Stomatal Parameters Research Articles

Thiourea Supplementation Improves Drought Stress Response of Ridge‐Sown and Mulch‐Applied Rainfed Maize (Zea mays L.) via Improved Leaf Source to Grain Sink Dynamics

ABSTRACTIn regions of South Asia where rainfed maize is grown, effective crop management during drought is essential for maximising yield. A variety of water‐conserving planting practices are used, and more recently, techniques such as foliar supplementation to maintain nutrients during drought have also shown promise. However, specific combinations of these approaches are often untested for optimality. Here, we explore the effects of two maize planting practices (ridge sowing and mulching) to conserve water, in combination with foliar thiourea. Drought stress response of crop was assessed at two experimental sites (L‐I and L‐II), through split‐plot design (main plots: flat sowing + mulch, flat sowing, ridge sowing + mulch [RS + M] and ridge sowing; sub‐plots: unsprayed, water spray, 500 ppm thiourea and 1000 ppm thiourea). Plant performance was assessed via dry matter accumulation, grain growth rate, stomatal parameters, grain yield, stover yield and nitrogen uptake. Rainfall breaks induced three dry spells during the pre‐anthesis and grain‐filling period. RS + M showed maximum drought tolerance by enhancing rainwater and nutrient use (N uptake [88.1 and 115.1 kg ha−1]) and recorded significantly higher periodic dry matter accumulation (149.2 and 156.8 g) along with higher 1000‐grain weight (181.0 and 196.6 g), grain‐filling duration (36.3 and 34.9 days) and leaf health parameters over the flat‐sown treatments. Furthermore, foliar supplementation of thiourea at 1000 ppm caused improved leaf health, likely through activation of a source to sink response (transfer of energy and materials from leaves to other plant organs) that alleviated moisture stress. Ultimately, the combination of RS + M and 1000 ppm thiourea led to the highest grain yields (32.1 and 39.5 qha−1).

Genome size variation in Cape schoenoid sedges (Schoeneae) and its ecophysiological consequences.

Increases in genome size in plants-often associated with larger, low-density stomata and greater water-use efficiency (WUE)-could affect plant ecophysiological and hydraulic function. Variation in plant genome size is often due to polyploidy, having occurred repeatedly in the austral sedge genus Schoenus in the Cape Floristic Region (CFR), while species in the other major schoenoid genus in the region, Tetraria, have smaller genomes. Comparing these genera is useful as they co-occur at the landscape level, under broadly similar bioclimatic conditions. We hypothesized that CFR Schoenus have greater WUE, with lower maximum stomatal conductance (gwmax) imposed by larger, less-dense stomata. We investigated relationships between genome size and stomatal parameters in a phylogenetic context, reconstructing a phylogeny of CFR-occurring Schoeneae (Cyperaceae). Species' stomatal and functional traits were measured from field-collected and herbarium specimens. Carbon stable isotopes were used as an index of WUE. Genome size was derived from flow-cytometric measurements of leafy shoots. Evolutionary regressions demonstrated that stomatal size and density covary with genome size, positively and negatively, respectively, with genome size explaining 72-75% of the variation in stomatal size. Larger-genomed species had lower gwmax and C:N ratios, particularly in culms. We interpret differences in vegetative physiology between the genera as evidence of more-conservative strategies in CFR Schoenus compared to the more-acquisitive Tetraria. Because Schoenus have smaller, reduced leaves, they likely rely more on culm photosynthesis than Tetraria. Across the CFR Schoeneae, ecophysiology correlates with genome size, but confounding sources of trait variation limit inferences about causal relationships between traits.

Comparing Methodologies for Stomatal Analyses in the Context of Elevated Modern CO2.

