Auxin is required for leaf vein pattern in Arabidopsis

To investigate possible roles of polar auxin transport in vein patterning, cotyledon and leaf vein patterns were compared for plants grown in medium containing polar auxin transport inhibitors (N-1-naphthylphthalamic acid, 9-hydroxyfluorene-9-carboxylic acid, and 2,3,5-triiodobenzoic acid) and in medium containing a less well-characterized inhibitor of auxin-mediated processes, 2-(p-chlorophynoxy)-2-methylpropionic acid. Cotyledon vein pattern was not affected by any inhibitor treatments, although vein morphology was altered. In contrast, leaf vein pattern was affected by inhibitor treatments. Growth in polar auxin transport inhibitors resulted in leaves that lacked vascular continuity through the petiole and had broad, loosely organized midveins, an increased number of secondary veins, and a dense band of misshapen tracheary elements adjacent to the leaf margin. Analysis of leaf vein pattern developmental time courses suggested that the primary vein did not develop in polar auxin transport inhibitor-grown plants, and that the broad midvein observed in these seedlings resulted from the coalescence of proximal regions of secondary veins. Possible models for leaf vein patterning that could account for these observations are discussed.     

Responses of plant vascular systems to auxin transport inhibition.

To assess the role of auxin flows in plant vascular patterning, the development of vascular systems under conditions of inhibited auxin transport was analyzed. In Arabidopsis, nearly identical responses evoked by three auxin transport inhibitor substances revealed an enormous plasticity of the vascular pattern and suggest an involvement of auxin flows in determining the sites of vascular differentiation and in promoting vascular tissue continuity. Organs formed under conditions of reduced auxin transport contained increased numbers of vascular strands and cells within those strands were improperly aligned. In leaves, vascular tissues became progressively confined towards the leaf margin as the concentration of auxin transport inhibitor was increased, suggesting that the leaf vascular system depends on inductive signals from the margin of the leaf. Staged application of auxin transport inhibitor demonstrated that primary, secondary and tertiary veins became unresponsive to further modulations of auxin transport at successive stages of early leaf development. Correlation of these stages to anatomical features in early leaf primordia indicated that the pattern of primary and secondary strands becomes fixed at the onset of lamina expansion. Similar alterations in the leaf vascular responses of alyssum, snapdragon and tobacco plants suggest common functions of auxin flows in vascular patterning in dicots, while two types of vascular pattern alterations in Arabidopsis auxin transport mutants suggest that at least two distinct primary defects can result in impaired auxin flow. We discuss these observations with regard to the relative contributions of auxin transport, auxin sensitivity and the cellular organisation of the developing organ on the vascular pattern.     

Computer simulation: The imaginary friend of auxin transport biology

Regulated transport of the plant hormone auxin is central to many aspects of plant development. Directional transport, mediated by membrane transporters, produces patterns of auxin distribution in tissues that trigger developmental processes, such as vascular patterning or leaf formation. Experimentation has produced many, largely qualitative, data providing strong evidence for multiple feedback systems between auxin and its transport. However, the exact mechanisms concerned remain elusive and the experiments required to evaluate alternative hypotheses are challenging. Because of this, computational modelling now plays an important role in auxin transport research. Here we review some current approaches and underlying assumptions of computational auxin transport models. We focus on self‐organising models for polar auxin transport and on recent attempts to unify conflicting mechanistic explanations. In addition, we discuss in general how these computer simulations are proving to be increasingly effective in hypothesis generation and testing, and how simulation can be used to direct future experiments. Editor's suggested further reading in BioEssays Local auxin production: a small contribution to a big field Abstract     

Control of vein patterning by intracellular auxin transport

The vein networks of plant leaves are among the most spectacular expressions of biological pattern, and the principles controlling their formation have continually inspired artists and scientists. Control of vein patterning by the polar, cell-to-cell transport of the plant signaling molecule auxin—mediated in Arabidopsis primarily by the plasma-membrane-localized PIN1—has long been known. By contrast, the existence of intracellular auxin transport and its contribution to vein patterning are recent discoveries. The endoplasmic-reticulum-localized PIN5, PIN6, and PIN8 of Arabidopsis define an intracellular auxin-transport pathway whose functions in vein patterning overlap with those of PIN1-mediated intercellular auxin transport. The genetic interaction between the components of the intracellular auxin-transport pathway is far from having been resolved. The study of vein patterning provides experimental access to gain such a resolution—a resolution that in turn holds the promise to improve our understanding of one of the most fascinating examples of biological pattern formation.     

