Multiplexed in situ hybridization reveals distinct lineage identities for major and minor vein initiation during maize leaf development.

Leaves of flowering plants are characterized by diverse venation patterns. Patterning begins with the selection of vein-forming procambial initial cells from within the ground meristem of a developing leaf, a process which is considered to be auxin-dependent, and continues until veins are anatomically differentiated with functional xylem and phloem. At present, the mechanisms responsible for leaf venation patterning are primarily characterized in the model eudicot Arabidopsis thaliana which displays a reticulate venation network. However, evidence suggests that vein development may proceed via a different mechanism in monocot leaves where venation patterning is parallel. Here, we employed Molecular Cartography, a multiplexed in situ hybridization technique, to analyze the spatiotemporal localization of a subset of auxin-related genes and candidate regulators of vein patterning in maize leaves. We show how different combinations of auxin influx and efflux transporters are recruited during leaf and vein specification and how major and minor vein ranks develop with distinct identities. The localization of the procambial marker PIN1a and the spatial arrangement of procambial initial cells that give rise to major and minor vein ranks further suggests that vein spacing is prepatterned across the medio-lateral leaf axis prior to accumulation of the PIN1a auxin transporter. In contrast, patterning in the adaxial-abaxial axis occurs progressively, with markers of xylem and phloem gradually becoming polarized as differentiation proceeds. Collectively, our data suggest that both lineage- and position-based mechanisms may underpin vein patterning in maize leaves.

A function of spalt major as a sequence-specific DNA binding transcription factor mediates repression of knirps in the Drosophila wing imaginal disc.

The Spalt transcriptional regulators participate in a variety of cell fate decisions during multicellular development. Vertebrate Spalt proteins have been mostly associated to the organization of heterochromatic regions, but they also contribute regulatory functions through binding to A/T rich motives present in their target genes. The developmental processes in which the Drosophila spalt genes participate are well known through genetic analysis, but the mechanism by which the Spalt proteins regulate transcription are still unknown. Furthermore, despite the prominent changes in gene expression associated to mutations in the spalt genes, the specific DNA sequences they bind are unknow. Here, we analyze a DNA fragment present in the regulatory region of the knirps gene. Spalt proteins are candidate repressors of knirps expression during the formation of the venation pattern in the wing disc, and we identified a minimal conserved 30bp sequence that binds to Spalt major both in vivo and in vitro. This sequence mediates transcriptional repression in the central region of the wing blade, constituting the first confirmed case of a direct regulatory interaction between Spalt major and its target DNA in Drosophila. Interestingly, we also find similar sequences in a set of eight novel candidate Spalt target genes, pointing to a common mechanism of transcriptional repression mediated by Spalt proteins.

The WIP6 transcription factor TOO MANY LATERALS specifies vein type in C4 and C3 grass leaves.

Grass leaves are invariantly strap shaped with an elongated distal blade and a proximal sheath that wraps around the stem. Underpinning this shape is a scaffold of leaf veins, most of which extend in parallel along the proximo-distal leaf axis. Differences between species are apparent both in the vein types that develop and in the distance between veins across the medio-lateral leaf axis. A prominent engineering goal is to increase vein density in leaves of C3 photosynthesizing species to facilitate the introduction of the more efficient C4 pathway. Here, we discover that the WIP6 transcription factor TOO MANY LATERALS (TML) specifies vein rank in both maize (C4) and rice (C3). Loss-of-function tml mutations cause large lateral veins to develop in positions normally occupied by smaller intermediate veins, and TML transcript localization in wild-type leaves is consistent with a role in suppressing lateral vein development in procambial cells that form intermediate veins. Attempts to manipulate TML function in rice were unsuccessful because transgene expression was silenced, suggesting that precise TML expression is essential for shoot viability. This finding may reflect the need to prevent the inappropriate activation of downstream targets or, given that transcriptome analysis revealed altered cytokinin and auxin signaling profiles in maize tml mutants, the need to prevent local or general hormonal imbalances. Importantly, rice tml mutants display an increased occupancy of veins in the leaf, providing a step toward an anatomical chassis for C4 engineering. Collectively, a conserved mechanism of vein rank specification in grass leaves has been revealed.

Connecting emerging with existing vasculature above and below ground.

