The activation of Arabidopsis axillary buds involves a switch from slow to rapid committed outgrowth regulated by auxin and strigolactone.

Arabidopsis thaliana (Arabidopsis) shoot architecture is largely determined by the pattern of axillary buds that grow into lateral branches, the regulation of which requires integrating both local and systemic signals. Nodal explants - stem explants each bearing one leaf and its associated axillary bud - are a simplified system to understand the regulation of bud activation. To explore signal integration in bud activation, we characterised the growth dynamics of buds in nodal explants in key mutants and under different treatments. We observed that isolated axillary buds activate in two genetically and physiologically separable phases: a slow-growing lag phase, followed by a switch to rapid outgrowth. Modifying BRANCHED1 expression or the properties of the auxin transport network, including via strigolactone application, changed the length of the lag phase. While most interventions affected only the length of the lag phase, strigolactone treatment and a second bud also affected the rapid growth phase. Our results are consistent with the hypothesis that the slow-growing lag phase corresponds to the time during which buds establish canalised auxin transport out of the bud, after which they enter a rapid growth phase. Our work also hints at a role for auxin transport in influencing the maximum growth rate of branches.

Tree architecture: A strigolactone-deficient mutant reveals a connection between branching order and auxin gradient along the tree stem.

Due to their long lifespan, trees and bushes develop higher order of branches in a perennial manner. In contrast to a tall tree, with a clearly defined main stem and branching order, a bush is shorter and has a less apparent main stem and branching pattern. To address the developmental basis of these two forms, we studied several naturally occurring architectural variants in silver birch (Betula pendula). Using a candidate gene approach, we identified a bushy kanttarelli variant with a loss-of-function mutation in the BpMAX1 gene required for strigolactone (SL) biosynthesis. While kanttarelli is shorter than the wild type (WT), it has the same number of primary branches, whereas the number of secondary branches is increased, contributing to its bush-like phenotype. To confirm that the identified mutation was responsible for the phenotype, we phenocopied kanttarelli in transgenic BpMAX1::RNAi birch lines. SL profiling confirmed that both kanttarelli and the transgenic lines produced very limited amounts of SL. Interestingly, the auxin (IAA) distribution along the main stem differed between WT and BpMAX1::RNAi. In the WT, the auxin concentration formed a gradient, being higher in the uppermost internodes and decreasing toward the basal part of the stem, whereas in the transgenic line, this gradient was not observed. Through modeling, we showed that the different IAA distribution patterns may result from the difference in the number of higher-order branches and plant height. Future studies will determine whether the IAA gradient itself regulates aspects of plant architecture.

Artificial selection reveals complex genetic architecture of shoot branching and its response to nitrate supply in Arabidopsis.

Quantitative traits may be controlled by many loci, many alleles at each locus, and subject to genotype-by-environment interactions, making them difficult to map. One example of such a complex trait is shoot branching in the model plant Arabidopsis, and its plasticity in response to nitrate. Here, we use artificial selection under contrasting nitrate supplies to dissect the genetic architecture of this complex trait, where loci identified by association mapping failed to explain heritability estimates. We found a consistent response to selection for high branching, with correlated responses in other traits such as plasticity and flowering time. Genome-wide scans for selection and simulations suggest that at least tens of loci control this trait, with a distinct genetic architecture between low and high nitrate treatments. While signals of selection could be detected in the populations selected for high branching on low nitrate, there was very little overlap in the regions selected in three independent populations. Thus the regulatory network controlling shoot branching can be tuned in different ways to give similar phenotypes.

The miR156 juvenility factor and PLETHORA 2 form a regulatory network and influence timing of meristem growth and lateral root emergence.

