Comparative leaf anatomical features influencing photosynthetic efficiency in tropical and temperate maize hybrids

Introduction

Around 1.15 billion tonnes of maize were produced globally in 2022 [1] . Maize uses C 4 Kranz anatomy enlarged bundle sheath cells surrounding vascular bundles to concentrate CO 2 near Rubisco and suppress photorespiration [2] . Tropical and temperate hybrids, bred under different photoperiod and temperature regimes, may differ in bundle sheath size, mesophyll density, and interveinal distance [3,4] . These traits influence CO 2 transfer rates from mesophyll to bundle sheath cells [5,6] . Tropical hybrids, adapted to high irradiance, might feature denser venation supporting higher photosynthetic throughput [7] . However, direct anatomical comparisons under uniform conditions remain scarce, especially in arid environments [8] . This research compared leaf anatomy and photosynthesis of tropical and temperate maize hybrids at Riyadh, Saudi Arabia, to identify which structural traits best explain C 4 efficiency differences.

Material and methods

Research area and climatic conditions

The trial was conducted at the Agricultural Research Station of King Saud University, Riyadh (24.7136°N, 46.6753°E, 612 m asl) during the winter planting season of November 2022 to March 2023. The site has an arid climate with mean winter temperatures of 16-24 °C and minimal rainfall (total 28 mm during the cropping period). Soil was sandy loam (68.4 % sand, 18.7% silt, 12.9 % clay) with pH 7.9 and organic matter of 0.63 %. Supplemental irrigation was provided through drip lines at 5-day intervals [9] .

Material

Three tropical hybrids (TH-1, TH-2, TH-3) sourced from CIMMYT tropical germplasm and three temperate hybrids (TeH-1, TeH-2, TeH-3) from European breeding programmes were grown in a randomized complete block design with four replications. Plot size was 5 m × 3.6 m with 60 cm row spacing and 25 cm plant-to-plant distance. Fertilizer was applied at 200 kg N/ha, 80 kg P 2 O 5 /ha, and 60 kg K 2 O/ha in split does [10] .

Methods

At silking, third leaves below the ear were sampled from five plants per plot. Leaf cross-sections (10 µm thick) were cut with a rotary microtome, stained with safranin-fast green, and examined under a compound microscope [11] . Leaf Thickness (LT), Bundle Sheath Area (BSA), Mesophyll Cell Density (MCD), Interveinal Distance (IVD), and Stomatal Density (SD)

were measured using impression technique [12] . Gas exchange (Pn, gs, Tr, Ci) was recorded with a LI-6800 system (LI-COR, USA) at 1,800 µmol/m²/s PPFD. Chlorophyll fluorescence (Fv/Fm) was measured after 30-min dark adaptation. Data were analysed by ANOVA in SAS 9.4 with Tukey HSD at P = 0.05 [13] .

Results

Comprehensive interpretation

Tropical hybrids showed consistently larger anatomical dimensions (Table 1). TH-3 had the highest BSA (1,916 µm²) and MCD (334 cells/mm²), 59.9% and 64.5% greater than TeH-3 respectively. Interveinal distance was 22-31 % shorter in tropical hybrids. TH-3 recorded the highest Pn at 41.2 µmol/m²/s, 64.1% above TeH-3. Stepwise regression identified BSA as the single largest contributor to photosynthetic variation (28.3%), followed by MCD (24.7%) and IVD (19.1%) (Figure 1). Together, BSA and MCD explained 82.3% of total Pn variation, confirming Kranz anatomy dimensions as main structural drivers of C4 efficiency. Gas exchange data reinforced the anatomical findings. Stomatal conductance was 26-56% higher in tropical hybrids, supporting greater CO 2 diffusion. Despite higher transpiration, tropical Hybrids maintained superior intrinsic water use efficiency (WUEi 7.83-8.05 µmol/mmol vs 7.21-7.51 in temperate types). Fv/Fm values were slightly higher in tropical hybrids (0.807-0.823 vs 0.773-0.789), pointing to better PSII integrity under warm, high-light conditions.

The grouped comparison highlights the consistent gap between tropical and temperate groups for both BSA and MCD (Figure 2). TH-3 topped both parameters, while TeH-3 was lowest.

Discussion

The anatomical superiority of tropical hybrids in bundle sheath area and mesophyll density aligns with the hypothesis that tropical C4 grasses invest more in Kranz anatomy to exploit high-irradiance environments [3,7] . Larger bundle sheaths provide more space for C4-cycle CO 2 fixation, while denser mesophyll increases PEP carboxylase activity surface [5] . The 22-31% shorter interveinal distance in tropical hybrids means CO 2 reaches the bundle sheath faster, reducing leakage [6,14] . That BSA and MCD together explained 82.3% of Pn variation suggests these two traits could serve as anatomical selection markers. The higher WUEi in tropical hybrids is relevant for arid agriculture [8,15] . Temperate hybrids still reached acceptable Fv/Fm values (>0.77), indicating PSII damage wasn't the bottleneck rather, smaller Kranz anatomy was the main structural constraint on carbon fixation.

Conclusion

Tropical maize hybrids had 37-60% larger bundle sheath areas, 28-65% higher mesophyll cell density, and 24-45 % greater stomatal density than temperate hybrids, translating into 35-64% higher net photosynthesis. Bundle sheath area and mesophyll cell density jointly explained 82.3 % of photosynthetic variation. These anatomical traits merit use as selection criteria in maize improvement programmes for warm, high-radiation environments. TH-3 was the best-performing genotype across all anatomical and physiological parameters.

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