Assessing the anatomical differences between C3 and C4 leaf cross sections helps understand photosynthesis efficiency. This paper describes free-hand and semi-thin leaf cross sections preparation and examination, along with the caveats in the preparation for the crop species Triticum aestivum and Zea mays.
The enhanced efficiency of C4 photosynthesis, compared to the C3 mechanism, arises from its ability to concentrate CO2 in bundle sheath cells. The effectiveness of C4 photosynthesis and intrinsic water use efficiency are directly linked to the share of mesophyll and bundle leaf cells, size and density of bundle sheaths, and size, density, and cell wall thickness of bundle sheath cells. Rapid microscopical analysis of these traits can be performed on free-hand and semi-thin sections using conventional light microscopy, providing valuable information about photosynthetic efficiency in C4 crops by means of identifying and examining specific cell types. Additionally, errors in freehand and semi-thin section preparation that affect anatomical measurements and cell type diagnoses are shown, as well as how to avoid these errors. This microscopical approach offers an efficient means of gathering insights into photosynthetic acclimation to environmental variation and aids in the rapid screening of crops for future climates.
Photosynthesis is a fundamental process where light energy is converted into chemical energy, serving as the cornerstone of terrestrial trophic networks. The majority of plants follow the C3 pathway of photosynthesis, where the primary photosynthetic product is the three-carbon compound glycerate 3-phosphate. C3 photosynthesis evolved over 2 billion years ago in the atmosphere abundant in CO2 and low in O21. The key photosynthetic enzyme ribulose 1,5 bisphosphate carboxylase/oxygenase (Rubisco), which evolved under these conditions, is suboptimal for current low CO2 high O2 conditions as it competitively reacts with O2, initiating photorespiration2. Photorespiration is a wasteful pathway that consumes energy instead of producing it and releasing CO2 as a byproduct. Consequently, it is crucial to maintain high CO2 concentration around Rubisco in chloroplasts to prevent oxygenation3,4. Due to the inability of C3 plants to concentrate CO2, there is a significant CO2 drawdown from ambient air to chloroplasts, curbing photosynthesis and affecting plant growth and biomass production2,5,6.
In C3 plants, photosynthesis is limited by the entrance of CO2 through stomata, its diffusion through the mesophyll, and the biochemical activity of photosynthetic enzymes. The entry of CO2 is first limited by stomatal conductance, which is controlled by environmental conditions such as air temperature and humidity. Then CO2 diffuses through the leaf's internal gaseous and liquid phase to Rubisco7. In C3 plants, all stages of photosynthesis occur in the chloroplasts of mesophyll cells, and plants need to maintain a constant influx of CO2 from the atmosphere into chloroplasts. The dependence of CO2 availability in chloroplasts on stomatal openness, mesophyll architecture, and individual cell and chloroplast characteristics leaves plants susceptible to environmental constraints that eventually affect photosynthesis, like low water availability and high temperatures7,8,9,10, particularly highlighting their vulnerability to climate change conditions11.
Given the challenges posed by the inefficiencies of the C3 pathway, as well as limitations in maintaining optimal CO2 levels and susceptibility to environmental factors, in certain plants, another pathway, the C4 photosynthesis pathway, has evolved. Characteristically, C4 plants have two spatially separated biochemical pathways; the initial CO2 fixation occurs in mesophyll cells by phosphoenolpyruvate carboxylase, which has a higher affinity for CO2 than Rubisco and also lacks oxygenation activity. The formed C4 product is further transported to bundle sheath cells, where it is decarboxylated, and CO2 is again released and fixed by Rubisco (C3 photosynthesis)12,13,14. The greater affinity of PEP carboxylase to CO2 and thick cell walls of bundle sheath cells allows CO2 concentration in bundle sheath cells, and thus, C4 plants minimize the photorespiration by spatially segregating CO2 fixation and the Calvin cycle. The adoption of the C4 pathway showcases nature's adaptive response to environmental constraints, offering insights into potential strategies for improving crop productivity and resilience in changing climate conditions15.
