Leaf Vein Patterning.

Leaves form veins whose patterns vary from a single vein running the length of the leaf to networks of staggering complexity where huge numbers of veins connect to other veins at both ends. For the longest time, vein formation was thought to be controlled only by the polar, cell-to-cell transport of the plant hormone auxin; recent evidence suggests that is not so. Instead, it turns out that vein patterning features are best accounted for by a combination of polar auxin transport, facilitated auxin diffusion through plasmodesmata intercellular channels, and auxin signal transduction-though the latter's precise contribution remains unclear. Equally unclear remain the sites of auxin production during leaf development, on which that vein patterning mechanism ought to depend. Finally, whether that vein patterning mechanism can account for the variety of vein arrangements found in nature remains unknown. Addressing those questions will be the exciting challenge of future research. Expected final online publication date for the Annual Review of Plant Biology, Volume 75 is May 2024. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.

Axes and polarities in leaf vein formation.

For multicellular organisms to develop, cells must grow, divide, and differentiate along preferential or exclusive orientations or directions. Moreover, those orientations, or axes, and directions, or polarities, must be coordinated between cells within and between tissues. Therefore, how axes and polarities are coordinated between cells is a key question in biology. In animals, such coordination mainly depends on cell migration and direct interaction between proteins protruding from the plasma membrane. Both cell movements and direct cell-cell interactions are prevented in plants by cell walls that surround plant cells and keep them apart and in place. Therefore, plants have evolved unique mechanisms to coordinate their cell axes and polarities. Here I will discuss evidence suggesting that understanding how leaf veins form may uncover those unique mechanisms. Indeed, unlike previously thought, the cell-to-cell polar transport of the plant hormone auxin along developing veins cannot account for many features of vein patterning. Instead, those features can be accounted for by models of vein patterning that combine polar auxin transport with auxin diffusion through plasmodesmata along the axis of developing veins. Though it remains unclear whether such a combination of polar transport and axial diffusion of auxin can account for the formation of the variety of vein patterns found in plant leaves, evidence suggests that such a combined mechanism may control plant developmental processes beyond vein patterning.

Correction: Leaf vein patterning is regulated by the aperture of plasmodesmata intercellular channels.

[This corrects the article DOI: 10.1371/journal.pbio.3001781.].

Leaf vein patterning is regulated by the aperture of plasmodesmata intercellular channels.

To form tissue networks, animal cells migrate and interact through proteins protruding from their plasma membranes. Plant cells can do neither, yet plants form vein networks. How plants do so is unclear, but veins are thought to form by the coordinated action of the polar transport and signal transduction of the plant hormone auxin. However, plants inhibited in both pathways still form veins. Patterning of vascular cells into veins is instead prevented in mutants lacking the function of the GNOM (GN) regulator of auxin transport and signaling, suggesting the existence of at least one more GN-dependent vein-patterning pathway. Here we show that in Arabidopsis such a pathway depends on the movement of auxin or an auxin-dependent signal through plasmodesmata (PDs) intercellular channels. PD permeability is high where veins are forming, lowers between veins and nonvascular tissues, but remains high between vein cells. Impaired ability to regulate PD aperture leads to defects in auxin transport and signaling, ultimately leading to vein patterning defects that are enhanced by inhibition of auxin transport or signaling. GN controls PD aperture regulation, and simultaneous inhibition of auxin signaling, auxin transport, and regulated PD aperture phenocopies null gn mutants. Therefore, veins are patterned by the coordinated action of three GN-dependent pathways: auxin signaling, polar auxin transport, and movement of auxin or an auxin-dependent signal through PDs. Such a mechanism of tissue network formation is unprecedented in multicellular organisms.

Transcriptional reprogramming during floral fate acquisition.

Coordinating growth and patterning is essential for eukaryote morphogenesis. In plants, auxin is a key regulator of morphogenesis implicated throughout development. Despite this central role, our understanding of how auxin coordinates cell fate and growth changes is still limited. Here, we addressed this question using a combination of genomic screens to delve into the transcriptional network induced by auxin at the earliest stage of flower development, prior to morphological changes. We identify a shoot-specific network suggesting that auxin initiates growth through an antagonistic regulation of growth-promoting and growth-repressive hormones, quasi-synchronously to floral fate specification. We further identify two DNA-binding One Zinc Finger (DOF) transcription factors acting in an auxin-dependent network that could interface growth and cell fate from the early stages of flower development onward.

Confocal Imaging of Developing Leaves.

