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.

Noncell-autonomous HSC70.1 chaperone displays homeostatic feedback regulation by binding its own mRNA.

The HSC70/HSP70 family of heat shock proteins are evolutionarily conserved chaperones involved in protein folding, protein transport, and RNA binding. Arabidopsis HSC70 chaperones are thought to act as housekeeping chaperones and as such are involved in many growth-related pathways. Whether Arabidopsis HSC70 binds RNA and whether this interaction is functional has remained an open question. We provide evidence that the HSC70.1 chaperone binds its own mRNA via its C-terminal short variable region (SVR) and inhibits its own translation. The SVR encoding mRNA region is necessary for HSC70.1 transcript mobility to distant tissues and that HSC70.1 transcript and not protein mobility is required to rescue root growth and flowering time of hsc70 mutants. We propose that this negative protein-transcript feedback loop may establish an on-demand chaperone pool that allows for a rapid response to stress. In summary, our data suggest that the Arabidopsis HSC70.1 chaperone can form a complex with its own transcript to regulate its translation and that both protein and transcript can act in a noncell-autonomous manner, potentially maintaining chaperone homeostasis between tissues.

Long distance RNA movement.

Contents Summary 29 I. Introduction 29 II. Phloem as a conduit for macromolecules 30 III. Classes of phloem transported RNAs and their function 32 IV. Mode of RNA transport 35 V. Conclusions 37 Acknowledgements 37 References 37 SUMMARY: In higher plants, small noncoding RNAs and large messenger RNA (mRNA) molecules are transported between cells and over long distances via the phloem. These large macromolecules are thought to get access to the sugar-conducting phloem vessels via specialized plasmodesmata (PD). Analyses of the phloem exudate suggest that all classes of RNA molecules, including silencing-induced RNAs (siRNAs), micro RNAs (miRNAs), transfer RNAs (tRNAs), ribosomal RNA (rRNAs) and mRNAs, are transported via the vasculature to distant tissues. Although the functions of mobile siRNAs and miRNAs as signalling molecules are well established, we lack a profound understanding of mobile mRNA function(s) in recipient cells and tissues, and how they are selected for transport. A surprisingly high number of up to thousands of mRNAs were described in diverse plant species such as cucumber, pumpkin, Arabidopsis and grapevine to move long distances over graft junctions to distinct body parts. In this review, we present an overview of the classes of mobile RNAs, the potential mechanisms facilitating RNA long-distance transport, and the roles of mobile RNAs in regulating transcription and translation. Furthermore, we address potential function(s) of mobile protein-encoding mRNAs with respect to their characteristics and evolutionary constraints.

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.

Effects of Fe deficiency on the protein profile of Brassica napus phloem sap.

The aim of this work was to study the effect of Fe deficiency on the protein profile of phloem sap exudates from Brassica napus using 2DE (IEF-SDS-PAGE). The experiment was repeated thrice and two technical replicates per treatment were done. Phloem sap purity was assessed by measuring sugar concentrations. Two hundred sixty-three spots were consistently detected and 15.6% (41) of them showed significant changes in relative abundance (22 decreasing and 19 increasing) as a result of Fe deficiency. Among them, 85% (35 spots), were unambiguously identified. Functional categories containing the largest number of protein species showing changes as a consequence of Fe deficiency were signaling and regulation (32%), and stress and redox homeostasis (17%). The Phloem sap showed a higher oxidative stress and significant changes in the hormonal profile as a result of Fe deficiency. Results indicate that Fe deficiency elicits major changes in signaling pathways involving Ca and hormones, which are generally associated with flowering and developmental processes, causes an alteration in ROS homeostasis processes, and induces decreases in the abundances of proteins involved in sieve element repair, suggesting that Fe-deficient plants may have an impaired capacity to heal sieve elements upon injury.

The distinct functional roles of the inner and outer chloroplast envelope of Pea (Pisum sativum) as revealed by proteomic approaches.

Protein profiles of inner (IE) and outer (OE) chloroplast envelope membrane preparations from pea were studied using shotgun nLC-MS/MS and two-dimensional electrophoresis, and 589 protein species (NCBI entries) were identified. The relative enrichment of each protein in the IE/OE pair of membranes was used to provide an integrated picture of the chloroplast envelope. From the 546 proteins identified with shotgun, 321 showed a significant differential distribution, with 180 being enriched in IE and 141 in OE. To avoid redundancy and facilitate in silico localization, Arabidopsis homologues were used to obtain a nonredundant list of 409 envelope proteins, with many showing significant OE or IE enrichment. Functional classification reveals that IE is a selective barrier for transport of many metabolites and plays a major role in controlling protein homeostasis, whereas proteins in OE are more heterogeneous and participate in a wide range of processes. Data support that metabolic processes previously described to occur in the envelope such as chlorophyll and tocopherol biosynthesis can be ascribed to the IE, whereas others such as carotenoid or lipid biosynthesis occur in both membranes. Furthermore, results allow empirical assignation to the IE and/or OE of many proteins previously assigned to the bulk chloroplast envelope proteome.

