DNA Barcoding and Fertilization Strategies in Sideritis syriaca subsp. syriaca, a Local Endemic Plant of Crete with High Medicinal Value.

Herein, we applied DNA barcoding for the genetic characterization of Sideritis syriaca subsp. syriaca (Lamiaceae; threatened local Cretan endemic plant) using seven molecular markers of cpDNA. Five fertilization schemes were evaluated comparatively in a pilot cultivation in Crete. Conventional inorganic fertilizers (ChFs), integrated nutrient management (INM) fertilizers, and two biostimulants were utilized (foliar and soil application). Plant growth, leaf chlorophyll fluorescence, and color were assessed and leaf content of chlorophyll, key antioxidants (carotenoids, flavonoids, phenols), and nutrients were evaluated. Fertilization schemes induced distinct differences in leaf shape, altering quality characteristics. INM-foliar and ChF-soil application promoted yield, without affecting tissue water content or biomass partitioning to inflorescences. ChF-foliar application was the most stimulatory treatment when the primary target was enhanced antioxidant contents while INM-biostimulant was the least effective one. However, when the primary target is yield, INM, especially by foliar application, and ChF, by soil application, ought to be employed. New DNA sequence datasets for the plastid regions of petB/petD, rpoC1, psbK-psbI, and atpF/atpH were deposited in the GenBank for S. syriaca subsp. syriaca while the molecular markers rbcL, trnL/trnF, and psbA/trnH were compared to those of another 15 Sideritis species retrieved from the GenBank, constructing a phylogenetic tree to show their genetic relatedness.

Blue Light Improves Photosynthetic Performance during Healing and Acclimatization of Grafted Watermelon Seedlings.

To investigate the importance of light on healing and acclimatization, in the present study, grafted watermelon seedlings were exposed to darkness (D) or light, provided by blue (B), red (R), a mixture of R (68%) and B (RB), or white (W; 35% B, 49% intermediate spectra, 16% R) LEDs for 12 days. Survival ratio, root and shoot growth, soluble carbohydrate content, photosynthetic pigments content, and photosynthetic performance were evaluated. Seedling survival was not only strongly limited in D but the survived seedlings had an inferior shoot and root development, reduced chlorophyll content, and attenuated photosynthetic efficiency. RB-exposed seedlings had a less-developed root system. R-exposed seedlings showed leaf epinasty, and had the smallest leaf area, reduced chlorophyll content, and suppressed photosynthetic apparatus performance. The R-exposed seedlings contained the highest amount of soluble carbohydrate and together with D-exposed seedlings the lowest amount of chlorophyll in their scions. B-exposed seedlings showed the highest chlorophyll content and improved overall PSII photosynthetic functioning. W-exposed seedling had the largest leaf area, and closely resembled the photosynthetic properties of RB-exposed seedlings. We assume that, during healing of grafted seedlings monochromatic R light should be avoided. Instead, W and monochromatic B light may be willingly adopted due to their promoting effect on shoot, pigments content, and photosynthetic efficiency.

Structural Diversity and Highly Specific Host-Pathogen Transcriptional Regulation of Defensin Genes Is Revealed in Tomato.

Defensins are small and rather ubiquitous cysteine-rich anti-microbial peptides. These proteins may act against pathogenic microorganisms either directly (by binding and disrupting membranes) or indirectly (as signaling molecules that participate in the organization of the cellular defense). Even though defensins are widespread across eukaryotes, still, extensive nucleotide and amino acid dissimilarities hamper the elucidation of their response to stimuli and mode of function. In the current study, we screened the Solanum lycopersicum genome for the identification of defensin genes, predicted the relating protein structures, and further studied their transcriptional responses to biotic (Verticillium dahliae, Meloidogyne javanica, Cucumber Mosaic Virus, and Potato Virus Y infections) and abiotic (cold stress) stimuli. Tomato defensin sequences were classified into two groups (C8 and C12). Our data indicate that the transcription of defensin coding genes primarily depends on the specific pathogen recognition patterns of V. dahliae and M. javanica. The immunodetection of plant defensin 1 protein was achieved only in the roots of plants inoculated with V. dahliae. In contrast, the almost null effects of viral infections and cold stress, and the failure to substantially induce the gene transcription suggest that these factors are probably not primarily targeted by the tomato defensin network.

The genetic architecture of photosynthesis and plant growth-related traits in tomato.

