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 Jo&#227;o 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.

1. Plant growth conditions
<ol>
	<li>Select wheat (Triticum aestivum L. Var. Winter Wheat, &#34;Fredis&#34;) and maize (Zea mays L. Var. Saccharata, &#34;Golden Bantam&#34;) as representative C3 and C4 plants, respectively. Grow both plant species in an environment-controlled growth chamber from seeds.</li>
	<li>Maintain relative humidity at 55% with the air temperature at 25 &#176;C/18 &#176;C (day/night) with a 12 h light period. Maintain photosynthetic photon ...

Microscopical Analysis for Enhancing Photosynthetic Efficiency in C4 Crops

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, <e...

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&#39;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&#39;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&#39;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.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Disodium hydrogen phosphate dihydrate (Na2HPO42H2O) pure</td>
			<td>PENTA, CZ</td>
			<td>10028-24-7</td>
			<td></td>
		</tr>
		<tr>
			<td>Embedding Film, 7.8 mil Thick, 8 x 12.5, (203 x 318mm)</td>
			<td>ACLAR, US</td>
			<td>10501-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol, abs. 100% a.r.</td>
			<td>Chem-Lab NV, BE</td>
			<td>CL00.0505.1000</td>
			<td>Danger: Highly inflammable liquid and vapour.</td>
		</tr>
		<tr>
			<td>EVOS Invitrogen FL Auto 2 Imaging System</td>
			<td>Thermo Fisher Scientific, US</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Flat Embedding PTFE Mold with Metal Frame, 16 cavities</td>
			<td>PELCO, US</td>
			<td>10501</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass vial 2 ml</td>
			<td>VWR Life Science, US</td>
			<td>548-0045</td>
			<td></td>
		</tr>
		<tr>
			<td>Glutaraldehyde 50% solution</td>
			<td>VWR Life Science, US</td>
			<td>23H2856331</td>
			<td>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.</td>
		</tr>
		<tr>
			<td>Histo diamond knife</td>
			<td>Diatome, US</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>LEICA EM UC7</td>
			<td>Leica Vienna, AT</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>LR White resin hard grade</td>
			<td>Electron Microscopy Sciences, US</td>
			<td>14383</td>
			<td>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.</td>
		</tr>
		<tr>
			<td>Microscope slides</td>
			<td>Normax, PT</td>
			<td>5470308A</td>
			<td></td>
		</tr>
		<tr>
			<td>Nikon Eclipse E600 and Nikon DS0Fi1 5 MP</td>
			<td>Nikon Corporation, JP</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Osmium Tetroxide (OsO4)</td>
			<td>Agar Scientific Ltd, GB</td>
			<td>R1019</td>
			<td>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.</td>
		</tr>
		<tr>
			<td>Pipette and pipette tips</td>
			<td>Thermo Scientific, FI</td>
			<td>KJ16047</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium dihydrogen phosphate dihydrate (NaH2PO4 . 2H2O) pure</td>
			<td>PENTA, CZ</td>
			<td>13472-35-0</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe 10 ml</td>
			<td>Ecoject, DE</td>
			<td>20010</td>
			<td></td>
		</tr>
		<tr>
			<td>Toluidine blue, general purpose grade</td>
			<td>Fisher Scientific, GB</td>
			<td>2045836</td>
			<td></td>
		</tr>
	</tbody>
</table>

assessing structural traits in triticum aestivum and zea mays for c3 and c4 photosynthetic differentiation using free-hand and semi-thin sections

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,&#160;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.

Author Spotlight: Innovative Approaches to Understanding Plant Structure-Function Relationships for Climate-Resilient Crops

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

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</em...

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.