Guard Cells Of Stomata Research Articles

Chromosome Study of Six Populations of Trifolium tumens Stev. ex M. B. in IRAN

Trifolium tumens Stev. ex M. B is a perennial legume with long‐lived and deep rooting from the Vesicaria section in Trifolium genus. It is distributed in Iran. The base chromosome number (x = 8) in the population, confirmed the views of the previous researchers. In this study, the number of chromosomes in the mitotic metaphase stage in the meristematic cells of the root ends were counted in six populations of Trifolium tumens. Also, chloroplast count of stomata guard cells, morphological studies, pollen grain size and flowcytometry were studied in six accessions. Our results showed that this species has ploidy level (2n=16, 2n=32). and the tetraploid state in the species T. tumens is reported. Through flowcytometry, chloroplast count of stomata guard cells and compare the pollen grain, different characters were showed between diploid and tetraploid populations, too. Based on the results, this new cytotype is reported from Iran.

Modulation of abscisic acid signaling via endosomal TOL proteins.

The phytohormone abscisic acid (ABA) functions in the control of plant stress responses, particularly in drought stress. A significant mechanism in attenuating and terminating ABA signals involves regulated protein turnover, with certain ABA receptors, despite their main presence in the cytosol and nucleus, subjected to vacuolar degradation via the Endosomal Sorting Complex Required for Transport (ESCRT) machinery. Collectively our findings show that discrete TOM1-LIKE (TOL) proteins, which are functional ESCRT-0 complex substitutes in plants, affect the trafficking for degradation of core components of the ABA signaling and transport machinery. TOL2,3,5 and 6 modulate ABA signaling where they function additively in degradation of ubiquitinated ABA receptors and transporters. TOLs colocalize with their cargo in different endocytic compartments in the root epidermis and in guard cells of stomata, where they potentially function in ABA-controlled stomatal aperture. Although the tol2/3/5/6 quadruple mutant plant line is significantly more drought-tolerant and has a higher ABA sensitivity than control plant lines, it has no obvious growth or development phenotype under standard conditions, making the TOL genes ideal candidates for engineering to improved plant performance.

A charged existence: A century of transmembrane ion transport in plants.

If the past century marked the birth of membrane transport as a focus for research in plants, the past 50 years has seen the field mature from arcane interest to a central pillar of plant physiology. Ion transport across plant membranes accounts for roughly 30% of the metabolic energy consumed by a plant cell, and it underpins virtually every aspect of plant biology, from mineral nutrition, cell expansion, and development to auxin polarity, fertilization, plant pathogen defense, and senescence. The means to quantify ion flux through individual transporters, even single channel proteins, became widely available as voltage clamp methods expanded from giant algal cells to the fungus Neurospora crassa in the 1970s and the cells of angiosperms in the 1980s. Here, I touch briefly on some key aspects of the development of modern electrophysiology with a focus on the guard cells of stomata, now without dispute the premier plant cell model for ion transport and its regulation. Guard cells have proven to be a crucible for many technical and conceptual developments that have since emerged into the mainstream of plant science. Their study continues to provide fundamental insights and carries much importance for the global challenges that face us today.

Phenotypic variability of epidermis structure and silicon inclusions in the leaves of Quercus robur in the Feofaniya Park

The micromorphology of the leaf epidermis, localization, and silicon content in the epidermal cells of Quercus robur leaves growing in the shade and under direct sunlight in the Feofaniya Park (Kyiv, Ukraine) were studied using scanning electron microscopy and laser confocal microscopy. Silicon inclusions were found in the anticlinal and periclinal walls of adaxial epidermal cells, trichomes, guard cells of stomata, and walls of regular epidermal cells on the abaxial leaf surface, the amount of which varied according to the conditions of growth. Natural shading and the intensity of solar irradiation were found affecting the size of leaf blades, the ultrastructure of the leaf epidermis, and changes in the silicon content of oak leaves. Studies have shown that the anticlinal walls of the adaxial epidermis and the trichomes and stomata of the abaxial epidermis of leaves are the main silicon accumulators. The findings suggest that changes in leaf microstructure and silicon content contribute to maintaining optimal water balance in plants and can be regarded as signs of phenotypic plasticity in plants and an adaptive marker depending on the sunlight conditions of oak growth.

