PROTOCOLS

ECOLOGICAL IMPLICATIONS: Grime's CSR triangle (Grime, 1977; Pierce t al., 2017), uses size and economic traits (i.e. traits related to acquisition, use, and allocation of resources) to describe three main plant evolutionary responses to disturbances (any factor removing plant biomass) and stresses (any factor hindering new biomass production): 

Competitor (C) strategists are tall and long-living plants that exhibit a resource-acquisitive strategy and invest in vegetative growth under productive and undisturbed conditions. They tend to have large (high LA) and short-lived leaves with low SLA and LDMC. 

Stress tolerant (S) are short, slow-growing and long-living plants that show a resource-conservative strategy and invest in survival under unproductive conditions. They tend to have small (low LA), long-lived, tough and dense leaves, with low SLA and high LDMC.

Ruderal (R) are short, fast-growing and short-living plants that invest mainly in reproduction to recover from frequent disturbances. They tend to have small (low LA), short-lived, and thin leaves with high SLA and low LDMC.

The percentage of CSR strategies of a plant species can be estimated using the ‘globally calibrated CSR strategy calculator tool StrateFy’ (Pierce et al., 2017). In this tool, three traits— leaf area (LA, to describe plant size spectrum), specific leaf area and leaf dry matter content (SLA and LDMC, both describing conservative- versus acquisitive-resource strategies)—of a target species are compared against the axes of a multivariate space occupied by 3,068 tracheophytes, thus returning species CSR% (competitiveness, C%; stress-tolerance, S%; ruderalism, R%).

PROTOCOLS:

Download Protocol [based on Pérez-Harguindeguy et al., 2023]

Download StrateFy (Excel) tool [Pierce et al., 2017]

Download test data (csv) 

Download Rscript [for plotting CSR triangle]

REFERENCES:

Grime, J. P. (1977). Evidence for the existence of three primary strategies in plants and its relevance to ecological and evolutionary theory. The American Naturalist, 111: 1169–1194. 

Pierce, S. et al. (2017), A global method for calculating plant CSR ecological strategies applied across biomes world-wide. Funct Ecol, 31: 444-457. 

Pierce, S.  et al. (2013), Allocating CSR plant functional types: the use of leaf economics and size traits to classify woody and herbaceous vascular plants. Funct Ecol, 27: 1002-1010. 

Pérez-Harguindeguy, N. et al. (2013) New handbook for standardised measurement of plant functional traits worldwide. Aus Bot, 61: 167-234.

ECOLOGICAL IMPLICATIONS: FWU was first observed nearly 300 years ago (Hales, 1727). Since then, overwhelming evidence has accumulated showing that water can enter the leaf through different pathways (e.g. trichomes, hydathodes, cuticle, via open stomata) and can have benefits for the whole-plant water balance, such as rehydrating plant tissues (Burgess & Dawson, 2004; Limm et al., 2009), preventing cavitation or repairing xylem embolisms (Laur & Hacke, 2014), and increasing photosynthesis, growth, and survival rates (e.g. Eller et al., 2013, 2016; Binks et al., 2019). Some reviews (e.g. Dawson & Goldsmith, 2018; Berry et al., 2019; Schreel & Steppe, 2020; Boanares et al., 2020) have highlighted the biophysical conditions necessary for FWU to occur, as well as the main pathways of water absorption and the need to revise current plant hydrological models (i.e. representations of the soil-plant-atmosphere continuum) to include FWU fluxes. A few studies have also estimated the impacts of FWU on the water and carbon fluxes at the ecosystem scale (e.g. Burgess & Dawson, 2004; Binks et al., 2019; Cavallaro et al., 2020).

PROTOCOLS: 

Download protocol [mass method and water potential method]

REFERENCES:

Hales, S. (1757). Vegetable Staticks. London, UK: Isaac Newton

Burgess, S.S.O., & Dawson, T.E. (2004). The contribution of fog to the water relations of Sequoia sempervirens (D.Don): foliar uptake and prevention of dehydration. Plant, Cell & Environment 27, 1023-1034.

Laur, J. & Hacke, U.G. (2014). Exploring Picea glauca aquaporins in the context of needle water uptake and xylem refilling. New Phytologist, 203, 388–400. https://doi.org/10.1111/nph.12806

Eller, C.B., Lima, A.L., & Oliveira, R.S. (2016). Cloud forest trees with higher foliar water uptake capacity and anisohydric behavior are more vulnerable to drought and climate change. New Phytologist, 211(2), 489-501.

Eller, C.B., Meireles, L.D., Sitch, S. et al. (2020). How Climate Shapes the Functioning of Tropical Montane Cloud. Forests Current Forestry Repository, 6, 97–114. https://doi.org/10.1007/s40725-020-00115-6

Binks, O., Mencuccini, M., Rowland, L, et al. (2019). Foliar water uptake in Amazonian trees: Evidence and consequences. Global Change Biology, 25, 2678– 2690. https://doi.org/10.1111/gcb.14666

Dawson, T.E. & Goldsmith, G.R. (2018). The value of wet leaves. New Phytologist, 219, 1156-1169. https://doi.org/10.1111/nph.15307

Berry, Z.C., Emery, N.C., Gotsch, S.G., & Goldsmith, G.R. (2019). Foliar water uptake: Processes, pathways, and integration into plant water budgets. Plant Cell & Environment, 42, 410– 423. https://doi.org/10.1111/pce.13439

Boanares, D., Oliveira, R.S., Isaias, R.M.S., França, M.G.C., & Penuelas, J. (2020). The Neglected Reverse Water Pathway: Atmosphere–Plant–Soil Continuum. Trends in Plant Science, 25(11), 1073-1075. https://doi.org/10.1016/j.tplants.2020.07.012

Cavallaro, A., Carbonell Silleta, L., Pereyra, D.A. et al. (2020). Foliar water uptake in arid ecosystems: seasonal variability and ecophysiological consequences. Oecologia, 193, 337–348. https://doi.org/10.1007/s00442-020-04673-1

