Plant Cuttings

Taxodium, a gymnosperm tree, here resplendent in its autumn colour, is one of Nature’s marvels. Built from a modicum of cell types and tissues, it represents a self-supporting and -repairing living structure that dwarfs the adjacent human-constructed Museum No. 1 [Ed. – as it was known before it was converted into a café…] at RBG Kew (London, UK) – in both stature and grandeur.

Chapter 3 considers the remarkable economy of design and structure that plants embody. All done with just three ‘tissue systems’ – dermal (covering the external surface of the plant this provides the important interface between plant and planet), vascular (the long-distance transport pathway for water, food, and other materials that support and fuel the plant), and ground (which embraces all of the cells between the other two systems). Nevertheless, as few as they are, this is sufficient to produce the tallest living structures on the planet, and diverse enough to provide for many of humanity’s material needs via cell types such as fibres. The utility of plant cells is showcased by a consideration of the great variety of fibres that plants produce and which people have exploited over millennia [Ed. – which section is not yet completed…].

It is a sobering thought that all of the tremendous external variation and diversity in form (morphology) – size, shape, colour, arrangement of parts – in the approx. 369,400 different species of flowering plants (which group represents, surely unarguably, the pinnacle of plant evolution) is achieved with a remarkable economy of internal structure (anatomy). Ultimately, higher plants are comprised of only three so-called tissue systems – Dermal (since this in most cases is just the epidermis, some sources refer to this tissue system simply as the epidermis), Vascular, and Ground (Melissa Ha et al.; Regina Bailey) – which perform all the essential life-sustaining functions.

[Ed. – you could reduce plant complexity even further, to just two organ systems – above-ground (‘shoot’), and below-ground root (MJ Farabee) – but that’s a reduction too far…]

And those tissue systems are in turn comprised of a comparatively modest number of different cell types (approx. 24 are ‘named’ in Chapter 24’s Summary table in Ray Evert & Susan Eichhorn, 2013); and >40 different cell types are listed by Alison Roberts [Ed. – although this sort of ‘stamp-collecting’ approach to cell types may be a little redundant for plants if we acknowledge that any cell with a nucleus can potentially differentiate into any cell type [the phenomenon of ‘totipotency’ (Srinibas Kum; Ying Hua Su et al., 2021)]; in other words, there is only one cell type, but it can be present in different cell states (Byron Rusnak et al., 2024…].

Human beings (which may be regarded as the apex of animal evolution), have 9, 10 (Jonny Docks), 11 (Carol Guze), or as many as 12 [Ed. – the consensus appears to be eleven (J Gordon Betts et al.; Rod Brouhard; Jana Vasković)] organ systems (made from a group of different organs, which all work together to do a particular job) – which comprise up to 411 cell types (Matthew Vickaryous & Brian Hall, 2006) (and maybe up to 10,000 (Andreas Bueckle et al., 2026).

In other words, compared to humans, plants are marvellously minimalist models of economical design and constrained construction.

Dermal, Vascular, and Ground Tissue Systems

These are the basic elements that make up the plant body.

The dermal system – the ‘epidermis’ – covers the exterior of the plant – both above-and below-ground (Beverley J Glover et al., 2016). For a tissue that is frequently only one cell thick it performs several remarkably important roles in the life of the plant: “The epidermis is the protecting layer of shoots, roots, leaves, flowers, and seeds. The protective role is against pathogens and mechanical insults. Other essential functions of the epidermis include regulation of transpiration or water loss, gas exchange, storage, secretion, repelling herbivores, attracting pollinating insects, absorption of water in the root, keeping the integrity of some organs, and protection against solar radiation” (quoted from here). Additionally, the epidermis forms a protective layer for the cells lying underneath, keeps the inner layers of cells in place, and helps maintain the internal temperature.

In some instances that protection is provided by different cell types found within the epidermis, in particular various hair-like structures (known technically as trichomes (Anu Sharma; Prateeksha L), or by a layer of waxy material – cuticle (Douglas Wilkin & Jean Brainard) – that covers the epidermis on above-ground parts of the plant. The cuticle helps prevent water loss, abrasions, infections, and damage from toxins (Douglas Wilkin & Jean Brainard). Absence of a cuticle from the below-ground parts of the plant, the root in particular, helps another extremely important role of the epidermis in water uptake. This is achieved primarily by specialist cells of the root epidermis known as root hairs (Anu Sharma; Prateeksha L), through which the plant absorbs water and dissolved minerals from the external environment, usually the soil.

Mr P Cuttings’ personal favourite amongst epidermal cell types is the bulliform cell (Prateeksha L). Where present – and only a few plant groups such as grasses (Ibrahim Khan et al., 2026) have them – this cell type is found in the epidermis of the upper leaf surface. Usually present as a cluster of cells they bring about closure or rolling inwards of the leaf when they lose water, e.g., when water is scarce in the environment. When they regain water, the leaf unfolds or unrolls. Such leaf closure is a method of reducing the surface area exposed to the atmosphere and is regarded as a water-saving mechanism that minimises uncontrolled water loss – technically known as transpiration (Melisa Petruzzello) – from both stomata (Srinibas Kum) and through the cuticle of the epidermis directly (Bristy Shrestha).