VAN3 ARF-GAP-mediated vesicle transport is involved in leaf vascular network formation

Within the leaf of an angiosperm, the vascular system is constructed in a complex network pattern called venation. The formation of this vein pattern has been widely studied as a paradigm of tissue pattern formation in plants. To elucidate the molecular mechanism controlling the vein patterning process, we previously isolated Arabidopsis mutants van1 to van7, which show a discontinuous vein pattern. Here we report the phenotypic analysis of the van3 mutant in relation to auxin signaling and polar transport, and the molecular characterization of the VAN3 gene and protein. Double mutant analyses with pin1, emb30-7/gn and mp, and physiological analyses using the auxin-inducible marker DR5::GUS and an auxin transport inhibitor indicated that VAN3 may be involved in auxin signal transduction, but not in polar auxin transport. Positional cloning identified VAN3 as a gene that encodes an adenosine diphosphate (ADP)-ribosylation factor-guanosine triphosphatase (GTPase) activating protein (ARF-GAP). It resembles animal ACAPs and contains four domains: a BAR (BIN/amphiphysin/RVS) domain, a pleckstrin homology (PH) domain, an ARF-GAP domain and an ankyrin (ANK)-repeat domain. Recombinant VAN3 protein showed GTPase-activating activity and a specific affinity for phosphatidylinositols. This protein can self-associate through the N-terminal BAR domain in the yeast two-hybrid system. Subcellular localization analysis by double staining for Venus-tagged VAN3 and several green-fluorescent-protein-tagged intracellular markers indicated that VAN3 is located in a subpopulation of the trans-Golgi network (TGN). Our results indicate that the expression of this gene is induced by auxin and positively regulated by VAN3 itself, and that a specific ACAP type of ARF-GAP functions in vein pattern formation by regulating auxin signaling via a TGN-mediated vesicle transport system.     

Dynamic integration of auxin transport and signalling

Recent years have seen rapid progress in our understanding of the mechanism of action of the plant hormone auxin. A major emerging theme is the central importance of the interplay between auxin signalling and the active transport of auxin through the plant to create dynamic patterns of auxin accumulation. Even in tissues where auxin distribution patterns appear stable, they are the product of standing waves, with auxin flowing through the tissue, maintaining local pockets of high and low concentration. The auxin distribution patterns result in changes in gene expression to trigger diverse, context-dependent growth and differentiation responses. Multi-level feedback loops between the signal transduction network and the auxin transport network provide self-stabilising patterns that remain sensitive to the external environment and to the developmental progression of the plant. The full biological implications of the behaviour of this system are only just beginning to be understood through a combination of experimental manipulation and mathematical modelling.     

Down-regulation of the 26S proteasome subunit RPN9 inhibits viral systemic transport and alters plant vascular development

Plant viruses utilize the vascular system for systemic movement. The plant vascular network also transports water, photosynthates, and signaling molecules and is essential for plant growth. However, the molecular mechanisms governing vascular development and patterning are still largely unknown. From viral transport suppressor screening using virus-induced gene silencing, we identified a 26S proteasome subunit, RPN9, which is required for broad-spectrum viral systemic transport. Silencing of RPN9 in Nicotiana benthamiana inhibits systemic spread of two taxonomically distinct viruses, Tobacco mosaic virus and Turnip mosaic virus. The 26S proteasome is a highly conserved eukaryotic protease complex controlling many fundamental biochemical processes, but the functions of many 26S proteasome regulatory subunits, especially in plants, are still poorly understood. We demonstrate that the inhibition of viral systemic transport after RPN9 silencing is largely due to alterations in the vascular tissue. RPN9-silenced plants display extra leaf vein formation with increased xylem and decreased phloem. We further illustrate that RPN9 functions at least in part through regulation of auxin transport and brassinosteroid signaling, two processes that are crucial for vascular formation. We propose that RPN9 regulates vascular formation by targeting a subset of regulatory proteins for degradation. The brassinosteroid-signaling protein BZR1 is one of the targets.     

