Lecture 1: Xylem, the vulnerable pipeline

1. Lecture 1: Xylem, the vulnerable pipeline • Teaching Aims: to introduce the structure and driving forces for transport of water in the xylem, and appreciate that plants operate a delicate balance between maximising water transport and the risk of cavitation and loss of hydraulic conductance • Learning outcomes: to understand that the function of xylem is a trade-off between structural support and water transport; criticisms of the cohesion-tension theory are not supported by recent evidence suggesting that living cell processes contribute to functioning and repair of cavitated vessels

2. 1.1 Soil-Plant-Atmosphere continuum • 1.2 Water Relations of cells and tissues: osmotic adjustment • 1.3 Xylem structure and function • 1.4 Cohesion- tension and rates of sap flow • 1.5 Cavitation and embolism: ecological trade-offs • 1.6 Repair of cavitation Key reference: Tyree MT and Sperry JS (1989) Vulnerability of xylem to cavitaion and embolism Ann. Rev. Pl. Physiol. Mol. Biol. 40, 19-38 Useful background text: Lambers, Chapin and Pons (1998) Chapter 3 Plant Physiological Ecology, Springer

3. 1.1 Soil-Plant-Atmosphere continuum

4. 1.1 Soil-Plant-Atmosphere continuum Water transport along the SPAC consists of a liquid water moving along a gradient with the following driving forces: • Into the roots- it is the water potential between soils and cells of the roots, with transport across a semi-permeable membrane • From roots to leaves- it is a gradient of negative hydrostatic pressure in the xylem • From leaves to the atmosphere- it is the vapour pressure gradient, which ultimately drives the whole process Components and conventions of water potential terminology: • w = s + p Water potential = [solute potential (-ve) + turgor potential (+ve)] • w = P - p Water potential = [Turgor pressure (+ve) minus osmotic pressure (+ve)]; try equations with w = -1.0 MPa ,s = -1.2 MPa or p = 1.2 Mpa: what are p and P equal to?

5. 1.1 Soil-Plant-Atmosphere continuum • Water flux, J, (mm3 s-1) reflects the rate of water movement between two points in the SPAC as a function of the gradient • Soil water availability depends on clay and organic content, with the hydrostatic pressure often described as a matric potential (-ve); this becomes more negative as water content decreases from field capacity towards permanent wilting point, as soil water becomes increasingly difficult for the plant to extract from soil pores • A positive “root” pressure can be generated by the transport of ions into the xylem, as described by Prof Leigh • Water movement through the xylem requires less pressure that movement through living cells: a typical sap flow rate in the apoplastic xylem of a tree will require a gradient of 0.02 MPa m-1; for symplastic flow across the membranes of plant cells, we would need a gradient of 2.108 MPa m-1; so the gradient would need to be 10 billion times greater- showing the efficiency of water flux in the xylem • The leaf to air vapour pressure difference is the driving force for transpiration

6. Data for Hammadia scoparia, a C4 plant • Plants re-equilibrate over night with soil water . • Pre-dawn  tracks soil  • Midday leaf  is not in equilibrium as recharge by xylem lags behind evaporation • Note that from June to August w is more negative than s by day…. what are the implications for leaf turgor?

7. 1.2 Water Relations of cells and tissues: osmotic adjustment

8. 1.2 Water Relations of cells and tissues: osmotic adjustment • For a drying soil,  progressively declines with time; • Each day,  leaf declines then recovers and re-equilibrates at night; it, too, progressively declines each day; • If s (or p in this diagram) does not adjust, turgor is zero by day 5 • If the cell sap solute concentration increases, leading to a decrease in s (or increase in p), then turgor loss point is not reached until day 7; • Osmotic adjustment can be brought about by the accumulation of ions and charged organic solutes in the vacuole, but the cytoplasm need s to accumulate compatible solutes • These include proline, glycine betaine and sugar alcohols (polyols); • 1.3 Xylem structure and function • Tracheids are elongated, spindle shaped cells, which overlap and transfer water by circular pits in lateral walls, often as “pit pairs” • Vessel elements are fond only in angiosperms and the Gnetales- shorter, wider with a perforated end plate stacked end to end = “vessel”; at end of vessel, transfer via pit pairs • Fibres evolved from tracheids • Stiffening prevents collapse under high tensions created by water transport

