Environmental signals such as photoperiod and temperature provide plants with seasonal information, allowing them to time flowering to occur in favourable conditions. Most ecotypes of the model plant Arabidopsis thaliana flower earlier in long photoperiods and after prolonged exposure to cold (vernalization). The vernalized state is stable through mitosis, but is not transmitted to progeny, suggesting that the vernalization signal may be transmitted via a modification of DNA such as cytosine methylation. The role of methylation in the vernalization response is investigated in this thesis. ¶ Arabidopsis plants transformed with an antisense construct to the cytosine methyltransferase METI (AMT) showed significant decreases in methylation. AMT plants flowered significantly earlier than unvernalized wildtype plants, and the promotion of flowering correlated with the extent of demethylation. The flowering time of mutants with decreased DNA methylation (ddm1) was promoted only in growth conditions in which wildtype plants showed a vernalization response, suggesting that the early flowering response to demethylation operated specifically through the vernalization pathway. ¶ The AMT construct was crossed into two late flowering mutants that differed in vernalization responsiveness. Demethylation promoted flowering of the vernalization responsive mutant fca, but not of the fe mutant, which has only a slight vernalization response. This supports the hypothesis that demethylation is a step in the vernalization pathway. ¶ The role of gibberellic acid (GA) in the early flowering response to demethylation was investigated by observing the effect of the gai mutation, which disrupts the GA signal transduction pathway, on flowering time in plants with demethylated DNA. The presence of a single gai allele delayed flowering, suggesting that the early flowering response to demethylation requires a functional GA signal transduction pathway, and that demethylation increases GA levels or responses, directly or indirectly. ¶ In most transgenic lines, AMT-mediated demethylation did not fully substitute for vernalization. This indicates that part of the response is not affected by METI-mediated methylation, and may involve a second methyltransferase or a factor other than methylation. A second Arabidopsis methyltransferase, METIIa, was characterized and compared to METI. The two genes are very similar throughout the coding region, and share the location of their eleven introns, indicating that they diverged relatively recently. Both are transcribed in all tissues and at all developmental stages assayed, but the level of expression of METI is significantly higher than that of METIIa. The possible functions of METI, METIIa, and other Arabidopsis cytosine methyltransferase genes recently identified are discussed.

This dissertation describes the cloning and characterization of the TOUSLEDlike kinases genes of maize (ZmTLKs). The TOUSLED-like kinases (TLKs) are a conserved family of nuclear Ser/Thr kinases in higher eukaryotes. The maize genome has three TOUSLED-like kinase genes (ZmTLK1, ZmTLK2, and ZmTLK3). Based upon sequence similarity, the ZmTLKs are divided into two classes, the ZmTLK1 and the ZmTLK2/3 class. The origins of these genes can be inferred from their map positions and relationships with TLKs in other Zea species. The ZmTLK1 and ZmTLK2 genes occupy syntenous positions on chromosome arms 1L and 5S in the maize genome. There are two equivalent classes of TLK genes in other Zea species, altogether indicating that the two ZmTLK classes are orthologous genes from the precursor species of maize, an ancient allotetraploid. Gene expression studies of ZmTLKs show that there is a higher level of expression in tissues undergoing DNA synthesis. This is consistent with studies of TLKs in animal systems that show involvement in chromatin assembly/remodeling activities during DNA replication and repair, as well as in transcription. The highest level of gene expression for the ZmTLK2/3 class was observed during development of the endosperm, in a period of massive nuclear endoreduplication. ZmTLK1 is not upregulated in endoreduplicating endosperm, suggesting functional divergence between the two classes of ZmTLK genes. The function of the ZmTLKs was examined by testing whether maize TLK genes could complement the tousled mutant of Arabidopsis. In Arabidopsis thaliana, recessive mutations in the single copy TOUSLED (TSL) gene cause moderate vegetative and severe floral defects, suggesting that TLKs may play a role in gene expression modulation through chromatin remodeling. The ZmTLK proteins are 84% identical to TSL in the catalytic region and 45 - 49% at the N-terminal regulatory domain. However, structural features of the N-terminal region domains of the ZmTLKs are similar to that of TSL. Arabidopsis tsl-1 mutant plants were transformed with ZmTLK2, under the control of the CaMV 35S promoter. These plants showed wild-type Arabidopsis phenotype, indicating that in spite of their sequence differences, ZmTLK2 and TSL interact with the same substrates and regulatory partners and are functionally equivalent. / Graduation date: 2005

Graduation date: 2001

Thesis (Ph. D.)--Ohio State University, 2006. / Title from first page of PDF file. Includes bibliographical references (p. 153-163).

