Sheath Cells Research Articles

Cyclic electron flow and Photosystem II-less photosynthesis.

Oxygenic photosynthesis is characterised by the cooperation of two photo-driven complexes, Photosystem II (PSII) and Photosystem I (PSI), sequentially linked through a series of redox-coupled intermediates. Divergent evolution has resulted in photosystems exhibiting complementary redox potentials, spanning the range necessary to oxidise water and reduce CO2 within a single system. Catalysing nature's most oxidising reaction to extract electrons from water is a highly specialised task that limits PSII's metabolic function. In contrast, potential electron donors in PSI span a range of redox potentials, enabling it to accept electrons from various metabolic processes. This metabolic flexibility of PSI underpins the capacity of photosynthetic organisms to balance energy supply with metabolic demands, which is key for adaptation to environmental changes. Here, we review the phenomenon of 'PSII-less photosynthesis' where PSI functions independently of PSII by operating cyclic electron flow using electrons derived from non-photochemical reactions. PSII-less photosynthesis enables supercharged ATP production and is employed, for example, by cyanobacteria's heterocysts to host nitrogen fixation and by bundle sheath cells of C4 plants to boost CO2 assimilation. We discuss the energetic benefits of this arrangement and the prospects of utilising it to improve the productivity and stress resilience of photosynthetic organisms.

A Rare Case Report of Malignant Peripheral Nerve Sheath Tumor in the Infratemporal Fossa.

Introduction: Malignant peripheral nerve sheath tumors (MPNSTs) are rare tumors that develop from peripheral nerve sheath cells and they account for approximately 5% to 10% of all soft-tissue sarcomas. MPNSTs in the head and neck region represent approximately 2% to 6% of all head and neck sarcomas and account for 12% to 19% of all MPNSTs, and the infratemporal fossa is a rare site for MPNSTs. MPNSTs originating from the trigeminal nerve are extremely rare. Case presentation: A 19-year-old female presented with a 6-month history of left-sided facial pain and paresthesia on the same side. On examination, there was left-sided facial paresthesia at the third trigeminal nerve (V3) areas; computed tomography scanning and magnetic resonance imaging showed an infratemporal lesion and surgical resection was done. Histological examination and the immunostaining finding showed high-grade MPNST. Conclusion: MPNSTs in the head and neck region may manifest with nonspecific symptoms. The diagnosis often requires a combination of clinical, pathological, and immunohistochemistry studies. Treatment involves total surgical resection with adjuvant radiotherapy.

Transcriptome dynamics in developing leaves from C3 and C4 Flaveria species.

C4 species have evolved more than 60 times independently from C3 ancestors. This multiple and parallel evolution of the complex C4 trait suggests common underlying evolutionary mechanisms, which could be identified by comparative analysis of closely related C3 and C4 species. Efficient C4 function depends on a distinctive leaf anatomy that is characterised by enlarged, chloroplast-rich bundle sheath cells and narrow vein spacing. To elucidate the molecular mechanisms that generate the Kranz anatomy, we analysed a developmental series of leaves from the C4 plant Flaveria bidentis and the closely related C3 species Flaveria robusta by comparing anatomies and transcriptomes. Vascular density measurements of all nine leaf developmental stages identified three leaf anatomical zones whose proportions vary with respect to the developmental stage. We then deconvoluted the transcriptome datasets using non-negative matrix factorisation, which identified four distinct transcriptome patterns in the growing leaves of both species. By integrating the leaf anatomy and transcriptome data, we were able to correlate the different transcriptional profiles with different developmental zones in the leaves. These comparisons revealed an important role for auxin metabolism, in particular auxin homeostasis (conjugation and deconjugation), in establishing the high vein density typical of C4 species.