Having mentioned transpiration above, it would be rather remiss of us not to mention one of the most important of epidermal cell types, the stomata (Anu Sharma). These cells – or rather the opening that’s created when the two guard cells are ‘pumped-up’ with water – are the major routes for exchange of gases between the plant and the atmosphere. For example, gases that leave the plant – e.g., oxygen in photosynthesis – or enter the plant – e.g., carbon dioxide in photosynthesis. Stomata are crucial to the ability of the plant to feed itself and produce the sugars and other organic compounds essential to plant growth and development. They are also the main route by which water leaves the plant in the phenomenon known as transpiration (Srinibas Kum; Bristy Shrestha). [Ed. – stomata are also a route by which harmful microbes gain entry to the inside of the plant (Sheilagh Molloy, 2006; Maeli Melotto et al., 2008; Weiqing Zeng et al., 2010), which is not such a good thing. But, that’s another story, for another place.]

All-in-all, the epidermis is a multi-functioning plant tissue par excellence.

The vascular system itself comprises two tissues which are specialised for long-distance transport – of water and dissolved materials (such as nutrients), the xylem (Alexander A Myburg et al. 2013) – and of photosynthate (foodstuffs derived from photosynthesis), the phloem (Alexander Schulz & Gary A Thompson, 2009). Development, and function, of such long-distance transport conduits – the xylem vessels and phloem sieve tubes in angiosperms (Anupama Sapkota) – which can extend in unbroken chains from near the apices of the roots to near the tips of the shoot and to the ends of even the longest branches – is assisted by their intimate association with a range of other cell types (Soumendra Kui). These additional cells provide important essential services such as: strengthening and support to the long vascular strands – the province of xylem (Alok Kumar Singh) and phloem fibres; storage – within xylem and phloem (Alok Kumar Singh) by parenchyma cells; and assistance in the loading and unloading of materials from the vessels and sieve tubes – parenchyma cells (in particular the companion cells of the phloem’s sieve tubes (Ji-Yun Kim & Wolf B Frommer, 2023).

Despite its rather unimpressive name, the ground tissue system (Paul E Berry) is also involved in extremely important roles within the plant. One of its most important components is the multifunctional parenchyma (Peter von Sengbusch, Undated a; Michael Pruyn & Rachel Spicer, 2012), one of the most underappreciated of all plant cell types. And, arguably, one of the most important services its constituent cells provides is photosynthesis. Photosynthesis primarily takes place in the chloroplast-bearing parenchyma cells that comprise the mesophyll – the middle layers – of the leaf (Peter von Sengbusch, Undated b), sandwiched between the outer epidermides [Ed. – although this word looks ‘odd’, it is the technically correct plural form for more than one epidermis].

Ground tissues also include the important strengthening and supportive cells of the collenchyma (Peter von Sengbusch, Undated c; Olivier Leroux, 2012) and sclerenchyma (Peter von Sengbusch, Undated d; Michael C Jarvis, 2012) – the latter of which is variously comprised of long fibres (Peter von Sengbusch, Undated e) and more irregularly-shaped sclereids (Peter von Sengbusch, Undated f). Fibres may be grouped together as stand-alone strengthening and supportive tissues – e.g., the bundles of fibres situated between the highly sought after food sources within the sieve tubes and the epidermis of the shoot in the stem of sunflower, or intermixed with other cells as in the xylem and phloem tissues within the vascular system.

The ground tissue system is alternatively known as the fundamental tissue. That name seems far more appropriate for this group of cells when we consider that, amongst its many roles, it participates in such fundamental processes as: photosynthesis; storage of nutrients and water; mechanical support; aeration [in aquatic plants, large air-filled parenchyma called aerenchyma provide buoyancy and facilitate gas exchange]; healing and regeneration; and formation of protective layers. An impressive catalogue of roles that are pretty fundamental to continued growth, development and life of the plant.

Mix-and-match

And those three tissue systems are all you need to construct the major non-reproductive organs of a flowering plant. Thus, leaves, roots and stems are all constructed from dermal, ground and vascular systems, differing principally in the arrangement of the vascular tissues.

Hence, roots tend to have a central column of vascular material surrounded by the ground tissue (primarily of parenchyma cells) with the epidermis to the outside of this organ (Nancy Kerk & Ian Sussex, 2012).

Stems tend to have bundles of phloem and xylem dispersed within the ground tissue of this organ, seemingly in a haphazard arrangement as in monocots (e.g., as seen in maize), or arranged in a circle towards the outside of the organ – as for dicots such as sunflower.

And leaves have modified this arrangement whereby the mesophyll of the flattened leaf-blade is permeated by strands of vascular tissue that frequently join up via cross-veins. In that way the more linear leaves of monocots appear to have parallel lines of major veins (as vascular strands are also known) running from their base towards the tip. Adjacent long, parallel major veins are connected to each other via cross-veins – rather like the rungs on a ladder (Andrew Hudson & Christopher Jeffree, 2001). Dicot leaves tend to have a much more net-like (or reticulate) arrangement of interconnecting major and minor veins extending throughout the often more equidimensional leaf blade.

And other organs such as floral structures – which are considered to be derived from highly modified leaves (Gordon Fraser Barclay, 2015) set upon a very short stem – are constructed using the same three basic components. Such simplicity and similarity of internal structure enables each organ to do what it needs to do; the physical continuity between organs is maintained via the all-permeating vascular system, transport of whose component compounds helps to co-ordinate and integrate growth and development of the whole organism.

Three tissue systems, a mere handful of basic building blocks, that generate seemingly infinite diversity and elegance.