Frequency and spectrum of M2 mutants and genetic variability in cyto-agronomic characteristics of fenugreek induced by caffeine and sodium azide.

Trigonella foenum graecum L. (Fenugreek) is a valuable medicinal plant cultivated for decades for its therapeutic characteristics. Still no pronounced improvement concerning wild form was accomplished as it is a self-pollinating crop. Induced mutagenesis is encouraged as a remarkable tool on this plant to circumvent the genetic bottleneck of cultivated germplasms. As a result, novel allelomorphic combinations for short-term agronomic attributes were developed. Fenugreek cultivar Pusa Early Bunching, selected for the present experiment, was mutagenized with five doses (0.2%, 0.4%, 0.6%, 0.8%, and 1.0%) of caffeine and sodium azide (SA) to evaluate its impact on the qualitative and quantitative traits of M1 and M2 generation conducted in a Complete Randomized Block Design (CRBD), replicated five times during 2019-2020 and 2020-2021, respectively. The frequency of induced phenotypic variations was assessed in M2 progenies, resulting in the identification and isolation of a broad spectrum of mutants with altered phenotypes. Mutagenic effectiveness and efficiency were found to be maximum at lower concentrations of the mutagen treatments and highest in SA, followed by caffeine. Various morphological mutants with modified characters were observed at different concentrations in M2 generation. The spectrum of mutations was wider in SA than in caffeine, as caffeine produced 51 while SA produced 54 individual mutants under seven major categories. The maximum frequency of morphological mutants was associated with leaf, followed by plant size, plant growth habit, pod, seed size, seed shape, and seed color. Morphological and structural variations in the guard cells of stomata and seeds were observed through scanning electron microscopy. The variations created in the economically important traits may enrich the genetic diversity of this plant species. Moreover, these morphological mutants may serve as a source of elite genes in further breeding programs of fenugreek.

Varijabilnost i diskriminacijska moć anatomskih parametara kod linija polusrodnika juglans regia podrijetlom iz prirodnih sastojina u Đerdapskoj klisuri, Srbija u rasadničkim uvjetima

This paper presents analysis of anatomical parameters of leaves in Juglans regia half-sib lines from natural stands in Đerdap Gorge in Serbia. Thirty-five half-sib lines were examined, designated according to the number of their mother trees from R1-R35. Twenty measured and derived anatomical parameters were examined, describing stomatal and leaf blade cross-section traits. According to Hierarchical analysis of variance, variation of the majority of characters describing stomatal properties were significantly affected by effect of half-sib lines. The effect of variation between plants within half-sib lines was significant for all examined stomatal traits. However, in cross-section characters, the effect of differences between half-sib lines was significant only for thickness of spongy mesophyll (SL). The discrimination model with seven selected traits achieved 60.5% of correct allocation, while the correct allocation of the model with all 20 examined traits was 81.6%. According to the results of stepwise discriminant analysis, the most powerful trait in discrimination of these half-sib lines were stomatal aperture width/stomatal aperture length ratio (Wb/La) and stomatal aperture length/ stomata guard cell length ratio (La/LA). Presented results strongly support implementation of stomatal and cross-section leaf traits in further studies that would deal with description of variability of Juglans regia.

Tetraploidy induction enhances morphological, physiological and biochemical characteristics of wallflower (Erysimum cheiri (L.) Crantz)

Wallflower (Erysimum cheiri (L.) Crantz is one of the most important bedding flowers and potted plants in the floriculture industry. Its widespread cultivation is hindered due to self-incompatibility and limited breeding. The present study was conducted to investigate the effects of polyploidy induction on different characteristics of wallflower. Seeds were treated with colchicine (0, 0.5, 0.75 and 1%) (w/v) and oryzalin (0, 50, 100 and 150 mgl−1) along various exposure times (24, 36 and 48 h). The highest tetraploidy induction (53%) was seen in 150 mgl−1 oryzalin treatment for 36 h. The chromosome doubled plants indicated increase in trichome number, pollen size, stomata size and number of chloroplasts in the stomata guard cells, while stomata density and pollen number were decreased. The new tetraploid induced wallflowers showed compact growth habit with shorter internodes and roots, while leaves were bigger (60%) and purplish, flowering period was prolonged (65%), and flowers showed greater longevity (41%), diameter (47%) and number (78%), compared to diploid controls. Their anthocyanin and total soluble solids contents were increased by 56% and 140%, respectively. UPLC-MS analysis indicated that phytohormone contents of Ze, SA, JA and ABA were significantly increased in the tetraploid induced plants than diploid controls, while IAA, GA3 and BR were less accumulated. The new ornamental features developed for the first time in wallflower proves that polyploidization is a promising tool to introduce a commercial flowering pot plant cultivar and a new genetic resource for future breeding programs.