9. 1.3 Xylem structure and function Perforation plates and pits in oak vessels

10. 1.4 Cohesion- tension and rates of sap flow • Cohesion-tension- proposed early in the last century, how trees up to 100m tall raise perhaps 50 to 100 litres per day to the canopy • A perfect vacuum pump can only raise water 10m, but a capillary 20mm in diameter supports a column 0.75m by capillary action…. • Water in stem in under tension- negative hydrostatic pressure- due to capillarity of xylem and cohesion of water molecules (remember the operation of the pressure bomb) • Can trees really support such tensions- Zimmerman says no, with evidence from the “xylem” pressure probe (but everyone else thinks there is a leak or the probe breaks the column); Canny also disagrees. • Latest evidence creates tensions in stem segments using a centrifuge, and checking the balance pressure with a pressure bomb- good agreement • Water column can in theory support a pressure difference of 30 Mpa; for a tree 100 m x 0.02 MPa m-1, 2 MPa required • In practice, the weight of the water column adds 1 MPa pressure at the bottom of the tree THEORETICAL TOTAL = 3 MPa

11. The hydraulic conductance is proportional to the fourth power of the vessel diameter; • Many small diameter vessels are therefore considerably less efficient than a few large diameter vessels; • Vessel length often correlates more with pore width than vessel diameter • Seasonal variations in vessel length and diameter give year rings in “ring-porous” trees, but are randomly produced in “diffuse porous” • The tradeoff is with structural support: the stem of a liana has the same hydraulic conductance as a tree with a tenfold greater sap area

12. 1.5 Cavitation and embolism • Pits provide the greatest resistance in the pathway • Most are simple, but a more complicated structure with a centrally thickened torus is found in conifers • Under high tensions, a bubble of air can enter through a pit membrane • Water then evaporates explosively into the bubble, registered as an acoustic event- cavitation • Surface tension in the pit membranes prevent the transmission of the gas –water meniscus- so cavitation is contained

13. 1.5 Cavitation and embolism: ecological trade-offs • Embolisms restrict conduction of water, and can limit growth of herbaceous plants and trees • In Zea mays, daily water flow can be reduced by 50% by embolisms; in Acer saccharum (sugar maple) flow in the main trunk was 31% at the end of summer and 60 % by the end of winter, with many small twigs completely blocked

14. In beech, seasonal cycle of cavitation in summer and winter • Maximum conductivity occurs in spring to coincide with budburst • Cavitation can be caused by drought and freeze-thaw cycles • Conifers from cold climates- more likely to be transpiring when freezing occurs • hence small tracheids are less vulnerable but restrict sap flux • Ring –porous cannot refill overwintering xylem –therefore leafing-out is later to avoid frosts • Pathogens induce embolisms

15. Vulnerability to cavitation is also related to desiccation tolerance • Plot loss of hydraulic conductance as a function of  in the xylem • Differences in xylem anatomy reflects a trade-off between large xylem diameter (maximising conductance) and strength (minimises cavitation) – remember the vines • For conifers: Juniper is less vulnerable than Abies (balsam fir) • For hardwoods: Acer (Maple) is more vulnerable than mangrove • The risk of cavitation differentiates mesic and drought adapted species, and investment in transport matches seasonal water supply

16. Cavitation: tradeoff between conductance and safety • “safe” xylem is less efficient at conducting water • Safety margins differ between species: in mesic habitats, plants operate close to tensions causing 100% cavitation (0.4 to 0.6 MPa); in arid habitats, where water deficits may last for months, (eg Larrea, creosote bush) 100% cavitiaon occurs at –16MPa, whereas minimum water potentials are closer to –9MPa • Pit membrane diameter is the key to reducing extent of cavitation, more than vessel diameter • Length of vessel also critical, so short and narrow conduits close to nodes or junctions in stems prevent “runaway embolism” and can be used as “safety zones” to isolate stems and twigs; • dieback is a common occurrence in semi-arid shrubs, due to cavitation • Gradation in height is also associated with increasing water deficits in semi-arid regions