To identify genomic regions involved in the regulation of fundamental physiological processes such as photosynthesis and respiration, a population of Solanum pennellii introgression lines was analyzed. We determined phenotypes for physiological, metabolic, and growth related traits, including gas exchange and chlorophyll fluorescence parameters. Data analysis allowed the identification of 208 physiological and metabolic quantitative trait loci with 33 of these being associated to smaller intervals of the genomic regions, termed BINs. Eight BINs were identified that were associated with higher assimilation rates than the recurrent parent M82. Two and 10 genomic regions were related to shoot and root dry matter accumulation, respectively. Nine genomic regions were associated with starch levels, whereas 12 BINs were associated with the levels of other metabolites. Additionally, a comprehensive and detailed annotation of the genomic regions spanning these quantitative trait loci allowed us to identify 87 candidate genes that putatively control the investigated traits. We confirmed 8 of these at the level of variance in gene expression. Taken together, our results allowed the identification of candidate genes that most likely regulate photosynthesis, primary metabolism, and plant growth and as such provide new avenues for crop improvement.

Pore size regulates operating stomatal conductance, while stomatal densities drive the partitioning of conductance between leaf sides.

BACKGROUND AND AIMS: Leaf gas exchange is influenced by stomatal size, density, distribution between the leaf adaxial and abaxial sides, as well as by pore dimensions. This study aims to quantify which of these traits mainly underlie genetic differences in operating stomatal conductance (gs) and addresses possible links between anatomical traits and regulation of pore width. METHODS: Stomatal responsiveness to desiccation, gs-related anatomical traits of each leaf side and estimated gs (based on these traits) were determined for 54 introgression lines (ILs) generated by introgressing segments of Solanum pennelli into the S. lycopersicum 'M82'. A quantitative trait locus (QTL) analysis for stomatal traits was also performed. KEY RESULTS: A wide genetic variation in stomatal responsiveness to desiccation was observed, a large part of which was explained by stomatal length. Operating gs ranged over a factor of five between ILs. The pore area per stomatal area varied 8-fold among ILs (2-16 %), and was the main determinant of differences in operating gs between ILs. Operating gs was primarily positioned on the abaxial surface (60-83 %), due to higher abaxial stomatal density and, secondarily, to larger abaxial pore area. An analysis revealed 64 QTLs for stomatal traits in the ILs, most of which were in the direction of S. pennellii. CONCLUSIONS: The data indicate that operating and maximum gs of non-stressed leaves maintained under stable conditions deviate considerably (by 45-91 %), because stomatal size inadequately reflects operating pore area (R(2) = 0·46). Furthermore, it was found that variation between ILs in both stomatal sensitivity to desiccation and operating gs is associated with features of individual stoma. In contrast, genotypic variation in gs partitioning depends on the distribution of stomata between the leaf adaxial and abaxial epidermis.

Threshold response of stomatal closing ability to leaf abscisic acid concentration during growth.

Leaf abscisic acid concentration ([ABA]) during growth influences morpho-physiological traits associated with the plant's ability to cope with stress. A dose-response curve between [ABA] during growth and the leaf's ability to regulate water loss during desiccation or rehydrate upon re-watering was obtained. Rosa hybrida plants were grown at two relative air humidities (RHs, 60% or 90%) under different soil water potentials (-0.01, -0.06, or -0.08MPa) or upon grafting onto the rootstock of a cultivar sustaining [ABA] at elevated RH. Measurements included [ABA], stomatal anatomical features, stomatal responsiveness to desiccation, and the ability of leaves, desiccated to varying degrees, to recover their weight (rehydrate) following re-watering. Transpiration efficiency (plant mass per transpired water) was also determined. Soil water deficit resulted in a lower transpiration rate and higher transpiration efficiency at both RHs. The lowest [ABA] was observed in well-watered plants grown at high RH. [ABA] was increased by soil water deficit or grafting, at both RHs. The growth environment-induced changes in stomatal size were mediated by [ABA]. When [ABA] was increased from the level of (well-watered) high RH-grown plants to the value of (well-watered) plants grown at moderate RH, stomatal responsiveness was proportionally improved. A further increase in [ABA] did not affect stomatal responsiveness to desiccation. [ABA] was positively related to the ability of dehydrated leaves to rehydrate. The data indicate a growth [ABA]-related threshold for stomatal sensitivity to desiccation, which was not apparent either for stomatal size or for recovery (rehydration) upon re-watering.