The vascular system was essential for plants to colonize land by facilitating the transport of water, nutrients, and minerals throughout the body. Our current knowledge on the molecular-genetic control of vascular tissue specification and differentiation is mostly based on studies in the Arabidopsis primary root. To what degree these regulatory mechanisms in the root meristem can be extrapolated to vascular tissue development in other organs is a question of great interest. In this review, we discuss the most recent progress on cotyledon vein formation, with a focus on polar auxin transport-dependent and -independent mechanisms. We also provide an overview of vasculature formation in postembryonic organs, namely lateral roots, which is more complex than anticipated as several tissues of the parent root must act in a spatio-temporally coordinated manner.

A genetic framework for proximal secondary vein branching in the Arabidopsis thaliana embryo.

Over time, plants have evolved flexible self-organizing patterning mechanisms to adapt tissue functionality for continuous organ growth. An example of this process is the multicellular organization of cells into a vascular network in foliar organs. An important, yet poorly understood component of this process is secondary vein branching, a mechanism employed to extend vascular tissues throughout the cotyledon surface. Here, we uncover two distinct branching mechanisms during embryogenesis by analyzing the discontinuous vein network of the double mutant cotyledon vascular pattern 2 (cvp2) cvp2-like 1 (cvl1). Similar to wild-type embryos, distal veins in cvp2 cvl1 embryos arise from the bifurcation of cell files contained in the midvein, whereas proximal branching is absent in this mutant. Restoration of this process can be achieved by increasing OCTOPUS dosage as well as by silencing RECEPTOR-LIKE PROTEIN KINASE 2 (RPK2) expression. Although RPK2-dependent rescue of cvp2 cvl1 is auxin- and CLE peptide-independent, distal branching involves polar auxin transport and follows a distinct regulatory mechanism. Our work defines a genetic network that confers plasticity to Arabidopsis embryos to spatially adapt vascular tissues to organ growth.

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.

OsARF11 Promotes Growth, Meristem, Seed, and Vein Formation during Rice Plant Development.

The plant hormone auxin acts as a mediator providing positional instructions in a range of developmental processes. Studies in Arabidopsis thaliana L. show that auxin acts in large part via activation of Auxin Response Factors (ARFs) that in turn regulate the expression of downstream genes. The rice (Oryza sativa L.) gene OsARF11 is of interest because of its expression in developing rice organs and its high sequence similarity with MONOPTEROS/ARF5, a gene with prominent roles in A. thaliana development. We have assessed the phenotype of homozygous insertion mutants in the OsARF11 gene and found that in relation to wildtype, osarf11 seedlings produced fewer and shorter roots as well as shorter and less wide leaves. Leaves developed fewer veins and larger areoles. Mature osarf11 plants had a reduced root system, fewer branches per panicle, fewer grains per panicle and fewer filled seeds. Mutants had a reduced sensitivity to auxin-mediated callus formation and inhibition of root elongation, and phenylboronic acid (PBA)-mediated inhibition of vein formation. Taken together, our results implicate OsARF11 in auxin-mediated growth of multiple organs and leaf veins. OsARF11 also appears to play a central role in the formation of lateral root, panicle branch, and grain meristems.

Auxin biosynthesis and cellular efflux act together to regulate leaf vein patterning.

Our current understanding of vein development in leaves is based on canalization of the plant hormone auxin into self-reinforcing streams which determine the sites of vascular cell differentiation. By comparison, how auxin biosynthesis affects leaf vein patterning is less well understood. Here, after observing that inhibiting polar auxin transport rescues the sparse leaf vein phenotype in auxin biosynthesis mutants, we propose that the processes of auxin biosynthesis and cellular auxin efflux work in concert during vein development. By using computational modeling, we show that localized auxin maxima are able to interact with mechanical forces generated by the morphological constraints which are imposed during early primordium development. This interaction is able to explain four fundamental characteristics of midvein morphology in a growing leaf: (i) distal cell division; (ii) coordinated cell elongation; (iii) a midvein positioned in the center of the primordium; and (iv) a midvein which is distally branched. Domains of auxin biosynthetic enzyme expression are not positioned by auxin canalization, as they are observed before auxin efflux proteins polarize. This suggests that the site-specific accumulation of auxin, as regulated by the balanced action of cellular auxin efflux and local auxin biosynthesis, is crucial for leaf vein formation.

