Stomatal Distribution Research Articles

Blocking IbmiR319a Impacts Plant Architecture and Reduces Drought Tolerance in Sweet Potato.

MicroRNA319 (miR319) plays a key role in plant growth, development, and multiple resistance by repressing the expression of targeted TEOSINTE BRANCHED/CYCLOIDEA/PCF (TCP) genes. Two members, IbmiR319a and IbmiR319c, were discovered in the miR319 gene family in sweet potato (Ipomoea batatas [L.] Lam). Here, we focused on the biological function and potential molecular mechanism of the response of IbmiR319a to drought stress in sweet potato. Blocking IbmiR319a in transgenic sweet potato (MIM319) resulted in a slim and tender phenotype and greater sensitivity to drought stress. Microscopic observations revealed that blocking IbmiR319a decreased the cell width and increased the stomatal distribution in the adaxial leaf epidermis, and also increased the intercellular space in the leaf and petiole. We also found that the lignin content was reduced, which led to increased brittleness in MIM319. Quantitative real-time PCR showed that the expression levels of key genes in the lignin biosynthesis pathway were much lower in the MIM319 lines than in the wild type. Ectopic expression of IbmiR319a-targeted genes IbTCP11 and IbTCP17 in Arabidopsis resulted in similar phenotypes to MIM319. We also showed that the expression of IbTCP11 and IbTCP17 was largely induced by drought stress. Transcriptome analysis indicated that cell growth-related pathways, such as plant hormonal signaling, were significantly downregulated with the blocking of IbmiR319a. Taken together, our findings suggest that IbmiR319a affects plant architecture by targeting IbTCP11/17 to control the response to drought stress in sweet potato.

Leaf epidermal and petiole anatomical features as taxonomic characters in some Vernonia species in Nigeria

Foliar and petiole micro-morphological characteristics of some members of the genus Vernonia namely; Vernonia amygdalina Del. (bitter variety), Vernonia amygdalina Del. (non-bitter variety), Vernonia cinerea (L) Less., Vernonia galamensis (Cass.) Less., and Vernonia adoensis Sch. Bip. in Jos, Nigeria was investigated to provide additional micro-morphological characters to support existing taxonomic information regarding the species. Epidermal peels of fresh leaves were made and transverse sections of petioles were cut using a rotary microtome. Staining was done using Safranin and slides observed using a light microscope. Quantitative characters were measured and subjected to Duncan Multiple Range Test. Stomatal distribution was amphistomatic except for V. galamensis and V. cinerea with hypostomatic distribution; stomata type was mostly anomocytic except V. galamensis with paracytic stomata. Stomata index varied among taxa with V. adoensis and V. amygdalina (bitter variety) having the highest (10.84 %) and lowest (0.67%) respectively. Anticlinal cell wall patterns were straight to slightly undulate while wavy anticlinal cell wall pattern was diagnostic to V. cinerea. Trichome types observed were glandular and multicellular uniseriate with highest and lowest trichome indices recorded in V. adoensis (2.80%) and V. galamensis (0.02%) respectively. Petiole outline was convex to concave, epidermis was uniseriate, trichome multicellular uniseriate and vascular bundles bicollateral. Variations observed in the stomatal complex, epidermal cell complex, petiole outline, petiole vasculature type, trichome and stomata indices could be employed for species identification and delimitation.

Changes of stomatal distribution and leaf thickness in response to transpiration rate in six dicot plant species

Changes in leaf anatomy can be influenced by the surrounding environment and several other factors. Shade plants or ornamental plants themselves also have their characteristics in responding to surrounding conditions through transpiration. This study aimed to measure the rate of transpiration related to changes in the distribution of stomata and leaf thickness of six types of dicot ornamental plants and to describe the leaf anatomy of each type. The experiment used Completely Randomized Design (CRD) with six types of plants as treatment with three replications. Transpiration rate was measured by modified lysimeter method, stomatal distribution was calculated from the ratio of the number of stomata to epidermal cells and leaf anatomy was observed using preserved slide made from paraffin method. Data were analyzed by SAS with CRD model and correlation was performed between transpiration rate and leaf thickness. The results showed that the transpiration rate, leaf thickness, and stomatal distribution were significantly different at α = 0.05 amongst plants. The transpiration rate was inversely proportional to leaf thickness with various correlation coefficients. The leaf anatomy of six plant species showed various thicknesses between tissues. Further research needs to be done to explain the rate of transpiration with changes in leaf anatomy.