In transgenic research the precise control of transgene expression is crucial in order to obtain transformed organisms with expected desirable traits. A broad range of transgenic plants use the constitutive cauliflower mosaic virus (CaMV) 35S promoter to drive expression of selectable marker genes. Due to its strong enhancer function, this promoter can disturb the specificity of nearby eukaryotic promoters. When inserted immediately downstream of the 35S promoter in transformation vectors, special DNA sequences called insulators can prevent the influence of the CaMV35S promoter/enhancer on adjacent tissue-specific promoters for the transgene. Insulators occur naturally in organisms such as yeasts and animals but few insulators have been found in plants. Therefore, the goal of this study is to identify DNA sequences with insulator activity in Arabidopsis thaliana. A random oligonucleotide library was designed as an initial step to obtain potential insulators capable of blocking enhancer-promoter interactions in transgenic plants. Fragments from this library with insulator activity were identified and re-cloned into pB31, in order to confirm their activity. To date, one insulator sequence (CLO I-3) has been identified as likely possessing enhancer-blocking activity. Also, two other oligonucleotide sequences (CLO II-10 and CLO III-78) may possess insulator activity but more sampling is needed to confirm their activity. Further studies are needed to validate the function of plant insulator(s) and characterize their associated proteins.

The secretory pathway of plant cells consists of several organelles that are connected by vesicle and tubular transport. Every compartment has a distinct function and the specificity of vesicle fusion is essential to maintain the organelles identity. N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) play a crucial role in the secretory pathway driving specific vesicle fusions. A vesicle SNARE (v-SNARE) on a vesicle specifically interacts with two or three target SNAREs (t-SNAREs) on the target compartment. This event leads to vesicle membrane fusion with the membrane of the target compartment and the release of cargo molecules into the organelle lumen.<p> The aim of this work was the characterization of two <i>Arabidopsis thaliana</i> SNAREs. The first one is a v-SNARE, Bet11 that is the Arabidopsis ortholog of the yeast and mammal ER-Golgi v-SNARE, Bet1. In these organisms, Bet1 is involved in trafficking between the ER and Golgi apparatus. The second protein studied is a putative SNARE called Bet12 that shares high sequence identity with Bet11. In particular, I was interested in studying the sorting of these two proteins and their role in the secretory pathway of plant cells. By confocal laser microscopy, I demonstrated that these two proteins have different intracellular localization: Bet11 was mainly localized on the ER, Golgi stacks and punctate structures that I have identified as endosomes. Bet12 was localized only on the Golgi stacks. The identification of signal(s) involved in targeting of Bet11 and Bet12 were studied. To reach this aim I generated different mutant chimeras of Bet11 and Bet12. The co-expression of these chimeras with specific protein markers suggested that the distribution of these proteins was the result of a combined influence of multiple domains.<p> A serine in the Bet11 sequence was identified as a putative phosphorylation site and appeared important for proper Bet11 intracellular distribution.<p> The different intracellular distributions of Bet11 and Bet12 suggest different biological roles for the two proteins. To functionally characterize these two proteins homozygous knock-down mutants of Bet11 were screened. These plants had no evident phenotype, suggesting a possible genetic redundancy in this SNARE family.

