Reconciling water-use efficiency estimates from carbon isotope discrimination of leaf biomass and tree rings: nonphotosynthetic fractionation matters.

Carbon isotope discrimination (∆) in leaf biomass (∆BL) and tree rings (∆TR) provides important proxies for plant responses to climate change, specifically in terms of intrinsic water-use efficiency (iWUE). However, the nonphotosynthetic 12C/13C fractionation in plant tissues has rarely been quantified and its influence on iWUE estimation remains uncertain. We derived a comprehensive, ∆ based iWUE model (iWUEcom) which includes nonphotosynthetic fractionations (d) and characterized tissue-specific d-values based on global compilations of data of ∆BL, ∆TR and real-time ∆ in leaf photosynthesis (∆online). iWUEcom was further validated with independent datasets. ∆BL was larger than ∆online by 2.53‰, while ∆BL and ∆TR showed a mean offset of 2.76‰, indicating that ∆TR is quantitatively very similar to ∆online. Applying the tissue-specific d-values (dBL = 2.5‰, dTR = 0‰), iWUE estimated from ∆BL aligned well with those estimated from ∆TR or gas exchange. ∆BL and ∆TR showed a consistent iWUE trend with an average CO2 sensitivity of 0.15 ppm ppm-1 during 1975-2015. Accounting for nonphotosynthetic fractionations improves the estimation of iWUE based on isotope records in leaf biomass and tree rings, which is ultimate for inferring changes in carbon and water cycles under historical and future climate.

Nanoceria-induced variations in leaf anatomy and cell wall composition drive the increase in mesophyll conductance of salt-stressed cotton leaves.

Nanomaterials as an emerging tool are being used to improve plant's net photosynthetic rate (AN) when suffering salt stress, but the underlying mechanisms remain unclear. To clarify this, a hydroponic experiment was conducted to study the effects of polyacrylic acid coated nanoceria (PNC) on the AN of salt-stressed cotton and related intrinsic mechanisms. Results showed that the PNC-induced AN enhancement of salt-stressed leaves was strongly facilitated by the mesophyll conductance to CO2 (gm). Further analysis showed that the PNC-induced improvement of gm was related to the increased chloroplast surface area exposed to intercellular airspaces, which was attribute to the increased mesophyll surface area exposed to intercellular airspaces and chloroplast number due to the increased K+ content and decreased reactive oxygen species level in salt-stressed leaves. Interestingly, our results also showed that PNC-induced variations in cell wall composition of salt-stressed cotton leaves strongly influenced gm, especially, hemicellulose and pectin. Moreover, the proportion of pectin in cell wall composition played a more important role in determining gm. Our study demonstrated for the first time that nanoceria, through alterations to anatomical traits and cell wall composition, drove gm enhancement, which ultimately increased AN of salt-stressed leaves.

Growing at the arid edge: Anatomical variations in leaves are more extensive than in stems of five Mediterranean species across contrasting moisture regimes.

PREMISE: Increasing aridity in the Mediterranean region affects ecosystems and plant life. Various anatomical changes in plants help them cope with dry conditions. This study focused on anatomical differences in leaves and xylem of five co-occurring Mediterranean plant species namely Quercus calliprinos, Pistacia palaestina, Pistacia lentiscus, Rhamnus lycioides, and Phillyrea latifolia in wet and dry sites. METHODS: Stomatal density, stomatal length, leaf mass area, lamina composition, percentage of intercellular air spaces, and mesophyll cell area in leaves of plants in wet and dry sites were analyzed. Xylem anatomy was assessed through vessel length and area in branches. RESULTS: In the dry site, three species had increased stomatal density and decreased stomatal length. Four species had increased palisade mesophyll and reduced air space volume. In contrast, phenotypic changes in the xylem were less pronounced; vessel length was unaffected by site conditions, but vessel diameter decreased in two species. Intercellular air spaces proved to be the most dynamic anatomical feature. Quercus calliprinos had the most extensive anatomical changes; Rhamnus lycioides had only minor changes. All these changes were observed in comparison to the species in the wet site. CONCLUSIONS: This study elucidated variations in anatomical responses in leaves among co-occurring Mediterranean plant species and identified the most dynamic traits. Understanding these adaptations provides valuable insights into the ability of plants to thrive under changing climate conditions.

