How to Recruit an Army

Plants primarily secrete nectar as an energy source to tempt pollinators to visit their flowers, but the secretion of this substance appears to have evolved long before flowering plants appeared. Many plants, including some ferns, secrete nectar from extrafloral nectaries - i.e. nectaries in other positions on the surface of the plant. 

Legumes, like the common vetch Vicia sativa in the image above, have extrafloral nectaries on their stipules (the small, leaf-like projections on either side of the base of a leaf stalk). The extrafloral nectary is the black spot on the image above and a closer look ....

... reveals what its function might be. Ants are famous for their attraction to sweet substances and regularly visit the plant for the sugar that leaks out of these locations. This might deliver two kinds of selective advantage to the plant that would outweigh the cost of using some of its assimilated sucrose in this way. In some plants it might deflect ants, which are usually very inefficient pollinators, away from the larger source of nectar that's there to service more efficient pollinators, like bees. In other plants it may be a way of recruiting  a defensive army of ants because they become aggressive towards herbivorous insects that might try to plunder their food supply; in Acacia trees for example, the defensive benefits of hosting ants are well documented.

Extrafloral nectaries are found in a wide variety of plants and are often located on leaf petioles and mid-ribs. This is a vertical section through an extrafloral nectary on the underside of the mid-rib of a cotton plant (Gossypium sp.), stained with fluorescent dyes. The bright yellow cells at the top are xylem vessels, conducting water to the leaf blade. The very small, brick shaped blue cells below are dividing cambial cells and also phloem sieve elements that are conducting assimilated sucrose away from the leaf blade. Below that are some larger, blue-stained parenchymatous cells and then, at the very bottom, there are thin-walled finger-shaped cells which constitute the extrafloral nectary tissue, on the lower surface of the leaf mid-rib.

The blue staining is due to cellulose in the cell walls binding to a dye called calcofluor, which then fluorescence blue in UV light. You can see from this image that there's a very thin cellulose cell wall in those finger-shaped extrafloral nectary cells, because they barely fluoresce. So they easily leak sucrose that accumulates in them. The other interesting feature of this section is the orange staining in the small cells immediately above those extra-floral nectary cells. This is the endoplasmic reticulum/ Golgi complex inside the cells - the membranes and secretory vesicles that manufacture substances and transport them between cells via channels in the cell walls called plasmodesmata; these brightly-fluorescing cells seem to be highly metabolically active, so maybe the nectary cells are secreting something else, as well as sucrose.

There are some scientific papers on cotton extrafloral nectaries, their role and how they might be exploited in biological control programmes in this crop here, here and here.

Breathing Space

This is a thin section of the lower stem of water milfoil Myriophyllum sp. , stained with the fluorochrome calcofluor which binds to the cellulose of the cell walls and is fluorescing brightly in ultraviolet light. Marsh plants tend to be rooted in anaerobic mud and so have air channels (aerenchyma) that conduct oxygen down to the roots. 


Working from the outside inwards in this section, there is a well defined single outer layer of very small cells forming the epidermis, then inside that lies the stem cortex with 17 air channels arranged around the central stele, which contains the phloem (brightest flourescence) and the xylem.

The stem and leaves of water milfoil. The small white structures in the leaf axils are the stigmas of the female flowers.

Banana Stellate Parenchyma

These beautiful cells come from the midrib of a banana leaf. Each is shaped like a 6- or 7-armed star, with its arms joined to the arms of surrounding cells, forming a lattice of cells. This form of tissue is known as stellate parenchyma and you can find another example here. The image was produced using polarised light and the brightly coloured birefringent objects inside the cells are calcium oxalate crystals inside the cell vacuole. You can see further examples of calcium oxalate crystals, including a video of their Brownian motion inside a cell, if you click here.

To find these cells you need to look inside the midrib of a banana (Musa sp.) leaf .....

by cutting transversely across the midrib, which reveals this internal pattern of strenthening tissue filled with very delicate, transverse plates of glassy cells ....

... then dissect out one of these plates of cells and mount it on a microscope slide.

Give Me Strength

This cross section of the stem of a soybean seedling shows the early stages in a developmental process that will produce a stem capable of supporting the mature plant. I stained the section with two fluorescent dyes - calcofluor, which binds to cellulose cell walls and fluorescences blue in ultraviolet light and auramine O, which binds to lignin and fluorescences yellow. It's the lignin laid down in cell walls that gives the stem the strength it will need to support the leaves and flower.

Working from the bottom left-hand corner towards top right, the core of the stem is filled with blue, thin-walled pith cells, which are simply packing tissue. Some of these have become slightly lignified and are fluorescing yellow and some, that are arranged in vertical rows of between two and five cells have distinctly thicker walls - these are xylem vessels, which are dead cells that form tubes that conduct water up the plant from the roots.

