3.3 - Leaf vein architecture and anatomy

A rapidly transpiring leaf can evaporate its own fresh weight of water in 10 to 20 min, though many plants such as cacti, mangroves and plants in deep shade have much smaller rates of water turnover. Leaf veins must carry this water to all parts of a leaf to replace evaporated water, and maintain cell hydration and turgor. When water supply fails to meet this demand, shoots wilt.


Figure 3.20 Typical leaf vein pattern of broad-leaf species versus grasses. Left: Leaf of Eucalyptus crenulata showing the arrangement of supply and distribution veins. Large veins with large vessels, in which water is moved rapidly across the lamina, surround islets of small veins with small vessels in which water is slowly distributed locally. Scale bar, 1 mm. Right: Leaf of wheat (Triticum aestivum) showing one large and three small longitudinal veins, with transverse veins connecting them. Scale bar, 0.1 mm. Distance between small veins is about 0.15 mm in both species. (Photographs courtesy of M. McCully and M. Canny)

Vein distribution patterns differ markedly between broad-leaf species and grasses. Broad-leaf species generally have a highly branched network while grass species have parallel veins (Figure 3.20).

Veins consist typically of tightly packed xylem and phloem tissues surrounded by a parenchymatous or fibrous sheath. Both xylem and the phloem contain living parenchyma cells as well as their characteristic transporting conduits: vessels and/or tracheids in the xylem tissue, plus sieve tubes in the phloem tissue. There are no intercellular air-spaces, or only very small ones.


Figure 3.21 Transverse section of small intermediate vein of wheat leaf, after being fed the fluorochrome sulphorodamine, which acts as a tracer for water movement. Water passes out of the xylem through paths in the walls of the mestome sheath cells (M) and enters the parenchymatous sheath cells (P) leaving red crystals in the intercellular spaces. Scale bar, 15 µm. Image, M. Canny. (Reproduced from New Phytologist, Tansley Review No 22, 1990)

The ring of cells forming the sheath around the xylem and phloem tissue acts both as a mechanical barrier that may confine pressure within the vein, and a permeability barrier that can control rates and places of entry and exit of materials (Figure 3.21).

3.3.1 - Vein architecture of conifers and angiosperms

The simplest vein architecture is found in conifer needles where a single unbranched strand of xylem and phloem is surrounded by mesophyll (Figure 3.22).


Figure 3.22 Transverse section of a pine needle. A single central vein has two strands of phloem (p) and xylem (x), embedded in transfusion tissue (t). These vascular tissues are separated from the chlorophyll-containing mesophyll (red) by an endodermis (e) with Casparian strips and suberised lamellae. Stomata in the epidermis appear bluish. Rhodamine B stain, fluorescence optics. Scale bar, 0.1 mm.  (Photograph courtesy M. McCully)


Figure 3.23 Wheat leaf showing one large supply vein and three small distribution veins, connected by transverse veins. Small veins could carry two thirds of the evaporated water. Dark-field optics, blue filter. Scale bar, 0.1 mm. (Photograph courtesy M. McCully)

Vascular strands are enclosed by an endodermis that separates them from the mesophyll, and are embedded in a mixture of parenchyma cells and tracheids called transfusion tissue. Water from the xylem permeates radially outward through transfusion tissue, endodermis and mesophyll to evaporate below lines of stomata in the epidermis.

Leaves of angiosperms have much more complicated venation than conifer needles. If you look at a grass leaf with your hand lens, parallel veins run the length of the leaf, but they are not all the same size. A few large veins have several small veins lying between them. On closer inspection with a light microscope, all these parallel veins are connected at intervals by very small transverse veins (Figure 3.23).

There are in fact two vein systems with different functions: large veins supply water rapidly to the whole length of a leaf blade while small veins and their transverse connections distribute water locally, drawing it from the large veins. Water in large veins flows only towards the tip, but in small veins it can flow either forwards or backwards along the leaf blade or transversely between adjacent parallel veins. The distinction of flow patterns in large and small veins arises as a result of different vessel sizes. Large veins have wide vessels (about 30 µm diameter), while small veins have narrow vessels (about 10 µm diameter). As the volume of water flowing along pipes is proportional to the fourth power of the radius (Equation 6; Poiseuille’s Law), volume flow in the larger vessels will be 3 to the fourth power (= 81) times greater than the flow in the smaller vessels. Put another way, pressure gradients along the leaf in large vessels will be very slight, but steep pressure gradients can develop locally in narrow vessels that will direct local flows into the mesophyll. Large veins supply water rapidly over the whole lamina while small veins distribute it locally and slowly. The slower flux along minor veins is compensated by their far greater number, which results in them having a greater length per unit leaf area than major veins.

