Thesis (Ph. D.)--University of Missouri-Columbia, 2001. / Typescript. Vita. Includes bibliographical references (leaves 231-256). Also available on the Internet.

Thesis (Ph.D.) - University of Glasgow, 2007. / Ph.D. thesis submitted to the Division of Biochemistry and Molecular Biology, Biomedical and Life Sciences (IBLS), University of Glasgow, 2007. Includes bibliographical references. Print version also available.

Thesis (Ph. D.)--University of Queensland, 2002. / Includes bibliographical references.

Thesis (Ph. D.)--Hong Kong University of Science and Technology, 2003. / Includes bibliographical references (leaves 197-236). Also available in electronic version. Access restricted to campus users.

published_or_final_version / Biological Sciences / Doctoral / Doctor of Philosophy

In Arabidopsis thaliana, a gene family encodes acyl-CoA-binding proteins (ACBPs) conserved at the acyl-CoA-binding domain which facilitates the binding to acyl-CoA esters. These ACBPs, designated ACBP1 to ACBP6, range in size from 10.4 to 73.1 kD. Previous studies have shown that the the overexpression of ACBP1 or ACBP2 in Arabidopsis likely promotes repair of lipid membranes and result in enhanced tolerance to lead and cadmium, respectively. Microarray data (http://bar.utoronto.ca/) revealed that the expression of ACBP1 and ACBP2 is also regulated by other abiotic stresses, such as cold and drought, suggestive of their association with these environmental pressures. The aim of this study is to investigate and better understand the roles of ACBP1 and ACBP2 in different stress responses. It has been previously observed that the expression of both ACBP1 and ACBP4 is lead [Pb(II)]-inducible and recombinant ACBP1 and ACBP4 bind Pb(II) in vitro. In this study, ACBP1 and ACBP4 were overexpressed in Brassica juncea to test if these ACBPs could be extended for application in Pb(II) phytoremediation in transgenic B. juncea. On freezing (-12 to -8 °C) treatment, ACBP1-overexpressing Arabidopsis was freezing sensitive and accumulated more phosphatidic acid (PA), but less phosphatidylcholine (PC), in contrast to acbp1 mutant plants which were freezing tolerant and had reduced PA and elevated PC levels. Such changes in PC and PA were consistent with the expression of the mRNA encoding phospholipase D1 (PLD1), a major enzyme that promotes the hydrolysis of PC to PA. In contrast, the expression of phospholipase D (PLD), which plays a positive role in freezing tolerance, was up-regulated in acbp1 mutant plants and down-regulated in ACBP1-overexpressing plants. Reduced PLD1 expression and decreased hydrolysis of PC to PA may enhance membrane stability in the acbp1 mutant plants. Given that recombinant ACBP1 binds PA and acyl-CoA esters in vitro, the expression of PLD1 and PLD could be regulated by PA or acyl-CoAs maintained by ACBP1, if ACBP1 were to resemble the yeast 10-kD ACBP by its capability to modulate gene expression during stress responses. Interestingly, another membrane-associated ACBP, ACBP2, which shows high (76.9%) conservation in amino acid homology to ACBP1, did not appear to be affected by freezing treatment. Besides freezing stress, ACBP1, as well as ACBP2, have been observed to participate in abscisic acid (ABA) signaling. They both promote ABA signaling in seed germination and seedling development, while only ACBP2 is involved in the drought response. The overexpression of ACBP2 in Arabidopsis up-regulated reactive oxygen species (ROS) production culminating in reduction in stomatal aperture and water loss in guard cells, thereby enhancing drought tolerance. For tests in phytoremediation, B. juncea was selected for overexpression of ACBP1 and ACBP4 because it is fast-growing, has a higher biomass than Arabidopsis, and is known to be a good accumulator of Pb(II). However, results of Pb(II) treatment for two days showed that the overexpression of ACBP1 or ACBP4 in B. juncea did not significantly improve Pb(II) tolerance. Nevertheless, B. juncea overexpressing ACBP1 did accumulate Pb(II) in roots whereas ACBP4-overexpressing B. juncea lines accumulated Pb(II) in both shoots and roots. Given that B. juncea has a larger biomass than Arabidopsis, it is likely that the duration of Pb(II)-incubation tested in this study was not drastic enough for comparison, and the incubation time should be further extended for Pb(II) translocation. In addition, future studies on Arabidopsis should be conducted to better understand the mechanism of ACBP4-mediated Pb(II) accumulation using Arabidopsis acbp4 mutant and ACBP4-overexpressing plants. / published_or_final_version / Biological Sciences / Doctoral / Doctor of Philosophy