Questions in developmental biology are most frequently addressed by using fluorescent markers of otherwise invisible cell states. In plants, such questions can be addressed most conveniently in leaves. Indeed, from the formation of stomata and trichomes within the leaf epidermis to that of vein networks deep into the leaf inner tissue, leaf cells and tissues differentiate anew during the development of each leaf. Moreover, leaves are produced in abundance and are easily accessible to visualization and perturbation. Yet a detailed procedure for the perturbation, dissection, mounting, and imaging of developing leaves has not been described. Here we address this limitation (1) by providing robust, step-by-step protocols for the local application of the plant hormone auxin to developing leaves and for the routine dissection and mounting of leaves and leaf primordia, and (2) by offering practical guidelines for the optimization of imaging parameters for confocal microscopy. We describe the procedure for the first leaves of Arabidopsis, but the same approach can be easily applied to other leaves of Arabidopsis or to leaves of other plants. © 2022 Wiley Periodicals LLC. Support Protocol 1: Preparation of plant growth medium Support Protocol 2: Preparation of growth medium plates Basic Protocol 1: Seed sterilization, sowing, and germination, and seedling growth Support Protocol 3: Preparation of IAA-lanolin paste Basic Protocol 2: Application of IAA-lanolin paste to 3.5-DAG first leaves Basic Protocol 3: Dissection of 3- to 6-DAG first leaves and leaf primordia Basic Protocol 4: Dissection of 1- and 2-DAG first-leaf primordia Basic Protocol 5: Mounting of dissected leaves and leaf primordia Support Protocol 4: Quality check of mounted leaves and leaf primordia by fluorescence microscopy Basic Protocol 6: Imaging of mounted leaves and leaf primordia by confocal microscopy.

Control of vein-forming, striped gene expression by auxin signaling.

BACKGROUND: Activation of gene expression in striped domains is a key building block of biological patterning, from the recursive formation of veins in plant leaves to that of ribs and vertebrae in our bodies. In animals, gene expression is activated in striped domains by the differential affinity of broadly expressed transcription factors for their target genes and the combinatorial interaction between such target genes. In plants, how gene expression is activated in striped domains is instead unknown. We address this question for the broadly expressed MONOPTEROS (MP) transcription factor and its target gene ARABIDOPSIS THALIANA HOMEOBOX FACTOR8 (ATHB8). RESULTS: We find that ATHB8 promotes vein formation and that such vein-forming function depends on both levels of ATHB8 expression and width of ATHB8 expression domains. We further find that ATHB8 expression is activated in striped domains by a combination of (1) activation of ATHB8 expression through binding of peak levels of MP to a low-affinity MP-binding site in the ATHB8 promoter and (2) repression of ATHB8 expression by MP target genes of the AUXIN/INDOLE-3-ACETIC-ACID-INDUCIBLE family. CONCLUSIONS: Our findings suggest that a common regulatory logic controls activation of gene expression in striped domains in both plants and animals despite the independent evolution of their multicellularity.

Of Cells, Strands, and Networks: Auxin and the Patterned Formation of the Vascular System.

Throughout plant development, vascular cells continually form from within a population of seemingly equivalent cells. Vascular cells connect end to end to form continuous strands, and vascular strands connect at both or either end to form networks of exquisite complexity and mesmerizing beauty. Here we argue that experimental evidence gained over the past few decades implicates the plant hormone auxin-its production, transport, perception, and response-in all the steps that lead to the patterned formation of the plant vascular system, from the formation of vascular cells to their connection into vascular networks. We emphasize the organizing principles of the cell- and tissue-patterning process, rather than its molecular subtleties. In the picture that emerges, cells compete for an auxin-dependent, cell-polarizing signal; positive feedback between cell polarization and cell-to-cell movement of the polarizing signal leads to gradual selection of cell files; and selected cell files differentiate into vascular strands that drain the polarizing signal from the neighboring cells. Although the logic of the patterning process has become increasingly clear, the molecular details remain blurry; the future challenge will be to bring them into razor-sharp focus.

Arabidopsis NHX5 and NHX6 regulate PIN6-mediated auxin homeostasis and growth.

NHX5 and NHX6, endosomal Na+,K+/H+ antiporters in Arabidopsis thaliana, play a vital role in growth and development. Our previous study has shown that NHX5 and NHX6 function as H+ leak to regulate auxin-mediated growth in Arabidopsis. In this report, we investigated the function of NHX5 and NHX6 in controlling PIN6-mediated auxin homeostasis and growth in Arabidopsis. Phenotypic analyses found that NHX5 and NHX6 were critical for the function of PIN6, an auxin transporter. We further showed that PIN6 depended on NHX5 and NHX6 in regulating auxin homeostasis. NHX5 and NHX6 were colocalized with PIN6, but they did not interact physically. The conserved acidic residues that are vital for the activity of NHX5 and NHX6 were critical for PIN6 function. Together, NHX5 and NHX6 may regulate PIN6 function by their transport activity.