High photosynthesis rates in Brassiceae species are mediated by leaf anatomy enabling high biochemical capacity, rapid CO2 diffusion and efficient light use.

Certain species in the Brassicaceae family exhibit high photosynthesis rates, potentially providing a valuable route toward improving agricultural productivity. However, factors contributing to their high photosynthesis rates are still unknown. We compared Hirschfeldia incana, Brassica nigra, Brassica rapa and Arabidopsis thaliana, grown under two contrasting light intensities. Hirschfeldia incana matched B. nigra and B. rapa in achieving very high photosynthesis rates under high growth-light condition, outperforming A. thaliana. Photosynthesis was relatively more limited by maximum photosynthesis capacity in H. incana and B. rapa and by mesophyll conductance in A. thaliana and B. nigra. Leaf traits such as greater exposed mesophyll specific surface enabled by thicker leaf or high-density small palisade cells contributed to the variation in mesophyll conductance among the species. The species exhibited contrasting leaf construction strategies and acclimation responses to low light intensity. High-light plants distributed Chl deeper in leaf tissue, ensuring even distribution of photosynthesis capacity, unlike low-light plants. Leaf anatomy of H. incana, B. nigra and B. rapa facilitated effective CO2 diffusion, efficient light use and provided ample volume for their high maximum photosynthetic capacity, indicating that a combination of adaptations is required to increase CO2-assimilation rates in plants.

Shorting the metaphorical circuit: vascular partitioning and stomatal patchiness can create apparent unsaturation and CO2 gradient inversion in the Ohmic analogy for leaf gas exchange.

Analyses of leaf gas exchange rely on an Ohmic analogy that arrays single stomatal, internal air space, and mesophyll conductances in series. Such models underlie inferences of mesophyll conductance and the relative humidity of leaf airspaces, reported to fall as low as 80%. An unresolved question is whether such series models are biased with respect to real leaves, whose internal air spaces are chambered at various scales by vasculature. To test whether unsaturation could emerge from modeling artifacts, we compared series model estimates with true parameter values for a chambered leaf with varying distributions and magnitudes of leaf surface conductance ('patchiness'). Distributions of surface conductance can create large biases in gas exchange calculations. Both apparent unsaturation and internal CO2 gradient inversion can be produced by the evolution of broader distributions of stomatal apertures consistent with a decrease in surface conductance, as might occur under increasing vapor pressure deficit. In gas exchange experiments, the behaviors of derived quantities defined by simple series models are highly sensitive to the true partitioning of flux and stomatal apertures across leaf surfaces. New methods are needed to disentangle model artifacts from real biological responses.

Comparative role of calcium oxide nanoparticles and calcium bulk fertilizer to alleviate cadmium toxicity by modulating oxidative stress, photosynthetic performance and antioxidant-defense genes expression in alfalfa.