Above those lies a broad band of blue-fluorescing thin-walled cells that are very small and arranged like piles of bricks. This is the cambium - the plant's stem cells that divide continuously to produce new xylem on the inside and new phloem elements on their outer surface. The small, bright blue-fluorescing cells on the outside of the cambium are the phloem sieve tubes and associated companion cells, which conduct sugars produced by photosynthesis in the leaves to other parts of the plant.

The sinuous layer of yellow-fluorescing cells above the phloem are becoming lignified and these will contribute major structural rigidity to the stem as it grows, forming a continuous cylinder inside the stem. Outside of these lies the stem cortex, with blue cells becoming smaller in the layers just below the epidermis - and then the outer epidermis of the stem is covered in the yellow-fluorescing cuticle, which restricts water loss and defends that plant against pathogens.

At the stage when this section was taken the stem was about 3mm. in diameter and about 10 centimetres tall.

Trees: the Inside Story

Almost as soon as plants colonised the land surface they began to compete for light, struggling to grow out of each other’s mutual shade. The ultimate solution, adopted by trees,  was to produce woody stems and grow tall, shading out competitors below. It's a very successful strategy - left to their own devices, many terrestrial ecosystems where water and warmth are adequate become forests. These (above) are cross sections of stems of two sycamore Acer pseudoplatanus seedlings, just a couple of weeks after germinating from a seed in spring, and already they have begun to produce woody thickening in some of their cells, visible here as the bright yellow fluorescent staining inside the stem (on the periphery of the large pith cells in its core). The very narrow yellow fluorescent line around the perimeter of the stem is the waxy cuticle secreted by the epidermal cells that protects the young stem – just a couple of millimetres in diameter at this stage - from water loss and invasion by pathogens. Double-click on the image for a clearer picture.

Fast-forward almost three years now and this seedling has grown into a sapling. In this cross section of a three year old lime (Tilia sp.) stem the big cells at the core are the pith. The three concentric rings of brown cells outside of that contain the xylem vessels that conduct water up and down the stem. They’re dead and their walls are strengthened with woody lignin, producing a strong, rigid support for the fast growing shoot and leaves. The width of those annual rings varies according the growing season – but I suspect that the outer, most recent ring is narrower because this shoot was harvested for microscopic sectioning sometime in mid-summer, before that year's annual growth was complete. Take a close look at the outer edge of the outer annual ring of xylem (double click the image to enlarge) and you may just be able to make out a distinct narrow zone of very small blue-stained cells, just a  few cells thick (at about 7 o'clock on the section). This is the cambium – the thin layer of living cells that divides to produce dead xylem cells on its inner face and living phloem cells, that conduct sugars from the leaves to the rest of the plant, on the outer side. Together the phloem and cambium are only a few cells thick and represent the most important living tissue inside the tree. Their protection is vital for the tree’s survival, so they are covered by a thick layer of bark tissue, also stained blue where the cells are alive but showing as grey-brown on the outer surface of the twig, where they are dying or dead. This is the tree’s waterproof,  self-repairing, insulating,  wound healing tissue, protecting the delicate living layer of cells inside. Growing tall by producing annual rings of growth is a long-term investment for a plant which only reaches full size after decade of growth, but the return on investment can then continue over centuries – and in some cases millennia - of annual flowering and seed production. As the stem adds annual rings, expanding in girth with every succeeding year, the outer dead bark layer splits into characteristic patterns, depending on the tree species.  The line of red cells in the bark tissues are fibres - dead cells that strengthen the young stem.

Plant Plumbing

Swiss cheese plant Monstera deliciosa is commonly grown as a decorative house plant but in its native Mexican rainforests it's a rampant climber, using its adventitious roots to cling to trees and climbing in much the same manner as ivy in temperate woodlands. Those holes in the Monstera leaf, whose resemblance to holes in Swiss cheese account for its name, let flecks of sunlight filter through to the layers of leaves below, all of which are transpiring water from their surface. If you cut a section through the stem, you can see the internal pipework that conducts water from the roots to the leaves.

In this transverse section of adventitious root, stained with fluorescent dyes that colour dead, woody cell walls yellow and living cellulose cell walls blue you can see the various cells that conduct liquids up and down the root. Embedded in that thick-walled strengthening tissue that gives the root (which in this case is used for clasping tree trunks and branches - this plant is a tropical climber)  rigidity and are fluorescing yellow, are large vessels that conduct water in a continuous tensile column from the roots to the leaf, pulled upwards by evaporation from the leaf surface. The smaller tubes, lined with a layer of blue-fluorescing cell walls, may be resin ducts. The outer cells on the left, part of the ring of small bundles of living cells that encircle the root, are the phloem cells that conduct sugars manufactured by photosynthesis in the leaf to other parts of the plant. Swiss cheese plants are such familiar items of interior decor that they hardly attract a second glance, but they have extraordinary hidden beauty, only visible under the microscope