Schreel, J. D. M., & Steppe, K. (2020). Foliar Water Uptake in Trees: Negligible or Necessary? Trends in Plant Science, 25(6), 590-603. https://doi.org/10.1016/j.tplants.2020.01.003

ECOLOGICAL IMPLICATIONS:  species range in its performance from isohydric to anisohydric strategies depending on whether they prioritize hydraulic safety or gas exchange under dry conditions. Isohydric plants avoid reaching low leaf water potentials by closing stomata earlier during drought. They have a strong stomatal conductance regulation and maintain nearly constant leaf water potentials despite of reductions in soil water potential. Typically, isohydric plants exhibit a high stomatal sensitivity to evaporative demand. This behavior limits xylem cavitation but simultaneously reduces carbon uptake during drought. 

Anisohydric plants, in turn, tend to keep their stomata open and endure lower leaf water potentials during drier conditions. These plants allow midday water potential to decline and maintain gas exchange even during drought conditions. This behavior promotes carbon uptake at the expense of decreased leaf water potential and increased probability of xylem cavitation. 

Therefore, isohydric species might be more susceptible to negative carbon balance and carbon starvation during drought, whereas anisohydric species might be more susceptible to cavitation and total hydraulic failure under drier conditions.

PROTOCOL:  there are many different methods to classify species into iso-or anisohydric categories. Two of the most commonly used methods are described below:

(1) Ψgs50 (MPa) = the leaf water potential when the stomatal conductance drops to 50% of its maximum. A lower (more negative) value indicates a more anisohydric behaviour (Scoffoni et al 2012).

Download Protocol [Scoffoni et al 2012 method]

Download Excel file [Scoffoni et al 2012 method]

Download Rscript [Scoffoni et al 2012 method]

(2) Hydrospace area (MPa2) = area of triangle bounded by the regression line, y-axis, and 1:1 line in a plot of midday leaf water potential versus pre-dawn leaf water potential (Meinzer et al 2016).

Download Protocol [in English/Portuguese]

Download Excel file [Meinzer et al 2016 method]

REFERENCES

Meinzer, F.C. et al. (2016), Mapping ‘hydroscapes’ along the iso- to anisohydric continuum of stomatal regulation of plant water status. Ecol Lett, 19: 1343-1352. https://doi.org/10.1111/ele.12670. 

Scoffoni, C. e t al (2012). Dynamics of leaf hydraulic conductance with water status: quantification and analysis of species differences under steady state, Journal of Experimental Botany, 63(2): 643–658. https://doi.org/10.1093/jxb/err270

ECOLOGICAL CONSIDERATIONS: steep leaf angles may be a selective adaptation in arid and semi-arid environments to minimize light interception during midday, hence reducing the probability of photodamage. Moreover, steeper leaf angles may facilitate water droplets being drained off leaf surfaces, thus decreasing rainfall interception and increasing throughfall to the soil.

PROTOCOLS: leaf angle can be measured using different methods listed below:

(1) Protractor method: using a transparent plastic protractor and a plumb line (Foot & Morgan 2005; Holder 2012);

(2) Cell phone method: using a cell phone and an app available at Escribano-Rocafort et al 2014.

(3) Picture method:  using a digital camera, a tripod, and the software ImageJ (Pisek et al 2011; Zou et al 2014); [Download protocol]

REFERENCES:

Escribano-Rocafort, A.G., Ventre-Lespiaucq, A.B., Granado-Yela, C., López-Pintor, A., Delgado, J. a., Muñoz, V., Dorado, G. a. & Balaguer, L. (2014) Simplifying data acquisition in plant canopies- Measurements of leaf angles with a cell phone. Methods in Ecology and Evolution, 5, 132–140.

Foot, K. & Morgan, R.P.C. (2005) The role of leaf inclination, leaf orientation and plant canopy architecture in soil particle detachment by raindrops. Earth Surface Processes and Landforms, 30, 1509–1520.

Holder, C.D. (2007) Leaf water repellency of species in Guatemala and Colorado (USA) and its significance to forest hydrology studies. Journal of Hydrology, 336, 147–154.

Holder, C.D. (2012) The relationship between leaf hydrophobicity, water droplet retention, and leaf angle of common species in a semi-arid region of the western United States. Agricultural and Forest Meteorology, 152, 11–16.

Pisek, J., Ryu, Y. & Alikas, K. (2011) Estimating leaf inclination and G-function from leveled digital camera photography in broadleaf canopies. Trees - Structure and Function, 25, 919–924.

Zou, X., Mõttus, M., Tammeorg, P., Torres, C.L., Takala, T., Pisek, J., Mäkelä, P., Stoddard, F.L. & Pellikka, P. (2014) Photographic measurement of leaf angles in field crops. Agricultural and Forest Meteorology, 184, 137–146.

Valiente-Banuet, A., Verdú, M., Valladares, F., García-Fayos, P., 2010. Functional and evolutionary correlations of steep leaf angles in the mexical shrubland. Oecolo- gia 163, 25–33

Aryal, B. & Neuner, G. (2010) Leaf wettability decreases along an extreme altitudinal gradient. Oecologia, 162, 1–9.

ECOLOGICAL CONSIDERATIONS: A chlorophyll meter indicates relative chlorophyll concentration, where higher readings suggest healthier plants with more chlorophyll, while lower readings suggest nutrient deficiency, stress, or disease. The specific readings that signal a problem vary by plant species, so it's important to compare measurements to the healthy range for that species or monitor trends over time. Chlorophyll levels are also closely tied to plant nitrogen status, since nitrogen is a key component of chlorophyll.  