Microstructural Feature of Salvia glutinosa L. vegetative and generative organs

 It has been studied perspective medicinal plant S. glutinosa  aboveground parts vegetative and generative organs internal diagnostic features. The aboveground vegetative and generative organs of the plant are lined with unicellular or multicellular, conical trichomes, glands, unicellular or multicellular, also conical, spherical, secretory trichomes; Basal layer cells of curved and curved wall epidermal tissue is characteristic feature for leaf. The leaf is bifacial, amphistomatic, dorsoventral structure. Folium and axial organs cover tissue are presented diacytic type stomata. Lentil-like Guard cells of stomata, subsidiary cells stomata are curvilinear structure.In the internal structure of aboveground part polygonal structure tissue and mechanical tissue are actively represented, fiber and collenchyma existence are particularly characteristic sign for mechanical tissue. The transport system is monocyclic, collateral vascular bundles reflected in the stem, leaf stalk, and midrib. Longitudinally arranged, spherically shaped vascular vessel and tracheid element is in the transport system cortex. The axis organs xylem vessel mainly is spiral type, rarely  porous wall thickening.

Functional characterization of 5'-regulatory region of flavonoid 3',5'-hydroxylase-1 gene of banana plants.

Generation of crops with broad-spectrum tolerance to biotic and abiotic stress conditions depends upon availability of genetic elements suitable for varied situations and diverse genotypes. Here, we characterize the 5'-upstream regulatory region of flavonoid 3'5'-hydroxylase-1 (F3'5'H-1) gene from banana and analyzed its tissue-specific and stress-mediated activation in genetic background of tobacco plants. MusaF3'5'H-1 is a stress-responsive gene as its expression is induced in banana after application of salicylic acid and methyl jasmonate while its transcript levels were drastically reduced in response to drought, high salinity and abscisic acid. PMusaF3'5'H-1 harbours cis-elements associated with stress conditions and those responsible for tissue-specific expression. Transgenic lines harbouring PMusaF3'5'H-1-GUS displays strong GUS expression in guard cells of stomata indicating guard cell preferred activity of PMusaF3'5'H-1 while its activity was undetectable in roots. Drought and high salinity induce strong expression of GUS in transgenic tobacco lines and exposure to abscisic acid, salicylic acid and methyl jasmonate revealed distinct profiles of GUS expression in transgenic lines confirming involvement of F3'5'H-1 in plant stress responses. Fluorescent β-galactosidase assay revealed induction profiles of PMusaF3'5'H-1 at different time points in transgenic lines exposed to salicylic acid and abscisic acid while strong suppression in GUS expression was observed after application of methyl jasmonate. The guard cell preferred activity of PMusaF3'5'H-1 and stress-mediated expression profiles of MusaF3'5'H-1 indicated the suitability of PMusaF3'5'H-1 for generating stress-enduring crops and analyzing guard cell functions.

Assessment of the phenotypic diversity of wild cherry (Prunus avium L.) populations and halfsib lines by multivariate statistical analyses

