The subject of this study is the evaluation of secondary metabolites production in Hypericum perforatum L. cultures in vitro after elicitor treatment. The aim was to find if orthosilicic acid as abiotic elicitor increases the flavonoid and hypericin production in Hypericum perforatum L. cultures in vitro. Experiment was carried out in callus and suspension cultures of H. perforatum using Murashige - Skoog nutrient medium78 supplemented with 10 mg. ml-1 α-naphtylacetic acid as growth regulator. The elicitor was added in the form of solution in 3 different concentrations (C1 = 10.4047∙10-3 mol l-1 , C2 = 10.4047∙10-4 mol l-1 , C3 = 10.4047∙10-5 mol l-1 ), it was affecting 6, 12, 24, 48, 72 and 168 hours. The content of flavonoids and hypericin was determined by HPLC. Secondary metabolites release into nutrient medium was also a part of this study. The increasing flavonoid and hypericin production in callus cultures after elicitor application at any concentrations was not observed. The maximum flavonoid content (0.04 mg g-1 DW) in suspension culture was detected after 72 h of elicitor treatment in concentration of C1 where the maximum hyperoside production was observed. The maximum hypericin production (0.21 mg g-1 DW) in suspension culture was detected after 12 h of elicitor application in...

In vitro cultures of medicinal plants - IXX The subject of this thesis is the evaluation of secondary metabolites production in in vitro cultures of Silybum marianum L. after elicitor treatment. In this study selenium dioxide as elicitor in concentrations of 9,012.10-3 mol/l; 9,012.10-4 mol/l; 9,012.10-5 mol/l was used. The samples were taken after 6, 12, 24, 48, 72 and 168 hours of elicitor treatment. The effect of elicitor was compared with control samples, which were cultured without elicitation. The content of taxifolin and flavonolignans was determined by the method of HPLC. The results showed, that almost all observed metabolites were released into a nutrient medium. Cells of callus and suspension cultures produced only small amounts of taxifolin (0.01 mg/g DW). Taxifolin and silymarin complex releasing into nutrient media was observed as in control and also in the elicitated samples of callus and suspension cultures. Selenium dioxide elicitation caused statistically significant increases in releasing taxifolin and silymarin complex into the nutrient medium. The statistically significant releasing of flavonolignans (2.2 mg/100 ml) to the medium of suspension culture was reached after 72 hours of treatment with selenium dioxide in concentration of c1 (9,012.10-3 mol/l). The statistically...

Doctor of Philosophy / Department of Agronomy / Allan K. Fritz / Heat and drought stress are two of the most significant abiotic stresses limiting wheat production in the Great Plains and worldwide. Introgression of novel tolerance genes from wild relatives is a strategy which presents promise. This study examined both heat and drought tolerance from the tetraploid species Aegilops geniculata (U[superscript g]U[superscript g]M[superscript g]M[superscript g]). Additional screening for heat tolerance was conducted with the US genome species Aegilops peregrina (Hack) and Aegilops kotschyi (Boiss). A comprehensive screening system for drought tolerance was also constructed to evaluate wheat and its wild relatives. Previous reports suggested that Ae. geniculata accession TA2899 was moderately tolerant to heat stress. It had also previously been used to develop a full set of wheat-Ae. geniculata chromosome addition lines in a Chinese Spring background. To identify the chromosome(s) carrying the heat tolerance, all addition lines, as well as wheat check genotypes, were screened for post-anthesis heat tolerance in two growth chamber experiments. No chromosome addition lines were significantly different (p<0.05) from Chinese Spring, and none were found to have superior performance to the positive check cultivars. Forty-five accessions of Ae. peregrina and its close relative, Ae. kotschyi were screened in a post-anthesis heat experiment. A follow-up experiment compared the genotypes in a split-plot temperature treatment with heat and optimal growth chambers. Many accessions were similar to the control genotypes for grain fill duration, and some exceeded the wheat controls for relative chlorophyll index values on Day 12 and Day 16. TA1889 and TA1904, both Ae. peregrina accessions originating from Israel, had a higher grain fill duration across experiments than the best wheat control, and warrant further investigation. Previous reports suggested drought tolerance in Ae. geniculata. After preliminary screenings, six genotypes were selected for advanced screening and compared with three wheat cultivars. The advanced greenhouse screening system was conducted in 152cm tall PVC growth tubes. The experiment measured multiple plant responses, and had a datalogging system automatically collecting water content and matric potential of the growth media. Multiple accessions warranted further investigation, and showed potentially different modes of drought tolerance, with varying levels of stomatal resistance, biomass, and osmotic adjustment.