Foliar abscisic acid content underlies genotypic variation in stomatal responsiveness after growth at high relative air humidity.

BACKGROUND AND AIMS: Stomata formed at high relative air humidity (RH) respond less to abscisic acid (ABA), an effect that varies widely between cultivars. This study tested the hypotheses that this genotypic variation in stomatal responsiveness originates from differential impairment in intermediates of the ABA signalling pathway during closure and differences in leaf ABA concentration during growth. METHODS: Stomatal anatomical features and stomatal responsiveness to desiccation, feeding with ABA, three transduction elements of its signalling pathway (H2O2, NO, Ca(2+)) and elicitors of these elements were determined in four rose cultivars grown at moderate (60 %) and high (90 %) RH. Leaf ABA concentration was assessed throughout the photoperiod and following mild desiccation (10 % leaf weight loss). KEY RESULTS: Stomatal responsiveness to desiccation and ABA feeding was little affected by high RH in two cultivars, whereas it was considerably attenuated in two other cultivars (thus termed sensitive). Leaf ABA concentration was lower in plants grown at high RH, an effect that was more pronounced in the sensitive cultivars. Mild desiccation triggered an increase in leaf ABA concentration and equalized differences between leaves grown at moderate and high RH. High RH impaired stomatal responses to all transduction elements, but cultivar differences were not observed. CONCLUSIONS: High RH resulted in decreased leaf ABA concentration during growth as a result of lack of water deficit, since desiccation induced ABA accumulation. Sensitive cultivars underwent a larger decrease in leaf ABA concentration rather than having a higher ABA concentration threshold for inducing stomatal functioning. However, cultivar differences in stomatal closure following ABA feeding were not apparent in response to H2O2 and downstream elements, indicating that signalling events prior to H2O2 generation are involved in the observed genotypic variation.

Plants exposed to titanium dioxide nanoparticles acquired contrasting photosynthetic and morphological strategies depending on the growing light intensity: a case study in radish.

Due to the photocatalytic property of titanium dioxide (TiO2), its application may be dependent on the growing light environment. In this study, radish plants were cultivated under four light intensities (75, 150, 300, and 600 µmol m-2 s-1 photosynthetic photon flux density, PPFD), and were weekly sprayed (three times in total) with TiO2 nanoparticles at different concentrations (0, 50, and 100 µmol L-1). Based on the obtained results, plants used two contrasting strategies depending on the growing PPFD. In the first strategy, as a result of exposure to high PPFD, plants limited their leaf area and send the biomass towards the underground parts to limit light-absorbing surface area, which was confirmed by thicker leaves (lower specific leaf area). TiO2 further improved the allocation of biomass to the underground parts when plants were exposed to higher PPFDs. In the second strategy, plants dissipated the absorbed light energy into the heat (NPQ) to protect the photosynthetic apparatus from high energy input due to carbohydrate and carotenoid accumulation as a result of exposure to higher PPFDs or TiO2 concentrations. TiO2 nanoparticle application up-regulated photosynthetic functionality under low, while down-regulated it under high PPFD. The best light use efficiency was noted at 300 m-2 s-1 PPFD, while TiO2 nanoparticle spray stimulated light use efficiency at 75 m-2 s-1 PPFD. In conclusion, TiO2 nanoparticle spray promotes plant growth and productivity, and this response is magnified as cultivation light intensity becomes limited.

Co-ordination of hydraulic and stomatal conductances across light qualities in cucumber leaves.

Long-term effects of light quality on leaf hydraulic conductance (K(leaf)) and stomatal conductance (g(s)) were studied in cucumber, and their joint impact on leaf photosynthesis in response to osmotic-induced water stress was assessed. Plants were grown under low intensity monochromatic red (R, 640 nm), blue (B, 420 nm) or combined red and blue (R:B, 70:30) light. K(leaf) and g(s) were much lower in leaves that developed without blue light. Differences in g(s) were caused by differences in stomatal aperture and stomatal density, of which the latter was largely due to differences in epidermal cell size and hardly due to stomatal development. Net photosynthesis (A(N)) was lowest in R-, intermediate in B-, and highest in RB- grown leaves. The low A(N) in R-grown leaves correlated with a low leaf internal CO(2) concentration and reduced PSII operating efficiency. In response to osmotic stress, all leaves showed similar degrees of stomatal closure, but the reduction in A(N) was larger in R- than in B- and RB-grown leaves. This was probably due to damage of the photosynthetic apparatus, which only occurred in R-grown leaves. The present study shows the co-ordination of K(leaf) and g(s) across different light qualities, while the presence of blue in the light spectrum seems to drive both K(leaf) and g(s) towards high, sun-type leaf values, as was previously reported for maximal photosynthetic capacity and leaf morphology. The present results suggest the involvement of blue light receptors in the usually harmonized development of leaf characteristics related to water relations and photosynthesis under different light environments.

