An important method for plant genetic modification is using peptide/pDNA complexes to transfer genes into plant cells. With conventional carrier peptides, the peptide sequence must contain a high amount of cationic amino acids to condense and introduce pDNA. As a result, the dissociation of pDNA from the complex is inefficient, often causing problems. Herein, we designed a new peptide carrier that mimics the basic leucine zipper (bZIP) domain of DNA-binding proteins, in which (LU) 4 is the leucine zipper motif and (KUA) 3 is the basic DNA-binding and cell-penetrating motif (U = α-aminoisobutyric acid). After (KUA) 3 -(LU) 4 peptide was mixed with pDNA, DNA molecules were condensed to form nanoparticles of approximately 130 nm. Furthermore, when complexes of (KUA) 3 -(LU) 4 peptide and pDNA were introduced into the leaves of Arabidopsis thaliana ( A. thaliana ), expression of the reporter protein was detected in the plant cells. Thus, (KUA) 3 -(LU) 4 peptide that mimics the bZIP domain is a novel and efficient carrier for pDNA with high dissociation efficiency.

Plants continuously remodel and degrade their organelles due to damage from their metabolic activities and environmental stressors, as well as an integral part of their cell differentiation programs. Whereas certain organelles use local hydrolytic enzymes for limited remodeling, most of the pathways that control the partial or complete dismantling of organelles rely on vacuolar degradation. Specifically, selective autophagic pathways play a crucial role in recognizing and sorting plant organelle cargo for vacuolar clearance, especially under cellular stress conditions induced by factors like heat, drought, and damaging light. In these short reviews, we discuss the mechanisms that control the vacuolar degradation of chloroplasts, mitochondria, endoplasmic reticulum, Golgi, and peroxisomes, with an emphasis on autophagy, recently discovered selective autophagy receptors for plant organelles, and crosstalk with other catabolic pathways.

Light is essential for plant growth, but excessive light energy produces reactive oxygen species (ROS), which can seriously damage cells. Mutants defective in ATG (autophagy related) genes show light intensity-dependent leaf damage and ROS accumulation. We found that autophagy is one of the crucial systems in protecting plants from ROS-induced damage by removing oxidative peroxisomes. Damaged peroxisomes are targeted by the PtdIns3P marker and specifically engulfed by phagophores labeled by ATG18a-GFP. Under high-intensity light, huge peroxisome aggregates are induced and captured by vacuolar membranes. Research provides a deeper understanding of plant stress response to light irradiation.

Studies on how exogenous molecules modulate properties of plant microtubules, such as their stability, structure, and dynamics, are important for understanding and modulating microtubule functions in plants. We have developed a Tau-derived peptide (TP) that binds to microtubules and modulates their properties by binding of TP-conjugated molecules in vitro . However, there was no investigation of TPs on microtubules in planta . Here, we generated transgenic Arabidopsis thaliana plants stably expressing TP-fused superfolder GFP (sfGFP-TP) and explored the binding properties and effects of sfGFP-TP on plant microtubules. Our results indicate that the expressed sfGFP-TP binds to the plant microtubules without inhibiting plant growth. A transgenic line strongly expressing sfGFP-TP produced thick fibrous structures that were stable under conditions where microtubules normally depolymerize. This study generates a new tool for analyzing and modulating plant microtubules.

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.

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.

Organelles contribute to plant growth via their movements and interactions, which ensure efficient metabolic flow and help plants adapt to environmental stress. Live-cell imaging of the interactions of organelles has been performed in yeast, plant, and animal cells. However, high-throughput quantitative methods are needed to simultaneously analyze the interactions of many organelles in living plant cells. Here, we developed a semi-automatic high-throughput method to quantitatively evaluate the interactions between peroxisomes and chloroplasts using a distance transformation algorithm and high-resolution 3D fluorescent images taken by confocal laser scanning microscopy. Using this method, we measured the 3D distance between the center of peroxisome and chloroplast surface in Arabidopsis thaliana. We then compared the distances between these organelles in leaf mesophyll cells under light and dark conditions. This distance was shorter in the light than in the dark, which is in agreement with the findings of previous studies. We used our method to evaluate peroxisome-chloroplast (plastid) interactions in different cell types in the light and dark, including guard, stem, and root cells. Like in mesophyll cells, the distance between the peroxisome and chloroplast was shorter in the light in guard and stem cells, but not in root cells, suggesting that photosynthetic plastids (chloroplasts) play important roles in these interactions. When leaf mesophyll cells were incubated under high-intensity light, the frequency of shorter distances between peroxisomes and chloroplasts significantly increased. Our high-throughput, semi-automatic method represents a powerful tool for evaluating peroxisome-chloroplast interactions in different types of plant cells under various environmental conditions.