Multifaceted analyses reveal sugar and organic acid metabolism affecting the palatability in mulched Phyllostachys violascens shoots with varied sheath colors

The color of bamboo shoot sheaths influences both appearance and palatability in mulched Phyllostachys violascens shoots, but the mechanisms linking sheath color to flavor and texture are unclear. This study combined metabolomics and transcriptomics to investigate the relationship between sheath color and palatability, focusing on sugar and organic acid metabolism. We identified 5698 metabolites and 33 differentially expressed genes across bamboo shoots with different sheath colors. Darker shoots (black-brown, BSD) exhibited enhanced organic acids and flavor amino acid synthesis, contributing to moderate sweetness. In contrast, as the sheath color lightens (bright-red, BSM; yellow-green, BSL), sugar degradation and cell wall metabolism (e.g., lignin, pectin, cellulose) became more pronounced. BSL shoots, with the lightest sheath color, demonstrated reduced sugar degradation and increased synthesis of pectin and cellulose, resulting in greater sweetness and improved texture. These findings demonstrate that variations in sheath color are closely linked to metabolic shifts that influence both flavor and texture, offering valuable insights for targeted breeding programs aimed at enhancing bamboo shoot quality.

Metabolomics of related C3 and C4 Flaveria species indicate differences in the operation of photorespiration under fluctuating light.

C3 photosynthesis can be complemented with a C4 carbon concentrating mechanism (CCM) to minimize photorespiratory losses. C4 photosynthesis is often more efficient than C3 under steady-state conditions. However, the C4 CCM depends on inter-cellular metabolite concentration gradients, which must increase following increases in light intensity and could decrease rates of C4 photosynthesis under fluctuating light. Additionally, incomplete flux through photorespiration could prove beneficial to C4 assimilation during light induction of the CCM. Here, we compare metabolic profiles in the closely related C3 Flaveria robusta and C4 Flaveria bidentis during a light transient from low to high light to determine if these non-steady state accumulation patterns provide insight to the induction of the metabolite gradients needed to drive C4 intermediate transport and if there is incomplete cycling of photorespiratory intermediates. In these C3 and C4 species, metabolite steady-state pool sizes suggest that C4 transport acids maintain concentration gradients across the bundle sheath and mesophyll cell types under these light fluctuations. However, there was incomplete flux through photorespiration in the C4 F. bidentis, which could reduce photorespiratory CO2 loss via glycine decarboxylation and help maintain higher rates of assimilation during following induction periods.

Mycoplasma synoviae surface-located elongation factor G functions as a cytoadhesin to promote adhesion to synovial sheath cells through binding to vimentin

Mycoplasma synoviae infection has caused serious economic losses to the poultry industry worldwide. The molecular mechanism by which M. synoviae colonizes the synovium and induces synovitis is unclear. In this study, desthiobiotin pull-down and liquid chromatography‐tandem mass spectrometry analyses were used to screen M. synoviae membrane proteins that bind the membrane proteins of synovial sheath cells (SSCs). Among the 128 screened proteins, elongation factor G (EF-G) of M. synoviae was identified as a surface-located protein using colony blotting and dual fluorescence analyses. The immunogenicity of EF-G was confirmed by the preparation of a rabbit polyclonal antibody. EF-G was identified as a cytoadhesin that directly binds to SSCs using indirect immunofluorescence assay and ELISA plate binding assay. In addition, antibody adhesion inhibition and protein adhesion inhibition demonstrated that EF-G could significantly promote the adhesion of M. synoviae to SSCs. Co-IP, GST pull-down, bacterial two-hybridization, and ELISA plate binding assays were performed to demonstrate the binding of EF-G and vimentin in vivo and in vitro. Antibody adhesion inhibition, protein adhesion inhibition, and siRNA interference adhesion inhibition assays demonstrated that vimentin significantly affected M. synoviae adhesion to SSCs. These studies indicate that two interacting proteins, EF-G, a novel cytoadhesin, and vimentin, an important cell surface receptor, play important roles in the adhesion of M. synoviae to SSCs, laying a foundation for subsequent studies on the mechanism of M. synoviae-induced synovitis and providing meaningful targets for screening target drugs against M. synoviae infection.