Plants develop throughout their lives: seeds become seedlings that mature and form fruits and seeds. Although the underlying mechanisms that drive these developmental phase transitions have been well elucidated for shoots, the extent to which they affect the root is less clear. However, root anatomy does change as some plants mature; meristems enlarge and radial thickening occurs. Here, in Arabidopsis thaliana, we show that overexpressing miR156A, a gene that promotes the juvenile phase, increased the density of the root system, even in grafted plants in which only the rootstock had the overexpression genotype. In the root, overexpression of miR156A resulted in lower levels of PLETHORA 2, a protein that affects formation of the meristem and elongation zone. Crossing in an extra copy of PLETHORA 2 partially rescued the effects of miR156A overexpression on traits affecting root architecture, including meristem length and the rate of lateral root emergence. Consistent with this, PLETHORA 2 also inhibited the root-tip expression of another miR156 gene, miR156C. We conclude that the system driving phase change in the shoot affects developmental progression in the root, and that PLETHORA 2 participates in this network.

KAI2 regulates seedling development by mediating light-induced remodelling of auxin transport.

Photomorphogenic remodelling of seedling growth is a key developmental transition in the plant life cycle. The α/ß-hydrolase signalling protein KARRIKIN-INSENSITIVE2 (KAI2), a close homologue of the strigolactone receptor DWARF14 (D14), is involved in this process, but it is unclear how the effects of KAI2 on development are mediated. Here, using a combination of physiological, pharmacological, genetic and imaging approaches in Arabidopsis thaliana (Heynh.) we show that kai2 phenotypes arise because of a failure to downregulate auxin transport from the seedling shoot apex towards the root system, rather than a failure to respond to light per se. We demonstrate that KAI2 controls the light-induced remodelling of the PIN-mediated auxin transport system in seedlings, promoting a reduction in PIN7 abundance in older tissues, and an increase of PIN1/PIN2 abundance in the root meristem. We show that removing PIN3, PIN4 and PIN7 from kai2 mutants, or pharmacological inhibition of auxin transport and synthesis, is sufficient to suppress most kai2 seedling phenotypes. We conclude that KAI2 regulates seedling morphogenesis by its effects on the auxin transport system. We propose that KAI2 is not required for the light-mediated changes in PIN gene expression but is required for the appropriate changes in PIN protein abundance within cells.

An ABA-GA bistable switch can account for natural variation in the variability of Arabidopsis seed germination time.

Genetically identical plants growing in the same conditions can display heterogeneous phenotypes. Here we use Arabidopsis seed germination time as a model system to examine phenotypic variability and its underlying mechanisms. We show extensive variation in seed germination time variability between Arabidopsis accessions and use a multiparent recombinant inbred population to identify two genetic loci involved in this trait. Both loci include genes implicated in modulating abscisic acid (ABA) sensitivity. Mutually antagonistic regulation between ABA, which represses germination, and gibberellic acid (GA), which promotes germination, underlies the decision to germinate and can act as a bistable switch. A simple stochastic model of the ABA-GA network shows that modulating ABA sensitivity can generate the range of germination time distributions we observe experimentally. We validate the model by testing its predictions on the effects of exogenous hormone addition. Our work provides a foundation for understanding the mechanism and functional role of phenotypic variability in germination time.

A plant's diet, surviving in a variable nutrient environment.

As primary producers, plants rely on a large aboveground surface area to collect carbon dioxide and sunlight and a large underground surface area to collect the water and mineral nutrients needed to support their growth and development. Accessibility of the essential nutrients nitrogen (N) and phosphorus (P) in the soil is affected by many factors that create a variable spatiotemporal landscape of their availability both at the local and global scale. Plants optimize uptake of the N and P available through modifications to their growth and development and engagement with microorganisms that facilitate their capture. The sensing of these nutrients, as well as the perception of overall nutrient status, shapes the plant's response to its nutrient environment, coordinating its development with microbial engagement to optimize N and P capture and regulate overall plant growth.

Network trade-offs and homeostasis in Arabidopsis shoot architectures.