>Magister Scientiae - MSc / Agricultural production is one of the most important sectors which provide food for the growing world population which is estimated to reach 9.7 billion by 2050, thus there is a need to produce more food. Climate change, on the other hand, is negatively affecting major global crops such as maize, sorghum, wheat and barley. Environmental factors such as salinity, drought, high temperatures and pathogens affect plant production by oxidatively damaging the physiological processes in plants, leading to plant death. Poor irrigation used to combat drought result in salinasation, which is estimated to affect 50% of arable land by 2050. Plants have developed several mechanisms that protect them against stress and these include overexpression of stress responsive genes and altered signal transduction to change the expression of stress responsive genes, among others. Cyclic 3’5’ guanosine monophosphate (cGMP), a second messenger that is synthesised by guanylyl cyclase (GC), transmit signals to various cellular functions in plants during plant development, growth and response to abiotic and biotic stresses. Arabidopsis thaliana nitric oxide guanylyl cyclase 1 (AtNOGC1) is a guanylyl cyclase which upon activation by nitric oxide (NO) leads to the production of more cGMP. Cyclic GMP further activates protein kinases, ion gated channels and phosphodiesterase which mediate response to various stresses. In this project the role of AtNOGC1 was investigated in response to abiotic and biotic stresses through analysis of its evolutionary relationships, promoter, gene expression and functional analysis via the viability assays in Escherichia coli (E.coli). Phylogenetic tree, exon-intron structure and conserved motifs were analysed using the Molecular Evolutionary Genetics Analysis (MEGA V.7), Gene Structure Display Server 2.0 (GSDS 2.0), and Multiple Expectation Maximisation for Motif Elicitation (MEME) tools respectively. AtNOGC1’s gene expression was analysed by the Real-Time Quantitative Reverse Transcription Polymerase Reaction (qRT-PCR), whereas functional analysis was carried out using the cell viability (liquid and spot) assays to determine its ability to confer stress tolerance to E. coli.

Current crop and climate modeling studies predict temperature extremes that may add future challenges to global food and agriculture production systems due to yield decreases in our staple cereal crops. Although there have been some temperature stress adaptation traits and a few associated genes discovered in plants for enhanced thermotolerance, very little is known about these traits in our major cereal crops, particularly in maize. Furthermore, the limited availability of appropriate selection environments and accurate phenotyping, including functional traits for selection, have been major hurdles to overcome in making meaningful gains towards improved thermotolerance in breeding programs. Previous reports have established that dynamic changes in leaf membrane lipids occur when exposed to temperature stress and many have tried to identify specific lipid classes, individual species, or levels of unsaturation as indicators of tolerance or susceptibility. In this study, several types of cereals, with special emphasis on maize, are studied with the goal to expand the understanding of the leaf lipid membrane characteristics and responses when exposed to temperature stress and to find evidence of heritable lipid biomarker(s) that could be used in breeding for enhanced thermotolerance. Leaf lipids for maize inbred lines and twelve hybrids exhibiting differential tolerance to high temperature stress were analyzed after growing in a controlled environment at optimal and high temperature stress conditions. It was hypothesized that the newly introduced ratios of monogalactosyldiacylglycerol (MGDG) and digalactosyldiacylglycerol (DGDG) containing acyl chains with total 36 carbons and 6 double bonds (36:6) compared to those with 36 carbons and 5 double bonds (36:5), here termed the MGDG and DGDG “Unsaturation Ratios” can be used to differentiate the changes in the plastidic lipid membrane unsaturation levels and to aid in identifying heat tolerant genotypes. An analysis of the MGDG and DGDG Unsaturation Ratios was performed on twenty-five diverse parents of the nested-association mapping (NAM) population, Mo17, and one hundred and ninety-one B73 x B97 recombinant inbred lines (RILs) grown in field conditions. The selected lipid phenotypes were found to be as diverse as the inbred lines in which they were measured and showed a large differential between the temperate inbred lines B73 and B97. Allelic variation controlling the differences in MGDG and DGDG Unsaturation Ratios was identified in the B73 x B97 RIL subpopulation through linkage mapping analysis. Finally, an analysis of the MGDG and DGDG Unsaturation Ratios was performed across eight of the world’s most important cereal crops. The results of these studies provide preliminary evidence that the MGDG and DGDG Unsaturation Ratios may be beneficial lipid biomarkers that can be used to screen germplasm in breeding programs for improved thermotolerance for our most important cereal crops with the potential to differentiate tolerance in germplasm even without the presence of the ideal selection environment.