Leaf stomata facilitate the exchange of water and CO2 during photosynthetic gas exchange. The shape, size, and density of leaf pores have not been constant over geologic time, and each morphological trait has potentially been impacted by changing environmental and climatic conditions, especially by changes in the concentration of atmospheric carbon dioxide. As such, stomatal parameters have been used in simple regressions to reconstruct ancient carbon dioxide, as well as incorporated into more complex gas-exchange models that also leverage plant carbon isotope ecology. Most of these proxy relationships are measured on chemically cleared leaves, although newer techniques such as creating stomatal impressions are being increasingly employed. Additionally, many of the proxy relationships use angiosperms with broad leaves, which have been increasingly abundant in the last 130 million years but are absent from the fossil record before this. We focus on the methodology to define stomatal parameters for paleo-CO2 studies using two separate methodologies (one corrosive, one non-destructive) to prepare leaves on both scale- and broad-leaves collected from herbaria with known global atmospheric CO2 levels. We find that the corrosive and non-corrosive methodologies give similar values for stomatal density, but that measurements of stomatal sizes, particularly guard cell width (GCW), for the two methodologies are not comparable. Using those measurements to reconstruct CO2 via the gas exchange model, we found that reconstructed CO2 based on stomatal impressions (due to inaccurate measurements in GCW) far exceeded measured CO2 for modern plants. This bias was observed in both coniferous (scale-shaped) and angiosperm (broad) leaves. Thus, we advise that applications of gas exchange models use cleared leaves rather than impressions.

A glossy mutant in onion (Allium cepa L.) shows decreased expression of wax biosynthesis genes.

Cuticular wax is a characteristic feature of land plants that provides protection against both biotic and abiotic stresses. In this study, a glossy mutant lacking an epicuticular wax layer was identified in the γ-irradiated M2 mutant population of the onion cultivar Bhima Super. The inheritance of the mutant's glossy phenotype was determined to be recessive and single locus. Scanning electron microscopy analysis showed poor accumulation of wax crystals in the glossy mutant, concentrated near the stomata. The plant height, number of leaves per plant, and stomatal parameters of the mutant were similar to the wild-type. RNA-seq was used to comprehend the expression variations of waxy cuticle-related genes in the glossy mutant and its wild-type waxy cultivars. Differential gene expression analysis of the RNA-seq data revealed that the genes involved in wax biosynthesis, such as AcCER1, AcCER26, AcMAH1, and AcWSD1, were downregulated by 2.72, 1.74, 2.59 and 2.12-fold, respectively, in the glossy mutant respectively. The expression patterns of these four unigenes were validated using semi-quantitative RT-PCR. The glossy mutant displayed a substantial 3.5-fold reduction in cuticular wax load compared to the wild-type due to the significant downregulation of these wax biosynthesis genes. These findings represent early advancements in understanding the molecular mechanisms of wax biosynthesis in onions. Furthermore, they provide a foundation for utilizing the glossy mutant trait in breeding programmes to enhance stress and pest resilience.

Correlation coefficient estimation of red chili (Capsicum annuum L) stomatal parameters under shade treatment condition

Stomatal is the most crucial part of plant organs that function as the gas exchange between CO2 and O2 in the environment through diffusion. Optimal stomatal performance can improve the quality of plant growth. This research aimed to determine the relationship between stomatal parameters and the production of red chili (Capsicum annuum L) under shade treatment conditions. This research was conducted in the field with three kinds of treatments, namely (1) control (without shade), (2) 50% shade, and (3) 80% shade. The parameters observed in this study were the number of stomatal, pore stomatal, stomatal area. The data were then analyzed using the correlation coefficient (r) calculation approach. The results showed that the closest relationship was found in the stomatal area variable with a pore area of 0.98. The stomatal characteristic that has the closest relationship with the production of red chili (Capsicum annuum L) is the pore area with a value of r = 0.73. These results indicate that pore area can be used as an indicator of crop production level.

Growth, physiology, and stomatal parameters of plant polyploids grown under ice age, present-day, and future CO2 concentrations.