Photosynthesis in higher plants relies upon collection of light by chlorophyll molecules associated with light harvesting chlorophyll a/b-binding (LHC) proteins. The two most abundant of these are Lhcb1 and Lhcb2, which make up light harvesting complex (LHC) II trimers. They are also involved in facilitating state transitions, a process during which energy balancing between photosystem (PS) II and I is achieved. Overexcitation of PSII reduces the plastoquinone pool which activates STN7, a kinase, that phosphorylates a threonine residue on Lhcb1 and Lhcb2. In order to studythe kinetics of this we developed antibodies capable of recognizingphosphorylated forms of each of these proteins. This showed that Lhcb2 is more rapidly phosphorylated than Lhcb1, that there are no differences in the migration of phosphorylated and non-phosphorylated forms of Lhcb1 and Lhcb2 and that the majority of phosphorylated LHCII (P-Lhcb1 and PLhcb2) are associated with super- and megacomplexes. Furthermore, a state 2-specific LHCII-PSI-LHCI band contains P-Lhcb2 but almost no P-Lhcb1, and a band corresponding to M trimers (band 4 from sucrose gradients, composed of LHCII, CP24 and CP29), contains only P-Lhcb1 but no P-Lhcb2. We also developed artificial microRNA lines specifically depleted in either Lhcb1 or Lhcb2, amiLhcb1 and amiLhcb2 respectively. We show that the roles of Lhcb1 and Lhcb2 in state transitions are complementary. Lhcb1 modulate the size of grana stacks. In the absence of Lhcb1 only a few LHC trimers are formed, while in the absence of Lhcb2, the antenna looks like in the wild type although the plants cannot perform state transitions normally. Trimers containing P-Lhcb2 functionally detach from PSII and connect to PSI to balance the relative excitation pressure. State transitions only occur when both Lhcb1 and Lhcb2 are present, presumably in a (Lhcb1)2 Lhcb2 heterotrimer. In absence of Lhcb2, the LHCII-PSI-LHCI supercomplex is not formed indicting that P-Lhcb2 mediates attachment of LHCII to PSI. We tried complementing amiLhcb2 with modified Lhcb2 genes coding for proteins with altered amino acids, Arg2 to Lys or the phosphorylatable Thr3 residue to Asn or Ser. Introduction of the additional gene often causes loss of amiRNA-inhibition, however we could confirm that substitution of the Thr3 with Asn led to the absence of Lhcb2 phosphorylation and thus no state transition. / Klorofyll a/b-bindande proteiner (s k light harvesting chlorophyll a/b-binding proteins eller LHC proteiner) är viktiga för högre växters fotosyntes, då deras klorofyllmolekyler skördar solljuset. Två av dessa proteiner, Lhcb1 och Lhcb2, bygger upp ”LHCII trimerer” och finns i större mängd än de andra och dessa är även viktiga för s k ”state transtions”, en process som ser till att fotosystem (PS) I och PSII exciteras lika mycket. Om PSII exciteras för mycket reduceras plastoquinon-poolen som i sin tur aktiverar ett proteinkinas, STN7, som fosforylerar en av Lhcb1/Lhcb2s treoniner. För att studera denna fosforylering har vi utvecklat antikroppar som är specifika för dessa fosforylerade former av proteinerna, och vi använde dem för att visa att Lhcb2 fosforyleras snabbare än Lhcb1, och att största delen av det fosforylerade proteinerna (P-Lhcb1 och P-Lhcb2) finns i s k super- eller megakomplex. Ett komplex som bara finns finns i ”state 2” består av LHCII, PSI och LHCI, och det innehåller endast P-Lhcb2 men nästan inget P-Lhcb1, och ett band som består av LHCII, CP24 och CP29 innehåller endast PLhcb1. Vi skapade artificiella mikro-RNA-linjer, amiLhcb1 och amiLhcb2, som saknade antingen Lhcb1 eller Lhcb2. Lhcb1 påverkar höjden av grana stackarna. Med hjälp av dessa visade vi att Lhcb1 och Lhcb2 har komplementära roller för state transitions, saknas Lhcb1 gör växten bara få LHCII trimerer, medan om Lhcb2 gör växten antennener som liknar vildtypens, men den kan inte utföra state transitions som den. Mängden Lhcb1 påverkar storleken av ”grana stacks”. Trimerer som innehåller PLhcb2 kopplas över från PSII till PSI för att balansera excitationstrycket. Både Lhcb1 och Lhcb2, antagligen i trimerer bestående av en Lhcb2 och the Lhcb1, behövs för state transitions. Saknas Lhcb2 bildas inga komplex bestående av LHCII, PSI och LHCI, vilket visar att P-Lhcb2 antagligen möjliggör LHCIIs bindning till PSI. Vi försökte komplementera amiLhcb2 med Lhcb2 gener där amino syror bytts ut, Arg2 till Lys eller den fosforylerbara Thr3 till Asn eller Ser. När denna gen introducerades försvann dock ofta amiRNA-inhiberingen, men vi kunde visa att om Thr3 ersattes med Asn skedde inga state transitions.

