@phdthesis{Huang2023,
  author    = {Huang, Shouguang},
  title     = {Role of ABA-induced Ca\(^{2+}\) signals, and the Ca\(^{2+}\)-controlled protein kinase CIPK23, in regulation of stomatal movements},
  doi       = {10.25972/OPUS-20473},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-204737},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2023},
  abstract  = {Stomata are pores in the leaf surface, formed by pairs of guard cells. The guard cells modulate the aperture of stomata, to balance uptake of CO2 and loss of water vapor to the atmosphere. During drought, the phytohormone abscisic acid (ABA) provokes stomatal closure, via a signaling chain with both Ca2+-dependent and Ca2+-independent branches. Both branches are likely to activate SLAC1-type (Slow Anion Channel Associated 1) anion channels that are essential for initiating the closure of stomata. However, the importance of the Ca2+-dependent signaling branch is still debated, as the core ABA signaling pathway only possesses Ca2+-independent components. Therefore, the aim of this thesis was to address the role of the Ca2+-dependent branch in the ABA signaling pathway of guard cells. In the first part of the thesis, the relation between ABA-induced Ca2+ signals and stomatal closure was studied, with guard cells that express the genetically encoded Ca2+-indicator R-GECO1-mTurquoise. Ejection of ABA into the guard cell wall rapidly induced stomatal closure, however, only in ¾ of the guard cells ABA evoked a cytosolic Ca2+ signal. A small subset of stomata (¼ of the experiments) closed without Ca2+ signals, showing that the Ca2+ signals are not essential for ABA-induced stomatal closure. However, stomata in which ABA evoked Ca2+ signals closed faster as those in which no Ca2+ signals were detected. Apparently, ABA-induced Ca2+ signals enhance the velocity of stomatal closure. In addition to ABA, hyperpolarizing voltage pulses could also trigger Ca2+ signals in wild type guard cells, which in turn activated S-type anion channels. However, these voltage pulses failed to elicit S-type anion currents in the slac1/slah3 guard cells, suggesting that SLAC1 and SLAH3 contribute to Ca2+-activated conductance. Taken together, our data indicate that ABA-induced Ca2+ signals enhance the activity of S-type anion channels, which accelerates stomatal closure. The second part of the thesis deals with the signaling pathway downstream of the Ca2+ signals. Two types of Ca2+-dependent protein kinase modules (CPKs and CBL/CIPKs) have been implicated in guard cells. We focused on the protein kinase CIPK23 (CBL-Interacting Protein Kinase 23), which is activated by the Ca2+-dependent protein CBL1 or 9 (Calcineurin B-Like protein 1 or 9) via interacting with the NAF domain of CIPK23. The CBL1/9-CIPK23 complex has been shown to affect stomatal movements, but the underlying molecular mechanisms remain largely unknown. We addressed this topic by using an estrogen-induced expression system, which specifically enhances the expression of wild type CIPK23, a phosphomimic CIPK23T190D and a kinase dead CIPK23K60N in guard cells. Our data show that guard cells expressing CIPK23T190D promoted stomatal opening, while CIPK23K60N enhanced ABA-induced stomatal closure, suggesting that CIPK23 is a negative regulator of stomatal closure. Electrophysiological measurements revealed that the inward K+ channel currents were similar in guard cells that expressed CIPK23, CIPK23T190D or CIPK23K60N, indicating that CIPK23-mediated inward K+ channel AKT1 does not contribute to stomatal movements. Expression of CIPK23K60N, or loss of CIPK23 in guard cells enhanced S-type anion activity, while the active CIPK23T190D inhibited the activity of these anion channels. These results are in line with the detected changes in stomatal movements and thus indicate that CIPK23 regulates stomatal movements by inhibiting S-type anion channels. CIPK23 thus serves as a brake to control anion channel activity. Overall, our findings demonstrate that CIPK23-mediated stomatal movements do not depend on CIPK23-AKT1 module, instead, it is achieved by regulating S-type anion channels SLAC1 and SLAH3. In sum, the data presented in this thesis give new insights into the Ca2+-dependent branch of ABA signaling, which may help to put forward new strategies to breed plants with enhanced drought stress tolerance, and in turn boost agricultural productivity in the future.},
  language  = {en}
}
@phdthesis{Demir2010,
  author    = {Demir, Fatih},
  title     = {Lipid rafts in Arabidopsis thaliana leaves},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-53223},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2010},