Primary and secondary growth

Those three multi-cellular tissue systems are produced by the activity of meristems located principally at the tips of roots and shoots. Within these apical meristems the plant equivalents of animal’s stem cells (Robert Sablowski, 2010; Renze Heidstra & Sabrina Sabatini, 2014) divide to produce a supply of new cells to fuel growth. Those so-called daughter cells subsequently increase in size, driven in large part by internal turgor pressure created as water is taken up into the vacuole of the cell (Daniel J Cosgrove, 2014), and differentiate as they become specialised for their particular role within the plant (Martin Hülskamp & Joachim Uhrig, 2007).

However, and as impressive as the foregoing is, it only accounts for part of the growth story of plants. The activity of those apical meristems – and the co-operation of the cells of the three tissue systems mentioned – gives rise to the so-called primary plant body (which all plants have) and results in primary growth – primarily, increase in length of organs, whether vertically as for roots and shoots, or at an angle to the main axis of the plant as for side branches and lateral roots.

But many plants also undergo secondary growth, which is additional to primary growth and which results in increase in diameter of roots and shoots (J Peter Etchells & Simon Turner, 2009). All plants that grow to any appreciable size undergo secondary growth (or secondary thickening as it is alternatively called), e.g., sunflowers which only grow for one season, as do those plants that are perennial (i.e., which grow for more than two seasons), e.g., trees and shrubs. Such secondary growth is the province of laterally-situated meristems such as the vascular cambium, which gives rise to secondary xylem (the main component of a tree’s trunk and ultimately the source of timber for construction, etc.) and secondary phloem (J Peter Etchells & Simon Turner, 2009). And this radially-additive secondary growth is essential to help support the long, heavy organs such as the trunks of trees derived from primary growth.

Modular design

There is another important economy underlying plant structure, that of a modular design. Essentially, each higher plant stem is built of one or more basic units of construction, phytomers (Gerd Bossinger, 2009). A phytomer comprises a node with its attached leaf, the internode below that leaf (which extends to the next node…), and the bud at the base of the internode. The more of these phytomers there are – essentially stacked one on top of another – and the greater the degree of internode extension that exists within each phytomer, the taller the resulting plant.

This modular organisation permits great flexibility in development allowing the plant to capitalise on availability of nutrients when times are good (and grow large – Fig. 3 at beginning of this chapter), or ‘contracting’ to be small – yet perfectly formed – when environmentally constrained – e.g., bonsai plants. As a result, plants are the biggest single living entities on the planet, regularly exceeding 110 metres in height in Sequoia sempervirens – the Coast Redwood (Christopher J Earle).

And much in the same manner as the Goldilocks Principle used in the search for habitable planets beyond Earth, if plants have conditions that are neither too hot nor too cold, and not too dry or too wet, they have a great potential to carry on growing in an indeterminate manner (as compared to their more constrained, growth-limited, determinant animal counterparts). However, even then, there are limits to upwards extension, of approx. 122-130 m for Sequoia sempervirens (George Koch et al., 2004), and 100-127 m for Douglas fir – Pseudotsuga menziesii (Jean-Christophe Domec et al., 2008), constrained in large part by the limits to which plants can draw water up their stems (Clare van der Willigen; Jacob Silverman).

Human exploitation ( a short thread about fibres…)

These fundamental units of plant construction are not without their resource value. Belying such simplicity of design is the range of uses that whole plants and their constituent tissues and cells have been put in providing some of humankind’s most basic requirements – such as the need to clothe naked bodies and to provide shelter from the extremes of temperature and climate, wild beasts, and murderous fellow humans. Examples are numerous and geographically diverse as befits the ingenuity of Mankind and the diversity of plant resources at its disposal.

Take for example the various types of fibres that plants contain, which have been exploited by Man for hundreds and thousands of years in providing fabrics used in clothing and for other purposes (Sara Buscaglia; here; here; BL Hardy et al., 2020; Stephanie Celiberti). This group embraces bast fibres (fibrous bundles in the ‘inner bark’ (phloem) of plant stems – James N BeMiller, 2001), e.g., flax, hemp, jute and ramie; leaf fibres (hard or cordage fibres – James N BeMiller, 2001) such as abacá and sisal; and seed-hair ‘fibres’ (James N BeMiller, 2001), which include kapok, coir from coconut husk, and cotton (“one of the most important fibers [sic.] in the world”, and “the most widely produced natural fiber [sic.] on the planet”).

In part due to the antiquity and multiplicity of human uses of plant fibres, and the biology of their formation within the plant, there are many ways in which the different fibres can be classified.

For example, a categorisation based on their use is the following: “Textile Fibers, the most important in that they are used for fabrics, cordage and netting; Brush Fibers are stiff tough fibers including small stems and twigs that are utilized for making brooms and brushes; Rough Weaving & Plaiting Fibers, flat and pliable strands that are interlaced to make straw hats, baskets, sandals, chair seats, etc.; Filling Fibers are used for stuffing mattresses, cushions and in upholstery; for caulking seams in boats and in casks and barrels; as stiffening in plaster and as packing material; Natural Fabrics, usually obtained from tree basts that are extracted from bark in layers or sheets and pounded into rough substitutes for lace or cloth; Fibers for Paper Manufacture includes textile fibers and wood fibers that are used in either the raw or manufactured state” (Erich Fred Legner)

Whereas a classification based on fibre source might look more like: Surface fibers, which grow from the surface of seeds (cotton), leaves, or fruits (coconut coir); Soft or bast fibers, found in the phloem (inner bark) of dicotyledonous stems (flax for linen; jute; hemp; ramie); Hard or leaf fibers, from monocot leaf vascular bundles (sisal, Manila hemp, pineapple) (Barnett).