植物叶脉发育的分子机制

叶脉在高等植物发育过程中扮演着为叶片输送水分和营养及支撑叶片的重要角色。植物叶片的形态构造看起来简单,而叶脉发育的分子机理却十分复杂。大量研究表明,植物叶脉发育与生长素的极性运输紧密相关,此外,还受到众多转录因子、小分子RNA以及细胞分裂素、油菜素内酯等因素的调控。综述了近年来叶脉发育的分子机制研究进展,以期了解叶脉发育的调控网络。     

Alignment between PIN1 polarity and microtubule orientation in the shoot apical meristem reveals a tight coupling between morphogenesis and auxin transport

Morphogenesis during multicellular development is regulated by intercellular signaling molecules as well as by the mechanical properties of individual cells. In particular, normal patterns of organogenesis in plants require coordination between growth direction and growth magnitude. How this is achieved remains unclear. Here we show that in Arabidopsis thaliana, auxin patterning and cellular growth are linked through a correlated pattern of auxin efflux carrier localization and cortical microtubule orientation. Our experiments reveal that both PIN1 localization and microtubule array orientation are likely to respond to a shared upstream regulator that appears to be biomechanical in nature. Lastly, through mathematical modeling we show that such a biophysical coupling could mediate the feedback loop between auxin and its transport that underlies plant phyllotaxis.     

植物叶缘和叶脉发育调控的研究进展

通常可通过植物叶片的形态来区分不同植物的种类。叶片由茎顶端分生组织侧翼发育而成,为多种多样大小和形状的扁平结构。叶片的结构看似简单,但调控叶片形态和结构发育的分子机理错综复杂,叶片的发育受植物激素、转录因子、一系列蛋白因子及环境的共同调控。本文回顾了叶片边缘形态和叶脉发育研究的最新进展。在叶边缘形态方面,Aux/IAA生长素响应抑制家族蛋白通过调节生长素浓度最大点的离散分布影响小叶的起始和生长以及叶边缘结构;NAM/CUC转录因子促进叶边缘锯齿的分离以及复叶中小叶的分离和分化,NAM/CUC和Aux/IAA通过不同通路实现对生长素的调控;拟南芥RAX1基因/番茄Potato-leaf基因和拟南芥JAG基因/番茄LYR基因促进叶边缘锯齿发育;RCO调控复叶小叶的发育不通过改变生长素的分布来实现;在番茄中反式小干扰RNA途径中的因子参与叶边缘形态发育;另外,在拟南芥中,mir164A、CUC2、PIN1、DPA4、SVR9-1及SVR9L-1构成复杂的调控网络影响叶边缘锯齿的发育。在叶脉发育方面,PIN1能否正确的定位会影响叶脉发育;AS1和AS2共同参与叶片远近轴极性的分化;另外AXR6、MP、BDL、CVP因子功能的缺失影响叶脉发育;生长素、PIN1、Aux/IAA、MP、ATHB8构成反馈循环调控子叶叶脉的形成。     

Feedback models for polarized auxin transport: an emerging trend

The phytohormone auxin is vital to plant growth and development. A unique property of auxin among all other plant hormones is its cell-to-cell polar transport that requires activity of polarly localized PIN-FORMED (PIN) auxin efflux transporters. Despite the substantial molecular insight into the cellular PIN polarization, the mechanistic understanding for developmentally and environmentally regulated PIN polarization is scarce. The long-standing belief that auxin modulates its own transport by means of a positive feedback mechanism has inspired both experimentalists and theoreticians for more than two decades. Recently, theoretical models for auxin-dependent patterning in plants include the feedback between auxin transport and the PIN protein localization. These computer models aid to assess the complexity of plant development by testing and predicting plausible scenarios for various developmental processes that occur in planta. Although the majority of these models rely on purely heuristic principles, the most recent mechanistic models tentatively integrate biologically testable components into known cellular processes that underlie the PIN polarity regulation. The existing and emerging computational approaches to describe PIN polarization are presented and discussed in the light of recent experimental data on the PIN polar targeting.     