Plant mitochondria move dynamically inside cells and this movement is classified into two types: directional movement, in which mitochondria travel long distances, and wiggling, in which mitochondria travel short distances. However, the underlying mechanisms and roles of both types of mitochondrial movement, especially wiggling, remain to be determined. Here, we used confocal laser-scanning microscopy to quantitatively characterize mitochondrial movement (rate and trajectory) in Arabidopsis thaliana mesophyll cells. Directional movement leading to long-distance migration occurred at high speed with a low angle-change rate, whereas wiggling leading to short-distance migration occurred at low speed with a high angle-change rate. The mean square displacement (MSD) analysis could separate these two movements. Directional movement was dependent on filamentous actin (F-actin), whereas mitochondrial wiggling was not, but slightly influenced by F-actin. In mesophyll cells, mitochondria could migrate by wiggling, and most of these mitochondria associated with chloroplasts. Thus, mitochondria migrate via F-actin-independent wiggling under the influence of F-actin during their association with chloroplasts in Arabidopsis.

Mitochondria-selective fluorescent probes such as MitoTracker are often used for mitochondria imaging in various plants. Although some of the probes are reported to induce mitochondria dysfunction in animal cells, the effect on plant cells remains to be determined. In the present study, we applied quantitative methods to analyze mitochondrial movement, speed frequency, and speed-angle changes, based on trajectory analysis of mitochondria in mesophyll protoplast cells of Arabidopsis thaliana expressing the mitochondria-localized fluorescent protein. Using the quantitative method, we assessed whether MitoTracker Red (FM and CMXRos) induce mitochondria dysfunction in A. thaliana. Although both the fluorescent probes well-stained mitochondria, the CMXRos probe, not the FM probe, gave a severe effect on mitochondrial movement at the low concentration (10 nM), indicating a MitoTracker-induced mitochondria dysfunction in A. thaliana. These results revealed that our quantitative method based on mitochondrial movement can be used to determine the appropriate concentrations of mitochondria-selective fluorescent probes in plants.

In the genetic modification of plant cells, the mitochondrion is an important target in addition to the nucleus and plastid. However, gene delivery into the mitochondria of plant cells has yet to be established by conventional methods, such as particle bombardment, because of the small size and high mobility of mitochondria. To develop an efficient mitochondria-targeting signal (MTS) that functions in plant cells, we designed the artificial peptide (LURL)3 and its analogues, which periodically feature hydrophobic α-aminoisobutyric acid (Aib, U) and cationic arginine (R), considering the consensus motif recognized by the mitochondrial import receptor Tom20. Circular dichroism measurements and molecular dynamics simulation studies revealed that (LURL)3 had a propensity to form a stable α-helix in 0.1 M phosphate buffer solution containing 1.0 wt % sodium dodecyl sulfate. After internalization into plant cells via particle bombardment, (LURL)3 revealed highly selective accumulation in the mitochondria, whereas its analogue (LARL)3 was predominantly located in the vacuoles in addition to mitochondria. The high selectivity of (LURL)3 can be attributed to the incorporation of Aib, which promotes the hydrophobic interaction between the MTS and Tom20 by increasing the hydrophobicity and helicity of (LURL)3. The present study provided a prospective mitochondrial targeting system using the simple design of artificial peptides.

Each plant cell has hundreds of copies of the chloroplast genome and chloroplast transgenes do not undergo silencing. Therefore, chloroplast transformation has many powerful potential agricultural and industrial applications. We previously succeeded in integrating exogenous genes into the chloroplast genome using peptide-DNA complexes composed of plasmid DNA and a fusion peptide consisting of a cell-penetrating peptide (CPP) and a chloroplast transit peptide (cpPD complex). However, how cpPD complexes are transported into the chloroplast from outside the cell remains unclear. Here, to characterize the route by which these cpPD complexes move into chloroplasts, we tracked their movement from the extracellular space to the chloroplast stroma using a fluorescent label and confocal laser scanning microscopy (CLSM). Upon infiltration of cpPD complexes into the extracellular space of Arabidopsis thaliana leaves, the complexes reached the chloroplast surface within 6h. The cpPD complexes reached were engulfed by the chloroplast outer envelope membrane and gradually integrated into the chloroplast. We detected several cpPD complexes localized around chloroplast nucleoids and observed the release of DNA from the cpPD. Our results thus define the route taken by the cpPD complexes for gene delivery from the extracellular space to the chloroplast stroma.