Edible Coatings from Opuntia ficus-indica Cladodes Alongside Chitosan on Quality and Antioxidants in Cherries during Storage.

The aim of this work was to investigate the effect of edible coatings (ECs) prepared from extracts of Opuntia ficus-indica (OFI) cladodes in comparison with a commercial chitosan formulation on the quality of 'Regina' cherries packaged in macro-perforated bags and stored for up to 28 d (1 °C, 90% RH). The coating concentrations were 25% and 50% aqueous OFI extract (approximately 0.59 and 1.18% dry matter, respectively), 1% OFI alcohol insoluble polysaccharide and 1% chitosan. The variables evaluated included weight loss (WL), respiration rates (RR), peel color, firmness, microbial decay, total antioxidants (phenolics, flavonoids, anthocyanins, antioxidant capacity), individual phenolic compounds (anthocyanins, hydroxycinnamic acids, flavan-3-O-ols), and pedicel removal force. The main results show that all coatings reduced WL and RR similarly, enhanced firmness throughout storage and antioxidants after 28 d of storage compared to the controls. Among treatments, chitosan resulted in much higher peel glossiness and firmness in comparison to OFI extracts. On day 28, all ECs resulted in higher antioxidants than controls, OFI extracts resulted in higher cyaniding-3-O-rutinoside than chitosan, while 50% OFI treatment resulted in the highest catechin concentration. Therefore, OFI extracts are promising ECs for cherry storage since they exhibited no negative effect, improved quality and extended storage life by one week compared to the controls.

The regulatory role of γ-aminobutyric acid in chickpea plants depends on drought tolerance and water scarcity level.

γ-Aminobutyric acid (GABA) is a non-protein amino acid with multifunctional roles in dynamic plant responses. To determine the effects of exogenous GABA application (0, 25 and 50 µM) on drought response, two chickpea cultivars with contrasting tolerance to water deficit were examined. Plants were exposed to four irrigation levels (irrigation to 100, 60, 40 and 20% field capacity). Water deficit decreased growth, chlorophyll content, and photosynthetic efficiency. It increased electrolyte leakage and lipid peroxidation owing to both higher ROS accumulation and lower antioxidant enzyme activity. These negative effects of water deficit and the alleviating role of GABA application were more prominent in the sensitive, as compared to the tolerant cultivar. Water deficit also increased proline and GABA contents more in the tolerant cultivar, whereas their content was more enhanced by GABA application in the sensitive one. This may confer an additional level of regulation that results in better alleviation of drought damage in tolerant chickpea cultivars. In conclusion, the stimulatory effect of GABA on growth and physiological modulation depends on both the water stress severity and the cultivar sensitivity to it, implying a probable unknown GABA-related mechanism established by tolerant chickpea cultivars; a lost or not gained mechanism in susceptible ones.

Avoiding high relative air humidity during critical stages of leaf ontogeny is decisive for stomatal functioning.

Plants of several species, if grown at high relative air humidity (RH ≥85%), develop stomata that fail to close fully in case of low leaf water potential. We studied the effect of a reciprocal change in RH, at different stages of leaf expansion of Rosa hybrida grown at moderate (60%) or high (95%) RH, on the stomatal closing ability. This was assessed by measuring the leaf transpiration rate in response to desiccation once the leaves had fully expanded. For leaves that started expanding at high RH but completed their expansion after transfer to moderate RH, the earlier this switch took place the better the stomatal functioning. Leaves initially expanding at moderate RH and transferred to high RH exhibited poor stomatal functioning, even when this transfer occurred very late during leaf expansion. Applying a daily abscisic acid (ABA) solution to the leaves of plants grown at continuous high RH was effective in inducing stomatal closure at low water potential, if done before full leaf expansion (FLE). After FLE, stomatal functioning was no longer affected either by the RH or ABA level. The results indicate that the degree of stomatal adaptation depends on both the timing and duration of exposure to high RH. It is concluded that stomatal functionality is strongly dependent on the humidity at which the leaf completed its expansion. The data also show that the effect of ambient RH and the alleviating role of ABA are restricted to the period of leaf expansion.

