHK1) produces pseudonodules in L. japonicus roots in the absence of rhizobia (Tirichine et al. 2007). The resulting spontaneous nodule formation 2 (snf2) mutants have increased cell layers in the roots caused by additional periclinal divisions. This is consistent with results showing that RNA interference of a homolog of another cytokinin receptor CYTOKININ RESPONSE 1 (MtCRE1) strongly reduces rhizobia-induced nodulation (Gonzalez-Rizzo et al. 2006). This close association between peaks of auxin and cytokinin action is reminiscent of recent work on embryonic root development in Arabidopsis (Muller and Sheen 2008). In this impressive study, root stem-cell specification was shown to require auxin-mediated down-regulation of cytokinin sensitivity in the basal cell lineage. Auxin exerted its effects through increasing the expression of antagonists of cytokinin signaling called A-type Arabidopsis Response Regulators. This type of cell-type specific secondary cross-regulation, in combination with additional auxin-mediated regulation of cytokinin biosynthesis (Nordstrom et al. 2004), may emerge as a common theme in organogenesis (Dello Ioio et al. 2008).
Careful examination of the architecture of signaling pathways will likely reveal general principles guiding the effective relay of biological information. As the details of the plant hormone network become known and local concentrations of key factors can be more accurately measured, models will be needed to evaluate which connections are most likely acting to control specific aspects of plant growth and development. Finding mutants that enhance or limit cross-regulation between pathways may ultimately answer the perennial question of how hormones manage to so accurately do so many different things in so many different contexts.
This work was supported by the University of Washington. CW is a trainee on the Developmental Biology Predoctoral Training Grant T32HD007183 from the National Institute of Child Health and Human Development.