Another ‘origin-based’ categorisation is this: Fruit: cotton, kapok; Stem: flax, hemp, nettle, ramie, lotus; Nuts: coconut; Leaves: pineapple, sisal (agave), raffia, aloe vera; and Bark: jute, baobab.

Finally, we have: Seed fibres, collected from seeds or seed cases. e.g., Cotton and Kapok; Bast fibres, from the inner bark or bast surrounding the stem of the plant, e.g., Flax, Jute, Kenaf, Hemp and Ramie; Hard fibres, collected from leaves (e.g., Sisal, Banana and Agave), or from fruit (e.g., Coir around the hard shell of coconuts) (Malgorzata Zimniewska). [Ed. – for pictures of some of the fibre-producing plants – and of the fibres extracted therefrom – see the ‘plant fibers factsheet’ produced by the Institute of Natural Fibres & Medicinal Plants here]

In terms of their utility, hemp (from Cannabis sativa) has been used for centuries to make rope, canvas and paper, and a linen-like fabric used in clothing, home furnishing textiles and floor coverings. Abacá – extracted from the banana relative Musa textilis (Alison Foster) – which, although having had a long history as rigging, amongst its more modern uses are as cigarette filter papers, tea-bags and sausage skins. Although coir (from coconut, Cocos nucifera) is probably best known for the sacking, brushes, and doormats that it produces, coir peat is gaining economic importance as a mulch (Mary H Dyer), soil treatment (Bonnie L Grant) and a hydroponic growth medium. As a final exemplar, I include silk here as an honorary plant fibre because the silkworms that produce the silk are fed almost exclusively on mulberry leaves (Ryan Egglestone).

So, just considering fibres as one category of many we can appreciate how plants can remedy the three dis-eases of this review’s title: Naked? Not when swathed in textiles from natural fibres. Miserable? Not when sipping the world’s most widely consumed beverage – tea [Ed. – strictly speaking water is the most drunk ‘beverage’ (“a drink of any type”), but is it really a beverage, “liquid which is essentially designed or developed for human consumption”..?] – brewed from abacá-encased tea leaves (Arnold Grummer)! Hungry? Not when feasting on fibre-laden bamboo shoots (Stacey Colino).

Inspired by – amongst other things – the lofty achievements of botanical architecture, the field of plant biomechanics (Julian FV Vincent, 2011) looks to plants – particularly their cell walls (Lorna Gibson, 2012) – and their products as inspiration for engineering problems, etc. Furthermore, such is the strength of natural materials – wood in particular – that they are being used as inspiration for the next generation of biocomposite materials (e.g., Maya Jacob John & Sabu Thomas, 2008; Brett C Suddall, 2008; Jitendra S Tate et al., 2009; Patrick Martone et al., 2010). And, a lot of this potential ultimately relies on the fact that plants contain cell walls that have a “fiber-reinforced-composite character” (Thomas Speck & Ingo Burgert, 2011).

We are right to celebrate the versatility of the resources that plants provide, and to applaud the great size they can attain using a handful of cellular systems and the simplest of organic architectural materials.

Conclusion

This chapter reveals the remarkable economy of design and structure that plants embody, but which is sufficient to produce the tallest living structures on the planet, and diverse enough to provide for many of Man’s material needs via cell types such as fibres. Botanical ingenuity is also a major source of inspiration for solutions to human design problems with the increasingly important field of biomimetics – “the emulation of the models, systems, and elements of nature for the purpose of solving complex human problems” (Bharat Bhushan, 2009; Janine Ungvarsky). And, a very good example of biomimetics is the inspiration from hinge-like bulliform cells in developing “the midrib of the facade shading system Flectofold [Richard James MacCowan] in which the bending of its midrib controls the hoisting of its wings” (Anja Mader et al., 2020).
 

A little bit extra: A structure-function case study

In this image of marsh fox-tail grass (Alopecurus geniculatus) leaves, the arrow indicates the ligule at the junction of the blade and the sheath. The picture was taken by Christian Fischer, and is licensed under the Creative Commons Attribution-Share Alike 3.0 Unported license.

Although plants have been studied by people for millennia there is still an awful lot we don’t known about how they work. And there are still structures we recognise in plants for which we don’t know the function. Take, for example, the grass ligule – or, more specifically, the membranous grass ligule. But, we’re getting ahead of ourselves. First some important context and background…

Where our story begins…

For his MSc research at Durham University [located in the city of Durham, in the county of Durham, in the north-east of England, Michael Lee investigated the transport of radioactive amino acids along the leaf of meadow fescue (Festuca pratensis). Longish story short, he found that, in grass leaves that had been detached from the plant by cutting them where the leaf joined a node on the stem, the compounds travelled all the way from the cut base of the sheath towards the tip of the blade (Erin Garrett) in young leaves. But, in older leaves, upward transport of the amino acids was progressively restricted at the blade/sheath junction [B-SJ] [reminder, a grass leaf has two main components – the sheath, and the blade (Horace Leithead et al.)…].

Although one may ask questions about the relevance of this system to an intact grass plant, the observed ‘block’ at the B-SJ was deemed sufficiently intriguing to the Agriculture and Food Research Council [AFRC] (now part of the BBSRC [Biotechnology and Biological Sciences Research Council) for them to provide a substantial grant to study grass leaf senescence [a technical term for ageing (Matin Miryeganeh, 2021). The task of undertaking that work fell to Mr P Cuttings as a Research Assistant at Durham University, working with his PhD supervisor, Dr Alan Pearson. [Ed. – for those who of you not in the knows, in those days a Research Assistant was a member of staff who was paid to study for a PhD [Doctor of Philosophy award (Hasna Haidar; David Higginbotham)]. It really was the best of both words getting a salary whilst being a post-graduate ‘student’ and immersing oneself in a research topic for three years – happy times!].