Distribusi dan Luas Stomata pada Enam Jenis Tanaman Dikotil

Stomatal distribution and area are related to transpiration rate. This study measured stomatal distribution and area in dicotyledon plants used as ornamental plants. The study used Factorial Completely Randomized Design (CRD) with the main factors were six types of dicotyledon plants and three plant parts (top, middle and bottom). Combination factors were the interaction of plant species and parts. By using the replica method and micrometer, the study used five replications with the measured parameters: the stomatal distribution and area. Data were analyzed using SAS with the Factorial RAL model and continued with the LSD test if the treatments were significant. Correlation coefficients between stomatal distribution and area were calculated to see the relationship between the two. The types and plant parts significantly affected the stomatal distribution, while the stomatal area was influenced by the main and combination factors. Syzygium paniculatum Gaertn had the highest stomatal distribution and was different from other plant types. Meanwhile, lower leaves had the lowest stomatal distribution and differed from the middle and upper. Aerva sanguinolenta Bl had the highest stomatal area (591.05) compared to other plant species. The stomatal area on the plant's upper leaves (272,71) was significantly lower than the middle and lower parts. The combination between species and the lower part of the plant produced the highest stomatal area compared to other combinations. Stomatal distribution was inversely proportional to the stomatal area. 
 
 Keywords: dicotyledon, part of plant, stomatal area, stomatal distribution

Distribusi dan Luas Stomata pada Tanaman Hias Monokotil

Ornamental plants give beauty to the surrounding environment. Fresh air is created around the plant. It is concerned with transpiration which relates to the distribution and extent of stomata. The study measured the distribution and extent of stomata in monocot plants used as houseplants. The experimental design used a complete randomized design (RAL) factorial with five replications. The main factors were six types of monocot plants and three parts of plants (top, middle, and bottom). The interaction between plant types and parts was a combination factor. The distribution and area of the stomata were measured using replica methods and micrometers. The data was analyzed using the factorial RAL model statistical analysis system and followed up with the smallest real different test for significant treatment. The distribution of stomata was correlated with the stomata area to observe the relationship between the two. Plant types, plant parts, and combinations of both have a noticeable effect on the stomata distribution and area. The Paris lilies (Chlorophytum comosum (Thumb) Jacq.) have the highest stomata distribution (20.53%) compared to others, with a stomata area of 291.89 μm2. Meanwhile, the top part of the plant has the highest distribution and differs from the middle and bottom. The highest stomata distribution is at the top of the plant (15.02%) and lowest at the bottom (11.96%), while the smallest stomata area is at the top of the plant (687.62 μm2) and the widest is at the bottom (813.88 μm2). The stomata distribution correlates inversely with the stomata area of both plant types, plant parts, and a combination of both.
 
 Keywords: ornamental plants, monocotyledon, stomatal area, stomatal distribution

Pharmacognostic Characterization of Cola millenii K. Schum. (Malvaceae)

