Hormones are in the air

Phytohormones act to control many aspects of plant growth, development, and responses to the environment. Genetic and biochemical studies have identified many of the factors that mediate hormonal control of cell division, expansion, differentiation, and, ultimately, death. However, despite major advances in our understanding of how individual hormones act, we know relatively little about how hormonal signals are integrated into overall responses. For example, the hormones indole-3-acetic acid (IAA), gibberellin (GA), brassinolide, and, in some circumstances, ethylene all promote stem elongation. But what is the role of each hormone, and how do they interact to mediate growth? An abundance of hormone-related mutants, particularly in Arabidopsis, has provided some insights into the mechanisms of cooperative hormone action. Note that the term “cross-talk” is frequently used in this context. However, classically, this term defines phenomena in which there is an unwanted transfer of signals from one circuit, channel, etc., to another (Oxford English Dictionary). These interactions are most certainly not “unwanted.” In a narrower context, crosstalk is generally limited to common elements within a signal transduction scheme. Such a definition clearly ignores a major source of phytohormone interactions: effects of one input signal on accumulation of other input signals. For example, genetic analyses have defined a complex relationship among sugars, abscisic acid (ABA), and ethylene (1, 2) that controls aspects of growth and development. This interaction is regulated at the level of both hormone synthesis and signal transduction. However, the molecular details of this and other interactions are far from understood.

Some of the best characterized examples of the complex interactions between phytohormones involve responses to biotic stress. For example, wound responses, such as those induced by chewing insects, integrate jasmonic acid (JA), ethylene, and systemin signaling pathways in a manner yet to be fully established (3, 4). Similarly, responses to pathogenic microbes involve integration of JA, ethylene, and salicylic acid (SA) signaling pathways (5). Plants clearly use a limited number of hormonal signals in a combinatorial manner to achieve distinct outcomes. Epistatic relationships between various mutants have defined a useful framework for testing molecular mechanisms integrating the various hormone pathways. However, genetic approaches can go only so far, and epistasis can be misleading. For hormones, purely genetic approaches do not address a major source of interactions; there is abundant evidence that perturbation of one hormone pathway can have profound effects on synthesis and accumulation of other hormones. Thus, a mutation in one pathway can lead to major alterations in other hormones. Of course, the obvious answer to this fundamental problem is to measure the levels of all hormones in a mutant. So why has chemical analysis of hormones lagged far behind genetic characterization? Very simply, accurate measurement of hormone levels in small amounts of tissues has historically been beyond the skill base of most laboratories. Few laboratories have had the capacity to measure multiple phytohormones. There is no doubt that molecular biology and genetics have, in the last 15 years, revolutionized the field of plant hormone biology, but we now find ourselves in the all too familiar position of being limited by biochemistry.

A robust, simple technique to measure multiple hormones in small amounts of tissue has been lacking.

It is in this context that the article by Schmelz et al. (6) in this issue of PNAS should be viewed. It is not by choice that most molecular geneticists have largely ignored a major mechanism whereby hormones interact; it has simply not been technically feasible for the average laboratory to tackle the issue. Rather, a relative handful of laboratories have used technically demanding protocols for measuring one or a few hormones. What has been lacking is a robust, simple technique to measure multiple hormones in small amounts of tissue. Although significant progress has been made recently (7), the work of Schmelz et al. (6) should be viewed as a major technical advance. The authors provide a simple and rapid procedure to simultaneously quantify multiple hormones. They have measured SA, JA, ABA, IAA, the phytotoxin coronatine, and a set of volatile organic compounds that function as ecological signals. The technique works with tissues from multiple plant species subjected to several biotic and abiotic stresses. It employs readily available chemicals and standards and relies on instrumentation available on most university campuses, chemical ionization gas chromatography mass spectrometry. The method is elegant in its simplicity. Target compounds are converted to their methyl esters and measured as volatiles. The technique is, in theory, applicable to a wide range of primary and secondary metabolites, including other hormones, the only limitation being availability of appropriate deuterated standards.