However, and for reasons that aren’t essential to this story, we used Lolium temulentum (darnel) [for more on this fascinating grass, see Howard Thomas et al., 2016; Howard Thomas, 2019] instead of F. pratensis. As for meadow fescue, darnel exhibited the same ‘amino acid blockage’ at the blade/sheath junction. That confirmation concentrated my interest in the anatomy of the cells, tissues, and organs at that region, which, inevitably, led me to take a closer look at the ligule [which is where our story really begins].

[Ed. – although much of my time was spent looking at ligules, I never abandoned study of this ‘blockage’ and did find tyloses at the blade/sheath junction in older leaves of darnel [presence of which features were also hinted at by Lee’s MSc work on meadow fescue]. Tyloses are balloon-like extensions of parenchyma cells that poke into adjacent cells. In this instance they infiltrated the protoxylem lacunae [spaces in the vascular bundle that remain after the first-formed xylem vessels have been destroyed during development]. It was suggested that the tyloses might be removing the radioactive amino acids at this site which could explain the apparent block to further upward movement of these compounds during senescence of the darnel grass leaf [for more, see Nigel Chaffey & J Alan Pearson, 1985].

What is the grass ligule?

Almost every source will define this structure as a membrane at the junction of the blade and the sheath of the grass leaf (Lizzie Harper; Jane Mangold; Daniel Murphy).

[Ed. – other ligules exist, e.g., in sedges and rushes (Lizzie Harper), and other monocots (Paula Rudall & M Buzgo, 2002). The strap-shaped structure on the ray florets, or ray flowers, of composites (Lizzie Harper) is known as a ligule, as is a feature on the leaves of extant species of the lycophytes Selaginella and Isoetes. And ligules also refer to “bell-shaped structures within the girdle bands” of diatom frustules (Daniel Zuluaga-Astudillo et al., 2023).]

Ligules of some species may have a variety of hairs or other structures associated with their outer surface or margins (Chaffey, 1984). In some grasses the ligule may be reduced to a ring of hairs (Peter Landschoot); in others – e.g. Echinochloa crus-galli – it may be absent.

[Ed. – for reasons that I now forget, I also looked at the ligule of Agrostis gigantea, where I was surprised to find stomata-like structures in the abaxial epidermis (Chaffey, 1982). In the absence of verification that they were actually functional stomata (Regina Bailey), I erred on the side of caution and called them stomata-like, although they were only seen in association with vascular tissues in this ligule. Yes, you read correctly, some ligules have vascular tissue in them, which is continuous with the vascular tissue of the rest of the grass leaf. Which revelation means that some so-called membranous ligules are not simple ‘membranes’. I therefore use the term veined ligule to distinguish those ligules with vascular tissue – such as rice, marram, cock’s foot, Yorkshire fog, creeping bent, and black bent – from ligules that are entirely membranous – like those in darnel. For more on veined ligules, see Chaffey, 1983; 1984; 1985c [NB, the latter paper complicates matters by identifying heteroligulate species, grasses that bear both membranous and veined ligules…]

The variation in structure and appearance of their ligules has considerable value as an aid to the identification of grasses (Erin Garrett; Lizzie Harper; Jane Mangold; Fionnuala O’Neill) – particularly in the absence of flowers.

What does the ligule do?

The longest-held view of ligule function [attributed to Schlechtendahl by Ernst Hackel (page 4 in Eds A Engler & K Prantl, 1887) – the one you’ll usually find in textbooks and on the ‘net – is that it excludes water [memorably phrased as “a means to thwart water molecules from invading the sheath” (Nathan Claxton et al.)], dust and spores from entering the interior of the grass plant [e.g., Grahame Hubbard; JY Kim et al., 2017], and thereby stops the plant becoming water-logged or infected.

[Ed. – seemingly discounting that notion is Siva Chudalayandi who opines that a “more plausible explanation for ligules and auricles is that they might act as a pivot to help position the leaf blade at the correct angle to receive appropriate amounts of solar radiation”.]

To my knowledge that proposal has never been tested experimentally. Rather, it’s a notion based upon the circumstantial evidence of the ligule’s position on the grass plant.

A deep dive into darnel ligule

The ligule of darnel is not much to look at with the naked eye or even with a hand-lens – it appears to be a whitish membrane-like structure situated at the junction between blade and sheath of a leaf, that’s a couple of millimetres tall [Fig. 1]. Because the sheath of the leaf to which it’s attached is closely pressed against the enclosed leaf or culm of the plant and partially wraps around it, the ligule – as an upward vertical extension of the sheath – also encircles the enclosed leaf or culm.

Fig. 1 When the blade of darnel is bent downwards the ligule is clearly seen as a wavy, translucent structure extending vertically upwards from the sheath. This is perennial ryegrass, which was used because I can’t find my pictures of darnel ligules. This image from Harry Rose is used under the Creative Commons Attribution 2.0 Generic license.