Characterization of human trabecular bone across multiple length scales using a correlative approach combining X-ray tomography with LaserFIB and plasma FIB-SEM

Three-dimensional correlative multimodal and multiscale imaging is an emerging method for investigating the complex hierarchical structure of biological materials such as bone. This approach synthesizes images acquired across multiple length scales, for the same region of interest, to provide a comprehensive view of the material structure of a sample. Here, we develop a workflow for the structural analysis of human trabecular bone using a femtosecond laser to produce a precise grid to facilitate correlation between imaging modalities and identification of structures of interest, in this case, a single trabecula within a volume of trabecular bone. Through such image registration, high resolution X-ray microscopy imaging revealed fine architectural details, including the cement sheath and bone cell lacunae of the selected bone trabecula. The selected bone volume was exposed with a combination of manual polishing and site-specific femtosecond laser ablation and then examined with plasma focused ion beam-scanning electron microscopy. This reliable and versatile correlation approach has the potential to be applied to a variety of biological tissues and traditional engineered materials. The proposed workflow has the enhanced capability for generating highly resolved and broadly contextualized structural data for a better understanding of the architectural features of a material spanning its macroscopic to nanoscopic levels.

Mesophyll airspace unsaturation drives C4 plant success under vapor pressure deficit stress

A fundamental assumption in plant science posits that leaf air spaces remain vapor saturated, leading to the predominant view that stomata alone control leaf water loss. This concept has been pivotal in photosynthesis and water-use efficiency research. However, recent evidence has refuted this longstanding assumption by providing evidence of unsaturation in the leaf air space of C3 plants under relatively mild vapor pressure deficit (VPD) stress. This phenomenon represents a nonstomatal mechanism restricting water loss from the mesophyll. The potential ubiquity and physiological implications of this phenomenon, its driving mechanisms in different plant species and habitats, and its interaction with other ecological adaptations have. In this context, C4 plants spark particular interest for their importance as crops, bundle sheath cells' unique anatomical characteristics and specialized functions, and notably higher water-use efficiency relative to C3 plants. Here, we confirm reduced relative humidities in the substomatal cavity of the C4 plants maize, sorghum, and proso millet down to 80% under mild VPD stress. We demonstrate the critical role of nonstomatal control in these plants, indicating that the role of the CO2 concentration mechanism in CO2 management at a high VPD may have been overestimated. Our findings offer a mechanistic reconciliation between discrepancies in CO2 and VPD responses reported in C4 species. They also reveal that nonstomatal control is integral to maintaining an advantageous microclimate of relatively higher CO2 concentrations in the mesophyll air space of C4 plants for carbon fixation, proving vital when these plants face VPD stress.

Regulatory Network of the Late-Recruited Primary Decarboxylase C4NADP-ME in Sugarcane.

In agronomically important C4 grasses, efficient CO2 delivery to Rubisco is facilitated by NADP-malic enzyme (C4NADP-ME), which decarboxylates malate in bundle sheath cells. However, understanding the molecular regulation of the C4NADP-ME gene in sugarcane (Saccharum spp.) is hindered by its complex genetic background. Enzymatic activity assays demonstrated that decarboxylation in sugarcane Saccharum spontaneum predominantly relies on the NADP-ME pathway, similar to sorghum (Sorghum bicolor) and maize (Zea mays). Comparative genomics analysis revealed the recruitment of eight core C4 shuttle genes, including C4NADP-ME (SsC4NADP-ME2), in the C4 pathway of sugarcane. Contrasting to sorghum and maize, the expression of SsC4NADP-ME2 in sugarcane is regulated by different transcription factors (TFs). We propose a gene regulatory network for SsC4NADP-ME2, involving candidate TFs identified through gene co-expression analysis and yeast one-hybrid experiment. Among these, ABA INSENSITIVE5 (ABI5) was validated as the predominant regulator of SsC4NADP-ME2 expression, binding to a G-box within its promoter region. Interestingly, the core element ACGT within the regulatory G-box was conserved in sugarcane, sorghum, maize, and rice (Oryza sativa), suggesting an ancient regulatory code utilized in C4 photosynthesis. This study offers insights into SsC4NADP-ME2 regulation, crucial for optimizing sugarcane as a bioenergy crop.