La tesi doctoral s'emmarca dins de l'àrea de la biologia molecular de plantes, incloent algunes consideracions de tipus fisiològic, i descriu la identificació i caracterització de 5 gens Arabidopsis que codifiquen proteïnes amb similitud estructural amb els transportadors de coure eucariotes de la família Ctr. Els resultats del treball s'han dividit en tres capítols. En el primer d'ells s'analitza a nivell teòric les seqüències dels polipèptids de la família COPT d'Arabidopsis, s'investiga la seua funcionalitat en un sistema d'expressió heteròleg i es caracteritza de forma global el perfil d'expressió dels gens de la família. En aquest apartat es mostra que COPT1 i COPT2 restableixen amb gran eficiència el creixement en medi de selecció d'una soca de llevat deficient en el transport de coure d'alta afinitat i s'estableix que l'expressió dels dos transportadors es reprimeix en resposta a un tractament amb dosis tòxiques de coure. Per un altre costat, COPT3 i COPT5 complementen amb baixa eficiència al mutant de llevat i no responen al tractament amb coure. Les conclusions d'aquest primer apartat permeten proposar un model sobre la localització i funció cel·lular dels transportadors de la família. Els altres dos capítols de la tesi es centren específicament en l'estudi del gen COPT1. En el segon capítol, es descriu l'obtenció de plantes transgèniques que expressen un gen quimèric format per la fusió del promotor de COPT1 al gen GUS. L'anàlisi histoquímic d'aquestes plantes permet dilucidar que el gen COPT1 s'expressa en embrions i plàntules d'Arabidopsis. A més, en plantes adultes, l'expressió de COPT1 es restringeix als àpex radiculars, als tricomes, a les cèl·lules guarda i al pol·len. En l'últim capítol de la tesi s'exposa l'anàlisi fenotípic de plantes transgèniques que sobreexpressen i silencien COPT1, focalitzant l'estudi en els teixits descrits en el capítol anterior. Així, es mostra que la sobreexpressió de COPT1 causa hipersensibilitat al coure i l'activació dels sistemes de segrest cel·lulars, representats per la metal·lotioneïna MT1a. D'altra banda, el silenciament postranscripcional de COPT1 causa una reducció en l'absorció de coure, hipersensibilitat al dèficit de coure, un allargament de l'arrel primari i irregularitats en la formació de la coberta d'exina. Globalment, els resultats de la tesi descriuen per primera vegada l'expressió d'un transportador de coure als arrels d'una planta superior i assenyalen la participació de COPT1 en l'absorció de coure i en altres processos fisiològics i del desenvolupament vegetatiu i reproductiu d'Arabidopsis thaliana. / The work presented in this thesis describes the identification and characterization of 5 genes (COPT1-5) encoding putative Ctr-like Arabidopsis copper transporters. The first chapter encompasses a theoretical comparison of the COPT protein sequences, a functional study in a heterologous system and a preliminary expression and copper-regulation pattern analysis. COPT1 and COPT2 restore with high efficiency the growth of a yeast strain deficient in high affinity copper uptake and both genes are downregulated by exposure of leaves to copper treatment. COPT3 and COPT5 poorly substitute for their yeast counterparts and do not respond to copper treatment. Based on these results, a model for the cellular function of the COPT family members is proposed. The second chapter describes the generation and analysis of transgenic plants expressing a GUS reporter gene under the control of the COPT1 promoter. GUS specific staining is specifically detected in embryos, cotyledons, root tips, tricomes, guard cells and pollen grains. The last chapter describes the generation and phenotypical analysis of Arabidopsis transgenic plants overexpressing and silencing the COPT1 gene. Overexpression of COPT1 causes enhanced copper sensitivity and constitutive activation of the metallothionein gene MT1a. Overexpressing the complementary sequence of COPT1 produces a reduction in copper absorption, enhanced sensitivity to copper deficiency, increased root length and abnormalities in the pollen exine layer. Overall, the results presented here describe for the first time the expression of a copper transporter in the roots of a higher plant and indicate the participation of COPT1 in copper uptake and other physiological and developmental processes of Arabidopsis thaliana