With a hand lens, you do not see the vessels, instead you see the sheaths that surround xylem and phloem, much as an endodermis surrounds vascular strands of a conifer needle. Grass leaf veins have two sheaths of cells surrounding the parallel veins and containing the xylem and phloem tissues. In the leaf anatomical development typical of C3 species (e.g. wheat), both sheaths are parenchymatous and lack chloroplasts (Figure 3.24).


Figure 3.24 Wheat leaf showing a single large (supply) vein comprising three large vessels (V). The vein is surrounded by two sheaths of living cells, the inner mestome sheath (arrowheads), and outer parenchyma sheath (stars). The mestome sheath of these veins is impermeable to water. Water enters the symplasm at the inner boundary of the parenchyma sheath (Canny 1990). Phloem (P). Transverse hand-section, Toluidine blue stain, bright-field optics. Scale bar, 0.1 mm. (Photograph courtesy M. McCully and M. Canny)


Figure 3.25 Minor vein of maize, consisting of one xylem vessel (v), five vascular parenchyma cells (vp), bundle sheath (bs), companion cell (cc), and sieve tube (st) with intercellular space (is). Scale bar, 4.2 µm. Transmission electron micrograph, R.F. Evert. (Reproduced from Planta 138: 279-294, 1978)

The large veins of a C3 species is surrounded by two sheaths of living cells, the inner mestome sheath and outer parenchyma sheath (Figure 3.24). The mestome sheath of these large veins is impermeable to water. There is no apoplastic path for water through the mestome sheath of large veins, except through a connecting transverse vein. In small veins, by contrast, two or three mestome sheath cells next to the xylem permit flow of water and solutes through the cell wall apoplasm.

In C4 species (e.g. maize), only the inner (mestome) sheath is without chloroplasts. The outer ring of sheath cells contains large chloroplasts and is known as the bundle sheath (Figure 3.25). This is the cell layer in which CO2 fixation in the Calvin cycle takes place in C4 plants. This is an essential part of the carbon concentrating mechanism, and the special anatomy of C4 photosynthesis, as detailed in Chapter 2, Section 2.2.2

A dicotyledonous leaf contains the same two vein systems as a grass leaf, but these are differently arranged. Large supply veins are prominent, comprising a midrib and two orders of branches off it, often standing out from the surface of the lamina. These contain wide vessels and carry water rapidly to the leaf margins. Between them lie distribution veins, another two branch orders of small veins dividing the mesophyll up into islets about 1–2 mm across (Figure 3.26), and within these islets a fifth and final order of branches of the finest veins. The fourth- and fifth-order branches have only narrow vessels (Figure 3.26).

In dicotyledenous leaves, as in grass leaves, these vascular tissues are enclosed by bundle sheath cells through which water and solutes must pass when leaving the xylem.


Figure 3.26 Whole mount of a cleared leaf of Eucalyptus crenulata showing the complicated arrangement of supply and distribution veins characteristic of a dicotyledonous leaf. Islands marked out by large veins with large vessels, in which water is moved rapidly all over the lamina, surround islets of small veins with small vessels in which water is slowly distributed locally to the mesophyll. Partial phase-contrast optics. Scale bar, 1 mm. (Photograph courtesy M. McCully and M. Canny)

Any distribution network such as the branching vessels of decreasing size in leaves is found to obey Murray’s Law. This states that the cube of the radius of a parent vessel is equal to the sum of cubes of the radii of the daughter vessels (e.g. a 50 µm vessel would branch into five 30 µm vessels). Such a pattern of branching produces optimal flow in several senses: minimum energy cost of driving that flow, minimum energy cost of maintaining the pipeline, constant shear stress at the walls of pipes, and rapid flow in supply pipes with slow flow in distributing pipes to permit exchange through the pipe walls (LaBarbera 1990).


Figure 3.27 Soybean leaf showing two of the smallest veins surrounded by bundle sheath cells. Veins of this size are the end of the branching network shown in Figure 3.26, and supply most water that is evaporated. This leaf was transpiring in a solution of fluorescent dye for 40 min. The small vessels in each vein contain dye solution which has become concentrated by water loss to the symplasm and out through the bundle sheath. Dye has started to diffuse away from small vessels in the cell wall apoplasm of bundle sheath cells. Anhydrous freeze-substitution and sectioning, fluorescence optics. Scale bar, 50 µm. (Photograph courtesy M. McCully and M. Canny)

The ring of cells forming the sheath around the xylem and phloem tissue (Figure 3.27) acts both as a mechanical barrier that may confine pressure within the vein, and a permeability barrier that can control rates and places of entry and exit of materials. Exceptions are to be found at the ultimate ends of some dicotyledonous fine veins where tracheids or sieve elements may be unaccompanied by other cells, in the transverse veins of grasses which have no sheath, and in some special veins at leaf margins where the sheath is absent on the xylem side. There is evidence of a suberised layer in walls of these sheaths cells in some species, but not in others. It is uncertain whether xylem sap must traverse the cell membranes, as in the suberised endodermis of roots, or if it can travel along the cell walls.