PROTOCOLS: The protocol below explain how to obtain atLEAF CHL PLUS is a powerful, handheld, easy to use device for noninvasively measuring the relative chlorophyll content of green leaf plants. Plant relative chlorophyll concentration is measured by inserting a leaf into the device aperture. Green leaves of up to 0.1 inch (3mm) thickness can be measured. The chlorophyll meter compares the transmission of light in red and near infra-red wavelengths in order to give a measure of chlorophyll content in green leaves. 

Download Protocol

Download Manual

ECOLOGICAL IMPLICATIONS: Kleaf summarizes the behavior of a complex system: water moves through the petiole and through several orders of veins, exits into the bundle sheath and passes through or around mesophyll cells before evaporating into the airspace and being transpired from the stomata to the surrounding atmosphere. Thus, Kleaf is determined by both the structure of the vein xylem (xylem conductance - Kx) and the ‘‘extra-xylem’’ (outside xylem conductance - Kox) pathways of water movement. Kleaf varies more than 65-fold across species, and this variation reflects differences in the anatomy of the petiole and the venation architecture, as well as pathways beyond the xylem through living tissues to sites of evaporation. Kleaf is highly dynamic over a range of time scales in response to changes in water supply, irradiance, temperature, circadian cycles, and leaf aging.

PROTOCOL: 

Kleaf can be measured through different methods that involve pushing (high pressure flowmeter method - HPFM), evaporating (evaporative flux method - EFM) and pulling (vacuum pump method - VPM) water out of the leaf. These standard methods all determine Kleaf directly as measured flow rate divided by the driving force for flow. In contrast, the rehydration kinetics method (RKM) estimates Kleaf by assuming leaf rehydration is equivalent to the discharging of a capacitor through a resistor, a formulation that requires knowledge of leaf water storage capacitance (Cleaf). The protocol below describe the measurement of Kleaf using the EFM method adapted for the use of pressure drop flow meter.

Download protocol

REFERENCES:

Melcher, P. J., Michele Holbrook, N. , Burns, M. J., Zwieniecki, M. A., Cobb, A. R., Brodribb, T. J., Choat, B. and Sack, L. (2012), Measurements of stem xylem hydraulic conductivity in the laboratory and field. Methods in Ecology and Evolution, 3: 685-694.

L., Sack and C., Scoffoni (2012), Measurement of Leaf Hydraulic Conductance and Stomatal Conductance and Their Responses to Irradiance and Dehydration Using the Evaporative Flux Method (EFM). Journal of Visualized Experiment (70); 2012PMC3577864

Constructing and operating a hydraulics flow meter:

http://prometheuswiki.org/tiki-print.php?page=Constructing%20and%20operating%20a%20hy

draulics%20flow%20meter

ECOLOGICAL IMPLICATIONS: leaves must be sufficiently stiff to support their own weight mechanically against the pull of gravity yet flexible enough to bend, twist or fold without breaking when subjected to large externally applied dynamic forces. They can also be tough to avoid being damaged by hervivores.

PROTOCOL: the techniques commonly used in ecological studies to assess leaf mechanical properties include fracture (strength and toughness) and flexure tests (stiffness). 

(1) Fracture tests: Fracture tests apply some kind of force, either tearing, shearing, punch, or a combination of these, to the test material until it fractures. The shearing test involves cutting across a leaf with a single blade (against an anvil) or with a pair of blades (i.e. instrumented scissors). It is the most meaningful leaf mechanical property in the case of herbivory by chewing insects or trampling by mammals. Four types of cutting devices have been used to conduct shearing tests (Pérez-Harguindeguy et al 2013). Although the design of these machines differs in terms of the number of blades (one or two), blade angle (relative to the anvil or lower blade), and sharpness, they produce similar results (Onoda et al 2011). The punch test involves forcing a rod of known cross-sectional area through part of a leaf. Results from punch tests are more likely to reflect mechanical defence against herbivory by chewing or sucking insects (Pérez-Harguindeguy et al 2013).  

(2) Flexural tests: such as bending or twisting/torsion tests, can be used to obtain flexural properties of leaves, such as flexural stiffness (‘structural’ properties, which represents the ability to resist bending), and Young’s modulus of elasticity (‘material’ properties, i.e. normalized per unit leaf thickness). 

The protocol below explains in detail how to conduct leaf fracture (punching and shearing tests) and flexural (3-point bending test) tests using a universal testing machine that simultaneously record the force applied and the displacement during the mechanical test. The protocol also shows how to measure a series of leaf mechanical traits (e.g. work to punch and shear a leaf, leaf modulus of elasticity) from the force-displacement curves.

Download protocol

REFERENCES:

Aranwela et al 1999 New Phytologist

Cornelissen et al 2003 Australian Journal of Botany

Kawai and Okada 2016 Functional Ecology

Kitajima and Poorter 2010 New Phytologist

Lucas and Pereira 1990 Functional Ecology

Lucas et al 2000 Annals of Botany

Onoda et al 2011 Ecology Letters

Onoda et al 2015 Journal of Experimental Botany

Pérez-Harguindeguy et al 2013 Australian Journal of Botany

Read 2001 Austral Ecology

Read and Sanson 2003 New Phytologist

Sanson et al 2001 Austral Ecology

Wright and Cannon 2001 Functional Ecology

ECOLOGICAL IMPLICATIONS: ΔKleaf depends on many factors, such as evaporative demand, leaf ability to reduce water loss (e.g. cuticle permeability), and leaf vascular architecture. Integrated redundancy in the venation (e.g. looping veins) can buffer Kleaf against damages by providing alternative pathways for continued water flow around damaged or blocked veins. Modular redundancy or sectoriality (e.g. veins clusters disconnected from each other) can increase resilience by reducing embolism spreading.

PROTOCOL:  The protocol below shows how to obtain ΔKleaf mesurements after severing the leaf midrib or lamina, using the EFM method (see above) to determine the changes in leaf hydraulic conductance after the damage.

