Leaf trait covariation and controls on leaf mass per area (LMA) following cotton domestication

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.Funding provided by: National Natural Science Foundation of ChinaCrossref Funder Registry ID: http://dx.doi.org/10.13039/501100001809Award Number: 31860355Funding provided by: National Natural Science Foundation of ChinaCrossref Funder Registry ID: http://dx.doi.org/10.13039/501100001809Award Number: U1803234Funding provided by: Plan for Training Youth Innovative Talent in Shihezi University*Crossref Funder Registry ID: Award Number: CXRC201701Funding provided by: The 111 Project*Crossref Funder Registry ID: Award Number: D20018Funding provided by: ARC Centre for Plant Success in Nature & Agriculture*Crossref Funder Registry ID: Award Number: CE200100015Funding provided by: China Scholarship CouncilCrossref Funder Registry ID: http://dx.doi.org/10.13039/501100004543Award Number: 201909505015Plant material 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-1 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-1 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-1 of compound fertilizer before sowing and an additional 375 kg ha-1 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-1 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. Gas exchange measurements The photosynthesis and stomatal conductance measurements were taken from a companion study, which focused on mesophyll conductance in wild and domesticated genotypes (Lei et al., 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–1. Light intensity in the leaf chamber was 2000 μmol m–2 s–1 photosynthetic photon flux density (PPFD) with 10 % blue light and 90 % red light, and ambient CO2 concentration was maintained at 400 μmol mol–1. 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. Measurement of anatomical traits Small leaf sections of approximately 4 × 1 mm2 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-1 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 et al., 2012). Leaf volume per area (Vleaf; μm3 μm-2) was equivalent to leaf thickness. Epidermis volume per area (Vepi), palisade mesophyll volume per area (Vpmes), palisade airspace volume per area (Vpair), vascular bundle volume per area (Vvas), spongy mesophyll volume per area (Vsmes) and spongy airspace volume per area (Vsair) 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 Vleaf (Triarhou, 1847). Epidermis cell size (Sepi), palisade mesophyll cell size (Spmes), spongy mesophyll cell size (Ssmes) were measured as its area in the leaf cross-section (a proxy of cell size). Palisade mesophyll cell length (Lpmes), palisade mesophyll cell width (Wpmes), spongy mesophyll cell length (Lsmes), spongy mesophyll cell width (Wsmes), epidermis cell number per area (Nepi), palisade mesophyll cell number per area (Npmes) and spongy mesophyll cell number per area (Nsmes) 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 (Tcw) 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 et al., 2017; Théroux-Rancourt and Gilbert. 2017; Harwood et al., 2021). Nonetheless, our comparisons of domesticated and wild genotypes are meaningful, given that we applied a consistent methodology across all our samples. Determination of leaf mass per area LMA measurements were taken from the companion study (Lei et al., 2021a) but briefly, eight randomly selected leaf discs that included major veins (1.56 cm2 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-2) was computed as the ratio of dry mass to leaf area. Leaf density (LD) was defined as LMA divided by Vleaf. We note that LMA was measured using a larger surface area than that used for the anatomical measurements (4 mm2), and this could potentially result in minor discordance between the traits. The relatively small difference in fertilizer amount (1350 kg ha-1 for domesticated genotypes VS 1275 kg ha-1 for wild genotypes) may, to some extent contribute to the differences in LMA, its underlying components and physiological traits