Cadmium (Cd) toxicity poses a significant threat to soil health and sustainable food production. Its bioaccumulation in plant tissues induces phytotoxicity by affecting physiological and biochemical attributes, leading to a reduction in plant biomass and production. Recently, nanotechnology has emerged as a promising approach for addressing heavy metal toxicity in an eco-friendly manner to enhance crop production. However, the comparative role of foliar applied calcium oxide nanoparticles (CaO-NPs) and bulk calcium fertilizer under Cd stress in alfalfa remains unexplored. Herein, we studied the ameliorative role of CaO-NPs and bulk calcium (50 and 100 mg L-1) to alleviate Cd stress (30 mg kg-1) in alfalfa seedlings. Plants exposed to Cd exhibited significant decreases in morpho-physiological traits, gas exchange attributes, and pigment contents as well as increase in Cd bioaccumulation in plant tissues. Notably, exogenous application of CaO-NPs ameliorates the toxic impact of Cd by enhancing plant biomass (45%), fluorescence efficiency and gaseous exchange attributes. The maximum dose of CaO-NPs induced Cd-tolerance response accompanied by a significant increase in antioxidative enzyme activities, such as superoxide dismutase (SOD; 29%), peroxidase (POD; 41%), catalase (CAT; 36%) and ascorbate peroxidase (APX; 49%), which play positive roles in ROS scavenging. TEM examination further revealed the protective role of these NPs in averting Cd-induced damage to leaf ultrastructure and mesophyll cells. Furthermore, CaO-NPs had a substantial influence on both Cd and Ca2+ accumulation in plant tissues, while qRT‒PCR analysis demonstrated higher expression of antioxidant defense genes viz. Cu/ZnSOD (0.38 fold change (FC)), MtPOD (0.51 FC), MtCAT (0.61 FC) and MtAPX (0.79 FC) under CaO-NPs application, over Cd control. Overall, our findings suggested that exogenous CaO-NPs could be effective in alleviating the adverse effects of Cd on alfalfa seedlings to ensure food safety and support sustainable agriculture.

Stomatal penetration: the cornerstone of plant resistance to the fungal pathogen Zymoseptoria tritici.

BACKGROUND: Septoria tritici blotch (STB), caused by the foliar fungus Zymoseptoria tritici, is one of the most damaging disease of wheat in Europe. Genetic resistance against this fungus relies on different types of resistance from non-host resistance (NHR) and host species specific resistance (HSSR) to host resistance mediated by quantitative trait loci (QTLs) or major resistance genes (Stb). Characterizing the diversity of theses resistances is of great importance for breeding wheat cultivars with efficient and durable resistance. While the functional mechanisms underlying these resistance types are not well understood, increasing piece of evidence suggest that fungus stomatal penetration and early establishment in the apoplast are both crucial for the outcome of some interactions between Z. tritici and plants. To validate and extend these previous observations, we conducted quantitative comparative phenotypical and cytological analyses of the infection process corresponding to 22 different interactions between plant species and Z. tritici isolates. These interactions included four major bread wheat Stb genes, four bread wheat accessions with contrasting quantitative resistance, two species resistant to Z. tritici isolates from bread wheat (HSSR) and four plant species resistant to all Z. tritici isolates (NHR). RESULTS: Infiltration of Z. tritici spores into plant leaves allowed the partial bypass of all bread wheat resistances and durum wheat resistance, but not resistances from other plants species. Quantitative comparative cytological analysis showed that in the non-grass plant Nicotiana benthamiana, Z. tritici was stopped before stomatal penetration. By contrast, in all resistant grass plants, Z. tritici was stopped, at least partly, during stomatal penetration. The intensity of this early plant control process varied depending on resistance types, quantitative resistances being the least effective. These analyses also demonstrated that Stb-mediated resistances, HSSR and NHR, but not quantitative resistances, relied on the strong growth inhibition of the few Z. tritici penetrating hyphae at their entry point in the sub-stomatal cavity. CONCLUSIONS: In addition to furnishing a robust quantitative cytological assessment system, our study uncovered three stopping patterns of Z. tritici by plant resistances. Stomatal resistance was found important for most resistances to Z. tritici, independently of its type (Stb, HSSR, NHR). These results provided a basis for the functional analysis of wheat resistance to Z. tritici and its improvement.

Carboxylation capacity is the main limitation of carbon assimilation in High Arctic shrubs.