The specialized anatomy of the leaf structure in C4 plants is characterized by veins surrounded by enlarged vascular bundle sheath cells containing chloroplasts and with a radial arrangement of mesophyll cells in an outer ring patterning around bundle sheath cells. The mesophyll cells are in close proximity to the bundle sheath cells, which enables a rapid and continuous transport of metabolites between the two cell types. This cell's arrangement is typical of C4 plants and is referred to as Kranz anatomy16. In C3 species, mesophyll cell specialization and disposition can vary, but bundle sheath cells are distinctly smaller and have a few chloroplasts or no chloroplasts at all. Specific Kranz anatomy allows concentrating CO2 in chloroplasts in bundle sheath cells where the C3-carboxylating enzyme Rubisco is located, effectively hindering photorespiration4, 17, 18. Despite its seemingly complex arrangement, these changes have occurred independently multiple times in the evolution of angiosperms, indicating that it is a relatively feasible evolutionary pathway19,20,21, and various taxa have been shown to be at an intermediate stage between C3 and C4 carbon metabolism, referred to as C3-C4 or C2, having abilities to concentrate and re-assimilate CO222,23,24,25.
Many C4 plants are crops with high economic importance, and genetically engineering C3 crops, like rice, to improve their climate resilience and secure the yield has been a topic of interest in the last decades26,27. However, the engineering efforts call for a detailed understanding of C4 specialized anatomy and how it controls photosynthesis2,28.
Establishing the C4 Kranz anatomy is a prerequisite to achieving the ambitious goal of engineering C4 photosynthesis into C3 crops25. However, the current understanding of the regulation of Kranz anatomy and methods for quickly screening its key anatomical traits is limited, making it difficult to identify hybrid species. Previous studies have shown that key traits regulating photosynthetic efficiency in C3 and C4 plants include the interveinal distance, the diameter of the bundle sheath complex, and the size of bundle sheath cells14,29. These traits can be easily screened using free-hand sections and quantitatively analyzed using semi-thin sections. Here, we describe the method of assessing the traits that allow for C3 and C4 anatomical differentiation diagnostics through free-hand cross and light microscopy, namely bundle sheath area, interveinal distance, and vein frequency.
1. Plant growth conditions
2. Preparation and viewing of free-hand sections
3. Preparation of semi-thin sections
4. Imaging of samples
Figure 1A shows the correct orientation for sectioning the leaf for both fresh sectioning and light microscopy. The method for cutting fresh sections using a single-sided razor and a dental wax sheet can be seen in Figure 1B. The resulting sections are shown in Figure 1C.
Figure 2 shows free-hand sections of representative leaves of the C3 plant T. aestivum and the C4 plant Z. mays. Bundle sheath cells are visible around the vein, with maize having rounder and greener cells, indicating a higher presence of chloroplasts. It is possible to obtain leaf thickness and total bundle sheath area from these images; however, further characterization of the mesophyll is not possible with this method.
Figure 3 shows semi-thin cross-sections of T. aestivum and Z. mays. With this method, all tissues of the leaf are visible and measurable. Kranz's anatomy is evident in Z. mays, with mesophyll cells arranged around the veins. Bundle sheaths are enlarged and filled with chloroplasts, notably concentrated towards the mesophyll cells. This allows a rapid exchange of metabolites between bundle sheath cells and mesophyll cells. From these images, the following leaf structural traits were measured: interveinal distance between small and large veins, bundle sheath diameter, total bundle sheath area, veins area, fraction of bundle sheath cells area, bundle sheath cells area, vein frequency within 500 µm, and leaf thickness. Box plots in Figure 4 show the difference in such traits between T. aestivum and Z. mays. All but one trait, the interveinal distance between large veins, are significantly different at a 95% confidence interval with a p-value < 0.05.