Background: Cola millenii K. Schum. is a member of Cola which is lesser-known in the family Malvaceae. The leaf is used in the treatment of ringworm, scabies, gonorrhoea, diarrhoea, cough and ophthalmia by the locals.
 Aim: The study determines various taxonomic, pharmacognostic and phytochemical standards helpful to ensure the identity, purity, safety and efficacy of the medicinal plant, C. millenii.
 Methodology: The pharmacognostic characterization was determined following the guidelines given by the World Health Organization (WHO). Parameters determined included macroscopy, microscopy, powder microscopy, petiole anatomy, chemomicroscopy, micromeritic properties, ash values, extractive values, fluorescence analysis and preliminary phytochemical screening.
 Results: Preliminary phytochemical screening for both the leaf and stem ethanol extracts showed the presence of phytochemicals. Epidermal cell shapes were polygonal with straight anticlinal walls. Stomatal distribution was hypostomatic with hexacytic, anisocytic and anomocytic stomata on the abaxial surface. Unicellular and glandular trichomes were observed on the abaxial surface. Areolation was quadrangular with calcium oxalate crystals observed along the veins. The chemomicroscopic study revealed the presence of lignin, starch, cellulose, oils, calcium oxalate crystals, mucilage and protein for both leaf and stem respectively. The fluorescence characteristics showed the presence of different colours supporting the presence of various phytoconstituents for both leaf and stem. The flow properties for both leaf and stem were fair and passable with the angle of repose of 40.6o and 36.8o respectively. The quantitative epidermal studies, chemomicroscopic and fluorescence characteristics revealed characteristic features for the drug. The physico-chemical results for leaf and stem gave total ash of 5.5 % and 12.07 %, water-soluble ash of 1.19 % and 3.39 %, acid-insoluble ash of 0.90 % and 2.29 % and moisture content of 4.49 % and 16.33 %, water-soluble extractive of 26.30 % and 22.50 %, ethanol-soluble extractive of 24.30 % and 16.80 %, methanol-soluble extractive of 20.90 % and 2.50 % and ethyl-acetate acetate-soluble extractive of 6.60 % and 1.10 % respectively. The GC-MS analysis revealed the presence of 31 and 45 phytochemical constituents for the leaf and stem respectively.
 Conclusion: The data generated from the present study would help to authenticate C. millenii and also affirm its folklore use in traditional medicine.

Activation of 1-Aminocyclopropane-1-Carboxylic Acid Synthases Sets Stomatal Density and Clustered Ratio on Leaf Epidermis of Arabidopsis in Response to Drought.

The adjustment of stomatal density and clustered ratio on the epidermis is the important strategy for plants to respond to drought, because the stoma-based water loss is directly related to plant growth and survival under drought conditions. But the relevant adjustment mechanism still needs to be explored. 1-Aminocyclopropane-1-carboxylate (ACC) is disclosed to promote stomatal development, while in vivo ACC levels depend on activation of ACC synthase (ACS) family members. Based on the findings of ACS expression involving in drought response and several ACS activity inhibitors reducing stomatal density and cluster in drought response, here we examined how ACS activation is involved in the establishment of stomatal density and cluster on the epidermis under drought conditions. Preliminary data indicated that activation of ACS2 and/or ACS6 (ACS2/6) increased stomatal density and clustered ratio on the Arabidopsis leaf epidermis by accumulating ACC under moderate drought, and raised the survival risk of seedlings under escalated drought. Further exploration indicated that, in Arabidopsis seedlings stressed by drought, the transcription factor SPEECHLESS (SPCH), the initiator of stomatal development, activates ACS2/6 expression and ACC production; and that ACC accumulation induces Ca2+ deficiency in stomatal lineage; this deficiency inactivates a subtilisin-like protease STOMATAL DENSITY AND DISTRIBUTION 1 (SDD1) by stabilizing the inhibition of the transcription factor GT-2 Like 1 (GTL1) on SDD1 expression, resulting in an increases of stomatal density and cluster ratio on the leaf epidermis. This work provides a novel evidence that ACS2/6 activation plays a key role in the establishment of stomatal density and cluster on the leaf epidermis of Arabidopsis in response to drought.

Callitriche as a potential model system for evolutionary studies on the dorsiventral distribution of stomata

ABSTRACT Controlling the distribution of stomata is crucial for the adaptation of plants to new, or changing environments. While many plant species produce stomata predominantly on the abaxial leaf surface (hypostomy), some produce stomata on both surfaces (amphistomy), and the remaining few produce them only on the adaxial surface (hyperstomy). Various selective pressures have driven the evolution of these three modes of stomatal distribution. Despite recent advances in our understanding of stomatal development and dorsiventral leaf polarity, the genetic basis for the evolution of different stomatal distributions is still unclear. Here, we propose the genus Callitriche as a new model system to investigate patterns in the evolution of stomatal distribution. Callitriche comprises species with diverse lifestyles, including terrestrial, amphibious, and obligately aquatic plants. We found that species in this genus cover all three modes of dorsiventral stomatal distribution, making it a desirable model for comparative and evolutionary analyses on distribution modes. We further characterized the genetic basis of the different distribution modes, focusing on the stomatal key transcription factor SPEECHLESS. Future research using the promising model system Callitriche would open a new direction for evolutionary developmental biology studies on stomata.

