Amphistomy increases leaf photosynthesis more in coastal than montane plants of Hawaiian ʻilima

Triplett, Genevieve1 ; Buckley, Thomas 2 ; Muir, Christopher 3

Published Jan 07, 2024 on Dryad. https://doi.org/10.5061/dryad.rxwdbrvfw

Data files

Jan 07, 2024 version files 44.23 KB

README.md

2.73 KB
stomata-ilima.csv

41.50 KB

Abstract

Premise of the study

The adaptive significance of stomata on both upper and lower leaf surfaces, called amphistomy, is unresolved. A widespread association between amphistomy and open, sunny habitats suggests the adaptive benefit of amphistomy may be greatest in these contexts, but this hypothesis has not been tested experimentally. Understanding amphistomy informs its potential as a target for crop improvement and paleoenvironment reconstruction.

Methods

We developed a method to quantify "amphistomy advantage", AA, as the log-ratio of photosynthesis in an amphistomatous leaf to that of the same leaf but with gas exchange blocked through the upper surface (pseudohypostomy). Humidity modulated stomatal conductance and thus enabled comparing photosynthesis at the same total stomatal conductance. We estimated AA and leaf traits in six coastal (open, sunny) and six montane (closed, shaded) populations of the indigenous Hawaiian species ʻilima (Sida fallax).

Key results

Coastal ʻilima benefits 4.04 times more from amphistomy than montane leaves. Evidence was equivocal with respect to two hypotheses – that coastal leaves benefit more because 1) they are thicker and have lower conductance through the internal airspace, and 2) they benefit more because they have similar conductance on each surface, as opposed to most conductance being through the lower surface.

Conclusions

This is the first direct experimental evidence that amphistomy increases photosynthesis, consistent with the hypothesis that parallel pathways through upper and lower mesophyll increase CO2 supply to chloroplasts. The prevalence of amphistomatous leaves in open, sunny habitats can partially be explained the increased benefit of amphistomy in 'sun' leaves, but the mechanistic basis remains uncertain.

https://doi.org/10.5061/dryad.rxwdbrvfw

This dataset contains leaf trait data for each individual 'ilima individual and/or leaf in our sample.

Description of the data and file structure

The data are structured as a comma separated value (CSV) file with 185 rows and 27 columns:

genus: genus name
species: specific epithet
authority: authority
exposition: sampled from 'natural environment' or 'experimental growth conditions'
plant_maturity: 'juvenile' or 'mature' plant
leaf_age: 'young', 'youthful', 'mature', 'old', or 'senescent' leaf according to Bowman and Box 1983
site: freeform site name
site_code: four-letter site code
site_type: 'coastal' or 'montane' site type
island: island where site is located (oahu = O'ahu; hawaii = Hawai'i, aka Big Island)
latitude_degree: approximate site latitude in decimal degrees
longitude_degree: approximate site longitude in decimal degrees
elevation_m_site: site elevation in meters above sea level
elevation_m_plant: plant elevation in meters above sea level
date_sampled: date of sampling in YYYY-MM-DD format
plant_id: id of individual plant within site (i1, i2, ...)
leaf_id: id of individual leaf within plant (l1, l2, ...)
lower_number_of_stomata: count of stomata on lower (abaxial) surface per 0.890 mm^2 field
upper_number_of_stomata: count of stomata on upper (adaxial) surface per 0.890 mm^2 field
lower_stomatal_density_mm2: stomatal density per mm^{2 on lower (abaxial) surface, calculated as lower_number_of_stomata / 0.890 mm}2
upper_stomatal_density_mm2: stomatal density per mm^{2 on upper (adaxial) surface, calculated as upper_number_of_stomata / 0.890 mm}2
lower_gcl_um: lower (abaxial) guard cell length (gcl) in um
upper_gcl_um: upper (adaxial) guard cell length (gcl) in um
leaf_thickness_um: leaf lamina thickness from upper cuticle to lower cuticle in um
Tleaf: leaf temperature for A and gsw in degree C
A: photosynthetic CO2 assimilation rate in umol / m^2 / s
gsw: stomatal conductance to water vapor in mol / m^2 / s

Missing data are indicated by NA.

