Structural and functional analysis of a plant nucleolar RNA chaperone-like protein.

Ribosome biogenesis is a key process in all eukaryotic cells that requires hundreds of ribosome biogenesis factors (RBFs), which are essential to build the mature ribosomes consisting of proteins and rRNAs. The processing of the required rRNAs has been studied extensively in yeast and mammals, but in plants much is still unknown. In this study, we focused on a RBF from A. thaliana that we named NUCLEOLAR RNA CHAPERONE-LIKE 1 (NURC1). NURC1 was localized in the nucleolus of plant cell nuclei, and other plant RBF candidates shared the same localization. SEC-SAXS experiments revealed that NURC1 has an elongated and flexible structure. In addition, SEC-MALLS experiments confirmed that NURC1 was present in its monomeric form with a molecular weight of around 28 kDa. RNA binding was assessed by performing microscale thermophoresis with the Arabidopsis internal transcribed spacer 2 (ITS2) of the polycistronic pre-rRNA precursor, which contains the 5.8S, 18S, and 25S rRNA. NURC1 showed binding activity to the ITS2 with a dissociation constant of 228 nM and exhibited RNA chaperone-like activity. Our data suggested that NURC1 may have a function in pre-rRNA processing and thus ribosome biogenesis.

Intrinsically disordered plant protein PARCL colocalizes with RNA in phase-separated condensates whose formation can be regulated by mutating the PLD.

In higher plants, long-distance RNA transport via the phloem is crucial for communication between distant plant tissues to align development with stress responses and reproduction. Several recent studies suggest that specific RNAs are among the potential long-distance information transmitters. However, it is yet not well understood how these RNAs enter the phloem stream, how they are transported, and how they are released at their destination. It was proposed that phloem RNA-binding proteins facilitate RNA translocation. In the present study, we characterized two orthologs of the phloem-associated RNA chaperone-like (PARCL) protein from Arabidopsis thaliana and Brassica napus at functional and structural levels. Microscale thermophoresis showed that these phloem-abundant proteins can bind a broad spectrum of RNAs and show RNA chaperone activity in FRET-based in vitro assays. Our SAXS experiments revealed a high degree of disorder, typical for RNA-binding proteins. In agroinfiltrated tobacco plants, eYFP-PARCL proteins mainly accumulated in nuclei and nucleoli and formed cytosolic and nuclear condensates. We found that formation of these condensates was impaired by tyrosine-to-glutamate mutations in the predicted prion-like domain (PLD), while C-terminal serine-to-glutamate mutations did not affect condensation but reduced RNA binding and chaperone activity. Furthermore, our in vitro experiments confirmed phase separation of PARCL and colocalization of RNA with the condensates, while mutation as well as phosphorylation of the PLD reduced phase separation. Together, our results suggest that RNA binding and condensate formation of PARCL can be regulated independently by modification of the C-terminus and/or the PLD.

Detection of RNA in Ribonucleoprotein Complexes by Blue Native Northern Blotting.

Northern blotting is a classical technique that allows the detection of specific nucleic acids using radioactive or non-radioactive probes. Normally, nucleic acids are denatured and separated by agarose or polyacrylamide gel electrophoresis and transferred and fixed to a membrane prior to detection. Here, we describe a method to analyze specific RNA in native ribonucleoprotein complexes using blue native PAGE with subsequent northern blotting, crosslinking of RNA onto a suitable membrane, and detection using non-radioactive probes.

Comparative proteomic analysis of salt-responsive proteins in canola roots by 2-DE and MALDI-TOF MS.

Salinity stress is a major abiotic stress that affects plant growth and limits crop production. Roots are the primary site of salinity perception, and salt sensitivity in roots limits the productivity of the entire plant. To better understand salt stress responses in canola, we performed a comparative proteomic analysis of roots from the salt-tolerant genotype Safi-7 and the salt-sensitive genotype Zafar. Plants were exposed to 0, 150, and 300 mM NaCl. Our physiological and morphological observations confirmed that Safi-7 was more salt-tolerant than Zafar. The root proteins were separated by two-dimensional gel electrophoresis and MALDI-TOF mass spectrometry was applied to identify proteins regulated in response to salt stress. We identified 36 and 25 protein spots whose abundance was significantly affected by salt stress in roots of plants from the tolerant and susceptible genotype, respectively. Functional classification analysis revealed that the differentially expressed proteins from the tolerant genotype could be assigned to 14 functional categories, while those from the susceptible genotype could be classified into 9 functional categories. The most significant differences concerned proteins involved in glycolysis (Glyceraldehyde-3-phosphate dehydrogenase, Fructose-bisphosphate aldolase, Phosphoglycerate kinase 3), stress (heat shock proteins), Redox regulation (Glutathione S-transferase DHAR1, L-ascorbate peroxidase), energy metabolism (ATP synthase subunit B), and transport (V-type proton ATPase subunit B1) which were increased only in the tolerant line under salt stress. Our results provide the basis for further elucidating the molecular mechanisms of salt-tolerance and will be helpful for breeding salt-tolerant canola cultivars.

Phloem Sap Sampling from Brassica napus for 3D-PAGE of Protein and Ribonucleoprotein Complexes.

