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Characterization of factors participating in regulation of intracellular dynamics of auxin carriers
Lacek, Jozef ; Zažímalová, Eva (advisor) ; Ibl, Verena (referee) ; Nodzynski, Tomasz (referee)
Plants are known to adjust the orientation of their organs, shoot and root, to ensure maximal energy generation and nutrient uptake, but also to avoid toxic growth conditions. Directional growth regulation depends on asymmetric plant organ growth and it is crucial to ensure plant survival. It is orchestrated on cellular level in concert with exogenous and intrinsic signals. Even though tropistic growth responses of plants were described by Darwin on macroscopic level already in 1880, now it is necessary to understand molecular mechanisms that underpin efficient modulation of directional plant growth. During my studies I focused on factors that modulate directional root growth regulation. The root is a complex, three-dimensional object, which continuously modifies its shape and growth path. Since the root needs to expand its surface to supply the plant with nutrients and water, it is important to understand how roots cope with changing growth conditions while exploring the soil. If the root cannot manage to grow through soil efficiently, mechanical impedance and lack of resources will also restrict shoot growth as well. Manifold signaling pathways coordinate the complex processes that underpin efficient root growth, including those modulated by phytohormones, sugars, flavonoids and other...
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Cellular determinants of the distribution of PIN auxin transporters in the plasma membrane
Stelate, Ayoub ; Petrášek, Jan (advisor) ; Mravec, Jozef (referee) ; Nodzynski, Tomasz (referee)
Asymmetric localization of auxin carriers has always attracted the attention of many scientists around the world. However, to address this topic while focusing on the plasma membrane (PM), requires advanced microscopy techniques and knowledge of biophysics and biology. This doctoral work bridges the two disciplines to contribute to our understanding of the dynamics and distribution of PIN-FORMED auxin carriers from tobacco (NtPINs) within the PM. I have developed a novel correlative light electron microscopy (CLEM) method using total internal reflection fluorescence microscopy (TIRFM) and advanced environmental scanning electron microscopy (A-ESEM). To my knowledge, this is the first effort to achieve a correlation between immunofluorescence and electron microscopy imaging of plant integral PM proteins. As I have shown, individual NtPINs are organized differently within the PM. Dynamic analyses that quantify individual nanodomains' diffusion rates allowed me to show that NtPINs have constraints behavior with different diffusion rates. I have investigated the role of the cell wall and cytoskeleton in the organization and dynamics of NtPINs. Using pharmacological treatments, I have shown that they differentially affect the mobility and organization of NtPINs within the PM. Complete removal of the...
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Mechanism of auxin transport across plasma membrane through PIN auxin efflux carriers
Lefnar, Radek ; Petrášek, Jan (advisor) ; Nodzynski, Tomasz (referee)
Phytohormone auxin and its directional distribution plays an essential role in the regulation of numerous processes during vegetative and reproductive plant development. Regulation of the expression, localization and activity of the PIN-FORMED (PIN) proteins is important for proper polar auxin transport in plant tissues. PIN proteins have been described as the major auxin efflux carriers regulating auxin's directional flow to build up gradients that provide information for the coordination of plant development. PIN protein structure topology prediction through bioinformatic analysis is still insufficient to understand their transport mechanism. Experimental analysis of PIN protein domains can provide valuable insight into understanding their role in mediating auxin transport. In this study, the C-terminal part of PINs have been modified by gradual trimming to determine the existence of relevant functional domains, which could be important for auxin transport. Seven modified PIN proteins from Arabidopsis thaliana and Nicotiana tabacum were prepared. Transiently transformed tobacco cell line Bright Yellow-2 (BY-2) was used to monitor differences in PIN transport activity. This approach allowed indirect monitoring of intracellular auxin levels using the DR5 reporter system. Transiently expressed...
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