But, when prepared, sectioned, and examined under the light microscope [LM], it’s a revelation. In longitudinal section, the surface of the ligule that is next to the enclosed leaf/culm [and which is designated the adaxial [“referring to the side of an organ facing toward the axis”], which is the enclosed leaf/stem in this case] epidermis because it faces towards the stem of the plant] is continuous with the inner [adaxial] epidermis of the sheath. The outward-facing – abaxial – epidermis of the ligule is continuous with the adaxial epidermis of the blade. In transverse section, the ligule has a three-layered [tripartite] structure: both epidermides are uniseriate [i.e. a layer that is one cell thick], and a mesophyll [so-called because it’s the middle layer of a leaf-attached organ] that’s one to a few cells wide [widest towards the base of the ligule, thinnest towards the tip and margins]. Cells of the abaxial epidermis appear quite ‘empty’ indicating a large vacuole. Cells of the mesophyll and adaxial epidermis are progressively more heavily-stained indicating an increasing amount of cytoplasmic material/decreasing volume of vacuole within them [Fig. 2] (Chaffey, 1985a).

Fig. 2 Black-and-white light micrograph of Toluidine Blue-stained transverse section of membranous ligule of Lolium perenne [so long after the work was done I can’t find one of L. temulentum…]. The main feature to note is the marked increase in staining/reduction in proportion of unstained vacuole from abaxial epidermis (ab) to chloroplast-bearing mesophyll (meso), to the densely-cytoplasmic adaxial (ad) epidermis. Image from Chaffey (2000). Note: indications of scale and size of structures for this figure will be found in the publication from which the images are sourced. I apologise for not having included an appropriate scale bar for this figure in this post.

In the transmission electron microscope [TEM] (David Joy et al.), the view is even more impressive (Chaffey, 1985a) Not only was the degree of vacuolation observed with the LM confirmed, but – and as you should expect from this instrument – important information about type and number of organelles was also revealed. The highly-vacuolate cells of the abaxial epidermis had a correspondingly-thin cytoplasm layer that contained few organelles (and no noticeable chloroplasts), and a thick cuticle over the external cell walls. The moderately-vacuolated mesophyll cells are characterised by prominent populations of chloroplasts.

[Ed. – discovery of the presence of chloroplasts in membranous ligules was a bit of a surprise. But, their presence raises the possibility that ligules may be photosynthetic organs. The narrow focus on ligule function proposed that mesophyll-produced photosynthetic products might fuel the energy-making activity of mitochondria for synthesis of the secretory product in the adaxial epidermal cells [remember the abundant plasmodesmata – channels that permit movement of material between cells – between the mesophyll and adaxial epidermis?]. However, the possibility that chloroplast-created sugars, etc. might be transported out of the ligule and may help the growth and development of the rest of the plant is also something to consider. To my knowledge that idea has never been followed-up – not even for human nutritionally-important wheat – despite evidence that photosynthesis of non-leaf plant parts can contribute to plant growth and development (Robert Henry et al., 2020). But, other questions arise if such ligules are to photosynthesise. For example, how do the chloroplasts get CO₂ through non-stomatous, thickly-cuticularised epidermis? In the absence of vascular connection with the rest of the leaf, how would any photosynthate get out from the ligule? And we don’t even know if the chloroplasts are photosynthetically active. They may look like photosynthetically-competent organelles, but are they functional? Proof that any piece of research should not only answer a question, but should raise others as well.]

The biggest surprise was the densely-cytoplasmic adaxial epidermal cells which had large numbers of mitochondria (Kara Rogers), and Golgi apparatus (or bodies) (Chris Hawes & Béatrice Satiat-Jeunemaitre, 1996; Paul Dupree & D Janine Sherrier, 1998), and abundant strands of rough endoplasmic reticulum [RER] [Fig. 3].

Fig. 3 TEM images of cells of the the adaxial epidermis of darnel ligule. Shown at left is the abundance of rough endoplasmic reticulum [RER] (the spirals of dots); on the right are several Golgi bodies and their empty-looking vesicles. Image from Chaffey (2000). Note: indications of scale and size of structures for this figure will be found in the publication from which the images are sourced. I apologise for not having included an appropriate scale bar for this figure in this post.

A cuticle was present over the outer cell walls of the adaxial epidermal cells, although this was thinner than that of the abaxial epidermis. Also of note was the abundance of plasmodesmata (Patricia Zambryski, 2008) in the walls between cells of the mesophyll and the adaxial epidermis. All of which catalogue of ultrastructure (see fig. 4) is not bad going for an organ that’s effectively written-off as a rather insignificant, small membranous structure.

Fig. 4 diagrammatic transverse section through the membranous ligule of darnel based on light and electron microscope observations. Abbreviations: abax, abaxial epidermis; adax, adaxial epidermis; ci, island of cytoplasm within cup-shaped depression of a plastid ; cl, chloroplast; cut, cuticle ; cv, coated vesicle ; d, dictyosome; dv, dictyosome-derived vesicle (not necessarily secretory); exp, extracellularly located product; mb, microbody; meso, mesophyll; mf, wall microfibril; ml, middle lamella; mt, mitochondrion; n, nucleus; nl, nucleolus; ol, oleosome; pd, plasmodesma; peri, periplasmic space; pl, plasmalemma; pmb, paramural body; po, polysome; pr, peripheral reticulum of chloroplast; pt, plastid ; rer, rough endoplasmic reticulum; ret, reticulate layer of cuticle ; st, starch; sv, secretory vesicle ; t, tonoplast; tr, transcellular strand; vac, vacuole; w, cell wall; 1, direct continuity between rough endoplasmic reticulum and cis aspect of dictyosome; 2, `smear’ of extracellularly located product over surface of cuticle ; 3, gaps in the cuticle. Image from Chaffey (2000).

