Assessing Structural Traits in Triticum aestivum and Zea mays for C3 and C4 Photosynthetic Differentiation Using Free-hand and Semi-thin Sections

Podsumowanie

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.

Streszczenie

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.

Wprowadzenie

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 oxygenation^3,4. 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 production^2,5,6.

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 temperatures^7,8,9,10, 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 photorespiration^{4, 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 pathway^19,20,21, 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 CO₂^22,23,24,25.

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 decades^26,27. However, the engineering efforts call for a detailed understanding of C₄ specialized anatomy and how it controls photosynthesis^2,28.

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 cells^14,29. 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.

Protokół

1. Plant growth conditions

1. Select wheat (Triticum aestivum L. Var. Winter Wheat, "Fredis") and maize (Zea mays L. Var. Saccharata, "Golden Bantam") as representative C₃ and C₄ plants, respectively. Grow both plant species in an environment-controlled growth chamber from seeds.
2. Maintain relative humidity at 55% with the air temperature at 25 °C/18 °C (day/night) with a 12 h light period. Maintain photosynthetic photon flux density (Q) at plant surface at 1200 µmol/m²/s to represent their natural growing conditions. Grow all plants at an ambient CO₂ concentration of 400 µmol/mol.
3. Water plants every 2^nd day with distilled water and fertilize with Hoagland's solution 10 days after seedling emergence. Grow the plants at the given conditions for 60 days and use fully expanded leaves for microscopic analyses.

2. Preparation and viewing of free-hand sections

1. Select 3 mature non-senescent leaves from each plant and keep them in distilled water. Using a new single-sided razor blade, make one leveling cut to form a right angle with the leaf surface to ensure the sections are transverse cross-sections made perpendicular to the leaf surface.
2. Place the leaf specimen on a glass plate on a piece of dental wax to stabilize the sample and avoid slipping. Cut by moving the blade straight down on the leaf specimen to cut cleanly through cells. Keep the cross-sections in distilled water.
3. Mount the samples on a glass slide by placing them on a water droplet and covering them with a glass coverslip for microscopical viewing and imaging. Avoid trapping air bubbles.
4. View the slide under a compound light microscope at 10x and 20x magnification. Save images as per the software guide, along with the relevant scale.

3. Preparation of semi-thin sections

1. Cut sections of approximately 3 mm x 5 mm in size from intercostal areas alongside the midrib from leaves from both Z. mays and T. aestivum on a glass plate as in step 2.2. Ensure that the length of the cut section runs along the grain of the leaf.
2. Place the plant material in a syringe with 1 mL of fixation buffer (4% glutaric aldehyde in 0.1 M phosphate buffer, pH = 6.9, Table 1).
3. Hold the syringe vertically, remove the air, and, holding a finger on the tip, pump the syringe to create a vacuum. Repeat until the samples lie at the bottom of the syringe, indicating successful infiltration.
4. Transfer the contents of the syringe to a labeled glass vial and store at 4 °C for 48 h before the next step (Table 2).
5. For each vial, gently remove the fixative with a pipette and add the phosphate buffer until the sample is covered. Leave for 30 min. Repeat this step 3x.
6. Remove the phosphate buffer with a pipette and add the osmium tetroxide (CAUTION) until the sample is covered. Samples and other organic matter will turn black. Use double gloves. Leave for 1 h.
7. Remove the previous solution with a pipette and cover the samples with distilled water. Leave for 30 min Repeat this step 3x and use double gloves.
8. 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 2x at 100 % ethanol. Samples in any of these solutions can be left overnight at 4 °C.
9. 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 2x at 100% resin. Samples in any of these solutions can be left overnight at 4 °C.
10. Cover the cavities of a flat embedding mold (16 cavities, cavity dimensions: 14 L x 4.8 W x 3 D (mm)) with 100% resin and place the samples on one end of the cavity. Prepare pencil-written paper labels for each sample and place them on the other end of the cavity.
11. Completely fill all the cavities with 100% resin and straighten the samples and labels if they moved. Cover the mold with embedding film and polymerize in an oven as per acrylic resin product guidelines.
12. Set the polymerized block with the sample in the specimen holder of the ultramicrotome. Trim the block using a rough glass knife until the tissue becomes visible and excess resin is eliminated.
13. Cut semi-thin sections transversely (1 µm) using a glass or diamond knife. Collect the sections using a metal inoculation loop from the water surface and place them on a glass slide. Dry the slide on a hot plate (60 °C) to fix the sections onto the glass.
14. Stain the fixed sections with toluidine blue (1% aqueous solution) for 5 s before rinsing with distilled water and drying again on the hot plate.
    NOTE: Ensure the chemical fixation process is performed under a fume hood. Glass knives should be replaced periodically to ensure sharp cuts that allow for sections without tears or distortions.

4. Imaging of samples

1. Imaging of fresh leaf sections
   1. Set up the compound light microscope with the accompanying software as per the manual.
   2. Place the glass slide on the stage. Adjust the eyepiece and view the section at 10x to obtain an overview, then at 20x and 40x objectives.
   3. At each objective, navigate the Live channel, focus on the area of interest, and locate the bundle sheath cells around the vein.
   4. Once focused, click the Freeze button and add the scale by pressing the Scale Bar button in the Tools tab. Save the image in .TIF format for image analysis.
2. Imaging of resin-infiltrated sections
   1. Set up the automated light microscope in connection with the accompanying software on the computer.
   2. Load the transparent channel and place the slide on the microscope stage. Focus on the section using a 40x objective magnification and adjust the brightness to ensure all cell structures are visible.
   3. Set the location area of the section using the navigator function to ensure the entire section is imaged and click the Stitch button.
   4. Click Done and then Start to allow the microscope to image the entire section. Open the image analysis software and open the tiles taken by the microscope.
   5. Using the Align feature, ensure that the tiles do not overlap. After generating the image, adjust the positioning, brightness, and rotation using the Adjust tab.
   6. Print the scale on the image before saving in .TIF format.
3. Anatomical measurements using ImageJ software
   1. Open ImageJ software and load the image by dragging it into the window.
   2. Using the Line tool, calibrate the scale as follows: Draw a line over the scale bar, choose Analyze > Set Scale, change the known distance to the length of the scale bar, and change the unit of length to the correct unit.
   3. Measure the prospective trait by selecting the Line Tool, drawing a line across the area of interest, and pressing the M key.
   4. For area measurements, select the Polygon Selection Tool and draw around the tissue of interest. Conclude by pressing the M key. The measurements show up as a pop-up window, which can be copied to a spreadsheet for further data analysis.

Wyniki

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

Dyskusje

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,

Materiały

Name	Company	Catalog Number	Comments
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