Local auxin biosynthesis is required for root regeneration after wounding.

The root meristem can regenerate following removal of its stem-cell niche by recruitment of remnant cells from the stump. Regeneration is initiated by rapid accumulation of auxin near the injury site but the source of this auxin is unknown. Here, we show that auxin accumulation arises from the activity of multiple auxin biosynthetic sources that are newly specified near the cut site and that their continuous activity is required for the regeneration process. Auxin synthesis is highly localized while PIN-mediated transport is dispensable for auxin accumulation and tip regeneration. Roots lacking the activity of the regeneration competence factor ERF115, or that are dissected at a zone of low regeneration potential, fail to activate local auxin sources. Remarkably, restoring auxin supply is sufficient to confer regeneration capacity to these recalcitrant tissues. We suggest that regeneration competence relies on the ability to specify new local auxin sources in a precise temporal pattern.

Vein patterning by tissue-specific auxin transport.

Unlike in animals, in plants, vein patterning does not rely on direct cell-cell interaction and cell migration; instead, it depends on the transport of the plant hormone auxin, which in turn depends on the activity of the PIN-FORMED1 (PIN1) auxin transporter. The current hypotheses of vein patterning by auxin transport propose that, in the epidermis of the developing leaf, PIN1-mediated auxin transport converges to peaks of auxin level. From those convergence points of epidermal PIN1 polarity, auxin would be transported in the inner tissues where it would give rise to major veins. Here, we have tested predictions of this hypothesis and have found them unsupported: epidermal PIN1 expression is neither required nor sufficient for auxin transport-dependent vein patterning, whereas inner-tissue PIN1 expression turns out to be both required and sufficient for auxin transport-dependent vein patterning. Our results refute all vein patterning hypotheses based on auxin transport from the epidermis and suggest alternatives for future tests.

The canalization hypothesis - challenges and alternatives.

The 'canalization hypothesis' was suggested 50 years ago by Tsvi Sachs to account for the formation of vascular strands in response to wounding or auxin application. The hypothesis proposes that positive feedback between auxin movement through a cell and the cell's auxin conductivity leads to the gradual selection of narrow 'canals' of polar auxin transport that will differentiate into vascular strands. Though the hypothesis has provided an invaluable conceptual framework to understand the patterned formation of vascular strands, evidence has been accumulating that seems to be incompatible with the hypothesis. We suggest that the challenging evidence is incompatible with current interpretations of the hypothesis but not with the concept at the core of the hypothesis' original formulation.

GAL4/GFP enhancer-trap lines for identification and manipulation of cells and tissues in developing Arabidopsis leaves.

BACKGROUND: Understanding developmental processes requires the unambiguous identification of cells and tissues, and the selective manipulation of the properties of those cells and tissues. Both requirements can most efficiently be satisfied through the use of GAL4/GFP enhancer-trap lines. No such lines, however, have been characterized for the study of early leaf development in the Columbia-0 reference genotype of Arabidopsis. RESULTS: Here we address this limitation by identifying and characterizing a set of GAL4/GFP enhancer-trap lines in the Columbia-0 background for the specific labeling of cells and tissues during early leaf development, and for the targeted expression of genes of interest in those cells and tissues. CONCLUSIONS: By using one line in our set to address outstanding questions in leaf vein patterning, we show that these lines can be used to address key questions in plant developmental biology.

A coherent feed-forward loop drives vascular regeneration in damaged aerial organs of plants growing in a normal developmental context.

Aerial organs of plants, being highly prone to local injuries, require tissue restoration to ensure their survival. However, knowledge of the underlying mechanism is sparse. In this study, we mimicked natural injuries in growing leaves and stems to study the reunion between mechanically disconnected tissues. We show that PLETHORA (PLT) and AINTEGUMENTA (ANT) genes, which encode stem cell-promoting factors, are activated and contribute to vascular regeneration in response to these injuries. PLT proteins bind to and activate the CUC2 promoter. PLT proteins and CUC2 regulate the transcription of the local auxin biosynthesis gene YUC4 in a coherent feed-forward loop, and this process is necessary to drive vascular regeneration. In the absence of this PLT-mediated regeneration response, leaf ground tissue cells can neither acquire the early vascular identity marker ATHB8, nor properly polarise auxin transporters to specify new venation paths. The PLT-CUC2 module is required for vascular regeneration, but is dispensable for midvein formation in leaves. We reveal the mechanisms of vascular regeneration in plants and distinguish between the wound-repair ability of the tissue and its formation during normal development.

Coordination of tissue cell polarity by auxin transport and signaling.

