Beyond the Receptor
Had this Special Issue on plant hormones been published 5 years ago, it is likely that details about biosynthetic pathways would have taken center stage. As articles in this issue show, however, the field of plant hormone research has progressed rapidly and is now moving beyond the search for receptors. Progress in research on the mechanism of action of plant hormones has been rapid; receptors for the main classes of hormones have been identified; and the search is on for players downstream in signal-transduction chains.
A new theme that is beginning to dominate the field is interactions between hormones—a topic that is the focus of several articles in this Special Issue. Klaus Palme and his colleagues review progress in the area of auxin action using a systems-biology approach. Systems biology may be uniquely suited to investigations of hormone interactions both because a large volume of data will be produced in the coming years and it must be integrated in a meaningful way. To illustrate the complexity inherent in interactions among plant hormones, Teale et al. focus on the action of auxin in the developing root of Arabidopsis. They review what is known about the interaction between auxin and the major classes of plant hormones and conclude that these interactions are so complex that only integrative analyses such as a systems-biology approach provides can begin to unravel their complexity.
The second article from Klaus Palme's group describes an analysis of auxin-regulated genes obtained from diverse RNA-profiling experiments in Arabidopsis accessed from online databases. This is an excellent example of the type of database that is invaluable for a systems-biology analysis. These researchers show that the primary effect of auxin is to up-regulate gene expression and that their expression is strongly tissue-specific. Perhaps of even greater interest is the effect that auxin has on the expression of genes that are involved in the synthesis of the other major classes of plant hormones. When these data are confirmed by proteomic and GCMS analyses of hormone concentration, they will provide perhaps the most definitive evidence for cross-talk among plant hormones.
Zhiyong Wang's group reports on the interaction between brassinosteroids (BR) and ethylene. Their approach serves to highlight the importance of using synthetic chemical libraries with the goal of targeting hormone action or biosynthesis. Other groups, most notably that of Tadao Asami, have shown that this approach has been enormously successful for investigating BR synthesis and signaling. In their report, Wang's group isolates a new class of compound—brassinopride (BRP)—by monitoring the growth response and the activity of a BR-repressed reporter gene of Arabidopsis seedlings grown on compounds of a chemical library. They conclude that BRP inhibits BR synthesis and activates ethylene responses. Using BRP and mutants in each pathway, they show that the effect of ethylene on apical hook is not mediated by BR, but rather is a consequence of downstream BR signaling, pointing to the very complex nature of hormone signal-transduction pathways.
BR signaling is also the subject of the article by Jiang Min Li's group. They have investigated how the levels of BIN2—one of 10 GSK-like kinases that negatively regulate the BR signaling pathway—is regulated in Arabidopsis. The current model for BR signaling suggests that binding of BR to its receptor BRI1 leads to activation of BRI1 and its co-receptor BAK1, resulting in inhibition of BIN2. Lowered BIN2 levels allow dephosphorylation of the transcription factors BES1 and BZR1 that regulate expression of BR-responsive genes. Li's group shows that BR specifically reduces BIN2 levels and that other plant hormones have no effect on BIN2. They present convincing biochemical evidence to show that the ubiqutin/proteasome system is responsible for BIN2 degradation in Arabidopsis. It remains to be determined whether phosphorylation of BIN2 by a yet to be identified kinase is required for its degradation.
Hahn et al., using de-capped maize roots, uncovered another aspect of cross-talk between ethylene and auxin. Removal of the root cap from maize seedlings allowed them to follow inhibition of root growth and stimulation of root hair formation by ethylene. They found that root cap removal prevents ethylene action but does not affect the ability of roots to respond to auxin or cytokinin. Using a combination of physiological and molecular approaches, they concluded that a mobile signal, probably auxin, is synthesized by the root cap and is transported to elongating cells of the root, where it confers ethylene responsiveness.
Jeff Leung and his colleagues provide a comprehensive and tantalizing review on ABA. In addition to describing progress in identifying enzymes involved in ABA synthesis and conjugation, they discuss the search for ABA receptors and the probability that additional receptors will be found, including those located at the cell surface. Perhaps the most tantalizing aspect of this excellent review is a summary of what is known about ABA in metazoans, especially the report that ABA acts as a pro-inflammatory cytokine in human granulocytes. Wasilewska et al. note that granulocytes produce several reactive oxygen species, including nitric oxide (NO), and they point to the interesting similarities between ABA's role in both plant and animal defense responses.
