The maize preligule band is subdivided into distinct domains with contrasting cellular properties prior to ligule outgrowth.

The maize ligule is an epidermis-derived structure that arises from the preligule band (PLB) at a boundary between the blade and sheath. A hinge-like auricle also develops immediately distal to the ligule and contributes to blade angle. Here, we characterize the stages of PLB and early ligule development in terms of topography, cell area, division orientation, cell wall rigidity and auxin response dynamics. Differential thickening of epidermal cells and localized periclinal divisions contributed to the formation of a ridge within the PLB, which ultimately produces the ligule fringe. Patterns in cell wall rigidity were consistent with the subdivision of the PLB into two regions along a distinct line positioned at the nascent ridge. The proximal region produces the ligule, while the distal region contributes to one epidermal face of the auricles. Although the auxin transporter PIN1 accumulated in the PLB, observed differential auxin transcriptional response did not underlie the partitioning of the PLB. Our data demonstrate that two zones with contrasting cellular properties, the preligule and preauricle, are specified within the ligular region before ligule outgrowth.

Brown algal cell walls and development.

Brown algae are complex multicellular eukaryotes whose cells possess a cell wall, which is an important structure that regulates cell size and shape. Alginate and fucose-containing sulfated polysaccharides (FCSPs) are two carbohydrate types that have major roles in influencing the mechanical properties of the cell wall (i.e. increasing or decreasing wall stiffness), which in turn regulate cell expansion, division, adhesion, and other processes; however, how brown algal cell wall structure regulates its mechanical properties, and how this relationship influences cellular growth and organismal development, is not well-understood. This chapter is focused on reviewing what we currently know about how the roles of alginates and FCSPs in brown algal developmental processes, as well as how they influence the structural and mechanical properties of cell walls. Additionally, we discuss how brown algal mutants may be leveraged to learn more about the underlying mechanisms that regulate cell wall structure, mechanics, and developmental processes, and finally we propose questions to guide future research with the use of emerging technologies.

Fake news blues: A GUS staining protocol to reduce false-negative data.

The ß-glucuronidase gene, uidA (GUS), has remained a favorite reporter gene in plants since its introduction in 1987 for its stability and versatility in a variety of fluorometric, spectrophotometric, and histochemical techniques. One of the most popular uses is as a reporter gene for visualizing endogenous promoter activities within plant tissues. Despite this popularity, specific protocols for minimizing nonrepresentative staining patterns, including false negatives, in challenging tissue types are not common. This became a large issue during our work on dark-grown Arabidopsis hypocotyls, and we set out to develop a protocol that would ensure accurate staining in a tissue that is biologically resistant to reagent penetration. Through extensive testing using a variety of constitutive and endogenous promoter::GUS fusion lines, we have developed an optimized GUS staining protocol that combines the use of acetone as a fixative, deliberate physical damage, and proper positive and negative controls to help ensure accurate staining along the hypocotyl while minimizing false negatives. Hopefully, our recommendations will allow for improved staining that more accurately reflects the true activity of cloned endogenous promoters and thus facilitate a more accurate understanding of promoter activity in Arabidopsis hypocotyls and other hard-to-stain tissues.

Identification and selection of optimal reference genes for qPCR-based gene expression analysis in Fucus distichus under various abiotic stresses.

Quantitative gene expression analysis is an important tool in the scientist's belt. The identification of evenly expressed reference genes is necessary for accurate quantitative gene expression analysis, whether by traditional RT-PCR (reverse-transcription polymerase chain reaction) or by qRT-PCR (quantitative real-time PCR; qPCR). In the Stramenopiles (the major line of eukaryotes that includes brown algae) there is a noted lack of known reference genes for such studies, largely due to the absence of available molecular tools. Here we present a set of nine reference genes (Elongation Factor 1 alpha (EF1A), Elongation Factor 2 alpha (EF2A), Elongation Factor 1 beta (EF1B), 14-3-3 Protein, Ubiquitin Conjugating Enzyme (UBCE2), Glyceraldehyde-3-phosphate Dehydrogenase (GAPDH), Actin Related Protein Complex (ARP2/3), Ribosomal Protein (40s; S23), and Actin) for the brown alga Fucus distichus. These reference genes were tested on adult sporophytes across six abiotic stress conditions (desiccation, light and temperature modification, hormone addition, pollutant exposure, nutrient addition, and wounding). Suitability of these genes as reference genes was quantitatively evaluated across conditions using standard methods and the majority of the tested genes were evaluated favorably. However, we show that normalization genes should be chosen on a condition-by-condition basis. We provide a recommendation that at least two reference genes be used per experiment, a list of recommended pairs for the conditions tested here, and a procedure for identifying a suitable set for an experimenter's unique design. With the recent expansion of interest in brown algal biology and accompanied molecular tools development, the variety of experimental conditions tested here makes this study a valuable resource for future work in basic biology and understanding stress responses in the brown algal lineage.

