Journal: Nature Communications

Article Title: Mesophyll porosity is modulated by the presence of functional stomata

doi: 10.1038/s41467-019-10826-5

Figure Lengend Snippet: Stomatal patterning shifts with ploidy level in wheat. Sample images of overall stomatal distribution along the adaxial epidermis ( a – c ) and individual stomatal complexes ( d – f ) in Triticum baeoticum (2n; a , d ), T. araraticum (4n; b , e ) and T. aestivum cv Cougar (6n; c , f ). Scale bar a – c = 80 µm; d – f = 20 µm. Stomatal width ( g ) (ANOVA, F (2,81) = 169.5, P < 0.0001), area ( h ) (ANOVA, F (2,81) = 218.7, P < 0.0001), length ( i ) (ANOVA, F (2,73) = 80.29 p < 0.0001), density ( j ) (ANOVA, F (2,81) = 61.21 P < 0.0001), and conductance, g s ( k ) (ANOVA, F (2,25) = 7.494, P = 0.0028) are shown for all analysed wheat lines. Results of a posthoc Tukey test comparing sequential ploidy levels are indicated within each analysis, with an asterisk when significant at the p < 0.05 level or NS when not significant. For g – k , data are shown as box plots (25th−75th percentile, horizontal line = median) with whiskers indicating maximum and minimum values, n = 6. l For each analysed wheat line, mean mesophyll porosity is plotted against mean stomatal conductance, g s , with ploidy level indicated for each point. Results of correlation analysis are presented (Pearson r value). Results for individual paired data are shown in Supplementary Fig.

Article Snippet: Seeds of Triticum baeoticum (2n) (TRI 18344), T. urartu (2n) (TRI 6735), T. araraticum (4n) (TRI 18513), T. dicoccoides (4n) (TRI 18465) (obtained from IPK Gatersleben, Germany ( https://www.ipk-gatersleben.de/en/gbisipk-gaterslebendegbis-i/ )), three cultivars of T. aestivum (6n) (cv. cougar, crusoe and shango) (provided by RAGT seeds, Cambridge, UK), and the wheat TaEPF1-OE1 line were germinated in 6:1 Levington M3: Perlite and grown in a controlled environment chamber (Conviron, Manitoba, Canada) with 16 h photoperiod set to 21 °C, 60% relative humidity (RH), and 400 μmol m 2 s −1 at bench level (425 μmol m 2 s −1 at the top of the canopy) photosynthetic photon flux density (PPFD).

Techniques:

Journal: Nature Communications

Article Title: Mesophyll porosity is modulated by the presence of functional stomata

doi: 10.1038/s41467-019-10826-5

Figure Lengend Snippet: MicroCT imaging reveals variation in wheat leaf airspace with ploidy. Sample leaf images of Triticum urartu (2n), T. araraticum (4n) and T. aestivum cv Cougar (6n) in 3D renderings of tissue blocks ( a – c ), transverse sections ( d – f ), longitudinal sections ( g – i ), and paradermal sections ( j – l ), with solid tissue in green and airspace in yellow. Mesophyll porosity (%) ( m – o ) is plotted along leaf depth from adaxial to abaxial surfaces in the diploid ( m ), tetraploid ( n ), and hexaploid ( o ) lines, as indicated. T. baoeticum –dark blue; T. urartu –light blue; T. dicoccoides –dark orange; T. araraticum –light orange; T. aestivum (Crusoe)–dark green; T. aestivum (Cougar)–mid-green; T. aestivum (Shango)–light green. For clarity, only mean values of 6 replicated samples are presented in panels m – o . Lines in a – c indicate plane of section in g – i , respectively, also indicated by vertical lines in j – l . Horizontal lines in j – l indicate plane of section for d – f , respectively. Image resolution = 2.75 µm. Scale bars a – l = 200 µm