Download protocol

REFERENCES:

Nardini and Salleo (2003) Effects of the experimental blockage of the major veins on hydraulics and gas exchange of Prunus laurocerasus L. leaves. Journal of Experimental Botany,54(385): 1213- 1219. 

Nardini et al (2001) Xylem Cavitation in the Leaf of Prunus laurocerasus and Its Impact on Leaf Hydraulics. Plant Physiol., 125: 1700-1709. 

Delaney et al (2008) Impairment of Leaf Photosynthesis After Insect Herbivory or Mechanical Injury on Common Milkweed, Asclepias syriaca. Environmental Entomology, 37(5): 1332-1343. 

Nabity et al (2009) Indirect suppression of photosynthesis on individual leaves by arthropod herbivory. Annals of Botany 103: 655–663. 

Sack et al (2008) Leaf palmate venation and vascular redundancy confer tolerance of hydraulic disruption, PNAS, 105: 1567–1572. 

Salleo et al (2003) Axial-to-radial water permeability of leaf major veins: a possible determinant of the impact of vein embolism on leaf hydraulics? Plant, Cell and Environment 26: 1749–1758. 

Baldwin (1990) Herbivory Simulations Ecological Research. TREE vol. 5, no. 3, March 1990 

Waterman et al (2019) Simulated Herbivory: The Key to Disentangling Plant Defence Responses. Trends in Ecology & Evolution, 34(5): 447-458. 

ECOLOGICAL IMPLICATIONS: leaf venation network architectural traits (e.g. vein density, degree of loopiness, and shape of loops) can influence different leaf functional axis, such as leaf flow efficiency, resistance and resilience to damages, mechanical support, and leaf construction cost [Read more].

PROTOCOL: the protocols below explain how to chemically clear the leaves and photograph them to obtain high-resolution images of the leaf venation network. And how to use the MATLAB app Leaf vein CNN (available here) to extract leaf venation networks from these cleared leaf images. Besides extracting the venation networks, the Leaf vein CNN app also calculates a series of multiscale leaf venation architecture traits, which describe how venation architecture features vary across vein sizes.

Download protocol [Producing cleared leaf images]

Download protocol [Extracting networks and calculating venation traits in MATLAB]

Download protocol [Hand-tracing venation networks]

Download manual [LeafVeinCNN manual]

REFERENCES:

Blonder et al 2020 New Phytologist

Xu et al 2021 New Phytologist

Niewiadomski et al 2025  Applications in Plant Sciences

ECOLOGICAL CONSIDERATIONS: One important symptom of water stress on plants is the formation and propagation of air bubbles (i.e. cavitation) within the xylem – the interconnected tubular network of cells that transport water from the roots to the rest of the plant. These bubbles tend to expand and block xylem cells (i.e. embolism) which progressively reduces the plant’s ability to transport water, which impacts plant productivity and eventually leads to death. The relationship between embolism formation and the degree of water stress experienced by the plant is known as xylem vulnerability, and is a key metric used to identify and compare drought tolerance between species and individuals. Knowing these differences, and understanding tolerances to levels of water availability, is critical in an age where the limits of agricultural and natural plant communities are being tested by rapid environmental change.

PROTOCOLS: In 2015, researchers at the University of Tasmania, in collaboration with the LiPhy national scientific research centre in Grenoble in France, realised that there was a subtle, but visible, change in the brightness of non-embolised (water-filled) xylem after it transitions to become embolised (air-filled). From this realisation was born a new and exciting, and relatively inexpensive and simple optical method for identifying embolism formation and assessing vulnerability. The optical method is based on the simple principle that light interacts differently with xylem that is water filled vs air filled. This is caused by the way light reflects and refracts as it encounters air-water interfaces. In air-filled xylem there are more air/water interfaces for light to interact with, and this results in more reflections, and a change in brightness, which can be captured using a light sensor. 

The protocol below describes how to obtain leaf vulnerability curves using the optical method, specifically using a custom-made camera (cavicam) and leaf psychrometers for determination of the leaf water potential.

Download Protocol

Download example images 

Download example excel file [Note that this example does not contain water potentials]

REFERENCES:

OpenSourceOV:  https://www.opensourceov.org/

Cavicam: https://cavicam.com/lander

Johnson, K. M., Lucani, C., & Brodribb, T. J. (2022). In vivo monitoring of drought-induced embolism in Callitris rhomboidea trees reveals wide variation in branchlet vulnerability and high resistance to tissue death. New Phytologist, 233(1), 207–218. https://doi.org/10.1111/nph.17786

Johnson, K. M., Brodersen, C., Carins-Murphy, M. R., Choat, B., & Brodribb, T. J. (2020). Xylem Embolism Spreads by Single-Conduit Events in Three Dry Forest Angiosperm Stems1[OPEN]. Plant Physiology, 184(1), 212–222. https://doi.org/10.1104/PP.20.00464

Sorek, Y., Grinshtein, S., Netzer, Y., Shtein, I., Jansen, S., & Hochberg, U. (2020). An increase in xylem embolism resistance of grapevine leaves during the growing season is coordinated with stomatal regulation, turgor loss point, and intervessel pit membranes. New Phytologist. https://doi.org/10.1111/nph.17025

Gauthey, A., Peters, J. M. R., Carins-Murphy, M. R., Rodriguez-Dominguez, C. M., Li, X., Delzon, S., … Choat, B. (2020). Visual and hydraulic techniques produce similar estimates of cavitation resistance in woody species. New Phytologist, 228(3), 884–897. https://doi.org/10.1111/nph.16746

Rodriguez-Dominguez CM, Carins Murphy MR, Lucani C, Brodribb TJ (2018) Mapping xylem failure in disparate organs of whole plants reveals extreme resistance in olive roots. New Phytologist.