<p>Adhezija i formiranje biofilma kvasaca na kontaktne povr&scaron;ine materijala je ozbiljan higijenski problem u prehrambenoj industriji, jer može biti izvor kontaminacije tokom proizvodnje, dovodeći do velikih ekonomskih gubitaka zbog kvarenja hrane i smanjenja kvaliteta proizvoda, a takođe može da &scaron;teti zdravlju potro&scaron;ača. S drge strane, mnoge infekcije u bolnici uzrokovane su adhezijom vrsta roda Candida na medicinska sredstva i implantate. Međutim, trenutno razumevanje adhezije i kolonizacije kvasaca na abiotičke povr&scaron;ine je ograničeno na samo neke vrste roda Candida, a naročito malo se zna o vrstama roda Pichia kao adhezivnim agensima.<br />Cilj istraživanja u okviru ove doktorske disertacije je bio da se proceni potencijal adhezije sojeva kvasaca Candida i Pichia na polistiren povr&scaron;inu, diskove od nerđajućeg čelika (AISI 304, Ra = 25.20 &ndash; 961.9 nm) i drveta sa različitim stepenom hrapavosti povr&scaron;ine, i ispita uticaj hrapavosti povr&scaron;ine na adheziju kvasaca. Hidrofobnost povr&scaron;ine ćelije (CSH) sojeva iz rodova Candida i Pichia je određena u cilju procene korelacije između hidrofobnosti povr&scaron;ine ćelije i adhezije kvasaca na polistiren. Pored toga, ispitan je uticaj različitih medijuma, temperatura, i različitih biljnih ekstrakata na adheziju odabranih sojeva iz rodova Candida i Pichia na povr&scaron;ine diskova od nerđajućeg čelika. Rezultati su pokazali da su svi testirani sojevi kvasaca Candida i Pichia su bili u stanju da adheriraju na polistiren, povr&scaron;ine od nerđajućeg čelika i drveta, ali u različitoj meri. Soj vrste C. albicans je imao veću sposobnost adhezije na polistiren i nerđajući čelik u odnosu na sojeve vrsta C. glabrata, C. parapsilosis i C. krusei. U slučaju kod roda Pichia, sojevi vrste P. membranifaciens su pokazali manju sposobnost adhezije na nerđajući čelik nego ispitivani soj vrste P. pijperi, &scaron;to nije slučaj kod adhezije na polistiren. Na osnovu dobijenih rezultata pokazano je da su sojevi vrsta C. glabrata, C. parapsilosis, C. krusei, P. pijperi i P. membranifaciens bili izrazito do umereno hidrofobni, dok je soj vrste C. albicans bio hidrofilan, &scaron;to ukazuje da hidrofobnost povr&scaron;ine ćelije (CSH) nije značajno u korelaciji sa količinom ćelija adheriranih na polistiren. Pored toga, na adheziju sojeva kvasaca Candida i Pichia značajno je uticala hrapavost povr&scaron;ine nerđajućeg čelika, dok hrapavost drvenih povr&scaron;ina nije značajno uticala. Dalje mi smo ispitali uticaj različitih medijuma za rast i temperature na adheziju odabranih kvasaca. Primećeno je da je količina adheriranih ćelija kvasaca C. albicans i C. glabrata na povr&scaron;inama od nerđajućeg čelika bila znatno veća u Malt Extract Broth (MEB) medijumu, dok je za C. parapsilosis, C. krusei, P. pijperi i P. membranifaciens bila u Yeast Peptone Dextrose (YPD) medijumu. Na&scaron;i podaci takođe ukazuju da je temperatura veoma važan faktor koji značajno utiče na adheziju ovih kvasaca. Problem adhezije i formiranje biofilma kvasaca na abiotičkim povr&scaron;inama i rastuća otpornost na postojeće antimikrobne agense predstavilo je izazov za nas za pronalaženjem novih alternativnih agenasa biljnog porekla. Stoga mi smo ispitali antimikrobnu i antibiofilm aktivnost biljnih ekstrakata kao &scaron;to su Humulus lupulus, Alpinia katsumadai i Evodia rutaecarpa prema C. albicans, C. glabrata i P. membranifaciens. Na osnovu dobijenih MIC vrednosti, testirani biljni ekstrakati su bili efikasni u inhibiciji kvasaca. Primećeno je da su biofilmovi C. glabrata pokazali veću otpornost na biljne ekstrakte u odnosu na C. albicans. Međutim, ekstrakti A. katsumadai i E. rutaecarpa su promovisani rast i razvoj formiranog biofilma P. membranifaciens.</p> / <p>The adhesion and biofilm formation of yeast on the contact surfaces of the material is a serious hygiene problem in the food industry, because it can be a source of contamination during production, leading to major economic losses due to food spoilage and reduced product quality, and can also affects the consumer&rsquo;s health. On the other hand, many infections in the hospital are caused by the adhesion of Candida spp. to medical devices and implants. However, the understanding of adhesion and colonization of yeasts on abiotic surfaces is limited to only some Candida species, and in particular little is known about Pichia species as adhesive agents.