Article Snippet: Seeds of Triticum baeoticum (2n) (TRI 18344), T. urartu (2n) (TRI 6735), T. araraticum (4n) (TRI 18513), T. dicoccoides (4n) (TRI 18465) (obtained from IPK Gatersleben, Germany ( https://www.ipk-gatersleben.de/en/gbisipk-gaterslebendegbis-i/ )), three cultivars of T. aestivum (6n) (cv. cougar, crusoe and shango) (provided by RAGT seeds, Cambridge, UK), and the wheat TaEPF1-OE1 line were germinated in 6:1 Levington M3: Perlite and grown in a controlled environment chamber (Conviron, Manitoba, Canada) with 16 h photoperiod set to 21 °C, 60% relative humidity (RH), and 400 μmol m 2 s −1 at bench level (425 μmol m 2 s −1 at the top of the canopy) photosynthetic photon flux density (PPFD).

Techniques: Imaging

Journal: Nature Communications

Article Title: Mesophyll porosity is modulated by the presence of functional stomata

doi: 10.1038/s41467-019-10826-5

Figure Lengend Snippet: EPF overexpression arrests sub-stomatal cavity development and decreases mesophyll porosity in wheat. a Confocal overview of a TaEPF1 OE wheat leaf showing epidermal layer (purple), subtending mesophyll cells (green), a stomate (St) consisting of guard cells and associated subsidiary cells and, in the same file, an arrested stomatal precursor (Ap). Scale bar = 60 µm. b , c Higher resolution images of ( b ) the stomate and ( c ) arrested stomatal precursor cell shown in a . Scale bars = 40 µm. d – g microCT images of a wild-type (WT) ( d , f ) and a TaEPF1 OE leaf ( e , g ) in a paradermal plane within the mesophyll directly subtending the epidermis ( d , e ) or deeper in the leaf ( f , g ), with solid tissue in green and airspace in yellow. The larger airspaces in d , e indicate sub-stomatal cavities. Note fewer sub-stomatal cavities in the TaEPF1 OE leaf. Scale bars = 100 µm. h – j In WT wheat and two independent lines of transgenic wheat overexpressing TaEPF1 (as indicated), ( h ) the density of stomata ( n = 87), sub-stomatal cavities ( n = 87) and arrested stomata precursor cells ( n = 52 from 5 independent leaves); ( i ) mesophyll porosity as measured from microCT analysis (ANOVA, F (2,12) = 4.977, p = 0.027); and ( j ) stomatal conductance, g s , are shown (ANOVA, F (2,12) = 46.86, p < 0.0001). For i and j a posthoc Tukey analysis was performed ( n = 5). Lines sharing the same letter are indistinguishable from each other at the p < 0.05 confidence limit. Data ( h – j ) are shown as box plots (25th–75th percentile, horizontal line = median) with whiskers indicating maximum and minimum values

Article Snippet: Seeds of Triticum baeoticum (2n) (TRI 18344), T. urartu (2n) (TRI 6735), T. araraticum (4n) (TRI 18513), T. dicoccoides (4n) (TRI 18465) (obtained from IPK Gatersleben, Germany ( https://www.ipk-gatersleben.de/en/gbisipk-gaterslebendegbis-i/ )), three cultivars of T. aestivum (6n) (cv. cougar, crusoe and shango) (provided by RAGT seeds, Cambridge, UK), and the wheat TaEPF1-OE1 line were germinated in 6:1 Levington M3: Perlite and grown in a controlled environment chamber (Conviron, Manitoba, Canada) with 16 h photoperiod set to 21 °C, 60% relative humidity (RH), and 400 μmol m 2 s −1 at bench level (425 μmol m 2 s −1 at the top of the canopy) photosynthetic photon flux density (PPFD).

