Telomeres are the ends of linear chromosomes tasked with preventing their recognition by the DNA damage machinery and providing a mechanism to solve the end replication problem. The telomeric DNA is mostly double-stranded, but it terminates in a 3’ protrusion termed the G-overhang. Telomeres utilize telomerase, a reverse transcriptase, to elongate the telomere, and thus, solve the end replication problem. Both the double strand region and the G-overhang are bound by specific proteins to facilitate the objectives of the telomere. First discovered in budding yeast, the CST (Cdc13(CTC1)/Stn1/Ten1) complex binds to the G-overhang and is important for both chromosome end protection and telomere replication. Work reported in this dissertation provided the first evidence that CST was present outside of yeast, which led to its subsequent identification in a number of vertebrates. Here I present the identification and characterization of the three components of CST in Arabidopsis thaliana. Similar to yeast, Arabidopsis CST is required for telomere length maintenance, for preventing telomere recombination and chromosome end-to-end fusions. Mutations in the CST complex result in severe genomic instability and stem cells defects. My research also shows that CST and telomerase act synergistically to maintain telomere length. Together these data provide evidence for an essential role for CST in maintaining telomere integrity. Unexpectedly, I discovered that the TEN1 component of CST may have a more complex role than other members of the heterotrimer. The majority of telomere-related functions we can assay using molecular and cytological approaches are shared by CTC1, STN1 and TEN1, though TEN1 has additional roles in maintaining genome stability, modulating telomerase activity and possibly non-telomeric functions in the chloroplast. I also present genetic evidence that TEN1 and STN1 act in the same pathway for the maintenance of telomere length and chromosome end protection. Interestingly, however, disrupting the STN1/TEN1 interaction reveals a separation of STN1 function for chromosome end protection versus telomere length maintenance. Finally, I describe the design and creation of a library of STN1 and TEN1 mutants that will be used to further characterize their functions and their interaction partners. By disrupting such interactions, it will be possible to elucidate the functional significance of these interactions, and thus, provide new insight into how CST functions in Arabidopsis.

Acid phosphatases (APases; E.C. 3.1.3.2) catalyze the hydrolysis of phosphate (Pi) from Pi monoesters and anhydrides within the acidic pH range. Induction of intracellular and secreted purple acid phosphatases (PAPs) is a widespread plant response to nutritional Pi-deficiency. The probable function of intracellular APases is to recycle Pi from expendable intracellular organophosphate pools, whereas secreted APases likely scavenge Pi from the organically bound Pi that is prevalent in most soils. Although the catalytic function and regulation of plant PAPs have been described, their physiological function in plants has not been fully established. Recent biochemical and proteomic studies indicated that AtPAP26 is the predominant intracellular (vacuolar) and a major secreted purple APase isozyme upregulated by Pi-starved (-Pi) Arabidopsis thaliana. The in planta function of AtPAP26 was assessed by molecular, biochemical, and phenotypic characterization of a homozygous Salk T-DNA insertion mutant. Loss of AtPAP26 expression resulted in the elimination of AtPAP26 transcripts and 55-kDa immunoreactive AtPAP26 polypeptides, correlated with a 9- and 5-fold decrease in extractable shoot and root APase activity, respectively, as well as a 40% reduction in secreted APase activity of –Pi seedlings. The results corroborate previous findings implying that AtPAP26 is: (i) the principal contributor to Pi starvation inducible APase activity in Arabidopsis, and (ii) controlled post-transcriptionally mainly at the level of protein accumulation. Total shoot free Pi level was about 40% lower in –Pi atpap26 mutants relative to wild-type controls, but unaffected under Pi-sufficient conditions. Moreover, shoot, root, inflorescence, and silique development of the atpap26 mutant was impaired during Pi deprivation, but unaffected under Pi-replete conditions, or during nitrogen or potassium-limited growth, or oxidative stress. The results suggest that the hydrolysis of Pi from organic-phosphate esters by AtPAP26 makes an important contribution to Pi-recycling and scavenging in –Pi Arabidopsis. / Thesis (Master, Biology) -- Queen's University, 2009-09-01 09:46:39.302

No description available.

No description available.

No description available.