Plant-specific Dof transcription factors VASCULAR-RELATED DOF1 and VASCULAR-RELATED DOF2 regulate vascular cell differentiation and lignin biosynthesis in Arabidopsis.

KEY MESSAGE: Plant-specific Dof transcription factors VDOF1 and VDOF2 are novel regulators of vascular cell differentiation through the course of a lifetime in Arabidopsis, with shifting their transcriptional target genes. Vascular system is one of critical tissues for vascular plants to transport low-molecular compounds, such as water, minerals, and the photosynthetic product, sucrose. Here, we report the involvement of two Dof transcription factors, named VASCULAR-RELATED DOF1 (VDOF1)/VDOF4.6 and VDOF2/VDOF1.8, in vascular cell differentiation and lignin biosynthesis in Arabidopsis. VDOF genes were expressed in vascular tissues, but the detailed expression sites were partly different between VDOF1 and VDOF2. Vein patterning and lignin analysis of VDOF overexpressors and double mutant vdof1 vdof2 suggested that VDOF1 and VDOF2 would function as negative regulators of vein formation in seedlings, and lignin deposition in inflorescence stems. Interestingly, effects of VDOF overexpression in lignin deposition were different by developmental stages of inflorescence stems, and total lignin contents were increased and decreased in VDOF1 and VDOF2 overexpressors, respectively. RNA-seq analysis of inducible VDOF overexpressors demonstrated that the genes for cell wall biosynthesis, including lignin biosynthetic genes, and the transcription factor genes related to stress response and brassinosteroid signaling were commonly affected by VDOF1 and VDOF2 overexpression. Taken together, we concluded that VDOF1 and VDOF2 are novel regulators of vascular cell differentiation through the course of a lifetime, with shifting their transcriptional target genes: in seedlings, the VDOF genes negatively regulate vein formation, while at reproductive stages, the VDOF proteins target lignin biosynthesis.

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.

The role of auxin in developmentally regulated programmed cell death in lace plant.

PREMISE: Lace plant (Aponogeton madagascariensis) leaves are remodeled via developmental programmed cell death (PCD) to produce perforations located equidistantly between longitudinal and transverse veins. Auxin has been implicated in other developmental PCD processes in plants; however, the role of auxin in perforation formation in lace plant is unknown. Here the role of auxin in developmental PCD in lace plant was studied using two auxin inhibitors N-1-naphthylphthalamic acid (NPA), an auxin transport inhibitor, and auxinole, a potent auxin antagonist. METHODS: Sterile cultures of lace plants were propagated and treated with NPA or auxinole. Leaf length, leaf width, and number of perforations were then analyzed. Vein patterning and perforation area were further examined in NPA-treated plants. Downstream PCD transduction events were investigated via spectrophotometric assays, histochemical staining, and immuno-probing. RESULTS: Lace plants treated with NPA or auxinole produced leaves with fewer perforations compared to their respective controls. Although NPA treatment was insufficient to completely alter vein patterning, NPA-treated leaves did have significantly more atypical areoles compared to control leaves. Events involved in perforation formation in lace plant leaves were altered following treatment with NPA, including anthocyanin production, reactive oxygen species (ROS) accumulation, and the release of mitochondrial cytochrome c. CONCLUSIONS: Our results indicated that inhibition of auxin signaling disrupts several downstream features of the lace plant PCD signaling cascade and results in fewer or no perforations. Therefore, we concluded that auxin signaling is important for developmentally regulated PCD in lace plant leaves.

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.

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.

Getting closer: vein density in C4 leaves.

Contents Summary 1260 I. Introduction 1260 II. Molecular and genetic mechanisms of C4 leaf venation 1262 III. Conclusions and future perspectives 1266 Acknowledgements 1266 References 1266 SUMMARY: C4 grasses are major contributors to the world's food supply. Their highly efficient method of carbon fixation is a unique adaptation that combines close vein spacing and distinct photosynthetic cell types. Despite its importance, the molecular genetic basis of C4 leaf development is still poorly understood. Here we summarize current knowledge of leaf venation and review recent progress in understanding molecular and genetic regulation of vascular patterning events in C4 plants. Evidence points to the interplay of auxin, brassinosteroids, SHORTROOT/SCARECROW and INDETERMINATE DOMAIN transcription factors. Identification and functional characterization of candidate regulators acting early in vascular development will be essential for further progress in understanding the precise regulation of these processes.

