Strigolactones and Shoot Branching: What Is the Real Hormone and How Does It Work?

There have been substantial advances in our understanding of many aspects of strigolactone regulation of branching since the discovery of strigolactones as phytohormones. These include further insights into the network of phytohormones and other signals that regulate branching, as well as deep insights into strigolactone biosynthesis, metabolism, transport, perception and downstream signaling. In this review, we provide an update on recent advances in our understanding of how the strigolactone pathway co-ordinately and dynamically regulates bud outgrowth and pose some important outstanding questions that are yet to be resolved.

Auxin-independent effects of apical dominance induce changes in phytohormones correlated with bud outgrowth.

The inhibition of shoot branching by the growing shoot tip of plants, termed apical dominance, was originally thought to be mediated by auxin. Recently, the importance of the shoot tip sink strength during apical dominance has re-emerged with recent studies highlighting roles for sugars in promoting branching. This raises many unanswered questions on the relative roles of auxin and sugars in apical dominance. Here we show that auxin depletion after decapitation is not always the initial trigger of rapid cytokinin (CK) increases in buds that are instead correlated with enhanced sugars. Auxin may also act through strigolactones (SLs) which have been shown to suppress branching after decapitation, but here we show that SLs do not have a significant effect on initial bud outgrowth after decapitation. We report here that when sucrose or CK is abundant, SLs are less inhibitory during the bud release stage compared to during later stages and that SL treatment rapidly inhibits CK accumulation in pea (Pisum sativum) axillary buds of intact plants. After initial bud release, we find an important role of gibberellin (GA) in promoting sustained bud growth downstream of auxin. We are, therefore, able to suggest a model of apical dominance that integrates auxin, sucrose, SLs, CKs, and GAs and describes differences in signalling across stages of bud release to sustained growth.

Integration of the SMXL/D53 strigolactone signalling repressors in the model of shoot branching regulation in Pisum sativum.

DWARF53 (D53) in rice (Oryza sativa) and its homologs in Arabidopsis (Arabidopsis thaliana), SUPPRESSOR OF MAX2-LIKE 6 (SMXL6), SMXL7 and SMXL8, are well established negative regulators of strigolactone (SL) signalling in shoot branching regulation. Little is known of pea (Pisum sativum) homologs and whether D53 and related SMXLs are specific to SL signalling pathways. Here, we identify two allelic pea mutants, dormant3 (dor3), and demonstrate through gene mapping and sequencing that DOR3 corresponds to a homolog of D53 and SMXL6/SMXL7, designated PsSMXL7. Phenotype analysis, gene expression, protein and hormone quantification assays were performed to determine the role of PsSMXL7 in regulation of bud outgrowth and the role of PsSMXL7 and D53 in integrating SL and cytokinin (CK) responses. Like D53 and related SMXLs, we show that PsSMXL7 can be degraded by SL and induces feedback upregulation of PsSMXL7 transcript. Here we reveal a system conserved in pea and rice, whereby CK also upregulates PsSMXL7/D53 transcripts, providing a clear mechanism for SL and CK cross-talk in the regulation of branching. To further deepen our understanding of the branching network in pea, we provide evidence that SL acts via PsSMXL7 to modulate auxin content via PsAFB5, which itself regulates expression of SL biosynthesis genes. We therefore show that PsSMXL7 is key to a triple hormone network involving an auxin-SL feedback mechanism and SL-CK cross-talk.

An Update on the Signals Controlling Shoot Branching.

Many new questions on the regulation of shoot branching have been raised in recent years, prompting a review and reassessment of the role of each signal involved. Sugars and their signaling networks have been attributed a major role in the early events of axillary bud outgrowth, whereas cytokinin appears to play a critical role in the modulation of this process in response to the environment. Perception of the recently discovered hormone strigolactone is now quite well understood, while the downstream targets remain largely unknown. Recent literature has highlighted that auxin export from a bud is important for its subsequent growth.

Apical dominance.

Barbier et al. give a quick guide to apical dominance, whereby a plant's main shoot dominates and inhibits the outgrowth of other shoots.

Phloem Transport of the Receptor DWARF14 Protein Is Required for Full Function of Strigolactones.