Techniques: Over Expression, Transgenic Assay

Journal: Nature Communications

Article Title: Mesophyll porosity is modulated by the presence of functional stomata

doi: 10.1038/s41467-019-10826-5

Figure Lengend Snippet: Developmental progression of stomatal differentiation and mesophyll airspace formation. a – f Confocal images of leaf 3 of wheat (6n) seedlings taken either at the distal tip region ( a – c ) or proximal base ( d – f ). Images are from the epidermis ( a , d ) or subtending mesophyll ( b , e ), with cell walls false-coloured and overlaid in ( c , f ). A mature stomata is visible in a , with two immature stomata in d . A large airspace subtends the stomata in a (indicated by asterisks in b , c ). Small airspaces (asterisks) are visible in e , f at cell junctions. g – l Confocal images of Arabidopsis leaves at maturity ( g – i ) or early development ( j – l ). Images are from the epidermis ( g , j ) or subtending mesophyll ( h , k ) with cell wall false-coloured and overlaid in i , l . A mature stomata is visible in centre ( g ), with numerous stomata at various developmental stages in j . A relatively large airspace (asterisks) is visible below the central stomate ( h ), whereas some very small airspaces (asterisks) are distributed within the immature mesophyll ( k , l ) at cell junctions. Scale bar c , f = 20 µm; i , l = 25 µm

Article Snippet: Seeds of Triticum baeoticum (2n) (TRI 18344), T. urartu (2n) (TRI 6735), T. araraticum (4n) (TRI 18513), T. dicoccoides (4n) (TRI 18465) (obtained from IPK Gatersleben, Germany ( https://www.ipk-gatersleben.de/en/gbisipk-gaterslebendegbis-i/ )), three cultivars of T. aestivum (6n) (cv. cougar, crusoe and shango) (provided by RAGT seeds, Cambridge, UK), and the wheat TaEPF1-OE1 line were germinated in 6:1 Levington M3: Perlite and grown in a controlled environment chamber (Conviron, Manitoba, Canada) with 16 h photoperiod set to 21 °C, 60% relative humidity (RH), and 400 μmol m 2 s −1 at bench level (425 μmol m 2 s −1 at the top of the canopy) photosynthetic photon flux density (PPFD).

Techniques:

Journal: Journal of Synchrotron Radiation

Article Title: Heitt Mjölnir: a heated miniature triaxial apparatus for 4D synchrotron microtomography

doi: 10.1107/S1600577523009876

Figure Lengend Snippet: ( a ) Cartoon schematic of the layout and connection between Heitt Mjölnir and the high-pressure syringes, LVDT (black), power cables (red), thermocouples (green) and fluid (blue) connections for routine operation. (1) Type-K thermocouple(s) fixed on the pressure vessel. (2) Linear variable displacement transducer. (3) Temperature controller with two channels for top and bottom cartridge heaters (red shaded areas inside the pressure vessel). (4) Three fluid manifolds composed of Top Industrie valves (TIV) to disconnect the cell from the pump and refill (refill syringe) without dropping the cell pressure. Each fluid line must be equipped with a proportional pressure relief valve (PRV). Connections to PEEK tubing are via Swagelok 1/16" bulkhead unions. (5) Three Cetoni Nemesys high-pressure syringe pumps provide independent fluid delivery for the confining pressure (sky blue), pore fluid pressure (blue) and axial load via the hydraulic actuator (purple). (6) CETONI I/O module. (7) Laptop PC with QmixElements software to control the high-pressure pumps (5) and log experimental data (7). Solid lines indicate external cable or tubing while dotted lines indicate their continuation inside the cell. (8) Intel cooling fan for the pressure vessel and its associated 12 V DC power supply (9). ( b ) Heitt Mjölnir in position and set up for operation on the rotary table of the TOMCAT beamline at the Swiss Light Source. Letters correspond to those in ( a ). The Heitt Mjölnir rig is fixed on the rotary table at approximately 1400 mm height above floor level.

Article Snippet: The fluid pressure for the actuator axial load, the confining and the pore fluid pressure are all delivered by high-pressure Cetoni Nemesys HP-syringe pump modules (see detail in Fig. 2 and Section 3 ).

Techniques: Software, Control