Didier Reinhardt

Regulation of phyllotaxis by polar auxin transport

The regular arrangement of leaves around a plants stem, called phyllotaxis, has for centuries attracted the attention of philosophers, mathematicians and natural scientists; however, to date, studies of phyllotaxis have been largely theoretical. Leaves and flowers are formed from the shoot apical meristem, triggered by the plant hormone auxin. Auxin is transported through plant tissues by specific cellular influx and efflux carrier proteins. Here we show that proteins involved in auxin transport regulate phyllotaxis. Our data indicate that auxin is transported upwards into the meristem through the epidermis and the outermost meristem cell layer. Existing leaf primordia act as sinks, redistributing auxin and creating its heterogeneous distribution in the meristem. Auxin accumulation occurs only at certain minimal distances from existing primordia, defining the position of future primordia. This model for phyllotaxis accounts for its reiterative nature, as well as its regularity and stability.

Auxin Regulates the Initiation and Radial Position of Plant Lateral Organs

Leaves originate from the shoot apical meristem, a small mound of undifferentiated tissue at the tip of the stem. Leaf formation begins with the selection of a group of founder cells in the so-called peripheral zone at the flank of the meristem, followed by the initiation of local growth and finally morphogenesis of the resulting bulge into a differentiated leaf. Whereas the mechanisms controlling the switch between meristem propagation and leaf initiation are being identified by genetic and molecular analyses, the radial positioning of leaves, known as phyllotaxis, remains poorly understood. Hormones, especially auxin and gibberellin, are known to influence phyllotaxis, but their specific role in the determination of organ position is not clear. We show that inhibition of polar auxin transport blocks leaf formation at the vegetative tomato meristem, resulting in pinlike naked stems with an intact meristem at the tip. Microapplication of the natural auxin indole-3-acetic acid (IAA) to the apex of such pins restores leaf formation. Similarly, exogenous IAA induces flower formation on Arabidopsis pin-formed1-1 inflorescence apices, which are blocked in flower formation because of a mutation in a putative auxin transport protein. Our results show that auxin is required for and sufficient to induce organogenesis both in the vegetative tomato meristem and in the Arabidopsis inflorescence meristem. In this study, organogenesis always strictly coincided with the site of IAA application in the radial dimension, whereas in the apical–basal dimension, organ formation always occurred at a fixed distance from the summit of the meristem. We propose that auxin determines the radial position and the size of lateral organs but not the apical–basal position or the identity of the induced structures.

A plausible model of phyllotaxis

A striking phenomenon unique to the kingdom of plants is the regular arrangement of lateral organs around a central axis, known as phyllotaxis. Recent molecular-genetic experiments indicate that active transport of the plant hormone auxin is the key process regulating phyllotaxis. A conceptual model based on these experiments, introduced by Reinhardt et al. [Reinhardt, D., Pesce, E. R., Stieger, P., Mandel, T., Baltensperger, K., et al. (2003) Nature 426, 255–260], provides an intuitively plausible interpretation of the data, but raises questions of whether the proposed mechanism is, in fact, capable of producing the observed temporal and spatial patterns, is robust, can start de novo, and can account for phyllotactic transitions, such as the frequently observed transition from decussate to spiral phyllotaxis. To answer these questions, we created a computer simulation model based on data described previously or in this paper and reasonable hypotheses. The model reproduces, within the standard error, the divergence angles measured in Arabidopsis seedlings and the effects of selected experimental manipulations. It also reproduces distichous, decussate, and tricussate patterns. The model thus offers a plausible link between molecular mechanisms of morphogenesis and the geometry of phyllotaxis.

A petunia ABC protein controls strigolactone-dependent symbiotic signalling and branching.