Changes in cellular redox status are implicated in the regulation of developmental and defence-related responses. The absence of S-nitrosoglutathione reductase (GSNOR) function in Arabidopsis leads to an accumulation of cellular S-nitrosoglutathione (GSNO), a mobile reservoir of nitric oxide (NO) which impacts the cellular redox tone. Consequently, the GSNOR knockout mutant, atgsnor1-3 displays defects in growth, time to flowering and pathogen resistance. Although it is now well established that GSNO is a key redox signalling molecule, the molecular mechanisms that underpin GSNO function remains largely unknown. RAP1 (REDOX-ASSOCIATED PROTEIN 1) was identified based on its dynamic changes of expression in atgsnor1-3 and sid2 plants upon avirulent Pseudomonas syringae pv. tomato (Pst) DC3000 (avrB) challenge. Pathogen-induced RAP1 expression was shown to be independent of the plant hormones salicylic acid, jasmonic acid, abscisic acid and ethylene. Recombinant RAP1 protein was shown to exhibit E3 ligase activity in vitro. Application of the NO donors (GSNO and Cysteine-NO (CysNO)) reduced the E3 ligase activity of RAP1 significantly. Biotinswitch analysis showed that RAP1 was S-nitrosylated and site-directed mutagenesis of RAP1 suggested that the S-nitrosylated site is the cysteine residue C325. The rap1 line does not show obvious developmental phenotypes, however, overexpressing RAP1 enhanced lateral root branching in young seedlings. Overexpression of a truncated RAP1 (RAP1ΔRING) led to a loss of apical dominance. In addition, rap1/rap2 double mutants showed delayed flowering, suggesting RAP1 might be involved in the regulation of plant growth and development. RAP1 may also be involved in plant defence, as rap1, rap2 and rap1/rap2 mutants exhibited increased susceptibility to PstDC3000 and Arabidopsis powdery mildew. Interestingly, rap1 plants showed enhanced resistance to methyl viologen (MV), which is in line with the phenotype of atgsnor mutants. Also, expression of RAP1 was rapidly inducible by ultraviolet-B (UV-B) light. As RAP1 expression and RAP1 E3 ligase activity are redox-related, it is speculated that RAP1 may be involved in redoxmediated regulation of a broad range of physiological responses.

Photosynthetic eukaryots emerged as a result of several billion years of evolution between proeukaryotic cell and ancestral cyanobacteria that formed modern chloroplasts. The symbiotic relationship led to significant rearrangements in the genomes of the plastid and the nucleus: as many as 90 % of all the plastid genes were transferred to the nucleus. The gene transfer has been accompanied by the development of sophisticated regulatory signaling networks originating in the organelle (retrograde) and in the nucleus (anterograde) that coordinate development of the plastid and ensure adequate cell responses to stress signals. In this thesis I have demonstrated that transcriptional activity of PEP in the chloroplast is essential for proper embryo and seedling development in Arabidopsis thaliana. The function of PEP is dependent on the nuclear encoded PEPassociated factor PRIN2 that is able to sense the redox status of the plastid during seedling development and different stress. In response to the plastid status PRIN2 modulates the transcription activity of the PEP enzyme complex. We further established that PRIN2, as an essential component for full PEP activity, is also required to emit the Plastid Gene Expression (PGE) retrograde signal to regulate the Photosynthesis-Associated Nuclear Genes (PhANG) in the nucleus during early seedling growth via GUN1. On the other hand, regulation of PhANG expression during the High Light (HL) conditions requires functional PRIN2 and PEP activity but is GUN1-independent. Another retrograde signal produced by the developing chloroplast is associated with the tetrapyrrole biosynthesis pathway. We have established that accumulation of the chlorophyll intermediate MgProtoIX-ME in the crd mutant triggers repression of the PhANG expression, and this negative signal is mediated by a cytoplasmic protein complex containing the PAPP5 phosphatase. The nuclear targets that receive the tetrapyrrole mediated signal are GLK1 and GLK2 transcription factors that control the PhANG expression and the expression of the enzymes involved in the biosynthesis of chlorophyll. / Fotosyntetiserande eukaryoter uppstod från en endosymbiotisk interaktion under några miljarder år mellan en ur-eukaryot och kloroplastens förfader, den prokaryota cyanobakterien. Den symbiotiska händelsen ledde till att kloroplastens och kärnans genom blev väsentligt förändrade. Så småningom överförde kloroplasten så många som 90 % av dess gener till cellkärnan. För att koordinera genutrycket från de två genomen utvecklade växtcellen ett sofistikerat signalsystemen som inkluderar: plastid-kärn (retrograd) och kärn-plastid (anterograd) signalering som styr kloroplastens utveckling och förmåga att anpassa sig till stressförhållanden. Den här avhandlingen beskriver kloroplastens maskineri för genuttryck (PEP) som en nödvändig komponent för embryo- och växtutvecklingen hos Arabidopsis thaliana. PEP funktionen är beroende av det kärnkodade kloroplastproteinet PRIN2 som är associerat med PEP. PRIN2 mottar redox signaler från plastiden och förändrar genuttrycksaktivitet under kloroplastens utvecklingen eller under olika stressförhållanden. Jag visar dessutom att PRIN2 spelar en viktig roll i överföring av kloroplastens signal som kommunicerar genuttrycksaktivitet (PGE) via GUN1 till kärnan där den styr uttryck av de kärnkodade fotosyntetesgenerna (PhANG). Under högljus stressförhållanden styrs dock PhANG-uttrycket av signaler som uppstår från PEP-aktivitet och PRIN2 men som är oberoende av GUN1. Vidare finns det en annan retrograd signal som har sitt ursprung i biosyntesen av tetrapyrroler. Jag har visat att ackumuleringen av tetrapyrrolen MgProtoIX-ME i crd-mutanten framkallar nedreglering av PhANG-uttryck genom interaktion med ett fosfatas (PAPP5) i cytosolen. GLK1 and GLK2 är två transkriptionsfaktorer som tar emot den tetrapyrrole-medierade signalen i sin tur styr biosyntes av chlorofyll och PhANG uttryck.

