TY - JOUR
AU1 - Nadal, Miquel
AU2 - Perera-Castro, Alicia V
AU3 - Gulías, Javier
AU4 - Farrant, Jill M
AU5 - Flexas, Jaume
AB - Abstract Resurrection plants are vascular species able to sustain extreme desiccation in their vegetative tissues. Despite its potential interest, the role of leaf anatomy in CO2 diffusion and photosynthesis under non-stressed conditions has not been explored in these species. Net CO2 assimilation (An) and its underlying diffusive, biochemical, and anatomical determinants were assessed in 10 resurrection species from diverse locations, including ferns, and homoiochlorophyllous and poikilochlorophyllous angiosperms. Data obtained were compared with previously published results in desiccation-sensitive ferns and angiosperms. An in resurrection plants was mostly driven by mesophyll conductance to CO2 (gm) and limited by CO2 diffusion. Resurrection species had a greater cell wall thickness (Tcw) than desiccation-sensitive plants, a feature associated with limited CO2 diffusion in the mesophyll, but also greater chloroplast exposure to intercellular spaces (Sc), which usually leads to higher gm. This combination enabled a higher An per Tcw compared with desiccation-sensitive species. Resurrection species possess unusual anatomical features that could confer stress tolerance (thick cell walls) without compromising the photosynthetic capacity (high chloroplast exposure). This mechanism is particularly successful in resurrection ferns, which display higher photosynthesis than their desiccation-sensitive counterparts. Cell wall thickness, chloroplast distribution, desiccation tolerance, leaf anatomy, mesophyll conductance, photosynthesis, resurrection plants Introduction Desiccation-tolerant or ‘resurrection’ plants are species that can withstand almost complete depletion of water (<10% relative water content) in their vegetative tissues for relatively long periods of time (Gaff, 1977; Gaff and Oliver, 2013). This unique trait is common among non-tracheophytes but is rare in the sporophytes of pteridophytes (64 species, or 1% of ferns) and angiosperms (135 species, or 0.04% of flowering plants) and is completely absent in extant gymnosperms (Gaff and Oliver, 2013). The mechanisms employed by resurrection plants to cope with this extreme water loss are complex and, while certain commonalities exist (accumulation of sugars, protective pigments, and proteins, high antioxidant potential), there is still considerable variation among species as to how desiccation tolerance is ultimately achieved (see Oliver et al., 2020). This is illustrated by the mechanisms shown to combat mechanical stress during desiccation, which range from species showing little wall folding but with considerable increases in vacuolation (these containing non-aqueous metabolites) to those with extremely flexible walls with little vacuolation (Farrant et al., 2007; Oliver et al., 2020). The ‘management’ of oxidative stress associated with photosynthesis is also executed differently among resurrection plants, differentiating between homoiochlorophyllous and poikilochlorophyllous species (Fernández-Marín et al., 2016). The former are typified by retention of chlorophyll and maintenance and protection of the photosynthetic apparatus during desiccation, thus enabling a rapid return of metabolic competence on rehydration. In the latter, chlorophyll is degraded, and thylakoids are dismantled during desiccation and regenerated upon rehydration. Notably, resurrection plants have been envisaged as potential sources for crop improvement to face growing water shortages in the future (Moore et al., 2009; Costa et al., 2017b; Hilhorst and Farrant 2018), and much of the current research focuses on the genomic and transcriptomic aspects of desiccation tolerance (Dinakar and Bartels, 2013; Costa et al., 2017a; Giarola et al., 2017; VanBuren et al., 2018). However, additional physiological processes associated with vegetative desiccation tolerance have not received much attention. For instance, photosynthesis in resurrection plants has been studied mostly in the context of water deficit stress and recovery upon rehydration (reviewed in Dinakar et al., 2012; Fernández-Marín et al., 2016). Only a few studies have addressed other aspects of photosynthesis, such as carbon balance differences among desiccation-tolerant and desiccation-sensitive species of Velloziaceae (Alcantara et al., 2015) and the response of Haberlea rhodopensis to irradiance regimes (Rapparini et al., 2015). Nonetheless, resurrection plants could constitute a key group to further our understanding of photosynthesis and its anatomical basis. Leaf photosynthetic capacity is usually measured as net CO2 assimilation rate (An), which is determined by both biophysical (CO2 diffusion through the stomata and the mesophyll) and biochemical processes (CO2 fixation and the associated photochemistry) (Sharkey et al., 2007). Mesophyll conductance to CO2 diffusion (gm), which integrates the CO2 path inside leaves from the substomatal air spaces to the sites of carboxylation inside chloroplasts, is one of the main parameters determining photosynthetic performance (Flexas et al., 2008, 2012; Niinemets et al., 2009; Nadal and Flexas, 2019; Flexas and Carriquí, 2020), and its structural drivers—mainly chloroplast distribution and cell wall thickness (Tcw)—are key features in shaping An across land plants (Onoda et al., 2017; Gago et al., 2019). To our knowledge, none of these traits has been characterized for any resurrection species under non-stressed conditions. In addition, some studies point towards a link between water stress tolerance and features such as Tcw and photosynthetic capacity (Peguero-Pina et al., 2017a; Nadal et al., 2018), although not at the extreme extent that desiccation implies. This potential trade-off may apply to desiccation-tolerant plants, which are allegedly limited in growth rates and size (Alpert, 2006). Hence, the main objective of the present work was to characterize photosynthesis characteristics and their possible anatomical basis in several C3 resurrection species. To broaden this objective, we utilized diverse species, including both resurrection ferns and angiosperms, and within the latter both homoiochlorophyllous