As surprising as it [and it most certainly was!] was to find this degree of cellular complexity in the ligule, the puzzle now was to work out what it all meant. At first sight, it looks like this organ is photosynthetic, and has an energy-requiring synthesis of something in the abaxial layer which involves the Golgi and/or RER: But, what?

That ‘light-bulb moment’

And this is where serendipity [which I once saw defined as ‘looking for earthworms and finding gold’] comes into play. I’d read somewhere about use of optical brighteners (Dr Pete), specifically Calcofluor White to reveal cell walls in the fluorescence microscope (Toshiyuki Nagata & Itaru Takebe, 1970; J Hughes & Margaret E McCully, 1975). This sounded like a nice and quick way to view cell arrangements and numbers of cell layers in thick-sections of ligules without all the fuss and time it took to produce semi-thin sections for light microscopy. It was. Using approx. 1 mm thick discs of material removed with a razor blade just above the B-SJ showed the ligule in transverse section and held in place between the base of its own leaf-blade and the enclosed leaf/stem. An example of the information this technique can give is shown in Fig. 5. Apart from the speed of preparation, a bonus of this procedure is that it avoids cellular collapse – especially of the highly-vacuolate adaxial epidermal cells – that often accompanies the aggressive fixation, etc. necessary for light and electron microscopy, i.e. the cell shapes are much more life-like (Chaffey, 1994).

[Ed. – readers of a certain age are probably familiar with optical brighteners because they were [still are..?] added to washing powders and detergents (Dr Pete) to give that brilliant blue-white appearance to washed clothing when held up against the daylight. This occurs because the brighteners bind to cellulose fibres – found in plant-derived materials such as cotton or linen – in the clothing and emit a bright whitish fluorescent light when exposed to ultra violet wavelengths. Which is why these compounds are particularly good at ‘staining’ the cellulose-rich cell walls of plants. And allow wearers of brightener-laundered cotton clothing to ‘light-up’ in a disco under UV illumination.]

Fig. 5 Transverse section of ligule of Milium effusum with calcofluor, which nicely outlines the cells by ‘staining’ their walls [seen as white lines and curves], and shows their undeformed shapes. Note the relatively smooth outlines of the outer walls of the adaxial (ad) epidermal cells compared to the more ‘corrugated’ surface of the abaxial epidermis, which is likely to ensure a tighter seal of ligule to enclosed leaf/stem. Scale bar represents 50 μm. Image from Chaffey (1994).

The brightener worked with darnel ligules – when viewed in transverse section. However, occasionally bad cover-slip technique meant that a ligule came adrift and ended up flattened on the slide. Although that was no use for looking at number of cell layers, it showed an intriguingly-different fluorescence pattern to that seen in transverse sections. Basically, when looking at the adaxial surface of the ligule the fluorescence was confined to the area between the cell walls, i.e. the cells appeared as white fluorescing lozenges surrounded by a non-fluorescing boundary that corresponded to the position of the cell walls [Fig. 6].

Fig. 6 Adaxial surface of Lolium temulentum ligule with calcofluor. In this image the cell walls are ‘unstained’ and appear as dark boundaries to the cell contents that fluoresce white. Scale bar represents 50 μm. Image from Chaffey (2000).

What was causing that? We’ve had serendipity with the use of brighteners, now came the mini-eureka moment: The brightener appeared to be ‘staining’ the region between the outside of the cell membrane and the outer wall in the cells of the adaxial epidermis. Which area – the so-called periplasmic space (Vincent Franceschi & William Lucas, 1981; Tatiane Maria Rodrigues et al., 2011) – was a region that had accumulations of circular profiles of membranes and other more fibrous looking material when seen in the TEM [the potential significance of which observation I hadn’t appreciated before hence its disclosure at this point in the story…]. Whatever the material was it appeared to have much greater affinity for the brightener than did the cellulose, but presumably had some chemical or physical characteristics in common with that cell wall polysaccharide to bind the calcofluor.

Could the material in the periplasmic space be something that the adaxial cells were synthesizing? If so, presumably it required energy – which might explain the large numbers of mitochondria in the cells. And, presumably also required the involvement of both the numerous Golgi bodies and abundant RER. Relating the carbohydrate-processing ability of the Golgi (Paul Dupree & D Janine Sherrier, 1998), and the protein synthesis capacity of the RER, maybe the material was a glycoprotein – a molecule that has both a protein and a carbohydrate component?

A new function for the grass ligule?

Rather than the purely observational nature of the work to this point, we now had an hypothesis. Testing that hypothesis led to much more targeted work looking at the connection – both figuratively and quite literally – between the Golgi and the RER, the chemical nature of the accumulated product and a potential pathway of its synthesis suggested that what was being made was indeed a glycoprotein-like material synthesized in co-operation between Golgi and RER, and transported by vesicles to the periplasmic space [for more, see Chaffey, 1985b; 1995]. But, the material didn’t appear to remain inside the ligule. In the TEM gaps in the cuticle of the adaxial epidermal cells could be seen with what looked like the material being released through the gaps to coat the outer surface of the cuticle [Fig. 7] – i.e., at the region between the ligule and the enclosed leaf/culm.

Fig. 7 Transmission electron micrograph of extracellular product in a cuticular gap and on the surface of the cuticle of an adaxial epidermal cell of darnel ligule. Image from Chaffey (2000). Note: indications of scale and size of structures for this figure will be found in the publication from which the images are sourced. I apologise for not having included an appropriate scale bar for this figure in this post.