Figure 4 shows a collection of box plots that represent the anatomical trait differences between T. aestivum and Z. mays, exemplifying the differences between C3 and C4 bundle sheath structures. There is a significant distinction between the interveinal distances between the crop species, the diameter of the bundle sheath (under the same growing conditions), the fraction of bundle sheath cells and non-photosynthetic material (i.e., veins), and vein frequency in a 500 µm section width – all of which are strongly indicative of what is expected between C3 and C4 leaf anatomy.
Figure 5 shows some examples of mistakes that are likely to occur when either cutting or preparing the samples. The leaf anatomy in these images is masked or deformed and is thus not reliable for characterization. Overstaining with toluidine blue can occur in sections cut with the ultramicrotome in which the stain is left for too long, thus obscuring the cell structure (Figure 5A,B). Common errors can occur during the infiltration protocol in which the resin does not fully penetrate the leaf tissues (Figure 5C,D). Leaf cross-sections that are cut along the grain result in oblong cells in which the mesophyll and bundle sheath cells cannot be located nor correctly measured (Figure 5F,G,H).
Statistical analyses were conducted in R (version 4.3.1) to create boxplots. Leaf anatomical traits were measured in triplicate (3 leaves per plant, 3 plants per species).
Figure 1: Correct method for cutting fresh leaf cross sections. (A) The orientation of leaf sections in order to obtain a cross-section. (B) Technique for sectioning leaves using a single-sided razor blade on a piece of flat dental wax. (C) The resulting cross-sections appear as thin leaf slivers that are ready for mounting on a glass slide and viewing under the microscope. Please click here to view a larger version of this figure.
Figure 2: Free-hand sections of representative leaves. (A) The C3 plant Triticum aestivum (400x magnification); and (B) the C4 plant Zea mays at (200x magnification). Cell type is indicated by arrows, where V = vein, BSC = bundle sheath cell, and MC = mesophyll cell. Please click here to view a larger version of this figure.
Figure 3: Semi-thin cross sections. (A) Zea mays and (B) Triticum aestivum to show the differences in bundle sheaths and chloroplast positions in C4 species with Kranz anatomy (A) and C3 species without Kranz anatomy (B). Abbreviations: Tl = leaf thickness, BS = bundle sheath, SV = small vein, ID = interveinal distance, and LV = large vein. Please click here to view a larger version of this figure.
Figure 4: Box-plots showing the difference in anatomical traits between Triticum aestivum and Zea mays. (A) interveinal distance between small veins, (B) interveinal distance between large veins, (C) bundle sheath diameter, (D) total bundle sheath area, (E) veins area, (F) fraction of bundle sheath cells area, (G) bundle sheath cells area, (H) vein frequency within 500 µm and (I) leaf thickness. Please click here to view a larger version of this figure.
Figure 5: Examples of common mistakes that might occur in tissue processing, indicated by red arrows. Overstaining in samples embedded in LR White acrylic resin (A) Triticum aestivum and (B) Zea mays; tears in resin in sections embedded in Spurr's epoxy resin (C) Ceratozamia robusta and (D) Dioon mejiae; T. aestivum free-hand sections showing (E) cross-section and (F) longitudinal section; bias in sectioning due to oblique cuts in (G) Annona muricata and (H) Encephalartos villosus. Please click here to view a larger version of this figure.
Solution | Preparation | Final volume | ||
Base for Buffer 1 | Dissolve 7.12 g of Na2HPO4 . 2H2O in 200 mL of distilled water | 200 mL | ||
Base for Buffer 2 | Dissolve 7.12 g of NaH2PO4 . 2H2O in 200 ml of distilled water | 200 mL | ||
Phosphate buffer solution | Mix 36 mL of Base 1, 14 mL of Base 2 and 50 mL of distilled water | 100 mL | ||
Fixative solution | Mix 9.25 mL of 5% glutaraldehyde (caution: see table of materials), 12.5 mL of phosphate buffer and 28.25 mL of distilled water. Store at 4 °C. | 50 mL |
Table 1: Recipe for the fixative solution.