Corso, D., Delzon, S., Lamarque, L. J., Cochard, H., Torres-Ruiz, J. M., King, A., & Brodribb, T. (2020). Neither xylem collapse, cavitation, or changing leaf conductance drive stomatal closure in wheat. Plant Cell and Environment, 43(4), 854–865. https://doi.org/10.1111/pce.13722

Cardoso, A. A., Visel, D., Kane, C. N., Batz, T. A., García Sánchez, C., Kaack, L., … McAdam, S. A. M. (2020). Drought-induced lacuna formation in the stem causes hydraulic conductance to decline before xylem embolism in Selaginella. New Phytologist, 227(6), 1804–1817. https://doi.org/10.1111/nph.16649

Suissa, J. S., & Friedman, W. E. (2021). From cells to stems: the effects of primary vascular construction on drought‐induced embolism in fern rhizomes. New Phytologist. https://doi.org/10.1111/nph.17629

Li, X., Blackman, C. J., Peters, J. M. R., Choat, B., Rymer, P. D., Medlyn, B. E., & Tissue, D. T. (2019). More than iso/anisohydry: Hydroscapes integrate plant water use and drought tolerance traits in 10 eucalypt species from contrasting climates. Functional Ecology, 33(6), 1035–1049. https://doi.org/10.1111/1365-2435.13320

Avila, R. T., Cardoso, A. A., Batz, T. A., Kane, C. N., DaMatta, F. M., & McAdam, S. A. M. (2021). Limited plasticity in embolism resistance in response to light in leaves and stems in species with considerable vulnerability segmentation. Physiologia Plantarum, 172(4), 2142–2152. https://doi.org/10.1111/ppl.13450

ECOLOGICAL CONSIDERATIONS: high leaf hydrophobicity can maintain the leaf dry during and/or after leaf wetting events (e.g. fog, mist, haze, rain) and thus prevent gas exchange reductions (CO2 diffusion is much lower in water than in the air), frost, pathogens development, pollutant deposition (pollutants dissolved in the atmospheric water), and photo destruction (water droplets can magnify sun flecks increase photodamage). Alternatively, low leaf hydrophobicity may facilitate foliar water uptake (leaf surface capacity to absorb atmospheric water sources), suppress transpiration (thus reducing leaf water loss during soil drought), and eases heat stress (help reducing leaf surface temperature). 

High angular values (>60°) of leaf retention indicate a greater tendency to retain droplets (i.e. higher leaf water retention). Low angular values (<20°) indicate leaf surfaces that readily shed droplets (i.e. lower leaf water retantion). Low leaf water retention can maintain the leaf dry during and/or after leaf wetting events, thereby preventing gas exchange reductions, frost, phatogens development, pollutant deposition, and pothodestruction. Alternativelly, high leaf water retention may facilitate foliar water uptake, suppress transpiration, and ease heat stress.

PROTOCOLS: The protocol below describes how to calculate the leaf water repellence and retention using a digital camera and the ImageJ software for image processing.

Download protocol

REFERENCES:

Matos IS, & Rosado, B. H. P. (2016) Retain or repel? Droplet volume does matter when measuring leaf wetness traits. Annals of Botany, 117(6): 1045-52.

Aryal, B. & Neuner, G. (2010) Leaf wettability decreases along an extreme altitudinal gradient. Oecologia, 162, 1–9.

Brewer, C.A. (1996) What is so bad about being wet all over. The American Biology Teacher, 58, 413–417.

Rosado, B.H.P. & Holder, C.D. (2013) The significance of leaf water repellency in ecohydrological research: A review. Ecohydrology, 6, 150–161.

Holder, C.D. (2012) The relationship between leaf hydrophobicity, water droplet retention, and leaf angle of common species in a semi-arid region of the western United States. Agricultural and Forest Meteorology, 152, 11–16.

PROTOCOL: The protocol below describes how to determine plant species cover using the pin point method.

Download protocol

REFERENCES:

Coulloudon, B., Eshelman, K., Gianola, J., Habich, N., Hughes, L., Johnson, C., Pellant, M., Podborny, P., Rasmussen, a, Robles, B., Shaver, P., Spehar, J. & Willoughby, J. (1996) Sampling vegetation attributes. BLM Technical Reference, 163.

Caratti, J.F. (2006) Point Intercept ( PO ) Sampling Method. IEEE Signal Processing Magazine, 27, 1–16.

Park, G.N. (1973) Point Height Intercept Analysis A Refinement of Point Analysis for Structural Quantification of Low Arboreal Vegetation. New Zealand Journal of Botany, 11, 103–114.

Hofmann, L. & Ries, R.E. (1990) :An evaluation of sample adequacy for point analysis of ground cover. :J. Range Manage., :43, 545–549.

Goodall, D.W. (1952) Some considerations in the use of point quadrats for the analysis of vegetation. Australian journal of scientific research. Ser. B: Biological sciences, 5, 1–41.

Levy, E. B. and Madden, E. A. (1933). The point method of pasture analysis. N. Z. Jour. Agr. 46: 267-279.

ECOLOGICAL IMPLICATIONS: resprout is a mechanism that allows an individual plant to regenerate after the elimination of its aboveground biomass, hence persist in ecosystems with recurrent disturbances,  caused by fire, herbivory, drought, insect attack. As a broad generalization, species growing in stressful sites or in sites with frequent disturbances are likely to resprout more vigorously and/or to retain their sprouting ability longer than species growing in less stressful sites or in sites with less frequent disturbance regimes.

PROTOCOL: The protocol below described a clipping experimental approach to determine species resprouting capacity, based on Moreira et al. (2012).

Download protocol

REFERENCES:

Bond, W.J. & Midgley, J.J. (2001) Ecology of sprouting in woody plants: The persistence niche. Trends in Ecology and Evolution, 16, 45–51.

Clarke, P.J., Lawes, M.J., Midgley, J.J., Lamont, B.B., Ojeda, F., Burrows, G.E., Enright, N.J. & Knox, K.J.E. (2013) Resprouting as a key functional trait: How buds, protection and resources drive persistence after fire. New Phytologist, 197, 19–35.