Anatomical Studies of the Foliar Epidermis and Petiole of some Lannea Species in Jos, Plateau State, Nigeria

Aims: Leaf epidermal morphology and petiole anatomy of seven Lannea species namely; Lannea velutina, Lannea kerstingii, Lannea egregia, Lannea schimperii, Lannea acida, Lannea microcarpa, and Lannea edulis in Nigeria were investigated to provide new taxonomic characters that could help in proper identification and delimitation of the taxa.
 Place and Duration of Study: The study was carried out in the Department of Plant Science and Biotechnology, University of Jos, Nigeria between August 2020 and March 2021.
 Methodology: Transverse sections of petioles were cut using a rotary microtome and epidermal peels were made. Staining was done using Safranin and slides observed using a light microscope.
 Results: Stomatal distribution was hypostomatic except for L. velutina and L. edulis with amphistomatic distribution. Three stomata types are found; paracytic occurring in L. velutina, L. edulis and L. egregia, cyclocytic in L. schimperii and L. microcarpa and anomocytic in L. kerstingii and L. acida. Stomata index vary among taxa with L. egregia and L. acida having the highest (22.43%) and lowest (12.17%) respectively. The epidermal cell shape was polygonal in all the species and anticlinal wall patterns were straight to slightly curved. Unicellular/multicellular uniseriate trichomes were observed in L. schimperii, L. egregia and L. kerstingii distinguishing them from other species. Petiole outlines were circular to oval, epidermis was uniseriate and vascular bundles are collateral. Presence of continuous sclerenchyma rings surrounding the vascular bundles in L. kerstingii and L. microcarpa serve as a distinguishing character. Other distinguishing characters are presence of crystals, druses and phenolic idioblasts. A taxonomic key was produced using the anatomical character as an aid to the identification of the species.
 Conclusion: Variations observed in the stomata types, petiole outline, petiole vasculature type, trichome type and stomata index could be employed for species identification and delimitation.

KELAYAKAN BOOKLET SUBMATERI STRUKTUR DAN FUNGSI JARINGAN TUMBUHAN PADA TANAMAN MONOKOTIL

This study aimed to determine the feasibility of booklet of plant structure and function tissue submateria lwith additional information about the stomatal distribution and area as teaching material. The research method used was descriptive. Validation by 5 validators consisting of 2 lecturers of the Faculty of Teacher Training and Education (FKIP) Tanjungpura University and 3 biology teachers of class XI SMA using the 2013 curriculum and school accreditation at least B. The assessment uses 5 aspects and 12 criteria. The results of the validation analysis of teaching materials obtained an average CVR and CVI value of 0.95. The booklet needs improvement on content graphing. Therefore, the booklet teaching submaterial with additional information based on the stomatal distribution and area in monocots plant was suitable to be used in the learning process of the structure and function of plant in class in XI SMA.

RSD1 Is Essential for Stomatal Patterning and Files in Rice

Stomatal density is an important factor that determines the efficiency of plant gas exchange and water transpiration. Through forward genetics, we screened a mutant rice stomata developmental defect 1 (rsd1-1) with decreased stomatal density and clustered stomata in rice (Oryza sativa). After the first asymmetric division, some of the larger sister cells undergo an extra asymmetric division to produce a small cell neighboring guard mother cell. Some of these small cells develop into stomata, which leads to stomatal clustering, and the rest arrested or developed into pavement cell. After map-based cloning, we found the protein encoded by this gene containing DUF630 and DUF632 domains. Evolutionary analysis showed that the DUF630/632 gene family differentiated earlier in land plants. It was found that the deletion of RSD1 would lead to the disorder of gene expression regarding stomatal development, especially the expression of stomatal density and distribution 1 (OsSDD1). Through the construction of OsSDD1 deletion mutants by CRISPR-Cas9, we found that, similar to rsd1 mutants, the ossdd1 mutants have clustered stomata and extra small cells adjacent to the stomata. OsSDD1 and RSD1 are both required for inhibiting ectopic asymmetric cell divisions (ACDs) and clustered stomata. By dehydration stress assay, the decreased stomatal density of rsd1 mutants enhanced their dehydration avoidance. This study characterized the functions of RSD1 and OsSDD1 in rice stomatal development. Our findings will be helpful in developing drought-resistant crops through controlling the stomatal density.

