Blue-Light Sensing in Arabidopsis : In Vivo Microscopy of Subcellular Localization, Endocytosis, and Vesicular Recycling of the Blue-Light Receptor Phototropin 1

In this thesis, the subcellular localization and possible functions of the blue light receptor, PHOT1, is examined. Results are presented, which indicate that blue lightinduced relocalization of auxin efflux carriers (PIN-proteins) is adjusted by PHOT1 via ARF-mediated and BFA-sensitive vesicle trafficking pathways. PHOT1 was visualized by expressing the PHOT1::GFP reporter in Arabidopsis mutants which contain a dysfunctional copy of the endogenous PHOT1-gene. It is shown that the subcellular localization of PHOT1 in plant cells reflects the physiological functions and development stages of special kinds of cells. For example in guard cells, PHOT1 appears only in the mature developmental stage, when these cells begin to react to blue light signals. Especially, the specific roles of PHOT1 were addressed in root and shoot apical meristems. The obtained data suggest that PHOT1 has different roles in root and shoot phototropic responses. PHOT1 in root tissues acts to adjust the polar auxin transport, while PHOT1 in shoot tissues has more abundant roles. It is also suggested that the mechanism of phototropic responses in shoots and roots might be different from each other. Interestingly, the PHOT1::GFP signals are quickly released from the plasma membrane and appear in intracellular vesicles, when cells are irradiated with blue laser light in the process of scanning in the confocal microscope. The level of PHOT1 relocalization reflects the amount of photons reaching the surface of the Arabidopsis cells. PHOT1-positive vesicles colocalize with the endosomal tracer, FM4-64, indicating that that light-induced movements of PHOT1-positive endosome are analogous to the ligand-induced receptor mediated endocytosis. PHOT1-mediated blue light sensing is sensitive to inhibitors of exocytosis and endocytosis and is also dependent on intact actin filaments. Endosomal recycling of PIN proteins is considered as the mechanism to adjust the levels of polar auxin transport. Here it is demonstrated that blue light affects the recycling of PIN1 and PIN2 between the plasma membrane and the endosomal compartment. The pin2 null mutant lacks phototropic responses in roots, while the shoot phototropism in this mutant is independent on PIN2. By taking into account the polar transport of auxin under asymmetric illumination at the root tip, a testable model for the mechanism of root phototropism is proposed, which will helpful in guiding future phototropism research

Arabidopsis blue light receptor phototropin 1 undergoes blue light-induced activation in membrane microdomains

Phototropin (phot)-mediated signaling initiated by blue light (BL) plays a critical role in optimizing photosynthetic light capture at the plasma membrane (PM) in plants. However, the mechanisms underlying the regulation of phot activity at the PM in response to BL remain largely unclear. In this study, by single-particle tracking and step-wise photobleaching analysis we demonstrated that in the dark phot1-GFP proteins remain in an inactive state and mostly present as a monomer. The phot1-GFP diffusion rate and its dimerization increased in a dose-dependent manner in response to BL. In contrast, BL did not affect the lateral diffusion of kinase-inactive phot1 -GFP, whereas it did enhance its dimerization, suggesting that phot1 dimerization is independent of its phosphorylation. Förster resonance energy transfer-fluorescence lifetime imaging microscopy (FRET-FLIM) analysis revealed that the interaction between phot1-GFP and AtRem1.3-mCherry was enhanced along with increased time of BL treatment. However, the BL-dependent interaction was not obvious in plants co-expressing phot1 -GFP and AtRem1.3-mCherry, implicating that BL facilitated the translocation of functional phot1-GFP into AtRem1.3-labeled microdomains to activate phot-mediated signaling. Conversely, sterol depletion attenuated phot1-GFP dynamics, dimerization, and phosphorylation. Taken together, these results indicate that membrane microdomains act as an organizing platform essential for proper function of activated phot1 at the PM

Screening and Validation of Housekeeping Genes of the Root and Cotyledon of Cunninghamia lanceolata under Abiotic Stresses by Using Quantitative Real-Time PCR

Cunninghamia lanceolata (Chinese fir) is a fast-growing and commercially important conifer of the Cupressaceae family. Due to the unavailability of complete genome sequences and relatively poor genetic background information of the Chinese fir, it is necessary to identify and analyze the expression levels of suitable housekeeping genes (HKGs) as internal reference for precise analysis. Based on the results of database analysis and transcriptome sequencing, we have chosen five candidate HKGs (Actin, GAPDH, EF1a, 18S rRNA, and UBQ) with conservative sequences in the Chinese fir and related species for quantitative analysis. The expression levels of these HKGs in roots and cotyledons under five different abiotic stresses in different time intervals were measured by qRT-PCR. The data were statistically analyzed using the following algorithms: NormFinder, BestKeeper, and geNorm. Finally, RankAggreg was applied to merge the sequences generated from three programs and rank these according to consensus sequences. The expression levels of these HKGs showed variable stabilities under different abiotic stresses. Among these, Actin was the most stable internal control in root, and GAPDH was the most stable housekeeping gene in cotyledon. We have also described an experimental procedure for selecting HKGs based on the de novo sequencing database of other non-model plants

Systematic Analysis of NRAMP Family Genes in <i>Areca catechu</i> and Its Response to Zn/Fe Deficiency Stress

Areca catechu is a commercially important medicinal plant widely cultivated in tropical regions. The natural resistance-associated macrophage protein (NRAMP) is widespread in plants and plays critical roles in transporting metal ions, plant growth, and development. However, the information on NRAMPs in A. catechu is quite limited. In this study, we identified 12 NRAMPs genes in the areca genome, which were classified into five groups by phylogenetic analysis. Subcellular localization analysis reveals that, except for NRAMP2, NRAMP3, and NRAMP11, which are localized in chloroplasts, all other NRAMPs are localized on the plasma membrane. Genomic distribution analysis shows that 12 NRAMPs genes are unevenly spread on seven chromosomes. Sequence analysis shows that motif 1 and motif 6 are highly conserved motifs in 12 NRAMPs. Synteny analysis provided deep insight into the evolutionary characteristics of AcNRAMP genes. Among the A. catechu and the other three representative species, we identified a total of 19 syntenic gene pairs. Analysis of Ka/Ks values indicates that AcNRAMP genes are subjected to purifying selection in the evolutionary process. Analysis of cis-acting elements reveals that AcNRAMP genes promoter sequences contain light-responsive elements, defense- and stress-responsive elements, and plant growth/development-responsive elements. Expression profiling confirms distinct expression patterns of AcNRAMP genes in different organs and responses to Zn/Fe deficiency stress in leaves and roots. Taken together, our results lay a foundation for further exploration of the AcNRAMPs regulatory function in areca response to Fe and Zn deficiency