<br />The aim of the research in this doctoral dissertation was to assess the potential of Candida and Pichia strains to adhere to polystyrene surface, stainless steel discs (AISI 304, Ra = 25.20 - 961.9 nm) and wood (beech) with different degrees of surface roughness, and examined the influence of surface roughness on yeast adhesion. Cell surface hydrophobicity (CSH) of Candida and Pichia strains was determined in order to assess the correlation between the cell surface hydrophobicity and the yeast adhesion to polystyrene. In addition, the influence of different medium, temperature, and various plant extracts on the adhesion of selected Candida and Pichia strains to to stainless steel surfaces was examined. Our results indicated that all Candida and Pichia strains were able to adhere to polystyrene, stainless steel surfaces and wood in a species- and strain-dependent manner. C. albicans had a higher ability to adhere to polystyrene and stainless steel compared to C. glabrata, C. parapsilosis and C. krusei. Regarding Pichia species, P. membranifaciens strains were less adherent to stainless steel than P. pijperi, what is not the case in the adhesion to polystyrene. Based on the results obtained, C. glabrata, C. parapsilosis, C. krusei, P. pijperi i P. membranifaciens strains were strongly to moderately hydrophobic while only C. albicans was moderately hydrophilic, indicating that the cell surface hydrophobicity is not significantly correlated with the amount of cells adhered to polystyrene. In addition, the adhesion of Candida and Pichia strains was significantly influenced by the roughness of stainless steel surface, while the roughness of wood surfaces did not significantly influence. Further on we examined the impact of different growth medium and temperature on adhesion of selected yeasts. It was noted that the amount of adhered cells of C. albicans and C. glabrata on stainless steel surfaces were significantly higher in the Malt Extract Broth (MEB) medium, whereas for C. parapsilosis, C. krusei, P. pijperi and P. membranifaciens it was Yeast Peptone Dextrose (YPD) medium. Our data also indicated that temperature is a very important factor which considerably affects the adhesion of these yeast. The problem of yeast adhesion and biofilm formation on abiotic surfaces and its growing resistance to existing antimicrobial agents, presented a challenge for us to find new alternative agents of plant origin. Hence, we investigated antimicrobial and antibiofilm activity of plant extracts such as Humulus lupulus, Alpinia katsumadai and Evodia rutaecarpa against C. albicans, C. glabrata and P. membranifaciens. According to the MIC values, all plant extracts were effective in the inhibition of yeast strains. It was observed that biofilms of C. glabrata are more resistance to plant extracts as compared to C. albicans. However, extracts of A. katsumadai and E. rutaecarpa were promoted the growth and development of a preformed biofilm of P. membranifaciens.</p>

Ce travail a porté sur l’étude de l’évolution des communautés zooplanctoniques à partir de séries temporelles de relevés effectués de 1995 à 2010 dans deux écosystèmes côtiers couplés, la Petite Rade de Toulon (PR) et Grande Rade de Toulon (GR) (Méditerranée Nord Occidentale, France) en relation avec les facteurs climatiques, les paramètres physiques et chimiques de l'eau et avec le phytoplancton. Le pas d’échantillonnage des relevés de zooplancton et du phytoplancton était d’un mois en moyenne. La maille du filet utilisé était de 90 µm, afin de cibler le mésozooplancton. La PR a différé de la GR dans son fonctionnement écologique, car elle est semi-fermée, mais aussi parce que l'activité anthropique y était beaucoup plus importante. Nos résultats ont montré que, de 1995 à 2010 dans les deux baies, l'abondance du zooplancton a sensiblement augmenté, surtout dans la PR. Il a été également établi, en utilisant différents outils statistiques, que la plus grande partie des espèces de zooplancton évolue de manière coordonnée chaque année, mais d’une manière différente d’une année à l’autre. C’est ce que nous avons appelé la signature annuelle, qui était très marquée dans la PR. Plusieurs paramètres environnementaux comme la température, l’oxygène, la salinité et l’ensoleillement, qui ont été simultanément enregistrés, expliquent cette signature annuelle. Il a été montré en effet qu’ils influencent très sensiblement la population de zooplancton, de manière instantanée ou avec un effet retardé. Les interactions responsables de cette évolution sont fort complexes, mais il a été aussi établi que ces facteurs sont plus forts lorsqu’ils agissaient de manière coordonnée. La répartition du zooplancton en groupes taxonomiques a montré que la diversité a augmenté jusqu’en 2005, puis a diminué légèrement, tout en restant à des niveaux plus élevés qu’en 1995. L’étude