Laser microdissection coupled to transcriptional profiling of Arabidopsis roots inoculated by Plasmodiophora brassicae indicates a role for brassinosteroids in clubroot formation.

The clubroot disease caused by the obligate biotrophic protist Plasmodiophora brassicae on host plants of the Brassicaceae family is characterized by enhanced cell division and cell expansion. Since a typical root section of an infected plant always includes different stages of the pathogen as well as uninfected cells, we were interested in investigating specific developmental stages of the pathogen and their effect on host transcriptional changes. We extended previous microarray studies on whole roots by using laser microdissection and pressure catapulting (LMPC) to isolate individual cells harboring defined developmental stages of the pathogen. In addition, we compared the central cylinder of infected plants with that of control plants. We were especially interested in elucidating the stage-specific hormonal network. The up-regulation of genes involved in auxin and cytokinin metabolism and signaling was confirmed. In addition, we found evidence that brassinosteroid (BR) synthesis and signal perception genes were in many cases up-regulated in enlarged cells and the central cylinder. This was confirmed by quantitative PCR. Treatment of wild-type plants with the BR biosynthesis inhibitor propiconazole reduced gall formation, and the analysis of the BR receptor mutant bri1-6 revealed less severe gall formation than in the respective wild type. Our results identify novel hormone pathways involved in clubroot development. Using LMPC to generate pools of homogeneous cell type populations combined with transcriptome analysis has been very useful to elucidate the regulation of gall growth by this obligate biotropic pathogen in a cell- and stage-specific manner.

The glycine-rich domain of GRP7 plays a crucial role in binding long RNAs and facilitating phase separation.

Microscale thermophoresis (MST) is a well-established method to quantify protein-RNA interactions. In this study, we employed MST to analyze the RNA binding properties of glycine-rich RNA binding protein 7 (GRP7), which is known to have multiple biological functions related to its ability to bind different types of RNA. However, the exact mechanism of GRP7's RNA binding is not fully understood. While the RNA-recognition motif of GRP7 is known to be involved in RNA binding, the glycine-rich region (known as arginine-glycine-glycine-domain or RGG-domain) also influences this interaction. To investigate to which extend the RGG-domain of GRP7 is involved in RNA binding, mutation studies on putative RNA interacting or modulating sites were performed. In addition to MST experiments, we examined liquid-liquid phase separation of GRP7 and its mutants, both with and without RNA. Furthermore, we systemically investigated factors that might affect RNA binding selectivity of GRP7 by testing RNAs of different sizes, structures, and modifications. Consequently, our study revealed that GRP7 exhibits a high affinity for a variety of RNAs, indicating a lack of pronounced selectivity. Moreover, we established that the RGG-domain plays a crucial role in binding longer RNAs and promoting phase separation.

Protein profile of Lupinus texensis phloem sap exudates: searching for Fe- and Zn-containing proteins.

The aim of this study was to obtain a comprehensive overview of the phloem sap protein profile of Lupinus texensis, with a special focus on proteins binding Fe and Zn. L. texensis was chosen as model plant given the simplicity to obtain exudates from sieve elements. Protein profiling by 2DE revealed 249 spots, and 54 of them were unambiguously identified by MALDI-MS and ESI-MS/MS. The largest number of identified protein species belongs to protein modification/turnover and general metabolism (19-21%), followed by redox homeostasis (9%) and defense and cell structural components (7%). This protein profile is similar to that reported in other plant species, suggesting that the phloem sap proteome is quite conserved. Staining of 2DE gels for Fe-containing proteins and affinity chromatography experiments revealed the presence of two low molecular weight Fe-binding proteins in phloem sap: a metallothionein-like protein type 2B identified in the Fe-affinity chromatography, and a second protein identified with both Fe staining methods. This protein species had a molecular weight of 13.5 kDa, a pI of 5.6 and 51% homology to a phloem-specific protein from Medicago truncatula. Zinc affinity chromatography revealed four Zn-binding proteins in phloem sap, one belonging to the dehydrin family and three Zn finger proteins.

Y3IP1, a nucleus-encoded thylakoid protein, cooperates with the plastid-encoded Ycf3 protein in photosystem I assembly of tobacco and Arabidopsis.

