Image-based analysis revealing the molecular mechanism of peroxisome dynamics in plants

Peroxisomes are present in eukaryotic cells and have essential roles in various biological processes. Plant peroxisomes proliferate by de novo biosynthesis or division of pre-existing peroxisomes, degrade, or replace metabolic enzymes, in response to developmental stages, environmental changes, or external stimuli. Defects of peroxisome functions and biogenesis alter a variety of biological processes and cause aberrant plant growth. Traditionally, peroxisomal function-based screening has been employed to isolate Arabidopsis thaliana mutants that are defective in peroxisomal metabolism, such as lipid degradation and photorespiration. These analyses have revealed that the number, subcellular localization, and activity of peroxisomes are closely related to their efficient function, and the molecular mechanisms underlying peroxisome dynamics including organelle biogenesis, protein transport, and organelle interactions must be understood. Various approaches have been adopted to identify factors involved in peroxisome dynamics. With the development of imaging techniques and fluorescent proteins, peroxisome research has been accelerated. Image-based analyses provide intriguing results concerning the movement, morphology, and number of peroxisomes that were hard to obtain by other approaches. This review addresses image-based analysis of peroxisome dynamics in plants, especially A. thaliana and Marchantia polymorpha

Pexophagy suppresses ROS-induced damage in leaf cells under high-intensity light

Although light is essential for photosynthesis, it has the potential to elevate intracellular levels of reactive oxygen species (ROS). Since high ROS levels are cytotoxic, plants must alleviate such damage. However, the cellular mechanism underlying ROS-induced leaf damage alleviation in peroxisomes was not fully explored. Here, we show that autophagy plays a pivotal role in the selective removal of ROS-generating peroxisomes, which protects plants from oxidative damage during photosynthesis. We present evidence that autophagy-deficient mutants show light intensity-dependent leaf damage and excess aggregation of ROS-accumulating peroxisomes. The peroxisome aggregates are specifically engulfed by pre-autophagosomal structures and vacuolar membranes in both leaf cells and isolated vacuoles, but they are not degraded in mutants. ATG18a-GFP and GFP-2×FYVE, which bind to phosphatidylinositol 3-phosphate, preferentially target the peroxisomal membranes and pre-autophagosomal structures near peroxisomes in ROS-accumulating cells under high-intensity light. Our findings provide deeper insights into the plant stress response caused by light irradiation

Gateway Vectors for Simultaneous Detection of Multiple Protein-Protein Interactions in Plant Cells Using Bimolecular Fluorescence Complementation.

Bimolecular fluorescence complementation (BiFC) is widely used to detect protein-protein interactions, because it is technically simple, convenient, and can be adapted for use with conventional fluorescence microscopy. We previously constructed enhanced yellow fluorescent protein (EYFP)-based Gateway cloning technology-compatible vectors. In the current study, we generated new Gateway cloning technology-compatible vectors to detect BiFC-based multiple protein-protein interactions using N- and C-terminal fragments of enhanced cyan fluorescent protein (ECFP), enhanced green fluorescent protein (EGFP), and monomeric red fluorescent protein (mRFP1). Using a combination of N- and C-terminal fragments from ECFP, EGFP and EYFP, we observed a shift in the emission wavelength, enabling the simultaneous detection of multiple protein-protein interactions. Moreover, we developed these vectors as binary vectors for use in Agrobacterium infiltration and for the generate transgenic plants. We verified that the binary vectors functioned well in tobacco cells. The results demonstrate that the BiFC vectors facilitate the design of various constructions and are convenient for the detection of multiple protein-protein interactions simultaneously in plant cells

Novel gateway binary vectors for rapid tripartite DNA assembly and promoter analysis with various reporters and tags in the liverwort Marchantia polymorpha

The liverwort Marchantia polymorpha is an emerging model species for basal lineage plant research. In this study, two Gateway cloning-compatible binary vector series, R4pMpGWB and R4L1pMpGWB, were generated to facilitate production of transgenic M. polymorpha. The R4pMpGWB series allows tripartite recombination of any promoter and any coding sequence with a specific reporter or tag. Reporters/tags for the R4pMpGWB series are GUS, ELuc(PEST), FLAG, 3×HA, 4×Myc, mRFP1, Citrine, mCitrine, ER-targeted mCitrine and nucleus-targeted mCitrine. The R4L1pMpGWB series is suitable for promoter analysis. R4L1pMpGWB vector structure is the same as that of R4pMpGWB vectors, except that the attR2 site is replaced with attL1, enabling bipartite recombination of any promoter with a reporter or tag. Reporters/tags for the R4L1pMpGWB series are GUS, G3GFP-GUS, LUC, ELuc(PEST), Citrine, mCitrine, ER-targeted mCitrine and mCitrine-NLS. Both vector series were functional in M. polymorpha cells. These vectors will facilitate the design and assembly of plasmid constructs and generation of transgenic M. polymorpha

Detection of multiple protein—protein interactions using transient expression assays.