and poikilochlorophyllous species. Furthermore, we compare data obtained from the selected species with those reported for desiccation-sensitive plant species to distinguish potential special features of resurrection ferns and angiosperms regarding their photosynthetic productivity under well-watered conditions. Materials and methods Plant material The present study combines measurements of 10 resurrection species from several locations, some of them analysed under natural field conditions and some in nurseries, greenhouses, and growth chambers. The detailed list of included species and their location is shown in Table 1. Most of the utilized species, and indeed the same individuals thereof, have been previously used in testing of a new method for assessing desiccation tolerance (López-Pozo et al., 2019) and in other recently published works (Nadal et al., 2018; Fernández-Marín et al., 2020). In this study, we completed the photosynthetic characterization of all species and present new complementary leaf anatomy data. Anemia caffrorum (previously named Mohria caffrorum) were collected from the field as described in Farrant et al. (2009) and measurements were taken within 2 weeks after acclimation in growth chambers (light intensity of 800 µmol m–2 s–1; 14/10 h photoperiod; 28/24 °C). Craterostigma plantagineum plants were grown from seeds and were maintained under optimum growth chamber conditions (light intensity of 400 µmol m–2 s–1; 12/12 h photoperiod; 25/20 °C). All measurements were conducted on young, fully expanded leaves of adult plants with no visual signals of stress. Table 1. Species description and location of the measured resurrection plants Group . Species . Family . Biome . Habitat . Location . Fern Anemia caffrorum (L.) Desv.a Mohriaceae Mediterranean ‘Semi- to fully exposed habitats on forest margins’ (Farrant et al., 2009) Table Mountain (Cape Town, South Africa) Fern Astrolepis sinuata (Lag. ex Sw.) Benham and Windham Pteridaceae Deserts and xeric shrublands ‘Rupestrian or (rarely) terrestrial habitats’ (Benham and Windham, 1992) Royal Botanic Garden Edinburgh (UK) Fern Bommeria hispida (Mett. ex Kuhn) Underw. Pteridaceae Deserts and xeric shrublands ‘Xeric and seasonally dry mountainous regions’ (Haufler, 1979) Royal Botanic Garden Edinburgh (UK) Fern Cheilanthes eatonii Baker Pteridaceae Deserts and xeric shrublands ‘Desert habitats, growing from rock cracks or specialized substrates’ (Windham and Yatskievych, 2003) Royal Botanic Garden Edinburgh (UK) Fern Asplenium ceterach L. Aspleniaceae Mediterranean Rocky, xeric habitats; cliffs (Terzi et al., 2018) Royal Botanic Garden Edinburgh (UK) Angiosperm Haberlea rhodopensis Friv. Gesneriaceae Temperate forests ‘Rock crevices, preferentially in sheltered, rather cool and humid places’ (Rakić et al., 2014) Royal Botanic Garden Edinburgh (UK) Angiosperm Ramonda myconi (L.) Rchb. Gesneriaceae Temperate forests ‘Rock crevices, preferentially in sheltered, rather cool and humid places’ (Rakić et al., 2014) Pre-Pyrenees (Embalse de la Peña, Spain) Angiosperm Craterostigma plantagineum Hochst. Linderniaceae Deserts and xeric shrublands ‘Shallow depressions on inselbergs’ (Porembski and Barthlott, 2000) Universitat de les Illes Balearsc Angiosperm Barbacenia purpurea Hook.b Velloziaceae Tropical and subtropical forests Rock outcrop exposed to frequent periods without precipitation (Suguiyama et al., 2014) Universitat de les Illes Balearsd Angiosperm Xerophyta viscosa Bakerb Velloziaceae Montane grasslands and shrublands ‘Mats and shallow depressions on inselbergs’ (Porembski and Barthlott, 2000) Universitat de les Illes Balearsd Group . Species . Family . Biome . Habitat . Location . Fern Anemia caffrorum (L.) Desv.a Mohriaceae Mediterranean ‘Semi- to fully exposed habitats on forest margins’ (Farrant et al., 2009) Table Mountain (Cape Town, South Africa) Fern Astrolepis sinuata (Lag. ex Sw.) Benham and Windham Pteridaceae Deserts and xeric shrublands ‘Rupestrian or (rarely) terrestrial habitats’ (Benham and Windham, 1992) Royal Botanic Garden Edinburgh (UK) Fern Bommeria hispida (Mett. ex Kuhn) Underw. Pteridaceae Deserts and xeric shrublands ‘Xeric and seasonally dry mountainous regions’ (Haufler, 1979) Royal Botanic Garden Edinburgh (UK) Fern Cheilanthes eatonii Baker Pteridaceae Deserts and xeric shrublands ‘Desert habitats, growing from rock cracks or specialized substrates’ (Windham and Yatskievych, 2003) Royal Botanic Garden Edinburgh (UK) Fern Asplenium ceterach L. Aspleniaceae Mediterranean Rocky, xeric habitats; cliffs (Terzi et al., 2018) Royal Botanic Garden Edinburgh (UK) Angiosperm Haberlea rhodopensis Friv. Gesneriaceae Temperate forests ‘Rock crevices, preferentially in sheltered, rather cool and humid places’ (Rakić et al., 2014) Royal Botanic Garden Edinburgh (UK) Angiosperm Ramonda myconi (L.) Rchb. Gesneriaceae Temperate forests ‘Rock crevices, preferentially in sheltered, rather cool and humid places’ (Rakić et al., 2014) Pre-Pyrenees (Embalse de la Peña, Spain) Angiosperm Craterostigma plantagineum Hochst. Linderniaceae Deserts and xeric shrublands ‘Shallow depressions on inselbergs’ (Porembski and Barthlott, 2000) Universitat de les Illes Balearsc Angiosperm Barbacenia purpurea Hook.b Velloziaceae Tropical and subtropical forests Rock outcrop exposed to frequent periods without precipitation (Suguiyama et al., 2014) Universitat de les Illes Balearsd Angiosperm Xerophyta viscosa Bakerb Velloziaceae Montane grasslands and shrublands ‘Mats and shallow depressions on inselbergs’ (Porembski and Barthlott, 2000) Universitat de les Illes Balearsd The biome nomenclature followed the categories described in Olson et al. (2001). aDesiccation-tolerant fronds (Farrant et al., 2009). bPoikilochlorophyllous. The rest of the species are homoiochlorophyllous. cSeeds obtained from plants at the University of Cape Town (South Africa). dSeeds obtained from commercial nurseries. Open in new tab Table 1. Species description and location of the measured resurrection plants Group . Species . Family . Biome . Habitat . Location . Fern Anemia caffrorum (L.) Desv.a Mohriaceae Mediterranean ‘Semi- to fully exposed habitats on forest margins’ (Farrant et al., 2009) Table Mountain (Cape Town, South Africa) Fern