The cell-to-cell transport of signaling molecules is essential for multicellular organisms to coordinate the action of their cells. Recent studies identified DWARF14 (D14) as a receptor of strigolactones (SLs), molecules that act as plant hormones and inhibit shoot branching. Here, we demonstrate that RAMOSUS3, a pea ortholog of D14, works as a graft-transmissible signal to suppress shoot branching. In addition, we show that D14 protein is contained in phloem sap and transported through the phloem to axillary buds in rice. SLs are not required for the transport of D14 protein. Disruption of D14 transport weakens the suppression of axillary bud outgrowth of rice. Taken together, we conclude that the D14 protein works as an intercellular signaling molecule to fine-tune SL function. Our findings provide evidence that the intercellular transport of a receptor can regulate the action of plant hormones.

LATERAL BRANCHING OXIDOREDUCTASE acts in the final stages of strigolactone biosynthesis in Arabidopsis.

Strigolactones are a group of plant compounds of diverse but related chemical structures. They have similar bioactivity across a broad range of plant species, act to optimize plant growth and development, and promote soil microbe interactions. Carlactone, a common precursor to strigolactones, is produced by conserved enzymes found in a number of diverse species. Versions of the MORE AXILLARY GROWTH1 (MAX1) cytochrome P450 from rice and Arabidopsis thaliana make specific subsets of strigolactones from carlactone. However, the diversity of natural strigolactones suggests that additional enzymes are involved and remain to be discovered. Here, we use an innovative method that has revealed a missing enzyme involved in strigolactone metabolism. By using a transcriptomics approach involving a range of treatments that modify strigolactone biosynthesis gene expression coupled with reverse genetics, we identified LATERAL BRANCHING OXIDOREDUCTASE (LBO), a gene encoding an oxidoreductase-like enzyme of the 2-oxoglutarate and Fe(II)-dependent dioxygenase superfamily. Arabidopsis lbo mutants exhibited increased shoot branching, but the lbo mutation did not enhance the max mutant phenotype. Grafting indicated that LBO is required for a graft-transmissible signal that, in turn, requires a product of MAX1. Mutant lbo backgrounds showed reduced responses to carlactone, the substrate of MAX1, and methyl carlactonoate (MeCLA), a product downstream of MAX1. Furthermore, lbo mutants contained increased amounts of these compounds, and the LBO protein specifically converts MeCLA to an unidentified strigolactone-like compound. Thus, LBO function may be important in the later steps of strigolactone biosynthesis to inhibit shoot branching in Arabidopsis and other seed plants.

Strigolactone Inhibition of Branching Independent of Polar Auxin Transport.

The outgrowth of axillary buds into branches is regulated systemically via plant hormones and the demand of growing shoot tips for sugars. The plant hormone auxin is thought to act via two mechanisms. One mechanism involves auxin regulation of systemic signals, cytokinins and strigolactones, which can move into axillary buds. The other involves suppression of auxin transport/canalization from axillary buds into the main stem and is enhanced by a low sink for auxin in the stem. In this theory, the relative ability of the buds and stem to transport auxin controls bud outgrowth. Here, we evaluate whether auxin transport is required or regulated during bud outgrowth in pea (Pisum sativum). The profound, systemic, and long-term effects of the auxin transport inhibitor N-1-naphthylphthalamic acid had very little inhibitory effect on bud outgrowth in strigolactone-deficient mutants. Strigolactones can also inhibit bud outgrowth in N-1-naphthylphthalamic acid-treated shoots that have greatly diminished auxin transport. Moreover, strigolactones can inhibit bud outgrowth despite a much diminished auxin supply in in vitro or decapitated plants. These findings demonstrate that auxin sink strength in the stem is not important for bud outgrowth in pea. Consistent with alternative mechanisms of auxin regulation of systemic signals, enhanced auxin biosynthesis in Arabidopsis (Arabidopsis thaliana) can suppress branching in yucca1D plants compared with wild-type plants, but has no effect on bud outgrowth in a strigolactone-deficient mutant background.

Strigolactones stimulate internode elongation independently of gibberellins.