Taking the Pith

Even as a dyed-in-the-wool botanist I have to admit that rushes (Juncus species) are pretty dull plants – just spiky bunches of cylindrical leaves growing in mud around pond margins (bottom picture) or dominating large areas of poorly drained upland pastures. But, as the old saying goes, ‘beauty is more than skin deep’ and to find the really attractive feature of this plant you have to delve below the leaf surface. Peeling back the outer layer of green photosynthetic tissue reveals a cylinder of spongy pith, as light as thistledown. Pith peeled from rushes and dipped in tallow was once used to form the wicks of rush-lights, a smoky-flamed form of interior lighting that was eventually replaced by gas mantles and then electric light. Take a look at these pith cells under the microscope, in a cross-section of the cylindrical leaf (see top picture) and their real beauty emerges. The individual cells, shaped like starfish, form three-dimensional interior scaffolding for the cylindrical leaf. Each ‘starfish-cell’ is joined to its neighbours by the tips of its arms, forming a three-dimensional lattice (see second picture from top). The top two photomicrographs were taken using fluorescence microscopy, where the section of the leaf was treated with a compound (in this case auramine) which fluoresces when illuminated with blue light, giving an very attractive green glow that shows the three dimensional structure of the plant tissue particularly well. Double-click the top image for a better view. To find out more about rush lights, visit http://pilgrim.ceredigion.gov.uk/index.cfm?articleid=1239

The Inner Workings of an Onion

Onions have long been a favourite source of material for microscopists who want to explore the inner workings of a cell. Peel apart the onion bulb scales and it's easy to strip away the skin of cells that coats the scales; mount these in water on a microscope slide and large, brick-shaped translucent cells are easily visible and reveal the nucleus, that contains the DNA and controls the life of the cell. The centre of the cell is occupied by a large fluid-filled vacuole, with cytoplasm squeezed between it and the cell walls. Watch for a while and it soon becomes apparent the the cytoplasm is constantly streaming around the cell walls, carrying with it minute organelles like the mitochondria, they provide the energy that keeps the cells alive. Sometimes the cytoplasm is drawn out in strings across the vacuole, like stretched-out chewing gum. The whole of the cell is in a constant state of motion. So, next time you're about to chop an onion and chuck it in the frying pan, pause for a moment and contemplate the marvellous process shown in these video clips, which is going on in hundreds of thousands of cells in the living onion in your hand.

Stung

I suffered my first sting of the season today while I was hacking down nettles in the garden, which offers a tenuous excuse to celebrate the achievements of Robert Hooke FRS, the father of English microscopy. Hooke was born in Freshwater on the Isle of Wight in 1635 and in a varied career, that included research in mechanics, astronomy and architecture, produced perhaps the most celebrated book on microscopy: Micrographica, or Some Philosophical Descriptions of Minute Bodies made by Magnifying Glasses withe Obervations and Inquiries thereupon. Samual Pepys bought a copy and commented ‘took home Hook’s book of microscopy, a most excellent piece’, as well he might, as some of Hooke’s descriptions and detailed, exquisite engravings would not be out of place in a biology text book today. Compare his engraving of the stinging hairs on a nettle leaf , above, with the photograph of the hairs on the leaf of the nettle that stung me today – astonishingly accurate, when you consider the rudimentary nature of the microcope lenses he was working with nearly four centuries ago. The nettle sting is a remarkable structure – a long, tapered hollow cell on a pediment (or a 'bladder' as Hooke called it), filled with irritants under pressure and tipped with a minute hooked glass bead that snaps off at the slightest touch, turning the hair into a hypodermic syringe. After watching the whole processes of stinging himself under his microscope, Hooke recorded the following:”The chief thing therefore is, how this plant comes, by so light a touch, to create so great a pain; and the reason of this seems to be nothing else, but the corrosive penetrant liquor contain’d in the little bags or bladders, upon which grow out those sharp syringe-pipes......”. All of Hooke's wonderful engravings from Micrographia can be viewed at http://archive.nlm.nih.gov/proj/ttp/hooke_home.html

Umbrellas to keep the water in

The leaves of the oleaster (Elaeagnus sp.) shrubs in my garden are glossy green above and dazzling white below. The cause of the highly reflective undersurface is revealed under the microscope – thousands of overlapping, flattened hairs, shaped like multi-armed starfish. Each is about a fifth of a millimetre in diameter and attached to the leaf surface by a short stalk. Imagine a surface covered in vast numbers of overlapping, flat, open umbrellas and you’ll have a pretty accurate mental picture of how they’re arranged. The hairs prevent excess water loss from the pores (stomata) on the leaf undersurface, while allowing free passage to the all-important carbon dioxide that the leaf needs for photosynthesis.