Abstract Wild cherry (Prunus avium L.) is a multi-purpose tree species with great ecological and economic importance for European forestry. Evaluating this species phenotypic diversity and quantitative traits characterization is of great importance to define its genetic resources conservation and breeding strategies. In this work, variations of physiological, biochemical, anatomical and morphological traits of one-year-old wild cherry seedlings were evaluated within and among populations to distinguish and characterize their phenotypic portfolio. We observed significant differences at the intra- and inter-population levels considering both biochemical and physiological leaf traits, whereas differences in morphological and anatomical traits were found to be significant only among half-sib lines within populations (i.e. intra-population level). With a multivariate approach, we explored the inter-population specificity and found out that the tiered approach spanning from organ morphology, across physiological scale, to the biochemical level gave out enough power to discriminate between different populations, and their acquisition and resource-use strategies. Moreover, stepwise discriminative analysis showed that radical scavenger capacity against 2,2’-azino-bis(3-ethylbenzothiazoline- 6-sulfonic acid) (ABTS•+) and water-use efficiency contributed to discrimination of studied populations to the largest extend. Lastly, our study highlights the robustness of certain functional traits, such as ABTS•+, water-use efficiency, net photosynthesis, total flavonoid content, width of stomata guard cell, and stomatal aperture length, which could be considered as a proxy to discriminate between wild cherry populations and assess phenotypic diversity.

The influence of powdery mildew on chlorophyll a fluorescence and stomatal characteristics of pedunculate oak (Quercus robur L.)

Oak powdery mildew (Erysiphe alphitoides (Griffon and Maubl.) U. Braun and S. Takam.)) is one of the most common foliar pathogenic organism of oaks, exploiting harmful effects, particularly on young seedlings. To assess and evaluate these negative effects, an experiment was conducted under semi-controlled conditions with 20 one-year-old seedlings of Quercus robur L. infected with E. alphitoides, half of them showing 50% of leaf coverage by epiphytic mycelia and the other half a coverage of 75%. The results of the present study have shown that all observed parameters of chlorophyll a fluorescence transient were highly effective in the detection of severe biotic stress. Moreover, the studied parameters of slow fluorescence kinetics were also considerably influenced, with the coefficient of non-photochemical fluorescence quenching and the Stem-Volmer type non-photochemical fluorescence quenching parameters showing the fastest responses. In case of leaf stomatal traits, the decrease of stomata guard cell width coupled with the increase of stomatal density was observed as the protective mechanism of Q. robur against the stressor. The overall results showed the adverse effects of powdery mildew infection on the photosynthesis of pedunculate oak seedlings, which progressed in time and depended on the severity of the infection. The importance of the results of the present study lays in evaluation and monitoring of the effects of powdery mildew development on the photosynthetic apparatus of one-year-old Q. robur seedlings, which is the most vulnerable stage for the infection by the mentioned pathogen.

A New Player in Jasmonate-Mediated Stomatal Closure: The Arabidopsis thaliana Copper Amine Oxidase β.

Plant defence responses to adverse environmental conditions include different stress signalling, allowing plant acclimation and survival. Among these responses one of the most common, immediate, and effective is the modulation of the stomatal aperture, which integrates different transduction pathways involving hydrogen peroxide (H2O2), calcium (Ca2+), nitric oxide (NO), phytohormones and other signalling components. The Arabidopsis thaliana copper amine oxidases β (AtCuAOβ) encodes an apoplastic CuAO expressed in guard cells and root protoxylem tissues which oxidizes polyamines to aminoaldehydes with the production of H2O2 and ammonia. Here, its role in stomatal closure, signalled by the wound-associated phytohormone methyl-jasmonate (MeJA) was explored by pharmacological and genetic approaches. Obtained data show that AtCuAOβ tissue-specific expression is induced by MeJA, especially in stomata guard cells. Interestingly, two Atcuaoβ T-DNA insertional mutants are unresponsive to this hormone, showing a compromised MeJA-mediated stomatal closure compared to the wild-type (WT) plants. Coherently, Atcuaoβ mutants also show compromised H2O2-production in guard cells upon MeJA treatment. Furthermore, the H2O2 scavenger N,N1-dimethylthiourea (DMTU) and the CuAO-specific inhibitor 2-bromoethylamine (2-BrEtA) both reversed the MeJA-induced stomatal closure and the H2O2 production in WT plants. Our data suggest that AtCuAOβ is involved in the H2O2 production implicated in MeJA-induced stomatal closure.