17. 1.6 Repair of cavitation • The cure: force the air back into solution • Root Pressure: at night, active ion influx continues though transpiration is reduced, generating 0.1-0.2MPa (also guttation) • In Betula (birch) and Vitis (grape) large root pressures develop in spring- leading to frothing and sap dripping from pruned vines! • Where root pressure is insufficient, in many woody species, there was some evidence that cavitation could be repaired on a daily basis, leading Canny to suggest a role or parenchyma in pressurisng tissues • Missy Holbrook and M Zwienicki (1999: Plant Physiology, 120, 7-10; see also paper that follows by Tyree et al) provide a solution: activity of living cells adjacent to the xylem is required • Girdling or MgCl2 in transpiration stream reduces recovery from embolism • Is there a role for a mechanism similar to that during root exudation? But xylem sap osmotic pressure in repairing elements not enough.

18. Integral membrane proteins (MIPS and TIPS)- water selective channels in plants- aquaporins • The key to the process is hydraulic isolation- which contrains the embolism, and allows local positive pressures to force gas back into solution • Xylem wall must be relatively impermeable, with droplets remaining attached to the wall by the contact angle generated • Influx of water compresses the gas phase (black arrows) with air forced into solution (yellow arrows) • Bordered pit geometry (inset), as an inverted funnel, prevents water from entering the pit channel until the lumen is entirely filled- thereby stabilising the restoration of hydraulic continuity and minimising the volume of undissolved gas

19. From Holbrook and Zwienicki, 1999

21. This mechanism allows the tension in adjacent vessels to be transmitted to the refilling conduit as soon as the advancing water contacts the pit membrane • The previous photograph, taken from Tyree et al 1999, Plant Physiol 120,11-21, shows the refilling process occurring in bay (Laurus nobilis)- using cryoscanning EM. (A) shows cavitated vessels; (B) shows one cavitated vessel and one with small air bubbles; (C) shows sap adhering to the cell wall and (D) shows water droplets entering the vessel • Reversibility of embolisms makes the balance between damage and repair a much more dynamic process • But what is the evidence for such an active process? Once again, Zimmerman tries to use it to disprove the overall cohesion tension theory in resurrection plants • But Missy strikes back Zwienecki et al 2001 Science 1059-1062- Hydrogel control of hydraulic resistance in plants……..

22. Hydraulic conductance is increased by adding salt solutions to xylem sap • Microchannels in the pit membranes are altered by the swelling and contraction of pectins (hydrogels)- allowing the xylem to alter internal rates of flow and highly relevant to root shoot signalling in response to drought, which we will meet later ….

23. Conclusions • Strictly speaking, the driving forces for movement of water through the SPAC vary between soil-root, xylem and leaf- air • Understand the different potential/pressure terminologies used to describe solute and turgor interactions with water potential- we’ll need it for guard cells! • The xylem, thought to be an inert, apoplastic pathway is the most effective way to transport water long distances with pit membranes and diameter of the conducting elements the most important resistance in the pathway • Plant water potential must track soil water supply, although osmotic adjustment can help to maintain gradients of water uptake uder drought and saline conditions • There is a trade-off between vessel diameter, trunk strength and susceptiblity to cavitation • Embolisms are acoustically detectable as the water column, seeded by air, snaps; water flow can proceed around the blockage via pits, but daily and seasonal restrictions in water flow are significant

24. Cavitation can be caused by drought and freeze-thaw cycles, with some plants capable of restoring the connection in spring using root pressure • The risk of cavitation marked driver of lifeform and ecological adaptations to drought- with a reduction in height, denser, smaller xylem conduits and the strategy of “segmentation” (shedding stems) common in semi-arid shrubs • Safety margins depend on how reliably a plant can refill or restore conductivity on a daily or seasonal basis • Facilitated movement of water through aquaporins may help to restore water content of cavitated vessels, with the isolation helping to force air back into solution and prevent runaway caviation • The xylem pathway is clearly not as inert as once thought!!!