Protocol

 

Author

This article is modified from Perez Harguindeguy et al. (2013). ” The New handbook for standardised measurement of plant functional traits worldwide” is a product of and is hosted by Nucleo Diversus (with additional Spanish translation). For more on this trait and on its context as part of the entire trait handbook visit its primary site Nucleo DiverSus at http://www.nucleodiversus.org/?lang=en

Contributing Author

Ian Wright

Summary

Leaf thickness (L_th, μm or mm) is one of the key components of SLA (Specific Leaf Area) and LDMC (Leaf dry-matter content), because SLA 1/(tissue density x L_th) (where density = dry mass/volume LDMC). L_th also plays a key role in determining the physical strength of leaves (see Fracture toughness). For example, leaf -work to shear’ is (by definition) the product of L_th and tissue toughness. Optimisation theory, balancing photosynthetic benefits against C costs of respiration and transpiration, predicts that L_th should be higher in sunnier, drier and less fertile habitats, as well as in longer-lived leaves. These patterns are indeed often observed, at least in interspecific studies. Within individuals, many studies have shown that outer-canopy -sun’ leaves tend to be thicker than those from more-shaded parts of the canopy. Both within and among species, the strongest anatomical driver of variation in L_th is the number and thickness of mesophyll layers. Consequently, L_th is a strong driver of leaf N per area. Although higher L_th should lead to faster photosynthetic rates per unit LA (via a higher N : area ratio), this relationship is often weak in interspecific studies, for a combination of reasons. First, because of covariance of SLA and %N, thicker leaves often have lower %N and longer leaf-lifespan (which are associated with lower photosynthetic rate per unit leaf mass). Second, thicker-leaved species may have slower CO₂ diffusion (lower mesophyll conductance) via longer diffusion pathways, greater internal self-shading of chloroplasts, or higher optical reflectivity in combination with lower internal transmittance. Thick leaves are also a feature of succulents.

Procedure

Follow similar procedures as for Specific leaf area In many cases, the same leaves will be used for the determination of SLA, L_th and LDMC (and perhaps Leaf fracture properties). For recommended sample size, see Table 1.

Storing and processing

Similarly as for SLA. L_th is strongly affected by LWC; hence, some form of rehydration should be seriously considered, as described for SLA, particularly if using a digital micrometer, where any slight loss of turgor results in an underestimation.

Measuring

Thickness tends to vary over the surface of the leaf, generally being thickest at the midrib, primary veins, margins and leaf base. Depending on the research question, you may be interested in the average thickness across the leaf, or the thickness at special locations or of special tissues. Often one measurement per leaf, at a position as standard as possible within the lamina (e.g. at an intermediate position between the border and the midrib, and between the tip and the base of the leaf, avoiding important secondary veins) is acceptable for broad interspecific comparisons. When more precision is needed, the average of thickness measurements at several points in the lamina will be more appropriate. Another way to estimate the average thickness over the entire leaf surface is to back-calculate it from the leaf volume divided by LA; however, it is laborious to accurately measure leaf volume, e.g. with a pycnometer. A relatively fast approximation of whole-leaf average L_th can be obtained by dividing leaf fresh mass by LA (which is the same as calculating 1/SLA x LDMC), i.e. by assuming that leaf fresh mass and volume are tightly related. This approach does not take into account the higher density of dry material in the leaf, or the lower density as a result of intercellular spaces; however, as an approximation it works well.

Other approaches are needed if one wants to distinguish between thickness of midrib, margin and intercostal regions of the leaf, or to compare replicates at a given point on the leaf, e.g. half-way between the leaf base and the tip, as is commonly done. One method is to measure these quantities directly from leaf cross-sections (hand-sections), or to use image analysis (see Specific leaf area for links to free software) to calculate average L_th across the section, by dividing the total cross-sectional area by the section width. On the positive side, this method enables reasonably accurate measurements to be made. On the down side, soft tissue may distort when hand-sectioned, and the method is relatively slow (e.g. 15 min per measurement).

Probably the fastest approach is to measure L_th using a dial-gauge or a digital micrometer (or even a linear variable displacement transducer; LVDT). Multiple measurements can be made within quick succession and averaged to give an indicative value of L_th for the feature in question (such as e.g. midrib or lamina between the main veins) or region of interest (e.g. near midpoint of leaf). If necessary, we recommend replacing the original contact points on the micrometer with contacts 2-3 mm in diameter; i.e. narrow enough to fit between major veins, but sufficiently broad so as not to dent the leaf surface when making measurements. However, for soft-leaved species such as Arabidopsis, permanent deformation is difficult to avoid.

