DOI: 10.5061/dryad.z08kprrft
title: Leaf trait covariation and controls on leaf mass per area (LMA) following cotton domestication

description: The process of domestication has driven dramatic shifts in plant
 functional traits including leaf mass per area (LMA). It remains unclear
 whether domestication has produced concerted shifts in the lower-level
 anatomical traits that underpin LMA and how these traits in turn affect
 photosynthesis. In this study, we investigated controls of LMA
 and leaf gas exchange by leaf anatomical properties at the cellular,
 tissue and whole leaf levels, comparing 26 wild and 31 domesticated
 genotypes of cotton (Gossypium). As expected, domesticated plants
 expressed lower LMA, higher photosynthesis and stomatal conductance,
 suggesting a shift towards the ‘faster’ end of the leaf economics
 spectrum. At whole-leaf level, variation in LMA was predominantly
 determined by leaf density (LD) both in wild and domesticated genotypes.
 At tissue level, higher leaf volume per area (Vleaf) in domesticated
 genotypes was driven by a simultaneous increase in the volume of
 epidermal, mesophyll and vascular bundle tissue and airspace, while lower
 LD resulted from a dilution effect of lower increased volume of palisade
 tissue and vascular bundle of high mass density by higher increased volume
 of epidermis and airspace of low mass density. The volume of spongy
 mesophyll exerted direct control on photosynthesis in domesticated
 genotypes but only indirect control in wild genotypes. At cellular level,
 a shift to larger but less numerous cells with thinner cell walls
 underpinned a lower proportion of cell wall mass, and thus a reduction in
 LD. Taken together, cotton domestication has triggered
 synergistic shifts in the underlying determinants of LMA but also
 photosynthesis, at cell, tissue and whole-leaf level, resulting in a
 marked shift in plant ecological strategy.
description: <em>Plant
 material</em> We performed a seven-month
 experiment within the National Wild Cotton Nursery, Sanya (18°
 40′ N, 109°17′ E), China. Wild genotypes (26
 perennial accessions) and seeds of domesticated genotypes (31 annual
 varieties/races; Table S1) were transplanted and sown in two identical,
 nearby experimental fields on October 2017,
 respectively. 750 kg ha<sup>-1
 </sup>of compound fertilizer (15% N, 15% P, 15% K) were applied
 uniformly to the plots of wild genotypes before transplanting seedlings,
 and an additional 600 kg ha<sup>-1 </sup>of compound
 fertilizer was concomitantly applied during the development of flowers and
 bolls. The plots of the domesticated genotypes were uniformly fertilized
 with 900 kg ha<sup>-1 </sup>of compound fertilizer before
 sowing and an additional 375 kg ha<sup>-1 </sup>of compound
 fertilizer during the development of flowers and bolls. The fertilizer
 amounts for wild and domesticated genotypes were chosen based on our
 relevant experience growing cotton for scientific purposes. Weed and pest
 control measurements were implemented following local management
 practices. Mean precipitation and temperature during the cotton growth
 period were 176 mm month<sup>-1</sup> and 22℃, respectively.
 We sampled only fully mature, uppermost leaves on the main stem, with 3-5
 replicates (i.e. individual) for each genotype at boll stage in January
 2018.   <em>Gas exchange
 measurements</em> The photosynthesis and stomatal
 conductance measurements were taken from a companion study, which focused
 on mesophyll conductance in wild and domesticated genotypes (Lei
 <em>et al</em>., 2021a) but in short, an open gas exchange
 system (Li-6400; Li-Cor, Inc., Lincoln, NE, USA) equipped with a leaf
 fluorometer chamber (Li-6400-40) was used to analyze steady-state gas
 exchange rates. Measurements were performed at a leaf temperature of 30 ±
 2 ℃ and vapor pressure deficit of 1.6 - 2.5 kPa. Gas flow rate was 400
 µmol s<sup>–1</sup>. Light intensity in the leaf chamber was
 2000 μmol m<sup>–2</sup> s<sup>–1</sup>
 photosynthetic photon flux density (PPFD) with 10 % blue light and 90 %
 red light, and ambient CO<sub>2</sub> concentration was
 maintained at 400 μmol mol<sup>–1</sup>. Measurements were
 recorded only after steady-state was reached (at least 2
 minutes). All gas exchange measurements
 were conducted in the middle of the leaf, avoiding the
 midrib. <strong> </strong> <em>Measurement of anatomical
 traits</em> Small leaf sections of
 approximately 4 × 1 mm<sup>2</sup>
 were excised from the central portion of each leaf. The tissue was
 immediately saturated in a solution of 2.5% glutaraldehyde
 and 3% paraformaldehyde in 0.1 mol L<sup>-1</sup> phosphate
 buffer (pH 7.2) at 4℃ using a centrifuge tube. The leaf sections were then
 fixed in 1% osmium tetroxide and dehydrated in a series of increasingly
 stronger ethanol solutions. After dehydration, the samples were embedded
 in Spurr’s resin (Spon 812) and subsequently polymerized for analyses via
 both light microscopy and transmission electron microscope (TEM).