Polyploidy plays an important role in plant evolution, but knowledge of its eco-physiological consequences, such as of the putatively enlarged stomata of polyploid plants, remains limited. Enlarged stomata should disadvantage polyploids at low CO2 concentrations (namely during the Quaternary glacial periods) because larger stomata are viewed as less effective at CO2 uptake. We observed the growth, physiology, and epidermal cell features of 15 diploids and their polyploid relatives cultivated under glacial, present-day, and potential future atmospheric CO2 concentrations (200, 400, and 800 ppm respectively). We demonstrated some well-known polyploidy effects, such as faster growth and larger leaves, seeds, stomata, and other epidermal cells. The stomata of polyploids, however, tended to be more elongated than those of diploids, and contrary to common belief, they had no negative effect on the CO2 uptake capacity of polyploids. Moreover, polyploids grew comparatively better than diploids even at low, glacial CO2 concentrations. Higher polyploids with large genomes also showed increased operational stomatal conductance and consequently, a lower water-use efficiency. Our results point to a possible decrease in growth superiority of polyploids over diploids in a current and future high CO2 climatic scenarios, as well as the possible water and/or nutrient dependency of higher polyploids.

Correlation of leaf trichome density and stomatal parameters of some commercial sugarcane genotypes to orange rust incidence and severity

Correlation of leaf trichome density and stomatal parameters of some commercial sugarcane genotypes to orange rust incidence and severity

Biochar Application Maintains Photosynthesis of Cabbage by Regulating Stomatal Parameters in Salt-Stressed Soil

Salinity is one of the main abiotic stresses, and the stomatal conductance (gs) is an important physiological index in plants that is used to measure their responses to salt stress, as stomatal closure leads to lower yields. However, the factors that affect the gs after biochar application in saline soil remain unclear. To explore the stomatal adaptation mechanisms of facility vegetables under salt stress after the addition of biochar, Chinese cabbage was selected for the pot experiment in this study. The soil and plant Na+ and K+ concentrations, water status, and plant stomatal parameters were measured following treatments with different salt concentrations (25, 50, and 100 mM) and biochar application rates (0, 2, and 4%). The results showed that salt stress induced the physiological closure of the stomata in Chinese cabbage. Compared with the salt-stress treatment without biochar, the biochar application significantly increased the plant gs (20.29–128.79%). Moreover, in the biochar treatment, the open state of the stomata was maintained by sustaining the plant osmotic adjustment, reducing the abscisic acid (ABA) content, and improving the water status. The Na+/K+ ratio had the most pronounced effect on the stomata (0.702). The actual photochemical efficiency of the photosystem II (ΦPSII) and electron transport rate (ETR) of the Chinese cabbage increased by 0.75–3.41% and 0.65–2.88%, respectively, after the biochar application, which supported the photosynthetic capacity and yield formation. According to the current findings, biochar application can mitigate salt stress and regulate stomatal opening, thereby improving the photosynthesis and the overall yield of Chinese cabbage. Therefore, the application of biochar is a promising method to maintain the productivity of Chinese cabbage under salt stress.

Estimating palaeoatmospheric CO2 levels based on fossil Ginkgoites cuticles from the Middle Jurassic of Northeast Iran

Fossils of Ginkgophyta are widely recognized as a suitable proxy to estimate Mesozoic and Cenozoic atmospheric CO2 levels. In this study, the fossil Ginkgoites species, Ginkgoites huttonii (Sternberg) Black is described based on a collection from the Middle Jurassic (Bajocian) deposits in Northeast Iran. The palaeoatmospheric CO2 level during Middle Jurassic in Iran is estimated for the first time based on the stomatal ratio (SR) method using this plant fossil. We show that the palaeoatmospheric CO2 concentration was about 1123 ppmv as calculated from stomatal parameters measured in this study. A comparison of our results with previously reported geochemical models indicates close accordance with the GEOCARB II model for Middle Jurassic.

Foliar Roughness and Water Content Impact on Escherichia coli Attachment in Baby Leafy Greens.

Understanding the relation between the susceptibility of different leafy greens to human pathogen contamination and leaf traits can contribute to increase the food safety of the fresh vegetable industry. The aim of this research was to evaluate the susceptibility to E. coli ATCC 35218 attachment in 30 accessions of baby leaves, and to identify leaf traits potentially involved in the contamination. The accessions were surface inoculated with a bacterial suspension containing 1 × 107 cells/mL and the attachment was measured 1.5 h after inoculation. Significant differences in attachment were detected between the accessions for p ≤ 0.05. The three most and the three least susceptible accessions were selected and characterized for leaf micro-morphological traits (stomata density and size, surface roughness) and water content. Scanning electron microscopy was used to analyse the stomatal parameters. Roughness was measured by an innovative portable 3D digital microscope. No significant correlation between the attachment of E. coli ATCC 35218 and stomatal parameters was detected, while the attachment was positively correlated with roughness and water content. The E. coli ATCC 35218 population in surface-inoculated leaves was also measured after a UV treatment, which was found to be less effective in reducing bacterial contamination in the rougher leaves. This result suggested that roughness offers UV protection, further highlighting its impact on the microbiological safety of baby leafy greens.

