Shedding light on an underground problem

In this issue of PNAS, Nishimura et al. demonstrate that ASTRAY, the legume homolog of HY5, a transcription factor that mediates light signaling and photomorphogenesis above ground, also affects the interaction between legume roots and their rhizobial endosymbionts underground (1). At first glance, this is a confusing result. What is a transcription factor that mediates light signaling doing regulating an underground (dark!) plant–microbe interaction? In fact, in Arabidopsis, HY5 regulates root architecture in addition to shoot photomorphogenetic events such as greening and hypocotyl elongation (2).

What is a transcription factor that mediates light signaling doing regulating an underground (dark!) plant–microbe interaction?

Still, fitting ASTRAY into the nodulation pathway is a puzzle. Nodulation-deficient mutants have allowed the identification of genes required for nodulation, but few of these genes have been cloned. Until recently, progress was hampered by the fact that these mutations were scattered throughout the crop legumes. With the advent of two model legumes, Lotus japonicus and Medicago truncatula, previously scattered efforts to investigate multiple crop legumes gradually turned into an intense focus on the genetics and genomics of these two species (3, 4). Extensive genetic screens of L. japonicus and M. truncatula have generated a large number of nodulation mutants, which, added to the wealth of nodulation mutants in pea (the original genetic organism!), have provided the foundation for a genetic analysis of nodulation (5–8). The recent cloning of LjNin1, which encodes a putative transcription factor required for L. japonicus nodulation (9), and an LRR-type receptor required for nodulation of L. japonicus, M. truncatula, alfalfa, and pea has provided the first molecular insight into this genetic pathway (10, 11).

The cloning of these nodulation genes has its roots in research begun more than 140 years ago. Legume root nodules first attracted the attention of researchers at about the same time that Darwin was investigating light signaling in oat seedlings and Koch was laying down his postulates. We now know that nodule formation is a complex morphogenetic process initiated as the result of a molecular conversation between plant and bacterial partners. A nodulation signal from the bacteria induces cells in the cortex of the plant root to start dividing to form a nodule primordium, which the bacteria subsequently infect. Cells of the nodule primordia differentiate into the many cell types that make up a mature nodule and organize into a radially symmetric organ containing a vascular system that connects with the root vasculature. Inside the nodule, the internalized bacteria differentiate into a specialized form that can fix atmospheric nitrogen. The decision of whether to nodulate is influenced by the availability of nitrogen in the soil, ethylene levels in the root, how many nodules it already has, and probably much more (12–14).

Interestingly, although the bacterial partner signals when and where to form a nodule, nodule formation is actually a plant developmental program. We know this because of two curious observations: (i) plant mutants exist that can nodulate in the absence of rhizobia (15, 16); and (ii) application of auxin transport inhibitors or localized cytokinin application can induce the formation of pseudonodules (17, 18).

How did legumes acquire a nodule developmental program? The fossil record is no help here, but looking at phylogenies can be. Thousands of legumes nodulate, but only one nonlegume. Are special genes that exist only in nodulating legumes required for nodulation? Or were genes required for other aspects of plant development recruited to function in a nodulation pathway? That nodulation may have evolved as many as three independent times within the legume family suggests existing genes or pathways may have predisposed legumes to form root nodules (19).

Symbiotic genes like astray that also have effects outside the nodule provide a glimpse of preexisting genes or pathways that were recruited to help form a nodule. It is the pleiotropic genes that, by maintaining an original function yet displaying their new role in nodulation, reveal their prenodule origins. Other pleiotropic symbiotic genes have been described, most notably the ethylene-insensitive mutant sickle (20) and the root architecture mutant har1 (21). The phenotypes of these mutants have demonstrated the link between other aspects of plant development and nodulation. astray is the first of these pleiotropic symbiotic genes to have been cloned (1).

Like its Arabidopsis homolog, HY5, ASTRAY mediates photomorphogenetic development. When the HY5 or ASTRAY genes are mutated, light-grown plants behave as if they were in the dark. In addition, astray mutants form excessive nodules. Several years ago, Kijne and colleagues observed that vetch roots grown in light were blocked from nodulating by an ethylene-mediated process (22, 23). This effect is less severe in other legumes but can occur there as well. Might astray mediate the light inhibition of vetch nodulation? This is a possibility because ethylene can inhibit the supernodulation of astray roots (24).