Increases in shrub height, biomass and canopy cover are key whole-plant features of warming-induced vegetation change in tundra. We investigated leaf functional traits underlying photosynthetic capacity of Arctic shrub species, particularly its main limiting processes such as mesophyll conductance. In this nutrient-limited ecosystem, we expect leaf nitrogen concentration to be the main limiting factor for photosynthesis. We measured the net photosynthetic rate at saturated light (Asat) in three Salix species throughout a glacial valley in High-Arctic tundra and used a causal approach to test relationships between leaf stomatal and mesophyll conductances (gsc, gm), carboxylation capacity (Vcmax), nitrogen and phosphorus concentration (Narea, Parea) and leaf mass ratio (LMA). Arctic Salix species showed no difference in Asat compared to a global data set, while being characterized by higher Narea, Parea and LMA. Vcmax, gsc and gm independently increased Asat, with Vcmax as its main limitation. We highlighted a nitrogen-influenced pathway for increasing photosynthesis in the two prostrate mesic habitat species. In contrast, the erect wetland habitat Salix richardsonii mainly increased Asat with increasing gsc. Overall, our study revealed high photosynthetic capacities of Arctic Salix species but contrasting regulatory pathways that may influence shrub ability to respond to environmental changes in High Arctic tundra.

Caught between two states: The compromise in acclimation of photosynthesis, transpiration and mesophyll conductance to different amplitudes of fluctuating irradiance.

While dynamic regulation of photosynthesis in fluctuating light is increasingly recognized as an important driver of carbon uptake, acclimation to realistic irradiance fluctuations is still largely unexplored. We subjected Arabidopsis thaliana (L.) wild-type and jac1 mutants to irradiance fluctuations with distinct amplitudes and average irradiance. We examined how irradiance fluctuations affected leaf structure, pigments and physiology. A wider amplitude of fluctuations produced a stronger acclimation response. Large reductions of leaf mass per area under fluctuating irradiance framed our interpretation of changes in photosynthetic capacity and mesophyll conductance as measured by three separate methods, in that photosynthetic investment increased markedly on a mass basis, but only a little on an area basis. Moreover, thermal imagery showed that leaf transpiration was four times higher under fluctuating irradiance. Leaves growing under fluctuating irradiance, although thinner, maintained their photosynthetic capacity, as measured through light- and CO2-response curves; suggesting their photosynthesis may be more cost-efficient than those under steady light, but overall may incur increased maintenance costs. This is especially relevant for plant performance globally because naturally fluctuating irradiance creates conflicting acclimation cues for photosynthesis and transpiration that may hinder progress towards ensuring food security under climate-related extremes of water stress.

Regional differences in leaf evolution facilitate photosynthesis following severe drought.

Characterizing physiological and anatomical changes that underlie rapid evolution following climatic perturbation can broaden our understanding of how climate change is affecting biodiversity. It can also provide evidence of cryptic adaptation despite stasis at higher levels of biological organization. Here, we compared evolutionary changes in populations of Mimulus cardinalis from historically different climates in the north and south of the species' range following an exceptional drought. We grew seeds produced from predrought ancestral plants alongside peak-drought descendants in a common glasshouse and exposed them to wet and dry conditions. Before the drought, northern ancestral populations expressed traits contributing to drought escape, while southern ancestral populations expressed drought avoidance. Following the drought, both regions evolved to reduce water loss and maintain photosynthesis in dry treatments (drought avoidance), but via different anatomical alterations in stomata, trichomes, and palisade mesophyll. Additionally, southern populations lost the ability to take advantage of wet conditions. These results reveal rapid evolution towards drought avoidance at an anatomical level following an exceptional drought, but suggest that differences in the mechanisms between regions incur different trade-offs. This sheds light on the importance of characterizing underlying mechanisms for downstream life-history and macromorphological traits.

Unsaturation in the air spaces of leaves and its implications.