Characterization and evaluation of putative mutant populations of polyembryonic mango genotype Nekkare for dwarfing rootstock traits

Availability of dwarfing rootstocks is an important pre-requisite for improving productivity of mango orchards in India as it facilitates high density planting as well as impart uniformity within an orchard. An attempt was made to induce variability in polyembryonic mango genotype Nekkare for dwarfness by treating kernels with different doses of gamma radiation ranging from 15 to 35 Gy. Irradiation created significant variation in plant height, stem girth, number of nodes, inter-nodal length, number of leaves, leaf blade length and leaf blade width. The highest reduction in seedling height along with highest variation was observed at 35 Gy where the seedling height ranged from 11.50 to 33 cm with a mean of 23.12 cm as compared to mean plant height of 44.55 cm in control ranging from 33.50 to 56 cm. Further, the effect of irradiation on stomatal parameters was also investigated and the highest stomatal length and width was recorded at 15 Gy (63.39 μm) and 20 Gy (63.12 μm) respectively while 30 Gy treatment produced maximum stomatal density (13.85 per μm2). Furthermore, theconcentration of ABA was found to be highest (429.1 ng/gm) in morphologically dwarf (putative mutant) progenies of Nekkare. The results suggest effectiveness of induced mutation for developing dwarfing rootstocks in mango to be used in high density planting.

Leaf wettability, anatomy and ultra-structure of Nothofagus antarctica and N. betuloides grown under a CO2 enriched atmosphere

Increasing CO2 air concentration may affect wettability, anatomy and ultra-structure of leaves of Patagonian forest species, evergreen and deciduous plants potentially responding differently to such CO2 increases. In this study, we analysed the wettability, anatomy and ultra-structure of leaves of Nothofagus antarctica (deciduous) and N. betuloides (evergreen) grown under high CO2 concentrations. Leaf wettability was affected by increasing CO2, in different directions depending on species and leaf side. In both species, soluble cuticular lipid concentrations per unit leaf area raised with higher CO2 levels. Stomatal parameters (density, size of guard cells and pores) showed different responses to CO2 increasing depending on the species examined. In both species, leaf tissues showed a general trend to diminish with higher CO2 concentration. Cuticle thickness was modified with higher CO2 concentration in N. betuloides, but not in N. antarctica leaves. In both species, chloroplasts were often damaged with the increase in CO2 concentration. Our results show that several surface and internal leaf parameters can be modified in association with an increase in atmospheric CO2 concentration which may very among plant species.

Variation, coordination, and trade-offs between needle structures and photosynthetic-related traits across five Picea species: consequences on plant growth

BackgroundPicea species are distributed and planted world-wide due to their great ecological and economic values. It has been reported that Picea species vary widely in growth traits in a given environment, which reflects genetic and phenotypic differences among species. However, key physiological processes underlying tree growth and the influencing factors on them are still unknown.ResultsHere, we examined needle structures, needle chemical components, physiological characteristics and growth traits across five Picea species in a common garden in Tianshui, Gansu province in China: Picea glauca, P. mariana, P. likiangensis, P. koraiensis, and P. crassifolia, among which P. glauca and P. mariana were introduced from North America, P. likiangensis was from Lijiang, Yunan province in China, P. koraiensis was from Yichun, Heilongjiang province in China, and P. crassifolia was native to the experimental site. It was found that nearly all traits varied significantly among species. Tissue-level anatomical characteristics and leaf mass per area (LMA) were affected by needle size, but the variations of them were not associated with the variations in photosynthetic and biochemical capacity among species. Variations in area-based maximum photosynthesis (Pnmax) were affected by stomatal conductance (gs), mesophyll conductance (gm) and biochemical parameters including maximum carboxylation rate (Vcmax), and maximum electron transport rate (Jmax). The fraction of N allocated to different photosynthetic apparatus displayed contrasting values among species, which contributed to the species variations in photosynthetic nitrogen use efficiency (PNUE) and Pnmax. Additionally, all growth traits were positively correlated with Pnmax and PNUE.ConclusionNeedle structures are less important than needle biochemical parameters in determining the variations in photosynthetic capacity across the five Picea species. Pnmax and PNUE are closedly associated with the fraction of N allocated to photosynthetic apparatus (Pphoto) compared with leaf N content per area (Narea). The tremendous growth differences among the five Picea species were substantially related to the interspecies variation in Pnmax and PNUE.