Moreira, B., Tormo, J. & Pausas, J.G. (2012) To resprout or not to resprout: Factors driving intraspecific variability in resprouting. Oikos, 121, 1577–1584.

Vesk, P. a. & Westoby, M. (2004) Sprouting ability across diverse disturbances and vegetation types worldwide. Journal of Ecology, 92, 310–320.

Zeppel, M.J.B., Harrison, S.P., Adams, H.D., Kelley, D.I., Li, G., Tissue, D.T., Dawson, T.E., Fensham, R., Belinda, E., Palmer, A., West, A.G. & Mcdowell, N.G. (2014) Drought and resprouting plants. New phytologist, 1–7.

Lloret, F., Verdu, M., Flores-Hernandez, N. & Valiente-Banuet, A. (1999) Fire and resprouting in Mediterranean ecosystems: insights from an external biogeographical region, the mexical shrubland. American Journal of Botany, 86, 1655– 1661

Bond, W. J. and Midgley, J. J. 2003. The evolutionary ecology of sprouting in woody plants. – Int. J. Plant Sci. 164: S103–S114

ECOLOGICAL IMPLICATIONS: plants can improve their drought tolerance by making their leaf water potential more negative as the drought progresses, which allow plants to continue extracting water from the soils. Such reductions in the leaf water potential under drought can be done in three possible ways: (1) osmotic adjustments, by accumulating cell solutes that decrease leaf osmotic potential; (2) apoplastic adjustments, by reducing symplastic or increasing apoplastic  water content via redistribution of water from inside to outside cell walls; and (3) elastic adjustment by increasing cell wall flexibility (i.e. decreasing bulk modulus of elasticity). 

PROTOCOL: The protocol below describes how to obtain pressure volume curves using the bench dry method abd the Sack and Pasquet Kok Excel spreadsheet.

Download protocol 

Download Excel sheet

REFERENCES:

Turner BL, Cernusak AE 2011 Ecology of Podocarpaceae Smithsonian Institute Scholarly Washington DC

Sack L Pasquet kok J 2013 Leaf pressure volume curves parametres Promethrus wiki

Maréchaux, I., Bartlett, M.K., Sack, L., Baraloto, C., Engel, J., Joetzjer, E. & Chave, J. (2015) Drought tolerance as predicted by leaf water potential at turgor loss point varies strongly across species within an Amazonian forest. Functional Ecology, n/a–n/a.

Schulte, P.J. & Hinckley, T.M. (1985) A comparison of pressure-volume curve data analysis techniques. Journal of Experimental Botany, 36, 1590–1602.

Tyree, M.T. & Hammel, H.T. (1972) The measurement of the turgor pressure and the water relations of plants by the pressure- bomb technique. Journal of Experimental Botany, 23, 267–282.

Parker W C and Pallardy S G 1987 The influence of resaturation method and tissue type on pressure-volume analysis of Quercus alba L. seedlings. J. Exp. Bot. 38(188), 535–54

Bartlett, M.K., Scoffoni, C. & Sack, L. (2012) The determinants of leaf turgor loss point and prediction of drought tolerance of species and biomes : a global meta-analysis. Ecology Letters, 15, 393–405. 

Bartlett, M.K., Scoffoni, C., Ardy, R., Zhang, Y., Sun, S., Cao, K. & Sack, L. (2012) Rapid determination of comparative drought tolerance traits: Using an osmometer to predict turgor loss point. Methods in Ecology and Evolution, 3, 880–888. 

Bartlett, M.K., Zhang, Y., Kreidler, N., Sun, S., Ardy, R., Cao, K. & Sack, L. (2014) Global analysis of plasticity in turgor loss point , a key drought tolerance trait. Ecology Letters, 17, 1580–1590. 

ECOLOGICAL IMPLICATIONS: Residual conductance (gres) varies many-fold across species, and can be a potentially strong determinant of drought tolerance, since a lower gres can better enables maintenance of leaf hydration during drought.

PROTOCOL: this protocol explains how to make basic residual conductance (gres) measurements using the method of mass loss of detached leaves.

Download Protocol 

Download Spreadsheet

Download data example

REFERENCES

Araus et al (1991) Epidermal conductance in different parts of durum wheat grown under Mediterranean conditions: the role of epicuticular waxes and stomata

Duursma et al (2018) On the minimum leaf conductance: its role in models of plant water use, and ecological and environmental controls

John et al (2018) Leaf rehydration capacity: associations with other indices of drought tolerance and environment

Kerstiens (1996) Cuticular water permeability and its physiological significance

Muchow & Sinclair (1989) Epidermal conductance stomatal density and stomatal size among genotypes of Sorghum bicoior {L.) Moench

PrometheusWiki

ECOLOGICAL IMPLICATIONS: seed mass and size are related to plant dispersal ability, colonization potential, seedling survival, seed longevity, seed production, and seed persintence in the soil bank.

PROTOCOL: The protocol below describes how to obtain seed traits (mass and size) following (Pérez-Harguindeguy et al 2013).

Download protocol

REFERENCES:

Mazer, S.J. (1989) Ecological, taxonomic, and life history correlates of seed mass among Indiana dune angiosperms. Ecological Monographs, 59, 153–175.

Moles, A.T., Ackerly, D.D., Tweddle, J.C., Dickie, J.B., Smith, R., Leishman, M.R., Mayfield, M.M., Pitman, A., Wood, J.T. & Westoby, M. (2007) Global patterns in seed size. Global Ecology and Biogeography, 16, 109–116.

Moles, A.T., Ackerly, D.D., Webb, C.O., Tweddle, J.C., Dickie, J.B., Pitman, A.J. & Westoby, M. (2005) Factors that shape seed mass evolution. Proceedings of the National Academy of Sciences of the United States of America, 102, 10540–10544.

