Cris Kuhlemeier

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.

Pectin-Induced Changes in Cell Wall Mechanics Underlie Organ Initiation in Arabidopsis

Tissue mechanics have been shown to play a key role in the regulation of morphogenesis in animals [1-4] and may have an equally important role in plants [5-9]. The aerial organs of plants are formed at the shoot apical meristem following a specific phyllotactic pattern [10]. The initiation of an organ from the meristem requires a highly localized irreversible surface deformation, which depends on the demethylesterification of cell wall pectins [11]. Here, we used atomic force microscopy (AFM) to investigate whether these chemical changes lead to changes in tissue mechanics. By mapping the viscoelasticity and elasticity in living meristems, we observed increases in tissue elasticity, correlated with pectin demethylesterification, in primordia and at the site of incipient organs. Measurements of tissue elasticity at various depths showed that, at the site of incipient primordia, the first increases occurred in subepidermal tissues. The results support the following causal sequence of events: (1) demethylesterification of pectin is triggered in subepidermal tissue layers, (2) this contributes to an increase in elasticity of these layers-the first observable mechanical event in organ initiation, and (3) the process propagates to the epidermis during the outgrowth of the organ.

Ethanolic fermentation: new functions for an old pathway

Ethanolic fermentation is an ancient metabolic pathway. In plants, it is a major route of ATP production under anaerobic conditions. In addition, recent developments suggest that the pathway has important functions in the presence of oxygen. Both of the enzymes required for the production of acetaldehyde and ethanol, pyruvate decarboxylase and alcohol dehydrogenase, are highly abundant in pollen, resulting in fermentation in fully oxygenated cells. Acetaldehyde toxicity is an inevitable side effect of aerobic fermentation. Could acetaldehyde be the elusive pollen factor that contributes to male sterility in cmsT maize? The versatility of this ancient pathway is also illustrated by the induction of aerobic fermentation by environmental stress and activation of a defense response by overexpression of pyruvate decarboxylase.

Single Gene–Mediated Shift in Pollinator Attraction in Petunia

Animal-mediated pollination is essential in plant reproductive biology and is often associated with pollination syndromes, sets of floral traits, such as color, scent, shape, or nectar content. Selection by pollinators is often considered a key factor in floral evolution and plant speciation. Our aim is the identification and characterization of the genetic changes that caused the evolution of divergent pollination syndromes in closely related plant species. We focus on ANTHOCYANIN2 (AN2), a well-defined myb-type transcription factor that is a major determinant of flower color variation between Petunia integrifolia and Petunia axillaris. Analysis of sequence variation in AN2 in wild P. axillaris accessions showed that loss-of-function alleles arose at least five times independently. DNA sequence analysis was complemented by functional assays for pollinator preference using genetic introgressions and transgenics. These results show that AN2 is a major determinant of pollinator attraction. Therefore, changes in a single gene cause a major shift in pollination biology and support the notion that the adaptation of a flowering plant to a new pollinator type may involve a limited number of genes of large effect. Gene identification and analysis of molecular evolution in combination with behavioral and ecological studies can ultimately unravel the evolutionary genetics of pollination syndromes.

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.

Auxin influx carriers stabilize phyllotactic patterning

One of the most striking features of plant architecture is the regular arrangement of leaves and flowers around the stem, known as phyllotaxis. Peaks in concentration of the plant hormone auxin, generated by the polar localization of the PIN1 auxin efflux carrier, provide the instructive signal for primordium initiation. This mechanism generates the spacing between neighboring primordia, which results in regular phyllotaxis. Studies of the role of auxin transport in phyllotactic patterning have focused on PIN1-mediated efflux. Recent computer simulations indicate an additional role for transporter-mediated auxin uptake. Mutations in the AUX1 auxin influx carrier have not, however, been reported to cause an aerial phenotype. Here, we study the role of AUX1 and its paralogs LAX1, LAX2, and LAX3. Analysis of the quadruple mutant reveals irregular divergence angles between successive primordia. A highly unusual aspect of the phenotype is the occurrence of clusters of primordia, in violation of classical theory. At the molecular level, the sharp peaks in auxin levels and coordinated PIN polarization are reduced or lost. In addition, the increased penetrance of the phenotype under short-day conditions suggests that the AUX LAX transporters act to buffer the PIN-mediated patterning mechanism against environmental or developmental influences.

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.