Dataset used in the paper Retta MA, Van Doorselaer L, Driever SM, Yin X, de Ruijter NCA, Verboven P, Nicolaï BM, Struik PC. High photosynthesis rates in Brassiceae species are mediated by leaf anatomy enabling high biochemical capacity, rapid CO2 diffusion and efficient light use. New Phytol. 2024 Sep 18. doi: 10.1111/nph.20136. PMID: 39294895. Please cite the paper presenting this datase. Plant Species and Inbred Lines: Hirschfeldia incana L. (7th generation inbred line 190003 HIN-NIJ-07-B) Brassica nigra L. (3rd generation inbred line 210093 BNI-DG1-03-B) Brassica rapa L. (inbred line ‘R-o-18’) Arabidopsis thaliana (accession Columbia) Growth Conditions: Media: Rock-wool blocks (Grodan Plantop, Roermond, Netherlands, 10×10×7.5 cm) Fertigation: Nitrogen-rich nutrient solution via automated dripping system. Light Conditions: 12 h day/12 h night, light intensity of 200 µmol m-2 s-1 and 1800 µmol m-2 s-1 Temperature: Day/Night temperatures of 23 °C and 20 °C, respectively. Relative Humidity: 70% Codes Species: Hirschfeldia incana L. - H. incana Brassica nigra L. - B. nigra Brassica rapa L. - B. rapa Arabidopsis thaliana - A. thaliana Light conditions: High light - HL Low light - LL Replicates: Biological replicates were labeled with numbers, e.g. replicate one from high light grown Hirschfeldia incana is referred to as HiHL1 Measurements Leaf Gas Exchange and Chlorophyll Fluorescence Measurements (GasExchangeData.zip): Four leaves per species per treatment. Conducted using a LI-6800 (LI-COR, Lincoln, NE, USA) on the mid-position of the youngest fully expanded leaf. The resoponse of photosynthesis to irradiance and external CO2 concentrations augumneted with multi-phase flash fluorescence were made Optical Properties Measurement (Absorbance & chlorophyll.zip): Leaves: Four leaves per species per treatment. Leaf transmittance and reflectance measured using a dual channel spectrophotometer (absorptance_reflac_data_355_750.xlsx) Chlorophyll content measured using a spectrophotometer (Chlorophyll.xlsx). Stomatal Density and Size Analysis (Stomata.zip): Sampling: Leaves: Four leaves per species per treatment. Plants: Samples taken from three different plants. Leaf-side: abaxial and adxial leaf side. Microscopy Setup: Stomatal imprints made using clear nail polish, imaged using a light microscope at 20x. Data Output: Imaging Results: .jpg files organised under folders for species e.g. AtHL\R1 T+B.zip contains images ofimprints of top (T) and bottom (B) leaf sides from replicate plant 1 (R1) of A. thaliana grown under high light (AtHL). The images are named as for example, AT_HL_BOTTOM_R1_A_stacked_minimum.jpg, The leters A to E label various imges made from one imprint. Light and Electron Microscopy of Leaf Sections (CellwallChloroplast.zip): Sampling: Leaves: Four leaves per species per treatment. Plants: Samples taken from four different plants. Sample preparation Leaf samples fixed, dehydrated, embedded in Araldite, and sectioned for imaging. 1 µm think sections were made for light microscopy Sections of 70 nm were double stained for TEM Microscopy Setup: Mesophyll cells imaged at 400x and 700x to measure chloroplast coverage. Electron microscopy performed with Zeiss EM900 electron microscope. Mesophyll Chlorophyll (ConfocalData.zip): Sampling: Leaves: Three leaves per species per treatment. Plants: Samples taken from three different plants. Thickness: 200 ± 10 µm sections prepared using a sliding microtome Microscopy Setup: Microscope: Leica DM8 inverted scope equipped with a Stellaris 5 confocal microscope (Leica Microsystems, Wetzlar, Germany). Excitation: 490 nm excitation laser line Fluorescence Recording: Chlorophyll autofluorescence recorded in a spectral range of 660−700 nm. Objective: Leica objective ×10/0.4 NA. Z-Stacks: 85–112 µm depth, two random positions per sample Data Output: Imaging Results: Z-stacks of chlorophyll autofluorescence in mesophyll cells. Spectral Information: Chlorophyll autofluorescence recorded in the 660−700 nm range. Analysis: Software: The confocal files are in .lif format and can be viewed using Leica application suite (LASx), ImageJ