Astrolepis sinuata (Lag. ex Sw.) Benham and Windham Pteridaceae Deserts and xeric shrublands ‘Rupestrian or (rarely) terrestrial habitats’ (Benham and Windham, 1992) Royal Botanic Garden Edinburgh (UK) Fern Bommeria hispida (Mett. ex Kuhn) Underw. Pteridaceae Deserts and xeric shrublands ‘Xeric and seasonally dry mountainous regions’ (Haufler, 1979) Royal Botanic Garden Edinburgh (UK) Fern Cheilanthes eatonii Baker Pteridaceae Deserts and xeric shrublands ‘Desert habitats, growing from rock cracks or specialized substrates’ (Windham and Yatskievych, 2003) Royal Botanic Garden Edinburgh (UK) Fern Asplenium ceterach L. Aspleniaceae Mediterranean Rocky, xeric habitats; cliffs (Terzi et al., 2018) Royal Botanic Garden Edinburgh (UK) Angiosperm Haberlea rhodopensis Friv. Gesneriaceae Temperate forests ‘Rock crevices, preferentially in sheltered, rather cool and humid places’ (Rakić et al., 2014) Royal Botanic Garden Edinburgh (UK) Angiosperm Ramonda myconi (L.) Rchb. Gesneriaceae Temperate forests ‘Rock crevices, preferentially in sheltered, rather cool and humid places’ (Rakić et al., 2014) Pre-Pyrenees (Embalse de la Peña, Spain) Angiosperm Craterostigma plantagineum Hochst. Linderniaceae Deserts and xeric shrublands ‘Shallow depressions on inselbergs’ (Porembski and Barthlott, 2000) Universitat de les Illes Balearsc Angiosperm Barbacenia purpurea Hook.b Velloziaceae Tropical and subtropical forests Rock outcrop exposed to frequent periods without precipitation (Suguiyama et al., 2014) Universitat de les Illes Balearsd Angiosperm Xerophyta viscosa Bakerb Velloziaceae Montane grasslands and shrublands ‘Mats and shallow depressions on inselbergs’ (Porembski and Barthlott, 2000) Universitat de les Illes Balearsd Group . Species . Family . Biome . Habitat . Location . Fern Anemia caffrorum (L.) Desv.a Mohriaceae Mediterranean ‘Semi- to fully exposed habitats on forest margins’ (Farrant et al., 2009) Table Mountain (Cape Town, South Africa) Fern Astrolepis sinuata (Lag. ex Sw.) Benham and Windham Pteridaceae Deserts and xeric shrublands ‘Rupestrian or (rarely) terrestrial habitats’ (Benham and Windham, 1992) Royal Botanic Garden Edinburgh (UK) Fern Bommeria hispida (Mett. ex Kuhn) Underw. Pteridaceae Deserts and xeric shrublands ‘Xeric and seasonally dry mountainous regions’ (Haufler, 1979) Royal Botanic Garden Edinburgh (UK) Fern Cheilanthes eatonii Baker Pteridaceae Deserts and xeric shrublands ‘Desert habitats, growing from rock cracks or specialized substrates’ (Windham and Yatskievych, 2003) Royal Botanic Garden Edinburgh (UK) Fern Asplenium ceterach L. Aspleniaceae Mediterranean Rocky, xeric habitats; cliffs (Terzi et al., 2018) Royal Botanic Garden Edinburgh (UK) Angiosperm Haberlea rhodopensis Friv. Gesneriaceae Temperate forests ‘Rock crevices, preferentially in sheltered, rather cool and humid places’ (Rakić et al., 2014) Royal Botanic Garden Edinburgh (UK) Angiosperm Ramonda myconi (L.) Rchb. Gesneriaceae Temperate forests ‘Rock crevices, preferentially in sheltered, rather cool and humid places’ (Rakić et al., 2014) Pre-Pyrenees (Embalse de la Peña, Spain) Angiosperm Craterostigma plantagineum Hochst. Linderniaceae Deserts and xeric shrublands ‘Shallow depressions on inselbergs’ (Porembski and Barthlott, 2000) Universitat de les Illes Balearsc Angiosperm Barbacenia purpurea Hook.b Velloziaceae Tropical and subtropical forests Rock outcrop exposed to frequent periods without precipitation (Suguiyama et al., 2014) Universitat de les Illes Balearsd Angiosperm Xerophyta viscosa Bakerb Velloziaceae Montane grasslands and shrublands ‘Mats and shallow depressions on inselbergs’ (Porembski and Barthlott, 2000) Universitat de les Illes Balearsd The biome nomenclature followed the categories described in Olson et al. (2001). aDesiccation-tolerant fronds (Farrant et al., 2009). bPoikilochlorophyllous. The rest of the species are homoiochlorophyllous. cSeeds obtained from plants at the University of Cape Town (South Africa). dSeeds obtained from commercial nurseries. Open in new tab Photosynthesis In the case of Ramonda myconi, Barbacenia purpurea, and Xerophyta viscosa, photosynthetic data were taken from previous publications (Nadal et al., 2018; Fernández-Marín et al., 2020). For the rest of the species, 3–6 replicates were taken from different individuals. Simultaneous measurements of gas exchange and chlorophyll fluorescence were conducted using an open gas exchange system with a coupled fluorescence chamber of 2 cm2 (Li-6400; Li-Cor Inc., Lincoln, NE, USA). Measurements were performed under ambient CO2 (400 µmol mol–1) and relative humidity, 25 °C (block temperature), and saturating photosynthetic photon flux density (PPFD). Light-saturated net CO2 assimilation (An), stomatal conductance to CO2 (gs), substomatal CO2 concentration (Ci), and photochemical yield of PSII (Φ PSII) were recorded after reaching steady-state conditions. All An values were corrected for CO2 leakage (Flexas et al., 2007a) and exposed area if applicable (when leaves did not completely cover the 2 cm2 chamber). Electron transport rate (Jflu) was estimated following Genty et al. (1989): Jflu=PPFD ΦPSII αβ(1) Where αβ is the product of leaf absorptance (α) and the electron partitioning between PSI and PSII (β). Due to logistical limitations in the field and the botanical gardens, αβ was assumed to be 0.45 as determined in Arabidopsis thaliana (Flexas et al., 2007b) in all newly measured species except for C. plantagineum, where it was possible to estimate αβ from low O2 conditions using the Yin method (Yin et al., 2004; Bellasio et al., 2016). Mesophyll conductance to CO2 (gm) was estimated using the variable J method (Harley et al., 1992): gm=AnCi− Γ∗(Jflu+8(An+Rd))J−4(An+Rd)(2) Averaged values of the CO2 compensation point in the absence of respiration (Γ*) for ferns (Gago et al., 2013) and values for Arabidopsis (Walker et al., 2013) were used for ferns and H. rhodopensis, respectively. Γ* was determined only for C. plantagineum following Bellasio et al. (2016). Dark (Rd) mitochondrial respiration measurements were used for gm calculation. See Nadal et al. (2018) and Fernández-Marín et al. (2020) for details regarding gm estimation in the rest of the species. A sensitivity analysis showed that