Automated Grapevine Cultivar Identification via Leaf Imaging and Deep Convolutional Neural Networks: A Proof-of-Concept Study Employing Primary Iranian Varieties.

Extending over millennia, grapevine cultivation encompasses several thousand cultivars. Cultivar (cultivated variety) identification is traditionally dealt by ampelography, requiring repeated observations by experts along the growth cycle of fruiting plants. For on-time evaluations, molecular genetics have been successfully performed, though in many instances, they are limited by the lack of referable data or the cost element. This paper presents a convolutional neural network (CNN) framework for automatic identification of grapevine cultivar by using leaf images in the visible spectrum (400-700 nm). The VGG16 architecture was modified by a global average pooling layer, dense layers, a batch normalization layer, and a dropout layer. Distinguishing the intricate visual features of diverse grapevine varieties, and recognizing them according to these features was conceivable by the obtained model. A five-fold cross-validation was performed to evaluate the uncertainty and predictive efficiency of the CNN model. The modified deep learning model was able to recognize different grapevine varieties with an average classification accuracy of over 99%. The obtained model offers a rapid, low-cost and high-throughput grapevine cultivar identification. The ambition of the obtained tool is not to substitute but complement ampelography and quantitative genetics, and in this way, assist cultivar identification services.

Automated In Situ Seed Variety Identification via Deep Learning: A Case Study in Chickpea.

On-time seed variety recognition is critical to limit qualitative and quantitative yield loss and asynchronous crop production. The conventional method is a subjective and error-prone process, since it relies on human experts and usually requires accredited seed material. This paper presents a convolutional neural network (CNN) framework for automatic identification of chickpea varieties by using seed images in the visible spectrum (400-700 nm). Two low-cost devices were employed for image acquisition. Lighting and imaging (background, focus, angle, and camera-to-sample distance) conditions were variable. The VGG16 architecture was modified by a global average pooling layer, dense layers, a batch normalization layer, and a dropout layer. Distinguishing the intricate visual features of the diverse chickpea varieties and recognizing them according to these features was conceivable by the obtained model. A five-fold cross-validation was performed to evaluate the uncertainty and predictive efficiency of the CNN model. The modified deep learning model was able to recognize different chickpea seed varieties with an average classification accuracy of over 94%. In addition, the proposed vision-based model was very robust in seed variety identification, and independent of image acquisition device, light environment, and imaging settings. This opens the avenue for the extension into novel applications using mobile phones to acquire and process information in situ. The proposed procedure derives possibilities for deployment in the seed industry and mobile applications for fast and robust automated seed identification practices.

Monochromatic red light during plant growth decreases the size and improves the functionality of stomata in chrysanthemum.

Light emitting diodes (LEDs) now enable precise light quality control. Prior to commercialisation however, the plant response to the resultant light quality regime ought to be addressed. The response was examined here in chrysanthemum by evaluating growth, chlorophyll fluorescence (before and following water deficit), as well as stomatal anatomy (density, size, pore dimensions and aperture heterogeneity) and closing ability. Plants were grown under blue (B), red (R), a mixture of R (70%) and B (RB), or white (W; 41% B, 39% intermediate spectrum, 20% R) light LEDs. Although R light promoted growth, it also caused leaf deformation (epinasty) and disturbed the photosynthetic electron transport system. The largest stomatal size was noted following growth under B light, whereas the smallest under R light. The largest stomatal density was observed under W light. Monochromatic R light stimulated both the rate and the degree of stomatal closure in response to desiccation compared with the other light regimes. We conclude that stomatal size is mainly controlled by the B spectrum, whereas a broader spectral range is important for determining stomatal density. Monochromatic R light enhanced stomatal ability to regulate water loss upon desiccation.

The Role of Proteases in Determining Stomatal Development and Tuning Pore Aperture: A Review.

Plant proteases, the proteolytic enzymes that catalyze protein breakdown and recycling, play an essential role in a variety of biological processes including stomatal development and distribution, as well as, systemic stress responses. In this review, we summarize what is known about the participation of proteases in both stomatal organogenesis and on the stomatal pore aperture tuning, with particular emphasis on their involvement in numerous signaling pathways triggered by abiotic and biotic stressors. There is a compelling body of evidence demonstrating that several proteases are directly or indirectly implicated in the process of stomatal development, affecting stomatal index, density, spacing, as well as, size. In addition, proteases are reported to be involved in a transient adjustment of stomatal aperture, thus orchestrating gas exchange. Consequently, the proteases-mediated regulation of stomatal movements considerably affects plants' ability to cope not only with abiotic stressors, but also to perceive and respond to biotic stimuli. Even though the determining role of proteases on stomatal development and functioning is just beginning to unfold, our understanding of the underlying processes and cellular mechanisms still remains far from being completed.