Strigolactone (SL) mutants in diverse species show reduced stature in addition to their extensive branching. Here, we show that this dwarfism in pea (Pisum sativum) is not attributable to the strong branching of the mutants. The continuous supply of the synthetic SL GR24 via the root system using hydroponics can restore internode length of the SL-deficient rms1 mutant but not of the SL-response rms4 mutant, indicating that SLs stimulate internode elongation via RMS4. Cytological analysis of internode epidermal cells indicates that SLs control cell number but not cell length, suggesting that SL may affect stem elongation by stimulating cell division. Consequently, SLs can repress (in axillary buds) or promote (in the stem) cell division in a tissue-dependent manner. Because gibberellins (GAs) increase internode length by affecting both cell division and cell length, we tested if SLs stimulate internode elongation by affecting GA metabolism or signaling. Genetic analyses using SL-deficient and GA-deficient or DELLA-deficient double mutants, together with molecular and physiological approaches, suggest that SLs act independently from GAs to stimulate internode elongation.

Dynamics of strigolactone function and shoot branching responses in Pisum sativum.

Strigolactones (SLs), or their metabolites, were recently identified as endogenous inhibitors of shoot branching. However, certain key features and dynamics of SL action remained to be physiologically characterized. Here we show that successive direct application of SL to axillary buds at every node along the stem can fully inhibit branching. The SL inhibition of early outgrowth did not require inhibitory signals from other growing buds or the shoot tip. In addition to this very early or initial suppression of outgrowth, we also found SL to be effective, up to a point, at moderating the continuing growth of axillary branches. The effectiveness of SL at affecting bud and branch growth correlated with the ability of SL to regulate expression of PsBRC1. PsBRC1 is a transcription factor that is expressed strongly in axillary buds and is required for SL inhibition of shoot branching. Consistent with a dynamic role of the hormone, SL inhibition of bud growth did not prevent buds from later responding to a decapitation treatment, even though SL treatment immediately after decapitation inhibits the outgrowth response. Also, as expected from the hypothesized branching control network in plants, treatment of exogenous SL caused feedback down-regulation of SL biosynthesis genes within 2 h. Altogether, these results reveal new insights into the dynamics of SL function and support the premise that SLs or SL-derived metabolites function dynamically as a shoot branching hormone and that they act directly in axillary buds.

Antagonistic action of strigolactone and cytokinin in bud outgrowth control.

Cytokinin (CK) has long been implicated as a promoter of bud outgrowth in plants, but exactly how this is achieved in coordination with other plant hormones is unclear. The recent discovery of strigolactones (SLs) as the long-sought branch-inhibiting hormone allowed us to test how CK and SL coordinately regulate bud outgrowth in pea (Pisum sativum). We found that SL-deficient plants are more sensitive to stimulation of bud growth by low concentrations of locally applied CK than wild-type plants. Furthermore, in contrast with SL mutant plants, buds of wild-type plants are almost completely resistant to stimulation by CK supplied to the vasculature. Regardless of whether the exogenous hormones were supplied locally or to the xylem stream, SL and CK acted antagonistically on bud outgrowth. These data suggest that SLs do not affect the delivery of CK to axillary buds and vice versa. Rather, these data combined with dose-response experiments suggest that SLs and CK can act directly in buds to control their outgrowth. These hormones may converge at a common point in the bud outgrowth regulatory pathway. The expression of pea BRANCHED1, a TCP transcription factor expressed strongly in buds and thought to act downstream of SLs in shoot branching, is regulated by CK and SL without a requirement for protein synthesis and in a manner that correlates with observed bud growth responses.

Strigolactone signaling is required for auxin-dependent stimulation of secondary growth in plants.

Long distance cell-to-cell communication is critical for the development of multicellular organisms. In this respect, plants are especially demanding as they constantly integrate environmental inputs to adjust growth processes to different conditions. One example is thickening of shoots and roots, also designated as secondary growth. Secondary growth is mediated by the vascular cambium, a stem cell-like tissue whose cell-proliferating activity is regulated over a long distance by the plant hormone auxin. How auxin signaling is integrated at the level of cambium cells and how cambium activity is coordinated with other growth processes are largely unknown. Here, we provide physiological, genetic, and pharmacological evidence that strigolactones (SLs), a group of plant hormones recently described to be involved in the repression of shoot branching, positively regulate cambial activity and that this function is conserved among species. We show that SL signaling in the vascular cambium itself is sufficient for cambium stimulation and that it interacts strongly with the auxin signaling pathway. Our results provide a model of how auxin-based long-distance signaling is translated into cambium activity and suggest that SLs act as general modulators of plant growth forms linking the control of shoot branching with the thickening of stems and roots.