SlSnRK2.3 interacts with SlSUI1 to modulate high temperature tolerance via Abscisic acid (ABA) controlling stomatal movement in tomato

Tomato is often exposed to high temperature stress during summer cultivation. Stomatal movement plays important roles in photosynthesis and transpiration which restricts the quality and yield of tomato under environmental stress. To elucidate the mechanism of stomatal movement in high temperature tolerance, SlSnRK2s (sucrose non-fermenting 1-related protein kinases) silenced plants were generated in tomato with CRISPR-Cas 9 gene editing techniques. Through the observation of stomatal parameters, SlSnRK2.3 regulated stomatal closure which was responded to ABA (abscisic acid) and activated signaling pathway of ROS (reactive oxygen species) in high temperature stress. Based on the positive functions of SlSnRK2.3, the cDNA library was generated to investigate interaction proteins of SlSnRK2s. The interaction between SlSnRK2.3 and SlSUI1 (protein translation factor SUI1 homolog) was employed by Yeast two hybrid assay (Y2H), Luciferase (LUC), and Bimolecular fluorescence complementation (BiFC). Finally, the specific interactive sites between SlSnRK2.3 and SlSUI1 were verified by site-directed mutagenesis. The consistent mechanism of SlSnRK2.3 and SlSUI1 in stomatal movement, indicating that SlSUI1 interacted with SlSnRK2.3 through ABA-dependent signaling pathway in high temperature stress. Our results provided evidence for improving the photosynthetic capacity of tomato under high temperature stress, and support the breeding and genetic engineering of tomato over summer facility cultivation.

Key traits of living fossil Ginkgo biloba are highly variable but not influenced by climate – Implications for palaeo-pCO2 reconstructions and climate sensitivity

The Ginkgoales, including the ‘living fossil’ Ginkgo biloba, are an important group for stomata-based palaeo-pCO2 reconstructions, with long evolutionary lineages and an extensive, abundant fossil record. The stomatal proxy for palaeo-pCO2 can improve our understanding of the exact relationship between pCO2 and temperatures – Earth's climate sensitivity: a key measure of global warming by pCO2. However, pCO2 records from future climate analogues in the past, such as the mid-Miocene Climatic Optimum, seemingly underestimate pCO2 – climate models cannot simulate the past temperatures with the only moderately elevated pCO2 reconstructed by proxies. Either climate sensitivity must have been elevated, which has implications for future climate forecasts, or proxies underestimate pCO2 due to additional environmental factors. Here we tested whether climate conditions impact stomatal parameters and thus pCO2 reconstruction on a large global database of G. biloba leaves from all continents except Antarctica, spanning 12 climate zones. We reconstructed ambient pCO2 using three stomatal proxy methods (stomatal ratio, transfer functions, Franks gas exchange model) and one stomata-independent isotope-based proxy for comparison (C3 proxy). We found that the stomatal proxy methods reconstructed ambient pCO2 reasonably well and uniformly, but that the C3 proxy underestimated pCO2. All the investigated stomatal parameters displayed an unexpectedly large variability, but no significant relationship with temperature, precipitation, or seasonality. Based on these results, the stomatal proxy is not influenced by climate and specifically does not systematically underestimate pCO2 under high temperatures. Climate sensitivity was likely instead elevated during past global warming episodes, an urgent consideration in climate forecasts for our rapidly warming Earth.