Weight-induced radial growth in plant stems depends on PIN3

How multiple growth programs coordinate during development is a fundamental question in biology. During plant stem development, radial growth is continuously adjusted in response to longitudinal-growth-derived weight increase to guarantee stability.1,2,3 Here, we demonstrate that weight-stimulated stem radial growth depends on the auxin efflux carrier PIN3, which, upon weight increase, expands its cellular localization from the lower to the lateral sides of xylem parenchyma, phloem, procambium, and starch sheath cells, imposing a radial auxin flux that results in radial growth. Using the protein synthesis inhibitor cycloheximide (CHX) or the fluorescent endocytic tracer FM4-64, we reveal that this expansion of the PIN3 cellular localization domain occurs because weight increase breaks the balance between PIN3 biosynthesis and removal, favoring PIN3 biosynthesis. Experimentation using brefeldin A (BFA) treatments or arg1 and arl2 mutants further supports this conclusion. Analyses of CRISPR-Cas9 lines for Populus PIN3 orthologs reveals that PIN3 dependence of weight-induced radial growth is conserved at least in these woody species. Altogether, our work sheds new light on how longitudinal and radial growth coordinate during stem development.

The Neuroprotective Effect of Erythropoietin on the Optic Nerve and Spinal Cord in Rats with Experimental Autoimmune Encephalomyelitis through the Activation of the Extracellular Signal-Regulated Kinase 1/2 Signaling Pathway.

Experimental autoimmune encephalomyelitis is a demyelinating disease that causes paralysis in laboratory rats. This condition lacks treatment that reverses damage to the myelin sheaths of neuronal cells. Therefore, in this study, treatment with EPO as a neuroprotective effect was established to evaluate the ERK 1/2 signaling pathway and its participation in the EAE model. EPO was administered in 5000 U/Kg Sprague Dawley rats. U0126 was used as an inhibitor of the ERK 1/2 pathway to demonstrate the possible activation of this pathway in the model. Spinal cord and optic nerve tissues were evaluated using staining techniques such as H&E and the Luxol Fast Blue myelin-specific technique, as well as immunohistochemistry of the ERK 1/2 protein. The EPO-treated groups showed a decrease in cellular sampling in the spinal cord tissues but mainly in the optic nerve, as well as an increase in the expression of the ERK 1/2 protein in both tissues. The findings of this study suggest that EPO treatment reduces cellular death in EAE-induced rats by regulating the ERK pathway.

The genome of Eleocharis vivipara elucidates the genetics of C3-C4 photosynthetic plasticity and karyotype evolution in the Cyperaceae.

Eleocharis vivipara, an amphibious sedge in the Cyperaceae family, has several remarkable properties, most notably its alternate use of C3 photosynthesis underwater and C4 photosynthesis on land. However, the absence of genomic data has hindered its utility for evolutionary and genetic research. Here, we present a high-quality genome for E. vivipara, representing the first chromosome-level genome for the Eleocharis genus, with an approximate size of 965.22 Mb mainly distributed across 10 chromosomes. Its Hi-C pattern, chromosome clustering results, and one-to-one genome synteny across two subgroups indicates a tetraploid structure with chromosome count 2n = 4x = 20. Phylogenetic analysis suggests that E. vivipara diverged from Cyperus esculentus approximately 32.96 million years ago (Mya), and underwent a whole-genome duplication (WGD) about 3.5 Mya. Numerous fusion and fission events were identified between the chromosomes of E. vivipara and its close relatives. We demonstrate that E. vivipara has holocentromeres, a chromosomal feature which can maintain the stability of such chromosomal rearrangements. Experimental transplantation and cross-section studies showed its terrestrial culms developed C4 Kranz anatomy with increased number of chloroplasts in the bundle sheath (BS) cells. Gene expression and weighted gene co-expression network analysis (WGCNA) showed overall elevated expression of core genes associated with the C4 pathway, and significant enrichment of genes related to modified culm anatomy and photosynthesis efficiency. We found evidence of mixed nicotinamide adenine dinucleotide - malic enzyme and phosphoenolpyruvate carboxykinase type C4 photosynthesis in E. vivipara, and hypothesize that the evolution of C4 photosynthesis predates the WGD event. The mixed type is dominated by subgenome A and supplemented by subgenome B. Collectively, our findings not only shed light on the evolution of E. vivipara and karyotype within the Cyperaceae family, but also provide valuable insights into the transition between C3 and C4 photosynthesis, offering promising avenues for crop improvement and breeding.

Chitinase 3-Like 1 and C-X-C motif chemokine ligand 5 proteins and the hair cycle.