The intricate assembly of photosystem I (PSI), a large multiprotein complex in the thylakoid membrane, depends on auxiliary protein factors. One of the essential assembly factors for PSI is encoded by ycf3 (hypothetical chloroplast reading frame number 3) in the chloroplast genome of algae and higher plants. To identify novel factors involved in PSI assembly, we constructed an epitope-tagged version of ycf3 from tobacco (Nicotiana tabacum) and introduced it into the tobacco chloroplast genome by genetic transformation. Immunoaffinity purification of Ycf3 complexes from the transplastomic plants identified a novel nucleus-encoded thylakoid protein, Y3IP1 (for Ycf3-interacting protein 1), that specifically interacts with the Ycf3 protein. Subsequent reverse genetics analysis of Y3IP1 function in tobacco and Arabidopsis thaliana revealed that knockdown of Y3IP1 leads to a specific deficiency in PSI but does not result in loss of Ycf3. Our data indicate that Y3IP1 represents a novel factor for PSI biogenesis that cooperates with the plastid genome-encoded Ycf3 in the assembly of stable PSI units in the thylakoid membrane.

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.

Long-Distance Transported RNAs: From Identity to Function.

There is now a wealth of data, from different plants and labs and spanning more than two decades, which unequivocally demonstrates that RNAs can be transported over long distances, from the cell where they are transcribed to distal cells in other tissues. Different types of RNA molecules are transported, including micro- and messenger RNAs. Whether these RNAs are selected for transport and, if so, how they are selected and transported remain, in general, open questions. This aspect is likely not independent of the biological function and relevance of the transported RNAs, which are in most cases still unclear. In this review, we summarize the experimental data supporting selectivity or nonselectivity of RNA translocation and review the evidence for biological functions. After discussing potential issues regarding the comparability between experiments, we propose criteria that need to be critically evaluated to identify important signaling RNAs.

Exact Bayesian inference for the detection of graft-mobile transcripts from sequencing data.

The long-distance transport of messenger RNAs (mRNAs) has been shown to be important for several developmental processes in plants. A popular method for identifying travelling mRNAs is to perform RNA-Seq on grafted plants. This approach depends on the ability to correctly assign sequenced mRNAs to the genetic background from which they originated. The assignment is often based on the identification of single-nucleotide polymorphisms (SNPs) between otherwise identical sequences. A major challenge is therefore to distinguish SNPs from sequencing errors. Here, we show how Bayes factors can be computed analytically using RNA-Seq data over all the SNPs in an mRNA. We used simulations to evaluate the performance of the proposed framework and demonstrate how Bayes factors accurately identify graft-mobile transcripts. The comparison with other detection methods using simulated data shows how not taking the variability in read depth, error rates and multiple SNPs per transcript into account can lead to incorrect classification. Our results suggest experimental design criteria for successful graft-mobile mRNA detection and show the pitfalls of filtering for sequencing errors or focusing on single SNPs within an mRNA.

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.

Phloem small RNAs, nutrient stress responses, and systemic mobility.

BACKGROUND: Nutrient availabilities and needs have to be tightly coordinated between organs to ensure a balance between uptake and consumption for metabolism, growth, and defense reactions. Since plants often have to grow in environments with sub-optimal nutrient availability, a fine tuning is vital. To achieve this, information has to flow cell-to-cell and over long-distance via xylem and phloem. Recently, specific miRNAs emerged as a new type of regulating molecules during stress and nutrient deficiency responses, and miR399 was suggested to be a phloem-mobile long-distance signal involved in the phosphate starvation response. RESULTS: We used miRNA microarrays containing all known plant miRNAs and a set of unknown small (s) RNAs earlier cloned from Brassica phloem sap 1, to comprehensively analyze the phloem response to nutrient deficiency by removing sulfate, copper or iron, respectively, from the growth medium. We show that phloem sap contains a specific set of sRNAs that is distinct from leaves and roots, and that the phloem also responds specifically to stress. Upon S and Cu deficiencies phloem sap reacts with an increase of the same miRNAs that were earlier characterized in other tissues, while no clear positive response to -Fe was observed. However, -Fe led to a reduction of Cu- and P-responsive miRNAs. We further demonstrate that under nutrient starvation miR399 and miR395 can be translocated through graft unions from wild type scions to rootstocks of the miRNA processing hen1-1 mutant. In contrast, miR171 was not transported. Translocation of miR395 led to a down-regulation of one of its targets in rootstocks, suggesting that this transport is of functional relevance, and that miR395, in addition to the well characterized miR399, could potentially act as a long-distance information transmitter. CONCLUSIONS: Phloem sap contains a specific set of sRNAs, of which some specifically accumulate in response to nutrient deprivation. From the observation that miR395 and miR399 are phloem-mobile in grafting experiments we conclude that translocatable miRNAs might be candidates for information-transmitting molecules, but that grafting experiments alone are not sufficient to convincingly assign a signaling function.

MicroRNA399 is a long-distance signal for the regulation of plant phosphate homeostasis.