Notes and troubleshooting tips

1. Needle leaves For needle leaves that are circular in cross-section, average L_th can be quickly estimated as Diameter x ℼ/4 (equivalent to cross-sectional area divided by cross-section width). Still, because needle leaves typically taper towards the leaf tip, several measurements would normally need to be made.

Literature references

References on theory, significance and large datasets:

Clements ES (1905) The relation of leaf structure to physical factors.Transactions of the American Microscopical Society 26, 19-102 doi:102307/3220956

Díaz S, Hodgson JG, Thompson K, Cabido M, Cornelissen JHC, Jalili A, Montserrat-Martí G, Grime JP, Zarrinkamar F, Asri Y, Band SR, Basconcelo S, Castro-Díez P, Funes G, Hamzehee B, Khoshnevi M, Pérez-Harguindeguy N, Pérez-Rontomé MC, Shirvany FA, Vendramini F, Yazdani S, Abbas-Azimi R, Bogaard A, Boustani S, Charles M, Dehghan M, de Torres-Espuny L, Falczuk V, Guerrero-Campo J, Hynd A, Jones G, Kowsary E, Kazemi-Saeed F, Maestro-Martínez M, Romo-Díez A, Shaw S, Siavash B, Villar-Salvador P, ZakMR (2004) The plant traits that drive ecosystems: evidence from three continents. Journal of Vegetation Science 15, 295-304

Enríquez S, Duarte CM, Sand-Jensen K, Nielsen SL (1996) Broad-scale comparison of photosynthetic rates across phototrophic organisms. Oecologia 108, 197-206.

Givnish TJ (1979) On the adaptive significance of leaf form. In –Topics in plant population biology‘. Ed. OT Solbrig, S Jain, GB Johnson, PH Raven. pp. 375-407. Macmillan: London

Green DS, Kruger EL (2001) Light-mediated constraints on leaf function correlate with leaf structure among deciduous and evergreen tree species.Tree Physiology 21, 1341-1346 doi:101093/treephys/21181341

Knapp AK, Carter GA (1998) Variability in leaf optical properties among 26 species from a broad range of habitats. American Journal of Botany 85, 940-946 doi:102307/2446360

Niinemets Ü (2001) Global-scale climatic controls of leaf dry mass per area,density and thickness in trees and shrubs. Ecology 82, 453-469 doi:101890/0012-9658(2001)0820453:GSCCOL20CO;2

Parkhurst DF (1994) Diffusion of CO₂and other gases inside leaves. New Phytologist 126, 449-479 doi:101111/j1469-81371994tb04244x

Smith WK, Bell DT, Shepherd KA (1998) Associations between leaf structure, orientation, and sunlight exposure in five Western Australian communities. American Journal of Botany 85, 56-63 doi:102307/2446554

Wilson PJ, Thompson K, Hodgson JG (1999) Specific leaf area and leaf dry matter content as alternative predictors of plant strategies. New Phytologist 143, 155-162 doi:101046/j1469-8137199900427x

More on methods:

Garnier E, Laurent G (1994) Leaf anatomy, specific mass and water content in congeneric annual and perennial grass species. New Phytologist 128, 725-736 doi:101111/j1469-81371994tb04036x

Hodgson JG, Montserrat-Martí G, Charles M, Jones G, Wilson P, Shipley B, Sharafi M, Cerabolini BEL, Cornelissen JHC, Band SR, Bogard A, Castro-Díez P, Guerrero-Campo J, Palmer C, Pérez-Rontomé MC, Carter G, Hynd A, Romo-Díez A, de Torres Espuny L, Royo Pla F(2011) Is leaf dry matter content a better predictor of soil fertility than specific leaf area Annals of Botany 108, 1337-1345 doi:101093/aob/mcr225

Poorter H, Niinemets Ü, Poorter L, Wright IJ, Villar R (2009) Causes and consequences of variation in leaf mass per area (LMA): a meta-analysis. New Phytologist 182, 565-588 doi:101111/j1469-8137200902830x

Shipley B (1995) Structured interspecific determinants of specific leaf area in 34 species of herbaceous angiosperms. Functional Ecology 9, 312-319 doi:102307/2390579

Vile D, Garnier E, Shipley B, Laurent G, Navas M-L, Roumet C, Lavorel S, Díaz S, Hodgson JG, Lloret F, Midgley GF, Poorter H, Rutherford MC, Wilson PJ, Wright IJ (2005) Specific leaf area and dry matter content estimate thickness in laminar leaves. Annals of Botany 96, 1129-1136 doi:101093/aob/mci264

Witkowski ETF, Lamont BB (1991) Leaf specific mass confounds leaf density and thickness. Oecologia 88, 486-493

Wright IJ, Westoby M (2002) Leaves at low versus high rainfall: coordination of structure, lifespan and physiology. New Phytologist 155, 403-416 doi:101046/j1469-8137200200479x