Modeling Auxin Transport and Plant Development

The plant hormone auxin plays a critical role in plant development. Central to its function is its distribution in plant tissues, which is, in turn, largely shaped by intercellular polar transport processes. Auxin transport relies on diffusive uptake as well as carrier-mediated transport via influx and efflux carriers. Mathematical models have been used to both refine our theoretical understanding of these processes and to test new hypotheses regarding the localization of efflux carriers to understand auxin patterning at the tissue level. Here we review models for auxin transport and how they have been applied to patterning processes, including the elaboration of plant vasculature and primordium positioning. Second, we investigate the possible role of auxin influx carriers such as AUX1 in patterning auxin in the shoot meristem. We find that AUX1 and its relatives are likely to play a crucial role in maintaining high auxin levels in the meristem epidermis. We also show that auxin influx carriers may play an important role in stabilizing auxin distribution patterns generated by auxin-gradient type models for phyllotaxis.     

VARICOSE, a WD-domain protein, is required for leaf blade development

To gain insight into the processes controlling leaf development, we characterized an Arabidopsis mutant, varicose (vcs), with leaf and shoot apical meristem defects. The vcs phenotype is temperature dependent; low temperature growth largely suppressed defects, whereas high growth temperatures resulted in severe leaf and meristem defects. VCS encodes a putative WD-domain containing protein, suggesting a function involving protein-protein interactions. Temperature shift experiments indicated that VCS is required throughout leaf development, but normal secondary vein patterning required low temperature early in leaf development. The low-temperature vcs phenotype is enhanced in axr1-3 vcs double mutants and in vcs mutants grown in the presence of polar auxin transport inhibitors, however, vcs has apparently normal auxin responses. Taken together, these observations suggest a role for VCS in leaf blade formation.     

Simulation of organ patterning on the floral meristem using a polar auxin transport model

An intriguing phenomenon in plant development is the timing and positioning of lateral organ initiation, which is a fundamental aspect of plant architecture. Although important progress has been made in elucidating the role of auxin transport in the vegetative shoot to explain the phyllotaxis of leaf formation in a spiral fashion, a model study of the role of auxin transport in whorled organ patterning in the expanding floral meristem is not available yet. We present an initial simulation approach to study the mechanisms that are expected to play an important role. Starting point is a confocal imaging study of Arabidopsis floral meristems at consecutive time points during flower development. These images reveal auxin accumulation patterns at the positions of the organs, which strongly suggests that the role of auxin in the floral meristem is similar to the role it plays in the shoot apical meristem. This is the basis for a simulation study of auxin transport through a growing floral meristem, which may answer the question whether auxin transport can in itself be responsible for the typical whorled floral pattern. We combined a cellular growth model for the meristem with a polar auxin transport model. The model predicts that sepals are initiated by auxin maxima arising early during meristem outgrowth. These form a pre-pattern relative to which a series of smaller auxin maxima are positioned, which partially overlap with the anlagen of petals, stamens, and carpels. We adjusted the model parameters corresponding to properties of floral mutants and found that the model predictions agree with the observed mutant patterns. The predicted timing of the primordia outgrowth and the timing and positioning of the sepal primordia show remarkable similarities with a developing flower in nature.     

Model for the role of auxin polar transport in patterning of the leaf adaxial–abaxial axis

Leaf adaxial–abaxial polarity refers to the two leaf faces, which have different types of cells performing distinct biological functions. In 1951, Ian Sussex reported that when an incipient leaf primordium was surgically isolated by an incision across the vegetative shoot apical meristem (SAM), a radialized structure without an adaxial domain would form. This led to the proposal that a signal, now called the Sussex signal, is transported from the SAM to emerging primordia to direct leaf adaxial–abaxial patterning. It was recently proposed that instead of the Sussex signal, polar transport of the plant hormone auxin is critical in leaf polarity formation. However, how auxin polar transport functions in the process is unknown. Through live imaging, we established a profile of auxin polar transport in and around young leaf primordia. Here we show that auxin polar transport in lateral regions of an incipient primordium forms auxin convergence points. We demonstrated that blocking auxin polar transport in the lateral regions of the incipient primordium by incisions abolished the auxin convergence points and caused abaxialized leaves to form. The lateral incisions also blocked the formation of leaf middle domain and margins and disrupted expression of the middle domain/margin‐associated marker gene WUSCHEL‐RELATED HOMEOBOX 1 (SlWOX1). Based on these results we propose that the auxin convergence points are required for the formation of leaf middle domain and margins, and the functional middle domain and margins ensure leaf adaxial–abaxial polarity. How middle domain and margins function in the process is discussed.