Induction of Autotetraploidy and its effects on morphophysiological traits in some annual and perennial medics

In order to study of the effects of polyploidy on morph-physiological traits in some annual and perennial medics, five annual and three perennial diploid medics were subjected to different concentrations of colchicine solution (0.1%, 0.5%, 1% and 2%) in mitosis stage. The induced tetraploids were identified by counting stomata guard cells chloroplasts. The highest survival rate and tetraploidy induction with average values of 80.2% and 74.3% were obtained using 0.1% colchicine concentration. All of the 21 entries (5 annual diploids and their induced tetraploids as M. lupulina, M. radiata, M. rijidula, M. truncatula, M. turbinata, and 3 populations of perennial diploid of M. sativa ssp. Caerulea (Karaj1, Karaj2 and Tehran) and their induced tetraploids and 5 commercial tetraploid alfalfa cultivars (Bami, Hamadani, Baghdadi, Ghareyongeh and Renger) grown in pots in a glasshouse experiment using completely randomized design with five replications in 2018 in Ahvaz, Iran. Data were collected for leaf length, leaf width, shoot and root length, seedling weight, shoot weight, root weight, branch number, leaf area index (LAI) and water use efficiency (WUE). Result of mean comparisons between three groups (2x, induced 4x and controls 4x), showed that both groups of induced and natural tetraploids had significantly higher mean values for all the traits except leaf length, shoot length and root length than that for diploids. For the latter traits there were no significant differences between 2x and induced 4x. On the overall, the induced 4x had 60%, 62%, 68%, 65%, 22%, 109% and 47% higher mean values than their parental 2x for seedling weight, shoot weight, root weight, LAI, WUE and branch number, respectively. It was concluded that increasing ploidy level provides plant breeders with a promising tool in the breeding improve new varieties suited for future climate scenarios.

Ozone Induced Stomatal Regulations, MAPK and Phytohormone Signaling in Plants.

Ozone (O3) is a gaseous environmental pollutant that can enter leaves through stomatal pores and cause damage to foliage. It can induce oxidative stress through the generation of reactive oxygen species (ROS) like hydrogen peroxide (H2O2) that can actively participate in stomatal closing or opening in plants. A number of phytohormones, including abscisic acid (ABA), ethylene (ET), salicylic acid (SA), and jasmonic acid (JA) are involved in stomatal regulation in plants. The effects of ozone on these phytohormones’ ability to regulate the guard cells of stomata have been little studied, however, and the goal of this paper is to explore and understand the effects of ozone on stomatal regulation through guard cell signaling by phytohormones. In this review, we updated the existing knowledge by considering several physiological mechanisms related to stomatal regulation after response to ozone. The collected information should deepen our understanding of the molecular pathways associated with response to ozone stress, in particular, how it influences stomatal regulation, mitogen-activated protein kinase (MAPK) activity, and phytohormone signaling. After summarizing the findings and noting the gaps in the literature, we present some ideas for future research on ozone stress in plants

Knockout of Auxin Response Factor SlARF4 Improves Tomato Resistance to Water Deficit.

Auxin response factors (ARFs) play important roles in various plant physiological processes; however, knowledge of the exact role of ARFs in plant responses to water deficit is limited. In this study, SlARF4, a member of the ARF family, was functionally characterized under water deficit. Real-time fluorescence quantitative polymerase chain reaction (PCR) and β-glucuronidase (GUS) staining showed that water deficit and abscisic acid (ABA) treatment reduced the expression of SlARF4. SlARF4 was expressed in the vascular bundles and guard cells of tomato stomata. Loss of function of SlARF4 (arf4) by using Clustered Regularly Interspaced Short Palindromic Repeats/Cas 9 (CRISPR/Cas 9) technology enhanced plant resistance to water stress and rehydration ability. The arf4 mutant plants exhibited curly leaves and a thick stem. Malondialdehyde content was significantly lower in arf4 mutants than in wildtype plants under water stress; furthermore, arf4 mutants showed higher content of antioxidant substances, superoxide dismutase, actual photochemical efficiency of photosystem II (PSII), and catalase activities. Stomatal and vascular bundle morphology was changed in arf4 mutants. We identified 628 differentially expressed genes specifically expressed under water deficit in arf4 mutants; six of these genes, including ABA signaling pathway-related genes, were differentially expressed between the wildtype and arf4 mutants under water deficit and unlimited water supply. Auxin responsive element (AuxRE) elements were found in these genes’ promoters indicating that SlARF4 participates in ABA signaling pathways by regulating the expression of SlABI5/ABF and SCL3, thereby influencing stomatal morphology and vascular bundle development and ultimately improving plant resistance to water deficit.