Dermal papilla cells (DPCs) exhibit self-recovery ability, which may be involved in hair growth. Therefore, we tested whether DPCs subjected to temporary growth-inhibiting stress (testosterone, 17β-estradiol, mitomycin C, or undernutrition) treatments exhibit self-recovery behavior that can activate hair follicle growth, and examined the changes in cell proliferation capacity and gene expression. Related proteins were identified and their relationships with the hair cycle was examined using a mouse model. Recovery-period DPCs (i.e., from day 3 after loading) were subjected to microarray analysis to detect genetic variations common to each stress treatment. Co-culture of recovery-period DPCs and outer root sheath cells (ORSCs) confirmed the promotion of ORSC proliferation, suggesting that the activation of hair follicle growth is promoted via signal transduction. Chitinase 3-like 1 (CHI3L1) and C-X-C motif chemokine 5 (CXCL5) exhibited ORSC proliferation-promoting effects. Measurement of protein content in the skin during each phase of the hair cycle in mice revealed that CHI3L1 and CXCL5 secretion increased immediately after anagen transition. In a hair-loss mouse model treated with testosterone or 17β-estradiol, CHI3L1 and CXCL5 secretion was lower in treated telogen skin than in untreated skin. Our results suggest that CHI3L1 and CXCL5 secreted by recovery-state DPCs promote hair growth.

Exploring the role of dermal sheath cells in wound healing and fibrosis.

Wound healing is a complex, dynamic process involving the coordinated interaction of diverse cell types, growth factors, cytokines, and extracellular matrix components. Despite emerging evidence highlighting their importance, dermal sheath cells remain a largely overlooked aspect of wound healing research. This review explores the multifunctional roles of dermal sheath cells in various phases of wound healing, including modulating inflammation, aiding in proliferation, and contributing to extracellular matrix remodelling. Special attention is devoted to the paracrine effects of dermal sheath cells and their role in fibrosis, highlighting their potential in improving healing outcomes, especially in differentiating between hairy and non-hairy skin sites. By drawing connections between dermal sheath cells activity and wound healing outcomes, this work proposes new insights into the mechanisms of tissue regeneration and repair, marking a step forward in our understanding of wound healing processes.

The Influence of SARS-CoV-2 Infection on the Development of Selected Neurological Diseases.

In 2024, over 775 million cases of COVID-19 were recorded, including approximately 7 million deaths, indicating its widespread and dangerous nature. The disease is caused by the SARS-CoV-2 virus, which can manifest a wide spectrum of symptoms, from mild infection to respiratory failure and even death. Neurological symptoms, such as headaches, confusion, and impaired consciousness, have also been reported in some COVID-19 patients. These observations suggest the potential of SARS-CoV-2 to invade the central nervous system and induce neuroinflammation during infection. This review specifically explores the relationship between SARS-CoV-2 infection and selected neurological diseases such as multiple sclerosis (MS), ischemic stroke (IS), and Alzheimer's disease (AD). It has been observed that the SARS-CoV-2 virus increases the production of cytokines whose action can cause the destruction of the myelin sheaths of nerve cells. Subsequently, the body may synthesize autoantibodies that attack nerve cells, resulting in damage to the brain's anatomical elements, potentially contributing to the onset of multiple sclerosis. Additionally, SARS-CoV-2 exacerbates inflammation, worsening the clinical condition in individuals already suffering from MS. Moreover, the secretion of pro-inflammatory cytokines may lead to an escalation in blood clot formation, which can result in thrombosis, obstructing blood flow to the brain and precipitating an ischemic stroke. AD is characterized by intense inflammation and heightened oxidative stress, both of which are exacerbated during SARS-CoV-2 infection. It has been observed that the SARS-CoV-2 demonstrates enhanced cell entry in the presence of both the ACE2 receptor, which is already elevated in AD and the ApoE ε4 allele. Consequently, the condition worsens and progresses more rapidly, increasing the mortality rate among AD patients. The above information underscores the numerous connections between SARS-CoV-2 infection and neurological diseases.

Glycogen Phosphorylase Inhibitor Promotes Hair Growth via Protecting from Oxidative-Stress and Regulating Glycogen Breakdown in Human Hair follicles.