The presence of microRNA species in plant phloem sap suggests potential signaling roles by long-distance regulation of gene expression. Proof for such a role for a phloem-mobile microRNA is lacking. Here we show that phosphate (Pi) starvation-induced microRNA399 (miR399) is present in the phloem sap of two diverse plant species, rapeseed and pumpkin, and levels are strongly and specifically increased in phloem sap during Pi deprivation. By performing micro-grafting experiments using Arabidopsis, we further show that chimeric plants constitutively over-expressing miR399 in the shoot accumulate mature miR399 species to very high levels in their wild-type roots, while corresponding primary transcripts are virtually absent in roots, demonstrating shoot-to-root transport. The chimeric plants exhibit (i) down-regulation of the miR399 target transcript (PHO2), which encodes a critical component for maintenance of Pi homeostasis, in the wild-type root, and (ii) Pi accumulation in the shoot, which is the phenotype of pho2 mutants, miR399 over-expressers or chimeric plants with a genetic knock-out of PHO2 in the root. Hence the transported miR399 molecules retain biological activity. This is a demonstration of systemic control of a biological process, i.e. maintenance of plant Pi homeostasis, by a phloem-mobile microRNA.

Identification and characterization of small RNAs from the phloem of Brassica napus.

Systemic signalling is indispensable for the coordination of diverse physiological processes during development, defence and nutrient allocation. Indirect evidence suggests that plant small RNAs (smRNAs) could be involved in long-distance information transfer via the vasculature of the plant. Analyses of the smRNA complements of vascular exudates from oilseed rape (Brassica napus) showed that xylem sap is devoid of RNA, whereas phloem sap contained a large number of smRNAs. In addition to 32 annotated microRNAs (miRNAs) from 18 different families that could be identified and approved, a set of unknown smRNAs, predominantly of 21 and 24 nucleotides in length, was obtained, and selected candidates were found to be highly abundant in phloem sap. Moreover, we could demonstrate that the levels of three miRNAs known to respond to nutrient deprivation in non-vascular tissue, miR395 (sulphate), miR398 (copper) and miR399 (phosphate), were increased in phloem sap during the growth of plants under the respective starvation conditions. Interestingly, only mature miRNA molecules were found to be stress responsive, demonstrating that single-stranded sense miRNAs are most likely to represent the physiologically relevant molecules. The strong responses in the phloem suggest a role of miRNAs in systemic information transfer via this long-distance transport system.

Identification of high levels of phytochelatins, glutathione and cadmium in the phloem sap of Brassica napus. A role for thiol-peptides in the long-distance transport of cadmium and the effect of cadmium on iron translocation.

Phytochelatins (PCs) are glutathione-derived peptides that function in heavy metal detoxification in plants and certain fungi. Recent research in Arabidopsis has shown that PCs undergo long-distance transport between roots and shoots. However, it remains unknown which tissues or vascular systems, xylem or phloem, mediate PC translocation and whether PC transport contributes to physiologically relevant long-distance transport of cadmium (Cd) between shoots and roots. To address these questions, xylem and phloem sap were obtained from Brassica napus to quantitatively analyze which thiol species are present in response to Cd exposure. High levels of PCs were identified in the phloem sap within 24 h of Cd exposure using combined mass spectrometry and fluorescence HPLC analyses. Unexpectedly, the concentration of Cd was more than four-fold higher in phloem sap compared to xylem sap. Cadmium exposure dramatically decreased iron levels in xylem and phloem sap whereas other essential heavy metals such as zinc and manganese remained unchanged. Data suggest that Cd inhibits vascular loading of iron but not nicotianamine. The high ratios [PCs]/[Cd] and [glutathione]/[Cd] in the phloem sap suggest that PCs and glutathione (GSH) can function as long-distance carriers of Cd. In contrast, only traces of PCs were detected in xylem sap. Our results suggest that, in addition to directional xylem Cd transport, the phloem is a major vascular system for long-distance source to sink transport of Cd as PC-Cd and glutathione-Cd complexes.

Enzyme activity and structural features of three single-domain phloem cyclophilins from Brassica napus.

Cyclophilins (CYPs) are a group of ubiquitous prolyl cis/trans isomerases (PPIases). It was shown that plants possess the most diverse CYP families and that these are abundant in the phloem long-distance translocation stream. Since phloem exudate showed PPIase activity, three single-domain CYPs that occur in phloem samples from Brassica napus were characterised on functional and structural levels. It could be shown that they exhibit isomerase activity and that this activity is controlled by a redox regulation mechanism, which has been postulated for divergent CYPs. The structure determination by small-angle X-ray scattering experiments revealed a conserved globular shape. In addition, the high-resolution crystal structure of BnCYP19-1 was resolved and refined to 2.0 Å resolution, and the active sites of related CYPs as well as substrate binding were modelled. The obtained data and results support the hypothesis that single domain phloem CYPs are active phloem PPIases that may function as chaperones.

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.