Stomatal guard cells and dormant seeds are two models that have been widely used to dissect ABA-signaling pathways. Sheng Luan and his colleagues focus on ABA and germination in Arabidopsis. They describe details of the interaction between a member of the calcineurin B-like (CBL) protein family and its target protein kinase, CIPK. CBLs act as Ca2+ sensors in plant cells and, following Ca2+ binding, they interact with a family of CIPKs. In the case of Arabidopsis seed germination, CBL9 activates CIPK3. Two aspects of this work merit special attention. First, it emphasizes the overlapping functions of various CBLs, explaining the functional redundancy of these Ca2+ sensors. Second, despite functional redundancy, the expression of constitutively active CIPK3 in cbl9 mutant rescued the mutant phenotype as far as ABA responsiveness is concerned, demonstrating that CBL9 is likely to be required for activation of CIPK3.
The guard cell has been the model of choice for Mike Blatt's group, and his elegant use of biophysical and cell biological approaches to ABA function are nicely illustrated by the cover illustration of this Special Issue. Blatt's group documents a functional relationship between a protein of the SNARE superfamily known to drive membrane vesicle traffic and Ca2+ homeostasis in stomatal guard cells. ABA regulates Ca2+ entry into guard cells by activating plasma membrane Ca2+ channels to trigger Ca2+-induced Ca2+ release from intracellular stores, and the increase in Ca2+ inactivates inwardly rectifying K+ channels. Expressing a fragment of the SNARE protein NtSyp121 in tobacco guard cells prevents stomatal closure by reducing Ca2+ entry across the plasma membrane in response to ABA, but, surprisingly, the SNARE fragment only partially suppresses Ca2+ entry in response to NO. Whereas these observations offer primary evidence for an action of NtSyp121 on Ca2+ channels at the guard cell plasma membrane, they also point to interesting differences in the roles of ABA and NO in Ca2+ homeostasis that reflect the physical separation between the two pathways for Ca2+ elevation—one associated with the plasma membrane and the other with endomembranes.
Xue Min Wang's group highlights the importance of membrane lipids in responses to ABA and drought. Wang's group has previously shown that phospholipase D
(PLD) plays an essential role in the response of stomatal guard cells to ABA. When plants have reduced PLD1 levels, they have reduced sensitivity to ABA, impaired stomatal closure and increased transpirational water loss in response to water stress. In this manuscript, they report on the overexpression of castor bean PLD1 in tobacco and show that in the early stage of drought, PLD overexpression leads to decreased water loss; however, at a later stage, plants are more susceptible to drought because of its negative effects on membranes. They conclude that PLD has multifaceted roles in the response of plants to water deficits—one relating to signaling, the other to the negative impact on membrane integrity. These conclusions are likely to have an important impact on the strategies for generating drought-tolerant plants.
A role for membrane lipids in the jasmonic acid (JA)-signaling pathway is a theme in the article by Ingo Heilmann's group. This group investigates wound responses in Arabidopsis and provides convincing roles for a phosphoinositide-signaling cascade. Mechanical wounding of Arabidopsis leaves results in a five-fold increase in JA within 5 min and a four-fold increase in phosphoinositide and InsP3 within 30 min. The increase in InsP3 coincided with a decrease in phosphoinositide levels. A link between elevated JA, InsP3, and wounding was established using Arabidopsis mutants with reduced JA and phosphoinositide synthesis. Mutants blocked in JA synthesis have lower levels of InsP3 and reduction in both JA and phosphoinositide concentrations show an attenuation of defence-related genes as well as reduced growth of herbivorous caterpillars. Whereas InsP3 has been implicated in Ca2+ signaling in some plant systems, Heilmann and his colleagues favor a link between inositol phosphate metabolism and auxin signalling in the JA-wounding response.
The cloning of the first ethylene receptor, ETR1, almost 15 years ago and its functional characterization 2 years later were milestones in plant hormone biology. We now know that Arabidopsis possesses at least five ethylene receptors. In this volume, Harter and colleagues provide definitive evidence that these receptors are localized to the endoplasmic reticulum (ER) of Arabidopsis and interact with each other in the ER as homo and heteromeric protein complexes. Taken together with the discovery of others that CTR1 (a MAPKK that is immediately downstream of the receptor) is also localized to the ER, this supports the notion that ethylene functions as a signaling molecule in the ER.