3.3.2 - Damage control

Transpiration operates by suction, but leaves are especially liable to damage by grazing, mechanical forces, and extreme negative pressures in the xylem water column due to high rates of transpiration. Leaf vessels therefore need special protection against air embolisms spreading in the vessel network that would block liquid flow. This is achieved at all points in the leaf distal to the node (i.e. petiole, large veins, small veins) by the vessels being very short. That is, files of vessel elements joined to make a single pipe with a terminal end-wall are much shorter in the leaf than in the rest of the plant. Water flows through vessel end-walls with little extra resistance, but an air–water interface cannot be pulled through an end-wall or pit membrane because the pores are so small. The force needed to curve the interface into a meniscus small enough to pass through the end-wall is similar to that generated by evaporation from wet cell walls (Section 3.1.3). From equation (4), (DP = 0.15/r) we see that a pressure of roughly 0.3/d (MPa) is required to pull the interface through a hole of diameter d (µm). While 0.1 MPa can pull an interface through a 3 µm hole, 6 MPa is required to pull air through a 0.05 µm hole. Suctions of 6 MPa are not known to occur in transpiring plants, and cell walls have pores much smaller than 0.05 µm - in the order of 0.003 µm. So an embolism formed from cavitating water fills one vessel but does not spread beyond it.

The length of xylem vessels is demonstrated by allowing a leaf to transpire in a fine colloidal suspension that cannot pass end-walls. Latex paint, diluted 100 times with water and allowed to settle for a week or two, provides such a suspension. Leaves that have drawn up this suspension for an hour or so during transpiration can be cleared by dissolving out the chlorophyll and soaking in lactic acid. Progress of the paint is then readily seen (Figure 3.28). Very few vessels exceed 1 cm in length.


Figure 3.28 Cleared whole mount of a wheat leaf demonstrating the frequent occurrence of end-walls in leaf vessels. The leaf was fed an emulsion of green latex paint in the transpiration fluid from a cut surface 6.5 mm to the right of the picture. At the right side of the picture, two vessels in the central large vein are carrying paint. Halfway across the picture (at arrowhead) the upper vessel is blocked by an end-wall through which the paint particles could not pass, although water continued to flow. The paint in the lower vessel continued for another 3 mm beyond the left of the picture, where an end-wall in that vessel limited its further progress. Note that paint has not passed out of the large vein into transverse veins where water flowed because pit membranes (Section 3.2) filtered out paint particles. Bright-field optics. Scale bar, 100 µm. (Photograph courtesy M. McCully and M. Canny)

3.3.3 - Water extraction from the xylem


Figure 3.11d SEM showing surface view of the pit membrane with secondary wall removed from most pits by sectioning. Tiny pores allow the movement of water between vessels but limit the movement of gas and pathogens. Bordered pits act as the safety valves of the plant hydraulic system. (Photograph courtesy B Choat and S Jansen)

Xylem vessels (and tracheids) are not just pipes to carry water, they are pipes with holes in them (pits) through which water can leak out, fulfilling a principal leaf function of water distribution through the transpiration stream to places where it will evaporate. The branching network of vessels is beautifully adapted to achieve this.

Think about flow in a leaky vessel. As explained earlier, the rate of volume flow varies as the fourth power of the radius (Poiseuille’s Law). The frequency of leaks through vessel walls varies with surface area of the walls, that is, as the first power of the radius (2πr). So if the vessel is wide, forward flow is much larger than leakage. The wide vessels in large veins supply water all over the leaf without losing much on the way. As the width of a vessel becomes smaller, the forward flow (a function of r4) is reduced much more strongly than the leaks (a function of r). The proportion of water lost to leakage therefore increases as vessels become smaller. Indeed, for a fixed pressure gradient there is a critical radius at which all water entering the vessels supplies leaks, and there is no forward flow at all. The finest veins of dicotyledenous leaves have vessels of a radius that is close to this critical value. As sap disperses into the fine ramifications of the network, it moves more and more slowly forward, and leaks increasingly outwards through the sheath to the mesophyll. This is the rationale of the distributing networks of the small branched veins of both dicotyledons and grasses.