Colchicine effects on the ploidy level and morphological characters of Katokkon pepper (Capsicum annuum L.) from North Toraja, Indonesia

BackgroundProductivity and quality of crops can be increased through polyploidy plants induced by colchicine. The use of colchicine has never been applied to Katokkon pepper, a local red pepper variety from North Toraja Indonesia. This pepper is characterized by its unique shape like the small-bell pod type of paprika and its strong spicy taste. Therefore, this study aimed to determine the effects of colchicine treatment on the ploidy level and morphological characters of Katokkon pepper. ResultsFlow cytometer analysis showed that all colchicine concentration treatments ranging from 0.025 to 0.1% with 24 h immersion time generated two plant groups based on their ploidy level; 50% of the total treatment plants was diploid while the rest plants were mixoploid. All colchicine treatment plants were significantly different in their width of stomata guard cells from the control plant. The effect of colchicine was also significantly on the plant height, thickness of fruit flesh, and the number of fruits per plant. ConclusionsThe results of this study showed that colchicine treatment had a significant effect on the ploidy level and several morphological characters of the Katokkon pepper. The colchicine treatment increased the number of fruits per plant and thicker flesh of fruits but reduced the size and weight of Katokkon pepper. Our findings provide essential information to obtain tetraploid Katokkon plants through colchicine treatment in further research. This study benefits as a preliminary step for increasing the productivity and quality of the local red peppers in Indonesia.

Micromorphology and monolignols of leaf epidermis in Phragmites australis (Poaceae) of air-aquatic and terrestrial ecotypes.

The microstructure of leaf epidermis, the localization, and content of syringyl (S) and guajacyl (G) monolignol in epidermal cells of Phragmites australis grown in water and moderate drought soil on the bank of the Venetian strait of the Dnipro River in Kiev (Ukraine) were studied with the electron microscopic method and laser confocal microscopy. The trichomes, guard cells of stomata, epidermal cells of vaulted zones over veins of adaxial and abaxial surface contained syringyl and guajacyl, the content of which depended on plant ecotype. We established that moisture soil influenced the leaf epidermis ultrastructure, on the change of monolignols content and S/G ratio in epidermis leaf of reed. The investigations show that trichomes and the cells of the vaulted zone in the leaf epidermis were the main accumulator of monolignols. The obtained results suggest that change of the microstructure of leaves, content of the monolignols, and S/G ratio in leaves contribute to the preservation of optimal water status of plants and can be considered as the signs of plant phenotypic plasticity and adaptive marker depending on the conditions of water supply.

Leaf transcriptomic response mediated by cold stress in two maize inbred lines with contrasting tolerance levels

Maize (Zea mays L.) is a thermophilic plant and a minor drop in temperature can prolong the maturity period. Plants respond to cold stress through structural and functional modification in cell membranes as well as changes in the photosynthesis and energy metabolism. In order to understand the molecular mechanisms underlying cold tolerance and adaptation, we employed leaf transcriptome sequencing together with leaf microstructure and relative electrical conductivity measurements in two maize inbred lines, having different cold stress tolerance potentials. The leaf physiological and transcriptomic responses of maize seedlings were studied after growing both inbred lines at 5 °C for 0, 12 and 24 h. Differentially expressed genes were enriched in photosynthesis antenna proteins, MAPK signaling pathway, plant hormone signal transduction, circadian rhythm, secondary metabolites related pathways, ribosome, and proteasome. The seedlings of both genotypes employed common stress responsive pathways to respond to cold stress. However, the cold tolerant line B144 protected its photosystem II from photooxidation by upregulating D1 proteins. The sensitive line Q319 was unable to close its stomata. Collectively, B144 exhibited a cold tolerance owing to its ability to mediate changes in stomata opening as well as protecting photosystem. These results increase our understanding on the cold stress tolerance in maize seedlings and propose multiple key regulators of stress responses such as modifications in photosystem II, stomata guard cell opening and closing, changes in secondary metabolite biosynthesis, and circadian rhythm. This study also presents the signal transduction related changes in MAPK and phytohormone signaling pathways in response to cold stress during seedling stage of maize.