Sampling the phloem of higher plants is often laborious and significantly dependent on the plant species. However, proteome studies under denaturing conditions could be achieved in different plant species. Native protein:protein and protein:nucleic acid complexes from phloem samples have as yet scarcely been analyzed, although they might play important roles in maintenance of this specialized compartment or in long-distance signaling. Large molecular assemblies can be isolated using a blue native gel electrophoresis (BN-PAGE). Their protein components can be separated by a subsequent sodium dodecyl sulfate PAGE (SDS-PAGE). However, proteins with similar molecular weights co-migrate, what can hinder protein identification by mass spectrometry. Combining BN-PAGE with two different denaturing gel electrophoresis steps, namely Tris-Tricine-urea and SDS-PAGE, enables the additional separation of proteins according to their hydrophilicity/hydrophobicity and thus increases resolution and the success of protein identification. It even allows distinguishing proteins that only differ in their posttranslational modifications. In addition, blue native northern blotting can be applied to identify the RNA components in macromolecular complexes. We show that our protocol is suitable to unravel the protein and RNA components of native protein:protein and ribonucleoprotein (RNP) complexes occurring in phloem samples. Combining a blue native PAGE with two different denaturing PAGE steps can help to separate different kinds of large protein complexes, and also enables an increased identification rate of their components by mass spectrometry. Furthermore, the protocol is robust enough to simultaneously detect potentially bound nucleic acids within single protein complexes.

Functional analysis of Brassica napus phloem protein and ribonucleoprotein complexes.

Phloem sap contains a large number of macromolecules, including proteins and RNAs from different classes. Proteome analyses of phloem samples from different plant species under denaturing conditions identified hundreds of proteins potentially involved in diverse processes. Surprisingly, these studies also found a significant number of ribosomal and proteasomal proteins. This led to the suggestion that active ribosome and proteasome complexes might be present in the phloem, challenging the paradigm that protein synthesis and turnover are absent from the enucleate sieve elements of angiosperms. However, the existence of such complexes has as yet not been demonstrated. In this study we used three-dimensional gel electrophoresis to separate several protein complexes from native phloem sap from Brassica napus. Matrix-assisted laser desorption ionization-time of flight MS analyses identified more than 100 proteins in the three major protein-containing complexes. All three complexes contained proteins belonging to different ribosomal fragments and blue native northern blot confirmed the existence of ribonucleoprotein complexes. In addition, one complex contained proteasome components and further functional analyses confirmed activity of a proteasomal degradation pathway and showed a large number of ubiquitinated phloem proteins. Our results suggest specialized roles for ubiquitin modification and proteasome-mediated degradation in the phloem.

Protocol: optimisation of a grafting protocol for oilseed rape (Brassica napus) for studying long-distance signalling.

BACKGROUND: Grafting is a well-established technique for studying long-distance transport and signalling processes in higher plants. While oilseed rape has been the subject of comprehensive analyses of xylem and phloem sap to identify macromolecules potentially involved in long-distance information transfer, there is currently no standardised grafting method for this species published. RESULTS: We developed a straightforward collar-free grafting protocol for Brassica napus plants with high reproducibility and success rates. Micrografting of seedlings was done on filter paper. Grafting success on different types of regeneration media was measured short-term after grafting and as the long-term survival rate (>14 days) of grafts after the transfer to hydroponic culture or soil. CONCLUSIONS: We compared different methods for grafting B. napus seedlings. Grafting on filter paper with removed cotyledons, a truncated hypocotyl and the addition of low levels of sucrose under long day conditions allowed the highest grafting success. A subsequent long-term hydroponic cultivation of merged grafts showed highest survival rates and best reproducibility.

Probing the Ca(2+) switch of the neuronal Ca(2+) sensor GCAP2 by time-resolved fluorescence spectroscopy.

We report fluorescence lifetime and rotational anisotropy measurements of the fluorescent dye Alexa647 attached to the guanylate cyclase-activating protein 2 (GCAP2), an intracellular myristoylated calcium sensor protein operating in photoreceptor cells. By linking the dye to different protein regions critical for monitoring calcium-induced conformational changes, we could measure fluorescence lifetimes and rotational correlation times as a function of myristoylation, calcium, and position of the attached dye, while GCAP2 was still able to regulate guanylate cyclase in a Ca(2+)-sensitive manner. We observe distinct site-specific variations in the fluorescence dynamics when externally changing the protein conformation. A clear reduction in fluorescence lifetime suggests that in the calcium-free state a dye marker in amino acid position 131 senses a more hydrophobic protein environment than in position 111. Saturating GCAP2 with calcium increases the fluorescence lifetime and hence leads to larger exposure of position 111 to the solvent and at the same time to a movement of position 131 into a hydrophobic protein cleft. In addition, we find distinct, biexponential anisotropy decays reflecting the reorientational motion of the fluorophore dipole and the dye/protein complex, respectively. Our experimental data are well described by a "wobbling-in-a-cone" model and reveal that for dye markers in position 111 of the GCAP2 protein both addition of calcium and myristoylation results in a pronounced increase in orientational flexibility of the fluorophore. Our results provide evidence that the up-and-down movement of an α-helix that is situated between position 111 and 131 is a key feature of the dynamics of the protein-dye complex. Operation of this piston-like movement is triggered by the intracellular messenger calcium.