Modern plant physiological theory stipulates that the resistance to water movement from plants to the atmosphere is overwhelmingly dominated by stomata. This conception necessitates a corollary assumption-that the air spaces in leaves must be nearly saturated with water vapour; that is, with a relative humidity that does not decline materially below unity. As this idea became progressively engrained in scientific discourse and textbooks over the last century, observations inconsistent with this corollary assumption were occasionally reported. Yet, evidence of unsaturation gained little traction, with acceptance of the prevailing framework motivated by three considerations: (1) leaf water potentials measured by either thermocouple psychrometry or the Scholander pressure chamber are largely consistent with the framework; (2) being able to assume near saturation of intercellular air spaces was transformational to leaf gas exchange analysis; and (3) there has been no obvious mechanism to explain a variable, liquid-phase resistance in the leaf mesophyll. Here, we review the evidence that refutes the assumption of universal, near saturation of air spaces in leaves. Refining the prevailing paradigm with respect to this assumption provides opportunities for identifying and developing mechanisms for increased plant productivity in the face of increasing evaporative demand imposed by global climate change.

Variation in photosynthetic capacity of Salvia przewalskii along elevational gradients on the eastern Qinghai-Tibetan Plateau, China.

Elevational variation in plant growing environment drives diversification of photosynthetic capacity, however, the mechanism behind this reaction is poorly understood. We measured leaf gas exchange, chlorophyll fluorescence, anatomical characteristics, and biochemical traits of Salvia przewalskii at elevations ranging from 2400 m to 3400 m above sea level (a.s.l) on the eastern Qinghai-Tibetan Plateau, China. We found that photosynthetic capacity showed an initial increase and then a decrease with rising elevation, and the best state observed at 2800 m a.s.l. Environmental factors indirectly regulated photosynthetic capacity by affecting stomatal conductance (gs), mesophyll conductance (gm), maximum velocity of carboxylation (Vc max), and maximum capacity for photosynthetic electron transport (Jmax). The average temperature (T) and total precipitation (P) during the growing season had the highest contribution to the variation of photosynthetic capacity of S. przewalskii in subalpine areas, which were 25% and 24%, respectively. Photosynthetic capacity was mainly affected by diffusional limitations (71%-89%), and mesophyll limitation (lm) played a leading role. The variation of gm was attributed to the effects of environmental factors on the volume fraction of intercellular air space (fias), the thickness of cell wall (Tcw), the surface of mesophyll cells and chloroplasts exposed to intercellular airspace (Sm, Sc), and plasma membrane intrinsic protein (PIPs, PIP1, PIP2), independent of carbonic anhydrase (CA). Optimization of leaf tissue structure and adaptive physiological responses enabled plants to efficiently cope with variable climate conditions of high-elevation areas, and the while maintaining high levels of carbon assimilation.

The long and tortuous path towards improving photosynthesis by engineering elevated mesophyll conductance.

The growing demand for global food production is likely to be a defining issue facing humanity over the next 50 years. To tackle this challenge, there is a desire to bioengineer crops with higher photosynthetic efficiencies, to increase yields. Recently, there has been a growing interest in engineering leaves with higher mesophyll conductance (gm), which would allow CO2 to move more efficiently from the substomatal cavities to the chloroplast stroma. However, if crop yield gains are to be realised through this approach, it is essential that the methodological limitations associated with estimating gm are fully appreciated. In this review, we summarise these limitations, and outline the uncertainties and assumptions that can affect the final estimation of gm. Furthermore, we critically assess the predicted quantitative effect that elevating gm will have on assimilation rates in crop species. We highlight the need for more theoretical modelling to determine whether altering gm is truly a viable route to improve crop performance. Finally, we offer suggestions to guide future research on gm, which will help mitigate the uncertainty inherently associated with estimating this parameter.

Structural changes of mesophyll cells in the rice leaf tissue in response to salinity stress based on the three-dimensional analysis.