Leaf senescence is the final stage of leaf development, during which the leaf relocates most of itsvaluable nutrients to developing or storing parts of the plant. As this process progresses, leaves losetheir green color and their capacity to perform photosynthesis. Shade and darkness are well-knownas factors inducing leaf senescence and it has been proposed that senescence can be initiated byreductions in photosynthesis, photomorphogenesis and transpiration. However, despite the fact thatthe signaling mechanisms regulating each of these processes have been extensively described,particularly in seedlings, their contribution to the initiation of senescence in mature leaves stillremains unclear. Furthermore, the use of different experimental systems to study shade-inducedleaf senescence has yielded several divergent results, which altogether complicate the overallunderstanding of leaf senescence. To address this, darkened plants and individually darkened leaves, which show different rates of leafsenescence, were studied. Comparing the transcriptome and metabolome of these two darktreatmentsrevealed that they differed distinctly with regard to their metabolic strategies. Wholedarkened plants were severely carbohydrate-starved, accumulated amino acids and slowed downtheir metabolism. In contrast, individually darkened leaves showed continued active metabolismcoupled to senescence-associated degradation and relocation of amino acids. This knowledge was used to set up a new system to study how shade affects leaf senescence in themodel plant Arabidopsis thaliana. Use of this system revealed that different senescence-associatedhallmarks appeared in response to different intensities of shade. Some of these hallmarks werefurther shown to be part of both leaf senescence and photosynthetic acclimation to low light. Finally, using this system on phytochrome mutants revealed that loss of phytochrome A increasedthe loss of chlorophyll under shade, without increasing the expression of senescence-associatedgenes. Together, these findings suggest that shade-induced leaf senescence, which is generally perceived asa single process, is actually an intricate network of different processes that work together tomaintain an optimal distribution of nutrients within the plant.