variation in αβ or Γ* did not significantly affect absolute gm estimates (Supplementary Fig. S1). An/Ci curves were performed to calculate the maximum rate of Rubisco carboxylation (Vcmax) from the Rubisco-limited part (Sharkey, 2016) and to estimate photosynthesis relative to stomatal (ls), mesophyll (lm), and biochemical (lb) limitations, calculated following the approach described in Grassi and Magnani (2005) using An/Cc as a proxy to ∂A/∂Cc, where Cc is the CO2 concentration at the Rubisco carboxylation site in the chloroplast. Anatomy Four small leaf or frond pieces per species were taken (avoiding larger veins) from the same tissues on which gas exchange was performed. These were immediately fixed under vacuum with 4% glutaraldehyde and 2% paraformaldehyde in 0.1 M phosphate buffer (pH 7.2) and further processed to obtain transverse semi-thin and ultra-thin sections of leaf mesophyll as detailed in Tomàs et al. (2013). Semi-thin sections were observed at ×100 and ×200 magnifications utilizing light microscopy (Olympus, Tokyo, Japan) and were photographed with a Moticam 3 (Motic Electric Group Co., Xiamen, China). Ultra-thin sections were viewed utilizing TEM (TEM H600, Hitachi, Tokyo, Japan). All images were analysed using the software ImageJ (Wayne Rasband/NIH). Leaf thickness (LT), palisade and spongy mesophyll thickness, and the fraction of intercellular air spaces (fias) were measured from images obtained by light microscopy. The mesophyll and chloroplast surface area exposed to intercellular air spaces per unit area (Sm and Sc, respectively) and chloroplast thickness were obtained from TEM images at ×1500 magnification (10–12 replicates). Sc and Sm were calculated as described in Evans et al. (1994) using the curvature correction factors from Thain (1983). Cell wall thickness of mesophyll cells (Tcw) and cytosol path length (from cell wall to chloroplast) were measured from 10–12 replicates of TEM images taken at ×30 000 magnification. Weighted averages based on tissue (palisade and spongy) fraction were calculated for all parameters. Leaf mass per area (LMA) was measured from neighbouring leaves on the same individuals. Leaf area was calculated using ImageJ (Wayne Rasband/NIH). Dry matter was obtained after 72 h oven drying at 70 °C. Leaf density (LD) was calculated as LMA/LT. gm modelling from anatomy The one-dimensional diffusion model of Niinemets and Reichstein (2003) and modified by Tomàs et al. (2013) as described in Tosens et al. (2016) was used to estimate gm from anatomical measurements, to calculate its limitations (air and gas phase) and to model the influence of Sc and Tcw on the liquid phase conductance to CO2 diffusion (gliq). Mesophyll conductance (gm) can be described considering CO2 diffusion in the gas and liquid phase components inside the leaf: gm=11gias+RTHgliq(3) Where gias and gliq are the gas and liquid phase conductances (m s–1), respectively, R is the gas constant (8.31 Pa m3 K–1 mol–1), T is the leaf temperature (K), and H is the Henry’s law constant for CO2 in water (3.4×10–4 mol Pa–1 m–3). Gas phase diffusion is determined as: gias=Dafias Δ Liasς(4) Where Da is the diffusion coefficient for CO2 in the gas phase (1.51×10–5 m2 s-1 at 22 °C), fias is the fraction of intercellular air spaces (the porosity of the CO2 pathway; in m3 m–3), ΔLias is the average gas phase thickness (taken as half the whole mesophyll thickness; in m), and ς is the diffusion path tortuosity, assumed to be 1.57 m m–1 (Niinemets and Reichstein, 2003). The liquid phase conductance results from the conductance at each step along the CO2 pathway from the cell wall to the sites of carboxylation inside the chloroplast stroma and the chloroplast surface exposed to intercellular air spaces per leaf area (Sc; Tosens et al., 2016): gliq=Sc1gcw+1gpl+1gcyt+1genv+1gstr(5) The sequential steps included in the model are the cell wall (cw), plasmalemma (pl), cytosol (cyt), chloroplast envelope (env), and stroma (str). gpl and genv are assumed to be 0.0035 m s–1 due to the difficulty for their correct estimation (Tosens et al., 2016). The conductance at each other step i (gi) is described as: gi=rf,iDwpi Δ Li(6) Where rf,i is a dimensionless factor accounting for the decrease of diffusion conductance compared with free diffusion in water (1 for cw, 0.3 for cyt and str; Tosens et al., 2016), Dw is the diffusion coefficient for CO2 in the liquid phase (1.90×10–9 m2 s–1 at 22 ºC), pi is the effective porosity (1 m3 m–3 for cyt and str), and ΔLi is the path length (m) measured for each step (taken as half chloroplast thickness for str). pi for cw was assumed to be 0.028 m3 m–3 as all species presented high cell wall thickness (Tomàs et al., 2013). Finally, the limitations to gm imposed by the gas and liquid phase diffusion conductances (lias and lliq, respectively) were calculated following the approach described in Tomàs et al. (2013). Statistical analysis Relationships among parameters were quantified using standardized major axis (SMA) slopes employing mean values for each species. Logarithmic (log10) fittings were used when appropriate. The Akaike information criterion (AIC) was used to assess the fitness of various models when comparing traits between resurrection and desiccation-sensitive species. All analyses were performed using R statistical software (R Core Team, 2016) and the ‘stats’ package (R Core Team); for SMA, the function ‘sma()’ from the ‘smatr’ package was used (Warton et al., 2012). Results Representative images of three species utilized in the current study can be found in Fig. 1. Data from photosynthetic analysis and leaf anatomy studies of all species utilized in the current study are given in Supplementary Tables S1 and S2. The mean assimilation rate of resurrection ferns was 9.81 µmol m–2 s–1 and that of angiosperms was 11.71 µmol m–2 s–1, although the latter displayed a greater range of values (from 5.02±0.42 µmol m–2 s–1 in R. myconi to 17.98±1.09 µmol m–2 s–1 in C. plantagineum). An showed no relationship to stomatal conductance across resurrection species (Fig. 2A), mainly due to the high gs in A. caffrorum (0.233±0.016 mol m–2 s–1), whereas it was tightly related to mesophyll conductance (Fig. 2B) and also to the biochemical parameters (Jflu and