Strigolactones were originally identified as stimulators of the germination of root-parasitic weeds that pose a serious threat to resource-limited agriculture. They are mostly exuded from roots and function as signalling compounds in the initiation of arbuscular mycorrhizae, which are plant–fungus symbionts with a global effect on carbon and phosphate cycling. Recently, strigolactones were established to be phytohormones that regulate plant shoot architecture by inhibiting the outgrowth of axillary buds. Despite their importance, it is not known how strigolactones are transported. ATP-binding cassette (ABC) transporters, however, are known to have functions in phytohormone translocation. Here we show that the Petunia hybrida ABC transporter PDR1 has a key role in regulating the development of arbuscular mycorrhizae and axillary branches, by functioning as a cellular strigolactone exporter. P. hybrida pdr1 mutants are defective in strigolactone exudation from their roots, resulting in reduced symbiotic interactions. Above ground, pdr1 mutants have an enhanced branching phenotype, which is indicative of impaired strigolactone allocation. Overexpression of Petunia axillaris PDR1 in Arabidopsis thaliana results in increased tolerance to high concentrations of a synthetic strigolactone, consistent with increased export of strigolactones from the roots. PDR1 is the first known component in strigolactone transport, providing new opportunities for investigating and manipulating strigolactone-dependent processes.

Localized upregulation of a new expansin gene predicts the site of leaf formation in the tomato meristem

Expansins are extracellular proteins that increase plant cell wall extensibility in vitro and are thought to be involved in cell expansion. We showed in a previous study that administration of an exogenous expansin protein can trigger the initiation of leaflike structures on the shoot apical meristem of tomato. Here, we studied the expression patterns of two tomato expansin genes, LeExp2 and LeExp18. LeExp2 is preferentially expressed in expanding tissues, whereas LeExp18 is expressed preferentially in tissues with meristematic activity. In situ hybridization experiments showed that LeExp18 expression is elevated in a group of cells, called I1, which is the site of incipient leaf primordium initiation. Thus, LeExp18 expression is a molecular marker for leaf initiation, predicting the site of primordium formation at a time before histological changes can be detected. We propose a model for the regulation of phyllotaxis that postulates a crucial role for expansin in leaf primordium initiation.

Phosphate systemically inhibits development of arbuscular mycorrhiza in Petunia hybrida and represses genes involved in mycorrhizal functioning

Most terrestrial plants form arbuscular mycorrhiza (AM), mutualistic associations with soil fungi of the order Glomeromycota. The obligate biotrophic fungi trade mineral nutrients, mainly phosphate (P(i) ), for carbohydrates from the plants. Under conditions of high exogenous phosphate supply, when the plant can meet its own P requirements without the fungus, AM are suppressed, an effect which could be interpreted as an active strategy of the plant to limit carbohydrate consumption of the fungus by inhibiting its proliferation in the roots. However, the mechanisms involved in fungal inhibition are poorly understood. Here, we employ a transcriptomic approach to get insight into potential shifts in metabolic activity and symbiotic signalling, and in the defence status of plants exposed to high P(i) levels. We show that in mycorrhizal roots of petunia, a similar set of symbiosis-related genes is expressed as in mycorrhizal roots of Medicago, Lotus and rice. P(i) acts systemically to repress symbiotic gene expression and AM colonization in the root. In established mycorrhizal roots, P(i) repressed symbiotic gene expression rapidly, whereas the inhibition of colonization followed with a lag of more than a week. Taken together, these results suggest that P(i) acts by repressing essential symbiotic genes, in particular genes encoding enzymes of carotenoid and strigolactone biosynthesis, and symbiosis-associated phosphate transporters. The role of these effects in the suppression of symbiosis under high P(i) conditions is discussed.

Elastic Domains Regulate Growth and Organogenesis in the Plant Shoot Apical Meristem

Shape-Shifting Signals Although orthogonal signaling systems seem to direct various developmental processes, few tissues remain in the same shape as they are at initiation to that of the final form. Arabidopsis leaves are free of the cell migrations that complicate animal development, and thus allowed Kuchen et al. (p. 1092) to track and model the trajectory of leaf growth under a variety of perturbations. Varying the values of parameters in their model produced outputs of different leaf shapes ranging from obcordate, ovate, and oval to elliptic, and offered predictions for genes that regulate the developmental process. The meristem at the growing tip of plants is home to stem cells and is the source of newly differentiating shoots and leaves. New leaves make their first appearance as bulges at the side of the dome-shaped meristem. Although these developmental events are under hormonal control, they also seem to be constrained by the physical properties of the meristem. Kierzkowski et al. (p. 1096) tested physical effects acting on the shoot apical meristem of growing tomato shoots that alter turgor pressure. Again, mathematical modeling combined with observations of plant tissue helped to define the different zones in the meristem that respond to diverse mechanical stimuli. New leaves emerge where they are allowed. Although genetic control of morphogenesis is well established, elaboration of complex shapes requires changes in the mechanical properties of cells. In plants, the first visible sign of leaf formation is a bulge on the flank of the shoot apical meristem. Bulging results from local relaxation of cell walls, which causes them to yield to internal hydrostatic pressure. By manipulation of tissue tension in combination with quantitative live imaging and finite-element modeling, we found that the slow-growing area at the shoot tip is substantially strain-stiffened compared with surrounding fast-growing tissue. We propose that strain stiffening limits growth, restricts organ bulging, and contributes to the meristems functional zonation. Thus, mechanical signals are not just passive readouts of gene action but feed back on morphogenesis.