Patterning of the Drosophila L2 vein is driven by regulatory interactions between region-specific transcription factors expressed in response to Dpp signalling.

Pattern formation relies on the generation of transcriptional landscapes regulated by signalling pathways. A paradigm of epithelial patterning is the distribution of vein territories in the Drosophila wing disc. In this tissue, Decapentaplegic signalling regulates its target genes at different distances from the source of the ligand. The transformation of signalling into coherent territories of gene expression requires regulatory cross-interactions between these target genes. Here, we analyse the mechanisms generating the domain of knirps expression in the presumptive L2 vein of the wing imaginal disc. We find that knirps is regulated by four Decapentaplegic target genes encoding the transcription factors aristaless, spalt major, spalt-related and optix The expression of optix is activated by Dpp and repressed by the Spalt proteins, becoming restricted to the most anterior region of the wing blade. In turn, the expression of knirps is activated by Aristaless and repressed by Optix and the Spalt proteins. In this manner, the expression of knirps becomes restricted to those cells where Spalt levels are sufficient to repress optix, but not sufficient to repress knirps.

Localization of Arabidopsis FORKED1 to a RABA-positive compartment suggests a role in secretion.

When FORKED1 (FKD1) is mutated, asymmetric localization of PINFORMED1 (PIN1), particularly to the apical side of cells, fails to occur properly in developing veins, resulting in an open vein pattern. FKD1 encodes a protein with a Pleckstrin homology-like (PL) domain, suggesting interaction with phosphoinositides. FKD1 has been previously found to interact with an ADP ribosylation factor GTPase-activating protein (ARF-GAP) important for vein patterning, SCARFACE/VAN3 (SFC). We find that FKD1-green fluorescent protein (GFP) localizes to the plasma membrane and to punctae labeled by SFC-yellow fluorescent protein (YFP). Supporting the idea that the FKD1 PL domain recognizes phosphatidylinositol 4-phosphate [PtdIns(4)P], FKD1-GFP co-localizes with PtdIns(4)P markers, and is more cytosolic when in a background mutant for the PtdIns(4,5)P2 hydrolases CVP2 and CVL1. Both FKD1 and SFC partially co-localize with markers for the trans-Golgi network (TGN), at which endocytic and secretory pathways merge. FKD1-labeled punctae rarely co-localize with the endocytic marker FM4-64, suggesting that FKD1 is not involved primarily in the endocytic pathway. FKD1 and SFC co-localize with members of the RABA group of RAB-GTPases, which are proposed to act in the post-Golgi secretory pathway. The compartments labeled by FKD1 and SFC do not localize to membrane compartments induced by the fungal toxin brefeldin A (BFA). Collectively, our data suggest that FKD1 and SFC act in a BFA-insensitive secretory pathway.

Overexpression of an F-box protein gene disrupts cotyledon vein patterning in Arabidopsis.

Plant vascular patterning is complex. However, the detailed molecular mechanism of vascular patterning is still unknown. In this study, FBXL, an Arabidopsis F-box motif gene, was isolated by using 3' rapid amplification of cDNA ends (RACE) technique. The gene contained a coding sequence of 1407 nucleotides coding 468 amino acid residues. Amino acid sequence analysis revealed that the gene encoded a protein harboring an F-box motif at the N terminus, an LRRs motif in the middle, and an FBD motif at the C terminus. FBXL promoter-ß-glucuronidase (GUS) and 35S promoter-FBXL vectors were constructed and transformed into Arabidopsis thaliana to understand the function of the FBXL gene. GUS expression analysis indicated that FBXL was specifically expressed in the vascular tissues of the root, stem, leaf, and inflorescence. FBXL overexpression in Arabidopsis displayed an abnormal venation pattern in cotyledons. Furthermore, FBXL expression was not induced by exogenous auxin and its transcript accumulation did not overlap with the distribution of endogenous auxin. These results suggested that FBXL may be involved in cotyledon vein patterning via auxin-independent pathway.

Leaf mass per area is independent of vein length per area: avoiding pitfalls when modelling phenotypic integration (reply to Blonder et al. 2014).