In vitro clonal propagation of Billbergia zebrina (Bromeliaceae) and analysis of anatomy and physiology of regenerated plantlets

Abstract During in vitro multiplication of horticulture plant species, synthetic cytokinins such as 6-benzylaminopurine (BA) can affect the microshoot formation. Murashige and Skoog culture medium (MS) can also act as a morphogenetic response modulator. The aim was to analyze the effects of BA and a gradient of MS salts on the morphophysiological status of Billbergia zebrina microshoots. Plantlets were cultured with a gradient of MS salts (50%, 100%, 150%, or 200%) supplemented with two BA levels (0 or 13 μM). After 50 days of culture, stomatal parameters, contents of photosynthetic pigments, and chlorophyll fluorescence were assessed. Plantlets cultured in all media without BA presented a low budding rate. In contrast, plantlets cultured with BA showed 100% budding. With 13 μM BA, the leaves presented bigger stomata and wider opening. Plantlets cultured with BA had lower contents of photosynthetic pigments. MS treatments without BA supplementation did not show any change in the pigments’ ratios. Plantlets cultured with BA had lower activity of the water-splitting complex and signs of photoinhibition damage. This resulted in the reduced performance of the photosynthetic apparatus. BA exposure can ensure the inducement of adventitious microshoots. BA at the tested concentration can induce morphophysiological disorders, irrespective of MS salt concentration.

Late-day measurement of excised branches results in uncertainty in the estimation of two stomatal parameters derived from response curves in Populus deltoides Bartr.×Populus nigra L.

Many terrestrial biosphere models depend on an understanding of the relationship between stomatal conductance and photosynthesis. However, unlike the measurement of photosynthetic parameters, such as the maximum carboxylation capacity, where standard methods (e.g., CO2 response or ACi curves) are widely accepted, a consensus method for empirically measuring parameters representing stomatal response has not yet emerged. Most models of stomatal response to environment represent stomatal conductance as being bounded by a lower intercept parameter (g0), and linearly scaled based on a multivariate term described by the stomatal slope parameter (g1). Here we employ the widely used Unified Stomatal Optimization model, to test whether g1 and g0 parameters are impacted by the choice of measurement method, either on an intact branch or a cut branch segment stored in water. We measured paired stomatal response curves on intact and excised branches of a hybrid poplar clone (Populus deltoides Bartr.×Populus nigra L. OP367), measured twice over a diurnal period. We found that predawn branch excision did not significantly affect measured g0 and g1 when measured within 4 h of excision. Measurement in the afternoon resulted in significantly higher values of g1 and lower values of g0, with values changing by 55% and 56%, respectively. Excision combined with afternoon measurement resulted in a marked effect on parameter estimates, with g1 increasing 89% from morning to afternoon and a 25% lower g1 for cut branches than those measured in situ. We also show that in hybrid poplar the differences in parameter estimates obtained from plants measured under different conditions can directly impact models of canopy function, reducing modeled transpiration by 18% over a simulated 12.5-h period. Although these results are only for a single isohydric woody species, our findings suggest that stomatal optimality parameters may not remain constant throughout the day.

Constraining conifer physiological parameters in leaf gas-exchange models for ancient CO2 reconstruction