No description available.

Plant non specific lipid transfer proteins (nsLTPs) enhance in vitro transfer of phospholipids between membranes. Our analysis exploited the large amount of Arabidopsis transcriptome data in public databases to learn more about the function of nsLTPs. The analysis revealed that some nsLTPs are expressed only in roots, some are seed specific, and others are specific for tissues above ground whereas certain nsLTPs show a more general expression pattern. Only few nsLTPs showed a strong up or downregulation after that the Arabidopsis plant had suffered from biotic or abiotic stresses. However, salt, high osmosis and UV-B radiation caused upregulation of some nsLTP genes. Further, when the coexpression pattern of the A.thaliana nsLTPs were investigated, we found that there were several modules of nsLTP genes that showed strong coexpression indicating an involvement in related biological processes. Our finding reveals that the nsLTPs gene was significantly correlated with lipase and peroxidase activity. Hence we concluded that the nsLTPs may play a role in seed germination, signalling and ligning biosynthesis.

Pseudomonas syringae delivers a plethora of effector proteins into host cells to sabotage host immune responses and physiology to favor infection. We have previously shown that P. syringae pv. tomato DC3000 effector HopF2 suppresses Arabidopsis innate immunity triggered by multiple pathogen-associated molecular patterns (PAMP) at the plasma membrane. We show here that HopF2 possesses distinct mechanisms in the suppression of two branches of PAMP-activated MAP kinase cascades. In contrast to blocking MKK5 in MEKK1-MKK4/5-MPK3/6 cascade, HopF2 targets additional component(s) upstream of MEKK1 in MEKK1-MKK1/2-MPK4 cascade and plasma membrane-localized receptor-like cytoplasmic kinase BIK1 and its homologs. We further show that HopF2 directly targets BAK1, a plasma membrane-localized receptor-like kinase involved in multiple PAMP signaling. The interaction between BAK1 and HopF2 or two additional P. syringae effectors AvrPto and AvrPtoB, was confirmed in vivo and in vitro. Consistent with BAK1 as a physiological target of HopF2, the lethality of overexpression of HopF2 in wild-type Arabidopsis transgenic plants was largely alleviated in bak1 mutant plants. Identification of BAK1 as an additional HopF2 virulence target not only explains HopF2 suppression of multiple PAMP signaling at the plasma membrane, but also supports the notion that pathogen virulence effectors have multiple targets in host cells.