The sequencing of ETR1 in 1993 showed that it shared sequence homology with bacterial two-component signaling kinases—an observation that fits well with ideas about signal perception and transduction. According to this model, the receptor would bind its ligand and initiate a signal transduction cascade via downstream protein kinases. ETR1 and another well characterized ethylene receptor (ESR1) both have histidine kinase domains. By cloning and expressing full-length ETR1, Georg Groth and his colleagues have provided evidence that the histidine kinase domain of ETR1 is autophosphorylated. What is especially exciting about this work is the demonstration that ethylene and cyanide (acting as an ethylene agonist) inhibit ETR1 autophosphorylation, whereas 1-methyl-cyclopropane, which functions as an ethylene antagonist and is used to inhibit ripening of banana, reverses the inhibitory effects of ethylene on autophosphorylation.
Cereals and grasses have proved to be excellent models for many aspects of hormone action, and work with rice has now led to the identification of GA receptors in plants. Based on this pioneering work of Makoto Matsuoka's laboratory in cloning Gid1 (a GA receptor from rice), Peter Chandler and his group report the isolation of a Gid1 orthologue from barley. By exploiting barley mutants showing reduced sensitivity to GA, Chandler and his group confirmed that the barley gene is indeed a bona fide GA receptor. An interesting aspect of the work with barley GA receptor mutants is the finding that all mutants show a response to GA3, albeit at high concentrations. Whether this response to added GA3 represents the presence of multiple GA receptors as opposed to the single receptor that work with rice suggests awaits further experimentation.
Despite the progress that has been made in understanding hormone biosynthesis, articles in this issue emphasize the interesting nuances in hormone biosynthesis that can have dramatic developmental consequences. An excellent example of this comes from research from Rod King's laboratory, documenting the interplay between hormone homeostasis and GA chemistry. GA5 is highly florigenic in the long-day grass Lolium temulentum, whereas GA1 and GA4 are much less effective in inducing flowering, but rather regulate vegetative development. The key to the difference in effectiveness of these GAs in floral induction rests in small differences in their chemistry. Whereas GA1 and GA4 can be readily catabolized by hydroxylation at the 2 position to biologically inactive GAs, GA5 contains a double bond between carbon atoms 2 and 3 that prevents hydroxylation. What is fascinating about the enzyme GA 2-oxidase that brings about hydroxylation at position 2 of the GA molecule is that it is preferentially localized in sub-apical tissue of Lolium, thus preventing the accumulation of GA1 and GA4 but not GA5 at the apex.
Nitric oxide is now widely recognized as a regulator of plant growth and development, and David Wendehenne and his group provide a timely update on the field of NO biology in plants. They point to the expanding list of responses that NO elicits in plants, including a crucial role in the regulation of Ca2+ fluxes and protein kinase activities in response to biotic and abiotic stresses. The source of NO in plants remains unresolved, although it is clear that it can be synthesized from nitrate by nitrate reductase and from nitrite by chemical reduction in an acidic environment. The puzzle at present for plant biologists is whether a bona fide NO synthase using arginine as a substrate occurs in plants.
The lack of suitable methods for capturing and quantitating NO has proved an obstacle to studies of NO synthesis, but Vitecek et al. describe a facile and inexpensive method for measuring NO. They present proof-of-concept data using well established plant models to illustrate the sensitivity and utility of this NO-measuring system. This method should be seen as a useful addition to the physicochemical approaches that rely on chemiluminescence, NO-specific electrodes, mass spectrometry, or photoacoustics.
Ottoline Leyser's group elegantly illustrates the power of simple anatomical manipulation of plants when coupled with the use of well defined mutants. They use several Arabidopsis mutants to explore how mobile signals regulate apical dominance. Using two-branch and W configuration systems in conjunction with max mutants, they show that an inhibitory signal can move in either direction in the shoot to inhibit axillary bud growth, and, more importantly, that movement of this signal does not correlate with vascular connectivity. They propose a model in which branch–branch communication involves both MAX and AXR1/TIR1/cytokinin pathways, whereas branch–accessory bud communication involves only the AXR1/TIR1/cytokinin pathway.
The task of organizing a Special Issue requires input from many individuals and I am indebted to my colleagues on the Editorial Committee for this volume, who helped to identify the authors who have contributed to this issue. They are: Yuji Kamiya, Jian Ming Li, Montse Pages, Klaus Palme, Julian Schroeder, Tony Trewavas and Zhi Yong Wang. I am very grateful for their help and for the opportunity to help to organize this issue.
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