F-box protein MAX2 has dual roles in karrikin and strigolactone signaling in Arabidopsis thaliana.

Smoke is an important abiotic cue for plant regeneration in postfire landscapes. Karrikins are a class of compounds discovered in smoke that promote seed germination and influence early development of many plants by an unknown mechanism. A genetic screen for karrikin-insensitive mutants in Arabidopsis thaliana revealed that karrikin signaling requires the F-box protein MAX2, which also mediates responses to the structurally-related strigolactone family of phytohormones. Karrikins and the synthetic strigolactone GR24 trigger similar effects on seed germination, seedling photomorphogenesis, and expression of a small set of genes during these developmental stages. Karrikins also repress MAX4 and IAA1 transcripts, which show negative feedback regulation by strigolactone. We demonstrate that all of these common responses are abolished in max2 mutants. Unlike strigolactones, however, karrikins do not inhibit shoot branching in Arabidopsis or pea, indicating that plants can distinguish between these signals. These results suggest that a MAX2-dependent signal transduction mechanism was adapted to mediate responses to two chemical cues with distinct roles in plant ecology and development.

Computational modeling and molecular physiology experiments reveal new insights into shoot branching in pea.

Bud outgrowth is regulated by the interplay of multiple hormones, including auxin, cytokinin, strigolactones, and an unidentified long-distance feedback signal that moves from shoot to root. The model of bud outgrowth regulation in pea (Pisum sativum) includes these signals and a network of five RAMOSUS (RMS) genes that operate in a shoot-root-shoot loop to regulate the synthesis of, and response to, strigolactones. The number of components in this network renders the integration of new and existing hypotheses both complex and cumbersome. A hypothesis-driven computational model was therefore developed to help understand regulation of shoot branching. The model evolved in parallel with stepwise laboratory research, helping to define and test key hypotheses. The computational model was used to verify new mechanisms involved in the regulation of shoot branching by confirming that the new hypotheses captured all relevant biological data sets. Based on cytokinin and RMS1 expression analyses, this model is extended to include subtle but important differences in the function of RMS3 and RMS4 genes in the shoot and rootstock. Additionally, this research indicates that a branch-derived signal upregulates RMS1 expression independent of the other feedback signal. Furthermore, we propose xylem-sap cytokinin promotes sustained bud outgrowth, rather than acting at the earlier stage of bud release.

Computational analysis of flowering in pea (Pisum sativum).

During plant development, the transition from a vegetative to reproductive state is a critical event. For decades, pea (Pisum sativum) has been used as a model species to study this transition. These studies have led to a conceptual, qualitative model for the control of flower initiation, referred to as the 'classical' model. This model involves many inputs, namely photoperiod, genetic states and two mobile signals which interact to determine the first node of flowering. Here, we developed a computational model based on the hypotheses of the classical model. Accordingly, we converted qualitative hypotheses into quantitative rules. We found that new hypotheses, in addition to those already described for the classical model, were required that explicitly described the signals. In particular, we hypothesized that the key flowering gene HR interacts with the photoperiod pathway to control flowering. The computational model was tested against a wide range of biological data, including pre-existing and new experimental results presented here, and was found to be accurate. This computational model, together with ongoing experimental advances, will assist future modelling efforts to increase our understanding of flowering in pea.

Strigolactones: discovery of the elusive shoot branching hormone.

The control of axillary bud outgrowth involves a network of hormonal signals and feedback regulation. A repressor of bud outgrowth that is central to the story has been missing since it was first postulated more than 70 years ago. This hormone moves upward in plant stems and can act as a long-distance messenger for auxin. Strigolactones, previously known as carotenoid-derived signals exuded from roots, fit the role of this elusive hormone. The discovery of branching inhibition by strigolactones will help solve many confusing aspects of branch control, including interactions with other signals, and is a great step forward toward uncovering the links between environment, genetics and plant form.