Step | Description | Duration | Repeat | |||||||
1 | Rinsing with buffer | Gently remove the fixative with a pipette and add the phosphate buffer until the sample is covered. Leave for 30 min. | 30 min | 3x | ||||||
2 | Staining with 1% osmium tetroxide | (Caution: see Table of Materials): Remove the phosphate buffer with a pipette and add the osmium tetroxide until the sample is covered. Samples and other organic matter will turn black. Use double gloves. | 60 min | 1 | ||||||
3 | Rinsing the samples with distilled water | Remove the previous solution with a pipette and cover the samples with distilled water. Leave for 30 min. Repeat this step 3x. Use double gloves. | 30 min | 3 times | ||||||
4 | Ethanol series infiltration | Remove the previous solution with a pipette and cover the samples with the 50/50 distilled water-ethanol solution. Leave for 30 min. Repeat this step with distilled water-ethanol solutions in the following series: 30/70, 20/80, 10/90, and 100% ethanol. | 30 min | 1 | ||||||
5 | Resin-ethanol series infiltration | Remove the previous solution with a pipette and cover the samples with 1/3 resin-ethanol solution. Put the samples on a mixer plate and leave for 2 h. Repeat this step with resin-ethanol solutions in the following series: 1/2, 1/1, 2/1, 3/1, and 100% resin. | 120 min | 1 | ||||||
6 | Store in the fridge for 72 h before polymerization | |||||||||
7 | Polymerize infiltrated leaf sections according to product specifications to obtain resin-embedded samples. |
Table 2: Resin infiltration protocol.
In this article, we discuss both the quantitative and qualitative methods of measuring leaf anatomy and ways in which they can be optimized. Furthermore, the methodology is applied to representative crop species so as to determine which anatomical traits are most useful in distinguishing between C3 and C4 cross-sections. Understanding these traits is essential as hybrid species, termed C2 photosynthesis, is becoming a more promising avenue of research. As of now, only one crop species, Diplotaxis tenuifolia (arugula), has been identified to use C2 photosynthesis, but it is likely that there are more than what current records indicate25. High levels of intraspecific photosynthetic diversity and plasticity are present within C2 lineages. This implies that confirming the occurrence of C2 photosynthesis necessitates multiple lines of evidence, such as CO2 compensation point, ultrastructural analyses, and immunohistochemistry30.
Free-hand and semi-thin sections have been used in identifying and measuring specific plant tissues for many years, but this article aims to discuss the particular use case of anatomical leaf structures that affect photosynthetic differentiation between C3 and C4 crop plants. There are caveats to both free-hand and semi-thin sectioning that ought to be considered. Free-hand sections, while quick to obtain, are likely to result in leaf sections with inconsistent thicknesses, hindering quantitative analyses. This can be avoided by using specialized equipment for the sectioning of fresh plant material (e.g., vibratome or cryostat). The thicker sections from hand-sectioning can result in multiple layers of cells atop each other, blurring the cells and, thus, the anatomy. If the purpose of sectioning is purely qualitative, freehand sections offer the advantage of chemical processing of plant tissues (i.e., histochemistry)31. This allows for the qualitative indication of the presence of particular compounds in plant cells. Conversely, semi-thin sections almost always result in consistently thick sections. However, the processes are longer, delicate, and thus prone to error. Issues that may arise, as well as ways to avoid them, are discussed further.
Fixing
Fresh material must be used for both free-hand and semi-thin sections, i.e., leaves from plants that are sufficiently hydrated. Sections from wilted leaves do not have structural integrity as the cells have a decreased turgor pressure. When obtaining semi-thin sections from resin-embedded material, it is critical to ensure that leaf sections are fixed under a vacuum so as to replace the air inside the leaf. This allows for structural integrity and osmosis during the infiltration process. If this step is not done correctly, cells are likely to collapse, leading to cross-sections that do not accurately depict the real leaf structure and subsequently incorrect anatomical measurements32.