détaillée de la diversité, avec une classification des indices eux-mêmes, a fait l’objet du dernier chapitre. Enfin, nous émettons l’hypothèse que la diminution des stocks de poissons au cours des dernières décennies dans toute la région a entraîné une diminution des taux de prédation sur les communautés zooplanctoniques, ce qui peut expliquer l’augmentation de peuplements zooplanctoniques au cours de ces dernières années. Cet accroissement de l’abondance du zooplancton a pu entraîner à son tour une diminution de la biomasse du phytoplancton. Cette diminution a été parallèlement observée par notre équipe. Ceci suggère un système de contrôle top-down du réseau trophique. / This work focused the study of the evolution of zooplankton communities from time series of surveys conducted from 1995 to 2010 in two coastal coupled ecosystems, Little Bay (PR) and Large Bay (GR) of Toulon (North Western Mediterranean Sea, France) in relation to climatic factors, physical and chemical water parameters and phytoplankton. The samplings surveys of zooplankton, and indeed also of phytoplankton, were a month, on average. The net mesh size used was 90 µm to target Mesozooplankton. The PR differed from the GR in its ecological functioning, because it is semi-closed, but also because human activity is much more important. Our results showed that, from 1995 to 2010 in both bays, zooplankton abundance increased substantially, especially in the PR. It was also established, using statistical tools, that most zooplankton species evolved coordinated each year, but in a different way from one year to another. This is what we call the annual signature, which was more pronounced in the PR. Several environmental parameters such as temperature, oxygen, salinity and sunlight, which were simultaneously recorded, explained this annual signature. It was shown that they significantly influenced the population of zooplankton, instantly or with a delayed effect. Interactions responsible for this development are complex, but it was also established that these factors were stronger when they acted in a coordinated manner. Distribution of zooplankton taxonomic groups showed diversity increases until 2005 and then decreased slightly, while remaining at levels higher than in 1995. The detailed study of diversity, with a classification of the clues themselves was the subject of the last chapter. Finally, we hypothesize that the decline of fish stocks in recent decades throughout the region, resulting in lower rate of predation on zooplankton communities, may explain the increase of zooplankton communities in recent years. This increase in zooplankton abundance could in turn lead to a decrease in phytoplankton biomass. The decrease of phytoplankton was at the same time observed by our team. The latter hypothesis suggests a top-down control of the the food web

Glutamine (Gln), a major nitrogen source in plants, is considered a central intermediate that coordinates carbon-nitrogen assembly for plant growth and development. To maintain a sufficient Gln supply, plant cells employ glutamine synthetases (GS), including cytosolic GS1 and plastidic GS2 for Gln production. Previous work has shown that the <i>GS1</i> is responsive to various environmental stresses. This study demonstrated the involvement of <i>GS1</i>s in Gln homeostasis and the role of GS1 in abiotic stress tolerance in <i>Arabidopsis</i>. The <i>GS1</i> family is comprised of five isoforms in <i>Arabidopsis thaliana</i>. Gene expression profiling showed that <i>GLN1;1, GLN1;3</i> and <i>GLN1;4</i> had similar expression patterns and were upregulated by abiotic (salinity and cold) stresses, whereas <i>GLN1;2</i> exhibited constitutive expression and no <i>GLN1;5</i> transcript was detected under any of the conditions tested. Null T-DNA insertion mutants for the five <i>GS1</i> genes were obtained. Only the <i>gln1;1</i> mutant displayed enhanced sensitivity to a GS inhibitor, phosphinothricin, and to cold and salinity treatments, suggesting a nonredundant role for GLN1;1. Increased stress sensitivity in <i>gln1;1</i> was associated with accelerated accumulation of reactive oxygen species (ROS), particularly in chloroplasts. To better understand the role of cytosolic GS isoforms, we generated two different triple mutant combinations. Triple mutant <i>gln1;1/gln1;2/gln1;3</i> showed reduced growth at an early stage. The <i>gln1;1/gln1;3/gln1;4</i> mutant is pollen lethal, indicating an essential role of Gln in plant gametophyte development. Collectively, our results establish a link between cytosolic Gln production, ROS accumulation, plant stress tolerance and development.