Understanding the optimization objectives that shape shoot architectures remains a critical problem in plant biology. Here, we performed 3D scanning of 152 Arabidopsis shoot architectures, including wildtype and 10 mutant strains, and we uncovered a design principle that describes how architectures make trade-offs between competing objectives. First, we used graph-theoretic analysis to show that Arabidopsis shoot architectures strike a Pareto optimal that can be captured as maximizing performance in transporting nutrients and minimizing costs in building the architecture. Second, we identify small sets of genes that can be mutated to shift the weight prioritizing one objective over the other. Third, we show that this prioritization weight feature is significantly less variable across replicates of the same genotype compared to other common plant traits (e.g., number of rosette leaves, total volume occupied). This suggests that this feature is a robust descriptor of a genotype, and that local variability in structure may be compensated for globally in a homeostatic manner. Overall, our work provides a framework to understand optimization trade-offs made by shoot architectures and provides evidence that these trade-offs can be modified genetically, which may aid plant breeding and selection efforts.

Natural variation in Arabidopsis shoot branching plasticity in response to nitrate supply affects fitness.

The capacity of organisms to tune their development in response to environmental cues is pervasive in nature. This phenotypic plasticity is particularly striking in plants, enabled by their modular and continuous development. A good example is the activation of lateral shoot branches in Arabidopsis, which develop from axillary meristems at the base of leaves. The activity and elongation of lateral shoots depends on the integration of many signals both external (e.g. light, nutrient supply) and internal (e.g. the phytohormones auxin, strigolactone and cytokinin). Here, we characterise natural variation in plasticity of shoot branching in response to nitrate supply using two diverse panels of Arabidopsis lines. We find extensive variation in nitrate sensitivity across these lines, suggesting a genetic basis for variation in branching plasticity. High plasticity is associated with extreme branching phenotypes such that lines with the most branches on high nitrate have the fewest under nitrate deficient conditions. Conversely, low plasticity is associated with a constitutively moderate level of branching. Furthermore, variation in plasticity is associated with alternative life histories with the low plasticity lines flowering significantly earlier than high plasticity lines. In Arabidopsis, branching is highly correlated with fruit yield, and thus low plasticity lines produce more fruit than high plasticity lines under nitrate deficient conditions, whereas highly plastic lines produce more fruit under high nitrate conditions. Low and high plasticity, associated with early and late flowering respectively, can therefore be interpreted alternative escape vs mitigate strategies to low N environments. The genetic architecture of these traits appears to be highly complex, with only a small proportion of the estimated genetic variance detected in association mapping.

Connective auxin transport contributes to strigolactone-mediated shoot branching control independent of the transcription factor BRC1.

The shoot systems of plants are built by the action of the primary shoot apical meristem, established during embryogenesis. In the axil of each leaf produced by the primary meristem, secondary axillary shoot apical meristems are established. The dynamic regulation of the activity of these axillary meristems gives shoot systems their extraordinary plasticity of form. The ability of plants to activate or repress these axillary meristems appropriately requires communication between meristems that is environmentally sensitive. The transport network of the plant hormone auxin has long been implicated as a central player in this tuneable communication system, with other systemically mobile hormones, such as strigolactone and cytokinin, acting in part by modulating auxin transport. Until recently, the polar auxin transport stream, which provides a high conductance auxin transport route down stems dominated by the auxin export protein PIN-FORMED1 (PIN1), has been the focus for understanding long range auxin transport in the shoot. However, recently additional auxin exporters with important roles in the shoot have been identified, including PIN3, PIN4 and PIN7. These proteins contribute to a wider less polar stem auxin transport regime, which we have termed connective auxin transport (CAT), because of its role in communication across the shoot system. Here we present a genetic analysis of the role of CAT in shoot branching. We demonstrate that in Arabidopsis, CAT plays an important role in strigolactone-mediated shoot branching control, with the triple pin3pin4pin7 mutant able to suppress partially the highly branched phenotype of strigolactone deficient mutants. In contrast, the branchy phenotype of mutants lacking the axillary meristem-expressed transcription factor, BRANCHED1 (BRC1) is unaffected by pin3pin4pin7. We further demonstrate that mutation in the ABCB19 auxin export protein, which like PIN3 PIN4 and PIN7 is widely expressed in stems, has very different effects, implicating ABCB19 in auxin loading at axillary bud apices.