Sharing/Access information

Links to other publicly accessible locations of the data:

https://www.try-db.org/

Code/Software

All code associated with these data and related publications is available on GitHub and archived on Zenodo:

Plant sampling and climate

We identified 7 suitable natural populations of ʻilima on Oʻahu and 5 on Hawaiʻi Island by consulting Yorkston and Daehler (2006) and citizen scientist records on iNaturalist (Anon, 2022). We avoided sites that appeared to be cultivated. We visited sites between August and November 2022. For logistical reasons, the sites on Hawaiʻi were sampled during one three-day trip. We haphazardly sampled eight plants distributed evenly between the highest and lowest elevation plants along a transect at each site. For safety and conservation reasons, transects were along a trail or road. We did not sample small individuals if there was risk removing leaves would cause mortality. From each plant, we collected two fully expanded leaves for trait measurements. We sampled stomatal traits on all leaves; leaf thickness on one leaf from three randomly selected plants per site; and, due to limited time, a single leaf from a single plant at the middle of each transect for gas exchange measurements.

Leaf traits

Stomata

We estimated the stomatal density and size on ab- and adaxial leaf surfaces from all leaves. For pubescent leaves (usually coastal), we dried and pressed leaves for 1 week (Hill et al., 2014), carefully scraped trichomes off with a razor blade, and rehydrated the leaf. Rehydration restores leaf area to its fresh value (Blonder et al., 2012). For glabrous leaves, we used fresh leaves. We applied clear nail polish to both leaf surfaces of fresh or rehydrated leaves in the middle of the lamina away from major veins. After nail polish dried, we mounted impressions on a microscope slide using transparent tape (Mott and Michaelson, 1991). We digitized a portion of each leaf surface impression using a brightfield microscope (Leica DM2000, Wetzlar, Germany). We counted all stomata and divided by the visible leaf area (0.890 mm2) to estimate density and measured guard cell length from five randomly chosen stomata per field using ImageJ (Schneider et al., 2012).

Leaf thickness

We cut thin sections using two razor blades taped together. We sectioned the leaf in a petri dish of water, wet-mounted sections onto a slide, and took digital micrographs using a brightfield microscope, as described above. Leaf thickness is measured as the length from upper cuticle to lower cuticle.

Gas exchange measurements

At each site, we selected one representative leaf from one plant near the middle of the transect for gas exchange measurements using a portable infrared gas analyzer (LI-6800PF, LI-COR Biosciences, Lincoln, Nebraska, USA). We estimated the photosynthetic rate (A) and stomatal conductance to water vapor (g_sw) at saturating light (photosynthetic photon flux density (PPFD) = 2000 μmol m^-2 s^-1}, ambient CO₂ (415 ppm), and T_leaf= 25.0-29.3 °C. The midday irradiance in coastal ʻilima typically meets or even exceeds a PPFD of 2000 μmol m-² s^-1 and previous experiments with sun leaves revealed that is always at or near saturating irradiance. Even though lower irradiance may be saturating for montane leaves, we used this higher value for all leaves to standardize conditions.

Literature Cited

Anon. 2022. iNaturalist.

Blonder, B., V. Buzzard, I. Simova, L. Sloat, B. Boyle, R. Lipson, B. Aguilar-Beaucage, et al. 2012. The leaf-area shrinkage effect can bias paleoclimate and ecology research. American Journal of Botany 99: 1756–1763.

Hill, K. E., G. R. Guerin, R. S. Hill, and J. R. Watling. 2014. Temperature influences stomatal density and maximum potential water loss through stomata of Dodonaea viscosa subsp. Angustissima along a latitude gradient in southern Australia. Australian Journal of Botany 62: 657.

Mott, K. A., and O. Michaelson. 1991. Amphistomy as an adaptation to high light intensity in Ambrosia cordifolia (Compositae). American Journal of Botany 78: 76–79.

Schneider, C. A., W. S. Rasband, and K. W. Eliceiri. 2012. NIH Image to ImageJ: 25 years of image analysis. Nature Methods 9: 671–675.

Yorkston, M., and C. C. Daehler. 2006. Interfertility between Hawaiian ecotypes of Sida fallax (Malvaceae) and evidence of a hybrid disadvantage. International Journal of Plant Sciences 167: 221–230.

Amphistomy increases leaf photosynthesis more in coastal than montane plants of Hawaiian ʻilima

Data files

Abstract

README: Data from: Amphistomy increases leaf photosynthesis more in coastal than montane plants of Hawaiian ʻilima (Sida fallax)

Description of the data and file structure

Sharing/Access information

Code/Software

Methods

Works referencing this dataset