This project was expected to contribute to the understanding of abscisic acid (ABA) perception in plants through identification of new ABA-binding proteins. The novel, biotinylated ABA derivative PBI686 (of biological activity comparable to natural ABA) has served as an affinity probe for isolation of ABA-binding proteins. Photoaffinity labeling in conjunction with affinity chromatography (streptavidin-biotin based) was used for specific identification of target proteins from complex mixtures of cytosolic and membrane-bound proteins. Proteins of interest were identified by Mass Spectrometry through peptide mass fingerprinting and MS/MS ion search.<p> Ribulose bisphosphate carboxylase/oxygenase (Rubisco) was identified as an ABA binding partner, and its interaction with ABA was initially confirmed by its ability to block the photoaffinity labeling reaction with PBI686. In addition, Surface Plasmon Resonance (SPR) experiments with ABA and Rubisco were performed, which provided further evidence for selective interaction between the two binding partners, with a very small preference towards (+)-ABA over (-)-ABA. SPR has also yielded the value of equilibrium dissociation constant (KD) being 5 nM for (+)-ABA and 7 nM for (-)-ABA. This was further confirmed by [3H] (±)-ABA binding assays, which have also shown that non-radiolabeled (+)-ABA and (-)-ABA (at concentration 1000 fold higher) were able to displace [3H] (±)-ABA from binding to Rubisco. Compounds other than ABA such as PA (phaseic acid) or trans-(+)-ABA were not able to displace [3H] (±)-ABA, which has suggested the selectivity of binding. Further, Rubisco enzymatic activity in the absence of ABA was compared to that in the presence of ABA at various concentrations. The results have clearly indicated the effect of ABA on Rubiscos enzymatic activity. This was reflected on the enzymes Km values being increased by seven fold in the presence of 10 mM ABA and 1 mM substrate (RuBP). The interpretation of changes in enzyme kinetics upon inhibition by ABA most resembles allosteric inhibition. The biological function of this newly discovered interaction is interpreted as ABAs ability to regulate plant growth during abiotic stress by its direct action on the photosynthetic machinery - hypothesis often suggested in the literature.

Glutamine (Gln), a major nitrogen source in plants, is considered a central intermediate that coordinates carbon-nitrogen assembly for plant growth and development. To maintain a sufficient Gln supply, plant cells employ glutamine synthetases (GS), including cytosolic GS1 and plastidic GS2 for Gln production. Previous work has shown that the <i>GS1</i> is responsive to various environmental stresses. This study demonstrated the involvement of <i>GS1</i>s in Gln homeostasis and the role of GS1 in abiotic stress tolerance in <i>Arabidopsis</i>. The <i>GS1</i> family is comprised of five isoforms in <i>Arabidopsis thaliana</i>. Gene expression profiling showed that <i>GLN1;1, GLN1;3</i> and <i>GLN1;4</i> had similar expression patterns and were upregulated by abiotic (salinity and cold) stresses, whereas <i>GLN1;2</i> exhibited constitutive expression and no <i>GLN1;5</i> transcript was detected under any of the conditions tested. Null T-DNA insertion mutants for the five <i>GS1</i> genes were obtained. Only the <i>gln1;1</i> mutant displayed enhanced sensitivity to a GS inhibitor, phosphinothricin, and to cold and salinity treatments, suggesting a nonredundant role for GLN1;1. Increased stress sensitivity in <i>gln1;1</i> was associated with accelerated accumulation of reactive oxygen species (ROS), particularly in chloroplasts. To better understand the role of cytosolic GS isoforms, we generated two different triple mutant combinations. Triple mutant <i>gln1;1/gln1;2/gln1;3</i> showed reduced growth at an early stage. The <i>gln1;1/gln1;3/gln1;4</i> mutant is pollen lethal, indicating an essential role of Gln in plant gametophyte development. Collectively, our results establish a link between cytosolic Gln production, ROS accumulation, plant stress tolerance and development.