When light energy input exceeds the capacity for photosynthesis the plant need to dissipate the excess energy and this is done through non-photo-chemical quenching (NPQ). Photochemical quenching (photosynthesis), NPQ and fluorescence are three alternative faiths of excited chlorophylls. PsbS associates to photosystem II and is involved in NPQ. The results presented in this thesis were generated on Arabidopsis plants and mainly based on wildtype Col-0 together with a mutant deficient in PsbS (npq4) and a transgene overexpressing PsbS (oePsbS). We connect light and herbivore stress and show that the level of PsbS influences the food preference of both a specialist (Plutella) and a generalist (Spodoptera) herbivore as well as oviposition of Plutella. Level of PsbS also affects both metabolomics and transcriptomics of the plant; up-regulation of genes in the jasmonic acid (JA) -pathway and amount of JA has been found in the npq4 plants after herbivory. Since many experiments were performed in field we have also characterized the field plant and how it differs from the commonly used lab plant. We have also studied the natural variation of NPQ in Arabidopsis plants both in the field and the lab. The results show surprisingly no correlation. / Överskottsenergi kan vara skadligt för en växts membran och fotosynteskomplex. Vid överskott av solenergi blir fotosystemen mättade och växten behöver därför ett sätt för att göra sig av med all överskottsenergi, detta kallas för ”icke-fotokemisk quenching” (NPQ). Fotokemisk quenching (fotosyntes), NPQ och fluoresens är tre alternativa vägar för exalterade klorofyller. PsbS är involverad i NPQ och associerar med fotosystem II. De resultat som presenteras i denna avhandling kommer från studier av modellväxten Arabidopsis thaliana (Backtrav), i huvudsak gjorda på vildtypen i jämförelse med en mutant som saknar PsbS (npq4) och en transgen som överuttrycker PsbS (oePsbS). Vi har försökt att undersöka kopplingen mellan ljus- och herbivoristress och visar här att mängden PsbS påverkar både en specialist (Plutella) och en generalist (Spodoptera) insekt vid val av föda, samt Plutella även vid äggläggning. Växternas nivå av PsbS visade sig även påverka metabolomet och transkriptomet, och vi fann en uppreglering av gener i biosyntesen för jasmonat samt mer av själva hormonet jasmonat i npq4 växter efter herbivori. Eftersom vi har gjort många av experimenten ute i fält har vi även karakteriserat en typisk Arabidopsis växt i fält samt hur denna skiljer sig från den vanligt använda lab-växten. Dessutom har vi även undersökt naturlig variation av NPQ av Arabidopsis både i fält och på lab och resultaten visar, till vår förvåning, att det inte går att finna någon korrelation mellan dessa.

Programmed Cell Death (PCD) describes an orderly cellular breakdown that occurs in both plants and animals throughout development and in response to biotic and abiotic stresses. The molecular machinery that functions in the induction and execution of animal PCD has been characterised in great detail. Conversely, few genes and proteins involved in plant PCD have been identified. While certain features of animal PCD may be conserved, the induction and execution of plant PCD is also likely to involve novel proteins and mechanisms. The aim of the work presented in this thesis was to investigate experimental approaches for studying plant PCD and to gain an understanding of the molecular mechanisms involved. To this end, an Arabidopsis thaliana cell suspension system was developed in which PCD could be induced by both a heat treatment (55°C, 10 min) and senescence (13 to 14 days-old). This system allowed for the molecular responses related to programmed cell death to be distinguished from those that were a specific response to the inducing stimulus. The Arabidopsis cell suspension system was utilised for an analysis of transcriptomic and proteomic changes that occur following the induction of PCD. A custom cDNA microarray analysis of ~100 putative cell death-related genes was used to measure the abundance of transcripts of these genes during PCD, and this work was extended to a whole-genome transcriptomic analysis of PCD. A number of candidate genes that may play a role in plant PCD were identified. These included those encoding antioxidant enzymes, cytosolic heat shock proteins, the mitochondrial adenine nucleotide translocase, ion transporters, a two-component response regulator (ARR4), several pathogenesis-related proteins, phospholipases and proteases, extracellular glycoproteins and enzymes (including a subtilisin-like protease, chitinases, and glucanases), and transcriptional regulators such as a homeobox leucine zipper and NAC-domain proteins. The induction and execution of plant PCD is also likely to involve mechanisms that are not transcriptionally regulated. A proteomic analysis of changes in the total cellular protein profile during heat- and senescence-induced PCD was therefore used to identify 12 proteins that are modulated in both systems and may play a PCD-specific role. These included the mitochondrial voltage-dependent anion channel (Athsr2), catalase, mitochondrial superoxide dismutase, an extracellular glycoprotein, and aconitase. Selected genes and proteins identified in the transcriptomic and proteomic analyses were further investigated in an attempt to define their role in plant PCD. Since PCD is difficult to quantitatively analyse at the whole-plant level, initially a strategy of transient expression of genes of interest in Arabidopsis protoplasts was adopted. However, it proved to be technically difficult to accurately quantify the number of dead cells in this system. As an alternative, Arabidopsis T-DNA insertional mutants within genes of interest were investigated for PCD-related phenotypes. Mutants in Senescence-Related Gene 3, the mitochondrial voltage-dependent anion channel (Athsr2), and cytosolic Heat shock protein 70-3 were isolated. The mutant lines were not visibly affected in their development, formation of xylem, onset and progression of senescence, or responses to abiotic and biotic stresses. This indicated that these genes are either not involved in the PCD pathway or that their functional role can be fulfilled by other gene products.