Rice leaf blades have intricate-shaped mesophyll cells (MCs) with a large volume of chloroplasts enhancing gas exchange between stroma and intercellular airspace (IAS). Since the rice MCs do not form palisade or spongy tissue cells and are considered monotypic cells, the structural analysis of MCs in the middle part of the leaf tissue has been done, neglecting the various shapes of MCs can be observed on the cross-section of rice leaves. Moreover, the middle MC layer is sandwiched between the upper and lower layers and is more restricted in its demand for light and CO2 entering from the outside. Therefore, the different layers of MCs may differ in their sensitivity to salt stress that causes structural changes in cells. This study aims to elucidate the intra- and extra-cellular structures of MC in different layers of leaf tissue and determine how salinity affects the MC structure in each layer. The mesophyll tissue was divided into adaxial, middle and abaxial layers, and eight MCs and chloroplast regions were selected from each layer and reconstructed into three-dimensional (3D) representations. The whole leaf anatomical and physiological parameters were measured to find the effects of salinity stress on the MC structures. As a result, the 3D analysis of rice leaf tissue revealed the different structures of MCs with greater diversity in the adaxial and abaxial layers than in the middle layer. Salinity stress reduced the size and height of the MCs and coverage of the chloroplast on the cytoplasm periphery of the adaxial and abaxial layers, as well as the chloroplast size of adaxial MCs. Overall, these results reveal the variation of rice MC in leaf tissue and suggest the higher sensitivity to salt stress in the adaxial mesophyll among the layers, which may partly account for the decrease in photosynthetic capacity.

Effect of light-induced changes in leaf anatomy on intercellular and cellular components of mesophyll resistance for CO2 in Fagus sylvatica.

Mesophyll resistance for CO2 diffusion (rm) is one of the main limitations for photosynthesis and plant growth. Breeding new varieties with lower rm requires knowledge of its distinct components. We tested new method for estimating the relative drawdowns of CO2 concentration (c) across hypostomatous leaves of Fagus sylvatica. This technique yields values of the ratio of the internal CO2 concentrations at the adaxial and abaxial leaf side, cd/cb, the drawdown in the intercellular air space (IAS), and intracellular drawdown between IAS and chloroplast stroma, cc/cbd. The method is based on carbon isotope composition of leaf dry matter and epicuticular wax isolated from upper and lower leaf sides. We investigated leaves from tree-canopy profile to analyse the effects of light and leaf anatomy on the drawdowns and partitioning of rm into its inter- (rIAS) and intracellular (rliq) components. Validity of the new method was tested by independent measurements of rm using conventional isotopic and gas exchange techniques. 73% of investigated leaves had adaxial epicuticular wax enriched in 13C compared to abaxial wax (by 0.50‰ on average), yielding 0.98 and 0.70 for average of cd/cb and cc/cbd, respectively. The rIAS to rliq proportion were 5.5:94.5% in sun-exposed and 14.8:85.2% in shaded leaves. cc dropped to less than half of the atmospheric value in the sunlit and to about two-thirds of it in shaded leaves. This method shows that rIAS is minor but not negligible part of rm and reflects leaf anatomy traits, i.e. leaf mass per area and thickness.

Greater mesophyll conductance and leaf photosynthesis in the field through modified cell wall porosity and thickness via AtCGR3 expression in tobacco.

Mesophyll conductance (gm) describes the ease with which CO2 passes from the sub-stomatal cavities of the leaf to the primary carboxylase of photosynthesis, Rubisco. Increasing gm is suggested as a means to engineer increases in photosynthesis by increasing [CO2] at Rubisco, inhibiting oxygenation and accelerating carboxylation. Here, tobacco was transgenically up-regulated with Arabidopsis Cotton Golgi-related 3 (CGR3), a gene controlling methylesterification of pectin, as a strategy to increase CO2 diffusion across the cell wall and thereby increase gm. Across three independent events in tobacco strongly expressing AtCGR3, mesophyll cell wall thickness was decreased by 7%-13%, wall porosity increased by 75% and gm measured by carbon isotope discrimination increased by 28%. Importantly, field-grown plants showed an average 8% increase in leaf photosynthetic CO2 uptake. Up-regulating CGR3 provides a new strategy for increasing gm in dicotyledonous crops, leading to higher CO2 assimilation and a potential means to sustainable crop yield improvement.