Automatic Stomatal Segmentation Based on Delaunay-Rayleigh Frequency Distance.

The CO2 and water vapor exchange between leaf and atmosphere are relevant for plant physiology. This process is done through the stomata. These structures are fundamental in the study of plants since their properties are linked to the evolutionary process of the plant, as well as its environmental and phytohormonal conditions. Stomatal detection is a complex task due to the noise and morphology of the microscopic images. Although in recent years segmentation algorithms have been developed that automate this process, they all use techniques that explore chromatic characteristics. This research explores a unique feature in plants, which corresponds to the stomatal spatial distribution within the leaf structure. Unlike segmentation techniques based on deep learning tools, we emphasize the search for an optimal threshold level, so that a high percentage of stomata can be detected, independent of the size and shape of the stomata. This last feature has not been reported in the literature, except for those results of geometric structure formation in the salt formation and other biological formations.

From one side to two sides: the effects of stomatal distribution on photosynthesis.

The functions of stomata have been studied for a long time; however, a clear understanding of the influences of stomatal distribution on photosynthesis, especially the CO2 diffusion, is still unclear. Here, we investigated the stomatal morphology, distribution on leaf surfaces, vein traits and gas exchange parameters of 61 species, of which 29 were amphistomatous, spanning 32 families. Photosynthesis (A) was tightly coupled with operational stomatal conductance (gs ) and mesophyll conductance (gm ) regardless of whether phylogenetic relationships were accounted for. Although the enhancement of gs from ferns and gymnosperms to angiosperms could largely be explained by the increase in leaf vein density (VLA) and stomatal density (SD), the gs was decoupled from VLA and SD across angiosperm species. Instead, A in angiosperms was further influenced by the allocation of stomatal pores on leaf surfaces, which dramatically increased gs and gm . Moreover, the ratio of gs to anatomically based maximum gs was, on average, 0.12 across species. Our results show that the shift of stomatal pores from one leaf side to both sides played an important role in regulating CO2 diffusion via both stomata and mesophyll tissues. Modifications of stomata distribution have potential as a functional trait for photosynthesis improvement.

Allometric co-variation of xylem and stomata across diverse woody seedlings.

Leaf stomatal density is known to co‐vary with leaf vein density. However, the functional underpinning of this relation, and how it scales to whole‐plant water transport anatomy, is still unresolved. We hypothesized that the balance of water exchange between the vapour phase (in stomata) and liquid phase (in vessels) depends on the consistent scaling between the summed stomatal areas and xylem cross‐sectional areas, both at the whole‐plant and single‐leaf level. This predicted size co‐variation should be driven by the co‐variation of numbers of stomata and terminal vessels. We examined the relationships of stomatal traits and xylem anatomical traits from the entire plant to individual leaves across seedlings of 53 European woody angiosperm species. There was strong and convergent scaling between total stomatal area and stem xylem area per plant and between leaf total stomatal area and midvein xylem area per leaf across all the species, irrespective of variation in leaf habit, growth‐form or relative growth rate. Moreover, strong scaling was found between stomatal number and terminal vessel number, whereas not in their respective average areas. Our findings have broad implications for integrating xylem architecture and stomatal distribution and deepen our understanding of the design rules of plants' water transport network.

N-glycosylation is involved in stomatal development by modulating the release of active abscisic acid and auxin in Arabidopsis.