Of puzzles and pavements: a quantitative exploration of leaf epidermal cell shape.

Epidermal cells of leaves are diverse: tabular pavement cells, trichomes, and stomatal complexes. Pavement cells from the monocot Zea mays (maize) and the eudicot Arabidopsis thaliana (Arabidopsis) have highly undulate anticlinal walls. The molecular basis for generating these undulating margins has been extensively investigated in these species. This has led to two assumptions: first, that particular plant lineages are characterized by particular pavement cell shapes; and second, that undulatory cell shapes are common enough to be model shapes. To test these assumptions, we quantified pavement cell shape in epidermides from the leaves of 278 vascular plant taxa. We found that monocot pavement cells tended to have weakly undulating margins, fern cells had strongly undulating margins, and eudicot cells showed no particular undulation degree. Cells with highly undulating margins, like those of Arabidopsis and maize, were in the minority. We also found a trend towards more undulating cell margins on abaxial leaf surfaces; and that highly elongated leaves in ferns, monocots and gymnosperms tended to have highly elongated cells. Our results reveal the diversity of pavement cell shapes, and lays the quantitative groundwork for testing hypotheses about pavement cell form and function within a phylogenetic context.

Anisotropic growth is achieved through the additive mechanical effect of material anisotropy and elastic asymmetry.

Fast directional growth is a necessity for the young seedling; after germination, it needs to quickly penetrate the soil to begin its autotrophic life. In most dicot plants, this rapid escape is due to the anisotropic elongation of the hypocotyl, the columnar organ between the root and the shoot meristems. Anisotropic growth is common in plant organs and is canonically attributed to cell wall anisotropy produced by oriented cellulose fibers. Recently, a mechanism based on asymmetric pectin-based cell wall elasticity has been proposed. Here we present a harmonizing model for anisotropic growth control in the dark-grown Arabidopsis thaliana hypocotyl: basic anisotropic information is provided by cellulose orientation) and additive anisotropic information is provided by pectin-based elastic asymmetry in the epidermis. We quantitatively show that hypocotyl elongation is anisotropic starting at germination. We present experimental evidence for pectin biochemical differences and wall mechanics providing important growth regulation in the hypocotyl. Lastly, our in silico modelling experiments indicate an additive collaboration between pectin biochemistry and cellulose orientation in promoting anisotropic growth.

Acid growth: an ongoing trip.

Since its first formulation almost 50 years ago, acid growth has had a chequered past complicated by utilization of diverse species and organs for testing alongside necessary but coarse methodology. Within the past 25 years, we have gained new insights into the molecular mechanisms behind the transduction of the signal auxin into the reality of an apoplastic pH shift as well as the effect on cell wall mechanics and the biochemical players within the wall contributing to the resultant growth. In this review, we begin by discussing the historical work and its complications, move on to the modern work and its addition to acid growth, which we finally summarize in an updated model which includes new postulations and questions.

Branched Pectic Galactan in Phloem-Sieve-Element Cell Walls: Implications for Cell Mechanics.

A major question in plant biology concerns the specification and functional differentiation of cell types. This is in the context of constraints imposed by networks of cell walls that both adhere cells and contribute to the form and function of developing organs. Here, we report the identification of a glycan epitope that is specific to phloem sieve element cell walls in several systems. A monoclonal antibody, designated LM26, binds to the cell wall of phloem sieve elements in stems of Arabidopsis (Arabidopsis thaliana), Miscanthus x giganteus, and notably sugar beet (Beta vulgaris) roots where phloem identification is an important factor for the study of phloem unloading of Suc. Using microarrays of synthetic oligosaccharides, the LM26 epitope has been identified as a ß-1,6-galactosyl substitution of ß-1,4-galactan requiring more than three backbone residues for optimized recognition. This branched galactan structure has previously been identified in garlic (Allium sativum) bulbs in which the LM26 epitope is widespread throughout most cell walls including those of phloem cells. Garlic bulb cell wall material has been used to confirm the association of the LM26 epitope with cell wall pectic rhamnogalacturonan-I polysaccharides. In the phloem tissues of grass stems, the LM26 epitope has a complementary pattern to that of the LM5 linear ß-1,4-galactan epitope, which is detected only in companion cell walls. Mechanical probing of transverse sections of M x giganteus stems and leaves by atomic force microscopy indicates that phloem sieve element cell walls have a lower indentation modulus (indicative of higher elasticity) than companion cell walls.

An Automated Confocal Micro-Extensometer Enables in Vivo Quantification of Mechanical Properties with Cellular Resolution.