Since a secretory role for the ligule was rather at odds with the prevailing view of what it’s supposed to do, I was keen to get more support for this notion and chose to examine the root cap of darnel. Famously, this part of the plant produces so-called root-cap ‘slime’ (Robert Paull et al., 1975), or mucigel, or mucilage which is released from the cells and – amongst a number of roles proposed for this material (Toshihiro Watanabe et al., 2008)  – acts as a lubricant for the growth of roots through the soil. In many respects the cytochemistry, calcofluor-staining and assemblage of organelles found in the ligule’s adaxial epidermis was similar to that in the cells of the root cap of darnel (Chaffey, 1996). Which was pretty good corroboration – and some sort of precedent – for my own view of ligule function.

Not one, but two functions for the ligule?

After three years or so of reasonably intensive study, what did I conclude about membranous ligule function?

[Ed. – I could (should..?) have said right at the start of this section, if you’d like a summary of – most – of Mr P Cuttings’ thoughts on the membranous grass ligule, you could just look at Chaffey (2000), which item is freely-accessible here, rather than wade through all of the other papers mentioned above. And, should anybody be that keen to know more, his PhD thesis is available here.]

Two things mainly. First, the ligule of darnel could act passively to exclude water, dust and harmful spores – as has always been proposed. Whilst that was not novel, we now had an explanation of how this might work. When the highly-vacuolate abaxial epidermis cells are fully turgid [as they would be in life, and frequently fail to be after processing for the TEM] this helps the ligule to ‘curl’. In so doing, the outer walls of the adaxial epidermis are closely-appressed against the enclosed leaf/stem to give a pretty tight seal at that region.

But, it was apparent that the darnel ligule might also have a more active role. It looked like it was synthesising and secreting a glycoprotein-like substance to the outside of its adaxial epidermis [Fig. 8]. Because that is taking place at the blade/sheath region where the enclosing leaf is tightly pressed against the enclosed leaf, and by analogy with the root cap, it is proposed that the extracellular material might act as a lubricant that eases the exsertion of the enclosed leaf/culm.

Fig. 8 Proposed route of synthesis, elaboration and secretion of the extracellular product of the adaxial epidermal cells of the ligule of Lolium temulentum. The protein component of the putative glycoprotein is synthesised on the ribosomes and polysomes (PO) and transferred to the lumen of the rough endoplasmic reticulum (RER) (1). Thence it is transported to the importing face of the Golgi [also known as dictyosome in plants ] (D) via the direct continuity between these two organelles (2). Further processing including the addition of carbohydrate to the RER-supplied product takes place during its passage across the Golgi (3). At the exporting face of the Golgi the ‘glycoprotein’ is packaged into smooth-membraned vesicles (DV) which bud off from the Golgi (4) and are transported to the outer cell membrane, the plasmalemma (PL) (5). At the plasmalemma the vesicles fuse to form paramural bodies (PMB) within the periplasmic space (6). The synthesised material is ultimately transported across the cell wall (7) and released to the outside of the ligule through gaps in the cuticle (C) over the outer tangential wall (8). Image from Chaffey (2000).

Is evidence from one species enough? Whilst it’s sufficient to challenge the orthodox view of a purely passive ligule function, it is always good to have supporting evidence from more than one taxon. Accordingly, I looked at a range of grasses (Chaffey, 1994). Although I didn’t have time to study them as intensively as darnel, I had a look at their ultrastructure and behaviour with calcofluor. That study suggested that potentially secretory ligules are also found in seven other species: Bromus ramosus, Festuca pratensis, Lolium x hybridum, L. multiflorum, L. perenne, Elytrigia repens L. Desv. ex Nevski ssp. repens [Fig. 9], and – possibly the most interesting of the species with such ligules, because its grain provides animal feed and nutritionally supports 3 billion people worldwide – Triticum aestivum., bread wheat.

Fig. 9 Transmission electron micrographs of transverse section of the membranous ligule of Elytrigia repens ssp. repens illustrating its tripartite structure of, from top to bottom of sequence: empty-looking abaxial epidermis (with thick cuticle), chloroplast-bearing mesophyll, and the densely-cytoplasmic adaxial epidermis with fibrillar material in the periplasmic space. Image from Chaffey (2000). Note: indications of scale and size of structures for this figure will be found in the publication from which the images are sourced. I apologise for not having included an appropriate scale bar for this figure in this post.

All good things come to an end…

Sadly, that was as far as I was able to get with my own studies of this fascinating plant organ. But, this was a great project to work on and gave me an in-depth knowledge of the minutiae of some grass ligules – and it was nice to find a little gold amongst the earthworms(!).

Well, anyway, that is why I find the grass ligule so fascinating, and why I’m always a little disappointed – although, sadly, not surprised – that nobody appears to have followed-up on this work. Certainly, ligule studies continue – e.g., rice (Dindin Hidayatul Mursyidin et al., 2021), Poaceae (Bruno Edson-Chaves et al., 2023), and even in the Cypeaceae (the sedge plant family (Anton Reznicek)) (Lucas Alves-dos-Santos et al., 2023) – but nobody seems to be looking at their function.

Oh well, that’s the way it goes. Whilst we still don’t know what exactly the membranous grass ligule is doing – i.e., we still have a structure (containing a lot of ultrastructure) in search of a function – we do now know a lot more about their structure than we did. And that’s how science progresses, each investigator doing his/her bit to add to the growing knowledge base.