It has been recently proposed that leaf vein length per area (VLA) is the major determinant of leaf mass per area ( MA), and would thereby determine other traits of the leaf economic spectrum (LES), such as photosynthetic rate per mass (A(mass)), nitrogen concentration per mass (N(mass)) and leaf lifespan (LL). In a previous paper we argued that this 'vein origin' hypothesis was supported only by a mathematical model with predestined outcomes, and that we found no support for the 'vein origin' hypothesis in our analyses of compiled data. In contrast to the 'vein origin' hypothesis, empirical evidence indicated that VLA and LMA are independent mechanistically, and VLA (among other vein traits) contributes to a higher photosynthetic rate per area (A(area)), which scales up to driving a higher A(mass), all independently of LMA, N(mass) and LL. In their reply to our paper, Blonder et al. (2014) raised questions about our analysis of their model, but did not address our main point, that the data did not support their hypothesis. In this paper we provide further analysis of an extended data set, which again robustly demonstrates the mechanistic independence of LMA from VLA, and thus does not support the 'vein origin' hypothesis. We also address the four specific points raised by Blonder et al. (2014) regarding our analyses. We additionally show how this debate provides critical guidance for improved modelling of LES traits and other networks of phenotypic traits that determine plant performance under contrasting environments.

Arabidopsis UNHINGED encodes a VPS51 homolog and reveals a role for the GARP complex in leaf shape and vein patterning.

Asymmetric localization of PIN proteins controls directionality of auxin transport and many aspects of plant development. Directionality of PIN1 within the marginal epidermis and the presumptive veins of developing leaf primordia is crucial for establishing leaf vein pattern. One mechanism that controls PIN protein distribution within the cell membranes is endocytosis and subsequent transport to the vacuole for degradation. The Arabidopsis mutant unhinged-1 (unh-1) has simpler leaf venation with distal non-meeting of the secondary veins and fewer higher order veins, a narrower leaf with prominent serrations, and reduced root and shoot growth. We identify UNH as the Arabidopsis vacuolar protein sorting 51 (VPS51) homolog, a member of the Arabidopsis Golgi-associated retrograde protein (GARP) complex, and show that UNH interacts with VPS52, another member of the complex and colocalizes with trans Golgi network and pre-vacuolar complex markers. The GARP complex in yeast and metazoans retrieves vacuolar sorting receptors to the trans-Golgi network and is important in sorting proteins for lysosomal degradation. We show that vacuolar targeting is reduced in unh-1. In the epidermal cells of unh-1 leaf margins, PIN1 expression is expanded. The unh-1 leaf phenotype is partially suppressed by pin1 and cuc2-3 mutations, supporting the idea that the phenotype results from expanded PIN1 expression in the marginal epidermis. Our results suggest that UNH is important for reducing expression of PIN1 within margin cells, possibly by targeting PIN1 to the lytic vacuole.

How do leaf veins influence the worldwide leaf economic spectrum? Review and synthesis.

Leaf vein traits are implicated in the determination of gas exchange rates and plant performance. These traits are increasingly considered as causal factors affecting the 'leaf economic spectrum' (LES), which includes the light-saturated rate of photosynthesis, dark respiration, foliar nitrogen concentration, leaf dry mass per area (LMA) and leaf longevity. This article reviews the support for two contrasting hypotheses regarding a key vein trait, vein length per unit leaf area (VLA). Recently, Blonder et al. (2011, 2013) proposed that vein traits, including VLA, can be described as the 'origin' of the LES by structurally determining LMA and leaf thickness, and thereby vein traits would predict LES traits according to specific equations. Careful re-examination of leaf anatomy, published datasets, and a newly compiled global database for diverse species did not support the 'vein origin' hypothesis, and moreover showed that the apparent power of those equations to predict LES traits arose from circularity. This review provides a 'flux trait network' hypothesis for the effects of vein traits on the LES and on plant performance, based on a synthesis of the previous literature. According to this hypothesis, VLA, while virtually independent of LMA, strongly influences hydraulic conductance, and thus stomatal conductance and photosynthetic rate. We also review (i) the specific physiological roles of VLA; (ii) the role of leaf major veins in influencing LES traits; and (iii) the role of VLA in determining photosynthetic rate per leaf dry mass and plant relative growth rate. A clear understanding of leaf vein traits provides a new perspective on plant function independently of the LES and can enhance the ability to explain and predict whole plant performance under dynamic conditions, with applications towards breeding improved crop varieties.