Figure 3.29 Fresh paradermal hand-section of a leaf of Eucalyptus crenulata which had been transpiring for 80 min in a solution of sulphorhodamine G. The dye solution is present at low concentration in the vessels of the larger veins, but is not visible at that concentration. In the smallest veins the dye has become so concentrated by loss of water to the symplasm that dye crystals have formed inside vessels. Sectioned in oil, bright-field optics. Scale bar, 100 µm. (Photograph courtesy M. McCully and M. Canny)

Extraction of water from fine veins can be readily demonstrated. Stand a cut leaf in an aqueous solution of dye, such as 0.1% sulphorhodamine G, and allow it to transpire for an hour. The solution moves rapidly through large veins all over the leaf in a few minutes. Then it moves increasingly slowly into the network of distributing small veins. By the end of an hour it has reached the ends of the finest veins and, as water is extracted from them, the dye becomes more and more concentrated (Figure 3.29). Movement of dyes from the finest veins to leaf surfaces 100 µm away takes about 30 min, suggesting that diffusion rather than mass flow is responsible for solute distribution to cells.

3.3.4 - Solute extraction from the xylem


Figure 3.33 Leaf of horse chestnut, Aesculus hippocastanum, showing as with all strongly-toothed leaves, conspicuous veins running to each tooth. (Photograph courtesy M. Waters)

(a) Phloem export

Potassium is known to be extracted from the xylem and re-exported via the phloem (Section 5.1). In leaves, the concentrated sap flowing through the narrow xylem vessels of fine veins is separated from sieve tubes of the phloem by only two or three parenchyma cells, frequently enhanced by transfer cells. Exchange from xylem to phloem is probably made by this direct path. Solutes travel in the phloem back to stems, either distally to younger developing leaves and the stem apex or proximally towards roots.

(b)  Scavenging cells  

Recycling of solutes out of leaves can also be fed by a variety of special structures adapted for processing rather larger volumes of sap, and so suited for collecting solutes present in the stream at quite low concentrations. The transfusion tissue of conifer needles is one of these structures. As sap leaves the xylem of a vascular strand it moves through a bed of tracheids mixed intimately with transfusion parenchyma cells (Figure 3.22 shown earlier). The endodermis acts as the ultrafiltration barrier, allowing water to pass through while leaving dilute solutes to accumulate in transfusion tracheids. Transfusion parenchyma cells have very active H+-ATPases in their cell membranes which accumulate selected solutes (certainly some amino acids) back into the symplasm for return to the phloem and re-export. Such actively accumulating cells are called scavenging cells.

A tissue that acts in the same way is a special layer of cells in the central plane of many legume leaves (extended bundle sheath system or paraveinal mesophyll). It consists of scavenging cells with active H+-ATPases and accumulates amino acids, stores them and forwards them to developing seeds via the phloem.

Jagged ‘teeth’ on the margins of many leaves also contain scavenging cells (Figure 3.33). Veins carry large volumes of the xylem sap to these points, where evaporation is especially rapid. Within a ‘tooth’, xylem strands end in a spray of small vessels among a bed of scavenging cells. Scavenging cells can thereby collect amino acids and load them into the phloem.

(c) Solute excretion

Not all the solutes of the transpiration stream are welcome back in the plant body. Some, such as calcium, are immobilised in insoluble compounds (calcium oxalate crystals) and shed when leaves fall. Others are excreted through the surface of living leaves. A striking excretion system is found along the margin of maize leaves. Here the outermost vein has a single very wide vessel. Rapid evaporation from the exposed leaf edge cooperates with this low-resistance vessel to draw to the leaf margin all residual solutes that have not been taken out of the stream by other veins. Thus foreign material (like dyes) accumulates in this outermost vessel. The vein sheath is missing from the outer part of this vein so that vessels abut directly the airspace at leaf margins (Canny 1990). Solutes are excreted from this marginal vein, dissolved out by rain and dew, and, more actively, at night time by guttation fluid when there is positive pressure in the xylem. A similar accumulation of dye from the transpiration stream is shown along the margin of a eucalyptus leaf in Figure 3.34.


Figure 3.34 Whole mount of the living margin of the eucalypt leaf sectioned in Figure 3.11 prepared after dye had been fed to the cut petiole for 90 min. Dye has spread to the leaf margin in large veins where it accumulates at high concentrations. By analogy with maize leaves (Canny 1990) this is likely to be a system for excreting unwanted solutes. Bright-field optics. Scale bar, 1 mm. (Photograph courtesy M. McCully and M. Canny)