To visualize and simultaneously quantify the necking behavior of crystalline polymer films during uniaxial stretching, tetraarylsuccinonitrile (TASN) moieties were introduced into polymers at the center of the main chain. TASN can produce a relatively stable radical that emits yellow fluorescence in response to mechanical stress. During the uniaxial elongation test of the TASN-centered crystalline polymers, the yellow fluorescence derived from the dissociated TASN radicals was used for microscale observations that showed the orientation of the polymer chains in the stretching direction. Furthermore, by comparing the radical generation in linear and star-shaped TASN-centered crystalline polymers during their tensile deformation, we found that the TASN dissociation ratio is higher in the star-shaped polymer, which has more chains connected to the lamellar crystal. Thus, the microforces generated in the amorphous region during uniaxial stretching were probed via the use of TASN, which allowed a direct visualization of the necking initiation and propagation processes as well as a quantification via electron paramagnetic resonance spectroscopy.

The growth of lamellar crystals has been studied in particular for spherulites in polymeric materials. Even though such spherulitic structures and their growth are of crucial importance for the mechanical and optical properties of the resulting polymeric materials, several issues regarding the residual stress remain unresolved in the wider context of crystal growth. To gain further insight into micro-mechanical forces during the crystallization process of lamellar crystals in polymeric materials, herein, we introduce tetraarylsuccinonitrile (TASN), which generates relatively stable radicals with yellow fluorescence upon homolytic cleavage at the central C–C bond in response to mechanical stress, into crystalline polymers. The obtained crystalline polymers with TASN at the center of the polymer chain allow not only to visualize the stress arising from micro-mechanical forces during polymer crystallization via fluorescence microscopy but also to evaluate the micro-mechanical forces upon growing polymer lamellar crystals by electron paramagnetic resonance, which is able to detect the radicals generated during polymer crystallization.

Light is essential for photosynthesis, but it has the potential to elevate intracellular levels of reactive oxygen species (ROS) during photosynthesis. Photorespiration is a metabolic pathway in photosynthesis to metabolise oxidised by products from chloroplasts, and it generates a high level of ROS in peroxisomes. Since high levels of ROS are toxic, plants must manage damage from ROS. However, the cellular mechanism to elude leaf damage from ROS in peroxisomes is 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 found that a series of peup mutants, which is a defect in autophagy degradation of peroxisomes, showed light-intensity-dependent leaf damage and excess aggregation of ROS-accumulating peroxisomes. The peroxisome aggregates were specifically engulfed by pre-autophagosomal structures and vacuolar membranes, but they were not degraded in the mutants. ATG18a-GFP and GFP-2 × FYVE, which both bind to phosphatidylinositol 3-phosphate, preferentially targeted the peroxisomal membranes and pre-autophagosomal structures near peroxisomes in ROS-accumulated cells under high-intensity light conditions. Our findings provide new information to better understand the plant stress response caused by light irradiation.

Cell-penetrating peptides (CPPs) have been widely utilized as efficient molecular tools for the delivery of bioactive cargoes such as peptides, proteins, and genetic material. However, to improve their versatility as tools in biological environments, the resistance of CPPs to enzymatic degradation and their structural stability must be improved to achieve long-term efficacy. Here we designed and synthesized novel artificial CPPs, poly(LysAibXaa), containing periodic α-aminoisobutyric acid (Aib) and l-lysine by chemoenzymatic polymerization. Poly(LysAibAla) tended to form 310- and α-helical structures under the amphiphilic cell-membrane-mimicking environment. Poly(LysAibXaa) exhibited long-term internalization and thus high accumulation in live cells, which is attributed to the improvement in the resistance to proteolytic digestion as a result of the incorporation of Aib residues into the peptide backbone. We presented a simple molecular design and synthesis of efficient CPPs applicable to both human and plant cells with long-term stability and negligible cytotoxicity.