Pérez-Harguindeguy, N., S., D., Garnier, E., Lavorel, S., Poorter, H., Jaureguiberry, P., Cornwell, W.K., Craine, J.M., Gurvich, D.E., Urcelay, C., Veneklaas, E.J., Reich, P.B., Poorter, L., Wright, I.J., Ray, P., Enrico, L., Pausas, J.G., Vos, A.C. De, Buchmann, N., Funes, G., Hodgson, J.G., Thompson, K., Morgan, H.D., Steege, H., Heijden, M.G.A. Van Der, Sack, L., Blonder, B., Poschlod, P., Vaieretti, M. V, Conti, G., Staver, A.C., Aquino, S. & Cornelissen, J.H.C. (2013) New handbook for standardised measurement of plant functional traits worldwide. Australian Journal of Botany, 61, 167–234.

Thompson, K, Band, S. & Hodgson, J.G. (1993) Seed size and shape predict persistence. Functional Ecology, 7, 236–241.

ECOLOGICAL IMPLICATIONS: water is normally under negative pressure (tension) as it moves through the stem xylem towards the leaves. Due to its meta-stable condition, water inside the xylem conduits is vulnerable to cavitation, i.e. breakage of the water column inside the xylem conduits due to the formation or air bubbles. The tension inside the conduits can promote the expansion of those air bubbles, resulting in embolisms (air blockage), that disrupt the water flow. Cavitation resistance is therefore an important trait, to allow continued water flow and hence continued photosynthesis during drought. 

PROTOCOL: The protocol below describes how to obtain the P50-stem using the bench dry method based on Melcher et al 2012.

Download protocol 

Download R-script

Download example excel file

REFERENCES:

Choat, B., Jansen, S., Brodribb, T.J., Cochard, H., Delzon, S., Bhaskar, R., Bucci, S.J., Feild, T.S., Gleason, S.M., Hacke, U.G., Jacobsen, A.L., Lens, F., Maherali, H., Martínez-Vilalta, J., Mayr, S., Mencuccini, M., Mitchell, P.J., Nardini, A., Pittermann, J., Pratt, R.B., Sperry, J.S., Westoby, M., Wright, I.J. & Zanne, A.E. (2012) Global convergence in the vulnerability of forests to drought. Nature, 491, 752–5.

Cochard, H., Badel, E., Herbette, S., Delzon, S., Choat, B. & Jansen, S. (2013) Methods for measuring plant vulnerability to cavitation: A critical review. Journal of Experimental Botany, 64, 4779–4791.

Zimmermann MH (1983) Xylem Structure and the Ascent of Sap. Springer, Berlin

Wheeler J.K., Sperry J.S., Hacke U.G. & Hoang N. (2005) Inter- vessel pitting and cavitation in Rosaceae and other vesselled plants: a basis for a safety versus efficiency trade-off in xylem transport. Plant, Cell & Environment 28, 800–812

Lo Gullo, M.A. & Salleo, S. (1991) Three different methods for measuring xylem cavitation and embolism: a comparison. Annals of Botany, 67, 417–424.

Maherali, H., Pockman, W.T. & Jackson, R.B. (2004) Adapttive variation in the vulnerability of woody plants to xylem cavitation. Ecology, 85, 2184–2199.

Markesteijn, L., Poorter, L., Paz, H., Sack, L. & Bongers, F. (2011) Ecological differentiation in xylem cavitation resistance is associated with stem and leaf structural traits. Plant, Cell and Environment, 34, 137–148.

Meinzer, F.C., Johnson, D.M., Lachenbruch, B., McCulloh, K. a. & Woodruff, D.R. (2009) Xylem hydraulic safety margins in woody plants: coordination of stomatal control of xylem tension with hydraulic capacitance. Functional Ecology, 23, 922–930.

Melcher, P.J., Holbrook, N.M., Burns, M.J., Zwieniecki, M. a., Cobb, A.R., Brodribb, T.J., Choat, B. & Sack, L. (2012) Measurements of stem xylem hydraulic conductivity in the laboratory and field. Methods in Ecology and Evolution, 3, 685–694.

Ogasa, M., Miki, N.H., Murakami, Y. & Yoshikawa, K. (2013) Recovery performance in xylem hydraulic conductivity is correlated with cavitation resistance for temperate deciduous tree species. Tree physiology, 33, 335–44.

Sperry, J.S., Meinzer, F.C. & McCulloh, K. a. (2008) Safety and efficiency conflicts in hydraulic architecture: Scaling from tissues to trees. Plant, Cell and Environment, 31, 632–645.

Tyree, M.T. & Sperry, J.S. (1989) Vulnerability of xylem to cavitation and embolism. Annual Review of Plant Physiology and Plant Molecular Biology, 40, 19–38.

ECOLOGICAL IMPLICATIONS: Stem specific density (SSD) is a core functional trait related to stability defence, architecture, hydraulics, carbon gain, and growth potential. SSD is also an indicator of the stem vulnerability to cavitation. Higher SSD values are usually linked to higher resistance to xylem embolisms under drought. High SSD  is achieved at the cost of reduced growth rate and stem storage capacity and capacitance, i.e. wood ability to store water, and release it under tension.

PROTOCOL: The protocol below describes how to obtain SSD using the water displacement method.

Download protocol

REFERENCES:

Pérez-Harguindeguy, N., S., D., Garnier, E., Lavorel, S., Poorter, H., Jaureguiberry, P., Cornwell, W.K., Craine, J.M., Gurvich, D.E., Urcelay, C., Veneklaas, E.J., Reich, P.B., Poorter, L., Wright, I.J., Ray, P., Enrico, L., Pausas, J.G., Vos, A.C. De, Buchmann, N., Funes, G., Hodgson, J.G., Thompson, K., Morgan, H.D., Steege, H., Heijden, M.G.A. Van Der, Sack, L., Blonder, B., Poschlod, P., Vaieretti, M. V, Conti, G., Staver, A.C., Aquino, S. & Cornelissen, J.H.C. (2013) New handbook for standardised measurement of plant functional traits worldwide. Australian Journal of Botany, 61, 167–234.