Vcmax), although to a lower extent (Fig. 2C, D). The limitation analysis (Fig. 2E) showed that photosynthesis was mainly limited by stomata (8–43% for ls) and mesophyll (20–60% for lm) diffusion, and only C. plantagineum was strongly limited by biochemistry (51±4% for lb). This predominance of ls and, especially, lm is reflected in the ratio between diffusive and biochemical limitations (Fig. 2F). gm showed a negative relationship with the parameters associated with higher structural investment (LMA and cell wall thickness) and a positive one with chloroplast exposure in the mesophyll, reflected by Sc (Supplementary Fig. S2). Sc was mainly driven by mesophyll cell exposure, which in turn was related to LMA through both leaf thickness and density (Supplementary Fig. S3). No differences in the Sc/Sm ratio were detected between ferns and angiosperms (t-test using means per species, P=0.3225), and fias did not show any significant relationship to any other anatomical parameter. Fig. 1. Open in new tabDownload slide Leaf anatomy of three representatives among the resurrection plants studied. The pictures for each species are depicted along each column. Images of the species during gas exchange measurements of the fern Bommeria hispida (A), the homoiochlorophyllous dicot Haberlea rhodopensis (B), and the poikilochlorophyllous monocot Xerophyta viscosa (C). Cross-sections of leaves viewed with light microscopy at ×200 (D), ×100 (E), and ×200 (F) magnifications. Cross-sections of mesophyll cells viewed utilizing TEM at ×1500 magnification (G–I). Detail of the cell wall (cw) and chloroplast (C) of mesophyll cells at ×30 000 magnification (J–L). Note the extent of cell wall thickness (>500 nm), especially in the case of H. rhodopensis, Fig. 1. Open in new tabDownload slide Leaf anatomy of three representatives among the resurrection plants studied. The pictures for each species are depicted along each column. Images of the species during gas exchange measurements of the fern Bommeria hispida (A), the homoiochlorophyllous dicot Haberlea rhodopensis (B), and the poikilochlorophyllous monocot Xerophyta viscosa (C). Cross-sections of leaves viewed with light microscopy at ×200 (D), ×100 (E), and ×200 (F) magnifications. Cross-sections of mesophyll cells viewed utilizing TEM at ×1500 magnification (G–I). Detail of the cell wall (cw) and chloroplast (C) of mesophyll cells at ×30 000 magnification (J–L). Note the extent of cell wall thickness (>500 nm), especially in the case of H. rhodopensis, Fig. 2. Open in new tabDownload slide Photosynthesis parameters and limitations of the resurrection plants utilized. Relationship between net CO2 assimilation (An) and stomatal conductance to CO2 (gs; A); no significant linear or logarithmic relationship was found (SMA, P>0.05). Relationship between An and mesophyll conductance to CO2 estimated using the variable J method (gm; B); significant logarithmic fit [An ~log10(gm), SMA, P=0.0001, r2=0.85]. Relationship between An and electron transport rate (Jflu; C); significant logarithmic fit [An ~log10(Jflu), SMA, P=0.0339, r2=0.45]. Relationship between An and maximum rate of Rubisco carboxylation (Vcmax; D); significant linear fit (SMA, P=0.0091, r2=0.70). Circles represent means ±SE (error bars) of measured fern (blue circles) and angiosperm (red circles) species. Accumulated photosynthesis relative limitations (E); stomatal (ls, white bars), mesophyll (lm, grey bars), and biochemical (lb, black bars) limitations. Ratio between diffusive (ls+lm) and biochemical limitations (ld/lb;F). Bars represents means ±SE (error bars) of A. caffrorum (Aca), A. sinuata (As), B. hispida (Bh), C. eatonii (Ce), A. ceterach (Ace), H. rhodopensis (Hr), R. myconi (Rm), C. plantagineum (Cp), B. purpurea (Bp), and X. viscosa (Xv). Fig. 2. Open in new tabDownload slide Photosynthesis parameters and limitations of the resurrection plants utilized. Relationship between net CO2 assimilation (An) and stomatal conductance to CO2 (gs; A); no significant linear or logarithmic relationship was found (SMA, P>0.05). Relationship between An and mesophyll conductance to CO2 estimated using the variable J method (gm; B); significant logarithmic fit [An ~log10(gm), SMA, P=0.0001, r2=0.85]. Relationship between An and electron transport rate (Jflu; C); significant logarithmic fit [An ~log10(Jflu), SMA, P=0.0339, r2=0.45]. Relationship between An and maximum rate of Rubisco carboxylation (Vcmax; D); significant linear fit (SMA, P=0.0091, r2=0.70). Circles represent means ±SE (error bars) of measured fern (blue circles) and angiosperm (red circles) species. Accumulated photosynthesis relative limitations (E); stomatal (ls, white bars), mesophyll (lm, grey bars), and biochemical (lb, black bars) limitations. Ratio between diffusive (ls+lm) and biochemical limitations (ld/lb;F). Bars represents means ±SE (error bars) of A. caffrorum (Aca), A. sinuata (As), B. hispida (Bh), C. eatonii (Ce), A. ceterach (Ace), H. rhodopensis (Hr), R. myconi (Rm), C. plantagineum (Cp), B. purpurea (Bp), and X. viscosa (Xv). Figure 3 presents data from the current study compared with those published on desiccation-sensitive ferns (Tosens et al., 2016) and angiosperms (Onoda et al., 2017). For both An and LMA (Fig. 3A, B), resurrection angiosperms fell within the range of their desiccation-sensitive counterparts whereas resurrection ferns displayed the higher-end values within their group. On the other hand, both resurrection ferns and angiosperms presented the largest values for Tcw and Sc within each group (Fig. 3C, D). The notable thickness of cell walls is illustrated in Fig. 1J–L; the measured resurrection species presented a mean Tcw of 0.667 µm, with H. rhodopensis and A. ceterach showing the thickest walls (>1 µm). Resurrection angiosperms had the highest Sc values among all species measured, except for R. myconi. Furthermore, C. plantagineum had the maximum Sc ever reported thus far (53.3±2.7 m2 m–2) (compiled in Gago et al., 2019). Notably, the An–Tcw relationship significantly differed between resurrection and desiccation-sensitive species, with resurrection plants presenting a similar slope but a higher intercept (Fig. 3F). On the other hand, when gm is normalized by Sc, both resurrection and