  abstract  = {Arabidopsis thaliana (A.th.) mesophyll cells play a pivotal role in the regulation of the drought stress response. The signaling \& transport components involved in drought stress regulation within lipid rafts of the plasma membrane were investigated by DRM isolation from highly purified plasma membranes. Detergent treatment with Brij-98 and Triton X-100 resulted in a total of 246 DRM proteins which were identified by nano HPLC-MS/MS. The majority of these proteins could be isolated by Triton X-100 treatment (78.5 \%) which remains the "golden" standard for the isolation of DRMs. Comparing in-gel and in-solution digestion approaches disclosed additional protein identifications for each method but the in-gel approach clearly delivered the majority of the identified proteins (81.8 \%). Functionally, a clear bias on signaling proteins was visible - almost 1/3 of the detected DRM proteins belonged to the group of kinases, phosphatases and other signaling proteins. Especially leucine-rich repeat receptor-like protein kinases and calcium-dependent protein kinases were present in Brij-98 \& Triton X-100 DRMs, for instance the calcium-dependent protein kinase CPK21. Another prominent member of DRMs was the protein phosphatase 2C 56, ABI1, which is a key regulator of the ABA-mediated drought stress response in A.th. The lipid raft localization of the identified DRM proteins was confirmed by sterol-depletion with the chemical drug MCD. Proteins which depend upon a sterol-rich environment are depleted from DRMs by MCD application. Especially signaling proteins exhibited a strong sterol-dependency. They represented the vast majority (41.5 \%) among the Triton X-100 DRM proteins which were no longer detected following MCD treatment. AtRem 1.2 \& 1.3 could be shown to be sterol-dependent in mesophyll cells as well as two CPKs (CPK10 \& CPK21) and the protein phosphatase ABI1. AtRem 1.2 \& 1.3 could be proven to represent ideal plant lipid raft marker proteins due to their strong presence in Triton X-100 DRMs and dependency upon a sterol-rich environment. When fluorescence labeled AtRem 1.2 \& 1.3 were transiently expressed in A.th. leaves, they localized to small, patchy structures at the plasma membrane. CPK21 was an intrinsic member of Triton X-100 DRMs and displayed extreme susceptibility to sterol-depletion by MCD in immunological and proteomic assays. Calcium-dependent protein kinases (CPKs) have already been studied to be involved in drought stress regulation, for instance at the regulation of S-type anion channels in guard cells. Hence, further transient expression studies with the anion channel SLAH3, protein kinase CPK21 and its counterpart, protein phosphatase ABI1 were performed in Nicotiana benthamiana. Transient co-expression of CPK21 and the anion channel SLAH3, a highly mesophyll- specific homologue of the guard cell anion channel SLAC1, resulted in a combined, sterol-dependent localization of both proteins in DRMs. Supplementary co-expression of the counterpart protein phosphatase ABI1 induced dislocation of SLAH3 from DRMs, probably by inactivation of the protein kinase CPK21. CPK21 is known to regulate the anion channel SLAH3 by phosphorylation. ABI1 dephosphorylates CPK21 thus leading to deactivation and dislocation of SLAH3 from DRMs. All this regulative events are taking place in DRMs of A.th. mesophyll cells. This study presents the first evidence for a lipid raft-resident protein complex combining signaling and transport functions in A.th. Future perspectives for lipid raft research might target investigations on the lipid raft localization of candidate DRM proteins under presence of abiotic and biotic stress factors. For instance, which alterations in the DRM protein composition are detectable upon exogenous application of the plant hormone ABA? Quantitative proteomics approaches will surely increase our knowledge of the post-transcriptional regulation of gene activity under drought stress conditions.},
  subject      = {Ackerschmalwand},
  language  = {en}
}
@phdthesis{EscalantePerez2010,
  author    = {Escalante Perez, Maria},
  title     = {Poplar responses to biotic and abiotic stress},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-46893},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2010},
  abstract  = {In this study poplar trees have been examined under different stress conditions. Apart from the detailed descriptions above two main conclusions might be drawn: i) A small plant like Arabidopsis thaliana is highly susceptible to stress situations that might become life-threatening compared to a tree that has extremely more biomass at its disposal. Such an organism might be able to compensate severe stress much longer than a smaller one. It seems therefore reasonable that a crop like Arabidopsis reacts earlier and faster to a massive threat. ii) In poplar both tested stress responses seemed to be regulated by hormones. The reactions to abiotic salt stress are mainly controlled by ABA, which also has a strong impact upon cold and drought stress situations. The term commonly used for ABA is "stress hormone" and is at least applicable to all abiotic stresses. In case of herbivory (biotic stress), jasmonic acid appears to be the key-player that coordinates the defence mechanism underlying extrafloral nectary and nectar production. Thus the presented work has gained a few more insights into the complex network of general stress induced processes of poplar trees. Future studies will help to understand the particular role of the intriguing indirect defence system of the extrafloral nectaries in more detail.},
  subject      = {abiotic stress},
  language  = {en}
}