Leaf gas-exchange models are increasingly used to reconstruct ancient atmospheric carbon dioxide (CO 2 ) concentrations. One of these widely used models, the Franks model, requires stomatal size (guard cell width and either guard cell length or pore length), whole-leaf stomatal density, and bulk-leaf carbon isotope composition (δ 13 C) from plant fossils. However, natural variations of these parameters within and across plant leaves have not been assessed closely, hindering the application of this model and the evaluation of its associated uncertainties. Here we investigate the range of variations of these parameters, and evaluate their impact on the output of the Franks model in three conifers ( Metasequoia, Sequoia , and Taxodium ). We introduce a modified cleared leaf method that allows accurate measurements of stomatal size. We show that among the stomatal size parameters, pore length is the most variable. Whole-leaf stomatal density can be accurately estimated in a representative area in the middle portion of a leaf. Variations of δ 13 C values are only slightly above analytical errors within a leaf and between leaves from a branchlet, but a ~ 1‰ negative shift of δ 13 C during early decay of Metasequoia leaf tissues was observed. Our measured ranges in pore length and whole-leaf stomatal density have the biggest influence on model estimated CO 2 . To improve model performance, we recommend (1) the use of our modified cleared leaf method to acquire accurate stomatal size and whole-leaf stomatal density measurements from the middle portion of a leaf located at the middle portion of a branchlet; (2) scaling pore length from guard cell length; and (3) a systematic correction of carbon isotope fractionation may be applicable if information regarding tissue decay and fossil preservation is available. We tested our recommendations by reconstructing CO 2 from both extant and fossil materials. Franks model-derived CO 2 based upon modern leaves collected in 2004 and 2020 (346 and 416 ppm) are close to their targets (378 and 414 ppm) whereas stomatal frequency methods substantially underestimate (285 and 341 ppm). Reconstructed CO 2 from the middle Miocene Clarkia deposit (505 and 507 ppm for Metasequoia and Taxodium ) are comparable with published results. We conclude that an improved cleared leaf method for accurate measurements of key stomatal parameters and a statistically-informed stomatal counting strategy will improve the performance of the Franks model for reconstructing CO 2 using these conifers with wide distributions of fossil records in the Northern Hemisphere since the Cretaceous. • Pore length and whole-leaf stomatal density vary the most among all parameters. • A modified cleared leaf method allows accurate stomatal size measurements. • The middle of a leaf at the middle of a branchlet is ideal for sampling. • δ 13 C varies only slightly but a ~ 1‰ negative shift can be corrected for fossils. • Constrained parameters yield ~400 and ~ 500 ppm CO 2 for modern and middle Miocene.

Automatic stomata recognition and measurement based on improved YOLO deep learning model and entropy rate superpixel algorithm

The traditional methods of analyzing stomatal traits are mostly manual observation and measurement. These methods are time-consuming, labor-intensive, and inefficient. Some methods have been proposed for the automatic recognition and counting of stomata, however most of those methods could not complete the automatic measurement of stomata parameters at the same time. Some non-deep learning methods could automatically measure the parameters of stomata, but they could not complete the automatic recognition and detection of stomata. In this paper, a deep learning-based method was proposed for automatically identifying, counting and measuring stomata of maize (Zea mays L.) leaves at the same time. An improved YOLO (You Only Look Once) deep learning model was proposed to identify stomata of maize leaves automatically, and an entropy rate superpixel algorithm was used for the accurate measurement of stomatal parameters. According to the characteristics of the stomata images data set, the network structure of YOLOv5 was modified, which greatly reduced the training time without affecting the recognition performance. The predictor in YOLO deep learning model was optimized, which reduced the false detection rate. At the same time, the 16-fold and 32-fold down-sampling layers were simplified according to the characteristics of stomatal objects, which improved the recognition efficiency. Experimental results showed that the recognition precision of the improved YOLO deep learning model reached 95.3% on the maize leaves stomatal data set, and the average accuracy of parameter measurement reached 90%. The proposed method could fully automatically complete the recognition, counting and measurement of stomata of plants, which can help agricultural scientists and botanists to conduct large-scale researches of stomatal morphology, structure and physiology, as well as the researches combined with genetic analysis or molecular-level analysis.

Leaves of more cold hardy grapes have a higher density of small, sunken stomata

Leaf stomatal density, index and size are known to be affected by the growing conditions, presumably to provide a better function for plant development. The question was whether there is a difference in stomatal parameters between grape species with different cold hardiness: V. riparia and V. vinifera; and the V. vinifera cultivars 'Riesling', 'Chardonnay', 'Sauvignon Blanc' and 'Merlot'. Analysis by scanning electron microscopy allowed the observation of 3 types of stomata in developing and mature leaves of all examined grape leaves. Stomatal parameters were found to be significantly affected by species or cultivar and growing conditions but not rootstock. A higher stomatal density and index were determined for the more cold hardy V. riparia and V. vinifera 'Riesling', whereby the higher number of stomata in 'Riesling' was found to be due to a higher number of small, sunken stomata. These findings might indicate a strategy of grape plants to optimize growth under low temperatures by using fast-acting stomata whose gas and water exchange are less affected than for larger stomata.