Asparagine-linked glycosylation (N-glycosylation) is one of the most important protein modifications in eukaryotes, affecting the folding, transport, and function of a wide range of proteins. However, little is known about the roles of N-glycosylation in the development of stomata in plants. In the present study, we provide evidence that the Arabidopsis stt3a-2 mutant, defective in oligosaccharyltransferase catalytic subunit STT3, has a greater transpirational water loss and weaker drought avoidance, accompanied by aberrant stomatal distribution. Through physiological, biochemical, and genetic analyses, we found that the abnormal stomatal density of stt3a-2 was partially attributed to low endogenous abscisic acid (ABA) and auxin (IAA) content. Exogenous application of ABA or IAA could partially rescue the mutant's salt-sensitive and abnormal stomatal phenotype. Further analyses revealed that the decrease of IAA or ABA in stt3a-2 seedlings was associated with the underglycosylation of β-glucosidase (AtBG1), catalysing the conversion of conjugated ABA/IAA to active hormone. Our results provide strong evidence that N-glycosylation is involved in stomatal development and participates in abiotic stress tolerance by modulating the release of active plant hormones.

With Over 60 Independent Losses, Stomata Are Expendable in Mosses.

Because stomata in bryophytes are uniquely located on sporangia, the physiological and evolutionary constraints placed on bryophyte stomata are fundamentally different from those on leaves of tracheophytes. Although losses of stomata have been documented in mosses, the extent to which this evolutionary process occurred remains relatively unexplored. We initiated this study by plotting the known occurrences of stomata loss and numbers per capsule on the most recent moss phylogeny. From this, we identified 40 families and 74 genera that lack stomata, of which at least 63 are independent losses. No trends in stomata losses or numbers are evident in any direction across moss diversity. Extant taxa in early divergent moss lineages either lack stomata or produce pseudostomata that do not form pores. The earliest land plant macrofossils from 400 ma exhibit similar sporangial morphologies and stomatal distribution to extant mosses, suggesting that the earliest mosses may have possessed and lost stomata as is common in the group. To understand why stomata are expendable in mosses, we conducted comparative anatomical studies on a range of mosses with and without stomata. We compared the anatomy of stomate and astomate taxa and the development of intercellular spaces, including substomatal cavities, across mosses. Two types of intercellular spaces that develop differently are seen in peristomate mosses, those associated with stomata and those that surround the spore sac. Capsule architecture in astomate mosses ranges from solid in the taxa in early divergent lineages to containing an internal space that is directly connected to the conducing tissue and is involved in capsule expansion and the nourishment, hydration and development of spores. This anatomy reveals there are different architectural arrangements of tissues within moss capsules that are equally effective in accomplishing the essential processes of sporogenesis and spore dispersal. Stomata are not foundational to these processes.

Elevated CO2 promotes satellite stomata production in young cotyledons of Arabidopsis thaliana.

Stomata are tiny pores on plant leaves and stems surrounded by a pair of differentiated epidermal cells known as guard cells. Plants undergo guard cell differentiation in response to environmental cues, including atmospheric CO2 . To quantitatively evaluate stomatal development in response to elevated CO2 , imaging analysis of stomata was conducted using young cotyledons of Arabidopsis thaliana grown under ambient (380ppm) and elevated (1,000ppm) CO2 conditions. Our analysis revealed that treatment with 1,000ppm CO2 did not affect stomatal numbers on abaxial sides of cotyledons but increased cotyledon area, resulting in decreased stomatal density, 7days after germination. Interestingly, this treatment also perturbed the uniform distribution of stomata via excess satellite stomata and stomatal precursor cells. We used overexpression lines of the DNA replication licensing factor gene CDC6, a reported positive regulator of satellite stomata production. CDC6 overexpression decreased the speed of cotyledon expansion, even under treatment with 1,000ppm CO2 , possibly by suppressing pavement cell maturation. In contrast, treatment with 1,000ppm CO2 induced stomatal distribution changes in the overexpressor. These results suggest that treatment with 1,000ppm CO2 enhances both cotyledon expansion and satellite stomata production via independent pathways, at least in young cotyledons of A.thaliana.

Effective range of non-cell autonomous activator and inhibitor peptides specifying plant stomatal patterning.