Santiago, L.S., Goldstein, G., Meinzer, F.C., Fisher, J.B., Machado, K., Woodruff, D. & Jones, T. (2004) Leaf photosynthetic traits scale with hydraulic conductivity and wood density in Panamanian forest canopy trees. Oecologia, 140, 543–550.

Goldstein, G., J.L. Andrade, F.C. Meinzer, N.M. Holbrook, J. Cav- elier, P. Jackson and A. Celis. 1998. Stem water storage and diurnal patterns of water use in tropical forest canopy trees. Plant Cell Environ. 21:397–406 

Chave, J., Coomes, D., Jansen, S., Lewis, S.L., Swenson, N.G. & Zanne, A.E. (2009) Towards a worldwide wood economics spectrum. Ecology Letters, 12, 351–366.

Hacke, U.G., Sperry, J.S., Pockman, W.T., Davis, S.D. & McCulloh, K. a. (2001) Trends in wood density and structure are linked to prevention of xylem implosion by negative pressure. Oecologia, 126, 457–461.

ECOLOGICAL IMPLICATIONS: stomata work as pressure regulators that modulate the water vapor exchange and carbon flux between the plant and the atmosphere. Stomatal closure is one of the earliest responses to drought, protecting the plants from extensive water loss, which could result in cell dehydrattion, run-away xylem cavitation, and ultimately plant death.

PROTOCOL: There are different methods to obtain leaf stomatal conductance (gs), the protocol below describes how to obtain gs using a portable leaf porometer.

Download protocol

Download manual

REFERENCES:

Damour, G., Simonneau, T., Cochard, H. & Urban, L. (2010) An overview of models of stomatal conductance at the leaf level. Plant, Cell and Environment, 33, 1419–1438.

Leaf porometer manual: https://www.decagon.com/en/canopy/canopy-measurements/sc-1-leaf-porometer-stomatal-conductance-measurements/

ECOLOGICAL IMPLICATIONS: stomatal density responses to various environmental factors, such as elevated CO2 concentration, heat stress, salt stress, drought, precipitation change, and plant density. Moreover, many studies have shown that water deficit leads to an increase in stomatal density, and a decrease in stomatal size, which may enhance plant capacity to resist drought (Xu & Zhou 2008).

PROTOCOL: The protocol below describes how to obtain stomatal density using the nail impression method.

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REFERENCES:

Xu, Z. & Zhou, G. (2008) Responses of leaf stomatal density to water status and its relationship with photosynthesis in a grass. Journal of Experimental Botany, 59, 3317–3325.

PROTOCOL: 

Download protocol [pressure chamber]

Download manual [pressure chamber]

Download protocol [psychrometer]

Download manual [psychrometer]

REFERENCES:

Bhaskar, R. & Ackerly, D.D. (2006) Ecological relevance of minimum seasonal water potentials. Physiologia Plantarum, 127, 353–359.

Donovan, L. a. (2003) Magnitude and Mechanisms of Disequilibrium Between Predawn Plant and Soil Water Potentials. Ecology, 84, 463–470.

Mitchell, P.J., Veneklaas, E.J., Lambers, H. & Burgess, S.S.O. (2008) Leaf water relations during summer water deficit: Differential responses in turgor maintenance and variation in leaf structure among different plant communities in south-western Australia. Plant, Cell and Environment, 31, 1791–1802.

Myers, A.B.J., Robichaux, R.H., Unwin, G.L. & Craig, I.E. (1987) Leaf Water Relations and Anatomy of a Tropical Rainforest Tree Species Vary with Crown Position. Oecologia, 74, 81–85.

Richter, H. (1997) Water relations of plants in the field : some comments on the measurement of selected parameters. Journal of Experimental Botany, 48, 1–7.

Choat B., Sack L. & Holbrook N.M. (2007) Diversity of hydraulic traits in nine Cordia species growing in tropical forests with contrasting precipitation. New Phytologist 175, 686–698.

Tyree MT, Davis SD, Cochard H (1994) Biophysical per- spectives of xylem evolution: is there a tradeoff of hy- draulic efficiency for vulnerability to dysfunction? Inter- national Association of Wood Anatomists Journal 15: 335–360 

Pockman, W.T. & Sperry, J.S. (2000) Vulnerability to xylem cavitation and the distribution of Sonoran Desert Vegetation. American Journal of Botany, 87, 1287–1299.

Pérez-Harguindeguy, N., S., D., Garnier, E., Lavorel, S., Poorter, H., Jaureguiberry, P., Cornwell, W.K., Craine, J.M., Gurvich, D.E., Urcelay, C., Veneklaas, E.J., Reich, P.B., Poorter, L., Wright, I.J., Ray, P., Enrico, L., Pausas, J.G., Vos, A.C. De, Buchmann, N., Funes, G., Hodgson, J.G., Thompson, K., Morgan, H.D., Steege, H., Heijden, M.G.A. Van Der, Sack, L., Blonder, B., Poschlod, P., Vaieretti, M. V, Conti, G., Staver, A.C., Aquino, S. & Cornelissen, J.H.C. (2013) New handbook for standardised measurement of plant functional traits worldwide. Australian Journal of Botany, 61, 167–234.

Rosado, B.H.P. & de Mattos, E. a. (2010) Interspecific variation of functional traits in a CAM-tree dominated sandy coastal plain. Journal of Vegetation Science, 21, 43–54.

PROTOCOL:  The protocol below describes all the steps to obtain leaf cross-sectional slides to be observed under light microscope. The protocol also describes how to image these slides and use image analysis software to obtain xylem implosion safety and other xylem anatomical features. A Standard Operating Procedure is also provided. 

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