Hair growth cycles are mainly regulated by human dermal papilla cells (hDPCs) and human outer root sheath cells (hORSCs). Protecting hDPCs from excessive oxidative stress and hORSCs from glycogen phosphorylase (PYGL) is crucial to maintaining the hair growth phase, anagen. In this study, we developed a new PYGL inhibitor, Hydroxytrimethylpyridinyl Methylindolecarboxamide (HTPI) and assessed its potential to prevent hair loss. HTPI reduced oxidative damage, preventing cell death and restored decreased level of anagen marker ALP and its related genes induced by hydrogen peroxide in hDPCs. Moreover, HTPI inhibited glycogen degradation and induced cell survival under glucose starvation in hORSCs. In ex-vivo culture, HTPI significantly enhanced hair growth compared to the control with minoxidil showing comparable results. Overall, these findings suggest that HTPI has significant potential as a therapeutic agent for the prevention and treatment of hair loss.

Detection of exogenous siRNA inside sweet corn bundle sheath cells and the RNAi dynamics in the early stage of Maize dwarf mosaic virus infection

Maize dwarf mosaic virus (MDMV) is one of the most serious viruses of sweet corn. Utilising the process of RNA interference, the exogenous introduction of small RNA molecules mimicking virus-derived small interfering RNA (siRNA) into the plant prior to infection triggers the antiviral RNA silencing effect, thereby promoting more effective antiviral protection. Hence, a treatment with MDMV-derived small RNA was applied to sweet corn plants one day before MDMV virus inoculation. ALEXA FLUOR®488 fluorophore-bound exogenous siRNA was successfully detected inside intact sweet corn cells using confocal fluorescence microscopy. Furthermore, it was demonstrated that the exogenous siRNA treatment led to a notable upregulation of the AGO1, AGO2b, AGO10b, AGO18a, DCL1, DCL3a, DCL4, RDR1, and MOP1 genes within 24 h of the treatment. Overall, exogenous siRNA treatment resulted in better virus control of infected sweet corn plants, as indicated by the lower viral RNA and coat protein levels compared to the infected group without pre-treatment.

18-β-Glycyrrhetinic Acid Promotes Hair Growth by Stimulating the Proliferation of Dermal Papilla Cells and Outer Root Sheath Cells, and Extends the Anagen Phase by Inhibiting 5α-Reductase.

18-β-Glycyrrhetinic acid, a major component of licorice, stimulated the proliferation of both dermal papilla cells and outer root sheath cells isolated from human hair follicles. Thus, suggesting that this compound promotes hair growth. Furthermore, this compound inhibited the activity of testosterone 5α-reductase, an enzyme responsible for converting androgen to dihydroandrogen, with an IC50 of 137.1 µM. 18-β-Glycyrrhetinic acid also suppressed the expression of transforming growth factor-β1 (TGF-β1), which shifts the hair cycle from the anagen phase to the telogen phase. It suggested that this compound may prolong the anagen phase. Based on these findings, this compound could be a potentially effective treatment for androgenetic alopecia.

Chloroplast NADH dehydrogenase-like complex-mediated cyclic electron flow is the main electron transport route in C4 bundle sheath cells.

The superior productivity of C4 plants is achieved via a metabolic C4 cycle which acts as a CO2 pump across mesophyll and bundle sheath (BS) cells and requires an additional input of energy in the form of ATP. The importance of chloroplast NADH dehydrogenase-like complex (NDH) operating cyclic electron flow (CEF) around Photosystem I (PSI) for C4 photosynthesis has been shown in reverse genetics studies but the contribution of CEF and NDH to cell-level electron fluxes remained unknown. We have created gene-edited Setaria viridis with null ndhO alleles lacking functional NDH and developed methods for quantification of electron flow through NDH in BS and mesophyll cells. We show that CEF accounts for 84% of electrons reducing PSI in BS cells and most of those electrons are delivered through NDH while the contribution of the complex to electron transport in mesophyll cells is minimal. A decreased leaf CO2 assimilation rate and growth of plants lacking NDH cannot be rescued by supplying additional CO2. Our results indicate that NDH-mediated CEF is the primary electron transport route in BS chloroplasts highlighting the essential role of NDH in generating ATP required for CO2 fixation by the C3 cycle in BS cells.

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,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.