Plants coordinate the polarity of hundreds of cells during vein formation, but how they do so is unclear. The prevailing hypothesis proposes that GNOM, a regulator of membrane trafficking, positions PIN-FORMED auxin transporters to the correct side of the plasma membrane; the resulting cell-to-cell, polar transport of auxin would coordinate tissue cell polarity and induce vein formation. Contrary to predictions of the hypothesis, we find that vein formation occurs in the absence of PIN-FORMED or any other intercellular auxin-transporter; that the residual auxin-transport-independent vein-patterning activity relies on auxin signaling; and that a GNOM-dependent signal acts upstream of both auxin transport and signaling to coordinate tissue cell polarity and induce vein formation. Our results reveal synergism between auxin transport and signaling, and their unsuspected control by GNOM in the coordination of tissue cell polarity during vein patterning, one of the most informative expressions of tissue cell polarization in plants.

Coordination of cell polarity and the patterning of leaf vein networks.

During development, the behavior of cells in tissues is coordinated along specific orientations or directions by coordinating the polar localization of components in those cells. The coordination of such cell polarity is perhaps nowhere more spectacular than in developing leaves, where the polarity of hundreds of cells is coordinated in the leaf epidermis and inner tissue to pattern vein networks. Available evidence suggests that the spectacular coordination of cell polarity that patterns vein networks is controlled by auxin transport and levels, and by genes that have been implicated in the polar localization of auxin transporters.

Reassessing the Roles of PIN Proteins and Anticlinal Microtubules during Pavement Cell Morphogenesis.

The leaf epidermis is a biomechanical shell that influences the size and shape of the organ. Its morphogenesis is a multiscale process in which nanometer-scale cytoskeletal protein complexes, individual cells, and groups of cells pattern growth and define macroscopic leaf traits. Interdigitated growth of neighboring cells is an evolutionarily conserved developmental strategy. Understanding how signaling pathways and cytoskeletal proteins pattern cell walls during this form of tissue morphogenesis is an important research challenge. The cellular and molecular control of a lobed cell morphology is currently thought to involve PIN-FORMED (PIN)-type plasma membrane efflux carriers that generate subcellular auxin gradients. Auxin gradients were proposed to function across cell boundaries to encode stable offset patterns of cortical microtubules and actin filaments between adjacent cells. Many models suggest that long-lived microtubules along the anticlinal cell wall generate local cell wall heterogeneities that restrict local growth and specify the timing and location of lobe formation. Here, we used Arabidopsis (Arabidopsis thaliana) reverse genetics and multivariate long-term time-lapse imaging to test current cell shape control models. We found that neither PIN proteins nor long-lived microtubules along the anticlinal wall predict the patterns of lobe formation. In fields of lobing cells, anticlinal microtubules are not correlated with cell shape and are unstable at the time scales of cell expansion. Our analyses indicate that anticlinal microtubules have multiple functions in pavement cells and that lobe initiation is likely controlled by complex interactions among cell geometry, cell wall stress patterns, and transient microtubule networks that span the anticlinal and periclinal walls.

The logic of plant vascular patterning. Polarity, continuity and plasticity in the formation of the veins and of their networks.

The problem of long-distance transport is solved in many multicellular organisms by tissue networks such as the vascular networks of plants. Because tissue networks transport from one tissue area to another, they are polar and continuous; most of them, including plant vascular networks, are also plastic. Surprisingly, the formation of tissue networks is in most cases just as polar, continuous and plastic. Available evidence suggests that the polarity, continuity and plasticity of plant vascular networks and their formation could be accounted for by a patterning process that combines: (i) excess of developmental alternatives competing for a limiting cell-polarizing signal; (ii) positive feedback between cell polarization and continuous, cell-to-cell transport of the cell-polarizing signal; and (iii) gradual restriction of differentiation that increasingly removes the cell-polarizing signal.

Connective Auxin Transport in the Shoot Facilitates Communication between Shoot Apices.

The bulk polar movement of the plant signaling molecule auxin through the stem is a long-recognized but poorly understood phenomenon. Here we show that the highly polar, high conductance polar auxin transport stream (PATS) is only part of a multimodal auxin transport network in the stem. The dynamics of auxin movement through stems are inconsistent with a single polar transport regime and instead suggest widespread low conductance, less polar auxin transport in the stem, which we term connective auxin transport (CAT). The bidirectional movement of auxin between the PATS and the surrounding tissues, mediated by CAT, can explain the complex auxin transport kinetics we observe. We show that the auxin efflux carriers PIN3, PIN4, and PIN7 are major contributors to this auxin transport connectivity and that their activity is important for communication between shoot apices in the regulation of shoot branching. We propose that the PATS provides a long-range, consolidated stream of information throughout the plant, while CAT acts locally, allowing tissues to modulate and be modulated by information in the PATS.