Structural plasticity of D3-D14 ubiquitin ligase in strigolactone signalling.

The strigolactones, a class of plant hormones, regulate many aspects of plant physiology. In the inhibition of shoot branching, the α/ß hydrolase D14-which metabolizes strigolactone-interacts with the F-box protein D3 to ubiquitinate and degrade the transcription repressor D53. Despite the fact that multiple modes of interaction between D14 and strigolactone have recently been determined, how the hydrolase functions with D3 to mediate hormone-dependent D53 ubiquitination remains unknown. Here we show that D3 has a C-terminal α-helix that can switch between two conformational states. The engaged form of this α-helix facilitates the binding of D3 and D14 with a hydrolysed strigolactone intermediate, whereas the dislodged form can recognize unmodified D14 in an open conformation and inhibits its enzymatic activity. The D3 C-terminal α-helix enables D14 to recruit D53 in a strigolactone-dependent manner, which in turn activates the hydrolase. By revealing the structural plasticity of the SCFD3-D14 ubiquitin ligase, our results suggest a mechanism by which the E3 coordinates strigolactone signalling and metabolism.

Cytokinin Targets Auxin Transport to Promote Shoot Branching.

Cytokinin promotes shoot branching by activating axillary buds, but its mechanism of action in Arabidopsis (Arabidopsis thaliana) in this process is unclear. We have shown previously that a hextuple mutant lacking a clade of type-A Arabidopsis Response Regulators (ARRs) known to act in cytokinin signaling has reduced shoot branching compared with the wild type. Since these proteins typically act as negative regulators of cytokinin signaling, this is an unexpected result. To explore this paradox more deeply, we characterized the effects of loss of function of the type-B ARR, ARR1, which positively regulates cytokinin-induced gene expression. The arr1 mutant has increased branching, consistent with a role antagonistic to the type-A ARRs but in apparent conflict with the known positive role for cytokinin in bud activation. We show that the arr branching phenotypes correlate with increases in stem auxin transport and steady-state levels of the auxin export proteins PIN3 and PIN7 on the plasma membrane of xylem-associated cells in the main stem. Cytokinin treatment results in an increased accumulation of PIN3, PIN7, and the closely related PIN4 within several hours, and loss of PIN3, PIN4, and PIN7 can partially rescue the arr1 branching phenotype. This suggests that there are multiple signaling pathways for cytokinin in bud outgrowth; one of these pathways regulates PIN proteins in shoots, independently of the canonical signaling function of the ARR genes tested here. A hypothesis consistent with the arr shoot phenotypes is that feedback control of biosynthesis leads to altered cytokinin accumulation, driving cytokinin signaling via this pathway.

The pea branching RMS2 gene encodes the PsAFB4/5 auxin receptor and is involved in an auxin-strigolactone regulation loop.

Strigolactones (SLs) are well known for their role in repressing shoot branching. In pea, increased transcript levels of SL biosynthesis genes are observed in stems of highly branched SL deficient (ramosus1 (rms1) and rms5) and SL response (rms3 and rms4) mutants indicative of negative feedback control. In contrast, the highly branched rms2 mutant has reduced transcript levels of SL biosynthesis genes. Grafting studies and hormone quantification led to a model where RMS2 mediates a shoot-to-root feedback signal that regulates both SL biosynthesis gene transcript levels and xylem sap levels of cytokinin exported from roots. Here we cloned RMS2 using synteny with Medicago truncatula and demonstrated that it encodes a putative auxin receptor of the AFB4/5 clade. Phenotypes similar to rms2 were found in Arabidopsis afb4/5 mutants, including increased shoot branching, low expression of SL biosynthesis genes and high auxin levels in stems. Moreover, afb4/5 and rms2 display a specific resistance to the herbicide picloram. Yeast-two-hybrid experiments supported the hypothesis that the RMS2 protein functions as an auxin receptor. SL root feeding using hydroponics repressed auxin levels in stems and down-regulated transcript levels of auxin biosynthesis genes within one hour. This auxin down-regulation was also observed in plants treated with the polar auxin transport inhibitor NPA. Together these data suggest a homeostatic feedback loop in which auxin up-regulates SL synthesis in an RMS2-dependent manner and SL down-regulates auxin synthesis in an RMS3 and RMS4-dependent manner.