desiccation-sensitive species did not differ significantly (Fig. 3G). This partially arises from resurrection plants presenting simultaneously very thick cell walls but also high chloroplast exposure (Fig. 3H). Fig. 3. Open in new tabDownload slide Comparison of photosynthetic and leaf anatomy traits between resurrection and desiccation-sensitive ferns and angiosperms. Means of measured ferns (blue circles) and angiosperms (red circles) against boxplots of published data for desiccation-sensitive species for net CO2 assimilation (An; A), leaf mass per area (LMA; B), mesophyll cell wall thickness (Tcw; C), and chloroplast surface area exposed to intercellular air spaces per area (Sc; D). Default boxplot quartile settings are from the R package ‘ggplot2’. Logarithmic (log10) relationships between means of measured ferns (blue circles) and angiosperms (red circles ) and published data of desiccation-sensitive species (small circles). Lines denote significant linear fittings [SMA; log10(Y)~log10(X)×R/DS) between parameters across resurrection (R) and/or desiccation-sensitive (DS) species. An and LMA (E): P=0.3059 for LMA, P=0.0113 for R/DS. An and Tcw (F): P<0.0001 for both Tcw and R/DS, no significant interaction (P=0.3263, equal slopes); separate linear SMA fit for R species (dashed line; P=0.0143, r2=0.49) and DS species (solid line; P<0.0001, r2=0.46). Mesophyll conductance to CO2 normalized to exposed chloroplast surface (gm/Sc) and Tcw (G): P<0.0001 for Tcw, no R/DS effect (P=0.6120); linear SMA fit for all data (solid line; P<0.0001, r2=0.37). Sc and Tcw (H): P=0.4774 for Tcw, P=0.0001 for R/DS. Published data from Tosens et al. (2016) for ferns and Onoda et al. (2017) for angiosperms. Fig. 3. Open in new tabDownload slide Comparison of photosynthetic and leaf anatomy traits between resurrection and desiccation-sensitive ferns and angiosperms. Means of measured ferns (blue circles) and angiosperms (red circles) against boxplots of published data for desiccation-sensitive species for net CO2 assimilation (An; A), leaf mass per area (LMA; B), mesophyll cell wall thickness (Tcw; C), and chloroplast surface area exposed to intercellular air spaces per area (Sc; D). Default boxplot quartile settings are from the R package ‘ggplot2’. Logarithmic (log10) relationships between means of measured ferns (blue circles) and angiosperms (red circles ) and published data of desiccation-sensitive species (small circles). Lines denote significant linear fittings [SMA; log10(Y)~log10(X)×R/DS) between parameters across resurrection (R) and/or desiccation-sensitive (DS) species. An and LMA (E): P=0.3059 for LMA, P=0.0113 for R/DS. An and Tcw (F): P<0.0001 for both Tcw and R/DS, no significant interaction (P=0.3263, equal slopes); separate linear SMA fit for R species (dashed line; P=0.0143, r2=0.49) and DS species (solid line; P<0.0001, r2=0.46). Mesophyll conductance to CO2 normalized to exposed chloroplast surface (gm/Sc) and Tcw (G): P<0.0001 for Tcw, no R/DS effect (P=0.6120); linear SMA fit for all data (solid line; P<0.0001, r2=0.37). Sc and Tcw (H): P=0.4774 for Tcw, P=0.0001 for R/DS. Published data from Tosens et al. (2016) for ferns and Onoda et al. (2017) for angiosperms. The interplay between cell wall thickness and chloroplast exposure in determining mesophyll conductance was further explored using the modelling approach described in Tosens et al. (2016). gm estimated from anatomical measurements showed a strong correlation (r2=0.92) with gm measured from gas exchange across the resurrection species (Fig. 4A), thus allowing extrapolation of the insights provided by the modelling from anatomical characteristics. Conductance in the liquid phase (gliq) was the largest limiting factor for CO2 diffusion in all species (lliq >80%) except for C. plantagineum (48±4% for lliq) (Fig. 4B). Considering the liquid phase only, the limitation imposed by high Tcw in resurrection ferns and angiosperms can be fully overcome by increasing Sc, thus achieving similar gliq (and hence gm) to their desiccation-sensitive counterparts (Fig. 4C). Fig. 4. Open in new tabDownload slide Mesophyll conductance (gm) modelling from leaf anatomical characteristics of the studied resurrection plants. Relationship between gm estimated from gas exchange (variable J method) and from anatomical measurements (A); significant linear fit (SMA, P<0.0001, r2=0.92); the dashed line denotes the 1:1 relationship. Circles represent means ±SE (error bars) of measured fern (blue circles ) and angiosperm (red circles) species. Accumulated gm limitations (B) imposed by gas (lias, white bars) and liquid phase (lliq, grey bars) diffusion conductances. Bars represents means ±SE (error bars) of A. caffrorum (Aca), A. sinuata (As), B. hispida (Bh), C. eatonii (Ce), A. ceterach (Ace), H. rhodopensis (Hr), R. myconi (Rm), C. plantagineum (Cp), B. purpurea (Bp), and X. viscosa (Xv). Effect of variable mesophyll cell wall thickness (Tcw) and chloroplast surface area exposed to intercellular air spaces per area (Sc) on the liquid phase conductance to CO2 (gliq; C) using the gm modelling from anatomy. Solid lines represent mean gliq of resurrection ferns (blue) and angiosperms (red) as a function of Sc. Dashed lines show the change in gliq when the mean Tcw values from desiccation-sensitive ferns (blue) and angiosperms (red) are used (0.422 µm and 0.300 µm, respectively). Circles for resurrection ferns (blue) and angiosperms (red), and triangles for desiccation-sensitive ferns (blue) and angiosperms (red) indicate their actual mean Sc values (7.3 m2 m–2 and 14.1 m2 m–2 for desiccation-sensitive ferns and angiosperms, respectively); arrows show the expected gliq value if the resurrection groups presented the same Sc as their desiccation-sensitive counterparts. Mean values for the desiccation-sensitive groups were obtained from Tosens et al. (2016) and Onoda et al. (2017). Fig. 4. Open in new tabDownload slide Mesophyll conductance (gm) modelling from leaf anatomical characteristics of the studied resurrection plants. Relationship between gm estimated from gas exchange (variable J method) and from anatomical measurements (A); significant linear fit (SMA, P<0.0001, r2=0.92); the dashed line denotes the 1:1 relationship. Circles represent means ±SE (error bars) of measured fern (blue circles ) and angiosperm (red circles) species. Accumulated gm limitations (B) imposed by gas (lias, white bars) and liquid phase (lliq, grey bars) diffusion conductances. Bars represents means ±SE (error bars) of A. caffrorum (Aca), A. sinuata (As), B. hispida (Bh), C. eatonii (Ce), A. ceterach (Ace), H. rhodopensis (Hr), R. myconi (Rm), C. plantagineum (Cp), B. purpurea (Bp), and X. viscosa (Xv). Effect of variable mesophyll cell wall thickness (Tcw) and chloroplast surface area exposed to intercellular air spaces per area (Sc) on the liquid phase conductance to CO2 (gliq; C) using the gm modelling from anatomy. Solid lines represent mean gliq of resurrection ferns (blue) and angiosperms (red) as a function of Sc. Dashed lines show the change in gliq when the mean Tcw values from desiccation-sensitive ferns (blue) and angiosperms (red) are used (0.422 µm and 0.300 µm, respectively). Circles for resurrection ferns (blue) and angiosperms (red), and triangles for desiccation-sensitive ferns (blue) and angiosperms (red) indicate their actual mean Sc values (7.3 m2 m–2 and 14.1 m2 m–2 for desiccation-sensitive ferns and angiosperms, respectively); arrows show the expected gliq value if the resurrection groups presented the same Sc as their desiccation-sensitive counterparts. Mean values for the desiccation-sensitive groups were obtained from Tosens et al. (2016) and Onoda et al. (2017). Discussion The present study provides data on photosynthesis and anatomy from 10 resurrection species and points to how these plants differ in the relationship between these two aspects of leaf physiology when compared with that of desiccation-sensitive ferns and angiosperms. Overall, except for C. plantagineum, the resurrection species appear to display a diffusion-limited photosynthesis when they are in their fully hydrated state. One of the most striking observations is the relative thickness of their cell walls, these being among the highest values reported for ferns and angiosperms (Gago et al., 2019) and in a similar range to those of gymnosperms (Veromann-Jürgenson et al., 2017). Among land plants, only lycophytes and bryophytes have thicker (0.5–3.4 µm) cell walls (Carriquí et al., 2019). It has been postulated that in such groups, thicker cell walls enabled tolerance of stresses such as high irradiance and, in particular, water deficit faced by early colonizers of land (Graham et al., 2014; Le Gall et al., 2015), possibly through an increase in modulus of elasticity (ε) and thus leaf rigidity (Peguero-Pina et al., 2017a). However, tissue elasticity is actually high (low ε) in bryophytes (Proctor et al., 1998) despite presenting large cell walls. In fact, high elasticity and/or plasticity are desirable features for resurrection plants to cope with the mechanical stress upon desiccation and avoid plasmalemma tearing (Vicré et al., 2004; Proctor et al., 2007; Moore et al., 2008; Oliver et al., 2020). Indeed, ɛ values reported for C. plantagineum, B. purpurea, and X. viscosa are 6.5, 11.7, and 6.3 MPa, respectively (Nadal et al., 2018; Perera-Castro et al., 2020), lower than those observed for temperate angiosperms (Bartlett et al., 2012), although more reports are needed for other resurrection species. Some resurrection plants, such as X. viscosa and other monocots, prevent volume loss and cell shrinking by vacuole refilling with water-replacing molecules (Farrant et al., 2015); however, in other cases, cell walls undergo considerable remodelling to prevent plasmolysis (Moore et al., 2008; Chen et al., 2020). For instance, expression of dehydrin proteins in the resurrection fern Polypodium polypodioides augments the flexibility of cell walls to sustain shrinkage and buckling (Layton et al., 2010). In Craterostigma wilmsii and C. plantagineum, changes in composition (increased arabinans and xyloglucans) and expansin activity increase cell wall extensibility and contribute to cell wall folding in the desiccated state (Vicré et al., 1999; Jones and McQueen-Mason, 2004). These short-term changes in cell wall volume through deformation may require thick cell walls with both high elasticity and plasticity. Recently, cell wall composition has been suggested to affect mesophyll conductance (Ellsworth et al., 2018; Clemente-Moreno et al., 2019; Roig-Oliver et al., 2020), and one of the considered hypotheses was that resurrection plants may present altered gm due to their distinct cell wall compositions, particularly the constitutive presence of hydroxyproline-rich glycosides such as arabinogalactans and extensins. (Moore et al., 2006, 2013; Farrant et al., 2015). However, our work shows that there was no difference in the gm/Sc–Tcw relationship among resurrection plants and desiccation-sensitive species (Fig. 3G), this being a proxy for the direct effect of cell wall properties on CO2 diffusion across the cell wall (Terashima et al., 2011). This in turn suggests that the limitation exerted by the cell wall can be attributed predominantly to the increased distance that CO2 molecules have to traverse through thick cell walls. On the other hand, this relationship also highlights the importance of chloroplast exposure in compensating for the high Tcw in resurrection plants (Fig. 4C). A similar pattern has also been observed in Mediterranean sclerophyllous oaks (Peguero-Pina et al., 2017b), but not at the extreme Sc and Tcw values reported here. Notably, the only resurrection species displaying an An–Tcw relationship similar to that of desiccation-sensitive species, R. myconi, unsurprisingly also has a lower Sc value and thus displays the largest mesophyll limitation among resurrection angiosperms. The high Sc values in resurrection plants appear to be driven by high mesophyll surface exposed to air spaces and leaf thickness (Supplementary Fig. S3A, C), which results in overall higher surface exchange for CO2 diffusion to chloroplasts per leaf area. Other factors could also be important in achieving higher Sc, such as increased leaf nitrogen content for its investment in chloroplast components (Onoda et al., 2017; Evans and Clarke, 2019). The photosynthetic and anatomical features presented here may also reflect some aspects of the ecophysiology of resurrection