<p>(A, B) Two types of vectors were introduced as controls. Representative images of interactions of nRFP-PEX7 with PTS2-cRFP (A) and AtSEC31A-nYFP with AtSEC13A-cYFP (B). (C-E) Four types of vectors, nRFP-PEX7, PTS2-cRFP, AtSEC31A-nYFP, and AtSEC13A-cYFP, were introduced simultaneously into onion epidermal cells. Representative images of interactions of nRFP-PEX7 with PTS2-cRFP (C) and AtSEC31A-nYFP with AtSEC13A-cYFP (D) in the same cell. (E) Merged image of (C) with (D). Bar: 50 μm.</p

Immunodetection of transiently expressed nRFP/cRFP-fused proteins.

<p>Using protein extracts prepared from tobacco leaves of Agrobacterium-infiltrated plants analyzed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0160717#pone.0160717.g002" target="_blank">Fig 2</a>, the accumulation of nRFP/cRFP-fused proteins by Agrobacterium infiltration was confirmed by immunoblot analysis with anti-Myc or anti-HA antibodies. Lane 1, nRFP; Lanes 2 and 5, PMP38-nRFP with PMP38-cRFP; Lanes 3 and 6, nRFP-PEX7 with PTS2-cRFP; Lane 4, cRFP. Arrowheads indicate the positions of untagged or tagged polypeptides. Asterisks represent extra polypeptides, which are considered to be degradation products of the fusion proteins.</p

Schematic diagrams of the Gateway cloning technology-compatible vectors.

<p>A. ECFP, EGFP, EYFP and mRFP1 can be divided into two fragments. The letters ‘n’ and ‘c’ represent N- or C-terminal fragments of a split fluorescent protein, and the letters ‘C’, ‘G’, ‘Y’ and ‘R’ represent the type of fluorescent protein (ECFP, EGFP, EYFP or mRFP1). Since the nucleotide sequences of C-terminal CFP and C-terminal GFP are identical, we designated the fragment cCG. The letters ‘myc’ and ‘HA’ in the N- and C-terminal fragment from mRFP1 represent myc- and hemagglutinin-epitope tags, respectively. B. The structures of the region indicated as ‘Gateway’ in (C) and (D). GWnX and GWcX contain N- or C-terminal split fluorescent protein downstream of the <i>att</i>R2 site, respectively, whereas nXGW and cXGW contain N- or C-terminal split fluorescent protein upstream of the <i>att</i>R1 site, respectively. C. Outline of the pUGW-based vectors for BiFC. pGWnX, pGWcX, pnXGW, and pcXGW vectors, which bear the DNA fragment shown in (B) downstream of the 35S promoter from cauliflower mosaic virus. D. Outline of the binary vectors for BiFC. The pB4 and pB5 series contain Km^r and Hyg^r markers, respectively, which are placed in reverse orientation to the genes cloned via LR recombination. Details of plasmid construction and the vector backbone are given in Materials and Methods. <i>Cm</i>^<i>r</i>, chloramphenicol-resistance marker; <i>ccd</i>B, negative selection marker used in bacteria; <i>35Sp</i>, 35S promoter; <i>Tnos</i>, nopaline synthase terminator; myc, c-myc affinity tag; HA, hemagglutinin affinity tag.</p

Fluorescence emission spectra of intact and reconstituted fluorescent proteins.

<p>Using the interaction between PEX7 and PTS2 as one of the split combinations transiently expressed in onion epidermal cells, florescence emission spectra were measured following excitation with an argon laser at 458 nm (A–L) or a HeNe laser at 543 nm (J, K) using the LSM510 META system. X- and Y-axes represent wavelength and relative fluorescence intensity, respectively. The maximum emission of each spectrum was taken as 1.0, and in each curve, fluorescence at a given wavelength was normalized relative to the maximum. (A) non-split ECFP, (B) nCFP with cCGFP, (C) nCFP with cYFP, (D) non-split EGFP, (E) nGFP with cCGFP, (F) nGFP with cYFP, (G) non-split EYFP, (H) nYFP with cCGFP, (I) nYFP with cYFP, (J) non-split mRFP1 and (K) nRFP with cRFP.</p