ASTRAY's dual role in light signaling and nodulation indicates participation in two pathways. Does it interact with the same pathway components when it functions in light signaling and nodulation? The role of HY5, ASTRAY's Arabidopsis homolog, is well understood in light signaling. In the dark, COP1 binds HY5, targeting it for degradation. When light is perceived by cryptochromes (blue/UV-A light) or phytochromes (red/far red light), COP1 levels in the nucleus fall and HY5 is free to bind promoters and activate transcription (25, 26). Does HY5 interact with COP1 in roots? When grown on Petri plates, roots are exposed to the light, but this is an artificial situation. In the soil, roots encounter little light. Might HY5 mediate another environmental signal in roots? COP1 has been shown to interact with other transcription factors as well as HY5. Perhaps in the root, HY5 can interact with other WD40 domain proteins besides COP1 (Fig. 1).

Fig 1.

A model for the role of ASTRAY in the regulation of nodule formation. Light activates ASTRAY, which inhibits nodule formation. On the basis of analogy to HY5, a COP1 homolog may mediate the light signal. In the soil, ASTRAY and HY5 regulate root-specific developmental events (nodulation and lateral root formation, respectively), even though there is little light. In the absence of light, ASTRAY may mediate a root-specific or even a legume-specific signal, via an unknown pathway, to inhibit nodulation.

If HY5 does mediate a nonlight signal in roots, do HY5 and ASTRAY mediate different signals in legume and Arabidopsis roots? Although they mediate similar photomorphogenetic events in the shoot, the roles of HY5 and ASTRAY diverge in roots. HY5 mutants grow excess lateral roots (2), whereas astray mutants form excess nodules (1). On the surface, this seems an odd difference, but the link between nodulation and lateral root formation has intrigued researchers for years.

Of all the plant structures, lateral roots have most often been proposed as the evolutionary origin of nodules. Although the structure and function of these two organs differ, nodules have to solve many of the same problems as developing roots. Unlike most plant organs, nodules and lateral roots form from already differentiated tissue, cortex for nodules and pericycle for lateral roots, not meristem. Thus the cells that will comprise the new nodule or lateral root must reenter the cell cycle, dedifferentiate, proliferate, and finally redifferentiate into the many cell types that make up the new organ. These newly differentiated cells must be organized into a radially patterned structure. Indeterminate nodules even form a meristem at the distal tip, just like lateral roots. The positioning of these structures along the primary root is also patterned in a similar way: both structures form adjacent to the xylem poles (27). In fact, in some legumes, rhizobia infect only at sites of lateral root emergence (28), and the two primordia are joined (29).

Interestingly, although nodule and lateral root formation are often regulated by the same pathways, the effects are often opposing. For example, both developmental processes are regulated by the auxin/cytokinin ratio but in opposite ways. Increasing auxin levels stimulates lateral root formation (30), whereas increasing local cytokinin concentrations or inhibiting auxin transport can trigger development of pseudonodules (17, 18). Similarly, ethylene appears to stimulate root growth (31) but inhibits nodulation (32–34). In addition, locally high levels of nitrate stimulate lateral root outgrowth (35) but inhibit nodule formation (36). The observation that pathways that regulate lateral root formation appear to have been recruited to regulate nodulation strengthens the argument that nodules and lateral roots may have had a common evolutionary origin.

Because many of the same pathways regulate nodulation and lateral root formation, but with opposite results, HY5 and ASTRAY could respond to the same signal in Arabidopsis and legume roots but activate different downstream genes. Alternatively, there could be a legume-specific signal that regulates nodule formation. Nishimura et al. found that ASTRAY has a legume-specific domain absent in HY5 but highly conserved in other legume sequences (1). What is the function of this legume-specific domain? Could it be involved in mediating a legume-specific signal? This domain is highly conserved in papillionoid legumes, all of which are proposed to arise from a single origin of nodulation (19). Is this domain conserved in more distantly related legumes that may have an independent origin of nodulation? What about the legumes that do not nodulate? Do they also have this domain, or do they have a more Arabidopsis-like Hy5 gene? Tracing the acquisition of the legume-specific domain of ASTRAY through the legume lineage may shed some light on the way in which legumes have altered their development to accommodate their rhizobial symbionts.

So far, only a handful of genes required for legume nodulation have been cloned. The cloning of ASTRAY has opened up an entirely new pathway of nodule regulation. The next few years promise to be an exciting ride as more and more genes required for nodulation are cloned. Still, identifying the sequence of these genes is just the first step. The homology of ASTRAY to HY5 has opened up a Pandora's box of questions. What signal does ASTRAY mediate in roots? What is the role of the legume-specific motifs in ASTRAY? How was the HY5 pathway recruited to help form nodules? The recruitment of HY5 from lateral root regulation to nodule regulation means there has been a loss of functionality as well as a gain. How has the lack of HY5 regulation of lateral root growth affected root architecture in legumes?

In the 140 years since light signaling and nodulation first attracted the attention of scientists, we have come full circle. The cloning of the L. japonicus ASTRAY gene has shed a glimmer of light on the way in which legumes have evolved to accommodate their rhizobial partner.