How complex developmental-genetic networks are translated into organs with specific 3D shapes remains an open question. This question is particularly challenging because the elaboration of specific shapes is in essence a question of mechanics. In plants, this means how the genetic circuitry affects the cell wall. The mechanical properties of the wall and their spatial variation are the key factors controlling morphogenesis in plants. However, these properties are difficult to measure and investigating their relation to genetic regulation is particularly challenging. To measure spatial variation of mechanical properties, one must determine the deformation of a tissue in response to a known force with cellular resolution. Here, we present an automated confocal micro-extensometer (ACME), which greatly expands the scope of existing methods for measuring mechanical properties. Unlike classical extensometers, ACME is mounted on a confocal microscope and uses confocal images to compute the deformation of the tissue directly from biological markers, thus providing 3D cellular scale information and improved accuracy. Additionally, ACME is suitable for measuring the mechanical responses in live tissue. As a proof of concept, we demonstrate that the plant hormone gibberellic acid induces a spatial gradient in mechanical properties along the length of the Arabidopsis thaliana hypocotyl.

Towards an understanding of spiral patterning in the Sargassum muticum shoot apex.

In plants and parenchymatous brown algae the body arises through the activity of an apical meristem (a niche of cells or a single cell). The meristem produces lateral organs in specific patterns, referred to as phyllotaxis. In plants, two different control mechanisms have been proposed: one is position-dependent and relies on morphogen accumulation at future organ sites; the other is a lineage-based system which links phyllotaxis to the apical cell division pattern. Here we examine the apical patterning of the brown alga, Sargassum muticum, which exhibits spiral phyllotaxis (137.5° angle) and an unlinked apical cell division pattern. The Sargassum apex presents characteristics of a self-organising system, similar to plant meristems. In contrast to complex plant meristems, we were unable to correlate the plant morphogen auxin with bud positioning in Sargassum, nor could we predict cell wall softening at new bud sites. Our data suggests that in Sargassum muticum there is no connection between phyllotaxis and the apical cell division pattern indicating a position-dependent patterning mechanism may be in place. The underlying mechanisms behind the phyllotactic patterning appear to be distinct from those seen in plants.

Plant Development: Lessons from Getting It Twisted.

In plants, one of the most understated developmental phenomena is that of straightness - a root will grow down, a petal will grow flat. A new mutant in Arabidopsis thaliana that displays twisting in petals and roots, at the organ and cell level, has been investigated. Strikingly, the twisting is always left-handed and is not due to underlying cytoskeletal skewing, as is the case in other known, phenotypically similar, mutants.

Morphological Plant Modeling: Unleashing Geometric and Topological Potential within the Plant Sciences.

The geometries and topologies of leaves, flowers, roots, shoots, and their arrangements have fascinated plant biologists and mathematicians alike. As such, plant morphology is inherently mathematical in that it describes plant form and architecture with geometrical and topological techniques. Gaining an understanding of how to modify plant morphology, through molecular biology and breeding, aided by a mathematical perspective, is critical to improving agriculture, and the monitoring of ecosystems is vital to modeling a future with fewer natural resources. In this white paper, we begin with an overview in quantifying the form of plants and mathematical models of patterning in plants. We then explore the fundamental challenges that remain unanswered concerning plant morphology, from the barriers preventing the prediction of phenotype from genotype to modeling the movement of leaves in air streams. We end with a discussion concerning the education of plant morphology synthesizing biological and mathematical approaches and ways to facilitate research advances through outreach, cross-disciplinary training, and open science. Unleashing the potential of geometric and topological approaches in the plant sciences promises to transform our understanding of both plants and mathematics.

Analyzing Cell Wall Elasticity After Hormone Treatment: An Example Using Tobacco BY-2 Cells and Auxin.

Atomic force microscopy, and related nano-indentation techniques, is a valuable tool for analyzing the elastic properties of plant cell walls as they relate to changes in cell wall chemistry, changes in development, and response to hormones. Within this chapter I will describe a method for analyzing the effect of the phytohormone auxin on the cell wall elasticity of tobacco BY-2 cells. This general method may be easily altered for different experimental systems and hormones of interest.

Shifting foundations: the mechanical cell wall and development.

The cell wall has long been acknowledged as an important physical mediator of growth in plants. Recent experimental and modelling work has brought the importance of cell wall mechanics into the forefront again. These data have challenged existing dogmas that relate cell wall structure to cell/organ growth, that uncouple elasticity from extensibility, and those which treat the cell wall as a passive and non-stressed material. Within this review we describe experiments and models which have changed the ways in which we view the mechanical cell wall, leading to new hypotheses and research avenues. It has become increasingly apparent that while we often wish to simplify our systems, we now require more complex multi-scale experiments and models in order to gain further insight into growth mechanics. We are currently experiencing an exciting and challenging shift in the foundations of our understanding of cell wall mechanics in growth and development.

