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

Summary

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.

Abstract

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.

Introduction

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 condi....

Protocol

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 .......

Discussion

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,

Materials

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