Using Carbon Stable Isotopes to Study C3 and C4 Photosynthesis: Models and Calculations.

Stable carbon isotopes are a powerful tool to study photosynthesis. Initial applications consisted of determining isotope ratios of plant biomass using mass spectrometry. Subsequently, theoretical models relating C isotope values to gas exchange characteristics were introduced and tested against instantaneous online measurements of 13C photosynthetic discrimination. Beginning in the twenty-first century, laser absorption spectroscopes with sufficient precision for determining isotope mixing ratios became commercially available. This has allowed collection of large data sets at lower cost and with unprecedented temporal resolution. More data and accompanying knowledge have permitted refinement of 13C discrimination model equations, but often at the expense of increased model complexity and difficult parametrization. This chapter describes instantaneous online measurements of 13C photosynthetic discrimination, provides recommendations for experimental setup, and presents a thorough compilation of equations available to researchers. We update our previous 2018 version of this chapter by including recently improved descriptions of (photo)respiratory processes and associated fractionations. We discuss the capabilities and limitations of the diverse 13C discrimination model equations and provide guidance for selecting the model complexity needed for different applications.

Mutation mapping of a variegated EMS tomato reveals an FtsH-like protein precursor potentially causing patches of four phenotype classes in the leaves with distinctive internal morphology.

BACKGROUND: Leaf variegation is an intriguing phenomenon observed in many plant species. However, questions remain on its mechanisms causing patterns of different colours. In this study, we describe a tomato plant detected in an M2 population of EMS mutagenised seeds, showing variegated leaves with sectors of dark green (DG), medium green (MG), light green (LG) hues, and white (WH). Cells and tissues of these classes, along with wild-type tomato plants, were studied by light, fluorescence, and transmission electron microscopy. We also measured chlorophyll a/b and carotene and quantified the variegation patterns with a machine-learning image analysis tool. We compared the genomes of pooled plants with wild-type-like and mutant phenotypes in a segregating F2 population to reveal candidate genes responsible for the variegation. RESULTS: A genetic test demonstrated a recessive nuclear mutation caused the variegated phenotype. Cross-sections displayed distinct anatomy of four-leaf phenotypes, suggesting a stepwise mesophyll degradation. DG sectors showed large spongy layers, MG presented intercellular spaces in palisade layers, and LG displayed deformed palisade cells. Electron photomicrographs of those mesophyll cells demonstrated a gradual breakdown of the chloroplasts. Chlorophyll a/b and carotene were proportionally reduced in the sectors with reduced green pigments, whereas white sectors have hardly any of these pigments. The colour segmentation system based on machine-learning image analysis was able to convert leaf variegation patterns into binary images for quantitative measurements. The bulk segregant analysis of pooled wild-type-like and variegated progeny enabled the identification of SNP and InDels via bioinformatic analysis. The mutation mapping bioinformatic pipeline revealed a region with three candidate genes in chromosome 4, of which the FtsH-like protein precursor (LOC100037730) carries an SNP that we consider the causal variegated phenotype mutation. Phylogenetic analysis shows the candidate is evolutionary closest to the Arabidopsis VAR1. The synonymous mutation created by the SNP generated a miRNA binding site, potentially disrupting the photoprotection mechanism and thylakoid development, resulting in leaf variegation. CONCLUSION: We described the histology, anatomy, physiology, and image analysis of four classes of cell layers and chloroplast degradation in a tomato plant with a variegated phenotype. The genomics and bioinformatics pipeline revealed a VAR1-related FtsH mutant, the first of its kind in tomato variegation phenotypes. The miRNA binding site of the mutated SNP opens the way to future studies on its epigenetic mechanism underlying the variegation.