What does the availability of this technique mean to the plant biologist? It should be possible to assess the effects of perturbing one hormone signaling system on a broad spectrum of other hormones. For many of the available mutants, this has never been done. However, there is ample evidence that alterations in one pathway have profound consequences on other signaling systems. An excellent example of hormone alterations initiating a chain reaction is found in deepwater rice (8). Flooding of the plant causes a 50-fold increase in internal ethylene, leading to rapid stem elongation, so ethylene causes stem elongation. But this is only a small part of the story. In fact, there is an increase in GA sensitivity mediated by reduction of ABA levels. Thus, stem elongation is actually the result of an interaction among three hormones. Only by measuring the effects of ethylene on other hormones in the target tissue does the complete story become evident.

Another illustration of the importance of hormone synthesis on signal integration is the example of the ein2 mutant. This mutant was originally isolated in screens for ethylene insensitivity and has been placed squarely within the ethylene signaling pathway (9). However, ein2 has been independently isolated in screens for cytokinin and ABA insensitivity as well as in screens for insensitivity to auxin transport inhibitors (10-13). Why does ein2 consistently show up in screens for so many different hormones? The answer lies in the fact that ethylene plays a critical role in mediating responses to many environmental stimuli. Its synthesis is highly regulated. The limiting step in synthesis is ACC synthase, an enzyme encoded by a gene family of at least 10 members in most plants (14). Different family members are induced by multiple factors, including cytokinin and auxin (15, 16). Thus, high levels of IAA or cytokinin induce ethylene synthesis. Many of the phenotypic effects associated with high IAA are actually ethylene effects (17). Alterations in multiple hormones are manifested as secondary effects associated with ethylene perturbation. Only because ethylene is a very simple hormone to assay do we know about these interactions.

The technique described by Schmelz et al. (6) is ideally suited for examination of plant-pathogen interactions. Genetic studies in Arabidopsis have defined parallel JA/ethylene and SA pathways mediating separate but overlapping defense responses (5). We know that these three hormones are important because mutants in their signaling pathways possess altered pathogen responses. However, few studies have actually examined the hormones directly. We have used the technique of Schmelz et al. (6) to examine the fluctuations in hormone levels in several wild-type and mutant lines after infection with a bacterial pathogen (18). Results indicate that the JA/ethylene and SA pathways are not entirely independent of one another. SA-deficient plants produced far less ethylene than wild-type plants after infection. An effect on ethylene synthesis was not predicted to be a consequence of SA deficiency. Without direct determination of hormone levels, it would be impossible to determine whether a phenotype was caused by loss of SA per se. A more complete hormone analysis also revealed a significant increase in IAA accumulation after infection. Although the role of this pathogenesis-related IAA has yet to be determined, its synthesis had not been previously reported, most likely because of the difficulties associated with IAA quantitation. The critical point in these studies is that simultaneous quantification of multiple biologically active substances should provide significant new insights into mechanisms of hormone interactions. It must be noted that the technology goes beyond plant hormones. For example, the authors have also measured accumulation of coronatine during infection with Pseudomonas syringae pv. tomato (Fig. 1), This compound is an important virulence factor that is believed to function as a mimic of JA or one of its precursors.

So what are the implications of the availability of a facile technique for quantitation of several hormones in a single assay? Hopefully, we are in a position to more fully elucidate mechanisms associated with cooperative hormone action. It has always been curious that a limited number of hormones can interact in different ways to mediate distinct environmental responses, e.g., pathogen and wound responses. Both timing and amplitude of hormone synthesis must surely be important contributors to the signal output. Availability of this technology will also justifiably raise the bar for publication. Just as microarrays have raised the barrier for gene expression studies, hormone analyses should also be an integral part of the characterization of a hormone-related mutant. Why look at one hormone when we can look at them all? Dare we call it “hormoneomics”? We have come full circle. Just as molecular biology reinvigorated hormone biology in the last decade, biochemistry must now reinvigorate molecular biology. We have the genes. Now what do they do? Chemical analysis has been a major barrier to hormone biologists. This is no longer the case.