The long-chain acyl-CoA synthetases (LACSs) are involved in lipid synthesis, fatty acid catabolism, and the transport of fatty acids between subcellular compartments. These enzymes catalyze the critical reaction of fatty acyl chains to fatty acyl-CoAs for the triacylglycerol biosynthesis used as carbon and energy reserves. In Arabidopsis, LACSs are encoded by a family of nine genes, with LACS9 being the only member located in the chloroplast envelope membrane. However, the comprehensive role of LACS9 and its contribution to plant metabolism have not been explored thoroughly. In this study, we report on the identification and characterization of LACS9 mutants in rice plants. Our results indicate that the loss-of-function mutations in OsLACS9 affect the architecture of internodes resulting in dwarf plants with large starch granules in the chloroplast, showing the suppression of starch degradation. Moreover, the plastid localization of α-amylase I-1 (AmyI-1)—a key enzyme involved in starch breakdown in plastids—was suppressed in the lacs9 mutant line. Immunological and confocal laser scanning microscopy analyses showed that OsLACS9-GFP is located in the chloroplast envelope in green tissue. Microscopic analysis showed that OsLACS9s interact with each other in the plastid envelope membrane. Furthermore, OsLACS9 is also one of the proteins transported to plastids without a transit peptide or involvement of the Toc/Tic complex system. To identify the plastid-targeting signal of OsLACS9, the transient expression and localization of a series of N-terminal truncated OsLACS9-green fluorescent protein (GFP) fusion proteins were examined. Truncation analyses identified the N-terminal 30 amino acid residues to be required for OsLACS9 plastid localization. Overall, the data in this study provide an advanced understanding of the function of OsLACS9 and its role in starch degradation and plant growth.

The highly efficient genetic transformation of cells is essential for synthetic biology procedures, especially for the transformation of large gene clusters. In this technical note, we present a novel cell-penetrating peptide (CPP)-mediated large-sized plasmid DNA transformation system for Escherichia coli. A large plasmid (pMSR227, 205 kb) was complexed with cationic peptides containing a CPP motif and was successfully transformed into E. coli cells. The transformants containing the plasmid DNA exhibited expression of a reporter gene encoding a red fluorescent protein. The transformation efficiency was significantly higher than that obtained using the heat-shock method and was similar to that of electroporation. This technique can be used as a platform for the simple and highly efficient transformation of large DNA molecules under mild conditions without causing significant damage to DNA, accelerating synthetic biology investigations for the design of genetically engineered microorganisms for industrial purposes.

Reactive oxygen species (ROS) function as key signaling molecules to inhibit stomatal opening and promote stomatal closure in response to diverse environmental stresses. However, how guard cells maintain basal intracellular ROS levels is not yet known. This study aimed to determine the role of autophagy in the maintenance of basal ROS levels in guard cells. We isolated the Arabidopsis autophagy-related 2 (atg2) mutant, which is impaired in stomatal opening in response to light and low CO2 concentrations. Disruption of other autophagy genes, including ATG5, ATG7, ATG10, and ATG12, also caused similar stomatal defects. The atg mutants constitutively accumulated high levels of ROS in guard cells, and antioxidants such as ascorbate and glutathione rescued ROS accumulation and stomatal opening. Furthermore, the atg mutations increased the number and aggregation of peroxisomes in guard cells, and these peroxisomes exhibited reduced activity of the ROS scavenger catalase and elevated hydrogen peroxide (H2O2) as visualized using the peroxisome-targeted H2O2 sensor HyPer. Moreover, such ROS accumulation decreased by the application of 2-hydroxy-3-butynoate, an inhibitor of peroxisomal H2O2-producing glycolate oxidase. Our results showed that autophagy controls guard cell ROS homeostasis by eliminating oxidized peroxisomes, thereby allowing stomatal opening.

The dynamic behavior of organelles is essential for plant survival under various environmental conditions. Plant organelles, with various functions, migrate along actin filaments and contact other types of organelles, leading to physical interactions at a specific site called the membrane contact site. Recent studies have revealed the importance of physical interactions in maintaining efficient metabolite flow between organelles. In this review, we first summarize peroxisome function under different environmental conditions and growth stages to understand organelle interactions. We then discuss current knowledge regarding the interactions between peroxisome and other organelles, i.e., the oil bodies, chloroplast, and mitochondria from the perspective of metabolic and physiological regulation, with reference to various organelle interactions and techniques for estimating organelle interactions occurring in plant cells.