Cross-species functional diversity within the PIN auxin efflux protein family.

In Arabidopsis, development during flowering is coordinated by transport of the hormone auxin mediated by polar-localized PIN-FORMED1 (AtPIN1). However Arabidopsis has lost a PIN clade sister to AtPIN1, Sister-of-PIN1 (SoPIN1), which is conserved in flowering plants. We previously proposed that the AtPIN1 organ initiation and vein patterning functions are split between the SoPIN1 and PIN1 clades in grasses. Here we show that in the grass Brachypodium sopin1 mutants have organ initiation defects similar to Arabidopsis atpin1, while loss of PIN1 function in Brachypodium has little effect on organ initiation but alters stem growth. Heterologous expression of Brachypodium SoPIN1 and PIN1b in Arabidopsis provides further evidence of functional specificity. SoPIN1 but not PIN1b can mediate flower formation in null atpin1 mutants, although both can complement a missense allele. The behavior of SoPIN1 and PIN1b in Arabidopsis illustrates how membrane and tissue-level accumulation, transport activity, and interaction contribute to PIN functional specificity.

BRC1 expression regulates bud activation potential but is not necessary or sufficient for bud growth inhibition in Arabidopsis.

The degree of shoot branching in Arabidopsis is determined by the activation of axillary buds. Bud activity is regulated by diverse environmental and developmental signals, often mediated via plant hormones, including auxin, strigolactone and cytokinin. The transcription factor BRANCHED1 (BRC1) has been proposed to integrate these regulatory signals. This idea is based on increased branching in brc1 mutants, the effects of bud-regulating hormones on BRC1 expression, and a general correlation between BRC1 expression and bud growth inhibition. These data demonstrate the important role of BRC1 in shoot branching, but here we show that in Arabidopsis this correlation can be broken. Buds lacking BRC1 expression can remain inhibited and sensitive to inhibition by strigolactone. Furthermore, buds with high BRC1 transcript levels can be active. Based on these data, we propose that BRC1 regulates bud activation potential in concert with an auxin transport-based mechanism underpinning bud activity. In the context of strigolactone-mediated bud regulation, our data suggest a coherent feed-forward loop in which strigolactone treatment reduces the probability of bud activation by parallel effects on BRC1 transcription and the shoot auxin transport network.

Strigolactone regulates shoot development through a core signalling pathway.

Strigolactones are a recently identified class of hormone that regulate multiple aspects of plant development. The DWARF14 (D14) α/ß fold protein has been identified as a strigolactone receptor, which can act through the SCFMAX2 ubiquitin ligase, but the universality of this mechanism is not clear. Multiple proteins have been suggested as targets for strigolactone signalling, including both direct proteolytic targets of SCFMAX2, and downstream targets. However, the relevance and importance of these proteins to strigolactone signalling in many cases has not been fully established. Here we assess the contribution of these targets to strigolactone signalling in adult shoot developmental responses. We find that all examined strigolactone responses are regulated by SCFMAX2 and D14, and not by other D14-like proteins. We further show that all examined strigolactone responses likely depend on degradation of SMXL proteins in the SMXL6 clade, and not on the other proposed proteolytic targets BES1 or DELLAs. Taken together, our results suggest that in the adult shoot, the dominant mode of strigolactone signalling is D14-initiated, MAX2-mediated degradation of SMXL6-related proteins. We confirm that the BRANCHED1 transcription factor and the PIN-FORMED1 auxin efflux carrier are plausible downstream targets of this pathway in the regulation of shoot branching, and show that BRC1 likely acts in parallel to PIN1.