Conversely, high vacuum pressure can damage leaves with soft tissues (like cotyledons), resulting in shrunk cells or the destruction of the samples. This can be avoided by using an appropriate vacuum pump setting or by skipping it altogether and just soaking the samples in the fixative solution. If the latter is preferred, samples can be cut at all sides, as to ease the infiltration of the fixative into the tissue.
Pre-Infiltration
Cell wall thickness and sclereid tissue are the most influential anatomical traits to the permeation of solutions that allow for optimal fixation and infiltration of the specimen. The purpose of sclereids in plant tissue is mechanical support and protection, which can impact the infiltration process33. As these tissues have highly thickened lignified cell walls, permeation of solutions can be hindered. Samples that have infiltration issues result in semi-thin sections with broken cells and tears in the resin (Figure 5C,D). Longitudinal sectioning results in distorted cells, causing biases in anatomical analyses and measuring of leaf traits such as cell size and cell density (Figure 5F-H).
Resin Infiltration
Quality sectioning is vital for the observation of anatomical tissues, which are directly affected by the medium in which the specimen is embedded. The most common embedding media used in plant histology are paraffin wax, epoxy, and acrylic resins34,35. Wax-embedding requires preparation of samples by chemical fixative, and infiltration with liquid wax, which are hardened at room temperature. These cuts typically result in sections that are 5-10 µm in thickness, which, as demonstrated here, can be achieved just as well by hand (Figure 2) and require just unfixed fresh tissue. In this instance, the arrangement of cells around the bundle sheath is vital to distinguishing between C3 and C4 plants, which requires the use of a more robust and rigid embedding media. Epoxy resin (Spurr's resin36) and acrylic resin (LR White) were tested both for hardness, longevity, sample infiltration, as well as sectioning durability in this study. Epoxy resin has been used in the embedding of biological material for decades37. Acrylic resins are relatively new and were initially considered for embedding plant specimens due to their resilience over time as, unlike epoxy resins, they do not discolor as a result of UV exposure and are less prone to shrinkage38. LR White acrylic resin (hard grade) was selected for its non-toxicity and UV-stable formula. Acrylic resins tend to be considerably less viscous than epoxy resins, which allows for quicker sample infiltration, and the hardness of the material allows for cleaner cutting with a microtome39. Figure 5C,D shows the results of epoxy resin in leaf specimens with a large amount of structural tissue, where tears are inevitable due to the softness of epoxy resin, as compared with acrylic resin (Figure 3).
Ultramicrotomy
Trimming away excess resin may create cracks in the resin that penetrate into the sample. To avoid this, it is vital to consider the density and brittleness of the resin chosen for embedding. Cutting deeper into the section should avoid any artifacts caused by hand or rough knife trimming. The sample should be aligned perpendicularly to the sample holder and validated by increasing the magnification through the eyepiece, as well as viewing a fixed and stained section. Plant cells not cut at a perfectly right angle appear as tapered cells whereby one end is larger than the other40.
Post-staining
Toluidine blue (1% in aqueous solution) was used to stain semi-thin sections cut on the ultramicrotome. If the stain is left to dry on the section too long, it results in overstaining, causing an oversaturation of the section when imaged (Figure 5A). When the stain is not filtered well, it results in undissolved particles drying on the section, hindering the visualization of the anatomical structures (Figure 5B). This issue can be avoided by using a fine filter when making toluidine blue solution as well as limiting exposure of the section to the stain. Samples are dried on a hot plate at 60 °C prior to staining to ensure that the section bonds to the glass microscope slide, but should not be exposed for more than 10 s to toluidine blue before being rinsed and dried again41.