plants. In the case of ferns, the resurrection species present overall larger An, LMA, and LT values than their desiccation-sensitive counterparts (Tosens et al., 2016). Taken together, these features may reflect acclimation and/or adaption to high irradiance environments, where a greater investment in cell layers results in an increase in mass and CO2 assimilation per unit area (Poorter et al., 2019). Most resurrection ferns, with the exception of Hymeophyllaceae (Cea et al., 2014), inhabit warm, xeric habitats prone to rapid desiccation and rehydration cycles (see Table 1). In their case, the adoption of desiccation tolerance mechanisms that enable them to live under these exposed conditions may, in turn, allow them to have a higher photosynthetic capacity in the hydrated state, which is achieved by a particular combination of high Tcw and Sc. On the other hand, resurrection angiosperms present a wider range of An and LMA, possibly reflecting their higher diversity in environments and tolerance mechanisms, as these plants inhabit both temperate and tropical habitats (see Table 1) and have evolved more derived adaptations such as poikilochlorophylly (Oliver et al., 2000; Tuba, 2008) and even freezing tolerance (Fernández-Marín et al., 2018). Yet they too combine simultaneously high Tcw and Sc to sustain a larger An per Tcw than their desiccation-sensitive counterparts. In conclusion, this study shows how the interplay between cell wall thickness and chloroplast exposure in the mesophyll is key in determining photosynthetic productivity in resurrection plants, where high Sc values enable compensation for high Tcw, this in turn allowing reasonable photosynthesis rates and, in the case of resurrection ferns, greater An compared with their desiccation-sensitive counterparts. We propose that understanding of the anatomical basis of photosynthesis is essential for future production of stress-tolerant plants that are not compromised in photosynthetic productivity. Our use of resurrection plants as models in this regard gives insight into mechanisms associated with tolerance of extreme water deficit, and how plants struggle to balance stress and productivity (Alpert, 2006). Future research is needed to close the gap between the molecular and ecophysiological aspects of this remarkable group of plants. Supplementary data The following supplementary data are available at JXB online. Table S1. Photosynthetic parameters of the measured resurrection ferns and angiosperms. Table S2. Leaf anatomy traits of the measured resurrection ferns and angiosperms. Fig. S1. Sensitivity analysis of mesophyll conductance to CO2 from the variable J method. Fig. S2. Structural and anatomical determinants of mesophyll conductance to CO2. Fig. S3. Structural and anatomical determinants of chloroplast surface area exposed to intercellular air spaces. Abbreviations Abbreviations An net CO2 assimilation rate Cc CO2 concentration at the Rubisco carboxylation site in the chloroplast Ci substomatal CO2 concentration fias fraction of mesophyll intercellular air spaces gliq liquid phase conductance to CO2 diffusion gm mesophyll conductance to CO2 diffusion gs stomatal conductance to CO2 diffusion J electron transport rate Jflu electron transport rate estimated from chlorophyll fluorescence lb relative biochemical limitation to photosynthesis LD leaf density lias fraction of gm limited by gas phase conductance to CO2 diffusion lliq fraction of gm limited by liquid phase conductance to CO2 diffusion lm relative mesophyll limitation to photosynthesis LMA leaf mass per area ls relative stomatal limitation to photosynthesis LT leaf thickness Rd mitochondrial respiration in the dark Sc chloroplast surface area exposed to intercellular air spaces Sm surface area of mesophyll cells exposed to intercellular air spaces Tcw mesophyll cell wall thickness Vcmax maximum rate of Rubisco carboxylation α leaf absorptance β electron partitioning between PSI and PSII Γ * CO2 compensation point in the absence of respiration Φ PSII photochemical yield of PSII. Acknowledgements This work was supported by the projects CTM2014-53902-C2-1-P from the Ministerio de Economía y Competitividad (MINECO, Spain) and the European Regional Development Fund (ERDF), and PGC2018-093824-B-C41 from the Ministerio de Ciencia, Innovación y Universidades (MICIU, Spain) and the ERDF. MN was supported by the MINECO and the European Social Fund (ESF) (pre-doctoral fellowship BES-2015-072578), and AVPC was supported by the Ministerio de Educación, Cultura y Deporte (MECD) (pre-doctoral fellowship FPU-02054). JMF acknowledges funding from the South African Department of Science and Innovation and National Research Foundation, grant no. 98406. We thank Miquel Truyols and collaborators of the UIB Experimental Field and Glasshouses for the use of their facilities, which are supported by the UIB Grant 15/2015. We also thank and acknowledge the technical support given in microscopical work by Universitat de València (Secció de Microscopia Electrònica, SCSIE), Universidad de Murcia (Servicio de Apoyo a las Ciencias Experimentales), Dr Ferran Hierro (UIB, Serveis Cientificotècnics), and Margalida Roig Oliver (UIB). We thank the Instituto Aragonés de Gestión Ambiental (INAGA), Departamento de Desarrollo Rural y Sostenibilidad, Gobierno de Aragón, for permission to study natural populations of R. myconi, and colleagues from Universidad del País Vasco (UPV/EHU) for organizing the field campaigns. Finally, we thank the Royal Botanic Garden Edinburgh for allowing access to its plant collection and nurseries. Author contributions MN, JG, JMF, and JF planned the research; MN, JG, and AVPC performed the measurements; MN analysed the data and drafted the manuscript. All authors contributed to subsequent revisions and discussion. 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TI - Resurrection plants optimize photosynthesis despite very thick cell walls by means of chloroplast distribution
JF - Journal of Experimental Botany
DO - 10.1093/jxb/erab022
DA - 2021-01-23
UR - https://www.deepdyve.com/lp/oxford-university-press/resurrection-plants-optimize-photosynthesis-despite-very-thick-cell-Ll00pa1jEu
SP - 1
EP - 1
VL - Advance Article
IS - 
DP - DeepDyve
ER -