Microsurgical and laser ablation analysis of interactions between the zones and layers of the tomato shoot apical meristem.

Plants exhibit life-long organogenic and histogenic activity in a specialised organ, the shoot apical meristem. Leaves and flowers are formed within the ring-shaped peripheral zone, which surrounds the central zone, the site of the stem cells. We have undertaken a series of high-precision laser ablation and microsurgical tissue removal experiments to test the functions of different parts of the tomato meristem, and to reveal their interactions. Ablation of the central zone led to ectopic expression of the WUSCHEL gene at the periphery, followed by the establishment of a new meristem centre. After the ablation of the central zone, organ formation continued without a lag. Thus, the central zone does not participate in organogenesis, except as the ultimate source of founder cells. Microsurgical removal of the external L1 layer induced periclinal cell divisions and terminal differentiation in the subtending layers. In addition, no organs were initiated in areas devoid of L1, demonstrating an important role of the L1 in organogenesis. L1 ablation had only local effects, an observation that is difficult to reconcile with phyllotaxis theories that invoke physical tension operating within the meristem as a whole. Finally, regeneration of L1 cells was never observed after ablation. This shows that while the zones of the meristem show a remarkable capacity to regenerate after interference, elimination of the L1 layer is irreparable and causes terminal differentiation.

Arabidopsis AXR6 encodes CUL1 implicating SCF E3 ligases in auxin regulation of embryogenesis

The AXR6 gene is required for auxin signaling in the Arabidopsis embryo and during postembryonic development. One of the effects of auxin is to stimulate degradation of the Aux/IAA auxin response proteins through the action of the ubiquitin protein ligase SCFTIR1. Here we show that AXR6 encodes the SCF subunit CUL1. The axr6 mutations affect the ability of mutant CUL1 to assemble into stable SCF complexes resulting in reduced degradation of the SCFTIR1 substrate AXR2/IAA7. In addition, we show that CUL1 is required for lateral organ initiation in the shoot apical meristem and the inflorescence meristem. These results indicate that the embryonic axr6 phenotype is related to a defect in SCF function and accumulation of Aux/IAA proteins such as BDL/IAA12. In addition, we show that CUL1 has a role in auxin response throughout the life cycle of the plant.

Starch Granule Biosynthesis in Arabidopsis Is Abolished by Removal of All Debranching Enzymes but Restored by the Subsequent Removal of an Endoamylase

Several studies have suggested that debranching enzymes (DBEs) are involved in the biosynthesis of amylopectin, the major constituent of starch granules. Our systematic analysis of all DBE mutants of Arabidopsis thaliana demonstrates that when any DBE activity remains, starch granules are still synthesized, albeit with altered amylopectin structure. Quadruple mutants lacking all four DBE proteins (Isoamylase1 [ISA1], ISA2, and ISA3, and Limit-Dextrinase) are devoid of starch granules and instead accumulate highly branched glucans, distinct from amylopectin and from previously described phytoglycogen. A fraction of these glucans are present as discrete, insoluble, nanometer-scale particles, but the structure and properties of this material are radically altered compared with wild-type amylopectin. Superficially, these data support the hypothesis that debranching is required for amylopectin synthesis. However, our analyses show that soluble glucans in the quadruple DBE mutant are degraded by α- and β-amylases during periods of net accumulation, giving rise to maltose and branched malto-oligosaccharides. The additional loss of the chloroplastic α-amylase AMY3 partially reverts the phenotype of the quadruple DBE mutant, restoring starch granule biosynthesis. We propose that DBEs function in normal amylopectin synthesis by promoting amylopectin crystallization but conclude that they are not mandatory for starch granule synthesis.