 Semi-thin leaf cross-sections of 1 µm were used for light microscopy and
 ultra-thin cross-sections of 70 nm were used for TEM, which were obtained
 using an ultramicrotome (Leica Ultracut, Wetzlar, Germany).
 For the light microscopy
 observations, semi-thin cross-sections were stained with 1% toluidine blue
 and photographed at 20× and 40× magnifications (Leica DM2500, Wetzlar,
 Germany). All abbreviations of the traits are listed in the Table 1. We
 analyzed all images using ImageJ software (Schneider <em>et
 al</em>., 2012). Leaf volume per area
 (<em>V</em><sub>leaf</sub>;
 μm<sup>3</sup> μm<sup>-2</sup>) was equivalent to
 leaf thickness. Epidermis volume per area
 (<em>V</em><sub>epi</sub>), palisade mesophyll
 volume per area (<em>V</em><sub>pmes</sub>),
 palisade airspace volume per area
 (<em>V</em><sub>pair</sub>), vascular bundle
 volume per area (<em>V</em><sub>vas</sub>), spongy
 mesophyll volume per area
 (<em>V</em><sub>smes</sub>) and spongy airspace
 volume per area (<em>V</em><sub>sair</sub>) were
 calculated as the proportion of epidermis, palisade mesophyll, palisade
 airspace, vascular bundle, spongy mesophyll and spongy airspace in the
 leaf cross-section, respectively, multiplied by
 <em>V</em><sub>leaf</sub> (Triarhou, 1847). Epidermis cell
 size (<em>S</em><sub>epi</sub>), palisade
 mesophyll cell size (<em>S</em><sub>pmes</sub>),
 spongy mesophyll cell size
 (<em>S</em><sub>smes</sub>) were measured as its
 area in the leaf cross-section (a proxy of cell size). Palisade mesophyll
 cell length (<em>L</em><sub>pmes</sub>), palisade
 mesophyll cell width (<em>W</em><sub>pmes</sub>),
 spongy mesophyll cell length
 (<em>L</em><sub>smes</sub>), spongy mesophyll cell
 width (<em>W</em><sub>smes</sub>), epidermis cell
 number per area (<em>N</em><sub>epi</sub>),
 palisade mesophyll cell number per area
 (<em>N</em><sub>pmes</sub>) and spongy mesophyll
 cell number per area (<em>N</em><sub>smes</sub>)
 were determined from the microscopic images of the leaf cross-section. For
 TEM, ultra-thin cross sections were stained with uranyl acetate and lead
 citrate double staining, and then observed under a transmission electron
 microscope (TEM HT7700, Tokyo, Japan), and images were
 photographed with a digital camera (BH-2, Olympus, Tokyo,
 Japan) at 3000×, 15000×
 and 30000× magnifications. Mesophyll cell wall thickness
 (<em>T</em><sub>cw</sub>) was determined from the
 TEM images. We note that using
 two-dimensional images for estimation of leaf volumetric properties has
 its limitations, as leaves are three-dimensional organs that can exhibit
 pronounced internal anatomical variation that is imperceptible using
 two-dimensional images (Théroux-Rancourt <em>et al</em>.,
 2017; Théroux-Rancourt and Gilbert. 2017; Harwood <em>et
 al</em>., 2021). Nonetheless, our comparisons of domesticated and
 wild genotypes are meaningful, given that we applied a consistent
 methodology across all our samples.   <em>Determination of leaf mass per
 area </em> LMA measurements were taken from
 the companion study (Lei <em>et al</em>., 2021a) but briefly,
 eight randomly selected leaf discs that included major veins (1.56
 cm<sup>2</sup> for each disc) were excised from entire leaves,
 and dried at 60°C for at least 48 h. Leaf mass per area (LMA; g
 m<sup>-2</sup>) was computed as the ratio of dry mass to leaf
 area. Leaf density (LD) was defined as LMA divided by
 <em>V</em><sub>leaf</sub>. We note that LMA was
 measured using a larger surface area than that used for the anatomical
 measurements (4 mm<sup>2</sup>), and
 this could potentially result in minor discordance between the traits.
 The relatively small difference
 in fertilizer amount (1350 kg ha<sup>-1</sup> for domesticated
 genotypes <em>VS</em> 1275 kg ha<sup>-1</sup> for
 wild genotypes) may, to some extent contribute to the differences in LMA,
 its underlying components and physiological traits.