Considerations on anatomical measurements
The measurements shown in this paper are just an example of the many anatomical traits that can be quantitatively assessed using semi-thin sections, including a fraction of intercellular airspace, average mesophyll thickness (without vascular tissue), and mesophyll surface area exposed to intercellular air spaces per unit of leaf area42. However, while bundle sheath cell size is an important indicator to differentiate between the C3 and C4 metabolism, bundle sheath cell wall thickness is another important trait that cannot be viewed based on light microscopy alone, as it determines the degree of leakiness of bundle sheath cells, i.e., the amount of CO2 back diffusing into mesophyll cells42,43. High leakiness reduces the photosynthetic efficiency, as a large fraction of energy used for CO2 fixation by PEP carboxylase is lost44,45,46. For such ultrastructural analyses, Transmission Electron Microscopy and image analyses are required. Although only light microscopy was considered for the present study, the protocol includes the treatment with osmium tetroxide, a contrasting and fixing agent which is a cornerstone for TEM microscopy. Polymerized samples can thus still be used for TEM analysis whenever the need arises, even if not considered at the time of preparation.
Although anatomical diagnosis can easily detect Kranz-like structures, the presence of C4 can further be diagnosed by gas-exchange measurements which can show typical C4 physiology, i.e., low CO2 compensation point, limited O2 inhibition of photosynthesis and different carbon isotope composition2,4,47.
It should also be noted that Kranz anatomy is not necessary for C4 photosynthesis to develop, as shown by the discovery of single-cell C4 plants48,49,50. Anatomical analysis alone may, therefore, not be sufficient to detect C4 physiology in less studied taxa. The coupling of anatomical characterization with physiological analysis offers powerful tools for investigating leaf traits and their structure-function relationship in both crops and wild plants.
The authors have nothing to disclose.
The authors acknowledge the European Union H2020 Program (project GAIN4CROPS, GA no. 862087). The Centre of Excellence AgroCropFuture Agroecology and new crops in future climates is financed by the Ministry of Education and Research, Estonia. We wish to thank Professor Evelin Loit-Harro for providing seeds of T. aestivum and Z. mays, Paula Palmet and Vaiko Vainola for their assistance in preparing leaf cross-sections, and João Paulo de Silva Souza for assistance with analysis. All images were obtained from the microscopy unit of the Estonian University of Life Sciences under various projects.
Disodium hydrogen phosphate dihydrate (Na2HPO42H2O) pure | PENTA, CZ | 10028-24-7 | |
Embedding Film, 7.8 mil Thick, 8 x 12.5, (203 x 318mm) | ACLAR, US | 10501-10 | |
Ethanol, abs. 100% a.r. | Chem-Lab NV, BE | CL00.0505.1000 | Danger: Highly inflammable liquid and vapour. |
EVOS Invitrogen FL Auto 2 Imaging System | Thermo Fisher Scientific, US | ||
Flat Embedding PTFE Mold with Metal Frame, 16 cavities | PELCO, US | 10501 | |
Glass vial 2 ml | VWR Life Science, US | 548-0045 | |
Glutaraldehyde 50% solution | VWR Life Science, US | 23H2856331 | Danger: Fatal if inhaled. Toxic if swallowed. Causes severe skin burns and eye damage. May cause respiratory irritation. Wear protective gloves, protective clothing, eyes and face protection. |
Histo diamond knife | Diatome, US | ||
LEICA EM UC7 | Leica Vienna, AT | ||
LR White resin hard grade | Electron Microscopy Sciences, US | 14383 | Danger: Causes skin irritation. Causes severe eye irritation May cause respiratory irritation. May cause drowsiness or dizziness Wear protective gloves, protective clothing, eyes and face protection. |
Microscope slides | Normax, PT | 5470308A | |
Nikon Eclipse E600 and Nikon DS0Fi1 5 MP | Nikon Corporation, JP | ||
Osmium Tetroxide (OsO4) | Agar Scientific Ltd, GB | R1019 | Danger: Fatal if swallowed, in contact with skin or if inhaled. Causes severe skin burns and eye damage Wear double protective gloves, protective clothing, eyes and face protection. |
Pipette and pipette tips | Thermo Scientific, FI | KJ16047 | |
Sodium dihydrogen phosphate dihydrate (NaH2PO4 . 2H2O) pure | PENTA, CZ | 13472-35-0 | |
Syringe 10 ml | Ecoject, DE | 20010 | |
Toluidine blue, general purpose grade | Fisher Scientific, GB | 2045836 |