Stomatal behavior following mid- or long-term exposure to high relative air humidity: A review.

High relative air humidity (RH ≥ 85%) is frequent in controlled environments, and not uncommon in nature. In this review, we examine the high RH effects on plants with a special focus on stomatal characters. All aspects of stomatal physiology are attenuated by elevated RH during leaf expansion (long-term) in C3 species. These include impaired opening and closing response, as well as weak diel oscillations. Consequently, the high RH-grown plants are not only vulnerable to biotic and abiotic stress, but also undergo a deregulation between CO2 uptake and water loss. Stomatal behavior of a single leaf is determined by the local microclimate during expansion, and may be different than the remaining leaves of the same plant. No effect of high RH is apparent in C4 and CAM species, while the same is expected for species with hydropassive stomatal closure. Formation of bigger stomata with larger pores is a universal response to high RH during leaf expansion, whereas the effect on stomatal density appears to be species- and leaf side-specific. Compelling evidence suggests that ABA mediates the high RH-induced stomatal malfunction, as well as the stomatal size increase. Although high RH stimulates leaf ethylene evolution, it remains elusive whether or not this contributes to stomatal malfunction. Most species lose stomatal function following mid-term (4-7 d) exposure to high RH following leaf expansion. Consequently, the regulatory role of ambient humidity on stomatal functionality is not limited to the period of leaf expansion, but holds throughout the leaf life span.

Improving stomatal functioning at elevated growth air humidity: A review.

Plants grown at high relative air humidity (RH≥85%) are prone to lethal wilting upon transfer to conditions of high evaporative demand. The reduced survival of these plants is related to (i) increased cuticular permeability, (ii) changed anatomical features (i.e., longer pore length and higher stomatal density), (iii) reduced rehydration ability, (iv) impaired water potential sensitivity to leaf dehydration and, most importantly, (v) compromised stomatal closing ability. This review presents a critical analysis of the strategies which stimulate stomatal functioning during plant development at high RH. These include (a) breeding for tolerant cultivars, (b) interventions with respect to the belowground environment (i.e., water deficit, increased salinity, nutrient culture and grafting) as well as (c) manipulation of the aerial environment [i.e., increased proportion of blue light, increased air movement, temporal temperature rise, and spraying with abscisic acid (ABA)]. Root hypoxia, mechanical disturbance, as well as spraying with compounds mimicking ABA, lessening its inactivation or stimulating its within-leaf redistribution are also expected to improve stomatal functioning of leaves expanded in humid air. Available evidence leaves little doubt that genotypic and phenotypic differences in stomatal functioning following cultivation at high RH are realized through the intermediacy of ABA.

Spatial heterogeneity in stomatal features during leaf elongation: an analysis using Rosa hybrida.

Within-leaf heterogeneity in stomatal traits poses a key uncertainty in determining a representative value for the whole leaf. Accounting for this heterogeneity, we studied stomatal initiation on expanding leaves and estimated stomatal conductance (gs) of mature leaves. The entire lamina was evaluated at four percentages of full leaflet elongation (FLE; leaflet length relative to its final length) in Rosa hybrida L. plants grown at 60% relative air humidity (RH), and at 100% FLE following cultivation at elevated (95%) RH. Over 80% of the stomata were initiated between 33 and 67% FLE, whereas stomatal growth mostly occurred afterwards. At 100% FLE, the heterogeneity in stomatal density was the result of uneven stomatal differentiation, while an uneven differentiation of epidermal cells contributed to this variation only at elevated RH. Noticeable within-leaf differences (up to 40%) in gs were calculated at 100% FLE. Avoiding leaflet periphery decreased this heterogeneity. Despite the large promotive effect of elevated RH on stomatal and pore dimensions, the within-leaf variation remained unaffected in all characters, besides pore aperture (and, thus, gs). The noted level of within-leaf variation in stomatal features demands a sampling scheme tailored to the leaf developmental stage, the feature per se and the evaporative demand during growth.