Melatonin and calcium function synergistically to promote the resilience through ROS metabolism under arsenic-induced stress

The interplay between melatonin (Mel) and calcium (Ca2+) in enhancing tolerance to metalloid toxicity and underlying physiological and biochemical mechanisms of this relationship still remains unknown. The present study reveals that the signaling molecules Mel and/or Ca2+ enhanced tolerance of Vicia faba (cv. Tara) plant to metalloid arsenic (As) toxicity. However, a combination of Mel and Ca2+ was more efficient than alone. Plants grew with As exhibited enhanced hydrogen peroxide, superoxide anion, electrolyte leakage, lipid peroxidation together with increased reactive oxygen species (ROS) producing enzymes, such as NADPH oxidase and glycolate oxidase (GOX). On the contrary, an inhibition in chlorophyll (Chl) biosynthesis and gas exchange parameters (net photosynthetic rate, stomatal conductance, intercellular carbon dioxide concentration) was observed. Under As toxicity conditions, the application of Mel and Ca2+ synergistically suppressed the plants’ program cell death features (nucleus condensation and nucleus fragmentation) in guard cells of stomata, DNA damage, and formation of ROS in guard cells, leaves and roots. Moreover, it enhanced gas exchange parameters and activity of enzymes involved in photosynthesis process (carbonic anhydrase and RuBisco), Chl biosynthesis (δ-aminolevulinic acid dehydratase), and decreased activity of Chl degrading enzyme (chlorophyllase) under As toxicity conditions. Our investigation evidently established that expression of ATP synthase, Ca2+-ATPase, Ca2+-DPKase, Hsp17.6 and Hsp40 was found maximum in the plants treated with Mel + Ca2+, resulting in higher tolerance of plants to As stress. Also, increased total soluble carbohydrates, cysteine, and Pro accumulation with increased Pro synthesizing enzyme (Δ1-pyrroline-5-carboxylate synthetase (P5CS) and decreased Pro degrading enzyme (proline dehydrogenase) in Mel + Ca2+ treated plants conferred As toxicity tolerance. The obtained results postulate strong evidence that the application of Mel along with Ca2+ enhances resilience against As toxicity by upregulating the activity of plasma membrane H+-ATPase, enzymes involved in antioxidant system, and ascorbate-glutathione pathway.

Cloning, Genetic Transformation and Cellular Localization of Abiotic Stress Responsive Universal Stress Protein Gene (GUSP1) in Gossypium hirsutum.

Background: Drought stress seriously affects the cotton fiber development. Universal stress protein gene isolated from native species Gossypium arboreum has the promising tolerance role against these stresses.Objectives: This study aimed to clone, characterize, and genetically transform the GUSP1 gene in local cotton and to observe its expression in transgenic plants under drought stress.Materials and Methods: Universal Stress Protein (GUSP1) gene from Gossypium arboreum was cloned in pCEMBIA (-) 1301plant expression vector by replacing Hygromycin and GUS exon with GUSP1-GFP fusion fragment. The construct was transformed into Agrobacterium tumefaciens and transient expression assay was confirmed by agro-infiltration of Nicotiana benthamiana leaves and green fluorescence under a confocal microscope. Gene integration and expression in transgenic plants was observed through Southern blot and real-time PCR analyses. Cellular localization was observed through a confocal microscope and the copy number of the transgene was observed in progeny plants. Results: Transformation efficiency was 1.9%. Developmental and spatial expression of GUSP1 was observed through Real-time PCR in stem, root, leaf, inflorescence, and seeds of transgenic plants at the vegetative and flowering stage. Integration of GUSP1 revealed a fragment of approximately 500 bp in Southern Blot analyses. Localization of GUSP1 was detected in the intact leaf of transgenic plants through GFP fluorescence in midrib, guard cells of stomata, and trichomes. Single gene copy was detected in the chromosome of transgenic seeds.Conclusion: GUSP1 has cloned from native species of local cotton and its integration and expression in transgenic plants confirmed that the role of GUSP1 will provide direction to breed economically important cotton varieties.