ABSTRACTStomata are epidermal valves that facilitate gas exchange between plants and their environment. Stomatal patterning is regulated by the EPIDERMAL PATTERING FACTOR (EPF) family of secreted peptides: EPF1 enforces stomatal spacing, whereas EPIDERMAL PATTERNING FACTOR-LIKE9 (EPFL9), also known as Stomagen, promotes stomatal development. It remains unknown, however, how far these signaling peptides act. Utilizing Cre-lox recombination-based mosaic sectors that overexpress either EPF1 or Stomagen in Arabidopsis cotyledons, we reveal a range within the epidermis and across the cell layers in which these peptides influence patterns. To determine their effective ranges quantitatively, we developed a computational pipeline, SPACE (stomata patterning autocorrelation on epidermis), that describes probabilistic two-dimensional stomatal distributions based upon spatial autocorrelation statistics used in astrophysics. The SPACE analysis shows that, whereas both peptides act locally, the inhibitor EPF1 exerts longer range effects than the activator Stomagen. Furthermore, local perturbation of stomatal development has little influence on global two-dimensional stomatal patterning. Our findings conclusively demonstrate the nature and extent of EPF peptides as non-cell autonomous local signals and provide a means for quantitative characterization of complex spatial patterns in development.This article has an associated ‘The people behind the papers’ interview.

A Ca2+/CaM-regulated transcriptional switch modulates stomatal development in response to water deficit

Calcium (Ca2+) signals are decoded by the Ca2+-sensor protein calmodulin (CaM) and are transduced to Ca2+/CaM-binding transcription factors to directly regulate gene expression necessary for acclimation responses in plants. The molecular mechanisms of Ca2+/CaM signal transduction processes and their functional significance remains enigmatic. Here we report a novel Ca2+/CaM signal transduction mechanism that allosterically regulates DNA-binding activity of GT2-LIKE 1 (GTL1), a transrepressor of STOMATAL DENSITY AND DISTRIBUTION 1 (SDD1), to repress stomatal development in response to water stress. We demonstrated that Ca2+/CaM interaction with the 2nd helix of the GTL1 N-terminal trihelix DNA-binding domain (GTL1N) destabilizes a hydrophobic core of GTL1N and allosterically inhibits 3rd helix docking to the SDD1 promoter, leading to osmotic stress-induced Ca2+/CaM-dependent activation (de-repression) of SDD1 expression. This resulted in GTL1-dependent repression of stomatal development in response to water-deficit stress. Together, our results demonstrate that a Ca2+/CaM-regulated transcriptional switch on a trihelix transrepressor directly transduces osmotic stress to repress stomatal development to improve plant water-use efficiency as an acclimation response.

EXOGENOUS CYTOKININS APPLICATION INDUCES CHANGES IN STOMATAL AND GLANDULAR TRICHOMES PARAMETERS IN ROSEMARY PLANTS REGENERATED IN VITRO

Rosemary (Rosmarinus officinalis L.) is a native Mediterranean herb, used to typically prevent oxidation of fats and oils in foods and cosmetics. Rosemary represents an effective source of natural antioxidants with analgesic, diuretic, digestive and anti-inflammatory effects. This study is carried out to properly evaluate if exogenous cytokinin applications typically led to hyperhydration symptoms or any histological variability in stomatal density, stomatal distribution, glandular trichomes density and size of leaf epidermis. The colloidal technique was used for observation and measurement of leaf epidermis characters. The plantlets during in vitro culture were developed on MS media and two different PGRs ratios (BAP) / auxin (NAA) were tested: (i) 1 mg l-1 / 0.1 mg l-1 (ii) 2 mg l-1 / 0.1 mg l-1. In vitro leaves of both categories were taken for analyses 4 weeks after the first subculture and were compared with in vivo ones. The study showed that in vivo plantlets have hypostomatic leaves, meanwhile, in vitro plantlets of both categories consist of amphistomatic leaves. In all cases, there are observed anisocytic stomata. In vitro leaves have a higher stomatal density, value which is increased in higher concentrations of BAP and thus transpiration rates are affected. In vitro specimens show about 3 times higher trichomes density in comparison with in vivo ones, but their diameter is about 2 times lower. Glandular trichomes size and density are affected by BAP concentration in nutrient media. These results convincingly show that in vitro culture can be used successfully to improve secondary metabolites production by intentionally altering the growing physical and chemical conditions.