How to let go: pectin and plant cell adhesion.

Plant cells do not, in general, migrate. They maintain a fixed position relative to their neighbors, intimately linked through growth and differentiation. The mediator of this connection, the pectin-rich middle lamella, is deposited during cell division and maintained throughout the cell's life to protect tissue integrity. The maintenance of adhesion requires cell wall modification and is dependent on the actin cytoskeleton. There are developmental processes that require cell separation, such as organ abscission, dehiscence, and ripening. In these instances, the pectin-rich middle lamella must be actively altered to allow cell separation, a process which also requires cell wall modification. In this review, we will focus on the role of pectin and its modification in cell adhesion and separation. Recent insights gained in pectin gel mechanics will be discussed in relation to existing knowledge of pectin chemistry as it relates to cell adhesion. As a whole, we hope to begin defining the physical mechanisms behind a cells' ability to hang on, and how it lets go.

Tuning of pectin methylesterification: consequences for cell wall biomechanics and development.

MAIN CONCLUSION: Recent publications have increased our knowledge of how pectin composition and the degree of homogalacturonan methylesterification impact the biochemical and biomechanical properties of plant cell walls, plant development, and plants' interactions with their abiotic and biotic environments. Experimental observations have shown that the relationships between the DM, the pattern of de-methylesterificaton, its effect on cell wall elasticity, other biomechanical parameters, and growth are not straightforward. Working towards a detailed understanding of these relationships at single cell resolution is one of the big tasks of pectin research. Pectins are highly complex polysaccharides abundant in plant primary cell walls. New analytical and microscopy techniques are revealing the composition and mechanical properties of the cell wall and increasing our knowledge on the topic. Progress in plant physiological research supports a link between cell wall pectin modifications and plant development and interactions with the environment. Homogalacturonan pectins, which are major components of the primary cell wall, have a potential for modifications such as methylesterification, as well as an ability to form cross-linked structures with divalent cations. This contributes to changing the mechanical properties of the cell wall. This review aims to give a comprehensive overview of the pectin component homogalacturonan, including its synthesis, modification, regulation and role in the plant cell wall.

Measuring the elasticity of plant cells with atomic force microscopy.

The physical properties of biological materials impact their functions. This is most evident in plants where the cell wall contains each cell's contents and connects each cell to its neighbors irreversibly. Examining the physical properties of the plant cell wall is key to understanding how plant cells, tissues, and organs grow and gain the shapes important for their respective functions. Here, we present an atomic force microscopy-based nanoindentation method for examining the elasticity of plant cells at the subcellular, cellular, and tissue level. We describe the important areas of experimental design to be considered when planning and executing these types of experiments and provide example data as illustration.

Shrinking the hammer: micromechanical approaches to morphogenesis.

Morphogenesis, the remarkable process by which a developing organism achieves its shape, relies on the coordinated growth of cells, tissues, and organs. While the molecular and genetic basis of morphogenesis is starting to be unravelled, understanding shape changes is lagging behind. Actually, shape is imposed by the structural elements of the organism, and the translation of cellular activity into morphogenesis must go through these elements. Therefore, many methods have been developed recently to quantify, at cellular resolution, the properties of the main structural element in plants, the cell wall. As plant cell growth is restrained by the cell wall and powered by turgor pressure, such methods also address the quantification of turgor. These different micromechanical approaches are reviewed here, with a critical assessment of their strengths and weaknesses, and a discussion of how they can help us understand the regulation of growth and morphogenesis.

Mechano-chemical aspects of organ formation in Arabidopsis thaliana: the relationship between auxin and pectin.

How instructive signals are translated into robust and predictable changes in growth is a central question in developmental biology. Recently, much interest has centered on the feedback between chemical instructions and mechanical changes for pattern formation in development. In plants, the patterned arrangement of aerial organs, or phyllotaxis, is instructed by the phytohormone auxin; however, it still remains to be seen how auxin is linked, at the apex, to the biochemical and mechanical changes of the cell wall required for organ outgrowth. Here, using Atomic Force Microscopy, we demonstrate that auxin reduces tissue rigidity prior to organ outgrowth in the shoot apex of Arabidopsis thaliana, and that the de-methyl-esterification of pectin is necessary for this reduction. We further show that development of functional organs produced by pectin-mediated ectopic wall softening requires auxin signaling. Lastly, we demonstrate that coordinated localization of the auxin transport protein, PIN1, is disrupted in a naked-apex produced by increasing cell wall rigidity. Our data indicates that a feedback loop between the instructive chemical auxin and cell wall mechanics may play a crucial role in phyllotactic patterning.