In sexually reproducing individuals, intraorganismal genetic heterogeneity (IGH) or mosaicism is thought to occur infrequently while genetic homogeneity is presumed the norm. In organisms that undergo modular development, such as long-lived plants, IGH has been substantially documented. In Arabidopsis thaliana we have shown that non-parental DNA that is inherited at low but detectable rates can also manifest on single plants as genotypically distinct somatic sectors suggesting that even short-lived annual plants show IGH. The underlying mechanism responsible for generating this type of IGH remains unknown. In order to better understand this phenomenon I have tested the hypothesis that among genome changes that occur in response to stress, these putative triggers also up-regulate IGH. Metabolic stress, cold stress, mechanical damage and ROS exposure were examined. To test for IGH, transgene markers and polymorphic molecular markers were used. Also, presented in this thesis is work investigating the effect of in vitro propagation through tissue culture on IGH frequencies. Regenerated plants as well as undifferentiated callus tissue were genotyped and assayed for sequence reversions. Molecular genotyping revealed an outcome contrary to that predicted by the initial hypothesis showing instead that a high frequency of restoration occurred in the progeny of un-treated control plants. With the exception of samples passed through tissue culture, molecular marker changes, including single and double reversions of alleles, were detected in every line at some low level Furthermore, many of the revertants were found to be genetic mosaics. DNA sequence analyses revealed that sequences flanking three molecular markers that had undergone reversion were near identical to the great-grandparent of the sequenced individual. These results suggest that stress is perhaps an inhibitor of restoration. Although there may be other explanations for the results described in this thesis, the evidence implicates genome restoration as a mechanism for generating IGH.

The secretory pathway of plant cells consists of several organelles that are connected by vesicle and tubular transport. Every compartment has a distinct function and the specificity of vesicle fusion is essential to maintain the organelles identity. N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) play a crucial role in the secretory pathway driving specific vesicle fusions. A vesicle SNARE (v-SNARE) on a vesicle specifically interacts with two or three target SNAREs (t-SNAREs) on the target compartment. This event leads to vesicle membrane fusion with the membrane of the target compartment and the release of cargo molecules into the organelle lumen.<p> The aim of this work was the characterization of two <i>Arabidopsis thaliana</i> SNAREs. The first one is a v-SNARE, Bet11 that is the Arabidopsis ortholog of the yeast and mammal ER-Golgi v-SNARE, Bet1. In these organisms, Bet1 is involved in trafficking between the ER and Golgi apparatus. The second protein studied is a putative SNARE called Bet12 that shares high sequence identity with Bet11. In particular, I was interested in studying the sorting of these two proteins and their role in the secretory pathway of plant cells. By confocal laser microscopy, I demonstrated that these two proteins have different intracellular localization: Bet11 was mainly localized on the ER, Golgi stacks and punctate structures that I have identified as endosomes. Bet12 was localized only on the Golgi stacks. The identification of signal(s) involved in targeting of Bet11 and Bet12 were studied. To reach this aim I generated different mutant chimeras of Bet11 and Bet12. The co-expression of these chimeras with specific protein markers suggested that the distribution of these proteins was the result of a combined influence of multiple domains.<p> A serine in the Bet11 sequence was identified as a putative phosphorylation site and appeared important for proper Bet11 intracellular distribution.<p> The different intracellular distributions of Bet11 and Bet12 suggest different biological roles for the two proteins. To functionally characterize these two proteins homozygous knock-down mutants of Bet11 were screened. These plants had no evident phenotype, suggesting a possible genetic redundancy in this SNARE family.

The adverse effect of Cu on growth is apparent from the reduction in root length of the Cu-treated Arabidopsis roots. Arabidopsis seems to be more resistant to Cu in comparison with that of soybean in terms of root growth. The increase of the levels of H2O2 was observed in Cu-treated Arabidopsis roots. The lignin biosynthesis related enzymes, PODs and laccases were enhanced during the Cu treatments. The lignin contents slightly increased in Cu-treated Arabidopsis roots. To our surprise, the Arabidopsis can be tolerant to high concentration of Cu (200 &#x00B5;M), because only part of high levels of H2O2 accumulated in Cu-treated tissues are used by PODs to synthesize the lignin.