Strigolactone acts downstream of auxin to regulate bud outgrowth in pea and Arabidopsis.

During the last century, two key hypotheses have been proposed to explain apical dominance in plants: auxin promotes the production of a second messenger that moves up into buds to repress their outgrowth, and auxin saturation in the stem inhibits auxin transport from buds, thereby inhibiting bud outgrowth. The recent discovery of strigolactone as the novel shoot-branching inhibitor allowed us to test its mode of action in relation to these hypotheses. We found that exogenously applied strigolactone inhibited bud outgrowth in pea (Pisum sativum) even when auxin was depleted after decapitation. We also found that strigolactone application reduced branching in Arabidopsis (Arabidopsis thaliana) auxin response mutants, suggesting that auxin may act through strigolactones to facilitate apical dominance. Moreover, strigolactone application to tiny buds of mutant or decapitated pea plants rapidly stopped outgrowth, in contrast to applying N-1-naphthylphthalamic acid (NPA), an auxin transport inhibitor, which significantly slowed growth only after several days. Whereas strigolactone or NPA applied to growing buds reduced bud length, only NPA blocked auxin transport in the bud. Wild-type and strigolactone biosynthesis mutant pea and Arabidopsis shoots were capable of instantly transporting additional amounts of auxin in excess of endogenous levels, contrary to predictions of auxin transport models. These data suggest that strigolactone does not act primarily by affecting auxin transport from buds. Rather, the primary repressor of bud outgrowth appears to be the auxin-dependent production of strigolactones.

Strigolactone inhibition of shoot branching.

A carotenoid-derived hormonal signal that inhibits shoot branching in plants has long escaped identification. Strigolactones are compounds thought to be derived from carotenoids and are known to trigger the germination of parasitic plant seeds and stimulate symbiotic fungi. Here we present evidence that carotenoid cleavage dioxygenase 8 shoot branching mutants of pea are strigolactone deficient and that strigolactone application restores the wild-type branching phenotype to ccd8 mutants. Moreover, we show that other branching mutants previously characterized as lacking a response to the branching inhibition signal also lack strigolactone response, and are not deficient in strigolactones. These responses are conserved in Arabidopsis. In agreement with the expected properties of the hormonal signal, exogenous strigolactone can be transported in shoots and act at low concentrations. We suggest that endogenous strigolactones or related compounds inhibit shoot branching in plants. Furthermore, ccd8 mutants demonstrate the diverse effects of strigolactones in shoot branching, mycorrhizal symbiosis and parasitic weed interaction.

Branching genes are conserved across species. Genes controlling a novel signal in pea are coregulated by other long-distance signals.

Physiological and genetic studies with the ramosus (rms) mutants in garden pea (Pisum sativum) and more axillary shoots (max) mutants in Arabidopsis (Arabidopsis thaliana) have shown that shoot branching is regulated by a network of long-distance signals. Orthologous genes RMS1 and MAX4 control the synthesis of a novel graft-transmissible branching signal that may be a carotenoid derivative and acts as a branching inhibitor. In this study, we demonstrate further conservation of the branching control system by showing that MAX2 and MAX3 are orthologous to RMS4 and RMS5, respectively. This is consistent with the long-standing hypothesis that branching in pea is regulated by a novel long-distance signal produced by RMS1 and RMS5 and that RMS4 is implicated in the response to this signal. We examine RMS5 expression and show that it is more highly expressed relative to RMS1, but under similar transcriptional regulation as RMS1. Further expression studies support the hypothesis that RMS4 functions in shoot and rootstock and participates in the feedback regulation of RMS1 and RMS5 expression. This feedback involves a second novel long-distance signal that is lacking in rms2 mutants. RMS1 and RMS5 are also independently regulated by indole-3-acetic acid. RMS1, rather than RMS5, appears to be a key regulator of the branching inhibitor. This study presents new interactions between RMS genes and provides further evidence toward the ongoing elucidation of a model of axillary bud outgrowth in pea.